Macrolide and lincosamide resistance of Streptococcus agalactiae in pregnant women in Poland.

Knowing about the antibiotic resistance, serotypes, and virulence-associated genes of Group B Streptococcus for epidemiological and vaccine development is very important. We have determined antimicrobial susceptibility patterns, serotype, and virulence profiles. The antibiotic susceptibility was assessed for a total of 421 Streptococcus agalactiae strains, isolated from pregnant women and neonates. Then, 89 erythromycin and/or clindamycin-resistant strains (82 isolates obtained from pregnant women and seven isolates derived from neonates) were assessed in detail. PCR techniques were used to identify the studied strains, perform serotyping, and assess genes encoding selected virulence factors. Phenotypic and genotypic methods determined the mechanisms of resistance. All tested strains were sensitive to penicillin and levofloxacin. The constitutive MLSB mechanism (78.2%), inducible MLSB mechanism (14.9%), and M phenotype (6.9%) were identified in the macrolide-resistant strains. It was found that macrolide resistance is strongly associated with the presence of the ermB gene and serotype V. FbsA, fbsB, fbsC, scpB, and lmb formed the most recurring pattern of genes among the nine surface proteins whose genes were analysed. A minority (7.9%) of the GBS isolates exhibited resistance to lincosamides and macrolides, or either, including those that comprised the hypervirulent clone ST-17. The representative antibiotic resistance pattern consisted of erythromycin, clindamycin, and tetracycline resistance (71.9%). An increase in the fraction of strains resistant to macrolides and lincosamides indicates the need for monitoring both the susceptibility of these strains and the presence of the ST-17 clone.

Increasing Resistance and Changes in Distribution of Serotypes of Streptococcus agalactiae in Poland.

Streptococcus agalactiae is responsible for serious infections in newborn babies, pregnant women, and other patients. The aim of this study was to evaluate antimicrobial susceptibility, serotype distribution, and virulence determinants of the S. agalactiae isolates derived from clinical specimens considering the global increase of both antibiotic resistance and virulence. A total of 165 isolates were identified and serotyped by PCR techniques. Antimicrobial susceptibility was assessed by disk diffusion method, gradient diffusion method and VITEK® System. Virulence associated genes were investigated by PCR; ability to form biofilm was assessed using a microtiter plate assay. The highest observed MIC value for penicillin G was 0.12 µg/mL, seen in 8.5% of isolates. Resistance to erythromycin and clindamycin were found in 30.38% and 24.8% of the strains, respectively. The serotype III (32.73%), V (25.45%), and Ia (18.18%) were found as the most frequently represented. Previously unidentified strains in Poland, belonging to serotypes VI (three strains) and VII (one strain) were recognized. The presence of genes encoding various virulence factors as well as diverse ability to form biofilm were found. In conclusion, macrolide-resistance and decreased susceptibility to penicillin G were revealed signifying the increasing resistance among group B streptococci. Moreover, the presence of genes encoding various virulence factors and the ability to form biofilm were confirmed indicating their role in the pathomechanisms of the evaluated GBS infections.

Can apoptosis and necrosis coexist in ejaculated human spermatozoa during in vitro semen bacterial infection?

PURPOSE: To evaluate whether ejaculated human spermatozoa undergo complete apoptosis or necrosis during experimental semen bacterial infection in vitro. METHODS: Apoptotic markers, including mitochondrial transmembrane potential (ΔΨm), phosphatidylserine (PS) externalization, and DNA fragmentation, have been detected simultaneously in ejaculated human sperm after their incubation with a known pathogenic (Escherichia coli), as well as with conditionally pathogenic bacterial strains (Staphylococcus haemolyticus, Bacteroides ureolyticus) and/or leukocytes. The ΔΨm and translocation of PS was evaluated using the JC-1 and Annexin V binding tests, respectively. A modified TUNEL assay with additional staining for sperm viability was used to detect the DNA fragmentation level. RESULTS: The exposure of ejaculated spermatozoa to bacterial strains was associated with a simultaneous decrease in the percentage of sperm with normal ΔΨm and an increase in the proportion of Annexin V-positive sperm. Additionally, in the presence of S. haemolyticus, B. ureolyticus and/or leukocytes, a significant increase in the percentage of live TUNEL-positive (apoptotic) as well as dead TUNEL-positive (necrotic) sperm cells was also observed. CONCLUSIONS: The cellular death observed in spermatozoa in the presence of inflammatory mediators may be due to both apoptosis and necrosis. Here, we demonstrate for the first time that direct contact of conditionally pathogenic bacteria with ejaculated human sperm may play an even greater role in the promotion of apoptosis than in case of some pathogenic bacterial strains. These findings suggest that significant bacteriospermia and leukocytospermia may be direct causes of subfertility or additional negative factors worsening the prognosis of fertility in natural and assisted procreation.

Fertilizing potential of ejaculated human spermatozoa during in vitro semen bacterial infection.

OBJECTIVE: To assess the in vitro effect of three bacterial isolates (Escherichia coli, serotype O75:HNT, Staphylococcus haemolyticus, and Bacteroides ureolyticus) and/or leukocytes on sperm motility, subcellular changes in sperm plasma membranes, and sperm fertilizing potential. DESIGN: An in vitro model of semen bacterial infection. SETTING: Basic research laboratory. PATIENT(S): Healthy normozoospermic volunteers and healthy blood donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm plasma membrane stability was evaluated with a LIVE/DEAD Sperm Viability Kit and with the merocyanine 540 (M540) test both performed using flow cytometry. An oxiSelect TBARS Assay Kit was used for quantitative measurement of malondialdehyde content. Functional ability of spermatozoa was assessed by hypo-osmotic swelling (HOS) test and sperm penetration assay (SPA). RESULT(S): The incubation of sperm with bacteria and/or leukocytes was associated with the reduction of their fertilizing potential demonstrated in both the HOS test and SPA, and this effect can be considered as a natural consequence of diminished motility and sperm membrane injury of lipid bilayers. Bacteroides ureolyticus demonstrated the most significant detrimental effect on sperm structure and function. CONCLUSION(S): Sperm motility and lipid sperm membrane status might be the earliest and the most sensitive indicators of sperm damage with negative consequences for male factor fertility, which can be attributed to both bacteria and leukocytes action.

Novel morphological findings of human sperm removal by leukocytes in in vivo and in vitro conditions: preliminary study.

PROBLEM: Biological significance of increased number of leukocytes in ejaculate remains a subject of controversy. The aim of this research was to describe the morphological characteristics of the sperm elimination by leukocytes in in vivo and in vitro conditions using natural stimulator of the immune system--uropathogenic Escherichia coli, O75:HNT, isolated from semen. METHOD OF STUDY: The study was performed on ejaculated spermatozoa from normozoospermic men with leukocytospermia (n=10, in vivo experiment) or without leukocytospermia (n=15, in vitro experiment). Morphological observations were performed using light and scanning electron microscopy. RESULTS: Sperm removal by active leukocytes mediated by traditional phagocytosis and generation of extracellular traps were observed in in vivo and in vitro experiments. CONCLUSION: Our morphological data suggest that human germ cells activate leukocytes triggering both traditional phagocytosis and a novel trapping mechanism, followed by extensive sperm elimination.

In vitro reconstruction of inflammatory reaction in human semen: effect on sperm DNA fragmentation.

The study was aimed at evaluating an in vitro induction of DNA damage in three sperm subpopulations exposed to selected inflammatory mediators, such as leukocytes, two combinations of pro-inflammatory cytokines (interleukin [IL]-6 + IL-8 and IL-12 + IL-18) and two bacterial strains (Escherichia coli and Bacteroides ureolyticus). Semen samples from normozoospermic volunteers were differentiated by swim-up (swim-up fraction) and Percoll gradient procedures (90% and 47% Percoll fractions). Leukocytes were isolated from the whole heparinized blood using the density gradient centrifugation technique. DNA fragmentation in sperm fractions was evaluated using flow cytometry with TUNEL labeling and Comet assay. Out of the inflammatory factors tested, bacteria were found to have a greatest toxic effect on sperm DNA, especially in fractions isolated by Percoll gradient, compared with untreated cells (P < 0.05). The results indicate that inflammatory mediators can be a direct cause of DNA fragmentation in ejaculated spermatozoa, which can ultimately lead to limited fertilizing abilities of the germ cells. In contrast to the swim-up technique, the selection of spermatozoa by gradient procedures increases the vulnerability of mature spermatozoa to the harmful effects of infectious agents on DNA integrity. This observation may have some meaning for recommendations concerning laboratory techniques used in assisted reproductive therapy.

[Occurrence of the hypervirulent ST-17 clone of Streptococcus agalactiae in pregnant women and newborns]. / Wystepowanie hiperwirulentnego klonu ST-17 Streptococcus agalactiae u kobiet ciezarnych oraz noworodków.

UNLABELLED: OBJECTIVES. The ST-17 clone of Streptococcus agalactiae (Group B Streptococcus, GBS) identified by Multi Locus Sequence Typing (MLST) is recognized as a hypervirulent international clone mainly associated with invasive neonatal infections. There is no data on the occurrence of the ST-17 clone of GBS in pregnant women and newborns in Poland. METHODS: 169 strains of GBS, isolated from pregnant women's carriage, and 42 from newborns with invasive infection or asymptotic carriage, which were derived from the collection of the Chair of Microbiology Jagiellonian University Medical College, collected between 2006-2010, were genetically characterized. The PCR with specific primers ST17S and ST17AS was adopted to characterize the gbs2018 gene variant specific to ST-17 clone. All positive results in direction of ST-17 were confirmed by MLST. The genetic relatedness of the ST-17 clone was determined by PFGE. RESULTS: The hypervirulent ST-17 clone was detected in 2.4% of women with GBS asymptotic carriage, whereas it was detected in 18.2% newborns with invasive infection. No ST-17 was isolated from healthy neonates. Statistically significant correlation between the presence of ST-17 and neonatal GBS infection was showed, in comparison to healthy neonates colonized by GBS (p = 0.0398) and to GBS-positive pregnant women (p <0.00001). The ST-17 strains belonged to 2 clearly distinguishable PFGE type, but were closely related to one another at a level of 60% similarity. CONCLUSIONS. The results emphasize the necessity of a regular study of the ST-17 GBS clone occurrence among pregnant women and newborns in Poland.

Clonal analysis of Staphylococcus aureus strains isolated in obstetric-gynaecological hospital.

Epidemiological studies were carried out on 135 isolates of Staphylococcus aureus strains originating from medical staff, patients, and hospital environment. Restriction fragment length polymorphism (RFLP) analysis revealed genetic diversity of S. aureus isolates. Some clones were transmitted among nurses, doctors and patients. Our studies also demonstrate contamination of the hospital environment with S. aureus strains and there is a possibility that the patients acquire staphylococci from the environment. Moreover, we found that many medical staff workers were colonized with S. aureus and the transmission of these strains to patients is possible.

Candida-associated denture stomatitis in type 2 diabetes mellitus.

OBJECTIVE: To describe the clinical appearance of Candida-associated denture stomatitis (DS) in subjects with type 2 diabetes (T2DM). The relationships between the types of DS, oral complaints and associated conditions were assessed in terms of glycemic control as determined by glycated hemoglobin (HbA1c) measurements. MATERIALS AND METHODS: Demographic and clinical data were obtained from questionnaires and oral examinations of 110 edentulous patients with T2DM and 50 control subjects. RESULTS: Type II DS commonly occurred in diabetics (57.3% vs 30%; p=0.002) together with DS related oral complaints (60.9% vs 24%; p<0.001) compared with controls. Burning sensation of the mouth (BS) was the most common complaint. Dryness of the oral mucosa (DOM) (50.9% vs 6%; p<0.001), angular cheilitis (26.4% vs 8%; p=0.01) and glossitis (27.3% vs 6%; p=0.003) occurred more frequently in diabetics. Oral complaints and associated conditions of DS coincided with elevated HbA1c levels (p<0.001). Diabetics with extensive type of inflammation had higher HbA1c levels than type I/III DS subjects (p<0.001). CONCLUSIONS: Diffuse type of inflammation was associated with T2DM. BS and DOM were the most common oral complaints. Inadequately controlled diabetes with Candida-associated DS was linked to a high incidence of an extensive type of inflammation, oral complaints and associated conditions.

Carriage of genes for various extended-spectrum beta-lactamases: a novel resistance strategy of Klebsiella pneumoniae in Poland.

Among 110 randomly sampled strains from a collection of 247 extended-spectrum beta-lactamase (ESBL)-producing clinical isolates of Klebsiella pneumoniae collected from hospitalised children in three paediatric hospitals in Poland, 64 strains (58.2%) with multiple ESBLs were found, including five non-clonal strains (4.5%) harbouring bla genes for ESBLs of three families (CTX-M, SHV and TEM). This is the first report of the emergence of triple ESBL-producing K. pneumoniae in Poland. In addition, K. pneumoniae strains harbouring bla genes for TEM-130 and TEM-132 ESBLs were detected in Poland for the first time. Epidemiological analysis of the multiple ESBL-producing K. pneumoniae isolates by pulsed-field gel electrophoresis (PFGE) revealed a relatively high genetic diversity between isolates producing the same combination of enzymes. Clonally related strains were uncommon.

Bacteria trigger oxygen radical release and sperm lipid peroxidation in in vitro model of semen inflammation.

OBJECTIVE: To assess the in vitro effect of five bacterial strains isolated from semen samples (Escherichia coli, Staphylococcus haemolyticus, Streptococcus oralis, Bacteroides ureolyticus, and Ureaplasma urealyticum) on reactive oxygen intermediate (ROI) release and lipid sperm membrane peroxidation in the coincubated suspensions of white blood cells (WBC) with spermatozoa. DESIGN: An in vitro model of semen infection. SETTING: Basic research laboratory. PATIENT(S): Healthy normozoospermic volunteers and healthy blood donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Chemiluminescent assay was used to evaluate ROI generation by WBC. Malondialdehyde (MDA) concentration was determined in sperm lysates using high-performance liquid chromatography. RESULT(S): Of the bacterial strains tested, B. ureolyticus, S. haemolyticus, and E. coli caused the greatest damage to sperm membrane lipids. An increase in MDA levels in sperm lysates was a natural consequence of bacteria-induced ROI generation. The WBC usually enhanced harmful activity of the infectious agent toward the cell membranes. CONCLUSION(S): The harmful effect of bacteria on spermatozoa depends on the type and species of microorganisms invading, colonizing, or infecting the male genital tract and is associated with the accompanying oxidative stress. The presence of leukocytes in semen appears to be the additional factor enhancing the sperm lipid membrane peroxidation, which may affect the fertility status.

Consequences of semen inflammation and lipid peroxidation on fertilization capacity of spermatozoa in in vitro conditions.

A body of data exists on reactive oxygen species (ROS) release, however, no direct correlation was found between the oxidative stress and infertility. The aim of the study was to measure semen oxidative stress and its correlation with classical in vitro fertilization (IVF) rate. A prospective study in academic non-profit institution where 79 infertile couples were subjected to IVF programme was conducted. Two infertile groups were discriminated according to the pronuclei presence in IVF. The main outcome measure (pronuclei presence) was then correlated with lipid peroxidation product in semen (ROS effect). Although the average IL-8 levels and malondialdehyde (MDA) content in semen did not differ between the studied subgroups (successful vs. non-successful fertilization), a statistically significant negative correlation was found between MDA level and fertilization rate in performed regression analysis. Thus we may suggest that MDA levels in seminal plasma may have prognostic value for IVF success.

Male genital tract infection: an influence of leukocytes and bacteria on semen.

We have studied the oxidative status of 155 semen samples, 95 originating from healthy individuals and 60 from infertile patients, which were subdivided into two groups: (a) normozoospermic with genitourinary tract infection (GTI); and (b) with pathological spermiogram and GTI. Several phases of infection were observed: with bacterial presence only, bacteria and leukocytes, and leukocytes only, following the routine inflammatory pattern. Leukocyte numbers, bacterial strains, pro- and anti-oxidants, and selected pro-inflammatory cytokines (IL-1 beta, IL-6, IL-8 and TNF-alpha) were studied. Additionally, two oxido-sensitive indices were created (SOD/XO and CAT/XO) in order to follow particular phases of semen infection in two subgroups of patients. Different patterns of activities of pro- and anti-oxidant substances, as well as cytokines, were observed in the studied populations. It was reflected mainly by elevated XO activity in a group of patients with a pathological spermiogram while, in a group of patients with GTI and normozoospermia, xanthine oxidase was normal. In the latter group, oxido-sensitive indices were elevated in favour of anti-oxidants; similarly, this occurred with IL-6 levels in comparison to healthy controls. It appears therefore that normozoospermic semen recovers better after infection than pathological semen. Perhaps, IL-6 secretion might be helpful in the observed recovery?