THE SUITABILITY OF CERTAIN OBLIGATELY ANAEROBIC NONSPOREFORMING ENTERIC BACTERIA, AS PART OF A MORE EXTENDED BACTERIAL ASSOCIATION, AS INDICATORS OF 'FAECAL CONTAMINATION'OF FOODS

SUMMARY: The suitability, as indicators of the faecal contamination of foods, of two types of obligately anaerobic nonsporeforming bacteria which are very numerous in faeces, viz. members of the genera Bacteroides and Bifidibacterium , was tested. For the counting of Bacteroides , Beerens'bile-azide agar (1957) enriched with 10% (v/v) of blood, and incubated in oval cross section tubes, was found useful. Bifidibacterium could be counted very well in Frisell's tomato-azide agar (1951), incubated in the same way . Both media being not too selective, confirmation of representative numbers of colonies was necessary. After microscopical examination, the biochemical study of properties such as catalase reaction, oxygen tolerance, carbohydrate fermentation and nitrate reduction may be required.
The 3 Bacteroides strains tested died off rather rapidly when present in sterile ice cream under conditions where Enterobacteriaceae and faecal streptococci increased in numbers. Accordingly they were never found in considerable numbers in some 25 samples of food which appeared to contain very high numbers of other bacteria of faecal origin. The 8 Bifidibacterium strains investigated appeared more resistant. However, under conditions permitting growth, they increased less than the Enterobacteriaceae and faecal streptococci; and where the latter did not increase but did survive, the Bifidibacterium decreased in numbers. In line with these findings Bifidibacterium was not even found in numbers of the order of 10/g in the 25 samples of food mentioned above. Neither Bacteroides nor Bifidibacterium can therefore be considered suitable as an indicator of the contamination of foods with bacteria of faecal origin and of the subsequent growth of these bacteria therein.     

Catalase activity in Paramecium aurelia]

The catalase activity of Paramecium aurelia was determined by the procedure of Sinha after bacteria elimination from culture medium. A significant level of catalase activity was shown, higher than in other cell kinds. The role of catalase activity in Paramecium sensitivity to low doses of ionizing radiations is discuted.     

Development and performance of an enzyme-linked amperometric immunosensor for the detection of Staphylococcus aureus in foods

An amperometric enzyme-linked immunosensor was developed to detect and quantify levels of Staphylococcus aureus electrically in pure cultures and in foods. The assay was a modification of a 'sandwich' ELISA for the protein A of Staph. aureus, employing catalase-labelled anti-protein A antibody. On addition of hydrogen peroxide to the assay system the catalase released O2 which was monitored using an amperometric oxygen electrode. The rate of current increase was proportional to the antigen concentration (protein A or Staph. aureus). Protein A was detected reliably at 0.1 ng/ml representing a 20-fold increase in sensitivity over the conventional ELISA that used horseradish peroxidase. Pure cultures of Staph. aureus were detected at 10(-3)-10(-4) cfu/ml with the amperometric electrode (cf greater than 10(5)/ml for conventional ELISA). The same level of sensitivity was achieved for inoculated food samples. Low levels of contamination (1 cfu/g) of Staph. aureus were detected after incubation at 37 degrees C for 18 h, and the immunosensor could from the basis of a test for screening and identification of protein A-bearing Staph. aureus in 24 h, although natural variations in protein A content between different strains could make the system unreliable in accurate quantification of cell numbers.     

Recovery of Campylobacter jejuni and Campylobacter coli from inoculated foods by selective enrichment.

A direct enrichment procedure was developed to selectively recover small numbers of Campylobacter jejuni, C. coli, and nalidixic acid-resistant thermophilic Campylobacter from foods. The procedure includes an enrichment medium composed of brucella broth, 7% lysed horse blood, 0.3% sodium succinate, 0.01% cysteine hydrochloride, vancomycin (15 micrograms/ml), trimethoprim (5 micrograms/ml), polymyxin B (20 IU/ml), and cycloheximide (50 micrograms/ml) that is inoculated with 10 or 25 g of food and incubated with agitation under microaerophilic conditions at 42 degrees C for 16 to 18 h. After incubation, the medium is plated directly onto Campy-BAP agar plates (M. J. Blaser et al., Ann. Intern. Med. 91:179-185, 1979), and resulting colonies that resemble Campylobacter are identified by conventional tests. The foods evaluated included raw milk, hamburger, and chicken skin which had aerobic plate counts of 10(5) to 10(9) bacteria/g. The procedure was effective in recovering as few as 0.1 cell of Campylobacter per g of food. Of the 50 isolates of Campylobacter evaluated, all were recovered from raw milk and hamburger at a level of 1 to 4 cells/g, and 41 and 40 isolaes were recovered from the hamburger and milk, respectively, at 0.1 to 0.4 cell/g. The enrichment was least effective for recovering campylobacters from chicken skin, as 7 and 26 of 50 isolates were not recovered at 1 to 4 and 0.1 to 0.4 cell/g, respectively. This new procedure is more rapid, direct, and effective than other enrichment or direct plating procedures for recovering small numbers of campylobacters from foods.     

pH-dependency of Escherichia coli catalase activity under modified culture conditions

In the previous work we have found two peaks of catalase activity at acid and neutral pH in partially destroyed bacteria E. coli K12 KS400. The present study indicates that catalase activity with two pH-optimums is sensitive to pH of cultivation medium. The relative catalase activity of frozen-thawed bacteria preparations measured at pH 3.5 increased two-fold and activity measured at pH 7.0 didn't change by shift of medium pH from value 5.5 to 7.0. In analogical preparations of bacteria grown in slightly alkaline media activity with acid maximum was not observed, but activity with neutral maximum rose to 130% in comparison with the intact cells was revealed. Two peaks of activity differed in their sensitivity to bacteria destruction, heating, inhibition by NaN3 and AMT, oxidative stress. The analysis of recent literature information and experimental data leads us to conclude that the activity with neutral pH-optimum consists of two known catalase forms HPI and HPII in E. coli. The ratio of HPI and HPII is 70 and 30%, respectively what was concluded from inhibition of catalase activity with neutral pH-optimum by AMT. Properties of catalase activity with acid pH-optimum didn't corresponding to any known enzyme forms. It is suggested the activity measured at pH 3.5 is results of some unstable activator which acts in acid pH range. It is possible that the described activity with acid pH-optimum is specific for the used E. coli strain. Investigation of another strain of E. coli K12 AB1157 confirmed this idea where the activity peak with acid pH-optimum was not detected.     

Development and application of an oligonucleotide microarray for the detection of food-borne bacterial pathogens

The rapid and accurate detection and identification of food-borne pathogenic bacteria is critical for food safety. In this paper, we describe a rapid (<4 h) high-throughput detection and identification system that uses universal polymerase chain reaction (PCR) primers to amplify a variable region of bacterial the 16S rRNA gene, followed by reverse hybridization of the products to species-specific oligonucleotide probes on a chip. This procedure was successful in discriminating 204 strains of bacteria from pure culture belonging to 13 genera of bacteria. When this method was applied directly to 115 strains of bacteria isolated from foods, 112/115 (97.4%) were correctly identified; two strains were indistinguishable due to weak signal, while one failed to produce a PCR product. The array was used to detect and successfully identify two strains of bacteria from food poisoning outbreak samples, giving results through hybridization that were identical to those obtained by traditional methods. The sensitivity of the microarray assay was 10² CFU of bacteria. Thus, the oligonucleotide microarray is a powerful tool for the detection and identification of pathogens from foods. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of garlic powder on the growth of commensal bacteria from the gastrointestinal tract

Garlic (Allium sativum) is considered one of the best disease-preventive foods. We evaluated in vitro the effect of a commercial garlic powder (GP), at concentrations of 0.1% and 1% (w/v), upon the viability of representative gut bacteria. In pure culture studies, Lactobacillus casei DSMZ 20011 was essentially found to be resistant to GP whereas a rapid killing effect of between 1 and 3 log CFU/ml reduction in cell numbers was observed with Bacteroides ovatus, Bifidobacterium longum DSMZ 20090 and Clostridium nexile A2-232. After 6h incubation, bacterial numbers increased steadily and once the strains became resistant they retained their resistant phenotype upon sub-culturing. A colonic model was also used to evaluate the effect of GP on a mixed bacterial population representing the microbiota of the distal colon. Lactic acid bacteria were found to be more resistant to GP compared to the clostridial members of the gut microbiota. While for most bacteria the antimicrobial effect was transient, the lactobacilli showed a degree of resistance to garlic, indicating that its consumption may favour the growth of these beneficial bacterial species in the gut. Garlic intake has the potential to temporarily modulate the gut microbiota.     

Rapid physicochemical detachment, separation and concentration of bacteria from beef surfaces

A simple, rapid, physicochemical treatment for the removal of viable bacteria from the surface of raw and cooked beef is described. The detachment method was linked to a differential centrifugation step which removed large amounts of particulate food matter and concentrated the detached bacteria. The method increased the numbers of bacteria released from beef surfaces and increased the numbers detected by at least one and a half orders of magnitude, when compared to the traditional 'stomaching' technique. This 1-h separation and concentration method produced cleaner suspensions of bacteria and improved the sensitivity of detection by DEFT and direct plate count.     

An improved selective and diagnostic medium for isolation and counting of Listeria spp. in heavily contaminated foods

Columbia agar base containing 0·001% acriflavine, 1·5% lithium chloride, 0·25% phenyl ethanol, 0·05% aesculin. 0·05% ferrous salt, 1% mannitol, 2·5% egg yolk emulsion and 0·008% phenol red (ALPAMY), recovered Listeria monocytogenes and some strains of Listeria seeligeri quantitatively, but suppressed Listeria ivanovii and virtually all other bacteria common in fresh foods. When used with foods processed for safety, repair on non-selective buffered glucose tryptone soya peptone yeast extract catalase agar must precede the use of ALPAMY.     

Biodeterioration of natural stone with special reference to nitrifying bacteria

An evaluation of field data from historical buildings in Germany showed that chemoorganotrophic bacteria are the most numerous microorganisms in building stones, followed by fungi and nitrifying bacteria. Chemoorganotrophic bacteria and fungi were present in almost every sample. Ammonia and nitrite oxidizers were found in 55 and 62% of the samples, respectively. Within months, natural stone was colonized by chemoorganotrophic microorganisms. The highest cell numbers were usually found near the surface. The colonization of natural stone by nitrifying bacteria took several years. The highest cell numbers were in some cases found underneath the surface. Nitrifying bacteria showed a preference for calcareous material with a medium pore radius between 1 and 10 m. Cell numbers of nitrifying bacteria did not correlate to the nitrate content of the stone material. We demonstrated that the stone inhabiting microflora can cause significant loss of nitrate by denitrification. Our data strongly suggested that microbial colonization of historical buildings was enhanced by anthropogenic air pollution. Samples taken from stone material with a pore radius 1 m had significantly higher cell numbers when they were covered with black crusts. A comparison of samples taken between 1990–1995 from buildings throughout Germany showed that in eastern Germany a significantly stronger colonization with facultatively methylotrophic bacteria and nitrifying bacteria existed. The same was true for natural stone from an urban exposure site when compared to material from a rural exposure site. Data from outdoor exposure and laboratory simulation experiments indicated that the colonization of calcareous stone by nitrifying bacteria was enhanced by chemical weathering.     

Survival of bacterial enteric pathogens in traditional fermented foods

The survival of strains of bacterial enteric pathogens was investigated in two traditional fermented foods (mahewu and sour porridge) and in unfermented porridge. The foods were inoculated with cell suspensions of Salmonella, Shigella, Campylobacter, Aeromonas species and pathogenic Escherichia coli which had a final concentration of 10⁶-10⁷ cfu/ml of food. None of the strains of Aeromonas and Campylobacter were detected in mahewu and sour porridge 20 min after inoculation. The salmonellas were not found 4 h after inoculation in either fermented foods but the shigellas and pathogenic E. coli strains were more tolerant to the low pH of the fermented foods. Some of the shigellas and pathogenic E. coli strains survived for 24 h after inoculation but showed a sharp decrease in numbers. All the strains of the enteric pathogens survived for 24 h in the unfermented porridge and increased in the numbers except for campylobacters, the numbers of which declined. These results suggest that the traditional fermented foods have bacteriostatic and bactericidal properties and are unlikely to play a major role in the transmission of bacterial enteric pathogens.     

The effect of step changes in sucrose concentration on the growth of Salmonella typhimurium LT2

Water activity is a method of preservation that can affect microbial growth in foods and that may fluctuate during their processing, distribution and storage. Sucrose has been used to change the water activity of microbiological culture media. Suspensions of Salmonella typhimurium LT2 in the exponential phase of growth have been subjected to step changes in sucrose concentration at 20°C. The changes in the numbers of viable bacteria were measured with time and the experimental growth curves compared with predictions based on growth data obtained at constant sucrose concentrations. Steps down in sucrose concentration showed some apparent loss of viability after the step followed by growth at a rate close to the expected value. Steps up in sucrose concentration resulted in a greater apparent loss of viability after the step and either growth or the inducement of lag, depending on the final concentration of sucrose. A series of small steps up in sucrose concentration to 45% (w/v) was able to sustain growth where it was not possible by inoculation directly into this concentration. Improved recovery of bacteria subject to osmotic stress was possible with a medium containing sodium chloride.     

Detection of Listeria monocytogenes in foods by immunomagnetic separation

Immunomagnetic separation with immunomagnetic beads was used to isolate strains of Listeria monocytogenes both from pure cultures and from heterogeneous suspensions. The monoclonal antibodies used recognized all six strains of serotype 4 but only one of three strains of serotype 1. Coating procedure, incubation time, and number of immunomagnetic beads influenced the sensitivity of the isolation method. Less than 1 x 10(2) bacteria per ml in pure cultures and less than 2 x 10(2) bacteria per ml in enriched foods could be detected. The method represents a new approach to extraction and isolation of pathogenic bacteria directly from foods, after resuscitation, or from enrichment broths.     

Detection of Listeria monocytogenes in foods by immunomagnetic separation.

Immunomagnetic separation with immunomagnetic beads was used to isolate strains of Listeria monocytogenes both from pure cultures and from heterogeneous suspensions. The monoclonal antibodies used recognized all six strains of serotype 4 but only one of three strains of serotype 1. Coating procedure, incubation time, and number of immunomagnetic beads influenced the sensitivity of the isolation method. Less than 1 x 10(2) bacteria per ml in pure cultures and less than 2 x 10(2) bacteria per ml in enriched foods could be detected. The method represents a new approach to extraction and isolation of pathogenic bacteria directly from foods, after resuscitation, or from enrichment broths.     

滩涂贝类养殖环境的细菌分布

从贝类养殖滩涂的底泥中共分离到2976株细菌,经鉴定可归于18个属与肠杆菌科的部分属,其中梭状芽孢杆菌属(Clostridium)、芽孢杆菌属(Bacillus)、棒状杆菌属(Corynebacterium)、肠杆菌科(Enterobacteriaceae)的部分属、发光杆菌属(Photobacterium)、黄杆菌属(Flavobacterium)和假单胞菌属(Pseudomonas)等为优势菌属.统计结果表明,在养殖滩涂的沉积物环境中,异养细菌的菌群组成具有明显的陆源性特点,而且在时空分布上有着较大的差异.在数量分布上,全年异养细菌、反硝化细菌、氨化细菌和硫酸还原菌的数量分别波动在1.62×103-2.00×105cfu/g、1.50×104-5.00×107个/g、9.00×104-9.0×107个/g和9.00×103-4.00×106个/g之间,此外,反硝化细菌、氨化细菌均与异养细菌在数量上呈正相关,而且氨化细菌与异养细菌之间的相关性极其显著,而硫酸还原菌与异养细菌在数量上呈负相关.     

Use of a microbial fuel cell for the rapid enumeration of bacteria

Summary The detection of bacteria using a thionine mediated microbial fuel cell was examined. On addition of bacteria to the anode compartment of a fuel cell, a rapid increase in the current output was observed. Both the total change in the steady state current (mA) and the initial rate of change of current were proportional to the numbers of bacteria added. Regression analysis of plots of log₁₀ mA against log₁₀ bacteria ml^-1 (final concentration) upon the addition of E. coli K12, Lactococcus lactis, coliform sp. A1, Micrococcus sp. M3 but not Pseudomonas sp. P5 gave reasonable correlation coefficients. Determination of the rates of respiration and thionine reduction by E. coli indicated that the transfer of metabolic electrons from the bacteria to the mediator was reasonably efficient (approx. 50%). These results are discussed with respect to the potential application of this technique for the rapid estimation of the bacterial contamination of foods.     

Enumeration of Vital and Thermally Stressed Staphylococcus aureus in Foods Using Baird-Parker Pig Plasma Agar (BPP)

Baird-Parker (BP), modified BP medium (egg yolk replaced with pig plasma, BPP) and modified Vogel and Johnson agar (phosphatidyl choline, deoxyribonucleic acid, with catalase added, PCVJ) were equally efficient for enumerating both non-stressed and thermally-stressed populations of Staphylococcus aureus from pure cultures and naturally contaminated foods. Vogel and Johnson agar was inferior. All colonies exhibiting coagulase activity on BPP were subsequently confirmed as Staph. aureus ; this was not the case with presumptive colonies from BP and PCVJ. Based on selectivity, definitive diagnostic characterization of colonies and increased sensitivity (more sample plated), BPP should be considered as the reference method for the routine enumeration of Staph. aureus in foods.     

Epiphytic bacteria: development of a method for determining respiring bacteria on leaves

A method was developed for studying the total numbers and the proportion of active bacteria on leaf surfaces. It involves staining gelatin impressions of leaves treated with an electron transport system indicator, 2-( p -iodophenyl)-3-( p -nitrophenyl)-5-phenyl tetrazolium chloride (INT). The method is rapid, inexpensive and allows simultaneous observation of numbers, ecology and respiratory activity of epiphytic bacteria. The total numbers of epiphytic bacteria for four species of aquatic plants varied between 0.6 to 10.2 times 10⁶/cm². The proportion of active bacteria on leaves of aquatic plants ranged from 2.2 to 42.9%. The method was also applied to a comparison of surface fouling of glass slides and aquatic leaf surfaces, indicating significant differences in numbers of bacteria but little difference in the proportion active on the two surfaces.     

Monitoring transfer of recombinant and nonrecombinant plasmids between Lactococcus lactis strains and members of the human gastrointestinal microbiota in vivo--impact of donor cell number and diet

AIMS: The generation of data of real relevance to the purported risks of DNA transfer from food-borne genetically modified microorganisms (GMMOs) using the human biota associated (HBA) rat model. Plasmid transfer between Lactococcus lactis strains and between donor strains and human gut bacteria was monitored. METHODS AND RESULTS: Transfer of the recombinant plasmid pCK1 and/or the promiscuous nonrecombinant plasmid pAMbeta1 between L. lactis strains was monitored in vivo in HBA rats. No transfer of pCK1 was observed. Transfer of pAMbeta1 was observed to Enterococcus spp. present in the HBA rats. Transconjugants persisted for 30 d and were distributed throughout the gastrointestinal tract. Both HBA rat diet and donor cell numbers impacted on transconjugant numbers. Fewer transconjugants were observed in animals fed a high-fat human type diet, while high levels of plasmid transfer were only observed at doses of donor L. lactis greater than 109 cfu. CONCLUSIONS: The utility of models of the human gut in monitoring DNA transfer events within the gut microbiota was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: Such findings give some confidence for the use of GMMOs with recombinant DNA borne on nonconjugative elements in fermented foods. HBA rats are a suitable model for monitoring the fate of food-borne GMMOs.