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Several products of industrial interest are produced by Bacillus , including enzymes, antibiotics, amino acids, insecticides, biosurfactants and bacteriocins. This study aimed to investigate the potential of two bacterial isolates (P5 and C3) from puba, a regional fermentation product from cassava, to produce multiple substances with antimicrobial and surface active properties. Phylogenetic analyses showed close relation of isolates P5 and C3 with Bacillus amyloliquefaciens and Bacillus thuringiensis , respectively. Notably, Bacillus sp. P5 showed antimicrobial activity against pathogens such as Listeria monocytogenes and Bacillus cereus , in addition to antifungal activity. The presence of genes encoding pre-subtilosin ( sboA ), malonyl CoA transacylase (ituD) , and the putative transcriptional terminator of surfactin ( sfp ) were detected in Bacillus sp. P5, suggesting the production of the bacteriocin subtilosin A and the lipopeptides iturin A and surfactin by this strain. For Bacillus sp. C3 the presence of sboA and spas (subtilin) genes was observed by the first time in members of B. cereus cluster. Bacillus sp. P5 showed emulsifying capability on mineral oil, soybean biodiesel and toluene, while Bacillus sp. C3 showed emulsifying capability only on mineral oil. The reduction of the surface tension in culture medium was also observed for strain P5, confirming the production of surface-active compounds by this bacterium. Monoprotonated molecular species and adducts of sodium and potassium ions of surfactin, iturin, and fengycin were detected in the P5 culture medium. Comparative MS/MS spectra of the peak m/z 1030 (C14 surfactin A or C15 surfactin B [M+Na] + ) and peak m/z 1079 (C15 iturin [M+Na] + ) showed the same fragmentation profile of standards, confirming the molecular identification. In conclusion, Bacillus sp. P5 showed the best potential for the production of antifungal, antibacterial, and biosurfactant substances.

Bacteria are assumed to efficiently remove organic pollutants from sewage in sewage treatment plants, where antibiotic-resistance genes can move between species via mobile genetic elements known as integrons. Nevertheless, few studies have addressed bacterial diversity and class 1 integron abundance in tropical sewage. Here, we describe the extant microbiota, using V6 tag sequencing, and quantify the class 1 integron-integrase gene (intI1) in raw sewage (RS) and activated sludge (AS). The analysis of 1,174,486 quality-filtered reads obtained from RS and AS samples revealed complex and distinct bacterial diversity in these samples. The RS sample, with 3,074 operational taxonomic units, exhibited the highest alpha-diversity indices. Among the 25 phyla, Proteobacteria, Bacteroidetes and Firmicutes represented 85% (AS) and 92% (RS) of all reads. Increased relative abundance of Micrococcales, Myxococcales, and Sphingobacteriales and reduced pathogen abundance were noted in AS. At the genus level, differences were observed for the dominant genera Simplicispira and Diaphorobacter (AS) as well as for Enhydrobacter (RS). The activated sludge process decreased (55%) the amount of bacteria harboring the intI1 gene in the RS sample. Altogether, our results emphasize the importance of biological treatment for diminishing pathogenic bacteria and those bearing the intI1 gene that arrive at a sewage treatment plant.

Puba or carimã is a Brazilian staple food obtained by spontaneous submerged fermentation of cassava roots. A total of 116 lactobacilli and three cocci isolates from 20 commercial puba samples were recovered on de Man, Rogosa and Sharpe agar (MRS); they were characterized for their antagonistic activity against foodborne pathogens and identified taxonomically by classical and molecular methods. In all samples, lactic acid bacteria were recovered as the dominant microbiota (7.86 ± 0.41 log10 CFU/g). 16S-23S rRNA ARDRA pattern assigned 116 isolates to the Lactobacillus genus, represented by the species Lactobacillus fermentum (59 isolates), Lactobacillus delbrueckii (18 isolates), Lactobacillus casei (9 isolates), Lactobacillus reuteri (6 isolates), Lactobacillus brevis (3 isolates), Lactobacillus gasseri (2 isolates), Lactobacillus nagelii (1 isolate), and Lactobacillus plantarum group (18 isolates). recA gene-multiplex PCR analysis revealed that L. plantarum group isolates belonged to Lactobacillus plantarum (15 isolates) and Lactobacillus paraplantarum (3 isolates). Genomic diversity was investigated by molecular typing with rep (repetitive sequence)-based PCR using the primer ERIC2 (enterobacterial repetitive intergenic consensus). The Lactobacillus isolates exhibited genetic heterogeneity and species-specific fingerprint patterns. All the isolates showed antagonistic activity against the foodborne pathogenic bacteria tested. This antibacterial effect was attributed to acid production, except in the cases of three isolates that apparently produced bacteriocin-like inhibitory substances. This study provides the first insight into the genetic diversity of Lactobacillus spp. of puba.

The aims of this study were to characterise the yeasts present in the reconstituted fruit juices and coconut water extracted with "coconut machines", both collected from commercial outlets in a Brazilian city, and to investigate the antifungal resistance of isolates from these beverages that were able to grow at 37°C. The yeast population counts in the coconut water samples ranged from 1.7 to >6.5logcfu/ml, and in the reconstituted fruit juices, the counts ranged from 1.5 to >5.5logcfu/ml. Aureobasidium pullulans, Candida boidinii, Candidaintermedia, Candidaoleophila, Candidaparapsilosis, Candidasantamariae, Candidatropicalis, Clavispora lusitaniae, Kloeckera apis, Lachancea fermentati, Pichia fermentans and Rhodotorula mucilaginosa were the most frequent species isolated from these beverages. At least 18 yeast species isolated from these beverages have been reported as opportunistic pathogens. Eight yeast isolates were resistant to fluconazole, seven were resistant to itraconazole, and 26 to amphotericin B. Some yeast species were resistant to more than one of the antifungal drugs tested. Two isolates of C. tropicalis from the reconstituted fruit juices exhibited resistance to all three drugs. The presence of yeast strains that are resistant to commonly used antifungal drugs suggests a potential risk, at least to immunocompromised individuals who consume these beverages., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In this study, we investigated the presence of plasmid-mediated quinolone resistance (PMQR) genes among 101 ciprofloxacin-resistant urinary Escherichia coli isolates and searched for mutations in the quinolone-resistance-determining regions (QRDRs) of the DNA gyrase and topoisomerase IV genes in PMQR-carrying isolates. Eight isolates harboured the qnr and aac(6')-Ib-cr genes (3 qnrS1, 1 qnrB19 and 4 aac(6')-Ib-cr). A mutational analysis of the QRDRs in qnr and aac(6')-Ib-cr-positive isolates revealed mutations in gyrA, parC and parE that might be associated with high levels of resistance to quinolones. No mutation was detected in gyrB. Rare gyrA, parC and parE mutations were detected outside of the QRDRs. This is the first report of qnrB19, qnrS1 and aac(6')-Ib-cr -carrying E. coli isolates in Brazil.

The aim of the present study was to compare the effect of intragastric administration with two strains of Bifidobacterium animalis subsp. lactis (Bifido A and Bifido B), in gnotobiotic and conventional mice, challenged with Salmonella Typhimurium. In vitro antagonism test showed that the two strains were able to produce antagonistic substances against various pathogenic microorganisms. In an ex vivo antagonism test the production of antagonistic substances was observed only against three out ten pathogens tested. Both Bifidobacterium strains were able to colonize and to maintain high population levels in the digestive tract of gnotobiotic mice. In addition, the two strains had low and limited translocation ability and did not cause any histological lesion in any of the organs analyzed. Both strains were able to reduce the fecal number of Salmonella in gnotobiotic mice challenged with the pathogen, but only Bifido B was able to confer a protection as demonstrated by a lower mortality. Higher levels of sIgA and IL-10 were observed only in Bifido B mono-associated mice when compared to germ free group. We could conclude that, among the parameters analyzed, the strain Bifido B exhibited the more desirable characteristics to be used as a probiotic.

Lactobacilli isolated from the vaginal tract of women with and without bacterial vaginosis (BV) were identified and characterized for the production of antagonists. Bacterial samples were isolated from healthy women (N = 16), from patients with clinical complaints but without BV (N = 30), and from patients with BV (N = 32). Identification was performed using amplified ribosomal DNA restriction analysis. Production of antagonistic compounds was evaluated by the double-layer diffusion technique using Gram-positive (N = 9) and Gram-negative bacteria (N = 6) as well as yeast (N = 5) as indicator strains. Of a total of 147 isolates, 133 were identified as pertaining to the genus Lactobacillus. Lactobacillus crispatus was the species most frequently recovered, followed by L. johnsonii and L. jensenii. Statistical analysis showed that L. crispatus was more frequent in individuals without BV (P < 0.05). A higher production of antagonists was noted in L. crispatus isolates from healthy women (P < 0.05). More acidic local pH and higher H2O2 production by isolated lactobacilli from healthy women suggest these mechanisms as the possible cause of this antagonism. In conclusion, a significant correlation was detected between the presence and antagonistic properties of certain species of Lactobacillus and the clinical status of the patients.

Aim: Lactobacillus sakei 2a isolated from sausage and presenting an in vitro antagonistic activity against Listeria monocytogenes Scott A was tested for a protective effect in mice experimentally challenged with the enterobacteria., Methods and Results: In the experimental group, germ-free mice (n = 24) were inoculated intragastrically with 0.1 ml of a suspension containing 10(8) colony forming units (CFU) of Lact. sakei and 4 days later the animals were challenged intragastrically with 0.1 ml of a suspension containing 10(8) CFU of L. monocytogenes. Control group (n = 24) was only inoculated with the bacterial pathogen. Faecal counts showed that L. monocytogenes reached similar population levels (10(9) CFU g(-1) of contents) in both the groups. Animals in the control group showed lower (P = 0.0004) survival frequency (58.3%) when compared with the experimental one (100%). Anatomopathological examination confirmed the mortality data., Conclusions: Lactobacillus sakei 2a can survive in the mammal digestive tract where showed a protective effect against L. monocytogenes. This phenomenon was not due to an antagonistic activity., Significance and Impact of the Study: Use of Lact. sakei 2a as a meat starter could inhibit not only L. monocytogenes growth in the fermented product but also pathogen virulence in the gastrointestinal tract.

A freshwater enterobacterial population (N = 111) was studied for antimicrobial and mercury resistance patterns, and for its possible association with biotic and abiotic factors in that environment. Conventional biochemical tests identified Klebsiella sp, Morganella sp, Serratia sp, Escherichia sp, Enterobacter sp, Edwarsiella sp, Proteus sp, Citrobacter sp, Providencia sp, and Kluyvera sp. There was no correlation between antimicrobial resistance patterns of isolates and bacterial genera, but resistance patterns varied among water samples and between seasons. Resistance to multiple antimicrobials was common (61%). The percentage of bacteria resistant to at least one antimicrobial differed between the rainy (100%) and dry seasons (89%). Resistance to beta-lactams and chloramphenicol was the most frequent and resistance to amikacin, gentamicin and kanamycin was less frequent. The main water variables examined (abiotic factors pH and temperature; biotic factor chlorophyll a concentration) did not influence antimicrobial resistance. Significant impact on freshwater enterobacteria, as evidenced by antimicrobial-multiple resistance and by the presence of bla(TEM) gene, may point to the fact that it has an important role in horizontal spread of resistance.

Aims: The antagonistic activity of the Escherichia coli strain H22 against enteric bacteria was studied both in vitro and in vivo., Methods and Results: In vitro, bacterial strains belonging to seven of nine genera of the family Enterobacteriaceae (Enterobacter, Escherichia, Klebsiella, Morganella, Salmonella, Shigella and Yersinia) were inhibited by the strain H22. Six days after simultaneous oral inoculation in germ-free mice, E. coli strain H22 reduced the faecal population of Shigella flexneri 4 to undetectable levels (P < 0.05). In ex vivo assay, inhibitory zones against Sh. flexneri 4 were observed around faecal samples from mice inoculated with E. coli strain H22. The in vitro inhibition of Sh. flexneri 4 was shown to be mediated by microcin C7. In addition to microcin C7, strain H22 was shown to produce aerobactin, new variants of colicins E1 and Ib, and bacteriophage particles with morphology similar to the phages of the family Myoviridae., Conclusions: Altogether, the properties of E. coli H22, observed both under in vitro and in vivo conditions, suggest its potential use as a probiotic strain for livestock and humans., Significance and Impact of the Study: The strain H22 was shown to produce several antimicrobial compounds with inhibitory capabilities against pathogenic or potentially pathogenic enterobacteria.

Sour cassava starch is a traditional fermented food used in the preparation of fried foods and baked goods such as traditional cheese breads in Brazil. Thirty samples of sour cassava starch were collected from two factories in the state of Minas Gerais. The samples were examined for the presence of lactic acid bacteria, yeasts, mesophilic microorganisms, Bacillus cereus and faecal coliforms. Lactic acid bacteria and yeasts isolates were identified by biochemical tests, and the identities were confirmed by molecular methods. Lactobacillus plantarum and Lactobacillus fermentum were the prevalent lactic acid bacteria in product from both factories, at numbers between 6.0 and 9.0 log cfu g(-)(1). Lactobacillus perolans and Lactobacillus brevis were minor fractions of the population. Galactomyces geothricum and Issatchenkia sp. were the prevalent yeasts at numbers of 5.0 log cfu g(-)(1). A species similar to Candida ethanolica was frequently isolated from one factory. Mesophilic bacteria and amylolytic microorganisms were recovered in high numbers at all stages of the fermentation. B. cereus was found at low numbers in product at both factories. The spontaneous fermentations associated with the production of sour cassava starch involve a few species of lactic acid bacteria at high numbers and a variety of yeasts at relatively low numbers.

Probiotics are defined as viable microorganisms that exhibit a beneficial effect on the host's health when they are ingested. Two important criteria are used for selection of probiotic microorganisms: they must be able to survive in the gastrointestinal environment and to present at least one beneficial function (colonization resistance, immunomodulation or nutritional contribution). Generally, in vitro assays demonstrating these properties were used to select probiotics but it is unclear if the data can be extrapolated to in vivo conditions. In the present work, twelve Saccharomyces cerevisiae strains isolated from different environments (insect association, tropical fruit, cheese and "aguardente" production) and pre-selected for in vitro resistance to simulated gastrointestinal conditions were inoculated in germ-free mice to evaluate their real capacity to colonize the mammal digestive tract. Using these data, one of the yeasts (S. cerevisiae 905) was selected and tested in gnotobiotic (GN) and conventional (CV) mice for its capacity to protect against oral challenge with two enteropathogenic bacteria (Salmonella Typhimurium and Clostridium difficile). The yeast reached populational levels potentially functional in the gastrointestinal portions where the enteropathogens tested act. No antagonism against either pathogenic bacterium by the yeast was observed in the digestive tract of GN mice but, after challenge with S. Typhimurium, mortality was lower and liver tissue was better preserved in CV animals treated with the yeast when compared with a control group (p<0.05). Histopathological results of intestines showed that the yeast also presented a good protective effect against oral challenge with C. difficile in GN mice (p<0.05). In conclusion, among the 12 S. cerevisiae tested, strain 905 showed the best characteristics to be used as a probiotic as demonstrated by survival capacity in the gastrointestinal tract and protective effect of animals during experimental infections.

In this study, the effect of Carica papaya seed macerate on conjugal R plasmid transfer from Salmonella typhimurium to Escherichia coli was investigated in vitro and in the digestive tract of gnotobiotic mice. Twenty-five micrograms per milliliter and 430 mg (administered intragastrically twice a day) of papaya seed macerate concentrations were used during conjugation for in vitro and in vivo assays, respectively. High frequency of conjugation inhibition by macerate was observed for both in vitro and in vivo experiments, independently of bacterial growth and mating conditions. Papaya seed macerate caused a reduction of the transconjugant population ranging from 71% to about 100%. There was no lethal effect of the seed macerate on donor or recipient cells in the concentrations used. Once the mechanisms and magnitude of resistance gene transfer are clearly understood, strategies to reduce or minimize the dissemination of these genes could be relevant. The data here obtained show a clinical potential use of papaya seed macerate on this transfer.

Aims: The aim of this work was to purify and characterize antibacterial compounds produced by Lactobacillus murinus strain L1., Methods and Results: Antagonistic activity was observed in a deferred agar-spot assay against spoilage and pathogenic bacteria, but not against lactobacilli. The inhibitory activity occurred between pH 3.0 and 5.0, and was heat stable. The active compounds were purified by gel filtration chromatography and two peaks of antibacterial activity were observed using Bacillus cereus ATCC 11778 and Shigella sonnei ATCC 11060 as indicator strains. Two active low molecular weight compounds were responsible for this phenomenon and UV spectroscopy, gas chromatography and mass spectrometry were used to characterize them. One of them is lactic acid, while the other is a mono-substituted aromatic ring apparently constituted by group residues of m/z 192 linked in tandem to phenylalanine., Conclusions: Lactobacillus murinus produces at least two low molecular weight compounds active against B. cereus and Sh. sonnei., Significance and Impact of the Study: This is the first purification of a new broad-spectrum antibacterial compound from Lact. murinus which inhibits various pathogenic and food spoilage bacteria without acting on other lactobacilli. Using it as a biotechnological control agent of bacterial spoilage may be a promising possibility for the food industry.

Saccharomyces boulardii was shown to be capable of inhibiting multiplication of enteropathogenic bacteria in vitro and is currently used for its anti-diarrhoea properties. We studied the capacity of this yeast to antagonize Salmonella typhimurium and Shigella flexneri in the intestinal tract of conventional or gnotobiotic NMRI mice. Conventional animals were given daily 10 mg doses of S. boulardii, whereas germ-free animals were given a single 10 mg dose. Both groups were challenged orally 5 d later with the pathogenic bacteria (10(8) or 10(2) viable cells, respectively). Control groups were treated with saline instead of S. boulardii. Mortality and/or histopathological data showed a protective effect against the pathogenic bacteria in yeast-treated mice. Saccharomyces boulardii colonized the digestive tract of gnotobiotic mice and the number of viable cells ranged around 10(10) g-1 of faeces. In experimental and control gnotobiotic animals, Salm. typhimurium and Sh. flexneri became rapidly established at a level of about 10(10) viable cells g-1 of faeces and remained at high levels until the animals died or were sacrificed. The protection against Salm. typhimurium and Sh. flexneri obtained in conventional and/or gnotobiotic mice previously associated with S. boulardii is not due to the reduction of the bacterial populations in the intestines.

Bacterial invasiveness and immunological responses were studied in germfree (GF) and conventional (CV) mice infected with Salmonella typhimurium. Bacterial counts of homogenates prepared from liver and spleen showed that the colony forming units (CFU) increased rapidly in GF mice and reached lethal proportions (10(9) cell per organ) by day 6. In CV mice, these counts increased to about 10(4.5) log cell per organ by day 6 and then declined slowly. An increase in serotype-specific IgM and IgG levels was noted in CV mice with a maximum by day 2. Very low values of these IgM and IgG were observed in GF mice during the course of infection. Delayed type hypersensitivity (DTH) response as measured by footpad swelling was higher in CV animals. Higher hypersensitivity to LPS during infection in GF animals resulted in death of all the animals tested for DTH after day 2. The data obtained suggest that during a rapid invasive bacterial infection, the slow development of immune response of GF mice may result in death of these animals.

The reactivity of 12 surgically removed uveal melanoma lesions with monoclonal antibodies (MoAb) to 14 membrane-bound and 2 cytoplasmic cutaneous melanoma-associated antigens (MAA), to the 2 subunits of HLA Class I antigens and to the gene products of the HLA-D region was compared with that of cutaneous melanoma lesions and correlated with their histiotype. The membrane-bound determinants defined by the anti-Mr 92,000 and 45,000 MAA MoAb TP39.1, anti-Mr 110,000 MAA MoAb M111, anti-Mr 118,000 MAA MoAb TP36.1, anti-Mr 115,000 MAA MoAb 345.134, anti-ICAM-1 MoAb CL203.4 and anti-Mr 31,000 MAA MoAb M2590, and the cytoplasmic determinants defined by the anti-MAA MoAb 465.12 and 2G-10 display a distribution in uveal melanoma lesions similar to that in cutaneous melanoma lesions. On the other hand, membrane-bound determinants defined by the anti-Mr 100,000 MAA MoAb 376.96, anti-9-O-acetyl-GD3 ganglioside MoAb ME311 and anti-GD2-GD3 ganglioside MoAb ME361 were not detected in the uveal melanoma lesions tested. Furthermore, the membrane-bound determinants defined by the anti-GD3 MoAb R24, anti-nerve growth factor receptor MoAb ME20.4, anti-Mr 97,000 MAA MoAb 140.240, anti-carcinoembryonic antigen MoAb B1.1 and anti-HMW-MAA 149.53, 225.28, and 763.74 have a markedly lower expression in uveal than in cutaneous melanoma lesions. Incubation of uveal melanoma lesions with the pool of the MoAb 149.53, 225.28, and 763.74 recognizing distinct and spatially distant determinants of the HMW-MAA increased the intensity of staining of six lesions and stained four lesions which were not stained by the individual monoclonal antibodies. The distribution of HLA Class I antigens in uveal melanoma lesions resembles that in cutaneous melanoma lesions, since they are expressed in all the lesions of the mixed and epithelioid type but were not detected in those of the spindle type, i.e., the counterparts of nevocellular nevi. HLA Class II antigens are expressed with a lower frequency in uveal than in cutaneous melanoma lesions, since they were detected only in 2 of the 12 lesions. One of them is of the mixed type and the other one of the epithelioid type. Besides HLA antigens the determinants defined by the anti-carcinoembryonic MoAb B1.1, anti-ICAM-1 MoAb CL203.4, and anti-GD3 MoAb R24 displayed a differential distribution in the different histiotypes of uveal melanoma, since they are preferentially expressed in lesions of the mixed and epithelioid type.

The present study investigates the lethality of intragastric Salmonella typhimurium infection of germfree (GF) and conventional (CV, not germfree) mice. The introduction of only 10 viable S. typhimurium into the gastrointestinal tract of GF mice resulted in death of all animals within 8 days of inoculation. In contrast, the 50% lethal dose for CV mice was 4.7 x 10(3) viable organisms. The results demonstrate that resident microbes protect mice from the pathogenic effect of S. typhimurium infection.