Elisabet Holst

Biofilm formation by Propionibacterium acnes is a characteristic of invasive isolates

Propionibacterium acnes is a common and probably underestimated cause of delayed joint prosthesis infection. Bacterial biofilm formation is central in the pathogenesis of infections related to foreign material, and P. acnes has been shown to form biofilm both in vitro and in vivo. Here, biofilm formation by 93 P. acnes isolates, either from invasive infections (n = 45) or from the skin of healthy people (n = 48), was analysed. The majority of isolates from deep infections produced biofilm in a microtitre model of biofilm formation, whereas the skin isolates were poor biofilm producers (p <0.001 for a difference). This indicates a role for biofilm formation in P. acnes virulence. The type distribution, as determined by sequencing of recA, was similar among isolates isolated from skin and from deep infections, demonstrating that P. acnes isolates with different genetic backgrounds have pathogenic potential. The biofilm formed on plastic and on bone cement was analysed by scanning electron microscopy (EM) and by transmission EM. The biofilm was seen as a 10-mum-thick layer covering the bacteria and was composed of filamentous as well as more amorphous structures. Interestingly, the presence of human plasma in solution or at the plastic surface inhibits biofilm formation, which could explain why P. acnes primarily infect plasma-poor environments of, for example, joint prostheses and cerebrospinal shunts. This work underlines the importance of biofilm formation in P. acnes pathogenesis, and shows that biofilm formation should be considered in the diagnosis and treatment of invasive P. acnes infections.

Bacterial vaginosis: Microbiological and clinical findings

A prospective study was performed involving 101 women who consecutively attended a primary health care unit for complaints of genital malodour and/or abnormal vaginal discharge. Bacterial vaginosis was diagnosed in 34 women on the basis of four diagnostic criteria: vaginal pH > 4.7, homogeneous vaginal discharge, a positive amine test and clue cells. The sensitivity of these criteria was > 90 % except for homogeneous discharge (82 %). Their specificity was > 90% except for vaginal pH > 4.7 (46%); a specificity of 87% could have been achieved by using the criterion for vaginal pH ⩾ 5.0. There was a strong association between diagnosis of bacterial vaginosis and the concomitant occurrence ofGardnerella vaginalis, Mobiluncusspp. andBacteroidesspp. There was no difference between women with or without bacterial vaginosis as regards contraception methods (except for use of an intrauterine device), age at first intercourse, or earlier episodes of vaginal discharge. Sexual transmission of the predominant bacteria was not supported by data collected from the male consorts.

Bacterial vaginosis - a microbiological and immunological enigma

The development of bacterial vaginosis (BV) among women of childbearing age and the resulting quantitative and qualitative shift from normally occurring lactobacilli in the vagina to a mixture of mainly anaerobic bacteria is a microbiological and immunological enigma that so far has precluded the formulation of a unifying generally accepted theory on the aetiology and clinical course of BV. This critical review highlights some of the more important aspects of BV research that could help in formulating new basic ideas respecting the biology of BV, not least the importance of the interleukin mediators of local inflammatory responses and the bacterial shift from the normally occurring lactobacilli species: L. crispatus, L. gasseri, L. jensenii, and L. iners to a mixed flora dominated by anaerobic bacteria.

Recovery of Clostridium difficile from children.

The occurrence of Clostridium difficile in faecal specimens of 218 children, aged 2 weeks to 15 years, was studied. The organism was recovered from 43 (20%) of the children (range 2 weeks to 10 years). The isolation frequency was significantly correlated to age. Thus, in children 1 to 8 months of age the organism occurred in 64%, while in children below and above that age C. difficile could only be recovered in 4%. No significant difference in the recovery frequency could be demonstrated between children with (23%) and without (17%) gastroenteritis. C. difficile occurred numerically more often in non-antibiotic treated children (22%) than in those given such drugs (13%). None of the children in the present study had evidence of pseudomembranous colitis. A comparative study of different selective media did not demonstrate any difference in the recovery frequency of C. difficile. The media used were Chopped Meat Glucose broth with cycloserine and either kanamycin or cefoxitin, and Cycloserine-Cefoxitin-Fructose agar.

Elastase-producing Pseudomonas aeruginosa degrade plasma proteins and extracellular products of human skin and fibroblasts, and inhibit fibroblast growth.

Leg ulcers of venous origin represent a disease affecting 0.1-0.2% of the population. It is known that almost all chronic ulcers are colonized by different bacteria, such as staphylococci, enterococci and Pseudomonas aeruginosa. We here report that P. aeruginosa, expressing the major metalloproteinase elastase, induces degradation of complement C3, various antiproteinases, kininogens, fibroblast proteins, and proteoglycans (PG) in vitro, thus mimicking proteolytic activity previously identified in chronic ulcer fluid in vivo. Elastase-producing P. aeruginosa isolates were shown to significantly degrade human wound fluid as well as human skin proteins ex vivo. Elastase-containing conditioned P. aeruginosa medium and purified elastase inhibited fibroblast cell growth. These effects, in conjunction with the finding that proteinase production was detected in wound fluid ex vivo, suggest that bacterial proteinases play a pathogenic role in chronic ulcers.

Relative neurotoxin gene expression in Clostridium botulinum type B, determined using quantitative reverse transcription-PCR

ABSTRACT A quantitative reverse transcription-PCR (qRT-PCR) method was developed to monitor the relative expression of the type B botulinum neurotoxin (BoNT/B) gene (cntB) in Clostridium botulinum. The levels of cntB mRNA in five type B strains were accurately monitored by using primers specific for cntB and for the reference gene encoding the 16S rRNA. The patterns and relative expression of cntB were different in the different strains. Except for one of the strains investigated, an increase in cntB expression was observed when the bacteria entered the early stationary growth phase. In the proteolytic strain C. botulinum ATCC 7949, the level of cntB mRNA was four- to fivefold higher than the corresponding levels in the other strains. This was confirmed when we quantified the production of extracellular BoNT/B by an enzyme-linked immunosorbent assay and measured the toxicity of BoNT/B by a mouse bioassay. When the effect of exposure to air on cntB expression was investigated, no decline in the relative expression was observed in spite of an 83% reduction in the viable count based on the initial cell number. Instead, the level of cntB mRNA remained the same. When there was an increase in the sodium nitrite concentration, the bacteria needed a longer adjustment time in the medium before exponential growth occurred. In addition, there was a reduction in the expression of cntB compared to the expression of the 16S rRNA gene at higher sodium nitrite concentrations. This was most obvious in the late exponential growth phase, but at the highest sodium nitrite concentration investigated, 45 ppm, a one- to threefold decline in the cntB mRNA level was observed in all growth phases.

Quantitative interaction effects of carbon dioxide, sodium chloride, and sodium nitrite on neurotoxin gene expression in nonproteolytic Clostridium botulinum type B.

ABSTRACT The effects of carbon dioxide, sodium chloride, and sodium nitrite on type B botulinum neurotoxin (BoNT/B) gene (cntB) expression in nonproteolytic Clostridium botulinum were investigated in a tryptone-peptone-yeast extract (TPY) medium. Various concentrations of these selected food preservatives were studied by using a complete factorial design in order to quantitatively study interaction effects, as well as main effects, on the following responses: lag phase duration (LPD), growth rate, relative cntB expression, and extracellular BoNT/B production. Multiple linear regression was used to set up six statistical models to quantify and predict these responses. All combinations of NaCl and NaNO2 in the growth medium resulted in a prolonged lag phase duration and in a reduction in the specific growth rate. In contrast, the relative BoNT/B gene expression was unchanged, as determined by the cntB-specific quantitative reverse transcription-PCR method. This was confirmed when we measured the extracellular BoNT/B concentration by an enzyme-linked immunosorbent assay. CO2 was found to have a major effect on gene expression when the cntB mRNA levels were monitored in the mid-exponential, late exponential, and late stationary growth phases. The expression of cntB relative to the expression of the 16S rRNA gene was stimulated by an elevated CO2 concentration; the cntB mRNA level was fivefold greater in a 70% CO2 atmosphere than in a 10% CO2 atmosphere. These findings were also confirmed when we analyzed the extracellular BoNT/B concentration; we found that the concentrations were 27 ng · ml−1 · unit of optical density−1 in the 10% CO2 atmosphere and 126 ng · ml−1 · unit of optical density−1 in the 70% CO2 atmosphere.

Effects of carbon dioxide on neurotoxin gene expression in nonproteolytic Clostridium botulinum type E

ABSTRACT Carbon dioxide is an antimicrobial gas commonly used in modified atmosphere packaging. In the present study, the effects of carbon dioxide on the growth of and neurotoxin production by nonproteolytic Clostridium botulinum type E were studied during the growth cycle. Quantitative reverse transcription-PCR and an enzyme-linked immunosorbent assay were used to quantify expression of the type E botulinum neurotoxin gene (cntE) and the formation of type E neurotoxin. The expression levels of cntE were similar in two strains, with relative expression peaking in the transition between exponential phase and stationary phase. In stationary phase, cntE mRNA expression declined rapidly. The cntE mRNA half-life was calculated to be approximately 9 minutes. Neurotoxin formation occurred in late exponential phase and stationary phase. High carbon dioxide concentrations delayed growth by increasing the lag time and decreasing the maximum growth rate. The effects of carbon dioxide concentration on relative neurotoxin gene expression and neurotoxin formation were significant. Expression of cntE mRNA and the formation of extracellular neurotoxin were twofold higher with a headspace carbon dioxide concentration of 70% (vol/vol) compared to 10% (vol/vol). This finding sheds a new, cautionary light on the potential risks of botulism associated with the use of modified atmosphere packaging.

Identification of a novel protein promoting the colonization and survival of Finegoldia magna, a bacterial commensal and opportunistic pathogen.

Anaerobic bacteria dominate the human normal microbiota, but strikingly little is known about these commensals. Finegoldia magna is a Gram‐positive anaerobe found in the skin and at other non‐sterile body surfaces, but it is also an opportunistic pathogen. This study describes a novel protein designated FAF (F. magna adhesion factor) and expressed by more than 90% of F. magna isolates. The protein is present in substantial quantities at the F. magna surface but is also released from the surface. FAF forms large protein aggregates in solution and surface‐associated FAF causes bacterial clumping. In skin F. magna bacteria were localized to the epidermis, where they adhere to basement membranes. FAF was found to mediate this adhesion via interactions with BM‐40, a basement membrane protein. The biological significance of FAF is further underlined by the observation that it blocks the activity of LL‐37, a major human antibacterial peptide. Altogether, the data demonstrate that FAF plays an important role in colonization and survival of F. magna in the human host.

CLOSTRIDIUM DIFFICILE TOXIN IN FAECAL SPECIMENS OF HEALTHY CHILDREN AND CHILDREN WITH DIARRHOEA

ABSTRACT. Presence of cytopathogenic effect (CPE) that could be inhibited by an antitoxin to Clostridium sordelli, known to cross‐react with Clostridium difficile toxin, was sought in faecal specimens from 101 infants. Of the children, 45 were healthy, while 56 had been hospitalized because of diarrhoea. CPE was found in 12 of the healthy infants and in 5 of those hospitalized. Faecal specimens of these 5 gave a CPE at titres of 103‐4, whereas in the 12 healthy infants the titres were 101‐2. Studies on consecutive samples showed that the CPE could persist for between 7‐11 weeks up to 9 months and more. Of the 45 healthy infants, 11 harboured C. difficile compared with 6 of the 56 with diarrhoea. In both groups, 3 CPE‐positive infants were culture‐negative for C. difficile. Four of those hospitalized had recently been given antibiotics; all were negative in both culture and CPE tests. The present study demonstrates that care should be exercised when interpreting the results of cultures for C. difficile and tests for CPE made on faecal specimens in order to establish a diagnosis of antibiotic‐associated enterocolitis in infants and children.