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Active efflux of ofloxacin by a highly quinolone-resistant strain of Proteus vulgaris.

A clinical isolate of Proteus vulgaris, strain 881051, highly resistant to ofloxacin (MIC for ofloxacin; 800 mg/L) possessed three types of resistance mechanisms to ofloxacin. The supercoiling activity of DNA gyrase from this strain was barely inhibited by ofloxacin, the ID50 value of ofloxacin against the enzyme was 2700 times higher than that against the enzyme from a susceptible strain, P. vulgaris 08602. In the outer membrane of this resistant strain, no porin protein of 37 kDa was detectable. In the presence of carbonylcyanide m-chloro-phenylhydrazone (CCCP), we found that the accumulation of ofloxacin in the cells of strain 881051, treated with the drug, was about 40-fold higher than that in the absence of CCCP. The CCCP-induced increase in the intracellular drug was 10 and 30 times higher than those in strain 880561 (a moderately resistant strain, MIC 50 mg/L) and 08602 (a fully sensitive strain), respectively. Simultaneous treatment of the strain 881051 with ofloxacin and CCCP resulted in bactericidal synergy. These results indicated that in addition to alterations of DNA gyrase and outer membrane proteins, the active efflux, as the third mechanism, could operate the resistance of strain 881051 to ofloxacin.

Anti-Infective Agents↗

Characterization of an inhibitor-resistant enzyme IRT-2 derived from TEM-2 beta-lactamase produced by Proteus mirabilis strains.

Ten clinical isolates of Proteus mirabilis were found to produce an inhibitor-resistant TEM beta-lactamase (IRT) in association with a TEM-1 enzyme. The IRT enzyme was derived from TEM-2. The blaIRT gene differs from blaTEM-2 gene by one point mutation which leads to the amino-acid substitution Arg-->Ser at position 244, as observed for the original IRT-2 enzyme derived from TEM-1 reported in Escherichia coli. This is the first report of an IRT beta-lactamase derived from TEM-2.

Microbial Sensitivity Tests↗

Permeability to carbapenems of Proteus mirabilis mutants selected for resistance to imipenem or other beta-lactams.

An imipenem-resistant mutant of Proteus mirabilis lacked a 26 kDa outer membrane protein (OMP). It has previously been postulated that this protein is a porin, but the present mutant, which was cross-resistant to mecillinam but not to other beta-lactams, proved as permeable to carbapenems as its parent. A mecillinam-selected mutant had similar cross-resistance yet retained the 26 kDa OMP, confirming that this protein was not important to resistance. In contrast, cefoxitin-selected mutants retained the 26 kDa protein but had diminished expression of major 41 and 44 kDa OMPs and showed reduced uptake of carbapenems, although this promoted resistance only when a carbapenemase was also present. We conclude that the imipenem-selected mutant owed its resistance to some factor other than porin loss, probably to a lesion in penicillin-binding protein 2.

Amdinocillin↗

Integron-located VEB-1 extended-spectrum beta-lactamase gene in a Proteus mirabilis clinical isolate from Vietnam.

A clinical isolate of Proteus mirabilis Lil-1 was obtained from a Vietnamese patient hospitalized in Paris, France. This isolate was resistant to cephalosporins, and there was marked synergy between cephalosporins and clavulanic acid together with unusual synergy between cefoxitin and cefuroxime. PCR analysis revealed the presence of blaVEB-1, an integron-located gene coding for an extended-spectrum beta-lactamase (ESBL) identified previously in an Escherichia coli isolate MG-1 from Vietnam. Using class 1 integron primers and blaVEB-1 intragenic primers, the insert region of the blaVEB-1-containing integron along with flanking sequences were amplified from P. mirabilis Lil-1 whole-cell DNA. A novel class 1 integron, In55, was identified that contained, in addition to intI1, qacEDelta1, sul1 and Orf5 genes, an 8 kb variable region. This region was comparable in size to that found previously in E. coli MG-1, but different from those previously identified in two Pseudomonas aeruginosa isolates from Thailand. In55 was located on a 190 kb self-transferable plasmid, which was different in size and structure from that found in E. coli MG-1. The finding of blaVEB-1 on different plasmids and integrons in enterobacterial isolates underlines the interspecies spread of this novel ESBL gene.

Chromosome Mapping↗

CMY-2-producing Salmonella enterica, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis and Escherichia coli strains isolated in Spain (October 1999-December 2000).

CMY-2 plasmid-mediated AmpC beta-lactamase (CMY-2) was detected in 21 isolates from two hospitals located in different geographical regions of Spain between October 1999 and December 2000. The isolates comprised two Salmonella enterica serovars (Mikawasima and Montevideo), 16 Escherichia coli, one Klebsiella pneumoniae, one Klebsiella oxytoca and one Proteus mirabilis. In addition to the expected resistance to beta-lactams, including extended-spectrum cephalosporins and cefoxitin, all isolates showed a broad spectrum of associated resistance. All were resistant to sulfamethoxazole, chloramphenicol, tetracycline and streptomycin, and all but two were also resistant to gentamicin. Five isolates were studied in detail and all transferred CMY-2 and other resistance determinants by conjugation. Genomic DNA restriction pattern analysis of the E. coli isolates excluded the dissemination of a single clone. To the best of our knowledge this is the first time that CMY-2 has been detected in P. mirabilis, K. oxytoca and S. enterica serovars Mikawasima and Montevideo. It is also the first time that CMY-2 has been described in Spain.

Anti-Bacterial Agents↗

Effects of mupirocin at subinhibitory concentrations on flagella formation in Pseudomonas aeruginosa and Proteus mirabilis.

Colonization of Pseudomonas aeruginosa at trachea, nares and oropharynx can cause ventilator-acquired pneumonia. To identify beneficial effects of antibiotics on expression of virulence factors related to colonization by such pathogens, we evaluated the effect of mupirocin on flagella formation in P. aeruginosa and on motility and flagella formation in Proteus mirabilis. In P. aeruginosa, subinhibitory concentrations of mupirocin inhibited flagella formation, which was associated with reduced flagellin expression. In P. mirabilis, subinhibitory concentrations of mupirocin dose-dependently suppressed bacterial motility and flagella formation, again with reduced flagellin expression. Our results indicate that subinhibitory concentrations of mupirocin can suppress expression of virulence factors in P. aeruginosa and P. mirabilis.

Anti-Bacterial Agents↗

Occurrence and molecular analysis of extended-spectrum {beta}-lactamase-producing Proteus mirabilis in Hong Kong, 1999-2002.

OBJECTIVES: A study was conducted to evaluate the occurrence and characterization of extended-spectrum beta-lactamases (ESBLs) among blood isolates of Proteus mirabilis collected over a 4 year period in Hong Kong. METHODS: Production of ESBLs among 99 consecutive and non-duplicate isolates was evaluated by the double-disc synergy test. The ESBLs were characterized by isoelectric focusing and PCR sequencing using specific primers. The epidemiological relationship of the isolates was studied by the Dienes test and PFGE. RESULTS: ESBLs were identified in 13 isolates, from none in 1999-2000 and up to 18.5% (5/27) in 2001 and 25.8% (8/31) in 2002. The ESBL-producing isolates were more resistant to ceftriaxone than to ceftazidime, and were more likely than non-ESBL-producers to have resistance to ciprofloxacin (76.9% versus 14%) and gentamicin (38.5% versus 9.3%). The ESBL content included CTX-M-13 (n=8), CTX-M-14 (n=3), SHV-5 (n=2), TEM-11 (n=1), and an unidentified ESBL with a pI of 7.5. The Dienes test revealed that the genetic background in the 99 isolates was highly heterogeneous, with 54 distinct types among 92 isolates and seven were non-typeable. Among the 13 ESBL-producing isolates, five different backgrounds, including one cluster (Dienes-pulsotype A) with nine isolates, were identified by both Dienes test and PFGE, thus suggesting both clonal and multi-clonal spread of the CTX-M enzymes. CONCLUSIONS: Our findings indicate the emergence of CTX-M enzymes among P. mirabilis in Hong Kong. More ESBL screening of this species is required to improve their recognition.

Drug Resistance, Bacterial↗

The sequence of the ribosomal 16S RNA from Proteus vulgaris. Sequence comparison with E. coli 16S RNA and its use in secondary model building.

The complete nucleotide sequence of the 16S RNA from Proteus vulgaris has been determined. The molecule (1544 nucleotides) shows 93% homology with the sequence of E. coli 16S RNA. Six methylated nucleotides have been localized in positions homologous to those observed in the E. coli RNA molecule. Both E. coli and P. vulgaris 16S RNA chains can be folded up into a common secondary structure scheme. Comparative sequence analysis of the two molecules has provided a valuable contribution to 16S RNA secondary structure model building.

Base Sequence↗

Two new restriction endonucleases from Proteus vulgaris.

Two novel sequence-specific endonucleases have been isolated from Proteus vulgaris, ATCC 13315. PvuI recognizes the sequence: 5' C G A T decrease C G 3' 3' G C increase T A G C 5' and PvuII recognizes the sequence: 5' C A G decrease C T G 3' 3' G T C increase G A C 5' and cleave as indicated by the arrow (decrease). PvuI is an isoschizomer of XorII, RshI, and XniI. No enzyme with the specificity of PvuII has been described previously.

Animals↗

Purification and characterization of the Proteus vulgaris BlaA protein, the activator of the beta-lactamase gene.

Induction of the expression of the beta-lactamase gene, blaB, is regulated by the blaA gene located just upstream of blaB in the opposite direction in Proteus vulgaris. The expression of the blaA gene is negatively autoregulated by its own product BlaA, the activator of the blaB gene. The P. vulgaris BlaA protein shares high amino acid homology with the LysR family members, which are prokaryotic transcriptional activators that possess a putative helix-turn-helix DNA binding motif. To characterize its function, we purified the BlaA protein to homogeneity from Escherichia coli carrying the expression plasmid of the blaA gene driven by the tac promoter. The gel shift assay and DNaseI footprinting showed that purified BlaA specifically bound to the blaA promoter region, which resides immediately upstream of that of blaB. The binding region contained an inverted repeat, including a T-N11-T sequence which is similar to the LysR motif (T-N11-A) that is conserved in some LysR family members [Goethals et al. (1992) Proc. Natl. Acad. Sci. USA 89, 1646-1650]. We also showed that the BlaA protein forms a dimer in solution, using glycerol gradient centrifugation and glutaraldehyde crosslinking.

Bacterial Proteins↗

The peptidoglycan-associated lipoprotein (PAL) of the Proteus mirabilis outer membrane: characterization of the peptidoglycan-associated region of PAL.

The peptidoglycan-associated lipoprotein (PAL) is present in the cell envelope in a form closely, but not covalently, associated with peptidoglycan in various Gram-negative bacteria. When the cell envelope or the isolated peptidoglycan-PAL complex from Proteus mirabilis, in which PAL maintains the interaction with peptidoglycan, was digested with trypsin, a polypeptide fragment with molecular weight 11,000 (11 K fragment) was obtained. However, when isolated PAL or the 11 K-fragment which had been dissociated from peptidoglycan was treated with trypsin, they were further digested. The 11 K-fragment maintained essentially the same tight interaction with peptidoglycan as intact PAL. These results indicate that the 11 K-fragment is probably derived from the peptidoglycan-associated region of the PAL molecule. The purified 11 K-fragment contained neither covalently-linked fatty acid nor glycerylcysteine, which are known to be present at the N-terminus of PAL. The N-terminal sequence of the 11 K-fragment was also determined.

Amino Acid Sequence↗

Antibodies to Klebsiella pneumoniae, Escherichia coli and Proteus mirabilis in the sera of patients with axial and peripheral form of ankylosing spondylitis.

IgM, IgG and IgA class serum antibodies against the whole Klebsiella pneumoniae, Escherichia coli and Proteus mirabilis bacteria, as well as against K. pneumoniae and E. coli lipopolysaccharides (LPSs) were studied earlier in two separate patient populations of 99 and 85 patients with ankylosing spondylitis (AS) and in 102 healthy blood donors by enzyme immunoassay. In this study the patients were divided into groups according to the presence or absence of peripheral arthritis. The patients with peripheral type AS had increased levels of IgM and IgA class antibodies against K. pneumoniae, whereas the patients with axial type AS had increased levels of IgG and IgA class antibodies to K. pneumoniae, as well as IgA class antibodies against E. coli and P. mirabilis bacteria. Sulphasalazine treatment decreased the IgM and IgA class antibodies in peripheral AS and IgA class antibodies in axial AS against K. pneumoniae LPS. The antibody levels were also decreased against E. coli and P. mirabilis bacteria in the sera of patients with axial AS. The immunological findings in patients with peripheral and axial form of AS were different from each other and thus may reflect different aetiopathogenetic mechanisms for these two types of AS.

Antibodies, Bacterial↗

Antibodies to Klebsiella pneumoniae, Escherichia coli and Proteus mirabilis in the sera of ankylosing spondylitis patients with/without iritis and enthesitis.

IgM, IgG and IgA class serum antibodies against the whole Klebsiella pneumoniae, Escherichia coli and Proteus mirabilis bacteria, as well as against K. pneumoniae and E. coli lipopolysaccharides (LPSs) were studied earlier in the sera of 98 patients with ankylosing spondylitis (AS) and in 102 healthy blood donors by enzyme immunoassay. In this study the patients were divided into groups according to the clinical picture, i.e. presence or absence of iritis and enthesitis. The previous major finding of increased IgA class antibody levels against the whole K. pneumoniae bacteria in AS patients when compared to the healthy controls was not specifically associated with any single patient group in the present study. However, the patients with iritis had higher levels of IgA class antibodies to LPS of K. pneumoniae and E. coli when compared to the patients without iritis. In addition, the patients without enthesitis had higher level of IgG class antibodies against whole K. pneumoniae bacteria compared to the patients with enthesitis. The increased IgA class antibody levels against K. pneumoniae and E. coli LPS in AS patients with iritis may reflect an inflammatory process in the gut area. Furthermore, there were certain other differences in the immunological parameters between the AS patients with and without iritis or enthesitis and the possibility that they reflect different mechanisms involved in the disease processes cannot be excluded.

Antibodies, Bacterial↗

Antibodies to Proteus in rheumatoid arthritis.

Increased levels of Proteus antibodies were found in patients with rheumatoid arthritis and coeliac disease, when compared to normal controls or patients with SLE and sarcoidosis.

Antibodies, Bacterial↗

Proteus syndrome.

Proteus syndrome is a rarely described dysplasia syndrome of the group of congenital hamartomas that arises from mosaic mutation. An extraordinary case history including imaging studies will be reported. This 17-year-old girl suffered from cachexia, lifelong chronic obstipation, different dysplasias, and lipomatous tumor-like lesions. The following findings were marked: macrodactyly, nevi, hemihypertrophy, aggressive lipomatosis, hemangiomas of the spleen, and skull and cerebral malformations. Additionally, an intestinal affection with fatty wall thickening was detected. In contrast to reports in the literature describing a reduced lifespan with a mean of few years, our patient is still alive. The treatment should take a palliative symptomatic approach considering the clinical situation.

Adipose Tissue↗

Histogenesis of vascular tumors in the Proteus syndrome.

Proteus syndrome (PS) is a congenital disorder manifesting with severe deformities, the salient features being gigantism and vascular tumors. The disorder is poorly understood, and there has been much discrepancy in the terminology regarding the vascular tumors in PS. The purpose of this study was to elucidate the histogenesis of these tumors by correlating microscopic observations with immunohistologic information. The value of immunoperoxidase studies in the pathologic evaluation of PS was also assessed. Fourteen formalin-fixed, paraffin-embedded tissue specimens obtained from vascular tumors of six children with PS were stained with Ulex europaeus agglutinin I (UEA-I) lectin and the following immunohistochemical reagents: anti-factor VIII-related antigen (FVIII-RAg) and anti-CD34. The tumors showed varied proportions of vascular, lipomatous, and fibrous tissue components consistent with vascular hamartomas. The predominant vascular channels of the tumors were morphologically consistent with lymphatic vessels. Immunostaining of the endothelium of these vessels was most consistently positive with UEA-I lectin. Although a color reaction product was present in small vessels and some larger blood vessels, anti-CD34 immunostaining spared the lumens of lymphatic channels. In addition, a striking population of dendritic spindle cells was noted with the anti-CD34 but was unnoticed with the other reagents. We concluded that the vascular tumors of PS are primarily lymphatic hamartomas. The spindle cells noted with anti-CD34 immunostaining may relate to angiogenesis and need further delineation.

Adult↗

Genotyping demonstrates that the strains of Proteus mirabilis from bladder stones and catheter encrustations of patients undergoing long-term bladder catheterization are identical.

PURPOSE: We established the incidence of bladder stones in patients who experienced recurrent encrustation and blockage of indwelling bladder catheters and examined the relationship between isolates of Proteus mirabilis from the stones and from the crystalline biofilms on the catheters. MATERIALS AND METHODS: The first 100 patients attending a clinic for patients experiencing problems with the management of long-term bladder catheters were studied. Flexible cystoscopy was used to detect bladder stones. Catheter encrustation was assessed visually and by electron microscopy. Bacteriological analysis was performed on the stones and catheter biofilms. P. mirabilis isolates were genotyped by pulsed field gel electrophoresis of restriction enzyme digests of bacterial DNA. RESULTS: Most patients (85%) had been referred because of catheter blockage and in 61 (72%) the catheters were encrusted. P. mirabilis was recovered from 37 of 47 encrusted catheters (79%) that were examined but not from any nonencrusted catheters. Of the 61 patients with encrusted catheters 38 (62%) had bladder stones. Pairs of isolates of P. mirabilis from the stones and the catheter biofilms from 6 patients were genotyped. The DNA profiles of each pair of isolates were identical. CONCLUSIONS: The majority of patients (62%) with recurrent catheter encrustation had bladder stones. The stones harbored the strains of P. mirabilis that rapidly colonize replacement catheters with crystalline biofilm. Flexible cystoscopy to detect and remove stones might help resolve the problem of recurrent catheter encrustation.

Adult↗

Does the valve regulated release of urine from the bladder decrease encrustation and blockage of indwelling catheters by crystalline proteus mirabilis biofilms?

PURPOSE: We tested whether valve regulated, intermittent flow of urine from catheterized bladders decreases catheter encrustation. MATERIALS AND METHODS: Laboratory models of the catheterized bladder were infected with Proteus mirabilis. Urine was allowed to drain continuously through the catheters or regulated by valves to drain intermittently at predetermined intervals. The time that catheters required to become blocked was recorded and encrustation was visualized by scanning electron microscopy. RESULTS: When a manual valve was used to drain urine from the bladder at 2-hour intervals 4 times during the day, catheters required significantly longer to become blocked than those on continuous drainage (mean 62.6 vs 35.9 hours, p = 0.039). A similar 1.7-fold increase occurred when urine was drained at 4-hour intervals 3 times daily. Experiments with an automatic valve in which urine was released at 2 or 4-hour intervals through the day and night also showed a significant increase in mean time to blockage compared with continuous drainage (p = 0.001). Scanning electron microscopy confirmed that crystalline biofilm was less extensive on valve regulated catheters. CONCLUSIONS: Valve regulated, intermittent flow of urine through catheters increases the time that catheters require to become blocked with crystalline biofilm. The most beneficial effect was recorded when urine was released from the bladder at 4-hour intervals throughout the day and night by an automatic valve.

Biofilms↗