femB Gene Research Articles

Characteristics of staphylococcal cassette chromosome mec and lugdunin operon genes in the complete genome of Staphylococcus lugdunensis.

Characteristics of staphylococcal cassette chromosome mec and lugdunin operon genes in the complete genome of Staphylococcus lugdunensis.

Membrane active 7-thiazoxime quinolones as novel DNA binding agents to decrease the genes expression and exert potent anti-methicillin-resistant Staphylococcus aureus activity.

A novel class of 7-thiazoxime quinolones was developed as potential antimicrobial agents for the sake of bypassing resistance of quinolones. Biological assays revealed that some constructed 7-thiazoxime quinolones possessed effective antibacterial efficiency. Methyl acetate oxime derivative 6l exhibited 32-fold more active than ciprofloxacin against MRSA, which also possessed rapidly bactericidal ability and low toxicity towards mammalian cells. The combination use of 7-thiazoxime quinolone 6l and ciprofloxacin was able to improve antibacterial potency and effectively alleviate bacterial resistance. The preliminarily mechanism exploration revealed that compound 6l could destroy the cell membrane and insert into MRSA DNA to bind with DNA gyrase, then decrease the expression of gyrB and femB genes. The above results strongly suggested that methyl acetate oxime derivative 6l held a promise for combating MRSA infection.

Avaliação genotípica da resistência antimicrobiana em Staphylococcus spp. Isolados de mastite clínica bovina

ABSTRACT Bovine clinical mastitis caused by Staphylococcus spp. is a serious and widespread disease in the world of dairy farming. Antimicrobial therapy is of fundamental importance in the prevention and treatment of infectious mastitis, but the indiscriminate use of antimicrobials acts as a determining factor for the spread of the disease. The present study evaluated the resistance profiles of 57 Staphylococcus spp. isolated from bovine clinical mastitis to beta-lactams and gentamicin, relating characteristics of phenotype (in vitro susceptibility tests) and genotype (detection and expression of genes encoding resistance - mecA, mecALGA251, blaZ, femA, femB, and aacA-aphD - using PCR and RT-PCR, respectively). One or more genes coding for resistance to different antimicrobials were detected in 50 Staphylococcus spp. isolates. The femA and femB genes were the most frequent (75.4% for both). The observed expression of the genes was as follows: blaZ (60%), femA (39.5%), aacA-aphD (50%), femB (32.6%), mecA (8.3%), and mecALGA251 (0%). Considering the relevance of the genus Staphylococcus to bovine mastitis, this study aimed to elucidate aspects regarding the genotypic and phenotypic profiles of these microorganisms so as to contribute to the development of effective strategies for mastitis control.

Expression of genes encoding resistance in Staphylococcus spp. isolated from bovine subclinical mastitis in Brazil.

Staphylococci are the most important agents associated with bovine mastitis. This study aimed at characterizing resistance factors to antimicrobials in Staphylococcus spp. isolated from the milk of cows with subclinical mastitis. In vitro resistance of 243 Staphylococcus spp. isolates to antimicrobials commonly used in clinical practice was evaluated. The detection and expression of genes encoding resistance mecA (gene encoding penicillin binding protein 2a) mecALGA251 (mecA homologue), blaZ (gene encoding penicillin resistance), femA and femB (genes encoding essential factors - A and B - for the expression of methicillin resistance) and aacA-aphD (gene encoding for a bifunctional enzyme that confers resistance to gentamicin) using PCR and RT-PCR was investigated. One or more genes encoding resistance to different antimicrobials were detected in 184 Staphylococcus spp. The femA and femB genes were the most frequent. Regarding the variables' detection (N = number of strains) and expression (% of strains), the following results were obtained: blaZ (N = 40 - 82.5%), femA (N = 147 - 47.6%), aacAaphD (N = 30 - 43.3%), femB (N = 138 - 29.7%), mecA (N = 33 - 27.3%), mecALGA251 (N = 01 - 0.0%). There was a higher occurrence of phenotypic resistant strains for amoxicillin, ampicillin and penicillin in isolates positive for detection and/or expression of blaZ gene when compared with the other genes. The present study provides new information on genotypic traits of Staphylococcus isolates from bovine subclinical mastitis especially regarding the evaluation of expression of genes associated with antimicrobial resistance in Staphylococcus spp. using molecular tools.

Staphylococcal Cassette Chromosome mec Typing of Meticillin-Resistant Staphylococcus aureus in Kermanshah Province, West of Iran

Background: Methicillin-resistant Staphylococcus aureus (MRSA) isolates are important agents of human bacterial infections. The mecA gene as the main cause of resistance against beta-lactams is located in genetic elements which are known as staphylococcal cassette chromosome mec (SCCmec). Objectives: The research aimed to evaluate the antibiotic susceptibility pattern and SCCmec typing of MRSA isolates in Kermanshah Province, West of Iran. Methods: Identification of MRSA isolates were done using phenotypic and genotypic methods. Antimicrobial susceptibility patterns were determined using the disk diffusion method and minimum inhibitory concentration (MIC) testing by agar dilution method. The SCCmec types of isolates were identified using PCR method. The results of the research were analyzed using SPSS.V16 software. Results: In this research, of 146 isolates, 126 isolates were confirmed as S. aureus using phenotypic methods and PCR analysis of femB gene. All isolates were sensitive to vancomycin by both methods: disk agar diffusion and MIC testing by agar dilution method. The highest resistance rate was related to erythromycin (75.4%) and ciprofloxacin (73%). Of 126 S. aureus isolates, 83 cases (65.9%) and 81 cases (64.3%) were MRSA based on the existence of mecA gene and cefoxitin diffusion disk test, respectively. There was a statistically significant difference in antibiotic susceptibility pattern of MRSA and methicillin susceptible Staphylococcus aureus (MSSA) isolates for some antibiotics such as gentamicin, amikacin, erythromycin, ciprofloxacin, and linezolid (P < 0.05). SCCmec types were detected as 20 cases (24.1%) type I, 5 cases (6%) type II, 37 cases (44.6%) were type III (the most prevalent type), 6 cases (7.2%) type IVa, and 3 cases (3.6%) type IV. The prevalence of HA-MRSA (types I, II, and III) and CA-MRSA (types IV and V) in this study were 74.7% and 10.8%, respectively. Conclusions: The prevalence of MRSA isolates is high in Kermanshah Province, West of Iran. The cefoxitin diffusion disk testing could be considered a simple, cheap and reliable test for identification of MRSA isolates in all laboratories. The most frequent type of SCCmec is type III. These findings could be due to an increase in antibiotic consumption and insufficient infection control systems.

Antibiotic Resistance Profile and Methicillin-Resistant Encoding Genes of Staphylococcus aureus Strains Isolated from Bloodstream Infection Patients in Northern Vietnam.

BACKGROUND:Evaluating the antibiotic susceptibility and resistance genes is essential in the clinical management of bloodstream infections (BSIs). Nevertheless, there are still limited studies in Northern Vietnam. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (CC BY-NC 4.0)AIM:This study aimed to determine the antibiotic resistance profile and methicillin-resistant encoding genes of Staphylococcus aureus (S. aureus) causing BSIs in Northern Vietnam.METHODS:The cross-sectional study was done from December 2012 to June 2014 in two tertiary hospitals in Northern Vietnam. Tests performed at the lab of the hospital.RESULTS:In 43 S. aureus strains isolating, 53.5 % were MRSA. Distribution of gene for overall, MRSA, and MSSA strains were following mecA gene (58.1 %; 95.7%, and 15%), femA gene (48.8%, 47.8%, and 50%), femB gene (88.4%, 82.6%, and 95%). Antibiotic resistance was highest in penicillin (100%), followed by erythromycin (65.1%) and clindamycin (60.5%). Several antibiotics were susceptible (100%), including vancomycin, tigecycline, linezolid, quinupristin/dalfopristin. Quinolone group was highly sensitive, include ciprofloxacin (83.7%), levofloxacin (86%) and moxifloxacin (86%).CONCLUSION:In S. aureus causing BSIs, antibiotic resistance was higher in penicillin, erythromycin, and clindamycin. All strains were utterly susceptible to vancomycin, tigecycline, linezolid, quinupristin/dalfopristin.

Establishment of rapid diagnostic method for the identification of Staphylococcus aureus in bacterial conjunctivitis

Staphylococcus aureus is the most frequent cause of conjunctivitis and may lead to severe visual impairment. However, the tools available for the diagnosis, such as bacterial culture and cytology, are often inadequate. Therefore, the goal of the present work was to establish a rapid detection method for S. aureus in the secretions of the conjunctival sac to provide a basis for the early diagnosis of acute bacterial conjunctivitis. The study included 32 patients with acute bacterial conjunctivitis, 30 patients with viral conjunctivitis, and 28 control subjects. Extracted DNA was analyzed by conventional PCR and RT-PCR using primers specific for S. aureus DNA and two antibiotic resistance genes, mecA and femB. By this technique, S. aureus was found in 37.5% of patients with bacterial conjunctivitis, 20% of patients with viral conjunctivitis, and in 7.1% normal control subjects. Except for the control group in which the femB gene was not found, in most other cases S. aureus carried mecA and femB genes. The developed molecular biology approach is faster and more sensitive than the methods requiring bacterial cultures and is uniquely suited for the detection of an S. aureus infection in the eye.

Identification of methicillin resistant staphylococcus aureus (mrsa) using cefoxitin disc diffusion test and dupleks polymerase chain reaction in Jambi city hospitals

Methicillin Resistant Staphylococcus aureus (MRSA) remains the important health problems because the frequency of isolate MRSA tends to increase in the world. Cefoxitin disc diffusion test and detection of MecA gene using PCR were used in MRSA identification. FemB gen is used for spesies SA confirmation. FemB encodes enzyme in cross-linking peptidoglycan in SA. This research aimed to identified MRSA from clinical samples of hospitalized patients in Jambi city using cefoxitin disc diffusion test and dupleks PCR. This research has been conducted in Biomedic Laboratory of Medical Faculty of Jambi University during periode June – November 2017. Swab of 76 festering wounds were grown on Mannitol Salt Agar medium which incubate at 30ºC 18–24 h. The Yellowish colony confirmed with gram staining and tested using Staphytect plus Test DR 850 M. Identified of MRSA using cefoxitin disc diffusion test and dupleks PCR. This research reported cefoxitin-RSA was detected in 41,67% of SA, MecA gene was detected in 45,83% of SA. MecA gene was detected in 100% of cefoxitin-RSA and 7,14% of cefoxitin-SSA. FemB gene was detected in 8,33% of cefoxitin-RSA, and 64,28% of cefoxitin-SSA. Detection of MecA and FemB genes by PCR for identification of MRSA is sensitif and fast.

Identification of Methicillin-Resistant Staphylococcus aureus (MRSA) Using Simultaneous Detection of mecA, nuc, and femB by Loop-Mediated Isothermal Amplification (LAMP).

The aim of this study was to develop a rapid detection assay to identify methicillin-resistant Staphylococcus aureus by simultaneous testing for the mecA, nuc, and femB genes using the loop-mediated isothermal amplification (LAMP) method. LAMP primers were designed using online bio-software ( http://primerexplorer.jp/e/ ), and amplification reactions were performed in an isothermal temperature bath. The products were then examined using 2% agarose gel electrophoresis. MecA, nuc, and femB were confirmed by triplex TaqMan real-time PCR. For better naked-eye inspection of the reaction result, hydroxy naphthol blue (HNB) was added to the amplification system. Within 60min, LAMP successfully amplified the genes of interest under isothermal conditions at 63°C. The results of 2% gel electrophoresis indicated that when the Mg2+ concentration in the reaction system was 6μmol, the amplification of the mecA gene was relatively good, while the amplification of the nuc and femB genes was better at an Mg2+ concentration of 8μmol. Obvious color differences were observed by adding 1μL (3.75mM) of HNB into 25μL reaction system. The LAMP assay was applied to 128 isolates cases of methicillin-resistant Staphylococcus aureus, which were separated from the daily specimens and identified by Vitek microbial identification instruments. The results were identical for both LAMP and PCR. LAMP offers an alternative detection assay for mecA, nuc, and femB and is faster than other methods.

Detection of MecA, MecC and Femb Genes by Multiplex Polymerase Chain Reaction

Staphylococcus aureus causing wide range of infectious diseases affecting livestock animals as well as human beings. The bacterium is highly potent to acquire antibiotic resistance, and it is considered a very important agent inducing mastitis in dairy farms in Egypt, as well as other parts of the world, methicillin resistant S. aureus (MRSA) is deliberated one of the most significant causative agents which having public health and economic impacts especially in developing countries, so the present study aimed broadly to obtain a snapshot on the genetic characterization of mecA gene which is responsible to induce the resistance against beta lactam antibiotics. The mecA gene sequence obtained through whole genome sequence process from locally isolated S. aureus from well-established Egyptian dairy farms as well as small scale raising ones. As a control measures, the femB gene was introduced in the multiplex PCR to avoid any contaminants from other staphylococci, also, the mecC gene which is discovered recently as a homologue of mecA gene; was investigated but we did not find any isolates having this homologue.

Detección de los genes mecA, mecRl y mecí en cepas de Staphylococcus aureus resistentes a meticilina de origen bovino aisladas en unidades de producción lechera familiar, México

SUMMARY In Mexico, as in other countries, Staphylococcus aureus is one of the main pathogens causing bovine Mastitis. This disease is usually treated with β-lactam antibiotics; however, in Mexico there are some reports of resistant Staphylococcus aureus to these antibiotics. The molecular mechanisms of methicillin-resistant Staphylococcus aureus have not been widely studied yet. In this study, we identified Staphylococcus aureus isolated from bovines by detecting the femB gene, while resistance to β-lactam antibiotics was determined by in vitro antimicrobial susceptibility, and the genes blaZ, mecA, as well as regulator genes mecR1 and mecI, were identified by PCR. The results showed Staphylococcus aureus presenting higher resistance to β-lactam antibiotics and to erythromycin. The blaZ gene was identified in only 38 isolates (44.7%). Four Staphylococcus aureus isolates carried the mecA gene and were considered as methicillin-resistant Staphylococcus aureus; they were grouped as follows according to their regulator genes: one carried both mecI and mecR1genes (group A); in two of them neither the mecI gene nor the mecR1 gene (PB domain) were found (group C); and one isolate did not carry any regulator gene (group D). In conclusion, we identified the presence of methicillin-resistant Staphylococcus aureus and Staphylococcus aureus resistant to β-lactam antibiotics in family dairy production in the State of Mexico. This resistance might be regulated by different genotypes of the mecA, mecR1 and mecI genes present in methicillin-resistant Staphylococcus aureus.

Development of a Duplex Real-Time PCR Method for the Pharmaceutical Rapid Microbial Detection of &amp;lt;i&amp;gt;Staphylococcus aureus&amp;lt;/i&amp;gt; and &amp;lt;i&amp;gt;Pseudomonas aeruginosa&amp;lt;/i&amp;gt;

Objective: To develop a duplex real-time PCR assay for pharmaceutical rapid microbial detection of Staphylococcus aureus and Pseudomonas aeruginosa. Methods: The specific primers and probes were designed to amplify the femB gene of S. aureus and the DNA gyrase subunit B gene of P. aeruginosa. The sensitivity of the system was detected by a multiple proportional dilution method. In order to examine the specificity of the system, other twenty-one bacteria strains were assayed simultaneously. Results: A highly sensitive and specific duplex real-time PCR assay for the detection of S. aureus and P. aeruginosa was established. The sensitivity was 50 copies/μL. The specificity was 100%. The whole detection procedure can be finished within 2.5 h. Conclusion: The duplex real-time PCR method is efficient in detecting with good sensitivity and specificity. There is a good prospect of this method applying in disease prevention and pharmaceutical industry due to the simultaneous detection of two pathogens.

English

The need for rapid methods in order to precisely detect methicillin-resistant Staphylococcus aureus (MRSA) is extensively acknowledged. This study evaluated a quantitative real-time PCR assay targeting mecA (encoding high level resistance to methicillin) and femB (a specific genomic marker for S. aureus) genes to detect MRSA from broth culture, from serum seeded with MRSA and straight from the patient's serum. One hundred and thirty-five clinical isolates of MRSA strains and different species were utilised in this study. In addition, a pilot study with 9 patients' serum samples was performed. The sensitivity and specificity values for this assay were 99% and 100% respectively. The detection limit for this method was 1.23×102 CFU/ml from the serum seeded with MRSA cells and the limiting concentration of DNA for detection was 18 fg, which equates to 5.14 genomic DNA copies. In addition, this assay detected MRSA from patient's serum (7 out of 9) with sensitivity of 77.8%. Overall, the assay was rapid, efficient, sensitive and easy to perform.

Molecular Epidemiological Analysis of Methicillin-Resistant Staphylococcus aureus Isolates From a Medical Intensive Care Unit: A Comparison of Nasal and Clinical Isolates

BackgroundThe control of nosocomial transmission of methicillin-resistant Staphylococcus aureus (MRSA) represents a significant challenge to infection control professionals. Nasal carriage colonization by MRSA plays a crucial role in the epidemiology and pathogenesis of this infection. MethodsPatients in the medical intensive care unit (ICU) between November 2010 and March 2011 were swabbed when hospitalized, reswabbed 1 week later and for a third time, when they were discharged from the ICU. All swabs were examined within 2 hours of collection using ChromID MRSA-Select agar plates to detect MRSA. Positive specimens were determined to have the mecA and femB gene through amplification with duplex polymerase chain reaction. Repetitive element sequence–based polymerase chain reaction was used to investigate the epidemiological types of MRSA isolates in the third screening and clinical isolates obtained from 2007 to 2010 in West China Hospital. A comparison of molecular types was performed to investigate the genetic relationship between nasal and clinical isolates. ResultsAfter the third screening, 16 nasal MRSA isolates were identified. Epidemiological analysis revealed that 16 nasal MRSA isolates and 37 clinical MRSA isolates differentiated into 2 clusters, comprising 9 subclusters. Of the 16 nasal strains, 11 (68.8%) belonged to subcluster I of cluster I; 3 of 9 subclusters consisted of both nasal and clinical isolates, while 4 of 9 subclusters consisted of clinical isolates and only 2 of 9 consisted of nasal isolates. ConclusionsOur study indicated a high degree of genetic relatedness between nasal and clinical MRSA isolates. The molecular typing of MRSA is critical for controlling the nosocomial transmission of this pathogen in ICU setting and defining a nosocomial infection control policy.

Loop-mediated isothermal amplification assays for identification of antiseptic- and methicillin-resistant Staphylococcus aureus

A method for rapid identification of antiseptic- and methicillin-resistant Staphylococcus aureus (MRSA) based on 3 loop-mediated isothermal amplification (LAMP) assays was developed. LAMP targeting the femB gene identified S. aureus with 100% specificity, and LAMP targeting the mecA gene associated with methicillin resistance identified methicillin-resistant staphylococci with 100% specificity. LAMP targeting the qacA/B gene encoding an efflux pump responsible for antiseptic resistance identified high-acriflavine-resistant (MIC ≥ 100 mg/L) MRSA (92.5% positive) and acriflavine-susceptible (MIC < 25 mg/L) MRSA (100% negative). They were performed under the same reaction conditions within 60 min at 63 °C. The combined LAMP assays will be useful for rapid identification of S. aureus isolates and determination of their antibiotic and antiseptic resistance patterns with regard to methicillin and organic cationic substrates.

Enhanced detection of staphylococcal genomes in positive blood cultures using a polymeric enzyme complex

This article describes a simple and inexpensive signal amplification method, termed polymeric enzyme detection (PED), which permits rapid and sensitive detection of conserved sequences in the tuf gene that identify Staphylococcus genus, conserved sequences in the femB gene that specifically detect Staphylococcus aureus species, and the methicillin resistance gene mecA directly from positive blood culture bottles. Microbe-specific capture probes were immobilized onto microtiter plates or silicon chips. Target sequences and biotin-labeled, target-specific probes were hybridized to complementary capture probes to create a biotin-labeled, surface-immobilized tripartite complex. In a two-step process, signal was amplified by incubating the surface-immobilized biotin with streptavidin followed by the addition of a 500-kDa dextran polymer conjugated with approximately 80 biotins. Signal was then developed by binding of a streptavidin–horseradish peroxidase conjugate followed by incubation with the substrate tetramethylbenzidine. Use of the PED method improved the lower limit of detection 10- to 100-fold in model DNA hybridization assays with limits of detection as low as 1fmol/L target DNA. This level of sensitivity permits detection of genomic DNA from methicillin-resistant S. aureus positive blood cultures within 25 to 35min using either a thin film biosensor chip or a microtiter plate-based assay.

Severe Atopic Dermatitis Is Associated With a High Burden of Environmental Staphylococcus aureus

Leung AD, Schiltz AM, Hall CF, Liu AH. Clin Exp Allergy. 2008;38(5):789–793 PURPOSE OF THE STUDY. It has been established that Staphylococcus aureus worsens atopic dermatitis (AD) by a variety of mechanisms. The purpose of this study was to quantify S aureus burden in the homes of participants with AD of varying severity. STUDY POPULATION. There were 62 volunteers aged 1 to 40 years. Participants were categorized as having mild (n = 18), moderate (n = 14), severe (n = 15), or no (n = 15) AD. METHODS. Participants completed questionnaires about their asthma, allergies, and home environment. Patients with AD completed a Lung-Browder diagram, documenting the area and intensity of erythema, excoriation, papulation, and lichenification. From these diagrams, AD severity was calculated by using the Eczema Area and Severity Index (EASI). Subjects collected dust samples from their bed, the floor next to their bed, and the home vacuum bag and sent them to the laboratory for analysis. S aureus DNA was extracted, and quantitative reverse-transcription polymerase chain reaction for the femB gene (an S aureus–specific genomic marker) was performed. Data were log-transformed and then analyzed with analysis of variance, student's t test, and Spearman's r. RESULTS. Bed dust yielded the highest S aureus concentrations. Participants with severe AD had significantly more S aureus DNA (14.67 pg/mg dust) in bed dust than those with moderate (0.41 pg/mg dust; P &amp;lt; .0001), mild (1.42 pg/mg; P = .0051), and no (0.09 pg/mg; P &amp;lt; .0001) AD. The concentration of S aureus DNA in bed dust strongly correlated with EASI scores. Similar patterns were observed for dust from bedroom floors for both DNA concentrations and EASI scores. The quantity of S aureus DNA from the vacuum samples was significantly higher in participants with severe AD versus moderate, mild, and no AD. However, there was no correlation between EASI scores and concentrations of S aureus DNA from vacuum dust samples. CONCLUSIONS. S aureus is ubiquitous and was detected in dust samples from almost all homes regardless of disease state. However, house dust from participants with severe AD contained the most S aureus DNA. The correlation between S aureus DNA levels and AD severity is driven by proximity to the patient, as shown by the fact that the bed and bedroom floors from the patients with AD yielded the highest levels of S aureus DNA. In the home and especially the bedroom, higher levels of S aureus may contribute to disease severity and persistence in patients with AD. REVIEWER COMMENTS. This is the first study to examine the environmental burden of S aureus in the homes of patients with AD. This study concludes that patients with severe AD have higher environmental burdens of S aureus. The source of the S aureus may be shed bacteria from the skin of the patient with AD, and if live organisms persist in house dust, then they may be the source of recolonization of patients’ skin. However, to prove whether the relationship between high environmental load of S aureus and severe AD is cause or effect, additional studies need to be performed, including quantification of bacterial load on the patient's skin as correlated with home environmental burden and quantification of live bacteria in house dust.

Development of a rapid diagnostic assay for methicillin-resistant Staphylococcus aureus and methicillin-resistant coagulase-negative Staphylococcus

We describe here a rapid assay for the detection of the tuf gene for the identification of Staphylococcus genus, the femB gene for the identification of Staphylococcus aureus species, and the mecA gene for the identification of methicillin resistance directly from BACTEC blood culture bottles showing Gram-positive cocci in clusters. The test, configured on a thin-film biosensor platform, allows for detection of genomic DNA from blood culture samples without the need for nucleic acid amplification. In an initial study to validate the technology, 107 consecutive positive blood cultures were tested on the thin-film biosensor, and the assay exhibited 100% concordance in comparison with standard microbiological methods for identifying methicillin-susceptible and methicillin-resistant S. aureus and for identifying methicillin-susceptible and methicillin-resistant coagulase-negative Staphylococcus. Results were obtained within 90 min directly from signal positive bottles with no instrumentation required.

Rapid and reliable detection of multiresistent Staphylococcus aureus (MRSA) by multiplex PCR

Staphylococci are widespread pathogens and are frequently associated with nosocomial infections. Many hospitals struggle with increasing amounts of methicillin-resistant Staphylococcus aureus (MRSA) which are "multiresistant" against all betalactam antibiotics. Often, applicable antibiotics for treatment are only glycopeptides like vancomycin and teicoplanin. In addition, MRSA infected patients require expensive intensive isolation measures and strict hygiene. To efficiently prevent dissemination of these pathogens rapid and reliable identification and a close collaboration between clinicians and microbiologists are required. The purpose of our study was to set up a rapid and reliable identification procedure for MRSA by the amplification of specific gene determinants by PCR in order to to efficiently support therapy and eradication of the pathogen. 153 strains of staphylococci isolated from in-patients of the hospital of the Justus-Liebig University of Giessen were examined. The femB gene was used to differentiate between Staphylococcus aureus (S. aureus) and coagulase-negative staphylococci (CNS), a gene which allows the species-specific identification of methicillin-resistant (MRSA) and -susceptible S. aureus (MSSA). Additionally, MRSA harbor the mecA gene encoding methicillin-resistance, which is absent in MSSA strains. Using a multiplex PCR with femB and mecA gene-specific oligonucleotides MRSA strains were unequivocally detected within 3 hours. The femB gene was detected in all 102 strains of S. aureus but in none of the 51 CNS. The mecA determinant was detected in 12 S. aureus. Among these, 11 strains were phenotypically methicillin-resistant and one strain was susceptible. The methicillin-resistance of this particular mecA-positive/methicillin-susceptible strain (cryptic MRSA) was inducible by cultivation on agar plates supplemented with flucloxacillin. The described method specifically detects S. aureus and identifies phenotypical and cryptic MRSA. These cryptic MRSA are of particular relevance since they are undetectable using common phenotypically based detection methods. It is conceivable that the methicillin resistance of these strains is induced under antibiotic therapy with flucloxacillin and that the mec-encoded feature of methicillin-resistance can be transferred to previously methicillin-susceptible strains. Using the reliable detection of these strains by PCR, failure of flucloxacillin therapy is avoidable.

Severe atopic dermatitis is associated with a high burden of environmental Staphylococcus aureus

About 90% of patients with atopic dermatitis (AD) are colonized with Staphylococcus aureus. S. aureus worsens AD by secreting superantigens and structural molecules within the cell wall that induce skin inflammation. Therefore, S. aureus in the home may contribute to persistent skin inflammation and disease severity. To quantify S. aureus burden in homes of participants with AD of varying severities. Participants with mild (n=18), moderate (n=14), severe (n=15), and no AD (n=15), collected dust from their bed and bedroom floor, and from their home vacuum cleaner bag. DNA was extracted from dust samples, and the S. aureus-specific femB gene was quantified using quantitative real-time PCR. Data was log-transformed, and then statistically analysed with anova, student's t-test, and Spearman's r. Participants with severe AD (geometric mean: 14.67 pg/mg dust) had significantly more S. aureus DNA in their bed dust than those with moderate (0.41 pg/mg dust, P<0.0001), mild (1.42 pg/mg dust, P=0.0051), and no AD [0.09 pg/mg dust, P<0.0001 (t-test)]. Similar patterns were observed for dust from the bedroom floors and vacuum bags. S. aureus DNA was highest in dust from beds as compared with bedroom floors or vacuum bags (medians: 1.51, 0.69, 0.21 pg/mg dust, respectively; P=0.007). Eczema Area and Severity Index scores correlated with S. aureus DNA from the bed (Spearman's r=0.7263; P=0.0004) and floor (0.6846; P=0.0002) dust, but not with the vacuum bag dust (0.3783; 0.0684). In the home and especially the bedroom, higher levels of S. aureus may contribute to disease severity and persistence in AD patients.