Study Design - Clinical Significance

Question 1

Biofilm producing organisms

Answer 1

P. aeruginosa and its small colony variants
Acinetobacter
S. aureus - dry surface biofilms
CPE

Colonisation >40% of ICU sinks -> tends to regrow quicly
In one study 90% of surfaces samples harboured DSBs -> detection not recovered by swabbing and less suceptible to physical and chemical disinfection and sterilisation

Question 2

Comment on growing threat of antibiotic resistance in general

Answer 2

One of the top global public health and development threats

Estimated that AMR was dorectly responsible for 1.27 million global deaths and contributed to 4.95 million deaths in 2019

Expected to contribute to 50 million deaths by 2050

Question 3

Comment on the growing threat of antimicrobial resistance, give specific tats

Answer 3

Third generation cephalosporin resistance in 42% of E. coli
Methicillin resistance in 35% of S. aureus
1 in 5 E. Coli UTIs show reduced susceptibility to standard antibiotics e.g. amipicillin, co-trimoxazole and fluoroquinolones
K. pneumonia reliance on last resort antibiotics rising
anticipated x2 surge in resistance to last resort antibiotics by 2035

Question 4

Resistance in Pseudomonas aeruginosa, mechanism ,detection etc

Answer 4

Fluoroquinolone resistance trhough efflux pumps, mutations in DNA gyrase and outer membrane changes

gyrA and parC genes

Question 5

Resistance in Klebsiella pneumonia, mechanisms and targets

Answer 5

resistance against beta lactams

beta-lactamase production, porin loss and efflux pumps

produces KPC - klebsiella pneumoniae carbapenemase and alters porins

Question 6

What resistant organisms are crossing into the community

Answer 6

MRSA
CPEs -> MDR R>3 antibiotic classes

HCAI crossing into the community

Question 7

Talk about resistance emerging in community pathogens

Answer 7

Resistance in traditionally community pathogens such as:
- S. pneumoniae
- N. gonothoea
- Shigella
- TB -> XDR

Question 8

Clinical significance of MRSA

Answer 8

Resistance and potential for cross-infection
Community acquired MRSA arround since the 1990s - displays less cross-resistance but retains virulence of mssa - often linked to production of panton-valentine leukocidin (PVL toxin)
Livestock associated MRSa also an emerging threat
Used to have high levels of MRSA but numbers have reduced
Outbreaks of CA reported in schools, sports teams and other community groups
Peaked at 42% in 2006 but now as low as 9.7% in 2023

Question 9

Target for MRSA and the significance of this

Answer 9

mecA/mecC gene

This leads to production of an altered penicillin-binding protein known as PBP2a

This has a reduced affinity for methicillin and other B-lactams, resulting in high-level resistance to these antibiotics

MecA is part of the staphylococcal chromosomal cassette mec (SCCmec), a larger mobile genetic element, allowing spread

Vancomycin resistant MRSA has been reported since 1990s but this hasnt been seen in ireland - vanA

Question 10

How do we screen for MRSA?

Answer 10

Screen using chromagar
Samples such as nasal, groin, auxilaa
Confirmation using commercial real-time PCR detection systems e.g. geneXpert

Question 11

What tests do we use to detect MRSA?

Answer 11

MIC for cefoxitin, >4mg/L = methicillin resistant
Disk diffusion 30ug cefoxitin, zone <22mm =resistant
MASTalex Latex agglutination to detect PBP2a -> will detect MecA and not MecC
Genotypically using PCR for MecA or MecC

Question 12

Clinical significance of VRE

Answer 12

Resistance to vanc and now other antimicrobials including quinolones, high level aminoglycosides (30-60%), macrolides, tetracyclines, linezolid, - virtually all
VRE spreads efficiently, persisting in hospital environments and colonizes many individuals but few develop infections
Has both intrinsic and mutational resistance

VRE decreased from 38.6% in 2019 to 21.4% in 2023, lowest level but still high - were above eu average of 16.8% though
Rates are increasing across europe

Question 13

B-lactam resistance in VREs

Answer 13

penicillin binding protein 5 production - which has low affinity for beta lactams

intinsic, chromosally encoded, low-level resistance or
Mutational - high level resistance

Rare in E. faecalis but common in E. faecium

Question 14

Glycopeptide resistance in VRE

Answer 14

Glycopeptides such as vancomycin bind to DalaDala preventing cross linking of peptidoglycan

VREs acquire vanA or vanB gene clusers which alter the target site of vancomycin and thus reduce its binding affinity - loss of H bond required for vanc binding

VanA confers high-level resistance to both vanc and teicoplanin while vanB are typically susceptible to teicoplanin

These genes are often located on mobile genetic elements, thus facilitating horizontal gene transfer among enterococci and other GPs

Question 15

Linezolid resistance in VRE

Answer 15

Linezolid was introduced in 2000 as a last resort antibiotic
Linezolid inhibits bacterial protein synthesis by binding to the 23S rRNA of the 50s ribosomal subunit

VRE has a point mutation, G2576T, in the 23S rRNA mutations which alters the linezolid binding site reducing its affinity for the ribosome

Question 16

How do we screen for VRE

Answer 16

Rectal swabs
chromaogenic agar
Confirm with real time PCR either directly from sample or a colony

Question 17

How do we test for VRE in the lab

Answer 17

MIC - agar dilution, broth microdilution, gradient MIC
-> high level van resist = Van A >64m/L
-> low level van resist = Van B, MIC = 4

Disk diffusion - vancomycin 5ug disk
-> <12 mm = resistant
-> fuzzy zone edges and/or colonies reported as resistant

VITEK/automation nont recommended
Confirm genotypically using PCR for VanA or vanB

Question 18

Strep pneumoniae resistance, clinical significance

Answer 18

Historically S. pneumoniae was suceptible to penicillin
Resistance appeared in the 1970s
From 1997 weve had abou 20% resitance
Considerable increase in incidence of invasive isolates
- significance increasing trend in AMR over lat 5 years

In 2023 17.5% of all invasive strains were Penicilin nonwild type representing a decrease form 23.6% in 2022

Question 19

Strep pneumoniae, resistance mechanism

Answer 19

Mediated by alterations in PBPs especially PBP2x, PBP2b or PBP1a

Resistance arises from mutations or horizontal acquisition of altered PBP genes via transformation from related bacterial species - results in reduced binding affinity for penicillin and other beta-lactam antibiotics

Most PNSP and Pen-I strains remain susc to broad spectrum B-lactams but Pen-HLR strains are often co-resistant to macrolides, cephalosporins and quinolones

Many strains exhibit MDR, defined as resistance to three or more antibiotic classes -> MDR is often associated with mobile genetic elements including transposons

Question 20

How do we test for Penicillin resistance in the lab

Answer 20

PNSP strains with reduced susceptiblity to penicillin -> will have MICs above those of wildtype >0.06 mg/L

Screen using 1 ug Oxacilin -> disk diffusion -> if oxacillin susceptible then penicillin susceptible

Confirm oxacillin non suscpetibles by performing penicillin and cefotaxime MIC

Question 21

Clinical significance of TB

Answer 21

8.6 million cases and 1.4 million deaths a year
high levels in southern africa
etc

Question 22

Detection of tuberculosis

Question 23

Resistance in TB

Question 24

Clinical significance of VTEC

Answer 24

Shiga toxin causes diarrhoea, haemorrhagic colitis, HUS

Question 25

Detection of VEC

Answer 25

Stx1 or Stx2