Miguel Prieto

Bacterial microflora of wild brown trout (Salmo trutta), wild pike (Esox lucius), and aquacultured rainbow trout (Oncorhynchus mykiss)

Initial numbers of bacteria associated with wild (brown trout and pike) and cultured (rainbow trout) freshwater fish as well as with the water in which they were caught were determined. Subsequently, a total of 979 randomly selected isolates were characterized and identified to the genus level. For all counts performed (aerobes, psychrotrophs, anaerobes, Enterobacteriaceae, and enterococci), no significant differences were observed in water samples, the highest level corresponding to psychrotrophs in pike environments (4.23 X 10(3) CFU/ml). Overall, the skin and intestinal content of brown trout were the most contaminated, while rainbow trout specimens (gills and gut) yielded the lowest numbers. For all bacterial groups, pike gills had the highest numbers. Counts for all of the sampling sites compare well with findings in other temperate geographical environments. Biological characteristics (feeding and skin properties) and the use of antimicrobials in aquaculture might have influenced these results. Motile and nonmotile aerobic gram-negative bacteria together with Enterobacteriaceae accounted for 50 to 70% of the psychrotrophs isolated from water. Micrococcaceae, lactic acid bacteria, Bacillus, and coryneforms were also found. The groups represented in psychrotrophic isolates from the outer surfaces do not reflected those detected in water, so it was common that those organisms recovered in significant numbers from fish were not detected in surrounding habitat of the fish. Motile aeromonads and Carnobacterium were the dominant psychrotrophs in the guts of pike and brown trout, respectively. The intestinal content of reared fish gave a high incidence of Bacillus and coryneforms, while Enterobacteriaceae was absent. Again, rearing practices could have influenced this finding. Listeria monocytogenes was not detected in any of the examined samples. Two strains of Salmonella, which belonged to the same serovar and lysotype, were recovered from pond-water samples taken from one facility on different sampling days. From the gut of a pike specimen and from the pikes environment, two Plesiomonas shigelloides strains of different serovars were recovered. These latter four strains were resistant to a considerable number of antimicrobial compounds (multiple antibiotic resistance indices > 0.2).

Fourier transform infrared spectroscopy as a tool to characterize molecular composition and stress response in foodborne pathogenic bacteria.

Vibrational spectroscopy techniques have shown capacity to provide non-destructive, rapid, relevant information on microbial systematics, useful for classification and identification. Infrared spectroscopy enables the biochemical signatures from microbiological structures to be extracted and analyzed, in conjunction with advanced chemometrics. In addition, a number of recent studies have shown that Fourier Transform Infrared (FT-IR) spectroscopy can help understand the molecular basis of events such as the adaptive tolerance responses expressed by bacteria when exposed to stress conditions in the environment (e.g. those that cells confront in food and during food processing). The current review gives an overview of the published experimental techniques, data-processing algorithms and approaches used in FT-IR spectroscopy to assess the mechanisms of bacterial inactivation by food processing technologies and antimicrobial compounds, to monitor the spore and membrane properties of foodborne pathogens in changing environments, to detect stress-injured microorganisms in food-related environments, to assess dynamic changes in bacterial populations, and to study bacterial tolerance responses.

Sampling Methods for Microbiological Analysis of Red Meat and Poultry Carcasses

Microbiological analysis of carcasses at slaughterhouses is required in the European Union for evaluating the hygienic performance of carcass production processes as required for effective hazard analysis critical control point implementation. The European Union microbial performance standards refer exclusively to the excision method, even though swabbing using the wet/dry technique is also permitted when correlation between both destructive and nondestructive methods can be established. For practical and economic reasons, the swab technique is the most extensively used carcass surface-sampling method. The main characteristics, advantages, and limitations of the common excision and swabbing methods are described here.

Discrimination of Enterobacterial Repetitive Intergenic Consensus PCR Types of Campylobacter coli and Campylobacter jejuni by Fourier Transform Infrared Spectroscopy

ABSTRACT Fourier transform infrared spectroscopy (FT-IR) has been used together with pattern recognition methodology to study isolates belonging to the species Campylobacter coli and Campylobacter jejuni and to compare FT-IR typing schemes with established genomic profiles based on enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). Seventeen isolates were cultivated under standardized conditions for 2, 3, and 4 days to study variability and improve reproducibility. ERIC-PCR profiles and FT-IR spectra were obtained from strains belonging to the species Campylobacter coli and C. jejuni, normalized, and explored by hierarchical clustering and stepwise discriminant analysis. Strains could be differentiated by using mainly the first-derivative FT-IR spectral range, 1,200 to 900 cm−1 (described as the carbohydrate region). The reproducibility index varied depending on the ages of the cultures and on the spectral ranges investigated. Classification obtained by FT-IR spectroscopy provided valuable taxonomic information and was mostly in agreement with data from the genotypic method, ERIC-PCR. The classification functions obtained from the discriminant analysis allowed the identification of 98.72% of isolates from the validation set. FT-IR can serve as a valuable tool in the classification, identification, and typing of thermophilic Campylobacter isolates, and a number of types can be differentiated by means of FT-IR spectroscopy.

Effectiveness of trisodium phosphate, acidified sodium chlorite, citric acid, and peroxyacids against pathogenic bacteria on poultry during refrigerated storage.

The effects of dipping treatments (15 min) in potable water or in solutions (wt/vol) of 12% trisodium phosphate (TSP), 1,200 ppm acidified sodium chlorite (ASC), 2% citric acid (CA), and 220 ppm peroxyacids (PA) on inoculated pathogenic bacteria (Listeria monocytogenes, Staphylococcus aureus, Bacillus cereus, Salmonella Enteritidis, Escherichia coli, and Yersinia enterocolitica) and skin pH were investigated throughout storage of chicken legs (days 0, 1, 3, and 5) at 3 +/- 1 degrees C. All chemical solutions reduced microbial populations (P < 0.001) as compared with the control (untreated) samples. Similar bacterial loads (P > 0.05) were observed on water-dipped and control legs. Type of treatment, microbial group, and sampling day influenced microbial counts (P < 0.001). Average reductions with regard to control samples were 0.28 to 2.41 log CFU/g with TSP, 0.33 to 3.15 log CFU/g with ASC, 0.82 to 1.97 log CFU/g with CA, and 0.07 to 0.96 log CFU/g with PA. Average reductions were lower (P < 0.001) for gram-positive (0.96 log CFU/g) than for gram-negative (1.33 log CFU/g) bacteria. CA and ASC were the most effective antimicrobial compounds against gram-positive and gram-negative bacteria, respectively. TSP was the second most effective compound for both bacterial groups. Average microbial reductions per gram of skin were 0.87 log CFU/g with TSP, 0.86 log CFU/g with ASC, 1.39 log CFU/g with CA, and 0.74 log CFU/g with PA for gram-positive bacteria, and 1.28 log CFU/g with TSP, 2.03 log CFU/g with ASC, 1.23 log CFU/g with CA, and 0.78 log CFU/g with PA for gram-negative bacteria. With only a few exceptions, microbial reductions in TSP- and ASC-treated samples decreased and those in samples treated with CA increased throughout storage. Samples treated with TSP and samples dipped in CA and ASC had the highest and lowest pH values, respectively, after treatment. The pH of the treated legs tended to return to normal (6.3 to 6.6) during storage. However, at the end of storage, the pH of legs treated with TSP remained higher and that of legs treated with CA remained lower than normal.

Salmonella spp. survival strategies within the host gastrointestinal tract

Human salmonellosis infections are usually acquired via the food chain as a result of the ability of Salmonella serovars to colonize and persist within the gastrointestinal tract of their hosts. In addition, after food ingestion and in order to cause foodborne disease in humans, Salmonella must be able to resist several deleterious stress conditions which are part of the host defence against infections. This review gives an overview of the main defensive mechanisms involved in the Salmonella response to the extreme acid conditions of the stomach, and the elevated concentrations of bile salts, osmolytes and commensal bacterial metabolites, and the low oxygen tension conditions of the mammalian and avian gastrointestinal tracts.

Prevalence of Salmonella enterica serovars and genovars from chicken carcasses in slaughterhouses in Spain

Aims: To determine the prevalence of Salmonella enterica serovars in chicken carcasses in slaughterhouses in Spain and to examine genotypic relations among these serovars.

Changes in Fourier transform infrared spectra of Salmonella enterica serovars Typhimurium and Enteritidis after adaptation to stressful growth conditions.

The effects of growth conditions (temperature in the range 10-45 degrees C, sodium chloride concentration in the range 0-4%, aerobic vs. anaerobic growth and acidification of the growth medium, up to pH 4.5) on the Fourier transform infrared (FT-IR) spectra of Salmonella enterica serovars Typhimurium and Enteritidis were studied using multivariate statistical methods (Hierarchical Cluster Analysis and Factor Analysis). Although all environmental factors tested affected S. Typhimurium and S. Enteritidis FT-IR spectra to some extent, growth temperature was the most influential factor within the five spectral regions. The w(4) spectral region (1200 to 900 cm(-1)) was the most variable region, suggesting that S. Typhimurium and S. Enteritidis modulate their cell wall and cell membrane composition in response to shifts in growth temperature. Changes in membrane fluidity were determined by monitoring the vibrational modes of the acyl chain v(s)CH(2) symmetric stretching band by FT-IR spectroscopy. For cells grown in unsupplemented media an increase in growth temperature was linked to a decrease in membrane fluidity. Even though the effect of NaCl concentration, pH and atmosphere was considered of less importance, cells grown in acidified media also showed a reduction in their membrane fluidity, and the addition of sodium chloride to the culture medium was associated with an increase in the bacterial membrane fluidity. These findings can help interpret how important adaptive mechanisms for the survival of pathogenic bacteria in foods are, and show that FT-IR spectroscopy is a useful tool to understand how environmental conditions mimicking those in certain food products affect the cell. Also, FT-IR can be used to perform a rapid discrimination between bacterial phenotypes with different adaptive tolerance responses to environmental stress.

Role of slaughtering in Salmonella spreading and control in pork production.

Salmonella is one of the major foodborne pathogens worldwide. Pork products are among the main sources of Salmonella infection in humans, and several countries have established Salmonella surveillance and control programs. The role of slaughtering in carcass contamination has been indicated by studies focused on the slaughterhouse environment. In this review, we examine and discuss the information available regarding the influence that farm status, pig transport, and lairage have on the carriage of Salmonella by pigs entering the slaughter line. The evolution of carcass contamination throughout the slaughtering process, the main sources of contamination in the dirty and clean zones of the slaughter line, and previously reported prevalence of Salmonella on carcasses and factors affecting this prevalence also are discussed. The importance of implementing interventions at the slaughter level is discussed briefly. Consistent with the information available, pigs from infected farms and newly acquired or recrudescent infections in pigs at the subsequent stages of transport and lairage are important sources of Salmonella at the slaughtering plant. The continuous introduction of Salmonella into the slaughterhouse and the potential for resident flora constitute a risk for carcass contamination. At the slaughterhouse, some dressing activities can reduce carcass contamination, but others are critical control points that jeopardize carcass hygiene. This information indicates the importance of considering slaughter and previous stages in the pork production chain for controlling Salmonella in swine production.

Changes in Ultrastructure and Fourier Transform Infrared Spectrum of Salmonella enterica Serovar Typhimurium Cells after Exposure to Stress Conditions

ABSTRACT The effect of exposure to acid (pH 2.5), alkaline (pH 11.0), heat (55°C), and oxidative (40 mM H2O2) lethal conditions on the ultrastructure and global chemical composition of Salmonella enterica serovar Typhimurium CECT 443 cells was studied using transmission electron microscopy and Fourier transform infrared spectroscopy (FT-IR) combined with multivariate statistical methods (hierarchical cluster analysis and factor analysis). Infrared spectra exhibited marked differences in the five spectral regions for all conditions tested compared to those of nontreated control cells, which suggests the existence of a complex bacterial stress response in which modifications in a wide variety of cellular compounds are involved. The visible spectral changes observed in all of the spectral regions, together with ultrastructural changes observed by transmission electron microscopy and data obtained from membrane integrity tests, indicate the existence of membrane damage or alterations in membrane composition after heat, acid, alkaline, and oxidative treatments. Results obtained in this study indicate the potential of FT-IR spectroscopy to discriminate between intact and injured bacterial cells and between treatment technologies, and they show the adequacy of this technique to study the molecular aspects of bacterial stress response.