Resistance Phenotype

Onehundred-and-ten (37%) Vietnamese Salmonella isolates were fully susceptible to all 15 antimicrobials tested (Table 1). No ceftazidime-resistant isolate was found. Nearly two thirds of the collection (187 isolates) showed resistance to at least one antimicrobial agent. More than 40% (n = 125) of the isolates belonging to 17 serovars were resistant against ≥2 antimicrobials. Resistance to six or more antimicrobials was found in 51 isolates (17%). Resistance to gentamicin was found among human (14%) and porcine (20%) salmonellae, especially S. Typhimurium isolates. Of the poultry isolates, 80% were resistant against nalidixic acid. Three norfloxacin-resistant S. Typhimurium isolates were found and all were isolates from humans (n = 3).

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Table 1. Number of resistant Salmonella isolates belonging to different serovars isolated from humans, cattle, pigs and poultry in Vietnam by an agar diffusion method^*.

https://doi.org/10.1371/journal.pone.0009440.t001

Integrons and Gene Cassettes

The prevalence of class 1 integrons was high (28%). Nine different profiles of class 1 integrons (Table 2) were detected in 83 isolates belonging to 11 serovars. The gene cassettes found in these integrons included the aadA1, aadA2 and aadA5 genes encoding resistance to streptomycin and spectinomycin, the bla_PSE-1 and bla_OXA-30 genes conferring resistance to β-lactams, the dfrA1, dfrA12 and dfrA17 genes encoding resistance against trimethoprim, the sat gene mediating streptothricin resistance and open reading frames encoding proteins of an unknown function. Phenotypic resistance to a certain antimicrobial drug was observed in all isolates carrying the corresponding gene cassettes. The transfer of the integrons and the antimicrobial resistance determinants (AAcGKSTSuTp, CSTSuTp, and CSSu) to E. coli was possible from 17 of the 83 integron-positive isolates of serovar Typhimurium, Anatum, and Agona, respectively (Table 3). Ten S. Typhimurium isolates from 17 isolates tested could transfer their integrons and resistance determinants to S. Enteritidis. This was demonstrated by the fact that the E. coli and S. Enteriditis transconjugants were int positive and obtained the phenotypic resistance patterns of the donors.

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Table 2. Characterization of class 1 integrons of Salmonella isolates from human and animal origin in Vietnam.

https://doi.org/10.1371/journal.pone.0009440.t002

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Table 3. Antimicrobial resistance characteristics of MDR Salmonella isolates from human and animal origin in Vietnam.

https://doi.org/10.1371/journal.pone.0009440.t003

Salmonella Genomic Island 1

In serovar Typhimurium, Derby, Albany, and Tallahassee, SGI1 and the variants SGI1-C and SGI1-F were found (Table 3). The three S. Emek isolates with the phenotypic resistance pattern CNaSSuTp, CNaTpSu, and AAcCeCNaTTpSu (V14, V28 and V116, respectively, were positive in the PCRs for SGI1 [14]. Remarkably, an integron structure was not present between SGI1 genes S027–S044, instead it was present in gene S023. PCRs specific for the genes commonly present in the integron structures of SGI1 showed the presence of a structure identical to SGI-J3 [16].

Circular Form of SGI1

Excision and circularization of a SGI is the first step in horizontal transfer to other bacteria. To study the possible excision of SGI1 from the genome as a circular form, a PCR specific for the circular form of SGI1 was used. The SGI1 in 3 S. Typhimurium isolates proved to be present in its circular form. Nucleotide sequencing showed that the fragment of ca. 430 bp obtained by SGIc-PCR from a SGI1 carrying S. Typhimurium isolate (V54) was identical to the sequence of the S004 gene, the right end of SGI1 up to the insertion site of the cryptic retronphage [18] in the sequence deposited in the GenBank under accession number AF261825.2. No PCR product indicating the presence of the circular form of SGI1 was obtained with DNA from other serovars.

Fluoroquinolone Resistance

No qnrA gene was found in the three norfloxacin/ciprofloxacin-resistant Salmonella Typhimurium isolates (V57, V58 and V60). However, AS-PCR-RFLP revealed that all 3 isolates had double point-mutations in their gyrA gene at Ser-83 and Asp-87. Nucleotide sequencing of the fragments, spanning the “Quinolone Resistance Determining Region” (QRDR) showed a substitution in the codon TCC (Ser) at position to 83 TTC (Phe) and in the codon GAC (Asp) at position 87 to AAC (Asn).

Isolates

A total of 297 epidemiologically unrelated isolates from Vietnam was investigated. The isolates originated from humans (n = 56), cattle (n = 63), pigs (n = 111), and poultry (n = 67). All animal isolates were collected during the year 2004. The animal isolates were cultured from faeces, carcasses and meat. Faecal samples from healthy animals were taken at slaughterhouses (78%) and from healthy or sick animals on farms (12%) as previously described [30]. The animal samples came from different flocks or herds. If more than one sample from a slaughterhouse, farm, market or supermarket was Salmonella positive, only one isolate was randomly chosen and included in this study. The 56 clinical human isolates of unrelated patients with diarrhoea and fever were obtained from five provincial hospitals and two Pasteur Institutes in Vietnam. These isolates had been isolated during the year 2004. The methods used for the isolation and identification of the isolates have been described [30]. The isolates included in the present study belonged to 38 serovars of Salmonella. S. Typhimurium, S. Anatum, S. Weltevreden, S. Emek and S. Rissen were the most prevalent serovars. S. Typhimurium phage type 90 (in the Dutch phage typing system), which has no recognized phage type in the English phage typing system, was predominant among the S. Typhimurium isolates [34].

Antimicrobial Susceptibility Testing

The antimicrobial susceptibility of the isolates was determined according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI) [35]. Agar diffusion assays were performed on Muller-Hinton agar and with disks containing 15 different antimicrobial agents (Oxoid, UK). The antimicrobials tested (Table 1) were ampicillin 10 µg (A), amoxicillin/clavulanic acid 30/15 µg (Ac), cefalothin 30 µg (Ce), ceftazidime 30 µg (Cf), chloramphenicol 30 µg (C), ciprofloxacin 5 µg (Ci), colistin 10 µg (Co), gentamicin 10 µg (G), kanamycin 30 µg (K), nalidixic acid 30 µg (Na), norfloxacin 10 µg (No), streptomycin 10 µg (S), tetracycline 30 µg (T), trimethoprim 5 µg (Tp), and sulphonamides 300 µg (Su). The interpretive categories susceptible, intermediate or resistant were used according to the CLSI guidelines [36] except for colistin where the zone criteria of ≤11 (resistant) and ≥14mm (susceptible) were used [37]. Escherichia coli ATCC 25922 and E. coli ATCC 35218 were used as quality control organisms.

Detection of Class 1 Integrons

All isolates were tested for the presence of class 1 integrons. The presence of integrons was determined by PCR amplification of the class 1 integrase specific int1 gene [38]. Template DNA was obtained by the whole cell boiled lysate method [39]. Integron gene cassettes were detected by PCR using the 5′-CS and 3′-CS primer set [39]. CS-PCR products were separated in 0.7% agarose gels for at least 3 hours at 100 V and visualized under UV-light after staining with ethidium bromide. Since the 3′-CS fragment of class 1 integrons is not always as conserved as its name indicates [40], an integrase-positive isolate does not always yield an amplicon in the CS-PCR. If this was the case, an integrase-inverted PCR was used to characterise the gene cassettes.

Briefly, 1 µg of genomic DNA of the isolate was cleaved with the restriction endonuclease SphI. The fragments were ligated and subjected to PCR using int-OUT and CS-F primers. Since the smallest size for a gene cassette inserted into an integron is about 400 bp only fragments larger than 500 bp generated from the inverted PCR were sequenced.

Characterization of Integrons

CS-PCR products with the same size were purified using the QiaQuick PCR purification kit (Qiagen, Germany) and analysed by restriction fragment length polymorphism (RFLP). The amplicons were digested with at least two different restriction endonucleases and the order and arrangement of the gene cassettes was considered identical if they showed the same RFLP patterns. The restriction endonucleases used were HpaII, HincII, BclI, NciI, and EcoRI.

Nucleotide Sequencing of Gene Cassettes

One representative of each RFLP type was randomly chosen for nucleotide sequencing. For isolates with a unique integron, purified CS-PCR products were cloned in the pGEM-T easy Vector (Promega, USA). Colonies carrying the inserted fragment were picked from Luria Bertani plates containing ampicillin (100 µg/ml), 40 µl (100 mM) IPTG and 40 µl (2%) X-Gal. The inserted fragments were obtained by PCR, using T7 and SP6 primers under the same conditions as for the CS-PCR. The amplification products were purified and sequenced. The T7 and SP6 primers were used for sequencing both ends of the different amplicons under study. In addition, for the 2000 bp amplicon obtained by CS-PCR, an internal primer was used to continue the sequencing until the resistance genes in each amplicon were identified. For isolates carrying two integrons which only differed about 50 bp in size, CS-PCR products were also cloned in the pGEM-T easy Vector. The two plasmids with different inserts were selected based on restriction enzyme (EcoRI or HpaII) analysis and used for sequencing. Dideoxy sequencing was performed on an ABI 3730 Sequencer. DNA sequences were analysed with the Clone Manager Suite and by consulting the GenBank database via the BLAST network service. The nucleotide sequences of the gene cassettes have been deposited in the GenBank database under the accession numbers shown in Table 2.

Bacterial Conjugation

A conjugation experiment was performed as described [41] to determine whether the integrons of the Salmonella isolates were on conjugative DNA elements and resistance determinants could be transferred to another Salmonella serovar or to another bacterial species (E. coli). Rifampicin-resistant (Rif^R) and sulfamethoxazole-susceptible (Sul^S) E. coli K12 was used as recipient. All 83 integron carrying Salmonella isolates were used as donor strains. In addition, a plasmid-free susceptible S. Enteritidis isolate, which was made resistant to rifampicin, was also used as recipient. In this second conjugation experiment, only Salmonella isolates which could transfer their resistance determinants to E. coli in the first conjugation experiment were used as donors. Both donor (int-positive sulfamethoxazole-resistant Salmonella isolates) and acceptor bacteria were cultured in LB broth until an OD₆₆₀ = 0.5–0.6 was reached. The mating process in which donor (sulfamethoxazole-resistant isolates) and recipient were present in a 1∶9 ratio (v/v) was performed in LB broth. Incubation took place overnight in a water-bath at 37°C. Transconjugants were selected by plating 50 µl of the mating culture on MacConkey (Oxoid, UK) agar plates containing both rifampicin (50 µg/ml) and sulfamethoxazole (512 µg/ml). Colonies were selected based on their resistance to both antimicrobials and purified by subculture on MacConkey agar containing antibiotics and then nutrient agar (NA, Oxoid, UK) without antibiotics. The transconjugants were tested for their biochemical characteristics, using the API 20E system (bioMérieux, France) and the Salmonella transconjugants were serotyped using antisera (Staten Serum Institute, Denmark) against antigens of S. Enteritidis. The transconjugants were tested as described above for their susceptibility patterns and the presence of class 1 integrons.

Detection of Salmonella Genomic Island 1 and Its Variants

Integron-positive isolates (n = 12) from various Salmonella serovars were selected for analysis of the presence of SGI1 on the basis of their integron profiles and antibiotic resistance patterns. First, the isolates were examined for the presence of the left and right junction of SGI1 by PCR. Then the order of the antibiotic resistance gene cluster was tested as described [12]. Template DNA was prepared using the High Pure PCR Preparation Kit (Roche, Germany). The primers used for amplification of the left and right junctions of SGI1 and the linking sequences in the antibiotic resistance gene cluster were previously described [12], [14]. Primers 5′-AATTATCTCGTTCTGCATCC-3′ and 5′-CTCTGTTGTCGCAAGAAATG-3′ were used to demonstrate integration into SGI1 gene S023. The PCRs were carried out in a total volume of 25 µl volumes containing 2.5 µl of 10× PCR buffer (HT Biotechnology, England), 0.5 µl 10× deoxynucleotide triphosphate mix (2mM each), 50 pmol of each primer, 1.25 U Taq DNA polymerase, and 1 µl of template DNA. To amplify fragments larger than 3.5 kb, Taq Plus polymerase was used instead of Taq DNA polymerase (HT Biotechnology, England). Thermal cycling conditions consisted of a hot start cycle of 94°C for 3 min, followed by 35 cycles with: 1 min at 94°C, 1 min at 50 to 65°C (depending on the primers), 1 to 5 min at 72°C (depending on the expected amplicon size) and a final step at 72°C for 10 min. The expected sizes of the PCR products were based on nucleotide sequences present in GenBank under accession number AF261825. S. Typhimurium N216 carrying SGI1 and S. Albany N107 containing SGI1-F [23] were included as positive controls. Amplification products of S. Emek V14 generated by PCR mapping were partially sequenced (Baseclear, The Netherlands).

Determination of the Circular Extrachromosomal form of SGI1

From isolates harbouring SGI1 or its variant types, 8 representative isolates carrying SGI1 or one of its variants were randomly chosen and examined for the presence of the circular extrachromosomal form of SGI1 by PCR (SGIc-PCR). The PCR was performed using primers oriented towards the left and right chromosomal SGI1 junctions and plasmid DNA extracted with the Qiagen plasmid midi kit (Qiagen, Germany) as template DNA [18]. The obtained PCR product was subsequently sequenced.

Fluoroquinolone Resistance

Three fluoroquinolone (norfloxacin/ciprofloxacin)-resistant Salmonella Typhimurium isolates (V57, V58 and V60) were further studied with respect to the resistance mechanism involved. A PCR described by Paauw et al [42] was used to investigate whether the class 1 integron-associated- qnrA gene cassette was present. A qnr-carrying Enterobacter cloacae strain [42] was used as positive control. Since a mutation in the target enzyme for fluoroquinolones, GyrA, is regularly found in Salmonellae [8], [43], [44], [45], the 3 fluoroquinolone-resistant isolates were subjected to allele-specific PCR and RFLP analysis (AS-PCR-RFLP) as described [43] to detect mutations related to quinolone resistance in codons 81, 83 and 87 of the gyrA gene. The gyrA mutations were confirmed by nucleotide sequencing of the products generated by PCR using GyrA-F and GyrA-R as primers [43].