M. Miragaia Laboratory of Molecular Genetics Instituto de Tecnologia Química e Biológica July 6, 2010

 Part of the human microflora  Primary ecological niche: anterior nares  Opportunistic bacteria  Wide range of infections: mild to life-threatening (skin and wound infections, food poisoning, endocardititis, osteomyelitis, pneumonia, brain abscesses, meningitis, bacteremia,)

Post-antibiotic era MRSA burden Penicillin resistance Penicillin introduction Penicillin introduction Methicillin introduction MRSA-I MRSA-II & III MRSA-IV VISA CA-MRSA VRSA Phage type 80/81 Phage type III/83A, PST Archaic clone

 The first CA-MRSA infection occurred in indigenous in Australia in early 1990s  The first CA-MRSA in USA: four death of children in Minnesota,

“New Bacteria Strain Is Striking Gay Men” Janeiro 2008 “Staph at the Gym? Not if You’re Careful” Novembro 2007 “Health Officials Try to Calm Parents About Staph” Outubro 2007 “Schools in Several States Report Staph Infections, and Deaths Raise the Alarm” Outubro 2007

 Neutrophil lysis after phagocytosis of USA300  Destruction of the human neutrophil at 6 h and survival of S. aureus  Production of higher amounts of PVL and PSMs Koboyashi et al., 2009

 High capability of dissemination (skin-to-skin contact) - ACME : survival at low pH  Skin and soft tissue infections  Fatal sepsis  Necrotizing pneumonia  Necrotizing fasciitis

 Skin trauma  Injection drug use  Poor personal hygiene  Populations at risk: closed healthy populations (children in DCC, prison inmates, homosexuals, athletes, military personnel, emergency departments)

1.Amplification by PCR of internal fragments of 7 housekeeping genes/ 1 virulence gene S. aureus genome arcCaroEglpFgmkptatpiyqiL 2. Sequencing of amplified fragments MLST spa typing

SmaI 5’ CCCGGG 3’ 3’ GGGCCC 5’ ~ 50 ~ 100 ~ 150 ~ 200 ~ 250 kb ~ 300 ~ 350 ~ 400

Type II Type III Type IV Type V Type I Type VI J3J2J1 pT181 tet Ψ-Tn554 ccrA3ccrB3 mec complex ccrA2ccrB2 mec complex B ccrChsdR/M/S mec complex C mec complex A Tn554 ccrA2ccrB2mecAmecR1mecI pUB110 bleaadBermspc mec complex A ccr complex orfX mecAmecR1Ψ-IS1272ccrA1ccrB1 mec complex B IS431 Tn554 mecAmecR1IS431 ccrA4ccrB4 mec complex B pI258 mer Milheirico et al IIIIIIIVVVI SCCmec typing

Portugal ? Portugal ? European cloneClone SouthWesth Pacific Clone USA400 Clone USA300Clone USA1000ST398 Europe 4-13% USA 50-75% USA 50-75% Australia 10% Australia 10%

CA-MRSA cloneNo of isolates USA3006 Multiresistants SW Pacific4 USA4002 USA7001 Sporadic MSSA/MRSA48 HA-MSSA/MRSA17 CA-MRSA/CA-S. aureus = 17% Population studied: isolates collected from patients hospitalized less than 48h and with no previous hospital contact Hospitals involved: 15 different Portuguese hospitals Typing methods: MLST, spa typing, PFGE, SCCmec typing

Hospital Community Veterinary ST398, PVL – Pigs col. 39% Farmers col. 20% Veterinary col.11% ST398 SCCmec IV, V Tet R CA-MRSA multi-resistant PVL +, ACME, PSM, α-toxin outbreaks/Infection SSTI PVL +, ACME, PSM, α-toxin

ITQB Hermínia de Lencastre Rosário Mato Ana Tavares Joana Rolo Ons Bouchami Collaborators Centro Hospitalar Lisboa Norte, Centro Hospitalar Lisboa Central, Centro Hospitalar Lisboa Ocidental, Centro Hospitalar do Barlavento Algarvio, Hospital do Espírito Santo-Évora, Hospital de São João do Porto, IPO Porto, Hospital de São Marcos-Braga, Hospital Pedro Hispano, Hospital da Luz, Hospital da Força Aérea, Hospital do SAMS, Clínica laboratorial Edgar Botelho Moniz, Centro de Saúde de Oeiras, Hospital de Cascais (HPP), Hospitais da Universidade de Coimbra, Hospital Fernando da Fonseca. Funding Fundação Calouste Gulbenkian (Ref. P-99911) European Union (Ref. CONCORD–HEALTH-F )