Biomedical Research

DOI: 10.4066/biomedicalresearch.29-18-794

Abstract

Objectives: Prevalence of multidrug-resistant (MDR) S. typhi in typhoid-positive patients, phenotypic screening and molecular detection of blaCTX-M2, and blaCTX-M9 ESBL coding genes.

Materials and Methods: Sixty-six of the 650 clinical blood samples collected have turned out to be positive for S. typhi. Antimicrobial susceptibility and MIC was done as recommended by CLSI. Phenotypic screening of ESBL production and the PCR amplification ESBL coding genes blaCTX-M2, and blaCTX-M9 was also achieved.

Results: Sixty-six out of 650 clinical blood samples have turned out to be positive for S. typhi with an incidence rate of 10.15%. Among 66 isolates (n=44) isolates were MDR. The significant increase in MIC was observed between 128-256 μg/ml to cefetoxime, among the isolates, 9 S. typhi isolates reported positive for cefetoxime-mediated ESBL production. The PCR amplification of ESBL coding genes blaCTX-M2 and blaCTXM-9 yielded amplicon size of 884 and 692 bp, respectively. The ciprofloxacin resistant and cefetoxime sensitive standard MTCC S. typhi 734 and S. typhi BST 63 strain showed absence of both blaCTX-M2 and blaCTXM-9 genes.

Conclusion: The study shows an increase in the incidence rate of MDR S. typhi infection and ESBL production in S. typhi clinical isolates.

Keywords

Salmonella typhi, Multidrug resistant (MDR), ESBL, blaCTX-M2, blaCTX-M9.

Introduction

Majority of Salmonella sp. causes various types of clinical syndromes in humans and have been broadly categorized into two groups. The first, enteric fever, is caused by Typhoidal salmonella (TS) like S. typhi (typhoid fever), S. paratyphi A, and S. paratyphi B (Paratyphoid fever) and are mostly transmitted through contaminated water and food. The second type of clinical syndromes like diarrhea and gastroenteritis are caused by non-typhoidal Salmonella serovars [1]. In India, enteric diseases are most prevalent in urban areas, with the incidence approaching one percent of the population annually in some endemic areas [2]. Earlier studies have shown that majority of enteric fevers are prevalent in most of the developing countries like India, Pakistan, Bangladesh, and several African nations. In India, some reports suggest the incidence of typhoid fever in urban slum areas was as high as 0.2% (2 typhoid positive patients per 1000 population) per year among children less than 5 years of age, and 0.51% (5.1 typhoid positive patients per 1000 population) per year among those under 10 years of age [3]. Incidence of typhoidal infection varies with age. Studies from Kolkata have shown that the incidence of typhoid fever is higher in persons with average age of 14.7 y [4]. In contrast, the incidence of paratyphoid fever is higher among persons mean age of 17.1 y [4]. A study conducted in north India shows that incidence ratio of typhoidal fever is higher in children between 5 and 12 y of age, and the ratio is far higher in children aged less than 5 y; 24.8% of cases per year were reported among the children aged less than 5 y [5].

Extended Spectrum Beta-Lactamases (ESBL) were originally recognized in the year 1983 [6]. They belong to group of amber-class A β-lactamase enzyme that potentially hydrolyze β-lactam antibiotics with an oximinino group such as thirdgeneration cephalosporin’s and aztreonem, and are completely inactivated by β-lactamase inhibitors. The screening of the positive ESBL production in S. enterica serovars was detected and reported from various countries like Italy, France, and Nepal [7]. The significant increase in the production of CTXM- β-lactamases, a family of ESBLs, preferably hydrolyze cefetoxime (CTX) has been recognized and reported with increasing frequency [8]. CTX-M type of ESBLs or cefotaximases encoded by blaCTX-M genes located in a plasmid or on the chromosome belongs to type of class A β- lactamases [9]. This widespread resistance mechanism across the most part of the globe, from Europe, Africa, Asia, South America, and most recently from North America with several reports of clinical isolates producing these lactamases [8,10].

The CTX-M-type ESBLs causing serious concerns by displaying significantly increased level of resistance to both cefetoxime (CTX) and ceftriaxone (CTR) than that to ceftazidime (Caz). Approximately, 40 various types of CTX-M enzymes have been identified, and classified into five groups based on their amino-acid sequence homology, subsequently, been reported in different enterobacteria [11]. The first cefotaximase to be reported in Salmonella sp. was CTX-M-2, subsequently, over ten other CTX-M enzymes have been identified and reported in different Salmonella serovars with S. enterica serovar typhimurium being the most frequent, producing CTX-M-type enzymes such as CTX-M-5 and CTXM- 15 [6,12,13].

Presently, ciprofloxacin is preferred as a drug of choice for the treatment of typhoid infections. Its frequent administration has resulted in dramatic emergence of S. typhi with reduced susceptibilities to fluoroquinolones [2]. A study conducted in Kolkata has given a strong evidence of fluoroquinoloneresistant S. typhi with the Minimum Inhibitory Concentration (MIC) as high as 16 μg/ml to ciprofloxacin and norfloxacin [14]. Fluoroquinolone resistance in India is similar in pattern to other Asian countries [15].

The principal objective of the conducted study was to determine the incidence of multidrug resistance (MDR) and screening of extended spectrum β-lactamase (ESBL) in S. typhi strains isolated from typhoid patients from Kalaburagi region, Karnataka, India, and PCR amplification of plasmid-encoded blaCTX-M2 and blaCTX-M9 genes majorly responsible for cefetoxime-mediated resistance in S. typhi isolates.

Materials and Methods

Isolation of S. typhi from clinical samples

In the present study, 650 blood samples were collected from typhoid-suspected patients confirmed by Widal test from Government Hospital, Kalaburagi, India and Pooja Diagnostic Centre, Kalaburagi, India during July 2013-December 2013. The samples were enriched in tryptic soy broth with 100 units of streptokinase to dissolve clot. They were processed and tested biochemically according to standard guidelines for S. typhi isolation [16]. The isolated S. typhi strains were screened in relation to Standard MTCC S. typhi 734 and genotypically confirmed by 16S rDNA sequencing at Chromous biotech laboratory, Bangalore, India, with universal primers.

Antimicrobial susceptibility testing

The S. typhi isolates and standard MTCC S. typhi 734 were tested for antibiotic susceptibility and resistance for various groups of antibiotics by Kirby-Bauer disk diffusion method as per the standard guidelines of Clinical Laboratory Standard Institute (CLSI) [17,18]. The 16 antibiotic disks (Himedia Pvt. Ltd., Mumbai, India) selected for this study are listed in Table 1. The zone of inhibition post incubation was measured using standard antibiogram scale.

Table 1: Antibiotics selected for the study.

Determination of MIC

MIC was determined by agar dilution method and broth dilution method following CLSI standard guidelines 2012 [18] of five selected antibiotics: ciprofloxacin, nalidixic acid, chloramphenicol, cefetoxime, ampicillin (commercially available as ciprofloxacin hydrochloride monohydrate), nalidixic acid-free acid, chloramphenicol, cefetoxime sodium salt, ampicillin sodium salt (Himedia laboratories, Mumbai, India). Stock solutions of the required antibiotics were prepared as follows: ciprofloxacin (2 mg/ml), nalidixic acid (2 mg/ml), chloramphenicol (1 mg/ml), cefetoxime (1 mg/ml), ampicillin (1 mg/ml) [19]. Significant MIC breaking points to the respective antibiotics were interpreted.

Screening of ESBL production in S. typhi (Extended spectrum ß-lactamases)

Combination disc method: ESBL production in S. typhi isolates was determined by combination disc method with reference to Drieux et al. [20] on MHA (Muller Hinton Agar) medium seeded with S. typhi test organism. A combination of cephalosporin’s/clavulanic acid, i.e. cefetoxime (30 μg) and ceftazidime (30 μg) in combination with clavulanic acid (30 μg +10 μg), was placed 30 mm apart as suggested in standard CLSI guidelines, 2012 [18] and incubated at 37°C for 24 h. A greater by 3 mm increase in zone diameter of either respective clavulanic acid combination discs versus its zone diameter when tested alone confirmed as a significant ESBL production.

Double disc synergetic test: Double disc synergy test is the confirmatory test to combination disc method and was performed by standard protocol of Drieux et al. [20]. This test was performed on MHA seeded with S. typhi isolates and impregnated with amoxiclav or augmentin (combination of amoxicillin with 10 μg of clavulanic acid) at the center surrounded by cephalosporin antibiotic discs like cefetoxime (30 μg), ceftazidime (30 μg), cefexime (30 μg) set 30 mm from the center (Standard CLSI Guidelines, 2012) and incubated at 37°C for 24 h. A significant enhancement in the inhibition zone with either one of the test antibiotics to Augmentin disc is confirms positive for ESBL production.

PCR detection of ESBL-resistant blaCTX-M2 and blaCTX-M9 genes

PCR analysis was carried out to detect plasmid-encoded blaCTX-M2 and blaCTX-M9 responsible for cefetoximemediated drug resistance in S. typhi. The primers, sourced from Chromous Biotech Pvt. Ltd, Bangalore, India, were for blaCTX-M2, 5’-CGGTGCTTAAACAGAGCGAG-3’ (Forward), blaCTX-M2 (reverse) 5’ CCATGAATAAGC AGCTGATTGCCC-3’, blaCTX-M9 (forward) 5’- GATTGACCGTATTGGGAGTTT-3’, blaCTX-M9 (reverse) 5’- CGGCTG GGTAAAATAGGTCA-3’ [21]. For PCR amplification, about 100 ng of DNA was added to 50 μl mixture containing 10 mM of dNTPs, 0.4 mM of each forward and reverse primer and 3 U of Taq polymerase in 10X PCR buffer containing 1.5 mM MgCl₂. Amplification was performed in a Corbett CGI-96 thermocycler with cycling parameters comprising the following steps, initial denaturation at 94°C for 5 min, sequentially followed by, denaturation 94°C for 30 s, annealing at 50°C for 30 s, extension at 72°C for 1 min and final extension at 72°C for 10 min for 35 cycles [22]. Finally, after accomplishing amplification, DNA ladder of 100 bp and 500 bp (Bangalore Genei Pvt Limited, India) were used to determine the size of amplicons.

Results

Of the total 650 samples, only 66 have shown culture positivity for S. enterica Serovar typhi with isolation rate of 10.15%. Results of 16S rDNA sequencing show 100% resemblance with S. typhi isolates in nucleotide BLAST analysis in comparison with existing databases. The sequences have been submitted to National Centre for Biotechnology Information through bankit and the accession number of three MDR isolates, namely, BST 51 (accession no. KR537431) BST 103 (accession no. KR537432) and BST 130 (accession no. KR537433) were obtained from the existing database through BLAST and phylogenetic tree constructed using bioinformatics tool (Figure 1).

Figure 1: Phylogenetic tree constructed with significantly aligned 16S rDNA sequence of Salmonella typhi in BLAST search with query sequence of BST 51, BST 103 and BST 130.

Antimicrobial susceptibility testing

Sixty-six positive S. typhi isolates were chosen for this study. Among them, 06.06% (n=04) were ampicillin resistant but 100% (n=66) of isolates showed reduced susceptibility to aztreonem. In addition to that 24.25% (n=16) strains were resistant to cefexime, ceftazidime respectively, in contrast only 13.63% (n=09) to ceftriaxone. However, 46.97% (n=31) isolates were cefetoxime resistant, comparatively higher to other cephalosporin’s. Subsequently, 42.42% (n=28), 46.97% (n=31) of isolates shown resistance to nalidixic acid, ciprofloxacin respectively in contrast 100% (n=66) isolates were showed reduced resistance against higher-generation quinolones such as levofloxacin and gatifloxacin. In addition to that, all n=66 isolates were entirely sensitive to cotrimoxizole and azithromycin. We observed decreased resistance in S. typhi isolates to aminoglycoside antibiotics; streptomycin and amikacin, i.e., 06.06% (n=04), 1.5% (n=01) respectively (Figure 2). Only 10.60% (n=7) isolates were resistant to tetracycline and 03.03% (n=2) to chloramphenicol. However, standard MTCC S. typhi 734 strain showed resistance to ciprofloxacin, in contrast it was susceptible to cefetoxime, cefexime and ceftriaxone belongs to group of cephalosporin’s (Table 2).

Table 2: Antibiotic resistance of Salmonella typhi isolates.

Figure 2: Graph showing the percentage of resistance against selected antibiotics by S. typhi isolates.

MIC of S. typhi isolates

Among n=31 ciprofloxacin-resistant strains; n=12 isolates showed a significantly increased MIC range of 128-256 μg/ml. Moreover, n=28 isolates have shown a remarkable increase in MIC range of 128-2048 μg/ml to nalidixic acid. However only one isolate S. typhi BST 174 has shown the maximum MIC of 512 μg/ml to chloramphenicol. Subsequently, strain S. typhi BST 43 has shown significantly increased MIC of 512 μg/ml to ampicillin and in contrast, S. typhi BST 45 and S. typhi BST 48 have shown MIC at 256 μg/ml. Consequently, n=4 strains, S. typhi BST 51, S. typhi BST 130, S. typhi BST 103, S. typhi BST 43, have shown a greater degree of MIC at 256 μg/ml to cefetoxime. In addition, the standard MTCC S. typhi 734 strain showed decreased MIC range to cefetoxime of 2 μg/ml and the high range of MIC of 128 μg/ml to ciprofloxacin (Table 3).

Table 3: Minimum inhibitory concentration (MIC) level of Salmonella typhi isolates.

Phenotypic screening of ESBL production in S. typhi

Of the 66 S. typhi isolates, 55% (n=36) showed resistance to third-generation cephalosporin’s such as ceftazidime, ceftriaxone and cefetoxime. Among 36 cephalosporin’s resistant S. typhi (CeRST) isolates, 25% (n=09) CeRST S. typhi isolates were positive ESBL producers. The ESBL production was evident in both combination disc method and double disc synergetic test. In addition to that entire n=9 S. typhi isolates were responsible for cefetoxime-mediated ESBL production (Figures 3a and 3b).

Figure 3: a. Increase in zone of inhibition with cefetoxime+clavulanic acid of ≥ 3 mm in comparison to cefetoxime alone. b. Enhancement in inhibition zone of cefetoxime, ceftazidime and cefexime towards β- lacatmase inhibitor clavulanic acid containing amoxiclav.

Detection of ESBL-resistant blaCTX-M2 and blaCTX-M9 genes

All the n=9 cefetoxime-mediated ESBL-producing S. typhi strains have been selected for genotypic study, i.e., PCR amplification of cefotaximase genes, blaCTX-M2 and blaCTXM9. The samples showed amplification of 884 bp and 692 bp, respectively (Figures 4a and 4b), with primers specific to blaCTX-M2 (accession no. KT277101) and blaCTX-M9 (accession no. KT277102). The blaCTX-M2 and blaCTX-M9 gene amplification was absent in ciprofloxacin resistant and cefetoxime sensitive standard MTCC S. typhi 734 and S. typhi BST 63.

Figure 4: a. PCR amplification of CTX-M2 gene. b. PCR amplification of CTX-M9 gene.

Discussion

Enteric fever has caused a major concern to developing countries like India, Pakistan, Nepal and African countries. The incidence of typhoid fever is higher in developing countries compared to developed ones. The prevalence of MDR S. typhi and ESBL-producing S. typhi has now become a challenging task to the field of medicine. Earlier, chloramphenicol was used extensively as a drug of choice to treat typhoid fever. Its extensive prescription triggered the emergence of chloramphenicol-resistant S. typhi in Aden, Chile, and Kuwait [23]. The first epidemic was caused by chloramphenicol-resistant S. typhi in the year 1972 in Mexico [23,24] and subsequent outbreaks were reported from Kerala, India [25], Vietnam, Indonesia and Korea [23,26]. In 1989, the first outbreak, caused by multidrug-resistant S. typhi (MDRST), which is resistant to chloramphenicol, ampicillin, trimethoprim, sulphonamides, and tetracycline, was reported from developing countries like India [26-29], Pakistan [30,31] and Bangladesh [32]. In 1991, ciprofloxacin has been recommended for the treatment of typhoid fever caused by chloramphenicol-resistant S. typhi. In 1992, chromosomal encoded resistance to ciprofloxacin with MIC range of 0.3 mg/L was reported [33].

The present study evaluates the incidence of MDR S. typhi and cefetoxime-mediated ESBL production in S. typhi strains isolated from Kalaburagi City, Karnataka, India. Of the total of 650 Widal-positive samples collected, n=66 have shown positivity for S. typhi at the incidence rate of 10.15%, which is significantly higher in comparison with earlier studies conducted in this area [34]. Sixty-seven percent (n=44) of isolates were MDR and have shown resistance to more than three antibiotics. Antibiotic susceptibility test has shown 46.97% (n=31) strains having reduced susceptibility to ciprofloxacin; in contrast, to all n=66 highly susceptible to third- and fourth-generation antibiotics like levofloxacin and gatifloxacin. Decreased resistance of 7.15% (n=02) was observed in ampicillin, chloramphenicol, streptomycin and 10.6% (n=07) in tetracycline, respectively. Altogether, 66 isolates have shown 100% susceptibility to both cotrimoxizole and azithromycin. Interestingly, 54.54% (n=36) have shown remarkably reduced susceptibility to third-generation cephalosporin’s. The percentage of resistance is relatively higher in cefetoxime 86.11% (n=31), cefexime 44.44% (n=16), ceftriaxone 25% (n=9) and ceftazidime 44.44% (n=16) respectively.

Twelve out of 31 ciprofloxacin-resistant S. typhi isolates have shown an elevated level of MIC in the range of 128-256 μg/ml. There is a substantial increase in MIC level to cefetoxime with four (n=4) isolates in the range of 256 μg/ml; the result is relatively similar in comparison with the studies conducted in China by Yu et al. [35] on S. typhimurium. Four isolates have shown MIC to ampicillin in the range 256-512 μg/ml and also we found relatively higher range of MIC to chloramphenicol of 512 μg/ml.

Phenotypic detection of ESBL production was made against cefetoxime and ceftazidime by two standard phenotypic ESBL detection methods. Among n=36 CeRST isolates, 25% (n=9) strains have shown positivity for ESBL production; remarkably, all n=9 isolates were cefetoxime-mediated ESBL producers. Prevalence of ESBL production in India is far higher in comparison to the studies conducted in Nepal (0.5%) and Poland (0.3%) (36). ESBL production in S. typhi was genotypically confirmed by PCR amplification of blaCTX-M2 and blaCTX-M9 genes with amplicon size of 884 and 692 bp, respectively. The absence blaCTX-M2 and blaCTX-M9 amplification in cefetoxime sensitive standard MTCC S. typhi 733 and S. typhi BST 63 isolate indicates the significance of blaCTX-M2 and blaCTX-M9 genes in cefetoxime mediated ESBL production. The similar studies carried out by Xie et al. also showed the significance blaCTX-M genes in cefetoxime mediated drug resistance in E. coli isolates. Ryoo et al. [21] found nearly similar product length on Klebsiella pneumonia. Earlier studies already reported the presence of blaCTX-M2 gene in S. typhimurium and S. oranienberg [36-38].

Conclusion

Our study shows that the significant increase in the level of drug resistance among S. typhi isolates to higher-generation antibiotics like cephalosporin’s and higher-generation fluoroquinolones with higher level of MIC. Emergence of ESBL in S. typhi majorly in developing countries reflects poorly on sanitary conditions, inadequate medical attention, and contamination of water and food. Antibiotic susceptibility test and ESBL detection are recommended prior to treatment.

Conflict of Interest

The authors declare of no conflict of interest in conducting this study.

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