Molecular Characterization of Multidrug-Resistant Escherichia coli Isolates from Bovine Clinical Mastitis and Pigs in the Vojvodina Province,Serbia

Animal farms and sampling

From 104 cases of clinical mastitis diagnosed during the years 2013–2014, E. coli was detected in nine samples. Among the nine isolates, seven were MDR (defined as resistant to ≥3 classes of antibiotics) and were included in this study. In addition, a total of 283 diagnostic samples from pigs were delivered to the laboratory during the years 2013–2014 for bacteriological, mycological, and parasitological investigations. The fecal samples were taken from diseased pigs, which were known to have received an antibiotic therapy (39 cases total). Only MDR E. coli isolates (15 isolates) collected on the basis of one isolate per pig farm were included in the current work. The diseased animals from which samples were taken for this work were raised on four small farms (up to 10 cows in barns), 3 larger farms (up to 300 cows on each farm), 15 pig farms (5 smaller farms with a total of 10–100 animals), and 10 large pig farms (with more than 100 animals). All farms are located in the northern part of Serbia, the Vojvodina Province, Serbia.

Isolation of E. coli and species confirmation

Milk samples for microbiological examinations were obtained separately from each of the four quarters. The disinfection of teats was performed using 70% ethanol, and the milk was collected into sterile plastic tubes, cooled, and directly transported to the laboratory. For isolation of bacteria, aliquots of 50 μL each were streaked onto a Columbia blood agar plate (CM0331; Oxoid, Basingstoke, United Kingdom) supplemented with 5% defibrinated sheep blood and onto a MacConkey agar plate (CM0007; Oxoid).

Rectal swabs from pigs were inoculated into a volume of 9 ml buffered peptone water (CM0509; Oxoid) and vigorously vortexed for 20–30 sec. Subsequently, each fecal homogenate was streaked onto a MacConkey agar plate (CM0007; Oxoid). The plates were incubated for 24–48 hr at 37°C.

The colonies with a typical appearance of E. coli were subcultured onto nutrient agar plates (CM0003; Oxoid) to obtain pure cultures. After overnight incubation at 37°C, the bacterial species was confirmed by biochemical assays, including an oxidase (negative) and catalase (positive) test; fermentation of glucose and lactose using triple sugar iron agar; production of indole; methyl red test (positive); Voges-Proskauer test (negative); and the inability to use citrate as a carbon source (IMViC test). Definitive confirmation of each E. coli isolate was performed by using the BBL Crystal Enteric/Nonfermenter ID Kit (Becton, Dickinson and Company, Sparks, MD).

Antimicrobial susceptibility testing and determination of minimal inhibitory concentrations

Susceptibility testing was performed by the disk diffusion method according to the recommendations of the Clinical and Laboratory Standards Institute.^9,10 The following discs and concentrations of antibiotics were used: ampicillin 10 μg (AMP), amoxicillin/clavulanic acid 20 + 10 μg (AMC), chloramphenicol 30 μg (CHL), ciprofloxacin 5 μg (CIP), gentamicin 10 μg (GEN), nalidixic acid 30 μg (NAL), streptomycin 10 μg (STR), sulfonamides 300 μg (SA), tetracycline 30 μg (TET), trimethoprim/sulfamethoxazole 1.25/23.75 μg (SXT), trimethoprim 5 μg (TMP), cefpodoxime 10 μg (CPD), cefotaxime 30 μg (CTX), and ceftazidime 30 μg (CAZ) (BioRad, Marnes-la-Coquette, France). For determinations of minimal inhibitory concentrations (MICs) of selected antibiotics, the broth microdilution method with polystyrene microtiter plates (Sarstedt, Nümbrecht, Germany) was used as recommended in the CLSI document M07-A10 (2015). ⁹ Antibiotics were purchased as powders from Sigma Aldrich (Taufkirchen, Germany). The E. coli strain ATCC 25922 was used for quality control purposes.

Isolation of whole-cell DNA, polymerase chain reaction, and sequence analysis

A whole-cell DNA was extracted by boiling bacteria for 5 min. After 10 min on ice, the suspension was centrifuged (Witeg Labortechnik GmbH, Korea) at 13,000 rpm and the supernatant was collected and stored in a freezer at −20°C. For PCR experiments, a commercially available kit (Qiagen, Hilden, Germany) was used. The master mix contained the following: 1.5 mM MgCl₂, 200 μM of each dNTP, 2.5 U of HotStartTaq DNA polymerase in a ready to go 1 × PCR buffer. PCR assays were performed as described earlier.^11–41 For the detection of the major E. coli phylogenetic groups (A, B1, B2, and D), a typing scheme based on a triplex PCR according to Clermont et al. ¹³ was used. Integrons of classes 1 and 2 and inserted gene cassettes were amplified by using the primer pairs 5′3′CS and Hep74/Hep51 and variable regions were sequenced as described earlier.^14,15 Furthermore, the previously described primers specific for the PCR detection of the quinolone resistance determining regions (QRDRs) of the topoisomerase genes gyrA, gyrB, parC, and parE were used.^16–19 Amplicons were sequenced and analyzed. The primer sequences, annealing temperatures, sizes of amplicons, and references are provided as Supplementary Data (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/mdr). The primer walking method was used for sequencing of PCR amplicons. Sequencing was performed at GATC Biotech AG (Cologne, Germany) and Eurofins Genomics (Ebersberg, Germany), and sequence alignments were done with the Basic Local Alignment Search Tool-BLAST program or with the DNAMAN version 7 software program (Lynnon Corporation, Quebec, Canada).

Pulsed-field gel electrophoresis

PFGE was performed with the CHEF DR-III (Bio-Rad Laboratories, Hercules, CA) system using a 1% SeaKem gold agarose gel (Lonza, Rockland, ME) and 2.0 L of 0.5 × Tris-EDTA running buffer. The electrophoresis conditions were chosen according to a 1-day (24–26 hr) standardized laboratory protocol for molecular subtyping of E. coli O157:H7, E. coli non-O157 STEC, and Salmonella, Shigella sonnei and Shigella flexneri (www.cdc.gov/pulsenet/pdf/ecoli-shigella-salmonella-pfge-protocol-508c.pdf). Fingerprints were based on the separation of XbaI (Thermo Scientific, Vilnius, Lithuania)-digested genomic DNA. The Salmonella enterica serotype Braenderup H9812 CDC PFGE marker was used as the molecular size standard.

Conjugation experiments

Conjugation experiments were done with the E. coli recipient strain HK225, which is resistant to rifampicin. The mating was performed by the filter method and during 8 hr of shaking bacterial suspensions in Luria Bertani (LB) broth (Becton Dickinson, Le Pont de Claix, France) at 37°C, followed by overnight incubation of the suspensions at 37°C. Transconjugants were selected on LB agar plates supplemented with 100 mg/L rifampicin (Sigma Aldrich, Munich, Germany) and 100 mg/L ampicillin or tetracycline (Sigma Aldrich, Munich, Germany). Presumptive transconjugants were confirmed by comparing PFGE patterns of donor strains, recipient strains, and transconjugants. In addition, transconjugants were subjected to susceptibility testing and resistance gene detection by PCR amplification.

Phylogenetic typing and PFGE analysis

Phylogenetic typing showed that six out of the seven E. coli isolates from cases of bovine clinical mastitis belong to the group A, while one isolate was classified in group B1. Of the 15 E. coli isolates from pigs, 11 were assigned to group A, and 2 isolates each were classified in groups B1 and D. Analysis of PFGE patterns showed unique fragment patterns for all isolates (Fig. 1).

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FIG. 1.

Macrorestriction profiles, phylogenetic groups, and resistance phenotype and genotype in Escherichia coli isolates from pigs and cow milk.

Resistance to fluoroquinolones

Among all of the 22 isolates included in further analysis, nine (41%) were resistant to CIP according to CLSI breakpoints with MICs ranging between 4 and ≥128 mg/L CIP. All but one CIP-resistant isolate exhibited mutations that resulted in changes of the amino acid sequences at positions Ser83→Leu and Asp87→Asn in GyrA and at position Ser80→Ile in ParC. The only exception was isolate number 6S-13 (from a pig), in which base pair exchanges leading to Ser83→Leu and Asp87→Gly amino acid substitutions were identified in gyrA, and a Glu84→Lys substitution was detected in ParC. In five isolates with MIC of ≥16 mg/L CIP, additional mutations were found in the parE gene, leading either to a Ser458→Ala or to a Ser458→Thr amino acid exchange. However, single-point mutations in gyrA were also detected in isolates with MIC values between 0.125 and 0.5 mg/L CIP. Three of these isolates had an alteration in the gyrA gene, resulting in a Ser83→Leu substitution, while in a single isolate, the Asp87→Asn transition in GyrA was identified (Table 1). In two isolates (isolate numbers 3M-13 and 7S-13) with a high level of CIP resistance (MICs of 32 and >128 mg/L CIP, respectively), the plasmid-borne quinolone resistance gene aac(6’)-Ib-cr was detected by PCR assays and sequencing, concomitantly with mutations in the target genes. In contrast, no mutations were found in the topoisomerase genes of E. coli isolate 16S-14, which carried the plasmid-borne qnrS gene and exhibited an MIC value of 0.125 mg/L CIP (Fig. 1 and Table 1 and Supplementary Table S2).

Resistance to β-lactam antibiotics, aminoglycosides, sulfonamides, chloramphenicol, and trimethoprim and transferability of resistance genes via conjugative plasmids

All E. coli isolates resistant to AMP (17 isolates) carried the bla_TEM-1 gene, which was located on a conjugative plasmid in 15 isolates. Phenotypic detection of ESBL production was done by using the screening and confirmatory tests with cefotaxime and cefotaxime with and without clavulanic acid. ¹⁰ An ESBL phenotype was confirmed in two isolates (No 7M-14 and 7S-13). Of these, the isolate number 7M-14 carried the bla_CTX-M-1 gene on a plasmid, which was transferable to a plasmid-free recipient strain. The genes bla_TEM-1 and sul2 were found to be colocated on the same plasmid in isolate number 7M-14 (Fig. 1 and Table 2).

Six isolates from pigs exhibited phenotypic resistance to gentamicin. The aac(3)-II gene encoding an aminoglycoside modifying enzyme was found in all cases (Fig. 1). Notably, in one isolate (isolate number 1M-13), the aac(3)-II gene was transferred together with the genes bla_TEM, aadA1, aadA2, cmlA, floR, sul2, and dfrA12 via the same plasmid to the plasmid-free recipient strain HK225, conferring an MDR phenotype (Table 2). In 17 isolates, the strA/strB pair of genes was detected, while the aadA1 gene, single or in combination with aadA2, was found in 11 isolates.

Resistance to sulfonamides was mediated either by the genes sul1 (five isolates), sul2 (six isolates) or the combinations of sul1 and sul2 (six isolates) or sul2 and sul3 (two isolates) (Fig. 1). The sul1 and sul2 genes from 11 isolates were transferable to the recipient strain by conjugation. In one isolate (isolate number 2M-13), resistance to sulfonamides was cotransferred to the recipient strain together with resistance to AMP (mediated by the bla_TEM gene), while the other 10 transconjugants revealed an MDR phenotype (Table 2).

Resistance to chloramphenicol was seen in 11 isolates, while combined resistance to CHL and its fluorinated derivative florfenicol (FLO) was detected in six isolates (Supplementary Table S2 and Fig. 1). In isolate number 1M-13, which was obtained from a cow milk sample, both genes, cmlA and floR, were detected and were cotransferred to the recipient strain HK225 via the same plasmid in the course of conjugation experiments. The cmlA- and floR-carrying isolate exhibited MIC values of 128 mg/L CHL and 512 mg/L florfenicol (Supplementary Table S2 and Table 2). Five isolates (one from a cow milk sample and four from pigs) carried a single floR gene, while the cat1 gene was detected in three isolates sampled from pigs (Fig. 1). Results from conjugation experiments revealed that the floR genes from other four isolates (isolate numbers 6S-13, 8S-13, 9S-13, and 12S-13) could be successfully transferred to the recipient strain (Table 2).

Resistance to trimethoprim was found in 12 out of 22 isolates (Supplementary Table S2) and was mediated by dihydrofolate reductase (dfr) genes. In five E. coli strains (isolates nos. 4M-13 and 7M-14) from cow milk samples and isolates 6S-13, 14S-14, and 15S-14 from pigs, the dfrA5/A14 gene was detected. In a single isolate obtained from a cow milk sample (isolate number 1M-13), the dfrA12 gene was found. Positive results from dfrA1-specific PCR assays were obtained from two isolates from cow milk (2M-13 and 6M-13) and from two isolates collected from pigs (5S-13 and 11S-13). In addition, the dfrA17 gene was found in isolate numbers 3S-13 and 7S-13 from pigs (Fig. 1). A plasmid localization of the genes dfrA12 and dfrA5/A14 was confirmed in two cases (isolate numbers 1M-13 and 4M-13) by conjugation experiments. In these cases, the genes could be easily transferred to the recipient strain via a plasmid that mediated multidrug resistance (Table 2).

Integron analysis

Altogether, class1 integrons were found in 12 E. coli isolates (three from cow milk samples and nine from pigs), while a single class 2 integron was detected in an isolate obtained from a pig. In the class 1 integron environment, the aadA1 gene cassette (isolate number 4M-13 and 1S-13), the aadA23 gene cassette (isolate number 13S-14 and 16S-14), or a combination of aadA and dfr gene cassettes was found. The dfrA1/aadA1 gene cassette array was observed in three isolates (isolate number 6M-13, 5S-13, and 11S-13), while the dfrA17/aadA5 gene cassette combination was present in four isolates (isolate number 3S-13, 7S-13, 14S-14, and 15S-14). In the class 1 integron of isolate 1M-13, the aadA2/linF gene cassette combination was found, while the aadA1/sat2 gene cassette array was identified in the class 2 integron environment of isolate number 16S-14. The sequences of the gene cassettes and arrays were submitted to the GenBank database and the following accession numbers were assigned: KU956956, KU956957, KU956958, KU948603, KU948604, KU948605, KU948606, KU948607, KU948608, KU948609, KU948610, KU886319, KY284051 (Table 1).