Research Article | Open Access
Carbapenem Resistance among Enterobacter Species in a Tertiary Care Hospital in Central India
Objective. To detect genes encoding carbapenem resistance among Enterobacter species in a tertiary care hospital in central India. Methods. Bacterial identification of Enterobacter spp. isolates from various clinical specimens in patients admitted to intensive care units was performed by routine conventional microbial culture and biochemical tests using standard recommended techniques. Antibiotic sensitivity test was performed by standard Kirby Bauer disc diffusion technique. PCR amplification and automated sequencing was carried out. Transfer of resistance genes was determined by conjugation. Results. A total of 70/130 (53.84%) isolates of Enterobacter spp. were found to exhibit reduced susceptibility to imipenem (diameter of zones of inhibition ≤13 mm) by disc diffusion method. Among 70 isolates tested, 48 (68.57%) isolates showed MIC values for imipenem and meropenem ranging from 32 to 64 mg/L as per CLSI breakpoints. All of these 70 isolates were found susceptible to colistin in vitro as per MIC breakpoints (<0.5 mg/L). PCR carried out on these 48 MBL (IP/IPI) -test positive isolates (12 Enterobacter aerogenes, 31 Enterobacter cloacae, and 05 Enterobacter cloacae complex) was validated by sequencing for beta-lactam resistance genes and result was interpreted accordingly. Conclusion. The study showed MBL production as an important mechanism in carbapenem resistance in Enterobacter spp. and interspecies transfer of these genes through plasmids suggesting early detection by molecular methods.
Beta-lactams are one of the most frequently used classes of antimicrobials in hospital settings, crucial for the treatment of infections caused by Gram-negative bacteria. Enterobacter spp. are common pathogens of Enterobacteriaceae family responsible for nosocomial infections, especially blood stream infections in intensive care units. Enterobacter may produce severe diseases including those of abdomen, lower respiratory tract, urinary tract, meningeal, eye, bone, and surgical site infections . As per National Nosocomial Infection Surveillance System, more than one-third of the Enterobacter spp. are resistant to extended-spectrum cephalosporins in intensive care units . However, of late due to the presence of extended-spectrum beta-lactamase (ESBL) and AmpC enzymes in Enterobacter spp., Carbapenems have become the drug of choice to treat such infections . There has been an increase in incidence of multidrug resistance in these organisms due to dissemination of resistance determinant genes mediated by transposons, plasmids, and gene cassettes in integrons. To understand the widespread occurrence of the beta-lactamases in Enterobacter spp., we conducted a study to detect beta-lactam resistance genes along with plasmid replicon typing of carbapenem resistant Enterobacter spp. isolates recovered from clinical specimens in a tertiary care hospital in central India.
2. Materials and Methods
2.1. The Bacterial Isolates
A prospective study was conducted in a 1000 bedded tertiary care centre in Pune, India, from October 2011 to May 2013. A total of 130 Enterobacter spp. isolates (45 Enterobacter aerogenes, 62 Enterobacter cloacae, and 23 Enterobacter cloacae complex) were recovered from clinical specimens from different patients (one isolate per patient) admitted to the medical and surgical intensive care units. Collection of sample was done using strict aseptic precautions and was immediately processed without any delay. The isolates were obtained from various clinical specimens such as cerebrospinal fluid, bone marrow, blood, pus, urine, lower respiratory secretions (endotracheal secretions, bronchoalveolar lavage, and bronchial wash), sputum, tissues, and other sterile body fluids. Bacterial identification was performed by routine conventional microbial culture and biochemical tests using standard recommended techniques . The organisms were identified up to the species level using VITEK-GNI cards (bioMérieux, Marcy l’Etoile, France).
2.2. Antimicrobial Susceptibility Testing
The antimicrobial susceptibility was performed by the Kirby Bauer’s disc diffusion technique on Mueller-Hinton agar, as per Clinical Laboratory Standard Institute (CLSI) guidelines . The antibiotics tested were as follows (potency in μg/disc): Ampicillin (10), Cefuroxime (30), Cefotaxime (30), Piperacillin (100), Ticarcillin (75), Piperacillin-Tazobactam (100/10), Ticarcillin-Clavulanic acid (75/10), Ceftazidime (30), Cefepime (30), Aztreonam (30), Imipenem (10), Meropenem (10), Ertapenem (10), Colistin (10), Gentamicin (10), Tobramycin (10), Amikacin (30), Netilmicin (30), Ciprofloxacin (5), Levofloxacin (5), Lomefloxacin (10), and Ofloxacin (5) (HiMedia Laboratories Pvt. Ltd., Mumbai, India). P. aeruginosa ATCC 27853, E. coli ATCC 25922, E. coli ATCC 35218, and K. pneumoniae ATCC 700603 were used as quality control strains.
2.3. MIC Determination
Minimum inhibitory concentrations (MICs) of antibiotics were determined by VITEK-2 AST-GN25 and AST-GN280 susceptibility cards in accordance with CLSI recommendations and manufacturers’ instructions, except tigecycline and colistin, for which the 2012 European Committee on Antimicrobial Susceptibility Testing break points were used [5, 6]. MICs were further determined by the -test (bioMérieux, Marcy l’Etoile, France). According to Centers for Disease Control and Prevention (CDC), CRE are defined as Enterobacteriaceae that are nonsusceptible to penicillins, third-generation cephalosporins (ceftriaxone, cefotaxime, and ceftazidime), and one of the Carbapenems (doripenem, meropenem, and imipenem).
2.4. Phenotypic Screening for the Carbapenemase Production
Isolates with reduced susceptibility to meropenem and imipenem (diameter of zones of inhibition ≤13 mm) by disc diffusion method were screened for the production of carbapenemase. The phenotypic detection of the carbapenemase production was performed by the modified Hodge test by using ertapenem and meropenem discs (10 μg) as per CLSI guidelines . For MHT K. pneumoniae ATCC BAA-1705 and BAA-1706 were used as positive and negative controls, respectively. Metallo-beta-lactamase production detected by double-disc synergy tests (DDST) with both imipenem and meropenem discs (10 ug) plus disc containing (750 ug) of EDTA as described earlier by Lee et al.  and combined-disc synergy test (CDST) as described previously by Franklin et al.  by using imipenem/meropenem (10 μg) discs and one disc with 292 μg EDTA. K. pneumoniae ATCC BAA-2146 and P. aeruginosa ATCC 27853 were used as positive and negative controls, respectively. Ratio of MICs of imipenem (IP) to IP plus EDTA (IPI) was carried out using MBL (IP/IPI) -test method as per manufacturer’s instructions.
2.5. DNA Extraction and Molecular Detection
DNA was extracted from the bacterial isolates using the spin column method (QIAGEN; GmbH, Hilden, Germany) as per manufacturer’s instructions. PCR-based detection of ESBL genes (, , , and ), Ambler class B MBLs (, , , , , and ), Ambler class D (, , and ), and serine class A carbapenemases (, , and ) were carried out on the isolates by using Gene Amp 9700 PCR System (Applied Biosystems, Singapore) [9–12]. PCR products were run on 1.5% agarose gel, stained with ethidium bromide visualized under UV light and photographed. The amplicons were purified using QIAquick PCR purification kit (QIAGEN; GmbH, Hilden, Germany).
2.6. DNA Sequencing and Sequence Analysis
Automated sequencing was performed on an ABI 3730XL DNA analyzer using the Big Dye system (Applied Biosystems Foster City, CA, USA). Sequences were compared with known sequences using the BLAST facility (http://blast.ncbi.nlm.nih.gov/).
2.7. Conjugation Experiments
Transfer of resistance genes by conjugation was assayed by mating experiments in Luria-Bertani broth using Enterobacter isolates (Parental strains) as donors and an azide-resistant E. coli J53 as the recipient strain using 1 : 10 ratio. The transconjugants were selected on Luria-Bertani agar with selection based on growth on agar in the presence of ceftazidime (30 μg/mL) and sodium azide (100 μg/mL). Plasmids were separated and compared by coelectrophoresis with plasmid of known sizes from E. coli (V517 and 39R861) on a horizontal 0.5% agarose gel at 50 volts for 3 hrs. Bands were visualized with UV transilluminator after staining with 0.05% ethidium bromide.
2.8. Strain Molecular Typing
Repetitive element based PCR (REP-PCR), Enterobacterial Repetitive Intergenic Consensus (ERIC-PCR), and Randomly Amplified Polymorphic DNA (RAPD) assays were performed to characterize Enterobacter strains recovered from patients [13, 14].
2.9. Plasmid Analysis
Plasmid from the parental strains and their transconjugants was extracted by using Qiagen plasmid mini kit (GmbH, Hilden, Germany) as per manufacturer’s instructions. Extracted plasmid DNA was subjected to plasmid based replicon incompatibility (Inc) typing by using eighteen pairs of primers to perform five multiplex and three single PCRs which recognized F, FIA, FIB, FIC, B/O, X, Y, N, P, W, T, A/C, HI1, HI2, I1-Ic, L/M, K, and FII replicons as described previously . Plasmid replicons were determined for the ESBL as well as carbapenemase producing clinical isolates.
3. Result and Discussion
A total of 70/130 (53.84%) isolates of Enterobacter spp. were found to exhibit reduced susceptibility to imipenem (diameter of zones of inhibition ≤13 mm) by disc diffusion method. Among 70 isolates tested, 48 (68.57%) isolates showed MIC values for imipenem and meropenem ranging from 32 to 64 mg/L as per CLSI breakpoints. Twenty-two, out of 70 isolates tested, showed MIC values below 8 mg/L. All of these 70 isolates were found susceptible to Colistin in vitro as per MIC breakpoints (<0.5 mg/L). Phenotypic characterization of Enterobacter spp. isolates () from clinical samples is shown in Table 1. PCR carried out on these 48 MBL (IP/IPI) -test positive isolates (12 Enterobacter aerogenes, 31 Enterobacter cloacae, and 05 Enterobacter cloacae complex) was validated by sequencing for beta-lactam resistant genes and results were interpreted accordingly. Distribution of carbapenem resistant genes among Enterobacter spp. depicted in [Table 2]. VIM-2, VIM-6, and NDM-1 genes were found in carbapenem resistance isolates. Among ESBLs , , , , , , , and were detected. These 48 isolates were further studied for conjugation assays and plasmid typing. Bacterial identification of the transconjugants from Luria-Bertani agar was performed by using VITEK-GNI cards and MICs of antibiotics were determined by VITEK-2 AST susceptibility cards. MICs values of ceftazidime (CAZ), ceftriaxone (CRO), cefepime (FEP), Piperacillin-Tazobactam (PIT), Piperacillin (PIP), Ticarcillin (TIC), Cefotaxime (CTX), Cefoxitin (FOX), imipenem (IMP), meropenem (MEM), and ertapenem (ETP) were high among transconjugants, whereas MICs of amikacin (AMK), gentamicin (GEN), tobramycin (TOB), ciprofloxacin (CIP), moxifloxacin (MXF), levofloxacin (LVX), tigecycline (TGC), and colistin (CST) fall within susceptible range. MICs of 42 clinical Enterobacter isolates recovered from Pus, Blood and urine along with their corresponding transconjugants were presented in the supplementary data file available online at http://dx.doi.org/10.1155/2014/972646. Conjugation experiments revealed that was transferable via a plasmid along with other beta-lactamase genes carried on other plasmids. Plasmid profiling of the isolates showed that was carried on plasmids ranging in sizes from 35 to 170 kb and was carried on 70 to 200 kb size plasmids.
3.1. Strain Molecular Typing
REP-PCR, ERIC-PCR, and RAPD assays as per banding pattern confirmed presence of eight, four, and three clones among E. cloacae(A–H), Enterobacter aerogenes(A–D), and Enterobacter cloacae complex(A–C), respectively. E. cloacae strain typing showed 8 clones, among them three in blood, two in urine, and three in pus, respectively, while in case of Enterobacter aerogenes, two clones were detected in medical and two were in surgical wards. Enterobacter cloacae complex showed two different clonality in Medical ICU where as in surgical ICU isolates were from single clone.
3.2. Plasmid Replicon Typing
Plasmids purified from the clinical isolates were typed by PCR based replicon typing. IncFIA, IncFIB, IncFIC replicons were associated with . Majority of showed association with multiple replicons (either IncFII, IncFIB or IncFIA, IncFIB); five isolates showed single replicon association (IncFIC). The gene in Enterobacter spp. was located on IncA/C, IncFII, and IncN plasmid. The was carried on plasmids belonging to IncP, IncW, IncFII, and IncFIB replicons. was associated with multiple replicons of plasmid (IncFIA, IncFIB). The identified on plasmids was associated with IncP, IncHI2, IncFIC, and IncW replicons. Enterobacter infections can be acquired from exogenous as well as endogenous sources being ubiquitous in nature as a saprophyte in soil and sewage and as a commensal in human gastrointestinal tract. It is present in the feces of humans, animal excreta, dairy products, plants, plant materials, insects, and water [16–18]. Outbreaks of Enterobacter infection associated with contaminated intravenous solutions, blood products, distilled water, endoscopes, stethoscopes and other health care devices have been reported [19–22], Enterobacter infections in a health care settings, seems to arise endogenously from a previously colonized site in an infective individual, mainly the colonization of the gastrointestinal tract with Enterobacter spp. in the debilitated patients. Sometimes colonization of more than one strain is seen among those patients who already have been hospitalized and were on antibiotic therapy. Colonization leads to infection by this organism. Prolonged hospital stay, debilitating underlying illnesses, immunosurveillance and indewelling devices/implants have been risk factors for Enterobacter spp. infection in hospital settings . E. cloacae and E. aerogenes are the two most common Enterobacter species causing nosocomial infections, most frequently associated with disease. Antimicrobial resistance in Enterobacter strains varies with geographic locations. Whereas resistance to betalactam antibiotics, aminoglycosides, trimethoprim-sulfamethoxazole, and quinolones is more prevalent in southern Europe, Belgium, and Israel, in Greece, resistance to cefotaxime, ceftazidime, ceftriaxone, and aminoglycosides is prevalent in 60 to 70% of strains. 2–10% resistance to fluoroquinolones have been documented in various reports [24–30]. The emergence of AmpC, ESBL, and carbapenemase producers along with multiple resistant isolates poses a serious problem in the hospital settings. In our study, among Enterobacter spp. 25.71% (18/70) metallo-beta-lactamase production seen in blood stream infections, followed by 18.57% (13/70) surgical site infections, 15.71% (11/70) urinary tract infections, 8.57% (6/70) respiratory secretions. In 2010, CDC first reported carriage of NDM-1 in E. cloacae from patients who received medical care in India , following which various reports for the same were published by various authors. Khan and Nordmann reported presence of from cases of diabetic foot ulcer . Lascols et al. and Castanheira et al. also reported carriage of among E. cloacae [33, 34]. Emergence of producing E. cloacae clinical isolates was reported from Singapore , China , Australia , United States , Kuwait , Turkey , and Canada . MBLs other than NDM-1 also have been reported by various authors in E. cloacae: from Turkey , from Taiwan , from Italy [44–46], from Korea , and from Greece . In our study, we detected and among Enterobacter spp. Presence of in E. cloacae have been reported in literature [49, 50]. However, our isolates were negative for OXA-48 like gene. Three studies from abroad by Brink et al. , Dai et al. , and Ageevets et al.  reported presence of in E. cloacae. Our study showed negative result for . Carbapenems are one of the important antibiotics used to treat serious infections caused by Enterobacteriaceae. Multidrug resistance in Enterobacteriaceae is associated with significant morbidity and mortality. Therefore, it is important to check constantly the prevalence of resistance to carbapenem in Gram-negative organisms. Multidrug resistance due to the presence of MBL carrying genes is a point of concern as few drugs can be used for the treatment. The transfer of these genes through plasmids increases the spread of drug resistance from one species to another. Hence, early detection of these drug resistance genes by molecular methods is essential in limiting the spread of infection due to these organisms.
Conflict of Interests
The authors declare that there is no conflict of interests regarding the publication of this paper.
MICs of 42 clinical Enterobacter isolates recovered from Pus, Blood and urine along with their corresponding transconjugants were presented in the supplementary data file.
- W. C. Washington Jr., S. D. Allen, W. M. Janda et al., “The Enterobacteriaceae,” in Color Atlas and Textbook of Diagnostic Microbiology, chapter 6, pp. 211–302, Lippincott Williams & Wilkins, Philadelphia, Pa, USA, 2006.
- M. S. Favero, R. P. Gaynes, T. C. Horan et al., “National nosocomial infections surveillance (NNIS) report, data summary from October 1986-April 1996, issued May 1996,” American Journal of Infection Control, vol. 24, no. 5, pp. 380–388, 1996.
- D. Paterson, W. Ko, A. Von Gottberg et al., “In vitro susceptibility and clinical outcomes of bacteremia due to extended-spectrum β-lactamase (ESBL)-producing Klebsiella pneumoniae,” Clinical Infectious Diseases, vol. 27, article 956, 1998.
- J. G. Collee, R. S. Miles, and B. Wan, “Tests for the identification of bacteria,” in Mackie and McCartney Practical Medical Microbiology, J. G. Collee, A. G. Fraser, B. P. Marmion, and A. Simmons, Eds., pp. 131–150, Churchill Livingstone, Edinburgh, UK, 14th edition, 1996.
- Clinical and Laboratory Standards Institute, Performance Standards for Antimicrobial Susceptibility Testing: Twenty Second Informational Supplement M100-S22, CLSI, Wayne, Pa, USA, 2012.
- European Committee on Antimicrobial Susceptibility Testing, Breakpoint tables for interpretation of MICs and zone diameters (Version 2), 2012, http://www.eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Breakpoint_tables/Breakpoint_table_v_2.0_120221.pdf.
- K. Lee, Y. S. Lim, D. Yong, J. H. Yum, and Y. Chong, “Evaluation of the Hodge test and the imipenem-EDTA double-disk synergy test for differentiating metallo-β-lactamase-producing isolates of Pseudomonas spp. and Acinetobacter spp.,” Journal of Clinical Microbiology, vol. 41, no. 10, pp. 4623–4629, 2003.
- C. Franklin, L. Liolios, and A. Y. Peleg, “Phenotypic detection of carbapenem-susceptible metallo-β-lactamase- producing gram-negative bacilli in the clinical laboratory,” Journal of Clinical Microbiology, vol. 44, no. 9, pp. 3139–3144, 2006.
- A. Oliver, L. M. Weigel, J. Kamile Rasheed, J. E. McGowan Jr., P. Raney, and F. C. Tenover, “Mechanisms of decreased susceptibility to cefpodoxime in Escherichia coli,” Antimicrobial Agents and Chemotherapy, vol. 46, no. 12, pp. 3829–3836, 2002.
- P. Villalón, S. Valdezate, M. J. Medina-Pascual, G. Carrasco, A. Vindel, and J. A. Saez-Nieto, “Epidemiology of the acinetobacter-derived cephalosporinase, carbapenem-hydrolysing oxacillinase and metallo-β-lactamase genes, and of common insertion sequences, in epidemic clones of acinetobacter baumannii from Spain,” Journal of Antimicrobial Chemotherapy, vol. 68, no. 3, pp. 550–553, 2013.
- L. Poirel, A. Potron, and P. Nordmann, “OXA-48-like carbapenemases: the phantom menace,” Journal of Antimicrobial Chemotherapy, vol. 67, pp. 1597–1606, 2012.
- S. S. Hong, K. Kim, J. Y. Huh, B. Jung, M. S. Kang, and S. G. Hong, “Multiplex PCR for rapid detection of genes encoding class A carbapenemases,” Annals of Laboratory Medicine, vol. 32, no. 5, pp. 359–361, 2012.
- J. Versalovic, T. Koeuth, and J. R. Lupski, “Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes,” Nucleic Acids Research, vol. 19, no. 24, pp. 6823–6831, 1991.
- L. Vogel, G. Jones, S. Triep, A. Koek, and L. Dijkshoorn, “RAPD typing of Klebsiella pneumoniae, Klebsiella oxytoca, Serratia marcescens and Pseudomonas aeruginosa isolates using standardized reagents,” Clinical Microbiology and Infection, vol. 5, no. 5, pp. 270–276, 1999.
- A. Carattoli, A. Bertini, L. Villa, V. Falbo, K. L. Hopkins, and E. J. Threlfall, “Identification of plasmids by PCR-based replicon typing,” Journal of Microbiological Methods, vol. 63, no. 3, pp. 219–228, 2005.
- P. M. A. Shanahan, B. A. Wylie, P. V. Adrian, H. J. Koornhof, C. J. Thomson, and S. G. B. Amyes, “The prevalence of antimicrobial resistance in human faecal flora in South Africa,” Epidemiology and Infection, vol. 111, no. 2, pp. 221–228, 1993.
- H. Srámová, M. Daniel, V. Absolonová et al., “Epidemiological role of arthropods detectable in health facilities,” Journal of Hospital Infection, vol. 20, no. 4, pp. 281–292, 1992.
- E. Lindh, P. Kjaeldgaard, W. Frederiksen, and J. Ursing, “Phenotypical properties of Enterobacter agglomerans (Pantoea agglomerans) from human, animal and plant sources,” Acta Pathologica, Microbiologica et Immunologica Scandinavica, vol. 99, no. 4, pp. 347–352, 1991.
- J. W. Chow, V. L. Yu, and D. M. Shlaes, “Epidemiologic perspectives on Enterobacter for the infection control professional,” The American Journal of Infection Control, vol. 22, no. 4, pp. 195–201, 1994, Review.
- N. S. Matsaniotis, V. P. Syriopoulou, M. C. Theodoridou, K. G. Tzanetou, and G. I. Mostrou, “Enterobacter sepsis in infants and children due to contaminated intravenous fluids,” Infection Control, vol. 5, no. 10, pp. 471–477, 1984.
- C. G. Mayhall, V. A. Lamb, W. E. Gayle Jr., and B. W. Haynes Jr., “Enterobacter cloacae septicemia in a burn center: epidemiology and control of an outbreak,” Journal of Infectious Diseases, vol. 139, no. 2, pp. 166–171, 1979.
- J. P. Flaherty, S. Garcia-Houchins, R. Chudy, and P. M. Arnow, “An outbreak of gram-negative bacteremia traced to contaminated O-rings in reprocessed dialyzers,” Annals of Internal Medicine, vol. 119, no. 11, pp. 1072–1078, 1993.
- S. J. McConkey, D. C. Coleman, F. R. Falkiner, S. R. McCann, and P. A. Daly, “Enterobacter cloacae in a haematology/oncology ward—first impressions,” Journal of Hospital Infection, vol. 14, no. 4, pp. 277–284, 1989.
- L. Verbist, “Epidemiology and sensitivity of 8625 ICU and hematology/oncology bacterial isolates in Europe,” Scandinavian Journal of Infectious Diseases, Supplement, no. 91, pp. 14–24, 1993.
- A. Tsakris, A. P. Johnson, R. C. George, S. Mehtar, and A. C. Vatopoulos, “Distribution and transferability of plasmids encoding trimethoprim resistance in urinary pathogens from Greece,” Journal of Medical Microbiology, vol. 34, no. 3, pp. 153–157, 1991.
- E. Tzelepi, L. S. Tzouvelekis, A. C. Vatopoulos, A. F. Mentis, A. Tsakris, and N. J. Legakis, “High prevalence of stably derepressed class-I β-lactamase expression in multiresistant clinical isolates of Enterobacter cloacae from Greek hospitals,” Journal of Medical Microbiology, vol. 37, no. 2, pp. 91–95, 1992.
- P. E. Varaldo, F. Biavasco, S. Mannelli, R. Pompei, and A. Proietti, “Distribution and antibiotic susceptibility of extraintestinal clinical isolates of Klebsiella, Enterobacter and Serratia species,” European Journal of Clinical Microbiology & Infectious Diseases, vol. 7, no. 4, pp. 495–500, 1988.
- F. Vázquez, M. C. Mendoza, M. H. Villar, F. Pérez, and F. J. Méndez, “Survey of bacteraemia in a Spanish hospital over a decade (1981–1990),” Journal of Hospital Infection, vol. 26, pp. 111–121, 1994.
- N. J. Legakis and A. Tsakris, “Antibiotic resistance mechanisms in gram-negative bacteria: the Greek experience,” International Journal of Experimental and Clinical Chemotherapy, vol. 5, no. 2, pp. 83–91, 1992.
- L. Leibovici, A. J. Wysenbeek, H. Konisberger, Z. Samra, S. D. Pitlik, and M. Drucker, “Patterns of multiple resistance to antibiotics in gram-negative bacteria demonstrated by factor analysis,” European Journal of Clinical Microbiology and Infectious Diseases, vol. 11, no. 9, pp. 782–788, 1992.
- Centers for Disease Control and Prevention (CDC), “Detection of Enterobacteriaceae isolates carrying metallo-beta-lactamase—United States, 2010,” Morbidity and Mortality Weekly Report, vol. 59, no. 24, p. 750, 2010.
- A. U. Khan and P. Nordmann, “NDM-1-producing enterobacter cloacae and klebsiella pneumoniae from diabetic foot ulcers in India,” Journal of Medical Microbiology, vol. 61, no. 3, pp. 454–456, 2012.
- C. Lascols, M. Hackel, S. H. Marshall et al., “Increasing prevalence and dissemination of NDM-1 metallo-β-lactamase in India: data from the SMART study (2009),” Journal of Antimicrobial Chemotherapy, vol. 66, no. 9, pp. 1992–1997, 2011.
- M. Castanheira, L. M. Deshpande, D. Mathai, J. M. Bell, R. N. Jones, and R. E. Mendes, “Early dissemination of NDM-1- and OXA-181-producing Enterobacteriaceae in Indian hospitals: report from the SENTRY Antimicrobial Surveillance Program, 2006-2007,” Antimicrobial Agents and Chemotherapy, vol. 55, no. 3, pp. 1274–1278, 2011.
- J. Teo, G. Ngan, M. Balm, R. Jureen, P. Krishnan, and R. Lin, “Molecular characterization of NDM-1 producing Enterobacteriaceae isolates in Singapore hospitals,” Western Pacific Surveillance and Response Journal, vol. 3, no. 1, pp. 19–24, 2012.
- W. Dai, S. Sun, P. Yang, S. Huang, X. Zhang, and L. Zhang, “Characterization of carbapenemases, extended spectrum β-lactamases and molecular epidemiology of carbapenem-non-susceptible Enterobacter cloacae in a Chinese hospital in Chongqing,” Infection, Genetics and Evolution, vol. 14, no. 1, pp. 1–7, 2013.
- B. A. Rogers, H. E. Sidjabat, A. Silvey et al., “Treatment options for New Delhi metallo-beta-lactamase-harboring enterobacteriaceae,” Microbial Drug Resistance, vol. 19, no. 2, pp. 100–103, 2013.
- J. K. Rasheed, B. Kitchel, W. Zhu et al., “New Delhi metallo-β-lactamase-producing enterobacteriaceae, United States,” Emerging Infectious Diseases, vol. 19, no. 6, pp. 870–878, 2013.
- G. Peirano, B. J. Ahmed, J. Fuller, J. E. Rubin, and J. D. Pitout, “Travel-related carbapenemase-producing Gram negatives in Alberta, Canada: the first three years,” Journal of Clinical Microbiology, 2014.
- L. Poirel, M. Yilmaz, A. Istanbullu et al., “Spread of NDM-1-producing Enterobacteriaceae in a neonatal intensive care unit, Istanbul, Turkey,” Antimicrobial Agents and Chemotherapy, 2014.
- W. Jamal, V. O. Rotimi, M. J. Albert, F. Khodakhast, P. Nordmann, and L. Poirel, “High prevalence of VIM-4 and NDM-1 metallo-β-lactamase among carbapenem-resistant enterobacteriaceae,” Journal of Medical Microbiology, vol. 62, no. 8, pp. 1239–1244, 2013.
- L. M. Deshpande, R. N. Jones, T. R. Fritsche, and H. S. Sader, “Occurrence and characterization of carbapenemase-producing enterobacteriaceae: report from the SENTRY Antimicrobial Surveillance Program (2000–2004),” Microbial Drug Resistance, vol. 12, no. 4, pp. 223–230, 2006.
- J. J. Yan, W. C. Ko, C. L. Chuang, and J. J. Wu, “Metallo-β-lactamase-producing Enterobacteriaceae isolates in a university hospital in Taiwan: prevalence of IMP-8 in Enterobacter cloacae and first identification of VIM-2 in Citrobacter freundii,” Journal of Antimicrobial Chemotherapy, vol. 50, no. 4, pp. 503–511, 2002.
- F. Luzzaro, J. D. Docquier, C. Colinon et al., “Emergence in Klebsiella pneumoniae and Enterobacter cloacae clinical isolates of the VIM-4 metallo-beta-lactamase encoded by a conjugative plasmid,” Antimicrobial Agents and Chemotherapy, vol. 48, no. 2, pp. 648–650, 2004.
- M. Perilli, M. L. Mezzatesta, M. Falcone et al., “Class I integron-borne blaVIM-1 carbapenemase in a strain of Enterobacter cloacae responsible for a case of fatal pneumonia,” Microbial Drug Resistance, vol. 14, no. 1, pp. 45–47, 2008.
- M. Falcone, M. L. Mezzatesta, M. Perilli et al., “Infections with VIM-1 metallo-β-lactamase-producing Enterobacter cloacae and their correlation with clinical outcome,” Journal of Clinical Microbiology, vol. 47, no. 11, pp. 3514–3519, 2009.
- M. F. Lee, C. F. Peng, H..J. Hsu, and Y. H. Chen, “Molecular characterisation of the metallo-beta-lactamase genes in imipenem-resistant Gram-negative bacteria from a university hospital in southern Taiwan,” International Journal of Antimicrobial Agents, vol. 32, no. 6, pp. 475–480, 2008.
- M. Panopoulou, E. Alepopoulou, A. Ikonomidis, A. Grapsa, E. Paspalidou, and S. Kartali-Ktenidou, “Emergence of VIM-12 in Enterobacter cloacae,” Journal of Clinical Microbiology, vol. 48, no. 9, pp. 3414–3415, 2010.
- L. Poirel, A. Ros, A. Carrër et al., “Cross-border transmission of OXA-48-producing Enterobacter cloacae from Morocco to France,” Journal of Antimicrobial Chemotherapy, vol. 66, no. 5, pp. 1181–1182, 2011.
- A. Carrër, L. Poirel, M. Yilmaz et al., “Spread of OXA-48-encoding plasmid in Turkey and beyond,” Antimicrobial Agents and Chemotherapy, vol. 54, no. 3, pp. 1369–1373, 2010.
- A. J. Brink, J. Coetzee, C. G. Clay et al., “Emergence of New Delhi metallo-beta-lactamase (NDM-1) and Klebsiella pneumoniae carbapenemase (KPC-2) in South Africa,” Journal of Clinical Microbiology, vol. 50, no. 2, pp. 525–527, 2012.
- V. A. Ageevets, I. V. Partina, E. S. Lisitsina et al., “Susceptibility of gramnegative carbapenemase-producing bacteria to various group antibiotics,” Antibiot Khimioter, vol. 58, pp. 10–13, 2013 (Russian).
Copyright © 2014 Atul Khajuria et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.