Seven-day triple therapy is sufficient to eradicate infection caused by Helicobacter pylori without 23S rRNA point mutation.

ABSTRACT: Tailored therapy based on dual priming oligonucleotide-based polymerase chain reaction (DPO-PCR) can be considered an alternative to overcome the low eradication rate in high clarithromycin-resistance areas. The triple therapy (TT) duration of the tailored approach in most studies was 7 days for patients without point mutation. However, recent western guidelines have recommended a treatment duration of 14 days. The aim of this study was to compare the success rate of 7 and 14 days of TT for eradicating Helicobacter pylori without point mutation, as determined by DPO-PCR.Between Feb 2016 and Feb 2019, medical records of patients who underwent DPO-PCR were reviewed. Patients without point mutation as determined by DPO-PCR were enrolled in this study. The eradication success rate and adverse events were evaluated.A total of 366 patients without A2142G and A2143G point mutation were enrolled. The success rates of 7-day and 14-day TT were 88.4% (168/190) and 85.9% (151/176) by intention to treat analysis (P = .453) and 90.8% (168/185) and 90.4% (151/167) by per-protocol analysis (P = .900), respectively. The adverse event rates showed no significant difference between the 2 groups.In patients without point mutation based on DPO-PCR results, 7-day TT is as effective as 14-day TT. Therefore, 7 days may be considered as a cost-effective treatment duration in Korea.

Macrolide and fluoroquinolone resistance of Mycoplasma genitalium in southern Spain, 2018-2019.

OBJECTIVES: In recent years, resistance in Mycoplasma genitalium (MG) to first-line (azithromycin) and second-line (moxifloxacin) treatment has been increasingly reported worldwide, however, no data regarding the south of Spain are available. METHODS: To determine resistance rates, MG-positive samples collected from June 2018 to June 2019 were analysed by sequencing the 23S rRNA and parC genes. RESULTS: A total of 77 patients (24 men having sex with men (MSM), 30 heterosexual men and 23 women) were included. Resistance-associated mutations against macrolide and fluoroquinolones were found in 36.4% (95% CI 25.7% to 48.1%) and 9.1% (95% CI 3.7% to 17.8%) of the patients, respectively. Being MSM and having had another STI in the last year were significantly associated with macrolide-resistant MG infection, while no associations were found with resistance to fluoroquinolones. CONCLUSIONS: Testing for resistance to first-line and second-line drugs against MG should be recommended for the general population and mandatory for the MSM population. We suggest that empiric azithromycin use for STI management should be avoided.

Empiric Versus Clarithromycin Resistance-Guided Therapy for Helicobacter pylori Based on Polymerase Chain Reaction Results in Patients With Gastric Neoplasms or Gastric Mucosa-Associated Lymphoid Tissue Lymphoma: A Randomized Controlled Trial.

INTRODUCTION: We investigated to compare the effect of empirical therapy vs clarithromycin resistance-guided tailored therapy (tailored therapy) for eradication of Helicobacter pylori. METHODS: In this prospective, single center, open-label randomized controlled trial, we enrolled 72 patients with H. pylori infection from January 2019 through June 2019 in Korea. The patients were randomly assigned to both groups received empirical (n = 36) or tailored therapy (n = 36). Empirical therapy was defined as triple therapy with esomeprazole, amoxicillin, and clarithromycin for 10 days irrespective of clarithromycin resistance. Tailored therapy was triple or quadruple therapy with esomeprazole, metronidazole, tetracycline, and bismuth for 10 days based on genotype markers of resistance determined by gastric biopsy. Resistance-associated mutations in 23S rRNA were confirmed by multiplex polymerase chain reaction. Eradication status was assessed by C-urea breath test, and the primary outcome was eradication rates. RESULTS: H. pylori was eradicated in 27 patients (75.0%), given empirical therapy and 32 patients (88.9%) treated with tailored therapy (P = 0.136) in intention-to-treat analysis. In per protocol analysis, the eradication rate was 97.0% and 81.8% in tailoredvs empirical groups (P = 0.046). Although clarithromycin-resistant H. pylori was eradicated in 3/9 (33.3%) with empirical therapy, it was treated in 11/12 (91.7%) with tailored therapy (P = 0.009). There was no difference in compliance between 2 groups. The rate of adverse events of the tailored group was higher than that of the empirical group (P = 0.036) because quadruple therapy had more side effects than those of triple therapy (P = 0.001). DISCUSSION: Tailored therapy based on polymerase chain reaction is a good alternative to increase eradication rates in a region of high prevalence of clarithromycin resistance (see Visual Abstract, Supplementary Digital Content 1, http://links.lww.com/CTG/A342).

The global prevalence ptxP3 lineage of Bordetella pertussis was rare in young children with the co-purified aPV vaccination: a 5 years retrospective study.

BACKGROUND: The global prevalent ptxP3 strains varies from about 10% to about 50% of circulating B. pertussis population in different areas of China. METHODS: To investigate the difference of vaccination status between different genotypes in the circulating B. pertussis after 10 years of acellular pertussis vaccine (aPV) used in China. The nasopharyngeal swabs and isolates of B. pertussis from these patients were used to perform genotyping of antigen genes. We use antibiotic susceptibility test against erythromycin and sequencing methods for site 2047 of 23S rRNA to determine the resistance status. RESULTS: The ptxP1 allele with erythromycin resistant (ER) B. pertussis infection (total of 449 subjects) consisted of 84.70 to 96.70% from 2012 to 2016 in this study. Vaccinated with co-purified aPV was found in 133(133/403,33.0%), 1(1/9,11.1%) and 2(2/21,9.5%) in ptxP1/fhaB3-ER, ptxP1/fhaB2-ES and ptxP3/fhaB2-ES B. pertussis infected children each, which showed a significant difference (χ2 = 6.87, P = 0.032). CONCLUSIONS: The ptxP3-ES B. pertussis was rare while the ptxP1-ER B. pertussis was steadily increased in Xi'an, China from 2012 to 2016, where co-purified aPV was prevalent used. This pose a hypothesis that the co-purified aPV might protect against ptxP3 strains more efficient, which generated a rare chance for ptxP3 strains to be under the antibiotic pressure and further developed to be erythromycin resistance. A further cohort study and the mechanisms of the additional antigen proteins of co-purified aPV protected against B. pertussis should be consideration.

Prevalence of mutations associated with resistance to macrolides and fluoroquinolones in Mycoplasma genitalium: a systematic review and meta-analysis.

BACKGROUND: Mycoplasma genitalium is now recognised as an important bacterial sexually transmitted infection. We summarised data from studies of mutations associated with macrolide and fluoroquinolone resistance in M genitalium to establish the prevalence of resistance. We also investigated temporal trends in resistance and aimed to establish the association between resistance and geographical location. METHODS: In this systematic review and meta-analysis, we searched PubMed, Embase, and MEDLINE for studies that included data for the prevalence of mutations associated with macrolide and fluoroquinolone resistance in M genitalium published in any language up to Jan 7, 2019. We defined prevalence as the proportion of M genitalium samples positive for key mutations associated with azithromycin resistance (23S rRNA gene, position 2058 or 2059) or moxifloxacin resistance (S83R, S83I, D87N, or D87Y in parC), or both, among all M genitalium samples that were successfully characterised. We used random-effects meta-analyses to calculate summary estimates of prevalence. Subgroup and meta-regression analyses by WHO region and time period were done. This study was registered with PROSPERO, number CRD42016050370. RESULTS: Overall, 59 studies from 21 countries met the inclusion criteria for our study: 57 studies of macrolide resistance (8966 samples), 25 of fluoroquinolone resistance (4003 samples), and 22 of dual resistance to macrolides and fluoroquinolones (3280 samples). The summary prevalence of mutations associated with macrolide resistance among M genitalium samples was 35·5% (95% CI 28·8-42·5); prevalence increased from 10·0% (95% CI 2·6-20·1%) before 2010, to 51·4% (40·3-62·4%) in 2016-17 (p<0·0001). Prevalence of mutations associated with macrolide resistance was significantly greater in samples in the WHO Western Pacific and Americas regions than in those from the WHO European region. The overall prevalence of mutations associated with fluoroquinolone resistance in M genitalium samples was 7·7% (95% CI 4·5-11·4%). Prevalence did not change significantly over time, but was significantly higher in the Western Pacific region than in the European region. Overall, the prevalence of both mutations associated with macrolide resistance and those associated with fluoroquinolone resistance among M genitalium samples was 2·8% (1·3-4·7%). The prevalence of dual resistance did not change significantly over time, and did not vary significantly by geographical region. INTERPRETATION: Global surveillance and measures to optimise the efficacy of treatments-including resistance-guided strategies, new antimicrobials, and antimicrobial combination approaches-are urgently needed to ensure cure in a high proportion of M genitalium infections and to prevent further spread of resistant strains. FUNDING: Australian National Health and Medical Research Council.

Oceanivirga miroungae sp. nov., isolated from oral cavity of northern elephant seal (Mirounga angustirostris).

Two independent strains of a Leptotrichia species (ES3154-GLUT and ES2714_GLU) were isolated from the oral cavity of northern elephant seals (Mirounga angustirostris) that were admitted to The Marine Mammal Centre facilities in California, USA. The strains were isolated from oral swabs by cultivation in PPLO broth supplemented with serum, penicillin and colistin in anaerobic conditions. The strains were Gram-negative, pleomorphic, indole-, oxidase- and catalase-negative, non-spore-forming, non-motile rods/coccobacilli in short chains. The 16S rRNA gene sequence of these strains shared 94.42 % nucleotide similarity with Oceanivirga salmonicida AVG 2115T but demonstrated ≤86.00-92.50 % nucleotide similarity to the 16S rRNA genes of other species of the family Leptotrichiaceae. The genome was sequenced for strain ES3154-GLUT. Average nucleotide identity values between strain ES3154-GLUT and 15 type strain genomes from the family Leptotrichiaceae ranged from 66.74 % vs. Sebaldella termitidis to 73.35 % vs. O. salmonicida. The whole genome phylogeny revealed that the novel species was most closely related to O. salmonicida AVG 2115T. This relationship was also confirmed by nucleotide similarity and multilocus phylogenetic analyses employing various housekeeping genes (partial 23S rRNA, rpoB, rpoC, rpoD, polC, adh, gyrA and gyrB genes). Chemotaxonomic and phenotypical features of strain ES3154-GLUT were in congruence with closely related members of the family Leptotrichiaceae, represented by similar enzyme profiles and fatty acid patterns. MALDI-TOF MS analysis was capable to clearly discriminate strain ES3154-GLUT from all currently described taxa of the family Leptotrichiaceae. Based on these data, we propose a novel species of the genus Oceanivirga, for which the name Oceanivirga miroungae sp. nov. is proposed with the type strain ES3154-GLUT (=DSM 109740T=CCUG 73653T=ATCC TSD-189T=NCTC 14411T) and one representative strain ES2714_GLU. The G+C content is 26.82 %, genome size is 1 356 983 bp.

High treatment failure rate is better explained by resistance gene detection than by minimum inhibitory concentration in patients with urogenital Chlamydia trachomatis infection.

OBJECTIVE: The aim of this study was to investigate the relationships between treatment outcomes of patients with urogenital Chlamydia trachomatis infections and minimum inhibitory concentrations (MICs) and drug resistance genes. METHODS: The clinical data of 92 patients diagnosed with Chlamydia trachomatis (C. trachomatis) infections were collected. Of these patients, 28 received regular treatment with azithromycin and 64 received minocycline. All patients underwent three monthly follow-ups after the completion of treatment. The microdilution method was used for the in vitro susceptibility tests. The acquisition of 23S rRNA mutations and presence of the tet(M) gene were detected by gene amplification and sequencing. RESULTS: The MICs of azithromycin, clarithromycin, erythromycin, tetracycline, doxycycline, and minocycline were comparable for isolates from the treatment failure and treatment success groups. Higher detection rates of 23S rRNA gene mutations and tet(M) were found in the treatment failure group (57.14% and 71.43%, respectively) than in the treatment success group (14.29% and 30.23%, respectively) (p < 0.05). The A2057G, C2452A, and T2611C gene mutations of 23S rRNA were detected in eight clinical isolates from the azithromycin treatment failure group, while the T2611C gene mutation was detected in one clinical strain from the treatment success group. CONCLUSIONS: The detection of resistance genes could better explain the high treatment failure rate than the MIC results in patients with urogenital C. trachomatis infections, highlighting the need for genetic antimicrobial resistance testing in infected patients.

Bacillus safensis JG-B5T affects the fate of selenium by extracellular production of colloidally less stable selenium nanoparticles.

Understanding the impact of microorganisms on the mobility of selenium (Se) is important for predicting the fate of toxic Se in the environment and improving wastewater treatment technologies. The bacteria strain Bacillus safensis JG-B5T, isolated from soil in a uranium mining waste pile, can influence the Se speciation in the environment and engineered systems. However, the mechanism and conditions of this process remain unknown. This study found that the B. safensis JG-B5T is an obligate aerobic microorganism with an ability to reduce 70% of 2.5 mM selenite to produce red spherical biogenic elemental selenium nanoparticles (BioSeNPs). Only extracellular production of BioSeNPs was observed using transmission electron microscopy. The two-chamber reactor experiments, genome analysis and corona proteins identified on BioSeNPs suggested that the selenite reduction process was primarily mediated through membrane-associated proteins, like succinate dehydrogenase. Extracellular presence and low colloidal stability of BioSeNPs as indicated by ζ-potential measurements, render B. safensis JG-B5T an attractive candidate in wastewater treatment as it provides easy way of recovering Se while maintaining low Se discharge. As this microorganism decreases Se mobility, it will affect Se bioavailability in the environment and decreases its toxicity.

Ureaplasma miroungigenitalium sp. nov. isolated from northern elephant seals (Mirounga angustirostris) and Ureaplasma zalophigenitalium sp. nov. isolated from California sea lions (Zalophus californianus).

Novel ureaplasma strains have been isolated from the genital tract of both sexes of northern elephant seals (Mirounga angustirostris; six strains) and California sea lions (Zalophus californianus; five strains) stranded along the Central California coast, USA. These strains were phenotypically and genetically characterized and compared to other seven known Ureaplasma species. All novel ureaplasma strains hydrolysed urea, but did not metabolize arginine, and all were isolated and propagated using PPLO medium supplemented with urea under aerobic, microaerophilic, and anaerobic atmospheric conditions at +35-37 °C. Transmission electron microscopy revealed typical mollicute cellular morphology. Molecular characterization included assessment of the following genetic loci: 16S rRNA, the 16S-23S ITS, 23S rRNA, rpoB, ftsH, tufB, rpoC, fusA and ureC. Complete 16S rRNA gene sequence analysis of these novel Ureaplasma species indicated that they were most closely related to each other with nucleotide identity 99.87 % and ≤93.08 % related to other known Ureaplasma species. The results of nucleotide analysis of the sequenced housekeeping genes revealed 71.68-93.02 % similarity to corresponding genes of other known Ureaplasma species. The multi-locus genetic characterization and the phylogenetic analysis of the 16S rRNA and rpoB genes of these Ureaplasma species clearly demonstrated their novelty and, reflecting their host specificites, the name Ureaplasma miroungigenitalium sp. nov. is proposed for the Ureaplasma species isolated from northern elephant seals, the type strain is ES2783-GENT (=DSM 24842T=ATCC BAA-2460T), and the name Ureaplasma zalophigenitalium sp. nov. is proposed for the Ureaplasma species isolated from California sea lions, the type strain is CSL7644-GENT (=DSM 24843T=ATCC BAA-2262T).

Effects of addition of probiotic and/or bismuth to triple therapy of H. pylori and analysis of genetic variation of 23S rRNA gene between patients with clarithromycin sensitivity and resistance.

The aim of the study was to compare the effects of three treatment regimens for H. pylori in patients sensitive to clarithromycin and analyze the polymorphism of 23S rRNA gene between patients who were sensitive or resistant to clarithromycin in a Chinese Han population. 204 H. pylori sensitive cases and 45 H. pylori resistant Han patients were selected as subjects of the research. All H. pylori sensitive cases were divided into three groups based on their different therapies. The polymerase chain reaction-ligase detection reaction (PCR) was used to identify the genotype at the A2143G of the 23S rRNA gene. SPSS18.0 software was applied to analyze the data statistically. The success rate of H. pylori eradication in the TTP (TT + probiotic) group was higher when compared with the triple therapy (TT) group, and the difference was statistically significant. The incidence of abdominal pain, headache and diarrhea in TTP group was significantly lower than that in the TT group and the TTB (TT+ bismuth) group. Moreover, patients in the TTP group suffered less taste impairment than patients in the other two groups. In addition, there was significant difference in genotype frequency distribution between the clarithromycin-resistant group and the clarithromycin-sensitive group. It was suggested in the results of Chinese Han population that the TTP regimen was significantly superior to the other two regimens in the treatment of clarithromycin-sensitive H.PYLORI infection. In addition, potential genotypic differences between clarithromycin-sensitive and drug-resistant patients provided a theoretical basis for gene therapy in patients with clarithromycin resistance.

Helicobacter pylori eradication rates with concomitant and tailored therapy based on 23S rRNA point mutation: A multicenter randomized controlled trial.

BACKGROUND: We evaluated the efficacy of tailored therapy based on point mutation presence identified with the dual-priming oligonucleotide (DPO)-based multiplex polymerase chain reaction (PCR) method compared with concomitant therapy. MATERIALS AND METHODS: Subjects were randomly assigned concomitant therapy (amoxicillin 1 g, clarithromycin 500 mg, metronidazole 500 mg, and lansoprazole 30 mg twice/day for 14 days) or tailored therapy (amoxicillin 1 g, clarithromycin 500 mg, and lansoprazole 30 mg twice/day for 14 days in point mutation-negative subjects; and amoxicillin 1 g, metronidazole 500 mg, and lansoprazole 30 mg twice/day for 14 days in point mutation-positive subjects). RESULTS: A total of 397 and 352 subjects were included in the intention-to-treat (ITT) and per-protocol (PP) analyses, respectively. Point mutations were identified in 25.9% of the subjects. The overall eradication rate was not significantly different between the groups by ITT (86.2% vs 81.6%, P = .132) and PP analyses (90.2% vs 86.5%, P = .179). There was no significant difference in the eradication rates between the groups in both the point mutation-negative subjects (91.7% vs 87.3%, P = .154) and the point mutation-positive subjects (71.2% vs 64.7%, P = .312). The eradication rates were significantly lower in the point mutation-positive subjects than in the point mutation-negative subjects in both the concomitant and tailored therapy groups. CONCLUSIONS: Tailored therapy based on point mutation presence identified with the DPO-based multiplex PCR method was as effective as concomitant therapy. The eradication rates of both therapy regimens were suboptimal in point mutation-positive subjects.

Helicobacter pylori Mutations Conferring Resistance to Fluoroquinolones and Clarithromycin among Dyspeptic Patients Attending a Tertiary Hospital, Tanzania.

Objectives. Helicobacter pylori (H. pylori) isolates resistant to clarithromycin and quinolones are increasing worldwide. Data regarding the magnitude of H. pylori resistance are limited in developing countries. Here, we report the prevalence of mutations conferring resistance to clarithromycin and fluoroquinolones among dyspeptic patients attending a tertiary hospital, Tanzania. Methods. Between August 2014 and August 2016, patients undergoing upper gastrointestinal endoscopy at the Bugando Medical Centre were enrolled. Biopsies were taken for polymerase chain reaction (PCR) and sequencing to detect mutations conferring resistance to clarithromycin and fluoroquinolones. Results. A total of 208 nonrepetitive biopsies were examined of which 188 (90.4%) tested positive for H. pylori specific 23S rRNA PCR. Clarithromycin resistance mutations were detected in 54/188 (28.7%) of patients tested. The most frequently detected mutation was A2143G (30) followed by A2142G (20). Out of 131 nonrepetitive biopsies tested for fluoroquinolones resistance mutations, 77/131 (58.8%) were positive, with N87I (20) mutation being the most frequently detected mutation followed by A92T mutation which was detected in 16 samples. Conclusion. A significant proportion of dyspeptic patients attending tertiary hospital in Tanzania are infected with H. pylori strains harbouring clarithromycin or fluoroquinolones resistance mutations. Detection of more than 50% of strains with fluoroquinolones resistance mutations makes the H. pylori second line treatment questionable in our setting. There is a need of surveillance of H. pylori resistance patterns in Tanzania to provide data that can guide empirical treatment to reduce associated morbidity of H. pylori infections. The correlation between A92T fluoroquinolone mutation and phenotypic resistance requires further investigations.

Selenium oxyanion bioconcentration in natural freshwater periphyton.

Selenium (Se) enrichment has been demonstrated to vary by several orders of magnitude among species of planktonic algae. This is a substantial source of uncertainty when modelling Se biodynamics in aquatic systems. In addition, Se bioconcentration data are largely lacking for periphytic species of algae, and for multi-species periphyton biofilms, adding to the challenge of modelling Se transfer in periphyton-based food webs. To better predict Se dynamics in periphyton dominated, freshwater ecosystems, the goal of this study was to assess the relative influence of periphyton community composition on the uptake of waterborne Se oxyanions. Naturally grown freshwater periphyton communities, sampled from five different water bodies, were exposed to environmentally relevant concentrations of selenite [Se(IV)] or selenate [Se(VI)] (nominal concentrations of 5 and 25 µg Se L-1) under similar, controlled laboratory conditions for a period of 8 days. Unique periphyton assemblages were derived from the five different field sites, as confirmed by light microscopy and targeted DNA sequencing of the plastid 23S rRNA gene in algae. Selenium accumulation demonstrated a maximum of 23.6-fold difference for Se(IV) enrichment and 2.1-fold difference for Se(VI) enrichment across the periphyton/biofilm assemblages tested. The assemblage from one field site demonstrated both high accumulation of Se(IV) and iron, and was subjected to additional experimentation to elucidate the mechanism(s) of Se accumulation. Selenite accumulation (at nominal concentrations of 5 and 25 µg Se L-1 and mean pH of 7.5 across all treatment replicates) was assessed in both unaltered and heat-killed periphyton, and in periphyton from the same site grown without light to exclude phototrophic organisms. Following an exposure length of 8 days, all periphyton treatments showed similar levels of Se accumulation, indicating that much of the apparent uptake of Se(IV) was due to non-biological processes (i.e., surface adsorption). The results of this study will help reduce uncertainty in the prediction of Se dynamics and food-chain transfer in freshwater environments. Further exploration of the ecological consequences of extracellular adsorption of Se(IV) to periphyton, rather than intracellular absorption, is recommended to further refine predictions related to Se biodynamics in freshwater food webs.

Quinolone and Macrolide-Resistant Campylobacter jejuni in Pediatric Gastroenteritis Patients from Central Iran.

Aims: To determine the prevalence and the antibiotic resistance patterns of Campylobacter jejuni isolated from pediatric diarrhea patients in central Iran. Materials and Methods: Stool specimens (n = 230) were investigated using a modified Gram stain, two specific culture media, and C. jejuni-specific PCR. Antibiotic resistance profiles and relevant resistance genes were determined. Genetic relationships among a selection of the isolates were studied by Fla typing. Results: Out of the 230 diarrhea samples, 48 (20.8%) cases of C. jejuni were identified using modified Gram stain, 45 (19.5%) using the culture media, and 76 (33%) cases were identified using PCR. The highest antibiotic resistance rates were observed in 37 (82.2%) strains against tetracycline, in 32 (71.1%) against ciprofloxacin, and in 31 (68.8%) against erythromycin. Twenty (44.4%) isolates were resistant to ciprofloxacin and erythromycin simultaneously. Genotypic investigations found 36 (97.3%) strains carrying the tet (o) gene, 31 (96.8%) harboring the cmeB gene, 22 (68.7%) strains with the gyrA6 gene, 20 (64.5%) strains containing a 23S rRNA mutation, and 21 (65.6%) strains with the qnrS gene. Fla typing of a random subset of 14 strains revealed 11 different types showing the genomic diversity of the isolates. Strains sharing the same Fla type could be easily distinguished by their resistance gene profile. Conclusions: This is the first study to demonstrate that genetically diverse quinolone-macrolide-resistant C. jejuni is an important cause of gastroenteritis in children from central Iran. Pediatricians should consider these resistance features once the antibiotic prescription is necessary for prevention of possible complications, especially in those under 5 years of age. Of note, most cases of Campylobacter diarrhea are self-limiting and antibiotics should only be prescribed in those cases where severe complications evolve.

Evidence of Recent Genomic Evolution in Gonococcal Strains With Decreased Susceptibility to Cephalosporins or Azithromycin in the United States, 2014-2016.

BACKGROUND: Given the lack of new antimicrobials or a vaccine, understanding the evolutionary dynamics of Neisseria gonorrhoeae is a significant public and global health priority. We investigated the emergence and spread of gonococcal strains with decreased susceptibility to cephalosporins and azithromycin using detailed genomic analyses of gonococcal isolates collected in the United States, 2014-2016. METHODS: We sequenced genomes of 649 isolates collected through the Gonococcal Isolate Surveillance Project. We examined the genetic relatedness of isolates and assessed associations between clades and various genotypic and phenotypic combinations. RESULTS: We identified a large and clonal lineage of strains (MLST ST9363) associated with elevated azithromycin minimum inhibitory concentration (AZIem), characterized by a mosaic mtr locus (C substitution in the mtrR promoter, mosaic mtrR and mtrD). Mutations in 23S rRNA were sporadically distributed among AZIem strains. Another clonal group (MLST ST1901) possessed 7 unique PBP2 patterns, and it shared common mutations in other genes associated with cephalosporin resistance. CONCLUSIONS: Whole-genome sequencing methods can enhance monitoring of antimicrobial resistant gonococcal strains by identifying gonococcal populations containing mutations of concern. These methods could inform the development of point-of-care diagnostic tests designed to determine the specific antibiotic susceptibility profile of a gonococcal infection in a patient.

Chemical probing for examining the structure of modified RNAs and ligand binding to RNA.

Among different RNA modifications, the helix 69 (H69) region of the bacterial ribosomal RNA (rRNA) contains three pseudouridines (Ψs). H69 is functionally important due to its location in the heart of the ribosome. Several structural and functional studies have shown the importance of Ψ modifications in influencing the H69 conformation as well as maintaining key interactions in the ribosome during protein synthesis. Therefore, a need exists to understand the influence of modified nucleosides on conformational dynamics of the ribosome under solution conditions that mimic the cellular environment. In this review on chemical probing, we provide detailed protocols for the use of dimethyl sulfate (DMS) to examine H69 conformational states and the influence of Ψ modifications under varying solution conditions in the context of both ribosomal subunits and full ribosomes. The use of DMS footprinting to study the binding of aminoglycosides to the H69 region of bacterial rRNA as a potential antibiotic target will also be discussed. As highlighted in this work, DMS probing and footprinting are versatile techniques that can be used to gain important insight into RNA local structure and RNA-ligand interactions, respectively.

Guided antibiotic therapy for Mycoplasma genitalium infections: Analysis of mutations associated with resistance to macrolides and fluoroquinolones. / Tratamiento antibiótico dirigido en infecciones por Mycoplasma genitalium: análisis de mutaciones asociadas con resistencia a macrólidos y fluoroquinolonas.

INTRODUCTION: The objective of this study was to analyse the susceptibility of Mycoplasma genitalium to macrolides and fluoroquinolones using molecular techniques. METHODS: Susceptibility to macrolides was tested (Gipuzkoa, 2014-2017) by a rapid probe-based real-time polymerase chain reaction assay (23S rRNA gene) and to fluoroquinolones by sequencing the parC and gyrA genes. RESULTS: Mutations associated with macrolide resistance were detected in 43/263 (16.3%) cases and potential fluoroquinolone resistance in 21/267 (7.9%). Macrolide resistance was more frequent in patients previously treated with azithromycin (76.5% vs 7.4%, P<.001) as well as in those treated with a single 1g dose (31.3%) vs the extended regimen (7%, P<.001). There were 5/245 (2%) cases with mutations probably associated with resistance to both antibiotics. CONCLUSIONS: The technique used for testing Mycoplasma genitalium susceptibility to azithromycin allowed the rapid implementation of resistance-guided antibiotic therapy. Moxifloxacin could be a good option in cases of macrolide resistance.

Punctual mutations in 23S rRNA gene of clarithromycin-resistant Helicobacter pylori in Colombian populations.

AIM: To characterize punctual mutations in 23S rRNA gene of clarithromycin-resistant Helicobacter pylori (H. pylori) and determine their association with therapeutic failure. METHODS: PCR products of 23S rRNA gene V domain of 74 H. pylori isolates; 34 resistant to clarithromycin (29 from a low-risk gastric cancer (GC) population: Tumaco-Colombia, and 5 from a high-risk population: Tuquerres-Colombia) and 40 from a susceptible population (28 from Tumaco and 12 from Túquerres) were sequenced using capillary electrophoresis. The concordance between mutations of V domain 23S rRNA gene of H. pylori and therapeutic failure was determined using the Kappa coefficient and McNemar's test was performed to determine the relationship between H. pylori mutations and clarithromycin resistance. RESULTS: 23S rRNA gene from H. pylori was amplified in 56/74 isolates, of which 25 were resistant to clarithromycin (20 from Tumaco and 5 from Túquerres, respectively). In 17 resistant isolates (13 from Tumaco and 4 from Túquerres) the following mutations were found: A1593T1, A1653G2, C1770T, C1954T1, and G1827C in isolates from Tumaco, and A2144G from Túquerres. The mutations T2183C, A2144G and C2196T in H. pylori isolates resistant to clarithromycin from Colombia are reported for the first time. No association between the H. pylori mutations and in vitro clarithromycin resistance was found. However, therapeutic failure of eradication treatment was associated with mutations of 23S rRNA gene in clarithromycin-resistant H. pylori (κ = 0.71). CONCLUSION: The therapeutic failure of eradication treatment in the two populations from Colombia was associated with mutations of the 23S rRNA gene in clarithromycin-resistant H. pylori.

Azithromycin-resistant Neisseria gonorrhoeae spreading amongst men who have sex with men (MSM) and heterosexuals in New South Wales, Australia, 2017.

Objectives: To identify the genetic basis of resistance as well as to better understand the epidemiology of a recent surge in azithromycin-resistant Neisseria gonorrhoeae in New South Wales, Australia. Methods: Azithromycin-resistant N. gonorrhoeae isolates (n = 118) collected from 107 males, 10 females and 1 transsexual between January and July 2017 were genotyped using a previously described iPLEX method. The results were compared with phenotypic resistance profiles and available patient data. Results: The iPLEX results revealed 10 different N. gonorrhoeae genotypes (designated AZI-G1 to AZI-G10) of which three were responsible for the majority of infections; AZI-G10 (74.6%, 88 isolates; 87 males and 1 transsexual), AZI-G4 (11.0%, 13 isolates; 7 males and 6 females) and AZI-G7 (6.8%, 8 isolates; 7 males and 1 female). The observed resistance was attributable to one of two different azithromycin resistance mechanisms; the 23S rRNA C2611T mutation was identified in 24% of isolates, whereas the majority of resistance (76%) was associated with a meningococcal-type mtrR variant. Additionally, one isolate was found to harbour both the 23S rRNA C2611T mutation and a type XXXIV mosaic penA sequence associated with cephalosporin resistance. Conclusions: These data indicate outbreaks of azithromycin-resistant gonococci amongst networks of MSM and heterosexuals in New South Wales. The results also provide further evidence that azithromycin may soon be an ineffective treatment option for gonococcal infection and highlight the urgent need to explore alternative therapies.

Long-term administration of oral macrolides for acne treatment increases macrolide-resistant Propionibacterium acnes.

Macrolide-resistant Propionibacterium acnes are frequently isolated from patients with acne vulgaris, and the most resistant isolates (>90% resistance) have the 23S rRNA mutation. An increase in resistant P. acnes with this mutation is thought to be caused by the inappropriate use of antimicrobials. Therefore, we studied the mutation frequency of macrolide resistance in P. acnes in vitro. When P. acnes mutants were exposed to clarithromycin after being incubated in broth without antimicrobials, resistant mutants with the 23S rRNA mutation were not isolated. However, the mutants were obtained at the frequency of 10-6 after being pre-incubated with 0.03 µg/mL of antimicrobials. This is the estimated epidermal concentration of clarithromycin after p.o. administration. The resistant mutants had the 23S rRNA mutations A2058G, A2059G and C2611G. When pre-incubated with clarithromycin, C2611G mutants which showed resistance to clarithromycin were obtained 32.1% more often than pre-incubated with clindamycin (P < 0.01). By contrast, when pre-incubated with clindamycin, A2058G mutants, which show high-level resistance to both clarithromycin and clindamycin, were more frequently obtained than pre-incubated with clarithromycin (87.5%, P < 0.01). No difference in the isolation rate of A2059G mutants, which show high-level resistance to macrolides but low-level resistance to clindamycin, was found with either treatment. These results indicate the possibility that long-term use of oral macrolides for acne treatment facilitate the increase of macrolide-resistant P. acnes.