Multiplex Real-Time PCR Assay for Simultaneous Identification of Neisseria gonorrhoeae and Its Ciprofloxacin Susceptibility Status.

A real-time PCR (RT-PCR) assay was designed for the simultaneous identification of Neisseria gonorrhoeae and its ciprofloxacin susceptibility status. A SYBR green-based multiplex RT-PCR format was used; it comprised two different forward primers and a common reverse primer to detect single nucleotide polymorphisms (SNPs) in gyrA of N. gonorrhoeae The primer pairs were evaluated for their sensitivity and specificity using genomic DNA from 254 N. gonorrhoeae isolates (82 were ciprofloxacin susceptible and 172 were ciprofloxacin resistant) and 23 non-N. gonorrhoeae species isolates. The performance of the primers was validated using genomic DNA from 100 different N. gonorrhoeae isolates (46 were ciprofloxacin susceptible and 54 were ciprofloxacin resistant) and 52 non-N. gonorrhoeae isolates. The latter panel was revalidated by testing 99 (46 isolates were ciprofloxacin susceptible and 53 isolates were ciprofloxacin resistant) of the N. gonorrhoeae isolates and 23 non-N. gonorrhoeae isolates. These primers detected N. gonorrhoeae and its ciprofloxacin susceptibility status with over 99% sensitivity and specificity for all panels tested. This assay has the potential to be an inexpensive and rapid test for the simultaneous identification of N. gonorrhoeae and its ciprofloxacin susceptibility status.

A minD mutant of enterohemorrhagic E. coli O157:H7 has reduced adherence to human epithelial cells.

Adherence to epithelial cells is a prerequisite for intestinal colonization by the bacterial pathogen, enterohemorrhagic Escherichia coli (EHEC). The deletion of minD, a cell division gene, in EHEC caused reduced adherence to human epithelioid cervical carcinoma (HeLa) and human colonic adenocarcinoma (Caco-2) cells as compared to wild-type. The minD mutant formed minicells and filaments owing to aberrant cytokinesis. Moreover, its ability to form microcolonies as typically seen in the co-cultures of wild-type with Caco-2 cells, was abolished. In conclusion, the present study highlights the importance of minD in regards to EHEC adherence to human epithelial cells.

Cytosolic phospholipase a2 activation by Candida albicans in alveolar macrophages: role of dectin-1.

Candida albicans is an increasingly important pulmonary fungal pathogen. Resident alveolar macrophages are important in host defense against opportunistic fungal infections. Activation of Group IVA cytosolic phospholipase A(2)alpha (cPLA(2)alpha) in macrophages initiates arachidonic acid (AA) release for production of eicosanoids, which regulate inflammation and immune responses. We investigated the ability of C. albicans to activate cPLA(2)alpha in unprimed alveolar macrophages and after priming with granulocyte macrophage colony-stimulating factor (GM-CSF), which regulates alveolar macrophage maturation. AA was released within minutes by GM-CSF-primed but not unprimed alveolar macrophages in response to C. albicans, and was blocked by soluble glucan phosphate (S-GP). The expression of the beta-glucan receptor dectin-1 was increased in GM-CSF-primed macrophages, and AA release from GM-CSF-primed dectin-1(-/-) alveolar macrophages was reduced to basal levels. The enhanced activation of extracellular signal-regulated kinases and phosphorylation of cPLA(2)alpha on Ser-505 that occurred in GM-CSF-primed macrophages were reduced by MEK1 and Syk inhibitors, which also suppressed AA release. At later times after C. albicans infection (6 h), unprimed and GM-CSF-primed macrophages released similar levels of AA. The expression of cyclooxygenase 2 and prostanoid production at 6 hours was higher in GM-CSF-primed macrophages, but the responses were not dependent on dectin-1. However, dectin-1 contributed to the C. albicans-stimulated increase in TNF-alpha production that occurred in GM-CSF-primed macrophages. The results demonstrate that dectin-1 mediates the acute activation of cPLA(2)alpha in GM-CSF-primed alveolar macrophages, but not in the more delayed phase of AA release and GM-CSF-dependent prostanoid production.

Evaluation of a Hydrogel-Based Diagnostic Approach for the Point-of-Care Based Detection of Neisseria gonorrhoeae.

Eleven primer pairs were developed for the identification of Neisseria gonorrhoeae. The sensitivity and specificity of these primers were evaluated by Real Time (RT)-PCR melt curve analyses with DNA from 145 N. gonorrhoeae isolates and 40 other Neisseria or non-Neisseria species. Three primer pairs were further evaluated in a hydrogel-based RT-PCR detection platform, using DNA extracted from 50 N. gonorrhoeae cultures. We observed 100% sensitivity and specificity in the hydrogel assay, confirming its potential as a point-of-care test (POCT) for N. gonorrhoeae diagnosis.

Murine infection model for Mycobacterium fortuitum.

Mycobacterium fortuitum is an atypical, non-tubercular, pathogenic, rapidly growing mycobacteria. As very little is known about its virulence determinants, the absence of an animal infection model was always sorely felt. A reliable and reproducible murine infection model has been developed in which non-replicating persistence of 10(5) CFU/g tissue in kidney was observed when a standardised dosage inoculum of 5x10(7) CFU was injected intravenously. The tissue bacillary load was determined at regular intervals (10, 25, 45 and 60 days post-inoculation) in different organs, viz., kidney, spleen, lung and liver. Histopathology of kidney revealed tissue damage and granuloma-like formations which appear to be part of the host's effort to combat the infection. As IFN-gamma is known to trigger antimycobacterial effects of murine macrophages, IFN-gamma was assayed to determine the correlation between host protective measures and bacillary load in kidney. Fifteen days after infection, the level of IFN-gamma secreted by CD4+ and CD8+ T lymphocytes was high, concomitant with high tissue bacillary load, while the level sharply declined as the number of bacilli in kidney decreased 45 days post-inoculation. The invasion and proliferation of M. fortuitum ATCC 6841, when incubated with non-phagocytic (recombination activating genes (RAG) murine kidney) and phagocytic (murine peritoneal macrophages) cell lines, was assessed. M. fortuitum did not invade RAG murine kidney cell line, while the bacilli infected and proliferated freely inside murine macrophages. In conclusion, we present a reproducible murine infection model that sustains a persistent infection, the progression of which correlates meaningfully with host protective response.

Suppression of ERK activation in urethral epithelial cells infected with Neisseria gonorrhoeae and its isogenic minD mutant contributes to anti-apoptosis.

In gonococci-infected transduced human urethral epithelial cells (THUEC), the role of ERK, a mitogen-activated protein kinase (MAPK), in apoptosis is unknown. We observed lowering of ERK activation in THUEC following infection with anti-apoptosis-inducing Neisseria gonorrhoeae strain CH811. An isogenic cell division mutant of this strain, Ng CJSD1 (minD deficient), which is large and abnormally shaped, reduced ERK phosphorylation levels even more than its parental strain in THUEC. This led to higher anti-apoptosis in mutant-infected cells as compared to the parental strain-infected cells. Our results suggest that N. gonorrhoeae infection reduces ERK activation in THUEC contributing to anti-apoptosis.

Regulation of minD by oxyR in Neisseria gonorrhoeae.

In Neisseria gonorrhoeae, cytokinesis involves Escherichia coli homologues of minC, minD and minE which are encoded as part of a min operon. MinD, a 30 kD protein component of the MinC-MinD septum inhibitory complex, together with MinE, mediates cell division site selection. Gonococci mutated in minD display aberrant cytokinesis, abnormal morphology, defective microcolony formation and virulence. minD is 274 bp upstream of oxyR, another min operon gene in N. gonorrhoeae, which encodes a redox-responsive transcriptional regulator implicated in responses to oxidative stress. In this study, we aimed to examine the oxyR-mediated regulation of minD. We observed the cotranscription of oxyR with the minCDE gene cluster. The mutation of oxyR resulted in non-midline formation of the division septum, anomalous DNA segregation, and increased aggregation of bacterial cells. qRT-PCR and Western Blot analysis revealed upregulation of minD in an oxyR mutant as compared to its isogenic wild-type N. gonorrhoeae strain in stationary phase. Furthermore, the exposure to oxidative stress in the form of H2O2 increased MinD expression levels in wild-type N. gonorrhoeae. Using ß-galactosidase activity-based promoter assays, we found that oxyR negatively regulates the promoter region (PminD) upstream of minD. Our results demonstrate the involvement of oxyR in cell division and minD expression in N. gonorrhoeae.

Attenuated virulence of min operon mutants of Neisseria gonorrhoeae and their interactions with human urethral epithelial cells.

Neisseria gonorrhoeae, a sexually-transmitted gram-negative bacterium, causes gonorrhoea in humans. The min genes of N. gonorrhoeae are involved in cell division site selection with oxyR co-transcribed with these genes. The mutation in min genes and oxy R cause aberrant cell morphology and aggregation patterns, respectively. Our objective was to assess the contribution of neisserial min operon cell division genes i.e. minC, minD and oxyR in virulence. Compared to the N. gonorrhoeae parental strain (Ng CH811Str(R)), its isogenic mutants with insertionally inactivated minC (Ng CSRC1), minD (Ng CJSD1) or oxyR (Ng KB1) showed reduced adherence to and invasion of urethral epithelial cells. This may be explained by defective microcolony formation in the mutant strains, possibly owing to abnormal morphology and aggregation. The expression levels of surface virulence factors like Opa, pilin and lipooligosaccharide in the mutants were unchanged relative to Ng CH811Str(R). Furthermore, in urethral epithelial cells, the min and oxyR mutants induced the release of proinflammatory cytokines like IL6 and IL8 to levels similar to that induced by the parental strain. Taken together, our studies indicate that inactivation of minC, minD or oxyR in N. gonorrhoeae attenuates its ability to bind to and invade urethral epithelial cells without altering its potential to induce IL6 and IL8 release.