Albumin reduces the antibacterial activity of polyhexanide-biguanide-based antiseptics against Staphylococcus aureus and MRSA.

BACKGROUND: Wound infection is one of the major complications in acute and chronic wound healing. Antiseptic solutions and wound irrigating agents are routinely used for therapy and prevention in healthcare today. Even if wound exudate contains total protein concentrations up to 9.3% and albumin concentrations up to 2.7% its influence to the antibacterial efficacy of these agents is barely investigated. MATERIALS AND METHODS: This study analyzed the antibacterial effect of polyhexanide biguanide (PHMB) agents (PHMB-concentration 0.005-0.1%) against Staphylococcus aureus and methicillin-resistant-S. aureus (MRSA) after 2min incubation in presents of albumin in different concentrations (0-3%) in a standardized quantitative suspension assay. RESULTS: A significant decrease of the antibacterial activity against S. aureus was shown for a PHMB-concentration of 0.005% from 0.3% albumin (p<0.05), respectively highly significant from 0.75% (p<0.01) on. Thereby the loss of antimicrobial effect was presented as a linear correlation to the rising concentration of albumin. Furthermore a reduction of the antibacterial activity against MRSA in comparison to S. aureus was presented, for albumin concentrations from 3% on highly significant (p<0.01). CONCLUSION: The study showed that albumin causes a significant decrease of the antibacterial potency of PHMB-based antiseptics. Furthermore a diminished potency of the investigated substances for MRSA-contaminated wounds must be taken in consideration. If in vitro experiments show a significant decrease of antibacterial efficacy in the presence of albumin a sufficient activity of PHMB-based agents in clinical practice, especially in cases of exuding wounds or dried-up exudates, cannot be expected.

Nugent score related to vaginal culture in pregnant women.

OBJECTIVE: To relate Gram-stained smears, using the Nugent criteria, to quantitative and qualitative vaginal cultures in pregnant women. METHODS: Two independent evaluators using the Nugent criteria, a standardized method of Gram-stain interpretation designed to detect bacterial vaginosis, scored 104 vaginal smears from pregnant women. The quantitative and qualitative vaginal cultures were assessed at the same time and the results expressed as log(10) colony-forming units per gram of vaginal secretion. The Nugent scores were compared with the microbiologic findings. RESULTS: The prevalence of normal, intermediate, or bacterial vaginosis vaginal flora as determined by Gram stain was 68%, 21%, and 11%, respectively. A comparison of the mean bacterial counts with the Nugent score showed a weak negative correlation for Lactobacillus species and a positive correlation for gram-variable and gram-negative rods. Additional analysis revealed a strong positive correlation between the mean bacterial counts analyses of Peptostreptococcus, a genus not included in the Nugent scoring system, and the Nugent score. In addition, the Prevotella counts correlated strongly with both the Nugent score and the Peptostreptococcus counts. The quantitative counts for Lactobacillus did not vary significantly among the three defined groups of vaginal microflora; however, significant increases in the concentrations of Gardnerella vaginalis and Prevotella were found as the Nugent score increased. CONCLUSION: A strong correlation was found among the gram-variable and gram-negative genera comprised by the Nugent score. Peptostreptococcus also correlated strongly with the Nugent score and with the Prevotella counts, suggesting that this genus may play a role in determining vaginal health.

Production of toxic shock syndrome toxin 1 by Staphylococcus aureus requires both oxygen and carbon dioxide.

The effect of O(2) and CO(2) on expression of toxic shock syndrome toxin 1 (TSST-1) by Staphylococcus aureus was investigated under controlled growth conditions with continuous-culture techniques. To stimulate TSST-1 production, air and anaerobic gas were premixed before delivery to the culture vessel. At a growth rate-or mass doubling time (t(d))-of 3 h, production of specific TSST-1 (expressed as micrograms per milligram of cell dry weight) was 5. 9-fold greater at an O(2) concentration of 4% than under anaerobic conditions. Increasing the O(2) concentration to 11% did not result in a significant increase (P> 0.05) in the rate of toxin production over that during growth in 4% O(2) but did result in a significant increase (4.9-fold; P<0.001) in the rate of toxin production over that during anaerobic growth. At a t(d) of 9 h, addition of 3.5% O(2) resulted in a 7.6-fold increase in specific TSST-1 production. When room air was sparged through a culture growing at a t(d) of 9 h, TSST-1 production increased significantly (by 3.4-fold) over that during anaerobic growth. When a growth environment of 4% O(2)-remainder N(2) was studied, no increase in TSST-1 production was observed; this was also the case with 8% O(2) at gas-flow rates of 0.1, 0.2, and 0.4 liters/min. In all experiments, production of biomass (expressed as milligrams of cell dry weight per milliliter) increased, indicating that O(2) was metabolized by S. aureus. Addition of CO(2) to the gas mix (4% O(2), 10% CO(2), 86% N(2)) resulted in a 5.1- to 6.8-fold increase in TSST-1 production over that during anaerobic growth and a 3.6-fold increase over that during growth in an environment of 4% O(2)-remainder N(2). The agr mutant strain tested produced 6.1-fold more specific TSST-1 in a growth environment of 4% O(2)-10% CO(2)-86% N(2) than during anaerobic growth. These data suggest that in this system, O(2) alone does not trigger production of TSST-1; rather, both CO(2) and O(2) are required.

T cells activated by zwitterionic molecules prevent abscesses induced by pathogenic bacteria.

Immunologic paradigms classify bacterial polysaccharides as T cell-independent antigens. However, these models fail to explain how zwitterionic polysaccharides (Zps) confer protection against intraabdominal abscess formation in a T cell-dependent manner. Here, we demonstrate that Zps elicit a potent CD4+ T cell response in vitro that requires available major histocompatibility complex class II molecules on antigen-presenting cells. Specific chemical modifications to Zps show that: 1) the activity is specific for carbohydrate structure, and 2) the proliferative response depends upon free amino and carboxyl groups on the repeating units of these polysaccharides. Peptides synthesized to mimic the zwitterionic charge motif associated with Zps also exhibited these biologic properties. Lysine-aspartic acid (KD) peptides with more than 15 repeating units stimulated CD4+ T cells in vitro and conferred protection against abscesses induced by bacteria such as Bacteroides fragilis and Staphylococcus aureus. Evidence for the biologic importance of T cell activation by these zwitterionic polymers was provided when human CD4+ T cells stimulated with these molecules in vitro and adoptively transferred to rats in vivo conferred protection against intraabdominal abscesses induced by viable bacterial challenge. These studies demonstrate that bacterial polysaccharides with a distinct charge motif activate T cells and that this activity confers immunity to a distinct pathologic response to bacterial infection.

Effect of molecular size on the ability of zwitterionic polysaccharides to stimulate cellular immunity.

The large-molecular-sized zwitterionic capsular polysaccharide of the anaerobe Bacteroides fragilis NCTC 9343, designated polysaccharide (PS) A, stimulates T cell proliferation in vitro and induces T cell-dependent protection against abscess formation in vivo. In the present study, we utilized a modification of a recently developed ozonolytic method for depolymerizing polysaccharides to examine the influence of the molecular size of PS A on cell-mediated immunity. Ozonolysis successfully depolymerized PS A into structurally intact fragments. PS A with average molecular sizes of 129.0 (native), 77.8, 46.9, and 17.1 kDa stimulated CD4+-cell proliferation in vitro to the same degree, whereas the 5.0-kDa fragment was much less stimulatory than the control 129.0-kDa PS A. Rats treated with 129.0-kDa, 46.9-kDa, and 17.1-kDa PS A molecules, but not those treated with the 5.0-kDa molecule, were protected against intraabdominal abscesses induced by challenge with viable B. fragilis. These results demonstrate that a zwitterionic polysaccharide as small as 22 repeating units (88 monosaccharides) elicits a T cell-dependent immune response. These findings clearly distinguish zwitterionic T cell-dependent polysaccharides from T cell-independent polysaccharides and give evidence of the existence of a novel mechanism for a polysaccharide-induced immune response.

Microbial interactions in the vaginal ecosystem, with emphasis on the pathogenesis of bacterial vaginosis.

During bacterial vaginosis (BV), populations of lactobacilli which are generally dominant in the vagina of overtly healthy women are replaced by other facultative and anaerobic microorganisms. Some Lactobacillus strains produce hydrogen peroxide and all produce lactic acid; however, the antagonistic role of these metabolites in vivo remains controversial. Positive interactions among BV-associated organisms may contribute to the pathogenesis of BV and its sequelae.

Effect of surgical adhesion reduction devices on the propagation of experimental intra-abdominal infection.

HYPOTHESIS: The use of certain surgical adhesion reduction devices where there is a risk of concomitant bacterial contamination potentiates intra-abdominal infection. DESIGN: Evaluation of adhesion reduction devices in an experimental model of intra-abdominal infection. SETTING: Experimental animal model. INTERVENTIONS: Adhesion reduction devices were administered at the time of bacterial challenge. MAIN OUTCOME MEASURES: Animal mortality rate, abscess formation, and bacterial counts in peritoneal fluid and blood cultures. RESULTS: The use of bioresorbable membrane adhesion reduction devices in the presence or absence of antibiotic therapy did not alter the disease process as compared with appropriate control groups. However, adhesion reduction gels prepared from sodium hyaluronate and carboxymethylcellulose chemically modified with carbodiimide or ferric ion complexed sodium hyaluronate increased the incidence of peritonitis in treated animals. Gel formulations containing diimide-modified carboxymethylcellulose did not have this effect. CONCLUSIONS: The use of certain adhesion reduction devices resulted in the propagation of intra-abdominal infection in an experimental rat model. This outcome was dependent on the composition of the device employed. The use of adhesion reduction devices should be tested in appropriate models of infection where there is the risk of concomitant bacterial contamination.

IL-2 mediates protection against abscess formation in an experimental model of sepsis.

Little is known regarding the mechanism by which T cells control intraabdominal abscess formation. Treating animals with polysaccharide A (PS A) from Bacteroides fragilis shortly before or after challenge protects against abscess formation subsequent to challenge with different abscess-inducing bacteria. Although bacterial polysaccharides are considered to be T cell-independent Ags, T cells from PS A-treated animals mediate this protective activity. In the present study, we demonstrate that CD4+ T cells transfer PS A-mediated protection against abscess formation, and that a soluble mediator produced by these cells confers this activity. Cytokine mRNA analysis showed that T cells from PS A-treated animals produced transcript for IL-2, IFN-gamma, and IL-10, but not for IL-4. The addition of IL-2-specific Ab to T cell lysates taken from PS A-treated animals abrogated the ability to transfer protection, whereas the addition of Abs specific for IFN-gamma and IL-10 did not affect protection. Finally, administration of rIL-2 to animals at the time of bacterial challenge prevented abscess formation in a dose-dependent manner. These data demonstrate that PS A-mediated protection against abscess formation is dependent upon a CD4+ T cell-dependent response, and that IL-2 is essential to this immune mechanism.

Analysis of a capsular polysaccharide biosynthesis locus of Bacteroides fragilis.

A major clinical manifestation of infection with Bacteroides fragilis is the formation of intra-abdominal abscesses, which are induced by the capsular polysaccharides of this organism. Transposon mutagenesis was used to locate genes involved in the synthesis of capsular polysaccharides. A 24,454-bp region was sequenced and found to contain a 15,379-bp locus (designated wcf) with 16 open reading frames (ORFs) encoding products similar to those encoded by genes of other bacterial polysaccharide biosynthesis loci. Four genes encode products that are similar to enzymes involved in nucleotide sugar biosynthesis. Seven genes encode products that are similar to sugar transferases. Two gene products are similar to O-acetyltransferases, and two products are probably involved in polysaccharide transport and polymerization. The product of one ORF, WcfH, is similar to a set of deacetylases of the NodB family. Deletion mutants demonstrated that the wcf locus is necessary for the synthesis of polysaccharide B, one of the two capsular polysaccharides of B. fragilis 9343. The virulence of the polysaccharide B-deficient mutant was comparable to that of the wild type in terms of its ability to induce abscesses in a rat model of intra-abdominal infection.

Nonlinear models for in vitro kill kinetics of antibiotics.

In this study, we developed nonlinear regression models to analyze the data generated from an in vitro continuous culture system to assess the kinetics of metronidazole and trovafloxacin in inhibiting the growth of Bacteroides fragilis. The model includes parameters describing the initial shock effect of an antibiotic pulse, the overall antibiotic wash-out rate from the system, and the long-term toxicity of the antibiotic in the environment after one pulse and before the next pulse.

Correlation of cecal microflora of HLA-B27 transgenic rats with inflammatory bowel disease.

Transgenic rats with a high level of expression of the human major histocompatibility complex class I molecule HLA-B27 develop chronic inflammatory bowel disease (IBD) and arthritis. Assessment of the cecal microflora showed a rise in numbers of Escherichia coli and Enterococcus spp., corresponding to the presence and severity of IBD in these rats.

Cellular mechanism of intraabdominal abscess formation by Bacteroides fragilis.

We investigated the cellular mechanism by which Bacteroides fragilis promotes the development of intraabdominal abscesses in experimental models of sepsis. B. fragilis, as well as purified capsular polysaccharide complex (CPC) from this organism, adhered to primary murine mesothelial cells (MMCs) in vitro. The binding of CPC to murine peritoneal macrophage stimulated TNF-alpha production, which when transferred to monolayers of MMCs elicited significant ICAM-1 expression by these cells. This response resulted in enhanced polymorphonuclear leukocyte attachment to MMCs that could be inhibited by Abs specific for TNF-alpha or ICAM-1. Mice treated with TNF-alpha- or ICAM-1-specific Abs failed to develop intraabdominal abscesses following challenge with purified CPC. These results illustrated the role of the CPC in promoting adhesion of B. fragilis to the peritoneal wall and coordinating the cellular events leading to the development of abscesses associated with experimental intraabdominal sepsis.

Pharmacodynamics and microbiology of trovafloxacin in animal models of surgical infection.

Trovafloxacin provides broad in vitro and in vivo coverage of the aerobic and anaerobic pathogens found frequently in surgical infections. In vitro susceptibility testing indicated that trovafloxacin inhibited gram-positive staphylococci and enterococci, numerous gram-negative organisms, including Escherichia coli, and anaerobic pathogens, such as Bacteroides fragilis. Trovafloxacin protected mice from lethal infections induced by gram-negative or gram-positive organisms, even when these organisms were inoculated in combination with B. fragilis. Trovafloxacin protected rats in models of intra-abdominal sepsis induced by inoculation with E. coli and B. fragilis or with multiple aerobic and anaerobic pathogens. In these experimental models, trovafloxacin protected rats from lethal infection, reduced intra-abdominal abscess formation, and inhibited bacterial growth. Drug concentrations were greater in intra-abdominal abscesses than in serum, reflecting the good tissue penetration of trovafloxacin. These results indicate that trovafloxacin may be effective in prophylaxis and treatment of mixed infections in surgical patients.

A commensal symbiosis between Prevotella bivia and Peptostreptococcus anaerobius involves amino acids: potential significance to the pathogenesis of bacterial vaginosis.

In both batch and continuous culture, Peptostreptococcus anaerobius was able to grow in vaginal defined medium with Prevotella bivia, but not in pure culture. Growth of P. anaerobius was increased by 238% (P < 0.001) in peptone-supplemented vaginal defined medium conditioned by prior growth of P. bivia. Analysis of P. bivia culture supernatants showed a net accumulation of amino acids and subsequent growth of P. anaerobius in the conditioned supernatants resulted generally in amino acid utilization. Supplementation of peptone-supplemented vaginal defined medium with amino acids in concentrations similar to those available after prior growth with P. bivia were growth-stimulatory (246%, P=0.006) for P. anaerobius. Increased availability of amino acids by P. bivia is proposed as a mechanism to support the observed in vitro commensal symbiosis between P. bivia and P. anaerobius.

Evaluation of the AnaeroPack system for growth of clinically significant anaerobes.

The AnaeroPack (Mitsubishi Gas Chemical America, Inc., New York, N.Y.) system was compared with the GasPak (Becton Dickinson Microbiology Systems, Cockeysville, Md.) system and a conventional anaerobe chamber to evaluate the ability of the AnaeroPack system to support the growth of clinically significant anaerobes. The AnaeroPack system requires no catalyst or water, produces no hydrogen, and is oxygen absorbing and carbon dioxide generating. It is simple to use and reduces preparation time to a minimum. One hundred forty clinical isolates obtained from various anatomic sites and 10 American Type Culture Collection type strains were evaluated. Isolates were plated on various media, and bacterial growth was examined after 24, 48, 72, and 168 h of incubation. Criteria for evaluation and comparison of systems included rate and quality of growth, colonial morphology, hemolytic reactions, and pigment production. Results indicate that the AnaeroPack system is highly effective in creating an anaerobic atmosphere. The AnaeroPack system never failed to reduce the methylene blue indicator, while the GasPak system failed 15% of the time. The rate or quality of growth achieved by the AnaeroPack system compared with that of established anaerobic culturing techniques was similar and significantly better for several genera including the Bacteroides fragilis group, Fusobacterium, Clostridium, and Peptostreptococcus. The AnaeroPack system appears to be an excellent alternative to established methods for generating an environment for anaerobic incubation.

Evidence for a commensal, symbiotic relationship between Gardnerella vaginalis and Prevotella bivia involving ammonia: potential significance for bacterial vaginosis.

Six strains of Prevotella bivia and 4 of Gardnerella vaginalis were examined for nutrient substrate utilization as part of ongoing studies on the pathogenesis of bacterial vaginosis. Addition of single amino acids to vaginal defined medium (VDM) was stimulatory to the growth of P. bivia but not to G. vaginalis. However, peptides significantly promoted the growth of both organisms. Growth of P. bivia in VDM and VDM supplemented with either amino acids or peptone was accompanied by net ammonia production, while growth of G. vaginalis under the same conditions resulted in net ammonia utilization. Ammonia-enriched supernatants from the growth of P. bivia in peptone-supplemented VDM were stimulatory to G. vaginalis growth. However, ammonia-reduced supernatants from G. vaginalis growth in peptone-supplemented VDM had a neutral effect on P. bivia growth. A commensal relationship between P. bivia to G. vaginalis is proposed, with ammonia flow as a mechanism to support this hypothesis.

Statistical models for vaginal microflora: identifying women at risk for group B streptococcus colonization as a test of concept.

OBJECTIVE: The purpose of this study was to formulate a statistical model that relates human microflora to probabilities for vaginal colonization by group B Streptococcus (GBS). METHODS: Longitudinal observations of total bacterial concentrations at various times during the menstrual cycle were obtained from overtly healthy, non-pregnant, menarcheal women. During each menstrual period and at appropriate intermenstrual times, the duplicate swab technique was used to sample the vaginal vault to obtain microbiologic samples. Women were identified as being colonized with GBS if their samples contained faculative gram-positive cocci. The method of generalized estimating equation (GEE) was used to model the longitudinal data set. RESULTS: Concentrations of Corynebacterium sp., Streptococcus spp., and total anaerobic bacteria were found to be risk factors for GBS colonization. The sensitivity of the predictive model is 84% and the specificity is 79%. CONCLUSIONS: Although vaginal cultures for GBS are routinely performed to detect colonization, the statistical model described identifies associated risk factors which may be important determinants for GBS colonization.

Evaluation of a new enzyme immunoassay for Clostridium difficile toxin A.

AIM: To evaluate a new enzyme immunoassay (EIA) method for detection of Clostridium difficile toxin by comparing it to cytotoxicity assay. To investigate the nature of false negative and false positive EIA results by evaluating clinical and therapeutic parameters. METHODS: 737 consecutive diarrhoeal specimens collected from patients clinically suspected of having C difficile colitis were tested for the presence of C difficile toxin by EIA for toxin A and by cytotoxicity assay. Clinical data were evaluated in all cases positive by either method. RESULTS: With the cytotoxicity assay as a gold standard, the specificity of EIA for toxin detection was 99.3% and the sensitivity was 62.2%. No false negative EIA specimens were obtained from patients already being treated for C difficile colitis. Among patients with cytotoxicity positive specimens, those with EIA positive samples had no clinical features distinguishing them from patients with EIA negative samples. CONCLUSIONS: Although specific, the new EIA method directed against toxin A lacks sensitivity compared to cytotoxicity. False negative EIA tests are not associated with concurrent treatment for C difficile colitis nor with any specific clinical features examined in our study.

Protein and lipid refeeding changes protein metabolism and colonic but not small intestinal morphology in protein-depleted rats.

In this study, we fed rats a 2% casein AIN 76 diet for 2 wk to produce protein malnutrition. We determined in these animals the effects of different concentrations of dietary protein refeeding (2% and 20% casein) on recovery and gut mucosal repletion and the potential role of type of dietary fat in the regulation of protein metabolism and mucosal growth by providing conventional long-chain triglyceride (LCT), a structured lipid composed of long-, medium- and short-chain fatty acids (SC/SL), or a physical mixture of the same components present in the structured lipid given as individual pure triglycerides (SC/PM) along with adequate amounts of protein and energy. The results confirmed that protein malnutrition can be reversed rapidly by protein refeeding, as indicated by an increase in body weight, positive nitrogen balance, liver growth and elevations in plasma concentrations of insulin-like growth factor-1, leucine and albumin. In the colon, crypt cell number, crypt depth and number of crypt cells in the rapidly proliferating fraction of the colon were greater in rats fed the higher protein diet. However, the general architecture of small intestinal mucosa, including duodenum, jejunum and ileum, was not affected by protein malnutrition. Although the number of colonic cells was similar with fat refeeding, there were significantly fewer displaying the proliferating cell nuclear antigen in the colonic epithelium when rats were fed SC/PM compared with SC/SL. Therefore, changes in colonic mucosal proliferation were only seen with repletion by adequate protein and by SC/SL feeding.