Cytotoxicity and Antimicrobial Resistance of Aeromonas Strains Isolated from Fresh Produce and Irrigation Water.

The genus Aeromonas has received constant attention in different areas, from aquaculture and veterinary medicine to food safety, where more and more frequent isolates are occurring with increased resistance to antibiotics. The present paper studied the interaction of Aeromonas strains isolated from fresh produce and water with different eukaryotic cell types with the aim of better understanding the cytotoxic capacity of these strains. To study host-cell pathogen interactions in Aeromonas, we used HT-29, Vero, J774A.1, and primary mouse embryonic fibroblasts. These interactions were analyzed by confocal microscopy to determine the cytotoxicity of the strains. We also used Galleria mellonella larvae to test their pathogenicity in this experimental model. Our results demonstrated that two strains showed high cytotoxicity in epithelial cells, fibroblasts, and macrophages. Furthermore, these strains showed high virulence using the G. mellonella model. All strains used in this paper generally showed low levels of resistance to the different families of the antibiotics being tested. These results indicated that some strains of Aeromonas present in vegetables and water pose a potential health hazard, displaying very high in vitro and in vivo virulence. This pathogenic potential, and some recent concerning findings on antimicrobial resistance in Aeromonas, encourage further efforts in examining the precise significance of Aeromonas strains isolated from foods for human consumption.

Behavior of Shiga-toxin-producing Escherichia coli in ewe milk stored at different temperatures and during the manufacture and ripening of a raw milk sheep cheese (Zamorano style).

This study was conducted to assess the survival of 2 wild Shiga toxin-producing Escherichia coli strains (one serotype O157:H7 and one non-O157:H7) in ewe milk stored at different conditions and to examine the fate of the O157 strain during the manufacture and ripening of a Spanish sheep hard variety of raw milk cheese (Zamorano). The strains were selected among a population of 50 isolates, which we obtained from ewe milk, because of their high resistance to 0.3% lactic acid. Both strains were inoculated (approximately 2 log10 cfu/mL) in raw and heat-treated (low-temperature holding, LTH; 63°C/30 min) ewe milk and stored for 5 d at 6, 8, and 10°C and also according to a simulation approach for assessing the effects of failures in the cold chain. The minimum growth temperature for the O157:H7 strain in LTH and raw ewe milk was 8°C. For the non-O157:H7 strain, the lowest temperature showing bacterial growth in LTH ewe milk was 6°C, but it did not grow at any of the tested conditions in raw milk. It appears that the O157 strain was more susceptible to cold stress but was likely a better competitor than the non-O157 strain against the milk autochthonous microbiota. For manufacture of Zamorano cheese, raw milk was inoculated with approximately 3 log10 cfu/mL, and after 2 mo of ripening at 10 to 12°C, the cheeses showed the expected general characteristics for this variety. The O157:H7 strain increased 0.9 log10 cfu/g after whey drainage and during ripening and storage decreased by 2.9 log10 cfu/g. Nevertheless, its detectable level (estimated at 6.2 cfu/g) after 2 mo of ripening suggests that Zamorano cheese manufactured from raw ewe milk contaminated with E. coli O157:H7 could represent a public health concern.

Extended-Spectrum Beta-Lactamase-Producing Enterobacteriaceae in Fresh Produce.

Fresh vegetables are an essential part of a healthy diet, but microbial contamination of fruits and vegetables is a serious concern to human health, not only for the presence of foodborne pathogens but because they can be a vehicle for the transmission of antibiotic-resistant bacteria. This work aimed to investigate the importance of fresh produce in the transmission of extended-spectrum ß-lactamases (ESBL)-producing Enterobacteriaceae. A total of 174 samples of vegetables (117) and farm environment (57) were analysed to determine enterobacterial contamination and presence of ESBL-producing Enterobacteriaceae. Enterobacterial counts above the detection limit were found in 82.9% vegetable samples and 36.8% environmental samples. The average count was 4.2 log cfu/g or mL, with a maximum value of 6.2 log cfu/g in a parsley sample. Leafy vegetables showed statistically significant higher mean counts than other vegetables. A total of 15 ESBL-producing isolates were obtained from vegetables (14) and water (1) samples and were identified as Serratia fonticola (11) and Rahnella aquatilis (4). Five isolates of S. fonticola were considered multi-drug resistant. Even though their implication in human infections is rare, they can become an environmental reservoir of antibiotic-resistance genes that can be further disseminated along the food chain.

The poly(I:C)-induced maternal immune activation model; a systematic review and meta-analysis of cytokine levels in the offspring.

The maternal polyinosinic:polycytidylic acid (poly(I:C)) animal model is frequently used to study how maternal immune activation may impact neuro development in the offspring. Here, we present the first systematic review and meta-analysis on the effects of maternal poly(I:C) injection on immune mediators in the offspring and provide an openly accessible systematic map of the data including methodological characteristics. Pubmed and EMBASE were searched for relevant publications, yielding 45 unique papers that met inclusion criteria. We extracted data on immune outcomes and methodological characteristics, and assessed the risk of bias. The descriptive summary showed that most studies reported an absence of effect, with an equal number of studies reporting an increase or decrease in the immune mediator being studied. Meta-analysis showed increased IL-6 concentrations in the offspring of poly(I:C) exposed mothers. This effect appeared larger prenatally than post-weaning. Furthermore, poly(I:C) administration during mid-gestation was associated with higher IL-6 concentrations in the offspring. Maternal poly(I:C) induced changes in IL-1ß, Il-10 and TNF-α concentrations were small and could not be associated with age of offspring, gestational period or sampling location. Finally, quality of reporting of potential measures to minimize bias was low, which stresses the importance of adherence to publication guidelines. Since neurodevelopmental disorders in humans tend to be associated with lifelong changes in cytokine concentrations, the absence of these effects as identified in this systematic review may suggest that combining the model with other etiological factors in future studies may provide further insight in the mechanisms through which maternal immune activation affects neurodevelopment.

Behaviour of Non-O157 STEC and Atypical EPEC during the Manufacturing and Ripening of Raw Milk Cheese.

This study was carried out to assess the survival of Shiga toxin-producing E. coli (STEC) and atypical enteropathogenic Escherichia coli (aEPEC) during the traditional manufacturing and ripening of Spanish hard cheese from raw cow's milk. Milk samples were spiked with up to 3.1-3.5 log cfu/mL of one strain of STEC (O140:H32 serotype) and one of aEPEC (serotype O25:H2). The first steps of cheesemaking allow for a STEC and aEPEC increase of more than 1 log cfu/mL (up to 4.74 log cfu/g and 4.55 log cfu/g, respectively). After cheese pressing, a steady reduction of both populations was observed, with the STEC strain being more sensitive. The studied pathogenic E. coli populations decreased by 1.32 log cfu/g in STEC and 0.59 log cfu/g in aEPEC in cheese ripened during a minimum period of 60 d. Therefore, a moderate contamination by these diarrhoeagenic E. coli pathotypes, in particular, with aEPEC, on cheese manufactured from raw milk may not be totally controlled through the cheesemaking process and during a maturation of 90 d. These findings remark the importance of improvement in bacteriological quality of raw milk and cross-contamination prevention with diarrhoeagenic E. coli in the dairy industry.

Molecular Diversity of ESBL-Producing Escherichia coli from Foods of Animal Origin and Human Patients.

Dissemination of enterobacteria that produce extended spectrum ß-lactamases (ESBL) throughout the food chain has become an important health concern. This work aimed to evaluate the occurrence of ESBL-producing bacteria in foods of animal origin and to investigate the similarities between food and human isolates. The presence of beta-lactam-resistant Enterobacteriaceae was analyzed in 108 food samples, isolating 10 strains of Escherichia coli, one strain of Citrobacter freundi, and one of Hafnia alvei. E. coli isolates were compared to a group of 15 strains isolated from human patients by antibiotic susceptibility testing, characterization of ESBL genes (blaTEM, blaCTX,), multilocus sequence typing (MLST) and pulse-field gel electrophoresis (PFGE). Nineteen (14 clinical and five food) isolates carried blaCTX, 14 (six clinical and eight food) carried blaTEM, and three (one clinical and two food) carried blaSHV gen. MLST analysis revealed the prevalence of ST131 among the clinical strains, which grouped together in a PFGE cluster. Food isolates showed higher diversity and two of them (ST57) grouped with clinical strains, whereas another two belonged to clonal groups with virulence potential (ST59). In conclusion, the results showed that foods of animal origin must be regarded as a reservoir of ESBL-producing bacteria of clinical relevance, which might spread through the food chain.

Partial replacement of glucose by galactose in the post-weaning diet improves parameters of hepatic health.

Replacing part of glucose with galactose in the post-weaning diet beneficially affects later life metabolic health in female mice. The liver is the main site of galactose metabolism, but the direct effects of this dietary intervention on the liver in the post-weaning period are not known. The aim of this study was to elucidate this. Weanling female mice (C57BL/6JRccHsd) were fed a starch containing diet with glucose (32 en%) monosaccharide (GLU), or a diet with glucose and galactose (1:1 both 16 en%) (GLU+GAL). Body weight, body composition, and food intake were determined weekly. After 3 weeks, mice were sacrificed, and serum and liver tissues were collected. Global hepatic mRNA expression was analyzed and hepatic triglyceride (TG) and glycogen contents were determined by enzymatic assays. Body weight and body composition were similar in both groups, despite higher food intake in mice on GLU+GAL diet. Hepatic TG content was lower in GLU+GAL-fed than GLU-fed females, while glycogen levels were unaffected. Analysis of global expression patterns of hepatic mRNA showed that mainly inflammation-related pathways were affected by the diet, which were predominantly downregulated in GLU+GAL-fed females compared to GLU-fed females. This reduction in inflammation in GLU+GAL-fed females was also reflected by decreased serum concentrations of acute phase protein Serum amyloid A 3. In conclusion, replacing part of glucose with galactose in the post-weaning diet reduces hepatic TG content and hepatic inflammation.

A Lowly Digestible-Starch Diet after Weaning Enhances Exogenous Glucose Oxidation Rate in Female, but Not in Male, Mice.

Starches of low digestibility are associated with improved glucose metabolism. We hypothesise that a lowly digestible-starch diet (LDD) versus a highly digestible-starch diet (HDD) improves the capacity to oxidise starch, and that this is sex-dependent. Mice were fed a LDD or a HDD for 3 weeks directly after weaning. Body weight (BW), body composition (BC), and digestible energy intake (dEI) were determined weekly. At the end of the intervention period, whole-body energy expenditure (EE), respiratory exchange ratio (RER), hydrogen production, and the oxidation of an oral 13C-labelled starch bolus were measured by extended indirect calorimetry. Pancreatic amylase activity and total 13C hepatic enrichment were determined in females immediately before and 4 h after administration of the starch bolus. For both sexes, BW, BC, and basal EE and RER were not affected by the type of starch, but dEI and hydrogen production were increased by the LDD. Only in females, total carbohydrate oxidation and starch-derived glucose oxidation in response to the starch bolus were higher in LDD versus HDD mice; this was not accompanied by differences in amylase activity or hepatic partitioning of the 13C label. These results show that starch digestibility impacts glucose metabolism differently in females versus males.

Extended indirect calorimetry with isotopic CO2 sensors for prolonged and continuous quantification of exogenous vs. total substrate oxidation in mice.

Indirect calorimetry (InCa) estimates whole-body energy expenditure and total substrate oxidation based on O2 consumption and CO2 production, but does not allow for the quantification of oxidation of exogenous substrates with time. To achieve this, we incorporated 13CO2 and 12CO2 gas sensors into a commercial InCa system and aimed to demonstrate their performance and added value. As a performance indicator, we showed the discriminative oscillations in 13CO2 enrichment associated with food intake in mice fed diets containing naturally low (wheat) vs high (maize) 13C enrichment. To demonstrate the physiological value, we quantified exogenous vs total carbohydrate and fat oxidation continuously, in real time in mice varying in fat mass. Diet-induced obese mice were fed a single liquid mixed meal containing 13C-isotopic tracers of glucose or palmitate. Over 13 h, ~70% glucose and ~48% palmitate ingested were oxidised. Exogenous palmitate oxidation depended on body fat mass, which was not the case for exogenous glucose oxidation. We conclude that extending an InCa system with 13CO2 and 12CO2 sensors provides an accessible and powerful technique for real-time continuous quantification of exogenous and whole-body substrate oxidation in mouse models of human metabolic physiology.

Replacing Part of Glucose with Galactose in the Postweaning Diet Protects Female But Not Male Mice from High-Fat Diet-Induced Adiposity in Later Life.

BACKGROUND: Duration of breastfeeding is positively associated with decreased adiposity and increased metabolic health in later life, which might be related to galactose. OBJECTIVE: The aim of this study was to investigate if partial replacement of glucose with galactose in the postweaning diet had a metabolic programming effect. METHODS: Male and female mice (C57BL/6JRccHsd) received an isocaloric diet (16 energy% fat; 64 energy% carbohydrates; 20 energy% protein) with either glucose (32 energy%) (GLU) or glucose + galactose (GLU + GAL, 16 energy% each) for 3 wk postweaning. Afterwards, all mice were switched to the same 40 energy% high-fat diet (HFD) for 9 wk to evaluate potential programming effects in an obesogenic environment. Data were analyzed within sex. RESULTS: Female body weight (-14%) and fat mass (-47%) were significantly lower at the end of the HFD period (both P < 0.001) among those fed GLU + GAL than among those fed GLU; effects in males were in line with these findings but nonsignificant. Food intake was affected in GLU + GAL-fed females (+8% on postweaning diet, -9% on HFD) compared with GLU-fed females, but not for hypothalamic transcript levels at endpoint. Also, in GLU + GAL-fed females, serum insulin concentrations (-48%, P  < 0.05) and the associated homeostasis model assessment of insulin resistance (HOMA-IR) were significantly lower ( P < 0.05) at endpoint, but there were no changes in pancreas morphology. In GLU + GAL-fed females, expression of insulin receptor substrate 2 (Irs2) (-27%, P  < 0.01 ; -44%, P  < 0.001) and the adipocyte size markers leptin (Lep) (-40%, P  < 0.05; -63% , P  < 0.05) and mesoderm-specific transcript homolog protein (Mest) (-80%, P < 0.05; -72%, P  < 0.05) was lower in gonadal and subcutaneous white adipose tissue (WAT), respectively. Expression of insulin receptor substrate1 (Irs1) (-24%, P  < 0.05) was only lower in subcutaneous WAT in GLU + GAL-fed females. CONCLUSIONS: Partial replacement of glucose with galactose, resulting in a 1:1 ratio mimicking lactose, in a 3-wk postweaning diet lowered body weight, adiposity, HOMA-IR, and expression of WAT insulin signaling in HFD-challenged female mice in later life. This suggests that prolonged galactose intake may improve metabolic and overall health in later life.

Direct and Long-Term Metabolic Consequences of Lowly vs. Highly-Digestible Starch in the Early Post-Weaning Diet of Mice.

Starches of low and high digestibility have different metabolic effects. Here, we examined whether this gives differential metabolic programming when fed in the immediate post-weaning period. Chow-fed mice were time-mated, and their nests were standardized and cross-fostered at postnatal days 1⁻2. After postnatal week (PW) 3, individually housed female and male offspring were switched to a lowly-digestible (LDD) or highly-digestible starch diet (HDD) for three weeks. All of the mice received the same high-fat diet (HFD) for nine weeks thereafter. Energy and substrate metabolism and carbohydrate fermentation were studied at the end of the HDD/LDD and HFD periods by extended indirect calorimetry. Glucose tolerance (PW 11) and metabolic flexibility (PW14) were analyzed. Directly in response to the LDD versus the HDD, females showed smaller adipocytes with less crown-like structures in gonadal white adipose tissue, while males had a lower fat mass and higher whole body fat oxidation levels. Both LDD-fed females and males showed an enlarged intestinal tract. Although most of the phenotypical differences disappeared in adulthood in both sexes, females exposed to LDD versus HDD in the early post-weaning period showed improved metabolic flexibility in adulthood. Cumulatively, these results suggest that the type of starch introduced after weaning could, at least in females, program later-life health.

Non-invasive continuous real-time in vivo analysis of microbial hydrogen production shows adaptation to fermentable carbohydrates in mice.

Real time in vivo methods are needed to better understand the interplay between diet and the gastrointestinal microbiota. Therefore, a rodent indirect calorimetry system was equipped with hydrogen (H2) and methane (CH4) sensors. H2 production was readily detected in C57BL/6J mice and followed a circadian rhythm. H2 production was increased within 12 hours after first exposure to a lowly-digestible starch diet (LDD) compared to a highly-digestible starch diet (HDD). Marked differences were observed in the faecal microbiota of animals fed the LDD and HDD diets. H2 was identified as a key variable explaining the variation in microbial communities, with specific taxa (including Bacteroides and Parasutterella) correlating with H2 production upon LDD-feeding. CH4 production was undetectable which was in line with absence of CH4 producers in the gut. We conclude that real-time in vivo monitoring of gases provides a non-invasive time-resolved system to explore the interplay between nutrition and gut microbes in a mouse model, and demonstrates potential for translation to other animal models and human studies.

No Adverse Programming by Post-Weaning Dietary Fructose of Body Weight, Adiposity, Glucose Tolerance, or Metabolic Flexibility.

SCOPE: Metabolic programming can occur not only in the perinatal period, but also post-weaning. This study aims to assess whether fructose, in comparison to glucose, in the post-weaning diet programs body weight, adiposity, glucose tolerance, metabolic flexibility, and health at adult age. METHODS AND RESULTS: Three-week-old male and female C57BL6/JRccHsd mice are given an intervention diet with 32 energy percent (en%) glucose or fructose for only 3 weeks. Next, all animals are switched to the same 40 en% high fat diet for 9 weeks. Neither body weight nor adiposity differs significantly between the animals fed with glucose or fructose diets at any point during the study in both sexes. Glucose tolerance in adulthood is not affected by the post-weaning diet, nor are activity, energy expenditure, and metabolic flexibility, as measured by indirect calorimetry. At the end of the study, only in females fasting serum insulin levels and HOMA-IR index are lower in post-weaning fructose versus glucose diet (p = 0.02), without differences in pancreatic ß-cell mass. CONCLUSIONS: Our present findings indicate no adverse programming of body weight, adiposity, glucose tolerance, and metabolic flexibility by dietary (solid) fructose in comparison to glucose in the post-weaning diet in mice.

Investigation of III-V nanowires by plan-view transmission electron microscopy: InN case study.

We discuss observations of InN nanowires (NWs) by plan-view high-resolution transmission electron microscopy (TEM). The main difficulties arise from suitable methods available for plan-view specimen preparation. We explore different approaches and find that the best results are obtained using a refined preparation method based on the conventional procedure for plan-view TEM of thin films, specifically modified for the NW morphology. The fundamental aspects of such a preparation are the initial mechanical stabilization of the NWs and the minimization of the ion-milling process after dimpling the samples until perforation. The combined analysis by plan-view and cross-sectional TEM of the NWs allows determination of the degree of strain relaxation and reveals the formation of an unintentional shell layer (2-3-nm thick) around the InN NWs. The shell layer is composed of bcc In2O3 nanocrystals with a preferred orientation with respect to the wurtzite InN: In2O3 [111] || InN [0001] and In2O3<110>||InN<1120>.

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.

Aneurysmal bone cyst of the mandible: case presentation and review of the literature

El quiste óseo aneurismático es una lesión ósea infrecuente que raramente se localiza a nivel del esqueleto craneofacial. Es una lesión de naturaleza benigna pero de comportamiento localmente agresivo. Del conjunto de lesiones quísticas de los maxilares, constituye una variedad muy poco frecuente, que presenta sin embargo características diferenciales, respecto a otro tipo de patologías con carácter de malignidad. Presentamos un caso clínico de un varón caucásico que presentaba dicha lesión a nivel mandibular y revisamos la literatura, centrándonos en el diagnóstico diferencial con lesiones neoplásicas como el ameloblastoma o el tumor de células gigantes. Así mismo, comentamos las diferentes posibilidades terapéuticas clásicamente descritas para el tratamiento de este tipo de patologías. En el caso que nos ocupa, el tratamiento fue quirúrgico, con cureteado exhaustivo de la lesión. Dicho tratamiento consiguió una extirpación completa y una posterior regeneración ósea local, que ha permitido una rehabilitación implantosoportada del segmento dentario sobre el que asentaba la lesión inicial

The aneurysmatic osseus cyst is a very infrequent bone lesion which in some occasions can be found at the craneofacial skeleton. Among all the cystic lesions that can be found at the mandible or the maxilla it is very rare. On the other side it is at the same time very interesting in terms of its differential diagnose with other types of maxillary bone lesions We present the case of a Caucasian male with an aneurysmatic cyst located at the right angle of the mandible and a review of the literature concerning the case. We have focused on the differential diagnose, mainly with the malignancies that can be found at this location. We also comment the therapeutic options clasically described for these kind of pathologies. In our patient, the surgical excision allowed a complete removal of the lesion and a posterior bone healing which made possible a implant-supported rehabilitation of the edentulous segment

Effect of different storage conditions on E. coli O157:H7 and the indigenous bacterial microflora on lamb meat.

Lamb chops inoculated with 2.23-2.83 log cfu/g of E. coli O157:H7 strain NCTC 12900 were packed in air (AP), vacuum (VP), and two modified atmospheres (MAP) consisting of 100% CO2 and a commercial mixture of 35% CO2/35% O2/30% N2. All samples (initial total counts <3.5 log cfu/g) were stored in a commercial cold storage facility set at 4 degrees C and one AP trial also at 12+/-1 degrees C in a temperature controlled incubator. Pathogen and indigenous flora evolution, physicochemical and sensory changes, surface packages temperature and MAP gas composition were monitored throughout the lamb meat shelf life. Temperature monitoring revealed that during chilled storage packed chops exceeded 7 degrees C about 3% of the time for periods of 10-20 min at 6 h intervals corresponding to defrosting cycles. In AP samples under these conditions, the E. coli O157:H7 strain had an overall increase of 0.48 log cfu/g by day 12. This increase, which may be regarded as an artefact of the sampling procedure, might also be a response to fluctuating temperatures. Regardless of rapid proliferation of the background microflora on AP lamb meat kept at 12+/-1 degrees C, the pathogen significantly increased by 2.35 log cfu/g after nine days. There was a slight decrease (0.20 log cfu/g) of the pathogen numbers after four weeks cold storage in VP despite a significant increase in lactic acid bacteria (LAB). With a relatively small outgrowth of LAB, chilled storage in 100% and 35% CO2 resulted in significant differences compared to similar conditions in air (decrease from initial numbers of 0.80 and 0.45 log cfu/g, respectively). Our data confirm the importance of effective temperature control to prevent pathogen growth on raw meat and also that contaminated meat remains hazardous regardless of refrigeration and protective packaging. Further studies are needed to determine the behaviour of E. coli O157:H7 at temperatures that fluctuate around the minimum for growth.

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.

Molecular and phenotypic typing of Staphylococcus aureus isolates from rabbit meat.

Twenty-six Staphylococcus aureus isolates were recovered from rabbit carcasses and cuts during a period of seven months. Samples from 51 different animals, flocks and farms were obtained from five establishments in four Spanish provinces. To determine their diversity and possible origin, isolates were typed by three molecular and three phenotypic methods. PFGE, with the highest discrimination index (D=0.966), identified 19 patterns (more than one band difference) and 10 types (more than three band differences). Based on > or = 90% similarity, RAPD (D=0.877) produced nine patterns while ribotyping (D=0.786) produced seven types. On the basis of biotyping (D=0.644), 11 isolates belonged to human ecovars and 15 to the non-host-specific crystal violet type C (NHS CV:C) biotypes. By direct phage typing (D=0.761), 17 isolates were lysed by human phages into groups II (8 isolates), III (5 isolates), I/III (2 isolates) and V (2 isolates). The overall resistance to antimicrobials (D=0.783) was 76.9%, with most isolates being resistant to tetracycline (61.5%) and penicillin G (26.9%). PFGE showed that samples from each processing plant carried different S. aureus types, some of them persisting over time. There also was evidence of interestablishment dissemination of genetically related clones, most of them belonging to the PFGE type A and phenotype "NHS CV:C biotypes-3A/3C/55/71 phage type", which is highly virulent for European commercial rabbitries. The ubiquity of the virulent phenotype, as well as the high incidence of resistance to antibiotics with application in human medicine, is a matter of concern in public and animal health.

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.