Jose M. Rodríguez-Calleja

Occurrence of motile Aeromonas in municipal drinking water and distribution of genes encoding virulence factors.

Aeromonas-associated cases of gastroenteritis are generally considered waterborne. The purpose of this study was to evaluate the potential microbiological risk associated with the presence of these bacteria in public drinking water. Over a period of one year, 132 drinking-water samples were monitored in León (NW of Spain, 137,000 inhabitants) for mandatory drinking-water standards and the occurrence of Aeromonas spp. Samples were taken at the municipal water treatment plant, one storage facility, and two public artesian drinking-water fountains. Because of low numbers of coliforms or Clostridium perfringens, the non-compliance rate with microbial standards was 3.8% whereas the percentage of positive samples for motile mesophilic Aeromonas was 26.5%. For all but two samples, Aeromonas was recovered between October and early March when the temperature was below 14 degrees C and the residual chlorine ranged from 0.21 to 0.72 mg/l. An apparent relationship was observed between rainfall and the incidence of Aeromonas. The 35 selected Aeromonas isolates were identified as A. caviae and A. media. The alt and laf genes were present in all isolates, the aerA gene was present in six isolates, and the four remaining genes investigated (hlyA, ast, stx1 and stx2) were absent. The combinations of putative virulence genes were: aerA(-)/hlyA(-)/alt(+)/ast(-)/laf(+)/stx1(-)/stx2(-) (82.9%) and aerA(+)/hlyA(-)/alt(+)/ast(-)/laf(+)/stx1(-)/stx2(-) (17.1%). None of the isolates bore plasmids. As Aeromonas strains harbouring two or more virulence-associated genes have the potential to cause disease by direct transmission via drinking water or by water use in food preparation, it would be advisable to control excessive numbers of these bacteria in drinking-water supplies.

Development of the aerobic spoilage flora of chilled rabbit meat

Even though worldwide production of rabbit meat is over 1,000,000ton, little information is available on rabbit meat microbiology. This paper reports on the microflora developing on chill-stored rabbit carcasses. Four different lots of 24h post-mortem rabbit carcasses dressed and kept at 0°C in a medium-size abattoir were collected and evaluated for sensory, physicochemical and microbiological changes during aerobic storage at 3±1°C. Mean initial pH value (pH(24)), extract-release volume (ERV) and lactate content of Biceps femoris muscle, were 6.26±0.20, 13.50±3.50ml and 0.70±0.07%, respectively. As with other muscle foods kept chilled in air, pH increased and ERV and lactate decreased as storage progressed. Initial levels (logcfu/g) of aerobes (APC), psychrotrophic flora, Pseudomonas spp., Brochothrix thermosphacta, lactic acid bacteria, Enterobacteriaceae and yeasts were 4.76±0.31, 4.81±0.81, 3.39±1.12, 2.01±0.92, 2.76±0.51, 0.49±0.45 and 3.46±0.32, respectively. Pseudomonads, most of them fluorescent, and to a lesser extent B. thermosphacta and yeasts grew faster than the remaining microorganisms and became predominant at the end of the shelf life. Carcasses spoiled when mean APC, psychrotrophic and pseudomonads numbers were ca. 8logcfu/g, their mean shelf life being estimated at 6.8 days. A lot of DFD-like rabbit carcasses, with higher pH and lower ERV values but similar microbial loads to normal meat, developed a strong putrid odour after 4 days.

Microbiological Quality of Rabbit Meat

World rabbit meat production is estimated to be over 1 million tons, and Spain is the third largest producer. Although rabbit meat is marketed and consumed worldwide, information on microbiological quality is very scarce. Here, we report indicator organisms, spoilage flora, sensory quality, and some physicochemical traits of 24 h postmortem chilled rabbit carcasses and prepackaged rabbit meat stored chilled in air for 0 to 3 days at the retail level. The mean total bacterial count (4.01 +/- 0.48 log CFU/g) for carcasses dressed at a small abattoir by a manual process was significantly lower (P < 0.05) than that (4.96 +/- 0.90 log CFU/g) for carcasses dressed at a large abattoir in automated slaughter lines. Both groups of carcasses had mean pH values of 5.98. The dominant contaminants on carcasses from the small abattoir were Pseudomonas, lactic acid bacteria, and yeasts. These microorganisms and Brochothrix thermosphacta were dominant on carcasses from the large abattoir. On prepacked hind legs (pH 6.26 +/- 0.18) stored at -1 to +1 degree C (supermarket 1), mean aerobic mesophilic count was 5.87 +/- 1.03 log CFU/g, and the major microbial groups were Pseudomonas, yeasts, lactic acid bacteria, and B. thermosphacta. On prepacked whole carcasses (pH 6.37 +/- 0.18) displayed at -1 to +5 degrees C (supermarket 2), mean aerobic mesophilic count was 6.60 +/- 1.18 and the same microbial groups were dominant. Relative Escherichia coli incidence was supermarket 2 > large abattoir > supermarket 1 > small abattoir. Overall, low numbers of coliforms, Enterobacteriaceae, psychrotrophic clostridia, coagulase-positive staphylococci, and molds were found. Sensory scores, pH values, and L-lactic acid content differentiated fresh carcasses from retail samples. Data obtained suggest that the microflora of chilled rabbit meat are different from those found on the meat of other animals.

Carnosic acid dietary supplementation at 0.12% rates slows down meat discoloration in gluteus medius of fattening lambs

Thirty-two Merino lambs fed barley straw and a concentrate alone (CONTROL) or enriched with vitamin E (VITE006) or carnosic acid (CARN006; CARN012) were used to assess the effect of these antioxidant compounds on meat quality attributes. The animals were slaughtered after being fed for at least 5weeks with the experimental diets. The longissimus lumborum samples of VITE006, CARN006 and CARN012 groups showed higher values (P<0.001) of L* (lightness) through the complete storage period under modified atmosphere when compared to the CONTROL group. Moreover, the VITE006 and CARN012 samples revealed lower discoloration when compared to the CONTROL group, these differences being more apparent in a less color stable muscle such as gluteus medius (P<0.05 for hue after 14days of refrigerated storage). Meat sensory traits were not significantly affected by carnosic acid and microbiological analyses were not conclusive at the doses administered.

Rabbit meat as a source of bacterial foodborne pathogens.

Even though worldwide production of rabbit meat is >1,000,000 tons, little information is available for rabbit meat microbiology. This study provides data on the prevalence of Salmonella, Escherichia coli O157:H7, Yersinia enterocolitica, Listeria spp., motile Aeromonas spp., and Staphylococcus aureus on rabbit meat. A total of 24 rabbit carcasses from two abattoirs and 27 rabbit meat packages from supermarket displays were examined. In addition to culturing methods, associated virulence genes were investigated by PCR in suspect isolates and samples. Neither Salmonella nor E. coli O157:H7 was detected. All samples were negative for virulence-associated invA, stx1, and stx2 genes. At one abattoir, two carcasses (3.9%) carried Y. enterocolitica yst-, and two were positive for the yst gene, although viable Y. enterocolitica cells were not recovered from these samples. Seven samples (13.7%) were contaminated with Listeria. Of them, three were positive for hly and iap genes (Listeria monocytogenes hly+ / iap+), two carried Listeria seeligeri, one carried Listeria ivanovii, and one carried Listeria innocua. For detectable motile Aeromonas spp. (average count, 1.77 +/- 0.62 log CFU/g), the contamination rate was 35.3%, although ca. 90% of the samples were positive for the aerA and/or hlyA genes. The majority of aeromonad isolates were Aeromonas hydrophila aerA+ / hlyA+. Aeromonas caviae, Aeromonas popoffii, Aeromonas schubertii, and the two biovars of Aeromonas veronii were also isolated. The prevalence of S. aureus contamination (average count, 1.37 +/- 0.79 log CFU/g) was 52.9%. Among 27 S. aureus isolates, two harbored genes for staphylococcal enterotoxin B (seb), and two harbored genes for staphylococcal enterotoxin C (sec). The remaining isolates were negative for sea, seb, sec, sed, and see.

Foodborne and indicator bacteria in farmed molluscan shellfish before and after depuration.

Galicias coast (northwestern Spain) is a major producer of bivalve molluscs. Over an 18-month period, the presence of Salmonella, Aeromonas, Plesiomonas shigelloides, Vibrio parahaemolyticus, and Clostridium botulinum was determined by PCR methods in mussels (22 batches) and infaunal bivalves (31 batches of clams and cockles) before and after depuration. All batches were harvested from Galician class B harvesting areas where bivalve molluscs must not exceed 4,600 Escherichia coli per 100 g of flesh and liquor in 90% of the samples. Virulence-associated genes of Salmonella (invA), Aeromonas (aerA, hlyA, alt, ast, and laf), P. shigelloides (hugA), V. parahaemolyticus (tdh and trh), and C. botulinum (BoNT) were not detected. The pR72H chromosomal DNA fragment, which is conservative in V. parahaemolyticus strains, was detected in five (4.7%) samples. A number of 192 suspect isolates did not fit the description of clinical Aeromonas phenospecies, pathogenic Vibrio spp., or P. shigelloides. The effectiveness of commercial depuration in reducing bacterial indicators was also examined. E. coli was reduced to < or = 230/100 g of flesh and liquor in 90.9% of mussel lots but in only 70.9% of infaunal bivalve lots. For total coliform elimination, mussels were also more effective. Total counts significantly (P < 0.005) correlated with numbers of Pseudomonas, Aeromonas, and Vibrio. Our data indicate that Salmonella and pathogenic bacteria indigenous to estuarine environments do not appear to be significant hazards in Galician molluscan shellfish. A reason for concern, however, is that clearance of E. coli to acceptable levels was not always achieved especially in infaunal bivalves.

Identification and epidemiological relationships of Aeromonas isolates from patients with diarrhea, drinking water and foods.

A collection of Aeromonas isolates obtained over a three-year period in the same geographic area (León, NW of Spain) was characterized by (GTG)₅-PCR fingerprinting, amplified fragment length polymorphism (AFLP) analysis and gyrB gene sequence analysis. The isolates originated from human diarrheal stools (29 isolates), potable water (13 isolates), rabbit meat (13 isolates) and marine fish (5 isolates). The distribution of Aeromonas species varied with the strain source. Aeromonas caviae HG4 and Aeromonas media HG5 were predominant in clinical and water isolates, respectively, whereas motile Aeromonas salmonicida HG3 strains were most frequently found in fish and meat. Molecular typing revealed several genotypic relationships among specific isolate subsets: (i) two clones of A. media HG5 persisted in drinking water over the study period, (ii) different patients harbored identical or closely related clones during several months, and (iii) clonal relatedness was observed in two sets of water and human isolates. The first of these sets comprised nine water isolates and two human A. media HG5 isolates, whereas the other one included a water isolate and a human isolate of A. caviae HG4. The latter finding suggests that Aeromonas transmission in the studied region followed a waterborne route. Interestingly, the three human isolates closely related to water isolates were recovered in a period of four days in June 2006 from non-related patients without underlying medical conditions that tested negative for other enteric pathogens. The data imply the transmission through contaminated water of strains of the A. caviae group that can produce disease in humans.

Incidence, radioresistance, and behavior of Psychrobacter spp. in rabbit meat.

The relative incidence of Psychrobacter spp. in rabbit meat, the radioresistance of these bacteria, and the growth of nonirradiated and irradiated psychrobacter isolates, alone and in coculture, during chilled storage of inoculated sterile rabbit meat was investigated. Psychrobacter spp. accounted for 4.2% of the storage psychrotrophic flora of 30 rabbit carcasses. The radiation D10-values of 10 Psychrobacter isolates, irradiated at 4 degrees C in minced rabbit meat, ranged from 0.8 to 2.0 kGy, with significant (P < 0.05) differences among strains. Over 12 days of storage at 4 degrees C, pure cultures of two nonirradiated psychrobacter strains (D10 = 2 kGy) were capable of substantial increases (up to 3 log CFU/g) in sterile rabbit meat, but when the fastest growing strain was cocultured with Pseudomonas fluorescens and Brochothrix thermosphacta isolates, maximum cell densities and growth rates were significantly (P < 0.01) lower. After irradiation (2.5 kGy) of pure cultures in sterile rabbit meat, surviving cells of both Psychrobacter strains decreased for a period of 5 to 7 days and then resumed multiplication that, at day 12, resulted in a similar increase (1.6 to 1.7 log CFU/g) over initial survivor numbers. When irradiated in combination with the spoilage bacteria, one of the strains required 12 days to reach initial numbers. In conclusion, Psychrobacter spp. are radioresistant nonsporeforming bacteria with a low relative incidence among the storage flora of rabbit meat, unable to compete with food spoilage bacteria in this ecosystem and apparently not a major contributor to the spoilage of rabbit meat after irradiation.