Effect of Selected Lactic Acid Bacteria on Growth of Staphylococcus aureus and Production of Enterotoxin

Representative strains of 15 species of lactic acid bacteria were examined for their ability to influence growth of Staphylococcus aureus and production of enterotoxin in associative culture. Among the organisms used as effectors the streptococci were most inhibitory, followed by Pediococcus cerevisiae. The lactobacilli and Leuconostoc citrovorum were not inhibitory to growth and only slightly inhibitory to production of enterotoxin. Enterotoxin was detected in all cultures in which the population of S. aureus reached 8 x 10(7) per ml. At lower S. aureus populations no enterotoxin was detected after incubation for 48 h. Mechanisms of inhibition of growth and enterotoxin production by S. aureus strain 243 grown in association with Streptococcus lactis A64 or P. cerevisiae 10791 in APT broth were investigated. Competition for vital nutrients, especially niacin and biotin, and probably production of hydrogen peroxide contribute to inhibition. Production of lactic acid appears to inhibit growth of S. aureus in the early but not the late stages of incubation.     

Transfer of plasmids RP4 and R68.45 and chromosomal mobilization in cowpea rhizobia

R-plasmids RP4 and its derivatives R68.45 were transferred from Escherichia coli to two cowpea rhizobia strains. The frequency of RP4 transfer in cowpea rhizobia strains JRC23-SM20 and IRC256-HA409 was 1,000-fold higher than transfer frequency of R68.45. The transconjugants were further used to transfer R-plasmids within (isogenic) and between (non-isogenic) cowpea rhizobia strains. The plasmid transfer frequency was higher in isogenic than non-isogenic strains. The ability of R-plasmids to mobilize chromosomal genes in cowpea rhizobia was also examined. R-plasmids mediated the chromosomal transfer; however, mobilization of chromosomal markers Sm^R and Met⁺ by RP4 in isogenic strains was more efficient than by R68.45. Chromosomal mobilization has not previously been reported in cowpea rhizobia.Abbreviations Ap ampicillin - Km kanamycin - Tc tetracycline - Rif rifampicin - TYS tryptone yeast-extract sodium chloride - YEMA yeast-extract mannitol agar - YEMB yeast-extract mannitol broth Part of the work was presented in 6th International Symposium on Nitrogen Fixation at Oregon State University, Corvallis, August 4–10, 1985     

Virulence of β-hemolytic and non-hemolytic Streptococcus mutans: lethal dose determinations in neonatal mice

The virulence of beta-hemolytic and non-hemolytic strains of Streptococcus mutans was studied in neonatal mice by LD50 determinations after intracerebral injection of the bacteria. Although the differences in LD50 values are small the results may indicate that beta-hemolytic S. mutans strains (mean LD50 of 6.3 X 10(7) c.f.u.) were more virulent than non-hemolytic S. mutans strains (mean LD50 of 47.7 X 10(7) c.f.u.). The LD50 values of other viridans streptococci varied between 4.8 and 10(7) c.f.u. Strains of S. pyogenes and S. agalactiae were highly virulent in this animal model with LD50 values lower than 10(5) c.f.u.     

Chemical composition of extracellular polysaccharides of cowpea rhizobia

The extracellular polysaccharides (EPS) of six strains of cowpea rhizobia were examined. The strains (MI50A, M6-7B, IRC253) produced polysaccharides containing glucose, galactose and mannose in a molar ratio of 2:1.1:1, 1:1.3:3.1 and 1:1.3:3.5 respectively. Two strains (513-B and Ez-Aesch) produced polysaccharides containing galactose and mannose in a molar ratio of 2:3. Mannose was the only sugar detected in the EPS of strain IRC291. Pyruvate, acetate, glucuronic acid and galacturonic acid were not detected in any strain.Abbreviations EPS Extracellular polysaccharide - YEMA yeast-extract mannitol agar - YEMB yeast extract mannitol broth     

The effect of condensed tannins from Lotus pedunculatus and Lotus corniculatus on the growth of proteolytic rumen bacteria in vitro and their possible mode of action

Five strains of proteolytic rumen bacteria were treated with condensed tannins (CT) purified from Lotus pedunculatus and Lotus corniculatus to investigate their effect on the growth of these bacteria in vitro. Streptococcus bovis NCFB 2476, Eubacterium sp. C124b, Prevotella bryantii B14, Butyrivibrio fibrisolvens H17c, and Clostridium proteoclasticum B316T were tested against 200, 400, and 600 microg CT x mL(-1) extracted from L. pedunculatus and L. corniculatus. In the absence of CT, all bacterial strains showed typical growth and reached maximum optical density (OD) after 6-8 h of incubation in a plant protein medium. Growth of Eubacterium sp., P. bryantii, and B. fibrisolvens was inhibited (P < 0.01-0.001) more by the CT from L. pedunculatus than by the CT from L. corniculatus. All strains continued to grow in the presence of 200 microg x mL(-1) of the CT from L. pedunculatus, but attained significantly (P < 0.05-0.01) lower maximum OD600 values than (minus CT) controls, except for S. bovis. At 400 and 600 microg x mL(-1), the addition of CT from L. pedunculatus inhibited (P < 0.05-0.001) the growth of all bacterial strains tested compared with controls. The growth of Eubacterium sp. and P. bryantii was stimulated for the first 4-6 h of incubation (P < 0.001) by 200 microg x mL(-1) of CT from L. corniculatus, but then declined leading to a significant difference in OD values compared with the controls. At 400 microg x mL(-1), the CT from L. corniculatus reduced (P < 0.05-0.01) the growth of all strains except S. bovis, while 600 microg x mL(-1) inhibited (P < 0.01-0.001) the growth of all strains. To study the mechanism of CT action, the degradation of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; Fraction 1 Leaf protein) was followed after bacterial cells or Rubisco were preincubated with CT extracted from L. corniculatus and L. pedunculatus. Both preincubations decreased LSU degradation, but they differed in their response to polyethylene glycol (PEG) addition. Addition of PEG to CT-Rubisco preincubations negated the effects of CT, while PEG addition to CT-bacteria preincubations did not. This implies that the CT-bacterial interaction is stronger than the CT-Rubisco interaction or the interaction is of a different type. Also, L. pedunculatus CT reduced the degradation of the LSU to a greater extent than the CT from L. corniculatus when preincubated with bacteria.     

A capillary polymerase chain reaction for Salmonella detection from poultry meat

AIMS: In this study, a capillary polymerase chain reaction (cPCR) was applied for Salmonella detection from poultry meat. METHODS AND RESULTS: Salmonella detection limits of the optimized cPCR were determined with DNA templates from the samples of tetrathionate broth (TTB), Rappaport Vassiliadis broth (RVB) and selenite cystine broth (SCB) artificially contaminated with 10-fold dilutions of 6 x 10(8) CFU ml(-1) of pure Salmonella enterica ssp. enterica serovar Enteritidis 64K stock culture. Detection limits of cPCR from TTB, RVB and SCB were found as 6, 6 x 10(1) and 6 x 10(4) CFU ml(-1), respectively. In addition, detection limits of bacteriology were also determined as 6 CFU ml(-1) with TTB and SCB, and 6 x 10(1) CFU ml(-1) with RVB. A total of 200 samples, consisting of 100 chicken and 100 turkey meat samples, were tested with optimized cPCR and bacteriology. Eight and six per cent of the chicken meat samples were found to harbour Salmonella by cPCR and standard bacteriology, respectively. Of six Salmonella isolates, four belonged to serogroup D, two to serogroup B. CONCLUSIONS: The TTB cultures of both artificially and naturally contaminated samples were found to be superior to those of RVB and SCB cultures in their cPCR results. This cPCR, utilizing template from 18-h TTB primary enrichment broth culture, takes approximately 40 min in the successful detection of Salmonella from poultry meat. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that cPCR from TTB enrichment culture of poultry meat would enable rapid detection of Salmonella in laboratories with low sample throughput and limited budget.     

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.     

Protection of Rhizobium by bentonite clay against predation by flagellates in liquid cultures

Abstract Bentonite clay strongly hampered the activity of the flagellate Bodo soltans in liquid culture, and thereby improved the survival of Rhizobium . A possible coating of bacteria and/or flagellates did not seem to play a part in protecting rhizobia from flagellates. Bentonite did not release any substances toxic to B. saltans during the incubation period. It is suggested that, in liquid cultures, bentonite clay increases the minimum level to which rhizobia can be predated upon, thereby giving rise to the presence of higher rhizobial cell concentrations at the end of the incubation period.     

Optimum Culture Conditions for Production of Exfoliative Toxin by Staphylococcus hyicus

Optimum culture conditions for the production of exfoliative toxin by Staphylococcus hyicus (shET) were examined. High shET activity was obtained from the culture filtrate of HI and TY broth inoculated with S. hyicus. The pH in these two media ranged from 7 to 8.5 during bacterial culture, while the lowest pH in TS and BHI broth was less than 6. shET activity in the culture filtrate from TY broth inoculated with 10⁷ CFU of S. hyicus per ml was higher than that in TY broth inoculated with 10⁶ and 10⁸ CFU of bacteria per ml. When shET activity in the culture filtrate was measured under various shaking conditions, the culture filtrate shaken at 75 oscillations per min had the highest shET activity of the five shaking conditions. shET activity of the culture filtrate of TY broth to which protease inhibitor had been added was the same as that of TY broth without inhibitor. shET activity in a shaking culture in an Erlenmeyer flask was also the same as that in sac culture and that in shaking culture using a shaking (Sakaguchi) flask. shET activity in TY broth supplemented with 100 mM glucose was significantly lower than that in TY broth without glucose. Based on the above results, the optimum culture conditions for the production of shET were as follows: inoculation of 3 × 10⁹ CFU of S. hyicus strain P-1 into 300 ml of TY broth in a 2,000-ml Erlenmeyer flask, and incubation at 37 C with shaking at 75 oscillations per min. Then shET activity of the culture filtrate under appropriate culture conditions was measured after various incubation periods. shET activity was detected 6 hr after inoculation, reached the maximum (2⁵³ exfoliative unit/0.1 ml) at 16 hr and decreased between 20 and 48 hr. Thus, the optimum incubation period was determined to be 16 hr. Then the optimum concentration of ammonium sulfate for isolation of shET from the culture filtrate under appropriate culture conditions was examined. The greatest shET activity was obtained from the fraction salted out with 90% saturated ammonium sulfate. Thus, the optimum concentration of ammonium sulfate for the isolation of shET was determined to be 90% saturation.     

A new approach of pellet formation of a filamentous fungus -Rhizopus oryzae

The effects of inoculum and medium composition (i.e. potato dextrose broth as carbon source, soybean peptone, calcium carbonate, and metal ions) on pellet formation of Rhizopus oryzae ATCC 20344 have been studied. Metal ions were found to have a significant negative effect on pellet formation while soybean peptone had a positive effect. In addition, potato dextrose broth and calcium carbonate were beneficial to R. oryzae for growing small, smooth pellets during the culture. The study also demonstrated that an inoculum size of less than 1.5x10(9)spores/L had no significant influence on pellet formation although it had large impacts on pellet growth. Thus, a new approach to form pellets has been developed using only potato dextrose broth, soybean peptone, and calcium carbonate allowing for pellet size to be controlled by adjusting inoculum size and the concentrations of potato dextrose broth, soybean peptone, and calcium carbonate in the medium.     

The correlation method for rapid monitoring of Escherichia coli in foods

AIMS: A new rapid method was developed to rapidly monitor Escherichia coli counts in foods. MATERIALS AND RESULTS: One ml of modified selective broth with 4-methylumbelliferyl beta-D-glucuronide and 1 ml of food sample were mixed in a sterile test tube and incubated at 37 degrees C. The positive reaction (fluorescence under u.v. light) was monitored at regular 30 min intervals. The positive reaction times in test tubes were compared with actual E. coli numbers from tested samples. The growth of E. coli in test tubes (broth) was much faster than growth on agar. The first experiment was performed to evaluate the rapid correlation method using pure E. coli cultures. The correlation between E. coli counts by the conventional plating method and positive reaction (fluorescence production) times in test tubes was highly agreeable (r(2) = 0 x 95). In the case of low E. coli numbers, such as 2 x 0 log10 cfu ml(-1), the rapid correlation method detected their presence after 10 h incubation. When highly contaminated samples were assayed (8 log10 cfu ml(-1)), the rapid correlation method detected the presence of E. coli after 4 h incubation. In the ground beef experiment, the correlation between fluorescence production time and actual E. coli numbers was also strongly agreeable (r(2) = 0 x 92). CONCLUSIONS: From these results, it is obvious that the new rapid method can rapidly monitor E. coli counts in foods. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicated that the new method saved about 10-14 h incubation time compared to conventional plating methods. The rapid correlation method required much shorter incubation times compared to conventional plating methods for monitoring E. coli.     

Isolation and partial characterization of normal and defective bacteriophages of gram-negative hydrogen bacteria

Widespread defective lysogeny was detected in Alcaligenes eutrophus by electron microscopic analysis of cultures. Mitomycin C treatment of the cultures resulted in the production of defective (inco-) particles. Polysheaths were produced both with and without induction.With the simultaneous isolation technique six phages were isolated for hydrogen-oxidizing strains of the new species Pseudomonas pseudoflava. The phages were able to replicate under autotrophic conditions and were found to have a very restricted host range. Electron microscopic analysis allowed classification into two structural groups. Group I contained phages with contractile tails; group II contained phages with flexible, noncontractile tails. All but one (gb) of the new phages were shown to be temperate by isolation of lysogens and induction with mitomycin C.Non-Standard Abbreviations OD optical density - SM-medium standard mineral medium - KS-medium Kaserer medium - NB nutrient broth - p.f.u. plaque forming unit - c.f.u. colony forming unit - e.o.p. efficiency of plating - Do-phage buffer Dopatka-phage buffer - PTA phosphotungstic acid     

Optimising the viability during storage of freeze-dried cell preparations of Campylobacter jejuni

Freeze-dried cultures of Campylobacter jejuni are used in the food and microbiological industry for reference materials and culture collections. However, C. jejuni is very susceptible to damage during freeze-drying and subsequent storage and it would be useful to have longer-lasting cultures. The survival of C. jejuni during freeze-drying and subsequent storage was investigated with the aim of optimising survival. C. jejuni was freeze-dried using cultures of different age (24-120 h), various lyoprotectants (10% phytone peptone, proteose peptone, peptonized milk, trehalose, soytone and sorbitol), various storage (air, nitrogen and vacuum) and re-hydration (media, temperature and time) conditions. One-day-old cultures had significantly greater survival after freeze-drying than older cultures. The addition of trehalose to inositol broth as a lyoprotectant resulted in almost 2 log(10) increase in survival after 2 months storage at 4 degrees C. Storage in a vacuum atmosphere and re-hydration in inositol broth at 37 degrees C increased recovery by 1-2 log(10) survival compared to re-hydration in maximal recovery diluent (MRD) after storage at 4 degrees C. Survival during storage was optimal when a one-day-old culture was freeze-dried in inositol broth plus 10% (w/v) trehalose, stored under vacuum at 4 degrees C and re-hydrated at the same incubation temperature (37 degrees C) in inositol broth for 30 min. The results demonstrate that the survival of freeze-dried cells of C. jejuni during storage can be significantly increased by optimising the culture age, the lyoprotectant, and the storage and re-hydration conditions. The logarithmic rate of loss of viability (K) followed very well an inverse dependence on the absolute temperature, i.e., the Arrhenius rate law. Extrapolation of the results to a more typical storage temperature (4 degrees C) predicted a very low K value of 1.5 x 10(-3). These results will be useful to the development of improved reference materials and samples held in culture collections.     

Molecular methods of analysing bacterial diversity: the case of Rhizobia

The distribution of rhizobia, bacteria which nodulate the roots of leguminous plants, was surveyed for 91 species of the familyLeguminosae. A total of 117 strains of rhizobia were isolated, and 28 strains were obtained from culture collections. The sample total of 145 strains was discriminated by rapid ribosomal RNA (rRNA) sequencing. The partial sequences (157 bases from position (inEscherichia coli) 1220 to 1377 from 5 end) of 16S rRNA revealed the presence of 16 groups in these rhizobia. Further, DNA-DNA homology studies suggested that the differences of the 16 groups were enough to justify establishing at least 16 species.     

The developmental cycle of Chlamydia trachomatis in McCoy cells treated with cytochalasin B.

The growth of a genital trachoma-inclusion conjunctivitis agent strain of Chlamydia trachomatis in McCoy cells treated with cytochalasin B was studied by quantitative infectivity estimations and by light and electron microscopy. Provided that infection of the monolayer was initiated by centrifuging the infectious particles on to the cells before incubation, this chlamydial strain grew as fast and to as high a titre [approximately 10(7) inclusion-forming units (i.f.u.) per culture] as those chlamydiae which infect cell cultures in vitro without centrifugation. Each i.f.u. inoculated yielded approximately 600 i.f.u., and extracellular infectivity was detected soon after intracellular infectivity appeared. Inclusions were recognized by fluorescent antibody staining techniques early in the developmental cycle when cultures were not infectious and when only reticulate bodies were seen by electron microscopy. Inclusions were recognized in Giemsa-stained preparations examined by dark ground microscopy only when elementary bodies appeared in the inclusions. Iodine staining was not a reliable indicator either of the number of inclusions present or of their infectivity.     

Novel potato micro-tuber-inducing compound, (3R,6S)-6-hydroxylasiodiplodin, from a strain of Lasiodiplodia theobromae

A novel potato micro-tuber-inducing compound was isolated from the culture broth of Lasiodiplodia theobromae Shimokita 2. The structure of the isolated compound was determined as (3R,6S)-6-hydroxylasiodiplodin by means of spectroscopic analyses, the modified Mosher method, and chemical conversion. The compound showed potato micro-tuber-inducing activity at a concentration of 10(-4) M, using the culture of single-node segments of potato stems in vitro.     

Dilution of Liquid Rhizobium Cultures To Increase Production Capacity of Inoculant Plants

Experiments were undertaken to test whether peat-based legume seed inoculants, which are prepared with liquid cultures that have been deliberately diluted, can attain and sustain acceptable numbers of viable rhizobia. Liquid cultures of Rhizobium japonicum and Rhizobium phaseoli were diluted to give 10⁸, 10⁷, or 10⁶ cells per ml, using either deionized water, quarter-strength yeast-mannitol broth, yeast-sucrose broth, or yeast-water. The variously diluted cultures were incorporated into gamma-irradiated peat, and the numbers of viable rhizobia were determined at intervals. In all of the inoculant formulations, the numbers of rhizobia reached similarly high ceiling values by 1 week after incorporation, irrespective not only of the number of cells added initially but also of the nature of the diluent. During week 1 of growth, similar multiplication patterns of the diluted liquid cultures were observed in two different peats. Numbers of rhizobia surviving in the various inoculant formulations were not markedly different after 6 months of storage at 28°C. The method of inoculant preparation did not affect the nitrogen fixation effectiveness of the Rhizobium strains.     

Growth and aflatoxin production by Aspergillus parasiticus when in the presence of Streptococcus lactis

Streptococcus lactis was grown with Aspergillus parasiticus in modified APT broth. Three inoculation procedures were used: (a) S. lactis was grown 3 days, then conidia of A. parasiticus were added (SLAP), (b) both organisms were added simultaneously (ST) and (c) A. parasiticus was grown 3 days, then S. lactis was added (APSL). At 3, 6 and 10 days of incubation, contents of flasks were analyzed for growth of each organism, pH of broth and aflatoxin content. S. lactis did not survive past 3 days when grown alone. In ST cultures, S. lactis grew to the same extent as in the control at 3 days; it remained viable at a low level through 10 days. In APSL cultures, S. lactis growth was inhibited at 3 days but the bacterium survived through 7 days (10 days of mold growth) at reduced numbers. At 3 days there were no appreciable differences in growth of A. parasiticus. At 6 days, in ST and SLAP cultures, growth of the mold was inhibited, while in the APSL culture growth increased over that in the control. At 10 days, growth of mold was somewhat increased over the control in all test conditions. The pH of broth in the A. parasiticus control and APSL culture was 6 at 3 days, dropped to 4.5–4.6 at 6 days and rose to 7 by 10 days. In ST and SLAP cultures, the pH was at 4.1 at 3 days and rose to pH 7 by 10 days. Aflatoxin (B₁ plus G₁) content was lowest at 3 days and increased at 6 days. Between 6 and 10 days two patterns were observed. In APSL and SLAP cultures, aflatoxin content decreased, while it increased in the ST culture. These patterns occurred when aflatoxin content was expressed on a total or per gram of dried mycelium basis. At 3 days the amounts of aflatoxin B₁ and G₁ were approximately equal. Between 3–6 days the amount of G₁ increased more rapidly than that of B₁. Between 6 and 10 days in the ST culture, the amount of G₁ increased at a slower rate than that of B₁ while in SLAP and APSL cultures, the amount of G₁ decreased more rapidly than that of B₁. When a different lot of the same medium was used, aflatoxin production was greatly reduced. The pH of broth at all test conditions rose through the incubation period.