Clinical Presentations and Treatment Outcomes of Mycoplasma genitalium Infections at a Large New York City Health Care System.

BACKGROUND: Mycoplasma genitalium (MG) is an emerging sexually transmitted infection. Treatment of MG is complicated by increasing resistance to primary treatment regimens, including macrolides and fluoroquinolones. Understanding the various clinical presentations and relative effectiveness of treatments for MG is crucial to optimizing care. METHODS: Patients with a positive MG nucleic acid amplification test between July 1, 2019, and June 30, 2021, at a large health system in New York City were included in a retrospective cohort. Demographics, clinical presentations, coinfections, treatment, and follow-up microbiologic tests were obtained from the electronic medical record. Associations with microbiologic cure were evaluated in bivariate and multivariable logistic regression models. RESULTS: Five hundred two unique patients had a positive MG nucleic acid amplification test result during the study period. Male individuals presented predominantly with urethritis (117 of 187 [63%]) and female individuals with vaginal symptoms (142 of 315 [45%]). Among patients with follow-up testing who received a single antibiotic at the time of treatment, 43% (90 of 210) had persistent infection and 57% (120 of 210) had microbiologic cure. Eighty-two percent of patients treated with moxifloxacin had microbiologic cure compared with 41% of patients receiving azithromycin regimens ( P < 0.001). In multivariable analysis, treatment with moxifloxacin was associated with 4 times the odds of microbiologic cure relative to low-dose azithromycin (adjusted odds ratio [aOR], 4.18; 95% confidence interval, 1.73-10.13; P < 0.01). CONCLUSIONS: Clinical presentations of MG vary, with urethritis or vaginal symptoms in most cases. Among patients who received a single antibiotic, only treatment with moxifloxacin was significantly associated with microbiologic cure relative to low-dose azithromycin.

Tea Polyphenols Protects Tracheal Epithelial Tight Junctions in Lung during Actinobacillus pleuropneumoniae Infection via Suppressing TLR-4/MAPK/PKC-MLCK Signaling.

Actinobacillus pleuropneumoniae (APP) is the causative pathogen of porcine pleuropneumonia, a highly contagious respiratory disease in the pig industry. The increasingly severe antimicrobial resistance in APP urgently requires novel antibacterial alternatives for the treatment of APP infection. In this study, we investigated the effect of tea polyphenols (TP) against APP. MIC and MBC of TP showed significant inhibitory effects on bacteria growth and caused cellular damage to APP. Furthermore, TP decreased adherent activity of APP to the newborn pig tracheal epithelial cells (NPTr) and the destruction of the tight adherence junction proteins ß-catenin and occludin. Moreover, TP improved the survival rate of APP infected mice but also attenuated the release of the inflammation-related cytokines IL-6, IL-8, and TNF-α. TP inhibited activation of the TLR/MAPK/PKC-MLCK signaling for down-regulated TLR-2, TLR4, p-JNK, p-p38, p-PKC-α, and MLCK in cells triggered by APP. Collectively, our data suggest that TP represents a promising therapeutic agent in the treatment of APP infection.

Development of a Serum-Free Medium To Aid Large-Scale Production of Mycoplasma-Based Therapies.

To assist in the advancement of the large-scale production of safe Mycoplasma vaccines and other Mycoplasma-based therapies, we developed a culture medium free of animal serum and other animal components for Mycoplasma pneumoniae growth. By establishing a workflow method to systematically test different compounds and concentrations, we provide optimized formulations capable of supporting serial passaging and robust growth reaching 60 to 70% of the biomass obtained in rich medium. Global transcriptomic and proteomic analysis showed minor physiological changes upon cell culture in the animal component-free medium, supporting its suitability for the production of M. pneumoniae-based therapies. The major contributors to growth performance were found to be glucose as a carbon source, glycerol, cholesterol, and phospholipids as a source of fatty acids. Bovine serum albumin or cyclodextrin (in the animal component-free medium) were required as lipid carriers to prevent lipid toxicity. Connaught Medical Research Laboratories medium (CMRL) used to simplify medium preparation as a source of amino acids, nucleotide precursors, vitamins, and other cofactors could be substituted by cysteine. In fact, the presence of protein hydrolysates such as yeastolate or peptones was found to be essential and preferred over free amino acids, except for the cysteine. Supplementation of nucleotide precursors and vitamins is not strictly necessary in the presence of yeastolate, suggesting that this animal origin-free hydrolysate serves as an efficient source for these compounds. Finally, we adapted the serum-free medium formulation to support growth of Mycoplasma hyopneumoniae, a swine pathogen for which inactivated whole-cell vaccines are available. IMPORTANCE Mycoplasma infections have a significant negative impact on both livestock production and human health. Vaccination is often the first option to control disease and alleviate the economic impact that some Mycoplasma infections cause on milk production, weight gain, and animal health. The fastidious nutrient requirements of these bacteria, however, challenges the industrial production of attenuated or inactivated whole-cell vaccines, which depends on the use of animal serum and other animal raw materials. Apart from their clinical relevance, some Mycoplasma species have become cellular models for systems and synthetic biology, owing to the small size of their genomes and the absence of a cell wall, which offers unique opportunities for the secretion and delivery of biotherapeutics. This study proposes medium formulations free of serum and animal components with the potential of supporting large-scale production upon industrial optimization, thus contributing to the development of safe vaccines and other Mycoplasma-based therapies.

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.

Mycoplasma hominis infections in solid organ transplant recipients: Clinical characteristics, treatment outcomes, and comparison of phenotypic and genotypic susceptibility profiles.

BACKGROUND: Mycoplasma hominis can cause significant infections after solid organ transplantation (SOT). Treatment should be guided by susceptibility testing, but conventional lab methods are laborious with prolonged turnaround time (TAT). This case series compares the phenotypic and genotypic susceptibility profiles of M. hominis isolates identified from SOT patients. METHODS: This is a single-center retrospective study evaluating SOT recipients with confirmed M. hominis infections. Patients' demographic, clinical, microbiological, and radiographic data were collected. Culture of M. hominis isolates was performed according to current Clinical and Laboratory Standards Institute guidelines. Phenotypic susceptibility testing was performed by University of Alabama Diagnostic Mycoplasma Laboratory. Whole genome sequencing (WGS) was performed followed by bioinformatic analysis of known genetic determinants of resistance. RESULTS: Seven SOT recipients with M. hominis infections were identified. Two out of seven (28.5%) patients had resistance detected by phenotypic susceptibility testing (Case 5 to levofloxacin and Case 7 to tetracycline). Genomic analyses confirmed the presence of mutations in the parC and parE topoisomerase genes at positions conferring to fluoroquinolone resistance in the isolate from Case 5, while the tetracycline-resistant isolate from Case 7 harbored the tetM gene. The median TAT from the date of specimen collection was 24 days for phenotypic susceptibility testing and 14 days for genotypic susceptibility testing. All seven patients received antimicrobials directed toward M. hominis and recovered with complete resolution of infection. CONCLUSIONS: WGS may offer a novel and more rapid methodology for M. hominis susceptibility testing to help optimize antimicrobial usage, but more data are needed.

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.

Antimicrobial resistance in Neisseria gonorrhoeae and Mycoplasma genitalium isolates from the private healthcare sector in South Africa: A pilot study.

BACKGROUND: Reports have emerged globally of antimicrobial resistance (AMR) in Neisseria gonorrhoeae and Mycoplasma genitalium infections. In South Africa (SA), there are substantial differences between private and public healthcare with regard to antimicrobial drug prescribing practice, which could affect AMR patterns of private and public healthcare patients. OBJECTIVES: To perform a pilot study to determine the frequency of AMR of N. gonorrhoeae and M. genitalium in patients accessing SA's private healthcare sector. METHODS: In this cross-sectional study, N. gonorrhoeae-positive cultures and M. genitalium DNA samples were collected from a private healthcare reference laboratory from August 2018 to August 2019. In N. gonorrhoeae-positive cultures, antimicrobial susceptibility testing was performed, followed by N. gonorrhoeae multiantigen sequence typing (NG-MAST) to determine genetic relatedness of the isolates. To determine macrolide and fluoroquinolone resistance rates, M. genitalium-positive samples were analysed by sequencing the 23S rRNA, gyrA and parC genes. RESULTS: Twenty-one N. gonorrhoeae- and 27 M. genitalium-positive specimens were included in this analysis. High rates of resistance were detected among gonococcal isolates, with 90% resistance to tetracycline, 86% to penicillin and 62% to ciprofloxacin, but no resistance to azithromycin, cefixime and ceftriaxone. NG-MAST revealed genetically diverse isolates with 83% novel NG-MAST sequence types. Macrolide and fluoroquinolone resistance-associated mutations were detected in 18.5% (n=5/27) and 7.4% (n=2/27) of M. genitalium strains, respectively. CONCLUSIONS: We observed high frequencies of ciprofloxacin, penicillin and tetracycline resistance in N. gonorrhoeae and macrolide resistance-associated mutations in M. genitalium in private healthcare sector patients in SA. This finding highlights the need to use diagnostics for sexually transmitted infections and to include the private healthcare sector in antimicrobial surveillance and stewardship programmes.

Evaluation of colonization, variable lipoprotein-based serological response, and cellular immune response of Mycoplasma hyorhinis in experimentally infected swine.

Mycoplasma hyorhinis (Mhr) is a commensal of the upper respiratory tract that can be shed by nasal secretions and transmitted by direct contact in neonatal and nursery pigs. Lesions associated with Mhr infection include polyserositis and arthritis; however, systemic Mhr disease pathogenesis is not well characterized. This study aimed to investigate the immunopathogenesis and bacterial dissemination pattern of Mhr using single and multiple inoculation approaches in a caesarian-derived colostrum-deprived (CDCD) pig model. Animals in three treatment groups were inoculated once (Mhr 1; n = 12) or four (Mhr 2; n = 8) times with Mhr or sham-inoculated (NC group; n = 3) nasally and by tonsillar painting. Inoculum consisted of a triple cloned Mhr field isolate (4.5 × 107 CFU/mL) in Friis medium. Clinical signs were evaluated daily during the study. Serum and oral fluid antibody (IgA and IgG) response and cellular immune response were assessed using a recombinant chimeric VlpA-G-based indirect ELISA and by ELISpot, respectively. The presence of Mhr in oral fluids, nasal and oropharyngeal swabs were evaluated by qPCR. At 6 wpi, pigs were euthanized and evaluated for gross lesions consistent with Mhr and bacterial colonization in tonsils by qPCR. No clinical signs or gross lesions consistent with Mhr-associated disease were observed throughout the study. For Mhr 2 group, the presence of IgA and IgG in serum and oral fluids were detected at 2 and 4 weeks post-inoculation (wpi), respectively, while in Mhr 1, only IgA was detected in oral fluids at 6 wpi. The proportion of animals shedding Mhr in nasal secretions varied from 20 to 40 % in the Mhr 1 and 62.5-100% in the Mhr 2 group. However, the proportion of animals shedding Mhr in oropharyngeal swabs was consistent through the study (60 %) in Mhr 1 and fluctuated from 20 % to 87.5 % in Mhr 2 group. The lack of clinical signs and the presence of Mhr specific humoral response and bacterial colonization indicates that the multiple inoculation experimental model may mimic subclinical natural infection in the field. In addition, the humoral and transient cellular response did not result in bacterial clearance. Based on these results, animals would have to be exposed multiple times to mount a detectable immune response.

Lipoic Acid Does Not Affect The Growth of Mycoplasma hominis Cells In Vitro.

Mycoplasma hominis is associated with various infections, for which the treatment can be complex. Lipoic acid (LA) plays a role as a cofactor in eukaryotes, most Bacteria, and some Archea. Research of recent years has increasingly pointed to the therapeutic properties of exogenously supplemented LA. The present study was conducted on 40 strains of M. hominis cultured with the following LA concentrations: 1,200 µg/ml, 120 µg/ml, and 12 µg/ml. The bacterial colonies of each strain were counted and expressed as the number of colony-forming units/ml (CFU). The number of CFU in M. hominis strains obtained in the presence of LA was compared with the number of CFU in the strains grown in the media without LA. The obtained results indicated that the presence of LA in the medium did not affect the growth of M. hominis. The investigation of the influence of LA on the growth and survival of microbial cells not only allows for obtaining an answer to the question of whether LA has antimicrobial activity and, therefore, can be used as a drug supporting the treatment of patients infected with a given pathogenic microorganism. Such studies are also crucial for a better understanding of LA metabolism in the microbial cells, which is also important for the search for new antimicrobial drugs. This research is, therefore, an introduction to such further studies.

Mycoplasma bovis and other Mollicutes in replacement dairy heifers from Mycoplasma bovis-infected and uninfected herds: A 2-year longitudinal study.

Replacement dairy heifers exposed to Mycoplasma bovis as calves may be at risk of future clinical disease and pathogen transmission, both within and between herds; however, little information is available about these risks. We conducted a 2-yr longitudinal (panel) study starting with 450 heifer calves reared to weaning in 8 herds (7 M. bovis infected with clinical disease, 1 uninfected) under the same ownership. After weaning, heifers were commingled and managed with non-study heifers at a single heifer rearing facility. Nose, conjunctival, and vaginal swabs were collected along with a blood sample at weaning, prebreeding, precalving, and approximately 1 mo postcalving. Additionally, a colostrum sample was collected upon calving and a composite milk sample was collected 1 mo postcalving. The swabs, colostrum, and milk samples were cultured for Mycoplasma spp., and serum from the blood was evaluated for serological evidence of exposure to M. bovis using an ELISA. Despite a high M. bovis ELISA seroprevalence at weaning in the heifers from the 7 M. bovis-infected herds with clinical disease [72% (289/400); range by herd: 28-98%], M. bovis was isolated from only 4% (16/400) of the same heifers at the same time. In heifers from the uninfected herd at weaning, M. bovis seroprevalence was 2% (1/50) and M. bovis was not detected by culture. Mycoplasma bovis was isolated from 0.5% (2/414) of heifers at prebreeding, 0% (0/374) of heifers at precalving, and 0.3% (1/356) of heifers 1 mo postcalving. The nose was the predominant anatomical site of M. bovis colonization (74%; 14/19 culture positives). A single heifer (from an M. bovis-infected herd with clinical disease) was repeatedly detected with M. bovis in its nose at weaning, prebreeding, and postcalving samplings. This demonstrates the possibility, albeit rare, of a long-term M. bovis carrier state in replacement heifers exposed to M. bovis as calves, up to at least 1 mo after entry into the milking herd. No M. bovis clinical disease was detected in any heifer from weaning through to the end of the study (approximately 1 mo after calving). Acholeplasma spp. were commonly isolated throughout the study. Mycoplasma bovigenitalium, Mycoplasma bovoculi, and Mycoplasma bovirhinis were isolated infrequently. Mycoplasma bovis seroprevalences at prebreeding, precalving, and postcalving samplings were 27% (112/414), 12% (46/374), and 18% (65/356), respectively. Overall, the results show that replacement heifers from groups exposed to M. bovis preweaning can become colonized with M. bovis and that colonization can, uncommonly, be present after their first calving. For groups of 50 or more heifers exposed to M. bovis preweaning, there is at least a nontrivial probability that the group will contain at least 1 shedding heifer postcalving.

Effects of dietary Original XPC on selected blood variables in layer pullets challenged with Mycoplasma gallisepticum,.

Effects of dietary Original XPC (XPC) on 17 selected blood variables in commercial layer pullets challenged with the virulent, low-passage R strain of Mycoplasma gallisepticum (RlowMG) were investigated. Hy-Line W-36 pullets sourced from M. gallisepticum-clean layer breeders were fed a basal diet with XPC (1.25 kg/metric ton) or without from hatch until 12 wk of age (woa). At 8 and 10 woa, half of the birds in each dietary treatment were challenged with RlowMG. Blood samples were taken immediately before the initial RlowMG challenge at 8 woa and again at 12 woa (4 wk after challenge). At 8 woa, blood pH was lower and glucose concentration was higher in the preassigned challenge treatment groups. At 12 woa, the concentration of oxygen dissolved in the blood was significantly lower in the RlowMG-challenged group than the unchallenged group of birds regardless of dietary treatment. The RlowMG challenge significantly increased blood carbon dioxide partial pressure, calcium, sodium, anion gap, osmolality, glucose, and corticosterone levels but significantly decreased blood oxygen partial pressure, oxyhemoglobin concentration, concentration of oxygen dissolved in the blood, chloride, and pH levels. Because blood pH and glucose concentration at 8 woa were examined before challenge, their baseline values were biased with respect to challenge treatment before treatment was applied. However, the lack of a significant main effect due to diet at 8 woa for blood pH and glucose concentration, along with the other 15 blood variables, indicate that the baseline data with respect to dietary treatment were unbiased, allowing for real dietary effects to be accurately assessed. In conclusion, layer pullets challenged with RlowMG undergo a stress response associated with changes in various physiological blood variables, and a decrease in pH and increase in carbon dioxide partial pressure, in association with a lack of change in bicarbonate, indicates that the stress response caused by the RlowMG challenge was associated with respiratory acidosis. Nevertheless, feeding XPC did not influence the effects of challenge treatment on these postchallenge physiological blood values.

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.

Combination of procedure for ultra rapid extraction (PURE) and loop-mediated isothermal amplification (LAMP) for rapid detection of Mycoplasma bovis in milk.

Polymerase chain reaction (PCR) is typically used for the early detection of mycoplasma in bovine milk; it requires 3 days to obtain results because of the necessary enrichment process. A more rapid, simple, and accurate detection method is required to directly detect the Mycoplasma bovis (M. bovis) gene in milk. In this study, we assess the utility of combining the following two methods to achieve this goal: the loop-mediated isothermal amplification (LAMP), which is more sensitive than PCR, and the procedure for ultra rapid extraction (PURE), which adsorbs and filters components that inhibit DNA amplification/detection. LAMP was examined using DNA extracts obtained by four methods. This showed that PURE had the highest sensitivity and specificity and that the combination of PURE and LAMP was able to detect M. bovis in milk. We then showed that the detection limit of M. bovis was 102 colony-forming units per milliliter of milk using the PURE-LAMP. Finally, the respective sensitivities of the PURE-LAMP and PCR were 57% and 86% for bulk tank milk, 89% and 74% for mature milk, 85% and 92% for colostrum/transitional milk, and 97% and 95% for mastitis milk. The specificity was 100% for all milk samples in both LAMP and PCR. We conclude that PCR was suitable for detecting mycoplasma in bulk tank milk and that the PURE-LAMP could detect mycoplasma within 2 hr and was also effective for mature and mastitis milk.

The presence of Mycoplasma bovis in colostrum.

In herds with Mycoplasma bovis circulation, colostrum is often considered infectious. However, in contrast to milk, the presence of M. bovis in colostrum was not previously evidenced. In this survey, the presence of M. bovis DNA was determined with real-time PCR in 368 colostrum samples from 17 herds, recently infected with M. bovis. Only 1.9% of the samples tested positive, with 13 herds having no positive samples and an overall within-herd prevalence of 3.2% (SD: 4.9%; Range: 0-30.0%). These results show that in infected herds M. bovis DNA can be retrieved in colostrum. To what extend colostrum is infectious remains to be determined.

Dynamics of the within-herd prevalence of Mycoplasma bovis intramammary infection in endemically infected dairy herds.

We aimed to identify the dynamics of the within-herd prevalence of Mycoplasma (M.) bovis intramammary infection (IMI) in four dairy herds, estimate prevalence of M. bovis in colostrum and clinical mastitis cases and compare M. bovis strains from calves' respiratory and cow clinical mastitis samples. Within a six-month study period, cow composite milk samples (CMS) were collected three times during routine milk recording, first milking colostrum samples from all calving cows and udder quarter milk samples from clinical mastitis cases. Calf respiratory samples were collected from calves with respiratory disease. Pooled milk samples were analysed for M. bovis with the Mastitis 4B polymerase chain reaction (PCR) test kit (DNA Diagnostic A/S). Prevalence estimates were calculated with Bayesian framework in R statistical programme. cg-MLST was used for M. bovis genotyping. In Herd I and II first testing M. bovis IMI within-herd prevalence (95 % credibility interval (CI)) was 4.7 % (2.9; 6.8) and 3.4 % (2.3; 4.6), changing to 1.0 % (0.1; 1.7) and 0.8 % (0.1; 1.4) in Herd I and 0.4 % (0.0; 0.7) in Herd II at the next samplings. In Herd III and IV first testing M. bovis IMI within-herd prevalence was 12.3 % (9.7; 15.2) and 7.8 % (6.2; 9.5), changing to 4.6 % (3.0; 6.4) and 3.2 % (1.9; 4.8) in Herd III and to 2.8 % (1.9; 3.8) and 4.9 % (3.6; 6.4) in Herd IV at the next samplings. The estimated prevalence of M. bovis in colostrum ranged between 1.7 % (0.2; 2.8) and 4.7 % (2.7; 7.1) and in clinical mastitis cases between 3.7 % (1.7; 6.4) and 11.0 % (7.5; 15.2) in the study herds. M. bovis strains isolated from cows and calves clustered within herds indicating possible transmission of M. bovis between dairy cows and calves. Prevalence of M. bovis in colostrum and clinical mastitis cases as well as the within-herd prevalence of M. bovis IMI was low in endemically infected dairy herds.

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.

Prenatal infection with Mycoplasma pulmonis in rats exaggerates the angiotensin II pressor response in adult offspring.

Exposure to different stressors in utero is linked to adult diseases such as obesity and hypertension. In this study, the impact of prenatal infection (PNI) on adult body weight and cardiovascular function was evaluated using a naturally occurring rodent pathogen, Mycoplasma pulmonis (MP). Pregnant Sprague-Dawley rats were infected with MP on gestationalday 14 and gave birth naturally. Adult PNI offspring weighed more than controls, but resting mean arterial pressure (MAP) was unchanged. Subcutaneous injection of angiotensin II (10 µg/kg) elicited a rise in MAP that was greater in both male and female PNI offspring compared with controls (P < 0.03). The accompanying reflex bradycardia was similar to the controls, suggesting that PNI induced baroreflex dysfunction. Subcutaneous nicotine administration, a potent cardiorespiratory stimulus, also elicited a transient rise in MAP that was generally greater in the PNI group, but the change in MAP from baseline was only significant in the PNI females compared with controls (P < 0.03). Elevated body weight and cardiovascular reactivity in the PNI offspring was associated with an increase in the ratio of hypothalamic corticotrophin-releasing hormone receptors type 1 to type 2 gene expression in both sexes compared with controls. These findings support previous studies demonstrating that PNI induces alterations in cardiovascular function and body weight. Yet, unlike previous studies utilizing other models of PNI (e.g., endotoxin), MP PNI did not induce resting hypertension. Thus, our study provides a foundation for future studies evaluating the cardiovascular risks of offspring exposed to microbial challenges in utero.

High rates of treatment failure for Mycoplasma genitalium among men and women attending a sexual health clinic.

BACKGROUND: Mycoplasma genitalium (Mgen) causes non-gonococcal urethritis (NGU) and is believed to cause pelvic inflammatory disease (PID). High rates of macrolide resistance are well documented globally for Mgen. In Brighton, patients with NGU and PID are tested for Mgen and test of cure (TOC) offered post-treatment. METHODS: Demographic, clinical and treatment history data were collected over a 12-month period for all Mgen-positive patients in a Brighton-based genitourinary clinic. RESULTS: There were 114 patients with Mgen. 18% (61/339) of men with NGU and 9% (15/160) of women with PID had Mgen. 62/114 (54%) returned for first test TOC 4 weeks after treatment. 27/62 (44%) had a positive TOC; 25/27 (92.6%) had received azithromycin first line (500 mg stat then 250 mg OD for 4 days), 1/27 (3.7%) had received moxifloxacin first line (400 mg OD for 14 days) and 1/27 (3.7%) had received doxycycline first line (100 mg BD for 7 days). 20/27 (74%) returned for a second TOC 4 weeks later. 5/20 (25%) patients were positive on second TOC; 3/5 (60%) had received azithromycin second line and 2/5 (40%) had received moxifloxacin second line. Patients were more likely to have a positive TOC if they were at risk of reinfection (9/27 positive TOC vs 3/35 negative TOC; p=0.02). Patients given moxifloxacin were more likely to have a negative TOC (1/27 positive TOC vs 9/35 negative TOC; p=0.03) than those who received other antibiotic regimens. CONCLUSIONS: Treatment failure rates for Mgen following azithromycin use are substantial, raising concerns regarding resistance. However, reinfection risk may contribute, suggesting a requirement for improved public awareness and clinician knowledge.

Resistance-Guided Antimicrobial Therapy Using Doxycycline-Moxifloxacin and Doxycycline-2.5 g Azithromycin for the Treatment of Mycoplasma genitalium Infection: Efficacy and Tolerability.

BACKGROUND: Macrolide resistance in Mycoplasma genitalium (MG) exceeds 50% in many regions, and quinolone resistance is increasing. We recently reported that resistance-guided therapy (RGT) using doxycycline followed by sitafloxacin or 2.5 g azithromycin cured 92% and 95% of macrolide-resistant and macrolide-susceptible infections, respectively. We present data on RGT using doxycycline-moxifloxacin, the regimen recommended in international guidelines, and extend data on the efficacy of doxycycline-2.5 g azithromycin and de novo macrolide resistance. METHODS: Patients attending Melbourne Sexual Health Centre between 2017 and 2018 with sexually transmitted infection syndromes were treated with doxycycline for 7 days and recalled if MG-positive. Macrolide-susceptible cases received 2.5 g azithromycin (1 g, then 500 mg daily for 3 days), and resistant cases moxifloxacin (400 mg daily, 7 days). Test of cure was recommended 14-28 days post-antimicrobials. RESULTS: There were 383 patients (81 females/106 heterosexual males/196 men who have sex with men) included. Microbial cure following doxycycline-azithromycin was 95.4% (95% confidence interval [CI], 89.7-98.0) and doxycycline-moxifloxacin was 92.0% (95% CI, 88.1-94.6). De novo macrolide resistance was detected in 4.6% of cases. Combining doxycycline-azithromycin data with our prior RGT study (n = 186) yielded a pooled cure of 95.7% (95% CI, 91.6-97.8). ParC mutations were present in 22% of macrolide-resistant cases. CONCLUSIONS: These findings support the inclusion of moxifloxacin in resistance-guided strategies and extend the evidence for 2.5 g azithromycin and presumptive use of doxycycline. These data provide an evidence base for current UK, Australian, and European guidelines for the treatment of MG.