Fecal Carriage Rates of Extended-Spectrum β-Lactamase-Producing Escherichia coli Among Antibiotic Naive Healthy Human Volunteers

Abstract

Introduction: Higher prevalence of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli fecal carriage has been reported in the nosocomial setting than in the community. We tried to determine the fecal carriage of ESBL-producing E. coli among healthy volunteers in a relatively isolated community. Materials and Methods: This study was conducted on 115 healthy adult volunteers from whom one fecal sample was collected and was plated on selective media. Each morphotypes were identified, characterized, and ESBL phenotype was confirmed by double-disk potentiation method. Molecular characterization of ESBL gene was done using multiplex polymerase chain reaction and pulse-field gel electrophoresis (PFGE) was done to identify their clonal relation. Results: ESBL-producing E. coli had a prevalence of 19% (22/115) among the healthy volunteers in the community. CTX-M was the predominant type, showed a presence 95.5% (21/22), TEM 63%, SHV 9%, and both TEM and CTX-M were present in 63.6% (14/22), all three present in 4.5% (1/22). The lineage using PFGE showed a single clone in 17 isolates. Seven isolates were type A (all TEM & CTX-M), six were type A1 (all TEM & CTX-M except 2), four were type A2 (all CTX-M), and three belonged to types B, C, and D respectively Conclusion: High prevalence rate of 19% in the community indicated by this study implies the possibility of sustained ESBL carriage even among isolated population, which could serve as a reservoir for enriching the ESBL pool in the hospital. Clonal relations also indicate a possible epidemiological source that needs to be evaluated.

Introduction

Bacterial resistance to β-lactam antibiotics is predominantly mediated by β-lactamase production. It has been noticed that with the introduction of each new β-lactam antibiotic, a new β-lactamase class causing resistance to that drug has emerged.⁴ Extended-spectrum β-lactamase (ESBL), which hydrolyzes oximino-β-lactams, such as the expanded-spectrum cephalosporins, but not carbapenems, belong to the Ambler class A and D β-lactamases¹ and of Bush-Jacoby Medeiros group 2be.¹⁹ Initially nosocomial outbreaks causing ESBLs were associated with single enzyme-producing strains, but recent studies have revealed more complex situations, with isolates harboring multiple ESBL enzymes and a significant increase in community isolates.¹⁰ Phenotypic and genotypic characterization of nosocomial extended-spectrum β-lactamase-producing enterobacteria (ESBLE) have been extensively carried out by many Indian studies and the overall prevalence was found to be 80–90%, which is the highest in the world, with CTX-M-15 as the most common genotype.^7,31 Community-acquired infections due to ESBLE has been reported from studies from Europe and India.^2,3 Despite the heavy burden of ESBLE in nosocomial settings in India, data on fecal carriage of ESBLE among healthy individuals is scarce.

A higher prevalence of ESBL-producing Escherichia coli fecal carriage has been reported in the nosocomial setting than in the community. Studies have shown that ESBL-producing uropathogens have their reservoir in the digestive tract.²⁸ Previous studies have documented 2–5 years of ESBL digestive carriage.^2,3 Finally, hospitalization of carriers increases the risk of infection for other hospitalized patients via crossinfection. Recent reports have implicated travel to the Indian subcontinent as a risk factor for asymptomatic fecal carriage of ESBLE with rates as high as 80% being reported from Netherlands.²⁹

The aim of the present study was to investigate the prevalence of E. coli producing different types of ESBLs in the human fecal flora among antibiotic naive healthy volunteers in a tribal area.

Materials and Methods

The study was conducted during January 2005 to January 2007 via eight health camps and 22 visits to Jawadhi Hills, a tribal hamlet 3,000 feet above sea level, to collect fecal samples from adult (>18 years) healthy volunteers with no prior history of hospitalization, antibiotic use, and comorbid conditions for a year. The study was approved by the institutional ethics committee. The only means for access to healthcare in this population was in the form of weekly visits by hospital ambulance from the department of community medicine, Christian Medical College, Vellore. The region has limited accessibility to urban areas due to the nonexistence of roads. People live in huts, along with their livestock, predominantly pigs, with no proper sanitation facilities. Water for consumption is obtained from wells or fresh water streams. Adults tend to migrate to the plains for work as agricultural laborers. The community is also known for its high incidence of neurocysticercosis due to pork consumption. The sample collection process was done from 115 healthy human volunteers with equal distribution of screening among both the male and female gender. One fecal sample/patient was collected after obtaining informed consent and processed within 24 hrs of sampling. A total of 0.5 g of each fecal sample was suspended in 5 ml of saline, and aliquots of 200 μl were seeded into two MacConkey agar plates one supplemented with 2 μg/ml of ceftazidime and the other without any antibiotics. A single colony representing each distinct colonial morphotype in the ceftazidime supplemented plate was regrown in the same selective plate and further analyzed. One isolate from each patient was selected for ESBL characterization. Klebsiella pneumoniae ATCC 700603 (positive control) and E. coli ATCC 25922 (negative control) were used for quality control of ESBL tests. Initial antibiotic susceptibility testing was performed by the standard CLSI disk diffusion method.¹⁸ ESBL production was screened by using both resistance phenotype and disk potentiation (Becton, Dickinson) and finally confirmed by Etest ESBL strips (AB Biodisk).

Polymerase chain reaction detection of ESBLs

Genomic DNA from these isolates was extracted after processing under standard conditions and used as the template for multiplex polymerase chain reaction (PCR). ESBL amplification was performed by multiplex PCR with the appropriate primers for the bla_TEM bla_SHV and bla_CTX-M ESBL types, as described previously.^20,26 The PCR products were separated using 1.5% agarose gels and visualized under UV light after the gels were stained with ethidium bromide. DNA amplification by PCR yielded a fragment ∼550 bp for CTX-M, 918 bp for TEM, and 850 bp for SHV.

Pulsed-field gel electrophoresis

Pulsed-field gel electrophoresis (PFGE) was done to evaluate the clonal lineage.

Bacterial DNA was prepared as described previously,¹² and XbaI (New England Bio labs) was used as the restriction enzyme. Digested DNA was separated in a GenePath system (Bio-Rad), and the conditions were as follows: 14°C, 6 V/cm, 10 to 40 sec, 27 h. The patterns obtained were interpreted according to the criteria established by Tenover et al.³⁰

Results

The rate of fecal carriage of ESBL-producing isolates of the E. coli in healthy volunteers was 19% (22 of 115 volunteers). The characterization of the ESBLs, the identities of the ESBL-producing isolates of the E. coli, are shown in Table 1. The distribution of ESBL-positive isolates by patient gender was similar among both males and females.

Table 1.

Phenotypic and Molecular Characteristics of 22 Extended-Spectrum β-Lactamase-Producing Escherichia coli Strains Recovered from Fecal Samples of 115 Antibiotic Naive Human Volunteers Residing in the Tribal Hamlet of Jawadhi Hills

Strain ID	Cefotaxime (μg/ml)	Ceftazidime (μg/ml)	Co-resistance ^a	CTX-M	TEM	SHV	PFGE clonal type
JH23	>16	>32	C, G, P/T, T/C	+ve	+ve	+ve	B
JH24	>16	12	C	+ve	+ve	−ve	A
JH26	>16	12	C	+ve	−ve	−ve	A1
JH50	>16	8	C, T/C	+ve	+ve	−ve	A
JH53	>16	8	C, P/T, T/C	+ve	−ve	−ve	A1
JH56	>16	>32	C, T/C	+ve	+ve	−ve	A
JH57	>16	8	C, T/C	+ve	+ve	−ve	A
JH58	>16	8	C, T/C	+ve	+ve	−ve	A
JH59	>16	6	C, T/C	+ve	+ve	−ve	A
JH60	>16	8	C, G, P/T, T/C	+ve	+ve	−ve	A
JH62	>16	4	C, G, T/C	−ve	+ve	−ve	C
JH68	>16	12	C, G, P/T, T/C	+ve	+ve	+ve	A1
JH71	>16	8	T/C	+ve	+ve	−ve	A
JH73	>16	12	T/C	+ve	+ve	−ve	A
JH80	>16	16	C, G, T/C	+ve	+ve	−ve	A1
JH89	>16	16	C, G, P/T, T/C	+ve	−ve	−ve	A1
JH97	>16	12	C, P/T, T/C	+ve	−ve	−ve	A2
JH102	>16	12	C, G, P/T, T/C	+ve	−ve	−ve	A2
JH105	>16	8	C, G, P/T, T/C	+ve	−ve	−ve	A2
JH108	>16	>32	C, G, P/T, T/C	+ve	−ve	−ve	A2
JH112	>16	8	C, G, P/T, T/C	+ve	+ve	−ve	D
JH113	>16	8	C, G, P/T, T/C	+ve	−ve	−ve	A1

Indicates co-resistance to various other antimicrobials; C, ciprofloxacin; G, gentamicin; P/T, piperacillin-tazobactam; T/C, ticarcillin-clavulanic acid.

PFGE, pulsed-field gel electrophoresis.

The β-lactamase susceptibility profiles and the associated antimicrobial resistance of the ESBL-producing isolates are shown in Table 1. When CLSI breakpoints are considered, all isolates were susceptible to imipenem and meropenem.¹⁸ Ceftazidime MIC showed that only 5 isolates were resistant (>16 μg/ml), 16 isolates were intermediate (8 μg/ml) while only 1 isolate was sensitive (≤4 μg/ml), whereas all of them were resistant to cefotaxime (>2 μg/ml). Tazobactam restored the piperacillin susceptibilities of 11 (50%) isolates, while clavulanic acid restored ticarcillin susceptibilities of only 2 (10%) isolates. Almost all (95.5%, 20/21) the CTX-M-producing isolates were resistant to ciprofloxacin while 41% (9/22) isolates were resistant to gentamicin. Multidrug resistance (MDR) phenotype was seen in 50% (11/22) of the isolates. The ESBL characterization (Table 1) revealed fecal carriage was mainly due to organisms producing CTX-M (95.5%) type enzyme cluster followed by TEM (63.6%). SHV was detected in two (9%) volunteers. All three enzymes were seen in two isolates that were also multidrug resistant. Both TEM and CTX-M were present in 63.6% (14/22) isolates, 36.4% (8/22) harbored only CTX-M, while only TEM was present in 4.5% (1/22) isolates. The ESBL-producing isolates obtained from healthy volunteers are described in Table 1. PFGE patterns of the E. coli isolates showed that a single clone was responsible for the colonization in 17 cases, denoting the presence of epidemic isolates within the population studied. PFGE patterns with difference of less than six fragments were interpreted as closely or possibly related to the outbreak isolate and were considered subtypes of A and are designated type A1, type A2 while the ones with ≥7 fragment difference were considered unrelated and were designated type B, C, and D. Nine isolates were type A (all TEM and CTX-M), six were type A1 (all CTX-M except for two isolates), four were type A2 (all CTX-M), and three belonged to types B, C, and D respectively. Interestingly, no evidence of any invasive infection in patients harboring ESBL-producing isolates was found in any of the colonized individuals.

Discussion

Asymptomatic colonization of the intestinal compartment with ESBL-producing isolates has been described previously.^2,15,32 Most of those studies were performed at the time of nosocomial outbreak situations and showed the dispersion and transmission of specific clones in specific wards or even in the same institution.

Colonization with multiresistant isolates, including ESBL-producing isolates, is considered a prerequisite for infection. The importance of detection of carriers of antimicrobial resistant bacteria has recently been highlighted, not only in patient populations but also in healthy people.^2,15,32 The reduction in the proportion of susceptible microbiota in the community reduces the possibility that the proportion of resistant bacteria in the nosocomial setting will decrease. Finally, the admission of carriers harboring resistant bacteria to hospitals increases the risk of infection for other hospitalized patients. Antibiotic selective pressure in hospitals may amplify the number of carriers harboring resistant bacteria and enhance the opportunity for these bacteria to cause infections.

In our study, 19% (22/115) of the healthy volunteers studied during 2005–2007 were colonized with E. coli isolates harboring ESBL enzymes. Of the 22 ESBL producing E. coli, 95.5% isolates had CTX-M ESBL type, demonstrating that the community compartment is essential for the maintenance of these enzymes. Moreover, the community can be a reservoir of ESBLs not yet detected in clinical isolates. The CTX-M enzymes usually have 4–16-fold more activity against cefotaxime than ceftazidime but some of them, such as CTX-M-15 and -19, also hydrolyze ceftazidime efficiently.²² CTX-M15 was first described in India in 2001 and the Indian population is a significant reservoir of CTX-M-15.¹¹

Muzaheed et al. found a 20% prevalence of ESBL-producing K. pneumoniae isolated from stool samples of patients with gastroenteritis.¹⁷ All were positive for CTX-M-15 gene. Sarma and Ahmed did a study to determine colonization of ESBLE among patients in different wards of a teaching hospital.²⁷ The prevalence was highest in orthopedics (41%) followed by surgery (23%) and medicine (14%). Rodrigues et al. found ESBLE colonization in healthy executives to be 11%.²⁴ Vandana et al. reported a ESBLE fecal carriage rate of 2.6% among newly admitted patients.³³ A recent study on neonatal fecal carriage by Kothari et al. found 20.6% prevalence of ESBLE with CTX-M-15 being the predominant gene.¹³ Two studies describing the spread of community-acquired bacteria producing CTX-M β-lactamases have recently appeared from the United Kingdom.^16,36 In one, ESBL-producing enterobacteriaceae were detected from the fecal flora of community and hospital-based patients from York.¹⁶ The second study from the United Kingdom was undertaken by Woodford et al. who investigated 291 CTX-M-producing E. coli from 42 centers.³⁶ The community isolates (24% of the 291) were mainly from urines and produced CTX-M-15 associated with the mobile element ISEcp1 or CTX-M-9. It is noteworthy that most of the CTX-M-15-producing strains were multiresistant to fluoroquinolones, trimethoprim, tetracycline, and aminoglycosides.¹⁴ In Barcelona, Spain a study carried out of stools revealed that the incidence of strains of E. coli-producing ESBLs, in hospitalized patients was 7.5% while healthy volunteers showed a prevalence of 3.7%.³²

Previously TEM-type ESBLs were found only in hospitalized patients, but our study showed a very high prevalence of 68% in our community isolates. On the contrary, the SHV enzyme, which was reported to be more prevalent in healthy volunteers, was seen only in 9% of the ESBL-producing E. coli isolates. Kothari et al. found that of the 22 TEM gene amplified from ESBLE from neonatal stool samples only 9% were ESBL on sequencing.¹³ Therefore, mere amplification of TEM and SHV genes does not necessarily mean that they are ESBL as they may encode the ubiquitous TEM-1, TEM-2, and SHV-1 enzymes. Sequencing them could have ascertained whether they were ESBL.

Fluoroquinolone resistance is becoming a common feature rather than an exception in ESBL-producing isolates. Previous studies have shown that all E. coli producing CTX-M-15 were resistant to ciprofloxacin.¹³ Recent studies have demonstrated co-transfer of the qnr determinant on ESBL-producing plasmids conferring resistance to nalidixic acid with reduced susceptibility to fluoroquinolones.^14,35 A population-based study from Canada showed that ciprofloxacin resistance was independently associated with the presence of CTX-M β-lactamase and these strains commonly caused community onset urinary tract infections.²¹ In our study, 95.5% (21/22) ESBLE isolates showed fluroquinolone resistance (Table 1). Previous fluoroquinolone use has been demonstrated to be a risk factor for the acquisition of ESBL-producing isolates, particularly isolates producing the CTX-M-type enzymes in the community setting. Plasmid-mediated Quinolone (qnr) resistance is on the rise; infact, qnrB plasmids reported from Indian isolates carried blaCTX-M-15 and SHV-12.⁹ Therefore, the high degree of fluroquinolone resistance in our isolates coupled with CTX-M production and clonality suggest the possibility of ST131 clone in the population.⁸ Thus, very broad antibiotic resistance extending to multiple antibiotic classes is now a frequent characteristic of ESBL-producing enterobacterial isolates. Fluroquinolone resistance in previous Indian studies on fecal carriage from Karnataka and Assam was 100%, while a study from Mumbai reported 45%.^17,24,27

The emergence of CTX-M type enzyme as the predominant ESBL in fecal carriers is not an isolated phenomenon.^25,32,36 In our study, a single epidemic clone was seen in 17 cases that may belong to the ST131 clone. It is a known fact that ESBL-producing strains can be transferred due to close contact, which, coupled with poor sanitation and common source exposure such as food and water, may have led to the spread of this clone in our study population. Similar findings have been reported by Pitout et al., who found two closely related restriction patterns among 67 (77%) CTX-M-14 producers that was responsible for a community-wide clonal outbreak of urinary tract infections during 2000 and 2001.²¹ However, studies from Spain and the United Kingdom, also using PFGE, showed that most E. coli producing CTX-M enzymes from the community were not clonally related. However, the U.K. study did suggest some evidence of genetic relatedness among strains producing CTX-M-15.³⁶ E. coli producing CTX-M β-lactamases seem to be true community ESBL-producers and the current emergence and spread of these bacteria is both intriguing and worrying. Muzaheed et al. from India too found two pulsotypes among the nine ESBL-producing K. pneumoniae from 45 patients with gastroenteritis.¹⁷ Prats et al. studied stool samples during an outbreak of acute gastroenteritis involving >100 patients and found CTX-M-9-producing E. coli in 11 patients with same clone present in 7 patients. They concluded that common source exposure such as food and water was linked to dissemination of ESBL-positive E. coli strains.²³ Tängdén et al. reported that foreign travel to areas with high prevalence of ESBL-producing strains is a risk factor for acquisition of ESBLE.²⁹ They concluded that participants visiting India were most likely to acquire ESBL-producing strains and all of them carried CTX-M-15.²⁹ Fecal carriage of 18.5% for metallobeta lactamase NDM-1 has been reported in a hospital setting from Pakistan.⁵ With NDM-1 being reported from sewage and environment, it is only a matter of time before fecal carriage of NDM-1 becomes a reality in the community.³⁴ However, a recent study by Rodrigues and colleagues from Mumbai failed to demonstrate any NDM-1 carriage in healthy individuals.⁶

In summary, a very high prevalence of fecal carriage of ESBL-producing E. coli has been observed in antibiotic naive healthy human volunteers. This increase was associated with the predominance of ESBLs with CTX-M-type enzymes. The reason for the high prevalence of ESBL in our population must be a consequence of feco-oral transmission due to poor sanitation. The migrant population in our study must have introduced the ESBL in the ecosystem that spread to the antibiotic naive individuals. The two main limitations of our study were that we did not investigate the presence of ST131 clone among the ESBL-producing E. coli isolates and that TEM and SHV genes were not sequenced to confirm that they encode for ESBL-producing enzymes.

Footnotes

Disclosure Statement

No competing financial interests exist.

References

Ambler

R.P.

, Coulson

A.F.

, Frere

J.M.

, et al. 1991. A standard numbering scheme for the class A b-lactamases. Biochem. J., 276:269–270.

Arpin

, Dubois

, Maugein

, Jullin

, Dutilh

, Brochet

J.P.

, Larribet

, Fischer

, and Quentin

. 2005. Clinical and molecular analysis of extended-spectrum {beta}-lactamase-producing enterobacteria in the community setting. J. Clin. Microbiol., 43:5048–5054.

Arpin

, Rogues

A.M.

, Kabouche

, Boulard

, Quesnel

, Gachie

J.P.

, and Quentin

. 2000. Prospective survey of colonization and infection caused by SHV-4 producing Klebsiella pneumoniae in a neurosurgical intensive care unit. Epidemiol. Infect., 124:401–408.

Asensio

, Oliver

, Gonzalez-Diego

, Baquero

, Pérez-Díaz

J.C.

, Ros

, Cobo

, Palacios

, Lasheras

, and Canton

. 2000. Outbreak of a multiresistant Klebsiella pneumoniae strain in an intensive care unit: antibiotic use as risk factor for colonization and infection. Clin. Infect. Dis., 30:55–60.

Day

K.M.

, Ali

, Mirza

I.A.

, Sidjabat

H.E.

, Silvey

, Lanyon

C.V.

, Cummings

S.P.

, Abbasi

S.A.

, Raza

M.W.

, Paterson

D.L.

, and Perry

J.D.

. 2013. Prevalence and molecular characterization of Enterobacteriaceae producing NDM-1 carbapenemase at a military hospital in Pakistan and evaluation of two chromogenic media. Diagn. Microbiol. Infect. Dis., 75:187–191.

Deshpande

, Vadwai

, Shetty

, Dalal

, Soman

, and Rodrigues

2012. No NDM-1 carriage in healthy persons from Mumbai: reassuring for now. J. Antimicrob. Chemother., 67:1046–1047.

Ensor

V.M.

, Shahid

, Evans

J.T.

, 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.

Hussain

, Ewers

, Nandanwar

, Guenther

, Jadhav

, Wieler

L.H.

, and Ahmed

. 2012. Multiresistant uropathogenic Escherichia coli from a region in India where urinary tract infections are endemic: genotypic and phenotypic characteristics of sequence type 131 isolates of the CTX-M-15 extended-spectrum-β-lactamase-producing lineage. Antimicrob. Agents Chemother., 56:6358–6365.

Jacoby

G.A.

, Walsh

K.E.

, Mills

D.M.

, Walker

V.J.

, Oh

, Robicsek

, and Hooper

D.C.

. 2006. qnrB, another plasmid-mediated gene for quinolone resistance. Antimicrob. Agents Chemother., 50:1178–1182.

10.

Jutersek

, Baraniak

, Zohar-Cretnik

, Storman

, Sadowy

, and Gniadkowski

2003. Complex endemic situation regarding extended-spectrum β-lactamase-producing Klebsiella pneumoniae in a hospital in Slovenia. Microb. Drug Resist., 9(Suppl. 1):S25–S33.

11.

Karim

, Poirel

, Nagarajan

, et al. 2001. Plasmid-mediated extendedspectrum b-lactamase (CTX-M-3 like) from India and gene association with insertion sequence ISEcp1. FEMS Microbiol. Lett., 201:237–241.

12.

Kaufmann

M.E.

1998. Pulsed-field-electrophoresis. In Woodford

and Johnson

A.P.

(eds.), Molecular Bacteriology: Protocols and Clinical Applications. Humana Press, Totowa, NJ, pp. 33–50.

13.

Kothari

, Gaind

, Chandreshwor Singh

, Sinha

, Kumari

, Arya

, Chellani

, Saxena

, and Deb

2013. Community acquisition of β-lactamase producing Enterobacteriaceae in neonatal gut. BMC Microbiol., 13:136.

14.

Mammeri

, Van De Loo

, Poirel

, et al. 2005. Emergence of plasmidmediated quinolone resistance in Escherichia coli in Europe. Antimicrob. Agents Chemother., 49:71–76.

15.

Miró

, Mirelis

, Navarro

, Rivera

, Mesa

R.J.

, Roig

M.C.

, Gómez

, and Coll

. 2005. Surveillance of extended-spectrum beta-lactamases from clinical samples and faecal carriers in Barcelona, Spain. J. Antimicrob. Chemother., 56:1152–1155. Epub 2005 Oct 21.

16.

Munday

C.J.

, Whitehead

G.M.

, Todd

N.J.

, Campbell

, and Hawkey

P.M.

. 2004. Predominance and genetic diversity of community- and hospital-acquired CTX-M extended-spectrum beta-lactamases in York, UK. J. Antimicrob. Chemother., 54:628–633.

17.

Muzaheed

, Doi

J.M.

, Adams-Haduch

C.T.

, Shivannavar

D.L.

, Paterson , and Gaddad

S.M.

. 2009. Faecal carriage of CTX-M-15-producing Klebsiella pneumoniae in patients with acute gastroenteritis. Indian J. Med. Res., 129:599–602.

18.

National Committee for Clinical Laboratory Standards. Performance Standards for Antimicrobial Susceptibility Testing: Twelfth Informational Supplement M100-S16. NCCLS, Wayne, PA, 2007.

19.

Paterson

D.L.

, and Bonomo

R.A.

. 2005. Extended-spectrum β-lactamases: a clinical update. Clin. Microbiol. Rev., 18:657–686.

20.

Paterson

D.L.

, Hujer

K.M.

, Hujer

A.M.

, Yeiser

, Bonomo

M.D.

, Rice

L.B.

, and Bonomo

R.A.

. 2003. International Klebsiella Study Group. 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.

21.

Pitout

J.D.

, Hanson

N.D.

, Church

D.L.

, et al. 2004. Population-based laboratory surveillance for Escherichia coli-producing extended-spectrum b-lactamases: importance of community isolates with blaCTX-M genes. Clin. Infect. Dis., 38:1736–1741.

22.

Poirel

, Naas

, Le Thomas

, et al. 2001. CTX-M-type extendedspectrum b-lactamase that hydrolyzes ceftazidime through a single amino acid substitution in the omega loop. Antimicrob. Agents Chemother., 45:3355–3361.

23.

Prats

, Mirelis

, Miro

, et al. 2003. Cephalosporin-resistant Escherichia coli among summer camp attendees with salmonellosis. Emerg. Infect. Dis., 9:1273–1280.

24.

Rodrigues

, Shukla

, Jog

, and Mehta

2005. Extended-spectrum β-lactamase-producing flora in healthy persons. Emerg. Infect. Dis., 11:981–982.

25.

Rodriguez-Baño

, Navarro

M.D.

, Romero

, et al. 2004. Epidemiology and clinical features of infections caused by extended-spectrum b-lactamase-producing Escherichia coli in nonhospitalized patients. J. Clin. Microbiol., 42:1089–1094.

26.

Sahly

, Aucken

, Benedí

V.J.

, Forestier

, Fussing

, Hansen

D.S.

, Ofek

, Podschun

, Sirot

, Tomás

J.M.

, Sandvang

, and Ullmann

. 2004. Increased serum resistance in Klebsiella pneumoniae strains producing extended-spectrum beta-lactamases. Antimicrob. Agents Chemother., 48:3477–3482.

27.

Sarma

J.B.

, and Ahmed

G.U.

. 2010. Prevalence and risk factors for colonisation with extended spectrum beta-lactamase producing enterobacteriacae vis-à-vis usage of antimicrobials. Indian J. Med. Microbiol., 28:217–220.

28.

Tan

B.H.

, and Tan

A L.

. 2002. Community-acquired bacteremia with an ESBL-producing Escherichia coli strain—a case report. Int. J. Infect. Dis., 6:318–319.

29.

Tängdén

, Cars

, Melhus

, and Löwdin

2010. Foreign travel is a major risk factor for colonization with Escherichia coli producing CTX-M-type extended-spectrum beta-lactamases: a prospective study with Swedish volunteers. Antimicrob. Agents Chemother., 54:3564–3568.

30.

Tenover

F.C.

, Arbeit

R.D.

, Goering

R.V.

, Mickelsen

P.A.

, Murray

B.E.

, Persing

D.H.

, and Swaminathan

. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol., 33:2233–2239.

31.

Toleman

M.A.

, Beidenbach

, Jones

R.N.

, et al. 2002. Molecular characterisation of b-lactamases from Escherichia coli isolated from a multicentre trial in India: benchmark report from the MYSTIC program. In Abstracts of the Forty-second Interscience Conference on Antimicrobial Agents and Chemotherapy, San Diego, CA. Abstract C2-1872, p. 114. American Society for Microbiology, Washington, DC.

32.

Valverde

, Coque

T.M.

, Sánchez-Moreno

M.P.

, et al. 2004. Dramatic increase in prevalence of fecal carriage of extended-spectrum blactamase-producing Enterobacteriaceae during nonoutbreak situations in Spain. J. Clin. Microbiol., 42:4769–4775.

33.

Vandana

K.E.

, Varghese

, Krishna

, Mukhopadhyay

, Kamath

, and Ajith

. 2010. Screening at admission for carrier prevalence of multidrug-resistant organisms in resource-constrained settings: a hospital-based observational study. J. Hosp. Infect., 76:180–181.

34.

Walsh

T.R.

, Weeks

, Livermore

D.M.

, and Toleman

M.A.

. 2011. Dissemination of NDM-1 positive bacteria in the New Delhi environment and its implications for human health: an environmental point prevalence study. Lancet Infect. Dis., 11:355–362.

35.

Wang

, Sahm

D.F.

, Jacoby

G.A.

, et al. 2004. Emerging plasmid-mediated quinolone resistance associated with the qnr gene in Klebsiella pneumoniae clinical isolates in the United States. Antimicrob. Agents Chemother., 48:1295–1299.

36.

Woodford

, Ward

M.E.

, Kaufmann

M.E.

, et al. 2004. Community and hospital spread of Escherichia coli producing CTX-M extended-spectrum b-lactamases in the UK. J. Antimicrob. Chemother., 54:735–743.