- Volume 51, Issue 1, 2002

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.

Coagulase-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.

Pyrazinamide (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.

A 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.

Black-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.

This 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.