Effect of different growth conditions on the discrimination of three bacteria by pyrolysis gas-liquid chromatography.

High-resolution pyrolysis gas-liquid chromatography was applied to three bacteria (Escherichia coli NCTC 9001, Pseudomonas putida (NCIB 9494, and Staphylococcus aureus NCTC 8532) grown under a variety of conditions. Changing the culture medium drastically altered the quantitative aspects of the pyrograms of all three organisms, but the effects of culture time and incubation temperature were less severe. Mathematical analysis of the relative peak heights showed that four peaks could be used to discriminate the three bacteria however they were cultured.     

Refinement of an adherence assay for the measurement of bacterial attachment to silica beads

An in vitro adherence assay [4] which utilised radiolabelled bacteria and silica beads was used to investigate firm and loose attachment of bacteria. A systematic evaluation of the effects of the assay conditions, including incubation vessel, bacteria to bead interaction, bead washing and bead weight, was carried out in an attempt to improve the reproducibility of adherence measurements. Four oral streptococcal strains (S. mutans NCTC 10449, S. sanguis NCTC 7863 and 10904, S. salivarius NCTC 8618) were studied with saliva coated and uncoated silica beads.Using the improved system, total bacterial adherence to uncoated beads was high (45–60% of bacteria present). While saliva coating of beads reduced total and firm adherence of all strains, these reductions were less marked (P > 0.005) for S. sanguis NCTC 10904.     

The effect of cooling rate, freeze-drying suspending fluid and culture age on the preservation of Campylobacter pylori

The effects of freezing rate, suspending fluid and age of culture on the ability of four strains of Campylobacter pylori to survive and recover from freeze-drying were examined. Freeze-drying by standard procedures generally resulted in an overall loss in viability of between 3 and 7 log units. The exact cause of poor recovery by C. pylori was not established but strain differences were detected, with NCTC 11637 (type strain) surviving better than NCTC 11638 and NCTC 11639. Recovery of the poorest growing strain (NE 26695) was notably more erratic. The largest loss in viability occurred at the primary drying stage. Losses resulting from freezing and secondary drying were less marked and the rate of freezing had only a marginal effect on recovery. Nineteen different freeze-drying suspending fluids were investigated. Overall the best recovery results were obtained with 5% inositol-broth (or horse serum) plus 25% glucose, at pH 7.0, in which loss of viability was typically about 4 log units. Other factors, such as age of culture and number of viable bacteria in the before-dry suspension, did not have a significant effect on survival. We conclude from these results that C. pylori can survive freeze-drying, albeit in small numbers, but the degree of recovery is apparently largely strain dependent.     

The Level of Expression of α-toxin by Different Strains ofClostridium perfringensis Dependent on Differences in Promoter Structure and Genetic Background

The control of expression of the α-toxin gene (cpaorplc) ofClostridium perfringenshas been studied in three strains shown to have high (NCTC8237), intermediate (strain 13) and low (NCTC8533) phospholipase C activity in the culture supernatant. The phospholipase C activity was shown to be related tocpamRNA levels. Primer extension studies were performed to locate thecpapromoter regions in strains NCTC8237 and 13. Differences in promoter sequences could account for the differences in α-toxin production between strains 13 and NCTC8237. In contrast, the differences in α-toxin production between strains NCTC8237 and NCTC8533 were unlikely to be due to promoter differences because the upstream promoter-containing sequences were identical in these strains. The recombinant plasmid carrying the NCTC8237cpagene was introduced into strains 13 and NCTC8533. The level of production of the α-toxin was 16-fold higher in strain 13, indicating the presence of strain-dependant regulatory systems.     

The effect of cooling rate, freeze-drying suspending fluid and culture age on the preservation of Campylobacter pylori

The effects of freezing rate, suspending fluid and age of culture on the ability of four strains of Campylobacter pylori to survive and recover from freeze-drying were examined. Freeze-drying by standard procedures generally resulted in an overall loss in viability of between 3 and 7 log units. The exact cause of poor recovery by C. pylori was not established but strain differences were detected, with NCTC 11637 (type strain) surviving better than NCTC 11638 and NCTC 11639. Recovery of the poorest growing strain (NE 26695) was notably more erratic. The largest loss in viability occurred at the primary drying stage. Losses resulting from freezing and secondary drying were less marked and the rate of freezing had only a marginal effect on recovery. Nineteen different freeze-drying suspending fluids were investigated. Overall the best recovery results were obtained with 5% inositol-broth (or horse serum) plus 25% glucose, at pH 7.0, in which loss of viability was typically about 4 log units. Other factors, such as age of culture and number of viable bacteria in the before-dry suspension, did not have a significant effect on survival. We conclude from these results that C. pylori can survive freeze-drying, albeit in small numbers, but the degree of recovery is apparently largely strain dependent.     

Lipoquinones of some spore-forming rods, lactic-acid bacteria and actinomycetes.

The respiratory quinones of 73 strains of Gram-positive bacteria including spore-forming rods, lactic-acid bacteria and actinomyctes were examined. Menaquinones with seven isoprenoid units (MK-7) were the main quinone type found in representatives of the genus Bacillus and in Sporolactobacillus inulinus. However, a strain of B. thuringiensis produced MK-8 in addition to MK-7, and strains of B. lentus and B. pantothenticus appeared to produce MK-9 and MK-8, respectively, with no MK-7. In the clostridia and lactic-acid bacteria, no quinones were found, except in Pediococcus cerevisiae NCTC 8066 and Lactobacillus casei subsp. rhamnosus ATCC 7469, which contained menaquinones, and Streptococcus faecalis NCTC 775 and HIM 478-1, which contained demethylmenaquinones, in relatively low concentrations. Menaquinones were also found in the actinomycetes (except Actinomyces odontolyticus and Bifidobacterium bifidum which did not produce any quinones) and in Protaminobacter alboflavus ATCC 8458, the so-called Actinobacillus actinoides ATCC 15900 and Noguchia granulosis NCTC 10559.     

Flavonols and an oxychromonol from Piliostigma reticulatum

The leaf extract from the plant Piliostigma reticulatum was found to exhibit antimicrobial activity against some bacteria and fungi such as Staphylococcus aureus (NCTC 6571), Escherichia coli (NCTC 10418), Bacillus subtilis (NCTC 8236), Proteus vulgaris (NCTC 4175), Aspergillus niger (ATCC 10578) and Candida albicans (ATCC 10231). Upon investigation of the chemical constituents present in the leaf extract, a total of seven compounds were isolated and their structures were unambiguously established by spectroscopic methods including HR-MS and NMR spectrometry. Four of the isolated compounds were novel, namely 6-C-methyl-2-p-hydroxyphenyloxychromonol (piliostigmol), 1, 6,8-di-C-methylquercetin-3,3',7-trimethyl ether, 2, 6,8-di-C-methylquercetin-3,3'-dimethyl ether, 3 and 3',6,8,-tri-C-methylquercetin-3,7-dimethyl ether, 4. The other three were known C-methylated flavonols and they were isolated from P. reticulatum for the first time. These were 6-C-methylquercetin-3-methyl ether, 5, 6,8-di-C-methylkaempferol-3-methyl ether, 6 and 6-C-methylquercetin-3,3',7-trimethyl ether 7. All the isolated compounds were tested for cytotoxicity using the brine shrimp toxicity assay and all of them were active albeit at different levels. With respect to antibacterial activity piliostigmol, 1 showed the highest activity against E. coli (MIC=2.57 microg/ml, 0.006 micromol), which is three times more that of Amoxicillin, where as 4 and 7 showed the least activity.     

Arginine deiminase system and acid adaptation of oral streptococci.

Streptococcus rattus FA-1 and Streptococcus sanguis NCTC 10904 underwent phenotypic acid adaptation in batch cultures toward the end of sugar-fueled growth after the culture pH had dropped to triggering values. The bacteria could be derepressed or induced for arginine deiminase independently of acid adaptation, and arginolysis afforded protection against acid killing over and above that of acid adaptation.     

Interstrain transfer of the prophage ϕNM2 in staphylococcal strains

Staphylococcus aureus is a successful pathogen in part because the bacterium can adapt rapidly to selective pressures imparted by the external environment. Horizontal gene transfer (HGT) plays an integral role in the evolution of bacterial genomes, and phage transduction is likely to be the most common and important HGT mechanism for S. aureus. Phage can transfer not only its own genome DNA but also host bacterial DNA with or without pathogenicity islands to other bacteria. Here, we demonstrate that the staphylococcal prophage ?NM2 could transfer between strains Newman and NCTC8325/NCTC8325-4 by simulating a natural situation in laboratory without mitomycin C or ultra-violet light treatment. This transference may be caused by direct contact between Newman and NCTC8325/NCTC8325-4 instead of phage particles released in Newman culture’s supernatant. The rates of successful horizontal genetic transfer in recipients NCTC8325 and NCTC8325-4 were 2.1% and 1.8%, respectively. Prophage ?NM2 was integrated with one direction at an intergenic region between rpmF and isdB in all 17 lysogenic isolates. Phage particles were spontaneously released from lysogenic strains again and had no noticeable influence on the growth of host cells. The results reported herein provide insight into how mobile genetic elements such as prophages can lead to the emergence of genetic diversity among S. aureus strains.     

反向间接胶乳凝集试验法检测粪便A型产气荚膜梭菌肠毒素的结果及评价

本文以１０种５２株供试菌分别与７个不同年龄组的健康人粪便混合,共配成３６４份模拟标本,采用反向间接胶乳凝集（ＲＰＬＡ）试验法与生物学试验法（小鼠致死试验、豚鼠皮肤血管透性因子试验,Ｖｅｒｏ细胞毒性试验）检测各标本中的Ａ型产气荚膜梭菌肠毒素（简称Ｃｐ－Ｅｎｔ）。除产气荚膜梭菌之外的其他菌种培养液２３８份标本（３４株）,ＲＰＬＡ与生物学试验结果完全一致,均为阴性。产气荚膜梭菌１２６份标本（１８株）中有７０份标本的ＲＰＬＡ同生物学诸法完全一致地检出了Ｃｐ－Ｅｎｔ．有１株７份标本（ＣｐＮＣＴＣ８７９７）的ＲＰＬＡ为阳性,而各生物学试验却均为阴性,该菌株经ＰＣＲ检查证明确为肠毒素原性产气荚膜梭菌,表明ＲＰＬＡ比生物学试验法更灵敏。有５株（ＣｐＮＣＴｃ８２３８,ＣｐＮＣＴＣ１０６１１,ＣｐＮＣＴＣ１０６１４,ＣｐＮＣＴＣ１０６１２,ＣｐＬ－５２）３５份标本ＲＰＬＡ与各生物学试验结果均为阴性,但经ＰＣＲ检吉证明该５株菌均为肠毒素原性产气荚膜梭菌,后经超声波破碎菌体提取物对其中部分菌株进行试验的结果仍然显示了ＲＰＬＡ与生物学法的一致性。有２株（ＣｐＮＣＴＣ８６８６,ＣｐＮＣＴＣ８４４９）１４份标本的所有结果均为阴性,ＰＣＲ     

Examining the genetic variation of reference microbial cultures used within food and environmental laboratories using fluorescent amplified fragment length polymorphism analysis

Fluorescent amplified fragment length polymorphism (FAFLP) analysis was applied to genetically fingerprint 'working culture control strains' used by accredited food microbiology laboratories. A working culture control strain is defined as a subculture from a strain initially obtained from an authenticated source [such as the National Collection of Type Cultures (NCTC)] that is maintained for use with routine testing within the laboratory. Working culture control strains from eight food examination laboratories, representing four bacterial species, were analysed by FAFLP; these were Salmonella Nottingham, Staphylococcus aureus, Listeria monocytogenes and Bacillus cereus. The resultant FAFLP profiles of the eight working culture control strains for each of these species were compared against the appropriate freeze-dried ampoules obtained directly from NCTC. FAFLP results demonstrated that within 50% of working cultures analysed, several laboratories were routinely using working cultures that were genetically different from the original reference NCTC strains. This study highlights the need for laboratories to review the protocols used to process and maintain control strains and working cultures, with a potential view to utilize single-use quality control materials.     

A new growth and in vitro sporulation medium for Clostridium perfringens

Growth and in vitro sporulation capabilities of three related Clostridium perfringens strains (NCTC 8798, 8-6 and R3) were followed in a new sporulation medium (NSM), with notable changes from a maintenance medium originally designed for strictly anaerobic bacteria. Compared with thioglycollate (FTG) medium, the new sporulation medium promoted growth of Cl. perfringens with a shorter lag phase and a 20% higher biomass production. The age of inoculum did not change Cl. perfringens growth kinetics. When compared with reference conditions, in vitro spore production kinetics were different in the new sporulation medium, but both conditions led routinely to 100% sporulation and spore counts of approximately 10(8) ml-1. The ease of preparation of the NSM, and the use of the same culture medium for good growth, high sporulation yields and spore production, represent an attractive alternative to the complex media routinely used for in vitro studies of Cl. perfringens physiology.     

An inexpensive infrared growth sensor array for detection of bacterial antibiotic susceptibility

Abstract An inexpensive infrared sensor was constructed and used for the rapid testing of bacterial antibiotic susceptibility by detection of changes in absorbance at 950 nm. By comparing cultures of clinical isolates together with control strains ( Escherichia coli NCTC 10418, Staphylococcus aureus NCTC 6571 or Pseudomonas aeruginosa NCTC 10662) after addition of an antibiotic, results on susceptibility were obtained within 3–5 h from the original plate culture. Representative strains of E. coli, P. aeruginosa , and S. aureus were tested successfully against ampicillin, penicillin, gentamicin or ciprofloxacin.     

Simultaneous analysis of cardiolipin and lipid A from Helicobacter pylori by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Cardiolipin (CL) is an anionic tetraacylphospholipid found in mammalian tissues, inner membrane of mitochondria and in the cytoplasmic membrane of Gram-positive and -negative bacteria. Lipid A is the principal structural component responsible for the range of biological activities of lipopolysaccharides. Here we report a MALDI-MS-based method for the sensitive simultaneous analysis of CL and lipid A from Helicobacter pylori cells. The sensitivity was demonstrated by the analysis of CL and lipid A from a single bacterial colony of in vitro grown H. pylori strain NCTC 11637 (ATCC 43504). We then characterized the CL and lipid A structures in H. pylori cells grown under three different conditions, on agar-horse blood plates, in liquid culture and ex vivo. The results revealed the presence of high amounts of myristic (C14:0) and 19-carbon cyclopropane (C19:0cyc) fatty acids. Alterations in CL structure were observed in H. pylori cells cultivated on plates as compared with the bacteria grown in broth culture. Furthermore, significant changes in lipid A acylation pattern were detected in H. pylori cells during formation of coccoids. In contrast, structural analysis of CL from ex vivo H. pylori cells recovered from the stomachs of infected Mongolian gerbils demonstrated only minor changes in acyl chain combination. This is the first report of simultaneous analysis of CL and lipid A from ex vivo cells of H. pylori.     

Interaction of cells of Helicobacter pylori with human polymorphonuclear leucocytes: Possible role of haemagglutinins

Abstract The interaction of fluorescein isothiocynate (FITC)-labelled cells of Helicobacter pylori with human polymorphonuclear leucocytes (PMNs) was studied. Two strains with surface haemagglutinins expressing different receptor specificity were used in order to decide if cell surface haemagglutinins of H. pylori may play a role in lectin-mediated binding to/uptake by phagocytes: (1) strain 17874 (NCTC 11637) which expresses sialic acid-specific haemagglutin; and (2) strain 17875 (NCTC 11638) which expresses a sialic acid-independent haemagglutinin. Cells of strain 17874 were poorly attached to/ingested by PMNs compared to cells of strain 17875. Pre-treatment of bacteria with fetuin or rabbit antibodies against partly purified sialic acid-specific haemagglutinin enhanced interaction of cells of strain 17874 with PMNs. The enhancement did not occur in the case of strain 17875. Phagocytosis of H. pylori 17874 bacteria was slightly increased by fresh human sera positive for anti- H. pylori antibodies. The results suggest that the sialic-acid-specific haemagglutinin complex of 17874 bacteria might disturb their uptake by human PMNs.     

Coccoid forms of Helicobacter pylori can be viable.

Controversy exists as to whether the coccoid form of Helicobacter pylori can exist in a viable form. Conversion of helical to coccoid morphology occurs in culture over several days. In this study, the morphology was correlated with parameters of genetic integrity in the reference NCTC 11637 strain over 21 days of culture. The capacity to regrow colonies of helical form was demonstrated from a culture where the coccoid form constituted up to 95% and negligible urease activity could be detected. Urease enzyme activity and its mRNA decreased between day 0 and 10 while 26 kD mRNA and 16S rRNA were expressed unchanged for up to 14 and 21 days of culture, respectively. Expression of mRNA for the Cag A gene behaved in a similar fashion to that of urease. No evidence of DNA fragmentation was detected. These data suggest that a viable form of non-urease producing H. pylori exists after short to intermediate culture and that some if not all of these viable bacteria have coccoid morphology.     

Mixed continuous culture experiments with an antibiotic-producing streptomycete andEscherichia coli

Four strains ofStreptomyces aureofaciens capable of producing different amounts of tetracycline have been grown in continuous culture with either of two strains ofEscherichia coli orBacillus pumillus. Each of the bacteria had faster specific growth rates than any of the streptomycetes.E. coli NTCT 5993 had a higher affinity for sucrose than didS. aureofaciens SR 11. In mixed culture experiments, the bacteria displaced the streptomycete under the following conditions: (a) when the streptomycete produced no tetracycline; (b) in tetracycline-producing cultures after a fraction of the bacteria had died—the bacterial population that subsequently developed was resistant to the drug; and (c) when using tetracycline-resistant bacteria. Under nutrient conditions leading to high antibiotic levels, it was possible to kill all the bacteria, and the streptomycete survived after a transient fall in mycelium and tetracycline levels. No stable mixed population was ever seen. Once tetracycline-resistant bacteria had displaced the streptomycete, and tetracycline concentrations had fallen, a sensitive bacterial population reappeared. Competition between sensitive and resistantE. coli NCTC 5993 in the chemostat confirmed the selective advantage of the sensitive strain. Results were discussed in terms of the role of antibiotics in nature.     

A phylogenetic analysis of Legionella

Four species of Legionella, L. pneumophila NCTC 11192, L. bozemanii NCTC 11368, L. micdadei NCTC 11371 and L. jordanis ATCC 33623 have been characterized by oligonucleotide cataloguing of their 16S ribosomal RNA. All four species are phylogenetically closely related, while no specific relationship could be detected with any other group of organisms investigated so far with respect to this method. At a low level of relationship legionellae are members of the broad group of purple photosynthetic bacteria and their non-phototrophic relatives, in which Legionella form an independent line of descent.     

The bactericidal and morphological effects of peroxynitrite on Helicobacter pylori

Peroxynitrite (ONOO-) is correlated with the pathogenesis of Helicobacter pylori-induced peptic ulcer diseases. We aimed to investigate the time- and concentration-dependent bactericidal and morphological effects of ONOO- on H. pylori. Authentic ONOO- was synthesized as quenched-flow method. A stock culture of H. pylori NCTC 11637 was exposed to different concentrations of ONOO- (0.1-40 micromol/L) or decomposed ONOO- or fresh medium. Samples were taken at 0, 15, 30, 60, and 120 minutes, for the evaluation of viable bacteria and bacterial morphology with gram strain and transmission electron microscopy. Decomposed ONOO- showed no bactericidal activity against H. pylori. ONOO- application caused a decrease in the number of viable bacteria within the first 15 minutes. The significant conversion of H. pylori from spiral form to coccoid form was determined with 10 micromol/L of ONOO-, and higher concentrations caused lysis of the cells. Separation of cell wall, bleb formation, vacuolization, decrease of secretory granules, and lysis of bacteria were the morphological effects of ONOO- on H. pylori. Because the morphology of the bacteria is one of the important factors in virulence; peroxynitrite-related morphological effects might have an impact in the progress of the H. pylori-induced peptic ulcer diseases.