Incidence of Clostridium perfrigens in fishes

Fish collected from local landing centres and also from local markets were examined for the presence and enumeration of Clostridium perfringens. A medium described by Beerens et al. (1982) was used for the detection and enumeration of C. perfringens. C. perfringens occurs in low numbers in fishes compared to prawns. Proper handling of fishes after landing can reduce the chance of any public health hazard by C. perfringens.

Comparison of media and methods for the detection and enumeration of Clostridium perfringens in seafoods

Three direct plating methods and two most probable number (MPN) procedures were compared for the enumeration of Clostridium perfringens in seafoods the sulfitecycloserine (SC) agar, sulfite-polymyxin-sulfadiazine (SPS) agar, tryptone-sulfite- neomycin (TSN) agar, LS medium MPN procedure and iron milk MPN procedure. Isolates were confirmed as C. perfringens. The two MPN procedures compared very well with the three plating media tested with stock culture of C. perfringens from our laboratory collection and the reference strain NCIB 6125. But in fish samples, the two liquid media were found to be more sensitive and hence the MPN procedure using LS medium for the detection of C. perfringens in seafoods is suggested.

Two different proteases produced by a deep-sea psychrotrophic bacterial strain, Pseudoaltermonas sp SM9913

A psychrotrophic bacterial strain, Pseudoaltermonas sp. SM9913, was isolated from deep-sea sediment collected at 1,855 m depth. Two proteases produced by Pseudoaltermonas sp. SM9913 were purified, MPC-01 and MCP-02. MCP-01 is a serine protease with a molecular weight of 60.7 kDa. It is cold-adapted with an optimum temperature of 30-35degreesC. Its K-m and E-a for the hydrolysis of casein were 0.18% and 39.1 kJ mol(-1), respectively. It had low thermostability, and its activity was reduced by 73% after incubation at 40degreesC for 10 min. MCP-02 is a mesophilic metalloprotease with a molecular weight of 36 kDa. Its optimum temperature for the hydrolysis of casein was 50-55degreesC. The K-m and E-a of MCP-02 for the hydrolysis of casein were 0.36% and 59.3 kJ mol(-1), respectively. MCP-02 had high thermostability, and its activity was reduced by only 30.5% after incubation at 60degreesC for 10 min. At low temperatures, Pseudoaltermonas sp. SM9913 mainly produced the psychrophilic protease MCP-01.

Analysis of the elements of catabolite repression in Clostridium acetobutylicum.

The ptsH gene, encoding the phosphotransferase protein HPr, from Clostridium acetobutylicum ATCC 824 was identified from the genome sequence, cloned and shown to complement a ptsH mutant of Escherichia coli. The deduced protein sequence shares significant homology with HPr proteins from other low-GC gram-positive bacteria, although the highly conserved sequence surrounding the Ser-46 phosphorylation site is not well preserved in the clostridial protein. Nevertheless, the HPr was phosphorylated in an ATP-dependent manner in cell-free extracts of C. acetobutylicum. Furthermore, purified His-tagged HPr from Bacillus subtilis was also a substrate for the clostridial HPr kinase/phosphorylase. This phosphorylation reaction is a key step in the mechanism of carbon catabolite repression proposed to operate in B. subtilis and other low-GC gram-positive bacteria. Putative genes encoding the HPr kinase/phosphorylase and the other element of this model, namely the catabolite control protein CcpA, were identified from the C. acetobutylicum genome sequence, suggesting that a similar mechanism of carbon catabolite repression may operate in this industrially important organism.

Analysis of the mechanism and regulation of lactose transport and metabolism in Clostridium acetobutylicum ATCC 824.

Although the acetone-butanol-ethanol (ABE) fermentation of Clostridium acetobutylicum is currently uneconomic, the ability of the bacterium to metabolise a wide range of carbohydrates offers the potential for revival based on the use of cheap, low grade substrates. We have investigated the uptake and metabolism of lactose, the major sugar in industrial whey waste, by C. acetobutylicum ATCC 824. Lactose is taken up via a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) comprising both soluble and membrane-associated components, and the resulting phosphorylated derivative is hydrolysed by a phospho--galactosidase. These activities are induced during growth on lactose, but are absent in glucose-grown cells. Analysis of the C. acetobutylicum genome sequence identified a gene system, lacRFEG, encoding a transcriptional regulator of the DeoR family, IIA and IICB components of a lactose PTS, and phospho--galactosidase. During growth in medium containing both glucose and lactose, C. acetobutylicum exhibited a classical diauxic growth, and the lac operon was not expressed until glucose was exhausted from the medium. The presence upstream of lacR of a potential catabolite responsive element (cre) encompassing the transcriptional start site is indicative of the mechanism of carbon catabolite repression characteristic of low-GC Gram-positive bacteria. A pathway for the uptake and metabolism of lactose by this industrially important organism is proposed.

As neurotoxinas de clostridium sp.

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Thuricin CD: a potential therapeutic targeted against Clostridium difficile-associated diarrhea (CDAD)

Clostridium difficile is mainly a nosocomial pathogen and is a significant cause of antibioticassociated diarrhea. It is also implicated in the majority of cases of pseudomembranous colitis. The main etiological agent of C. difficile-associated diarrhea (CDAD) is perturbations to the gut microbiota by broad-spectrum antibiotics. Recently, thuricin CD, a two-peptide narrow spectrum sactibiotic bacteriocin with potent activity against C. difficile has been discovered. It is produced by Bacillus thuringiensis DPC6431. The efficacy of thuricin CD against a range of C. difficile clinical isolates has been determined in the form of minimum inhibitory concentration (MIC) values and compared to metronidazole, vancomycin, ramoplanin and actagardine in this thesis. Furthermore, by assessing paired combinations of the above-mentioned antimicrobials, it was determined that ramoplanin and actagardine function in a synergistic manner against the majority of C. difficile isolates. The functions of the genes in the thuricin CD gene cluster have also been elucidated by cloning the cluster and expressing thuricin CD in a heterologous Bacillus subtilis host and are described herein. In addition, the immunity mechanisms employed by the B. thuringiensis DPC6431 producer to protect itself from the antimicrobial actions of thuricin CD have also been elucidated. It has been shown that a small immunity peptide, TrnI, is involved in thuricin CD immunity, most likely by intercepting the thuricin CD peptides and/or blocking their access to the thuricin CD receptor. This immunity peptide and also the ABC-transporter system TrnFG serve to protect the B. thuringiensis host against thuricin CD.

Risk factors for and estimated incidence of community-associated Clostridium difficile infection, North Carolina, USA.

We determined estimated incidence of and risk factors for community-associated Clostridium difficile infection (CA-CDI) among patients treated at 6 North Carolina hospitals. CA-CDI case-patients were defined as adults (>18 years of age) with a positive stool test result for C. difficile toxin and no hospitalization within the prior 8 weeks. CA-CDI incidence was 21 and 46 per 100,000 person-years in Veterans Affairs (VA) outpatients and Durham County populations, respectively. VA case-patients were more likely than controls to have received antimicrobial drugs (adjusted odds ratio [aOR] 17.8, 95% confidence interval [CI] 6.6-48] and to have had a recent outpatient visit (aOR 5.1, 95% CI 1.5-17.9). County case-patients were more likely than controls to have received antimicrobial drugs (aOR 9.1, 95% CI 2.9-28.9), to have gastroesophageal reflux disease (aOR 11.2, 95% CI 1.9-64.2), and to have cardiac failure (aOR 3.8, 95% CI 1.1-13.7). Risk factors for CA-CDI overlap with those for healthcare-associated infection.

Quasiexperimental Study of the Effects of Antibiotic Use, Gastric Acid-Suppressive Agents, and Infection Control Practices on the Incidence of Clostridium difficile-Associated Diarrhea in Hospitalized Patients

The objective of this study was to evaluate the effects of antimicrobial drug use, gastric acid-suppressive agent use, and infection control practices on the incidence of Clostridium difficile-associated diarrhea (CDAD) in a 426-bed general teaching hospital in Northern Ireland. The study was retrospective and ecological in design. A multivariate autoregressive integrated moving average (time-series analysis) model was built to relate CDAD incidence with antibiotic use, gastric acid-suppressive agent use, and infection control practices within the hospital over a 5-year period (February 2002 to March 2007). The findings of this study showed that temporal variation in CDAD incidence followed temporal variations in expanded-spectrum cephalosporin use (average delay = 2 months; variation of CDAD incidence = 0.01/100 bed-days), broad-spectrum cephalosporin use (average delay = 2 months; variation of CDAD incidence = 0.02/100 bed-days), fluoroquinolone use (average delay = 3 months; variation of CDAD incidence = 0.004/100 bed-days), amoxicillin-clavulanic acid use (average delay = 1 month; variation of CDAD incidence = 0.002/100 bed-days), and macrolide use (average delay = 5 months; variation of CDAD incidence = 0.002/100 bed-days). Temporal relationships were also observed between CDAD incidence and use of histamine-2 receptor antagonists (H2RAs; average delay = 1 month; variation of CDAD incidence = 0.001/100 bed-days). The model explained 78% of the variance in the monthly incidence of CDAD. The findings of this study highlight a temporal relationship between certain classes of antibiotics, H2RAs, and CDAD incidence. The results of this research can help hospitals to set priorities for restricting the use of specific antibiotic classes, based on the size-effect of each class and the delay necessary to observe an effect.

An evaluation of the effectiveness of an algorithm intervention in reducing inappropriate faecal samples sent for Clostridium difficile testing.

Background: Clostridium difficile (C. difficile) is a leading cause of infectious diarrhoea in hospitals. Sending faecal samples for testing expedites diagnosis and appropriate treatment. Clinical suspicion of C. difficile based on patient history, signs and symptoms is the basis for sampling. Sending faecal samples from patients with diarrhoea ‘just in case’ the patient has C. difficile may be an indication of poor clinical management.

Aim: To evaluate the effectiveness of an intervention by an Infection Prevention and Control Team (IPCT) in reducing inappropriate faecal samples sent for C. difficile testing.

Method: An audit of numbers of faecal samples sent before and after a decision-making algorithm was introduced. The number of samples received in the laboratory was retrospectively counted for 12-week periods before and after an algorithm was introduced.
Findings: There was a statistically significant reduction in the mean number of faecal samples sent post the algorithm. Results were compared to a similar intervention carried out in 2009 in which the same message was delivered by a memorandum. In 2009 the memorandum had no effect on the overall number of weekly samples being sent.

Conclusion: An algorithm intervention had an effect on the number of faecal samples being sent for C. difficile testing and thus contributed to the effective use of the laboratory service.