Staphylococcus aureus Microbiology, Pathology,

Immunology, Therapy and Prophylaxis

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Editors
Fabio Bagnoli Guido Grandi
GSK Vaccines Center for Integrative Biology (CIBIO)
Siena University of Trento
Italy Trento
Italy
Rino Rappuoli
GSK Vaccines
Siena
Italy

ISSN 0070-217X ISSN 2196-9965 (electronic)

Current Topics in Microbiology and Immunology
ISBN 978-3-319-72061-6 ISBN 978-3-319-72063-0 (eBook)
https://doi.org/10.1007/978-3-319-72063-0
Library of Congress Control Number: 2017963738

© Springer International Publishing AG 2017

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Declaration of Interest

Fabio Bagnoli and Rino Rappuoli are employees of GSK Vaccines and own GSK
stocks. Fabio Bagnoli owns patents on S. aureus vaccine candidates. The authors
have no other relevant affiliations or financial involvement with any organization or
entity with a financial interest in or financial conflict with the subject matter or
materials discussed in the manuscript apart from those disclosed.

Authorship

Fabio Bagnoli and Rino Rappuoli were involved in the conception and design
of the book and approved its content before publication.

v
Preface

Staphylococcus aureus is a leading pathogen in surgical site, intensive care unit,

and skin infections as well as health-care associated pneumonias. These infections
are associated with an enormous burden of morbidity, mortality and increase of
hospital length of stay and patient cost. S. aureus is impressively fast in acquiring
antibiotic resistance and multidrug resistant strains are a serious threat to human
health. It has been recently estimated that deaths attributable to antibiotic resis-
tant infections will exceed the ones caused by cancer by 2050 (https://amr-review.
org/Publications). S. aureus, was included among the ESKAPE pathogens
(Enterococcus faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter bau-
mannii, Pseudomonas aeruginosa, and Enterobacter species) recognized as the
leading cause of antibiotic-resistant infections occurring worldwide in hospitals.
Due to resistance or insufficient effectiveness, antibiotics and bundle measures leave
a tremendous unmet medical need worldwide. In addition there are no licensed
vaccines or immunotherapies on the market despite the significant efforts done by
public and private initiatives.
This book includes 16 chapters spanning from basic Microbiology and
Immunology aspects to Pathology of key disease manifestations as well as a review
of current standard of care. Furthermore, front-edge discoveries on therapeutic and
prophylactic approaches alternative to antibiotics are reviewed.
Given the complexity of the Microbiology of this pathogen we decided to give
significant emphasis to this aspect. We started describing conventional and
molecular diagnostics-based identification methods of S. aureus in the microbiol-
ogy laboratory. Rapid and more informative typization tests are likely to represent a
significant benefit for improving clinical practice and containing the emergence of
antimicrobial resistance. Methicillin-resistant S. aureus (MRSA) is a global issue
causing increase of mortality and the need to use last-resource antibiotics.
Predominant clones circulating worldwide and the associated antibiotic resistance
are described.
Sugar and protein surface structures of the bacterium are comprehensively dis-
cussed. These components play key roles in cell viability, virulence and evasion of
host defences. The major surface polysaccharides include the capsular

vii
viii Preface

polysaccharide (CP), cell wall teichoic acid (WTA), and polysaccharide intercel-
lular adhesin/poly-b(1–6)-N-acetylglucosamine (PIA/PNAG). They play distinct
roles in colonization and pathogenesis and are being explored as targets for
antimicrobial interventions.
Surface proteins have very diverse functions (e.g., adhesion, invasion, sig-
nalling, conjugation, interaction with the environment and immune-evasion). They
have been categorized into distinct classes based on structural and functional
analysis. We provide the defining features associated with cell wall-anchored sur-
face proteins and a framework for their categorization based on the current
knowledge of structure and function.
On top of surface virulence factors, S. aureus secretes pore-forming toxins that
kill eukaryotic immune and non-immune cells. Here we provide an update on the
various toxins, the identification of its receptors on host cells, and their roles in
pathogenesis.
S. aureus pathogenicity is driven by the wealth of virulence factors and its ability
to adapt to different environments. The latter is due to the presence of complex
regulatory networks fine-tuning metabolic and virulence gene expression. One
of the most widely distributed mechanisms is the two-component signal trans-
duction system (TCS) that can reveal an environmental signal and trigger an
adaptive gene expression response. It encodes a total of 16 conserved pairs of TCS
that are involved in diverse signalling cascades ranging from global virulence gene
regulation such as quorum sensing by the Agr system, the bacterial response to
antimicrobial agents, cell wall metabolism, respiration and nutrient sensing. Herein
we give an overview of the current knowledge on TCS and its influence on viru-
lence gene expression.
The versatility of S. aureus is reflected by the wide range of disease that it can
cause. It’s a leading cause of bacteraemia, infective endocarditis, osteomyelitis,
pneumonia, indwelling medical device related infections, as well as skin and soft
tissue infections (SSTIs). SSTIs are among the most common infections worldwide.
They range in severity from minor, self-limiting, superficial infections to
life-threatening diseases requiring all the resources of modern medicine. They have
variable presentations ranging from impetigo and folliculitis to surgical site infec-
tions (SSIs). Here we describe the anatomical localization of the different SSTI
associated with S. aureus, the virulence factors known to play a role in these
infections, their current epidemiology as well as the standard of care and potential
prophylaxis.
Musculoskeletal infections, bacteremia and infective endocarditis associated to
S. aureus infections are very difficult to treat and important causes of morbidity and
mortality. Osteomyelitis can cause long-term relapses and functional deficits and
bacteremia and infective endocarditis are associated with excess mortality when
compared to other pathogens. Although considerable advances have been achieved
in their diagnosis, prevention and treatment, the management remains challenging
and impact on the healthcare system is still very high.
S. aureus can also infect several animal species (e.g., cattle, poultry and pigs)
and transmission from animals to humans and vice versa has been observed. This
Preface ix

represents an important threat to public health, as animal strains can adapt to the
human population and spread additional antibiotic resistance.
Medical need associated to S. aureus infections is enhanced by raising preva-
lence of multidrug resistant strains and acquisition of resistance to last resort
antibiotics. Therefore, alternative medical interventions are urgently needed.
Vaccines certainly represent one of the most important options. Unfortunately a
correlate of protection against S. aureus is not known and this represents a sig-
nificant issue for developing vaccines. Herein, we review what is known and
unknown about innate and adaptive immunity against this complex pathogen. We
provide an overview on the major cell types involved in innate immune defence and
major differences of the immune response during colonization versus infection.
Although the contribution of adaptive immunity against S. aureus is not yet clear,
there are accumulating evidence both from animal models and from human data
that T cell- and B cell-mediated adaptive immunity can control the infection.
Unfortunately S. aureus has evolved several mechanisms to manipulate innate and
adaptive immune responses to its advantage. Indeed, it expresses factors able to
interfere with many critical components of the immune system and hamper proper
immune functioning. In recent years research, including structural and functional
studies, has fundamentally contributed to our understanding of the mechanisms of
action of the individual factors.
In addition to the lack of a known correlate of protection, failure of developing
an effective vaccine against this pathogen is likely due to several other reasons.
Indeed. all attempts so far targeted single antigens, contained no adjuvants and
efficacy trials were performed in severely ill subjects. We show the link between
Phase III clinical trial data of failed vaccines with their preclinical observations and
we provide a comprehensive evaluation of potential target populations for efficacy
trials taking into account key factors such as population size, incidence of S. aureus
infection, disease outcome, primary endpoints as well as practical advantages and
disadvantages.
The last chapter provides an overview of a promising new therapeutic approach.
Lysins are a new class of anti-infectives derived from bacteriophage, which cleave
cell wall peptidoglycan causing immediate bacterial lysis. Importantly, lysins have
high specificity for the pathogen and low chance of bacterial resistance.
In conclusion, this volume gives a comprehensive overview of the
Microbiology, Pathology, Immunology, Therapy and Prophylaxis of S. aureus
reviewing recent findings and knowledge on very diverse arguments and at the
same time linked to each other. That is the uniqueness behind a book like this and
the added value towards a search in literature databases.

Siena, Italy Fabio Bagnoli

Rino Rappuoli
Contents

Carriage, Clinical Microbiology and Transmission

of Staphylococcus aureus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Anna Aryee and Jonathan D. Edgeworth
Worldwide Epidemiology and Antibiotic Resistance
of Staphylococcus aureus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Monica Monaco, Fernanda Pimentel de Araujo, Melania Cruciani,
Eliana M. Coccia and Annalisa Pantosti
Structure and Function of Surface Polysaccharides
of Staphylococcus aureus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
Christopher Weidenmaier and Jean C. Lee
Cell Wall-Anchored Surface Proteins of Staphylococcus aureus:
Many Proteins, Multiple Functions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Joan A. Geoghegan and Timothy J. Foster
Staphylococcus aureus Pore-Forming Toxins . . . . . . . . . . . . . . . . . . . . . 121
Tamara Reyes-Robles and Victor J. Torres
The Role of Two-Component Signal Transduction Systems
in Staphylococcus aureus Virulence Regulation . . . . . . . . . . . . . . . . . . . 145
Andreas F. Haag and Fabio Bagnoli
Staphylococcus aureus-Associated Skin and Soft Tissue
Infections: Anatomical Localization, Epidemiology,
Therapy and Potential Prophylaxis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Reuben Olaniyi, Clarissa Pozzi, Luca Grimaldi and Fabio Bagnoli
Staphylococcus aureus-Associated Musculoskeletal Infections . . . . . . . . . 229
Evgeny A. Idelevich, Carolin Kreis, Bettina Löffler and Georg Peters

xi
xii Contents

Bacteremia, Sepsis, and Infective Endocarditis Associated

with Staphylococcus aureus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263
Stephen P. Bergin, Thomas L. Holland,
Vance G. Fowler Jr. and Steven Y.C. Tong
Amphixenosic Aspects of Staphylococcus aureus Infection
in Man and Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
Giacomo Rossi, Matteo Cerquetella and Anna Rita Attili
Treatment of Staphylococcus aureus Infections . . . . . . . . . . . . . . . . . . . . 325
Michael Z. David and Robert S. Daum
The Innate Immune Response Against Staphylococcus aureus . . . . . . . . 385
Isabelle Bekeredjian-Ding, Christoph Stein and Julia Uebele
Adaptive Immunity Against Staphylococcus aureus . . . . . . . . . . . . . . . . 419
Hatice Karauzum and Sandip K. Datta
Staphylococcal Immune Evasion Proteins: Structure, Function,
and Host Adaptation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441
Kirsten J. Koymans, Manouk Vrieling, Ronald D. Gorham Jr.
and Jos A.G. van Strijp
Vaccines for Staphylococcus aureus and Target Populations . . . . . . . . . 491
Clarissa Pozzi, Reuben Olaniyi, Lassi Liljeroos, Ilaria Galgani,
Rino Rappuoli and Fabio Bagnoli
Lysin Therapy for Staphylococcus aureus
and Other Bacterial Pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529
Vincent A. Fischetti
Carriage, Clinical Microbiology
and Transmission of Staphylococcus
aureus

Anna Aryee and Jonathan D. Edgeworth

Abstract Staphylococcus aureus is one of the most important bacterial pathogens

in clinical practice and a major diagnostic focus for the routine microbiology lab-
oratory. It is carried as a harmless commensal in up to two-thirds of the population
at any one time predominantly not only in the anterior nares, but also in multiple
other sites such as the groin, axilla, throat, perineum, vagina and rectum. It colo-
nizes skin breach sites, such as ulcers and wounds, and causes superficial and deep
skin and soft tissue infections and life-threatening deep seated infections particu-
larly endocarditis and osteomyelitis. S. aureus is constantly evolving through
mutation and uptake of mobile genetic elements that confer increasing resistance
and virulence. Since the 1960s, hospitals have had to contend with emergence of
methicillin-resistant S. aureus (MRSA) strains that spread better in hospitals than
methicillin-susceptible S. aureus (MSSA) and are harder to treat. Since the 1980s,
distinct community MRSA strains have also emerged that cause severe skin and
respiratory infections. Conventional identification of MSSA and MRSA in the
microbiology laboratory involves microscopy, culture and biochemical analysis that
for most samples is straightforward but slow, taking at least 48 h. This delay has
significant consequences for individual patient care and public health, through
inadequate or excessive empiric antibiotic use, and failure to implement appropriate
infection control measures for MRSA-colonized patients during those first 48 h.
This unmet need has driven development of rapid molecular diagnostics that either
complement or replace conventional culture techniques in the laboratory, or can be
placed in the clinical environment as point-of-care (POC) devices. These new
technologies provide results to clinicians anything from within an hour to 24 h,
depending on sample and clinical setting, and should transform management of
patients with S. aureus and other bacterial diseases; however, uptake is often slow
due to the disruptive effect of new technologies, costs of transition and uncertainty

A. Aryee
J.D. Edgeworth (&)

Centre for Clinical Infection and Diagnostics Research,
Department of Infectious Diseases, Kings College London
and Guy’s and St. Thomas’ NHS Foundation Trust, 5th Floor North Wing,
Westminster Bridge Road, London SE1 7EH, UK
e-mail: [email protected]

Current Topics in Microbiology and Immunology (2017) 409:1–19

DOI 10.1007/82_2016_5
© Springer International Publishing Switzerland 2016
Published Online: 21 April 2016
2 A. Aryee and J.D. Edgeworth

of the optimal solution given successive advances. More evidence of the health
economic, clinical and antimicrobial resistance benefit will help support introduc-
tion of these new technologies. Finally, preventing MRSA transmission has been a
priority for healthcare organizations for many years. There have been significant
recent reductions in transmission following local and national campaigns to
re-enforce basic and heightened infection control interventions such as universal
hand hygiene, barrier nursing, decolonization and isolation of MRSA-colonized
patients detected through routine culture or screening policies. Developments in
whole genome sequencing are providing greater insight into reservoirs and routes of
transmission that should help better target interventions to ensure sustainable
control of endemic strains and to identify and prevent emergence of new strains.

Contents
1 Clinical Microbiology.......................................................................................................... 2
1.1 Introduction of Rapid Molecular Detection Methodologies...................................... 4
1.2 Enhancing Culture-based Techniques ........................................................................ 4
1.3 Replacing Culture-based Techniques ......................................................................... 5
1.4 Point-of-Care Technologies........................................................................................ 7
2 S. aureus Carriage ............................................................................................................... 7
3 S. aureus Transmission ....................................................................................................... 9
3.1 MRSA Transmission in the Hospital ......................................................................... 10
3.2 Preventing MRSA Transmission................................................................................ 11
3.3 MRSA Transmission in the Community ................................................................... 12
4 Summary.............................................................................................................................. 14
References .................................................................................................................................. 14

1 Clinical Microbiology

Staphylococcus aureus is a facultative anaerobe belonging to the genus

Staphylococcus within the family of Staphylococcae. It is one of the most com-
monly identified clinically significant bacteria in a routine microbiology laboratory,
and its identification by traditional techniques is a straightforward, albeit slow
process, which is becoming more rapid with the introduction of molecular
techniques.
Upon receipt of samples in the laboratory, Gram staining can be performed on
sterile site samples such as pus and deep respiratory specimens to identify the
presence of bacteria by light microscope. Staphylococci appear as irregular small
clusters of Gram-positive cocci and traditionally no further information is available
to the clinician on the first day. Samples are cultured on blood agar for 18–24 h
when S. aureus colonies appear glistening, smooth and translucent, often with a
golden pigment. Presumptive colonies are confirmed as S. aureus at this point using
the techniques described below, although plates are usually re-incubated for a
Carriage, Clinical Microbiology and Transmission … 3

further 24 h to detect slower growing colonies. Antibiotic susceptibility testing can

also be set up on colonies identified at 24 h. By 48 h, colonies are approximately 1–
2 mm in diameter and often exhibit a small zone of b-haemolysis. Thus, in a
traditional laboratory, the clinician can expect to be told if staphylococci are present
in important sterile site samples on the day of sample collection, whether S. aureus
is present in the sample the following day, and receive a final report with antibiotic
susceptibilities the day after.
A variety of biochemical tests are used to identify S. aureus colonies based on
production of coagulase and deoxyribonuclease, presence of S. aureus specific
antigens or the ability to ferment mannitol. The tube coagulase test is the traditional
gold standard for discriminating between S. aureus and other staphylococci, usually
referred to as coagulase-negative staphylococci (CoNS). This is a clinically
important distinction because CoNS are rarely pathogenic in the absence of pros-
thetic material upon which they can reside in biofilm, although it is recognized that
some CoNS are coagulase positive and some coagulase-negative S. aureus isolates
have been reported (Vandenesch et al. 1993). The slide coagulase test is a more
rapid test based on the presence of clumping factor, but up to 15 % of S. aureus
isolates are negative. Latex agglutination tests detecting protein A, clumping factor
and other surface antigens are also sensitive although less specific due to
cross-reactivity with various CoNS.
Antimicrobial susceptibility testing is set up at the same time as identification of
S. aureus using a number of culture-based methodologies. Disc diffusion testing is
often used to assess simultaneous susceptibility to a variety of antibiotics. A key
focus is to distinguish between methicillin-susceptible and methicillin-resistant S.
aureus. This can be done using an oxacillin or cefoxitin disc, which has been shown
to be an accurate surrogate marker for methicillin resistance (Skov et al. 2006).
Antibiotic susceptibilities can also be performed using commercially available
automated platforms such as the Vitek®2, BD Phoenix™ or MicroScan WalkAway
systems.
An additional important focus for the microbiology laboratory is the specific
detection of methicillin-resistant S. aureus (MRSA) in screening swabs from car-
riage and clinical sites, particularly the anterior nares, to identify colonized patients
and institute infection control precautions (Coia et al. 2006). Many laboratories
inoculate screening swabs directly onto selective agar, particularly chromogenic
agars that provide a presumptive positive identification of MRSA within 24 h of
sample receipt in the laboratory (Nahimana et al. 2006; Denys et al. 2013).
Excluding presence of MRSA requires a further 24 h, and presumptively positive
samples should be confirmed by antimicrobial susceptibility testing.
The analysis of blood cultures differs from other samples. 10–15 ml of blood is
inoculated into media bottles immediately after collection from the patient and sent
to the laboratory where they are placed into automated incubators. Positive cultures
are flagged when bacterial growth is detected usually by continuous monitoring of
changes in pH due to CO2 production. The time taken for automated systems to
detect bacteria depends on the number of bacteria in the sample (which can be up to
200 CFU/ml for endovascular infection down to <10 bacteria per ml of blood) and
4 A. Aryee and J.D. Edgeworth

the initial viability of bacteria that may either be intracellular or dormant. For
Staphylococcus aureus bacteriaemia (SAB), over 80 % of positive culture bottles
flag within 24 h (Khatib et al. 2005). Gram staining is performed on an aliquot of a
flagged bottle to identify staphylococci, although this information has only limited
clinical benefit because CoNS are more frequently identified in blood cultures.
Conventionally, flagged blood culture media is plated onto agar that provides
identification and disc diffusion susceptibility testing results the following day.

1.1 Introduction of Rapid Molecular Detection

Methodologies

The slow nature of culture and biochemical-based detection methods means that
identification and antimicrobial susceptibility of S. aureus only becomes available
about 48 h after initial key clinical management decisions are made, and this is
recognized as a major clinical and public health problem. For the individual patient,
if serious S. aureus infection was not clinically suspected, then the patient may not
be started on appropriate initial antibiotic therapy, particularly if the S. aureus is
methicillin resistant, and this delay has been associated with higher mortality in
some studies (González et al. 1999; Soriano et al. 2000). At a population level,
uncertainty about whether an acute illness is bacterial or the likely antimicrobial
susceptibilities prompts empiric treatment with broad-spectrum antibiotics to cover
a range of potential bacterial causes including MRSA. This presents a public health
problem due to overuse of empiric antibiotics that drives antibiotic resistance. There
is also delay in identifying MRSA-colonized patients and instituting infection
control precautions, which increases the potential for nosocomial transmission.
These unmet clinical needs have driven the development of rapid molecular
diagnostics throughout the patient pathway to speed up time to detection and
reporting of pathogenic bacteria including S. aureus. In the laboratory, these
molecular methods can either enhance traditional culture-based processing or
completely replace culture-based techniques.

1.2 Enhancing Culture-based Techniques

This involves rapid laboratory-based molecular analysis of S. aureus colonies or

flagged positive blood culture bottle after initial culture of specimens for 24 h or
more. Many laboratories have introduced matrix-assisted laser desorption
ionization-time-of-flight mass spectrometry (MALDI-TOF MS), which identifies
bacterial colonies by analysing the protein composition of the bacterial cell (Wieser
et al. 2012). This new technology has transformed species identification in
Carriage, Clinical Microbiology and Transmission … 5

microbiology laboratories allowing bacterial identification within minutes: it is

cheaper, more accurate and usually faster than biochemical-based methodologies
and can replace most traditional biochemical tests. It was initially applied to bac-
terial colonies but has also been successfully applied to aliquots of blood culture
sample that have flagged as positive (Mestas et al. 2014). Results are available
within an hour although identification of Gram-positive bacteria is less effective
than Gram-negative bacteria (78 % vs. 90 %). Additionally, rapid latex aggluti-
nation tests can be performed on single colonies or positive blood culture bottles to
detect PBP2a as a marker of MRSA (Brown and Walpole 2001; Chapin and
Musgnug 2004).
There have also been advances in the rapid nucleic acid based detection of
organisms including MSSA and MRSA from flagged positive blood culture bottles
(Opota et al. 2015). The Cepheid Xpert system uses PCR to identify S. aureus and
MRSA direct from positive blood culture samples in about 2 h. PCR correlated
with culture results in 80/82 (97.5 %) flagged blood culture bottles containing GPC
in clusters by microscopy (Ratnayake and Olver 2011). Nanosphere’s Verigene
Gram-positive blood culture test allows rapid identification of both MSSA and
MRSA from positive blood culture samples in less than 3 h. Mono-microbial
bacterial isolates were correctly identified in 147 of 148 flagged blood culture
bottles containing Gram-positive bacteria (38 MRSA or MSSA) (Beal et al. 2013).
The FilmArray Blood Culture ID panel identifies 24 organisms and 3 antibiotic
resistance genes including S. aureus and mecA in positive blood culture samples in
approximately 1 h. The FilmArray correctly identified 19 S. aureus isolates from
167 mono-microbial flagged blood culture bottles and 156 (91.6 %) isolates overall
(Altun et al. 2013). These technologies reduce by about 24 h the time to provide
clinicians with a S. aureus identification and methicillin susceptibility result from
blood cultures.
Rapid non-nucleic acid-based technologies are also under development.
Accelerate diagnostics have a platform that uses automated digital microscopy and
high-resolution growth analysis to provide identification and antimicrobial sus-
ceptibility data from blood and other sterile samples in approximately 5 h. It cor-
rectly identified all 77 MRSA and 54 MSSA mostly reference isolates in one study
(Price et al. 2014a). Specific Technologies are developing a system that detects
mixtures of volatile organic compounds using a colorimetric array integrated into a
blood culture bottle, allowing faster detection and identification than traditional
methods, although it does not provide susceptibility data (Lim et al. 2014).

1.3 Replacing Culture-based Techniques

Technologies are being developed to provide bacterial identification and genotypic

prediction of antimicrobial susceptibilities directly on primary samples. Some are
designed to analyse clinical samples including blood cultures and include a broad
6 A. Aryee and J.D. Edgeworth

range of bacteria. For example, Abbott’s Iridica system identifies over 750 bacteria
and 4 antibiotic resistance genes (including mecA) from a range of samples
including whole blood and respiratory specimens in under 6 h. The Mobidiag
Prove-It™ Bone & Joint StripArray system identifies over 30 Gram-positive and
Gram-negative bacterial species and various genotypic resistance determinants
including the mecA gene from synovial fluid, bone biopsy and tissue in 3.5 h from
DNA extraction. In one study, 8 of 38 prosthetic joint infection samples culture
positive for S. aureus were also identified by PCR and there was one additional
PCR positive sample in a patient who had received antibiotics before sample col-
lection that was culture negative (Metso et al. 2014). The Curetis Unyvero pneu-
monia platform detects 18 bacteria including S. aureus and the mecA gene directly
from respiratory samples in 4–5 h (Jamal et al. 2014). Direct molecular analysis of
clinical samples rather than a colony or suspension after culture allows same day
detection of pathogens including MSSA and MRSA and rapid targeting of
appropriate therapy.
Diagnostics have also been developed for the specific detection of S. aureus and
the mecA gene including the Cepheid Xpert systems, the LightCycler MRSA
Advanced and BD Max MRSA. These PCR tests take about 2 h (Rossney et al.
2008; Peterson et al. 2010; Widen et al. 2014) and have comparable sensitivity and
specificity to enrichment and plating on different chromogenic agars (all > 92 %)
whilst saving 24–72 h (Lee et al. 2013). Agreement between enriched culture and
PCR was 96 % in this study. Although these S. aureus specific tests have been
predominantly applied to MRSA screening swabs to target infection control
interventions, they could also be used on clinical samples such as skin and soft
tissue samples (Wolk et al. 2009) and respiratory specimens (Cercenado et al. 2012)
to target early appropriate therapy.
Although molecular diagnostics dramatically reduce analysis time and can
provide same day identification of MSSA and MRSA, the adoption into routine
laboratory service is not straightforward. Molecular technologies are usually more
expensive than traditional techniques, require a period of double running during
evaluation and are then often used alongside rather than completely replacing the
routine culture bench, so fixed costs remain. The time taken to transport specimens
to the laboratory, particularly when the laboratory is off-site, can make a same-day
test into a next-day test for a significant proportion of specimens, particularly if
batch processing rather than random access platforms is used (Jeyaratnam et al.
2008). Samples submitted in the afternoon may provide results in the middle of the
night when specialists who advise on result interpretation and patient management
may not be available; hence, decisions may be postponed until the following day,
when results would have become available using culture-based techniques: this may
be particularly relevant if the advice is to narrow antibiotic spectrum. Molecular
diagnostics are therefore disruptive for the laboratory and clinical teams, and
adoption will require evidence of clinical benefit and cost effectiveness, and edu-
cation of staff across the clinical pathway to realize the anticipated benefits of rapid
diagnostics (Wassenberg et al. 2011; Van Der Zee et al. 2013).
Carriage, Clinical Microbiology and Transmission … 7

1.4 Point-of-Care Technologies

An even more radical advance for clinical microbiology is the movement of diag-
nostics out of the laboratory to the ward or bedside. Laboratory platforms or new
point-of-care (POC) devices that can rapidly identify pathogens including MSSA and
MRSA are being evaluated on the wards. Unlike laboratory-based testing, sample
analysis at the bedside can influence initial empiric treatment and infection control
decisions. Some studies have assessed laboratory platforms such as the Cepheid
Xpert system to detect MRSA on the ICU, in general ward and in outpatient clinics
(Leone et al. 2013; Parcell and Phillips 2014). Many companies are also developing
small bench top devices that are specifically designed for use by non-laboratory
personnel on the wards; for example, Cobas and Alere-I, Atlas Diagnostics, Enigma
Diagnostics, BioCartis Idylla, Orion Diagnostica and GNA Biosolutions and clinical
utility studies are being performed for some pathogens (Binnicker et al. 2015;
Goldenberg and Edgeworth 2015; van den Kieboom et al. 2015). Even closer to the
patient from a ward-based to bedside-based system, a hand-held device is being
developed that can identify MSSA or MRSA within 30 min (www.quantumdx.com).
This field is in its infancy, and the technology advancing so rapidly, it is unclear what,
where and when rapid POC infectious diseases diagnostics will enter into routine
clinical practice. There will be many factors to consider including training front-line
staff, quality control, accreditation, regulatory and legal constraints, linking results to
hospital health records, resolving discrepancies between POC- and
laboratory-generated results, and having mechanisms to alert specialist teams for
advice and follow-up. At an organizational level, there will need to be strong gov-
ernance processes to ensure POC devices are introduced safely and consistently,
recognizing that the clinical environment is more complex than the laboratory, where
there is a tradition of high-quality process control. Health economic evaluations that
incorporate all the costs and benefits of laboratory versus POC-based testing will be
needed to support decision-making of clinicians and managers.

2 S. aureus Carriage

Staphylococcus aureus is part of the commensal flora of human skin and mucosal
surfaces, in addition to being a pathogen capable of causing both superficial
infections and invasive disease with considerable associated morbidity and mor-
tality. The anterior nares are the main reservoir of S. aureus carriage in humans.
Other carriage sites include the skin, perineum, pharynx, gastrointestinal tract,
vagina and axillae (Wertheim et al. 2005). About one-third of the population carry
S. aureus on skin and mucosal sites at any one time (Kluytmans et al. 1997). Some
individuals harbour the same strain over an extended period of time, whereas others
carry different strains. S. aureus may also be present at different anatomical sites
with varying frequency in different populations (Wertheim et al. 2005).
8 A. Aryee and J.D. Edgeworth

The variability in the detection of S. aureus at carriage sites has led to the
description of distinct states, with potentially distinct underlying mechanisms. In
the early 1960s, carriage was designated into four groups, persistent, intermittent,
occasional and non-carriage (Williams 1963), but most studies now recognize three
states: persistent, intermittent and non-carriage (Nouwen et al. 2004). It has been
reported that approximately 60 % of the population are intermittent carriers, whilst
20 % each are either persistent carriers or non-carriers (Kluytmans et al. 1997).
A large longitudinal survey published in 1997 analysing nasal swabs from staff at a
university hospital found the same strain of S. aureus (confirmed by PGFE) in the
same individuals on two occasions eight years apart in 3 out of 17 (18 %) staff
members, suggesting that persistence reflects a stable host strain relationship
(VandenBergh et al. 1999). However, a longitudinal study of 109 healthy indi-
viduals over a period of up to three years found persistent carriers having a resident
persistent strain for most of the time but with additional distinct strains at other
times (Muthukrishnan et al. 2013).
The prevalence of transient and persistent S. aureus nasal carriage varies by
geographical location, age, gender and ethnicity. Studies have shown carriage
ranges from 9 % in Indonesia to 37 % in Mexico (Lestari et al. 2008;
Hamdan-Partida et al. 2010). Carriage is highest amongst newborns (up to 70 %)
but steadily decreases in childhood. It has been posited, but not proven, that this
may be due to pneumococcal competition or interference by other bacteria present
in the nasopharynx in childhood (Lebon et al. 2008). There is another peak at
adolescence followed by a decrease in early adulthood. Persistent carriage is seen
more frequently in children than adults, and a conversion from persistent to tran-
sient or non-carriage most commonly occurs in adolescence (Williams 1963;
Kluytmans et al. 1997; Wertheim et al. 2005). Rates of carriage have also been
found to be higher in patients with Type 1 Diabetes Mellitus, intravenous drug
users, haemodialysis patients, surgical patients, AIDS patients and patients with
qualitative or quantitative defects in leucocyte function (Lowy 1998).
The fact that S. aureus is found at multiple body sites, that many studies have
only looked for nasal carriage, and that detection methodologies are of variable
sensitivity complicates our understanding of the significance of carriage states.
Evidence from longitudinal studies does imply that persistent carriers and persistent
non-carriers are distinct and likely therefore to have an underlying biological
explanation, but the significance of transient carriage is less clear. Defining the host
and bacterial factors involved in carriage should help resolve this issue. A feature of
persistent carriers identified in a number of studies is that they carry a higher
bacterial load than intermittent carriers (Nouwen et al. 2004; Van Belkum et al.
2009). This higher bacterial load may mean that persistent carriers are also more
likely to be implicated in transmission of S. aureus. This also has implications for
autoinfection—with persistent carriers at significantly higher risk of this than
transient and non-carriers (Von Eiff et al. 2001; Wertheim et al. 2004b, 2005).
Studies have also specifically investigated carriage of MRSA in hospitalized
patients, to determine the optimal sites for screening programmes. Screening is
often performed at the anterior nares alone but this can miss up to a third of
Carriage, Clinical Microbiology and Transmission … 9

MRSA-colonized patients (Meurman et al. 2005), particularly those with throat or

rectal carriage, and the latter may be particularly important for hospital transmission
(Boyce et al. 2007). Screening programmes in high-risk areas often take swabs from
multiple carriage sites to ensure colonized patients are detected (Batra et al. 2008).
It is, however, unclear whether MRSA has a differential propensity for carriage at
particular sites compared with MSSA.
A number of studies have attempted to identify human genetic factors associated
with carriage. A study in 2007, conducted as part of the Rotterdam study (a
prospective, population-based study of the incidence and risk factors of disease in
an elderly population), sought to identify polymorphisms in host inflammatory
response genes associated with susceptibility to S. aureus carriage and infection.
They found the Interleukin 4 (IL4)–524 C/C host genotype was associated with
increased risk of S. aureus carriage, irrespective of organism genotype. They also
found that individuals with the C-reactive protein (CRP) haplotype 1184C; 2042C;
2911C were less likely to be colonized, and that individuals with boils were more
likely to be carriers of the CFH Tyr402 variant and the CRP 2911 C/C genotype
(Emonts et al. 2008). A study carried out in 2006 and 2008 compared the genetics
of S. aureus strains, epidemiological risk factors, antibiotic exposure and allelic
polymorphisms of human genes posited to be involved in carriage of persistent
carriers as compared to those of volunteers in an isolated population of adult
Wayampi Amerindians living in an village in the Amazonian forest. The authors
concluded that a specific set of host genetic polymorphisms were the main deter-
minants of S. aureus persistent nasal carriage, namely single nucleotide polymor-
phisms (SNPs) for CRP genes (C2042T and C1184T) and IL4 genes (IL4 C524T)
(Ruimy et al. 2010). A further study published in 2006, also as part of the
Rotterdam study, examined the role of host polymorphisms in the glucocorticoid
receptor gene in persistent S. aureus carriage. They found GG homozygotes of the
exon 9b polymorphism had a 68 % reduced risk of persistent carriage, whereas
carriers of the codon 23 lysine allele had 80 % increased risk (Van den Akker et al.
2006).

3 S. aureus Transmission

The high prevalence of transient or persistent carriage with genetically diverse S.

aureus strains in all human populations makes the epidemiology of S. aureus
complex. Most attention has focused on transmission during outbreaks, particularly
with MRSA or clones that are associated with more frequent and severe disease;
however, it is important to also focus on transmission of endemic MSSA clones not
least to define the mechanistic basis for successful and outbreak strains. Our
understanding of S. aureus transmission has advanced dramatically with recent
developments in whole genome sequencing (WGS) supported by advances in
bioinformatics, mathematical modelling and social network analysis. Sequencing
and interpreting hundreds of bacterial genomes is now feasible in some centres