GyrA Substitutions Research Articles

Flumequine, a fluoroquinolone in disguise

Fluoroquinolone resistance in E. coli isolates from livestock in Europe remains high despite EMA restrictions on fluoroquinolone use in animals. However, flumequine, a quinolone not classified as a fluoroquinolone by various regulatory bodies, is still used in livestock in the Netherlands, Belgium, Greece and France. We investigated whether flumequine selects for the same resistance mechanisms in E. coli. Resistant and non-resistant E. coli isolates were obtained from caecal fermentation assays and broilers exposed to concentrations of flumequine and enrofloxacin. Flumequine usage leads to an approximately 3-fold increase in resistant E. coli in the caecal fermentation, similar to enrofloxacin. In vitro exposure to both flumequine and enrofloxacin revealed the same amino acid substitutions (S83L, D87G) in GyrA. Additionally, the same resistance-causing substitutions were found in phenotypically resistant E. coli isolates from broilers treated with either enrofloxacin or flumequine. Flumequine induces similar resistance mechanisms as enrofloxacin, warranting equivalent restrictions on its use.

Comparison of Resistance Acquisition and Mechanisms in Erwinia amylovora against Agrochemicals Used for Fire Blight Control.

Agrochemicals containing antibiotics are authorized to manage fire blight that has been occurring in Korea since 2015. The minimum inhibitory concentration (MIC) of each antibiotic against Erwinia amylovora, the causal pathogen of fire blight, has increased over the years due to the pathogen's frequent exposure to antibiotics, indicating the necessity to prepare for the emergence of antibiotic resistance. In this study, E. amylovora was exposed to stepwise increasing concentrations of eight different agrochemicals, each containing single or mixed antibiotics, and gene mutation and changes in MIC were assessed. Streptomycin and oxolinic acid induced an amino acid substitution in RpsL and GyrA, respectively, resulting in a rapid increase in MIC. Oxytetracycline initially induced amino acid substitutions or frameshifts in AcrR, followed by substitutions of 30S small ribosomal protein subunit S10 or AcrB, further increasing MIC. E. amylovora acquired resistance in the order of oxolinic acid, streptomycin, and oxytetracycline at varying exposure frequencies. Resistance acquisition was slower against agrochemicals containing mixed antibiotics than those with single antibiotics. However, gene mutations conferring antibiotic resistance emerged sequentially to both antibiotics in the mixed formulations. Results suggested that frequent application of mixed antibiotics could lead to the emergence of multidrug-resistant E. amylovora isolates. This study provided essential insights into preventing the emergence of antibiotic-resistant E. amylovora and understanding the underlying mechanisms of resistance acquisition.

Genotypic characterization and antimicrobial susceptibility of human Campylobacter jejuni isolates in Southern Spain.

Campylobacter jejuni is the main cause of bacterial gastroenteritis and a public health problem worldwide. Little information is available on the genotypic characteristics of human C. jejuni in Spain. This study is based on an analysis of the resistome, virulome, and phylogenetic relationship, antibiogram prediction, and antimicrobial susceptibility of 114 human isolates of C. jejuni from a tertiary hospital in southern Spain from October 2020 to June 2023. The isolates were sequenced using Illumina technology, and a bioinformatic analysis was subsequently performed. The susceptibility of C. jejuni isolates to ciprofloxacin, tetracycline, and erythromycin was also tested. The resistance rates for each antibiotic were 90.3% for ciprofloxacin, 66.7% for tetracycline, and 0.88% for erythromycin. The fluoroquinolone resistance rate obtained is well above the European average (69.1%). CC-21 (n = 23), ST-572 (n = 13), and ST-6532 (n = 13) were the most prevalent clonal complexes (CCs) and sequence types (STs). In the virulome, the cadF, ciaB, and cdtABC genes were detected in all the isolates. A prevalence of 20.1% was obtained for the genes wlaN and cstIII, which are related to the pathogenesis of Guillain-Barré syndrome (GBS). The prevalence of the main antimicrobial resistance markers detected were CmeABC (92.1%), RE-cmeABC (7.9%), the T86I substitution in gyrA (88.9%), blaOXA-61 (72.6%), tet(O) (65.8%), and ant (6)-Ia (17.1%). High antibiogram prediction rates (>97%) were obtained, except for in the case of the erythromycin-resistant phenotype. This study contributes significantly to the knowledge of C. jejuni genomics for the prevention, treatment, and control of infections caused by this pathogen.IMPORTANCEDespite being the pathogen with the greatest number of gastroenteritis cases worldwide, Campylobacter jejuni remains a poorly studied microorganism. A sustained increase in fluoroquinolone resistance in human isolates is a problem when treating Campylobacter infections. The development of whole genome sequencing (WGS) techniques has allowed us to better understand the genotypic characteristics of this pathogen and relate them to antibiotic resistance phenotypes. These techniques complement the data obtained from the phenotypic analysis of C. jejuni isolates. The zoonotic transmission of C. jejuni through the consumption of contaminated poultry supports approaching the study of this pathogen through "One Health" approach. In addition, due to the limited information on the genomic characteristics of C. jejuni in Spain, this study provides important data and allows us to compare the results with those obtained in other countries.

Interplay between Amino Acid Substitution in GyrA and QnrB19: Elevating Fluoroquinolone Resistance in Salmonella Typhimurium.

Globally, there have been increasing reports of antimicrobial resistance in nontyphoidal Salmonella (NTS), which can develop into severe and potentially life-threatening diarrhea. This study focuses on the synergistic effects of DNA gyrase mutations and plasmid-mediated quinolone resistance (PMQR) genes, specifically qnrB19, on fluoroquinolone (FQ) resistance in Salmonella Typhimurium. By utilizing recombinant mutants, GyrAS83F and GyrAD87N, and QnrB19's, we discovered a significant increase in fluoroquinolones resistance when QnrB19 is present. Specifically, ciprofloxacin and moxifloxacin's inhibitory concentrations rose 10- and 8-fold, respectively. QnrB19 was found to enhance the resistance capacity of mutant DNA gyrases, leading to high-level FQ resistance. Additionally, we observed that the ratio of QnrB19 to DNA gyrase played a critical role in determining whether QnrB19 could protect DNA gyrase against FQ inhibition. Our findings underscore the critical need to understand these resistance mechanisms, as their coexistence enables bacteria to withstand therapeutic FQ levels, posing a significant challenge to treatment efficacy.

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.

In silico gepotidacin target mining among 33 213 global Neisseria gonorrhoeae genomes from 1928 to 2023 combined with gepotidacin MIC testing of 22 gonococcal isolates with different GyrA and ParC substitutions.

The novel dual-target triazaacenaphthylene, gepotidacin, recently showed promising results in its Phase III randomized controlled trial for the treatment of gonorrhoea. We investigated alterations in the gepotidacin GyrA and ParC targets in gonococci by in silico mining of publicly available global genomes (n = 33 213) and determined gepotidacin MICs in isolates with GyrA A92 alterations combined with other GyrA and/or ParC alterations. We examined gonococcal gyrA and parC alleles available at the European Nucleotide Archive. MICs were determined using the agar dilution method (gepotidacin) or Etest (four antimicrobials). Models of DNA gyrase and topoisomerase IV were obtained from AlphaFold and used to model gepotidacin in the binding site. GyrA A92 alterations were identified in 0.24% of genomes: GyrA A92P/S/V + S91F + D95Y/A/N (0.208%), A92P + S91F (0.024%) and A92P (0.003%), but no A92T (previously associated with gepotidacin resistance) was found. ParC D86 alterations were found in 10.6% of genomes: ParC D86N/G (10.5%), D86N + S87I (0.051%), D86N + S88P (0.012%) and D86G + E91G (0.003%). One isolate had GyrA A92P + ParC D86N alterations, but remained susceptible to gepotidacin (MIC = 0.125 mg/L). No GyrA plus ParC alterations resulted in a gepotidacin MIC > 4 mg/L. Modelling of gepotidacin binding to GyrA A92/A92T/A92P suggested that gepotidacin resistance due to GyrA A92T might be linked to the formation of a new polar contact with DNA. In silico mining of 33 213 global gonococcal genomes (isolates from 1928 to 2023) showed that A92 is highly conserved in GyrA, while alterations in D86 of ParC are common. No GyrA plus ParC alterations caused gepotidacin resistance. MIC determination and genomic surveillance of potential antimicrobial resistance determinants are imperative.

Emergence of Plasmid-Mediated Quinolone Resistance (PMQR) Genes in Campylobacter coli in Tunisia and Detection of New Sequence Type ST13450.

The aim of this study is to investigate the occurrence of plasmid mediated quinolone resistance (PMQR) determinants in Campylobacter coli isolates collected from broilers, laying hens and poultry farm environments. One hundred and thirty-nine C. coli isolates were isolated from broilers (n = 41), laying hens (n = 53), eggs (n = 4) and the environment (n = 41) of 23 poultry farms located in northeastern of Tunisia. Antimicrobial susceptibility testing was performed on all isolates according to the recommendation of the European Committee on Antimicrobial Susceptibility Testing guidelines. The detection of PMQR genes: qnrA, qnrB, qnrC, qnrD, qnrS, qepA, and aac(6)-Ib gene was performed using polymerase chain reaction (PCR) and specific primers. aac(6')-Ib amplicons were further analyzed by digestion with BtsCI to identify the aac(6')-Ib-cr variant. Mutations in GyrA and the occurrence of RE-CmeABC efflux pump were determined by mismatch amplification mutation assay (MAMA) PCR and PCR, respectively. In addition, eleven isolates were selected to determine their clonal lineage by MLST. The 139 C. coli isolates were resistant to ciprofloxacin, and 86 (61.8%) were resistant to nalidixic acid. High rates of resistance were also observed toward erythromycin (100%), azithromycin (96.4%), tetracycline (100%), chloramphenicol (98.56%), ampicillin (66.1%), amoxicillin-clavulanic acid (55.39%), and kanamycin (57.55%). However, moderate resistance rates were observed for gentamicin (9.35%) and streptomycin (22.3%). All quinolone-resistant isolates harbored the Thr-86-Ile amino acid substitution in GyrA, and the RE-CmeABC efflux pump was detected in 40.28% of isolates. Interestingly, the qnrB, qnrS, qepA, and aac(6')-Ib-cr were detected in 57.7%, 61.15%, 21.58%, and 10% of isolates, respectively. The eleven isolates studied by MLST belonged to a new sequence type ST13450. This study described for the first time the occurrence of PMQR genes in C. coli isolates in Tunisia and globally.

The Prevalence, Risk Factors, and Antimicrobial Resistance Determinants of Helicobacter pylori Detected in Dyspeptic Patients in North-Central Bangladesh.

Chronic infection of Helicobacter pylori represents a key factor in the etiology of gastrointestinal diseases, with high endemicity in South Asia. The present study aimed to determine the prevalence of H. pylori among dyspeptic patients in north-central Bangladesh (Mymensingh) and analyze risk factors of infection and antimicrobial resistance (AMR) determinants in the pathogen. Endoscopic gastrointestinal biopsy samples were collected from dyspeptic patients for a one-year period from March 2022 and were checked for the presence of H. pylori via the rapid urease test and PCR and further analyzed for the status of virulence factors vacA/cagA and genetic determinants related to AMR via PCR with direct sequencing or RFLP. Among a total of 221 samples collected, 80 (36%) were positive for H. pylori, with the vacA+/cagA+ genotype being detected in almost half of them. H. pylori was most prevalent in the age group of 41-50-year-olds, with it being more common in males and rural residents with a lower economic status and using nonfiltered water, though the rates of these factors were not significantly different from those of the H. pylori-negative group. Relatively higher frequency was noted for the A2147G mutation in 23S rRNA, related to clarithromycin resistance (18%, 7/39). Amino acid substitutions in PBP-1A (T556S) and GyrA (N87K and D91N) and a 200 bp deletion in rdxA were detected in samples from some patients with recurrence after treatment with amoxicillin, levofloxacin, and metronidazole, respectively. The present study describes the epidemiological features of H. pylori infection in the area outside the capital in Bangladesh, revealing the spread of AMR-associated mutations.

Detection of CTX-M-15 ESBL in XDR Haemophilus parainfluenzae from a urethral swab.

Haemophilus parainfluenzae is an opportunistic pathogen causing respiratory tract infection and sexually transmitted diseases. The emergence of multidrug resistance in this species is particularly worrisome, especially since the recent description of CTX-M-15 ESBL-producing isolates in Spain. The aim of this study was to characterize a CTX-M-15-producing H. parainfluenzae clinical isolate, HP01, obtained from a urethral swab. MICs were determined with gradient strips for this isolate. Hydrolysis assays were performed with the β LACTA test. Genomic DNA from HP01 was subjected to Illumina and Oxford Nanopore sequencing to investigate the genetic environment of blaCTX-M-15. Phylogenetic analysis was performed with available H. parainfluenzae genomes from the NCBI database, including CTX-M-15 producers. HP01, an XDR isolate, was resistant to penicillin, third-generation cephalosporins, fluoroquinolones, macrolides, cyclines and co-trimoxazole and susceptible only to carbapenems and rifampicin. HP01 carried blaTEM-1, blaCTX-M-15, tet(M), catS and mef(E)/mel and harboured amino acid substitutions in PBP3, PBP5, GyrA, ParC and FolA implicated in resistance. Genomic analysis revealed that blaCTX-M-15 was carried by a Tn3-like transposon inserted into a novel integrative and conjugative element (ICE), ICEHpaSLS, present on the chromosome and belonging to the ICEHin1056 family described in Haemophilus influenzae. The tet(M)-MEGA element was also detected on the chromosome. No plasmid was found. The phylogenetic analysis showed that four H. parainfluenzae producing CTX-M-15 clustered in the same clade. Here we report the description of an XDR H. parainfluenzae producing blaCTX-M-15 isolated from a urethral swab. The blaCTX-M-15 gene was inserted into an ICE structure similar to those recently described in CTX-M-15 producers in Spain. The emergence of XDR H. parainfluenzae producing blaCTX-M-15 is a matter of great concern. Careful surveillance is required to prevent its spread.

Сравнительный биоинформатический анализ состава генов антимикробной устойчивости в геномах представителей рода Corynebacterium

Currently, multidrug resistance of bacterial infectious agents poses a serious threat to the global public health. The following Corynebacterium strains are of special importance for infections, including hospital-acquired ones: C. amycolatum, C. urealyticum, C. striatum, C. jeikeium, C. aurimucosum, C. genitalium that are resistant to the broad spectrum of antimicrobial drugs. The study was aimed to conduct bioinformatics analysis of the pool of antimicrobial resistance genes in the published genomes of some members of the genus Corynebacterium. The data on the whole genome nucleotide sequences of 22 Corynebacterium isolates readily available from NCBI GenBank were assessed. Bioinformatics analysis of the whole genome sequences conducted in order to search for antimicrobial resistance genes in the specified genomes was performed using the PATRIC online resource. It was found that the genomes provided comprised various combinations of 25 antimicrobial drug resistance genes. Amino acid substitutions in GyrA (positions 87, 88 and 91) were revealed in some Corynebacterium strains, through which quinolone/fluoroquinolone resistance could be realized.

Amino acid 17 in QRDR of Gyrase A plays a key role in fluoroquinolones susceptibility in mycobacteria.

The polymorphism at amino acid 17 of quinolone resistance-determining region of GyrA has been stated with a potential role in fluoroquinolone susceptibility in different mycobacterial species. However, no study has provided dependable evidence so far. Here, we verified that gene-edited Mycobacterium abscessus mutants bearing Ser/Gly at this position were more susceptible to fluoroquinolones than their parent strain and the revertant that supports mycobacteria containing Ser/Gly at this position were more susceptible to fluoroquinolones than those containing Ala. IMPORTANCE Fluoroquinolones (FQs) play a key role in the treatment regimens against tuberculosis and non-tuberculous mycobacterial infections. However, there are significant differences in the sensitivities of different mycobacteria to FQs. In this study, we proved that this is associated with the polymorphism at amino acid 17 of quinolone resistance-determining region of Gyrase A by gene editing. This is the first study using CRISPR-associated recombination for gene editing in Mycobacterium abscessus to underscore the contribution of the amino acid substitutions in GyrA to FQ susceptibilities in mycobacteria.

Characterization of DNA Gyrase Activity and Elucidation of the Impact of Amino Acid Substitution in GyrA on Fluoroquinolone Resistance in Mycobacterium avium.

Mycobacterium avium, a member of the M. avium complex (MAC), is the major pathogen contributing to nontuberculous mycobacteria (NTM) infections worldwide. Fluoroquinolones (FQs) are recommended for the treatment of macrolide-resistant MACs. The association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA of M. avium is not yet clearly understood, as many FQ-resistant clinical M. avium isolates do not have such mutations. This study aimed to elucidate the role of amino acid substitution in the QRDR of M. avium GyrA in the development of FQ resistance. We found four clinical M. avium subsp. hominissuis isolates with Asp-to-Gly change at position 95 (Asp95Gly) and Asp95Tyr mutations in gyrA that were highly resistant to FQs and had 2- to 32-fold-higher MICs than the wild-type (WT) isolates. To clarify the contribution of amino acid substitutions to FQ resistance, we produced recombinant WT GyrA, GyrB, and four GyrA mutant proteins (Ala91Val, Asp95Ala, Asp95Gly, and Asp95Tyr) to elucidate their potential role in FQ resistance, using them to perform FQ-inhibited DNA supercoiling assays. While all the mutant GyrAs contributed to the higher (1.3- to 35.6-fold) FQ 50% inhibitory concentration (IC50) than the WT, Asp95Tyr was the most resistant mutant, with an IC50 15- to 35.6-higher than that of the WT, followed by the Asp95Gly mutant, with an IC50 12.5- to 17.6-fold higher than that of the WT, indicating that these amino acid substitutions significantly reduced the inhibitory activity of FQs. Our results showed that amino acid substitutions in the gyrA of M. avium contribute to FQ resistance. IMPORTANCE The emergence of fluoroquinolone (FQ) resistance has further compounded the control of emerging Mycobacterium avium-associated nontuberculous mycobacteria infections worldwide. For M. avium, the association of FQ resistance and mutations in the quinolone resistance-determining region (QRDR) of gyrA is not yet clearly understood. Here, we report that four clinical M. avium isolates with a mutation in the QRDR of gyrA were highly resistant to FQs. We further clarified the impact of mutations in the QRDR of GyrA proteins by performing in vitro FQ-inhibited DNA supercoiling assays. These results confirmed that, like in Mycobacterium tuberculosis, mutations in the QRDR of gyrA also strongly contribute to FQ resistance in M. avium. Since many FQ-resistant M. avium isolates do have these mutations, the detailed molecular mechanism of FQ resistance in M. avium needs further exploration.

Neisseria gonorrhoeae diagnostic escape from a gyrA-based test for ciprofloxacin susceptibility and the effect on zoliflodacin resistance: a bacterial genetics and experimental evolution study.

The aetiological bacterial agent of gonorrhoea, Neisseria gonorrhoeae, has become resistant to each of the first-line antibiotics used to treat it, including ciprofloxacin. One diagnostic approach to identify ciprofloxacin-susceptible isolates is to determine codon 91 in the gene encoding the A subunit of DNA gyrase, gyrA, where coding for the wild-type serine (gyrA91S) is associated with ciprofloxacin susceptibility and phenylalanine (gyrA91F) with resistance. The aim of this study was to investigate the possibility of diagnostic escape from gyrA susceptibility testing. We used bacterial genetics to introduce pairwise substitutions in GyrA positions 91 (S or F) and 95 (D, G, or N), which is a second site in GyrA associated with ciprofloxacin resistance, into five clinical isolates of N gonorrhoeae. All five isolates encoded GyrA S91F, an additional substitution in GyrA at position 95, substitutions in ParC that are known to cause an increased minimum inhibitory concentration (MIC) to ciprofloxacin, and GyrB 429D, which is associated with susceptibility to zoliflodacin (a spiropyrimidinetrione-class antibiotic in phase 3 trials for treatment of gonorrhoea). We evolved these isolates to assess for the existence of pathways to ciprofloxacin resistance (MIC ≥1 μg/mL) and measured MICs for ciprofloxacin and zoliflodacin. In parallel, we searched metagenomic data for 11 355 N gonorrhoeae clinical isolates with reported ciprofloxacin MICs that were publicly available from the European Nucleotide Archive for strains that would be identified as susceptible by gyrA codon 91-based assays. Three clinical isolates of N gonorrhoeae with substitutions in GyrA position 95 associated with resistance (G or N) maintained intermediate ciprofloxacin MICs (0·125-0·5 μg/mL), which has been associated with treatment failure, despite reversion of GyrA position 91 from phenylalanine to serine. From an in-silico analysis of the 11 355 genomes from N gonorrhoeae clinical isolates, we identified 30 isolates with gyrA codon 91 encoding a serine and a ciprofloxacin resistance-associated mutation at codon 95. The reported MICs for these isolates varied from 0·023 μg/mL to 0·25 μg/mL, including four with intermediate ciprofloxacin MICs (associated with substantially increased risk of treatment failure). Finally, through experimental evolution, one clinical isolate of N gonorrhoeae bearing GyrA 91S acquired ciprofloxacin resistance through mutations in the gene encoding for the B subunit of DNA gyrase (gyrB) that also conferred reduced susceptibility to zoliflodacin (ie, MIC ≥2 μg/mL). Diagnostic escape from gyrA codon 91 diagnostics could occur through either reversion of the gyrA allele or expansion of circulating lineages. N gonorrhoeae genomic surveillance efforts might benefit from including gyrB, given its potential for contributing to ciprofloxacin and zoliflodacin resistance, and diagnostic strategies that reduce the likelihood of escape, such as the incorporation of multiple target sites, should be investigated. Diagnostics that guide antibiotic therapy can have unintended consequences, including novel resistance determinants and antibiotic cross-resistance. US National Institutes of Health National Institute of Allergy and Infectious Diseases, National Institute of General Medical Sciences, and the Smith Family Foundation.

Contribution of amino acid substitutions in ParE to quinolone resistance in Haemophilus haemolyticus revealed through a horizontal transfer assay using Haemophilus influenzae.

In 2019, a high-level quinolone-resistant Haemophilus haemolyticus strain (levofloxacin MIC = 16 mg/L) was isolated from a paediatric patient. In this study, we aimed to determine whether the quinolone resistance of H. haemolyticus could be transferred to Haemophilus influenzae and to identify the mechanism underlying the high-level quinolone resistance of H. haemolyticus. A horizontal gene transfer assay to H. influenzae was performed using genomic DNA or PCR-amplified quinolone-targeting genes from the high-level quinolone-resistant H. haemolyticus 2019-19 strain. The amino acids responsible for conferring quinolone resistance were identified through site-directed mutagenesis. By adding the genomic DNA of H. haemolyticus 2019-19, resistant colonies were obtained on agar plates containing quinolones. Notably, H. influenzae grown on levofloxacin agar showed the same level of resistance as H. haemolyticus. Sequencing analysis showed that gyrA, parC and parE of H. influenzae were replaced by those of H. haemolyticus, suggesting that horizontal transfer occurred between the two strains. When the quinolone-targeting gene fragments were added sequentially, the addition of parE, as well as gyrA and parC, contributed to high-level resistance. In particular, amino acid substitutions at both the 439th and 502nd residues of ParE were associated with high-level resistance. These findings indicate that quinolone resistance can be transferred between species and that amino acid substitutions at the 439th and 502nd residues of ParE, in addition to amino acid substitutions in both GyrA and ParC, contribute to high-level quinolone resistance.

Antimicrobial resistance in Campylobacter fetus: emergence and genomic evolution.

Campylobacter fetus is a pathogen, which is primarily associated with fertility problems in sheep and cattle. In humans, it can cause severe infections that require antimicrobial treatment. However, knowledge on the development of antimicrobial resistance in C. fetus is limited. Moreover, the lack of epidemiological cut-off values (ECOFFs) and clinical breakpoints for C. fetus hinders consistent reporting about wild-type and non-wild-type susceptibility. The aim of this study was to determine the phenotypic susceptibility pattern of C. fetus and to determine the C. fetus resistome [the collection of all antimicrobial resistance genes (ARGs) and their precursors] to describe the genomic basis of antimicrobial resistance in C. fetus isolates over time. Whole-genome sequences of 295 C. fetus isolates, including isolates that were isolated in the period 1939 till the mid 1940s, before the usage of non-synthetic antimicrobials, were analysed for the presence of resistance markers, and phenotypic antimicrobial susceptibility was obtained for a selection of 47 isolates. C. fetus subspecies fetus (Cff) isolates showed multiple phenotypic antimicrobial resistances compared to C. fetus subspecies venerealis (Cfv) isolates that were only intrinsic resistant to nalidixic acid and trimethoprim. Cff isolates showed elevated minimal inhibitory concentrations for cefotaxime and cefquinome that were observed in isolates from 1943 onwards, and Cff isolates contained gyrA substitutions, which conferred resistance to ciprofloxacin. Resistances to aminoglycosides, tetracycline and phenicols were linked to acquired ARGs on mobile genetic elements. A plasmid-derived tet(O) gene in a bovine Cff isolate in 1999 was the first mobile genetic element observed, followed by detection of mobile elements containing tet(O)-aph(3')-III and tet(44)-ant(6)-Ib genes, and a plasmid from a single human isolate in 2003, carrying aph(3')-III-ant(6)-Ib and a chloramphenicol resistance gene (cat). The presence of ARGs in multiple mobile elements distributed among different Cff lineages highlights the risk for spread and further emergence of AMR in C. fetus. Surveillance for these resistances requires the establishment of ECOFFs for C. fetus.

A mismatch amplification mutation assay for specific detection of ciprofloxacin-resistant Neisseria meningitidis

Meningococcal chemoprophylaxis for people in close contact with patients with invasive meningococcal disease (IMD) is necessary for preventing the spread of Neisseria meningitidis. Ciprofloxacin (CIP) is commonly used to treat IMD. However, CIP-resistant N. meningitidis isolates have rapidly evolved worldwide; therefore, rapid and accurate detection of CIP-resistant N. meningitidis is essential. We developed a mismatch amplification mutation assay for identifying gyrA substitutions T91I and D95Y, associated with reduced CIP susceptibility, using two primer sets to detect these variants. Comparison with gyrA sequencing data showed complete congruency. This method enables reliable detection of CIP-resistant N. meningitidis, thus leading to efficient management and control of IMD infections.

Antibiotic Susceptibility and Molecular Typing of Invasive Haemophilus influenzae Isolates, with Emergence of Ciprofloxacin Resistance, 2017–2021, Italy

Haemophilus influenzae invasive disease is a severe infection that needs rapid antibiotic therapy. The aim of the study was to perform and evaluate the serotype distribution, antibiotic susceptibility and molecular characteristics of 392 H. influenzae invasive isolates collected during 2017-2021 in Italy. The majority of isolates were NTHi (305/392, 77.8%), followed by Hib (49/392, 12.5%). Ampicillin resistance was frequently detected (85/392, 21.7%): 12.2% were β-lactamase producers (all blaTEM except one blaROB), 9.4% were β-lactamase-negative ampicillin-resistant (BLNAR), with mutations in the ftsI gene. Six isolates were resistant to ciprofloxacin, with substitutions in GyrA and ParC. An MLST analysis revealed the occurrence of international resistant clones, such as ST103 and ST14, highlighting the importance of molecular surveillance.

Relationships between Virulence Genes and Antibiotic Resistance Phenotypes/Genotypes in Campylobacter spp. Isolated from Layer Hens and Eggs in the North of Tunisia: Statistical and Computational Insights.

Globally, Campylobacter is a significant contributor to gastroenteritis. Efficient pathogens are qualified by their virulence power, resistance to antibiotics and epidemic spread. However, the correlation between antimicrobial resistance (AR) and the pathogenicity power of pathogens is complex and poorly understood. In this study, we aimed to investigate genes encoding virulence and AR mechanisms in 177 Campylobacter isolates collected from layer hens and eggs in Tunisia and to assess associations between AR and virulence characteristics. Virulotyping was determined by searching 13 virulence genes and AR-encoding genes were investigated by PCR and MAMA-PCR. The following genes were detected in C. jejuni and C. coli isolates: tet(O) (100%/100%), blaOXA-61 (18.82%/6.25%), and cmeB (100%/100%). All quinolone-resistant isolates harbored the Thr-86-Ile substitution in GyrA. Both the A2074C and A2075G mutations in 23S rRNA were found in all erythromycin-resistant isolates; however, the erm(B) gene was detected in 48.38% and 64.15% of the C. jejuni and C. coli isolates, respectively. The machine learning algorithm Random Forest was used to determine the association of virulence genes with AR phenotypes. This analysis showed that C. jejuni virulotypes with gene clusters encompassing the racR, ceuE, virB11, and pldA genes were strongly associated with the majority of phenotypic resistance. Our findings showed high rates of AR and virulence genes among poultry Campylobacter, which is a cause of concern to human health. In addition, the correlations of specific virulence genes with AR phenotypes were established by statistical analysis.

Quantitative detection and genetic characterization of thermotolerant Campylobacter spp. in fresh chicken meats at retail in Japan.

Campylobacter jejuni and C. coli are one of the leading causes of gastrointestinal illnesses, and which are considered to be transmitted to humans mainly from chicken meats. Considering the less availability of quantitative contamination data in the retail chicken meats in Japan, 510 fresh chicken meats retailed at five distinct regions in Japan between June 2019 and March 2021 were examined. The quantitative testing resulted that 45.7% of the samples (254/510) were positive at mean ± standard deviation of 1.15 ± 1.03 logCFU/g, whereas 43 samples (8.4%) exceeded 3.0 logCFU/g. Seasonal comparison revealed increased bacterial counts in fall compared with spring and summer. As for the chicken slaughter age, those slaughtered at >75 days old were less contaminated than those at <75 days old. Genome sequencing analyses of 111 representative C. jejuni isolates resulted in the detection of three antimicrobial resistance genes (gyrA substitution T86I, tetO and blaOXA-61) at 25.2, 27.9 and 42.3%, respectively. In silico MLST analysis revealed the predominance of sequence types (ST)-21 clonal complex (CC), followed by ST-45CC and ST-464CC. The single nucleotide polymorphism (SNP)-based phylogenetic tree largely classified the sequenced C. jejuni isolates into two clusters (I and II), where all C. jejuni from highly contaminated samples (STs-21CC, -22CC and -45CC) belonged to cluster I, independent of both season and slaughter age. To our knowledge, this is the first example to study the current status of Campylobacter contamination levels in fresh chicken meats retailed in Japan. Our data would be contributable to future quantitative microbial risk assessment, to establish effective control measures for campylobacteriosis.

Alternative quinolone-resistance pathway caused by simultaneous horizontal gene transfer in Haemophilus influenzae.

Quinolone-resistant bacteria are known to emerge via the accumulation of mutations in a stepwise manner. Recent studies reported the emergence of quinolone low-susceptible Haemophilus influenzae ST422 isolates harbouring two relevant mutations, although ST422 isolates harbouring one mutation were never identified. To investigate if GyrA and ParC from quinolone low-susceptible isolates can be transferred horizontally and simultaneously to susceptible isolates. Genomic DNA was extracted from an H. influenzae isolate harbouring amino acid substitutions in both gyrA and parC and mixed with clinical isolates. The emergence of resistant isolates was compared, and WGS analysis was performed. By adding the genomic DNA harbouring both mutated gyrA and parC, resistant bacteria exhibiting recombination at gyrA only or both gyrA and parC loci were obtained on nalidixic acid and pipemidic acid plates, and the frequency was found to increase with the amount of DNA. Recombination events in gyrA only and in both gyrA and parC occurred with at least 1 and 1-100 ng of DNA, respectively. The genome sequence of a representative strain showed recombination events throughout the genome. The MIC of quinolone for the resulting strains was found to be similar to that of the donor. Although the recombination efficacy was different among the various strains, all strains used in this study obtained multiple genes simultaneously. These findings indicate that H. influenzae can simultaneously obtain more than two mutated genes. This mechanism of horizontal transfer could be an alternative pathway for attaining quinolone resistance.