Bacterial Bioluminescence: Applications in Food Microbiology.

Many marine microorganisms ( Vibrio , Photobacterium ) are capable of emitting light, that is, they are bioluminescent. The light-yielding reaction is catalyzed by a luciferase, and it involves the oxidation of reduced riboflavin phosphate and a long-chain aldehyde in the presence of oxygen to produce a blue green light. The genes responsible for the luciferase production, (lux A and lux B), aldehyde synthesis (lux C, D, and E), and regulation of luminescence (lux I and lux R) have all been identified, and recent research has resulted in the discovery of three new genes (lux F, G, and H). The ability to genetically engineer dark microorganisms to become light emitting by introducing the lux genes into them has opened up a wide range of applications of bioluminescence. Assays using bacterial bioluminescence for the detection and enumeration of microorganisms are rapid, sensitive, accurate, and can be made specific. It is these attributes that are making in vivo bioluminescent assays so attractive to the food industry.

Use of low molecular mass RNA profiles to identify lactic acid bacteria and related organisms associated with foods.

Fourteen strains of lactic acid bacteria and species of Brochothrix, Carnobacterium, Enterococcus, Erysipelothrix, Kurthia and Listeria were examined using low molecular mass RNA (5S rRNA and tRNA) profiles. These profiles were developed on denaturing polyacrylamide gels. Gel strengths between 9 and 14% were tested to improve resolution of distinct bands for densitometrical analysis. Profiles generated on 12% gels proved to be the best for scanning. Scans of class 2 tRNAs by densitometry showed a characteristic profile for each genus. In the case of Lactobacillus each species studied gave a unique profile. The technique of low molecular mass RNA profiling may provide a useful means for identifying different bacteria from ecosystems such as meats.

Differentiation of the genus Listeria from other gram-positive species based on low molecular weight (LMW) RNA profiles.

Low molecular weight RNA (LMW RNA; 5S rRNA and tRNAs) profiles of several Gram-positive species were generated on 9% denaturing polyacrylamide gels. The profiles of five Listeria spp. (L. innocua, L. ivanovii, L. monocytogenes, L. seeligeri and L. welshimeri) were identical when cultured in three different media (APT, BHI and TSBYE), either shaken or statically, both at 30 and 37 degrees C. Twenty-six strains from 15 other species representing seven different genera were also compared. Each species exhibited a characteristic profile. Strain variants of the same species gave identical profiles. The technique represents a simple, reproducible approach to the identification of species and possibly of relationships between species. The taxonomic and phylogenetic implications, particularly with respect to Listeria spp., Brochothrix thermosphacta and the lactic acid bacteria, are considered.

Influence of freezing-thawing and refrigeration on R-plasmid (pRPJ24) stability in Enterobacter cloacae 94R.

The effect of twenty freezing (-20 degrees C) and thawing cycles of Enterobacter cloacae 94R cells containing the R-plasmid pRPJ24 inoculated into broth and ground beef meat samples revealed no loss of resistance due to plasmid instability. In addition, low temperature storage at 4 degrees C did not produce any significant loss of the tetracycline and kanamycin resistances encoded on the pRPJ24 plasmid. The results of this study indicated that indigenous R-plasmids like pRPJ24 are stable in resident recipients like E. cloacae 94R in ground beef. However, the proportion of viable cells containing the pRPJ24 plasmid decreased significantly after 20 freezing-thawing cycles over 14 days incubation at 4 degrees C.

Occurrence of aminoglycoside phosphotransferase subclass I and II structural genes among Enterobacteriaceae spp. isolated from meat samples.

3'-Aminoglycoside phosphotransferase [APH(3')] enzymes are a group responsible for resistance to the antibiotics kanamycin (Km) and neomycin (Nm) in bacteria. Escherichia coli ECT24, originally isolated from a meat sample, harboured an 83-kb conjugative R-plasmid (pRPJ24) that carries transferable resistance to Km and Nm. Plasmid pRPJ24 was transferred by conjugation to Enterobacter cloacae 94R, which was used as the source of plasmid DNA in development of a probe for the Km-resistance determinant. Random cloning of BamHI and HindIII double-digest restriction fragments of pRPJ24 in the pUC18 vector plasmid produced clones resistant to both Nm and Km carrying a 1.9-kb DNA insert. Southern hybridization of pRPJ24 cloned chimeric plasmid DNA (pKPJ94) showed homology with the APH(3')II gene from transposon Tn5. A PstI digest of pKPJ94 produced a 920-bp fragment which hybridized with the APH(3')II structural gene, and was used as a DNA probe for the APH(3')II subclass gene. A 980-bp BamHI fragment from plasmid pGH54 carrying the APH(3')I gene from transposon Tn903 was used as a subclass I probe. Total DNA from 206 randomly screened Km-resistant Enterobacteriaceae isolates from raw ground beef and chicken meat samples were examined for the occurrence of APH(3') subclass I and II using non-radioactively-labelled DNA probes. Thirty-six percent and 60% of the isolates examined carried subclass I and II resistances, respectively, in the isolates from chicken meat samples. The corresponding values for bacterial strains from raw ground beef samples were 51% and 72%, respectively. Four percent of the resistant bacterial isolates from chicken samples did not display homology to either probe.(ABSTRACT TRUNCATED AT 250 WORDS)

Additive Effects of Alcohols, Their Acidic By-Products, and Temperature on the Yeast Pachysolen tannophilus.

The effects of alcohols on the growth and fermentation of the yeast Pachysolen tannophilus were investigated at both 30 and 35 degrees C. Addition of alcohols to the culture medium decreased both the growth rate and the final cell yield in a dose-dependent manner, and this decrease was more severe at 35 degrees C. The concentration for 50% growth rate inhibition decreased as the chain length of the alcohol increased. In fermentations using a high initial cell density, production of acids was always observed when the medium was supplemented with alcohols. Supplementation of the culture medium with a short-chain alcohol plus the corresponding acid was shown to exert an additive deleterious effect on fermentation, and this effect increased with temperature. Production of acids was associated with the presence of alcohol dehydrogenase activity in cell extracts.

Use of plasmid profiles and restriction endonuclease digest in environmental studies of Listeria spp. from raw milk.

Forty-eight isolates of Listeria spp. (19 L. monocytogenes, 27 L. innocua, 2 L. welshimeri) from bulk raw milk were screened for plasmid DNA. These isolates were collected over a period of 1 year. Only L. innocua harboured plasmids (8/27 strains) which ranged in size 10-44 megadaltons. The plasmid bearing strains could be arranged into three groups 10 Md, 44 Md and 44 + 10 Md. In two farms where Listeria innocua were persistent in bulk raw milk different plasmids profiles were found (farm 1 and 3). In species carrying similar plasmid profiles (44 Md) isolated from the same bulk tank raw milk samples over a period of 2-4 months (farm 2) no similarities in restriction endonuclease digest patterns of the plasmids were observed. This study suggests there may be a constant influx of Listeria spp. into raw milk supplies on the farm.

Persistence at source of Listeria spp. in raw milk.

From 36 of 315 bulk tank sources of raw milk found to harbour Listeria spp., 34 were available for resampling at intervals to determine persistence of the organisms. Listeriae were reisolated from 21 sources. In 16 Listeria spp. were isolated in one retest. From the other five listeriae were obtained in more than one retest. Listerial populations were not particularly persistent. In all but one instance listeriae were not reisolated more than 5 months after initial sampling. Intermittent variations in Listeria spp. isolated were observed. Some repeat samples yielded the same species as originally identified, but sometimes only one of originally two species was isolated. On occasions completely different or additional species were found. The aetiology of listeriosis in cattle and contamination of raw milk is discussed.

Detection of Antibiotic Residues in Consumer Milk Supplies in North America using the Charm Test II Procedure.

Two hundred and fourteen consumer milk samples from across North America were examined for antibiotics residues by means of the Bacillus stearothermophilus disc assay (Charm test) and the competitive isotopic (Charm Test II) procedure. Of the 174 samples taken from 16 states, 150 results were positive for one or more antibiotics. The greatest number of positives were sulphamethazine, 82, and tetracyline, 48. Candian samples, 40, also showed the same problem related to tetracycline, 12, and sulphamethazine residues, 12. The Bacillus stearothermophilus disc assay procedure was unable in most cases to detect these residues possibly due to the lower sensitivity of this test. Further comparative tests between Charm Test II and other methods of similar sensitivity are recommended to confirm these findings.

Resistance and Sensitivity of Meat Lactic Acid Bacteria to Antibiotics.

Sixty-seven strains of lactic acid bacteria (LAB) isolated from raw ground pork and beef were studied. Morphological and biochemical studies enabled the strains to be classified in four broad groups. These were atypical streptobacteria, Lactobacillus plantarum , Lactobacillus brevis and Leuconostoc mesenteroides . Antibiotic resistance/sensitivity patterns of these strains to 17 common antibiotics are reported. All 67 strains were sensitive to penicillin, ampicillin, cephaloridine and erythromycin. Most strains were resistant to cloxacillin, methicillin, gentamicin, kanamycin, neomycin, streptomycin, tetracycline, polymyxin B, colistin, novobiocin, and bacitracin. Most strains were sensitive to chloramphenicol and rifampin. MIC studies on tetracycline and chloramphenicol resistant strains showed them to be highly resistant. Population studies of LAB in raw meat were shown to vary in tetracycline resistance (12-58% of total LAB as measured by MRS).

Two-Stage Enrichment Procedures for Isolating Listeria monocytogenes from Raw Milk.

Recovery of two strains of Listeria monocytogenes (Scott A and V7) inoculated into raw milk, and of strains indigenous to milk was investigated. Isolation of the organisms from the milk was attempted using pre-enrichment broths [nutrient broth no. 2 (NB2) and tryptose broth (TB)] at 1:5 and 1:10 dilutions of milk. The broths were incubated at 4°C for 0, 7, 14, 21, and 28 d. Recoveries were compared by direct plating onto acriflavine-nalidixic acid agar (AN), McBride Listeria agar (MLA) and tryptose agar (TA), and after selective enrichment in thiocyanate-nalidixic acid broth, with and without acriflavine. Favorable recoveries were obtained using a two-stage protocol consisting of cold enrichment of the sample diluted 1:10 in TB, followed by plating 0.1 ml of this pre-enrichment to MLA, and transfer of 1 ml to 9 ml Thio-Nal-Acri broth. Selectively-enriched cultures streaked to MLA and TA yielded optimal isolations after 7-14 of cold enrichment.

An Evaluation of Antibotulinal Activity in Nitrite-Free Curing Systems Containing Dinitrosyl Ferrohemochrome.

The ability of the pigment dinitrosyl ferrohemochrome to mimic the cured meat color function attributed to nitrite, was evaluated in a number of nitrite-free, model meat systems. In addition, compounds with reported antibotulinal properties were compared to the antibotulinal effect of nitrite. Fifteen treatments were evaluated and compared to 50 and 150 ppm nitrite. Two processing conditions (short and extended heating) were also compared for their ability to enhance pigment color and eliminate the natural meat microbial population. Meat slurries varying in cure composition were inoculated with a composite of six different strains of Clostridium botulinum , types A and B. After processing, the packages were incubated at 10 and 27°C, and were analyzed for toxin. The treatment containing 3000 ppm sodium hypophosphite most closely resembled the 150 ppm nitrite control in its ability to prevent spore outgrowth and toxin production. The treatment containing 1250 ppm monomethyl fumerate also scored better than the other treatments including ethylene diamine tetraacetic acid (EDTA), potassium sorbate and tertiary butyl hydroquinome (TBHQ), but was slightly less inhibitory than sodium hypophosphite. The longer heat treatment eliminated all the natural meat flora (lactic acid bacteria) and enhanced the color production of the pigment.

Changes in Manganese Content in Lactobacillus plantarum During Inhibition with Sodium Nitrite.

Lactobacillus plantarum ATCC e8014 and L. plantarum (MC) were sensitive to NO2- under anaerobic conditions. This sensitivity was linked to an increase in cell-associated manganese levels. Normal manganese levels under aerobic conditions in the presence of NO2- were 1 × 107 atoms/cell. Under anaerobic conditions with NO2-, these levels increased to 3 × 108 to 2 × 109 atoms/cell depending on the sensitivity of lactobacilli to NO2-. This study suggests that in trace metal metabolism, NO2- may stimulate uptake or transport of ions such as manganese.

Production of N2O and CO2 during the reduction of NO2- by Lactobacillus lactis TS4.

N2O was produced during the reduction of NO2- by resting cells of Lactobacillus lactis TS4. At an initial NO2- concentration of 69 micrograms/ml, the rate of N2O production was 1.97 nmol/min per mg of protein, and the recovery of reduced NO2- -N as N2O-N after 24 h was 77%. Higher initial NO2- concentrations decreased both the rate of production of N2O and the recovery of reduced NO2- -N. CO2 production increased during NO2- reduction.

Nisin Sensitivity of Lactic Acid Bacteria Isolated from Cured and Fermented Meat Products.

Thirty strains of lactic acid bacteria from different meat sources (bologna, summer sausage, thurlinger sausage, chicken loaf and bacon) were tested for nisin sensitivity. The maximum concentration of nisin permitting growth for 20 strains was 50 IU/ml. Lactobacilli classified as atypical were sensitive to <5 IU nisin/ml. These strains could not be induced to increase resistance by five transfers to media with increased nisin concentrations. The ten strains with the higher resistance to nisin were checked for nisinase activity. One strain, Lactobacillus brevis , showed weak nisinase activity and the rest were negative.

Shelf-Life Studies of Vacuum-Packaged Bacon Treated with Nisin.

The effect of various concentrations of nisin (250, 500 or 750 IU/g) combined with 50 ppm sodium nitrite on the shelf-life of vacuum-packaged bacon was evaluated. Control packages of bacon containing 50 and 150 ppm nitrite were included. Total numbers of lactic acid bacteria (LAB) (as measured on MRS medium) was used as a criterion for shelf-life. Treated bacon samples were stored at 30 and 5°C for 4 d or 6 wk, respectively. Bacon stored at 30°C showed a 1-d extension of shelf-life at nisin levels of 500 and 750 IU/g. Lowest counts at 6 wk were in bacon treated with 750 IU nisin and stored at 5°C. The LAB count was 1.5-log10 CFU/g lower than the controls. A 1-wk extension of storage life was predicted for nisin-treated (750 IU) bacon.

Source and Persistence of Salmonella muenster in Naturally Contaminated Cheddar Cheese.

Public health authorities in Oxford, Middlesex and Elgin Counties, Ontario, seized raw milk Cheddar cheese due to presence of Salmonella muenster . Investigations by these units and the University of Guelph traced the source of Salmonella to one particular milk supplier shipping to a cheese factory. Analysis of milk samples from a herd of 35 cattle revealed only one cow shedding S. muenster directly into the milk (ca. 200 CFU/ml). Eleven of 181 vats of cheese, produced at the factory between May and October 1982, were positive for Salmonella at the curd stage. Only 2 vats of the finished raw milk Cheddar, however, were positive. One lot of Salmonella -positive cheese was still positive after the legally required 60-d holding period and remained so for 125 d.

Characteristics of the Inhibition of Brochothrix thermosphacta by Lactobacillus brevis.

When cultures of Brochothrix thermosphacta and Lactobacillus brevis were grown together or separated by a dialysis membrane (M.W. cut off = 3,500 daltons), the growth of Brochothrix was inhibited. This phenomena occurred under both aerobic and anaerobic conditions and was unaffected by the presence of catalase (412 units/ml). The antagonism appeared to be pH-mediated since it depended on glucose concentration, but low pH (4.5) alone did not directly affect the viability or salt tolerance of singular cultures of B. thermosphacta . Electron microscopy of thin sections of B. thermosphacta after 24 to 48 h of exposure to L. brevis revealed distinct lesions within the peripheral cell wall fabric. These were not seen in control cells of the same age or in cells exposed to 0.01 to 0.1 M acetic acid. Induction of autolysis in B. thermosphacta by cell wall metabolism imbalance was believed to be the cause of the growth inhibition.

Factors Controlling the Growth of Gram-Negative Bacteria in Vacuum-Packed Bologna.

Gram-negative organisms were isolated from vacuum-packed bologna only after 7-8 weeks of storage at 5 C. These isolates were identified as Acinetobacter sp., Enterobacter hafniae , Enterobacter aerogenes , Serratia marcescens and Serratia liquefaciens . Nitrite tolerance tests on these isolates at pH 5, 5.5 and 6 showed that only Acinetobacter sp. may be controlled by nitrite and pH levels associated with bologna during the initial storage period. The nitrite tolerance of these organisms was linked to their ability to deplete nitrite at 5 C in broth cultures under anaerobic conditions. Associated growth experiments were done at 15 C in APT and BHI broths under anaerobic conditions. These studies with E. aerogenes and S. liquefaciens in the presence of Lactobacillus brevis or Lactobacillus buchneri showed varying degrees of inhibition of the gram-negative isolates. The major factor which appeared to influence this inhibition in both broth cultures and bologna was glucose levels.