Tralokinumab treatment improves the skin microbiota by increasing the microbial diversity in adults with moderate-to-severe atopic dermatitis: Analysis of microbial diversity in ECZTRA 1, a randomized controlled trial.

BACKGROUND: Atopic dermatitis (AD) is characterized by microbial dysbiosis, immune dysregulation, and an impaired skin barrier. Microbial dysbiosis in AD involves a reduction in diversity primarily driven by an increased abundance of Staphylococcus aureus. Tralokinumab, an approved treatment for adults with moderate-to-severe AD, improves the skin barrier and immune abnormalities by specifically targeting the interleukin 13 cytokine, but its impact on the skin microbiome is unknown. OBJECTIVE: To investigate how tralokinumab affects the skin microbiome by examining the lesional skin of adults with moderate-to-severe AD from the phase 3 ECZTRA 1 trial (NCT03131648). METHODS: Microbiome profiling, S aureus abundance, and biomarker data were assessed in a subset of ECZTRA 1 participants (S aureus abundance at baseline and week 16; microbiome profiling at baseline, and week 8/16; and serum sampling before dose and week 4/8/16/28/52). RESULTS: Tralokinumab treatment led to increased microbial diversity, reduced S aureus abundance, and increased abundance of the commensal coagulase-negative Staphylococci. LIMITATIONS: Limitations include a lack of S aureus abundance data at week 8, sampling site variation between participants, and possible influence from concomitant systemic antiinfectives. CONCLUSION: Our findings indicate specific targeting of the interleukin 13 cytokine with tralokinumab can directly and/or indirectly improve microbial dysbiosis seen in AD skin.

Staphylococcal Enterotoxin Genes in Coagulase-Negative Staphylococci-Stability, Expression, and Genomic Context.

In the current study, we screened a collection of coagulase-negative staphylococci (CoNS) isolates for orthologues of staphylococcal enterotoxins (SEs) involved in S. aureus-related staphylococcal food poisoning (SFP). The amplicons corresponding to SEs were detected in S. chromogenes, S. epidermidis, S. haemolyticus, S. borealis, S. pasteuri, S. saprophyticus, S. vitulinus, S. warneri, and S. xylosus. All amplicons were sequenced and identified as parts of known S. aureus or S. epidermidis SE genes. Quantitative real-time PCR allowed determining the relative copy number of each SE amplicon. A significant portion of the amplicons of the sea, seb, sec, and seh genes occurred at low copy numbers. Only the amplicons of the sec gene identified in three isolates of S. epidermidis displayed relative copy numbers comparable to sec in the reference enterotoxigenic S. aureus and S. epidermidis strains. Consecutive passages in microbiological media of selected CoNS isolates carrying low copy numbers of sea, seb, sec, and seh genes resulted in a decrease of gene copy number. S. epidermidis isolates harbored a high copy number of sec, which remained stable over the passages. We demonstrated that enterotoxin genes may occur at highly variable copy numbers in CoNS. However, we could identify enterotoxin genes only in whole-genome sequences of CoNS carrying them in a stable form at high copy numbers. Only those enterotoxins were expressed at the protein level. Our results indicate that PCR-based detection of enterotoxin genes in CoNS should always require an additional control, like analysis of their presence in the bacterial genome. We also demonstrate S. epidermidis as a CoNS species harboring SE genes in a stable form at a specific chromosome site and expressing them as a protein.

Characterizing a Novel Staphylococcal Cassette Chromosome mec with a Composite Structure from a Clinical Strain of Staphylococcus hominis, C34847.

Staphylococcal cassette chromosome mec (SCCmec) has predominantly been described in methicillin-resistant Staphylococcus aureus. However, studies have indicated that coagulase-negative staphylococci (CoNS) carry a larger diversity of SCC elements. We characterized a composite SCCmec element carrying an uncharacterized ccr1 and type A mec gene combination, in conjunction with a secondary element bearing ccr4, from a clinical strain of Staphylococcus hominis. The element's complex structure points to a high degree of recombination occurring in SCCmec in CoNS.

Virulence and toxigenicity of coagulase-negative staphylococci in Nigerian traditional fermented foods.

The incidence of coagulase-negative staphylococci (CoNS) may render food unsafe, as the clinical isolates have been reported to exude virulent traits. A total of 255 CoNS isolates from 6 traditional fermented foods (nono, kunu, wara, iru, ogi, and kindirmo) from North Central Nigeria, identified as Staphylococcus epidermidis, Staphylococcus simulans, Staphylococcus xylosus, Staphylococcus kloosii, and Staphylococcus caprae, were investigated for virulence traits. The strains were examined for biofilm formation and production of hyaluronidase, DNase, TNase, haemolysins, and superantigenic toxins (SEA, SEB, SEC, SED, and TSST-1) using standard and genotypic methods. The analysis of virulence factors revealed the production of slime in 200 isolates (78.4%); α-haemolysin in 136 (53.3%); ß-haemolysin in 43 (16.9%); DNase in 199 (78.0%); TNase in 29 (11.4%); hyaluronidase in 125 (49.0%); TSST-1 in 119 (46.7%); and enterotoxin-producing isolates SEA, SEB, SEC, and SED in 61 (23.9%), 19 (7.5%), 9 (3.5%), and 8 (3.1%), respectively. PCR analysis detected tsst-1, sea, seb, and sec genes. The ability of these microorganisms to exhibit virulence evokes the potential to cause disease especially under determinate conditions or in immune-compromised patients. The occurrence of CoNS in food should not be ignored nor their pathogenic potential considered as insignificant, rather safety measures should be taken to reduce or totally eliminate their occurrence in foods.

Species distribution and antimicrobial resistance pattern of Coagulase-negative Staphylococci at a tertiary care centre.

BACKGROUND: Coagulase-negative Staphylococci (CoNS), previously dismissed at contaminants, have now emerged as an important cause of nosocomial infections especially in patients with implants and prosthetic devices. They are a well-known cause of bloodstream infections, urinary tract infections, wound infections, prosthetic valve endocarditis and eye infections. This study was conducted with an aim to identify CoNS at the species level from various clinical samples and determine the antimicrobial resistance pattern of these isolates. METHODS: This cross sectional study was carried out from September 2011 to February 2014 in which 150 non-repetitive clinical isolates of CoNS were identified at the species level by conventional phenotypic methods. Complete antimicrobial susceptibility profile was also determined by Kirby Bauer disc diffusion method. Susceptibility testing to vancomycin was done by E-test method. RESULTS: Only three species of CoNS were isolated, the most common being Staphylococcusepidermidis (60%) followed by Staphylococcussaprophyticus (27.3%) and Staphylococcushemolyticus (12.7%). Most S. epidermidis were isolated from blood and intravascular catheter tip samples, whereas all S. saprophyticus were isolated from urine samples of female patients. All isolates were found to be resistant to penicillin, but were susceptible to glycopeptides and linezolid and showed variable resistance to fluoroquinolones, aminoglycosides and macrolides. CONCLUSION: CoNS are emerging nosocomial pathogens and should not always be overlooked as contaminants. However, growth of CoNS from blood cultures and intravascular catheter tips should be clinically correlated and carefully interpreted. As many CoNS strains exhibit drug resistance, antimicrobial susceptibility profile should be determined prior to treatment of these infections.

Phenotypic and Genotypic Assays to Evaluate Coagulase-Negative Staphylococci Biofilm Production in Bloodstream Infections.

Coagulase-negative staphylococci (CoNS) are commensal on human body surfaces and, for years, they were not considered a cause of bloodstream infection and were often regarded as contamination. However, the involvement of CoNS in nosocomial infection is increasingly being recognized. The insertion of cannulas and intravascular catheters represents the primary source of CoNS entry into the bloodstream, causing bacteremia and sepsis. They owe their pathogenic role to their ability to produce biofilms on surfaces, such as medical devices. In this study, we evaluate the adhesive capacity of CoNS isolated from blood cultures by comparing a spectrophotometric phenotypic assay with genotypic analysis based on the evidence of the ica operon. We retrospectively reviewed the database of CoNS isolated from blood cultures from January to December 2021 that were considered responsible for 361 bloodstream infections. Eighty-nine CoNS were selected among these. Our data show that Staphylococcus epidermidis was the predominant species isolated, expressing greater adhesive capacities, especially those with the complete operon. Knowledge of the adhesive capabilities of a microorganism responsible for sepsis can be useful in implementing appropriate corrective and preventive measures, since conventional antibiotic therapy cannot effectively eradicate biofilms.

Identification of staphyloxanthin and derivates in yellow-pigmented Staphylococcus capitis subsp. capitis.

Introduction: Staphylococcus capitis naturally colonizes the human skin but as an opportunistic pathogen, it can also cause biofilm-associated infections and bloodstream infections in newborns. Previously, we found that two strains from the subspecies S. capitis subsp. capitis produce yellow carotenoids despite the initial species description, reporting this subspecies as non-pigmented. In Staphylococcus aureus, the golden pigment staphyloxanthin is an important virulence factor, protecting cells against reactive oxygen species and modulating membrane fluidity. Methods: In this study, we used two pigmented (DSM 111179 and DSM 113836) and two non-pigmented S. capitis subsp. capitis strains (DSM 20326T and DSM 31028) to identify the pigment, determine conditions under which pigment-production occurs and investigate whether pigmented strains show increased resistance to ROS and temperature stress. Results: We found that the non-pigmented strains remained colorless regardless of the type of medium, whereas intensity of pigmentation in the two pigmented strains increased under low nutrient conditions and with longer incubation times. We were able to detect and identify staphyloxanthin and its derivates in the two pigmented strains but found that methanol cell extracts from all four strains showed ROS scavenging activity regardless of staphyloxanthin production. Increased survival to cold temperatures (-20°C) was detected in the two pigmented strains only after long-term storage compared to the non-pigmented strains. Conclusion: The identification of staphyloxanthin in S. capitis is of clinical relevance and could be used, in the same way as in S. aureus, as a possible target for anti-virulence drug design.

A Comprehensive Study on Antibiotic Resistance among Coagulase-Negative Staphylococci (CoNS) Strains Isolated from Ready-to-Eat Food Served in Bars and Restaurants.

The present study aimed to characterize and assess the diversity of CoNS strains as potential vectors for the spread of resistance to antimicrobial agents from RTE foods served in bars and restaurants. Eighty-five CoNS strains, obtained from 198 RTE food samples, were investigated. Sixty-seven CoNS isolates (78.8%) were resistant to at least one antibiotic tested, and 37 (43.5%) were multidrug resistant (MDR-CoNS). Moreover, CoNS strains contained genes conferring resistance to antibiotics critically important in medicine, i.e., ß-lactams [mecA (29.4%); blaZ (84.7%)], aminoglycosides [aac(6')-Ie-aph(2″)-Ia (45.9%); aph(2″)-Ic (3.5%)], macrolides, lincosamides and streptogramin B-MLSB [msrA/B (68.2%); ermB (40%) and mphC (4.7%)], tetracyclines [tetK (31.8%); tetM (16.5%) and/or tetL (2.35%)]. We also found the fusB/C/D genes responsible for the acquired low-level fusidic acid resistance (17.6%) and streptogramin resistance determinant vgaA in 30.6% of isolates. In three linezolid resistant strains (2 S. epidermidis and 1 S. warneri), mutation was detected, as demonstrated by L101V and V188I changes in the L3 protein amino acid sequences. The high frequency in RTE food of MDR-CoNS including methicillin-resistant (MR-CoNS) strains constitutes a direct risk to public health as they increase the gene pool from which pathogenic bacteria can pick up resistance traits.

Exploring the Biofilm Formation Capacity in S. pseudintermedius and Coagulase-Negative Staphylococci Species.

The ability of biofilm formation seems to play an important role in the virulence of staphylococci. However, studies reporting biofilm formation of coagulase-negative staphylococci isolated from animals are still very scarce. Thus, we aimed to evaluate the biofilm-forming capacity of CoNS and S. pseudintermedius isolated from several animal species and to investigate the effect of conventional antimicrobials on biofilm reduction. A total of 35 S. pseudintermedius and 192 CoNS were included. Biofilm formation was accessed by the microtiter plate assay and the biofilms were stained by crystal violet. Association between biofilm formation and staphylococci species and antimicrobial resistance was also performed. Biofilm susceptibility testing was performed with tetracycline and amikacin at the minimum inhibitory concentration (MIC) and 10 × MIC. The metabolic activity of the biofilm cells after antimicrobial treatment was accessed by the XTT assay. All isolates formed biofilm, with S. urealyticus producing the most biofilm biomass and S. pseudintermedius producing the least biomass. There was a positive association between biofilm formation and multidrug resistance as well as resistance to individual antimicrobials. Neither tetracycline nor amikacin were able to eradicate the biofilm, not even at the highest concentration used. This study provides new insights into biofilm formation and the effects of antimicrobials on CoNS species.

Analysis of enterotoxigenic effect of Staphylococcus aureus and Staphylococcus epidermidis enterotoxins C and L on mice.

Pathogenic properties of orthologues to S. aureus staphylococcal enterotoxin C (SEC) and staphylococcal enterotoxin L (SEL) produced by S. epidermidis are largely unexplored. We assessed the enteropathogenic effects of S. epidermidis SECepi and SELepi and S. aureus SEC3 and SEL after oral administration to Balb/c mice. Intestinal sections from SE-treated mice were analyzed histopathologically. The T cell lineage markers (αß and γδ TCR CD3, CD4, CD8), T-cell activation marker CD69 and proliferation-related marker CD71 were assessed in intraepithelial lymphocytes (IEL), mesenteric lymph nodes (MLN) and spleens (SPL). Serum concentrations of SEC and SEL were determined. Ortologous S. epidermidis and S. aureus SEs exerted a number of common histopathological changes in the mouse gut. Atrophy, generation of villi gap and edema of the villi were the most prominent effects of SE treatment observed in mouse gut sections. The most marked effect of ortologous S. epidermidis and S. aureus SEs on the number of goblet cells, crypt depth and villi height was noted in the mice duodenum and jejunum. We indicate early changes of TCRαß CD4-CD8a+ T and TCRαß CD4+CD8a+ T cells in response to both S. aureus and S. epidermidis SEs. Upon the treatment with SEs, markers of T cell activation and proliferation were upregulated in both αß and γδ T cell populations derived from IEL and MLN. We demonstrated that S. epidermidis-encoded SEs applied via oral route exert pathological changes in mice gut similarly to S. aureus-encoded SEs. For the first time we indicated that SEL co-produced together with SEC by both S. aureus and S. epidermidis induces some elements of mice gut immune response and contributes to gastrointestinal tract damage. Our results indicate the potential involvement of CoNS-encoded enterotoxins in the pathogenesis of SFP.

Anti-staphylococcal lysin, LSVT-1701, activity: In vitro susceptibility of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) clinical isolates from around the world collected from 2002 to 2019.

LSVT-1701 (previously known as SAL200), is a novel, recombinantly-produced, bacteriophage-encoded lysin that specifically targets staphylococci via cell wall enzymatic hydrolysis. In vitro activities of LSVT-1701 and comparators were tested against 415 Staphylococcus aureus and coagulase-negative staphylococci (CoNS) clinical isolates expressing various resistance phenotypes. The isolates were collected worldwide from 2002 to 2019 and tested for in vitro susceptibility using broth microdilution methodology performed according to the Clinical and Laboratory Standards Institute (CLSI) guidelines. Minimum inhibitory concentration (MIC) interpretations were based on CLSI and EUCAST criteria. MIC90 for all S. aureus, methicillin-susceptible S. aureus, methicillin-resistant S. aureus, and CoNS, were 2, 2, 2 and 2 µg/ml, respectively. LSVT-1701's activity was not adversely affected by resistance to antimicrobial comparators against this worldwide collection of S. aureus and CoNS clinical isolates. The results of this study support further clinical development of LSVT-1701 to treat staphylococcal infections, including those caused by multidrug resistance isolates.

Analysis of Genome Sequences of Coagulase-Negative Staphylococci Isolates from South Africa and Nigeria Highlighted Environmentally Driven Heterogeneity.

Here, we report high-quality annotated draft genomes of eight coagulase-negative staphylococci (CoNS) isolates obtained from South Africa and Nigeria. We explored the prevalence of antibiotic resistance and virulence genes, their association with mobile genetic elements. The pan-genomic analysis highlighted the environmentally driven heterogeneity of the isolates. Isolates from Nigeria had at least one gene for cadmium resistance/tolerance, these genes were not detected in isolates from South Africa. In contrast, isolates from South Africa had a tetM gene, which was not detected among the isolates from Nigeria. The observed genomic heterogeneity correlates with anthropogenic activities in the area where the isolates were collected. Moreover, the isolates used in this study possess an open pan-genome, which could easily explain the environmentally driven heterogeneity.

A Novel Assay for Detection of Methicillin-Resistant Staphylococcus aureus Directly From Clinical Samples.

The timely detection of Methicillin-resistant Staphylococcus aureus (MRSA) is crucial for antimicrobial therapy and a key factor to limit the hospital spread of MRSA. Currently available commercial MRSA detection assays target the 3' end of the orfX gene and the right extremity of Staphylococcal Cassette Chromosome mec (SCCmec). These assays suffer from both false positive due to SCC-like elements that lack mecA and false negative results due to the inability to detect new or variant SCCmec cassettes with the existing primers. We developed a novel MRSA detection scheme, designed to circumvent issues present in the existing commercial assays. Our assay demonstrated specificity and accuracy, capable of detecting prototypic strains of SCCmec types I-XIII [C(t) values ranged 8.58-26.29]. Previous false positive isolates (N = 19) by Xpert MRSA nasal assay were accurately classified with our assay. Further validation with 218 randomly selected clinical isolates (73 MRSA, 75 MSSA, 43 MR-CoNS, and 27 MS-CoNS) confirmed its feasibility and practicality. Testing assay performance with 88 direct clinical swabs from 33 patients showed that the assay was 96.6% in agreement with clinical culture results. Our novel MRSA detection assay targets both the S. aureus specific sequence and the mecA/mecC genes simultaneously to overcome the false positive and false negative deficits of currently available commercial assays. The results validate our assay and confirmed its feasibility and practicality. The assay is not affected by SCCmec types and only needs modification if new mec homologs emerge and establishes a new platform for other emerging SCCmec types.

Prevalence of mecA gene among staphylococci from clinical samples of a tertiary hospital in Benin City, Nigeria.

BACKGROUND: The staphylococci have increasingly been associated with infections worldwide and anti-microbial resistance has made these versatile pathogens more recalcitrant in the hospital setting. OBJECTIVES: This study sought to investigate the occurrence and distribution of Staphylococcus species as well as determine the prevalence of methicillin resistant Staphylococcus aureus (MRSA) and methicillin resistant coagulase negative staphylococci (MRCoNS) among clinical samples from University of Benin Teaching Hospital (UBTH) in Benin City. METHODS: Ninety one (91) clinical isolates comprising S. aureus and Coagulase Negative staphylococci (CoNS) were recovered from routine clinical specimens and anti-microbial susceptibility tests were carried out. Polymerase Chain Reaction (PCR) was thereafter carried out on these isolates to detect mecA gene. RESULTS: Staphylococcus species had its highest prevalence from infected wounds of patients (28.8%) while urine samples showed the least (5.4%). The highest level of resistance was to ceftazidime (S. aureus - 68%, CoNS - 75.6%) while the least resistance was observed for meropenem (S. aureus- 26%, CoNS- 46.3%). Using phenotypic method (with 1µg oxacillin antibiotic disc), the distribution of MRSA and MRCoNS was 44.0% and 46.3% respectively. PCR analysis showed that 38.0% of S. aureus and 41.5% of the CoNS had mecA gene respectively; wound swabs showed the highest prevalence with 30.5% of staphylococcal isolates being mecA gene positive. There was also no significant association between the Staphylococcal isolates and their isolation rate, isolation site and mecA gene distribution (p > 0.05). CONCLUSION: This study draws attention on the increase in the prevalence of mecA gene (39.6%) and an increase in multidrug resistant staphylococci when compared to previous studies in our country; it recommends laboratory guidance and periodic review to stem the tide of resistance.

Prevalence of methicillin resistance and macrolide-lincosamide-streptogramin B resistance in Staphylococcus haemolyticus among clinical strains at a tertiary-care hospital in Thailand.

Staphylococcus spp. is a major cause of nosocomial infection and sepsis. However, increasing drug resistance is becoming a challenge to microbiologists. The purpose of this study was to identify and determine antimicrobial resistance phenotypes and drug resistance genes of clinical coagulase-negative staphylococci (CoNS) isolates at Mae Sot Hospital in Tak province, Thailand. A total of 229 CoNS isolates were collected from clinical specimens during two periods in 2014 and in 2015. Staphylococcus haemolyticus was the most prevalent species (37.55%), followed by S. epidermidis (21.83%), S. saprophyticus (11.79%) and S. hominis (11.35%) respectively. The remaining 17.48% of the organisms comprised S. capitis, S. arlettae, S. cohnii, S. equorum, S. xylosus, S. warneri, S. sciuri, S. pettenkoferi, S. kloosii and S. lugdunensis. Methicillin-resistant CoNS (MRCoNS), containing the mecA gene, were detected in 145 of 229 isolates, mostly found in S. haemolyticus and S. epidermidis. In addition, the differentiation of their macrolide-lincosamide-streptogramin B (MLSB) resistance phenotypes was determined by the D-test and corresponding resistance genes. Among 125 erythromycin-resistant CoNS, the prevalence of constitutive type of MLSB, inducible clindamycin resistance and macrolide-streptogramin B resistance phenotypes were 72, 13.60 and 14.40% respectively. These phenotypes were expressed in 80% of MRCoNS strains. In addition, the ermC gene (79.20%) was found to be more prevalent than the ermA gene (22.40%), especially among MRCoNS. These results indicate that CoNS may play an important role in spreading of drug resistance genes. More attention to these organisms in surveillance and monitoring programs is needed.

Virulence, Speciation and Antibiotic Susceptibility of Ocular Coagualase Negative Staphylococci (CoNS).

BACKGROUND: Coagulase negative Staphylococci (CoNS) are common inhabitants of human skin and mucous membranes. With the emergence of these organisms as prominent pathogens in patients with ocular infections, investigation has intensified in an effort to identify important virulence factors and to inform new approaches to treatment and prevention. AIM: To isolate CoNS from ocular specimens; to study the possible virulence factors; speciation of coagulase negative staphylococci (CoNS) which were isolated from ocular complications; antibiotic susceptibility testing of ocular CoNS. MATERIALS AND METHODS: The specimens were collected from the target patients who attended the Microbiology Laboratory of a tertiary care eye hospital in Coimbatore, Tamilnadu state, India. The isolates were subjected to tube and slide coagulase tests for the identification of CoNS. All the isolates were subjected to screening for lipase and protease activities. Screening for other virulence factors viz., slime production on Congo red agar medium and haemagglutination assay with use of 96-well microtitre plates. These isolates were identified upto species level by performing biochemical tests such as phosphatase test, arginine test, maltose and trehalose fermentation tests and novobiocin sensitivity test. The isolates were subjected to antibiotic susceptibility studies, based on the revised standards of Clinical and Laboratory Standards Institutes (CLSI). RESULTS: During the one year of study, among the total 260 individuals who were screened, 100 isolates of CoNS were obtained. Lipolytic activity was seen in all the isolates, whereas 38 isolates showed a positive result for protease. A total of 63 isolates showed slime production. Of 100 isolates, 30 isolates were analyzed for haemagglutination, where 4 isolates showed the capacity to agglutinate the erythrocytes. The results of the biochemical analysis revealed that of the 100 isolates of CoNS, 43% were Staphylococcus epidermidis. The other isolates were identified as S. xylosus (n=8), S. captis (n=16), S. haemolyticus (n=10), S. saccharolyticus (n=2), S. hominis (n=5), S. saprophyticus (n=6) and S. intermedius (n=1). On the other hand, 9 isolates were not identified. In the antibiotic susceptibility analysis, it was found that most of the isolates were sensitive to vancomycin, amikacin and linczolid and resistant to cefatoxime, oxacillin, bacitracin and nalidixic acid. CONCLUSION: S. epidermidis was found to be predominant in causing the ocular complications. Slime production, heamagglutination, protease and lipase activities could be the putative virulence factors of CoNS. Antibiotic susceptibility patterns of CoNS against antibacterial agents revealed maximum resistance to beta lactam groups, and the resistance was found to be higher to oxacillin, and lowest to vancomycin.

Evaluation of fuur different dna extraction metods in coagulase-negative staphhylococci clinical isolates / Avaliacao de quatro metodos diferentes de extracao de DNA em isolados clinicos de estafilococos coagulase negativos (SCoN)

Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.

Atualmente, para extrair o DNA bacteriano, existem diversos métodos baseados em diferentes princípios. Entretanto, a quantidade e qualidade do DNA obtido por cada um destes métodos é variável e depende do tipo de micro-organismo em questão; os estafilococos coagulase-negativos (CoNS), por exemplo, possuem parede celular espessa difícil de lisar. O objetivo deste estudo foi comparar a quantidade e a qualidade do DNA extraído de isolados clínicos de CoNS utilizando quatro metodologias diferentes: dois protocolos caseiros e dois kits comerciais. A determinação da quantidade e da qualidade do DNA foi realizada por espectrofotometria. O DNA extraído também foi analisado em eletroforese em gel de agarose e por PCR. A concentração média de DNA foi mais alta no método de lise térmica (). Entretanto, com relação à qualidade do DNA, o kit comercial que utiliza um método de extração baseado em uma coluna de separação apresentou melhor resultado (média da relação A260/280 = 1,95). As quatro técnicas testadas forneceram DNA passível de amplificação por PCR, porém com diferentes rendimentos. A qualidade do DNA extraído de bactérias é importante, pois possibilita a realização de maior número de técnicas de biologia molecular e também armazenamento do material por maior período de tempo. Neste sentido, a técnica de extração por coluna de separação apresentou melhor desempenho frente aos CoNS.

Urinary Tract Infection Due to Coagulase-negative Staphylococci: Species Identification, Antimicrobial Resistance and Clinical Characteristics / 대한임상미생물학회지

BACKGROUND: Previously considered as nonpathogenic contaminants, coagulase-negative staphylococci (CoNS) are now a major cause of nosocomial infections as increased use of prosthetic devices, intravascular catheters, and other invasive technology in more immunosuppressed patients and urinary tract infections (UTI) due to CoNS have been reported. In present study, species frequency, antimicrobial susceptability and clinical characteristics of CoNS UTI were evaluated. METHOD: We performed species identification and antimicrobial susceptibility of 109 CoNS strains isolated from urine in Ewha Womans University hospital from January 1998 to December 2002 and analysed clinical characteristics of 57 cases with CoNS UTI cases, retrospectively. RESULTS: Among 13,336 strains isolated from urine, 109 strains were CoNS showing 0.8%. The most common species of CoNS from urine were S. epidermidis (49.5%) followed by S. haemolyticus (16.5%), S. saprophyticus (13.8%), S. auricularis (1.8%), S. simulans (0.9%) and unidentified CoNS represented 17.4%. The antimicrobial susceptibility showed high resistance to multiple drugs. Among CoNS, S. haemolyticus showed the highest resistance whereas S. saprophyticus showed the highest susceptibility to multiple drugs. The patients isolated S. saprophyticus were younger (mean age: 41 yrs) than those isolated other CoNS (mean age: 53 yrs) and more frequently female (9/14 vs 19/43). The hospitalized patients (74.4% vs 21.4%), bacteriuria more than 10 5 CFU/ml (83.7% vs 64.3%), indwelling catheter (34.9% vs 7.1%) and other risk factors (48.8% vs 35.7%) were more common in patients isolated other CoNS than those isolated S. saprophyticus and no significant differences were noted in pyuria (51.2% vs 57.1%). The symptomatic presentations were more common in patients isolated S. saprophyticus than those isolated other CoNS (71.4% vs 9.3%) and so were treatment (85.7% vs 44.2%). CONCLUSIONS: When CoNS other than S. saprophyticus isolated from urine in hospitalized patientes with risk factors, identification and antimicrobial susceptability test is necessary for proper management.