Novel biochemical aspects of lugdulysin, a Staphylococcus lugdunensis metalloprotease that inhibits formation and disrupts protein biofilm of methicillin-resistant Staphylococcus aureus.

Staphylococcus lugdunensis produces lugdulysin, a metalloprotease that may contribute to its virulence. This study aimed to evaluate the biochemical aspects of lugdulysin and investigate its effect on Staphylococcus aureus biofilms. The protease was isolated and characterized for its optimal pH and temperature, hydrolysis kinetics, and influence of metal cofactor supplementation. The protein structure was determined via homology modeling. The effect on S. aureus biofilms was assessed by the micromethod technique. The protease optimal pH and temperature were 7.0 and 37 °C, respectively. EDTA inhibited protease activity, confirming it as a metalloprotease. Lugdulysin activity was not recovered by divalent ion supplementation post-inhibition, and supplementation with divalent ions did not change enzymatic activity. The isolated enzyme was stable for up to 3 h. Lugdulysin significantly inhibited the formation and disrupted preestablished protein-matrix MRSA biofilm. This preliminary study indicates that lugdulysin has a potential role as a competition mechanism and/or modulation of staphylococcal biofilm.

Methicillin-resistant Staphylococcus aureus and coagulase-negative Staphylococcus produce antimicrobial substances against members of the skin microbiota in children with atopic dermatitis.

Coagulase-negative Staphylococcus (CoNS) species inhibiting Staphylococcus aureus has been described in the skin of atopic dermatitis (AD) patients. This study evaluated whether Staphylococcus spp. from the skin and nares of AD and non-AD children produced antimicrobial substances (AMS). AMS production was screened by an overlay method and tested against NaOH, proteases and 30 indicator strains. Clonality was assessed by pulsed-field gel electrophoresis. Proteinaceous AMS-producers were investigated for autoimmunity by the overlay method and presence of bacteriocin genes by polymerase chain reaction. Two AMS-producers had their genome screened for AMS genes. A methicillin-resistant S. aureus (MRSA) produced proteinaceous AMS that inhibited 51.7% of the staphylococcal indicator strains, and it was active against 60% of the colonies selected from the AD child where it was isolated. On the other hand, 57 (8.8%) CoNS from the nares and skin of AD and non-AD children, most of them S. epidermidis (45.6%), reduced the growth of S. aureus and other CoNS species. Bacteriocin-related genes were detected in the genomes of AMS-producers. AMS production by CoNS inhibited S. aureus and other skin microbiota species from children with AD. Furthermore, an MRSA colonizing a child with AD produced AMS, reinforcing its contribution to dysbiosis and disease severity.

Methicillin-resistant and methicillin-sensitive Staphylococcus aureus isolates from skin and nares of Brazilian children with atopic dermatitis demonstrate high level of clonal diversity.

BACKGROUND: Atopic dermatitis (AD) primarily affects the pediatric population, which is highly colonized by S. aureus. However, little is known about the genetic features of this microorganism and other staphylococcal species that colonize AD patients. OBJECTIVE: This study aimed to characterize Staphylococcus spp. isolated from the nares and skin (with and without lesion) of 30 AD and 12 non-AD Brazilian children. METHODS: Skin and nasal swabs were cultured onto mannitol salt agar, and bacterial colonies were counted and identified by matrix assisted laser desorption ionization time of flight mass spectrometry and polymerase chain reaction (PCR). Antimicrobial susceptibility was evaluated by phenotypic and genotypic tests. In S. aureus isolates, Panton-Valentine leukocidin genes were detected by PCR, and their clonality was assessed by pulsed-field gel electrophoresis and multilocus sequence typing. RESULTS: S. aureus was more prevalent in the nares (P = 0.005) and lesional skin (P = 0.0002) of children with AD, while S. hominis was more frequent in the skin of non-AD children (P < 0.0001). All children in the study, except one from each group, were colonized by methicillin-resistant coagulase-negative Staphylococcus and 24% by methicillin-resistant S. aureus. Despite the great clonal diversity of S. aureus (18 sequence types identified), most AD children (74.1%) were colonized by the same genotype in both niches. CONCLUSION: High colonization by polyclonal S. aureus isolates was found among children with AD, while S. hominis was more frequent among non-AD children. The high prevalence of methicillin-resistant staphylococcal isolates highlights the importance of continued surveillance, especially when considering empiric antibiotic therapy for the treatment of skin infections in these patients.

Methicillin-resistant Staphylococcus aureus from infected skin lesions present several virulence genes and are associated with the CC30 in Brazilian children with atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease and colonization by Staphylococcus aureus may affect up to 100% of these patients. Virulent and resistant isolates can worsen AD patient clinical condition and jeopardize the treatment. We aimed to detect virulence genes and to evaluate the biofilm production of S. aureus isolates from infected skin lesions of children with AD. Methicillin resistance was detected by phenotypic and molecular tests and the virulence genes were detected by PCR. Biofilm formation was assessed by bacterial growing on microtiter plates and later stained with safranin. Genotyping was performed by Pulsed-Field Gel Electrophoresis and Multilocus Sequence Typing. Among 106 AD patients, 55 (51.8%) had developed S. aureus cutaneous infections and 23 (41.6%) were methicillin-resistant (MRSA). All 55 isolates carried the fnbA, hla, icaA, sasG, and seu genes, and more than 70% presented cna, eap, ebpS, hlg, and pvl genes. Clonal complex (CC) 30 was the main lineage found (34.5%), especially among MRSA isolates (52.2%). The egc cluster and the bbp gene were significantly the most frequent in MRSA isolates and in USA1100/ST30/CC30 lineage. Most of the isolates (74.5%) were non-biofilm producers and many of them only started to produce it in the presence of fibrinogen. There was no significant association between S. aureus isolates features and the AD severity. This study demonstrated a high frequency of CC30 MRSA isolates presenting several virulence genes in infected skin lesions of AD children in Brazil, that may influence the severity of the disease and the treatments required.

Prevalence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus nasal carriage and their clonal diversity among patients attending public health-care facilities.

Context: Nosocomial infections arise from many microorganisms, including Staphylococcus aureus. Aims: The aim of this study is to determine the molecular epidemiology of circulating methicillin-resistant S. aureus (MRSA) clones among patients attending community and health-care facilities in Nova Friburgo, RJ, Brazil. Methods: A total of 1002 nasal swab samples were collected from May 2010 to September 2015. S. aureus isolates were identified through phenotypic tests, submitted to antimicrobial susceptibility tests and genotypic analysis to detect mecA, panton-valentine leucocidin (PVL) genes, SCCmec, SPA and multilocus sequencing typing (MLST) typing. Results: We identified 294 (29.3%) isolates as S. aureus and 91 (9.1%) as MRSA. A total of 17 isolates did not present a correlation between phenotypic and genotypic resistance profiles. Among MRSA isolates, 17 (18.7%) carried PVL genes. A total of 20 different SPA types were determined, being grouped by MLST into eight different sequence types. ST5/t002 was the most prevalent genotype found among these isolates. Conclusions: There is a gradual colonisation shift happening in the infection pattern by S. aureus in Brazil. The Brazilian Epidemic Clone (ST239-SCCmec IIIa-PVL-) seems to be substituted by isolates from different clonal complexes, such as ST5, ST8 and ST30. The non-correlation between phenotypic/genotypic resistance profile observed in some isolates suggests the presence of other methicillin resistance mechanisms different from mecA presence or a difference in the nucleotide sequence, which prevents the primers to identify the specific region during polymerase chain reaction reactions. MRSA identification should be based on phenotypic and genotypic testing to ensure the various types of resistance mechanisms.

Osmotic stress induces biofilm production by Staphylococcus epidermidis isolates from neonates.

Staphylococcus epidermidis is one of the leading causes of bloodstream infections, particularly in premature neonates, and biofilm formation is a major virulence factor. We characterized biofilm formation by 50 S. epidermidis neonatal isolates under osmotic stress and evaluated the expression of biofilm-associated genes. Phenotypical analyses of biofilm production were performed in culture medium with or without addition of NaCl or glucose. In control medium (no additions), most isolates (84%) were nonproducers or weak biofilm producers. Growth in NaCl-containing medium increased the number of moderate/strong producers, and this increase was even greater in medium containing glucose. Most of the protein-enriched biofilms (60%) could be observed only during growth in glucose, whereas 50% of the polysaccharide-enriched biofilms were observed during growth in NaCl. Studies that evaluate the conditions used to characterize biofilm production are important to help us understand the dynamics of this important virulence factor in S. epidermidis and their impact on neonatal infections.

Daptomycin and vancomycin non-susceptible methicillin-resistant Staphylococcus aureus clonal lineages from bloodstream infection in a Brazilian teaching hospital.

INTRODUCTION: This study aimed to characterize Staphylococcus aureus isolates from bloodstream infections in patients attending a teaching hospital, between 2011 and 2015. METHODS: The minimum inhibitory concentration for daptomycin, linezolid, oxacillin, teicoplanin, vancomycin, and trimethoprim/sulfamethoxazole was accessed by broth microdilution. SCCmec type and clonal profile were determined by molecular tests. Vancomycin heteroresistance was evaluated using screening tests and by population analysis profile/area under the curve. RESULTS: Among 200 S. aureus isolates, 55 (27.5%) were MRSA, carrying SCCmec II (45.5%) or IV (54.5%). The most frequent MRSA lineages were USA100 (ST5-II) (45.5%) and USA800 (ST5-IV) (30.9%). Six isolates were confirmed as vancomycin heteroresistant, showing area under the curve ratio 1.1, 1.2 or 1.3 (four USA100, one USA800 and one USA1100 isolates). CONCLUSIONS: Daptomycin and vancomycin non-susceptible MRSA clonal lineages were found in bloodstream infections over five years, highlighting the importance of continuous surveillance of multiresistant bacteria in hospitals.

High rate of neonates colonized by methicillin-resistant Staphylococcus species in an Intensive Care Unit.

INTRODUCTION: Staphylococcal colonization is a risk factor for healthcare-associated infections, which are frequent in Neonatal Intensive Care Units (NICU). This study analyzed microbiology, epidemiology and clinical aspects of Staphylococcus spp. colonizing neonates. METHODOLOGY: Nasal or periumbilical swabs were evaluated from 175 newborns admitted to a NICU of a Rio de Janeiro hospital from March to September 2009. Clinical data were obtained from the medical records. SCCmec typing and the mecA and Panton-Valentine Leukocidin (PVL) genes were detected by PCR. Clonal diversity was evaluated by pulsed-field gel electrophoresis. RESULTS: Staphylococcus spp. isolates were detected in 98 (56%) neonates, 66.3% of them had birth weight ≤ 2500 g, 62.2% were preterm (˂ 37 weeks) and the mean length of hospitalization was 14.9 days. Among the 133 isolates identified, 48.1% were S. epidermidis, 23.3% S. haemolyticus and 13.5% S. aureus. Methicillin-resistant Staphylococcus isolate was detected in 77.6% of neonates. The methicillin-resistant S. aureus isolates carried the SCCmec type IV, while 94.6% of S. epidermidis and 85.7% of S. haemolyticus presented non-typeable cassettes. Among the S. aureus, 55.6% had PVL genes and the USA800 genotype was prevalent. Two genotypes of S. epidermidis and one of S. haemolyticus clustered 42.2% and 25.8% of the isolates, respectively. S haemolyticus colonization was associated with the use of parenteral nutrition and mechanical ventilation. CONCLUSION: High rate of neonates colonized by methicillin-resistant Staphylococcus species and the permanence of clones circulating in the NICU highlight the importance for continuous and preventive surveillance in this high-risk population.

Daptomycin and vancomycin non-susceptible methicillin-resistant Staphylococcus aureus clonal lineages from bloodstream infection in a Brazilian teaching hospital

ABSTRACT Introduction: This study aimed to characterize Staphylococcus aureus isolates from bloodstream infections in patients attending a teaching hospital, between 2011 and 2015. Methods: The minimum inhibitory concentration for daptomycin, linezolid, oxacillin, teicoplanin, vancomycin, and trimethoprim/sulfamethoxazole was accessed by broth microdilution. SCCmec type and clonal profile were determined by molecular tests. Vancomycin heteroresistance was evaluated using screening tests and by population analysis profile/area under the curve. Results: Among 200 S. aureus isolates, 55 (27.5%) were MRSA, carrying SCCmec II (45.5%) or IV (54.5%). The most frequent MRSA lineages were USA100 (ST5-II) (45.5%) and USA800 (ST5-IV) (30.9%). Six isolates were confirmed as vancomycin heteroresistant, showing area under the curve ratio 1.1, 1.2 or 1.3 (four USA100, one USA800 and one USA1100 isolates). Conclusions: Daptomycin and vancomycin non-susceptible MRSA clonal lineages were found in bloodstream infections over five years, highlighting the importance of continuous surveillance of multiresistant bacteria in hospitals.