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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 5  |  Issue : 2  |  Page : 62-68

Molecular characterisation of beta-lactamases-producing Enterobacteriaceae members from critical care patients


1 Department of Microbiology, JNMC, AMU, Aligarh, Uttar Pradesh, India
2 Department of Anaesthesiology, JNMC, AMU, Aligarh, Uttar Pradesh, India

Date of Web Publication19-Jan-2018

Correspondence Address:
Dr. Richa Gupta
Department of Microbiology, JNMC, AMU, Aligarh, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jpsic.jpsic_20_17

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  Abstract 


Background: This study was done to assess the prevalence of beta-lactamases and biofilm production in general and blaCTX-M, blaTEM, blaSHV and blaAmpC in particular amongst Enterobacteriaceae members in Intensive Care Unit (ICU) patients.
Materials and Methods: Samples were collected aseptically from 210 ICU patients from February 2012 to December 2015. Culture, identification, antimicrobial susceptibility, extended-spectrum beta-lactamases (ESBLs), AmpC, metallo-beta-lactamase (MBL) and biofilm detection were done according to the standard protocol. Polymerase chain reaction analysis for beta-lactamase genes of the family CTX-M, TEM, SHV and AmpC was carried out.
Results: Amongst 200 pathogens, most commonly isolated Enterobacteriaceae member was Escherichia coli (2 [26%]), Klebsiella pneumonia (37 [18.5%]), Klebsiella oxytoca (12 [6%]), followed by Citrobacter species (33 [16.5%]) and Serratia species (15 [8%]). E. coli (29 [55.7%]) was most commonly associated with urinary tract infection; however, the frequency of K. pneumoniae (18 [48.6%]), K. oxytoca (5 [41.6%]) and Citrobacter koseri (20 [33%]) was higher in lower respiratory tract, bloodstream and surgical site infections, respectively. Phenotypically, prevalence of ESBL, AmpC and MBL amongst Enterobacteriaceae members was 50.2%, 36.6% and 12.3%, respectively. However, blaCTX-M and blaAmpC genes were detected in 48% and 32.4% of members, respectively. BlaTEM and blaSHV were not detected in any of the isolates. The average hospital stay of ICU patients was 21 days and was associated with 48.5% mortality.
Conclusion: There is a great need for informed antibiotic treatment guided by not only routine antimicrobial susceptibility but also by knowledge of ESBL, AmpC and MBL status of the isolate.

Keywords: Enterobacteriaceae members, Intensive Care Unit, molecular characterisation


How to cite this article:
Gupta R, Malik A, Rizvi M, Ahmed SM. Molecular characterisation of beta-lactamases-producing Enterobacteriaceae members from critical care patients. J Patient Saf Infect Control 2017;5:62-8

How to cite this URL:
Gupta R, Malik A, Rizvi M, Ahmed SM. Molecular characterisation of beta-lactamases-producing Enterobacteriaceae members from critical care patients. J Patient Saf Infect Control [serial online] 2017 [cited 2018 Apr 25];5:62-8. Available from: http://www.jpsiconline.com/text.asp?2017/5/2/62/223691




  Introduction Top


Antimicrobial resistance is proven to develop when an organism is exposed to an antimicrobial substance for a given period of time as they develop adaptability to the compound. The resistance acquired by extended-spectrum beta-lactamase (ESBL)-producing strains stems from genetic point mutation, and it is an unsolved, growing problem. In the near future, both clinicians and microbiologist will be battling against ESBL-producing strains due to their dynamic evolution and epidemiology in preventing and controlling them. Bali et al., 2010,[1] reported that ESBL-producing strains are undergoing continuous mutations, causing the development of new enzymes showing expanded substrate profiles. One of the major reasons for their resistance is inappropriate usage of antibiotics in treatment of infection and rapid detection of the antibiotic pattern is crucial for treatment.[2] The prevalence of ESBLs such as TEM, SHV, CTX, OXA and AmpC amongst clinical isolates varies from country to country and from institution to institution.[3]

Beta-lactamases have been frequently identified in Klebsiella pneumoniae and  Escherichia More Details coli but also in other species such as Citrobacter spp., Enterobacter spp., Serratia spp. and Pseudomonas aeruginosa. ESBL producers have been found worldwide.[4]

Survey regarding ESBL-producing isolates' detection is crucial for infection control measure and the choice of antimicrobial therapy chosen there upon. The detection of ESBL-producing bacteria in laboratories is a vital step for appropriate management of patients, but genotypic identification of these enzymes provides essential information for infection prevention and control efforts.[5] Keeping in view about the complexity of ESBL-producing strains, the current study was undertaken to examine the prevalence of ESBL-producing strains in Aligarh district and occurrence of CTX-M, TEM, SHV and AmpC gene amongst Enterobacteriaceae members.


  Materials and Methods Top


The study was conducted in the Department of Microbiology on Intensive Care Unit (ICU) patients of Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, from February 2012 to December 2014. A total of 160 isolates were obtained from clinical samples during this study admitted into various units such as medical, surgical, obstetrics and gynaecology as well as orthopaedic units. J. N. Medical College is 1050-bed tertiary-level university hospital. The mixed medical-surgical ICU is a 10-bed unit with one 6-bed, one 2-bed and two single-bed rooms. The isolates were obtained from the cultures of specimens from patients hospitalised for >48 h. The sources of these isolates were endotracheal aspirate, urine, pus, blood, eye discharges and body fluids. Demographic and other relevant details were retrieved from patient record files. Informed written consent was taken before the study from all the patients, and the study was performed after approval from the Institutional Ethics Committee. All the specimens were quickly sent to the laboratory to be processed for microbial pathogens.

Patients were chosen consecutively, and clinical samples were obtained from each patient (endotracheal aspirate, blood, pus and urine). Single isolate per patient was included in the study. Wounds (surgical site infections) have been classified according to the Southampton grading. Majority of the cases belonged to Grade IV (purulent discharge along the wound) and Grade V (wound dehiscence). Standard methods for isolation and identification of Enterobacteriaceae were used.[6],[7]

Antimicrobial Susceptibility Testing

Susceptibility testing of bacterial isolates was performed using the disc diffusion method as described by the Clinical and Laboratory Standards Institute.[8] Antimicrobial disc used was imipenem (10 μg), cefpodoxime (10 μg), cefotaxime (30 μg), cefepime (30 μg), cefixime (5 μg), cefoperazone (75 μg), cefoperazone/sulbactam (75/10 μg), ticarcillin (75 μg), piperacillin (100 μg), piperacillin/tazobactam (100/10 μg), ceftazidime (30 μg), ceftazidime/clavulanic acid (30/10 μg), cefotaxime/clavulanic acid (30/10 μg), ceftriaxone (30 μg), amikacin (30 μg), gentamicin (10 μg), tobramycin (10 μg), ofloxacin (5 μg), levofloxacin (5 μg), polymyxin B (300 units) and colistin (10 μg). All discs were obtained from HiMedia Laboratories, Mumbai, India.

Phenotypic methods for extended-spectrum beta-lactamases detection

Enterobacteriaceae isolates were first screened for the production of ESBL by the disc diffusion method (screening test) using cefotaxime, ceftriaxone, cefepime and ceftazidime [8] and later on confirmed by the cephalosporin/clavulanate combination disc (disc potentiation test) method using ceftazidime, ceftazidime + clavulanic acid, cefotaxime, cefotaxime + clavulanic acid, cefoperazone, cefoperazone + sulbactam and piperacillin, piperacillin + tazobactam. A difference of 5 mm between the zone diameters of either of the cephalosporin discs and their respective cephalosporin/clavulanate disc is taken to be phenotypic confirmation of ESBL production.[9]

Double-disc synergy test [10] was also used for phenotypic confirmation of ESBL producers, in which the test strains were adjusted to the 0.5 McFarland standard and swabbed on a Mueller-Hinton agar plate. A susceptibility disc containing piperacillin-tazobactam was placed in the centre of the plate. Discs containing one of the oxyimino-beta-lactam antibiotics (cefotaxime, ceftazidime and ceftriaxone) were placed 20 mm (centre to centre) from piperacillin-tazobactam disc. A clear extension of the edge of the oxyimino-β-lactam inhibition zone towards the disc containing clavulanate was interpreted as synergy indicating a positive result. E. coli ATCC 25922 (non-ESBL producer) was used as a control strain.

Phenotypic methods for AmpC detection

Cefoxitin discs were used to screen AmpC producers by disc diffusion method.[11] Those isolates which were resistant to cefoxitin were considered as potential AmpC producers.

Phenotypic methods for MBL detection

The isolates were tested for sensitivity to imipenem (10 μg) using Kirby–Bauer method as recommended by CLSI.[7] All the isolates with zone of inhibition ≤16 or which demonstrated heaping, or if the zone was >16 but ≤20, were tested for metallo-beta-lactamase (MBL) production.

Modified Hodge test

The indicator organism, E. coli ATCC 25922, at a turbidity of 0.5 McFarland standard, was used to swab inoculate the surface of a Mueller-Hinton agar plate (Becton Dickinson, Cockeysville, MD, USA), and the test strain was heavily streaked from the centre to the plate periphery. After the plate was allowed to stand for 15 min at room temperature, a 10 μg IPM disc was placed at the centre, and the plate was incubated overnight. The presence of distorted inhibition zone (cloverleaf) was interpreted as a positive result for carbapenem hydrolysis.[12]

The imipenem-ethylenediaminetetraacetic acid double-disc synergy test

The test strains were adjusted to the McFarland 0.5 standard and used to inoculate Mueller-Hinton agar plates. Disc containing imipenem 10 μg was placed on the plate and a blank filter paper soaked with 10 μl of 0.5 M ethylenediaminetetraacetic acid solution was placed at a distance of 10 mm (edge to edge). After overnight incubation, the presence of synergistic inhibition zone was interpreted as positive for MBL production.[12]

Genotypic Methods for the detection of extended-spectrum beta-lactamase and AmpC production

Preparation of DNA template

Template DNA was prepared from freshly cultured bacterial isolates by suspending bacterial colonies in 50 μL of molecular-grade water and then heating at 95°C for 5 min and immediately chilling at 4°C.

Detection of bla genes by polymerase chain reaction

Molecular detection of blaCTX-M, blaTEM, blaSHV and blaAmpC was performed using polymerase chain reaction (PCR) according to methods described.[13],[14],[15] The quality control strain K. pneumoniae ATCC 700603 (ESBL producer) was used. PCR amplification was carried out using the 2x PCR Master Mix (Fermentas, Thermo scientific, USA) on Gradient Thermo Cycler, Le cycler, LABNIC, USA, with primers targeting bla CTX-M, bla SHV, bla TEM and bla Amp-C genes as given below.


  Results Top


Amongst two hundred isolates, commonly isolated Enterobacteriaceae members were E. coli (52 [26%]), K. pneumoniae (37 [18.5%]), Klebsiella oxytoca (12 [6%]), followed by Citrobacter species (33 [16.5%]), Citrobacter freundii (20 [10%]) and Serratia species (15 [8%]).

The risk factors associated with ICU patients were observed as elderly age (>60 years); 143 (56.2%), endotracheal tube/tracheostomy (126 [60%]), parenteral nutrition (210 [100%]), central venous line (84 [40%]), urinary catheter (210 [100%]), nasogastric tube (151 [72%]), mechanical ventilation (126 [60%]), comatosed (Glasgow Coma Scale [GCS[ <8) patients (110 [50%]) and prior antibiotic treatment taken (192 [91%]). Demographic profile of the ICU patients is depicted in [Table 1]. E. coli (29 [55.7%]) was most commonly associated with urinary tract infection; however, the frequency of K. pneumoniae (18 [48.6%]), K. oxytoca (5 [41.6%]) and Citrobacter koseri (20 [33%]) was higher in lower respiratory tract, bloodstream and surgical site infections, respectively. Distribution of Enterobacteriaceae members isolated from different samples in ICU patients is shown in [Table 2].
Table 1: Demographic profile of the Intensive Care Unit patients

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Table 2: Distribution of Enterobacteriaceae members isolated from different samples in Intensive Care Unit patients

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All the isolates of Enterobacteriaceae were multidrug resistance (MDR). Antimicrobial resistance pattern of Enterobacteriaceae members isolated from ICU patients is depicted in [Figure 1].
Figure 1: Antimicrobial resistance pattern of members of Enterobacteriaceae isolated from Intensive Care Unit Patients

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Phenotypically, prevalence of ESBL, AmpC and MBL amongst Enterobacteriaceae members was 50.2%, 36.6% and 12.3%, respectively. Only those isolates which were confirmed phenotypically as ESBL and AmpC producers were subjected to bla (CTX-M, TEM and SHV) ESBLs and blaAmpC gene study, respectively. BlaCTX-M and blaAmpC gene was detected in 48% and 32.4% of Enterobacteriaceae members, respectively. BlaTEM and blaSHV were not detected in any of the isolates [Figure 2].
Figure 2: Distribution of blaCTX-Mand blaAmpCGene amongst Gram-negative bacterial isolates

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  Discussion Top


In the current era of increasing use of broad-spectrum antimicrobial agents, the incidence of ESBL- and AmpC-producing Enterobacteriaceae has increased worldwide at an alarming rate.[16] At present, the major challenge to infection control teams is the prevention of the emergence and spread of ESBL- and AmpC-producing Enterobacteriaceae.

Besides being associated with high morbidity and mortality, therapeutic options for the treatment of infections involving ESBLs and AmpCs have also become increasingly limited. The efficacy of extended-spectrum cephalosporins is compromised while co-resistance to co-trimoxazole, aminoglycosides and fluoroquinolones has been reported.[17]

Epidemiologic and descriptive data on ESBL- and AmpC-producing isolates in hospitals from India, especially from ICU patients, are limited. The current study is one of the few reports focussing on both the distribution of ESBL- and AmpC-producing Enterobacteriaceae and detection of ESBL and AmpC genes in the North Indian ICU.

Patients in ICU are at a higher risk of acquiring nosocomial infections compared with patients in general wards due to the severity of the underlying illnesses and iatrogenic factors related to the high frequency of invasive procedures needed for the monitoring and treatment which include insertion of intravascular catheters, endotracheal intubation and positive pressure ventilation, urinary catheterisation and surgical operations. Studies from different parts of the world on nosocomial infections have shown that patient and treatment factors are risk factors for the development of nosocomial infections. These risk factors were also seen in our cases as elderly age (>60 years); 143 (56.2%), endotracheal tube/tracheostomy (126 [60%]), parenteral nutrition (210 [100%]), central venous line (4 [40%]), urinary catheter (210 [100%]), nasogastric tube (151 [72%]), mechanical ventilation (126 [60%]), comatosed (GCS <8) patients (110 [50%]) and prior antibiotic treatment taken (192 [91%]). Similar correlations were also observed in other studies as well.[18] Antibiotic therapy is one of the most important risk factors contributed to nosocomial fungal colonisations/infections by suppressing endogenous bacterial flora and decrease of non-bacterial emerging flora, mainly in colorectal tract and in areas close to the ureteral meatus.[19] In the current study, antibiotic therapy was the principal (94.3%) predisposing factor, and a total of 198 patients (out of 210) used broad-spectrum antibiotics. In support of our study, other researchers from different countries concluded that broad-spectrum antibiotic therapy can be one of the most important predisposing factors for the development of nosocomial infections in patients admitted to ICUs.[20],[21] Nonetheless, some other studies found a greater prevalence of this factor and showed that all critically ill patients with nosocomial infections had received antibiotics.[22] According to Sydnor and Perl, admission to ICU itself was a significant risk factor; roughly 25% of nosocomial infections occur in ICUs, which have been estimated to increase ICU length of stay by 4.3–15.6 days.[23] Length of stay in the ICU is also associated with increased risk for fungal infections, which rises rapidly after 7–10 days.[24] In our study also, a maximum number of patients had a hospital stay of 7–15 days.

In this study, amongst 200 isolates, commonly isolated Enterobacteriaceae members were E. coli (52 [26%]), K. pneumoniae (37 [18.5%]), K. oxytoca (12 [6%]), followed by Citrobacter species (33 [16.5%]), C. freundii (20 [10%]) and Serratia species (15 [8%]).

E. coli 29 (55.7%) was most commonly associated with urinary tract infection; however, the frequency of K. pneumoniae (18 [48.6%]), K. oxytoca (5 [41.6%]) and C. koseri (20 [33%]) was higher in lower respiratory tract, bloodstream and surgical site infections, respectively. Isolation patterns of infecting Enterobacteriaceae members were similar to those in previous studies.[25],[26],[27],[28],[29]

In the current study, all the isolates of Enterobacteriaceae were MDR. Eighty-eight percent of E. coli isolates were resistant to cefoperazone and piperacillin, followed by cefixime (80%), ofloxacin (80%), levofloxacin (72%), cefotaxime (60%), cefepime (60%), amikacin (48%) and imipenem (8.0%). In that study by Kiffer et al., 2005, E. coli species were fully susceptible to imipenem and amikacin, while our results show a relatively significant resistance to amikacin (48%) and imipenem (8%).[30] In the present study, phenotypically, E. coli (47.3%) and Klebsiella spp.(43.6%) were the predominant ESBL producers amongst Enterobacteriaceae members. Baby Padmini et al., 2008,[30] showed that isolates 41% of E. coli and a higher percentage of 40% of K. pneumoniae isolates were ESBL producers in their study cohort.

In the present study, the prevalence of ESBL producers was found to be amongst E. coli (47.3%) and K. pneumoniae (43.6%) isolates. The alarming rate of resistance noted amongst these isolates in the present study is of concern. Resistance of ESBL-producing isolates to third-generation cephalosporins (3GCs) was found to coexist with resistance to two or more antibiotics such as piperacillin (88%), levofloxacin (72%) and gentamicin (60%). This coexistence of MDR has been reported earlier.[31],[32] Mechanisms of co-resistance are not clear, but one possible mechanism is the co-transmission of beta-lactamases and resistance to other antimicrobials within the same conjugative plasmids. The same has been demonstrated in a study by Mishra et al. which showed plasmid-mediated resistance in Enterobacteriaceae isolates to multiple antibiotics including cephalosporins and aminoglycosides.[33]

Our study is in accordance with Lewis et al., 2007, in which the most common ESBL-producing gene was CTX-M in the critically ill ICU patients.[34] In the present study, TEM and SHV genes were not detected in any of the Gram-negative isolates. However, Sharma et al., 2010,[35] demonstrated the presence of TEM gene (50% E coli and 60% K. pneumoniae) and SHV gene (72% K. pneumoniae and 48% E. coli). A study by Baby Padmini et al., 2008, also shows that CTX-M group is the predominant form in Enterobacteriaceae.

Organisms overexpressing AmpC beta-lactamases are of major clinical concern because these are usually resistant to all beta-lactam antimicrobials, except for cefepime, cefpirome and carbapenems. There is a paucity of data from Indian laboratories on the coexistence of multiple beta-lactamases in individual isolates. Studies from various parts of India have reported the prevalence of AmpC in clinical isolates of Enterobacteriaceae as varying from 2.2% to 20.7%.[36],[37] In the present study, phenotypically AmpC production was observed in 43 (35.5%) of the total isolates. Amongst these isolates, PCR detected blaAmpC gene in 35 (81.4%) isolates using the PCR. Maximum AmpC production was observed amongst K. pneumoniae ( 31.6%), K. oxytoca (20.9%), E. coli (24.1%), followed by Citrobacter species (23.3%) and Serratia marcescens (11.8%). Similarly, in a study conducted by Khanal et al., 2013,[26] AmpC β-lactamase was confirmed in 51 (37.2%) of the screen-positive isolates and E. coli constituted 22 (43.1%) followed by K. pneumoniae with 13 (25.5%). Phenotypic method detected beta-lactamases in larger number of isolates as compared to molecular detection of blaAmpC genes. This variation could be due to the presence of some other enzymes/proteins, which functions as AmpC beta-lactamases.


  Conclusion Top


This study demonstrates the prevalence of ESBL, AmpC and MBL-mediated drug resistance to 3GC by Gram-negative bacilli belonging to the Enterobacteriaceae family in the critically ill patients admitted in ICU of a tertiary hospital. ESBL and AmpC detection is not routinely carried out in many microbiology units of service laboratories. This could be attributed to lack of awareness or lack of resources and facilities to conduct ESBL, AmpC and MBL identification.

Notwithstanding the fact that genotypic tests are gold standards for detecting ESBLs, AmpC and MBLs, it is also true that in resource-constrained settings, molecular tests are not available, are expensive, technologically demanding and labour intensive. Results of our study suggest that phenotypic screening tests should be utilised routinely as they are easy to perform, discriminatory, reproducible and cost-effective tool for the detection of antimicrobial resistance.

Acknowledgement

I am indebted to Prof. Mohd Shahid and Dr. Anuradha Singh for the help in molecular work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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