Cellular impermeability and uptake of biocides and antibiotics in Gram-positive bacteria and mycobacteria

Gram-positive bacteria possess a permeable cell wall that usually does not restrict the penetration of antimicrobials. However, resistance due to restricted penetration can occur, as illustrated by vancomycin-intermediate resistant Staphylococcus aureus strains (VISA) which produce a markedly thickened cell wall. Alterations in these strains include increased amounts of nonamidated glutamine residues in the peptidoglycan and it is suggested that the resistance mechanism involves 'affinity trapping' of vancomycin in the thickened cell wall. VISA strains have reduced doubling times, lower sensitivity to lysostaphin and reduced autolytic activity, which may reflect changes in the D-alanyl ester content of the wall and membrane teichoic acids. Mycobacterial cell walls have a high lipid content, which is assumed to act as a major barrier to the penetration of antimicrobial agents. Relatively hydrophobic antibiotics such as rifampicin and fluoroquinolones may be able to cross the cell wall by diffusion through the hydrophobic bilayer composed of long chain length mycolic acids and glycolipids. Hydrophilic antibiotics and nutrients cannot diffuse across this layer and are thought to use porin channels which have been reported in many species of mycobacteria. The occurrence of porins in a lipid bilayer supports the view that the mycobacterial wall has an outer membrane analogous to that of gram-negative bacteria. However, mycobacterial porins are much less abundant than in the gram-negative outer membrane and allow only low rates of uptake for small hydrophilic nutrients and antibiotics.

Lactobacillus bacteremia, with special focus on the safety of probiotic Lactobacillus rhamnosus GG

Lactobacilli are gram positive rods, which belong to normal oropharyngeal, gastrointestinal and urogenital flora. They are widely used in food industry and as food additives. Although their virulence is presumed to be very low, opportunistic bacteremic infections, especially in immunocompromised hosts, have been detected. In the present study, the possible effects of increasing probiotic use of Lactobacillus rhamnosus GG (LGG) on the occurrence of bacteremia due to lactobacilli was evaluated on population level. In Finland, 90 Lactobacillus bacteremia cases were reported to the National Infectious Disease Register maintained by National Public Health Institute, during 1995-2000. Their proportion of all bacteremia cases was on average 0.24%, corresponding to 0.3 cases/100 000 inhabitants annually. In the Helsinki University Central Hospital district the corresponding proportion of all bacteremia cases was observed during 1990-2000. Despite LGG intake increased six folded no increasing trend in the occurrence of lactobacilli bacteremia was seen. A total of 85 Lactobacillus sp. blood isolates collected from different human bacteremic cases were characterised and compared with the commercial probiotic LGG strain. In species characterisation 46 L. rhamnosus strains, 12 L. fermentum and L. casei strains each, three each of L. gasseri, L. salivarius and L. jensenii species, two L. curvatus, and one each of L. plantarum, L. sakei, L. zeae and L. reuteri species were detected. Nearly half of the L. rhamnosus findings turned out to be indistinguishable from the probiotic LGG strain. Common predisposing factors to Lactobacillus bacteremia were immunosuppression, prior prolonged hospitalisation and prior surgical interventions. Severe or fatal comorbidities were found in 82% of the patients. Mortality at one month was 26% and severe underlying diseases were a significant predictor of death (OR 15.8). Antimicrobial susceptibility of Lactobacillus strains was species dependent. The Lactobacillus isolates were generally susceptible to imipenem, piperacillin-tazobactam, clindamycin and erythromycin, whereas all other than L. gasseri and L. jensenii species were not at all susceptible to vancomycin. The susceptibility to cephalosporin varied greatly even within species why they might not be recommended for treatment of Lactobacillus infections. The effect and safety of probiotic LGG preparation in amelioration of gastric symptoms and diarrhea in HIV-infected patients was evaluated. No significant differences in gastrointestinal symptoms or diarrhoea in LGG treated patients compared to placebo could be found. LGG was well tolerated with no adverse effects including bacteremic outbreaks could be observed. The use of probiotic LGG can be regarded safe in this immunocompromised patient group.

Eficácia de um protocolo de higiene bucal com utilização de solução de clorexidina a 0,12% na prevenção de pneumonias associadas à ventilação mecânica (PAVM) e os efeitos sobre a microbiota da mucosa bucal de pacientes internados em unidades de terapia intensiva

A presente investigação teve como objetivo avaliar a prática de cirurgiões dentistas em uma unidade de terapia intensiva (UTI) de um hospital militar, o estabelecimento de um protocolo de higiene oral e os seus efeitos sobre a redução de pneumonias associadas à ventilação mecânica (PAVM). As percepções da equipe da UTI sobre as atividades dos cirurgiões dentistas também foram avaliadas por meio de um questionário. O perfil de colonização microbiana da mucosa oral antes e depois do estabelecimento das medidas de higiene oral também foi avaliado tanto por diluição e plaqueamento em meios de cultura microbiológicos seletivos e enriquecidos e através da amplificação pelo método de PCR e eletroforese em gel desnaturante em gradiente (DGGE), subsequente ao sequenciamento dos amplicons. A carga microbiana foi avaliada após a contagem de placas de agar e através da amplificação por PCR em tempo real (qPCR) do gene rrs nas amostras. O protocolo de higiene oral, realizado pelos cirurgiões dentistas, foi capaz de reduzir a incidência de PAVM (p <0,05). O questionário revelou que a modificação da halitose foi percebida por 93,33% dos participantes. A redução da ocorrência das úlceras orais e dos lábios durante a internação dos pacientes foi observada por 80% da equipe da UTI. Foi observada a redução da produção das secreções nasais e bucais por 70% da equipe dos profissionais da UTI. Para 86,66% dos participantes a assistência aos pacientes tornou-se mais agradável após a instituição dos cuidados bucais. O protocolo, realizado com a utilização de solução 0,12% de clorexidina, não foi capaz de evitar a colonização da mucosa oral por patógenos microbianos usualmente encontrados no ambiente hospitalar tais como os bastonetes Gram-negativos entéricos e não fermentadores, nem foi capaz de eliminá-los quando tais micro-organismos já se encontravam presentes antes dos procedimentos de higiene bucal. Alguns Bastonetes Gram-positivos (Lactobacillus sp e corinebactérias) e Staphylococcus epidermidis permaneceram após a realização dos procedimentos. O protocolo de higiene oral permitiu a redução da carga microbiana na mucosa oral de 50% dos pacientes considerando-se o método de contagem microbiana e para 35% dos pacientes pela avaliação dos números de cópias de genes rrs através de qPCR. Em conclusão, o protocolo de higiene oral desenvolvido pelos cirurgiões dentistas foi capaz de reduzir a incidência de PAV na UTI, embora não tenha sido capaz de prevenir a colonização da mucosa oral por supostos patógenos microbianos. O protocolo de higiene oral com a participação ativa dos cirurgiões dentistas foi bem aceito pelos profissionais da UTI e foi capaz de melhorar a qualidade da assistência aos pacientes críticos.

Bacterial flora associated with diseased fish and their antibiogram

Bacterial flora associated with tail rot/fin rot of Carassius auratus, Xiphophorus helleri and hemorrhagic ulcers of Clarias spp were studied. Sensitivity pattern of 33 isolates comprising Aeromonas spp, Pseudomonas spp and Gram-positive rods from diseased C. auratus, X. helleri and Clarias spp were screened against six broad-spectrum antibiotics viz. ciprofloxacin, chloramphenicol, co-trimoxazole, gentamycin, nitro-furantoin and oxytetracycline. Ciprofloxacin was the most effective in inhibiting bacteria at 0.05-0.10 µg/ml level. About 44% of Pseudomonas spp. was resistant to nitrofurantoin. Resistance to oxytetracycline was seen in 27% of Aeromonas spp Gram-positive rods were comparatively more resistant to antibiotics. The multiple antibiotic resistances were seen in 21% of the bacterial isolates of diseased fish.

Caractérisation et évaluation de la virulence de souches cliniques de Clostridium perfringens chez le poulet à griller élevé sans antibiotique.

réalisé en cotutelle avec Marie Archambault

Bacterial diversity in soil in response to different plans, phosphate fertilizers and liming

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

MICROBIOLOGICAL CONTAMINATION of A HEMODIALYSIS CENTER WATER DISTRIBUTION SYSTEM

The microbiological monitoring of the water used for hemodialysis is extremely important, especially because of the debilitated immune system of patients suffering from chronic renal insufficiency. To investigate the occurrence and species diversity of bacteria in waters, water samples were collected monthly from a hemodialysis center in upstate São Paulo and tap water samples at the terminal sites of the distribution system was sampled repeatedly (22 times) at each of five points in the distribution system; a further 36 samples were taken from cannulae in 19 hemodialysis machines that were ready for the next patient, four samples from the reuse system and 13 from the water storage system. To identify bacteria, samples were filtered through 0.22 mu m-pore membranes; for mycobacteria, 0.45 mu m pores were used. Conventional microbiological and molecular methods were used in the analysis. Bacteria were isolated from the distribution system (128 isolates), kidney machine water (43) and reuse system (3). Among these isolates, 32 were Gram-positive rods, 120 Gram-negative rods, 20 Gram-positive cocci and 11 mycobacteria. We propose the continual monitoring of the water supplies in hemodialysis centers and the adoption of effective prophylactic measures that minimize the exposure of these immunodeficient patients to contaminated sources of water.

Honey consumption in the state of São Paulo: a risk to human health?

Infantile botulism was recognized in 1976 as a paralyzing disease caused by the ingestion of viable spores that would germinate and colonize the intestinal tract of infants, with local production and absorption of Clostridium botulinum toxin. The possible origins of botulinic spores are dust and honey, which has been identified as a dietary risk factor for infantile botulism. The objectives of the present study were to investigate 100 honey samples obtained in the state of São Paulo (Brazil) in terms of incidence of botulinic spores and of microbiologic quality, in agreement with Decree 367/9. All 100 samples analysed were negative for the presence of Salmonella, Shigella, total coliforms. C botulinum spores were present in 3 samples (3%) and molds and yeasts, in 64 samples (64%), but only 25 (25%) exceeded established criteria, with counts ranging from zero to 1.5 x 10(5) CFU/g. The presence of small sporogenic Gram-positive rods was observed in 42 (42%) of the 100 samples tested but these bacteria were not identified. (C) 2003 Elsevier Ltd. All rights reserved.

Efficacy of antibiotic therapy for peritoneal dialysis-associated peritonitis: a proportional meta-analysis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Biological Hydrogen production from environmental sample in tropical countries

The hydrogen gas is regarded as clean and renewable energy source, since it generates only water during combustion when used as fuel. It shows 2.75 times more energy content than any hydrocarbon and it can be converted into electrical, mechanical energy or heat. Inoculum sources have been successfully tested for hydrogen biological production in temperate climate countries as sludge treatment plants sewage, sludge treatment plant wastewater, landfill sample, among others. However, hydrogen biologic production with inoculum from environmental samples such as sediment reservoirs, especially in tropical countries like Brazil, is rarely investigated. Reservoirs and fresh water lake sediment may contain conditions for the survival of a wide variety of microorganisms which use different carbon sources mainly glucose and xylose, in the fermentation. Glucose is an easily biodegradable, present in most of the industrial effluents and can be obtained abundantly from agricultural wastes. A wide variety of wastewater resulting from agriculture, industry and pulp and paper processed from wood may contain xylose in its constitution. Such effluent contains glucose and xylose concentrations of about 2 g/L. In this sense, this work verified hydrogen biological production in anaerobic batch reactor (1L), at 37 ° C, initial pH 5.5, headspace with N2 (100%), Del Nery medium, vitamins and peptone (1 g/L), fed separately with glucose (2g/L) and xylose (2 g/L). The inoculum was taken from environmental sample (sediment reservoir Itupararanga - Ibiúna - SP-Brazil). It was previously purified in serial dilutions at H2 generation (10-5, 10-7, 10-10), and heat treated (90º C - 10 min) later to inhibited the H2 consumers. The maximum H2 generations obtained in both tests were observed at 552 h, as described below. At the reactors fed with glucose and xylose were observed, respectively, 9.1 and 8.6 mmol H2/L, biomass growth (0.2 and 0.2 nm); consumption of sugar concentrations 53.6% (1.1 glucose g/L) and 90.5% (1.8 xylose g/L); acetic acid generation (124.7 mg/L and 82.7 mg/L), butyric acid (134.0 mg/L and 230.4 mg/L) and there wasn’t methane generation in the reactors. Microscopic analysis of biomass in anaerobic reactors showed the predominance of Gram positive rods and rods with endospores, whose morphology is characteristic of H2-generating bacteria, in both tests. These species were selected from the natural environment. In DGGE analysis performed difference were observed between populations from inoculum and in tests. This analysis confirmed that some species of bacteria were selected which remained under the conditions imposed on the experiment. The efficiency of the pre-treatment of inoculum and the imposition of pH 5.5 inhibited methane-producing microorganisms and the consumers of H2. Therefore, the experimental conditions imposed allowed the attainment of bacterial consortium of producer H2 taken from an environmental sample with concentration of xylose and glucose similar to the ones of the industrial effluents.

Evidence-based medicine: an update on treatments for peritoneal dialysis-related peritonitis

Peritonitis continues to be a major complication of peritoneal dialysis (PD), and adequate treatment is crucial for a favorable outcome. There is no consensus regarding the optimal therapeutic regimen, and few prospective controlled studies have been published. The objective of this manuscript is to review the results of PD peritonitis treatment reported in narrative reviews, systematic reviews, and proportional meta-analyses. Two narrative reviews, the only existing systematic review and its update published between 1991 and 2014 were included. In addition, we reported the results of a proportional meta-analysis published by our group. Results from systematic reviews of randomized control trials (RCT) and quasi-RCT were not able to identify any optimal antimicrobial treatment, but glycopeptide regimens were more likely to achieve a complete cure than a first generation cephalosporin. Compared to urokinase, simultaneous catheter removal and replacement resulted in better outcomes. Continuous and intermittent IP antibiotic use had similar outcomes. Intraperitoneal antibiotics were superior to intravenous antibiotics in reducing treatment failure. In the proportional meta-analysis of RCTs and the case series, the resolution rate (86%) of ceftazidime plus glycopeptide as initial treatment was significantly higher than first generation cephalosporin plus aminoglycosides (66%) and glycopeptides plus aminoglycosides (75%). Other comparisons of regimens used for either initial treatment or treatment of gram-positive rods or gram-negative rods did not show statistically significant differences. The superiority of a combination of a glycopeptide and a third generation cephalosporin was also reported by a narrative review study published in 1991, which reported an 88% resolution rate.

Actinobaculum schaalii an emerging pediatric pathogen?

Background Actinobaculum schaalii was first described as a causative agent for human infection in 1997. Since then it has mainly been reported causing urinary tract infections (UTI) in elderly individuals with underlying urological diseases. Isolation and identification is challenging and often needs molecular techniques. A. schaalii is increasingly reported as a cause of infection in humans, however data in children is very limited. Case presentation We present the case of an 8-month-old Caucasian boy suffering from myelomeningocele and neurogenic bladder who presented with a UTI. An ultrasound of the urinary tract was unremarkable. Urinalysis and microscopy showed an elevated leukocyte esterase test, pyuria and a high number of bacteria. Empiric treatment with oral co-trimoxazole was started. Growth of small colonies of Gram-positive rods was observed after 48 h. Sequencing of the 16S rRNA gene confirmed an A. schaalii infection 9 days later. Treatment was changed to oral amoxicillin for 14 days. On follow-up urinalysis was normal and urine cultures were negative. Conclusions A.schaalii is an emerging pathogen in adults and children. Colonization and subsequent infection seem to be influenced by the age of the patient. In young children with high suspicion of UTI who use diapers or in children who have known abnormalities of their urogenital tract, infection with A. schaalii should be considered and empiric antimicrobial therapy chosen accordingly.

Staphylococcus spp., Enterobacteriaceae and Pseudomonadaceae oral isolates from Brazilian HIV-positive patients. Correlation with CD4 cell counts and viral load

The aim was to evaluate the presence of Staphylococcus spp., Enterobacteriaceae and Pseudomonadaceae in the oral cavities of HIV-positive patients. Forty-five individuals diagnosed as HIV-positive by ELISA and Western-blot, and under anti-retroviral therapy for at least 1 year, were included in the study. The control group constituted 45 systemically healthy individuals matched to the HIV patients to gender, age and oral conditions. Oral rinses were collected and isolates were identified by API system. Counts of microorganisms from HIV and control groups were compared statistically by a Mann-Whitney test (alpha = 5%). The percentages of individuals positive for staphylococci were similar between the groups (p = 0.764), whereas for Gram-negative rods, a higher percentage was observed amongst HIV-positive (p = 0.001).There was no difference in Staphylococcus counts between HIV and control groups (p = 0.1008). Counts were lower in the oral cavities of patients with low viral load (p = 0.021), and no difference was observed in relation to CD4 counts (p = 0.929). Staphylococcus aureus was the most frequently isolated species in HIV group, and Staphylococcus epidermidis was the prevalent species in the control group. Significantly higher numbers of enteric bacteria and pseudomonas were detected in the oral cavities of the HIV group than in the control (p = 0.0001). Enterobacter cloacae was the most frequently isolated species in both groups. Counts of enteric bacteria and pseudomonas were significantly lower in patients with low CD4 counts (p = 0.011); however, there was no difference relating to viral load. It may be concluded that HIV group showed greater species diversity and a higher prevalence of Enterobacteriaceae/Pseudomonadaceae. (C) 2011 Elsevier Ltd. All rights reserved.

Oral Actinomyces Species in Health and Disease: Identification, Occurence and Importance of Early Colonization

The genus Actinomyces consists of a heterogeneous group of gram-positive, mainly facultatively anaerobic or microaerobic rods showing various degrees of branching. In the oral cavity, streptococci and Actinomyces form a fundamental component of the indigenous microbiota, being among initial colonizers in polymicrobial biofilms. The significance of the genus Actinomyces is based on the capability of species to adhere to surfaces such as on teeth and to co-aggregate with other bacteria. Identification of Actinomyces species has mainly been based on only a few biochemical characteristics, such as pigmentation and catalase production, or on the use of a single commercial kit. The limited identification of oral Actinomyces isolates to species level has hampered knowledge of their role both in health and disease. In recent years, Actinomyces and related organisms have attracted the attention of clinical microbiologists because of a growing awareness of their presence in clinical specimens and their association with disease. This series of studies aimed to amplify the identification methods for Actinomyces species. With the newly developed identification scheme, the age-related occurrence of Actinomyces in healthy mouths of infants and their distribution in failed dental implants was investigated. Adhesion of Actinomyces species to titanium surfaces processed in various ways was studied in vitro. The results of phenotypic identification methods indicated a relatively low applicability of commercially available test kits for reliable identification within the genus Actinomyces. However, in the study of conventional phenotypic methods, it was possible to develop an identification scheme that resulted in accurate differentiation of Actinomyces and closely related species, using various different test methods. Genotypic methods based on 16S rRNA sequence analysis of Actinomyces proved to be a useful method for genus level identification and further clarified the species level identification with phenotypic methods. The results of the study of infants showed that the isolation frequency of salivary Actinomyces species increased according to age: thirty-one percent of the infants at 2 months but 97% at 2 years of age were positive for Actinomyces. A. odontolyticus was the most prominent Actinomyces colonizer during the study period followed in frequency by A. naeslundii and A. viscosus. In the study of explanted dental implants, Actinomyces was the most prevalent bacterial genus, colonizing 94% of the fixtures. Also in the implants A. odontolyticus was revealed as the most common Actinomyces species. It was present in 84% of Actinomyces -positive fixtures followed in frequency by A. naeslundii, A. viscosus and A. israelii. In an in vitro study of titanium surfaces, different Actinomyces species showed variation regarding their adhesion to titanium. Surface roughness as well as albumin coating of titanium had significant effects on adhesion. The use of improved phenotypic and molecular diagnostic methods increased the accuracy of the identification of the Actinomyces to species level. This facilitated an investigation of their occurrence and distribution in oral specimens in both health and disease.

The bacterial flora and their possible association with spoilage in a variety of fresh-water fish: Cyprinus carpio. var. communis

The bacteria from a variety of fresh-water fish, Cyprinus carpio. var. communis, showed the presence of micrococci, Gram positive and Gram negative rods. These have been characterized as far as was possible. Of thirty-eight strains of bacteria used, only six strains were considered as causing spoilage of fish flesh in experiments where flesh was incubated with individual cultures of the bacteria. These six strains had been found on the surface and/or intestine of the fish and support the suggestions that, after death, invasion of flesh by bacteria from the surface and intestine could be the cause of bacterial spoilage of fish.