Diagnostic Microbiology and Infectious Disease xxx (2014) xxx–xxx

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NDM-1–producing Enterobacteriaceae from South Africa: moving towards endemicity? Joseph E. Rubin a, b, d, Gisele Peirano a, b, Abdool K. Peer e, Chetna N. Govind e, Johann D.D. Pitout a, b, c,⁎ a

Division of Microbiology, Calgary Laboratory Services, University of Calgary, Calgary, Alberta, Canada Departments of Pathology & Laboratory Medicine, University of Calgary, Calgary, Alberta, Canada Microbiology, Immunology and Infectious Diseases, University of Calgary, Calgary, Alberta, Canada d Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK, Canada e Department of Clinical Microbiology, Lancet laboratories, Durban, KwaZulu-Natal, South Africa b c

a r t i c l e

i n f o

Article history: Received 1 November 2013 Received in revised form 10 April 2014 Accepted 16 April 2014 Available online xxxx

a b s t r a c t We describe 4 patients infected with NDM-1 Enterobacter cloacae, Citrobacter freundii, and Serratia marcescens from South Africa that co-produced SHV-12, CTX-M-3, and CTX-M-15 and were positive for qnrS, qnrA, aac(6′)-Ib-cr, rmtF, rmtC, and armA. Plasmids belonged to IncN, IncA/C replicon types with ccdAB and vagC/D addiction factors. Local and imported cases of NDM-producing bacteria co-exist within South Africa. © 2014 Elsevier Inc. All rights reserved.

Keywords: Metallo- β-lactamases NDM Molecular epidemiology Enterobacteriaceae

Carbapenemases in Enterobacteriaceae can be broadly divided into the class A β-lactamases (i.e., KPC types), the class B metalloβ-lactamases (MBLs) (i.e., VIMs, IMPs, and NDMs) and the class D oxacilinases (i.e., OXA-48–like enzymes) (Nordmann et al., 2011). Bacteria with NDM-1 have been recognized in over 40 countries on every continent, except Antarctica (Johnson and Woodford, 2013). NDM-1 has most commonly been reported in Escherichia coli and Klebsiella pneumoniae but also been found in a variety of other members of the Enterobacteriaceae, Acinetobacter spp., Pseudomonas spp., and Vibrio cholerae (Poirel et al., 2011; Walsh et al., 2011). During November 2012, a carbapenem-resistant Enterobacter cloacae was isolated at Lancet laboratories from the urine of a patient from Durban, South Africa, who had recently returned from India. In the subsequent weeks, 3 additional carbapenem-resistant Enterobacteriaceae (E. cloacae, Citrobacter freundii, and Serratia marcescens) were isolated at the same laboratory within the Durban metropolitan area. Since carbapenemase-producing Enterobacteriaceae have not previously been detected in the Durban region, a study was designed to characterize these isolates. The isolates were identified using the VITEK 2 instrument (Vitek AMS; bioMerieux Vitek Systems Inc., Hazelwood, MO, USA). MICs were determined using E-tests (bioMerieux Inc.) according to the manufacturer's instructions and interpreted by using CLSI guidelines (CLSI, 2012). Fosfomycin (FOS) susceptibility was determined using the CLSI disk methodology. The European Committee for Antimicrobial Suscep⁎ Corresponding author. Tel.: +1-403-770-3309; fax: +1-403-770-3347. E-mail address: [email protected] (J.D.D. Pitout).

tibility Testing breakpoint was used for colistin (COL), and the Food and Drug Administration breakpoint was used for tigecycline (TGC). The presence of carbapenemases was detected using the Modified Hodge test (MHT) and the MASTDISCS™ ID inhibitor combination disks (Doyle et al., 2012) (Mast Group Ltd., Merseyside, United Kingdom). PCR and sequencing of the blaCTX-M, blaNDM, blaSHV, and blaTEM was done using conditions and primers as previously described (Peirano et al., 2011a, 2011b, 2012). The presence of ISABA125 and bleMB was determined using PCR conditions and primers as previously described (Poirel et al., 2011). Conjugation experiments were performed by mating-out assays with nutrient agar containing meropenem (MEM) 1 μg/mL and using E. coli J53 (azide 100 μg/mL) as recipient. The presence of blaNDM was confirmed in the transconjugants using PCR. Plasmid sizes were determined as previously described (Boyd et al., 2004) and assigned to plasmid incompatibility groups by PCR-based replicon typing (Carattoli et al., 2005; Villa et al., 2010) http://pubmlst.org/plasmid/ primers/incF.shtml. Plasmid addiction systems were determined using PCR as described before (Mnif et al., 2010). The amplification of plasmid quinolone determinants and RNA methylases was undertaken as described before (Doi and Arakawa, 2007; Hidalgo et al., 2013; Peirano et al., 2011a, 2011b). Genetic relatedness of the E. cloacae isolates was examined by pulsed field gel electrophoresis (PFGE) following the extraction of genomic DNA and digestion with XbaI using the standardized E. coli (O157:H7) protocol established by the CDC, Atlanta, GA (Hunter et al., 2005). A female from Durban, South Africa, presented in November 2012 to her family physician for the removal of a urinary catheter (Govind

http://dx.doi.org/10.1016/j.diagmicrobio.2014.04.003 0732-8893/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Rubin JE, et al, NDM-1–producing Enterobacteriaceae from South Africa: moving towards endemicity? Diagn Microbiol Infect Dis (2014), http://dx.doi.org/10.1016/j.diagmicrobio.2014.04.003


J.E. Rubin et al. / Diagnostic Microbiology and Infectious Disease xxx (2014) xxx–xxx

et al., 2013). She had just returned from India after being admitted to a hospital in that country during October 2012. A multi-resistant E. cloacae (EcSA01) at N10 8 CFUs/L was cultured from a midstream urine sent to Lancet laboratories in Durban. During the subsequent 3 weeks, the laboratory also isolated 3 multi-resistant isolates of C. freundii (CfSA01), S. marcescens (SmSA01), and E. cloacae (EcSA02) from respiratory specimens of 3 separate patients admitted to 3 different community hospitals in the Durban metropolitan area. CfSA01 and EcSA02 were isolated from the sputa of patients with hospital-acquired pneumonia in community hospitals A and B while SmSA01 was obtained from a tracheal specimen of a patient with ventilator-associated pneumonia in community hospital C. Active surveillance was performed in the respective units after the isolation of CfSA01, SmSA01, and EcSA02 that included rectal swabs from patients in the same wards and ICU but failed to identify additional cases. We were unable to establish any clinical or epidemiological links among the 4 patients. Patients infected with CfSA01, SmSA01, and EcSA02 had no history of recent travel (i.e. within the last 12 months) outside the Durban metropolitan area. No other carbapenem-resistant Enterobacteriaceae (n = 547) were isolated during that time period. The susceptibilities, characteristics, and plasmids of EcSA01, CfSA01, SmSA01, and EcSA02 are shown in Table 1. PFGE showed that EcSA01 was not related to EcSA02. We describe the characteristics of NDM-1–producing E. cloacae (EcSA01) that was isolated in Durban, South Africa, from an outpatient with recent hospitalization in India. The molecular characteristics of this isolate (i.e., the presence of CTX-M-15, aac (6′)-Ib-cr, ISABA125, bleMBL, IncA/C plasmid with NDM-1, and the recent RNA methylase rmtF) are very similar to the features previously described in other NDM-1–producing Enterobacteriaceae that had

been linked to travel in the Indian Subcontinent (Ahmed-Bentley et al., 2013; Johnson and Woodford, 2013; Peirano et al., 2011b; Poirel et al., 2011). Of interest was that the molecular characteristics of EcSA01 were substantially different to the other NDM-1–producing Enterobacteriaceae that were isolated in the same geographical area during the same time period (Table 1). In South Africa, NDM-1 was first recognized in 2010 in an E. cloacae causing a respiratory tract infection (Lowman et al., 2011). Interestingly, neither this first NDM-1–producing organism nor the subsequent NDM-1–producing K. pneumoniae were isolated from patients with a recent travel history, suggesting local acquisition of these organisms (Brink et al., 2012; Lowman et al., 2011). We report 3 cases of hospital-acquired infections due to NDM-producing Enterobacteriaceae without the history of recent travel outside the Durban metropolitan area. These isolates shared the same plasmid-mediated quinolone resistance (PMQR) determinants and 16S RNA methylases, but the NDM-1 containing plasmids had different characteristics (Table 1). The identification of NDM-1 in our study on at least 3 different plasmid scaffolds (IncN, IncA/C, and non-typable) suggests that the epidemiology of these enzymes in South Africa is more complex than merely imported cases from other countries. It is, therefore, possible that bacteria with NDMs might be endemic in South Africa, and it seems that local and imported cases of NDMproducing bacteria co-exist within this country. The results from our study as well as data from previous studies (Brink et al., 2012; Lowman et al., 2011) that described case reports about NDMs in South Africa indicated that such publically funded surveillance studies are desperately needed in this country to determine the magnitude of imported and local carbapenemase-producing bacteria in the health care system.

Table 1 Characteristics of NDM-1–producing Enterobacteriaceae from South Africa.

Location Travel history Specimen Susceptibilities TZP FOX CRO CAZ FEP ATM MEM ERT AMK GEN TOB CIP SXT TGC COL MHT MASTDISCS™ β-Lactamases PMQR determinants 16S RNA methylases ISAba125 bleMBL Plasmids (sizes [kb]) Transconjugant (plasmid size) Replicon typing Addiction systems

E. cloacae (EcSA01)

C. freundii (CfSA01)

S. marcescens (SmSA01)

E. cloacae (EcSA02)

Outpatient Yes (India) Urine

Hospital A (medical ward) No Sputum

Hospital B (ICU) No Tracheal aspirate

Hospital C (medical ward) No Sputum

N256/4 N256 N256 N256 N256 N256 32 N32 128 N256 N256 N32 N2/38 ≤1 0.25 pos MBL pos NDM-1, CTX-M-15, TEM-1 aac(6)-Ib-cr, qnrA armA rmtF + + 150, 180 EcSA01T (180 kb) IncA/C neg

N256/4 N256 N256 N256 N256 N256 16 N32 64 N256 N256 N32 N2/38 ≤1 0.12 pos MBL pos NDM-1, CTX-M-3, TEM-1 qnrS rmtC + + 60, 80, 130, 220, 250 CfSA01T (80 kb) neg neg

N256/4 N256 N256 N256 N256 N256 32 N32 128 N256 N256 N32 N2/38 ≤1 48 pos MBL pos NDM-1 qnrS rmtC + + 50, 60, 100, 110, 180 SmSA01T (110 kb) IncN ccdAB vagC/D

N256/4 N256 N256 N256 N256 N256 16 N32 128 N256 N256 N32 N2/38 ≤1 48 pos MBL pos NDM-1, SHV-12, TEM-1 qnrS rmtC + + 50, 100, 120, 250 EcSA02T (120 kb) IncN ccdAB

TZP = piperacillin-tazobactam; FOX = cefoxitin; CRO = ceftriaxone; CAZ = ceftazidime; FEP = cefepime; ATM = aztreonam; MEM = meropenem; ERT = ertapenem; AMK = amikacin; GEN = gentamicin; TOB = tobramycin; CIP = ciprofloxacin; SXT = trimethoprim-sulfamethoxazole; vagCD = virulence-associated protein; ccdAB = coupled cell division locus. All the isolates were susceptible to FOS. The following susceptibilities were co-transferred with the blaNDM: FOX, CAZ, CRO, FEP, ERT, GEN, TOB, AMK, and SXT. The transconjugants were positive for NDM and negative for CTX-M, SHV, TEM, PMQR determinants, and 16S RNA methylases.

Please cite this article as: Rubin JE, et al, NDM-1–producing Enterobacteriaceae from South Africa: moving towards endemicity? Diagn Microbiol Infect Dis (2014), http://dx.doi.org/10.1016/j.diagmicrobio.2014.04.003

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Funding This work was supported by a research grant from the Calgary Laboratory Services (#10006465) and was presented at the 53rd ICAAC in Denver, CO, USA (abstract no C2-904). Transparency declaration JDDP had previously received research funds from Merck and Astra Zeneca. All other authors have nothing to declare. Acknowledgements We would like to thank Dr Krishnee Moodley for his contribution to this manuscript. References Ahmed-Bentley J, Chandran AU, Joffe AM, French D, Peirano G, Pitout JD. Gram-negative bacteria that produce carbapenemases causing death attributed to recent foreign hospitalization. Antimicrob Agents Chemother 2013;57:3085–91. Boyd DA, Tyler S, Christianson S, McGeer A, Muller MP, Willey BM, et al. Complete nucleotide sequence of a 92-kilobase plasmid harboring the CTX-M-15 extendedspectrum beta-lactamase involved in an outbreak in long-term-care facilities in Toronto, Canada. Antimicrob Agents Chemother 2004;48:3758–64. Brink A, Coetzee J, Clay C, Corcoran C, van Greune J, Deetlefs JD, et al. The spread of carbapenem-resistant Enterobacteriaceae in South Africa: risk factors for acquisition and prevention. S Afr Med J 2012;102:599–601. Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 2005;63:219–28. Clinical and Laboratory Standards Institute (CLSI). Performance Standards for Antimicrobial Susceptibility Testing: Twenty second Informational Supplements. M100-S22. Wayne: CLSI; 2012. Doi Y, Arakawa Y. 16S ribosomal RNA methylation: emerging resistance mechanism against aminoglycosides. Clin Infect Dis 2007;45:88–94.


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Please cite this article as: Rubin JE, et al, NDM-1–producing Enterobacteriaceae from South Africa: moving towards endemicity? Diagn Microbiol Infect Dis (2014), http://dx.doi.org/10.1016/j.diagmicrobio.2014.04.003

NDM-1-producing Enterobacteriaceae from South Africa: moving towards endemicity?

We describe 4 patients infected with NDM-1 Enterobacter cloacae, Citrobacter freundii, and Serratia marcescens from South Africa that co-produced SHV-...
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