Molecular Characterization of Cephalosporin and Fluoroquinolone Resistant Salmonella Choleraesuis Isolated from Patients with Systemic Salmonellosis in Thailand

The antimicrobial resistance of nontyphoidal Salmonella has become a major clinical and public health problem. Southeast Asia has a high level of multidrug-resistant Salmonella and isolates resistant to both fluoroquinolone and third-generation cephalosporins. The incidence of co-resistance to both drug classes is a serious therapeutic problem in Thailand. The aim of this study was to determine the antimicrobial resistance patterns, antimicrobial resistance genes and genotypic relatedness of third-generation cephalosporins and/or fluoroquinolone-resistant Salmonella Choleraesuis isolated from patients with systemic salmonellosis in Thailand. Antimicrobial susceptibility testing was performed using the agar disk diffusion method, and ESBL production was detected by the combination disc method. A molecular evaluation of S. Choleraesuis isolates was performed using PCR and DNA sequencing. Then, a genotypic relatedness study of S. Choleraesuis was performed by pulse field gel electrophoresis. All 62 cefotaxime-resistant S. Choleraesuis isolates obtained from 61 clinical specimens were multidrug resistant. Forty-four isolates (44/62, 71.0%) were positive for ESBL phenotypes. Based on the PCR sequencing, 21, 1, 13, 23, 20 and 6 ESBL-producing isolates harboured the ESBL genes blaCTX-M-14, blaCTX-M-15, blaCTX-M-55, blaCMY-2, blaACC-1 and blaTEM-1, respectively. This study also found that nine (9/62, 14.5%) isolates exhibited co-resistance to ciprofloxacin and cefotaxime. All of the co-resistant isolates harboured at least one PMQR gene. The qnr genes and the aac(6′)-Ib-cr gene were the most prevalent genes detected. The QRDR mutation, including the gyrA (D87Y and D87G) and parC (T57S) genes, was also detected. PFGE patterns revealed a high degree of clonal diversity among the ESBL-producing isolates.


Introduction
Salmonella is one of the most common types of bacteria; it is a human pathogen that causes 'Salmonellosis' and some types of gastroenteritis, including self-limiting enterocolitis, bacteremia, meningitis, and osteomyelitis [1]. More than 2600 different serovars of S. enterica have been identified. The microorganisms are transmitted by contaminated food or inadequate food hygiene and the fecal-oral route. Southeast Asia has a high level of multidrug-resistant nontyphoidal Salmonella and isolates resistant to both fluoroquinolone and third-generation cephalosporins [2,3]. Compared to previous susceptibility patterns among isolates from Southeast Asia, current nontyphoidal Salmonella infections in humans in Thailand are more resistant to fluoroquinolone and cephalosporins [4]. The incidence of co-resistance to both drugs classes is a serious therapeutic problem in Thailand.
The production of extended-spectrum β-lactamases (ESBLs) is an important resistance mechanism to extended-spectrum cephalosporins (ESCs) in nontyphoidal Salmonella [5]. Salmonella spp. have been found to express a wide variety of ESBL genes, including bla TEM , bla CTX-M , bla SHV , bla ACC , and bla CMY [5]. A previous study reported that most ESBLs are coded by plasmids, some of which also carry genes conferring resistance to fluoroquinolones [6]. To date, several plasmid-mediated quinolone resistance (PMQR) mechanisms have been identified: qnr gene families encode Qnr protection proteins, aac(6 )-Ib-cr gene encodes variant of an aminoglycoside acetyltransferase, and oqxA and oqxAB genes encode a multidrug efflux pump [7]. In addition, the mechanisms of quinolone resistance are chromosomal mutations in the quinolone resistance-determining regions (QRDRs) and consist of either the modification of the quinolone targets with changes in the genes encoding DNA gyrase (gyrA and gyrB) and/or topoisomerase IV (parC and parE) [8]. A high level of drug resistance arises due to alterations in amino acids on each subunit.
In Thailand, the number of patients who are infected with Salmonella enterica serovar Choleraesuis has been increasing. The data show that the prevalence increased from 1.5% (n = 87) in 1994 to 9.2% (n = 190) in 2006. People between the ages of 6 and 40 years in Thailand have the highest risk of infection [4]. In 2007, an increased number of S. Choleraesuis isolates from Thailand were fluoroquinolone-and ceftriaxone-resistant [9]. Lee et al. [10] found that isolates from Thailand and Taiwan demonstrated an alarmingly high frequency of resistance to fluoroquinolones and third-generation cephalosporins. A recent Thai study found that the rate of ESC resistance for S. Choleraesuis isolated from bacteremic patients increased from 66.7% in 2010 to 94.1% in 2014 [11]. However, few studies have reported data on antimicrobial resistance to third-generation cephalosporins and fluoroquinolones in S. Choleraesuis in Thailand. Therefore, this study aimed to evaluate the antimicrobial resistance patterns, antimicrobial resistance genes and genotypic relatedness of 62 S. Choleraesuis isolates collected from patients with systemic salmonellosis in Thailand during 2010-2015.

Antimicrobial Susceptibility and ESBL-Producing S. Choleraesuis Isolates
A total of 62 S. Choleraesuis isolates were randomly collected from 61 clinical specimens and used in this study. The majority of S. Choleraesuis isolates in this study were recovered from blood specimens which accounted for 83.87% of all strains. According to data obtained from Thailand, S. Weltevreden, S. Stanley, S. Anatum, and S. Rissen were the most common serovars in patient stool samples. Conversely, S. Choleraesuis, S. Enteritidis, S. Typhimurium, and S. Typhi were observed in blood samples [12]. In a previous study in Thailand during 2005, 135 nontyphoidal Salmonella isolated from blood in Siriraj Hospital showed that Salmonella group C (47%) was the most common serogroup and 17.8%, 14.1%, 17.8% and 5.8% of isolates were resistant to ceftriaxone, ceftazidime cefotaxime and ciprofloxacin, respectively [13]. Furthermore, S. Choleraesuis was the most common serovar (49.3%) in 414 nontyphoid Salmonella isolates from bacteremic patients in Thailand between 2005 and 2016 [11].

Characterization of the ESBL, QRDR and PMQR Genes
This study provides a description of the diversity of β-lactamase-resistant gene patterns in S. Choleraesuis isolates from patients with systemic infection in Thailand. The characteristics of plasmid-mediated ESBL-producing S. Choleraesuis isolates are shown in Figure 1. All ESBL-producing isolates have 13 patterns of β-lactamase-resistant genes. Based on PCR sequencing, 35, 23, 20 and 6 ESBL-producing isolates harboured the ESBL genes encoding CTX-M, CMY-2, ACC and TEM-1, respectively. Within the CTX-M family, the CTX-M-9 group is more prevalent than the CTX-M-1 group. The CTX-M-1 and CTX-M-9 groups were represented by CTX-M-55 (13 isolates) or CTX-M-15 (1 isolates) and CTX-M-14 (21 isolates), respectively. The bla OXA and bla SHV genes were also targeted, but there were no positive results. The occurrence of ESBL phenotypes and genotypes in this study was similar to those of other studies in Taiwan and Thailand [16,17]. The bla CTX-M-14 and bla CMY-2 genes were found in extended-spectrum cephalosporinaseproducing S. Choleraesuis isolates recovered from Thai patients [17]. The bla CMY-2 gene for ampicillin resistance was also reported in ESC-resistant S. Choleraesuis strains isolated in Taiwan in 2006 [14] and Thailand during 2005-2007 [11]. A previous paper has reported that plasmids harbouring the bla CTX-M-14 , bla CTX-M-15 and bla CTX-M-55 genes were found in S. Typhimurium isolates from paediatric patients in China [18]. To our knowledge, there was only one previous article that reported the dissemination of CTX-M-55-producing S. Choleraesuis isolates from ESC-resistant S. Choleraesuis clinical isolates in Thailand during 2012-2016 [11]. In the past, plasmids carrying bla CTX-M-55 and bla CTX-M-15 have been found in ESBL-producing E. coli and Klebsiella in China and Thailand [19,20]. The molecular characterization showed that bla CTX-M-55 is a derivative of bla CTX-M-15 , in which the Ala in position 77 is substituted with a Val. With regard to antibiotic resistance, the MIC ranges for cefotaxime, ceftazidime, ceftriaxone and cefpodoxime of isolates carrying the bla CTX-M-55 and bla CTX-M-15 genes were significantly higher than those of isolates carrying bla CTX-M-14 .
The bla CTX-M-15 and bla CTX-M-55 are the main genes that cause ceftazidime resistance [19]. Ceftazidime-hydrolysing CTX-M-15 and CTX-M-55 enzymes possess a Gly substitution in position 240, which may be associated with a reduced susceptibility to ceftazidime [21]. The data suggest that ESBL-producing isolates have emerged in Thailand on several plasmids and in multiple clones of S. Choleraesuis. The clonal spread of CTX-M-mediated resistance may be due to horizontal transfer, but this study did not perform gene or plasmid transfer experiments on our ESBL-producing isolates. However, an epidemiological study of plasmid transfer between bacteria in the Enterobacteriaceae family has been reported previously [19,20]. In intensive health-care hospital settings, the development and spread of ESBL genes has most likely been caused or at least facilitated by the overuse of antibiotic drugs [22].
In addition, 20.5% (9/44 isolates) of the ESBL-producing isolates were also co-resistant to ciprofloxacin (Table 3). All co-resistant isolates displayed a QRDR mutation in the gyrA (D87Y and D87G) and parC (T57S) genes. The most frequently observed point mutations of the gyrA gene in fluoroquinolone-resistant Salmonella are the amino acid changes at serine-83 to phenylalanine, tyrosine, or alanine, or at aspartic acid-87 to glycine, asparagine, or tyrosine [23]. Mutations in parC genes have been found in fluoroquinolone-resistant S. Choleraesuis [14]. Of the nine isolates, seven patterns of PMQR genes were identified, all of the co-resistant isolates harboured at least one PMQR gene; the qnr genes (qnrA or/and qnrB or/and qnrS) and the aac(6 )-Ib-cr gene were the most prevalent resistance genes detected. Based on the MICs of the tested cephalosporins, the MIC values of coresistant isolates for cefotaxime, ceftazidime, ceftriaxone, and cefpodoxime ranged from 64 to ≥256 µg/mL, 1 to 48 µg/mL, ≥256 µg/mL, and 32 to ≥256 µg/mL, respectively. In addition, four strains isolated from the blood samples showed high MIC levels for ciprofloxacin (2 µg/mL). Only one strain in a 40-year-old patient, SH575/12, showed high MIC levels for both ciprofloxaxin (MIC 2 µg/mL) and cephalosporins (cefotaxime ≥ 256 µg/mL, ceftazidime = 48 µg/mL, ceftriaxone ≥ 256 µg/mL and cefpodoxime ≥ 256 µg/mL). This strain contained two types of ESBL genes (bla TEM-1 and bla CTX-M-55 ), four types of PMQR genes (qnrA, qnrB, qnrS and aac(6 )-lb-cr) and presented a mutation in both gyrA and parC. However, an earlier study demonstrated fluoroquinolone and ESC resistance in S. Choleraesuis isolated from bacteremic patients in Thailand in 2003-2005 [9]. In this study, four co-resistant isolates obtained from three patients with a systemic infection showed a higher MIC for ciprofloxacin (2 µg/mL) than previously reported, which reduced the susceptibility to ciprofloxacin to ≥0.125 µg/mL [9].     CTX, cefotaxime; CAZ, ceftazidime; CRO, ceftriaxone; and CPD, cefpodoxim; D, aspartic acid; G, glycine; S, serine; T, threonine; Y, tyrosine.

PFGE Analysis
After digestion by the XbaI enzyme, the genetic relatedness of ESBL-producing S. Choleraesuis isolates was evaluated by PFGE. The dendrograms in Figure 1 demonstrate the banding patterns and the genetic relatedness of the isolates evaluated by PFGE. Multidrug antimicrobial resistance patterns and ESBL genes were also reported. Thirty-one fingerprint-patterns generated by PFGE were identified amongst the strains recovered from various locations at different times and source isolation, providing information on associations amongst the ESBL-producing strains and evidence of the diversity of ESBL genes harboured on plasmids in Thailand. Forty-four isolates were clustered into six predominant PFGE pulsotypes with ≥2 indistinguishable isolates, which showed a close relationship (Dice correlation coefficient of 95%) based on Tenover criteria [24]. However, this study represented a high diversity of PFGE fingerprint patterns of ESBL-producing S. Choleraesuis isolates in Thailand. The PFGE and antimicrobial resistance patterns revealed a high degree of clonal diversity amongst the 44 ESBL-producing isolates. All of the analyses indicate that multiple clones and multiple resistance genes on the plasmids are responsible for the extended-spectrum cephalosporin resistance amongst the S. Choleraesuis isolates obtained from patients in Thailand. Similarly, Sirichote et al. [16] reported multiple clones and multiple ESBL resistance genes of S. Choleraesuis isolates obtained from patients in Thailand and one patient in Denmark. In addition, 16 unique XbaI PFGE patterns of 22 S. Choleraesuis isolates were obtained.

Data Source and Salmonella Strains
Pure cultures of cefotaxime-resistant S. Choleraesuis were obtained from the WHO National Salmonella and Shigella Center in Thailand, which were collected from S. Choleraesuis isolated on all regions of Thailand. They were selected isolates from patients with systemic salmonellosis in all regions of Thailand during 2010-2015. A total of 62 cefotaxime-resistant S. Choleraesuis isolates used in this study were isolated from 61 clinical specimens. S. Choleraesuis isolates were obtained from blood (n = 53) and other sources (knee joint aspiration (n = 3), pus (n = 2), stool (n = 2), urine (n = 1) and thigh swabs (n = 1)). The stock culture of S. Choleraesuis isolates were re-subcultured to check the purity and were confirmed serovar according to the Kauffmann-White antigen schema [25]. The colonies of S. Choleraesuis on xylose lysine deoxycholate agar (XLD agar) appeared colourless, without a black centre. The biochemical test, lysine indole motility (LIM) medium and triple sugar ion (TSI) agar were used for the preliminary identification of S. Choleraesuis. The LIM results for S. Choleraesuis revealed positive results for lysine decarboxylase and motility, but negative results for indole. The TSI results were presented as alkaline (red) slant and acid butt (yellow), gas positive, and hydrogen sulphide negative.

Characterization of ESBL, QRDR and PMQR Genes
DNA was extracted by the Wizard ® Genomic DNA purification kit (Promega, Madison, WI, USA). All primers used for this study are listed in Table 1. The Ex Taq DNA polymerase (Takara Bio Inc., Japan) was used for the detection of ESBL, QRDR and PMQR genes. The ESBL genes, including bla TEM , bla SHV , bla CTX-M , bla ACC , and bla CMY-2 , were amplified by PCR [27][28][29]. The amplification conditions for the bla TEM , bla SHV , bla CTX-M , bla ACC , and bla CMY-2 genes were standardised as follows: an initial denaturation at 95 • C for 5 min, followed by 30 cycles of denaturation at 98 • C for 10 s, annealing at 55 • C for 1 min and DNA extension at 72 • C for 1.30 min, with a final extension at 72 • C for 10 min. For confirmation, the CTX group was amplified and sequenced by the bla CTX-M-1 group, the bla CTX-M-2 group, the bla CTX-M-8/25 group, the bla CTX-M-9 group and the bla CTX-M-15 primer [30,31]. The PCR program consisted of an initial denaturation at 95 • C for 5 min, followed by 30 cycles of denaturation at 98 • C for 10 s, annealing at 55 • C for the bla CTX-M-1 , bla CTX-M-2 , bla CTX-M-9 groups and bla CTX-M-8/25 groups, and 50 • C for bla CTX-M-15 for 1 min with a DNA extension at 72 • C for 1.30 min, followed by a final extension at 72 • C for 10 min. The presence of QRDR mutations in gyrA and parC genes and screening for PMQR, including the qnrA, qnrB, qnrS, and aac(6 )-lb-cr genes, were amplified by PCR [32][33][34]. The amplification conditions for all the PCRs were as follows: an initial denaturation at 95 • C for 5 min, followed by 30 cycles of denaturation at 98 • C for 10 s, annealing at 55 • C (parC, qnrA, qnrS, and aac(6 )-lb-cr) and 50 • C (gyrA and qnrB) for 1 min and DNA extension at 72 • C for 1.30 min, with a final extension at 72 • C for 10 min. All PCR products were purified for sequencing using nucleospin gel extraction (Macherey-Nagel, GmbH & Co. KG, Düren, Germany) and were ran using an automatic sequencer at the Macrogen Inc. (Seoul, Korea). The nucleotide sequence analyses were compared to nucleotide database sequences using the megablast search and to published protein databases using Blastx from the National Center for Biotechnology Information website (https://blast.ncbi.nlm.nih.gov/Blast.cgi, accessed on 21 January 2021).

Pulse-Field gel Electrophoresis (PFGE)
All ESBL-producing isolates included in this study were analysed for epidemiological relatedness by PFGE using XbaI according to the Centers for Disease Control and Prevention (CDC) PulseNet protocol [35]. S. Braenderup H9812 was used as a standard marker. The electrophoresis was performed with a CHEF-DR III instrument using the following condition: one pulse time of 2.2 to 63.8 s for 19 h. The gel was stained with ethidium bromide, and the DNA bands were visualised with a UV transilluminator. The PFGE profile was analysed by BioNumerics software version 3.0 (Bio-Rad). The similarity index was calculated using the Dice correlation coefficient option of the software with a position tolerance of 1% and an optimization of 0.5%. The dendrogram was created using the unweighted-pair group method with average linkages (UPGMA).

Conclusions
The present study showed a high occurrence of ESBL producers among S. Choleraesuis isolated from patients with systemic salmonellosis in Thailand during 2010-2015. In addition, ESBL-producing isolates showed co-resistance to ESCs and fluoroquinolone. The current study describes the diversity of ESBL genes and identifies QRDR mutations and PMQR genes in ESBL-producing S. Choleraesuis. The PFGE typing suggests that ESBLproducing S. Choleraesuis strains isolated from patients in different regions in Thailand are multiple clones, which may indicate that isolates of this serovar have spread and that resistance has evolved locally among the isolates in Thailand.