Antimicrobial resistance and molecular subtypes of Salmonella enterica serovar Typhi isolates from Kolkata, India over a 15 years period 1998-2012.

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), remains an unresolved public health problem in India. Emergence of antimicrobial resistant strains poses a great concern for typhoid treatment and influences reshaping of current S. Typhi population. We included representative S. Typhi strains (n=164) from retrospective studies, both community and hospital based, conducted at National Institute of Cholera and Enteric Diseases, Kolkata during 15 years period (1998-2012) to analyze their antimicrobial resistance (AMR) profiles, mechanism of AMR and molecular subtypes of the strains. More than 60% of the S. Typhi isolates were obtained from community based studies. During the study period, steady decline (46.4%-15.6%) in isolation of multidrug-resistant (MDR, resistant to ampicillin, chloramphenicol and co-trimoxazole) S. Typhi was noticed with parallel increase of nalidixic acid-resistant (NALR) strains (60.7%-93.8%) and ciprofloxacin resistant (CIPR) strains (0%-25%). Of 53 MDR strains, 46 (86.8%) were NALR showing decreased ciprofloxacin susceptible (DCS) (MIC for ciprofloxacin 0.12-0.5µg/ml) phenotype. Conjugative IncHI1 (230kb) and non-conjugative non-IncHI1 (180kb) plasmids were found in 23 (43.4%) and 14 (26.4%) MDR strains respectively, plasmid was absent in 16 (30.2%) MDR strains. MDR strains with or without plasmid shared the same set of resistance genes (blaTEM-1, catA1, sul1, sul2, strA and strB) and class 1 integron possessing dfrA7 gene cassette. Two S. Typhi strains harbored 50kb transferrable plasmids carrying dfrA15 and aadA1 gene cassettes in class 1 integron. The majority of the strains (135/164, 82.3%) belonged to H58 haplotype. Among the MDR isolates, fluoroquinolone resistant or combined resistant isolates (n=147), 127 (86.4%) were H58 and 20 (13.6%) belonged to non-H58. NALRS. Typhi strains with decreased susceptibility or resistance to ciprofloxacin had point mutation(s) in quinolone resistance-determining region of gyrA and parC genes. Pulsed-field gel electrophoresis showed more diversity among NALRS. Typhi than MDR strains. Results of this study generated information useful for better understanding of the disease epidemiology and its control in endemic settings.

Emergence of ceftriaxone resistant Salmonella enterica serovar Typhi in Eastern India.

In view of widespread isolation of fluoroquinolone (FQ) resistant Salmonella enterica serovar Typhi globally, third generation cephalosporins (ceftriaxone) are used as alternative drugs for treatment of typhoid fever in recent years. But reports on emergence of third generation cephalosporin resistant S. Typhi have been documented from various countries including India posing threat in future use of this drug for typhoid treatment. Here, we report on genomic analysis of a third generation cephalosporin resistant S. Typhi strain isolated for the first time from Eastern India, Kolkata during 2019. The study strain was phenotypically resistant to ceftriaxone, ampicillin. Whole genome sequencing revealed the presence of conjugative IncX3 plasmid carrying blaSHV-12 gene on it. The study strain belongs to H58 haplotype (4.3.1.2) and ST1 type. Comparison of phylogenetic analysis of the study strain with other cephalosporin resistant S. Typhi strains across the world revealed that three strains isolated from Western part of India during 2016 were its closest neighbours. Hence close monitoring of cephalosporin resistant S. Typhi strains are of great importance to control the furure use of this antibiotic for the treatment of typhoid fever.

Characterization of non-typhoidal Salmonella isolates from children with acute gastroenteritis, Kolkata, India, during 2000-2016.

Non-typhoidal Salmonella (NTS) is an important cause of acute gastroenteritis in children. The study was undertaken to determine the isolation rate, serovar prevalence, antimicrobial resistance (AMR) profiles, and molecular subtypes of NTS from a hospital-based diarrheal disease surveillance in Kolkata, India. Rectal swabs were collected from children (< 5 years of age) with acute gastroenteritis from 2000 to 2016. Samples were processed following standard procedures for identification of NTS. The isolates were tested for antimicrobial susceptibility, AMR genes, plasmid profiles, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE) subtypes. A total of 99 (1.0%) Salmonella isolates were recovered from 9957 samples processed. Of the 17 Salmonella serovars identified, S. Worthington (33%) was predominant followed by S. Enteritidis (13%), S. Typhimurium (12%), and others. The isolates showed high resistance towards nalidixic acid (43%), ampicillin (34%), third-generation cephalosporins (32%), and azithromycin (25%), while low resistance was observed for fluoroquinolones (2%). Extended-spectrum beta-lactamase production (blaCTX-M-15 and blaSHV-12 genes) and azithromycin resistance (mphA gene) were common in S. Worthington, while fluoroquinolone resistance (gyrA and parC mutations) was found in S. Kentucky. Diverse plasmid profiles were observed among the isolates. PFGE analysis identified genetically related strains of each serovar in circulation. MLST also revealed phylogenetically clonal isolates of which S. Worthington ST592 and ciprofloxacin-resistant S. Kentucky ST198 were not reported earlier from India. NTS resistant to current drugs of choice poses a potential public health problem. Continuous monitoring of AMR profiles and molecular subtypes of NTS serovars is recommended for controlling the spread of resistant organisms.

Profiling Virulence and Antimicrobial Resistance Markers of Enterovirulent Escherichia Coli from Fecal Isolates of Adult Patients with Enteric Infections in West Cameroon.

OBJECTIVES: This study aimed to identify virulent and antimicrobial resistant genes in fecal E. coli in Mbouda, Cameroon. METHODS: A total of 599 fecal samples were collected from patients with enteric infections who were ≥ 20 years old. E. coli was isolated on the MacConkey agar and virulent genes were detected by multiplex/simplex PCR. Isolates in which ≥ 1 virulent gene was detected were subjected to antibiotic susceptibility testing. The resulting resistant isolates were subjected to PCR, followed by sequencing for resistant genes detection. RESULTS: There were 119 enterovirulent E. coli identified, amongst which 47.05% were atypical enteropathogenic E. coli (EPEC), 36.97% enterotoxigenic E. coli, 10.08% Shiga toxin producing E. coli (STEC) and 5.88% were enteroinvasive E. coli (EIEC). The occurrence of the eae gene (47.06%) was higher compared with CVD432 (33.61%), aaic (13.45%), stx2 (10.08%) and stx1 (0.84%). High resistance rates were noted for ampicillin (94.64% EPEC, 91.67% STEC, 59.09% EAEC, and 57.14% EIEC) and sulfamethoxazole-trimethoprim (100% EPEC and 83.33% STEC, 81.82% EAEC and 71.43% EIEC). sul2 (71.43%), tetB (64.71%), tetA (59.94%) and blaTEM (52.10%) were detected. A double mutation (S83L; D87N) was seen in gyrA and a single mutation (S80I) was observed in parC. CONCLUSION: These findings suggested that measures should be taken to reduce the harm of E. coli to public health.

Changes in antimicrobial resistance and molecular attributes of Salmonellae causing enteric fever in Kolkata, India, 2014-2018.

Globally, enteric fever caused by Salmonella Typhi (S. Typhi, ST) and S. Paratyphi A (SPA) remain one of the major diseases of public health importance. In this study, a total of 457 (380 ST, 77 SPA) blood isolates were collected from three tertiary care hospitals in Kolkata during 2014-18. Additionally, 66 (3.4%) ST and 5 (0.25%) SPA were recovered from blood culture of 1962 patients attending OPD of one pediatric hospital during 2016-18. The study isolates were tested for antimicrobial resistance (AMR) profiles; AMR genes; molecular sub-types by PFGE, MLVA and CRISPR. Among the total 446 ST and 82 SPA isolates, fluoroquinolone (FQ) resistance was very common in both serovars. Ciprofloxacin resistance of 24.9% and 9.8% & ofloxacin resistance of 20.9% and 87.8% were found in ST and SPA respectively. Majority (>70%) of the isolates showed decreased susceptibility to ciprofloxacin (DCS). A single point mutation in gyrA gene (S83F) was responsible for causing DCS in 37.5% (n = 42/112) ST and 63% (n = 46/73) SPA isolates. Multidrug resistance (MDR) was found only in 3.4% ST isolates and encoded the genes blaTEM-1, catA, sul, strA-strB, class 1 integron with dfrA7. All MDR ST (n = 15) possessed non-conjugative non-IncHI1 (180 kb) plasmid except one having conjugative IncHI1 (230 kb) plasmid and one without plasmid. The MDR genes were integrated near chromosomal cyaA gene site in ST with/without the presence of plasmid (nonIncH1). Almost 65.7% resistant ST belonged to H58 haplotype. PFGE showed clonally related isolates with 81% similarity in ST and 87% in SPA. Similarly, CRISPR typing showed less diversity among the isolates. However, the isolates (ST and SPA) were found to be more diverse by MLVA typing (D value 0.987 and 0.938). The study reports decrease in MDR and increase in FQ resistance among typhoidal Salmonella isolates over the years giving interesting information for enteric fever treatment.

Report of Relapse Typhoid Fever Cases from Kolkata, India: Recrudescence or Reinfection?

Three relapse cases were reported out of 107 hospital-attending typhoid cases within a period of 2 years (2014-2016) from Apollo Gleneagles Hospital, Kolkata, India. During the first episode of typhoid fever, 2 of the 3 cases were treated with ceftriaxone (CRO) for 7 days, and 1 was treated for 14 days. Six Salmonella Typhi (S. Typhi) isolates, obtained from the 3 patients during both typhoid episodes, were subjected to antimicrobial susceptibility testing, detection of quinolone resistance-determining region (QRDR) mutation and molecular subtyping by pulsed-field gel electrophoresis (PFGE), multiple-locus variable number tandem repeat analysis (MLVA), multilocus sequence typing (MLST), clustered regularly interspaced short palindromic repeats (CRISPR), and H58 haplotyping. Pairs of the S. Typhi strains isolated from two of the patients during the 1st and 2nd episodes were similar with respect to the antimicrobial resistance (AMR) profiles, QRDR mutations, and molecular subtypes; whereas, the S. Typhi strain pair isolated from the 3rd patient were different in their AMR profiles, QRDR mutations, and MLVA profiles. From these observations, it may be concluded that in spite of treating typhoid cases with CRO for 7-14 days, relapse of typhoid fever might occur. The article also showed the advantage of MLVA typing over PFGE, MLST, and CRISPR typing for the discrimination of strains isolated from the same patient in case of relapse of typhoid fever.

Molecular Subtyping of Salmonella enterica Serovar Typhi by Pulsed-Field Gel Electrophoresis and Multiple-Locus Variable-Number Tandem-Repeat Analysis in India: Their Association with Antimicrobial Resistance Profiles.

Molecular subtyping and DNA sequencing-based methods, which are commonly used for discriminating Salmonella enterica serovar Typhi (S. Typhi) isolates, lead to improved molecular epidemiological investigations for prevention and control of typhoid fever. We obtained S. Typhi blood isolates (n = 66) from India during 2007-14 for molecular subtyping by pulsed-field gel electrophoresis (PFGE) and multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) in association with antibiotic resistance profiles. Genotypic diversity was observed more by MLVA (Simpson's index of diversity, D value = 0.997) than PFGE (D value = 0.864). Two prevalent pulsotypes containing nalidixic acid-resistant (NALR) and NALR-ciprofloxacin-resistant (CIPR) S. Typhi isolates circulated in India. Multidrug-resistant (MDR), NALR-CIPR, and most NALR isolates were found to be clonal by PFGE. MLVA could differentiate the clonal isolates. Most of the MDR and NALR-CIPR isolates showed variation in single or double VNTR loci, whereas NALR isolates varied in more than 2 loci, reflecting higher genetic diversity among the NALR isolates. Of the 6 VNTR loci, TR4,699 (D value = 0.838) and Sal02 (D value = 0.890) loci played important roles as MLVA cluster-supporting alleles. The rapid turnaround time and high-level discriminatory power of MLVA may be useful for tracking and controlling the transmission of S. Typhi isolates during epidemiological investigations.