Pseudomonas aeruginosa and Escherichia coli were exposed to nocardicin A, and were subsequently observed with transmission and scanning electron microscopes. Although the nocardicin A-induced morphological alterations such as bulges and spheroplast formations were observed both in P. aeruginosa and E. coli, their positions on the cell surface were different in the two species.
Purpose: To examine bacterial virulence factors in Pseudomonas aeruginosa isolates from contact lens (CL) wearers and non–CL wearers with P. aeruginosa keratitis, and to investigate relationships between virulence factors and clinical features of keratitis. Methods: The study involved 25 subjects including 18 CL and 7 non–CL-related P. aeruginosa keratitis patients. Slit-lamp photographs of all subjects were captured, and the focus occupancy ratio (FOR) was defined as the total focus area/entire cornea area, using image processing software. Twenty-five clinical P. aeruginosa isolates from keratitis were assessed for protease production, elastase production, biofilm formation, bacterial swimming and swarming motility, cell surface hydrophobicity, and genes encoding the type III secretion system (TTSS) effectors (ExoU and ExoS). Results: Ring abscess was found in 9 of 18 CL-related P. aeruginosa keratitis cases (CL[+] ring[+] group) but not in another 9 cases (CL[+] ring[−] group). Expression or prevalence of virulence factors in P. aeruginosa isolates from the CL(+) ring(+) group, CL(+) ring(−) group, and CL(−) group were compared. The FOR for CL(+) ring(+) or CL(−) was higher than for CL(+) ring(−) (P < 0.05 and P < 0.01, respectively). The rate of positive swimming motility for CL(+) ring(+) or CL(−) was higher than for CL(+) ring(−) (P < 0.05), whereas the rate of positive swarming motility for CL(+) ring(+) was higher than for CL(+) ring(−) or CL(−) (P < 0.05). Prevalence of an exoS+/exoU-genotype for CL(+) ring(+) or CL(−) was higher than for CL(+) ring(−) (P < 0.05). In the CL-related group, expression of elastase and swarming motility significantly correlated with FOR. Conclusions: Swimming motility, swarming motility, and TTSS ExoS could play a major role in the determination of clinical features of P. aeruginosa keratitis.
Pseudomonas aeruginosa and Escherichia coli were exposed to nocardicin A, and were subsequently observed with transmission and scanning electron microscopes. Although the nocardicin A-induced morphological alterations such as bulges and spheroplast formations were observed both in P. aeruginosa and E. coli, their positions on the cell surface were different in the two species.
Pseudomonas aeruginosa showing resistance to imipenem were found in 100 of 1,058 strains (9.5%) from six hospitals (a-f) in Hiroshima City, Japan. Of the 100 strains, 14 (14%) were double disk synergy test positive using sodium mercaptoacetic acid disks, and 18 (18%) were bla(IMP-1) or bla(VIM-2) allele positive by polymerase chain reaction (PCR). Among 100 imipenem-resistant strains, 32 were categorized into multi-drug resistant strains, in which 13 were positive for the metallo-beta-lactamase gene. Fifty-one strains (51%) among the 100 imipenem-resistant strains had elevated RND efflux pump activity against levofloxacin. But only 6 of 51 strains were classified as multi-drug resistant strains. The pulsed field gel electrophoresis analysis of the Spe I-digested DNA from the 100 isolates suggested not only clonal spread but spread of heterogeneous clones started to contribute to the prevalence of metallo-beta-lactamase producing P. aeruginosa strains in Japanese hospitals.
Multiple antibiotic resistance(MDR) in bacteria was at first thought to be caused exclusively by the combination of several resistance mechanisms including membrane impermeability, the outer membrane barrier in gram-negative bacteria. More recently, it became clear that MDR are often achieved by interplay between impermeability and multidrug efflux pumps. Streptococcus pneumoniae MefA, Staphylococcus aureus NorA and Pseudomonas aeruginosa Mex of these pumps are significant, because those bacteria are more frequently isolated in clinical. In this review, we described on characteristics and clinical significance of P. aeruginosa Mex systems, MexA-MexB-OprM, MexC-MexD-OprJ, MexE-MexF-OprN and MexX-MexY-OprM, and furthermore, on interplay between the efflux systems, the outer membrane barrier, hydrolyzing enzyme and mutated target.
Pseudomonas aeruginosa and Escherichia coli were exposed to nocardicin A, and were subsequently observed with transmission and scanning electron microscopes. Although the nocardicin A-induced morphological alterations such as bulges and spheroplast formations were observed both in P. aeruginosa and E. coli, their positions on the cell surface were different in the two species.
Antibiotic‐resistant mutants of Pseudomonas aeruginosa were generated using chloramphenicol and ciprofloxacin as selective agents. These mutants displayed a multidrug phenotype and overexpressed an outer membrane protein of 50kDa, which was shown by Western blot analysis to correspond to OprN. A cosmid clone harbouring the oprN gene was isolated by partial complementation of a mutant deficient in OprM, the outer membrane component of the mexAB–oprM efflux operon. Antibiotic‐accumulation studies indicated that OprN was part of an energy‐dependent antibiotic‐efflux system. Sequencing of a 6180bp fragment from the complementing cosmid revealed the presence of three open reading frames (ORFs), which exhibited amino acid similarity to the components of the mexAB–oprM and mexCD–oprJ efflux operons of P. aeruginosa . The ORFs were designated MexE, MexF and OprN. Mutation of the mexE gene eliminated the multidrug‐resistance phenotype in an OprN‐overexpressing strain, but did not affect the susceptibility profile of the wild‐type strain. Expression of the mexEF–oprN operon was shown to be positively regulated by a protein encoded on a 1.5 kb DNA fragment located upstream of mexE and belonging to the LysR family of transcriptional activators. The presence of a plasmid containing this DNA fragment was sufficient to confer a multidrug phenotype onto the wild‐type strain but not onto the mexE mutant. Evidence is provided to show that the mexEF–oprN operon may be involved in the excretion of intermediates for the biosynthesis of pyocyanin, a typical secondary metabolite of P. aeruginosa .
The 81 kDa protein (designated OpcPO) which forms a diffusion pore in the outer membrane of Burkholderia(formerly Pseudomonas) cepaciahas a unique characteristic in that when the purified protein is heated it yields a major 36 kDa protein (designated OpcP1) and a minor 27 kDa protein (designated OpcP2). Moreover, incubation of OpcPO in citrate buffer at pH 3.0 produced an unusual dissociation into 72 kDa and 27 kDa proteins. For the characterization of OpcPO and its derivatives, OpcP1 and OpcP2 from purified OpcPO were isolated by preparative SDS-PAGE. Reconstitution of OpcPO using purified preparations of OpcP1 and OpcP2 indicated that these derivatives were not proteolytic fragments of OpcPO. Moreover, immunoblot assays with murine polyclonal antisera specific for OpcP1 and OpcP2 yielded the following results: (i) OpcP1 and OpcP2 are immunologically distinguishable proteins; (ii) the unusual dissociation of OpcPO in citrate buffer at pH 3.0 resulted in the release of OpcP2 from OpcPO, and the resulting 72 kDa protein was probably an oligomer of OpcP1; (iii) purified OpcP1 itself produced two additional 53 kDa and 72 kDa proteins spontaneously following elution from the bottom of the SDS-PAGE gel. From these findings, it was concluded that OpcPO is formed by the non-covalent association of OpcP2 with an oligomer of OpcP1 that has the ability to self-assemble.
