A genomic approach to explore the development and evolution of methicillin-resistant staphylococci

Transcription

1 A genomic approach to explore the development and evolution of methicillin-resistant staphylococci Neeltje Carpaij

2 ISBN: Lay-out and printed by: Gildeprint Drukkerijen - Enschede, the Netherlands

3 A genomic approach to explore the development and evolution of methicillin-resistant staphylococci Onderzoek naar evolutie en ontwikkeling van methicilline-resistente staphylococci door bestudering van hun genoom (met een samenvatting in het nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit van Utrecht op gezag van rector magnificus prof. dr. G.J. van der Zwaan, ingevolge het besluit van het college van promoties in het openbaar te verdedigen op dinsdag 22 november 2011 des middags om uur. door Neeltje Carpaij geboren op 20 april 1981 te Eindhoven

4 Promotor: Co-promotoren: Prof. dr. M.J.M. Bonten Dr. A.C. Fluit Dr. R.J.L. Willems

5 Table of contents Chapter 1 General introduction 7 Chapter 2 New methods to analyse microarray data that partially lack 27 a reference signal Chapter 3 Genetic variation in spatio-temporal confined USA community-associated MRSA isolates: a shift from clonal dispersion to genetic evolution? Chapter 4 The first sequence type 80 community-associated 57 methicillin-resistant Staphylococcus aureus strain in the USA Chapter 5 Comparison of the identification of coagulase-negative 73 staphylococci by matrix-assisted laser desorption ionization time-of-flight mass spectrometry and tuf sequencing Chapter 6 Shared reservoir of ccrb sequences between coagulase- 81 negative staphylococci and methicillin-resistant Staphylococcus aureus Chapter 7 General discussion 113 Chapter 8 Nederlandse samenvatting 125 Publications 133 Dankwoord 137 Curriculum vitae 143 Color figures 147

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7 1 General introduction

8 Chapter 1 8

9 The genus Staphylococcus (from the Greek: σταφυλή, staphylē, bunch of grapes and κόκκος, kókkos, granule ) belongs to the class Bacilli, order Bacillales and family Staphylococcae. Staphylococci are low GC Gram-positive bacteria that appear under the microscope as round (cocci) spheres that form grape-like clusters. More than thirty species of staphylococci have been described, and eleven commonly colonize the skin and mucous membranes of humans (1). Based on the presence of coagulase, an enzyme that clots plasma, and colony morphology, staphylococci are divided in Staphylococcus aureus (coagulase positive and yellow hemolytic colonies) and coagulase-negative staphylococci (CoNS) (grey-white colonies). Staphylococcus colonization and infections S. aureus is a common colonizer of healthy persons. About 20% of healthy individuals carry S. aureus persistently, 30% are intermittent carriers and 50% are never colonized with S. aureus. S. aureus is also a potential pathogen of humans and animals, such as poultry, pigs, horse, sheep, bovines, and pigs (2-7). In humans multiple body sites can be colonized, however, the anterior nares are the most frequently colonized sites (Figure 1) (8). The risk of developing an infection with S. aureus depends on the intrinsic virulence of the bacterium, but also on the ability of the host to control invasion of the bacteria. Furthermore, S. aureus carriership is a risk factor for acquiring S. aureus infection, ranging from local skin infections to severe systemic diseases both in hospitalized and non-hospitalized patients. Examples of infections caused by S. aureus are impetigo, abscesses, bacteraemia, endocarditis, osteomyelitis and necrotizing pneumonia (Table 1) (9). CoNS hold a more benign relationship with their host and are only considered pathogenic when natural barriers are damaged, usually due to trauma or implantation of medical devices (10;11). Yet, rates of CoNS infections in hospitals have increased in the last decade. CoNS that are commonly isolated from humans are Staphylococcus epidermidis, Staphylococcus capitis, Staphylococcus hominis, Staphylococcus haemolyticus, Staphylococcus warneri, Staphylococcus caprae, Staphylococcus saccharolyticus, Staphylococcus pasteuri, Staphylococcus saprophyticus, and Staphylococcus lugdunensis (12-14). However, some species (i.e., S. epidermidis, S. haemolyticus, S. lugdunensis, S. warneri, and Staphylococcus schleiferi) are more likely to cause severe human infections such as endocarditis (13;14) The different infections caused per species are shown in Table 1. Infections caused by CoNS other than S. epidermidis, S. saprophyitcus and S. lugdunensis are usually found in patients with underlying conditions and/or immunocompromised patients (9;15;16). General introduction 9

10 Table 1. Infections caused per different Staphylococcus species Species Infections S. aureus Skin and soft tissue infections: furuncles, carbuncles, paronychia, wound infections, cellulitis, impetigo. Intravascular: bacteraemia, endocarditis. Central nervous system: brain abscess, meningitis. Respiratory: pneumonia, otitis media, empyema, sinusitis. Bone and joints: osteomyelitis, arthritis. Toxin mediated diseases: food poisoning, toxic shock syndrome. S. epidermidis Endocarditis, cystitis, phylonephritis, meningitis, osteomyelitis, joint infections, surgical site infections, implant device infections. S. saprophyticus Urinary tract infections. S. lugdunensis Joint infections, endocarditis, myocarditis, sepsis, osteomyelitis, skin and surgical wound infections. S. haemolyticus Sepsis in neonates, septic pulmonary embolism. S. warneri Endocarditis, orthopaedic infections, meningitis, central nervous system shunt infections. S. hominis Endocarditis, pyomyositis, sacroiliitis, spondylodiscitis. S. intermedius Brain abscess, endocarditis. S. capitis Endocarditis, sepsis. S. pettenkoferi Osteomyelitis. Chapter 1 10 Figure 1. S. aureus carriage rates per body site in adults in the general population (a) and S. aureus nasal carriers (b). See page 149 for color figure

11 Antibiotic treatment of staphylococcal infections and antibiotic resistance Originally, staphylococci were susceptible to penicillin, however they became rapidly resistant in the 1940s due to the acquisition of penicillinase-encoding plasmids. Nowadays, most staphylococcal strains produce penicilinases. Therefore, penicillnase-stable antibiotics, such as methicillin, flucloxacillin and cephalosporins, are the preferred antibiotics for the treatment of infections, caused by methicillin-susceptible staphylococci. Aminoglycosides (i.e., gentamicin), in combination with these beta-lactam antibiotics and rifampicin are frequently used to treat S. arueus endocarditis. Treatment of S. aureus infections, however, is increasingly hampered by the emergence of antibiotic resistance to all beta-lactam antibiotics, which is called methicillin-resistant. The major challenge in antibiotic treatment of staphylococci is methicillin resistance. Methicillin-resistant S. aureus (MRSA) strains are of particular clinical importance because they are a leading cause of health care-associated (HA) infections world-wide, and have also emerged as a major cause of community-associated (CA) infections (17-22). The first MRSA strains were found in 1962, soon after methicillin was introduced, but infections caused by MRSA occurred only sporadically. However, in the late 1970s MRSA emerged as a major pathogen of hospital infections worldwide. Nowadays, up to 30-50% of invasive hospital-acquired S. aureus isolates in the USA and many European countries are resistant to methicillin. Exceptions to these high rates are the Netherlands and Scandinavian countries in which <3% of S. aureus bloodstream infections are methicillin resistant. In the USA, MRSA account for an estimated 19,000 hospital deaths per year, which is comparable to the number of annual deaths caused by HIV/AIDS, viral hepatitis and tuberculosis. The mortality correlated with MRSA infections is higher than MSSA infections. This might be due to the necessary use of less effective drugs, such as vancomycin and teicoplanin, to treat the MRSA infections or to the fact that patients with MRSA related infections are more ill than patients with MSSA infections (23;24). Vancomycin and teicoplanin are the first choice antibiotics for MRSA and MR-CoNS infections. Although these antibiotics are increasingly used in clinical practice since the 1980s, resistance to these glycopeptides in staphylococci remains rare. Vancomycin-resistant and vancomycin-intermediate S. aureus (VRSA and VISA, respectively) have been described in the last decade, but numbers of reported infections has remained extremely low (25-27). General introduction 11 Methicillin-resistant staphylococci Methicillin resistance is caused by the acquisition of penicillin-binding protein 2a (PBP2a), with low affinity for beta-lactam antibiotics, rendering such bacteria resistant to almost all beta-lactam antibiotics. The gene encoding PBP2a, meca, is located on a genomic element called SCCmec (staphylococcal cassette chromosome mec) that is integrated into the staphylococcal chromosome at a specific site, attbscc, located downstream of an open

12 reading frame of unknown function, orfx (28;29). SCCmec elements are highly diverse in their structural organization and genetic content and have been classified into types and subtypes. SCCmec elements have two characteristic components: the meca gene complex, which is responsible for methicillin resistance and the ccr genes, involved in mobilization/ integration (Figure 2) (29;30). Besides these two characteristic parts, the SCCmec element also contains three highly variable regions, so called J-regions (joining regions). J1 is the region between the right chromosomal junction and the ccr complex, J2 between the ccr gene complex and the mec gene complex, and J3 between the mec complex and the left chromosomal junction (Figure 2). Chapter 1 12 Figure 2. Genetic structure of the first eight SCCmec types. The ccr complex is indicated in blue and the mec gene complex in red. See page 150 for color figure

13 The mec gene complex is composed of meca, its regulatory genes and associated insertion sequences. There are five different common mec class gene complexes (A, B, C, D and E). The differences between these complexes are shown in Table 2 (29;30, org/pages/scc_typesen.html). Table 2. Different mec gene complexes described so far Class mec gene complex A IS431-mecA-mecR1-mecI B IS431-mecA- mecr1-meci-is1272 C IS431-mecA- mecr1-meci-is431 D IS431-mecA- mecr1 E blaz-mecalga251-mecr1lga251-mecilga251 The ccr genes encode site-specific recombinases of the resolvase family, which mediate excision and integration of the element into the bacterial chromosome and are thus responsible for the mobilization of SCCmec (28;29;31;32). At present, three phylogenetically distinct ccr genes, ccra, ccrb, and ccrc, are classified in methicillin-resistant S. aureus isolates. These three genes have DNA sequence similarities below 50% with each other. So far, five different allotypes of ccra and ccrb have been classified. In general, ccr genes with nucleotide identities of more than 85% are assigned to the same allotype, whereas ccr genes that belong to different allotypes show nucleotide identities between 60% and 82%. All ccrc variants identified to date have >87% similarity; indicating the presence of only one ccrc allotype. CcrB alone can mediate excision, but ccrb and ccra are both necessary to promote excision at the specific SCCmec place, the orfx att site. The combination of ccrb and ccra gene of each SCCmec type can excise the SCCmec from any of the others, despite the considerable variation between the different ccr types (33). In contrast, ccrc can mediate both excision and integration and does not need the activity of a second recombinase, however it can only excise the homologous SCCmec type. SCCmec elements are categorized by the combination of the ccr gene allotype and the class of the mec gene complex (Table 3) (30, html). SCCmec types are further subtypes by differences in the J regions, but with the same mec-ccr complex which for the first eight (of the in total eleven) SCCmec types is shown in Figure 2 (30). Because of the high similarity (over 99%) of meca, it is assumed that all meca variants in staphylococci descended from one common ancestor (34). Since the meca gene is more prevalent among CoNS than among S. aureus strains, CoNS could be a potential reservoir of meca for S. aureus (29). There are two reported events of presumed in vivo transfers of SCCmec from CoNS to S. aureus. In one event a type V SCCmec was transferred from methicillin-resistant S. haemolyticus to S. aureus in a neonatal intensive care unit, and in the General introduction 13

14 other event a SCCmec type IV was transferred from a S. epidermidis to S. aureus Furthermore, CoNS and S. aureus often occupy the same niche, providing ample opportunities for genetic exchange (35-37). Table 3. SCCmec types identified in S. aureus SCCmec type ccr gene complex a mec gene complex I 1 (A1B1) B II 2 (A2B2) A III 3 (A3B3) A IV 2 (A2B2) B V 5 (C) C2 VI 4 (A4B4) B VII 5 (C) C1 VIII 4 (A4B4) b A IX 1 (A1B1) C2 X 7 (A1B6) C1 XI 8 (A1B3) E a ccr genes in the gene complex are indicated in parenthesis. b ccrab4 genes found in type VIII SCCmec were nearly identical to those in the S. epidermidis SCC-VI element and showed nucleotide identities of 89.6% and 94.5% to those found in type VI SCCmec. Chapter 1 14 Identification of Staphylococcus species The increasing importance of hospital- and community-associated infections with Staphylococcus species necessitates the need for accurate and simple identification of Staphylococcus isolates at species level (13;38;39). Several identification methods have been developed to improve identification of the different species. These identification methods can be divided in phenotypic and genotypic methods. Phenotypic methods classify bacteria on the level of gene expression. These methods use phenotypic properties, such as biochemical profiles, antigens present on the cell s surface, whole protein analysis and antimicrobial susceptibility patterns. Since gene expression can vary extensively, reproducibility, and thus reliability of phenotypic typing methods is limited (40). Genotypic methods are based on DNA sequences and have been introduced and developed in the early nineteen nineties. These methods are based on differences in sequences of one particular gene, like for instance tuf, soda or rpob. At present, methods targeting the 16S rrna, hsp60, fema, rpob, gap, tuf, and soda genes are favoured for accurate bacterial as well as Staphylococcus identification, but these methods are time consuming and expensive. For that reason phenotypic methods are still used, although increasingly being replaced by DNA-based techniques in clinical laboratories (41;42).

15 Strain typing and evolution Several molecular typing methods are used to study the genetic relatedness of staphylococci and S. aureus isolates in particular, such as Pulsed-Field Gel Electrophoresis (PFGE), Multilocus Sequence Typing (MLST) and Staphylococcus protein A (spa)-typing. PFGE is based on the macro digestion by restriction endonucleases of the bacterial genomic DNA followed by separation of the DNA fragments on an agarose gel by an alternating electric field. The resulting banding pattern is used to compare isolates (43). This system has a high resolution, but comparison of patterns between different laboratories may be difficult. In MLST approximately 500bp fragments of 7 household genes (loci) dispersed throughout the genome are sequenced and compared. Each unique sequence for a locus is assigned a different number and the combination of the numbers for the seven loci is given a sequence type (ST). Closely related STs are combined in clonal complexes (CC). The sequence results can be easily compared between laboratories, using web-based databases ( MLST schemes exist for both S. aureus and S. epidermidis. The method is less discriminatory than PFGE, but more suited for studying the long term and global epidemiology, as well as population structures (44-46). Spa-typing is specific for S. aureus and involves DNA sequencing of the polymorphic X region of the S. aureus protein A gene (spa). This region consists of variable repeats which differ due to deletion and duplication of the units, as well as by point mutations within the repeats (47). Spa-typing has slightly inferior discriminatory power compared to PFGE, but is very well suited for multicentre studies as DNA sequence data quality can be controlled and checked and the results can be stored digitally in online databases that can be queried remotely (spa.ridom.de) (48). Epidemiology of hospital, community and animal associated MRSA Molecular typing methods have been essential in understanding the epidemiology of S. aureus and MRSA. MRSA have been strongly associated with nosocomial infections and during the last 45 years hospital-associated MRSA (HA-MRSA) clones have emerged worldwide. Since the late 1960s five main MLST-defined MRSA clones (CC5, CC8, CC22, CC30 and CC45) spread successfully in different regions of the world (49;50). With CC22 as an exception, because it has only been described to contain SCCmec IV, the other four clonal complexes (CC5, CC8, CC30 and CC45) contained more than one SCCmec type. The last decade, though, the epidemiology of MRSA has changed dramatically with MRSA increasingly being isolated from skin and soft tissue infections in patients without predisposing risk factors for HA-MRSA. The first reports of these so-called communityassociated MRSA (CA-MRSA) infections were from Western Australia in the early 1990s, followed by an epidemic rise of CA-MRSA infections in the USA (51). Clustered infections due to CA-MRSA were first reported from patients in remote regions, such as aboriginals General introduction 15

16 Chapter 1 16 in North-Western Australia, Native Americans in Nebraska, Wisconsin, and Alaska (52-56) Although skin and soft tissue infections are the most common manifestations, CA-MRSA have been associated with a wide range of clinical manifestations, such as fasciitis necroticans, pyomyositis, pyoderma, liver-abcess, psoas abcess, sepsis, endocarditis, prosthetic joint infections, meningitis, arthritis, pyomyositis and myositis (57-64). Nowadays, CA-MRSA outbreaks are reported from all over the world. The first widely recognized CA-MRSA clone in the USA was the Midwestern clone (USA400), named after its unique PFGE type, or ST1. After the turn of the century, USA300 (ST8) emerged and replaced USA400 as the dominant clone in the USA responsible for the majority of skin and soft tissue infections. Subsequently USA300 isolates have been increasingly found outside the USA in Central America, Europe, Australia and Asia indicating pandemic spread of this CA-MRSA clone (Figure 3). In other countries than the USA the molecular epidemiology of CA-MRSA is more diverse, which means that more different clones circulate, although certain clones appear to dominate on every continent. The predominant CA-MRSA clones per continent cluster in 6 lineages: ST1/USA400, the USA Midwest clone; ST8/USA300, the epidemic American clone; ST30/USA1100, the Southwest Pacific Oceania clone; ST93, the Queensland clone; ST59, the Asian clone; and ST80, the European clone. Although not as frequent as USA300, most of these clones have also been observed on continents other than where they were first described and now dominate (Figure 3) (19;65). CA-MRSA isolates usually carry SCCmec IV, while HA-MRSA typically harboring SCCmec type I, II or III. SCCmec IV seems to be highly promiscuous and presumably moved repeatedly into diverse lineages of MSSA (66). Recently, MRSA has also been isolated from animals, such as dogs, cats, horses, sheep, pigs, dairy cows and veal calves (67). At least some of these animal-associated MRSA are, based on PFGE, characterized as HA-MRSA, and the route of transfer was presumably from humans to animals (68-70). Vice versa, several reports have suggested that animals may serve as reservoirs for MRSA infection in humans (67;71-73). The possibility that dogs and cats could act as the source for staphylococcal infections in humans was already suggested 50 years ago (74). In 2005 MRSA (ST398:IV) was reported for the first time among pigs in France and this MRSA clone was widespread among pigs and pig farmers in The Netherlands (73;75). In addition to ST398:IV, ST398 isolates with SCCmec V have been described (76;77). ST398 MRSA isolates are now also emerging in other countries and other animals, such as horses, poultry and bovines. The MRSA ST398 is able to cause disease in both humans and animals (70;78).

17 Figure 3. Global distribution of major community-associated methicillin-resistant Staphylococcus aureus Sequence Types. See page 151 for color figure S. aureus genomics Studies applying microarrays and whole genome sequencing, have improved our understanding of in the genetic make up, and evolution and epidemic spread of S. aureus (79-88). Yet, a comparative genomics study of 61 CA S. aureus and 100 nasal carriage isolates, using a microarray of seven whole genome S. aureus sequences, revealed no correlation between the presence of specific genes or lineages and invasive disease (84;89). In April 2011 there are 22 completed S. aureus genomes. So far, whole genome sequencing has taught us that about 80% of the S. aureus genome is conserved and, apparently essential for central metabolism, growth and replication. N315 and Mu50 were the two first completely sequenced S. aureus isolates (Kuroda et al, Lancet ). Their sequences revealed that most of the antibiotic resistance genes and virulence genes reside on mobile genetic elements, such as plasmids, transposons and prophages. This suggested that genes important for the infectious lifestyle of S. aureus can be horizontally transferred. After this work, genomes of other clinical S. aureus isolates (MW2, MSSA476, MRSA252 and USA300 FPR3747) were sequenced. In doing so a new SCCmec element (SCCmec IV) was identified (90). This SCCmec element is much smaller than the SCCmec elements originally found in the HA-MRSA clones and appears not to impose a considerable fitness cost (1). In addition novel virulence determinants were identified, including the genes encoding PVL toxin and enterotoxin G and K. However, the contribution of these genes to human disease remains uncertain (82). Whole genome sequencing of USA300 revealed its relatedness to the COL strain, which is one of the first MRSA clinical isolates. The small number of difference between COL and USA300 were also quite General introduction 17

18 remarkable, because of the difference in virulence between the two strains, COL has limited virulence in mice and is not a major cause of human disease while USA300 is rapidly lethal in mice and is the leading cause of CA-MRSA infections in the USA (83;91-93). From the SNP analysis of 63 ST239 isolates a rate of core genome divergence of 1 SNP per 6 weeks was estimated. This information, in combination with the variation in non-core regions, can improve contact tracing which can lead to better interventions (81). Further delineation of this lineage based on 4310 SNPs in 63 isolates using a Bayesian framework revealed that the most recent ancestor of ST239 probably occurred shortly after the introduction of methicillin in 1958 and spread world wide for almost twenty years before the first isolate was identified (79). This analysis also revealed pandemic spread of this clone and a significant association between human migration and current HA-MRSA pandemic spread. This contrasts with the findings for clone ST5, which is associated with only limited geographical spread (85). By using mutation discovery at 269 genetic loci, Nübel concluded that clone ST225 had diverged since the late 1980s/early 1990s and that expansion of this clone began in the mid 1990s, several years before this new clone was isolated for the first time (86). The independent finding of unnoticed spread of MRSA clones (ST239 and ST225) for many years is alarming since it suggests that antibiotic-resistant clones can circulate globally undetected for a relatively long time (79). Chapter 1 18 Outline and aim of this thesis The aim of this thesis was to investigate genomic differences between CA-MRSA and HA- MRSA to provide our understanding of the factors favouring the recent emergence of CA- MRSA. Since MRSA may result from de novo acquisition of SCCmec by methicillin-susceptible isolates the potential role of methicillin-resistant CoNS as a source of SCCmec for these MRSA strains was also assessed. The first part of this thesis focuses on genomic differences between CA-MRSA and HA- MRSA and within these groups. For this purpose we compared the genetic repertoire of different CA-MRSA clones to that of HA-MRSA from the USA and Europe through comparative genomic hybridization (CGH). In chapter two new methods to analyze microarrays were investigated, in particular when a reference signal is absent for a number of probes on the microarray. These methods were used for identifying genetic differences between and within HA- and CA-MRSA isolates. Comparative genomic hybridization results from HA- and CA-MRSA isolates obtained with this microarray showed unexpected genetic differences among 14 USA300 CA-MRSA isolates. The 14 CA-MRSA isolates were recovered within one month and originated from a confined geographic area (Chicago) (chapter three). The last chapter of the first part, chapter four, describes the results of whole genome sequencing of the first ST80 CA-MRSA isolate from the USA, and its comparison to the European clone.

19 The second part focuses on the epidemiology of SCCmec in staphylococci. CoNS are thought to be the potential reservoir of the methicillin resistance determinant meca for S. aureus. The aim was to assess the potential role of CoNS as a reservoir of SCCmec elements for MRSA. First we evaluated a reliable and easy CoNS species determination method (MALDI- TOF MS), (chapter five). Oliveira et al. showed that DNA sequencing of an internal fragment of ccrb enables the characterization of the ccrab allotype present in MRSA strains and, after cluster analyses, the prediction of SCCmec types I-IV and VI. In chapter six we determined the genetic relatedness of ccrb sequences of a large, diverse, Europe-wide collection of meca-positive CoNS with ccrb sequences from MRSA isolates. General introduction 19

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22 Chapter Bloemendaal AL, Brouwer EC, Fluit AC. Methicillin resistance transfer from Staphylocccus epidermidis to methicillin-susceptible Staphylococcus aureus in a patient during antibiotic therapy. PLoS One 2010;5:e Wielders CL, Vriens MR, Brisse S, et al. In-vivo transfer of meca DNA to Staphylococcus aureus [corrected]. Lancet 2001;357: Hidron AI, Edwards JR, Patel J, et al. NHSN annual update: antimicrobial-resistant pathogens associated with healthcare-associated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, Infect Control Hosp Epidemiol 2008;29: Spanu T, De CE, Fiori B, et al. Evaluation of matrix-assisted laser desorption ionization-time-of-flight mass spectrometry in comparison to rpob gene sequencing for species identification of bloodstream infection staphylococcal isolates. Clin Microbiol Infect 2011;17: van Leeuwen WB. Molecular approaches for the epidemiological characterization of Staphylococcus aureus strains, p In A.C. Fluit and J. Schmitz (eds.), MRSA: Current Perspectives Caister Academic Press, Wymondham, Norfolk, England. 41. Deurenberg RH, Stobberingh EE. The evolution of Staphylococcus aureus. Infect Genet Evol 2008;8: Heikens E, Fleer A, Paauw A, Florijn A, Fluit AC. Comparison of genotypic and phenotypic methods for species-level identification of clinical isolates of coagulase-negative staphylococci. J Clin Microbiol 2005;43: McDougal LK, Steward CD, Killgore GE, Chaitram JM, McAllister SK, Tenover FC. Pulsed-field gel electrophoresis typing of oxacillin-resistant Staphylococcus aureus isolates from the United States: establishing a national database. J Clin Microbiol 2003;41: Maiden MC. Multilocus sequence typing of bacteria. Annu Rev Microbiol 2006; Thomas JC, Vargas MR, Miragaia M, Peacock SJ, Archer GL, Enright MC. Improved multilocus sequence typing scheme for Staphylococcus epidermidis. J Clin Microbiol 2007;45: Enright MC, Day NP, Davies CE, Peacock SJ, Spratt BG. Multilocus sequence typing for characterization of methicillin-resistant and methicillin-susceptible clones of Staphylococcus aureus. J Clin Microbiol 2000;38: Shopsin B, Gomez M, Montgomery SO, et al. Evaluation of protein A gene polymorphic region DNA sequencing for typing of Staphylococcus aureus strains. J Clin Microbiol 1999;37: Harmsen D, Claus H, Witte W, et al. Typing of methicillin-resistant Staphylococcus aureus in a university hospital setting by using novel software for spa repeat determination and database management. J Clin Microbiol 2003;41: Morgan M, Evans-Williams D, Salmon R, Hosein I, Looker DN, Howard A. The population impact of MRSA in a country: the national survey of MRSA in Wales, J Hosp Infect 2000;44: Tiemersma EW, Bronzwaer SL, Lyytikainen O, et al. Methicillin-resistant Staphylococcus aureus in Europe, Emerg Infect Dis 2004;10:

23 51. Udo EE, Pearman JW, Grubb WB. Genetic analysis of community isolates of methicillin-resistant Staphylococcus aureus in Western Australia. J Hosp Infect 1993;25: Adhikari RP, Cook GM, Lamont I, Lang S, Heffernan H, Smith JM. Phenotypic and molecular characterization of community occurring, Western Samoan phage pattern methicillin-resistant Staphylococcus aureus. J Antimicrob Chemother 2002;50: Baggett HC, Hennessy TW, Rudolph K, et al. Community-onset methicillin-resistant Staphylococcus aureus associated with antibiotic use and the cytotoxin Panton-Valentine leukocidin during a furunculosis outbreak in rural Alaska. J Infect Dis 2004;189: Munckhof WJ, Schooneveldt J, Coombs GW, Hoare J, Nimmo GR. Emergence of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) infection in Queensland, Australia. Int J Infect Dis 2003;7: O Brien FG, Lim TT, Chong FN, et al. Diversity among community isolates of methicillin-resistant Staphylococcus aureus in Australia. J Clin Microbiol 2004;42: Stemper ME, Shukla SK, Reed KD. Emergence and spread of community-associated methicillin-resistant Staphylococcus aureus in rural Wisconsin, 1989 to J Clin Microbiol 2004;42: Bahrain M, Vasiliades M, Wolff M, Younus F. Five cases of bacterial endocarditis after furunculosis and the ongoing saga of community-acquired methicillin-resistant Staphylococcus aureus infections. Scand J Infect Dis 2006;38: Chi CY, Kuo BI, Fung CP, Liu CY. Community-acquired methicillin-resistant Staphylococcus aureus liver abscess in a patient with end-stage renal disease. J Microbiol Immunol Infect 2004;37: Crum NF. The emergence of severe, community-acquired methicillin-resistant Staphylococcus aureus infections. Scand J Infect Dis 2005;37: Kourbatova EV, Halvosa JS, King MD, Ray SM, White N, Blumberg HM. Emergence of communityassociated methicillin-resistant Staphylococcus aureus USA 300 clone as a cause of health care-associated infections among patients with prosthetic joint infections. Am J Infect Control 2005;33: Miller LG, Perdreau-Remington F, Rieg G, et al. Necrotizing fasciitis caused by community-associated methicillin-resistant Staphylococcus aureus in Los Angeles. N Engl J Med 2005;352: Nagaraju U, Bhat G, Kuruvila M, Pai GS, Jayalakshmi, Babu RP. Methicillin-resistant Staphylococcus aureus in community-acquired pyoderma. Int J Dermatol 2004;43: Ruiz ME, Yohannes S, Wladyka CG. Pyomyositis caused by methicillin-resistant Staphylococcus aureus. N Engl J Med 2005;352: Siddiqui MA, Richards PJ, Ahmed EB. Falling downstairs does not mean a fracture: the 1st case report of an immunocompetent community acquired MRSA disc/psoas abscess. Injury 2005;36: Otter JA, French GL. Molecular epidemiology of community-associated meticillin-resistant Staphylococcus aureus in Europe. Lancet Infect Dis 2010;10: Daum RS, Ito T, Hiramatsu K, et al. A novel methicillin-resistance cassette in community-acquired methicillin-resistant Staphylococcus aureus isolates of diverse genetic backgrounds. J Infect Dis 2002;86: General introduction 23

24 Chapter Leonard FC, Markey BK. Meticillin-resistant Staphylococcus aureus in animals: a review. Vet J 2008;175: Hanselman BA, Kruth SA, Rousseau J, et al. Methicillin-resistant Staphylococcus aureus colonization in veterinary personnel. Emerg Infect Dis 2006;12: van Duijkeren E, Box AT, Heck ME, Wannet WJ, Fluit AC. Methicillin-resistant staphylococci isolated from animals. Vet Microbiol 2004;103: van Duijkeren E, Wolfhagen MJ, Heck ME, Wannet WJ. Transmission of a Panton-Valentine leucocidinpositive, methicillin-resistant Staphylococcus aureus strain between humans and a dog. J Clin Microbiol 2005;43: Boag A, Loeffler A, Lloyd DH. Methicillin-resistant Staphylococcus aureus isolates from companion animals. Vet Rec 2004;154: Rich M, Roberts L. Methicillin-resistant Staphylococcus aureus isolates from companion animals. Vet Rec 2004;154: Armand-Lefevre L, Ruimy R, Andremont A. Clonal comparison of Staphylococcus aureus isolates from healthy pig farmers, human controls, and pigs. Emerg Infect Dis 2005;11: MANN PH. Antibiotic sensitivity testing and bacteriophage typing of staphylococci found in the nostrils of dogs and cats. J Am Vet Med Assoc 1959;134: Huijsdens XW, van Dijke BJ, Spalburg E, et al. Community-acquired MRSA and pig-farming. Ann Clin Microbiol Antimicrob 2006;5: Schijffelen MJ, Boel CH, van Strijp JA, Fluit AC. Whole genome analysis of a livestock-associated methicillin-resistant Staphylococcus aureus ST398 isolate from a case of human endocarditis. BMC Genomics 2010;11: Witte W, Strommenger B, Stanek C, Cuny C. Methicillin-resistant Staphylococcus aureus ST398 in humans and animals, Central Europe. Emerg Infect Dis 2007;13: Ekkelenkamp MB, Sekkat M, Carpaij N, Troelstra A, Bonten MJ. [Endocarditis due to meticillin-resistant Staphylococcus aureus originating from pigs]. Ned Tijdschr Geneeskd 2006;150: Gray RR, Tatem AJ, Johnson JA, et al. Testing spatiotemporal hypothesis of bacterial evolution using methicillin-resistant Staphylococcus aureus ST239 genome-wide data within a bayesian framework. Mol Biol Evol 2011;28: Guinane CM, Ben Zakour NL, Tormo-Mas MA, et al. Evolutionary genomics of Staphylococcus aureus reveals insights into the origin and molecular basis of ruminant host adaptation. Genome Biol Evol 2010;2: Harris SR, Feil EJ, Holden MT, et al. Evolution of MRSA during hospital transmission and intercontinental spread. Science 2010;327: Holden MT, Feil EJ, Lindsay JA, et al. Complete genomes of two clinical Staphylococcus aureus strains: evidence for the rapid evolution of virulence and drug resistance. Proc Natl Acad Sci U S A 2004;101:

25 83. Kennedy AD, Otto M, Braughton KR, et al. Epidemic community-associated methicillin-resistant Staphylococcus aureus: recent clonal expansion and diversification. Proc Natl Acad Sci U S A 2008;105: Lindsay JA, Moore CE, Day NP, et al. Microarrays reveal that each of the ten dominant lineages of Staphylococcus aureus has a unique combination of surface-associated and regulatory genes. J Bacteriol 2006;188: Nübel U, Roumagnac P, Feldkamp M, et al. Frequent emergence and limited geographic dispersal of methicillin-resistant Staphylococcus aureus. Proc Natl Acad Sci U S A 2008;105: Nübel U, Dordel J, Kurt K, et al. A timescale for evolution, population expansion, and spatial spread of an emerging clone of methicillin-resistant Staphylococcus aureus. PLoS Pathog 2010;6:e Shore AC, Deasy EC, Slickers P, et al. Detection of Staphylococcal Cassette Chromosome mec Type XI Carrying Highly Divergent meca, meci, mecr1, blaz, and ccr Genes in Human Clinical Isolates of Clonal Complex 130 Methicillin-Resistant Staphylococcus aureus. Antimicrob Agents Chemother 2011;55: Kennedy AD, Porcella SF, Martens C, et al. Complete nucleotide sequence analysis of plasmids in strains of Staphylococcus aureus clone USA300 reveals a high level of identity among isolates with closely related core genome sequences. J Clin Microbiol 2010 Dec;48: Witney AA, Marsden GL, Holden MT, et al. Design, validation, and application of a seven-strain Staphylococcus aureus PCR product microarray for comparative genomics. Appl Environ Microbiol 2005;71: Baba T, Takeuchi F, Kuroda M, et al. Genome and virulence determinants of high virulence communityacquired MRSA. Lancet 2002;359: Diep BA, Gill SR, Chang RF, et al. Complete genome sequence of USA300, an epidemic clone of communityacquired meticillin-resistant Staphylococcus aureus. Lancet 2006;367: Fridkin SK, Hageman JC, Morrison M, et al. Methicillin-resistant Staphylococcus aureus disease in three communities. N Engl J Med 2005;352: Voyich JM, Otto M, Mathema B, et al. Is Panton-Valentine leukocidin the major virulence determinant in community-associated methicillin-resistant Staphylococcus aureus disease? J Infect Dis 2006;194: General introduction 25

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27 2 New methods to analyse microarray data that partially lack a reference signal Neeltje Carpaij 1, Ad C. Fluit 1, Jodi A. Lindsay 2, Marc J.M. Bonten 1 and Rob J.L. Willems 1 1 Department of Medical Microbiology, University Medical Centre Utrecht, room G PO BOX 85500, 3508 GA Utrecht, The Netherlands. 2 Department of Cellular and Molecular Medicine, St. George s, University of London, London, United Kingdom. BMC genomics 2009;10:522

28 Abstract Background Microarray-based Comparative Genomic Hybridisation (CGH) has been used to assess genetic variability between bacterial strains. Crucial for interpretation of microarray data is the availability of a reference to compare signal intensities to reliable determine presence or divergence each DNA fragment. However, the production of a good reference becomes unfeasible when microarrays are based on pan-genomes. When only a single strain is used as a reference for a multistrain array, the accessory gene pool will be partially represented by reference DNA, although these genes represent the genomic repertoire that can explain differences in virulence, pathogenicity or transmissibility between strains. The lack of a reference makes interpretation of the data for these genes difficult and, if the test signal is low, they are often deleted from the analysis. We aimed to develop novel methods to determine the presence or divergence of genes in a Staphylococcus aureus multistrain PCR product microarray-based CGH approach for which reference DNA was not available for some probes. Chapter 2 28 Results In this study we have developed 6 new methods to predict divergence and presence of all genes spotted on a multistrain Staphylococcus aureus DNA microarray, published previously, including those gene spots that lack reference signals. When considering specificity and PPV (i.e. the false-positive rate) as the most important criteria for evaluating these methods, the method that defined gene presence based on a signal at least twice as high as the background and higher than the reference signal (method 4) had the best test characteristics. For this method specificity was 100% and 82% for MRSA252 (compared to the GACK method) and all spots (compared to sequence data), respectively, and PPV were 100% and 76% for MRSA252 (compared to the GACK method) and all spots (compared to sequence data), respectively. Conclusions A definition of gene presence based on signal at least twice as high as the background and higher than the reference signal (method 4) had the best test characteristics, allowing the analysis of 6-17% more of the genes not present in the reference strain. This method is recommended to analyse microarray data that partially lack a reference signal.

29 Background Comparative Genomic Hybridisation (CGH) microarray studies are applied to identify genetic diversity in both eukaryotes and prokaryotes (1-8). In bacteria microarray-based CGH has been used in genome typing and comparative phylogenomic analyses to assess genomic regions or genes involved in bacterial adaptation (9). The relationship between the intensity of a hybridised probe and the presence or divergence of a gene is crucial in microarray-based CGH (10). Features such as secondary structure, melting temperature, and even target characteristics make it difficult to define a cut-off intensity for gene presence (11,12). In general, DNA from a test strain is co-hybridised with differently labelled DNA from a reference strain in order to sidestep these issues in microarray analysis. The use of a reference allows the comparison of signal intensities and determination, for each DNA fragment in the reference strain, whether it is present or divergent in the test strain (13). The reference strain serves also as quality control for spots on microarray slides. In principle all spots should yield a signal for the reference, as it contains all genes. When a spot does not yield a signal with the DNA of both the reference strain and the test strain, the spot will be deleted from the analysis. The production of a good reference becomes more difficult or even unfeasible when the probes present on the microarray are not based on a single strain, but represent multiple genomes or even the pan-genome of a species. It is to be expected that the number of pan-genome arrays built from multiple strains will only increase with the rapid expansion of available (bacterial) whole genome sequences (9). There are already several methods to analyse microarrays, which partly lack a reference (8,14-16). However, in these approaches spots without reference and lacking a test signal are flagged as poorly performing, and removed from the analysis. Consequently, these genes cannot be classified with certainty as present or divergent. In this study we developed novel methods to determine the presence or divergence of all genes in a Staphylococcus aureus multistrain PCR product microarray-based CGH approach, including those that lack a reference signal by using performance data from all the spots on the microarray. New methods to analyse microarray data that partially lack a reference signal 29 Methods Description of the DNA microarray and its use All laboratory protocols have been described in detail by Witney et al. (16) and are registered at BmG@Sbase ( The S. aureus DNA microarray used in this study, which consists of PCR-based probes for all open reading frames (ORFs) of seven S. aureus strains, have been described and

30 Chapter 2 30 validated previously and are summarised here (16). In short, all ORFs of MRSA252, which served as base strain, were added to the microarray design followed by the addition of probes for genes from the other strains that are absent in, or show significant divergence from the genes of MRSA252 based on BLAST bit scores. The order in which the probes for the genes of the strains were added to the array was: MRSA252 (base strain), N315, Mu50, COL, NCTC8325, MW2, and MSSA476 (16). In total, the microarray consisted of 3623 PCR products spotted in duplicate representing every predicted open reading frame of the seven strains (8,16). Around 75% of the PCR products (n = 5478 in duplo) represent MRSA252, while around 25% of the PCR products (n = 1768 in duplo) were obtained from the other six strains. All strains were cultured on tryptic soy agar sheep blood plates at 37 C overnight. DNA of the reference strain and the test strains was isolated using the QIAGEN genomic-tip 100/G column and an Edge Biosystems Bactererial Genomic DNA purification kit (Edge Biosystems, Gateshead, United Kingdom). DNA of all seven sequenced S. aureus strains, labelled with Cy3, was hybdrised in duplicate on an array, with DNA of MRSA252, which was labelled with Cy5 as reference signal. Labeling was performed as described previously (8,16). Microarray images were quantified with ImaGene software (Biodiscovery, biodiscovery.com, El Segundo, California, United States). The two pictures per slide, one for every dye, were analysed separately in ImaGene. Fully annotated microarray data are deposited in BmG@Sbase ( and has been retrieved for this study. The background was calculated automatically and separately per colour and spot by ImaGene software. Data-processing Since, a reference signal was available for 75% of the microarray spots, only for this part hybridisation data could be analysed using ratios of test, Cy3, and reference, Cy5, signal. Therefore, the data were dissected into two different data sets. The first data set consisted of the hybridisation results of the MRSA252 specific spots (75% of the spots in duplicate) containing a reference signal (further indicated as MRSA252 spots). Hybridisation signals for these MRSA252 spots were analysed using GACK (further referred to as GACK method), which is one of the well-documented standard analysis methods (4,17,18) and also the new analysis methods developed in this study. The second data set that includes all spots, i.e. the non-mrsa252 spots that lack a reference signal as well as the MRSA252 spots, were only analysed using the new developed methods. The MRSA252 spots were also analysed with the new analysis methods to compare the outcome of the methods with that of the GACK method.