Multidrug-resistant (MDR) Corynebacterium striatum has been cited with increased frequency as pathogen of nosocomial infections. In this study, we report the draft genome of a C. striatum isolated from a patient with bloodstream infection in a hospital of Rio de Janeiro, Brazil. The isolate presented susceptibility only to tetracycline, vancomycin and linezolid. The detection of various antibiotic resistance genes is fully consistent with previously observed multidrug-resistant pattern in Corynebacterium spp. A large part of the pTP10 plasmid of MDR C. striatum M82B is present in the genome of our isolate. A SpaDEF cluster and seven arrays of CRISPR-Cas were found.

BACKGROUND The association between Staphylococcus haemolyticus and severe nosocomial infections is increasing. However, the extent to which fomites contribute to the dissemination of this pathogen through patients and hospital wards remains unknown. OBJECTIVES In the present study, sphygmomanometers and thermometers were evaluated as potential fomites of oxacillin-resistant S. haemolyticus (ORSH). The influence of oxacillin and vancomycin on biofilm formation by ORSH strains isolated from fomites was also investigated. METHODS The presence of ORSH on swabs taken from fomite surfaces in a Brazilian hospital was assessed using standard microbiological procedures. Antibiotic susceptibility profiles were determined by the disk diffusion method, and clonal distribution was assessed in pulsed-field gel electrophoresis (PFGE) assays. Minimum inhibitory concentrations (MICs) of oxacillin and vancomycin were evaluated via the broth microdilution method. Polymerase chain reaction (PCR) assays were performed to detect the mecA and icaAD genes. ORSH strains grown in media containing 1/4 MIC of vancomycin or oxacillin were investigated for slime production and biofilm formation on glass, polystyrene and polyurethane catheter surfaces. FINDINGS ORSH strains comprising five distinct PFGE types were isolated from sphygmomanometers (n = 5) and a thermometer (n = 1) used in intensive care units and surgical wards. ORSH strains isolated from fomites showed susceptibility to only linezolid and vancomycin and were characterised as multi-drug resistant (MDR). Slime production, biofilm formation and the survival of sessile bacteria differed and were independent of the presence of the icaAD and mecA genes, PFGE type and subtype. Vancomycin and oxacillin did not inhibit biofilm formation by vancomycin-susceptible ORSH strains on abiotic surfaces, including on the catheter surface. Enhanced biofilm formation was observed in some situations. Moreover, a sub-lethal dose of vancomycin induced biofilm formation by an ORSH strain on polystyrene. MAIN CONCLUSIONS Sphygmomanometers and thermometers are fomites for the transmission of ORSH. A sub-lethal dose of vancomycin may favor biofilm formation by ORSH on fomites and catheter surfaces.

Corynebacterium diphtheriae, the aetiologic agent of diphtheria, also represents a global medical challenge because of the existence of invasive strains as causative agents of systemic infections. Although tellurite (TeO32-) is toxic to most microorganisms, TeO32--resistant bacteria, including C. diphtheriae, exist in nature. The presence of TeO32--resistance (TeR) determinants in pathogenic bacteria might provide selective advantages in the natural environment. In the present study, we investigated the role of the putative TeR determinant (CDCE8392_813 gene) in the virulence attributes of diphtheria bacilli. The disruption of CDCE8392_0813 gene expression in the LDCIC-L1 mutant increased susceptibility to TeO32- and reactive oxygen species (hydrogen peroxide), but not to other antimicrobial agents. The LDCIC-L1 mutant also showed a decrease in both the lethality of Caenorhabditis elegans and the survival inside of human epithelial cells compared to wild-type strain. Conversely, the haemagglutinating activity and adherence to and formation of biofilms on different abiotic surfaces were not regulated through the CDCE8392_0813 gene. In conclusion, the CDCE8392_813 gene contributes to the TeR and pathogenic potential of C. diphtheriae.

Corynebacterium striatum is a potentially pathogenic microorganism that causes nosocomial outbreaks. However, little is known about its virulence factors that may contribute to healthcare-associated infections (HAIs). We investigated the biofilm production on abiotic surfaces of multidrug-resistant (MDR) and multidrug-susceptible (MDS) strains of C. striatum of pulsed-field gel electrophoresis types I-MDR, II-MDR, III-MDS and IV-MDS isolated during a nosocomial outbreak in Rio de Janeiro, Brazil. The results showed that C. striatum was able to adhere to hydrophilic and hydrophobic abiotic surfaces. The C. striatum1987/I-MDR strain, predominantly isolated from patients undergoing endotracheal intubation procedures, showed the greatest ability to adhere to all surfaces. C. striatumbound fibrinogen to its surface, which contributed to biofilm formation. Scanning electron microscopy showed the production of mature biofilms on polyurethane catheters by all pulsotypes. In conclusion, biofilm production may contribute to the establishment of HAIs caused by C. striatum.

The aim of this study was to compare the efficacy of chemochemical methods (Carisolv™ and Papacárie®) versus the manual method (excavators) in reducing the cariogenic microbiota in dentine caries of primary teeth. Forty-six healthy children (5 to 9 years old) having at least one primary tooth with a cavitated dentine carious lesion were included in the study. The teeth presented no clinical or radiographic signs of pulpal involvement. The sample of 74 teeth was randomly divided into three different groups: Papacárie® (n = 25), Carisolv™ (n = 27) and Manual (n = 22). Samples of carious and sound dentine were collected with sterile excavators before and after caries removal in the three groups. The dentine samples were transferred to glass tubes containing a 1mL thioglycollate medium used as a carrier and enriched for microbiological detection of mutans streptococci and Lactobacillus spp, after incubation for 6h at room temperature. The minimum detection value for colony forming units (CFU) was 3.3 x 102 CFU/ml, and the results were converted into scores from 0 to 4. A significant difference was observed in relation to the microbiological scores before and after caries removal for all methods (Wilcoxon test; p < 0.001). The use of chemomechanical methods for caries removal did not improve the reduction of cariogenic microorganisms in dentine caries lesions, in comparison with manual excavation.

Corynebacterium pseudodiphtheriticum is a well-known human pathogen that mainly causes respiratory disease and is associated with high mortality in compromised hosts. Little is known about the virulence factors and pathogenesis of C. pseudodiphtheriticum. In this study, cultured human epithelial (HEp-2) cells were used to analyse the adherence pattern, internalisation and intracellular survival of the ATCC 10700 type strain and two additional clinical isolates. These microorganisms exhibited an aggregative adherence-like pattern to HEp-2 cells characterised by clumps of bacteria with a "stacked-brick" appearance. The differences in the ability of these microorganisms to invade and survive within HEp-2 cells and replicate in the extracellular environment up to 24 h post infection were evaluated. The fluorescent actin staining test demonstrated that actin polymerisation is involved in the internalisation of the C. pseudodiphtheriticum strains. The depolymerisation of microfilaments by cytochalasin E significantly reduced the internalisation of C. pseudodiphtheriticum by HEp-2 cells. Bacterial internalisation and cytoskeletal rearrangement seemed to be partially triggered by the activation of tyrosine kinase activity. Although C. pseudodiphtheriticum strains did not demonstrate an ability to replicate intracellularly, HEp-2 cells were unable to fully clear the pathogen within 24 h. These characteristics may explain how some C. pseudodiphtheriticum strains cause severe infection in human patients.

The production of fibrinous exudates may play an important role in determining the outcome of bacterial infection. Although pseudomembrane formation is a characteristic feature of diphtheria, little is known about the fibrinogen (Fbn)-binding properties of Corynebacterium diphtheriae strains and the influence of the gene that codes for diphtheria toxin (tox gene) in this process. In this study we demonstrated the ability of C. diphtheriae strains to bind to Fbn and to convert Fbn to fibrin. Bacterial interaction with rabbit plasma was evaluated by both slide and tube tests. Interaction of microorganisms with human Fbn was evaluated by both enzyme linked immunosorbent assay (ELISA) and fluorescein isothiocyanate-conjugated (FITC) Fbn binding assays. Nontoxigenic and toxigenic strains formed bacterial aggregates in the presence of plasma in the slide tests. The ability to convert Fbn to a loose web of fibrin in the plasma solution in the tube tests appeared to be a common characteristic of the species, including strains that do not carry the tox gene. Fbn binding to C. diphtheriae strains occurred at varying intensities, as demonstrated by the FITC-Fbn and ELISA binding assays. Our data suggest that the capacity to bind to Fbn and to convert Fbn to fibrin may play a role in pseudomembrane formation and act as virulence determinants of both nontoxigenic and toxigenic strains.

A eficácia das pastas de hidróxido de cálcio: Calen® e Calen-PMCC® associadas ao preparo químico-mecânico, foi avaliada sobre Enterococcus faecalis cultivados no interior dos canais radiculares. Setenta incisivos foram inseridos em caldo TSB, esterilizados e contaminados com E. faecalis. O meio de cultivo foi substituído a cada 24 h, sendo incubados a 37oC por 72 h. Após o preparo químico-mecânico, os canais radiculares foram preenchidos com Calen® ou Calen-PMCC® (7 ou 14 dias). As pastas foram removidas e os dentes inseridos em tubos contendo caldo Enterococcosel. A pasta Calen® (mantida por 7 ou 14 dias) induziu a eliminação dos enterococos em 70% dos dentes, enquanto a pasta Calen-PMCC® induziu a eliminação em 100% dos dentes, somente após a manutenção por 14 dias. Tais medicações foram significativamente mais efetivas (p<0,001) do que o protocolo do preparo químico-mecânico e o Calen-PMCC® mantido por 7 dias, ambos incapazes de eliminar os enterococos. As pastas de hidróxido de cálcio demonstraram efeitos adjuvantes importantes na eliminação dos enterococos durante o preparo químico-mecânico dos sistemas de canais radiculares. Quando associada ao PMCC, as pastas de hidróxido de cálcio devem ser mantidas por pelo menos 14 dias.

The effectiveness of calcium hydroxide pastes: Calen™ and PMCC-Calen™ associated to chemo-mechanical preparation was assessed on Enterococcus faecalis grown within root canals. Seventy incisors were inserted into TSB medium, sterilized and contaminated with E. faecalis. Culture medium was replaced each 24 h and incubated at 37oC for 72 h. After chemo-mechanical preparation, root canals were filled with Calen™ or PMCC-Calen™ (7 or 14 days). Pastes were removed and teeth were inserted into test tubes containing Enterococcosel broth. Calen™ paste (maintained for 7 and 14 days) induced 70% elimination of enterococci and PMCC-Calen™ 100% elimination only after maintenance for 14 days. These medications were significantly more effective (p<0.001) than chemo-mechanical protocol alone and PMCC-Calen™ maintained for 7 days, both incapable to eliminate the viability of enterococci. Calcium hydroxide pastes demonstrated important adjuvant effects in the elimination of enterococci during chemo-mechanical preparation of root canal systems. When associated with PMCC, calcium hydroxide pastes should be maintained for at least 14 days.

The increasing problems with multidrug resistance in relation to Corynebacterium, including C. diphtheriae, are examples of challenges confronting many countries. For this reason, Brazilian C. diphtheriae strains were evaluated by the E-Test for their susceptibility to nine antibacterial drugs used in therapy. Resistance (MIC < 0.002; 0.38 µg/ml) to penicillin G was found in 14.8% of the strains tested. Although erythromycin (MIC90 0.75 µg/ml) and azithromycin (MIC90 0.064 µg/ml) were active against C. diphtheriae in this study, 4.2% of the strains showed decreased susceptibility (MIC 1.0 µg/ml) to erythromycin. Multiple resistance profiles were determined by the disk diffusion method using 31 antibiotics. Most C. diphtheriae strains (95.74%) showed resistance to mupirocin, aztreonam, ceftazidime, and/or oxacillin, ampicillin, penicillin, tetracycline, clindamycin, lincomycin, and erythromycin. This study presents the antimicrobial susceptibility profiles of Brazilian C. diphtheriae isolates. The data are of value to practitioners, and suggest that some concern exists regarding the use of penicillin.

O objetivo do presente trabalho foi avaliar e comparar o efeito antimicrobiano do MTA Dentsply, MTA Angelus, hidróxido de cálcio e cimento Portland sobre quatro cepas bacterianas: Pseudomonas aeruginosa, Escherichia coli, Bacteroides fragilis, e Enterococcus faecalis. Placas contendo agar Muller-Hinton suplementadas com 5% de sangue de carneiro, hemina e menadiona foram inoculadas com as suspensões bacterianas. Poços foram confeccionados com auxílio de perfuradores e imediatamente preenchidos com os materiais, e incubados a 37ºC por 48 horas em atmosfera de anaerobiose, exceto P. aeruginosa. O diâmetro dos halos de inibição foi medido e os dados analisados usando o teste estatístico ANOVA e o de Tukey com nível de significância de 1%. O MTA Dentsply, MTA Angelus e Cimento Portland inibiram o crescimento da P.aeruginosa. O hidróxido de cálcio foi efetivo contra P. aeruginosa e B. fragillis. Sob atmosfera de anaerobiose, condição que pode impedir a formação de espécies reativas do oxigênio, nenhum dos materiais foi capaz de exercer efeitos sobre E. faecalis e E. coli.

The present study aimed to evaluate and compare the antimicrobial effect of MTA Dentsply, MTA Angelus, Calcium Hydroxide and Portland cement. Four reference bacterial strains were used: Pseudomonas aeruginosa, Escherichia coli, Bacteroides fragilis, and Enterococcus faecalis. Plates containing Mueller-Hinton agar supplemented with 5% sheep blood, hemin, and menadione were inoculated with the bacterial suspensions. Subsequently, wells were prepared and immediately filled with materials and incubated at 37ºC for 48 hours under anaerobic conditions, except P. aeruginosa. The diameters of inhibition zones were measured, and data analyzed using ANOVA and the Tukey test with 1% level of significance. MTA Dentsply, MTA Angelus and Portland cement inhibited the growth of P. aeruginosa. Calcium Hydroxide was effective against P. aeruginosa and B. fragillis. Under anaerobic conditions, which may hamper the formation of reactive oxygen species, the materials failed to inhibit E. faecalis, and E. coli.

Changes in the epidemiology of diphtheria are occurring worldwide. A large proportion of adults in many industrialized and developing countries are now susceptible to diphtheria. Vaccine-induced immunity wanes over time unless periodic booster is given or exposure to toxigenic Corynebacterium diphtheriae occurs. Immunity gap in adults coupled with large numbers of susceptible children creates the potential for new extensive epidemics. Epidemic emergencies may not be long in coming in countries experiencing rapid industrialization or undergoing sociopolitical instability where many of the factors thought to be important in producing epidemic such as mass population movements and difficult hygienic and economic conditions are present. The continuous circulation of toxigenic C. diphtheriae emphasizes the need to be aware of epidemiological features, clinical signs, and symptoms of diphtheria in vaccine era so that cases can be promptly diagnosed and treated, and further public health measures can be taken to contain this serious disease. This overview focused on worldwide data obtained from diphtheria with particular emphasis to main factors leading to recent epidemics, new clinical forms of C. diphtheriae infections, expression of virulence factors, other than toxin production, control strategies, and laboratory diagnosis procedures.