J Antimicrob Chemother 2011; 66: 512 – 516 doi:10.1093/jac/dkq472 Advance Access publication 15 December 2010
Isolation of fluoroquinolone-resistant O25b:H4-ST131 Escherichia coli with CTX-M-14 extended-spectrum b-lactamase from UK river water Hiran Dhanji1*, Niamh M. Murphy2, Christine Akhigbe3, Michel Doumith1, Russell Hope1, David M. Livermore1 and Neil Woodford1 1
Antibiotic Resistance Monitoring and Reference Laboratory, Microbiology Services—Colindale, Health Protection Agency, 61 Colindale Avenue, London NW9 5EQ, UK; 2Food Water and Environmental Network London Laboratory, Microbiology Services—Colindale, Health Protection Agency, 61 Colindale Avenue, London NW9 5EQ, UK; 3School of Life Sciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK *Corresponding author. Tel: +44-(0)20-8327-6180; Fax: +44-(0)20-8327-6264; E-mail:
[email protected]
Received 25 August 2010; returned 1 November 2010; revised 10 November 2010; accepted 12 November 2010 Objectives: We analysed water sampled from the River Thames in London for Escherichia coli resistant to oxyimino-cephalosporins and/or fluoroquinolones, particularly seeking isolates with CTX-M extended-spectrum b-lactamases (ESBLs) and members of the clinically important O25b:H4-ST131 lineage. Methods: River water was collected from three urban sites on the River Thames by the City of London Port Health Authority on two occasions 1 week apart. Coliforms and E. coli were identified by the Quanti-TrayTM method. Disc susceptibility tests were performed and MICs were determined for E. coli isolates resistant to either ciprofloxacin or cefpodoxime and genetic relatedness was determined by PFGE and real-time PCR. PCR was used for phylogenetic and plasmid typing, to detect antibiotic resistance genes and to detect ISEcp1 upstream of blaCTX-M genes. blaCTX-M alleles were identified by sequencing. Results: The mean E. coli count, as the most probable number, from the first river samples, taken on a falling tide on 23 March 2010, was 4.7×104/100 mL and 30 ciprofloxacin-resistant colonies were isolated. Twenty of the 30 colonies belonged to clone ST131; 10 of these had blaCTX-M-14 whereas the remaining 10 lacked ESBLs. The ST131 isolates represented two different PFGE types. No ciprofloxacin- or cefpodoxime-resistant E. coli were isolated from the second river sample taken at low tide. CTX-M-15, the most common ESBL in clinical E. coli, was not detected in the river samples. Conclusions: Water from the River Thames in West London is contaminated, perhaps transiently, with antibiotic-resistant E. coli belonging to the clinically important O25b:H4-ST131 lineage. Keywords: plasmids, ISEcp1, ESBLs, antibiotic resistance
Introduction Clinical Escherichia coli isolates with CTX-M-15 extendedspectrum b-lactamase (ESBL) encoded by multidrug-resistance IncF plasmids have become frequent in the UK since 2003.1 Many belong to the internationally disseminated O25b:H4ST131 lineage. This clone, which belongs to phylogenetic group B2,2 is frequently resistant to fluoroquinolones, and is frequent also as a fluoroquinolone-resistant ESBL-negative organism.3 The reasons underlying the successful dissemination of ST131 are unknown, as is the extent to which it has contaminated the wider environment, although it is clear that human carriage is frequent in some groups; thus 40.5% of Belfast care home residents were found to be carriers.4 To explore environmental contamination, we conducted a small study to examine water from
the River Thames in urban London for E. coli producing CTX-M-15 ESBL and belonging to the O25b:H4-ST131 lineage. The Thames potentially has a significant influx of raw sewage contamination when sewage treatment plants are not able to cope with excess surface water due to heavy rainfall.5
Materials and methods Sampling of river water The river water samples were provided to the Food Water and Environmental Network London Laboratory by the City of London Port Health Authority. Six 100 mL midstream samples (two per site) were taken on 23 March 2010 between 09: 00 and 10: 00 h from Pu tney, Barnes and Kew Bridges, all in suburban West London and on the uppermost part
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of the river to be affected by tides, with six further, similar samples taken from the same sites between 09:00 and 10:00 h on 30 March 2010.
Identification of coliforms and E. coli The 12×100 mL samples of river water were tested for the presence of E. coli with total coliform count determined according to National Standard Method W18.6 Each river water sample was tested using the Quanti-TrayTM system (Idexx Laboratories, Suffolk, UK), which is a multiwell most-probable-number (MPN) method using O-nitrophenylb-D-galactopyranoside and 4-methyl-umbelliferyl-b-D-glucuronide as growth indicators. Twelve Quanti-Trays, each containing 100 mL of river water, were incubated at 378C for 18– 22 h and were then read; wells turned yellow in the presence of coliforms and fluoresced blue under UV light (14VA lamp, 245 nm; Omicron Research, Hungerford, UK), indicating the presence of E. coli. The bacterial count was then estimated based upon the number of positive wells, using standard tables supplied by the manufacturer.
Antibiotic susceptibility, PCR testing and PFGE From each of the Quanti-Trays (one per sample) a 100 mL aliquot from a single fluorescent well (chosen randomly by eye) was withdrawn and spread onto Iso-Sensitest agar with ciprofloxacin (1 mg) and cefpodoxime (10 mg) discs according to BSAC methodology.7 Colonies growing within the inhibition zones were confirmed as E. coli based upon a positive indole test and production of gas when incubated in Brilliant Green Bile broth at 448C for 24 h. MICs for resistant E. coli isolates were determined by BSAC agar dilution.8 PCR was used to seek antibiotic resistance genes, including blaCTX-M,9 blaTEM-1 and aac(3)-IIa,10 and also to assign E. coli isolates to phylogenetic groups.11 The relatedness of resistant isolates was assessed by PFGE of XbaI-digested genomic DNA.1 Isolates belonging to phylogenetic group B2 were screened by real-time PCR to identify members of the ST131 clone.12
Genetic environment of blaCTX-M and plasmid transfer studies Primers ISEcp1 for 5′ -CCT AGA TTC TAC GTC AGT ACT TCA AAA A-3 and CTX-M-9rev 5′ -TTA CAG CCC TTC GGC GAT GAT TC-3′ were used to detect the presence of ISEcp1 upstream of group 9 blaCTX-M alleles by PCR using the following cycling conditions: initial denaturation for 5 min at 958C; 30 cycles of 20 s at 958C, 40 s at 608C and 3 min at 728C; followed by 5 min at 728C. The same primers were employed to sequence group 9 blaCTX-M alleles using an ABI Genetic Analyser capillary platform 3130XL (Applied Biosystems, CA, USA). Plasmids encoding CTX-M ESBL were transferred by plate mating to rifampicin-resistant E. coli K12 J53-2 or, where this conjugation failed, by electroporation into E. coli DH5a (Bioline, London, UK) according to the supplier’s instructions. Transformants were selected on nutrient agar supplemented with 2 mg/L cefotaxime, with further addition of rifampicin 250 mg/L (Sigma, Poole, UK) to select for transconjugants. Transferred plasmids were extracted using an alkaline lysis method and assigned to plasmid incompatibility groups by PCR.13
from fluorescent Quanti-Tray wells containing river water collected on this date were inoculated on to Iso-Sensitest agar and tested for ciprofloxacin and cefpodoxime susceptibility; 20 colonies resistant to ciprofloxacin were obtained along with another 10 resistant to cefpodoxime. These latter organisms proved resistant also to ciprofloxacin. The mean coliform count present among the six river water samples collected 1 week later (30 March) from the same three river sites was only 4.5×104 MPN/100 mL, with an E. coli count of 3.5×103 MPN/100 mL, and no colonies resistant to ciprofloxacin or cefpodoxime were isolated from the six 100 mL aliquots tested.
Molecular epidemiology The 30 E. coli colonies that were resistant to cefpodoxime and/or ciprofloxacin in disc tests all belonged to the extra-intestinal virulent phylogenetic group B2. Twenty, including all 10 cefpodoxime-resistant colonies, belonged to the uropathogenic ST131 clone as confirmed using the ST131 real-time PCR assay.12 PFGE analysis, based on ≥85% banding pattern similarity, grouped the 30 isolates into three clusters: cluster 1 included the 10 cefpodoxime- and ciprofloxacin-resistant ST131 isolates; cluster 2 included the 10 ST131 ciprofloxacinresistant, cefpodoxime-susceptible isolates; and cluster 3 included the 10 non-ST131 ciprofloxacin-resistant, cefpodoximesusceptible isolates (Figure 1). Isolates belonging to clusters 1 and 2 shared 78% banding pattern similarity, consistent with their ST131 status. None of the isolates belonged to the PFGEdefined variants of ST131 prevalent in the UK, namely strains A, C and D.1
Antibiotic susceptibility and resistance genes All 30 selected colonies were highly resistant to ciprofloxacin (MIC .8 mg/L) (Table 1) and to ampicillin (MIC .64 mg/L), with blaTEM detected by PCR. All were susceptible to piperacillin/ tazobactam (2 –8 mg/L), carbapenems (0.06– 0.5 mg/L), amikacin (2 –8 mg/L), minocycline (1 –4 mg/L), colistin (,0.5– 2 mg/L) and tigecycline (,0.25 mg/L). The 10 ST131 isolates with blaCTX-M-14 were highly resistant also to cefotaxime and cefpirome (MIC 16 –64 mg/L), whereas MICs of ceftazidime were only 2 –4 mg/L. The organisms thus exhibited a typical CTX-M phenotype with synergy between cephalosporins and clavulanic acid. All 10 harboured an intact copy of ISEcp1 42 bp upstream of blaCTX-M-14. They were susceptible to gentamicin and tobramycin, whereas the 10 ESBLnegative ST131 colonies were resistant to these aminoglycosides and harboured an aac(3)-IIa acetyltransferase gene (Table 1). The 10 non-ST131 colonies were fully susceptible to both oxyimino-cephalosporins and aminoglycosides (Table 1).
Plasmid transfer studies
Results Isolation of coliforms and E. coli in river water The mean coliform count present among the six river water samples collected on 23 March 2010 was 1.0×106 MPN/100 mL, with an E. coli count of 4.7×104 MPN/100 mL. Six 100 mL aliquots
blaCTX-M-14 could not be transferred by conjugation, but was transformed from each of the 10 cefpodoxime-resistant colonies by electroporation. It was located on plasmids of 100 kb. The transformants exhibited a typical CTX-M phenotype, and PCR rep typing showed that blaCTX-M-14 was located on IncFIA-FIB plasmids in all cases.
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gentamicin/ tobramycin susceptibilities
8
+ve
+ve
S
9
+ve
+ve
S
6
+ve
+ve
S
+ve
+ve
S
+ve
+ve
S
2
+ve
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S
3
+ve
+ve
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4
+ve
+ve
S
1
+ve
+ve
S
5
+ve
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S
14
+ve
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R
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+ve
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15
+ve
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17
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R
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12
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–ve
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13
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–ve
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29
–ve
–ve
S
30
–ve
–ve
S
28
–ve
–ve
S
21
–ve
–ve
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23
–ve
S
26
Cluster 3 –ve –ve
–ve
S
25
–ve
–ve
S
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–ve
–ve
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24
–ve
–ve
S
22
–ve
–ve
S
100
85% 90
80
70
60
50
40
78%
10 7
11
Cluster 1
Cluster 2
Figure 1. Dendrogram showing the relatedness of the 30 ciprofloxacin-resistant E. coli isolated from the River Thames. S, susceptible; R, resistant. Table 1. Antibiotic resistance profiles of E. coli colonies isolated from the River Thames CTX-M-14 ESBL-producing ST131 (n¼10) MIC breakpoint (mg/L) Antibiotic Ciprofloxacin Cefotaxime Cefotaxime+clavulanate Ceftazidime Ceftazidime+clavulanate Gentamicin Tobramycin
R.
I
1 2 1 8 1 4 4
1 2 — 2– 8 — 4 4
R, resistant; I, intermediate; S, susceptible.
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S≤
ESBL-non-producing ST131 (n¼10)
number MIC range
0.5 .8 1 256 1 0.06– 0.125 1 2– 4 1 0.25– 0.5 2 1– 2 2 1– 2
R
I
10 10 0 0 0 0 0
0 0 0 10 0 0 0
ESBL-non-producing non-ST131 (n¼10)
number S
MIC range
0 .8 0 0.125–1 10 0.06 0 0.25– 2 10 0.125–0.25 10 32 10 32
R
I
10 0 0 0 0 10 10
0 0 0 0 0 0 0
number S
MIC range
0 .8 10 0.125 10 0.06 10 0.25 –0.5 10 0.125– 0.25 0 1 –2 0 1 –2
R
I
S
10 0 0 0 0 0 0
0 0 0 0 0 0 0
0 10 10 10 10 10 10
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Discussion We have shown that fluoroquinolone-resistant E. coli could be recovered from the River Thames in London. The isolates included representatives of the uropathogenic ST131 lineage harbouring blaCTX-M-14 on IncF type plasmids, and those resistant to gentamicin and tobramycin. We did not isolate any E. coli with CTX-M-15 ESBL, which is the clinically dominant ESBL in this and other clones in the UK;1 nevertheless, CTX-M-14 ESBL too is common, occurring in 10% of clinical ESBL E. coli in the UK.13 It is also the dominant ESBL in Spain and China and is globally the second most prevalent CTX-M ESBL after CTX-M-15.14 Clinical E. coli belonging to ST131 harbouring CTX-M-14 are frequent in Canada.15 E. coli producing CTX-M-14 ESBL harboured ISEcp1 upstream of blaCTX-M-14. This insertion sequence has been reported to facilitate the mobilization of blaCTX-M genes.14 Interestingly all the ST131 isolates lacking ESBL were resistant to gentamicin and tobramycin, whereas those with CTX-M-14 enzyme were susceptible. The subgroups of ST131 isolates formed distinct clusters by PFGE, although they were related at 78% banding pattern similarity. It should be added that the sampling strategy, which examined 100 mL river water samples taken from just one positive well from each of 12 Quanti-Trays, almost certainly underestimated the diversity of resistant clones and variants present in the river water and was biased towards the repeated recovery of just a few types, and therefore is unlikely to be a true representation of strain and resistance gene diversity in the River Thames; this requires larger-scale sampling. Antibiotic-resistant bacteria have previously been recovered from rivers in China, France16,17 and the USA18 and have harboured diverse genes on mobile genetic elements, including blaTEM, blaSHV, blaOXA-23, blaIMI-2, drfA, aadA, aac(6)-Ib, aac(3)-IIa, tet(A)/(B)/(C)/(D), aadB, cmlA and arr3, variously conferring resistance to aminoglycosides, fluoroquinolones, tetracyclines, b-lactams (including carbapenems), trimethoprim, chloramphenicol and rifampicin. We sampled river water from the same three locations on two occasions 1 week apart (23 and 30 March), but isolated ciprofloxacin- and cefpodoxime-resistant E. coli only on 23 March, when they were recovered at all three sampling sites. There could be many explanations for this difference, including (i) sensitivity of sampling, since we tested water only from one positive well within each Quanti-Tray; (ii) the level of rainfall during the days prior to collection, which might influence the level of raw sewage contamination and/or agricultural run-off into the Thames; and (iii) tidal conditions at the time of collection. There were no significant differences in rainfall during the days prior to sampling; i.e. average rainfall between 17 and 23 March was 0.06 inches and between 24 and 30 March it was 0.09 inches.19 However, there was a difference in the tides. On 23 March the tide was high at 07:13 h (4.3 m) and low at 14:42 h (0.2 m), whereas on 30 March the tide was high at 02:39 h (4.8 m) and low at 10:00 h (0.2 m).20 Thus, the former samples were collected on a falling tide and the latter almost exactly at low tide. High tides might wash contaminants towards the sampling areas and would allow intertidal sediments to become resuspended in the water. A previous study conducted by Boehm et al.21 found that tides impacted on bacterial concentrations recovered from river water. The river shores
associated with the collection sites are vegetated and gently slope towards the water, allowing waste from the towpath and vegetation to enter the river. Potential contamination from river wildlife and companion pets such as dogs is also possible because antibiotic-resistant E. coli, including members of the ST131 clone, have been isolated from seagull, rat and dog faeces.22 – 24 In summary, we have shown that the River Thames in suburban London is contaminated, perhaps transiently, with antibiotic-resistant E. coli belonging to the O25b:H4-ST131 clone. Some isolates harboured CTX-M-14 enzyme but none was found to carry CTX-M-15, which is the most common ESBL among clinical E. coli in the UK. Consequently, the implications of our findings are not straightforward in relation to public health; nevertheless, our study does highlight the need for more extensive investigation of the potential role of rivers and surface waters in the dissemination of antibiotic resistance genes and successful E. coli clones in the UK.
Acknowledgements We wish to thank the City of London Port Health Authority for collection of samples.
Funding No external funding sources.
Transparency declarations N. W. and D. M. L. have independently accepted research grants and speaking invitations from various pharmaceutical companies, though none poses a conflict of interest with the work presented here. D. M. L. has shares in GlaxoSmithKline, Merck, Pfizer and Dechra, and, as enduring attorney, manages holdings in Eco Animal Health and GlaxoSmithKline within diversified portfolios. All other authors have no interests to declare.
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