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Volume 51,
Issue 1,
2002
Volume 51, Issue 1, 2002
- Editorial
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- Host Response To Infection
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Elimination of Salmonella enterica serotype Enteritidis in intestinal epithelial cells by mechanisms other than nitric oxide
Production of nitric oxide (NO) by intestinal epithelial cells is induced after infection with Salmonella spp. or some other enteroinvasive bacteria. However, direct evidence of the role of NO in the elimination of intracellular pathogens in intestinal mucosa has not been established. This study investigated whether NO mediates killing of Salmonella enterica serovar Enteritidis in human intestinal epithelial cells by using parent Henle-407 cell line and a transfected cell line not capable of induced NO production (Henle-NOdef). NO synthesis was studied as combined accumulation of nitrite and nitrate, as inducible nitric oxide synthase (iNOS) protein determined by Western blotting and as iNOS mRNA detected by reverse transcription (RT)-PCR. Although parent and Henle-NOdef cells differed markedly in their ability to produce NO after infection, they eliminated S. Enteritidis equally, as determined by cfu counts. The presence of aminoguanidine, a selective iNOS inhibitor, during the infection blocked the production of NO but did not affect the elimination of the bacteria. These data suggest that NO does not have a direct role in the elimination of intracellular Salmonellae by human intestinal epithelial cells.
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Induction of a Th1-type of immune response but not protective immunity by intramuscular DNA immunisation with Brucella abortus GroEL heat-shock gene
More LessThe immunogenicity and protective efficacy of a DNA vaccine encoding the GroEL heat-shock gene from Brucella abortus was tested in BALB/c mice immunised by intramuscular (i.m.) needle injection or epidermally by gene gun. The Brucella GroEL gene was amplified by PCR and cloned into two different mammalian expression vectors pCMV-link and pCMV-tPA. The D17 cell line was transfected with both constructs and GroEL transcripts were detected by Northern blot. To determine the level of protein synthesised, transfected cell lysates were then submitted to Western blot. The non-secreted form of the recombinant GroEL produced by the pCMV-link construct was detected in much greater amount than the secreted form of the protein produced by the pCMV-tPA construct. After immunisation, a strong anti-GroEL IgG response was detected in mice vaccinated by i.m. injection or gene gun only when the pCMV-link/GroEL plasmid was used. Regarding the pattern of immune response induced, i.m. needle injection raised a predominantly Th1 response with mostly IgG2a-specific anti-GroEL and high levels of IFN-γ produced by splenic T cells. Gene gun immunisation induced a Th0 type of immune response in mice characterised by a high IgG1/IgG2a ratio, and IL-4 and interferon (IFN)-γ production. Even though a distinct pattern of immune response was generated depending upon the immunisation route used, neither method engendered a significant level of protection with the GroEL DNA vaccine.
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- Diagnostic Microbiology
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A comparison of PCR detection of mecA with two standard methods of oxacillin disk susceptibility testing for coagulase-negative staphylococci
More LessCoagulase-negative staphylococci (CNS) are common isolates from blood cultures, and an increasing proportion is now methicillin resistant. The National Committee for Clinical Laboratory Standards (NCCLS) recently issued new criteria for zone sizes applicable to oxacillin disk sensitivity testing for CNS and the British Society of Antimicrobial Chemotherapy (BSAC) has also issued guidelines. This study evaluated two standard methods for oxacillin disk sensitivity testing of 67 CNS isolates from blood cultures, and compared these results with detection of the mecA gene by PCR. Over 94% of mecA-positive isolates were detected by conventional disk testing, with no significant differences between the two methods. In this study, the clinical utility of mecA detection in CNS for the determination of methicillin resistance appears to be limited.
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Development of a combined filtration-enrichment culture followed by a one-step duplex PCR technique for the rapid detection of Campylobacter jejuni and C. coli in human faecal samples
More LessA new combined filtration-enrichment culture followed by a PCR technique for the rapid detection of Campylobacter jejuni and C. coli in human faeces has been developed. Only bacteria that passed through the membrane could multiply in the enrichment culture; target bacteria were detected by a one-step duplex PCR technique with combinations of primers that are specific for different Campylobacter spp., which should allow for the detection of a mixed infection in a single patient. A Falcon ® cell-culture insert and 24-well tissue-culture plates were used. After 2 days, both C. jejuni and C. coli were reliably detected in diluted faeces that were seeded with as few as 10 cells which corresponds to a concentration of 1035mucfu/g. Even allowing for the dilution of faecal samples, this represents an increase in sensitivity of two-to-three orders of magnitude over the conventional filtration method.
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- Antimicrobial Agents
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Conditions that may affect the results of susceptibility testing of Mycobacterium tuberculosis to pyrazinamide
More LessPyrazinamide (PZA) is an important front-line anti-tuberculosis drug that is active only at acid pH. However, acid pH causes significant difficulty for PZA susceptibility testing. A common problem in PZA testing is false resistance caused by large bacterial inocula. This study investigated the relationship of false resistance to numbers of bacilli, pH and other factors that potentially affect susceptibility to PZA. Large inocula (107−−8 bacilli/ml) of M. tuberculosis H37Ra caused significant increase in medium pH from 5.5 towards neutrality, and thus produced false resistance results. The increase in medium pH was determined to be a function of live bacilli; heat-killed bacilli had little or no effect. Susceptibility to PZA and its active derivative pyrazinoic acid (POA) was comparable on 7H11 agar medium, but POA was less active than PZA in liquid medium containing bovine serum albumin (BSA), suggesting that susceptibility to PZA or POA was reduced in the presence of BSA, because of its neutralising effect on medium pH and significant POA binding. A 3-month-old H37Ra culture was shown to be more susceptible to PZA exposure than a 4-day log-phase culture, suggesting that PZA is more active for non-growing bacilli. Finally, reserpine, an inhibitor of POA efflux pump, increased susceptibility to PZA even near neutral pH 6.8, with an MIC of 400 mg/L compared with 1000 mg/L without reserpine. These findings should have implications for understanding the mode of action of PZA and for PZA susceptibility testing.
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Effects of silver sulphadiazine on the production of exoproteins by Staphylococcus aureus
More LessThe effects of subinhibitory concentrations of silver sulphadiazine (AgSD) on exoprotein production in Staphylococcus aureus strains T1, T4, RN4282 and RN 4282agr were studied. AgSD markedly increased levels of toxic shock syndrome toxin (TSST)-1 in strains T4 and RN4282. This effect was independent of agr and AgSD restored TSST-1 production to the wild-type level in RN 4282agr. AgSD had no effect on enterotoxin A or coagulase activity in strains T1 or T4. Strain T4 produced enterotoxin C at high levels and no effect was observed with AgSD. AgSD repressed metalloprotease production in strain T4 but the overall protease activity remained the same. No change in proteolytic activities was seen in strain T1 with AgSD. Molecular mechanisms for these observations are discussed.
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Natural antibiotic susceptibility and biochemical profiles of Yersinia enterocolitica-like strains: Y. bercovieri, Y. mollaretii, Y. aldovae and ‘Y. ruckeri'
More LessThe natural susceptibility of 54 Yersinia enterocolitica-like strains of Y. bercovieri (formerly Y. enterocolitica biovar 3B, n=17), Y. mollaretii (formerly Y. enterocolitica biovar 3A, n=12), Y. aldovae (formerly Y. enterocolitica-like group X2, n=10) and ‘Y. ruckeri' (n=15) was tested to 69 antibiotics. MIC values were determined with a microdilution procedure in IsoSensitest broth for all strains and in cation-adjusted Mueller Hinton broth for some strains. All yersiniae tested showed uniform MIC distributions to most antibiotics and were naturally sensitive or intermediate to aminoglycosides, several cephalosporins, and penicillins, carbapenems, aztreonam, quinolones, tetracyclines, antifolates, chloramphenicol and nitrofurantoin, and naturally resistant to benzylpenicillin, oxacillin, all macrolides except azithromycin, lincosamides, streptogramins, glycopeptides, rifampicin and fusidic acid. Significant differences in susceptibility affecting clinical assessment criteria were seen with aminopenicillins (in the presence and absence of β-lactamase inhibitors), some cephalosporins (e.g., cefoxitin) and fosfomycin. Whereas strains of Y. aldovae and ‘Y. ruckeri’ were naturally sensitive or intermediate to amoxicillin and amoxicillin/clavulanate, strains of Y. bercovieri and Y. mollaretii were naturally resistant or naturally resistant or intermediate, respectively. Strains of the two latter species were also highly susceptible to fosfomycin. These data can be valuable for the validation of routine susceptibility test results. β-Lactam MICs suggest that Y. bercovieri and Y. mollaretii strains express chromosomally encoded AmpC β-lactamases and that most Y. aldovae and `Y. ruckeri' strains express no, or only small amounts, of enzyme. An evaluation of 30 biochemical tests that determined phenotypic identification to the Yersinia species level is presented.
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- Epidemiological Typing
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Identification of a novel repetitive DNA element and its use as a molecular marker for strain typing and discrimination of ara− from ara+ Burkholderia pseudomallei isolates
More LessA novel 10-bp repeat (5′−CGACGCAGGC−3′)34 was identified in a strain of Burkholderia pseudomallei, the first repetitive element found in this species. A pair of primers, based on the flanking sequences of the repetitive region, was used in PCR and DNA sequence analysis to determine the genomic structure and distribution of the repetitive element in 76 arabinose− (ara−) and 7 ara+ B. pseudomallei isolates. DNA fragments of 400–700 bp were amplified in all ara− isolates. Ara+ isolates were characterised by a uniform fragment of 402 bp. Nucleotide sequence analysis of these fragments revealed broad heterogeneity of the variable-number tandem repeats with 26 distinct alleles ranging between (5′−CGACGCAGGC−3′)13 and (5′−CGACGCAGGC−3′)45 identified in the ara− isolates. In contrast, a novel non-repetitive sequence was identified in each of the ara+ isolates. This was confirmed by Southern blot analysis. Such biotype-specific variable-number tandem repeats may be useful as genetic markers for rapid strain differentiation of ara− isolates.
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- Correspondence
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- Bacterial Pathogenicity
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Activation of human gingival epithelial cells by cell-surface components of black-pigmented bacteria: augmentation of production of interleukin-8, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor and expression of intercellular adhesion molecule 1
More LessBlack-pigmented anaerobic bacteria, such as Porphyromonas gingivalis and Prevotella intermedia, are amongst the predominant bacteria in periodontal pockets and have been implicated in periodontal diseases. To elucidate the roles of gingival keratinocytes, which are the first cells encountered by oral bacteria in periodontal diseases, human gingival keratinocytes in primary culture were stimulated with cell-surface components of P. gingivalis and Pr. intermedia. A glycoprotein fraction from Pr. intermedia (PGP) clearly augmented the release of interleukin-8, granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor, as determined by enzyme-linked immunosorbent assay. This PGP also induced expression of intercellular adhesion molecule-1 (ICAM-1), as determined by flow cytometry. The augmentation of mRNA expression for these molecules was also confirmed by reverse transcription PCR. In contrast, lipopolysaccharide (LPS) from Pr. intermedia and Escherichia coli was completely inactive in these assays. LPS fraction and purified fimbriae from P. gingivalis exhibited weak activities. Cytokine production and ICAM-1 expression by gingival keratinocytes might cause accumulation and activation of neutrophils in the epithelium and, therefore, may be involved in the initiation and development of inflammation in periodontal tissues.
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Linkage between toxin production and purine biosynthesis in Clostridium difficile
The production of toxins A and B by Clostridium difficile was greatly enhanced under biotin-limited conditions, in which a 140-kDa protein was expressed strongly. Gene cloning revealed that this protein was a homologue of formylglycinamidine ribonucleotide synthetase (FGAM synthetase, EC 6.3.5.3), which is known as PurL in Escherichia coli and catalyses the fourth step of the de novo purine biosynthesis pathway. This enzyme consisted of a single polypeptide, although FGAM synthetases of gram-positive bacteria usually consist of two subunits. Inhibition of the enzymic activity of C. difficile PurL by O-diazoacetyl-l-serine (azaserine) resulted in enhanced toxin B production even in biotin-sufficient conditions. In contrast, blockade of the preceding step of the PurL catalysing step by sulfamethoxazole inhibited toxin B production almost completely. These results suggest that accumulation of formylglycinamide ribonucleotide (FGAR), a substrate of FGAM synthetase, enhances toxin production by C. difficile and depletion of FGAR reduces toxin production.
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- Mycology
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Massive intracerebral aspergillosis responding to combination high dose liposomal amphotericin B and cytokine therapy without surgery
M. ELLIS, R. WATSON, A. MCNABB, M.L. LUKIC and M. NORKThis report describes a patient with intracranial Aspergillus flavus infection in whom it was impossible to remove the fungal mass surgically. Progressive fungal infiltration of the optic nerves was reversed and the extensive intracranial fungal burden was managed successfully with combination antifungal-immunomodulatory therapy alone.
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Volumes and issues
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Volume 74 (2025)
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Volume 73 (2024)
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Volume 72 (2023 - 2024)
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Volume 71 (2022)
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Volume 70 (2021)
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Volume 69 (2020)
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Volume 68 (2019)
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Volume 67 (2018)
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Volume 66 (2017)
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Volume 65 (2016)
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Volume 54 (2005)
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Volume 52 (2003)
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Volume 51 (2002)
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Volume 50 (2001)
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Volume 49 (2000)
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Volume 47 (1998)
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Volume 45 (1996)
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Volume 35 (1991)
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Volume 34 (1991)
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Volume 33 (1990)
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Volume 18 (1984)
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Volume 17 (1984)
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Volume 16 (1983)
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Volume 15 (1982)
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Volume 14 (1981)
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Volume 13 (1980)
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Volume 12 (1979)
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Volume 11 (1978)
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Volume 10 (1977)
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Volume 9 (1976)
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Volume 8 (1975)
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Volume 7 (1974)
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Volume 6 (1973)
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Volume 5 (1972)
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Volume 4 (1971)
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Volume 3 (1970)
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Volume 2 (1969)
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Volume 1 (1968)
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