Presence and Role of the Type 3 Fimbria in the Adherence Capacity of Enterobacter hormaechei subsp. hoffmannii.

Enterobacter hormaechei, one of the species within the Enterobacter cloacae complex, is a relevant agent of healthcare-associated infections. In addition, it has gained relevance because isolates have shown the capacity to resist several antibiotics, particularly carbapenems. However, knowledge regarding colonization and virulence mechanisms of E. hormaechei has not progressed to the same extent as other Enterobacteriaceae species as Escherichia coli or Klebsiella pneumoniae. Here, we describe the presence and role of the type 3 fimbria, a chaperone-usher assembled fimbria, which was first described in Klebsiella spp., and which has been detected in other representatives of the Enterobacteriaceae family. Eight Chilean E. cloacae isolates were examined, and among them, four E. hormaechei isolates were found to produce the type 3 fimbria. These isolates were identified as E. hormaechei subsp. hoffmannii, one of the five subspecies known. A mutant E. hormaechei subsp. hoffmannii strain lacking the mrkA gene, encoding the major structural subunit, displayed a significantly reduced adherence capacity to a plastic surface and to Caco-2 cells, compared to the wild-type strain. This phenotype of reduced adherence capacity was not observed in the mutant strains complemented with the mrkA gene under the control of an inducible promoter. Therefore, these data suggest a role of the type 3 fimbria in the adherence capacity of E. hormaechei subsp. hoffmannii. A screening in E. hormaechei genomes contained in the NCBI RefSeq Assembly database indicated that the overall presence of the type 3 fimbria is uncommon (5.94-7.37%), although genes encoding the structure were detected in representatives of the five E. hormaechei subspecies. Exploration of complete genomes indicates that, in most of the cases, the mrkABCDF locus, encoding the type 3 fimbria, is located in plasmids. Furthermore, sequence types currently found in healthcare-associated infections were found to harbor genes encoding the type 3 fimbria, mainly ST145, ST78, ST118, ST168, ST66, ST93, and ST171. Thus, although the type 3 fimbria is not widespread among the species, it might be a determinant of fitness for a subset of E. hormaechei representatives.

Disinfection efficacy of ozone on ESKAPE bacteria biofilms: Potential use in difficult-to-access medical devices.

BACKGROUND: Medical devices can be reservoirs of multidrug-resistant bacteria that may be involved in the acquisition of infections since bacteria with the ability to form biofilms that are difficult to eradicate, mainly in mechanical ventilators. The aim of this work was to evaluate the efficacy of O3 against biofilms of bacteria ESKAPE group through disinfection studies. METHODS: The formation of biofilms of ESKAPE group bacteria was induced in vitro. O3 was injected at different exposure times at a constant dose of 600 mg/h. The recovery of surviving bacteria after O3 treatment was assessed by bacterial counts and biofilm disruption was analyzed. Finally, the viability and integrity of biofilms after O3 treatment was determined by confocal laser scanning microscopy (CLSM). RESULTS: O3 showed bactericidal activity on biofilms from 12 min/7.68 ppm for A. baumannii and C. freundii. P. aeruginosa, K. pneumoniae and S. aureus were killed after 15 min/9.60 ppm. Correlation analyses showed inversely proportional relationships between the variables "disruption versus O3". CLSM revealed that death was time-dependent of biofilms upon O3 exposure. Orthogonal plane analysis showed that bacteria located in the outer region of the biofilms were the ones that initially suffered damage from O3 exposure. CONCLUSIONS: Our findings suggest that this method could be an alternative for the disinfection in mechanical ventilators colonized by bacteria biofilm forming.

In vitro bacterial adherence on teeth submitted to whitening with musa paradisiaca / Adherencia bacteriana in vitro en dientes sometidos a blanqueamiento con musa paradisiaca

Objetive: To compare in vitro bacterial adherence on teeth submitted to whitening with 50% ethanolic extract of Musa paradisiaca and 35% hydrogen peroxide. Material and Methods: The study was experimental and used 18 premolars that were grouped into: G1 (control), G2 (50% ethanol extract of Musa paradisiaca) and G3 (35% hydrogen peroxide). The teeth were then exposed to a Streptococcus mutans culture for 24 hours, followed by centrifugation in thioglycolate broth. A culture on trypticase soy agar was done with a 1 in 100 dilution, and after 48 hours colony forming units (CFU) were counted. Statistical analysis was performed using the ANOVA test, complemented by the Bonferroni post-hoc. Results: Bacterial adherence was 77x105 CFU/ml in Group 3 using 35% hydrogen peroxide, 40x105 CFU/ml in Group 2 using 50% ethanol extract of Musa paradisiaca, and 89x104 CFU/ml in Group 1 (control). The difference between the three groups was significant (p=0.000). Conclusion: Both whitening methods cause bacterial adherence to the tooth surface, although to a lower degree with Musa paradisiaca.eses.

Objetivo: Comparar la adherencia bacteriana in vitro en dientes sometidos a blanqueamiento con extracto etanólico de Musa paradisiaca al 50% y con peróxido de hidrógeno al 35%. Material y Métodos: Comparar la adherencia bacteriana in vitro en dientes sometidos a blanqueamiento con extracto etanólico de Musa paradisiaca al 50% y con peróxido de hidrógeno al 35%.Resultados: La adherencia bacteriana fue de 77x105 UFC/ml con el peróxido de hidrógeno al 35%, de 40x105 UFC/ml con el extracto etanólico de Musa paradisiaca al 50% y de 89x104 UFC/ml con el control. La diferencia fue significativa entre los tres grupos (p=0.000). Conclusión: Ambos métodos de blanqueamiento causan adherencia bacteriana en la superficie dental, siendo menor con Musa paradisiaca.

Coli Surface Antigen 26 Acts as an Adherence Determinant of Enterotoxigenic Escherichia coli and Is Cross-Recognized by Anti-CS20 Antibodies.

The coli surface antigen 26 (CS26) of enterotoxigenic Escherichia coli (ETEC) had been described as a putative adhesive pilus based on the partial sequence of the crsH gene, detected in isolates from children with diarrhea in Egypt. However, its production and activity as adherence determinant has not been experimentally addressed. The crsH was identified as a homolog of genes encoding structural subunits of ETEC colonization factors (CFs) CS12, CS18, and CS20. These CFs, along with the recently discovered CS30, belong to the γ2 family of pili assembled by the chaperone-usher pathway (CU pili). Further, the complete CS26 locus, crsHBCDEFG, was described in an O141 ETEC strain (ETEC 100664) obtained from a diarrhea case in The Gambia, during the Global Enterics Multicenter Study. Here, we report that CS26 is a pilus of ∼10 nm in diameter, with the capacity to increase the cell adherence of the non-pathogenic strain E. coli DH10B. As for other related pili, production of CS26 seems to be regulated by phase variation. Deletion of crsHBCDEFG in ETEC 100664 significantly decreased its adherence capacity, which was recovered by in trans complementation. Furthermore, CrsH was cross-recognized by polyclonal antibodies directed against the major structural subunit of CS20, CsnA, as determined by Western blotting and immunogold labeling. ETEC CS26+ strains were found to harbor the heat-labile enterotoxin only, within three different sequence types of phylogroups A and B1, the latter suggesting acquisition through independent events of horizontal transfer. Overall, our results demonstrate that CS26 is an adhesive pilus of human ETEC. In addition, cross-reactivity with anti-CsnA antibodies indicate presence of common epitopes in γ2-CFs.

Streptococcus Mutans Biofilm Influences on the Antimicrobial Properties of Glass Ionomer Cements

Abstract The aim of this study was to evaluate the in vitro antibacterial and biofilm inhibition properties of glass ionomer restorative cements. Ketac Nano, Vitremer, Ketac Molar Easymix and Fuji IX were analyzed using the following tests: a) agar plate diffusion test to evaluate the inhibitory activity of cements against S. mutans (n=8); b) S. mutans adherence test by counting colony-forming units after 2 h of material/bacteria exposure (n=10); c) biofilm wet weight after seven days of bacterial accumulation on material disks, with growth medium renewed every 48 h (n=10); d) pH and fluoride measurements from the medium aspired at 48 h intervals during the 7-day biofilm development (n=10). Data from the a, b and c tests were submitted to Kruskal-Wallis and Mann-Whitney tests and the fluoride-release and pH data were submitted to two-way ANOVA and Tukey tests (a=5%). Vitremer followed by Ketac Nano showed the greatest inhibitory zone against S. mutans than the conventional ionomers. Vitremer also showed higher pH values than Ketac Nano and Fuji IX in the first 48 h and released higher fluoride amount than Ketac Nano e Ketac Molar Easymix throughout the experimental period. The chemical composition of restorative glass ionomer materials influenced the antibacterial properties. The resin modified glass ionomer (Vitremer) was more effective for inhibition of S. mutans and allowed greater neutralization of the pH in the first 48 h. However, the type of glass ionomer (resin modified or conventional) did not influence the weight and adherence of the biofilm and fluoride release.

Resumo O objetivo neste estudo foi avaliar in vitro as propriedades antibacterianas e a inibição do biofilme de cimentos de ionômero de vidro restauradores. Ketac Nano, Vitremer, Ketac Molar Easymix and Fuji IX foram avaliados através dos seguintes testes: a) teste de difusão em ágar para avaliar a inibição de S. mutans nos cimentos (n=8); b) adesão de S. mutans pela contagem de unidades formadoras de colônia após 2h de exposição material/bactéria (n=10); c) peso do biofilme úmido após sete dias de acúmulo bacteriano nos discos do material, com meio de cultura renovado após 48 h (n=10); d) mensuração do pH e liberação de flúor do meio aspirado nos intervalos de 48 h durante 7 dias de crescimento do biofilme (n=10). Os dados dos testes a, b e c foram submetidos aos testes Kruskal-Wallis e Mann-Whitney e os dados de liberação de flúor e pH a ANOVA dois fatores e Tukey (a = 5%). Vitremer seguido pelo Ketac Nano mostrou maior zona de inibição contra S. mutans quando comparados aos ionômeros convencionais. Vitremer também apresentou valores de pH mais elevados do que Ketac Nano e Fuji IX nas primeiras 48 h e liberou maior quantidade de flúor do que Ketac Nano e Ketac Molar Easymix durante todo o período experimental. A composição química dos ionômeros de vidro restauradores influenciou nas propriedades antibacterianas. O ionômero de vidro modificado por resina (Vitremer) foi mais eficaz na inibição de S. mutans e permitiu maior neutralização do pH nas primeiras 48 h. No entanto, o tipo de ionômero de vidro (modificado por resina ou convencional) não influenciou no peso e adesão do biofilme e na liberação de flúor.

Estudo de atributos de virulência e resistência a antimicrobianos em amostras de P. aeruginosa / Study of attributes of virulence and antimicrobial resistance in P. aeruginosa isolates

P. aeruginosa é um importante agente de infecções relacionadas à assistência em saúde. Habitualmente, o estabelecimento de infecções agudas é precedido pela colonização das mucosas dos pacientes. Não se sabe, porém, se os processos infecciosos são causados pelas próprias cepas bacterianas colonizadoras ou por outras com que os pacientes entrem em contato, dotadas ou não de maior potencial de virulência ou de resistência a antimicrobianos que as tornem mais eficientes como agentes infecciosos. Assim, este estudo teve como objetivos i) investigar a existência de potenciais diferenças entre amostras de P. aeruginosa que causaram apenas colonização e aquelas responsáveis por infecção, isoladas de um mesmo paciente, quanto a seus fenótipos de virulência e de não susceptibilidade a antimicrobiamos; ii) pesquisar a existência de associação entre características dos paciente, incluindo o tipo de evolução clínica, com as demais variáveis estudadas. No estudo foram incluídos 21 pacientes que desenvolveram infecção por P. aeruginosa durante sua internação no Centro de Terapia Intensiva do Hospital Universitário Clementino Fraga Filho, entre abril de 2007 e abril de 2008. De cada paciente foram selecionadas duas amostras bacterianas: a primeira isolada durante o episódio de infecção e a amostra colonizadora obtida imediatamente antes da ocorrência da infecção. As amostras selecionadas foram estudadas quanto a i) expressão de três mecanismos de virulência (citotoxicidade, aderência a células epiteliais respiratórias humanas e capacidade de formação de biofilme); ii) presença de genes codificadores das proteínas efetoras do sistema de secreção do tipo 3 (SST3 - exoS, exoT, exoU e exoY); iii) perfil de susceptibilidade a antimicrobianos, iv) perfil de fragmentação do DNA cromossômico por eletroforese em gel de campo pulsado (PFGE). As amostras bacterianas obtidas de infecções agudas foram significativamente mais citotóxicas que aquelas obtidas de colonização...

P. aeruginosa is an important agent of healthcare-associated infections. The establishment of acute infectious episodes is usually preceded by colonization of patient mucosa. However, it remains unknown whether the infectious processes are caused by bacterial strains previously colonizing the patient or by additional strains the patient may come into contact. These new isolates may carry greater virulence potential or antibiotic resistance that makes them more efficient as an infecting agent. Thus, the objetives of the present study were i) to investigate the existence of potential differences between P. aeruginosa isolates obtained from a colonized mucosa and isolates accounting for infectious processes, recovered from the same patient, with respect to virulence phenotypes and non-susceptibility to antimicrobial agents; ii) to investigate the existence of association between patient features, including the type of clinical outcome, with bacterial characteristics. The study included 21 patients who developed P. aeruginosa infection during their stay in the Intensive Care Unit of the University Hospital Clementino Fraga Filho, from April 2007 to April 2008. Two P. aeruginosa isolates were selected from each patient: the first isolate recovered from the infectious episode and the colonizing isolate obtained immediately before the onset of the infection. Features from the isolates investigated included: i) expression of three virulence mechanisms (cytotoxicity, adherence to human respiratory epithelial cells and biofilm formation); ii) presence of the genes encoding type III secretion system effector proteins (TTSS, exoS , exoT , exoU and exoY); iii) antimicrobial susceptibility profile; iv) profile of the bacterial chromossomic DNA fragmentation following analysis by pulsed-field gel electrophoresis (PFGE). The bacterial isolates obtained from acute infections were significantly more cytotoxic than colonizing strains...