Antimicrobial drug resistance of Salmonella enterica serovar typhi in asia and molecular mechanism of reduced susceptibility to the fluoroquinolones.
ABSTRACT: This study describes the pattern and extent of drug resistance in 1,774 strains of Salmonella enterica serovar Typhi isolated across Asia between 1993 and 2005 and characterizes the molecular mechanisms underlying the reduced susceptibilities to fluoroquinolones of these strains. For 1,393 serovar Typhi strains collected in southern Vietnam, the proportion of multidrug resistance has remained high since 1993 (50% in 2004) and there was a dramatic increase in nalidixic acid resistance between 1993 (4%) and 2005 (97%). In a cross-sectional sample of 381 serovar Typhi strains from 8 Asian countries, Bangladesh, China, India, Indonesia, Laos, Nepal, Pakistan, and central Vietnam, collected in 2002 to 2004, various rates of multidrug resistance (16 to 37%) and nalidixic acid resistance (5 to 51%) were found. The eight Asian countries involved in this study are home to approximately 80% of the world's typhoid fever cases. These results document the scale of drug resistance across Asia. The Ser83-->Phe substitution in GyrA was the predominant alteration in serovar Typhi strains from Vietnam (117/127 isolates; 92.1%). No mutations in gyrB, parC, or parE were detected in 55 of these strains. In vitro time-kill experiments showed a reduction in the efficacy of ofloxacin against strains harboring a single-amino-acid substitution at codon 83 or 87 of GyrA; this effect was more marked against a strain with a double substitution. The 8-methoxy fluoroquinolone gatifloxacin showed rapid killing of serovar Typhi harboring both the single- and double-amino-acid substitutions.
Project description:BACKGROUND: Typhoid and paratyphoid fever are endemic in China. The objective of this investigation was to determine the molecular features of nalidixic acid-resistant Salmonella enteric serovar Typhi (S. typhi) and Paratyphi (S. paratyphi) from blood isolates in Shenzhen, China. RESULTS: Twenty-five S. typhi and 66 S. paratyphi were isolated from 91 bacteremic patients between 2002 and 2007 at a hospital in Shenzhen, Southern China. Fifty-two percent (13/25) of S. typhi and 95.3% (61/64) of S. paratyphi A were resistant to nalidixic acid. Sixty-seven isolates of nalidixic acid-resistant Salmonella (NARS) showed decreased susceptibility to ciprofloxacin (MICs of 0.125-1 microg/mL). All 75 NARS isolates had a single substitution in the quinolone resistance-determining region (QRDR) of GyrA (Ser83-->Phe/Pro/Tyr, or Asp87-->Gly/Asn), and 90.7% of these isolates carried the substitution Ser83Phe in GyrA. No mutation was found in the QRDR of gyrB, parC, or parE. Plasmid mediated quinolone resistance genes including qnr and aac(6')-Ib-cr were not detected in any isolate. Twenty-two distinct pulsed field gel electrophoresis (PFGE) patterns were observed among S. typhi. Sixty-four isolates of S. paratyphi A belonged to one clone. Eighty-seven investigated inpatients were infected in the community. Six patients infected by S. paratyphi A had a travel history before infection. CONCLUSIONS: Nalidixic acid-resistant S. typhi and S. paratyphi A blood isolates were highly prevalent in Shenzhen, China. PFGE showed the variable genetic diversity of nalidixic acid-resistant S. typhi and limited genetic diversity of nalidixic acid -resistant S. paratyphi A.
Project description:typhoid fever remains a public health problem in Vietnam, with a significant burden in the Mekong River delta region. Typhoid fever is caused by the bacterial pathogen Salmonella enterica serovar Typhi (S. Typhi), which is frequently multidrug resistant with reduced susceptibility to fluoroquinolone-based drugs, the first choice for the treatment of typhoid fever. We used a GoldenGate (Illumina) assay to type 1,500 single nucleotide polymorphisms (SNPs) and analyse the genetic variation of S. Typhi isolated from 267 typhoid fever patients in the Mekong delta region participating in a randomized trial conducted between 2004 and 2005.the population of S. Typhi circulating during the study was highly clonal, with 91% of isolates belonging to a single clonal complex of the S. Typhi H58 haplogroup. The patterns of disease were consistent with the presence of an endemic haplotype H58-C and a localised outbreak of S. Typhi haplotype H58-E2 in 2004. H58-E2-associated typhoid fever cases exhibited evidence of significant geo-spatial clustering along the Sông H u branch of the Mekong River. Multidrug resistance was common in the established clone H58-C but not in the outbreak clone H58-E2, however all H58 S. Typhi were nalidixic acid resistant and carried a Ser83Phe amino acid substitution in the gyrA gene.the H58 haplogroup dominates S. Typhi populations in other endemic areas, but the population described here was more homogeneous than previously examined populations, and the dominant clonal complex (H58-C, -E1, -E2) observed in this study has not been detected outside Vietnam. IncHI1 plasmid-bearing S. Typhi H58-C was endemic during the study period whilst H58-E2, which rarely carried the plasmid, was only transient, suggesting a selective advantage for the plasmid. These data add insight into the outbreak dynamics and local molecular epidemiology of S. Typhi in southern Vietnam.
Project description:We have observed a high incidence of isolated nalidixic acid resistance in Salmonella enterica serovar Enteritidis isolates in Ireland, particularly isolates of phage type 1 (PT1). A group of nalidixic acid-resistant (n = 22) and nalidixic acid-susceptible (n = 28) isolates of serovar Enteritidis from multiple sites in Ireland were selected. Isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI, and the MICs for nalidixic acid and ciprofloxacin were determined. Mutations associated with nalidixic acid resistance in clinical isolates and laboratory mutants of serovar Enteritidis and 32 nalidixic acid-resistant isolates of 15 other salmonella serovars were identified. PFGE had limited discriminatory power. A specific point mutation (G246T) associated with amino acid substitution Asp87Tyr in the quinolone resistance determining region of the gyrA gene accounted for 95% of all mutations in serovar Enteritidis and for all mutations in PT1 isolates. Greater diversity of mutations was observed among all non-Enteritidis salmonella serovars studied. Rates of nalidixic acid resistance in serovar Enteritidis may predominantly reflect clonal expansion after infrequent mutation or selection events.
Project description:We characterized by antibiotic susceptibility, plasmid analysis, incompatibility grouping, and pulsed-field gel electrophoresis (PFGE) of XbaI- and SpeI-digested DNA 102 Salmonella enterica serovar Typhi (serovar Typhi) isolated from recent outbreaks of typhoid in three different parts of Kenya. Only 13.7% were fully susceptible, whereas another 82.4% were resistant to each of the five commonly available drugs: ampicillin, chloramphenicol, and tetracycline (MICs of >256 microg/ml); streptomycin (MIC, >1,024 microg/ml); and cotrimoxazole (MIC of >32 microg/ml). Resistance to these antibiotics was encoded on a 110-kb self-transferable plasmid of IncHI1 incompatibility group. The MICs of nalidixic acid (MIC, 8 to 16 micro g/ml) and ciprofloxacin (MIC of 0.25 to 0.38 micro g/ml) for 41.7% of the 102 serovar Typhi isolates were 5- and 10-fold higher, respectively, than for sensitive strains. Amplification by PCR and sequencing of the genes coding for gyrase (gyrA and gyrB) and topoisomerase IV (parE and parC) within the quinolone resistance-determining region revealed that the increase in the MICs of the quinolones had not resulted from any significant mutation. Analysis of genomic DNA from both antimicrobial agent-sensitive and multidrug-resistant serovar Typhi by PFGE identified two distinct subtypes that were in circulation in the three different parts of Kenya. As the prevalence of multidrug-resistant serovar Typhi increases, newer, more expensive, and less readily available antimicrobial agents will be required for the treatment of typhoid in Kenya.
Project description:OBJECTIVE:To establish the relative importance of Salmonella enterica serovar Typhi with non-classical quinolone resistance. METHODS:Eight hundred and ninety-one isolates of S. Typhi, isolated between 2004 and 2011, were tested for antibiotic susceptibility determination using disc diffusion and E-test. The mechanisms of fluoroquinolone resistance were studied in a sub-set of the NALS (nalidixic acid susceptible) isolates by wave nucleic acid fragment analysis of PCR products from gyrA, gyrB, parC and parE and from the plasmid borne determinants: qnrA,B,S; aac(6')-Ib-cr and qepA. To assess genetic relatedness multi-locus variable number tandem repeat analysis was carried out using five loci. RESULTS:Eighty isolates with a nalidixic acid MIC of <32 mg/L (NALS) and a ciprofloxacin MIC of >0.064 mg/L CIPI (ciprofloxacin reduced susceptibility) were found. In 36 NALS CIPI isolates two distinct genotypes were identified when compared with 16 susceptible controls: Group B (n = 34), mutation in gyrB at codon 464, NAL MIC of 3-12 mg/L and CIP MIC of 0.064-0.5 mg/L.; and Group C, mutation in gyrA at codon 83 (n = 2) NAL MIC of 16 mg/L and CIP MIC of 0.25-0.38 mg/L. Group B isolates were found in different strain backgrounds as defined by MLVA. CONCLUSION:The use of nalidixic acid to screen for reduced susceptibility to fluoroquinolones in S. Typhi misses CIPI-NALS isolates, an established phenotype in India.
Project description:In Salmonella enterica serovar Typhimurium, an S431P substitution in the B subunit of gyrase (allele gyrB651) confers resistance to nalidixic acid and causes reduced DNA superhelicity and hypersensitivity to novobiocin. Selection for novobiocin resistance allowed isolation of a mutation in the gyrA gene (allele gyrA659), a T467S substitution, which partially suppresses the supercoiling defect of gyrB651. Modeling analysis suggests that this mutation acts by destabilizing the GyrA bottom dimer interface. This is the first example of a gyrA mutation that compensates for a gyrB defect.
Project description:Salmonella enterica serotype Typhi clinical isolates (n = 91) resistant to nalidixic acid (Nal(r)) were collected from sporadic cases and minor outbreaks throughout Vietnam between 1996 and 2004. These isolates were typed and compared by four methods: Vi phage typing, PstI ribotyping, XbaI and SpeI pulsed-field gel electrophoresis (PFGE), and single-nucleotide polymorphism (SNP) analysis. The results indicated that 65% of the isolates were not typeable by Vi phage typing. In contrast, the ribotyping and, with more accuracy, the SNP analysis methods indicated that all Nal(r) isolates belonged to a single clone (ribotype 3a, haplotype H58) that was found previously and that largely consisted of plasmid-encoded multidrug-resistant serotype Typhi isolates. PFGE demonstrated the occurrence of microevolution within this clone. We identified two major combined PFGE profiles: X1-S1 and X3-S6. X3-S6 predominated between 1996 and 2002 but was replaced by X1-S1 after 2002. Nevertheless, PFGE, with a Simpson's index of 0.78, was not considered an optimal discriminatory method for investigating typhoid fever outbreaks in Vietnam. The rate of quinolone resistance increased and the rate of multidrug resistance decreased during the study period. From 2002 to 2004, 80.6% of the isolates from South Vietnam were resistant only to Nal. The mechanism of Nal resistance in most of the isolates (94%) was a mutation in the quinolone resistance-determining chromosomal region of gyrA that led to the amino acid substitution Ser83Phe. No plasmid-located qnrA, qnrB, or qnrS was detected.
Project description:Salmonella enterica serovar Virchow is highly prevalent in humans and farm animals in Israel. In addition to high rates of resistance to multiple antibiotics, this serovar exhibits a high incidence of resistance to nalidixic acid. More than 90% of Salmonella serovar Virchow isolates of human and poultry origin obtained from 1997 to 2004 were resistant to nalidixic acid (MIC > or = 128 microg/ml), with reduced susceptibility to ciprofloxacin (MIC between 0.125 and 0.250 microg/ml). Most isolates belonged to two predominant, closely related pulsed-field gel electrophoresis image types. Investigation of the mechanisms of quinolone resistance revealed that this pathogen probably emerged from a parental clone that overproduced the AcrAB efflux pump and had a single point mutation in gyrA leading to the Asp87Tyr substitution. The close resemblance between human and poultry isolates points to poultry as a likely source of Salmonella serovar Virchow in the food chain.
Project description:Infections with Salmonella enterica serovar Typhi isolates that have reduced susceptibility to ofloxacin (MIC ? 0.25 ?g/ml) or ciprofloxacin (MIC ? 0.125 ?g/ml) have been associated with a delayed response or clinical failure following treatment with these antimicrobials. These isolates are not detected as resistant using current disk susceptibility breakpoints. We examined 816 isolates of S. Typhi from seven Asian countries. Screening for nalidixic acid resistance (MIC ? 16 ?g/ml) identified isolates with an ofloxacin MIC of ?0.25 ?g/ml with a sensitivity of 97.3% (253/260) and specificity of 99.3% (552/556). For isolates with a ciprofloxacin MIC of ?0.125 ?g/ml, the sensitivity was 92.9% (248/267) and specificity was 98.4% (540/549). A zone of inhibition of ?28 mm around a 5-?g ofloxacin disc detected strains with an ofloxacin MIC of ?0.25 ?g/ml with a sensitivity of 94.6% (246/260) and specificity of 94.2% (524/556). A zone of inhibition of ?30 mm detected isolates with a ciprofloxacin MIC of ?0.125 ?g/ml with a sensitivity of 94.0% (251/267) and specificity of 94.2% (517/549). An ofloxacin MIC of ?0.25 ?g/ml and a ciprofloxacin MIC of ?0.125 ?g/ml detected 74.5% (341/460) of isolates with an identified quinolone resistance-inducing mutation and 81.5% (331/406) of the most common mutant (carrying a serine-to-phenylalanine mutation at codon 83 in the gyrA gene). Screening for nalidixic acid resistance or ciprofloxacin and ofloxacin disk inhibition zone are suitable for detecting S. Typhi isolates with reduced fluoroquinolone susceptibility.
Project description:Background:Little is known about the evolutionary process and emergence time of resistance mutations to fluoroquinolone in Salmonella enterica serovar Typhi. Methods:We analyzed S. Typhi isolates collected from returned travelers between 2001 and 2016. Based on ciprofloxacin susceptibility, isolates were categorized as highly resistant (minimum inhibitory concentration [MIC] ? 4 ?g/mL [CIPHR]), resistant (MIC = 1-2 ?g/mL [CIPR]), intermediate susceptible (MIC = 0.12-0.5 ?g/mL [CIPI]), and susceptible (MIC ? 0.06 ?g/mL [CIPS]). Results:A total of 107 isolates (33 CIPHR, 14 CIPR, 30 CIPI, and 30 CIPS) were analyzed by whole-genome sequencing; 2461 single nucleotide polymorphisms (SNPs) were identified. CIPS had no mutations in the gyrA or parC genes, while each CIPI had 1 of 3 single mutations in gyrA (encoding Ser83Phe [63.3%], Ser83Tyr [33.3%], or Asp87Asn [3.3%]). CIPHR had the same 3 mutations: 2 SNPs in gyrA (encoding Ser83Phe and Asp87Asn) and a third in parC (encoding Ser80Ile). CIPHR shared a common ancestor with CIPR and CIPI isolates harboring a single mutation in gyrA encoding Ser83Phe, suggesting that CIPHR emerged 16 to 23 years ago. Conclusions:Three SNPs-2 in gyrA and 1 in parC-are present in S. Typhi strains highly resistant to fluoroquinolone, which were found to have evolved in 1993-2000, approximately 10 years after the beginning of the ciprofloxacin era. Highly resistant strains with survival advantages arose from strains harboring a single mutation in gyrA encoding Ser83Phe. Judicious use of fluoroquinolones is warranted to prevent acceleration of such resistance mechanisms in the future.