Mutations In gyrA Genes Research Articles

Survey of Helicobacter pylori levofloxacin and clarithromycin resistance rates and drug resistance genes in Ningxia, 2020-2022

Objective: To explore the rate of Helicobacter pylori (Hp) resistance to levofloxacin and clarithromycin and the common mutation patterns of resistance genes in Ningxia, and to assess the concordance between phenotypic resistance and genotypic resistance. Methods: Cross-sectional study. Patients diagnosed with Hp infection in 14 hospitals in Ningxia region from February 2020 to May 2022 were retrospectively selected. Hp strains were isolated from gastric biopsy specimens of Hp-infected patients and subjected to phenotypic drug sensitivity testing and detection of resistance genes to analyze the rate of Hp resistance to levofloxacin and clarithromycin and the common mutation patterns of resistance genes in Ningxia region; and the concordance rate and Kappa concordance test were used to assess the concordance between phenotypic resistance and genotypic resistance. Results: A total of 1 942 Hp strains were isolated and cultured, and among the infections, 1 069 cases (55.0%) were male and 873 cases (45.0%) were female, aged (50.0±12.5) years (15-86 years). The rates of Hp resistance to levofloxacin and clarithromycin in Ningxia were 42.1% (818/1 942) and 40.1% (779/1 942), respectively, and the rate of dual resistance to both was 22.8% (443/1 942). The rate of resistance to levofloxacin and clarithromycin of Hp strains from female patients was higher than in male patients (levofloxacin: 50.4%(440/873) vs 35.4%(378/1 069); clarithromycin: 44.4%(388/873) vs 36.6%(391/1 069), both P<0.001). Among the GyrA gene mutations associated with levofloxacin resistance, the differences in mutation rate of amino acid at positions 87 and 91 were statistically significant in both drug-resistant and sensitive strains(both P<0.001), except for Asn87Thr. Hp strains were statistically significant for levofloxacin (Kappa=0.834, P<0.001) and clarithromycin (Kappa=0.829, P<0.001) had good concordance in resistance at the phenotypic and genotypic levels. Conclusion: The resistance of Hp to levofloxacin and clarithromycin in Ningxia region is severe, and there is good consistency between genotypic and phenotypic resistance.

Prevalence and mechanisms of ciprofloxacin resistance in Escherichia coli isolated from hospitalized patients, healthy carriers, and wastewaters in Iran

BackgroundThis study was aimed to evaluate the prevalence and molecular characteristics of ciprofloxacin resistance among 346 Escherichia coli isolates collected from clinical specimens (n = 82), healthy children (n = 176), municipal wastewater (n = 34), hospital wastewater (n = 33), poultry slaughterhouse wastewater (n = 12) and livestock (n = 9) slaughterhouse wastewater in Iran.MethodsCiprofloxacin minimum inhibitory concentration (MIC) was determined by agar dilution assay. Phylogroups and plasmid-mediated quinolone resistance (PMQR) genes were identified using PCR. Mutations in gyrA, gyrB, parC, and parE genes and amino acid alterations were screened through sequencing assay. The effect of efflux pump inhibitor (PAβN) on ciprofloxacin MICs in ciprofloxacin-resistant isolates was investigated using the microdilution method.ResultsIn total, 28.03% of E. coli isolates were phenotypically resistant to ciprofloxacin. Based on sources of isolation, 64.63%, 51.51%, 33.33%, 14.70%, 10.22% and 8.33% of isolates from clinical specimens, hospital wastewater, livestock wastewater, municipal wastewater, healthy children and poultry wastewater were ciprofloxacin-resistant, respectively. Eighty-one point eighty-one percent (Ser-83 → Leu + Asp-87 → Asn; 78.78% and Ser-83 → Leu only; 3.03% (of ciprofloxacin-resistant E. coli isolates showed missense mutation in GyrA subunit of DNA gyrase, while no amino-acid substitution was noted in the GyrB subunit. DNA sequence analyses of the ParC and ParE subunits of topoisomerase IV exhibited amino-acid changes in 30.30% (Ser-80 → Ile + Glu-84 → Val; 18.18%, Ser-80 → Ile only; 9.10% and Glu-84 → Val only; 3.03%0 (and 15.38% (Ser-458 → Ala) of ciprofloxacin-resistant E. coli isolates, respectively. The PMQR genes, aac(6')-Ib-cr, qnrS, qnrB, oqxA, oqxB, and qepA were detected in 43.29%, 74.22%, 9.27%, 14.43%, 30.92% and 1.03% of ciprofloxacin-resistant isolates, respectively. No isolate was found to be positive for qnrA and qnrD genes. In isolates harboring the OqxA/B efflux pump, the MIC of ciprofloxacin was reduced twofold in the presence of PAβN, as an efflux pump inhibitor. The phylogroups B2 (48.45%) and A (20.65%) were the most predominant groups identified in ciprofloxacin-resistant isolates.ConclusionsThis study proved the high incidence of ciprofloxacin-resistant E. coli isolates in both clinical and non-clinical settings in Iran. Chromosomal gene mutations and PMQR genes were identified in ciprofloxacin resistance among E. coli population.

Characterization of Genetic Mutations in Multi-Drug-Resistant Isolates of Mycobacterium tuberculosis Bacilli Conferring Resistance to a Second-Line Anti-tuberculosis Drug.

Multi-drug-resistant tuberculosis (MDR-TB) has become a major public health concern globally. Mutations in first- and second-line drug targets such as katG, inhA, rpoB, rrs, eis, gyrA, and gyrB have been associated with drug resistance. Monitoring predominant mutations in the MDR-TB patient population is essential to monitor and devise future therapeutic regimes. The present study is aimed to characterize genetic mutations in MDR isolates of Mycobacterium tuberculosis (MTB)bacilli conferring resistance to a second-line anti-tuberculosis drug in the Eastern Indian population. This cross-sectional study was conducted in the Department of Microbiology, Indira Gandhi Institute of Medical Sciences, Patna, Bihar, and in the Tuberculosis Demonstration & Training Centre, Agamkuan, Patna. A total of 3270 patients suspected to have MDR-TB were recruited in the study. Two sputum samples, one on the spot, and the other in the morning were collected from each patient and the diagnosis ofrifampicin-sensitive (RS)/rifampicin-resistant (RR/MDR) TB was done by Gene-Xpert test. One hundred fifty RS-TB samples and 150 RR/MDR-TBsamples were considered for line probe assay (LPA). RSsamples were subjected to first-line LPA using Genotype® MTBDR Plus ver 2.0 and RR/MDRsamples were considered for second-line LPA using Genotype®MTBDRslver 2.0. All sputum samples were subjected to sputum smear microscopy using the Ziehl-Neelsen staining method.Statistical analysis was done using Statistical Package for Social Sciences (SPSS) version 26.0 (IBM Corp. Armonk, NY) and R (version 4.1; R Core Team 2021). In the present study, out of 3270 patients, we detected RR/MDR-TB in 235 patients (7.19%),RS-TB in 812 patients (24.83%), the rest of the patients negative for MTB (2223, 67.98%). Out of 150 RR/MDR-TB sputum samples tested, resistance to fluoroquinolone (FQ) was observed in 41 samples. The selected patients had predominantly FQ resistance due to the gyrA gene mutations (97.56%, n=40) compared to the gyrB gene mutations (2.44%, n=1). We observed >60% of the mutations in the gyrA gene in codon 94 (MUT3C (D94G), MUT3A (D94A), and MUT3D (D94H).In addition, we found the mutations MUT1 (A90V) and MUT2 (S91P)in the codons 90 and 91 of the gyrA gene in the considered MTB patient population. The identified genes can be further validated to be considered as therapeutic targets, but more therapeutics and advanced strategies should be applied in the management of MTB.

Association of Mutations in gyrA Gene with Resistance to Fluoroquinolones in Clinical Isolates of Multidrug-Resistant Mycobacterium tuberculosis

Association of Mutations in gyrA Gene with Resistance to Fluoroquinolones in Clinical Isolates of Multidrug-Resistant Mycobacterium tuberculosis

Prevalence, genomic characterization and antimicrobial resistance of Campylobacter spp. isolates in pets in Shenzhen, China.

The prevalence of Campylobacter spp.in pets is a potential concern for human health. However, little is known about the pet-related Campylobacter spp. in China. A total of 325 fecal samples were collected from dogs, cats, and pet foxes. Campylobacter spp. were isolated by culture, and MALDI-TOF MS was used to identify 110 Campylobacter spp. isolates in total. C. upsaliensis (30.2%, 98/325), C. helveticus (2.5%, 8/325), and C. jejuni (1.2%, 4/325) were the three found species. In dogs and cats, the prevalence of Campylobacter spp. was 35.0% and 30.1%, respectively. A panel of 11 antimicrobials was used to evaluate the antimicrobial susceptibility by the agar dilution method. Among C. upsaliensis isolates, ciprofloxacin had the highest rate of resistance (94.9%), followed by nalidixic acid (77.6%) and streptomycin (60.2%). Multidrug resistance (MDR) was found in 55.1% (54/98) of the C. upsaliensis isolates. Moreover, 100 isolates, including 88 C. upsaliensis, 8 C. helveticus, and 4 C. jejuni, had their whole genomes sequenced. By blasting the sequence against the VFDB database, virulence factors were identified. In total, 100% of C. upsaliensis isolates carried the cadF, porA, pebA, cdtA, cdtB, and cdtC genes. The flaA gene was present in only 13.6% (12/88) of the isolates, while the flaB gene was absent. By analyzing the sequence against the CARD database, we found that 89.8% (79/88) of C. upsaliensis isolates had antibiotic target alteration in the gyrA gene conferring resistance to fluoroquinolone, 36.4% (32/88) had the aminoglycoside resistance gene, and 19.3% (17/88) had the tetracycline resistance gene. The phylogenetic analysis using the K-mer tree method obtained two major clades among the C. upsaliensis isolates. All eight isolates in subclade 1 possessed the gyrA gene mutation, the aminoglycoside and tetracycline resistance genes, and were phenotypically resistant to six classes of antimicrobials. It has been established that pets are a significant source of Campylobacter spp. strains and a reservoir for them. This study is the first to have documented the presence of Campylobacter spp. in pets in Shenzhen, China. In this study, C. upsaliensis of subclade 1 required additional attention due to its broad MDR phenotype and relatively high flaA gene prevalence.

Emergence and evolution of drug-resistant Mycobacterium tuberculosis in eastern China: A six-year prospective study

Understanding the emergence and evolution of drug resistance can inform public health intervention to combat tuberculosis (TB). In this prospective molecular epidemiological surveillance study from 2015 to 2021 in eastern China, we prospectively collected whole-genome sequencing and epidemiological data on TB patients. We dissect the ordering of drug resistance mutation acquisition for nine commonly used anti-TB drugs, and we found that the katG S315T mutation first appeared around 1959, followed by rpoB S450L (1969), rpsL L43A (1972), embB M306V (1978), rrs 1401 (1981), fabG1 (1982), pncA (1985) and folC (1988) mutations. GyrA gene mutations appeared after the year of 2000. We observed that the first expansion of Mycobacterium tuberculosis (M.tb) resistance population among eastern China appeared after the introduction of isoniazid, streptomycin and para-amino salicylic acid, and the second expansion after the ethambutol, rifampicin, pyrazinamide, ethionamide and aminoglycosides. We speculate these two expansions are linked with population shift historically. By geospatial analysis, we found drug-resistant isolates migrated within eastern China. With epidemiological data of clonal strains, we observed some strains can evolve continuously in individuals and transmit readily in a population. In conclusion, this study mirrored the emergence and evolution of drug-resistant M.tb in eastern China were linked to the sequence and timing of introduction of anti-TB drugs, and multiple factors may contribute to the resistant population enlarged. To resolve the epidemic of drug-resistant TB, it requires applying anti-TB drugs carefully and/or identifying resistant patients timely to prevent them from developing high-level resistance and transmitting to others.

The first evidence of blaCTX-M-55, QnrVC5, and novel insight into the genome of MDR Vibrio vulnificus isolated from Asian sea bass (Lates calcarifer) identified by resistome analysis

Vibrio vulnificus is a bacterium that causes vibriosis and exhibits resistance to many antimicrobials. To comprehensively understand V. vulnificus resistance, four MDRs isolates were selected for resistome analysis. Based on the Average Nucleotide Identity (ANI) and DNA-DNA hybridization (DDH), all isolates showed a high identity score to the V. vulnificus ATCC 33147. Likewise, multilocus sequence typing (MLST) showed that CUVETSS48 isolate was founded closer to two STs (409 and 570), while the others (CUVETCC1, CUVETCC2, and CUVETSS2) were founded closer to a single ST (209, 426, and 372, respectively). MIC determination showed that all isolates were resistant to Amoxicillin and Colistin sulfate, but only two isolates (CUVETCC1 and CUVETSS48) were resistant to all tested antimicrobials. Referring to resistome analysis, multiple antibiotic resistance genes (ARGs) were found, such as QnrVC1, QnrVC7, tetR, tetB, and blaCTX-M-55 (CUVETCC1); tet(59), sul2, and QnrVC5 (CUVETSS48). Moreover, a mutation was founded within the QRDR region of gyrA; S83I (CUVETCC1, CUVETCC2, and CUVETSS48), gyrB; K87N (CUVETCC2, SS2, and SS48), and parC; S80Y (CUVETCC1 and CUVETCC2). In conclusion, this study showed the first evidence for the presence of blaCTX-M-55, QnrVC5 antimicrobial resistance genes, as well as mutations in gyrA, gyrB, and parC genes (at positions 83, 87, and 80) among V. vulnificus isolates. The presence of multiple ARGs may contribute to the MDR phenotype of V. vulnificus.

Prevalence of Mycoplasma genitalium infection with antimicrobial resistance mutations among gay sex workers in China.

Gay sex workers (GSWs) within the population of men who have sex with men in China are known as money boys (MBs). Limited research has been conducted to investigate the infection rate and antimicrobial resistance of Mycoplasma genitalium (M. genitalium) among GSWs in China. This study aimed to evaluate the status of M. genitalium infection among in GSWs. This study was performed among 349 GSWs who were followed up for four years by internet-based sampling collection. The participants were asked to complete an online questionnaire using a mobile app, and trained interviewers took urethral, anorectal, and saliva swab specimens. STIs, including HIV and M. genitalium, were detected. Detection of resistance-associated mutations (RAMs) to macrolides and fluoroquinolones was performed via Sanger sequencing of the 23S rRNA, parC and gyrA genes. GSWs were enrolled by identifying 10 initial "seeds" from the Blued and WeChat apps. Face-to-face interviews were conducted with 349 GSWs from June 2017 to July 2021. The prevalence of M. genitalium and HIV positivity was 92/349 (26.4%, 95% confidence interval [CI] 21.7-31.0) and 71/349 (20.3%, 95% CI 16.3-24.4), respectively. The proportion of GSWs with M. genitalium infection alone in urethral swabs was 16, and the proportion with symptoms was 2/16 (12.5%). The proportion of GSWs with M. genitalium infection alone in anorectal swabs was 36, and the proportion with symptoms was 3/36 (8.3%). Multivariate regression analysis showed that using new types of drugs in the past 3 months and inconsistent condom usage with clients in the past 30 days were associated with M. genitalium infection. Macrolide resistance within the 23S rRNA gene was detected in 73/88 (83.0%) of the M. genitalium-positive GSWs. Moreover, 79.8% (71/89) of parC and 21.1% (19/90) of gyrA genes had mutations responsible for fluoroquinolone resistance. Three cases had no mutations in any of the three genes, 11 cases had mutations in all three genes, five cases had gyrA and parC gene mutations with no mutation in the 23S rRNA gene, and 42 cases had 23S rRNA and parC gene mutations with no mutation in the gyrA gene. M. genitalium infections in our study displayed a high prevalence and very high levels of macrolide and fluoroquinolone resistance among GSWs in China. Asymptomatic M. genitalium infections are quite common among GSWs. Routine resistance testing of M. genitalium-positive specimens and antimicrobial resistance surveillance are crucial.

Mutations in gyrA and parC genes in fluoroquinolone-resistant Acinetobacter baumannii that causes hospital acquired infection

Mutations in gyrA and parC genes in fluoroquinolone-resistant Acinetobacter baumannii that causes hospital acquired infection

Molecular Characterization of Resistance to Second-Line Anti-Mycobacterial Drugs among Clinical Isolates of Multidrug-Resistant Mycobacterium tuberculosis

The emergence of multidrug resistance and extensively drug-resistant tuberculosis is a serious public health crisis. Using rapid and inexpensive molecular methods such as HRM assay in the detection of second-line drugs resistance in M. tuberculosis would be helpful in the treatment and control of XDR tuberculosis cases. MDR-TB isolates were collected from Iranian tuberculosis laboratories. Drug susceptibility test performed via the indirect proportion method utilizing LJ Medium. Susceptibility to ciprofloxacin, ofloxacin, amikacin, kanamycin, and capreomycin, as second-line anti-tuberculosis agents were assessed. Single point mutations in gyrA, rrs and eis genes were detected via HRM assay and DNA sequencing. A DST test was performed for 56 MDR isolates and at least 27 (48.2%) isolates were resistant to CIP or OFL. Also, 14 (25%), 12 (21.4%), and 15 (26.7%) isolates were resistant to capreomycin, amikacin, and kanamycin, respectively. D94G, A90V, and G88C mutations were the most frequent mutations in gyrA gene. Also, A1401G mutation was detected more than the other mutations in rrs gene. The frequency of CIP/OFL and AMK/CAP/KAN-resistant TB is considerable among Iranian tuberculosis cases. HRM assay is a rapid and inexpensive test and can detect important mutation-based drug resistance in MDR-TB and XDR-TB isolates.

Genomic characterization of molecular markers associated with antimicrobial resistance and virulence of the prevalent Campylobacter coli isolated from retail chicken meat in the United Arab Emirates

Campylobacter is a major cause of gastroenteritis worldwide, with broiler meat accounting for most illnesses. Antimicrobial intervention is recommended in severe cases of campylobacteriosis. The emergence of antimicrobial resistance (AMR) in Campylobacter is a concerning food safety challenge, and monitoring the trends of AMR is vital for a better risk assessment. This study aimed to characterize the phenotypic profiles and molecular markers of AMR and virulence in the prevalent Campylobacter species contaminating chilled chicken carcasses sampled from supermarkets in the United Arab Emirates (UAE). Campylobacter was detected in 90 (28.6%) out of 315 tested samples, and up to five isolates from each were confirmed using multiplex PCR. The species C. coli was detected in 83% (75/90) of the positive samples. Whole-genome sequencing was used to characterize the determinants of AMR and potential virulence genes in 45 non-redundant C. coli isolates. We identified nine resistance genes, including four associated with resistance to aminoglycoside (aph(3′)-III, ant(6)-Ia, aph(2″)-Ib, and aac(6′)-Im), and three associated with Beta-lactam resistance (blaOXA-61, blaOXA-193, and blaOXA-489), and two linked to tetracycline resistance (tet(O/32/O), and tet(O)), as well as point mutations in gyrA (fluoroquinolones resistance), 23S rRNA (macrolides resistance), and rpsL (streptomycin resistance) genes. A mutation in gyrA 2 p.T86I, conferring resistance to fluoroquinolones, was detected in 93% (42/45) of the isolates and showed a perfect match with the phenotype results. The simultaneous presence of blaOXA-61 and blaOXA-193 genes was identified in 86.6% (39/45) of the isolates. In silico analysis identified 7 to 11 virulence factors per each C. coli isolate. Some of these factors were prevalent in all examined strains and were associated with adherence (cadF, and jlpA), colonization and immune evasion (capsule biosynthesis and transport, lipooligosaccharide), and invasion (ciaB). This study provides the first published evidence from the UAE characterizing Campylobacter virulence, antimicrobial resistance genotype, and phenotype analysis from retail chicken. The prevalent C. coli in the UAE retail chicken carries multiple virulence genes and antimicrobial resistance markers and exhibits frequent phenotype resistance to macrolides, quinolones, and tetracyclines. The present investigation adds to the current knowledge on molecular epidemiology and AMR development in non-jejuni Campylobacter species in the Middle East and globally.

Evaluation of a Molecular Mosprie Assay for Detection of Helicobacter pylori and Resistance to Clarithromycin and Levofloxacin

Helicobacter pylori eradication regimens should be guided by antimicrobial susceptibility testing. The objective of this study was to evaluate the molecular-based Mosprie assay for detecting H. pylori resistance to clarithromycin and levofloxacin using gastric biopsies. A total of 185 culture-positive frozen gastric biopsies were included for Mosprie assay and also for 23S rRNA and gyrA gene sequencing. The susceptibility results by the Mosprie assay were compared with the E-test results retrospectively retrieved. The discordant results were analyzed by sequencing of the 23S rRNA and gyrA genes. Susceptibility concordance between the Mosprie assay and E-test for clarithromycin and levofloxacin was 97.30% (180/185) and 88.11% (163/185), respectively. The full agreement between clarithromycin genotypes by Mosprie assay and the 23S rRNA sequencing results was observed in the 5 samples with discordant Mosprie assay and E-test results. However, for levofloxacin, of the 16 discordant samples with resistant phenotype but a susceptible genotype by Mosprie assay, 6 were found to have levofloxacin resistance-related gyrA gene mutations. The rapid and reliable Mosprie assay can be recommended for H. pylori susceptibility testing of clarithromycin and levofloxacin on gastric biopsies. Future technical improvements are needed in detecting levofloxacin resistance-associated gene mutations.

Correlating clinical breakpoint concentration of moxifloxacin with gyrA mutations using the GenoType MTBDRsl assay Version 2.0

IntroductionWidespread use of Fluoroquinolones (FQs) has led to the development of its resistance in clinical isolates of Mycobacterium tuberculosis. However, in Mycobacterium tuberculosis, phenotypic resistance to FQs has been shown to be heterogeneous, ranging from low-level resistance to high-level resistance. This stratification in resistance has important implications for the inclusion of moxifloxacin (Mfx) in the treatment regimen. The World Health Organization recommends the use of GenoType MTBDRsl assay as the initial test for detecting resistance conferring mutations (both high and low) to FQs in patients with confirmed MDR-RR TB. The present study was conducted to explore the relationship of MTBDRsl Version 2.0 detected mutations in gyrA gene and genotypic DST of Mfx at WHO defined Clinical Breakpoint (CB). Materials and methodsA total of 200 sputum samples from Confirmed MDR/RR TB patients were included in this study. All of these samples had mutations conferring resistance to FQ confirmed by GenoType MTBDRsl assay. These samples were further subjected to Phenotypic DST against moxifloxacin using the Bactec MGIT-960 system. ResultsAll of the 200 representative FQ resistant isolates had mutations in gyrA gene only with no detectable mutation in gyrB gene. 109 (54.5%) of the isolates had mutations associated with high-level increase in MIC while 91 (45.5%) isolates had mutations associated with low-level increase in MIC. Phenotypic DST of these 200 isolates against Mfx at CB (1.0μg/ml) revealed that of the 109 isolates with mutations associated with high-level increase in MIC and expected to be resistant at CB, only 34 (31.2%) were resistant and the remaining 75 (68.8%) were sensitive. ConclusionMoxifloxacin is an important drug in the regimen for treating Drug-resistant TB and the decision to exclude this drug from the regimen should not be taken merely on the basis of mutational patterns. It should rather be taken after considering the combined results of mutational analysis and phenotypic DST.

Molecular characterization of extended-spectrum cephalosporin and fluoroquinolone resistance genes in Salmonella and Shigella isolated from clinical specimens in Thailand

Antimicrobial resistance of Salmonella and Shigella has become a major clinical and public health problem. The incident of co-resistance to third generation cephalosporins and fluoroquinolone is a serious therapeutic issue in Thailand. The present study aimed to investigate the antimicrobial resistance and molecular character of clinical Shigella and Salmonella isolates. A total of 33 Salmonella and 53 Shigella cefotaxime-resistant isolates were collected from human clinical cases in Thailand during the period from 2011–2018. The antimicrobial susceptibility of Salmonella and Shigella was determined by the disk diffusion method, and extended-spectrum beta-lactamase (ESBL) production was characterized by the double-disk synergy test. Genotype characterization was performed by PCR and DNA sequencing. Thirty-two (97.0%) and fifty-two (98.1%) isolates of cefotaxime-resistant Salmonella and Shigella, respectively, were identified as ESBL producers. Shigella sonnei (4 isolates), Salmonella serovar 4,5,12:i:- (6 isolates), Salmonella serovar Agona (2 isolates) and Salmonella serovar Rissen (2 isolates) showed co-resistance to ciprofloxacin and cefotaxime or ceftriaxone. The combination of blaCTX-M-15 plus other ESBL and/or AmpC β-lactamase genes was the most dominant of the genotype patterns in ESBL-producing isolates. The plasmid harbouring the aac(6′)-Ib-cr gene and mutations of gyrA (S83F, D87Y or D87G) and parC (T57S) genes was found in 2 ESBL-producing Salmonella isolates. Three Shigella sonnei isolates harboured mutations in gyrA (S83L, D87Y or D87G), and only one Shigella sonnei phase I isolate showed mutations in both gyrA (S83L and D87G) and parC (S80I) genes. Among these clinical Shigella sonnei isolates, qnrS determinants were identified. Production of ESBLs is an important mechanism for resistance to extended-spectrum cephalosporins in Salmonella and Shigella. The emergence of a decreased susceptibility to extended-spectrum cephalosporins and fluoroquinolone in ESBL-producing isolates has important clinical and therapeutic implications.

Lower Susceptibility to Fluoroquinolones and a gyrA Gene Mutation in Salmonella Typhi Isolates from Enteric Fever Patients.

Enteric fever remains a major cause of morbidity and mortality in Nepal. The emergence of multi-drug resistant Salmonella is a challenge to the clinician to care for patients with enteric fever. This study assessed the antibiotic susceptibility of Salmonella Typhi isolated from enteric fever and the presence of gyrA? ?gene mutation at ser83 of S. Typhi. Blood samples (n = 834) from suspected enteric fever patients were collected and cultured to identify Salmonella Typhi. Antimicrobial sensitivity test was performed by the modified Kirby Bauer disc diffusion method. The minimum ?inhibitory concentration (MIC) tests for ofloxacin and ciprofloxacin were examined by the agar dilution method. The gyrA gene was amplified by PCR and restriction enzyme digestion was performed to evaluate the ser83 mutation. Among 824 blood samples analyzed, 5.1% (42/824) were culture positive for S. Typhi. First-line antibiotics chloramphenicol and co-trimoxazole showed higher in-vitro efficacy compared to amoxicillin. Macrolides (azithromycin) and third-generation cephalosporins (ceftriaxone, cefixime, and cefotaxime) were highly effective against S. Typhi. Nalidixic acid resistance (NAR) was observed in 95.2% (40/42) isolates, among them, all (40/40) isolates harbored mutant gyrA gene at ser83. However, none of the nalidixic acid-sensitive Salmonella? isolates was positive for gyrA? mutation at ser83. This study showed decreased susceptibility to fluoroquinolones and the presence of gyrA? mutation at ser83 position in majority of S. Typhi isolates which highlights the importance of alternate drugs as empirical therapy for the treatment of enteric fever patients. So, the clinician should focus on prescribing conventional first-line antibiotics for the treatment of typhoid patients after higher cohort and extended follow-up studies.

Antimicrobial resistance and genomic characterization of Salmonella enterica serovar Senftenberg isolates in production animals from the United States.

In the USA, Salmonella enterica subspecies enterica serovar Senftenberg is among the top five serovars isolated from food and the top 11 serovars isolated from clinically ill animals. Human infections are associated with exposure to farm environments or contaminated food. The objective of this study was to characterize S. Senftenberg isolates from production animals by analyzing phenotypic antimicrobial resistance profiles, genomic features and phylogeny. Salmonella Senftenberg isolates (n = 94) from 20 US states were selected from NVSL submissions (2014-2017), tested against 14 antimicrobial drugs, and resistance phenotypes determined. Resistance genotypes were determined using whole genome sequencing analysis with AMRFinder and the NCBI and ResFinder databases with ABRicate. Plasmids were detected using PlasmidFinder. Integrons were detected using IntFinder and manual alignment with reference genes. Multilocus-sequence-typing (MLST) was determined using ABRicate with PubMLST database, and phylogeny was determined using vSNP. Among 94 isolates, 60.6% were resistant to at least one antimicrobial and 39.4% showed multidrug resistance. The most prevalent resistance findings were for streptomycin (44.7%), tetracycline (42.6%), ampicillin (36.2%) and sulfisoxazole (32.9%). The most commonly found antimicrobial resistance genes were aac(6')-Iaa (100%), aph(3″)-Ib and aph(6)-Id (29.8%) for aminoglycosides, followed by bla TEM-1 (26.6%) for penicillins, sul1 (25.5%) and sul2 (23.4%) for sulfonamides and tetA (23.4%) for tetracyclines. Quinolone-resistant isolates presented mutations in gyrA and/or parC genes. Class 1 integrons were found in 37 isolates. Thirty-six plasmid types were identified among 77.7% of the isolates. Phylogenetic analysis identified two distinct lineages of S. Senftenberg that correlated with the MLST results. Isolates were classified into two distinct sequence types (ST): ST14 (97.9%) and ST 185 (2.1%). The diversity of this serotype suggests multiple introductions into animal populations from outside sources. This study provided antimicrobial susceptibility and genomic characteristics of S. Senftenberg clinical isolates from production animals in the USA during 2014 to 2017. This study will serve as a base for future studies focused on the phenotypic and molecular antimicrobial characterization of S. Senftenberg isolates in animals. Monitoring of antimicrobial resistance to detect emergence of multidrug-resistant strains is critical.

Molecular Detection of gyrA Mutation in Clinical Strains of Klebsiella pneumoniae

Multi-drug resistant (MDR) Klebsiella pneumoniae strains cause the majority of community acquired and life-threatening infections. We aimed to detect the gyrA mutations in the clinical strains of nalidixic acid and ciprofloxacin resistant K. pneumoniae isolated from the patients with urinary tract infections. Bacterial strains were isolated from the patients with urinary tract infections admitted to a major hospital in Tehran, Iran (2017-2018). Bacterial identification was done according to standard microbiological tests. Antimicrobial susceptibility testing for quinolones and fluoroquinolone antibiotics was done using both disc diffusion and minimal inhibitory concentrations (MICs) methods. PCR-RFLP was used to detect the probable mutation in the gyrA gene in nalidixic acid and ciprofloxacin resistant strains. Finally sequencing was performed to detect point mutations in isolated K. pneumoniae strains. One hundred K. pneumonia isolates were recovered from the urine samples of the clinical cases. Antibiotic resistance testing showed that among all K. pneumoniae isolates, 26% and 19% of the strains were resistant to nalidixic acid and ciprofloxacin respectively. MIC value was ≥4 μg/ml for ciprofloxacin resistant isolates. The results of RFLP on gyrA PCR amplicons using HinfI restriction enzyme showed point mutation in this gene in 46% of nalidixic acid and ciprofloxacin resistant K. pneumonia. The data obtained from the sequencing confirmed the RFLP results and indicated the presence of point mutations in codons 83 and 87 in the gyrA gene which leads to the substitution of different amino acids in gyrA protein. Our findings indicated a relative increased rate of resistance against quinolones and fluoroquinolone antibiotics that raised a concern about extensive dissemination of clinical strains of nalidixic acid and ciprofloxacin resistant K. pneumonia. Point mutation of gyrA gene was responsible for the resistance in our strains however to gain more insight into the molecular characterization of quinolone-resistant isolates, other possible mechanisms of the resistance should also be investigated.

Comparison of Antimicrobial Susceptibility Profiles of Thermotolerant Campylobacter spp. Isolated from Human and Poultry Samples in Georgia (Caucasus).

Antimicrobial resistance remains a public health concern globally. This study presents antimicrobial resistance by microdilution and genetic diversity by the whole-genome sequencing of Campylobacter spp. from human and poultry samples isolated in Georgia in 2020/2021. The major species in poultry samples was C. coli, while C. jejuni was preferentially isolated from human samples. Resistance against tetracycline was highest (100%) in C. coli from industrial chicken and lowest in C. jejuni from clinical isolates (36%), while resistance against ciprofloxacin varied from 80% in C. jejuni from backyard chicken to 100% in C. jejuni and C. coli from industrial chicken. The point mutations in gyrA (T86I) and tet (O) genes were detected as resistance determinants for (fluoro-)quinolone or tetracycline resistance, respectively. Ertapenem resistance is still enigmatic. All isolates displayed sensitivity towards erythromycin, gentamicin and chloramphenicol. Multi-resistance was more frequently observed in C. coli than in C. jejuni, irrespective of the isolation matrix, and in chicken isolates compared to human isolates, independent of the Campylobacter species. The Georgian strains showed high variability of multi-locus sequence types (ST), including novel STs. This study provides the first antibiotic resistance data from Campylobacter spp. in Georgia and addresses the need for follow-up monitoring programs.

Drug susceptibility profiling and genetic determinants of drug resistance in Mycobacterium simiae isolates obtained from regional tuberculosis reference laboratories of Iran.

BackgroundAmong Non-tuberculous mycobacteria (NTM) which generally cause opportunistic infections, especially in immunocompromised hosts, Mycobacterium simiae (M. simiae) is one of the most important NTM, associated with pulmonary disease. The main concern about M. simiae infections is the extreme resistance of this NTM to antibiotics. There are limited studies about drug susceptibility testing (DST) and the causes of drug resistance in M. simiae. Hence, the current study aimed to identify the M. simiae isolates and to assess the drug resistance of the isolates using phenotypic and molecular methods.Materials and methodsIn this study, 50 clinical pulmonary isolates suspected of NTM were collected from regional tuberculosis reference laboratories in Iran. The isolates were identified as M. simiae by using standard biochemical tests and molecular methods. DST was performed for identified M. simiae isolates and additional 35 M. simiae isolates from the department archive, against eight drugs. The mutations in gyrA, gyrB, and rrl genes in clarithromycin and moxifloxacin resistant isolates were investigated by polymerase chain reaction (PCR) followed by sequencing.ResultsOut of 50 suspected NTM isolates, 25 isolates were detected as M. simiae species based on the biochemical tests, and 18 isolates were verified based on the rpoB gene sequence analysis to achieve a total of 53 isolates when the archive isolates were included. DST results showed that all 53 isolates were resistant to isoniazid, rifampin, and clofazimine. The rate of resistance to ethambutol and linezolid were 34 (64%), and 40 (76%) respectively. The highest susceptibility rate was demonstrated for amikacin 53 (100%) and clarithromycin 45(85%), followed by moxifloxacin 35(66%). Sequence analysis showed mutations in positions 2058 and 2059 of the rrl gene, as well non-synonymous mutation at codons 389, 444, and 571 of the gyrB gene. Sequence analysis showed no mutation in the gyrA gene. drug-resistant isolates with mutations showed higher MICs compared to non-mutant resistant isolates.ConclusionsThis study revealed amikacin, clarithromycin, and moxifloxacin as the most effective antibiotics. However, since M. simiae exhibited a high level of antibiotic resistance in vitro, therefore, species identification and determining the antibiotic susceptibility pattern of the isolates are essential before treatment.

Genomic Epidemiology of ST34 Monophasic Salmonella enterica Serovar Typhimurium from Clinical Patients from 2008 to 2017 in Henan, China

Salmonella enterica serovar 4,[5],12:i:- ( S. 4,[5],12:i:-) is a monophasic variant of Salmonella enterica serovar Typhimurium that has emerged as a global serovar causing public health concern. To date, the epidemiology and genomic characterization of this pathogen in China have not been well described. We investigated the prevalence, antimicrobial resistance (AMR) phenotypes, and population genomics of sequence type 34 (ST34) S. 4,[5],12:i:- among cases of human salmonellosis in Henan Province, China. A total of 100 ST34 S. 4,[5],12:i:- isolates were studied from 2008 to 2017 and found mostly resistant to ampicillin (AMP), streptomycin (STR), sulfonamides (SUL), and tetracycline (TET) (ASSuT). Bayesian phylogenetic analysis demonstrated that isolates identified in China were mostly related to the European lineage and evolved into two major clades with different resistance genes and plasmid profiles. Notably, clade 1 showed a significantly higher rate of mutations in gyrA and plasmid-mediated quinolone resistance genes. The carrying of the resistance-containing region (encoding R-type ASSuT), including bla TEM-1B (conferring resistance to AMP), strAB (STR), sul2 (SUL), and tet (B) (TET) inserted into the fljBA operon, was responsible for most of the monophasic variants in clade 2. IncHI2 plasmids were the dominant multi-drug resistance mobile genetic elements accounting for the transmission of acquired resistance genes in this serovar, and these were more prevalent in clade 1. Our findings highlighted the increasing prevalence of multi-drug resistant S. 4,[5],12:i:- in China, along with the differential characteristics of resistance gene acquisition among various lineages. Based on our data, control measures are required to address the spread of this zoonotic pathogen. Further owing to its potential origin in food-producing animals, a “One Health” approach, should be implemented to support surveillance whilst informing interventional strategies.