Occurrence, diversity and temperature-dependent growth kinetics of Aeromonas spp. in lettuce.

Bagged, pre-cut and prewashed lettuce products are marketed as ready to eat. This concept poses a food safety concern, due to lack of efficient hurdles to eliminate possible microbial contaminants from the fresh produce and/or the processing itself. Aeromonas spp. are potential foodborne pathogens that are frequently isolated from lettuce. High counts of, e.g., A. hydrophila have been found in retail ready-to-eat (RTE) vegetable salads. The aim of this study was to assess the general microbiological quality, the occurrence and diversity of potential human pathogenic mesophilic Aeromonas spp. of retail RTE lettuce products. Additionally, temperature-dependent growth kinetic parameters of Aerobic Plate Counts (APC) and Aeromonas spp. in one selected RTE lettuce product, rocket lettuce, were quantified by performing storage experiments at 4 °C, 8 °C and 12 °C. The Aeromonas isolates were further characterized regarding pathogenic traits and phylogenetic relationship. The overall hygienic quality of the lettuce products was unsatisfactory, as 60% of the products had an APC level higher than 7.0 log CFU/g. Presumptive Aeromonas spp. were detected in 52% of the samples, levels ranging from approximately 2.0-6.0 log CFU/g. Significantly lower counts of APC and Aeromonas spp. were found in uncut and unwashed products. Presumptive Aeromonas spp. were able to proliferate in rocket lettuce stored at 4 °C (µmax = 0.39 ± 0.06/d and µmax = 0.43 ± 0.05/d for lettuce from producers A and B, respectively), and µmax was approximately 2× higher at 8 °C and 3× higher at 12 °C. Eighty-four percent of the collected isolates were identified as A. media, based on partial gyrB sequencing. Additionally A. salmonicida and A. bestiarum were detected. The pathogenic potential in this material was high, most of the isolates harbored at least one of the toxin genes, act, ast, alt.

The Significance of Mesophilic Aeromonas spp. in Minimally Processed Ready-to-Eat Seafood.

Minimally processed and ready-to-eat (RTE) seafood products are gaining popularity because of their availability in retail stores and the consumers' perception of convenience. Products that are subjected to mild processing and products that do not require additional heating prior to consumption are eaten by an increasing proportion of the population, including people that are more susceptible to foodborne disease. Worldwide, seafood is an important source of foodborne outbreaks, but the exact burden is not known. The increased interest in seafood products for raw consumption introduces new food safety issues that must be addressed by all actors in the food chain. Bacteria belonging to genus Aeromonas are ubiquitous in marine environments, and Aeromonas spp. has held the title "emerging foodborne pathogen" for more than a decade. Given its high prevalence in seafood and in vegetables included in many RTE seafood meals, the significance of Aeromonas as a potential foodborne pathogen and a food spoilage organism increases. Some Aeromonas spp. can grow relatively uninhibited in food during refrigeration under a broad range of pH and NaCl concentrations, and in various packaging atmospheres. Strains of several Aeromonas species have shown spoilage potential by the production of spoilage associated metabolites in various seafood products, but the knowledge on spoilage in cold water fish species is scarce. The question about the significance of Aeromonas spp. in RTE seafood products is challenged by the limited knowledge on how to identify the truly virulent strains. The limited information on clinically relevant strains is partly due to few registered outbreaks, and to the disputed role as a true foodborne pathogen. However, it is likely that illness caused by Aeromonas might go on undetected due to unreported cases and a lack of adequate identification schemes. A rather confusing taxonomy and inadequate biochemical tests for species identification has led to a biased focus towards some Aeromonas species. Over the last ten years, several housekeeping genes has replaced the 16S rRNA gene as suitable genetic markers for phylogenetic analysis. The result is a more clear and robust taxonomy and updated knowledge on the currently circulating environmental strains. Nevertheless, more knowledge on which factors that contribute to virulence and how to control the potential pathogenic strains of Aeromonas in perishable RTE seafood products are needed.

Growth of mesophilic Aeromonas salmonicida in an experimental model of nigiri sushi during cold storage.

The genus Aeromonas includes human pathogenic bacteria frequently isolated from seafood, and the increased consumption of ready-to-eat seafood poses new food safety issues regarding the presence of potentially pathogenic Aeromonas spp. in stored products for raw consumption, such as retail sushi with a shelf life of up to three days. This study assessed 1) the growth kinetics of a mesophilic A. salmonicida strain during storage at 4 °C and 8 °C in a nigiri sushi model, and 2) the strain variability in growth at pH ranging from 3.5 to 10 for a subset of mesophilic Aeromonas strains previously isolated from sushi. Inoculated slices of raw salmon were compared with and without rice. A predictive model for A. hydrophila (ComBase Predictor) did not sufficiently predict growth of the tested strain under the intrinsic conditions of nigiri sushi or salmon at both temperatures. Refrigeration alone (4 °C) did not inhibit growth of A. salmonicida on salmon. Within the first 72 h, representing the typical shelf life of retail sushi products, we observed a 10-fold increase in the concentration of the inoculated strain (including a lag-phase of approximately 34 h). Contact with acidified rice, resulting in a pH drop in the salmon, was the reason for the decreased bacterial viability in the nigiri sushi samples. However, the effect of acidification decreased at 8 °C, resulting in a 2-fold increase in the growth rate and a reduced lag-phase compared to refrigeration. Variability in the ability to grow in different pH levels was observed between strains. The highest color formation rates, representing cellular respiration analyzed in a phenotypic microarray system, were observed between pH 5 and 8. A few strains, including the A. salmonicida strain applied in the nigiri sushi model, were able to grow at pH 4.5 (at optimal temperature). The results demonstrated that mesophilic Aeromonas spp. can represent a microbiological hazard in retail sushi products during cold storage. Rice acidification in combination with low storage temperature (≤4 °C) are prerequisites to prevent growth of potentially pathogenic Aeromonas species during the relatively short shelf life.

Species Distribution and Prevalence of Putative Virulence Factors in Mesophilic Aeromonas spp. Isolated from Fresh Retail Sushi.

Aeromonas spp. are ubiquitous bacteria that have received increasing attention as human pathogens because of their widespread occurrence in food, especially seafood and vegetables. The aim of this work was to assess the species identity and phylogenetic relationship of 118 Aeromonas strains isolated from fresh retail sushi from three producers, and to characterize the isolates with respect to genetic and phenotypic virulence factors. We also evaluate the potential hazard associated with their presence in ready-to-eat seafood not subjected to heat treatment. Mesophilic Aeromonas salmonicida was most prevalent (74%), followed by A. bestiarum (9%), A. dhakensis (5%), A. caviae (5%), A. media (4%), A. hydrophila (2%), and A. piscicola (1%). All isolates were considered potentially pathogenic due to the high prevalence of genes encoding hemolysin (hlyA) (99%), aerolysin (aerA) (98%), cytotoxic enterotoxin (act) (86%), heat-labile cytotonic enterotoxin (alt) (99%), and heat-stable cytotonic enterotoxin (ast) (31%). The shiga-like toxins 1 and 2 (stx-1 and stx-2) were not detected. Moreover, there was heterogeneity in toxin gene distribution among the isolates, and the combination of act/alt/hlyA/aerA was most commonly detected (63%). ß-hemolysis was species-dependent and observed in 91% of the isolates. All A. media and A. caviae strains were non-hemolytic. For isolates belonging to this group, lack of hemolysis was possibly related to the absence of the act gene. Swimming motility, linked to adhesion and host invasion, occurred in 65% of the isolates. Partial sequencing of the gyrB gene demonstrated its suitability as a genetic marker for Aeromonas species identification and for assessment of the phylogenetic relationship between the isolates. The gyrB sequence divergence within a given species ranged from 1.3 to 2.9%. A. bestiarum, A. salmonicida, and A. piscicola were the most closely related species; their sequences differed by 2.7-3.4%. The average gyrB sequence similarity between all species was 93%, demonstrating its acceptable taxonomic resolution. The presence of multiple species of potential pathogenic Aeromonas in fresh retail sushi raises new food safety issues related to the increased consumption of ready-to-eat food composed of raw ingredients.

Accumulation of docosahexaenoic acid-rich lipid in thraustochytrid Aurantiochytrium sp. strain T66: effects of N and P starvation and O2 limitation.

Aurantiochytrium sp. strain T66 was grown in batch bioreactor cultures in a defined glutamate- and glycerol-containing growth medium. Exponentially growing cells had a lipid content of 13% (w/w) of dry weight. A fattening of cells fed excess glycerol occurred in the post-exponential growth phase, after the medium was depleted of N or P. Lipid accumulation was also initiated by O2 limitation (below 1% of saturation). N starvation per se, or in combination with O2 limitation, gave the highest lipid content, i.e., 54% to 63% (w/w) of dry weight. The corresponding maximum culture density was 90 to 100 g/l dry biomass. The content of docosahexaenoic acid (22:6n-3) in N starved, well-oxygenated cells reached 29% (w/w) of total fatty acids but increased to 36% to 52% in O2-limited cells, depending on the time span of the limitation. O2-limited cells did not accumulate the monounsaturated fatty acids that were normally present. We inferred that the biological explanation is that O2 limitation hindered the O2-dependent desaturase(s) and favored the O2-independent polyunsaturated fatty acid synthase. The highest overall volumetric productivity of docosahexaenoic acid observed was 93 mg/l/h. Additionally, we present a protocol for quantitative lipid extraction, involving heat and protease treatment of freeze-dried thraustochytrids.

Endogenously synthesized (-)-proto-quercitol and glycine betaine are principal compatible solutes of Schizochytrium sp. strain S8 (ATCC 20889) and three new isolates of phylogenetically related thraustochytrids.

We report that endogenously synthesized (-)-proto-quercitol (1D-1,3,4/2,5-cyclohexanepentol) and glycine betaine were the principal compatible solutes of Schizochytrium sp. strain S8 (ATCC 20889) and three new osmotolerant isolates of thraustochytrids (strains T65, T66, and T67). The compatible solutes were identified and quantified by use of nuclear magnetic resonance spectroscopy, and their identity was confirmed by mass spectroscopy and measurement of the specific optical rotation. The cellular content of compatible solutes increased with increasing NaCl concentration of a defined medium. (-)-proto-Quercitol was the dominating solute at all NaCl concentrations tested (0.25 to 1.0 M), e.g., cells of S8 and T66 stressed with 1.0 M NaCl accumulated about 500 micromol (-)-proto-quercitol and 100 micromol glycine betaine per g dry weight. To our knowledge, (-)-proto-quercitol has previously been found only in eucalyptus. The 18S rRNA gene sequences of the four (-)-proto-quercitol-producing strains showed 99% identity, and they displayed the same fatty acid profile. The only polyunsaturated fatty acids accumulated were docosahexaenoic acid (78%) and docosapentaenoic acid (22%). A less osmotolerant isolate (strain T29), which was closely phylogenetically related to Thraustochytrium aureum (ATCC 34304), did not contain (-)-proto-quercitol or glycine betaine. Thus, the level of osmotolerance and the osmolyte systems vary among thraustochytrids.