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Genetic diversity and antibiotic resistance in Escherichia coli from environmental surface water in Dhaka City, Bangladesh
Diagnostic Microbiology and Infectious Disease 76 (2013) 222–226
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Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Genetic diversity and antibiotic resistance in Escherichia coli from environmental surface water in Dhaka City, Bangladesh☆ M. Kamruzzaman a, b, c, 1, Shereen Shoma a, b, d, 1, S.M. Naymul Bari c, Andrew N. Ginn a, b, Agnieszka M. Wiklendt a, e, Sally R. Partridge a, b, e, Shah M. Faruque c, Jonathan R. Iredell a, b, e,⁎ a b c d e
Centre for Research Excellence in Critical Infection and Sydney Institute for Emerging Infections and Biosecurity, University of Sydney, Sydney, New South Wales, Australia Westmead Millennium Institute, Westmead, New South Wales, Australia Centre for Food and Water Borne Diseases, International Centre for Diarrhoeal Disease Research, Bangladesh, Mohakhali, Dhaka-1212, Bangladesh Centre for Communicable Diseases, International Centre for Diarrhoeal Disease Research, Bangladesh, Mohakhali, Dhaka-1212, Bangladesh Centre for Infectious Diseases and Microbiology, Westmead Hospital, Westmead, New South Wales, Australia
a r t i c l e
i n f o
Article history: Received 9 December 2012 Received in revised form 21 February 2013 Accepted 21 February 2013 Available online 28 March 2013 Keywords: Environment MLST Plasmid
a b s t r a c t The extended-spectrum β-lactamase gene blaCTX-M-15 was almost ubiquitous in diverse antibiotic-resistant Escherichia coli isolated from surface water around Dhaka City, Bangladesh. Forty-eight isolates represented 34 multi-locus sequence types and a variety of plasmid replicons were identified in association with blaCTX-M-15 and other resistance genes. This water is likely to be an important source of transmissible antibiotic resistance in Bangladesh. © 2013 Elsevier Inc. All rights reserved.
Escherichia coli, a dominant facultative aerobe in the gut and an important human pathogen, may be a major reservoir of antibiotic resistance determinants on conjugative plasmids. Multi-resistant E. coli have been isolated from a variety of water sources in India and China (Diwan et al., 2010; Hu et al., 2008; Lu et al., 2010; Ram et al., 2007), but to our knowledge there are no studies of antibioticresistant E. coli in Bangladesh environmental waters. Here we characterized E. coli isolated from environmental surface water in Bangladesh and screened for genes conferring resistance to clinically important antibiotics (third generation cephalosporins, carbapenems, aminoglycosides, and quinolones). Water samples were collected from eight different sites in and around Dhaka (2 major rivers, 2 lakes; numbered 1–8, see Fig. 1) on June 6 (A), July 4 (B), and August 1 (C), 2011. A 5.0-mL aliquot from each sample was added to 2.5-mL 3× peptone water (1% peptone,
☆ This work was funded in part through NHMRC grant 1001021 and a research grant from the International Centre for Diarrhoeal Diseases Research, Bangladesh, which is supported by countries and agencies which share its concern for the health problems of developing countries. The MLST data are publicly available at http://mlst.ucc.ie, which was supported by a grant from the Science Foundation of Ireland (05/FE1/B882). ⁎ Corresponding author. Tel.: +61-2-9845-6012; fax: +61-2-9891-5317. E-mail address: [email protected] (J.R. Iredell). 1 M. Kamruzzaman and Shereen Shoma contributed equally. 0732-8893/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2013.02.016
1% NaCl; pH 8.0) within 2 h of collection, incubated (4 h, 37 °C) and dilutions (up to 10 −3) in phosphate-buffered saline were plated onto MacConkey agar (Oxoid, Basingstoke, England) without antibiotic selection. Two suspected E. coli colonies (designated 1 and 2) were picked at random for each sample, giving 6 isolates from each site and a total of 48 isolates, designated 1A1, 2B2, etc. MALDI-TOF MS (Kok et al., 2011), and the Phoenix automated microbiology system (BD Diagnostic Systems, Sparks, MD) confirmed all 48 isolates as E. coli. Multi-locus sequence typing (http://mlst.ucc.ie/mlst/dbs/Ecoli) identified 34 different sequence types (ST) among the 48 isolates. Most of the previously described ST (25/28) include putative human and/or animal pathogens (Table S1). Nine isolates with novel combinations of known alleles were assigned to 4 new STs (Table 1 and Table S1). Two isolates with a novel adk or gyrB allele were most closely related to STs that include human isolates (Table 2). Isolates at each site were diverse; some ST were identified in 2 to 3 sites and ST38 included an antibiotic-resistant and a susceptible isolate (Table 1). Isolates were classified into phylogenetic groups (A, B1, B2, D) using a polymerase chain reaction (PCR) method based on the chuA, yjaA, and TSPE4.C2 markers (Doumith et al., 2012). Isolates negative for all 3 markers were classified as A0, those positive for yjaA only as A1, isolates having chuA only were classified as D1 and those having chuA with TSPE4.C2 were classified as D2, as suggested by Escobar-
M. Kamruzzaman et al. / Diagnostic Microbiology and Infectious Disease 76 (2013) 222–226
223
Fig. 1. Sampling sites in and around Dhaka City, Bangladesh (from Google Maps). “A” indicates the main centre of Dhaka. Swarighat (1), Kamrangirchar (2), Gabtoli (3), Aminbazaar (4), and Tongi Bridge (5) are on the same river system, listed upstream to downstream. Gulshan Lake (6) and Rampura Bridge (7) are part of a separate lake system. Balu River (8) is a separate river system.
Paramo et al. (2004). Most isolates (39/48) belong to groups A or B1, which are generally more prevalent among human commensal isolates (Table 1). The remaining 9 isolates belonged to groups B2 or D, which are more prevalent amongst pathogenic bacteria. Antimicrobial susceptibility testing using the Phoenix automated microbiology system (Becton Dickinson, Sparks, MD, USA) and/or by disk diffusion (CLSI, 2012) revealed widespread resistance to ampicillin (n = 30/48), and third-generation cephalosporins (n = 20/48) (Table 1). Resistance to ciprofloxacin (CIP; n = 19) and trimethoprim/sulfamethoxazole (SXT; n = 19) was similarly common, with no evident correlation between antibiotic resistance phenotypes. Seventeen isolates were resistant to tetracycline (TET), 4 were resistant to chloramphenicol (CML), and none were resistant to kanamycin. Eighteen of 20 isolates growing in the presence of 2 μg/ mL cefotaxime (CTX) and/or ceftazidime (CAZ) had a classical extended-spectrum β-lactamase (ESBL) phenotype in double disk synergy tests (DDST) (Sabia et al., 2012), 10 were resistant to CIP (N2 μg/mL), 9 to SXT (N4 μg/mL), and only 4 to gentamicin (GEN) and tobramycin (TOB) (N8 μg/mL). All isolates were susceptible to imipenem and meropenem (MICs b1.0 μg/mL). Selected antibiotic resistance genes were identified by multiplex PCR-reverse line blot hybridization (Ginn et al., 2013). Confirmatory PCR was performed for blaTEM (Caniça et al., 1997), blaOXA-30 (Aubert et al., 2001), blaCTX-M-1 group, blaCTX-M-9 group, blaSHV-5/SHV-12, blaVEB (Ellem et al., 2011) and qnrS (QnrS1-OF, 5′-AATGTGTTGATGTAACAGGCTTT-3′; QnrS1-OR 5′-AGACGCCTGAGGGTAAAAAT-3′) genes. The blaNDM-1 gene was screened for by PCR (Nordmann et al., 2011). All 19 blaCTX-M-1 group genes (from 7/8 sites) were identified as the globally dominant blaCTX-M-15 by PCR with flanking primers and sequencing (Partridge et al., 2011). One isolate also carried a blaCMY-2like gene, expected to mask a classical ESBL phenotype. Another isolate, resistant to GEN, TOB, and amikacin (AMK), had armA (conferring broad high-level aminoglycoside resistance). All of the 6 isolates resistant to GEN and TOB but not AMK had an aac(3)-II gene.
Plasmid-mediated quinolone resistance determinants (PMQR) were detected in 20 isolates aacA4cr (also called aac(6')-Ib-cr), n = 4; aacA4cr+qnrB, n = 1; qnrS, n = 15). One isolate with qnrS was susceptible to all antibiotics and only aacA4cr correlated with CIP resistance. This is not unexpected, as PMQR alone are generally not sufficient to mediate high-level quinolone resistance (Strahilevitz et al., 2009). One isolate carried a blaDHA-like gene with qnrB, a common combination (Mata et al., 2011). blaNDM-1 was not detected in any isolate. PCR-based replicon typing (PBRT) (Johnson et al., 2007), plus primers to detect E. coli-type IncFII plasmids (Villa et al., 2010), identified incompatibility types in 32/48 isolates. IncFIA (n = 17), IncI1 (n = 11), IncFIB (n = 10), and IncFII (n = 10) were most common and most isolates carried more than one replicon type (Table 1). IncA/C, IncY, IncP, and IncN replicon types were identified in multi-resistant isolates only, but IncFIA, IncFIB, IncFII, and IncI1 plasmids were present in both sensitive and resistant isolates. S1 nuclease (Promega, Madison, WI, USA) digestion and pulsed-field gel electrophoresis (PFGE) (Barton et al., 1995) revealed plasmids of 35– 240 kb. Several isolates yielded more plasmid bands by S1/PFGE than replicon types by PBRT, 8 had plasmids evident by S1/PFGE only and no plasmids were apparent in 10 isolates (Table 1). Small (2–8 kb) plasmids were identified (Quick plasmid miniprep kit, Invitrogen, Carlsbad, CA) in 2 of these, while 3 others had resistance genes, including blaCTX-M-15 (Table 1) which may be on the chromosome. Unlike other countries (Fam et al., 2011; Zong et al., 2008) only 3/ 19 isolates with blaCTX-M-15 were GEN resistant. This may relate to the relative availability of oral β-lactam antibiotics in Bangladesh, whereas aminoglycosides are generally administered only parenterally in hospitals. blaCTX-M-15 was linked to ISEcp1 and orf477Δ in all isolates but the associated genes commonly found on IncF plasmids (blaTEM, aac(3)-II and blaOXA-30 plus aac(6′)-Ib-cr) (Partridge, et al., 2011) were generally not present, so the wider genetic context of blaCTX-M-15 in these isolates is of particular interest.
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Table 1 Characteristics of Bangladeshi environmental E. coli isolates. Phylo group
STb
CCb
1C1 1C2 2A1, 2C2 2A2 3C1 3C2 5A1 5C1 6B1 7A1 8A2 8B2 8C1, 6C2 8C2 4B1 4C2 8A1 8B1 3A1 3B1 7B1 7C1 4A2 6B2 5B2 5C2 1B1f 6A1 6A2 7A2 4A1 5B1 4B2 5A2 1A1 3A2 3B2 1A2 2B1 2B2 7C2 1B2 2C1 4C1 6C1f 7B2
B1 B1 B1 B1 A1 A0 B1 B2 A0 B1 B1 B1 B1 D2 B2 B1 A1 D1 A1 B1 A1 B1 D2 B2 A1 B1 A1 B1 D2 A1 B1 A0 A0 A1 A1 B1 D2 A0 A1 A0 A0 B1 A1 D2 A1 A1
155 2280 155 162 949 2854 366 2960 409 58 10 155 2853 2961 38 394 206 1140 48 609 48 156 648 95 206 443 167 156 315 617 542 226 2852 215 2851 2450 38 1408 2851 46 226 2852 2851 405 167 2851
155 155 469 -
Phenotypec
Resistance genesd
Plasmidse
bla
aac
qnr
S 155 10 155 38 398 206 10 46 10 156 95 206 205 10 156 38 10 226 10 38 46 226 405 10 -
TIM TIM TIM TIM TIM TIM TIM TIM TIM TIM TIM TIM TIM TIM
TIM
TIM TIM
TIM TIM TIM
CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX
CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ
GEN GEN GEN GEN
FOX FEP FEP FEP FEP FEP FEP FEP FEP FEP FEP FEP FEP FEP FEP FEP FEP FEP
TOB TOB TOB TOB TOB
CIP CIP CIP CIP CIP CIP CIP CIP CIP CIP CIP
FOX CIP
SXT SXT SXT SXT SXT SXT SXT SXT SXT SXT SXT SXT SXT
TET TET TET TET TET TET
CML
CML CML
TEM TEM TEM
TET TET TET TET
OXA-30
TET SXT
TET
CIP SXT
FOX FOX AMK
GEN GEN GEN
TOB TOB TOB TOB
CIP CIP CIP CIP
TET TET SXT SXT SXT
OXA-30
TET TET
SXT
CIP CIP
TEM TEM TEM TEM TEM TEM
CML TET
CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15
DHA
S S A4cr (3)-II (3)-II (3)-II A4cr, (3)-II
TEM TEM TEM TEM TEM TEM
S S B
S S S S
S S S S S OXA-30 OXA-30 OXA-30
TEM CMY-2
A4cr A4cr, (3)-II A4cr, (3)-II armA
S
Inc
S1 (kb)
FIB, FII FIB FIC FIA, FIB FII FII FIA, I1 -
~120 ~120 ~60, 100 -g ~60 ~100 ~195 ~100 ~48 -g ~48 ~60 ~50 ~35, 130 ~100 ~35, 100 ~150 ~80, 130 ~120 ~70 ~80, 97 ~80, 200 ~65 ~35 ~35, 50, 80, 195, 240 ~48, 120 ~80, 190 ~140 ~35, 97, 200 ~95 ~35 ~50, 70, 190 ~70 ~200 ~60, 70, 200 ~145 ~35, 190 ~70, 145 ~50, 220
FIA, FII P FIA, FIB FIB, P FIB FIA, FIB, A/C FIA, FIB FIA, I1 FII FIC FIA, I1 I1 FIA, I1 I1 FII FIA FIA, FII, I1 Y FIA, FII, I1 FIA, FII, I1 FIA FIA, FIB, N FIA, FIB, FII, I1 A/C
M. Kamruzzaman et al. / Diagnostic Microbiology and Infectious Disease 76 (2013) 222–226
Isolatea
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Table 2 Closest relatives of novel STs identified in this study. ST
410 2851a 649 2852a 2937 2960a 2473 2853a 694 2854a 2622 2961a
Allelea
Source
adk
fumC
gyrB
icd
mdh
purA
recA
6 6 6 6 58 301 6 6 10 10 13 40
4 4 65 65 53 53 19 19 11 11 363 363
12 12 65 5 1 70 3 3 4 4 10 302
1 1 1 1 58 58 18 18 12 12 97 97
20 20 11 11 24 24 9 9 8 8 17 17
18 18 13 13 1 1 13 13 18 1 94 94
7 6 6 6 42 42 6 156 2 2 93 6
Human Water Human Water Water Water Livestock Water Human Water Human Water
a
New ST, this study alleles that vary between pairs of ST are shaded.
Although the ST seen may be biased towards more environmentally hardy types, or a wider range of hosts, including livestock and birds, most have previously been reported in humans. Despite the marked dominance of (usually plasmid-borne) blaCTX-M-15 no E. coli ST131, an important vector of this gene in humans, was found and only one ST405 isolate, also associated with blaCTX-M-15 and other important resistance genes (Woodford et al., 2011), was detected. Although previously reported in Dhaka (Islam et al., 2012) and in Indian environmental waters (Walsh et al., 2011), blaNDM-1 was not found in these isolates collected in 2011. Sites 1–5 are part of the same river system into which hospital, industrial and household waste drains directly and from which residents of slum areas may take untreated water for cooking and bathing. Such waste includes antibiotics and antibiotic resistant bacteria and multidrug-resistant bacteria appear to be abundant in the surface water where this type of contamination occurs (Hu et al., 2008). Balu River (Site 8) is part of a separate river system in a less populated and presumably less contaminated area; it is noteworthy that antibiotic resistance was less prevalent there and that no isolate carried blaCTX-M-15. Excessive use of antibiotics in medicine and agriculture is problematic in some countries (Ahmed and Islam, 2012; Guyon et al., 1994) and the wide distribution of antibiotic-resistant bacteria in aquatic environments (Baquero et al., 2008; Reinthaler et al., 2003; Watkinson et al., 2007; Xi et al., 2009) is a major public health concern. More than 200,000 drug stores in Bangladesh allow relatively unrestricted access to antibiotics (Ahmed and Islam, 2012; Kamat and Nichter, 1998) and waste from households, hospitals, and sewage treatment plants drains into rivers that provide bathing and drinking water for large populations in Dhaka and other major cities. Our study shows that Bangladesh surface water is a reservoir of diverse antibiotic resistant E. coli and may play an important role in dissemination of antibiotic resistance genes such as blaCTX-M-15, which appear to be highly mobile. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.diagmicrobio.2013.02.016.
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Notes to Table 1 a Isolates are coded to indicate their origin as follows: the first number (1–8) indicates the sampling site (see Fig. 1). The letter indicates the date of isolation, June 6 (A), July 4 (B), or August 1 (C), 2011. The second number indicates colony 1 or colony 2. b ST and clonal complex (CC) according to MLST data. c All isolates resistant to any antibiotic as indicated (standard nomenclature) are also resistant to ampicillin. Resistance was determined by BD Phoenix and disk diffusion method according to CLSI guidelines (CLSI, M100-S22, 2012). Resistance to CML, TET and KAN were determined by disk diffusion only (not included in BD phoenix panel). d Genes detected were CTX-M-15, blaCTX-M-15 (determined by sequencing); OXA-30, blaOXA-30-like genes; DHA, blaDHA-like genes; CMY-2, blaCMY-2-like genes; TEM, blaTEM genes; (3)-II, aac(3)-II-like genes; A4cr, aacA4cr (simplified name for aac(6')-Ib-cr) (differentiated by mPCR/RLB); B, qnrB gene; S, qnrS gene. e Inc (FIA etc.) = incompatibility type by PBRT; S1 = plasmid sizes (kb) estimated from S1/PFGE; − = no plasmid detected. f All CTX and/or CAZ-resistant isolates had a classical ESBL phenotype on DDST except 6C1 which has a blaCMY-2-like gene that may have masked the ESBL phenotype conferred by blaCTX-M-15 and 1B1, which had a blaDHA-like gene. g Only isolates with no large plasmids evident (by PBRT and/or S1 PFGE) were tested for small plasmids by alkaline lysis: plasmids of ~2, 3, and 8 kb were detected in 3C1 and a single ~4 kb plasmid in 8A2.
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Contents lists available at SciVerse ScienceDirect
Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Genetic diversity and antibiotic resistance in Escherichia coli from environmental surface water in Dhaka City, Bangladesh☆ M. Kamruzzaman a, b, c, 1, Shereen Shoma a, b, d, 1, S.M. Naymul Bari c, Andrew N. Ginn a, b, Agnieszka M. Wiklendt a, e, Sally R. Partridge a, b, e, Shah M. Faruque c, Jonathan R. Iredell a, b, e,⁎ a b c d e
Centre for Research Excellence in Critical Infection and Sydney Institute for Emerging Infections and Biosecurity, University of Sydney, Sydney, New South Wales, Australia Westmead Millennium Institute, Westmead, New South Wales, Australia Centre for Food and Water Borne Diseases, International Centre for Diarrhoeal Disease Research, Bangladesh, Mohakhali, Dhaka-1212, Bangladesh Centre for Communicable Diseases, International Centre for Diarrhoeal Disease Research, Bangladesh, Mohakhali, Dhaka-1212, Bangladesh Centre for Infectious Diseases and Microbiology, Westmead Hospital, Westmead, New South Wales, Australia
a r t i c l e
i n f o
Article history: Received 9 December 2012 Received in revised form 21 February 2013 Accepted 21 February 2013 Available online 28 March 2013 Keywords: Environment MLST Plasmid
a b s t r a c t The extended-spectrum β-lactamase gene blaCTX-M-15 was almost ubiquitous in diverse antibiotic-resistant Escherichia coli isolated from surface water around Dhaka City, Bangladesh. Forty-eight isolates represented 34 multi-locus sequence types and a variety of plasmid replicons were identified in association with blaCTX-M-15 and other resistance genes. This water is likely to be an important source of transmissible antibiotic resistance in Bangladesh. © 2013 Elsevier Inc. All rights reserved.
Escherichia coli, a dominant facultative aerobe in the gut and an important human pathogen, may be a major reservoir of antibiotic resistance determinants on conjugative plasmids. Multi-resistant E. coli have been isolated from a variety of water sources in India and China (Diwan et al., 2010; Hu et al., 2008; Lu et al., 2010; Ram et al., 2007), but to our knowledge there are no studies of antibioticresistant E. coli in Bangladesh environmental waters. Here we characterized E. coli isolated from environmental surface water in Bangladesh and screened for genes conferring resistance to clinically important antibiotics (third generation cephalosporins, carbapenems, aminoglycosides, and quinolones). Water samples were collected from eight different sites in and around Dhaka (2 major rivers, 2 lakes; numbered 1–8, see Fig. 1) on June 6 (A), July 4 (B), and August 1 (C), 2011. A 5.0-mL aliquot from each sample was added to 2.5-mL 3× peptone water (1% peptone,
☆ This work was funded in part through NHMRC grant 1001021 and a research grant from the International Centre for Diarrhoeal Diseases Research, Bangladesh, which is supported by countries and agencies which share its concern for the health problems of developing countries. The MLST data are publicly available at http://mlst.ucc.ie, which was supported by a grant from the Science Foundation of Ireland (05/FE1/B882). ⁎ Corresponding author. Tel.: +61-2-9845-6012; fax: +61-2-9891-5317. E-mail address: [email protected] (J.R. Iredell). 1 M. Kamruzzaman and Shereen Shoma contributed equally. 0732-8893/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.diagmicrobio.2013.02.016
1% NaCl; pH 8.0) within 2 h of collection, incubated (4 h, 37 °C) and dilutions (up to 10 −3) in phosphate-buffered saline were plated onto MacConkey agar (Oxoid, Basingstoke, England) without antibiotic selection. Two suspected E. coli colonies (designated 1 and 2) were picked at random for each sample, giving 6 isolates from each site and a total of 48 isolates, designated 1A1, 2B2, etc. MALDI-TOF MS (Kok et al., 2011), and the Phoenix automated microbiology system (BD Diagnostic Systems, Sparks, MD) confirmed all 48 isolates as E. coli. Multi-locus sequence typing (http://mlst.ucc.ie/mlst/dbs/Ecoli) identified 34 different sequence types (ST) among the 48 isolates. Most of the previously described ST (25/28) include putative human and/or animal pathogens (Table S1). Nine isolates with novel combinations of known alleles were assigned to 4 new STs (Table 1 and Table S1). Two isolates with a novel adk or gyrB allele were most closely related to STs that include human isolates (Table 2). Isolates at each site were diverse; some ST were identified in 2 to 3 sites and ST38 included an antibiotic-resistant and a susceptible isolate (Table 1). Isolates were classified into phylogenetic groups (A, B1, B2, D) using a polymerase chain reaction (PCR) method based on the chuA, yjaA, and TSPE4.C2 markers (Doumith et al., 2012). Isolates negative for all 3 markers were classified as A0, those positive for yjaA only as A1, isolates having chuA only were classified as D1 and those having chuA with TSPE4.C2 were classified as D2, as suggested by Escobar-
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Fig. 1. Sampling sites in and around Dhaka City, Bangladesh (from Google Maps). “A” indicates the main centre of Dhaka. Swarighat (1), Kamrangirchar (2), Gabtoli (3), Aminbazaar (4), and Tongi Bridge (5) are on the same river system, listed upstream to downstream. Gulshan Lake (6) and Rampura Bridge (7) are part of a separate lake system. Balu River (8) is a separate river system.
Paramo et al. (2004). Most isolates (39/48) belong to groups A or B1, which are generally more prevalent among human commensal isolates (Table 1). The remaining 9 isolates belonged to groups B2 or D, which are more prevalent amongst pathogenic bacteria. Antimicrobial susceptibility testing using the Phoenix automated microbiology system (Becton Dickinson, Sparks, MD, USA) and/or by disk diffusion (CLSI, 2012) revealed widespread resistance to ampicillin (n = 30/48), and third-generation cephalosporins (n = 20/48) (Table 1). Resistance to ciprofloxacin (CIP; n = 19) and trimethoprim/sulfamethoxazole (SXT; n = 19) was similarly common, with no evident correlation between antibiotic resistance phenotypes. Seventeen isolates were resistant to tetracycline (TET), 4 were resistant to chloramphenicol (CML), and none were resistant to kanamycin. Eighteen of 20 isolates growing in the presence of 2 μg/ mL cefotaxime (CTX) and/or ceftazidime (CAZ) had a classical extended-spectrum β-lactamase (ESBL) phenotype in double disk synergy tests (DDST) (Sabia et al., 2012), 10 were resistant to CIP (N2 μg/mL), 9 to SXT (N4 μg/mL), and only 4 to gentamicin (GEN) and tobramycin (TOB) (N8 μg/mL). All isolates were susceptible to imipenem and meropenem (MICs b1.0 μg/mL). Selected antibiotic resistance genes were identified by multiplex PCR-reverse line blot hybridization (Ginn et al., 2013). Confirmatory PCR was performed for blaTEM (Caniça et al., 1997), blaOXA-30 (Aubert et al., 2001), blaCTX-M-1 group, blaCTX-M-9 group, blaSHV-5/SHV-12, blaVEB (Ellem et al., 2011) and qnrS (QnrS1-OF, 5′-AATGTGTTGATGTAACAGGCTTT-3′; QnrS1-OR 5′-AGACGCCTGAGGGTAAAAAT-3′) genes. The blaNDM-1 gene was screened for by PCR (Nordmann et al., 2011). All 19 blaCTX-M-1 group genes (from 7/8 sites) were identified as the globally dominant blaCTX-M-15 by PCR with flanking primers and sequencing (Partridge et al., 2011). One isolate also carried a blaCMY-2like gene, expected to mask a classical ESBL phenotype. Another isolate, resistant to GEN, TOB, and amikacin (AMK), had armA (conferring broad high-level aminoglycoside resistance). All of the 6 isolates resistant to GEN and TOB but not AMK had an aac(3)-II gene.
Plasmid-mediated quinolone resistance determinants (PMQR) were detected in 20 isolates aacA4cr (also called aac(6')-Ib-cr), n = 4; aacA4cr+qnrB, n = 1; qnrS, n = 15). One isolate with qnrS was susceptible to all antibiotics and only aacA4cr correlated with CIP resistance. This is not unexpected, as PMQR alone are generally not sufficient to mediate high-level quinolone resistance (Strahilevitz et al., 2009). One isolate carried a blaDHA-like gene with qnrB, a common combination (Mata et al., 2011). blaNDM-1 was not detected in any isolate. PCR-based replicon typing (PBRT) (Johnson et al., 2007), plus primers to detect E. coli-type IncFII plasmids (Villa et al., 2010), identified incompatibility types in 32/48 isolates. IncFIA (n = 17), IncI1 (n = 11), IncFIB (n = 10), and IncFII (n = 10) were most common and most isolates carried more than one replicon type (Table 1). IncA/C, IncY, IncP, and IncN replicon types were identified in multi-resistant isolates only, but IncFIA, IncFIB, IncFII, and IncI1 plasmids were present in both sensitive and resistant isolates. S1 nuclease (Promega, Madison, WI, USA) digestion and pulsed-field gel electrophoresis (PFGE) (Barton et al., 1995) revealed plasmids of 35– 240 kb. Several isolates yielded more plasmid bands by S1/PFGE than replicon types by PBRT, 8 had plasmids evident by S1/PFGE only and no plasmids were apparent in 10 isolates (Table 1). Small (2–8 kb) plasmids were identified (Quick plasmid miniprep kit, Invitrogen, Carlsbad, CA) in 2 of these, while 3 others had resistance genes, including blaCTX-M-15 (Table 1) which may be on the chromosome. Unlike other countries (Fam et al., 2011; Zong et al., 2008) only 3/ 19 isolates with blaCTX-M-15 were GEN resistant. This may relate to the relative availability of oral β-lactam antibiotics in Bangladesh, whereas aminoglycosides are generally administered only parenterally in hospitals. blaCTX-M-15 was linked to ISEcp1 and orf477Δ in all isolates but the associated genes commonly found on IncF plasmids (blaTEM, aac(3)-II and blaOXA-30 plus aac(6′)-Ib-cr) (Partridge, et al., 2011) were generally not present, so the wider genetic context of blaCTX-M-15 in these isolates is of particular interest.
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Table 1 Characteristics of Bangladeshi environmental E. coli isolates. Phylo group
STb
CCb
1C1 1C2 2A1, 2C2 2A2 3C1 3C2 5A1 5C1 6B1 7A1 8A2 8B2 8C1, 6C2 8C2 4B1 4C2 8A1 8B1 3A1 3B1 7B1 7C1 4A2 6B2 5B2 5C2 1B1f 6A1 6A2 7A2 4A1 5B1 4B2 5A2 1A1 3A2 3B2 1A2 2B1 2B2 7C2 1B2 2C1 4C1 6C1f 7B2
B1 B1 B1 B1 A1 A0 B1 B2 A0 B1 B1 B1 B1 D2 B2 B1 A1 D1 A1 B1 A1 B1 D2 B2 A1 B1 A1 B1 D2 A1 B1 A0 A0 A1 A1 B1 D2 A0 A1 A0 A0 B1 A1 D2 A1 A1
155 2280 155 162 949 2854 366 2960 409 58 10 155 2853 2961 38 394 206 1140 48 609 48 156 648 95 206 443 167 156 315 617 542 226 2852 215 2851 2450 38 1408 2851 46 226 2852 2851 405 167 2851
155 155 469 -
Phenotypec
Resistance genesd
Plasmidse
bla
aac
qnr
S 155 10 155 38 398 206 10 46 10 156 95 206 205 10 156 38 10 226 10 38 46 226 405 10 -
TIM TIM TIM TIM TIM TIM TIM TIM TIM TIM TIM TIM TIM TIM
TIM
TIM TIM
TIM TIM TIM
CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX CTX
CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ CAZ
GEN GEN GEN GEN
FOX FEP FEP FEP FEP FEP FEP FEP FEP FEP FEP FEP FEP FEP FEP FEP FEP FEP
TOB TOB TOB TOB TOB
CIP CIP CIP CIP CIP CIP CIP CIP CIP CIP CIP
FOX CIP
SXT SXT SXT SXT SXT SXT SXT SXT SXT SXT SXT SXT SXT
TET TET TET TET TET TET
CML
CML CML
TEM TEM TEM
TET TET TET TET
OXA-30
TET SXT
TET
CIP SXT
FOX FOX AMK
GEN GEN GEN
TOB TOB TOB TOB
CIP CIP CIP CIP
TET TET SXT SXT SXT
OXA-30
TET TET
SXT
CIP CIP
TEM TEM TEM TEM TEM TEM
CML TET
CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15 CTX-M-15
DHA
S S A4cr (3)-II (3)-II (3)-II A4cr, (3)-II
TEM TEM TEM TEM TEM TEM
S S B
S S S S
S S S S S OXA-30 OXA-30 OXA-30
TEM CMY-2
A4cr A4cr, (3)-II A4cr, (3)-II armA
S
Inc
S1 (kb)
FIB, FII FIB FIC FIA, FIB FII FII FIA, I1 -
~120 ~120 ~60, 100 -g ~60 ~100 ~195 ~100 ~48 -g ~48 ~60 ~50 ~35, 130 ~100 ~35, 100 ~150 ~80, 130 ~120 ~70 ~80, 97 ~80, 200 ~65 ~35 ~35, 50, 80, 195, 240 ~48, 120 ~80, 190 ~140 ~35, 97, 200 ~95 ~35 ~50, 70, 190 ~70 ~200 ~60, 70, 200 ~145 ~35, 190 ~70, 145 ~50, 220
FIA, FII P FIA, FIB FIB, P FIB FIA, FIB, A/C FIA, FIB FIA, I1 FII FIC FIA, I1 I1 FIA, I1 I1 FII FIA FIA, FII, I1 Y FIA, FII, I1 FIA, FII, I1 FIA FIA, FIB, N FIA, FIB, FII, I1 A/C
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Isolatea
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Table 2 Closest relatives of novel STs identified in this study. ST
410 2851a 649 2852a 2937 2960a 2473 2853a 694 2854a 2622 2961a
Allelea
Source
adk
fumC
gyrB
icd
mdh
purA
recA
6 6 6 6 58 301 6 6 10 10 13 40
4 4 65 65 53 53 19 19 11 11 363 363
12 12 65 5 1 70 3 3 4 4 10 302
1 1 1 1 58 58 18 18 12 12 97 97
20 20 11 11 24 24 9 9 8 8 17 17
18 18 13 13 1 1 13 13 18 1 94 94
7 6 6 6 42 42 6 156 2 2 93 6
Human Water Human Water Water Water Livestock Water Human Water Human Water
a
New ST, this study alleles that vary between pairs of ST are shaded.
Although the ST seen may be biased towards more environmentally hardy types, or a wider range of hosts, including livestock and birds, most have previously been reported in humans. Despite the marked dominance of (usually plasmid-borne) blaCTX-M-15 no E. coli ST131, an important vector of this gene in humans, was found and only one ST405 isolate, also associated with blaCTX-M-15 and other important resistance genes (Woodford et al., 2011), was detected. Although previously reported in Dhaka (Islam et al., 2012) and in Indian environmental waters (Walsh et al., 2011), blaNDM-1 was not found in these isolates collected in 2011. Sites 1–5 are part of the same river system into which hospital, industrial and household waste drains directly and from which residents of slum areas may take untreated water for cooking and bathing. Such waste includes antibiotics and antibiotic resistant bacteria and multidrug-resistant bacteria appear to be abundant in the surface water where this type of contamination occurs (Hu et al., 2008). Balu River (Site 8) is part of a separate river system in a less populated and presumably less contaminated area; it is noteworthy that antibiotic resistance was less prevalent there and that no isolate carried blaCTX-M-15. Excessive use of antibiotics in medicine and agriculture is problematic in some countries (Ahmed and Islam, 2012; Guyon et al., 1994) and the wide distribution of antibiotic-resistant bacteria in aquatic environments (Baquero et al., 2008; Reinthaler et al., 2003; Watkinson et al., 2007; Xi et al., 2009) is a major public health concern. More than 200,000 drug stores in Bangladesh allow relatively unrestricted access to antibiotics (Ahmed and Islam, 2012; Kamat and Nichter, 1998) and waste from households, hospitals, and sewage treatment plants drains into rivers that provide bathing and drinking water for large populations in Dhaka and other major cities. Our study shows that Bangladesh surface water is a reservoir of diverse antibiotic resistant E. coli and may play an important role in dissemination of antibiotic resistance genes such as blaCTX-M-15, which appear to be highly mobile. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.diagmicrobio.2013.02.016.
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Notes to Table 1 a Isolates are coded to indicate their origin as follows: the first number (1–8) indicates the sampling site (see Fig. 1). The letter indicates the date of isolation, June 6 (A), July 4 (B), or August 1 (C), 2011. The second number indicates colony 1 or colony 2. b ST and clonal complex (CC) according to MLST data. c All isolates resistant to any antibiotic as indicated (standard nomenclature) are also resistant to ampicillin. Resistance was determined by BD Phoenix and disk diffusion method according to CLSI guidelines (CLSI, M100-S22, 2012). Resistance to CML, TET and KAN were determined by disk diffusion only (not included in BD phoenix panel). d Genes detected were CTX-M-15, blaCTX-M-15 (determined by sequencing); OXA-30, blaOXA-30-like genes; DHA, blaDHA-like genes; CMY-2, blaCMY-2-like genes; TEM, blaTEM genes; (3)-II, aac(3)-II-like genes; A4cr, aacA4cr (simplified name for aac(6')-Ib-cr) (differentiated by mPCR/RLB); B, qnrB gene; S, qnrS gene. e Inc (FIA etc.) = incompatibility type by PBRT; S1 = plasmid sizes (kb) estimated from S1/PFGE; − = no plasmid detected. f All CTX and/or CAZ-resistant isolates had a classical ESBL phenotype on DDST except 6C1 which has a blaCMY-2-like gene that may have masked the ESBL phenotype conferred by blaCTX-M-15 and 1B1, which had a blaDHA-like gene. g Only isolates with no large plasmids evident (by PBRT and/or S1 PFGE) were tested for small plasmids by alkaline lysis: plasmids of ~2, 3, and 8 kb were detected in 3C1 and a single ~4 kb plasmid in 8A2.
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