Recognition of greater diversity of Bacillus species and related bacteria in human faeces

In a study looking at the culturable, aerobic Actinobacteria associated with the human gastrointestinal tract, the vast majority of isolates obtained from dried human faeces belonged to the genus Bacillus and related bacteria. A total of 124 isolates were recovered from the faeces of 10 healthy adult donors. 16S rRNA gene sequence analyses showed the majority belonged to the families Bacillaceae (n = 81) and Paenibacillaceae (n = 3), with Bacillus species isolated from all donors. Isolates tentatively identified as Bacillus clausii (n = 32) and B. licheniformis (n = 28) were recovered most frequently, with the genera Lysinibacillus, Ureibacillus, Oceanobacillus, Ornithinibacillus and Virgibacillus represented in some donors. Phenotypic data confirmed the identities of isolates belonging to well-characterized species. Representatives of the phylum Actinobacteria were recovered in much lower numbers (n = 11). Many of the bacilli exhibited antimicrobial activity against one or more strains of Clostridium difficile, C. perfringens, Listeria monocytogenes and Staphylococcus aureus, with some (n = 12) found to have no detectable cytopathic effect on HEp-2 cells. This study has revealed greater diversity within gut-associated aerobic spore-formers than previous studies, and suggests that bacilli with potential as probiotics could be isolated from the human gut.

In vitro evaluation of the antimicrobial activity of a range of probiotics against pathogens: Evidence for the effects of organic acids

The aim of this study was to investigate the antimicrobial properties of fifteen selected strains belonging to the Lactobacillus, Bifidobacterium, Lactococcus, Streptococcus and Bacillus genera against Gram-positive and Gram-negative pathogenic bacteria. In vitro antibacterial activity was initially investigated by an agar spot method. Results from the agar spot test showed that most of the selected strains were able to produce active compounds on solid media with antagonistic properties against Salmonella Typhimurium, Escherichia coli, Enterococcus faecalis, Staphylococcus aureus and Clostridium difficile. These results were also confirmed when cell-free culture supernatants (CFCS) from the putative probiotics were used in an agar well diffusion assay. Neutralization of the culture supernatants with alkali reduced the antagonistic effects. These experiments are able to confirm the capacity of potential probiotics to inhibit selected pathogens. One of the main inhibitory mechanisms may result from the production of organic acids from glucose fermentation and consequent lowering of culture pH. This observation was confirmed when the profile of organic acids was analysed demonstrating that lactic and acetic acid were the principal end products of probiotic metabolism. Furthermore, the assessment of the haemolytic activity and the susceptibility of the strains to the most commonly used antimicrobials, considered as basic safety aspects, were also studied. The observed antimicrobial activity was mainly genus-specific, additionally significant differences could be observed among species.

GTPases RhoA and Rac1 are important for amelogenin and DSPP expression during differentiation of ameloblasts and odontoblasts

Morphogenesis and cytodifferentiation are distinct processes in tooth development. Cell proliferation predominates in morphogenesis; differentiation involves changes in form and gene expression. The cytoskeleton is essential for both processes, being regulated by Rho GTPases. The aim of this study was to verify the expression, distribution, and role of Rho GTPases in ameloblasts and odontoblasts during tooth development in correlation with actin and tubulin arrangements and amelogenin and dentin sialophosphoprotein (DSPP) expression. RhoA, Rac1, and Cdc42 were strongly expressed during morphogenesis; during cytodifferentiation, RhoA was present in ameloblasts and odontoblasts, Rac1 and its effector Pak3 were observed in ameloblasts; and Cdc42 was present in all cells of the tooth germ and mesenchyme. The expression of RhoA mRNA and its effectors RockI and RockII, Rac1 and Pak3, as analyzed by real-time polymerase chain reaction, increased after ameloblast and odontoblast differentiation, according to the mRNA expression of amelogenin and DSPP. The inhibition of all Rho GTPases by Clostridium difficile toxin A completely abolished amelogenin and DSPP expression in tooth germs cultured in anterior eye chamber, whereas the specific inhibition of the Rocks showed only a partial effect. Thus, both GTPases are important during tooth morphogenesis. During cytodifferentiation, Rho proteins are essential for the complete differentiation of ameloblasts and odontoblasts by regulating the expression of amelogenin and DSPP. RhoA and its effector RockI contribute to this role. A specific function for Rac1 in ameloblasts remains to be elucidated; its punctate distribution indicates its possible role in exocytosis/endocytosis.

Meropenem versus piperacillin-tazobactam for definitive treatment of bloodstream infections due to ceftriaxone non-susceptible Escherichia coli and Klebsiella spp (the MERINO trial): study protocol for a randomised controlled trial

BACKGROUND: Gram-negative bacteria such as Escherichia coli or Klebsiella spp. frequently cause bloodstream infections. There has been a worldwide increase in resistance in these species to antibiotics such as third generation cephalosporins, largely driven by the acquisition of extended-spectrum beta-lactamase or plasmid-mediated AmpC enzymes. Carbapenems have been considered the most effective therapy for serious infections caused by such resistant bacteria; however, increased use creates selection pressure for carbapenem resistance, an emerging threat arising predominantly from the dissemination of genes encoding carbapenemases. Recent retrospective data suggest that beta-lactam/beta-lactamase inhibitor combinations, such as piperacillin-tazobactam, may be non-inferior to carbapenems for the treatment of bloodstream infection caused by extended-spectrum beta-lactamase-producers, if susceptible in vitro. This study aims to test this hypothesis in an effort to define carbapenem-sparing alternatives for these infections.

METHODS/DESIGN: The study will use a multicentre randomised controlled open-label non-inferiority trial design comparing two treatments, meropenem (standard arm) and piperacillin-tazobactam (carbapenem-sparing arm) in adult patients with bacteraemia caused by E. coli or Klebsiella spp. demonstrating non-susceptibility to third generation cephalosporins. Recruitment is planned to occur in sites across three countries (Australia, New Zealand and Singapore). A total sample size of 454 patients will be required to achieve 80% power to determine non-inferiority with a margin of 5%. Once randomised, definitive treatment will be for a minimum of 4 days, but up to 14 days with total duration determined by treating clinicians. Data describing demographic information, antibiotic use, co-morbid conditions, illness severity, source of infection and other risk factors will be collected. Vital signs, white cell count, use of vasopressors and days to bacteraemia clearance will be recorded up to day 7. The primary outcome measure will be mortality at 30 days, with secondary outcomes including days to clinical and microbiological resolution, microbiological failure or relapse, isolation of a multi-resistant organism or Clostridium difficile infection.

Ocorrência e investigação de fatores de virulência em enteropatógenos de origem bacteriana em potros até três meses de idade, com e sem diarréia, criados no interior do Estado de São Paulo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo clínico e etiológico da diarréia em potros

Pós-graduação em Medicina Veterinária - FMVZ

Desenvolvimento e validação de método analítico para a quantificação de teicoplanina injetável por turbidimetria

A teicoplanina é um complexo antibiótico glicopeptídico derivado do Actinoplanes teichomyceticus, ativo contra bactérias Gram-positivas resistentes a outros antibióticos. Seu espectro de ação é similar ao da vancomicina, sendo, porém mais ativo para Streptococcus faecalis e Clostridium difficile. Seu uso é indicado para profilaxia de endocardite, peritonite, osteomielite e para septicemia estafilocócica. No Brasil, a teicoplanina é comercializada sob a forma farmacêutica de pó liofilizado, que deve ser reconstituído antes da administração. O medicamento de referência é o Targocid®, produzido pelo laboratório Sanofi-Aventis, em duas apresentações, 66 mg/mL e 133 mg/mL. A teicoplanina, bem como a vancomicina, inibe a síntese da parede celular bacteriana, pois a molécula se liga ao precursor da parede D-alanil-D-alanina, formando um complexo, impedindo a ligação à porção terminal do peptidoglicano, que é o alvo das enzimas transglicolase e transpeptidase. Desse modo, não há incorporação de aminoácidos aos glicopeptídeos integrantes da parede celular das bactérias Gram-positivas. No estudo de validação, foram aplicados os parâmetros de linearidade, intervalo, precisão, sensibilidade, especificidade, exatidão e robustez. O método desenvolvido e validado para a quantificação de teicoplanina pó liofilizado foi: Ensaio microbiológico por turbidimetria na faixa de concentração de 20,0 a 80,0 μg/mL, utilizando o micro-organismo Staphylococcus epidermidis ATCC 12228 IAL 2150. Os parâmetros estudados para a validação do método turbidimétrico atenderam a todas as especificações para a adequada quantificação de teicoplanina na forma farmacêutica pó liofilizado

A Pilot Study to Determine the Effectiveness of Probiotic Use in Elderly Patients with Antibiotic-Associated Diarrhea

Long-term care settings have the majority of their patients on multiple antibiotics, and outbreaks of antibiotic-associated diarrhea and Clostridium difficile are common. Probiotics have been used with these patients to reduce these side effects. Probiotics can re-establish the composition of intestinal microflora, enhance immune response, and clear pathogens from the host which may reduce the symptoms of antibiotic-associated diarrhea. Therefore, the goal of this study was to conduct a retrospective study of the effectiveness of using probiotic in elderly patients in a long-term care facility in a Midwestern city who suffered from antibiotic-associated diarrhea. The probiotic, CulturelleTM had been administered once a day to eight males and twelve female patients who were taking antibiotics and stool consistency and number were recorded. Out of the original group, seven of the patients receiving the probiotic appeared to have positive effects while two patients had negative effects on stools. Thirteen patients showed no change in stool consistency and number. It was difficult to determine the effects of the probiotic due to the use by the facility of a bowel movement protocol for preventing constipation and impaction, and the lack of dietary records. Published studies in patients in long-term facilities vary greatly in terms of trial design, type and dose of probiotic and duration of treatment, which may explain why probiotics work for some patients and not for others. Probiotic use is becoming more accepted with antibiotic-associated diarrhea but due to the lack of definitive evidence about efficacy and the safety of probiotic use, more studies need to be conducted. Advisors: Kaye Stanek Krogstrand and Julie Albrecht

Establishment of the bacterial fecal community during the first month of life in Brazilian newborns

OBJECTIVE: The establishment of the intestinal microbiota in newborns is a critical period with possible long-term consequences for human health. In this research, the development of the fecal microbiota of a group of exclusively breastfed neonates living in low socio-economic conditions in the city of Sao Paulo, Brazil, during the first month of life, was studied. METHODS: Fecal samples were collected from ten neonates on the second, seventh, and 30th days after birth. One of the neonates underwent antibiotic therapy. Molecular techniques were used for analysis; DNA was extracted from the samples, and 16S rRNA libraries were sequenced and phylogenetically analyzed after construction. A real-time polymerase chain reaction (PCR) was performed on the samples taken from the 30th day to amplify DNA from Bifidobacterium sp. RESULTS: The primary phylogenetic groups identified in the samples were Escherichia and Clostridium. Staphylococcus was identified at a low rate. Bifidobacterium sp. was detected in all of the samples collected on the 30th day. In the child who received antibiotics, a reduction in anaerobes and Escherichia, which was associated with an overgrowth of Klebsiella, was observed throughout the experimental period. CONCLUSION: The observed pattern of Escherichia predominance and reduced Staphylococcus colonization is in contrast with the patterns observed in neonates living in developed countries.

Bedeutung von Rho-GTPasen bei der Regulation zellulärer Antworten auf genotoxischen Stress

SAPK/JNK regulieren nach genotoxischem Stress eine Vielzahl von Zielsubstraten, die bedeutsam für Reparatur und Überleben der Zelle sind, somit nehmen sie Einfluss auf das zelluläre Schicksal der Zelle. Ob DNA-Schäden eine Phosphorylierung von Stress-Kinasen nach sich ziehen ist bisher noch wenig untersucht. Mit reparaturdefizienten Zellen wurde der Einfluss von DNA-Schäden, durch Cisplatin/Transplatin/UV-C, auf die SAPK/JNK Aktivierung untersucht. Die Aktivierung der Stress-Kinasen erfolgte agenzspezifisch und abhängig von verschieden Reparaturfaktoren. Die Aktivierung korrelierte in reparaturdefizienten Zellen teilweise mit dem späten Auftreten von DNA-Strangbrüchen, war jedoch unabhängig von erhöhten initialen DNA-Schäden. Diese Befunde zeigten, dass die späte Aktivierung der SAPK/JNK DNA-schadensabhängig verläuft und das Cisplatin und Transplatin bei Verwendung von äquitoxischen Dosen zu einer vergleichbaren Aktivierung von SAPK/JNK führten. Die Hemmung der Rho-GTPasen sowohl durch Statine als auch mittels Clostridium difficile Toxin B zeigte weiterhin, dass Rho-GTPasen möglicherweise die späte DNA-schadensabhängige Aktivierung der Stress-Kinasen vermitteln. Die Hemmung von Rho-GTPasen durch physiologisch relevante Konzentrationen von Statinen führte in primären humanen Endothelzellen (HUVECs) zu einer Protektion vor IR-Strahlung und Doxorubicin. In beiden Fällen konnte eine Hemmung des pro-apoptotischen Transkriptionsfaktors p53 sowie der Chk1, welche einen Zellzyklusarrest reguliert, mit der Statin-Behandlung erreicht werden. Effektor-Caspasen wurden dabei durch den HMG-CoA-Reduktase Hemmer nicht beeinflusst. Ausschließlich bei dem Statin-vermittelten Schutz vor Doxorubicin kam es zu einer Reduktion von initialen DNA-Schäden, in Form von DNA-Strangbrüchen. Die IR-induzierten Strangbrüche in der DNA blieben von der Statin-Inkubation hingegen unbeeinflusst. Aufgrund ihrer protektiven Eigenschaften gegenüber IR- und Doxorubicin-induzierter Zytotoxizität in Endothelzellen und ihrer pro-apoptotischen Wirkung auf Tumorzellen könnten Statine möglicherweise die unerwünschten Nebenwirkungen von Zytostatika und einer Strahlentherapie günstig beeinflussen

[Right-sided lower abdominal pain and diarrhea of a young diabetic woman]

We report the case of a 24-years old diabetic women hospitalised because of right-sided lower abdominal pain and diarrhea. She fulminantly developed shock before appendectomy could be performed and was transferred to intensive care unit. Hypotension remained and laparoscopy revealed primary peritonitis and toxic shock syndrome by Group A Streptococcus which was cultivated in blood and ascites. Therapy with penicilline and clindamycine resolved symptoms. During hospitalisation Clostridium difficile colitis occurred. This complication leaded to prolonged hospitalisation.

Genome sequence of Fusobacterium nucleatum subspecies polymorphum - a genetically tractable fusobacterium.

Fusobacterium nucleatum is a prominent member of the oral microbiota and is a common cause of human infection. F. nucleatum includes five subspecies: polymorphum, nucleatum, vincentii, fusiforme, and animalis. F. nucleatum subsp. polymorphum ATCC 10953 has been well characterized phenotypically and, in contrast to previously sequenced strains, is amenable to gene transfer. We sequenced and annotated the 2,429,698 bp genome of F. nucleatum subsp. polymorphum ATCC 10953. Plasmid pFN3 from the strain was also sequenced and analyzed. When compared to the other two available fusobacterial genomes (F. nucleatum subsp. nucleatum, and F. nucleatum subsp. vincentii) 627 open reading frames unique to F. nucleatum subsp. polymorphum ATCC 10953 were identified. A large percentage of these mapped within one of 28 regions or islands containing five or more genes. Seventeen percent of the clustered proteins that demonstrated similarity were most similar to proteins from the clostridia, with others being most similar to proteins from other gram-positive organisms such as Bacillus and Streptococcus. A ten kilobase region homologous to the Salmonella typhimurium propanediol utilization locus was identified, as was a prophage and integrated conjugal plasmid. The genome contains five composite ribozyme/transposons, similar to the CdISt IStrons described in Clostridium difficile. IStrons are not present in the other fusobacterial genomes. These findings indicate that F. nucleatum subsp. polymorphum is proficient at horizontal gene transfer and that exchange with the Firmicutes, particularly the Clostridia, is common.

Pathogenesis of antibiotic-associated diarrhea (AAD)---a systematic review

A common complication of antibiotic use is the development of diarrheal illness. The pathogenesis of antibiotic associated diarrhea (AAD) may be mediated through alteration of intestinal microbiota, overgrowth of opportunistic pathogens, and direct drug toxicity on the gut. Alterations in the intestinal microbiota result in metabolic imbalances, loss of colonization resistance and in turn allow proliferation of opportunistic pathogens. Currently less than 33% of AAD cases can be attributable to Clostridium difficile leaving a large number of cases undiagnosed and poorly treated. Although the pathogenesis of Clostridium difficile infection (CDI) has been well documented, the role of other putative microbial etiologies (Clostridium perfringens, Staphylococcus aureus, Klebsiella oxytoca, Candida species) and their pathogenic mechanisms in AAD has been unclear. This review provides a comprehensive and systematic approach to the existing data on AAD and includes concise descriptions of the pathogenesis of CDI and non-CDI AAD in the form of figures.^

Delivery of proteins into living cells by reversible membrane permeabilization with streptolysin-O

The pore-forming toxin streptolysin O (SLO) can be used to reversibly permeabilize adherent and nonadherent cells, allowing delivery of molecules with up to 100 kDa mass to the cytosol. Using FITC-labeled albumin, 105–106 molecules were estimated to be entrapped per cell. Repair of toxin lesions depended on Ca2+-calmodulin and on intact microtubules, but was not sensitive to actin disruption or to inhibition of protein synthesis. Resealed cells were viable for days and retained the capacity to endocytose and to proliferate. The active domains of large clostridial toxins were introduced into three different cell lines. The domains were derived from Clostridium difficile B-toxin and Clostridium sordelli lethal toxin, which glycosylate small G-proteins, and from Clostridium botulinum C2 toxin, which ADP-ribosylates actin. After delivery with SLO, all three toxins disrupted the actin cytoskeleton to cause rounding up of the cells. Glucosylation assays demonstrated that G-proteins Rho and Ras were retained in the permeabilized cells and were modified by the respective toxins. Inactivation of these G-proteins resulted in reduced stimulus-dependent granule secretion, whereas ADP-ribosylation of actin by the C. botulinum C2-toxin resulted in enhanced secretion in cells. The presented method for introducing proteins into living cells should find multifaceted application in cell biology.

Gram-positive infections in patients with end-stage renal disease

Gram-positive microorganisms, specifically coagulase-negative staphylococci are the most common species recovered from clinical culture specimens of patients with end-stage renal disease. The propensity of coagulase-negative staphylococci (CNS) to cause infection in this patient group has been widely debated. However, it is still unclear how this usually avirulent commensal microorganism produces infection that contributes to high rates of morbidity and mortality in patients with end-stage renal disease. The aim of this thesis was to investigate the rate, geographical distribution, molecular and phenotypic mechanisms of Gram-positive microorganisms associated with infection in renal dialysis patients. In addition, it sought to assess the value of early serological diagnosis of dialysis catheter-associated infection and the effect of antimicrobial treatment regimens on the faecal carriage of enteric microorganisms. In this study, the incidence of haemodialysis catheter-associated infection was established with the Meditrend audit tool. This tool was used to assess the infection outcomes of catheter insertion and management procedures until the catheter was explanted. Introduction of a catheter management protocol decreased the incidence of catheter-related infection. Staphylococcal species recovered from episodes of haemodialysis catheter-associated infection and continuous ambulatory peritoneal dialysis (CAPD)-associated peritonitis were genotyped by determination of macrorestriction profiles with pulsed-field gel electrophoresis. This highlighted horizontal transfer of microorganisms between different patients and the environment. The phenotypic characteristics of these strains were also investigated to determine characteristics that could be used as markers for dialysis catheter-associated infection. The expression of elastase, lipase and esterase by CNS was significantly associated with infection. A rapid enzyme-linked immunosorbent assay incorporating a novel staphylococcal antigen (lipid S) was used to evaluate the early detection of anti-staphylococcal immunoglobulin gamma in patient sera. The comparison of culture positive and culture negative patients demonstrated a steady state of immune activation in both groups. However anti-lipid S serum antibody titres > 1000 were found to be a predictor of infection. The effect on faecal carriage of vancomycin resistant enterococci (VRE) and Clostridium difficile toxins in patients treated with CAPD when empiric cephalosporin therapy was substituted for piperacillin/tazobactam was investigated. The introduction of piperacillin/tazobactam demonstrated a decrease in the faecal carriage of VRE.