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.

Study of the pathological antibacterial effects of tea extract and its role in reducing hypertension in pregnant women

Aims@#Due to the world's direction of alternative medicine and herbal medication, tea leaves have been employed to inhibit certain bacteria that cause urinary tract infections (UTIs). This study aimed to evaluate the effect of green, red and black tea as antibacterial against UTIs in pregnant women and changes in blood pressure and iron level in the blood of their women.@*Methodology and results@#Forty-eight isolates were isolated from 50 women suffering from urinary tract infections, Staphylococcus aureus (18) 37.5%, Escherichia coli (15) 31.25%, Pseudomonas aeruginosa (8) 16.6%, Klebsiella sp. (5) 10.4% and Enterobacter sp. (2) 4.16%. The sensitivity of bacteria to the antibiotics Amikacin, Amoxicillin/Clavulanic, Ampicillin/Sulbactam, Cefixime, Ceftriaxone, Ciprofloxacin, Imipenem, Nitrofurantion, Penicillin and Tetracycline were tested, while E. coli and P. aeruginosa (8), Enterobacter sp. were resistance for Ceftriaxone and Amoxicillin /Clavulanic (100%). While Enterobacter sp. is sensitive to Nitrofurantoin and Imipenem (100%). The ability of the isolates to form biofilms was tested using the Congo red agar method and the micro titrations plate method. The results showed that not all isolates have the ability to produce biofilms and red tea is the most powerful antibacterial under study. Drinking green tea for two weeks regularly in pregnant women who suffer from high blood pressure showed an improvement in blood pressure, as it became normal 118/78 and with the normal iron level in the blood at a rate of hemoglobin = 11.8, while drinking red tea did not change blood pressure measurements in pregnant women with high blood pressure.@*Conclusion, significance and impact of study@#The effect of red tea extract was stronger than other teas used in the study as an antibacterial against urinary tract bacteria. Regular consumed of green tea helps regulate blood pressure, especially for pregnant women who are at risk of hypertension during pregnancy.

Comparison of the effect of two disinfectants on the independent waterway disinfection of a periodontal ultrasonic scaler / 口腔疾病防治

Objective @#To compare the disinfection effect of 3% (v/v) hydrogen peroxide and 500 mg/L chlorine-containing disinfectants in the independent waterway of a periodontal ultrasonic scaler to provide a reference for clinical waterway disinfection management in stomatology departments.@*Methods @#The 18 ultrasonic scalers were randomly divided into 3 groups of 6 units: the control group, experimental group 1 (3% hydrogen peroxide disinfectant group), and experimental group 2 (500 mg/L chlorine-containing disinfectant group). The replaceable parts of the independent waterway pipes of the 3 groups of ultrasonic scalers were replaced, and the water supply was supplied with sterile distilled water (DW). In the control group, special treatment was not applied to the nonreplaceable pipe part. In experimental group 1, the 3% hydrogen peroxide was used to disinfect nonreplaceable pipelines. In experimental group 2, the nonreplaceable part was disinfected with the 500 mg/L chlorine-containing disinfectant. The water sample was taken from the outlet of the scaler working part in the three groups for monitoring before disinfection, immediately after disinfection and 10 consecutive days after disinfection. Bacteria in the water samples were cultured for the colony counts. Then, the bacterial culture data were compared between groups. The qualified criterion of the water sample was that the number of bacterial colonies was less than or equal to 100 CFU/mL. After disinfection, a bacterial species mass spectrometry identification analysis was carried out when the number of bacterial colonies in each group exceeded the standard for the first time. Biofilms from the inner wall of the tube in the three groups were observed under an electron microscope on the 10th day after disinfection.@*Results @#There were no significant differences between the three groups before disinfection (F = 2.549, P = 0.111). The number of bacterial colonies in the spout of 6 scalers in the control group all exceeded the standard, and three kinds of bacteria were cultured: Sphingomonas melonis, Herbaspirillum huttiense, and Ralstonia pickettii. Compared with those in the control group, the number of bacterial colonies in experimental group 1 decreased significantly for 1-2 days after disinfection (P<0.05) and reached the standard. On the 3rd day after disinfection, the number of bacterial colonies of group 1 increased rapidly and exceeded the standard, and three kinds of bacteria were cultured: Sphingomonas, Herbaspirillum huttiense, and Ralstonia pickettii. For experimental group 2, the number of bacterial colonies decreased significantly compared to the control group on Days 1 to 6 after disinfection, but the number of bacterial colonies increased slightly from the 7th day after disinfection and exceeded the standard. Two kinds of bacteria were cultured: Herbaspirillum huttiense and Ralstonia pickettii. The average number of bacterial colonies 10-day after disinfection in experimental group 2 was lower than that in experimental group 1(P<0.001). Under an electron microscope, the biofilm thickness of the two experimental groups was significantly lower than that of the control group. @* Conclusion @# There is water pollution in the independent waterway of a periodontal ultrasound scaler. Three percent hydrogen peroxide and 500 mg/L chlorine disinfectant both have effective disinfection effects on the outlet water of scalers, and the effect of 500 mg/L chlorine disinfectant is better than that of 3% hydrogen peroxide. The use of 3% hydrogen peroxide to disinfect periodontal ultrasound scaler-independent waterways is recommended for disinfection every other day, and disinfection once a week is recommended for the use of 500 mg/L chlorine disinfectant.

Research progress on the relationship between free sugars and caries / 口腔疾病防治

@#Caries is a chronic infectious disease that occurs in the hard tissues of teeth. The interaction of oral microorganisms, host, food and time leads to the occurrence and progression of caries. Free sugar is an important food factor in the occurrence of dental caries. This paper reviews the research progress on the relationship between free sugar and caries in recent years, providing a reference for further clarifying the mechanism of the occurrence and progression of caries caused by free sugar and exploring the methods of caries prevention. The cariogenic mechanism of free sugar is multifaceted. Free sugar can not only be used by oral microorganisms in dental plaque biofilms to produce acid and synthesize intracellular and extracellular polysaccharides but also cause an imbalance in oral microecology and enhance gene expression related to the cariogenic toxicity of oral microorganisms. Based on the correlation between free sugar and caries, it is important to limit the intake amount and frequency of free sugar to prevent caries. This can be achieved mainly by formulating public health policies for reducing sugar, creating a low-sugar family environment with good eating habits, using sugar substitutes and using fluoride. There are few studies on the relationship between free sugar and oral microecology. More research is needed to better understand the effect of free sugar on oral microecology and its mechanism and to validate the caries-preventing effect of various sugar reduction measures. We believe such studies would open up new avenues for the effective prevention of caries.

Research progress on antimicrobial peptides in terms of the prevention and treatment of dental caries / 口腔疾病防治

@#Antimicrobial peptides have antibacterial effects on various pathogenic microorganisms, including natural antimicrobial peptides and synthetic antimicrobial peptides. According to the structure of natural antimicrobial peptides, synthetic antimicrobial peptides can be obtained by recombining different functional domains, adjusting the original amino acid sequence, or completely redesigning the peptides from scratch. Antimicrobial peptides can inhibit the growth of various cariogenic microorganisms and the formation of microbial biofilms. They also reduce acid production and acid resistance of microorganisms. Natural antimicrobial peptide genes can be used as genetic susceptibility markers for predicting the development of caries, thus, showing potential applications in the prevention and treatment of dental caries. The instability of natural antimicrobial peptides and the inability to achieve targeted sustained release limit their application in the prevention and treatment of oral caries. Synthetic antimicrobial peptides can enhance their stability and the antibacterial effect. Synthetic antimicrobial peptides can also be polymerized with common oral adhesives to reduce the incidence of microleakage after filling treatment for caries and to prevent the occurrence of secondary caries. The pH-sensitive antimicrobial peptides are slowly released to promote remineralization in the process of caries. However, the safety and biocompatibility of synthetic antimicrobial peptides are worse than those of natural antimicrobial peptides. Moreover, the combined effect of antibacterial peptides and anticaries drugs, such as fluoride, is still uncertain. Therefore, in this paper, we will review the design methods, application and underlying mechanisms of antimicrobial peptides to introduce novel methods and ideas for the prevention and treatment of dental caries.

Research progress on oxidative stress regulatory mechanisms in Streptococcus mutans / 口腔疾病防治

@#The oral microecological balance is closely associated with the development of dental caries. Oxidative stress is one of the important factors regulating the composition and structure of the oral microbial community. Streptococcus mutans is closely related to the occurrence and development of dental caries. The ability of S. mutans to withstand oxidative stress affects its survival competitiveness in biofilms. The oxidative stress regulatory mechanisms of S. mutans include the synthesis of reductase, the regulation of iron and manganese uptake by metalloregulatory proteins, transcription regulator Spx, extracellular uptake of glutathione and other related signal transduction systems. The current research focuses on how S. mutans adapts to a complex external environment through an oxidative stress response and its influence on oral microecology. We can design targeted small molecular compounds for key signaling pathways to inhibit oxidative stress and weaken the virulence of S. mutans, which is important for oral microecological modulation and dental caries prevention and treatment.

A short-term analysis of the modification process of the plaque microbial community before and after treatment in children with S-ECC / 口腔疾病防治

Objective @# To explore from the perspective of microorganisms the changes in plaque microbial community of children with severe early childhood caries (S-ECC) before and 3 months after dental treatment. Meanwhile to show the effect of treatment on the maintenance of long- term caries-free state. @*Methods@# S-ECC children completed dental treatment under general anesthesia. We collected plaque from caries-free dental surfaces before treatment (caries, C) and at the postoperative follow-up review time points of 7 days (C-7D), 1 month (C-1 M), and 3 months (C-3 M). We included caries-free children (caries free, CF) as the control group to analyze the dynamic modification process of the plaque microbial community in the short-term pre- and postdental treatment.@*Results@#Species clustering analysis showed that the compositions of the microbial communities of the S-ECC and CF groups were highly similar. The α diversity index was not statistically significant (P>0.05). From the analysis of the relative abundance, Leptotrichia spp. and Aggregatibacter spp. decreased after treatment compared with before treatment (P<0.05). Streptococcus sanguinis in the C-7D group increased compared with that in the C group and gradually decreased within 3 months. Veillonella spp., Actinomyces spp., Allprevotella spp., Capnocytophaga spp., and Streptococcus mutans differed between the C and CF groups (P<0.05), Streptococcus mutans did not differ significantly between the C-7D and C-1 M groups and the CF group after treatment, while C-3 M showed an increase compared with the CF group (P<0.01). @*Conclusion@#The rapid change in the structure of the flora of children with S-ECC after treatment. The plaque microbial community structure in a caries-free state gradually starts to be established 1-3 months after treatment. There is a "core microbiota" in the oral plaque community that jointly maintains microecological stability. Veillonella spp., Allprevotella spp. and Streptococcus mutans have potential as possible microbial markers.