Biofilm production by coagulase-negative Staphylococcus: a review / Produção de biofilme por Staphylococcus coagulase-negativo: uma revisão

This review aimed to describe the biofilm formation ability of coagulase-negative Staphylococcus, addressing its impact to the food industry. Coagulase-negative Staphylococcus have the ability to produce enterotoxins in food, making it an important line of study, as it constitutes a risk to public health. The biofilm formation by these microorganisms requires physicochemical processes, such as hydrophobic forces, which are essential for the first phase of fixing the biofilm on the surface. In industrial facilities, stainless steel equipment is the most associated with the formation of biofilms, due to the presence grooves and cracks. Many species of coagulase-negative Staphylococcus produce biofilm, but the most studied is S. epidermidis, as it is the most frequently isolated from food. Coagulase-negative Staphylococcus form biofilm on different surfaces in the food industry, and can become a source of permanent contamination, that can be present in the final product, intended for human consumption. Among other alternatives to combat the formation of biofilm in industrial food facilities, there is the implementation of Good Manufacturing Practices, which is effective in preventing bacterial adhesion, and therefore, the formation of biofilm. However, further studies are needed in order to quantify the occurrence of coagulase-negative Staphylococcus biofilms in the food industry.(AU)

Esta revisão bibliográfica teve como objetivo descrever a capacidade de formação de biofilme por Staphylococcus coagulase-negativo, relacionando seu impacto na indústria alimentícia. Os Staphylococcus coagulase-negativos possuem a capacidade de produzir enterotoxina nos alimentos, tornando-se uma importante linha de estudo, pois constitui um risco para a saúde pública. A formação do biofilme por esses micro-organismos requer processos físico-químicos, como forças hidrofóbicas, essenciais para a primeira fase de fixação do biofilme na superfície. Nas indústrias, equipamentos de aço inoxidável são os mais associados à formação de biofilmes, em decorrência de possuírem ranhuras e fendas. Muitas espécies de Staphylococcus coagulase-negativo produzem biofilme, porém, o mais estudado é o S. epidermidis, por ser o mais frequentemente isolado de alimentos. Os Staphylococcus coagulase-negativos formam biofilme em diferentes superfícies de indústrias alimentícias, podendo se tornar uma fonte de contaminação permanente, contaminando o produto final destinado ao consumo humano. Dentre outras alternativas para combater a formação do biofilme nas plantas alimentícias, a implantação das Boas Práticas de Fabricação é eficaz para prevenir a adesão bacteriana, evitando a formação do biofilme. No entanto, são necessários estudos para quantificar a ocorrência de biofilmes de Staphylococcus coagulase-negativos em indústrias alimentícias.(AU)

Resistencia a la meticilina y producción de biopelícula en aislamientos clínicos de Staphylococcus aureus y Staphylococcus coagulasa negativa en México / Methicillin resistance and biofilm production in clinical isolates of Staphylococcus aureus and coagulase-negative Staphylococcus in México

Resumen Introducción. Las infecciones por Staphylococcus aureus y Staphylococcus coagulasa negativa multirresistentes a los antibióticos y asociadas con la atención en salud tienen un gran impacto epidemiológico por su alta morbimortalidad; además, se han relacionado con la formación de biopelículas, lo cual también se asocia con la resistencia a los antimicrobianos. Objetivo. Determinar la resistencia a la meticilina y cuantificar la producción de biopelículas para establecer su posible relación con los aislamientos clínicos de S. aureus y Staphylococcus coagulasa negativa. Materiales y métodos. Se estudiaron 11 cepas de S. aureus y 12 de Staphylococcus coagulasa negativa. La resistencia a la meticilina se determinó con discos de cefoxitina tomando como valores de referencia los estándares del Clinical Laboratory Standards Institute (CLSI) de 2018. La producción de biopelícula se cuantificó con cristal violeta. Los genes mecA e icaADBC se identificaron mediante reacción en cadena de la polimerasa (PCR), y se hizo un análisis bivariado con la prueba de ji al cuadrado y el coeficiente V de Cramér, utilizando el programa SPSS™, versión 20.0. Resultados. Nueve cepas de S. aureus fueron resistentes a la meticilina (SARM) y dos fueron sensibles. Ocho cepas de Staphylococcus coagulasa negativa fueron resistentes y cuatro fueron sensibles. El genotipo mecA se encontró en ocho de las nueve cepas de S. aureus y en seis de las ocho de Staphylococcus coagulasa negativa resistentes a meticilina. Todas las cepas formaron biopelícula. Diez cepas de S. aureus y 11 de Staphylococcus coagulasa negativa presentaron el genotipo icaADCB. No se encontró asociación entre la resistencia a meticilina y la formación de biopelícula. Conclusiones. La cefoxitina es suficiente para determinar el fenotipo resistente a meticilina y se asoció con el genotipo mecA. Las cepas resistentes a la meticilina y poseedoras del gen mecA pueden presentar un mecanismo de resistencia alterno. Los dos grupos de cepas formadoras de biopelícula se relacionaron con la presencia del operón icaADCB. La formación de biopelícula y la resistencia a la meticilina se expresaron como características independientes en los dos grupos de cepas.

Abstract Introduction: Infections associated with health care caused by S. aureus and coagulase- negative Staphylococci multi-resistant to antibiotics cause a high epidemiological impact due to their high morbidity and mortality. Biofilm formation, which has been associated with antimicrobial resistance, can also occur. Objectives: To determine methicillin resistance and to quantify the biofilm production to establish if there is a relationship in clinical isolates of S. aureus and coagulase-negative Staphylococci. Material and methods: A total of 11 strains of S. aureus and 12 of coagulase-negative Staphylococci were studied. Methicillin resistance was determined with cefoxitin discs and the Clinical Laboratory Standards Institute (CSLI), 2018 reference values. Biofilm production was quantified by the crystal violet method. The mecA and icaADBC genes were identified by PCR. A bivariate analysis was performed with chi-square (c2) and Cramér's V statistical tests, using SPSS™, version 20.0 software. Results: Nine S. aureus strains were methicillin-resistant and two were sensitive. Eight coagulase-negative Staphylococci strains were resistant and four were sensitive. The mecA genotype was found in eight of the nine S. aureus resistant strains and six of eight resistant coagulase-negative Staphylococci. All strains formed biofilms. Ten strains of S. aureus and 11 of coagulase-negative Staphylococci presented the icaADCB genotype. No association was found between methicillin-resistance and biofilm formation. Conclusions: Cefoxitin is enough to define the resistance phenotype and is associated with the mecA genotype. All strains formed biofilms and were related to the presence of the icaADCB operon. Biofilm formation and methicillin resistance were independent features in both groups of strains.

Produção de biofilme por Staphylococcus coagulase negativa isolados de queijos e equipamentos / Biofilm production by coagulase-negative Staphylococci isolated from cheese and equipments

Biofilme é uma comunidade organizada de microrganismos que se forma em superfícies mal higienizadas, constituindo um mecanismo de defesa microbiana para permanência no ambiente. O objetivo do presente estudo foi investigar a capacidade de formação de biofilme de espécies de Staphylococcus coagulase negativa (SCN) isoladas de queijo Mussarela fatiado e de fatiadores de frios de estabelecimentos do município de Garanhuns-PE. De 103 isolados de SCN 56 (54,4%) foram negativos para produção de biofilme e 47 (45,6%) positivos, com a maior frequência de detecção nas espécies S. saprophyticus e S. cohnii subsp. urealyticum. Os resultados apontam para o potencial risco de contaminação cruzada de outros alimentos, uma vez que cepas bacterianas produtoras de biofilmes podem colonizar e persistir em superfícies de diversos equipamentos.

INVESTIGATION OF BIOFILM FORMATION IN COAGULASE-NEGATIVE STAPHYLOCOCCI ISOLATED FROM PLATELET CONCENTRATE BAGS

Platelet Concentrates (PCs) are the blood components with the highest rate of bacterial contamination, and coagulase-negative staphylococci (CoNS) are the most frequently isolated contaminants. This study investigated the biofilm formation of 16 contaminated units out of 691 PCs tested by phenotypic and genotypic methods. Adhesion in Borosilicate Tube (ABT) and Congo Red Agar (CRA) tests were used to assess the presence of biofilm. The presence of icaADC genes was assessed by means of the Polymerase Chain Reaction (PCR) technique. With Vitek(r)2, Staphylococcus haemolyticus was considered the most prevalent CoNS (31.25%). The CRA characterized 43.8% as probable biofilm producers, and for the ABT test, 37.5%. The icaADC genes were identified in seven samples by the PCR. The ABT technique showed 85.7% sensitivity and 100% specificity when compared to the reference method (PCR), and presented strong agreement (k = 0.8). This study shows that species identified as PCs contaminants are considered inhabitants of the normal skin flora and they might become important pathogens. The results also lead to the recommendation of ABT use in laboratory routine for detecting biofilm in CoNS contaminants of PCs.

The Effect of Temperature on Nitrate and Phosphate Uptake from Synthetic Wastewater by Selected Bacteria Species.

Aim: The aim of this study was to ascertain the effect of temperature on nutrient uptake ability of four bacterial species. Methodology: A total of four bacterial species (Klebsiella sp., Pseudomonas sp., Lysinibacillus sp. and Staphylococcus sp.) were used for the study. The media used for the investigation was synthetic wastewater. Four different temperatures (25ºC, 30ºC, 35ºC and 40ºC) were used for the investigation. The study was carried out under shake flasks conditions. Immediate after inoculation with the respective test bacterial species and every 24 h for a 96 h incubation time, aliquot wastewater samples were removed from the flasks for the estimation of total phosphate, nitrate, pH and growth rate, using standard procedures. Results: The results revealed phosphate and nitrate removal ranges of 10.84 % to 55.55 % and 90.67 % to 97.27 %, respectively in the presence of the Klebsiella sp. In the presence of the Pseudomonas sp, Lysinibacillus sp. and Staphylococcus sp., phosphate removals ranges of 0.36 % to 46.98 %, 11.89 % to 50.80 % and 2.74 % to 51.21 % were observed, respectively. For nitrate concentrations, removal levels that ranged from 2.19 % to 92.95 %, 0.97 % to 23.12 % and 7.56 % to 91.66 % were observed in the presence of Pseudomonas sp, Lysinibacillu ssp. and Staphylococcus sp., respectively. All the test bacterial species showed some measure of efficiency in phosphate removal. For nitrate removal, the Lysinibacillus sp. did not exhibit remarkable nitrate removal ability at any of the temperatures. In addition, the optimum temperatures for phosphate removals were observed to be 30ºC to 40ºC for the Klebsiella sp. and Pseudomonas sp; and 30ºC to 35ºC for the Lysinibacillus sp. and Staphylococcus sp. For nitrate removal, optimum temperatures for removal were observed to be 25ºC to 40ºC, for the Klebsiella sp and 25ºC to 35ºC, for the Pseudomonas sp. and Staphylococcus sp. Conclusion: The study was able to reveal the optimum temperatures for phosphate and nitrate uptake in synthetic wastewater by the test bacterial species.

Bactericidia de hipoclorito de sodio sobre Staphylococcus cohnii productor de biofilm en una fabrica / Bactericidal activity of sodium hypochlorite against biofilm-producing Staphylococcus cohnii in a factory / Atividade bactericida de hipoclorito de sodio sobre Staphylococcus cohnii produtor de biofilme numa fabrica

En equipamientos industriales la persistencia de las bacterias se debe al desarrollo de biofilms. El hipoclorito de sodio (NaClO) es usado rutinariamente como desinfectante. El objetivo del trabajo fue determinar el efecto inhibitorio del NaClO sobre S. cohnii productoras y no productoras de biofilms aisladas de una fabrica de pastas a partir de maquinarias de un establecimiento de Tucuman, Argentina. Se estudio produccion de biofilm, concentracion inhibitoria minima (CIM), concentracion bactericida minima (CBM) y curva de tiempo de muerte con NaClO frente a S. cohnii productora y no productora de biofilm. Los resultados obtenidos demostraron que de los ocho cocos grampositivos aislados, cuatro correspondieron a S. cohnii. Los valores CIM y CBM para S. cohnii productoras y no productoras de biofilm resultaron entre 0,05 y 0,2 g/dL. Mediante la curva de muerte se determino efecto bactericida observando una disminucion de ≥4 log de ufc (1h) tratado con 0,2 y 0,4% de NaClO. La formacion de biofilm para la cepa no tratada fue de DO: 0,12 y para la cepa tratada DO: 0,07 a las 18 h. Aplicar desinfectantes con amplio espectro de accion es importante, debido a que la eliminacion de bacterias puede prevenir su diseminacion y evitar la formacion de biofilms.

Persistence of bacteria in industrial equipment is favoured by biofilm formation. Sodium hypochlorite (NaClO) is routinely used as a disinfectant. The objective of the current study was to determine the inhibitory effect of NaClO on biofilm-producing and non-biofilm-producing S. cohnii strains, isolated from equipment in a fresh pasta factory in Tucuman, Argentina. The following parameters were assayed: production of biofilm, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and NaClO death curves for both biofilm-producing and non-biofilm-producing S. cohnii strains. Four of the eight isolated Gram-positive cocci corresponded to S. cohnii. MIC and MBC values for biofilm-producing and non-biofilm-producing S. cohnii strains were between 0.05 and 0.2 g/dL. A bactericidal effect of 0.2 and 0.4% NaClO for 1 h was established with the death curve, showing a decrease in cfu of ≥4 log units. Biofilm production after 18 h for untreated strains gave an OD of 0.12, whereas the OD of treated strains was 0.07. The use of broad spectrum disinfectants is important, because elimination of bacteria helps prevent their propagation and avoid the formation of biofilms.

A persistência da bactéria em equipamento industrial e resultado do desenvolvimento de biofilmes. O hipoclorito de sodio (NaOCl) e rotineiramente utilizado como desinfetante. O objetivo do trabalho foi determinar o efeito inibitorio do NaClO sobre S. cohnii produtoras e nao produtoras de biofilmes isoladas de uma fabrica de massas frescas a partir de maquinarias de um estabelecimento de Tucuman, Argentina. Foram estudados: producao de biofilme, concentracao inibitoria minima (CIM), concentracao bactericida minima (CBM) e curva de tempo de morte com NaClO perante S. cohnii produtora e naoprodutora de biofilme. Os resultados obtidos demonstraram que quatro dos oito cocos Gram-positivos isolados corresponderam a S. cohnii. Os valores CIM e CBM para S. cohnii produtoras e nao produtoras de biofilme foram entre 0,05 e 0,2 g/dL. Atraves da curva de morte foi determinado um efeito bactericida observando uma diminuicao de ≥ 4 log de ufc (1h) tratado com 0,2 e 0,4% de NaClO. A formacao de biofilme, apos 18 h, para a cepa nao tratada foi de DO: 0,12, ao passo que para a cepa tratada foi de 0,07. A utilizacao de desinfetantes de amplo espectro de acao e importante, visto que a eliminação de bacterias pode prevenir sua propagação e evitar a formacao de biofilmes.

High vancomycin resistance among biofilms produced by Staphylococcus species isolated from central venous catheters

Biofilm production is an important mechanism that allows microbes to escape host defences and antimicrobial therapy. Vancomycin has been used largely for the treatment of methicillin-resistant staphylococcal infections. Here, we determined the minimal inhibitory concentration (MIC) and minimal biofilm eradication concentration (MBEC) for 82 Staphylococcus species isolated from central venous catheters (CVC). Our results showed that the 41 strong and moderate-biofilm-producing isolates presented a higher MBEC/MIC ratio for vancomycin than the 24 weak-biofilm-producing isolates, illustrating the importance of biofilm production ability and the difficulty in treating biofilm-related infections. The MBEC was significantly higher in moderate-biofilm-producing isolates than in weak-biofilm-producing isolates (p < 0.001) and in strong-biofilm-producing isolates than in weak-biofilm-producing isolates (p = 0.001). The correlation between the MIC and the MBEC was poor. Based on our results, we recommend that bacterial biofilms be suspected in all cases of CVC infection.

Atividade anti-estafilócica em extratos de plantas com indicativo medicinal ou condimentar / Anti-staphylococci activity in extracts from plants presenting medicinal and spice indicative

O gênero Staphylococcus permanece em evidência no contexto das doenças transmissíveis considerando-se os portadores humanos e animais como a sua principal fonte de infecção. Por sua vez, a pesquisa de fatores de proteção antibacteriana em recursos naturais como plantas com indicativo medicinal ou condimentar vem sendo recomendado como prioridade pelas Conferências Mundiais de Saúde. A presente revisão descreve a triagem in vitro, através de testes de diluição em sistema de tubos múltiplos, de atividade anti-estafilococo de diferentes extratos de 80 plantas com indicativo etnográfico medicinal, condimentar ou aromático, acessadas na região metropolitana de Porto Alegre, RS. Demonstrou-se atividade seletiva em 39 espécies, enquanto as demais 41 não apresentaram atividade.

The genus Staphylococcus remains in evidence regarding transmissible diseases since human and animal carriers are its main infection source. Studies on antibacterial protection factors in natural resources, such as plants presenting medicinal or spice indicative, have been recommended by World Health Conferences as a priority. Dilution tests in multiple tube system were used in this review to describe the in vitro sorting of anti-staphylococci activity of different extracts from 80 plants presenting medicinal, spice or aromatic ethnographic indicatives at the metropolitan region of Porto Alegre, Rio Grande do Sul State, Brazil. Selective activity was observed for 39 species, whereas the remaining 41 species did not show activity.

Biofilms: microbes and disease: [review]

Bacteria that attach to surface aggregate in a hydrated polymeric matrix of their own synthesis to form biofilms. These represent microbial societies with their own defense and communication system. Transitioning from acute to chronic infection is frequently associated with biofilm formation.Bacteria in biofilms are innately more resistant to antimicrobial agents. The presence of indwelling medical devices increases the risk for biofilm formation and subsequent infection. The current antibiotic therapies are of limited effectiveness in resolving biofilms infection.This review attempts to discuss the stages in biofilm formation, their pathogenic mechanisms, effect of antimicrobial agents, detection and eradication of the biofilms.

Slime production and antibiotic susceptibility in staphylococci isolated from clinical samples

A total of 187 isolates from several clinical specimens were identified to species level as 129 Staphylococcus aureus strains and 58 coagulase-negative staphylococci (CNS) strains by the API Staph System (Biomerieux). Slime production was detected both by the conventional Christensen's method as well as by the Congo red agar method. Seventy-two strains of staphylococci isolates (38.5 percent) were found to be slime producers by Christensen's test tube method whereas 58 strains (31 percent) were slime positive with Congo red agar method. There was no statistically significant difference between the two methods for the detection of slime production (P > 0.05). Susceptibility of isolates against antimicrobial agents was tested by the disk diffusion method. Staphylococcal species had resistance to one or more antibiotics. Among the various antimicrobial agents, oxacillin (71.1 percent) and erythromycin (47.1 percent) showed higher resistance than most of the agents used against all isolates. Oxacillin resistant S. aureus (ORSA) and oxacillin resistant coagulase-negative staphylococci (ORCNS), 97 (75.2 percent) and 36 (62.1 percent) respectively were frequently observed in strains isolated from clinical materials. Among the ORSA strains, two strains were resistant to vancomycin. Moreover, 96 (74.4 percent) of 129 S. aureus strains were positive for blactamase enzyme. However, 78 (81.25 percent) of 96 b-lactamase positive S. aureus strains were b-lactamase positive ORSA isolates, but none of them had vancomycin resistance.

Determinaçao de especies de estafilococos coagulase-negativos de significado clinico / Species identification of coagulase-negative staphylococci of clinical significance

Uma coleçao de 141 amostras de estafilococos coagulase-negativos, isoladas de especimes clinicos, assim como de portadores e ambiente hospitalar, foram analisadas quanto as caracteristicas fisiologicas e propriedades biologicas. Oito especies foram identificadas pelos metodos convencionais segundo Kloos & Schleifer, e corresponderam a S. saprophyticus, S. epidermis, S. cohnil, S. simulans, S. hominis, S. warneri, S. haemolyticus e S. xylosus. Quatorze amostras nao foram identificadas. Atraves da aplicaçao da tecnica de taxonomia numerica se observou uma tendencia para a separaçao em dois grandes grupos, compreendendo S. saprophyticus e S. epidermidis. A partir dai as amostras restantes se identificaram com as outras especies de referencia de diferentes graus de similaridade. O emprego do micrometodo API no estudo de 39 das amostras ja identificadas so permitiu a classificaçao de 17 cepas em especies.

Adherence of bacteria and Candida albicans to polyethylene catheters

The ability to adhere to polyethylene catheters is important in the mechanism of catheter-associated infecctions caused by bacteria and fungi. We observed greater capacity of attachment in strains of Candida albicans, Staphylococcus epidermidis and Staphylococcus aureus

Bacterial Adherence to Human Buccal Epitheliald Cells and Its Possible Role in Bacterial Colonization in Human Oral Cavity

The ability of several species of streptococcus and staphylococcus to adhere to human buccal epithelial cells was studied in vitro by using bacteria and epithelial cells isolated from human buccal cavity. Viridans streptococci were found adhering in highest numbers(65 +/- 8 bacteria per epithelial cell) to epithelial cells. Streptococcus pyogenes adhered in great numbers (44 +/- 4), whereas Streptococcus pneumoniae (26 +/- 2), Staphylococcus aureus (21 +/- 2), Staphylococcus epidermidis (14 +/- 2) adhered poorly. These data showed that bacteria differed in their ability to adhere to human buccal epithelial cells. This difference in adhesive ability between bacterial species may correlate with the ability of the bacteria to colonize oral surface of human.