Queensland University of Technology School of Life Sciences

Mutation Frequency of Non-ESBL Phenotype SENTRY (Asia-Pacific) Isolates of Klebsiella pneumoniae

Conversion to an ESBL Positive Phenotype

Submitted by Farshid Dakh, M.Sc, School of Life Sciences, Queensland University

of Technology in partial fulfillment of the requirements of the degree of

Master of Applied Science (Research)

December 2008

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Keywords β-lactamase, extended spectrum β-lactamase, ESBL, SENTRY, SHV, blaSHV,

IS26, mutation frequency, mutation rate, Klebsiella pneumoniae, real-time

PCR, MSS maximum likelihood

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Abstract

Extended spectrum β-lactamases or ESBLs, which are derived from non-

ESBL precursors by point mutation of β-lactamase genes (bla), are

spreading rapidly all over the world and have caused considerable problems

in the treatment of infections caused by bacteria which harbour them. The

mechanism of this resistance is not fully understood and a better

understanding of these mechanisms might significantly impact on choosing

proper diagnostic and treatment strategies. Previous work on SHV β-

lactamase gene, blaSHV, has shown that only Klebsiella pneumoniae strains

which contain plasmid-borne blaSHV are able to mutate to phenotypically

ESBL-positive strains and there was also evidence of an increase in blaSHV

copy number. Therefore, it was hypothesised that although specific point

mutation is essential for acquisition of ESBL activity, it is not yet enough, and

blaSHV copy number amplification is also essential for an ESBL-positive

phenotype, with homologous recombination being the likely mechanism of

blaSHV copy number expansion. In this study, we investigated the mutation

rate of non-ESBL expressing K. pneumoniae isolates to an ESBL-positive

status by using the MSS-maximum likelihood method. Our data showed that

blaSHV mutation rate of a non-ESBL expressing isolate is lower than the

mutation rate of the other single base changes on the chromosome, even

with a plasmid-borne blaSHV gene. On the other hand, mutation rate from a

low MIC ESBL-positive (≤ 8 µg/mL for cefotaxime) to high MIC ESBL-positive

(≥16 µg/mL for cefotaxime) is very high. This is because only gene copy

number increase is needed which is probably mediated by homologous

recombination that typically takes place at a much higher frequencies than

point mutations. Using a subinhibitory concentration of novobiocin, as a

homologous recombination inhibitor, revealed that this is the case.

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Contents

1 Introduction and History ...................................................................... 1

1.1 β-lactams .......................................................................................... 2

1.2 Classes of β-lactams ........................................................................ 2

1.3 β-lactamses ...................................................................................... 5

1.4 Nomenclature and Classification of β-lactamses .............................. 5

1.5 ESBLs ............................................................................................. 11

1.6 SHV β-lactamases .......................................................................... 12

1.7 ESBL Detection Methods ................................................................ 16

1.7.1 Phenotypic Tests or Clinical Microbiology Techniques ............. 16

1.7.2 Genotypic Tests or Molecular Detection Methods .................... 21

1.8 Epidemiology and Dissemination of β-lactamase ........................... 24

1.8.1 Global Epidemiology ................................................................ 24

1.8.2 Molecular Epidemiology of Nosocomial Infections ................... 26

1.9 Bacterial Resistance to β-lactam Antibiotics ................................... 27

1.10 blaSHV Gene Evolution ..................................................................... 27

1.11 Origin of Mutations .......................................................................... 29

1.12 Gene Amplification .......................................................................... 32

1.13 Heteroresistance ............................................................................. 33

1.14 Treatment of Infections with ESBL-Producing Organisms .............. 34

1.15 Mutation Rate Determination .......................................................... 35

1.15.1 Mutation Rate versus Mutation Frequency ............................... 35

1.15.2 Poisson Distribution .................................................................. 37

1.15.3 The MSS Maximum-Likelihood (MSS-MLL) Method ................ 38

1.15.4 Calculation of Mutation Rate .................................................... 40

1.16 Summary ........................................................................................ 42

1.17 Aims ................................................................................................ 44

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2 Mutation Rate Determination ............................................................. 45

2.1 Materials and Methods .................................................................... 45

2.1.1 Bacterial Isolates ...................................................................... 45

2.1.2 Determination of Mutation Rates .............................................. 47

2.1.3 ESBL Phenotypic Detection ..................................................... 49

2.1.4 DNA Extraction for Real-Time PCR Analysis ........................... 49

2.1.5 Real-Time PCR and Relative Quantitation of blaSHV ................ 49

2.2 Results ............................................................................................ 51

2.3 Mutation Rate from a Low MIC ESBL-Positive Phenotype to a High

MIC ESBL-Positive Phenotype .......................................................... 56

3 Detection of Heteroresistance to Cefotaxime in K. pneumoniae

Isolates by Population Analysis Profile (PAP) Method ....................... 59

3.1 PAP Experiments for Cefotaxime-Resistant K. pneumoniae Strains ....... 59

3.2 Results ............................................................................................ 60

4 Evaluation of the Effect of Novobiocin on the blaSHV Copy Number Expansion ............................................................................................... 69

5 Discussion ........................................................................................... 73

Appendices ................................................................................................. 79

A. MSS-MLE Program in MATLAB R2007a Software ......................... 79

B. nllike_m.m Program ........................................................................ 80

References.................................................................................................. 81

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List of Figures Figure 1. A β-lactam ring ................................................................................ 3

Figure 2. A cephem nucleus .......................................................................... 3

Figure 3. Tertiary structure of a class A TEM β-lactamase .......................... 10

Figure 4. ESBL Etest® results ...................................................................... 19

Figure 5. Comparison between mutation frequency and mutation rate ........ 36

Figure 6. Maximum likelihood estimate of one mutant colony in twenty

parallel cultures ............................................................................................ 41

Figure 7. Determination of mutation rate by fluctuation analysis method ..... 48

Figure 8. ESBL Etest® experiments of K2 three mutant colonies ................. 55

Figure 9. F1 mutant colonies in different cefotaxime concentration ............. 58

Figure 10. Phenotypic (Etest®) and genotypic (blaSHV relative copy number)

experiments of F1 and B1 isolates of the PAH collection ............................. 62

Figure 11. Total blaSHV relative copy number of B1 and F1 isolates ............ 64

Figure 12. PAP results of isolates A1 and D1 of the PAH collection ............ 66

Figure 13. PAP results of isolates E1 and F1 of the PAH collection ............ 67

Figure 14. Population analysis profile for cefotaxime-resistant Klebsiella

pneumoniae strains from PAH isolates ........................................................ 68

Figure 15. Effect of novobiocin on the colony size and distribution of F1 strain

..................................................................................................................... 71

Figure 16. Effect of novobiocin on PAH F1 isolate growth and blaSHV copy

number ......................................................................................................... 72

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List of Tables Table 1. Nomenclature of some of the β-lactamases ..................................... 6

Table 2. β-lactamases classification ............................................................ 8

Table 3. A sample of SHV β-lactamases .................................................... 15

Table 4. K. pneumoniae PAH and SENTRY isolates genotype and phenotype

..................................................................................................................... 46

Table 5. Primers for kinetic or real-time PCR ............................................... 50

Table 6. Number of the mutant colonies in each 20 parallel culture plate of

different isolates ........................................................................................... 52

Table 7. Mutation rates of PAH and SENTRY Klebsiella pneumoniae isolates

..................................................................................................................... 53

Table 8. Quantification of blaSHV 238 mutant alleles of K2 mutant colonies . 55

Table 9. Total blaSHV relative copy number of K2 mutant colonies ............... 55

Table 10. Total blaSHV relative copy number of B1 and F1 isolates .............. 63

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Statement of original authorship

“The work contained in this thesis has not been previously submitted to meet

requirements for an award at this or any other higher education institution. To

the best of my knowledge and belief, the thesis contains no material

previously published or written by another person except where due

reference is made.”

Farshid Dakh

15/12/2008

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Acknowledgments

My thanks go to:

• My supervisors, Prof Philip Giffard and Dr Mark Turner, for their

guidance and encouragement

• A/Prof Terry Walsh, for his great support

• Dr Jan Bell and Dr John Turnidge, for the bacterial isolates

• Joshua Chan and Dr David Hammond, for the computer program

• All of our research team, Tegan Harris, Alex Stephens, Daniel Barry,

Raquel Lo, Shreema Merchant, for their great help

• My lovely family, Fariba ,Kosar, my Mum and Dad, for their support

and patience

And special thanks to QUT Research Students Centre and School of Life

Sciences, for supporting me by scholarships

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Chapter 1

1 Introduction and History

Penicillin, the first β-lactam antibiotic, was discovered by chance by Sir

Alexander Fleming, the Scottish microbiologist, at St. Mary’s Hospital in

London in 1928 (Fleming 1929) but it was not used in clinics until 1940. The

main development of penicillin as a clinically relevant chemotherapeutic was

done in 1939 by an Australian scientist Howard Florey and his team. They

published a report describing “penicillin as a chemotherapeutic agent” (Chain

et al. 1940; Florey and Abraham 1951). During World War II, penicillin made

a major difference in the number of deaths and amputations caused by

infected wounds in Allied Forces. Before penicillin became clinically used as

an antibiotic, in 1940, Abraham and Chain identified the ability of bacteria to

produce an enzyme that destroys penicillin. They called it penicillinase

(Abraham and Chain 1940; Mascaretti 2003; Turner 2005). Since these

enzymes can inactivate other β-lactams such as cephalosporins,

carbapenems, and monobactams, they are now called β-lactamases. These

groups of enzymes hydrolyse the β-lactam ring of β-lactam antibiotics.

Bacterial resistance to antibiotics and worldwide dissemination of these

resistant strains is one of the major problems encountered in clinics.

Production of β-lactamases is one of the most common sources of resistance

in bacteria against these types of antimicrobial agents. The history of the

development of antibiotics shows an “arms race” in which resistance is

circumvented by new β-lactams which in turn are circumvented by the

evolution of new β-lactamases.

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1.1 β-lactams

A β-lactam is a four member cyclic amide and the common structural feature

among β-lactam antibiotics and β-lactamase inhibitors (Demarco and

Nagarajan 1972; Mascaretti 2003). The major resistance mechanism to

β-lactam antibiotics is the enzymatic cleavage of the β-lactam ring by

β-lactamases (Figure 1).

1.2 Classes of β-lactams

β-lactam antibiotics are divided into two groups: traditional group (penicillins

and cephalosporins) and non-classical or non-traditional group

(cephamycins, carbapenems and monobactams) with the latter being

discovered since 1971 (Mascaretti 2003). In this literature review, because of

the usage, only cephalosporins are described below.

Cephalosporins have a cephem nucleus (7-aminocephalosporanic acid),

which consists of a β-lactam ring fused to a dihydrothiazine ring (Figure 2).

Cephalosporins are not inactivated by many bacterial β-lactamases.

Therefore they have a broader spectrum of activity than other low spectrum

β-lactams.

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Figure 1. A β-lactam ring

Figure 2. A cephem nucleus

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Depending on the spectrum of antimicrobial activity, cephalosporins can be

classified into four major groups or generations which are summarized below

(Chambers 2007; Mascaretti 2003).

1. First generation cephalosporins include cefadroxyl, cefazolin,

cephalexin, cephalothin, cephapirin and cephradine. They have a narrow

spectrum of activity and relatively nontoxic.

2. Second generation cephalosporins include cefaclor, cefamandole,

cefonicid, cefuroxime, cefprozil, loracarbef, and ceforanide and structurally

related cephamycins such as cefoxitin, cefmetazole, and cefotetan. They are

less effective than first generation against Gram-positive bacteria but have

extended activity against Gram-negative bacteria.

3. Third generation cephalosporins include cefoperazone, cefotaxime,

ceftazidime, ceftizoxime, ceftriaxone, cefixime, cefpodoxime proxetil, cefdinir,

cefditoren pivoxil, ceftibuten, and moxalactam. These drugs have more

extended Gram-negative coverage than second generation and some are

able to cross blood brain barrier. They are used to treat a wide variety of

serious infections caused by organisms that are resistant to most other

drugs.

4. Fourth generation cephalosporins include cefepime and cefpirome.

Their potencies against members of Enterobacteriaceae are higher than

those of the earlier broad-spectrum cephalosporins. In addition, both

antibiotics remain effective against β-lactamase-overproducing gram-

negative bacterial strains resistant to other extended-spectrum

cephalosporins.

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1.3 β-lactamses

Production of β-lactamases is one of the important bacterial defense

mechanisms against β-lactam antibiotics. The number, diversity, and

substrate specificity of β-lactamases have been increased considerably over

60 years and the problem of resistance to β-lactam antibiotics has been

expanded. β-lactamases (EC 3.5.2.6) has been designated by the

Nomenclature Committee of International Union of Biochemistry and

Molecular Biology (NC-IUBMB) as a group of enzymes, hydrolysing cyclic

amides, amidines and other C-N bonds (Bush et al. 1995). The first

β-lactamase was identified by Abraham and Chain in 1940 before penicillins

became clinically used as antibiotics (Abraham and Chain 1940; Mascaretti

2003; Turner 2005). They termed it penicillinase. Since these enzymes can

inactivate other β-lactams such as cephalosporins, carbapenems, and

monobactams, they are now called β-lactamases. β-lactamases are plasmid

or chromosomally encoded enzymes which hydrolyse the β-lactam C-N bond

of β-lactam antibiotics. They are secreted into the periplasmic space of

Gram negative bacteria or are attached to the cell envelope in Gram positive

bacteria (Lai et al. 1981; Mascaretti 2003; Nielsen et al. 1981; Sarvas and

Palva 1983). Many of the Gram negative bacteria produce a natural

chromosomal mediated β-lactamase, which may help the bacteria in finding a

niche when faced with competition from other bacteria that naturally produce

β-lactams (Turner 2005). The known number, diversity, and substrate

specifity of β-lactamases has increased considerably over 60 years, and

resistance to β-lactam antibiotics has continued to be problematic.

1.4 Nomenclature and Classification of β-lactamses

β-lactamase nomenclature has neither been rational nor has followed a

consistent rule. Some have three and the others have four-letter

abbreviations. Table 1 shows the reasons of nomenclature of some different

β-lactamases (Helfand and Bonomo 2003; Jacoby 2006; Mascaretti 2003).

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Table 1. Nomenclature of some of the β-lactamases

β-lactamase Reason of nomenclature CARB Substrate they hydrolyse (carbenicillin) OXA Substrate they hydrolyse (oxacillin) IMP Substrate they hydrolyse (imipenem) CTX-M Substrate they hydrolyse (cefotaxime), first isolated at Munich IMI An imipenemase from Enterobacter cloacae TEM First three letters of the patient’s surname (Temoniera) SHV Biochemical properties (sulphydryl variable) NMC-A Abbr. of nonmetallo carbapenemase of Class A IBC Abbr. of integron-borne cephalosporinase OKP Abbr. of other Klebsiella pneumoniae β-Lactamase ACT AmpC type KPC Klebsiella pneumoniae carbapenemase PSE Refers to Pseudomonas-specific enzymes Sme Refers to Serratia marcescens enzyme SFO Refers to Serratia fonticola enzyme MIR Refers to a name of a hospital (Miriam Hospital) OHIO Refers to a particular state in US (discovered in Cleveland, Ohio) VEB Abbr. of Vietnam extended spectrum β-lactamase BES Abbr. of Brazilian extended spectrum β-lactamase GES Abbr. of (French)Guiana extended spectrum β-lactamase VIM Verona integron-encoded metallo-β-lactamase (most common in Taiwan) FPM Refers to a pharmaceutical company (Fujisawa’s Proteus mirabilis) HMS Refers to researchers who discovered them (Harris, Matthew, Sykes) TLA A β-lactamase found in Escherichia coli named after Tlahuicas Indians LEN A β-lactamase found in Klebsiella pneumoniae strain LEN-1 GC1 A β-lactamase found in Enterobacter cloacae strain GC1 PC1 A β-lactamase found in Staphylococcus aureus strain PC1

Adapted from Jacoby GA. (2006)

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Now more than 700 β-lactamases have been characterised. To organise the

nomenclature of β-lactamases, Bush and Jacoby have maintained an

Internet database initially for extended-spectrum and inhibitor-resistant

β-lactamases (http://www.lahey.org/studies/webt.html) (Bush and Jacoby

1997b, 1997a; Jacoby and Bush 2005). Since 1968, several classification

methods have been used as follow:

Sawai et al. 1968; Jack and Richmond, 1970; Richmond and Sykes, 1973;

Sykes and Matthew, 1976; Ambler 1980; Bush 1989 and Bush–Jacoby-

Medeiros functional classification (Bush et al. 1995).

β-lactamases have been classified according to the following aspects:

1. Function, especially substrate profile and sensitivity to inhibitors

2. Physical properties such as isoelectric point and molecular weight

3. Genetic location: plasmid or chromosome

β-lactamases have been commonly classified according to two general

schemes: the Ambler molecular classification scheme and the more recent,

Bush–Jacoby-Medeiros functional classification system (Ambler 1980; Bush

et al. 1995). Molecular structure classification of β-lactamases was first

proposed by Ambler in 1980 on the basis of their primary structure and amino

acid sequence identity. He divided β-lactamases into four classes: A, B, C,

and D (Table 2). Class A, C, and D β-lactamases have serine in their active

sites, while class B enzymes are metalloproteins containing Zn2+ ions in their

active subunits. According to Bush–Jacoby-Medeiros functional classification

β-lactamases are classified into four groups based on their functional

characteristics and substrate/inhibitor profiles (Table 2). Group 2 is the

largest category and contains six subgroups based on preferential hydrolysis

of penicillins, cephalosporins, oxyimino β-lactams, cloxacillin, carbenicillins,

or carbapenems. Over half of the β-lactamases are plasmid encoded.

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Table 2. β-lactamases classification (Bush et al. 1995)

Bush-Jacoby-Medeiros group

Molecular class

Preferred substrates

Inhibited by: Representative enzymes Clavulanic acid EDTA

1 C Cephalosporines - - AmpC β-lactamases from gram negative

bacteria; MIR-1 2a A Penicillins + - Penicillinases from gram-positive bacteria 2b A Penicillins, cephalosporins + - TEM-1, TEM-2, SHV-1 2be A Penicillins, narrow-spectrum + - TEM-3 to TEM-26, SHV-2 to SHV-6, and extended- spectrum K. oxytoca K1 cephalosporins and monobactams 2br A Penicillins ± - TEM-30 to TEM-36, TCR-1 2c A Penicillins, carbenicillin + - PSE-1, PSE-3, PSE-4 2d D Penicillin, cloxacillin ± - OXA-1 to OXA-11, PSE-2 (OXA-10) 2e A Cephalosporins + - Inducible cephalosporinases from

Proteus vulgaris 2f A Penicillins, cephalosporins,

carbapenems + - NMC-A from E. cloacae, Sme-1 from

S. marcescens3 B β-lactams including

carbapenems - + L1 from S. maltophila, CcrA from B. fragilis

4 ND Penicillin - ? Penicillinase from P. cepacia ND, not determined Printed with permission from the publisher and the author

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Regardless of the huge amino acid variability of β-lactamases, their globular

structure is similar and consists of alpha-helices and beta-plated sheets

(Knox 1995). X-ray crystallography of class A β-lactamases reveals an α/β

domain (a five-stranded antiparallel β-sheet surrounded by α-helices on both

sides) and an all-α domain with an active site in the groove between domains

(Figure 3) (Kuzin et al. 1999; Matagne et al. 1998).

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Figure 3. Tertiary structure of a class A TEM β-lactamase from Matagne et al. (1998)

[Printed with the publisher permission]

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1.5 ESBLs Extended Spectrum β-lactamases or ESBLs are enzymes which hydrolyse

extended-spectrum β-lactam antibiotics such as oxyimino cephalosporins

(e.g. cefotaxime, ceftazidime, ceftriaxone, cefepime, and cefpirome), as well

as oxyimino monobactam (aztreonam), therefore providing resistance to

these antibiotics. ESBLs are located in 2be subgroup (Ambler’s class A) and

subgroup 2d (Ambler’s class D) of Bush-Jacoby-Medeiros functional

classification (Bush et al. 1995). Cephamycins and carbapenems are stable

to ESBLs. They are inhibited by β-lactamases inhibitors such as clavulanic

acid (Mascaretti 2003; Paterson and Bonomo 2005). ESBL refers to enzyme

activity which can be found in several β-lactamase classes. In some classes

(e.g. TEM and SHV) it arises from point mutation in their β-lactamase genes

(bla), while others, in particular CTX-M, have inherent ESBL activity. ESBLs

and AmpC-type β-lactamases have a similar activity but AmpCs are not

inhibited by clavulanic acid (Paterson and Bonomo 2005). The first ESBL

was reported in 1983 in Frankfurt, Germany (Knothe et al. 1983). ESBL

production is also often accompanied by resistance to other antibiotic classes

such as aminoglycosides, trimethoprim/sulfamethoxazole and

floroquinolones (Colodner 2005).

Some β-lactamases such as TEM-1, TEM-2, SHV-1, SHV-11, OXA-2 and

OXA-10 are not ESBLs and have evolved from chromosomal penicillinases.

They do not have the mutations required for ESBL-production but show a

broader spectrum activity than other low spectrum β-lactams (Haeggman et

al. 1997). The majority of ESBLs are derivatives of these broad spectrum

β-lactamases (Gniadkowski 2001). The difference between ESBLs and their

progenitors arise from one or more amino acid substitution. Point mutation is

the most common cause of these substitutions; however insertions and

deletions have been reported either (Arpin et al. 2001; Perilli et al. 1997).

Mutation of the amino acid residue 164 [according to Ambler’s numbering

scheme] in TEMs, 179 in SHVs and 238 in both, which are the most common

point mutations, lead to enlarging the binding site of the β-lactam antibiotics

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to accommodate the bulky side chain of third generation cephalosporins, and

expanding the spectrum of action of the β-lactamases.

In ESBLs, due to the substitution of Gly238→Ser because of the existence of

two large side chains in positions Met69 and Ser238 the lower part of the β3

β-strand is pushed away from the active site. Consequently active sites of

these β-lactamases become larger and they become capable to hydrolyse

β-lactams with bulky oxyimino side chains (Huletsky et al. 1993).

Furthermore amino acid substitutions of 104 in TEMs and 240 in TEMs and

SHVs increase the interaction of β-lactamases with oxyimino side chains

(Gniadkowski 2001). In another words, strong electrostatic bond between the

lysine as a substituent at position 240 with the carboxylate residue of these

side chains improves the level of resistance of the bacterium against

oxyimino cephalosporins (Tzouvelekis and Bonomo 1999).

In 2002, Hujer showed that mutation at 238 residue leads to a lower

expression level of the gene in comparison to the wild type (SHV-1) because

of a decrease in translation. For compensation, bacteria began to improve

their resistance status by hyperproduction of the enzyme in different ways

such as mutation in the promoter sequences or insertion sequences

integrated upstream of the gene, resulting in more potent promoters

(Heritage et al. 1999).

1.6 SHV β-lactamases The SHV β-lactamases comprises one of the most clinically significant

classes of the β-lactamase families. Because of their importance, our

research team focused on this class. Therefore, the other β-lactamases have

not been explained in this literature review. This group of β-lactamases was

first detected by Pitton in 1972 and was named Pitton’s type-2 β-lactamases

(PIT-2) (Barthelemy et al. 1986; Pitton 1972). In 1979, Matthew et al. found a

unique biochemical property in this group in comparison to the other

β-lactamases. They designated this group, SHV-1 (means sufhydryl

variable). SHV β-lactamases hydrolysed cephaloridine but not

benzylpenicillin in the presence of the inhibitory sufhydryl binding chemical,

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p-chloromercuribenzoate [PCMB]. This suggested that cephaloridine and

benzylpenicillin bind to different parts of the active site in SHV and the active

site for cephaloridine but not benzylpenicillin contains a sulfhydryl group,

essential for substrate binding (Matthew et al. 1979; Tzouvelekis and

Bonomo 1999). This property, which was the basis of SHV β-lactamase

nomenclature, was never confirmed later. Other research groups tried to

obtain the same results by using purified enzyme, but this was never

achieved later (Paterson and Bonomo 2005).

SHV β-lactamases include SHV-1 and nearly 100 variants

(http://www.lahey.org/studies/webt.html). They belong to group 2be

β-lactamases (Ambler’s class A). SHV-1 is both encoded chromosomally on

plasmids. LEN-1 (a chomosomally encoded β-lactamase and K.pneumoniae

species-specific penicillinase) and OHIO-1 (a plasmid encoded β-lactamase)

have 84-88.9% and 91.8% amino acid sequence similarity with SHV-1,

respectively, whereas SHV-1 has only 63.7-67% identity with TEM-1

(Heritage et al. 1999; Tzouvelekis and Bonomo 1999). The kinetics of these

enzymes and their biochemical properties are quite similar.

PCR and hybridisation studies revealed that the SHV β-lactamase gene or

blaSHV that encode these enzymes are native to the Klebsiella pneumoniae

chromosome (Ford and Avison 2004; Haeggman et al. 1997). It has also

been found on plasmids of K. pneumoniae, and a smaller but still significant

proportion of other species in the Enterobacteriaceae family such as

Escherechia coli, Klebsiella oxytoca, Pseudomonas aeruginosa, Proteus

mirabilis and nontyphoid Salmonella spp. (Hirakata et al. 2005; Poirel et al.

2004; Politi et al. 2005; Sabate et al. 2002; Vercauteren et al. 1997).

In the early 1980’s, to overcome the resistance of bacteria against β-lactams

antibiotics, long side chains were added to cephalosporins, not to let them fit

in the active site of β-lactamases. Third generation cephalosporins or

oxyimino cephalosporins such as cefotaxime, ceftazidime and ceftriaxone are

the product of these modifications. Unfortunately, overuse of these antibiotics

led to the rapid emergence of the resistance to these antibiotics (Heritage et

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al. 1999). SHV-2 was the first ESBL which was reported by Konthe et al.

(Kliebe et al. 1985; Knothe et al. 1983).

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Table 3. A sample of SHV β-lactamases (Bush and Jacoby 1997a)

β-lactamase pI Amino acid residue ESBL comment 8 35 43 130 140 141 179 205 238* 240* Pos Neg SHV-1 7.6 Ile Leu Arg Ser Ala/Thr Thr/Ala Asp Arg Gly Glu X SHV-2 7.6 Thr/Ala Ala/Thr Ser X

SHV-2a 7.6 Gln Ser X SHV-3 7.0 Leu Ser X SHV-4 7.8 Leu Ser Lys X SHV-5 8.2 Ser Lys X SHV-6 7.6 Ala X Only hydrolyse

ceftazidime

SHV-7 7.6 Phe Ser Ser Lys X Substitution 8 is cleared in mature enzyme

SHV-8 7.6 Asn X SHV-9 8.2 Arg Ser Lys X Loss of aa 54

SHV-10 8.2 Gly Arg Ser Lys X Inhibitor resistant

SHV-11 7.6 Gln X SHV-12 8.2 Gln Ser Lys X

* Important point mutations for ESBL activity

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Substitutions G238S and E240K are the most important ones for ESBL

activity in SHVs, which confer the highest levels of resistance, while

microorganisms containing D179N substitution express the weakest SHV

ESBLs, such as SHV-6 [Table 3] (Randegger et al. 2000). Replacement of

glycine by serine at residue 238 (i.e. substitution of a G for an A nucleotide)

changes the spectrum of activity of SHV-1 or SHV-11, and leads to the

production of SHV ESBLs. In addition, replacement of glutamic acid by lysine

at residue 240 (i.e. substitution of a G for an A nucleotide) increases the

activity of the enzyme against ceftazidime.

1.7 ESBL Detection Methods ESBL expressing status of an organism is determined by phenotypic and

genotypic tests.

1.7.1 Phenotypic Tests or Clinical Microbiology Techniques

Phenotypic tests are divided into two categories; screening tests and

confirmatory tests. It is very important to know that in both types of tests, the

size of inoculum can affect MICs, as a lower inoculums size leads to a false

negative result, while a higher inoculum size may lead to a higher MIC value.

The Clinical and Laboratory Standards Institute (CLSI, formerly NCCLS)

recommends 5×105 CFU/mL as a standard inoculum (Queenan et al. 2004).

Screening Tests

Broth Microdilution Method (BMD) This method is recommended by CLSI for screening for ESBL producers

including K. pneumoniae, K. oxytoca and E.coli. CLSI recommended

screening criteria are as follows (AB-BIODISK 2007):

MIC ≥8 μg/ml to cefpodoxime

MIC ≥2 μg/ml to ceftazidime, cefotaxime, ceftriaxone and aztreonam

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Disk Diffusion Methods

These methods are recommended by CLSI for screening for ESBL producers

including K. pneumoniae, K. oxytoca, E.coli and Proteus mirabilis.

Recommended screening criteria by CLSI for Klebsiellae and E.coli are as

follows (AB-BIODISK 2007). The numbers show the size of the inhibition

zones in millimitre:

Cefpodoxime ≤17 mm

Ceftazidime ≤22 mm

Cefotaxime ≤27 mm

Ceftriaxone ≤25 mm

Aztreonam ≤27 mm

But the criterion for P. mirabilis for cefpodoxime is ≤22 mm (MIC ≥2 μg/ml).

Phenotypic Confirmatory Tests

These tests should be used for the confirmation of ESBL production for

screening-positive or suspicious results. All of these methods are based on

the augmentation of inhibition zones of third generation cephalosporins and

monobactams such as cefotaxime, ceftazidime, ceftriaxone, cefpodoxime

and aztreonam in the presence of clavulanate. There are several different

phenotypic confirmatory tests as follow (Carter et al. 2000; Ho et al. 1998;

M'Zali et al. 2000; Paterson and Bonomo 2005; Schooneveldt et al. 1998;

Thomson and Sanders 1992; Vercauteren et al. 1997; Woodford et al. 1990;

Jarlier et al. 1988; Kader et al. 2006).

1. Cehalosporin/clavulanate combination disk

2. Etest®

3. MAST double disc (MDD) test

4. Double-disk diffusion (DDD) Test

5. Agar supplemented with clavulanate

6. Disk replacement method

7. Three-dimensional tests

18

A positive confirmatory test isolate should be reported to the clinician as

being resistant to all penicillins, cephalosporins and aztreonam but not to

cephamycins. Susceptibility to cephamycins (cefoxitin and cefotetan) should

be reported based on their results (Paterson and Bonomo 2005; Queenan et

al. 2004). Since Etest® has been chosen as the confirmatory test in this

project, it is described below.

Etest®

ESBL Etest® strips produced by AB Biodisk (Solna, Sweden), are one of the

best methods for ESBL screening and phenotypic confirmation. There are

different types of Etest® strips, such as ESBL Etest® strips for cefotaxime and

ceftazidime. Cefotaxime Etest®s are plastic strips calibrated with MIC in

μg/ml on one side and on the reverse surface of the strip contain exponential

gradient of cefotaxime with or without 4 μg/ml clavulanic acid as an ESBL

inhibitor at the CTL and CT sides ,respectively (Paterson and Bonomo 2005).

According to the manufacturer’s recommendation, the criteria for ESBL-

positive phenotype are as follows (Figure 4):

1. an eight-fold or more reduction in the cefotaxime/clavulante MIC (CTL

side) in comparison to the cefotaxime MIC alone (CT side)

[i.e. CTL/CT ratio ≥ 8]

2. presence of a phantom inhibition zone

3. any deformation of cephalosporin inhibition ellipse

Presence of each one of these criteria indicates ESBL production

(MacKenzie et al. 2002).

Although it was thought that the ceftazidime is the best oxyimino compound

in resistance detection, it is not able to detect CTX-M-producing bacteria.

Though, manufacturers recommend to use both ceftazidime and cefotaxime

strips for ESBL detection (Brenwald et al. 2003; Katsanis et al. 1994;

MacKenzie et al. 2002). However data from Howard et al. showed that

cefotaxime distinguishes ESBLs better than ceftazidime (2002).

19

Figure 4. ESBL Etest® results

20

Other Methods

There are also some other methods for ESBL detection, based on the

augmentation of inhibition in the presence of clavulanate, which are not

explained in this thesis.

1. VITEK ESBL cards

2. Microscan panels

3. BD Phoenix automated microbiology system

21

1.7.2 Genotypic Tests or Molecular Detection Methods

The fact that some ESBL expressing isolates may be missed by phenotypic

diagnostic methods suggests that the using of phenotypic methods alone is

not sufficient in ESBL identification and DNA-based methods are required, as

well. Although genotypic methods are not as rapid as phenotypic methods,

attempts are done to find new rapid genotypic techniques as a substitution

for conventional methods. On the other hand, genotypic techniques only

reveal the genetic potential of resistance expression in microorganisms, not

an actual response to an antibiotic (Hammond 2004). These methods are

also useful in epidemiological investigations.

Direct PCR Sequencing

Direct sequencing of the PCR-amplified bla genes were used traditionally for

ESBL detection but the presence of multiple bla genes can confound this

approach (Kim and Lee 2000; Perilli et al. 2002).

PCR-Single Strand Conformational Polymorphism (PCR-SSCP) and

PCR-Restriction Fragment Length Polymorphism (PCR-RFLP)

PCR-SSCP was first described by Orita et al. (1989), which is useful for

strain typing and detection of SNPs. The PCR-SSCP relies on the basis that

SNPs can confer different migration patterns of small single stranded DNA

molecules from their paternal DNA in non denaturing gels. M’Zali et al. used

this method for identification of SHV genes by comparison of the patterns

with the known SHV variants patterns as standard genes (M'Zali et al. 1996;

M'Zali et al. 1998). PCR-SSCP can be used as a rapid screening test and

also for detection of novel SHV-β-lactamases by finding an undescribed

pattern. By using PCR-SSCP, more than one β-lactamase could be found in

a single isolate. Kim et al. have reported that by using this technique, SHV-2a

and SHV-12 were not characterised from SHV-2 and SHV-5, respectively

(2000). By adding PCR-RFLP techniques to PCR-SSCP, the reliability of

SHV β-lactamase identification was improved and resulted in an increase in

the sensitivity of the test (Chanawong et al. 2000). However, PCR-RFLP has

22

also some limitations due to the absence of the restriction enzymes for some

SNPs within blaSHV (Chanawong et al. 2001).

Ligase Chain Reaction (LCR)

LCR was first used by Kim et al. (2000) for characterisation of SNPs in blaSHV

genes of ESBL positive organisms. This method is based on the using of a

thermostable ligase for ligation of consensus primers containing a phosphate

at their 5' end. After doing a SHV-specific PCR, four different sets of

consensus and biotinylated mutant-specific primers are added to 870bp

amplimers. Consensus primers are amplified exponentially, while mutant

ones will not be amplified due to the absence of phosphate at their 5' end.

One of the mutant-specific primers, containing a biotin instead of the

phosphate, is captured on a streptavidin-coated microwell, and the other

primer, containing an additional 21-base sequence, is complemented by an

alkaline-phophatase-conjugated oligonucleotide which in turn is used in a

color change reaction (Kim and Lee 2000). This method is easier and more

rapid than the direct PCR sequencing method.

Restriction Site Insertion-PCR (RSI-PCR)

In this technique, mismatch primers with one or two modified nucleotides

near their 3' end, which are specially designed for different blaSHV SNPs,

create or remove restriction sites on amplimers of the blaSHV gene.

Afterwards, the relevant restriction enzymes are used for PCR product

digestion and the result is compared to a DNA size marker (Chanawong et al.

2001; Haliassos et al. 1989). By combination of this method and PCR-RFLP

analysis, Chanawong et al. differentiated 27 SHV variants (2001).

First Nucleotide Change (FNC) Method FNC is based on the interrogation of a specific nucleotide in the gene. Four

different flourescein-labaled ddNTPs are added in separate microwells,

containing the amplicon of the target gene, which has been previously

hybridised with biotinylated primers (exactly 1 base upstream of the SNPs).

Afterwards, antiflourescein-alkaline phasphatase conjugate is added followed

by p-nitrophenyl phosphate as the reaction substrate. An ELISA plate reader

23

is applied for reading the results (Howard et al. 2002). By using this

technique, Howard et al. designed a quantitative minisequencing-plate-based

method for blaSHV discrimination without involvement of electrophoresis. They

analysed 21 K. pneumoniae isolates from Princess Alexandra Hospital

outbreak (1991-1995) in Brisbane, Australia. They also reported a correlation

between the MIC data and the relative copy number of blaSHV gene.

Real-time PCR or Kinetic PCR Method

Real-time PCR was first described by Higuchi et al. (1992), which was

recognised later as the best technique for nucleic acid analysis quantification.

One of the limitations of the original end-point PCR method is the difficulty of

quantification of the primary DNA template, but Real-time PCR technique is

based on the continuous monitoring of PCR product by determination of the

fluorescence of introduced dyes or probes. Detection and quantification of

SNPs are among the most common usages of this method (Kubista et al.

2006). There are several real-time PCR methods for SNP detection. The

SHV melting curve mutation detection method was described by Randegger

et al. (2001) and fluorescence resonance transfer (FRET) real-time PCR

method was applied by Szabó et al. (2005). Hammond et al. (2004)

developed an allele specific PCR (AS-PCR) for blaSHV SNP detection, which

was a variant of a technique used by Germer et al. (2000). AS-PCR

technique is based on the less efficiency extension of DNA polymerases from

3´ mismatches than matches. The real-time PCR SNP assay is a useful

technique in situations where there is more than one β-lactamase in a

bacterial strain. In this project, genetic determination of ESBL production and

blaSHV gene copy number quantification were based on Hammond’s allele

specific PCR technique.

24

1.8 Epidemiology and Dissemination of β-lactamase

1.8.1 Global Epidemiology The history of antibiotics shows a great challenge between the usage of new

developed antibiotics and the emergence of the bacterial resistance which

would make the need for new agents. In the early 1980’s, to overcome the

resistance of bacteria against β-lactams antibiotics, third generation

cephalosporins such as cefotaxime, ceftazidime and ceftriaxone were used in

hospitals and subsequently the first wave resistance to these new drugs

emerged later by producing ESBLs such as SHV and TEM type ESBLs

(Heritage et al. 1999). More recently CTX-M ESBLs are a particular problem

in East Asia, but cause problems just about everywhere.

The first ESBL was detected in 1983 in Germany, Europe (Knothe et al.

1983). During the first decade, ESBLs proliferated dramatically in France.

However, recently the incidence of ESBL-producing K. pneumoniae das been

decreased in France. In contrast, there is a significant increase in Western

Europe (Paterson and Bonomo 2005).

TEM-4 was the first reported ESBL in the United States in 1988 (Jacoby et al.

1988). Although SHV-type ESBLs have also been reported but TEM-

producing organisms have been the main cause of the most outbreaks in the

US. However, recently CTX-M-type ESBLs have been reported in the US and

Canada (Paterson and Bonomo 2005).

There are some reports about SHV-2 and SHV-5 ESBL producing organisms

in Chile and Argentina during 1988 and 1989. In 1989, an outbreak of

Salmonella enterica serovar Typhimurium infection, harboring CTX-M-2

ESBL, was reported in Argentinean provinces. Other CTX-M-type ESBLs

have been also reported from Brazil. There are rare reports of TEM-type

ESBLs from South America (Paterson and Bonomo 2005).

25

Several outbreaks of ESBL-producing organisms have been also reported

from Israel, Saudi Arabia, South Africa and different North African countries.

ESBL production rate has been described much lower in Japan in

comparison to other parts of Asia. Appearance of CTX-M-type ESBLs has

been recently reported in different countries in Asia (Paterson and Bonomo

2005).

Although ESBL producing organisms were detected in every state of

Australia and in the Northern Territory but only two outbreaks have been

reported in Australian hospitals. The first case occurred from a gentamicin-

resistant Klebsiella spp. in Perth, Western Australia between 1986 and 1988

(Mulgrave and Attwood 1993). Later, Howard et al. found that the type of

ESBL was SHV-12 (2002). The second case occurred at Princess Alexandra

Hospital (PAH) in Brisbane, Australia between 1991 and 1995 (Schooneveldt

et al. 1998). The types of ESBLs were SHV-2a and SHV-12 and the majority

of the non-ESBL ones were SHV-11. It was suggested that ESBLs may have

been arisen from an SHV-11 ancestor in Australia (Howard et al. 2002).

The SENTRY Antimicrobial Surveillance Program started in 1997 to monitor

main pathogens and manage antibiotic resistance in Asia-Pacific region and

South Africa by using valid and reference methods in a central laboratory

from the first of 1998 till the end of 1999 (Bell and Turnidge 2003). Bell et al.

performed a survey about the frequency of occurrence of the ESBL-

producing isolates through this program (2002). They also described the

preferred substrates for ESBL detection and the co-resistance patterns of the

microorganisms, as well. The frequency for E.coli strains varied from 0-1%for

medical centers in Australia to 13-35% for mainland Chinese centers. The

prevalence of ESBL positive K. pneumoniae in Australia showed lower rates

than the other countries (<20%). They also reported that ceftriaxone and

aztreonam were the best substrates for ESBL detection and co-resistance to

other antibiotics were common in most centers.

26

1.8.2 Molecular Epidemiology of Nosocomial Infections K. pneumoniae have been reported as the cause of more than 75% of ESBL-

producing infections. All of K. pneumoniae isolates are almost naturally

resistant to ampicillin and amoxicillin due to their chromosomally mediated

SHV-1 β-lactamase. This leads to the colonisation of the bacteria on the skin

and mucosa of hospitalised patients. Although these patients may be

asymptomatic, but they may transfer it to compromised patients such as

neonates or the microorganisms can be transferred on staff hands. Intensive

care units have been recognised as the epicenter of ESBL-producing

organism in hospital outbreaks (French et al. 1996; Paterson and Bonomo

2005).

Among Enterobacteriacea, Klebsiella has the longest survival on the hospital

environment. Clonal persistence of more than three years has been reported.

Intercontinental transfer has been reported as well (Paterson and Bonomo

2005). There are also some reports of more complex epidemiologic pattern

such as simultaneously clonal dissemination of five different ESBL-positive

Klebsiella strains in the same hospital unit, occurrence of different ESBL

genes in a single epidemic, production of the same ESBL by different strains

(plasmid transfer inter different species), and finally dissemination of the

same ESBL by different types of plasmid (Bradford et al. 1994; Fiett et al.

2000).

Although, most of the reports are about hospital-acquired infections or related

to nursing homes, in recent years, true community-acquired colonisation or

infections have been described (Paterson and Bonomo 2005). CTX-M-type

β-lactamases, which have naturally the potential hydrolytic activity against

cefotaxime, are identified as the cause of these community-acquired

infections and are rapidly spreading all over the world. In several areas, CTX-

M-type ESBLs are more prevalent than TEM or SHV ESBLs (Rossolini et al.

2008).

27

1.9 Bacterial Resistance to β-lactam Antibiotics

As discussed before, mutation at residue 238 leads to a lower expression

level of the gene in comparison to the wild type but there are other

mechanisms which increase ESBL-related resistance (Gniadkowski 2008).

1. A reduction in accumulation of β-lactam in cell due to lowering the outer

membrane permeability, known as porin deficiency

2. Higher expression of ESBLs by promoter mutation or replacements,

mediated by insertion sequences such as IS26 or gene copy

amplification

3. Increasing ESBL activity by further evolution such as further mutation at

residue 240

1.10 blaSHV Gene Evolution The story of the evolution of SHV gene begins with translocation of the

chromosomal blaSHV gene on to the plasmids. Evidences have shown that

plasmid-borne blaSHV genes are transferred by transposons; and Insertion

sequences such as IS26 has played an important role in this translocation.

The SHV β-lactamase gene, blaSHV, is a normal part of the K. pneumoniae

chromosome and has been found on the plasmids in K. pneumoniae strains,

as well as other members of the Enterobacteriaceae family. The plasmid-

borne blaSHV carries IS26 insertions (an 820bp IS) either upstream of the

promoter or in the promoter, which in turn reinforces promoter strength. Two

single, separate mobilisation events of blaSHV from the K. pneumoniae

chromosomes, containing blaSHV-1 and blaSHV-11, onto the plasmids appear

to have been mediated by insertion sequence IS26 (Ford and Avison 2004).

The case of blaSHV-1, the mobilisation led to an IS26 insertion approximately

two kbp upstream of the blaSHV coding sequence and the mobilisation of

blaSHV-11 resulted in an IS26 insertion in the blaSHV promoter (termed

pr::IS26-blaSHV). Other blaSHV types have been emerged from these two

types of plasmid-borne blaSHV genes later.

28

An intriguing aspect of the biology of the SHV β-lactamases in K.

pneumoniae is that plasmid-borne ESBL encoding blaSHV genes are found

commonly, while reports of ESBLs encoded by the K. pneumoniae

chromosome located blaSHV remain vanishingly rare. Three likely (and non-

mutually exclusive) explanations for this are:

1. Plasmid- borne blaSHV genes are expressed more highly because they

possess more powerful promoters than the chromosomal blaSHV, so the

ESBL-activity conferring mutations are more likely to be fixed by evolution if

they occur in a plasmid-borne gene (Hammond et al. 2005; Podbielski et al.

1991; Rice et al. 2000).

2. Plasmid-borne blaSHV genes are expressed more highly because they are

present in the higher copy number than the chromosomal gene. Evidence

suggests that the conversion to an ESBL-positive phenotype occurs following

an increase in the number of mutation targets (i.e. blaSHV gene copy number)

either by plasmid or IS26-mediated amplification (Hammond et al. 2005).

3. Mutations conferring ESBL activity are exceedingly rare, while

dissemination of plasmid-borne ESBL-expressing mutant forms of blaSHV is

exceedingly efficient. . Recent studies have shed light on this issue. It has

been shown that the strains which only harbour the chromosomal blaSHV

gene - IS26 negative isolates - cannot express an ESBL phenotype, even

under selective pressure which maybe due to the affect of the gene

regulatory mechanisms, but strains containing plasmid-borne blaSHV are

capable of acquisition of an ESBL-positive phenotype (Ford and Avison

2004; Hammond et al. 2008). Therefore, the presence of a plasmid-borne

non-ESBL-encoding blaSHV is essential for ESBL expression.

Further studies on strains with IS26 upstream of the promoter of blaSHV gene

showed that there is a single nucleotide polymorphism or SNP (C→A) in the

second position of the -10 region of the blaSHV of ESBL positive

K. pneumoniae isolates (Rice et al. 2000). Recent research conducted at

QUT revealed that in these cases, the A allele only exists on the plasmid-

29

borne blaSHV, and not on the chromosomal-borne blaSHV (Turner, unpublished

data). On the other hand, Hammond et al. (2005) suggested that IS26

insertion in the blaSHV promoter maybe associated with the selection for an

ESBL-positive phenotype. These findings explain that why the promoter of a

plasmid-borne blaSHV is more efficient than a chromosomal-borne blaSHV. Hammond et al. (2008) also found a correlation between the copy number of

the blaSHV gene and the resistance level. This suggests that the evolution of

widespread clinically significant SHV-ESBL has required gene mobilisation

on to plasmids plus the appearance of variant promoters that are stronger

than the chromosomal promoter plus the presence of IS26 copies that flank

blaSHV and facilitate its multimerisation.

Theses findings have also an important clinical usage, as specific IS26

insertions can be used as a marker for the presence of plasmid-borne blaSHV.

In the absence of IS26, blaSHV only exists on the chromosome and never

expresses ESBL activity, but in the presence of IS26 and no ESBL activity,

ESBL expression can occur in response to a selective pressure. Therefore, in

the latter, usage of third generation cephalosporins in the treatment of the

patients is not effective.

1.11 Origin of Mutations

Over 60 years ago, Luria and Delbrück (1943) proposed a fluctuation

analysis to determine the origin of mutation in bacteria. They tried to find out

whether mutations occur spontaneously and randomly prior to exposure to

selective agents, or occur in response to exogenous environment (induced or

directed mutations). It was hypothesised that circumstances has no effect on

mutation rate. To determine this, Luria and Delbrück inoculated E. coli into a

culture media and dispensed a known cell volume into parallel identical tubes

containing broth culture media without any selective pressure and incubated

them. After reaching a suitable concentration, they transferred an equal

amount of broth cultures onto the agar plates containing 1010 lethal

bacteriophage T1. In the case of induced mutation theory, it was expected to

30

have equal probabilities of resistant development and therefore equal

number of resistant colonies from parallel broth cultures. But there was a

wide variation between the results of parallel cultures. This was in

accordance with the spontaneous mutation theory and indicated the

presence of different number of mutants before bacteriophage exposure.

According to this theory, mutation had occurred non-selectively and

independently sometimes during the growth of the bacteria in broth cultures

from the first cell divisions to the last ones. If it happens in the early stages,

the number of mutants will be higher than when it occurs in the last stages

(Angerer 2001; Dewanji et al. 2005; Jones et al. 1994; Ma et al. 1992). Luria

and Delbrück concluded that mutation rates are not influenced by selective

pressures (Cairns and Foster 1991).

In 1988, Cairns et al. proposed induced mutation theory again. This time,

they used a non-lethal selective agent, lactose, and E. coli Lac- mutants. This

experimental system also gives the opportunity of directed mutation for other

non-mutant cells. In this system, Lac- mutants with a revertible Lac mutation

are plated on the media containing lactose as the only carbon source of

energy. Looking at the genotype of the strains in these experiments

conducted by Cairns et al (1988) they used E. coli FC40 mutants harboring a

LacI-LacZ fusion that lacks the Lac regulatory region and containing a

frameshift mutation that encodes a translational fusion between LacI and

LacZ (Muller-Hill and Kania 1974). The frameshift (+1) is located in the LacI

portion. Although, transcription is initiated constitutively from the Lac

promoter (LacP) but the construct is Lac- because of a polar +1 frameshift

changing CCC to CCCC at the 320th codon of LacI (Calos and Miller 1981).

In other words, a frameshift in the upstream LacI part of the fused genes

prevents expression of the downstream lower LacZ part and transforms the

strain to Lac-. Compensating (-1) frameshift mutations lead to the expression

of LacZ and revert the strain to Lac+. Revertant colonies can grow on lactose

medium by either compensating (-1) frameshift mutation or increasing mutant

gene copy number (Roth et al. 2006).

31

After a few days, some Lac+ revertants formed visible colonies during the

exponential growth (spontaneous mutation) and Lac+ revertants continued to

appear in a stationary phase with a higher mutation rate due to Lac+ mutation

stimulation (induced or adaptive mutation) (Cairns and Foster 1991; Cairns et

al. 1988). Therefore, Cairns et al (1988) reopened the debate of the origin of

the genetic variation and the controversy was still remaining. They showed

that the majority of mutants/revertants emerge after exposure to lactose.

Their results also revealed that without other requirements for growth such as

RecA and LexA, adaptive mutants cannot emerge in the presence of lactose

and this has not any effect on the spontaneous mutation rate (Cairns and

Foster 1991).

Several models have been proposed to describe the mechanism of stress-

induced mutation of non-growing cells. In functional direction model, cells

change sites in their genome to overcome the situation and survive under

selection pressure (Foster and Cairns 1992). In another model called

hypermutable state (HMS) model, stress induces a general or genome-wide

hypermutable state in a subpopulation of non-growing cells. Only cells that

acquire proper mutation relive the stress (Hall 1990). Positional direction

model is a combination of the previous two models and states that stress

induces dinB, which is located 16 kb from Lac gene in plasmids, encodes

DNA Pol IV and associates mutagenesis. Stress only mutagenises regions

with recombinational replication. According to this model, mutagenesis is

directed to plasmids because of their intense recombinational events.

It seems that to obtain a revertant phenotype, the cells have to undergo two

processes: mutational process and amplification process, which in the first

process true revertants are formed and in the latter, slowly growing colonies

are produced.

The amplification model proposes that mutations emerge in a growing subset

of cells. It is known that there are some cells with a Lac duplication in the

whole population (about 1%). Formation of these duplications is RecA-

independent. These cells start slowly growing clones under selective

32

pressure. In these developing clones, the Lac region is amplified by a heavily

RecA-dependent recombination process. These copies are unstable. After

the occurrence of Lac+ mutation, these mutants proliferate and the extra Lac-

copies are eliminated by RecA. During this process the Lac+ copy, which is a

useful genes, is retained and leads to a pure Lac+ colony. Recombination

provides Lac+ higher copy numbers that might increase the mutation rate

(Foster 2004; Roth et al. 2006).

1.12 Gene Amplification

It has been known for many years that antibiotic-mediated selective pressure

can result in an increase in the copy number of the corresponding resistance

gene. It has also been established that this process is dependent upon the

homologous recombination pathway, and sequence repeats flanking the

resistance gene. Recent findings have lead to a renewed interest in the

contribution of gene copy number effects to adaptive processes in bacteria.

Kugelberg et al. (2006) and Slechta et al. (2003) have demonstrated that

amplification of the LacZ gene is the mechanistic basis for apparent, but now

disproved, events adaptive mutation in enteric bacteria, and Bergthorsson et

al. (2007) have posited that gene amplification facilitates the evolution of new

functions of pre-existing genes. Of more relevance to antibiotic resistance, it

has recently been found that duplication of a 13.5 kbp chromosomal

fragment in Streptococcus agalactiae leads to resistance to sulphonamide

and trimethoprim, by increasing expression of genes involved in folate

biosynthesis (Brochet et al. 2008). A broadly similar mechanism contributes

to linezolid resistance in Staphylococcus aureus (Tsakris et al. 2007).

A remaining enigma is the role of copy number plasticity of plasmid-borne

blaSHV. There is some evidence that, even allowing for the effect of the strong

promoters driving plasmid-borne blaSHV, copy number plasticity of plasmid-

borne blaSHV is central to the evolution of SHV ESBL–dependent resistance

to third generation cephalosporins. There are increasing reports of copy

number amplification of bla genes, and causative associations with increased

33

expression. One of the earliest was by Xiang et al. (1997) who described

amplification of plasmid-borne blaSHV-5. Three possible mechanisms of SHV-5

β-lactamase hyperproduction were investigated: a more powerful promoter,

an increase in plasmid copy number or blaSHV-5 gene copy number

amplification on a plasmid. They found that the selection of high resistance

levels to CTX in a blaSHV-5- expressing K. pneumoniae is associated with

blaSHV copy number amplification. Similar results were obtained by Hammond

et al. (2005). They reported an association between cefotaxime resistance

level and blaSHV copy number amplification in the PAH ESBL-positive

isolates. The examination of closely related K. pneumoniae clinical isolates

that carried blaSHV, but differed in their ESBL expression status revealed that

the ESBL-expressing strains had higher blaSHV copy numbers than the ESBL

non expressers. Finally, blaSHV copy number amplification via the formation of

tandem arrays has been directly detected in a fully characterised plasmid

from a K. pneumoniae clinical isolate by Zienkiewicz et al. (2007). They

sequenced the blaSHV-5- carrying plasmid p1658/97 and found tandem

repeats of the blaSHV-5 containing region bounded by copies of IS26. They

termed this region an amplimer. Copy number amplification is not confined to

SHV family genes, with Bertini et al. (2007) reporting an association between

duplication of blaoxa-58 and high level carbapenem resistance in Acinetobacter

baumannii. Remarkably, this duplication is also of an IS26 delineated

cassette.

1.13 Heteroresistance

The term “heteroresistance” is difficult to define precisely but has been used

to describe microbial populations that generate higher MIC derivatives at

such a high frequency that essentially any significant population will contain

such derivatives. Classically, the term has been applied to vancomycin

resistance in Gram-positive bacteria, in particular S. aureus (Falagas et al.

2008). The concept has now been extended to colistin resistance in

Acinetobacter baumannii-calcoaceticus complex (Hawley et al. 2008), and to

penicillin resistance in Streptococcus pneumoniae (Morand and Muhlemann

34

2007). It seems that heteroresistance provides bacteria with an opportunity to

explore growth in the presence of antibiotics before organism is completely

evolved to a resistant strain.

According to Clinical Laboratory Standard Institute (CLSI) criteria, Gram

negative microorganisms with an MIC value ≥ 16 µg/mL for third generation

cephalosporins such as cefotaxime and ceftazidime are considered resistant.

However, there are too many treatment failure reports due to infections

caused by ESBL producers with MIC values between 2 and 8 µg/mL (Perez

et al. 2007). It was hypothesised that the heteroresistance in these bacteria

may provide the opportunity for the organism to amplify the blaSHV gene in a

selective environment, which in turn leads to the emergence of resistant

phenotypes. This might explain the reason of the reported treatment failures.

1.14 Treatment of Infections with ESBL-Producing Organisms

According to the results of the SENTRY Antimicrobial Surveillance Program,

the best treatment results were obtained with imipenem as the first choice,

amikacin as the second choice followed by piperacillin/tazobactam as the

third choice with 0%, 6% and 10% resistance, respectively (Bell et al. 2002).

ESBLs are frequently plasmid encoded which may carry other genes

encoding resistance to other drugs (e.g. aminoglycosides). Therefore

antibiotic options in the treatment are extremely limited. Carbapenems are

also suggested as the drug of choice for the treatment of the infections

caused by ESBL-producing organisms (Chambers 2007). Clinicians should

avoid cephalosporins if drug penetration to infection site is poor (e.g.

meningitis, endocarditis and osteomyelitis) or at the presence of high-

inoculum infections such as intra-abdominal abscess (Paterson and Bonomo

2005). There are also some reports that show ESBL-producing strains of

K. pneumoniae can become resistant to cephamycins due to the loss of outer

membrane porin proteins, OmpK35 and OmpK36 (Domenech-Sanchez et al.

2003; Hernandez-Alles et al. 1999; Martinez-Martinez et al. 1996).

35

1.15 Mutation Rate Determination

1.15.1 Mutation Rate versus Mutation Frequency

“Mutation rate” and “mutant frequency” are terms that are sometimes

confused, but they mean very different things. Mutant frequency is the

proportion of mutants to the total number of cells. Because the number of

specific mutants in a culture depends on when the mutation occurred during

growth of the culture, simply counting the mutants in a single culture may not

be very useful (Figure 5) (Pope et al. 2008; Rosche and Foster 2000). In

contrast, the mutation rate is the probability of a mutation occurring during a

single growth and division cycle of a single cell.

This is regarded as the more useful descriptor because it is not a function of

the number of generations of growth and so is inherently more absolute.

Two methods for the mutation rate determination are mutation accumulation

method and fluctuation analysis method. Although, mutation accumulation

method is more accurate than the other method, it is difficult and time-

consuming. Therefore, fluctuation analysis was preferred. In antibiotic

research, fluctuation analysis methods are more common than mutation

accumulation methods. In fluctuation methods, mutation rate is calculated by

analysing the distribution of mutants in parallel cultures. This method was

first introduced by Luria and Delbrück in 1943 (Rosche and Foster 2000).

36

Figure 5. Comparison between mutation frequency and mutation rate; Mutation frequency is 7 in 16 cells but mutation rate is 3 in 15 cell divisions (mutants are in gray color)

Figure 5. Comparison between mutation frequency and mutation rate; Mutation frequency is 7 in 16 cells but mutation rate is 3 in 15 cell divisions (mutants are in gray color)

37

1.15.2 Poisson Distribution

The Poisson distribution is the probability distribution of random events in

time or random distribution of small particles in space (Armitage 1952). It is

useful for determination of the probability of random mutations of a particular

gene in a cell. Poisson equation for the probability of the random mutations in

a cell is as follow:

Px = mx e-m / x!

Where:

Px = probability that a culture will have exactly x mutations

m = average number of mutations per culture (i.e. per cell divisions)

“Estimator” is a traditional word meaning a method of calculation of m. This

parameter is used in mutation rate calculations. There are 8 different

estimators to determine Px.

1. Po method

2. Luria-Delbrück method of the mean

3. Lea-Coulson method of the mean

4. Drake formula using the median

5. Jones median estimator

6. Koch’s quartiles method

7. Lea-Coulson maximal likelihood method

8. Ma-Sandri-Sarkar maximum-likelihood method (MSS-MLL)

The simplest one is Po method which was applied by Luria and Delbrück.

However, the frequency of zero mutations has to be calculated:

38

Po = m0 e-m/0!

Po = 1 × e-m / 1

Po = e-m

m = -ln Po

Where:

Po = proportion of cultures without mutants (number of cultures with 0

mutants / total number of cultures)

After determining m value, it could be used for determination of Px. Then, this

calculated result, which is a prediction of randomness, can be compared to

the observed values. The Po method estimator is only valid when Po is

between 0.1 and 0.7 (0.3 ≤ m ≤ 2.3) (Rosche and Foster 2000).

1.15.3 The MSS Maximum-Likelihood (MSS-MLL) Method

MSS maximum-likelihood method is the gold standard estimator for m

estimation for all ranges of m (Rosche and Foster 2000). In 1992, Ma et al.

described a new recursion relation for computing of the Luria-Delbrück

distribution using the Lea-Coulson generating function

The MSS-MLL algorithm is as follows (Hammond 2004; Rosche and Foster

2000):

1. Estimation of a preliminary m using Lea-Coulson method of the

median equation ( r~ is the median number of mutants in a culture):

024.1)ln(/~ =−− mmr

39

2. Obtaining a maximum –likelihood estimation of m, calculation of Po and

Pr for the calculation of the total probability (Pr) for a given m:

∑ −

=−

+−==

1

00 )1(; r

ii

rm

irp

rmpep

Where:

Po = proportion of cultures with no mutants

Pr = proportion of cultures with observed r mutants in a culture

Calculation of the Pr’s for r = 0 to 150 for a given m.

3. Calculation of a likelihood value from the following equation:

∏=

=C

ii mrfmrf

1

)()(

Where:

rpmrf =)( and C = The number of parallel cultures in the experiment

Recalculation of the Pr’s, using adjacent m’s, until obtaining of an m that

maximises the likelihood.

4. Plotting the value of the likelihood function against m and determine the m

for the maximum value

5. Using m for the mutation rate calculation

As a summary, maximum likelihood is in essence a “trial and error” method in

which the likelihood of obtaining the results is calculated for different values

of m, the number of mutations in culture. Maximum likelihood differs from

probability. A probability refers events that occur in future, while likelihood

refers to the events in the past which have known outcomes (Weisstein

2003). In another words, likelihood is a conditional probability. Figure 6

shows the maximum-likelihood of one mutant colony in 20 parallel cultures.

40

The maximum likelihood estimate is obtained by finding the value of m that

maximises the log-likelihood. In this example, it is 0.05.

1.15.4 Calculation of Mutation Rate

Mutation rate (µ) is the probability of mutation of a cell during one cell cycle.

The amount of growth has a great influence on the parameter m. Therefore,

mutation rate is obtained by dividing m by the some measure of the number

of cells at the risk of mutation (Rosche and Foster 2000). According to

Armitage, mutation can occur at two points of the cell division, at the

beginning and the end of cell division (1952). At the beginning of the cell

division, the number of the total cells at risk is the total number of cells that

ever existed in the culture, i.e. 2Nt. Nt is the final number of viable cells in

culture. So, the sum of Nt cells with their Nt/2 parents and Nt/4 grandparents,

etc., is 2Nt. Therefore the mutation rate per cell per generation is calculated

as follows:

µ = m/2Nt

Since at the end of the cell division, the number of the cells at risk is the

same as the number of divisions that occurred, therefore, the calculation of

the mutation rate per cell per division is:

µ = m/Nt

Determination of Nt should be done with a high level of accuracy.

Confidence limits for m are calculated as (Rosche and Foster 2000):

315.096.1

95 )(96.1)ln( −+=+σσ emCL

315.096.195 )(96.1)ln( ++=−

σσ emCL Where:

.225.1 315.0 Cm−≈σ

41

Figure 6. Maximum likelihood estimate of one mutant colony in twenty parallel cultures

42

1.16 Summary

Bacterial resistance to antibiotics and worldwide dissemination of these

resistant strains is one of the major problems encountered in clinics. The

history of antibiotics shows a great challenge between the usage of new

developed antibiotics and the emergence of the bacterial resistance which

would make the need for new agents. In the early 1980’s, to overcome the

resistance of bacteria against β-lactams antibiotics, third generation

cephalosporins such as cefotaxime, ceftazidime and ceftriaxone were used in

hospitals and subsequently the resistance to these new drugs emerged later

by producing ESBLs (Heritage et al. 1999). There are different types of

ESBLs. The SHV ESBLs comprise one of the most clinically significant types

of the β-lactamases. The SHV β-lactamase gene, blaSHV, is a normal part of

the K. pneumoniae chromosome and has been found on the plasmids in

K. pneumoniae strains, as well as other members of the Enterobacteriaceae

family. K. pneumoniae have been reported as the cause of more than 75% of

ESBL-producing infections. Because of its importance, our research team

only focused their work on this group. ESBL production is conferred by point

mutation but this mutation leads to a lower expression level of the gene in

comparison to the wild type (Hujer et al. 2002). To overcome this problem, it

seems that the bacterial cells improve their resistance status by an increase

in blaSHV gene copy number.

The hypothesis of this project is that when there is a blaSHV on a plasmid,

mutation rate to ESBL positive phenotype will be consistent with the

requirement for two genetic events, the mutation and the copy number

expansion of blaSHV. In other words, mutation is only effective when there

happens to have been prior blaSHV copy number expansion and the mutation

is essential but not sufficient for acquisition of ESBL activity. A remaining

enigma is the role of copy number plasticity of plasmid-borne blaSHV.

Evidence suggests that the conversion to an ESBL-positive phenotype

occurs following an increase in the number of mutation targets (i.e. blaSHV

gene copy number) either by plasmid or IS26-mediated amplification

(Hammond et al. 2005). There is some evidence that, even allowing for the

43

effect of the strong promoters driving plasmid-borne blaSHV, copy number

plasticity of plasmid-borne blaSHV is central to the evolution of SHV ESBL–

dependent resistance to third generation cephalosporins. If this model is

correct, then the mutation rate from a non-ESBL to ESBL-positive phenotype

would be very low, even with a plasmid-borne blaSHV gene. On the other

hand, the mutation rate from a low MIC ESBL-positive ( ≤ 8 µg/mL for CTX)

to high MIC ESBL-positive ( ≥16 µg/mL for CTX) was predicted to be very

high. This is because only blaSHV gene copy number increase is needed,

which is probably mediated by homologous recombination that typically takes

place at a much higher frequency than point mutation.

According to Clinical Laboratory Standard Institute (CLSI) criteria, Gram

negative microorganisms with an MIC value ≥ 16 µg/mL for third generation

cephalosporins such as cefotaxime and ceftazidime are considered resistant.

However, there are too many treatment failure reports due to infections

caused by ESBL producers with MIC values between 2 and 8 µg/mL (Perez

et al. 2007). It was hypothesised that the heteroresistance in these bacteria

may provide the opportunity for the organism to amplify the blaSHV gene in a

selective environment, which in turn leads to the emergence of resistant

phenotypes. This might explain the reason for the reported treatment failures.

In this study, the aim was to shed light on the role of blaSHV copy number

plasticity on the evolution of SHV ESBL activity.

44

1.17 Aims

According to these hypotheses, three aims were designed for this study.

1. Determination of blaSHV mutation rate in K. pneumoniae isolates from a

non-ESBL status to an ESBL positive status and comparison to the

mutation rate of rpoB gene, which is responsible for rifampicin resistance,

as a control. Also, determination of blaSHV mutation rate in

K. pneumoniae isolates from a low MIC ESBL status to a high MIC ESBL

status.

2. Detection of heterogenous phenotype with regards to cephalosporin

resistance in K. pneumoniae strains

3. Determination of the mechanism of blaSHV copy number amplification.

45

Chapter 2

2 Mutation Rate Determination

It has previously been reported that the selection of SHV ESBL expressing

derivatives of non-ESBL expressing K. pneumoniae is associated with blaSHV

copy number expansion (Hammond et al. 2005). This experiment was carried

out in liquid medium, so determination of the mutation frequency was not

possible. However, it suggests that selection of an SHV-dependent ESBL-

expressing derivative of a non-ESBL expressing K. pneumoniae requires the

simultaneous selection of a point mutant and copy number variant. It may

therefore be predicted that the observed mutation frequency will be very low,

as it would be the product of the frequencies of appropriate blaSHV point

mutants and copy number variants. This was tested using six K. pneumoniae

clinical isolates.

2.1 Materials and Methods

2.1.1 Bacterial Isolates

The isolates 18, 33, 54, 110, 113, 114 and 119 were from the SENTRY

Antimicrobial Surveillance Program collection and were collected from the

Asia-Pacific region and South Africa between January 1998 and December

1999, and isolates A1, B1, D1, E1, F1 and K2 were from the Princess

Alexander Hospital (PAH), Brisbane (Table 4) (Howard et al. 2002;

Schooneveldt et al. 1998).

46

Table 4. K. pneumoniae PAH and SENTRY isolates genotype and phenotype

Isolate Origin blaSHV Pr::IS26-blaSHV U/S IS26 ESBL A1 Australia Pos Pos Neg Pos B1 Australia Pos Pos Neg Pos D1 Australia Pos Pos Neg Pos E1 Australia Pos Pos Neg Pos F1 Australia Pos Pos Neg Pos K2 Australia Pos Pos Neg Neg 33 Philippines Pos Pos Neg Neg*

119 Hong Kong Pos Pos Neg Neg 18 South Africa Pos Neg Pos Neg*

54 Philippines Pos Neg Pos Neg 110 Taiwan Pos Neg Pos Neg 113 Taiwan Pos Neg Neg Neg 114 Philippines Pos Neg Neg Neg

pr::IS26-blaSHV, IS26 in the blaSHV promoter U/S IS26, IS26 approximately 2kbp upper blaSHV promoter * These isolates found to be ESBL-positive or contaminated with ESBL-positive samples.

47

2.1.2 Determination of Mutation Rates

A single colony of each isolate, streaked on MHII agar plates was used to

make a 0.5-McFarland unit suspension for the following procedure (Figure 7).

5 microliter of a 1 in 150 dilution of the suspension in sterile physiological

saline, which approximately contains 10,000 cells, was used for inoculating

each of 20 parallel MHII broth cultures (to rule out jackpot mutations). After

20 hours incubation at 37 °C with agitation, 225 RPM (I 26 incubator shaker

series, New Brunswick Scientific), 50 microliter from each tube was plated

out onto MHII-cefotaxime (1µg/mL) and MHII-rifampicin (150 µg/mL) agar

plates. This low concentration of cefotaxime only prevents the growth of non-

ESBL strains and has not any selection pressure on the growth of the

bacteria. Following 18-24 hours incubation at 37 °C the number of CTXr

(cefotaxime resistant) and Rif r (rifampicin resistant) mutant colonies were

counted. The total number of viable cells (Nt) was determined by plating 50

microliter of a diluted suspension from 5 parallel broth cultures of an isolate

onto free antibiotic MHII agar plates. For this purpose 10-6 dilution was used

and the results were multiplied by 106 (Hammond 2004). The mean of the

five numbers of viable cells is considered as the number of viable cells (Nt).

48

Figure 7. Determination of mutation rate by fluctuation analysis method

49

Mutation rates were calculated by using the MSS-maximum likelihood

method according to Rosche et al. (2000) A computer-based program was

designed for the convenience (appendices A and B). This algorithm is

discussed in section 15.3 of the the first chapter.

2.1.3 ESBL Phenotypic Detection

The Etest® ESBL screening strips were used for the detection of ESBL-

expression and determination of CTX MICs, according to the manufacturer’s

instructions (AB Biodisk, Solna, Sweden).

2.1.4 DNA Extraction for Real-Time PCR Analysis

The DNA extraction was done by using a DNeasy tissue kit (Qiagen, Hilden,

Germany) in accordance with manufacturer’s instructions and DNA

concentration was checked by agarose gel electrophoresis.

2.1.5 Real-Time PCR and Relative Quantitation of blaSHV

Allele specific real-time PCR, which is sometimes known as kinetic PCR, was

used to interrogate the codon 238 SNP associated with acquisition and of

SHV ESBL activity. This was carried out essentially as previously described

(Hammond et al. 2005). The assays were performed in duplicate in a Corbett

6000 Rotorgene, and Platinum ® SYBR® Green qPCR SuperMix (Invitrogen)

was used as the Mastermix. The primer sequences are shown in Table 5.

The reactions contained 5 pmol of each primer, 1µL of bacterial cell lysate,

and master mix according to the manufacturer’s instructions. The final

reaction volumes were 10 µL. The thermocycling protocol was an initial

denaturation at 95°C for 60 s, followed by 40 cycles of 94°C for 15 s, 60°C

for 20 s, and 72°C for 30 s. Fluorescence readings were acquired during the

72°C step.

50

Table 5. Primers for kinetic or real-time PCR (Hammond et al. 2005)

Primer name Gene Primer sequence (5'→3')a

16sAllBactF 16s rRNA TCCATGAAGTCGGAATCGCTAG

16sAllBactR 16s rRNA CACTCCCATGGTGTGACGG

SHVquantF blaSHV TGCTTGGCCCGAATAACAA

SHVquantR blaSHV GCGTATCCCGCAGATAAATCA

Shv238mt blaSHV CGCCGATAAGACCGGAGCTA

Shv238wt blaSHV CGCCGATAAGACCGGAGCTG

Shv238reverse blaSHV CGGCGTATCCCGCAGATAA

Shv240mt blaSHV GCGCGCACCCCGCTT

Shv240wt blaSHV GCGCGCACCCCGCTC

Shv240reverse blaSHV CCGGCGGGCTGGTTTAT a underlined oligonucleotides are considered for allele specific PCR for SHV 238 and 240 residues

51

Real-time PCR was also used for total blaSHV relative copy number

determination by the comparative CT method with the 16s rRNA-encoding

gene as the endogenous control, as previously described (Hammond et al.

2005).

2.2 Results

The mutation rates to resistance to 1 µg/mL CTX were determined, and

compared with rates of mutation to 150 µg/mL rifampicin, which is conferred

by a small number of point mutations in the chromosomally located rpoB

gene (Tables 6 and 7).

52

Table 6. Number of the mutant colonies in each 20 parallel culture plate of different isolates

Plate No. K2 54 110 113 114 119 CTX r Rif r CTX r Rif r CTX r Rif r CTX r Rif r CTX r Rif r CTX r Rif r

1 0 2 0 4 0 0 0 2 0 3 0 1 2 0 6 0 4 0 2 0 1 0 1 0 1 3 0 34 0 22 0 0 0 1 0 1 0 2 4 0 1 0 5 0 0 0 0 0 0 0 4 5 0 18 0 0 0 4 0 0 0 2 0 0 6 0 1 0 6 0 7 0 0 0 7 0 12 7 0 10 0 3 0 5 0 0 0 4 0 2 8 0 3 0 4 0 9 0 1 0 21 0 0 9 0 2 0 21 0 11 0 0 0 7 0 0 10 1 1 0 0 0 1 0 0 0 0 0 0 11 0 5 0 2 0 13 0 0 0 4 0 4 12 0 8 0 0 0 0 0 0 0 16 0 0 13 2 5 0 3 0 0 0 2 0 2 0 5 14 0 3 0 0 0 2 0 0 0 0 0 1 15 0 6 0 7 0 17 0 0 0 85 0 1 16 0 4 0 3 0 1 0 0 0 4 0 2 17 0 1 0 4 0 1 0 0 0 7 0 2 18 0 1 0 3 0 0 0 4 0 3 0 7 19 0 1 0 3 0 1 0 2 0 0 0 4 20 0 1 0 1 0 2 0 0 0 2 0 18 Nt 1.75 × 108 1.70 × 108 1.76 × 109 8.4 × 108 8.16 × 108 1.18 × 109

CTX r, the number of mutant colonies in the presence of 1µg/mL cefotaxime Rif r, the number of mutant colonies in the presence of 150µg/mL rifampicin Nt, number of viable cells cultured on each plate

53

Table 7. Mutation rates of PAH and SENTRY Klebsiella pneumoniae isolates

54

In this experimental system, colonies grew on between eight and 20 of the 20

rifampicin plates used for each isolate, with the highest number of colonies

on any one plate being 85, and the calculated mutation rates to rifampicin

resistance are between 10-10 and 10-9. Although the mutation rate to

rifampicin resistance was easily determined, the mutation to CTX resistance

was almost undetectable in this system. Preliminary experiments showed

that isolates 18 and 33 are ESBL-positive or had been contaminated with

ESBL-positive samples. Therefore, they were not incorporated in mutation

rate determinations. Isolates 113 and 114 do not possess a plasmid-borne

blaSHV and only have the chromosomal blaSHV. It has previously been shown

in step-wise selection experiments that plasmid-borne blaSHV is required for

the selection of ESBL expressing mutants (Hammond et al. 2008). As

expected, these strains yielded no mutants. However, the same result was

obtained from three of the four isolates that possess plasmid-borne blaSHV,

and only three ESBL-expressing colonies were obtained in 20 parallel

cultures from just one strain (K2). All three mutant derivatives of K2 were

shown to be ESBL positive according to the cefotaxime E-test (Figure 8), and

to be mutated at codon 238 as determined by kinetic PCR (Table 8).

Maximum likelihood calculations indicated that the rifampicin resistance

mutation rate is at least 10-100 times higher than the cefotaxime resistance

mutation rate, and the mutation rates or µ values (probabilities of mutation

per cell per generation) for mutation to cefotaxime resistance are less than

10-11 and probably much lower (less than the frequency of a single specific

point mutation) (Table 7). The generally accepted mutation rate for a specific

base change in non-mutator enteric bacteria is approximately 10-9. While the

rifampicin resistance mutation rate indicates that this figure may be slightly

lower in K. pneumoniae, the data indicate that the mutation rate to

cefotaxime resistance is less than the frequency of a single specific point

mutation. blaSHV relative copy number analysis of three mutant colonies also

revealed a consistent small increase in the amount of blaSHV gene copy

number (Table 9).

55

Figure 8. ESBL Etest® experiments of K2 three mutant colonies

CT/CTL = 6 / 0.125 = 48 CT/CTL = 6 / 0.125 = 48 CT/CTL = 2 / 0.125 = 16

Criterion for ESBL production: CT/CTL ≥ 8 → ESBL-positive

Table 8. Quantification of blaSHV 238 mutant alleles of K2 mutant colonies

Colony Mean wild type shv238

allele CT Mean mutant shv 238

allele CT ΔCT TCΔ2N1 15.42 18.46 -3.04 0.1 C1 14.7 13.4 1.35 2.5 C2 14.54 14.09 0.45 1.4 C3 14.0 12.77 1.24 2.4

CT, The comparative threshold cycle ΔCT = CT wild type – CT mutant

TCΔ2 , quantity of blaSHV 238 mutant alleles

Table 9. Total blaSHV relative copy number of K2 mutant colonies

Colony 16S SHV TCΔ

TCΔΔ TCΔΔ−2 * N1 10.12 15.64 4.53 C1 10.77 14.07 3.31 -1.22 2.33 C2 10.91 13.96 3.05 -1.48 2.79 C3 10.80 14.18 3.39 -1.14 2.20

CT, The comparative threshold cycle ΔCT = blaSHV CT – 16S rRNA encoding gene CT ΔΔCT was calculated by subtracting the starting culture ΔCT from the calculated ΔCT *, Total blaSHV relative copy number

56

2.3 Mutation Rate from a Low MIC ESBL-Positive Phenotype to a High

MIC ESBL-Positive Phenotype

The selections were carried out using two PAH low MIC ESBL-expressing

K. pneumoniae clinical isolates, B1 and F1, which had previously been

determined to have CTX MIC values of 1 μg/ml. This is towards the low end

of the range of what is found in ESBL-expressing isolates.

The current model for the mechanism of acquisition of high CTX MIC values

is copy number amplification mediated by the generation of tandem repeats

that is in turn mediated by homologous recombination involving the IS26

elements that flank plasmid-borne blaSHV. This would be predicted to take

place at a very high frequency in comparison to point mutation, so the

frequency with which derivatives with increased CTX MIC values can be

selected would be predicted to be very high.

Preliminary experiments indicated that this is indeed the case. Plating of

approximately 109 cells on to CTX concentrations from 2-32 μg/ml yielded a

confluent growth (data not shown). It was decided that this reflected such an

extreme and obvious difference from the frequency of mutation from non-

ESBL expressing to ESBL-expressing that frequency determination using

parallel cultures and maximum likelihood analysis was unnecessary to prove

that the mutation rate was high than the mutation rate from ESBL non-

expressing to ESBL positive. Also, the presence of very large numbers of

mutation events in the cultures essentially eliminates the Luria fluctuation and

makes parallel cultures and statistical analyses unnecessary. Rather, it was

reasoned that more insight would be obtained by plating relatively low

numbers of cells from ESBL-expressing isolates onto a range of CTX

concentrations above the nominal CTX MICs of the isolates, and observing

the numbers of colonies that grow.

Isolates F1 and B1 were grown overnight form single colonies in MHII broth

containing 1µg/mL CTX. Approximately 106 cells of each of these isolates

was plated out onto a 2 fold increasing series of cefotaxime concentrations

57

(Figure 9), and colonies that grew were analysed for CTX MIC as determined

by the E-test and blaSHV copy number amplification as compared with the

original isolate.

In comparison to the previous experiment results, the number of mutant

colonies in the plates was much more and instead of having a number of

mutant colonies, we had a confluent growth of the mutant colonies in the first

plates. Therefore, the MSS maximum likelihood method was not used for the

mutation rate determination, because it was obvious that the mutation rate

from a low MIC ESBL status to a high MIC ESBL status was very high. The

rate for the appearance of higher MIC cells (i.e analogous to the mutation

rate) was approximately 10 million-fold higher than the mutation frequency of

an ESBL-negative strain.

58

Figure 9. F1 mutant colonies in different cefotaxime concentration

A 10-2 dilution is cultured on each plate.

59

Chapter 3

3 Detection of Heteroresistance to Cefotaxime in

K. pneumoniae Isolates by Population Analysis Profile (PAP) Method

3.1 PAP Experiments for Cefotaxime-Resistant K. pneumoniae Strains According to Wootton et al. (2001) the population analysis profiles were

performed for large and distinct colonies as follows. Stock isolates were

cultured on MHII agar plates containing CTX in accordance with their

predetermined MICs. A single separate colony was cultured overnight in

MHII-cefotaxime broths, containing the same CTX concentration. The broth

was diluted 10-2 in saline and 100 µL of the diluted sample plated out by a

spreader onto MHII agar plates containing 1, 2, 4, 8, 16, 32, 64 and 128

µg/mL cefotaxime and incubated at 37 °C for 24 hours. In later experiments

more diluted samples (10-4 and 10-6) were also used to obtain countable

colonies. ESBL-production of the mutant colonies was determined

phenotypically and genotypically as described before. Total blaSHV gene copy

number was determined, as well. The experiment repeated for F1 isolate in a

second run.

Our current model is that plasmid-borne blaSHV exhibits considerable copy

number heterogeneity within K. pneumoniae cells, and that this confers

heterogeneity in resistance phenotype. It was therefore predicted that any

population of K. pneumoniae cells with a plasmid-borne blaSHV would contain

a significant proportion of cells that have an elevated β-lactam MIC as

compared to the population as a whole. This conjecture was tested using two

isolates F1 and B1. These were chosen because they are ESBL expressers

according to the E-test, and this phenotype is conferred by a mutated blaSHV

(Hammond et al. 2005). However, they both have CTX MIC values at the low

end of the range commonly observed for ESBL expressing K. pneumoniae,

60

and this was seen as desirable for this experiment because it provides a wide

range of higher, biologically plausible MICs that can be selected for.

3.2 Results The first quantitative experiments were carried out using inocula of 106

CFU/mL per plate. There were several significant observations. It was found

that isolates B1 and F1 behaved very differently on the selective media.

Firstly, it was observed that two different colony types appeared on the plates

containing different CTX concentrations (Figure 10); large colonies: that

appeared as normal K. pneumoniae colonies, albeit somewhat variable in

size, and were scattered evenly over the plates, and small colonies: that

were tiny and “unhealthy” looking, growing in clumps. They appeared to be

dependent for growth on other nearby colonies, and would have been termed

satellite colonies except that they frequently grew on plates devoid of

colonies of normal morphology i.e. they were not satellites of anything except

each other. In general, F1 yielded primarily the colonies of usual normal

morphology, while isolate B1 yielded the very small “clumpy” colonies. It was

hypothesised that the large colonies represented genuine variants with

higher CTX MIC values, whereas small colonies were growing by virtue of

secreted and carried over β-lactamase activity and grew only in regions of

the plate where sufficient cells had been inoculated to reduce the localised

CTX concentration below a threshold value. The predictions of this

hypothesis would be that large colonies would exhibit an elevated CTX MIC

on the basis of the E-test and also blaSHV copy number amplification with

respect to the original isolate, whereas small colonies would exhibit neither of

these. This was tested with duplicate colonies of both normal and small

morphologies from each plate containing different CTX concentration and

found to be the case (Figure 11 and Table 10). Remarkably, isolate B1

yielded only small colonies when subject to the same selection procedure. It

was concluded that this isolate does not encompass or generate blaSHV copy

number variants in a similar manner to isolate F1. The mechanistic basis for

61

the difference between F1 and B1 is unknown and is worthy of further

investigation.

In addition, the number of colonies that appeared on the selective plates from

isolate F1 indicated that the rate of copy number expansion (i.e. analogous to

the mutation rate) is of the order of 1-10 million times higher than the

frequency of point mutation.

62

Figure 10. Phenotypic (Etest®) and genotypic (blaSHV relative copy number) experiments of F1 and B1 isolates of the PAH collection

(The numbers indicate blaSHV copy number of duplicate colonies of each plate)

63

Table 10. Total blaSHV relative copy number of B1 and F1 isolates

Isolate (in μg/mL CTX) blaSHV TC a 16S rRNA TC TCΔ

b TCΔΔ c TCΔΔ−2 d

First experiment F1 colonies (first colony):

F1(1) 13.15 10.41 2.74 F1(2) 11.12 10.00 1.12 -1.62 3.07 F1(4) 10.22 9.63 0.59 -2.16 4.45 F1(8) 10.65 10.46 0.19 -2.55 5.86

F1(16) 10.36 10.95 -0.59 -3.34 10.09

F1 colonies (second colony): F1(1) 14.27 9.21 5.07 F1(2) 15.94 10.60 5.35 0.28 0.82 F1(4) 10.56 8.92 1.64 -3.43 10.74 F1(8) 11.71 9.50 2.21 -2.86 7.24

F1(16) 11.93 9.94 1.99 -3.08 8.43

B1 colonies (first colony): B1(1) 17.64 10.85 6.79 B1(2) 19.06 10.76 8.30 1.51 0.35 B1(4) 21.22 11.12 10.11 3.32 0.10 B1(8) 18.06 10.62 7.44 0.65 0.64 B1(16) 18.88 11.59 7.30 0.51 0.70

B1 colonies (second colony):

B1(1) 17.80 13.70 4.10 B1(2) 18.11 14.34 3.77 -0.33 1.26 B1(4) 20.00 14.81 5.19 1.09 0.47 B1(8) 18.51 14.32 4.20 0.09 0.94 B1(16) 18.90 15.19 3.72 -0.39 1.31

Second experiment F1 colonies (first colony):

F1(1) 14.15 11.98 2.18 F1(2) 14.58 10.83 3.75 1.58 0.34 F1(4) 13.16 11.69 1.47 -0.70 1.63 F1(8) 11.01 11.28 -0.27 -2.45 5.45

F1(16) 10.98 11.71 -0.73 -2.91 7.52

F1 colonies (second colony):

F1(1) 14.23 11.46 2.78 F1(2) 13.96 11.55 2.41 -0.37 1.29 F1(4) 11.25 10.59 0.65 -2.12 4.35 F1(8) 10.50 10.87 -0.38 -3.15 8.88

F1(16) 8.96 11.51 -2.56 -5.33 40.22

a, The comparative threshold cycle b, ΔCT = blaSHV CT – 16S rRNA encoding gene CT c, ΔΔCT was calculated by subtracting the starting culture ΔCT from the calculated ΔCT d, Total blaSHV relative copy number

64

Figure 11. Total blaSHV relative copy number of B1 and F1 isolates

R1, first set of experiments; R2, second set of experiments

65

Turning to the details of data in figure 11, Etest® results of isolate F1 in

higher cefotaxime concentrations showed more resistant colonies with

increased blaSHV copy numbers, while in B1 isolate, Etest® results of the

colonies of higher cefotaxime concentrations did not show more resistant

variants and it also did not generate blaSHV copy number variants in a similar

manner to isolate F1. This suggests that F1 colonies are genuine variants,

while B1 colonies are not and grow by virtue of secreted and carried over β-

lactamase activity.

Population analysis profile analysis revealed copy number heterogeneity. In

order to expand and refine these findings, the experiments were repeated

with more isolates, and different numbers of inoculated cells per plate, so as

to yield PAPs over as wide a range of CTX concentrations as practically

possible. The number of viable colonies was counted after 24 hours

incubation at 37°C and the logarithm of the colony numbers plotted against

cefotaxime concentration using SAS 9.1 software. It can be seen that all

isolates tested contained substantial populations of cells that grew at CTX

concentrations 16 times higher than the published MIC values (Figures 12-

14). These figures also show the growth of clumpy colonies growing by virtue

of secreted and carried over β-lactamase activity and grew only in regions of

the plate where sufficient cells had been inoculated. Of course, these

colonies are not genuine and were not counted in our results. In some

instances, growth of undiluted samples on higher concentration CTX yielded

to a non-ESBL background growth, whereas a 10-2 dilution resulted in no

growth of the bacteria.

It was concluded that it is possible to select at a very high frequency

derivatives of ESBL expressing K. pneumoniae with stable increases in CTX

MIC, although this may be strain dependent.

66

Figure 12. PAP results of isolates A1 and D1 of the PAH collection

* 10x corresponds to the dilution plated.

67

Figure 13. PAP results of isolates E1 and F1 of the PAH collection

* 10x corresponds to the dilution plated.

68

Figure 14. Population analysis profile for cefotaxime-resistant Klebsiella pneumoniae strains

from PAH isolates

[D1 isolate had a previously determined CTX MIC of 4µg/mL, while the other isolates had CTX MICs of 1µg/mL.]

69

Chapter 4

4 Evaluation of the Effect of Novobiocin on the blaSHV Copy Number Expansion

During the project, it was hypothesised that homologous recombination is the

mechanism of multimrisation of blaSHV gene in plasmids. The amplification of

blaSHV gene results in emergence of resistant strains and over expression of

β-lactamases.

It has recently been demonstrated that variation in resistance to linezolid in

Gram-positive bacteria is due to variations in copy number of the rRNA

encoding operon (Tsakris et al. 2007). These workers reasoned that DNA

gyrase inhibitors such as novobiocin would inhibit the homologous

recombination reactions that are thought to generate copy number

amplification, and so prevent the appearance of variants with higher MIC

values. Novobiocin inhibits bacterial DNA gyrase activity by attaching to the

GyrB subunit of the enzyme. This conjecture was supported by their data.

We hypothesised that novobiocin would have a similar effect for a similar

reason in our experimental system. Since, a sub-inhibitory concentration of

novobiocin is a homologous recombination inhibitor, it can be used as a

substance to delay or prevent the emergence of resistant strains. According

to Tsakris et al. (2007), a selection method was developed. A low MIC ESBL-

positive strain (F1) from PAH isolates was cultured in 1mL MHII broth,

containing 1µg/mL cefotaxime, and incubated overnight at 37 °C with

agitation, 225 RPM (I 26 incubator shaker series, New Brunswick Scientific).

106 cells of each isolate (0.1mL of a 10-2 dilution in sterile physiological

saline) were plated out on a series of MHII agar plates, containing 1, 2, 4, 8,

16, 32, 64 and 128µg/mL cefotaxime, with and without 15µg/mL novobiocin.

A sub-inhibitory concentration of 15 µg/mL novobiocin was arrived at

empirically. After 24 hours incubation at 37 °C, the mutant colonies were

examined phenotypically and genotypically for ESBL production. The total

blaSHV copy number was quantitated by real-time PCR.

70

Results The results were as expected. It was shown that subinhibitory concentration

of novobiocin has no effect on colony phenotype in the absence of CTX

(Figure 15). This supports that novobiocin is only effective on the gene

amplification phase conducted by homologous recombination. The

novobiocin prevented the selection of the well separated colonies with stably

increased CTX MICs and expanded blaSHV copy numbers. The colonies that

grew were all of the unhealthy clumpy “satellite” colony morphology, and no

evidence for blaSHV copy number expansion could be found (Figure 16). One

unexpected result was that the satellite colonies were more numerous on the

novobiocin containing plates than on those without novobiocin. This is not

fully understood, but may indicate a higher rate of cell lysis or leakage.

It was concluded that a known inhibitor of homologous recombination

prevents the selection of F1 variants with higher CTX MIC, and that this

suggests that recombination is required for blaSHV copy number expansion.

71

Figure 15. Effect of novobiocin on the colony size and distribution of F1 strain

72

Figure 16. Effect of novobiocin on PAH F1 isolate growth and blaSHV copy number

73

Chapter 5

5 Discussion

The concept of increase in the copy number of related genes as a result of

selective pressure has been reported as a mechanism of the resistance of

different bacteria against antibiotics. This process occurs as a result of the

homologous recombination phenomenon following sequence repeats that

flank the resistance gene. This has led to the reintroduction of adaptive

mutation theory. Recently, Kugelberg et al. (2006) and Slechta et al. (2003)

showed that the mechanistic basis of adaptive mutation in gram negative

bacteria is multimerisation of the LacZ gene. However, this concept has been

disproved by Bergthorsson et al. (2007) who demonstrated that

multimerisation expedites the evolution of pre-existing genes. Recent findings

have also attached greater importance to gene amplification in antibiotic

resistance. Brochet et al. (2008) reported a 13.5 kbp chromosomal fragment

duplication in Streptococcus agalactiae, which through higher expression of

genes involved in folate biosynthesis, results in resistant strains to

sulphonamide and trimethoprim. Research conducted by Tsakris et al. (2007)

revealed a similar mechanism for linezolid resistance in Staphylococcus

aureus. The mechanistic basis for linezolid resistance is a point mutation in

the 23S rRNA encoding gene and multimerisation of the same gene results in

more resistant levels. They showed that the selection for high levels of

resistance can be prevented by using a subinhibitory concentration of

novobiocin, as a homologous recombination inhibitor. They proposed that the

mutation is detrimental and multimerisation of the gene compensates for this.

There are increasing reports of copy number amplification of bla genes, and

causative associations with increased expression. One of the earliest was by

Xiang et al. (1997) who described amplification of plasmid-borne blaSHV-5.

Three possible mechanisms of SHV-5 β-lactamase hyperproduction were

investigated: the effect of a powerful promoter, an increase in plasmid copy

number or blaSHV-5 gene copy number amplification on a plasmid. They found

74

that the selection of high resistance levels to CTX in a blaSHV-5- expressing

K. pneumoniae is associated with blaSHV copy number amplification. Similar

results were obtained by Hammond et al. (2005). They reported an

association between cefotaxime resistance level and blaSHV copy number

amplification in the PAH ESBL-positive isolates. The examination of

epidemiologically closely related K. pneumoniae clinical isolates that carried

blaSHV, but differed in their ESBL expression status revealed that the ESBL-

expressing strains had higher blaSHV copy numbers than the ESBL non

expressers. Finally, blaSHV copy number amplification due to the formation of

tandem arrays has been directly detected in a fully characterised plasmid

from a K. pneumoniae clinical isolate by Zienkiewicz et al. (2007). They

sequenced the blaSHV-5-carrying plasmid p1658/97 and found tandem repeats

of the blaSHV-5 containing region bounded by copies of IS26. They termed this

region an amplimer. Copy number amplification is not confined to SHV family

genes, and Bertini et al. (2007) reported an association between duplication

of blaoxa-58 and high level carbapenem resistance in Acinetobacter baumannii.

Interestingly, this duplication is also of an IS26 delineated cassette.

The results of this study help to clarify the selection model for SHV-ESBL

dependent resistance to CTX and other similar antibiotics. As discussed in

the literature review, plasmid-borne blaSHV is derived from the chromosome

of K. pneumoniae, and mobilised by IS26 through transposition-like events

involving enzyme IS26 transposase. Research showed that the different

types of blaSHV promoters on plasmids are more powerful than the

chromosomal one (Ford and Avison 2004; Podbielski et al. 1991). Therefore,

it is obvious that blaSHV gene copy number is not the only parameter that

expedites the selection of ESBL expression in K. pneumoniae. According to

our data, mutation rate for the selection of ESBL expressers is very low. This

result in conjunction with other findings which revealed a correlation between

the higher blaSHV gene copy numbers and higher MIC values for ESBL

expressers strongly indicates that copy number plasticity is still of central

importance. The story of the evolution of blaSHV gene suggests the

requirement of more powerful promoters than chromosomal one plus blaSHV

mobilisation on to plasmids by IS26 copies that flank blaSHV gene and

75

facilitate multimerisation of the gene. IS26 is an intriguing element which is

820 bp and plays an important role with plasmids or gene cassettes

containing resistance genes. Hammond et al. (2005) showed the association

of IS26 with amplification of blaSHV gene copy number in K.pneumoniae

isolates. The mechanistic basis for this homologous repeats is gene

amplification involving two IS26 copies (Zienkiewicz et al. 2007). Our

experiments confirmed that the mechanistic basis for blaSHV gene

amplification is homologous recombination. Our results showed that as a

gyrase inhibitor, novobiocin could prevent the appearance of blaSHV copy

number amplified derivatives with increased MIC values. Although,

novobiocin is no longer used clinically, these results suggest that other

recombination inhibitors could have the potential to inhibit the evolution of

high CTX MICs SHV-ESBL expressers and may be useful adjuncts in the

treatment of infections with ESBL expressing bacteria. As homologous

recombination is a RecA protein dependent process, recently, a research

group at University of North Carolina (UNC) is working to find molecules

which are inhibitors of RecA (Lee et al. 2007; Wigle and Singleton 2007).

The term “heteroresistance” is difficult to define precisely but it is explained

as the presence of microbial subpopulations with higher MIC values at a

frequency of 10-7 to 10-3 in the whole population (Morand and Muhlemann

2007). This is very important from the evolutionary point of view because it

may give the opportunity of bacterial growth on higher concentrations of

antimicrobials to survive without acquisition of resistant genes for subsequent

emergence of resistant strains. This phenomenon was detected twenty years

ago and the term was firstly used for methicillin and vancomycin resistance in

Staphylococcus aureus strains (Falagas et al. 2008). This phenomenon has

now been extended to other microorganisms, including collistin resistance in

Acinetobacter baumanni-calcoaceticus complex (Hawley et al. 2008) and

penicillin resistance in Streptococcus pneumoniae (Morand and Muhlemann

2007). The CTX K. pneumoniae PAP profiles obtained in the current study

are very similar to the PAP profiles of S. pneumoniae obtained by Morand

and Mühlemann. In general, our results show the existence of the resistance

over a wider range of antibiotic concentrations. They indicate that there is

76

sufficient justification regarding K. pneumoniae strains carrying plasmid-

borne blaSHV as heteroresistant with respect to the substrates for the

encoded enzymes. Relative copy number analyses revealed that blaSHV copy

number plasticity is the cause of this heteroresistance. This has not

previously been identified as a mechanism of heteroresistance, even though

its role in heteroresistance is not surprising. This may provide further insight

in understanding the mechanism of the resistance which in turn would have a

great impact on diagnostic and treatment strategies, not just for SHV ESBLs,

but also for other β-lactamases.

This project has also thrown light on the concept of “inoculum effect, which

refers to an increase in MIC associated with a higher inoculum which may

contain higher number of cells used for MIC determinations, particularly in

MIC determination against β-lactam antibiotics. Previously, this phenomenon

was explained as the transfer of excess β-lactamase into the media along

with the inoculum and depletion of more β-lactam being used as the selective

agent. Apart from this reason, our results suggest an alternative mechanism

for inoculum effect which is the high level of heteroresistance. In other words,

larger inoculums have a higher probability of having cells that have higher

blaSHV gene copy number and consequently higher MIC values. These cells

over-express the β-lactamase which in turn may interfere in MIC

determination. The morphology of the cells that grow for the first reason is

small and clumpy on the selective solid media, while cells with the second

reason are stable and can produce normal morphology colonies. Analyses of

the results obtained from B1 and F1 isolates revealed that their behaviour is

completely different on the selective media. B1 isolate could only produce

small clumpy colonies without the ability to increase blaSHV gene copy

number, while F1 isolate mainly produced separate normal morphology

colonies and showed blaSHV gene copy number increase in higher CTX

concentration (Figure 10). It can be seen in figure 10 that B1 yielded more

colonies. It could be the result of a very high carryover of β-lactamase or it is

also possible that B1 is not heteroresistant in the same way as F1. The

results also show that determination of MIC alone is not sufficient for ESBL

77

detection and resistant strains are likely to emerge. This explains treatment

failures in Klebsiella infections with low CTX MIC values.

In summary, the findings presented in this thesis provide a valuable insight in

understanding the evolutionary mechanism of the resistance of non-ESBL

K. pneumoniae strains. Additional studies could be performed to build on the

outcomes of this thesis. It has been shown that RecA protein has a key role

in recombination stage during adaptive mutation and as a result in gene copy

number (Lee et al. 2007). The novobiocin experiment results also indicate

that the mechanism of blaSHV copy number increase is homologous

recombination. However, novobiocin is not used clinically anymore. Showing

a clinically used RecA inhibitor with the same effect on blaSHV copy number

increase, as shown by novobiocin, may lead to a new treatment strategy for

infections, caused by microorganisms producing ESBLs.

78

79

Appendices1

A. MSS-MLE Program in MATLAB R2007a Software

Since the likelihood is a univariate function of m, the maximum likelihood

estimate can be obtained simply by a grid search. More specifically, we first

build a grid for various values of m. For each of these values, we compute

the corresponding log-likelihood value. In this way we construct a graph of

log-likelihood against m. The maximum likelihood estimate is obtained by

finding the value of m that maximizes the log-likelihood. The following

program gives an answer that is accurate up to 4 significant figures.

load CTX.csv; Y = CTX; ngrid = 200; m = linspace(.01, 2, ngrid)'; C = sum( Y(:,2)); llike = zeros(ngrid,1); for i=1:ngrid llike(i,1) = llike_m(m(i),Y); end plot(m,llike); [valid] = max(llike); options = optimset('Display','off'); [m_MLE fval flag output grad Hess] = fminunc(@nllike_m, m(id) ,options, Y); sig = 1.225 * m(id) ^ (-.315) / sqrt(C); ulim = log(m(id)) + 1.96 * sig * exp(1.96 * sig) ^ (-.315); llim = log(m(id)) - 1.96 * sig * exp(1.96 * sig) ^ (.315); disp( ['MLE using the Newtons method is ' num2str(m_MLE)]) disp( ['The confidence interval is (' num2str(exp(llim)) ',' num2str(exp(ulim)) ').' ]) %disp( ['MLE using the grid-search method is ' num2str(m(id))]) %disp( ['The confidence interval is (' num2str(exp(llim)) ',' num2str(exp(ulim)) ').'])

1 The computer programs were designed by Joshua C. C. Chan whose work was in turn supported by the Australian Research Council (Discovery Grant DP0558957).

80

B. nllike_m.m Program % the log likelihood of the MSS method function llike = nllike_m(m,Y); n = length(Y); pr = zeros(500,1); p0 = exp(-m); for r = 1:500 pr(r) = m/r * (sum( pr(1:r-1) ./ (r:-1:2)') + p0/(r+1)); end logpr = log(pr); r = Y(2:end,1); llike = Y(2:end,2)' * logpr(r,1) + Y(1,2) * log(p0); llike = -llike;

81

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