Mathematically modeling the repair of heat-injured Listeria monocytogenes as affected by temperature, pH, and salt concentration

Heat-injured cells of Listeria monocytogenes were inoculated into Listeria repair broth (LRB) adjusted to various pH levels (4.2, 5.0, 6.6, 8.0 and 9.6) and salt concentrations (0.5%, 2.5%, 5.0%, 7.5% and 10.0% w/v) at controlled temperatures (4, 10, 22, 37 and 43 degrees C) in a complete factorial manner (5(3)). Repair of the injured microorganisms was evaluated using selective and non-selective plating media. The Gompertz parameters, which were generated by fitting the equation with the bacterial counts, were used to calculate the repair percentage as a function of time from which the repair time was estimated. All growth curves fit the Gompertz equation well (R(2) > or = 0.972). A first-order model described the repair trend closely (R2 = 0.989 +/- 0.011). Heat-injured Listeria could fully repair in LRB only under 63 of 125 conditions tested during 21 days of incubation. Refrigeration temperature was the most effective means to prevent the repair of heat-injured Listeria. The minimum temperature required for repair increased with an increase in NaCl concentration. The pH ranges at which the repair could occur were narrower at 4 and 10 degrees C than those at higher temperature. The repair was observed in media containing 10% NaCl between temperatures of 22 and 43 degrees C at pH 6.6.     

Culture of dialysis fluids on nutrient-rich media for short periods at elevated temperatures underestimate microbial contamination

Recommended culture methods for monitoring bacterial contamination of H2O, dialysate and bicarbonate concentrate in dialysis centers in the USA involves culturing these fluids for 48 h at 37 degrees C. A variety of media and commercial culture methods are accepted for monitoring these fluids. Over a 3-month a comparison was made between an acceptable culture method, tryptic soy agar (TSA) employing the pour plate (PP) technique at 37 degrees C for 48 h, and PP cultures on standard methods agar (SMA) and R2A agar, incubated at ambient temperature (23 degrees C) for 48, 72, 168 h. Increases in the colony counts over time occurred for all three fluids. However, counts wee greater on SMA and R2A than on TSA. The increases over the standard 48-hour TSA cultures ranged as high as 10(4) times for 23 degrees C cultures at 7 days of incubation. Endotoxin levels even in the most contaminated samples were found to be below the acceptable 5 EU/ml recommended for reprocessor water. Bacterial colonies that appeared at 48, 72 and 168 h were isolated and identified. Pseudomonas, Moraxella, Acinetobacter and CDC group VI C-2 were among some of the common bacteria isolated. This study indicates that the media utilized, the time and temperature of incubation may result in a significant underestimation of the bacterial population of water and dialysis fluids, thus potentially placing the patient at a higher risk.     

Studies on the rod-coccus life cycle of Rhodococcus equi

In the present study all 19 Rhodococcus equi cultures isolated from horses and 19 of 22 R. equi cultures isolated from human patients displayed a rod-coccus life cycle after cultivation under defined growth conditions. A bacillary growth could be observed after cultivation of the bacteria in fluid media for 4 h at 37 degrees C, a coccoid morphology after cultivation of the bacteria for 24 h either on sheep blood agar plates or in fluid media. The different morphological features did not significantly influence the typability of the bacteria or the expression of surface proteins including 15-17 kDa virulence proteins. Studies on further surface characteristics revealed a relation between haemagglutinating properties, the surface hydrophobicity and adherence properties of the bacteria to HeLa cells. These properties seemed to be influenced by the cultivation conditions but not by the different morphological forms of the bacteria. A haemagglutination reaction, a hydrophobic surface and an enhanced adherence to HeLa cells could be observed with coccoid bacteria after cultivation in fluid media for 24 h at 37 degrees C but not with coccoid bacteria harvested from sheep blood agar plates or with bacillary bacteria after 4 h growth in fluid media. This difference might possibly be caused by the degree of encapsulation of the bacteria after various cultivation conditions and a subsequent masking effect of the hydrophilic polysaccharide capsule of R. equi.     

Studies on bactericidal activities of beta-lactam antibiotics on agar plates: the correlation with the antibacterial activities determined by the conventional methods

A simplified method to determine minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of beta-lactam antibiotics on agar plates is described. MIC values were determined on agar plates for benzylpenicillin, methicillin and cephalothin using Staphylococcus aureus and Klebsiella pneumoniae. A beta-lactamase solution was then sprayed onto the plates to inactivate the drug(s). After further incubation at 37 degrees C overnight, the minimal concentration at which no test bacteria were visible on the plates was defined as MBC. Both MIC and MBC values decreased with decreased inoculum size. The two values were almost coincidental when high dilutions were used as the inocula. These values were compared with those obtained by the conventional broth dilution method. In this study, MIC as well as MBC values determined by the simplified method were generally smaller than the values determined by the broth dilution technique.     

Bacillus subtilis GSY 1057 assay for aflatoxin B activation by rainbow trout (Salmo gairdneri)

A rapid and sensitive microbial assay was developed to detect lethal products of aflatoxin B metabolism by rainbow trout (Salmon gairdneri) Mt. Shasta strain. Bacillus subtilis GSY 1057 (hisA1, uvr-1, metB4), a DNA repair deficient strain, was incubated for 20 min in the 20,000 times g supernate from trout liver homogenates which had been preincubated for 10 min with various levels of aflatoxin B. Serial dilutions of the incubation mixture were plated in triplicate on tryptose blood agar base plates and colonies were counted after 12 hr at 37 degrees C. One mumole aflatoxin B in 3.2 ml incubation mixture reduced viability 60%.     

Comparison of two enrichment methods and five selective media for the isolation of Yersinia enterocolitica from tonsils of slaughter pigs (author's transl)

115 tonsils of healthy slaughter pigs were culturally examined for presence of Yersinia (Y.) enterocolitica. For this purpose each sample was enriched both in phosphate buffered saline solution (pH 7.6; stored at 4 degrees C and plated every week, thrice all together) and modified Rappaport broth (plated after an incubation of two days at 22 degrees C). Each such enrichment was plated on 5 different selective media: Yersinia selective agar proposed by Wauters (1973), deoxycholate-citrate-mannitol agar (Saari and Jansen, 1979), pectin agar (Bowen and Kominos), MacConkey and Leifson agar as used in the routine, diagnostic of Enterobacteriaceae. Each agar plate was incubated at 28 degrees C for two days. By cold enrichment method were isolated 11 strains of human pathogenic Y. enterocolitica (9 X O-group I syn. serotype O:3; 2X O-group V syn. serotype O:9). With the modified Rappaport medium were recovered 33 strains (24X O-group I, 9 X O O-group V). The most recoveries were done over the Yersinia selective agar with 65.2%, then followed deoxycholate-citrate-mannitol agar with 57.6%, Leifson agar with 45.5%, MacConkey agar with 42.3% and pectin agar only with 18.2% of the isolations. Not only the type of enrichment medium has a marked effect in the recovery efficiency of Y. enterocolitica out of samples but also number and type of the used selective media on which the enrichment is plated.     

Identification of mecA-related oxacillin resistance in staphylococci by the E test and the broth microdilution method

A set of 165 strains of different staphylococcal species, 67 Staphylococcus aureus, 71 novobiocin-sensitive coagulase-negative staphylococci (CNS) and 27 novobiocin-resistant CNS was used. The oxacillin and methicillin MICs were recorded after 24 and 42 h of incubation at 35 degrees C and at 30 degrees C. Significantly higher MICs were recorded at 30 degrees C compared with 35 degrees C. While a poor discrimination between mecA-positive and mecA-negative strains was obtained with methicillin, the oxacillin MICs enabled identification of resistant strains under certain conditions. The distribution of MICs differed between the three groups of species. Separation of uninduced mecA-positive (> or = 4.0 mg oxacillin/L) and mecA-negative (< or = 2.0 mg oxacillin/L) strains of S. aureus was only achieved with the E test and after 42 h of incubation. Oxacillin-induction yielded higher MICs for mecA-positive strains of S. aureus, and a separation from mecA-negative strains was achieved with the E test after 24 h and with the broth microdilution method after 42 h. Separation of mecA-positive and mecA-negative strains of novobiocin-sensitive CNS required agar supplemented with 5% blood, incubation of MIC trays and E test for 42 h, and species-specific oxacillin MIC breakpoints (S < or = 0.5 mg/L and R > or = 1.0 mg/L). The mecA-positive and mecA-negative strains of novobiocin-resistant CNS were clearly separated after 24 h of incubation by either method.     

Dermatophilus chelonae sp. nov., isolated from chelonids in Australia

Three isolates of a previously undescribed Dermatophilus sp. obtained from chelonids (two strains obtained from turtles and one strain obtained from a tortoise) were compared with 30 Dermatophilus congolensis isolates obtained from Australian mammals. The microscopic appearance, the colony morphology, and most biochemical test results for the chelonid isolates were characteristic of the genus Dermatophilus. Our isolates differed from the mammalian D. congolensis isolates in a number of cultural characteristics, including faster growth at 27 degrees C than at 37 degrees C, formation of two hemolysis zones around colonies on blood agar at 37 degrees C in the presence of 10% CO2, poor motility, and production of a distinctive odor. The DNA restriction enzyme digestion and protein electrophoresis patterns of our strains were distinct. The electrophoretic mobilities of 11 enzymes differed from the mobilities observed with D. congolensis strains. A monoclonal antibody to a surface antigen of an ovine isolate did not react with zoospores or filaments of the chelonid isolates. Biochemical differences between our isolates and D. congolensis included the ability of the chelonid isolates to reduce nitrate to nitrate and the fact that the chelonid isolates exhibit collagenase activity in vitro. We propose that the chelonid isolates should be placed in a new species, Dermatophilus chelonae. Strain W16, which was isolated from a nose scab on a snapping turtle, is the type strain; a culture of this strain has been deposited in the American Type Culture Collection as strain ATCC 51576.     

Use of the bacille Calmette-Guérin vaccination for the prevention of tuberculosis: renewed interest in an old vaccine

A method for the enumeration of colony-forming units of oral anaerobic spirochetes in new oral spirochete agarose (NOS-A) medium was described recently. However, the high cost of agarose limits the extent to which large assays can be carried out. Accordingly, a search for an inexpensive gelling agent that remains molten at 37 degrees C and gels at 25 degrees C was undertaken. Varying amounts of Noble agar or Bacto agar (0.5 to 1.5%, w/v) were mixed with varying amounts of gelatin (0.5 to 1.0%, w/v) in NOS medium. NOS medium containing 0.5% gelatin-0.5% Noble agar (NOS-GN) or 0.5% gelatin-0.5% Bacto agar (NOS-GB) met the above criteria. NOS-GN and NOS-GB media yielded higher colony-forming units with Treponema denticola than NOS-A medium in that order. However, all three media, NOS-GN, NOS-GB and NOS-A, performed equally well in the recovery of viable counts of T. vincentii. The NOS-GN medium was not liquefied by subgingival bacteria or two gelatinase-producing species of bacteria, Bacillus subtilis and Staphylococcus aureus. Thus NOS-GN medium is the recommended medium both in cost and performance for obtaining colony counts of spirochetes.     

A comparison of brain heart infusion blood agar sterilized by filtration and heat on the growth of Neisseria gonorrhoeae

The growth of Neisseria gonorrhoeae on brain heart infusion blood agar in which the base was sterilized by filtration has been compared with growth on the same medium sterilized by heat. Colonies were larger on the unheated medium, and autoclaving at 115 degrees C of 121 degrees C for 15 minutes was accompanied by a progressive decrease in colony size. Viable counts on the three media showed no difference in end points. Colonies on the unheated medium were usually large enough to be easily recognizable after overnight incubation.     

Simultaneous isolation of MRSA and Pseudomonas aeruginosa using a novel selective and differential PMAC agar

PMAC agar, a novel, selective and differential medium has been developed and was subjected for evaluation of its selective and differential capability of methicillin resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa from other bacteria such as Bacillus, Micrococcus, Gram-negative bacteria and drug resistant ones. Growth of MRSA and P. aeruginosa on PMAC agar was facilitated and their colonies were easily differentiated. Colonies of MRSA after 24 approximately 48 h incubation at 35 degrees C were small (2 to 4 mm in diameter), smooth and egg-yolk reaction positive. On the other hand, P. aeruginosa with pigment production (pyocianin, fluorescin or pyomelanin) formed large (2.5 to 7.0 mm in diameter), brownish black or brown colonies with a creamy edge. PMAC agar did not allow to grow unwanted bacteria tested except certain species formerly classified to Pseudomonas such as Burkholderia and Stenotrophomonas. However multi-drug resistant strains such as Enterobacter cloacae, Serratia marcescens and Acinetobacter calcoaceticus formed extremely small colonies. PMAC agar is recommended as a novel, useful medium for isolation, differentiation and presumptive identification of MRSA and P. aeruginosa from clinical and environmental sources.     

Long-term survival of Streptococcus pneumoniae at room temperature on Dorset egg medium

Forty-five isolates of Streptococcus pneumoniae were inoculated on Dorset egg and supplemented Columbia agar base media, incubated overnight at 37 degrees C, and then kept at room temperature (RT; 21 degrees C) or 4 degrees C. Long-term viability was best at RT for both media, with all isolates remaining viable on Dorset egg medium for 44 days; viability was 90 and 57% on Columbia agar base medium after 7 and 30 days. We recommend the use of Dorset egg medium for the maintenance of pneumococci at RT.     

Limited access to a dietary fat option affects ingestive behavior but not body composition in male rats

Survival, recoverability and sublethal injury of two strains of Listeria monocytogenes, Scott A and an environmental strain KM, on exposure to sea water at 12.8 or 20.8 degrees C was determined using in situ diffusion chambers. Plate counts were used to assess recoverability and injury while 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) reduction was used to determine respiratory activity. T90 values (times for 10-fold decreases in numbers of recoverable cells) on non-selective medium (trypticase soya agar with 0.6% yeast extract) at 12.8 and 20.8 degrees C were 61.7 and 69.2 h for L. monocytogenes Scott A, and 103.0 and 67.0 h for L. monocytogenes KM, respectively. On selective medium (Oxford agar), T90 values at 12.8 and 20.8 degrees C were 60.6 and 56.9 h for L. monocytogenes Scott A, and 83.0 and 65.9 h for L. monocytogenes KM, respectively. With Scott A, the percentage of sublethally injured cells at 12.8 and 20.8 degrees C was 1.7 and 17.7%, respectively, while for KM the values were 19.0 and 1.6%, respectively. The fraction of cells reducing CTC but which were not recoverable on plating progressively increased on exposure to sea water. Listeria monocytogenes KM challenged at 58 degrees C showed an apparent increase in heat resistance after exposure to sea water at 20.8 degrees C for 7 d (D58 = 2.64 min) compared with before exposure (D58 = 1.24). This increase in thermal resistance was not apparent at temperatures greater than 63 degrees C, and analysis of the best-fit regression lines fitted to the thermal data obtained from the two cell populations indicated that their thermal resistance was not significantly different (P > 0.05) over the temperature range tested (58-62 degrees C).     

Effects of above-optimum growth temperature and cell morphology on thermotolerance of Listeria monocytogenes cells suspended in bovine milk

The thermotolerances of two different cell forms of Listeria monocytogenes (serotype 4b) grown at 37 and 42.8 degrees C in commercially pasteurized and laboratory-tyndallized whole milk (WM) were investigated. Test strains, after growth at 37 or 42.8 degreesC, were suspended in WM at concentrations of approximately 1.5 x 10(8) to 3.0 x 10(8) cells/ml and were then heated at 56, 60, and 63 degrees C for various exposure times. Survival was determined by enumeration on tryptone-soya-yeast extract agar and Listeria selective agar, and D values (decimal reduction times) and Z values (numbers of degrees Celsius required to cause a 10-fold change in the D value) were calculated. Higher average recovery and higher D values (i.e., seen as a 2.5- to 3-fold increase in thermotolerance) were obtained when cells were grown at 42.8 degrees C prior to heat treatment. A relationship was observed between thermotolerance and cell morphology of L. monocytogenes. Atypical Listeria cell types (consisting predominantly of long cell chains measuring up to 60 micron in length) associated with rough (R) culture variants were shown to be 1.2-fold more thermotolerant than the typical dispersed cell form associated with normal smooth (S) cultures (P 相似文献   

Cobalt-chromium-molybdenum but not titanium-6aluminium-4vanadium alloy discs inhibit human T cell activation in vitro

The Authors studied the morphological, biochemical, physico-chemical and biological characteristics of Vibrio anguillarum cultured on different growth conditions, characterized by low osmolarity and high temperature (37 degrees). One culture was subcultured for several days in tryptone soya agar with 0.5% Nacl at 37 degrees C incubation until the cell morphology was stabilized. The low osmolarity, through an osmotic shock, induced remarkable morphological modifications in the strain, evidenced by optic and electron microscopic studied; in addition SDS-PAGE analysis of saline extracts from the culture at 37 degrees C showed a specific new protein band of about 66KDa. This band was correlated with remarkable differences in outer membrane protein composition (OMPs) evidenced by Ag/Ah cross-reactions with rabbit hyperimmune sera against the modified and the reference V. anguillarum strains. Finally, the modified strain proved to be non pathogenic for trout and sea bass.     

Influence of endotoxin on contractility in the rat gastric fundus

Cefoperazone amphotericin teicoplanin (CAT) agar was developed from cefoperazone deoxycholate (mCCD) agar by modification of the selective antibiotics in order to permit growth of strains of Campylobacter upsaliensis. In this study, 35 strains of Campylobacter and Arcobacter were tested for their ability to grow on CAT and mCCD media using the ecometric method. Six of these strains were also tested using the modified Miles-Misra method. Overall, nineteen strains out of the 35 tested grew better on CAT than on mCCD agar, although for eight strains, the difference was slight. These differences could not be attributed solely to poorer growth of C. upsaliensis on mCCD agar. No strain grew better on mCCD than CAT agar. Eight of the 35 strains tested did not grow on mCCD agar at all, however, only one strain failed to grow on CAT medium. The two methods of testing gave similar results, although the Miles-Misra method was found to be more sensitive and less prone to subjective interpretation. All four CNUPC (catalase negative, urease positive campylobacter-like) strains, one strain of C. sputorum biovar, fecalis, one of two Arcobacter cryaerophilus strains (incubated at 30 degrees C, aerobically) could be detected only using CAT agar. In addition, for some strains of A. butzleri, C. upsaliensis and C. hyointestinalis, CAT medium gave better growth scores than mCCD agar. The level of cefoperazone in mCCDA is inhibitory to some campylobacter strains, but suboptimal growth of Arcobacter strains is more probably due to synergistic interaction between deoxycholate and cefoperazone. CAT agar supports the growth of a wider variety of Campylobacter and Arcobacter species than mCCD agar.     

Enumeration of intestinal enterococci and interfering organisms with Slanetz-Bartley agar, KF streptococcus agar and the MUST method

The recovery of intestinal species of enterococci and streptococci and potentially interfering nonfaecal species was measured on KF streptococcus agar, Slanetz-Bartley agar and in a medium based on 4-methylumbelliferyl-beta-D-glucoside (MUST method) using pure cultures. Both of the solid media yielded high recoveries of the target species. Their selectivity was better at elevated incubation temperature but nonfaecal Enterococcus and Staphylococcus species were not eliminated even at the elevated temperature. The MUST method tended to give slightly lower recoveries than the agar cultivation methods with some target species at 44 degrees C but recoveries were better at 41 degrees C.     

A highly adherent phenotype associated with virulent Bvg+-phase swine isolates of Bordetella bronchiseptica grown under modulating conditions

The ability of Bvg(-)-phase and Bvg(+)-phase Bordetella bronchiseptica swine isolates, grown under modulating or nonmodulating conditions, to adhere to swine ciliated nasal epithelial cells was determined. When virulent strains were cultivated at 37 degrees C in the Bvg+ phase, numerous adherent bacteria (approximately eight per cell, depending on the strain used) were observed. However, when such strains were grown under modulating conditions (23 degrees C), a significant increase in the level of attachment was seen, suggesting that B. bronchiseptica produces a Bvg-repressed adhesin under these conditions. bvg mutant strains, including an isogenic bvgS mutant, adhered minimally. Western blots indicated that two putative B. bronchiseptica adhesins, filamentous hemagglutinin and pertactin, were not detectable in cultures displaying the highly adherent phenotype. Several proteins apparent in Western blots obtained by using bacterial extracts enriched in outer membrane proteins derived from B. bronchiseptica grown at 23 degrees C were not present in similar extracts prepared from an isogenic bvgS mutant grown at 23 degrees C or from the parent strain grown at 37 degrees C. Adherence of bacteria cultivated at 23 degrees C was almost completely abolished by pretreatment of organisms at 60 degrees C; adherence was reduced by 57% when bacteria were pretreated with pronase E. Temperature shift experiments revealed that the heightened level of adhesion that occurs following growth at 23 degrees C was maintained for up to 18 h when bacteria were subsequently incubated at 37 degrees C. We propose that a Bvg-repressed adhesin, expressed only by modulated bvg+ strains of B. bronchiseptica, may play a key role in the initial colonization of naturally infected swine.     

H-NS regulates DNA repair in Shigella

We report a new role for H-NS in Shigella spp.: suppression of repair of DNA damage after UV irradiation. H-NS-mediated suppression of virulence gene expression is thermoregulated in Shigella, being functional at 30 degrees C and nonfunctional at 37 to 40 degrees C. We find that H-NS-mediated suppression of DNA repair after UV irradiation is also thermoregulated. Thus, Shigella flexneri M90T, incubated at 37 or 40 degrees C postirradiation, shows up to 30-fold higher survival than when incubated at 30 degrees C postirradiation. The hns mutants BS189 and BS208, both of which lack functional H-NS, show a high rate of survival (no repression) whether incubated at 30 or 40 degrees C postirradiation. Suppression of DNA repair by H-NS is not mediated through genes on the invasion plasmid of S. flexneri M90T, since BS176, cured of plasmid, behaves identically to the parental M90T. Thus, in Shigella the nonfunctionality of H-NS permits enhanced DNA repair at temperatures encountered in the human host. However, pathogenic Escherichia coli strains (enteroinvasive and enterohemorrhagic E. coli) show low survival whether incubated at 30 or 40 degrees C postirradiation. E. coli K-12 shows markedly different behavior; high survival postirradiation at both 30 and 40 degrees C. These K-12 strains were originally selected from E. coli organisms subjected to both UV and X irradiation. Therefore, our data suggest that repair processes, extensively described for laboratory strains of E. coli, require experimental verification in pathogenic strains which were not adapted to irradiation.     

Evaluation of MUG-supplemented media for the detection of E. coli in recreational water surveillance

Four media containing 4-methylumbelliferyl-beta-D-glucuronide were evaluated as a non-confirmatory procedure for E. coli detection in recreational water surveillance. The media included ECD-Agar for membrane filtration and laurylsulphate-tryptose, brilliant-green-bile and lactose as broth media in a three tube most probable number procedure. From six representative water sites, samples were collected weekly over a typical summer season (17.05-27.09.1994) and processed as parallels, using each media at two different incubation temperatures (36 degrees/44 degrees C). Results showed that incubation temperature had no impact on E. coli counts. Each media at a given temperature could be regarded as individual enrichment procedure. None of these enrichment procedures showed a constant and predictable higher sensitivity during the sampling period at all sites compared to the others tested. For parallel results, the rate of agreement, based upon EC-guideline (76/160/EWG) staging of recreational water quality, was 85% for membrane filtration and 75% for the MPN-procedure results. Marked differences could be observed in false-positive specificity showing correlation to the selective characteristics of the media. Subsequently lactose-broth at 44 degrees C performed worst with 30% non verifiable results, while ECD-agar and laurysulphate-tryptose-broth, both at 44 degrees C, had a nearly 100% confirmation rate. Thus, combining high specificity with no lack in sensitivity these two MUG-supplemented media seem to be best suited for E. coli detection in routine recreational water surveillance.