Production of histamine and tyramine by lactic acid bacteria isolated from vacuum-packed sugar-salted fish

The incidence of histamine- or tyramine-producing lactic acid bacteria was examined in several products of vacuum-packed sugar-salted fish (salmon, halibut, mackerel). No histamine-producing isolates were observed, whereas the majority of tyramine-producing isolates were identified as Carnobacterium spp. These organisms were shown to be important members of the microbial flora during storage of vacuum-packed sugar-salted salmon at 5°C. The amount of tyramine produced was reduced by lowering the temperature from 9°C to 4°C for all of five strains of carnobacteria or lactobacilli. The majority of tyramine was produced during the exponential growth phase for Carnobacterium piscicola N 5 and Lactobacillus viridescens N 69. The ability of these bacteria to produce tyramine may be used as an index of microbial quality/acceptability of stored vacuum-packed sugar-salted fish.     

Genotypic characterization of Brochothrix spp. isolated from meat,poultry and fish

Aim: To study genotypic diversity of isolates of Brochothrix thermosphacta recovered from meat, poultry and fish. Methods and Results: A total of 27 bacteria isolated from 19 samples of meat, poultry and fish were identified phenotypically and genotypically using PCR amplification of 16S‐23S rDNA intergenic transcribed spacer (ITS‐PCR), repetitive sequence‐based PCR (rep‐PCR) and 16S rDNA sequencing. Using ITS‐PCR, all bacteria showed the same DNA profile as the reference strains of Br. thermosphacta, allowing typing of the isolates at species level. Using 16S rDNA sequencing, all isolates were identified, at genus and species level, as Br. thermosphacta. Identification as Br. campestris was observed with a lower, but very close, level of similarity. Rep‐PCR was more discriminatory than ITS‐PCR and allowed differentiation of four subgroups among the isolates. Conclusion: Minor genotypic differences among Br. thermosphacta strains from meat, poultry and fish were observed. Significance and Impact of the Study: A rudimentary exploration of genotypic differences of Br. thermosphacta from meat, poultry and fish resulted in preliminary confirmation of the suitability of ITS‐PCR for typing Br. thermosphacta and confirmed the value of rep‐PCR fingerprinting to discriminate between Br. thermosphacta strains.     

The activity of ceftiofur sodium for Aeromonas spp. isolated from ornamental fish.

Our objective was to determine the activity of ceftiofur sodium against Aeromonas hydrophila and A. sobria isolated from a variety of domestic and imported tropical fish. Twelve antimicrobial drugs were tested for effectiveness against these aeromonads using the Kirby-Bauer Disk Diffusion technique and minimum inhibitory concentration determinations. Ceftiofur sodium was highly effective in vitro against aeromonads isolated from ornamental fish. Of the 42 isolates of Aeromonas spp. tested, none were resistant to ceftiofur sodium; however, all isolates were resistant to ampicillin, and 71% were resistant to tetracycline.     

Characterisation ofPseudomonas spp. isolated from foods

PutativePseudomonas spp. (102 isolates) from different foods were first characterised by API 20NE and then tested for some enzymatic activities (lipase and lecithinase production, starch hydrolysis and proteolytic activity). However subsequent molecular tests did not always confirm the results obtained, thus highlighting the limits of API 20NE. Instead RFLP ITS1 and the sequencing of 16S rRNA gene grouped the isolates into 6 clusters:Pseudomonas fluorescens (cluster I),Pseudomonas fragi (duster II and V)Pseudomonas migulae (cluster III),Pseudomonas aeruginosa (cluster IV) andPseudomonas chicorii (cluster VI). The pectinolytic activity was typical of species isolated from vegetable products, especiallyPseudomonas fluorescens. InsteadPseudomonas fragi, predominantly isolated from meat was characterised by proteolytic and lipolytic activities.     

Kazachstania psychrophila sp. nov., a novel psychrophilic yeast isolated from vacuum-packed beef

Five novel ascosporogenous yeast strains (H382, H396, H409, H433^T and H441) were found through a survey of vacuum-packed beef microbiota. Sequence analysis of ITS domain and LSU rRNA genes showed that the new strains represent a distinct lineage within the genus Kazachstania, closely related to Kazachstania lodderae (97.0 % identity) and Kazachstania ichnusensis (96.1 % identity). The main difference of strains H382, H396, H409, H433^T and H441 to strains of known Kazachstania species is the maximum growth temperature, which is below 20 °C for the new strains, whereas related species grow at 25 °C. Furthermore, the strains differed from known Kazachstania species in assimilation and fermentation patterns of carbon sources. Based on these characteristics, the five strains are considered to represent a novel species of the genus Kazachstania for which the name Kazachstania psychrophila sp. nov. is proposed. The type strain is H433^T (DSM 26230^T=CBS 12689^T). The Mycobank number of the type strain is MB 803980.     

Virulence ofPythium spp. isolated from pond water

Pre-emergence damping-off tests indicate that Pythiumspp. from pond water can be divided into two categories: avirulent Pythium (P. diclinum, P. marsipium, P. middletonii, P. monospermum, P. pleroticum, P. undulatum)and weakly virulent Pythium (P. catenulatum, P. coloratum, P. dissotocum, P. papillatum, Pythium Group F, Pythium group HS, Pythium group P, Pythiumgroup T). Post-emergence damping-off tests indicate that the pythia tested can also be divided into three categories: avirulent Pythium (P. catenulatum, P. dissotocum, P. marsipium, P. monospermum, P, papillatum, P. pleroticum, P. undulatum, Pythium group F, Pythium group T),weakly virulent Pythium (P. coloratum, P. diclinum, P. middletonii, Pythiumgroup P and moderately virulent Pythium (Pythium group HS).     

Catabolism of 5-chlorosalicylate by a Bacillus isolated from the Mississippi River.

A strain of Bacillus brevis isolated from a polluted section of the Mississippi River was shown to utilize 5-chloro-2-hydroxybenzoate (5-chlorosalicylate) as a sale source of carbon and energy. Enzymic analyses of cell-free extracts prepared from 5-chlorosalicylate-grown cells demonstrated that the initial step in the pathway involved cleavage of the aromatic ring between C1 and C2 by a specific 5-chlorosalicylate 1,2-dioxygenase. Loss of chloride from the growth substrate occurred after ring fission and was probably enzyme mediated. An intermediate chlorolactone apparently lost chloride by enzymatic hydrolysis with formation of maleylpyruvate. Maleylpyruvate was further degraded by both glutathione-dependent and glutathione-independent mechanisms, with these reactions being identical to the terminal reactions of the gentisate pathway. It was suggested that this novel 5-chlorosalicylate pathway may have evolved by recruitment of enzymes from an ancestral gentisate pathway.     

Epidemiological typing of Bacillus spp. isolated from food.

Biotypes, fatty acid profiles, and restriction fragment length polymorphisms of a PCR product (PCR-RFLP of the cereolysin AB gene) were compared for 62 isolates of the Bacillus cereus group. Eleven isolates originated from various foods, and 51 isolates were obtained from pasteurized milk which had been processed by two different dairies. The isolates were clustered into 6 biotypes, 10 fatty acid groups, or 7 PCR-RFLP clusters. Isolates with mesophilic or psychrotrophic characteristics were preferentially distributed into specific fatty acid or PCR-RFLP groups (P = 0.004). Unique fatty acid clusters were predominantly found in milk samples of each dairy (P < 0.0001), suggesting that certain dairy plants may harbor plant-specific B. cereus which might constantly contribute to postpasteurization contamination.     

Antimicrobial susceptibilities of Aeromonas spp. isolated from environmental sources

Aeromonas spp. are ubiquitous aquatic bacteria that cause serious infections in both poikilothermic and endothermic animals, including humans. Clinical isolates have shown an increasing incidence of antibiotic and antimicrobial drug resistance since the widespread use of antibiotics began. A total of 282 Aeromonas pure cultures were isolated from both urban and rural playa lakes in the vicinity of Lubbock, Texas, and several rivers in West Texas and New Mexico. Of these, at least 104 were subsequently confirmed to be independent isolates. The 104 isolates were identified by Biolog and belonged to 11 different species. The MICs of six metals, one metalloid, five antibiotics, and two antimicrobial drugs were determined. All aeromonads were sensitive to chromate, cobalt, copper, nickel, zinc, cefuroxime, kanamycin, nalidixic acid, ofloxacin, tetracycline, and sulfamethoxazole. Low incidences of trimethoprim resistance, mercury resistance, and arsenite resistance were found. Dual resistances were found in 5 of the 104 Aeromonas isolates. Greater numbers of resistant isolates were obtained from samples taken in March versus July 2002 and from sediment versus water. Plasmids were isolated from selected strains of the arsenite- and mercury-resistant organisms and were transformed into Escherichia coli XL1-Blue MRF'. Acquisition of the resistance phenotypes by the new host showed that these resistance genes were carried on the plasmids. Mercury resistance was found to be encoded on a conjugative plasmid. Despite the low incidence of resistant isolates, the six playa lakes and three rivers that were sampled in this study can be considered a reservoir for antimicrobial resistance genes.     

Characteristic of Listeria spp. bacteria isolated from food products

The frequency of occurrance of Listeria strains in different food products was determined. Biochemical characteristic of the isolated strains was achieved in accordance with procedure included in PNEN ISO 11290 standard, genus was determined byApiListeria (bioMéieux) test. Sensitivity to selected antibacterial medicines was investigated using disck method and Mueller-Hinton 2 Agar medium. From the 577 examinated food samples 126 strains of Listeria were isolated and among them: 34.1% L. monocytogenes, 36,5% L. welshimeri, 19.0% L. innocua, 3.17% L. grayi, 0.79% L. seeligeri, 0.79% L. seeligeri/welshinmeri and 5.56% L. ivelshimeri/innocua. L. monocytogenes strains most often were found in minced pork, culinary products and in frozen vegetables. On the base of ApiListeria (bioMéieux) test the isolated L. monocytogenes strains were qualified into 2 biochemical types. It was found that all L. monocytogenes were sensitive to sulphametaksazol/trimetoprim and ampicyllin, 25% of strains were moderatety sensitive to penicillin and only 2 L. monocytogenes strains were resistant to gentamicin. Presence of Listeria spp. microorganisms in food products may be an production hygiene indicator for critical control point and show the possibility of contamination with L. monocytogenes strains.     

PCR detection of psychrophilic Clostridium spp. causing 'blown pack' spoilage of vacuum-packed chilled meats

AIMS: To develop a practical molecular procedure that directly, without isolation, and specifically detects the presence of clostridia which cause 'blown pack' spoilage of vacuum-packed meat. METHODS AND RESULTS: Primer sets and PCR amplification procedures were developed that detect the presence of 16S rDNA gene and/or 16S-23S rDNA internal transcribed spacer fragments of 'blown pack' causing clostridia in meat. The specificity of the developed procedures was evaluated with DNA obtained from close phylogenetic neighbours of 'blown pack' causing clostridia, food clostridia and common meat spoilage microorganisms. The sensitivity of detection was assessed in non-enriched and low-temperature-enriched beef mince inoculated with serially diluted pure cultures of Clostridium estertheticum DSMZ 8809T and Cl. gasigenes DB1AT. The efficacy of detection procedures was evaluated for naturally contaminated vacuum-packed meat samples. Three primer sets, 16SE, 16SDB and EISR, produced amplicons of the expected size with DNA templates from target clostridia, but failed to yield PCR products with DNAs from any other microorganisms tested. With 16SE and 16SDB primers, minimum levels of detection were 104 CFU g(-1) for non-enriched, and 102 CFU g(-1) for enriched meat samples. Based on the established specificity of these primers, as well as DNA sequencing of amplicons, Cl. gasigenes was confirmed as the causative agent of 'blown pack' spoilage in two packs, and Cl. estertheticum as the causative agent in the third. CONCLUSIONS: The developed method can be used for rapid detection of 'blown pack' causing clostridia in commercial blown packs, or following low temperature enrichment, for detection of these microorganisms in meat containing as few as 100 clostridial cells per gram. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper reports practical procedures that can be used for rapid confirmation of the causative agents of clostridial 'blown pack' spoilage in commercial spoiled packs, or for detection of psychrophilic clostridia in epidemiological trace back of 'blown pack' spoilage incidents in meat processing plants.     

Heat resistance of Lactobacillus spp. isolated from Cheddar cheese

Mesophilic Lactobacillus spp. are the dominant organisms in mature Cheddar cheese. The heat resistance of broth grown cultures of Lactobacillus plantarum DPC1919 at temperatures between 50 and 57.5 degrees C, Lact. plantarum DPC2102 at temperatures between 48 and 56 degrees C and Lact. paracasei DPC2103 at temperatures between 50 and 67.5 degrees C was determined. The z-values for Lact. plantarum DPC1919, Lact. Plantarum DPC2102 and Lact. paracasei DPC2103 were 6.7 degrees C, 6.2 degrees C and 5.3 degrees C, respectively. Lactobacillus paracasei DPC2103 showed evidence of injury and recovery, especially at higher temperatures. Milk grown cultures of strains DPC2102 and DPC2103 showed greater heat resistance than broth grown cultures, tailing of the death curves and a nonlinear z-curve. Of the three strains, Lact. paracasei DPC2103 had the potential to survive pasteurization temperatures, whether grown in milk or broth.     

Pantothenate auxotrophy of Methylobacterium spp. isolated from living plants

A number of pink-pigmented facultative methylotrophs (PPFMs) belonging to Methylobacterium spp. isolated from living plant samples were found to require B vitamins for their growth in minimal medium, and most B vitamin-auxotrophic PPFMs required pantothenate (vitamin B_⁵). Further investigation of pantothenate auxotrophy using the representative strain Methylobacterium sp. OR01 demonstrated that this strain cannot synthesize β-alanine, one of the precursors of pantothenate. β-alanine and several precursors of pantothenate restored the growth of Methylobacterium sp. OR01 in minimal medium. Furthermore, this strain could colonize leaves of Arabidopsis thaliana cultivated in medium without pantothenate or its precursors. Pantothenate, β-alanine and several precursors were detected in the suspension of A. thaliana leaves. These results suggest that pantothenate-auxotrophic PPFMs can symbiotically colonize the surface of plant leaves by acquiring β-alanine and other precursors, in addition to pantothenate. Finally, the fitness advantage of B vitamin auxotrophy of PPFMs in the phyllosphere environment is discussed.     

Transferable erythromycin resistance in Listeria spp. isolated from food.

An erythromycin-resistant (Emr) Listeria innocua and an Emr Listeria monocytogenes isolate both carried ermC genes, which code for rRNA methylases. The ermC genes were transferable by conjugation to recipient L. monocytogenes, Listeria ivanovii, and Enterococcus faecalis but did not appear to be associated with conjugative plasmids.     

Characterization of cellulases of fungal endophytes isolated from Espeletia spp.

Endophytes are microorganisms that asymptomatically invade plant tissues. They can stimulate plant growth and/or provide defense against pathogen attacks through the production of secondary metabolites. Most endophyte species are still unknown, and because they may have several applications, the study of their metabolic capabilities is essential. We characterized 100 endophytes isolated from Espeletia spp., a genus unique to the paramo ecosystem, an extreme environment in the Andean mountain range. We evaluated the cellulolytic potential of these endophytes on the saccharification of the oil palm empty fruit bunch (OPEFB). The total cellulolytic activity was measured for each endophyte on filter paper (FPA). In addition, the specific carboxymethyl cellulase (CMCase), exoglucanase, and β-glucosidase activities were determined. We found four fungi positive for cellulases. Of these fungi, Penicillium glabrum had the highest cellulolytic activity after partial purification, with maximal CMCase, exoglucanase and β-glucosidase enzyme activities of 44.5, 48.3, and 0.45 U/ml, respectively. Our data showed that the bioprospection of fungi and the characterization of their enzymes may facilitate the process of biofuel production.     

Characteristics of Yersinia spp. isolated from wild and zoo animals

Thirteen strains of Yersinia spp. were isolated at the Rome zoo and at Castelporziano, a game preserve near Rome. The strains were tested for calcium dependency, autoagglutination, heat-stable toxin production, 50% minimum lethal dose in mice (LD50), pyrazinamidase activity and content of plasmids by electrophoresis in agarose gel. The former three tests were negative for all strains, the LD50 was always greater than or equal to 1 X 10(7.6) CFU/ml and pyrazinamidase activity was positive for all strains. Electrophoresis revealed the presence of two plasmids of 27 and 66 megadaltons (MDa) in the two strains of Y. enterocolitica of serotype 027 isolated from animals in the zoo. The two strains of the same species and serotype, isolated from wild animals harboured a 42-MDa plasmid. A small plasmid of 2 MDa was found in two strains of Y. enterocolitica of serotype 07.8 from two subsequent samples of a zoo animal.     

Susceptibility to antibiotics of Enterobacter spp. rods isolated from urine

Enterobacter spp. rods are opportunistic microorganisms which cause of urinary tract infections. The aim of this study was the evaluation of the susceptibility to antimicrobial agents antibiotics of Enterobacter spp. rods isolated from urine. The study was carried 50 of Enterobacter spp strains isolated in the Clinical Microbiology Department of dr. A. Jurasz University Hospital. Antibiotic susceptibility was tested by disk diffusion method. All of strains were susceptible to imipenem and meropenem. There was 87,5% of strains sensitive to doripenem, 79,2% to ertapenem, 54,0% to piperacillin/tazobactam and 50,0% to cephepime. The relatively high percentage (62,0%) of Enterobacter spp. was sensitive to fluoroquinolones. Extended spectrum beta-lactamases were produced by 24 (48,0%) strains.