Proteus appendicitidis sp. nov., isolated from the appendiceal pus of an appendicitis patient in Yongzhou, China.

A Gram-negative, facultatively anaerobic, short rod-shaped bacterium, designated as strain HZ0627T, was isolated from the appendiceal pus of a patient with appendicitis in Yongzhou, Hunan, China. This strain was subjected to comprehensive phenotypic, phylogenetic, and genomic analyses using polyphasic taxonomic methods. Phylogenetic analysis of the 16S rRNA gene sequence revealed that this strain belonged to the genus Proteus and the family Morganellaceae, whereas that based on the rpoB gene sequence and phylogenomic analysis demonstrated that this strain was distinctly separated from other type strains of Proteus species. Moreover, whole-genome-based analyses, including in silico DNA-DNA hybridization (isDDH) and average nucleotide identity (ANI), revealed that strain HZ0627T had much lower isDDH rates (24.5-55.6%) and ANI (82.04-93.90%) than those of the thresholds (i.e., 70% and 95%, respectively) for species delineation, when compared to the type strains of other Proteus species. The cellular fatty acid profile of strain HZ0627T was dominated by C16:0 (34.5%), cyclo C17:0 (25.8%), C14:0 (12.6%), C16:1 iso I/14:0 3-OH (7.7%), C18:1ω7c/18:1ω6c (6.5%), and C16:1ω7c/16:1ω6c (4.9%), which clearly differentiated it from the documented type strains of Proteus species. In addition, several specific physiological traits, including optimal growth temperature, tolerance to sodium chloride, and carbon source utilization, differed from those of other Proteus species. Therefore, we propose the name Proteus appendicitidis sp. nov. for strain HZ0627T (= CCTCC AB 2022380T = KCTC 92986T), which represents the type strain of this novel Proteus species.

Complicated pyelonephritis caused by Proteus alimentorum in a woman with peritoneal cancer: a case report.

BACKGROUND: Proteus spp. are widespread in the environment and comprise a part of the normal flora of the human gastrointestinal tract. Only six species in this genus, including Proteus mirabilis, Proteus vulgaris, Proteus terrae, Proteus penneri, Proteus hauseri, and Proteus faecis, have been isolated from human clinical specimens. However, there are no reports of Proteus alimentorum isolated from humans, and the clinical characteristics of P. alimentorum infection are unknown. CASE PRESENTATION: An 85-year-old female patient with peritoneal cancer was hospitalized for complicated pyelonephritis and bacteremia caused by P. alimentorum. The patient received antimicrobial therapy and was discharged on day 7 of hospitalization. No recurrence was observed 14 days after the treatment. Various methods were used to identify the Proteus sp. Furthermore, the VITEK-2 GN ID card resulted in low discrimination between P. hauseri and P. penneri. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry showed P. hauseri with a spectral score of 2.22 as the best match. Nevertheless, the pathogen was identified as P. alimentorum based on genetic investigation using 16 S rRNA gene sequencing and biochemical tests. CONCLUSION: Proteus alimentorum is a human pathogen, and its infection has an excellent therapeutic response to antimicrobials based on antimicrobial susceptibility. Genomic methods may be helpful for the precise identification of P. alimentorum.

Blowing epithelial cell bubbles with GumB: ShlA-family pore-forming toxins induce blebbing and rapid cellular death in corneal epithelial cells.

Medical devices, such as contact lenses, bring bacteria in direct contact with human cells. Consequences of these host-pathogen interactions include the alteration of mammalian cell surface architecture and induction of cellular death that renders tissues more susceptible to infection. Gram-negative bacteria known to induce cellular blebbing by mammalian cells, Pseudomonas and Vibrio species, do so through a type III secretion system-dependent mechanism. This study demonstrates that a subset of bacteria from the Enterobacteriaceae bacterial family induce cellular death and membrane blebs in a variety of cell types via a type V secretion-system dependent mechanism. Here, we report that ShlA-family cytolysins from Proteus mirabilis and Serratia marcescens were required to induce membrane blebbling and cell death. Blebbing and cellular death were blocked by an antioxidant and RIP-1 and MLKL inhibitors, implicating necroptosis in the observed phenotypes. Additional genetic studies determined that an IgaA family stress-response protein, GumB, was necessary to induce blebs. Data supported a model where GumB and shlBA are in a regulatory circuit through the Rcs stress response phosphorelay system required for bleb formation and pathogenesis in an invertebrate model of infection and proliferation in a phagocytic cell line. This study introduces GumB as a regulator of S. marcescens host-pathogen interactions and demonstrates a common type V secretion system-dependent mechanism by which bacteria elicit surface morphological changes on mammalian cells. This type V secretion-system mechanism likely contributes bacterial damage to the corneal epithelial layer, and enables access to deeper parts of the tissue that are more susceptible to infection.

Proteus alimentorum sp. nov., isolated from pork and lobster in Ma'anshan city, China.

Two strains of Gram-stain-negative, facultatively anaerobic short-rod bacteria were recovered from two different food samples in Ma'anshan city, Anhui province, China in 2008. The bacteria were characterized in a polyphasic taxonomic study that included phenotypic, phylogenetic and genotypic methodologies. Phylogenetic analysis of the 16S rRNA gene demonstrated that the two strains belonged to the genus Proteus and were most similar to Proteus vulgaris ATCC 29905T with a score of 99.7 %. Phylogenetic analysis of the rpoB gene placed the two strains into a cluster with a distinctly interspecies phylogenetic branch that was clearly separated from six type strains of the genus Proteus, with the most closely related species being Proteus mirabilis ATCC 29906T. In silico genomic comparisons, including in silico DNA-DNA hybridization (isDDH) and average nucleotide identity (ANI) analysis showed that the representative strain, 08MAS0041T, and all six Proteus species share less than 70 % isDDH and have a 95 % ANI cutoff level, supporting the designation of the two strains as a novel species of the genus Proteus. The predominant cellular fatty acids of strain 08MAS0041T were C16 : 0 (24.8 %), C16 : 1ω7c/16 : 1ω6c (16.5 %), C18 : 1ω6c/C18 : 1ω7c (14.5 %), C17 : 0 cyclo (12.6 %) and C16 : 1iso I/C14 : 0 3-OH (10.6 %). The analysis of biochemical, phylogenetic and genomic data confirmed that the two strains were clearly different from all recognized species of the genus Proteus and represent a novel Proteus species, for which the name Proteus alimentorum sp. nov. is proposed. The type strain is 08MAS0041T (=DSM 104685T=CGMCC 1.15939T).

The association between ethnicity and vaginal microbiota composition in Amsterdam, the Netherlands.

OBJECTIVE: To evaluate whether ethnicity is independently associated with vaginal microbiota (VMB) composition in women living in Amsterdam, the Netherlands, as has been shown for American women. METHODS: Women (18-34 years, non-pregnant, N = 610) representing the six largest ethnic groups (Dutch, African Surinamese, South-Asian Surinamese, Turkish, Moroccan, and Ghanaian) were sampled from the population-based HELIUS study. Sampling was performed irrespective of health status or healthcare seeking behavior. DNA was extracted from self-sampled vaginal swabs and sequenced by Illumina MiSeq (16S rRNA gene V3-V4 region). RESULTS: The overall prevalence of VMBs not dominated by lactobacilli was 38.5%: 32.2% had a VMB resembling bacterial vaginosis and another 6.2% had a VMB dominated by Bifidobacteriaceae (not including Gardnerella vaginalis), Corynebacterium, or pathobionts (streptococci, staphylococci, Proteus or Enterobacteriaceae). The most prevalent VMB in ethnically Dutch women was a Lactobacillus crispatus-dominated VMB, in African Surinamese and Ghanaian women a polybacterial G. vaginalis-containing VMB, and in the other ethnic groups a L. iners-dominated VMB. After adjustment for sociodemographic, behavioral and clinical factors, African Surinamese ethnicity (adjusted odds ratio (aOR) 5.1, 95% confidence interval (CI) 2.1-12.0) and Ghanaian ethnicity (aOR 4.8, 95% CI 1.8-12.6) were associated with having a polybacterial G. vaginalis-containing VMB, and African Surinamese ethnicity with a L. iners-dominated VMB (aOR 2.8, 95% CI 1.2-6.2). Shorter steady relationship duration, inconsistent condom use with casual partners, and not using hormonal contraception were also associated with having a polybacterial G. vaginalis-containing VMB, but human papillomavirus infection was not. Other sexually transmitted infections were uncommon. CONCLUSIONS: The overall prevalence of having a VMB not dominated by lactobacilli in this population-based cohort of women aged 18-34 years in Amsterdam was high (38.5%), and women of sub-Saharan African descent were significantly more likely to have a polybacterial G. vaginalis-containing VMB than Dutch women independent of modifiable behaviors.

Caracterización clínica y molecular de bacteriemias causadas por enterobacterias productoras de β -lactamasas de espectro extendido: 2004-2007 / Clinical and molecular characterization of ESBL-producing enterobacteria isolated from bacteremia in a university hospital

Introduction: Extended-spectrum-β-lactamases (ESBL) are plasmid-encoded enzymes that confer resistance to multiple antimicrobials. ESBL-producing enterobacteria that cause bacteremia limit therapeutic options and increase mortality. Objective: To perform a clinical and molecular description of bacteremia caused by ESBL-producing enterobacteria. Method: We retrospectively studied the cases of bacteremia due to ESBL-producing Escherichia coli, Klebsiella pneumoniae and Proteus spp in adults admitted to a university hospital during the years 2004-2007. We reviewed the clinical records and antimicrobial susceptibility patterns. Molecular typing was performed by polymerase chain reaction and study of clonality by pulsed-field electrophoresis. Results: We found a prevalence of 9.8 percent ESBL in enterobacteria causing bacteremia. Decreased susceptibility to quinolones and aminoglycosides was observed, without resistance to carbapenems. The predominant ESBL types were CTX-M (96 percent), TEM (62 percent) and GES (28 percent). 79 percent of the strains presented more than one type of ESBL. Clinical analysis revealed high prevalence of risk factors, previous use of antimicrobials and of invasive devices. There was no significant clonality. Conclusion: The presence of ESBLs in bloodstream infections is a clinical problem that must be considered when choosing empiric therapy.

Introducción: β-lactamasas de espectro extendido (BLEE) son enzimas plasmidiales que confieren resistencia a múltiples antimicrobianos. Las bacteriemias por enterobacterias productoras de BLEE restringen las opciones terapéuticas y aumentan la mortalidad. Objetivo: Realizar una descripción clínica y molecular de las bacteriemias causadas por enterobacterias productoras de BLEE. Método: Se estudiaron retrospectivamente los casos de bacteriemia por Escherichia coli, Klebsiella pneumoniae y Proteus spp. confirmadas para BLEE, en adultos ingresados en un hospital universitario durante los años 2004-2007. Se revisaron los registros clínicos y de susceptibilidad. Se realizó tipificación molecular por reacción de polimerasa en cadena y estudio de clonalidad por electroforesis de campo pulsado. Resultados: Se identificó una prevalencia de BLEE de 9,8 por ciento en enterobacterias causantes de bacteriemias. Se observó susceptibilidad disminuida a quinolonas y aminoglucósidos, sin resistencia a carbapenémicos. Los tipos de BLEE predominantes fueron CTX-M (96 por ciento), TEM (62 por ciento) y GES (28 por ciento). El 79 por ciento de las cepas presentó más de un tipo de BLEE. El análisis clínico reveló alta frecuencia de patologías de riesgo, uso previo de antimicrobianos y uso de dispositivos invasores. No se encontró clonalidad significativa. Conclusión: La presencia de BLEE en bacteriemias constituye un problema clínico que debe ser considerado al elegir la terapia empírica.

Rheumatoid arthritis is caused by Proteus: the molecular mimicry theory and Karl Popper.

Rheumatoid arthritis is a crippling and disabling joint disease affecting over 20 million people. It occurs predominantly in women and smokers, and affects the HLA-DR1/4 individuals who carry the "shared epitope" of amino acids EQRRAA. The cause of this disease was investigated by the methods of the philosopher of science Karl Popper who suggested that scientific research should be based on bold conjectures and critical refutations. The "Popper sequences" generate new facts which then change or alter the original problem. The new facts must then be explained by any new theory. Using the "molecular mimicry" model, it was found that Proteus bacteria possess an amino acid sequence ESRRAL in haemolysin which resembles the, shared epitope, and another sequence in urease which resembles type XI collagen. Antibodies to Proteus bacteria have been found in 14 different countries. It would appear that rheumatoid arthritis is caused by an upper urinary tract infection by Proteus bacteria. Anti-Proteus therapy should be assessed in the management of this disease separately or in conjunction with existing modalities of therapy.

Identification and functional characterisation of genes and corresponding enzymes involved in carnitine metabolism of Proteus sp.

Enzymes involved in carnitine metabolism of Proteus sp. are encoded by the cai genes organised as the caiTABCDEF operon. The complete operon could be sequenced from the genomic DNA of Proteus sp. Amino acid sequence similarities and/or enzymatic analysis confirmed the function assigned to each protein involved in carnitine metabolism. CaiT was suggested to be an integral membrane protein responsible for the transport of betaines. The caiA gene product was shown to be a crotonobetainyl-CoA reductase catalysing the irreversible reduction of crotonobetainyl-CoA to gamma-butyrobetainyl-CoA. CaiB and CaiD were identified to be the two components of the crotonobetaine hydrating system, already described. CaiB and caiD were cloned and expressed in Escherichia coli. After purification of both proteins, their individual enzymatic functions were solved. CaiB acts as betainyl-CoA transferase specific for carnitine, crotonobetaine, gamma-butyrobetaine and its CoA derivatives. Transferase reaction proceeds, following a sequential bisubstrate mechanism. CaiD was identified to be a crotonobetainyl-CoA hydratase belonging to the crotononase superfamily. Because of amino acid sequence similarities, CaiC was suggested to be a betainyl-CoA ligase. Taken together, these results show that the metabolism of carnitine and crotonobetaine in Proteus sp. proceeds at the CoA level.

Assay method for antitumor L-methionine gamma-lyase: comprehensive kinetic analysis of the complex reaction with L-methionine.

L-Methionine gamma-lyase (EC 4.4.1.11) is a pyridoxal 5'-phosphate-dependent multifunctional enzyme. Measuring the initial velocity of alpha-ketobutyrate production by alpha,gamma-elimination of L-methionine catalyzed by L-methionine gamma-lyase is not very feasible, because the enzyme simultaneously catalyzes both gamma-replacement and alpha,gamma-elimination. To develop an accurate enzyme assay, the comprehensive enzyme kinetics needed to be elucidated by progress curve analysis on the basis of a reaction model for conversion of L-methionine to alpha-ketobutyrate, methanethiol, and ammonia with pyridoxal 5'-phosphate as a cofactor. Kinetic parameters were determined by linear transformation using an approximation of a Maclaurin series from the whole velocity of alpha-ketobutyrate production including alpha,gamma-elimination and gamma-replacement. The significance of gamma-replacement was revealed both theoretically and practically by the kinetic analysis. The enzyme activity was standardized and represented as the Vmax value taking into consideration gamma-replacement in the presence of L-methionine at 37 degrees C and pH 8.0. The novel method that we proposed is accurate, sensitive, reproducible, and linear over a wide range for the determination of L-methionine gamma-lyase activity.

Epigenetic regulation of carnitine metabolising enzymes in Proteus sp. under aerobic conditions.

Proteus sp. is able to catalyse the reversible transformation of crotonobetaine into L(-)-carnitine during aerobic growth. Contrary to other Enterobacteriaceae no reduction of crotonobetaine into gamma-butyrobetaine could be detected in the culture supernatants. Activities of L(-)-carnitine dehydratase, carnitine racemasing system and crotonobetaine reductase could be determined enzymatically in cell-free extracts of Proteus sp. Small amounts of gamma-butyrobetaine were found in cell-free extracts, indicating that it accumulates in the cell and inhibits the crotonobetaine reductase. Crotonobetaine and L(-)-carnitine were able to induce enzymes of carnitine metabolism. gamma-Butyrobetaine and glucose repress carnitine metabolism in Proteus sp. Other betaines are neither inducers nor repressors. Monoclonal antibodies against purified CaiA from Escherichia coli O44K74 recognise an analogous protein in cell-free extract of Proteus sp. No cross-reactivity could be detected with monoclonal antibodies against purified CaiB and CaiD from E. coli O44K74.

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.

Interspecies compatibility of selenoprotein biosynthesis in Enterobacteriaceae.

Several species of Enterobacteriaceae were investigated for their ability to synthesize selenium-containing macromolecules. Seleniated tRNA species as well as seleniated polypeptides were formed by all organisms tested. Two selenopolypeptides could be identified in most of the organisms which correspond to the 80 kDa and 110 kDa subunits of the anaerobically induced formate dehydrogenase isoenzymes of E. coli. In those organisms possessing both isoenzymes, their synthesis was induced in a mutually exclusive manner dependent upon whether nitrate was present during anaerobic growth. The similarity of the 80 kDa selenopolypeptide among the different species was assessed by immunological and genetic analyses. Antibodies raised against the 80 kDa selenopolypeptide from E. coli cross-reacted with an 80 kDa polypeptide in those organisms which exhibited fermentative formate dehydrogenase activity. These organisms also contained genes which hybridised with the fdhF gene from E. coli. In an attempt to identify the signals responsible for incorporation of selenium into the selenopolypeptides in these organisms we cloned a portion of the fdhF gene homologue from Enterobacter aerogenes. The nucleotide sequence of the cloned 723 bp fragment was determined and it was shown to contain an in-frame TGA (stop) codon at the position corresponding to that present in the E. coli gene. This fragment was able to direct incorporation of selenocysteine when expressed in the heterologous host, E. coli. Moreover, the E. coli fdhF gene was expressed in Salmonella typhimurium, Serratia marcescens and Proteus mirabilis, indicating a high degree of conservation of the seleniating system throughout the enterobacteria.

Extracellular haemolysin of Proteus penneri coded by chromosomal hly genes is similar to the alpha-haemolysin of Escherichia coli.

Extracellular haemolysin of four Proteus penneri strains was characterized as a polypeptide of approximately 110 kD. The chromosomal DNAs of these strains cleaved with Hind III showed three fragments hybridizing with a DNA probe containing cloned haemolysin (hly) genes of Escherichia coli. The results presented here suggested that the haemolysin of P. penneri strains is chromosomally determined and similar to the alpha-haemolysin of E. coli.

Identification of two different hemolysin determinants in uropathogenic Proteus isolates.

DNA sequences similar to those of the Escherichia coli hemolysin genes were detected among uropathogenic isolates of Proteus vulgaris and Morganella morganii by using the Southern blotting technique and hly gene-specific DNA probe. Immunoblotting revealed that among the hemolytic P. vulgaris and M. morganii isolates there was expressed a polypeptide species similar in molecular size (110 kilodaltons) and antigenicity to Escherichia coli HlyA. A plasmid-mediated P. vulgaris hemolysin determinant identified by Southern blotting analysis was molecularly cloned, and the recombinant plasmid (pWPV100) was characterized by restriction endonuclease fragment mapping. A second recombinant library of genomic DNA prepared from a hemolytic, urinary tract isolate of Proteus mirabilis was constructed in E. coli. A 5.5-kilobase XhoI fragment encoding an extracellular hemolytic activity was molecularly cloned (pWPM100), and this plasmid was subjected to transposon-mediated mutagenesis with TnphoA. The P. mirabilis hemolytic phenotype was determined to be encoded by a polypeptide species (HpmA) with an estimated molecular size of 140 kilodaltons based on minicell polypeptide analysis of pWPM100 and its mutant derivatives. Southern blotting analysis with a HpmA-specific DNA probe revealed that this novel determinant is commonly found in both Proteus species but is not present in hemolytic isolates of M. morganii, E. coli, Citrobacter freundii, and Serratia marcescens.

Mode of action of morganocin 174.

Morganocin 174-induced lethality was characterized by one-hit kinetics. Although it induced simultaneous inhibition of deoxyribonucleic acid, ribonucleic acid, and protein syntheses, the most striking effect of morganocin was a rapid reduction of intracellular adenosine 5'-triphosphate to less than 10% of the initial values within 2 min of addition. Accumulation of thymidine, uridine, glutamine, and proline was also inhibited. A gradual efflux of K(+) was observed, but the lethal effects of morganocin 174 could not be prevented by maintaining a high K(+) or Mg(2+) concentration. The Mor174 plasmid codes for an immunity substance that protects cells against the effect of morganocin 174.