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The spread of methicillin-resistant Staphylococcus aureus (MRSA) in the community, hospitals and in livestock is mediated by highly diverse virulence factors that include secreted toxins, superantigens, enzymes and surface-associated adhesins allowing host adaptation and colonization. Here, we combined proteogenomics, secretome and phenotype analyses to compare the secreted virulence factors in selected S. aureus isolates of the dominant human- and livestock-associated genetic lineages CC8, CC22, and CC398. The proteogenomic comparison revealed 2181 core genes and 1306 accessory genes in 18 S. aureus isolates reflecting the high genome diversity. Using secretome analysis, we identified 869 secreted proteins with 538 commons in eight isolates of CC8, CC22, and CC398. These include 64 predicted extracellular and 37 cell surface proteins that account for 82.4% of total secretome abundance. Among the top 10 most abundantly secreted virulence factors are the major autolysins (Atl, IsaA, Sle1, SAUPAN006375000), lipases and lipoteichoic acid hydrolases (Lip, Geh, LtaS), cytolytic toxins (Hla, Hlb, PSMβ1) and proteases (SspB). The CC398 isolates showed lower secretion of cell wall proteins, but higher secretion of α- and β-hemolysins (Hla, Hlb) which correlated with an increased Agr activity and strong hemolysis. CC398 strains were further characterized by lower biofilm formation and staphyloxanthin levels because of decreased SigB activity. Overall, comparative secretome analyses revealed CC8- or CC22-specific enterotoxin and Spl protease secretion as well as Agr- and SigB-controlled differences in exotoxin and surface protein secretion between human-specific and zoonotic lineages of S. aureus.

The evolution and global transmission of antimicrobial resistance have been well documented for Gram-negative bacteria and health care-associated epidemic pathogens, often emerging from regions with heavy antimicrobial use. However, the degree to which similar processes occur with Gram-positive bacteria in the community setting is less well understood. In this study, we traced the recent origins and global spread of a multidrug-resistant, community-associated Staphylococcus aureus lineage from the Indian subcontinent, the Bengal Bay clone (ST772). We generated whole-genome sequence data of 340 isolates from 14 countries, including the first isolates from Bangladesh and India, to reconstruct the evolutionary history and genomic epidemiology of the lineage. Our data show that the clone emerged on the Indian subcontinent in the early 1960s and disseminated rapidly in the 1990s. Short-term outbreaks in community and health care settings occurred following intercontinental transmission, typically associated with travel and family contacts on the subcontinent, but ongoing endemic transmission was uncommon. Acquisition of a multidrug resistance integrated plasmid was instrumental in the emergence of a single dominant and globally disseminated clade in the early 1990s. Phenotypic data on biofilm, growth, and toxicity point to antimicrobial resistance as the driving force in the evolution of ST772. The Bengal Bay clone therefore combines the multidrug resistance of traditional health care-associated clones with the epidemiological transmission of community-associated methicillin-resistant S. aureus (MRSA). Our study demonstrates the importance of whole-genome sequencing for tracking the evolution of emerging and resistant pathogens. It provides a critical framework for ongoing surveillance of the clone on the Indian subcontinent and elsewhere.IMPORTANCE The Bengal Bay clone (ST772) is a community-associated and multidrug-resistant Staphylococcus aureus lineage first isolated from Bangladesh and India in 2004. In this study, we showed that the Bengal Bay clone emerged from a virulent progenitor circulating on the Indian subcontinent. Its subsequent global transmission was associated with travel or family contact in the region. ST772 progressively acquired specific resistance elements at limited cost to its fitness and continues to be exported globally, resulting in small-scale community and health care outbreaks. The Bengal Bay clone therefore combines the virulence potential and epidemiology of community-associated clones with the multidrug resistance of health care-associated S. aureus lineages. This study demonstrates the importance of whole-genome sequencing for the surveillance of highly antibiotic-resistant pathogens, which may emerge in the community setting of regions with poor antibiotic stewardship and rapidly spread into hospitals and communities across the world.

The evolution and global transmission of antimicrobial resistance have been well documented for Gram-negative bacteria and health care-associated epidemic pathogens, often emerging from regions with heavy antimicrobial use. However, the degree to which similar processes occur with Gram-positive bacteria in the community setting is less well understood. In this study, we traced the recent origins and global spread of a multidrug-resistant, community-associated Staphylococcus aureus lineage from the Indian subcontinent, the Bengal Bay clone (ST772). We generated whole-genome sequence data of 340 isolates from 14 countries, including the first isolates from Bangladesh and India, to reconstruct the evolutionary history and genomic epidemiology of the lineage. Our data show that the clone emerged on the Indian subcontinent in the early 1960s and disseminated rapidly in the 1990s. Short-term outbreaks in community and health care settings occurred following intercontinental transmission, typically associated with travel and family contacts on the subcontinent, but ongoing endemic transmission was uncommon. Acquisition of a multidrug resistance integrated plasmid was instrumental in the emergence of a single dominant and globally disseminated clade in the early 1990s. Phenotypic data on biofilm, growth, and toxicity point to antimicrobial resistance as the driving force in the evolution of ST772. The Bengal Bay clone therefore combines the multidrug resistance of traditional health care-associated clones with the epidemiological transmission of community-associated methicillin-resistant S. aureus (MRSA). Our study demonstrates the importance of whole-genome sequencing for tracking the evolution of emerging and resistant pathogens. It provides a critical framework for ongoing surveillance of the clone on the Indian subcontinent and elsewhere.IMPORTANCE The Bengal Bay clone (ST772) is a community-associated and multidrug-resistant Staphylococcus aureus lineage first isolated from Bangladesh and India in 2004. In this study, we showed that the Bengal Bay clone emerged from a virulent progenitor circulating on the Indian subcontinent. Its subsequent global transmission was associated with travel or family contact in the region. ST772 progressively acquired specific resistance elements at limited cost to its fitness and continues to be exported globally, resulting in small-scale community and health care outbreaks. The Bengal Bay clone therefore combines the virulence potential and epidemiology of community-associated clones with the multidrug resistance of health care-associated S. aureus lineages. This study demonstrates the importance of whole-genome sequencing for the surveillance of highly antibiotic-resistant pathogens, which may emerge in the community setting of regions with poor antibiotic stewardship and rapidly spread into hospitals and communities across the world.

Zur Untersuchung alternativer genetischer Determinanten, die eine β-Laktam-Resistenz verursachen, wurden vergleichende, genomische Analysen sowie genomweite Assoziationsstudien (GWAS) verwendet, um genetische Polymorphismen zu ermitteln, die mit dem MRLM (Methicillin-resistent, lacking mec) -Phänotyp in klinischen S. aureus Stämmen in Verbindung stehen. Zu diesem Zweck wurde eine repräsentative Stammsammlung aus 141 MRLM Isolaten und 142 genetisch eng verwandten, Methicillin-empfindlichen Kontrollstämmen zusammengestellt. Die Presence/Absence-basierte SCOARY GWAS Analyse konnte Mutationen im Phosphodiesterase-Gen gdpP signifikant mit dem MRLM-Phänotyp assoziieren. 131 MRLM Isolate (92,9 %) trugen gdpP-Mutationen, die zu Trunkierungen, Insertionen, Deletionen und Aminosäuresubstitutionen führten. Darüber hinaus bestätigte eine Gen-Enrichment Analyse, dass das gdpP Gen in MRLM-Stämmen im Durchschnitt 4,6-mal häufiger mutiert war als in MSSA Kontrollen. Die Mutationen in gdpP hatten keine Auswirkungen auf die Transkriptionslevel von pbp4, es konnten jedoch verringerte Zellgrößen bei MRLM Isolaten nachgewiesen werden. Vergleichende SNP-Analysen der nativen pbp1-4-Gene zeigten außerdem 57 verschiedene Aminosäuresubstitutionen in 44 Isolaten (31,2 %), die ausschließlich in MRLM-Stämmen vorkamen. Bei zwei weiteren Isolaten wurden Deletionen im pbp4-Promotor festgestellt, die bei diesen Isolaten zu einer 15-fachen bzw. 7,5-fachen Erhöhung der Transkription von pbp4 führten. Im zweiten Teil der Arbeit wurde ein Ausbruchsszenario mit MRLM Isolaten analysiert. Es wurden insgesamt neun S. aureus-Stämme (MRLM (n=6); MSSA (n=3)) untersucht, die bei vier Frühgeborenen während eines routinemäßigen Nasenscreenings auf der neonatalen Intensivstation (NICU) eines Krankenhauses in Nordrhein-Westfalen isoliert wurden. Alle Isolate wurden dem spa-Typen t3338, dem Multi-Locus-Sequenz-Typen (MLST) ST7 und dem core-Genom MLST (cgMLST) Komplex-Typen CT20916 zugeordnet. Sie unterschieden sich voneinander in maximal 8 der 2249 genomischen Loci, die in die cgMLST-Analyse einbezogen wurden, was auf einen gemeinsamen Ursprung der Isolate hinweist. Sechs Isolate zeigten erhöhte MHKs für Oxacillin (MHK ≥ 2 mg/L) und Cefoxitin (MHK > 4 mg/L), wurden jedoch in der PCR negativ auf mec-Determinanten getestet. Fünf der MRLM Isolate hatten unterschiedliche Mutationen in gdpP, was darauf hindeutet, dass sich diese Polymorphismen bei einzelnen Patienten unabhängig voneinander entwickelt hatten und dass das ursprüngliche Indexisolat wahrscheinlich ein MSSA war. Dieses MSSA Isolat scheint in jedem Patienten unabhängig voneinander unter dem Einfluss eines bisher unbekannten Selektionsdrucks durch verschiedene Mutationen in gdpP eine Resistenz gegen β-Laktame erworben zu haben. Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass Mutationen in gdpP einen klinisch relevanten Mechanismus für β-Laktam-Resistenz in MRLM Isolaten darstellen. Diese Beobachtung ist von besonderer klinischer Relevanz, da MRLM leicht als MSSA fehlklassifiziert werden, was zu einer unbemerkten Ausbreitung von β-Laktam-resistenten Isolaten und anschließendem Therapieversagen führen kann., To investigate alternative genetic determinants causing β-lactam resistance, comparative genomic analyses and genome-wide association studies (GWAS) were used to identify genetic polymorphisms associated with the MRLM (methicillin-resistant, lacking mec) -phenotype in clinical S. aureus strains. To this end, a representative strain collection was assembled from 141 MRLM isolates and 142 genetically closely related, methicillin-susceptible control strains. The presence/absence-based SCOARY GWAS analysis was able to significantly associate mutations in the phosphodiesterase gene gdpP with the MRLM phenotype. 131 MRLM isolates (92.9 %) carried gdpP mutations resulting in truncations, insertions, deletions, and amino acid substitutions. In addition, a gene-enrichment analysis confirmed that the gdpP gene was mutated on average 4.6-fold more frequently in MRLM strains than in MSSA controls. Mutations in gdpP did not affect pbp4 transcript levels, but decreased cell sizes were detected in MRLM isolates. Comparative SNP analysis of the native pbp1-4 genes also revealed 57 different amino acid substitutions in 44 isolates (31.2 %), that were exclusive to MRLM strains. Deletions in the pbp4 promoter were detected in two other isolates, resulting in a 15-fold and 7.5-fold increase in transcription of pbp4 in these isolates, respectively. In the second part of the work, an outbreak scenario with MRLM isolates was analyzed. A total of nine S. aureus strains (MRLM (n=6); MSSA (n=3)) isolated from four preterm infants during routine nasal screening in the neonatal intensive care unit (NICU) of a hospital in North Rhine-Westphalia were studied. All isolates were assigned to spa type t3338, multi-locus sequence type (MLST) ST7, and core-genome MLST (cgMLST) complex type CT20916. They differed from each other in a maximum of 8 of the 2249 genomic loci included in the cgMLST analysis, indicating a common origin of the isolates. Six isolates showed elevated MICs for oxacillin (MIC ≥ 2 mg/L) and cefoxitin (MIC > 4 mg/L) but were tested negative for mec determinants by PCR. Five of the MRLM isolates showed different mutations in gdpP, suggesting that these polymorphisms had developed independently in the individual patients and that the original index isolate was probably an MSSA. This MSSA isolate appears to have acquired resistance to β-lactams in each patient independently under the influence of a previously unknown selection pressure by different mutations in gdpP. In summary, this work demonstrated that mutations in gdpP represent a clinically relevant mechanism for β-lactam resistance in MRLM isolates. This observation is of particular clinical relevance, as MRLM are easily misclassified as MSSA, which can lead to unnoticed spread of β-lactam-resistant isolates and subsequent treatment failure.

Hastanelerde ve toplum içinde, metisiline dirençli Staphylococcus aureus (MRSA) epidemik kökenlerinin prevalansındaki artış, enfeksiyon kontrol önlemleri açısından büyük önem taşımaktadır. Bu çalışmanın amacı, Türkiye’nin Orta Karadeniz bölgesindeki klinik MRSA izolatlarının, kısa ve uzun dönemli epidemiyolojik özelliklerinin anlaşılabilmesi için, patojenik fenotip (enfeksiyözite, resiztotip) ve genotip (PFGE pulsotipi, SLST spa tipi, MLST dizi tipi, eBURST klonal kompleks tipi) özelliklerinin belirlenmesi ve olası bir kümelenme varlığının araştırılmasıdır. Çalışmaya, prospektif olarak, Aralık 2006-Şubat 2007 tarihleri arasında hastanede yatan hastalardan izole edilen, ardışık ve tekrarlardan arındırılmış, 48 MRSA izolatı (18’i kolonizasyon, 30’u hastane enfeksiyonu etkeni) ile tümü hastane enfeksiyonu etkeni olarak tanımlanan yedi metisiline duyarlı S.aureus (MSSA) izolatı alınmıştır. İzolatların tanımlaması Vitek-2 otomatize sistemi (BioMérieux, ABD) ile, in vitro antimikrobiyal duyarlılıkları testleri ise sıvı mikrodilüsyon yöntemi ve Vitek-2 otomatize sistemiyle yapılmıştır. Eritromisine duyarlı bulunan MRSA izolatlarında (n 10), PCR yöntemi ile ermA geni araştırılmıştır. Tüm izolatlar, spa tiplendirme ve PFGE pulsotiplendirme yöntemleriyle tiplendirilmiştir. Farklı spa tiplerini temsilen seçilen üç MRSA izolatı ile tüm (n 7) MSSA izolatlarına MLST yöntemiyle tiplendirme yapılmıştır. Farklı spa tiplerine ait izolatlar içinde, antibiyotik a duyarlılık paternleri birbirinden farklı olan MRSA izolatları arasından seçilen temsilcilerin (n 8) SCCmec tipleri, multipleks PCR yöntemiyle belirlenmiştir. İzolatların, antimikrobiyal direnç paternleri sayısallaştı- rılarak, standardize antimikrobiyal direnç fenotipleri belirlenmiştir. MRSA izolatlarının, ayırt edici özel- liklerinden olan enfeksiyözite, fenotipik ve genotipik özellikleri temelinde oluşan patern gruplarındaki kümelenmeleri, belirli içerme ve dışlama ölçütleri ile istatistiksel analize alınmış ve anlamlılık bakımından incelenmiştir. Çalışmamızda, MSSA izolatlarında üç, MRSA izolatlarında 13 farklı antimikrobiyal direnç fenotipi belirlenmiş ve MRSA izolatlarında 14, MSSA izolatlarında yedi farklı PFGE pulsotipi saptanmıştır. MRSA izolatlarının 10’u, PFGE-pulsotipi ile sporadik tekil kökenler olarak saptanmıştır. MRSA izolatlarının, ikisi (t459, t632) hariç, diğerlerinin t030 spa tipinde olduğu belirlenmiş; farklı spa tipi temsilcilerinin (n 3) analizi sonucunda, tek MLST klonal kompleks tipi (CC8) ve tek MLST dizi tipi (ST239) bulunmuştur. Yedi MSSA izolatının ise her birisinin spa tipi, MLST dizi tipi ve MLST klonal kompleks tipinin farklı olduğu izlenmiştir (t777-ST5-CC5; t660-ST25-CC5; t153-ST34-CC30; t015-ST45-CC45; t267-ST97-CC97; t377- ST360-CC8; t084-ST15-C15). Sporadik olmayan 38 MRSA izolatının istatistiksel analizinde, Grup-13’te yer alan izolatların (n 16; enfeksiyöz, rezistotip 14, pulsotip 4; antimikrobiyal direnç skoru 24), anlamlı düzeyde enfeksiyözite-fenotip-genotip kümelenmesi sergilediği saptanmıştır (p 0.001). MRSA izolat- larının 27‘sinde azalmış teikoplanin duyarlılığı (MİK 4 ?g/mL) saptanmıştır. Global olarak, MLST-CC8, MLST-ST239, t030 spa tipi MRSA izolatlarının, genellikle eritromisine dirençli olması beklenmesine karşın, duyarlı olan 10 izolatın altısında ermA geninin bulunduğu gösterilmiş; bu genlerin nokta mutasyonu veya delesyon nedeniyle işlevsiz olabileceği düşünülmüştür. Antimikrobiyal duyarlılık paternleri birbirinden farklı olan MRSA izolatları arasından seçilen sekiz temsilci suşun SCCmec tipleri, tip III olarak saptanmıştır. Sonuç olarak, Orta Karadeniz bölgesi hasta popülasyonunu temsil eden hastanemizde saptanan S.aureus izolatlarının, enfeksiyözite, antimikrobiyal direnç fenotipi ve klonal genotip bakımından, istatistiksel ola- rak anlamlı kümelenme sergilediği ortaya konmuştur (p 0.001). Baskın olan MRSA klonunun, beş büyük pandemik MRSA klonundan biri olan, epidemik ST239 klonu olduğu ve nozokomiyal MSSA izolatlarının uzun dönemli klonal çeşitlilik sergilediği belirlenmiştir. Bu çalışmanın sonuçları, hastane enfeksiyonlarına yol açan çeşitli S.aureus kökenlerinin, bölgesel, ulusal ve uluslararası uzun dönemli sürveyans çalışmaları için veri sağlamaktadır. The increase in the prevalence of epidemic strains of methicillin resistant Staphylococcus aureus (MRSA) in hospitals and community requires special attention of infection control. The aim of this study was to determine the pathogenic phenotype (i.e. infectivity and resistotype) and genotypic character- istics (i.e. PFGE-pulsotyping, SLST-spa typing, MLST-sequence typing, eBURST-clonal complex detection algorithm) of clinical MRSA isolates in the Central Blacksea region of Turkey, in order to understand their short- and long-term epidemiological and evolutionary dynamics, and to investigate any probable presence of a significant clustering. This prospective study included consecutive but non-repetitive 48 MRSA isolates (of them 18 were colonized strains and 30 were causes of nosocomial infection) and seven methicillin-susceptible S.aureus (MSSA, all were isolated from nosocomial infection), collected between December 2006-February 2007 period from hospitalized patients. Identification of the isolates were per- formed by Vitek-2 automated system (BioMérieux, USA), and in vitro antimicrobial susceptibility testing by broth microdilution method and Vitek-2 automated system. The MRSA isolates found susceptible to erythromycin (n 10) were further investigated for the presence of ermA gene by the PCR method. All the strains were typed by spa-typing and PFGE-pulsotyping methods. Among the isolates with differ- ent spa-types, representatives were selected (3 MRSA, 7 MSSA) and typed with MLST typing method. Among the isolates with different spa-types, representatives with different antimicrobial susceptibility patterns were selected (n 8), and SCCmec types were determined by the multiplex PCR method. Antimicrobial resistance patterns of the isolates were digitized to get standardized antimicrobial resist- ance phenotypes. Clustering of MRSA isolates in pattern groups on the basis of discriminatory character- istics, namely infectivity, phenotype and genotype were statistically analyzed with specific inclusion and exclusion criteria. As a result, three different antimicrobial resistance phenotypes were found in MSSA isolates, whereas 13 were identified in MRSA isolates. In MSSA isolates, seven different PFGE-pulsotypes were detected, as compared to 14 pulsotypes in MRSA isolates. Among MRSA isolates, 10 sporadic strains with single PFGE-pulsotypes were detected. All MRSA isolates, with two exceptions (t459, t632), were of t030 spa-type; in the MLST analysis of the representatives of different spa-types (n 3), a single type of MLST-clonal complex (CC8) and single MLST-sequence type (ST239) were identified. Each of the seven MSSA isolates yielded different spa-types, MLST-clonal complex types and MLST-sequence types (t777-ST5-CC5; t660-ST25-CC5; t153-ST34-CC30; t015-ST45-CC45; t267-ST97-CC97; t377- ST360-CC8; t084-ST15-C15). In the statistical analysis of 38 non-sporadic MRSA isolates, the isolates in Group-13 (n 16; infectious, resistotype 14, pulsotype 4; antimicrobial resistance score 24) displayed significant infectivity-phenotype-genotype clustering (p< 0.001). In 27 of the MRSA isolates, decreased susceptibility to teicoplanin (MIC 4 μg/mL) was detected. Although, global MRSA isolates belonging to MLST-CC8, MLST-ST239, t030 spa-type were usually expected to be resistant to erythromycin, 10 such strains were erythromycin susceptible. However, ermA gene was found in six of these 10 strains, leading to a conclusion that the ermA gene of these isolates might be dysfunctional due to a point mutation or deletion. Selected representatives of MRSA isolates with different antimicrobial susceptibility patterns (n 8) were detected to be SCCmec type III. In conclusion, S.aureus isolates in the patient population of our hospital representing the Central Blacksea region showed statistically significant clustering in infectivity, antimicrobial resistance phenotype and clonal genotype (p< 0.001). The dominant MRSA clone was ST239 which was one of the five major pandemic MRSA clones. Nosocomial MSSA isolates displayed long-term clonal diversity. This study produced regional evolutionary-epidemiological data that may support further regional, national and international long-term surveillance studies of S.aureus strains.