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Monoclonal antibody-mediated protection and neutralization of motility in experimental Proteus mirabilis infection.

A panel of monoclonal antibodies with specificity for a wound isolate of Proteus mirabilis was established. Of nine antibodies studied in detail, three were broadly reactive with various Proteus isolates, while six reacted in a serotype-specific fashion with the strain used for immunization. Five of the six serotype-specific antibodies were reactive with lipopolysaccharide. The sixth serotype-specific antibody, 4-F (immunoglobulin G1 [IgG1]), was potently protective in a burn wound sepsis model and recognized a protein antigen. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analysis were used to determine that 4-F was reactive with flagellar protein. Approximately 1.3 micrograms of the antibody was sufficient to provide protection against 8 50% lethal doses of wound isolate, and approximately 26 micrograms provided full protection against challenge with 333 50% lethal doses. In vitro test results indicated that 4-F inhibited the motility of the wound isolate, and in vivo testing showed that it inhibited dissemination of the inoculum from the burn site to the liver and spleen. Whereas the antibody was highly effective in preventing the death of mice subsequent to challenge at a burn site, no protection was seen following an intraperitoneal challenge. These results may therefore indicate that the protection observed in the burn model is solely a reflection of the capacity of 4-F to neutralize bacterial motility.

Animals↗

Proteinases of Proteus spp.: purification, properties, and detection in urine of infected patients.

The proteinases secreted by pathogenic strains of Proteus mirabilis, P. vulgaris biotype 2, P. vulgaris biotype 3, and P. penneri were purified with almost 100% recovery by affinity chromatography on phenyl-Sepharose followed by anion-exchange chromatography. The proteinase purified from the urinary tract pathogen P. mirabilis, which we had previously shown to degrade immunoglobulins A and G, appeared as a composite of a single band and a double band (53 and 50 kDa, respectively) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The other Proteus proteinases had similar patterns but slightly different mobilities. In each case all proteinase activity in culture supernatants was demonstrated by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be associated with only the triple-band complex; all three bands were proteolytically active. The P. mirabilis proteinase was resistant to inhibitors of both serine and thiol proteinases but strongly inhibited by metal chelators, although it was not affected by phosphoramidon, an inhibitor of the thermolysin group of bacterial metalloproteinases. Active proteinase was detected in urine samples from P. mirabilis-infected patients; this is consistent with our detection of immunoglobulin A fragments of a size suggestive of P. mirabilis proteinase activity.

Chromatography, Affinity↗

Pathological and therapeutic significance of cellular invasion by Proteus mirabilis in an enterocystoplasty infection stone model.

Proteus mirabilis infection often leads to stone formation. We evaluated how bacterium-mucin adhesion, invasion, and intracellular crystal formation are related to antibiotic sensitivity and may cause frequent stone formation in enterocystoplasties. Five intestinal (Caco-2, HT29, HT29-18N2, HT29-FU, and HT29-MTX) and one ureter cell line (SV-HUC-1) were incubated in artificial urine with five Proteus mirabilis strains. Fluorescence-activated cell sorting (FACS), laser scanning microscopy, and electron microscopy evaluated cellular adhesion and/or invasion, pathologic changes to mitochondria, and P. mirabilis-mucin colocalization (MUC2 and MUC5AC). An MTT (thiazolyl blue tetrazolium bromide) assay and FACS analysis of caspase-3 evaluated the cellular response. Infected cells were incubated with antibiotics at dosages representing the expected urinary concentrations in a 10-year-old, 30-kg child to evaluate bacterial invasion and survival. All cell lines showed colocalization of P. mirabilis with human colonic mucin (i.e., MUC2) and human gastric mucin (i.e., MUC5AC). The correlation between membrane mucin expression and invasion was significant and opposite for SV-HUC-1 and HT29-MTX. Microscopically, invasion by P. mirabilis with intracellular crystal formation and mitochondrial damage was found. Double membranes surrounded bacteria in intestinal cells. Relative resistance to cotrimoxazole and augmentin was found in the presence of epithelial cells. Ciprofloxacin and gentamicin remained effective. Membrane mucin expression was correlated with relative antibiotic resistance. Cell invasion by P. mirabilis and mucin- and cell type-related distribution and response differences indicate bacterial tropism that affects crystal formation and mucosal presence. Bacterial invasion seems to have cell type-dependent mechanisms and prolong bacterial survival in antibiotic therapy, giving a new target for therapeutic optimalization of antibiotic treatment.

Anti-Bacterial Agents↗

Development of an intranasal vaccine to prevent urinary tract infection by Proteus mirabilis.

Proteus mirabilis commonly infects the complicated urinary tract and is associated with urolithiasis. Stone formation is caused by bacterial urease, which hydrolyzes urea to ammonia, causing local pH to rise, and leads to the subsequent precipitation of magnesium ammonium phosphate (struvite) and calcium phosphate (apatite) crystals. To prevent these infections, we vaccinated CBA mice with formalin-killed bacteria or purified mannose-resistant, Proteus-like (MR/P) fimbriae, a surface antigen expressed by P. mirabilis during experimental urinary tract infection, via four routes of immunization: subcutaneous, intranasal, transurethral, and oral. We assessed the efficacy of vaccination using the CBA mouse model of ascending urinary tract infection. Subcutaneous or intranasal immunization with formalin-killed bacteria and intranasal or transurethral immunization with purified MR/P fimbriae significantly protected CBA mice from ascending urinary tract infection by P. mirabilis (P < 0.05). To investigate the potential of MrpH, the MR/P fimbrial tip adhesin, as a vaccine, the mature MrpH peptide (residues 23 to 275, excluding the signal peptide), and the N-terminal receptor-binding domain of MrpH (residues 23 to 157) were overexpressed as C-terminal fusions to maltose-binding protein (MBP) and purified on amylose resins. Intranasal immunization of CBA mice with MBP-MrpH (residues 23 to 157) conferred effective protection against urinary tract infection by P. mirabilis (P < 0.002).

Adhesins, Bacterial↗

Proteus mirabilis amino acid deaminase: cloning, nucleotide sequence, and characterization of aad.

Proteus, Providencia, and Morganella species produce deaminases that generate alpha-keto acids from amino acids. The alpha-keto acid products are detected by the formation of colored iron complexes, raising the possibility that the enzyme functions to secure iron for these species, which do not produce traditional siderophores. A gene encoding an amino acid deaminase of uropathogenic Proteus mirabilis was identified by screening a genomic library hosted in Escherichia coli DH5 alpha for amino acid deaminase activity. The deaminase gene, localized on a cosmid clone by subcloning and Tn5::751 mutagenesis, was subjected to nucleotide sequencing. A single open reading frame, designated aad (amino acid deaminase), which appears to be both necessary and sufficient for deaminase activity, predicts a 473-amino-acid polypeptide (51,151 Da) encoded within an area mapped by transposon mutagenesis. The predicted amino acid sequence of Aad did not share significant amino acid sequence similarity with any other polypeptide in the PIR or SwissProt database. Amino acid deaminase activity in both P. mirabilis and E. coli transformed with aad-encoding plasmids was not affected by medium iron concentration or expression of genes in multicopy in fur, cya, or crp E. coli backgrounds. Enzyme expression was negatively affected by growth with glucose or glycerol as the sole carbon source but was not consistent with catabolite repression.

Amino Acid Oxidoreductases↗

COMPARISON OF LIPIDS AND LIPOPOLYSACCHARIDE FROM THE BACILLARY AND L FORMS OF PROTEUS P18.

Nesbitt, J. A., III (The Johns Hopkins University School of Medicine, Baltimore, Md.), and W. J. Lennarz. Comparison of lipids and lipopolysaccharides from the bacillary and L forms of Proteus P18. J. Bacteriol. 89:1020-1025. 1965.-Comparative studies on the L form of Proteus P18 and the parent bacterium grown in a defined medium showed that the L form contained 1.5 times as much extractable lipid (dry weight) as the bacillary form. The composition of the lipids extractable by chloroform-methanol was quite similar in the two bacterial forms. The occurrence of myristate and beta-hydroxymyristate in the bound, nonextractable lipid was found to be a reflection of the presence of lipopolysaccharide (LPS) in each organism. The bacillary organism contains three to four times as much LPS as the L form. The LPS isolated from both organisms contains heptose, hexosamine, phosphorus, 3-deoxyoctulosonate, glucose, galactose, and fatty acids.

Bacillus↗

Development of specific tests for rapid detection of Escherichia coli and all species of Proteus in urine.

Proteus mirabilis was distinguishable from Escherichia coli and from several other species that may be associated with urinary tract infections when grown in a nutrient medium supplemented with 0.1 M L-methionine by the formation of large amounts of dimethyl disulfide and methyl mercaptan, which were detected by head-space gas-liquid chromatography (HS-GLC). E. coli could be detected by the same HS-GLC technique by ethanol production from methionine peptone water enriched with 1% either lactose or arabinose but not by any product from 10 amino acids tested. Ethanol from lactose was detected early in the exponential phase of growth. Significant numbers, 10(5) or more per ml, of E. coli in urine could be detected in about 5 h by ethanol production from an unshaken culture of urine in lactose methionine peptone water buffered at pH 7.2 (urine test medium); only a trace of dimethyl disulfide was produced. Significant numbers of P. mirabilis in urine could be detected in 4 h by dimethyl disulfide production and in 5 h by methyl mercaptan production from a shaken culture of urine in urine test medium; no ethanol was produced. Incubation of urine specimens in the test medium followed by examination by HS-GLC is proposed as a rapid method of detecting whether or not the urine contains significant numbers of E. coli or a species of Proteus.

Bacteriological Techniques↗

Circumcision and periurethral carriage of Proteus mirabilis in boys.

Swabs were taken for culture from the periurethral area and urethral meatus in 124 uncircumcised and 60 circumcised boys. Proteus mirabilis was grown from 28 (22.6%) swabs from uncircumcised boys and from only one (1.7%) swab from circumcised boys. This supports the idea that the prepuce may be the source of proteus urinary tract infection.

Adolescent↗

ANTIBIOTIC SENSITIVITY OF PROTEUS SPECIES.

A study has been made of the antibiotic sensitivity pattern of 96 strains of Proteus isolated from clinical material and a further 29 strains kindly supplied by Dr. Patricia Carpenter. The results have been analysed in relation to the different species. The effect of electrolytes on the penicillin sensitivity of Proteus species has also been examined.

Ammonia↗

Serotyping and the Dienes reaction on Proteus mirabilis from hospital infections.

The serotype of 320 strains of Proteus mirabilis from clinical material was determined. Using 20 O antisera and four H antisera 61% of strains could be fully identified and 90% partially identified. A large number of serotypes were recognized but no difference was found between the serotype of organisms infecting the urinary tract and those from other infections. Biochemically identical organisms found in the same ward generally differed in serology. Proteus mirabilis was isolated from the faeces of 84.5% of 84 patients with urinary infection and from none of 20 normal controls. By serology and the Dienes test 61% of the organisms isolated from the urine and faeces of a single patient were identical, indicating that infection arose from the intestine. Most groups of serologically identical strains could, by the Dienes test, be further divided into a number of subtypes indicating that the strains were different and that cross infection had not been responsible for their spread. With three serological groups, however, the majority of strains belonged to a single Dienes type and it was concluded that these organisms had been spread from a common reservoir or carrier. Because of the unreliability of the Dienes test when carried out on random organisms it is suggested that reliable results can only be obtained by combining the Dienes test with serotyping.

Antigens↗

Bacteriocine typing of Proteus.

A method of typing isolates of Proteus mirabilis and Proteus vulgaris is described based on the sensitivity of the organisms to bacteriocine. Twelve standard proteocine producing strains were selected from a large number of isolates tested, and from these liquid proteocine preparations were prepared. The sensitivities of 1805 isolates to these 12 preparations were then determined and none was found to be untypable. From the results a bacteriocine typing system has been developed.

Bacteriocins↗

An experimental model for ascending acute pyelonephritis caused by Escherichia coli or proteus in rats.

Experimental, ascending acute pyelonephritis in rats was produced by injecting 0 x 5 ml of 10(9) bacteria/ml into the urinary bladder via the urethra. No traumatic manipulation of the ureters of kidneys was necessary. A grading system for kidney lesions based on macro- and microscopical examination was used. The capacity of different Escherichia coli and proteus strains to induce acute pyelonephritis was tested, and the E. coli 06K13H1 strain and the Proteus mirabilis 03H1 strain were especially capable of causing urinary tract infection. For the P. mirabilis 03H1 strain, a dominance of right kidney lesions was noted in contrast to the E. coli 06K13H1 strain which did not show any side preference.

Animals↗

Pneumatocele formation in a patient with Proteus mirabilis pneumonia.

A rare case of pulmonary pneumatoceles complicating the course of Proteus pneumonia in an adult is described. The pneumatoceles which appeared early in the course of the pneumonia resolved completely without complications. A brief review of the literature regarding formation of cavities in Proteus pneumonia is given.

Female↗

Siderophore production by Proteus mirabilis.

Studies on the isolation and characterization of Proteus mirabilis siderophores provided no evidence that these bacteria synthesize catechol- or hydroxamate-type siderophores. However, gas chromatograph analysis in conjunction with mass spectroscopy revealed the presence of alpha-hydroxyisovaleric acid, a previously unknown metabolite. Additional substantiating evidence for the presence of alpha-hydroxyisovaleric acid in these bacteria was obtained from experiments involving the use of thin-layer chromatography and an ultraviolet absorption spectrum. This compound was found to be capable of removing iron from the synthetic chelator, ethylene-diamine-di-orthohydroxyphenyl acetic acid, and supplying that iron to the bacteria both in a solid agar medium and in a liquid medium. Proteus mirabilis was found to possess an enzyme capable of catalyzing the reaction by which alpha-hydroxyisovaleric acid is converted to alpha-ketoisovaleric acid, an intermediate in the valine biosynthetic pathway.

Animals↗

[Proteus penneri].

Proteus penneri, formerly P. vulgaris biogroup 1, was recognized as a new species in 1982. This species is associated with clinical processes similar to those involving P. mirabilis and P. vulgaris and expresses similar pathogenic determinants. In clinical samples, P. penneri is mainly isolated from urine (50%), wound and soft tissue exudates (25%), and blood cultures (15%), mostly of nosocomial origin. Although P. penneri is easy to identify, it can be misidentified as P. vulgaris by automatic systems that do not include the indol test result in the identification process. This species has a characteristic susceptibility profile, essentially due to the production of the chromosomal inducible beta-lactamase HugA, which presents a high homology (86%) with CumA from P. vulgaris. HugA is inhibited by clavulanic acid and determines resistance to aminopenicillins and first- and second-generation cephalosporins, including cefuroxime, but does not affect cephamycins or carbapenems, and is inhibited by clavulanic acid. HugA is derepressed due to mutational processes in gene regulators, affecting the activity of cefotaxime and, to a much lesser extent, that of ceftazidime and aztreonam. This phenotype resembles the production of an extended spectrum beta-lactamase. Like other Proteus species, P. penneri is resistant to tetracyclines and should be considered resistant to nitrofurantoin.

Drug Resistance, Microbial↗

Evaluation of a method using Proteus mirabilis and Pseudomonas aeruginosa to experimentally induce dual infection of the urinary bladder in dogs.

OBJECTIVE: To evaluate a method using Proteus mirabilis and Pseudomonas aeruginosa to experimentally induce dual infection of the urinary bladder in dogs. ANIMALS: 6 healthy mixed-breed dogs. PROCEDURE: Dogs were anesthetized, and cystitis was induced by infusing a solution of salicylic acid in ethanol into the bladder, followed by an inoculum containing field isolates of P. mirabilis and P. aeruginosa. Dogs were examined daily for 21 days after induction of cystitis. On day 21, dogs were euthanatized, and urinary bladder, renal pelvis, and prostate specimens were submitted for bacterial culture. RESULTS: After induction of cystitis, all dogs had evidence of thickening of the bladder wall, dysuria, tenesmus, and hematuria. Urinalysis revealed proteinuria, hematuria, and pyuria. All urine samples obtained on day 21 yielded growth of P. mirabilis, but P. aeruginosa was not cultured from any of these samples. Proteus mirabilis was isolated from bladder, renal pelvis, or prostate specimens from 4 dogs; P. aeruginosa was not isolated from any of the tissue specimens. CONCLUSION: Results suggest that the method used in the present study fails to induce dual infection of the urinary bladder with P. mirabilis and P. aeruginosa. The inability to establish a persistent dual infection with this method may have been a result of insufficient pathogenicity of the Pseudomonas isolate or an inadequacy of the experimental design.

Animals↗