Molecular Characterization of β-Lactamase-Producing Escherichia coli Collected from 2001 to 2011 from Pigs in Korea

Abstract

To survey the diversity of plasmid-mediated β-lactamases, and the replicon types of the plasmids in Escherichia coli in the community, 95 multidrug-resistant E. coli isolates from stool samples of pigs in Korea from 2001 to 2011 were analyzed, and all isolates were confirmed as β-lactamase-producing bacteria, which mostly carried genes encoding TEM (100%), ACC (95%), SHV (62%), OXA (28%), CTX-M (26%), and NDM-1 (15%). Moreover, each isolate contained at least three β-lactamases genes on its plasmid. The polymerase chain reaction–based replicon typing method revealed that among the 18 replicon types, 15 types were identified in this work, and IncF groups (FIB; 100%, Frep; 99%) and HI1 (98%) type were predominant. Other replicon types such as Y, B/O, A/C, P, FIA, N, HI2, I1, and W were also detected; moreover at least three types were identified in each organism. In conclusion, a high prevalence of various types of β-lactamases genes were identified in E. coli from pigs in Korea, and these genes appeared to be mainly associated with IncF group, IncHI1, and IncY plasmids.

Introduction

β-Lactamase-producing Enterobacteriaceae have emerged as a great concern in public health worldwide (Nordmann et al., 2012; Wu et al., 2015). Infections with these bacteria result in increased morbidity, mortality, and cost of therapy (Hawkey and Jones, 2009). Moreover, they have real risks of disseminating resistance among other bacteria species in animals and humans (Wellington et al., 2013). One of the most significant reservoirs for β-lactamases transmission is the guts of humans and food-producing animals, whether or not they were treated with antibiotics before (Machado et al., 2008; Wellington et al., 2013). Moreover, these bacteria have been detected in healthy food-producing animals in Europe (Cortes et al., 2010; Geser et al., 2012) and Asia (Wu et al., 2012). Antibiotics have been used in the animal industry therapeutically, preventively, and as growth promoters (Shea, 2004), especially in swine production (Aarestrup et al., 2008). In the United States, 40–80% of the antimicrobial agents are used for subtherapeutic purposes in food animals each year (Shea, 2004). However, low doses of antibiotics used for a prolonged period not only help to produce larger animals by enhancing feed efficacy but also produce large reservoirs of multidrug-resistant Enterobacteriaceae (Shea, 2004). As a consequence of extensive use of antibiotics in swine production, multiple antimicrobial resistances have emerged among common swine pathogens (Aarestrup et al., 2008). Among Enterobacteriaeceae, Escherichia coli is a very common pathogen of swine disease, such as neonatal scours, postweaning diarrhea, arthritis, cystitis, and navel infections. Thus, in swine farms, application of antibiotics for controlling E. coli was started in 1-day-old pigs, which caused occurrence of multidrug-resistant strains, as well as resistance to β-lactams (Aarestrup et al., 2008). In E. coli, production of β-lactamases is the most common mechanism of resistance against β-lactams, including carbapenems, and several of these enzymes are encoded by bla genes, which often are associated with plasmids (Carattoli, 2009). Plasmid-encoding β-lactamases have been classified into four Ambler classes: A, B, C, and D (Ambler, 1980). Class A β-lactamases include KPC, TEM, SHV, and CTX-M enzymes. With a serine active site at position 70, they can hydrolyze a variety of β-lactams and carbapenems. They can be partially inhibited by clavulanic acid. Class B metallo-β-lactamases (MBL) are mostly of VIM, IMP, and NDM types. These enzymes can hydrolyze all β-lactams except aztreonam. They are not inhibited by clavulanic acid. However, they are inhibited by ethylene diaminetetraacetic acid. Class C β-lactamases have little sequence similarities to class A. They include CMY, FOX, and ACC. Class D β-lactamases are recently identified enzymes known as OXA type β-lactamases (Ambler, 1980). Some bla genes are present in different plasmids and some often coexist on the same plasmid (Carattoli, 2009). Bacterial plasmid, extrachromosomal DNA are not essential for survival or growth of bacteria but encode various traits such as virulence, heavy metal and antibiotic resistance, and horizontal gene transfer by encoding mobile genetic elements (Carattoli, 2009). Plasmids also contain the replication system called replicon, which consist of an origin of replication (cop), genes involved in control of the initiation of replication (inc), and genes encoding proteins required for replication and its control (rep) (Couturier et al., 1988). Classification and identification of plasmids are based on the differences found in the replication system and by studying incompatibility (Inc) type (Couturier et al., 1988). In the same cell line, Inc is defined as the inability of coexistence of plasmids, which share common elements involved in plasmid replication control or partitioning system (Novick, 1987).

Among Inc type plasmids, particular types are more frequently identified in multidrug-resistant E. coli strains and these play a role in dissemination of resistance (Antunes et al., 2010). IncI1, IncN type plasmids encoding β-lactamases (CTX-M, CMY-2, SHV-12, and TEM-1) were identified in Europe and the United States (Moodley et al., 2009; Antunes et al., 2010), which meant that resistant plasmids could transfer between food-producing animals and humans (Antunes et al., 2010). Recently, IncFII, IncA/C, IncN, and IncI1 plasmids carrying ESBL genes are considered epidemic-resistant plasmids in Enterobacteriaeceae (Carattoli, 2009). For Inc type grouping, using polymerase chain reaction–based replicon typing (PBRT) methods (Carattoli et al., 2005), 18 pairs of primers (FIA, FIB, FIC, HI1, HI2, I1-Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, and FII replicon) are designed for 5-multiplex and 3-simplex PCRs to detect the major plasmid Inc groups among Enterobacteriaceae (Couturier et al., 1988).

Bacteria have been evolving an ability to resist antibiotics along with the discovery of new antibiotics by transfer of antibiotic resistance genes (Toleman et al., 2006a). The transfer of antibiotic resistance genes among bacteria is mostly driven by transposons-bearing wide host ranging plasmids (Toleman et al., 2006b). Resistance genes are linked to special sequences, called common regions (CRs), which are often found beyond but close to the 3′ conserved sequences of class 1 integrons (Batchelor et al., 2005; Toleman et al., 2006a). These CRs, designated ISCR elements (Toleman et al., 2006b), are related and resemble an unusual class of insertion sequences (IS) such as IS91 (Batchelor et al., 2005; Toleman et al., 2006a). ISCR elements are transposed by a process called rolling-circle replication and, consequently, they can transpose adjacent DNA sequences found upstream of their transposase genes (Toleman et al., 2006a). Studies about the role of ISCR and other mobile elements in plasmids, which have a wide range of hosts and contain multiple antibiotic resistance genes such as tetA (tetracycline), sul1, 2 (sulfanilamide), FloR (chloramphenicol/florfenicol), and bla _NDM-1, have revealed that mobilization and duplication of the β-lactamase genes are mediated by ISEcp1 (Arduino et al., 2002; Bratu et al., 2005; Call et al., 2010). Modules in these plasmids containing antibiotic resistance genes also contained mobile elements, including the Tn21 transposon and the ISCR2, ISCR16, and IS26 insertion sequences (Qi et al., 2008). Consequently, plasmids seem to use mobile elements to gain and accumulate different antibiotic resistance genes (Féria et al., 2002).

The primary aim of this work was to investigate the presence and the distribution of plasmid Inc groups and plasmid-mediated β-lactamase genes in multidrug-resistant E. coli isolates collected from food-producing animals (e.g., pigs) in Korea, and furthermore, to elucidate the phylogenetic relationships among β-lactamase gene-encoding plasmids by PBRT methods. Therefore, this research endeavors to better understand the relationship of these E. coli isolates in pigs and the possible transmission to humans.

Materials and Methods

E. coli isolates and susceptibility testing

A total of 469 E. coli isolates (329 from Chonnam, 50 from Gyeongsang, 50 from Chungcheong, and 40 from Gyeonggi Province) were obtained from fecal samples of pigs in Korea between 2001 and 2011. They were directly collected from fecal samples of rectums of farmed pigs (two or three randomly selected at each farm). All isolates were identified using standard biochemical testing (Gram staining, TSI, indole, methyl-red-Voges-Proskauer, and urease). Antibiotic susceptibility tests were performed for each isolate using the disk-diffusion method on Müller-Hinton agar with β-lactam antibiotics (penicillin, P; ampicillin, AM; amoxicillin, AMC; ticarcillin, TIC; ticarcillin/clavulanic acid, TIM; cephalothin, CF; cefepime, FEP; cefoxitin, FOX; cefotaxime, CTX; imipenem, IPM; aztreonam, ATM; ceftazidime, CAZ; ceftiofur, CFT) and non-β-lactam antibiotics (spectinomycin, SP; erythromycin, E; lincomycin, LI; lincomycin/spectinomycin, LI+SP; kanamycin, K; gentamycin, GM; amikacin, AN; neomycin, N; streptomycin, S; trimethoprim/sulfamethoxazole, SXT; ciprofloxacin, CIP; enrofloxacin, ENO; tetracycline, TE; tylosin, TYLO; and tiamulin, TIA). For colistin (CL), minimum inhibitory concentrations were determined by broth microdilution method. All performance and evaluation were followed the guidelines of the Clinical and Laboratory Standards Institute (CLSI, 2011).

Resistance transfer assay

Conjugation experiments were carried out with donor isolates, which were resistant to β-lactam antibiotics and sodium azide–resistant E. coli J-53 as the recipient strain. Transconjugants were selected on MacConkey agar plates containing sodium azide (100 μg/mL) and cefotaxime (1 μg/mL) (Kim et al., 2014). Not only was the β-lactamase production of transconjugants confirmed by PCR amplification and sequencing analysis, but also its replicon typing by the PCR-based method.

Molecular analysis of β-lactamase

Genomic DNA was extracted using PureLink™ Quick Genomic DNA Mini Kits (Invitrogen, Carlsbad, CA), according to the manufacturer's recommendations. All isolates were screened by PCR amplification and sequencing for the Ambler classes A, B, C, and D of β-lactamase-encoding genes such as bla _KPC, bla _IMP, bla _VIM, bla _NDM, and bla _OXA, bla _TEM, bla _SHV, bla _CTX, and bla _ACC (Table 1).

Table 1.

Primers for Phylogenetic Grouping and Plasmid Characterization

Target genes	Primer	Sequence (5′-3′)	Size (bp)	Annealingtemp (°C)	References
Phylogenetic grouping
ChuA	F	GACGAACCAACGGTCAGGAT	279	59	Clermont et al., 2000
	R	TGCCGCCAGTACCAAAGACA
YjaA	F	TGAAGTGTCAGGAGACGCTG	211
	R	ATGGAGAATGCGTTCCTCAAC
TspE4C2	F	GAGTAATGTCGGGGCATTCA	152
	R	CGCGCCAACAAAGTATTACG
Plasmid typing
Int1	F	CTGCGTTCGGTCAAGGTTCT	882	68	Maze et al., 2000
	R	GGAATGGCCGAGCAGATCCT
Int2	F	CACGGATATGCGACAAAAAGGT	746	59	Sandvang, 1999
	R	GTAGCAAACGAGTGACGA AATG
bla _KPC	F	ATGTCACTGTATCGCCGTCT	892	55	Bratu et al., 2005
	R	TTTTCAGAGCCTTACTGCCC
bla _IMP	F	CATGGTTTGGTGGTTCTTGT	488	53	Qi et al., 2008
	R	ATAATTTGGCGGACTTTGGC
bla _VIM	F	AGTGGTGAGTATCCGACA	280	51	Tsakris et al., 2000
	R	ATGAAAGTGCGTGGAGAC
bla _SHV	F	TCGCCTGTGTATTATCTCCC	768	50	Van et al., 2008
	R	CGCAGATAAATCACCACAATG
bla _NDM-1	F	GGCGGAATGGCTCATCACGA	282	60	Yong et al., 2009
	R	CGCAACACAGCCTGACTTTC
bla _CMY-2	F	AACACACTGATTGCGTCTGAC	1226	60	Pérez-Pérez et al., 2002
	R	CTGGGCCTCATCGTCAGTTA
Bla _OXA	F	TTTTCTGTTGTTTGGGTTTT	519	50	Poirel et al., 2004
	R	TTTCTTGGCTTTTATGCTTG
bla _CTX	F	CGATGTGCAGTACCAGTAA	585	60	Batchelor et al., 2005
	R	TTAGTGACCAGAATCAGCGG
bla _TEM	F	GAGTATTCAACATTTTCGT	857	50	Van et al., 2008
	R	ACCAATGCTTAATCAGTGAG
bla _ACC	F	CACCGAAGCCGTTAGTTGAT	900	60	Doloy et al., 2006
	R	GACACCGTTGATGACCTGAT
ISCR1	F	GCTCGAAGATCACATGGGTG	120	55	In this study
	R	GTCGCTACATTAGTCGGCCA
ISCR2	F	ATGTGCAACGGGAATTTGAA	233	55	In this study
	R	CTGAAACGGGAAGCTCAACA
IS26	F	GCACGCATCACCTCAATACC	121	55	In this study
	R	ACCGGAACAACGTGATTGAA
IS3	F	TTCGAGGACAACTACCGCTG	240	55	In this study
	R	CAGATCGGTCAACCATTTCG
IS6	F	ATTTCAGGTGTTGCAGGCAG	148	55	In this study
	R	GCCGCGTATCTTGAAATTGA
tpnA	F	GAATGCAAAAAGCAATGGGA	170	55	In this study
	R	TGACGAGGGGATCATTTTGA

Plasmid characterization

Plasmid DNA was extracted using PureLink™ Quick Plasmid Miniprep Kits (Invitrogen) according to manufacturer's recommendations. The presence of mobile genetic elements, associated with the dissemination of β-lactamase genes, was identified using primers (Table 1), which were designed using NCBI/Primer_BLAST (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). The following conditions were used for PCR: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 30 s, annealing for 30 s at each annealing temperature, extension at 72°C for 1 min; and a final extension at 72°C for 10 min. For PBRT, 18 pairs of primers were used to multiplex PCRs, for a total of 8 reactions for each isolate (5 multiplex and 3 simplex reactions; Table 2) (Carattoli et al., 2005). Multiplex PCR reaction was designed to identify each nucleotide sequence of three different replicons. Primer pairs of HI1, HI2, I1 plasmids were used in multiplex 1. Primer pairs of X, L/M, and N were used in multiplex 2. Primer pairs of FIA, FIB, and W were used in multiplex 3. Multiplex 4 primer pairs of Y, P, and FIC were used in multiplex 4. Primer pairs of A/C, T, and FIIA were used in multiplex 5. The three simplex PCR used primer pairs for I1, K, and B/O replicons. After denaturation at 94°C for 5 min, all PCRs were performed with 30 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 30 s, and extension at 72°C for 1 min. A final extension was performed at 72°C for 5 min.

Table 2.

Primers for Plasmid Replicon Typing (Carattoli et al., 2005 )

Name	Primer	Sequence (5′-3′)	Size (bp)	Target site
HI1	F	GGAGCGATGGATTACTTCAGTAC	471	parA-parB
	R	TGCCGTTTCACCTCGTGAGTA
HI2	F	TTTCTCCTGAGTCACCTGTTAACAC	644	iterons
	R	GGCTCACTACCGTTGTCATCCT
I1	F	CGAAAGCCGGACGGCAGAA	139	RNAI
	R	TCGTCGTTCCGCCAAGTTCGT
X	F	AACCTTAGAGGCTATTTAAGTTGCTGAT	376	ori γ
	R	TGAGAGTCAATTTTTATCTCATGTTTTAGC
L/M	F	GGATGAAAACTATCAGCATCTGAAG	785	repA,B,C
	R	CTGCAGGGGCGATTCTTTAGG-
N	F	GTCTAACGAGCTTACCGAAG	559	repA
	R	GTTTCAACTCTGCCAAGTTC
FIA	F	CCATGCTGGTTCTAGAGAAGGTG	462	iterons
	R	-GTATATCCTTACTGGCTTCCGCAG
FIB	F	GGAGTTCTGACACACGATTTTCTG	702	repA
	R	CTCCCGTCGCTTCAGGGCATT-
W	F	CCTAAGAACAACAAAGCCCCCG	242	repA
	R	GGTGCGCGGCATAGAACCGT
Y	F	AATTCAAACAACACTGTGCAGCCTG	765	repA
	R	GCGAGAATGGACGATTACAAAACTTT
P	F	CTATGGCCCTGCAAACGCGCCAGAAA	534	iterons
	R	TCACGCGCCAGGGCGCAGCC
FIC	F	GTGAACTGGCAGATGAGGAAGG	262	repA2
	R	TTCTCCTCGTCGCCAAACTAGAT
A/C	F	GAGAACCAAAGACAAAGACCTGGA	465	repA
	R	ACGACAAACCTGAATTGCCTCCTT
T	F	TTGGCCTGTTTGTGCCTAAACCAT	750	repA
	R	CGTTGATTACACTTAGCTTTGGAC
FII	F	CTGTCGTAAGCTGATGGC	270	repA
	R	CTCTGCCACAAACTTCAGC
Frep	F	TGATCGTTTAAGGAATTTTG	270	RNAI/repA
	R	GAAGATCAGTCACACCATCC
K/B FW	F	GCGGTCCGGAAAGCCAGAAAAC	160	RNAI
K RV	R	TCTTTCACGAGCCCGCCAAA
B/O RV	R	TCTGCGTTCCGCCAAGTTCGA	159	RNAI

Phylogenetic grouping

Genomic DNA was extracted using PureLink™ Genomic DNA mini kits (Invitrogen) according to the manufacturer's instructions. Tranconjugants were phylogenetically grouped into A, B1, B2, or D using a triplex PCR to detect genes chuA, yjaA, and a DNA fragment of TspE4C2 (Clermont et al., 2000). Primers used for triplex PCR are listed in Table 1.

Results and Discussion

Genotypes of β-lactam resistance

The status of β-lactam antibiotic resistance of the 469 donor E. coli isolates was as follows: P (460/469, 98%), CF (450/469, 96%), TIC (425/469, 91%), CFT (420/469, 90%), AMC (400/469, 85%), TIM (300/469, 64%), FEP (280/469, 60%), FOX (175/469, 37%), CTX (135/469, 29%), AMT (31/469, 7%), CAZ (19/469, 4%), and IPM (9/469, 2%). Transfer of β-lactam antibiotic resistance was detected in 95 isolates out of 469 E. coli isolates in this study. Among the 95 isolates, the distribution of resistance was as follows: P (94/95, 99%), CF (91/95, 96%), CFT (83/95, 87%), TIC (81/95, 85%), AMC (70/95, 74%), TIM (51/95, 54%), FEP (42/95, 44%), FOX (34/95, 36%), CTX (24/95, 25%), ATM (7/95, 7%), CAZ (5/95, 5%), and IPM (2/95, 2%).

By PCR amplification, multiple classes of β-lactamase genes were identified on the plasmids in all isolates. The distribution of plasmid-mediated β-lactamase genes of our multidrug-resistant E. coli isolates is shown in Figure 1. TEM type was identified on all plasmids, ACC type was detected in 90 (n = 95, 95%) and followed by SHV (62%), OXA (28%), CTX (26%), NDM (15%), KPC (13%), CMY-2 (7%), VIM (5%), and CARB type was just detected in one isolate. Two or more β-lactamase genes were present in all isolates; bla _TEM + bla _ACC was the most common combination (by 95%), followed bla _ACC + bla _TEM + bla _SHV (51%), bla _ACC + bla _TEM + bla _CTX (26%), bla _ACC + bla _TEM + bla _OXA (25%), and bla _ACC + bla _TEM + bla _NDM (15%).

FIG. 1.

A heat-map summary of the results; β-lactamase present/absent profiles; the mobile genetic elements present/absent profiles; the non-β-lactamase antibiotics resistance profile for all 95 β-lactamase-positive Escherichia coli. Gray squares indicate feature present in that isolate, while white squares denote features that are lacking in the corresponding bacterial isolate.

Among plasmid-encoded β-lactamases, TEM, CTX-M, and SHV types are widely distributed in human and animals (Carattoli, 2008). The prevalent genotypes vary by country; the TEM type is predominant in the United States and United Kingdom (Jacoby and Medeiros, 1991), and in France the SHV and TEM types were prevalent enzymes (Coque et al., 2008). In Korea, SHV, TEM, and CTX-M types were the main types detected, with the CTX-M type being the most prevalent (≈90%) ESBLs (Kim et al., 2005; Lee et al., 2009). In this study, three types were also detected, but main types were different as TEM, SHV, and ACC. Plasmid-mediated AmpC also has been founded globally (Philippon et al., 2002), and is increasing in livestock in Europe (Ewers et al., 2012; Hammerum et al., 2014). However, in Korea, reports of the prevalence of AmpC encoding plasmids in E. coli were rare, while plasmid-mediated AmpC in Salmonella isolates were reported in 2014 (Lee et al., 2014). Among 95 recipient plasmids in this study, the ACC type was identified at a high rate (95%). KPC-producing Enterobacteriaeceae also have disseminated globally (Bush, 2010), in addition, KPC-encoding plasmids are almost carrying other β-lactamase types, such as TEM, SHV, CTX-M, and CMY types (Bush, 2010). In this study, all 11 KPC-encoding plasmids were co-carrying other β-lactamases, such as TEM (11), ACC (10), CTX-M, SHV and IMP (4), OXA and NDM-1 (3), and VIM (1). Recently, IMP, VIM, and NDM-1 types, which are MBLs, are identified on transferable plasmids of Enterobacteriaceae worldwide (Bush, 2010). Of more than 80 ESBLs identified, 75% were plasmid-mediated enzymes (Bush and Jacoby, 2010). MBLs can hydrolyze all β-lactams without being inhibited easily. In addition, they often co-exist with other β-lactamases. Therefore, MBLs are global concerns (Bush, 2010). Ever since plasmid-mediated MBLs have been identified in 1994 (IMP in Japan, Osano et al., 1994), VIM types were isolated in Korea (in 2001; Yan et al., 2001). In this study, they were also identified in eight plasmids. The most prominent member of this class is NDM-1 type (Kumarasamy et al., 2010; Nordmann et al., 2011b), which was first discovered in a Klebsiella spp. isolate from an Indian patient previously hospitalized in New Delhi, India (Nordmann et al., 2011b). Since 2010, bacteria in the family Enterobacteriaceae encoding the bla _NDM-1 gene have been detected in every continent except Central and South America (Nordmann et al., 2011b). Plasmids carrying the bla _NDM-1 gene are diverse and associated with other β-lactamases genes (OXA types, VIM types, CTX-M types), AmpC cephalosporinase (CMY types), and aminoglycoside resistance genes (Kumarasamy et al., 2010; Nordmann et al., 2011b). Among our 95 isolates, 16 NDM-harboring plasmids were identified, and 15 plasmids of them co-carried the bla _ACC gene, 12 plasmids co-carried the bla _SHV gene, and 6 plasmids co-carried bla _OXA gene. All 16 NDM-harboring plasmids co-carried more than 3 other β-lactamase genes with the NDM gene (Fig. 1). OXA types are one of the most common β-lactamases, which were first identified in a K. pneumoniae isolate from Turkey in 2003 (Poirel et al., 2004). Since then, they have been reported as a source of nosocomial outbreaks worldwide including Southern Europe and Africa, but not in the United States (Nordmann et al., 2011a). Identifications of OXA-producing Enterobacteriaceae are increasing in countries such as France, Germany, Spain, the Netherlands, and the United Kingdom (Nordmann et al., 2011a). In this study, the OXA-type β-lactamases gene was detected in 24 plasmids of the E. coli isolates.

Plasmid characterization

Distribution of replicon types of 95 β-lactamase gene-encoding plasmids is also shown in Figure 1. Among the known 18 replicon types, 15 types were detected in this study. The most common replicon type was FIB (n = 95, 100%), the second predominant type was Frep (n = 94, 99%), followed by HI1 (98%), Y (43%), B/O (35%), A/C (34%), P (26%), FIA (20%), N (19%), HI2 (8%), I1 (8%), and W (5%).

F family and HI1 types, which were predominant in this study, have been reported to be harbored by plasmids of E. coli, K. pneumoniae, and some Salmonella enterica, worldwide, and they were associated with ESBL genes, or plasmid-mediated quinolone or aminoglycoside resistance genes (Carattoli, 2009). IncF group plasmids (FIA, FIB, and Frep) have been associated with the spread of resistance genes such as bla _KPC, quinolone, and plasmid-mediated aminoglycoside genes, and other Inc types (IncN, IncA/C, IncHI1, IncP) were also the vehicles for the spread of β-lactamase genes such as bla _CTX, bla _VIM, and bla _CMY (Carattoli, 2009).

By PCR analysis, multiple patterns of mobile elements were detected in the plasmid collection studied here. Int1, Int2, ISCR1, ISCR2, IS3, and IS26 were detected in all isolates, and ISEcp1 (97%) and IS6 (46%) followed. Moreover, in all plasmids, at least five elements were co-existing. The transfer of their resistance traits in multidrug-resistant E. coli to other microorganisms could be explained with this result.

Susceptibility to non-β-lactamase antibiotics

All E. coli isolates were resistant to at least eight non-β-lactamase antibiotic agents. Among the 95 isolates, the distribution of antibiotic resistance was as follows: TIA, TYLO (95/95), E, S, N (94/95), K (93/95), TE (91/95), GM, SXT (78/95), CIP (75/95), NOR, ENO (70/95), AN (65/95), and CL (40/95).

Phylogenetic group analysis

E. coli strains can be divided into four main phylogenetic groups (A, B1, B2, and D) by phylogenetic analysis (Clermont et al., 2000). Virulent extraintestinal strains mainly belong to group B2 and, to a lesser extent, to group D (Clermont et al., 2000), whereas most commensal strains belong to groups A and B1. In this study, the less virulent group B1 and most commensal group A were predominant (43% and 26%), while more virulent groups, D and B2, were detected at the same rate as 15%, which was a similar result to human study (Jakobsen et al., 2010).

In conclusion, we demonstrated a high rate prevalence of plasmid-mediated β-lactamases in the fecal flora of pigs, which is an obvious risk for increasing prevalence in humans. Because E. coli is a zoonotic pathogen, genes encoding β-lactamase enzymes can be present in humans. The plasmids harboring these resistance genes co-carried mobile genetic elements, which can facilitate the transfer of drug-resistance genes among bacterial species. Consequently, the occurrence and spread of multidrug-resistant bacteria in humans and animals should be controlled by appropriate use of antibiotics in veterinary medicine and a prohibition on the subtherapeutic use of antibiotics in the animal industry.

Footnotes

Disclosure Statement

No competing financial interests exist.

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