23S rRNA Mutations Research Articles

Molecular Detection of Metronidazole and Tetracycline Resistance Genes in Helicobacter pylori-Like Positive Gastric Samples from Pigs.

Antimicrobial resistance is a major public health concern. The aim of this study was to assess the presence of antibiotic resistance genes, previously reported in Helicobacter pylori, in gastric samples of 36 pigs, in which DNA of H. pylori-like organisms had been detected. Based on PCR and sequencing analysis, two samples were positive for the 16S rRNA mutation gene, conferring tetracycline resistance, and one sample was positive for the frxA gene with a single nucleotide polymorphism, conferring metronidazole resistance. All three amplicons showed the highest homology with H. pylori-associated antibiotic resistance gene sequences. These findings indicate that acquired antimicrobial resistance may occur in H. pylori-like organisms associated with pigs.

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.

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.

Whole genome sequencing reveals novel genetic mutations of Helicobacter pylori associating with resistance to clarithromycin and levofloxacin.

Detection of mutations in one or a couple of genes may not provide enough data or cover all the genomic DNA variance related to antibiotic resistance of Helicobacter pylori to clarithromycin (CLA) and levofloxacin (LVX). We aimed to perform whole genome sequencing to explore novel antibiotic resistance-related genes to increase predictive accuracy for future targeted sequencing tests. Gastric mucosal biopsies were taken during upper endoscopy in 27 H. pylori-infected patients. According to culture-based antibacterial susceptibility test, H. pylori strains were divided into three groups, with nine strains in each group: CLA single-drug resistance (group C), LVX single-drug resistance (group L), and strains sensitive to all antibacterial drugs (group S). Based on whole genome sequencing with group S being the control, group C and group L group-specific single nucleotide variants and amino acid mutations were screened, and potential candidate genes related to CLA and LVX resistance were identified. The median age of study subjects was 35 years (IQR: 31-40), and 17 (63.0%) were male. All nine CLA-resistant strains had A2143G mutations in 23S rRNA, while none of nine sensitive strains had the mutation. Six of nine strains in group L and six of nine strains in group S had 87th or 91st mutation in gyrA. After comparing sequencing data of strains among the three groups, we identified five mutated positions belonging to four genes related to CLA resistance, and 31 mutated positions belonging to 20 genes related to LVX resistance. Novel genetic mutations were detected for CLA resistance (including fliJ and clpX) and LVX resistance (including fliJ, cheA, hemE, Val360Ile, and HP0568). Missense mutations in fliJ and cheA gene were mainly involved in chemotaxis and flagellar motility to facilitate bacterial escape of antibiotics, while the functions of other novel gene mutations underpinning antibiotic resistance remain to be investigated. Whole genome sequencing detected potential novel genetic mutations conferring resistance of H. pylori to CLA and LVX including fliJ and cheA. Further studies to correlate these findings with treatment outcome should be performed.

Prevalence of Mycoplasma genitalium infection with antimicrobial resistance mutations among gay sex workers in China.

Gay sex workers (GSWs) within the population of men who have sex with men in China are known as money boys (MBs). Limited research has been conducted to investigate the infection rate and antimicrobial resistance of Mycoplasma genitalium (M. genitalium) among GSWs in China. This study aimed to evaluate the status of M. genitalium infection among in GSWs. This study was performed among 349 GSWs who were followed up for four years by internet-based sampling collection. The participants were asked to complete an online questionnaire using a mobile app, and trained interviewers took urethral, anorectal, and saliva swab specimens. STIs, including HIV and M. genitalium, were detected. Detection of resistance-associated mutations (RAMs) to macrolides and fluoroquinolones was performed via Sanger sequencing of the 23S rRNA, parC and gyrA genes. GSWs were enrolled by identifying 10 initial "seeds" from the Blued and WeChat apps. Face-to-face interviews were conducted with 349 GSWs from June 2017 to July 2021. The prevalence of M. genitalium and HIV positivity was 92/349 (26.4%, 95% confidence interval [CI] 21.7-31.0) and 71/349 (20.3%, 95% CI 16.3-24.4), respectively. The proportion of GSWs with M. genitalium infection alone in urethral swabs was 16, and the proportion with symptoms was 2/16 (12.5%). The proportion of GSWs with M. genitalium infection alone in anorectal swabs was 36, and the proportion with symptoms was 3/36 (8.3%). Multivariate regression analysis showed that using new types of drugs in the past 3 months and inconsistent condom usage with clients in the past 30 days were associated with M. genitalium infection. Macrolide resistance within the 23S rRNA gene was detected in 73/88 (83.0%) of the M. genitalium-positive GSWs. Moreover, 79.8% (71/89) of parC and 21.1% (19/90) of gyrA genes had mutations responsible for fluoroquinolone resistance. Three cases had no mutations in any of the three genes, 11 cases had mutations in all three genes, five cases had gyrA and parC gene mutations with no mutation in the 23S rRNA gene, and 42 cases had 23S rRNA and parC gene mutations with no mutation in the gyrA gene. M. genitalium infections in our study displayed a high prevalence and very high levels of macrolide and fluoroquinolone resistance among GSWs in China. Asymptomatic M. genitalium infections are quite common among GSWs. Routine resistance testing of M. genitalium-positive specimens and antimicrobial resistance surveillance are crucial.

Epidemiology and prevalence of mutations associated with resistance to macrolides and fluoroquinolones in Mycoplasma genitalium in a tertiary hospital from Madrid, Spain

Mycoplasma genitalium causes persistent sexually transmitted infections. The aims of this study were to estimate the prevalence of resistances to macrolides and fluoroquinolones in M. genitalium and the sexually transmitted coinfections in patients at Hospital Universitario La Paz (Madrid, Spain). Patients attended between January and October 2021 were studied. Screening for sexually transmitted pathogens and detection of 23S rRNA and parC genes mutations were performed by real-time PCR (Allplex,SeegeneTM). A total of 1,518 females and 1,136 males were studied. The prevalence of M. genitalium was 2.1%. The macrolides resistance rate was 51.8%. The mutations found were A2059G, A2058T and A2058G. The rate of resistance to fluoroquinolones was 17.8% being the G248T mutation (S83I) the most frequent. Seven males had some sexual transmitted coinfection. Although the percentage of M. genitalium infections is low, the high rate of resistance to macrolides makes it necessary to revise the protocols for diagnosis and empirical treatment of sexually transmitted infections. The use of fluoroquinolones is appropriate after screening of macrolide resistance profile.

Mycoplasma genitalium prevalence, antimicrobial resistance-associated mutations, and coinfections with non-viral sexually transmitted infections in high-risk populations in Guatemala, Malta, Morocco, Peru and South Africa, 2019-2021.

The prevalence of Mycoplasma genitalium (MG) and MG antimicrobial resistance (AMR) appear to be high internationally, however, prevalence data remain lacking globally. We evaluated the prevalence of MG and MG AMR-associated mutations in men who have sex with men (MSM) in Malta and Peru and women at-risk for sexually transmitted infections in Guatemala, South Africa, and Morocco; five countries in four WHO regions mostly lacking MG prevalence and AMR data, and estimated MG coinfections with Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and Trichomonas vaginalis (TV). Male urine and anorectal samples, and vaginal samples were tested for MG, CT, NG, and TV (only vaginal samples) using Aptima assays (Hologic). AMR-associated mutations in the MG 23S rRNA gene and parC gene were identified using ResistancePlus MG kit (SpeeDx) or Sanger sequencing. In total, 1,425 MSM and 1,398 women at-risk were recruited. MG was detected in 14.7% of MSM (10.0% in Malta and 20.0% Peru) and in 19.1% of women at-risk (12.4% in Guatemala, 16.0% Morocco, 22.1% South Africa). The prevalence of 23S rRNA and parC mutations among MSM was 68.1 and 29.0% (Malta), and 65.9 and 5.6% (Peru), respectively. Among women at-risk, 23S rRNA and parC mutations were revealed in 4.8 and 0% (Guatemala), 11.6 and 6.7% (Morocco), and 2.4 and 3.7% (South Africa), respectively. CT was the most frequent single coinfection with MG (in 2.6% of MSM and 4.5% of women at-risk), compared to NG + MG found in 1.3 and 1.0%, respectively, and TV + MG detected in 2.8% of women at-risk. In conclusion, MG is prevalent worldwide and enhanced aetiological MG diagnosis, linked to clinical routine detection of 23S rRNA mutations, in symptomatic patients should be implemented, where feasible. Surveillance of MG AMR and treatment outcome would be exceedingly valuable, nationally and internationally. High levels of AMR in MSM support avoiding screening for and treatment of MG in asymptomatic MSM and general population. Ultimately, novel therapeutic antimicrobials and/or strategies, such as resistance-guided sequential therapy, and ideally an effective MG vaccine are essential.

Course of Lower Respiratory Tract Infection in Young People Treated at the Military Hospital of Smolensk Garrison with Detected Mycoplasma pneumoniae Carrying a Macrolide-Resistant Mutation in 23S rRNA Gene.

We evaluated the effect of macrolide-resistant mutations in the Mycoplasma pneumoniae 23S rRNA gene on the severity of lower respiratory tract infections in immunocompetent young adults treated at the Smolensk Military Hospital between 25 October 2017, and 17 November 2021. All analyzed cases represented a non-severe infection of the lower respiratory tract: 44 case histories with community-acquired pneumonia and 20 cases with acute bronchitis. The presence of mutations in the gene 23S rRNA of M. pneumoniae was determined with standard Sanger sequencing. The macrolide-resistant genotype was found in 4/44 (9.1%) of the samples of the patients with pneumonia and in 3/20 (15%) of the samples of the patients with acute bronchitis. The analyzed cases with identified M. pneumoniae carrying a mutation in the 23S rRNA gene did not show any differences in the clinical presentation in terms of disease severity caused by M. pneumoniae with the wild-type (WT) phenotype.

Genomic characterization of molecular markers associated with antimicrobial resistance and virulence of the prevalent Campylobacter coli isolated from retail chicken meat in the United Arab Emirates

Campylobacter is a major cause of gastroenteritis worldwide, with broiler meat accounting for most illnesses. Antimicrobial intervention is recommended in severe cases of campylobacteriosis. The emergence of antimicrobial resistance (AMR) in Campylobacter is a concerning food safety challenge, and monitoring the trends of AMR is vital for a better risk assessment. This study aimed to characterize the phenotypic profiles and molecular markers of AMR and virulence in the prevalent Campylobacter species contaminating chilled chicken carcasses sampled from supermarkets in the United Arab Emirates (UAE). Campylobacter was detected in 90 (28.6%) out of 315 tested samples, and up to five isolates from each were confirmed using multiplex PCR. The species C. coli was detected in 83% (75/90) of the positive samples. Whole-genome sequencing was used to characterize the determinants of AMR and potential virulence genes in 45 non-redundant C. coli isolates. We identified nine resistance genes, including four associated with resistance to aminoglycoside (aph(3′)-III, ant(6)-Ia, aph(2″)-Ib, and aac(6′)-Im), and three associated with Beta-lactam resistance (blaOXA-61, blaOXA-193, and blaOXA-489), and two linked to tetracycline resistance (tet(O/32/O), and tet(O)), as well as point mutations in gyrA (fluoroquinolones resistance), 23S rRNA (macrolides resistance), and rpsL (streptomycin resistance) genes. A mutation in gyrA 2 p.T86I, conferring resistance to fluoroquinolones, was detected in 93% (42/45) of the isolates and showed a perfect match with the phenotype results. The simultaneous presence of blaOXA-61 and blaOXA-193 genes was identified in 86.6% (39/45) of the isolates. In silico analysis identified 7 to 11 virulence factors per each C. coli isolate. Some of these factors were prevalent in all examined strains and were associated with adherence (cadF, and jlpA), colonization and immune evasion (capsule biosynthesis and transport, lipooligosaccharide), and invasion (ciaB). This study provides the first published evidence from the UAE characterizing Campylobacter virulence, antimicrobial resistance genotype, and phenotype analysis from retail chicken. The prevalent C. coli in the UAE retail chicken carries multiple virulence genes and antimicrobial resistance markers and exhibits frequent phenotype resistance to macrolides, quinolones, and tetracyclines. The present investigation adds to the current knowledge on molecular epidemiology and AMR development in non-jejuni Campylobacter species in the Middle East and globally.

Emergence of high-level azithromycin-resistant Neisseria gonorrhoeae causing male urethritis in Johannesburg, South Africa, 2021.

In South Africa, Neisseria gonorrhoeae , which is the predominant cause of male urethritis, is treated syndromically using dual ceftriaxone and azithromycin therapy. We determined antimicrobial susceptibilities of N. gonorrhoeae isolates from urethral discharge specimens, and genetically characterised those with elevated minimum inhibitory concentrations (MICs) for first-line antimicrobials. Routine antimicrobial susceptibility testing (AST) of N. gonorrhoeae isolates included E-test for ceftriaxone, cefixime and gentamicin and agar dilution for azithromycin and spectinomycin. Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR) was performed for isolates with elevated MICs to identify antimicrobial resistance (AMR) determinants, and Neisseria gonorrhoeae Multi-Antigen Sequence Typing (NG-MAST) was used to determine strain relatedness. N. gonorrhoeae was cultured from urethral discharge swab specimens obtained from 196 of 238 (82.4%) men presenting to a primary healthcare facility in Johannesburg in 2021. All viable isolates were susceptible to extended-spectrum cephalosporins. Four isolates had high azithromycin MICs ranging from 32mg/L to >256mg/L and grouped into two novel NG-MAST and NG-STAR groups. Two isolates from Group 1 (NG-MAST ST20366, NG-STAR ST4322) contained mutated mtrR (G45D) and 23S rRNA (A2059G) alleles, while the two isolates from Group 2 (NG-MAST ST20367, NG-STAR ST4323) had different mutations in mtrR (A39T) and 23S rRNA (C2611T). We report the first cases of high-level azithromycin resistance in N. gonorrhoeae from South Africa. Continued AMR surveillance is critical to detect increasing azithromycin resistance prevalence in N. gonorrhoeae , which may justify future modifications to the STI syndromic management guidelines.

Genetic Adaptation and Acquisition of Macrolide Resistance in Haemophilus spp. during Persistent Respiratory Tract Colonization in Chronic Obstructive Pulmonary Disease (COPD) Patients Receiving Long-Term Azithromycin Treatment.

Patients with chronic obstructive pulmonary disease (COPD) benefit from the immunomodulatory effect of azithromycin, but long-term administration may alter colonizing bacteria. Our goal was to identify changes in Haemophilus influenzae and Haemophilus parainfluenzae during azithromycin treatment. Fifteen patients were followed while receiving prolonged azithromycin treatment (Hospital Universitari de Bellvitge, Spain). Four patients (P02, P08, P11, and P13) were persistently colonized by H. influenzae for at least 3 months and two (P04 and P11) by H. parainfluenzae. Isolates from these patients (53 H. influenzae and 18 H. parainfluenzae) were included to identify, by whole-genome sequencing, antimicrobial resistance changes and genetic variation accumulated during persistent colonization. All persistent lineages isolated before treatment were azithromycin-susceptible but developed resistance within the first months, apart from those belonging to P02, who discontinued the treatment. H. influenzae isolates from P08-ST107 acquired mutations in 23S rRNA, and those from P11-ST2480 and P13-ST165 had changes in L4 and L22. In H. parainfluenzae, P04 persistent isolates acquired changes in rlmC, and P11 carried genes encoding MefE/MsrD efflux pumps in an integrative conjugative element, which was also identified in H. influenzae P11-ST147. Other genetic variation occurred in genes associated with cell wall and inorganic ion metabolism. Persistent H. influenzae strains all showed changes in licA and hgpB genes. Other genes (lex1, lic3A, hgpC, and fadL) had variation in multiple lineages. Furthermore, persistent strains showed loss, acquisition, or genetic changes in prophage-associated regions. Long-term azithromycin therapy results in macrolide resistance, as well as genetic changes that likely favor bacterial adaptation during persistent respiratory colonization. IMPORTANCE The immunomodulatory properties of azithromycin reduce the frequency of exacerbations and improve the quality of life of COPD patients. However, long-term administration may alter the respiratory microbiota, such as Haemophilus influenzae, an opportunistic respiratory colonizing bacteria that play an important role in exacerbations. This study contributes to a better understanding of COPD progression by characterizing the clinical evolution of H. influenzae in a cohort of patients with prolonged azithromycin treatment. The emergence of macrolide resistance during the first months, combined with the role of Haemophilus parainfluenzae as a reservoir and source of resistance dissemination, is a cause for concern that may lead to therapeutic failure. Furthermore, genetic variations in cell wall and inorganic ion metabolism coding genes likely favor bacterial adaptation to host selective pressures. Therefore, the bacterial pathoadaptive evolution in these severe COPD patients raise our awareness of the possible spread of macrolide resistance and selection of host-adapted clones.

Helicobacter pylori resistance to clarithromycin and quinolones in patients with dyspepsia in Tuzla Canton, Bosnia and Herzegovina.

Aim To evaluate Helicobacter pylori (H. pylori) resistance to clarithromycin and quinolones in patients with dyspepsia in Tuzla Canton, Bosnia and Herzegovina, a region with no data on clarithromycin or quinolones resistance. Methods A prospective cross-sectional study was conducted at the Department of Gastroenterology and Hepatology at University Clinical Centre Tuzla between January 2021 and June 2022. The study included 99 patients who underwent esophagogastroduodenoscopy (EGDS) due to dyspepsia. In all patients biopsies were taken for rapid urease test (RUT) and histology findings, concomitantly with blood samples for IgG serology. All RUT positive patient samples were tested for clarithromycin and quinolones susceptibility with GenoType HelicoDr, a PCR method which detects point mutations in 23S rRNA and mutations in the gyrA gene. Results Out of 99 dyspeptic patients, 67 (67.7%) were serologically positive to H. pylori, 46 (46.4.%) were RUT positive, and 19 (19.2 %) had a positive histology finding. Antibiotic (AB) resistance was tested in the total of 46/99 (46.4%) patients. Resistance to clarithromycin was detected in 28.26% (13/46), quinolones resistance in 36.96% (17/46) , and resistance to both AB was detected in 8.69% (4/46) tested biopsies. Conclusions Due to high clarithromycin and quinolones resistance rates, we recommend the use of bismuth quadruple or non-bismuth concomitant quadruple therapy for H. pylori eradication in Tuzla Canton, Bosnia and Herzegovina.

Characterization of antibiotic resistance and genotyping of Helicobacter pylori isolates in patients with gastroduodenal disease

Background: In this study, we assessed the status of antibiotic resistance of H. pylori isolates to antimicrobial agents at Hospital of Hue University of Medicine and Pharmacy in Central Vietnam and identified the underlying molecular mechanisms related to tetracycline and amoxicillin resistance. In addition, the cagA, cagE, cagT, vacA, iceA genotypes of H. pylori strains were investigated to predict clinical outcomes. Materials and methods: H. pylori was successfully cultured in 52 patients with different gastrointestinal disorders at the Hospital of Hue University of Medicine and Pharmacy in Central Vietnam. The minimum inhibitory concentrations (MICs) of five antimicrobials; clarithromycin (CLR), metronidazole (MTZ), levofloxacin (LE), amoxicillin (AMX) and tetracycline (TE) were determined by the E-test method. Genetic determinants of AMX and TE resistance were identified with the polymerase chain reaction (PCR), followed by sequencing and gene analysis. Allelic variants of cagA, cagE, cagT, vacA, iceA were identified by the PCR. Results: The resistance to CLR, MTZ, LE, AMX, and TE were 90.4%, 86.5%, 65.4%, 40.4% and 0%, respectively. Multidrug resistance was observed in 88.5% of the isolates investigated. Several known AMX resistance mutations were identified in PBP1A (A369T, V374L, S543R, T556S, N562Y), whereas a known mutation in 16S rRNA (A926G) was detected in strains with higher MIC level (TE MICs of 0.25 and 0.5 mg/L). The cagA, cagE and cagT genotypes were found together in 46 isolates (88.5%), vacAs- region genotype in 51 (98.1%, predominantly vacAs1), vacAmregion genotype in all strains studied (vacAm1 - 51.9%, vacAm2 - 40.4%, vacAm1 and vacAm2 - 7.7%), iceA1 in 22 (42.3%) and iceA2 in 20 (38.5%) of strains. The allelic variant vacAs1m1 was prominent (57.4%), and vacAs1m2 (42.6%). Conclusion: Overall, resistance rates to CLR, MTZ, LE and AMX were high in H. pylori from Central Vietnam, except for TE, which serves as a foundation for developing local guidelines for more effective therapeutic strategies. Neither the single genes nor the combination of genes was significantly helpful in predicting the clinical outcome of H. pylori infection in patients in our study. Key words: H. pylori, antibiotic resistance, genotype.

Evaluation of clarithromycin and levofloxacin resistance of Helicobacter pylori strains isolated from patients at the Hospital for Tropical Diseases, Ho Chi Minh City

Clarithromycin (CLA) and levofloxacin (LFX) have been recommended as the most effective antibiotics for treating Helicobacter pylori infection. However, the increase in H. pylori’s resistance to antibiotics is an alarming and growing challenge. The study aimed to determine the antimicrobial resistance profiles of H. pylori strains isolated from patients at the Hospital for Tropical Diseases and to detect point mutations in 23S ribosomal RNA (23S rRNA) and gyraseA (gyrA) genes. Point mutations in 23S rRNA and gyrA were detected using Sanger sequencing. Antibiotic resistance was tested by the microdilution method. Out of the 45 isolates, 44 (97.8%) were found to be resistant to at least one antibiotic, 38 (84.4%) resistant to metronidazole (MTZ), and all were sensitive to amoxicillin (AMX) and tetracycline (TET). Sixty percent of the isolated strains were resistant to 3-6 antibiotics; among them, multiple drug resistance (MDR) strains presented twenty percent resistance to more than two classes of antibiotics. Eleven strains (24.4%) carried two mutations associated with CLA and LFX but only nine of them were dual resistant to CLA and LFX, and twenty-four (53.3%) carried either CLA or LFX mutation. The point mutations A2143G in the 23S rRNA and N87K (Asn87Lys) in the gyrA were detected in the CLA and LFX resistant strains, respectively. The prevalence of MDR, especially CLA, MTZ, and LFX resistance, in the H. pylori isolates suggests that the use of these antibiotics need to be more considerable and cautious.

Prediction of functional proteins associated with the gut microbiome of an adult population in Lagos State, Nigeria

BackgroundThe human gut microbiome is implicated in health and disease. Yet, its functions in healthy adults from a divergent population like those seen in Africa are quite unclear. We set to reveal the roles of the gut microbiome of a healthy rural and urban Yoruba population of Lagos State. MethodologySociodemographic and clinical data, and fecal samples were obtained from rural (n = 10) and urban (n = 10) participants. fecal DNA extraction was done using ZR Fecal DNA MiniPrep™ D6010 and metagenomic next generation sequencing performed on Illumina Miseq platform. Functional roles prediction and antibiotics resistance genes (ARGs) were done using the Clusters of Orthologous Groups (COG) and Comprehensive Antibiotic Resistance Database (CARD) tools, respectively. The potential link between phenotypes and COG abundance were inferred using principal component analysis (PCA). ResultsWe found 12 (urban) and 11 (rural) COG classes of proteins from the gut microbes. Class E protein which denotes amino acid transport and metabolism was unique to urban respondents and was dominated by 5-enolpyruvylshikimate-3-phosphate synthase (45%) and asparagine synthase (30%), implying a high protein-based diet. Class S was found in both groups. COG families of proteins revealed the presence of exo-beta-1,3-glucanase (31%), pyruvate-decarboxylase, and thiamine pyrophosphate-requiring enzymes (22%) among the rural respondents’ indicative of carbohydrate-rich diets. In addition, uncharacterized protein linked to hypothetical functions was found in both. The COG proteins clustered with female sex, BMI (body mass index) and BP (blood pressure) for urban while for the rural populace, it clustered with height, BMI, and BP. CARD analysis showed ARGs in both groups (urban 90%; rural 10%) and all were Escherichia coli 16S rRNA (rrsB) mutation conferring resistance to streptomycin with gene variant model A523C n/a to aminoglycoside antibiotics. ConclusionThe study defined the classes of proteins from gut microbes of urban and rural respondents and highlighted the presence of uncharacterized proteins linked to hypothetical and unknown functions. Differences seen among the predicted roles appear to be driven by diet, clinical and sociodemographic factors. The higher abundance of ARGs observed in the urban population poses a serious public health concern with regards to the spread of antibiotic resistance.

Emergence and Genomic Characterization of Neisseria gonorrhoeae Isolates with High Levels of Ceftriaxone and Azithromycin Resistance in Guangdong, China, from 2016 to 2019.

Currently, antibiotic resistance (especially ceftriaxone and azithromycin dual resistance) in Neisseria gonorrhoeae is the main obstacle affecting the efficacy of treatment. As analysis of drug sensitivity, molecular features, and dissemination of dual-resistant strains is important for gonococcal prevention and control, MIC, genotyping, and genome analysis were conducted to reveal the molecular characteristics and phylogeny of N. gonorrhoeae isolates. During 2016 to 2019, 5 out of 4,113 strains were defined as dual-resistant clones, with ceftriaxone MICs of 0.25 to ≥1 mg/L and azithromycin MICs of 2 to ≥2,048 mg/L. In particular, two strains with a ceftriaxone MIC above 0.5 mg/L were characterized as penA-60.001 FC428-related clones, and two isolates with a high-level azithromycin MIC above 1,024 mg/L featuring a 23S rRNA mutation were identified. Furthermore, phylogenetic analysis confirmed that the dual-resistant strains were closer to the evolutionary origin of F89 in France, global FC428-related clones, and high-level dual-resistant clones in Australia and the United Kingdom. Dual-resistant strains, including FC428-related clones and high-level azithromycin-resistant clones, have circulated in Guangdong, China. The ability of laboratories to perform real-time drug susceptibility and genetic analyses should be strengthened to monitor the spread of threatening strains. IMPORTANCE Here, we report five sporadic dual-resistant isolates, including FC428-related ceftriaxone-resistant clones with MICs of ≥0.5 mg/L and high-level azithromycin resistance with MICs of ≥1,024 mg/L. This study highlights that dual-resistant clones with the same evolutionary origin as FC428, A2735, and F89 have circulated in Guangdong, China, which suggests that the capacity for antibiotic resistance testing and genome analysis should be strengthened in daily epidemiological surveillance.

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

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

Prevalence of antimicrobial-resistant Cutibacterium isolates and development of multiplex PCR method for Cutibacterium species identification

IntroductionCutibacterium species such as C. acnes, C. avidum, and C. granulosum are known anaerobic skin inhabitants and often cause surgical site infections. These species are genetically similar and are difficult to identify rapidly. In addition, their pathogenicity and antimicrobial resistance remain unknown. In this study, antimicrobial resistance in Cutibacterium isolates was studied and a multiplex PCR method for their identification was developed. MethodsA total of 497 C. acnes, 71 C. avidum, and 25 C. granulosum strains which were isolated from the acne pustule and infectious regions, were used. ResultsThe antimicrobial resistance rates of C. acnes, C. avidum, and C. granulosum strains isolated from patients with acne vulgaris were higher than those of strains isolated from patients with infectious diseases. In particular, macrolide-clindamycin-resistant strains were isolated most frequently from all species. Among the resistant strains, strains with 23S rRNA mutations were the most common in C. acnes (24.3%, 71/292), whereas C. avidum and C. granulosum strains were most frequently found with erm(X). For the first time, a C. granulosum strain carrying pTZC1, which codes erm(50) and tet(W), was isolated from patients with acne vulgaris. Regarding the rapid identification of causative pathogens from infectious regions, three Cutibacterium species were identified with 100% sensitivity and specificity using multiplex PCR method. ConclusionsOur data showed that antimicrobial resistance differed among Cutibacterium species. The multiplex PCR method may contribute to the rapid detection of Cutibacterium species and selection of appropriate antimicrobial agents.

Increases in the Macrolide Resistance of Mycoplasma genitalium and the Emergence of the A2058T Mutation in the 23S rRNA Gene: Clonal Spread?

The management of Mycoplasma genitalium sexually transmitted infection (STI) is hindered by increasing resistance to the recommended antibiotics, macrolides and quinolones, worldwide. In Gipuzkoa (Basque Country, Spain), macrolide and quinolone resistance rates in 2014-2018 were reported as <20% and <10%, respectively. The aims of this study were to compare these rates with those in 2019-2021 and analyse the genetic and epidemiological features of the strains and cases associated with striking changes in the resistance trends. Resistance to macrolides (n = 1019) and quinolones (n = 958) was studied, analysing mutations in 23S rRNA and parC/gyrA genes, respectively. The rate of macrolide resistance increased from 17.3% in 2014-2018 to 32.1% in 2019-2021, as much in the more prevalent A2058/2059G mutations (16.6-27.8%) as in the emergent A2058T mutations (0.5-4.1%) but with differences in the odds ratios and the relative risk increase between A2058T and A2058/2059G mutations. MG191 adhesin and MG309 lipoprotein of the 27 emergent strains detected with A2058T mutations were amplified, sequenced, and typed using phylogenetic and variable number tandem repeat analysis, respectively. Genetic clonal spread was ruled out, but most of the A2058T cases were men who had sex with men (24/27) with a history of STI and antibiotic treatments (19/27). No changes were observed in quinolone resistance trends, but the rate of resistance to both antibiotics rose from 2.9% to 8.3%, especially in cases with A2058T mutations. The genetic characterisation of strains and epidemiological surveillance of cases are needed to detect populations at increased risk of treatment failure in this infection.

Real-Time Polymerase Chain Reaction for the Detection of Helicobacter pylori and Clarithromycin Resistance.

Real-time polymerase chain reaction (RT-PCR) is a fast and simple method for the simultaneous detection of clarithromycin (CLR) resistance and Helicobacter pylori. We evaluated the effectiveness of RT-PCR compared to that of the rapid urease test (RUT) and assessed its value in verifying CLR resistance. A total of 70 specimens with confirmed H. pylori infection in culture were enrolled and analyzed in this prospective study. All specimens were subjected to RT-PCR assay using fluorescence melting peak signals to detect H. pylori infection and CLR resistances caused by either A2142G or A2143G mutations in the 23S ribosomal RNA gene (23S rRNA). The results were compared to those of RUT and antimicrobial susceptibility culturing tests to investigate the efficacy of RT-PCR. Among the 70 specimens analyzed, the positivity rate was 97.1% (68/70) with RT-PCR and 82.9% (58/70) with RUT. CLR resistance (minimum inhibitory concentration >1.0 μg/mL) was confirmed in 18.6% (13/70), and fluorescence melting curve analysis showed that 84.6% (11/13) had point mutations in 23S rRNA. Ten specimens had only A2143G mutation, and one specimen contained both A2142G and A2143G mutations. RT-PCR assay was found to be more efficient than RUT in detecting H. pylori infection and could effectively verify CLR resistance compared to the antimicrobial susceptibility culturing test. Considering the high sensitivity and accessibility of RT-PCR method, it could be used to easily detect CLR-resistant H. pylori, thus helping clinicians select suitable treatment regimen and improve the eradication rate.