Expression of icaA and icaD genes in biofilm formation in Staphylococcus aureus isolates from bovine subclinical mastitis

ABSTRACT: Staphylococcus spp. plays a significant role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most important species due to the high prevalence and the difficulty of in vivo treatment that is related to the expression of virulence factors and biofilm formation. This study aimed to detect the phenotypic expression of the biofilm formation in 20 S. aureus isolated from bovine mastitis and to evaluate the expression and regulation of genes involved in its production. MALDI-TOF and phenogenotypic identification assays were performed to characterize the isolates. The phenotypic biofilm production and the presence of icaA and icaD and bap genes were evaluated. The Agr system was typified (agr I, agr II, agr III and agr IV) and its regulator (agr RNAIII) was detected. Furtherly, Real-time PCR (qPCR) was performed at chosen times to quantify the expression of icaA, icaD and hld genes in three selected isolates. All 20 strains were biofilm producers and most presented icaA and icaD genes. Only one isolate presented the bap gene. The agr gene type II showed a prevalence of 70%. Transcriptional analysis revealed increased expression of ica genes at eight hours of growth. These results confirm that polysaccharides production mediated by the icaADBC operon genes is an essential mechanism to the biofilm formation and contributes to the early stages of bacterial growth.

RESUMO: Staphylococcus spp. desempenham um papel significativo na etiologia da mastite bovina. Staphylococcus aureus é considerada a espécie mais importante devido a alta prevalência e a dificuldade de tratamento in vivo que está relacionado à expressão dos fatores de virulência e formação de biofilme. Este estudo teve como objetivo detectar a expressão fenotípica da formação de biofilme em 20 cepas de S. aureus isoladas de mastite bovina e avaliar a expressão e regulação de genes envolvidos em sua produção. MALDI-TOF e ensaios de identificação fenogenotípica foram realizados para caracterizar os isolados. A produção fenotípica de biofilme e a presença dos genes icaA, icaD e bap foram avaliadas. O sistema Agr foi tipificado (agr I, agr II, agr III e agr IV) e seu regulador (agr RNAIII) foi detectado. Além disso, a PCR em tempo real (qPCR) foi realizada nos tempos determinados para quantificar a expressão dos genes icaA, icaD e hld em três isolados selecionados. Todas as 20 linhagens foram produtoras de biofilme e a maioria apresentava os genes icaA e icaD. Apenas um isolado apresentou o gene bap. O gene agr do tipo II mostrou uma prevalência de 70%. A análise transcricional revelou aumento da expressão de genes ica às oito horas de crescimento. Estes resultados confirmam que a produção de polissacarídeos mediada pelos genes do operon icaADBC é um mecanismo essencial para a formação do biofilme e contribui para os estágios iniciais do crescimento bacteriano.

Expression of icaA and icaD genes in biofilm formation in Staphylococcus aureus isolates from bovine subclinical mastitis / Expressão dos genes icaA e icaD na formação de biofilme em isolados de Staphylococcus aureus de mastite subclínica bovina

Staphylococcus spp. plays a significant role in the etiology of bovine mastitis. Staphylococcus aureus is considered the most important species due to the high prevalence and the difficulty of in vivo treatment that is related to the expression of virulence factors and biofilm formation. This study aimed to detect the phenotypic expression of the biofilm formation in 20 S. aureus isolated from bovine mastitis and to evaluate the expression and regulation of genes involved in its production. MALDI-TOF and phenogenotypic identification assays were performed to characterize the isolates. The phenotypic biofilm production and the presence of icaA and icaD and bap genes were evaluated. The Agr system was typified (agr I, agr II, agr III and agr IV) and its regulator (agr RNAIII) was detected. Furtherly, Real-time PCR (qPCR) was performed at chosen times to quantify the expression of icaA, icaD and hld genes in three selected isolates. All 20 strains were biofilm producers and most presented icaA and icaD genes. Only one isolate presented the bap gene. The agr gene type II showed a prevalence of 70%. Transcriptional analysis revealed increased expression of ica genes at eight hours of growth. These results confirm that polysaccharides production mediated by the icaADBC operon genes is an essential mechanism to the biofilm formation and contributes to the early stages of bacterial growth.(AU)

Staphylococcus spp. desempenham um papel significativo na etiologia da mastite bovina. Staphylococcus aureus é considerada a espécie mais importante devido a alta prevalência e a dificuldade de tratamento in vivo que está relacionado à expressão dos fatores de virulência e formação de biofilme. Este estudo teve como objetivo detectar a expressão fenotípica da formação de biofilme em 20 cepas de S. aureus isoladas de mastite bovina e avaliar a expressão e regulação de genes envolvidos em sua produção. MALDI-TOF e ensaios de identificação fenogenotípica foram realizados para caracterizar os isolados. A produção fenotípica de biofilme e a presença dos genes icaA, icaD e bap foram avaliadas. O sistema Agr foi tipificado (agr I, agr II, agr III e agr IV) e seu regulador (agr RNAIII) foi detectado. Além disso, a PCR em tempo real (qPCR) foi realizada nos tempos determinados para quantificar a expressão dos genes icaA, icaD e hld em três isolados selecionados. Todas as 20 linhagens foram produtoras de biofilme e a maioria apresentava os genes icaA e icaD. Apenas um isolado apresentou o gene bap. O gene agr do tipo II mostrou uma prevalência de 70%. A análise transcricional revelou aumento da expressão de genes ica às oito horas de crescimento. Estes resultados confirmam que a produção de polissacarídeos mediada pelos genes do operon icaADBC é um mecanismo essencial para a formação do biofilme e contribui para os estágios iniciais do crescimento bacteriano.(AU)

Capacidad de formación de biopelícula y limo en Staphylococcus que causan mastitis subclínica bovina en el estado Zulia-Venezuela / Biofilm formation and slime producing ability in Staphylococci causing subclinic bovine mastitis in the Zulia state-Venezuela

Un total de 79 cepas de S. aureus y 47 cepas de Staphylococcus coagulasa negativa (SCN) aislados de muestras de leche de vaca con mastitis subclínica fueron evaluadas para establecer su propiedad para formar biopelícula como uno de los factores de virulencia más importantes. Usando el método de Rojo Congo Agar, 80% de las cepas de S. aureus fueron productores de limo, mientras que en las cepas de SCN el porcentaje fue de 32%. Por el método de microplaca, 55%, 17% y 28% de los aislamientos de S. aureus fueron fuerte, moderadas y débiles productoras de biopelícula, mientras en los SCN el porcentaje fue 43%, 17% y 40%, respectivamente. Se realizó un ensayo de Reacción en Cadena de la Polimerasa (PCR) a todos los aislamientos con la finalidad de identificar el gen A de adhesión intracelular (icaA). En las cepas de S. aureus el gen icaA estuvo presente en el 65% de los aislamientos, y en los SCN en el 11%. La mayoría de las cepas de S. aureus caracterizados en el estudio fueron formadores de biopelícula, lo cual sugiere que está tiene un importante papel en la virulencia de S. aureus aislados de infecciones intramamarias en bovinos del estado Zulia.

A total of 79 S. aureus strains and 47 coagulase negative Staphylococcus (CNS) isolates from cow milk suffering subclinical mastitis were investigated for their ability to form biofilm as one of the most important virulence factors. Using Congo Red Agar method, 80% of S. aureus strains were slime producers, while in CNS was 32%. By microtiter plate method, 55%, 17%, and 28% of S. aureus isolates were strong, moderate, and weak biofilm producers, respectively, while in CNS the percentages were 43%, 17%, and 40%, respectively. All isolates were screened by Polymerase chain reaction (PCR) for amplification of intercellular adhesion gene A (icaA). In S. aureus isolates the icaA gene was present in 65 % while in CNS was 11%. The majority of S. aureus characterized in this study formed biofilm, which suggests that biofilm formation has an important role in the virulence of S. aureus isolated from bovine intramammary infections in Zulia state.

Preliminary study of biofilm formation properties and antibiotic susceptibility pattern of MRSA and MSSA isolates obtained in Yogyakarta, Indonesia

Aims: Nowadays, Staphylococcus aureus, especially methicillin resistant Staphylococcus aureus (MRSA), emerged as a major pathogenic agent of nosocomial infection and sepsis worldwide. Infections caused by these bacteria are often difficult to treat because of the development of antibiotic resistance. Biofilm formation is an important factor in the pathogenicity of staphylococcal infections and one of the reason of antibiotic treatment failure. In this study, the relationship between biofilm formation properties, the presence of mecA, icaA/D genes and antimicrobial resistance pattern were investigated in 10 methicillin sensitive Staphylococcus aureus (MSSA) and 10 MRSA clinical isolates. Methodology and results: Staphylococcal strains were identified by conventional microbiological methods, while determination of methicillin susceptibility was distinguished by the presence of mecA gene. To investigate biofilm production, congo red agar and microtiter plate test were performed. PCR was done to detect the presence of icaA/D genes, which responsible for biofilm production. Antibiotic sensitivity was carried out by agar diffusion method. The majority of MRSA isolates (90%) were not able to form biofilm, only one isolate (10%) showed capability of weak biofilm producer. Meanwhile, fully established biofilms were formed by all of MSSA isolates (100%). In addition, all MRSA and almost MSSA isolates (90%) harboured both icaA/D genes in their chromosomes. Antibiotic resistance profile of MRSA was more dominant than MSSA isolates. Conclusion, significance, and impact of study: Biofilm production of staphylococci showed difference regulation with regard to methicillin susceptibility. Antibiotic resistance profile was more dominant in MRSA, however biofilm production was found mostly in MSSA isolates.

Correlation bewteen associated genes and biofilm phenotype in Staphylococcus epidermidis / 国际检验医学杂志

Objective To investigate the relationship of icaA,aap ,atlE,sarA gene with biofilm phenotype in Staphyloccus epi-dermidis .Methods Tissue culture plates assays were used to assess biofilm-forming ability of 78 Staphyloccus epidermidis strains.The presence of icaA,atlE,aap ,sarA gene was amplified with PCR method,the relationship between these genes and bio-film phenotype was evaluated by χ2 test and the difference of the biofilm OD′s value in TSB and TSB+3% NaCl by icaA + strains and icaA - strains was evaluated by Wilcoxon sign test,respectively.Results The positive rates of icaA,atlE,aap ,sarA gene were 24.4%(19/78),79.5%(62/78),73.8%(57/78),82.1%(64/78),respectively.40 biofilm-positive strains were detected(51.3%), among which 16 strains carried icaA gene,24 strains showed weak biofilm-forming ability.Those genes above with biofilm forma-tion were statistically correlated.Moreover,there was a relation between the icaA gene and the high biofilm-producing phenotype. There was a significant difference between the biofilm OD values in TSB and TSB+3% NaCl by icaA + strains and icaA - strains, respectively.Conclusion Multiple genes are involved in Staphylococcus epidermidis biofilm-positive phenotype,but the icaA gene contributes to the high biofilm-forming phenotype.Biofilm phenotype is also influenced by environmental factors.

Análise fenotípica e molecular da produção de biofilmes por estirpes de Staphylococcus aureus isoladas de casos de mastite subclínica bovina / Phenotypic and molecular analysis of biofilm production by strains of Staphylococcus aureus isolated from cases of bovine subclinical mastitis

A formação de biofilmes é considerada uma vantagem que alguns Staphylococcus aureus isolados de mastite bovina possuem, facilitando a permanência dos mesmos no úbere. Isto requer a adesão das bactérias no epitélio mamário com proliferação e formação de multicamadas de células envolvidas por uma matriz polimérica conhecida como exopolissacarídeo. O objetivo deste estudo foi avaliar a produção de biofilmes das estirpes de S. aureus isoladas de casos mastite subclínica bovina. Estudou-se 94 estirpes de S. aureus obtidas do leite de vacas com mastite subclínica oriundas de duas propriedades rurais no estado de São Paulo. Essas estirpes foram caracterizadas quanto a produção de biofilmes pelos testes de aderência em microplacas e pela presença dos genes icaA e icaD responsáveis pela produção do polissacarídeo de adesão intercelular. Os resultados obtidos revelaram que no teste de aderência em placas, 98,9% das estirpes produziram biofilme e a presença dos genes icaA e icaD foi encontrada em 95,7% das estirpes de S. aureus. Bactérias produtoras de biofilmes no epitélio mamário diminuem a efetividade dos antimicrobianos, por isso a detecção de S. aureus produtoras de biofilmes têm importância no entendimento da patogenia desse agente, além de permitir uma melhor adoção de medidas de prevenção e controle.

Biofilm formation is considered an advantage for Staphylococcus aureus mastitis isolates, facilitating bacterial persistence in the udder. It requires attachment to mammary epithelium, proliferation and accumulation of cells in multilayers and enclosing in a polymeric matrix known as exopolysaccharide. The objective of this study was to evaluate the ability of Staphylococcus aureus isolated from bovine subclinical mastitis for formation of biofilms. A total of 94 Staphylococcus aureus strains obtained from milk samples of cows suffering from subclinical mastitis in dairy herds on two properties in the state of São Paulo were evaluated. These strains were characterized by in vitro biofilm formation, and by the presence of icaA and icaD genes which are responsible for intercellular adhesion. The results revealed that 98.9% of the isolates produced biofilm in vitro by adherence in sterile 96-well "U" bottom polystyrene tissue culture plates; 95.7% of the isolates possessed the icaA and icaD genes. These bacterial isolates biofilm producers may impair eradication of chronic mastitis, rendering antibiotherapy less effective. The detection of biofilm forming ability in mastitis isolates may provide useful information for more adequate therapeutic regimen and for preventive actions in the control of those bacterial isolates in bovine herds.

Detection of methicillin resistance and slime factor production of Staphylococcus aureus in bovine mastitis / Detecção de resistência a meticilina e produção do fator slime por Staphylococcus aureus em mastite bovina

This study aimed to detect methicillin resistant and slime producing Staphylococcus aureus in cases of bovine mastitis. A triplex PCR was optimized targetting 16S rRNA, nuc and mecA genes for detection of Staphylococcus species, S. aureus and methicillin resistance, respectively. Furthermore, for detection of slime producing strains, a PCR assay targetting icaA and icaD genes was performed. In this study, 59 strains were detected as S. aureus by both conventional tests and PCR, and 13 of them were found to be methicillinresistant and 4 (30.7%) were positive for mecA gene. Although 22 of 59 (37.2%) S. aureus isolates were slimeproducing in Congo Red Agar, in PCR analysis only 15 were positive for both icaA and icaD genes. Sixteen and 38 out of 59 strains were positive for icaA and icaD gene, respectively. Only 2 of 59 strains were positive for both methicillin resistance and slime producing, phenotypically, suggesting lack of correlation between methicillin resistance and slime production in these isolates. In conclusion, the optimized triplex PCR in this study was useful for rapid and reliable detection of methicillin resistant S. aureus. Furthermore, only PCR targetting icaA and icaD may not sufficient to detect slime production and further studies targetting other ica genes should be conducted for accurate evaluation of slime production characters of S. aureus strains.

Este estudo objetivou a detecção de Staphylococcus aureus resistente a meticilina e produtor do fator slime em casos de mastite bovina. Um PCR triplex foi otimizado, com alvo no genes 16SrRNA, nuc e mecA para detecção de Staphylococcus spp, S. aureus e resistencia a meticilina, respectivamente. Para detecção das cepas produtoras do fator slime, empregou-se um PCR com alvo nos genes icaA e icaD. No estudo, 59 cepas foram identificadas como S. aureus por testes convencionais e PCR, sendo 13 resistentes a meticilina e quatro positivas para o gene mecA. Embora 22 das 59 cepas tenham sido produtoras do fator slime em Agar Vermelho Congo, no teste PCR somente 15 foram positivas para os genes icaA e icaD. Dezesseis e 38 das 59 cepas foram positivas para os genes icaA e icaD, respectivamente. Somente duas das 59 cepas foram positivas simultaneamente para resistência a meticilina e produção do fator slime, sugerindo falta de correlação entre estas características. Em conclusão, o PCR triplex otimizado neste trabalho mostrou-se ser um método rápido e confiável para detecção de S.aureus meticilina resistente. Por outro lado, somente PCR para os genes icaA e icaD pode não ser suficiente para detectar produção de fator slime e outros estudos com alvo em outros genes ica são necessários para um avaliação correta da produção do fator slime por S. aureus.

Analysis of the biofilm formation and icaA genes of Staphylococcus epidermidis isolates / 中华微生物学和免疫学杂志

Objective To determine the ability of biofilm formation of Staphylococcus ephtermidis isolates and analyze the correlation between the icaA gene and its expression and biofilm formation. Methods Collecting 205 Staphylococcus epidermidis isolates identified with normal laboratory tests (coagulase-negative, biochemical identification, polymyxin-resistant and novobiocin-sensitive ), the suspected isolates were con-formed with API-Staph. Biofilm production was assessed by incubating the strains on Congo Red Agar (CRA) plates and quantitative biofilm production determined by a 96-well tissue culture plate and biofilm morphous were detected by scanning electron microscope ( SEM ) ; Amplifying partial fragments of icaA genes with PCR; Analyzing the expression levels of icaA gene with RT-PCR through Bio-Rad system and Quantity One software. Results 24 isolates showed positive in CRA tests, 22 isolates were positive in semiquantita- tive adhesion assays and 28 isolates existed icaA gene among 205 isolates of Staphylococcus epidermidis. The icaA-positive strains demonstrated biofilm formation (microcolonies on silica films ) while icaA-negative strains only adhered as individual cells under scanning electron microscope. All 22 strains which showed positive in semiquantitative adhesion assays harbored the icaA gene. The expression levels of icaA gene with RT-PCR in 6 Staphylococcus epidermidis isolates showed a higher tendency in 4 strains which demonstrated positive in semiquantitative adhesion assays than 2 negative strains in semiquantitative adhesion assays. Conclusion The isolations of Staphylococcus epidermidis have the abilities of forming biofilm, and the icaA gene and its normal expression is the important molecular biology foundation of biofilm formation. Other fac-tors maybe involve in the expression of icaA gene in Staphylococcus epidermidis isolates.

Detection of Virulence Genes of Staphyloccus aureus and Staphylococcus epidermidis Isolated from Suprapubic Urine from Infants with Fever

While methicillin-resistant Staphylococcus aureus (MRSA) isolated from urinary tract infection (UTI) has recently increased in elderly adult urology patients, it has been only rarely reported in infants. Therefore, in this study to understand whether MRSA may be involved in UTI of infants who run fever without other apparent causes, we identified and counted S. aureus and S. epidermidis in suprapubic urine from 750 febrile infants via microbiological methods, and confirmed the counts via multiplex PCR. And we also detected four virulence genes, mecA, PVL, bbp and icaA genes for S. aureus and S. epidermidis via multiplex PCR in the same specimens. S. aureus (28 cases) counts were as follows: >10(4) CFU/ml (3/28), 10(2)~10(3) CFU/ml (1/28) and 10(4) CFU/ml (2/26), 10(2)~10(3) CFU/ml (4/26) and 10(2)~10(3) CFU/ml (20/26). S. aureus virulence genes were detected in 26 cases as mecA (16/26, 59.3%), PVL (17/26, 63.0%), bbp (7/26, 26.9%) and icaA (20/26, 76.9%). S. epidermidis virulence genes were detected in 22 cases as mecA (17/22, 81.0%), PVL (15/22, 71.4%), bbp (3/22, 13.6%) and icaA (13/22, 50.1%). Therefore, mecA, PVL and icaA genes of MRSA and MRSE were detected with high positivity in urines from infants with fever. The results demonstrate that community-acquired MRSA or MRSE may be responsible for UTI incidence in febrile infants.

Relationship of macrolide-resistant phenotypes with biofilm formation and icaA-genetypes of clinically isolated Staphylococcus epidermidis / 第三军医大学学报

Objective To study the macrolide-resistance,ability of biofilm formation and icaA-genetypes of 68 stains of clinically isolated Staphylococcus epidermidis(S.epidermidis) so as to explore the efficacy of macrolide to prevent biofilm-associated infections caused by S.epidermidis.Methods Minimum inhibitory concentration(MIC) were detected by agar-plate dilution method,and microtiter-plate assay was used to investigate the ability of biofilm formation,and icaA genetype was identified by PCR.Results High macrolide-resistant rate(88.2%) was showed for clinical isolated S.epidermidis,and biofilms for macrolide-resistant strains were significantly stronger than that of macrolide-sensitive strains,but there was no dependability found between these two for icaA positive rate.Conclusion Frequently-used macrolide such as erythromycin,azithromycin,and clarithromycin may incompetence to prevent biofilm-associated infections caused by S.epidermidis.