Salmonella is an important foodborne pathogen, whose ability to resist stress and survive can vary among strains. This variability is normally not taken into account when predictions are made about survival in foods with negative consequences. Therefore, we examined the contribution of variable phenotypic properties to survival under stress in 10 Salmonella serovars. One strain (Typhimurium 10) was intentionally RpoS-negative; however, another strain (Heidelberg) showed an rpoS mutation, rendering it inactive. We assessed an array of characteristics (motility, biofilm formation, bile resistance, acid resistance, and colony morphology) that show major variability among strains associated with a 10- to 19-fold difference between the highest and the lowest strain for most characteristics. The RpoS status of isolates did not affect variability in the characteristics, with the exception of resistance to NaCl, acetic acid, lactic acid, and the combination of acetic acid and salt, where the variability between the highest and the lowest strain was reduced to 3.1-fold, 1.7-fold, 2-fold, and 1.7-fold, respectively, showing that variability was significant among RpoS-positive strains. Furthermore, we also found a good correlation between acid resistance and lysine decarboxylase activity, showing its importance for acid resistance, and demonstrated a possible role of RpoS in the lysine decarboxylase activity in Salmonella.
Oxalic acid was evaluated as a treatment for reducing populations of naturally occurring microorganisms on raw chicken. Raw chicken breasts were dipped in solutions of oxalic acid (0, 0.5, 1.0, 1.5, and 2.0%, wt/vol) for 10, 20, and 30 min, individually packed in oxygen-permeable polyethylene bags, and stored at 4 degrees C. Total plate counts of aerobic bacteria and populations of Pseudomonas spp. and Enterobacteriaceae on breasts were determined before treatment and after storage for 1, 3, 7, 10, and 14 days. The pH and Hunter L, a, and b values of the breast surface were measured. Total plate counts were ca. 1.5 and 4.0 log CFU/g higher on untreated chicken breasts after storage for 7 and 14 days, respectively, than on breasts treated with 0.5% oxalic acid, regardless of dip time. Differences in counts on chicken breasts treated with water and 1.0 to 2.0% of oxalic acid were greater. Populations of Pseudomonas spp. on chicken breasts treated with 0.5 to 2.0% oxalic acid and stored at 4 degrees C for 1 day were less than 2 log CFU/g (detection limit), compared with 5.14 log CFU/g on untreated breasts. Pseudomonas grew on chicken breasts treated with 0.5% oxalic acid to reach counts not exceeding 3.88 log CFU/g after storage for 14 days. Counts on untreated chicken exceeded 8.83 log CFU/g at 14 days. Treatment with oxalic acid caused similar reductions in Enterobacteriaceae counts. Kocuria rhizophila was the predominant bacterium isolated from treated chicken. Other common bacteria included Escherichia coli and Empedobacter brevis. Treatment with oxalic acid caused a slight darkening in color (decreased Hunter L value), retention of redness (increased Hunter a value), and increase in yellowness (increased Hunter b value). Oxalic acid has potential for use as a sanitizer to reduce populations of spoilage microorganisms naturally occurring on raw chicken, thereby extending chicken shelf life.
Experiments were performed to determine the thermal resistance of hepatitis A virus (HAV) in three types of dairy products containing increased amounts of fat content (skim milk, homogenized milk; 3.5% MFG, and table cream; 18% MFG). HAV-inoculated dairy products were introduced into custom-made U-shaped microcapillary tubes that in turn were simultaneously immersed in a waterbath, using custom-made floating boats and a carrying platform. Following exposure to the desired time and temperature combinations, the contents of each of the tubes was retrieved and was tested by plaque assay to determine the reduction in virus titer. Our data indicated that < 0.5 min at 85 degrees C was sufficient to cause a 5-log reduction in HAV titer in all three dairy products, whereas at 80 degrees C, < or = 0.68 min (for skim and homogenized milk), and 1.24 min (for cream) were needed to cause a similar log reduction. Using a nonlinear two-phase negative exponential model (two-compartment model) to analyze the data, it was found that at temperatures of 65, 67, 69, 71, and 75 degrees C, significantly (P < 0.05) higher exposure times were needed to achieve a 1-log reduction in virus titer in cream, as compared to skim and homogenized milk. For example, at 71 degrees C, a significantly (P < 0.05) higher exposure time of 0.52 min (for cream) was needed as compared to < or = 0.18 min (for skim and homogenized milk) to achieve a 1-log reduction in virus titer. A similar trend of inactivation was observed at 73 and 75 degrees C where significantly (P < 0.05) higher exposure times of 0.29 to 0.36 min for cream were needed to cause a 1-log reduction in HAV in cream, as compared to < or = 0.17 min for skim and homogenized milk. This study has provided information on the heat resistance of HAV in skim milk, homogenized milk, and table cream and demonstrated that an increase in fat content appears to play a protective role and contributes to the heat stability of HAV.
While there is good epidemiological evidence for foods as vehicles for norovirus transmission, the precise means of spread and its control remain unknown. The feline calicivirus was used as a surrogate for noroviruses to study infectious virus transfer between hands and selected types of foods and environmental surfaces. Assessment of the potential of selected topicals in interrupting such virus transfer was also made. Ten microliters of inoculum of feline calicivirus deposited onto each fingerpad of adult subjects was allowed to air dry and the contaminated area on individual fingerpads was pressed (10 s at a pressure of 0.2 to 0.4 kg/cm2) onto 1-cm-diameter disks of ham, lettuce, or brushed stainless steel. The virus remaining on the donor and that transferred to the recipient surfaces was eluted and plaque assayed. Virus transfer to clean hands from experimentally contaminated disks of ham, lettuce, and stainless steel was also tested. Nearly 46 +/- 20.3, 18 +/- 5.7, and 13 +/- 3.6% of infectious virus was transferred from contaminated fingerpads to ham, lettuce, and metal disks, respectively. In contrast, approximately 6 +/- 1.8, 14 +/- 3.5, and 7 +/- 1.9% virus transfer occurred, respectively, from ham, lettuce, and metal disks to hands. One-way analysis of variance test showed that pretreatment (washing) of the fingerpads either with water or with both topical agent and water significantly (P < 0.05) reduced virus transfer to < or = 0.9%, as compared with < or = 2.3 and < or = 3.4% transfer following treatments with either 75% (vol/vol) ethanol or a commercial hand gel containing 62% ethanol, respectively. Despite wide variations in virus transfer among the targeted items used, intervention agents tested reduced virus transfer significantly (P < 0.05) when compared with that without such treatments (71 +/- 8.9%). These findings should help in a better assessment of the potential for cross-contamination of foods during handling and also assist in developing more effective approaches to foodborne spread of norovirus infections.
A total of 43 Salmonella enterica isolates belonging to different serovars (Salmonella Albany, Salmonella Agona, Salmonella Corvallis, Salmonella Stanley, Salmonella Typhimurium, Salmonella Mikawasima, and Salmonella Bovismorbificans) were isolated from catfish (Clarias gariepinus) and tilapia (Tilapia mossambica) obtained from nine wet markets and eight ponds in Penang, Malaysia. Thirteen, 19, and 11 isolates were isolated from 9 of 32 catfish, 14 of 32 tilapia, and 11 of 44 water samples, respectively. Fish reared in ponds were fed chicken offal, spoiled eggs, and commercial fish feed. The genetic relatedness of these Salmonella isolates was determined by random amplified polymorphic DNA PCR (RAPD-PCR) using primer OPC2, repetitive extragenic palindromic PCR (REP-PCR), and pulsed-field gel electrophoresis (PFGE). Composite analysis of the RAPD-PCR, REP-PCR, and PFGE results showed that the Salmonella serovars could be differentiated into six clusters and 15 singletons. RAPD-PCR differentiated the Salmonella isolates into 11 clusters and 10 singletons, while REP-PCR differentiated them into 4 clusters and 1 singleton. PFGE differentiated the Salmonella isolates into seven clusters and seven singletons. The close genetic relationship of Salmonella isolates from catfish or tilapia obtained from different ponds, irrespective of the type of feed given, may be caused by several factors, such as the quality of the water, density of fish, and size of ponds.
Campylobacter jejuni was found to occur at high prevalence in the raw salad vegetables examined. Previous reports describe cross-contamination involving meat; here we investigated the occurrence of cross-contamination and decontamination events in the domestic kitchen via C. jejuni-contaminated vegetables during salad preparation. This is the first report concerning quantitative cross-contamination and decontamination involving naturally contaminated produce. The study was designed to simulate the real preparation of salad in a household kitchen, starting with washing the vegetables in tap water, then cutting the vegetables on a cutting board, followed by slicing cucumber and blanching (heating in hot water) the vegetables in 85 degrees C water. Vegetables naturally contaminated with C. jejuni were used throughout the simulation to attain realistic quantitative data. The mean of the percent transfer rates for C. jejuni from vegetable to wash water was 30.1 to 38.2%; from wash water to cucumber, it was 26.3 to 47.2%; from vegetables to cutting board, it was 1.6 to 10.3%; and from cutting board to cucumber, it was 22.6 to 73.3%. The data suggest the wash water and plastic cutting board as potential risk factors in C. jejuni transmission to consumers. Washing of the vegetables with tap water caused a 0.4-log reduction of C. jejuni attached to the vegetables (most probable number/gram), while rapid blanching reduced the number of C. jejuni organisms to an undetectable level.
The antioxidant and microbial stabilities of galangal (Alpinia galanga) extract in raw minced beef were examined at 4 +/- 1 degree C. Raw minced beef containing galangal extracts (0 to 0.10%, wt/wt) were prepared. Lipid oxidation during refrigerated storage was assessed by monitoring malonaldehyde formation, using the thiobarbituric acid reactive substances method. In minced beef, added galangal extract improved oxidative stability. Galangal extract at higher concentrations of 0.05% and 0.10% (wt/wt) were also found to extend the shelf-life of minced beef. Addition of alpha-tocopherol (0.02%, wt/wt) to galangal extract (0.05%, wt/wt) were observed to increase the oxidative but not the microbial stability of minced beef during the storage of 7 days. Galangal extract may prove useful in inhibiting lipid oxidation and increasing microbial stability of minced meat.
Listeria spp. are ubiquitous in nature and can be found in various environmental niches such as soil, sewage, river water, plants, and foods, but the most frequently isolated species are Listeria monocytogenes and Listeria innocua. In this study, the presence of Listeria spp. in raw chicken meat and chicken-related products sold in local markets in Klang Valley, Malaysia was investigated. A total of 44 Listeria strains (42 L. innocua and 2 L. welshimeri) were isolated from 106 samples. Antibiotic susceptibility tests of the L. innocua strains revealed a high prevalence of resistance to clindamycin (92.9%), ceftriaxone (76.2%), ampicillin (73.8%), tetracycline (69%), and penicillin G (66.7%). Overall, 31 L. innocua and 1 L. welshimeri strain were multidrug resistant, i.e., nonsusceptible to at least one antimicrobial agent in three or more antibiotic classes. The majority of the L. innocua strains were placed into five AscI pulsogroups, and overall 26 distinct AscI pulsotypes were identified. The detection of multidrug-resistant Listeria strains from different food sources and locations warrants attention because these strains could serve as reservoirs for antimicrobial resistance genes and may facilitate the spread and emergence of other drug-resistant strains.
ABSTRACT: Environmental hygiene monitoring in the food processing environment has become important in current food safety programs to ensure safe food production. However, conventional monitoring of surface hygiene based on visual inspection and microbial counts is slow, tedious, and thus unable to support the current risk-based management system. Therefore, this study was conducted to assess the performance of a real-time total adenylate assay that detected ATP+ADP+AMP (A3) for food contact surface hygiene in 13 food processing plants and two commercial kitchens in Malaysia. The A3 value was compared with the microbial count (aerobic plate count [APC]) on food contact surfaces. Receiver-operating characteristic (ROC) analysis was performed to assess the reliability of the data and to determine the optimal threshold value for hygiene indication of food contact surfaces. Overall, the A3 value demonstrated a weak positive relationship with APC. However, the A3 value significantly correlated with APC for food processing environments associated with raw meat and raw food ingredients such as fruit that harbor a high microbial load. ROC analysis suggested an optimal threshold for the A3 value of 500 relative light units to balance the sensitivity and specificity at 0.728 and 0.719, respectively. The A3 assay as a hygiene indicator for food contact surfaces had an efficiency of 72.1%, indicating its reliability as a general hygiene indicator.
Seafood samples obtained in seafood markets and supermarkets at 11 sites selected from four states in Malaysia were examined for the presence of nine potentially pathogenic species from the genus Vibrio between July 1998 and June 1999. We examined 768 sample sets that included shrimp, squid, crab, cockles, and mussels. We extensively examined shrimp samples from Selangor State to determine seasonal variation of Vibrio populations. Eight potentially pathogenic Vibrio species were detected, with overall incidence in the samples at 4.6% for V. cholerae, 4.7% for V. parahaemolyticus, 6.0% for V. vulnificus, 11% for V. alginolyticus, 9.9% for V. metschnikovii, 1.3% for V. mimicus, 13% for V. damsela, 7.6% for V. fluvialis, and 52% for a combined population of all of the above. As many as eight Vibrio species were detected in shrimp and only four in squid and peel mussels. The overall percent incidence of any of the eight vibrios was highest (82%) in cockles (Anadara granosa) among the seafoods examined and was highest (100%) in Kuching, Sarawak State, and lowest (25%) in Penang, Pulau Penang State, among the sampling sites. Of 97 strains of V. cholerae isolated, one strain belonged to the O1 serotype and 14 to the O139 serotype. The results indicate that the various seafood markets in Malaysia are contaminated with potentially pathogenic Vibrio species regardless of the season and suggest that there is a need for adequate consumer protection measures.
To determine the occurrence of Salmonella and Shigella in infant formula from Southeast Asia, 74 packages of dehydrated powdered infant follow-on formula (recommended age, > 4 months) from five different manufacturers, four from Indonesia and one from Malaysia, were analyzed. None of the 25-g test portions yielded Salmonella or Shigella. However, further identification of colonies growing on selective media used for Salmonella and Shigella detection revealed the frequent occurrence of several other Enterobacteriaceae species. A total of 35 samples (47%) were positive for Enterobacteriaceae. Ten samples (13.5%) from two Indonesian manufacturers yielded Enterobacter sakazakii. Other Enterobacteriaceae isolated included Pantoea spp. (n = 12), Escherichia hermanii (n = 10), Enterobacter cloacae (n = 8), Klebsiella pneumoniae subsp. pneumoniae (n = 3), Citrobacter spp. (n = 2), Serratia spp. (n = 2), and Escherichia coli (n = 2). To our knowledge, this is the first report to describe the contamination of dehydrated powdered infant formula from Indonesia with E. sakazakii and several other Enterobacteriaceae that could be opportunistic pathogens. Improper preparation and conservation of these products could result in a health risk for infants in Indonesia.
Twenty-two strains of vancomycin-resistant Enterococcus faecalis were isolated from 9 (6%) of 150 samples of frozen beef and beef products imported to Malaysia. The isolates were obtained from eight samples of beef and one sample of minced beef patty. No E. faecalis was isolated from frankfurters. Twelve of the 22 isolates (54.5%) were beta-hemolytic, and all isolates harbored the vanA gene. All vancomycin-resistant isolates were also resistant to streptomycin, erythromycin, kanamycin, bacitracin, ceftazimide, gentamycin, tetracycline, nalidixic acid, and teicoplanin; 95.4% were resistant to trimethoprimsulfamethoxazole; 68.8% were resistant to chloramphenicol; and 41% were resistant to ampicillin and penicillin. Small plasmids ranging in size from 1.5 to 5.8 kb were detected in 8 (36.4%) of 22 strains. The 22 isolates were classified into 20 random amplified polymorphic DNA types. Isolates were divided into two groups, each containing subclusters, that may reflect their clonal lineages. It is concluded that several clones of vancomycin-resistant E. faecalis are represented in the isolates obtained from beef imported to Malaysia.
A 2-year nationwide survey was conducted by the Food and Drug Administration to provide current information about filth, decomposition, and microbiological contamination of domestic and imported fresh and frozen shrimp. Whole or equivalent filth insects, mostly ants, were found in 14.4% of the samples. Of countries contributing at least 10 samples for filth analysis, India had the highest percentage positive for filth insects (45.5%); the United States had the lowest (6.3%). Filth insect fragments were present in 5.4% of the samples. Incidental insects were present in 6.3% of the samples, with flies the most commonly found. Of countries contributing at least 10 samples for filth analysis, India had the highest percentage positive for incidental insects (27.3%); Ecuador had the lowest (2.3%). Unidentified insect fragments were found in 33.3% of the samples; cockroach excreta pellets were present in 2.1%, rat or mouse hairs in 5.7%, and other striated animal hairs in 15.3%. Of countries contributing at least 10 samples for filth analysis, the Philippines had the highest percentages positive (30.0 and 50.0%) for rat or mouse hairs and other striated hairs, respectively; Malaysia was close behind with 23.1 and 46.2%. Samples from the United States had the lowest percentage positive for rat or mouse hairs (0.9%) and other striated hairs (7.2%). Listeria spp. were found in 6.8% of the samples; Salmonella spp. were found in 8.1%. Some level of decomposition was observed in 15.4% of the samples tested organoleptically.
High-quality fish oil for human consumption requires low levels of toxic elements. The aim of this study was to compare different oil extraction methods to identify the most efficient method for extracting fish oil of high quality with the least contamination. The methods used in this study were Soxhlet extraction, enzymatic extraction, wet reduction, and supercritical fluid extraction. The results showed that toxic elements in fish oil could be reduced using supercritical CO2 at a modest temperature (60°C) and pressure (35 MPa) with little reduction in the oil yield. There were significant reductions in mercury (85 to 100%), cadmium (97 to 100%), and lead (100%) content of the fish oil extracted using the supercritical fluid extraction method. The fish oil extracted using conventional methods contained toxic elements at levels much higher than the accepted limits of 0.1 μg/g.
This study examined whether the survival of Vibrio cholerae O1 on contaminated cooked rice was influenced by the type of rice. Vibrios survived unchanged on clumps of glutinous white rice (wet, grains adhered) held at room temperature for 24 h. On nonglutinous white rice (slightly moist, grains separate), 30% viable vibrios remained at 24 h. On nonglutinous brown rice (moist, separate, covered with a mucus-like substance), the number of vibrios increased 2.7-fold at 24 h. Survival rates of vibrios on the surfaces of a row of five cooked rice grains after 2 h of exposure at room temperature were 86, 29, 12, and 4% for glutinous rice, white rice, and the endosperm and pericarp of brown rice, respectively. (Each boiled brown rice grain surface was partly pericarp and partly endosperm, which became exposed by a rupture of the pericarp.) Covering each inoculated grain with a similar cooked rice grain surface increased the corresponding figures to 93, 99, 60, and 94%. Scanning electron microscopy revealed that each type of cooked grain surface possessed a distinct microtopography. For example, the surfaces of glutinous rice grains consisted of separated overlapping strips with many holes, while the pericarps of brown rice were flat interspersed with small pits. In conclusion, each type of boiled rice produced a distinct survival pattern of V. cholerae O1 caused by both the distinct gross features and the fine surface characteristics of the rice. The significance of this finding is that the type of rice consumed can be a factor in cholera transmission by contaminated rice.
The use of simple crude water extracts of common herbs to reduce bacterial attachment may be a cost-effective way to control bacterial foodborne pathogens, particularly in developing countries. The ability of water extracts of three common Malaysian herbs (Andrographis paniculata, Eurycoma longifolia, and Garcinia atroviridis) to modulate hydrophobicity and attachment to surfaces of five food-related bacterial strains (Bacillus cereus ATCC 14576, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 10145, Salmonella Enteritidis ATCC 13076, Staphylococcus aureus ATCC 25923) were determined. The bacterial attachment to hydrocarbon assay was used to determine bacterial hydrophobicity. Staining and direct microscopic counts were used to determine attachment of bacteria to glass and stainless steel. Plating on selective media was used to determine attachment of bacteria to shrimp. All extracts were capable of either significantly ( P < 0.05) increasing or decreasing bacterial surface hydrophobicity, depending on the herb extract and bacteria combination. Bacterial attachment to all surfaces was either significantly (P < 0.05) increased or decreased, depending on the herb extract and bacteria combination. Overall, hydrophobicity did not show a significant correlation (P > 0.05) to bacterial attachment. For specific combinations of bacteria, surface material, and plant extract, significant correlations (R > 0.80) between hydrophobicity and attachment were observed. The highest of these was observed for S. aureus attachment to stainless steel and glass after treatment with the E. longifolia extract (R = 0.99, P < 0.01). The crude water herb extracts in this study were shown to have the potential to modulate specific bacterial and surface interactions and may, with further work, be useful for the simple and practical control of foodborne pathogens.
Two hundred ten samples of selected vegetables (okra, pumpkin, tomato, potato, eggplant, spinach, and cabbage) from Faisalabad, Pakistan, were analyzed for the analysis of heavy metals: cadmium (Cd), lead (Pb), arsenic (As), and mercury (Hg). Inductively coupled plasma optical emission spectrometry was used for the analysis of heavy metals. The mean levels of Cd, Pb, As, and Hg were 0.24, 2.23, 0.58, and 7.98 mg/kg, respectively. The samples with Cd (27%), Pb (50%), and Hg (63%) exceeded the maximum residual levels set by the European Commission. The mean levels of heavy metals found in the current study are high and may pose significant health concerns for consumers. Furthermore, considerable attention should be paid to implement comprehensive monitoring and regulations.
ABSTRACT: A total of 133 samples of whole wheat and barley grains and wheat and barley flour collected from retail markets in the main cities of Punjab, Pakistan, were analyzed for the mycotoxin fumonisin B1 (FB1) using reverse phase high-performance liquid chromatography with fluorescence detection. Of these samples, 120 (90%) were positive for FB1, and 75 (63%) of the 120 positive samples had FB1 concentrations higher than the European Union maximum (200 μg/kg). The limit of detection was 4 μg/kg. The highest mean (±SD) concentration of FB1 was found in whole wheat samples, 980.5 ± 211.4 μg/kg. The calculated dietary intakes of FB1 from wheat and barley flours were 4,456 and 503.7 ng/g of body weight per day, respectively.
This work presents current information on the presence of aflatoxins (AFs) and zearalenone (ZEN) in feed and feed ingredients from Punjab, Pakistan. The 105 samples tested were concentrated feed, i.e., cotton seed meal (18 samples) and soybean meal (14), and feed ingredients, i.e., crushed corn (17), crushed wheat (15), barley (17). and poultry feed (24). Samples were analyzed using high-performance liquid chromatography equipped with a fluorescence detector. Analysis revealed that 69 of 105 samples were contaminated with AFs, and the highest mean concentrations of AFB1 (6.20 μg/kg) and total AFs (9.30 μg/kg) were found in poultry feed samples. The mean total AF concentrations ranged from the limit of quantification to 165.5 μg/kg. However, 75 of the 105 samples were positive for ZEN. The highest mean concentration (19.45 μg/kg) was found in poultry feed samples. The mean ZEN concentrations were 0.15 to 145.30 μg/kg. The prevalence of AFs and ZEN was high in feed and feed ingredients and needs urgent attention.
The present study was conducted to investigate the prevalence and antibiotic resistance among Campylobacter jejuni in ulam at farms and retail outlets located in Kuala Terengganu, Malaysia. A total of 526 samples (ulam, soil, and fertilizer) were investigated for the presence of C. jejuni and the gene for cytolethal distending toxin (cdt) by using a multiplex PCR method. Antibiotic susceptibility to 10 types of antibiotics was determined using the disk diffusion method for 33 C. jejuni isolates. The average prevalence of contaminated samples from farms, wet markets, and supermarkets was 35.29, 52.66, and 69.88%, respectively. The cdt gene was not detected in 24 of the 33 C. jejuni isolates, but 9 isolates harbored cdtC. Antibiotic resistance in C. jejuni isolates was highest to penicillin G (96.97% of isolates) followed by vancomycin (87.88%), ampicillin (75.76%), erythromycin (60.61%), tetracycline (9.09%), amikacin (6.06%), and norfloxacin (3.03%); none of the isolates were resistant to ciprofloxacin, enrofloxacin, and gentamicin. In this study, C. jejuni was present in ulam, and some isolates were highly resistant to some antibiotics but not to quinolones. Thus, appropriate attention and measures are required to prevent C. jejuni contamination on farms and at retail outlets.