VICTOR TORNAMORELL GARCIA wrote:
> i´m studing the caracteristics of S.aureus. I'm from spain and i need help.
> if you have any information about
> this bacteria i'll be pleased. thanks.
Best place is a textbook, but try these study notes of mine which I
obtained from common textbooks like the Manual of Clinical Microbiology
(American Society for Microbiology), Topley and Wilsons, Mandels
Principles and practice of infectious diseases.
CHAPTER 1
Staphylococcus
Minidefinition
· Gram Positive Coccus
· Aerobic
· Catalase positive
· Oxidase negative (some exceptions)
· Indole and H2S not produced
Staphylococcus
· GPC in pairs and clusters
· Variation in size and Gram reaction
· Nonmotile
· Nonsporing
· Aerobic and facultatively anaerobic
· Catalase positive
· Oxidase negative
· Ferments sugars
Micrococcus
· GPC in pairs, fours and small clusters
· Uniform size
· Nonmotile
· Nonsporing
· Aerobic
· Catalase positive
· Oxidises sugars or does not attack them at all
Some Exceptions to definition
· S. saccharolyticus and S. aureus subsp. anaerobius grow better
anaerobically and are catalase negative
Cell wall
· contains peptidoglycan and teichoic acid
· diamino acid in peptidoglycan is L-lysin
· interpeptide bridge of peptidoglycan consists of oligoglycine peptides
· Micrococcus has no glycine in interpeptide bridge and does not have
teichoic acids
· Staphylococcus has a and b type cytochromes
· Micrococcus has c and d type cytochromes as well as a and b types
Basic differences
· Staphylococcus are resistant to bacitracin 0.04U disc
· Staphylococcus are susceptible to furazolidine 100 mg disc
· there are 27 species and 3 subspecies
Natural Habitat
· widespread in nature
· occurs mainly on skin, in skin glands, and mucous membranes of mammals
and avians
· S. captitis
adult human head, especially, scalp and forehead
®sebaceous glands and well developed
· S. auricularis
external auditory meatus
· S. hominis and S. haemolyticus
apocrine glands of axillae and pubis
· S. aureus
anterior nares
Clinical Significance
· skin infections most commonly caused by S. aureus
J folliculitis
J furuncles
J carbuncles
J cellulitis
J impetigo
J scalded skin syndrome
J post operative skin/wound infections
· common community disorders
J food poisoning due to thermostable enterotoxins elaborated during
growth
· serious processes
L bacteraemia
L endocarditis
L meningitis
L pneumonia
L pyoarthrosis (joints)
L osteomyelitis (bones)
· MRSA emerged as a serious problem during the 1980s
· most are resistant to several of the most commonly used antimicrobial
agents, e.g., macrolides, aminoglycosides, b-lactams
· most successfully treated with vancomycin, rifampicin, fusidic acid,
and ciprofloxacin
· toxic shock syndrome (TSS)
· community acquired
· potentially serious
· young menstruating females
· highly absorbant tampons
· TSST-1 from S. aureus has been elaborated at other sites as well as in
males
· coagulase negative staphylococci may also be able to elaborate TSST-1
· methods of determining TSST-1
· RIA
· EIA
· reverse passive latex agglutination
Coagluase Negative Staphylococci (CNS)
· opportunistic pathogens
· especially with invasive procedures and indwelling foreign materials
· S. epidermidis greatest pathogenic role and diversity
· major agent in oncology and neonatology
· 74 to 92 percent of hospital CNS bacteraemias after cardiac procedures
· mediastinitis
· pace maker infection
· vascular graft infection
· prosthetic valve infection
· mitral valve prolapse
· CSF shunts®S. epidermidis is often the primary pathogen present
· S. epidermidis causes 40 percent of prosthetic joint infections,
rarely causes osteomyelitis
· S. epidermidis is the primary pathogen in CAPD peritonitis
· many CNS are resistant to methicillin
· resistance is heterogenic and may extend to cephalosporin
antimicrobial agents
· in vitro tests for cephalosporin resistance is difficult
· usual therapy is vancomycin±rifampicin or ???an aminoglycoside
· S. haemolyticus is the second most commonly encountered species of CNS
· native valve endocarditis
· septicaemia
· peritonitis
· urinary tract infections
· wound infections
· S. lugdunensis and S. schleiferi
· emerging as opportunistic pathogens in the USA
· colonise catheters and drains
· not common on human skin
· native valve endocarditis
· septicaemia
· brain abscess
· deep tissue infection
· osteitis
· chronic osteoarthritis
· vascular prostheses
· wound and skin infections
· peritoneal fluid
®predisposers ® diabetes mellitus
surgery/trauma
renal failure
cancer
acquired immunodeficiency syndrome
· S. saprophyticus
· common in UTI especially in sexually active females
· occasionally causes wound infections
· occasionally septicaemia
· S. hominis, S. warneri, S. simulans, S. cohnii, S. saccharolyticus, S.
capitis, S. xylosus
· occur in low incidence of infections in humans
Culture and Isolation
· inoculate blood agar and thioglycolate
· after 18 to 24 hours
· colonies will be 1 to 3 mm
· circular/smoothed/raised
· butyrous consistency
Identification
· colonial morphology
· coagulase production
· oxygen requirements
· haemolysins
· antimicrobial resistance
· enzyme activity
· aerobic acid production
Colonial appearance
¨ 1-3 mm diameter at 24 hours
¨ 3-10 mm diameter at 5 days
· S. aureus
· large 6-8 mm, smooth, entire edge
· slightly raised, translucent
· pigmented cream yellow to orange
· some strains produce dwarf colonies
· rare strains with large capsules—small and more convex and glistening
wet appearance
· S. epidermidis
· relatively small, 2.5-6.0 mm
· no pigment
· with age, an increase in temperature or crowding
· translucent to transparent with dark centres
· "sticky" consistency
· some are "slime producers"
· S. haemolyticus
· larger, 5-9 mm
· smooth, butyrous, opaque
· S. saprophyticus
· large, 5-8 mm
· entire edge
· very glossy
· opaque
· smooth
· butyrous
· more convex
Coagulase Production
· ability to clot plasma
· S. aureus Humans
· S. intermedius and S. hyicus Animals
· FREE COAGULASE
· single colony from a 24 hour blood agar plate is rubbed up in 1 mL
fresh citrated rabbit plasma diluted 1 in 6 with physiological saline;
incubate in water bath at 37oC and read after 1, 2, 4, 8, and 14 hours;
include a weak positive strain (NCTC 6571)
· BOUND COAGULASE
· detected by slide tests, coagulase must be confirmed with tube assay
which will also detect free coagulase
Heat Stable Nuclease
· Thermonuclease—endo- and exonucleolytic properties
· able to cleave DNA or RNA
· present in MOST strains of
· S. aureus
· S. schleiferi
· S. intermedius
· S. hyicus
· present in SOME strains of
· S. epidermidis
· S. simulans
· S. carnosus
· DNA-toludine blue agar, metachromatic agar diffusion
· a seroinhibition test has been developed to distinguished S. aureus
from the others
Phosphate Activity
· hydrolysis of p-nitrophenylphosphate into Pi and p-nitrophenol by
alkaline phosphatase
· activity indicated by release of yellow p-nitrophenol from colourless
substrate
· strains of S. aureus, S. schleiferi, S. intermedius and S. hyicus; and
most S. epidermidis are POSITIVE
· 10-15% of S. epidermidis are NEGATIVE
Pyrrolidonyl Arylamidase Activity
· hydrolysis of pyroglutamyl-b-naphthylamide into L-pyrrolidone and
b-naphthylamine which combines with p-dimethyl aminocinnamaldehyde (PYR)
to produce a red colour
· POSITIVE
· S. haemolyticus, S. lugdunensis, S. schleiferi, S. intermedius
· NEGATIVE
· S. aureus, S. epidermidis, S. saprophyticus, S. hyicus
Ornithine Decarboxylase
· POSITIVE
· S. lugdunensis
Urea Hydrolysis
· POSITIVE
· S. epidermidis, S. intermedius, most S. saprophyticus
· VARIABLE
· S. aureus, S. lugdunensis, S. hyicus
· NEGATIVE
· S. haemolyticus, S. schleiferi
b-Galactosidase
· POSITIVE
· S. intermedius, most S. saprophyticus
· DELAYED
· S. schleiferi
· NEGATIVE
· S. aureus, S. epidermidis, S. haemolyticus, S. lugdunensis, S. hyicus
Acetylmethylcarbinol Production
· POSITIVE
· S. aureus only
Novobiocin Resistance
· 5mg novobiocin disc
· S. saprophyticus is intrinsically resistant on P agar, MH agar,
tryptic soy sheep blood agar
Acid Production from Carbohydrates
· results will depend on the indicator used
· SEE INDICATOR CHART
Methicillin Resistant Staphylococci
· MRSA can range from 10-50 percent in hospitals in USA
· usually resistant to erythromycin, clindamycin, tetracycline,
chloramphenicol, gentamicin
· heteroresistant strains exist which show heteroresistance to b-lactam
antimicrobials
· 2 subpopulations
· one resistant, one susceptible
· coexist in culture
· each cell has genetic information for resistance but only a small
fraction, usually 10-6 to 10-4, can express resistance phenotype under
in vitro conditions
· the resistant subpopulation usually grows slowly
· its growth is favoured by
1. neutral pH 7.0 to 7.4
2. cooler temperature 30-35oC
3. NaCl 2-4 percent
4. ±prolonged incubation up to 48 hours
Epidemiological Typing Systems
Reasons
· monitoring staphylococcal community structure of patients and
facilities
· tracing source of infection
· strain identification to monitor distribution of potential pathogens
or antimicrobial resistance reservoirs in a hospital or community
Methods
· bacteriophage typing
· colony morphology
· biotyping
· antibiograms/resistograms
· plasmid composition
· restriction fragment length polymorphism (RFLP) of plasmid and
chromosomal DNA
· pulse field gel electrophoresis (PFGE)
S. aureus
· phage typing—a dynamic system
· international standards and procedure exist
S. epidermidis
· phage typing—system exists
· no international standards
· colonial morphology can be useful
· needs extended incubation at 35oC for a few days and then at room
temperature for 2 days to enhance strain characteristics
· colonies of same strain show consistencies in:
· size
· consistency
· profile
· edge
· lustre
· colour
· biochemistry is NOT reliable
Antibiograms
· are useful and easy to assess
· can be standardised
· can be used as a strain marker
· results can vary from one community to another
· valid only "in house" during a short period of time
· patterns can change over time
· strains may also change because of plasmid instability or high
mutation rate
· plasmid composition and RE analysis are valuable molecular typing
tools
· best for species that contain several different plasmids
Other Clinical Syndromes
Staphylococcal Scalded Skin Syndrome
· a disease of infants and young children
· large bullae
· separated epidermis from dermis
· Nikolsky sign- friction to apparently "healthy" skin wrinkles and
displaces
· leaves well demarcated denuded areas
· originally Ritter’s syndrome
· confused with toxic epidermal necrolysis (TEN), which is a drug
hypersensitivity reaction that occurs in older children and adults
· SSSS is intraepidermal splitting
· TEN is splitting of dermoepidermal junction
· SSSS starts abruptly, as perioral oedema that spreads over the entire
body
· can lead to partial or complete desquamation lasting 3-5 days
· new epidermis appears after 10 days
· staphylococci isolated from skin and nasopharynx but rarely blood
Kawasaki Mucocutaneous Disorder
· differentiated by negative Nikolsky’s sign and negative bacterial
culture of skin lesions
· most SSSS is due to phage group II, phage type 71 in the USA but this
is different in other countries
Toxic Shock Syndrome
· high fever ³40oC
· profound refractory hypotension
· profuse diarrhoea
· intense myalgias
· vomiting
· erythroderma
· headache
· mental confusion
· renal failure-oliguric and non-oliguric, usually reversible
· vaginal hyperaemia with discharge, from which staphylococci can be
isolated
· associated with hyperabsorbant tampons in 1980-81
· presently occurs in 3-15 per 100,000 women of menstrual age per year
Criteria for diagnosis:
· T>38.9oC
· Systolic BP <90mmHg
· Rash with desquamation especially on palms and soles
· Involvement of ³3 of the following
· Gastrointestinal Tract-vomiting, diarrhoea
· Muscular-myalgias or >5-fold increase in CPK
· Mucous membranes-hyperaemia
· Renal function-creatinine twice normal
· Hepatic-bilirubin or transaminases twice normal
· Blood-thrombocytopaenia
· CNS-mental confusion
STAPHYLOCOCCAL TOXINS
a-toxin
· a heterogeneous protein by electrophoresis
· acts on a wide variety of membranes-RBC, WBC, PLTs, fibroblasts, HeLa
cells
· but NOT on bacterial cytoplasmic membranes
· most active against RBC, causing haemolysis
· when injected it is dermonecrotic
b-toxin
· cytotoxic by degrading sphyngomyelin
· \active on great number of cells
· including RBC, WBC, fibroblasts
· on RBC haemolysis dependent on amount of sphyngomyelin present in the
cell membrane
g-toxin
· lysis RBC of many species including humans by unknown mechanisms
d-toxin
· electrophoretically heterogeneous
· dissociates into many subunits when treated with detergents
· it is hydrophobic and thermostable
· in animal ileum models it inhibits water absorption and stimulates
cAMP, hence it may have a role in staphylococcal diarrhoea
Leucocidin
· affects humans and rabbits only
· injection produces deep reversible granulocytopaenia (¯ neutrophils)
· with membrane damaging component that can be observed within 60
minutes
· act synergistically at phagocyte membrane to form pores with
permeability to cations
Exfoliatin A and B
· two serologically and biologically distinct proteins
· responsible for major skin findings in SSSS
· Exf A is chromosomal, thermostable, inactivated by EDTA
· Exf B is plasmid, relatively thermolabile, stable in EDTA
· evidence of pathological role-neutralised by specific neutralising
antibody, not seen in adults who have antibody due to maturation
Toxic shock syndrome toxin
· TSST-1
· protein
· strong inducer of IL-1 in monocyte cultures
· a SUPERANTIGEN
Enterotoxins
· 5 distinct serological groups
· causative factor in staph food poisoning
· mechanism of action unknown, but definitely increase peristalsis,
possibly by sympathetic activation
AGAR SCREENS FOR MRSA
Resistance phenotype mec gene encoded Mechanism Borderline resistance
b-lactamase inhibitor effect b-lactam cross-resistance Multiple
resistance to non-b-lactams
Classic
Homogeneous + supplemental PBP (PBP 2a) – – + (+)
Heterogeneous + supplemental PBP (PBP 2a) ± – + (+)
Inactivation by b-lactamase – Increased b-lactamase production + + – –
MOD-SA – Modified preexisting PBP 1, 2, and 4 + – – –
· three different resistance mechanisms exist
· clinically important to differentiate mec producers from the others
· strains that possess the mec gene are either heterogeneous or
homogeneous in their expression
· with homogeneous all cells express resistance
· in heterogeneous cells only 1 in 104 to 1 in 105 mec positive cells
expresses resistance
· isolates with classic resistance usually are resistant to other
antimicrobial agents, e.g., erythromycin, clindamycin, chloramphenicol,
tetracycline, TMP-SMX, a quinolone, or an aminoglycoside
· resistance due to b-lactamase or the presence of MOD-SAs results in
borderline resistance
· b-lactamase types can be distinguished from classic type or MOD-SA
resistance by addition of a b-lactamase inhibitor, e.g., clavulanic
acid, to oxacillin lowers the MIC by ³2 dilutions
· isolates that are resistant by either b-lactamase or the MOD-SA
mechanism usually do not have multiple drug resistance
· presence of the classic phenotype can be detected reliably with the
agar screening test
· b-lactamase or MOD-SA strains are unlikely to grow on agar screen
plate
· the mec gene has been identified in coagulase negative staphylococci
and the agar screening test can also detect mec-positive resistance
Test Method
· MHA supplemented with 4% NaCl and 6 mg/L as recommended by NCCLS
· oxacillin is the preferred substrate because of its stability
· prepare direct inoculum by selecting colonies and transferring to
saline to produce a suspension equivalent to a 0.5 MacFarland standard
· a cotton swab is dipped into this and wrung out on the side of the
tube
· the swab is spotted onto the screening plate
· alternatively 10 mL of a 1:100 dilution of the 0.5 MacFarland
standardised suspension can be used resulting in a 104 CFU per spot
· test plates incubated for a full 24 hours at 35°C (no higher) in
ambient air and examined for any evidence of growth which indicates
resistance
· growth of a staphylococcal isolate on the oxacillin agar screen
generally means the isolate is mec positive
· occasionally a heteroresistant isolate may not grow on the agar
screening plate
· it generally does not detect borderline resistance except for some
MOD-SA strains with MICs ~8 mg/L
· NCCLS recommends that all classically resistant staphylococci be
regarded as resistant to all b-lactam agents
· do not add NaCl nor increase length of time nor increase inoculum,
these methods may produce false resistant results
If you want a formatted document E-mail me and I can send one.
Gary
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********************************************************
Dr Gary Lum
Director of Microbiology
Royal Darwin Hospital
Microbiologists do it with culture and sensitivity
Meet me at http://www.ozemail.com.au/~glum/index.html
E-mail me at glum at ozemail.com.au or gary.lum at nt.gov.au
