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The effect of polyethylene packaging (PEP) in air cushion and vacuum packaging (VP) on histamine related to the quality of Japanese Spanish mackerel (JS mackerel) was studied with samples stored at -20, 4, 15, and 25°C. The aerobic plate count (APC), total volatile basic nitrogen (TVBN), and histamine concentrations of the PEP and VP samples stored at 25°C increased as the storage time continued. The PEP and VP samples stored at temperatures below 15°C showed lower levels of APC, TVBN, and histamine, with VP samples having considerably lower levels of APC, TVBN, and histamine than PEP samples. For the frozen JS mackerel stored at -20°C for 2 months and then thawed and stored at 25°C, the VP treatment delayed the increases of TVBN and histamine longer than did the PEP treatment. Thus, the storage of VP JS mackerel at temperatures below 4°C could prevent quality deterioration and extend shelf life.

Bubble tea beverages ( n = 105) purchased from vendors in Taiwan were tested to determine their microbiological and chemical quality. Nearly half of the tested samples (48.6%, 51 of 105) had aerobic plate counts (APCs) higher than the Taiwan Food and Drug Administration guideline of 4.0 log CFU/mL, and 55 (52.4%) had coliform counts (most probable number [MPN]) higher than the 10 MPN/mL guideline. Escherichia coli, Salmonella, Staphylococcus aureus, sweeteners, preservatives, maleic acid, and coumarin were not detected in any sample. However, catechins were not detected to 188 mg/mL, and caffeine was 10.1 to 457.6 mg/mL. Bubble tea samples obtained from vendors in southern Taiwan had a mean APC of 2.6 log CFU/mL and a mean coliform count of 61.7 MPN/mL; these values were significantly lower ( P < 0.05) than those from samples collected from vendors in northern, eastern, or central Taiwan. Samples obtained from southern Taiwan had the highest mean catechin concentrations of 21.3 mg/mL ( P < 0.05). About 60% (63 of 105) of the bubble tea samples were not labeled with the origin of the tea leaves, which is in violation of Taiwanese food labeling regulations. In general, the bubble tea beverages tested had satisfactory microbial and chemical qualities.

The molecular mechanism of biofilm formation by Salmonella Typhimuriun DT104 was characterized for a better understanding of its attachment and colonization in food processing environments. A library of random mutagenized clones was screened for phenotypic analyses of their ability to form biofilm, pellicle, curli, and cellulose. The genes identified were involved in lipopolysaccharide synthesis, assembly of flagella, regulation of rRNA biosynthesis, and outer membrane transportation and signaling. The insertion of transposon in flgK, rfbA, nusB, and pnp genes resulted in decreased biofilm formation. Alterations of flagellar and lipopolysaccharide production were confirmed in the flgK mutant and rfbA mutant, respectively. Biofilm formation by these four mutants in meat and poultry broths and their attachment on surfaces of stainless steel and glass were significantly reduced compared with those of the wild-type strain (P < 0.05). On the contrary, the mutation of STM4263 and yjcC genes in Salmonella Typhimuriun DT104 resulted in increased biofilm formation and attachment of the species in tested broths and on contact surfaces. Our findings suggest that many factors, such as production of exopolymeric substances and their efficient transportation through outer membrane, expression of flagella, and regulation of exoribonucleases and RNA-binding protein, could be involved in biofilm formation and attachment of Salmonella Typhimurium DT104 on contact surfaces.

Multistate outbreaks of Escherichia coli O157:H7 infections through consumption of contaminated foods including produce products have brought a great safety concern. The objectives of this study were to determine the effect of biofilm and quorum sensing production on the attachment of E. coli O157:H7 on food contact surfaces and to evaluate the transfer of the pathogen from the food contact to various food products. E. coli O157:H7 produced maximum levels of AI-2 signals in 12 h of incubation in tested meat, poultry, and produce broths and subsequently formed strong biofilm in 24 h of incubation. In general, E. coli O157:H7 formed stronger biofilm on stainless steel than glass. Furthermore, E. coli O157:H7 that had attached on the surface of stainless steel was able to transfer to meat, poultry, ready-to-eat deli, and produce products. Strong attachment of the transferred pathogen on produce products (cantaloupe, lettuce, carrot, and spinach) was detected (>10(3) CFU/cm2) even after washing these products with water. Our findings suggest that biofilm formation by E. coli O157:H7 on food contact surfaces can be a concern for efficient control of the pathogen particularly in produce products that require no heating or cooking prior to consumption.

Interaction of Escherichia coli O157:H7/pGFP with hydroponically grown lettuce plants was evaluated in this study. Lettuce seedlings were planted in contaminated Hoagland's nutrient solution and thereafter subjected to gamma radiation at 0.25, 0.5, and 0.75 kGy, and aqueous chlorine at 200 ppm. There was no trace of E. coli O157:H7/pGFP in lettuce leaves harvested from noncontaminated nutrient solution (control); however, for plants grown in contaminated nutrient solution, the pathogen was recovered from the leaves disinfected with 80% ethanol and 0.1% mercuric chloride. Most of the lettuce seedlings grown in contaminated nutrient solution tested negative for E. coli O157:H7/pGFP under controlled conditions. Gamma radiation at 0.25 and 0.5 kGy, and aqueous chlorine at 200 ppm failed to eliminate E. coli O157:H7/pGFP in lettuce tissue completely; however, the bacteria were not detected in 0.75-kGy treated plants. The presence of E. coli O157:H7/pGFP in lettuce leaves is an indication that the pathogen migrated from the contaminated hydroponic system through the roots to the internal locations of lettuce tissue. Due to inaccessibility and limited penetrating power, aqueous chlorine could not eliminate the bacteria localized in the internal tissue. Findings from this study suggest that gamma irradiation was more efficacious than was aqueous chlorine to control internal contamination in hydroponically grown lettuce. Gamma irradiation is a process that processors can use to inactivate E. coli O157:H7 and therefore, consumers benefit from a safer food product [corrected]

The biofilm-forming capability of Salmonella enterica serotypes Typhimurium and Heidelberg, Pseudomonas aeruginosa, Listeria monocytogenes, Escherichia coli O157:H7, Klebsiella pneumoniae, and Acinetobacter baumannii isolated from humans, animal farms, and retail meat products was evaluated by using a microplate assay. The tested bacterial species showed interstrain variation in their capabilities to form biofilms. Strong biofilm-forming strains of S. enterica serotypes, E. coli O157: H7, P. aeruginosa, K. pneumoniae, and A. baumannii were resistant to at least four of the tested antibiotics. To understand their potential in forming biofilms in food-processing environments, the strong biofilm formers grown in beef, turkey, and lettuce broths were further investigated on stainless steel and glass surfaces. Among the tested strains, Salmonella Typhimurium phage type DT104 (Salmonella Typhimurium DT104) isolated from retail beef formed the strongest biofilm on stainless steel and glass in beef and turkey broths. K. pneumoniae, L. monocytogenes, and P. aeruginosa were also able to form strong biofilms on the tested surface materials. Salmonella Typhimurium DT104 developed a biofilm on stainless steel in beef and turkey broths through (i) initial attachment to the surface, (ii) formation of microcolonies, and (iii) biofilm maturation. These findings indicated that Salmonella Typhimurium DT104 alongwith other bacterial pathogens could be a source of cross-contamination during handling and processing of food.

Multidrug-resistant enteric bacteria were isolated from turkey, cattle, and chicken farms and retail meat products in Oklahoma. Among the isolated species, multidrug-resistant Klebsiella pneumoniae was prevalently isolated from most of the collected samples. Therefore, a total of 132 isolates of K. pneumoniae were characterized to understand their potential roles in the dissemination of antibiotic-resistance genes in the food chains. Multidrug-resistant K. pneumoniae was most frequently recovered from a turkey farm and ground turkey products among the tested samples. All isolates were resistant to ampicillin, tetracycline, streptomycin, gentamycin, and kanamycin. Class 1 integrons located in plasmids were identified as a common carrier of the aadA1 gene, encoding resistance to streptomycin and spectinomycin. Production of beta-lactamase in the K. pneumoniae isolates played a major role in the resistance to beta-lactam agents. Most isolates (96%) possessed bla(SHV1). Five strains were able to express both SHV-11 (pI 6.2) and TEM-1 (pI 5.2) beta-lactamase. Transfer of these antibiotic-resistance genes to Escherichia coli was demonstrated by transconjugation. The bacterial genomic DNA restriction patterns by pulsed-field gel electrophoresis showed that the same clones of multidrug-resistant K. pneumoniae remained in feathers, feed, feces, and drinking water in turkey environments, indicating the possible dissemination of antibiotic-resistance genes in the ecosystem and cross-contamination of antibiotic-resistant bacteria during processing and distribution of products.

An immunoassay system was developed for efficient detection of prohibited meat and bone meal (MBM) in animal feed. Monoclonal antibodies (MAbs) were raised against bovine smooth muscle autoclaved at 130 degrees C for 20 min. Among the 1,500 supernatants of hybridoma cells screened, MAbs 3E1, 1G3, and 3E10 were selected and characterized in this study. The first set of MAbs produced, 3E1 and 1G3, had stronger reactivity against MBM than against smooth muscle that was heat treated at 90 degrees C for 10 min. However, reactivity gradually increased against smooth muscle that was autoclaved at 130 degrees C for up to 1 h. The enzyme-linked immunosorbent assay for detection of MBM in animal feed was optimized with the MAb 3E10 because of its superior performance. MAb 3E10 diluted to 100-fold was used to differentiate bovine MBM from that of other species in ingredients used for commercial animal feeds and could detect down to 0.05% MBM mixed in animal feed.

Enterobacter aerogenes was studied for its growth and ability to promote the formation of total volatile base nitrogen (TVBN) and histamine in sailfish (Istiophorus platypterus) and milkfish (Chanos chanos) stored at various temperatures from -20 to 37 degrees C. The optimal temperature for bacterial growth in both fish species was 25 degrees C, whereas the optimal temperature for histamine formation was 37 degrees C. The two fish species inoculated with E. aerogenes, when not properly stored at low temperatures such as 15 degrees C for 36 h, formed histamine at above the U.S. Food and Drug Administration hazardous guideline level of 50 mg/100 g. Milkfish was a better substrate than sailfish for histamine formation by bacterial histidine decarboxylation at elevated temperatures (> 15 degrees C). Although higher contents of TVBN were detected in the spiked sailfish than milkfish during the same storage time at temperatures above 15 degrees C, the use of the 30-mg/100 g level of TVBN as a determination index for fish quality and decomposition was not a good criterion for assessing potential histamine hazard for both fish species. Bacterial growth was controlled by cold storage of the fish at 4 degrees C or below, but histamine formation was stopped only by frozen storage. Once the frozen fish samples were thawed and stored at 25 degrees C, histamine started to accumulate rapidly and reached levels greater than the hazardous action level in 36 h.

Sixty-four multidrug-resistant isolates of Proteus mirabilis were collected from retail meat products in Oklahoma. The isolates showed four different patterns of antibiotic resistance based on their resistant phenotype and genotypes. Most of these isolates were resistant to ampicillin, tetracycline, gentamycin, and kanamycin. Class 1 integrons were detected as a common carrier of the antibiotic-resistant genes, such as aadA1, aadB, and aadA2. A few isolates (9%) contained class 2 integrons with three gene cassettes included: dhfr1, sat1, and aadA1. These isolates were even resistant to nalidixic acid due to mutations in gyrA and parC. All ampicillin-resistant isolates contained blaTEM-1. Plasmids that contained class 1 or 2 integrons and blaTEM-1 were able to be transferred from P. mirabilis isolates into Escherichia coli by conjugation, indicating that conjugal transfer could contribute to the dissemination of antibiotic resistance genes between the Enterobacteriaceae species.

A polymerase chain reaction (PCR) assay for the rapid and sensitive detection of the most prolific histamine former, Morganella morganii, was developed. 16S rDNA targeted PCR primers were designed, and the primer specificity and sensitivity of the PCR assay were evaluated. The 16S rDNA sequence (1,503 bp) for M. morganii showed 95% identity to those for enteric bacteria, i.e., Enterobacter spp., Klebsiella spp., Citrobacter spp., Hafnia alvei, Proteus spp., and Providencia spp. The unique primers for M. morganii were designed on the basis of the variable regions in the 16S rDNA sequence. The primers showed positive reactions with all M. morganii strains tested. However, PCR amplification was not detected when the primers were tested with other enteric or marine bacteria. When the sensitivity of the assay was evaluated, M. morganii was detected at levels ranging from 10(6) to 10(8) CFU/ml in albacore homogenate after the PCR amplification. The sensitivity of the assay was greatly improved with the enrichment of samples, and 9 CFU of M. morganii per ml of albacore homogenate was detected after 6 h of enrichment at 37 degrees C.

Salmonella contamination of precut watermelon, tomatoes, and cantaloupes was responsible for several outbreaks of salmonellosis. To better understand the relationship between bacterial doses and their transfer onto cut surfaces by using a knife, rifampicin-resistant Salmonella montevideo at 7, 70, 700, or 7,000 CFU in Butterfield's buffer (BPB) or tryptic soy broth (TSB) was added to the stem scars of tomatoes. Tomatoes were cut from the stem scar to blossom end using a sterilized knife. After stem scars were aseptically removed, cut surfaces were placed on tryptic soy agar-rifampicin (TSA-RIF) plates or processed by a broth enrichment method to determine if S. montevideo had been transferred to the cut surface. S. montevideo was recovered in a dose-related fashion using both methods. A higher recovery rate was obtained with bacterial inocula in TSB than in BPB, and also with broth enrichment rather than the direct plating method. The distribution of the transferred S. montevideo on the cut surface of contaminated and noncontaminated tomatoes with a knife was related to the inoculum dose added to the stem scars. S. montevideo colonies were found to cluster at the stem scar region with the lower inoculum dose. However, when a higher inoculum dose was used, the colonies spread from the stem scar region to the center and bottom of cut tomatoes, or they were transferred to another uninoculated tomato by the contaminated knife. Therefore, the safety operation criteria recommended by FDA to wash fruits before cutting, to use clean and sanitized utensils and surfaces when preparing cut fruits, and to store the cut fruits below 7°C should be followed in preparing tomato slices to minimize salmonellosis outbreaks caused by this product.

Alternaria alternata RL671-2 was cultivated in synthetic, semisynthetic or a rice culture medium to study its growth, and production of the mycotoxins alternariol (AOH), alternariol methyl ether (AME), and alteneune (ALT). Toxins were produced during the later stage of growth in both liquid media. Greater toxin production was found in semisynthetic (560 μg/100 ml of medium) than in synthetic medium (135 μg/100 ml). The pH decreased from 4.0 to 2.1 during the cultivation period in the synthetic medium, while it increased from 5.1 to 6.8 in the semisynthetic medium. Reduction of carbon source (glucose) levels in the synthetic medium to change the C/N ratios from 12:1 to 6:1 or 3:1, greatly increased both production of toxins and fungal mycelial weights. At a C/N ratio of 6:1, the fungus produced toxins at a level close to that produced in the semisynthetic medium. The rice culture was more efficient for production of large quantities of toxin.

Enrichment techniques using five broths (gram-negative broth, alkaline peptone water, tetrathionate broth without iodine and two Plesiomonas broths) were compared to direct plating methods using freshwater samples to determine their ability to increase the isolation rate of Plesiomonas shigelloides , a suspected food and waterbome pathogen. Tetrathionate broth consistently gave significantly (p<0.05) greater recovery of P. shigelloides than the other four broths tested as well as by direct plating. Incubation of the enrichment broths at 40°C also resulted in significantly higher recovery of Plesiomonas than at 35°C. It is therefore suggested that for routine monitoring of P. shigelloides , tetrathionate broth incubated at 40°C be used for enrichment before plating.

The volatile ketone (β-ionone showed a dose-related inhibition of fungal growth and aflatoxin production on peanuts after they were soaked in distilled water for 25 or 50 min, inoculated with spores, and incubated at 28°C for up to 2 weeks. For example, aflatoxin B 1 (AFB 1 ) production after 1 week of incubation was reduced to less than 11.0 and 6.7% of the control when 2.5 or 5 ml of (β-ionone/100 g of peanuts, respectively, was added to water-soaked (25 min) peanuts. For AFG 1 , production was reduced to 4.7 (2.5 ml) or 3.3% (5.0 ml) under the same treatment conditions. Unlike controls or those treated with less than 0.1 ml of β-ionone, peanuts treated with more than 0.25 ml of β-ionone had only sparse mycelial growth and supported only limited sporulation. The mycelia, after being transferred to fresh Mycological or Fluorescent Agar plates, still had the ability to form normal colonies and produce aflatoxins. This temporary limitation of fungal growth was also noticed for those Aspergillus cultures on Mycological Agar that had been treated with (β-ionone either by direct contact or volatile bioassay procedures. The fungus was still able to grow of Fluorescent Agar even after the infected peanuts were treated with sodium hypochlorite for 15 or 30 min, indicating that mycelial penetration into peanut tissues occurs. This may confer protection from the action of various antifungal compounds. This observtion is further supported by microscopic detection of mycelial fragments in peanut tissues.

Effects of culture media, near-ultraviolet exposure time, and temperature on sporulation of Alternaria alternata were investigated. Strains RL 671-2 and ATCC 36068 were cultivated on Potato Dextrose Agar (PDA), V8 Juice Agar (V8 Agar) and Mycological Agar (MA). The best culture medium for sporulation of strain RL 671-2 was PDA, followed by V8 agar, with only negligible numbers of spores appearing on MA. Near-UV exposure significantly increased sporulation in strain RL 671-2 on PDA and V8 agar. Significantly higher (P<0.01) spore counts were found in PDA cultures of this strain exposed to near-UV at 35 than at 20°C. On V8 agar significantly more spores were observed at 20 than at 35°C. MA was not a satisfactory medium for sporulation of ATCC 36068. Both PDA and V8 agar equally supported sporulation for this strain (ATCC 36068) at all exposure times.

In a study to compare five enrichment broths for enhancing the recovery of Plesiomonas shigelloides from water samples, tetrathionate broth without the addition of iodine exhibited better (P<0.05) recovery of this microorganism than did alkaline peptone water, gram negative broth, plesiomonas enrichment broth (PLE), a modified PLE, or direct plating. The established enrichment parameters were applied to test raw packaged and freshly shucked oyster meats, as well as bacterial mixtures. The enrichment process enhanced the detection of P. shigelloides in a Plesiomonas-Klebsiella mixture but not in a Plesiomonas-Pseudomonas mixture. Recovery of P. shigelloides in oyster samples was found to be affected by both the number and type of competing bacteria present as well as incubation temperature used for enrichment.