Bianca Mendes Maciel

High prevalence of Salmonella in tegu lizards (Tupinambis merianae), and susceptibility of the serotypes to antibiotics.

Species of tegu (Tupinambis) are the largest lizards in South America. Large numbers of these lizards are hunted; there is a vigorous trade in their skins and the meat is consumed by rural and native peoples. The animals are also bred in captivity, an economic activity for rural populations which can help in the animals’ conservation. Faecal samples from 30 captive‐born tegus were analysed for the presence of Salmonella in two separate samplings. In the first analysis, samples from 26 animals (87%) yielded Salmonella enterica of which 23% were of Rubislaw serotype; 20% Carrau and Agona serotypes; 7% Infantis and Saint‐Paul serotypes; 3% Panama and Brandenburg serotypes; 10% were S. enterica subsp. enterica and 7% were rough form. In the second analysis, four tegus (13%) which had been negative in the first sampling were positive, thus, 100% of the animals studied carried the bacterium. Antibiotic susceptibility showed resistance to sulfonamide in 82% of the isolates, streptomycin in 64%, tetracycline in 6% and Chloramphenicol in 20%. Two animals carried strains of the same serotype with different patterns of antibiotic susceptibility. Although it is well known that reptiles are a significant source of Salmonella, to our knowledge, its prevalence in tegu has not been studied previously.

Microbial surfactant activities from a petrochemical landfarm in a humid tropical region of Brazil

The goal of this study was to assess the presence and surfactant potential of naturally occurring microbes from a tropical soil with petrochemical contamination. Microorganisms in a soil sample from a Brazilian landfarm were isolated and grown on petroleum as the sole carbon source. Of 60 isolates screened for petroleum-based growth, 7 demonstrated surfactant activities by the drop-collapse methodology over various types of oils. From their growth profiles in liquid culture during 132 h, all had their first detection of surfactant activity after 96 h. Little is currently known about biosurfactant-producing microorganisms in tropical environments contaminated by hydrophobic compounds, and the search for them is essential for bioremediation and for oil recovery enhanced by microbes. Our results indicate that different petroleum-grown microorganisms showing surfactant activity can be recovered from landfarm soil in a tropical environment.

Canine parvovirus infection in puppies with gastroenteritis in Niterói, Rio de Janeiro, Brazil from 1995 to 1997.

Amostras fecais de caes com gastrenterite, ate 6 meses de idade, foram testadas para a presenca do parvovirus canino (CPV-2) pela reacao de hemaglutinacao (HA) e confirmadas pela reacao de inibicao da hemaglutinacao (HI). Quarenta das 79 amostras, recebidas no periodo de abril de 1995 a junho de 1997, foram consideradas positivas. Aproximadamente 70% destas amostras foram obtidas de animais entre 2 e 4 meses de idade, epoca em que o risco de contrairem a infeccao pelo CPV-2 e alto apesar da vacinacao. Nenhuma variacao sazonal da infeccao pelo parvovirus canino pode ser observada, e um estudo retrospectivo realizado na PolVet - UFF mostrou que em um periodo de 6 anos (1991-97), casos de gastrenterite ocorreram durante todos os anos em Niteroi, sem uma sazonalidade definida.

Identification and discrimination of Toxoplasma gondii, Sarcocystis spp., Neospora spp., and Cryptosporidium spp. by righ-resolution melting analysis

The objective of this study was to standardize the high-resolution melting method for identification and discrimination of Toxoplasma gondii, Sarcocystis spp., Neospora spp., and Cryptosporidium spp. by amplification of 18S ribosomal DNA (rDNA) using a single primer pair. The analyses were performed on individual reactions (containing DNA from a single species of a protozoan), on duplex reactions (containing DNA from two species of protozoa in each reaction), and on a multiplex reaction (containing DNA of four parasites in a single reaction). The proposed method allowed us to identify and discriminate the four species by analyzing the derivative, normalized, and difference melting curves, with high reproducibility among and within the experiments, as demonstrated by low coefficients of variation (less than 2.2% and 2.0%, respectively). This is the first study where this method is used for discrimination of these four species of protozoa in a single reaction.

Application of multiplex real-time polymerase chain reaction assay for simultaneous quantification of Escherichia coli virulence genes in oysters

Abstract Strains of diarrheagenic Escherichia coli (DEC) are involved in foodborne disease outbreaks worldwide, especially the enterohemorrhagic E. coli O157:H7. This study describes two multiplex quantitative real time PCR (qPCR) assays for simultaneous identification and quantification of genes related to virulence of DEC; a triplex reaction for detection and quantification of stxA1, stxA2, and eaeA genes, and a duplex reaction for detection and quantification of eaeA and virA genes. The technique was applied in raw oyster samples for direct quantification of DEC, thereby evaluating the applicability of this methodology for microbiological quality assessment of food. Using custom designed primers and specific MGB probes, a triplex qPCR assay was performed to quantify stxA1, stxA2, and eaeA, and a duplex reaction was performed to quantify virA and eaeA genes. The assays showed high sensitivity, with the detection limit varying between 5 and 17 copies of the genes. The coefficient of determination (R2) of the standard curves was 0.99. The coefficient of variation was < 1% indicated high intra- and inter-assay reproducibilities. The application of this methodology in oyster samples from tropical environment provided direct quantitative data that determined the presence of the genes stxA1 (32.1%), eaeA (28.6%), stxA2 (3.6%), and virA (3.6%). This would prove critical for immediate intervention of control strategies, particularly in oysters that are often ingested as raw food.

Isolation and Genotyping of Toxoplasma gondii in Brazilian Dogs

Strains of Toxoplasma gondii in Brazil are highly genetically diverse compared to strains from North America and Europe. Dogs are epidemiologically important because they act as sentinels for T. gondii infections in humans and are good indicators of environmental contamination. The aim of this study was to isolate and genetically characterize T. gondii strains from tissues of naturally infected Brazilian dogs. For this study, 21 blood samples were collected from dogs at the Zoonosis Control Centers of Ilhéus and Itabuna cities, Bahia, Brazil. The sera were examined for T. gondii antibodies using the indirect hemagglutination test. Brains and hearts of seropositive dogs were bioassayed in mice to isolate and characterize T. gondii parasites by PCR-RFLP using 10 genetic markers (SAG1, newSAG2, SAG3, BTUB, c22-8, c29-2, GRA6, PK1, APICO, and L358). However, T. gondii was isolated from only 4 (57.1%) dogs, designated TgDgBr6, 13, 17, and 21. All strains were virulent, causing clinical changes (rough hair coat, lethargy, and abdominal distention) and the death of all mice within 8–20 days after inoculation. Genetic analysis of these 4 T. gondii isolates revealed 4 distinct genotypes with different clonal lineage combinations (types I, II, and III) and 2 atypical alleles. Using PCR-RFLP with several markers, this study contributes to evaluations of the genetic diversity of strains circulating in Brazil.

Microbiological Quality and Prevalence of β-Lactam Antibiotic Resistance Genes in Oysters (Crassostrea rhizophorae)

The microbiological quality of oysters reflects the microbiological quality of their habitats because they are filter feeders. The objective of this study was to assess the bacterial composition of the edible oyster Crassostrea rhizophorae in urban and preserved estuaries. Particularly, we assessed the presence of pathogenic bacteria, investigated antibiotic susceptibility in bacterial isolates, and quantified β-lactam antibiotic resistance genes (blaTEM, blaSHV, and blaKPC) via quantitative PCR of oyster DNA. Our results detected total coliforms, Escherichia coli , and enterobacteria in the oysters from urban estuaries, which is indicative of poor water quality. In addition, our detection of the eaeA and stxA2 virulence genes in 16.7% of E. coli isolates from oysters from this region suggests the presence of multiantibiotic-resistant enteropathogenic and enterohemorrhagic E. coli strains. During periods of low precipitation, increased contamination by E. coli (in winter) and Vibrio parahaemolyticus (in autumn) was observed. In contrast, cultivated oysters inhabiting monitored farms in preserved areas had low levels of bacterial contamination, emphasizing that oyster culture monitoring enhances food quality and makes oysters fit for human consumption. Distinct antibiotic resistance profiles were observed in bacteria isolated from oysters collected from different areas, including resistance to β-lactam antibiotics. The presence of the blaTEM gene in 91.3% of oyster samples indicated that microorganisms in estuarine water conferred the capability to produce β-lactamase. To our knowledge, this is the first study to directly quantify and detect β-lactam antibiotic resistance genes in oysters. We believe our study provides baseline data for bacterial dynamics in estuarine oysters; such knowledge contributes to developing risk assessments to determine the associated hazards and consequences of consuming oysters from aquatic environments containing pathogenic bacteria that may possess antibiotic resistance genes.

Genetic Diversity of Toxoplasma gondii Isolates From Free-Range Chickens In Bahia, Brazil

Abstract The genotyping of 25 isolates of Toxoplasma gondii from free-range chickens in the state of Bahia, Brazil, was performed by PCR-restriction fragment length polymorphism using 11 genetic markers: SAG1, 5′+3′SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, Apico, and CS3. The analysis revealed 8 genotypes, 3 of which had not been previously reported. Four genotypes were represented by single isolates, whereas the other genotypes were represented by 2 or more isolates. Five isolates showed mixed infections, and 2 of them were identical. None of the clonal types I, II, or III were found, but 2 isolates corresponded to the Brazilian clonal lineage BrIII. There was a single allele for the c22-8 marker. The CS3 marker demonstrated efficiency in the evaluation of virulence in mice. This study reaffirms the diverse genetic variability of T. gondii in Brazil.

Multiplex Real-Time Polymerase Chain Reaction for Simultaneous Quantification of Salmonella spp., Escherichia coli, and Staphylococcus aureus in Different Food Matrices: Advantages and Disadvantages

Quantitative real-time polymerase chain reactions (qPCRs) of the most prevalent bacteria causing foodborne diseases worldwide, such as Salmonella spp., Escherichia coli, and Staphylococcus aureus, can be an important tool for quantitative microbial risk assessment, which requires numerical data to determine the level of contamination at a specific stage of food production. However, most of qPCR assays described in the literature for these pathogens are qualitative; their objective is pathogen detection and not pathogen quantification. Thus, the aim of our work was to develop a qPCR for the simultaneous quantification of Salmonella spp., E. coli, and S. aureus and to propose its use in the analysis of foods, as a tool for microbiological quality monitoring. For this, a multiplex qPCR was standardized for the simultaneous quantification of specific fragments of target genes (ssf, phoA, and nuc) corresponding to each one of the mentioned bacteria. The limit of detection of the technique was 13, 10, and 12 gene copies for ssf, phoA, and nuc, respectively; standard curves showed R2 > 0.99, with efficiencies ranging from 99 to 110%, and inter- and intraexperiment reproducibility presented a low coefficient of variation in all trials. This methodology was applied in different food matrices (milk, ground beef, and oyster meat), and the results were compared with official microbiological culture methodology and with ready-to-use test. Advantages and disadvantages of each methodology used in this study are pointed out. We suggest that this multiplex qPCR can be used as a rapid screening technique for the analysis of food microbiological quality.

Salmonella enterica: Latency

Infection caused by more than 1500 serotypes of Salmonella enterica subsp. enterica is one of the most common food-borne diseases, prevalent worldwide. Concerning public health, Salmonella latent carrier animals represent an important source of transmission of the disease. They are responsible for silent introduction of the bacteria into the food chain and the environment. Most pathogenesis studies of salmonellosis are focused on events that lead to clinical disease. Researchers have been unable to clearly discern the interaction between intracellular microorganisms and their resistant hosts in latency. However, understanding this interaction is essential for the proper employment of the control and eradication strategies. Thus, the objective of this article is to present an overview of some important events that occur during the infection cycle of S. enterica in latent carriers.