Methicillin-Resistant Staphylococcus aureus ST80 Induce Lower Cytokine Production by Monocytes as Compared to Other Sequence Types

Methicillin-resistant Staphylococcus aureus (MRSA) remains an important cause of nosocomial and community-associated infections due to its ability to produce toxins and evade host’s immune responses. The aim of the present study was to investigate the association of monocytes immune response in terms of cytokines produced after inoculation with different MRSA clones. Thirty-one clinical MRSA strains were selected on the basis of clonal types, accessory gene regulator (agr) groups and toxin genes carriage. Isolates were identified as S. aureus by Gram stain, catalase, coagulase production and PCR for nuc gene. The presence of mecA, lukS/lukF-PV (Panton-Valentine Leukocidin) and tst (Toxic Shock Syndrome Toxin-1) genes, as well as, the determination of agr groups was performed by PCR. Clonality was investigated by means of multi-locus sequence typing (MLST). Peripheral blood mononuclear cells were stimulated with live bacterial cells for 45 min at a ratio of 1:10. Cells were incubated for 10 h and supernatants were collected. The levels of Tumor Necrosis Factor alpha (TNFa), IL-1b, IL-8, IL-6, IL-12p40, IL-10, interferon-gamma (IFN-γ) and IL-2, were measured by Human Cytokine Multiplex Immunoassay kit. Thirteen strains were tst and 12 lukS/lukF-PV-positive. Seven strains belonged to ST80 and ST225, five to ST30 and ST239, while the remaining seven isolates were grouped together as “other.” Strains belonging to ST80 induced statistically lower levels of TNFa, IL-1b, IL-8, IL-6, IL-10, IFN-γ, and IL-2. PVL-positive strains classified into ST80 clone induced statistically lower concentrations of most cytokines as compared to PVL-positive strains belonging to other clones, tst-positive strains and toxin-negative ones. Strains of agr3 group belonging to ST80 induced statistically lower concentrations of most tested cytokines as compared to agr3 strains not-belonging to ST80, agr2 or agr1. This low induction of immune response by MRSA ST80 cannot be attributed to the presence of neither lukS/lukF-PV nor agr3

Rapid detection of Staphylococcus aureus Panton-Valentine leukocidin in clinical specimens by enzyme-linked immunosorbent assay and immunochromatographic tests

10.1128/JCM.02274-09Journal of Clinical Microbiology4841384-139

High Genetic Diversity among Community-Associated Staphylococcus aureus in Europe: Results from a Multicenter Study

Background: Several studies have addressed the epidemiology of community-associated Staphylococcus aureus (CA-SA) in Europe; nonetheless, a comprehensive perspective remains unclear. In this study, we aimed to describe the population structure of CA-SA and to shed light on the origin of methicillin-resistant S. aureus (MRSA) in this continent. Methods and Findings: A total of 568 colonization and infection isolates, comprising both MRSA and methicillin-susceptible S. aureus (MSSA), were recovered in 16 European countries, from community and community-onset infections. The genetic background of isolates was characterized by molecular typing techniques (spa typing, pulsed-field gel electrophoresis and multilocus sequence typing) and the presence of PVL and ACME was tested by PCR. MRSA were further characterized by SCCmec typing. We found that 59 % of all isolates were associated with community-associated clones. Most MRSA were related with USA300 (ST8-IVa and variants) (40%), followed by the European clone (ST80-IVc and derivatives) (28%) and the Taiwan clone (ST59-IVa and related clonal types) (15%). A total of 83 % of MRSA carried Panton-Valentine leukocidin (PVL) and 14 % carried the arginine catabolic mobile element (ACME). Surprisingly, we found a high genetic diversity among MRSA clonal types (ST-SCCmec), Simpson’s index of diversity = 0.852 (0.788–0.916). Specifically, about half of the isolates carried novel associations between genetic background and SCCmec. Analysis by BURP showed that some CA-MSSA and CA-MRS

Study of the epidemiology of carriafe and infections caused by Staphylococcus aureus in animals ad humans: SCCmec types, toxin genes, agr types, clones defined by PFGE and MLST

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of both healthcare (HA-MRSA) and community-associated infections (CA-MRSA). Severe MRSA infections have been associated with the virulence factor Panton-Valentine leukocidin (PVL). Part of this study was to investigate susceptibility patterns, presence of toxin genes including PVL and clonality among MRSA collected from patients in Greece over a six-year period. A total of 1678 MRSA were collected from January 2007 till December 2012 from two hospitals in Western Greece confirmed by the detection of nuc and mecA genes by PCR. Antibiotic susceptibility testing was performed by the disk diffusion method and E-test. The presence of genes encoding toxic shock syndrome toxin, enterotoxin gene cluster and PVL was investigated by PCR. The genotypic characteristics of strains were analyzed including SCCmec and agr typing. Clonality of MRSA was determined by Pulsed Field Gel Electrophoresis and Multi Locus Sequence Typing. The majority of PVL-positive MRSA belonged to a single clone, ST80-IV, which was disseminated in both community and hospitals. CA-MRSA were recovered mainly from SSTIs, while HA-MRSA were associated with surgical and wound infections. During the period 2007-2012, ST80-IV dominated in both the community and hospital settings in Greece and successfully undermined the presence of other PVL-positive clones. The second part of the study focused on the prevalence of S. aureus and MRSA among companion animals and veterinary personnel (VP) in Nothern Greece. Strains’ molecular characteristics were evaluated in order to assess S. aureus transmission. Specimens (224) from colonized and infected sites of 102 animals (92 dogs, 10 cats) and 18 VP were collected during 2012 and 2013. Antibiotic susceptibility was performed by the disk diffusion method and E-test. mecA, mecC, tst (toxic shock syndrome toxin) and lukF/lukS-PV (PVL) genes were investigated by PCRs. Genotypes were identified by MLST, SCCmec, agr, spa and PFGE. S. aureus prevalence among pets and VP was 36.3% (37/102) and 38.9% (7/18), respectively. Younger companion animals, those living in rural areas, having a disease upon admission or CNS co-carriage showed significantly increased risk for S. aureus isolation (p<0.05). Twenty-six pets and five VP carried PVL-positive S. aureus. In total, 60 S. aureus strains were recovered (53 from pets, seven from VP) of which 16 were MRSA (26.7%), 12 mecA- and four mecC-positives. MRSA showed higher resistance rates against antimicrobials as compared to methicillin-susceptible ones. Strains were classified by MLST in 13 STs, with the predominance of ST80 and ST15. In MRSA, SCCmec types II, IV and XI were identified. The most frequent spa types were t5559 and t7558. Fifty-six strains were classified into 15 PFGE types. Comparison of genetic markers showed that identical or very similar strains disseminated among animals and VP. Companion animals harbor PVL-positive clones constituting a possible source for cross-transmission to humans. Because S. aureus is part of microbiota flora in many animal species we also investigated the clonal spread of S. aureus among animals and personnel in a Zoological Park. Samples were collected from colonized and infected sites among 32 mammals, 11 birds and eight humans. The genes mecA, mecC, lukF/lukS-PV and tst were investigated by PCRs. Clones were defined by MLST, spa type and PFGE. Seven S. aureus isolates were recovered from four animals and one from an employee. All were mecA, mecC and tst–negative, whereas, one carried the PVL genes and was isolated from an infected Squirrel monkey. Clonal analysis revealed the occurrence of seven STs, eight PFGE and five spa types including ones of human origin. Even though a variety of genotypes were identified among S. aureus strains colonizing zoo park residents, our results indicate that colonization with human lineages occurred. The last part of this thesis involved S. aureus isolates that were recovered from food chain animals, meat products, personnel and equipment of a Greek abattoir and were molecularly analyzed in order to gain a deeper insight into their diversity and epidemiology. S. aureus isolates were genotyped using MLST, spa, agr and SCCmec. Antimicrobial susceptibility was determined according to EUCAST guidelines. Genes encoding PVL and TSST-1 were detected by PCR. Differentiation between livestock-associated and human CC398 clade was investigated. From 392 samples, 46 S. aureus were recovered. Ten were of human, six of animals’ origin, ten were isolated from meat products and 20 from the equipment’s surfaces. Eleven isolates (23.9%) were MRSA; eight mecA- and three mecC-positives, the latter ones belonging to the livestock-associated clade. MRSA were assigned into SCCmec types I, III, IV and XI. The highest resistance rate was expressed to tetracycline (37/46, 80.4%), whereas, 27 isolates were multi-drug resistant (MDR). Strains were classified into ten sequence types (STs). ST80 predominated among MSSA, followed by ST15 and ST30 of human origin. The main clone among MRSA was ST88-IV. A great distribution of 14 spa types was detected (predominant: t7558, t094). Fifteen strains (32.6%) carried PVL genes (four MRSA and 11 MSSA), classified into six clones. S. aureus and specifically MDR strains were prevalent in animals’ nares, resulted meat products, humans and abattoir’s environment. Human-associated clones are disseminated, but also livestock-associated lineages are transferred to humans. Abattoirs may serve as a transmission route for S. aureus strains.Ο ανθεκτικός στη μεθικιλλίνη Staphylococcus aureus (MRSA) αποτελεί σημαντικό αίτιο ενδονοσοκομειακών λοιμώξεων (HA-MRSA) καθώς επίσης και της κοινότητας (CA-MRSA). Οι σοβαρές λοιμώξεις από MRSA έχουν συσχετιστεί με τον λοιμογόνο παράγοντα Panton-Valentine leukocidin, (PVL). Μέρος αυτής της μελέτης ήταν να διερευνήσει τα πρότυπα ευαισθησίας, την παρουσία γονιδίων τοξινών συμπεριλαμβανομένης και της PVL και την κλωνικότητα μεταξύ MRSA που συλλέχθηκαν από ασθενείς στην Ελλάδα κατά τη διάρκεια μιας περιόδου έξι ετών. Συνολικά, 1678 MRSA συλλέχθηκαν από τον Ιανουάριο του 2007 μέχρι το Δεκέμβριο του 2012 από δύο διαφορετικά νοσοκομεία στη Δυτική Ελλάδα και ταυτοποιήθηκαν με βάση την ανίχνευση των γονιδίων nuc και mecA με PCR. Η αντοχή στα αντιβιοτικά πραγματοποιήθηκε με τη μέθοδο διάχυσης των δίσκων και το E-test. Η παρουσία των γονιδίων που κωδικοποιούν την τοξίνη TSST-1, την PVL και ορισμένα γονίδια των εντεροτοξινών ελέγχθηκε με PCR. Τα γονοτυπικά χαρακτηριστικά των στελεχών αναλύθηκαν με την ταυτοποίηση των τύπων SCCmec και agr. Η κλωνικότητα καθορίστηκε μέσω PFGE και MLST. Η πλειοψηφία των PVL-θετικών MRSA ανήκε στον ST80-IV κλώνο ο οποίος διεσπάρη στην κοινότητα αλλά και τα νοσοκομεία. CA-MRSA απομονώθηκαν κυρίως από SSTIs, ενώ HA-MRSA συνδέθηκαν με χειρουργικές λοιμώξεις και μολύνσεις τραυμάτων. Κατά την περίοδο 2007-2012, ο ST80-IV έχει κυριαρχήσει στην Ελλάδα και έχει περιορίσει με επιτυχία την παρουσία άλλων PVL-θετικών κλώνων. Το δεύτερο μέρος της παρούσας μελέτης επικεντρώνεται στην μελέτη διασποράς των S. aureus και MRSA μεταξύ των ζώων συντροφιάς και του κτηνιατρικού προσωπικού (ΚΠ) στην Βόρεια Ελλάδα. Τα μοριακά χαρακτηριστικά των στελεχών αξιολογήθηκαν, προκειμένου να εκτιμηθεί εάν υφίσταται μετάδοση του S. aureus. Δείγματα (224) που αποίκισαν και μόλυναν 102 ζώα (92 σκυλιά, 10 γάτες) και 18 ΚΠ, συλλέχθηκαν κατά τη διάρκεια του 2012 και του 2013. Η ευαισθησία στα αντιβιοτικά διερευνήθηκε με τη μέθοδο διάχυσης των δίσκων και E-test. Τα mecA, mecC, tst και lukF/lukS-PV γονίδια μελετήθηκαν με PCRs. Οι γονότυποι προσδιορίστηκαν με την ταυτοποίηση των τύπων MLST, SCCmec, agr, spa και PFGE. Το ποσοστό απομόνωσης S. aureus μεταξύ των κατοικίδιων ζώων και του ΚΠ ήταν 36.3% (37/102) και 38.9% (7/18), αντίστοιχα. Τα νεώτερα ζώα συντροφιάς, όσοι ζουν σε αγροτικές περιοχές, νοσούσαν κατά την εισαγωγή ή ήταν ήδη αποικισμένοι με CNS έδειξαν στατιστικά σημαντικά αυξημένο κίνδυνο για προσβολή από S. aureus (p <0,05). Είκοσι-έξι κατοικίδια ζώα και πέντε ΚΠ έφεραν PVL-θετικά στελέχη. Συνολικά, απομονώθηκαν 60 S. aureus (53 από τα κατοικίδια ζώα, επτά από το ΚΠ) εκ των οποίων 16 ήταν MRSA (26.7%), 12 ήταν mecA-θετικά και τέσσερα mecC-θετικά αντίστοιχα. Τα MRSA έδειξαν υψηλότερα ποσοστά αντοχής στα αντιβιοτικά σε σύγκριση με τα MSSA.Τα στελέχη ταξινομήθηκαν μέσω της MLST σε 13 STs, με τους ST80 και ST15 να είναι οι επικρατέστεροι τύποι. Στα MRSA, εντοπίστηκαν οι SCCmec τύποι II, IV και XI. Οι πιο συχνοί τύποι spa ήταν οι t5559 και t7558. Πενήντα έξι στελέχη ταξινομήθηκαν σε 15 τύπους PFGE. Σύγκριση των γενετικών δεικτών έδειξε ότι τα ίδια ή παρόμοια στελέχη μεταδίδονται μεταξύ των ζώων και του ΚΠ. Τα ζώα συντροφιάς αποτελούν δεξαμενή PVL-θετικών κλώνων που συνιστούν πιθανή πηγή διασταυρούμενης μετάδοσης στον άνθρωπο. Επειδή ο S. aureus είναι μέρος της μικροβιακής χλωρίδας σε πολλά είδη ζώων, στο τρίτο μέρος της μελέτης διερευνήθηκε η κλωνική εξάπλωση του S. aureus μεταξύ των ζώων και του προσωπικού σε ένα Ζωολογικό Πάρκο. Τα δείγματα συλλέχθηκαν από αποικισμένα ή μολυσμένα ζώα τα οποία περιελάμβαναν 32 θηλαστικά, 11 πουλιά και οκτώ ανθρώπους. Τα γονίδια mecA, mecC, lukF/lukS-PV και tst διερευνήθηκαν με PCRs. Οι κλώνοι καθορίστηκαν με την MLST, την τυποποίηση spa και την PFGE. Επτά S. aureus απομονώθηκαν από τέσσερα ζώα και ένα από έναν υπάλληλο. Όλα ήταν mecA, mecC και tst αρνητικά, ενώ ένα έφερε τα γονίδια της PVL που απομονώθηκε από μολυσμένο πίθηκο. Η κλωνική ανάλυση αποκάλυψε την εμφάνιση επτά STs, οκτώ PFGE και πέντε τύπων spa συμπεριλαμβανομένων και εκείνων ανθρώπινης προέλευσης. Παρά το γεγονός ότι ανιχνεύθηκε ποικιλία γονότυπων μεταξύ των στελεχών S. aureus που είχαν αποικίσει τα μέλη του Ζωολογικού Πάρκου, τα αποτελέσματα δείχνουν ότι μάλλον πρόκειται για αποικισμό με στελέχη ανθρώπινης προέλευσης. Το τελευταίο μέρος της διατριβής αφορά στελέχη S. aureus που απομονώθηκαν από ζώα της τροφικής αλυσίδας, τα προϊόντα τους με βάση το κρέας, το προσωπικό και τον εξοπλισμό ενός ελληνικού σφαγείου. Πραγματοποιήθηκε μοριακή ανάλυση προκειμένου να αποκτηθεί βαθύτερη κατανόηση της πολυμορφίας και επιδημιολογίας τους. Από 392 δείγματα απομονώθηκαν 46 S. aureus. Δέκα ήταν ανθρώπινης προέλευσης, έξι ζωικής, δέκα απομονώθηκαν από προϊόντα κρέατος και 20 από τις επιφάνειες του εξοπλισμού. Έντεκα (23.9%) ήταν MRSA εκ των οποίων οκτώ ήταν mecA και τρία mecC θετικά. Τα MRSA ταξινομήθηκαν στους τύπους SCCmec I, III, IV και XI. Το υψηλότερο ποσοστό αντοχής εκφράστηκε στην τετρακυκλίνη (37/46, 80.4%), ενώ 27 στελέχη ήταν πολυανθεκτικά (MDR). Τα στελέχη ταξινομήθηκαν σε δέκα STs. Ο ST80 ήταν ο κυρίαρχος κλώνος μεταξύ των MSSA, ακολουθούμενος από τους ανθρώπινης προέλευσης ST15 και ST30. Ο κύριος κλώνος μεταξύ των MRSA ήταν ο ST88-IV. Επίσης, ανιχνεύθηκε μεγάλη ποικιλία τύπων spa (14 τύποι) (κυρίαρχοι: t7558, t094). Δεκαπέντε στελέχη (32.6%) έφεραν τα γονίδια της PVL (τέσσερα MRSA και 11 MSSA) τα οποία κατατάχθηκαν σε έξι κλώνους. Τα στελέχη S. aureus και ειδικά τα MDR επικρατούσαν στα ζώα, στα προϊόντα κρέατος, στους ανθρώπους και στο περιβάλλον του σφαγείου. Συμπερασματικά, τα σφαγεία μπορεί να χρησιμεύσουν ως πηγή μετάδοσης στελεχών S. aureus

Biofilm synthesis and its relationship with genetic characteristics in clinical methicillin-resistant staphylococci

Staphylococcus aureus can cause a broad range of infections, including skin infections, pneumonia and bacteraemia. Coagulase-negative staphylococci (CNS), mainly S. epidermidis, have also emerged as important pathogens, especially in immunocompromised patients or those with prosthetic devices, such as intravascular catheters or biomaterials. Of great importance in the initiation of these infections is the ability of staphylococci to adhere to various surfaces, such as host tissues and prosthetic devices and to form biofilm. The staphylococcal adhesins are encoded by a number of genes such as fnbA (S. aureus fibronectin binding protein A), sasG (S. aureus surface protein G), aap (S. epidermidis accumulation associated protein), bhp (Bap homologue protein) and fbe (fibrinogen binding protein epidermidis). In this study, 106 methicillin-resistant S. aureus (MRSA), 145 methicillin-resistant S. epidermidis (MRSE) and 70 non-epidermidis methicillin-resistant CNS (MR-CNS; 58 S. haemolyticus, 10 S. hominis and two S. lugdunensis) were compared in terms of biofilm formation, antimicrobial resistance, clonal distribution and adhesin genes carriage. Isolates were classified into pulsotypes by PFGE and assigned to sequence types by MLST. In total, 121/321 isolates (37.7%) produced biofilm and 219 (68.2%) carried ica operon. The majority was multidrug resistant (94.7%) and carried one or more adhesin genes. MRSE and all other MR-CNS prevailed in biofilm formation (P &lt; 0.001) and antimicrobial resistance (P &lt; 0.05) as compared to MRSA. MRSE also prevailed in ica carriage compared to the other methicillin-resistant staphylococci (P ≤ 0.007) Among MRSE, isolates from bacteraemias prevailed in biofilm formation (P = 0.031), whereas, strains from prosthetic device-associated infections carried more frequently aap (P = 0.003). Even though PFGE showed genetic diversity among MRSE, MLST revealed three major clones (ST2, ST5, ST16). MRSA isolates were less diverse, with five PFGE types and, among them, one major PFGE type (C) consisting of 77/106 strains (72.6%). MLST identified five sequence types: ST5, ST30, ST80, ST225 and ST239. One major PFGE type (h) was identified in S. haemolyticus. A clonal relationship was found concerning fnbA carriage in MRSA, ica carriage in MRSE, and antimicrobial susceptibility in both groups reinforcing the aspect of clonal expansion in hospital settings

Spread of Tst–Positive Staphylococcus aureus Strains Belonging to ST30 Clone among Patients and Healthcare Workers in Two Intensive Care Units

Staphylococcus aureus is a major cause of infections. Toxic shock syndrome toxin (TSST-1) and Panton–Valentine leukocidin (PVL) are associated with severe clinical syndromes. S. aureus colonizing isolates recovered from healthcare workers and patients in the intensive care unit (ICU) of a university hospital comprising Group A were compared with those from adult non-ICU carriers (Group B). mecA, lukS/lukF-PV (Panton–Valentine leukocidin, PVL), and tst (toxic shock syndrome toxin) gene carriage was detected by PCR. Clones were identified in all methicillin-resistant S. aureus (MRSA) and toxin-positive methicillin-susceptible strains (MSSA) by pulsed-field gel electrophoresis (PFGE), agr groups, and multi locus sequencing typing (MLST). Group A included 90 S. aureus isolates, whereas Group B 53. PVL was more frequently found among MRSA vs. MSSA (p &lt; 0.001) and in strains of Group B as compared to Group A (p &lt; 0.001), consistent with the spread of ST80-IV. Higher incidence of tst gene carriage was identified among MSSA vs. MRSA (P 0.005) belonging mainly to ST30, and Group A vs. Group B (P 0.002). The wide dissemination of ST80-IV mainly in the community is responsible for a high percentage of PVL-positive MRSA, while silent spread of tst-positive S. aureus clones among ICU patients and personnel poses a threat of hospital transmission and possible severe infections

Coagulase-negative staphylococcal bloodstream and prosthetic-device-associated infections: the role of biofilm formation and distribution of adhesin and toxin genes

Coagulase-negative staphylococci (CNS), especially Staphylococcus epidermidis and Staphylococcus haemolyticus, have emerged as opportunistic pathogens in immunocompromised patients and those with indwelling medical devices. In this study, CNS recovered from patients with bloodstream infections (BSIs) or prosthetic-device-associated infections (PDAls) were compared in terms of biofilm formation, antimicrobial resistance, clonal distribution, and carriage of adhesin and toxin genes. A total of 226 CNS isolates (168 S. epidermidis and 58 S. haemolyticus) recovered from hospital inpatients with BSIs (100 isolates) or PDAls (126 isolates) were tested for biofilm formation, antimicrobial susceptibility, and mecA, ica operon, adhesin (aap, bap, fnbA, atlE, fbe) and toxin (tst, sea, sec) genes. The selected CNS were classified into pulsotypes by PFGE and assigned to sequence types by multilocus sequence typing. In total, 106/226 isolates (46.9%) produced biofilm, whereas 150 (66.4 %) carried the ica operon. Most isolates carried mecA and were multidrug resistant (90.7 %). CNS recovered from BSIs were significantly more likely to produce biofilm (P=0.003), be resistant to antimicrobials and carry mecA (P0.05). Although PFGE revealed genetic diversity, especially among S. epidermidis, analysis of representative strains from the main PFGE types by multilocus sequence typing revealed three major clones (ST2, ST5 and ST16). A clonal relationship was found with respect to antimicrobial susceptibility and ica and aap gene carriage, reinforcing the premise of clonal expansion in hospital settings. The results of this study suggest that the pathogenesis of BSIs is associated with biofilm formation and high-level antimicrobial resistance, whereas PDAls are related to the adhesion capabilities of S. epidermidis and S. haemolyticus strains