Col V plasmids and the resistance of Escherichia coli to divalent copper ions

Free organisms of both plasmid-free and plasmid-carrying strains of Escherichia coli were killed by incubation in water containing low levels of cupric ions. Sensitivity was temperature-dependent with killing being more marked at 20° or 25°C than at 10° or 15°C. In contrast to the effects of other inhibitors from natural waters (which affect free Col V⁺organisms more than Col^-ones), free Col^-and Col V⁺organisms were equally sensitive to kill by Cu²⁺. Attachment to glass beads essentially abolished sensitivity to cupric ions with full survival after exposure to 15 μ g/ml. This applied to both p⁺and p^-strains but attachment would have more effect on the survival of p⁺organisms in natural waters because some plasmids markedly enhance attachment.     

Heat resistance in Salmonella enteritidis phage type 4: the influence of storage temperatures before heating

H umphrey , T.J. 1990. Heat resistance in Salmonella enteritidis phage type 4: the influence of storage temperatures before heating. Journal of Applied Bacteriology 69 493–497.
Storage of cultures of Salmonella enteritidis PT4 at either 4° or 8°C before heating significantly increased heat sensitivity. The differences between fresh and stored cultures, which became apparent after 4–7 h, were more pronounced with cultures stored at the lower temperature and in those heated at 60° rather than 55°C. Incubation of the stored cultures in either egg or Lemco broth for 30 min at 37°C prior to heating enabled the organisms to recover heat resistance.     

Virulence plasmid-associated adhesion of Escherichia coli and its significance for chlorine resistance

Introduction of the ColV, I-K94 virulence plasmid into strains of Escherichia coli led (for four out of five strains tested) to a marked increase in the ability of organisms to adhere to glass beads. For strain 1829, the plasmid led to increased attachment to other materials including sand, agar, agarose, chitin and cellulose. The increased adhesion to glass beads was due to the presence of the plasmid and not to its introduction into a variant with altered adhesive properties. The plasmid-encoded VmpA protein did not appear to be necessary for the ColV, I-K94-promoted adhesion but adhesion was absolutely dependent on the presence of derepressed levels of transfer components in the ColV+ strains and partially dependent on the presence of colicin components. The extent of the plasmid-promoted adhesion was greatest for organisms grown at 30 degrees, 37 degrees or 42 decrees C and adhesion was almost abolished by growth at 21 degrees or 25 degrees C; this finding is in accord with transfer and colicin components being involved in adhesion. Of several other plasmids tested for their effects on adhesion, those with derepressed transfer properties showed a marked effect as did the RI resistance plasmid. Because of the ease of handling glass bead-attached organisms, such preparations were used as a model for studying the relevance of attachment to the resistance of E. coli to chlorination in the water purification process. Organisms of 1829 ColV, I-K94, attached to glass beads, were more resistant to damage and killing by chlorine than were unattached organisms. Three findings suggest that such chlorine resistance may be significant for survival during water chlorination. Firstly, ColV, I-K94+ bacteria became attached if incubated in sewage effluent with glass beads at 20 degrees C. Secondly, ColV+ organisms already attached to glass beads maintained their attachment during 24 h incubation in effluent at 20 degrees C and thirdly such effluent incubated organisms remained chlorine resistant provided that they retained their attachment.     

A study of the growth kinetics of Yersinia enterocolitica serotype O:3 in pure and meat culture systems

The growth kinetics of a virulence plasmid-bearing (P+) and a plasmid-cured (P−) strain of Yersinia enterocolitica serotype O:3 in pure and meat culture were investigated. Growth studies were carried out at 25 and 37 °C in supplemented phosphate-buffered saline, buffered peptone water , cefsulodin-irgasan-novobiocin broth base or supplemented broth base (CIN). The lag phase durations and growth rates under these conditions were determined by linear regression analysis. In pure culture, under most sets of equivalent conditions, P+ and P− strains had similar lag phase durations. However, under one set of conditions, i.e. CIN broth at 37 °C, the lag phase duration of the P+ strain was significantly longer than P−. In all but the most selective medium, P+ strains had slower growth rates than P− strains at 37 °C, probably due to the increased metabolic burden entailed in the maintenance of the virulence plasmid. In the most selective medium, i.e. CIN broth, P+ strains grew significantly faster than P−. This finding suggests that possession of virulence plasmid confers an enhanced ability to grow in the presence of selective agents. In meat cultures, both strains had longer lag phases than in equivalent pure cultures, with longer lag phases noted at 37 than at 25 °C. No significant differences were observed between the length of lag phases of P+ and P− strains in meat culture. Both strains of Y. enterocolitica displayed faster growth rates in meat cultures than in pure cultures, indicating that one or more components of meat enhanced the growth of this organism. The effects and interaction of incubation temperature, enrichment broth and meat on the growth kinetics of plasmid-bearing and plasmid-cured Y. enterocolitica strains are discussed.     

THE EFFECT OF VARIOUS WATER BACTERIA ON THE GROWTH OF ESCHERICHIA COLI I IN MACCONKEY'S BROTH AT 37° AND 42°

SUMMARY: The growth of a streptomycin resistant strain of Escherichia coli I in MacConkey's broth at 37° or 42° was not inhibited by water organisms of the genera Micrococcus, Proteus, Achromobacter and Pseudomonas , but the presence of large numbers of other members of the coli-aerogenes group resulted in retardation or inhibition of its growth.     

Conditions affecting growth and enterotoxin production by Staphylococcus aureus on temperature-abused chicken meat

Growth of Staphylococcus aureus at 15°C, with and without addition of representative spoilage bacteria, was studied in cooked, whole chicken meat and chicken broth. In the absence of competitors, the organism grew better in broth culture than on whole meat, but multiplied more slowly in broth when other organisms were present, even from twice the previous level of inoculum. The presence of competitors had no marked effect on the growth of Staph. aureus on whole meat. Enterotoxin A was not produced at 15°C on either whole meat or in broth, and occurred at 20°C only in pure culture. At 30° and 37°C, toxin was produced whether or not competitors were present. Toxin production by Staph. aureus appeared to be influenced more by growth temperature than by bacterial competition.     

Factors affecting the adhesion and uptake of an oculo-genital strain of Chlamydia trachomatis to McCoy cells

Abstract We have studied adhesion and uptake of C. trachomatis serovar E in McCoy cells under various infection conditions. Adhesion and uptake of chlamydiae was completed about 3 h after the initiation of stationary infection at 37°C, but ingestion of cell membrane-attached organisms was finished within 0.5 h at 37°C. Reincubated chlamydiae, not attached after 3 h at 37°C, attached readily to fresh McCoy cell monolayers, but to a lesser extent than the original inoculum. Our results indicate that the lack of further attachment after 3 h incubation at 37°C under stationary infection conditions has complex causes, involving both host cell and parasite. Centrifugation did not affect the uptake of chlamydiae already bound to the cell membrane, suggesting that the uptake phase of C. trachomatis serovar E by McCoy cells is unaffected by centrifugation.     

Relationship between variations in pathogenicity and lag phase at 37°C of Listeria monocytogenes previously stored at 4°C

s. BUNCIC AND S.M. AVERY. 1996. Three haemolytic, pathogenic strains of Listeria monocytogenes (a reference strain, a food-derived strain and a human strain) were held at 4°C for 4 weeks in phosphate-buffered saline pH 5.5 or 7.0, with and without 0.2% potassium sorbate or 0.3% sodium acetate. The number of viable cells did not change significantly during this storage. Pathogenicity of non-growing L. monocytogenes cells for 14-d-old chick embryos was determined before and after storage. Storage at 4°C resulted in decreased pathogenicity, but effects were strain-, pH- and substrate-dependent. After 4 weeks storage at 4°C non-growing bacterial cells were transferred to Brain Heart Infusion broth and growth characteristics were determined during incubation at 37°C. Strains that showed decreased pathogenicity had significantly longer lag phases at 37°C than strains that maintained pathogenicity. It is concluded that decreased pathogenicity of L. monocytogenes stored without growth at 4°C for 4 weeks and subsequent long lag phase at 37°C are correlated.     

Techniques for the optimum recovery of cold injured Campylobacter jejuni from milk or water

When broth was inoculated with cells of Campylobacter jejuni that had been injured by chilling there was a fall in the viable population of up to 90%. It was greater at 43° than 37°C and in the presence of certain antibiotics and in some cases resulted in a surviving population that was below the minimum inoculum for subsequent growth. A technique of pre-enrichment in non-selective culture broth at 37°C for 2 h before the addition of antibiotics and incubation at 43°C was found to significantly reduce the fall in numbers and to improve the detection of C. jejuni in samples of raw milk and water.     

Chemotherapeutic sensitivity and viability of Nocardia asteroides and Nocardia brasiliensis in drugs

Procedures for testing drug sensitivity ofNocardia were standardized.Five strains each ofN. asteroides (NA) andN. brasiliensis (NB) isolated from pathogenic materials were tested for drug sensitivity in Sabouraud's glucose agar (SGA), Sabouraud's glucose broth (SGB) and SGB with addition of 10 % horse serum against sulphadiazine (S), penicillin (P), streptomycin (St), chloramphenicol (C), tetracycline (T) and 4-4-diamino diphenyl sulphone (DDS). The viability of strains at minimum inhibitory concentrations (MIC) of different drugs were also tested.Results showed that liquid medium is preferable to solid medium for drug testing as pellicle formation observed on the surface of liquid medium helps in accurate assessment of MIC. Serum addition did not appear to be necessary. Ten days incubation at 37 °C gives optimum results for determination of MIC. Taking the maximum drug concentration attainable in blood during therapy, the strains were classified as sensitive and resistant. All strains were resistant to DDS and P. One strain of NB was sensitive to St. One strain each of NA and NB was borderline sensitive to C. Four out of 5 strains in both species were sensitive to S. All strains were sensitive to T. The organisms remained viable in drugs, thus suggesting the limitations of antinocardial therapy.From Mycology Research Unit, Department of Dermatology, Seth G.S. Medical College and K.E.M. Hospital, Bombay-12.Hon. Professor Dermato-Venereology.Research Fellow.     

Heat resistance of different serovars of Listeria monocytogenes

Twelve Listeria monocytogenes strains representing seven serovars were heat-treated in physiological saline by a glass capillary tube method. Five strains were treated at 58°, 60° and 62°C, three at 60°, 62° and 64°C and four at 60°C. Heat-treated bacteria were recovered on blood agar in two ways: (1) incubation at 37°C for 7 d; and (2) preincubation at 4°C for 5 d, followed by incubation at 37°C for 7 d. D and z values were determined. Better average recovery and higher D values were obtained when the preincubation procedure was used. The final evaluations of the heat resistance properties of the strains were therefore based on values for preincubated samples. D values recorded at 58°, 60°, 62° and 64°C for preincubated samples were 1.7–3.4, 0.72–3.1, 0.30–1.3 and 0.33–0.68 min, respectively. z values determined were 5.2–6.9°C. D values were compared statistically. Significant differences in heat resistance were noted both between serovars and between strains belonging to the same serovar.     

A Comparison of Methods for the Enrichment of Salmonellae from Sheep Faeces

S ummary : A 10-tube MPN technique was used to test the efficiency of nutrient, tetrathionate, mannitol-selenite and mannitol-selenite-cystine broths as enrichment media for detecting salmonellae. Small numbers of broth grown salmonellae could be detected in all 4 media in the presence of 5% of sheep faeces. In naturally infected sheep faeces small numbers of salmonellae were not detected with either nutrient or tetrathionate broths. With mannitol-selenite the sensitivity of salmonella detection increased with both incubation temperature (37–43°) and the addition of faeces. The most sensitive and reliable medium for detecting salmonellae in naturally infected sheep faeces was mannitol-selenite-cystine broth. Neither incubation temperature (37–43°) nor the addition of faeces had a statistically significant effect on its sensitivity.     

Evaluation of the Growth of Salmonellae in Rappaport's Broth and in Muller-Kauffmann's Tetrathionate Broth

S ummary . The behaviour of 70 strains of salmonellae belonging to 44 serotypes in Rappaport's broth and in Muller-Kauffmann's tetrathionate broth was examined. With an inoculum of 5–25 cells, 5 strains did not grow in Rappaport's medium, 2 multiplied slowly and 63 grew strongly in 24 h. With an inoculum of 100–500 organisms all but one strain grew readily in 24 h. In Muller–Kauffmann's tetrathionate broth inoculated with pure cultures of salmonellae, growth of many strains was markedly inhibited, in the absence of added faeces, at 37° and 43°. This inhibition was more severe with light inocula at 43°. The addition of 0.05% (w/v) of salmonella-free human faeces to Muller–Kauffmann's tetrathionate broth, did not stimulate growth of salmonellae. In contrast, the addition of 5% (w/v) of human stools to this medium resulted in a heavy growth of the added salmonellae, especially at 43°.     

Minimal Medium Recovery of Chilled Salmonella Heidelberg

1. Salmonella heidelberg , chilled from 37 to 5°C in glucose-salts broth, grew better on a simple medium (glucose-salts agar) than on a complex medium (Tryptic Soy Agar + 0·5% yeast extract).
2. The organisms recovered the ability to grow on the complex medium after a further 8 h incubation at 5°C.
3. RNA synthesis would appear to be a critical factor in the recovery process.     

Methods used to investigate a possible environmental source of Mycobacterium avium-intracellulare-scrofulaceum (MAIS) infection in farmed deer

Currently used methods for the culture of mycobacteria from contaminated material were found to be unsatisfactory in an investigation of a possible environmental source of Mycobacterium avium-intracellulare-scrofulaceum (MAIS) infection in a New Zealand deer farm. Different combinations of established procedures were investigated using soil spiked with a laboratory strain of M. avium.
The most successful combination involved mixing the soil in nutrient broth (pH 8mD0) containing Tween 80, incubating at 37°C for 1 h to germinate sporing contaminants, treatment for 24 h with 1% cetylpyridinium chloride, followed by washing and culture on Lowenstein-Jensen slopes and incubation at 37°C and 42°C in approximately 5% CO₂ atmosphere. This procedure allowed good recovery of M. avium while successfully inhibiting saprophytic mycobacteria and other soil organisms, and was chosen to process the deer farm samples. No mycobacteria resembling the deer strains were found in these samples.     

Combining heat treatment and subsequent incubation temperature to prevent growth from spores of non-proteolytic Clostridium botulinum

Refrigerated processed foods of extended durability rely on a mild heat treatment combined with refrigerated storage to ensure microbiological safety and quality. The principal microbiological safety risk in foods of this type is non-proteolytic Clostridium botulinum. In this article the combined effect of mild heat treatment and refrigerated storage on the time to growth and probability of growth from spores of non-proteolytic Cl. botulinum is described. Spores of non-proteolytic Cl. botulinum (two strains each of type B, E and F) were heated at 90°C for between 0 and 60 min and subsequently incubated at 5°, 10° or 30°C in PYGS broth in the presence or absence of lysozyme. The number of spores that resulted in turbidity depended on the combination of heat treatment, incubation time and incubation temperature they received. Heating at 90°C for 1 or more min ensured a 10⁶ reduction when spores were subsequently incubated at 5°C for up to 23 weeks. Heating at 90°C for 60 min ensured a 10⁶ reduction over 23 weeks when subsequent incubation was at 10°C in the presence of added lysozyme. The same treatment did not reduce the spore population by 10⁶ when subsequent incubation was at 30°C.     

THE RATES OF GROWTH OF SOME THERMODURIC BACTERIA IN PURE CULTURE AND THEIR EFFECTS ON TESTS FOR THE KEEPING QUALITY OF MILK

SUMMARY: The growth rates of eleven representative thermoduric bacteria, comprising 3 aerobic spore formers, 3 streptococci, 1 Corynebacterium lacticum and 4 micrococci, have been determined in glucose broth and sterile pasteurized milk at 37·5°, 26° and 15°. The spore formers and streptococci were generally not affected by the presence of inhibitory factors in pasteurized milk. When multiplication of micrococci and C. lacticum occurred in milk this was only after a lag period. One micrococcus showed an unusual series of growth phases in glucose broth at 37·5°, possibly due to the appearance of mutants or to adaptation of the organism to growth at that temperature. This was not observed in pasteurized milk. C. lacticum died off when incubated in glucose broth at 37·5°.
None of the keeping quality tests was more effective than any other in detecting these organisms in milk. The micrococci and C. lacticum had little effect on the keeping quality of pasteurized milk within the period of 'commercial life'. Some of the spore formers and streptococci showed marked differences in the end-points with the clot-on-boiling and the alcohol precipitation tests.     

COLD-TOLERANT FERMENTATIVE GRAM-NEGATIVE ORGANISMS FROM MEAT AND OTHER SOURCES

SUMMARY: From various chilled meats, twenty-eight strains of coli-aerogenes bacteria and one Aeromonas were isolated which grew well at +1±5° and some at −1±5°. The optimum growth temperature for most of these strains was nearer 37° than 30°. Nine strains (including the Aeromonas ) fermented lactose rapidly, the remainder slowly or not at all. All the strains which fermented lactose rapidly with the production of gas gave positive presumptive coli-aerogenes tests in MacConkey's broth at 30°, but only five were positive at 37°; none was positive at 44°. Because such organisms can attain populations of millions/cm², they could confuse the interpretation of presumptive coli-aerogenes tests made on chilled meat.     

The enumeration of thermotrophic types amongst the Enterobacteriaceae colonizing perishable foods

A total of 41 pure cultures of Enterobacteriaceae, comprising 32 thermotrophic and nine psychrotrophic strains, pathogens or marker organisms, were examined for numbers of colony forming units obtained at 37° and 42°5°C (thermotrophs) and 30°C (psychrotrophs), when surface-plated on a rich infusion agar and violet red bile agar. In addition 42 food and water samples, collected in a rural area of the Philippines, were examined by surface inoculating violet red bile AIPC (agar immersion plating and contact; 'dip') slides and incubating at 37° and 42°5°C. At 42°5°C there was almost total recovery of the thermotrophic Enterobacteriaceae, whereas the psychrotrophic strains were completely suppressed. At 37°C the psychrotrophs were only slightly inhibited. The Philippine foods, predominantly cooked meals, milk and drinking water, appeared to be significantly colonized by thermotrophic Enterobacteriaceae. It is concluded that incubation at 42°5°C satisfactorily selects enteropathogenic and other enteric Enterobacteriaceae while suppressing the psychrotrophic types which are mainly of vegetable origin. It is emphasized that, regardless of the temperature used, a resuscitation procedure for Enterobacteriaceae populations that have incurred sublethal injury in food has to precede counts on or in the usual selective media.     

Comparison of most probable number and pour plate procedures for isolation and enumeration of sulphite-reducing Clostridium spores and Group D faecal streptococci from oysters

A comparative study of methods to enumerate sulphite-reducing Clostridium spores and Group D faecal streptococci in oysters demonstrated that pour plate solid agar techniques gave higher counts than liquid broth most probable number procedures. Reinforced clostridial broth with supplements to detect sulphite reduction was compared with pour plates of egg yolk-free tryptose sulphite cycloserine agar incubated at 37°C for 24 h. Azide dextrose broth was compared with pour plates using Slanetz and Bartley (SB) agar or KF-streptococcus agar at 37°C. Most probable number procedures used for both groups of organisms gave excessive numbers of improbable tube combinations. For enumeration of Group D faecal streptococci, a pour plate technique using SB agar incubated at 37°C for 48 h is recommended.