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.

Fezes de galinhas poedeiras na alimentação de ruminantes análises microbiológicas

The objectives of the present study were: to evaluate the variation in total number of microorganisms in laying hen fecal samples and to identify the pathogenic microorganisms possibly present such as Staphylococcus aureus, Salmonella sp., Clostridium botulinum, C chauvoei, Campylobacter sp., Escherichia coli, and Corynebacterium sp. The experiment was conducted at the School of Agrarian and Veterinarian Sciences of Jaboticabal, - UNESP. Fecal samples were collected from the aviary. A fully randomized experimental design was used with 7 treatments and 4 replications, for a total of 28 samples. A decrease in total number of microorganisms was detected when treatment T0 (zero days of storage) was compared with treatment T6 (42 days of storage), with oscillations in numbers being observed during this period of time. Pathogenic bacteria such as Clostridium noyvi, C perfringens, C sordelli, C septicum and C noyvi type B were detected, as well as non-pathogenic bacteria such as Bacillus sp. and Staphylococcus epidermidis.

Microbiological analyses of poultry litter used for ruminant feeding

The aims of this study were to estimate the changes in total bacterial counts (TBC) in poultry litter samples, consisting of rice hulls, after storage, and to identify pathogenic bacteria. For the countings Plate Count agar (Difco) was used. Enrichment and selective media such as blood agar, MacConkey, Baird Parker, brain and heart agar, and egg yolk solid media, and cooked meat and brain and heart infusion, incubated under aerobic or anaerobic conditions were used to isolate Staphylococcus aureus, Salmonella sp, Clostridium perfringens, C. botulinum, C. chauvoei, Campylobacter sp, Escherichia coli, and Corynebacterium sp. Litter samples were collected from the houses of the Veterinary School experimental aviary. A fully randomized experimental design was used with four treatments and four replications, for a total of 16 samples. A decrease in TBC was detected when treatment T1 (zero days of storage) was compared with treatments T2 (14 days of storage). on the other hand the treatments T3 (28 days of storage) and T4 (42 days of storage) presented significantly superior counting in relation to treatment T1. Some pathogenic bacteria of Enterobacteriaceae such as Escherichia coil, Proteus, Arizona, Providencia, Edwardsiella, as well as Staphylococcus aureus, S. epidermidis, different species of genus Clostridium as C. perfringens, C. sordelli, C. chauvoei, C. tetani and C. novyi as well as some strains of Corynebacterium pyogenes were isolated.

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.

[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.^

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.

Multiple component patient safety intervention in English hospitals:controlled evaluation of second phase

Objective: To independently evaluate the impact of the second phase of the Health Foundation's Safer Patients Initiative (SPI2) on a range of patient safety measures. Design: A controlled before and after design. Five substudies: survey of staff attitudes; review of case notes from high risk (respiratory) patients in medical wards; review of case notes from surgical patients; indirect evaluation of hand hygiene by measuring hospital use of handwashing materials; measurement of outcomes (adverse events, mortality among high risk patients admitted to medical wards, patients' satisfaction, mortality in intensive care, rates of hospital acquired infection). Setting: NHS hospitals in England. Participants: Nine hospitals participating in SPI2 and nine matched control hospitals. Intervention The SPI2 intervention was similar to the SPI1, with somewhat modified goals, a slightly longer intervention period, and a smaller budget per hospital. Results: One of the scores (organisational climate) showed a significant (P=0.009) difference in rate of change over time, which favoured the control hospitals, though the difference was only 0.07 points on a five point scale. Results of the explicit case note reviews of high risk medical patients showed that certain practices improved over time in both control and SPI2 hospitals (and none deteriorated), but there were no significant differences between control and SPI2 hospitals. Monitoring of vital signs improved across control and SPI2 sites. This temporal effect was significant for monitoring the respiratory rate at both the six hour (adjusted odds ratio 2.1, 99% confidence interval 1.0 to 4.3; P=0.010) and 12 hour (2.4, 1.1 to 5.0; P=0.002) periods after admission. There was no significant effect of SPI for any of the measures of vital signs. Use of a recommended system for scoring the severity of pneumonia improved from 1.9% (1/52) to 21.4% (12/56) of control and from 2.0% (1/50) to 41.7% (25/60) of SPI2 patients. This temporal change was significant (7.3, 1.4 to 37.7; P=0.002), but the difference in difference was not significant (2.1, 0.4 to 11.1; P=0.236). There were no notable or significant changes in the pattern of prescribing errors, either over time or between control and SPI2 hospitals. Two items of medical history taking (exercise tolerance and occupation) showed significant improvement over time, across both control and SPI2 hospitals, but no additional SPI2 effect. The holistic review showed no significant changes in error rates either over time or between control and SPI2 hospitals. The explicit case note review of perioperative care showed that adherence rates for two of the four perioperative standards targeted by SPI2 were already good at baseline, exceeding 94% for antibiotic prophylaxis and 98% for deep vein thrombosis prophylaxis. Intraoperative monitoring of temperature improved over time in both groups, but this was not significant (1.8, 0.4 to 7.6; P=0.279), and there were no additional effects of SPI2. A dramatic rise in consumption of soap and alcohol hand rub was similar in control and SPI2 hospitals (P=0.760 and P=0.889, respectively), as was the corresponding decrease in rates of Clostridium difficile and meticillin resistant Staphylococcus aureus infection (P=0.652 and P=0.693, respectively). Mortality rates of medical patients included in the case note reviews in control hospitals increased from 17.3% (42/243) to 21.4% (24/112), while in SPI2 hospitals they fell from 10.3% (24/233) to 6.1% (7/114) (P=0.043). Fewer than 8% of deaths were classed as avoidable; changes in proportions could not explain the divergence of overall death rates between control and SPI2 hospitals. There was no significant difference in the rate of change in mortality in intensive care. Patients' satisfaction improved in both control and SPI2 hospitals on all dimensions, but again there were no significant changes between the two groups of hospitals. Conclusions: Many aspects of care are already good or improving across the NHS in England, suggesting considerable improvements in quality across the board. These improvements are probably due to contemporaneous policy activities relating to patient safety, including those with features similar to the SPI, and the emergence of professional consensus on some clinical processes. This phenomenon might have attenuated the incremental effect of the SPI, making it difficult to detect. Alternatively, the full impact of the SPI might be observable only in the longer term. The conclusion of this study could have been different if concurrent controls had not been used.

Metabolomics as a tool to explore the staphylococcal biofilm

Orthopaedic infections can be polymicrobial existing as a microbiome. Infections often incorporate staphylococcal species, including Staphylococcus aureus. Such infections can lead to life threatening illness and implant failure. Furthermore, biofilm formation on the implant surface can occur, increasing pathogenicity, exacerbating antibiotic resistance and altering antimicrobial mechanism of action. Bacteria change dramatically during the transition to a biofilm growth state: phenotypically; transcriptionally; and metabolically, highlighting the need for research into molecular mechanisms involved in biofilm formation. Metabolomics can provide a tool to analyse metabolic changes which are directly related to the expressed phenotype. Here, we aimed to provide greater understanding of orthopaedic infection caused by S. aureus and biofilm formation on the implant surface. Through metagenome analysis by employing: implant material extraction; DNA extraction; microbial enrichment; and whole genome sequencing, we present a microbiome study of the infected prosthesis to resolve the causative species of orthopaedic hip infection. Results highlight the presence of S. aureus as a primary cause of orthopaedic infection along with Enterococcus faecium and the presence of secondary pathogen Clostridium difficile. Although results were hindered by the presence of host contaminating DNA even after microbial enrichment, conclusions could be made over the potential increased pathogenicity caused by the presence of a secondary pathogen and highlight method and sample preparation considerations when undertaking such a study. Following this finding, studies were focused on an orthopaedic clinical isolate of S. aureus and a metabolome extraction method for staphylococcal biofilms was developed using cell lysis through bead beating and solvent metabolome extraction. The method was found to be reproducible when coupled with liquid chromatography-mass spectrometry (LC-MS) and bioinformatics, allowing for the detection of significant changes in metabolism between planktonic and biofilm cultures to be identified and drug mechanism of actions (MOA) to be studied. Metabolomics results highlight significant changes in a number of metabolic pathways including arginine biosynthesis and purine metabolism between the two cell populations, evidence of S. aureus responding to their changing environment, including oxygen availability and a decrease in pH. Focused investigations on purine metabolism looking for biofilm modulation effects were carried out. Modulation of the S. aureus biofilm phenotype was observed through the addition of exogenous metabolites. Inosine increased biofilm biomass while formycin B, an inosine analogue, showed a dispersal effect and a potential synergistic effect in biofilm dispersal when coupled with gentamycin. Changes in metabolism between planktonic cells and biofilms highlight the requirement for antimicrobial testing to be carried out against planktonic cells and biofilms. Untargeted metabolomics was used to study the MOA of triclosan in S. aureus. The triclosan target and MOA in bacteria has already been characterised, however, questions remain over its effects in bacteria. Although the use of triclosan has come under increasing speculation, its full effects are still largely unknown. Results show that triclosan can induce a cascade of detrimental events in the cell metabolism including significant changes in amino acid metabolism, affecting planktonic cells and biofilms. Results and conclusions provide greater understanding of orthopaedic infections and specifically focus on the S. aureus biofilm, confirming S. aureus as a primary cause of orthopaedic infection and using metabolomic analysis to look at the changing state of metabolism between the different growth states. Metabolomics is a valuable tool for biofilm and drug MOA studies, helping understand orthopaedic infection and implant failure, providing crucial insight into the biochemistry of bacteria for the potential for inferences to be gained, such as the MOA of antimicrobials and the identification of novel metabolic drug targets.

Diarrea crónica por Salmonella typhimurium en paciente inmunocompentente

La diarrea crónica de origen infeccioso en pacientes inmunocompetentes es un cuadro poco frecuente en países desarrollados, aunque ciertos patógenos, generalmente parásitos (Giardia lamblia, Isospora belli, Cryptosporidium, Cyclospora, Strongyloides, Ameba, Trichuris y Schistosoma) y algunas bacterias (Aeromonas, Plesiomonas, Campylobacter, Clostridium difficile, Salmonella o Mycobacterium tuberculosis) pueden ser causantes de diarrea persistente. Se presenta un caso de un paciente que presentó Salmonella typhimunium en el coprocultivo y se recuperó tras tratamiento con levofloxacino durante 7 días.

Involvement of Clostridium gasigenes and C. algidicarnis in `blown pack` spoilage of Brazilian vacuum-packed beef

The objectives of this study were to isolate psychrotrophic clostridia from Brazilian vacuum-packed beef cuts (spoiled or not) and to identify the isolates by using 16S rRNA gene sequencing. Anaerobic psychrotrophic microorganisms were also enumerated and samples were collected to verify the incidence of psychrotrophic clostridia in the abattoir environment. Vacuum-packed beef cuts (n = 8 grossly distended and n = 5 non-spoiled) and environmental samples were obtained from a beef packing plant located in the state of Sao Paulo, Brazil. Each sample was divided in three subsamples (exudate, beef surface and beef core) that were analyzed for vegetative forms, total spore-forming, and sulfide reducing spore-forming, both activated by alcohol and heat. Biochemical profiles of the isolates were obtained using API20A, with further identification using 16S rRNA gene sequencing. The growth temperature and the pH range were also assessed. Populations of psychrotrophic anaerobic vegetative microorganisms of up to 10(10) CFU/(g, mL or 100 cm(2)) were found in `blown pack` samples, while in non-spoiled samples populations of 10(5) CFU/(g, CFU/mL or CFU/100cm(2)) was found. Overall, a higher population of total spores and sulfide reducing spores activated by heat in spoiled samples was found. Clostridium gasigenes (n = 10) and C. algidicarnis (n = 2) were identified using 16S rRNA gene sequencing. Among the ten C. gasigenes isolates, six were from spoiled samples (C1, C2 and C9), two were isolated from non-spoiled samples (C4 and C5) and two were isolated from the hide and the abattoir corridor/beef cut conveyor belt. C. algidicarnis was recovered from spoiled beef packs (C2). Although some samples (C3, C7, C10 and C14) presented signs of `blown pack` spoilage, Clostridium was not recovered. C. algidicarnis (n = 1) and C. gasigenes (n = 9) isolates have shown a psychrotrophic behavior, grew in the range 6.2-8.2. This is the first report on the isolation of psychrotrophic Clostridium (C. gasigenes and C. algidicarnis) in Brazil. This study shows that psychrotrophic Clostridium may pose a risk for the stability of vacuum-packed beef produced in tropical countries during shelf-life and highlights the need of adopting control measures to reduce their incidence in abattoir and the occurrence of `blown pack` spoilage. (C) 2011 Elsevier B.V. All rights reserved.