MRSA: Current Perspectives
Chapter Abstracts

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Chapter 1
Introduction to MRSA

Barry Cookson, Franz-Josef Schmitz, and Ad C. Fluit

No abstract is available for this chapter. Therefore we have provided the first paragraph:
Staphylococci were first observed and cultured by Pasteur and Koch, but the initial detailed studies on staphylococci were made by Ogston in 1881 and Rosenbach in 1884 (Ogston, 1881; Rosenbach, 1884). The genus Staphylococcus was given its name by Ogston in 1881 when he observed grape-like clusters of bacteria in pus from human abscesses (Ogsten, 1881). Three years later, Rosenbach was able to isolate and grow these micro-organisms in pure culture. He gave these bacteria the specific epithet Staphylococcus aureus because of the yellow-to-orange pigmented appearance of their colonies. Rosenbach showed that S. aureus was responsible for wound infections and furunculosis, and that Staphylococcus epidermidis was a normal colonizer of the skin (Rosenbach, 1884). Ever since Rosenbach first described the growth of this 'golden' coccus, surgeons have feared staphylococcal wound infections after surgery. Staphylococci also caused life-threatening disease after trauma and fatal pneumonia during the influenza season. Therefore, in the pre-antibiotic era, S. aureus was known as a major life-threatening pathogen. Staphylococcal infections are characterized by intense suppurative inflammation of local tissues with a tendency for the infected area to become encapsulated, leading to abscess formation. The most common staphylococcal infection is the furuncle or boil, which is a localized painful superficial skin infection that develops in a hair follicle or gland. Similar infections at the base of the eyelashes are the common styes. The most usual sites for boils are the neck and the buttocks, often as a result of wearing tight clothes. The infecting organism is usually identical to that carried in the patients' anterior nares. Approximately 2­3% of the population have chronic furunculosis. Staphylococcal infection may spread from a furuncle to the deeper subcutaneous tissues resulting in the development of one or more abscesses known as carbuncles. These are now less common, but such patients were often suffering from diabetes mellitus, and a carbuncle may have been the initial presentation of the condition. These abscesses occur mostly on the back of the neck, but may involve other skin sites. They may result in bloodstream invasion.


Chapter 2
Detection of MRSA

Derek Brown and Barry Cookson

Abstract
The detection of MRSA in clinical specimens is a challenge to any microbiology laboratory. We describe the various aspects of this process: isolation media, confirmation of species, determination of methicillin-resistance and the many factors that can affect this. The phenotypic and genotypic laboratory techniques, their advantages and various pitfalls are outlined as well as current and promising rapid methods. We also discuss the cost effectiveness of these methods and propose a categorisation of MRSA screening strategies. Mathematical modelling to evaluate cost effectiveness is needed so that informed choices of the most appropriate technique for each screening strategy can be made.


Chapter 3
Mechanisms of Methicillin Resistance

Susanne Rohrer, Markus Bischoff, Jutta Rossi, and Brigitte Berger-Bächi

Abstract
Methicillin-resistant staphylococci have been on the rise since shortly after the introduction of penicillinase-resistant b -lactams in 1960. The methicillin resistance is due to the acquisition of the low-affinity penicillin binding protein, PBP2', encoded by mecA. This gene resides on a large, chromosomally inserted element termed SCCmec (staphylococcal cassette chromosome mec) from an extraspecies source. It has been possible to trace the evolution and dissemination of methicillin resistance within the genus Staphylococcus, but the origin of SCCmec is still unclear. Although mecA is the sole prerequisite for methicillin resistance, the resulting phenotype is highly complex.

A characteristic of methicillin resistance is its heterogeneous expression ­ highly resistant subclones arising under antibiotic pressure at a frequency that lies above that of spontaneous mutation. This phenomenon has puzzled clinicians and researchers for the past 30 years. Although different approaches have yielded a great number of genetic factors that influence the expression of methicillin resistance and have given us insight into a wide variety of processes in the staphylococcal cell, the underlying mechanism causing heteroresistance has not been found. It is to be hoped that modern techniques enabling the analysis of cellular metabolism on a global scale will unravel the "mystery" of heterogeneous resistance to methicillin.


Capter 4
MRSA Resistance Mechanisms and Surveillance Data for Non-betalactams and Non-glycopeptides

Ian Morrissey and David J. Farrell

Abstract
This chapter discusses the genetics and regulation of resistance mechanisms to the major classes of anti-staphylococcal antibiotics in methicillin-resistant Staphylococcus aureus (MRSA) with the exception of betalactam agents and glycopeptides, which are dealt with elsewhere. To summarise, it can be seen that MRSA from almost all regions of the world appear to be cross-resistant to many other classes of antibiotic and this emphasises the need to find new antibiotics to treat this difficult organism.


Chapter 5
Molecular approaches for the epidemiological characterization of Staphylococcus aureus strains

Willem B. van Leeuwen

Abstract
Staphylococcus aureus has remained a significant cause of nosocomial morbidity and mortality. For almost four decades the increasing prevalence of methicillin-resistant S. aureus (MRSA) has posed an additional major clinical threat worldwide. During this period, multiple DNA-based methods have been introduced to genetically type S. aureus strains, but not a single technique appeared to be universally applicable. Most of the current image-based approaches generate complex banding patterns and lack generally accepted interpretation criteria. The need for straightforward and reproducible techniques generating simple output that can be used for computerized data-management, still is an important research topic.

This review summarizes the chronology of development and the qualities of the various laboratory techniques and includes the description of the generation, application and validation of novel DNA-based strategies rendering a binary outcome, such as multilocus sequence typing (MLST), binary typing (BT) or Micro Array technologies. The value of these novel typing procedures, with respect to simplicity and performance in the elucidation of complex biological phenomena such as epidemicity, relevance to pathogenesis of both human and bovine strains, antimicrobial resistance and genomic evolution of S. aureus strains, will be discussed.


Chapter 6
The Molecular Evolution of Methicillin-Resistant Staphylococcus aureus

J. Ross Fitzgerald and James M. Musser

Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) are a major problem in the treatment of nosocomial infections. Recent discoveries have contributed important insights into the molecular evolution and dissemination of MRSA. This chapter summarizes information regarding the nature of the chromosomal elements encoding resistance to methicillin, and the molecular evolution of the limited number of clones responsible for MRSA infections, worldwide. We also discuss data concerning the recent emergence of MRSA as a significant cause of community-acquired infections. Finally we discuss the implications of the availability of the complete genome sequence of MRSA strains, and suggest important directions for future research.


Chapter 7
Population Structure of MRSA

Ad C. Fluit and Franz-Josef Schmitz

Abstract
This chapter describes the spread of clonal lineages, and the distribution of different clonal lineages of MRSA strains in hospitals, cities and countries. One or two clonal lineages often dominate in a hospital or sometimes even in individual cities, or countries, however many lineages are found only infrequently. Some clones have a pandemic spread. Directly related to the description of clonal spread is presence of different mecA Staphylococcal Cassette Chromosomes (SCCmec) types. Four different types, that vary in composition, have been described. The data obtained from the evolutionary appearance in time of different SCCmec types, demonstrate a link between the SCCmec type and the clonal lineages present. This demonstrates that horizontal transfer of SCCmec between staphylococci took place on least several occasions and may be more common than previously believed. The mechanism of this transfer is still unknown, but the genes necessary for the excision and integration of SCCmec into the staphylococcal chromosome have been identified.


Chapter 8
Vancomycin-Resistant Staphylococcus aureus

Longzhu Cui and Keiichi Hiramatsu

Abstract
The glycopeptide antibiotics, vancomycin and teicoplanin, were the only licensed antibacterial compounds, until recently, to which methicillin-resistant Staphylococcus aureus clinical isolates have remained uniformly susceptible. However, the worldwide increase in the incidence of S. aureus clinical isolates with reduced susceptibility to vancomycin and teicoplanin raises the possibility that glycopeptide resistance in S. aureus is becoming an important clinical problem. S. aureus has evolved genetic and biochemical ways of resisting these antimicrobial actions. Genetic mechanisms include multistep mutations in bacteria chromosome without acquisition of new DNA. A review of available biochemical studies suggests that S. aureus does not resist glycopeptides by inactivating the drugs or altering the drug target, but reducing drug access to bacterial cell membrane by thickened cell wall and changed cell wall components. This article outlines the background to this developing issue with a focus on the mechanisms of resistance. The geographic prevalence, potential for continued spread, and proposed strategies for prevention and control are also discussed.


Chapter 9
Virulence Mechanisms in MRSA Pathogenesis

Jesse S. Wright III and Richard P. Novick

Abstract
The multifactorial virlulence mechanisms of Staphylococcus aureus represent the evolution of a well-adapted human pathogen. The ability of S. aureus to colonize the host safely and its capacity to acquire and exchange genetic information are elements that contribute to its success as a pathogen. Here we discuss the factors that contribute to S.aureus pathogenesis and the potential impact of methicillin resistance on virulence. Virulence factors can be generally separated into two main classes; surface-associated factors, providing the mechanisms for adherence, attachment and immune evasion and secreted factors, which cause tissue destruction and are used to counteract host cell responses. While the roles of several virulence factors in pathogenesis have been elucidated, many more play an unknown role in the infectious process. Elaboration of these virulence determinants occurs in a preprogrammed fashion in vitro by an extensive regulatory network. These regulatory factors integrate environmental parameters to coordinate the expression of several, if not all, virulence genes. While the acquisition of antibiotic resistance, and most notably, methicillin resistance, by S. aureus has had an alarming impact on the treatment of staphylococcal infections, there is little information that links these elements with increased virulence. Complicating the relationship between resistance and virulence is the high degree of genomic diversity found among both antibiotic sensitive and resistance S. aureus isolates. Although gain of antibiotic resistance elements by themselves does not appear to be a major factor in the overall pathogenesis of the organism, three specific areas have been identified in which methicillin resistance may contribute indirectly to virulence.


Chapter 10
Small Colony Variants - Another Mechanism by Which Staphylococcus aureus Can Evade the Immune response and Antimicrobial Therapy

Christof von Eiff and Karsten Becker

Abstract
In the past, most studies have addressed the problem of antibiotic-resistant infectious diseases from the standpoint of classic forms of antibiotic resistance mainly founded on the possession of resistance genes. However, bacteria such as Staphylococcus aureus may have additional mechanisms for resisting therapy that extend beyond these classic mechanisms. In patients whose acute infection initially responded to antimicrobial treatment and which recured after long disease-free intervals or with infections that persisted despite appropriate antibiotic treatment, small colony variants (SCVs) of S. aureus were recovered. A wide variety of bacterial species are known to form SCVs, but in particular in staphyloccoal infections a renewed interest due to SCVs emerged in the last decade following the first description as dwarf-colony or G variants, since an association of the occuurence of S. aureus SCVs and persistent and relapsing infection was described. SCVs are a naturally occuring subpopulation which may be identified in the microbiological laboratory as nonpigmented, nonhemolytic, slow-growing pinpoint colonies. In addition, the often relatively unstable SCVs demonstrate a number of other characteristics that are atypical for S. aureus making the correct identification difficult, including: reduced a-toxin production, delayed coagulase activity, failure to use mannitol and increased resistance to aminoglycosides and cell-wall active antibiotics. Most characteristics of the SCVs can be tied together by a common thread, which is alterations in electron transport. S. aureus SCVs from clinical material are commonly auxotrophic for menadione and hemin, which are key co-factors for the formation of menaquinone and cytochromes, respectively, and are thus important components of the electron transport chain. While studies with clinical isolates of SCVs suggested a link between persistent infections and electron transport defective strains or thymidine-auxotrophic SCVs, a defined hemB mutant mimicing the SCV phenotype provided additional evidence for these connections. In a model of endovascular infection to determine the intracellular persistence, it was demonstrated that > 200-fold more hemB-mutant cells persisted intracellularly after 24 or 48 h incubation relative to the parent strain. The intracellular location of the SCV phenotype may shield this subpopulation from host defenses and antibiotics, thus providing one explanation for the difficulty in clearing S. aureus SCVs from host tissues.


Chapter 11
Treatment of MRSA infections

Debby Ben-David and Ethan Rubinstein

Abstract
Staphylococcus aureus is a major cause of nosocomial infections. In recent years, the prevalence of methicillin-resistant S. aureus (MRSA) has increased considerably. Glycopeptides, the agents of choice for MRSA infections, have a relatively slow bactericidal effect and have been associated with clinical and bacteriological failures. Fusidic acid, rifampin, fosfomycin, quinolones and trimethoprim-sulfamethoxazole all have potential activity against MRSA. However, the increasing cross-resistance to these agents has restricted their treatment efficacy. The emergence of clinical isolates of S. aureus with reduced susceptibility to vancomycin (VISA) is of great concern. Several new antimicrobial agents, including streptogramins, oxazolidinones, daptomycin, glycylcyclines, oritavancin, and peptides, have proved useful against MRSA and VISA strains and are under rapid development. Currently, there are no recommended therapy guidelines for VISA infections. Treatment options include new in vitro active antimicrobial agents against VISA strains, and different existing antibiotic combinations.


Chapter 12
Prevention and Control of Methicillin-Resistant Staphylococcus aureus (MRSA)

Uwe Frank

Abstrach
This chapter is intended to clarify controversial infection control issues in the management of methicillin-resistant Staphylococcus aureus (MRSA). For more than three decades, MRSA strains have been identified as a major source of nosocomial infections and outbreaks in the health-care setting. MRSA presents a challenge to infection control departments in hospitals of varying size attempting to control and eradicate this micro-organism. Controlling the transmission of MRSA is primarily the responsibility of healthcare workers providing direct bedside care to in-patients. Hand-washing and appropriate application of barrier precautions are the two primary transmission prevention measures. The use of single rooms is strongly recommended. If resources are limited, cohorting MRSA colonised/infected patients in one room, ward or area may be a consideration. Unfortunately all health care facilities, regardless of location or the complexity of care rendered will experience sporadic or endemic MRSA over the next few years.


Chapter 13
Concluding Remarks

A.C. Fluit and F.J. Schmitz

No abstract is available for this chapter. Therefore we have provided the first paragraph:
Staphylococcus aureus is an important human pathogen capable of causing a variety of diseases (Chapter 1). This variety of diseases is made possible by an armamentarium of virulence factors, which include adhesins, immune system evasion factors, enzymes, and toxins. It has frequently been suggested that MRSA are more pathogenic than MSSA, but are they? To answer this question one has to consider two possibilities. First, the mobile genetic element SCCmec harboring mecA entered only the more virulent strains. Second, SCCmec contributes to pathogenicity. No conclusive evidence for the first hypothesis is available. Some studies suggest that MRSA are indeed more virulent, whereas other studies contradict this. No studies have been published on the contribution of SCCmec to the virulence of MRSA. Only one possible virulence factor involved in adhesion is encoded on some SCCmec, but its role as a virulence factor is still a matter of debate. So, despite a large volume of research it still not clear whether MRSA are more pathogenic than their methicillin-susceptible counterparts. However, one remarkable finding favors the hypothesis that at least some MRSA are more virulent. All cases of necrotizing pneumonia in youngsters are caused by Panton-Valentine Leukotoxin (PVL) encoding S. aureus, which were all methicillin-resistant (See Wright and Novick, Chapter 9).

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