Detection of Escherichia albertii in retail oysters

2021 ◽  
Author(s):  
Sakura Arai ◽  
Satoko Yamaya ◽  
Kayoko Ohtsuka ◽  
Noriko Konishi ◽  
Hiromi Obata ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Pacific Oyster ◽  
Genetic Characterization ◽  
Foodborne Pathogen ◽  
Most Probable Number ◽  
Macconkey Agar ◽  
Pcr Assay ◽  
Probable Number ◽  
Pacific Oysters ◽  
Rock Oyster

Escherichia albertii  is an emerging foodborne pathogen. Owing to its distribution in river water,  it is important to determine the presence of  E. albertii  in aquaculture-related foods. In this study, we investigated the distribution of  E. albertii  in retail oyster samples.  A total of  427 raw oyster samples (385 Pacific oysters, and 42 Japanese rock oysters) were enriched in  modified Escherichia coli  broth (mEC) or mEC supplemented with novobiocin (NmEC) at 42 °C. The cultures were used for  E. albertii -specific nested PCR assay, as well as for  E. albertii  isolation using  deoxycholate hydrogen sulfide lactose agar  (DHL), DHL supplemented with rhamnose and xylose (RX-DHL), and MacConkey agar supplemented with rhamnose and xylose (RX-MAC). The population of  E. albertii  in nested PCR-positive samples was  determined using the  most probable number  (MPN) method.  E. albertii  isolates were subjected to biochemical and genetic characterization.  E. albertii   was detected in 5 of 315 (1.6%) Pacific oyster samples  (one piece each), 2 of 70 (2.9 %)  Pacific oyster samples  (25 g each), and 2 of 42 (4.8 %) Japanese rock oyster samples  procured from four geographically distant regions. A total of 64  E. albertii  strains were isolated from eight of the nine nested PCR assay-positive oyster samples, and  the MPN value was under the detection limit (< 3 MPN/10 g).  A specific season or month for detecting  E. albertii  was not observed in this study, suggesting that the pathogen is present in seawater.   All the  E. albertii  isolates, except one, were positive for the virulence factor  eae,  indicating that these isolates have  the potential to infect humans.

Evaluating methods for detecting Escherichia albertii in chicken meat

2020 ◽  
Author(s):  
Sakura Arai ◽  
Kayoko Ohtsuka ◽  
Noriko Konishi ◽  
Kenji Ohya ◽  
Takayuki Konno ◽  
...  
Keyword(s):  
Nested Pcr ◽  
Enrichment Culture ◽  
Foodborne Pathogen ◽  
Chicken Meat ◽  
Most Probable Number ◽  
Broad Host Range ◽  
Probable Number ◽  
Infectious Dose ◽  
Source Of Infection ◽  
Evaluating Methods

Escherichia albertii is an emerging foodborne pathogen. The source of infection in most foodborne outbreaks is unknown, as it is difficult to isolate E. albertii from suspected foods or water. E. albertii has a broad host range among birds and can be isolated from chicken meat. In this study, PCR assay, enrichment and isolation conditions for detecting E. albertii in chicken meat were evaluated. The growth of 48 E. albertii strains isolated in Japan between 1994 and 2018 was evaluated in modified EC broth (mEC) and mEC supplemented with novobiocin (NmEC) and on different media containing carbohydrates. In addition, the enzyme for nested PCR, the enrichment condition, the most probable number (MPN) method, and agar media were also evaluated for chicken meat. To distinguish E. albertii from presumptive non-E. albertii bacteria, desoxycholate hydrogen sulfide lactose agar (DHL), MacConkey agar (MAC), and these agars supplemented with rhamnose and xylose (RX-DHL and RX-MAC, respectively) were used. All E. albertii strains grew in mEC and NmEC at both 36°C and 42°C and did not utilize rhamnose, sucrose, or xylose. Both the first PCR and nested PCR, using TaKaRa Ex Taq which was 10–100 times superior to the other enzymes, showed positive results in enrichment culture of 25 g of chicken meat inoculated with >20 CFU of E. albertii in mEC and NmEC at 42°C for 22 ± 2 h. Thus, the first PCR was sensitive enough to detect E. albertii in chicken meat. The MPN values in mEC and NmEC were 0.5- and 2.3-fold of inoculated bacterial concentration, respectively. E. albertii in chicken meat was more efficiently isolated with enrichment in NmEC (70.1-100%) and plating onto RX-DHL (85.4%) and RX-MAC (100%) compared to enrichment in mEC (53.5-83.3%) and plating onto DHL (70.1%) and MAC (92.4%), respectively. Thus, optimized conditions for the surveillance of E. albertii contamination in food and investigations of E. albertii outbreaks, including the infectious dose, were clarified.

Refrigerated Seawater Depuration for Reducing Vibrio parahaemolyticus Contamination in Pacific Oyster (Crassostrea gigas)

2010 ◽  
Vol 73 (6) ◽  
pp. 1111-1115 ◽  
Author(s):  
YI-CHENG SU ◽  
QIANRU YANG ◽  
CLAUDIA HÄSE
Keyword(s):  
Crassostrea Gigas ◽  
Vibrio Parahaemolyticus ◽  
Pacific Oyster ◽  
Most Probable Number ◽  
Probable Number ◽  
Log Reduction ◽  
Pacific Oysters ◽  
The Pacific ◽  
Postharvest Treatment ◽  
Depuration Process

The efficacy of refrigerated-seawater depuration for reducing Vibrio parahaemolyticus levels in Pacific oyster (Crassostrea gigas) was investigated. Raw Pacific oysters were inoculated with a mixed culture of five clinical strains of V. parahaemolyticus (105 to 106 most probable number [MPN] per g) and depurated with refrigerated seawater (5°C) in a laboratory-scale recirculation system equipped with a 15-W gamma UV sterilizer. Depuration with refrigerated seawater for 96 h reduced V. parahaemolyticus populations by >3.0 log MPN/g in oysters harvested in the winter. However, 144 h of depuration at 5°C was required to achieve a 3-log reduction in oysters harvested in the summer. Depuration with refrigerated seawater at 5°C for up to 144 h caused no significant fatality in the Pacific oyster and could be applied as a postharvest treatment to reduce V. parahaemolyticus contamination in Pacific oysters. Further studies are needed to validate the efficacy of the depuration process for reducing naturally accumulated V. parahaemolyticus in oysters.

Development of Rapid Real-Time PCR and Most-Probable-Number Real-Time PCR Assays To Quantify Enterotoxigenic Strains of the Species in the Bacillus cereus Group

2007 ◽  
Vol 70 (12) ◽  
pp. 2774-2781 ◽  
Author(s):  
I-CHEN YANG ◽  
DANIEL YANG-CHIH SHIH ◽  
JAN-YI WANG ◽  
TZU-MING PAN
Keyword(s):  
Bacillus Cereus ◽  
Real Time ◽  
Real Time Pcr ◽  
Food Samples ◽  
Most Probable Number ◽  
Pcr Assay ◽  
Bacillus Cereus Group ◽  
Probable Number ◽  
Cooked Rice ◽  
Group Detection

Members of the Bacillus cereus group may produce diarrheal enterotoxins and could be potential hazards if they enter the food chain. Therefore, a method capable of detecting all the species in the B. cereus group rather than B. cereus alone is important. We selected nhe as the target and developed a real-time PCR assay to quantify enterotoxigenic strains of the B. cereus group. The real-time PCR assay was evaluated with 60 B. cereus group strains and 28 others. The assay was also used to construct calibration curves for different food matrices and feces. The assay has an excellent quantification capacity, as proved by its linearity (R2 > 0.993), wide dynamic quantification range (102 to 107 CFU/g for cooked rice and chicken, 103 to 107 CFU/ml for milk, and 104 to 107 CFU/g for feces), and adequate relative accuracy (85.5 to 101.1%). For the low-level contaminations, a most-probable-number real-time PCR assay was developed that could detect as low as 100 CFU/ml. Both assays were tested with real food samples and shown to be considerably appropriate for B. cereus group detection and quantification.

Effects of Flash Freezing, Followed by Frozen Storage, on Reducing Vibrio parahaemolyticus in Pacific Raw Oysters (Crassostrea gigas)

2009 ◽  
Vol 72 (1) ◽  
pp. 174-177 ◽  
Author(s):  
CHENGCHU LIU ◽  
JIANZHANG LU ◽  
YI-CHENG SU
Keyword(s):  
Crassostrea Gigas ◽  
Vibrio Parahaemolyticus ◽  
Vibrio Vulnificus ◽  
Frozen Storage ◽  
Most Probable Number ◽  
Freezing Process ◽  
Probable Number ◽  
Pacific Oysters ◽  
Subsequent Storage ◽  
Shellfish Sanitation

This study investigated the effects of flash freezing, followed by frozen storage, on reducing Vibrio parahaemolyticus in Pacific raw oysters. Raw Pacific oysters were inoculated with a five-strain cocktail of V. parahaemolyticus at a total level of approximately 3.5 × 105 most probable number (MPN) per gram. Inoculated oysters were subjected to an ultralow flash-freezing process (−95.5°C for 12 min) and stored at −10, −20, and −30°C for 6 months. Populations of V. parahaemolyticus in the oysters declined slightly by 0.22 log MPN/g after the freezing process. Subsequent storage of frozen oysters at −10, −20, and −30°C resulted in considerable reductions of V. parahaemolyticus in the oysters. Storing oysters at −10°C was more effective in inactivating V. parahaemolyticus than was storage at −20 or −30°C. Populations of V. parahaemolyticus in the oysters declined by 2.45, 1.71, and 1.45 log MPN/g after 1 month of storage at −10, −20, and −30°C, respectively, and continued to decline during the storage. The levels of V. parahaemolyticus in oysters were reduced by 4.55, 4.13, and 2.53 log MPN/g after 6 months of storage at −10, −20, and −30°C, respectively. Three process validations, each separated by 1 week and conducted according to the National Shellfish Sanitation Program's postharvest processing validation–verification interim guidance for Vibrio vulnificus and Vibrio parahaemolyticus, confirmed that a process of flash freezing, followed by storage at −21 ± 2°C for 5 months, was capable of achieving greater than 3.52-log (MPN/g) reductions of V. parahaemolyticus in half-shell Pacific oysters.

Occurrence and Characterization of Aeromonas Species in Pasteurized Milk and White Cheese in Rio De Janeiro, Brazil

1993 ◽  
Vol 56 (1) ◽  
pp. 62-65 ◽  
Author(s):  
ANGELA CÔRREA FREITAS ◽  
MARLY PAIVA NUNES ◽  
ARLETE MOREIRA MILHOMEM ◽  
ILVAN DELGADO RICCIARDI
Keyword(s):  
Rio De Janeiro ◽  
Foodborne Pathogen ◽  
Food Samples ◽  
High Rate ◽  
Most Probable Number ◽  
Aeromonas Species ◽  
Probable Number ◽  
Pasteurized Milk ◽  
Milk Samples ◽  
White Cheese

A total of 35 samples (1000 ml each) of pasteurized milk and 25 samples (100 g each) of white cheese purchased at supermarkets in Rio de Janeiro were analyzed for the presence of Aeromonas. Strains of Aeromonas were isolated from 28.5% of pasteurized milk and 32% of white cheese samples. Standard Plate counts in the pasteurized milk samples ranged from 7.2 × 10* to 2.5 × 105 CFU/ml. Total and fecal coliform counts in white cheese samples ranged from 1.9 × 10* to 2.4 × 105 most probable number per g and 3.2 × 102 to 1.2 × 105 most probable number per g, respectively. It was possible to identify Aeromonas caviae (58.9%), Aeromonas hydrophila (12.8%), and Aeromonas schubertii (2.5%) among the cultures isolated from pasteurized milk samples. Twenty-five percent of the strains could only be classified as Aeromonas spp. In white cheese samples, unclassified strains were the most frequent isolates (61.5%) followed by A. hydrophila (26.9%), A. caviae (7.6%) and Aeromonas sobria (3.8%). Only strains of A. hydrophila and A. sobria showed high rate of positive results when tested for the production of hemolysin, cytotoxin, and staphylolytic activity. Heat-stable enterotoxin and autoagglutination test did not correlate as virulence factors. The presence of Aeromonas species in refrigerated food samples suggests that this microorganism could be a potential foodborne pathogen, and dairy products may represent an important vehicle of its transmission.

scholarly journals Prevalence of Bacillus cereus s.l. in ultra-high temperature chocolate milk from selected milk manufacturers in Malaysia

Food Research ◽  
2020 ◽  
Vol 4 (4) ◽  
pp. 982-990
Author(s):  
Ubong Anyi ◽  
C.Y. New ◽  
L.C. Chai ◽  
Y.Y. Loo ◽  
Nor Khaizura M.A.R. ◽  
...  
Keyword(s):  
Bacillus Cereus ◽  
Foodborne Pathogen ◽  
Most Probable Number ◽  
Contamination Level ◽  
Chocolate Milk ◽  
Probable Number ◽  
Potential Source ◽  
Milk Contamination ◽  
Bacillus Spp ◽  
Polymerase Chain

Bacillus cereus is a major foodborne pathogen of great concern to the dairy industry owing to its resilient spores as well as the adverse effect of its toxins. At present, there is no informational study available to solve or pinpoint the UHT chocolate milk contamination issue in Malaysia. This work aimed to investigate the prevalence and contamination level of B. cereus s.l. in UHT chocolate milk and to suggest the appropriate solution for the issue. In the present study, B. cereus s.l. prevalence and contamination level in individually packed UHT chocolate milk from processing factories was evaluated. The prevalence and concentration of B. cereus s.l. were determined via MPN-PCR (Most Probable Number-Polymerase Chain Reaction) assay. Results showed that 31.11% from 220 of UHT chocolate milk tested contained Bacillus spp.; of this Bacillus spp. positive samples, 24.30% were also positive for B. cereus s.l. with concentration ranging from less than 3 to more than 1100 MPN/mL. Findings from this study highlighted the possibility of UHT chocolate milk as a potential source of B. cereus s.l. infection. Therefore, findings emphasized the needs to revise, monitor and improve UHT sterilization process to reduce infection risk. Furthermore, it is also essential to maintain the hygiene to minimize initial microbial load and contamination of UHT chocolate milk, beginning from production to retail.

Enumerative Analysis of Salmonella in Outbreak-Associated Breaded and Frozen Comminuted Raw Chicken Products

2017 ◽  
Vol 80 (5) ◽  
pp. 814-818 ◽  
Author(s):  
Angela Catford ◽  
Kyle Ganz ◽  
Sandeep Tamber
Keyword(s):  
Foodborne Pathogen ◽  
Food Samples ◽  
Most Probable Number ◽  
Distribution Analysis ◽  
Probable Number ◽  
Contaminated Food ◽  
Foodborne Outbreaks ◽  
Low Levels ◽  
Data Gap ◽  
Pathogen Exposure

ABSTRACT A significant data gap exists with respect to the levels of pathogens in foods implicated in foodborne outbreaks. These data are essential for the quantification of pathogen exposure via the ingestion of contaminated food. Here we report the levels of the foodborne pathogen Salmonella in comminuted raw chicken products that had been breaded and then frozen. The products investigated were collected during four food safety investigations of foodborne outbreaks that occurred in Canada from 2014 to 2016. Most-probable-number (MPN) distribution analysis of the food samples revealed Salmonella levels of 0.0018 to 3 MPN/g, which is equivalent to 1 MPN per 0.33 to 556 g of product. These data suggest low levels of Salmonella may be associated with foodborne outbreaks.

scholarly journals Development and Application of a Most-Probable-Number–PCR Assay To Quantify Flagellate Populations in Soil Samples

2001 ◽  
Vol 67 (4) ◽  
pp. 1613-1618 ◽  
Author(s):  
Line Fredslund ◽  
Flemming Ekelund ◽  
Carsten Suhr Jacobsen ◽  
Kaare Johnsen
Keyword(s):  
Dna Sequences ◽  
Sequence Data ◽  
Small Subunit ◽  
Most Probable Number ◽  
Rrna Gene ◽  
Heterotrophic Flagellates ◽  
Small Subunit Rrna ◽  
Pcr Assay ◽  
Probable Number ◽  
Soil Protozoa

ABSTRACT This paper reports on the first successful molecular detection and quantification of soil protozoa. Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations. Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellate Heteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene. The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacterium Pseudomonas putida OUS82 UCB55 as prey. There was satisfactory overall agreement between H. globosa population estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.

scholarly journals Prevalence, characterization, and antibiotic susceptibility of Vibrio parahaemolyticus isolated from retail aquatic products in North China

2016 ◽  
Vol 16 (1) ◽  
Author(s):  
Xiaoke Xu ◽  
Jianheng Cheng ◽  
Qingping Wu ◽  
Jumei Zhang ◽  
Tengfei Xie
Keyword(s):  
Vibrio Parahaemolyticus ◽  
North China ◽  
Antimicrobial Agents ◽  
Foodborne Pathogen ◽  
Eastern Coast ◽  
Most Probable Number ◽  
Antibiotic Resistant ◽  
Probable Number ◽  
Aquatic Products ◽  
Resistance Patterns

Abstract Background Vibrio parahaemolyticus is a major foodborne pathogen, particularly in Asian countries. Increased occurrence of outbreaks of V. parahaemolyticus gastroenteritis in China indicates the need to evaluation of the prevalence of this pathogenic species. V. parahaemolyticus distribution in shellfish from the eastern coast of China has been reported previously. However, to date, the prevalence of V. parahaemolyticus in retail aquatic products in North China has not been determined. To investigate the prevalence of V. parahaemolyticus in aquatic products in North China, 260 aquatic product samples were obtained from retail markets in 6 provinces of North China from November to December in 2012 and July to August in 2013. Results V. parahaemolyticus was detected in 94 (36.2 %) of the samples by the most probable number method. The density of V. parahaemolyticus ranged from 1.50 to 1100 MPN/g. V. parahaemolyticus was detected at a rate of 50.0 % and 22.7 % in summer and in winter, respectively. The density of V. parahaemolyticus was significantly higher in summer than in winter, with mean levels of 16.5 MPN/g and 5.0 MPN/g, respectively. Among 145 V. parahaemolyticus isolates examined, none of the isolates possessed tdh and trh. In multiplex PCR-based O-antigen serotyping of these 145 isolates, all serotypes, other than O6, O7, and O9, were detected, and serotype O2 was found to be the most prevalent (detected in 54 isolates). The 145 isolates were grouped into 7 clusters by enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) at a similarity coefficient of 0.66. The antimicrobial resistance patterns of these 145 isolates to 12 antimicrobial agents revealed that most of the isolates were resistant to streptomycin (86.2 %), while fewer were resistant to ampicillin (49.6 %), cefazolin (43.5 %), cephalothin (35.9 %), and kanamycin (22.1 %). All of the examined isolates were susceptible to azithromycin and chloramphenicol. Conclusions The findings of this study will help in defining appropriate monitoring programs, understanding of the dissemination of antibiotic resistant strains, and providing information for the assessment of exposure to this microorganism at the consumption level.

scholarly journals Development of a Multiplex Real-Time PCR Assay with an Internal Amplification Control for the Detection of Total and Pathogenic Vibrio parahaemolyticus Bacteria in Oysters

2007 ◽  
Vol 73 (18) ◽  
pp. 5840-5847 ◽  
Author(s):  
Jessica L. Nordstrom ◽  
Michael C. L. Vickery ◽  
George M. Blackstone ◽  
Shelley L. Murray ◽  
Angelo DePaola
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Vibrio Parahaemolyticus ◽  
The United States ◽  
Internal Amplification Control ◽  
Most Probable Number ◽  
Specific Marker ◽  
Pcr Assay ◽  
Probable Number ◽  
The Real

ABSTRACT Vibrio parahaemolyticus is an estuarine bacterium that is the leading cause of shellfish-associated cases of bacterial gastroenteritis in the United States. Our laboratory developed a real-time multiplex PCR assay for the simultaneous detection of the thermolabile hemolysin (tlh), thermostable direct hemolysin (tdh), and thermostable-related hemolysin (trh) genes of V. parahaemolyticus. The tlh gene is a species-specific marker, while the tdh and trh genes are pathogenicity markers. An internal amplification control (IAC) was incorporated to ensure PCR integrity and eliminate false-negative reporting. The assay was tested for specificity against >150 strains representing eight bacterial species. Only V. parahaemolyticus strains possessing the appropriate target genes generated a fluorescent signal, except for a late tdh signal generated by three strains of V. hollisae. The multiplex assay detected <10 CFU/reaction of pathogenic V. parahaemolyticus in the presence of >104 CFU/reaction of total V. parahaemolyticus bacteria. The real-time PCR assay was utilized with a most-probable-number format, and its results were compared to standard V. parahaemolyticus isolation methodology during an environmental survey of Alaskan oysters. The IAC was occasionally inhibited by the oyster matrix, and this usually corresponded to negative results for V. parahaemolyticus targets. V. parahaemolyticus tlh, tdh, and trh were detected in 44, 44, and 52% of the oyster samples, respectively. V. parahaemolyticus was isolated from 33% of the samples, and tdh + and trh + strains were isolated from 19 and 26%, respectively. These results demonstrate the utility of the real-time PCR assay in environmental surveys and its possible application to outbreak investigations for the detection of total and pathogenic V. parahaemolyticus.

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