Comparison of Two Culture-Based Detection Systems for the Isolation of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii in Raw Ground Poultry.

Arcobacters are emerging pathogens that have been underestimated due to a lack of a standardized isolation method. The aim of this research was to evaluate the ability to isolate Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii using two Arcobacter-specific culture detection systems: (i) the Houf broth and modified charcoal cefoperazone deoxycholate agar supplemented with cefoperazone, amphotericin B, and teicoplanin (HB/mCCDA+CAT), and (ii) the Nguyen-Restaino-Juárez Arcobacter enrichment broth and chromogenic agar (NRJ-B/M). Both detection systems were evaluated for productivity ratio, sensitivity, and specificity. As a result, the productivity ratio for both plating agars were >90%, which indicates that the selective agents used in the two plating agars did not inhibit Arcobacter growth. Moreover, sensitivity evaluations using artificially inoculated retail ground poultry (n = 780) determined that both detection systems were able to isolate A. butlzeri with >95% sensitivity at the 0.1 and 1.0-2.0 CFU/g detection level. The sensitivity in A. cryaerophilus isolation was higher for NRJ-B/M (78.0% at 0.1 CFU/g; 95.1% at 1.0-2.0 CFU/g) when compared with HB/mCCDA+CAT (34.1% at 0.1 CFU/g; 51.2% at 1.0-2.0 CFU/g). Both detection systems resulted in <50% sensitivity when isolating A. skirrowii at 0.1 and 1.0-2.0 CFU/g; however, the sensitivity for NRJ-B/M was significantly higher than HB/mCCDA+CAT. At the detection level of 5.0 CFU/g, both detection systems were able to isolate A. skirrowii with 100% sensitivity. Specificity comparisons using uninoculated ground poultry samples (n = 40) indicated the growth of background microbiota were significantly inhibited or could be easily differentiated on NRJ-B/M (90.0%, specificity) when compared with HB/mCCDA+CAT (30.0%, specificity). Overall, these results show that the NRJ-B/M detection system is a more sensitive and specific detection system when isolating Arcobacter spp. from ground chicken.

NRJ Media as the Gold-Standard Arcobacter-Specific Detection System: Applications in Poultry Testing.

Arcobacter species are ubiquitous emerging pathogens with an impact that has been underestimated due to limitations in isolation and detection methods. Our group recently developed the novel NRJ Arcobacter-detection system, with major improvements in specificity and selectivity compared to other culture-based methods. In this work, the NRJ detection system was evaluated using retail whole broiler chicken carcass. Nanopore 16S rRNA gene amplicon sequencing demonstrated that Arcobacter species are found in very low abundance in retail chicken and that indigenous microbiota could be a major factor interfering with detection. Comparison of the microbiome obtained from modified Houf broth (HB) method, as the standard detection system, and the novel NRJ method, showed Arcobacter abundances of <15% and >97%, respectively. The NRJ system significantly inhibits the growth of non-target microbiota, and specifically allows the multiplication of Arcobacter species. In this report, we describe the gold-standard of Arcobacter-specific culture-based method to test food matrices, which can be used for other applications, such as clinical and environmental sampling.

A New Method for Detection of Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii Using a Novel Chromogenic Agar.

ABSTRACT: Arcobacter species are gram-negative rods that have been implicated in food- and waterborne illness. Although various cultural isolation methods have been proposed, the current procedures are unable to fully suppress the growth of background microbiota present in food samples, which inhibits Arcobacter isolation. The purpose of this study was to develop a selective enrichment broth and chromogenic plating medium to detect three Arcobacter species that have been recognized as emerging foodborne pathogens: Arcobacter butzleri, Arcobacter cryaerophilus, and Arcobacter skirrowii. The developed Nguyen-Restaino-Juárez (NRJ) Arcobacter detection system consists of a selective enrichment broth (NRJ-B) and a selective-differential plating medium (NRJ-M). The protocol of the detection method was determined by evaluating the growth of A. butzleri, A. cryaerophilus, and A. skirrowii under various temperatures (30, 35, and 42°C) and incubation (aerobic, microaerophilic, and anaerobic) conditions. Additionally, 47 Arcobacter strains and 39 non-Arcobacter strains were tested in inclusivity and exclusivity evaluations of NRJ-B and NRJ-M. Overall, the study determined that the optimal growth conditions of Arcobacter species using the NRJ Arcobacter detection system were aerobic incubation at 30°C. NRJ-B supported good growth of A. butzleri, A. cryaerophilus, and A. skirrowii while effectively suppressing the growth of non-Arcobacter strains after 48 h. Furthermore, NRJ-M yielded 97.8% inclusivity and 100.0% exclusivity using the tested strains and resulted in salmon-pigmented Arcobacter colonies (1.0 to 1.5 mm in diameter) after 72 h. The novel protocol is the first to develop a chromogenic plating medium for the isolation of Arcobacter species. This simple and accurate test method would greatly contribute to understanding the distribution of pathogenic Arcobacter species in food samples.

A selective chromogenic agar that distinguishes Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis.

A selective and differential plating medium, R & F anthracis chromogenic agar (ACA), has been developed for isolating and identifying presumptive colonies of Bacillus anthracis. ACA contains the chromogenic substrate 5-bromo-4-chloro-3-indoxyl-choline phosphate that upon hydrolysis yields teal (blue green) colonies indicating the presence of phosphatidylcholine-specific phospholipase C (PC-PLC) activity. Among seven Bacillus species tested on ACA, only members of the Bacillus cereus group (B. anthracis, B. cereus, and B. thuringiensis) produced teal colonies (PC-PLC positive) having cream rings. Examination of colony morphology in 18 pure culture strains of B. anthracis (15 ATCC strains plus AMES-1-RIID, ANR-1, and AMED-RIID), with one exception, required 48 h at 35 to 37 degrees C for significant color production, whereas only 24 h was required for B. cereus and B. thuringiensis. This differential rate of PC-PLC synthesis in B. anthracis (due to the truncated plcR gene and PlcR regulator in B. anthracis) allowed for the rapid differentiation on ACA of presumptive colonies of B. anthracis from B. cereus and B. thuringiensis in both pure and mixed cultures. Effective recovery of B. anthracis from a variety of matrices having both high (soil and sewage) and low microbial backgrounds (cloth, paper, and blood) spiked with B. anthracis ANR-1 spores suggests the probable utility of ACA plating for B. anthracis recovery in a diversity of applications.

Antimicrobial Effectiveness of Potassium Sorbate and Sodium Benzoate against Zygosaccharomyces bailii in a Salsa Mayonnaise.

The antimicrobial effectiveness of sodium benzoate and potassium sorbate (0.05, 0.10, 0.20 and 0.30%, wt/wt), separately and in equal combinations, were evaluated against the growth of Zygosaccharomyces bailii in an artificially inoculated salsa mayonnaise stored at room temperature (23 to 25°C). Potassium sorbate was able to suppress the growth of Z. bailii significantly (P < 0.05) more than sodium benzoate did, whereas no significant difference in growth was calculated between potassium sorbate and the combination in equal amounts of the two preservatives. Equal concentrations of the two preservatives were, however, significantly (P < 0.05) more effective than sodium benzoate in suppressing the growth of the yeast in a salsa mayonnaise. At the investigated concentrations, the preservative systems did not prevent spoilage of the product by Z. bailii . Therefore, the use of yeast-free ingredients, clean and sanitary equipment, and strict adherence to good manufacturing practices during manufacture and packaging is required to produce a salsa mayonnaise free of spoilage microorganisms.

Efficacy of Petrifilm™ VRB for Enumerating Coliforms and Escherichia coli from Frozen Raw Beef.

Petrifilm™ violet red bile (PVRB) compared favorably to the most probable number method (MPN) and violet red bile agar (VRBA) methods for enumerating coliforms from frozen raw ground beef. When comparing PVRB and VRBA incubated at 35°C, coliform enumeration displayed a linear relationship (correlation coefficient of 0.932). However, by analyzing 64 ground beef samples, PVRB enumerated 41% more coliforms/g than did VRBA. Two distinct colony types were observed on PVRB: (a) type I (butterfly in appearence) with a colony diameter equal to or greater than 1 mm and gas bubbles 2-4 mm in diameter touching the associated colony; and (b) type II with a colony diameter less than 1 mm in diameter and gas bubbles of the associated colony not necessarily touching the colony but within a colony diameter. The disparity between PVRB and VRBA for enumerating coliforms was attributed to non-coliforms representing approximately 50% of the type II coliform colonies. At 35°C, 83.7% of the type I colonies were Escherichia coli , whereas only 10.9%, of the type II colonies were E. coli . By elevating the incubation temperature from 35°C to 44.5°C, over 90% of the colonies in the counting dilution were type I of which 99.2% were E. coli . At 44.5°C, 39.4% of the type II colonies were E. coli ; however, this colony type represented only 9.5% of the total colonies on PVRB. Therefore, a reliable method for enumerating E. coli from raw meat was developed by counting only the type I colonies on PVRB incubated at 44.5°C.

Evaluation of the ß-Glucuronidase Substrate 5-Bromo-4-Chloro-3-Indolyl-ß-D-Glucuronide (X-GLUC) in a 24-Hour Direct Plating Method for Escherichia coli.

A 24-h direct plating method for Escherichia coli using Peptone-Tergitol agar was used to compare the effectiveness of the chromogenic substrate 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide (X-GLUC) with the fluorogenic substrate 4-methylumbelliferyl-ß-D-glucuronide (MUG) for ß-glucuronidase activity. Values obtained for enumeration of two strains of E. coli recovered from artificially inoculated raw minced chicken (i.e., plating efficiencies on the inoculum, cells per g, and recovery percentages elated to those on Plate Count Agar) indicate that X-GLUC at 50 µg/ml was as effective as MUG in an agar medium. Unlike MUG, X-GLUC does not require ultraviolet light illumination, and the color reaction produced remains localized in the positive colonies.

Effect of Acids and Sorbate Combinations on the Growth of Four Osmophilic Yeasts.

The effects of hydrochloric, citric, lactic, phosphoric and malic acids in combination with potassium sorbate on the growth of Saccharomyces bailii , Saccharomyces acidifaciens ( Saccharomyces bailii var. osmophilus ), Saccharomyces rouxii and Saccharomyces bisporus were evaluated. Double strength potato dextrose broth supplemented with 58% (wt/vol) sucrose, 14% (wt/vol) glucose, and 0.2% agar acidulated to a pH of 5.0 to a final aw of 0.88 to 0.89 was used as the growth medium. In general, at 0.05% potassium sorbate, S. rouxii and S. bisporus were more resistant than S. bailii and S. acidifaciens to the antimycotic agent independent of the acid used to acidulate the growth medium, whereas 0.1% potassium sorbate inhibited the growth of the four yeast strains. At 0.05% potassium sorbate, growth occurred (1 log number yeast cells/ml) for S. acidifaciens in the lactic acid/sorbate combination after 36 h of incubation, whereas a bacteriostatic relationship existed for the other acids employed. Citric acid potentiated the antimicrobial effectiveness of 0.05% potassium sorbate at pH 5.0 against the growth of S. rouxii and S. bisporus by either delaying the lag phase or reducing the growth rate.

Use of the Chromogenic Substrate 5-Bromo-4-Chloro-3-Indolyl-B-D-Glucuronide (X-GLUC) for Enumerating Escherichia coli in 24 H from Ground Beef.

A 24-h direct plating method for Escherichia coli using the chromogenic substrate 5-bromo-4-chloro-3-indolyl-B-D-glucuronide (X-GLUC) incorporated into a Peptone-tergitol agar base (PTX) was compared with the standard 3-tube Most Probable Number (MPN) method on 50 naturally contaminated ground beef samples. A paired-comparisons t-test showed no significant difference between the two methods. A positive linear correlation between the two methods was observed over the entire range of values. Ninety-seven percent of the positive colonies (blue colonies) on PTX agar were indentified as E. coli , whereas no atypical colonies (nonblue) were characterized as such. Thus, a simple and reliable enumeration of E. coli can be made within 24 h using the X-GLUC substrate in a selective agar as an indicator of B-glucuronidase activity.

Comparison of ß-Glucuronidase and Indole-Based Direct Plating Methods for Enumerating Escherichia coli in Artificially Inoculated Ground Meats.

Peptone tergitol glucuronide (PTG) agar containing 4-methylumbelliferyl-ß-D glucuronide (MUG) (for ß-glucuronidase activity), the Holbrook, Anderson, Baird-Parker (HABP) method (for detecting indole production), and the standard 3-tube most probable number (MPN) method were compared with plate count agar (PCA) for enumerating three strains of unstressed Escherichia coli artificially inoculated into ground beef and chicken at 1-6 × 106 cells/g. No significant difference (P>0.05) was determined between PTG agar and PCA in the recovery of E. coli . The MPN method enumerated a significantly greater (P<0.05) number of E. coli cells than PCA. Compared with PCA, the HABP method recovered a significantly lower (P<0.05) number of E. coli cells from chicken, whereas no significant difference (P>0.05) was obtained with ground beef. When combining all data from chicken and beef, the recovery of E. coli cells by the HABP method was also significantly lower (P<0.05). Overall, based on the enumeration of E. coli on PCA, the HABP method, PTG agar, and MPN method recovered 57, 102, and 144%, respectively.

Antimicrobial Efficacy of a New Organic Acid Anionic Surfactant Against Various Bacterial Strains.

The surface sanitizing properties of a buffered organic acid anionic surfactant (BOAAS) was compared with six traditional sanitizers (organic chlorine - 100 ppm, two iodophors - 25 ppm, peroxyacetic acid - 483 ppm, acid anionic - 230 ppm, and a quaternary ammonium compound - 150 ppm) in its ability to reduce Staphylococcus aureus on an inoculated Formica surface. In the absence of organic material, the traditional sanitizers were not significantly different (P > 0.05) from water in reducing S. aureus at time 0. whereas ≥ 1.2% of the BOAAS reduced a significantly greater (P < 0.05) number of bacteria. When compared with water over 60 min, only the BOAAS significantly reduced (P < 0.05) S. aureus cells. Sixty minutes after exposure, a 1.75% concentration of the BOAAS was > 100× more effective than organic chlorine. Overall, the organic material reduced the effectiveness of the traditional sanitizers and BOAAS. In the presence of 0.5% protein, BOAAS levels ≥ 0.6% significantly (P < 0.05) reduced more S. aureus cells than the quaternary ammonium sanitizer immediately after application. BOAAS concentrations ≥ 0.6% were significantly (P < 0.05) more effective in reducing S. aureus during a 60 min exposure than the organic chlorine sanitizer. In a separate efficacy study, a BOAAS concentration of 0.6% killed >5 logs of S. aureus , Salmonella typhimurium , Pseudomonas aeruginosa and Listeria monocytogenes cells after 30 s exposure.

Antibody-Direct Epifluorescent Filter Technique and Immunomagnetic Separation for 10-h Screening and 24-h Confirmation of Escherichia coli O157:H7 in Beef.

Rapid screening of beef for the presence of Escherichia coli O157:H7 was shown to be feasible using a 10-h enrichment in modified buffered peptone water and the antibody-direct epifluorescent filter technique (Ab-DEFT). The Ab-DEFT involved membrane filtration, fluorescent antibody staining and epifluorescence microscopy and was accomplished in less than 1 h. The procedure allowed detection of the pathogen artificially inoculated into beef patties at 0.1 CFU/g. The 10-h nonselective enrichment broth supported rapid growth, which provided sufficient numbers of cells for a positive determination by the Ab-DEFT after fewer than 10 microscope fields were scanned using a 40× objective lens. Immunomagnetic separation using anti- E. coli O157 Dynabeads® was used to confirm presumptively positive cultures within 24 h. The ease and rapidity of the Ab-DEFT may provide a substantial time and cost savings to the beef industry for screening beef for the presence of E. coli O157:H7.