Emerging Carbapenem-Resistant Enterobacter cloacae Producing OXA-48-, VIM- and IMP-Type-β-Lactamases in Eastern Cape Hospitals in South Africa

Abstract

Introduction: Enterobacter cloacae strains have been isolated from Eastern Cape hospitalised patients. Methodology: We have molecularly characterised blaOXA-48-, blaIMP- and blaVIM-expressing E. cloacae isolates demonstrating resistance to carbapenems from five hospitals by multilocus sequence typing. Organism identification and antimicrobial susceptibility testing was done using automated systems and the isolates were screened for carbapenemases using either conventional or real-time PCR and then typed using multilocus sequence typing. Further characterisation of IMP-type-producing E. cloacae isolates, an unusual occurrence in South Africa, was performed by pulsed-field gel electrophoresis. Results and Conclusion: Twenty-five E. cloacae isolates from 24 patients were investigated. Eighteen (72%) isolates harboured either one of the following genes: blaIMP, blaVIM or blaOXA-48. Multilocus sequence typing data and pulsed-field gel electrophoresis showed that several strains from the same geographical region and hospitals were genetically related.

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Singh-Moodley, A. , Ekermans, P. and Perovic, O. (2015) Emerging Carbapenem-Resistant Enterobacter cloacae Producing OXA-48-, VIM- and IMP-Type-β-Lactamases in Eastern Cape Hospitals in South Africa. Open Journal of Medical Microbiology, 5, 246-253. doi: 10.4236/ojmm.2015.54030.

Received 18 September 2015; accepted 20 December 2015; published 23 December 2015

1. Introduction

The increase in the rate of antibiotic resistance is a major concern in isolates from the enterobacteriaceae family. Serious infections caused by extended spectrum β-lactamase-producing enterobacteriaceae are treated with carbapenems, the broadest spectrum of β-lactam antimicrobial agents. Resistance to carbapenems including carbapenemase production is emerging, creating difficulties in the management of life-threatening infections [1] . Amongst the enterobacteriaceae family, Enterobacter species cause a number of hospital acquired infections [2] and Enterobacter cloacae is the most resistant pathogen [3] . The dominant genes contributing to drug resistance in E. cloacae are the plasmid-mediated serine extended spectrum β-lactamase (ESBL) blaCTX-M; the serine carbapenemase blaKPC and the metallo-β-lacatamases (MBLs) blaNDM, blaIMP and blaVIM [4] [5] . The acquired MBL, blaIMP-1 emerged and spread in Gram-negative bacilli in Japan in the early 1990s [6] and together with its variants have since been detected in other countries worldwide [7] -[12] . blaVIM-1 was first reported in Italy, also in the 1990s [13] . Like blaIMP, variants of this gene have been detected globally [14] -[18] . These genes are located on plasmids that enhance their ability to spread horizontally making treatment of infections more challenging. The production of IMP and VIM β-lactamases in the hospital environment has become a serious concern [19] [20] . In South Africa, the emergence of blaNDM, blaKPC, blaVIM and blaOXA-48 and its variants produced by enterobacteriaceae have been reported [21] -[23] . However, to our knowledge there are no studies that document blaIMP genes in E. cloacae in South Africa. In this study, we identified E. cloacae isolates harbouring IMP, VIM and OXA-48 enzymes and assessed strain clonality to determine whether they were genetically related.

2. Methodology

Carbapenem non-susceptible enterobacteriaceae isolates were submitted to the Antimicrobial Resistance Laboratory at the National Institute for Communicable Diseases for confirmation of carbapenemase-producing enterobacteriaceae (CPE) genes. Upon receipt, organisms were retested for identification and antimicrobial susceptibility testing using automated systems (VITEK® II (bioMèrieux, France) and/or the Microflex MALDI-ToF (BrukerDaltonik, GmbH) and the MicroScan® Walkaway system (Siemens, USA) respectively). The interpretation of susceptibility was done according to the Clinical and Laboratory Standards Institute (CLSI) guidelines [24] . DNA was extracted using a crude boiling method at 95˚C for 25 minutes. The supernatant was harvested and screened for blaNDM, blaKPC and blaOXA-48 and its variants using real-time polymerase chain reaction (PCR) (LightCycler 480 II, Roche Applied Science, LightCycler 480 Probes Master kit, Roche Diagnostics, IN USA) and the Centre for Disease Control and Prevention (CDC) protocol primers for detection of blaNDM and blaKPC and the following primers and probes for blaOXA-48 which was designed for this study: OXA-48Fvariant5’- gCgTggTTAAggATgAACAC-3’,OXA-48Svariant5’-CATYTCgggCAATgTAgACAg-3’, OXA-48Rvariant 5’- gATgTgggCATATCCATATTCATCgCA-3’ and OXA-48probe 5’-CY5-CATTggCTTCggTCAgCATggCT- BBQ-3’. The screening of blaGES, blaIMP and blaVIM was done using conventional PCR (GStorm Thermal Cycler, Somerton Biotechnology Centre, UK and the Qiagen multiplex PCR kit, Qiagen, Germany) and the primers from previous publications [25] [26] . Multilocus sequencing (MLST) was performed on these isolates using previously published primers and conventional typing methods [27] . Conventional PCR was performed for each of the seven reference/house-keeping genes and the products were purified (Qiagen Purification kit; Qiagen, Germany) and sequenced (Inqaba Biotech, South Africa). Sequences were analysed using the online database (http://pubmlst.org/) and analysis tools (http://pubmlst.org/analysis/). For the 15 IMP-producing E. cloacae isolates identified by conventional PCR, pulsed-field gel electrophoresis (PFGE) using the XbaI restriction enzyme (Thermo Scientific, MA USA) was performed. Electrophoresis was performed on 1% PFGE agarose gel with a CHEF-DR III electrophoresis system (Bio-Rad Laboratories, Richmond, CA, USA). Clustering was done according to Tenover criteria [28] , and a cluster was defined as unique PFGE patterns differing by 3 or less bands.

3. Results and Discussion

From January 2013 to April 2014, eighteen of twenty-five E. cloacae isolates from twenty-four patients at five hospitals in the Eastern Cape harboured either one of the following genes: blaIMP, blaVIM or blaOXA-48 resulting in further investigation. Isolates were obtained from clinical specimens: urine (n = 8), sputum (n = 1), pus swab (n = 7), catheter (n = 1) and other sterile body fluids (n = 8). Demographic analysis showed that 13 (54%) were male and 11 (46%) were female. The mean age was 40.75 years (including one neonate). Based on antimicrobial susceptibility testing, majority of the isolates were non-susceptible to ertapenem (96%) and all were susceptible to imipenem and meropenem. The MIC50 and MIC90 for ertapenem were 2 and ≥4 respectively and ≤1 for imipenem and meropenem for both values. Fifteen isolates harboured blaIMP, two contained blaVIM and one contained blaOXA-48. The remaining seven did not express any of the genes tested. All seven of the PCR-negative isolates were non-susceptible to ertapenem. One isolate positive for blaVIM was fully susceptible to the carbapenem group of antibiotics.

The MLST scheme performed in this study is a new scheme developed and published in 2013 [27] and we were not able to compare the sequence types (ST) obtained in this study to previous sequence types obtained for this organism in South Africa. Five isolates resulted in new sequence types as they have not been located on the database although allelic profiles were generated for all seven reference genes. To our knowledge, this study provides novel information regarding MLST data for E. cloacae in South Africa. MLST results show that horizontal transmission of bacteria in the hospital setting is potentially an important factor to consider. Figure 1 illustrates a timeline describing the isolates from each hospital over a 16 month period and based on the STs observed, horizontal transmission is possible. Phylogenetic analysis revealed that all strains shared a common ancestor and were distantly related as evidenced by the different number of STs detected. Those belonging to the same ST clustered together. Identical STs within the same hospital and among different hospitals could indicate intra- and inter-clonal spread. This information therefore suggests that the isolates are related phylogenetically but does not necessarily infer transmission. The limitation of using a methodology like MLST which investigates a relatively small number of reference genes is that there is substantial genetic diversity within a single species group. Therefore, the use of whole genome sequencing would provide a better understanding of transmission events and genetic relatedness. Another limitation of this study is that we had little epidemiological and clinical data. We were not able to obtain information regarding treatment regimens for the patients and therefore cannot comment on previous exposure to carbapenems.

IMP-type-producing E. cloacae have been documented globally, for example in the United Kingdom, the Far East and Australia [8] [11] [29] . To the best of our knowledge, the occurrence of blaIMP genes in E. cloacae isolates is an unusual finding in South Africa. Majority (n = 15) of the 18 isolates that were PCR-positive for carbapenemase-producing genes, harboured blaIMP. MLST data for three isolates was not obtained due to sequencing technicalities, four belonged to ST124, four belonged to ST90, two belonged to a new sequence type and displayed identical allelic profiles and, the remaining two belonged to ST93 and ST108. Relatively little is known about IMP-type MBL-producing E. cloacae with regards to prevalence, risk factors and clinical effects [8] and although our clinical and epidemiological data is scarce some information can be utilised. The specimen types for these infections were fluid/aspirate (n = 5; 33%), pus swab (n = 5; 33%), urine (n = 3; 20%) and catheter tip and sputum (n = 1; 7% each). Eight (53%) patients had bacteria other than IMP-type-producing E. cloacae isolated from the same culture specimen i.e. polymicrobial isolation. Susceptibility to antimicrobial agents is presented in Table 1.The minimal inhibitory concentration (MIC) breakpoints for imipenem and meropenem were not elevated and was in the susceptible range < 1 for these isolates. Similar findings were observed in the study by Hayakawa et al., in 2014 underscoring the difficulties in identifying metallo-β-carbapenemase-pro- ducing organisms primarily based on MIC results [8] . This could also be used as a possible explanation for the one blaVIM-positive isolate that was fully susceptible to the carbapenem group of antibiotics in our study.

Further investigation of the IMP-type-producing E. cloacae isolates using PFGE for strain comparison was performed using the Bionumerics v 6.5 software (Applied Maths, Belgium). Three major clusters were identified. Clustering was done according to Tenover criteria [28] ), and a cluster was defined as PFGE patterns differing by 3 or less bands. The isolates from Cluster A were indistinguishable (no band differences). These included ML0087, ML0279 and ML0207. Cluster B consisted of three isolates that were indistinguishable (ML0197, ML0198 and ML0203). Cluster C consisted of ML0269, ML0280, ML0299 and ML0330. Slight band differences are seen but the patterns were highly similar (more than 90%) (Figure 2). The remaining five isolates are related but do not fall within the three major clusters (Figure 2).

Moreover, these 15 IMP-producing E. cloacae strains were analysed on the Microflex MALDI-ToF instrument and compared using the Flex analysis software (Bruker Daltonik, GmbH). All 15 isolates identified with the same E. cloacae reference strain with high confidence. The spectra generated showed that all 15 isolates were similar. Mass peaks of the proteins were comparable and there were no mass protein shifts identified between the isolates. Additionally, the dendograms generated showed that all 15 isolates have the same origin. Two major clusters could be seen with further sub-clustering (Figure 1 and Figure 3).

Figure 1.Timeline of Enterobacter cloacae isolates from five hospitals in the Eastern Cape from January 2013 to April 2014.

Table 1.Antimicrobial susceptibility testing for 15 IMP-type-producing Enterobacter cloacae isolates.

Figure 2. PFGE dendogram and electrophoresis gel pattern for IMP-type-producing Enterobacter cloacae isolates.

Figure 3. MALDI-ToF dendogram for 15 IMP-type-producing Enterobacter cloacae isolates.

The majority of the isolates appear clonally related by MLST. The dendogram generated by mass spectrometry of the 15 IMP-producing isolates indicate that the strains are highly related and PFGE of these 15 isolates identified three major clusters indicating that the strains were related. The possibility that mobile genetic elements may have been transferred among strains possessing the same drug-resistant gene does exist but the exact mechanisms of acquisition are not certain. It is also not known if the strains possessed the same subtype i.e. IMP-type genes or variants thereof.

Moreover, it is interesting to note that when the 15 IMP-type-producing E. cloacae isolates were screened for blaIMP using real-time PCR, LightCycler 480 II, Roche Applied Science, LightCycler 480 Probes Master kit and the LightMix Modular IMP (ESBL) kit(Roche Diagnostics, IN, USA), blaIMP was not detected in any of these isolates. This may be because the kit is able to detect at least IMP-9, 16, 18, 22 and 25 only. With the conventional PCR used originally in this study, primers from a previous publication were used and were designed from reference sequences downloaded from Genbank to obtain multiple variants [25] . It appears that the latter method is more sensitive to a wider range of IMP variants. Should this prove true, IMP variants are not being detected due to limitations in the methodology resulting in an under-reporting of IMP-type producing E. cloacae variants in South Africa.

4. Conclusion

From this study, we can conclude that MBL-producing strains are prevalent in the Eastern Cape hospitals. Further investigation of 15 IMP-producing E. cloacae isolates, currently an unusual occurrence in South Africa, revealed that majority of the isolates did appear related within three major clusters.

Acknowledgements

We thank Ms. Marshagne Smith, Ms. Ruth Mohlabeng, Ms. Rubeina Badat, Ms. Gloria Molaba and Ms. Naseema Bulbulia for assistance with the laboratory work and Ms. Penny Crowther for assistance with the database. We also thank the Centre for Enteric Diseases, National Institute for Communicable Diseases for the use of the PFGE equipment.

NOTES

*Corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1] Yong, D., et al. (2009) Characterization of a New Metallo-Beta-Lactamase Gene, bla(NDM-1), and a Novel Erythromycin Esterase Gene Carried on a Unique Genetic Structure in Klebsiella pneumoniae Sequence Type 14 from India. Antimicrobial Agents and Chemotherapy, 53, 5046-5054.
http://dx.doi.org/10.1128/AAC.00774-09
[2] Ren, Y., et al. (2010) Complete Genome Sequence of Enterobacter cloacae Subsp. Cloacae Type Strain ATCC 13047. Journal of Bacteriology, 192, 2463-2464.
http://dx.doi.org/10.1128/JB.00067-10
[3] Mezzatesta, M.L., Gona, F. and Stefani, S. (2012) Enterobacter cloacae Complex: Clinical Impact and Emerging Antibiotic Resistance. Future Microbiology, 7, 887-902.
http://dx.doi.org/10.2217/fmb.12.61
[4] Bush, K. (2010) Alarming Beta-Lactamase-Mediated Resistance in Multidrug-Resistant Enterobacteriaceae. Current Opinion in Microbiology, 13, 558-564.
http://dx.doi.org/10.1016/j.mib.2010.09.006
[5] Heller, I., Grif, K. and Orth, D. (2012) Emergence of VIM-1-Carbapenemase-Producing Enterobacter cloacae in Tyrol, Austria. Journal of Medical Microbiology, 61, 567-571.
http://dx.doi.org/10.1099/jmm.0.038646-0
[6] Osano, E., et al. (1994) Molecular Characterization of an Enterobacterial Metallo Beta-Lactamase Found in a Clinical Isolate of Serratia marcescens That Shows Imipenem Resistance. Antimicrobial Agents and Chemotherapy, 38, 71-78.
http://dx.doi.org/10.1128/AAC.38.1.71
[7] Gibb, A.P., et al. (2002) Nosocomial Outbreak of Carbapenem-Resistant Pseudomonas aeruginosa with a New bla(IMP) Allele, bla(IMP-7). Antimicrobial Agents and Chemotherapy, 46, 255-258.
http://dx.doi.org/10.1128/AAC.46.1.255-258.2002
[8] Hayakawa, K., et al. (2014) Molecular and Epidemiological Characterization of IMP-Type Metallo-Beta-Lactamase-Producing Enterobacter cloacae in a Large Tertiary Care Hospital in Japan. Antimicrobial Agents and Chemotherapy, 58, 3441-3450.
http://dx.doi.org/10.1128/AAC.02652-13
[9] Ho, S.E., et al. (2002) Carbapenem-Resistant Pseudomonas aeruginosa in Malaysia Producing IMP-7 Beta-Lactamase. Antimicrobial Agents and Chemotherapy, 46, 3286-3287.
http://dx.doi.org/10.1128/AAC.46.10.3286-3287.2002
[10] Hrabák, J. and Cervená, D. (2011) Regional Spread of Pseudomonas aeruginosa ST357 Producing IMP-7 Metallo-β-Lactamase in Central Europe. Journal of Clinical Microbiology, 49, 474-475.
http://dx.doi.org/10.1128/JCM.00684-10
[11] Leung, G.H., Gray, T.J., Cheong, E.Y., Haertsch, P. and Gottlieb, T. (2013) Persistence of Related bla-IMP-4 Metallo-Betalactamase Producing Enterobacteriaceae from Clinical and Environmental Specimens within a Burns Unit in Australia—A Six-Year Retrospective Study. Antimicrobial Resistance and Infection Control, 2, 35.
[12] Naas, T., Cuzon, G., Bogaerts, P., Glupczynski, Y. and Nordmann, P. (2011) Evaluation of a DNA Microarray (Check-MDR CT102) for Rapid Detection of TEM, SHV, and CTX-M Extended-Spectrum Beta-Lactamases and of KPC, OXA-48, VIM, IMP, and NDM-1 Carbapenemases. Journal of Clinical Microbiology, 49, 1608-1613.
http://dx.doi.org/10.1128/JCM.02607-10
[13] Lauretti, L., Riccio, M.L., Mazzariol, A., Cornaglia, G., Amicosante, G., Fontana, R. and Rossolini, G.M. (1999) Cloning and Characterization of blaVIM, a New Integron-Borne Metallo-Beta-Lactamase Gene from a Pseudomonas aeruginosa Clinical Isolate. Antimicrobial Agents and Chemotherapy, 43, 1584-1590.
[14] Cardoso, O., Leitão, R., Figueiredo, A., Sousa, J.C., Duarte, A. and Peixe, L.V. (2002) Metallo-Beta-Lactamase VIM-2 in Clinical Isolates of Pseudomonas aeruginosa from Portugal. Microbial Drug Resistance, 8, 93-97.
http://dx.doi.org/10.1089/107662902760190635
[15] Lee, K., Lim, J.B., Yum, J.H., Yong, D., Chong, Y., Kim, J.M. and Livermore, D.M. (2002) blaVIM-2 Cassette-Containing Novel Integrons in Metallo-Beta-Lactamase-Producing Pseudomonas aeruginosa and Pseudomonas putida Isolates Disseminated in a Korean Hospital. Antimicrobial Agents and Chemotherapy, 46, 1053-1058.
http://dx.doi.org/10.1128/AAC.46.4.1053-1058.2002
[16] Pournaras, S., Tsakris, A., Maniati, M., Tzouvelekis, L.S. and Maniatis, A.N. (2002) Novel Variant (blaVIM-4) of the Metallo-Beta-Lactamase Gene blaVIM-1 in a Clinical Strain of Pseudomonas aeruginosa. Antimicrobial Agents and Chemotherapy, 46, 4026-4028.
http://dx.doi.org/10.1128/AAC.46.12.4026-4028.2002
[17] Toleman, M.A., Rolston, K., Jones, R.N. and Walsh, T.R. (2002) Molecular Characterization of VIM-4, a Novel Metallo-Beta-Lactamaseisolated from Texas: Report from the Cancer Surveillance Program (2001). 42nd Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, 27-30 September 2002.
[18] Yan, J.-J., Hsueh, P.-R., Ko, W.-C., Luh, K.-T., Tsai, S.-H., Wu, H.-M. and Wu, J.-J. (2001) Metallo-Beta-Lactamases in Clinical Pseudomonas Isolates in Taiwan and Identification of VIM-3, a Novel Variant of the VIM-2 Enzyme. Antimicrobial Agents and Chemotherapy, 45, 2224-2228.
http://dx.doi.org/10.1128/AAC.45.8.2224-2228.2001
[19] Poirel, L., Naas, T., Nicolas, D., Collet, L., Bellais, S., Cavallo, J.-D. and Nordmann, P. (2000) Characterization of VIM-2, a Carbapenem-Hydrolyzing Metallo-Beta-Lactamase and Its Plasmid- and Integron-Borne Gene from a Pseudomonas aeruginosa Clinical Isolate in France. Antimicrobial Agents and Chemotherapy, 44, 891-897.
http://dx.doi.org/10.1128/AAC.44.4.891-897.2000
[20] Richet, H.M., Mohammed, J., McDonald, L.C. and Jarvis, W.R. (2001) Building Communication Networks: International Network for the Study and Prevention of Emerging Antimicrobial Resistance. Emerging Infectious Diseases, 7, 319-322.
http://dx.doi.org/10.3201/eid0702.010235
[21] Brink, A.J., Coetzee, J., Clay, C.G., Sithole, S., Richards, G.A., Poirel, L. and Nordmann, P. (2011) Emergence of New Delhi Metallo-Beta-Lactamase (NDM-1) and Klebsiella pneumoniae Carbapenemase (KPC-2) in South Africa. Journal of Clinical Microbiology, 50, 525-527.
http://dx.doi.org/10.1128/JCM.05956-11
[22] Brink, A.J., Coetzee, J., Corcoran, C., Clay, C.G., Hari-Makkan, D., Jacobson, R.K., et al. (2013) Emergence of OXA-48 and OXA-181 Carbapenemases among Enterobacteriaceae in South Africa and Evidence of in Vivo Selection of Colistin Resistance as a Consequence of Selective Decontamination of the Gastrointestinal Tract. Journal of Clinical Microbiology, 51, 369-372.
http://dx.doi.org/10.1128/JCM.02234-12
[23] Peirano, G., Moolman, J., Pitondo-Silva, A. and Pitout, J.D.D. (2012) The Characteristics of VIM-1-Producing Klebsiella pneumoniae from South Africa. Scandinavian Journal of Infectious Diseases, 44, 74-78.
http://dx.doi.org/10.3109/00365548.2011.614276
[24] CLSI (2013) Clinical and Laboratory Standards Institute (CLSI) Guidelines—Performance Standards for Antimicrobial Susceptibility Testing. CLSI Document M100.
[25] Mendes, R.E., Kiyota, K.A., Monteiro, J., Castanheira, M., Andrade, S.S., Gales, A.C., Pignatari, A.C.C. and Tufik, S. (2007) Rapid Detection and Identification of Metallo-Beta-Lactamase-Encoding Genes by Multiplex Real-Time PCR Assay and Melt Curve Analysis. Journal of Clinical Microbiology, 45, 544-547.
http://dx.doi.org/10.1128/JCM.01728-06
[26] Queenan, A.M. and Bush, K. (2007) Carbapenemases: The Versatile Beta-Lactamases. Clinical Microbiology Reviews, 20, 440-458.
http://dx.doi.org/10.1128/CMR.00001-07
[27] Miyoshi-Akiyama, T., Hayakawa, K., Ohmagari, N., Shimojima, M. and Kirikae, T. (2013) Multilocus Sequence Typing (MLST) for Characterization of Enterobacter cloacae. PLoS ONE, 8, e66358.
http://dx.doi.org/10.1371/journal.pone.0066358
[28] Tenover, F.C., Arbeit, R.D., Goering, R.V., Mickelsen, P.A., Murray, B.E., Persing, D.H. and Swaminathan, B. (1995) Interpreting Chromosomal DNA Restriction Patterns Produced by Pulsed-Field Gel Electrophoresis: Criteria for Bacterial Strain Typing. Journal of Clinical Microbiology, 33, 2233-2239.
[29] Shet, V., Gouliouris, T., Brown, N.M., Turton, J.F., Zhang, J. and Woodford, N. (2011) IMP Metallo-β-Lactamase-Producing Clinical Isolates of Enterobacter cloacae in the UK. Journal of Antimicrobial Chemotherapy, 66, 1408-1409.
http://dx.doi.org/10.1093/jac/dkr078

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