A novel murine model mimicking male genital Neisseria species infection using Neisseria musculi†.

With ~78 million cases yearly, the sexually transmitted bacterium Neisseria gonorrhoeae is an urgent threat to global public health due to continued emergence of antimicrobial resistance. In the male reproductive tract, untreated infections may cause permanent damage, poor sperm quality, and subsequently subfertility. Currently, few animal models exist for N. gonorrhoeae infection, which has strict human tropism, and available models have limited translatability to human disease. The absence of appropriate models inhibits the development of vital new diagnostics and treatments. However, the discovery of Neisseria musculi, a mouse oral cavity bacterium, offers much promise. This bacterium has already been used to develop an oral Neisseria infection model, but the feasibility of establishing urogenital gonococcal models is unexplored. We inoculated mice via the intrapenile route with N. musculi. We assessed bacterial burden throughout the male reproductive tract, the systemic and tissue-specific immune response 2-weeks postinfection, and the effect of infection on sperm health. Neisseria musculi was found in penis (2/5) and vas deferens (3/5) tissues. Infection altered immune cell counts: CD19+ (spleen, lymph node, penis), F4/80+ (spleen, lymph node, epididymus), and Gr1+ (penis) compared with noninfected mice. This culminated in sperm from infected mice having poor viability, motility, and morphology. We hypothesize that in the absence of testis infection, infection and inflammation in other reproductive is sufficient to damage sperm quality. Many results herein are consistent with outcomes of gonorrhoea infection, indicating the potential of this model as a tool for enhancing the understanding of Neisseria infections of the human male reproductive tract.

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.

Decreased Neisseria gonorrhoeae genotypic diversity following COVID-19 restrictions in Queensland, Australia 2020.

We investigated the potential effects of COVID-19 public health restrictions on the prevalence and distribution of Neisseria gonorrhoeae (NG) genotypes in our Queensland isolate population in the first half of the year 2020. A total of 763 NG isolates were genotyped to examine gonococcal strain distribution and prevalence for the first 6 months of 2020, with 1 January 2020 to 31 March 2020 classified as 'pre' COVID-19 restrictions (n = 463) and 1 April 2020 to 30 June 2020 classified as 'post' COVID-19 restrictions (n = 300). Genotypes most prevalent 'pre' restrictions remained proportionally high 'post' restrictions, with some significantly increasing 'post' restrictions. However, genotype diversity was significantly reduced 'post' restrictions. Overall, it seems public health restrictions (9-10 weeks) were not sufficient to affect rates of infection or reduce the prevalence of well-established genotypes in our population, potentially due to reduced access to services or health-seeking behaviours.

Mycoplasma hominis and Ureaplasma urealyticum infections in the immediate post-lung transplant period: A case series and literature review.

Mycoplasma hominis and Ureaplasma species infections in the post-transplant setting are believed to be donor-derived and can be associated with poor outcomes. Difficulty in culturing and identifying these organisms is a significant barrier to diagnosis and early intervention. Tetracyclines, macrolides and fluoroquinolones are the mainstay treatments to cure these infections; however, there are increasing reports of antibiotic resistance. In this case series, we report our single-centre experience with M. hominis and U. urealyticum infection after lung transplantation (9 recipients, all men, mean age 56 years). Delayed diagnosis was common. Young donor age (mean age 23 yrs) and high-risk donor social history (67%) were repeatedly noted in these cases, and all infections were associated with significant morbidity (anastomosis and sternal wound infection, empyema, mediastinitis, pericarditis). Two patients died; with one directly related to Ureaplasma urealyticum infection. In conclusion post lung transplant M. hominis, and U. urealyticum infections are challenging and carry high morbidity. More prospective studies are required to assess the true prevalence, full spectrum of complications and utility of molecular diagnostics to aid early diagnosis and identify antibiotic susceptibility of Mycoplasma and Ureaplasma infections in the post-lung transplant setting.

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.

parC Variants in Mycoplasma genitalium: Trends over Time and Association with Moxifloxacin Failure.

Prevalence, trends, and treatment outcome estimates were generated for parC variants in macrolide-resistant Mycoplasma genitalium. Among 539 cases, the most common amino acid change was S83I, which increased from 13% in 2012 to 2013, to 23% in 2019 to 2020 (Ptrend = 0.046). From 381 moxifloxacin treatments, failure occurred in 58.7% (95% confidence interval [CI], 46.7 to 69.9) of cases with S83I. Other changes affecting S83 or D87 were uncommon and minor contributors to failure. The absence of S83I was highly predictive of moxifloxacin cure (96.4%; 95% CI, 93.7 to 98.2), highlighting diagnostic potential.

Novel probe-based melting curve assays for the characterization of fluoroquinolone resistance in Mycoplasma genitalium.

BACKGROUND: Mycoplasma genitalium infection is a sexually transmitted infection that has rapidly become resistant to mainstay treatments. While individualized treatment approaches have been recommended and adopted for macrolides, individualized therapy for fluoroquinolones has not yet been explored, due to a lack of commercial molecular assays and a lack of confidence in specific mutations associated with resistance. In another recent study, we defined a clear role and diagnostic utility in focusing on the absence of resistance mutations to inform microbial cure with fluoroquinolone antimicrobials. METHODS: We developed two proof-of-concept molecular tests that focus on detection of M. genitalium and characterization of WT parC sequences that are strongly linked to fluoroquinolone susceptibility. RESULTS: We screened a total of 227 M. genitalium-positive samples using novel molecular beacon and dual hybridization probe assays. These assays were able to detect M. genitalium and characterize fluoroquinolone susceptibility in 143/227 (63%) samples, based on clear differences in melting peak temperatures. The results of these molecular assays were in 100% agreement with 'gold standard' Sanger sequencing. Additionally, WT parC sequences were readily distinguished from M. genitalium samples harbouring parC mutations of known or suspected clinical significance. The ability of the assays to successfully characterize fluoroquinolone susceptibility and resistance was reduced in low M. genitalium load samples. CONCLUSIONS: These proof-of-concept assays have considerable potential to improve individualized treatment approaches and rationalize tests of cure for M. genitalium infection. The ability to initiate individualized treatment in up to two-thirds of cases will enhance antimicrobial stewardship for this challenging pathogen.

In-Orchard Population Dynamics of Erwinia amylovora on Apple Flower Stigmas.

Populations of the fire blight pathogen Erwinia amylovora Ea110 on apple flower stigmas were tracked over the course of apple bloom in field studies conducted between 2016 and 2019. In 18 of 23 experiments, flower stigmas inoculated on the first day of opening were found to harbor large (106 to 107 cells per flower) populations of E. amylovora when assessed 3 to 5 days postinoculation. However, populations inoculated on stigmas of flowers that were already open for 3 days did not reach 106 cells per flower, and populations inoculated on stigmas of flowers that were already open for 5 days never exceeded 104 cells per flower. During this study, ≥10-fold increases in E. amylovora stigma populations in a 24-h time period (termed population surges) were observed on 34.8, 20.0, and 4.0% of possible days on 1-, 3-, and 5-day-open flowers, respectively. Population surges occurred on days with average temperatures as high as 24.5 and as low as 6.1°C. Experiments incorporating more frequent sampling during days and overnight revealed that many population surges occurred between 10:00 p.m. and 2:00 a.m. A Pearson's correlation analysis of weather parameters occurring during surge events indicated that population surges were significantly associated with situations in which overnight temperatures increased or remained constant, in which wind speed decreased, and in which relative humidity increased. This study refines our knowledge of E. amylovora population dynamics and further indicates that E. amylovora is able to infect flowers during exposure to colder field temperatures than previously reported.

Mycoplasma genitalium: enhanced management using expanded resistance-guided treatment strategies.

Mycoplasma genitalium is an emerging sexually transmitted bacterium that is gaining attention because of the impact escalating antimicrobial resistance (AMR) is having on patient management. Of additional concern is that increased availability of testing appears to be resulting in screening practices that are not supported by clinical guidelines. This results in increasing numbers of asymptomatic M. genitalium infections being identified, which when combined with AMR issues, creates significant challenges for patients and clinicians. Rapidly rising levels of AMR, coupled with limited alternative treatment options, means patients can enter cycles of complex antimicrobial regimens that may cause more harm than the infection itself. In this review, we discuss the emergence of AMR and the implication for treatment practices, highlight the recommendations for testing but not screening for M. genitalium , and discuss expansion of individualised treatment strategies, to curb the emergence of resistance and improve outcomes for patients. We also provide suggestions for future research on the transmission and spread of resistance, to enhance global surveillance of this antimicrobial resistant pathogen and inform the revision of local and international treatment strategies.

Mycoplasma genitalium infections can comprise a mixture of both fluoroquinolone-susceptible and fluoroquinolone-resistant strains.

BACKGROUND: Mycoplasma genitalium was recently added to the CDC's antimicrobial resistance threats 'watch list', as it has rapidly become resistant to mainstay treatments. In Australia, treatment failure with fluoroquinolones remain commonplace, even when Sanger sequencing fails to identify evidence of resistance mutations. METHODS: Suspecting that Sanger sequencing may miss low-load mixed infections, we applied three additional PCR-based approaches (allele-specific primer-based PCR, probe-based PCR and amplicon deep sequencing) to detect mutations associated with fluoroquinolone susceptibility/resistance. We focused on resistance mutations at amino acid positions 83 and 87 of parC, as these were previously shown to be common in Australia. RESULTS: Our results showed evidence of mixtures of fluoroquinolone-susceptible and -resistant strains in up to 27/423 samples (6.4%). These included 1 sample that was indicated to be mixed by Sanger sequencing and all three additional PCR methods, 6 samples detected by two or more of the additional PCRs but not by Sanger sequencing and finally 20 samples that were detected by only one of the additional PCR methods. A key question was whether Sanger sequencing failed to detect fluoroquinolone resistance in any samples; overall, we observed that Sanger sequencing failed to detect fluoroquinolone resistance in up to 3.8% (16/423) of samples. CONCLUSIONS: The presence of mixed susceptibility infections may have important implications for clinical patient management and stresses the need for appropriate detection of resistance and selection of antimicrobials to ensure appropriate treatment of M. genitalium infections.

Impaired Insulin Profiles Following a Single Night of Sleep Restriction: The Impact of Acute Sprint Interval Exercise.

Experimental sleep restriction (SR) has demonstrated reduced insulin sensitivity in healthy individuals. Exercise is well-known to be beneficial for metabolic health. A single bout of exercise has the capacity to increase insulin sensitivity for up to 2 days. Therefore, the current study aimed to determine if sprint interval exercise could attenuate the impairment in insulin sensitivity after one night of SR in healthy males. Nineteen males were recruited for this randomized crossover study which consisted of four conditions-control, SR, control plus exercise, and sleep restriction plus exercise. Time in bed was 8 hr (2300-0700) in the control conditions and 4 hr (0300-0700) in the SR conditions. Conditions were separated by a 1-week entraining period. Participants slept at home, and compliance was assessed using wrist actigraphy. Following the night of experimental sleep, participants either conducted sprint interval exercise or rested for the equivalent duration. An oral glucose tolerance test was then conducted. Blood samples were obtained at regular intervals for measurement of glucose and insulin. Insulin concentrations were higher in SR than control (p = .022). Late-phase insulin area under the curve was significantly lower in sleep restriction plus exercise than SR (862 ± 589 and 1,267 ± 558; p = .004). Glucose area under the curve was not different between conditions (p = .207). These findings suggest that exercise improves the late postprandial response following a single night of SR.

Response to Daly-Smith et al.'s commentary on 'The Daily Mile makes primary school children more active, less sedentary and improves their fitness and body composition: a quasi-experimental pilot study'.

We thank Daly-Smith et al. for taking the time to read the results of our pilot research study, describing it as an important and welcome contribution. Nonetheless, the authors argue six points against our conclusion. We contend that we addressed three of these points in our original discussion and disagree with their remaining points. Overall, their Commentary adds little to the topic of research into the Daily Mile™ that we had not already raised in our discussion. Additionally, they attribute statements to us that we did not make and ignore the raising of key issues in our original article. Given this, we stand by our original peer-reviewed conclusion that introducing the Daily Mile™ to the primary school day appears to be an effective intervention for increasing levels of moderate to vigorous physical activity, reducing sedentary time, increasing physical fitness and improving body composition, and that these findings have relevance for teachers, policy-makers, public health practitioners and health researchers.

False-negative Chlamydia polymerase chain reaction result caused by a cryptic plasmid-deficient Chlamydia trachomatis strain in Australia.

Background The 7.5-kb chlamydial cryptic plasmid remains a widely used sequence target for Chlamydia trachomatis nucleic acid amplification tests, but sequence variation in this plasmid, particularly a previously reported 377-bp deletion, can cause false-negative results. Here we report the presence in Australia of a C. trachomatis strain lacking the cryptic plasmid. METHODS: A rectal swab from a male in his 50s provided a positive result for C. trachomatis using the Roche Cobas 4800 test, but a negative result in our confirmatory in-house polymerase chain reaction (PCR) method targeting the chlamydial cryptic plasmid. This result was unexpected given our in-house PCR assay targeted a region of sequence outside the recognised 377-bp deletion. To further investigate this discrepancy, the sample was retested using a second in-house PCR targeting a chromosomal (ompA) gene as well as six primer sets flanking various regions of the cryptic plasmid. RESULTS: The sample provided positive results in the second in-house method, confirming the presence of C. trachomatis DNA. All other primer sets targeting the cryptic plasmid failed to amplify, indicating a lack of the chlamydial cryptic plasmid in this sample. CONCLUSIONS: The recognition of a plasmid-deficient strain of C. trachomatis within Australia highlights further limitations of using the chlamydial cryptic plasmid for C. trachomatis diagnostics and re-emphasises the benefits of using multitarget assays to avoid false-negative results.

The Human Ureaplasma Species as Causative Agents of Chorioamnionitis.

The human Ureaplasma species are the most frequently isolated microorganisms from the amniotic fluid and placentae of women who deliver preterm and are also associated with spontaneous abortions or miscarriages, neonatal respiratory diseases, and chorioamnionitis. Despite the fact that these microorganisms have been habitually found within placentae of pregnancies with chorioamnionitis, the role of Ureaplasma species as a causative agent has not been satisfactorily explained. There is also controversy surrounding their role in disease, particularly as not all women infected with Ureaplasma spp. develop chorioamnionitis. In this review, we provide evidence that Ureaplasma spp. are associated with diseases of pregnancy and discuss recent findings which demonstrate that Ureaplasma spp. are associated with chorioamnionitis, regardless of gestational age at the time of delivery. Here, we also discuss the proposed major virulence factors of Ureaplasma spp., with a focus on the multiple-banded antigen (MBA), which may facilitate modulation/alteration of the host immune response and potentially explain why only subpopulations of infected women experience adverse pregnancy outcomes. The information presented within this review confirms that Ureaplasma spp. are not simply "innocent bystanders" in disease and highlights that these microorganisms are an often underestimated pathogen of pregnancy.

The Daily Mile makes primary school children more active, less sedentary and improves their fitness and body composition: a quasi-experimental pilot study.

BACKGROUND: The Daily Mile is a physical activity programme made popular by a school in Stirling, Scotland. It is promoted by the Scottish Government and is growing in popularity nationally and internationally. The aim is that each day, during class time, pupils run or walk outside for 15 min (~1 mile) at a self-selected pace. It is anecdotally reported to have a number of physiological benefits including increased physical activity, reduced sedentary behaviour, increased fitness and improved body composition. This study aimed to investigate these reports. METHODS: We conducted a quasi-experimental repeated measures pilot study in two primary schools in the Stirling Council area: one school with, and one without, intention to introduce the Daily Mile. Pupils at the control school followed their usual curriculum. Of the 504 children attending the schools, 391 children in primary classes 1-7 (age 4-12 years) at the baseline assessment took part. The follow-up assessment was in the same academic year. Outcomes were accelerometer-assessed average daily moderate to vigorous intensity physical activity (MVPA) and average daily sedentary behaviour, 20-m shuttle run fitness test performance and adiposity assessed by the sum of skinfolds at four sites. Valid data at both time points were collected for 118, 118, 357 and 327 children, respectively, for each outcome. RESULTS: After correction for age and gender, significant improvements were observed in the intervention school relative to the control school for MVPA, sedentary time, fitness and body composition. For MVPA, a relative increase of 9.1 min per day (95% confidence interval or 95%CI 5.1-13.2 min, standardised mean difference SMD = 0.407, p = 0.027) was observed. For sedentary time, there was a relative decrease of 18.2 min per day (10.7-25.7 min, SMD = 0.437, p = 0.017). For the shuttle run, there was a relative increase of 39.1 m (21.9-56.3, SMD = 0.236, p = 0.037). For the skinfolds, there was a relative decrease of 1.4 mm (0.8-2.0 mm, SMD = 0.246, p = 0.036). Similar results were obtained when a correction for socioeconomic groupings was included. CONCLUSIONS: The findings show that in primary school children, the Daily Mile intervention is effective at increasing levels of MVPA, reducing sedentary time, increasing physical fitness and improving body composition. These findings have relevance for teachers, policymakers, public health practitioners, and health researchers.

A retrospective pilot study to determine whether the reproductive tract microbiota differs between women with a history of infertility and fertile women.

BACKGROUND: We know very little about the microbiota inhabiting the upper female reproductive tract and how it impacts on fertility. AIMS: This pilot study aimed to examine the vaginal, cervical and endometrial microbiota for women with a history of infertility compared to women with a history of fertility. MATERIALS AND METHODS: Using a retrospective case-control study design, women were recruited for collection of vaginal, cervical and endometrial samples. The microbiota composition was analysed by 16S ribosomal RNA (rRNA) gene amplification and endometrial expression of selected human genes by quantitative reverse transcription polymerase chain reaction. RESULTS: Sixty-five specimens from the reproductive tract of 31 women were successfully analysed using 16S rRNA gene amplicon sequencing (16 controls and 15 cases). The dominant microbial community members were consistent in the vagina and cervix, and generally consistent with the endometrium although the relative proportions varied. We detected three major microbiota clusters that did not group by tissue location or case-control status. There was a trend that infertile women more often had Ureaplasma in the vagina and Gardnerella in the cervix. Testing for the expression of selected genes in the endometrium did not show evidence of correlation with case-control status, or with microbial community composition, although Tenascin-C expression correlated with a history of miscarriage. CONCLUSIONS: There is a need for further exploration of the endometrial microbiota, and how the microbiota members or profile interplays with fertility or assisted reproductive technologies.

Placental Infection With Ureaplasma species Is Associated With Histologic Chorioamnionitis and Adverse Outcomes in Moderately Preterm and Late-Preterm Infants.

OBJECTIVE: The human Ureaplasma species are the microbes most frequently isolated from placentae of women who deliver preterm. The role of Ureaplasma species has been investigated in pregnancies at <32 weeks of gestation, but currently no studies have determined the prevalence of ureaplasmas in moderately preterm and late-preterm (hereafter, "moderate/late preterm") infants, the largest cohort of preterm infants. METHODS: Women delivering moderate/late preterm infants (n = 477) and their infants/placentae (n = 535) were recruited, and swab specimens of chorioamnion tissue, chorioamnion tissue specimens, and cord blood specimens were obtained at delivery. Swab and tissue specimens were cultured and analyzed by 16S ribosomal RNA polymerase chain reaction (PCR) for the presence of microorganisms, while cord blood specimens were analyzed for the presence of cytokines, chemokines, and growth factors. RESULTS: We detected microorganisms in 10.6% of 535 placentae (443 were delivered late preterm and 92 were delivered at term). Significantly, Ureaplasma species were the most prevalent microorganisms, and their presence alone was associated with histologically confirmed chorioamnionitis in moderate/late preterm and term placentae (P < .001). The presence of ureaplasmas in the chorioamnion was also associated with elevated levels of granulocyte colony-stimulating factor (P = .02). CONCLUSIONS: These findings have important implications for infection and adverse pregnancy outcomes throughout gestation and should be of major consideration for obstetricians and neonatologists.