EVALUATION OF CULTURE PROTOCOLS AND OXYRASETM SUPPLEMENTATION FOR ARCOBACTER SPP.

Growth of Arcobacter butzleri was evaluated in Brain Heart Infusion broth incubated aerobically, microaerobically, and with Oxyrase^TM supplementation (anaerobically). At initial concentrations of10¹ to 10³ cells/mL, A. butzleri populations reached 7.5 to 8.0 log CFU/ml in 48h at 37C in Oxyrase^TM -supplemented broth. The organism quickly declined in the other two systems to undetectable levels during the initial 24h of incubation. Only moderate population levels (ca. 3 log CFU/ml) could be detected in aerobic and microaerobic systems after 56h incubation. Growth of five Arcobacter spp. strains was evaluated at 30C in Brucella-blood broth, modified Cary and Blair Transport Medium, and a biphase cultural system. Strains were inoculated at a level of ca. 10³ cells/ml and incubated with and without Oxyrase^TM supplementation to the system for 36h. Both the Brucella-blood broth and the biphase system supported good growth of the strains, with counts reaching ca. 8 to 9 log CFU/ml. Modified Cary and Blair Transport Medium was the least effective cultural system. Oxyrase^TM provided slight to moderate stimulation of growth for most strains.     

INFLUENCE OF GLUCOSE OXIDASE ON THE GROWTH OF ESCHERICHIA COLI 0157:H7, LISTERIA MONOCYTOGENES, AND SALMONELLA TYPHIMURIUM IN UNIVERSAL PREENRICHMENT BROTH

Glucose oxidase (GO), a food-grade enzyme, was compared with Oxyrase^TM oxygen reducing membrane fraction in Universal Preenrichment Broth (UPB) for enhancement of the growth of the facultatively anaerobic pathogens Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes Scott A. Oxidation-reduction potential (ORP) and pH changes in UPB following the addition of GO (4 units/ml) or Oxyrase^TM (0.5 units/ml) were measured. Microbial growth was evaluated at 0, 3, 6, 18, and 24 h of incubation using spiral plating. Nonenzyme supplemented UPB served as the control. Oxyrase^TM provided a higher oxygen scavenging action in terms of ORP decrease during the initial 6 h of incubation. However, no difference occurred in Eh between Oxyrase^TM and GO by 18 h, with both enzyme systems effectively reducing the Eh compared to that of the control. A 1.0 pH unit reduction was observed in GO-supplemented UPB after 18 h, indicating production of gluconic acid. The pH decrease in Oxyrase^TM - supplemented media was 0.2 units. By 6 h, the E. coli O157:H7 population was enhanced by 0.6 and 1.4 log CFU/ml in Oxyrase^TM -supplemented media, compared to the control and GO-supplemented media, respectively. By 18 h, 0.4 and 0.9 log CFU/ml growth enhancements of the E. coli O157:H7 populations were seen in GO- and Oxyrase^TM -supplemented media, respectively, compared to the control. By the end of 18 h, counts of S. typhimurium and L. monocytogenes increased by 0.6 and 0.2 log units, respectively, in GO-supplemented media compared to the control.     

USE OF UNIVERSAL PREENRICHMENT MEDIUM SUPPLEMENTED WITH OXYRASETM FOR THE SIMULTANEOUS RECOVERY OF ESCHERICHIA COLI O157:H7 AND YERSINIA ENTEROCOLITICA

Universal Preenrichment (UP) medium was used successfully for the simultaneous recovery of two strains each of Escherichia coli O157:H7 and Yersinia enterocolitica in the presence of Listeria monocytogenes and Salmonella typhimurium. E. coli O157:H7 and Y. enterocolitica populations reached ca. 10⁸ CFU/ml in UP medium in 18 h from an initial level ofca. 10² CFU/ml. Addition of Oxyrase_TM enhanced the growth of both E. coli O157:H7 strains and one strain of Y. enterocolitica. These three strains were able to recover from heat injury by 6 h when 24-h cultures were tested, but not when 18-h cultures were used. Injured and noninjured E. coli O157:H7 could be recovered from artificially inoculated food samples (shredded cheddar cheese, turkey ham, hot dogs, mayonnaise, and ground beef) in UP medium supplemented with Oxyrase^TM (UPO) by 18 h using immunoblotting. Y. enterocolitica could be recovered from turkey ham, hog dogs, and mayonnaise by direct plating on CIN agar from UPO medium. However, recovery of Y. enterocolitica from shredded cheddar cheese and ground beef required subsequent selective enrichment in sorbitol bile broth and isolation on Cefsulodin Irgasan Novobiocin agar (CIN). UPO medium can be used for simultaneous detection of E. coli O157:H7 and Y. enterocolitica from foods. However, subsequent selective enrichment and isolation on selective plating media are required for isolation of Y. enterocolitca from raw foods containing high population levels of background microflora.     

Gastrointestinal colonisation of BALB/cA mice by Helicobacter pylori monitored by heparin magnetic separation

Abstract Immunocompetent and immunodeficient BALB/cA mice were fed orally with 10⁸ colony forming units of 2-day-old spiral or coccoid (12 days old) Helicobacter pylori strain NCTC 11637. Immunocompetent BALB/cA mice were also fed orally with decreasing numbers of spiral or coccoid forms of H. pylori . The gastrointestinal colonisation process was monitored for 34 days post-infection by heparin magnetic separation and subsequent enzyme immunoassay (EIA) for the detection of the H. pylori cells. Both mice types were colonised with H. pylori . The coccoid form of H. pylori gave higher EIA absorbance values and more efficient colonisation in the mice than the spiral form. Immunocompetent BALB/cA mice fed with the coccoid form of H. pylori exhibited an acute inflammation process in histopathological samples from the stomachs. In conclusion, H. pylori can infect both immunocompetent as well as immunodeficient BALB/cA mice and coccoids (viable but non-culturable) obtained after 12 days of culturing can infect BALB/cA mice.     

Development of a 24 h screen to detect viable salmonellas in faeces

A 24 h screen to detect viable salmonellas in faeces was developed by studying growth dynamics of salmonellas and competing flora in combinations of enrichment media and artificially-inoculated pig faeces. Muller-Kauffmann tetrathionate (MK) broth, incubated overnight at 42°C, maintained the lowest ratio of salmonella: competing flora and identified all inoculated samples. A 4 h postenrichment in M broth plus novobiocin reduced the number of false-positive results in subsequent ELISAs. Adjusting the negative cut-off values and incubation time of the chromogenic substrate from that recommended in the ELISA instructions reduced the rate of false-positive results further and allowed the detection of 10³ salmonellas per ml in the presence of up to 10⁷ ml⁻¹ aerobic-competing cells. Suspension of faeces diluted 1 in 2 and 1 in 5, rather than 1 in 10 in MK broth did not necessitate further adjustments to the ELISA baseline values. The proposed screen protocol is an overnight incubation of faeces suspended 1 in 10 in MK broth, a 1 in 100 subculture into M broth plus 10 μg ml⁻¹ novobiocin (MbN) for 4 h, steam inactivation of MbN cultures and testing by ELISA, and can detect three salmonella cells per g faeces.     

Rapid Confirmation of Suspect Salmonella Colonies by Use of the Minitek System in Conjunction with Serological Tests

Colonies of 40 members of the Enterobacteriaceae family (26 Salmonella serotypes and 14 other organisms) were picked from selective agar plates and inoculated into Minitek inoculum broth (BBL) and then onto Minitek discs of dextrose, lactose, sucrose, mannitol, maltose, dulcitol, lysine and H₂S. After incubation for 6 h, the inoculum broth was tested with salmonella Poly O and after 24 h with salmonella Poly H antisera. The results of the biochemical tests were read after 24 h incubation. With this procedure, all the salmonella cultures used in this study were confirmed as salmonella and differentiated from all the other organisms, which were rejected. This procedure provides an alternative to the time consuming conventional procedures for the biochemical and serological confirmation of suspect salmonella colonies on selective agar plates.     

Unchanged characteristics of Helicobacter pylori during its morphological conversion.

Helicobacter pylori strains RH 54 and NCTC 11637 were grown in brain-heart infusion broth up to 56 days, and the coccoid form was obtained during prolonged incubation. Two morphological types of coccoids were observed, one of which was electron-dense and had an intact cellular membrane and flagella, indicating that it was likely to be viable. The other coccoid form was sphaeroblast-like and weakly stained, showing features of degeneration. Catalase activity was positive for aged cultures even up to 160 days. Sodium dodecyl sulphate polyacrylamide gel electrophoresis showed that most of the protein bands appeared to be similar in both the spiral and coccoid forms. In addition, Lewis blood group antigens were detected in cultures of up to 8 weeks. Furthermore, two sets of primers for the vacA and cagA genes were used in polymerase chain reaction, and these two important genes remained conserved in both the spiral and coccoid forms. The present study shows that the coccoid form of H. pylori retained many important characteristics present in the spiral form despite the morphological conversion, and thus supports the notion that some of the coccoid forms of H. pylori are likely to be viable.     

Comparison of classical isolation protocols with a 24 h screen to detect viable salmonellas in faeces

A 24 h screen which detects three viable salmonella cells per g of faeces was compared with classical isolation procedures for their ability to identify salmonella-positive samples from a pig rearing unit. The screen involved an overnight enrichment in Muller-Kauffmann tetrathionate (MK) broth, subculture for 4 h in M broth containing 10 μg ml⁻¹ novobiocin, followed by detection of the presence of salmonellas by BacTrace and Salmonella-tek ELISAs. The classical protocols were: (1) an overnight and 48 h incubation in MK or selenite cysteine broth; or (2) overnight incubation in buffered peptone water and 24 h subculture in Rappaport-Vassiliadis broth (BPW-RV). Salmonellas were isolated from the broth cultures on xylose lysine deoxycholate and brilliant green agars. Thirty four of 100 samples were positive for salmonellas but no single isolation protocol identified all of them. The best of the classical isolation protocols, 48 h incubation in MK broth, identified 27 (79%) of the 34 positive samples whilst the screen identified 26 (76%) of the 34 positive samples. False-positive results were obtained from all isolation protocols except BPW-RV.     

Helicobacter pylori: A Fickle Germ

The morphologic changes from bacillary to coccoid forms of Helicobacter pylori were studied. These form changes were analyzed by bacterial growth in Brucella broth plus 2% fetal calf serum. The coccoid forms were observed at five days of incubation and a rapid decrease of CFU/ml was recorded. At two weeks of microaerophilic incubation, all coccoid forms observed were not culturable in vitro. The coccoid morphology was observed earlier when the culture of H. pylori was incubated in aerobic conditions and with subinhibitory concentrations of omeprazole and roxithromycin. To evaluate the possibility of resistance of coccal forms, before plating, the cultures were heated to 80 C for 10 min and sonicated. In the absence of these treatments the cultures did not show growth in vitro. The proteic patterns of the same strains of two different morphologies were studied revealing significant differences.     

Sandwich capture ELISA by a murine monoclonal antibody against a genus-specific LPS epitope for the detection of different common serotypes of salmonellas

D.CHOI, R.S.W. TSANG AND M.H. NG. 1992. A sandwich capture ELISA based on a murine monoclonal antibody against a genus-specific epitope in the outer core region of the Salmonella lipopolysaccharide is described for the detection of different common serotypes of salmonellas. Four h broth cultures of seven standard and 24 wild strains of salmonellas were all detected by the capture ELISA while overnight broth cultures of 21 non-salmonella standard strains were all negative. The capture ELISA detected 1 ng/ml of Ra lipopolysaccharide, 10⁶/ml of a smooth wild strain of Salm. typhimurium , and 1120 cells of Salm. heidelberg after enrichment culture for 4 h.     

Oxygen tension regulates reactive oxygen generation and mutation of Helicobacter pylori

Although both bacillary and coccoid forms of Helicobacter pylori reside in human stomach, the pathophysiological significance of the two forms remains obscure. The present work describes the effect of oxygen tension on the transformation and reactive oxygen species (ROS) metabolism of this pathogen. Most H. pylori cultured under an optimum O2 concentration (7%) were the bacillary form, whereas about 80% of cells cultured under aerobic or anaerobic conditions were the coccoid form. The colony-forming unit of H. pylori decreased significantly under both aerobic and anaerobic culture conditions. The bacillary form of H. pylori generated predominantly superoxide radical, whereas the coccoid form generated preferentially hydroxyl radical. Specific activities of cellular respiration, urease, and superoxide dismatase decreased markedly after transformation of the bacillary form to the coccoid form, with concomitant generation of protein carbonyls and 8-hydroxyguanine. The frequency of mutation of cells increased significantly during culture under nonoptimum O2 conditions. These results indicate that ROS generated by H. pylori catalyze the oxidative modification of cellular DNA, thereby enhancing the transformation from the bacillary to the coccoid form. The enhanced generation of mutagenic hydroxyl radicals in the coccoid form might accelerate mutation and increase the genetic diversity of H. pylori.     

DETECTION OF SALMONELLA SPP. CONTAMINATION IN RAW MATERIALS AND COSMETICS/PHARMACEUTICAL PRODUCTS USING THE BAXTM SYSTEM, A PCR-BASED ASSAY

Abstract A system utilizing the polymerase chain reaction (PCR), the BAX ^TM , was compared and validated against standard selective/enrichment assays to detect the presence of Salmonella spp. in artificially contaminated samples of raw materials and cosmetic/pharmaceutical products. After a 24 h incubation in lactose broth or lactose broth with Tween 20, the inoculated samples were analyzed both by the BAX ^TM system and by standard enrichment/selective methods. Standard enrichment assays required 5–7 days to confirm the presence and identification of Salmonella typhimurium , while the BAX ^TM system reduced the detection time to 30 h. The BAX ^TM system allowed a faster quality control evaluation of those raw materials and cosmetic/pharmaceutical formulations that require Salmonella spp. screening.     

Characteristics of Helicobacter pylori attachment to human primary antral epithelial cells

Conventional cell lines are commonly used to study infection characteristics of the human gastric pathogen Helicobacter pylori. We sought to investigate bacterial attachment to human antral primary epithelial cells, a cell model that more closely resembles the human stomach than transformed cell lines. Primary cells were infected for 24 and 48 h with H. pylori. Morphological appearance of both the pathogen and the cells as well as features of colonization, attachment and internalization were evaluated by electron microscopy and compared to features observed with cultured AGS cells. H. pylori exhibited various shapes during colonization including the spiral, U-shaped, donut, and coccoid forms. The prevalence of each form seemed to be dependent on the infected donor tissue but, in general, changed with time to the coccoid form. Bacterial cell membranes progressively enlarged and appeared at times to be connected with microvilli. Bacterial attachment occurred to cells that were either unchanged, or had formed cup-like structures. Simultaneously, outer membrane vesicles were increasingly secreted from the bacteria, coinciding with increased cellular damage. We conclude that bacterial shape conversion, adherence and secretion of outer membrane vesicles are features of H. pylori infection. Primary gastric cell cultures closely imitate the antral environment and present an appropriate and useful model to study H. pylori pathogenesis.     

Sporulation of Clostridium perfringens (welchii) in Four Laboratory Media

S ummary . Sporulation of 7 strains of Clostridium perfringens ( welchii ) was investigated in 4 laboratory media. A method to induce rapid and simultaneous sporulation was attempted which involved obtaining a purely vegetative culture to inoculate the test media. Heat resistance of spores produced in the individual media by each of 4 selected strains was investigated. The clean spores for the heating tests were obtained by a special procedure which included chilling to 6° for a minimum of 1 week immediately following the usual incubation period, then centrifuging, resuspending to volume in 0.85% NaCl solution and pasteurizing at 75° for 20 min before subjecting to the heating tests. Morphology of each strain was studied using stained microscopic preparations from the 24 h sporulating cultures.
In the Ellner medium spore counts approaching 10⁷/ml were recorded and this medium appeared to be the most efficient when judged in terms of numbers of spores produced. In other media the counts were in the range 10⁴-10⁵ spores/ml. Cooked meat medium yielded slightly higher spore counts than did either SEC broth or modified Wagenaar & Dack medium, the latter contained in a dialysis sac apparatus. A period of chilling to 6° for a minimum of 1 week following incubation enhanced maturation in all cultures except those grown in SEC broth for 24 h or 15 days and those grown 15 days in the modified Wagenaar & Dack medium.
Considerable heat resistance, expressed as percentage spore survival, was recorded for spores of 4 strains when heated at 80°, and heat resistance generally increased with lengthening of incubation time for the culture. Survival of spores heated at 100° for 10 min was usually less than 0.01% but spores in SEC broth after 15 days showed a somewhat greater heat resistance than the others. In no instance did total destruction of spores occur at 100°.     

Population dynamics in ageing Helicobacter pylori

The aim of this work was to characterize population changes occurring in aged broth cultures of Helicobacter pylori. Experiments were performed using clinical strains cultured immediately after isolation and after multiple subcultures in solid medium. Morphological changes in the ageing bacteria during a 7-day broth culture were analysed by optical and electron microscopy. The expression of the virulence factor, CagA, together with the presence of the cell cycle regulator, cGMP, were also assessed. The transition from bacillary to coccoid forms was the main morphological change observed in freshly isolated bacteria, together with the increase in cGMP from 1 to 2.25 nmoles/mg of proteins within the first 7 days of broth culture. A similar trend of morphological and physiological changes was observed in cells after multiple subcultures in solid medium with a major presence of large cell clusters. The cagA gene product was always expressed in all experimental conditions evaluated. These data show a significant morphological and physiological diversity in fresh, ageing and aged cultures of H. pylori.     

Reduced intracellular survival of Helicobacter pylori vacA mutants in comparison with their wild-types indicates the role of VacA in pathogenesis

The vacuolating cytotoxin VacA of Helicobacter pylori plays an important but yet unknown role in pathogenesis. We studied the impact of the vacuolating cytotoxin on H. pylori invasion of and survival within AGS cells (human gastric cell line derived from an antral adenocarcinoma). Isogenic vacA and cagA mutants were constructed in a wild-type clinical isolate H. pylori, AF4. An H. pylori VacA-deficient mutant, AF4(vacA::kan), was cultured in significantly lower numbers from AGS cells after 24 h incubation with gentamicin added to the culture medium than were the type I wild-type strain AF4 (P<0.03) and an isogenic cagA mutant (P<0.01). Complementation of the AF4 vacA mutant with broth culture supernatant from wild-type AF4 improved the intracellular survival of the vacA mutant. We conclude that H. pylori's vacuolating cytotoxin improves the intracellular survival of H. pylori within AGS cells, suggesting the role of the vacuolating cytotoxin in H. pylori pathogenesis.     

Efflux of ions and ATP depletion induced by pediocin PA-1 are concomitant with cell death in Listeria monocytogenes Scott A

Domination of Carnobacterium divergens LV13 by a bacteriocin-producing (bac⁺) organism Carnobacterium piscicola LV17 was dependent on the level of inoculum of the producer strain and its bacteriocin production. When C. piscicola LV17 was grown in APT broth from an initial inoculum of α-10⁴ cfu ml^-1, bacteriocin was not produced (bac^-) although maximum population was reached. The culture remained bac^- during subsequent inoculation at 10²-10⁷ cfu ml^-1 unless it was first grown on solid medium or if heat-treated supernatant fluids from a bac⁺ culture of C. piscicola LV17, LV17A or LV17B were added to the culture prior to the stationary phase of growth. Use of purified carnobacteriocins from C. piscicola LV17A and LV17B confirmed their role in regulation of the bac⁺ phenotype. The need for induction might account in part for differences in bacteriocin production by cultures in liquid and on solid growth media.