Optimization of a propidium monoazide-qPCR method for Escherichia coli quantification in raw seafood.

The present study compared different concentrations of propidium monoazide (PMA), time of exposure to light and different light intensities to determine the optimal conditions for the quantification of viable Escherichia coli in cell suspension and in food matrix. The influence of cell density and the effectiveness of PMA in viable but non-culturable (VBNC) E. coli cells were evaluated and also applied in food matrix. For that purpose, different concentrations of PMA (20 µM, 40 µM, 50 µM, 60 µM and 80 µM) under different times of exposure (5 min, 10 min, 15 min, 20 min and 30 min) to lights of different intensities (500 W and 650 W) were evaluated. After determining the optimal conditions, the PMA-qPCR methods were applied to different compositions of live and heat-killed E. coli suspensions (v:v; 0:1; 1:0; 1:1) in concentrations ranging from 3 Log to 7 Log CFU/mL. The same dilutions were prepared with E. coli in VBNC state and applied in food matrix. The results obtained from qPCR, PMA-qPCR and plate counts were compared. The results suggested that a PMA treatment of 50 µM PMA for 15 min under 650 W light intensity was optimal under our conditions. For E. coli cell suspensions, the amplification of heat-killed cells was inhibited greatly by PMA when concentrations were ≤ 5 Log CFU/mL. For the samples of oyster inoculated with heat-killed cells, E. coli was not detected by PMA-qPCR in concentrations ≤4 Log CFU/g. Regarding the results with VBNC state, we considered the PMA-qPCR method to be applicable for enumerating E. coli VBNC cells in oyster samples. Based on our findings, we further recommend the use of PMA-qPCR with the aim of reducing the amplification of dead cells for improving its performance, since false-positives could still occur depending on the level of E. coli in the sample. The application of the PMA-qPCR for quantification of bacteria, compared to the use of culture-dependent methods, is quite promising. However, further studies are recommended, especially using different food matrices.

Evaluation of the interaction between microencapsulated Bifidobacterium BB-12 added in goat's milk Frozen Yogurt and Escherichia coli in the large intestine.

Goat milk and goat milk and inulin were used as encapsulating agents of Bifidobacterium BB-12 and applied in Frozen Yogurt (GF2 and GF3, respectively) in order to evaluate the antagonistic effect against Escherichia coli. GF1 is a control containing only Escherichia coli. Simulation of gastrointestinal digestion occurred sequentially. Quantification of Bifidobacterium BB-12 was performed using plate counting while E. coli count was compared with quantification by qPCR with viable and nonviable cell differentiation. The Bifidobacterium BB-12 count was <1.0, 9.23 and 9.11 log CFU g-1 for GF1, GF2 and GF3, respectively. In the ascending colon, all samples showed E. coli counts of approximately 5 log CFU g-1 by plate counting and by qPCR. Throughout the transverse (24 h) and descending colon (48 h) samples GF2 and GF3 showed decrease in E. coli number. GF3 showed higher decrease of E. coli in the descending colon because of inulin bifidogenic characteristic. The production of organic acids by bifidobacteria was directly related to the decrease in the E. coli count. In plate counts, E. coli was not detected for the GF3 sample in the descending colon. However, when quantified by qPCR the sample presented amplification that corresponded to 3 log CFU g-1. In this way, it was possible to observe the phenomenon of the viable but not-culturable cells of E. coli. Finally, it is recommended the microcapsule produced with goat milk and the inulin for application in goat milk products, due to the better antagonist effect against E. coli.

Development and application of a real-time polymerase chain reaction method for quantification of Escherichia coli in oysters (Crassostrea gigas).

Oysters are important mariculture species worldwide. Because of their filter-feeding behaviors, oysters can accumulate microorganisms, including pathogens, from surrounding water and concentrate bacteria in high numbers. Rapid and suitable methods for quantification of Escherichia coli in oysters are necessary considering that oysters are perishable foods often consumed raw and some countries use E. coli as the regulatory limit. The objective of this study was to develop a qPCR method for quantification of E. coli in oysters. Additionally, different methods were evaluated for DNA extraction from oyster samples and the more reliable method was chosen. Primers and probe were designed targeting uidA gene of E. coli and shown to specifically amplify DNA from E. coli. Standard curves with bacterial DNA extracted from oysters samples artificially inoculated with E. coli were conducted. A good correlation was noticed when the qPCR method was compared to a culture method in oyster samples. This is the first report of a method exclusively developed for direct quantification of E. coli in oyster, the method showed to be suitable for quantification of E. coli in oysters and could be useful in routine analyses, as it requires less time than the culture method.

Genotypic and phenotypic characterization of Escherichia coli isolated from mollusks in Brazil and the United States.

The aim of this study was to determine the serogroups, antimicrobial resistance and genetic diversity of Escherichia coli isolates from samples of bivalve mollusks collected along Santa Catarina coast, Brazil, and from the Chesapeake Bay, Maryland, USA. One hundred forty-one E. coli isolates were characterized for serogroups with 181 specific O antisera and antimicrobial susceptibility using the disk diffusion method. The genetic diversity was assessed using pulsed-field gel electrophoresis (PFGE). The results showed that among the isolates, 19.9% were classified as multi-drug resistant (MDR) and resistance was most frequently observed to cephalothin, nitrofurantoin, and ampicillin. The predominant serogroups were O6, O8, and O38. Some serogroups were recognized as pathogenic E. coli. PFGE dendrograms indicated extensive genetic diversity among the isolates. Although characteristics of the E. coli isolates were highly variable, it is important to note that E. coli belonging to pathogenic serogroups and MDR isolates are present in mollusks of both study areas. This is the first report on the phenotypic and genotypic characterization of E. coli from mollusks from Santa Catarina and the Chesapeake Bay that should encourage studies focusing on comparison of isolates across countries.

Prevalência de Cryptosporidium spp e Giardia sp em eqüinos estabulados no Jockey Club de Santa Maria - RS, Brasil / Prevalence of Cryptosporidium spp. and Giardia sp. infection in horses stabled in the Jockey Club of Santa Maria - RS, Brazil

O Cryptosporidium spp. e a Giardia sp. são atualmente reconhecidos como os principais patógenos entéricos com potencial zoonótico. O presente estudo visou estabelecer a prevalência desses protozoários em eqüinos hospedados no Jockey Club de Santa Maria, RS, Brasil, no período de 19 de maio a 30 de junho de 2007. Foram coletadas amostras de fezes, diretamente da ampola retal, de 64 animais. As amostras de fezes foram processadas por meio do método de centrifugação-flutuação de Faust modificado. Posteriormente essas amostras foram visualizadas ao microscópio óptico para a pesquisa de cistos e oocistos. Os resultados encontrados revelaram a presença de Cryptosporidium spp. em 75% (48/64) das amostras. Cistos de Giardia sp. não foram encontrados nas amostras de fezes analisadas. A freqüência de Cryptosporidium spp. nas diferentes faixas etárias foi de 83,3% (15/18) nos potros até dois anos de idade, 71% (22/31) nos jovens entre dois e cinco anos e 80% (12/15) nos adultos. Os resultados demonstram que o Cryptosporidium spp. está amplamente disseminado na população de eqüinos do Jockey Club de Santa Maria e pode representar uma fonte de infecção significativa para a população da região.

Cryptosporidium spp. and Giardia sp. are currently recognised as the main enteric pathogens with potential zoonotic transmission risk. The present study aimed to investigate the prevalence of these parasites in horses stabled in the Santa Maria Jockey Club between May 19 and June 30, 2007. Fecal samples from 64 horses were collected directly from the animals’ rectal ampoule. The 64 fecal samples were processed using modified Faust’s method through the centrifugation-floatation technique, and were then later visualized under optical microscope for detection of Cryptosporidium spp. oocysts and Giardia sp. cysts. The results showed the occurrence of Cryptosporidium spp. in 75% (48/64) of the samples. Giardia sp. cysts were not found in the fecal samples analysed. The prevalence of Cryptosporidium spp. in foals below two years of age was 83.3% (15/18); 71% (22/31) in young foals aged between three and five years of age, and 80 percent (12/15) in adult horses. These results show that Cryptosporidium spp. is widely disseminated in this population, and it can represent an important source of infection for the population in the region.

Phenotypical assays and partial sequencing of the hsp60 gene for identification of Streptococcus equi.

Strangles is an acute and contagious disease characterized by inflammation of the upper respiratory tract of horses. The etiological agent of strangles is the bacteria S. equi subsp. equi, which belongs to the Lancefield group C. Opportunistic agents from the same group are frequently isolated from horses with strangles and may induce mistaken diagnoses. Among the subspecies of S. equi, the phenotypic features are almost undistinguishable; however, the pathogenic potential is widely differentiated. The aim of this study was to characterize S. equi isolates obtained from clinical samples of strangles by phenotypic tests and to analyze the partial sequences obtained from fragments of the hsp60 gene. In this work, 26 strains of Streptococcus spp. isolated from horse clinical samples were analyzed. By phenotypical assays, 18 were characterized as S. equi subsp. equi, five as S. equi subsp. zooepidemicus, two as S. dysgalactiae subsp. equisimilis, and one as Streptococcus sp. However 21 isolates were identified as S. equi subsp. equi and five as S. equi subsp. zooepidemicus by DNA sequencing. The sequencing of the partial hsp60 gene was demonstrated to be an alternative method to analyze and differentiate strains of Streptococcus spp. In addition, this method can be useful as a discriminatory tool for characterization of atypical isolates.