Determination of the antimicrobial susceptibility of

Neisseria gonorrhoeae

Trevor Winstanley Rebecca Clarke

Department of MicrobiologyRoyal Hallamshire Hospital

Sheffield UK29 July 2003 Freeman Hospital

Gonorrhoea 2nd most common bacterial STD

> 22,500 episodes diagnosed in GUM clinics in England & Wales (2001)

Highest incidence Males 20-24 256/100,000 Females 16-19 198/100,000

Epidemiology Concentrated within demographic

and behavioural risk groups High-levels of

re-infection concurrent STDs asymptomatic infection

Public health concern Increasing incidence Poor reproductive and sexual

health outcomes High prevalence of resistance

onward transmission adverse clinical sequelae

Antimicrobial resistance 10 -12% of gonococcal isolates are

resistant to some degree inner cities those acquiring infections abroad gay and bisexual men

Plasmid-mediated

ß-lactamase

Tetracycline

PPNG Positive < 16 mg/L

TRNG Negative 16 mg/L

PP/TRNG Positive 16 mg/L

Chromosomal

ß-lactamase Penicillin Tetracyclin

e

PenR Negative 1 mg/L < 2 mg/L

TetR Negative < 1 mg/L 2 - 8 mg/L

CMRNG Negative 1 mg/L 2 - 8 mg/L

Chromosomal

CiprofloxacinIntermedia

te 0.125 < 1

mg/LResistant 1 mg/L

Spectinomycin Resistant 128 mg/L

Ceftriaxone Decreased 0.5 mg/LAzithromycin Resistant 1 mg/L

U.K. guidelines Easily treatable

95% cure from 1st line therapy Penicillins Fluoroquinolones

ciprofloxacin, ofloxacin Cephalosporins

ceftriaxone, cefixime (Doxycycline / tetracycline)

GRASP Gonococcal Resistance to

Antimicrobials Surveillance Programme DoH sentinel surveillance

PHLS Communicable Disease Surveillance Centre (CDSC)

PHLS Genitourinary Infections Reference Laboratory (GUIRL)

Department of Infectious Diseases & Microbiology at Imperial College

GRASP 2001 (n = 2666)

Penicillin Plasmid 3.0%Chromosome 5.1%

Tetracycline Plasmid 4.6%Chromosome 27.9%

Ciprofloxacin Intermediate 2.6%Resistant 3.1%

Azithromycin Resistant 0.3%Spectinomycin Resistant NDCeftriaxone

Cure ratesUncomplicated genital gonorrhoeaRecommended dosage

Susceptible > 95% Intermediate 90-95% Resistant < 90%

WHO; SRGA; NCCLS

Media

NCCLS SRGA BSAC ARMRL GRASP

Objectives To validate the BSAC disc diffusion

method for N.gonorrhoeae to translate reference into routine

To confirm or refute tentative breakpoints

To extend the range of interpretive criteria

Methodology 222 distinct isolates from 5

geographical regions 5 WHO control strains Disc diffusion tests, MICs

BSAC methodology ß-lactamase

Nitrocefin

0

5

10

15

20

25

30

48464442403836343230282624222018161412108642

Zone diameter (mm)

Num

ber

of is

olat

es

32

16

8

4

2

1

0.5

0.25

0.125

0.064

0.032

0.016

0.008

0.004

MI C(mg/ L)

Penicillin

Amoxycillin

0

5

10

15

20

25

30

464442403836343230282624222018161412108642

Zone diameter (mm)

Num

ber

of is

olat

es

321684210.50.250.1250.0640.0320.0160.0080.004

MIC(mg/ L)

Co-amoxyclav

0

5

10

15

20

25

30

50484644424038363432302826242220181614121086

Zone diameter (mm)

Num

ber

of is

olat

es

321684210.50.250.1250.0640.0320.0160.0080.004

MIC(mg/ L)

Ciprofloxacin

0

5

10

15

20

25

30

35

585654525048464442403836343230282624222018161412108642

Zone diameter (mm)

Num

ber

of is

olat

es

32

16

84

2

1

0.50.25

0.125

0.064

0.032

0.0160.008

0.004

MIC(mg/ L)

Nalidixic acid

0

5

10

15

20

25

30

35

40

45

50

48464442403836343230282624222018161412108642

Zone diameter (mm)

Num

ber

of is

olat

es

32

16

8

4

2

1

0.5

0.25

0.125

0.064

0.032

0.016

0.008

0.004

MIC(mg/L)

Tetracycline

0

5

10

15

20

25

30

5048464442403836343230282624222018161412108642

Zone diameter (mm)

Num

ber

of is

olat

es

16

84

2

1

0.50.25

MIC (mg/ L)

Metzler & DeHaan analysis

AntibioticMIC breakpoint (mg/L)

Disc content

Zone diameter (mm)

R S R S Spectinomycin 128 64 25 13 14

Antibiotic

MIC breakpoint (mg/L)Disc

content

Zone diameter (mm)

R Low R S R

Low R S

Spectinomycin 128 - 64 25 13 - 14

Ciprofloxacin 2 0.12-1 0.06 1 14 15-28 29

The MIC breakpoint has been lowered to ensure that isolates with reduced susceptibility to ciprofloxacin are detected.

AntibioticMIC breakpoint (mg/L)

Disc content

Zone diameter (mm)

R S R S Spectinomycin 128 64 25 13 14

Ciprofloxacin 2 0.12-1 0.06 1 14 15-28 29

Nalidixic acid - - 30 - -

Quinolone resistance is most reliably detected with nalidixic acid. Strains with reduced susceptibility to fluoroquinolones have no zone of inhibition with nalidixic acid.

Antibiotic

MIC breakpoint (mg/L)Disc

content

Zone diameter (mm)

R Low R S R Low

RS

Spectinomycin 128 - 64 25 13 - 14

Ciprofloxacin 2 0.12-1 0.06 1 14 15-28 29

Nalidixic acid - - - 30 - - -

Penicillin 2 0.12-1 0.06 1 unit 17 18-25 26

Test for -lactamase.

Antibiotic

MIC breakpoint (mg/L)Disc

content

Zone diameter (mm)

R Low R S R Low

RS

Spectinomycin 128 - 64 25 13 - 14

Ciprofloxacin 2 0.12-1 0.06 1 14 15-28 29

Nalidixic acid - - - 30 - - -

Penicillin 2 0.12-1 0.06 1 unit 17 18-25 26

Cefuroxime 2 - 1 5 19 - 20

Ceftriaxone 0.5 - 0.25 5 34 - 35

Resistance to ceftriaxone has not been described. Isolates with chromosomally encoded penicillin resistance (low level) have slightly reduced zones of inhibition with ceftriaxone but remain susceptible. Confirm by MIC.

Antibiotic

MIC breakpoint (mg/L)Disc

content

Zone diameter (mm)

R Low R S R Low

RS

Spectinomycin 128 - 64 25 13 - 14

Ciprofloxacin 2 0.12-1 0.06 1 14 15-28 29

Nalidixic acid - - - 30 - - -

Penicillin 2 0.12-1 0.06 1 unit 17 18-25 26

Cefuroxime 2 - 1 5 19 - 20

Ceftriaxone 0.5 - 0.25 5 34 - 35

Tetracycline 2 - 1 10 13 14-26 27

Use tetracycline result to infer susceptibility to doxycycline. Isolates with plasmid-mediated resistance have no zones of inhibition and those with low-level chromosomal resistance have zones 14-26 mm

Antibiotic

MIC breakpoint (mg/L)Disc

content

Zone diameter (mm)

R Low R S R Low

RS

Spectinomycin 128 - 64 25 13 - 14

Ciprofloxacin 2 0.12-1 0.06 1 14 15-28 29

Nalidixic acid - - - 30 - - -

Penicillin 2 0.12-1 0.06 1 unit 17 18-25 26

Cefuroxime 2 - 1 5 19 - 20

Ceftriaxone 0.5 - 0.25 5 34 - 35

Tetracycline 2 - 1 10 13 14-26 27

Erythromycin 1 - 0.5 5 11 - 12

Azithromycin 2 - 1 15 27 - 28

Rifampicin 2 - 1 2 20 - 21

Antibiotic

MIC breakpoint (mg/L)Disc

content

Zone diameter (mm)

R Low R S R Low

RS

Spectinomycin 128 - 64 25 13 - 14

Ciprofloxacin 2 0.12-1 0.06 1 14 15-28 29

Nalidixic acid - - - 30 - - -

Penicillin 2 0.12-1 0.06 1 unit 17 18-25 26

Cefuroxime 2 - 1 5 19 - 20

Ceftriaxone 0.5 - 0.25 5 34 - 35

Tetracycline 2 - 1 10 13 14-26 27

Erythromycin 1 - 0.5 5 11 - 12

Azithromycin 2 - 1 15 27 - 28

Rifampicin 2 - 1 2 20 - 21

Next?

Cefixime

Clap!