Sílvia Dias de Oliveira

Detection and identification of salmonellas from poultry-related samples by PCR.

A polymerase chain reaction (PCR) assay was developed for the generic detection of Salmonella sp. and the identification of S. Enteritidis (SE), S. Gallinarum (SG), S. Pullorum (SP) and S. Typhimurium (ST) in material collected in the field from poultry. The specificity and sensitivity of the assay combined with Rappaport-Vassiliadis selective enrichment broth (PCR-RV) were determined, and field samples were analyzed to verify the validity of the method application. Specificity of the assay was tested using 29 SE, 11 SG, 10 ST and 10 SP strains, along with 75 strains of 28 other Salmonella serovars and 21 strains of other bacterial genera. The assay was 100% specific for Salmonella detection and ST identification. The primer pair for SE, SG and SP also detected S. Berta. PCR detection limits for Salmonella at the genus level were 2 ST, 8 SE, 1.1x10(3) SG and 1.8x10(5) SP cells. At the serovar level, detection limits were 7 ST, 1.2x10(3) SE, 4.4x10(7) SG and 1.8x10(6) SP cells. At the genus level, PCR-RV detected approximately 128% more positive field samples than the standard microbiological techniques and results were ready in 48h instead of 7 days. PCR-RV method is diagnostic of Salmonella at the genus level and ST at the serovar level, although other tests are needed to identify SE, SG and SP to serovar level.

Evaluation of selective and non-selective enrichment PCR procedures for Salmonella detection

Aims: To compare PCR combined with enrichment media with the standard microbiological techniques (SMT) and to determine the most sensitive method for the detection of Salmonella and the identification of Salm. typhimurium (ST), Salm. enteritidis (SE), Salm. gallinarum (SG) and Salm. pullorum (SP).

Phage types of Salmonella enteritidis isolated from clinical and food samples, and from broiler carcasses in Southern Brazil

272 isolates of Salmonella Enteritidis (111 isolated from frozen broiler chicken carcasses, 126 from human food and other biological materials involved in food poisoning outbreaks and 35 from different poultry materials) were selected for phage typing. From these, 111 were phage typed, 57.65% being classified as phage type 4, 32.43% as phage type 4a, 3.60% as phage type 6a and 0.90% as phage type 7, whereas 5.40% samples were not phage typeable. The predominance of phage type 4 is in agreement with the results published worldwide, and reinforces the need for studies related to the epidemiological meaning of these findings.

Detection of virulence genes in Salmonella Enteritidis isolated from different sources

The presence of three virulence genes, invA, spvR, and spvC, was determined in Salmonella Enteritidis isolated from poultry, pigs, humans and food. All isolates were positive for the invA gene, with 91.2% being positive for spvR and 90.2% for spvC. There was no significant difference in the prevalence of the virulence genes between isolates from different sources. The results indicate that there is a putative high virulence potential for the S. Enteritidis isolates characterized.

Polymerase chain reaction (PCR) for the detection of Salmonella in artificially inoculated chicken meat

The aim of this study was to develop a polymerase chain reaction (PCR) protocol for the detection of Salmonella in artificially contaminated chicken meat. Tests were performed with different dilutions of Salmonella Typhimurium or Salmonella Enteritidis cells (10(-7), 10(-8) or 10(-9) CFU/mL) inoculated in chicken meat samples, in order to establish the limits of detection, incubation times (0, 6, 8 and 24 hours of pre-enrichment in PBW 1%) and three DNA extraction protocols (phenol-chloroform, thermal treatment and thermal treatment and Sephaglass). The assay was able to detect until 10(-9) CFU/mL of initial dilution of Salmonella cells inoculated in chicken meat, which allows detection of Salmonella within 48 hours, including 24 hours of pre-enrichment and using the phenol-chloroform DNA extraction protocol. As the results are obtained in a shorter time period than that of microbiological culture, this procedure will be useful in the methodology for detection of Salmonella in chicken.

Phenotypic and genotypic characterization of Salmonella enteritidis isolates

In order to study the epidemiology of Salmonella Enteritidis outbreaks and determine the source of contamination so that a recurrence can be avoided, detailed characterization is necessary. Thus, the purpose of this study was to verify whether rep-PCR was able to discriminate among Salmonella Enteritidis isolates. Phage typing, detection of virulence genes and antimicrobial resistance testing were also associated to repPCR results. One hundred and two S. Enteritidis isolates from broiler carcasses, food, human, pigs, poultryrelated samples, and nine isolates from other countries were genotypically typed by REP-PCR, ERIC-PCR and BOX-PCR, collectively called rep-PCR. Phage typing, detection of virulence genes and antimicrobial resistance testing were also performed. Only three fingerprinting profiles were obtained with each rep-PCR method, with the majority of isolates belonging to the same profile. No relationship was observed between genotypic profile and year, place of isolation or source of infection. However, the less frequent rep-PCR profiles showed single antimicrobial resistance patterns. Although few strains isolated from swine were analyzed, different antimicrobial resistance patterns were observed. Furthermore, phage type 4 was not found in swine isolates. rep-PCR showed a lower discriminatory power as compared with antimicrobial resistance and phage typing, but the combination of genotypic and phenotypic methods was more discriminatory than any method alone, resulting in 48 different types.

Detecção de Ornithobacterium rhinotracheale (ORT) por meio da reação em cadeia da polimerase (PCR)

Ornithobacterium rhinotracheale (ORT) is a recently discovered Gram negative bacterium that has been associated with respiratory diseases in commercial poultry and wild birds from many countries. In Brazil, antibodies were detected in some broiler and breeder flocks from the States of Sao Paulo and Minas Gerais. Because the bacteria is difficult to grow, the Polymerase Chain Reaction (PCR) has been found to be suitable for identification and diagnostic purposes. The aim of the present work was to verify the occurrence of ORT in Rio Grande do Sul through the detection of the bacteria DNA. Tracheal swabs (84) were collected from 14 broiler flocks of distinct companies. DNA was purified and PCR performed with species specific primers from the ORT 16S ribosomal RNA gene. Amplification products with 784 base pairs were obtained from 10 out of the 84 samples. The positive samples were from four flocks of tree companies established in different regions of the state. The results indicate that this respiratory pathogen occurs in major broiler producing areas from the State of Rio Grande do Sul. Further studies are under way to determine the prevalence of this pathogen and to characterize the strains isolated.

Potentiation of the action of calcium hydroxide on Enterococcus faecalis by proton pump inhibitor omeprazole

Purpose: Calcium hydroxide is not fully effective against Enterococcus faecalis (E. faecalis), axa0facultative anaerobic bacterium proven to be resistant to most conventional disinfection processes.xa0The aim of this in vitro study was to evaluate the effect of calcium hydroxide, omeprazole and thexa0association of these substances against Enterococcus faecalis, as well as to evaluate if the acidcatalysationxa0of the omeprazole had any influence in the results. Methods: The Minimum Inhibitory Concentration (MIC) of these drugs against E. faecalis (ATCCxa029212) was determined using macrodilution test adapted from the CLSI (Clinical Laboratory andxa0Standards Institute). Solutions with different concentrations of calcium hydroxide, associated or notxa0to omeprazole, were tested. Data were statistically analyzed using ANOVA test with Tukey post-hoc,xa0with a level of significance of 5%. Results: The MIC to calcium hydroxide was 32 mg mL-1 and, when associated with omeprazole, thisxa0was reduced reduced to 16 mg mL-1. The omeprazole and acidified omeprazole had similar activity.xa0 Conclusions: Omeprazole potentiated the effect of calcium hydroxide, since the association ofxa0these drugs reduced the MIC for E. faecalis. The acidification of omeprazole, when associated withxa0calcium hydroxide in different concentrations, did not influence its effect.