Mutations In Quinolone Resistance-determining Regions Research Articles

Defining Fluoroquinolone Resistance-Mediating Mutations from Non-Resistance Polymorphisms in Mycoplasma hominis Topoisomerases.

Often dismissed as a commensal, Mycoplasma hominis is an increasingly prominent target of research due to its role in septic arthritis and organ transplant failure in immunosuppressed patients, particularly lung transplantation. As a mollicute, its highly reductive genome and structure render it refractile to most forms of treatment and growing levels of resistance to the few sources of treatment left, such as fluoroquinolones. We examined antimicrobial susceptibility (AST) to fluoroquinolones on 72 isolates and observed resistance in three (4.1%), with corresponding mutations in the quinolone resistance-determining region (QRDR) of S83L or E87G in gyrA and S81I or E85V in parC. However, there were high levels of polymorphism identified between all isolates outside of the QRDR, indicating caution for a genomics-led approach for resistance screening, particularly as we observed a further two quinolone-susceptible isolates solely containing gyrA mutation S83L. However, both isolates spontaneously developed a second spontaneous E85K parC mutation and resistance following prolonged incubation in 4 mg/L levofloxacin for an extra 24–48 h. Continued AST surveillance and investigation is required to understand how gyrA QRDR mutations predispose M. hominis to rapid spontaneous mutation and fluoroquinolone resistance, absent from other susceptible isolates. The unusually high prevalence of polymorphisms in M. hominis also warrants increased genomics’ surveillance.

Isolation and Characterization of Shiga Toxin-Producing Escherichia coli from Retail Beef Samples from Eight Provinces in China.

While Shiga toxin-producing Escherichia coli (STEC) is a major foodborne pathogen worldwide, data on the molecular and phylogenetic properties of STEC isolates from retail beef samples in China remain scant. Fresh retail beef samples (n = 1062) were collected from eight provinces, and STEC isolates were recovered and characterized. PCR data showed that more than 50% of the samples were stx positive, and 82 STEC isolates were recovered from 14.8% (79/535) stx-positive enriched broths. In contrast, all ciprofloxacin resistant isolates (n = 19) and 13 cefotaxime (CTX) resistant isolates were eae positive and belonged to three serotypes: O111:H8, O26:H11, or O157:H7. Point mutations in quinolone resistance-determining regions and plasmid-mediated quinolone resistance determinants were identified in 16 and 20 isolates, respectively. BlaCTX-M and a point mutation (C-42T) in ampC promoter were detected in 15 and 8 of the CTX resistant isolates, respectively. In addition, macrolide resistance gene mphA was identified in eight azithromycin resistant O111:H8 isolates and one O26:H11 isolate. Single nucleotide polymorphism analysis demonstrated that the O26 and O157 isolates had multiple origins, but the O111 isolates were closely related. Taken together, our data demonstrated that several sequence types associated with hemolytic uremic syndrome from the retail beef samples in China had developed into dangerous multidrug resistant pathogens. The resistant phenotype can facilitate their transmission among the farm animals and human beings when there is an antimicrobial selective pressure.

Detection of unprecedented level of antibiotic resistance and identification of antibiotic resistance factors, including QRDR mutations in Escherichia coli isolated from commercial chickens from North India.

This study aimed to investigate the occurrence of antibiotic resistance phenotype and simultaneously understand its genetic basis in Escherichia coli isolated from the cloacal swabs of commercial chickens from north India. Escherichia coli isolates were assessed for susceptibility to 14 different antibiotics using the disc-diffusion technique and were screened for the presence of 22 antibiotic resistance genes (ARGs) by employing PCR. Isolates were found to be highly resistant to fluoroquinolones (nalidixic acid 91%, norfloxacin 73% and ciprofloxacin 66%), tetracycline (71%), beta-lactams (ampicillin 49% and amoxicillin/clavulanic acid 37%), co-trimoxazole (48%), streptomycin (31%) and chloramphenicol (28%); and comparatively less resistant to cefazolin (13%), amikacin (10%), aztreonam (4%), gentamicin (4%) and ceftriaxone (3%). Sixty-three percent of isolates were resistant to more than four different drugs. Abundance of plasmid-borne ARGs like tetA (83%), sul3 (44%), aadA1 (44%), strA (43%), strB (41%), qnrS (38%), sul2 (28%) and aac(6)-Ib-cr (15%) was observed among the isolates. Forty-five percent of isolates possessed more than five different ARGs. Quinolone resistance-determining region (QRDR) mutations within gyrA and parC genes were found to be the major determiners of quinolone resistance. QRDR mutations included leu83, asn87 and gly87 within gyrase-A polypeptide and ile80 and lys84 within topoisomerase IV (encoded by parC). Our findings suggest the abuse of antibiotics as feed additives and prophylactic drugs in Indian poultry sector. It also projects this industry as an active hotspot for the replication and selection of ARGs. Our findings would provide evidence to the authorities for formulating effective strategies for restricting antibiotic usage as non-therapeutic agents in food animals. Occurrence of both plasmid-borne and chromosome-borne resistance towards quinolones can drive movement of resistance phenotype across bacterial species and vertical movement of resistance along the bacterial generations, respectively, which can pose mitigation challenges.

Resistome characterization of Flavobacterium columnare isolated from freshwater cultured Asian sea bass (Lates calcarifer) revealed diversity of quinolone resistance associated genes

Flavobacterium columnare is a Gram-negative bacterium which causes columnaris disease in freshwater fish species worldwide. Antibiotics, especially quinolone, is a primary therapeutic option to mitigate the economic loss caused by this disease. Phenotypic characterization and susceptibility testing were performed to identify quinolone resistance (QR)-associated genes in F. columnare isolated from freshwater cultured Asian sea bass. A total of 15 F. columnare isolates were collected from moribund fish that exhibited columnaris-like clinical signs and lesions including gill necrosis, saddleback lesion, and fin erosion. Phylogenetic analysis based on 16S rRNA and dnaK revealed that these isolates belonged to genetic group (GG) 2 and 4. Disk diffusion test results indicated that the 15 isolates were susceptible to all tested antibiotics except quinolones. Five and six isolates were resistant to oxolinic (OA) (33.33%) and nalidixic acid (NA) (40%) respectively. Broth microdilution indicated that highest OA, NA and enrofloxacin (ENR) minimum inhibitory concentration (MIC) values were 16, 64 and 1 μg/mL respectively. The MIC level to quinolone of GG 2 was higher for the GG 4 counterparts. Sequence analysis revealed a serine:phenylalanine amino acid substitution at position 83 (S83F) and a glutamine:histidine amino acid substitution at position 458 (Q458H) of quinolone resistance-determining regions (QRDRs) of gyrA and gyrB respectively. The Q458H in gyrB was identified as a novel non-synonymous mutation of the Flavobacteriaceae family. A genome-scale resistome analysis revealed that connections between GG and QR level potentially correlated with the presence or absence of chromosomal antimicrobial resistance genes and accumulated mutations in QRDRs. This study provide a guidance for choosing antibiotics for treatment columnaris disease and point out the important quinolone resistance mechanisms in F. columnare.

Profile of gyrA gene mutation in clinical isolate of levofloxacin resistant Escherichia coli.

Escherichia coli is one of the pathogenic bacteria that caused a nosocomial infection. Levofloxacin is one of the fluoroquinolones group antibiotics which is a broad-spectrum antibiotic that works effectively against E.coli. The mutation can happen in the bacteria which caused a resistant effect in the use of antibacterial. This study aimed at identifying mutation in gene gyrA among E.coli that were resistant to levofloxacin. The susceptibility of E . coli was determined by disk diffusion. PCR and sequencing were performed to identify the mutation in gyrA. A total of 10 isolates showed result resistance to levofloxacin and gyrA gene mutation in the amino acid changes. Nucleotide sequence analysis revealed a point mutation in QRDR (quinolone resistance determining region) of gyrA Ser83→Leu, Asp87→Asn. The silent mutation was also found at codon Val85, Arg91, Ser111, Thr123. Mutation in the gyrA gene affect the occurrence of bacterial resistance of E.coli to levofloxacin.

Discovery and Optimization of DNA Gyrase and Topoisomerase IV Inhibitors with Potent Activity against Fluoroquinolone-Resistant Gram-Positive Bacteria.

Herein, we describe the discovery and optimization of a novel series that inhibits bacterial DNA gyrase and topoisomerase IV via binding to, and stabilization of, DNA cleavage complexes. Optimization of this series led to the identification of compound 25, which has potent activity against Gram-positive bacteria, a favorable in vitro safety profile, and excellent in vivo pharmacokinetic properties. Compound 25 was found to be efficacious against fluoroquinolone-sensitive Staphylococcus aureus infection in a mouse thigh model at lower doses than moxifloxacin. An X-ray crystal structure of the ternary complex formed by topoisomerase IV from Klebsiella pneumoniae, compound 25, and cleaved DNA indicates that this compound does not engage in a water-metal ion bridge interaction and forms no direct contacts with residues in the quinolone resistance determining region (QRDR). This suggests a structural basis for the reduced impact of QRDR mutations on antibacterial activity of 25 compared to fluoroquinolones.

Prevalence and Characterization of Quinolone-Resistance Determinants in Escherichia coli Isolated from Food-Producing Animals and Animal-Derived Food in the Philippines.

Antimicrobial resistance to quinolones, which constitutes a threat to public health, has been increasing worldwide. In this study, we investigated the prevalence of quinolone-resistant determinants in Escherichia coli not susceptible to quinolones and isolated from food-producing animals and food derived from them, in the Philippines. A total of 791 E. coli strains were isolated in 56.4% of 601 beef, chicken, pork, egg, and milk samples, as well as environmental, cloacal, and rectal swab-collected samples from supermarkets, open markets, abattoirs, and poultry, swine, and buffalo farms. Using the disc diffusion method, it was determined that 78.6% and 55.4% of the isolates were resistant to at least one antimicrobial and multiple drugs, respectively. In 141 isolates not susceptible to quinolones, 115 (81.6%) harbored quinolone-resistant determinants and had mutations predominantly in the quinolone-resistance determining regions (QRDRs) of gyrA and parC. Plasmid-mediated, quinolone resistance (PMQR) and Qnr family (qnrA1, qnrB4, and qnrS1) genes were detected in all isolates. Forty-eight sequence types were identified in isolates harboring mutations in QRDR and/or PMQR genes by multilocus sequence typing analysis. Moreover, 26 isolates harboring mutations in QRDR and/or PMQR genes belonged mostly to phylogroup B1 and Enteroaggregative E. coli. In conclusion, a high prevalence of E. coli was found in food-producing animals and products derived from them, which could potentially spread high-risk clones harboring quinolone-resistance determinants.

Exposure to Sublethal Ciprofloxacin Induces Resistance to Ciprofloxacin and Cross-Antibiotics, and Reduction of Fitness, Biofilm Formation, and Apx Toxin Secretion in Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is increasingly resistant to antibiotics. However, little is known about the mechanisms of antibiotic resistance in this pathogen. In this study, we experimentally evolved the reference strain of both A. pleuropneumoniae serovar 1 and serovar 7, the most prevalent serovars worldwide, to quinolone resistance by sequential exposure to subinhibitory concentrations of ciprofloxacin. The adaptive ciprofloxacin-resistant mutants of A. pleuropneumoniae serovar 1 and serovar 7 had a minimum inhibitory concentration (MIC) increment from 0.004 to 1 or 2 μg/mL, respectively. Adaptation to ciprofloxacin was shown to confer quinolone resistance with a 32- to 512-fold increase (serovars 1 and 7, respectively) as well as cross-resistance to ampicillin with an increased MIC by 16,384- and 64-fold (serovars 1 and 7, respectively). The genetic analysis of quinolone resistance-determining region mutations showed that substitutions occurred in gyrA (S83A) and parC (D84N) of serovar 1, and gyrA (D87N) of serovar 7. The ciprofloxacin-resistant mutants showed significantly reduced bacterial fitness. The mutants also showed changes in efflux ability and biofilm formation. Notably, the transcription and secretion levels of Apx toxins were dramatically reduced in ciprofloxacin-resistant mutants compared with their wild-type strains. Altogether, these results demonstrated marked phenotypic changes in ciprofloxacin-resistant mutants of A. pleuropneumoniae. The results stress the need for further studies on the impact of both the genotypic and phenotypic characteristics of A. pleuropneumoniae following exposure to subinhibitory concentrations of antibiotics.

Predominance of III/ST19 and Ib/ST10 Lineages With High Multidrug Resistance in Fluoroquinolone-Resistant Group B Streptococci Isolates in Which a New Integrative and Conjugative Element Was Identified.

Fluoroquinolone (FQ)-resistant Group B Streptococcus (GBS) has been reported with considerable cross-resistance, worsening the crisis of multidrug-resistant (MDR) GBS in clinical settings. However, national epidemiological data on FQ-resistant GBS in mainland China have not been well-characterized. This study aimed to determine the prevalence of FQ resistance among GBS from neonatal invasive infections and maternal colonization in northern and southern China, to investigate the serotyping, multilocus sequence typing, and antibiotic cross-resistance, and to characterize the mutations in gyrA and parC genes in quinolone resistance-determining region (QRDR). In order to provide a comprehensive view of the location and structure of resistance genes, whole-genome sequencing on III/ST19 MDR isolates were performed. Among 426 GBS, 138 (32.4%) were FQ resistant, with higher prevalence in northern China than in southern China in both neonates (57.8%, 37/64 vs. 21.7%, 39/180) and pregnant women (50.9%, 29/57 vs. 26.4%, 33/125). Serotypes were distributed as III (48.5%), Ib (39.9%), V (6.5%), and Ia (5.1%). Sequence types were mainly ST19 (53.6%) and ST10 (39.1%), followed by ST12 (1.4%), ST17 (1.4%), ST23 (1.4%), and 0.7% each of ST27, ST188, ST197, and ST597. ST19 isolates were more prevalent in southern China than in northern China in both neonates (64.1%, 25/39 vs. 27.0%, 10/37) and pregnant women (81.8%, 27/33 vs. 41.4%, 12/29), whereas ST10 isolates were more common in northern China than in southern China in both neonates (64.9%, 24/37 vs. 20.5%, 8/39) and pregnant women (58.6%, 17/29 vs. 15.2%, 5/33). Serotype III isolates were mainly ST19 (89.6%, 60/67), while Ib isolates were largely ST10 (94.5%, 52/55). Sequencing data revealed several mutations in QRDR, including Ser81Leu in gyrA (99.2%, 130/131), Ser79Phe or Tyr in parC (76.2%, 48/63), and a previously unreported Ile218Thr and Ile219Phe double mutation pattern (49.2%, 31/63) in parC. ST10 isolates were associated with Ser79Phe (84%, 21/25), while ST19 isolates were limited to Ser79Tyr (95.7%, 22/23). A new integrative and conjugative element (ICE) harboring tetM and gyrA genes was identified in a III/ST19 isolate. This study investigates the molecular characteristics of FQ-resistant GBS in northern and southern China, emphasizing the need for continuous surveillance geographically and further research to characterize the mechanisms of ICE transfer.

Genomic Analysis of CTX-M-Group-1-Producing Extraintestinal Pathogenic E. coli (ExPEc) from Patients with Urinary Tract Infections (UTI) from Colombia.

Background: The dissemination of the uropathogenic O25b-ST131 Escherichia coli clone constitutes a threat to public health. We aimed to determine the circulation of E. coli strains belonging to O25b:H4-B2-ST131 and the H30-Rx epidemic subclone causing hospital and community-acquired urinary tract infections (UTI) in Colombia. Methods: Twenty-six nonduplicate, CTX-M group-1-producing isolates causing UTI in the hospital and community were selected for this study. Results: Twenty-two E. coli isolates harboring CTX-M-15, one CTX-M-3, and three CTX-M-55 were identified. Multilocus Sequence Typing (MLST) showed a variety of sequence types (STs), among which, ST131, ST405, and ST648 were reported as epidemic clones. All the E. coli ST131 sequences carried CTX-M-15, from which 80% belonged to the O25b:H4-B2 and H30-Rx pandemic subclones and were associated with virulence factors iss, iha, and sat. E. coli isolates (23/26) were resistant to ciprofloxacin and associated with amino acid substitutions in quinolone resistance-determining regions (QRDR). We detected two carbapenem-resistant E. coli isolates, one coproducing CTX-M-15, KPC-2, and NDM-1 while the other presented mutations in ompC. Additionally, one isolate harbored the gene mcr-1. Conclusions: Our study revealed the circulation of the E. coli ST131, O25b:H4-B2-H30-Rx subclone, harboring CTX-M-15, QRDR mutations, and other resistant genes. The association of the H30-Rx subclone with sepsis and rapid dissemination warrants attention from the public health and infections control.

Changing trends of antimicrobial susceptibility and resistance mechanisms to quinolones in typhoidal salmonellae isolated from India in last 5 years

Background: Antimicrobial resistance in enteric fever is a major therapeutic challenge and there is a need to monitor the pattern of resistance to the antityphoidal agents and detection of molecular mechanisms of resistance.The study was designed to monitor the antimicrobial susceptibility of Salmonella enterica serovars Typhi and Paratyphi A isolated from patients of enteric fever during 2014 to 2019 and detection of antimicrobial resistance mechanisms to quinolones. Methods and materials: Total 281 strains isolated from January 2014 to September 2019 were selected for the study. Antimicrobial susceptibility was done as per CLSI guidelines (2019) for amoxicillin, chloramphenicol, co-trimoxazole, ciprofloxacin, ceftriaxone and azithromycin. Minimum inhibitory concentration (MIC) determination for ciprofloxacin, ceftriaxone and azithromycin were done by E-Test method according to manufacturer's (ABs Biodisk, Sweden) instructions. DNA sequencing was done for gyrA, gyrB, parC and parE genes to find mutations in quinolone resistance determining region (QRDR). Results: Out of total 281 strains included in the study 214 (76.2%) were S. Typhi and 67 (23.8%) were S. Paratyphi A. As per CLSI 2019, antimicrobial susceptibility for chloramphenicol, ampicillin and cotrimoxazole was 0.7% (2/281), 1.42% (4/281) and 1.06% (3/281) respectively. Only 6/214 (2.8%) were susceptible for S. Typhi and no strain was found to be susceptible in S. Paratyphi A. Azithromycin susceptibility was 100% in S. Typhi, for S. Paratyphi there is no breakpoint available. Ciprofloxacin MIC50 and MIC90 values found to be 0.25 μg/mL and 1 μg/mL in S. Typhi and 0.5 μg/mL and 6 μg/mL in S. Paratyphi A. Ceftriaxone MIC50 and MIC90 values found to be 0.023 μg/mL and 0.5 μg/mL in both S. Typhi and S. Paratyphi A. For azithromycin MIC50 and MIC90 were 2 μg/mL and 6 μg/mL for S. Typhi and 4 μg/mL and 12 μg/mL for S. Paratyphi A respectively. All quinolone resistance strains had mutations in QRDR of gyrA and parC genes. Conclusion: Ciprofloxacin is no longer a drug of choice for treatment of enteric fever in India. Although susceptibility to ceftriaxone is 100% but MIC is creeping towards resistance. Azithromycin can be a promising treatment option for uncomplicated enteric fever.

Whole-Genome Sequence of Fluoroquinolone-Resistant Escherichia coli HUE1, Isolated in Hokkaido, Japan.

We report the complete genome sequence of Escherichia coli strain HUE1, isolated from the urinary catheter of a female patient, showing fluoroquinolone resistance without quinolone resistance-determining region mutations. To facilitate the exploration of the molecular characteristics of HUE1, the whole genome was sequenced using long- and short-read platforms.

Genotypic antimicrobial resistance characterization of E. coli from dairy calves at high risk of respiratory disease administered enrofloxacin or tulathromycin

The objective of this study was to evaluate the longitudinal effect of enrofloxacin or tulathromycin use in calves at high risk of bovine respiratory disease (BRD) on antimicrobial resistance genes and mutation in quinolone resistance-determining regions (QRDR) in fecal E. coli. Calves at high risk of developing BRD were randomly enrolled in one of three groups receiving: (1) enrofloxacin (ENR; n = 22); (2) tulathromycin (TUL; n = 24); or (3) no treatment (CTL; n = 21). Fecal samples were collected at enrollment and at 7, 28, and 56 days after beginning treatment, cultured for Escherichiacoli (EC) and DNA extracted. Isolates were screened for cephalosporin, quinolone and tetracycline resistance genes using PCR. QRDR screening was conducted using Sanger sequencing. The only resistance genes detected were aac(6′)Ib-cr (n = 13), bla-CTX-M (n = 51), bla-TEM (n = 117), tetA (n = 142) and tetB (n = 101). A significantly higher detection of gyrA mutated at position 248 at time points 7 (OR = 11.5; P value = 0.03) and 28 (OR = 9.0; P value = 0.05) was observed in the ENR group when compared to calves in the control group. Our findings support a better understanding of the potential impacts from the use of enrofloxacin in calves on the selection and persistence of resistance.

Fluoroquinolone resistance of Staphylococcus epidermidis isolated from healthy conjunctiva and analysis of their mutations in quinolone-resistance determining region

BackgroundStaphylococcus epidermidis is the most common pathogen in postoperative endophthalmitis and causes various infectious eye diseases. However, there is very little information on fluoroquinolone antibiotic resistance to S. epidermidis identified in conjunctival microbe and analysis of related genes. Here, the authors investigated the rate of resistance to fluoroquinolones of Staphylococcus epidermidis isolated from normal conjunctival microbes and mutations in the quinolone-resistance determining region (QRDR).Methods377 eye samples from 187 patients who underwent intravitreal injection and cataract surgery were included. Specimens were taken from the bilateral lower conjunctival sacs using a cotton swab and cultured. The cultures were identified using MALDI-TOP MS and gyrA, gyrB, parC, and parE gene mutations of QRDR were confirmed by DNA extraction from resistant strains of S. epidermidis with a micro-dilution method using ciprofloxacin, levofloxacin, and moxifloxacin.ResultsThe culture positive rate was 61.8% (231) for 374 eye samples. Of the 303 total strains cultured, S. epidermidis was the most common with 33.7% (102). Ten types of gene mutations were observed in the resistant S. epidermidis of 21 strains. One-point mutation was observed mainly in gyrA and parC, and a small number of mutations were observed in parE in the form of a double point mutations. When there were multiple point mutations in both gyrA and parC, the highest minimum inhibitory concentration was observed.ConclusionsThe quinolone resistance rate of S. epidermidis increased in comparison with previous studies, and resistant S. epidermidis showed mostly QRDR mutations, which were mainly found in gyrA and parC, and showed strong resistance when mutated in both genes.

Acquired fluoroquinolone resistance genes in corneal isolates of Pseudomonas aeruginosa.

Fluoroquinolones are widely used as an empirical therapy for pseudomonal ocular infections. Based on increasing reports on acquired fluoroquinolone resistance genes in clinical isolates of Pseudomonas aeruginosa, we investigated 33 strains of P. aeruginosa isolated from the cornea of microbial keratitis patients in India and Australia between 1992 and 2018 to understand the prevalence of acquired fluoroquinolone resistance genes in ocular isolates and to assess whether the possession of those genes was associated with fluoroquinolone susceptibility. Fourteen out of 33 strains were resistant to at least one fluoroquinolone. We obtained the whole genome sequence of 33 isolates using Illumina MiSeq platform and investigated the prevalence of two fluoroquinolone resistance genes crpP and qnrVC1. To examine the associated mobile genetic elements of qnrVC1 positive strains, we obtained long read sequences using Oxford Nanopore MinION and performed hybrid assembly to combine long reads with Illumina short sequence reads. We further assessed mutations in quinolone resistance determining regions (QRDRs) and antibiotic susceptibilities to ciprofloxacin, levofloxacin and moxifloxacin to examine the association between resistance genes and phenotype. Twenty strains possessed crpP in genetic islands characterised by possession of integrative conjugative elements. The qnrVC1 gene was carried by four isolates on class I integrons and Tn3 transposons along with aminoglycoside and beta-lactam resistance genes. We did not observe any evidence of plasmids carrying fluoroquinolone resistance genes. Resistance to fluoroquinolones was observed in those strains which possessed crpP, qnrVC1 and that had QRDRs mutations. The presence of crpP on its own was not associated with increased resistance to fluoroquinolones.

In vitro and in vivo persistence of IncN plasmids carrying qnr genes in uropathogenic Escherichia coli isolates

ObjectivesThis study aimed to evaluate the persistence of the plasmid-mediated quinolone resistance (PMQR) among uropathogenic Escherichia coli strains grown under or without exposure to subinhibitory concentrations of ciprofloxacin. Based on that, we evaluated the possible spontaneous loss or maintenance of PMQR and the possible appearance of compensatory mutations in gyrA and parC genes. MethodsThree uropathogenic E. coli strains harbouring chromosomal mutations in the gyrA and/or parC genes coupled with qnrS1 or qnrB2 determinants carried by distinct plasmid sizes and incompatibility N groups (IncN/ST1, IncN/ST5) were evaluated using in vitro and in vivo assays. ResultsPMQRs remained stable in all strains throughout the generations evaluated, independently of exposure to ciprofloxacin in both in vivo and in vitro assays. Analysis of gyrA and parC genes after in vivo and in vitro assays revealed that no changes occurred in quinolone-resistance determining regions (QRDR). ConclusionWe demonstrated that IncN plasmids were persistent over 14 days in E. coli clinical strains independently of exposure to ciprofloxacin, as well as previous mutations in QRDR.

Mycoplasma bovis in Spanish Cattle Herds: Two Groups of Multiresistant Isolates Predominate, with One Remaining Susceptible to Fluoroquinolones.

Mycoplasma bovis is an important bovine pathogen causing pneumonia, mastitis, and arthritis and is responsible for major economic losses worldwide. In the absence of an efficient vaccine, control of M. bovis infections mainly relies on antimicrobial treatments, but resistance is reported in an increasing number of countries. To address the situation in Spain, M. bovis was searched in 436 samples collected from beef and dairy cattle (2016–2019) and 28% were positive. Single-locus typing using polC sequences further revealed that two subtypes ST2 and ST3, circulate in Spain both in beef and dairy cattle, regardless of the regions or the clinical signs. Monitoring of ST2 and ST3 isolates minimum inhibitory concentration (MIC) to a panel of antimicrobials revealed one major difference when using fluoroquinolones (FQL): ST2 is more susceptible than ST3. Accordingly, whole-genome sequencing (WGS) further identified mutations in the gyrA and parC regions, encoding quinolone resistance-determining regions (QRDR) only in ST3 isolates. This situation shows the capacity of ST3 to accumulate mutations in QRDR and might reflect the selective pressure imposed by the extensive use of these antimicrobials. MIC values and detection of mutations by WGS also showed that most Spanish isolates are resistant to macrolides, lincosamides, and tetracyclines. Valnemulin was the only one effective, at least in vitro, against both STs.

Detection of S83V GyrA mutation in quinolone-resistant Shewanella algae using comparative genomics.

Shewanella algae is a zoonotic pathogen that poses a serious health threat to immunocompromised hosts. Treatment of S. algae infections is challenging due to the pathogen's intrinsic resistance to a variety of β-lactam antibiotics. Therapeutic options have become further limited by the emergence of quinolone-resistant strains. Currently, there are few studies concerning the genetic and molecular mechanisms underlying acquired quinolones resistance in S. algae. qnrA was once proposed as the candidate gene related to quinolones resistance in S. algae. However, recent studies demonstrated qnrA are highly conservative and does not confer resistance to quinolones in S. algae. A total of 27 non-duplicated isolates of S. algae strains were examined. MICs of ciprofloxacin were determined using Vitek 2. Whole genome sequencing was performed using MiSeq platform. Comprehensive Antibiotic Resistance Database and ResFinder were used for annotation of quinolones resistance genes. Multiple sequence alignment by EMBOSS Clustal Omega were used to identified mutation in quinolone resistance-determining regions. To investigation of the alteration of protein structure induced by mutation, in silico molecular docking studies was conducted using Accryl Discovery studio visualizer. All S. algae harbored the quinolone-resistance associated genes (qnrA, gyrA, gyrB, parC, and parE) regardless its resistance to ciprofloxacin. Comparison of these genomes identified a nonsynonymous mutation (S83V) in chromosome-encoded gyrase subunits (GyrA) in quinolone-resistant strain. We found this mutation disrupts the water-metal ion bridge, reduces the affinity of the quinolone-enzyme complex for the metal ions and therefore decrease the capability of quinolones to stabilize cleavage complexes. The study provides insight into the quinolone resistance mechanisms in S. algae, which would be helpful for the evolution of antibiotic resistance in this bacterium.

Novel Quinolone Nonsusceptible Streptococcus canis Strains with Point Mutations in Quinolone Resistance-Determining Regions and Their Related Factors.

This study investigated quinolone nonsusceptible Streptococcus canis with point mutations in quinolone resistance-determining regions (QRDRs). After selecting targets from 185 isolates, we tested antimicrobial susceptibility using levofloxacin, ciprofloxacin, norfloxacin, and moxifloxacin. We also determined the amino acid sequences of QRDRs in gyrA/gyrB/parC/parE genes and their point mutations. Finally, we performed S. canis-derived M-like protein (SCM) allele typing, multilocus sequence typing, and antimicrobial resistance genotyping. Correlations between nonsusceptible strains and their related factors were examined. We found 13 (7.0%) nonsusceptible isolates consisting of two classes, high-level minimum inhibitory concentrations (MICs) (n = 7, 3.8%) and low-level MICs (n = 6, 3.2%). Mutations Ser81Phe/Ser81Tyr/Glu85Lys in gyrA, Ser67Phe/Ser67Tyr/Asp71Tyr in parC, Asp438Asn in parE, and Gly408Asp in gyrB were observed in these nonsusceptible strains. Common mutations included Ser81 and Ser67/Asp71; additionally, we found one strain each with Glu85, Asp438, and Gly408 mutations. There was a significant correlation between nonsusceptible isolates and the presence of SCM allele type 2, sequence type 46, tetracyclineresistance genes, and macrolide/lincosamide-resistance genes. These results could be used in future, by veterinarians while treating companion animals with clinical symptoms of streptococcal infections.

Genetic Diversity of Multidrug-Resistant CMY-Producing Escherichia coli from Feces and Soil in a Small-Scale Pig Farm.

Diarrheagenic Escherichia coli cause diarrheal diseases, which are a public health concern and affect mainly developing countries. Multidrug-resistant (MDR) pathogens have been spreading in different sources, including animals and the environment. E. coli strains were obtained from a small-scale pig farm and 33 antimicrobials were tested. All strains were classified as MDR and harbored several antimicrobial resistance genes (ARGs) [blaCMY, blaOXA-1-like, blaSHV, tet(A), tet(B), aadA, aac(6')-Ib, aph(3')-Ia, sul1, sul2, sul3, floR, and cmlA] and plasmids. Besides, mutations in quinolone resistance-determining region of GyrA (Ser83Leu and Asp87Asn) and ParC (Glu84Asp) were detected. Among the MDR E. coli, nine strains (52%) presented diarrheagenic virulence genes, including genes related to Shiga toxin-producing E. coli (STEC), enteroinvasive E. coli (EIEC), and enteroaggregative E. coli (EAEC). The pulsed-field gel electrophoresis results showed a high genetic diversity among the MDR E. coli strains. Multilocus sequence typing (MLST) analyses revealed different sequence types phylogenetically related to each other, including ST10 and ST56. Subtyping of MLST by fimH gene showed different fimH type. This study shows a high genetic diversity among MDR ARG-producing E. coli belonging to STEC, EIEC, and EAEC pathotypes obtained from a small-scale pig farm and contributes to the monitoring of antimicrobial-resistant pathogens worldwide, mainly in environmental samples, which are associated with One Health framework.