Prevalence and Genetic Relatedness of Extended Spectrum-β-Lactamase-Producing Escherichia coli Among Humans,Cattle,and Poultry in Pakistan

Abstract

Objective:

To determine the prevalence and genetic relatedness of bla_CTX-M-type extended spectrum-β-lactamase (ESBL)-producing Escherichia coli at the human–animal interface in Pakistan.

Materials and Methods:

A total of 150 human, cattle, and poultry fecal samples (50 each) were screened for ESBL-producing E. coli using ESBL CHROMagar^®. Bacterial species confirmation as well as determination of minimum inhibitory concentrations (μg/mL) to different antibiotics was performed using the automated VITEK^®-2 compact system. Phenotypic confirmation of ESBL production was performed according to the Clinical Laboratory Standards Institute (CLSI) guidelines. Genetic analysis of bla_CTX-M was carried out by PCR and DNA sequencing. Plasmids and clonal similarity of the E. coli strains were determined by PCR-based replicon typing and pulsed-field gel electrophoresis (PFGE), respectively.

Results:

Of 150 samples, 29 (19.3%) ESBL-producing E. coli were recovered, and majority of them originated from human (n = 16; 55%), followed by cattle (n = 9; 31%) and poultry (n = 4; 13.7%). bla_CTX-M-15 was predominant ESBL genotype (n = 25; 86.2%), mainly identified from human (n = 15) and cattle (n = 9). This is also the first report of the occurrence of CTX-M-15 and CTX-M-55 in cattle and poultry E. coli isolates of Pakistan, respectively. The majority of the ESBL-producing E. coli (96.5%) showed a multidrug resistance phenotype. All isolates carried IncFII or IncFIA plasmids, and the phylogroup B1 was dominant (44.8%) followed by phylogroups A (31%), D (17.2%), and B2 (6.8%). PFGE revealed that isolates from different hosts were genetically unrelated.

Conclusion:

Presence of CTX-M-15-type ESBL-producing E. coli in different reservoirs is alarming and has the potential to impact both veterinary and human therapeutic treatment options.

Introduction

Antimicrobial resistance (AMR) is becoming an emerging threat to public health sector due to excessive antimicrobial exposure in human and veterinary medicine, particularly in developing countries such as Pakistan.¹ Globally, ∼63,000 tons of antibiotics are being used in livestock, which will further increase to 1,05,000 tons in 2030.² These practices contribute to the widespread increase of AMR pathogens in human, livestock, and the environment, which consequently leads to the prolonged hospital stay for patients, financial burden to the society, and even fatal consequences.² Infections of multidrug-resistant Escherichia coli are of great concern and challenge in medical and veterinary settings.³ In particular, extended spectrum-β-lactamase (ESBL)-producing pathogenic E. coli can lead to common community-acquired infections, which often require hospitalization and enable resistance genes to move between different environments and hospital settings.⁴ Clinically relevant ESBL-producing E. coli strains are not only restricted to humans, but they have also been reported in food-producing animals and the environment.⁵ Dissemination of ESBL-producing E. coli in clinical and extra-clinical settings is mainly driven by plasmid and clonal expansion of certain lineages.⁶ Furthermore, it has been shown that part of the burden associated with ESBL-producing E. coli in human is linked to antibiotic usage in food-producing animals.^7,8

Among different types of ESBL, CTX-M types are considered the most common genetic variant among clinical and nonclinical isolates around the globe.⁹ In particular, CTX-M-15 type of ESBL-producing E. coli is epidemiologically important in clinical settings, chicken, environment, and wildlife.^10,11 In Pakistan, CTX-M has been reported as the most common ESBL genotype from clinical settings^12,13 and poultry.¹⁴ One Health approach has been proposed as a key to study transmission dynamics of AMR. Therefore, this study was designed to identify CTX-M-producing E. coli from humans, cattle, and poultry in Pakistan and to determine the genetic relatedness of isolates from different hosts.

Materials and Methods

Sample collection

The Institutional Bioethics/Biosafety Committee of the University of Agriculture, Faisalabad, has approved this study (D. No. 109/ORIC). A total of 150 convenience sample of humans and animals were collected from different locations from Faisalabad metropolis during February to March 2016. Pus (n = 25) and urine (n = 25) human samples were collected from Burn unit and Urology ward, respectively, from Allied Hospital, Faisalabad using aseptic techniques.¹⁵ The mean age group of patients from urology and burn wards were 49.5 and 35.8, respectively. All the patients were under antibiotic therapy with the most commonly administered antibiotics were ceftriaxone and nitrofurantoin. Fecal swabs (n = 50) were collected from the rectum of 10 healthy cows from each of 5 cattle farms. In addition, cloacal swabs (n = 50) were randomly collected from free-range poultry found in the proximity of these farms. Geographical locations of these farms are displayed in Fig. 1. All the samples were collected and transported in Amies transport media under refrigerated conditions.

FIG. 1.

Geographical location of livestock sampling.

Identification of ESBL-producing E. coli

All the samples (n = 150) were primarily streaked on CHROMagar ESBL (CHROMagar, Paris, France) for presumptive detection of ESBL-producing E. coli. Biochemical confirmation of E. coli was done by the VITEK^® 2 compact system using GN cards (bioMérieux, Nürtingen, Germany).

Antimicrobial susceptibility testing

Minimum inhibitory concentration (MIC; μg/mL) of 16 different antibiotics, including ampicillin, cefalexin, cefpirome, cefpodoxime, ceftiofur, amoxicillin/clavulanic acid, chloramphenicol, enrofloxacin, gentamicin, marbofloxacin, piperacillin, tetracycline, tobramycin, trimethoprim/sulfamethoxazole, imipenem, and polymyxin B, was determined against E. coli isolates using AST GN38 cards in VITEK 2 compact system (bioMérieux). Results of susceptibility testing were interpreted per Clinical Laboratory Standards Institute (CLSI) guidelines, 2016. Multidrug resistance (MDR) was defined as resistance to three or more different classes of antimicrobials.

Phenotypic confirmation of ESBL

Phenotypic confirmation of ESBL-producing E. coli was carried out with double disk synergy test per CLSI guidelines 2016. In brief, E. coli was inoculated onto Muller-Hinton agar (MHA) (Oxoid, Hampshire, United Kingdom) plate, and ceftazidime (30 μg) and ceftazidime with clavulanic acid (30/10 μg) disks were implanted. Bacteria will be considered the ESBL producer if ceftazidime in combination with clavulanic acid disk showed ≥5 mm zone of inhibition as compared with ceftazidime alone.

Phenotypic confirmation of carbapenamase activity

Phenotypic detection of carbapenemase activity was performed by Modified Hodge's test per CLSI guidelines 2016. Shortly, 0.5 McFarland E. coli (ATCC 25922) bacterial suspension was lawned on MHA plate. Ten micrograms of meropenem disk was placed in the middle of MHA plate, and tested E. coli, positive and negative bacterial controls were streaked from the margin of the disk to the margin of the MHA plate. Carbapenamase-positive bacteria permit meropenem-sensitive E. coli (ATCC 25922) to grow against the meropenem disk, which leads to a cloverleaf-like indentation.¹

Molecular detection of ESBL-encoding genes

Bacterial DNA was extracted using commercially available kit MasterPure™ Purification (Epicenter Biotechnologies, WI) per manufacturer's instruction. ESBL-encoding genes, bla_TEM, bla_SHV, and bla_CTX-M, were identified by PCR as described previously.¹⁶ The amplicon was separated on ethidium-bromide-stained 1.5% agarose gel and visualized under UV light by gel documentation instruments.

Bacterial DNA sequencing

The CTX-M-producing E. coli isolates were processed further using the PCR methodology described by Guerra and colleagues,¹⁷ and PCR products were sequenced commercially by AGOWA GmbH (Berlin, Germany). Raw sequences were checked for quality and assigned to a CTX-M type using Ridom Seq Sphere 0.9.19.

Phylogenetic grouping of E. coli

PCR-based phylogenetic grouping of ESBL-producing E. coli was carried out by amplifying three genetic markers, chuA, yjaA, and DNA fragment TSPE4.C2 as described earlier.¹⁸ E. coli are mainly divided into four main phylogenetic groups such as A, B1, B2, and D.

Plasmid conjugation experiment

Transferability of the bla_CTX-M was carried out for randomly selected 10 ESBL-producing E. coli in this study. Conjugation experiment was performed using the sodium azide-resistant E. coli J53 strains as recipients.¹⁹ The transconjugates were finally obtained on MHA supplemented with cefotaxime (2 mg/L) and sodium azide (150 mg/L). All transconjugates were subjected to PCR to confirm bla_CTX-M gene.

Replicon typing of plasmids

Plasmid incompatibility (Inc) types were determined by PCR-based replicon typing as described earlier.²⁰

Pulsed-field gel electrophoresis

All the ESBL-producing E. coli were subjected to pulsed-field gel electrophoresis (PFGE) analysis using CHEF DRIII System (BioRad, Munich, Germany) as described in Pulse Net protocol of the Centers for Disease Control and Prevention, Atlanta, United States.¹ In brief, agarose-embedded DNA was digested with the restriction endonuclease XbaI at 37°C for 16 hr. Electrophoresis conditions were 6 V, with 2.2–54 sec pulses for 20 hr. Strain differentiation by PFGE analysis was achieved by comparison of band patterns using BioNumerics software (Version 7.1; Applied Math, Belgium). Dendrogram was generated according to the unweighted pair group method with arithmetic mean (UPGMA) based on Dice coefficients. Isolates were considered to belong to the same PFGE group if their Dice similarity index was ≥85%.

Results

ESBL-producing E. coli carriage

Of 150 samples, 29 (19.3%) E. coli were positive for ESBL production; among these 16 (55%) were from humans, 9 (31%) from cattle, and 4 (13.7%) from poultry (Table 1).

Table 1.

Phenotypic and Genotypic Characteristics of Extended Spectrum-β-Lactamase-Producing Escherichia coli from Human, Cattle, and Poultry

Sample ID	Origin	ESBL type	Incompatibility group	Non β-lactam resistance (except carbapenems)
X-1	Poultry	CTX-M-1, TEM	Fll	AMP, AMC, CPO, C, CPD, CFT, ENR, CN, TE, PIP, GM, MRB, SXT
X-2	Poultry	CTX-M-15, TEM	Fll	AMP, CPO, CPD, CFT, CN, TE, ENR, MRB, PIP, GM, SXT
X-3	Poultry	CTX-M-55	Fll	AMP, CN, GM, CPO, CPD, CFT, TE, ENR, MRB, PIP, SXT
X-4	Poultry	CTX-M-55	Fll	AMP, CN, GM, CPO, CPD, CFT, TE, ENR, MRB, PIP, SXT
X-5	Human	CTX-M-15	Fll	AMP, CN, CPO, CPD, CFT, GM, TE, ENR, MRB, SXT, PIP
X-6	Human	CTX-M-15	Fll	AMP, AMC, CN, CPO, CPD, CFT, GM, TE, ENR, MRB, SXT, IPM, PIP
X-7	Human	CTX-M-1	Fll	AMP, AMC, CN, C, GM, PIP TE, ENR, MRB, SXT, IPM
X-8	Human	CTX-M-15	Fll	AMP, CN, AMC, C, GM, TE, ENR, MRB, SXT, IPM, PIP
X-9	Human	CTX-M-15	Fll	AMP, CN, AMC, GM, C, TE, ENR, MRB, SXT, IPM, PIP
X-10	Human	CTX-M-15	FIA	AMP, CN, GM, AMC, TM, ENR, MRB, SXT, IMP, PIP
X-11	Human	CTX-M-15, TEM	FIA	AMP, AMC, CN, GM, C, TM, ENR, MRB, SXT, IMP, PIP, SXT
X-12	Human	CTX-M-15, TEM	Fll	AMP, CN, C, TE, C, ENR, MRB, SXT, PIP, IMP, GM
X-13	Human	CTX-M-15, TEM	Fll	AMP, AMC, CN, PIP, GM, TM, ENR, C, MRB, SXT, IMP
X-14	Human	CTX-M-15, TEM	FIA	AMP, PIP, AMC, C, CN, TM, TE, ENR, MRB, GM, SXT
X-15	Human	CTX-M-15	FIA	AMP, CN, PIP, AMC, C, GM, TM, TE, ENR, MRB, SXT
X-16	Cattle	CTX-M-15, TEM	Fll	AMP, PIP, CN, GM, ENR, SXT, MRB, TM
X-17	Cattle	CTX-M-15	Fll	AMP, CN, AMC, C, GM, TE, PIP, ENR, MRB, SXT, IMP, TM, SXT
X-18	Cattle	CTX-M-15, TEM	FIA	AMP, CN, TE, ENR, C, MRB, PIP, SXT, IMP, GM, TM
X-19	Cattle	CTX-M-15, TEM	Fll	AMP, CN, TE, C, SXT, GM, IMP, PIP, ENR, MRB, TM
X-20	Cattle	CTX-M-15, TEM	Fll	AMP, CN, TE, C, SXT, IMP, MRB, GM, PIP, ENR, TM
X-21	Cattle	CTX-M-15	Fll	AMP, CN, TE, C, SXT, PIP, GM, ENR, MRB, TM
X-22	Cattle	CTX-M-15	Fll	AMP, CN, TE, SXT, MRB, TM, PIP, ENR, GM
X-24	Cattle	CTX-M-15, TEM	Fll	AMP, CN, TE, SXT, PIP, MRB, ENR, GM
X-23	Cattle	CTX-M-15, TEM	Fll	AMP, CN, TE, C, SXT, IMP, MRB, PIP, ENR, GM
X-25	Human	CTX-M-15	FIA	AMP, AMC, CN, GM, C, TM, TE, ENR, PIP, MRB, SXT, C, IMP
X-26	Human	CTX-M-15, TEM	FIA	AMP, AMC, CN, GM, TM, C, PO, ENR, MRB, PIP, SXT, IMP
X-27	Human	CTX-M-15, TEM	FIA	AMP, AMC, PIP, CN, GM, TM, TE, ENR, MRB, SXT
X-28	Human	CTX-M-15, TEM	FIA	AMP, AMC, PIP, CN, GM, TE, ENR, MRB, SXT
X-29	Human	CTX-M-15	FIA	AMP, AMC, PIP, CN, GM, C, TE, ENR, MRB, SXT

AMC, amoxicillin/clavulanic acid; AMP, ampicillin; C, chloramphenicol; CFT, ceftiofur; CN, cefalexin; CPD, cefpodoxime; CPO, cefpirome; ENR, enrofloxacin; ESBL, extended spectrum-β-lactamase; GM, gentamicin; IPM, imipenem; MRB, marbofloxacin; PB, polymyxin B; PIP, piperacillin; SXT, trimethoprim/sulfamethoxazole; TE, tetracycline; TM, tobramycin.

Phenotypic resistance of ESBL-producing E. coli

Of 29 ESBL-producing E. coli isolates, 28/29 (96.5%) showed an MDR phenotype. The most common non β-lactam resistance was found toward tetracycline (89.6%) and doxycycline (86.2%) (Table 1).

MIC of ESBL-producing E. coli

MIC testing (μg/mL) for all ESBL-producing E. coli resulted in high MICs not only among β-lactam antibiotics, including ampicillin (≥32 μg/mL), piperacillin (≥128 μg/mL), cefalexin (≥64 μg/mL), cefpodoxime (≥8 μg/mL), ceftiofur (≥8 μg/mL), but also among non-β-lactams such as enrofloxacin (≥4 μg/mL), gentamicin (≥16 μg/mL), marbofloxacin (≥4 μg/mL), and trimethoprim/sulfamethoxazole (≥320 μg/mL). The MIC values for the both chloramphenicol and tetracycline were ≥64 and ≥16 μg/mL against 17 and 25 isolates, respectively. Furthermore, the MIC values for imipenem were ≥4 and 2 μg/mL against 15 and 8 isolates, respectively. However, least resistance was observed against polymyxin B with MIC value <0.5 μg/mL (Table 2). Moreover, all the imipenem-resistant E. coli (n = 15) with MICs ≥4 μg/mL were positive for carbapenemase activity.

Table 2.

Minimum Inhibitory Concentrations (μg/mL) of Extended Spectrum-β-Lactamase-Producing Escherichia coli

Isolates	AMP	CN	CPO	CPD	CFT	AMC	C	ENR	GM	MRB	PIP	TE	TM	SXT	IPM	PB
X1	≥32	≥64	≥64	≥8	≥8	≥32/16	≥64	≥4	≥16	≥4	≥128	≥16	8	≥320	1	<0.5
X2	≥32	≥64	≥64	≥8	≥8	16/8	4	≥4	≥16	≥4	≥128	≥16	8	≥320	1	<0.5
X3	≥32	≥64	≥64	≥8	≥8	4/2	4	≥4	≥16	≥4	≥128	≥16	8	≥320	2	<0.5
X4	≥32	≥64	≥64	≥8	≥8	4/2	8	≥4	≥16	≥4	≥128	≥16	8	≥320	1	<0.5
X5	≥32	≥64	≥64	≥8	≥8	16/8	8	≥4	≥16	≥4	≥128	≥16	8	≥320	2	<0.5
X6	≥32	≥64	≥64	≥8	≥8	≥32/16	8	≥4	≥16	≥4	≥128	≥16	8	≥320	4	<0.5
X7	≥32	≥64	≥64	≥8	≥8	≥32/16	≥64	≥4	≥16	≥4	≥128	≥16	8	≥320	≥4	<0.5
X8	≥32	≥64	≥64	≥8	≥8	≥32/16	≥64	≥4	≥16	≥4	≥128	≥16	8	≥320	4	<0.5
X9	≥32	≥64	≥64	≥8	≥8	≥32/16	≥64	≥4	≥16	≥4	≥128	≥16	8	≥320	4	<0.5
X10	≥32	≥64	≥64	≥8	≥8	≥32/16	8	≥4	≥16	≥4	≥128	8	≥16	≥320	4	<0.5
X11	≥32	≥64	≥64	≥8	≥8	≥32/16	≥64	≥4	≥16	≥4	≥128	≥16	≥16	≥320	4	<0.5
X12	≥32	≥64	64	≥8	≥8	16/8	≥64	≥4	≥16	≥4	≥128	≥16	≥16	≥320	4	<0.5
X13	≥32	≥64	≥64	≥8	≥8	≥32/16	≥64	≥4	≥16	≥4	≥128	8	16	≥320	4	<0.5
X14	≥32	≥64	≥64	≥8	≥8	≥32/16	≥64	≥4	≥16	≥4	≥128	≥16	≥16	≥320	2	<0.5
X15	≥32	≥64	≥64	≥8	≥8	≥32/16	≥64	≥4	≥16	≥4	≥128	≥16	≥16	≥320	2	<0.5
X16	≥32	≥64	64	≥8	≥8	4/2	8	≥4	≥16	≥4	≥128	8	≥16	≥320	2	<0.5
X17	≥32	≥64	≥64	≥8	≥8	≥32/16	≥64	≥4	≥16	≥4	≥128	≥16	≥16	≥320	4	<0.5
X18	≥32	≥64	64	≥8	≥8	4/2	32	≥4	≥16	≥4	≥128	≥16	≥16	≥320	4	<0.5
X19	≥32	≥64	≥64	≥8	≥8	8/4	≥64	≥4	≥16	≥4	≥128	≥16	≥16	≥320	4	<0.5
X20	≥32	≥64	64	≥8	≥8	8/4	≥64	≥4	≥16	≥4	≥128	≥16	≥16	≥320	4	<0.5
X21	≥32	≥64	64	≥8	≥8	8/4	≥64	≥4	≥16	≥4	≥128	≥16	≥16	≥320	2	<0.5
X22	≥32	≥64	64	≥8	≥8	4/2	16	≥4	≥16	≥4	≥128	≥16	≥16	≥320	2	<0.5
X23	≥32	≥64	64	≥8	≥8	8/4	16	≥4	≥16	≥4	≥128	≥16	8	≥320	2	<0.5
X24	≥32	≥64	64	≥8	≥8	4/2	>64	≥4	≥16	≥4	≥128	≥16	8	≥320	4	<0.5
X25	≥32	≥64	≥64	≥8	≥8	≥32/16	>64	≥4	≥16	≥4	≥128	≥16	≥16	≥320	4	<0.5
X26	≥32	≥64	≥64	≥8	≥8	≥32/16	≥64	≥4	≥16	≥4	≥128	8	≥16	≥320	4	<0.5
X27	≥32	≥64	≥64	≥8	≥8	≥32/16	8	≥4	≥16	≥4	≥128	≥16	≥16	≥320	1	<0.5
X28	≥32	≥64	≥64	≥8	≥8	≥32/16	16	≥4	≥16	≥4	≥128	≥16	8	≥320	1	<0.5
X29	≥32	≥64	≥64	≥8	≥8	≥32/16	>64	≥4	≥16	≥4	≥128	≥16	8	≥320	1	<0.5

Molecular identification of ESBL-producing E. coli

All the ESBL-producing E. coli carried bla_CTX-M; however, 15 (51%) isolates carried the combination of bla_CTX-M and bla_TEM. DNA sequencing data of CTX-M revealed the presence of three different types of genotypes: bla_CTX-M-15 (n = 25), bla_CTX-M-1 (n = 2), and bla_CTX-M-55 (n = 2). Subsequently, bla_CTX-M-15-producing E. coli were mainly identified in human samples (n = 15), followed by cattle (n = 9) and poultry (n = 1). bla_CTX-M-1-producing E. coli were found in one human and one poultry isolate, while two bla_CTX-M-55-producing E. coli were only identified in poultry isolates (Table 1).

Plasmid conjugation and replicon typing

All the tested bla_CTX-M-positive isolates demonstrated transferability to recipient strain. bla_CTX-M gene was detected by PCR in all the transconjugates. PCR-based replicon typing of ESBL-producing E. coli revealed two major types of plasmids: IncFII and IncFIA. The IncFII was the most common (n = 19) plasmid type present in ESBL-producing E. coli isolates from all 3 hosts and IncFIA plasmid type was detected in 10 isolates (1 from cattle and 9 from humans), while none of the chicken isolates carried IncFIA plasmid replicon (Table 1).

Phylogenetic groups

PCR-based phylogenetic analysis showed that 13 (44.8%) ESBL-producing E. coli belonged to B1 phylogenetic group, 11 (37.9%) to group A, 4 (13.8%) to group D, and 1 (3.4%) to group B2. The phylogenetic groups B1 and A were detected in ESBL-producing E. coli isolates from both human and animal, while group B2 was only detected in two human isolates (Fig. 2).

FIG. 2.

Dendrogram of PFGE patterns of 29 ESBL-producing Escherichia coli isolates. Dotted box indicates clonal group defined at 85% Dice similarity. ESBL, extended spectrum-β-lactamase; PFGE, pulsed-field gel electrophoresis.

PFGE analysis

PFGE analysis showed that most of the ESBL-producing E. coli were genetically unrelated. However, three pairs of human clinical isolates (X6 and X7; X25 and X27; X28 and X29) showed clonal relatedness. Each pair of the isolates in these clones shared common plasmid replicon type, phylogenetic group, and AMR profile (Fig. 2 and Table 1).

Discussion

Despite the highest burden of infectious diseases in low- and middle-income countries, surveillance of AMR is limited.²¹ To address the challenges of AMR, Government of Pakistan has drafted its National Action Plan on AMR in 2017.²² Studies focused on surveillance of AMR at human–animal interface using a One Health approach have been proposed as one of the main objectives of its action plan.²³ To the best of our knowledge, such studies are currently lacking in Pakistan.

Infections caused by ESBL-producing E. coli are a serious issue to both public health and veterinary medicine. In this study, high prevalence of ESBL-producing E. coli (19.3%) has been identified mainly in human and cattle. Recently, a meta-analysis on ESBL from different regions of Pakistan revealed that overall pooled proportion of ESBL-producing bacteria was 40%. Most of the cases (41.8%) have been reported from Islamabad/Rawalpindi followed by Punjab (25.4%) and KPK (16.4%), while no case has been reported from the province of Baluchistan.¹² However, these estimates did not include studies from animals. Similar prevalence of ESBL-producing E. coli (17%) has been found in migratory birds in Pakistan.²⁴ There is a dearth of studies on the key drivers of AMR in Pakistan. A situation analysis report suggested irrational use of antibiotics in human and veterinary settings, over-the-counter drugs availability, and lack of surveillance systems as the possible reasons for rise in AMR in Pakistan.²⁵

DNA sequencing revealed the predominant presence of bla_CTX-M-15 ESBL genotype. These results are in agreement with various previously reported studies from humans, animals, chickens, and wild birds.^{16,24,26–31} To the best of our knowledge, this is the first report on the presence of bla_CTX-M-15 from cattle and bla_CTX-M-55 from poultry isolates in Pakistan. However, these genotypes have been previously reported in E. coli isolates from food-producing animals from other countries.^32,33

Transmission of ESBL-producing Enterobacteriaceae is further complicated by ESBL genes being encoded on self-transmissible plasmids, which can be exchanged among the same and different species of Enterobacteriaceae. In this study, plasmid replicon type IncFII was found in both human and animal isolates. Although limited due to reduced sample size, our results agree with a French study that found bla_CTX-M-15 on IncFII plasmids from dogs and human E. coli isolates.³⁴ Similarly, a Kenyan study also documented the spread of bla_CTX-M-15-positive E. coli containing IncFII plasmids in human and dog samples.³⁵ South Korea and China have also reported IncFII in both human and animal E. coli isolates.^36,37

AMR patterns of ESBL-producing E. coli depicted high MIC against commonly used antibiotics, including β-lactam and β-lactam inhibitors, and least resistance to polymyxin B. These findings are in accordance with previous studies reported on the prevalence of MDR E. coli from Australia and India.^38,39 Moreover, two studies from Pakistan revealed high resistant patterns against β-lactam drugs and quinolones.^40,41 Lack of effective infections control committee in clinical and veterinary settings, unhygienic practices, and over-the-counter availability of drugs are likely factors for dissemination of AMR in Pakistan.^40,42

There is a lack of clear evidence on zoonotic spread of ESBL-producing bacteria. However, few previous studies indicated presence of identical ESBL genotype, clones, and multilocus sequence types.^43,44 In this study, we did not find evidence of clonal spread of ESBL-producing E. coli at human–animal interface. However, we found identical ESBL genes, plasmids, and resistance profile among isolates from animals and humans. Interestingly, we found high rates of CTX-M-15 (25/29; 86.2%) in this study, which is alarming as this genotype is emerging worldwide and associated with multidrug resistance pathogens causing both community and hospital-acquired infections.^45,46 Furthermore, we found CTX-M gene on transferable plasmids. This agrees with a previous report in which no evidence of ESBL zoonosis was found between farm animals, foods, and humans.⁴⁷

Conclusion

In summary, CTX-M-15 is the most frequent ESBL produced by E. coli isolated from animals and human clinical settings. Our findings underscore the need to further investigate the molecular epidemiology and zoonosis of CTX-M-producing E. coli at human–animal intersection. In future, the spread of AMR should be closely monitored using One Health approach.

Footnotes

Acknowledgments

This work was supported by the funds from the Higher Education Commission, Pakistan (NRPU Grant No. 4681) and by the AIP/CIMMYT Project, Pakistan Agricultural Research Council, Pakistan (Grant No. AS04).

Disclosure Statement

No competing financial interests exist.

References

Qamar

M.U.

, Saleem

, Toleman

M.A

, Saqalein

, Waseem

, Nisar

M.A

, Khurshid

, Taj

, and Jahan

2018. In vitro and in vivo activity of Manuka honey against NDM-1-producing Klebsiella pneumoniae ST11. Future. Microbiol. 13:13–26.

Van Boeckel

T.P.

, Brower

, Gilbert

, Grenfell

B.T

, Levin

S.A

, Robinson

T.P

, Teillant

, and Laxminarayan

2015. Global trends in antimicrobial use in food animals. Proc. Natl. Acad. Sci. U. S. A. 112:5649–5654.

Wieler, L.H. 2014. “One Health”—linking human, animal and environmental health. Int. J. Med. Microbiol. 304:775–776.

Leser

T.D.

, and Molbak

2009. Better living through microbial action: the benefits of the mammalian gastrointestinal microbiota on the host. Environ. Microbiol. 11:2194–2206.

Bevan

E.R.

, Jones

A.M

, and Hawkey

P.M.

2017. Global epidemiology of CTX-M β-lactamases: temporal and geographical shifts in genotype. J. Antimicrob. Chemother. 72:2145–2155.

Davies

, and Davies

2010. Origins and evolution of antibiotic resistance. Microbiol. Mol. Biol. Rev. 74:417–433.

Collignon

, Aarestrup

F.M

, Irwin

, and McEwen

2013. Human deaths and third-generation cephalosporin use in poultry, Europe. Emerg. Infect. Dis. 19:1339–1340.

Tang

K.L.

, Caffrey

N.P

, Nóbrega

D.B

, Cork

S.C

, Ronksley

P.E

, Barkema

H.W

, Polachek

A.J

, Ganshorn

, Sharma

, and Kellner

J.D.

2017. Restricting the use of antibiotics in food-producing animals and its associations with antibiotic resistance in food-producing animals and human beings: a systematic review and meta-analysis. Lancet Planet. Health, 1:e316–e327.

Paterson

D.L.

, and Bonomo

R.A.

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

10.

Silva

K.C.

, Moreno

, Cabrera

, Spira

, Cerdeira

, Lincopan

, and Moreno

A.M

. 2016. First characterization of CTX-M-15-producing Escherichia coli strains belonging to sequence type (ST) 410, ST224, and ST1284 from commercial swine in South America. Antimicrob. Agents Chemother. 60:2505–2508.

11.

Kim

, Kang

H.W

, and Woo

G.J.

2015. Prevalence of CTX-M-15 extended-spectrum beta-lactamase-producing Salmonella isolated from chicken in Korea. Foodborne Pathog. Dis. 12:661–663.

12.

Abrar

, Hussain

, Khan

R.A

, Ul Ain

, Haider

, and Riaz

2018. Prevalence of extended-spectrum-β-lactamase-producing Enterobacteriaceae: first systematic meta-analysis report from Pakistan. Antimicrob. Resist. Infect. Control, 7:1–11.

13.

Abbas

, Khan

, and Mohsin

2019. High rates of CTX-M group-1 extended-spectrum β-lactamases producing Escherichia coli from pets and their owners in Faisalabad, Pakistan. Infect. Drug. Resist. 12:571–578.

14.

Ur Rahman

, Ahmad

, and Khan

2018. Incidence of ESBL-producing-Escherichia coli in poultry farm environment and retail poultry meat. Pak. Vet. J. 39:116–120.

15.

Bonham, P.A. 2009. Swab cultures for diagnosing wound infections: a literature review and clinical guideline. J. Wound Ostomy Continence Nurs. 36:389–395.

16.

Raza

, Mohsin

, Madni

W.A

, Sarwar

, Saqib

, and Aslam

2017. First report of blaCTX-M-15-type ESBL-producing Klebsiella pneumoniae in wild migratory birds in Pakistan. Ecohealth, 14:182–186.

17.

Rodríguez

, Barownick

, Helmuth

, Mendoza

M.C

, Rodicio

M.R

, Schroeter

, and Guerra

2009. Extended-spectrum β-lactamases and AmpC β-lactamases in ceftiofur-resistant Salmonella enterica isolates from food and livestock obtained in Germany during 2003–07. J. Antimicrob. Chemother. 64:301–309.

18.

Clermont

, Bonacorsi

, and Bingen

2000. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl. Environ. Microbiol. 66:4555–4558.

19.

Collingwood

, Kemmett

, Williams

, and Wigley

2014. Is the concept of avian pathogenic Escherichia coli as a single pathotype fundamentally flawed? Front. Vet. Sci. 1:1–5.

20.

Carattoli

, Bertini

, Villa

, Falbo

, Hopkins

K.L

, and Threlfall

E.J.

2005. Identification of plasmids by PCR-based replicon typing. J. Microbiol. Methods, 63:219–228.

21.

Seale

A.C.

, Gordon

N.C

, Islam

, Peacock

S.J

, and Scott

J.A.G.

2017. AMR Surveillance in low and middle-income settings-A roadmap for participation in the Global Antimicrobial Surveillance System (GLASS). Wellcome Open Res. 2:1–17.

22.

Government of Pakistan. 2017. Antimicrobial Resistance National Action Plan Pakistan. Available at https://www.nih.org.pk/wp-content/uploads/2018/08/AMR-National-Action-Plan-Pakistan.pdf, accessed May 18, 2018.

23.

Saleem

, Hassali

M.A

, and Hashmi

F.K.

2018. Pakistan's national action plan for antimicrobial resistance: translating ideas into reality. Lancet Infect Dis. 18:1066–1067.

24.

Mohsin

, Raza

, Schaufler

, Roschanski

, Sarwar

, Semmler

, Schierack

, and Guenther

2017. High prevalence of CTX-M-15-type ESBL-producing E. coli from migratory avian species in Pakistan. Front. Microbiol. 8:2476.

25.

Centre for Disease Dynamics, Economics & Policy. Situation analysis report on antimicrobial resistance in Pakistan. 2018. Available at https://cddep.org/wp-content/uploads/2018/03/Situational-Analysis-Report-on-Antimicrobial-Resistance-in-Pakistan.pdf, accessed June 2, 2018.

26.

Pesesky

M.W.

, Hussain

, Wallace

, Wang

, Andleeb

, Burnham

C.A

, and Dantas

2015. KPC and NDM-1 genes in related Enterobacteriaceae strains and plasmids from Pakistan and the United States. Emerging Infect. Dis. 21:1034–1037.

27.

Guenther

, Ewers

, and Wieler

L.H.

2011. Extended-spectrum beta-lactamases producing E. coli in wildlife, yet another form of environmental pollution?. Front. Microbiol. 2:1–13.

28.

Maciuca

I.E.

, Williams

N.J

, Tuchilus

, Dorneanu

, Guguianu

, Carp-Carare

, Rimbu

, and Timofte

2015. High prevalence of Escherichia coli-producing CTX-M-15 extended-spectrum beta-lactamases in poultry and human clinical isolates in Romania. Microb. Drug Resist. 21:651–662.

29.

Fallah

, Abdolghafoorian

, Hashemi

, Goudarzi

, Gachkar

, and Hamedany

2016. Antibiotic susceptibility patterns in CTX-M-15-producing Enterobacteraceae isolated from healthy Afghan refugees in Iran. Afr. J. Microbiol. Res. 10:357–362.

30.

Hasman

, Hammerum

A.M

, Hansen

, Hendriksen

R.S

, Olesen

, Agerso

, Zankari

, Leekitcharoenphon

, Stegger

, Kaas

R.S

, Cavaco

L.M

, Hansen

D.S

, Aarestrup

F.M

, and Skov

R.L.

2015. Detection of mcr-1 encoding plasmid-mediated colistin-resistant Escherichia coli isolates from human bloodstream infection and imported chicken meat, Denmark 2015. Euro Surveill. 20:1–5.

31.

Xia

, Fan

, Huang

, Xia

, Xiao

, Chen

, Xu

, and Zhuo

2014. Dominance of CTX-M-type extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolated from patients with community-onset and hospital-onset infection in China. PLoS One, 9:e100707.

32.

Liebana

, Carattoli

, Coque

T.M

, Hasman

, Magiorakos

A.P

, Mevius

, Peixe

, Poirel

, Schuepbach-Regula

, Torneke

, Torren-Edo

, Torres

, and Threlfall

2013. Public health risks of enterobacterial isolates producing extended-spectrum beta-lactamases or AmpC beta-lactamases in food and food-producing animals: an EU perspective of epidemiology, analytical methods, risk factors, and control options. Clin. Infect. Dis. 56:1030–1037.

33.

Parker

, Lemma

, and Randall

2016. Industry survey of Great Britain-sourced broilers for extended-spectrum beta-lactamase and Amp C beta-lactamase-producing Escherichia coli. Vet. Rec. 178:474.

34.

Dahmen

, Haenni

, Châtre

, and Madec

J.Y.

2013. Characterization of blaCTX-M IncFII plasmids and clones of Escherichia coli from pets in France. J. Antimicrob. Chemother. 68:2797–2801.

35.

Albrechtova

, Dolejska

, Cizek

, Tausova

, Klimes

, Bebora

, and Literak

2012. Dogs of nomadic pastoralists in northern Kenya are reservoirs of plasmid-mediated cephalosporin-and quinolone-resistant Escherichia coli, including pandemic clone B2-O25-ST131. Antimicrob. Agents Chemother. 56:4013–4017.

36.

J.H.

, Kim

, Bae

I.K

, Jeong

S.H

, Kim

S.H

, Lim

S.K

, Park

Y.H

, and Lee

2012. Dissemination of multidrug-resistant Escherichia coli in Korean veterinary hospitals. Diagn. Microbiol. Infect. Dis. 73:195–199.

37.

Kim

, Bae

I.K

, Jeong

S.H

, Chang

C.L

, Lee

C.H

, and Lee

2011. Characterization of IncF plasmids carrying the blaCTX-M-14 gene in clinical isolates of Escherichia coli from Korea. J. Antimicrob. Chemother. 66:1263–1268.

38.

Sidjabat

H.E.

, Paterson

D.L

, Adams-Haduch

J.M

, Ewan

, Pasculle

A.W

, Muto

C.A

, Tian

G.B

, and Doi

2009. Molecular epidemiology of CTX-M-producing Escherichia coli isolates at a tertiary medical center in western Pennsylvania. Antimicrob. Agents Chemother. 53:4733–4739.

39.

Sanjit

S.A.

, Lekshmi

, Prakasan

, Nayak

B.B

, and Kumar

2017. Multiple antibiotic-resistant, extended spectrum-beta-lactamase (ESBL)-producing Enterobacteria in fresh seafood. Microorganisms, 5:1–10.

40.

Qamar

M.U.

, Hannan

, Arshad

M.U

, and Arshad

2014. Metallo-β-lactamase producing Enterobacter cloacae: an emerging threat in neonates. Afr. J. Microbiol. Res. 8:525–528.

41.

Qamar

M.U.

, Nahid

, Walsh

T.R

, Kamran

, and Zahra

2015. Prevalence and clinical burden of NDM-1 positive infections in pediatric and neonatal patients in Pakistan. Pediatr. Infect. Dis. J. 34:452–454.

42.

Ur Rahman

, and Mohsin

2019. The under reported issue of antibiotic-resistance in food-producing animals in Pakistan. Pak. Vet. J. 1:1–16.

43.

Dahms

, Hübner

O.N

, Kossow

, Mellmann

, Dittmann

, and Kramer

2015. Occurrence of ESBL-producing Escherichia coli in livestock and farm workers in Mecklenburg-Western Pomerania, Germany. PLoS One, 10:e0143326.

44.

Schaufler

, Bethe

, Lubke-Becker

, Ewers

, Kohn

, Wieler

L.H

, and Guenther

2015. Putative connection between zoonotic multiresistant extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in dog feces from a veterinary campus and clinical isolates from dogs. Infect. Ecol. Epidemiol. 5:25334.

45.

Coque

T.M.

, Novais

, Carattoli

, Poirel

, Pitout

, Peixe

, Baquero

, Canton

, and Nordmann

2008. Dissemination of clonally related Escherichia coli strains expressing extended-spectrum beta-lactamase CTX-M-15. Emerg. Infect. Dis. 14:195–200.

46.

Peirano

, Richardson

, Nigrin

, McGeer

, Loo

, Toye

, Alfa

, Pienaar

, Kibsey

, and Pitout

J.D

. 2010. High prevalence of ST131 isolates producing CTX-M-15 and CTX-M-14 among extended-spectrum-β-lactamase-producing Escherichia coli isolates from Canada. Antimicrob. Agents Chemother. 54:1327–1330.

47.

Borjesson

, Ny

, Egervarn

, Bergstrom

, Rosengren

, Englund

, Lofmark

, and Byfors

2016. Limited dissemination of extended-spectrum beta-lactamase and plasmid-encoded AmpC-producing Escherichia coli from food and farm animals, Sweden. Emerg. Infect. Dis. 22:634–640.