gyrA Gene Research Articles

Microbial Induced Calcite Precipitation (MICP) Potential of Ureolytic Bacillus sp. Isolated from the Soil of Eroded Ecosystems for Stabilizing and Improving the Fertility of Eroded Soils

The loss of soil from lands due to erosion has a negative effect on ecosystems and food security. Bacillus due to high catabolic capability is an appropriate candidate for application in biocementation process. The aim of this study is isolate and characterize Bacillus sp. with biocementation capability from various ecosystems. The isolates were separated from 400 samples, and characterized by biochemical and molecular methods include the amplification and sequencing analysis of gyrA and 16S rRNA genes. Growth in presence of urea, in different salinity, pH, and temperature, also scanning electron microscope (SEM), X-ray diffraction (XRD), and wind tunnel analysis were applied to determine biocementation ability. A total number of 195 isolates were recovered from environmental samples, of which 25 isolates (12.82%) were identified as urease-positive Bacillus which belonged to 10 species consisting of Bacillus subtilis five strains (20%), Bacillus vallismortis and Bacillus seohaeanensis four strains (16%) each, Bacillus mobilis, Bacillus pseudofirmus, Bacillus cohnii, Bacillus cereus, Bacillus alkalinitrilicus two strains (8%) each, and Bacillus sphaericus and Bacillus megaterium one strain (4%) each. Moreover, 15 urease-positive isolates (7.7%) belonging to Ralostenia, Actinomycete, and Halomonas genera were identified. Optimum conditions for microbial induced calcite precipitation (MICP) by isolates are 30 °C, pH 9, and 6% salinity. The highest rate of calcium carbonate formation and urease activity recorded in B. subtilis with 24.15 mg/mL of calcium carbonate and 4.40 × 103 unit/L of urease, followed by B. mobilis and B. alkalinitrilicus with 22.85 mg/mL of calcium carbonate and 3.93 × 103 unit/L of urease. After MICP the lowest soil loss ratio at a flow rate of 90 km/h, was observed in B. subtilis 100-fold reduction, followed by B. seohaeanensis, B. cereus, B. vallismortis, with 90, 85, 80-folds reduction, respectively. Results indicate that the diversity of Bacillus sp. offers the potential ability for adaptation to harsh and untapped environments, also showed that the use of MICP on the soil surface can have a very significant role in reducing soil losses due to wind erosion.

Comparison of the Diagnostic Accuracy of Three Real-Time PCR Assays for the Detection of Arcobacter butzleri in Human Stool Samples Targeting Different Genes in a Test Comparison without a Reference Standard

Potential etiological relevance for gastroenteric disorders including diarrhea has been assigned to Arcobacter butzleri. However, standard routine diagnostic algorithms for stool samples of patients with diarrhea are rarely adapted to the detection of this pathogen and so, A. butzleri is likely to go undetected unless it is specifically addressed, e.g., by applying pathogen-specific molecular diagnostic approaches. In the study presented here, we compared three real-time PCR assays targeting the genes hsp60, rpoB/C (both hybridization probe assays) and gyrA (fluorescence resonance energy transfer assay) of A. butzleri in a test comparison without a reference standard using a stool sample collection with a high pretest probability from the Ghanaian endemicity setting. Latent class analysis was applied with the PCR results obtained with a collection of 1495 stool samples showing no signs of PCR inhibition to assess the real-time PCR assays’ diagnostic accuracy. Calculated sensitivity and specificity were 93.0% and 96.9% for the hsp60-PCR, 100% and 98.2% for the rpoB/C-PCR, as well as 12.7% and 99.8% for the gyrA-PCR, respectively. The calculated A. butzleri prevalence within the assessed Ghanaian population was 14.7%. As indicated by test results obtained with high-titer spiked samples, cross-reactions of the hsp60-assay and rpoB/C-assay with phylogenetically related species such as A. cryaerophilus can occur but are less likely with phylogenetically more distant species like, e.g., A. lanthieri. In conclusion, the rpoB/C-assay showed the most promising performance characteristics as the only assay with sensitivity >95%, albeit associated with a broad 95%-confidence interval. In addition, this assay showed still-acceptable specificity of >98% in spite of the known cross-reactivity with phylogenetically closely related species such as A. cryaerophilus. If higher certainty is desired, the gyrA-assay with specificity close to 100% can be applied for confirmation testing with samples showing positive rpoB/C-PCR results. However, in case of a negative result in the gyrA-assay, this cannot reliably exclude the detection of A. butzleri in the rpoB/C-assay due to the gyrA-assay’s very low sensitivity.

Culturable bacterial endophytes of Aconitum carmichaelii Debx. were diverse in phylogeny, plant growth promotion, and antifungal potential.

Medicinal plants harbor tremendously diverse bacterial endophytes that maintain plant growth and health. In the present study, a total of 124 culturable bacterial endophytes were isolated from healthy Aconitum carmichaelii Debx. plants. These strains were clustered into 10 genera based on full-length 16S rDNA sequences, among which Bacillus and Pseudomonas were the dominant genera. In addition, A. carmichaelii may capture 10 potential new bacterial species based on multi-locus sequence analysis of three housekeeping genes (gyrA, rpoB, and atpD). The majority of these bacterial endophytes exhibited plant growth-promoting ability through diverse actions including the production of either indole acetic acid and siderophore or hydrolytic enzymes (glucanase, cellulose, and protease) and solubilization of phosphate or potassium. A total of 20 strains inhibited hyphal growth of fungal pathogens Sclerotium rolfsii and Fusarium oxysporum in vitro on root slices of A. carmichaelii by the dual-culture method, among which Pseudomonas sp. SWUSTb-19 showed the best antagonistic activity. Field experiment confirmed that Pseudomonas sp. SWUSTb-19 significantly reduced the occurrence of southern blight and promoted plant biomass compared with non-inoculation treatment. The possible mode of actions for Pseudomonas sp. SWUSTb-19 to antagonize against S. rolfsii involved the production of glucanase, siderophore, lipopeptides, and antimicrobial volatile compounds. Altogether, this study revealed that A. carmichaelii harbored diverse plant growth-promoting bacterial endophytes, and Pseudomonas sp. SWUSTb-19 could be served as a potential biocontrol agent against southern blight.

Distinct subunit architecture and assembly pattern of DNA gyrase from mycobacteria.

DNA gyrase, the sole negative supercoiling type II topoisomerase, is composed of two subunits, GyrA and GyrB, encoded by the gyrA and gyrB genes, respectively, that form a quaternary complex of A2 B2 . In this study, we have investigated the assembly of mycobacterial DNA gyrase from its individual subunits, a step prerequisite for its activity. Using analytical size-exclusion chromatography, we show that GyrA from Mycobacterium tuberculosis and Mycobacterium smegmatis forms tetramers (A4 ) in solution unlike in Escherichia coli and other bacteria where GyrA exists as a dimer. GyrB, however, persists as a monomer, resembling the pattern found in E. coli. GyrB in both mycobacterial species interacts with GyrA and triggers the dissociation of the GyrA tetramer to facilitate the formation of catalytically active A2 B2 . Despite oligomerisation, the GyrA tetramer retained its DNA binding ability, and DNA binding had no effect on GyrA's oligomeric state in both species. Moreover, the presence of DNA facilitated the assembly of holoenzyme in the case of M. smegmatis by stabilising the GyrA2 B2 tetramer but with little effect in M. tuberculosis. Thus, in addition to the distinct organisation and regulation of the gyr locus in mycobacteria, the enzyme assembly also follows a different pattern.

Molecular testing-guided therapy versus susceptibility testing-guided therapy in first-line and third-line Helicobacter pylori eradication: two multicentre, open-label, randomised controlled, non-inferiority trials

Helicobacter pylori infection is an important causal factor of gastric cancer and peptic ulcer disease and is associated with immune thrombocytopenic purpura and functional dyspepsia. In H pylori strains, point mutations in the 23S rRNA and gyrA genes are associated with clarithromycin resistance and levofloxacin resistance, respectively. Whether the efficacy of molecular testing-guided therapy is non-inferior to that of susceptibility testing-guided therapy for H pylori eradication is unclear. Therefore, we aimed to compare the efficacy and safety of molecular testing-guided therapy and traditional culture-based susceptibility testing-guided therapy in first-line and third-line treatment of H pylori infection. We did two multicentre, open-label randomised trials in Taiwan. In trial 1 (done at seven hospitals), treatment-naive individuals infected with H pylori who were aged 20 years or older were eligible for study inclusion. In trial 2 (done at six hospitals), individuals aged 20 years or older who failed treatment after two or more eradication therapies for H pylori infection were eligible for enrolment. Eligible patients were randomly assigned (1:1) to receive either molecular testing-guided therapy or susceptibility testing-guided therapy. The randomisation sequence was generated by computer using permuted block randomisation with a block size of 4. All investigators were masked to the randomisation sequence. Clarithromycin and levofloxacin resistance were determined by agar dilution test for measuring minimum inhibitory concentrations in the susceptibility testing-guided therapy group, and by PCR and direct sequencing for detection of 23S rRNA and gyrA mutations in the molecular testing-guided therapy group. Study participants received clarithromycin sequential therapy, levofloxacin sequential therapy, or bismuth quadruple therapy according to the resistance status to clarithromycin and levofloxacin. The 13C-urease breath test was used to determine the status of H pylori infection at least 6 weeks after eradication therapy. The primary outcome was the eradication rate by intention-to-treat analysis. The frequency of adverse effects was analysed in patients with available data. The prespecified margins for non-inferiority were 5% for trial 1 and 10% for trial 2. The trials are ongoing for post-eradication follow-up and registered with ClinicalTrials.gov, NCT03556254 for trial 1, and NCT03555526 for trial 2. Between March 28, 2018, and April 23, 2021, 560 eligible treatment-naive patients with H pylori infection were recruited and randomly assigned to the molecular testing-guided therapy group or the susceptibility testing-guided therapy group in trial 1. Between Dec 28, 2017, and Oct 27, 2020, 320 eligible patients with refractory H pylori infection were recruited and randomly assigned to the molecular testing-guided therapy group or the susceptibility testing-guided therapy group in trial 2. 272 men and 288 women were recruited for trial 1, and 98 men and 222 women were recruited for trial 2. In first-line H pylori treatment, infection was eradicated in 241 (86%, 95% CI 82-90) of 280 patients in the molecular testing-guided therapy group and 243 (87%, 83-91) of 280 patients in the susceptibility testing-guided therapy group by intention-to-treat analysis (p=0·81). In third-line H pylori treatment, infection was eradicated in 141 (88%, 83-93) of 160 patients in the molecular testing-guided therapy group and 139 (87%, 82-92) of 160 patients in the susceptibility testing-guided therapy group by intention-to-treat analysis (p=0·74). The difference in the eradication rate between the molecular testing-guided therapy group and the susceptibility testing-guided therapy group was -0·7% (95% CI -6·4 to 5·0; non-inferiority p=0·071) in trial 1 and 1·3% (-6·0 to 8·5; non-inferiority p=0·0018 in trial 2 by intention-to-treat analysis. We found no difference in adverse effects across both treatment groups in trial 1 and trial 2. Molecular testing-guided therapy was similar to susceptibility testing-guided therapy in first-line therapy and non-inferior to susceptibility testing guided therapy in third-line treatment of H pylori infection, supporting the use of molecular testing-guided therapy for H pylori eradication. Ministry of Science and Technology of Taiwan, and Centre of Precision Medicine of the Higher Education Sprout Project by the Ministry of Education of Taiwan.

High macrolides and fluoroquinolones resistance rate in Mycoplasma genitalium in southern Tenerife

Mycoplasma genitalium (MG) is a recognized sexually transmitted pathogen. Increasing resistance to main lines of treatment (macrolides and quinolones) justifies a genetic study of mutations to improve cure rates. A total of 8,508 samples from April 2018 to July 2022 were processed using AllplexTM STI Essential Assay. In MG positive cases 23S rRNA V domain, gyrA and parC genes were studied. Mutations detected were checked to assess their clinical significance and medical records were reviewed to obtain demographic and treatment information. Resistance study was performed on 92 samples (65 men and 27 women). In relation to the genotypic study, 28 patients presented mutations to macrolides (30.43%). Most common was A2059G (18.48%). For quinolones, 5 patients (5.43%) had clinically relevant mutations in parC gene. Of note was a patient with G295 mutation in gyrA associated with G248T in parC. Thirty subjects underwent a test of cure (TOC). Azithromycin was the most common empirical regimen and moxifloxacin the main alternative. High rate of resistance in our environment evidences the need for targeted therapy by genotypic study of macrolide resistance, supported by the detection of mutations in parC and gyrA to predict quinolones susceptibility and the use of TOC to evaluate treatment response.

Mycobacterium tuberculosis Intra-Host Evolution Among Drug-Resistant Tuberculosis Patients Failing Treatment.

Understanding Mycobacterium tuberculosis (Mtb) intra-host evolution of drug resistance is important for successful drug-resistant tuberculosis (DR-TB) treatment and control strategies. This study aimed to characterise the acquisition of genetic mutations and low-frequency variants associated with treatment-emergent Mtb drug resistance in longitudinally profiled clinical isolates from patients who experienced DR-TB treatment failure. We performed deep Whole Genome Sequencing on 23 clinical isolates obtained longitudinally across nine timepoints from five patients who experienced DR-TB treatment failure enrolled in the CAPRISA 020 InDEX study. The minimum inhibitory concentrations (MICs) were established on the BACTEC™ MGIT 960™ instrument on 15/23 longitudinal clinical isolates for eight anti-TB drugs (rifampicin, isoniazid, ethambutol, levofloxacin, moxifloxacin, linezolid, clofazimine, bedaquiline). In total, 22 resistance associated mutations/variants were detected. We observed four treatment-emergent mutations in two out of the five patients. Emerging resistance to the fluoroquinolones was associated with 16- and 64-fold elevated levofloxacin (2-8 mg/L) and moxifloxacin (1-2 mg/L) MICs, respectively, resulting from the D94G/N and A90V variants in the gyrA gene. We identified two novel mutations associated with elevated bedaquiline MICs (>66-fold): an emerging frameshift variant (D165) on the Rv0678 gene and R409Q variant on the Rv1979c gene present from baseline. Genotypic and phenotypic resistance to the fluoroquinolones and bedaquiline was acquired in two out of five patients who experienced DR-TB treatment failure. Deep sequencing of multiple longitudinal clinical isolates for resistance-associated mutations coupled with phenotypic MIC testing confirmed intra-host Mtb evolution.

Identification of Antibiotic Resistance Pattern and Antibiotic Resistance Gene of Staphylococcus aureus from Broiler Chicken Farm in Sukabumi and Cianjur Regency, West Java Province, Indonesia

Broiler chicken is one of the most common forms of protein in Indonesia. Sukabumi and Cianjur are two of the province of West Java’s top producers of broiler chickens. Farmers nonetheless use antibiotics for prevention, treatment, and growth enhancement. The objective of this investigation is to detect antibiotic resistance and the related resistance gene. The cloacal swab samples were determined to be Staphylococcus aureus by biochemical and molecular assays. Against the positive S. aureus strains, the antibiotics tetracycline, ampicillin, gentamicin, nalidixic acid, ciprofloxacin, erythromycin, and chloramphenicol were evaluated. The colony’s obvious inhibitory zone was evaluated in accordance with the 2018 Clinical and Laboratory Standard Institute recommendations (CLSI 2018). After extracting the DNA from resistant strains, the polymerase chain reaction (PCR) method was employed to identify the resistance gene. The total number of S. aureus isolates from both regions was 26, with 14 coming from Sukabumi and 12 from Cianjur. The vast majority were resistant to the vast majority of antibiotics used in broiler farms including several medicines. These samples carried the genes blaTEM, gyrA, tetA, and ermB for antibiotic resistance.

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

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

Analysing the Impact of Bedaquiline Resistance in Fluoroquinolone-Resistant Clinical Isolates and Investigating the Molecular Interactions of BDQ with the atpE Gene

The progress of the tuberculosis eradication programme is hindered by the global health issue of drug-resistant tuberculosis, which is a significant threat to the population. Resistance-associated mutations vary geographically, so investigate the prevalence of resistance to fluoroquinolone (anti-TB) drugs and its association with resistance-related mutations in clinical isolates of Tamilnadu (north zone) and Puducherry was carried out. And the resultant fluoroquinolone drug-resistant tuberculosis strains will be subjected to the molecular analysis of bedaquiline resistance to study the mutation in the atpE gene. The study includes 430 specimens received at Intermediate Reference Laboratory, State TB training and Demonstration Centre in the Government Hospital for Chest Diseases for Molecular testing between January 2020 and December 2021. Samples were analysed by GenoType MTBDRslV.2 for the molecular detection of mutations in the gyrA and gyrB genes. FQ resistance was observed in 32 samples (7.4%), most of which were naïve and previous treatment failure cases. Twenty-five isolates had mutations in DNA gyrase subunit -A (gyrA), while mutations in gyrB were observed in only 7 isolates. Mutational analysis revealed that the mutations mainly alter codon 94 (replacing aspartic acid with glycine, D94G), and 90 (replacing alanine with valine, A90V). In Isoniazid Resistance, MDR, and treatment failure cases, the resistance to Fluoroquinolones was most commonly associated with the D94G mutation. A molecular stimulation study evaluates the effect of BDQ on atpE gene by docking, using AutoDock 4.2, showing hydrogen, hydrophobic and electrostatic interactions.

Strain diversity in Mycobacterium avium subsp. paratuberculosis-positive bovine fecal samples collected in Switzerland.

Paratuberculosis or Johne's disease is a chronic intestinal disease in domestic and wild ruminants. It affects global dairy economy and is caused by Mycobacterium avium subsp. paratuberculosis (MAP). The objective of this study was to analyze strain diversity in MAP-positive fecal samples by using a particular single nucleotide polymorphism (SNP) distinguishing between cattle (C-) and sheep (S-) type MAP and analysis of SNPs within gyrA and gyrB genes differentiating between Types I, II, and III. Moreover, mycobacterial interspersed repetitive unit and variable-number tandem repeat (MIRU-VNTR) analysis using eight established loci was performed. A total of 90 fecal samples from diseased animals presenting diarrhea and/or weight loss, originating from 59 bovine herds across 16 cantons of Switzerland were screened by PCR for the MAP-specific F57 and IS900 genes and were further subtyped. 96.7% and 3.3% of the samples contained C- and S-type MAP, respectively. Ten INRA Nouzilly MIRU-VNTR (INMV) profiles, with a discriminatory index of 0.802, calculated based on 65 epidemiological independent genotypes, were detected: INMV 1 (33.8%), INMV 2 (23.1%), INMV 6 (16.9%), INMV 9 (9.2%), INMV 116 (4.6%), INMV 3 (3.1%), INMV 5 (3.1%) and INMV 72 (1.5%), including two novel INMV profiles, namely INMV 253 (3.1%; S-type III) and INMV 252 (1.5%; C-type). INMV 1, INMV 2, and INMV 6 comprised almost 75% of the F57- and IS900-positive samples. Typing data from 11 herds suggest that there are some herds with intra-herd diversity of genotypes. The results of this study indicate a heterogeneity of MAP in Switzerland.

Genomic analysis of fluoroquinolone-resistant Leclercia adecarboxylata carrying the ISKpn19-orf-qnrS1-ΔIS3-blaLAP-2 module in a synanthropic pigeon, Brazil

The aim of this study was to perform a genomic investigation of a multiple fluoroquinolone-resistant Leclercia adecarboxylata strain isolated from a synanthropic pigeon in São Paulo, Brazil. Whole-genome sequencing was performed using an Illumina platform, and in silico deep analyses of the resistome were performed. Comparative phylogenomics was conducted using a global collection of publicly available genomes of L. adecarboxylata strains isolated from human and animal hosts. L. adecarboxylata strain P62P1 displayed resistance to human (norfloxacin, ofloxacin, ciprofloxacin, and levofloxacin) and veterinary (enrofloxacin) fluoroquinolones. This multiple quinolone-resistant profile was associated with mutations in the gyrA (S83I) and parC (S80I) genes and the presence of the qnrS gene within an ISKpn19-orf-qnrS1-ΔIS3-blaLAP-2 module, previously identified in L. adecarboxylata strains isolated from pig feed and faeces in China. Genes associated with arsenic, silver, copper, and mercury resistance were also predicted. Phylogenomic analysis revealed clustering (378-496 single nucleotide polymorphism differences) with two L. adecarboxylata strains isolated from human and fish sources in China and Portugal, respectively. L. adecarboxylata is a Gram-negative bacterium of the Enterobacterales order and is considered an emergent opportunistic pathogen. Since L. adecarboxylata has adapted to human and animal hosts, genomic surveillance is highly recommended, in order to identify the emergence and spread of resistant lineages and high-risk clones. In this regard, this study provides genomic data that can help clarify the role of synanthropic animals in the dissemination of clinically relevant L. adecarboxylata within a One Health context.

Genetic diversity and antimicrobial resistance profiles of Staphylococcus pseudintermedius associated with skin and soft-tissue infections in companion animals in Lisbon, Portugal.

Staphylococcus pseudintermedius is the main bacterial pathogen of skin and soft-tissue infections (SSTIs) in companion animals. Antimicrobial resistance in this species is a growing public health concern. This study aims to characterize a collection of S. pseudintermedius causing SSTIs in companion animals, establishing the main clonal lineages and antimicrobial resistance traits. The collection corresponded to all S. pseudintermedius (n = 155) causing SSTIs in companion animals (dogs, cats and one rabbit) collected between 2014 and 2018 at two laboratories in Lisbon, Portugal. Susceptibility patterns were established by disk diffusion for 28 antimicrobials (15 classes). For antimicrobials without clinical breakpoints available, a cut-off value (COWT) was estimated, based on the distribution of the zones of inhibition. The blaZ and mecA genes were screened for the entire collection. Other resistance genes (e.g., erm, tet, aadD, vga(C), dfrA(S1)) were searched only for those isolates showing an intermediate/resistance phenotype. For fluoroquinolone resistance, we determined the chromosomal mutations in the target genes grlA and gyrA. All the isolates were typed by PFGE following SmaI macrorestriction and isolates representative of each PFGE type were further typed by MLST. Forty-eight out of the 155 S. pseudintermedius isolates (31.0%) were methicillin-resistant (mecA +, MRSP). Multidrug-resistant (MDR) phenotypes were detected for 95.8% of the MRSP and 22.4% of the methicillin-susceptible (MSSP) isolates. Of particular concern, only 19 isolates (12.3%) were susceptible to all antimicrobials tested. In total, 43 different antimicrobial resistance profiles were detected, mostly associated with the carriage of blaZ, mecA, erm(B), aph3-IIIa, aacA-aphD, cat pC221, tet(M) and dfr(G) genes. The 155 isolates were distributed within 129 PFGE clusters, grouped by MLST in 42 clonal lineages, 25 of which correspond to new sequence types (STs). While ST71 remains the most frequent S. pseudintermedius lineage, other lineages that have been replacing ST71 in other countries were detected, including ST258, described for the first time in Portugal. This study revealed a high frequency of MRSP and MDR profiles among S. pseudintermedius associated with SSTIs in companion animals in our setting. Additionally, several clonal lineages with different resistance profiles were described, evidencing the importance of a correct diagnosis and selection of the therapy.

Genomic investigation of antimicrobial resistance in Brucella abortus strains isolated from cattle in Brazil

The emergence of resistance to antimicrobials among Brucella spp. has highlighted the need to understand the genetic determinants responsible for these phenotypes. We investigated the gene targets associated with antimicrobial resistance in 53 genomes of B. abortus strains, isolated from cattle in Brazil, with resistance or intermediate susceptibility to antimicrobials. The genetic diversity of the genomes was evaluated using single-nucleotide polymorphism (SNP) differences among the isolates. We compared 18 genes (gyrB, bepD, bepE, norMII, norMI, parC, bepC, folP, gyrA, rpoB, folA, rsmG, marR, parE, mprF, oxyR, bepF, and bepG) previously described as related to antimicrobial resistance in Brucella spp. to those of the reference strain B. abortus 2308, which was susceptible to all tested antimicrobials. Eight genes had nonsynonymous mutations, deletions, or stop codons in at least one of the analyzed genomes. However, we did not observe any association between the genetic polymorphisms in the evaluated genes and the antimicrobial resistance phenotypes observed in the Brazilian analyzed strains (46 intermediate susceptible to rifampicin, 6 resistance to at least one antimicrobial and 1 multidrug resistance).

The Effects of Antiperspirant Aluminum Chlorohydrate on the Development of Antibiotic Resistance in Staphylococcus epidermidis.

This study investigates the effects of the antiperspirant aluminum chlorohydrate on the development of antibiotic resistance in commensal Staphylococcus epidermidis isolates. The isolates were exposed to aluminum chlorohydrate for 30 days. The bacteria that developed resistance to oxacillin and ciprofloxacin were isolated, and the expression levels of some antibiotic resistance genes were determined using quantitative reverse transcriptase PCR. Before and after exposure, the minimum inhibitory concentration (MIC) values of the bacteria were determined using the microdilution method. A time-dependent increase was observed in the number of bacteria that developed resistance and increased MIC values. Consistent with the ciprofloxacin resistance observed after exposure, an increase in norA, norB/C, gyrA, gyrB, parC, and parE gene expression was observed. In addition to aluminum chlorohydrate exposure, oxacillin resistance was observed in all test bacteria in the group only subcultured in the medium, suggesting that phenotypic resistance cannot be correlated with chemical exposure in light of these data. The increase in mecA gene expression in selected test bacteria that acquired resistance to oxacillin after exposure compared with control groups suggests that the observed resistance may have been related to aluminum chlorohydrate exposure. To our knowledge, this is the first time in the literature that the effects of aluminum chlorohydrate as an antiperspirant on the development of antibiotic resistance in Staphylococcus epidermidis have been reported.

Mycoplasma genitalium antibiotic resistance-associated mutations in genital and extragenital samples from men-who-have-sex-with-men attending a STI clinic in Verona, Italy.

Mycoplasma genitalium (MG) is one of the most warning emerging sexually transmitted pathogens also due to its ability in developing resistance to antibiotics. MG causes different conditions ranging from asymptomatic infections to acute mucous inflammation. Resistance-guided therapy has demonstrated the best cure rates and macrolide resistance testing is recommended in many international guidelines. However, diagnostic and resistance testing can only be based on molecular methods, and the gap between genotypic resistance and microbiological clearance has not been fully evaluated yet. This study aims at finding mutations associated with MG antibiotic resistance and investigating the relationship with microbiological clearance amongst MSM. From 2017 to 2021, genital (urine) and extragenital (pharyngeal and anorectal swabs) biological specimens were provided by men-who-have-sex-with-men (MSM) attending the STI clinic of the Infectious Disease Unit at the Verona University Hospital, Verona, Italy. A total of 1040 MSM were evaluated and 107 samples from 96 subjects resulted positive for MG. Among the MG-positive samples, all those available for further analysis (n=47) were considered for detection of mutations known to be associated with macrolide and quinolone resistance. 23S rRNA, gyrA and parC genes were analyzed by Sanger sequencing and Allplex™ MG and AziR Assay (Seegene). A total of 96/1040 (9.2%) subjects tested positive for MG in at least one anatomical site. MG was detected in 107 specimens: 33 urine samples, 72 rectal swabs and 2 pharyngeal swabs. Among them, 47 samples from 42 MSM were available for investigating the presence of mutations associated with macrolide and quinolone resistance: 30/47 (63.8%) showed mutations in 23S rRNA while 10/47 (21.3%) in parC or gyrA genes. All patients with positive Test of Cure (ToC) after first-line treatment with azithromycin (n=15) were infected with 23S rRNA-mutated MG strains. All patients undergoing second-line moxifloxacin treatment (n=13) resulted negative at ToC, even those carrying MG strains with mutations in parC gene (n=6). Our observations confirm that mutations in 23S rRNA gene are associated with azithromycin treatment failure and that mutations in parC gene alone are not always associated with phenotypic resistance to moxifloxacin. This reinforces the importance of macrolide resistance testing to guide the treatment and reduce antibiotic pressure on MG strains.

Genomic Characterization and Genetic Profiles of Salmonella Gallinarum Strains Isolated from Layers with Fowl Typhoid in Colombia.

Salmonella Gallinarum (SG) is the causative agent of fowl typhoid (FT), a disease that is harmful to the poultry industry. Despite sanitation and prophylactic measures, this pathogen is associated with frequent disease outbreaks in developing countries, causing high morbidity and mortality. We characterized the complete genome sequence of Colombian SG strains and then performed a comparative genome analysis with other SG strains found in different regions worldwide. Eight field strains of SG plus a 9R-derived vaccine were subjected to whole-genome sequencing (WGS) and bioinformatics analysis, and the results were used for subsequent molecular typing; virulome, resistome, and mobilome characterization; and a comparative genome study. We identified 26 chromosome-located resistance genes that mostly encode efflux pumps, and point mutations were found in gyrase genes (gyrA and gyrB), with the gyrB mutation S464T frequently found in the Colombian strains. Moreover, we detected 135 virulence genes, mainly in 15 different Salmonella pathogenicity islands (SPIs). We generated an SPI profile for SG, including C63PI, CS54, ssaD, SPI-1, SPI-2, SPI-3, SPI-4, SPI-5, SPI-6, SPI-9, SPI-10, SPI-11, SPI-12, SPI-13, and SPI-14. Regarding mobile genetic elements, we found the plasmids Col(pHAD28) and IncFII(S) in most of the strains and 13 different prophage sequences, indicating a frequently obtained profile that included the complete phage Gifsy_2 and incomplete phage sequences resembling Escher_500465_2, Shigel_SfIV, Entero_mEp237, and Salmon_SJ46. This study presents, for the first time, the genomic content of Colombian SG strains and a profile of the genetic elements frequently found in SG, which can be further studied to clarify the pathogenicity and evolutionary characteristics of this serotype.

Advantages of analysing both pairwise SNV-distance and differing SNVs between Mycobacterium tuberculosis isolates for recurrent tuberculosis cause determination.

Endogenous reactivation and exogenous reinfection are two possible causes of recurrent tuberculosis (TB). However, in some cases, precise cause determination can be challenging. In this study, we used whole genome sequencing to determine pairwise SNV distances and detect differing SNVs in initial and subsequent isolates for recurrent TB cases when the first and second episodes were caused by Mycobacterium tuberculosis (Mtb) strains with an identical spoligotype pattern. In total, 104 Mtb isolates from 36 recurrent TB and 16 single TB episode patients were included in the study. Most isolate pairs belonged to the SIT1 (n=21), SIT42 (n=9), SIT53 (n=9), and SIT254 (n=7) spoligotypes, and in 27 cases, resistance to at least one anti-TB drug was found in either isolate. Drug susceptibility was more common in the recurrent TB patient cohort, and longitudinal single TB episode isolates were more prone to be drug-resistant (p=0.03), while the association between patient cohort and spoligotype was not statistically significant (p=0.07). The pairwise SNV-distance between the longitudinal single TB episode isolates was small (0-7 SNVs). Among the recurrent TB isolates, based on the high SNV-distance (38-273 SNVs), six reinfection cases (16.7%) were identified. This distance was small (<10 SNVs) in the remaining 30 isolate pairs. Further analysis of differing SNVs revealed that 22 (61.1%) cases could be classified as possible reactivation. Notably, despite the small distance of 2-7 SNVs, initial isolates of eight patients (22.2%) had several SNVs that were not found in the second isolates; therefore, these cases were classified as reinfection with a closely related Mtb strain. No statistically significant difference in the time interval between specimen collection in the reactivation and reinfection Mtb sample groups (p=0.13) or an association between recurrence cause and drug resistance status (p=0.62) or spoligotype (p=0.79) could be detected. The mycobacterial median mutation rate of longitudinal single TB episodes and possible reactivation isolate pairs (n=37) was 0.12 SNVs/genome/year (IQR 0-0.39), and in 18 cases (48.6%), it was equal to zero. No statistically significant differences in mutation rate were found between recurrent TB and longitudinal single TB episode isolates (p=0.087), drug-susceptible and resistant isolates (p=0.37) or isolates of Beijing and other genotype families (p=0.33). Furthermore, four cases of fluoroquinolone resistance development through the acquired SNVs in the gyrA gene were identified. To conclude, this study highlighted the complexity of recurrent episode cause determination and showed the usefulness of differing SNV identification in both Mtb isolates in such cases. Expected drug susceptibility was the only discriminative factor for recurrent TB episode-causing mycobacterial strains, while no differences between reactivation and reinfection sample groups could be identified.

Genetic Determinants of Macrolide and Fluoroquinolone Resistance in Mycoplasma genitalium and Their Prevalence in Moscow, Russia

Macrolide (MLR) and fluoroquinolone (FQR) resistance in Mycoplasma genitalium (MG) has recently become a major problem worldwide. The available data on the prevalence of MLR and FQR in MG in Russia are limited. In this study, we aimed to evaluate the prevalence and pattern of mutations in 213 MG-positive urogenital swabs from patients in Moscow between March 2021 and March 2022. MLR- and FQR-associated mutations were searched in 23S rRNA as well as in the parC and gyrA genes using Sanger sequencing. The prevalence of MLR was 55/213 (26%), with A2059G and A2058G substitutions being the two most common variants (36/55, 65%, and 19/55, 35%, respectively). FQR detection showed 17% (37/213); two major variants were D84N (20/37, 54%) and S80I (12/37, 32.4%) and three minor variants were S80N (3/37, 8.1%), D84G (1/37, 2.7%), and D84Y (1/37, 2.7%). Fifteen of the fifty-five MLR cases (27%) simultaneously harbored FQR. This study revealed the high frequency of MLR and FQR. We conclude that the improvement of patient examination algorithms and therapeutic approaches should be combined with the routine monitoring of antibiotic resistance based on the sensitivity profiles presented. Such a complex approach will be essential for restraining the development of treatment resistance in MG.

Gene network interaction analysis to elucidate the antimicrobial resistance mechanisms in the Clostridiumdifficile

Antimicrobial resistance has caused chaos worldwide due to the depiction of multidrug-resistant (MDR) infective microorganisms. A thorough examination of antimicrobial resistance (AMR) genes and associated resistant mechanisms is vital to solving this problem. Clostridium difficile (C. difficile) is an opportunistic nosocomial bacterial strain that has acquired exogenous AMR genes that confer resistance to antimicrobials such as erythromycin, azithromycin, clarithromycin, rifampicin, moxifloxacin, fluoroquinolones, vancomycin, and others. A network of interactions, including 20 AMR genes, was created and analyzed. In functional enrichment analysis, Cellular components (CC), Molecular Functions (MF), and Biological Processes (BP) were discovered to have substantial involvement. Mutations in the rpl genes, which encode ribosomal proteins, confer resistance in Gram-positive bacteria. Full erythromycin and azithromycin cross-resistance can be conferred if more than one of the abovementioned genes is present. In the enriched BP, rps genes related to transcriptional regulation and biosynthesis were found. The genes belong to the rpoB gene family, which has previously been related to rifampicin resistance. The genes rpoB, gyrA, gyrB, rpoS, rpl genes, rps genes, and Van genes are thought to be the hub genes implicated in resistance in C. difficile. As a result, new medications could be developed using these genes. Overall, our observations provide a thorough understanding of C. difficile AMR mechanisms.