Sterile Tryptic Soy Broth Research Articles

Ileal and cecal microbiota response to Salmonella Typhimurium challenge in conventional and slow-growing broilers.

Despite the negative impacts of Salmonella intestinal colonization on human health, Salmonella is a natural colonizer of the gastrointestinal tract and is not overtly pathogenic to the avian host. It is of interest to understand the impacts and colonization rates of Salmonella across selected genetic lines such as slow-growing (SG) and conventional (CONV) broilers. The objective of this study was to characterize the relationship between Salmonella enterica serovar Typhimurium challenge and selected broiler genetic lines on the ileal and cecal microbiome. Male chicks of two broiler breeds (n = 156/breed) were cohoused in an open floor pen until day 7. On day 13, the chicks were then separated into 12 isolators per breed (4 rooms, 6 isolators/room, 11 chicks/isolator). On day 14, chicks in the 12 treatment isolators (6 isolators/breed, 108 total) were challenged with Salmonella Typhimurium (ST) (1 × CFU/ml) via oral gavage while the remaining chicks (n = 108) were given an oral gavage of sterile tryptic soy broth control (C). Ileal and cecal contents were collected on day 7 from 24 chicks of each breed, and on days 13, 17, 21, and 24 from two chicks per isolator. Samples underwent DNA extraction and PCR amplification to obtain 16S rRNA amplicons that were sequenced with Illumina MiSeq. Salmonella Typhimurium colonization in the cecum was not different in the two broiler breeds. The main effect of breed had the greatest impact on the ileum microbiota of broilers 7 days of age where SG broilers had significantly lower diversity and richness compared to CONV broilers (p < 0.05). Salmonella Typhimurium challenge consistently caused a change in beta diversity. Regardless of day or intestinal location, challenged broilers had many amplicon sequence variants (ASVs) with decreased abundance of likely beneficial bacteria such as Mollicutes RF39, Shuttleworthia, Flavonifractor, and Oscillibacter compared to broilers that were unchallenged with Salmonella Typhimurium (p < 0.05). Additionally, there was a difference in the timing of when the microbiota alpha and beta diversity of each breed responded to Salmonella Typhimurium challenge. Thus, both broiler breed and Salmonella Typhimurium can impact the intestinal microbiota.

Bacterial profile and antimicrobial susceptibility patterns in cancer patients.

Bloodstream infections have been the leading complications in cancer patients because they are at high risk for antibiotic-resistant bacterial infections. There is increasing evidence from different parts of the world of the high prevalence of antimicrobial-resistant bacterial strains in cancer patients. The burden of the infection is high in developing countries, especially in Ethiopia. Data on bacterial profile and antimicrobial susceptibility patterns among cancer patients in Ethiopia is limited. Thus, this study aimed to determine the predominant bacterial species causing bacteremia and their antibiotic resistance pattern among cancer patients at University of Gondar comprehensive specialized hospital. A hospital-based, cross-sectional study was conducted on 200 study participants from March to July 2021. All cancer patients who developed a fever at the time of hospital visit were included in this study, and their socio-demographic and clinical data were collected using a structured questionnaire. Blood samples (10 mL for adults and 4 mL for children) were collected from each patient, and the collected blood samples were transferred into sterile tryptic soy broth, then incubated at 37°C for 7 days. Tryptic soy broth which showed signs of growth were Gram-stained and sub-cultured on blood agar, chocolate agar, MacConkey agar, and mannitol salt agar. The inoculated plates were then aerobically incubated at 37°C for 18-24 hours and the isolates obtained were identified using standard microbiological methods. Antimicrobial susceptibility tests were done using a modified Kirby-Bauer disk diffusion technique following CLSI 2021 guidelines. Data were entered using EPI data version 4.6 and analyzed with SPSS version 20. In this study, out of 200 cancer patients included and 67.5% (135/200) of them were males. The majorities of study participants, 56% (113/200) of cancer patients were pediatrics and 26.5% (53/200) of them belong under five years of age. Out of 200 patient samples that had undergone culture, 27% (54/200) samples had bacterial growth. Gram-positive bacterial isolates were predominant, 61.1%, and S. aureus was the predominant Gram-positive isolate, (51.5.6%), followed by coagulase-negative staphylococci (48.5%). Moreover, K. pneumoniae (47%) and P. aeruginosa (29.5%) were the most common Gram-negative bacterial isolates. Among patients who had BSIs, the highest prevalence of BSIs was observed among males (66.7%), and in pediatrics cancer patients (44.2%). Pediatric study participants were more venerable to bloodstream infection (P = 0.000) compared to adult participants. Meropenem (100%), amikacin (100%), piperacillin/tazobactam (72.3%), and ceftazidime (73.5%) were effective against for Gram-negative isolates while cefoxitin (81.2%) and penicillin (70.5%) were effective for Gram-positive isolates. Additionally, most Gram-negative and Gram-positive bacterial isolates were sensitive for gentamycin (75.9%). Multidrug resistance was seen among 17.1% bacterial isolates, and MDR in Gram-negative and Gram-positive bacteria were 83.3% and 16.7%, respectively. Gram-negative bacterial isolates showed a high prevalence of MDR than Gram-positive isolates. BSI's remains an important health problem in cancer patients, and Gram-positive bacteria were more common as etiologic agents of BSIs in cancer patients. S. aureus was the dominant bacteria followed by CoNS, K. pneumoniae, and P. aeruginosa. Multidrug-resistant isolates found in cancer patients and routine bacterial surveillance and study of their resistance patterns may guide successful antimicrobial therapy and improve the quality of care. Therefore, strict regulation of antibiotic stewardship and infection control programs should be considered in the study area.

Method of Inoculum Preparation Impacts Efficiency of E. Coli O157:H7 and Salmonella Surrogate Organism Inoculation onto Intact Beef During Antimicrobial Intervention Validation

ObjectivesMeat processors must validate the efficacy of antimicrobial interventions for food safety verification purposes. The methods by which pathogen surrogates are prepared for in-plant challenge trials may impact their attachment onto meat surfaces, potentially impacting challenge trial results. This study compared 2 procedures for preparing an inoculum of non-pathogenic Escherichia coli for completion of antimicrobial intervention validation.Materials and MethodsChilled beef bottom rounds were purchased from a local purveyor; each was halved into 2 portions and assigned to 1 of 2 inoculum preparation methods. Five non-pathogenic E. coli Biotype I strains were activated from culture stocks by loop transfer of each strain from a tryptic soy agar (TSA) slant to 10 mL sterile tryptic soy broth and incubating aerobically at 35°C (18 to 24 h). This process was completed in identical fashion twice sequentially. Upon activation completion, tubes from each culture were combined together into a 50 mL vol. mixture (Preparation Method 1). For Preparation Method 2, cells produced in identical fashion to those for Method 1 were at this point collected by centrifugation. The supernatant was decanted and pellet resuspended in 50 mL 0.1% (w/v) peptone diluent. Washing and resuspension procedures were repeated thrice identically. Final pellets were stored in insulated coolers containing coolant packs for 48 h prior to resuspending in 50 mL 0.1% diluent. Inocula were sprayed onto beef (2 mL delivered) using a hand-held sprayer. After application, E. coli cells were allowed to attach to meat surfaces for 30 min (25°C). Following attachment, 3.5% lactic acid (15 mL, 25°C) or Citrilow (15 mL; pH 1.05, 25°C) were sprayed onto inoculated beef. Prior to, and following intervention application, surface tissue excisions (5 × 10 cm2 each; 2 mm depth) were collected via sterile implements. Excisions were composited, and stomached in 100 mL sterile diluent. Decimal dilutions in 0.1% peptone diluent were prepared and spread on TSA surfaces, then overlaid with 12 mL MacConkey Agar. Plates were incubated 48 h (35°C) prior to colony inspection and enumeration. Data were collected from 2 identically completed replications (n = 12). Mean E. coli attaching to beef, and E. coli mean reductions as a function of inoculum preparation and antimicrobial treatment, were then compared.ResultsMethod of cocktail preparation impacted numbers of E. coli cells recovered from inoculated beef samples following post-attachment (P = 0.003). Mean numbers of E. coli on beef samples via inoculation methods 1 and 2 were 6.5 and 6.1 log10 CFU/cm2, respectively. However, no differences in E. coli reductions as a function of the interaction of main effects (inoculation method*antimicrobial treatment) were detected (P = 0.407).ConclusionAnalysis of data indicates that method of pathogen surrogate preparation for in-plant food safety intervention validation will impact the numbers of cells successfully inoculated onto experimental product surfaces. However, method of inoculum preparation did not impact observed surrogate reductions following treatment. Researchers and industry specialists engaged in food safety intervention validation should carefully consider all aspects of challenge trial development, execution, and the potential impacts on resulting data.

Rapid Detection of Pediatric Bacteriuria Using Narrow Angle Forward Laser Scattering Technology (NAFLST) with Bacterioscan

BackgroundPediatric urinary tract infections (UTI) are common, but culture-based diagnosis can take up to 48 hours. This time delay means patients are exposed to potentially unnecessary antibiotics. The sensitivity of screening urinalysis can vary, so rapid detection of UTI by another means would be beneficial. Narrow Angle Forward Laser Scattering Technology (NAFLST) with Bacterioscan can rapidly detect bacteriuria by shining a laser continuously through a liquid sample containing replicating bacteria, and graphing the degree of light refraction over time. Higher degrees of light refraction represent higher initial bacterial load and continued bacterial growth. After 3 hours, the optical scatter classifies a sample as either Likely Positive or Likely Negative. We compared Bacterioscan results to culture data in pediatric patients to assess the ability to diagnose UTI and avoid unnecessary urine culture.MethodsThis protocol was approved by the UNC Biomedical Institutional Review Board. Over one month, 169 pediatric (<18 yo) urine cultures were collected as part of routine patient care. An individual urine sample and 2.5mL of Sterile Tryptic Soy Broth were pipetted into a Bacterioscan micro-curette. Bacterioscan labeled the specimen as Likely Positive or Likely Negative after a 3 hour period. Results were then compared with urine culture results obtained by routine microbiologic methods.ResultsOf the 169 urine cultures, 96 were positive, but only 27 were positive for uropathogens. Bacterioscan was 100% sensitive and 58.4% specific in predicting clinically relevant/pathogenic bacterial growth in culture (PPV 31.3%, NPV 100%), and 70.8% sensitive and 75.3% specific in predicting any bacterial growth (PPV 79.0%, NPV 66.2%). If a “Likely Positive” Bacterioscan result had been used in our study population to screen urine samples for culture, then 58% (83/142) of negative urine cultures would have been eliminated with no UTIs missed.ConclusionBy rapidly identifying urine cultures likely to be positive, NAFLST with Bacterioscan can safely obviate the plating of every urine sample and reduce empiric antibiotic use while waiting for culture results. Larger studies are required to confirm these results.Disclosures A. Cline, Bacterioscan: Equipment necessary to perform this study was provided by Manufacturer, Equipment necessary to perform this study; R. Jhaveri, Gilead: Investigator, Research support; Abbvie: Investigator, Research support; Merck: Grant Investigator, Research grant

Thermotolerance of Rifampicin‐Resistant Escherichia coli O157:H7 Derivatives and Their Parental Strains in a Broth System

AbstractRifampicin‐resistant (RifR) strains have often been used in studies of Escherichia coli O157:H7 to assure more specific recovery. The present study compared the heat inactivation kinetics of rifampicin‐resistant variants of pathogenic and nonpathogenic E. coli O157 strains with those of the wild‐type parental strains. Spontaneous RifR derivatives of 11 pathogenic and six nonpathogenic E. coli O157 strains were selected. Stationary‐phase cells of each strain in sterile tryptic soy broth were heated at 60C for 0, 30, 60, 90, 120, 150, 180 or 240 s, and aliquots were plated on tryptic soy agar supplemented with 0.1% pyruvate + 100 μg/mL rifampicin. D values of three pathogenic and two nonpathogenic RifR E. coli O157 strains were 35–70% lower (P &lt; 0.05) than those of their wild‐type counterparts. However, the heat resistance of most of the RifR E. coli O157:H7 strains (12 strains) was similar (P ≥ 0.05) to that of their parental counterparts. Therefore, the findings of this study demonstrated that the majority of the rifampicin‐resistant E. coli O157:H7 variants that were evaluated to assure more reliable recovery in microbial inactivation studies are suitable for use in various heat challenge studies.Practical ApplicationsMore reliable media are needed for the isolation or recovery of E. coli O157:H7 in inoculated challenge studies. Prior to use of rifampicin‐resistant E. coli O157:H7 strains in heat challenge studies, it is important that their thermal inactivation kinetics be compared with those of their wild‐type parental strains. The results of this study showed that the thermotolerance at 60C of the majority of the tested rifampicin‐resistant E. coli O157:H7 variants was not different than that of the corresponding wild‐type strains. These rifampicin‐resistant strains can, therefore, be used in heat challenge studies and can be recovered with non‐selective culture media supplemented with rifampicin.

Media Fill Test for validation of autologous leukocytes separation and labelling by 99mTc-HmPAO

Manufacturing of sterile products must be carried out in order to minimize risks of microbiological contamination. White blood cells (WBC) labelled with (99m)Tc-exametazime ((99m)Tc-hexamethylpropyleneamine oxime; (99m)Tc-HMPAO) are being successfully applied in the field of infection/inflammation scintigraphy for many years. In our radiopharmacy lab, separation and labelling of autologous leukocytes with (99m)Tc-HMPAO were performed in a laminar flow cabinet not classified and placed in a controlled area, whereas (99m)Tc-HMPAO radiolabelling procedure was carried out in a hot cell with manipulator gloves. This study was conducted to validate this process using a Media Fill simulation test. The study was performed using sterile Tryptic Soy Broth (TSB) in place of active product, reproducing as closely as possible the routine aseptic production process with all the critical steps, as described in the our internal standard operative procedures (SOP). The final vials containing the media of each processed step were then incubated for 14 days and examined for the evidence of microbial growth. No evidence of turbidity was observed in all the steps assayed by the Media Fill. In the separation and labelling of autologous leukocytes with (99m)Tc-HmPAO, Media-Fill test represents a reliable tool to validate the aseptic process.

Attachment and biofilm formation by Escherichia coli O157:H7 at different temperatures, on various food-contact surfaces encountered in beef processing

Escherichia coli O157:H7 attached to beef-contact surfaces found in beef fabrication facilities may serve as a source of cross-contamination. This study evaluated E. coli O157:H7 attachment, survival and growth on food-contact surfaces under simulated beef processing conditions. Stainless steel and high-density polyethylene surfaces (2×5cm) were individually suspended into each of three substrates inoculated (6log CFU/ml or g) with E. coli O157:H7 (rifampicin-resistant, six-strain composite) and then incubated (168h) statically at 4 or 15°C. The three tested soiling substrates included sterile tryptic soy broth (TSB), unsterilized beef fat-lean tissue (1:1 [wt/wt]) homogenate (10% [wt/wt] with sterile distilled water) and unsterilized ground beef. Initial adherence/attachment of E. coli O157:H7 (0.9 to 2.9log CFU/cm(2)) on stainless steel and high-density polyethylene was not affected by the type of food-contact surface but was greater (p<0.05) through ground beef. Adherent and suspended E. coli O157:H7 counts increased during storage at 15°C (168h) by 2.2 to 5.4log CFU/cm(2) and 1.0 to 2.8log CFU/ml or g, respectively. At 4°C (168h), although pathogen levels decreased slightly in the substrates, numbers of adherent cells remained constant on coupons in ground beef (2.4 to 2.5log CFU/cm(2)) and increased on coupons in TSB and fat-lean tissue homogenate by 0.9 to 1.0and 1.7 to 2.0log CFU/cm(2), respectively, suggesting further cell attachment. The results of this study indicate that E. coli O157:H7 attachment to beef-contact surfaces was influenced by the type of soiling substrate and temperature. Notably, attachment occurred not only at a temperature representative of beef fabrication areas during non-production hours (15°C), but also during cold storage (4°C) temperatures, thus, rendering the design of more effective sanitation programs necessary.

Protection against heterologous Streptococcus iniae isolates using a modified bacterin vaccine in Nile tilapia, Oreochromis niloticus (L.)

Streptococcus iniae is a significant pathogen impacting aquaculture production worldwide. The objectives of this study were to determine whether a developed modified S. iniae (ARS-98-60) bacterin vaccine is efficacious in Nile tilapia, Oreochromis niloticus (L.), against challenge with heterologous isolates from diverse geographical locations and to evaluate protein and antigenic variability among the isolates tested. Two groups of tilapia (approximately 5 g) were intraperitoneally (IP) vaccinated with 100 microL of the vaccine or sham vaccinated with 100 microL of sterile tryptic soy broth and held for 28 days. Fish were challenged with each isolate by IP injection of 2-3 x 10(7) CFU per fish using calcein to mark fish prior to cohabitation for challenge. The results demonstrated significant protection against all challenge isolates, and relative percent survivals ranged from 79% to 100%. SDS-PAGE analysis of whole-cell lysate proteins from the S. iniae isolates demonstrated similar protein profiles between 10 and 31 kDa and variation in profiles between 35 and 100 kDa. Western blot analysis using antiserum from vaccinated fish (ARS-98-60) demonstrated shared immunogenic proteins among all isolates in the molecular mass range of 22-35 kDa and high molecular mass material >150 kDa. The results suggest that the developed S. iniae vaccine has broad ranging protection among isolates exhibiting different protein profiles.

Antibacterial effect of bioactive glass in combination with powdered enamel and dentin

In endodontics, various intracanal medications have been advocated to eliminate bacteria after root canal instrumentation. A recent study has revealed that addition of powdered dentin to bioactive glass (BAG) led to increased glass dissolution, and an increased antibacterial efficacy. Therefore, the present study was undertaken to compare the effect of enamel and dentin powder on the antibacterial efficacy of a commercially available BAG. Dentin blocks (dbs) were prepared from single rooted human teeth. These dbs were infected with Enterococcus faecalis for two weeks in Tryptic Soy Broth (TSB), while negative controls were kept in sterile TSB. In group I, the infected dbs were filled with BAG, in group II with BAG + Enamel powder and group III with BAG + Dentin powder. Dentin samples were harvested from the dbs and cultured. Kruskal-Wallis ANOVA was used for multiple group comparison followed by Scheffe's post hoc test for pair-wise comparisons. All the combinations of BAG evaluated significantly reduced the bacterial counts compared to the control group. However, at the end of 24 hours, three days, and five days BAG + Dentin powder showed significant reduction ( P < 0.01) in bacterial counts compared to the other experimental groups. Among the various materials evaluated, it appeared that though BAG exhibits antimicrobial efficacy, the addition of powdered enamel and dentin in aqueous suspension definitely enhanced this property. However, the addition of enamel powder BAG did not significantly alter its antimicrobial efficacy compared to BAG + dentin powder.

Application of Colicin E1 as a Prefabrication Intervention Strategy

Colicin E1 (ColE1) is a bacteriocin produced by and effective against Escherichia coli and related species. The current study examined ColE1 as a potential intervention strategy for controlling E. coli O157:H7 contamination on beef carcasses. Untrimmed beef round roasts were cut into sample sizes of 5.08 by 2.52 by 5.08 cm, with an adipose layer covering an entire surface of lean beef. Samples were placed on sterile metal hooks and inoculated with E. coli O157:H7 at a level of 5 log CFU/ml in sterile tryptic soy broth. After inoculum attachment, ColE1 in doses of 0, 100 μg, 500 μg, and 1 mg/ml of 10mM Tris, pH 7.6, was sprayed on the samples for a period of 10 min. Samples were evaluated at 0 and 30 min, 1, 2, 3, 4, and 5 days post-spraying at 10°C for E. coli O157:H7 inhibition. Treating samples with 500 μg and 1 mg of ColE1 effectively inhibited E. coli O157:H7 growth. When these doses were applied to samples inoculated with E. coli WS 3331, E. coli contamination was reduced by 4 and 7 log CFU/cm2, respectively, compared with the untreated control samples. In strain WS 3331, treatment with 1 mg ColE1 significantly inhibited growth of E. coli O157:H7 compared with the untreated control during the entire study. ColE1 provided powerful reduction of E. coli O157:H7 as a beef carcass spray intervention.

A comparative study on the disinfection potentials of bioactive glass S53P4 and calcium hydroxide in contra‐lateral human premolars ex vivo

To evaluate the effects of bioactive glass S53P4 versus calcium hydroxide when used as dressings in contra-lateral human premolars infected with Enterococcus faecalis ATCC 29212. Pairs of contra-lateral premolar teeth plus single control premolars were obtained from 23 individuals aged 10-26 years undergoing orthodontic treatment. Root canals of teeth with fully formed apices (nine contra-lateral pairs, seven controls) were instrumented using a size 60 FlexoFiles 2 mm short of canal length. Canals with open apices (six contra-lateral pairs, four controls) were circumferentially instrumented using a FlexoFile. Root canals were rinsed with 1% sodium hypochlorite and 10% citric acid. Teeth were then suspended in tryptic soy broth (TSB) and autoclaved. Positive controls and study teeth were infected with E. faecalis ATCC 29212 for 2 weeks in TSB, while negative controls were kept in sterile TSB. Subsequently, contra-lateral premolars were dressed with bioactive glass S53P4 (BAG) or calcium hydroxide suspensions for 10 days. Dentine samples were obtained from teeth with fully formed apices using ISO-size 70, 80 and 90 FlexoFiles to working length and cultured. Teeth with open apices were fixed, fractured and examined using scanning electron microscopy (SEM). Calcium hydroxide had a strong antibacterial effect and was significantly more effective than BAG in preventing residual bacterial growth (P < 0.01). SEM analysis revealed apparent substance-specific modes of action. Calcium hydroxide was an effective disinfectant in human teeth.

Local Delivery of Vancomycin for the Prophylaxis of Prosthetic Device-Related Infections

To evaluate the in vivo efficacy and pharmacokinetics of vancomycin delivered from glycerylmonostearate (GMS) implants in a prosthetic-device based biofilm infection model. A biofilm infection model was developed in male Sprague-Dawley rats by implanting a vascular graft on the dorsal side of each rat and infecting it with 1.5 x 10(8) cfu/ml Staphylococcus epidermidis. The rats were divided into 3 groups of 6 rats each: 1) the control group that received no antibiotics, 2) the IM group that received multiple IM injections of vancomycin at a dose of 25 mg/kg every 6 h for a total of 12 doses, and 3) the implant group that received GMS implants designed to deliver vancomycin at a total dose of 300 mg/kg for a period of 4 days. The pharmacokinetics of vancomycin was determined from IM and implant groups by analyzing for vancomycin in blood using HPLC. In vivo efficacy was studied by evaluation of the wound site and the prosthetic device upon excision, for evidence of infection in the form of purulent discharge at the wound site and yellowish discoloration of the prosthetic device and inflammation as sign of biofilm formation. Microbiological evaluation on the wound site and the prosthetic device was performed by culturing the swabs at the wound site and the prosthetic device in sterile tryptic soy broth for 36-48 h at 37 degrees C. Vancomycin was successfully delivered in a sustained manner for 100 h from GMS implants and the resulting plasma profile showed that the concentrations, after an initial burst, plateaued at about of 4.77 +/- 1.43 mug/ml with less fluctuations than the IM group in which the plasma concentrations fluctuated between 2.73 +/- 0.94 mug/ml and 19.26 +/- 3.67 mug/ml. Upon excision of the wound site, all the animals in the control group developed infection in the form of purulent discharge and yellowish discoloration of the prosthetic device. However, none of the rats in the implant group showed evidence of infection clearly demonstrating the efficacy of the local delivery system in preventing infection. Systemically delivered vancomycin by IM injections failed to prevent infection in four out of six rats. Microbiological evaluation of the wound site and prosthetic device resulted in isolation of biofilm-producing organisms such as Staphylococcus epidermidis, Enterococcus faecalis, and Staphylococcus aureus. These organisms were isolated in greater number of animals in the control group compared to the IM and implant groups. The GMS implants as a delivery system for vancomycin were successful in preventing infection in all the animals compared to the IM and control groups demonstrating the efficacy of a local delivery system in a prosthetic device related biofilm infection model.

Changes in Heat Resistance Resulting from pH and Nutritional Shifts of Acid-Adapted and Non–Acid-Adapted Listeria monocytogenes Scott A

Stationary-phase Listeria monocytogenes cells that were either pH dependent acid adapted or not acid adapted were heat challenged at 60°C in a two-level full factorial design for three variables. The three variables and the levels consisted of tryptic soy broth (TSB) and sterile cell-free culture supernatant (sterile TSB), the presence and absence of 1% added glucose, and pH 4.8 and pH 7. Non–acid-adapted cells were most heat resistant when challenged in TSB (mean decimal reduction times at 60°C: D60 = 1.16 min). In the absence of added glucose, non–acid-adapted cells had similar D60-values for inactivations at pH 4.8 and pH 7; however, the presence of glucose caused non–acid-adapted cells challenged at pH 4.8 to be more heat sensitive (D60 = 0.65 min) than those inactivated at pH 7 (D60 = 1.03 min), indicating an interaction between glucose and pH. Overall, the significantly decreased heat resistance of the acid-adapted cells was due to the presence of glucose (D60 = 0.78 min without glucose, D60 = 0.59 min with glucose). Acid-adapted cells heat challenged in TSB had similar D60-values for inactivations at pH 4.8 and pH 7; however, acid-adapted cells in sterile TSB challenged at pH 4.8 (D60 = 0.52 min) had significantly lower heat resistance than did cells challenged at pH 7 (D60 = 0.76 min), indicating an interaction between the medium and pH. The L. monocytogenes survivor data were modeled to extract information on the frequency distribution of heat resistance within heat-challenged populations, and the frequency distribution characteristics of mean, mode, and variance were compared among treatment conditions. Significant differences in the frequency distribution data were compared with the D60-values. These data indicated that the presence and level of cross-protection is highly dependent on the physiological state of the cells and nutrient availability at the time of heat challenge. Such conditions should be considered to ensure that stressed pathogens in foods are destroyed or inactivated.

Spread and survival of a genetically modified Pseudomonas aureofaciens in the phytosphere of wheat and in soil

An isolate of Pseudomonoas aureofaciens indigenus to the UK, which was chromosomally modified by the insertion of two gene cassettes ( xy/ E in combination with the gene for kanamycin resistance and lacZY) for the sensitive monitoring purposes was provided by IVEM (Oxford). Growth of this organism and the non-modified wildtype strain were not significantly different in sterile tryptic soy broth. Spread and survival of the marked bacterium in the phytosphere of spring wheat and in soil was quantified in a number of glasshouse and laboratory experiments. Six days after vacuum infiltration of wheat seeds (growth stage (GS) 11; seedling) with bacteria, most (approximately 99%) of the bacteria stayed associated with the seed, while a small proportion was recovered from the roots and leaves (0.8% and 0.2%, respectively). When seeds were inoculated with 5 × 10 8 recombinant colony forming units (cfu) per seed the bacterium was able to survive up to levels of 10 5 cfu g −1 root during the whole of the plant cycle (4 months), but the bacterium established on the first emerging leaves only in low number (approximately 1000 cfu g −1 tissue). The bacterium could be recovered from roots extracted from a depth of 60 cm, but most (> 95%) of the bacteria stayed associated with roots in the top 15 cm the soil profile. When bacteria were sprayed onto wheat plants at GS 22 (tillering), they could be recovered 1 month later (GS 69; flowering) from all leaves at levels greater than 10 5 cfu g −1 tissue, but not from the ear. Seventy days after spray application (GS 92; ripening) most of the bacteria had disappeared from the phylloplane. After incorporation in soil at rates of 10 6 recombinant cfu g −1 soil, bacterial numbers of P. aureofaciens declined by 0.028 log units/d at a temperature of 5°C and 0.07 log units per day at 18°C. The isolate of P. aureofaciens used in this study was able to survive desiccation at 60% r.h. for 1 month, but was unable to survive for more than 1 month when exposed to high levels of microbial competition at 100% r.h.