Differential susceptibility of invasive Haemophilus influenzae serotype a and serotype b to ampicillin and other commonly prescribed antibiotics

ORIGINAL ARTICLE

Differential susceptibility of invasive Haemophilusinfluenzae serotype a and serotype b to ampicillin andother commonly prescribed antibioticsM. Shuel1, K. Whyte1, T. Drew1, J. Wylie2, B. Lefebvre3, L. Hoang4 and R.S.W. Tsang1

1 Vaccine Preventable Bacterial Diseases Division, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, MB, Canada

2 Cadham Provincial Laboratory, Manitoba Health, Winnipeg, MB, Canada

3 Laboratoire de sant�e publique du Qu�ebec, Sainte-Anne-de-Bellevue, QC, Canada

4 Public Health Microbiology and Reference Laboratory, BC Public Health Microbiology and Reference Laboratory, Vancouver, BC, Canada

Significance and Impact of the Study: Despite H. influenzae serotype b (Hib) vaccine programs, invasivedisease due to Hib still exists in Canada and is either second or third most common behind nontypeableand/or serotype a (Hia). Many previous studies on antibiotic resistance have focussed on respiratory iso-lates, and few have looked at resistance with regard to serotype. This study analysed antibiotic resis-tance in invasive Hia and Hib collected over 20 years from three provinces, and results found thatsignificantly more Hib showed resistance compared to Hia. This provides a small snapshot of H. influen-zae disease in Canada and highlights the importance to continually monitor antibiotic resistanceprofiles.

Keywords

antibiotic resistance, beta-lactamase,

Haemophilus influenzae,

serotype a, serotype b.

Correspondence

Raymond Tsang, Public Health Agency of

Canada, National Microbiology Laboratory,

1015 Arlington Street, Winnipeg, Manitoba

R3E 3R2, Canada.

E-mail: [email protected]

2013/2482: received 10 December 2013,

revised 3 April 2014 and accepted 3 April

2014

doi:10.1111/lam.12265

Abstract

Haemophilus influenzae serotype a (Hia) has become an important pathogen in

the post-H. influenzae serotype b (Hib) vaccine era. Antibiotic resistance in

H. influenzae is a global phenomenon, but few studies have looked at antibiotic

resistance profiles with regard to serotype. Invasive Hia (n = 157), noninvasive

Hia (n = 2) and invasive Hib (n = 42) collected over the last two decades from

three Canadian Provinces were examined for resistance to several commonly

prescribed antibiotics, and sequence types (STs) were determined by MLST.

Only 1�9% of Hia showed antibiotic resistance, while 31% of Hib were resistant

to one or more antibiotic. Resistance to ampicillin, sulfamethoxazole–trimethoprim, chloramphenicol and tetracycline was observed, with b-lactamase-mediated ampicillin resistance being the most common. Nine STs

were identified for Hia with 7 STs belonging to the same clonal complex. Ten

STs were observed in Hib strains, and all of them belonged to a single clonal

complex. A possible correlation between sequence type and ampicillin resistance

was observed for Hib, while no correlations were observed for Hia.

Introduction

Haemophilus influenzae is an important bacterial pathogen

causing a wide variety of respiratory and invasive infec-

tions. Six serotypes, a through f, have been identified

based on polysaccharide capsular antigens, and strains not

exhibiting a capsule are termed nontypeable. Since the

introduction of the H. influenzae serotype b (Hib) vac-

cine, there has been a dramatic decrease in disease due to

Hib and several reports of increase in nontypeable and

nontype b infections (Peltola 2000; Dworkin et al. 2007;

Tsang et al. 2007; Adam et al. 2010; Ulanova et al. 2012).

Despite the Hib vaccine programs, invasive disease due to

Hib still exists in small numbers in Canada and has been

reported to be either the second most common type

behind nontypeable (Adam et al. 2010; Shuel et al. 2011)

or third behind nontypeable and Hia infections (Tsang

et al. 2007), depending on the province.

Ampicillin resistance was first described in H. influen-

zae in the 1970s (Gunn et al. 1974) and is now a global

Letters in Applied Microbiology 59, 193--199 © 2014 Her Majesty the Queen in Right of Canada Letter in Applied Microbiology © 2014 Society for Applied Microbiology

Reproduced with the permission of the Minister of Public Health Agency of Canada 193

Letters in Applied Microbiology ISSN 0266-8254

phenomenon with resistance rates ranging from 8% up to

almost 40% (Jacobs 2003; Sahm et al. 2008; Hoshino

et al. 2013). The American Academy of Pediatrics as well

as the Canadian Paediatric Society recommends amoxicil-

lin as the first-line drug of choice for treatment of upper

respiratory tract infections such as otitis media and sinus-

itis. If the upper respiratory tract infection is caused by

b-lactamase producing ampicillin-resistant strains, amoxi-

cillin–clavulanate, azithromycin or oral cephalosporins

such as cefdinir, cefaclor or cefpodoxime are suggested

(American Academy of Pediatrics 2012; http://www.cps.

ca/documents/position/acute-otitis-media. Accessed 2013-

11-04). For initial treatment of Hib meningitis infections,

cefotaxime or ceftriaxone is recommended; however,

ampicillin can also be used, provided that the isolate is b-lactamase negative and susceptible to ampicillin (Ameri-

can Academy of Pediatrics 2012; http://www.macpeds.

com/documents/CPSmeningitisguideline2007.pdf. Accessed

2013-11-04). In North America and Europe, production

of a b-lactamase is responsible for the majority of ampi-

cillin-resistant strains (b-lactamase positive, ampicillin

resistant or BLPAR). However, in Japan, ampicillin resis-

tance is mainly caused by mutations in the ftsI gene

which encodes an altered penicillin-binding protein 3 and

results in strains that are b-lactamase negative, ampicillin

resistant (BLNAR) (Jacobs 2003; Hoshino et al. 2013).

Over 30% of the ampicillin-resistant strains in Japan are

BLNAR compared to <10% that are resistant due to b-lactamase production (Hoshino et al. 2013).

Many of the previous studies in Canada on antibiotic

resistance in H. influenzae were carried out on respiratory

isolates, and only a few of those studies further character-

ize the isolates based on serotype. Studies on invasive

H. influenzae disease have shown that antibiotic resistance

is most often seen in nontypeable strains (Sill and Tsang

2008; Resman et al. 2012). However, in the post-Hib con-

jugate vaccine era, Hia has emerged to become an

increasingly important cause of invasive infections in the

indigenous populations in North America, South Amer-

ica, Australia and New Zealand (Bruce et al. 2013; Ulano-

va and Tsang 2014), and few studies have looked at

susceptibility of Hia isolates to antibiotics. Therefore, this

study compares the sensitivity of Hia and Hib to com-

monly prescribed antibiotics for treatment of invasive

H. influenzae infections. Here, we report a differential

susceptibility of invasive Hia and Hib strains to ampicillin

and other antibiotics.

Results and discussion

A sample of 159 Hia (157 recovered from normally sterile

body sites such as blood and CSF, and 2 from ear speci-

mens) and 42 Hib invasive isolates from individual

disease cases obtained over the last 2 decades (1991 to

2013) from the provinces of British Columbia, Manitoba

and Quebec were used in this study. These strains were

provided to us for confirmation of serotypes and other

studies as part of our laboratory surveillance of invasive

H. influenzae disease. Table 1 shows the breakdown of

isolates by province and year of isolation. Over 70% of

both Hia (n = 116) and Hib (n = 31) were recovered

from blood, followed by CSF (12% of Hia [n = 19] and

14% of Hib [n = 6]), and isolates that were submitted

from both blood and CSF were approx. 7% for both Hia

(n = 11) and Hib (n = 3). The remaining 15 isolates were

from other sources such as ear, joint fluid and synovial

fluid. The age range of patients with Hia infections was

1 month to 101 years and 1 month to 87 years for Hib.

The median age of patients with Hia was 1 year in both

Manitoba and British Columbia, while the median age in

Quebec was 4 years. Like Hib in the prevaccine era, Hia

mainly affected children under 5 years old, especially

those under 2 (Jordens and Slack 1995). In the post-Hib

vaccine era, Hib disease is now very rare in those under

5 years old, except in those who are incompletely immu-

nized or not immunized at all. The overall median age

for patients with Hib was 38�5 years. In British Columbia,

the median age was 46 years, with 21 cases in adults aged

24–86 years and three recovered from patients who

were 1 year old. In Manitoba, the median age for Hib

Table 1 Distribution of Haemophilus influenzae serotype a and b iso-

lates by province and year of isolation

Year

Manitoba

British

Columbia Quebec

Hia Hib Hia Hib Hia Hib

1991 1

1995 1

1996 2

1997 1

1999 6

2000 3

2001 9 2

2002 7 1

2003 6 1 1

2004 4 1 1

2005 7 1

2006 2 2 2

2007 2 2

2008 12 1 5 9 1

2009 5 4 3 1

2010 14 1 2 1 7

2011 11 3 1 5 1

2012 10 2 3 4 12 1

2013 5 3 1 2 6

Total 108 15 16 24 35 3


Reproduced with the permission of the Minister of Public Health Agency of Canada194

Antibiotic resistance of H. influenzae M. Shuel et al.

infections was 10 months, with 11 isolates from patients

1 year or under and only four found in adults aged 25–87 years. There were only 3 Hib isolates from Quebec,

aged 3 months, 3 and 27 years. Although vaccination his-

tory was not provided for any of the cases, there were 10

cases from Manitoba and 1 case from Quebec in patients

under 12 months who were too young to have received

the booster dose of Hib conjugate vaccine. Exact birth

dates were not given for the 1-year-old patients from

British Columbia and Manitoba, so it is unknown

whether or not they had received the booster dose (Forgie

et al. 2009; http://www.phac-aspc.gc.ca/publicat/cig-gci/

p01-12-eng.php#tab1. Accessed 2014-01-29). Medical his-

tories were also not provided, so it is unknown whether

the adult cases occurred in high-risk or immunocompr-

omized patients.

Serotyping results for slide agglutination and PCR

methods were consistent in 199 of the 201 strains. One

Hia and one Hib strains were detected by PCR only,

while bacterial agglutination tests for these two strains

were negative.

Biotype II was the most common biotype seen among

the Hia isolates, and biotype I was most common among

Hib. Of the 159 Hia, 156 were biotype II (98�1%) and 3

were biotype I (1�9%). Of the 42 Hib, 36 were biotype I

(85�7%), 5 were biotype II (11�9%), and 1 was biotype IV

(2�4%). Multilocus sequence typing (MLST) results iden-

tified 9 different sequence types (STs) among the Hia iso-

lates. ST-23 (n = 131) was most common, followed by

ST-56 (n = 11), ST-576 (n = 10), and ST-62 (n = 2), and

there was one isolate each belonging to ST-4, ST-397, ST-

405, ST-529 and ST-1035. For Hib, there were 10 STs

found: ST-231 (n = 17), ST-95 (n = 11), ST-6 (n = 4),

ST-190 (n = 4) and 1 isolate each of ST-44, ST-53, ST-

54, ST-92, ST-157 and ST-641. A diagram of the clonal

relationships between STs is shown in Fig. 1. For our col-

lection of strains, the founding STs for Hia and Hib iso-

lates were ST-23 and ST-6, respectively.

The majority (98�1%) of Hia strains were b-lactamase

negative and susceptible to all antibiotics tested by the disc

diffusion method. There were only 3 Hia strains found to

be BLPAR (1�9%). Two were found to produce the TEM-

1 type b-lactamae, and 1 isolate from Manitoba recovered

in 2002 produced a ROB-1 type b-lactamase. Resistance to

other antibiotics was not observed, and none of the Hia

strains harboured mutations in the ftsI gene that may be

associated with decreased susceptibility to b-lactam antibi-

otics such as ampicillin (Table 2). On the other hand, Hib

strains showed a much higher rate of ampicillin resistance

due to b-lactamase production. Of the 42 Hib strains

studied, 10 (23�8%) were BLPAR and all had TEM-1 type

b-lactamases. Resistance to other antibiotics was also

observed, including sulfamethoxazole–trimethoprim (9�5%

or 4 isolates), chloramphenicol (9�5% or 4 isolates) and

tetracycline (2�4% or 1 isolate) (Table 2). Overall, 28�6%of the Hib isolates in this study showed resistance to at

least one antibiotic. In addition, 3 Hib strains (7�1%)

were found to have the N526K substitution in PBP3: two

were b-lactamase positive (PBP3 substitutions also

included D350N G490E A530S V547I and N569S for the

ampicillin-resistant strain and A502V V547I and N569S

for the strain resistant to ampicillin, chloramphenicol and

tetracycline), and the other was b-lactamase negative

(PBP3 substitutions also included K344R D350N

T352G K355T L356V M377I I449V and V569S). The

23

56

1035

397

576

405

529

62

4

6

231

641

95

190

92

44

53

157

54

(a)

(b)

Figure 1 Clonal analysis and comparison of multilocus sequence typ-

ing data by presentation of minimum spanning tree diagram of

Haemophilus influenzae serotype a (a) and serotype b (b) isolates.

Each node represents each sequence type identified, and clonal com-

plexes are shown with shaded backgrounds.



M. Shuel et al. Antibiotic resistance of H. influenzae

b-lactamase-negative strain with PBP3 substitutions

showed a disc diffusion zone size resulting in intermediate

resistance to ampicillin for both the 2- and 10-lg discs

(Karpanoja et al. 2004). Besides the TEM-1 and ROB b-lactamases, we did not detect the newly defined TEM var-

iant b-lactamases (Tristram et al. 2012).

Besides the antibiotic resistance profiles we have

described here for Hia and Hib, we have also reported on

antibiotic resistance of nontypeable H. influenzae. Our

previous study on 147 invasive nontypeable isolates from

1990 to 2006 in Canada found that 31�3% were resistant

to one or more antibiotic, 20�4% were BLPAR, and

11�6% had the N526K substitution in PBP3 and were

classified as BLNAR (Sill and Tsang 2008; Shuel and

Tsang 2009).

Previous reports have shown a significant change in the

frequency of b-lactamase-producing H. influenzae in Can-

ada, with rates of 28�4% in 1992–1993 (Scriver et al.

1994), 32�2% in 1993–1994 (Blondeau et al. 1999), 24�2%in 1997–1998 and decreasing to 18�6% in 2001–2002(Zhanel et al. 2003). These studies focused on respiratory

isolates and did not report the serotype nature of the

strains. Our current study with invasive Hia and Hib iso-

lates also supports this decreasing trend if considering

results for all 201 isolates together, which have an overall

rate of b-lactamase production of 6�5%. However, when

looking at Hia and Hib separately, our study found that

significantly more Hib were BLPAR compared to Hia (P

value <0�001), with 23�8% of Hib and only 1�9% of Hia

producing b-lactamase. The rate of BLPAR Hib strains

observed in this study was nearly as high as the overall

rate observed by Zhanel et al. in 1997–1998 (Zhanel et al.

2003). We do not believe this was related to strain selec-

tion in our study as none of the strains were submitted

for confirmation of ampicillin resistance or b-lactamase

detection.

Mutations involving the N526K substitutions in the ftsI

gene were only observed in Hib and included 2 b-lactamase-

positive and 1 b-lactamase-negative strain. The b-lactam-

ase-negative isolate showed reduced susceptibility to ampi-

cillin (MIC = 1�0 lg ml�1) and was consistent with

previous findings of low-BLNAR or gBLNAR strains in

Japan, Spain and Canada that have PBP3 substitutions and

show reduced susceptibility to ampicillin (Hasegawa et al.

2004; Garcia-Cobos et al. 2008; Shuel and Tsang 2009).

Resistance to antibiotics other than ampicillin, whether in

combination with b-lactamase production or not, was only

observed in Hib. Multidrug resistance was also only

observed in Hib and included a combination of chloram-

phenicol, tetracycline and sulfamethoxazole–trimethoprim

in addition to ampicillin resistance due to b-lactamase.

Although this was not a systematic study and the observa-

tions may not represent the complete picture, it is interest-

ing to note that of the four multidrug-resistant strains,

three were collected within the past 18 months (1 from

2011, 1 from 2012 and 1 from 2013) and came from two of

the three provinces included in this study, Manitoba and

British Columbia.

MLST data show that all Hib isolates belonged to the

same clonal complex, and the majority of Hia was within

one clonal complex with the exception of ST-4 and ST-

62. Even though the Hib isolates all belonged to the same

clonal complex, Hib appears to be more diverse with

more single locus variants and more double locus variants

compared to Hia.

There did not appear to be a correlation between b-lac-tamase producing Hia and biotype or ST. The 3 BLPAR

isolates belonged to 2 related STs, ST-23 (n = 2) and ST-

1035 (n = 1) and were all biotype II, the most common

biotype seen in Hia. Of the 9 STs identified among the

Hia isolates, 7 were related by MLST and were either a

single, double or, in the case of ST-529, a triple locus var-

iant of ST-23. All Hia belonging to these 7 STs were clas-

sified into clonal division I (Tsang et al. 2013). Another

clonal division I Hia belonged to biotype I and ST-4.

Two biotype I Hia were classified as clonal division II

Table 2 Antibiotic resistance profiles of 201 serotype a and serotype b Haemophilus influenzae strains

Serotype

Number of

isolates

b-Lactamase

production

ftsI Gene

mutations Antibiotic resistance profile* Sequence types

a 156 � No Susceptible to all antibiotics tested ST-4, 23, 56, 62, 397, 405, 529, 576

3 + No Amp R ST-23, 1035

b 29 � No Susceptible to all antibiotics tested ST-6, 95, 157, 190, 231, 641

2 � No Sxt R ST-53, 54

1 � Yes Amp I ST-44

5 + No Amp R ST-95

1 + No Amp R, Chl R, Sxt I ST-190

2 + No Amp R, Chl R, Sxt R ST-190

1 + Yes Amp R ST-92

1 + Yes Amp R, Chl R, Tet R ST-95

*Ampicillin (Amp), trimethoprim–sulfamethoxazole (Sxt), chloramphenicol (Chl), tetracycline (Tet), resistant (R), intermediate (I).




and belonged to ST-62, which shared no common alleles

with any of the clonal division I strains (Tsang et al.

2013). Hia has been described as a recently emerged path-

ogen (Bruce et al. 2013), and with time, whether they will

acquire resistance to ampicillin due to b-lactamase pro-

duction and/or PBP3 substitutions should be carefully

monitored in the future.

The 3 Hib strains displaying PBP3 substitutions

belonged to ST-44, ST-92 and ST-95 and were all biotype

I. The 10 BLPAR Hib isolates belonged to ST-92, ST-95

and ST-190 and were again all biotype I. Six of 11 ST-95

isolates and 3 of 4 ST-190 isolates were b-lactamase posi-

tive, suggesting a possible correlation between ST and

ampicillin resistance. In the National Microbiology Labo-

ratory’s H. influenzae culture collection, there were a total

of 34 Hib belonging to ST-95 and 7 belonging to ST-190.

The number of BLPAR strains within these sequence

types were 22 (64�7%) and 6 (85�7%), respectively (NML

unpublished data). Although the numbers are small and

more isolates should be studied to confirm this finding, it

is important to note this connection between ST and b-lactamase production, especially because ST-95 is one of

the more common Hib STs.

With recent reports suggesting nontypeable and non-

type b serotypes as the main causes of invasive H. influen-

zae disease in the post-Hib vaccine era, in addition to the

continued presence of Hib infections, it is important to

continually monitor the antibiotic susceptibility patterns

of strains responsible for disease. This study on Hia and

Hib from 3 Canadian provinces provides a small snapshot

of H. influenzae disease in Canada, and future studies

should be expanded to include isolates from all serotypes

as well as from other provinces.

Materials and methods

Isolates of H. influenzae were serotyped by bacterial slide

agglutination using commercial antisera (Difco, Oakville,

Ontario, Canada) and confirmed by PCR detection of

serotype-specific genes and the capsule transport gene,

bexA (Falla et al. 1994). Biotypes were determined accord-

ing to Kilian (1976), and MLST was carried out as previ-

ously described (Meats et al. 2003) using the MLST

online database, http://haemophilus.mlst.net, for allele

number and sequence type (ST) assignment. eBURST

(ver. 3) was used to determine the founder STs in our

collection of strains (Feil et al. 2004).

Production of b-lactamase was detected using DrySlide

nitrocefin (BBL, Becton Dickinson, Oakville, Ontario).

PCR detection of TEM-1 and ROB type b-lactamases was

performed using previously described methods (Molina

et al. 2003). Disc diffusion was performed using guide-

lines set out by the Clinical Laboratory Standards

Institute (CLSI), and control strain ATCC 49247 was used

in each experiment (CLSI 2013). The following antibiotics

discs (Oxoid, Nepean, Ontario) were tested: ampicillin (2

and 10 lg), amoxicillin–clavulanic acid (30 lg), azithro-mycin (15 lg), ceftriaxone (30 lg), cefuroxime (30 lg),chloramphenicol (30 lg), ciprofloxacin (5 lg), clarithro-mycin (15 lg), imipenem (10 lg), levofloxacin (5 l),meropenem (10 lg), moxifloxacin (5 lg), tetracycline

(30 lg) and trimethoprim–sulfamethoxazole (25 lg).Both the 2-lg and 10-lg ampicillin discs (Karpanoja

et al. 2004) were used in addition to partial gene sequenc-

ing of the ftsI gene (Ubukata et al. 2001) to detect

BLNAR strains.

Acknowledgements

The authors would like to thank the staff at the National

Microbiology Laboratory’s DNA Core Facility for the pri-

mer synthesis and the DNA sequencing work, and Dennis

Law for confirmation of the serotypes by PCR.

Conflict of Interest

No conflict of interest to declare.

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Differential susceptibility of invasive Haemophilus influenzae serotype a and serotype b to ampicillin and other commonly prescribed antibiotics