Waddlia chondrophila, a Chlamydia-related bacterium, has a negative impact on human spermatozoa.

STUDY QUESTION: What is the impact of Waddlia chondrophila, an emerging Chlamydia-related bacterium associated with miscarriage, on human spermatozoa? SUMMARY ANSWER: W. chondrophila had a negative impact on human spermatozoa (decrease in viability and mitochondrial membrane potential) and was not entirely removed from infected samples by density gradient centrifugation. WHAT IS KNOWN ALREADY: Bacterial infection or colonization might have a deleterious effect on male fertility. Waddlia chondrophila was previously associated with miscarriage, but its impact on male reproductive function has never been studied. STUDY DESIGN SIZE, DURATION: An in vitro model of human spermatozoa infection was used to assess the effects of W. chondrophila infection. Controls included Chlamydia trachomatis serovar D and latex beads with similar size to bacteria. PARTICIPANTS/MATERIALS, SETTING, METHODS: Purified motile spermatozoa were infected with W. chondrophila (multiplicity of infection of 1). Immunohistochemistry combined with confocal microscopy was used to evaluate how bacteria interact with spermatozoa. The impact on physiology was assessed by monitoring cell viability, mitochondrial membrane potential and DNA fragmentation. MAIN RESULTS AND THE ROLE OF CHANCE: Using super-resolution confocal microscopy, bacteria were localized on spermatozoa surface, as well as inside the cytoplasm. Compared to controls, W. chondrophila caused a 20% increase in mortality over 72 h of incubation (P < 0.05). Moreover, higher bacterial loads significantly reduced mitochondrial membrane potential. Bacteria present on spermatozoa surface were able to further infect a cell-monolayer, indicating that sperm might vector bacteria during sexual intercourse. LIMITATIONS REASONS FOR CAUTION: The main limitation of the study is the use of an in vitro model of infection, which might be too simplistic compared to an actual infection. An animal model of infection should be developed to better evaluate the in vivo impact of W. chondrophila. WIDER IMPLICATIONS OF THE FINDINGS: Intracellular bacteria, including C. trachomatis, Mycoplasma spp. and Ureaplasma spp., are associated with male infertility. Waddlia chondrophila might represent yet another member of this group, highlighting the need for more rigorous microbiological analysis during investigations for male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work has been funded by the Department of Obstetrics and Gynecology, Lausanne University Hospital, Switzerland, and by the Swiss National Science Foundation (Grant nos. 310030-156169/1, 320030-169853/1 and 320030-169853/2 attributed to D.B.). D.B. is also supported by the 'Fondation Leenaards' through the 'Bourse pour la relève académique', by the 'Fondation Divesa' and by the 'Loterie Romande'. No conflicts of interest to declare.

Development and evaluation of double locus sequence typing for molecular epidemiological investigations of Clostridium difficile.

Despite the development of novel typing methods based on whole genome sequencing, most laboratories still rely on classical molecular methods for outbreak investigation or surveillance. Reference methods for Clostridium difficile include ribotyping and pulsed-field gel electrophoresis, which are band-comparing methods often difficult to establish and which require reference strain collections. Here, we present the double locus sequence typing (DLST) scheme as a tool to analyse C. difficile isolates. Using a collection of clinical C. difficile isolates recovered during a 1-year period, we evaluated the performance of DLST and compared the results to multilocus sequence typing (MLST), a sequence-based method that has been used to study the structure of bacterial populations and highlight major clones. DLST had a higher discriminatory power compared to MLST (Simpson's index of diversity of 0.979 versus 0.965) and successfully identified all isolates of the study (100 % typeability). Previous studies showed that the discriminatory power of ribotyping was comparable to that of MLST; thus, DLST might be more discriminatory than ribotyping. DLST is easy to establish and provides several advantages, including absence of DNA extraction [polymerase chain reaction (PCR) is performed on colonies], no specific instrumentation, low cost and unambiguous definition of types. Moreover, the implementation of a DLST typing scheme on an Internet database, such as that previously done for Staphylococcus aureus and Pseudomonas aeruginosa ( http://www.dlst.org ), will allow users to easily obtain the DLST type by submitting directly sequencing files and will avoid problems associated with multiple databases.

[Will a vaccine against Chlamydia trachomatis be available soon?]. / Bientôt un vaccin contre Chlamydia trachomatis?

Chlamydia trachomatis is the most prevalent cause of sexually transmitted bacterial infections worldwide, with more than 100 million estimated cases annually. This obligate intracellular pathogen is known to cause pelvic inflammatory disease (PID) and chronic infections resulting in tubal factor infertility and ectopic pregnancy. However, the majority of the infections remains asymptomatic and thus untreated. For this reason, the ultimate goal for the prevention C. trachomatis infections is an effective vaccine. Here we review the major challenges and the different strategies associated with the development of an anti-Chlamydial vaccine. Even if an effective vaccine is not available yet, recent advances in the understanding of C. trachomatis pathogenesis and mucosal immune system are promising for its future development.

Characterization of the staphylococcal cassette chromosome mec insertion site in 108 isolates lacking the mecA gene and identified as methicillin-resistant Staphylococcus aureus by the Xpert MRSA assay.

During a 3-year period, 848 patients were detected as carriers of methicillin-resistant Staphylococcus aureus (MRSA) by the Xpert MRSA assay (Cepheid). Among them, 108 patients (12.7 %) were colonized with strains showing methicillin-susceptible phenotypes and absence of the mecA gene, despite being positive with the rapid polymerase chain reaction (PCR) assay. DNA sequences of the staphylococcal cassette chromosome mec (SCCmec) insertion site of these "false-positive" strains was determined by direct sequencing of the genomic DNA. More than half (53.7 %) of the strains had DNA sequences unrelated to either SCC or SCCmec and one-third had DNA sequences related to non-mec SCC. Only 10.2 % of the strains carried sequences related to SCCmec, suggesting that a sequence containing the mecA gene was lost from an SCCmec. These findings differ from the general idea that all methicillin-susceptible S. aureus having positive Xpert MRSA assay results are essentially MRSA that lost the mecA gene.

Impact of semen microbiota on the composition of seminal plasma.

Several studies have found associations between specific bacterial genera and semen parameters. Bacteria are known to influence the composition of their niche and, consequently, could affect the composition of the seminal plasma. This study integrated microbiota profiling and metabolomics to explore the influence of seminal bacteria on semen metabolite composition in infertile couples, revealing associations between specific bacterial genera and metabolite profiles. Amino acids and acylcarnitines were the predominant metabolite groups identified in seminal plasma. Different microbiota profiles did not result in globally diverse metabolite compositions in seminal plasma. Nevertheless, levels of specific metabolites increased in the presence of a dysbiotic microbiota. Urocanate was significantly increased in abnormal semen samples (adjusted P-value < 0.001) and enriched in samples dominated by Prevotella spp. (P-value < 0.05), which was previously linked to a negative impact on semen. Therefore, varying microbiota profiles can influence the abundance of certain metabolites, potentially having an immunomodulatory effect, as seen with urocanate.IMPORTANCEMale infertility is often considered idiopathic since the specific cause of infertility often remains unidentified. Recently, variations in the seminal microbiota composition have been associated with normal and abnormal semen parameters and may, therefore, influence male infertility. Bacteria are known to alter the metabolite composition of their ecological niches, and thus, seminal bacteria might affect the composition of the seminal fluid, crucial in the fertilization process. Our research indicates that distinct seminal microbiota profiles are not associated with widespread changes in the metabolite composition of the seminal fluid. Instead, the presence of particular metabolites with immunomodulatory functions, such as urocanate, could shed light on the interplay between seminal microbiota and variations in semen parameters.

Expression of SCCmec cassette chromosome recombinases in methicillin-resistant Staphylococcus aureus and Staphylococcus epidermidis.

OBJECTIVES: Methicillin resistance in staphylococci is mediated by the mecA gene, which is carried on the staphylococcal cassette chromosome mec (SCCmec). SCCmec is responsible for vertical and horizontal transfer of methicillin resistance. Horizontal transfer implies first SCCmec excision from the chromosome. Site-specific excision is catalysed by the Ccr recombinases, which are encoded by ccrAB genes located on the cassette. The aim of this study is to determine the promoter activity of ccrAB genes in individual cells of methicillin-resistant Staphylococcus aureus (N315, COL and MW2) and Staphylococcus epidermidis (RP62A). One mutant cured of its SCCmec (N315EX) was also used. Exposure to various stresses was included in the study. METHODS: For each strain, translational promoter-green fluorescent protein (gfp) fusions were used to assess the levels of ccr promoter activity in individual cells. Analyses were performed using epifluorescence microscopy and flow cytometry. RESULTS: ccr promoter activity was observed in only a small percentage of cell populations. This 'bistable' phenotype was strain dependent (GFP was expressed in N315 and RP62A, but not in COL and MW2) and growth dependent (GFP-expressing cells decreased from approximately 3% to 1% between logarithmic and stationary growth phases). The ccr promoter of strain N315 displayed normal promoter activity when expressed in SCCmec-negative N315EX. Likewise, the ccr promoter of strain COL (which was inactive in COL) showed normal N315-like activity when transformed into N315 and N315EX. CONCLUSIONS: SCCmec excision operates through bistability, favouring a small fraction of cells to 'sacrifice' their genomic islands for transfer, while the rest of the population remains intact. Determinants responsible for the activity of the ccr promoter were not located on SCCmec, but were elsewhere on the genome. Thus, the staphylococcal chromosome plays a key role in determining SCCmec stability and transferability.

Absence of Zika virus among pregnant women in Vietnam in 2008.

BACKGROUND: Despite being first identified in 1947, Zika virus-related outbreaks were first described starting from 2007 culminating with the 2015 Latin American outbreak. Hypotheses indicate that the virus has been circulating in Asia for decades, but reports are scarce. METHODS: We performed serological analysis and screened placental samples isolated in 2008 for the presence of Zika virus from pregnant women in Ho Chi Minh City (Vietnam). RESULTS: None of the placental samples was positive for Zika virus. Four serum samples out of 176 (2.3%) specifically inhibited Zika virus, with variable degrees of cross-reactivity with other flaviviruses. While one of the four samples inhibited only Zika virus, cross-reactivity with other flaviviruses not included in the study could not be ruled out. CONCLUSION: Our results support the conclusion that the virus was not present among pregnant women in the Vietnamese largest city during the initial phases of the epidemic wave.

Impact of Waddlia chondrophila infection on pregnancy in the mouse.

The intracellular bacterium Waddlia chondrophila, which belongs to the Chlamydiales order, was found to be associated with miscarriage in humans. There is little to no knowledge regarding the mode of infection, impact on the neonate and pathophysiology of this emerging bacterium. We have previously shown that W. chondrophila induces a systemic infection, organ pathology and elicits T helper type 1-associated humoral immunity in a murine model of genital infection. In the present study, we took advantage of this model of infection to evaluate the impact of this bacterium on the mouse pregnancy. We used two routes of inoculation, vaginal and intrauterine, to introduce infection before and after mating. Our results show that genital infection by W. chondrophila did not have any significant impact on gestation length and maternal weight gain, nor on the number of offspring and their weight. This observation indicates that the mouse model of infection is not suitable to study the effect of W. chondrophila on pregnancy and alternative models of infection, including in vitro ones, should be used. Moreover, an indirect immunopathological mechanism activated by this bacterium should be further explored.

Male infertility: the intracellular bacterial hypothesis.

Infertility is a disease that affects one in seven couples. As male infertility affects approximately 30% of these couples with an unknown cause in half the cases, it represents a major public health concern. The classic treatment of male infertility involves intrauterine insemination, with modest outcome, and in vitro fertilization with intracytoplasmic sperm injection, which is known to be invasive and expensive, without treating the specific cause of infertility. Male fertility is mainly evaluated through a semen assessment where abnormal parameters such as concentration and motility can be associated with a decreased chance of conception. Infectious processes represent plausible candidates for male infertility. Chlamydia trachomatis is well known to cause female infertility through tubal damage but its role in male infertility remains controversial. The link between ureaplasmas/mycoplasmas and male infertility is also debatable. The potential negative impact of these bacteria on male fertility might not only involve semen parameters but also, as with C. trachomatis, include important physiological mechanisms such as fertilization processes that are not routinely assessed during infertility investigation. Basic research is important to help determine the exact effect of these bacteria on male fertility to develop targeted treatment and go beyond in vitro fertilization with intracytoplasmic sperm injection.

Exfoliation of the beta-adrenergic receptor and the regulatory components of adenylate cyclase by cultured rat glioma C6 cells.

Cultured rat glioma C6 cells exfoliate membrane vesicles which have been termed 'exosomes' into the culture medium. The exosomes contained both stimulatory and inhibitory GTP-binding components of adenylate cyclase (the stimulatory, Gs, and the inhibitory, Gi, regulatory components) and beta-adrenergic receptors but were devoid of adenylate cyclase activity. It was therefore apparent that the catalytic component of adenylate cyclase was either not exfoliated or was inactivated during the exfoliation process. The presence of Gs or Gi in the exosomes was detected by ADP ribosylation using [alpha-32P]NAD in the presence of cholera or pertussis toxins, respectively. The exosomal concentration of each of the two components was estimated to be about one fifth of that of the cell membrane when expressed on a per mg protein basis. Exosomal Gs was almost as active as the membrane-derived Gs in its ability to reconstitute NaF- and guanine nucleotide-stimulated adenylate cyclase activity in membranes of S49 cyc- cells, which lack a functional Gs. The ability of exosomal Gs to reconstitute isoproterenol-stimulated activity, however, was much lower than that of membrane Gs. The density of beta-adrenergic receptors in the exosomes was much less than that found in the membranes. Although the exosomal receptors bound the antagonist iodocyanopindolol with the same affinity as receptors from the cell membrane, the affinity for the agonist isoproterenol was 13- to 18-fold lower in the exosomes. In addition, this affinity was not modulated by GTP in the exosomes. Thus, exfoliated beta-adrenergic receptors seem to be impaired in their ability to couple to and activate Gs. This was directly tested by coupling the receptors to a foreign adenylate cyclase using membrane fusion. The fusates were then assayed for agonist-stimulated activity. While significant stimulation of the acceptor adenylate cyclase was obtained using C6 membrane receptors, the exosomal receptors were completely inactive. Thus during exfoliation, there appear to be changes in the components of the beta-adrenergic-sensitive adenylate cyclase that results in a nonfunctional system in the exosomes.

Antihypertensive action of heparin: role of the renin-angiotensin aldosterone system and prostaglandins.

Chronic subcutaneous administration of heparin consistently lowers blood pressure in hypertensive rats. This antihypertensive effect is related at least in part to a concomitant decrease in hematocrit. Groups of spontaneously hypertensive (SHR) and normotensive Wistar (NWR) rats were treated with subcutaneous heparin (700 U/d) for 6 weeks. Weekly determinations of systolic blood pressure (tail-cuff) and hematocrit were done. Peripheral plasma renin activity, plasma aldosterone, plasma prostaglandins (PGs) (PGF2 alpha, PGI2), thromboxane A2, and urinary kallikrein were measured. Blood pressure responses of acute and chronic heparin treatment to vasoconstrictor substances, including angiotensin I, angiotensin II, and norepinephrine, were determined. As before, heparin produced a significant (P < .01) decrease in hematocrit in both SHRs and NWRs, but a parallel decrease in blood pressure was noted only in SHRs. A significant (P < .001) increase in plasma renin activity was found in heparin-treated SHRs and NWRs; however, a corresponding elevation of plasma aldosterone level was noted only in heparin-treated NWR. Plasma aldosterone level significantly (P < .01) decreased in heparin-treated SHRs. Plasma PGs and urinary kallikrein levels were not different among the groups. The blood pressure responses to vasoconstrictor substances were essentially similar among the heparin-treated and control groups. These findings suggest that PGs or kallikrein have a slight or no role in determining the antihypertensive effect of heparin. Conversely, the results suggest that a reduced aldosterone level contributes to the antihypertensive mechanism of heparin.

Oxytocinase (CAP) activity in serum during normal pregnancy.

Reference values for the activity of oxytocinase were determined in the sera of 371 women during normal pregnancy. An exponential relationship between enzyme activities and gestational age was found. The activity of oxytocinase (CAP) increased progressively from the beginning to the end of pregnancy. Statistical evaluation showed a significant difference between the 5th and 2nd months, as well as after the 20th week of pregnancy.

Determination of free and esterified cholesterol by a kinetic method. II. Evaluation of the enzymic method which uses 2,2-azino-di(3-ethyl-benzthiazoline-6-sulfonate) (ABTS) as chromogen.

A simple kinetic method for the determination of free and esterified serum cholesterol based on the oxidation of 2,2'-azino-di(3-ethyl-benzthiazoline-6-sulfonate) (ABTS) by use of choilesterol esterase, cholesterol oxidase and peroxidase has already been reported. Here the method is statistically examined. The method is very sensitive and precise (C.V. below 5%). The standard curve is linear up to 25.9 mmol/l. Comparison with results by Abell's method gave a linear regression of Yx = 0.2025 + 1.0043X with a correlation coefficient (r) of 0.983. Comparison with the enzymic methods of Roeschlau et al., Trinder, and Allain et al. gave Yx = 0.2037 + 0.9549X (r = 0.975), Yx = 0.4777 +0.8857X (r = 0.958) and Yx = 0.244 + 0.932X (r = 0.970), respectively. The effects of haemoglobin and bilirubin were studied and normal ranges for the method were determined on 150 healthy mature subjects of both sexes between 20 and 45 years of age. They are 3.375 to 6.948 mmol/l for total cholesterol, and 0.7196 to 2.089 mmol/l for free cholesterol.

The estimation of fructosamine and HbAlc in pregnant women with diabetes mellitus.

Fructosamine, HbAlc, glucose, albumins and total proteins were estimated in 40 healthy pregnant women and 80 pregnant women with insulin dependent diabetes mellitus. Fructosamine was estimated by the NBT method with "Fructosamine test" commercially available kit on Technicom automatic analyser RA-1000. Glucose was determined on Beckman glucose analyser. HbAlc was assayed by the Bio-Rad test, while albumin and total proteins by Beckman tests. For all estimated parameters no significant differences were found between healthy pregnant women and pregnant women with insulin dependent diabetes mellitus.

[Changes in N-acetyl-beta-D-glucosaminidase in the urine of patients with pyelonephritis and glomerulonephritis]. / Promena aktivnosti N-acetil-beta-D glukozaminidaze u urinu pacijenata obolelih od pijelonefritisa i glomerulonefritisa.

The activity of N-acetyl-beta-D-glucosaminidase (NAGase) activity was determined in urine of patients with pyelonephritis and glomerulonephritis. Determination was done with the non-commercial "Boehringer-Mannheim" test reagents for research purposes only. The protein concentrations were measured in the same samples. The obtained results were compared with results obtained in the control group and the significant increase (p less than 0.001) in NAGase activity was found in both groups of patients. At the same time normal protein values were found in 33% of all cases. It can be concluded that NAGase is a more sensitive parameter for early detection of renal disease.

Kinetic assay of aldehyde oxidase with 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonate) as chromogen.

A new kinetic method for the assay of aldehyde oxidase (EC 1.2.3.1) is described. By the oxidation of pyridoxal as a substrate, pyridoxic acid and hydrogen peroxide are formed. Hydrogen peroxide is subsequently reduced to water by the action of peroxidase, while the chromogen 2,2'-azino-di-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) is oxidized to a blue-greenish-dyed product. The absorbance increase of the oxidized form of ABTS, registered every minute at 410 nm, is proportional to aldehyde oxidase activity.

Effect of ethanol on prostacyclin and thromboxane A2 synthesis in rat aortic rings in vitro.

Spontaneous 6-keto-PGF1 alpha and TXB2 levels in isolated rat aortic rings increased in a concentration dependent manner after a 0.5 hour incubation with moderate or high ethanol concentrations (11 mM to 218 mM). After a 1 hour incubation with moderate concentrations of ethanol less than or equal to 22 mM) spontaneous prostaglandin (PG) production did not increase although high concentrations (87 mM and 218 mM) increased both 6-keto-PGF1 alpha and TXB2 levels. Similarly, in the presence of 40 microM Na-arachidonate, high ethanol concentrations increased PG production after 0.5 and 1 hour incubation. In addition, either a 4 or an 8 hour exposure to high ethanol concentrations increased spontaneous PG production. A moderate concentration of ethanol (22 mM) increased the 6-keto-PGF1 alpha/TXB2 ratio whereas high levels (greater than or equal to 87 mM) depressed the ratio after 0.5 and 1 hour exposure. This effect was short-lived since after 4 or 8 hours incubation with high ethanol concentrations the 6-keto-PGF1 alpha/TXB2 ratio was markedly increased. The alcohol-induced changes in both spontaneous and arachidonate-stimulated PG levels were concentration dependent and related to the incubation time. Furthermore, these data suggest that there may be unbalanced production of PGI2 and thromboxane A2 in vascular tissue exposed to alcohol.

[Correlation of glycosylated hemoglobin and fructosamine in pregnant women with diabetes mellitus]. / Korelacija glikolizovanog hemoglobina i fruktozamina kod trudnica s dijabetesom melitusom.

Glycolisated hemoglobin (HBA1c), fructosamine, glucose, albumin and total proteins were estimated 40 healthy pregnant women and 90 pregnant women with diabetes mellitus. Fructosamine was estimated by NBT method with "Fructosamine test" commercially available kit on Technicom automatic analyser RA-1000. Glucose was determined on Beckmman glucose analyser. HBA1c was assayed by Bio-Rad test, while albumin and total proteins by Beckmman tests. We found best correlation between fructosamine and HBA1c at pregnant women who were on dietary therapy worst at pregnancy women on insulin therapy.