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Characteristics of infections caused by extended-spectrum β-lactamase–producing Escherichia coli from community hospitals in South Africa
Available online at www.sciencedirect.com
Diagnostic Microbiology and Infectious Disease 69 (2011) 449 – 453 www.elsevier.com/locate/diagmicrobio
Characteristics of infections caused by extended-spectrum β-lactamase–producing Escherichia coli from community hospitals in South Africa☆,☆☆ Gisele Peiranoa,c , Cornelius H.J. van Greuneb , Johann D.D. Pitouta,c,d,⁎ a
Division of Microbiology, Calgary Laboratory Services, Calgary, AB T2L 2K8, Canada b Ampath Pathology Laboratories, Cape Town, South Africa c Department of Pathology & Laboratory Medicine, University of Calgary, Calgary, AB, Canada d Microbiology and Infectious Diseases, University of Calgary, Calgary, AB, Canada Received 23 September 2010; accepted 17 November 2010
Abstract A study was designed to characterize 22 nonrepeat extended-spectrum β-lactamase (ESBL)–producing Escherichia coli clinical isolates recovered from specimens originating from doctor's consultation rooms and several private and a state hospital in the Cape Town metropolitan area during 2008–2009. Characterization was done by using isoelectric focusing, PCR, sequencing of blaCTX-M, blaTEM, blaSHV, and blaOXA as well as PCR for plasmid-mediated quinolone resistance determinants, ST131, phylogenetic groups, and plasmid replicon typing. Genetic relatedness was determined with pulsed-field gel electrophoresis using XbaI and multilocus sequencing typing. The majority of patients (17/22 [77%]) presented with urinary tract infections (UTIs) originating from the hospital setting. Thirteen (59%) of the isolates produced CTX-M-15, 7 produced CTX-M-14, and 1 isolate each produced CTX-M-3 and SHV-2, respectively. Sixteen (73%) isolates were nonsusceptible to ciprofloxacin and 8 (36%) were positive for aac(6′)-Ib-cr. Overall, 10/22 (45%) of ESBL producers belonged to clonal complex ST131 that produced CTX-M-15 or CTX-M-14. Molecular characteristics of ST131 showed that this clone belonged to phylogenetic group B2. Our study illustrated that clonal complex ST131 isolates producing CTX-M-15 and CTX-M-14 had emerged as an important cause of UTIs due to ESBL-producing E. coli in the Cape Town area. This is the first report to identify ST131 in ESBL-producing E. coli from Southern Africa. © 2011 Elsevier Inc. All rights reserved. Keywords: CTX-M; Urinary tract infections; ST131; E. coli
1. Introduction CTX-M enzymes are currently the most prevalent extended-spectrum β-lactamase (ESBL) type detected on a worldwide basis and are identified mainly in Escherichia coli. Currently, the most widely distributed CTX-M enzyme is CTX-M-15, and bacteria that produce this enzyme have
☆
This work was supported by research grants from the Calgary Laboratory Services (no. 73-4063) and from Merck (Canada). ☆☆ Transparency declaration: JDDP has previously received research funds from Merck and Astra Zeneca. ⁎ Corresponding author. Tel.: +1-(403)-770-3309; fax: +1-(403)770-3347. E-mail address: [email protected] (J.D.D. Pitout). 0732-8893/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2010.11.011
been isolated from all corners of the world (Pitout & Laupland, 2008). Recent studies using multilocus sequence typing (MLST) identified a single clone of CTX-M-15– producing E. coli, named ST131, in isolates from several countries, including Spain, France, Canada, Portugal, Switzerland, Lebanon, India, Kuwait, and Korea (Coque et al., 2008; Nicolas-Chanoine et al., 2008). This clone is characterized by co-resistance to several classes of antibiotics; it belongs to highly virulent phylogenetic type B2, serotype O25:H4, and harbors the multidrug-resistant narrow host range type IncFII plasmids. Clone ST131 that produces CTX-M-15 often co-produce OXA-1 and TEM-1 β-lactamases as well as aac(6′)-Ib-cr, a variant of an aminoglycoside modifying enzyme aac(6′)-Ib, which is also responsible for reduced susceptibility to certain fluoroquinolones
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such as ciprofloxacin. The molecular epidemiology of ST131 CTX-M-15–producing E. coli has since been described in several countries across different continents (Peirano & Pitout, 2010). In South Africa, the cefotaximases CTX-M-2 and CTXM-3 were first detected in Klebsiella pneumoniae clinical isolates, as part of a worldwide study (Paterson et al., 2003), and CTX-M-15 and CTX-M-37 were identified among Salmonella enterica serotypes (Govinden et al., 2006; Usha et al., 2008). Reports about the prevalence of CTX-M– producing E. coli, especially in the community settings from South Africa, are still rare. This study was undertaken to characterize infections caused by ESBL-producing E. coli, collected in the Cape Town metropolitan area, and to investigate the occurrence of clone ST131 in this collection.
2. Materials and methods 2.1. Patients, bacterial isolates, and antimicrobial susceptibility Twenty-two nonrepeat ESBL-producing E. coli clinical isolates recovered from specimens originating from doctor's consultation rooms and several private and a state hospital in the Cape Town metropolitan area during 2008 and 2009 were included in the study. These hospitals provide emergency and several speciality care services. The majority of isolates (n = 17) were recovered from urines and 5 from pus swab cultures. ESBL production was confirmed phenotypically by using the Clinical and Laboratory Standards Institute (CLSI, 2009) criteria for ESBL screening and disk confirmation tests. Antimicrobial susceptibility was determined with the VITEK 2 instrument (Vitek AMS; bioMerieux Vitek Systems, Hazelwood, MO). The MICs of the following drugs were determined: amoxycillin/clavulanic acid (AMC), piperacillin–tazobactam (TZP), cefoxitin (FOX), ertapenem (ERT), amikacin (AMK), gentamicin (GEN), tobramycin (TOB), ciprofloxacin (CIP), tigecycline (TIG), and trimethoprim–sulfamethoxazole (SXT). Throughout this study, results were interpreted using CLSI (2009) criteria for broth dilution. 2.2. β-Lactamase identification Isoelectric focusing, which included cefotaxime hydrolysis and determination of inhibitor profiles on polyacrylamide gels, was performed on freeze–thaw extracts as previously described (Pitout et al., 2007). PCR amplification and sequencing for blaCTX-Ms, blaOXAs, blaTEMs, and blaSHV was carried out on the isolates with a GeneAmp 9700 ThermoCycler instrument (Applied Biosystems, Norwalk, CT) using PCR conditions and primers as previously described (Pitout et al., 2007).
2.3. Plasmid-mediated quinolone resistance determinants The amplification of the qnrA, qnrS, and qnrB genes was undertaken in all ESBL-positive isolates with multiplex PCR (Robicsek et al., 2006b). aac(6′)-Ib and qepA were amplified in a separate PCR using primers and conditions as previously described (Robicsek et al., 2006a; Yamane et al., 2008). The variant aac(6′)-Ib-cr was further identified by digestion with BstF5I (Park et al., 2006) (New England Biolabs, Ipswich, MA). 2.4. Pulsed-field gel electrophoresis Genetic relatedness of the ESBL-producing 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 Centers for Disease Control and Prevention, Atlanta, GA (Hunter et al., 2005). Cluster designation was based on isolates showing approximately 80% or greater relatedness, which corresponds to the “possibly related (4–6 bands difference)” criteria of Tenover et al. (1995). 2.5. Identification of clone ST131 All the ESBL-producing isolates were screened for ST131 using a PCR for the pabB allele, recently described by Clermont et al. (2009). MLST was performed on those isolates that tested positive for ST131 using 7 conserved housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, recA). A detailed protocol of the MLST procedure, including allelic type and sequence type assignment methods, is available at MLST Databases at the ERI, University College Cork website (http://www.mlst.ucc.ie/mlst/dbs/Ecoli). 2.6. Phylogenetic grouping The ESBL-positive isolates were assigned to 1 of the 4 main E. coli phylogenetic groups (A, B1, B2, and D) by the use of a multiplex PCR-based method (Clermont et al., 2000). 3. Results 3.1. Patients, bacterial isolates, and susceptibilities The majority of patients (17/22 [77%]) presented with urinary tract infections (UTIs), 4 patients presented with intra-abdominal abscesses (including perforated diverticle [n = 1], post intra-abdominal surgery [n = 3]), while 1 patient had an infected burn wound. The majority of patients (12 [55%]) were inpatients. The prevalence of ESBL-producing E. coli isolated in the community hospital included in this study was less than 7%. Of the 22 isolates, 20 (91%) were nonsusceptible (i.e., intermediate or resistant) to SXT, 17 (77%) to AMC, 15 (68%) to TZP, 16 (73%) to CIP, 13 (59%) to TOB, 12 (55%) to GEN, and 9 (41%) to AMK. No resistance to ERT or TIG was detected.
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3.2. β-Lactamase gene identification
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ST131. ST131 status was confirmed by MLST. The PCR for the pabB allele was also performed on the remaining ESBLproducing E. coli, and none tested positive for ST131 (Table 1). Overall, 10 isolates belonged to clone ST131; 7 producing CTX-M-15 and 3 producing CTX-M-14 (Table 1).
Of the 22 E. coli isolates, 21 (95%) were positive for blaCTX-M genes: 13 produced CTX-M-15, 7 produced CTXM-14, and 1 produced CTX-M-3, while the remaining isolate produced SHV-2. Some of the CTX-M–producing isolates also produced TEM-1 (i.e., those with CTX-M-14 and CTX-M-15) and OXA-1 (only those with CTX-M-15) β-lactamases (Table 1).
3.6. Phylogenetic grouping Eleven isolates (including the 10 that tested positive for ST131) belonged to phylogenetic group B2, while the remaining ESBL-producing E. coli isolates belonged to phylogenetic groups D and A (9 and 2 isolates, respectively) (Table 1). We were unable to determine the phylogenetic group in 1 isolate (SA 9).
3.3. Plasmid-mediated quinolone resistance determinants Eight (28%) of the ESBL-producing E. coli isolates (all producing CTX-M-15) were positive for aac(6′)-Ib-cr. None of the other PMQR genes were detected. 3.4. Pulsed-field gel electrophoresis
4. Discussion
PFGE identified a closely related group of E. coli isolates that was designated as clone A (n = 6), as well as 4 isolates (SA1, SA15, SA17, and SA20) that were related to clone A (Fig. 1). The isolates that belonged to clone A had N80% similar PFGE profiles while SA1, SA15, SA17, and SA20 exhibited N60% similarity of profiles to clone A. The remaining ESBL-producing isolates were not clonally related, that is, exhibited b80% similar PFGE profiles and did not show patterns similar to those from clone A.
The SENTRY Antimicrobial Surveillance Program from 1998 to 1999 showed that 36% of K. pneumoniae and 5% of E. coli from South Africa were positive for ESBL production (Bell et al., 2002; Hirakata et al., 2005), and the PEARLS study from 2001 to 2002 showed similar results, that is, 34% of K. pneumoniae and 4.6% of E. coli from South Africa were positive for ESBL production (Bouchillon et al., 2004). According to the National Antibiotic Surveillance Forum, the prevalence of ESBL production in E. coli from private laboratories that serves community hospitals similar to the institutions included in this study was 5%, and the figure did not differ significantly between the cities within South Africa
3.5. Identification of clone ST131 The PCR for the pabB allele of ST131 status identified PFGE clone A and the 4 related isolates as belonging to Table 1 Characteristics of ESBL-producing E. coli isolated from the Cape Town area Isolate no.
Specimen
Antimicrobial nonsusceptibilitya
PMQR
β-Lactamases
Phylogenetic group
ST131 PCR
SA 1 SA 2 SA 3 SA 4 SA 5 SA 6 SA 7 SA 8 SA 9 SA 10 SA 11 SA 12 SA 13 SA 14 SA 15 SA 16 SA 17 SA 18 SA 19 SA 20 SA 21 SA 22
Urine Pus swab Pus swab Urine Urine Urine Urine Urine Pus swab Urine Urine Pus swab Urine Urine Urine Urine Urine Urine Urine Pus swab Urine Urine
AMC, TZP, CIP, SXT FOX, CIP, SXT CIP AMC, TZP, GEN, TOB, SXT AMC, FOX, GEN, TOB, SXT AMC, TZP, AMK, TOB, CIP, SXT AMC, TZP, FOX, AMK, GEN, TOB, CIP, SXT AMC, TZP, GEN, TOB, CIP, SXT AMC, TZP, AMK, GEN, TOB, CIP, SXT SXT AMC, TZP, FOX, CIP, SXT AMC, TZP, AMK, GEN, TOB, CIP, SXT AMC, TZP, AMK, GEN, TOB, CIP, SXT AMC, TZP, AMK, GEN, TOB, CIP, SXT AMC, TZP, CIP SXT AMC, TZP, CIP, SXT SXT AMC, TZP, AMK, GEN, TOB, CIP, SXT AMC, AMK, GEN, TOB, CIP, SXT AMC, TZP, AMK, GEN, TOB, CIP, SXT AMC, TZP, FOX, GEN, TOB, SXT
– – – – – aac(6′)-lb-cr aac(6′)-lb-cr – aac(6′)-lb-cr – – aac(6′)-lb-cr aac(6′)-lb-cr aac(6′)-lb-cr – – – – aac(6′)-lb-cr – aac(6′)-lb-cr –
TEM-1, CTX-M-14 CTX-M-14 CTX-M-15 TEM-1, SHV-2 TEM-1, CTX-M-3 TEM-1, OXA-1, CTX-M-15 TEM-1, OXA-1, CTX-M-15 TEM-1, CTX-M-14 TEM-1, OXA-1, CTX-M-15 CTX-M-14 TEM-1, OXA-1, CTX-M-15 OXA-1, CTX-M-15 OXA-1, CTX-M-15 OXA-1, CTX-M-15 TEM-1, CTX-M-15 CTX-M-14 CTX-M-15 CTX-M-14 TEM-1, OXA-1, CTX-M-15 TEM-1, CTX-M-14 TEM-2, OXA-1, CTX-M-15 TEM-1, CTX-M-15
B2 A D B2 D B2 D B2 NT D D B2 A B2 B2 D B2 D B2 B2 B2 D
+ − − − − + − + − − − + − + + − + − + + + −
AMC = amoxycillin/clavulanic acid; TZP = piperacillin/tazobactam; FOX = cefoxitin; CIP = ciprofloxacin; GEN = gentamicin; TOB = tobramycin; AMK = amikacin; SXT = trimethoprim–sulfamethoxazole; PMQR = plasmid-mediated quinolone resistance determinants. a Antimicrobial nonsusceptibility (i.e., resistant or intermediate).
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Fig. 1. PFGE of XbaI-digested DNA of ESBL-producing E. coli isolates from the Cape Town area.
(i.e., Johannesburg, Pretoria, Durban, Cape Town, and Bloemfontein) (Brink et al., 2007). The CTX-M enzymes, particularly those belonging to CTX-M group 1, were first identified in South Africa during the mid-2009s, in K. pneumoniae isolated from a patient with hospital-acquired pneumonia (Elliott et al., 2006). Enterobacteriaceae that produce CTX-M-15 were recently described from academic hospitals in Durban and Pretoria, respectively (Ehlers et al., 2009; Usha et al., 2008). In our collection of E. coli isolated from several community hospitals in the Cape Town metropolitan area, blaCTX-Ms was present in 95% of isolates, with CTX-M-15 and CTX-M-14 being the most common types. Most of the E. coli that produce CTX-M-15 co-produced TEM-1 and OXA-1 β-lactamases, some were positive for aac(6′)-lb-cr, while MLST identified ST131 among 7/13 (54%) of isolates that also belonged to the phylogenetic group B2 (Table 1) . Of particular interest was that nearly half of the E. coli that produced CTX-M-14 also belonged to clone ST131 (3/7
[43%]). These isolates also belonged to phylogenetic group B2 and co-produced TEM-1. Two previous studies, one from Calgary, Canada, and the other from Chicago, USA, showed that E. coli clone ST131 producing CTX-M-15 and CTX-M-14 had emerged as an important cause of community-onset bacteremia and UTIs (Peirano et al., 2010; Pitout et al., 2009). The present study shows that these isolates are also present in South Africa and caused UTIs among hospitalized patients in community hospitals. Reports of E. coli clone ST131 that produce CTXM-15 from Africa are rare and had been described from Tunisia and Central African Republic (Clermont et al., 2008; Dahmen et al.). To our knowledge, the present study is the first to identify ST131 in ESBL-producing E. coli from Southern Africa and the first to show the presence of aac (6′)-lb-cr among Enterobacteriaceae isolates. The only previous PMQR described from South Africa was qnrB from a single isolate of Salmonella typhimurium (Govender et al., 2009).
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In conclusion, our study illustrated that clonal complex ST131 isolates producing CTX-M-15, CTX-M-14, OXA-1, and TEM-1 β-lactamases are present among inpatients with UTIs due to ESBL-producing E. coli in community hospitals from the Cape Town area. The emergence of E. coli ST131 clonal complex producing CTX-M-14 and CTX-M-15 should raise concern among clinicians, infection control practitioners, and clinical microbiologists. Acknowledgments This work will be presented at the 50th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, September 13, 2010, Boston, NE. Gisele Peirano was supported by a postdoctoral research fellowship from the Foreign Affairs and International Trade Canada (DFAIT). References Bell JM, Turnidge JD, Gales AC, Pfaller MA, Jones RN (2002) Prevalence of extended spectrum beta-lactamase (ESBL)–producing clinical isolates in the Asia-Pacific region and South Africa: regional results from SENTRY Antimicrobial Surveillance Program (1998–99). Diagn Microbiol Infect Dis 42:193–198. Bouchillon SK, Johnson BM, Hoban DJ, Johnson JL, Dowzicky MJ, Wu DH, Visalli MA, Bradford PA (2004) Determining incidence of extended spectrum beta-lactamase producing Enterobacteriaceae, vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus in 38 centres from 17 countries: the PEARLS study 2001–2002. Int J Antimicrob Agents 24:119–124. Brink A, Moolman J, da Silva MC, Botha M (2007) Antimicrobial susceptibility profile of selected bacteraemic pathogens from private institutions in South Africa. S Afr Med J 97:273–279. Clermont O, Bonacorsi S, Bingen E (2000) Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol 66:4555–4558. Clermont O, Lavollay M, Vimont S, Deschamps C, Forestier C, Branger C, Denamur E, Arlet G (2008) The CTX-M-15–producing Escherichia coli diffusing clone belongs to a highly virulent B2 phylogenetic subgroup. J Antimicrob Chemother 61:1024–1028. Clermont O, Dhanji H, Upton M, Gibreel T, Fox A, Boyd D, Mulvey MR, Nordmann P, Ruppé E, Sarthou JL, Frank T, Vimont S, Arlet G, Branger C, Woodford N, Denamur E (2009) Rapid detection of the O25b-ST131 clone of Escherichia coli encompassing the CTX-M-15–producing strains. J Antimicrob Chemother 64:274–277. CLSI (2009) Performance standards for antimicrobial susceptibility testing: nineteenth informational supplement. Wayne: CLSI. Coque TM, Novais A, Carattoli A, Poirel L, Pitout J, Peixe L, Baquero F, Canton R, Nordmann P (2008) Dissemination of clonally related Escherichia coli strains expressing extended-spectrum beta-lactamase CTX-M-15. Emerg Infect Dis 14:195–200. Dahmen S, Bettaieb D, Mansour W, Boujaafar N, Bouallegue O, Arlet G (2010) Characterization and molecular epidemiology of extendedspectrum beta-lactamases in clinical isolates of Enterobacteriaceae in a Tunisian University Hospital. Microb Drug Resist 16:163–170. Ehlers MM, Veldsman C, Makgotlho EP, Dove MG, Hoosen AA, Kock MM (2009) Detection of blaSHV, blaTEM and blaCTX-M antibiotic resistance genes in randomly selected bacterial pathogens from the Steve Biko Academic Hospital. FEMS Immunol Med Microbiol 56:191–196. Elliott E, Brink AJ, van Greune J, Els Z, Woodford N, Turton J, Warner M, Livermore DM (2006) In vivo development of ertapenem resistance in a patient with pneumonia caused by Klebsiella pneumoniae with an extended-spectrum beta-lactamase. Clin Infect Dis 42:e95–e98.
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Govender N, Smith AM, Karstaedt AS, Keddy KH (2009) Plasmidmediated quinolone resistance in Salmonella from South Africa. J Med Microbiol 58:1393–1394. Govinden U, Mocktar C, Moodley P, Sturm AW, Essack SY (2006) CTXM-37 in Salmonella enterica serotype Isangi from Durban, South Africa. Int J Antimicrob Agents 28:288–291. Hirakata Y, Matsuda J, Miyazaki Y, Kamihira S, Kawakami S, Miyazawa Y, Ono Y, Nakazaki N, Hirata Y, Inoue M, Turnidge JD, Bell JM, Jone RN, Kohno S, SENTRY Asia-Pacific Participants (2005) Regional variation in the prevalence of extended-spectrum beta-lactamase–producing clinical isolates in the Asia-Pacific region (SENTRY 1998–2002). Diagn Microbiol Infect Dis 52:323–329. Hunter SB, Vauterin P, Lambert-Fair MA, Van Duyne MS, Kubota K, Graves L, Wrigley D, Barrett T, Ribot E (2005) Establishment of a universal size standard strain for use with the PulseNet standardized pulsed-field gel electrophoresis protocols: converting the national databases to the new size standard. J Clin Microbiol 43:1045–1050. Nicolas-Chanoine MH, Blanco J, Leflon-Guibout V, Demarthy R, Alonso MP, Caniça MM, Park YJ, Lavigne JP, Pitout J, Johnson JR (2008) Intercontinental emergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15. J Antimicrob Chemother 61:273–281. Park CH, Robicsek A, Jacoby GA, Sahm D, Hooper DC (2006) Prevalence in the United States of aac(6′)-Ib-cr encoding a ciprofloxacin-modifying enzyme. Antimicrob Agents Chemother 50:3953–3955. Paterson DL, Hujer KM, Hujer AM, Yeiser B, Bonomo MD, Rice LB, Bonomo RA (2003) Extended-spectrum beta-lactamases in Klebsiella pneumoniae bloodstream isolates from seven countries: dominance and widespread prevalence of SHV- and CTX-M–type beta-lactamases. Antimicrob Agents Chemother 47:3554–3560. Peirano G, Costello M, Pitout JD (2010) Molecular characteristics of extended-spectrum beta-lactamase–producing Escherichia coli from the Chicago area: high prevalence of ST131 producing CTX-M-15 in community hospitals. Int J Antimicrob Agents 36:19–23. Peirano G, Pitout JD (2010) Molecular epidemiology of Escherichia coli producing CTX-M beta-lactamases: the worldwide emergence of clone ST131 O25:H4. Int J Antimicrob Agents 35:316–321. Pitout JD, Church DL, Gregson DB, Chow BL, McCracken M, Mulvey MR, Laupland KB (2007) Molecular epidemiology of CTX-M– producing Escherichia coli in the Calgary Health Region: emergence of CTX-M-15–producing isolates. Antimicrob Agents Chemother 51: 1281–1286. Pitout JD, Laupland KB (2008) Extended-spectrum beta-lactamase– producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis 8:159–166. Pitout JD, Gregson DB, Campbell L, Laupland KB (2009) Molecular characteristics of extended-spectrum-beta-lactamase-producing Escherichia coli isolates causing bacteremia in the Calgary Health Region from 2000 to 2007: emergence of clone ST131 as a cause of communityacquired infections. Antimicrob Agents Chemother 53:2846–2851. Robicsek A, Strahilevitz J, Jacoby GA, Macielag M, Abbanat D, Park CH, Bush K, Hooper DC (2006a) Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med 12:83–88. Robicsek A, Strahilevitz J, Sahm DF, Jacoby GA, Hooper DC (2006b) qnr prevalence in ceftazidime-resistant Enterobacteriaceae isolates from the United States. Antimicrob Agents Chemother 50:2872–2874. Tenover FC, Arbeit RD, Goering RV, Mickelsen PA, Murray BE, Persing DH, Swaminathan B (1995) Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol 33:2233–2239. Usha G, Chunderika M, Prashini M, Willem SA, Yusuf ES (2008) Characterization of extended-spectrum beta-lactamases in Salmonella spp. at a tertiary hospital in Durban, South Africa. Diagn Microbiol Infect Dis 62:86–91. Yamane K, Wachino J, Suzuki S, Arakawa Y (2008) Plasmid-mediated qepA gene among Escherichia coli clinical isolates from Japan. Antimicrob Agents Chemother 52:1564–1566.
Diagnostic Microbiology and Infectious Disease 69 (2011) 449 – 453 www.elsevier.com/locate/diagmicrobio
Characteristics of infections caused by extended-spectrum β-lactamase–producing Escherichia coli from community hospitals in South Africa☆,☆☆ Gisele Peiranoa,c , Cornelius H.J. van Greuneb , Johann D.D. Pitouta,c,d,⁎ a
Division of Microbiology, Calgary Laboratory Services, Calgary, AB T2L 2K8, Canada b Ampath Pathology Laboratories, Cape Town, South Africa c Department of Pathology & Laboratory Medicine, University of Calgary, Calgary, AB, Canada d Microbiology and Infectious Diseases, University of Calgary, Calgary, AB, Canada Received 23 September 2010; accepted 17 November 2010
Abstract A study was designed to characterize 22 nonrepeat extended-spectrum β-lactamase (ESBL)–producing Escherichia coli clinical isolates recovered from specimens originating from doctor's consultation rooms and several private and a state hospital in the Cape Town metropolitan area during 2008–2009. Characterization was done by using isoelectric focusing, PCR, sequencing of blaCTX-M, blaTEM, blaSHV, and blaOXA as well as PCR for plasmid-mediated quinolone resistance determinants, ST131, phylogenetic groups, and plasmid replicon typing. Genetic relatedness was determined with pulsed-field gel electrophoresis using XbaI and multilocus sequencing typing. The majority of patients (17/22 [77%]) presented with urinary tract infections (UTIs) originating from the hospital setting. Thirteen (59%) of the isolates produced CTX-M-15, 7 produced CTX-M-14, and 1 isolate each produced CTX-M-3 and SHV-2, respectively. Sixteen (73%) isolates were nonsusceptible to ciprofloxacin and 8 (36%) were positive for aac(6′)-Ib-cr. Overall, 10/22 (45%) of ESBL producers belonged to clonal complex ST131 that produced CTX-M-15 or CTX-M-14. Molecular characteristics of ST131 showed that this clone belonged to phylogenetic group B2. Our study illustrated that clonal complex ST131 isolates producing CTX-M-15 and CTX-M-14 had emerged as an important cause of UTIs due to ESBL-producing E. coli in the Cape Town area. This is the first report to identify ST131 in ESBL-producing E. coli from Southern Africa. © 2011 Elsevier Inc. All rights reserved. Keywords: CTX-M; Urinary tract infections; ST131; E. coli
1. Introduction CTX-M enzymes are currently the most prevalent extended-spectrum β-lactamase (ESBL) type detected on a worldwide basis and are identified mainly in Escherichia coli. Currently, the most widely distributed CTX-M enzyme is CTX-M-15, and bacteria that produce this enzyme have
☆
This work was supported by research grants from the Calgary Laboratory Services (no. 73-4063) and from Merck (Canada). ☆☆ Transparency declaration: JDDP has previously received research funds from Merck and Astra Zeneca. ⁎ Corresponding author. Tel.: +1-(403)-770-3309; fax: +1-(403)770-3347. E-mail address: [email protected] (J.D.D. Pitout). 0732-8893/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2010.11.011
been isolated from all corners of the world (Pitout & Laupland, 2008). Recent studies using multilocus sequence typing (MLST) identified a single clone of CTX-M-15– producing E. coli, named ST131, in isolates from several countries, including Spain, France, Canada, Portugal, Switzerland, Lebanon, India, Kuwait, and Korea (Coque et al., 2008; Nicolas-Chanoine et al., 2008). This clone is characterized by co-resistance to several classes of antibiotics; it belongs to highly virulent phylogenetic type B2, serotype O25:H4, and harbors the multidrug-resistant narrow host range type IncFII plasmids. Clone ST131 that produces CTX-M-15 often co-produce OXA-1 and TEM-1 β-lactamases as well as aac(6′)-Ib-cr, a variant of an aminoglycoside modifying enzyme aac(6′)-Ib, which is also responsible for reduced susceptibility to certain fluoroquinolones
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such as ciprofloxacin. The molecular epidemiology of ST131 CTX-M-15–producing E. coli has since been described in several countries across different continents (Peirano & Pitout, 2010). In South Africa, the cefotaximases CTX-M-2 and CTXM-3 were first detected in Klebsiella pneumoniae clinical isolates, as part of a worldwide study (Paterson et al., 2003), and CTX-M-15 and CTX-M-37 were identified among Salmonella enterica serotypes (Govinden et al., 2006; Usha et al., 2008). Reports about the prevalence of CTX-M– producing E. coli, especially in the community settings from South Africa, are still rare. This study was undertaken to characterize infections caused by ESBL-producing E. coli, collected in the Cape Town metropolitan area, and to investigate the occurrence of clone ST131 in this collection.
2. Materials and methods 2.1. Patients, bacterial isolates, and antimicrobial susceptibility Twenty-two nonrepeat ESBL-producing E. coli clinical isolates recovered from specimens originating from doctor's consultation rooms and several private and a state hospital in the Cape Town metropolitan area during 2008 and 2009 were included in the study. These hospitals provide emergency and several speciality care services. The majority of isolates (n = 17) were recovered from urines and 5 from pus swab cultures. ESBL production was confirmed phenotypically by using the Clinical and Laboratory Standards Institute (CLSI, 2009) criteria for ESBL screening and disk confirmation tests. Antimicrobial susceptibility was determined with the VITEK 2 instrument (Vitek AMS; bioMerieux Vitek Systems, Hazelwood, MO). The MICs of the following drugs were determined: amoxycillin/clavulanic acid (AMC), piperacillin–tazobactam (TZP), cefoxitin (FOX), ertapenem (ERT), amikacin (AMK), gentamicin (GEN), tobramycin (TOB), ciprofloxacin (CIP), tigecycline (TIG), and trimethoprim–sulfamethoxazole (SXT). Throughout this study, results were interpreted using CLSI (2009) criteria for broth dilution. 2.2. β-Lactamase identification Isoelectric focusing, which included cefotaxime hydrolysis and determination of inhibitor profiles on polyacrylamide gels, was performed on freeze–thaw extracts as previously described (Pitout et al., 2007). PCR amplification and sequencing for blaCTX-Ms, blaOXAs, blaTEMs, and blaSHV was carried out on the isolates with a GeneAmp 9700 ThermoCycler instrument (Applied Biosystems, Norwalk, CT) using PCR conditions and primers as previously described (Pitout et al., 2007).
2.3. Plasmid-mediated quinolone resistance determinants The amplification of the qnrA, qnrS, and qnrB genes was undertaken in all ESBL-positive isolates with multiplex PCR (Robicsek et al., 2006b). aac(6′)-Ib and qepA were amplified in a separate PCR using primers and conditions as previously described (Robicsek et al., 2006a; Yamane et al., 2008). The variant aac(6′)-Ib-cr was further identified by digestion with BstF5I (Park et al., 2006) (New England Biolabs, Ipswich, MA). 2.4. Pulsed-field gel electrophoresis Genetic relatedness of the ESBL-producing 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 Centers for Disease Control and Prevention, Atlanta, GA (Hunter et al., 2005). Cluster designation was based on isolates showing approximately 80% or greater relatedness, which corresponds to the “possibly related (4–6 bands difference)” criteria of Tenover et al. (1995). 2.5. Identification of clone ST131 All the ESBL-producing isolates were screened for ST131 using a PCR for the pabB allele, recently described by Clermont et al. (2009). MLST was performed on those isolates that tested positive for ST131 using 7 conserved housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, recA). A detailed protocol of the MLST procedure, including allelic type and sequence type assignment methods, is available at MLST Databases at the ERI, University College Cork website (http://www.mlst.ucc.ie/mlst/dbs/Ecoli). 2.6. Phylogenetic grouping The ESBL-positive isolates were assigned to 1 of the 4 main E. coli phylogenetic groups (A, B1, B2, and D) by the use of a multiplex PCR-based method (Clermont et al., 2000). 3. Results 3.1. Patients, bacterial isolates, and susceptibilities The majority of patients (17/22 [77%]) presented with urinary tract infections (UTIs), 4 patients presented with intra-abdominal abscesses (including perforated diverticle [n = 1], post intra-abdominal surgery [n = 3]), while 1 patient had an infected burn wound. The majority of patients (12 [55%]) were inpatients. The prevalence of ESBL-producing E. coli isolated in the community hospital included in this study was less than 7%. Of the 22 isolates, 20 (91%) were nonsusceptible (i.e., intermediate or resistant) to SXT, 17 (77%) to AMC, 15 (68%) to TZP, 16 (73%) to CIP, 13 (59%) to TOB, 12 (55%) to GEN, and 9 (41%) to AMK. No resistance to ERT or TIG was detected.
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3.2. β-Lactamase gene identification
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ST131. ST131 status was confirmed by MLST. The PCR for the pabB allele was also performed on the remaining ESBLproducing E. coli, and none tested positive for ST131 (Table 1). Overall, 10 isolates belonged to clone ST131; 7 producing CTX-M-15 and 3 producing CTX-M-14 (Table 1).
Of the 22 E. coli isolates, 21 (95%) were positive for blaCTX-M genes: 13 produced CTX-M-15, 7 produced CTXM-14, and 1 produced CTX-M-3, while the remaining isolate produced SHV-2. Some of the CTX-M–producing isolates also produced TEM-1 (i.e., those with CTX-M-14 and CTX-M-15) and OXA-1 (only those with CTX-M-15) β-lactamases (Table 1).
3.6. Phylogenetic grouping Eleven isolates (including the 10 that tested positive for ST131) belonged to phylogenetic group B2, while the remaining ESBL-producing E. coli isolates belonged to phylogenetic groups D and A (9 and 2 isolates, respectively) (Table 1). We were unable to determine the phylogenetic group in 1 isolate (SA 9).
3.3. Plasmid-mediated quinolone resistance determinants Eight (28%) of the ESBL-producing E. coli isolates (all producing CTX-M-15) were positive for aac(6′)-Ib-cr. None of the other PMQR genes were detected. 3.4. Pulsed-field gel electrophoresis
4. Discussion
PFGE identified a closely related group of E. coli isolates that was designated as clone A (n = 6), as well as 4 isolates (SA1, SA15, SA17, and SA20) that were related to clone A (Fig. 1). The isolates that belonged to clone A had N80% similar PFGE profiles while SA1, SA15, SA17, and SA20 exhibited N60% similarity of profiles to clone A. The remaining ESBL-producing isolates were not clonally related, that is, exhibited b80% similar PFGE profiles and did not show patterns similar to those from clone A.
The SENTRY Antimicrobial Surveillance Program from 1998 to 1999 showed that 36% of K. pneumoniae and 5% of E. coli from South Africa were positive for ESBL production (Bell et al., 2002; Hirakata et al., 2005), and the PEARLS study from 2001 to 2002 showed similar results, that is, 34% of K. pneumoniae and 4.6% of E. coli from South Africa were positive for ESBL production (Bouchillon et al., 2004). According to the National Antibiotic Surveillance Forum, the prevalence of ESBL production in E. coli from private laboratories that serves community hospitals similar to the institutions included in this study was 5%, and the figure did not differ significantly between the cities within South Africa
3.5. Identification of clone ST131 The PCR for the pabB allele of ST131 status identified PFGE clone A and the 4 related isolates as belonging to Table 1 Characteristics of ESBL-producing E. coli isolated from the Cape Town area Isolate no.
Specimen
Antimicrobial nonsusceptibilitya
PMQR
β-Lactamases
Phylogenetic group
ST131 PCR
SA 1 SA 2 SA 3 SA 4 SA 5 SA 6 SA 7 SA 8 SA 9 SA 10 SA 11 SA 12 SA 13 SA 14 SA 15 SA 16 SA 17 SA 18 SA 19 SA 20 SA 21 SA 22
Urine Pus swab Pus swab Urine Urine Urine Urine Urine Pus swab Urine Urine Pus swab Urine Urine Urine Urine Urine Urine Urine Pus swab Urine Urine
AMC, TZP, CIP, SXT FOX, CIP, SXT CIP AMC, TZP, GEN, TOB, SXT AMC, FOX, GEN, TOB, SXT AMC, TZP, AMK, TOB, CIP, SXT AMC, TZP, FOX, AMK, GEN, TOB, CIP, SXT AMC, TZP, GEN, TOB, CIP, SXT AMC, TZP, AMK, GEN, TOB, CIP, SXT SXT AMC, TZP, FOX, CIP, SXT AMC, TZP, AMK, GEN, TOB, CIP, SXT AMC, TZP, AMK, GEN, TOB, CIP, SXT AMC, TZP, AMK, GEN, TOB, CIP, SXT AMC, TZP, CIP SXT AMC, TZP, CIP, SXT SXT AMC, TZP, AMK, GEN, TOB, CIP, SXT AMC, AMK, GEN, TOB, CIP, SXT AMC, TZP, AMK, GEN, TOB, CIP, SXT AMC, TZP, FOX, GEN, TOB, SXT
– – – – – aac(6′)-lb-cr aac(6′)-lb-cr – aac(6′)-lb-cr – – aac(6′)-lb-cr aac(6′)-lb-cr aac(6′)-lb-cr – – – – aac(6′)-lb-cr – aac(6′)-lb-cr –
TEM-1, CTX-M-14 CTX-M-14 CTX-M-15 TEM-1, SHV-2 TEM-1, CTX-M-3 TEM-1, OXA-1, CTX-M-15 TEM-1, OXA-1, CTX-M-15 TEM-1, CTX-M-14 TEM-1, OXA-1, CTX-M-15 CTX-M-14 TEM-1, OXA-1, CTX-M-15 OXA-1, CTX-M-15 OXA-1, CTX-M-15 OXA-1, CTX-M-15 TEM-1, CTX-M-15 CTX-M-14 CTX-M-15 CTX-M-14 TEM-1, OXA-1, CTX-M-15 TEM-1, CTX-M-14 TEM-2, OXA-1, CTX-M-15 TEM-1, CTX-M-15
B2 A D B2 D B2 D B2 NT D D B2 A B2 B2 D B2 D B2 B2 B2 D
+ − − − − + − + − − − + − + + − + − + + + −
AMC = amoxycillin/clavulanic acid; TZP = piperacillin/tazobactam; FOX = cefoxitin; CIP = ciprofloxacin; GEN = gentamicin; TOB = tobramycin; AMK = amikacin; SXT = trimethoprim–sulfamethoxazole; PMQR = plasmid-mediated quinolone resistance determinants. a Antimicrobial nonsusceptibility (i.e., resistant or intermediate).
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Fig. 1. PFGE of XbaI-digested DNA of ESBL-producing E. coli isolates from the Cape Town area.
(i.e., Johannesburg, Pretoria, Durban, Cape Town, and Bloemfontein) (Brink et al., 2007). The CTX-M enzymes, particularly those belonging to CTX-M group 1, were first identified in South Africa during the mid-2009s, in K. pneumoniae isolated from a patient with hospital-acquired pneumonia (Elliott et al., 2006). Enterobacteriaceae that produce CTX-M-15 were recently described from academic hospitals in Durban and Pretoria, respectively (Ehlers et al., 2009; Usha et al., 2008). In our collection of E. coli isolated from several community hospitals in the Cape Town metropolitan area, blaCTX-Ms was present in 95% of isolates, with CTX-M-15 and CTX-M-14 being the most common types. Most of the E. coli that produce CTX-M-15 co-produced TEM-1 and OXA-1 β-lactamases, some were positive for aac(6′)-lb-cr, while MLST identified ST131 among 7/13 (54%) of isolates that also belonged to the phylogenetic group B2 (Table 1) . Of particular interest was that nearly half of the E. coli that produced CTX-M-14 also belonged to clone ST131 (3/7
[43%]). These isolates also belonged to phylogenetic group B2 and co-produced TEM-1. Two previous studies, one from Calgary, Canada, and the other from Chicago, USA, showed that E. coli clone ST131 producing CTX-M-15 and CTX-M-14 had emerged as an important cause of community-onset bacteremia and UTIs (Peirano et al., 2010; Pitout et al., 2009). The present study shows that these isolates are also present in South Africa and caused UTIs among hospitalized patients in community hospitals. Reports of E. coli clone ST131 that produce CTXM-15 from Africa are rare and had been described from Tunisia and Central African Republic (Clermont et al., 2008; Dahmen et al.). To our knowledge, the present study is the first to identify ST131 in ESBL-producing E. coli from Southern Africa and the first to show the presence of aac (6′)-lb-cr among Enterobacteriaceae isolates. The only previous PMQR described from South Africa was qnrB from a single isolate of Salmonella typhimurium (Govender et al., 2009).
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