Cefsulodin-irgasan-novobiocin Research Articles

Immuno-Magnetic Separation and Agar Layer Methods for the Isolation of Freeze-Injured Yersinia enterocolitica O:8 from Water

To develop an effective method to isolate an injured pathogenic Yersinia enterocolitica O:8 organism from environmental samples, we compared the isolation of freeze-injured and non-injured Y. enterocolitica O:8 and found that the isolation was more successful when immuno-magnetic separation (IMS) with anti-Y. enterocolitica O:8 antibody was used. Plating onto cefsulodin-irgasan-novobiocin (CIN) agar and Virulent Yersinia enterocolitica (VYE) agar by means of the agar layer method was found to be effective in isolating the injured cells. The alkali treatment which is generally used for selective detection of Yersinia organism failed to isolate freeze-injured pathogenic Y. enterocolitica O:8 cells. Recovery methods without using the alkali treatment were superior for detecting freeze-injured Y. enterocolitica O:8. Our results demonstrate that the IMS and the agar layer methods should be used to isolate injured pathogenic Yersinia organisms from environmental samples such as water.

Evaluation of a 5′ -Nuclease (TaqMan) Assay with the Thin Agar Layer Oxyrase Method for the Detection of Yersinia enterocolitica in Ground Pork Samples

A 5′-nuclease (TaqMan) assay was evaluated for its capability to recover and detect stressed Yersinia enterocolitica. Sensitivity studies of a 5′-nuclease assay for detecting Y. enterocolitica O:8 in a pure culture system and spiked ground pork samples demonstrated that the assay has reliable sensitivity with a detection limit of 3 to 4 log CFU/ml or CFU/g. The PCR 5′-nuclease (TaqMan) assay was evaluated with the Thin Agar Layer Oxyrase method (TALO, overlaying 14 ml of Trypticase soy agar with a 1:30 dilution of “Oxyrase® for Agar” onto a prepoured pathogen-specific, selective medium), and it was compared against the selective medium cefsulodin-irgasan-novobiocin (CIN) for recovering and detecting Y. enterocolitica from inoculated nonfrozen and frozen (−15°C, 2 days) ground pork samples. The TALO method showed more sensitivity (detection limit, 2 log CFU/ml), and it has greater recovery capability (0.5 to 1 log CFU/ml) than CIN (P < 0.05). The 5′-nuclease assay provided rapid detection processing (5 versus 24 h after an 18-h enrichment). The sensitivity per PCR was calculated to as low as 0 to 1 log CFU per PCR reaction; however, in the method's current developmental stage, target pathogens should be enriched to 3 to 4 log CFU/ml or CFU/g to show consistent results. In a survey of 100 ground pork samples using TALO, CIN, and PCR methods, no Y. enterocolitica was recovered. A combined cultivation and an automated PCR TaqMan could be used as a presumptive screening test for detecting Y. enterocolitica in food samples.

GASTROENTERITIS - A PROSPECTIVE STUDY

Two thousand faecal samples were collected from diarrhoeal patients of all age groups and processed by using MacConkey agar and Yersinia selective agar [Cefsulodin-Irgasan-Novobiocin (CIN) media]. One hundred and seventy faecal samples from non-diarrhoeal (healthy controls) were also processed. Samples were subjected to cold enrichment before processing. Three hundred and seventy-four (18.7%) faecal samples were positive for bacterial pathogens, of which 41 (2.05%) had Y.enterocolitica infection. Y.enterocolitica was more common in patients above 16 years of age 23/41 (56%). Higher rates of isolation were recorded during winter months. Of the typable isolates 21.9% belonged to 0:3 serotype followed by 01, 2a, 3 (17%) serotype. Twelve (29.2%) isolates were untypable. 51.2% isolates of Y.enterocolitica belonged to biotype IV whereas 24.4%, 19.5%, 4.9% belonged to type I, II and III respectively. Majority of the isolates were found sensitive to chloramphenicol, gentamicin, amikacin, ciprofloxacin, ampicillin, netromycin, cefoperazone, tobramycin and pefloxacin. All isolates were resistant to ofloxacin.

Rapid urease screening of Yersinia on CIN agar plates

Yersinia enterocolitica is an important foodborne pathogen, but isolation of virulent Yersinia from food sources is still time consuming and requires skills. In this article, we describe a rapid urease screening on cefsulodin–irgasan–novobiocin (CIN) agar plates with an agar overlay assay. This test is simple to perform, all colonies on a plate can be checked simultaneously, it only takes minutes for detection of urease-positive colonies and the colonies survive for transfer, further characterisation, and storage. Additionally, this method is useful to isolate virulent (urease-positive and pYV harbouring) Y. enterocolitica from foodstuffs.

Incidence and pathogenicity of Yersinia spp. isolates from poultry in Spain

Forty eviscerated and refrigerated chicken carcasses were collected from retail outlets in León (Spain) and investigated for the presence of Yersinia spp. Following a two-step enrichment procedure (phosphate buffer saline was used as primary enrichment and bile oxalate sorbose as secondary enrichment), yersiniae were isolated on cefsulodin-irgasan-novobiocin (CIN) and MacConkey agar. Yersinia spp., Y. enterocolitica and Y. frederiksenii were detected in 26 (65%), 22 (55%) and six (15%) samples, respectively. The incidence of Yersinia and Y. enterocolitica was greater than that obtained in poultry meat by the majority of the consulted authors. Of the 68 Yersinia isolates, 52 were identified as Y. enterocolitica and 16 as Y. frederiksenii. Biotyping of Y. enterocolitica revealed that 45 (86·5%) strains corresponded to biotype 1A, and three (5·8%) to biotype 3. Four (7·7%) strains could not be classified. Two biotype 3 strains were found to be presumptively virulent according to the dye (Congo red and crystal violet) binding, low-calcium response, and pyrazinamidase activity techniques. It is suggested from results that the presence of Yersinia in poultry could represent a health risk for consumers in Spain. The education of people involved in production, processing and final preparation of poultry products are required in order to assure adequate cooking and to avoid cross-contamination.

Evaluation of a 5′-Nuclease (TaqMan) Assay for the Detection of Virulent Strains of Yersinia enterocolitica in Raw Meat and Tofu Samples

Culture methods for detecting virulent Yersinia enterocolitica require selective enrichment and a series of confirmatory tests that are time-consuming, costly, and laborious. The objective of this study was to evaluate a fluorogenic 5′-nuclease assay for detecting the enterotoxin yst gene of virulent Y. enterocolitica in pure cultures, inoculated ground pork samples, and naturally contaminated food samples. These results were then compared with “gold standard” methods recommended by the U.S. Food and Drug Administration in the Bacteriological Analytical Manual for detecting pathogenic Y. enterocolitica. The 5′-nuclease assay was able to identify the organism in 100% of the repetitions when 102 CFU/ml or more organisms were present in pure cultures and 103 CFU/g or more organisms were present in ground pork. Similar recovery efficiency on cefsulodin-irgasan-novobiocin (CIN) agar plates was only evident when 105 CFU/ml or more organisms were present in pure culture and 106 CFU/g or more organisms were present in inoculated ground pork. The 5′-nuclease assay indicated a contamination rate of 35.5% (94/265) in various meats and tofu, whereas the CIN plating method indicated a contamination rate of 28.3% (75/265). This resulted in 100% sensitivity and 64.5% specificity for the 5′-nuclease assay when compared with the standard culture recovery method. Only 75% (60/80) of the Yersinia spp. isolated on CIN was identified as containing a virulence plasmid by autoagglutination and crystal violet binding tests. These results indicate that the true rate of contamination of virulent Y. enterocolitica in pork and other processed meats and foods is being underestimated using current detection methods. This study demonstrates the potential of the 5′-nuclease assay for rapidly and specifically detecting virulent Y. enterocolitica in processed foods with the added advantage of being an automated detection system with high-throughput capability.

Detection of Arcobacter spp. in Ground Pork by Modified Plating Methods

A modified cefsulodin-irgasan-novobiocin (CIN) medium was developed for the recovery of Arcobacter spp. from meats. Modified CIN was compared to brain heart infusion agar supplemented with 10% bovine blood and cephalothin, vancomycin, and amphotericin B (CVA) as well as brain heart infusion agar supplemented with 10% bovine blood and no antibiotics. The three media were used to recover Arcobacter spp. in a survey of pork-processing plants. Examination of ground pork (149 samples) from one Iowa slaughter facility (Plant #1) revealed that 89 percent of the samples were positive for Arcobacter spp. In a second survey conducted 9 months later involving that same plant and four others, only 5% of the samples from the four plants were found to be positive for Arcobacter spp. Again, 90% of the samples were positive from Plant #1. It was not determined whether the sanitary practices during slaughter or the rearing of pigs on the source farms contributed to the prevalence of Arcobacter spp. in one plant versus another.

USE OF UNIVERSAL PREENRICHMENT MEDIUM SUPPLEMENTED WITH OXYRASETM FOR THE SIMULTANEOUS RECOVERY OF ESCHERICHIA COLI O157:H7 AND YERSINIA ENTEROCOLITICA

Universal Preenrichment (UP) medium was used successfully for the simultaneous recovery of two strains each of Escherichia coli O157:H7 and Yersinia enterocolitica in the presence of Listeria monocytogenes and Salmonella typhimurium. E. coli O157:H7 and Y. enterocolitica populations reached ca. 108 CFU/ml in UP medium in 18 h from an initial level ofca. 102 CFU/ml. Addition of OxyraseTM enhanced the growth of both E. coli O157:H7 strains and one strain of Y. enterocolitica. These three strains were able to recover from heat injury by 6 h when 24‐h cultures were tested, but not when 18‐h cultures were used. Injured and noninjured E. coli O157:H7 could be recovered from artificially inoculated food samples (shredded cheddar cheese, turkey ham, hot dogs, mayonnaise, and ground beef) in UP medium supplemented with OxyraseTM (UPO) by 18 h using immunoblotting. Y. enterocolitica could be recovered from turkey ham, hog dogs, and mayonnaise by direct plating on CIN agar from UPO medium. However, recovery of Y. enterocolitica from shredded cheddar cheese and ground beef required subsequent selective enrichment in sorbitol bile broth and isolation on Cefsulodin Irgasan Novobiocin agar (CIN). UPO medium can be used for simultaneous detection of E. coli O157:H7 and Y. enterocolitica from foods. However, subsequent selective enrichment and isolation on selective plating media are required for isolation of Y. enterocolitca from raw foods containing high population levels of background microflora.

Recognition of pathogenic Yersinia enterocolitica by crystal violet binding and polymerase chain reaction.

Cefsulodin-Irgasan-Novobiocin (CIN) agar is used for the selective isolation and enumeration of Yersinia enterocolitica from clinical specimens and food. The medium contains crystal violet and about 1 mmol l-1 calcium and can be used for the phenotypic characterization of strains that carry a virulence plasmid. At 32 degrees C, irrespective of pathogenicity, colonies are translucent with a pale pink centre surrounded by a transparent border ('bullseye'), while at 37 degrees C pathogenic strains grow as calcium-dependent microcolonies which, because of crystal violet binding, are intensely coloured. These results were confirmed by the polymerase chain reaction with primers directed at the virF gene, which is present only in pathogenic strains of Y. enterocolitica. Pathogenic strains of Y. enterocolitica can be recognized by growth at 37 degrees C on Yersinia selective agar.

Isolation of Yersinia enterocolitica from foods

Many selective enrichment and plating media for the isolation of Yersinia enterocolitica from foods are described. However, at present no single isolation procedure is available for the recovery of all pathogenic strains of Yersinia enterocolitica. Cold enrichment in phosphate-buffered saline plus 1% sorbitol and 0.15% bile salts (PBSSB) and two-step enrichment with tryptone soy broth (TSB) and bile oxalate sorbose (BOS) broth are very efficient methods for the recovery of a wide spectrum of serotypes of Y. enterocolitica. Enrichment in irgasan ticarcillin chlorate (ITC) broth was found to be the most efficient method for the recovery of strains of serotype 0:3, which is the most common clinical serotype of Y. enterocolitica in Europe. Post-enrichment alkali treatment often results in higher isolation rates. Cefsulodin irgasan novobiocin (CIN) agar and Salmonella-Shigella deoxycholate calcium chloride (SSDC) agar are the most commonly used plating media. For the recovery of serotype 0:8 strains, the common clinical isolates in North America, enrichment in BOS and plating on CIN seems the most efficient procedure. Selection of the proper enrichment procedure will depend on the bio/serotypes of Yersinia spp. sought and on the type of food to be examined. The use of more than one medium for both enrichment and plating will result in higher recovery rates of Yersinia spp. from foods. Parallel use of the following two isolation procedures is recommended. (1) Enrichment in ITC for 2 days at 24°C; plating on SSDC agar (2 days at 30°C). (2) Pre-enrichment in TSB for 1 day at 24°C; enrichment in BOS for 5 days at 24°C; alkali treatment (mixing 0.5 ml enriched broth with 4.5 ml of 0.5% KOH in 0.5% NaCl for 5 s); plating on CIN agar (2 days at 24°C).

Comparison of Enrichment and Plating Media for Isolation of Yersinia

Yersinia enterocolitica (Serotypes 0:3 or 0:8), Yersinia frederiksenii, Yersinia kristensenii, or Yersinia intermedia along with 108 cells of each of three extraneous organisms (Escherichia coli, Enterobacter aerogenes, Pseudomonas fragi), all commonly found on market poultry, were inoculated into five enrichment media followed by streaking onto 11 plating media to determine the most-efficacious combinations for future surveys or assessment studies. For Yersinia enterocolitica (0:8), infrequent recoveries were made using yeast extract-rosebengal-bile oxalate sorbose broth and phosphate-buffered saline (4 C) followed by plating onto pectin, DNase-Tween 80 (polyoxyethylene sorbitan monooleate)-sorbitol, MacConkey-Tween 80, or cefsulodin-irgasan-novobiocin (CIN) agars. With Y. enterocolitica (0:3), recoveries were most frequently made using phosphate-buffered saline, sorbitol bile (incubated for 17 days) and yeast extract-rosebengal-bile oxalate sorbose broth followed by plating onto pectin, CIN, bismuth sulfite (Difco Laboratories, Detroit, MI), or modified Rimler-Shotts agar. For Y. frederiksenii, Y. kristensenii, and Y. intermedia, incubation in sorbitol bile for 17 days or in yeast extract-rosebengal-bile oxalate sorbose broth, followed by plating onto CIN, pectin, DNase-Tween/80-sorbitol, cellobiose-arginine-lysine agar, or MacConkey-Tween 80 agar yielded the most-frequent recoveries. Overall, the CIN and pectin agars performed best for the recovery of the Yersinia bacterium; the modified selenite broth and the bismuth-sulfite plating agars were unsatisfactory in the present study.

Research Note: The Presence of Yersinia enterocolitica and Other Yersinia Species on the Carcasses of Market Broilers

Sixty ready-to-cook broiler carcasses obtained from several local supermarkets were tested for the presence of Yersinia enterocolitica and other Yersinia species. In the present study, the authors used two enrichment broths, yeast-extract/rosebengal-bile oxalate sorbose (YER-BOS) and phosphate-buffered saline with a postenrichment KOH treatment (PBS-KOH), and two plating media, cefsulodin-irgasan-novobiocin (CIN) and pectin agar. Yersinia organisms were found on 34 of 60 carcasses (56.7%) and Y. enterocolitica, on 16 of 60 carcasses (26.7%). There was no significant difference between CIN and pectin agar; however, PBS-KOH yielded a significantly higher (P≤.05) detection rate than YER-BOS, regardless of the plating media used. In addition to Y. enterocolitica, Y. frederiksenii and Y. intermedia were also isolated from the market broilers. None of the Y. enterocolitica isolates were found to be presumptively virulent.

Improved methods for the isolation of Yersinia species from milk and foods

Recovery of Yersinia spp. following enrichment of milk samples in tris-buffered peptone water pH 8.0 (TPW) incubated at 9 °C was compared with recovery from buffered peptone water pH 7.2 (BPW) incubated at 4 °C. The number of isolations obtained from TPW/9 °C was approximately twice the number obtained from BPW/4 °C. Weekly subculture to Cefsulodin-Irgasan-Novobiocin (CIN) agar for three weeks was found to achieve greatest recovery of Yersinia , but this might be replaced by a single subculture after 11–14 days' enrichment with little loss of recovery. When enrichment of milk and food samples in TPW incubated at 9 °C was followed by post-enrichment alkali treatment, the detection of Yersinia spp. was again improved, particularly from food samples. A regime of enrichment at 9 °C in TPW followed by post-enrichment alkali treatment immediately before subculture to CIN agar is recommended for the recovery of Yersinia spp. from milk and foods.

Comparison of methods for the isolation of Yersinia enterocolitica from porcine tonsils and pork

Three enrichment procedures and three plating media were evaluated for their efficiency in isolating Yersinia enterocolitica from porcine tonsils and pork. Cold enrichment in phosphate-sorbitol-bile medium (PBSSB) with alkali treatment before plating resulted in higher isolation rates than enrichment in modified Rappaport broth (MRB) and bile-oxalate-sorbose broth (BOS). Post-enrichment alkali treatment gave a considerable increase in isolation rate with all the enrichment media tested. A sample inoculum of 10 g showed a better recovery than 0.2 g. Cefsulodin-irgasan-novobiocin (CIN) agar was slightly better than MacConkey agar and MacConkey agar + Tween 80 as isolation medium for Yersinia spp. from porcine tonsils. Most of the serotype 0:3 and 0:9 strains were isolated after enrichment in MRB and PBSSB. Enrichment in PBSSB resulted in the isolation of some other potentially virulent Yersinia strains. For the isolation of Yersinia spp. from pork and porcine tonsils an inoculum of 10 g, parallel use of MRB and PBSSB, post-enrichment alkali treatment and isolation on CIN agar is recommended.

Growth Characteristics of Yersinia enterocolitica in Pasteurized Skim Milk

Experiments were undertaken to determine the growth characteristics of five strains of Yersinia enterocolitica in pasteurized milk at 4°C. Pasteurized milk was inoculated with approximately 10 or 1000 cells/ml of Y. enterocolitica, and was incubated at 4°C for 0, 3, 7, 14 and 21 d. Each sample was spread-plated in duplicate on Tryptone Soya Agar, MacConkey Agar and Cefsulodin-Irgasan-Novobiocin (CIN) agar. Plates were incubated at 25°C for 48 h or at 32°C for 24 h and enumerated for total and Yersinia plate count. All five strains of Y. enterocolitica competed very well with background microflora of pasteurized milk and reached levels of log 5.0 to 7.0/ml after 7 d at 4°C. Level of inoculation had little or no effect on the total number of Y. enterocolitica after 14 or 21 d in pasteurized milk at 4°C. Generation times at 4°C were highly strain-dependent.

Differentiation of Kluyvera cryocrescens from Kluyvera ascorbata by irgasan susceptibility testing

From the faeces of a diarrhoeic patient, we isolated a strain of Kluyvera ascorbata on cefsulodin-irgasan-novobiocin (CIN) agar, a highly selective medium used for Yersinia enterocolitica. Since no selective media have been described for Kluyvera spp., we tested 5 strains each of K. ascorbata and K. cryocrescens for their ability to grow on CIN agar. K. ascorbata exhibited an inhibition on CIN of less than 10% CFU compared to blood agar, whereas K. cryocrescens did not grow at all even with inocula as high as 10(5) CFU per plate. This phenomenon correlated very well with MIC values to irgasan, which were greater than 128 mg/l for K. ascorbata and less than or equal to 0.25 mg/l for K. cryocrescens. No significant differences between the two species were observed for the other two antibiotics contained in CIN. This phenotypic character has been used to differentiate 18 strains belonging to either species on the basis of a simple disk test: with disks containing 5, 25, or 125 micrograms irgasan, K. ascorbata never showed zones of inhibition, whereas K. cryocrescens produced zones of greater than or equal to 15, greater than or equal to 21.5 and greater than or equal to 25.0 mm in diameter, respectively.

Isolation ofAeromonas spp. from human feces

Following our observation that Aeromonas spp. grow on Cefsulodin-Irgasan-Novobiocin (CIN) agar (Altorfer et al., Ahstr. A. Meet. Am. Soc. Microbiol. (1985)) we looked for the presence of Aeromonas spp. in stool specimens using CIN medium during 3 months in summer 1984. From 1282 specimens plated on CIN directly and after enrichment in PBS and Rappaport broth (the same procedur as used for recovery of yersiniae) 31 strains of Aeromonas spp. were isolated (2.4%). In addition, one strain from water was found. Of these 32 strains, 21 were A. eaviae, 5 were A. sobria, 1 was A. hydrophila, and 5 were atypical isolates. These findings were surprising in two ways: 1) A. caviae, our most frequent isolate, is often considered not to be pathogenic, 2) most of our isolates were from patients with intestinal disorders from whom no other enteropathogenic organisms could be isolated. So far, the role of Aeromonas spp. in diarrheal disease is controversial, but our results imply a clinical significance of isolates of all three species.

Yersinia spp. in foods and related environments

Food samples, porcine swabs and environmental swabs were examined for the presence of Yersinia spp. over a 3-year period. A total of 118 strains were isolated from 3717 samples. Most isolates belonged to Y. enterocolitica biotype 1 or Y. intermedia. The most commonly detected source of Yersinia organisms in foods was pasteurized milk. Investigations at one local dairy indicated a need for improved hygiene at the processing plant. Enrichment at 4°C in 1% buffered peptone water followed by subculture to Cefsulodin-Irgasan-Novobiocin (CIN) agar appears suitable for the isolation of Yersinia spp. from a variety of food commodities and environments.

Keeping quality of cefsulodin-irgasan-novobiocin (CIN) medium for detection and enumeration of Yersinia enterocolitica

Freshly produced CIN-agar was stored at +2°C for 14 and 32 days. Both the recovery rate and the colony size of Yersinia enterocolitica were reduced when cultures were drown on media stored for 32 days. The selective principle was not reduced during storage.

Occurrence of Yersinia enterocolitica in Raw and Pasteurized Milk

Raw milk (100 samples) and pasteurized milk (100 samples) were assayed for presence of Yersinia enterocolitica, using four different enrichment and post-enrichment techniques. Isolation of Y. enterocolitica was made on MacConkey agar and Cefsulodin-Irgasan-Novobiocin (CIN) agar directly from incubated broths containing the milk, and after treatment of enrichment broths with a potassium hydroxide solution. Twelve samples of raw milk and one of pasteurized milk contained Y. enterocolitica. Nearly all isolates were found after KOH treatment. All isolates except that obtained from the pasteurized milk sample produced detectable levels of heat-stable enterotoxin. However, none of the 13 isolates agglutinated in WA-SAA, an antiserum specific for virulent Y. enterocolitica.