The Gull ( Chroicocephalus brunnicephalus ) as an Environmental Bioindicator and Reservoir for Antibiotic Resistance on the Coastlines of the Bay of Bengal

Abstract

The presence and frequency of multiresistant bacteria in wild birds act as indicators of the environmental contamination of antibiotic resistance. To explore the rate of contamination mediated by Escherichia coli, 150 fecal samples from the brown-headed gull (Chroicocephalus brunnicephalus) and 8 water samples from the Bay of Bengal area were collected, cultured, and tested for antibiotic susceptibility. Special attention was paid to extended-spectrum beta-lactamase (ESBL)-producing isolates, which were further characterized genetically. Antibiotic resistance was found in 42.3% (36/85) of the E. coli isolates and multidrug resistance in 11.8%. Isolates from the area with a higher human activity were more resistant than those from an area with a lower level of activity. Most frequent was resistance to ampicillin (29.4%), followed by trimethoprim-sulfamethoxazole (24.7%) and quinolones (22.4%). Carriage of ESBL-producing E. coli was relatively high (17.3%) in the gulls, whereas no ESBL producers were found in the water. All ESBL-producing E. coli isolates, but one, carried bla_CTX-M-15 or bla_CTX-M-15-like genes. A bla_CTX-M-14-like enzyme was found as an exception. Gulls from two different colonies shared E. coli clones and harbored the clinically relevant sequence types ST10, ST48, and ST131. The high frequency of antibiotic resistance and ESBL production among E. coli isolates from gulls indicates that the environmental contamination of antibiotic resistance has already gone far on the coastlines of the Bay of Bengal. Considering the limited control over the antibiotic consumption and waste from human activities in Bangladesh, there is no easy solution in sight.

Introduction

The emergence and rapid dissemination of resistance against antibiotics is a worldwide problem. The overuse of antibiotics is one of the main sources behind the emergence of antibiotic resistance,²³ and through the selective pressure, the transmission of plasmids with resistance genes has become much quicker than the rate at which new antibiotics are developed. Various gram-negative bacteria harbor beta-lactamases in their chromosome. In some instances, genes encoding beta-lactamases with a broader spectrum have been mobilized and successfully transmitted to different bacterial species, such as TEM, SHV, CTX-M,⁵ and, most recently, NDM. The latter enzyme hydrolyzes not only penicillins and cephalosporins but also carbapenems,²⁰ and is a serious human threat and medical challenge. The first extended-spectrum beta-lactamase (ESBL) of the CTX-M type was reported in a clinical setting in Germany during 1989.² Within a few years, it had spread widely into different bacterial populations, becoming the most frequent ESBL type in hospitals, domesticated animals, the environment, and wildlife^3,6,15

Antibiotic resistance is a major problem in the clinical, veterinary, and environmental settings of Bangladesh.^13–15 ESBL-producing Escherichia coli and Klebsiella pneumoniae are highly prevalent in healthcare settings,²⁶ and E. coli isolates from commercial or free-range poultry and wild ducks tend to carry antibiotic-resistant phenotypes and genotypes associated with humans.^14,15 Migratory birds like seagulls have been reported as potential reservoirs and vectors of human-associated ESBL-producing bacteria in France³ and Portugal.²⁵ They may also reflect the spectrum of environmental antibiotic resistance.^15,28 The brown-headed gull (Chroicocephalus brunnicepahalus), known as an opportunistic and adaptive feeder, migrates between Tajikistan, Southern China, Pakistan, India, Bangladesh, Myanmar, Sri Lanka, Vietnam, and Thailand. It has a wider presence in the environmental matrix than other wild avian species with a migratory behavior.

The aim of this study was to investigate the frequency of antibiotic resistance among E. coli isolates derived from brown-headed gulls and the surrounding natural water reserves to gain more detailed knowledge about the environmental contamination of multiresistant bacteria in the Bay of Bengal area. Special attention was paid to ESBL-producing isolates, which were further characterized.

Materials and Methods

Collection of samples

From January to February 2010, a total of 150 fecal samples from brown-headed gulls were collected from Kuakata (n=97) and Cox's Bazar (n=53) beaches of Bangladesh. The distance between two beaches was more than 250 km. Of the two beaches, Cox's Bazar is associated with the highest level of human activities like tourism, medical, livestock, aquaculture, and pharmaceutical industries. Fresh fecal droppings were collected by sterile cotton swabs, which were immediately stored at −80°C in bacterial freeze media containing the Luria-Bertani broth (Becton, Dickinson and Company, Sparks, MD), phosphate-buffered saline, and 4.4% glycerol. In addition, water samples were obtained. Four water samples were collected from the Bay of Bengal in the Cox's Bazar beach area, and another four from the Karnafuli River, the closest major river where sea gulls visit regularly. Forty milliliters of sampled water was mixed with 10 ml of bacterial freeze media and immediately stored at −80°C. All samples were thereafter shipped to Sweden for analysis.

Isolation of E. coli with and without ESBL production

The fecal samples were plated on Uriselect 4 agar plates (Bio-Rad Laboratories Ltd., Hemel Hempstead, United Kingdom) or cysteine lactose-electrolyte-deficient (CLED) agar (Lab M Ltd., Lancashire, United Kingdom). The water samples were thawed, vortexed, and filtered (35 ml) through Millipore filters (0.45 μM; SAS, Molsheim, France). The filters were placed on CLED agar. Finally, each fecal sample and the remaining 15 ml of each water sample were enriched in a brain-heart infusion broth (Becton Dickinson, Franklin Lakes, NJ) supplemented with vancomycin (16 mg/L; ICN Biomedicals, Inc., Aurora, OH) for 18 hr at 37°C. The broth was thereafter inoculated onto chromID™ ESBL plates (bioMérieux SA, Marcy-l'Etoile, France) with sterile cotton swabs. ESBL production from growing colonies was confirmed with the cefpodoxime/cefpodoxime+clavulanic acid double disk test (MAST Diagnostics, Bootle, United Kingdom).All plates were inoculated overnight at 37°C, and identification to the species level was performed by conventional biochemical tests or API 20E biochemical strips (bioMérieux SA).

Antibiotic susceptibility testing

Resistance to antibiotics was determined by disk diffusion according to the recommendation of the European Committee on Antimicrobial Susceptibility Testing.¹¹ Only one E. coli isolate per sample was tested unless there was an ESBL producer in the sample. A panel of antibiotics that essentially mirror the antibiotic consumption in aquaculture, human, and veterinary medicine in Bangladesh were used: ampicillin, cefadroxil, cefuroxime, mecillinam, trimethoprim-sulfhamethoxazole, nalidixic acid, ciprofloxacin, chloramphenicol, gentamicin, nitrofurantoin, and tigecycline. E. coli ATCC 25922 was included as a quality control strain. Isolates with resistance to three or more classes of antibiotics were considered to be multidrug resistant (MDR).

Genetic characterization of ESBL producers

The presence of bla_CTX-M genotypes was detected by using an earlier described polymerase chain reaction (PCR) method.²⁴ Positive control strains for CTX-M-I and CTX-M-IV group enzymes were kindly provided by David Livermore and Neil Woodford (London, United Kingdom). Sequencing of genes encoding CTX-M-type enzymes was carried out as described previously.¹⁹

Epidemiological typing

The genetic profiles of the ESBL-producing E. coli isolates were determined by repetitive element PCR (rep-PCR). Bacterial DNA was prepared from overnight cultures by heating the bacterial suspension to 95°C for 10 min. followed by centrifugation. Amplification was conducted in a total volume of 25 μl containing 2 mM dNTP, 10× PCR buffer, 25 mM MgCl₂, 5 U/μl of HotStar taq polymerase (QIAGEN GmbH, Hilden, Germany), and primer ERIC1R (5′-ATGTAAGCTCCTGG GGATTCAC-3′) and 5 μl of the DNA template. Cycling parameters were as follows: 1 min at 94°C, 1 min at 36°C, and 2 min at 72°C for 45 cycles. A final extension at 72°C for 5 min was performed afterward. The amplified products were visualized in a 1% agarose gel stained with ethidium bromide. To control the sizes of the products, an express DNA ladder was used (GeneRuler DNA Ladder; Fermentas, Hanover, MD). The gel was photographed and the DNA bands were analyzed visually. Isolates differing by one strong band or more were assigned to different genotypes,²⁹ whereas isolates differing with one weak band from their genotype were assigned different subtypes.

Multilocus sequence typing

A representative from each rep-PCR genotype was characterized by multilocus sequence typing (MLST), using specified primers for seven standard housekeeping genes (adk, fumC, gyrB, icd, mdh, purA, and recA) and an earlier described protocol.³⁰ Cycling parameters were as follows: 30 sec at 95°C, 30 sec at 60°C, and 1.5 min at 72°C for 30 cycles. A final extension at 72°C for 5 min was performed afterward. The PCR products were purified using a PCR purification kit (Fermentas, St. Leon-Rot, Germany). PCR products were sequenced at Eurofins MWG Operon, Germany and allele profiles and sequence types (STs) were determined using the E. coli MLST database (http://mlst.ucc.ie/mlst/dbs/Ecoli/#). The E. coli ST131 isolate identified by MLST analysis was also run in an O25b-ST131 PCR assay, as described previously.⁸

Results

E. coli isolates

The 150 bird samples yielded a total of 85 E. coli isolates when there was no enrichment or selective pressure. The isolation rate was thereby 56.7%. Fifty-nine of the isolates originated from the Kuakata beach and 26 from the Cox's Bazar beach. Of the eight water samples, E. coli was only recovered from the four samples from the Karnafuli River.

Antibiotic susceptibility of E. coli not selected for ESBL production

Resistance to all antibiotics except gentamicin and tigecycline was noted. Nearly half (36 out of 85, 42.3%) of the E. coli isolates from gulls were found resistant to one or more antibiotics. MDR was found in 10 isolates (11.8%). The most widely spread resistance was to ampicillin (29.4%), followed by resistance to trimethoprim- sulfamethoxazole (24.7%), nalidixic acid (22.4%), and ciprofloxacin (16.5%). Lower frequencies of resistance were found to chloramphenicol (2.4%), nitrofurantoin (3.5%), cefuroxime (2.4%), cefadroxil (2.4%), and mecillinam (1.2%). E. coli isolates resistant to cephalosporines were confirmed ESBL producers. There was a difference in resistance frequency between the two gull colonies: 18 of 26 isolates (69.2%) from Cox's Bazar were resistant (see Table 1), whereas the corresponding figure for Kuakata was 18 of 59 (30.5%). Furthermore, seven MDR isolates were isolated from Cox's Bazar and only three MDR isolates from Kuakata. Of the four E. coli isolates derived from water, two were resistant to ampicillin and trimethoprim-sulfamethoxazole. One of these two was, in addition, resistant to ciprofloxacin.

Table 1.

Antibiotic Resistance in Escherichia coli from Brown-Headed Gulls Visiting Kuakata and Cox's Bazar Beach Areas

Name of the antibiotic	Kuakata (n=59)	Cox's bazar (n=26)
Ampicillin	11	14
Cefuroxime	0	2
Cefadroxil	0	2
Mecillinam	1	0
Trimethoprim-sulfamethoxazole	10	11
Nalidixic acid	9	10
Ciprofloxacin	5	9
Chloramphenicol	2	0
Nitrofurantoin	2	1
Gentamicin	0	0
Tigecycline	0	0

Antibiotic susceptibility of E. coli isolates selected for ESBL production

There were no ESBL producers in the water samples. The ESBL-producing E. coli isolates from the gulls displayed resistance to all antibiotics but three: mecillinam, nitrofurantoin, and tigecycline. All isolates were completely resistant to ampicillin and the included cephalosporins. Additional resistance was exhibited to nalidixic acid (n=11, 37.9%), trimethoprim-sulfamethoxazole (n=9, 31%), ciprofloxacin (n=9, 24.1%), chloramphenicol (n=2; 6.8%), and gentamicin (n=2, 6.8%). The diversity of antibiotic-resistant profiles of the ESBL-producing isolates is shown in Table 2.

Table 2.

Characterization of Extended-Spectrum Beta-Lactamase-Producing Escherichia coli Isolated from Brown-Headed Gulls Visiting Two Different Beach Areas in Bangladesh

Sample	Location	ESBL-type	Antibiotic resistance profile	Rep-PCR genotype	Sequence type
110547	Kuakata	CTX-M-15	AMP-CFR-CXM	D	ST1727
110552	Kuakata	CTX-M-15 like	AMP-CFR-CXM	B
110553	Kuakata	CTX-M-15	AMP-CFR-CXM-NA-CIP-CN	C	ST648
110555	Kuakata	CTX-M-15	AMP-CFR-CXM	A	ST2687^a
	Kuakata	CTX-M-15	AMP-CFR-CXM	B
110558	Kuakata	CTX-M-15 like	AMP-CFR-CXM-NA-CIP	C
110559	Kuakata	CTX-M-15	AMP-CFR-CXM-NA-CIP-SXT	A1^b	ST853
	Kuakata	CTX-M-15	AMP-CFR-CXM	A
110564	Kuakata	CTX-M-15	AMP-CFR-CXM	A
110579	Kuakata	CTX-M-15	AMP-CFR-CXM-SXT	A3^b	ST48 (ST48 complex)
110582	Kuakata	CTX-M-15	AMP-CFR-CXM	B	ST2688^a
110583	Kuakata	CTX-M-15	AMP-CFR-CXM-NA-SXT-C	A
110586	Kuakata	CTX-M-15	AMP-CFR-CXM	B
110587	Kuakata	CTX-M-15	AMP-CFR-CXM	A
110593	Kuakata	CTX-M-15	AMP-CFR-CXM	A2^b	ST2689^a
110596	Kuakata	CTX-M-15	AMP-CFR-CXM	A
110606	Kuakata	CTX-M-15	AMP-CFR-CXM	A
110616	Kuakata	CTX-M-15	AMP-CFR-CXM-NA-CIP-SXT	A
110629	Kuakata	CTX-M-15	AMP-CFR-CXM	A
110632	Kuakata	CTX-M-15	AMP-CFR-CXM	A2^b
110899	Cox's bazar	CTX-M-15	AMP-CFR-CXM-NA-SXT	E	ST349 (ST349 complex)
110903	Cox's bazar	CTX-M-15	AMP-CFR-CXM	A
110908	Cox's bazar	CTX-M-15	AMP-CFR-CXM	A
	Cox's bazar	CTX-M-14 like	AMP-CFR-CXM-NA-CIP-SXT	F	ST131
110920	Cox's bazar	CTX-M-15	AMP-CFR-CXM-NA-SXT	A
110924	Cox's bazar	CTX-M-15	AMP-CFR-CXM-NA-CIP-SXT	C1^b	ST345 (ST345 complex)
110934	Cox's bazar	CTX-M-15 like	AMP-CFR-CXM-NA	A
110949	Cox's bazar	CTX-M-15 like	AMP-CFR-CXM	A	ST10 (ST10 complex)
	Cox's bazar	CTX-M-15	AMP-CFR-CXM-NA-CIP-SXT-C-CN	C

New type.

Arabic numeral indicates a difference of one weak band.

NA, nalidixic acid; AMP, ampicillin; C, chloramphenicol; CFR, cefadroxil; F, nitrofurantoin; SXT, trimethoprim-sulfamethoxazole; CIP, ciprofloxacin; CN, gentamicin; CXM, cefuroxime; ESBL, extended-spectrum beta-lactamase; PCR, polymerase chain reaction.

Characterization of ESBL-producing bacteria

Thirty-one ESBL-producing members of the Enterobacteriaceae family were obtained from 26 different samples with the selective isolation method. Of these, 29 were E. coli. Twenty-eight E. coli isolates were confirmed to harbor bla_CTX-M-15 or bla_CTX-M-15-like genes. Only one E. coli isolate carried bla_CTX-M-14 (see Table 2 for details). The remaining two consisted of a CTX-M-15-producing K. pneumoniae isolate and a CTX-M-55/79-producing Escherichia albertii isolate. The rep-PCR yielded six genotypes (Table 2), and almost half (n=14) of the ESBL-producing E. coli isolates belonged to genotype A. Birds from the two beaches shared genotypes A and C. Both these genotypes had subtypes. MLST was performed on isolates from both beach areas showing different rep-PCR profiles. The 11 different genotypes/subtypes generated the same number of STs (Table 2). Three isolates had new combinations of allele types and were given the novel STs 2687–2689. The isolate from the Cox's Bazar beach area with production of a CTX-M-14-like enzyme belonged to the O25b-ST131 clone.

Discussion

The present study explored the rate of environmental contamination of antibiotic resistance mediated by E. coli in gulls and water samples from two different beach areas with different levels of human activity. The study showed that the environmental contamination has already extended over some distance in these beach areas: over 40% of the E. coli isolates from gulls were resistant to one or more of the tested compounds, and the number of MDR isolates exceeded 10%. Compared with other gull studies from the Czech Republic, France, and Sweden,^3,4,9 the antibiotic resistance was at a higher level and was common and more diverse. Furthermore, the number of ESBL-producing isolates was relatively high, and they carried two of the currently and globally most successful types of ESBLs, that is, CTX-M-15 and CTX-M-14. The antibiograms of some of these isolates suggested a risk of therapeutic failure with several of the commonly used empiric drugs for human gram-negative infections.

The brown-headed gull harbored predominantly bla_CTX-M-15. Several gull species, including black-headed gulls (Chroicocephalus ridibundus), yellow-legged gulls (Larus michahellis), and glaucous-winged gulls (Larus glaucescens), from different countries have been reported to be carriers of this globally endemic genotype.^3,4,16 CTX-M-15 is also widely distributed in clinical settings of neighboring India¹⁰ and Thailand.¹ Some birds had picked up more than one ESBL-producing strain. Most extreme in this context was the gull that simultaneously carried two different E. coli isolates with bla_CTX-M-15 and bla_CTX-M-14 genotypes. K. pneumoniae, an additional species carried a bla_CTX-M-15 genotype. Apart from E. coli and K. pneumonie, one more species carried ESBL. This was E. albertii. Of all the ESBL producers, only the E. albertii isolate carried a CTX-M-type typical for Asia, CTX-M-55/79, which has been isolated from humans, poultry, and fish.^17,21,31

Wild birds, such as gulls, are hardly ever treated with antibiotics. The resistance in their fecal flora can exhibit therefore only a mirror of what they come in contact with through their environment. Interestingly, the resistance phenotypes of E. coli isolates from gulls were similar to earlier studied isolates from both poultry and wild ducks in Bangladesh,^14,15 indicating a certain proximity to human activities for both domesticated and wild birds. The human influence on the bird flora was made obvious when the two beaches were compared with each other. The Cox's Bazar area where more antibiotic-resistant Enterobacteriaceae isolates were found in gulls has a higher number of shrimp hatcheries, fish processing plants, touristic activities, hospitals/clinics, poultry farms, and pharmaceutical industries than the Kuakata area.

There was also a frequency difference in antibiotic resistance when it came to the water samples: the fresh water was more polluted with resistant bacteria than the sea water. Considering how the human activities are concentrated to the rivers, and that the dilution capacity of the sea is much greater, this is not surprising. The result suggests, however, that surveillance of wild birds is a better choice than either sea or fresh water, if there is a need for an early indicator of environmental contamination of multiresistant bacteria.

The epidemiological typing of the ESBL-producing E. coli isolates from both colonies showed that there was one dominating cluster, designated genotype A. This genotype together with another genotype (C), indicated that gulls may act not only as bioindicators and reservoirs but also as vectors. However, in the genotype A cluster, two human-associated STs were found (ST10 and ST48), which can be explained by a closer and a repeated contact with human activities instead of an intraspecies spread. ST10 has been reported from several Chinese hospitals,⁷ and ST48 from humans and poultry in Denmark¹⁸ and the Netherlands.²² A bird with the human-associated O25b-ST131 clone was also noted, but there were also more bird-associated STs. For example, ST648 with CTX-M-15, the most common ST and ESBL type among wild birds in a recent German study.¹²

Like other developing countries of the world, the usage of antibiotics is not closely monitored in Bangladesh. In coastal aquacultures, 80% of the hatchery owners have been shown to use antibiotics like chloramphenicol, erythromycin, and tetracycline without prescription or proper clinical indications.²⁷ The corresponding figure for poultry farmers is 83%.¹⁴ Gulls have an opportunistic feeding behavior, and they often collect food from hospital or city dumps, polluted rivers, sewage treatment plants, farms, and shrimp hatcheries. Without doubt, human activities have had a major impact on the intestinal flora of these wild birds.

In conclusion, the present study showed a relatively high frequency of antibiotic resistance and ESBL production among E. coli isolates from gulls. This suggests that the environmental contamination of antibiotic resistance on the coastlines of the Bay of Bengal is a growing problem that must be attended to. Nationwide programs are necessary to control the irrational use of antibiotics and improve the management of sewage and human wastes to prevent increased morbidity and mortality among humans and animals in the future.

Footnotes

Acknowledgments

The authors would like to thank Abbtesaim Jawad, Justin Makii, Bawantee Dudhee, and Dr. Monjurul Islam Talukdar for their technical support during work on the field and in the laboratory. This work was supported by The Swedish Institute (Ref: 00559/2008), the Marcus Borgström Foundation, Lindahls Ester Foundation, Lundells PO foundation, Bergmark Foundation, Olle Engkvist Byggmästare Foundation, and the Medical Faculty of Uppsala University and Karin Korsner's foundation. Munirul Alam from ICDDR, B also assisted the author in the publication.

Disclosure Statement

No competing financial interests exist.

References

Archambault

, Petrov

, Hendriksen

R.S.

, Asseva

, Bangtrakulnonth

, Hasman

, and Aarestrup

F.M.

. 2006. Molecular characterization and occurrence of extended-spectrum beta-lactamase resistance genes among Salmonella enterica serovar Corvallis from Thailand, Bulgaria, and Denmark. Microb. Drug Resist. (Larchmont, NY), 12:192–198.

Bauernfeind

, Grimm

, and Schweighart

1990. A new plasmidic cefotaximase in a clinical isolate of Escherichia coli. Infection, 18:294–298.

Bonnedahl

, Drobni

, Gauthier-Clerc

, Hernandez

, Granholm

, Kayser

, Melhus

, Kahlmeter

, Waldenstrom

, Johansson

, et al. 2009. Dissemination of Escherichia coli with CTX-M type ESBL between humans and yellow-legged gulls in the south of France. PLoS One, 4:e5958.

Bonnedahl

, Drobni

, Johansson

, Hernandez

, Melhus

, Stedt

, Olsen

, and Drobni

2010. Characterization, and comparison, of human clinical and black-headed gull (Larus ridibundus) extended-spectrum beta-lactamase-producing bacterial isolates from Kalmar, on the southeast coast of Sweden. J. Antimicrob. Chemother., 65:1939–1944.

Bonnet

2004. Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes. Antimicrob. Agents Chemother., 48:1–14.

Canton

, and Coque

T.M.

2006. The CTX-M beta-lactamase pandemic. Curr. Opin. Microbiol., 9:466–475.

Cao

, Cavaco

L.M.

, Lv

, Li

, Zheng

, Wang

, Hasman

, Liu

, and Aarestrup

F.M.

2011. Molecular characterization and antimicrobial susceptibility testing of Escherichia coli isolates from patients with urinary tract infections in 20 Chinese hospitals. J. Clin. Microbiol., 49:2496–2501.

Clermont

, Dhanji

, Upton

, Gibreel

, Fox

, Boyd

, Mulvey

M.R.

, Nordmann

, Ruppe

, Sarthou

J.L.

, et al. 2009. Rapid detection of the O25b-ST131 clone of Escherichia coli encompassing the CTX-M-15-producing strains. J. Antimicrob. Chemother. 64:274–277.

Dolejska

, Bierosova

, Kohoutova

, Literak

, and Cizek

2009. Antibiotic-resistant Salmonella and Escherichia coli isolates with integrons and extended-spectrum beta-lactamases in surface water and sympatric black-headed gulls. J. Appl. Microbiol., 106:1941–1950.

10.

Ensor

V.M.

, M. Shahid

J.T

. Evans, and Hawkey

P.M.

. 2006. Occurrence, prevalence and genetic environment of CTX-M beta-lactamases in Enterobacteriaceae from Indian hospitals. J. Antimicrob. Chemother., 58:1260–1263.

11.

European Committee on Antimicrobial Susceptibility Testing (EUCAST). Available at www.eucast.org (Online, accessed December 31, 2012)

12.

Guenther

, Grobbel

, Beutlich

, A. Bethe

N.D.

, Friedrich , Goedecke

, Lubke-Becker

, Guerra

, Wieler

L.H.

, and Ewers

. 2010. CTX-M-15-type extended-spectrum beta-lactamases-producing Escherichia coli from wild birds in Germany. Environ. Microbiol. Rep., 2:641–645.

13.

Hasan

, Drobni

, Alam

, and Olsen

2012. Dissemination of NDM-1. Lancet Infect. Dis., 12:99–100; author reply 101–102.

14.

Hasan

, Faruque

, Drobni

, Waldenstrom

, Sadique

, Ahmed

K.U.

, Islam

. Parvez

M.B.

, Olsen

, and Alam

. 2011. High prevalence of antibiotic resistance in pathogenic Escherichia coli from large- and small-scale poultry farms in Bangladesh. Avian Dis., 55:689–692.

15.

Hasan

, Sandegren

, Melhus

, Drobni

, Hernandez

, Waldenstrom

, Alam

, and Olsen

2012. Antimicrobial drug-resistant Escherichia coli in wild birds and free-range poultry, Bangladesh. Emerg. Infect. Dis., 18:2055–2058.

16.

Hernandez

, Bonnedahl

, Eliasson

, Wallensten

, Comstedt

, Johansson

, Granholm

, Melhus

, Olsen

, and Drobni

2010. Globally disseminated human pathogenic Escherichia coli of O25b-ST131 clone, harbouring blaCTX-M-15, found in Glaucous-winged gull at remote Commander Islands, Russia. Environ. Microbiol. Rep., 2:329–332.

17.

Jiang

H.X.

, Tang

, Liu

Y.H.

, Zhang

X.H.

, Zeng

Z.L.

, Xu

, and Hawkey

P.M.

. 2012. Prevalence and characteristics of beta-lactamase and plasmid-mediated quinolone resistance genes in Escherichia coli isolated from farmed fish in China. J. Antimicrob. Chemother., 67:2350–2353.

18.

Jorgensen

R.L.

, Nielsen

J.B.

, Friis-Moller

, Fjeldsoe-Nielsen

, and Schonning

. 2010. Prevalence and molecular characterization of clinical isolates of Escherichia coli expressing an AmpC phenotype. J. Antimicrob. Chemother., 65:460–464.

19.

Klint

, Lofdahl

, Ek

, Airell

, Berglund

, and Herrmann

2006. Lymphogranuloma venereum prevalence in Sweden among men who have sex with men and characterization of Chlamydia trachomatis ompA genotypes. J. Clin. Microbiol., 44:4066–4071.

20.

Kumarasamy

K.K.

, Toleman

M.A.

, Walsh

T.R.

, Bagaria

, Butt

, Balakrishnan

, Chaudhary

, Doumith

, Giske

C.G.

, Irfan

, et al. 2010. Emergence of a new antibiotic resistance mechanism in India, Pakistan, and the UK: a molecular, biological, and epidemiological study. Lancet Infect. Dis., 10:597–602.

21.

Lee

, Chung

H.S.

, Lee

, Yum

J.H.

, Yong

, Jeong

S.H.

, Lee

, and Chong

. 2013. CTX-M-55-type extended-spectrum beta-lactamase-producing Shigella sonnei isolated from a Korean patient who had travelled to China. Ann. Lab. Med., 33:141–144.

22.

Leverstein-van Hall

M.A.

, Dierikx

C.M.

, Cohen Stuart

, Voets

G.M.

, van den Munckhof

M.P.

, van Essen-Zandbergen

, Platteel

, Fluit

, van de Sande-Bruinsma

, Scharinga

, et al. 2011. Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains. Clin. Microbiol. Infect., 17:873–880.

23.

Martinez

J.L.

2008. Antibiotics and antibiotic resistance genes in natural environments. Science (New York, NY), 321:365–367.

24.

Pitout

J.D.

, Hossain

, and Hanson

N.D.

. 2004. Phenotypic and molecular detection of CTX-M-beta-lactamases produced by Escherichia coli and Klebsiella spp. J. Clin. Microbiol., 42:5715–5721.

25.

Poeta

, Radhouani

, Igrejas

, Goncalves

, Carvalho

, Rodrigues

, Vinue

, Somalo

, and Torres

2008. Seagulls of the Berlengas natural reserve of Portugal as carriers of fecal Escherichia coli harboring CTX-M and TEM extended-spectrum beta-lactamases. Appl. Environ. Microbiol., 74:7439–7441.

26.

Rahman

M.M.

, Haq

J.A.

, Hossain

M.A.

, Sultana

, Islam

, and Islam

A.H.

. 2004. Prevalence of extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae in an urban hospital in Dhaka, Bangladesh. Int. J. Antimicrob. Agents, 24:508–510.

27.

Sheikh

, and Abdul

2006. The use of antibiotics in shrimp hatcheries in Bangladesh. J. Fish. Aquat. Sci., 1:64–67.

28.

Sjolund

, Bonnedahl

, Hernandez

, Bengtsson

, Cederbrant

, Pinhassi

, Kahlmeter

, and Olsen

2008. Dissemination of multidrug-resistant bacteria into the Arctic. Emerg. Infect. Dis., 14:70–72.

29.

van Belkum

, Kluytmans

, van Leeuwen

, Bax

, Quint

, Peters

, Fluit

, Vandenbroucke-Grauls

, van den Brule

, Koeleman

, et al. 1995. Multicenter evaluation of arbitrarily primed PCR for typing of Staphylococcus aureus strains. J. Clin. Microbiol. 33:1537–1547.

30.

Wirth

, Falush

, Lan

, Colles

, Mensa

, Wieler

L.H.

, Karch

, Reeves

P.R.

, Maiden

M.C.

, Ochman

, et al. 2006. Sex and virulence in Escherichia coli: an evolutionary perspective. Mol. Microbiol., 60:1136–1151.

31.

Zheng

, Zeng

, Chen

, Liu

, Yao

, Deng

, Chen

, Lv

, Zhuo

, Chen

, et al. 2012. Prevalence and characterisation of CTX-M beta-lactamases amongst Escherichia coli isolates from healthy food animals in China. Int. J. Antimicrob. Agents, 39:305–310.