Prevalence of Extended-Spectrum Beta-Lactamase-Producing Gram-Negative Bacilli and Emergence of mcr-1 Colistin Resistance Gene in Lebanese Swine Farms

Abstract

Livestock are considered reservoirs of multidrug-resistant organisms that can be transferred to humans through direct/indirect routes. Once transmitted, these organisms can be responsible for infections with therapeutic challenges. The aim of this study was to determine the prevalence of extended-spectrum cephalosporin and colistin-resistant Gram-negative bacilli in Lebanese swine farms. In May 2017, 114 fecal samples were collected from swine farms in south Lebanon. Separate media supplemented with cefotaxime, ertapenem, and colistin were used for the screening of resistant organisms. Double-disk synergy test and ampC disk test were performed to detect extended-spectrum beta-lactamase (ESBL) and ampC producers, respectively. Detection of beta-lactamase and mcr genes was performed using real time PCR. Of 114 fecal samples, 76 showed growth on the medium with cefotaxime. In total, 111 strains were isolated with 94.5% being Escherichia coli. Phenotypic tests showed that 98, 6, and 7 strains were ESBL, ampC, and ESBL/ampC producers, respectively. CTX-M and CMY were the main beta-lactamase genes detected. On the medium with colistin, 19 samples showed growth. In total, 23 colistin-resistant E. coli strains harboring the mcr-1 gene were isolated. This is the first study in Lebanon determining multidrug resistance epidemiology in pigs. The prevalence of ESBLs is high and the emergence of colistin resistance is alarming.

Introduction

Resistance in Gram-negative bacilli toward the most common antibiotics administered in the human medicine, that is, beta-lactams has significantly increased in the past decade.¹ Resistance to beta-lactams and carbapenems in Gram-negative bacteria is mainly mediated through the production of extended-spectrum beta-lactamases (ESBLs), ampC beta-lactamases, and carbapenemases.¹ Genes encoding these enzymes are often colocalized on plasmids harboring resistance genes to other commonly prescribed antibiotics in human medicine such as aminoglycosides and quinolones.¹ Dissemination of resistant organisms often results in reducing the efficacy of beta-lactam antibiotics, thus limiting treatment options of infectious diseases.²

This is currently emphasized with the recent emergence of colistin resistance in Gram-negative bacilli. Colistin belongs to the polymyxin antibiotics family that acts on the lipopolysaccharide (LPS) chain of the bacteria and leads to increased permeability of the outer membrane and subsequent cellular leakage followed by cell death.³ In human medicine history, colistin was abandoned because of its nephrotoxicity and neurotoxicity inside human body.⁴ However, because of the widespread multidrug-resistant (MDR) organisms, mainly carbapenem-resistant organisms, colistin was reintroduced in clinical settings.⁵ This antibiotic revival had to face the emergence of colistin resistance in bacteria of human and animal origin.⁶

Before 2015, colistin resistance was thought to be only mediated through chromosomal mutations that leads to the alteration of the lipid A subunit of the LPS chain by the addition of 4-amino-4-deoxy-l-arabinose (l-Ara4N) and/or phosphoethanolamine (PEtN),⁶ thus resulting in a reduced binding to colistin and subsequently bacterial resistance.⁶ However, in 2016, Liu et al. reported the first detection of a transferable phosphoenolamine transferase named mcr-1 gene in Escherichia coli strains isolated from pigs and meat.⁷ In this context, mcr-1 was reported from clinical and animal isolates across all continents. Furthermore, mcr variants, that is, mcr-2,⁸ mcr-3,⁹ mcr-4,¹⁰ and mcr-5¹¹ have also emerged.

Nowadays, farm animals are considered reservoirs of antimicrobial resistance.¹² The unregulated use of antibiotics is considered among the most common drivers for the emergence of resistance in livestock.¹³ Indeed, antibiotics are given not only for treatment but are also prescribed for prophylaxis and administered as growth promoters.¹³ The major public health concern about multidrug resistance spread in animals is the potential transmission to humans through direct contact or indirectly through the consumption of undercooked/uncooked animal-origin food.¹⁴ Once transmitted, these organisms can cause infections with limited therapeutic options, especially those cross-resistant to antibiotics frequently used in the human medicine.¹⁵

In Lebanon, the dissemination of MDR organisms in the clinical settings is well documented^16–20; however, studies addressing multidrug resistance in animals remain scarce. One study carried by Diab et al. showed a relatively high prevalence of the CTX-M-15 ESBL type in E. coli of cattle origin in Lebanon.²¹ More recently, a nationwide study conducted in Lebanese chicken farms reported an elevated level of ESBL/ampC-producing Gram-negative bacilli intestinal carriage.²² Recently, our group reported the first detection of an E. coli isolated from poultry in south Lebanon harboring the mcr-1 colistin resistance gene in addition to the TEM-135-like ESBL gene.²³ In pigs, only one study reported the detection of an OXA-23-producing Acinetobacter baumannii in northern Lebanon.²⁴ The prevalence of MDR organisms in the Lebanese swine farms remains unknown. In collaboration with the ministry of agriculture, the aim of this study was to determine the prevalence of extended-spectrum cephalosporin and colistin-resistant Gram-negative bacilli in Lebanese swine farms.

Materials and Methods

Ethics statement and collection of samples

The Ministry of Agriculture in Lebanon approved the collection of fecal samples from swine farms. The sampling was realized in compliance with the national guidelines for animal safety. On May 30, 2017, 111 fecal samples were randomly collected from 3 different swine farms located in south Lebanon. In addition, three fecal samples were taken from three wild pigs living in the same region. The number of samples collected was relatively proportional to the farms size that ranged from 20 to 120 pigs per farm (Table 1). The fecal samples were collected using sterile urine cups and directly placed in a portable refrigerator; then when taken to the university laboratory, they were stored at −80°C until being used.

Table 1.

Distribution of Extended-Spectrum Beta-Lactamase/ampC-Producing and Colistin-Resistant Gram-Negative Bacilli per Farm

	AB used	Collected samples, n	ESBLs/ampCs samples, n	ESBLs/ampCs isolates, n	Species	Col/R samples, n	Col/R isolates, n	Species
Farm 1 (n = 120)	Enrofloxacin	60	42	65	60 Escherichia coli, 4 Escherichia fergusonii, 1 Klebsiella pneumoniae	8	8	E. coli
Farm 2 (n = 20)	Unknown	15	8	9	8 E. coli, 1 K. pneumoniae	4	5	E. coli
Farm 3 (n = 100)	Unknown	36	24	35	E. coli	7	10	E. coli
WP (n = 3)	Unknown	3	2	2	E. coli	0	0

AB, antibiotic; Col/R, colistin resistant; ESBLs, extended-spectrum beta-lactamases; WP, wild pigs.

Screening of resistant organisms and identification

Each fecal sample was mixed in a sterile container and then a swab was used to subculture a considerable amount on MacConkey agars supplemented separately with cefotaxime (2 μg/mL), ertapenem (1 μg/mL), and colistin (4 mg/L) for the screening of resistant Gram-negative bacilli. After overnight incubation at 37°C, isolated colonies with different morphologies were separately taken from each plate and identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with a score value ≥1.9 using the Microflex LT spectrometer (Bruker Daltonics, Bremen, Germany).²⁵ Thereafter, the strains were conserved in 40% glycerol aliquots at −80°C for further tests.

Antibiotic susceptibility testing

Antibiotic susceptibility testing was performed using the Kirby–Bauer disk diffusion method and interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines 2017.²⁶ A total of 16 antibiotics were tested involving 11 beta-lactams (ampicillin, amoxicillin–clavulanic acid, aztreonam, cefotaxime, ceftazidime, cefoxitin [FOX], cefepime, piperacillin–tazobactam, ertapenem, meropenem, imipenem) and 5 non-beta-lactams (colistin, gentamicin, ciprofloxacin, trimethoprim–sulfamethoxazole and tigecycline; Bio-Rad, Marnes-la-Coquette, France). The phenotypic detection of ESBL was performed using the double-disk synergy test by placing an amoxicillin–clavulanic acid disk between cefepime, ceftazidime, and aztreonam. Formation of a keyhole effect was considered as a phenotypic indication of ESBL production. Regarding screening of ampC beta-lactamase and carbapenemase production, ampC disk test and carba NP test were performed, respectively, as previously described.^27,28 Furthermore, all isolates having a narrow diameter zone of inhibition around the colistin disk were subjected to colistin broth microdilution test as previously described.²⁶ An isolate is termed as MDR if this latter was resistant to three different classes of antibiotics at least.²⁹

PCR identification of beta-lactamase genes

All isolates showing a keyhole effect or having resistance to both cefoxitin and cefepime were subjected to real-time PCR analysis for blaCTX-M, blaSHV, and blaTEM genes screening.³⁰ Furthermore, all strains found positive to the ampC disk test were also tested for genes encoding ampC beta-lactamases FOX, MOX, ACC, EBC, DHA, and CMY using simplex PCRs.³¹ DNA extraction was performed using EZ1 DNA extraction kit (Qiagen, Courtaboeuf, France), following manufacturer instructions with an EZ1 Advanced XL biorobot.

Molecular characterization of mcr-1 colistin resistance gene

All strains having a colistin minimum inhibitory concentration (MIC) ≥2 μg/mL were subjected to standard PCR amplification and sequencing for the detection of mcr-1 colistin resistance gene. DNA extraction was carried out using an EZ1 DNA extraction kit (Qiagen) with an EZ1 Advanced XL biorobot. Primers used in molecular analysis were previously described in other studies.³²

Results

Prevalence of beta-lactamase producers and colistin-resistant Gram-negative bacilli

Of 114 fecal samples collected, 76 (66.5%) showed positive growth on the selective medium supplemented with cefotaxime. In total, 111 MDR strains were isolated according to the following distribution: 65 strains in farm 1, 9 in farm 2, 35 in farm 3, and 2 isolates from the wild pigs. MALDI-TOF MS identification revealed that E. coli made up to 94.5% of isolated MDR strains, Escherichia fergusonii 3.5%, and Klebsiella pneumoniae 2% (Table 1). Besides, 23 colistin-resistant E. coli strains isolated from 19 fecal samples were obtained. No carbapenemase producers were detected in this study.

Phenotypic profiles of beta-lactamase producers

The resistance profiles of isolated ESBL and/or ampC-producing Gram-negative bacilli are summarized in Table 2. All ESBL/ampC-producing strains were susceptible to colistin and carbapenems. Carba np test, double-disk synergy test, and ampC disk test revealed the absence of carbapenemase producers, 98 isolates (88.5%) were categorized as ESBL producers, 7 (6%) as ESBL/ampC coproducers, and 6 strains (5.5%) as solely ampC producers. K. pneumoniae isolates were only ESBL producers, whereas three E. fergusonii were categorized as ampC producers and one as an ESBL producer. Coproduction of ESBL and ampC was only detected in E. coli isolates. Regarding non-beta-lactam antibiotics resistance in the aforementioned strains, one isolate was coresistant to all non-beta-lactams tested: tigecycline, gentamicin, ciprofloxacin, and trimethoprim–sulfamethoxazole, 32 (29%) were co-resistant to 3 non-beta-lactams, 59 (53%) to 2 non-beta-lactams, 16 (14%) to 1 non-beta-lactam, and three strains were susceptible to all non-beta-lactam antibiotics. Overall, 83% of beta-lactamase-producing Gram-negative bacilli in this study were co-resistant to at least two non-beta-lactams.

Table 2.

Resistance Profiles of Extended-Spectrum Beta-Lactamase/ampC-Producing Gram-Negative Bacilli

Antibiotic susceptibility testing													Phenotype
Species	AMP	CTX	AZT	FOX	CAZ	AUG	FEP	PTZ	TGC	SXT	CIP	GNT	% of ESBL	% of ampC	% of ESBL/ampC
E. coli (n = 105)	103 (98)	70 (67)	45 (43)	25 (24)	44 (42)	48 (46)	57 (54)	1 (1)	1 (1)	97 (92)	82 (78)	44 (42)	90	3	7
E. fergusonii (n = 4)	4 (100)	2 (50)	3 (80)	4 (100)	4 (100)	4 (100)	0 (0)	0 (0)	0 (0)	1 (20)	4 (100)	3 (80)	20	80
K. pneumoniae (n = 2)	2 (100)	2 (100)	1 (50)	0 (0)	1 (50)	1 (50)	2 (100)	0 (0)	0 (0)	1 (50)	1 (50)	0 (0)	100

Resistance profiles are presented as number (percentage).

AMP, ampicillin; AUG, amoxicillin–clavulanic acid; AZT, aztreonam; CAZ, ceftazidime; CIP, ciprofloxacin; CTX, cefotaxime; FEP, cefepime; FOX, cefoxitin; GNT, gentamicin; SXT, trimethoprim–sulfamethoxazole; TGC, tigecycline; PTZ, piperacillin–tazobactam.

Molecular characterization of beta-lactamase genes

A total of 105 Gram-negative bacilli having ESBL phenotypes were subjected to real-time PCR analysis for the screening of CTX-M-, TEM-, and SHV-encoding genes. CTX-M was detected in 83 (79%) ESBL isolates, TEM in 57 (54%), and SHV in 9 (8.5%). In total, 12 strains (11%) showed the coexistence of the 3 bla genes together, 43 (41%) showed the coexistence of 2 bla genes, and 57 (54%) harbored only 1 beta-lactamase gene. In addition, CMY was the only ampC-encoding gene detected in ampC and ESBL/ampC coproducers.

Colistin-resistant isolates: resistance profiles and genotype

The detailed profile of the resistance of E. coli colistin-resistant strains isolated in this study is given in Fig. 1. To summarize, 4 of the 23 strains were colistin resistant and also ESBL producers, whereas the remaining strains (19 isolates) were susceptible to all beta-lactams tested, except for ampicillin. Resistance rates toward non-beta-lactam antibiotics varied: eight strains were co-resistant to gentamicin, ciprofloxacin, and trimethoprim–sulfamethoxazole, seven strains were resistant to two non-beta-lactams, two were resistant to only one non-beta-lactam antibiotic and six strains were susceptible to all non-beta-lactams tested. Colistin MICs of the 23 E. coli isolates ranged between 4 and 16 μg/mL except 1 strain having an MIC of 256 μg/mL. Standard PCR and sequencing revealed that all the strains were mcr-1 positive. In the four ESBL mcr-1-positive resistant isolates, CTX-M was detected in two strains, whereas SHV and TEM were detected in all four strains (Fig. 1).

FIG. 1.

Resistance profiles of mcr-1 colistin-resistant Escherichia coli isolates. AMP, ampicillin; AUG, amoxicillin–clavulanic acid; AZT, aztreonam; bla, beta-lactamase; Carb, carbapenems; CAZ, ceftazidime; CIP, ciprofloxacin; CTX, cefotaxime; FEP, cefepime; FOX, cefoxitin; GNT, gentamicin; R, resistant; S, sensitive; SXT, trimethoprim–sulfamethoxazole; TGC, tigecycline; PTZ, piperacillin–tazobactam.

Discussion

Antimicrobial resistance is rapidly evolving and disseminating worldwide. In the context of antimicrobial resistance in the one health concept, livestock (i.e., pigs, poultry, and cattle) is now considered as a major reservoir of MDR organisms and antibiotic resistance genes.¹² In Lebanon, few studies have been conducted to determine the prevalence of MDR organisms in Lebanese livestock²¹; however in pork, only one study reported the detection of a carbapenemase-producing A. baumannii isolate from a pig in northern Lebanon.²⁴ To the best of our knowledge, our study is the first in Lebanon to describe the epidemiology of beta-lactamase-producing Gram-negative bacilli in Lebanese swine farms. It is worth mentioning that the number of samples collected was not relatively high because only few swine farms are accessible in Lebanon. The role of the Ministry of Agriculture was essential to carry out this study because it provided the legal permission to access and sample the different sites.

In our investigation, ESBL/ampC-producing Gram-negative bacilli were detected in 66.5% of the collected fecal samples (Table 1). Compared with other epidemiological studies investigating pigs worldwide, the prevalence in Lebanon is not far from what is reported in Belgium (75%)³³ and Germany (88%)³⁴ but is still much higher than those reported in China (32%),³⁵ United Kingdom (23%),³⁶ Denmark (18.5%),³⁷ Switzerland (15%),³⁸ and Thailand (2.4%).³⁹ Differences in the number of samples and screening methodologies, in addition to the level and type of antibiotics prescribed in the farms of each country could explain these differences.³ The aforementioned concept applies also to prevalence of mcr-1-positive E. coli strains detected in our previous study (17%) compared with other international studies: Portugal (98%),⁴⁰ Vietnam (37.5%),⁴¹ China (20.6%),⁷ Japan (1%),⁴² France (0.5%)⁴³ and United States (0.35%).⁴⁴

In this study, 83% of ESBL/ampC producers were co-resistant to at least two non-beta-lactam antibiotics with the highest level of resistance being observed against trimethoprim–sulfamethoxazole and ciprofloxacin. During our samples collection, we tried hard to collect correct data on the types and quantities of antibiotics used in the different farms, a mission nearly impossible. Indeed, despite the official presence of the Ministry of Agriculture, the cooperation of the farm owners was not easy to get, and there was no clear distinction between different uses of antibiotic in farms investigated (treatment of infections, prevention on infection, and growth enhancement). Unofficially, we were informed that enrofloxacin is frequently administered to pork in Lebanon. In fact, it has been reported that in pigs, penicillins are used to treat necrotic enteritis, whereas cephalosporins such as cefquinome and ceftiofur are prescribed for polyarthritis, septicemia, polyserositis, and respiratory infections.² Use of non-beta-lactams such as gentamicin, fluoroquinolones, aminoglycosides, and colistin was also reported.^45,46

On the contrary, it is not clear to us to which extent international guidelines and recommendations for hygiene and waste management in pig farms are applied in our country. Questionable hygiene, poor feed quality, and bad waste management imply another important drive in the emergence of multidrug resistance in pigs in addition to the overuse of antibiotics that facilitates the transmission of resistant organisms from pigs to their surrounding environment and vice versa. At the molecular level, the most commonly detected beta-lactamase gene was the CTX-M. This gene was highly reported in Lebanon in the clinical settings^16,47 and in cattle²¹ and poultry.²² CTX-M is also the main ESBL type reported globally in farm animals.^36,37,39,48 As for ampC producers, this study showed that CMY was the only ampC beta-lactamase gene detected in swine farms in Lebanon. The same observation was also made in chicken farms (data not given). It has been shown worldwide that this gene is the most common ampC beta-lactamase gene detected in poultry,^49,50 food-producing animals,^51,52 and healthy pets.^53,54 In this study, it has not escaped our attention that no carbapenemase producers were detected. This is in accordance with another study performed by our group in poultry farms²² reflecting that carbapenemase producers are really scarce in Lebanese livestock.

Furthermore, in this study we report for the first time the detection of mcr-1 in pork in Lebanon. In this country, mcr-1 gene was first reported in chicken during an epidemiological study aiming at determining the prevalence of MDR organisms in Lebanese chicken farms.²³ The MIC values of colistin in mcr-1-producing E. coli isolates in this study range between 4 and 16 μg/mL. These results are in accordance with other studies showing that mcr-1-harboring isolates do not usually have elevated colistin MICs.^55,56 Some reports showed that mcr-1-positive E. coli isolate could have a colistin MIC as low as 2 μg/mL.⁵⁷ In our collection of mcr-1 strains, only one ESBL-producing E. coli had a colistin MIC of 256 μg/mL. This elevated MIC might be attributed to additional chromosomal mutations in the phoP/Q, pmrA/B, and mgrB genes as reported previously in the literature.⁵ However, further genomic analysis is needed to explore this possibility. Delannoy et al. reported the isolation of E. coli strains harboring mcr-1 and having amino acid mutations in the phoP/Q, pmrA/B, and mgrB genes from diseased pigs in France.⁵⁷

Furthermore, it is worth mentioning that, as given in Fig. 1, none of the colistin-resistant isolates was pandrug resistant, but rather remained susceptible to the majority of the tested antibiotics, except four strains that were ESBL producers. The coexistence of mcr-1- and ESBL/carbapenemase-encoding genes was previously reported in several studies in the literature.^58,59 Resistance profiles of mcr-1 strains in this study possibly illustrate an overestimated fear of colistin resistance. E. coli colistin-resistant isolates will pose therapeutic challenges only if transmission of MDR strains to humans occurs.

In conclusion, this study describes the epidemiology of ESBL/ampC-producing Gram-negative bacilli in Lebanese swine farms. The emergence of mcr-1 in pigs is alarming. The level of antibiotic consumption in Lebanese swine farms remains unknown; a more transparent policy should be adopted in this context. Therefore, the surveillance and control programs addressing antibiotic consumption in Lebanese farms, especially in pigs, are urgently needed. Future studies should not only focus on antimicrobials usage but also on the risk factors associated with the carriage of MDR organisms in pigs.

Footnotes

Acknowledgments

The authors thank CookieTrad for English corrections. This work was supported by the Lebanese Council for Research and the French Government under the “Investissements d'avenir” (Investments for the Future) program managed by the Agence Nationale de la Recherche (ANR, fr: National Agency for Research), reference: Méditerranée Infection 10-IAHU-03.

Disclosure Statement

No competing financial interests exist.

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