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Volume 142,
Issue 10,
1996
Volume 142, Issue 10, 1996
- Pathogenicity And Medical Microbiology
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Multiply antibiotic-resistant Streptococcus pneumoniae recovered from Spanish hospitals (1988-1994): novel major clones of serotypes 14, 19F and 15F
We analysed a collection of 95 multiply antibiotic-resistant pneumococci, recovered since 1988 from 14 Spanish hospitals, that have MICs ≥ 0·25 µg benzylpenicillin ml−1. The majority of the isolates were of serogroups 14, 23, 6, 19 and 15, which are currently the serogroups mainly associated with multiresistance in Spain. All of the serogroup 23 isolates were members of the major Spanish serotype 23F multiresistant clone. Similarly, most of the serogroup 6 isolates were members of the major multiresistant serotype 6B clone, or variants of this clone. Eighteen of the 24 isolates of serogroup 19 were members of a highly penicillin-resistant clone that appears to be a serotype 19F variant of the major Spanish serotype 23F multiresistant clone. Eighteen of the 25 isolates of serotype 14 were members of a previously uncharacterized highly penicillin-resistant clone. Thirteen of the 16 isolates of serogroup 15 were members of a single previously unreported clone of serotype 15F that had moderate levels of resistance to penicillin. Approximately 65% of the multiresistant pneumococci that are currently circulating in Spain were members of the three new clones of serotype 14, 15F and 19F that we describe here, or the previously described serotype 6B and 23F clones. The other 35% of isolates were minor variants of the major clones, unrelated minor clones, and unique isolates, many of which appeared to have arisen by horizontal gene transfer events.
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P-fimbriae-producing septicaemic Escherichia coli from poultry possess fel-related gene clusters whereas pap-hybridizing P-fimbriae-negative strains have partial or divergent P fimbrial gene clusters
More LessThe organization of P fimbrial gene clusters of 13 papC-hybridizing Escherichia coli strains isolated from poultry with colisepticaemia, five P-fimbriae-expressing (P-positive) and eight P-fimbriae-non-expressing (P-negative), were examined by PCR and by Southern blot hybridization using primers or gene probes specific to the I, B, A, C or G genes. The absence of P fimbrial expression was associated with lack of PCR amplification of one or more of these genes, most commonly the I gene. Restriction endonuclease EcoRI, BamHI or Pstl digests of genomic DNA from all strains hybridized with each of the gene probes and demonstrated polymorphisms between P-positive and P-negative strains. Pstl digests of DNA from 12 of the 13 strains, when hybridized with the A gene probe, demonstrated a 0·1 kb fragment specific to the felA gene which encodes the major structural protein of F11 fimbriae. Hence, only the P-positive strains contained complete copies of fel-related gene clusters. In contrast, most of the pap-hybridizing P-negative strains contained partial or divergent P fimbrial gene clusters, which explains the lack of P fimbrial expression by these strains.
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Motility mutants of Vibrio cholerae 01 have reduced adherence in vitro to human small intestinal epithelial cells as demonstrated by ELISA
More LessVibrio cholerae must colonize the human small intestine to cause diarrhoeal disease. V. cholerae strains N16961 (El Tor, Inaba) and 395 (classical, Ogawa) adhered to the epithelial cell surface and the mucus layer of isolated human small intestinal epithelial cells. They adhered specifically to the mucosa and apical membrane in thin sections of small intestine. No binding to the basolateral membrane of dissected epithelial tissue or to intracellular components of the epithelial cells was observed by either light or indirect immunofluorescence microscopy. Based on these results, a modified ELISA was developed to quantitatively study adherence of V. cholerae to human small intestinal epithelial cells. The assay used homogenized human small intestinal mucosal tissue as the substrate for binding. Treatment of the epithelial cell homogenate with 2-mercaptoethanol to disrupt protein and glycoprotein secondary structure inhibited the binding of V. cholerae strains, suggesting that binding was to specific receptors. Several V. cholerae strains and mutants from both biotypes were tested for adherence in the modified ELISA. Wild-type strains of both biotypes and non-enterotoxigenic strains, which were known to colonize humans, adhered. V. cholerae mutants defective in motility, flagellar structure or chemotaxis, which were known to exhibit reduced colonization in animal models, exhibited decreased adherence. The specificity of the assay and its ability to quantify binding should facilitate identification and the study of adherence factors involved in the colonization of human small intestinal epithelial cells by V. cholerae.
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- Physiology And Growth
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Chemotactic responses to an attractant and a repellent by the polar and lateral flagellar systems of Vibrio alginolyticus
More LessChemotactic responses in Vibrio alginolyticus, which has lateral and polar flagellar systems in one cell, were investigated. A lateral-flagella-defective (Pof+ Laf−) mutant, which has only a polar flagellum, usually swam forward by the pushing action of its flagellum and occasionally changed direction by backward swimming. When the repellent phenol was added, Pof+ Laf− cells moved frequently forward and backward (tumbling state). The tumbling was derived from the frequent changing between counter-clockwise and clockwise (CW) rotation of the flagellar motor, as was confirmed by the tethered-cell method. Furthermore, we found that the tumbling cells did not adapt to the phenol stimulus. When the attractant serine was added, the phenol-treated cells ceased tumbling and swam smoothly, adapting to the attractant stimulus after several minutes. We isolated chemotaxis-defective (Che−) mutants from the Pof+ Laf− mutant; the tumbling mutants were not isolated. One interesting mutant swam backwards continuously, with its flagellum leading the cell and its flagellar motor rotating CW continuously. A polar-flagella-defective mutant (Pof− Laf+) stopped swimming after phenol addition and then recovered swimming ability within 10 min, indicating that lateral flagella can adapt to the repellent stimulus. This may represent a functional difference between the two flagellar systems in Vibrio cells, and between the chemotaxis systems affecting the two types of flagella.
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Generation of a transient non-culturable state in Pseudomonas putida during detoxification of N-ethylmaleimide
More LessThe response of Pseudomonas putida to the electrophile N-ethylmaleimide (NEM) has been investigated. Treatment with low levels of NEM (20–30 μM) led to transient growth inhibition followed by recovery of normal growth. Stationary phase cells acquired resistance to NEM; one component of this tolerance was a more rapid metabolism of NEM. In exponential phase cells, the period of growth inhibition was associated with detoxification of NEM. Detoxification was biphasic and cells lost the ability to form colonies on minimal agar plates during the first phase of detoxification. Full viability was retained on MacConkey agar. Peptones are the active components in MacConkey medium. Addition of peptones to minimal agar restored colony-forming ability, but each peptone source had a different efficiency. These data suggest that a deficiency in the ability to assimilate nitrogen is a consequence of NEM treatment.
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Dependence of peptidoglycan metabolism on phospholipid synthesis during growth of Escherichia coli
More LessThe role of phospholipid synthesis in peptidoglycan metabolism during growth of Escherichia coli was determined. The inhibition of phospholipid synthesis, achieved by inhibiting fatty acid synthesis with cerulenin or by glycerol deprivation of gpsA mutant strains, resulted in the concomitant inhibition of peptidoglycan synthesis. These effects on peptidoglycan synthesis were relatively specific in that the treatments did not cause a general inhibition of macromolecular synthesis. Furthermore, the inhibition of phospholipid synthesis also resulted in the rapid development of penicillin tolerance. It was unlikely that penicillin tolerance in these cases were simply due to the inhibition of growth caused by cerulenin treatment or glycerol deprivation because treatments with more effective growth inhibitors, e.g. chloramphenicol or norfloxacin, did not confer penicillin tolerance. Penicillin tolerance was shown to be a direct consequence of the inhibition of phospholipid synthesis and not due to the possible accumulation of guanosine-3′,5′-bispyrophosphate (ppGpp), the starvation stress signal molecule known to be responsible for the development of penicillin tolerance in amino-acid-deprived bacteria. Therefore, peptidoglycan metabolism is coupled to phospholipid synthesis during growth of E. coli, and this may represent an important means to ensure the coordination of cell envelope synthesis in growing bacteria.
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The thiJ locus and its relation to phosphorylation of hydroxymethylpyrimidine in Escherichia coli
More LessExtracts from Escherichia coli K-12 contained two distinct enzymes capable of catalysing the phosphorylation of hydroxymethylpyrimidine (HMP) to HMP monophosphate: pyridoxine kinase (EC 2.7.1.35) and an enzyme that has not previously been genetically analysed, HMP kinase (EC 2.7.1.49). Two distinct genes, pdxL and thiJ, specify the activities of the former and latter enzymes, respectively. The inactivation of both genes by independent mutations in the same cell resulted in the complete loss of HMP kinase activity. Experiments with a series of strains that carry mutations in thiC, thiC pdxB, thiC pdxB pdxL and thiC pdxB pdxL thiJ revealed that the ability of the double mutant (pdxL thiJ) to utilize HMP in thiamin pyrophosphate biosynthesis was restored by introducing the wild-type allele corresponding to the thiJ mutation. The thiJ locus was mapped on the chromosome near the thiD and thiM loci, which govern the activities of phosphomethylpyrimidine kinase (EC 2.7.4.7) and hydroxyethylthiazole kinase (EC 2.7.1.50), respectively.
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Acid tolerance in Listeria monocytogenes: the adaptive acid tolerance response (ATR) and growth-phase-dependent acid resistance
More LessListeria monocytogenes acquired increased acid tolerance during exponential growth upon exposure to sublethal acid stress, a response designated the acid tolerance response (ATR). Maximal acid resistance was seen when the organism was exposed to pH 5·0 for 1 h prior to challenge at pH 3·0, although intermediate levels of protection were afforded by exposure to pH values ranging from 4·0 to 6·0. A 60 min adaptive period was required for the development of maximal acid tolerance; during this period the level of acid tolerance increased gradually. Full expression of the ATR required de novo protein synthesis; chloramphenicol, a protein synthesis inhibitor, prevented full induction of acid tolerance. Analysis of protein expression during the adaptive period by two-dimensional gel electrophoresis revealed a change in the expression of at least 23 proteins compared to the non-adapted culture. Eleven proteins showed induced expression while 12 were repressed, implying that the ATR is a complex response involving a modulation in the expression of a large number of genes. In addition to the exponential phase ATR, L. monocytogenes also developed increased acid resistance upon entry into the stationary phase; this response appeared to be independent of the pH-dependent ATR seen during exponential growth.
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- Plant-Microbe Interactions
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Transcriptional activity of the symbiotic plasmid of Rhizobium etli is affected by different environmental conditions
More LessGlobal patterns of transcriptional activity of the symbiotic plasmid (pSym) of Rhizobium etli were studied under a variety of environmental conditions, including some relevant to the symbiotic process. 32P-labelled single-stranded complementary DNAs synthesized from total RNA were used as hybridization probes against an ordered collection of cosmid clones that covered the whole pSym. Our results showed that, under aerobic conditions, discrete regions of the pSym are differentially transcribed depending on the carbon and nitrogen sources employed. In general, poor carbon or nitrogen sources allowed greater expression than rich ones. Time-course experiments with the nod gene inducer genistein led us to the identification of new regions responsive to this flavonoid. Widespread transcription was observed during microaerobiosis, but not under aerobic conditions, indicating that oxygen concentration is a major effector of transcriptional activity in the pSym. This response was reduced, but not suppressed, in a nifA mutant, indicating the location of regions whose transcription may depend on other oxygen-sensitive regulators. During symbiosis, almost the entire pSym was actively transcribed and the transcription pattern was similar to that observed during microaerobiosis. The experimental approach described allowed the identification and localization of specific regions in the pSym whose expression depends on defined environmental stimuli.
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- Systematics
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Molecular biological evidence for the occurrence of uncultured members of the actinomycete line of descent in different environments and geographical locations
More LessA 16S rDNA based molecular ecological study was performed on a sample taken from a peat bog in Germany. Total DNA was extracted by directly lysing micro-organisms in the peat matrix. The 5′ 1400 nucleotides of the bacterial 16S rDNA were amplified using conserved bacterial PCR primers. A clone library was generated by blunt-end cloning and 262 16S rDNA clones were analysed. Of these, 37 were located in the Gram-positive phylum, as determined by hybridization to an oligonucleotide probe specific for Gram-positive bacteria. Analysis of 17 of these clones by sequence analysis and their comparison with published sequences representing all of the main bacterial phyla indicated their membership of the actinomycete line of descent. These peat clones were found to represent three novel lineages, two of which appear to be related to the species Acidimicrobium ferrooxidans, and ‘Candidatus Microthrix parvicella’. Clone sequences of the third group are phylogenetically related to Rubrobacter radiotolerans. Comparison with short 16S rDNA clone sequences obtained from DNA isolated from a geothermally heated soil in New Zealand, and from DNA isolated from soil in Australia, Japan and Finland and marine environments from the Atlantic and the Pacific Oceans, suggests that members of these three groups occur in very different environments across the world.
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- Genome Analysis
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