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Proteus mirabilis urease: transcriptional regulation by UreR.

Proteus mirabilis urease catalyzes the hydrolysis of urea, initiating the formation of urinary stones. The enzyme is critical for kidney colonization and the development of acute pyelonephritis. Urease is induced by urea and is not controlled by the nitrogen regulatory system (ntr) or catabolite repression. Purified whole-cell RNA from induced and uninduced cultures of P. mirabilis and Escherichia coli harboring cloned urease sequences was probed with a 4.2-kb BglI fragment from within the urease operon. Autoradiographs of slot blots demonstrated 4.2- and 5.8-fold increases, respectively, in urease-specific RNA upon induction with urea. Structural and accessory genes necessary for urease activity, ureD, A, B, C, E, and F, were previously cloned and sequenced (B. D. Jones and H. L. T. Mobley, J. Bacteriol. 171:6414-6422, 1989). A 1.2-kb EcoRV-BamHI restriction fragment upstream of these sequences confers inducibility upon the operon in trans. Nucleotide sequencing of this fragment revealed a single open reading frame of 882 nucleotides, designated ureR, which is transcribed in the direction opposite that of the urease structural and accessory genes and encodes a 293-amino-acid polypeptide predicted to be 33,415 Da in size. Autoradiographs of sodium dodecyl sulfate-polyacrylamide gels of [35S]methionine-labeled polypeptides obtained by in vitro transcription-translation of the PCR fragments carrying only ureR yielded a single band with an apparent molecular size of 32 kDa. Fragments carrying an in-frame deletion within ureR synthesized a truncated product. The predicted UreR amino acid sequence contains a potential helix-turn-helix motif and an associated AraC family signature and is similar to that predicted for a number of DNA-binding proteins, including E. coli proteins that regulate acid phosphatase synthesis (AppY), porin synthesis (EnvY), and rhamnose utilization (RhaR). These data suggest that UreR governs the inducibility of P. mirabilis urease.

Base Sequence↗

Biotypes of Proteus rettgeri.

Examination of 729 isolates of Proteus rettgeri showed that 674 reacted positively in tests for phenylalanine deamination, indole production, growth on citrate, and acid production from meso-inositol, and negatively for L-ornithine decarboxylation and acid production from lactose, maltose, D-xylose, and L-arabinose. Only 51 isolates differed in one, and four differed in two of these ten reactions, which were taken as the core characteristics of the species. On the basis of additional tests (acid production from salicin, L-rhamnose, D-mannitol, adonitol, and D-arabitol), the 729 isolates could be separated into five groups. Goups 1, 2, 3, and 4 could be further separated on the basis of the reaction with meso-erythritol, and group 5 could be subdivided on the basis of reaction with D-mannitol. Two metabolically distinct kinds of P. rettgeri were recognized. Isolates of the first kind (groups 1, 2, 3, and 4) each utilized both adonitol and D-arabitol and most utilized meso-erythritol. Isolates of the other kind (group 5) were negative with the three polyhydric alcohols but resembled, in their reactions, some strains of Providencia stuartii. These may be intermediates between P. rettgeri that catabolize these substrates and the Providencia.

Acids↗

O antigen grouping of Morganella morganii (Proteus morganii) by slide agglutination.

Antisera were prepared against Morganella morganii (Proteus morganii) type strains from the scheme described by Rauss et al. (Acta Microbiol. Acad. Sci. Hung. 22:315--321, 1975) and Raus and Vörös (Acta Microbiol. Acad. Sci. Hung. 6:233--248, 1959; Acta Microbiol. Acad. Sci. Hung. 14:195--198, 1967). The specificities of the somatic (O) antigens were studied using the passive hemagglutination and slide agglutination techniques. Previously unreported interstrain relations were observed, and O groups were provisionally defined on the basis of related strains. Of 143 isolates, collected mostly from two hospitals, 96% could be placed in one or another of the O groups. Forty-eight percent belonged to O group 1 and most of these were of two serotypes O1ab, 2 (20%) and O1ad, 2 (12%).

Agglutination Tests↗

Application of O-serotyping in a study of Providencia rettgeri (Proteus rettgeri) isolated from human and nonhuman sources.

A somatic (O) antigen serotyping scheme for Providencia rettgeri (Proteus rettgeri) was modified to exclude O-type strains recently reclassified as urea-positive Providencia stuartii and was extended to include new serotypes to provide for serotyping on the basis of 93 O-antigens. Isolates from two hospitals, five public health laboratories, and nonhuman sources (polluted water and frogs) were serotyped. The 112 isolates collected from a large general hospital over a 99-month period were distributed among 42 O-serotypes. No serotype showed significant predominance that would suggest the occurrence of human strains that are more prone than others to cause human infections, but in an institution experiencing cross-infection, 11 of the 22 (50%) isolates belonged to one serotype. The 54 isolates from the five public health laboratories belonged to 33 serotypes, 15 of which were found also among hospital isolates. All but 5 of 99 frog isolates were typable, and the 94 typable isolates were separated into 25 serotypes. Each of the four isolates from polluted water samples was of a different serotype. Sixteen of the serotypes found in frogs and three found in water were also identified among human isolates.

Animals↗

Unusual groups of Morganella ("Proteus") morganii isolated from clinical specimens: lysine-positive and ornithine-negative biogroups.

Morganella ("Proteus") morganii is the only species in the recently proposed genus Morganella, but we suspect there are yet undescribed species in the genus. Two candidates for new species were recently investigated. Nineteen strains isolated from clinical specimens resembled M. morganii but were lysine positive and nonmotile and fermented glycerol within 24 h. Typical M. morganii are lysine negative and motile and ferment glycerol slowly or not at all. The three-test variance from typical M. morganii suggested that this new group might be a new Morganella species; however, strains of the group were closely related to typical M. morganii by deoxyribonucleic acid (DNA)-DNA hybridization. A second group of 14 strains isolated from clinical specimens was ornithine negative but was otherwise very similar to M. morganii. This group was also closely related to M. morganii by DNA hybridization. Both sets of strains clearly belong in the species Morganella morganii as distinct biogroups; they are not separate species as originally suspected. Initially both biogroups posed a problem in identification until their true relationship to M. morganii was determined.

Anti-Bacterial Agents↗

Proteus penneri and urinary calculi formation.

The clinical significance of Proteus penneri, a newly described species, is unknown. A case report is presented, which is to the best of our knowledge the first description of this organism causing a urinary tract infection and bladder calculi.

Aged↗

Expanded clinical spectrum of infections caused by Proteus penneri.

Strains of Proteus penneri from seven abdominal wounds (after bowel resection), five urine samples, and eight other sites were isolated in mixed cultures. Seven urine isolates were in pure cultures. All infections were nosocomially acquired, indicating that complete identification of P. penneri in the clinical laboratory is warranted.

Aged↗

Differentiation of Proteus mirabilis by bacteriophage typing and the Dienes reaction.

A provisional typing schema based on sensitivity to 23 bacteriophages has been established for Proteus mirabilis. Seventy-three bacteriophages were isolated on strains of P. mirabilis (64), P. vulgaris (1), P. morganii (7), and P. rettgeri (1), but those isolated on P. mirabilis were the most useful in differentiating other strains of . mirabilis. From the 73 phages studied, the best 23 were chosen by computer analysis for the provisional system, which was then used to study P. mirabilis infections in a 500-bed general hospital. All patient isolates for 19 months were saved and then compared by bacteriophage typing and the Dienes reaction in a retrospective study. There was evidence for only three instances of cross-infection or -colonization during this time. Bacteriophage typing was very sensitive in differentiating strains, since 200 strains were differentiated into 113 different lysis patterns and 94% were typable. The Dienes reaction was useful at times but often gave reactions that were difficult to read or that changed when the tests were repeated. The bacteriophages described by Schmidt and Jeffries were also evaluated and proved useful in combination with ours. The value of bacteriophage typing was clearly established, and work toward a standardized schema for P. mirabilis should continue.

Bacteriophage Typing↗

Evaluation of a Proteus rettgeri O-serotyping system for epidemiological investigation.

An evaluation of the serotyping system for Proteus rettgeri has been performed through its application to 561 human isolates of this species. Of these, 545 were typable and could be separated into 58 O serotypes. A group of isolates from one hospital, and clinically implicated in cross-infections, was found to belong the same O serotype, confirming the clinical findings. Isolates with more than one specificity of thermostable antigen, in most cases, were found to have combinations of such specificities restricted to combinations previously defined for the O-type strains. Furthermore, the use of pooled antisera was investigated and system of pooling was devised to retain the specificity of the system and to increase the facility of the typing procedure.

Antigens, Bacterial↗

Arbitrarily primed polymerase chain reaction provides rapid differentiation of Proteus mirabilis isolates from a pediatric hospital.

During a systematic survey, maternal carriage of Proteus mirabilis was found over a 25-day period in 18 pregnant women admitted to the delivery ward of our hospital maternity. Five neonates born to these mothers were found to be colonized with P. mirabilis. We report here on the use of DNA fingerprinting by the arbitrarily primed polymerase chain reaction technique (AP-PCR) for the epidemiological investigation of this sudden outbreak. This approach was compared with the analysis of restriction fragment length polymorphisms of ribosomal DNA regions (ribotyping). Results of the AP-PCR and of ribotyping were in complete agreement in showing the genetic unrelatedness of the isolates obtained from each mother. Moreover, the results showed mother-to-infant vertical transmission of P. mirabilis in the neonates. AP-PCR is a rapid and discriminative method which seems particularly well suited to the epidemiological study of P. mirabilis.

Bacterial Typing Techniques↗

Problems related to determination of MICs of oximino-type expanded-spectrum cephems for Proteus vulgaris.

During in vitro susceptibility testing of clinical isolates of Proteus vulgaris, we noted that the MICs of several expanded-spectrum cephems were much higher in the broth microdilution method than in the agar dilution method (termed the MIC gap phenomenon). Here we investigated the mechanism of the MIC gap phenomenon. Cephems with the MIC gap phenomenon were of the oximino type, such as cefotaxime, cefteram, and cefpodoxime, which serve as good substrates for inducible class A beta-lactamase (CumA) enzymes produced by P. vulgaris; this finding suggests a relationship between the MIC gap phenomenon and CumA. Since peptidoglycan recycling shares a system common to that inducing CumA, we analyzed the mechanism of the MIC gap phenomenon using P. vulgaris B317 and isogenic mutants with mutations in the peptidoglycan recycling and beta-lactamase induction systems. The MIC gap phenomenon was observed in the parent strain B317 but not in B317G (cumG-defective mutant; defective peptidoglycan recycling) and B317R (cumR-defective mutant; defective CumA transcriptional regulator). No beta-lactamase activity was detected in B317G and B317R. beta-Lactamase activity and the MIC gap phenomenon were restored in B317G/pMD301 (strain transcomplemented by a cloned cumG gene) and B317R/pMD501 (strain transcomplemented by a cloned cumR gene). MICs determined by the agar dilution method increased when lower agar concentrations were used. Our results indicated that the mechanism of the MIC gap phenomenon is related to peptidoglycan recycling and CumA induction systems. However, it remains unclear how beta-lactamase induction of P. vulgaris is suppressed on agar plates.

Cephalosporins↗

Evaluation of the discriminatory powers of the Dienes test and ribotyping as typing methods for Proteus mirabilis.

A total of 63 clinical isolates of Proteus mirabilis collected over a 19-month period were typed by the Dienes test and ribotyping. Ribotyping was performed using the fully automated RiboPrinter Microbial Characterization System (Qualicon, Wilmington, Del.). Isolates that were indistinguishable by the Dienes test and/or ribotyping were characterized further by pulsed-field gel electrophoresis (PFGE). Most of the isolates represented unique strains as judged by the Dienes test and ribotyping. Forty isolates represented 40 different ribotypes and Dienes types. The remaining 23 isolates were grouped into 13 Dienes types, 12 ribotypes, and 14 PFGE types. The index of discrimination was 0.980 for the Dienes test, 0.979 for ribotyping, and 0.992 for PFGE. Both the Dienes test and ribotyping are useful methods for identifying individual strains of P. mirabilis. The Dienes test is simple, inexpensive, and easy to perform. It can be performed in virtually any laboratory and should be used in the initial epidemiologic characterization of P. mirabilis isolates.

Automation↗

Biochemical identification and characterization of DNA groups within the Proteus vulgaris complex.

We placed 43 isolates belonging to the Proteus vulgaris complex into proposed DNA groups (genomovars) using five previously recommended tests (tests for esculin hydrolysis, production of acid from salicin, L-rhamnose fermentation, and elaboration of DNase and lipase). On the basis of the results of these five tests, 49% of the isolates fell into DNA groups 5 and 6, 37% fell into DNA group 2, and the remaining 14% fell into DNA groups 3 and 4. Sequencing of the 16S rRNA genes of 11 members of DNA groups 5 and 6 indicated that 10 of these isolates (91%) could be unambiguously assigned to one of these two genomospecies. Overall expression of selected enzymatic and virulence-associated characteristics did not differ significantly among DNA groups.

Bacterial Typing Techniques↗

Emerging extended-spectrum beta-lactamases in Proteus mirabilis.

Beta-lactamase production was detected in 147 (52%) of 282 consecutive nonduplicate Proteus mirabilis isolates obtained over a 1-year period from the S. Matteo Hospital of Pavia (northern Italy). Seventy isolates (48% of the beta-lactamase producers) were found to produce extended-spectrum beta-lactamases (ESBLs), identified as PER-1 (first report in this species) and TEM-52 in 52 and 18 isolates, respectively. Analysis of clonal diversity of the ESBL producers suggested different spreading patterns for the two ESBL determinants.

Humans↗

Molecular epidemiology of Proteus mirabilis infections of the catheterized urinary tract.

Proteus mirabilis compromises the care of many patients undergoing long-term indwelling bladder catheterization. It forms crystalline bacterial biofilms in catheters which block the flow of urine, causing either incontinence due to leakage or painful distention of the bladder due to urinary retention. If it is not dealt with, catheter blockage can lead to pyelonephritis and septicemia. We have examined the epidemiology of catheter-associated P. mirabilis infections by use of pulsed-field gel electrophoresis (PFGE) of NotI restriction enzyme digests of bacterial DNA. This technique was shown to be more discriminatory than the classical phenotypic Dienes typing technique. We demonstrated that each of 42 isolates from diverse environmental sources and 10 of 12 isolates from blood, wound swabs, and mid-stream urine samples of hospitalized patients had distinct genotypes. Examination of a set of 55 isolates of P. mirabilis, each from a different clinical or environmental source, identified 49 distinct genotypes and 43 Dienes types. The index of discrimination was 0.993 for the PFGE method and 0.988 for the Dienes method. Applying the PFGE method to isolates from catheter-associated urinary tract infections confirmed that the strains present in the crystalline catheter biofilms were identical to those isolated from the same patient's urine. An analysis of samples taken during a prospective study of infections in catheterized nursing home patients revealed that a single genotype of P. mirabilis can persist in the urinary tract despite many changes of catheter, periods of noncatheterization, and antibiotic therapy.

Biofilms↗

Nosocomial outbreak of infections by Proteus mirabilis that produces extended-spectrum CTX-M-2 type beta-lactamase.

Nineteen multidrug-resistant Proteus mirabilis strains were isolated from 19 patients suffering from infections probably caused by P. mirabilis. These strains were recovered from urine or other urogenital specimens of 16 inpatients and three outpatients with a hospitalization history in a urology ward of Funabashi Medical Center, from July 2001 to August 2002. These strains demonstrated resistance to cefotaxime, ceftriaxone, cefpodoxime, and aztreonam, while they were highly susceptible to ceftazidime (MIC, </=0.5 micro g/ml). The resistance level of these strains to cefotaxime was decreased by the presence of clavulanic acid. Therefore, the strains were speculated to produce extended-spectrum class A beta-lactamases. These strains were later found to carry bla(CTX-M-2) genes by both PCR and sequencing analyses. The profiles of SmaI-digested genomic DNA of 19 isolates were distinguished into five different clusters by biased sinusoidal field gel electrophoresis. Four of them, consisting of 18 isolates, were suggested to be a clonal expansion. These findings suggested that a nosocomial outbreak of infections by CTX-M-2-producing P. mirabilis had occurred in our medical center. Most patients suffered from urogenital malignancies with long-term catheterization. Cefazolin, cefoperazone-sulbactam, and/or levofloxacin were mostly administered to the patients, but these agents seemed ineffective for eradication of CTX-M-2 producers. Early recognition and rapid identification of colonizing antimicrobial-resistant bacteria, including CTX-M-2-producing P. mirabilis, would be the most effective measures to cope with further spread of this kind of hazardous microorganism in clinical environments.

Aged↗

Tandem tetramer-based microsatellite fingerprinting for typing of Proteus mirabilis strains.

Two microsatellite tandem repeated tetramers, (GACA)(4) and (CAAT)(4), were used for Proteus mirabilis strain differentiation. The microsatellite-based PCR tests were applied for the examination of interstrain diversity for 87 P. mirabilis strains. Forty-six of the investigated strains were clinical isolates (5 were hospital isolates and 39 were outpatient clinic isolates); 42 strains were derived from the Kauffmann-Perch collection of laboratory strains. Fingerprinting done with the tetramers had a high discrimination ability [0.992 and 0.940 for (GACA)(4) and (CAAT)(4), respectively]. The distributions of clinical isolates among well-defined laboratory strains, determined by numerical analysis (unweighted pair-group method with arithmetic averages; Dice similarity coefficient), proved their genetic similarity to reference strains in the Kauffmann-Perch collection. This analysis also indicated that it is possible to estimate some phenotypic properties of P. mirabilis clinical isolates solely on the basis of microsatellite fingerprinting.

Bacterial Typing Techniques↗

Typing methods for Proteus rettgeri: comparison of biotype, antibiograms, serotype, and bacteriocin production.

Two hunderd five strains of Proteus rettgeri from epidemic and nonepidemic sources were differentiated by a new biotyping scheme, agglutination in O antisera, antimicrobial resistance patterns, and a new scheme based on bacteriocin production. The P. rettgeri were divided into 10 groups by their fermentation of lactose, sucrose, D-mannitol, and salicin. These groups were then subdivided into 19 biotypes by other biochemical reactions. Bacteriocin production was tested by the cross-streak method. Thirty-four bacteriocin-sensitive indicator strains were evaluated, and 16 were selected for the final scheme and used to type the 205 P. rettgeri, which were divided into 15 bacteriocin types. Serologically, 43% of the P. rettgeri were O42, 13% were untypable, 4% were O15, and 3% each were O33, O64, and O84 in addition to 31 remaining serotypes. Strains of P. rettgeri from known outbreaks contained fewer biotypes, O groups, and bacteriocin types and were more resistant to antimicrobial agents than endemic strains. Strains with common patterns with all four marker systems were frequently associated with outbreaks. A strong correlation between multiple antibiotic resistance and bacteriocin production was shown.

Anti-Bacterial Agents↗