Identification of Enterococcus spp. by MALDI-TOF mass spectrometry isolated from clinical mastitis and bulk tank milk samples.

BACKGROUND: Throughout a three-year study period, 1,577 bovine clinical mastitis samples and 302 bulk tank samples were analyzed from ten Brazilian dairy herds. Enterococcus spp. was isolated and identified in 93 (5.9%) clinical mastitis samples. In addition, 258 Enterococcus spp. were isolated from the bulk tank samples of the same herds. The identification of Enterococcus spp. isolated from bulk tanks and milk samples of clinical mastitis were accomplished by phenotypic characteristics and confirmed by MALDI-TOF Mass Spectrometry (MS). Fisher test was performed to verify the difference between bulk tanks and mastitis samples. RESULTS: The following species were identified from clinical mastitis: E. saccharolyticus (62.4%), E. faecalis (19.4%), E. faecium (15.1%), E. hirae (1.1%), E. mundtii (1.1%), E. durans (1.1%). Furthermore, from 258 bulk tank milk samples, eight enterococci species were isolated: E. faecalis (67.8%), E. hirae (15.1%), E. faecium (4.6%), E. saccharolyticus (4.6%), E. mundtii (3.1%), E. caseliflavus ( 2.7%), E. durans (1.2%), E. galinarum (0.8%). CONCLUSIONS: The difference in species predominance in bulk tank samples (67.8% of E. faecalis) and clinical mastitis (62.4% of E. saccharolyticus) was unexpected and caught our attention. Although Enterococcus spp. are traditionally classified as an environmental mastitis agent, in the present study, E. saccharolyticus behaved as a contagious agent of mastitis, which consequently changed the control patterns to be implemented.

Comparación de la actividad antimicrobiana de cementos selladores en endodoncia / Comparison of the antimicrobial activity of sealing cements in endodontics

Introducción: la persistencia de microorganismos en los conductos radiculares es uno de los principales factores del fracaso endodóncico. Por ello la importancia de conocer las propiedades antimicrobianas de los distintos tipos de selladores. Objetivo: realizar una comparación con base en la evidencia disponible sobre la actividad antimicrobiana de los diferentes cementos selladores en endodoncia. Material y métodos: la información fue recopilada de las bases de datos PubMed y Google Académico en el idioma inglés y español, publicados en el periodo 2014-2023. Resultados: un gran número de microorganismos se encuentran presentes en los diferentes tipos de infecciones de origen endodóncico, se han identificado más de 500 especies microbianas, entre ellas bacterias, hongos, arqueas y virus. Los cementos selladores se pueden clasificar según su composición química, en cementos a base de óxido de zinc-eugenol, hidróxido de calcio, a base de ionómero de vidrio, silicona, resina y biocerámicos. Conclusión: los cementos selladores que mostraron mayor actividad antimicrobiana contra los microorganismos persistentes fueron los cementos a base de óxido de zinc-eugenol, resina y biocerámicos. Sin embargo, se identificó que cada autor utilizó diferentes métodos y tiempos, por lo tanto, no es posible lograr definir con exactitud qué cemento sellador posee la mejor capacidad antimicrobiana (AU)

Introduction: the persistence of microorganisms in root canals is one of the main factors of endodontic failure. Therefore, the importance of knowing the antimicrobial properties of the different types of sealants. Objective: to make a comparison based on the available evidence on the antimicrobial activity of the different endodontics sealers. Material and methods: the information was collected from PubMed and Google Academic databases in English and Spanish, published in the period 2014-2023. Results: a large number of microorganisms are present in the different types of infections of endodontic origin, more than 500 microbiological species have been identified, including bacteria, fungi, archaea and viruses. Sealer cements can be classified according to their chemical composition, into cements based on zinc oxide-eugenol, calcium hydroxide, based on glass ionomer, silicone, resin and bioceramics. Conclusion: sealer cements that showed the highest antimicrobial activity against persistent microorganisms were zinc oxide-eugenol, resin, and bioceramic-based cements. However, it was identified that each author used different methods and times, therefore, it is not possible to accurately define which sealer cement has the best antimicrobial capacity (AU)

Propylene Glycol Potentiates the Inhibitory Action of CTZ Paste on Antibiotic-Resistant Enterococcus faecalis Isolated from the Root Canal: An In Vitro Study.

This study aimed to evaluate if the change of vehicle for CTZ (Chloramphenicol, Tetracycline, zinc oxide, and Eugenol) paste improves the inhibition of Enterococcus faecalis in vitro. The vehicles evaluated alone and mixed with CTZ were Eugenol, propylene glycol (PG), super-oxidized solution (SOS), grapefruit-seed extract (GSE), and 0.9% saline solution as a negative control. A clinical isolate of E. faecalis was morphologically and biochemically characterized, and its antimicrobial susceptibility was tested using 20 antimicrobial agents. Once characterized, the clinical isolate was cultivated to perform the Kirby-Bauer disc diffusion method with paper discs embedded with the different vehicles mixed or used alone, and incubated at 37 °C for 24 h. Data were analyzed using one-way ANOVA, and the means were compared using Tukey test with a significance level of p < 0.05. For vehicles used alone, GSE presented the greatest inhibition showing a statistically significant difference with the rest of the vehicles. When vehicles were mixed with the CTZ paste, PG showed a greater inhibition with a statistically significant difference from the rest of the vehicles. In conclusion, the vehicle used to mix the CTZ paste plays an important role in the inhibition of E. faecalis in vitro; therefore, we consider that this can be an important factor to achieve success in the use of this technique.

The Emergence of Antimicrobial Resistance and Virulence Characteristics in Enterococcus Species Isolated from Bovine Milk.

Enterococcus spp., including E. faecalis and E. faecium, pose risks to dairy farms as opportunistic pathogens. The study evaluates antimicrobial resistance (AMR) and virulence characteristics of Enterococcus spp. isolated from bovine milk. Bile esculin agar was used to assess 1471 milk samples, followed by colony identification, gram staining, catalase tests, and 45 °C incubation. PCR analysis targeted E. faecalis and E. faecium in characteristic Enterococcus spp. colonies, with MALDI-TOF used for negative samples. Multiple tests, including disk diffusion, chromogenic VRE agar for vancomycin resistance, Vancomycin Etest® for MIC determination, and PCR for virulence factors (cylA, esp, efaA, ace, asa1, gelE, and hyl genes), were performed. Out of 100 identified strains, E. durans (30.66%), E. faecium (26.28%), and E. faecalis (18.25%) were predominant. AMR in Enterococcus spp. varied, with the highest rates against rifampicin (27%), tetracycline (20%), and erythromycin (18%). Linezolid (5%), vancomycin, ciprofloxacin, and teicoplanin (3% each) had lower prevalence. E. faecium and E. faecalis showed high AMR to rifampicin, erythromycin, and tetracycline. Thirty-two strains (18.98%) grew on VRE Chromoselect agar, while 4 (2 E. faecalis and 2 E. faecium) showed vancomycin resistance by MIC values. E. faecalis carried gelE (45.5%) and asa1 (36%), and E. gallinarum had 9.1% with the asa1 gene. Detecting resistant Enterococcus in bovine milk supports control strategies for enterococci on dairy farms, highlighting AMR concerns in the food chain.

Comparative evaluation of antimicrobial potential of herbal extracts on Streptococcus mutans and Enterococcus faecalis An in vitro study / Avaliação comparativa do potencial antimicrobiano de extratos de ervas em Streptococcus mutans e Enterococcus faecalis: estudo in vitro

Though aloe vera extract, green tea extract and coriander oil are proven antimicrobial agents, very little information is available regarding its effects on oral bacteria, Streptococcus mutans, which is responsible for initiating caries and Enterococcus faecalis, responsible for failure of root canal treatment. Objective: To find the antimicrobial activity of aloe vera extract, black tea extract and coriander oil against S. mutans and E. faecalis. Materials and Methods: The agar well diffusion method was used to determine the antibacterial activity of Aloe vera extract, black tea extract and coriander oil. Different concentration of prepared plant extracts and coriander seed oil (50 & 100 µl) was incorporated into the wells and the plates containing S. mutans and E. faecalis were incubated at 37 °C for 24 h. The antibiotic (amoxicillin 30 µl) was used as positive control. Zone Of Inhibition (ZOI) was recorded in each plate. Results: For S. mutans, the maximum ZOI was created by coriander oil with a diameter of 25.00±0.58 mm at 50 µl and for E. faecalis, maximum ZOI was created by aloe vera extract 16.00±0.58 mm at 100 µl concentration which were far better than the control: amoxicillin 30 µl concentration. Conclusion: The extracts of Aloe vera, black tea and coriander oil, showed significant activity against the investigated microbial strains, Streptococcus mutans and Enterococcus faecalis which further helps in the development of new topical agents that help in reducing the numbers of these organisms present in the oral cavity. (AU)

Embora o extrato de aloe vera, extrato de chá verde e óleo de coentro sejam agentes antimicrobianos comprovados, há pouca informação disponível sobre seus efeitos nas bactérias orais, Streptococcus mutans, que é responsável por iniciar cáries e Enterococcus faecalis, responsável pela falha do tratamento de canal radicular. Objetivo: Avaliar a atividade antimicrobiana do extrato de aloe vera, extrato de chá preto e óleo de coentro contra S. mutans e E. faecalis. Materiais e Métodos: O método de difusão em agar foi usado para determinar a atividade antibacteriana do extrato de Aloe vera, extrato de chá preto e óleo de coentro. Diferentes concentrações dos extratos de plantas e óleo de semente de coentro (50 e 100 µl) foram preparados e colocados nos poços e nas placas contendo S. mutans e E. faecalis e foram incubadas a 37°C por 24 h. O antibiótico (amoxicilina 30 µl) foi utilizado como controle positivo. A zona de inibição (ZOI) foi registrada em cada placa. Resultados: Para S. mutans, a ZOI máxima foi obtida com o óleo de coentro com um diâmetro de 25,00 ± 0,58 mm a 50 µl e para E. faecalis, a ZOI máxima foi obtiada pelo extrato de aloe vera 16,00 ± 0,58 mm na concentração de 100 µl, as quais foram melhores do que o controle: concentração de 30 µl de amoxicilina. Conclusão: Os extratos de Aloe vera, chá preto e óleo de coentro apresentaram atividade significativa contra as cepas microbianas investigadas, Streptococcus mutans e Enterococcus faecalis auxiliando no desenvolvimento de novos agentes tópicos visando a redução do número desses organismos presentes no cavidade oral. (AU)

Effective control of biofilms by photothermal therapy using a gold nanorod hydrogel.

Biofilms are matrices synthesized by bacteria containing polysaccharides, DNA, and proteins. The development of biofilms in infectious processes can induce a chronic inflammatory response that may progress to the destruction of tissues. The treatment of biofilms is difficult because they serve as a bacterial mechanism of defense and high doses of antibiotics are necessary to treat these infections with limited positive results. It has been demonstrated that photothermal therapy using gold nanorods (AuNRs) is an attractive treatment because of its anti-biofilm activity. The purpose of this work was to generate a novel chitosan-based hydrogel embedded with AuNRs to evaluate its anti-biofilm activity. AuNRs were synthesized by the seed-mediated growth method and mixed with the chitosan-based hydrogel. Hydrogels were characterized and tested against two bacterial strains by irradiating the produced biofilm in the presence of the nanoformulation with a laser adjusted at the near infrared spectrum. In addition, the safety of the nanoformulation was assessed with normal human gingival fibroblasts. Results showed that a significant bacterial killing was measured when biofilms were exposed to an increase of 10°C for a short time of 2 min. Moreover, no cytotoxicity was measured when normal gingival fibroblasts were exposed to the nanoformulation using the bactericidal conditions. The development of the reported formulation can be used as a direct application to treat periodontal diseases or biofilm-produced bacteria that colonize the oral cavity.

Antibiotic resistance of Enterococcus faecalis from persistent endodontic infections / Resistencia antibiótica de Enterococcus faecalis provenientes de infecciones endodónticas persistentes

Enterococci are important nosocomial pathogens due to their intrinsic multiresistance and the acquisition of new antibiotic resistance genes (ARG). Enterococcus faecalis has been shown to be one of the main pathogens in persistent endodontic infections, therefore, the main objective of this study was to evaluate the phenotype and resistance genotype of strains of E. faecalis isolated from teeth with persistent endodontic lesions, to the most commonly prescribed antibiotics in dentistry. Thirteen strains of E. faecalis of different pulsotype were analyzed to evaluate the susceptibility to antibiotics, amoxicillin, amoxicillin/clavulanic acid, tetracycline, erythromycin and metronidazole, using the Epsilometer test (E- test) and the presence of beta-lactamases with nitrocefin test. Finally, the detection of ARG was performed with a molecular polymerase chain reaction (PCR) technique and confirmed by the sequencing of the amplification products. Fisher's exact test was used, using 95 % confidence. Regarding the phenotype of resistance, the evaluated strains, independent of the pulsotype, were totally resistant to the action of metronidazole. Antibiotics with higher minimum inhibitory concentration (MIC) after metronidazole include tetracycline and erythromycin. In contrast, lower MIC are applied to the combination of amoxicillin with clavulanic acid. The nitrocefin test was positive only in one strain. Genotypically, two genetically distant strains isolated from a single patient, presented a genotype of resistance to erythromycin, determined by the presence of the ermB gene. No statistically significant relationship was found between phenotypic resistance and the presence of ARG in relation to erythromycin (p> 0.05). It was concluded that isolates of E. faecalis from persistent endodontic infections showed phenotypes of resistance to several antimicrobial agents, all of which were susceptible to amoxicillin/clavulanic acid. Periodic evaluation of susceptibility to antibiotics is suggested as an important practice for the surveillance of antibiotic resistance in oral strains.

Los enterococos son importantes patógenos nosocomiales debido a su multi resistencia intrínseca y la adquisición de nuevos genes de resistencia a los antibióticos (ARG). Enterococcus faecalis es uno de los principales patógenos en infecciones endodónticas persistentes, por lo tanto, el objetivo principal de este estudio fue evaluar el fenotipo y el genotipo de resistencia de cepas de E. faecalis aisladas de dientes con lesiones endodóncicas persistentes, a los antibióticos comúnmente recetados en odontología. Se analizaron 13 cepas de E. faecalis de diferentes pulsotipos para evaluar la susceptibilidad a los antibióticos, amoxicilina, amoxicilina / ácido clavulánico, tetraciclina, eritromicina y metronidazol, utilizando la prueba de Epsilometría (E-test) y la presencia de beta-lactamasas con prueba de nitrocefina. Finalmente, la detección de ARG se realizó con una técnica molecular de reacción en cadena de la polimerasa (PCR) y se confirmó mediante la secuenciación de los productos de amplificación. Se utilizó la prueba exacta de Fisher, con un 95 % de confianza. En cuanto al fenotipo de resistencia, las cepas evaluadas, independientes del pulsotipo, fueron totalmente resistentes a la acción del metronidazol. Los antibióticos con los valores más altos de concentración mínima inibitoria (CMI) después del metronidazol incluyen tetraciclina y eritromicina. En contraste, las CMI mas bajas se aplican a la combinación de amoxicilina con ácido clavulánico. La prueba de nitrocefina fue positiva solo en una cepa. Genotípicamente, dos cepas distantes genéticamente, aisladas de un mismo paciente fueron positivas para el gen ermB. No se encontró una relación estadísticamente significativa entre la resistencia fenotípica y la presencia de ARG en relación con la eritromicina (p> 0,05). Se concluyó que los aislamientos de E. faecalis de infecciones endodónticas persistentes mostraron fenotipos de resistencia a varios agentes antimicrobianos, todos los cuales fueron susceptibles a amoxicilina / ácido clavulánico. Se sugiere una evaluación periódica de la susceptibilidad a los antibióticos como una práctica importante para la vigilancia de la resistencia a los antibióticos en las cepas orales.

Microbiological water quality and sources of contamination along the coast of the Department of Atlántico (Caribbean Sea of Colombia). Preliminary results.

Microbiological sea water quality is a public health problem that has serious repercussions in the tourism and economy of Colombia. This study determines the concentrations of Escherichia coli, Enterococcus faecalis and Clostridium perfringens at eleven beach water points and seven streams along the coast of the Department of Atlántico, Colombia. In seawater, total E. coli, E. faecalis and C. perfringens concentrations were found between 16 and 572 cfu/100 mL, 7-450 cfu/100 ml and 2-125 cfu/100 ml, respectively. The highest counts were observed mainly on urbanised beaches and in correspondence with streams whose waters had a high concentration of faecal origin microorganisms, which represent a serious health risk factor for bathers. Relevant efforts have to be addressed to improve the microbiological quality of these beaches by the establishment of efficient wastewater management programs aimed at enhancing the efficiency of the local treatment plant and the control of illegal sewage pouring onto the coast.

Comparación de la eficacia de los irrigantes OxOral ® y NaOCl en la eliminación de Enterococcus faecalis / Comparison of OxOral ® and NaOCl irrigants efficiency in Enterococcus faecalis elimination

Resumen Objetivo: Comparar la eficacia en la eliminación de Enterococcus faecalis con OxOral® versus hipoclorito de sodio a los 15 y 60 segundos. Material y métodos: Se incluyeron 36 cultivos de E. faecalis ATCC 29212 asignados en dos grupos; OxOral® e hipoclorito de sodio al 5.25% que a su vez fueron divididos en 15 y 60 segundos. Se colocaron 8 mL de agua peptonada, 1 mL del irrigante y 1 mL de la cepa, se dejó reposar. A cada tiempo se extrajo 1 mL y se sembró en agar sangre por 24 horas. Se empleó и de Mann-Whitney. Resultados: Con hipoclorito de sodio a 15 segundos hubo tres cultivos con crecimiento aceptable y seis extendido; a los 60 segúndos, cuatro tuvieron resultado eficaz, tres aceptable, uno extendido. Con OxOral® hubo crecimiento extendido en los nueve cultivos, en ambos tiempos, encontrando diferencias estadísticamente significativas a los 60 segundos (p < 0.01). Conclusión: La eliminación de E. faecalis fue mejor con hipoclorito de sodio a los 60 segundos.

ABSTRACT Objective: To compare effectiveness of ОхОrаl® versus sodium hypochlorite in Enterococcus faecalis elimination at 15 and 60 seconds. Material and methods: Material used in the study was 36 E, faecalis ATCC 29212 cultures assigned to two groups: ОхОrаl® and 5.25% sodium hypochlorite. Both groups were in turn divided into 15 and 60 second samples. Samples were placed in peptone water, 1 mL of irrigating solution and 1 mL of strain were left to rest. 1 mL was extracted at each time, samples were seeded into blood agar for 24 hours. Mann-Whitney и test was applied. Results: With sodium hypochlorite at 15 seconds, there were three cultures with acceptable growth and six with extended growth; at 60 seconds four cultures exhibited effective result, three acceptable and one extended. With ОхОrаl® there was extended growth in all nine cultures at both established times, significant statistical differences were found at the 60 seconds time (p < 0.01). Conclusion: E, faecalis elimination was better with sodium hypochlorite at 60 seconds.

Dissemination of Enterococcus faecalis and Enterococcus faecium in a ricotta processing plant and evaluation of pathogenic and antibiotic resistance profiles.

In this work, the sources of contamination by Enterococcus spp. in a ricotta processing line were evaluated. The isolated strains were tested for virulence genes (gelE, cylA,B, M, esp, agg, ace, efaA, vanB), expression of virulence factors (hemolysin and gelatinase), and the resistance to 10 different antibiotics. Enterococcus faecium and Enterococcus faecalis were subjected to discriminatory identification by intergenic spacer region (ITS)-polymerase chain reaction and sequencing of the ITS region. The results showed that Enterococcus spp. was detected in the raw materials, environment samples and the final product. None of the 107 Enterococcus isolates were completely free from all virulence genes considered. A fraction of 21.5% of the isolates containing all of the genes of the cylA, B, M operon also expressed ß-hemolysis. Most of the isolates showed the gelE gene, but only 9.3% were able to hydrolyze gelatin. In addition, 23.5% of the observed Enterococcus isolates had the vanB gene but were susceptible to vancomycin in vitro. The dissemination of antibiotic-resistant enterococci was revealed in this study: 19.3% of the E. faecium samples and 78.0% of the E. faecalis samples were resistant to at least one of the antibiotics tested. Sequencing of region discriminated 5 and 7 distinct groups among E. faecalis and E. faecium, respectively. Although some similarity was observed among some of the isolates, all E. faecalis and E. faecium isolates had genetic differences both in the ITS region and in the virulence profile, which makes them different from each other.

Efficacy of 3D conforming nickel titanium rotary instruments in eliminating canal wall bacteria from oval-shaped root canals.

OBJECTIVES: To evaluate the effectiveness of TRUShape® 3D Conforming Files, compared with Twisted Files, in reducing bacteria load from root canal walls, in the presence or absence of irrigant agitation. METHODS: Extracted human premolars with single oval-shaped canals were infected with Enterococcus faecalis. Teeth in Group I (N=10; NaOCl and QMix® 2in1 as respective initial and final irrigants) were subdivided into 4 subgroups: (A) TRUShape® instrumentation without irrigant activation; (B) TRUShape® instrumentation with sonic irrigant agitation; (C) Twisted Files without irrigant agitation; (D) Twisted Files with sonic irrigant agitation. To remove confounding factor (antimicrobial irrigants), teeth in Group II (N=10) were irrigated with sterile saline, using the same subgroup designations. Specimens before and after chemomechanical débridement were cultured for quantification of colony-forming units (CFUs). Data from each group were analyzed separately using two-factor ANOVA and Holm-Sidak multiple comparison (α=0.05). Canal wall bacteria were qualitatively examined using scanning electron microscopy (SEM) and light microscopy of Taylor-modified Brown and Brenn-stained demineralised sections. RESULTS: CFUs from subgroups in Group I were not significantly different (P=0.935). For Group II, both file type (P<0.001) and irrigant agitation (P<0.001) significantly affected log-reduction in CFU concentrations. The interaction of these two factors was not significant (P=0.601). Although SEM showed reduced canal wall bacteria, bacteria were present within dentinal tubules after rotary instrumentation, as revealed by light microscopy of longitudinal root sections. CONCLUSIONS: TRUShape® files removed significantly more canal wall bacteria than Twisted Files when used without an antibacterial irrigant; the latter is required to decontaminate dentinal tubules. CLINICAL SIGNIFICANCE: Root canal disinfection should not be focused only on a mechanistic approach. Rather, the rational choice of a rotary instrumentation system should be combined with the use of well-tested antimicrobial irrigants and delivery/agitation techniques to establish a clinically realistic chemomechanical débridement protocol.

A liaR deletion restores susceptibility to daptomycin and antimicrobial peptides in multidrug-resistant Enterococcus faecalis.

Daptomycin is a lipopeptide antibiotic that is used clinically against many gram-positive bacterial pathogens and is considered a key frontline bactericidal antibiotic to treat multidrug-resistant enterococci. Emergence of daptomycin resistance during therapy of serious enterococcal infections is a major clinical issue. In this work, we show that deletion of the gene encoding the response regulator, LiaR (a member of the LiaFSR system that controls cell envelope homeostasis), from daptomycin-resistant Enterococcus faecalis not only reversed resistance to 2 clinically available cell membrane-targeting antimicrobials (daptomycin and telavancin), but also resulted in hypersusceptibility to these antibiotics and to a variety of antimicrobial peptides of diverse origin and with different mechanisms of action. The changes in susceptibility to these antibiotics and antimicrobial peptides correlated with in vivo attenuation in a Caenorhabditis elegans model. Mechanistically, deletion of liaR altered the localization of cardiolipin microdomains in the cell membrane. Our findings suggest that LiaR is a master regulator of the enterococcal cell membrane response to diverse antimicrobial agents and peptides; as such, LiaR represents a novel target to restore the activity of clinically useful antimicrobials against these organisms and, potentially, increase susceptibility to endogenous antimicrobial peptides.

Penicillin-resistant, ampicillin-susceptible Enterococcus faecalis of hospital origin: pbp4 gene polymorphism and genetic diversity.

Despite the spread of penicillin-resistant, ampicillin-susceptible Enterococcus faecalis (PRASEF) isolates in diverse countries, the mechanisms leading to this unusual resistance phenotype have not yet been investigated. The aim of this study was to evaluate whether polymorphism in the pbp4 gene is associated with penicillin resistance in PRASEF isolates and to determine their genetic diversity. E. faecalis isolates were recovered from different clinical specimens of hospitalized patients from February 2006 to June 2010. The ß-lactam minimal inhibitory concentrations (MICs) were determined by E-test®. The PCR-amplified pbp4 gene was sequenced with an automated sequencer. The genetic diversities of the isolates were established by PFGE (pulsed-field gel electrophoresis) and MLST (multilocus sequencing typing). Seventeen non-producing ß-lactamase PRASEF and 10 penicillin-susceptible, ampicillin-susceptible E. faecalis (PSASEF) strains were analyzed. A single-amino-acid substitution (Asp-573→Glu) in the penicillin-binding domain was significantly found in all PRASEF isolates by sequencing of the pbp4 gene but not in the penicillin-susceptible isolates. In contrast to the PSASEF isolates, a majority of the PRASEFs had similar PFGE profiles. Six representative PRASEF isolates were resolved by MLST into ST9 and ST524 and belong to the globally dispersed clonal complex 9 (CC9). In conclusion, it appears quite likely that the amino acid alteration (Asp-573→Glu) found in the PBP4 of the Brazilian PRASEF isolates may account for their reduced susceptibility to penicillin, although other resistance mechanisms remain to be investigated.

Immunomodulatory properties of cell wall extract from Enterococcus faecalis CECT7121.

The aim of this study was to investigate the immunomodulatory properties of cell wall extract from Enterococcus faecalis CECT7121, measuring the induction of cytokines TNF-α, IL-6, IL-10 and IL-12 from human peripheral blood mononuclear cells (PBMCs). Cell wall extract was prepared from their growth in brain heart infusion broth (18h, 35°C). Subsequently, toxicity of the obtained cell wall extract was tested in Balb-C mice. PBMCs were isolated from buffy coats at the Blood Transfusion Service of Hospital Ramón Santamarina (Tandil, Argentina). PBMCs were purified using standard Ficoll-Paque gradient centrifugation. Aliquots of purified leukocytes were incubated at 37°C for 24h with heat-killed E. faecalis CECT7121 and cell wall extract. Concentrations of IL-6, TNFα, IL-10 and IL-12 (p70) were measured by solid phase sandwich ELISA. Changes in appearance and behavior of mice were evidenced only in the group with the maximal concentration of wall cell extract used (10,000µg). Cell wall extract and heat-killed E. faecalis CECT7121 induced the production of significantly higher amounts of Il-12, IL-6, TNF-α and IL-10 cytokines compared to the nonstimulated PBMCs. These findings provide helpful information on immunomodulation activity by cell wall extract in sight of the application of this compound in controlling certain infectious diseases.

Investigation of virulence factors of Enterococcus faecalis strains isolated in secondary/ persistent infections / Investigação de fatores de virulência de estirpes de Enterococcus faecalis isoladas em infecções secundárias persistentes

More virulent strains may result from the acquisition of genes by genetic exchange, pathogenicity islands in several species encoding toxins, adhesion factors and other factors associated with virulence. The aim of this study was to investigate the prevalence of E. faecalis strains in secondary endodontic/ persistent using endodontic infection by culture and PCR technqiues; and to investigate for the presence of virulence factor genes of gelatinase (gelE), cytolysin activator (Cyla), surface adhesion of Enterococcus (ESP) and collagen adhesin of Enterococcus (ACE). Material and methods: Microbial samples were obtained from 12 teeth with secondary/ persistent endodontic infection showing apical periodontitis. Culture techniques were used including serial dilution, plating, incubation, and biochemical identification. For PCR detection, samples were analyzed using a species-specific primer of the 16S rDNA and the downstream intergenic spacer region. Results: Culture and PCR detected the test species in 3/12 (25%) and 5/12 (41.6%) of teeth,respectively. A total of 38 Enterococcus faecalis strains were isolated and submitted to the virulence factor genes analysis. PCR products consistent with genes encoding surface adhesion (ESP), gelatinase (gelE) and collagen binding antigen (ACE) were found in 26/38 (68%), 31/38 (81%) and 38/38 (100%) of the isolates. The Cytolysin activator (Cyla) gene was not recovered from E. faecalis isolates. Conclusions: In conclusion, the present study revealed by culture and molecular methods revealed a high prevalence of E. faecalis in teeth with secondary/ persistent endodontic infection. Moreover, of a clinical relevance, we found different E. faecalis strains carrying different virulence determinants.

Objetivos: O objetivo deste estudo foi investigar a prevalência de cepas de E. faecalis em canais com infecções endodônticas secundárias/persistentes por meio de cultura e PCR, além de analisar a presença de fatores de virulência genéticos como: gelatinase (gelE), ativador de citolisina (Cyla), adesina de superfície (ESP) e adesina de colágeno (ACE). Material e métodos: Foram coletadas amostras de 12 canais radiculares com infecção endodôntica secundária/persistente e presença de lesão periapical. Para a cultura microbiológica foi realizada diluição em série, incubação e identificação bioquímica dos microrganismos, enquanto que no PCR as amostras foram analisadas através de primers específicos 16S rDNA. Os casos com presença de Enterococcus faecalis foram selecionadas para realização de análise quanto aos fatores de virulência: gelE, Cyla, ESP e ACE. Resultados: Enterococcus faecalis foi detectado através de cultura e PCR em 3/12 (25%) e 5/12 (41,6%) dos casos, respectivamente. No total, foram isoladas 38 amostras com presença de E. faecalis. Os produtos de PCR consistentes com os genes ESP, gelE e ACE foram encontrados em 26 /38 (68%), 31 /38 (81%) e 38/38 (100%) dos isolados. Cyla não foi recuperado a partir de E. faecalis em nenhum dos isolados. Conclusões: O presente estudo revelou alta prevalência de E. faecalis em dentes com infecção endodôntica secundária/ persistente. Estes microrganismos apresentaram elevado índice de diferentes fatores de virulência.

Virulence, bacterocin genes and antibacterial susceptibility in Enterococcus faecalis strains isolated from water wells for human consumption.

The objectives of this study were to detect genes for virulence and bacteriocins in addition to studying the antimicrobial susceptibility of 78 strains of E. faecalis isolated from water wells for human consumption. The virulence and bacteriocin genes of 78 E. faecalis were amplified by PCR and visualized in agarose gels. The antimicrobial susceptibility was determined through diffusion agar tests and the MIC through microdilution. It was observed that the major percentage of virulence genes in the E. faecalis strains corresponds to aggA (93.5%). The bacteriocin gene entA (64.1%) is the most frequently detected. The studied strains exhibited different virulence and bacteriocin genes, and an important antibacterial resistance. The most common resistant phenotype (n = 14) corresponds to tetracycline and chloramphenicol and the less frequent (n = 2) to ciprofloxacin and moxifloxacin. Eight different genetic profiles were observed for virulence y bacteriocin genes. It was determined a statistical association between the bacterial resistance and some of the genetic profiles detected.

Enterococcus spp.: Resistencia antimicrobiana en infecciones intrahospitalarias / Enterococcus spp.: Antimicrobial resistance in hospital- acquired infections / Enterococcus spp.: ResistÛncia antimicrobiana em infecþ§es intra-hospitalares

Entre los años 1996 y 2010 se estudiaron 1873 aislamientos de Enterococcus spp. pertenecientes a pacientes internados en un hospital universitario de la Ciudad Autónoma de Buenos Aires con infección intrahospitalaria. El 64,2% y el 30,4% de los aislamientos correspondieron a E. faecalis y E. faecium, respectivamente. En el periodo estudiado las infecciones por Enterococcus spp. representaron entre el 8% y el 10% del total de las infecciones nosocomiales. La prevalencia de E. faecium aumentó de un 1,5% en el año 1996 a un 4% en 2010. El primer aislamiento de enterococo resistente a vancomicina se detectó en el año 1998 y correspondió a un E. faecium y en el año 2004 se halló en E. faecalis. Actualmente más del 70% de los aislamientos de E. faecium son resistentes a vancomicina, no así en E. faecalis donde la resistencia es ocasional. No se detectó resistencia a linezolid ni a tigeciclina en Enterococcus spp.(AU)

One thousand eight hundred and seventy-three Enterococcus spp. isolates belonging to patients undergoing hospital-acquired infections at the University Hospital of Buenos Aires city were studied between the years 1996 and 2010. A total of 64.2% and 30.4% of the isolates were identified as E. faecalis y E. faecium respectively. The infections caused by Enterococcus spp. represented from 8% to 10% of the total number of the nosocomial infections in the period studied. The E. faecium prevalence increased from 1.5% in 1996 to 4% in 2010. The first vancomycin-resistant Enterococcus faecium was detected in the year 1998 and in the year 2004 this resistance was detected in E. faecalis as well. At present, more than 70% of the E. faecium isolates show vancomycin resistance; on the contrary, in the case of E. faecalis, this resistance is unusual. Resistance to linezolid or to tigecycline has not been detected in Enterococcus spp. so far.(AU)

Entre os anos 1996 e 2010 estudaram-se 1873 isolamentos de Enterococcus spp. pertencentes a pacientes internados num Hospital Universitário da Cidade Aut¶noma de Buenos Aires com infecþÒo intra-hospitalar. 64,2% e 30,4% dos isolamentos corresponderam a E. faecalis e E. faecium respectivamente. No estudado as infecþ§es por Enterococcus spp. representaram entre 8% e 10% do total das infecþ§es nosocomiais. A prevalÛncia de E. faecium aumentou de 1,5% no ano 1996 para 4% em 2010. O primeiro isolamento de enterococo resistente a vancomicina foi detectado no ano 1998 e correspondeu a um E. faecium e no ano 2004 foi achado em E. faecalis. Atualmente mais de 70% dos isolamentos de E. faecium sÒo resistentes a vancomicina, mas nÒo é assim em E. faecalis onde a resistÛncia é ocasional. NÒo se detectou resistÛncia a linezolid nem a tigeciclina em Enterococcus spp.(AU)

A preliminary study of the antibacterial potential of cetylpyridinium chloride in root canals infected by E. faecalis

The aim of this preliminary study was to verify the antibacterial potential of cetylpyridinium chloride (CPC) in root canals infected by Enterococcus faecalis. Forty human maxillary anterior teeth were prepared and inoculated with E. faecalis for 60 days. The teeth were randomly assigned to the following groups: 1: Root canal preparation (RCP) + 0.1% CPC with positive-pressure irrigation (PPI, Conventional, NaviTip®); 2: RCP + 0.2% CPC PPI; 3: RCP + 2.5% NaOCl PPI; 4: RCP + 2.5% NaOCl with negative-pressure irrigation system (NPI, EndoVac®); 5: Positive control; and 6: Negative control. Four teeth of each experimental group were evaluated by culture and 4 by scanning electron microscopy (SEM). In all teeth, the root canals were dried and filled with 17% EDTA (pH 7.2) for 3 min for smear layer removal. Samples from the infected root canals were collected and immersed in 7 mL of Letheen Broth (LB), followed by incubation at 37°C for 48 h. Bacterial growth was analyzed by turbidity of culture medium and then observed with a UV spectrophotometer. The irrigating solutions were further evaluated for antimicrobial effect by an agar diffusion test.The statistical data were treated by means, standard deviation, Kruskal-Wallis test and analysis of variance. Significance level was set at 5%. The results showed the presence of E. faecalis after root canal sanitization. The number of bacteria decreased after the use of CPC. In the agar diffusion test, CPC induced large microbial inhibition zones, similar to 2% chlorhexidine and large than 2.5% NaOCl. In conclusion, cetylpyridinium chloride showed antibacterial potential in endodontic infection with E. faecalis.

O objetivo deste estudo preliminar foi verificar o potencial antibacteriano de cloreto de cetilpiridínio (CCP) em canais radiculares infectados por E. faecalis. Quarenta dentes anteriores de humanos foram preparados e inoculados com E. faecalis por 60 dias. Os dentes foram aleatoriamente distribuídos como se segue: 1. Preparo do canal radicular (PCR) + CCP 0,1% com sistema de pressão positiva de irrigação (PPI, convencional, Navitip®); 2. PCR + CPC 0,2% PPI; 3. PCR + NaOCl 2,5% PPI, 4. PCR + NaOCl 2,5% com sistema de pressão negativa de irrigação (PNI, EndoVac®); 5 e 6. Controles positivos e negativos. Quatro dentes de cada grupo experimental foram avaliados por cultura e quatro por microscopia eletrônica de varredura (MEV). Em todos os dentes, os canais foram secos e preenchidos com EDTA 17% (pH 7,2) durante 3 min. As amostras dos canais radiculares infectados foram coletadas e imersas em 7 mL Letheen Broth (LB), seguido de incubação a 37° C durante 48 h. O crescimento bacteriano foi analisado pela turvação do meio de cultura, e mensurados por meio de um espectrofotometro (UV). As soluções irrigantes foram ainda avaliadas em teste de difusão em ágar. A análise estatística utilizou de média, desvio padrão,teste de Kruskal-Wallis e análise de variância. O nível de significância foi de 5%. Os resultados mostraram a presença de E. faecalis posterior ao processo de desinfecção do canal radicular. O cloreto de cetilpiridínio mostrou reduzir o número de bactérias. No teste de difusão em ágar, o CPC determinou inibição microbiana, com resultados semelhantes à CHX a 2% e maiores do que o hipoclorito de sódio a 2,5%. O cloreto de cetilpiridínio demonstrou potencial antibacteriano em infecção endodôntica por E. faecalis.

Isolation and characterization of potential lactic acid bacteria (LAB) from freshwater fish processing wastes for application in fermentative utilisation of fish processing waste

Proteolytic and/or lipolytic lactic acid bacteria (LAB) were isolated from visceral wastes of different fresh water fishes. LAB count was found to be highest in case of visceral wastes of Mrigal (5.88 log cfu/g) and lowest in that of tilapia (4.22 log cfu/g). Morphological, biochemical and molecular characterization of the selected LAB isolates were carried out. Two isolates FJ1 (E. faecalis NCIM5367) and LP3 (P. acidilactici NCIM5368) showed both proteolytic and lipolytic properties. All the six native isolates selected for characterization showed antagonistic properties against several human pathogens. All the native isolates were sensitive to antibiotics cephalothin and clindamycin; and, resistant to cotrimoxazole and vancomycin. Considering individually, P. acidilactici FM37, P. acidilactici MW2 and E. faecalis FD3 were sensitive to erythromycin. The two strains FJ1 (E. faecalis NCIM 5367) and LP3 (P. acidilactici NCIM 5368) that had both proteolytic and lipolytic properties have the potential for application in fermentative recovery of lipids and proteins from fish processing wastes.