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Rising rates of multidrug-resistant Klebsiella infections necessitate a comprehensive understanding of the major strains and plasmids driving spread of resistance elements. Here, we analysed 540 clinical, screen and environmental Klebsiella isolates recovered from across Wales between 2007 and 2020 using combined short- and long-read sequencing approaches. We identified resistant clones that have spread within and between hospitals including the high-risk strain sequence type (ST)307, which acquired the bla OXA-244 carbapenemase gene on a pOXA-48-like plasmid. We found evidence that this strain, which caused an acute outbreak largely centred on a single hospital in 2019, had been circulating undetected across South Wales for several years prior to the outbreak. In addition to clonal transmission, our analyses revealed evidence for substantial plasmid spread, mostly notably involving bla KPC-2 and bla OXA-48-like (including bla OXA-244) carbapenemase genes that were found among many species and strain backgrounds. Two thirds (20/30) of the bla KPC-2 genes were carried on the Tn4401a transposon and associated with IncF plasmids. These were mostly recovered from patients in North Wales, reflecting an outward expansion of the plasmid-driven outbreak of bla KPC-2-producing Enterobacteriaceae in North-West England. A total of 92.1 % (105/114) of isolates with a bla OXA-48-like carbapenemase carried the gene on a pOXA-48-like plasmid. While this plasmid family is highly conserved, our analyses revealed novel accessory variation including integrations of additional resistance genes. We also identified multiple independent deletions involving the tra gene cluster among pOXA-48-like plasmids in the ST307 outbreak lineage. These resulted in loss of conjugative ability and signal adaptation of the plasmids to carriage by the host strain. Altogether, our study provides, to our knowledge, the first high resolution view of the diversity, transmission and evolutionary dynamics of major resistant clones and plasmids of Klebsiella in Wales, and forms an important basis for ongoing surveillance efforts. This article contains data hosted by Microreact.

Ureaplasma species (spp.) are considered commensals of the adult genitourinary tract, but have been associated with chorioamnionitis, preterm birth, and invasive infections in neonates, including meningitis. Data on mechanisms involved in Ureaplasma-driven neuroinflammation are scarce. The present study addressed brain inflammatory responses in preterm lambs exposed to Ureaplasma parvum (UP) in utero. 7 days after intra-amniotic injection of UP (n = 10) or saline (n = 11), lambs were surgically delivered at gestational day 128–129. Expression of inflammatory markers was assessed in different brain regions using qRT-PCR and in cerebrospinal fluid (CSF) by multiplex immunoassay. CSF was analyzed for UP presence using ureB-based real-time PCR, and MRI scans documented cerebral white matter area and cortical folding. Cerebral tissue levels of atypical chemokine receptor (ACKR) 3, caspases 1-like, 2, 7, and C–X–C chemokine receptor (CXCR) 4 mRNA, as well as CSF interleukin-8 protein concentrations were significantly increased in UP-exposed lambs. UP presence in CSF was confirmed in one animal. Cortical folding and white matter area did not differ among groups. The present study confirms a role of caspases and the transmembrane receptors ACKR3 and CXCR4 in Ureaplasma-driven neuroinflammation. Enhanced caspase 1-like, 2, and 7 expression may reflect cell death. Increased ACKR3 and CXCR4 expression has been associated with inflammatory central nervous system (CNS) diseases and impaired blood–brain barrier function. According to these data and previous in vitro findings from our group, we speculate that Ureaplasma-induced caspase and receptor responses affect CNS barrier properties and thus facilitate neuroinflammation.

Objectives To evaluate the accuracy, susceptibility and specificity of MYCOPLASMA IST3, the next generation of the most popular culture-based in vitro diagnostic device designed to detect, identify and test the susceptibility of urogenital mycoplasma infections. Methods MYCOPLASMA IST3 was evaluated against culture- and molecular-based gold standard methodologies to detect, identify, enumerate and determine antimicrobial resistance for Mycoplasma hominis and Ureaplasma species in 516 clinical samples collected across France, Serbia and the UK. Sample types included vulvovaginal/endocervical or urethral swabs (dry swab or eSwab®), semen and urine samples, which included blinded analysis following addition of a panel of 80 characterized control strains. Results Overall species identification was excellent for both Ureaplasma spp. (98.4% sensitivity, 99.7% specificity) and M. hominis (95.7% sensitivity, 100% specificity) relative to combined colony morphology on agar and quantitative PCR standards. Non-dilution-based bacterial load estimation by the assay was accurate between 83.7% (M. hominis) and 86.3% (Ureaplasma spp.) of the time (increased to 94.2% and 100%, respectively, if ±10-fold variance was allowed) relative to colonies counted on agar. Resistance accuracy for Ureaplasma spp. varied from gold standards for only 11/605 of individual tests (major error rate = 1.8%) and for 14/917 individual tests for M. hominis (major error rate = 1.5%). Conclusions The redesigned MYCOPLASMA IST3 assay eliminated previous shortcomings by providing independent accurate resistance screening of M. hominis and Ureaplasma species, even in mixed infections, with CLSI-compliant thresholds. Specificity, sensitivity and enumeration estimates correlated closely with the confirmatory methods.

We studied the prevalent cytotoxic CD8 T cell response mounted against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike glycoprotein269-277 epitope (sequence YLQPRTFLL) via the most frequent human leukocyte antigen (HLA) class I worldwide, HLA A∗02. The Spike P272L mutation that has arisen in at least 112 different SARS-CoV-2 lineages to date, including in lineages classified as “variants of concern,” was not recognized by the large CD8 T cell response seen across cohorts of HLA A∗02+ convalescent patients and individuals vaccinated against SARS-CoV-2, despite these responses comprising of over 175 different individual T cell receptors. Viral escape at prevalent T cell epitopes restricted by high frequency HLAs may be particularly problematic when vaccine immunity is focused on a single protein such as SARS-CoV-2 Spike, providing a strong argument for inclusion of multiple viral proteins in next generation vaccines and highlighting the need for monitoring T cell escape in new SARS-CoV-2 variants.

Rising rates of multi-drug resistant Klebsiella infections necessitate a comprehensive understanding of the major strains and plasmids driving spread of resistance elements. Here we analysed 540 Klebsiella isolates recovered from patients across Wales between 2007 and 2020 using combined short- and long-read sequencing approaches. We identified resistant clones that have spread within and between hospitals including the high-risk strain, sequence type (ST) 307, which acquired the blaOXA-244 carbapenemase gene on a pOXA-48-like plasmid. We found evidence that this strain, which caused an acute outbreak largely centred on a single hospital in 2019, had been circulating undetected across South Wales for several years prior to the outbreak. In addition to clonal transmission, our analyses revealed evidence for substantial plasmid spread, mostly notably involving blaKPC-2 and blaOXA-48-like (including blaOXA-244) carbapenemase genes that were found among many species and strain backgrounds. Two thirds (20/30) of the blaKPC-2 genes were carried on the Tn4401a transposon and associated with IncF plasmids. These were mostly recovered from patients in North Wales, reflecting an outward expansion of the plasmid-driven outbreak of blaKPC-2-producing Enterobacteriaceae in North-West England. 92.1% (105/114) of isolates with a blaOXA-48-like carbapenemase carried the gene on a pOXA-48-like plasmid. While this plasmid family is highly conserved, our analyses revealed novel accessory variation including integrations of additional resistance genes. We also identified multiple independent deletions involving the tra gene cluster among pOXA-48-like plasmids in the ST307 outbreak lineage. These resulted in loss of conjugative ability and signal adaptation of the plasmids to carriage by the host strain. Altogether, our study provides the first high resolution view of the diversity, transmission and evolutionary dynamics of major resistant clones and plasmids of Klebsiella in Wales and forms an important basis for ongoing surveillance efforts.Data SummaryAll raw short read sequence data and hybrid assemblies are available in the European Nucleotide Archive (ENA) under project accession PRJEB48990.

Cases of invasive group B streptococcal infection in the adult UK population have steadily increased over recent years, with the most common serotypes being V, III and Ia, but less is known of the genetic background of these strains. We have carried out in-depth analysis of the whole-genome sequences of 193 clinically important group B

Neonatal sepsis is defined as a systemic infection within the first 28 days of life, with early-onset sepsis (EOS) occurring within the first 72h, although the definition of EOS varies in literature. Whilst the global incidence has dramatically reduced over the last decade, neonatal sepsis remains an important cause of neonatal mortality, highest in low- and middle-income countries (LMICs). Symptoms at the onset of neonatal sepsis can be subtle, and therefore EOS is often difficult to diagnose from clinical presentation and laboratory testing and blood cultures are not always conclusive or accessible, especially in resource limited countries. Although the World Health Organisation (WHO) currently advocates a ß-lactam, and gentamicin for first line treatment, availability and cost influence the empirical antibiotic therapy administered. Antibiotic treatment of neonatal sepsis in LMICs is highly variable, partially caused by factors such as cost of antibiotics (and who pays for them) and access to certain antibiotics. Antimicrobial resistance (AMR) has increased considerably over the past decade and this review discusses current microbiology data available in the context of the diagnosis, and treatment for EOS. Importantly, this review highlights a large variability in data availability, methodology, availability of diagnostics, and aetiology of sepsis pathogens.

The genital mycoplasmas are a unique group of inherently antibiotic-resistant sexually transmitted bacteria, often associated with non-gonococcal urethritis and bacterial vaginosis. The MYCO WELL D-ONE is a culture-based assay that aims to detect these organisms whilst concurrently screening them for antibiotic resistance. Urine and/or swabs from 856 informed and consented participants attending Welsh sexual health clinics were subjected to MYCO WELL D-ONE analysis, alongside qPCR and culture titration methodologies to determine sensitivity, specificity, PPV, NPV and accuracy. Resistance was confirmed by CLSI-compliant susceptibility testing and genetic mechanisms determined. The MYCO WELL D-ONE displayed a sensitivity and specificity of 91.98% and 96.44% for the detection of Ureaplasma spp., with sensitivity and specificity values of 78.23% and 98.84% for Mycoplasma hominis, compared with qPCR. Swabs harboured significantly greater bacterial loads than urine samples for both Ureaplasma spp. and M. hominis. Levofloxacin resistance rates, mediated by Ser83Leu mutation in ParC, for Ureaplasma spp. were 0.54%. Tetracycline resistance rates, mediated by tet(M), were 0.54% and 2% for Ureaplasma spp. and M. hominis, respectively; sequence analysis of tet(M)-positive Ureaplasma spp. and M. hominis strains isolated from a single individual confirmed separate resistance gene origins. The MYCO WELL D-ONE is a sensitive and specific assay for the detection of Ureaplasma spp. and M. hominis in genitourinary medicine samples, facilitating the accurate detection of these organisms within low-technology environments. While good for antibiotic resistance screening, accurate confirmation by MIC determination or molecular methods are required, and more optimally performed on urine samples.

Often dismissed as a commensal, Mycoplasma hominis is an increasingly prominent target of research due to its role in septic arthritis and organ transplant failure in immunosuppressed patients, particularly lung transplantation. As a mollicute, its highly reductive genome and structure render it refractile to most forms of treatment and growing levels of resistance to the few sources of treatment left, such as fluoroquinolones. We examined antimicrobial susceptibility (AST) to fluoroquinolones on 72 isolates and observed resistance in three (4.1%), with corresponding mutations in the quinolone resistance-determining region (QRDR) of S83L or E87G in gyrA and S81I or E85V in parC. However, there were high levels of polymorphism identified between all isolates outside of the QRDR, indicating caution for a genomics-led approach for resistance screening, particularly as we observed a further two quinolone-susceptible isolates solely containing gyrA mutation S83L. However, both isolates spontaneously developed a second spontaneous E85K parC mutation and resistance following prolonged incubation in 4 mg/L levofloxacin for an extra 24–48 h. Continued AST surveillance and investigation is required to understand how gyrA QRDR mutations predispose M. hominis to rapid spontaneous mutation and fluoroquinolone resistance, absent from other susceptible isolates. The unusually high prevalence of polymorphisms in M. hominis also warrants increased genomics’ surveillance.

Lefamulin is the first of the pleuromutilin class of antimicrobials to be available for therapeutic use in humans. Minimum inhibitory concentrations of lefamulin were determined by microbroth dilution for 90 characterised clinical isolates (25 Ureaplasma&nbsp, parvum, 25 Ureaplasma urealyticum, and 40 Mycoplasma hominis). All Mycoplasma hominis isolates possessed lefamulin MICs of ≤0.25 mg/L after 48 h (MIC50/90 of 0.06/0.12 mg/L), despite an inherent resistance to macrolides, while Ureaplasma isolates had MICs of ≤2 mg/L after 24 h (MIC50/90 of 0.25/1 mg/L), despite inherent resistance to clindamycin. Two U. urealyticum isolates with additional A2058G mutations of 23S rRNA, and one U. parvum isolate with a R66Q67 deletion (all of which had a combined resistance to macrolides and clindamycin) only showed a 2-fold increase in lefamulin MIC (1–2 mg/L) relative to macrolide-susceptible strains. Lefamulin could be an effective alternative antimicrobial for treating Ureaplasma spp. and Mycoplasma hominis infections irrespective of intrinsic or acquired resistance to macrolides, lincosamides, and ketolides. Based on this potent in vitro activity and the known good, rapid, and homogenous tissue penetration of female and male urogenital tissues and glands, further exploration of clinical efficacy of lefamulin for the treatment of Mycoplasma and Ureaplasma urogenital infections is warranted.

Objectives There is a lack of international unification for AST methodology for Legionella pneumophila. Current literature contains multiple possible methods and this study compares each of them to determine methodological concordance. Methods Antibiotic susceptibility of 50 L. pneumophila strains was determined using broth microdilution (BMD), serial antimicrobial dilution in traditional buffered charcoal yeast extract (BCYE) agar (as well as comparison with gradient strip overlay on BCYE) and in a novel charcoal-free agar (LASARUS) for rifampicin, azithromycin, levofloxacin and doxycycline. Results The deviation of tested media relative to BMD highlighted the overall similarity of BMD and LASARUS across all antimicrobials tested (within one serial dilution). BCYE agar dilution showed an increased MIC of up to five serial dilutions relative to BMD, while MICs by gradient strip overlay on BCYE were elevated by two to three serial dilutions, with the exception of doxycycline, which was decreased by three serial dilutions relative to MIC values determined by BMD. The MIC range for azithromycin was wider than for other antimicrobials tested and found to be caused by the presence or absence of the lpeAB gene. Conclusions BMD-based antimicrobial susceptibility testing (AST) methodology should be the internationally agreed gold standard for Legionella spp. AST, as is common for other bacterial species. Traditional BCYE gave significantly elevated MIC results and its use should be discontinued for Legionella spp., while MIC determination using LASARUS solid medium gave results concordant (within one serial dilution) with BMD for all antimicrobials tested. To the best of our knowledge, this study is the first to identify the lpeAB gene in UK isolates.

Aims: To use a flow-based method to establish, quantify and visualize biofilms of Ureaplasma parvum. Methods and Results: Absorbance readings of a U. parvum HPA5 culture were taken at 550 nm every 3 h for 30 h in order to establish a growth curve, with viability determined by the number of colour changing units (CCUs). Biofilms were established using the DTU flow-cell with a flow rate of 0·01 ml min−1 and compared to the static control. Titres of bacteria were determined by CCU and biofilm biomass was quantified by Syto9 staining and COMSTAT analysis. High-resolution images were obtained by scanning electron microscopy (SEM). Flow resulted in significantly more biofilm and higher cell titre (0·599 µm3/µm2 ± 0·152 and 4 × 108 CCU per ml, respectively) compared with static conditions (0·008 µm3/µm2 ± 0·010 and no recoverable cells, respectively). SEM revealed pleomorphic cells, with signs of budding and possible membrane vesicle formation. Conclusions: Flow is an essential requirement for the establishment of U. parvum biofilms. Significance and Impact of the Study: This is the first quantification of biofilm biomass formed by U. parvum. It is now possible to establish viable biofilms of U. parvum which will allow for future testing of antimicrobial agents and understanding of virulence-associated with adhesion.

A minimal genome and absent bacterial cell wall renders Mycoplasma hominis inherently resistant to most antimicrobials except lincosamides, tetracyclines and fluoroquinionlones. Often dismissed as a commensal (except where linked to preterm birth), it causes septic arthritis in immunodeficient patients and is increasingly associated with transplant failure (particularly lung) accompanying immunosuppression. We examined antimicrobial susceptibility (AST) on strains archived between 2005-2015 submitted to the Public Health England reference laboratory and determined the underlying mechanism of resistance by whole genome sequencing (WGS). Archived M. hominis strains included 32/115 from invasive infection (sepsis, CSF, peritoneal and pleural fluid) over the 10-year period (6.4% of all samples submitted between 2010-2015 were positive). No clindamycin resistance was detected, while two strains were resistant to moxifloxacin and levofloxacin (resistance mutations: S83L or E87G in gyrA and S81I or E84V in parC). One of these strains and 11 additional strains were tetracycline resistant, mediated by tet(M) carried within an integrative conjugative element (ICE) consistently integrated at the somatic rumA gene; however, the ICEs varied widely in 5-19 associated accessory genes. WGS analysis showed tet(M)-carrying strains were not clonal, refuting previous speculation that the ICE was broken and immobile. We found tet(M)-positive and -negative strains (including the multi-resistant 2015 strain) to be equally susceptible to tigecycline and josamycin, however, the British National Formulary does not include guidance for these. Continued M. hominis investigation and AST surveillance (especially immunocompromised patients) is warranted, and expansion of the limited therapeutics needs to be expanded in the UK.

A minimal genome and absent bacterial cell wall render Mycoplasma hominis inherently resistant to most antimicrobials except lincosamides, tetracyclines, and fluoroquinolones. Often dismissed as a commensal (except where linked to preterm birth), it causes septic arthritis in immunodeficient patients and is increasingly associated with transplant failure (particularly lung) accompanying immunosuppression. We examined antimicrobial susceptibility (AST) on strains archived from 2005 to 2015 submitted to the Public Health England reference laboratory and determined the underlying mechanism of resistance by whole-genome sequencing (WGS). Archived M. hominis strains included 32/115 from invasive infection (sepsis, cerebrospinal [CSF], peritoneal, and pleural fluid) over the 10-year period (6.4% of all samples submitted from 2010 to 2015 were positive). No clindamycin resistance was detected, while two strains were resistant to moxifloxacin and levofloxacin (resistance mutations S83L or E87G in gyrA and S81I or E84V in parC). One of these strains and 11 additional strains were tetracycline resistant, mediated by tet(M) carried within an integrative conjugative element (ICE) consistently integrated at the somatic rumA gene; however, the ICEs varied widely in 5 to 19 associated accessory genes. WGS analysis showed that tet(M)-carrying strains were not clonal, refuting previous speculation that the ICE was broken and immobile. We found tet(M)-positive and -negative strains (including the multiresistant 2015 strain) to be equally susceptible to tigecycline and josamycin; however, the British National Formulary does not include guidance for these. Continued M. hominis investigation and AST surveillance (especially immunocompromised patients) is warranted, and the limited number of therapeutics needs to be expanded in the United Kingdom.

Chorioamnionitis can lead to inflammation and injury of the liver and gut, thereby predisposing patients to adverse outcomes such as necrotizing enterocolitis (NEC). In addition, intestinal bile acids (BAs) accumulation is causally linked to NEC development. Plant sterols are a promising intervention to prevent NEC development, considering their anti-inflammatory properties in the liver. Therefore, we investigated whether an intra-amniotic (IA) Ureaplasma parvum (UP) infection affected the liver and enterohepatic circulation (EHC) and evaluated whether an IA administered plant sterol mixture dissolved in &beta, cyclodextrin exerted prophylactic effects. An ovine chorioamnionitis model was used in which liver inflammation and the EHC were assessed following IA UP exposure in the presence or absence of IA prophylactic plant sterols (a mixture of &beta, sitosterol and campesterol dissolved in &beta, cyclodextrin (carrier)) or carrier alone. IA UP exposure caused an inflammatory reaction in the liver, histologically seen as clustered and conflated hepatic erythropoiesis in the parenchyma, which was partially prevented by IA administration of sterol + &beta, cyclodextrin, or &beta, cyclodextrin alone. In addition, IA administration of &beta, cyclodextrin prior to UP caused changes in the expression of several hepatic BAs transporters, without causing alterations in other aspects of the EHC. Thereby, the addition of plant sterols to the carrier &beta, cyclodextrin did not have additional effects.

Around 40% of preterm births are attributed to ascending intrauterine infection, and Ureaplasma parvum (UP) is commonly isolated in these cases. Here we present a mouse model of ascending UP infection that resembles human disease, using vaginal inoculation combined with mild cervical injury induced by a common spermicide (Nonoxynol-9, as a surrogate for any mechanism of cervical epithelial damage). We measure bacterial load in a non-invasive manner using a luciferase-expressing UP strain, and post-mortem by qPCR and bacterial titration. Cervical exposure to Nonoxynol-9, 24 h pre-inoculation, facilitates intrauterine UP infection, upregulates pro-inflammatory cytokines, and increases preterm birth rates from 13 to 28%. Our results highlight the crucial role of the cervical epithelium as a barrier against ascending infection. In addition, we expect the mouse model will facilitate further research on the potential links between UP infection and preterm birth., Ureaplasma parvum is often isolated from intrauterine infections, which are associated with 40% of preterm births. Here, Pavlidis et al. present a mouse model of ascending U. parvum infection that resembles human disease, and show that mild cervical damage promotes intrauterine infection, inflammation and preterm birth.

Objectives Mycoplasma genitalium (MG) is a common cause of sexually transmitted infection, however no prevalence data is available for Wales. MG was detected by qPCR (quantitative) as well as two separate SpeeDx commercial assays, and related to clinical symptoms, age, gender and sample type. Methods Cervical swabs, urethral swabs and/or urine were collected from 1000 patients at walk-in sexual health clinics at 3 Welsh health centres from October 2017–October 2018. Extracted DNA was investigated to determine concordance between an in-house quantitative PCR, SpeeDx ResistancePlus® MG and the SpeeDx MG + parC (beta 2) assays; mutations in parC were substantiated by Sanger sequencing. Results MG was detected in 17/600 female patients (2.7%) and 13/400 (3.5%) male patients, with a 100% concordance between in-house qPCR and both SpeeDx assays. Macrolide resistance was low (relative to other studies), but more common in males (4/13; 30.8%) than females (2/17; 11.8%) and the only fluoroquinolone resistant sample (3.4% overall) was also macrolide resistant and detected from an MSM. Vaginitis was clinically apparent in 12/17 MG-positive females (2 with additional cervicitis, 1 with additional pelvic inflammatory disease), while 7 MG-positive males were asymptomatic. MG bacterial load did not correlate to clinical symptoms and females (4559 ± 1646/ml) had significantly lower MG load than males (84,714 ± 41,813/ml; p = 0.0429). Conclusions MG prevalence and antibiotic resistance in Welsh sexual health clinics is low. MG bacterial load did not correlate to clinical presentation, men have higher MG load/ml in urine than women, genders have different age bias for MG prevalence and urine and swabs are equivalent for detecting MG.

The susceptibility of the cell-wall free bacterial pathogens Ureaplasma spp. to Manuka honey was examined. The minimum inhibitory concentration (MIC) of Manuka honey for four Ureaplasma urealyticum and four Ureaplasma parvum isolates was determined. Sensitivity to honey was also compared to clinical isolates with resistance to tetracycline, macrolide and fluoroquinolone antibiotics. Finally step-wise resistance training was utilised in an attempt to induce increased tolerance to honey. The MIC was dependent on the initial bacterial load with 7.5 % and 18.0 % w/v honey required to inhibit U. urealyticum at 1 and 10 6 colour changing units (CCU), respectively, and 4.8 % and 15.3 % w/v required to inhibit U. parvum at 1 and 10 6 CCU, respectively. MIC values were consistently lower for U. parvum compared with U. urealyticum. Antimicrobial activity was seen against tetracycline resistant, erythromycin resistant and ciprofloxacin resistant isolates at 10 5 CCU. No resistance to honey was observed with fifty consecutive challenges at increasing concentrations of honey. This is the first report of the antimicrobial activity of Manuka honey against a cell-wall free bacterial pathogen. The antimicrobial activity was retained against antibiotic resistant strains and it was not possible to generate resistant mutants.

Chorioamnionitis is associated with adverse neurodevelopmental outcomes in preterm infants. Ureaplasma spp. are the microorganisms most frequently isolated from the amniotic fluid of women diagnosed with chorioamnionitis. However, controversy remains concerning the role of Ureaplasma spp. in the pathogenesis of neonatal brain injury. We hypothesize that reexposure to an inflammatory trigger during the perinatal period might be responsible for the variation in brain outcomes of preterms following Ureaplasma-driven chorioamnionitis. To investigate these clinical scenarios, we performed a detailed multimodal study in which ovine neurodevelopmental outcomes were assessed following chronic intra-amniotic Ureaplasma parvum (UP) infection either alone or combined with subsequent lipopolysaccharide (LPS) exposure. We show that chronic intra-amniotic UP exposure during the second trimester provoked a decrease in astrocytes, increased oligodendrocyte numbers, and elevated 5-methylcytosine levels. In contrast, short-term LPS exposure before preterm birth induced increased microglial activation, myelin loss, elevation of 5-hydroxymethylcytosine levels, and lipid profile changes. These LPS-induced changes were prevented by chronic preexposure to UP (preconditioning). These data indicate that chronic UP exposure has dual effects on preterm brain development in utero. On the one hand, prolonged UP exposure causes detrimental cerebral changes that may predispose to adverse postnatal clinical outcomes. On the other, chronic intra-amniotic UP exposure preconditions the brain against a second inflammatory hit. This study demonstrates that microbial interactions and the timing and duration of the inflammatory insults determine the effects on the fetal brain. Therefore, this study helps to understand the complex and diverse postnatal neurological outcomes following UP driven chorioamnionitis.

Chorioamnionitis, clinically most frequently associated with Ureaplasma, is linked to intestinal inflammation and subsequent gut injury. No treatment is available to prevent chorioamnionitis-driven adverse intestinal outcomes. Evidence is increasing that plant sterols possess immune-modulatory properties. Therefore, we investigated the potential therapeutic effects of plant sterols in lambs intra-amniotically (IA) exposed to Ureaplasma. Fetal lambs were IA exposed to Ureaplasma parvum (U. parvum, UP) for six days from 127 d&ndash, 133 d of gestational age (GA). The plant sterols &beta, sitosterol and campesterol, dissolved with &beta, cyclodextrin (carrier), were given IA every two days from 122 d&ndash, 131 d GA. Fetal circulatory cytokine levels, gut inflammation, intestinal injury, enterocyte maturation, and mucosal phospholipid and bile acid profiles were measured at 133 d GA (term 150 d). IA plant sterol administration blocked a fetal inflammatory response syndrome. Plant sterols reduced intestinal accumulation of proinflammatory phospholipids and tended to prevent mucosal myeloperoxidase-positive (MPO) cell influx, indicating an inhibition of gut inflammation. IA administration of plant sterols and carrier diminished intestinal mucosal damage, stimulated maturation of the immature epithelium, and partially prevented U. parvum-driven reduction of mucosal bile acids. In conclusion, we show that &beta, sitosterol and campesterol administration protected the fetus against adverse gut outcomes following UP-driven chorioamnionitis by preventing intestinal and systemic inflammation.

There is growing global concern regarding the rise of antibiotic-resistant organisms. Many of these reports have focused on various Gram-positive and Gram-negative pathogens, with little attention to the genus Ureaplasma. Ureaplasma spp. are associated with numerous infectious diseases affecting pregnant women, neonates and the immunocompromised. Treatment options are extremely limited due to high levels of intrinsic resistance resulting from the unique physiology of these organisms and further restricted in cases of the developing fetus or neonate, often limiting therapeutic options to predominantly macrolides or rarely fluoroquinolones. The increasing presence of macrolide- and fluoroquinolone-resistant strains among neonatal infections may result in pan-drug resistance and potentially untreatable conditions. Here, we review the requirements for accurate measurement of antimicrobial susceptibility, provide a comprehensive review of the antimicrobial resistance (AMR) for Ureaplasma species in the literature and contextualize these results relative to some investigators' reliance on commercial kits that are not CLSI compliant when determining AMR. The dramatic variation in the resistance patterns and impact of high levels of AMR amongst neonatal populations suggests the need for continued surveillance. Commercial kits represent an excellent tool for initial antibiotic susceptibility determination and screening. However, AMR reporting must utilize internationally standardized methods, as high-titre samples, or Mycoplasma hominis-contaminated samples routinely give false AMR results. Furthermore, there is a requirement for future reports to determine the underlying AMR mechanisms and determine whether expanding AMR is due to spontaneous mutation, transmission of resistance genes on mobile elements or selection and expansion of resistant clones.

Introduction: Several recent studies have identified a potential role for intrauterine Candida albicans in adverse pregnancy outcomes, including preterm birth. There is, however, a limited understanding of the impact of intrauterine candida infection on fetal well-being in early pregnancy. Using a sheep model of early pregnancy, the aims of this study were to determine (1) the ability of experimentally induced intrauterine C albicans to infect the fetus and (2) whether C albicans exposure in early pregnancy is associated with alterations in fetal cardiac function, as measured by spectral tissue Doppler imaging analysis of fetal cardiac function. Methods: Merino ewes carrying singleton pregnancies at 89 days’ gestation (term is ∼150 days) received C albicans (n = 8) via ultrasound-guided intra-amniotic injection. Saline-exposed fetuses served as controls (n = 6). Spectral tissue Doppler imaging echocardiography and amniotic fluid collection were performed at baseline and 24 and 72 hours after intrauterine C albicans injection. Fetal tissues were collected at postmortem for analysis of infection and inflammation. Results: Relative to saline control, intrauterine C albicans infection resulted in pronounced increases in amniotic fluid tumor necrosis factor α (TNF-α; P < .05) and cytokine/chemokine messenger RNA (interleukin [IL] 1β, IL-6, TNF-α, and monocyte chemoattractant protein 1; P < .05) in the fetal myocardium, lung, skin, and liver at 72 and 96 hours postinfection. Spectral tissue Doppler imaging showed diastolic dysfunction at 24 hours and severe biventricular diastolic dysfunction 72 hours postinfection. Conclusion: Intrauterine C albicans infection in a sheep model of early pregnancy causes systemic fetal candidiasis, which is associated with a robust systemic inflammatory response and progressive cardiac dysfunction detectable by spectral tissue Doppler imaging.

Background\ud \ud Chorioamnionitis can induce pulmonary inflammation and promote bronchopulmonary dysplasia development, distinguished by alveolar simplification and impaired vascular growth. Chorioamnionitis is more common during the extremely preterm canalicular lung stage (crucial for vascular development); and increases the risk for subsequent sepsis. We hypothesized that single/combined exposure to chronic and/or acute inflammation induces pulmonary inflammatory responses and vascular changes.\ud \ud Methods\ud \ud Ovine fetuses were intra-amniotically exposed to chronic Ureaplasma parvum (UP) at 24 days (d) before extreme preterm delivery at 94d (term 147d) and/or to lipopolysaccharide (LPS) 7 or 2d before delivery. Pulmonary inflammation, vascular remodeling and angiogenic factors were assessed.\ud \ud Results\ud \ud LPS exposure increased CD3-positive and myeloperoxidase-positive cells. Combined UP-LPS exposure increased pulmonary inflammation compared with 2d LPS or UP groups. The UP+2d LPS group had an increased adventitial fibrosis score when compared with UP-treated animals. A reduced wall-to-lumen ratio was found in the 7d LPS animals when compared to the 2d LPS-treated animals. Exposure to UP+2d LPS reduced VEGF and VEGFR-2 levels compared with 2d LPS-treated animals. Angiopoietin-1 (Ang1) and tunica interna endothelial cell kinase 2 (Tie-2) levels were decreased after UP+7d LPS as well as after 7d LPS, but not with UP alone.\ud \ud Conclusion\ud \ud Chronic UP and subsequent LPS exposure increased pulmonary inflammation and decreased expression of angiogenic growth factors and receptors when compared to single hit-exposed animals.\ud \ud Figures

Background: Previous meta-analyses have reported a significant association between pulmonary colonization with Ureaplasma and development of bronchopulmonary dysplasia (BPD). However, because few studies reporting oxygen dependency at 36 weeks corrected gestation were previously available, we updated the systematic review and meta-analyses to evaluate the association between presence of pulmonary Ureaplasma and development of BPD. Methods: Five databases were searched for articles reporting the incidence of BPD at 36 weeks postmenstrual age (BPD36) and/or BPD at 28 days of life (BPD28) in Ureaplasma colonized and noncolonized groups. Pooled estimates were produced using random effects meta-analysis. Meta-regression was used to assess the influence of difference in gestational age between the Ureaplasma-positive and Ureaplasma-negative groups. The effects of potential sources of heterogeneity were also investigated. Results: Of 39 studies included, 8 reported BPD36, 22 reported BPD28 and 9 reported both. The quality of studies was assessed as moderate to good. There was a significant association between Ureaplasma and development of BPD36 (odds ratio = 2.22; 95% confidence intervals: 1.42–3.47) and BPD28 (odds ratio = 3.04; 95% confidence intervals: 2.41–3.83). Sample size influenced the odds ratio, but no significant association was noted between BPD28 rates and difference in gestational age between Ureaplasma colonized and noncolonized infants (P = 0.96). Conclusions: Pulmonary colonization with Ureaplasma continues to be significantly associated with development of BPD in preterm infants at both 36 weeks postmenstrual age and at 28 days of life. This association at BPD28 persists regardless of difference in gestational age.

Mycoplasma hominis is a member of the genus mycoplasma and has only been isolated from humans. It is most frequently isolated from the urogenital tract in the absence of symptoms, but has been isolated from wounds, brain abscess, inflamed joints, blood and placenta from pregnancy with adverse outcomes (especially preterm birth and occasionally term stillbirth). Controversy surrounds whether this organism is a commensal or a pathogen; however, Mycoplasma hominis has been shown to induce preterm birth and foetal lung injury in an experimental primate model as a sole pathogen. These bacteria are known to exist as a parasitic infection, due to a number of missing synthetic and metabolism pathway enzymes from their minimal genome; therefore, the ability to adhere to host cells is important. Here we provide a review that clarifies the different nomenclature (variable adherence-associated antigen and P50) that has been used to investigate the major surface adhesin for this organism, as well as reported mechanisms responsible for turning off its expression. Variation in the structure of this protein can be used to separate strains into six categories, a method that we were able to use to distinguish and characterise 12 UK strains isolated from between 1983 and 2012. We propose that the Vaa should be used in further investigations to determine if commensal populations and those that are associated with disease utilise different forms of this adhesin, as this is under-studied and identification of pathogenic determinants is overdue for this organism.

Background Mycoplasma hominis is an opportunistic human pathogen, associated with clinically diverse disease. Currently, there is no standardised method for typing M. hominis, which would aid in understanding pathogen epidemiology and transmission. Due to availability and costs of whole genome sequencing and the challenges in obtaining adequate M. hominis DNA, the use of whole genome sequence analysis to provide clinical guidance is unpractical for this bacterial species as well as other fastidious organisms. Results This study identified pan-genome set of 700 genes found to be present in four published reference genomes. A subset of 417 genes was identified to be core genome for 18 isolates and 1 reference. Leave-one-out analysis of the core genes highlighted set of 48 genes that are required to recapture the original phylogenetic relationships observed using whole genome SNP analysis. Three 7-locus MLST schemas with high diversity index (97%) and low dN/dS ratios (0.1, 0.13, and 0.11) were derived that could be used to confer good discrimination between strains and could be of practical use in future studies direct on clinical specimens. Conclusions The genes proposed in this study could be utilised to design a cost-effective and rapid PCR-based MLST assay that could be applied directly to clinical isolates, without prior isolation. This study includes additional genomic analysis revealing high levels of genetic heterogeneity among this species. This provides a novel and evidence based approach for the development of MLST schema that accurately represent genomic phylogeny for use in epidemiology and transmission studies. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-3284-z) contains supplementary material, which is available to authorized users.

Two separate species of Ureaplasma have been identified that infect humans: Ureaplasma parvum and Ureaplasma urealyticum. Most notably, these bacteria lack a cell wall and are the leading infectious organism associated with infection-related induction of preterm birth. Fourteen separate representative prototype bacterial strains, called serovars, are largely differentiated by the sequence of repeating units in the C-terminus of the major surface protein: multiple-banded antigen (MBA). Monoclonal antibodies that recognise single or small groups of serovars have been previously reported, but these reagents remain sequestered in individual research laboratories. Here we characterise a panel of commercially available monoclonal antibodies raised against the MBA and describe the first monoclonal antibody that cross-reacts by immunoblot with all serovars of U. parvum and U. urealyticum species. We also describe a recombinant MBA expressed by Escherichia coli which facilitated further characterisation by immunoblot and demonstrate immunohistochemistry of paraffin-embedded antigens. Immunoblot reactivity was validated against well characterised previously published monoclonal antibodies and individual commercial antibodies were found to recognise all U. parvum strains, only serovars 3 and 14 or only serovars 1 and 6, or all strains belonging to U. parvum and U. urealyticum. MBA mass was highly variable between strains, consistent with variation in the number of C-terminal repeats between strains. Antibody characterisation will enable future investigations to correlate severity of pathogenicity to MBA isoform number or mass, in addition to development of antibody-based diagnostics that will detect infection by all Ureaplasma species or alternately be able to differentiate between U. parvum, U. urealyticum or mixed infections.

We studied the role of ante- and post-natal infection in the development of chronic lung disease (CLD) of prematurity. 192 newborn infants (61 term and 131 pre-term of

Background— Group B coxsackieviruses (CVBs) are the prototypical agents of acute myocarditis and chronic dilated cardiomyopathy, but an effective targeted therapy is still not available. Here, we analyze the therapeutic potential of a soluble (s) virus receptor molecule against CVB3 myocarditis using a gene therapy approach. Methods and Results— We generated an inducible adenoviral vector (AdG12) for strict drug-dependent delivery of sCAR-Fc, a fusion protein composed of the coxsackievirus-adenovirus receptor (CAR) extracellular domains and the carboxyl terminus of human IgG1-Fc. Decoy receptor expression was strictly doxycycline dependent, with no expression in the absence of an inducer. CVB3 infection of HeLa cells was efficiently blocked by supernatant from AdG12-transduced cells, but only in the presence of doxycycline. After liver-specific transfer, AdG12 (plus doxycycline) significantly improved cardiac contractility and diastolic relaxation compared with a control vector in CVB3-infected mice if sCAR-Fc was induced before infection (left ventricular pressure 59±3.8 versus 45.4±2.7 mm Hg, median 59 versus 45.8 mm Hg, P max 3645.1±443.6 versus 2057.9±490.2 mm Hg/s, median 3526.6 versus 2072 mm Hg/s, P min −2125.5±330.5 versus −1310.2±330.3 mm Hg/s, median −2083.7 versus −1295.9 mm Hg/s, P P max 5214.2±1786.2 versus 3011.6±918.3 mm Hg/s, median 5182.1 versus 3106.6 mm Hg/s, P Conclusion— AdG12-mediated sCAR-Fc delivery prevents cardiac dysfunction in CVB3 myocarditis under prophylactic and therapeutic conditions.

Antibiotic resistance determination of Ureaplasma spp. ( Ureaplasma parvum and Ureaplasma urealyticum ) usually requires predetermination of bacterial titer, followed by antibiotic interrogation using a set bacterial input. This 96-well method allows simultaneous quantification of bacteria in the presence and absence of antibiotics. A method for determining precise MICs and a method for screening against multiple antibiotics using breakpoint thresholds are detailed. Of the 61 Ureaplasma -positive clinical isolates screened, one (1.6%) was resistant to erythromycin (MIC, >64 mg/liter) and clarithromycin (MIC, 4 mg/liter), one to ciprofloxacin (1.6%), and one to tetracycline/doxycycline (1.6%). Five isolates were also consistently found to have an elevated MIC of 8 mg/liter for erythromycin, but this may not represent true antibiotic resistance, as no mutations were found in the 23S rRNA operons or ribosome-associated L4 and L22 proteins for these strains. However, two amino acids (R66Q67) were deleted from the L4 protein of the erythromycin-/clarithromycin-resistant strain. The tetM genetic element was detected in the tetracycline-resistant clinical isolate as well as in the positive control Vancouver strain serotype 9. The tetM gene was also found in a fully tetracycline-susceptible Ureaplasma clinical isolate, and no mutations were found in the coding region that would explain its failure to mediate tetracycline resistance. An amino acid substitution (D82N) was found in the ParC subunit of the ciprofloxacin-resistant isolate, adjacent to the S83L mutation reported by other investigators in many ciprofloxacin-resistant Ureaplasma isolates. It is now possible to detect antibiotic resistance in Ureaplasma within 48 h of positive culture without prior knowledge of bacterial load, identifying them for further molecular analysis.