Cold Enrichment Method Research Articles

Epidemiological characteristics of Yersinia enterocolitica in patients with diarrhea from 2013 to 2019 in Pudong New Area, Shanghai

Objective: To investigate the epidemiological characteristics of Yersinia enterocolitica in patients with diarrhea in Pudong New Area, Shanghai. Methods: Active surveillance of diarrhea was conducted in 14 sentinel hospitals (three tertiary-level hospitals, nine secondary-level hospitals, and two primary-level hospitals) from January 2013 to December 2019 in Pudong New Area of Shanghai, China base on their location, catchment area, and patient volume. Cold enrichment method was used to isolate Y. enterocolitica and further detection of bioserotype, virulence genes and antimicrobial susceptibility of the isolates were conducted. The difference of rates was determined using chi-square test or Fisher's exact test. Results: A total of 12 941 diarrhea cases were included, and 0.7% (88/12 941) cases were confirmed with Yersinia enterocolitica infection. 67.0% (59/88) cases were single infection, 33.0% (29/88) cases were mixed infections. Detection rates of Y. enterocolitica increased annually (0.3%-1.2%) and were highest in children<5 years of age (1.1%, 37/3 218) and in spring (1.1%, 32/2 998) (χ2 were 18.64 and 9.76, respectively, P<0.05). 58.0% (51/88) cases had watery diarrhea, 15.9% (14/88) had fever and 14.8% (13/88) had vomiting. The predominant bioserotypes were 3/O:3 (53.4%, 47/88), followed by 1A/O:8 (15.9%, 14/88) and 1A/O:5(6.8%, 6/88), respectively. Bioserotype 3/O:3 counted for the highest proportions (89.2%, 33/37) in children <5 years of age. All the strains of bioserotype 3/O:3 harbored ail, ystA, yadA and virF genes, which encoded pathogenic Y. enterocolitica. 11/14 strain of 1A/O:8 and 4/6 strains of 1A/O:5 harbored ystB gene. Most strains were resistant to ampicillin (80.7%,71/88) and amoxicillin/clavulanic acid (71.6%,63/88), and 63.8% (56/88) strains were multidrug resistance (MDR). The difference of antimicrobial resistance rates between 3/O:3 and non 3/O:3 was statistically significant in ampicillin, cefoxitin, nalidixic acid, tetracycline and ampicillin/sulbactam (χ2 was 14.68, 43.80, 41.86, 30.54 and 5.07, respectively, P<0.05). Conclusion: The detection rate of Yersinia enterocolitica was higher in children than in adults in Pudong New Area, Shanghai. The predominant bioserotype was pathogenic 3/O:3 with multidrug resistance.

Antimicrobial resistance and molecular epidemiology of foodborne Yersinia enterocolitica in Pudong New District, Shanghai

Objective: To investigate the antimicrobial resistance and molecular epidemiology of foodborne Yersinia (Y.) enterocolitica in Pudong New District of Shanghai. Methods: Four kinds of raw food samples were collected in retail circulation sites in Pudong from 2012 to 2016. Cold enrichment method was used to isolate Y. enterocolitica and further detection of biotype, serotype, virulent genes, antimicrobial susceptibility of the isolates and pulsed field gel electrophoresis (PFGE) were conducted. Results: A total of 3 900 raw food samples were collected during this period, including poultry product (n=590), livestock product (n=1 074), aquatic product (n=1 488), vegetable (n=748), in which 111 (2.8%) were contaminated by Y. enterocolitica. The detection rates of Y. enterocolitica in poultry product samples (5.3%, 31/590) and livestock product samples (4.5%, 48/1 074) were higher than those in aquatic product samples (1.6%, 24/1 488) and vegetable samples (1.1%, 8/748). The predominant biotype was 1A (95.5%) and predominant serotype was O∶8 (42.3%). All the strains lacked ail, ystA, yadA and virF genes, which encoded pathogenic Y. enterocolitica. Seventy six (68.5%) strains harbored ystB gene, in which 35 (31.5%) belonged to 1A/O∶8/ystB pattern. Most strains were resistant to ampicillin (74.8%) and amoxicillin/clavulanic acid (70.3%), and non-sensitive rate to Cefoxitin was over 50.0%. No third generation cephalosporin or fluoroquinolone resistant strains were detected, but 38.7% (43/111) strains were multidrug resistant (MDR). Serotype O∶8 and O∶5 strains had 44 and 18 PFGE patterns, respectively. Conclusions: The main foodborne exposure sources of Y. enterocolitica in raw food were poultry and livestock products in Pudong New District. 1A/O∶8/ystB was the predominant pattern with potential pathogenicity despite lacks of typical pathogenic virulent genes. The antimicrobial resistant rates of Y. enterocolitica were at a low level, but MDR strains still existed. Molecular types of the isolates showed highly genetic diversity.

Genotypic and phenotypic virulence characteristics and antimicrobial resistance of Yersinia spp. isolated from meat and milk products.

A total of 300 food samples including 180 milk and 120 meat products have been examined for the presence of Yersinia spp. using the ISO 10273 and the cold enrichment method. The overall prevalence of Yersinia spp. was 84 (28%). Yersinia enterocolitica was isolated from 18 (6%) of the 300 samples. The other Yersinia species were detected in the samples Yersinia rohdei 15 (5%), Yersinia intermedia 14 (4.7%), Yersinia pseudotuberculosis 12 (4%), Yersinia ruckeri 12 (4%), Yersinia mollaretii 5 (1.7%), Yersinia bercovieri 4 (1.3%), and atypical Yersinia spp. 4 (1.3%). The conventionally identified Y. enterocolitica strains were also confirmed by the 16S rRNA gene sequencing. All Y. enterocolitica strains biotyped as 1A had negative results in the phenotypic virulence tests. The 84 Yersinia strains were also examined genotypically for the presence of virulence genes. None of the Y. enterocolitica and other Yersinia strains contained the ail, ystA, yadA, and virF except only 1 Y. intermedia and 2 Y. enterocolitica strains that were found to be positive for ystB. Antimicrobial resistance of 84 Yersinia to 16 antimicrobial agents was determined by the disk diffusion method. All strains were sensitive to tobramycine and imipenem while resistant to clindamycin. Although 84.5% of the strains were resistant to at least 3 or more antimicrobial agents, 64.3% of them were resistant to 4 or more antimicrobial agents.

English

Listeria monocytogenes is a ubiquitous Gram positive organism causative agent of many hazardous conditions like meningitis and sepsis. To isolate L. monocytogenes from various food samples collected from different markets in Iran and evaluate pathogenic potential of the isolates by determining two virulence associated genes (hly and plc-A) and sequencing them, a total of 130 samples including dairy and meat products were collected. Cold enrichment method was used for the isolation of Listeria monocytogenes. The isolated pathogens were identified and confirmed by biochemical and polymerase chain reaction (PCR). PCR method was done to amplify two gene fragments, a 1590 and 954 bp for hly and plc-A genes, respectively by using sequencing primers. Listeria monocytogenes was isolated from 3.1% of all samples. The bacterium was isolated 2.5% in dairy and 4% in meat samples. All isolates were hemolytic and positive for hly gene. Also 100% of isolates had plc-A gene. The results of this study indicates that consumption of ready to eat food is hazardous specially for high risk persons .Besides that, pathogenic potential of isolated strains is important because of existences of virulence gene in all of the isolates. &nbsp; Key words: Listeria monocytogenes, hly and plc-A genes, polymerase chain reaction (PCR), dairy, meat.

Detection of Yersinia enterocolitica in diarrhoiec stools and environmental samples

Aim: To detect distribution of Yersinia enterocolitica in gastroenteritis cases, in environmental samples and food samples consumed by humans. Materials and Methods: Stool samples, environmental samples comprising water from water pools on the bank of river 'Kabini,' fruit juices from commercial fresh juice centers, pork intestinal contents, drinking water from public eateries, ('paani puri' outlets), public tap water samples and commercial prepacked juices. All samples were cultured by four different methods; direct plating on MacConkey agar, plating following cold enrichment, single alkali and double alkali treatment with KOH & PBS. Results: A total of 181 stool samples and 15 food and environmental samples were processed. Y. enterocolitica was isolated from one stool sample (0.55%) by cold enrichment method and from one water sample collected from river bank (6.6%). Conclusion: Y. enterocolitica is found in humans and in the environment in this region. Adopting a rapid isolation method may infuse interest among clinical microbiologists to undertake an epidemiological study on Y. enterocolitica and Yersiniosis. Unhygienic defaecation habits of the people and movement of swine herds along the river bank may be the source for river water contamination.

Investigation on distribution of Yersinia enterocolitica in Henan province between 2005 and 2011

To investigate the distribution of Yersinia enterocolitica in Henan province from 2005 to 2011. A total of 6700 samples of stool specimen were collected from diarrhea patients and different domestic animals between 2005 and 2011 from Zhengzhou, Suixian and Dengfeng, as well as flies and the daub specimens of raw and cooked meat products. The bacteria were isolated by cold enrichment method, analyzed by the systematic biochemistry to determine the serotypes and bio-types, and tested the virulence genes by PCR method. A total of 216 strains of Yersinia enterocolitica were isolated from 11 kinds of animal hosts and foods, while 29.63% (64/216) of them were from swine. The dominant epidemic serotypes of the Yersinia enterocolitica were O: 5 and O: 8, accounted for 23.2% (50/216) and 20.4% (44/216), respectively; type 1A was the dominant bio-type, accounted for 84.7% (183/216). The dominant serotype and bio-type differed a lot among various hosts.16 pathogenic strains were isolated from swine, followed by diarrhea patients (6 strains) and dogs (6 strains). The distribution of the host of Yersinia enterocolitica was widespread, while swine was the dominant animal host.

Isolation and characterization of &lt;em&gt;Yersinia intermedia&lt;/em&gt; strains from pig tonsils in Abidjan, Côte d’Ivoire

A total of 150 samples of pig tonsils were collected from slaughterhouse in Abidjan; the pigs were from pig farms located in different areas of C&#244;te d&#8217;Ivoire. Samples were examined for the presence of Yersinia intermedia. Optimal recovery of Y. intermedia was achieved using two step enrichment procedures based on pre-enrichment in trypticase soya broth at 28 &#176;C for 24 h, followed by cold enrichment method at 4 &#176;C for 21 days in phosphate buffered saline broth. Then, Aulisio&#8217;s alkali treatment method was performed before streaking onto MacConkey agar. Six strains of Yersinia intermedia were isolated and tested for the following characteristics associated to the virulence in Y. enterocolitica such as pyrazinamidase activity and autoagglutinability. All the six strains were all positive for the pyrazinamidase test and four of them were autoagglutinable. Four strains were biotype 4/autoagglutinable and two were biotype 5/O:7,8-8-8,19. All the six strains of Yersinia intermedia were rhamnose negative and not motile at 25 &#176;C. The results of antimicrobial resistance showed that all the strains presented multiple antibiotic resistance. The results indicate that 4% of pig tonsils from different farms collected at the slaughterhouse were contaminated with Yersinia intermedia. This study is the first which shows the presence of Yersinia intermedia in pigs in C&#244;te d&#8217;Ivoire.Keywords: Yersinia intermedia, pig tonsils, prevalence, biotype, antibiotic resistance, C&#244;te d&#8217;Ivoire

The presence of Listeria monocytogenes in raw milk samples in Mashhad, Iran

The purpose of this preliminary study was to determine the prevalence of raw milk contamination with Listeria monocytogenes. In this study, 100 bulk tank milk samples were collected randomly and delivered to Pegah Pasteurization Factory in Mashhad. For isolation and identification of L. monocytogenes, the samples were first enriched using cold enrichment method in Listeria enrichment broth, followed by plating onto supplemented Oxford agar. For final identification of suspected colonies a multiplex-PCR assay, using two pair of primers was employed. The prs primers are specific for putative phophoribosyl pyrophosphate synthetase (prs) gene of Listeria spp. and the LM lip1 primers are specific for prf A gene of its monocytogenes serovar. Using this method, the contamination of raw milk with L. monocytogenes was determined to be 4% and the sensitivity of the primers was 3.5 × 10 3 cfu ml -1 , and the specificity was determined to be 100%. Considering the high specificity and sensitivity of the employed multiplex-PCR assay, it is recommended to use this method for the identification of suspected colonies of Listeria spp. and L. monocytogenes.

The prevalence of pathogenic Yersinia enterocolitica among diarrhea patients in Jos, Nigeria

One hundred and fifty (150) stool samples from diarrhoeic children and adults seeking for medical attention (including hospitalized patients) in Vom Christain Hospital (VCH), Mandela Clinic (MC) Vom and Dagott Family Health Clinic (DFHC) Vom were screened for Yersinia enterocolitica infection between August 2005 and August 2006. The isolation methods adopted were direct plating on MacConkey Agar (MCA), Deoxycholate Citrate Agar (DCA) and cold enrichment method using phosphate buffered saline prior to subculture onto selective solid culture media (Cefsulodin Irgasan Novobiocin [CIN] agar). Out of the 150 samples screened, 6 (15%) were positive. The incidence of the infection was highest among those aged 1 - 10 years 3 (7.5%), followed by 21 - 31 years 2 (5%) and 11 - 20 years 1 (2.5%). Serotyped and biotyped, pathogenic Y. enterocolitica (2/O: 9. 4/O: 9) were susceptible to ciprofloxin, floxavid, streptomycin and tetracycline.

The prevalence of pathogenic Yersinia enterocolitica among diarrhea patients in Jos, Nigeria

One hundred and fifty (150) stool samples from diarrhoeic children and adults seeking for medical attention (including hospitalized patients) in Vom Christain Hospital (VCH), Mandela Clinic (MC) Vom and Dagott Family Health Clinic (DFHC) Vom were screened for Yersinia enterocolitica infection between August 2005 and August 2006. The isolation methods adopted were direct plating on MacConkey Agar (MCA), Deoxycholate Citrate Agar (DCA) and cold enrichment method using phosphate buffered saline prior to subculture onto selective solid culture media (Cefsulodin Irgasan Novobiocin [CIN] agar). Out of the 150 samples screened, 6 (15%) were positive. The incidence of the infection was highest among those aged 1 - 10 years 3 (7.5%), followed by 21 - 31 years 2 (5%) and 11 - 20 years 1 (2.5%). Serotyped and biotyped, pathogenic Y. enterocolitica (2/O: 9. 4/O: 9) were susceptible to ciprofloxin, floxavid, streptomycin and tetracycline.

Pathological and epidemiological investigations into listerial encephalitis in sheep

Three outbreaks of encephalitis caused by Listeria monocytogenes were investigated in the migratory flocks of sheep in Punjab, India. Sixty-nine sheep were affected out of 930 animals (875 sheep and 55 goats) during these outbreaks, with cumulative morbidity, mortality and case fatality rate of 7.89, 7.08 and 89.85%, respectively. Incidence of disease was low in sheep below 6 months of age with cumulative morbidity and mortality rate of 3.75% as compared to 9.5 and 8.3%, respectively in sheep above 6 months of age. Case fatality was highest (100%) in young sheep below 6 months of age when compared to 88.3% in adult sheep above 6 months of age. Clinical signs included circling movements, unilateral facial paralysis, twisting of neck, stiffening and opisthotonus, blindness, corneal opacity and retention of food in mouth. Histopathologically, extensive perivascular cuffing, microabscesses and meningitis were observed in medulla oblongata, pons and anterior part of spinal cord. In few cases, microabscesses were also present in liver. Cerebrospinal fluid revealed higher concentration of total proteins with increase in globulins. Isolation of L. monocytogenes was made from hind brain and anterior part of spinal cord using cold enrichment method. There was no symptom of disease in the goats.

Comparison of a cold enrichment method and the IDF method for isolation of Listeria monocytogenes from animal autopsy material

The method most often used in Sweden for isolation of Listeria monocytogenes from animal autopsy material is a cold enrichment method. This method is very slow. The International Dairy Federation (IDF) has recently presented a method for detection of L. monocytogenes in milk and milk products that is complete in one week. During a two year period 69 specimens from dead animals with suspected listeriosis were examined for L. monocytogenes in parallel analyses with both the cold enrichment method and the IDF method. Samples derived from different autopsy material representing a variety of animals. L. monocytogenes was isolated in 27.5% of the samples with the IDF method but only in 4.3% with the cold enrichment method. It is concluded that the IDF method was more sensitive than the cold enrichment method.

Comparison of cold enrichment and U.S. Department of Agriculture methods for isolating Listeria monocytogenes from naturally contaminated foods. The Listeria Study Group

We compared the cold enrichment (CE) and U.S. Department of Agriculture (USDA) methods for isolating Listeria monocytogenes by examining 402 food samples. The food samples were collected from refrigerators of listeriosis patients as part of a multistate active surveillance project to determine the role of foods in sporadic listeriosis in the United States. L. monocytogenes was isolated from 51 food samples (13%). The USDA method was significantly better (P less than 0.001) than the CE method. The isolation efficiencies of the USDA and CE methods were 96 and 59%, respectively. Quantitation of L. monocytogenes in the food samples revealed that many food samples containing less than 0.3 CFU/g were negative as determined by the CE method but positive as determined by the USDA method.

Survey of the incidence of Listeria monocytogenes and other Listeria spp. in experimentally irradiated and in matched unirradiated raw chickens

Listeria spp. were sought in 32 chickens experimentally irradiated (2.5 kGy) and in 25 matched unirradiated chickens. Using the modified Food and Drug Administration method only three of the chickens were positive for L. monocytogenes. Using the more sensitive cold enrichment method 30 were found positive. The proportion of carcasses contaminated with L. monocytogenes was lower in the irradiated birds than in the unirradiated birds. The results also indicated that the numbers of L. monocytogenes in the irradiated birds were lower than in the unirradiated birds. The only other species of Listeria isolated was L. innocua, which was found on 44% of unirradiated chickens, but on none of the irradiated chickens.

A comparison of two procedures for the isolation of Listeria monocytogenes from raw chickens and soft cheeses

A cold enrichment method and a modified FDA procedure were compared for the isolation of Listeria monocytogenes from raw chickens and soft cheeses. L. monocytogenes was isolated from a total of 23 of 222 cheese and 70 of 160 chicken samples by either one or both methods. Neither method alone yielded all isolates from the two food types. Only 12 cheese and 13 chicken samples were shown to be positive by both methods, although the serotypes isolated were not always identical. On some occasions one method yielded L. monocytogenes while the other produced a different Listeria sp. Reasons for differences in the performance of the two procedures and various points of technical interest are discussed.

Isolation ofYersinia enterocolitica andYersinia enterocolitica-like organisms from raw milk in Italy

Thirty samples of raw milk, originating from individual producers in the Turin area, were examined for the presence ofYersinia enterocolitica. A cold enrichment method with phosphate-buffered saline (PBS) 1/15M, pH 7.6, and sorbitol-bile-salts broth (SB) was used. After 7, 14, or 21 days at 4°–5°C, plating was performed on selective agar media directly (MacConkey agar andSalmonella-Shigella agar) after the alkali method was used. Six strains ofY. enterocolitica (biotype 1) and 32 strainsY. enterocolitica-like (threeY. fredericksenii; nineYersinia rhamnose-, melibiose+, α-methyl-d-glucoside+, raffinose+, probablyYersinia intermedia biotype rhamnose-; and 20Y. intermedia) were isolated.Yersinia strains were found in 11 samples of raw milk, andY. enterocolitica in four samples.

Vergleichende Anwendung von zwei Anreicherungsverfahren and fünf Selektivnährböden zur Isolierung von Yersinia enterocolitica aus Tonsillen von Schlachtschweinen

115 tonsils of healthy slaughter pigs were culturally examined for presence of Yersinia (Y.) enterocolitica. For this purpose each sample was enriched both in phosphate buffered saline solution (pH 7.6; stored at 4 °C and plated every week, thrice all together) and modified Rappaport broth (plated after an incubation of two days at 22 °C). Each such enrichment was plated on 5 different selective media: Yersinia selective agar proposed by Wauters (1973), deoxycholate-citrate-mannitol agar ( Saari and Jansen, 1979), pectin agar ( Bowen and Kominos), Mac Conkey and Leifson agar as used in the routine diagnostic of Enterobacteriaceae. Each agar plate was incubated at 28 °C for two days. By cold enrichment method were isolated 11 strains of human pathogenic Y. enterocolitica (9 X 0-group I syn. serotype O : 3 ; 2 X 0-group V syn. serotype O:9). With the modified Rappaport medium were recovered 33 strains (24 X O-group I, 9 X O-group V). The most recoveries were done over the Yersinia selective agar with 65.2%, then followed deoxycholate-citrate-mannitol agar with 57.6%, Leisfon agar with 45.5%, Mac Conkey agar with 42.3% and pectin agar only with 18.2% of the isolations. Not only the type of enrichment medium has a marked effect in the recovery efficiency of Y. enterocolitica out of samples but also number and type of the used selective media on which the enrichment is plated.

Alkali method for rapid recovery of Yersinia enterocolitica and Yersinia pseudotuberculosis from foods.

A new culture method employing a potassium hydroxide treatment was compared with the conventional cold enrichment method for efficacy in recovering Yersinia sp. from naturally and artificially contaminated food. The new method increased the yield of Yersinia sp. fourfold and the sensitivity 100-fold, shortened the incubation period, and appreciably decreased the growth of non-Yersinia bacteria from a variety of meats, shellfish, and vegetables.

Isolation of Yersinia enterocolitica from swine.

Data pertaining to 20 strains of Yensinia enterocolitica isolated from the cecal content of swine slaughtered at Toronto Abattoirs are presented. Fifteen of 20 cultures belonged to the two predominant human serotypes in Ontario (i.e., serotype O:3 and O:5,27.) Seven cultures of Y. enterocolitica O:3 belonged to the "Canadian" human phage type 9b. These findings are further proof that swine are reservoirs for Y. enterocolitica human infection. Seventeen Y. enterocolitica cultures were isolated by the cold enrichment method only. Difficulties encountered in isolating Y. enterocolitica from feces specimens are discussed. It would appear that the reported rate of Y. enterocolitica isolation from feces specimens is far below the actual incidence. Fourteen isolates of Y. pseudotuberculosis serotype III were isolated during the same survey.