Genotypic assessment of a dichotomous key to identify Vibrio coralliilyticus, a coral pathogen.

Vibrio coralliilyticus is a known pathogen to corals and larvae of bivalves. Its identification is made based on phenotypic and genotypic characters of isolated strains. To evaluate the efficiency of the phenotypic identification, 21 strains identified as V. coralliilyticus using a widely used dichotomous key were analyzed by qualitative PCR and sequencing of the 16S rDNA region. The results obtained by the behavioral test, amino acids usage, allow us to distinguish 3 A/L/O profiles: (1) A+/L-/O+; (2) A+/L+/O+; and (3) A-/L+/O+. In the genotypic tests, all strains tested positive with primers specific for the Vibrio genus. However, when primers were used for species identification, the results did not match those obtained with the dichotomous key chosen. The phenotypic characteristics taken into account to set apart V. coralliilyticus and other species were not proven to be efficient. More information about the morphological diversity of colonies and enzymatic activities should be considered in the formulation of phenotypic keys for V. coralliilyticus and related species.

Pathogenic Vibrio species isolated from estuarine environments (Ceará, Brazil) - antimicrobial resistance and virulence potential profiles.

Detection of virulent strains associated with aquatic environment is a current concern for the management and control of human and animal health. Thus, Vibrio diversity was investigated in four estuaries from state of Ceará (Pacoti, Choró, Pirangi and Jaguaribe) followed by antimicrobial susceptibility to different antimicrobials used in aquaculture and detection of main virulence factors to human health. Isolation and identification were performed on TCBS agar (selective medium) and dichotomous key based on biochemical characteristics, respectively. Nineteen strains of genus Vibrio were catalogued. Vibrio parahaemolyticus (Choró River) and V. alginolyticus (Pacoti River) were the most abundant species in the four estuaries. All strains were submitted to disk diffusion technique (15 antimicrobials were tested). Resistance was found to: penicillin (82%), ampicillin (54%), cephalotin (7%), aztreonan (1%), gentamicin, cefotaxime and ceftriaxone (0.5%). Five pathogenic strains were chosen to verification of virulence factors. Four estuaries showed a high abundance of species. High number of tested positive strains for virulence is concerning, since some of those strains are associated to human diseases, while others are known pathogens of aquatic organisms.

Serotypes and Pulsotypes diversity of Listeria monocytogenes in a beef-processing environment.

Utensils and equipment from meat-processing facilities are considered relevant cross-contamination points of Listeria monocytogenes to foods, demanding tracking studies to identify their specific origins, and predict proper control. The present study aimed to detect L. monocytogenes in a beef-processing facility, investigating the diversity of serotypes and pulsotypes in order to identify the possible contamination routes. Surface samples from knives (n=26), tables (n=78), and employees hands (n=74) were collected before and during the procedures from a beef-processing facility, in addition to surface samples of end cuts: round (n=32), loin (n=30), and chuck (n=32). All samples were subjected to L. monocytogenes screening according ISO 11.290-1, and the obtained isolates were subjected to serotyping and pulsed-field gel electrophoresis. Listeria spp. were identified in all processing steps, in 61 samples, and L. monocytogenes was detected in 17 samples, not being found only in knives. Eighty-five isolates were identified as L. monocytogenes, from serotypes 1/2c (n=65), 4b (n=13), and 1/2b (n=7), being grouped in 19 pulsotypes. Considering these results, cross-contamination among hands, tables, and beef cuts could be identified. The obtained data indicated the relevance of cross-contamination in the beef-processing facility, and the occurrence of serotypes 1/2b and 4b in beef cuts distributed for retail sale is a public health concern.

Characterization of Yersinia enterocolitica biotype 1A strains isolated from swine slaughterhouses and markets.

Yersinia enterocolitica is an important foodborne pathogen that causes illness in humans and animals. Y. enterocolitica is also the most heterogeneous species of the genus and is divided into distinct serotypes and over six biotypes. Y. enterocolitica biotype 1A strains are classically considered as nonpathogenic; however, some biotype 1A isolates have been considered as causative of gastrointestinal disease, yielding symptoms indistinguishable from those produced by pathogenic biotypes. Even after decades of isolation of clinical strains, the pathogenic mechanisms of these isolates are still not fully understood. In the present study, 122 Yersinia enterocolitica biotype 1A strains isolated from swine slaughterhouses and meat markets in Sao Paulo, Brazil, were characterized according to the presence of the virulence genes ail, virF, and ystA. A total of 94 strains were positive to at least one virulence gene (77.05%), and 67 were positive to all of them (54.92%). Twenty-two strains were submitted to PFGE genotyping resulting in 22 distinct pulsotypes, varying from 50% to 84% of genetic similarity. Any clustering tendency among pulsotypes related to origin, isolation site, or even virulence profile was not observed. The present study reports an important contamination of the environment in swine slaughterhouses, meat markets, and pork, by potentially virulent Y. enterocolitica biotype 1A.

The aquatic environment as a reservoir of Vibrio cholerae O1 in hydrographic basins of the state of Pernambuco, Brazil.

After the worldwide cholera epidemic in 1993, permanent environmental monitoring of hydrographic basins was established in Pernambuco, Brazil, where cholera is endemic. After a quiescent period, 4 rfbN (serogroup O1) positive water samples that were culture negative were detected by multiplex single-tube nested PCR (MSTNPCR); 2 of these were also ctxA (cholera toxin) positive. From May to June 2012, 30 V. cholerae O1 isolates were obtained by culturing samples. These isolates were analyzed for the presence of virulence genes by PCR, intergenic spacer region 16S-23S PCR (ISR-PCR), and pulsed field gel electrophoresis (PFGE). The isolates were positive for the rfbN gene and negative for the assessed pathogenic genes and were classified into 2 groups by ISR and the same profile by PFGE. Close genetic similarity was observed between them (2012) and environmental strains from 2004 to 2005, indicating the permanence of endemic V. cholerae O1 in the region.

Heavy metal tolerance (Cr, Ag AND Hg) in bacteria isolated from sewage.

Samples of sewage from a university hospital and a chemistry technical school were analysed for the percentage of bacterial tolerance to chromium (Cr), silver (Ag) and mercury (Hg). Additionally, we investigated the effect of these metals on pigmentation and on some enzymatic activities of the metal tolerant strains isolated, as well as antimicrobial resistance in some metal tolerant Enterobacteriaceae strains. Tolerance to Cr was observed mainly in Gram positive bacteria while in the case of Ag and Hg the tolerant bacteria were predominately Gram negative. Hg was the metal for which the percentage of tolerance was significantly higher, especially in samples from the hospital sewage (4.1%). Mercury also had the most discernible effect on color of the colonies. Considering the effect of metals on the respiratory enzymes, one strain of Ag-tolerant Bacillus sp. and one of Hg-tolerant P. aeruginosa were unable to produce oxidase in the presence of Ag and Hg, respectively, while the expression of gelatinase was largely inhibited in various Gram negative strains (66% by Cr). Drug resistance in Hg-tolerant Enterobacteriaceae strains isolated from the university hospital sewage was greater than 80%, with prevalence of multiple resistance, while the Ag-tolerant strains from the same source showed about 34% of resistance, with the predominance of mono-resistance. Our results showed that, despite the ability of metal tolerant strains to survive and grow in the presence of these elements, the interactions with these metals may result in metabolic or phisiological changes in this group of bacteria.

Prevalence of R-type ACSSuT in strains of Salmonella serovar Typhimurium DT193 isolated from human infections in Brazil.

OBJECTIVE: To determine the prevalence of resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracyclines (ACSSuT) in Salmonella serovar Typhimurium definitive [phage] type (DT) 193 strains isolated from human sources over the last four decades. METHODS: From 2008 to 2010, 553 DT193 isolates out of 810 human-origin Salmonella ser. Typhimurium phage-typed strains isolated from the 1970s through 2008 were selected and tested for ACSSuT resistance: 91 strains isolated during the 1970s, 65 from the 1980s, 70 from the 1990s, and 327 from 2000-2008. Resistance profiles were determined using the disk diffusion method. RESULTS: An antimicrobial susceptibility assay indicated 20.9%, or 116, of all isolates tested were ACSSuT-resistant, 52.0% (287) were resistant to one or more drugs in the ACSSuT profile, and 27.1% (150) were nonresistant (susceptible to antimicrobials). Based on the assay, overall antimicrobial resistance was extremely high in the 1970s (affecting 99.0% of isolates from that period) and remained high during the 1980s, when 95.4% of isolates had some type of antimicrobial resistance and incidence of Salmonella ser. Typhimurium DT193 R-type ACSSuT increased to 73.8%. R-type ACSSuT dropped to 27.1% (19 isolates) during the 1990s, and to 5.2% (17) during 2000-2008, despite a substantial increase in the number of isolates tested (397 versus 204, 111, and 98, respectively, for the previous three decades). CONCLUSIONS: Although prevalence of Salmonella ser. Typhimurium DT193 R-type ACSSuT in Brazil has decreased since the 1970s, ACSSuT resistance markers continue to circulate. Therefore, continuous surveillance should be conducted to evaluate the occurrence of Salmonella ser. Typhimurium DT193 and its antimicrobial resistance.

Antibiotic resistance of Vibrio parahaemolyticus isolated from pond-reared Litopenaeus vannamei marketed in natal, brazil.

Ten out of fifty fresh and refrigerated samples of shrimp (Litopenaeus vannamei) collected from retailers in Natal (Rio Grande do Norte, Northeastern Brazil) tested positive for Vibrio parahaemolyticus. The Kanagawa test and multiplex PCR assays were used to detect TDH and TRH hemolysins and the tdh, trh and tlh genes, respectively. All strains were Kanagawa-negative and tlh-positive. Antibiotic susceptibility testing was done for seven antibiotics by the agar diffusion technique. Five strains (50%) presented multiple antibiotic resistance to ampicillin (90%) and amikacin (60%), while two strains (20%) displayed intermediate-level resistance to amikacin. All strains were sensitive to chloramphenicol. Intermediate-level susceptibility and/or resistance to other antibiotics ranged from 10 to 90%, with emphasis on the observed growing intermediate-level resistance to ciprofloxacin. Half our isolates yielded a multiple antibiotic resistance index above 0.2 (range: 0.14-0.29), indicating a considerable risk of propagation of antibiotic resistance throughout the food chain.

Prevalence of Listeria monocytogenes fecal carriers in HIV-infected and -uninfected pregnant women from Brazil.

OBJECTIVE: The aim of this study is to describe the prevalence of Listeria spp. in feces of HIV-infected and -uninfected pregnant women in Brazil. METHODS: Cross-sectional study. Women on their second or third trimester of pregnancy were submitted to a clinical questionnaire and feces collection. The feces were inoculated on selective media and identification by biochemical tests combined with PCR. RESULTS: A total of 213 pregnant women were enrolled: 73 (34%) HIV-infected and 140 (66%) -non-infected. The prevalence of Listeria spp. and L. monocytogenes in feces of HIV-infected women were 8.2% and 2.7%. In the HIV-uninfected were 8.6% and 2.9% (p-values = 0.98 and 0.66, respectively). CONCLUSION: The prevalence of fecal carriers of Listeria spp. and L. monocytogenes was not associated with HIV infection during pregnancy.

Seroprevalence of Listeria monocytogenes in HIV infected pregnant women from Brazil.

OBJECTIVE: To describe the prevalence and factors associated with serologic response to Listeria monocytogenes in HIV infected and uninfected pregnant women in Brazil. METHODS: Cross-sectional study, pregnant women after 14 weeks of gestational age were enrolled. Positive serologic test for L. monocytogenes was defined as titers >1:80 (agglutination test). Comparisons were performed using logistic regression. RESULTS: A total of 213 women were enrolled, 73 (34%) were HIV infected. 55 women were seroreactive for L. monocytogenes, 27 (37%) HIV-infected and 28 (20%) HIV-uninfected (p < 0.01). Considering the diet record, white cheese consumption was associated with seroreactivity (p < 0.01). In the group of pregnant women living with HIV, the variables associated with L. monocytogenes positive serology were: lower CD4+ cells count at study entry OR=4.8 (95%CI=1.1-19.8) and having neonates admitted to the intensive care unit OR=5.9 (95%CI=1.01-34.9). CONCLUSION: Positive serology for Listeria monocytogenes was associated with HIV infection. Brazilian women should avoid white cheese during pregnancy.

Evaluation of MALDI-TOF MS as a tool for detection of Listeria spp. directly from selective enrichment broth from food and stool samples.

We evaluated the detection of Listeria spp. using MALDI-TOF MS directly in enrichment broths, without isolated colonies, with naturally contaminated food and stool samples. The success rate was 77%. Considering the reduced time for diagnosis and the success rate, this is a promising screening tool, but more tests are needed to determine its viability.

Characterization of Vibrio cholerae isolated from the aquatic basins of the State of Pernambuco, Brazil.

Through a continuous bacteriological monitoring programme carried out by the Health Secretariat of the State of Pernambuco, Brazil, two isolates of Vibrio cholerae O1 El Tor Ogawa were discovered in an endemic area in 2001, during a cholera inactive period, along with six V. cholerae non-O1/non-O139 strains and two Aeromonas veronii biovar sobria strains showing an unusual characteristic of agglutination with O1 antiserum. Between that time and 2005, eight other O1 isolates were found. The virulence genes present in the V. cholerae differed among strains, with only three O1 strains harboring the ctxA gene. The O1 and some non-O1/non-O139 strains displayed identical patterns of amplification of the 16S-23S intergenic spacer region. RAPD of the 10 V. cholerae O1 strains, with the two primers used, revealed heterogeneity. The presence of V. cholerae carrying virulence genes in the aquatic basins examined confirms that they constitute a vibrio reservoir during a cholera inactive period, thus strengthening the argument for a continuous monitoring programme and preventative measures for cholera, mainly in the areas where the supply of drinking water is deficient.

Multiplex-PCR serotyping of Listeria monocytogenes isolated from human clinical specimens.

The genus Listeria is composed of six species of which Listeria monocytogenes is considered the single pathogenic species that causes listeriosis in humans. Of the 13 serovars of L. monocytogenes, 1/2a, 1/2b and 4b are responsible for the majority of clinical cases. The aim of this work was to detect L. monocytogenes in the cerebrospinal fluid sample of premature newborns and to characterize this sample using biotyping, serotyping and molecular typing. The results indicated the presence of L. monocytogenesin the clinical sample studied. Moreover, the isolate was identified as the 4b serovar that was characterized by the presence of a unique 691 bp band after analysis using the Multiplex-PCR technique. The results of repeated Multiplex-PCR and sequencing have indicated that the L. monocytogenes isolate was an atypical 4b serovar, which is the first time this finding has been reported.

Rapid detection of Yersinia enterocolitica serotype O:3 using a duplex PCR assay.

Yersinia enterocolitica, a member of the Enterobacteriaceae family, is a zoonotic agent that causes gastrointestinal diseases and some extraintestinal disorders in humans. Y. enterocolitica ssp. palearctica bioserotype 4/O:3 is the primary pathogenic bioserotype in Europe, where it has a high public health relevance. The isolation and identification of Y. enterocolitica from various sources on selective media have been seldom successful due to several reasons. In an attempt to overcome the problems associated with traditional culture-based methods, we developed a single duplex PCR assay for the detection of Y. enterocolitica ssp. palearctica bioserotype 4/O:3 using DNA extracted from a source. We combined the primer for tufA (elongation factor Tu) with the primer for rfbC (the biosynthesis of the O side chain) in one single reaction, which showed good results when we analyzed 88 Yersinia strains and when it was tested in the DNA from stool samples of two groups of pregnant women, one comprising HIV-positive women and the other comprising of HIV-negative women. Furthermore, the duplex PCR assay was found to be 16 times better in detecting Yersinia spp. in stool samples than the culture-based method. In addition, it was found to be a rapid screening method for the detection of Y. enterocolitica serotype O:3, and it could still detect other Y. enterocolitica serotypes and Yersinia species as well. We anticipate that the duplex PCR assay could be a useful tool for hospital and veterinary surveillance studies on Yersinia worldwide.

Listeria monocytogenes: Occurrence in beef and identification of the main contamination points in processing plants.

This study aimed to establish the occurrence of Listeria spp., especially L. monocytogenes and its main serotypes, in beef and processing plants. A total of 443 samples were obtained from equipment, installations and products from 11 meat processing establishments from Paraná state, Brazil. All samples were analyzed using USDA methodology for Listeria spp. detection, followed by species identification. The occurrence of Listeria spp. in the samples was 38.1% of which 51.4% were from equipment, 35.4% from installations and 30.2% from products. The identified species were: L. monocytogenes (12.6%), L. innocua (78.4%), L. seeligeri (1.2%), L. welshimeri (7.2%) and L. grayi (0.6%). The identified serotypes of L. monocytogenes were 1/2a and 4b. The results demonstrate the significance of equipment and installations as sources of contamination by Listeria spp. and L. monocytogenes in the processing of beef and meat products.

[The importance of circulating vibriocidal antibodies in a pos-epidemic region of diarrhea, São Bento do Una, State of Pernambuco]. / Significância de anticorpos vibriocidas circulantes em área pós-epidêmica de diarréia, São Bento do Una, Estado de Pernambuco.

The levels of vibriocidal antibodies were investigated among 41 adults without any past or present history of diarrhea due to Vibrio cholerae O1 who were living in the municipality of São Bento do Una, Pernambuco. A diarrhea outbreak occurred in this locality at the beginning of 2004, involving multiple bacterial agents, including Vibrio cholerae. The microtitration test was used to investigate the presence of anti-Ogawa and anti-Inaba vibriocidal serum antibodies. Vibriocidal titers e" 1:640 were considered indicative of infection by Vibrio cholerae O1. The frequency of the reagents was 36 (87.8%) for the Ogawa serovar, which showed that Vibrio cholerae O1 was possibly circulating during and/or after the diarrhea epidemic.

Next-generation sequencing virulome analysis of a Yersinia enterocolitica subsp. palearctica bioserotype 4/O:3 ST18 isolated from human blood in Brazil.

Yersinia enterocolitica is a widespread Gram-negative bacterium that causes gastrointestinal disease and other clinical manifestations in humans. Potentially pathogenic Y. enterocolitica has been isolated in Brazil, from human, environmental, food, and animal sources. Herein we report a genome sequence of Y. enterocolitica subsp. palearctica strain YE 19, serotype O:3, biotype 4, sequence type 18, with virulence determinants isolated from human blood in Rio de Janeiro in 2005. The results corroborate other findings that this strain harbors a set of virulence determinants that could play a role in host pathoadaptation and may also justify the successful dissemination of bioserotype 4/O:3 in Brazil. The presence of strains harboring all of these virulence genes in Brazil is a potential threat to young children and immunocompromised individuals, for whom yersiniosis are a significant source of morbidity and mortality. The results of a genomic data analysis will help understand the virulence of Brazilian strains and provide data for Y. enterocolitica studies worldwide.

Thiocarbamates from Moringa oleifera Seeds Bioactive against Virulent and Multidrug-Resistant Vibrio Species.

Prospect of antibacterial agents may provide an alternative therapy for diseases caused by multidrug-resistant bacteria. This study aimed to evaluate the in vitro bioactivity of Moringa oleifera seed extracts against 100 vibrios isolated from the marine shrimp Litopenaeus vannamei. Ethanol extracts at low (MOS-E) and hot (MOS-ES) temperature are shown to be bioactive against 92% and 90% of the strains, respectively. The most efficient Minimum Inhibitory Concentration (MIC) levels of MOS-E and MOS-ES against a high percentage of strains were 32 µg mL-1. Bioguided screening of bioactive compounds showed that the ethyl acetate fraction from both extracts was the only one that showed antibacterial activity. Vibriocidal substances, niazirine and niazimicine, were isolated from the aforementioned fraction through chromatographic fractionation.

Virulence genes and genetic relationship of L. monocytogenes isolated from human and food sources in Brazil.

The herein presented assay provided a bacteriological and molecular characterization of 100 samples of L. monocytogenes isolated from human (43) and food (57) sources, from several regions of Brazil, and collected between 1975 and 2013. Antigenic characterization defined 49% of serotype 4b samples, followed by 28% of serotype 1/2b, 14% of serotype 1/2c, 8% of serotype 1/2a, and 1% of serotype 3b. Both type of samples from human and food origin express the same serotype distribution. Multiplex PCR analysis showed 13 strains of type 4b with the amplification profile 4b-VI (Variant I). Virulence genes hly, inlA, inlB, inlC, inlJ, actA, plcA, and prfA were detected in all samples, highlighting a deletion of 105pb on the actA gene in 23% of serotype 4b samples. Macrorestriction profile with ApaI at PFGE showed 55 pulsotypes, with the occurrence of the same pulsotype in hospitalized patients in São Paulo in 1992 and 1997, and two other highly related pulsotypes in patients hospitalized in Rio de Janeiro in 2008. Recognized pulsotypes in listeriosis cases have also been detected in food. Thus, the prevalence of a serotype and the persistence of certain pulsotypes herald future problems.