Multispecies biofilm formation by the contaminating microbiota in raw milk.

Biofilms can be formed on the surfaces of dairy processing equipment and are a potential source of product contamination. This study evaluated the diversity of multispecies biofilms formed on stainless steel (SS) due to the contaminating microbiota in raw milk. Samples of raw milk were used: one was fresh milk and the other maintained in refrigerated bulk tanks for up to 48 h. The mesophilic aerobic contamination was ∼104 CFU ml-1 in fresh milk and 106 CFU ml-1 in bulk milk. SS coupons were kept immersed in the milk at 7 ±2 °C for 10 days, and every two days, the raw milk was changed for samples of the same origin collected on the current day. After incubation for 10 days, sessile cells in the biofilm reached 105 CFU cm-2 in the presence of fresh milk, and 106 CFU cm-2 in the presence of bulk milk. The genetic diversity analysis showed that Gammaproteobacteria and Bacilli predominated in the biofilms throughout the incubation of both milk samples and these biofilms showed a reduction in diversity over time. The main classes of bacteria found in these biofilms have representatives of great importance since many of them have spoilage potential.

Potential virulence of Klebsiella sp. isolates from enteral diets.

We aimed to evaluate the potential virulence of Klebsiella isolates from enteral diets in hospitals, to support nosocomial infection control measures, especially among critical-care patients. Phenotypic determination of virulence factors, such as capsular expression on the external membrane, production of aerobactin siderophore, synthesis of capsular polysaccharide, hemolytic and phospholipase activity, and resistance to antibiotics, which are used therapeutically, were investigated in strains of Klebsiella pneumoniae and K. oxytoca. Modular industrialized enteral diets (30 samples) as used in two public hospitals were analyzed, and Klebsiella isolates were obtained from six (20%) of them. The hypermucoviscous phenotype was observed in one of the K. pneumoniae isolates (6.7%). Capsular serotypes K1 to K6 were present, namely K5 and K4. Under the conditions of this study, no aerobactin production, hemolytic activity or lecithinase activity was observed in the isolates. All isolates were resistant to amoxicillin and ampicillin and sensitive to cefetamet, imipenem, chloramphenicol, gentamicin and sulfamethoxazole-trimethoprim. Most K. pneumoniae isolates (6/7, 85.7%) from hospital B presented with a higher frequency of resistance to the antibiotics tested in this study, and multiple resistance to at least four antibiotics (3/8; 37.5%) compared with isolates from Hospital A. The variations observed in the antibiotic resistance profiles allowed us to classify the Klebsiella isolates as eight antibiotypes. No production of broad-spectrum ß-lactamases was observed among the isolates. Our data favor the hypothesis that Klebsiella isolates from enteral diets are potential pathogens for nosocomial infections.

A new repertoire of informations about the quorum sensing system in Salmonella enterica serovar Enteritidis PT4.

Salmonella spp are among the main causative agents of foodborne diseases. Some phenotypes associated with increased drug resistance and virulence are regulated by quorum sensing (QS). In the present study, the autoinducer (AI)-1- and -2-mediated QS mechanisms were characterized in Salmonella enterica serovar Enteritidis PT4 for the first time. Salmonella Enteritidis did not produce AI-1. Phylogenetic analysis of nucleotides encoding the SdiA protein, the response regulator of AI-1-mediated QS, and comparative alignment of its amino acids showed that the gene and protein are conserved within the same bacterial genus. Thus, bacteria of the same genus respond to the same AIs. However, this finding did not preclude the possibility that Salmonella Enteritidis might respond to AIs released from bacteria of a different genus, which might confer a competitive advantage to this pathogen. We found that the regulation of AI-2-mediated QS in Salmonella Enteritidis is similar to that in serovar Typhimurium. The elucidation of the AI-1- and AI-2-mediated QS mechanisms in Salmonella Enteritidis will contribute to the development of new control strategies for this pathogen by indicating new targets for antimicrobial drugs.

Stress enhances the sensitivity of Salmonella enterica serovar Typhimurium to bacteriocins.

AIMS: The aim of this study was to evaluate the potential application of bacteriocins against Gram-negative bacteria when associated with others food preservation methods. METHODS AND RESULTS: Salmonella was subjected to heat, cold, acid and chemical (with ethylenediaminetetracetate and trisodium phosphate) stresses. Then, the cells were recovered and subjected to treatment with bacteriocins (500 AU ml(-1) ) for 6 h. Heat and cold stress were those that promoted more sensitization to bactericidal activity of nisin. Under the same conditions, bovicin HC5 acted more rapidly than nisin reducing the number of viable cells to undetectable levels after 20 min of treatment. Similar results with use of nisin only were observed after 6 h of treatment. CONCLUSIONS: Stress conditions used in food industry, such as temperature and pH, and use of chelating agents or membrane disruptors, sensitized Salmonella Typhimurium cells to bacteriocins produced by lactic acid bacteria, such as nisin and bovicin HC5. SIGNIFICANCE AND IMPACT OF THE STUDY: Food preservation methods sensitized Gram-negative bacteria to bacteriocins activity, which demonstrate the potential of nisin and bovicin HC5 to inhibit the growth of Salmonella.

Differences in the antibacterial activity of nisin and bovicin HC5 against Salmonella Typhimurium under different temperature and pH conditions.

AIMS: To compare the action of nisin and bovicin HC5 in combination with EDTA on Salmonella Typhimurium under different environmental conditions. METHODS AND RESULTS: Salmonella Typhimurium was treated in BHI broth containing EDTA (1·5 mmol l(-1)) and nisin or bovicin HC5 (200 AU ml(-1)) under different pH and temperature conditions, and according to a central composite design with two factors (temperature and pH). Cell viability was evaluated on plate count agar for 48 h. The combination of nisin or bovicin HC5 with EDTA was able to inhibit the growth of Salmonella, but the temperature and pH conditions promoting inhibition were distinct for each bacteriocin. Nisin was bactericidal over a broad range of temperature and pH, while bovicin HC5 was bacteriostatic in most conditions and bactericidal only in specific conditions (pH >6·0 and temperature >30°C). Salmonella Typhimurium did not show tolerance to bovicin HC5 or cross-tolerance between these lantibiotics. CONCLUSIONS: Nisin and bovicin HC5 both inhibited the growth of Salmonella, but the activity of each bacteriocin was differently influenced by environmental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Nisin and bovicin HC5 have the potential to inhibit the growth of Salmonella, but environmental conditions should be considered to establish optimal conditions for its application.

Evaluation of a polymerase chain reaction protocol for the detection of Salmonella species directly from superficial samples of chicken carcasses and preenrichment broth.

Chicken meat is an important vehicle of foodborne pathogens, such as Salmonella spp., and demands a systematic control of microbiological contamination during industrial processing. This control occurs by the adoption of quality control systems in slaughters based on the microbiological investigation on hygiene indicators and pathogens, requiring the development of fast, trustable, and precise methodologies. The objective of this study was to compare the Salmonella spp. conventional methodology to a protocol of PCR in chicken carcass surface samples. The PCR protocol was developed directly from the collected samples and from preenrichment broth before and after incubation. The obtained results were compared by chi(2) and McNemar tests (P < 0.05), and the values of concordance, sensitivity, and specificity of PCR variations were calculated considering the conventional methodology as a parameter. The obtained results indicated that although some similarities between the methodologies were observed when positive results were considered (P > 0.05), the PCR developed from preenrichment after incubation presented significant differences from all the other methodologies (P < 0.05). Wide variations were observed in the PCR performance for Salmonella spp. detection in chicken carcasses, which can be due to intrinsic factors inherent to the achievement of this food. Further studies are necessary to elucidate the applicability of the PCR as a tool for microbiological monitoring in quality control systems for chicken processing.

The proteolytic activity of Pseudomonas fluorescens 07A isolated from milk is not regulated by quorum sensing signals

The proteolytic activity of Pseudomonas fluorescens 07A was investigated, and was optimal on tryptone-calcium medium. N-acyl-homoserine lactones (AHLs) were not detected on supernatants of late-exponential and stationary-phase culture broths. Synthetic AHLs or bacterial cell extracts added to the medium did not influence growth or proteolytic activity suggesting that quorum sensing might not regulate protease production in this strain.

The proteolytic activity of Pseudomonas Fluorescens 07A isolated from milk is not regulated by quorum sensing signals.

The proteolytic activity of Pseudomonas fluorescens 07A was investigated, and was optimal on tryptone-calcium medium. N-acyl-homoserine lactones (AHLs) were not detected on supernatants of late-exponential and stationary-phase culture broths. Synthetic AHLs or bacterial cell extracts added to the medium did not influence growth or proteolytic activity suggesting that quorum sensing might not regulate protease production in this strain.

Bovicin HC5 reduces thermal resistance of Alicyclobacillus acidoterrestris in acidic mango pulp.

AIMS: To test the effect of bovicin HC5 against vegetative cells and endospores of Alicyclobacillus acidoterrestris DSMZ 2498 in synthetic media and in acidic mango pulp. METHODS AND RESULTS: Alicyclobacillus acidoterrestris was grown in synthetic medium at 40 degrees C and pH 4.0. The effect on vegetative cells was assayed by adding bovicin HC5 to synthetic medium (40-160 AU ml(-1)) or to mango pulp (100 AU ml(-1)) at various pH values and determining the effect on growth (OD(600 nm)) and viable cell number, respectively. The effect of bovicin HC5 on spore germination and thermal sensitivity of A. acidoterrestris was tested in mango pulp (pH 4.0) containing 80 AU ml(-1) of bovicin HC5. Bovicin HC5 was bactericidal against vegetative cells of A. acidoterrestris at different pH values and showed sporicidal activity against endospores of this bacterium. When spores of A. acidoterrestris were heat treated in the presence of bovicin HC5, D-values decreased 77% to 95% compared to untreated controls at temperatures ranging from 80 to 95 degrees C. CONCLUSION: Bovicin HC5 was bactericidal and sporicidal against A. acidoterrestrsi DSMZ 2498. SIGNIFICANCE AND IMPACT OF THE STUDY: These results indicated that bovicin HC5 has potential to prevent spoilage of acidic fruit juices by thermocidophilic spore-forming bacteria.

Bactericidal effect of bovicin HC5 and nisin against Clostridium tyrobutyricum isolated from spoiled mango pulp.

AIMS: To test the effect of bovicin HC5--a bacteriocin from Streptococcus bovis HC5--against the strains of Clostridium tyrobutyricum isolated from canned spoiled mango pulp. METHODS AND RESULTS: Bovicin HC5 [40-160 arbitrary unit (AU) ml(-1)] reduced the specific growth rate and increased the lag phase duration of the bacterial isolates inoculated in brain heart infusion media at 30 degrees C. The inhibitory activity of bovicin HC5 (100 AU ml(-1)) in mango pulp was bactericidal and more pronounced at acidic conditions. When C. tyrobutyricum was inoculated into mango pulp with bovicin HC5, gas production was not observed. Cultures that were successively transferred in the presence of sublethal doses of bovicin HC5 did not become resistant. CONCLUSIONS: The addition of bovicin HC5 to mango pulp might be effective in preventing deterioration by spoilage bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: Bovicin HC5 and nisin have the potential to increase the shelf life of canned fruit pulps.

Effect of bovicin HC5 on growth and spore germination of Bacillus cereus and Bacillus thuringiensis isolated from spoiled mango pulp.

AIMS: To use bovicin HC5 to inhibit predominant bacteria isolated from spoiled mango pulp. METHODS AND RESULTS: Bovicin HC5 and nisin were added to brain heart infusion (BHI) medium (40-160 AU ml(-1)) or mango pulp (100 AU ml(-1)) and the growth of Bacillus cereus and Bacillus thuringiensis was monitored. Cultures treated with bovicin HC5 or nisin showed longer lag phases and grew slower in BHI medium. Bovicin HC5 and nisin were bactericidal and showed higher activity in mango pulp at acidic pH values. To determine the effect on spore germination and D values, mango pulp containing bovicin HC5 was inoculated with 10(6) and 10(9) spores per ml(-1), respectively, from each strain tested. Bovicin HC5 reduced the outgrowth of spores from B. cereus and B. thuringiensis, but thermal sensitivity was not affected. CONCLUSIONS: Bovicin HC5 was bactericidal against B. cereus and B. thuringiensis isolated from spoiled mango pulp. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus cereus and B. thuringiensis had not been previously isolated from spoiled mango pulp and bovicin HC5 has the potential to inhibit such bacteria in fruit pulps.

Aplicação da técnica de PCR na detecção de Yersinia enterocolitica em suínos abatidos sem inspeção / PCR technique application in Yersinia enterocolitica detection in non-inspected swine

Avaliou-se a contaminação de carcaças e tonsilas de suínos por Y. enterocolitica em estabelecimentos de abate não inspecionados, comparando a pesquisa microbiológica convencional com a técnica da reação em cadeia de polimerase (PCR) e o tipo de amostra analisada (de tonsila ou de carcaça), como subsídio ao monitoramento microbiano em sistemas de análise de perigos e pontos críticos de controle. Calcularam-se os custos dos dois testes. Não se detectou Y. enterocolitica pela técnica microbiológica convencional, mas, pela técnica de PCR foi possível detectar a bactéria em 40 por cento das carcaças e em 43 por cento das tonsilas, incluindo cepas patogênicas nas tonsilas. Não houve diferença entre os resultados positivos para as amostras de tonsilas e esfregaços de superfície das carcaças. A PCR apresentou-se como uma alternativa na detecção do agente e uma técnica aparentemente mais eficaz, econômica e rápida. Os resultados indicam a PCR como um importante recurso para o controle de qualidade da carne suína.

The contamination of swine carcasses and tonsils by Yersinia enterocolitica in slaughterhouses without inspection was evaluated. The conventional microbiological analysis was compared with the PCR technique of carcass or tonsil swabs, as a subsidy to the microbiological evaluation in the HACCP system. The costs of the two techniques were also calculated. Y. enterocolitica was not detected by the conventional microbiological analysis. Using the PCR, it was possible to detect this bacterium in carcass (40 percent) and tonsil (43 percent) samples. There was no difference between the positive results for the carcass and tonsil samples. The PCR showed to be a more effective, fast and economic alternative for the Y. enterocolitica detection, as compared to the conventional microbiological analysis.

Effect of modified atmospheres on microbiological, color and sensory properties of refrigerated pork.

Pork loin samples were stored (4°C) in nylon polyethylene plastic bags using different modified atmospheres packaging (MAP): vacuum, 100% CO(2) 99% CO(2)+1% CO, 100% O(2) or 100% CO followed by vacuum. Throughout the storage period Pseudomonas growth was limited in loins packaged in all MAPs evaluated, except for 100% O(2). Psychrotrophs reached 10(7)CFUg(-1) after 20 days of storage except for the loin samples in 100% O(2) MAP that present count above 10(8)CFUg(-1). The 1%CO/99%CO(2) atmosphere was best for preserving the desirable pork loin color and the L* and a* values remained similar to the fresh meat values using this MAP. Pork loins in 99%CO(2)/1%CO MAP obtained the highest consumer acceptance scores after 24h of storage. These samples and those treated with CO and then vacuum packaged received the greatest acceptance scores even after 20 days of storage.

Detection of Lawsonia intracellularis in faeces of swine from the main producing regions in Brazil.

Swine proliferative enteropathy is an enteric disease caused by Lawsonia intracellularis which affects animals between 6 and 20 weeks of age, causing diarrhea, anorexia, and poor growth. The presence of L. intracellularis was evaluated in the faecal samples of 636 swine from 75 randomly chosen herds in the main swine-producing regions of Brazil. The pathogen was detected by the polymerase chain reaction method (PCR) using L. intracellularis specific primers. A 319-bp DNA fragment specific for L. intracellularis was produced on amplification of DNA from the faeces of pigs with proliferative enteropathy. Equal amounts of DNA extracted from the faeces of animals from the same herd were pooled together and, once L. intracellularis was detected, the faecal material of each animal was analyzed separately. The incidence of L. intracellularis was 33.4% in the state of Santa Catarina, 29.4% in Paraná, 26.3% in Minas Gerais, 16.7% in Mato Grosso, and 7.1% in São Paulo. The presence of the pathogenic agent was detected in samples from 15 farms, representing a total incidence of 20%. Although 46 animals (7.2%) were shown to be infected, 11% did not present any symptoms of swine proliferative enteropathy. The use of PCR allowed the detection of L. intracellularis in swine farms and the evaluation of the incidence of proliferative enteropathy in different regions of Brazil.

mRNA distribution of two isoforms of somatostatin receptor 2 (mSSTR2A and mSSTR2B) in mouse brain.

The primary gene transcript of the mouse somatostatin receptor 2 is alternatively spliced giving rise to two isoforms (mSSTR2A and mSSTR2B) which differ at the C-terminus. Using reverse transcription polymerase chain reaction (RT-PCR), both mRNAs were found in the cortex, hippocampus, hypothalamus, striatum, mesencephalon, cerebellum, medulla oblongata, pituitary and in testis, however with different ratios between mSSTR2A and mSSTR2B, implicating a tissue-specific control of transcription and splicing. Among the analyzed tissues, cortex contained the highest amounts of mSSTR2A but only little mSSTR2B, whereas the pons/medulla oblongata expressed both isoforms to an equal extent. Northern blot analysis of these tissues revealed a single mRNA of about 2.4 kb using a mSSTR2A-specific hybridization probe. No additional signal was seen using a probe which hybridizes to both mSSTR2A and mSSTR2B, suggesting that the two mRNAs may be nearly identical in length. In addition, in situ hybridization indicated that mSSTR2A is predominantly expressed in mouse brain, and mSSTR2B is never expressed independently from mSSTR2A.

Molecular cloning of a bovine MSH receptor which is highly expressed in the testis.

A novel G protein-coupled receptor (BDF3) was isolated from bovine genomic DNA by a combined approach of polymerase chain reaction (PCR) and hybridization techniques. The predicted amino acid sequence is 317 amino acids in length and displays 80% homology to the human alpha-MSH receptor MC1. Stably transfected into CHO-K1 cells, BDF3 mediates an increase of intracellular cAMP-levels following incubation with NLE-alpha-MSH, a potent alpha-MSH analog. The stimulation with ACTH1-10 is only moderate and gamma-MSH is ineffective. Northern blot analysis of bovine tissues revealed that the BDF3 gene is highly expressed in the testis as a single 2.3 kb mRNA species, suggesting an involvement of the BDF3 receptor in spermatogenesis.

The two isoforms of the mouse somatostatin receptor (mSSTR2A and mSSTR2B) differ in coupling efficiency to adenylate cyclase and in agonist-induced receptor desensitization.

The somatostatin receptor 2 (mSSTR2) is alternatively spliced into two isoforms (mSSTR2A and mSSTR2B) which differ at the C-terminus. Both receptors bind somatostatin peptides with a similar high affinity when stably expressed in CHO-K1 cells. However, the spliced form (mSSTR2B) mediates a more efficient inhibition of adenylate cyclase and is much more resistant to agonist-induced reduction of binding than the longer form (mSSTR2A). These findings indicate that alternative splicing may be a physiological mechanism to modulate receptor desensitization and G-protein coupling of mSSTR2.

Cloning of a novel putative G-protein-coupled receptor (NLR) which is expressed in neuronal and lymphatic tissue.

A novel G-protein-coupled receptor was isolated from mouse and rat neuronal and lymphatic tissues. The amino acid sequence of the rat receptor (rNLR) shows an overall homology of 80% to a recently cloned receptor from Burkitt's lymphoma cells (BLR1) which is exclusively expressed in lymphatic tissues [(1992) Eur. J. Immunol. 22, 2795]. Much less homology between rNLR and BLR1 was observed at the N-terminus (about 40%), whereas rNLR and the mouse homologue mNLR show 92% amino acid identity. Northern blot analysis of NLR revealed a predominant 5.5 kb mRNA species in various brain regions and neuronal cell lines, whereas in the spleen a 3 kb transcript is predominant. This distribution suggests a role of NLR in the nervous and immune systems.

Cloning and expression of a novel mouse somatostatin receptor (SSTR2B).

A mouse somatostatin (SS) receptor cDNA was cloned from neuroblastoma x glioma (NG108-15) cells. The sequence is almost identical to that of the mouse SSTR2 receptor [(1992) Proc. Natl. Acad. Sci. USA 89, 251)] but lacks about 300 nucleotides between transmembrane domain VII and the C-terminus. This spliced variant of SSTR2 (designated SSTR2B) encodes a protein which is 23 residues shorter than that predicted from the SSTR2 sequence, and differs in 15 amino acids at the C-terminus. mRNA corresponding to SSTR2B occurs in mouse tissues in higher abundance than that of SSTR2. SSTR2B binds SS peptides with high affinity when expressed in mammalian cells.

Riboflavin excretion byPachysolen tannophilus grown in synthetic medium supplemented withD-xylose.

Pachysolen tannophilus excreted riboflavin (12.7 m g/ml) into a synthetic medium withD-xylose as carbon source, when the pH was below 2.0. The addition of glucose enhanced the quantity of riboflavin excreted. The greatest riboflavin production was at pH 1.6 after 5 days at 30°C.