aureus infections in both community and hospital settings. S. aureus causes a number of diseases, which vary from carbuncles and food poisoning to would related infections. These can all potentially lead to life threatening situations such as bacteremia, necrotizing pneumonia and endocarditis.
Though most strains seem to show variation, whether by genotype or phenotype (Baba et al. 2002), it has been shown strains such as MRSA252 and EMRSA-16 are almost identical to one another and are responsible for half of the MRSA infections in the United Kingdom (Johnson et al. 2001). MRSA252 is also one of the major MRSA strains found in the United States, which is also known as USA200 (McDougal et al. 2003). Studies assessing genetic changes in Vancomycin Intermediate Staphylococcus aureus (VISA) isolates have shown a number of metabolic pathways and regulatory genes that may be key contributors to resistance (Cui et al. 2005, Kuroda et al. 2000, Mongodin et al. 2003, Sakoulas et al. 2003, Walsh et al. 2002).
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This study will examine molecular changes in isolates from patients with persistent S. aureus infections. This may lead to the development of a diagnostic test, which could identify strains associated with persistence and guide more aggressive forms of treatment of such persistent infections.
Introduction to Staphylococcus aureus infections 20
S. aureus is a gram-positive, facultatively anaerobic coccus. Being coagulase-positive it is able to cause clots in vitro and also separates itself from other common staphylococci (e.g., S. epidermidis) (27). S. aureus is a normal commensal found in human nares. Approximately 20% of the population are consistently colonized with this bacterium, while 60% of the population are transient carriers (28). Even though it remains harmless in most cases, S. aureus infection can lead to several diseases, ranging from minor skin infections such as furuncles and boils and eye infections (e.g., keratitis) to more serious illnesses, including bacteremia, endocarditis, septic arthritis, wound infections, pneumonia and toxic shock syndrome (20).
Though S. aureus infections have usually been manageable and easily treatable, doctors and patients alike become increasingly worried with the emergence of multiple antibiotic-resistant strains, such as MRSA. Approximately 40 to 60% of all nosocomially acquired S. aureus strains are methicillin-resistant, and these strains are now considered endemic in hospitals (29). S. aureus avoids being beaten by the body's natural defenses by using a vast variety of virulence factors, which involve attachment, biofilm formation, colonization, immune response evasion and tissue damage. The evolution of its persistence and resistance capabilities has reduced the number and effectiveness of a number of antibiotics. Penicillin, methicillin and in recent years, vancomycin, have all slowly become in-effective in treating patients when used as a mono-therapy.
Though these infections tend to be skin related, community-acquired strains also acquire Methicillin resistance and are of greater concern, not only because they are becoming more virulent (29), but also because these strains can infect hosts outside of the hospital setting who have no predisposing risk factors. S. aureus is able to cause acute osteomyelitis. The disease progresses to a chronic, biofilm-mediated infection because of the ability of S. aureus to rapidly develop antibiotic resistance and the timed expression of its arsenal of virulence factors.
20 S. aureus has an array of virulence factors that have roles in infection and that may be classified as being responsible for adherence, direct host damage, or immuno-avoidance. These factors have specific roles in the colonization and infection processes involved, their expression is coordinated throughout the various stages of infection. During early exponential growth, when cell density is low, proteins that promote adherence and colonization (such as fibronectin-binding protein, protein A, staphylokinase, and coagulase) are expressed. When cell growth reaches high densities, the production of the adherence and colonization factors is suppressed, while secreted toxins and enzymes are expressed, such as enterotoxins; alpha, beta, and delta hemolysin; clumping factor; leukocidin; and toxic shock syndrome toxin 1. 20
Many of these post-exponential-phase proteins are involved in damaging the host, obtaining nutrients from the host for bacterial growth, and dissemination once the staphylococci have adequately colonized and increased in number to promote an active infection. The expression of most of these staphylococcal products is under partial or complete control of the staphylococcal accessory regulator (sar) and the accessory gene regulator (agr) quorum-sensing systems.
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Many of the reactions of S. aureus to these environmental cues are classed as stress responses, and they are believed to be regulated by sigma factors that control gene expression.
FIG 1. Virulence factors produced by S aureus.
The organism produces cell surface virulence factors during the exponential phase and exoproteins and exopolysaccharides during the post-exponential/stationary phase. 49
20 Antibiotic resistance in the pre-antibiotic era, bacteremia with S. aureus resulted in a 90% death rate (31). Though considerably better today, with the ever-increasing level of antibiotic resistance and the evolution of community-acquired resistant strains, the health risk associated with this pathogen is sure to rise. β-lactam antibiotics, such as methicillin, oxacillin, and penicillin, act on the bacterial cell wall. These antibiotics work by inactivating transpeptidases, so that the peptidoglycan of the cell wall cannot be synthesized. In MRSA, the mecA gene, which encodes the penicillin-binding protein PBP2a, is horizontally acquired, and this bacterial product takes over the functions of
the inactivated transpeptidases and is naturally resistant to β-lactams (32). Because β-lactams are ineffective against MRSA, glycopeptides, such as vancomycin and teicoplanin, have become the drugs of choice for treatment (33).
However, as mentioned above, S. aureus strains with an intermediate susceptibility to glycopeptides are beginning to emerge, as are strains that are resistant to the antibiotic (34). Vancomycin-resistant S. aureus strains have gained the vanA resistance gene from enterococci that are also resistant to vancomycin (29), while in strains showing reduced susceptibility to vancomycin, the cell wall is thicker than in susceptible strains (34). The rather rapid acquisition of both methicillin and vancomycin resistance underscores the fact that S. aureus has been capable of becoming resistant to any drug it has been faced with, making current and future treatment difficult. Antimicrobial resistance, particularly methicillin resistance, usually results in a delay of appropriate and effective antimicrobial therapy.
G MRSA strains appear to have become established within the community. There is little doubt that if vancomycin resistance (vanA) genes become established in S. aureus, they would also spread into successful hospital MRSA lineages. A better understanding of the features that make MSSA and MRSA strains successful in both community and hospital settings is urgently needed.
26 Persistence of antibiotic-resistant S. aureus strains, and the scope for spread of resistances among populations, is strongly dependent on biological costs associated with resistance determinants or phenotypes. Resistances are generally unstable unless these ï¬tness costs are offset by either tight regulation of resistance genes, compensatory mutations or heterogeneous expression of resistance. Antibiotic resistances can be controlled by dedicated cognate regulators, but almost all resistance phenotypes are also strongly inï¬‚uenced by additional regulatory mechanisms. S. aureus employs multiple layers of gene regulation to ensure protection against environmental stresses and host
defences; metabolic adaptation to changing nutrient conditions; and the coordinated expression of virulence factors. Resistances to cell wall-active antibiotics are especially sensitive to any regulatory changes inï¬‚uencing peptidoglycan metabolism and cell
envelope properties. Core genomic regulatory networks can also be modiï¬ed by imported regulators present on mobile elements such as plasmids, phages and genomic islands. S. aureus genomic strain backgrounds can vary by over 20%, which may contribute to vast strain-speciï¬c differences in resistance levels, resistance phenotypes and resistance gene expression levels, as exempliï¬ed by the wide range of different oxacillin MICs and resistance proï¬les seen in different MRSA isolates.
web Phylogenetic classification places S aureus in the Bacillus/Staphylococcus group, up to 52% of predicted proteins encoded by the N315 strain genome are similar to those in Bacillus substilis and Bacillus halodurans. They typically contain housekeeping genes involved in essential functions of the vegetative life of the bacteria, such as DNA replication, protein synthesis, and carbohydrate metabolism.
The 8 S. aureus sequenced genomes range in size from 2.820Mb to 2.903Mb[16,21,24] and is composed of core and auxiliary (accessory) genes. The majority of genes comprising the core genome are those associated with central metabolism and other housekeeping functions. Approximately 75% of the S. aureus genome comprises a core component of genes present in all of the strains. Other genes within the core genome that are not essential for growth and survival include virulence genes that are not carried by other staphylococcal species, surface binding proteins, toxins, exoenzymes, and the capsule biosynthetic cluster.
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e It has been proposed that the value and stability of typing and taxonomy of bacteria
are highly dependent on mechanisms of genome evolution, with diï¬€erent regions of the bacterial chromosome undergoing different rates of genome evolution (8). The data
presented here suggest that at least three mechanisms of genome evolution in MRSA
exist including (i) recombination and or gene conversion of the rrn operon resulting in sequence homogeneity in some regions and rearrangements, insertions or deletions in
others (5, 17), (ii) recombination of essential housekeeping genes (4) and (iii) horizontal
transfer (2, 13, 15, 16). Based on sequence data currently available, the mecA gene is a
poor candidate for typing because the sequence is highly stable between S. aureus, S.
epidermidis and S. sciuri. A more variable region such as the ISR, which may undergo
frequent rearrangements, may be more suitable. Some of these questions may be resolved
by performing experiments (in S. aureus) similar to those performed in Escherichia coli
to measure the mutation rates within diï¬€erent genes (18) as a function of bacterial generation times. Analysis of SNPs in diï¬€erent regions of the S. aureus genome suggest that diï¬€erent evolutionary mechanisms apply to diï¬€erent regions of the genome and further studies to elucidate these mechanisms may shed light on the origin of MRSA. E
Whole genome sequences for MRSA strain N315 and a vancomycin-resistant S. aureus strain (Mu50) have been reported recently (16), and a number of partial genome sequences for other MRSA strains are also available (1, 19, 20), allowing comparison of specific known sequences studied, and the frequency of mutations within them.
2.1 Sequence variation within ISR types between MRSA strains
A further contribution to diï¬€erences in ISRs between the whole genomes of the ®ve strains is from single nucleotide polymorphisms (SNPs) between identical ISR types when in an individual strain. Additionally, diï¬€erences between alleles of the same ISR type may occur within diï¬€erent strains. In a previous study a `T' at number 162 of rrnE was found in 92% of MRSA strains (7) ; this `T' was found in rrnA1 of COL and rrnE of H11, 252 and A48074 but not in strains Mu50 and N315. Two copies of the same ISR type that diï¬€er by a single nucleotide diï¬€erence isolated from one strain could be explained by (i) the presence of diï¬€erent alleles in the one genome or (ii) possibly by the occurrence of diï¬€erent bacterial cells with diï¬€erent ISR types within the one culture of one strain. Thus, further strain diï¬€erences are re¯ected in SNPs within strain-speci®c ISR types. It can be seen that the ISR, a constant and possibly obligatory part of the genome, is genetically dynamic and subject to homogenization, consistent with concerted evolution (5, 9, 10), which is only possible because multiple copies of it occur in the one genome. E
2.2 GraRS Two-component regulatory system
In this study, we showed that isogenic strains with mutations in genes encoding the GraRS TCRS and the VraFG ABC transporter are hypersensitive to vancomycin as well as polymyxin B. Moreover, GraRS regulates the expression of the adjacent VraFG pump, reminiscent of gram-positive bacteriocin-immunity regulons. Mutations of graRS and vraFG also led to increased autolytic rates and a more negative net surface charge, which may explain, in part, to their increased sensitivity to cationic antimicrobial peptides. Taken together, these data reveal an important genetic mediator to the vancomycin-intermediate S. aureus phenotype and may hold clues to the selective pressures on staphylococci upon exposure to selective cationic peptide antibiotics used in clinical practice.
In this study, we have described two genes that likely mediate the VAN-intermediate resistance phenotype in S. aureus. Although graRS and vraFG have been previously linked to over expression in VISA strains by microarray analyses, we have genetically linked their activity to VAN resistance in this report.More speciï¬cally, we found that the isogenic mutants of graR and vraG in the VISA strain Mu50 had increased susceptibility to the clinically relevant antibiotics VAN and PMB (both having a net positive charge). These mutant strains exhibited increased autolysis rates and enhanced net negative surface charges compared to the wild-type and wild-type revertant strains. The observation that GraRS regulated the expression of vraFG links their phenotypes to an expression cascade, but partial phenotypes observed in Mu50 graR mutants complemented with vraFG genes suggest that other genetic elements controlled by GraRS may affect VISA phenotypes. Of note, we did not visualize considerable alterations in RNA transcript levels of sarA, RNAIII of agr, pbp2,or pbp4 using Northern analysis (data not shown). Moreover, the mutant phenotypes in non-VISA strains (COL and RN6390) also conï¬rmed our observation that these genes are important in mediating basal resistance to VAN and PMB.
Pic of genomes
3 Laboratory diagnosis of Staphylococcus aureus infections
Diagnostic microbiology laboratories and reference laboratories are key for identifying outbreaks of MRSA. New rapid techniques for the identification and characterization of MRSA have been developed. These techniques include Real-time PCR and Quantitative PCR and are increasingly being employed in clinical laboratories for the rapid detection and identification of MRSA strains.
To determine minimum inhibitory concentration (MIC) for confirmation of resistance, and necessary concentrations of the various S. aureus strains to vancomycin along with methicillin and penicillin to act as controls, the Epsilomer test (E-test) will be used. After a 24hour incubation period with the E-test strip, which has been inoculated with the specific antibiotic, the MIC will be determined.
For the Population Analysis Profile (PAP) I will be using a broth dilution method. For basic culture blood heart infusion agar (BHI) will be used. However, when running tests on the vancomycin resistant Mu50 strain, the BHI again will be inoculated with 4ug/ml of vancomycin. For routine culture agar plates will be used, though for a population analysis Mueller-Hilton broth (Howden et al. 2010) will be used.
3.1 Microbiology and microscopy
Current methods used to identify S. aureus include Gram stain morphology, cell morphology, production of catalase, coagulase production, pigment production, susceptibility to lysostaphin and lysozyme, and anaerobic production of acid from glucose (4). In addition, there are several commercially available systems that allow
strains to be biochemically characterized. Among Staphylococcus species associated with human infections, S. aureus is unique in its ability to clot plasma (coagulase). Two different coagulase tests are commonly used to identify S. aureus. One is a tube test for free coagulase and the other is a slide test for bound coagulase. The tube coagulase test is thought to be the more definitive of the two, however, it can take several hours to overnight to produce a result. The slide coagulase test may yield a negative result for up to 10 to 15 percent of S. aureus strains (2).
Colonies may be tested from any of the following culture media: Tryptone Soya Agar (Tryptic Soy Agar or TSA) with 5% sheep blood (Blood Agar), Columbia Agar with 5% sheep blood, and Mueller Hinton Agar. The use of fresh (18-24 hours) cultures (grown at 35 degrees C) is recommended.
After extraction and preparation of the test specimen, perform the test the same day. The test specimen may be stored refrigerated at 2-10 degrees C for batch testing later in the day or at -80 degrees C for long-term storage.
3.2 Agar and broth mediums
Various types of agar plates are used which allow growth of morphologically suggestive staphylococci. Incubation of cultures is usually between 18 to 24 hours but in some case, respective of the strain and type of agar it may take up to 48 hours. For basic culture blood heart infusion agar (BHI) is be used. However, when running tests on the vancomycin resistant Mu50 strain, the BHI wiould be inoculated with 4ug/ml of vancomycin.
Currently the majority of screening is carried out using plate based methods. However a number of alternative methods including broth based methods, chromogenic media, rapid screening kits, molecular assays and automated systems are increasingly being used. Isolation from screening swabs can be a lengthy procedure, due to the number of 'contaminating' organisms that are present in swabs from non-sterile sites.
Broth based enrichment media are commonly employed to increase sensitivity. However this is at the expense of speed of result. NaCl is generally added to the base broth along with either Methicillin or oxacillin.
Recently developed chromogenic media combine primary growth and selectivity with differentiation from coagulase negative staphylococci. These media show improved specificity when compared with traditional media. Sensitivity is also improved but requires 48hrs incubation to achieve >85%.
4 Molecular methods for detection of Staphylococcus aureus
4.1 Real-time Polymerase Chain Reaction
DNA extraction may be done with a variety of methods however Phenol-Chloroform extraction method is used well on S. aureus PCR will then be run on the extracted DNA. PCR is rapid, specific and sensitive identification for any microorganisms based of the detection of specific DNA segments. By using the enzyme DNA polymerase and a pair of forward and reverse oligonucleotide primers, specifically targetted DNA sequences can be amplified. This is achieved by a number of heating and cooling cycles done in a thermocycler.
Many techniques which are now in use rely on PCR amplification of targeted sequences, which are followed up by a number of post amplification analysis procedures, to support and aid the confirmation of these results. A melt-curve analysis of the PCR products will be used to determine if SNPs are present.
4.2 High resolution melt curve analysis of single nucleotide polymorphisms
60 Single nucleotide polymorphisms (SNPs) comprise the most abundant category of
DNA sequence variation, occurring at a rate of 1 per 500 nucleotides in coding sequences and at a higher rate in non-coding sequences. The continuing requirement for detection of rare SNPs will maintain the need for high-throughput, inexpensive methods for sequence variant detection.
60 Several methods for detection of SNPs are based on the principle of different melting temperatures of DNA. Using a double-stranded DNA-specific fluorescent dye such as SYBR Green I (SYBR) in a PCR thermo-cycler capable of high resolution melt (HRM) curve analysis in which DNA melting would be controlled.
DNA melting analysis (DMA) has been used successfully for variant detection, and discovering previously unknown SNPs by this approach. Concentrations of DNA amplicons are readily monitored by SYBR fluorescence, and DNA amplicon concentrations are highly reproducible. Differences in the melting temperature are easily detected in fragments 15-167 bp in length and differ by only a single nucleotide substitution. Therefore the efficiency and sensitivity of DMA make it highly suitable for the large-scale detection of sequence variants.
5 Rationale of study
Certain genes have been reported as being responsible for certain virulence factors, yet the difference in characteristics between the strains are due to the expression of certain genes which may be due to the SNPs. Though different genetic variations have been identified which code for intermediate vancomycin resistance, many functional implications are not yet known (Cui et al. 2005, Kuroda et al. 2000, Mongodin et al. 2003). For example; the two-component regulatory system (TCRS) SACOL0716 to 0717, also known as graRS, has been linked to the VISA phenotype, yet its function is still undefined (Cui et al. 2005).
Many studies support previous results, suggesting that different transcriptional pathways and presumably different mutations can be linked to resistance and persistence of infections (Howden et al. 2008).
By answering these questions and aims, we will be able to target and look for certain genes responsible for persistence in chronically infected patients. Using this information as a diagnostic test may result in different treatment plans for different patients. Early aggressive treatment may result in reduced hospital stays for patients.
The key aim of this project will be to identify changes in genes which may be associated with persistence rather than resistance, i.e. in comparison to the work done by Howden et al. (2008) concentrating on resistance. The aims of this project will be;
To examine clinical isolates of MRSA for genetic changes occurring over time in present infections
To test clinical isolates for single nucleotide polymorphisms (SNPs) reported as associated with persistent infections
To optimise identification of SNPs using melt-curve analysis of PCR products