gyrA Mutations in Mycoplasma genitalium and Their Contribution to Moxifloxacin Failure: Time for the Next Generation of Resistance-Guided Therapy.

BACKGROUND: Although single nucleotide polymorphisms (SNPs) in Mycoplasma genitalium parC contribute to fluoroquinolone treatment failure, data are limited for the homologous gene, gyrA. This study investigated the prevalence of gyrA SNPs and their contribution to fluoroquinolone failure. METHODS: Samples from 411 patients (male and female) undergoing treatment for M. genitalium infection (Melbourne Sexual Health Centre, March 2019-February 2020) were analyzed by Sanger sequencing (gyrA and parC). For patients treated with moxifloxacin (n = 194), the association between SNPs and microbiologic treatment outcome was analyzed. RESULTS: The most common parC SNP was G248T/S83I (21.1% of samples), followed by D87N (2.3%). The most common gyrA SNP was G285A/M95I (7.1%). Dual parC/gyrA SNPs were found in 8.6% of cases. One third of infections harboring parC G248T/S83I SNP had a concurrent SNP in gyrA conferring M95I. SNPs in gyrA cooccurred with parC S83I variations. Treatment failure was higher in patients with parC S83I/gyrA dual SNPs when compared with infections with single S83I SNP alone from analysis of (1) 194 cases in this study (81.2% vs 45.8%, P = .047), and (2) pooled analysis of a larger population of 535 cases (80.6% vs 43.2%; P = .0027), indicating a strong additive effect. CONCLUSIONS: Compared with parC S83I SNP alone, M. genitalium infections with dual mutations affecting parC/gyrA had twice the likelihood of failing moxifloxacin. Although antimicrobial resistance varies by region globally, these data indicate that gyrA should be considered as a target for future resistance assays in Australasia. We propose a strategy for the next generation of resistance-guided therapy incorporating parC and gyrA testing.

Combination Therapy for Mycoplasma genitalium, and New Insights Into the Utility of parC Mutant Detection to Improve Cure.

BACKGROUND: Mycoplasma genitalium (MG) infection is challenging to cure because of rising antimicrobial resistance and limited treatment options. METHODS: This was a prospective evaluation of the efficacy and tolerability of resistance-guided combination antimicrobial therapy for MG treatment at Melbourne Sexual Health Centre (August 2019-December 2020). All patients received 7 days of doxycycline before combination therapy based on the macrolide-resistant profile. Macrolide-susceptible infections received combination doxycycline + azithromycin (1 g, day 1; 500 mg, days 2-4) and macrolide-resistant infections combination doxycycline + moxifloxacin (400 mg daily for 7 days). Adherence and adverse effects were recorded at test-of-cure, recommended 14-28 days after antimicrobial completion. Sequencing was performed to determine the prevalence of single nucleotide polymorphisms (SNPs) in the parC gene and their association with moxifloxacin treatment outcomes in macrolide-resistant infections. RESULTS: Of 100 patients with macrolide-susceptible MG treated with doxycycline + azithromycin, 93 were cured (93.0%; 95% confidence interval [CI], 86.1-97.1). Of 247 patients with macrolide-resistant MG receiving doxycycline + moxifloxacin, 210 were cured (85.0%; 95% CI, 80.0-89.2). parC sequencing was available for 164 (66%) macrolide-resistant infections; 29% had SNPs at parC S83 or D87 (23% S83I). The absence of SNPs at parC S83/D87 was associated with 98.3% cure (95% CI, 93.9-99.8) following doxycycline + moxifloxacin. The presence of the parC S83I-SNP was associated with failure in 62.5% (95% CI, 45.8-77.3). Side effects were common (40%-46%) and predominantly mild and gastrointestinal. CONCLUSIONS: Combination doxycycline + azithromycin achieved high cure for macrolide-susceptible infections. However, in the context of a high prevalence of the parC S83I mutation (23%) in macrolide-resistant infections, doxycycline + moxifloxacin cured only 85%. Infections that were wild-type for S83/D87 experienced high cure following doxycycline + moxifloxacin, supporting the use of a parC-resistance/susceptibility testing strategy in clinical care.

The effect of probiotic supplementation on the gut microbiota of preterm infants.

Introduction. Probiotic supplementation of preterm infants may prevent serious morbidities associated with prematurity.Aim. To investigate the impact of probiotic supplementation on the gut microbiota and determine factors associated with detection of probiotic species in the infant gut.Hypothesis/Gap Statement. Probiotic supplementation increases the long-term colonization of probiotic species in the gut of preterm infants.Methodology. Longitudinal stool samples were collected from a cohort of very preterm infants participating in a blinded randomized controlled trial investigating the impact of probiotic supplementation (containing Bifidobacterium longum subsp. infantis BB-02, Bifidobacterium animalis subsp. lactis BB-12 and Streptococcus thermophilus TH-4) for prevention of late-onset sepsis. The presence of B. longum subsp. infantis, B. animalis subsp. lactis and S. thermophilus was determined for up to 23 months after supplementation ended using real-time PCR. Logistic regression was used to investigate the impact of probiotic supplementation on the presence of each species.Results. Detection of B. longum subsp. infantis [odds ratio (OR): 53.1; 95 % confidence interval (CI): 35.6-79.1; P < 0.001], B. animalis subsp. lactis (OR: 89.1; 95 % CI: 59.0-134.5; P < 0.001) and S. thermophilus (OR: 5.66; 95 % CI: 4.35-7.37; P < 0.001) was increased during the supplementation period in infants receiving probiotic supplementation. Post-supplementation, probiotic-supplemented infants had increased detection of B. longum subsp. infantis (OR: 2.53; 95 % CI: 1.64-3.90; P < 0.001) and B. animalis subsp. lactis (OR: 1.59; 95 % CI: 1.05-2.41; P=0.030). Commencing probiotic supplementation before 5 days from birth was associated with increased detection of the probiotic species over the study period (B. longum subsp. infantis, OR: 1.20; B. animalis subsp. lactis, OR: 1.28; S. thermophilus, OR: 1.45).Conclusion. Probiotic supplementation with B. longum subsp. infantis BB-02, B. animalis subsp. lactis BB-12 and S. thermophilus TH-4 enhances the presence of probiotic species in the gut microbiota of very preterm infants during and after supplementation. Commencing probiotic supplementation shortly after birth may be important for improving the long-term colonization of probiotic species.

Antiseptic mouthwash for gonorrhoea prevention (OMEGA): a randomised, double-blind, parallel-group, multicentre trial.

BACKGROUND: To address the increasing incidence of gonorrhoea and antimicrobial resistance, we compared the efficacy of Listerine and Biotène mouthwashes for preventing gonorrhoea among men who have sex with men (MSM). METHODS: The OMEGA trial was a multicentre, parallel-group, double-blind randomised controlled trial among MSM, done at three urban sexual health clinics and one general practice clinic in Australia. Men were eligible if they were diagnosed with oropharyngeal gonorrhoea by nucleic acid amplification test (NAAT) in the previous 30 days or were aged 16-24 years. They were randomly assigned to receive Listerine (intervention) or Biotène (control) via a computer-generated sequence (1:1 ratio, block size of four). Participants, clinicians, data collectors, data analysts, and outcome adjudicators were masked to the interventions after assignment. Participants were instructed to rinse and gargle with 20 mL of mouthwash for 60 s at least once daily for 12 weeks. Oropharyngeal swabs were collected by research nurses every 6 weeks, and participants provided saliva samples every 3 weeks, to be tested for Neisseria gonorrhoeae with NAAT and quantitative PCR. The primary outcome was proportion of MSM diagnosed with oropharyngeal N gonorrhoeae infection at any point over the 12-week period, defined as a positive result for either oropharyngeal swabs or saliva samples by NAAT, and the cumulative incidence of oropharyngeal gonorrhoea at the week 12 visit. A modified intention-to-treat analysis for the primary outcome was done that included men who provided at least one follow-up specimen over the 12-week study period. The trial was registered on the Australian and New Zealand Clinical Trials Registry (ACTRN12616000247471). FINDINGS: Between March 30, 2016, and Oct 26, 2018, 786 MSM were screened and 256 were excluded. 264 MSM were randomly assigned to the Biotène group and 266 to the Listerine group. The analysis population included 227 (86%) men in the Biotène group and 219 (82%) in the Listerine group. Oropharyngeal gonorrhoea was detected in ten (4%) of 227 of MSM in the Biotène group and in 15 (7%) of 219 in the Listerine group (adjusted risk difference 2·5%, 95% CI -1·8 to 6·8). The cumulative incidence of oropharyngeal gonorrhoea at the week 12 visit did not differ between the two mouthwash groups (adjusted risk difference 3·1%, 95% CI -1·4 to 7·7). INTERPRETATION: Listerine did not reduce the incidence of oropharyngeal gonorrhoea compared with Biotène. However, previous research suggests that mouthwash might reduce the infectivity of oropharyngeal gonorrhoea; therefore, further studies of mouthwash examining its inhibitory effect on N gonorrhoeae are warranted to determine if it has a potential role for the prevention of transmission. FUNDING: Australian National Health and Medical Research Council.

Moxifloxacin and Sitafloxacin Treatment Failure in Mycoplasma genitalium Infection: Association with parC Mutation G248T (S83I) and Concurrent gyrA Mutations.

BACKGROUND: The basis of fluoroquinolone treatment failure for Mycoplasma genitalium is poorly understood. METHODS: To identify mutations associated with failure we sequenced key regions of the M. genitalium parC and gyrA genes for patients undergoing sequential therapy with doxycycline-moxifloxacin (201 patients, including 21 with failure) or doxycycline-sitafloxacin (126 patients, including 13 with failure). RESULTS: The parC G248T/S83I mutation was more common among patients with failed sequential doxycycline-moxifloxacin (present in 76.2% of failures vs 7.8% cures, P < .001) or doxycycline-sitafloxacin (50% vs 16.8%, respectively; P = .01) treatment. Doxycycline-sitafloxacin was more efficacious than doxycycline-moxifloxacin against infections carrying the parC mutation conferring S83I amino acid change. Treatment was more likely to fail in these infections if they had a concurrent gyrA mutation (M95I or D99N) (P = .07 for doxycycline-moxifloxacin group and P = .009 for doxycycline-sitafloxacin group), suggesting an additive effect. CONCLUSIONS: This study indicates that parC G248T/S83I mutations contribute to failure of moxifloxacin and sitafloxacin, and the findings will inform the development of quinolone resistance assays needed to ensure optimal selection of antimicrobials for M. genitalium.

Gut microbiota of preterm infants supplemented with probiotics: sub-study of the ProPrems trial.

BACKGROUND: The ProPrems trial, a multi-center, double-blind, placebo-controlled randomized trial, previously reported a 54% reduction in necrotizing enterocolitis (NEC) of Bell stage 2 or more from 4.4 to 2.0% in 1099 infants born before 32 completed weeks' gestation and weighing < 1500 g, receiving probiotic supplementation (with Bifidobacterium longum subsp. infantis BB-02, Streptococcus thermophilus TH-4 and Bifidobacterium animalis subsp. lactis BB-12). This sub-study investigated the effect of probiotic supplementation on the gut microbiota in a cohort of very preterm infants in ProPrems. RESULTS: Bifidobacterium was found in higher abundance in infants who received the probiotics (AOR 17.22; 95% CI, 3.49-84.99, p < 0.001) as compared to the placebo group, and Enterococcus was reduced in infants receiving the probiotic during the supplementation period (AOR 0.27; 95% CI, 0.09-0.82, p = 0.02). CONCLUSION: Probiotic supplementation with BB-02, TH-4 and BB-12 from soon after birth increased the abundance of Bifidobacterium in the gut microbiota of very preterm infants. Increased abundance of Bifidobacterium soon after birth may be associated with reducing the risk of NEC in very preterm infants.

Leptospira interrogans requires a functional heme oxygenase to scavenge iron from hemoglobin.

The transposon TnSC189 was used to construct a mutant in the putative heme oxygenase gene hemO (LB186) of Leptospira interrogans. Unlike its parent strain, the mutant grew poorly in medium in which hemoglobin was the sole iron source. The putative heme oxygenase was over expressed in a His-tagged form, purified and was demonstrated to degrade heme in vitro. Unexpectedly, it was also found that the L. interrogans growth rate was significantly increased when medium was supplemented with hemoglobin, but only if ferrous iron sources were absent. This result was mirrored in the expression of some iron-related genes and suggests the presence of regulatory mechanisms detecting Fe2+ and hemoglobin. This is the first demonstration of a functional heme oxygenase from a spirochete.