Current types of staphylococcal cassette chromosome mec (SCCmec) in clinically relevant coagulase-negative staphylococcal (CoNS) species.

Coagulase-negative staphylococci (CoNS) colonize human skin and mucosal membranes, which is why they are considered harmless commensal bacteria. Two species, Staphylococcus epidermidis and Staphylococcus haemolyticus belong to the group of CoNS species and are most frequently isolated from nosocomial infections, including device-associated healthcare-associated infections (DA-HAIs) and local or systemic body-related infections (FBRIs). Methicillin resistance, initially described in Staphylococcus aureus, has also been reported in CoNS species. It is mediated by the mecA gene within the staphylococcal cassette chromosome (SCCmec). SCCmec typing, primarily using PCR-based methods, has been employed as a molecular epidemiological tool. However, the introduction of whole genome sequencing (WGS) and next-generation sequencing (NGS) has enabled the identification and verification of new SCCmec types. This review describes the current distribution of SCCmec types, subtypes, and variants among CoNS species, including S. epidermidis, S. haemolyticus, and S. capitis. The literature review focuses on recent research articles from the past decade that discuss new combinations of SCCmec in coagulase-negative Staphylococcus. The high genetic diversity and gaps in CoNS SCCmec annotation rules underscore the need for an efficient typing system. Typing SCCmec cassettes in CoNS strains is crucial to continuously updating databases and developing a unified classification system.

Staphylococcal saoABC Operon Codes for a DNA-Binding Protein SaoC Implicated in the Response to Nutrient Deficit.

Whilst a large number of regulatory mechanisms for gene expression have been characterised to date, transcription regulation in bacteria still remains an open subject. In clinically relevant and opportunistic pathogens, such as Staphylococcus aureus, transcription regulation is of great importance for host-pathogen interactions. In our study we investigated an operon, exclusive to staphylococci, that we name saoABC. We showed that SaoC binds to a conserved sequence motif present upstream of the saoC gene, which likely provides a negative feedback loop. We have also demonstrated that S. aureus ΔsaoB and ΔsaoC mutants display altered growth dynamics in non-optimal media; ΔsaoC exhibits decreased intracellular survival in human dermal fibroblasts, whereas ΔsaoB produces an elevated number of persisters, which is also elicited by inducible production of SaoC in ΔsaoBΔsaoC double mutant. Moreover, we have observed changes in the expression of saoABC operon genes during either depletion of the preferential carbon or the amino acid source as well as during acidification. Comparative RNA-Seq of the wild type and ΔsaoC mutant demonstrated that SaoC influences transcription of genes involved in amino acid transport and metabolism, and notably of those coding for virulence factors. Our results suggest compellingly that saoABC operon codes for a DNA-binding protein SaoC, a novel staphylococcal transcription factor, and its antagonist SaoB. We linked SaoC to the response to nutrient deficiency, a stress that has a great impact on host-pathogen interactions. That impact manifests in SaoC influence on persister formation and survival during internalisation to host cells, as well as on the expression of genes of virulence factors that may potentially result in profound alternations in the pathogenic phenotype. Investigation of such novel regulatory mechanisms is crucial for our understanding of the dynamics of interactions between pathogenic bacteria and host cells, particularly in the case of clinically relevant, opportunistic pathogens such as Staphylococcus aureus.

May Staphylococcus lugdunensis Be an Etiological Factor of Chronic Maxillary Sinuses Infection?

Staphylococcus lugdunensis is an opportunistic pathogen found in the healthy human skin microbiome bacterial community that is able to cause infections of diverse localization, manifestation, and course, including laryngological infections, such as necrotizing sinusitis. Chronic maxillary sinusitis is a disease present in up to one third of European and American populations, and its etiology is not fully described. Within this study, we aimed to characterize 18 S. lugdunensis strains recovered from maxillary sinuses and evaluate them as etiological agents of chronic disease. We performed MLST analysis, the complex analysis of both phenotypic and genetic virulence factors, antibiotic susceptibility profiles, and biofilm formation assay for the detection of biofilm-associated genes. Altogether, S. lugdunensis strains were clustered into eight different STs, and we demonstrated several virulence factors associated with the chronic disease. All tested strains were able to produce biofilm in vitro with numerous strains with a very strong ability, and overall, they were mostly susceptible to antibiotics, although we found resistance to fosfomycin, erythromycin, and clindamycin in several strains. We believe that further in-depth analysis of S. lugdunensis strains from different niches, including the nasal one, should be performed in the future in order to reduce infection rate and broaden the knowledge about this opportunistic pathogen that is gaining attention.

Distribution and antibiotic-resistance of different Staphylococcus species identified by matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) isolated from the oral cavity.

BACKGROUND: The use of antibiotics in dentistry is associated with the emergence and spread of antibiotic-resistant microorganisms, including commensal staphylococci. METHODS: A total of 367 oral samples were collected, from which staphylococci were isolated and identified by using matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF). The antibiotic susceptibility of the isolates was determined and molecular characteristics for methicillin-resistant staphylococci was performed. RESULTS: A total of 103 coagulase-negative staphylococci (CoNS), among them S. warneri, S. haemolyticus, S. saprophyticus, S. pasteuri, S. epidermidis, S. hominis, S. xylosus, S. equorum, S. kloosii, S. succinus, S. cohnii, and S. simulans, were confirmed by MALDI-TOF. Resistance to most tested antibiotics was statistically higher in CoNS than in S. aureus isolates (P-value < 0.05). CoNS isolates showed high resistance to penicillin (S. saprophyticus 88.9%), erythromycin (S. haemolyticus 84.6%), fusidic acid (S. saprophyticus 77.8%), co-trimoxazole (S. epidermidis 71.4%), gentamicin (S. warneri 63.8%), and tetracycline (S. saprophyticus 55.6%). Multidrug resistance was largely observed, especially among S. haemolyticus and S. saprophyticus species. Methicillin-resistance in S. haemolyticus (38.5%), S. saprophyticus (22.2%) and S. aureus (13.5%) was associated with the presence of the mecA gene and SCCmec type IV or V. CONCLUSION: Coagulase-negative staphylococci, especially S. haemolyticus and S. saprophyticus, seem to be a reservoir of methicillin resistance and multidrug resistance in the oral cavity.

Effect of Staphylococcus aureus infection on the heat stress protein 70 (HSP70) level in chicken embryo tissues.

Staphylococcus aureus bacteria are components of physiological biocenosis of skin or mucous membranes in some animals' genera but also they are dangerous opportunistic pathogens responsible for infections of various localization, course or manifestations. Proteins produced by these bacteria destroy tissues, leukocytes and cause haemolysis of erythrocytes. Host organisms respond by defence mechanisms. Production of heat stress proteins (HSPs) is one of defence responses of infected host organism. To evaluate infection and host defence mechanisms some animal models of experimental infection are reported. Use of chick embryo model allows demonstrating adequate differences in staphylococcal virulence depending on the strain genotype. The aim of the study was to examine the changes in heat shock protein HSP70 levels in chick embryo tissues after infection caused by S. aureus strains no. tu2, pa3, ch5, ch10, ch24, and ch25 isolated from chickens. The bacteria were injected directly into fluid of amnion cavity and incubated for 10 days. The mortality of particular chick embryos was reported and the tissues for further analysis were taken every day from day 13 to day 19. The levels of heat stress protein HSP70 were determined by dot-blot method. Results showed that the strains no. ch5, ch24, and ch25 were the most virulent. HSP70 levels increased in all groups of injected embryos at the same time the hatching process was started. The presented study showed that the infected chick embryos were characterized by higher HSP level from 12.3% up to 19.7% compared to the control group. The morphological analysis showed numerous erythrocytes with damaged cell membranes and morphological changes of erythrocytes. Changes in the level of HSP70 protein can be a useful indicator of infection caused by S. aureus bacteria. Additionally, chicken embryo is a helpful research model in studies of pathogenesis of diseases caused by bacteria.

Human skin microbiota-friendly lysostaphin.

Growing antibiotic resistance of bacteria is a burning problem of human and veterinary medicine. Expansion and introduction of novel microbicidal therapeutics is highly desirable. However, antibiotic treatment disturbs the balance of physiological microbiota by changing its qualitative and/or quantitative composition, resulting in a number of adverse effects that include secondary infections. Although such dysbiosis may be reversed by the treatment with probiotics, a more attractive alternative is the use of antibiotics that target only pathogens, while sparing the commensals. Here, we describe lysostaphin LSp222, an enzyme produced naturally by Staphylococcus pseudintermedius 222. LSp222 is highly effective against S. aureus, including its multi-drug resistant strains. Importantly, the inhibitory concentration for S. epidermidis, the predominant commensal in healthy human skin, is at least two orders of magnitude higher compared to S. aureus. Such significant therapeutic window makes LSp222 a microbiota-friendly antibacterial agent with a potential application in the treatment of S. aureus-driven skin infections.

Epidemiology and Pathogenesis of Staphylococcus Bloodstream Infections in Humans: a Review.

Staphylococci are among the most frequent human microbiota components associated with the high level of bloodstream infection (BSI) episodes. In predisposed patients, there is a high risk of transformation of BSI episodes to sepsis. Both bacterial and host factors are crucial for the outcomes of BSI and sepsis. The highest rates of BSI episodes were reported in Africa, where these infections were up to twice as high as the European rates. However, there remains a great need to analyze African data for comprehensive quantification of staphylococcal BSI prevalence. The lowest rates of BSI exist in Australia. Asian, European, and North American data showed similar frequency values. Worldwide analysis indicated that both Staphylococcus aureus and coagulase-negative staphylococci (CoNS) are the most frequent BSI agents. In the second group, the most prevalent species was Staphylococcus epidermidis, although CoNS were not identified at the species level in many studies. The lack of a significant worldwide decrease in BSI episodes indicates a great need to implement standardized diagnostic methods and research etiological factors using advanced genetic methods.

First Case of Staphylococci Carrying Linezolid Resistance Genes from Laryngological Infections in Poland.

Linezolid is currently used to treat infections caused by multidrug-resistant Gram-positive cocci. Both linezolid-resistant S. aureus (LRSA) and coagulase-negative staphylococci (CoNS) strains have been collected worldwide. Two isolates carrying linezolid resistance genes were recovered from laryngological patients and characterized by determining their antimicrobial resistance patterns and using molecular methods such as spa typing, MLST, SCCmec typing, detection of virulence genes and ica operon expression, and analysis of antimicrobial resistance determinants. Both isolates were multidrug resistant, including resistance to methicillin. The S. aureus strain was identified as ST-398/t4474/SCCmec IVe, harboring adhesin, hemolysin genes, and the ica operon. The S. haemolyticus strain was identified as ST-42/mecA-positive and harbored hemolysin genes. Linezolid resistance in S. aureus strain was associated with the mutations in the ribosomal proteins L3 and L4, and in S. haemolyticus, resistance was associated with the presence of cfr gene. Moreover, S. aureus strain harbored optrA and poxtA genes. We identified the first case of staphylococci carrying linezolid resistance genes from patients with chronic sinusitis in Poland. Since both S. aureus and CoNS are the most common etiological factors in laryngological infections, monitoring of such infections combined with surveillance and infection prevention programs is important to decrease the number of linezolid-resistant staphylococcal strains.

Identification of Clinically Relevant Streptococcus and Enterococcus Species Based on Biochemical Methods and 16S rRNA, sodA, tuf, rpoB, and recA Gene Sequencing.

Streptococci and enterococci are significant opportunistic pathogens in epidemiology and infectious medicine. High genetic and taxonomic similarities and several reclassifications within genera are the most challenging in species identification. The aim of this study was to identify Streptococcus and Enterococcus species using genetic and phenotypic methods and to determine the most discriminatory identification method. Thirty strains recovered from clinical samples representing 15 streptococcal species, five enterococcal species, and four nonstreptococcal species were subjected to bacterial identification by the Vitek® 2 system and Sanger-based sequencing methods targeting the 16S rRNA, sodA, tuf, rpoB, and recA genes. Phenotypic methods allowed the identification of 10 streptococcal strains, five enterococcal strains, and four nonstreptococcal strains (Leuconostoc, Granulicatella, and Globicatella genera). The combination of sequencing methods allowed the identification of 21 streptococcal strains, five enterococcal strains, and four nonstreptococcal strains. The 16S rRNA and rpoB genes had the highest identification potential. Only a combination of several molecular methods was sufficient for unambiguous confirmation of species identity. This study will be useful for comparison of several identification methods, both those used as a first choice in routine microbiology and those used for final confirmation.

Presence of egc-positive major clones ST 45, 30 and 22 among methicillin-resistant and methicillin-susceptible oral Staphylococcus aureus strains.

The oral cavity may comprise a significant reservoir for Staphylococcus aureus but the data on molecular epidemiology and clonal distribution of oral strains are really scarce. This study aimed to evaluate the clonal relatedness in S. aureus isolated from oral cavity and their relationship with carriage of virulence genes, and antimicrobial resistance profiles. A total of 139 oral S. aureus isolates were obtained from 2327 analysed oral samples of dental patients. Antimicrobial susceptibility testing was performed. Isolates were characterized using protein A gene (spa) typing, spa-CC clonal complexes, toxin genes and SCCmec typing for MRSA. High resistance rates for penicillin, tetracycline and gentamicin were detected, respectively 58.3%, 42.4%, and 35.2%. Twelve (8.6%) S. aureus isolates were identified as MRSA. All of MRSA isolates were mecA-positive and mecC-negative. SCCmec IV was the most common type (66.7%), which was typical for community-acquired MRSA (CA-MRSA). Overall, the enterotoxin gene cluster (egc) was the most frequent detected virulence factor (44.9%), both in MSSA and MRSA isolates. Presence of genes encoding for the enterotoxins (sea, seb, sec, seh, sek), exfoliative toxin A (eta), and toxic shock syndrome toxin-1 (tst) was also observed. Strains carrying lukS-PV/lukF-PV genes belonged to SCCmecV- spa type t437. The most prevalent spa types were t091, t015, t084, t002, t571, and t026 among all 57 identified. Spa types, including 3 new ones, grouped in 6 different spa-CC clonal complexes, with four major dominated; CC45, CC30, CC5, and CC15. This study demonstrated that both methicillin-susceptible and methicillin-resistant major European clones of S. aureus could be isolated from the oral cavity of dental patients, with the emergence of PVL-positive CA-MRSA strains. The oral cavity should be considered as a possible source of toxigenic egc-positive S. aureus strains, in terms of potential risk of cross-infection and dissemination to other body sites.

Coagulase-negative staphylococci (CoNS) as a significant etiological factor of laryngological infections: a review.

This review article shows that coagulase-negative staphylococci (CoNS) are widely responsible for laryngological diseases. General characteristics of CoNS infections are shown in the introduction, and the pathogenicity in terms of virulence determinants, biofilm formation and genetic regulation mechanisms of these bacteria is presented in the first part of the paper to better display the virulence potential of staphylococci. The PubMed search keywords were as follows: CoNS and: nares infections, nasal polyps, rhinosinusitis, necrosing sinusitis, periprosthetic joint infection, pharyngitis, osteomyelitis of skull and neck bones, tonsillitis and recurrent tonsillitis. A list of laryngological infections and those related to skull and neck bones was presented with descriptions of the following diseases: rhinosinusitis, necrotizing sinusitis, nasal polyps, nares and nasal skin infections, periprosthetic joint infections, osteomyelitis, pharyngitis, and tonsillitis. Species identification and diagnostic problems challenging for diagnosticians are presented. Concluding remarks regarding the presence of CoNS in humans and their distribution, particularly under the effect of facilitating factors, are mentioned.

Emergence of linezolid-resistant Staphylococcus epidermidis in the tertiary children's hospital in Cracow, Poland.

Coagulase-negative staphylococci, ubiquitous commensals of human skin, and mucous membranes represent important pathogens for immunocompromised patients and neonates. The increasing antibiotic resistance among Staphylococcus epidermidis is an emerging problem worldwide. In particular, the linezolid-resistant S. epidermidis (LRSE) strains are observed in Europe since 2014. The aim of our study was to genetically characterize 11 LRSE isolates, recovered mostly from blood in the University Children's Hospital in Krakow, Poland, between 2015 and 2017. For identification of the isolates at the species level, we used 16S rRNA sequencing and RFLP of the saoC gene. Isolates were characterized phenotypically by determining their antimicrobial resistance patterns and using molecular methods such as PFGE, MLST, SCCmec typing, detection of the ica operon, and analysis of antimicrobial resistance determinants. All isolates were multidrug-resistant, including resistance to methicillin, and exhibited so-called PhLOPSA phenotype. In PFGE, all isolates (excluding one from a catheter) represented identical patterns, were identified as ST2, and harbored the ica operon, responsible for biofilm formation. Linezolid resistance was associated with acquisition of A157R mutation in the ribosomal protein L3 and the presence of cfr gene. All isolates revealed new SCCmec cassette element composition. Recently, pediatric patients with serious staphylococcal infections are often treated with linezolid. The increasing linezolid resistance in bacterial strains becomes a real threat for patients, and monitoring such infections combined with surveillance and infection prevention programs is very important to decrease number of linezolid-resistant staphylococcal strains.

Preliminary evaluation of application of a 3-dimensional network structure of siloxanes Dergall preparation on chick embryo development and microbiological status of eggshells.

The spatial network structure of Dergall is based on substances nontoxic to humans and the environment which, when applied on solid surfaces, creates a coating that reduces bacterial cell adhesion. The bacteriostatic properties of siloxanes are based on a purely physical action mechanism which excludes development of drug-resistant microorganisms. The aims of the present study were to 1) evaluate a Dergall layer formed on the eggshell surface regarding the potential harmful effects on the chick embryo; 2) evaluate antimicrobial activity and estimate the prolongation time of Dergall's potential antimicrobial activity. Dergall at a concentration of 0.6% formed a layer on the eggshell surface. In vitro testing of the potential harmful effects of Dergall by means of a hen embryo test of the chorioallantoic membrane showed no irritation reaction at a concentration of 3% and lower. The hatchability of the groups sprayed with a Dergall water solution with a concentration of 0 to 5% was 89.1 to 93.8% for fertilized eggs (P > 0.05) but decreased to 63.7% (P < 0.05) in the group sprayed with a 6% concentration of the solution. This phenomenon was caused by embryo mortality in the first week of incubation. At the concentration of 0.6%, Dergall exhibited strong antibacterial properties against bacteria such as Staphylococcus aureus, Escherichia coli, Shigella dysenteriae, Shigella flexneri, and Salmonella typhimurium. For Streptococcus pyogenes, the highest antibacterial activity of Dergall was reported in the concentrations of 100 and 50%. For Pseudomonas aeruginosa, no antibacterial activity of Dergall was generally observed, but in vivo testing showed a strong decrease of all gram-negative bacteria growth. Moreover, a prolonged antimicrobial effect lasting until 3 D after disinfection was observed, which makes Dergall a safe and efficient disinfectant.

Development of a reference data set for assigning Streptococcus and Enterococcus species based on next generation sequencing of the 16S-23S rRNA region.

Background: Many members of Streptococcus and Enterococcus genera are clinically relevant opportunistic pathogens warranting accurate and rapid identification for targeted therapy. Currently, the developed method based on next generation sequencing (NGS) of the 16S-23S rRNA region proved to be a rapid, reliable and precise approach for species identification directly from polymicrobial and challenging clinical samples. The introduction of this new method to routine diagnostics is hindered by a lack of the reference sequences for the 16S-23S rRNA region for many bacterial species. The aim of this study was to develop a careful assignment for streptococcal and enterococcal species based on NGS of the 16S-23S rRNA region. Methods: Thirty two strains recovered from clinical samples and 19 reference strains representing 42 streptococcal species and nine enterococcal species were subjected to bacterial identification by four Sanger-based sequencing methods targeting the genes encoding (i) 16S rRNA, (ii) sodA, (iii) tuf and (iv) rpoB; and NGS of the 16S-23S rRNA region. Results: This study allowed obtainment and deposition of reference sequences of the 16S-23S rRNA region for 15 streptococcal and 3 enterococcal species followed by enrichment for 27 and 6 species, respectively, for which reference sequences were available in the databases. For Streptococcus, NGS of the 16S-23S rRNA region was as discriminative as Sanger sequencing of the tuf and rpoB genes allowing for an unambiguous identification of 93% of analyzed species. For Enterococcus, sodA, tuf and rpoB genes sequencing allowed for identification of all species, while the NGS-based method did not allow for identification of only one enterococcal species. For both genera, the sequence analysis of the 16S rRNA gene was endowed with a low identification potential and was inferior to that of other tested identification methods. Moreover, in case of phylogenetically related species the sequence analysis of only the intergenic spacer region was not sufficient enough to precisely identify Streptococcus strains at the species level. Conclusions: Based on the developed reference dataset, clinically relevant streptococcal and enterococcal species can now be reliably identified by 16S-23S rRNA sequences in samples. This study will be useful for introduction of a novel diagnostic tool, NGS of the 16S-23S rRNA region, which undoubtedly is an improvement for reliable culture-independent species identification directly from polymicrobially constituted clinical samples.

Development and Validation of a Reference Data Set for Assigning Staphylococcus Species Based on Next-Generation Sequencing of the 16S-23S rRNA Region.

Many members of the Staphylococcus genus are clinically relevant opportunistic pathogens that warrant accurate and rapid identification for targeted therapy. The aim of this study was to develop a careful assignment scheme for staphylococcal species based on next-generation sequencing (NGS) of the 16S-23S rRNA region. All reference staphylococcal strains were identified at the species level using Sanger sequencing of the 16S rRNA, sodA, tuf, and rpoB genes and NGS of the 16S-23S rRNA region. To broaden the database, an additional 100 staphylococcal strains, including 29 species, were identified by routine diagnostic methods, 16S rRNA Sanger sequencing and NGS of the 16S-23S rRNA region. The results enabled development of reference sequences encompassing the 16S-23S rRNA region for 50 species (including one newly proposed species) and 6 subspecies of the Staphylococcus genus. This study showed sodA and rpoB targets were the most discriminative but NGS of the 16S-23S rRNA region was more discriminative than tuf gene sequencing and much more discriminative than 16S rRNA gene sequencing. Almost all Staphylococcus species could be distinguished when the max score was 99.0% or higher and the sequence similarity between the best and second best species was equal to or >0.2% (min. 9 nucleotides). This study allowed development of reference sequences for 21 staphylococcal species and enrichment for 29 species for which sequences were publicly available. We confirmed the usefulness of NGS of the 16S-23S rRNA region by identifying the whole species content in 45 clinical samples and comparing the results to those obtained using routine diagnostic methods. Based on the developed reference database, all staphylococcal species can be reliably detected based on the 16S-23S rRNA sequences in samples composed of both single species and more complex polymicrobial communities. This study will be useful for introduction of a novel diagnostic tool, which undoubtedly is an improvement for reliable species identification in polymicrobial samples. The introduction of this new method is hindered by a lack of reference sequences for the 16S-23S rRNA region for many bacterial species. The results will allow identification of all Staphylococcus species, which are clinically relevant pathogens.

New Insight into Genotypic and Phenotypic Relatedness of Staphylococcus aureus Strains from Human Infections or Animal Reservoirs.

Staphylococcus aureus is a common human and livestock opportunistic pathogen, and there is evidence of animal to human transmission. This paper aimed to recognize properties of the isolates from collections of human and livestock S. aureus strains and to estimate compatibility of results based on phenotypic tests, microarrays and the spa typing methods. The second goal was to study differences between human and animal isolates in terms of specificity of their hosts and the strain transmission among various hosts. Most strains showed multi-susceptible profiles and produced enzymes on a high level, and they were phenotypically and genetically similar. However, in contrast to the Polish bovine mastitis strains, the Slovakian strains were multi-resistant. In this research, the strains showed significant differences in terms of their phenotypic manifestations and the presence of hemolysins genes; however, other enzyme-encoding genes correlated to a higher extent with the microarrays results. Interestingly, there was a lack of enterotoxin genes in human Poultry-like protein A+ strains in comparison to other human strains. Our study showed that differences between virulence profiles of the human and animal strains correlated with their origin rather than their hosts, and any trait allowed clearly distinguishing between them based on the microarray results.Staphylococcus aureus is a common human and livestock opportunistic pathogen, and there is evidence of animal to human transmission. This paper aimed to recognize properties of the isolates from collections of human and livestock S. aureus strains and to estimate compatibility of results based on phenotypic tests, microarrays and the spa typing methods. The second goal was to study differences between human and animal isolates in terms of specificity of their hosts and the strain transmission among various hosts. Most strains showed multi-susceptible profiles and produced enzymes on a high level, and they were phenotypically and genetically similar. However, in contrast to the Polish bovine mastitis strains, the Slovakian strains were multi-resistant. In this research, the strains showed significant differences in terms of their phenotypic manifestations and the presence of hemolysins genes; however, other enzyme-encoding genes correlated to a higher extent with the microarrays results. Interestingly, there was a lack of enterotoxin genes in human Poultry-like protein A+ strains in comparison to other human strains. Our study showed that differences between virulence profiles of the human and animal strains correlated with their origin rather than their hosts, and any trait allowed clearly distinguishing between them based on the microarray results.

Multiple-locus variable-number tandem repeat fingerprinting as a method for rapid and cost-effective typing of animal-associated Staphylococcus aureus strains from lineages other than sequence type 398.

In veterinary medicine, Staphylococcus aureus is associated with a range of mild to severe infections. The high density of livestock in intensive farming systems increases the risk of disease spread and hampers its control and measures of prevention, making S. aureus one of the most important animal pathogens. Multiple-locus variable-number tandem repeat fingerprinting (MLVF) has been successfully applied to the characterization of livestock-associated meticillin-resistant Staphylococcus aureus (MRSA) ST398 but not to the characterization of a wide range of other animal isolates. The objective of the current study was to examine the effectiveness of MLVF for studying S. aureus strains isolated from households, farms and exotic animals in three regions of Poland. MLVF, random amplification of polymorphic DNA (RAPD), spa typing and diagnostic microarrays were compared to determine the most suitable combination of methods for veterinary purposes. MLVF generated results consistent with host and geographic origins, reflecting population structures with a high concordance to spa typing results. MLVF has been proven to be a rapid, highly discriminatory and cost-effective method suitable for molecular typing in veterinary settings.

Identification of Secreted Exoproteome Fingerprints of Highly-Virulent and Non-Virulent Staphylococcus aureus Strains.

Staphylococcus aureus is a commensal inhabitant of skin and mucous membranes in nose vestibule but also an important opportunistic pathogen of humans and livestock. The extracellular proteome as a whole constitutes its major virulence determinant; however, the involvement of particular proteins is still relatively poorly understood. In this study, we compared the extracellular proteomes of poultry-derived S. aureus strains exhibiting a virulent (VIR) and non-virulent (NVIR) phenotype in a chicken embryo experimental infection model with the aim to identify proteomic signatures associated with the particular phenotypes. Despite significant heterogeneity within the analyzed proteomes, we identified alpha-haemolysin and bifunctional autolysin as indicators of virulence, whereas glutamylendopeptidase production was characteristic for non-virulent strains. Staphopain C (StpC) was identified in both the VIR and NVIR proteomes and the latter fact contradicted previous findings suggesting its involvement in virulence. By supplementing NVIR, StpC-negative strains with StpC, and comparing the virulence of parental and supplemented strains, we demonstrated that staphopain C alone does not affect staphylococcal virulence in a chicken embryo model.

Clonal Structure and Characterization of Staphylococcus aureus Strains from Invasive Infections in Paediatric Patients from South Poland: Association between Age, spa Types, Clonal Complexes, and Genetic Markers.

The aim of current study was to examine clonal structure and genetic profile of invasive Staphylococcus aureus isolates recovered from infants and children treated at the Jagiellonian University Children's Hospital of Krakow, Poland. The 107 invasive S. aureus isolates, collected between February 2012 and August 2014, were analysed retrospectively. Antimicrobial susceptibility testing, spa typing and DNA microarray analysis were performed to determine clonal distribution, diversity and gene content in regard to patients characteristics. In total, 107 isolates were recovered from 88 patients with clinical symptoms of invasive bacterial infection. The final set of 92 non-duplicate samples included 38 MRSA isolates. Additionally, a set of 54 S. aureus isolates collected during epidemiological screening was genotyped and analysed. There were 72 healthcare-associated (HCA) and 20 community-onset (CO) infection events caused by 33 and 5 MRSA isolates, respectively. The majority of isolates were affiliated with the major European clonal complexes CC5 (t003, spa-CC 002), CC45 (spa-CC 015), CC7 or CC15 (t084, t091, spa-CC 084). Two epidemic clones (CC5-MRSA-II or CC45-MRSA-IV) dominated among MRSA isolates, while MSSA population contained 15 different CCs. The epidemiological screening isolates belonged to similar genetic lineages as those collected from invasive infection cases. The HCA infection events, spa types t003, t2642 or CC5 were significantly associated with infections occurring in neonates and children under 5 years of age. Moreover, carriage of several genetic markers, including erm(A), sea (N315), egc-cluster, chp was significantly higher in isolates obtained from children in this age group. The spa types t091 and t008 were underrepresented among patients aged 5 years or younger, whereas spa type t008, CC8 and presence of splE was associated with infection in children aged 10 years or older. The HCA-MRSA strains were most frequently found in children under 5 years, although the majority of invasive infections was associated with MSSA strains. Moreover, an association between age group of children from the study population and a specific strain genotype (spa type, clonal complex or genetic content) was observed among the patients.