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)

Trans-Sacral Epiduroscopic Laser Decompression (SELD) for Low back pain secondary to herniated lumbar disc in the Philippines: A case report and review of literature

@#LBP has a lifetime prevalence ranging from 54% to 80%, an annual prevalence of 15-45%, and a point prevalence of 30%. Lumbar radicular pain often results from a Lumbar disc herniation. With recent advances in technique and access in instrumentation, minimally invasive spine surgery has ushered in a renaissance of spine care. SELD has promising positive effects in controlling LBP following HLD. This is a case of a 26 year old with radicular LBP of 2 years duration secondary to HLD, underwent conservative management of LBP but offered no relief and improvement, hence he became the case for the pioneering procedure of SELD in the Philippines. Immediate and significant improvement in the patient was noted. SELD was proven to be an effective therapeutic modality for patients with LBP secondary to HLD.

Impact of Antimicrobial Agents on Transcription of Gene hld in Forming Process of Staphylococcus epidermidis Biofilm / 中华医院感染学杂志

OBJECTIVE To investigate the transcription level of gene hld of Staphylococcus epidermidis in the biofilm forming and detaching under MIC antibiotic and explore the relationship between biofilm-related drug resistance and persistant infection.METHODS The transcription level of gene hld of S.epidermidis under MIC concentration of 4 antibiotics was compared with those of the control group without antibiotics by SYBR real-time fluorescent quantitative RT-PCR at the different time point of biofilm formation and detachment.RESULTS The transcription of gene hld decreased rapidly from initial adherence,and droped continuously for few hours.There was an increase from 24 hours to 72 hours in groups without antibiotics but not in antibiotics groups,the differenet was significant.CONCLUSIONS Antibiotics improve adherence at first and then prevent matrix decomposition water-conducting tube and detachment of cells by impact of transcription of gene hld,it can protect cells from killing by inhibiting the penetration of biotics and prevent them become planktonic cells after detachment from biofilm.

Minimaly Invasive Spinal Surgery: Cinical analysis of Chemonucleolysis & Percutaneous Automated Lumbar Discectomy

The authors retrospectively analized the radiographs and medical records of 135 consecutive patients who underwent minimaly invasive spinal surgery from January, 1990 to December, 1994 at our institution. Among these, 72 patients have had chemonucleolysis and others percutaneous automated lumbar discectomy(PALD). The success rate was similar in both groups, ie, 75% in chemonucleolysis and 74.6% in PALD by Macnab's criteria. The higher success rate could be obtained in 1) younger age below 30, 2) predominant sciatica than lower back pain, 3) eccentric bulged disc with minimal degeneration, 4) abscent or minimal neurologic deficits. In conclusion, provided patients are carefully screened, minimal invasive spinal surgery is more preferable over conventional surgery, providing a safe and effective therapeutic alternative to many patients suffering from herniated lumbar disc.

Modality of malocclusion according to regional difference using HLD(handicapping labiolingual deviation), TPI(treatment priority index) and HMAR(handicapped malocclusion assessing record) / 대한치과교정학회지

We tried to evaluate frequencies and severities of malocclusion of various socioeconomic areas according to growth and development. To obtain objective validity, we used Handicapping Labiolingual Deviation (HLD) suggested by Draker, Treatment Priority Index (TPI) by Grainger, Handicapped Malocclusion Assessing Record (HMAR) by American Dental Association and American Association of Orthodontist. Seoul and Kwangju were selected as an urban group, Ahnyang as an middle socioeconomic group and Hwasun near the Kwangju area as an rural group. 140 (male 63, female 73) of 5th grade in 'E' elementary school students in Seoul, 202 (male 101, female 101) of 'S' elementary school students in Kwangju, 207(male 105, female 102) of 'H' elementary school students in Hwasun, and 100(male 49, female 51) of 'M' elementary school students of in Ahnyang of all the same grade were analyzed and we obtained the results as follows; 1. TPI, HLD, and HMAR showed high coincidence and reproducibility between different observer. (p<0.01) 2. In comparison of TPI, HLD, and HMAR according to clinical decision, there was statistically difference between each other. (p<0.01) 3. As the severity of malocclusion increases there was high correlation between TPI and HLD, and between HLD and HMAR (p<0.05) 4. With respect to differences between areas in TPI and HLD, there was statistically difference between Ahnyang and other areas. 5. In Seoul as an urban group there was a high incidence of periodic checkup and history of orthodontic treatment than other area. 6. Mode of feeding and posture during sleeping did not affect the severity of malocclusion. As a conclusion, TPI, HLD and HMAR are so reproducible and coincident that they can be used as a guide on evaluating the frequency and severity of malocclusion and determining the priority of orthodontic treatment to determine the need and supply of orthodontic treatment. Furthermore these indices can provide objectively valid data for establishing public health problem solution.