gyrA Gene Research Articles

Evaluation of genetic correlation with fluoroquinolones resistance in rifampicin-resistant Mycobacterium tuberculosis isolates

ObjectiveTo detect levofloxacin (LFX) and moxifloxacin (MFX) resistance among rifampicin-resistant tuberculosis (RR-TB) isolates, and predict the resistance level based on specific mutations in gyrA and gyrB genes. MethodsA total of 686 RR-TB isolates were collected from Chinese Drug Resistance Surveillance Program from 2013 to 2020. The minimum inhibitory concentrations (MICs) of 12 anti-TB drugs were acquired using the broth microdilution method, followed by whole genome sequencing (WGS) analysis. ResultsAmong the 686 RR isolates, the most prevalent resistance was to isoniazid (80.5 %) and ethambutol (28.4 %), followed by LFX (26.1 %) and MFX (21.9 %). The resistance rate of LFX (26.1%–99.4 %) was higher than that of MFX (21.9%–83.3 %) across various drug resistance patterns. Of the 180 fluoroquinolones (FQs) resistant isolates, 168 (93.3 %) had mutations in quinolone-resistant determining regions (QRDRs) with 21 mutation types, and Asp94Gly (32.7 %, 55/168) was the predominant mutation. Isolates with mutations in Asp94Asn and Asp94Gly were associated with high levels of resistance to LFX and MFX. Using broth microdilution method as gold standard, the sensitivities of WGS for LFX and MFX were 93.3 % and 98.0 %, and the specificities were 98.6 % and 95.0 %, respectively. ConclusionThe resistance rate of LFX was higher than that of MFX among various drug resistance patterns in RR-TB isolates. The gyrA Asp94Gly was the predominant mutation type underlying FQs resistance. However, no significant difference was observed between mutation patterns in gyrA gene and resistance level of FQs.

Characterization of Bacillus velezensis DCJ 2 isolated from the rhizosphere soil of aglaonema ‘Maria’ and investigation of its plant growth-promoting effects

Aglaonema ‘Maria’ is one of the key foliage plants that has gained high demand in foreign destinations in the last few years. This study was conducted to characterize Bacillus velezensis DCJ 2, assess its plant growth promotion effect and investigate the effectiveness of different delivery methods of the bacterial inoculum for the growth promotion of Aglaonema ‘Maria’. In this context, DCJ 2, isolated from the rhizosphere soil of A. Maria was screened to investigate its potential as a plant growth-promoting bacterium. The pure culture of DCJ 2 maintained on nutrient agar was utilized for morphological, biochemical and physiological examinations. The molecular characterization of the strain was performed with different primer pairs targeting 16SrRNA, gyrA, pgsB and sugar kinase genes of DCJ 2. A pot experiment was carried out to investigate the plant growth promotion effect. It was conducted in the greenhouse as per the complete randomized block design with eight treatments and five replicates. . Healthy plants of A. ‘Maria’ established in field soil and solarized soil were treated with 100 ml of the suspension of the DCJ 2 at the rate of 10 8 CFU/ml either as a soil drench or foliar spray. The partial 16SrRNA, gyrA, pgsB and sugar kinase gene sequences of DCJ 2, BLAST searched in the NCBI database revealed that it has a high similarity to Bacillus velezensis. The pot experiment showed that the application of strain DCJ 2 as a soil drench to solarized soil (Treatment 6) yielded a significant increase in growth parameters at p=0.05. Based on molecular analysis, morphological, biochemical, and physiological reactions, DCJ 2 was identified as Bacillus velezensis. This study revealed that B. velezensis DCJ 2 has the potential to boost the growth of A. Maria and hence serve as a base for developing a biofertilizer.

Epidemiological, Virulence, and Antibiotic Resistance Analysis of Klebsiella pneumoniae, a Major Source of Threat to Livestock and Poultry in Some Regions of Xinjiang, China.

Klebsiella pneumoniae (K. pneumoniae) is recognized as a zoonotic pathogen with an increasing threat to livestock and poultry. However, research on K. pneumoniae of animal origin remains limited. To address the gap, a comprehensive investigation was carried out by collecting a total of 311 samples from the farms of four animal species (dairy cow, chicken, sheep, and pig) in selected areas of Xinjiang, China. Isolates were identified by khe gene amplification and 16S rRNA gene sequencing. Genotyping of K. pneumonia isolates was performed using wzi typing and multilocus sequence typing (MLST). PCR was employed to identify virulence and resistance genes. An antibiotic susceptibility test was conducted using the Kirby-Bauer method. The findings revealed an isolation of 62 K. pneumoniae strains, with an average isolation rate of 19.94%, with the highest proportion originating from cattle sources (33.33%). Over 85.00% of these isolates harbored six virulence genes (wabG, uge, fimH, markD, entB, and ureA); while more than 75.00% of isolates possessed four resistance genes (blaTEM, blaSHV, oqxA, and gyrA). All isolates exhibited complete resistance to ampicillin and demonstrated substantial resistance to sulfisoxazole, amoxicillin/clavulanic acid, and enrofloxacin, with an antibiotic resistance rate of more than 50%. Furthermore, 48.39% (30/62) of isolates were classified as multidrug-resistant (MDR) strains, with a significantly higher isolation rate observed in the swine farms (66.67%) compared to other farms. Genetic characterization revealed the classification of the 62 isolates into 30 distinct wzi allele types or 35 different sequence types (STs). Notably, we identified K. pneumoniae strains of dairy and swine origin belonging to the same ST42 and wzi33-KL64 types, as well as strains of dairy and chicken origin belonging to the same wzi31-KL31-K31 type. These findings emphasize the widespread occurrence of drug-resistant K. pneumoniae across diverse animal sources in Xinjiang, underscoring the high prevalence of multidrug resistance. Additionally, our results suggest the potential for animal-to-animal transmission of K. pneumoniae and there was a correlation between virulence genes and antibiotic resistance genes. Moreover, the current study provides valuable data on the prevalence, antibiotic resistance, and genetic diversity of K. pneumoniae originating from diverse animal sources in Xinjiang, China.

Phenotypic and genomic characterization of a multidrug-resistant Salmonella enterica serovar Kentucky ST198 isolated from a patient in China

ObjectivesThe objective of this study was to investigate the resistance mechanisms of a multidrug-resistant Salmonella Kentucky ST198 FJ-2064 isolated from a patient in China. MethodsThe antimicrobial susceptibility of FJ-2064 was determined by the standard disc dilution and broth microdilution methods. The complete genome of FJ-2064 was sequenced using PacBio and Illumina MiSeq platforms. Polymerase chain reaction (PCR) and S1-PFGE were utilized to confirm the mutation sites and the genomic plasmids, respectively. ResultsIsolate FJ-2064 belongs to sequence type ST198 and harboured no visible large plasmids, but was concurrent resistant to 22 detected antimicrobial agents including cefotaxime, ciprofloxacin, and azithromycin. The complete genome sequence identified 20 acquired antibiotic resistance genes (ARGs) and five chromosomal mutations in the gyrA and parC genes of the quinolone resistance determining regions (QRDRs) in FJ-2064. In addition, PCR sequencing confirmed that most of the ARGs were clustered on one multidrug-resistant region and a variant of SGI1-K. In particular, the bla-TEM-1 and bla-CTX−M-55, qnrS1, mph(A) genes, which confer resistance to cephalosporins, quinolones, and macrolides respectively, were all located on the multidrug-resistant region. ConclusionsWe have demonstrated one multidrug-resistant region and a variant of SGI1-K in a Salmonella Kentucky ST198 that is co-resistant to cefotaxime, ciprofloxacin, and azithromycin.

Drug-resistant Mycobacterium tuberculosis among Nepalese patients at a tuberculosis referral center.

Multidrug-resistant tuberculosis (MDR-TB), characterized by isoniazid and rifampicin resistance, is caused by chromosomal mutations that restrict treatment options and complicate tuberculosis management. This study sought to investigate the prevalence of pre-extensively drug-resistant (pre-XDR) and extensively drug-resistant (XDR) tuberculosis, as well as mutation pattern, in Nepalese patients with MDR/rifampicin-resistant (RR)-TB strains. A cross-sectional study was conducted on MDR/RR-TB patients at the German Nepal Tuberculosis Project from June 2017 to June 2018. The MTBDRsl line probe assay identified pre-XDR-TB and XDR-TB. Pre-XDR-TB included MDR/RR-TB with resistance to any fluoroquinolone (FLQ), while XDR-TB included MDR/RR-TB with resistance to any FLQ and at least one additional group A drug. Mutation status was determined by comparing bands on reaction zones [gyrA and gyrB for FLQ resistance, rrs for SILD resistance, and eis for low-level kanamycin resistance, according to the GenoType MTBDRsl VER 2.0, Hain Lifescience GmbH, Nehren, Germany definition of pre-XDR and XDR] to the evaluation sheet. SPSS version 17.0 was used for data analysis. Out of a total of 171 patients with MDR/RR-TB, 160 had (93.57%) had MTBC, of whom 57 (35.63%) had pre-XDR-TB and 10 (6.25%) had XDR-TB. Among the pre-XDR-TB strains, 56 (98.25%) were FLQ resistant, while 1 (1.75%) was SLID resistant. The most frequent mutations were found at codons MUT3C (57.14%, 32/56) and MUT1 (23.21%, 13/56) of the gyrA gene. One patient had SLID resistant genotype at the MUT1 codon of the rrs gene (100%, 1/1). XDR-TB mutation bands were mostly detected on MUT1 (30%, 3/10) of the gyrA and rrs, MUT3C (30%, 3/10) of the gyrA, and MUT1 (30%, 3/10) of the rrs. Pre-XDR-TB had a significantly higher likelihood than XDR-TB, with different specific mutation bands present in gyrA and rrs genes.

Uncovering the growing burden of enteric fever: A molecular analysis of Salmonella Typhi antimicrobial resistance

Enteric fever, a persistent public health challenge in developing regions, is exacerbated by suboptimal socioeconomic conditions, contaminated water and food sources, and insufficient sanitation. This study delves into the antimicrobial susceptibility of Salmonella Typhi, uncovering the genetic underpinnings of its resistance. Analyzing 897 suspected cases, we identified a significant prevalence of typhoid fever, predominantly in males (58.3 %) and younger demographics. Alarmingly, our data reveals an escalation in resistance to both primary and secondary antibiotics, with cases of multi-drug resistant (MDR) and extensively drug-resistant (XDR) S. Typhi reaching 14.7 % and 43.4 %, respectively, in 2021. The Multiple Antibiotic Resistance (MAR) index exceeded 0.2 in over half of the isolates, signaling widespread antibiotic misuse. The study discerned 47 unique antibiotic resistance patterns and pinpointed carbapenem and macrolide antibiotics as the remaining effective treatments against XDR strains, underlining the critical need to preserve these drugs for severe cases. Molecular examinations identified blaTEM, blaSHV, and blaCTX-M genes in ceftriaxone-resistant strains, while qnrS was specific to ciprofloxacin-resistant variants. Notably, all examined strains exhibited a singular mutation in the gyrA gene, maintaining wild-type gyrB and parC genes. The erm(B) gene emerged as the primary determinant of azithromycin resistance. Furthermore, a distressing increase in resistance genes was observed over three years, with erm(B), blaTEM and qnrS showing significant upward trends. These findings are a clarion call for robust antimicrobial stewardship programs to curtail inappropriate antibiotic use and forestall the burgeoning threat of antibiotic resistance in S. Typhi.

Antimicrobial resistance by mutation in the genes folp1, rpob and GYRA in patients with lepra diagnosed between 2017 and 2020 at the center of dermatological specialties-paraguay

Antimicrobial resistance by mutation in the genes folp1, rpob and GYRA in patients with lepra diagnosed between 2017 and 2020 at the center of dermatological specialties-paraguay

Molecular analysis of bedaquiline resistance and genetic variations in clinical isolates of multidrug and fluoroquinolones-resistant Mycobacterium tuberculosis

BackgroundMycobacterium tuberculosis developing resistance to anti-tubercular therapy leads to pioneering innovative treatment protocols featuring carefully curated combinations of potent medications. The aim of this study was to analyze the frequency and pattern of fluoroquinolone (FQ) resistant mutations among the first-line drug-resistant specimens. By employing an elective sampling strategy, we deliberately focused our investigation on a targeted subset of multidrug resistant (MDR) and FQ resistant (FQR) TB cases. MethodSputum samples from first-line drug-resistant patients were subjected to a GenoType MTBDRsl VER 2.0 assay to determine FQR. To investigate the effect of bedaquiline (BDQ) on these mutation-conferring genes (Rv0678, pepQ, and atpE), sanger sequencing was carried out on the MDR + FQR subset that was obtained. We have also conducted a computational analysis of the gene products of Rv0678 and pepQ with BDQ using molecular docking methods. ResultsEighty-three samples were observed to have FQR, and the majority of these specimens were associated with naïve and treatment failure cases. Among them, 64 isolates exhibited mutations in gyrA gene, while 19 isolates showed mutations in gyrB gene. The predominant occurrence of genetic variations, particularly in D94G and A90V, was observed within the specimens displaying resistance in gyrA gene, and the same has been confirmed by PCR-based detection. And the sequence analysis of BDQ genes among MDR + FQR subset indicated that the majority of the samples did not exhibit mutations in pepQ and atpE genes. However, only 2 samples were found to have the genetic variant G337A, which involves replacing the amino acid glutamic acid (E) at codon position 113 of Rv0678 gene with lysine (K). ConclusionsMost of the mutations were harbored in rpoB + katG + gyrA gene pattern. These findings pave the way for the development of databases and rapid diagnostic probes.

Integrative and Conjugative Elements and Prophage DNA as Carriers of Resistance Genes in Erysipelothrix rhusiopathiae Strains from Domestic Geese in Poland.

Goose erysipelas is a serious problem in waterfowl breeding in Poland. However, knowledge of the characteristics of Erysipelothrix rhusiopathiae strains causing this disease is limited. In this study, the antimicrobial susceptibility and serotypes of four E. rhusiopathiae strains from domestic geese were determined, and their whole-genome sequences (WGSs) were analyzed to detect resistance genes, integrative and conjugative elements (ICEs), and prophage DNA. Sequence type and the presence of resistance genes and transposons were compared with 363 publicly available E. rhusiopathiae strains, as well as 13 strains of other Erysipelothrix species. Four strains tested represented serotypes 2 and 5 and the MLST groups ST 4, 32, 242, and 243. Their assembled circular genomes ranged from 1.8 to 1.9 kb with a GC content of 36-37%; a small plasmid was detected in strain 1023. Strains 1023 and 267 were multidrug-resistant. The resistance genes detected in the genome of strain 1023 were erm47, tetM, and lsaE-lnuB-ant(6)-Ia-spw cluster, while strain 267 contained the tetM and ermB genes. Mutations in the gyrA gene were detected in both strains. The tetM gene was embedded in a Tn916-like transposon, which in strain 1023, together with the other resistance genes, was located on a large integrative and conjugative-like element of 130 kb designated as ICEEr1023. A minor integrative element of 74 kb was identified in strain 1012 (ICEEr1012). This work contributes to knowledge about the characteristics of E. rhusiopathiae bacteria and, for the first time, reveals the occurrence of erm47 and ermB resistance genes in strains of this species. Phage infection appears to be responsible for the introduction of the ermB gene into the genome of strain 267, while ICEs most likely play a key role in the spread of the other resistance genes identified in E. rhusiopathiae.

A Set of Multiresistant Isolates of Mycoplasma bovis Subtype ST-1 with a Variable Susceptibility to Quinolones Are Also Circulating in Spain.

Mycoplasma bovis (M. bovis) is one of the worldwide most important infectious agents involved in respiratory complex diseases (RCD). In Spain, the endemic presence of subtypes ST-2 and ST-3 with phenotypic differences linked to their susceptibility to fluoroquinolones opened the way to develop control strategies focused on previous diagnosis of the subtype and the use of directed therapies when M. bovis were involved in RCD. Surprisingly, microbiological studies conducted during 2023 evidenced for the first time the presence of Spanish isolates of a new polC-subtype, previously classified as ST-1, recovered from calves with respiratory symptoms and pneumonia in different areas of the country (n = 16). Curiously, the minimum inhibitory concentration (MIC) to a panel of antimicrobials revealed phenotypic differences between these ST-1 isolates when using fluoroquinolones (FLQ). There is no geographical correlation between MIC profiles even for a set of 8 isolates recovered from different animals in the same flock. Sequencing of 4 genes (gyrA, gyrB, parC and parE) encoding quinolone resistance-determining regions (QRDR) evidenced the presence of accumulate mutations in 2 ST-1 isolates with high FLQ MICs, but not in all them (n = 3), thus suggesting that, as previously recorded for ST-2 isolates, other mechanisms should be involved in the acquisition of resistence to these antimicrobials. Additionally, as previously detected in the Spanish ST-2 and ST-3, subtype ST-1 isolates are also resistant to macrolides or lincosamides.

Identification of positively selected genes in Mycobacterium tuberculosis from southern Xinjiang Uygur autonomous region of China.

Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis (Mtb), remains a serious public health problem. Increasing evidence supports that selective evolution is an important force affecting genomic determinants of Mtb phenotypes. It is necessary to further understand the Mtb selective evolution and identify the positively selected genes that probably drive the phenotype of Mtb. This study mainly focused on the positive selection of 807 Mtb strains from Southern Xinjiang of China using whole genome sequencing (WGS). PAML software was used for identifying the genes and sites under positive selection in 807 Mtb strains. Lineage 2 (62.70%) strains were the dominant strains in this area, followed by lineage 3 (19.45%) and lineage 4 (17.84%) strains. There were 239 codons in 47 genes under positive selection, and the genes were majorly associated with the functions of transcription, defense mechanisms, and cell wall/membrane/envelope biogenesis. There were 28 codons (43 mutations) in eight genes (gyrA, rpoB, rpoC, katG, pncA, embB, gid, and cut1) under positive selection in multi-drug resistance (MDR) strains but not in drug-susceptible (DS) strains, in which 27 mutations were drug-resistant loci, 9 mutations were non-drug-resistant loci but were in drug-resistant genes, 2 mutations were compensatory mutations, and 5 mutations were in unknown drug-resistant gene of cut1. There was a codon in Rv0336 under positive selection in L3 strains but not in L2 and L4 strains. The epitopes of T and B cells were both hyper-conserved, particularly in the T-cell epitopes. This study revealed the ongoing selective evolution of Mtb. We found some special genes and sites under positive selection which may contribute to the advantage of MDR and L3 strains. It is necessary to further study these mutations to understand their impact on phenotypes for providing more useful information to develop new TB interventions.

Rapid detection of multidrug resistance in tuberculosis using nanopore-based targeted next-generation sequencing: a multicenter, double-blind study.

Resistance to anti-tuberculous drugs is a major challenge in the treatment of tuberculosis (TB). We aimed to evaluate the clinical availability of nanopore-based targeted next-generation sequencing (NanoTNGS) for the diagnosis of drug-resistant tuberculosis (DR-TB). This study enrolled 253 patients with suspected DR-TB from six hospitals. The diagnostic efficacy of NanoTNGS for detecting Mycobacterium tuberculosis and its susceptibility or resistance to first- and second-line anti-tuberculosis drugs was assessed by comparing conventional phenotypic drug susceptibility testing (pDST) and Xpert MTB/RIF assays. NanoTNGS can be performed within 12 hours from DNA extraction to the result delivery. NanoTNGS showed a remarkable concordance rate of 99.44% (179/180) with the culture assay for identifying the Mycobacterium tuberculosis complex. The sensitivity of NanoTNGS for detecting drug resistance was 93.53% for rifampicin, 89.72% for isoniazid, 85.45% for ethambutol, 74.00% for streptomycin, and 88.89% for fluoroquinolones. Specificities ranged from 83.33% to 100% for all drugs tested. Sensitivity for rifampicin-resistant tuberculosis using NanoTNGS increased by 9.73% compared to Xpert MTB/RIF. The most common mutations were S531L (codon in E. coli) in the rpoB gene, S315T in the katG gene, and M306V in the embB gene, conferring resistance to rifampicin, isoniazid, and ethambutol, respectively. In addition, mutations in the pncA gene, potentially contributing to pyrazinamide resistance, were detected in 32 patients. Other prevalent variants, including D94G in the gyrA gene and K43R in the rpsL gene, conferred resistance to fluoroquinolones and streptomycin, respectively. Furthermore, the rv0678 R94Q mutation was detected in one sample, indicating potential resistance to bedaquiline. NanoTNGS rapidly and accurately identifies resistance or susceptibility to anti-TB drugs, outperforming traditional methods. Clinical implementation of the technique can recognize DR-TB in time and provide guidance for choosing appropriate antituberculosis agents.

First Report of Dickeya dadantii Causing Soft Rot in Cariru in Brazil.

Cariru (Talinum fruticosum) is a non-conventional food plant and a source of vitamins A, B2, B5, and C, calcium, phosphorus, and iron. It is an important crop for producers of the Amazon region and it is used as a leafy green vegetable, similar to spinach, as well as for medicinal purposes. It is cultivated by producers in the states of Pará and Amazonas (Amorim et al. 2014). In February 2020, in gardens of approximately 1.0 and 0.18 ha in the municipality of Ananindeua (01° 34' 12" S - 48° 38' 11" W), Pará, Brazil, we observed approximately 10% of the plants exhibited soft rot symptoms, bacterial oozing, and stem pith disintegration. From cariru infected leaves, the bacteria were selectively isolated using healthy pepper fruit, as described by Moraes et al. (2017). Two strain (UFRADD17 and UFRADD18) were obtained and both showed white, small, "broken glass" colonies on CPG (peptone-casamino acid-glucose) medium when observed in a stereoscope under oblique lighting. Pathogenicity tests were performed on 20-day-old cariru seedlings by depositing 10 ul of the bacterial suspension (106 CFU/ml) onto a wound made on the leaf blade with an entomological pin. After inoculation, seedlings were incubated in a greenhouse for 48 h at 30 ± 2°C and 90 ± 2% RH. Plants treated similarly with sterile distilled water were used as a negative control. Symptoms of soft rot were observed between 12 and 24 h after inoculation. Seedlings used as negative control remained symptomless. Forty-eight hours after inoculation, we reisolated the pathogen and performed rep-PCR (REP, ERIC, and BOX-PCR) analyses (Gama et al. 2018) with the strains used for inoculation and those reisolated from the pathogenicity test to confirm the identity of the strains and to fulfill Koch's postulates. The two re-isolated strains showed the same REP, ERIC, and BOX-PCR profiles as the strains used for inoculation. In addition, there were no differences between the rep-PCR profiles of the isolates UFRADD17 and UFRADD18. These isolates were Gram-negative, grew at 37°C, and were positive for maceration in pepper fruit and potato tubers. Molecular identification of the isolate UFRADD18 was performed from sequencing fragments from the 16S rDNA region and dnaX, fusA, gyrA, and mdH housekeeping genes as previously described (Van der Wolf et al. 2014; Ma et al. 2007). Sequences were deposited in Genbank under accessions OP142347, OP191704, OP191705, OP191706, and OP191707. Blastn analysis showed 96.15% identity with the 16S rDNA region of accession KY231142.1 (400/416 bp), 99.5% identity with dnaX of accession KC844490.1 (396/398 bp), 99.86% identity with fusA of accession CP023467.1 (714/715 bp), 99.74% identity with gyrA of accession KC844598 (387/388 bp), and 99.10% identity with mdH of accession GQ891979.1 (563/560 bp) from the type strain of Dickeya dadantii. A phylogenetic analysis performed by Bayesian inference with the dnaX, fusA, gyrA, and mdH genes grouped the isolate UFRADD18 along with NCPPB898T, with a 1.00 posterior probability. To our knowledge, this is the first report of D. dadantii causing soft rot in cariru in the Brazilian territory. In addition, this report increases understanding of the host range of this bacterium, which is important for adopting management strategies based on the control of alternative hosts.

Identification of knowledge gaps in whole-genome sequence analysis of multi-resistant thermotolerant Campylobacter spp.

BackgroundCampylobacter spp. is the most frequent cause of bacterial food-borne gastroenteritis and a high priority antibiotic resistant bacterium according to the World Health Organization (WHO). European monitoring of thermotolerant Campylobacter spp. does not reflect the global burden of resistances already circulating within the bacterial population worldwide.MethodsWe systematically compared whole genome sequencing with comprehensive phenotypic antimicrobial susceptibility, analyzing 494 thermotolerant Campylobacter poultry isolates from Vietnam and Germany. Any discrepancy was checked by repeating the wet lab and improving the dry lab part. Selected isolates were additionally analyzed via long-read Oxford Nanopore technology, leading to closed chromosomes and plasmids.ResultsOverall, 22 different resistance genes and gene variants (e. g. erm(B), aph(3’)-IIIa, aph(2’’)-If, catA, lnu(C), blaOXA, sat4) and point mutations in three distinct genes (gyrA, 23S rRNA, rpsL) associated with AMR were present in the Campylobacter isolates. Two AMR genes were missing in the database and one falsely associated with resistance. Bioinformatic analysis based on short-read data partly failed to identify tet(O) and aadE, when the genes were present as duplicate or homologous gene variants. Intriguingly, isolates also contained different determinants, redundantly conferring resistance to chloramphenicol, gentamicin, kanamycin, lincomycin and streptomycin. We found a novel tet(W) in tetracycline sensitive strains, harboring point mutations. Furthermore, analysis based on assemblies from short-read data was impaired to identify full length phase variable aad9, due to variations of the poly-C tract within the gene. The genetic determinant responsible for gentamicin resistance of one isolate from Germany could not be identified. GyrT86I, presenting the main determinant for (fluoro-)quinolone resistance led to a rare atypical phenotype of ciprofloxacin resistance but nalidixic acid sensitivity. Long-read sequencing predicted AMR genes were mainly located on the chromosome, and rarely on plasmids. Predictions from long- and short-read sequencing, respectively, often differed. AMR genes were often organized in multidrug resistance islands (MDRI) and partially located in proximity to transposase genes, suggesting main mobilization of resistance determinants is via natural transformation and transposition in Campylobacter.ConclusionsThe results of this study suggest that there is frequent resistance gene duplication, mosaicism, and mutation leading to gene variation and truncation in Campylobacter strains that have not been reported in previous studies and are missing from databases. Furthermore, there is a need for deciphering yet unknown resistance mechanisms and resistance spread in thermotolerant Campylobacter spp. that may pose a challenge to global food safety.

Ciprofloxacin- and levofloxacin-loaded nanoparticles efficiently suppressed fluoroquinolone resistance and biofilm formation in Acinetobacter baumannii

The spread of fluoroquinolone (FQ) resistance in Acinetobacter baumannii represents a critical health threat. This study aims to overcome FQ resistance in A. baumannii via the formulation of polymeric nanoFQs. Herein, 80 A. baumannii isolates were obtained from diverse clinical sources. All A. baumannii isolates showed high resistance to most of the investigated antimicrobials, including ciprofloxacin (CIP) and levofloxacin (LEV) (97.5%). FQ resistance-determining regions of the gyrA and parC genes were the most predominant resistant mechanism, harbored by 69 (86.3%) and 75 (93.8%) of the isolates, respectively. Additionally, plasmid-mediated quinolone resistance genes aac(6′)-Ib and qnrS were detected in 61 (76.3%) and 2 (2.5%) of the 80 isolates, respectively. The CIP- and LEV-loaded poly ε-caprolactone (PCL) nanoparticles, FCIP and FLEV, respectively, showed a 1.5–6- and 6–12-fold decrease in the MIC, respectively, against the tested isolates. Interestingly, the time kill assay demonstrated that MICs of FCIP and FLEV completely killed A. baumannii isolates after 5–6 h of treatment. Furthermore, FCIP and FLEV were found to be efficient in overcoming the FQ resistance mediated by the efflux pumps in A. baumannii isolates as revealed by decreasing the MIC four-fold lower than that of free CIP and LEV, respectively. Moreover, FCIP and FLEV at 1/2 and 1/4 MIC significantly decreased biofilm formation by 47–93% and 69–91%, respectively. These findings suggest that polymeric nanoparticles can restore the effectiveness of FQs and represent a paradigm shift in the fight against A. baumannii isolates.

Prevalence and Antimicrobial Resistance of Campylobacter in Conventional and No Antibiotics Ever Broiler Farms

Campylobacter is a leading cause of enteric disease worldwide. No antibiotics ever (NAE) poultry has become increasingly popular, yet little is known about the incidence and antimicrobial resistance (AMR) of Campylobacter in this production system. This study was conducted to determine the prevalence, concentration, and AMR of Campylobacter in conventional (CV) and NAE-raised broilers. Two CV and two NAE commercial broiler flocks were included in this study. Cecum (n = 420) and ileum (n = 420) of chickens were collected at different stages during the broiler grow-out phase and following transportation to the processing plant. Samples of litter (n = 24), feed (n = 24), and water (n = 24) were also collected. Screening for Campylobacter was conducted using real-time PCR assay, and enumeration was performed by direct plating on Campy Cefex agar. Campylobacter isolates were confirmed by real-time PCR, and antimicrobial susceptibility was evaluated following the National Antimicrobial Resistance Monitoring System (NARMS) methods. Whole Genome Sequencing (WGS) was used to identify AMR genes carried by the resistant isolates. Campylobacter prevalence reached 100% within the first 3 weeks of summer production under both NAE and CV rearing. A lower Campylobacter prevalence was detected in conventionally raised broilers during fall (P ≤ 0.05), yet no change in prevalence was observed in NAE birds (P > 0.05). Populations were high in the cecum, carrying an average of 6.6 Log10 CFU/g after transportation, and antimicrobial-resistant Campylobacter was isolated from CV broilers during the fall. Three isolates (1.2%), identified as C. coli, carrying the gyrA and tet(O) genes, exhibited simultaneous resistance to ciprofloxacin, tetracycline, and nalidixic acid. Results from this study can help identify important shifts in gut microbial community dynamics and Campylobacter prevalence associated with antibiotic administration within commercial poultry operations.

Molecular mechanisms of quinolone resistance in non-typhoidal Salmonella

By conducting retrospective analysis, this study aim to investigate the resistance mechanism of quinolones in non-typhoidal Salmonella (NTS). A total of 105 strains of NTS isolated from clinical specimens from the Fifth Affiliated Hospital of Southern Medical University from May 2020 to February 2021 were used as research objects. VITEK2 Compact automatic identification drug sensitivity analysis system and serological test were used to identify the strains. The sensitivity of the strains to ciprofloxacin, levofloxacin and nalidixic acid was detected by AGAR dilution method. The whole genome of 105 strains of NTS was sequenced. Abricate and other softwares were used to analyze drug-resistant genes, including plasmid-mediated quinolone resistance gene (PMQR) and Quinolone resistance determination region (QRDR). Serotypes and ST types were analyzed using SISTR and MLST, and phylogenetic trees were constructed. The results showed that the NTS isolated in this region were mainly ST34 Salmonella typhimurium (53.3%). The drug sensitivity results showed that the drug resistance rates of NTS to ciprofloxacin, levofloxacin and nalidixic acid were 30.4%, 1.9% and 22.0%, respectively, and the intermediate rates of ciprofloxacin and levofloxacin were 27.6% and 54.2%.A total of 46 (74.2%) of the 62 quinolone non-susceptible strains carried the PMQR gene, mainly qnrS1 (80.4%), followed by aac(6')-Ib-cr(15.2%); there were 14 NTS and 8 NTS had gyrA and parC gene mutations, respectively. The gyrA was mutations at the amino acid position 87, Asp87Tyr, Asp87Asn, Asp87Gly, and Thr57Ser mutations were detected in parC. In conclusion, this study found that NTS had relatively high resistance to quinolones, carrying qnrS1 gene mainly resulted in decreased sensitivity of NTS to ciprofloxacin and levofloxacin, and gyrA:87 mutation mainly resulted in NTS resistance to Nalidixic acid; Salmonella typhimurium in clinical isolates showed clonal transmission and required further epidemiological surveillance.

Multi-drug-resistant Escherichia coli in adult male patients with enlarged prostate attending general hospitals in Benue state.

The aim of this study was to investigate multi-drug-resistant (MDR) Escherichia coli in urine of adult male patients with enlarged prostate. Three hundred and sixty-eight samples of urine and blood were collected. Escherichia coli was isolated, purified, and identified and prostate-specific antigen (PSA) was determined. Multi-drug resistance test and specific drug resistance genes were assessed. Prevalence of Escherichia coli was high (38.5%) in patients with PSA of 60-79ngml-1 and 60% were MDR. The isolates showed highest resistance to tetracycline (53.3.0%) and least to cephalosporins (5%). They had intL and gyrA genes, which are integron, and quinolone resistance genes and sul1 and sul2 which are sulphonamide resistance-associated genes. Levofloxacin, ertapenem, and Augmentin (100% susceptibilities) were considered choice drugs for treatment of Escherichia coli infection in patients with elevated PSA.

Molecular Analysis of Pseudomonas aeruginosa Isolates with Mutant gyrA Gene and Development of a New Ciprofloxacin Derivative for Antimicrobial Therapy.

This study focuses on the prevalence of Pseudomonas aeruginosa in various medical specimens. In addition, the investigates of this research shows the genetic analysis of pathogen-resistant isolates and chemical modifications to ciprofloxacin. A total of 225 specimens from men and women aged 30 to 60 were carefully collected and examined, including samples from wound, burn, urine, sputum, and ear samples. The data were obtained from AL Muthanna hospitals. PCR-RFLP and gene expression analysis were used to identify resistant strains and explore the genetic basis of antibiotic resistance. A ciprofloxacin derivative was synthesized and confirmed through FT-IR, 1H-NMR, and mass spectroscopy techniques then it was tested as antibacterial agent. Also, molecular docking study was conducted to predict the mechanism of action for the synthesized derivative. The results demonstrated that wound samples had the highest positive rate (33.7%) of P. aeruginosa isolates. The PCR-RFLP testing correlated ciprofloxacin resistance with gyrA gene mutation. Gene expression analysis revealed significant changes in the gyrA gene expression in comparison to the reference rpsL gene subsequent to exposure to the synthesized derivative. Furthermore, the molecular docking investigation illustrated the strategic positioning of the ciprofloxacin derivative within the DNA-binding site of the gyrA enzyme. The examination of genetic expression patterns manifested diverse effects attributed to the CIP derivative on P. aeruginosa, thus portraying it as a viable candidate in the quest for the development of novel antimicrobial agents. Ciprofloxacin derivative may offer new antimicrobial therapeutic options for treating Pseudomonas aeruginosa infections in wound specimens, addressing resistance and gyrA gene mutations.

Genomic Sequencing of Clinical Cupriavidus gilardii Isolates Revealed Their Diverse Antimicrobial Resistance Mechanisms.

Cupriavidus gilardii is an emerging multidrug-resistant pathogen found in many environments and few clinical samples. The clinical infectiousness, pathogenicity, and resistance mechanisms of C. gilardii are still unclear due to the lack of clinical and sequencing data. We need to obtain insight into the clinical characteristics, virulence, and resistance mechanisms of C. gilardii. We isolated five C. gilardii isolates from hospitalized patients and carried out assay, culture and genome sequencing. We analyzed the genomic features of clinical C. gilardii isolates and took insight into their clinical characteristics, virulence, and resistance mechanisms. These isolates were resistant to meropenem, gentamicin, and other antimicrobials due to intrinsic resistance genes. Furthermore, the sequencing results revealed the widespread presence of the MCR-5.1 gene in C. gilardii. The virulence magnitude of C. gilardii is closely correlated with the number of virulence factors they carry. Some C. gilardii strains can acquire resistance to levofloxacin through gyrA gene mutation during treatment. The diverse antimicrobial resistance mechanisms challenge the treatment of C. gilardii infections. We present the genomic characteristics of clinically isolated C. gilardii to improve (i) our understanding of this pathogen and (ii) treatment options.