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.

Inflammatory response to Porphyromonas gingivalis partially requires interferon regulatory factor (IRF) 3.

Innate immune activation with expression of pro-inflammatory molecules such as TNF-α is a hallmark of the chronic inflammation associated with periodontal disease (PD). Porphyromonas gingivalis, a bacterium associated with PD, engages TLRs and activates MyD88-dependent and TIR-domain-containing adapter-inducing IFN-ß (TRIF)-dependent signaling pathways. IFN regulatory factor (IRF) 3 is activated in a TRIF-dependent manner and participates in production of cytokines such as TNF-α; however, little is known regarding IRF3 and the host response to PD pathogens. We speculated that IRF3 participates in the host inflammatory response to P. gingivalis. Our results show that bone marrow macrophages (MØ) from WT mice respond to P. gingivalis with activation and nuclear translocation of IRF3. Compared with WT, MØ from IRF3(-/-), TRIF(-/-), and TLR4(-/-) mice responded with reduced levels of TNF-α on P. gingivalis challenge. In addition, full expression of IL-6 and RANTES by MØ to P. gingivalis was dependent on IRF3. Lastly, employing MØ from IRF3(-/-) and IRF7(-/-) mice we observed a significant role for IRF3 and a modest role for IRF7 in the P. gingivalis-elicited TNF-α response. These studies identify a role for IRF3 in the inflammatory response by MØ to the periodontal pathogen P. gingivalis.

Membrane stability and mitochondrial activity of human-ejaculated spermatozoa during in vitro experimental infection with Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus.

The aim of the study was to examine an in vitro effect of the three bacterial strains (Escherichia coli, Staphylococcus haemolyticus and Bacteroides ureolyticus) on ejaculated spermatozoa with reference to sperm membrane integrity and mitochondrial activity. The study was carried out on swim-up-separated spermatozoa from 12 normozoospermic volunteers. Sperm plasma membrane stability was evaluated by the LIVE/DEAD Sperm Viability Kit and by the merocyanine 540 test. Mitochondrial activity was evaluated using the JC-1 test as well as the NADH-dependent NBT assay. The percentage of dead cells was significantly higher in spermatozoa treated with B. ureolyticus as compared to that of control spermatozoa (P < 0.01). All the bacterial strains applied affected sperm plasma membrane architecture measured by M540 test (P < 0.01). Moreover, the presence of E. coli or B. ureolyticus was connected with significant decrease in both the number of cells with high mitochondrial transmembrane potential (ΔΨm) and the cells with normal oxidoreductive function of mitochondria (P < 0.05 as compared to untreated cells). To conclude, the contact of bacteria with ejaculated spermatozoa can be a reason for severe injury of sperm membrane stability and mitochondrial activity with potential consequences for male fertility.

Proteolytic inactivation of LL-37 by karilysin, a novel virulence mechanism of Tannerella forsythia.

Tannerella forsythia is a gram-negative bacterium strongly associated with the development and/or progression of periodontal disease. Here, we have shown that a newly characterized matrix metalloprotease-like enzyme, referred to as karilysin, efficiently cleaved the antimicrobial peptide LL-37, significantly reducing its bactericidal activity. This may contribute to the resistance of T. forsythia to the antibacterial activity of LL-37, since their vitality was found not to be affected by LL-37 at concentrations up to 2.2 muM. Furthermore, proteolysis of LL-37 by karilysin not only abolished its ability to bind lipopolysaccharide (LPS) to quench endotoxin-induced proinflammatory activity, but LL-37 cleavage also caused the release of active endotoxin from the LPS/LL-37 complex. Proteolytic inactivation of LL-37 bactericidal activity by karilysin may protect LL-37-sensitive species in the subgingival plaque and maintain the local inflammatory reaction driven by LPS from gram-negative bacteria. Consequently, the karilysin protease may directly contribute to periodontal tissue damage and the development and/or progression of chronic periodontitis.

Incidence, microbiology and clinical history of peritonsillar abscesses.

The objective of this study was to describe the incidence and microbiology of peritonsillar abscesses (PTA) and to study the clinical history with special reference to primary care management of these patients. We performed a retrospective study of hospital records to determine the incidence of PTA in 2000-2006, and a prospective study of consecutive PTA patients to study the microbiology of PTA, the clinical history and previous management in primary care of these patients. The incidence of PTA varied between 19 and 37/100,000 per y in the y 2000-2006. In total, 89 patients were included in the prospective study (54M, 35F), with a median age of 22 y (range 13-83 y). The most frequent single bacterial agent found was group A beta- haemolytic Streptococcus (GAS), identified in 18% of throat swabs and 24% of aspirates. The majority of PTA developed within 5 d of onset of sore throat and 54% of patients presented without prior consultation for sore throat. In the studied population the patient that first presented to primary care seems to have been appropriately managed and referred.

Bacteremic sepsis disturbs alveolar perfusion distribution in the lungs of rats.

OBJECTIVE: Sepsis often leads to lung injury, although the mechanisms that initiate this are unclear. One preinjury phenomenon that has not been explored previously is the effect of bacterial (nonlipopolysaccharide) sepsis on the distribution of alveolar perfusion. The goals of our studies were to measure this. DESIGN: Randomized, controlled, prospective animal study. SETTING: University animal laboratory. SUBJECTS: Male Sprague-Dawley rats (450-550 g). INTERVENTIONS: We induced sepsis by placing gelatin capsules containing Escherichia coli and Bacteroides fragilis into the abdomens of rats (n = 9). Empty capsules (n = 6) were placed into the abdomens of controls. After 24 hrs, 4-microm-diameter fluorescent latex particles (2 x 10(8)) were infused into the pulmonary circulation. Sepsis was induced in additional rats and controls to assess lung injury, as follows: Lung histology was performed on eight septic rats and on seven controls; lung lavage was performed on three septic rats and three controls after their plasma albumin had been labeled with Evans blue dye. MEASUREMENTS AND MAIN RESULTS: Confocal microscopy was used to prepare digital maps of latex particle trapping patterns (eight per lung). Analysis of these patterns revealed statistically more clustering (perfusion inhomogeneity) down to tissue volumes less than that of ten alveoli in septic lungs compared with controls (p < or = .05). Bacterial counts and neutrophil counts were significantly higher in the circulation of septic rats (p < or = .05). Blood pressures and arterial PO2s were unchanged. Cell counts in histological images were three-fold higher in septic lungs than in controls (p < or = .05). Lung lavage revealed 0.41 +/- 0.03 mL of plasma in the lungs of septic rats, and 0.06 +/- 0.05 mL in the lungs of controls (p < or = .05). CONCLUSIONS: Bacterial sepsis caused significant maldistribution of interalveolar perfusion in the lungs of rats in the absence of significant lung injury.

Altered expression of adhesion molecules in inflammatory cervical smears.

OBJECTIVE: The aim of the present study was to evaluate the expression of pan-cadherin and beta-catenin in cervical smears with various types of infectious agents. PATIENTS AND METHODS: Cervical smears obtained from 53 women, aged 21-65 years, with a diagnosis of specific inflammation were examined in our study. Eighteen subjects were infected by Candida albicans, 18 by Gardnerella vaginalis, nine by Bacteroides spp. and eight by Chlamydia trachomatis. All infectious agents found in the smears were at the same time confirmed by the microbiological laboratory methods. We performed a biotin-streptavidin-peroxidase immunocytochemical method using anti-beta-catenin (Clone 12F7) and anti-pan-cadherin (pan, polyclonal) antibodies. RESULTS: Aberrant expression of pan-cadherin was found in the cytoplasmic membrane of glandular, metaplastic, superficial and intermediate squamous cells in all types of infections. With regard to beta-catenin, this was expressed in majority (90%) of glandular and metaplastic cells in all types of infections and in a small proportion (15%) of superficial and intermediate squamous cells in infections caused by C. albicans and G. vaginalis. CONCLUSION: Our data show that infectious agents may cause alterations in the expression and distribution of these adhesive molecules, which can be recognized in cervical smears. Additional studies in larger sets of patients should help clarify this issue further.

Induction of colonic transmural inflammation by Bacteroides fragilis: implication of matrix metalloproteinases.

BACKGROUND: Commensal bacteria are implicated in the pathophysiology of intestinal inflammation, but the precise pathogenetic mechanisms are not known. We hypothesized that Bacteroides fragilis-produced metalloproteinases (MMPs) are responsible for bacterial migration through the intestinal wall and transmural inflammation. AIM: To investigate the role of bacterial-MMP activity in an experimental model of colitis induced by the intramural injection of bacteria. METHODS: Suspensions of viable B. fragilis or Escherichia coli were injected into the colonic wall, and the effect of the MMP inhibitor (phenantroline) on histologic lesion scores was tested. MMP activity in bacterial suspensions was measured by azocoll assay. RESULTS: The inoculation with B. fragilis induced chronic inflammatory lesions that were preferentially located in the subserosa, whereas inoculation with E. coli induced acute-type inflammatory reactions, evenly distributed in both the submucosa and subserosa. Treatment with phenantroline significantly decreased subserosal lesion scores in rats inoculated with B. fragilis, but not in rats inoculated with E. coli. Bacterial suspensions of B. fragilis showed MMP activity, but E. coli suspensions did not. Sonication of B. fragilis reduced MMP activity and virulence to induce serosal lesions. CONCLUSION: Our data suggest that bacterial MMPs may be implicated in the serosal migration of B. fragilis and in the induction of transmural inflammation.

Sepsis alters vessel contraction by adrenoceptor-induced nitric oxide and prostanoid.

BACKGROUND: Alpha-adrenergic agents contract vascular smooth muscle (VSM) and stimulate endothelial release of secondary factors which modulate VSM contraction. Our study examined constrictor prostanoid (cPN) and nitric oxide (NO) as secondary factors which could alter alpha-1 adrenoceptor-mediated contraction during sepsis. METHODS: Sepsis was induced in rats by inoculation of an implanted sponge with Escherichia coli and Bacteroides fragilis. Aortic rings at 24 h from septic (n = 21) and control (n = 21) rats were suspended in physiological salt solution (PSS) with or without blockers to NO (N(G)-monomethylarginine), cPN (mefenamic acid, MFA), or thromboxane A2 (SQ29548). Contraction dose-response curves were generated to determine maximal contraction force (F(max)) and pD2 (sensitivity) to phenylephrine in each experimental group. RESULTS: Sepsis increased F(max) to phenylephrine (PHE) (1.18 vs 0.90 g, SEM 0.0703). COX inhibition reduced the F(max) in control (0.63 vs 0.90 g, SEM 0.0675) but not in septic animals (1.19 vs 1.18 g, SEM 0.0433). TXA2 receptor inhibition did not alter F(max) in control (1.017 vs 0.973 g, SEM 0.0959) or septic animals (1.28 vs 1.12 g, SEM 0.0823). NOS inhibition enhanced the F(max) in both nonseptic (2.03 vs 0.83 g, SEM 0.0523) and septic rats (1.96 vs 1.15 g, SEM 0.0526), but did less so in the septic animals. CONCLUSIONS: PHE-induced F(max) is determined by a balance between PHE-stimulated VSM alpha-adrenoceptor activity, and PHE-stimulated endothelial release of cPN and NO. Sepsis enhances total PHE-induced F(max) by increasing VSM alpha-adrenoceptor activity and reducing PHE-stimulated endothelial release of dilator NO. Sepsis abolishes the PHE-stimulated endothelial release of cPN. PHE-stimulated cPN is not thromboxane A2, but could be a nonprostanoid dilator in the lipoxygenase (HETE) or cytochrome P450 (EET) pathways.

Anaerobic infections in the surgical patient: microbial etiology and therapy.

Anaerobic infections occur in surgical patients in part because of structural or functional defects in the host that (1) cause a breech in the normal mucosal barriers, (2) create localized vascular insufficiencies, or (3) produce an obstruction. Any or all of these events may compromise the oxidation-reduction potential within the tissues, encouraging rapid anaerobic growth. Although diverse anaerobic populations are spread throughout the gastrointestinal tract, a relatively limited number of organisms are responsible for clinical infection in the surgical patient. Many of these offending organisms express overt virulence factors that enhance microbial adherence, tissue destruction, and, in the case of Bacteroides fragilis, facilitate abscess formation. The selection of an appropriate perioperative or therapeutic agent requires a fundamental knowledge of the microbial ecology of this microbial population. The failure to consider the anaerobic flora as a component in the etiology of mixed surgical infections is associated with a high rate of perioperative and therapeutic failures.

Sustained infection induces 2 distinct microvascular mechanisms in the splanchnic circulation.

BACKGROUND: Altered intestinal blood flow during systemic inflammation leads to organ dysfunction. Mucosal ischemia occurs during sepsis despite an increase in portal blood flow. We hypothesized that separate mechanisms are active in the large resistance and small mucosal microvessels to account for this dichotomy. METHODS: Chronic infection was induced in rats by bacterial inoculation (Escherichia coli and Bacteroides fragilis) of an implanted subcutaneous sponge. Separate groups were studied at 24 and 72 hours after a single inoculation of bacterium or 24 hours after a second inoculation (ie, 72 hours of sepsis). Time-matched controls were used for each group. Intravital microscopy of the terminal ileum was used to assess endothelial-dependent vasodilation to acetylcholine (10(-9) to 10(-5) mol/L) in resistance (A(1)) and premucosal (A(3)) arterioles. Threshold sensitivity (-log of 20% response dose) was calculated from dose response curves for each animal. RESULTS: Vasodilator sensitivity to acetylcholine in A(1) arterioles was significantly decreased at 24 hours, and these changes persisted up to 72 hours after a single bacterial inoculation. There was no change in the dilator sensitivity of A(3) arterioles after a single inoculation. When there was a challenge with a second bacterial inoculation, there was a reversal of the A(1) dilator response and an increase in A(3) sensitivity. CONCLUSIONS: An initial septic event results in a decrease in dilator reactivity in the resistance A1 arterioles that persists for at least 72 hours. A sustained septic challenge results in increased dilator reactivity in both A(1) and A(3) vessels. This enhanced sensitivity during sepsis suggests that more than 1 therapeutic approach to preservation of intestinal blood flow will be necessary.

Effect of metronidazole on the pathogenicity of resistant Bacteroides strains in gnotobiotic mice.

Metronidazole is widely used to treat protozoan and fungal infections. As an antibacterial drug, it is used mainly against anaerobes. Among anaerobes, the Bacteroides fragilis group is the most relevant in terms of frequency of recovery and antimicrobial resistance patterns. The use of metronidazole and other antimicrobial drugs induces morphological changes in this bacterial group. The present study investigated in vivo if these morphological modifications were accompanied by changes in virulence patterns by using germfree mice experimentally challenged with metronidazole-resistant Bacteroides strains before and after exposure to metronidazole. It was observed that metronidazole-resistant strains were more virulent after contact with the drug, as demonstrated by anatomicopathologic data for spleen, liver, and small intestine samples. These results suggest that long-term therapy and high metronidazole concentrations could interfere with microbial pathogenicity, resulting in changes to host-bacterium relationships.

Reduced bacterial dissemination and liver injury in CD14-deficient mice following a chronic abscess-forming peritonitis induced by Bacteroides fragilis.

The CD14 myelomonocytic differentiation antigen plays a major role in acute Gram-negative infections with Escherichia coli; however, its role in chronic infections has not yet been analyzed. To address this question, we studied the role of CD14 in a chronic abscess-forming peritonitis, induced by Bacteroides fragilis. B. fragilis (3x10(8) CFU/ml) were resuspended in a liquid nutrient agar and injected into the peritoneal cavity of CD14-deficient (CD 14-/-) and normal C57BL/6J (CD 14+/+) mice, respectively. After 3 days there was a severe phlegmonous intra-abdominal inflammation in both groups. After 7 days an abscess-forming peritonitis developed and by 14 days the infectious foci were compartimentalized. These observations were indistinguishable between CD14-/- and CD14+/+ mice. Although no differences were seen in abscess formation, CD14-/- mice were able to clear B. fragilis more efficiently from the blood than CD14+/+ mice. After 3, 7, and 14 days blood cultures were B. fragilis positive in 11% (1/9), 20% (2/10), and 0% (0/9) in CD14-/-compared with 90% (9/10), 78% (7/9), and 20% (2/10) in CD14+/+ mice, respectively (P<0.05). Furthermore, although the infection resulted in hepatocellular necrosis and severe hepatitis in both groups, at day 14 the liver cell damage was more severe in CD14+/+ than in CD14-/- mice (P<0.05). These results show that the chronic abscess formation induced by B. fragilis capsular polysaccharides is CD14 independent; however, bacterial clearance and/or dissemination and liver cell damage are at least partially influenced by CD14-dependent mechanisms.

TNF-binding protein ameliorates inhibition of skeletal muscle protein synthesis during sepsis.

We examined the effects of TNF-binding protein (TNFBP) on regulatory mechanisms of muscle protein synthesis during sepsis in four groups of rats: Control; Control+TNFBP; Septic; and Septic+TNFBP. Saline (1. 0 ml) or TNFBP (1 mg/kg, 1.0 ml) was injected daily starting 4 h before the induction of sepsis. The effect of TNFBP on gastrocnemius weight, protein content, and the rate of protein synthesis was examined 5 days later. Sepsis reduced the rate of protein synthesis by 35% relative to controls by depressing translational efficiency. Decreases in protein synthesis were accompanied by similar reductions in protein content and muscle weight. Treatment of septic animals with TNFBP for 5 days prevented the sepsis-induced inhibition of protein synthesis and restored translational efficiency to control values. TNFBP treatment of Control rats for 5 days was without effect on muscle protein content or protein synthesis. We also assessed potential mechanisms regulating translational efficiency. The phosphorylation state of p70(S6) kinase was not altered by sepsis. Sepsis reduced the gastrocnemius content of eukaryotic initiation factor 2Bepsilon (eIF2Bepsilon), but not eIF2alpha. The decrease in eIF2Bepsilon content was prevented by treatment of septic rats with TNFBP. TNFBP ameliorates the sepsis-induced changes in protein metabolism in gastrocnemius, indicating a role for TNF in the septic process. The data suggest that TNF may impair muscle protein synthesis by reducing expression of specific initiation factors during sepsis.

A rat model of non-lethal bacterial infection.

The purpose of the study was to develop a small animal model of intraperitoneal infection without mortality and with a catabolic response to the infection, viz., to mimic the clinical situation in man. Intraperitoneal infection was induced in female Wistar rats by deposition of a gelatin capsule containing a mixture of Escherichia coli and Bacteroides fragilis and adjuvant substances. Seven groups of animals were infected with different bacterial inocula (0.2-4.3 x 10(6) CFU) to establish reproducible and dose-dependent changes in mortality, body weight in relation to food intake, blood cultures, peripheral blood leukocyte counts, and abscess formation on autopsy. No mortality was observed in animals with an inoculum below 2.2 x 10(6) CFU in spite of positive blood cultures. Initial weight loss was followed by weight gain in all animals except the group infected with the low inoculum (0.2 x 10(6) CFU). This group had no mortality, was in a catabolic state for three days, indicated by weight loss in spite of nearly normal food intake, and the infectious state was supported by intraperitoneal dissemination of small abscesses. The low-grade character of the infection was reflected by changes in peripheral blood lymphocyte and neutrophil granulocyte concentrations. In conclusion, this study presents a small animal model with a reproducible dose response to the bacterial challenge, allowing prolonged studies of metabolic changes following infection.

Bacteroides fragilis pyomyositis in a patient with multiple myeloma.

Pyomyositis is a bacterial infection with abscess formation affecting large skeletal muscles. It is predominantly caused by Staphylococcus aureus. The disease is common in tropical areas, but rare in temperate climates. We report a patient with multiple myeloma who developed a giant elastic tumor on the right thigh and a hen egg-sized tumor on the right upper arm. MR imaging revealed cystic spaces in the femoral quadriceps and brachial biceps muscles. A large amount of pus with foul smell was removed by incision, drainage and aspiration of the two tumors. The lesions were successfully treated with intravenous administration of antibiotics. Repeated bacterial cultures yielded only Bacteroids fragilis. To our knowledge, this is the first report of pyomyositis due to Bacteroides fragilis.

Renovascular interaction of epinephrine, dopamine, and intraperitoneal sepsis.

OBJECTIVE: To determine the effect of intraperitoneal sepsis on the systemic and renal actions of the continous infusion of epinephrine or dopamine, and during the concurrent administration of both drugs. DESIGN: Prospective, randomized study. SETTING: Laboratory at a university hospital. SUBJECTS: Seven conscious, chronically catheterized, adult merino sheep. INTERVENTIONS: Epinephrine at 40 micrograms/min or dopamine at 2 micrograms/kg/min, or both drugs concurrently were infused for 4 hrs on separate study days in healthy sheep. This protocol was then repeated following the induction of sepsis after the intraperitoneal injection of 10(11) Escherichia coli, 10(12) Bacteroides fragilis, and bran. MEASUREMENTS AND MAIN RESULTS: Systemic oxygen delivery (DO2) and consumption were measured using thermodilution cardiac output and measured oxygen content. Renal blood flow was measured using an electromagnetic flow transducer, and creatinine clearance was calculated as the quotient of renal blood flow and the renal extraction ratio of creatinine. Infusion of epinephrine augmented systemic DO2 and mean arterial pressure (MAP) during both healthy and septic studies. Systemic oxygen consumption was only increased during epinephrine infusion in the septic study. During the healthy animal study, renal blood flow was initially decreased during epinephrine infusion, but increased to 36% above baseline (p = .003). However, creatinine clearance remained unchanged. During the experimental sepsis study, the infusion of epinephrine had less marked effects on renal blood flow (unchanged from baseline), while an initial reduction (15 mins) in creatinine clearance (p = .04) was not sustained and had returned to baseline by 3 hrs. Dopamine alone produced no change in systemic oxygen variables or MAP during the studies on healthy or septic animals. Although dopamine produced renal vasodilation and an increase in renal blood flow in the healthy state, these results were not found during the septic state. In addition, concurrent infusion of dopamine with epinephrine did not alter the systemic or renal effects of epinephrine during the healthy or septic states. CONCLUSIONS: These results do not support the routine use of low-dose dopamine, and demonstrate a change in renovascular responses to catecholamines during intraperitoneal sepsis. The infusion of epinephrine at 40 micrograms/min had few deleterious effects on the kidney, and augmented both MAP and systemic DO2. Its role as a catecholamine in the management of sepsis may need to be reconsidered.