Maintenance of Carbapenemase-Producing Enterobacteriaceae in a Farrow-to-Finish Swine Production System

Abstract

Carbapenemase-producing Enterobacteriaceae (CPE) threaten both agriculture and public health. While carbapenems are restricted in food-producing animals, other β-lactams, such as ceftiofur, are frequently applied in livestock. While the relationship is not fully elucidated, ceftiofur use may provide selective pressure that promotes carbapenem resistance. Recently reported in U.S. livestock, plasmid-mediated CPE are also present in livestock in Europe and Asia. We previously reported the rare carbapenemase gene, bla _IMP-64, in the environment of a large farrow-to-finish swine operation. To better understand CPE in this swine production system, in 2016 we followed a cohort of 350+ pigs over 5 months from late sow gestation to the final finishing phase. We screened both environmental and fecal samples for CPE using our selective enrichment protocol, with resulting phenotypic CPE isolates further characterized. Of 55 environmental and 109 sow fecal samples collected from a farrowing barn on our initial visit, 35 (64%) environmental and 15 (14%) sow fecal samples yielded isolates of multiple Enterobacteriaceae species carrying the metallo-β-lactamase gene bla _IMP-64 on an IncQ plasmid. The frequency of IMP-64-positive environmental (n = 32), sow fecal (n = 30), and piglet fecal swab (n = 120) samples was highest for all groups when the market pig cohort was between 1 and 10 days, with observed prevalence of 97%, 28%, and 18%, respectively. After weaning, bla _IMP-64 was detected in a single environmental sample from a nursery pen, with no CPE recovered in the finishing phase. Used in U.S. swine production to treat and control disease, ceftiofur is administered to piglets on this farm at birth, with males receiving a second dose at castration (≈day 7). Once introduced into animal agriculture, the common use of ceftiofur may provide the selection pressure required for CPE dissemination throughout large, intensively managed food animal populations housed in animal-dense environments.

Introduction

The global prevalence of carbapenemase-producing Enterobacteriaceae (CPE) is increasing. The expansion of this phenotype threatens the efficacy of multiple drug classes, including the critically important carbapenems. Both the CDC and WHO have listed carbapenem-resistant infections as highest level concerns to human health (Centers for Disease Control and Prevention, 2016; World Health Organization, 2017).

Problematically, plasmid-mediated CPE are often highly mobile, able to disseminate beyond the realm of human healthcare, and have been reported to cause community-acquired infections (Dortet et al., 2014; Khatri et al., 2015). CPE have also been reported in both companion animals (Shaheen et al., 2013; Abraham et al., 2016; Liu et al., 2016) and in livestock. Nine bla _OXA-23-harboring Acinetobacter genomospecies 15TU have been recovered from the enteric flora of dairy cattle in France (Poirel et al., 2012). A screening of animal diagnostic submissions at the Foshan University, Guangdong Province, China, revealed six Enterobacteriaceae isolates carrying bla _NDM-1 from diseased pig lung samples and included one ST48 Escherichia coli isolate, a common sequence type associated with human infection (Zhang et al., 2015). VIM-2-producing Pseudomonas aeruginosa and OXA-48-producing Acinetobacter baumannii have been detected in livestock fecal samples collected from farms in North Lebanon (Al Bayssari et al., 2015). Salmonella Infantis with bla _VIM-1 was found in both environmental and fecal samples from three swine farms and a poultry farm in Germany in 2011 and 2012. Later, two additional VIM-1 Salmonella Infantis isolates were identified from 2015/2016 submissions to the German National Reference Laboratory for Salmonella. These isolates, cultured from minced pork meat and a sick piglet, appeared clonally related based on pulsotype analysis, suggesting vertical transmission of the bla _VIM-1 genotype (Borowiak et al., 2017). In 2016, we reported a highly mobile IncQ1 plasmid carrying bla _IMP-64 in multiple Enterobacteriaceae species from the environment of a U.S. swine production system (Mollenkopf et al., 2017). Recently, 8 fecal samples from a sampling of 673 diarrheic and nondiarrheic piglets from 10 government pig farms in India were found to carry E. coli with bla _NDM (Pruthvishree et al., 2017).

Although still rare, CPE are now reported in livestock populations worldwide, but little is known about the epidemiology of carbapenemase-producing bacteria in these food animal settings. A better understanding of the on-farm epidemiology and transmission dynamics of CPE may lead to the development of effective prevention and control strategies. Therefore, our objective was to describe the dissemination and sample-level prevalence of Enterobacteriaceae harboring bla _IMP-64, from fecal and environmental samples in a cohort of market pigs, which we followed through a swine production flow.

Materials and Methods

All sampling occurred at a single multibarn swine farrow-to-finish production operation with high biosecurity located in the United States. We previously reported the presence of CPE harboring the single-base-change bla _IMP-27 variant, later identified as IMP-64 (GenBank accession number KX949735.2; Benson et al., 2017), in the environment of the farrowing and nursery barns at this farm (Mollenkopf et al., 2017). However, CPE were not reported from pig fecal samples at that time. Subsequent to the previous report, we identified a cohort of market pigs housed in the same barns to sample repeatedly over the entire production cycle.

On our initial visit, composite environmental and individual sow fecal samples were collected from all rooms in a single farrowing barn to confirm the presence of Enterobacteriaceae carrying bla _IMP-64 on an IncQ1 mobile plasmid. From each room, five composite environmental samples were collected using electrostatic cloths (Proctor & Gamble, Cincinnati, OH), which were then placed in sterile Whirl-paks. The individual environmental samples represented the room's exhaust fans, farrowing crate walls, mats, and overhead bars, and sow feeders.

After our initial visit in May 2016, two temperature-controlled farrowing rooms in the same barn with a total of 30 bred sows were identified and sampled on the day the rooms were filled (visit 2, July 5). The rooms were cleaned and disinfected using Tek-Trol^® (Bio-Tek Industries, Inc., Atlanta, GA) and bleach just before sow entry. Fecal samples were collected from these late-gestation sows. Environmental samples were collected with each electrostatic cloth representing an individual farrowing crate and an additional cloth used to sample the exhaust fans in each room. The progenies of these 30 sows were followed through the production flow to the finishing phase. For visits 3, 4, and 5 (July 15, 22, and 29), fecal samples were collected from the individual sows. Environmental samples of each farrowing crate and the barn exhaust fans were also collected as well as 120 piglet fecal swabs (liquid Stuart medium, BBL CultureSwab™, Becton Dickinson, Sparks, MD), with 2 gilts and 2 boars/barrows sampled from each crate to avoid duplicating samples. These piglets received ceftiofur at birth, with boars receiving a second dose at castration (≈ day 7).

At weaning, the piglets were moved to 15 pens in 2 rooms of a single temperature-controlled nursery barn. Each nursery room was cleaned using Tek-Trol and bleach before it was filled. Zinc (318 ppm) and copper (40 ppm) were included in the nursery diet, with newly weaned pigs receiving carbadox in feed for 10 days and neomycin sulfate in drinking water for 7 days. We sampled the pigs and their environment on August 9, the day after weaning, and September 6 (visits 6 and 7), collecting eight individual fecal swabs and two environmental cloth samples per pen. Sampled pigs were marked with paint sticks to prevent sample duplication. Exhaust fans in both rooms were also sampled with electrostatic cloths. In the finishing phase, the market pigs were housed in three consecutive pens of 100+ pigs each in a single partially open finishing barn. Twenty-five individual fecal samples and nine environmental electrostatic cloth samples were collected from each pen on September 28, 2016.

For the recovery of bla _IMP-64-harboring Enterobacteriaceae, all samples were processed in our laboratory on the day of sampling, with fecal samples reduced to 4 g and homogenized 1:9 with 36 mL MacConkey broth modified with 2 μg/mL cefotaxime. Fecal swabs were added to 9 mL MacConkey broth modified with 2 μg/mL cefotaxime, and 90 mL nutrient broth modified with 2 μg/mL cefotaxime was added to each environmental electrostatic cloth sample. All samples were incubated overnight at 37°C and inoculated to MacConkey agar supplemented with 0.5 μg/mL meropenem and 70 μg/mL zinc sulfate to identify the metallo-β-lactamase phenotype. After overnight incubation, up to three isolates representing unique morphologies from each plate were conserved for further characterization, with preference given to lactose-positive isolates. Conventional PCR of boiled-lysate template, utilizing previously reported primers (Mollenkopf et al., 2017), was used to detect bla _IMP-64 and IncQ alleles.

Results

We have previously reported the presence of the rare metallo-β-lactamase gene, bla _IMP-64, carried by multiple Enterobacteriaceae species, in the environment of this farm (Mollenkopf et al., 2017). For this study, we followed a cohort of 350+ market pigs from late sow gestation to the final finishing phase. We collected over 1100 samples (n = 1178) from pigs and their environment over the 5 months of this project. These samples comprised 274 environmental samples, 229 sow fecal samples, 600 pig fecal swabs, and 75 finisher pig fecal samples collected from the farrowing, nursery, and finishing barns over 8 farm visits (Fig. 1). From the 1178 samples collected from this swine production system, 301 carbapenem-resistant isolates were further characterized to determine their resistance genotype. After characterization, 286 Enterobacteriaceae isolates were confirmed to harbor the bla _IMP-64/IncQ1 gene/plasmid resistance genotype. These CPE were cultured from 195 positive samples (16.5%). Of these, the majority was recovered from environmental samples (n = 142), with an additional 30 positive samples from sows and 23 positive samples from the pig cohort. These bla _IMP-64/IncQ1-harboring isolates were composed of multiple unique morphologies suggestive of different Enterobacteriaceae species, with the majority, 99 environmental, 13 sow fecal, and 3 pig fecal swab isolates, being lactose positive. We did not further identify the species of bacteria recovered.

FIG. 1.

Sample prevalence of bla _IMP-64-harboring Enterobacteriaceae from sow fecal samples, piglet fecal swabs, finisher pig fecal samples, and electrostatic cloth environmental samples collected from a farrowing, nursery, and finishing barn of a single swine production flow over a 5-month period.

Our initial visit, in May 2016, confirmed that Enterobacteriaceae harboring bla _IMP-64 were present in the environment of the farrowing barn and in the fecal flora of the sows (Fig. 1). Of the 55 electrostatic cloth samples collected from throughout the farrowing environment, 60% were positive for isolates with IMP-64. Our convenience sampling of 109 sows, which ranged from late gestation to 26 days postfarrowing, yielded 15 samples (14%) with IMP-64 isolates.

After confirming the presence of IMP-64-bearing Enterobacteriaceae throughout the farrowing barn, we identified 30 late gestation sows housed in two adjacent rooms (Rooms A and B) of the same barn. On visit 2 (July 5, 2016), fecal samples were collected from sows within a few hours of being moved into the barn and loaded into farrowing crates with the exception of one farrowing sow that was not sampled. All sow fecal samples were negative for the presence of CPE. We were, however, able to detect bla _IMP-64 isolates in 28 of 32 (88%) electrostatic cloth samples collected, with two environmental samples from farrowing crates in Room A and a single crate in Room B negative.

All sows had farrowed by visit 3 (July 15, 2016), with the last litter born on the day of sampling. Of the 30 lactating sows, we collected fecal samples from 25, but were unable to collect a sample from 1 sow in Room A and 4 sows in Room B. Of the 25 sampled sows, 7 (28%) harbored bla _IMP-64 in their enteric flora. In addition, 21 of 120 (18%) piglet fecal swabs and 31 of 32 (97%) environmental cloths were positive for bacterial isolates carrying bla _IMP-64. Positive piglet swabs were originally collected from 5 litters in Room A and 8 litters in Room B, which included samples from the piglets farrowed earlier on the morning of sampling.

The following week, July 22, 2016 (visit 4), we again sampled sows, piglets, and their environment with similar results for the sows and their environment. On this visit, we collected 30 sow fecal samples with 7 (23%) of those positive for bacteria with bla _IMP-64. Of the environmental cloth samples, 28 of 32 (88%) yielded IMP-64-bearing Enterobacteriaceae. However, we only cultured isolates with bla _IMP-64 from 1 of 120 (0.8%) piglet fecal swabs.

Our last farrowing barn sampling, visit 5 (July 29, 2016), found only 1 sow fecal and 1 piglet fecal swab collected from that sow's litter carrying bacteria with bla _IMP-64. We also collected fewer positive environmental samples, with 22 of 32 (69%) electrostatic cloths positive. Interestingly, while no fecal samples or fecal swabs from Room A were found to carry IMP-64, the majority of positive environmental samples (64%) were collected in that room.

At weaning (August 8, 2016), the cohort of 350+ pigs was moved to two rooms in a single nursery barn. On visits 6 and 7 (August 9 and September 6, 2016), we collected 55 individual fecal swabs from pigs in Room A and 65 from pigs in Room B, along with 16 environmental samples from each room. Of these, only a single electrostatic cloth sample from the back wall of a pen in Room B (visit 6) yielded Enterobacteriaceae with bla _IMP-64.

Our final visit was September 28, 2016, approximately 3 weeks after the pigs were moved to three pens in a single finishing barn. We collected 25 fresh fecal samples and 9 environmental samples from upright surfaces in each pen. Of these 75 fecal and 27 electrostatic cloth samples, we did not recover any CPE isolates. Our study design included plans for one additional sampling of pigs and the environment in the finishing barn in December 2016 just before harvest, but the farm withdrew from the study before that final sampling was completed.

Discussion

Although believed to be extremely rare, the presence of CPE in intensively managed food animal populations is not surprising. The population-dense environments of modern livestock production systems lend to the dissemination of enteric flora from one animal to another, with each herd, barn, or flock essentially developing common flora. Once carbapenemase-producing bacteria are introduced into a livestock population, it can be hypothesized that the use of antimicrobials such as the veterinary extended-spectrum cephalosporin, ceftiofur, may help create an ecological niche favoring the maintenance of this β-lactamase-mediated resistance phenotype.

Piglets in this production system are treated for respiratory disease with ceftiofur at processing (day 1) with males treated again at castration (≈ day 7) with veterinary oversight under the control label. We collected fecal swabs from four piglets in each farrowing crate on July 15, 2016 (visit 3). The piglets were 1–10 days old and all litters had been processed and received ceftiofur with the exception of the litter farrowed early that day. Of the 120 piglet fecal swabs, 21 (17.5%) produced bla _IMP-64-positive isolates, including two samples from the piglets that had not yet received antimicrobial therapy. In contrast, fecal swabs again collected from 120 piglets 1 and 2 weeks later (7/22 and 7/29, visits 4 and 5), after boar castration and ceftiofur therapy on July 19, 2016, yielded only single IMP-64-positive sample (0.83%) at each visit. Although we do not know which animals were resampled from visit to visit, we would expect antimicrobial selection pressure to impact our ability to recover CPE from the enteric flora of these piglets on both sampling days.

We did not recover CPE from fecal swabs or samples after visit 5. Weaned pigs entering the nursery barn receive both carbadox in feed for 10 days and neomycin sulfate in drinking water for 7 days to control for swine dysentery and bacterial enteritis, suggesting that these antibiotics do not provide selection pressure favoring CPE. Also, the nursery diet is enhanced with zinc, which is required by metallo-β-lactamases for β-lactam hydrolysis (Palzkill, 2013). These observations suggest transient carriage and shedding of IMP-64 Enterobacteriaceae in these pigs rather than colonization.

The ability of ceftiofur selection pressure to amplify CPE has been hypothesized (Mollenkopf et al., 2017), but has not been established. Our observation that CPE were frequently present in sow fecal samples and common in the environment of this farrowing barn where ceftiour was routinely used, but not present in barns where ceftiofur use was rare, is consistent with the hypothesis that ceftiofur selection pressure can both maintain and amplify CPE. Conversely, our relatively low recovery of CPE from piglet samples following ceftiofur therapy suggests that other environmental factors present in this farrowing barn may be important in the maintenance of these CPE.

Transient carriage of IMP-64 could be a function of age as neonates have been shown to maintain unique microflora compared to older pigs (Mathew et al., 1998) and are at higher risk for carriage of some resistant Enterobacteriaceae species (Scott et al., 2005). The use of ceftiofur in piglets has been demonstrated to impact extended-spectrum cephalosporin resistance in E. coli recovered from the enteric flora of suckling piglets (Callens et al., 2015). However, applied studies in swine populations have demonstrated a rapid increase in resistant bacterial prevalence following antimicrobial therapy as well as a rapid decrease in resistance following drug withdrawal (Mathew et al., 2007). In the case of IMP-64, this rapid decrease may be further heightened by the dynamic nature of the neonatal intestinal microbiota and the maintenance of specific bacterial phenotypes such as found in E. coli for a few days to weeks (Katouli et al., 1995).

In this swine population, we found CPE throughout the farrowing barn environment with the exception of a single empty room sampled on visit 1. This room had been cleaned and disinfected using Tek-Trol and bleach on the day of sampling and would be filled with bred sows later that day. Environmental samples collected from the farrowing crate walls, mats, and overhead bars, and sow feeders in this room were all found to be negative for the presence of IMP-64. However, we cultured IMP-64 Enterobacteriaceae from the vents of the electric exhaust fans, which were not washed or disinfected.

If the farm's cleaning and disinfection routine consistently removes viable CPE from the pig environment, the IMP-64 isolates may be disseminated through the farrowing barn by air circulation or by the sows acting as either biological or mechanical vectors. In this study, we did not collect samples from the sow gestation barns, but these environments could serve as potential CPE reservoirs for this farm. Some proportion of the sow herd may be colonized with this resistance genotype and others may transiently harbor these CPE and be intermittently reinfected by the contaminated gestation barn environment.

After identifying the bred dams of the pig cohort to be followed, we sampled these bred sows and the farrowing room environment, a few hours after the rooms were filled. All but four environmental samples (28/32) were IMP-64 positive, but we did not recover CPE from any sow fecal samples (n = 29). We sampled the sows again 10 days later when they were 1–10 days postfarrowing with 28% (7/25) of fecal samples IMP-64 positive. While we did not measure stress indicators such as cortisol levels in the sows, parturition and the transition to a farrowing crate environment may play a role in our ability to detect carbapenemase producers in this population (Lawrence et al., 1994; Isaacson and Kim, 2012).

Although no intervention was used, the movement of piglets at weaning from the contaminated barn to a new environment, where there was no ceftiofur selection pressure, appeared to reduce the prevalence of IMP-64-bearing Enterobacteriaceae to below our detection limits. This is in direct contrast to the movement of sows into the apparently clean farrowing rooms, after which the environment in the rooms became contaminated by CPE, even before the application of ceftiofur. This result suggests the potential role of the sows, but not the piglets, as the reservoir of CPE at this farm. However, the finding of CPE in any segment of a healthy and well-managed pig production system indicates a clear need for remediation strategies for colonized farms. Reliable and economical antimicrobial alternatives may help reduce, if not eliminate, carbapenem-resistant phenotypes from livestock populations. While there are still relatively few reports of CPE in livestock populations worldwide, the increased prevalence of CPE in community-associated human infections (Dortet et al., 2014; Khatri et al., 2015), companion animals (Woodford et al., 2014; Abraham et al., 2016; Liu et al., 2016), and in the environment (Nordmann et al., 2011; Woodford et al., 2014) should signal that the prevalence of CPE in food-producing animals may be underestimated.

Footnotes

Acknowledgments

This work was supported by the National Institute of Food and Agriculture, U.S. Department of Agriculture, under award 2014-67005-21709, and by The Ohio State University Public Health Preparedness for Infectious Diseases Program.

Disclosure Statement

No competing financial interests exist.

References

Abraham

, O'Dea

, Trott

, Abraham

, Hughes

, Pang

, McKew

, Cheong

, Merlino

, Saputra

, Malik

, Gottlieb

. Isolation and plasmid characterization of carbapenemase (IMP-4) producing Salmonella enterica Typhimurium from cats. Sci Rep, 2016; 6:35527.

Al Bayssari

, Dabboussi

, Hamze

, Rolain

. Emergence of carbapenemase-producing Pseudomonas aeruginosa and Acinetobacter baumannii in livestock animals in Lebanon. J Antimicrob Chemother, 2015; 70:950–951.

Benson

, Cavanaugh

, Clark

, Karsch-Mizrachi

, Lipman

, Ostell

, Sayers

. GenBank. Nucleic Acids Res, 2017; 45:D37–D42.

Borowiak

, Szabo

, Baumann

, Junker

, Hammerl

, Kaesbohrer

, Malorny

, Fischer

. VIM-1-producing Salmonella Infantis isolated from swine and minced pork meat in Germany. J Antimicrob Chemother, 2017; 72:2131–2133.

Callens

, Faes

, Maes

, Catry

, Boyen

, Francoys

, de Jong

, Haesebrouck

, Dewulf

. Presence of antimicrobial resistance and antimicrobial use in sows are risk factors for antimicrobial resistance in their offspring. Microb Drug Resist, 2015; 21:50–58.

Centers for Disease Control and Prevention. Antibiotic Resistance Threats in the United States, 2013. Centers for Disease Control and Prevention, 2016. https://www.cdc.gov/drugresistance/threat-report-2013/pdf/ar-threats-2013-508.pdf, accessed May 23, 2017 .

Dortet

, Poirel

, Nordmann

. Worldwide dissemination of the NDM-type carbapenemases in Gram-negative bacteria. Biomed Res Int, 2014; 2014:249856.

Isaacson

, Kim

. The intestinal microbiome of the pig. Anim Health Res Rev, 2012; 13:100–109.

Katouli

, Lund

, Wallgren

, Kuhn

, Soderlind

, Mollby

. Phenotypic characterization of intestinal Escherichia coli of pigs during suckling, postweaning, and fattening periods. Appl Environ Microbiol, 1995; 61:778–783.

10.

Khatri

, Naeger Murphy

, Wiest

, Osborn

, Garber

, Hecker

, Hurless

, Rudin

, Jacobs

, Kalayjian

, Salata

, van Duin

, Perez

, Bonomo

, Paterson

, Harris

. Community-Acquired Pyelonephritis in Pregnancy Caused by KPC-Producing Klebsiella pneumoniae . Antimicrob Agents Chemother, 2015; 59:4375–4378.

11.

Lawrence

, Petherick

, McLean

, Deans

, Chirnside

, Gaughan

, Clutton

, Terlouw

. The effect of environment on behaviour, plasma cortisol and prolactin in parturient sows. Appl Anim Behav Sci, 1994; 39:313–330.

12.

Liu

, Thungrat

, Boothe

. Occurrence of OXA-48 carbapenemase and other β-lactamase genes in ESBL-producing multidrug resistant Escherichia coli from dogs and cats in the United States, 2009–2013. Front Microbiol, 2016; 7:1057.

13.

Mathew

, Cissell

, Liamthong

. Antibiotic resistance in bacteria associated with food animals: A United States perspective of livestock production. Foodborne Pathog Dis, 2007; 4:115–133.

14.

Mathew

, Upchurch

, Chattin

. Incidence of antibiotic resistance in fecal Escherichia coli isolated from commercial swine farms. J Anim Sci, 1998; 76:429–434.

15.

Mollenkopf

, Stull

, Mathys

, Bowman

, Feicht

, Grooters

, Daniels

, Wittum

. Carbapenemase-producing Enterobacteriaceae recovered from the environment of a swine farrow-to-finish operation in the United States. Antimicrob Agents Chemother, 2017; 61:e02348–16.

16.

Nordmann

, Naas

, Poirel

. Global spread of Carbapenemase-producing Enterobacteriaceae . Emerg Infect Dis, 2011; 17:1791–1798.

17.

Palzkill

. Metallo-β-lactamase structure and function. Ann N Y Acad Sci, 2013; 1277:91–104.

18.

Poirel

, Bercot

, Millemann

, Bonnin

, Pannaux

, Nordmann

. Carbapenemase-producing Acinetobacter spp. in Cattle, France. Emerg Infect Dis, 2012; 18:523–525.

19.

Pruthvishree

, Vinodh Kumar

, Sinha

, Malik

, Dubal

, Desingu

, Shivakumar

, Krishnaswamy

, Singh

. Spatial molecular epidemiology of carbapenem-resistant and New Delhi metallo beta-lactamase bla _NDM-producing Escherichia coli in the piglets of organized farms in India. J Appl Microbiol, 2017; 122:1537–1546.

20.

Scott

, Campbell

, Harvey

, Bischoff

, Alali

, Barling

, Anderson

. Patterns of antimicrobial resistance among commensal Escherichia coli isolated from integrated multi-site housing and worker cohorts of humans and swine. Foodborne Pathog Dis, 2005; 2:24–37.

21.

Shaheen

, Nayak

, Boothe

. Emergence of a New Delhi metallo-beta-lactamase (NDM-1)-encoding gene in clinical Escherichia coli isolates recovered from companion animals in the United States. Antimicrob Agents Chemother, 2013; 57:2902–2903.

22.

Woodford

, Wareham

, Guerra

, Teale

. Carbapenemase-producing Enterobacteriaceae and non-Enterobacteriaceae from animals and the environment: An emerging public health risk of our own making?. J Antimicrob Chemother, 2014; 69:287–291.

23.

World Health Organization. Global Priority List of Antibiotic-Resistant Bacteria to Guide Research, Discovery, and Development of New Antibiotics. Essential Medicines and Health Products. World Health Organization, 2017. http://www.who.int/medicines/publications/WHO-PPL-Short_Summary_25Feb-ET_NM_WHO.pdf?ua=1, accessed May 16, 2017 .

24.

Zhang

, Yang

, Zhihai

, Jiyun

, Wenjuan

, Lei

, Congming

, Jianzhong

. Characterization of NDM-1-producing carbapenemase in Acinetobacter spp. and E. coli isolates from diseased pigs. Front Agr Sci Eng, 2015; 2:223–229.