IJARET_06_10_015

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International Journal of Advanced Research in Engineering and Technology

(IJARET) Volume 6, Issue 10, Oct 2015, pp. 105-112, Article ID: IJARET_06_10_015

Available online at

http://www.iaeme.com/IJARET/issues.asp?JType=IJARET&VType=6&IType=10

ISSN Print: 0976-6480 and ISSN Online: 0976-6499

© IAEME Publication

___________________________________________________________________________

Β-LACTAMASE GENES AMONG

NOSOCOMIAL MULTI DRUG RESISTANT

ACINETOBACTER BAUMANNII ISOLATED

IN CLINICAL SAMPLES

Bosco Dhanaseeli. P, V.Balasubramanian and John Maria Louis. P

Department of chemistry, AMET University, Kanathur, Chennai

ABSTRACT

Antibiotic resistance in Acinetobacter spp., particularly Acinetobacter

baumannii, is increasing rapidly. A. baumannii possesses two intrinsic b-

lactamase genes, in addition to weak permeability and efflux systems, that

together confer a natural reduced susceptibility to antibiotics. In addition,

numerous acquired mechanisms of resistance have been identified in A.

baumannii. The very high genetic plasticity of A. baumannii allows an

accumulation of resistance determinants that give rise to multidrug resistance

at an alarming rate. The objective of the present study was to identify the

presence of A. baumannii carbapenem-resistant encoding genes by real time

PCR targeting blaOXA51-like gene. The role of novel genetic elements, such

as resistance islands; in concentrating antibiotic resistance genes in A.

baumannii requires detailed investigation in the near future.

Key words: Antibiotic Resistance, Acinetobacter Baumannii, Β-Lactamases,

R-Plasmids, R-PCR.

Cite this Article: Bosco Dhanaseeli. P, V. Balasubramanian and John Maria

Louis. P. Β-Lactamase Genes Among Nosocomial Multi Drug Resistant

Acinetobacter Baumannii Isolated In Clinical Samples. International Journal

of Advanced Research in Engineering and Technology, 6(10), 2015, pp. 105-

112. http://www.iaeme.com/IJARET/issues.asp?JType=IJARET&VType=6&IType=10

INTRODUCTION

Nowadays, Anti microbial resistance in growing problem in modern hospitals the

increasing severity of illness and compromised immunity of patients treated for

cancer and other leads to frequent use of broad spectrum antibiotic agents. There are

over 20 species of acinetobacter, though the acinetobacter baumannii accounts for



more than 80% of isolates causing human disease. Acinetobacter species are

ubiquitous gram negative bacteria wide spread in nature.

Acinetobacter baumannii has emerged as an important nosocomial pathogen (1–

5). Hospital outbreaks have been described from various geographic areas (6–9), and

this organism has become endemic in some of them. The role of the environmental

contamination in the transmission of nosocomial infections in general and in A.

baumannii infections in particular is well recognized (10, 11). A. baumannii does not

have fastidious growth requirements and is able to grow at various temperatures and

pH conditions (12). The versatile organism exploits a variety of both carbon and

energy sources. These properties explain the ability of Acinetobacter species to persist

in either moist or dry conditions in the hospital environment, thereby contributing to

transmission (13, 14). This hardiness, combined with its intrinsic resistance to many

antimicrobial agents, contributes to the organism’s fitness and enables it to spread in

the hospital setting.

The nosocomial epidemiology of this organism is complex. Villegas and Hart

stein reviewed Acinetobacter outbreaks occurring from 1977 to 2000 and

hypothesized that endemicity, increasing rate, and increasing or new resistance to

antimicrobial drugs in a collection of isolates suggest transmission. These authors

suggested that transmission should be confirmed by using a discriminatory

genotyping test (15). The importance of genotyping tests is illustrated by outbreaks

that were shown by classic epidemiologic methods and were thought to be caused by

a single isolate transmitted between patients; however, when molecular typing of the

organisms was performed, a more complex situation of multiple unrelated strains

causing the increasing rates of infections by A. baumannii was discovered (16–17).

Almost 25 years ago, researchers observed acquired resistance of A. baumannii to

antimicrobial drugs commonly used at that time, among them aminopenicillins,

ureidopenicillins, first and second-generation cephalosporins, cephamycins, most

aminoglycosides, chloramphenicol, and tetracyclines (19).

Since then, strains of A. baumannii have also gained resistance to newly

developed antimicrobial drugs. Although multidrug-resistant (MDR) A. baumannii is

rarely found in community isolates, it became prevalent in many hospitals (23). MDR

A. baumannii has recently been established as a leading nosocomial pathogen in

several Israeli hospitals, including our institution (20,21). Several locally contained

small outbreaks of MDR A. baumannii occurred in our institution during the late

1990s. In 1999, however, the incidence of MDR A. baumannii isolation had doubled

compared to the previous 2 years, and the organism became endemic in many wards

(unpub. data). The likelihood of isolation of A. baumannii from a hospitalized patient

is related to temporospatial (extrinsic, ecologic characteristics) factors such as

colonizationpressure (22), nurse-to-patient ratio, and other ward characteristics and to

individual patient risk factors (characteristics). The current study was designed to

examine the occurrence and spread of A. baumannii within our institution, as well as

to define individual risk factors for isolation of this organism.

MATERIALS AND METHODS

Isolation and Traditional Identification of Isolates

Collection of samples: Three hundred Acinetobacter samples were collected during

2012 from different labs located at Chennai (which was isolated and screened from

the clinical samples of the patients admitted in the Intensive Care Units). Clinical

Β-Lactamase Genes Among Nosocomial Multi Drug Resistant Acinetobacter Baumannii

Isolated In Clinical Samples


samples included in this study - urine, blood, pus, wound swab, sputum, ET

(Endotrachial aspirate).

ISOLATION OF SAMPLES

Preliminary identification was performed by gram staining, culturing on

MacConkey's agar (Himedia, India) and incubated for overnight at 37°C for 24 hrs,

non lactose fermenting bacteria or colorless were sub-cultured and incubated for

additional overnights. Suspected bacterial isolates which their cells are Gram negative

coccobacillary and negative to oxidase, positive to catalase further identified by the

traditional biochemical test according to15,16

then additional confirmed by Vitek 2

system. Informed consent was obtained from the patients prior to sample collection.

The identified Acinetobacter spp. isolates were confirmed by API 20E and API20 NE

multi-test systems (BioMerieux, France). These tests were used according to

manufacturer's protocol for Enterobacteriaceae and non enteric bacteria. Wells of

biochemical test were inoculated with overnight 0.5 McFarland bacterial suspensions

and incubated at 37°C for 24 hrs. The results were read after addition of reagents, as 7

digit number that identify by API 20 analytical index.17

CONFIRMATION

The organisms were further confirmed as Acinetobacter by performing various

biochemical tests such as Oxidase, Catalase and culturing in McConkey agar medium

(Purchased from Sisco Research Laboratores Pvt. Ltd., Mumbai, India.

Figure 1

The ability of A.bauminnii to survive for extended periods on environmental

surfaces is notorious and is likely important for transmission within the health care

setting. Multidrug resistance is common with health care- associated A.bauminnii

infections. The impressive number of acquired mechanisms of resistance makes

selection of an appropriate empirical antimicrobial agent exceeding difficult. The

diversity of resistance mechanisms is beyond the scope of this article but can be

reviewed in. 18

Acinetobacter species has rapidly become the most common ESBL

producing organism, making it difficult to eradicate this organism from the high risk

wards such as Intensive Care Units. It is considered to be dangerous species due to its

multidrug resistance capacity. A. baumanii strains presented a large metabolic

activity. They had the capacity to produce acid from glucose, xylose, galactose,

manose, rhamnose and lactose.

The production of acid from maltose and urea test are variable reactions. All

strains were positive to Simmons citrate. The negative reactions: the acid production



from manitol and sucrose, esculin hydrolisis, H2S on TSI, nitrate reduction, methyl

red and Voges-Proskauer.19

Biochemical Identification tests for Acinetobacter

Figure 2

ISOLATION OF Acinetobacter Species

The Organism which was received from different hospital laboratories located in

Chennai was further confirmed by the observation of growth in Mc Conkey Agar

Medium and various Biochemical assays (Table No: 1 ). The organisms were Gram

negative, rod shaped bacteria, Hence the organisms were confirmed to be

Acinetobacter species by referring Bergey’s Manual of Bacteriology.

Table-1

Samples No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Gram strain - - - - - - - - - - - - - - - - - -

Glucose + + + + + + + + + + + + + + + + + +

Luctose + + + + + + + + + + + + + + + + + +

Surcose + + + + + + + + + + + + + + + + + +

Caesin - - - - - - - - - - - - - - - - - -

Urease - - - - - - - - - - - - - - - - - -

Strach - - - - - - - - - - - - - - - - - -

M R - - - - - - - - - - - - - - - - - -

V P - - - - - - - - - - - - - - - - - -

Indole - - - - - - - - - - - - - - - - - -

Citrate + + + + + + + + + + + + + + + + + +

Oxidase - - - - - - - - - - - - - - - - - -

Gelatin + + + + + + + + + + + + + + + + + +

Catalase + + + + + + + + + + + + + + + + + +

Plasmid isolation and its molecular weight by agarose gel electrophoresis

Plasmid isolation and determination of its molecular weight

Plasmid isolation: The plasmids from A. baumannii were isolated by following the

standard procedure with slight modifications.31, 32 This

study was carried out to

investigate the presence of resistant plasmids (R-plasmids) among the clinical isolates

of A. baumannii. In addition, the study was performed to check the presence of

common β-lactamases encoding genes on this plasmid.31, 32

Fig. no. 8, pictures the

documented gel for the plasmid isolated and separated. The organism which was




considered to be the ESBL producers [i.e. resistant to ceftazidime(CAZ) and sensitive

to ceftazidime clavulinic acid(CAC)] was subjected for plasmid isolation and

separated using Agarose Gel Electrophoresis.

Lane-1 Lane-2 Lane-3 Lane-4 Lane-5 Lane-6

1 kb Ladder

Fig. no. 3. Agarose gel electrophoresis containing I kb ladder and plasmid DNA

Lane-1- Marker(1kb ladder) , Lan-2- plasmid(strain-1), Lane 4 and 5- Plasmid (strain

11), Lan-6- plasmid(strain-15) In the figure Lane 1 represents the marker of molecular

weight ranging from 14000kb – 67000kb. In the lanes 2, 4 and 6 the plasmid which

was isolated from strains 1, 11, 15 was separated. The molecular weight which

corresponding to the marker was calculated to be 14000kb.33

To identify the gene sequence using RT-PCR techniques

Characterization of antimicrobial resistance

All Multi drug resistance A. baumannii isolates were sequenced by PCR to detect

antibiotic resistance determinants and to identify mutations associated with

fluoroquinolone resistance (Ciprofloxacin). The specific primers used in detecting

antibiotic resistance determinants are described in Table 7. PCR and sequencing

analysis of antibiotic resistance determinants, excluding integron, was performed by

the same method as probe gene analysis with the exception of primer annealing 560C

for 1min. Integron detection, characterization, and gene cassette amplification were

carried out using previously described. 33

Primer Sequence: [bla-OXA gene] PCR Product size: 580

F-5’-CTTTCCCATGATGAGCACCT-3’

R-5’-CAATGCGCTCTGCTTTGTTA-3’

PCR Procedure

Components Quantity

In PCR vial Master mix 25µl

MupA Primer Mix (10pmoles/ µl) 1µl

MupA Primer Mix (10pmoles/ µl) 1µl

Genomic DNA 1µl

Water, nuclease free 22µl

Toatal volume 50µl



Reactions set up as follows;

Mixed gently and spin down briefly.

Place into PCR machine and program it as follows;

Initial Denaturation: 94ºC for 3 min Denaturation: 94ºC for 1 min Annealing:

56ºC for 1min 35 cycles Extension: 72ºC for 1min Final extension: 72º C for 5 min.

A real-time PCR assay was developed using TaqMan chemistry for the

amplification of nucleotide sequence. The probe 5’ is labeled with 6-

carboxyflurescein and the 3’ labeled with 6- carboxytetramethylrhodamine were used.

The blaOXA gene was chosen because it is present in all sequenced genome of

A.baumannii spp. The A.baumannii blaOXA sequences 100% identity. The primer

blaOXA forward F-5’- CTTTCCCATGATGACACCT-3’ and blaOXA reverse R- 5’-

CAATGCGCTCTGCTTTGTTA-3’. The specificity of the assay for A.baumannii was

determined using genomic DNA from a clinical isolates.

Table 3

Details of Primer used for the detection of blaoxa gene in this study Amplicon size

Target gene Primer Sequence Amplicon

size(bp) Source/Reference

blaoxa F-5’CTTTCCCATGATGAGCACCT-3’

R-5’-CAATGCGCTCTGCTTTGTTA-3’

580 Present study

S- 2 S-3 LADER S-4 DNA Lader S -7 S -8

1Kb

Figure 4 PCR Product size: ~580bp

HELINI 250bp DNA Ladder [1000bp, 750bp, 500bp, 250bp and 100bp

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