Monika A. Chlebowicz

Proteomics uncovers extreme heterogeneity in the Staphylococcus aureus exoproteome due to genomic plasticity and variant gene regulation

Sequencing of at least 13 Staphylococcus aureus isolates has shown that genomic plasticity impacts significantly on the repertoire of virulence factors. However, genome sequencing does not reveal which genes are expressed by individual isolates. Here, we have therefore performed a comprehensive survey of the composition and variability of the S. aureus exoproteome. This involved multilocus sequence typing, virulence gene, and prophage profiling by multiplex PCR, and proteomic analyses of secreted proteins using 2‐DE. Dissection of the exoproteomes of 25 clinical isolates revealed that only seven out of 63 identified secreted proteins were produced by all isolates, indicating a remarkably high exoproteome heterogeneity within one bacterial species. Most interesting, the observed variations were caused not only by genome plasticity, but also by an unprecedented variation in secretory protein production due to differences in transcriptional and post‐transcriptional regulation. Our data imply that genomic studies on virulence gene conservation patterns need to be complemented by analyses of the extracellular protein pattern to assess the full virulence potential of bacterial pathogens like S. aureus. Importantly, the extensive variability of secreted virulence factors in S. aureus also suggests that development of protective vaccines against this pathogen requires a carefully selected combination of invariably produced antigens.

Whole-Genome Sequencing for Routine Pathogen Surveillance in Public Health: a Population Snapshot of Invasive Staphylococcus aureus in Europe

ABSTRACT The implementation of routine whole-genome sequencing (WGS) promises to transform our ability to monitor the emergence and spread of bacterial pathogens. Here we combined WGS data from 308 invasive Staphylococcus aureus isolates corresponding to a pan-European population snapshot, with epidemiological and resistance data. Geospatial visualization of the data is made possible by a generic software tool designed for public health purposes that is available at the project URL (http://www.microreact.org/project/EkUvg9uY?tt=rc). Our analysis demonstrates that high-risk clones can be identified on the basis of population level properties such as clonal relatedness, abundance, and spatial structuring and by inferring virulence and resistance properties on the basis of gene content. We also show that in silico predictions of antibiotic resistance profiles are at least as reliable as phenotypic testing. We argue that this work provides a comprehensive road map illustrating the three vital components for future molecular epidemiological surveillance: (i) large-scale structured surveys, (ii) WGS, and (iii) community-oriented database infrastructure and analysis tools. IMPORTANCE The spread of antibiotic-resistant bacteria is a public health emergency of global concern, threatening medical intervention at every level of health care delivery. Several recent studies have demonstrated the promise of routine whole-genome sequencing (WGS) of bacterial pathogens for epidemiological surveillance, outbreak detection, and infection control. However, as this technology becomes more widely adopted, the key challenges of generating representative national and international data sets and the development of bioinformatic tools to manage and interpret the data become increasingly pertinent. This study provides a road map for the integration of WGS data into routine pathogen surveillance. We emphasize the importance of large-scale routine surveys to provide the population context for more targeted or localized investigation and the development of open-access bioinformatic tools to provide the means to combine and compare independently generated data with publicly available data sets. The spread of antibiotic-resistant bacteria is a public health emergency of global concern, threatening medical intervention at every level of health care delivery. Several recent studies have demonstrated the promise of routine whole-genome sequencing (WGS) of bacterial pathogens for epidemiological surveillance, outbreak detection, and infection control. However, as this technology becomes more widely adopted, the key challenges of generating representative national and international data sets and the development of bioinformatic tools to manage and interpret the data become increasingly pertinent. This study provides a road map for the integration of WGS data into routine pathogen surveillance. We emphasize the importance of large-scale routine surveys to provide the population context for more targeted or localized investigation and the development of open-access bioinformatic tools to provide the means to combine and compare independently generated data with publicly available data sets.

Application of next generation sequencing in clinical microbiology and infection prevention

Current molecular diagnostics of human pathogens provide limited information that is often not sufficient for outbreak and transmission investigation. Next generation sequencing (NGS) determines the DNA sequence of a complete bacterial genome in a single sequence run, and from these data, information on resistance and virulence, as well as information for typing is obtained, useful for outbreak investigation. The obtained genome data can be further used for the development of an outbreak-specific screening test. In this review, a general introduction to NGS is presented, including the library preparation and the major characteristics of the most common NGS platforms, such as the MiSeq (Illumina) and the Ion PGM™ (ThermoFisher). An overview of the software used for NGS data analyses used at the medical microbiology diagnostic laboratory in the University Medical Center Groningen in The Netherlands is given. Furthermore, applications of NGS in the clinical setting are described, such as outbreak management, molecular case finding, characterization and surveillance of pathogens, rapid identification of bacteria using the 16S-23S rRNA region, taxonomy, metagenomics approaches on clinical samples, and the determination of the transmission of zoonotic micro-organisms from animals to humans. Finally, we share our vision on the use of NGS in personalised microbiology in the near future, pointing out specific requirements.

The dynamic changes of dominant clones of Staphylococcus aureus causing bloodstream infections in the European region: Results of a second structured survey

Staphylococcus aureus is one of the most important human pathogens and meticillin-resistant S. aureus (MRSA) presents a major cause of healthcare- and community-acquired infections. This study investigated the spatial and temporal changes of S. aureus causing bacteraemia in Europe over a five-year interval and explored the possibility of integrating pathogen-based typing data with epidemiological and clinical information at a European level. Between January 2011 and July 2011, 350 laboratories serving 453 hospitals in 25 countries collected 3,753 isolates (meticillin-sensitive S. aureus (MSSA) and MRSA) from patients with S. aureus bloodstream infections. All isolates were sent to the national staphylococcal reference laboratories and characterised by quality-controlled spa typing. Data were uploaded to an interactive web-based mapping tool. A wide geographical distribution of spa types was found, with some prevalent in all European countries. MSSA was more diverse than MRSA. MRSA differed considerably between countries with major international clones expanding or receding when compared to a 2006 survey. We provide evidence that a network approach of decentralised typing and visualisation of aggregated data using an interactive mapping tool can provide important information on the dynamics of S. aureus populations such as early signalling of emerging strains, cross-border spread and importation by travel.

Requirement of the agr Locus for Colony Spreading of Staphylococcus aureus

The important human pathogen Staphylococcus aureus is known to spread on soft agar plates. Here, we show that colony spreading of S. aureus involves the agr quorum-sensing system. This finding can be related to the agr-dependent expression of biosurfactants, such as phenol-soluble modulins, suggesting a connection between spreading motility and virulence.

Evolutionary dynamics of methicillin-resistant Staphylococcus aureus within a healthcare system

BackgroundIn the past decade, several countries have seen gradual replacement of endemic multi-resistant healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) with clones that are more susceptible to antibiotic treatment. One example is Singapore, where MRSA ST239, the dominant clone since molecular profiling of MRSA began in the mid-1980s, has been replaced by ST22 isolates belonging to EMRSA-15, a recently emerged pandemic lineage originating from Europe.ResultsWe investigated the population structure of MRSA in Singaporean hospitals spanning three decades, using whole genome sequencing. Applying Bayesian phylogenetic methods we report that prior to the introduction of ST22, the ST239 MRSA population in Singapore originated from multiple introductions from the surrounding region; it was frequently transferred within the healthcare system resulting in a heterogeneous hospital population. Following the introduction of ST22 around the beginning of the millennium, this clone spread rapidly through Singaporean hospitals, supplanting the endemic ST239 population. Coalescent analysis revealed that although the genetic diversity of ST239 initially decreased as ST22 became more dominant, from 2007 onwards the genetic diversity of ST239 began to increase once more, which was not associated with the emergence of a sub-clone of ST239. Comparative genomic analysis of the accessory genome of the extant ST239 population identified that the Arginine Catabolic Mobile Element arose multiple times, thereby introducing genes associated with enhanced skin colonization into this population.ConclusionsOur results clearly demonstrate that, alongside clinical practice and antibiotic usage, competition between clones also has an important role in driving the evolution of nosocomial pathogen populations.

Recombination between ccrC Genes in a Type V (5C2&5) Staphylococcal Cassette Chromosome mec (SCCmec) of Staphylococcus aureus ST398 Leads to Conversion from Methicillin Resistance to Methicillin Susceptibility In Vivo

ABSTRACT Various types of the staphylococcal cassette chromosome mec (SCCmec) are known to confer methicillin resistance on the human pathogen Staphylococcus aureus. Such cassettes are not always stably maintained. The present studies were aimed at identifying the mechanism underlying the in vivo conversion of methicillin-resistant S. aureus (MRSA) to methicillin-susceptible S. aureus (MSSA) derivatives as encountered in two patients suffering from pneumonia and an umbilicus infection, respectively. All MRSA and MSSA isolates identified belong to multilocus sequence type (MLST) 398, have spa type t034, and are Panton-Valentine leukocidin positive. Sequencing of 27,616 nucleotides from the chromosomal SCCmec insertion site in orfX to the hsdR gene for a restriction enzyme revealed a type V (5C2&5) SCCmec. Sequence comparisons show that parts of the cassette are highly similar to sequences within SCCmec elements from coagulase-negative staphylococci, indicating a possible common origin. The cassette investigated contains ccrC-carrying units on either side of its class C2b mec gene complex. In vivo loss of the mec gene complex was caused by recombination between the recombinase genes ccrC1 allele 8 and ccrC1 allele 10. In vitro, the SCCmec was very stable, and low-frequency MRSA-to-MSSA conversion was only observed when MRSA isolates were cultivated at 41°C for prolonged periods of time. In this case also, loss of the mec complex was due to ccrC gene recombination. Interestingly, the MRSA and MSSA isolates studied displayed no detectable differences in competitive growth and virulence, suggesting that the presence of the intact type V (5C2&5) SCCmec has no negative bearing on staphylococcal fitness under the conditions used.

Microfluidic-Chip-Based Multiple-Locus Variable-Number Tandem-Repeat Fingerprinting with New Primer Sets for Methicillin-Resistant Staphylococcus aureus

ABSTRACT The detection of outbreaks of methicillin-resistant Staphylococcus aureus (MRSA) infections and a rapid and accurate identification of sources and routes of transmission should be conducted in hospital settings as early and swiftly as possible. In this study, we investigated the application potential of a new approach based on multiple-locus variable-number tandem-repeat fingerprinting (MLVF) and microfluidics technology for a rapid discrimination of MRSA lineages in outbreak settings. A total of 206 nonrepetitive MRSA isolates recovered from infected patients at the University Medical Center Groningen between 2000 and 2010 were tested. The results obtained by MLVF using microcapillary electrophoresis with newly designed primers were compared to those obtained by spa typing and multiple-locus variable-number tandem-repeat analysis (MLVA). The discriminatory power was 0.980 (107 patterns), 0.969 (85 allelic profiles), and 0.959 (66 types) for MLVF, MLVA, and spa typing, respectively. All methods tested showed a good concordance of results calculated by the adjusted Rands coefficient method. Comparisons of data obtained by the three approaches allowed us to propose an 88% cutoff value for the similarity between any two MLVF patterns, which can be used in S. aureus epidemiological studies, including analyses of outbreaks and strain transmission events. Of the three tested methods, MLVF is the cheapest, fastest, and easiest to perform. MLVF applied to microfluidic polymer chips is a rapid, cheap, reproducible, and highly discriminating tool to determine the clonality of MRSA isolates and to trace the spread of MRSA strains over periods of many years. Although spa typing should be used due to its portability of data, MLVF has a high added value because it is more discriminatory.

The Staphylococcal Cassette Chromosome mec type V from Staphylococcus aureus ST398 is packaged into bacteriophage capsids

The Staphylococcal Cassette Chromosome mec (SCCmec) confers methicillin resistance to Staphylococcus aureus. While SCCmec is generally regarded as a mobile genetic element, the precise mechanisms by which large SCCmec elements are exchanged between staphylococci have remained enigmatic. In the present studies, we observed that the clinical methicillin-resistant S. aureus (MRSA) isolate UMCG-M4 with the sequence type 398 contains four prophages belonging to the serological groups A, B and Fa. Previous studies have shown that certain serological group B bacteriophages of S. aureus are capable of generalized transduction. We therefore assessed the transducing capabilities of the phages from strain UMCG-M4. The results show that some of these phages can indeed transduce plasmid pT181 to the recipient S. aureus strain RN4220. Therefore, we also investigated the possible involvement of these transducing phages in the transmission of the large SCCmec type V (5C2&5) element of S. aureus UMCG-M4. While no transduction of the complete SCCmec element was observed, we were able to demonstrate that purified phage particles did contain large parts of the SCCmec element of the donor strain, including the methicillin resistance gene mecA. This shows that staphylococcal phages can encapsulate the resistance determinant mecA of a large SCCmec type V (5C2&5) element, which may lead to its transfer to other staphylococci.

Considerations for the Distinction of ccrC-Containing Staphylococcal Cassette Chromosome mec Elements

Staphylococcal cassette chromosome mec (SCCmec) elements confer methicillin resistance to the human pathogen Staphylococcus aureus (methicillin-resistant S. aureus [MRSA]). Recently, the International Working Group on the Classification of Staphylococcal Cassette Chromosome Elements (IWG-SCC) has published new guidelines for classifying different SCCmec elements (3). The new classification of SCCmec is based on the mec gene complex (classes A, B, C1, and C2) that confers methicillin resistance, the ccr gene complex composed of the recombinase-encoding ccr gene(s) (ccrA, ccrB, or ccrC) with surrounding open reading frames (ORFs) (4, 5), and joining regions (J1 to J3). The new guidelines were urgently needed due to the emergence of mosaic-structured SCCmec elements that could not be classified by the previous classification scheme. Interestingly, the classification of SCCmec elements carrying a ccrC recombinase gene appears to be more complicated than the classification of other known SCCmec elements. This is illustrated in Fig. ​Fig.1,1, which shows a comparison of all sequenced ccrC-carrying SCCmec elements. The region containing a ccrC gene flanked by three conserved upstream genes and three conserved downstream genes was denoted as a “type 5 ccr gene complex” by Ito and coworkers (4, 5) and the IWG-SCC (3), but it has also been referred to as a “ccrC-carrying unit” (1, 2). Such complexes are located either after the mec complex (e.g., in type V [5C2]), or between orfX and the mec complex (e.g., in type VII [5C1]; Fig. ​Fig.1).1). The presence of a second type 5 ccr gene complex located between orfX and the mec complex is indicated by “&5” in types V (5C2&5) (1, 2) and IV (2B&5). FIG. 1. Schematic representation of ccrC-containing SCCmec elements (adapted from reference 1). On the left, the classification proposed by the IWG-SCC is indicated. The strains carrying specific SCCmec types are indicated in the second set of parentheses. The ... Here we note that it is not easy to understand the structural differences of ccrC-carrying SCCmec elements if these are indicated only by “5” or “&5,” which may be a complication for epidemiological studies. However, by sequence comparisons, we identified several distinguishing features among the SCCmec elements carrying ccrC. Most importantly, the type 5 ccr gene complexes can be classified into two major groups (groups I and II) on the basis of the sequences of the ccrC genes and surrounding ORFs. The “group I” ccr gene complex 5 is always located between orfX and the mec complex, and it is preceded by three additional conserved genes of unknown function (Fig. ​(Fig.1).1). Comparisons of the group I regions plus the three preceding genes from SCCHg and of SCCmec elements IV (2B&5), V (5C2&5), and VII (5C1) revealed that they share more than 97% nucleotide sequence identity, underpinning the high conservation of this “extended group I” region. The group II ccr gene complex 5 is always located after the mec complex. The sequence identity within this group is about 93% due to variations in the ccrC alleles. It is thus possible to distinguish ccrC-containing SCCmec elements on the basis of these differences, and this may be more robust than a distinction based on allelic differences of ccrC (Fig. ​(Fig.11). In conclusion, we believe that the IWG-SCC recommendations provide an excellent starting point for improved SCCmec classification. However, certain refinements in the distinction of SCCmec elements as described here can possibly be applied for detecting MRSA in the years to come. Clearly, any adjustments to the SCCmec classification should be made on the basis of agreed consensus.