A Core Genome Multilocus Sequence Typing Scheme for Proteus mirabilis / 生物医学与环境科学（英文）

OBJECTIVE@#A core genome multilocus sequence typing (cgMLST) scheme to genotype and identify potential risk clonal groups (CGs) in Proteus mirabilis.@*METHODS@#In this work, we propose a publicly available cgMLST scheme for P. mirabilis using chewBBACA. In total 72 complete P. mirabilis genomes, representing the diversity of this species, were used to set up a cgMLST scheme targeting 1,842 genes, 635 unfinished (contig, chromosome, and scaffold) genomes were used for its validation.@*RESULTS@#We identified a total of 205 CGs from 695 P. mirabilis strains with regional distribution characteristics. Of these, 159 unique CGs were distributed in 16 countries. CG20 and CG3 carried large numbers of shared and unique antibiotic resistance genes. Nine virulence genes ( papC, papD, papE, papF, papG, papH, papI, papJ, and papK) related to the P fimbrial operon that cause severe urinary tract infections were only found in CG20. These CGs require attention due to potential risks.@*CONCLUSION@#This research innovatively performs high-resolution molecular typing of P. mirabilis using whole-genome sequencing technology combined with a bioinformatics pipeline (chewBBACA). We found that the CGs of P. mirabilis showed regional distribution differences. We expect that our research will contribute to the establishment of cgMLST for P. mirabilis.

Construction and biological characterization of a Proteus mirabilis strain with modABC gene deletion / 南方医科大学学报

OBJECTIVE@#To construct a modABC gene knockout strain of Proteus mirabilis and explore the effect of modABC gene deletion on biological characteristics of Proteus mirabilis.@*METHODS@#Fusion PCR was used to obtain the fusion gene of modABC and the kanamycin-resistant gene Kn, which was ligated with the suicide vector pCVD442 and transduced into Proteus mirabilis. The modABC gene knockout strain of Proteus mirabilis was obtained after homologous recombination with the suicide vector. PCR and Sanger sequencing were used to identify genomic deletion of modABC gene in the genetically modified strain. The concentration of molybdate in the wild-type and gene knockout strains was determined using inductively coupled plasma mass spectrometry (ICP-MS), and their survival ability in LB medium was compared under both aerobic and anaerobic conditions.@*RESULTS@#PCR and sanger sequencing confirmed genomic deletion of modABC gene in the obtained Proteus mirabilis strain. The concentration of intracellular molybdenum in the modABC gene knockout strain was 1.22 mg/kg, significantly lower than that in the wild-type strain (1.46 mg/kg, P < 0.001). Under the aerobic condition, the modABC gene knockout strain grown in LB medium showed no significant changes in survival ability compared with the wild-type strain, but its proliferation rate decreased significantly under the anaerobic condition and also when cultured in nitrate-containing LB medium under anaerobic condition.@*CONCLUSION@#Homologous recombination with the suicide vector can be used for modABC gene knockout in Proteus mirabilis. modABC gene participates in molybdate uptake and is associated with anaerobic growth of Proteus mirabilis in the presence of nitrate.

First report of bla GES-1 in Proteus mirabilis clinical isolates

Abstract Proteus mirabilis is one of the main pathogens causing urinary tract infections and sepsis. To our knowledge, this is the first report of a P. mirabilis hosting bla GES. The presence of these genes was determined using PCR and sequencing. We identified the presence of bla GES-1 in all three isolates. In addition, we identified the bla KPC-2 and bla NDM-1 genes in the two strains. These data emphasize the importance of monitoring and surveillance of all enterobacteria. The circulation of P. mirabilis strains carrying bla GES-1 constitutes a new scenario of resistance in this species and should be an epidemiological alert for global health.

Perfil de sensibilidad antimicrobiana de microorganismos causantes de infecciones urinarias adquiridas en la comunidad en pacientes con diabetes mellitus en Colombia / Antimicrobial susceptibility profile in urinary pathogens causing community-acquired infections in diabetic patients in Colombia

Resumen Introducción. La infección de las vías urinarias es la más frecuente en pacientes diabéticos, y es un factor determinante de la morbilidad y la mortalidad en este grupo de pacientes. El aumento de la resistencia de los microorganismos adquiridos en la comunidad a los antibióticos comúnmente utilizados para combatirla es alarmante. Objetivo. Determinar el perfil de sensibilidad a los antibióticos de los microorganismos responsables de infecciones urinarias adquiridas en la comunidad en pacientes diabéticos atendidos en algunos hospitales de Colombia. Materiales y métodos. Se hizo un estudio descriptivo de un subgrupo de pacientes diabéticos en el marco de una investigación en adultos con infección de origen comunitario de las vías urinarias. Durante un año, se recolectaron aislamientos de Escherichia coli, Klebsiella spp. y Proteus mirabilis en nueve hospitales de Colombia y se determinó su perfil de sensibilidad mediante métodos microbiológicos y moleculares, para establecer la presencia de betalactamasas de espectro extendido del tipo AmpC y de carbapenemasas del tipo KPC. Resultados. Se recolectaron 68 aislamientos (58 de E. coli, nueve de Klebsiella spp. y uno de P. mirabilis). Cuatro (6,9 %) de los aislamientos de E. coli expresaron dichas betalactamasas, en dos (3,4 %) de ellos, pertenecientes al grupo filogenético B2 y al clon ST131, se detectaron las betalactamasas TEM-1 y CTM-X-15. En otros cuatro (6,9 %) aislamientos de E. coli se encontró el fenotipo AmpC, y en tres de ellos se produjeron las betalactamasas TEM-1 y CMY-2. Un aislamiento de K. pneumoniae expresó la carbapenemasa KPC-3. Conclusión. Se confirmó la presencia de cepas productoras de betalactamasas de espectro extendido y carbapenemasas en microorganismos responsables de infección urinaria adquirida en la comunidad en pacientes diabéticos.

Abstract Introduction: Urinary tract infection is the most common pathology in diabetic patients, and an important determinant of morbidity and mortality among them. The increasing resistance of uropathogens acquired in the community to commonly used antibiotics is alarming. Objective: To identify the profile of antibiotic susceptibility of uropathogens responsible for community-acquired infections among diabetic patients in hospitals in Colombia. Materials and methods: We conducted a descriptive study in a subgroup of diabetic patients in the framework of a larger study in adults with urinary tract infection acquired in the community. Over one year, we collected Escherichia coli, Klebsiella spp. and Proteus mirabilis isolates from nine hospitals in Colombia. Their susceptibility profile was determined using microbiological and molecular methods to establish the presence of extended-spectrum AmpC betalactamases and KPC carbapenemases. Results: We collected 68 isolates (58 E. coli, nineKlebsiella spp. and oneProteus mirabilis). Four (6.9%) of the E. coli isolates expressed extended spectrum betalactamases,two (3.4%) of thembelonged to the phylogenetic group B2 andto ST131 clone and expressed the TEM-1 and CTM-X-15 betalactamases. The AmpC phenotype was found in four(6.9%) of the E. coli isolates, three of which producedTEM-1 and CMY-2 betalactamases. One K. pneumoniaeisolate expressed the KPC-3 carbapenemase. Conclusion: The presence of extended spectrum betalactamases and carbapenemases in uropathogens responsible for community-acquired infection was confirmed in diabetic patients.

Resistencia a cefalosporinas de espectro extendido en enterobacterias sin AmpC inducible: Evaluación de los nuevos puntos de corte / Resistance to extended-spectrum cephalosporins in non-inducible AmpC enterobacteria: Evaluation of the new MIC breakpoints

Los objetivos de este estudio fueron determinar la actividad in vitro de las cefalosporinas de espectro extendido frente a aislamientos clínicos de enterobacterias sin AmpC inducible y evaluar la utilidad de las normativas propuestas por el CLSI 2009 y de los puntos de corte recomendados por el CLSI 2010 y el EUCAST 2010. El análisis incluye la caracterización feno y genotípica de los mecanismos de resistencia. En todos los aislamientos se realizó un antibiograma semicuantitativo y se determinó la CIM por dilución en agar. Asimismo, se realizó la detección fenotípica de p-lactamasas de espectro extendido (BLEE), de AmpC plasmídica (AmpCp) y de carbapenemasas de tipo KPC. En los aislamientos que fueron resistentes a las cefalosporinas de espectro extendido (CEE) se evaluó, mediante PCR múltiple para b/aSHV y b/aCTX-M y PCR con cebadores específicos, el tipo de p-lactamasa pre-valente y la presencia de KPC. Se recuperaron de pacientes 169 aislamientos resistentes a CEE: 95 de K/ebsie//a pneumoniae, 55 de Escherichia co/i y 19 de Proteus mirabi/is. La resistencia a CEE se verificó en el 56,2 %; 32,6 % y 11,2 % de estos conjuntos de aislamientos, respectivamente. Se detectó el fenotipo BLEE en 152 aislamientos (90 %), el fenotipo AmpCp en 12 (7 %) y el KPC en 5 (3 %). Las recomendaciones del CLSI 2009 y los puntos de corte del CLSI 2010 y del EUCAST 2010 para la ceftriaxona permitieron detectar eficientemente las BLEE, mientras que para la ceftacidima, con los puntos de corte del CLSI 2010 solo se detectó el 55 % de las BLEE. Esta discrepancia en los porcentajes de resistencia a ceftriaxona y a ceftacidima se relaciona con la presencia de CTX-M en nuestro medio. Los nuevos puntos de corte detectaron con mayor eficiencia las enzimas de tipo AmpCp.

The aims of this study were to evaluate the in vitro activity of extended-spectrum cephalosporins (ESC) in non-inducible AmpC enterobacteria throµgh phenotypic and genotypic characterization of the mechanisms of resistance (ESBL, plasmid-mediated AmpC and KPC) and to evaluate the interpretation criteria proposed by the existing recommendations and the new breakpoints established by the CLSI and the EUCAST. Susceptibility tests and PCR multiplex for b/aSHV and b/aCTX-M and amplification using specific primers was performed. One hundred sixty nine resistant isolates: K/ebsie//a pneumoniae (95), Escherichia co/i (55), and Proteus mirabi/is (19) were recovered. ESC resistance was 56.2 %, 32.6%, and 11.2 %, respectively. ESBL was detected in 152 (90 %) isolates, plasmid-mediated AmpC in 12 (7 %) and KPC in 5 (3 %). The CLSI 2009 recommendations and the breakpoints sµggested by the CLSI 2010 and the EUCAST for ceftriaxone were efficacious to detect ESBL, whereas the different breakpoints for ceftazidime presented discrepancies. The CLSI 2010 breakpoints only detected 55 % of the ESBL-producing isolates due to the endemic presence of CTX-M ESBLs in our country. Regarding the plasmid-mediated AmpC producers, the recommendations of the CLSI 2010 and the EUCAST 2010 proved to be more efficient than the old ones.

Comparison of 3 Phenotypic-detection Methods for Identifying Plasmid-mediated AmpC beta-lactamase-producing Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis Strains / 대한진단검사의학회지

BACKGROUND: Plasmid-mediated AmpC beta-lactamases (PABLs) have been detected in the strains of Escherichia coli, Klebsiella spp., Proteus mirabilis, and Salmonella spp. PABLs may be difficult to detect and might interfere in the therapeutic and infection-control processes. Although several PABL-detection methods based on phenotypes have been reported, the Clinical and Laboratory Standards Institute currently does not recommend a routine detection method for PABLs. The aim of this study is to compare the performances of 3 phenotypic PABL detection methods. METHODS: Total 276 non-duplicated clinical isolates of E. coli (N=97), K. pneumoniae (N=136), and P. mirabilis (N=43) were collected from 14 hospitals in Korea between April and June 2007 in a non-consecutive and non-random manner. Multiplex PCR was performed to detect the PABL genes. Further, 3 phenotypic detection methods-cephamycin-Hodge test, Tris-EDTA (TE) disk test, and combination-disk test with 3-aminophenylboronic acid (BA)-were performed using cefoxitin and cefotetan disks. RESULTS: PABL genes were detected by multiplex PCR in 122/276 isolates, including 14/97 E. coli, 105/136 K. pneumoniae, and 3/43 P. mirabilis isolates. The combination-disk test with BA showed higher sensitivity (98.4%), specificity (92.2%), and efficiency (96.3%) than the cephamycin-Hodge (76.2%, 96.1%, and 88.6%, respectively) and the TE-disk (80.3%, 91.6%, and 87.9%, respectively) tests. CONCLUSIONS: The combination-disk test with BA is a simple, efficient, and interpretable test that can be applicable in clinical laboratories involved in the detection of PABLs in clinical isolates of E. coli, K. pneumoniae, and P. mirabilis.

Comparison of PCR-based molecular markers for the characterization of Proteus mirabilis clinical isolates

Proteus mirabilis is one of the most important pathogens associated with complicated urinary tract infections (acute pyelonephritis, bladder infections, kidney stones) and bacteremia, affecting patients with anatomical abnormalities, immunodeficiency, and long-term urinary catheterization. For epidemiological purposes, various molecular typing methods, such as pulse-field gel electrophoresis (PFGE) or ribotyping, have been developed for this pathogen. However, these methods are labor intensive and time-consuming. We evaluated the discriminatory power of several PCR-based fingerprinting methods (RAPD, ISSR, ERIC-PCR, BOX-PCR and rep-PCR) for P. mirabilis clinical isolates. Typing patterns and clustering analysis indicated that RAPD, BOX-PCR and ERIC-PCR differentiated P. mirabilis strains from Escherichia coli, Hafnia alvei, and Morganella morganii. With the exception of rep-PCR, the methods gave medium to high discriminatory efficiency in P. mirabilis. In general, the results obtained with RAPD, BOX-PCR and ERIC-PCR were in good agreement. We concluded that a combination of ERIC-PCR and BOX-PCR results is a rapid and reliable alternative for discrimination among P. mirabilis clinical isolates, contributing to epidemiological studies.

Clonal study of avian Escherichia coli strains by fliC conserved-DNA-sequence regions analysis / Estudo clonal de Escherichia coli aviário por análise de seqüências de DNA conservadas do gene fliC

The clonal relationship among avian Escherichia coli strains and their genetic proximity with human pathogenic E. coli, Salmonela enterica, Yersinia enterocolitica and Proteus mirabilis, was determined by the DNA sequencing of the conserved 5' and 3'regions fliC gene (flagellin encoded gene). Among 30 commensal avian E. coli strains and 49 pathogenic avian E. coli strains (APEC), 24 commensal and 39 APEC strains harbored fliC gene with fragments size varying from 670bp to 1,900bp. The comparative analysis of these regions allowed the construction of a dendrogram of similarity possessing two main clusters: one compounded mainly by APEC strains and by H-antigens from human E. coli, and another one compounded by commensal avian E. coli strains, S. enterica, and by other H-antigens from human E. coli. Overall, this work demonstrated that fliC conserved regions may be associated with pathogenic clones of APEC strains, and also shows a great similarity among APEC and H-antigens of E. coli strains isolated from humans. These data, can add evidence that APEC strains can exhibit a zoonotic risk.(AU)

A relação clonal entre linhagens de Escherichia coli de origem aviária e sua proximidade genética com E. coli patogênica para humanos, Salmonella enterica, Yersinia enterocolitica e Proteus mirabilis foi determinada através da utilização das seqüências conservadas 5' e 3' do gene fliC (responsável pela codificação da flagelina). Entre as 30 linhagens comensais de E. coli aviária e as 49 linhagens patogênicas de E. coli para aves (APEC), 24 linhagens comensais e 39 APEC apresentaram o gene fliC, que foi encontrado em tamanhos que variam de 670pb a 1900pb. Um dendrograma representando similaridade genética foi obtido a partir do seqüenciamento das regiões 5' e 3' conservadas do gene fliC das linhagens de E. coli de origem aviária, das seqüências dos antígenos H de E. coli de origem humana, de S. enterica, Y. enterocolitica e de P. mirabilis. A análise do dendrograma demonstrou que este apresenta dois grupos principais: um composto principalmente por isolados APEC e por antígenos H de E. coli de origem humana e outro formado por isolados comensais de E. coli aviária, S. enterica e por antígenos H de E. coli. No geral, o presente trabalho demonstrou que as regiões conservadas do gene fliC podem estar associadas à diferenciação clonal de linhagens de E. coli aviária, e que existe uma grande similaridade genética entre estas linhagens e antígenos H de E. coli humana. Estes dados podem adicionar evidências de que linhagens APEC podem apresentar riscos zoonóticos.(AU)

Occurrence of Proteus mirabilis associated with two species of venezuelan oysters

The fecal contamination of raw seafood by indicators and opportunistic pathogenic microorganisms represents a public health concern. The objective of this study was to investigate the presence of enteric bacteria colonizing oysters collected from a Venezuelan touristic area. Oyster samples were collected at the northwestern coast of Venezuela and local salinity, pH, temperature, and dissolved oxygen of seawater were recorded. Total and fecal coliforms were measured for the assessment of the microbiological quality of water and oysters, using the Multiple Tube Fermentation technique. Analyses were made using cultures and 16S rRNA gene sequencing. Diverse enrichment and selective culture methods were used to isolate enteric bacteria. We obtained pure cultures of Gram-negative straight rods with fimbriae from Isognomon alatus and Crassostrea rhizophorae. Our results show that P. mirabilis was predominant under our culture conditions. We confirmed the identity of the cultures by biochemical tests, 16S rRNA gene sequencing, and data analysis. Other enterobacteria such as Escherichia coli, Morganella morganii and Klebsiella pneumoniae were also isolated from seawater and oysters. The presence of pathogenic bacteria in oysters could have serious epidemiological implications and a potential human health risk associated with consumption of raw seafood.

A contaminação fecal de frutos do mar crus por microrganismos oportunistas patogênicos representa problema de saúde pública. O objetivo deste estudo é investigar a presença de bactérias entéricas que colonizam ostras coletadas em área turística da Venezuela. Amostras de ostras foram coletadas na costa noroeste da Venezuela e foram registrados a salinidade local, pH, temperatura e o oxigênio dissolvido na água do mar. O total de coliformes fecais foi medido para a avaliação da qualidade microbiológica da água e das ostras, usando a técnica de fermentação em tubos múltiplos. Análises foram feitas usando culturas e seqüência do gene 16S rRNA. Enriquecimento diversificado e métodos de cultura seletivos foram usados para isolar a bactéria entérica. Obtivemos culturas puras de bastões retos Gram negativos com fímbrias de Isognomon alatus e Crassostrea rhizophorae. Nossos resultados mostram que P. mirabilis foi predominante nas nossas condições de cultura. Confirmamos a identidade das culturas por testes bioquímicos, seqüência do gene 16rRNA e a análise de dados. Outras enterobactérias como Escherichia coli, Morganella morganii e Klebsiella pneumoniae foram também isoladas da água do mar e ostras. A presença de bactérias patogênicas em ostras podem ter implicações epidemiológicas e potencial risco para a saúde humana quando do consumo de frutos do mar crus.

Development of an operational substrate for ZapA, a metalloprotease secreted by the bacterium Proteus mirabilis

The protease ZapA, secreted by Proteus mirabilis, has been considered to be a virulence factor of this opportunistic bacterium. The control of its expression requires the use of an appropriate methodology, which until now has not been developed. The present study focused on the replacement of azocasein with fluorogenic substrates, and on the definition of enzyme specificity. Eight fluorogenic substrates were tested, and the peptide Abz-Ala-Phe-Arg-Ser-Ala-Ala-Gln-EDDnp was found to be the most convenient for use as an operational substrate for ZapA. A single peptide bond (Arg-Ser) was cleaved with a Km of 4.6 µM, a k cat of 1.73 s-1, and a catalytic efficiency of 376 (mM s)-1. Another good substrate for ZapA was peptide 6 (Abz-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Gln-EDDnp) which was cleaved at a single bond (Phe-Ser) with a Km of 13.6 µM, a k cat of 3.96 s-1 and a catalytic efficiency of 291 (mM s)-1. The properties of the amino acids flanking the scissile bonds were also evaluated, and no clear requirement for the amino acid residue at P1 was found, although the enzyme seems to have a preference for a hydrophobic residue at P2.

High frequency of auxotrophy in clinical isolates of Proteus mirabilis harboring an R plasmid

We report a novel phenomenon of high genetic instability, related to auxotrophy, in strains of Proteus mirabilis. Among P. mirabilis strains harboring the R plasmid Kept in our laboratory collection, and some freshly isolated strians from clinical material, 54 per cent of the samples presented auxotrophy at frequencies higher than 10(-3). Prototrophic closes gave rise to auxotrophic ones at frequencies not explainable by the usual mutation mechanisms. The instability mainly affected the carbamoyl phosphate synthetase gene (car), which leads to a double requirement for arginine and uracil for growth in minimal medium. Other genes were also affected, at a lower frequency. The car mutation does not revert to prototrophy. A similar phenomenon of instability was induced in Escherichia coli strain HB 101 upon introduction of a drug-resistance plasmid from P. mirabilis. We have ruled out the hypothesis of a transposon in the generation of auxotrophy.

A review of protease secretion instability in Proteus mirabilis

Proteus mirabilis secreta uma única protease que é uma metalo-enzima alcalina. Descrevemos aqui alguns fatores fisiológicos que afetam a secreçäo de protease em clones isolados. Mais de cem diferentes isolados clínicos foram examinados quanto a capacidade de secretar protease. Verificou-se que algumas linhagens herdam o determinante de protease como um caráter estável (säo denominadas linhagens estáveis); outras linhagens exigem um fenótipo muito instável (denominadas linhagens instáveis) com 10% das colônias-filhas perdendo o caráter de produçäo de protease. Nunca foi observada a reversäo do fenótipo protease negativa. Por outro lado, a introduçäo de fatores R em células produtoras de protease reduziu a atividade proteásica em cerca de 50%, o que sugere uma interferência na expressäo e/ou secreçäo de protease. Embora haja evidências de que um locus da protease tenha localizaçäo extracromossômica, näo foi demosntrada a presença de plasmídios nas linhagens instáveis. Um modo que pode explicar os vários dados é apresentado

Behavior of transposon Tn1 IN Proteus mirabilis

A freqüência de transposiçäo do Tn1 do plasmídio pTH10 para o cromossomo da célula hospedeira foi maior em Proteus mirabilis que em Escherichia coli. Contudo pode-se supor que os valores obtidos em P. mirabilis foram subestimados desde que o plamídio doador do transposoma é instavél na linhagem PG1342. Experimentos de hibridaçäo sugerem que existem mais do que uma inserçäo por cromossomo de P. mirabilis, mas análises adicionais säo necessárias para a localizaçäo dessas inserçöes. Se o transposon T n1 se insere em um ponto quente do cromossomo da célula hospedeira, esse alvo näo causou auxotrofia em nenhum dos clones testados de PG1342::Tn1. Transposiçöes entre plasmídios albergados por P. mirabilis parecem näo ocorrer em níveis maiores do que para o cromossomo, confrome relatados para E. coli. Outrossim, as freqüências de inserçäo do Tn1 variam de acordo com o DNA alvo. A seleçäo de um sítio de inserçäo ocorre diferentemente nos dois gêneros, sugerindo que fatores dos hospedeiros têm uma participaçäo importante nessa seleçäo. Por outro lado, Tn1 mostrou inserçäo preferencial em outrom transposon (Tn402), independentemente do hospedeiro. Neste caso a inserçäo do Tn1 näo é estável, levando à excisäo do transposon ampicilina