|Year : 2015 | Volume
| Issue : 2 | Page : 221-224
Utility of whole-cell repetitive extragenic palindromic sequence-based PCR (REP-PCR) for the rapid detection of nosocomial outbreaks of multidrug resistant organisms: Experience at a tertiary care center in North India
G Singh1, M Biswal1, V Hallur1, KLN Rao2, P Ray1, V Gautam1, SB Appannanavar1, N Taneja1
1 Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh, India
2 Department of Paediatric Surgery, Postgraduate Institute of Medical Education and Research, Chandigarh, India
|Date of Submission||11-Jan-2014|
|Date of Acceptance||19-Mar-2014|
|Date of Web Publication||10-Apr-2015|
Department of Medical Microbiology, Postgraduate Institute of Medical Education and Research, Chandigarh
Source of Support: This study was funded by the Departments of
Medical Microbiology, Postgraduate Institute of Medical Education and
Research, Chandigarh, Conflict of Interest: None
Background: There is a huge need to develop molecular typing methods which are simple to perform, rapid and cost effective to confirm clonality of nosocomial isolates in outbreak situations. Objectives: The aim of the study was to investigate a hospital outbreak of multi-drug resistant (MDR) Klebsiellapneumoniae septicemia in a paediatric surgery intensive care unit (PSICU) using a repetitive extragenic palindromic polymerase chain reaction (REP-PCR). Materials and Methods: MDR Klebsiella pneumoniae isolates from an outbreak of nosocomial sepsis were typed byREP-PCR using consensus primers. Isolates from different intensive care units (ICUs) but with similar antibiogram were also genotyped for comparison. Results and Conclusion: A cluster of twelve MDR K Pneumoniae septicemia cases was identiﬁed at the PSICU by genotyping using REP-PCR. Surveillance cultures failed to pick up any source of infection. REP-PCR was found to be a rapid and simple tool for investigation outbreaks in hospitals. Due to early detection we could initiate infection control practices with focus on hand washing and prevent the further transmission of the organism.
Keywords: Diagnosis, nosocomial, outbreak, repetitive extragenic palindromic polymerase chain reaction, rapid
|How to cite this article:|
Singh G, Biswal M, Hallur V, Rao K, Ray P, Gautam V, Appannanavar S B, Taneja N. Utility of whole-cell repetitive extragenic palindromic sequence-based PCR (REP-PCR) for the rapid detection of nosocomial outbreaks of multidrug resistant organisms: Experience at a tertiary care center in North India. Indian J Med Microbiol 2015;33:221-4
|How to cite this URL:|
Singh G, Biswal M, Hallur V, Rao K, Ray P, Gautam V, Appannanavar S B, Taneja N. Utility of whole-cell repetitive extragenic palindromic sequence-based PCR (REP-PCR) for the rapid detection of nosocomial outbreaks of multidrug resistant organisms: Experience at a tertiary care center in North India. Indian J Med Microbiol [serial online] 2015 [cited 2020 Jan 19];33:221-4. Available from: http://www.ijmm.org/text.asp?2015/33/2/221/154857
| ~ Introduction|| |
Nosocomial infections due to multidrug resistant members of family Enterobacteriaceae are a major cause of morbidity and mortality in most of the hospitals in the developing countries. ,, Outbreaks due to such strains spread swiftly in any hospital through contaminated hands, fomites, contaminated fluids etc., It is imperative to identify the existence of such strains in the hospital environment and keep a close watch on the patterns of spread. K. pneumonia is an opportunistic pathogenthat are being implicated in sporadic epidemics of hospital infection with increasing frequency. Molecular typing techniques have been used in the past to help identify clusters of infections in hospitals viz. pulsed field gel electrophoresis (PFGE), random amplified polymorphic DNA (RAPD) etc., Eukaryotic and prokaryotic DNA contains so-called repetitive DNA elements distributed more or less randomly over the genome. A PCR-based ﬁngerprinting system that uses consensus primers for the repetitive extragenic palindromic (REP) sequences found in many bacterial chromosomes has been shown to be applicable to a wide range of bacterial species and is known as repetitive extragenic palindromic polymerase chain reaction (REP-PCR). ,,, The PCR-products are separated using agarose gel electrophoresis. These patterns can be analysed manually or by using softwares e.g. BioNumerics. Several publications have shown the application of this technique in rapid typing of isolate from hospital based outbreaks. The aim of the present study was to assessthe utility of REP-PCR to investigate a hospital outbreak of multi-drug resistant (MDR) K. pneumoniae septicemia in a paediatric surgery ICU (PSICU).
| ~ Material and Methods|| |
Laboratory based surveillance performed regularly in our clinical bacteriology laboratory was able to detect clustering of cases of K. pneumoniae septicemia in patients admitted to the PSICU. Retrospective data was collected of all paediatric patients reported to have K. pneumoniae septicemia with similar antibiogram. All isolates were identified using standard identification protocols.  Antimicrobial susceptibility was performed by the Kirby Bauer's disc diffusion method following the Clinical and Laboratory Standards Institute (CLSI) guidelines using the Muller Hinton agar (Difco) and antimicrobial discs (Oxoid and Hi-Media).  Tigecycline disc diffusion criteria are not yet established by CLSI or EUCAST, therefore the FDA criteria were folllowed. , The following antimicrobial agents were used-cefotaxime (30 μg), cefoperazone (75 μg), gentamicin (10 μg), amikacin (30 μg), ciprofloxacin (5 μg), nalidixic acid (30 μg), norfloxacin (10 μg), cotrimoxazole (25 μg), nitrofurantoin (300 μg), imipenem (10 μg), piperacillinandtazobactam (75 μg + 10 μg), cefoperazoneandsulbactam (75 μg + 30 μg), tetracycline (30 μg) and tigecycline (15 μg). In addition, surveillance cultures from the environment including all high touch surfaces (bed rails, door knobs, Mayo tables, medicine trolleys, pediatric incubators, monitors, stethoscopes etc.) as well as swabs from health care workers (nasal, oral, axilla, webs of hands and feet, rectal) working in the PSICU were also taken to identify the source of infection.
Whole-cell repetitive extragenic palindromic sequence-based PCR (REP-PCR)
Five isolates of K. pneumoniae with similar antibiogram which could be retrieved were genotyped by using REP-PCR by the method described earlier with few modifications.  Briefly, bacterial isolates were inoculated on nutrient agar plates and incubated at 37°C. To prepare genomic DNA, bacterial isolates were collected, re-suspended in 600 mL of lysisbuffer [20 mmol Tris-ClpH 7.5, 10 mmolethylenediaminetetraacetic acid (EDTA), 40 mmol NaCl, and 0.2% sodium dodecyl sulfate (SDS)] and boiled for 15 min. The supernatant was used as the DNA template. REP-PCR uses consensus primers for the REP sequences found in many bacterial chromosomes, including those of K. pneumoniae. The paired primers REP 1 (5'- IIIGCGCCGICATCAGGC-3') and REP 2 (5'-ACGTCTTATCAGGCCTAC-3') were used to amplify putative REP-like elements in the bacterial DNA. In addition one isolate from the PSICU environment and one from the Respiratory Intensive Care Unit (RICU) with similar antibiograms were also included. We also included four isolates of K. pneumoniae isolated from the blood of patients admitted to PSICU with different antibiograms. A negative control containing all components except the DNA extract, which was replaced with 5μl of sterile distilled H 2 O, was included in each PCR run, to rule out reagent contamination. The procedures for amplification by PCR were followed as described elsewhere.  Aliquots (12 μl) of each sample were subjected to electrophoresis in 1.5% agarose gels. Ampliﬁed products were detected by being stained with ethidium bromide (EtBr). Strains showing similar banding patterns with not more than three band differences were considered belonging to the same clone.
| ~ Results|| |
A cluster of twelve multi drug resistant K. pneumoniaesepticemia caseswas identiﬁed at the PSICU from March 2012 to April 2012 based on the antibiogram of the isolates. All the isolates were susceptible to only tetracycline and resistant to cefotaxime, cefoperazone, cefoperazoneandsulbactam, gentamicin, amikacin, ciprofloxacin, nalidixic acid, norfloxacin, cotrimoxazole, nitrofurantoin, imipenem, piperacillinandtazobactam and tigecycline.
Majority of affected patients were infants (58.3%). The commonest underlying disease was tracheo-oesophageal fistula. All the patients had undergone some surgical procedure. Details of the patients are mentioned in [Table 1]. Genotyping of five isolates that were available revealed a similar clone as shown in [Figure 1]. Surveillance culture from the bed rails picked up a K. pneumoniae isolate with similar antibiogram, however, on REP-PCR it was found to be different strain. The isolate from a patient in RICU with a similar antibiogram was also found to belong to a different genotype. Other surveillance cultures from either the healthcare workers or the environment failed to pick up any additional source of infection. However, due to immediate action in terms of stringent hand hygiene, surface disinfection with 70% alcohol of all high touch surfaces led to the cessation of the outbreak and no further cases could be picked up in the weeks that followed the spread of this organism.
|Figure 1: Gel electrophoresis results following REP-PCR of different isolates of Klebsiellapneumoniae. Lane 1 and 15: molecular marker; Lanes 2-10 indicate K. pneumoniae isolated from blood of patients admitted to PSICU, Lanes 5,6,8,9 and 10 show similar banding pattern. Lane 11: K. pneumoniae isolated from environment of PSICU, Lane 12: K. pneumoniae isolated from blood of patients admitted to Respiratory ICU; Lanes 13 and 14: Negative Control|
Click here to view
|Table 1: Clinical details of patients with K. pneumoniaesepsis in PSICU |
Click here to view
| ~ Discussion|| |
One of the most essential responsibilities of the hospital infection surveillance laboratory is to investigate hospital outbreaks. The benefits of the early detection of an outbreak are decreased morbidity and mortality, lower hospital costs, less number of hospital days etc., After an outbreak is confirmed, stringent measures can be taken to prevent the spread of the infectious agent, better still once the source has been determined. The easiest method for the detection of an outbreak scenario is the laboratory based surveillance in a microbiology laboratory by observing the type of organism being isolated from a particular sample and its similar antibiogram. This however is a crude method, as in any hospital set up there can be multiple strains of the same species with similar antibiogram. Therefore, it is essential to have a robust, highly discriminatory, reproducible and inexpensive typing method with a short turnaround time.
Although, the PFGE is considered the "gold standard" method for molecular typing but is time-consuming, expensive, requires dedicated machines and is very laborious. REP-PCR, however, is a simple, rapid, and less expensive method that has been used to study nosocomial outbreaks of organisms with acceptable reproducibility and discrimination.  Hahm and colleagues (2003) compared REP-PCR, multiplex-PCR, PFGE, ribotyping and amplified fragment length polymorphism (AFLP) to characterise Escherichia More Details coli strains, and concluded that REP-PCR and PFGE were the most discriminative DNA fingerprinting methods available.  Similarly, Foley and colleagues (2006) reported that REP-PCR, PFGE and multilocus sequence typing (MLST) had better discriminant power than did plasmid profiling and antimicrobial susceptibility testing when subtyping Salmonella More Details enterica serovar Typhimurium strains obtained from various animals.  Recently, a semi-automated REP-PCR DNA fingerprinting system has become commercially available. The DiversiLab system (bioMérieux, Marcy e'Etoile, France) uses a microfluidics device to separate REP-PCR amplicon rapidly and reliably, which can be subsequently analysed using web-based DiversiLab software and libraries of fingerprints from several bacteria. ,
In the present study there was a cluster of twelve cases of K. pneumonia septicemia in a PSICU. All the patients had undergone some surgical procedure. All our patients had intravenous lines in place. As twelve cases had occurred in a short span of time, we wanted to investigate this unusual clustering of cases. Only five isolates could be retrieved from our clinical bacteriology laboratory. We extracted the DNA by boiling method, which is rapid, cheap and yields a good quantity of genomic DNA. The supernatant was directly utilised for performing the REP-PCR thereby, saving further time. In the setting of a single day we were able to label the clustering due to a single strain of K. pneumoniae. The other isolates with similar antibiograms but from other units or the environment showed a different pattern by this method.
To conclude, in REP-PCR proved to be a rapid, economical and very useful confirmatory test for strain differentiation with acceptable discrimination and reproducibility. In resource poor settings this molecular method can be put to routine use for the detection and hence, control of nosocomial outbreaks by MDR organisms.
| ~ References|| |
Kumarasamy KK, Toleman MA, Walsh TR, Bagaria J, Butt F, Balakrishnan R, et al
. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: A molecular, biological, and epidemiological study. Lancet Infect Dis 2010;10:597-602.
Sarma JB, Bhattacharya PK, Kalita D, Rajbangshi M. Multidrug-resistant Enterobacteriaceae including metallo-beta-lactamase producers are predominant pathogens of healthcare-associated infections in an Indian teaching hospital. Indian J Med Microbiol 2011;29:22-7.
Thapa B, Adhikari P, Mahat K, Chhetri MR, Joshi LN. Multidrug-resistant nosocomial Citrobacter in a hospital in Kathmandu. Nepal Med Coll J 2009;11:195-9.
Chang HL, Tang CH, Hsu YM, Wan L, Chang YF, Lin CT, et al
. Nosocomial outbreak of infection with multidrug-resistant Acinetobacter baumannii in a medical center in Taiwan. Infect Control Hosp Epidemiol 2009;30:34-8.
Martin-Lozano D, Cisneros JM, Becerril B, Cuberos L, Prados T, Ortiz-Leyba C, et al
. Comparison of a repetitive extragenic palindromic sequence-based PCR method and clinical and microbiological methods for determining strain sources in cases of nosocomial Acinetobacter baumannii bacteremia. J Clin Microbiol 2002;40:4571-5.
Snelling AM, Gerner-Smidt P, Hawkey PM, Heritage J, Parnell P, Porter C, et al
. Validation of use of whole-cell repetitive extragenic palindromic sequence-based PCR (REP-PCR) for typing strains belonging to the Acinetobactercal coaceticus-Acinetobacterbaumannii complex and application of the method to the investigation of a hospital outbreak. J Clin Microbiol 1996;34:1193-202.
Viau RA, Hujer AM, Marshall SH, Perez F, Hujer KM, Briceno DF, et al
. "Silent" dissemination of Klebsiellapneumoniae isolates bearing K. pneumoniaecarbapenemase in a long-term care facility for children and young adults in Northeast Ohio. Clin Infect Dis 2012;54:1314-21.
Koneman E, Allen D, Janda W, Schreckenberger P, Winn W. Color atlas and Textbook of diagnostic microbiology. 5 th
ed. Philadelphia: Lippincott; 1997.
CLSI. Performance Standards for Antimicrobial Susceptibility Testing; 18 th
Informational Supplement, M100-S18, vol. 28, no. 1. Wayne, PA: Clinical and Laboratory Standards Institute; 2008.
Behera B, Das A, Mathur P, Kapil A, Gadepalli R, Dhawan B. Tigecycline susceptibility report from an Indian tertiary care hospital. Indian J Med Res 2009;129:446-50.
Hahm BK, Maldonado Y, Schreiber E, Bhunia AK, Nakatsu CH. Subtyping of foodborne and environmental isolates of Escherichia coli
by multiplex-PCR, rep-PCR, PFGE, ribotyping and AFLP. J Microbiol Methods 2003;53:387-99.
Foley SL, White DG, McDermott PF, Walker RD, Rhodes B, Fedorka-Cray PJ, et al
. Comparison of subtyping methods for differentiating Salmonella entericaserovar Typhimurium isolates obtained from food animal sources. J Clin Microbiol 2006;44:3569-77.
Healy M, Huong J, Bittner T, Lising M, Frye S, Raza S, et al
. Microbial DNA typing by automated repetitive-sequence-based PCR. J Clin Microbiol 2005;43:199-207.
Healy M, Reece K, Walton D, Huong J, Frye S, Raad II, et al
. Use of the Diversi Lab System for species and strain differentiation of Fusarium species isolates. J Clin Microbiol 2005;43:5278-80.