MECHANISM OF ANTIMICROBIAL RESISTANCE:

STAPHYLOCOCCUS AUREUS IN FOCUS

M.Sc. SEMINAR
BY
TESFAYE GUJA
ADVISOR:DR.TEKLU WEGAYEHU
APRIL,2024
ARBA MINCH,ETHIOPIA
 Out line of presentation
1. Introduction
2. Biology of Staphylococcus aureus
The pathogen
The genome
3. Mechanism of antimicrobial resistance
4. Therapeutic options
5. Conclusion

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 1. Introduction
• Gram positive pathogenic bacteria
• It found as commensal in natural flora
• It can also found in environment setting
• Transmission via direct contact
• It spread in both human and animal
• Cause of infection: gets trauma or compromised immune
• Infections include, skin or soft tissue , urinary tract, respiratory, etc..
• More serious and even fatal

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 Cont..

• Antiseptics and disinfectants reduce pathogen burden

• Long time or excess usage promoted resistance
• Staphylococcus aureus strain identified
•Penicillin treat infections, resistance developed
•Methicillin used but, resistance developed
• Beta-lactam and non-beta-lactam antimicrobial produced
• But, easily bacteria developed resistance

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 Cont..
Time line of antibiotics used in therapy and resistance developed
Pencillin 1959 1932 1945 1952 1962 1990 2003
1940 methicilli sulfinami Tetracy Macrolid lincosam Vancomyci Daptomy
Antibioti
In n ds clin es ides n cin
cs in
therapy therapy

Staphylococcus aureus

Resistan Mid
ce 1940 or 1942 1953 1953 1979 2005
develop 1941
ed time
1961- first Hospital acquired methicillin 1997- Vancomycin intermediate
resistant Staphylococcus aureus(MRSA) Staphylococcus aureus(VISA)
1990- community acquired MRSA 2002
2000-Livestock acquired-MRSA Vancomycin resistant
Staphylococcus aureus (VRSA)

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 Cont..
• Over 148,000 infections and 7000 deaths European Union
• In Africa, Ethiopia infection is high
• CDC declared that we are facing infections
• resistant to every available treatment options
• Seminar focused to provide brief explanation
on mechanism of antimicrobial resistance

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 2. Biology of Staphylococcus aureus
• Non-motile, non-spore forming
• Spherical in shape with 1μm in diameter
• Cell wall single lipid membrane contain peptidoglycan, lipid membrane acid and
proteins
• Cytoplasm enclosed by the cytoplasmic membrane
• Peptidoglycan constitutes teichoic acids

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 2.2 The pathogen
• Family staphylococcal, genus Staphylococcus
• In genus more than 32 species and 8 sub-species
• Colonize the human body
• Only 3 are medical important
• Staphylococcus aureus(human), Staphylococcus epidermidis and
• Staphylococcus saprophytics(animal pathogen)
• Staphylococcus aureus form grape-like colony with golden or yellowish
• It is coagulase positive, above 2 were negative

Staphylococcus aureus colonies on agar

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 The genome
• Single circular chromosome approximately 2.8 mega base pairs, but can exceed
• Genome contains the core and accessory genome regions
• All sequenced strains similar chromosome architecture but, variability in genome
sequences
• Newman strain 2.8 mega base pairs with a G+C content of 32.9%.
• Single or multiple nucleotide changes in 2.8 mega base pairs genome
• Brings antibiotics resistance
• Figure shows, genome sequences shaded differently
• Areas not shaded are lacking in the strain and
shows variations in the accessory genome

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Cont..

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 3. Resistance mechanisms
3.1. Limiting up take of antimicrobial
• Preventing the entrance of antimicrobials to bacterial target
• widely observed by gram negative bacteria
• Staphylococcus aureus acquired resistance from vancomycin resistance
enterococci species
• Thickened outer cell wall
• First Vancomycin intermediate resistance developed
• Aminoglycosides entrance also difficult
• Due to polar molecules of cell wall rigidity to penetrate

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 Cont..
• Biofilm formation protects to access the target cells by
residing inside the biofilm
• Biofilms makes strong adhesion to antimicrobial
• penetrance is less, due to polymeric subs, act as glue
• Poly saccharides woven together to form matrix, thicken up to 300µm
• Tetracycline, benzyl penicillin, and vancomycin decreased in biofilm setting, than
planktonic

Mature colony

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 Cont..

Mechanisms of antibiotic resistance in biofilms

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 3.2. Enzymatic inactivation
• Beta-lactams have 3-carbon, 1-nitrogen beta lactam ring
• Helps to bind native penicillin binding protein structure
• Resistance development, bacteria used two mechanisms
• Production of penicillinase enzyme or beta-lactamase enzymes(blaZ) gene and
mecA gene
• Penicillin resistance is due to production pencillinase enzymes
• Breaks down beta-lactam ring of penicillin with amide bond of 4-membered
penicillin binding protein structure

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 3.3. Target modification
• Methicillin best Pencillinase tolerant antibiotics targets
penicillin binding protein structure
• Resistance developed due to acquisition of mec A gene
• Codes for expression of alternative protein penicillin binding protein 2A
• Promote cross-linkage of peptidoglycan
• After native penicillin binding proteins are inactivated by methicillin

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 Cont..
• Vancomycin targets cell wall synthesis
• Cell wall made from cross-linked peptidoglycan strands
• Strands are made up of glycan chains of N-acetyl glucosamine (NAG) and N-acetyl
muramic acid (NAM)
• Vancomycin binding to the final D-Ala-D-Ala residue
• Interferes peptidoglycan synthesis
• It prevents transglycosylation and trans peptidation
• Resistance developed by acquisition of the vanA operon
• VanA operon altering the synthesis cell wall to remove the vancomycin binding
site

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 Cont..
• Resistance developed due to
• VanA, VanH, VanX and VanY
• Coded by the vanA operon remove the vancomycin binding site
• By two steeps
• 1. Dipeptide D-Alanine-D-Alanine peptidoglycan precursors hydrolyzed
by VanX (a D,D-dipeptidase) and
VanY (a D,D-carboxylpeptidase)
• 2. VanA (a ligase that catalyses ester bond formation) and
VanH (a dehydrogenase that forms D-Lactate by reduction of pyruvate)
synthesise D-Alanine-D-Lactate (that vancomycin cannot bind)

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Cont..

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 Conti..
• Macrolide, lincosamide and streptogramin B antibiotics are a broad spectrum
• Targets protein synthesis and inhibits translation
• Macrolide antibiotics internalize, the protein exit channels in the 50S subunit are
blocked
• Leads to inhibition of polypeptide chain elongation
• Formation of new 50S ribosome subunits disrupt
• Acquisition of erm A/B/C genes pressured resistance to antibiotics
• Genes codes for methylase (erm) enzymes
• Change of target site is mediated by the adenyl‐N‐methyltransferase

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 Cont..
• Methylation of adenine results formation of N‐methyl adenine or
• N, N‐dimethyl adenine and consequently post‐transcriptional modification of the
23S rRNA structure
• Interferes with antibiotic bind ing and result resistance

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 Cont..
• Daptomycin is the recent lipopeptide antibiotics
• Characterized with positively charged calcium complex
• Attach the cell wall of bacteria negatively charged phospholipids
• Phosphatidylglycerol, lipoteichoic acids and lipopolysaccharides
• Causing depolarisation, permeabilisation and ion leakage
• Resistance developed due to acquisition of mprF gene
• makes several point mutations and change negative charge to positive
• Repels the positively charged daptomycin

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Cont..

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 3.4. Efflux pump
• Is a normal physiological structure helps to eliminate
endogenous metabolites toxin out of cell
• By default when antimicrobials enter cell efflux system encoding genes to be
activate
• Substrates transporters active and exclude antimicrobials
• Efflux pumps are substrate-specific or multi-drug resistant efflux pumps
• Recognizes and export a broad spectrum of structurally-unrelated substrates
• 5 efflux pump system exhibited by Staphylococcus aureus
• ATP-binding cassette superfamily, major facilitator superfamily, multidrug and
toxin extrusion family, small multidrug resistance family, and resistance
nodulation division superfamily

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 Cont..
• Chlorhexidine antiseptic: multidrug and toxin extrusion family or major
facilitator superfamily of transport proteins
• Encoded by plasmid-mediated qacA/B genes
• Quaternary ammonium compound : Small multidrug resistance family or
multidrug and toxic extrusion family
• Encoded by qacA, qacB and qacC genes
or acquisition of MepA gene
• Fluoroquinolones: major facilitator super family of membrane protein
• Encoded by NorA/B/C chromosomal genes

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 Cont..
• Tetracyclines: major facilitator super family encoded tetK gene: carried plasmid
• Macrolide: ATP-binding-cassette family proteins encoded smr gene: carried
plasmid

Tet(K)
qacA/B MepA
norA,B, smr

Tetracycli Chlorhe Disinfecta

ne xidine nt(QACs) Macroli
fluoroqui de
noles

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 4. Therapeutic options
•Sulfamethoxazole + Trimethoprim= co-trimoxazole
Urinary tract infections and uncomplicated soft-tissue infections
•Ceftobiprole 5th generation of Cephalosporin
demonstrated in vitro activity against methicillin resistant
Staphylococcus aureus and vancomycin resistant Staphylococcus
•Ceftaroline also approved to treat severe cases
•Ceftaroline fosamil- approved to treat complicated skin and soft- tissue infections
and pneumonia
•Delafloxacin 4th generation of fluoroquinolone markedly contribute
against Staphylococcus aureus
•Oritavancin active to against some vancomycin-resistant staphylococcus aureus

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 5. Conclusion
• Staphylococcus aureus developed resistance
• Due to its unique mechanism treatment options limited
• Understanding mechanism of antimicrobial resistance
• Help to focus on development of new alternative drugs

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Thank you

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