Sensitivity testing (following BSAC) Plate selection ISA Staphylococcus (routine sensitivity and when using cefoxitin to test

for MRSA). Enterococci. Enterobactericeae Pseudomonas, Stenotrophomonas Staphylococci (when using methicillin/oxacillin to detect MRSA) Strep pneumonia, beta haemolytic streptococcus, Moraxella catarrhalis, Neisseria Campylobacter Alpha haemolytic streptococcus. Haemophilus sp Pasteurella Bacteroides Clostridia Coryneform

Columbia agar with 2% NaCl ISA + blood (ISOB)
(5% defibrinated horse blood) Or

ISA + Blood + NAD (20mg/L) ISA + Blood + NAD (20mg/L) (ISON)

Preparation of inoculum The inoculum should give semi-confluent growth of colonies after overnight incubation. Use of an inoculum that yields semi-confluent growth has the advantage that an incorrect inoculum can easily be observed. A denser inoculum will result in reduced zones of inhibition and a lighter inoculum will have the opposite effect. The following methods reliably give semi-confluent growth with most isolates.

Prepare a 0.5 McFarland solution by picking up colonies and mixing with distilled water. To make sure it is 0.5 McF – compared with the standard given in the exam or use a densitometer. Once you have a 0.5 McF standard dilute it 1:100 BHS Enterococci Enterobactericeae Pseudomonas Stenotrophomonas Acinetobacter Haemophilus Pasteurella Bacteroides 1:10 Staphylococci Serratia Streptcoccus pneumonia Neisseria meningitides Moraxella catarrhalis Alpha HS Clostridium perfringens Coryneform No dilution Neisseria Campylobacter

Direct sensitivity Blood culture GNR Put 1 drop of blood in 5 ml sterile water, dip a cotton swab, remove the excess water and by turning the swab against the inside of the container and then inoculate Put 3 drops in 5 ml sterile water. In exam – you will get broth If not sure which type of strep – just put a drop of broth on agar and lawn

Suspecting staphylococci/enterococci Pneumococci/viridans Put a drop directly onto the agar plate and lawn strep/coryne

Urine 1 Or 2 Take 10mcl loopful of urine, put at the centre of plate. Lawn with a sterile swab. Dip a swab, remove excess by turning, and make a cross on the agar plate, lawn with another swab.

Incubation 35 degC, air, 20h Enterobactericeae Pseudomonas, Acinetobacter, Staphylococci (Except when screening for MRSA with methicillin/oxacillin) Staphylococci – screen for MRSA using cefoxitin Moraxella BHS Enterococci Stenotrophomonas Staphylococci ( When screening for MRSA using methicillin/Oxacillin) AHS Pneumococci Neisseria Haemophilus Pasteurella Coryneform Campylobacter Bacteroides, clostridia If not sure, try this.

35 deg C, air, 24 hr 30 deg C, air, 20 h 30 deg C, air, 24 h 35 deg C, 4-6% CO2, 20h

42 degC, microaerophilic, 24hr 35 degC, anaerobic, 20h 35 deg C, 4-6% CO2, 24 h

Proteus – ignore any spreading, minute colonies near the zone, and few colonies growing inside trimethoprim and sulphonamide zone. Staph aureus cefoxitin screen – measure the obvious zone, not the inner zone. Other – if any growth occurs inside, culture them and repeat sens if necessary.

What sensitivity to put in 1. The examination centre might provide stamper – Staph1, staph 2, Coli 1(GN1), Coli2 (GN2), Strep, Entero, Pseudo, coryne1, coryne 2 etc 2. BSAC recommendations are here. – check AST testing and reporting guidance 3. You might want to make your own panel. If you are doing it try to keep it as minimum as possible (6 discs), it will save time. But make a panel 2 with other antibiotic which you should use if a resistant bug suspected/seen. If you have some sen from nonselective panel (eg GN), don’t put that again in selective panel (eg Salmonella – amox , piptazo, mero common) GPC /staph 1 GPC/staph 2 Staph E test Cefoxitin Neomycin Teic Linezolid Chloramphenicol Vanc Gentamicin Tigecycline dapto Erythromycin Trimethoprim Clindamycin Mupirocin Tetracycline Rifampicin Pneumo 1st line Pneumo 2 Enterococcus panel Optochin Pen E test Amoxicillin Oxacillin Ceftriaxone E test Linezolid Tetracycline Meropenem E test Vancomycin Moxifloxacin Teicoplanin Linezolid Gentamicin 200 Amox (for entero) Qui-Dalfo st nd GN 1 line GN 2 line Salmonella (exclude if Amoxicillin Ceftazidime anything already done in GN) Coamoxiclav Ciprofloxacin Amox/ampi Piptazo Chloramphenicol Cotri Meropenem Tetracycline Pip-taz Cefpodoxime Trimethoprim Meropenem Gentamicin Nalidixic acid Colistin E test Azithromycin Cipro E test (invasive) Strep1 Corynebacterium Campylobacter Pen Pen Cipro Ery Cip Nail Clind Vanc Ery Linezolid Tetra Or consider putting strep and Trim ent panel Haemophilus Listeria Acinetobacter Amox Ampi E test Gentamicin Coamox Gent E test Pip-Taz Ceftriaxone Cotri E test Meropenem Tetra Meropenem E tets Ciprofloxacin Mero (Amikacin) Cotri Colistin E test Mero E test (meningitis)

Pseudomonas Gent Tobra Pip-taz Mero Cefta Cipro

Pseudomonas 2 Tobra Amik Aztreonam Coli MIC

Stenotrophomonas Cotrimoxazole

Points to remember while reporting Salmonella Aminoglycosides always report resistant Cefuroxime always report resistant Do cipro E test for all invasive salmonella infection. Amoxicillin always report resistant Chromosomal penicillinase(Kleb), inducible ampC (ESC) Inducible amp C

Klebsiella, Enterobacter, Serratia, Citrobacter Enterobacter, Serratia, Citrobacter (ESC) ESC and Morganella Tigecycline

Coamoxiclav always report resistant

Colistin Daptomycin

Sta aureus

Suppress cefotaxime, ceftazidime, ceftriaxone. Or put a comment to discourage monotherapy E coli – do disc test If R/I – do MIC Non E coli – need MIC (if you want to check it) Prot/Providentia/Morganella – always R Always do E test Need calcium for e test. Culture plate with calcium supplement or E test strip with calcium supplement is available. Cefoxitin/Oxacillin R - Test for MRSA Suspecting vanc/teic R – send to ref lab Southmead/cardiff Ery S would be sen to other macrolide Ery R, clinda S – test for dissociative resistance (if making panel put ery and clinda next to each other)

Risk of selection of resistance

Prot always R Disc testing not done

If screening with oxacillin remember to use MHA/Columbia agar with 2% NaCl and incubate at 30 degC If screening by cefoxitin – just incubate with other ab disc at 35 degC

CoNS (no s sapro) Cefoxitin intermediate and sensitivity needed (ie deep seated infection), do E tets Strep pneumo Put an oxacillin disc to test for beta-lactams. If sensitive – report S for all beta lactam abx If Oxacillin R – do pen MIC

MIC <0.06 – CNS /other inf can be treated with pen <2

Test for ESBL (Not for non-fermenters) Screen Ceftazidime, cefotaxime resistant/ Cefpodoxime resistant.

Use – ISA, 35, air - put Ceftazidime and ceftazidime+coamoxiclav Cefotaxime and cefotaxime + coamoxiclav Cefpodoxime and cefpodoxime + coamoxiclav For Enterobacter, C freundii , Morganella, Providential, Serratia (AmpC producers – hence use cephalosporins that are AmpC resistant) usecefpirome/clavulanate or cefepime/clavulanate

Reading - - zone diameter increases of >5mm or >50% in the presence of the clavulanate implying ESBL production If using E test for ESBL detection – use heavier inoculum then disc test. ESBL production is inferred if the MIC ratio for cephalosporin alone: cephalosporin + clavulanate MIC is >8 Diagnosing Hyperproduction of K1 Klebsiella, spot indole +, R – pip-taz, aztreonam, cefuroxime Mod susceptible to Cefotaxime (23-30) S to ceftazidime OXA1 ESBLs may be susceptible to clavulinate/tzobactam, but in UK often it is associated with OXA1 which has a penicillinase resistant inhibitor and converts resistance to PT/AMC. Carbapenemase Intrinsic – Stenotrophomonas maltophilia, Aeromonas sp., and ‘chryseobacteria’, including Elizabethkingia meningoseptica. Acinetobacter baumannii (OXA like) Non fermentors are resistant to Ertapenem. Proteus, serratia are resistant to Imipenem. Acquired Class A Class B KPC, IMI NDM, VIM, IMP

MBL, needs Zn, inhibited by EDTA Enterobact, acineto, pseudo Porin loss – affect erta mostly, OprD in psA affect imi.

Class D OXA Other method of R

Screening of carbapenemase Enterobactericeae Test Erta (more sensitive), mero (more specific) Non fermentor Test Mero, imi, dori Acinetobacter – If R – just report as R, if travelled abroad test for NDM (Strong EDTA synergy (>8-fold) correlates well with MBL) Pseudomonas – R to carbapenems only – do not test further R to ceph and taz – test for NDM

Confirmatory test
Increase in zone>5 mm/ In etest – 8mm ROSCO disc test 0.5 McF, Mueller Hinton agar, Meropenem and Meropenem + Cloxacillin ( Meropenem and Meropenem + DPA Meropenem and Meropenem + Boronic Acid 35 degree/20 hrs/ air Difference between M and MC M and MD Amp C ≥ 5 mm ≤ 3 mm KPC ≤ 3 mm ≤ 3 mm MBL ≤ 3 mm ≥ 5 mm 2 mechanism can be present and expect ≥ 5 mm value in more than one pair. Other tests – Consider doing mero and imi e-test – mero R, imi S – efflux pump Modified Hodge test MALDI-ToF Carba NP Molecular method Chromogenica agar / Mac with carbapenem disc – Can be used for rectal screening. M and MB ≤ 3 mm ≥ 5 mm ≤ 3 mm