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Staphylococcus aureus - Biology

Staphylococcus aureus - Biology


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Staphylococcus aureus

Scientists reveal silver-based antimicrobials can be utilized as antibiotic adjuvants to combat antibiotic-resistant Staphylococcus aureus

A research team led by Professor Hongzhe SUN, Norman & Cecilia Yip Professor in Bioinorganic Chemistry and Chair Professor from Research Divison for Chemistry and Department of Chemistry, Faculty of Science, in collaboration with Dr Richard Yi-Ysun KAO, Associate Professor from the Department of Microbiology, Li Ka Shing Faculty of Medicine, and Dr Aixin YAN, Associate Professor from School of Biological Sciences, the University of Hong Kong (HKU), discovers that silver (Ag)-based antimicrobials can effectively combat antibiotic resistant Staphylococcus aureus by targeting multiple biological pathways via functional disruption of key proteins and can be further exploited to enhance the efficacy of conventional antibiotics as well as to resensitise methicillin-resistant Staphylococcus aureus (MRSA) to antibiotics.

The study resolves the long-standing question of the molecular targets of silver in Staphylococcus aureus and offers insights into the sustainable bacterial susceptibility of silver, providing a new approach for combating antimicrobial resistance. The ground-breaking findings are now published in a leading multidisciplinary science journal, Nature Communications.

Antibiotics are medicines designed to kill bacteria and treat bacterial infections. Antibiotic resistance occurs when bacteria adjust in response to the misuse or overuse of these medicines, and it has become one of the biggest public health challenges in this era. At least 2.8 million people get an antibiotic-resistant infection annually in the US, and more than 35,000 people die from it.

Staphylococcus aureus, a round-shaped Gram-positive bacterium, is a dangerous and versatile pathogen for humans and is estimated that approximately 30% of the human population are asymptomatic nasal and long-term carriers. Staphylococcus is the causative agent of a variety of diseases, such as skin infection, food poisoning, bone/joint infection, and bacteremia, ranging from subacute superficial skin infection to life-threatening septicemia. The rise in incidence has been accompanied by an increase in antibiotic-resistant strains, especially MRSA. Moreover, the outbreak of the Coronavirus Disease 2019 (COVID-19) pandemic may further increase antimicrobial resistance due to the heavy use of antibiotics to treat patients infected with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-COV-2). Given the rapid emergence of drug-resistant Staphylococcus aureus but a lack of antibiotic-development pipeline, alternative strategies are urgently needed to combat antibiotic-resistant Staphylococcus aureus.

Key findings

Metal ions have been historically used as antimicrobial agents owing to their inherent broad-spectrum antimicrobial properties and less chance of resistance. There is a growing interest in revitalising metal-based compounds as promising alternatives to tackle the antimicrobial resistance crisis. Silver ions (Ag+) and silver nanoparticles (AgNPs) have been used as antimicrobial agents for centuries and are still being widely used in the healthcare and food industry. Previously, the team has built a technical platform named LC-GE-ICP-MS to systematically identify Ag+-proteome in Escherichia coli and developed a strategy named metabolome reprogramming to enhance the efficacy of antibacterial metallodrugs (PLoS Biol., 2019, 17, e3000292 Chem. Sci., 2019, 10, 7193-7199 Chem. Sci., 2020, 11, 11714-11719).

In this study, using the customised approach of LC-GE-ICP-MS, the team successfully separated and identified 38 authentic Ag+-binding proteins (Ag+-proteome) in Staphylococcus aureus at the whole-cell scale. In combination with bioinformatics analysis and systematic biochemical characterisation, they demonstrate that Ag+ exploits a shotgun action through targeting multiple proteins, thus interfering with multiple pathways, including glycolysis, oxidative pentose phosphate pathway (oxPPP), and reactive oxygen species (ROS) stress defence system, to exert its bactericidal effect against Staphylococcus aureus. Further studies unveiled that oxPPP served as a vital pathway targeted by Ag+ in Staphylococcus aureus, with 6PGDH identified as the key enzyme involved in the inhibitory effects of Ag+ against Staphylococcus aureus. They resolved the first crystal structures of 6PGDH from Staphylococcus aureus both in substrate-bound and Ag-bound forms and revealed that Ag+ abolished the enzymatic activity of 6PGDH through targeting Histidine 185 in the active site and morphing its catalytic pocket. This study resolves the long-standing question on the molecular targets and mode of action of silver against Staphylococcus aureus. Such a unique mode of action of silver via targeting multiple pathways confers the inability to select silver-resistant Staphylococcus aureus and endows it with the sustainable efficacy against Staphylococcus aureus.

Based on the uncovered molecular mechanism, they further demonstrate that Ag+/AgNP can potentiate the efficacy of a broad range of antibiotics, resensitise MRSA to antibiotics, and slow down the evolution of antibiotic resistance in Staphylococcus aureus. Therefore, a combination of antibiotics with silver or other metal-based compounds or nanomaterials could serve as a promising strategy to suppress the selection effects of antibiotics, thus preventing the occurrence of primary antibiotic resistance and extending the lifespan of conventional antibiotics to relieve the current crisis of antibiotic resistance.


Background

Methicillin-resistant Staphylococcus aureus (MRSA) has been identified as one of the major risk pathogens associated with the development of antimicrobial resistance (AMR). The emergence of AMR in S. aureus is well documented and the species has proven particularly adept at evolving resistance in the face of new antibiotic challenges. The introduction of penicillin in the 1940s heralded a revolution in the treatment of infectious diseases. However, at the same time as its use was becoming more widespread following advances in the scaling up of production, evidence of penicillin resistance in S. aureus was already being uncovered [1].

Methicillin (Celbenin), a semi-synthetic β-lactam, was introduced in the UK in 1959 to circumvent growing penicillin resistance in S. aureus, associated with the acquisition of a β-lactamase enzyme, blaZ [2]. As a second-generation β-lactam antibiotic, methicillin was insensitive to breakdown by BlaZ. Following the introduction of methicillin into clinical practice in the UK, the Staphylococcal Reference Laboratory in Colindale (London, England) screened S. aureus isolates for evidence of resistance to this antibiotic [3]. More than 5000 S. aureus strains were assessed in the period between October 1959 and November 1960, and in October 1960 three isolates showing increased minimum inhibitory concentrations (MICs) to the new drug, methicillin, were identified. The isolates originated from the same hospital and shared a common phage type and resistance profile (penicillin, streptomycin, and tetracycline), suggesting that they were related. In the description of these isolates it was noted that methicillin had been used only once previously at this hospital, and that none of the individuals from whom MRSA was isolated had been exposed to the drug. Within 2 years MRSA was being detected elsewhere in Europe, with invasive infections being identified in Denmark [4]. These MRSA isolates from the UK and Denmark in the early 1960s constitute the very first epidemic MRSA clone.

The genetic basis of methicillin resistance in S. aureus is associated with carriage of a mobile cassette of genes known as the staphylococcal cassette chromosome mec (SCCmec) [5]. Within this cassette is the mecA gene that is responsible for resistance to β-lactams including methicillin. The product of mecA is the peptidoglycan synthesis enzyme penicillin binding protein (PBP) 2a involved in cross-linking of peptidoglycan in the bacterial cell wall [6, 7]. PBP2a has a lower binding affinity for β-lactam antibiotics than the native PBP proteins encoded in the core genome of S. aureus. The subsequent combination of reduced penicillin-binding affinity and increased production of PBP2a accounts for the observed resistance to β-lactam antibiotics.

Genetic analyses of the first MRSA by multi-locus sequence typing (MLST) demonstrated that they were sequence type (ST) 250, a lineage belonging to clonal complex (CC) 8 and carried the type I SCCmec element [8, 9]. After emerging in the UK, this first epidemic MRSA clone (ST250-MRSA-I) spread across Europe during the 1960s and 70s, but by the late 1980s had become less prevalent and is now rarely reported [9,10,11]. The single locus variant and close relative of ST250-MRSA-I, ST247-MRSA-I was first detected in Denmark in 1964 [8] and has been more successful, spreading globally and persisting as a source of outbreaks in Europe into the late 1990s [10, 11], but this too has been superseded by more successful contemporary clones [10]. Five decades on since the appearance of the first MRSA, multiple MRSA lineages have emerged which have acquired different variants of SCCmec elements.

Epidemiological evidence has always suggested that MRSA arose as a consequence of the introduction of methicillin into clinical practice. Here we have used whole genome sequencing of a collection of 209 of the earliest MRSA isolates recovered in Europe between 1960 and 1989 to reconstruct the evolutionary history of methicillin resistance. Using Bayesian phylogenetic reconstruction we have identified the likely time point at which this early lineage arose and also predicted the time around which SCCmec was acquired.


STAPHYLOCOCCUS AUREUS

Staphylococcus aureusis a Gram-positive, coagulase-positive, catalase-positive, non-motile coccus found in the genus Staphylococcus and family Staphylococcaceae. They are facultative anaerobic organisms, and they cause haemolysis on blood agar. Staphylococcus species are usually arranged in groups, in pairs, as well as in tetrads. They can also occur singly or as single cells. S. aureus usually appear as grape-like clusters under the microscope. They are asporogenous or non-sporulating in nature. Asporogenous bacteria are organisms that do not produce spores. S. aureus are habitually found in the nose of humans but it may be found regularly in most other anatomical sites of the body such as the respiratory tract, mucous membranes, GIT and skin. They can also be found on fomites including tables, clothing materials, and bed linens. Out of all the species of Staphylococcus, only Staphylococcus epidermidis (a normal flora of the human skin) and S. aureus are more clinically important to humans. S. aureus normally exist as either resident or transient microorganism on the human skin.

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In the microbiology laboratory, S. aureus can easily be isolated by taking samples from the nose and skin of volunteers and culturing same on growth/bacteriological culture media that supports their growth. It is noteworthy that a good percentage of healthy individuals carry S. aureus in their nose, and these persons can be infectious to susceptible hosts by transferring the microbe via their hands (after picking their noses) or through body contacts. S. aureus cause gastroenteritis and they can also colonize the female vagina especially during menstruation to cause infection. Thus, most infections of S. aureus are due to poor personal and environmental hygiene. S. aureus causes a variety of infections/diseases in humans and these include abscesses, wound infections, gastroenteritis, toxic shock syndrome toxin (TSST) disease, pneumonia, burns, and septicaemia. S. aureus produce a range of enzymes and toxins which aids in its pathogenicity or disease course. S. aureus is mostly implicated in human pyogenic infections such as boils, pimples, impetigo and pustules.

PATHOGENESIS OF STAPHYLOCOCCUS AUREUS INFECTION

S. aureus is notorious in causing a variety of invasive and mixed infections in humans including pus-forming infections, food-poisoning (gastroenteritis) characterized by vomiting, skin infections and blood-borne related diseases. Urinary tract infections (UTIs), pneumonia, mastitis, endocarditis, meningitis and osteomyelitis are some of the serious infections or diseases in which S. aureus is implicated as a causative agent. S. aureus is also amongst the causative agents implicated in most nosocomial infections especially postoperative wound infections. Community-acquired methicillin resistant S. aureus (CA-MRSA) and hospital-acquired methicillin resistant S. aureus (HA-MRSA) are other important infections caused by resistant strains of pathogenic S. aureus.It is noteworthy that only pathogenic S. aureus strains are invasive in nature, as well as being haemolytic and coagulase-producing organisms. They are largely responsible for the multitude of infections caused by Staphylococcus species.

Non-pathogenic Staphylococcus species such as S. epidermidis are noninvasive, non-haemolytic and they do not produce coagulase enzymes. The invasiveness of S. aureus, as well as their virulence and pathogenicity is founded on the ability of the pathogen to produce a variety of toxins and extracellular enzymes that determine the course of the infection. These toxins and enzymes produced by the microbe help to increase the severity of infection during S. aureus invasion. In general, the virulence and/or pathogenicity of pathogenic S. aureus are largely dependent on the invasiveness of the invading strain, and the shared or cooperative action of the toxins and extracellular enzymes they produce. Thus, the pathogenesis of S. aureus will be expanded here based on the virulence factors (i.e., enzymes and toxins) that they produce. The toxins produced by S. aureus are also elucidated in this section.

  • EXFOLIATIN: Exfoliatin or exfoliative toxins (ET) are protein toxins produced by Staphylococcus aureus strains that cause staphylococcal scalded skin syndrome (SSSS) in humans. Two serological types of exfoliative toxins are known, and they are ETA (a chromosomally-mediated toxin) and ETB (a plasmid-mediated toxin). ETA is heat-stable while ETB is a heat-labile toxin and both toxins exhibit esterase and protease activity that have an impact on the integrity of the skin. SSSS is a staphylococcal disease caused by the plasmid-mediated toxin (ETB) produced by S. aureus and it isclinically experienced as a desquamation of the upper skin layer. In SSSS, there is usually a broad loss of the epidermis (a condition known as epidermolysis), and this exposes the red area beneath the skin surface. SSSS is commonly experienced in neonates and young children below 5 years of age and the disease can also occur in monkeys and mice but not in rats. Exfoliatin toxins are superantigens and they are also known as epidermolytic toxins due to their ability to cause an extensive peeling of the human skin (i.e., a loss of the skin’s epidermis). These toxins are also implicated in causing staphylococcal impetigo disease in humans.
  • TOXIC SHOCK SYNDROME TOXIN-1 (TSST-1): Toxic shock syndrome toxin-1 (TSST-1) is another class of superantigens produced by S. aureus, and it causes toxic shock syndrome disease in humans. In toxic shock syndrome disease, there is a profound release of staphylococcal superantigens into the bloodstream of the infected individuals and this leads to toxaemia. Toxaemia is a medical condition in which there are high amounts of toxins in the blood. Patients suffering from toxic shock syndrome disease usually experience skin desquamation (Figure 1), and this can progress throughout the body just like in cases of SSSS. Toxic shock syndrome disease is a fatal and severe disease characterized clinically by widespread desquamated skin rash, vomiting, muscle ache, high fever and diarrhea as well as renal and hepatic injury in some complicated cases. Young children and women who use certain types of tampons during their menses are mostly affected by the disease. Toxic shock syndrome disease can also be experienced in non-menstruating women and men who may have other infections such as wound infections. Specifically, TSST-1 stimulates massive production of cytokines (e.g., interleukins, tumour necrosis factor, and interferons) without the availability of any processed antigen to attack, and this result in toxic shock due to toxaemia. People without the appropriate neutralizing antibody against TSST-1 have recurrence of toxic shock syndrome disease due to the massive production of cytokines which ignites TSST-1 in infected individuals.
Figure 1. Desquamation of palm and thumb (arrows) due to toxic shock syndrome toxin-1 (TSST-1) staphylococcal disease. CDC
  • ENTEROTOXINS: Enterotoxins are superantigens produced by pathogenic S. aureus strains that cause toxicoses, a type of food poisoning in humans. The enterotoxins of S. aureus are designated as A, B, C, D, and E proteins. These classes of toxins are notorious in stimulating massive production of cytokines that cause staphylococcal food poisoning in humans who consume S. aureus contaminated food. Produced or preformed in food, staphylococcal enterotoxins are plasmid-mediated and chromosomally-mediated, and some are phage-borne (e.g., enterotoxin A). Staphylococcal enterotoxins can also be produced by pathogenic S. aureus in the intestinal tract of infected humans. Enterotoxins of pathogenic S. aureus are genetically related to TSST-1, and both share many characteristics together. However, enterotoxins are more heat-stable and resistant to enzymes in the digestive tract of affected people than TSST-1. Staphylococcal food poisoning is usually characterized by non-bloody diarrhea, abdominal cramp and vomiting (also known clinically as emesis). Enterotoxins like TSST-1 stimulate massive production of cytokines that activate the vomiting centre in the brain, and this causes gastroenteritis.
  • STAPHYLOCOCCAL ALPHA (α) TOXIN: Staphylococcal alpha (α) toxins are cytolytic toxins produced by pathogenic S. aureus, and which has killing effect on the cell membranes of eukaryotic cells. They are the most potent membrane-damaging haemolysins produced by S. aureus. These toxins basically act on a wide variety of cell membranes where they cause haemolysis and necrosis. Alpha toxins cause lysis by producing small pores or holes in the cell membranes of cells (e.g., erythrocytes), thus leading to the massive loss of cell materials from the damaged cells.
  • STAPHYLOCOCCAL BETA (β) TOXIN: Staphylococcal beta (β)toxins are less cytotoxic than α toxin but they also attack erythrocytic cells and some cells of the nerves (e.g., sphingomyelin). Beta toxins have high affinity for lipid-rich cells where they cause haemolysis.
  • STAPHYLOCOCCAL GAMMA (γ) TOXIN: Staphylococcal gamma (γ) toxins are produced by both S. aureus and S. epidermidis. Gamma toxins disrupt the integrity of cell membranes like the other haemolysins produced by S. aureus.
  • LEUKOCIDIN: Leukocidin is a cell membrane damaging toxin produced by pathogenic strains of S. aureus. They specifically kill leukocytes by creating small pores or holes that increases loss of materials from the damaged cell, thus inhibiting the process of phagocytosis in the infected human host. Panton-valentine (P-V) leukocidin as they are often called are haemolytic in nature, and they are mostly implicated in the majority of resistant Staphylococcus aureus infections (e.g., methicillin resistant Staphylococcus aureus, MRSA).

EXTRACELLULAR ENZYMES PRODUCED BY S. AUREUS

    It enhances the survival of the pathogen in phagocytes through the production of the enzyme, catalase. Catalase production is used for the biochemical identification of S. aureus in the laboratory, and it converts hydrogen peroxide (H2O2) to water and oxygen thereby walling off or protecting the infected body sites from phagocytic cells.
  1. Proteases: Proteases or proteinases are extracellular enzymes produced by pathogenic S. aureus, and which assist the pathogen in breaking down protein molecules.
  2. Nuclease: Nuclease is an enzyme produced by pathogenic S. aureus, and whichbreaks down nucleic acids of infected cells in human host. DNase enzymes produced by pathogenic S. aureus perform a similar function with nucleases in that they destroy the host cell DNA.
  3. Beta-lactamase: Beta lactamase is an enzyme produced by pathogenic S. aureus, and theyare of clinical significance in that this class of enzymes confers on the pathogen the capacity to develop resistance to a range of synthetic antibiotics and other antimicrobial agents. Beta lactamase enzymes of pathogenic S. aureus degrade beta-lactam antibiotics such as penicillins.
  4. Lipase: Lipases are fat or lipid destroying enzymes produced by pathogenic S. aureus. The production of this enzyme by pathogenic S. aureus strains inhibits the process of phagocytosis in the affected human host cells. Coagulase enzymes converts fibrinogen to fibrin clot which surrounds and protect infected sites from the action of phagocytes. The production of coagulase (a blood clotting factor) is used to identify pathogenic S. aureus in the clinical microbiology laboratory.
  5. Staphylokinase: Staphylokinase is an extracellular enzyme produced by pathogenic S. aureus, and it is a plasminogen activator (i.e., the enzyme stimulates a plasmin-like proteolytic activity that lyses fibrin). Streptokinase may aid in the spreading of the pathogen within the host due to its ability to degrade fibrin clots.
  6. Hyaluronidase: Hyaluronidase is an enzyme that breaks down hyaluronic acid that makes up the host connective tissues. Hyaluronic acid is defined as tissue cement. The ability of pathogenic S. aureus to produce hyaluronidase encourages the spread of the pathogen in host tissues.
  7. Protein A: Protein A is found in the cell wall of most S. aureus strains, and they prevent the activation of complement in the host cell. Protein A is anti-phagocytic in nature, and it binds to the crystallizable fragment (Fc portion) of antibody molecules (e.g., IgG), and thus prevents phagocytosis and opsonization. In this way, protein A (also known as staphylococcal surface protein) contributes to the virulence of pathogenic S. aureus.

LABORATORY DIAGNOSIS OF STAPHYLOCOCCUS AUREUS INFECTION The laboratory diagnosis of staphylococcal disease is based mainly on the isolation and identification of the invading pathogen through microscopy and culture. Serological and biochemical tests (e.g., catalase, DNase and coagulase tests) are also employed in typing the strain of S. aureus implicated in the disease process – since S. aureus is also a member of the normal flora of the human body. Blood, CSF, sputum, tracheal aspirate, pus, and surface swab specimens from infected sites (e.g., wound and burns) are examples of clinical specimens collected for laboratory investigations. Gram staining reveals Gram-positive grape-like cocci in clusters, tetrads or pairs under the microscope. Mannitol salt agar (MSA) is a selective medium (that contains NaCl which inhibit other normal flora and non-staphylococcal organisms) used to screen for S. aureus and recover the pathogen from clinical specimens resulting from a mixed bacterial/microbial infection. S. aureus produces several types of haemolysis including beta-haemolysis, alpha haemolysis and gamma haemolysis on blood agar media (Figure 2). S. aureus can also be cultured and isolated successfully on blood agar – where it produces white or pale haemolytic colonies (Figure 3). It also grows on chocolate agar and MacConkey agar. S. aureus grow aerobically at 35-37 o C.

Figure 2. Blood culture plate showingthe different types of haemolysis produced by pathogenic S. aureus on blood-enriched culture media plates.Photo courtesy: https://www.microbiologyclass.com Figure 3. Colonies of S. aureus (arrows) on blood agar plate. S. aureus produces small pale colonies on blood agar. Photo courtesy: https://www.microbiologyclass.com

IMMUNITY TO STAPHYLOCOCCUS AUREUS INFECTION

Host defense against staphylococcal diseases or infections is based on the action of phagocytic cells against the invading pathogens. Though phagocytosis plays active role in inhibiting the progression of the infection, pathogenic S. aureus is ingenious in producing toxins and extracellular enzymes that disrupts the course of action of phagocytes. No active immunity is developed in the host against a futuristic staphylococcal infection after previous infections with pathogenic S. aureus.

TREATMENT OF STAPHYLOCOCCUS AUREUS INFECTION

Therapy for staphylococcal disease is based on the administration of specific class of antibiotics to which the pathogen is susceptible to. All isolated Staphylococcus species should be subjected to antimicrobial susceptibility testing so as to guide treatment. Though pathogenic S. aureus is resistant to some known potent antibiotics cephalosporins, vancomycin, methicillin, oxacillin and penicillins are some of the drug of choice used for treating staphylococcal disease or infections. Nonetheless, strains of S. aureus resistant to methicillin, penicillin and vancomycin now exist in both the community and hospital environment. Drainage of the fluids or pus in abscess caused by S. aureus can be employed in the management of pus-infections mediated by the pathogen. Staphylococcal food poisoning should be treated through fluid and electrolyte replacement by the administration of the correct amount of a salt-sugar-solution (SSS) to the affected patients since the disease normally leads to a considerable loss of fluid from the body.

PREVENTION AND CONTROL OF STAPHYLOCOCCUS AUREUS INFECTION

Staphylococcus species are habitual inhabitants of the human body especially the nares or nose and skin where they are resident as normal microflora. Most individuals who harbour pathogenic S. aureus are asymptomatic, and they shed the pathogen to susceptible hosts around them. The control and prevention of staphylococcal infections in the hospital environment should be based on the practice of proper hospital infection control measures. No vaccine currently exist to prevent staphylococcal diseases or infections, thus it is vital for individuals and hospital institutions to maintain and imbibe personal and environmental hygienic practices such as hand washing and proper disinfection of surfaces. Doctors, nurses and other health personnel should maintain aseptic technique as they do their job especially around newborn wards and operating rooms in order to avoid spreading infections due to pathogenic S, aureus in the hospital environment. Wounds and burns should be properly dressed and disinfected with antiseptics too. The removal of superabsorbent tampons from general circulation has helped to reduce the incidence of toxic shock syndrome disease in menstruating women. Contaminated foods should be avoided and food handlers should always observe proper hygiene in the handling, processing, preparation and distribution of food in order to avoid the outbreak of food poisoning due to Staphylococcus aureus.

OTHER SPECIES OF STAPHYLOCOCCI

  • S. saprophyticus is a common cause of community acquired urinary tract infection. It is a leading cause of cystitis in young women and it is second only to E. coli as the most frequent causative organism of uncomplicated UTI in women.It is found as a normal flora in the female genital tract and perineum. S. saprophyticus is also found in vegetables and the environment, as well as in the gastrointestinal tract of animals.
  • S. epidermidis is part of the human normal flora, and it is abundantly found on the skin. It is not pathogenic in nature but it can cause infection in immunocompromised patients. S. epidermidis is an opportunistic bacterium that requires a breach in the host’s innate defenses to cause infection.
  • S. saccharolyticus causes infective endocarditis and it is known to contaminate samples of platelets taken from humans.
  • S. lugdunensis is a common cause of skin and soft tissue infections in the community. It occurs as a normal flora on human skin, but it has been recorded as a cause of serious human infections including osteomyelitis, arthritis, septicaemia, wound infections, and aggressive endocarditis.
  • S. warneri is found on the skin of humans and animals as a normal flora. It rarely causes disease in humans, but it may occasionally cause infection in patients whose immune system is compromised.
  • S. intermedius is isolated from the anterior nares of pigeons, dogs, mink, and horses. It is a part of the normal flora in the oral cavity, upper respiratory tract, female urogenital tract and gastrointestinal tracts and it can be found in human feaces. S. intermedius is an opportunistic organism that can cause infection in compromised human hosts.
  • S. capitis is a coagulase-negative species of Staphylococcus. It is part of the normal flora of the skin of the human scalp, face, neck, and ears and it has been associated with prosthetic valve endocarditis.
  • S. caprae is implicated in infections of the bloodstream, urinary tract, bones, and joints. It was originally isolated from goats, but it has also been isolated from human samples.
  • S. haemolyticus is an opportunistic organism abundantly found in the axillae, perineum, and inguinal regions as normal flora and it also colonizes primates and other domestic animals.

Further reading

Brooks G.F., Butel J.S and Morse S.A (2004). Medical Microbiology, 23 rd edition. McGraw Hill Publishers. USA.

Gilligan P.H, Shapiro D.S and Miller M.B (2014). Cases in Medical Microbiology and Infectious Diseases. Third edition. American Society of Microbiology Press, USA.

Madigan M.T., Martinko J.M., Dunlap P.V and Clark D.P (2009). Brock Biology of Microorganisms, 12 th edition. Pearson Benjamin Cummings Inc, USA.

Mahon C. R, Lehman D.C and Manuselis G (2011). Textbook of Diagnostic Microbiology. Fourth edition. Saunders Publishers, USA.

Patrick R. Murray, Ellen Jo Baron, James H. Jorgensen, Marie Louise Landry, Michael A. Pfaller (2007). Manual of Clinical Microbiology, 9th ed.: American Society for Microbiology.

Wilson B. A, Salyers A.A, Whitt D.D and Winkler M.E (2011). Bacterial Pathogenesis: A molecular Approach. Third edition. American Society of Microbiology Press, USA.

Woods GL and Washington JA (1995). The Clinician and the Microbiology Laboratory. Mandell GL, Bennett JE, Dolin R (eds): Principles and Practice of Infectious Diseases. 4th ed. Churchill Livingstone, New York.


Staphylococcus aureus - Biology

Tag words: Staphylococcus aureus, Staphylococcus, staph, staphylococcal, S. aureus, MRSA, CA-MRSA, superbug, staph infection, wound infection, food poisoning, toxic shock syndrome, antibiotic resistance, Staph epidermidis, normal flora, skin bacteria, bacteriology, microbiology

Staphylococcus aureus

Kingdom: Bacteria
Phylum: Firmicutes
Class: Bacilli
Order: Bacillales
Family: Staphylococcaceae
Genus: Staphylococcus
Species: S. aureus


Common References: Staphylococcus, Staph, MRSA, Superbug

Pathogenesis of S. aureus infections

Staphylococcus aureus causes a variety of suppurative (pus-forming) infections and toxinoses in humans. It causes superficial skin lesions such as boils, styes and furuncules more serious infections such as pneumonia, mastitis, phlebitis, meningitis, and urinary tract infections and deep-seated infections, such as osteomyelitis and endocarditis. S. aureus is a major cause of hospital acquired (nosocomial) infection of surgical wounds and infections associated with indwelling medical devices. S. aureus causes food poisoning by releasing enterotoxins into food, and toxic shock syndrome by release of superantigens into the blood stream.

Although methicillin-resistant Staph aureus (MRSA) have been entrenched in hospital settings for several decades, MRSA strains have recently emerged outside the hospital becoming known as community associated- MRSA( (CA-MRSA) or superbug strains of the organism, which now account for the majority of staphylococcal infections seen in the ER or clinic.

S. aureus expresses many potential virulence factors: (1) surface proteins that promote colonization of host tissues (2) invasins that promote bacterial spread in tissues (leukocidin, kinases, hyaluronidase) (3) surface factors that inhibit phagocytic engulfment (capsule, Protein A) (4) biochemical properties that enhance their survival in phagocytes (carotenoids, catalase production) (5) immunological disguises (Protein A, coagulase) (6) membrane-damaging toxins that lyse eucaryotic cell membranes (hemolysins, leukotoxin, leukocidin (7) exotoxins that damage host tissues or otherwise provoke symptoms of disease (SEA-G, TSST, ET) and (8) inherent and acquired resistance to antimicrobial agents.

FIGURE 2. Virulence determinants of Staphylococcus aureus

For the majority of diseases caused by S. aureus, pathogenesis is multifactorial, so it is difficult to determine precisely the role of any given factor. However, there are correlations between strains isolated from particular diseases and expression of particular virulence determinants, which suggests their role in a particular diseases. The application of molecular biology has led to advances in unraveling the pathogenesis of staphylococcal diseases. Genes encoding potential virulence factors have been cloned and sequenced, and many protein toxins have been purified. With some staphylococcal toxins, symptoms of human disease can be reproduced in animals with the purified protein toxins, lending an understanding of their mechanism of action.

Human staphylococcal infections are frequent, but usually remain localized at the portal of entry by the normal host defenses. The portal may be a hair follicle, but usually it is a break in the skin which may be a minute needle-stick or a surgical wound. Foreign bodies, including sutures, are readily colonized by staphylococci, which may make infections difficult to control. Another portal of entry is the respiratory tract. Staphylococcal pneumonia is a frequent complication of influenza. The localized host response to staphylococcal infection is inflammation, characterized by an elevated temperature at the site, swelling, the accumulation of pus, and necrosis of tissue. Around the inflamed area, a fibrin clot may form, walling off the bacteria and leukocytes as a characteristic pus-filled boil or abscess. More serious infections of the skin may occur, such as furuncles or impetigo. Localized infection of the bone is called osteomyelitis. Serious consequences of staphylococcal infections occur when the bacteria invade the blood stream. A resulting septicemia may be rapidly fatal a bacteremia may result in seeding other internal abscesses, other skin lesions, or infections in the lung, kidney, heart, skeletal muscle or meninges.

FIGURE 3. Sites of infection and diseases caused by Staphylococcus aureus


Materials and methods

Cell types used

Fig. 1a (luciferase) Cell type: HEK293T
Fig. 1b (NNGRR(T/V)) Cell type: HEK293
Fig. 1c (SaCas9 vs SpCas9) Cell type: HEK293FT
Fig. 1d (top) (gRNA length — VEGFA) Cell type: HEK293
Fig. 1d (mid) (gRNA length — GFP) Cell type: HEK293-GFP
Fig. 1d (bottom) (gRNA length — CCR5) Cell type: HEK293
Fig. 2b–d (nickases) Cell type: HEK293FT
Fig. 3a–c (AAV transduction) Cell type: HEK293
Fig. 3d–f (AAV transduction) Cell type: HEK293FT
Fig. 4a, b (GUIDE-seq) Cell type: U-2 OS

Cell culture

HEK293, HEK293FT (Life Technologies, catalog #R700-07), HEK293-GFP (GenTarget, catalog #SC001), and U2-OS (ATCC #HTB-96) cells were maintained in Dulbecco’s modified Eagle medium (DMEM Life Technologies) supplemented with 10 % fetal bovine serum (FBS), 5 % penicillin/streptomycin, and 2 mM Glutamax. Cells were kept at 37 °C in a 5 % CO2 incubator.

Plasmid and gRNA construction

The pCMVSau plasmid expressing a human codon optimized SaCas9 and a customizable U6-driven gRNA scaffold have been previously described [18]. Cognate luciferase indicator constructs were generated as previously described [14]. Maps of these plasmids and all other SaCas9 plasmids are shown in Figure S1 in Additional file 1.

gRNA used in Fig. 1a was generated by cloning annealed oligos containing the target sequence into pCMVSau. gRNAs used for data shown in Figs. 1b–d and 2d were generated by PCR and transfected as amplicons containing U6 promoter, spacer sequence, and TRACR scaffold. gRNAs used for data shown in Figs. 2b, c and 4a, b were generated by ligating either one or two of these into a pUC19 backbone vector via Gibson Assembly (New England Biolabs).

AAV vectors used in Fig. 3a–c were constructed by Gibson Assembly of one or two gRNA cassettes into SaCas9-containing AAV backbone pSS3. Vectors used in Fig. 3d–f were constructed by subcloning gRNA cassette pairs from vectors pAF089, pAF091, pAF092 into pSS60. Inverted terminal repeats (ITRs) were confirmed by XmaI digest of the vectors.

Transfections

Cells were seeded at a density of 100,000 cells/well in 24-well plates. After 24 hours, cells were transfected with 250 ng of gRNA plasmid or amplicon and 750 ng of either wild-type Cas9 plasmid, Cas9-D10A nickase plasmid, or Cas9-N580A nickase plasmid. All transfections were performed in duplicate using either Lipofectamine 3000 (Life Technologies) or MirusTransIT-293 reagent (Mirus Bio).

Luciferase analysis

293T cells were seeded at 1.25 × 10 5 cells per well in 12-well plates. Cells were transfected using the calcium phosphate method with 1 μg of the SaCas9/gRNA expression vector, 250 ng of a cognate gRNA firefly luciferase indicator plasmid, and 10 ng of a renilla luciferase internal control plasmid. Transfected cells were harvested 72 hours post-tranfection and lysed in Passive Lysis Buffer (Promega) and then assayed for luciferase activity using a Dual Luciferase Assay Kit (Promega).

GFP analysis

At 3.5 days post-transfection, cells had their media removed and were washed with 500 μl of phosphate-buffered saline (PBS). Next, 200 μl of trypsin was added to the cells and they were incubated at 37 °C with 5 % CO2 for 5 min. Trypsinization was halted by adding 500 μl of complete media to each well. Cells were collected from each well and transferred to eppendorf tubes, spun down at 3000 rpm for 7 min, washed with 1 ml fluorescence-activated cell sorting (FACS) buffer (PBS with 3 % FBS) and spun down again, and finally resuspended in 200 μl FACS buffer. Cells were then analyzed with a BD Accuri C6 flow cytometer.

DNA analysis

DNA was harvested 72 hours post-transfection or post-infection using an Agencourt DNAdvance genomic DNA isolation kit (Beckman) with a 4 hour lysing period, according to the manufacturer’s directions. Genomic DNA was then purified using Agencourt AMPure XP beads (Beckman) as per the manufacturer’s protocol.

For T7E1 assays, locus PCRs were performed to amplify regions of VEGF A, CCR5, and B2M. All reactions were performed with Phusion high-fidelity DNA polymerase (New England Biolabs) with resulting products purified by Agencourt AMPure XP beads (Beckman) according to the manufacturer’s instructions. T7E1 digestion was then performed in NEB Buffer 2 according to manufacturer’s instructions and resulting cleavage products were analyzed on a Qiagen QIAxcel Advanced System (Qiagen).

PCR conditions (Table S3 in Additional file 1).

Locus: VEGF(1) Primers: OME6/OME8 Annealing Temp: 67.5 °C
Locus: VEGF(2) Primers: AF116/AF117 Annealing Temp: 64 °C
Locus: CCR5(1) Primers: AF205/AF208 Annealing Temp: 64 °C
Locus: CCR5(2) Primers: AF209/AF211 Annealing Temp: 64 °C
Locus: B2M Primers: GWED67/68 Annealing Temp: 65 °C

For nickase assays, amplified VEGF A locus fragments were cloned into pCR4-TOPO vector using ZeroBlunt TOPO Cloning Kit (Life Technologies). TOPO reaction products were then transformed in One Shot Top10 chemically competent Escherichia coli cells. Cells were plated on carbenicillin LB agar plates and incubated overnight at 37 °C. Plasmid DNA was sequenced by Macrogen Corp. and Genewiz, Inc. using an M13 forward primer.

Viral vector production and titration

HEK293 cells were maintained in DMEM supplemented with 10 % FBS, 100 U/ml penicillin, and 100 U/ml streptomycin on 150-mm petri dishes in 5 % CO2 at 37 °C incubation. HEK293 cells were split 1:3 at 18 hours prior to transfection. AAV2 vectors were packaged with the “triple transfection” method using three plasmids: (1) 60 μg of pHelper (Cell Biolabs, Inc., San Diego, CA, USA) expressing E2A, E4, and VA from adenovirus (2) 50 μg of pRC2 expressing Rep2 and Cap2 from AAV2 (Cell Biolabs, Inc.) and (3) 30 μg of pSS/pAF plasmids with ITRs from wild-type AAV2 and CRISPR components. Mirus TransIT-293 reagent (420 μl Mirus Bio LLC, Madison, WI, USA) was mixed with 14 ml of OptiMEM and incubated at room temperature for 10 min before being added to the mixture of three packaging plasmids. After another 10-min incubation, the transfection mix was evenly distributed to five plates of HEK293 cells. At 70 hours post-transfection, supernatants and HEK293 production cells were collected by pelleting and centrifugation. Cell pellets underwent sonication, CsCl ultracentrifugation, and dialysis with 1× PBS to yield recombinant AAV2 viral particles.

To titrate AAV2 preparations, 10 μl of dialyzed viral vector was incubated in 90 μl of DNaseI solution at 37 °C for 1 hour, followed by serial dilution with ddH2O. Droplets were generated with Bio-Rad QX200 using 70 μl of droplet generation oil and 20 μl of samples including probe, saCas9-1-Probe (5′-6FAM-catcgggattacaagcgtggggtatggg-MGB-NFQ-3′), and primers, OliSS67 (5′-gaactacattctggggctgg-3′) and OliSS68 (5′-acgttggcctccttgaacag-3′). PCR reactions were carried out with 40 μl of droplet mix on a regular thermocycler. Droplets were read with Bio-Rad QX200 system to quantify positive and negative droplets. Viral vector titers were obtained by multiplying ddPCR readouts and dilution factors.

Vector transduction and western blotting

HEK293 cells were plated at a density of 100,000 cells/well in a 24-well plate and transduced with AAV2 vectors packaging U6-driven gRNA and EFS-driven SaCas9 at a multiplicity of infection (MOI) of 10,000 viral genome (vg)/cell. Growth medium was aspirated off the 24-well plate 72 hours post-transduction and cells were lysed with lysis buffer from the Agencourt DNAdvance kit (Beckman Coulter, Brea, CA, USA) followed by genomic DNA (gDNA) extraction, locus PCR (VEGF and CCR5 loci), and T7E1 assay to quantify genomic modification.

For western blotting, cells were lysed with 1× RIPA buffer with 1× cOmplete ULTRA protease inhibitor cocktail (Roche Diagnostics Corporation, Indianapolis, IN, USA) and 1× PhosSTOP phosphatase inhibitor cocktail (Roche Diagnostics Corporation, Indianapolis, IN, SUA) at 72 hours post-transduction. Cells were lysed at 4 °C for 15 min and lysates were spun down at 13.3 krpm for 15 min at 4 °C. Supernatants were collected and protein concentrations were quantified using Pierce BCA protein assay kit (Life Technologies, Carlsbad, CA, USA). Total protein (41.7 μg) was subjected to 4–12 % NuPAGE Bis-Tris gel electrophoresis at 150 V for 75 min. Gel transfer was performed using High Molecular Weight program on the Trans-Blot Turbo Transfer System (BioRad, Hercules, CA, USA). After blotting with 5 % milk in 1× PBS-T, western blots were incubated separately with corresponding primary antibodies overnight: (1) mouse-anti-Flag (clone m2, F3165, Sigma-Aldrich, St Louis, MO, USA) at 1:1000 dilution in 5 % milk in PBS-T, and (2) mouse-anti-alpha tubulin (clone B7, sc-5286, Santa Cruz Biotechnology, Dallas, TX, USA) at 1:200 dilution in 5 % milk in PBS-T. Blots were washed with PBS-T three times prior to incubation with secondary antibody, goat-anti-mouse IgG-HRP (sc-2005, Santa Cruz Biotechnology, Dallas, TX, USA), at 1:5000 dilution in 5 % milk in TBS-T at room temperature for 1 hour. After four washes with 1× PBS-T, western blots were developed with Western Lightning Plus-ECL (Perkin Elmer, Waltham, MA, USA) and imaged.

Guide-seq

U-2 OS cells were maintained in DMEM (Life Technologies) supplemented with 10 % FBS, 1 % penicillin/streptomycin. Cells were kept at 37 °C in a 5 % CO2 incubator. Cells were nucleofected at a density of 200,000/well with 250 ng of gRNA plasmid (pAF015), 500 ng SaCas9 plasmid (pAF003), and 100 pmol dsODN [19] using SE Cell line nucleofection solution and the DN-100 program on a Lonza 4D- nulceofector (V02.16). The nucleofected cells were seeded in 1 ml media in a 24-well plate and media was changed 12 hours post-nucleofection. Cells were grown for 72 hours post-nucleofection and gDNA was harvested using an Agencourt DNAdvance gDNA extraction kit. dsODN integration at the target site was confirmed by restriction fragment length polymorphism assay with NdeI.

gDNA was quantified with the qubit high sensitivity dsDNA assay kit. Roughly 400 ng of gDNA from SpCas9-treated cells and 180 ng of gDNA from SaCas9-treated cells were sheared acoustically via the Covaris m220 instrument to an average length of 500 bp in a total volume of 130 μl 1× TE. The sheared product was concentrated by AMPure (1× ratio) according to the manufacturer's protocol and eluted in 15 μl of 1× TE. One microliter of the product was run on the Agilent Tapestation system using the D1000 tape to confirm appropriate sizing. The remaining 14 μl of the sheared DNA was end-repaired, A-tailed, and adapter ligated. Adapter-ligated product was cleaned via AMPure (0.9×), eluted in 10 μl 1× TE, and split into sense and anti-sense PCR reactions. Post-PCR products were cleaned via AMPure (1.2×) and eluted in 15 μl of 1× TE. A second round of PCR was then conducted to incorporate the P7 illumina adapter and capture bi-directionality of off-target sites based on dsODN incorporated at each site. The final PCR product was cleaned via AMPure (0.7×) and eluted in 30 μl 1× TE. One microliter of each reaction was analyzed via Agilent Tapestation system using the D1000 screen tape and quantified using the qubit high sensitivity dsDNA assay kit. Finally, each reaction was normalized into one library pool and sequenced on the Illumina Miseq according to the manufacturer's protocols.

We analyzed GUIDE-seq data following the method described in Tsai et al. [19]. Reads were aligned to the UCSC hg19 genome assembly using bowtie2 (PMID:22388286). We selected regions passing the bidirectional filter [19] or with reads originating at the presumptive cutting site (three bases away from the PAM).

Supporting data

MiSeq sequence data gathered for the GUIDE-seq experiment (Fig. 4) were deposited in the Sequence Read Archive (SRA) at NCBI with BioProject number PRJNA298919. The SpCas9 sense, antisense, and barcode data can be accessed via accession numbers SRX1341497, SRX1341608, and SRX1341607, respectively. The SaCas9 sense, antisense, and barcode data can be accessed via accession numbers SRX1341609, SRX1341611, and SRX1341610, respectively.


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The history of MRSA infection goes back to 1961 when it was first described. Since then, the incidence and prevalence of MRSA infection have been increasing dramatically across the United States. Recently, some population studies have hintedਊt reducing HA-MRSA incidences in the United States but at the expense of a growing prevalence of CA-MRSA. The reported incidence of  MRSA infection ranges from 7% to 60%[4][5][6].

The commonly associated risk factors for MRSA infection are prolonged hospitalization, intensive care admission, recent hospitalization, recent antibiotic use, MRSA colonization, invasive procedures, HIV infection, admission to nursing homes, open wounds, hemodialysis, and discharge with long-term central venous access or long-term indwelling urinary catheter. A higher incidence of MRSA infection is also seen among healthcare workers who come in direct contact with patients infected with this organism.

Although advancing age by itself is not consideredਊ risk factor for MRSA infection, age more than 65 years is a significant risk factor for hospitalization. Hence, advancing age is indirectly linked to MRSA acquisition. Living in an area with a high prevalence of CA-MRSA or admission to a hospital withਊ high prevalence of HA-MRSA also is considered a significant risk factor for MRSA colonization[7].


6 CLINICAL IMPLICATIONS AND TREATMENT

6.1 Risk factors for and impact of S. aureus in CF lung disease

Methicillin sensitive S. aureus is one of the earliest bacteria occurring in the first year of life. Due to its ubiquitous nature, and nasal colonization in up to 30% of healthy people, search for risk factors is very difficult. For infants, birth by C-section is one reported factor associated with higher S. aureus carriage. This has not been studied specifically for CF. A study comparing naso-pharyngeal microbiome in CF and non-CF infants during the first 6 months of life, showed differences in microbiome emerge within the first few months, where S. aureus was one of the taxa more dominant in CF. 81 Potential explanations could be the CF specific environment, that is, increased mucus, hypoxia, and inflammation occurring already in early life. More studies have examined risk factors for MRSA using CF Patient Registry data. A summary is provided in Table 2.

Patient related Environment related and other
Pancreatic insufficiency 82 Winter season 53
CF-related diabetes 2 Southern latitude 83, 84
Younger age/higher FEV1 82 PM2.5 concentration in the year prior to birth 85
F508 del homozygote 82 MRSA exposure at work or home 86
Co-infection with P aeruginosa* * Also associated with risk to develop chronic infection.
82, 87
CF center with high MRSA prevalence* * Also associated with risk to develop chronic infection.
87
Increased hospitalizations/year* * Also associated with risk to develop chronic infection.
82, 87, 88
More CF clinic visits 84
Low Socioeconomic status* * Also associated with risk to develop chronic infection.
82, 87
More frequent cultures taken per year* * Also associated with risk to develop chronic infection.
87
  • PM2.5, particulate matter of 2.5 μm.
  • * Also associated with risk to develop chronic infection.

Studies in pediatric CF patients showed increased airway inflammation with MSSA or MRSA 89-91 and lower lung function than those without S. aureus. 92 Therefore S. aureus prophylaxis is recommended in the first few years of life for all children with CF in several countries (eg, Australia, UK, Germany) and treatment of incident S. aureus is recommended whenever recovered. 93 This contrasts with the US guidelines of treating S. aureus (MSSA) only during exacerbations. 94 Although no specific guidelines exist for MRSA, many physicians treat MRSA aggressively.

Although in vitro studies on virulence factors do not necessarily explain the differences in clinical outcomes with MSSA versus MRSA infection, several epidemiologic studies showed worse outcomes with MRSA compared to MSSA, 95, 96 and reviewed by Zemanick and Hoffman. 91 The causative role of MRSA, that is, does lung function decline after acquisition of MRSA, showed different results depending on study population and the definitions of MRSA infection that were used. Sawicki et al found that subjects with subsequent acquisition of MRSA had faster FEV1 decline before and after incident MRSA than subjects who remained MRSA free. 88 This study included any incident MRSA case and excluded FEV1 obtained 90 days before and after the incident culture. In contrast, Dasenbrook et al using only persistent MRSA (≥3 positive cultures) found a faster rate of FEV1 decline after incident MRSA compared to before MRSA or those remaining MRSA free. 82 Such findings suggest that outcomes differ between chronic compared to intermittent MRSA infection.

6.2 Eradication of MRSA in people with CF

Attempts to eradicate MRSA early in the course of infection may be warranted to avoid or delay chronic infection. Several CF centers have reported successful eradication of MRSA using a variety of regimens yet only two randomized controlled trials have been conducted one in the United States 97 and one in Italy. 98 Both trials used nasal mupirocin for 5 days and trimethoprim-sulfamethoxazole (TMP-SMX) in combination with rifampin. Differences between the trials were duration of oral antibiotics of 14 days in the United States compared to 21 days in the Italian trial and additional skin and surface decontamination in the US trial. The US trial showed significantly lower MRSA positivity in the treated compared to the observational arm at day 28 (primary endpoint): 26% versus 82%, P < 0.001. In contrast the Italian study, examining chronic MRSA negativity, that is, three negative cultures after 6 months, showed trends toward lower MRSA positivity with treatment however, this did not reach significance. Jointly these data indicate that early MRSA eradication is possible but duration and exact treatment regimen need to be defined.

The most frequently used antibiotics in non-randomized trials include fusidic acid (not licensed in the United States) and TMP-SMX mostly in combination with rifampin. More aggressive approaches using either a step-wise protocol to include an IV antibiotic if oral fusidic acid failed 99 or an a priori course of 3-week IV antibiotics followed by 6 weeks dual oral antibiotics based on susceptibility and inhaled vancomycin 100 have also been reported.

Two case series also reported eradication success of chronic MRSA infection using a 6 month therapy with fusidic acid and rifampin. 101, 102 A more recent trial in the United States compared dual oral antibiotics (rifampin with either TMP-SMX or minocycline) with versus without addition of inhaled vancomycin for 28 days [ClinicalTrials.gov NCT01594827]. Twenty-nine subjects were enrolled and results are pending publication.

A novel, dry powder formulation of inhaled vancomycin is being developed for chronic MRSA infections in CF. The phase 2 trial showed a significant reduction in CFU/g sputum, that is, the primary endpoint. A placebo controlled phase 3 study evaluating FEV1 and exacerbation frequency in subjects >6 years is under way [ClinicalTrials.gov NCT03181932].


Staphylococcus: From Harmless Skin Bacteria to Deadly Pathogen

An international research team, led by scientists from Tübingen and the German Center for Infection Research (DZIF) discovers additional component in staphylococcal cell wall that turns the bacterium potentially deadly.

The bacterium Staphylococcus epidermidisis primarily a harmless microbe found on the skin and in the noses of humans. Yet some strains of this species can cause infections – in catheters, artificial joints, heart valves, and in the bloodstream – which are difficult to treat. These bacteria are often resistant to a particularly effective antibiotic, methicillin, and are among the most feared germs in hospitals. How these usually harmless skin microbes become deadly pathogens has been unclear up to now.

An international research team has now discovered what distinguishes peaceful S. epidermidis microorganisms from the many dangerous invaders. The scientists have identified a new gene cluster that enables the more aggressive bacteria to produce additional structures in their cell walls. This morphological alteration allows the staphylococci to attach more easily to human cells forming the blood vessels, a process via which they can persist in the bloodstream to become pathogens. These new cell wall structures may also allow the spread of methicillin resistance, by transferring it, for example, from Staphylococcus epidermidis to its more dangerous relative Staphylococcus aureus.

The study was carried out under the direction of researchers of the Cluster of Excellence “Controlling Microbes to Fight Infections” (CMFI) of the University of Tübingen and the German Center for Infection Research (DZIF) in cooperation with universities in Copenhagen, Hamburg, Shanghai and Hanover as well as the German Center for Lung Research (DZL) in Borstel. The results are being published in the journal Nature Microbiology.

Set apart by structure

A considerable portion of the cell walls of Staphylococci – like other gram-positive bacteria – is made up of teichoic acids. Chain-like, these polymers cover the bacterial surface. Their chemical structures vary according to species.

“During our examination we determined that many pathogenic strains of S. epidermidis have an additional gene cluster that contains information for the synthesis of wall teichoic acids that are actually typical of S. aureus,” says researcher Dr. Xin Du of the Cluster of Excellence of the CMFI and DZIF. She adds that experiments have shown S. epidermidis bacteria with only species-specific teichoic acids in their walls are not very invasive, colonizing the surfaces of the skin and mucous membranes. If the wall teichoic acids for S. aureus are also present, Xin Du explains, they are unable to attach effectively to those surfaces. Instead, they are more successful in penetrating the tissues of their human host.

“At some point, a few S. epidermidis clones took on the corresponding genes from S. aureus and became threatening pathogens as a result,” says Professor Andreas Peschel of the Cluster of Excellence CMFI and of the DZIF.

It’s long been known that bacteria can share genetic material through gene transfer. Bacteriophages – viruses that infect bacteria – carry out the transfer. Mostly, this takes place within one species and requires similar surface structures to which the bacteriophages bind.

“Differing cell wall structures normally prevent gene transfer between S. epidermidis and S. aureus. But in S. epidermidis strains that can also produce the wall teichoic acids of S. aureus, that type of gene transfer suddenly becomes possible between different species,” explains Peschel. That would explain, he continues, how S. epidermidis could transfer methicillin resistance to even more threatening – and then methicillin-resistant – S. aureus, adding that more investigation is still needed.

The new findings are an important step, says Peschel, towards developing better treatments or vaccinations against dangerous pathogens such as S. epidermidis ST 23, which has been known for fifteen years and belongs to the group of HA-MRSE (healthcare-associated methicillin-resistant S. epidermidis).

Reference: “Staphylococcus epidermidis clones express Staphylococcus aureus-type wall teichoic acid to shift from a commensal to pathogen lifestyle” by Xin Du, Jesper Larsen, Min Li, Axel Walter, Christoph Slavetinsky, Anna Both, Patricia M. Sanchez Carballo, Marc Stegger, Esther Lehmann, Yao Liu, Junlan Liu, Jessica Slavetinsky, Katarzyna A. Duda, Bernhard Krismer, Simon Heilbronner, Christopher Weidenmaier, Christoph Mayer, Holger Rohde, Volker Winstel and Andreas Peschel, 24 May 2021, Nature Microbiology.
DOI: 10.1038/s41564-021-00913-z


Watch the video: Staphylococcus aureus (September 2022).


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