Quantitative PCR analysis of genes expressed during biofilm development of methicillin resistant Staphylococcus aureus (MRSA)

Staphylococcus aureus biofilm associated infections remains a major clinical concern in patients with indwelling devices. Quantitative real-time PCR (qPCR) can be used to investigate the pathogenic role of such biofilms. We describe qPCRs for 12 adhesion and biofilm-related genes of four S. aureus isolates which were applied during in vitro biofilm development. An endogenous control (16S rRNA) was used for signal normalization. We compared the qPCR results with structural analysis using scanning electron microscopy (SEM). The SEM studies showed different cellular products surrounding the aggregated cells at different times of biofilm formation. Using qPCR, we found that expression levels of the gene encoding fibronectin binding protein A and B and clumping factor B (fnbA/B and clfB), which involves in primary adherence of S. aureus, were significantly increased at 24 h and decreased slightly and variably at 48 h when all 4 isolates were considered. The elastin binding protein (ebps) RNA expression level was significantly enhanced more than 6-fold at 24 and 48 h compared to 12 h. Similar results were obtained for the intercellular adhesion biofilm required genes type C (icaC). In addition, qPCR revealed a fluctuation in expression levels at different time points of biofilm growth of other genes, indicating that different parameter modes of growth processes are operating at different times.     

Assessment of eugenol inhibitory effect on biofilm formation and biofilm gene expression in methicillin resistant Staphylococcus aureus clinical isolates in Egypt

Methicillin-resistant Staphylococcus aureus (MRSA) biofilm infection is a major threat in Healthcare facilities. The search for biofilm inhibitors is essential to overcome the antibiotic resistance. Eugenol is a phyto-compound that possesses many biological properties. In this study, the aim was to estimate the effect of eugenol on biofilms of MRSA through quantifying the level of gene expression of three genes (IcaA, IcaD and SarA) involved in biofilm development.. Fifty MRSA biofilm producers collected from the microbiology lab at Theodor Bilharz Research Institute were incubated with different concentrations of eugenol for 24 h. The minimum inhibitory concentration of eugenol (MIC) that eradicates the biofilms growth was detected. mRNA was extracted from all isolates before and after the application of eugenol at 0.5 x MIC, and then subjected to quantitative real-time PCR (qPCR). Results showed that fourteen isolates out of 50 (28%) exhibited intermediate biofilm formation ability, and 36 out of 50 (72%) were strong biofilm producers. The MIC values of eugenol for MRSA ranged from 3.125% to 0.01%. The mean values of MIC in both strong and intermediate biofilm forming MRSA isolates were statistically comparable (p = 0.202). qPCR results revealed that the levels of expression of the studied genes IcaA, IcaD, and SarA were decreased after eugenol treatment when compared with their corresponding values before treatment (p = 0.001). Eugenol inhibited the formation of biofilm of MRSA isolates, indicating it could be used to control infections associated with MRSA biofilms.     

The effect of Staphylococcus aureus on the antibiotic resistance and pathogenicity of Pseudomonas aeruginosa based on crc gene as a metabolism regulator: An in vitro wound model study

BackgroundThe cooperation of Pseudomonas aeruginosa and Staphylococcus aureus in various infections results in increased pathogenicity and antibiotic resistance. However, the mechanism controlling such a phenomenon is still unclear. In this study, the effects of S. aureus on the metabolism, antibiotic resistance, and pathogenicity of P. aeruginosa were investigated.Material and methodsThe biofilm and the planktonic states of growth of P. aeruginosa and S. aureus were investigated using the co-culture method in the L929 cell line. Then, the antibiotic resistance and virulence factors production of the recovered colonies of P. aeruginosa were examined by phenotypic methods. Quantitative Real-Time PCR was used to determine the expression level of crc, lasI/R, and rhlI/R genes. Two way ANOVA test and student's t-test were used to analyze the effect of S.aureus on metabolism, virulence, and resistance of P.aeruginosa.ResultsP. aeruginosa strains in a single-species planktonic culture on the L929 cell line indicated higher CFU counts than the biofilm. Conversely, in the biofilm state of co-culture, the CFU counts increased in comparison to the planktonic condition. Also, the expression level of crc increased two fold in the PA-1 and PA-2 strains compared to the single-species cultures on the L929 cell line. However, the PA-3 strain indicated a sharp decrease in the expression of crc (3 fold decrease). Besides, a 3–4 fold increase in susceptibility to amikacin was observed as the expression level of crc declined. The QS-regulated factors were diminished as rhlR and lasI were downregulated in both states of growth.ConclusionIn polymicrobial wound infection, Staphylococcus aureus plays a vital role in the metabolic changes of Pseudomonas aeruginosa. However, the levels of antibiotic susceptibility and pathogenicity of Pseudomonas aeruginosa also changed due to metabolism.     

Detection of the intercellular adhesion gene cluster (ica) in clinical Staphylococcus aureus isolates

Staphylococcus aureus is a major hospital and community pathogen having the aptitude to cause a wide variety of infections in men. The ability of microorganisms to produce biofilm facilitates them to withstand the host immune response and is recognized as one factor contributing to chronic or persistent infections. It was demonstrated that the ica-encoded genes lead to the biosynthesis of polysaccharide intercellular adhesion (PIA) molecules, and may be involved in the accumulation phase of biofilm formation. Different studies have shown the decisive role of the ica gene as virulence factors in staphylococcal infections. This study was carried out to demonstrate the relationship between ica gene and production of slime layer in S. aureus strains. Sixty S. aureus strains were isolated from patients. The isolates were identified morphologically and biochemically following standard laboratory methods. After identification, the staphylococcal isolates were maintained in trypticase soy broth (TSB), to which 15% glycerol was added, and stored at –20°C. Slime formation and biofilm assay was monitored. A PCR assay was developed to identify the presence of icaD (intercellular adhesion gene) gene in all isolates. Thirty-nine slime producing colonies with CRA plates (65%) formed black colors, the remaining 21 isolates were pink (35%). In the quantitative biofilm assay 35 (58%) produced biofilm while 25 (42%) isolates did not exhibit this property. All isolates were positive for detection of icaD gene by PCR method. The interaction of icaA and icaD in the investigated isolates may be important in slime layer formation and biofilm phenomena.We propose PCR detection of the ica gene locus as a rapid and effective method to be used for discrimination between potentially virulent and nonvirulent isolates, with implications for therapeutic and preventive measures pertainin to the management of colonized indwelling catheters.     

金黄色葡萄球菌生物被膜耐药机制研究进展

金黄色葡萄球菌是常见的引起医院感染和社区感染的致病菌之一。近年来, 临床上金黄色葡萄球菌抗感染治疗失败的病例越来越多, 生物被膜的形成被认为是导致抗菌药物治疗失败的主要原因。然而, 金黄色葡萄球菌生物被膜的耐药机制并未完全阐明。证据表明, 金黄色葡萄球菌生物被膜感染难以治愈且容易反复, 感染后反复治疗将大大增加患者的痛苦和经济负担。本文对金黄色葡萄球菌生物被膜耐药机制的研究进展进行综述, 以期为开发新的抗菌药物提供参考依据。     

Molecular characterization of invasive Staphylococcus aureus strains isolated from patients with diabetes in Iran: USA300 emerges as the major type

There have been few studies focused on the molecular characterization of invasive Staphylococcus aureus strains in patients with diabetes in Iran. In the present study, 20 invasive S. aureus strains recovered from the patients with diabetes characterized by the virulence and resistance analysis, biofilm formation, staphylocoagulase (SC) typing, S. aureus protein A locus (spa) typing staphylococcal cassette chromosome mec (SCCmec) typing, and multilocus sequence typing (MLST). Virulence gene detection indicated a high prevalence of strains encoding the pvl genes (50%), a low prevalence of the tst and seg gene (each of them was 5%) and a markedly high prevalence of fnbB (95%), fnbA (85%), icaD (75%), icaA (65%). A total of 3 coagulase types (III, 85%; II, 10%; V, 5%), 2 agr types (I, 90%; II 10%) and 2 SCCmec types (IV, 65%; III, 35%) and four different clones namely ST8-MRSA-IV/t008 (50%) (USA300), ST239-MRSA-III/t030 (35%), ST5-MRSA-IV/t002 (10%), and ST45-MRSA-IV/t038 (5%) were detected in this study. Eighty-five percent of the isolates were biofilm producers. All the 4 high-level mupirocin resistance (HLMUPR) strains belonged to CC/ST8-MRSA-IV/t008 clone and carried mupA gene. Fusidic acid-resistant isolate belonged to ST239-SCCmec III/t030 clone. One vancomycin-intermediate resistance isolates was detected in our study, which belonged to ST5-MRSA-IVt002. Circulating clone in MRSA strains (USA300) isolated from the patients with diabetes highlighting the possibility of transmission of these microorganisms' clones between hospital, community, and environments. However, further studies require providing critical insights into the importance of continued controlling and treatment of S. aureus infections in patients with diabetes.     

Population genetics and the evolution of virulence in Staphylococcus aureus

Staphylococcus aureus is one of the most important human pathogens, causing life-threatening infection in the community and hospital setting. The population genetics of S. aureus and the evolution of virulence is the focus of this review. We describe the various techniques in determining S. aureus population structure and discuss the insights gained from whole genome sequencing of various S. aureus strains. The emergence of community-acquired, methicillin-resistant S. aureus provides a framework for the discussion on evolution of virulence, and the role of horizontal gene transfer in the development of virulence and antibiotic resistance is explored. The knowledge generated from population genetics has the potential to inform strategies to assist in the prevention or treatment of this highly successful human pathogen.     

Probing vaccine antigens against bovine mastitis caused by Streptococcus uberis

Streptococcus uberis is a worldwide pathogen that causes intramammary infections in dairy cattle. Because virulence factors determining the pathogenicity of S. uberis have not been clearly identified so far, a commercial vaccine is not yet available. Different S. uberis strains have the ability to form biofilm in vitro, although the association of this kind of growth with the development of mastitis is unknown. The objective of this study was to evaluate the potential use as vaccine antigens of proteins from S. uberis biofilms, previously identified by proteomic and immunological analyses. The capability of eliciting a protective immune response by targeted candidates was assayed on a murine model. Sera from rabbits immunized with S. uberis biofilm preparations and a convalescent cow intra-mammary infected with S. uberis were probed against cell wall proteins from biofilm and planktonic cells previously separated by two-dimensional gel electrophoresis. Using rabbit immunized serum, two proteins were found to be up-regulated in biofilm cells as compared to planktonic cells; when serum from the convalescent cow was used, up to sixteen biofilm proteins were detected. From these proteins, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), fructose-biphosphate aldolase (FBA), and elongation factor Ts (EFTs) were chosen to be tested as vaccine antigen candidates. For this purpose, different groups of mice were immunized with the three recombinant-expressed proteins (each one formulated separately in a vaccine), and thereafter intraperitoneally challenged with S. uberis. The three proteins induced specific IgG antibodies, but a significant reduction of mortality was only observed in the groups of mice vaccinated with FBA or EFTs. These results suggest that FBA and EFTs might be considered as strong antigenic candidates for a vaccine against S. uberis bovine mastitis. Moreover, this is the first study to indicate that also in S. uberis, GAPDH, FBA and EFTs, as proteins detected in both cytoplasm and cell wall fractions, can play a second function (moonlighting), the latter being particularly involved in the virulence of such a pathogen organism.     

Increase in IS256 transposition in invasive vancomycin heteroresistant Staphylococcus aureus isolate belonging to ST100 and its derived VISA mutants

In Staphylococcus aureus, transposition of IS256 has been described to play an important role in biofilm formation and antibiotic resistance. This study describes the molecular characterization of two clinical heterogeneous vancomycin-intermediate S. aureus (hVISA) isolates recovered from the same patient (before and after antibiotic treatment) and two VISA derivatives obtained by serial passages in the presence of vancomycin. Our results showed that antibiotic treatment (in vivo and in vitro) could enhance IS256 transposition, being responsible for the eventual loss of agr function. As far as we know this is the first study that reports the increase of IS256 transposition in isogenic strains after antibiotic treatment in a clinical setting.     

Evolution of Staphylococcus aureus and MRSA during outbreaks

Investigation of Staphylococcus aureus outbreaks, and particularly those due to methicillin-resistant S. aureus (MRSA) in hospitals, can identify infection reservoirs and prevent further colonization and infection. During outbreaks, S. aureus genomes develop single nucleotide polymorphisms (SNPs), small genetic rearrangements, and/or acquire and lose mobile genetic elements (MGE) encoding resistance and virulence genes. Whole genome sequencing (WGS) is the most powerful method for discriminating between related isolates and deciding which are involved in an outbreak. Isolates with only minor variations are detectable and can identify MRSA transmission routes and identify reservoirs. Some patients may carry ‘clouds’ of related isolates, and this has consequences for how we interpret the data from outbreak investigations. Different clones of MRSA are evolving at different rates, influencing their typability. S. aureus genome variation reveals the importance of antibiotic resistance in the long term evolution of successful hospital clones, contributing to strategies to prevent the spread of successful MRSA clones.     

铜绿假单胞菌lasI/lasR群体感应系统与生物膜形成相关基因表达调控的研究

目的 了解铜绿假单胞菌(PAE)生物膜形成过程中多糖合成相关基因,lasI/lasR群体感应系统及鼠李糖脂转移酶合成相关基因rhlA在生物膜形成过程中的表达,探讨其在PAE生物膜形成中的关系及调控作用.方法 分别收集非黏液型铜绿假单胞菌PAO1的浮游菌及生物被膜菌,用实时荧光定量RT-PCR的方法,对基因的表达进行相对定量分析.结果 pslAld的浮游菌表达量略高于6d生物膜菌外,algD、pelA、LasI、LasR、RhlA生物被膜菌的mRNA相对表达量均高于浮游菌,并且pslA、pelA、RhlA的mRNA相对表达量趋势一致,均是培养1d生物膜菌表达最高.结论 algD、pslA、pelA、LasI,LasR、RhlA的表达与铜绿假单胞菌生物膜形成密切相关,在生物膜形成中具有重要作用,同时群体感应系统很可能参与pslA、pelA、RhlA的正向转录调节.     

In vitro efficacy of cold atmospheric pressure plasma on S. sanguinis biofilms in comparison of two test models

Dental plaque critically affects the etiology of caries, periodontitis and periimplantitis. The mechanical removal of plaque can only be performed partially due to limited accessibility. Therefore, plaque still represents one of the major therapeutic challenges. Even though antiseptic mouth rinses reduce the extent of biofilm temporarily, plaque removal remains incomplete and continuous usage can even result in side effects. Here we tested argon plasma produced by kinpen09 as one option to inactivate microorganisms and to eliminate plaque. S. sanguinis biofilms cultivated in either the European Biofilm Reactor (EUREBI) or in 24 well plates were treated with argon plasma. In both test systems a homogeneous, good analyzable and stable biofilm was produced on the surface of titan plates within 72 h (>6,9 log₁₀ CFU/ml). Despite the significantly more powerful biofilm production in EUREBI, the difference of 0.4 log₁₀ CFU/ml between EUREBI and the 24 well plates was practically not relevant. For that reason both test models were equally qualified for the analysis of efficacy of cold atmospheric pressure plasma. We demonstrate a significant reduction of the biofilm compared to the control in both test models. After plasma application of 180 s the biofilm produced in EUREBI or in 24 well plates was decreased by 0.6 log₁₀ CFU/ml or 0.5 log₁₀ CFU/ml, respectively. In comparison to recently published studies analyzing the efficacy of kinpen09, S. sanguinis produces a hardly removable biofilm. Future investigations using reduced distances between plasma source and biofilm, various compositions of plasma and alternative plasma sources will contribute to further optimization of the efficacy against S. sanguinis biofilms.     

土槿乙酸对葡萄球菌生物膜形成的抑制作用

目的:研究中药活性成分土槿乙酸对葡萄球菌生物膜形成的抑制作用。方法:采用肉汤培养法检测土槿乙酸对金黄色葡萄球菌和表皮葡萄球菌浮游细菌生长的影响;采用分光光度计检测法分析土槿乙酸对上述二菌株生物膜形成的影响。结果:30μg/ml土槿乙酸对两株葡萄球菌浮游细菌数量没有明显影响,但同样浓度下土槿乙酸能明显抑制细菌形成生物膜。金黄色葡萄球菌生物膜试验组与对照组A550值分别为1.03±0.12和0.26±0.03(P<0.05),表皮葡萄球菌生物膜相应数值为1.36±0.08和0.41±0.05(P<0.05),有统计学差异。结论:尽管土槿乙酸不能直接杀灭葡萄球菌,但对其生物膜形成有抑制作用,为进一步开发中药抗感染药物奠定基础。     

Vancomycin resistance in Staphylococcus aureus: a new challenge for infection control and antibiotic prescribing

There have been several reported cases to date of Staphylococcus aureus (S. aureus) with decreased susceptibility to the glycopeptide antibiotics, particularly vancomycin. Such isolates have been termed VISA (vancomycin-intermediate S. aureus) or GISA (glycopeptide-intermediate S. aureus). These strains have developed in patients with pre-existing methicillin-resistant S. aureus (MRSA) infections treated with prolonged courses of the drug of choice, vancomycin. Resistance to vancomycin severely limits treatment options for these patients, highlighting the need to preserve the utility of the drug through restricted usage and the need for increased awareness of infection control measures to prevent acquisition of MRSA in the first instance. While vancomycin-resistant Enterococcus (VRE) has received significant attention in the scientific literature and the media, the clinical implications of a vancomycin-resistant strain of S. aureus, a more pathogenic and virulent organism, are far more significant. This review details the clinical features and microbiology of the isolates found so far, the use of vancomycin in staphylococcal infections and some of the measures necessary to prevent the development and spread of vancomycin resistance in Australia. [AIC Aust Infect Control 2000; 5(4):9-16].     

Disinfection of meticillin-resistant Staphylococcus aureus and Staphylococcus epidermidis biofilms using a remote non-thermal gas plasma

The effective disinfection of hospital surfaces is recognised as an important factor in preventing hospital-acquired infections. The purpose of this study was to quantify the disinfection rate of a novel gas plasma system on clinically relevant biofilms. Clinical isolates of Staphylococcus epidermidis and meticillin-resistant Staphylococcus aureus (MRSA) were grown as biofilms on glass surfaces and tested in a disinfection container remote from the plasma source. The strains used in this study were known to produce substantial quantities of biofilm and average log?? counts were 9.0 and 9.1 cfu/cm(2) for S. epidermidis and MRSA respectively. Counts were reduced by between 4 and 4.5 log?? after 1h of exposure for MRSA and S. epidermidis respectively. More prolonged treatment in the case of MRSA biofilms resulted in a 5.5 log?? reduction after 90 min. Biofilm samples were also placed in medical device packaging bags and similar rates of disinfection were observed.     

Isolation and antimicrobial susceptibility pattern of Staphylococcus aureus in patients with surgical site infection at Debre Markos Referral Hospital,Amhara Region,Ethiopia

Background

Staphylococcus aureus, especially Methicillin Resistant Staphylococcus Aureus (MRSA) is a major health problem recognized as the most important nosocomial pathogen, often causing postoperative wound infections. Antibiotic resistance by MRSA has grown to be common, and resistance to almost all antibiotics has been found among these strains. The aim of this study was to determine the prevalence, antimicrobial susceptibility patterns and associated risk factors of S. aureus in patients with surgical site infections in an Ethiopian hospital.

Methods

A cross-sectional study was conducted from December 1, 2011 to March 30, 2012 among patients with surgical site infections at Debre Markos Referral Hospital, Debre Markos, Ethiopia. All wound swabs obtained from patients with surgical site infections during the study period were cultured on mannitol salt agar media which is selective for S. aureus. Isolated strains of S. aureus were tested for antibiotic susceptibility patterns using standard disc diffusion technique, and interpretation of resistance was done based on Clinical and Laboratory Standard Institute criteria. Univariate and multivariable analyses were used to assess the risk factors.

Results

Of the 184 surgical patients who had developed surgical site infection, S. aureus was isolated from 73 (39.7%) cases. Out of the 73 isolates of S. aureus, 36 (49.7%) were MRSA. Among the study participants, prevalence of MRSA was found to be 19.6%. The clinical isolates showed >80% level of resistance to ampicillin, amoxicillin, penicillin G, erythromycin, gentamicin and cotrimoxazole whereas <50% level of resistance was observed against clindamycin, oxacillin, tetracycline and vancomycin. MRSA strains showed resistance ranging from 5.6% (vancomycin) to 100% (cotrimoxazole). Of the following risk factors: sex, age, pus consistency, duration of operation, type of surgery, ward and hospital stay, laparotomy type of surgery was identified as a risk factor for infection by S. aureus.

Conclusion

The prevalence of S. aureus and/or MRSA infection in surgical and gynaecology & obstetrics wards of Debre Markos Referral Hospital was found to be high. The majority of isolates were highly resistant to major antimicrobial agents.     

Virulence Factors of Staphylococcus aureus Isolates in an Iranian Referral Children's Hospital

Objectives

The clinical importance of Staphylococcus aureus (S. aureus) is attributed to notable virulence factors, surface proteins, toxins, and enzymes as well as the rapid development of drug resistance. The aim of this study was to compare the occurrence of virulence factors produced by S. aureus strains isolated from children in an Iranian referral children''s hospital.

Methods

The presence of genes encoding for the enterotoxins A (sea), B (seb), C (sec), D (sed), TSST-1 (tsst), exfoliative toxin A (eta), and exfoliative toxin B (etb) were detected by Multiplex polymerase chain reaction (PCR) using specific primers. In addition, the standardized Kirby-Bauer disc-diffusion method was performed on Mueller-Hinton agar.

Results

In total, 133 S. aureus isolates were obtained from different patients. Of these S. aureus isolates, 64 (48%) were methicillin-resistant S. aureus (MRSA), and all of these tested positive for the mecA gene. Regarding the classical enterotoxin genes, sea gene (40.6%) was the most prevalent followed by seb (19.6%), tsst (12.8%), eta (11.3%), etb (9%), sed (4.5%), and sec (3%). Among methicillin-susceptible S. aureus (MSSA) isolates, seb and tsst were the more prevalent toxins in comparison with MRSA isolates (p < 0.05), while the frequency of sea, sed, eta, and etb genes were higher among MRSA isolates (p > 0.05).

Conclusion

In our study enterotoxin A was produced by 40.6% of the isolates (48% from MRSA and 33% from MSSA isolates) which was higher than in previous reports. According to our results, strict hygiene and preventative measures during food processing are highly recommended.     

Prevalence of community acquired methicillin resistant Staphylococcus aureus (CA-MRSA) in skin and soft tissue infections among cases from Puducherry, India

Aim

Methicillin resistant Staphylococcus aureus (S. aureus) is a major human pathogen which causes a wide range of hospital and community-acquired infections worldwide. Our objective is to describe the prevalence of MRSA within Puducherry, India.

Methods

A total of 172 patients treated with incision and drainage of soft tissue infections from March 2009 to September 2010 were included in the present study. We examined the antibiotic sensitivity pattern and the distribution of genes among MRSA isolates.

Results

In total, 72 isolates were obtained of which 51 were MRSA and 21 were methicillin sensitive S. aureus (MSSA). All the isolates showed absolute resistance pattern against beta lactam antibiotics (penicillin, ampicillin, amoxicillin) and were found to be sensitive towards macrolide and lincosamide antibiotics. The gene distribution among MRSA isolates showed the presence of femA, mecA, and lukS in 100, 94.4 and 69.4?% of the isolates respectively. The antibiotic sensitivity pattern and molecular characterization showed an increased prevalence of community acquired MRSA (CA-MRSA) in the study population.

Conclusion

The MRSA isolates were found to be sensitive to macrolide and lincosamide antibiotics but MSSA showed resistance pattern to the same. In this regard these antibiotics can be used for the effective treatment of MRSA at the expense of MSSA among the study population.