Jesús A. Santos

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.

Mesophilic Aeromonads in Wild and Aquacultured Freshwater Fish

Numbers and species of motile Aeromonas were determined in freshly caught freshwater fish, in the surrounding environment, and also during iced chilled storage of fish specimens. Although no significant differences were observed in water samples, initial levels for skin, gill, and intestines were significantly lower in farmed rainbow trout (Oncorhynchus mykiss) than in wild brown trout (Salmo trutta) and pike (Esox lucius). During storage of wild specimens, naturally occurring aeromonads grew fairly well on the surfaces of skin and body cavity. Of 171 strains assigned to the genus Aeromonas, 88% were identified to phenospecies and putative genospecies level by using comprehensive biochemical schemes. The isolates were allocated to putative hybridization groups (HGs) 1 and 3 Aeromonas hydrophila (29%); putative HG 8 Aeromonas veronii biovar sobria (19%); putative HG 2 Aeromonas bestiarum (18%); putative HG 9 Aeromonas jandaei (16%); putative HGs 4 and 5a Aeromonas caviae (2%); putative HG 12 Aeromonas schubertii (2%); and putative HG 11 (unnamed, 0.6%). The remaining 20 isolates (12%) resembled A. schubertii but could not be allocated to currently recognized phenospecies or to putative HGs. Although cultured rainbow trout yielded strains of putative HGs 1, 4, and 8, which appear to be of major clinical importance, most isolates assigned to putative HGs 1 and 8 were recovered from pike. Differences among HGs found in wild animals could be related to their origin (unpolluted rivers for brown trout and urban rivers for pike). The recovery of these aeromonads species was not related to sampling site. The initial levels of motile aeromonads, their behavior during storage, and the strong potential spoilage activity of most isolates confirm that these bacteria can contribute to deterioration of iced wild freshwater fish. Although adequate cooking would inactivate motile aeromonads, the high incidence of isolates belonging to gastroenteritis-associated HGs should be regarded as a potential health concern, particularly for susceptible populations when there is a possibility of cross-contamination.

Psychrobacters and Related Bacteria in Freshwater Fish

Three phenotypic identification systems were employed to identify 106 strains of gram-negative, nonmotile, aerobic bacteria obtained during iced storage of wild (Salmo trutta and Esox lucius) and farmed (Oncorhynchus mykiss) freshwater fish. Using diagnostic tables and computer-assisted identification, the isolates were Psychrobacter (64 strains), Acinetobacter (24 strains), Moraxella (6 strains), Chryseobacterium (5 strains), Myroides odoratus (2 strains), Flavobacterium (1 strain), Empedobacter (1 strain), and unidentified (3 strains). Overall similarities of all strains were determined for 108 characters by numerical analysis (simple matching coefficient of similarity [S] and clustering by unweighted pair group average linkage [UPGMA]). At the 77% similarity level, 92 strains formed nine major clusters (3 or more strains) and four small clusters (2 strains). Cluster 1 (25 isolates divided into two main subclusters) could be assigned to Psychrobacter phenylpyruvicus, clusters 2 and 3 (26 isolates) were designated as Psychrobacter immobilis, and clusters 4 (3 isolates) and 7 (4 isolates) were identified as Psychrobacter urativorans and Psychrobacter spp., respectively. Clusters 5 (five isolates), 6 (three isolates), and 9 (five isolates) were labeled as Acinetobacter spp., Acinetobacter johnsonii, and Acinetobacter lwoffii, respectively. Cluster 8 (12 isolates), with a high resemblance to Thornleys phenon 4 (a heterogeneous group of bacteria isolated from poultry and related to Acinetobacter), remained unnamed. The restriction pattern was identical for strains grouped into clusters 2 and 3 (P. immobilis) but was different for the remaining Psychrobacter isolates. A large proportion of isolates belonging to the family Moraxellaceae were closely related. Psychrobacters and A. johnsonii were present in freshly caught fish and river water. In the latter stages of storage, P. phenylpyruvicus and acinetobacters tended to decrease, whereas P. immobilis increased.

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.

Identity, virulence genes, and clonal relatedness of Aeromonas isolates from patients with diarrhea and drinking water

Among 800 stool specimens from patients with diarrhea submitted by Primary Care Centers for routine analysis to the Hospital of León (NW Spain) Microbiology and Parasitology Service, 32 (4%) were tested positive for Aeromonas spp. Mixed infections with other enteric pathogens occurred in 12 patients. A. caviae was isolated from 23 clinical specimens. There were also patients infected with A. media, A. hydrophila, A. bestiarum, and A. veronii biovar veronii. All but three isolates carried one or more of the virulence genes. The incidence of the alt, hlyA, aerA, ast, and laf genes was 71.9, 28.1, 25.0, 18.8, and 9.4%, respectively. The alt+/ast+ combination was detected in four isolates and the aerA+/hlyA+ combination was detected in the two A. hydrophila isolates. None of the strains harbored the TTSS, stx1, or stx2 genes and nine bore plasmids. Thirty clinical isolates and a collection of 12 A. caviae and A. media strains obtained from León municipal drinking water over the study period were typed by pulsed-field gel electrophoresis (PFGE). PFGE patterns revealed genetic relatedness and persistence over time among water isolates and some clinical isolates. Interestingly, one A. caviae (aerA−/hlyA−/alt+/ast−/laf+) human isolate and two A. caviae (aerA−/hlyA−/alt+/ast−/laf+) drinking water isolates had indistinguishable PFGE patterns, suggesting waterborne infection.

Hemolytic and elastolytic activities influenced by iron in Plesiomonas shigelloides.

The aim of this study was to determine the presence of hemolytic and elastolytic enzymes in several strains of Plesiomonas shigelloides in relation to the availability of iron in culture media. Hemolytic activity and elastolytic activity were detected in strains of P. shigelloides and were enhanced when the strains were grown in an iron-depleted medium and lost after thermal treatment at 100 degrees C for 10 min. Also, elastolytic activity was inactivated by phenylmethyl sulfonyl fluoride, an inhibitor of serine proteases. Hemolytic activity was detected extracellularly in cell-free supernatants, whereas elastin degradation activity was cell associated. Both activities may be related to the virulence of P. shigelloides.

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.

Short communication: Influence of storage and preservation on microbiological quality of silo ovine milk

This study was designed to analyze the effects of the storage and preservation conditions on counts of mesophilic, thermoduric, psychotrophic, coliform, Escherichia coli, Streptococcus agalactiae, and Staphylococcus aureus organisms in silo ovine milk. A total of 910 analytical determinations were conducted from aliquots of 10 silo ovine milks. The conditions tested were unpreserved and azidiol-preserved milk stored at 4°C, and unpreserved milk stored at -20°C. Milk aged 2, 24, 48, 72, and 96 h post-collection for refrigerated aliquots, and 7, 15, and 30 d post-collection for frozen aliquots. The factors silo and storage conditions significantly contributed to variation of all microbiological variables, although milk age effect within storage was only significant for mesophilic, psychrotrophic, and coliform bacteria counts. In refrigerated raw milk, mesophile, psychrotroph, and coliform counts significantly increased over 96 h post-collection, whereas the other groups and bacteria species tested maintained their initial concentration. In all cases, azidiol preservation maintained the initial bacterial concentration in raw sheep milk under refrigeration throughout 96 h. Thus, azidiol was a suitable preservative for microbiological studies in sheep milk. Smallest counts were registered for frozen samples, particularly for coliforms, E. coli, Strep. agalactiae and Staph. aureus. Estimates of mesophilic, thermoduric and psychrotrophic organisms showed similar values on both azidiol-preserved and frozen milk samples. Coliforms and E. coli counts significantly decrease over time after freezing. Consequently, freezing at -20°C could also be appropriate for analysis of mesophilic, thermoduric, and psychrotrophic bacterial groups, but not for coliforms or mammary pathogens.