A chromogenic plating medium for isolating Escherichia coli O157:H7 from beef

There was no significant difference (P > 0.05) in the percentages of Escherichia coli O157:H7 cells recovered on BCM O157:H7 (+) agar (69.7%) and MacConkey sorbitol agar containing 5-bromo-4-chloro-3-indoxyl-beta-D-glucuronic acid (MSA-BCIG) (76.8%) vs Tryptic soy agar. Three E. coli O157:H7 strains (ATCC 35150, 43890 and 43894) were separately inoculated into raw ground beef at low (mean 0.32 cfu g-1) and high (mean 3.12 cfu g-1) levels. Using the United States Department of Agriculture (USDA) m-EC + novobiocin enrichment broth, BCM O157:H7 (+) medium surpassed MSA-BCIG agar with overall percentage sensitivities for BCM O157:H7 (+) of 92.1 and 94.4 compared with 52.6 and 84.7 for MSA-BCIG at low and high levels, respectively. A comparison of BCM O157:H7 (+) and MSA-BCIG agars using naturally contaminated beef samples was made utilizing presumptively positive enrichment broths previously identified by rapid methods. The E. coli O157:H7 cells in these broths were concentrated with Dynabeads anti-E. coli O157 before inoculating the agars. The respective percentage sensitivity and specificity values were 90.0 and 78.5 for BCM O157:H7 (+) and 70.0 and 46.4 for MSA-BCIG. Thus, under identical pre-plating conditions, BCM O157:H7 (+) medium displayed a greater sensitivity than MSA-BCIG for detecting E. coli O157:H7 in artificially inoculated beef, and both greater sensitivity and specificity upon examining naturally contaminated beef samples.     

Suitability of selective plating media for recovering heat- or freeze-stressed Escherichia coli O157:H7 from tryptic soy broth and ground beef.

The efficacy of tryptic soy agar (TSA), modified sorbitol MacConkey agar (MSMA), modified eosin methylene blue (MEMB) agar, and modified SD-39 (MSD) agar in recovering a five-strain mixture of enterohemorrhagic Escherichia coli O157:H7 and five non-O157 strains of E. coli heated in tryptic soy broth at 52, 54, or 56 degrees C for 10, 20, and 30 min was determined. Nonselective TSA supported the highest recovery of heated cells. Significantly (P < or = 0.05) lower recovery of heat-stressed cells was observed on MSMA than on TSA, MEMB agar, or MSD agar. The suitability of MEMB agar or MSD agar for recovery of E. coli O157:H7 from heated or frozen (-20 degrees C) low- or high-fat ground beef was determined. Recovery of E. coli O157:H7 from heated ground beef was significantly (P < or = 0.05) higher on TSA than on MEMB agar, which in turn supported higher recovery than MSD agar did; MSMA was inferior. Recovery from frozen ground beef was also higher on MEMB and MSD agars than on MSMA. Higher populations were generally recovered from high-fat beef than from low-fat beef, but the relative performance of the recovery media was the same. The inability of MSMA to recover stressed cells of E. coli O157:H7 underscores the need to develop a better selective medium for enumerating E. coli O157:H7.     

Culture medium for selective isolation and enumeration of Gram-negative bacteria from ground meats.

We developed a new medium, designated peptone bile amphotericin cycloheximide (PBAC) agar, which contains (per liter) 10 g of peptone, 300 mg of bile salts, 1 mg of amphotericin B, 1 g of cycloheximide, and 15 g of agar. When 21 samples of fresh ground beef were studied and plate count agar counts were used as references, we obtained a mean recovery of 28% of total counts with violet red bile agar overlay, whereas we obtained 48% recovery with PBAC agar. With 12 samples of frozen ground beef, recovery on violet red bile agar overlay was 29% of the recovery on plate count agar, whereas the corresponding value on PBAC agar was 45%. PBAC agar allowed the enumeration of 1.4 times as many gram-negative bacteria as violet red bile agar overlay. None of eight strains of gram-positive bacteria and none of eight strains of yeasts grew on PBAC agar. Of 158 colonies randomly selected from pour plates of eight fresh ground meat samples, 95% stained gram negative. In comparison, only 70% of 151 colonies selected from corresponding plate count agar plates were gram negative. The lack of background color, turbidity, and ease of use make PBAC agar easier to handle than other media used for gram-negative bacteria, such as violet red bile agar, violet red bile agar overlay, and crystal violet tetrazolium agar. In the preparation PBAC agar, all ingredients are autoclaved together except amphotericin B, which is filter sterilized and added before the plates are poured.     

Comparative Study of Two Methods for Detection of Clostridium perfringens in Ground Beef

The tryptose-sulfite-cycloserine agar pour plate method was superior to selective enrichment in liquid sulfite medium for isolation of small numbers of Clostridium perfringens from frozen ground beef.     

Antifungal properties of 2-alkynoic acids and their methyl esters.

Thirteen 2-alkynoic acids and their methyl esters (C3--C12, C14, C16, and C18) were tested against Aspergillus niger, Trichoderma viride, and Myrothecium verrucaria in Sabouraud dextrose agar at pH 4.0 and 5.6. Toxicity to Candida albicans, Trichophyton mentagrophytes, and Mucro mucedo was determined in the same medium at pH 5.6 and 7.0 in the absence and presence of 10% beef serum. The fungitoxicity of the acids was influenced by chain length, pH of the medium, and absence or presence of adsorbents. The toxicity of the esters was influenced primarily by chain length and to a lesser extent by the pH of the medium and the presence of beef serum. The order of activity of the 2-alkynoic acids is C10=C11=C12 greater than C14=C16 greater than C9 greater than C8 greater than C7. When compared with other fatty acid analogs, the order of fungitoxicity on a weight basis is 2-alkynoic acids greater than 2-alkenoic acids greater than alkanoic acids greater than 2-bromoalkanoic acids greater than 2-fluoroalkanoic acids. There is an inverse relationship between chain length and pKa of the acids, suggesting that partition behavior is a fundamental determinant of fungitoxicity along with the effect of adsorbents.     

实验室薄口螨的大量培养及休眠体的诱导

用玉米粉培养基、BY(牛肉膏+酵母膏)软琼脂培养基、BY培养液3种培养基对实验室薄口螨(Histiostoma laboratorium)进行了培养。其最适培养基是玉米粉培养基;该螨在BY软琼脂培养基上也能生长,但生长速度比较缓慢,经过BY软琼脂培养基的培养,能够收集到大量干净的个体,为DNA提取和分子生物学研究提供了方便;在BY培养液中,实验室薄口螨不能进行继代生长,但能够产生大量的卵,可收集卵做更进一步的深入研究。休眠体是该螨生活史中的重要阶段,是借助携播者进行传播的特殊形式。对孳生于培养有果蝇的玻璃指管中的实验室薄口螨产生的休眠体及其在果蝇体表的吸附状况进行了观察,利用较高温度(30～35℃)培养基逐步干燥、较低温度(10～15℃)、BY液体培养3种方法,可以诱导该螨休眠体集中大量地形成,方便收集休眠体,是对其进行生理生化、基因表达、疾病传播机理等方面研究的先决条件。     

DETECTION of ENTEROTOXIGENIC CLOSTRIDIUM PERFRINGENS IN GROUND BEEF

Clostridium perfringens is widely distributed in foods. This experiment was performed to assess occurrence of C. perfringens cultures and toxigenic strains isolated from ground beef Samples (118) were collected from 20 locations in Northeast Kansas and the number of C. perfringens was enumerated in these samples by Fung's Double-tube method with tryptose sulfite cycloserine agar medium. Out of 118 samples, 54 (46%) were found positive for C. perfringens. Pure isolates of C. perfringens were further grown in cooked meat medium for 24 h at 42C then heat shocked at 75C for 20 min and inoculated into modified Duncan and Strong medium for production of C. perfringens enterotoxin. Presence of enterotoxin was tested by the reverse passive latex agglutination test (Oxoid), which can detect enterotoxin up to a minimum level of 2 ng/mL. the data indicate that 46% of the beef samples harbored C. perfringens , but only 32 (6%) of 525 isolates were found to produce enterotoxin. This study emphasized the importance of continued surveillance of C. perfringens in meats and meat products and assessment of the toxigenesis of isolates.     

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 Oxyrase_TM 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 Oxyrase^TM (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.     

Antifungal properties of alpha,omega-alkanedicarboxylic acids and their dimethyl esters.

Thirteen alpha, omega-alkanedicarboxylic acids (C2-C12, C14, and C16) and their dimethyl esters were tested against Aspergillus niger, Trichoderma viride, and Myrothecium verrucaria in Sabourauc dextrose agar at pH 4.0 AND 5.6. Toxicity to Canadida albicans, Trichophyton mentagrophytes, and Mucor mucedo was determined in the same medium at pH 5.6 and 7.0 in the absence and presence of 10% beef serum. The dicarboxylic acids possessed very poor to no antifungal activity against all six fungi. The fungitoxicity of the dimethyl esters to A. niger, T. viride, and M. verrucaria was C8 = C9 greater than C7 greater than C6 = C5 greater than C10 greater than C4 greater than C11 and to C. albicans, T. mentagrophytes, and M. mucedo C9 greater than C10 greater than C11 greater than C12 = C8 greater than C7 greater than C6 greater than C5 greater than C4 greater than C3. The fungitoxicity of the esters of fatty acids and alpha-omega-alkanedicarboxylic acids was influenced by chain length and not by the pH of the medium or the absence or presence of beef serum.     

Detection and production of verotoxin 1 of Escherichia coli O157:H7 in food.

Verotoxin 1 (VT1) is a recognized virulence factor of Escherichia coli O157:H7, a cause of severe food-borne disease. The public health significance of preformed verotoxin in food is unknown, and relatively little research has been done to determine the production of VT1 in food. The purposes of this study were to develop a sensitive method to detect VT1 in milk and in ground beef and to determine the conditions for VT1 production in these foods. A sandwich enzyme-linked immunosorbent assay in which we used VT1-specific monoclonal antibody 9C9F5 as the capture antibody and a rabbit polyclonal antibody raised against VT2 as the detection antibody was developed for the detection and quantification of VT1 in milk and in ground beef. The enzyme-linked immunosorbent assay was sensitive to a minimum of 0.5 ng of VT1 per ml of milk and 1.0 ng of VT1 per g of ground beef. The greatest amount of VT1 detected in milk (306 ng/ml) was detected in samples that were incubated at 37 degrees C with agitation (160 rpm) for 48 h. Very little toxin (1 ng/ml) was produced at 25 or 30 degrees C within 96 h. VT1 production was greater in ground beef than in milk; 452 ng of VT1 per g was produced in beef at 37 degrees C in 48 h. Relatively little VT1 was produced in beef within 96 h at 25 and 30 degrees C (2.1 and 9.8 ng of VT1 per g, respectively). Our results indicate that ground beef is a better medium for VT1 production than milk.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection and production of verotoxin 1 of Escherichia coli O157:H7 in food.

Verotoxin 1 (VT1) is a recognized virulence factor of Escherichia coli O157:H7, a cause of severe food-borne disease. The public health significance of preformed verotoxin in food is unknown, and relatively little research has been done to determine the production of VT1 in food. The purposes of this study were to develop a sensitive method to detect VT1 in milk and in ground beef and to determine the conditions for VT1 production in these foods. A sandwich enzyme-linked immunosorbent assay in which we used VT1-specific monoclonal antibody 9C9F5 as the capture antibody and a rabbit polyclonal antibody raised against VT2 as the detection antibody was developed for the detection and quantification of VT1 in milk and in ground beef. The enzyme-linked immunosorbent assay was sensitive to a minimum of 0.5 ng of VT1 per ml of milk and 1.0 ng of VT1 per g of ground beef. The greatest amount of VT1 detected in milk (306 ng/ml) was detected in samples that were incubated at 37 degrees C with agitation (160 rpm) for 48 h. Very little toxin (1 ng/ml) was produced at 25 or 30 degrees C within 96 h. VT1 production was greater in ground beef than in milk; 452 ng of VT1 per g was produced in beef at 37 degrees C in 48 h. Relatively little VT1 was produced in beef within 96 h at 25 and 30 degrees C (2.1 and 9.8 ng of VT1 per g, respectively). Our results indicate that ground beef is a better medium for VT1 production than milk.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oil Cakes as Media for Growing Catenaria anguillulae Sorokin, a Facultative Endoparasite of Nematodes

Summary Growth, morphology, visibility of sporangia and colony colour of 10 isolates of Catenaria anguillulae were compared on six media: linseed oil-cake agar, mustard oil-cake agar, neem oil-cake agar, beef extract agar, Emerson agar and YPSS agar with a view to selecting the best growth medium. In general, maximum radial growth of most of the isolates was recorded on linseed oil-cake agar medium, whereas neem oil-cake agar medium supported least growth of all the isolates of C. anguillulae. Linseed oil-cake agar medium also maintained the typical characters of the fungus and clear visibility of morphological details.     

Gentamicin-thallous-carbonate medium for isolation of fecal streptococci from foods.

Gentamicin-thallous-carbonate (GTC) agar was formulated by Donnelly and Hartman (Appl. Environ. Microbiol. 35:576-581, 1978) to select for fecal streptococci in sewage and water samples. The present study was conducted to determine the usefulness of GTC agar for the enumeration of fecal streptococci in foods. Comparisons were made with KF streptococcal (KF), Pfizer selective enterococcus (PSE), and thallous acetate (TA) agars. Samples of ground beef pork sausage, frozen broccoli, frozen fish, and ice cream were examined. Presumptive streptococcal counts obtained on GTC agar were significantly higher than those obtained on KF and PSE agars and were comparable to those obtained on TA agar. GTC was more sensitive than KF or PSE agars primarily because of the recovery of greater numbers of Streptococcus bovis and Streptococcus equinus strains. Percentages of confirmed fecal streptococci obtained on GTC, KF, PSE, and TA agars were 70, 95, 80, and 74, respectively. Differences between these percentages were not statistically significant, but they indicated that selectivity of GTC agar could be improved. Advantages of using GTC agar to isolate fecal streptococci from foods include a short incubation time (16 to 18 h) and large, distinct colonies that facilitate rapid enumeration and subsequent confirmation.     

Gentamicin-thallous-carbonate medium for isolation of fecal streptococci from foods

Gentamicin-thallous-carbonate (GTC) agar was formulated by Donnelly and Hartman (Appl. Environ. Microbiol. 35:576-581, 1978) to select for fecal streptococci in sewage and water samples. The present study was conducted to determine the usefulness of GTC agar for the enumeration of fecal streptococci in foods. Comparisons were made with KF streptococcal (KF), Pfizer selective enterococcus (PSE), and thallous acetate (TA) agars. Samples of ground beef pork sausage, frozen broccoli, frozen fish, and ice cream were examined. Presumptive streptococcal counts obtained on GTC agar were significantly higher than those obtained on KF and PSE agars and were comparable to those obtained on TA agar. GTC was more sensitive than KF or PSE agars primarily because of the recovery of greater numbers of Streptococcus bovis and Streptococcus equinus strains. Percentages of confirmed fecal streptococci obtained on GTC, KF, PSE, and TA agars were 70, 95, 80, and 74, respectively. Differences between these percentages were not statistically significant, but they indicated that selectivity of GTC agar could be improved. Advantages of using GTC agar to isolate fecal streptococci from foods include a short incubation time (16 to 18 h) and large, distinct colonies that facilitate rapid enumeration and subsequent confirmation.     

A New Immunoassay for Detecting All Subtypes of Shiga Toxins Produced by Shiga Toxin-Producing E. coli in Ground Beef

Background

Shiga toxin (Stx) is a common virulence factor of all Shiga toxin producing E. coli (STEC) that cause a wide spectrum of disease, including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Although several commercial kits are available for detection of Stx produced by STEC, none of them are capable of recognizing all subtypes of Stxs, which include three subtypes of Stx1 and seven subtypes of Stx2.

Methods and Findings

New monoclonal and polyclonal antibodies against Stx1 and Stx2 were developed. A universal sandwich ELISA capable of detecting all known subtypes of Stx1 and Stx2 was established using a pool of newly developed antibodies. To precisely monitor the sensitivity of the assay for each subtype of Stxs, recombinant toxoids were created and used as standards in ELISAs. Because of the high affinity of the antibodies incorporated, the ELISA assay is highly sensitive with a limit of detection for the different subtypes of Stx1a and Stx2a between 10 and 50 pg/mL in phosphate buffered saline (PBS). The assay was also able to identify STEC based on the production of Stxs using the supernatants of culture fluids or even single colonies on agar plates without lengthy enrichment in liquid medium. When applied to ground beef samples, this newly developed ELISA was capable of distinguishing beef samples spiked with a single bacterial cell.

Conclusions

A highly sensitive and universal assay for all subtypes of Stx1 and Stx2 was developed. It has significantly improved upon the current technologies by avoiding false negative results due to the narrow detection range of the assay. The assay developed in this study can be useful for prompt detection of new and emerging serotypes and screening ground beef samples for contamination of STEC at an early stage in the food supply chain, thus avoiding the need for possible recall.     

Gamma Radiation Inactivation of Coxsackievirus B-2

The radioresistance of coxsackievirus B-2 was studied when the virus was suspended in Eagle minimal essential medium, distilled water, cooked ground beef, and raw ground beef and irradiated at various temperatures in a cobalt-60 gamma radiation source. The number of surviving viruses at given doses of radiation was determined by a plaque assay system. All destruction curves indicated a first-order reaction. When the virus was irradiated in minimal essential medium at temperatures of -30, -60, and -90 C, D values (in Mrad) were 0.69, 0.59, and 0.64, respectively. When the virus was suspended in water and irradiated at -90 C, the D value was 0.53. Cooked ground beef containing the virus was irradiated at temperatures ranging from 16 to -90 C. The D values were 0.70 (16 C), 0.76 (0.5 C), 0.68 (-30 C), 0.78 (-60 C), and 0.81 (-90 C). Raw ground beef containing the virus was irradiated at -30, -60, and -90 C, and the D values were respectively 0.75, 0.71, and 0.68. The D values indicate that the rate of viral inactivation was dependent on the suspending menstrum.     

Comparison of Methods for the Recovery of Virus Inoculated into Ground Beef

Various methods for the recovery of virus inoculated into ground beef were investigated in an attempt to develop a sensitive system that could be used to detect viral contaminants in market foods. A 100-g sample, inoculated with poliovirus 1, was suspended in 150 to 900 ml of Eagle minimum essential medium, pH 8.5, and mixed in either plastic bags or plastic cups on a mechanical shaker. The particulate materials were removed by means of cheese cloth, glass wool, woven fiber glass, or low-speed centrifugation. Large volumes of fluid were concentrated by ultrafiltration. Microbiological contamination was controlled by high antibiotic concentrations or by filtration. Quantitative plaque-forming-unit recovery of the virus was determined by utilizing an agar overlay technique on Vero cell cultures. The data indicated that from 20 to 50% of the seeded virus could be recovered from a 100-g sample of ground beef. The glass wool and woven fiber glass methods were the most effective, with recovery of approximately 50% of the inoculated virus.     

Substituted Diazenes: Effect on the Growth of Enterobacteria and Possible Use as Selective Agents for Isolation of Pseudomonads

Incorporation of various diazenes into Trypticase soy media appeared selectively to permit the growth of pseudomonads while inhibiting the growth of a variety of enterobacteria. One of these diazenes, diamide (diazenedicarboxylic acid bisdimethylamide), was shown to be bactericidal for pure cultures of Escherichia coli, Proteus sp., and Salmonella enteritidis and to cause a 1- to 2-hr delay in the growth of Pseudomonas aeruginosa. When mixtures of these four organisms were inoculated into Trypticase soy broth or Trypticase soy agar (TSA) containing diamide, P. aeruginosa grew in overnight cultures. TSA containing diamide was also used successfully to isolate pseudomonads from soil, clinical urine specimens, fish, ground beef, ground pork, and ground veal.     

Polymyxin-based enzyme-linked immunosorbent assay for the detection of Escherichia coli O111 and O26

Polymyxin-based enzyme-linked immunosorbent assay (polymyxin-ELISA) systems were developed for the detection of Escherichia coli O111 and O26 in ground beef after enrichment. Polymyxin immobilized in the wells of a microtiter plate served as a high affinity adsorbent for lipopolysaccharide (LPS) antigens, which were detected immunoenzymatically using commercially available anti-E. coli O111 or anti-E. coli O26 antisera. The polymyxin-ELISA sensitively detected E. coli strains bearing the O111 and O26 LPS antigens, discriminating between these target strains and a panel of various non-target Gram negative and Gram positive bacteria. The detection of E. coli O111 and O26 strains inoculated into ground beef was achieved after enrichment in either modified trypticase soy broth (TSB) with novobiocin, or the serotype-specific medium TSB supplemented with cefixime and vancomycin (E. coli O111), and the same medium containing potassium tellurite (E. coli O26). The polymyxin-ELISA shows promise as a rapid, simple and inexpensive screening tool for E. coli O111 and O26 in enrichment cultures of ground beef.     

MINIATURIZED ANAEROBIC CULTIVATION METHODS FOR RECOVERY OF CLOSTRIDIUM SPOROGENES FROM MEAT

Two new miniaturized methods (sandwiched microtiter plate [SMP] and mini-tube) for recovery of Clostridium sporogenes from food samples were developed and evaluated. One hundred microliter of SFP (Shahidi Ferguson Perfringens) agar and 10 üL of diluted food sample were used in the SMP anaerobic system. The samples were sandwiched between two sterile microtiter plates to create an anaerobic environment. Black colonies in the sandwiched wells were counted as C. sporogenes. In case of mini-tube system, 1 mL of SFP agar (45C) containing diluted food sample was aspirated into a 1.2 mL sterile pipet and allowed to solidify in place. The two ends are sealed to create an anaerobic environment. Black colonies were counted directly through the plastic tube. C. sporogenes was inoculated into ground beef samples for recovery study. The recovery rates in SFP agar, using the SMP and mini-tube method were compared with the double-tube method. Organisms were recovered more in the double-tube than with SMP method and mini-tube method after 24–30 h incubation. However, the final counts at 48 h were similar in all methods from the food samples. These new simple methods have potential use for recovery of Clostridium spp.