GONORRHOEAE.
Patterns of antimicrobial susceptibility vary greatly from one geographic area or population to another and flunctuate over time. An incurable case of gonorrhea has public health implications, because of the potential for continued transmission and for rapid emergence and propagation of antimicrobial resistant Neisseria gonorrhoeae strains.
Before 1976, antimicrobial susceptibility testing of Neisseria gonorrhoeae was not generally performed. Unfortunately, penicillinase- producing strains of Neisseria gonorrhoeae (PPNG) emerged. These isolates contain a plasmid (extrachromosomal DNA) that carries the gene for a B – lactamase. In 1977, the prevalence of PPNG strains was 0% 87. As at 1987 Osoba et al 88 showed a PPNG prevalence of 85.5% in Ibadan, Nigeria. By 1997, a study done by Bakare et al showed that the prevalence of PPNG strains had increased considerably to 94.6% 87. Also a study done, in 1998, by Arowojolo et al , where the microbiological features of acute salpingitis was investigated, showed that 95% of the Neisseria gonorrhoeae isolated were of the
Neisseria gonorrhoeae from patients that attended Special Treatment Clinic, University College Hospital, Ibadan, Nigeria between 1997 and 1999 showed a PPNG prevalence of 98.6% 9. These studies negates the usefulness of penicillin and ampicillin as the drugs of choice in gonorrhea therapy.
Spectinomycin emerged as an alternative antibiotic for the treatment of gonorrhea. By 1981 however, four spectinomycin – resistant Neisseria gonorrhoeae had been reported 90, although a study by Osoba et al , in 1987, showed that 100% of Neisseria gonorrhoeae isolates in Ibadan, Nigeria (including PPNG strains) were sensitive to spectinomycin 88. Resistance to spectinomycin is due to a single-step ribosomal mutation that results in high-level resistance (i.e. MICs of 256g/ml or higher). Due to reports of emergence of resistant strains and most importantly, the need to administer spectinomycin as intra-muscular injections, it’s usage in Nigeria became unpopular.
Emergence of PPNG also led to the use of tetracycline as a replacement for penicillin but unfortunately by 1985, the CDC reported the isolation of 12 Neisseria gonorrhoea strains from Georgia, Pennsylvania and New Hampshire that were penicillin susceptible but showed high-level tetracycline resistance 23. Studies have now determined that tetracycline resistant -Neisseria gonorrhoea (TRNG) harbor a 25.2MDa plasmid resulting from insertion of a tetracycline resistant determinant (tet M) into the 24.5MDa conjugative plasmid found in some Neisseria gonorrhoeae strains 91. The tetM conjugative plasmid can be transferred to suitable recipient strains of Neisseria gonorrhoeae by both genetic transformation and conjugation. The tet M determinant is believed to code for a protein or proteins that interact with the gonococcal ribosome to prevent inhibition of protein synthesis by tetracycline.
To further characterize these emerging multiple antibiotic resistant Neisseria gonorrhoeae strains, the CDC delineated 5 resistance phenotypes 92. These include:
1. Penicillin susceptible strains (MICs lower than 2.0g/ml)
2. Penicillinase producing Neisseria gonorrhoea strains (PPNG, B- lactamase positive)
3. High level, plasmid – mediated tetracycline resistant strains (TRNG, MIC’s of 2.0g/ml or higher) possessing the tetM determinants.
4. Strains with chromosomally mediated resistance to penicillin (CMRNG, MICs of 2.0g/ml or higher).
5. Strains with high level, plasmid-mediated resistance to both penicillin and tetracycline (PPNG B-lactamase positive and having the tetM determinant).
The appearance and spread of other resistance determinants among these five resistance phenotypes forms the basis of the gonococcal surveillance conducted throughout the United States by the CDC and the Gonococcal Isolate Surveillance Project (GISP).
As mentioned earlier, antimicrobial susceptibility pattern of Neisseria gonorrhoeae varies from region to region. In Jos, Nigeria, a study by Banwat et al showed that of the 31 strains of Neisseria gonorrhoeae isolated in 2001, 93.6% were susceptible to azithromycin, 90.3% to ciprofloxacin, 87.1% to ceftriaxone, 80.7% and 64.3% to erythromycin and ofloxacin respectively, less than half were each susceptible to spectinomycin (38.7%) and gentamicin (32.8%), while 12.9% and 4.6% were susceptible to tetracycline and penicillin respectively 93. These findings suggest that the use of gentamicin, tetracycline and penicillin were absolutely not recommended in Jos, Nigeria for the treatment of gonococcal infections. Azithromycin, Ceftriaxone and Ciprofloxacin, a quinolone antibiotic, were the better alternatives.
Quinolone antibiotics have been quite effective in the treatment of gonococcal infections. They are synthetic analogues of nalidixic acid. The earlier quinolones (nalidixic acid, oxolinic, acid, and cinoxacin) did not achieve systemic antibacterial levels after oral intake and thus were useful only as urinary antiseptics. The fluorinated derivatives (e.g. ciprofloxacin, norfloxacin, enoxacin, pefloxacin, lomefloxacin and others) have greater antibacterial activity and low toxicity and achieve clinically useful levels in blood and tissues.
Quinolones block bacterial DNA synthesis by inhibiting DNA gyrase, thus preventing the relaxation of supercoiled DNA, which is required for normal transcription and duplication. After oral administration, fluoroquinolones are well absorbed and widely distributed in body fluids and tissue to varying degrees, but they do not reach the central nervous system to a significant extent. The serum half-life is variable (3-8 hours) and can be prolonged in renal failure depending on the specific drug used. The fluoroquinolones are mainly excreted into the urine via the kidney, but some of it may be metabolized in the liver 11.
Worth mentioning are the most prominent side effects of quinolones, which include nausea, insomnia, headache and dizziness. Occasionally, there are other gastrointestinal disturbances, impaired liver function, skin rashes and super infections, particularly with Enterococci spp. and Staphylococci spp. In animals, prolonged administration of fluoroquinolones produce joint damage, and for that reason, fluoroquinolones have been seldom prescribed for children under 18 years but are used as needed in cystic fibrosis patients 11.
Table 2.3 below shows the generas of quinolones with examples:
Table 2.3: Classification of quinolones with examples
1st Generations 2nd Generations 3rd & 4th Generations Nalidixic acid
Conoxacin Oxolinic acid
Ciprofloxacin Enoxacin Lomefloxacin Ofloxacin
Clinafloxacin Gatifloxacin Gemifloxacin Levofloxacin Moxifloxacin Sparfloxacin
Courtesy: Brooks G.F, Butkel J.S, Morse S.A. Jawetz, Melnick and Adelberg’s Medical Microbiology.
22nd ed; Lange: 2001.
Subsequent to the publication of the PHS Treatment Guidelines where recommendation of the fluoroquinolones as acceptable therapy for uncomplicated gonorrhea was suggested, single dose oral quinolones have been recommended for gonorrhea treatment since 1989 94. Because of the ease of oral administration, low cost, apparent low prevalence of quinolone resistant Neisseria gonorrhoeae (QRNG) and at the suggestion
of the World Health Organisation (WHO), ciprofloxacin was adapted for the syndromic management of gonorrhoea in Nigeria 13. As mentioned earlier, Otubu et al in Jos, Nigeria, reported that the in-vitro sensitivity of Neisseria gonorrhoeae isolates to ciprofloxacin (using the disc diffusion sensitivity test) was 100% in 1992 16. But unfortunately, Neisseria gonorrhoeae isolates with decreased susceptibility to ciprofloxacin and ofloxacin began to emerge 95. By 1991, the Gonococcal Isolate Surveillance Project (GISP), a division of the CDC, identified the first ciprofloxacin resistant Neisseria gonorrhoeae in the United States 96. In 1997, Bakare et al observed a 15.6% prevalence of Neisseria gonorrhoeae strains resistant to pefloxacin 8. A later study by Banwat et al in Jos, Nigeria showed a 9.7% and 36.6% prevalence of Neisseria gonorrhoeae isolates resistant to ciprofloxacin and ofloxacin, respectively, in 2001 93. This rapid emergence of QRNG strains in Nigeria suggests that the usefulness of these agents for the syndromic management of gonorrhea will need to be reassessed in the not too distant future.
Presently, the CDC recommends that all patients with uncomplicated gonococcal infection receive one of the four single – dose treatment regimes, i.e. ceftriaxone (125mg/IM), cefixime (400mg orally), ciprofloxacin (500mg orally) or ofloxacin (400mg orally) 97, 98. Azithromycin (1.0g given orally, single dose) is also active against both Neisseria gonorrhoeae and Chlamydia trachomatis 97.
Mechanism of quinolone resistance in Neisseria gonorrheae
In bacteria, antimicrobial resistance is facilitated through their ability to quickly adapt to new environment and along with their ability to replicate very quickly, comes the aptitude to mutate their DNA or acquire DNA from another drug-resistant bacteria through transformation, transduction or conjugation 99,100. In order to decrease the spread of resistance among bacteria, physicians and patients alike need to be more
aware of selective pressures driving these bacteria to decrease their susceptibility to antibiotics.
Quinolone resistance in Neisseria gonorrhoeae can happen by alteration of drug targets, restriction of access to bacterial targets or both. Fluoroquinolone potency is determined by its ability to reach and act on their target enzyme, DNA gyrase and topoisomerase IV, which are both required for bacterial DNA replication. There are two subunits of DNA gyrase and topoisomerase IV genes: gyr A and gyr B; and par C and par E respectively. A reduction in the porin diffusion channels in the outer membrane of the gram negative bacteria cell envelope or an increase in the efflux mechanism activity, may decrease drug permeation or its ability to reach bacterial targets. Double mutations in gyrA (the DNA gyrase gene) have been correlated with high-level resistance in isolates with MIC’s greater than 1g/l 101, along with additional mutations in some isolates in the topoisomerase gene, par C. Gyr A is the primary target in Neisseria gonorrhoeae causing most of the low level resistance (ciprofloxacin MIC, 0.06 to 0.5g/ml), with par C changes secondary but associated with high level QRNG 102. Gyrase B subunit (gyr B) changes and other influences may also be present and increase quinolones MICs, but they are of additive nature rather than being a major influence. Corraborated by the study of Shultz et al, where DNA extraction and PCR analysis to demonstrate the sequence changes in the Quinolone Resistance Determining Region (QRDR) was done, the most common alterations in the gyr A QRDR was a serine exchanged for either a tyrosine or phenylalanine residue at position 91 (Ser 91-to-Tyr or Ser 91-to-phe, respectively) and aspartate for either an asparagine or glycine residue at position 95 (Asp 95-to Asn or Asp 95 to Gly) 103. Gyr A changes were confined to amino acid position 91 and 95, parC point mutations encoded amino acid changes to asparagine at position 86 (Asp
proline at position 88 (Ser 88-to-Pro) and glutamine or lysine at position 91 (Glu 91-to-Gln or Gly 91-to-lys).
Newer quinolones with differential activities against gyr A and par C, and overall enhanced activities have been developed 104, 105. For example, moxifloxacin and grepafloxacin have greater activity against topoisomerase IV while trovafloxacin principally targets gyrase activity. It has been suggested that these newer quinolones could provide more suitable treatments for infections caused by ciprofloxacin – resistant Neisseria gonorrhoeae 106. Earlier studies of the QRDR were restricted to low-level resistance and QRDR changes were related to ciprofloxacin only 107. Subsequently, more resistant strains have been investigated with QRDR changes related to newer quinolones 108,109. The study by Shultz et al suggests that newer quinolones (trovafloxacin, moxifloxacin and grepafloxacin) are unlikely to be useful replacements for ciprofloxacin in the treatment of gonorrhea, particularly where ciprofloxacin MICs are high or where resistance is widespread 103.
2.9. ANTIMICROBIAL SUSCEPTIBILITY TESTING OF NEISSERIA GONORRHOEAE
The National Committee on Clinical Laboratory Standards (NCCLS), now renamed the Clinical and Laboratory Standards Institute (CLSI), recommends the use of agar disk diffusion test and determination of minimal inhibitory concentration (MIC) by agar dilution technique to determine the susceptibility of a cultured Neisseria gonorrhoeae strain to an antibiotics. The more convienient broth dilution test (micro- or macro-) can be used but it is suggested that supplements like lysed horse blood or any haemoglobin containing supplement should be added to the broth 110. A multicenter study has been published in which disk diffusion and agar dilution
interpretive criteria for these antibiotics were determined and the guidelines for testing and interpretation has been published by the CLSI 111,112. The agar dilution test is performed only in reference centres.
In addition to macro- or micro- broth and agar dilution technique, the Epsilometer (E) test has also been used to determine the MIC of Neisseria gonorrhoeae 113. The E test strips consist of a plastic carrier strip with a predefined continuous antibiotic gradient immobilized on one side. When placed on a agar plate with a confluent growth of the test Neisseria gonorrhoeae strain and incubated at 37 0C in CO2 enriched environment for 16 to 18 hrs, the point and corresponding MIC value at which the line of inhibition meets the plastic strip is noted and recorded. The E test is easy to perform and the results are easy to interprete but the strips are expensive, making it’s use unpopular in developing countries like Nigeria.
A table of the 2002 interpretative criteria for Neisseria gonorrhoeae susceptibility testing is showed below.
Table 2.4: 2002 Interpretive Criteria for Neissseria gonorrhoeae Susceptibility Testing.
The following are zone diameter interpretive standards and equivalently MIC breakpoints for Neisseria gonorrhoeae 114.
At this time, no interpretation criteria exist for Azithromycin and Levofloxacin.
Antimicrobial Agent Zone Diameter ( mm) MIC Breakpoints (g/ml) Fluoroquinolones R I S R I
Ciprofloxacin or Gatifloxacin or Ofloxacin
<=27
<=33
<=24
28-40 34-37 25-30
>=41
>=38
>=31
>=1
>=0.5
>=2
<=0.06
<=0.12
<=0.25
Enoxacin <=31 32-35 >=36 >=2 <=0.5
Grepafloxacin <=27 28-36 >=38 >=1 <0.06
Lomefloxacin <=26 27-37 >38 >=2 <=0.12.
Trovafloxacin - - >=34 - <=0.25
Fleroxacin <=28 29-34 >=35 >=1 <=0.25
Courtesy: National Commitee for Clinical Laboratory Standard Performance Standard for Antimicrobial Susceptibility Testing; Twelfth International supplement; 2002.
To ascertain the susceptibility of Neisseria gonorrhoeae isolate to an antibiotic, determination of the MIC is more appropriate because it’s interpretation is less subjective than the disk diffusion test, the distinction between high and low level resistance can be easily determined and the optimal dose of the antibiotic for adequate treatment of the infection can be easily calculated. But due to the high cost and tedious nature of MIC determination, most laboratory centres usually use the cheaper, more convienient and but also reliable disk diffusion test.
CHAPTER 3
MATERIALS AND METHODS.
The study area was Ibadan North Local Government area of Oyo State, Nigeria.
The study was a prospective one and the normal procedure for approval by the Research and Ethical Committee of the University Teaching Hospital was followed.
Sample size determination.
Due to the high prevalence of patients with Sexually Transmitted Diseases (STD) not attending STD clinics and preferring self medication in Ibadan, a one and half week outreach programme was conducted at the motor parks at Ojo, Mokola, Bodija and Agodi areas of Ibadan North Local Government in February 2005. The outreach was aimed at encouraging workers, drivers and their cohorts in the parks with suspected STDs to attend the Special Treatment Clinic and Veneral Disease and Treponematoses Research Laboratory of the University College Hospital, Ibadan, Oyo State and Adeoyo Sexually Transmitted Disease Clinic, Ibadan, Oyo State, by promising them free diagnosis and treatment.
A total of 669 patients (186 male and 483 female patients) attended the Special Treatment Clinic and Veneral Disease and Treponematoses Research Laboratory of the University College Hospital, Ibadan, Oyo State and Adeoyo Sexually Transmitted Disease Clinic, Ibadan, Oyo State, from January 2005 to December 2005. A standard medical history concerning symptoms, marital status, previous genito-urinary symptoms, date of last coitus and sexual consorts (whether casual or regular) was obtained from them. A clinical examination of the lower genito-urinary tract for signs of infection such as vaginal, endocervical and urethral discharge was performed.
Patients recruited into the study were those who gave an approved consent and presented with a confirmed genital discharge. Exclusion criteria included the absence an observable discharge and declined consent to participate in the study. One hundred and fifty males and 475 females (total of 625 patients) met the inclusion criteria.
Sample size of Neisseria gonorrhoeae isolates required for this study, with given degree of accuracy at a given level of statistical significance was determined using a simple formula:
n z2 p q d2 where
n the desired sample size (when population is greater than 10,000).
z the standard normal deviate, set at 1.96, which corresponds to 95 percent confidence level.
p the proportion in the target population estimated to have a particular characteristics (percentage of Neisseria gonorrhoeae strains estimated to be resistant to ciprofloxacin), therefore 9.7% 93 was used.
q 1.0 – p.
d degree of accuracy desired, usually set at 0.05.
Therefore sample size for Neisseria gonorrhoeae isolates required was:
n (1.96) 2 (0.097)(0.903) (0.05) 2
= 134.6
Therefore approximate least sample size of Neisseria gonorrhoeae isolates appropriate for this study was 135.
Method for sample collection and laboratory procedures
Specimens were obtained using a pair of sterile swab sticks applied in the endocervix (endocervical swab) and another pair applied in the posterior fornix (high vaginal swab) of female participants. In male participants, a pair of sterile swab sticks was also inserted 2 – 2.5 cm into the urethra. The first of each pair of swab sticks were inoculated immediately on Modified Thayer – Martins (MTM) agar plates and the plates were incubated initially in a portable candle extinction jar and later promptly transfered to a CO2 incubator for incubation at 37 oC for 24hrs.
Smears were made from the second of the pair of swabsticks and stained by gram’s method (direct gram stain) to screen for presence of intracellular gram-negative diplococci and also for clue cells. Wet preparations of the swabs were also examined for Trichomonas vaginalis, Candida albicans, clue cells and other parasites. Samples from which clue cells were identified were regared as positive for Gardnerella vaginalis.
After 24hrs incubation, the plates were examined for characteristic Neisseria gonorrheae colonies and the suspicious ones were stained by gram’s method. All
gram-negative diplococci were identified as Neisseria gonorrheae by oxidase test and sugar utilization test.
Procedure for oxidase test
Oxidase reagent (N’, N’, N’, N’- tetramethyl –P- phenylenediamine dihydrochloride, 1% aqueous solution) was applied on a piece of white filter paper and a portion of the growth was rubbed onto the reagent with a wooden applicator.
A positive test was indicated by a dark purple coloration within 10 seconds and a negative result was reported when no coloration development within 10 secs.
The procedure was controlled with a confirmed Escherichia coli strain as negative control and a confirmed Pseudomonas aeroginosa strain as a positive control.
Procedure for sugar utilization test
Sugars used were glucose, sucrose, maltose and lactose.
Brain Heart Infusion (BHI) agar slants containing 1% sugar and phenol red, as acid production indicator, were made in 2ml bijou bottles. Inoculi of Neisseria gonorrhoeae isolates to be tested were stabbed into and streaked on the surface of the BHI agar slants containing the desired sugars. The same isolate was also inoculated into a BHI agar slant without any added sugar, to serve as a negative control. Then the bijoh bottles were incubated in a CO2 incubator at 37 oC and bottles were observed after 3hrs incubation. A postive result is indicated by yellow coloration of the media, indicating a pH drop i.e. acid production. The media in the negative control should show no colour change.
A control Neisseria gonorrhoeae (ATC 49226) was not available to control the procedure.
Colonies that were oxidase positive and fermented only glucose were identified as Neisseria gonorrheae species.
Venous blood was also taken from the participants and a Rapid Plasma Reagent (RPR) Card Test was performed, to rule out an associated or previous syphilitic infection.
Procedure for Rapid Plasma Reagent (RPR) Card Test (BD Macro - VueTM RPR Card, Becton Dickson and Company, 7 Loveton Circle Sparks, MD 21152, USA).
Principle of procedure
This is a macroscopic, non-treponemal flocculation card test which detects “reagin”, an antibody like substance present in serum or plasma from syphilitic persons and occasionally in serum or plasma of persons with other acute or chronic conditions.
The reagin binds to the antigen which is prepared from a modified Veneral Disease Research Laboratory (VDRL) antigen suspension containing choline chloride to eliminate the need to heat inactivate serum, ethylenediaminetetraacetic acid (EDTA) to enhance the stability of the suspension and finely divided charcoal particles as a visualizing agent and the reagin / antigen complex causes a macroscopic flocculation.
Procedure
1. Venous blood was taken from the participants, spun at 1000rpm for 30secs and the serum collected. The serum samples were stored and pooled in groups/multiples of eight because each RPR 18mm cards could be used for only 8 samples, with one positive and one negative control i.e. total of 10 spots.
2. Using a dispenstirTM , 50µl of each participant’s serum was placed into the first eight 18mm circle of the RPR card, making sure a different dispenstirTM was used for each sample. In the 9th 18mm circle, 50µl of positive control serum (i.e. human serum samples containing antibodies to Treponema pallidum, which is provided in the kit) and in the last (10th) circle, 50µl of
3. With each sample, the inverted dispenstirTM (closed end) was used to spread the serum to fill the entire circle, but not beyond the confinements.
4. The provided antigen dispensing bottle was gently shaken to resuspend the particles, and then one free falling drop (17µl) was added to each circle containg test and control serum.
5. The card was then placed on a mechanical rotator under a humidifying cover and rotated for 8 mins at 100rpm.
6. The card was then immediately removed from the rotator, briefly rotated and tilted by hand to aid in differentiating nonreactive from minimal reactive results.
A reactive (positive) result was the presence of characteristic flocculation, ranging from slight to intense black clumping and a nonreactive (negative) result was the absence of visible clumping. The positive control should be reactive and the negative control should be nonreactive.
Antimicrobial susceptibility testing on all confirmed Neisseria gonorrheae isolates was done using the disk diffusion test (modified Kirby Bauer test) and determination of minimal inhibitory concentration (MIC) using the broth macro-dilution technique.
Modified Kirby Bauer (Disk diffusion test)
All procedures below were carried out under strict aseptic condition.
1. The inoculum of each confirmed Neisseria gonorrheae isolate in Brain Heart Infusion (BHI) broth was adjusted to 0.5 Mc Farlands standard (107/108 CFU/ml). .
2. Using a sterile swab, the above broth suspension for each sample was smeared on a dry chocolate agar plate in 3 directions to ensure a confluent growth.
3. Antibiotic disks for ampicillin (Abtek Biologicals Ltd), ceftriaxone (Abtek Biologicals Ltd), ciprofloxacin, ofloxacin, pefloxacin and sparfloxacin (V.S International PVT, Ltd, Hind Cycle Rd, Worli, Mumbai) were then applied on the agar surface.
4. The agar plate was then placed in a CO2 incubator at 37 oC.
5. After overnight incubation (18 to 24hrs), the diameter of zones of inhibition of the different antibiotic disks were measured and compared with the National Committee on Clinical Laboratory Standards (NCCLS), thus the Neisseria gonorrheae isolate was categorized as either sensitive, intermediate or resistant to the antimicrobial agent.
No control Neisseria gonorrheae strain (ATC 49226) was available to control the procedure.
In the disk diffusion method, resistant strains were those with zone diameter ≤ 26mm (for ampicillin), ≤ 30mm (for tetracycline), ≤ 27mm (for ciprofloxacin), ≤ 24mm (for ofloxacin), ≤ 27mm (for pefloxacin), ≤ 35mm (for ceftriaxone) 114 and sparfloxacin (≤
15mm) 115.
Those that were resistant to the ampicillin disc were assumed to be Penicillinase producing strains (PPNG) and were confirmed by the starch paper technique 116. Starch paper technique for detection of beta-lactamase production
Procedure
1. Depending on the number of Neisseria gonorrhoeae isolates available for the procedure, strips of starch paper (one strip was used to test 4 isolates including the control) were soaked for 10 minutes in 100,000µg/ml of benzylpenicillin and spread smoothly in a petri dish.
2. Using a fine bacteriological loop, colonies of the test isolates were
test paper and spread over an area of 2 - 3mm (the inocula were placed at least 1.5cm apart).
3. The plates were incubated at 37 oC for 30mins, after which the paper was flooded with iodine solution (i.e gram’s iodine diluted 1 in 2) and observed in 30 seconds and after 5 mins.
The adding of iodine to the strip causes the paper to turn blue-black immediately because the starch in the paper binds with the iodine to form a blue-black complex.
If the test strain is beta-lactamase producing, the enzyme produced will degrade the beta lactam ring of the benzylpenicillin in the strip and lead to the production of penicilloic acid., which then reacts with the iodine to cause dissociation of the starch- iodine complex. Hence penicillinase producing Neisseria gonorrhoeae (PPNG) strains were detected by the decoloration of the blue-black color around the organism.
The whitish halo widens in the course of the ensuing five minutes, while the surface of the inoculum remains whitish. Penicillinase-negative strains will not produce any decoloration of the surrounding area and the organism retains a yellow tinge due to unchanged iodine.
Confirmed Staphylococcus aureus strains were used as positive control, but no negative control was available for the procedure.
Minimal inhibitory concentration (MIC) determination (Macro-broth dilution technique).
Preparation of Brain Heart Infusion (BHI) broth
All procedures below were carried out under strict aseptic condition.
To ensure sustained Neisseria gonorrhoeae growth, soluble haemoglobin powder was added as a supplement to the BHI broth 110.