Effect of N-acetyl cysteine, rifampicin, and ozone on biofilm formation in pan-resistant Klebsiella pneumoniae: an experimental study

ABSTRACT BACKGROUND: To the best of our knowledge, this is the first study to evaluate the effectiveness of specific concentrations of antibiofilm agents, such as N-acetyl cysteine (NAC), rifampicin, and ozone, for the treatment of pan-resistant Klebsiella pneumoniae (PRKp). OBJECTIVES: We evaluated the effectiveness of antibiofilm agents, such as NAC, rifampicin, and ozone, on biofilm formation in PRKp at 2, 6, 24, and 72 h. DESIGN AND SETTING: This single-center experimental study was conducted on June 15, 2017, and July 15, 2018, at Istanbul Faculty of Medicine, Istanbul University, Turkey. METHODS: Biofilm formation and the efficacy of these agents on the biofilm layer were demonstrated using colony counting and laser-screened confocal microscopy. RESULTS: NAC at a final concentration of 2 μg/mL was administered to bacteria that formed biofilms (24 h), and no significant decrease was detected in the bacterial counts of all isolates (all P > 0.05). Rifampicin with a final concentration of 0.1 μg/mL was administered to bacteria that formed biofilm (24 h), and no significant decrease was detected in bacterial count (all P > 0.05). Notably, ozonated water of even 4.78 mg/L concentration for 72 h decreased the bacterial count by ≥ 2 log10. CONCLUSION: Different approaches are needed for treating PRKp isolates. We demonstrate that PRKp isolates can be successfully treated with higher concentrations of ozone.

Antimicrobial activity of Er, Cr: YSGG and Ultrasonic on E. faecalisbiofilm in the mesial root canal systems of lower molars / Atividade antimicrobiana de Er, Cr: YSGG e Ultrassônico no biofilme de E. faecalis nos sistemas de canais radiculares mesiais de molares inferiores

The internal topography of the root canal is complex, especially for the permanent molar's mesial root. In response to such issues, improved irrigation techniques have been created, which use laser pulses to agitate fluids and improve microbial deposit removal. Objective: To assess the effectiveness of the Er,Cr:YSGG laser with a wavelength of 2,780 nm via photon-induced photoacoustic streaming (PIPS) protocol which agitated of 2% chlorohexidine (CHX) in removing mature Enterococcus faecalis (E. faecalis) biofilm in root canal systems of lower molars. Material and Methods: The mesial roots of lower first and second molars were separated and inoculated with E. faecalis bacterial suspension for 30 days. The roots were irrigated with CHX, some of them were agitated with a passive ultrasonic device (PUI), while the other roots were agitated by an Er,Cr:YSGG laser in PIPS at 60 µs/pulse, 5 Hz, (0.25, 0.5, 0.75, and 1) W. An atomic force microscope (AFM) was used as a new method to get the results in the isthmus area; the obtained results from each group were compared with each other. Results: Based on the AFM and SEM analyses, laser and ultrasonic activation groups showed higher antimicrobial efficacy than the conventional syringe irrigation group (P<0.05). Conclusion: Based on the investigation's findings, the activation of 2% CHX solution by Er,Cr:YSGG laser in PIPS and PUI offers better mature bacterial biofilm removal in the mesial root of lower human molars than the same irrigant with the SI technique (AU)

A topografia interna do canal radicular é complexa, especialmente para a raiz mesial do molar permanente. Em resposta a esses problemas, foram criadas técnicas aprimoradas de irrigação, que utilizam pulsos de laser para agitar fluidos e melhorar a remoção de depósitos microbianos. Objetivo: Avaliar a eficácia do laser Er,Cr:YSGG com comprimento de onda de 2.780 nm via protocolo de streaming fotoacústico induzido por fótons (PIPS) que agitou clorohexidina a 2% (CHX) na remoção de Enterococcus faecalis maduro (E. faecalis) biofilme em sistemas de canais radiculares de molares inferiores. Material e Métodos: As raízes mesiais de 28 primeiros e segundos molares inferiores foram separadas e inoculadas com suspensão bacteriana de E. faecalis por 30 dias. As raízes foram irrigadas com CHX, sendo algumas delas agitadas com aparelho ultrassônico passivo (PUI), enquanto as demais raízes foram agitadas com laser Er,Cr:YSGG em PIPS a 60 µs/pulso, 5 Hz (0,25, 0,5, 0,75 e 1) W. Um microscópio de força atômica (AFM) foi utilizado como um novo método para obter os resultados na área do istmo; os resultados obtidos de cada grupo foram comparados entre si. Resultados: Com base nas análises de AFM e SEM, os grupos de ativação por laser e ultrassom apresentaram maior eficácia antimicrobiana do que o grupo de irrigação com seringa convencional (P<0.05). Conclusão: Com base nos achados da investigação, a ativação da solução de CHX a 2% pelo laser Er,Cr:YSGG em PIPS a (60 µs/pulso, 5 Hz, 0,75 W) oferece melhor remoção de biofilme (AU)

Biological control of foodborne pathogens by lacticacid bacteria: A focus on juice processing industries

Abstract The use of lactic acid bacteria (LAB) in foods as biocontrol agents against foodborne pathogens has become increasingly known. Under the premise that controlling the adhesion of microorganisms to food contact surfaces is an essential step for meeting the goals of food processing, the aim of this work was to investigate the inhibitory and anti-biofilm effectiveness of Lactobacillus rhamnosus GG (ATCC 53103) and Lactobacillus casei (ATCC 393) against Escherichia coli O157:H7, Salmonella enterica and Listeria monocytogenes. Lactobacillus strains (108UFCCFU/ml) and pathogens (104UFCCFU/ml) were evaluated to monitor LAB anti-adhesive and antibiofilm effect, in two main scenarios: (i) co-adhesion and (ii) pathogen incorporation to stainless steel surfaces with a protective biofilm of Lactobacillus cells. In (i) the predominant effect was observed in L. rhamnosus against S. enterica and L. monocytogenes, whereas in (ii) both LAB significantly reduced the number of pathogenic adherent cells. The effect of pre-established LAB biofilms was more successful in displacing the three pathogens than when they were evaluated under co-adhesion. These findings show that both LAB can be considered good candidates to prevent or inhibit the adhesion and colonization of L. monocytogenes, S. enterica and E. coli O157:H7 on surfaces and conditions of relevance for juice processing industries, offering alternatives for improving the safety and quality of fruit-based products.

Resumen Existe un creciente interés en el uso de bacterias ácido lácticas (BAL) como agentes de biocontrol frente a patógenos de transmisión alimentaria. Bajo la premisa de que el control de la adhesión de microorganismos a superficies de contacto con alimentos es el paso esencial para evitar su contaminación, el objetivo de este trabajo fue investigar la efectividad inhibitoria y antibiofilm de Lactobacillus rhamnosus GG (ATCC 53103) y Lactobacillus casei (ATCC 393) frente a Escherichia coli O157:H7, Salmonella enterica y Listeria monocytogenes. A fin de cumplir con el objetivo propuesto, las cepas de Lactobacillus (108UFCUFC/ml) y los patógenos (104UFCUFC/ml) se ensayaron en 2 escenarios: (1) coadhesión, y (2) incorporación de los patógenos a las superficies de acero inoxidable con un biofilm preformado de Lactobacillus. En (1), el efecto predominante se observó con L. rhamnosus frente a S. enterica y L. monocytogenes, mientras que en (2), ambas BAL redujeron significativamente el número de células patógenas adheridas. En función de estos resultados, concluimos que el efecto de un biofilm preformado de ambas BAL fue más exitoso en el desplazamiento de los 3 patógenos que en coadhesión. Ambas BAL pueden considerarse buenas candidatas para mitigar la adhesión y colonización de L. monocytogenes, S. enterica y E. coli O157:H7 en superficies en condiciones de relevancia para la industria procesadora de jugos, y, de esta manera, ofrecer alternativas para mejorar la seguridad y calidad de los alimentos a base de frutas.

Role of Proteus mirabilis flagella in biofilm formation / Papel de los flagelos de Proteus mirabilis en la formación de biofilms

Abstract Proteus mirabilis (P. mirabilis) is a common etiological agent of urinary tract infec-tions, particularly those associated with catheterization. P. mirabilis efficiently forms biofilms on different surfaces and shows a multicellular behavior called 'swarming', mediated by flagella. To date, the role of flagella in P. mirabilis biofilm formation has been under debate. In this study, we assessed the role of P. mirabilis flagella in biofilm formation using an isogenic allelic replacement mutant unable to express flagellin. Different approaches were used, such as the evaluation of cell surface hydrophobicity, bacterial motility and migration across catheter sections, measurements of biofilm biomass and biofilm dynamics by immunofluorescence and confocal microscopy in static and flow models. Our findings indicate that P. mirabilis flagella play a role in biofilm formation, although their lack does not completely avoid biofilm genera-tion. Our data suggest that impairment of flagellar function can contribute to biofilm prevention in the context of strategies focused on particular bacterial targets.

Resumen Proteus mirabilis (P mirabilis) es un agente etiológico común de infecciones del tracto urinario, en particular de aquellas asociadas con cateterización. P. mirabilis forma biofilms eficientemente en diferentes superficies y muestra un comportamiento multicelular llamado swarming, mediado por flagelos. Hasta el momento, el papel de los flagelos en la formación de biofilms de P. mirabilis ha estado en discusión. En este estudio, se evaluó el papel de los flagelos de P. mirabilis en la formación de biofilms, utilizando una mutante isogénica generada por reemplazo alélico, incapaz de expresar flagelina. Se utilizaron diferentes enfoques, como la evaluación de la hidrofobicidad de la superficie celular, de la movilidad y la migración bacteriana sobre secciones de catéteres y medidas de biomasa y de la dinámica del biofilm mediante inmunofluorescencia y microscopia confocal, tanto en modelos estáticos como de flujo. Nuestros hallazgos indican que los flagelos de P. mirabilis desempeñan un papel en la formación de biofilms, aunque su falta no suprime por completo su generación. Asimismo, evidencian que la interferencia de la función flagelar puede contribuir a evitar la formación de biofilms en el contexto de estrategias centradas en blancos bacterianos particulares.

Enterotoxigenic profiles and submerged and interface biofilms in Bacillus cereus group isolates from foods / Perfiles enterotoxigénicos y biopelículas de superficie y sumergidas en aislados del grupo Bacillus cereus de alimentos

Abstract Biofilm formation by Bacillus cereus strains is now recognized as a systematic contaminaron mechanism in foods; the aim of this study was to evaluate the production of submerged and interface biofilms in strains of B. cereus group in different materials, the effect of dex-trose, motility, the presence of genes related to biofilms and the enterotoxigenic profile of the strains. We determine biofilm production by safranin assay, motility on semi-solid medium, toxin gene profiling and genes related to biofilm production by PCR in B. cereus group iso-lated from food. In this study, we observe strains used a higher production of biofilms in PVC; in the BHI broth, no submerged biofilms were found compared to phenol red broth and phenol red broth supplemented with dextrose; no strains with the ces gene were found, the enterotoxin profile was the most common the profile that includes genes for the three enterotoxins. We observed a different distribution of tasA and sipW with the origin of isolation of the strain, being more frequent in the strains isolated from eggshell. The production and type of biofilms are differential according to the type of material and culture medium used.

Resumen La formación de biopelículas por cepas de Bacillus cereus es reconocida como un mecanismo de contaminación sistemática en alimentos; el objetivo del estudio fue evaluar la producción de biopelículas sumergidas y de superficie en cepas del grupo de Bacillus cereus en diferentes materiales, el efecto de la dextrosa, la motilidad, la presencia de genes relacionados a biopelículas y el perfil enterotoxigénico de las cepas. Determinamos la producción de biopelículas por el ensayo de safranina, motilidad en medio sólido, perfil enterotoxigénico y genes relacionados a producción de biopelículas por PCR en aislados del grupo de Bacillus cereus de alimentos. En este estudio, observamos en las cepas utilizadas una alta producción de biopelículas en PVC; en caldo BHI, no se encontraron biopelículas sumergidas en comparación con el caldo rojo de fenol y caldo rojo de fenol suplementando con dextrosa; no se encontraron cepas con el gen ces, el perfil de enterotoxinas más común fue el perfil que incluía los genes de las tres enterotoxinas, también observamos una distribución diferente de tasA y sipW con relación al origen de la cepa, siendo más frecuente estos genes en las cepas aisladas de huevos. La producción y el tipo de biopelículas es diferente de acuerdo con el tipo de material y el medio de cultivo utilizado.

Sonication as a tool for disrupting biofilms and recovering microorganisms in bladder catheters / Sonicação como uma ferramenta para romper biofilmes e recuperar microrganismos em cateteres vesicais

Abstract Introduction: Urinary catheter-related infection is commonly associated with bacterial biofilm. The impact of anaerobes is unknown, but their detection in the biofilm on this device has not been previously reported. This study aimed to evaluate the capability to recovery strict, facultative, and aerobic microorganisms in patients using bladder catheters from ICUs using conventional culture, sonication, urinary analysis, and mass spectrometry. Methods: Parallel, sonicated bladder catheters from 29 critically ill patients were compared with their routine urine culture. Identification was performed using matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry. Results: The positivity rate in urine (n = 2, 3.4%) was lower than that in sonicated catheters (n = 7, 13.8%). Conclusion: Bladder catheter sonication showed more positive culture results than urine samples for anaerobic and aerobic microorganisms. The role of anaerobes in urinary tract infection and catheter biofilm is discussed.

Resumo Introdução: A infecção relacionada ao cateter urinário é comumente associada ao biofilme bacteriano. O impacto dos anaeróbios é desconhecido, mas sua detecção no biofilme deste dispositivo não foi relatada anteriormente. Este estudo teve como objetivo avaliar a capacidade de recuperar microrganismos estritos, facultativos e aeróbios em pacientes que utilizam cateteres vesicais de UTIs utilizando cultura convencional, sonicação, análise urinária e espectrometria de massa. Métodos: Paralelamente, foram comparados cateteres vesicais sonicados de 29 pacientes gravemente enfermos com sua urocultura de rotina. A identificação foi realizada utilizando dessorção/ionização a laser assistida por matriz com espectrometria de massa por tempo de voo. Resultados: A taxa de positividade na urina (n = 2; 3,4%) foi inferior à dos cateteres sonicados (n = 7; 13,8%). Conclusão: A sonicação do cateter vesical apresentou resultados de cultura mais positivos do que as amostras de urina para microrganismos anaeróbios e aeróbios. É discutido o papel dos anaeróbios na infecção do trato urinário e no biofilme do cateter.

In vitro and in situ caries-preventive effect of a new combined fluoride and calcium experimental nanocomposite solution

Abstract To assess the in vitro and in situ effect of experimental combined fluoride and calcium nanocomposite solutions on dental caries prevention. Nanocompound mesoporous silica (MS) with calcium (Ca) and sodium fluoride (NaF) - (MSCaNaF); MS with NaF (MSNaF), NaF solution (positive control), and deionized water (negative control - CG) were studied. The specimens (n=130) were submitted in vitro to a multispecies biofilm in the presence of 2% sucrose. After 24 h and 48 h, the culture medium pH, the percent of surface mineral loss (%SML), and lesion depth (ΔZ) were analyzed. In the in situ study, 10 volunteers participated in four phases of 7-days each. The products were applied on the specimens (n=240) before 20% sucrose solution drips. The polysaccharides (SEPS and IEPS), %SML and roughness (Sa) were evaluated. There was an in vitro decrease in pH values in 24h and 48h, compared to baseline. The MSCaNaF and MSNaF groups obtained lower values of %SML and ΔZ (p < 0.05) than CG and NaF after 24h and were similar to NaF after 48h (p<0.05). In situ results showed similar SEPS and IEPS among all groups after 48h. An after 7-days, the nanocomposites had similar values (p>0.05), while NaF was similar to CG (p>0.05). After 48h, the MSCaNaF and MSNaF reduced the %SML (p<0.05). After 7-days, both experimental nanocomposites were similar to NaF (p>0.05). Regarding Sa, MSCaNaF was better than NaF for both periods (p<0.05). The nanocomposites controlled the in vitro and in situ enamel demineralization, mainly in the initial periods.

Analysis of antimicrobial efficacy of sodium hypochlorite and ozonated water against biofilm in oval canals

Abstract This in vitro study compared the antimicrobial efficacy of 2.5% sodium hypochlorite (NaOCl) and 8 µg/mL ozonated water agitated by passive ultrasonic irrigation (PUI) or PUI combined with EndoActivator (EA) against mature multispecies biofilm. One hundred and five oval-shaped mandibular premolars were instrumented, sterilized, and inoculated with Enterococcus faecalis, Candida albicans, and Staphylococcus aureus, divided into: control group - saline; O3 group - ozonated water; O3 PUI group - ozonated water with PUI agitation; O3 PUI+EA group - ozonated water with PUI+EA agitation; NaOCl group - NaOCl; NaOCl PUI group - NaOCl with PUI agitation; and NaOCl PUI+EA group - NaOCl with PUI+EA agitation. Microbiological samples were collected before (S1) and after (S2) the disinfection procedures and the data were statistically analyzed using the Kruskal-Wallis test. In the culture method, there was significant disinfection in the O3 PUI+EA, NaOCl, NaOCl PUI, and NaOCl PUI+EA groups (p˂0.05). The combination of NaOCl with PUI+EA reduced microbial counts to zero (p˂0.05). In the qPCR method, there was a significant reduction in the total count of viable microorganisms in the O3 PUI, O3 PUI+EA, NaOCl, NaOCl PUI, and NaOCl PUI+EA groups (p˂0.05). It can be concluded that 2.5% NaOCl with and without agitation, as well as 8 µg/mL ozonated water with its action enhanced by the agitation techniques, were effective in root canal disinfection, and their antimicrobial efficacy is related to the microorganisms present in the biofilm.

Resumo Este estudo in vitro comparou a desinfecção do hipoclorito de sódio 2,5% (NaOCl) e da água ozonizada 8 µg/mL agitados pela irrigação ultrassônica passiva (PUI) e por associação da PUI com EndoActivator (EA) na redução de biofilme misto maduro. Cento e cinco pré-molares inferiores ovalados foram instrumentados, esterilizados e inoculados com Enterococcus faecalis, Candida albicans e Staphylococcus aureus, divididos em: Grupo controle: soro; Grupo O3: água ozonizada; Grupo O3 PUI: água ozonizada agitada por PUI; Grupo O3 PUI + EA: água ozonizada agitada por PUI e EA: Grupo NaOCl: hipoclorito de sódio; Grupo NaOCl PUI: hipoclorito de sódio agitado por PUI; Grupo NaOCl PUI + EA: hipoclorito de sódio agitado por PUI e EA. Amostras microbiológicas foram coletadas antes (S1) e após (S2) os procedimentos de desinfecção e os dados foram analisados estatisticamente pelo teste de Kruskal-Wallis. No método de cultura, houve desinfecção significativa nos grupos O3 PUI + EA, NaOCl, NaOCl PUI e NaOCl PUI + EA (p˂0.05), sendo que no grupo NaOCl PUI + EA não houve crescimento de microrganismo (p˂0.05). No método de qPCR, nas contagens dos microrganismos antes e após os protocolos de desinfecção, houve redução microbiana nos grupos O3 PUI, O3 PUI + EA, NaOCl, NaOCl PUI, NaOCl PUI + EA (p˂0.05). Concluiu-se que o NaOCl 2,5% com e sem agitação foi eficiente, assim como a água ozonizada 8 µg/mL potencializada pelos métodos de agitação na desinfecção e que a mesma está relacionada com os microrganismos presentes no biofilme.

In vitro evaluation of membranes for regenerative procedures against oral bacteria

Abstract The current literature on guided bone regeneration (GBR) and guided tissue regeneration (GTR) membrane contamination reports that the physicochemical characteristics of these biomaterials might influence affinity to bacteria, which appears to be a major drawback for the clinical outcome of the regenerative procedures. Thus, this study aimed to evaluate, in vitro, a multispecies biofilm adherence and passage of bacteria through different types of commercially available membranes for GTR/GBR. Four types of membranes were tested (n=12): LC) Lumina Coat®; JS) Jason®; BG) Biogide®; and LP) Lumina PTFE®. Aluminum foil (AL) simulated an impermeable barrier and was used as the control. The membranes were adapted to specific apparatus and challenged with a mixed bacterial culture composed of A. actinomycetemcomitans b, S. mutans, S. mitis, and A. israelii. After 2 h or 7 days, bacterial adhesion and passage of bacteria were evaluated through CFU counting, which was analyzed by two-way ANOVA e post hoc Tukey, at a 5% significance level. Representative areas of two membranes of each group were analyzed through scanning electron microscopy (SEM) to assess the morphology and organization of the biofilm over the membrane fibers. LC and LP presented similar values of adhered bacterial cells (p > 0.05), significantly inferior when compared to the other groups, in both time points (p < 0.05). All the tested groups were permeable to bacterial cells, with no significant difference between the trial period of 2 h and 7 days (p > 0.05). SEM analyses demonstrated that adhered bacteria number increased throughout the time points (2 h < 7 days). Commercially available biological membranes demonstrated intense bacterial adherence and passage of bacteria, which increased throughout the trial period.

Resumo O objetivo deste estudo foi avaliar, in vitro, a aderência do biofilme multiespécie e a passagem de bactérias através dos diferentes tipos de membranas disponíveis comercialmente para RTG/ROG. Quatro tipos de membranas foram testados (n=12): LC) Lumina Coat®; JS) Jason®; BG) Biogide®; e LP) Lumina PTFE®. Papel alumínio (AL) simulou uma barreira impermeável e foi usado como controle negativo. As membranas foram adaptadas à um aparato específico e desafiadas com uma cultura bacteriana mista composta de A. actinomycetemcomitans b, S. mutans, S. mitis, e A. israelii. Após 2 h ou 7 dias, a aderência e passagem bacteriana foi avaliada através da contagem de UFCs. Duas membranas de cada grupo foram analisadas através da microscopia eletrônica de varredura (MEV). LC e LP apresentaram valores semelhantes de células bacterianas aderidas (p < 0.05), significativamente inferiores quando comparados aos outros grupos, em ambos os períodos experimentais (p < 0.05). Desde a análise inicial, todos os grupos testados foram permeáveis às células bacterianas, sem diferença significativa entre o período experimental de 2 h e 7 dias (p > 0.05). As análises em MEV demonstraram que o número de bactérias aderidas aumentou com o tempo (2 h < 7 days). Membranas biológicas comercialmente disponíveis demonstraram intensa aderência bacteriana e passagem de bactérias, que aumentou durante os períodos experimentais.

Calidad microbiológica: detección de Aeromonas sp y Pseudomonas sp en garrafones provenientes de pequeñas plantas purificadoras de agua / Microbiological quality: detection of Aeromonas sp and Pseudomonas sp in jugs from small water purification establishments

RESUMEN Las plantas purificadoras de agua que carecen de un adecuado sistema de control de calidad pueden generar problemas de salud pública. El objetivo de este estudio fue examinar la calidad microbiológica del agua proveniente de pequeñas plantas purificadoras de la ciudad de Puebla, así como, determinar la existencia de bacterias Aeromonas sp y Pseudomonas sp, y caracterizar si presentan un fenotipo patógeno oportunista. Se recolectaron 70 muestras de garrafones de agua de 25 establecimientos. La cuantificación bacteriana se realizó mediante el método de goteo en placa. Se comprobaron los géneros microbianos mediante análisis bioquímico. En las cepas que mostraron discrepancia se utilizó la identificación molecular con base a secuencias parciales del gen 16S rRNA para confirmar su especie y se les evaluaron sus características de patogenicidad: multirresistencia a antibióticos, producción de biopelícula y actividad hemolítica. El 40 % de las plantas purificadoras no cumplieron con la calidad microbiológica del agua para consumo humano. El 41.4 % de los garrafones de agua muestreados incumplió la normativa, presentando coliformes totales 35.7 %, Pseudomonas 30 %, Enterococcus faecalis 8.6 % y bacterias coliformes fecales el 5.7 %. Se obtuvieron 56 aislados, provenientes de los 29 garrafones contaminados; 10 de ellos se caracterizaron molecularmente, resultando 7 aislados relacionados con especies diferentes de P. aeruginosa y 3 con especies de Aeromonas. De los aislados de Pseudomonas, 5 presentaron resistencia a 2 familias de antibióticos y 2 mostraron multirresistencia. El 36 % de los 10 aislados produjeron hemólisis y biopelícula. Dos cepas de Aeromonas mostraron resistencia a Cefalosporina 3a generación pero no produjeron hemólisis. Los 10 aislados analizados fueron clasificados como no patógenos. Es necesario un seguimiento sanitario más estricto para lograr el cumplimiento de las normas nacionales e internacionales relacionadas con el consumo de agua purificada, para evitar dañar la salud de los consumidores.

ABSTRACT Water purification establishments that lack an adequate quality control system can cause public health problems. The objective of this study was to examine the microbiological quality of water from small purification establishments in the city of Puebla, as well as to determine the existence of Aeromonas sp and Pseudomonas sp bacteria, and to characterize whether they present an opportunistic pathogenic phenotype. 70 water jug samples were collected from 25 establishments. Bacterial quantification was performed using the drop plate method. Microbial genera were determined by biochemical analysis using the standard methodology. In the strains that showed discrepancy, molecular identification based on partial sequences of the 16S rRNA gene was used to confirm their species, and their pathogenic characteristics were evaluated: multiresistance to antibiotics, biofilm production, and hemolytic activity. The results showed that 40 % of the purification establishments did not comply with the microbiological quality of water for human consumption. Similarly, 41.4 % of the jugs of water sampled failed to comply with the regulations, presenting total coliforms 35.7 %, Pseudomonas 30 %, Enterococcus faecalis 8.6 % and fecal coliform bacteria 5.7 %. Likewise, 56 isolates were obtained from the 29 contaminated jugs, of which 10 were molecularly characterized, resulting in 4 different species for P. aeruginosa and 3 for Aeromonas. Of the 7 Pseudomonas isolates, 5 presented resistance to 2 families of antibiotics and 2 showed multiresistance. In total, 36 % of the 10 isolates produced hemolysis and biofilm. Two Aeromonas strains showed resistance to 3rd generation Cephalosporin but did not produce hemolysis. The 10 isolates analyzed were classified as non-pathogenic. A stricter sanitary monitoring is necessary to achieve compliance with national and international standards related to the consumption of purified water, to avoid harming the health of consumers.

Detection of Antibiotic Resistance and Biofilm-Producing Ability of Staphylococcus Species in Clinical Isolates

Background: Staphylococci are responsible for life-threatening infections in hospitals and community. Their ability to produce multiple virulence factors and antibiotic resistance is an important reason of high mortality in staphylococcal infections. Biofilm production by these organisms makes it difficult to treat. Most of the treating antibiotics are failing and making it a matter of concern. Aims?This study aims to detect the increased antibiotic resistance in biofilm-producing Staphylococcus and to compare the performance of three potential methods of detection. Methods?A total of 81 isolates of staphylococci including coagulase negative staphylococci (CoNs), methicillin resistant S. aureus (MRSA), and methicillin sensitive S. aureus (MSSA) are included in this study. After the identification, an antibiotic sensitivity test was performed. Biofilm detection was done by three different methods: Congo red agar method, tube adherence method, and microtiter plate method. Result?Out of the 81 samples, 37 CoNs, 17 MRSA, and 27 MSSA were identified. Out of them we got 43 (53%) biofilm producers by Congo red agar method, 40 (49%) by tube adherence method, and 52 (64%) producers by tissue culture plate/microtiter plate method. Most of the biofilm producers showed multiple drug resistance. Conclusion?We found out that the microtiter plate method is sensitive and reliable as compared with the other two methods. Antibiotic resistance was found to be very common in biofilm producers. This was due to the resistance developed as a result of the matrix that does not let the antibiotic bind with the organisms. This can make the treatment of Staphylococcus very difficult in the future as the rate of drug resistance is faster as compared with newly emerging antibiotics.

Escherichia coli productora de toxina Shiga: el desafío de adherirse para sobrevivir / Shiga toxin producing Escherichia coli: the challenge of adherence to survive

Resumen Las cepas de Escherichia coli productoras de toxina Shiga (STEC) son reconocidas como responsables de un alto número de casos de enfermedades de transmisión alimentaria a nivel mundial. Su patogenicidad ha sido vinculada directamente con la actividad de las toxinas (Stx); sin embargo, la habilidad de estas bacterias para colonizar al huésped y otras superficies puede ser esencial para desarrollar su poder patogénico. La gran plasticidad genómica de cepas STEC se infiere de la variabilidad de perfiles de virulencia, con la frecuente emergencia de cepas con nuevos genes, codificados en nuevas islas de patogenicidad vinculadas al metabolismo y la adherencia. La formación de biofilm es un mecanismo espontáneo por el cual las cepas STEC resisten en un ambiente hostil, lo que les permite sobrevivir y, de esa forma, llegar al huésped, a través de los alimentos o de las superficies que están en contacto con ellos. Este mecanismo presenta una alta variabilidad intra e interserotipo y su desarrollo no depende solo de los microorganismos que lo conforman. Factores inherentes al ambiente (pH, temperatura) y la superficie (acero inoxidable, poliestireno) a la que pueden adherirse influyen en la expresión de biofilm. El concepto «una salud¼ implica la interrelación entre los actores de salud pública, animal y ambiental para lograr alimentos inocuos y evitar contaminación cruzada y resistencia a sanitizantes, lo cual pone de manifiesto la necesidad de identificar patógenos emergentes a través de nuevos marcadores moleculares, que detecten cepas STEC portadoras del denominado locus for enterocyte effacement (LEE) o del locus de adherencia y autoagregación (LAA).

Abstract Shiga Toxin-producing Escherichia coli (STEC) is recognized as being responsible for a large number of foodborne illnesses around the world. The pathogenicity of STEC has been related to Stx toxins. However, the ability of STEC to colonize the host and other surfaces can be essential for developing its pathogenicity. Different virulence profiles detected in STEC could cause the emergence of strains carrying new genes codified in new pathogenicity islands linked to metabolism and adherence. Biofilm formation is a spontaneous mechanism whereby STEC strains resist in a hostile environment being able to survive and consequently infect the host through contaminated food and food contact surfaces. Biofilm formation shows intra-and inter-serotype variability, and its formation does not depend only on the microorganisms involved. Other factors related to the environment (such as pH, temperature) and the surface (stainless steel and polystyrene) influence biofilm expression. The «One Health¼ concept implies the interrelation between public, animal, and environmental health actors to ensure food safety, prevent cross-contamination and resistance to sanitizers, highlighting the need to identify emerging pathogens through new molecular markers of rapid detection that involve STEC strains carrying the Locus of Enterocyte Effacement or Locus of Adhesion and Autoaggregation.

Effects of BlueM® against Streptococcus mutans biofilm and its virulence gene expression

Abstract This study evaluated the antimicrobial capacity of BlueM® mouthwash against the bacterium Streptococcus mutans and its influence on gbpA gene expression as well as its cytotoxic effect on fibroblast cells. BlueM® showed antimicrobial activity, with MIC and MBC values of 0.005% and 0.01%, respectively. The MBIC was 6.25% for S. mutans. CFU count and confocal microscopy revealed significant effect of BlueM® on S. mutans biofilm pre-formed on dentin surfaces. Interestingly, the analysis of gbpA gene expression indicated a decrease in gene expression after 15 min of treatment with BlueM® at a concentration of 25%. Moreover, BlueM® exhibited low levels of cytotoxicity. In conclusion, our results showed the antimicrobial effectiveness of BlueM® against S. mutans, its ability to modulate the expression of the gbpA gene and its low cytotoxicity. This study supports the therapeutic potential of BlueM® as an alternative agent for the control of oral biofilm.

Resumo Este estudo avaliou a capacidade antimicrobiana do enxaguatório BlueM® contra a bactéria Streptococcus mutans e sua influência na expressão do gene gbpA, bem como seu efeito citotóxico em células de fibroblastos. BlueM® apresentou atividade antimicrobiana, com valores de CIM e CBM de 0,005% e 0,01%, respectivamente. O MBIC foi de 6,25% para S. mutans. A contagem de UFC e a microscopia confocal revelaram efeito significativo do BlueM® no biofilme de S. mutans pré-formado nas superfícies de dentinas. Curiosamente, a análise da expressão do gene gbpA, indicou uma diminuição na expressão do gene após 15 min de tratamento com BlueM® na concentração de 25%. Além disso, BlueM® exibiu baixos níveis de citotoxicidade. Em conclusão, nossos resultados mostraram a eficácia antimicrobiana do BlueM® contra S. mutans, sua capacidade de modular a expressão do gene gbpA e sua baixa citotoxicidade. Este estudo apoia o potencial terapêutico do BlueM® como agente alternativo para o controle do biofilme oral.

Antimicrobial Resistance, Phenotypic Characteristics, and Biofilm Production in Citrobacter freundii Isolates Obtained from Urinary Tract Infections

Objective To evaluate the link between phenotypic traits, antimicrobial resistance, and biofilm-producing capacity of urinary isolates of Citrobacter freundii (C. Freundii). Methods Both pan-antibiotic-susceptible and -resistant C. freundii isolates (n = 120) obtained from laboratory-confirmed urinary tract infections were analyzed for their link between antimicrobial resistance, phenotypic characteristics, and biofilm production. Results Of the total C. freundii isolates (n = 120), 30% (37/120) of them formed large colonies. Among the total large colonies produced (n = 37), they were present in 21.62%, 10.81%, 13.5%, 16.2%, 21.62%, and 16.21% in the control group, CIP-group, FOS-group, COT-group, Gent-group, and ESBL groups, respectively. Compared to the pan-susceptible isolates, the occurrence of large-sized-colony-forming strains was relatively reduced in most of the drug-resistant groups. The overall distribution of mucoid colonies produced (n = 54) includes 9.25%, 18.51%, 16.66%, 18.51%, 20.3%, and 16.66% in the control group, CIP-group, FOS-group, COT-group, Gent-group, and ESBL groups, respectively. Of the total isolates that produced biofilm (n = 51), 11.76% of isolates showed biofilm formation in the control group. Alternatively, the rate was found to be 15.68%, 11.76%, 25.49%, 19.6%, and 15.68% in the CIP-group, FOS-group, SXT-group, Gen-group, and ESBL-groups, respectively. Conclusion This study correlates the association between phenotypic characteristics, antimicrobial resistance, and biofilm production, the three main characteristics of C. Freundii.

Biofilm dysbiosis and caries activity: a surface or an individual issue?

Abstract Objective This study aimed to analyze the functional profile of supragingival biofilm from sound (CAs), active (CAa), and inactive (CAi) enamel caries lesions from caries-active individuals to provide insights into the diversity of biological processes regarding biofilm dysbiosis. Methodology A metatranscriptome analysis was performed in biofilm samples collected from five caries-active individuals. Total RNA was extracted, and the microbial cDNAs were obtained and sequenced (Illumina HiSeq3000). Trimmed data were submitted to the SqueezeMeta pipeline in the co-assembly mode for functional analysis and further differential gene expression analysis (DESeq2). Results Bioinformatics analysis of mRNAs revealed a similar functional profile related to all analyzed conditions (CAa, CAi, and CAs). However, active and inactive surfaces share up-regulated genes (gtsA; qrtT; tqsA; pimB; EPHX1) related to virulence traits that were not overrepresented in sound surfaces. From a functional perspective, what matters most is the individual carious status rather than the surface condition. Therefore, pooling samples from various sites can be carried out using naturally developed oral biofilms but should preferably include carious surfaces. Conclusion Metatranscriptome data from subjects with caries activity have shown that biofilms from sound, arrested, and active lesions are similar in composition and function.

In-vitro effect of a single application of CPP-ACP pastes and different fluoridated solutions on the prevention of dental caries around orthodontic brackets

ABSTRACT Objective: To assess the in-vitro effect of single applications of CPP-ACP pastes and different fluoridated solutions on the prevention of dental caries around orthodontic brackets. Material and Methods: Tooth/bracket sets (n=65) were immersed in artificial saliva (1h at 37ºC) and randomly subjected to single applications (100µL; 1min) of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP emulsion), CPP-ACP with fluoride (CPP-ACPF emulsion), solutions of titanium tetrafluoride (TiF4) or sodium fluoride (NaF), or no treatment (CG). Multispecies biofilm (5 x 105 CFU/mL) was formed in the presence of 2% sucrose. After 24 h, the pH and the concentration of total soluble fluoride (TSF) were analyzed by culture medium. The presence of active white spot lesions (WSL) evaluated by macroscopic examination and the percent surface mineral loss (%SML) were analyzed. Also, the topography of enamel was detected by analysis of scanning electron microscopy (SEM). The data was assessed by chi-square, Kruskal-Wallis, and Mann-Whitney tests (p < 0.05). Results: Fluoride-containing compounds led to a smaller pH reduction than did CPP-ACP and CG (p<0.05). There was difference in TSF between the groups (p<0.05), denoted as TiF4> NaF > CPP-ACPF > CPP-ACP > CG. Regarding the presence of WSL and %SML, the NaF group obtained lower values (p<0.05), while TiF4 and CPP-ACPF were similar (p>0.05). SEM demonstrated that fluoride-free groups had a larger surface dissolution. Conclusion: Fluoridated groups including solutions and CPP-ACPF were more effective than CPP-ACP in reducing enamel demineralization around orthodontic brackets after a single application.

RESUMO Objetivo: Avaliar in-vitro o efeito de uma aplicação única de cremes dentais de CPP-ACP e diferentes soluções fluoretadas na prevenção da cárie dentária ao redor de braquetes ortodônticos Material e Métodos: O conjunto dentes/braquetes (n=65) foi imerso em saliva artificial (1h em 37°C) randomizado e submetido a tratamento único (100µL; 1 min) de emulsão de fosfopeptídeo de caseína-fosfato de cálcio amorfo (CPP-ACP) e CPP-ACP associado ao flúor (CPP-ACPF); soluções de tetrafluoreto de titânio (TiF4) e fluoreto de sódio (NaF); e ausência de tratamento (GC). Biofilmes multiespécie (5 x 105 CFU/mL) foram formados na presença de sacarose a 2%. Após 24h, o pH e a concentração de fluoreto solúvel total (FST) foram analisados pelo meio de cultura. Foram avaliadas a presença de lesões de mancha branca (LMB), por meio da análise de macroscopia visual, e a porcentagem de perda de dureza (%PD). Também foi verificada a topografia do esmalte, usando microscopia eletrônica de varredura (MEV). Os dados foram analisados pelos testes Qui-quadrado, Kruskal-Wallis e Mann-Whitney (p < 0,05). Resultados: Os compostos contendo flúor levaram a uma redução do pH menor do que o CPP-ACP e GC (p<0,05). Houve diferença no FST entre os grupos (p <0,05), sendo TiF4> NaF > CPP-ACPF > CPP-ACP > GC. Quanto à presença de LMB e à %PD, o grupo NaF obteve os menores valores (p<0,05), enquanto TiF4 e CPP-ACPF foram semelhantes (p> 0,05). A MEV demonstrou que os grupos sem flúor tiveram uma dissolução superficial maior. Conclusão: Os grupos fluoretados, incluindo soluções e CPP-ACPF, foram mais eficazes do que o CPP-ACP sem flúor na redução da desmineralização do esmalte ao redor dos braquetes ortodônticos após uma única aplicação.

Extratos hidroetanólicos de canela (Cinnamomum verum) e romã (Punica granatum L) promovem ação antibacteriana frente cepas resistentes a antibióticos de Acinetobacter baumannii e Pseudomonas aeruginosa / Cinnamon (Cinnamomum verum) and pomegranate (Punica granatum L) hydroethanolic extracts promote antibacterial action against antibiotic-resistant strains of Acinetobacter baumannii and Pseudomonas aeruginosa

A resistência bacteriana tem aumentado progressivamente no mundo, assim, há necessidade de novas opções de tratamentos. A fitoterapia tem ganhado notoriedade para combater infecções, principalmente as causadas por bactérias resistentes aos antibacterianos disponíveis. Diante do exposto, o presente estudo teve como objetivo preparar e analisar a composição fitoquímica e a ação antibacteriana dos extratos hidroetanólicos de canela (EHC) e romã (EHR) isolados e associados frente culturas planctônicas e biofilmes de cepas padrão e clínicas de Acinetobacter baumannii e Pseudomonas aeruginosa, além disso, analisar a ação citotóxica dos extratos em queratinócitos humanos (HaCat). Para isso, os EHC e EHR foram preparados e quantificado o teor de sólidos solúveis. Posteriormente, foi quantificado o teor de flavonoides e fenóis totais, análise antioxidante por meio da redução do radical 2,2'-difenil-1-picrilhidrazila (DPPH), e a fitoquímica por cromatografia líquida (HPLC). Em relação a ação antibacteriana dos extratos, foi aplicado o teste de microdiluição em caldo (CLSI ­ M7-A9) e a ação sinérgica realizada por meio do ensaio de checkerboard. As concentrações mais efetivas foram analisadas sobre biofilmes em formação (prevenção) e biofilmes formados (tratamento de 24 h), e quantificada a viabilidade por meio do teste colorimétrico MTT. Para avaliar a citotoxidade, os tratamentos foram aplicados sobre cultura celular de HaCat por 24 h e analisados por meio do teste colorimétrico MTT. A análise estatística foi realizada com 5% de significância (p<0.05), analisados pelo método ANOVA complementado pelo Teste de Tukey. Os resultados demonstraram que os EHC e EHR possuem ação antioxidante e presença de fitocompostos. Os extratos apresentaram ação antibacteriana para todas as cepas avaliadas, quando os mesmos foram associados, obteve-se concentrações sinérgicas para as cepas clínicas de A. baumannii. Em relação a ação antibiofilme, o EHC inibiu a formação em 95% e EHR em 96% do biofilme de #Ab 1, enquanto a cepa #Pa 2 teve 92% e 93% de inibição quando em contato com EHC e EHR, respectivamente. Após tratamento de 24 h em biofilmes formados, as reduções da viabilidade foram de 72% para as cepas #Ab 2 e #Ab 3 quando em contato com o EHC, já EHR inibiu em 83% a viabilidade da cepa #Ab ATCC. Para P. aeruginosa (#Pa 2), as reduções da viabilidade foram de 84% e 88,5% quando tratados com EHC e EHR, respectivamente. A avaliação da citotoxicidade em HaCat demonstrou que após tratamentos com diferentes concentrações dos extratos a viabilidade celular se manteve acima de 70% em todos os grupos. Diante disso, conclui-se que os EHC e EHR apresentam importante ação antioxidante e antibacteriana, tanto em culturas planctônicas quanto em biofilmes, e não apresentaram efeitos citotóxicos na faixa de concentração testada. (AU)

Bacterial resistance has progressively increased in the world, thus, there is a need for new treatment options. Phytotherapy has gained notoriety for fighting infections, mainly those caused by bacteria resistant to available antibacterials. In view of the above, the present study aimed to prepare and analyze the phytochemical composition and antimicrobial action of hydroethanolic extracts of cinnamon (EHC) and pomegranate (EHR) isolated and associated against planktonic cultures and biofilms of standard and clinical strains of Acinetobacter baumannii and Pseudomonas aeruginosa, in addition, analyze the cytotoxic action of the extracts on human keratinocytes (HaCat). For this, the EHC and EHR were prepared and the soluble solids content was quantified. Subsequently, the content of flavonoids and total phenols, antioxidant analysis through the reduction of the radical 2,2'-diphenyl1-picrylhydrazyl (DPPH), and phytochemistry by liquid chromatography (HPLC) were quantified. Regarding the antimicrobial action of the extracts, the broth microdilution test (CLSI ­ M7-A9) was applied and the synergistic action was performed through the checkerboard test. The most effective concentrations were analyzed on forming biofilms (prevention) and formed biofilms (24 h treatment), and viability was quantified using the MTT colorimetric test. To evaluate the cytotoxicity, the treatments were applied on HaCat cell culture for 24 h and analyzed using the MTT colorimetric test. Statistical analysis was performed with 5% significance (p<0.05), analyzed by the ANOVA method complemented by the Tukey test. The results showed that the EHC and EHR have antioxidant action and presence of phytocompounds. The extracts showed antibacterial action for all evaluated strains, when they were associated, synergistic concentrations were obtained for the clinical strains of A. baumannii. Regarding the antibiofilm action, EHC inhibited formation by 95% and EHR by 96% of the #Ab 1 biofilm, while the #Pa 2 strain had 92% and 93% inhibition when in contact with EHC and EHR, respectively. After 24 h treatment in formed biofilms, viability reductions were 72% for strains #Ab 2 and #Ab 3 when in contact with EHC, whereas EHR inhibited the viability of strain #Ab ATCC by 83%. For P. aeruginosa (#Pa 2), viability reductions were 84% and 88.5% when treated with EHC and EHR, respectively. The evaluation of cytotoxicity in HaCat showed that after treatments with different concentrations of extracts, cell viability remained above 70% in all groups. Therefore, it is concluded that EHC and EHR have important antioxidant and antibacterial action, both in planktonic cultures and in biofilms, and did not show cytotoxic effects in the tested concentration range. (AU)

Asociación entre obesidad y caries dental en niños y adolescentes. Revisión

Obesidad y caries dental comparten factores de riesgo comunes y modificables: dieta y estilo de vida. Objetivo: Identificar y analizar la literatura disponible sobre la posible asociación entre obesidad y caries dental en niños y adolescentes. Método: Dos investigadoras realizaron una revisión de la literatura en idiomas español, inglés y portugués utilizando Pubmed, SciELO, Latindex y Cochrane (obesidad AND índice de masa corporal AND caries AND niños OR adolescentes). Resultados: Se identificaron 115 artículos y fueron incluidos 28 luego del análisis a texto completo (21 estudios transversales, 4 longitudinales, 3 revisiones sistemáticas). Cuatro estudios transversales y uno longitudinal mostraron asociación entre obesidad y presencia de caries. Conclusiones: Los estudios analizados sobre asociación entre obesidad y caries presentan resultados inconsistentes. El origen multifactorial de las patologías analizadas puede contribuir a rechazar la hipótesis de asociación de ambas patologías a partir del consumo excesivo de carbohidratos y azúcares fermentables.

A obesidade e a cárie dentária compartilham fatores de risco comuns e modificáveis: dieta e estilo de vida. Objetivo: Identificar e analisar a literatura disponível sobre a possível associação entre obesidade e cárie dentária em crianças e adolescentes. Método: Dois pesquisadoras realizaram uma revisão da literatura em espanhol, inglês e português usando Pubmed, SciELO, Latindex e Cochrane (obesidade E índice de massa corporal E cárie E crianças ou adolescentes). Resultados: 115 artigos foram identificados e 28 foram incluídos após análise do texto completo (21 estudos transversais, 4 estudos longitudinais, 3 revisões sistemáticas). Quatro estudos transversais e um longitudinal demonstraram a associação entre obesidade e presença de cárie. Conclusões: Os estudos analisados ​​sobre a associação entre obesidade e cárie apresentam resultados inconsistentes. A origem multifatorial das patologias analisadas pode contribuir para rejeitar a hipótese de associação entre elas a partir do consumo excessivo de carboidratos e açúcares fermentescíveis.

Establishment of a saliva donor selection for in vitro biofilm growth / Seleção de doadores de saliva para crescimento de biofilme in vitro

Introdução: O emprego de biofilmes polimicrobianos, utilizando a saliva como inóculo, é um modelo promissor para o estudo de biofilmes cariogênicos in vitro. Entretanto, ainda não existe uma padronização para seleção de doadores de saliva. Objetivo: O objetivo deste estudo foi estabelecer uma metodologia para seleção de doadores de saliva utilizando fatores salivares microbianos e características in vitro do biofilme. Material e método: Para doação de saliva foram selecionados vinte voluntários. Os voluntários permaneceram 24 horas sem escovar os dentes e ficaram em jejum por 2 horas antes da coleta da saliva. Foram avaliados os seguintes parâmetros: viabilidade das bactérias anaeróbias totais e mutans streptococci; concentração inibitória mínima (CIM) e concentração bactericida mínima (CBM) da clorexidina; capacidade de formação de biofilme por meio da biomassa; e a suscetibilidade dos biofilmes à clorexidina. Resultado: A viabilidade bacteriana da saliva, a capacidade de formação de biofilme e a suscetibilidade do biofilme à clorexidina foram apresentadas como média e intervalo de confiança (95%). A diferença entre a viabilidade do biofilme (mutans streptococci e bactérias totais) após tratamento com NaCl 0,9% e diacetato de clorexidina 0,2% foi comparada pelo teste t de Student com nível de significância estabelecido em 5%. A viabilidade total de bactérias anaeróbias (mediana) foi de 7,28 log 1+UFC/mL (unidades formadoras de colônia/mL). A viabilidade dos mutans streptococci na saliva apresentou mediana de 5,47 log 1+UFC/mL. Para capacidade de formação de biofilme a mediana da biomassa foi de 0,1172 A570. Conclusão: O tratamento com clorexidina reduziu significativamente os mutans streptococci e a viabilidade total das bactérias. A metodologia para seleção do doador de saliva foi estabelecida com sucesso.

Introduction: The utilization of polymicrobial biofilms, with saliva as an inoculum, represents a promising model for in vitro studies on cariogenic biofilms. However, there is still no standardization for selecting saliva donors. Objective: The aim of this study is to establish a methodology for the selection of saliva donors using microbial salivary factors and in vitro biofilm characteristics. Material and method: For saliva donation, twenty volunteers were selected. Volunteers remained 24 h without brushing their teeth and fasted for 2 h before saliva collection. The following parameters were evaluated: total anaerobic bacteria and mutans streptococci viability; minimum inhibitory concentration (MIC) and minimum bactericide concentration (MBC) of chlorhexidine; biofilm forming capacity by biomass assessment; and the susceptibility of biofilms to chlorhexidine. Result: Saliva bacterial viability, biofilm forming capacity and biofilm susceptibility to chlorhexidine were presented as mean and confidence interval (95%). The difference between biofilm (mutans streptococci and Total bacteria) viability after treatment with NaCl 0.9% and 0.2% chlorhexidine diacetate was compared using the Student t-test with a significance level established at 5%. Total anaerobic bacteria viability (median) was 7.28 log 1+CFU/mL (colony forming units/ mL). Mutans streptococci viability in the saliva showed a median of 5.47 log 1+CFU/mL. Biofilm forming capacity showed that biomass had a median of 0.1172 A570. Conclusion: Treatment with chlorhexidine significantly reduced mutans streptococci and total bacteria viability. The methodology for the selection of the saliva donor was successfully established.

Bilayer hydrogel dressing with lysozyme-enhanced photothermal therapy for biofilm eradication and accelerated chronic wound repair

Biofilms are closely associated with the tough healing and dysfunctional inflammation of chronic wounds. Photothermal therapy (PTT) emerged as a suitable alternative which could destroy the structure of biofilms with local physical heat. However, the efficacy of PTT is limited because the excessive hyperthermia could damage surrounding tissues. Besides, the difficult reserve and delivery of photothermal agents makes PTT hard to eradicate biofilms as expectation. Herein, we present a GelMA-EGF/Gelatin-MPDA-LZM bilayer hydrogel dressing to perform lysozyme-enhanced PTT for biofilms eradication and a further acceleration to the repair of chronic wounds. Gelatin was used as inner layer hydrogel to reserve lysozyme (LZM) loaded mesoporous polydopamine (MPDA) (MPDA-LZM) nanoparticles, which could rapidly liquefy while temperature rising so as to achieve a bulk release of nanoparticles. MPDA-LZM nanoparticles serve as photothermal agents with antibacterial capability, could deeply penetrate and destroy biofilms. In addition, the outer layer hydrogel consisted of gelatin methacryloyl (GelMA) and epidermal growth factor (EGF) promoted wound healing and tissue regeneration. It displayed remarkable efficacy on alleviating infection and accelerating wound healing in vivo. Overall, the innovative therapeutic strategy we came up with has significant effect on biofilms eradication and shows promising application in promoting the repair of clinical chronic wounds.