Mutations In Quinolone Resistance-determining Regions Research Articles

P-1511. In Vitro Activity of Gepotidacin Against Molecularly Characterized Neisseria gonorrhoeae Isolates Exhibiting Reduced Susceptibility to Antibacterial Agents, 2018-2021

Abstract Background Gepotidacin (GEP) is a novel, bactericidal, first-in-class triazaacenaphthylene antibacterial that inhibits bacterial DNA replication by a unique mechanism of action, distinct binding site, and provides a well-balanced inhibition (for most uncomplicated urinary tract infection (uUTI) uropathogens and N. gonorrhoeae)) of two different Type II topoisomerase enzymes. We present the activity of GEP against molecularly characterized N. gonorrhoeae isolates collected from patients in Australia, India, and the United States from 2018-2021 as part of a GEP global gonococcal surveillance study. Methods N. gonorrhoeae isolates were tested for susceptibility to GEP and comparator agents by CLSI agar dilution method and analyzed using CLSI 2024 breakpoints. Isolates that met predefined selection criteria, which included GEP MIC values >1 µg/mL or nonsusceptible or resistant (R) to select comparators, were characterized by whole genome sequencing. Results A total 711 N. gonorrhoeae isolates were collected and of these 341 (48%) were characterized. The GEP MICs for both populations of isolates ranged from ≤0.06 to 2 µg/mL; MIC90 of 1 µg/mL (Table 1). Of 324 ciprofloxacin-R isolates, 105 (32.4%) had a mutation of D86N in ParC; this mutation was associated with a GEP MIC90 of 2 µg/mL (range= 0.25-2 µg/mL). Isolates with this mutation also carried mutations in the quinolone-resistance determining region (QRDR) of GyrA as well as mutation(s) suggesting the upregulation of the MtrCDE efflux pump. When categorizing isolates by QRDR mutation, ParC D86N was the only mutation associated with a GEP MIC90 >1 µg/mL. A myriad of resistance mechanisms relevant to penicillin, azithromycin, and tetracycline, were identified among the characterized isolates, against which GEP MIC90s ranged from 1-2 µg/mL (Table 2). Conclusion GEP demonstrated potent in vitro activity against N. gonorrhoeae isolates, including those that were not susceptible to comparator agents. GEP’s MIC90 value relative to all isolates, and those with other QRDR mutations in this study, was one dilution higher (2 versus 1 µg/mL) against isolates that carried ParC D86N, which is known to be important for GEP binding. Disclosures Mark Estabrook, MS, Pfizer, Inc.: Advisor/Consultant Renuka Kapoor, PhD, GSK: Employee|GSK: Stocks/Bonds (Public Company) Didem Torumkuney, PhD, GSK: Employee|GSK: Stocks/Bonds (Public Company) Henry Li, MS in Biotechnology, Pfizer, Inc.: Advisor/Consultant Daniel F. Sahm, PhD, Pfizer, Inc.: Advisor/Consultant

Detection of Delafloxacin Resistance Mechanisms in Multidrug-Resistant Klebsiella pneumoniae.

Background: In this study, the mechanisms implicated in delafloxacin resistance in Klebsiella pneumoniae strains were investigated. Delafloxacin is a novel, broad-spectrum fluoroquinolone that has been approved for clinical application. Methods: In our study, 43 K. pneumoniae strains were assessed, antimicrobial susceptibility testing was performed via the broth microdilution method, and the minimum inhibitory concentration (MIC) values for ciprofloxacin, delafloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, and imipenem were determined. Four delafloxacin-resistant K. pneumoniae strains were selected for whole-genome sequencing (WGS). Results: The MIC50 values for the 43 K. pneumoniae strains were as follows: ciprofloxacin 0.5 mg/L, levofloxacin 0.25 mg/L, moxifloxacin 0.5 mg/L, and delafloxacin 0.25 mg/L. All four selected delafloxacin-resistant K. pneumoniae strains showed extended-spectrum beta-lactamase production, and one strain exhibited carbapenem resistance. WGS enabled us to determine the sequence types (STs) of these strains, namely, ST307 (two strains), ST377, and ST147. Multiple mutations in quinolone-resistance-determining regions (QRDRs) were detected in all the delafloxacin-resistant K. pneumoniae strains; specifically, gyrA Ser83Ile and parC Ser80Ile were uniformly present in the strains of ST307 and ST147. However, in the ST377 strain, gyrA Ser83Tyr, Asp87Ala, and parC Ser80Ile, amino acid substitutions were detected. We also identified OqxAB and AcrAB efflux pumps in all delafloxacin-resistant K. pneumoniae strains. The association between beta-lactamase production and delafloxacin resistance was determined; specifically, CTX-M-15 production was detected in the ST147, ST307, and ST377 strains. Moreover, NDM-1 was detected in ST147. Conclusions: We conclude that multiple mutations in QRDRs, in combination with OqxAB and AcrAB efflux pumps, achieved delafloxacin resistance in K. pneumoniae. In our study, we report on NDM-1-producing K. pneumoniae ST147 in Hungary.

Genome and antibiotic resistance characteristics of Shigella clinical isolates in Fujian Province, Southeast China, 2005-2019.

Shigellosis is a serious public health issue in many developing countries. The emergence of multidrug-resistant (MDR) Shigella isolates has deepened the treatment difficulty and health burden of shigellosis. China is the largest developing country in the world, but so far, the genome of MDR Shigella isolates has not been well characterized. In this study, 60 clinical isolates of Shigella spp. in Fujian Province, southeast China, from 2005 to 2019 were characterized for drug resistance phenotype, whole-genome sequencing and bioinformatics analysis. The results showed that the MDR rate of Shigella isolates was 100%, among which the resistance rates of cefotaxime, ciprofloxacin and azithromycin were 36.67, 21.67 and 10.00 %, respectively. The positive rate of extended-spectrum beta-lactamase (ESBL)-producing strains was 23.33%. The resistance profiles of Shigella flexneri and Shigella sonnei to some antimicrobials differed. The MDR isolates carried multiple antimicrobial resistance genes, among which blaCTX-M-14 and blaCTX-M-15 mediated ESBL resistance. 'ISEcp1 -blaCTX-M -IS903' (type I) and 'ISEcp1 -blaCTX-M' (type II) were the most common genetic environments around the blaCTX-M genes, and plasmids containing these structures included IncFII, IncI1, IncI2 and IncN. The double gene mutation pattern of gyrA and parC resulted in a significant decrease in the sensitivity of S. flexneri to ciprofloxacin. The overall resistance phenotype and genotype concordance rate was 88.50%, and the sensitivity and specificity of the genotype antimicrobial susceptibility test (AST) were 93.35 and 82.53 %, respectively. However, inconsistency occurred between phenotypic and genotype profiles for a few antibiotics. Phylogenomic investigation with core genome multi-locus sequence typing (cgMLST) and SNPs were used to characterize the endemic transmission of these infections in Fujian and their evolutionary origin within the global context. For S. flexneri, Fujian isolates were all limited to PG3 and could be divided into three phylogenetic clusters. The ciprofloxacin-resistant strains were mainly F2a and FXv and assigned to the three clusters with different quinolone resistance-determining region mutation patterns. For S. sonnei, most Fujian strains belonged to Lineage III with genotype 3.7.6, except three isolates of Lineage I with genotype 1.3. The strains carrying the blaCTX-M genes were dispersed, indicating different origins of gene acquisition. Most of the circulating isolates in Fujian Province were not related to major international outbreak lineages and were only endemic to the country. In conclusion, multi-drug resistance of Shigella isolates in Fujian Province was serious, and genome-based laboratory surveillance will be crucial to the clinical treatment and public health measures for shigellosis.

Analysis of molecular mechanisms of delafloxacin resistance in Escherichia coli

In this study delafloxacin resistance mechanisms in Escherichia coli strains were analyzed. Delafloxacin is a new fluoroquinolone, that is approved for clinical application however, resistance against this agent is scarcely reported. In our study 37 E. coli strains were included and antimicrobial susceptibility testing was performed for ciprofloxacin, delafloxacin, levofloxacin, moxifloxacin, ceftazidime, cefotaxime, imipenem. Six delafloxacin resistant E. coli strains were selected for whole-genome sequencing and all of them exhibited resistance to other fluoroquinonlones and showed an extended-spectrum beta-lactamase phenotype. The six delafloxacin resistant E. coli strains belonged to different sequence types (STs) namely, ST131 (2 strains), ST57 (2 strains), ST162 and ST15840. Each delafloxacin resistant strain possessed multiple mutations in quinolone resistance-determining regions (QRDRs). Notably, three mutations in gyrA Ser83Leu, Asp87Asn and parC Ser80Ile were in strains of ST162, ST57 and ST15840. However, the two strains of ST131 carried five combined mutations namely, gyrA Ser83Leu, Asp87Asn, parC Ser80Ile, Glu84Val, parE Ile549Leu. Association of delafloxacin resistance and production of CTX-M-15 in ST131, CMY-2 in ST162 and ST15840 was detected. In this study a new ST, ST15840 of clonal complex 69 was identified. Our results demonstrate, that at least three mutations in QRDRs are required for delafloxacin resistance in E. coli.

Genetic and in-silico approaches for investigating the mechanisms of ciprofloxacin resistance in Salmonella typhi: Mutations, extrusion, and antimicrobial resistance

Salmonella enterica serovar Typhi spreads typhoid infection in humans through the consumption of contaminated food and water. Poor sanitation plays a pivotal role in its dissemination. Over time, the bacterium has acquired resistance to many promising antibiotics, posing a growing global health concern and hindering the achievement of sustainable development goals. This study aims to elucidate the molecular complexity of fluoroquinolone resistance, a first-line treatment for typhoid infection. To achieve this aim, 80 clinical isolates were collected from various diagnostic laboratories. These isolates were confirmed based on morphological characteristics and biochemical tests. Multidrug-resistant (MDR) and extensively drug-resistant (XDR) isolates were identified using the Kirby-Bauer disc diffusion method. The mechanism of ciprofloxacin resistance was investigated by sequencing the quinolone resistance-determining region (QRDR) genes and identifying the presence of the qnrS1 gene. As a result of this study, 60 % of isolates showed resistance to ciprofloxacin. At the same time, the qnrS1 gene was present in all the selected strains while mutation analysis identified significant mutation in QRDR of DNA gyrase subunit A (gyrA) and Topoisomerase IV (parC) gene. The combinatorial effect was further investigated by downloading 286 draft genomes. The Mutation analysis reveals significant mutations at gyrA S83F, gyrA D87N, gyrA S83Y, gyrB S464F, parC S80I, and parE L416F. Additionally, docking analysis indicates reduced binding affinity and altered solvent accessibility, which show the structural changes at mutation sites. This study provides crucial insights that mutation reduces the binding affinity while qnrS1 acts as a transport channel to extrude the ciprofloxacin. In the future, further validation through experimental mutagenesis is recommended, for targeted therapeutic interventions against the mounting threat of antibiotic-resistant S. Typhi.

Relationship Between Fluoroquinolone Resistance and Mutations in the Quinolone Resistance-Determining Region in Corynebacterium macginleyi.

The purpose of this study was to investigate the bacterial composition in the conjunctiva and to determine the relationship between fluoroquinolone resistance and mutations in the quinolone resistance-determining region (QRDR) in Corynebacterium macginleyi (C. macginleyi). Bacteria isolated from conjunctival swabs of patients awaiting ophthalmic surgery or patients with presumed keratoconjunctivitis were included in this study. For C. macginleyi isolates from 49 samples, the minimum inhibitory concentrations (MICs) of second- to fourth-generation fluoroquinolones were determined by broth microdilution. Additionally, we determined the sequence of the QRDR in the gyrA gene of C. macginleyi-positive isolates by direct sequencing and investigated the relationship between the QRDR mutation and the MICs of fluoroquinolones for C. macginleyi. Among 423 eyes of 296 preoperative patients who underwent conjunctival culture testing, 105 eyes of 89 patients were culture-positive, and among 148 eyes of 147 patients with keratoconjunctivitis, 55 eyes of 54 patients were culture-positive. C. macginleyi accounted for the largest proportion of cultured organisms (34.8%). C. macginleyi-positive isolates were found in 45 eyes of 37 preoperative patients and in 4 eyes of 4 patients with keratoconjunctivitis. Direct sequencing revealed that 91.8% of C. macginleyi-positive isolates had amino acid mutations in the QRDR and 95.5% of mutations were found at Ser-87 and Asp-91. Isolates harboring double mutations at Ser-87 and Asp-91 were resistant to second- to fourth-generation fluoroquinolones. One isolate with double mutations at Ser-87 and Ala-88 but no mutation in Asp-91 showed intermediate susceptibility to moxifloxacin, a fourth-generation fluoroquinolone. C. macginleyi isolated from conjunctiva harboring QRDR amino acid mutations were resistant to second- to fourth-generation fluoroquinolones.

Investigation of gyrA and parC mutations and the prevalence of plasmid-mediated quinolone resistance genes in Klebsiella pneumoniae clinical isolates

BackgroundThe emergence of fluoroquinolone resistance in clinical isolates of Klebsiella pneumoniae is a growing concern. To investigate the mechanisms behind this resistance, we studied a total of 215 K. pneumoniae isolates from hospitals in Bushehr province, Iran, collected between 2017 and 2019. Antimicrobial susceptibility test for fluoroquinolones was determined. The presence of plasmid mediated quinolone resistance (PMQR) and mutations in quinolone resistance-determining region (QRDR) of gyrA and parC genes in ciprofloxacin-resistant K. pneumoniae isolates were identified by PCR and sequencing.ResultsOut of 215 K. pneumoniae isolates, 40 were resistant to ciprofloxacin as determined by E-test method. PCR analysis revealed that among these ciprofloxacin-resistant isolates, 13 (32.5%), 7 (17.5%), 40 (100%), and 25 (62.5%) isolates harbored qnrB, qnrS, oqxA and aac(6’)-Ib-cr genes, respectively. Mutation analysis of gyrA and parC genes showed that 35 (87.5%) and 34 (85%) of the ciprofloxacin-resistant isolates had mutations in these genes, respectively. The most frequent mutations were observed in codon 83 of gyrA and codon 80 of parC gene. Single gyrA substitution, Ser83→ Ile and Asp87→Gly, and double substitutions, Ser83→Phe plus Asp87→Ala, Ser83→Tyr plus Asp87→Ala, Ser83→Ile plus Asp87→Tyr, Ser83→Phe plus Asp87→Asn and Ser83→Ile plus Asp87→Gly were detected. In addition, Ser80→Ile and Glu84→Lys single substitution were found in parC gene.ConclusionsOur results indicated that 90% of isolates have at least one mutation in QRDR of gyrA orparC genes, thus the frequency of mutations was very significant and alarming in our region.

Identification of novel resistance-associated mutations and discrimination within whole-genome sequences of fluoroquinolone-resistant Mycobacterium tuberculosis isolates.

This study aims to elucidate additional mutation loci associated with fluoroquinolone (FQ) resistance and evaluate the discriminatory capacity of mutation loci and allele mutation frequencies in identifying FQ-resistant Mycobacterium tuberculosis (MTB) isolates. A random selection of isolates was extracted from an ongoing collection. Drug resistance was determined using the resazurin microtiter assay (REMA) as the gold standard. Mutation loci and the burden of mutations in the quinolone resistance-determining region (QRDR) were elucidated through whole-genome sequencing (WGS). Novel amino acid mutations, namely, G520D and G520T, were identified in the gyrB and associated with FQ resistance. In the context of distinguishing FQ-resistant isolates, the AUC for the QRDR mutation frequency burden (0.969) surpassed that of the mutation locus (0.929), and this difference was statistically significant (P = 0.03). Furthermore, using the resistance mutation locus as a reference, setting the QRDR mutation frequency burden threshold at 1.31% resulted in a 3.60% increase in the accuracy of classifying FQ-resistant isolates (NRI = 3.60%, P < 0.001). The QRDR mutation frequency burden appears to offer superior diagnostic efficacy in discriminating FQ-resistant isolates compared to qualitative detection of mutant loci.IMPORTANCEFluoroquinolone (FQ) drugs are recommended as second-line drugs for the treatment of multidrug-resistant tuberculosis. With the massive use of FQ drugs in the clinical treatment of tuberculosis (TB), there is an increasing rate of drug resistance to FQ drugs. In this study, we identified and demonstrated novel amino acid mutations associated with FQ resistance in Mycobacterium tuberculosis (MTB), and we quantified the mutation sites and identified the quinolone resistance-determining region (QRDR) mutation frequency burden as a novel diagnostic method for FQ resistance. We hope that the results of this study will provide data support and a theoretical basis for the rapid diagnosis of FQ-resistant MTB.

Genetic characterization and drug resistance analysis of Salmonella Kentucky ST314 in Shenzhen in 2010-2021

To understand the prevalence, genetic characteristics and drug resistance features of Salmonella Kentucky ST314 in Shenzhen. Whole genome sequencing of 14 strains of Salmonella Kentucky ST314 collected from 2010-2021 by the Foodborne Disease Surveillance Network of Shenzhen Center for Disease Control and Prevention for phylogenetic evolutionary analysis, drug resistance gene and plasmid detection; drug susceptibility experiments were performed by micro-broth dilution method. A total of 57 strains of Salmonella Kentucky were collected from the foodborne disease surveillance network, 14 of which were ST314. The Shenzhen isolates were clustered with isolates from Southeast Asian countries such as Vietnam and Thailand on clade 314.2, and the single nucleotide polymorphism distance between local strains in Shenzhen was large, indicating dissemination. In this study, a total of 17 drug resistance genes/mutations in 9 categories were detected in the genome of Salmonella Kentucky ST314, carrying 3 extended spectrum beta-lactamases(ESBLs), including bla_(CTX-M-24)(14.3%, 2/14), bla_(CTX-M-55)(7.1%, 1/14), and bla_(CTX-M-130)(14.3%, 2/14), all located on plasmids. Regarding quinolone resistance factors, two plasmid-mediated quinolone resistance(PMQR) genes were identified in the genome: qnrB6(71.4%, 10/14) and aac(6')Ib-cr(78.6%, 11/14), a quinolone resistance quinolone resistance-determining regions(QRDR) mutation T57 S(100%, 14/14). The multi-drug resistance rate of Salmonella Kentucky ST314 in Shenzhen was 92.86%(13/14)with the highest rate of resistance to tetracycline and cotrimoxazole(100%, 14/14), followed by chloramphenicol(92.86%, 13/14), cefotaxime and ampicillin(78.57%, 11/14), ciprofloxacin and nalidixic acid(71.43%, 10/14), and ampicillin-sulbactam had the lowest resistance rate(21.43%, 3/14). ST314 is the second most prevalent ST type among Salmonella Kentucky in Shenzhen, mainly isolated from food, especially poultry; phylogenetic analysis suggests that ST314 is a disseminated infection and the genome shows a highly genetically conserved phenotype. Drug resistance of Salmonella Kentucky ST314 is very serious, especially QRDR mutation, PMQR gene co-mediated quinolone resistance and plasmid-mediated cephalosporin resistance are prominent and deserve extensive attention.

Evaluating the relationship between ciprofloxacin prescription and non-susceptibility in Salmonella Typhi in Blantyre, Malawi: an observational study

Ciprofloxacin is the first-line drug for treating typhoid fever in many countries in Africa with a high disease burden, but the emergence of non-susceptibility poses a challenge to public health programmes. Through enhanced surveillance as part of vaccine evaluation, we investigated the occurrence and potential determinants of ciprofloxacin non-susceptibility in Blantyre, Malawi. We conducted systematic surveillance of typhoid fever cases and antibiotic prescription in two health centres in Blantyre, Malawi, between Oct 1, 2016, and Oct 31, 2019, as part of the STRATAA and TyVAC studies. In addition, blood cultures were taken from eligible patients presenting at Queen Elizabeth Central Hospital, Blantyre, as part of routine diagnosis. Inclusion criteria were measured or reported fever, or clinical suspicion of sepsis. Microbiologically, we identified Salmonella enterica serotype Typhi (S Typhi) isolates with a ciprofloxacin non-susceptible phenotype from blood cultures, and used whole-genome sequencing to identify drug-resistance mutations and phylogenetic relationships. We constructed generalised linear regression models to investigate associations between the number of ciprofloxacin prescriptions given per month to study participants and the proportion of S Typhi isolates with quinolone resistance-determining region (QRDR) mutations in the following month. From 46 989 blood cultures from Queen Elizabeth Central Hospital, 502 S Typhi isolates were obtained, 30(6%) of which had either decreased ciprofloxacin susceptibility, or ciprofloxacin resistance. From 11 295 blood cultures from STRATAA and TyVAC studies, 241 microbiologically confirmed cases of typhoid fever were identified, and 198isolates from 195 participants sequenced (mean age 12·8 years [SD 10·2], 53% female, 47% male). Between Oct1,2016, and Aug31, 2019, of 177 typhoid fever cases confirmed by whole-genome sequencing, four (2%) were caused by S Typhi with QRDR mutations, compared with six (33%) of 18 cases between Sept 1 and Oct 31, 2019. This increase wasassociated with a preceding spike in ciprofloxacin prescriptions. Every additional prescription of ciprofloxacin given to study participants in the preceding month was associated with a 4·2% increase (95% CI 1·8-7·0) in the relative risk of isolating S Typhi with a QRDR mutation (p=0·0008). Phylogenetic analysis showed that S Typhi isolates with QRDR mutations from September and October, 2019, belonged to two distinct subclades encoding two different QRDR mutations, and were closely related (4-10 single-nucleotide polymorphisms) to susceptible S Typhi endemic to Blantyre. We postulate a causal relationship between increased ciprofloxacin prescriptions and an increase in fluoroquinolone non-susceptibility in S Typhi. Decreasing ciprofloxacin use by improving typhoid diagnostics, and reducing typhoid fever cases through the use of an efficacious vaccine, could help to limit the emergence of resistance. Wellcome Trust, Bill & Melinda Gates Foundation, and National Institute for Health and Care Research (UK).

Antibiotic Combination to Effectively Postpone or Inhibit the In Vitro Induction and Selection of Levofloxacin-Resistant Mutants in Elizabethkingia anophelis.

Fluoroquinolones are potentially active against Elizabethkingia anophelis. Rapidly increased minimum inhibitory concentrations (MICs) and emerging point mutations in the quinolone resistance-determining regions (QRDRs) following exposure to fluoroquinolones have been reported in E. anophelis. We aimed to investigate point mutations in QRDRs through exposure to levofloxacin (1 × MIC) combinations with different concentrations (0.5× and 1 × MIC) of minocycline, rifampin, cefoperazone/sulbactam, or sulfamethoxazole/trimethoprim in comparison with exposure to levofloxacin alone. Of the four E. anophelis isolates that were clinically collected, lower MICs of levofloxacin were disclosed in cycle 2 and 3 of induction and selection in all levofloxacin combination groups other than levofloxacin alone (all p = 0.04). Overall, no mutations were discovered in parC and parE throughout the multicycles inducted by levofloxacin and all its combinations. Regarding the vastly increased MICs, the second point mutations in gyrA and/or gyrB in one isolate (strain no. 1) occurred in cycle 2 following exposure to levofloxacin plus 0.5 × MIC minocycline, but they were delayed appearing in cycle 5 following exposure to levofloxacin plus 1 × MIC minocycline. Similarly, the second point mutation in gyrA and/or gyrB occurred in another isolate (strain no. 3) in cycle 4 following exposure to levofloxacin plus 0.5 × MIC sulfamethoxazole/trimethoprim, but no mutation following exposure to levofloxacin plus 1 × MIC sulfamethoxazole/trimethoprim was disclosed. In conclusion, the rapid selection of E. anophelis mutants with high MICs after levofloxacin exposure could be effectively delayed or postponed by antimicrobial combination with other in vitro active antibiotics.

Characterization of the resistome and predominant genetic lineages of Gram-positive bacteria causing keratitis.

Bacterial keratitis is a vision-threatening infection mainly caused by Gram-positive bacteria (GPB). Antimicrobial therapy is commonly empirical using broad-spectrum agents with efficacy increasingly compromised by the emergence of antimicrobial resistance. We used a combination of phenotypic tests and genome sequencing to identify the predominant lineages of GPB causing keratitis and to characterize their antimicrobial resistance patterns. A total of 161 isolates, including Staphylococcus aureus (n = 86), coagulase-negative staphylococci (CoNS; n = 34), Streptococcus spp. (n = 34), and Enterococcus faecalis (n = 7), were included. The population of S. aureus isolates consisted mainly of clonal complex 5 (CC5) (30.2%). Similarly, the population of Staphylococcus epidermidis was homogenous with most of them belonging to CC2 (78.3%). Conversely, the genetic population of Streptococcus pneumoniae was highly diverse. Resistance to first-line antibiotics was common among staphylococci, especially among CC5 S. aureus. Methicillin-resistant S. aureus was commonly resistant to fluoroquinolones and azithromycin (78.6%) and tobramycin (57%). One-third of the CoNS were resistant to fluoroquinolones and 53% to azithromycin. Macrolide resistance was commonly caused by erm genes in S. aureus, mphC and msrA in CoNS, and mefA and msr(D) in streptococci. Aminoglycoside resistance in staphylococci was mainly associated with genes commonly found in mobile genetic elements and that encode for nucleotidyltransferases like ant(4')-Ib and ant(9)-Ia. Fluroquinolone-resistant staphylococci carried from 1 to 4 quinolone resistance-determining region mutations, mainly in the gyrA and parC genes. We found that GPB causing keratitis are associated with strains commonly resistant to first-line topical therapies, especially staphylococcal isolates that are frequently multidrug-resistant and associated with major hospital-adapted epidemic lineages.

Evaluation and Characterization of Quinolone-Resistant Escherichia coli in Wastewater Treatment Plant Effluents

The increasing global incidence of quinolone antimicrobial resistance poses a considerable public health concern. The aquatic environment, particularly wastewater treatment plants (WWTPs), serves as a major reservoir for antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs), leading to the dissemination of antibiotic resistance. This study aimed to assess the prevalence and factors contributing to quinolone antibiotic resistance in Escherichia coli isolates obtained from effluents of 33 WWTPs. A total of 1082 E. coli isolates were analyzed, 32.6% and 17.1% of which showed resistance to nalidixic acid and ciprofloxacin, respectively. Phenotypic and genotypic analyses of antibiotic resistance demonstrated that quinolone resistance primarily originated from chromosomal mutations in the gyrA, parC, and parE genes, known as quinolone resistance-determining regions (QRDRs). The amino acid substitution at codon 83 in gyrA was closely associated with nalidixic acid resistance, whereas substitutions at codon 87 in gyrA and codon 80 in parC were significantly associated with ciprofloxacin resistance. The plasmid-mediated quinolone resistance (PMQR) genes qnrS and qnrB were identified in 41 isolates (11.5%) and 15 isolates (4.2%), respectively. Thus, we confirmed that the quinolone resistance in E. coli in WWTPs primarily occurs through QRDR mutations rather than through the acquisition of PMQR genes. Phylogenetic analysis revealed that most quinolone-resistant isolates belonged to the B1, A, B2, and D phylogenetic groups. Notably, the B2 group, which is responsible for extraintestinal infections, exhibited the highest rate of quinolone resistance. These findings provide novel insights into the presence and mechanisms of quinolone resistance in E. coli isolates from WWTPs, emphasizing the need for further research and understanding of quinolone resistance in the environment.

Characterization of Transferrable Mechanisms of Quinolone Resistance (TMQR) among Quinolone-resistant Escherichia coli and Klebsiella pneumoniae causing Urinary Tract Infection in Nepalese Children

BackgroundTransferrable mechanisms of quinolone resistance (TMQR) can lead to fluoroquinolone non-susceptibility in addition to chromosomal mechanisms. Some evidence suggests that fluoroquinolone resistance is increasing among the pediatric population. We sought to determine the occurrence of TMQR genes among quinolone-resistant E. coli and K. pneumoniae causing urinary tract infections among Nepalese outpatient children (< 18 years) and identify molecular characteristics of TMQR-harboring isolates.MethodsWe performed antimicrobial susceptibility testing, phenotypic extended-spectrum β-lactamase (ESBL) and modified carbapenem inactivation method tests, and investigated the presence of six TMQR genes (qnrA, qnrB, qnrS, aac(6’)-Ib-cr, oqxAB, qepA), three ESBL genes (blaCTX−M, blaTEM, blaSHV), and five carbapenemase genes (blaNDM, blaOXA−48, blaKPC, blaIMP, blaVIM). The quinolone resistance-determining region (QRDR) of gyrA and parC were sequenced for 35 TMQR-positive isolates.ResultsA total of 74/147 (50.3%) isolates were TMQR positive by multiplex PCR [aac(6’)-Ib-cr in 48 (32.7%), qnrB in 23 (15.7%), qnrS in 18 (12.3%), qnrA in 1 (0.7%), and oqxAB in 1 (0.7%) isolate]. The median ciprofloxacin minimum inhibitory concentration of TMQR-positive isolates (64 µg/mL) was two-fold higher than those without TMQR (32 µg/mL) (p = 0.004). Ser-83→Leu and Asp-87→Asn in GyrA and Ser-80→Ile in ParC were the most common QRDR mutations (23 of 35). In addition, there was a statistically significant association between TMQR and two β-lactamase genes; blaCTX−M (p = 0.037) and blaTEM (p = 0.000).ConclusionThis study suggests a high prevalence of TMQR among the quinolone-resistant E. coli and K. pneumoniae isolates causing urinary tract infection in children in this area of Nepal and an association with the carriage of ESBL gene. This is a challenge for the management of urinary infections in children. Comprehensive prospective surveillance of antimicrobial resistance in these common pathogens will be necessary to devise strategies to mitigate the emergence of further resistance.

The Drug-Specific Propensity Regarding the Acquisition of Fluoroquinolone Resistance in Escherichia coli: An in vitro Challenge and DNA Mutation Analysis.

Many fluoroquinolones, such as ciprofloxacin, are used clinically. We investigated the relationship between resistance acquisition and exposure duration in each drug through the exposure of fluoroquinolone to Escherichia coli clinical isolates in vitro. Eleven E. coli clinical isolates were exposed to each fluoroquinolone, ie, ciprofloxacin, levofloxacin, sitafloxacin, garenoxacin, and lascufloxacin, with the concentration of the mutant selection window for 5 days; these procedures were repeated 5-times. In addition, the DNA sequence in the quinolone-resistance determining region (QRDR) and the expression level in the drug efflux pump acrA were analyzed to determine the resistance mechanism. Although resistant strains were not detected after 5 to 10 days of exposure to fluoroquinolone, after 25 days of exposure to ciprofloxacin and levofloxacin, 100% and 45% of isolates acquired resistance, respectively. Due to 25 days of exposure to sitafloxacin, garenoxacin, and lascufloxacin, MIC measurement was elevated 2- to 4096-fold for those of the parental strain, and the cross-resistance rate to levofloxacin was 72%, 54%, and 27%, respectively. In strains with high fluoroquinolone resistance, acrA overexpression was observed in addition to QRDR mutation. In our findings, fluoroquinolone resistance was not observed in the E. coli strain after 5- to 10-days of exposure. However, resistance acquisition was detected frequently after 15- to 25-days of exposure. Among fluoroquinolones, lascufloxacn had the least impact on the resistance acquisition in E. coli.

Population structure and antimicrobial resistance patterns of Salmonella Typhi and Paratyphi A amid a phased municipal vaccination campaign in Navi Mumbai, India.

We performed whole-genome sequencing of 174 Salmonella Typhi and 54 Salmonella Paratyphi A isolates collected through prospective surveillance in the context of a phased typhoid conjugate vaccine introduction in Navi Mumbai, India. We investigate the temporal and geographical patterns of emergence and spread of antimicrobial resistance. We evaluated the relationship between the spatial distance between households and genetic clustering of isolates. Most isolates were non-susceptible to fluoroquinolones, with nearly 20% containing ≥3 quinolone resistance-determining region mutations. Two H58 isolates carried an IncX3 plasmid containing blaSHV-12, associated with ceftriaxone resistance, suggesting that the ceftriaxone-resistant isolates from India independently evolved on multiple occasions. Among S. Typhi, we identified two main clades circulating (2.2 and 4.3.1 [H58]); 2.2 isolates were closely related following a single introduction around 2007, whereas H58 isolates had been introduced multiple times to the city. Increasing geographic distance between isolates was strongly associated with genetic clustering (odds ratio [OR] = 0.72 per km; 95% credible interval [CrI]: 0.66-0.79). This effect was seen for distances up to 5 km (OR = 0.65 per km; 95% CrI: 0.59-0.73) but not seen for distances beyond 5 km (OR = 1.02 per km; 95% CrI: 0.83-1.26). There was a non-significant reduction in odds of clustering for pairs of isolates in vaccination communities compared with non-vaccination communities or mixed pairs compared with non-vaccination communities. Our findings indicate that S. Typhi was repeatedly introduced into Navi Mumbai and then spread locally, with strong evidence of spatial genetic clustering. In addition to vaccination, local interventions to improve water and sanitation will be critical to interrupt transmission. IMPORTANCE Enteric fever remains a major public health concern in many low- and middle-income countries, as antimicrobial resistance (AMR) continues to emerge. Geographical patterns of typhoidal Salmonella spread, critical to monitoring AMR and planning interventions, are poorly understood. We performed whole-genome sequencing of S. Typhi and S. Paratyphi A isolates collected in Navi Mumbai, India before and after a typhoid conjugate vaccine introduction. From timed phylogenies, we found two dominant circulating lineages of S. Typhi in Navi Mumbai-lineage 2.2, which expanded following a single introduction a decade prior, and 4.3.1 (H58), which had been introduced repeatedly from other parts of India, frequently containing "triple mutations" conferring high-level ciprofloxacin resistance. Using Bayesian hierarchical statistical models, we found that spatial distance between cases was strongly associated with genetic clustering at a fine scale (<5 km). Together, these findings suggest that antimicrobial-resistant S. Typhi frequently flows between cities and then spreads highly locally, which may inform surveillance and prevention strategies.

Prevalence, antibiotic susceptibility and genomic analysis of Salmonella from retail meats in Shaanxi, China

Salmonella is a major foodborne pathogen that poses a substantial risk to food safety and public health. This study aimed to assess the prevalence, antibiotic susceptibility, and genomic features of Salmonella isolates recovered from 600 retail meat samples (300 pork, 150 chicken and 150 beef) from August 2018 to October 2019 in Shaanxi, China. Overall, 40 (6.67 %) of 600 samples were positive to Salmonella, with the highest prevalence in chicken (21.33 %, 32/150), followed in pork (2.67 %, 8/300), while no Salmonella was detected in beef. A total of 10 serotypes and 11 sequence types (STs) were detected in 40 Salmonella isolates, with the most common being ST198 S. Kentucky (n = 15), ST13 S. Agona (n = 6), and ST17 S. Indiana (n = 5). Resistance was most commonly found to tetracycline (82.50 %), followed by to ampicillin (77.50 %), nalidixic acid (70.00 %), kanamycin (57.50 %), ceftriaxone (55.00 %), cefotaxime (52.50 %), cefoperazone (52.50 %), chloramphenicol (50.00 %), levofloxacin (57.50 %), cefotaxime (52.50 %), kanamycin (52.50 %), chloramphenicol (50.00 %), ciprofloxacin (50.00 %), and levofloxacin (50.00 %). All ST198 S. Kentucky isolates showed multi-drug resistance (MDR; ≥3 antimicrobial categories) pattern. Genomic analysis showed 56 distinct antibiotic resistance genes (ARGs) and 6 target gene mutations of quinolone resistance determining regions (QRDRs) in 40 Salmonella isolates, among which, the most prevalent ARG types were related to aminoglycosides and β-lactams resistance, and the most frequent mutation in QRDRs was GyrA (S83F) (47.5 %). The number of ARGs in Salmonella isolates showed a significant positive correlation with the numbers of insert sequences (ISs) and plasmid replicons. Taken together, our findings indicated retail chickens were seriously contaminated, while pork and beef are rarely contaminated by Salmonella. Antibiotic resistance determinants and genetic relationships of the isolates provide crucial data for food safety and public health safeguarding.

BlaTEM-positive Salmonella enterica serovars Agona and Derby are prevalent among food-producing animals in Chongqing, China.

Salmonella is one of the most important foodborne zoonotic pathogens, causing global morbidity and mortality in both humans and animals. Due to the extensive use of antimicrobials in food-producing animals, the antimicrobial resistance of Salmonella has attracted increasing attention globally. There have been many reports concerning the antimicrobial resistance of Salmonella from food-producing animals, meats and the environment. However, few studies on Salmonella from food-producing animals have been reported in Chongqing municipality, China. The aim of the present study was to determine the prevalence, serovar diversity, sequence types, and antimicrobial resistance of Salmonella isolated from livestock and poultry in Chongqing. Meanwhile, we also want to know the presence of β-lactamase genes, plasmid-mediated quinolone resistance (PMQR) genes and quinolone resistance-determining region (QRDR) mutations of Salmonella isolates. A total of 129 Salmonella strains were recovered from 2,500 fecal samples at 41 farms from pigs, goats, beef cattle, rabbits, chickens, and ducks. Fourteen serovars were identified, with S. Agona and S. Derby being the dominant serovars. The 129 isolates had high resistance to doxycycline (87.6%), ampicillin (80.6%), tetracycline (79.8%), trimethoprim (77.5%), florfenicol (76.7%) chloramphenicol (72.9%), and trimethoprim-sulfamethoxazole (71.3%), but were susceptible to cefepime. A total of 114 (88.4%) isolates showed multidrug resistant phenotypes. The prevalence of β-lactamase genes in Salmonella isolates was 89.9% (116/129), and among these isolates, 107 (82.9%) harbored blaTEM, followed by blaOXA (26, 20.2%), blaCTX-M (8, 6.2%), and blaCMY (3, 2.3%). In addition, qnrB, qnrD, qnrS, oqxA, oqxB, and aac(6')-Ib-cr were detected in 11, 2, 34, 34, 43, and 72 PMQR-producing isolates, respectively. Moreover, QRDR mutations were very common in PMQR-positive Salmonella isolates (97.2%, 70/72) with mutation(s) in parC or combinative mutations in gyrA and parC. More significantly, 32 extended spectrum beta-lactamase (ESBL)-producing isolates were identified, and 62.5% of them were found to harbor one to four PMQR genes. Furthermore, 11 sequence types were identified from the isolates, and most of ESBL-producing isolates were attributed to ST34 (15.6%) and ST40 (62.5%). The coexistence of PMQR genes with β-lactamase genes and the extensive mutations in QRDR present in Salmonella isolates from food-producing animals suggest a potential threat to public health. Reasonable utilization and strict control strategies for antimicrobials in animal husbandry and animal treatment are necessary to reduce the emergence and dissemination of drug-resistant Salmonella isolates.

Comparison of the Diagnostic Performance of MeltPro and Next-Generation Sequencing in Determining Fluoroquinolone Resistance in Multidrug-Resistant Tuberculosis Isolates

This study systematically investigated the performance of MeltPro and next-generation sequencing in the diagnosis of fluoroquinolone (FQ) resistance among multidrug-resistant tuberculosis patients and explored the relationship between nucleotide alteration and the level of phenotypic susceptibility to FQs. From March 2019 to June 2020, a feasibility and validation study with both MeltPro and next-generation sequencing was performed in 126 patients with multidrug-resistant tuberculosis. Using phenotypic drug susceptibility testing as the gold standard, 95.3% (82 of 86) of ofloxacin-resistant isolates were identified correctly by MeltPro. In addition, whole-genome sequencing was able to detect 83 phenotypically ofloxacin-resistant isolates. The isolates with an individual gyrB mutation outside the quinolone resistance-determining region (QRDR) had minimum inhibitory concentrations (MICs) of ≤2 μg/mL. Despite showing low MICs close to the breakpoint for isolates carrying only gyrA_Ala90Val, the combined mutation gyrB_Asp461Asn caused the ofloxacin MIC to be eight higher than that obtained in Mycobacterium tuberculosis (MTB) isolates with the Ala90Val mutation alone (median, 32 μg/mL; P=0.038). Heteroresistance was observed in 12 of 88 isolates harboring mutations in the QRDRs. In conclusion, our data show that MeltPro and the whole-genome sequencing assay correctly can identify FQ resistance caused by mutations in the gyrA QRDR. The combined gyrB_Asp461Asn mutation may significantly decrease invitro FQ susceptibility of MTB isolates with low-level-resistance-associated gyrA mutations.

Quinolone Resistance in Gallibacterium anatis Determined by Mutations in Quinolone Resistance-Determining Region.

Control of the important pathogen, Gallibacterium anatis, which causes salpingitis and peritonitis in poultry, relies on treatment using antimicrobial compounds. Among these, quinolones and fluoroquinolones have been used extensively, leading to a rise in the prevalence of resistant strains. The molecular mechanisms leading to quinolone resistance, however, have not previously been described for G. anatis, which is the aim of this study. The present study combines phenotypic antimicrobial resistance data with genomic sequence data from a collection of G. anatis strains isolated from avian hosts between 1979 and 2020. Minimum inhibitory concentrations were determined for nalidixic acid, as well as for enrofloxacin for each included strain. In silico analyses included genome-wide queries for genes known to convey resistance towards quinolones, identification of variable positions in the primary structure of quinolone protein targets and structural prediction models. No resistance genes known to confer resistance to quinolones were identified. Yet, a total of nine positions in the quinolone target protein subunits (GyrA, GyrB, ParC and ParE) displayed substantial variation and were further analyzed. By combining variation patterns with observed resistance patterns, positions 83 and 87 in GyrA, as well as position 88 in ParC, appeared to be linked to increased resistance towards both quinolones included. As no notable differences in tertiary structure were observed between subunits of resistant and sensitive strains, the mechanism behind the observed resistance is likely due to subtle shifts in amino acid side chain properties.