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Background Antimicrobial resistance in N. gonorrhoeae occurs as chromosomally mediated resistance to a variety of agents including penicillin, tetracycline, spectinomycin, and fluoroquinolones. In addition, high-level, plasmid-mediated resistance to penicillin and tetracycline has been described in N. gonorrhoeae. The resistance of strains of Neisseria gonorrhoeae to antimicrobial agents has usually been detected within a few years of their introduction for gonorrhea therapy. In the Gonococcal Isolate Surveillance Project (GISP), the Centers for Disease Control and Prevention (CDC) conducts routine surveillance to monitor antimicrobial resistance in N. gonorrhoeae in approximately 26 cities in the United States. Susceptibilities of gonococcal isolates to penicillin, tetracycline, spectinomycin, ceftriaxone, ciprofloxacin, and azithromycin are currently measured. Resistance of gonococci to penicillin and tetracycline are measured as plasmid- or chromosomally mediated resistance. Chromosomal resistance to spectinomycin and ciprofloxacin are measured as are decreased susceptibility to ciprofloxacin, ceftriaxone, and cefixime. The distribution of strains resistant to antimicrobial agents varies geographically; local surveillance is required to determine the distribution of antimicrobial resistance in individual communities. CDC currently recommends that broad spectrum cephalosporins or fluoroquinolones be used to treat uncomplicated gonorrhea. At this time, no failures of gonococcal infections to respond to therapy with broad spectrum cephalosporins have been documented. Recently, however, the failure of gonococcal infections caused by fluoroquinolone-resistant strains of N. gonorrhoeae to respond to ciprofloxacin has been documented in Australia, the United Kingdom, Canada, and the United States. Fluoroquinolone-resistant strains of N. gonorrhoeae now account for as many as 10% of isolates in Hong Kong and Japan, and for as many as 62% of isolates in the Republic of the Philippines. Although the agar dilution method is the reference method for measuring antimicrobial susceptibilities of N. gonorrhoeae strains to antimicrobial agents, when performed correctly, a disk diffusion susceptibility test may be used to reliably identify antimicrobial-resistant isolates of N. gonorrhoeae.

Principal In the disk diffusion susceptibility test, disks containing known amounts of an antimicrobial agent are placed on the surface of an agar plate that has been inoculated confluently with a standardized suspension of a strain of N. gonorrhoeae . The antimicrobial agent diffuses into the medium causing a zone of inhibition of growth of the strain around the disk corresponding to the susceptibility of the strain to that agent. Interpretative zone diameters have been established to permit classifying an isolate as belonging to the susceptible, intermediate resistance, or resistant categories of susceptibilities to an antimicrobial agent. Reference Strains Gonococcal reference strains may be provided either by the Neisseria Department, Statens Seruminstitut, Copenhagen, Denmark, or selected from a strain collection maintained in the Bacterial STD Branch (BSTDB), Division of AIDS, STD, and TB Laboratory Research (DASTLR), National Center for Infectious Diseases, CDC. These strains have been used as controls in disk-diffusion and agar-dilution susceptibility procedures for several years. Strains have been grown in large volumes, dispensed in 0.5 ml volumes, lyophilized, and stored at 4 C. The strains have the zone inhibition sizes shown in Table 1. Reference strains are tested to confirm the quality of disk diffusion and agar dilution susceptibility test procedures which are used to determine the antimicrobial susceptibilities of isolates of N. gonorrhoeae. These strains may be used to define zones of inhibition corresponding to resistance of N. gonorrhoeae isolates to penicillin, tetracycline, spectinomycin, ceftriaxone, ciprofloxacin, and ofloxacin. It is intended that these strains be used in conjunction with strains recommended by the NCCLS when determining the susceptibilities of gonococcal isolates. Recovery, Maintenance, and Storage of Reference Strains The strains may be rehydrated in 0.5 to 1.0 ml of Mueller-Hinton broth or sterile saline; phosphate buffered saline, pH 7.2, may also be used. Inoculate the suspension of rehydrated organisms onto plates of a selective medium by the streak plate method to allow growth of individual colonies. Examine plates for growth after incubation at 35C to 36.5C in 5% carbon dioxide for 24 to 48 hours. Subculture isolated colonies resembling N. gonorrhoeae onto a non-selective medium (e.g., chocolate agar) and incubate plates at 35C to 36.5C in 5% carbon dioxide for 24 hours. Confirm that the culture is pure, and composed of gram-negative, oxidase-positive diplococci. (Although vials of each strain have been tested, it is possible that the strain may have been contaminated during preparation or rehydration.) To store strains for future use, prepare a dense suspension with growth from a 24-h culture (on a nonselective medium) in 0.5- to 1.0-ml trypicase soy broth containing 20% glycerol. Strains should be stored at -70C; gonococcal isolates do not survive when stored for long periods of time at -20C. Please note that cultures for susceptibility testing must be subcultured every 24 h or prepared from a frozen culture each week of use. Notes: Do not incubate cultures for susceptibility testing for 48 h. Chromosomal resistance to penicillin, particularly in strain F-45, may be lost if this strain is incubated for 48 h. Do not use 24-hour cultures of strains from the frozen preparation for susceptibility testing; strains must be subcultured once before use. Materials -N. gonorrhoeae reference strains: ATCC 49226, NCCLS-recommended QC strain F-28, F-45, CMRNG (Chromosomally mediated resistance to penicillin and tetracyline P681E, PPNG (Plasmid-mediated resistance to penicillin and tetracycline CDC 10,328, Strain exhibiting intermediate resistance to ciprofloxacin and ofloxacin CDC 10,329 -Selective medium (Martin-Lewis, modified Thayer-Martin, GC-Lect, or equivalent medium). -Nonselective medium (Chocolate agar supplemented with 1% IsoVitaleX or equivalent medium) -Mueller-Hinton broth, saline, or phosphate buffered saline, pH 7.2. -Pasteur pipettes and rubber bulb or tuberculin syringes/27 gauge needles. -Microbiological loop. -Incubator at 35C to 36.5C; use an incubator supplied with 5% carbon dioxide or candle extinction jars to provide supplemental carbon dioxide. -GC agar base medium containing 1% (v/v) IsoVitaleX (or equivalent medium; 60 ml of medium in a 150 mm Petri dish). -Antibiotic disks. -McFarland Turbidity Standard Procedure Suspend isolated colonies from an overnight culture on supplemented chocolate agar medium in 0.5 to 1.0 ml. of Mueller-Hinton broth. Mix the suspension thoroughly on a vortex mixer to break up all clumps of growth. Adjust the turbidity of the cell suspension by adding additional Mueller-Hinton broth, as required, until the turbidity of the cell suspension is equivalent to the turbidity of a McFarland 0.5 barium sulphate standard. Discard the cell suspension if it is not used within 15 to 20 min. after preparation. Prewarm, to room temperature, plates of supplemented GC agar for each strain to be tested. The number of plates required per strain will depend on the number of antimicrobial agents to be tested and their anticipated zone inhibition diameters; disks should be placed on plates so that zones of inhibition do not overlap. The surface of the plates should be dry. If not, dry the plates (with lids ajar) in a 35C to 36.5C incubator just prior to inoculation. There should be no visible droplets of moisture on the surface of the agar or on the lids of the plates when they are inoculated. Moisten a sterile applicator swab in the standardized cell suspension and express any excess moisture by rotating the swab against the glass above the liquid in the tube. Inoculate the entire surface of each plate, inoculating the surface completely in three different directions to ensure uniform growth (Figure 1). It is recommended that cotton swabs with wooden handles be used for this procedure. Swabs made of synthetic materials do not soak up sufficient suspension to inoculate the entire surface of the plate. Swabs with plastic handles bend when excess suspension is being expressed and may splatter liquid out of the tube. Figure 1. The entire surface of the plate is inoculated in three directions as indicated to ensure uniform growth. Repeat step 4 to inoculate additional plates as needed. Store the inoculated plates at room temperature for 3 to 5 min. to allow the medium to absorb the moisture from the inoculum. Apply disks of the selected antimicrobial agents to the surface of the inoculated medium and tamp them with a sterile loop or forceps to ensure that they are in complete contact with the agar surface. All disks should be approximately the same distance from the edge of the plate and from each other (Figure 2). Invert the inoculated plates and incubate them at 35 C in 3-5% carbon dioxide for 20-24 hours. Figure 2. Antibiotic disks are placed approximately the same distance from the edge of the plate and from each other. Ensure that the disks are in complete contact with the surface of the agar; tamp with a sterile loop or forceps if necessary. The zones of inhibition are measured as shown in the figure. Examine the plates from the back, viewed against a black background and illuminated with reflected light. With calipers, measure the diameter of each zone of inhibition to the nearest whole millimeter. The NCCLS has recommended a range of values for the susceptibility of N. gonorrhoeae strain ATCC 49226 to a variety of antimicrobial agents. The values given for the other reference strains in this protocol were obtained when strains were tested on GC II agar base plus 1% IsoVitaleX; values may vary slightly when strains are tested on other media (Table 1). If the results for the reference strains meet those recommended by the NCCLS and those listed in Table 1, the results for the test strains may be interpreted according to the criteria listed in Table 2

Table 1. Quality Control Values for Reference Strains of Neisseria gonorrhoeae

Abbreviations. Susc., susceptible; SpcR, spectinomycin-resistant; CMRNG, strains resistant to penicillin and tetracycline (MICs >=2.0 mg/L); PP/TR, strains with plasmid-mediated resistance to penicillin and tetracycline; CipI, strains with intermediate resistance to ciprofloxacin and ofloxacin; CipR, resistant to ciprofloxacin and ofloxacin; NT, not tested. *Values for strain ATCC 49226 are those published by the National Committee for Clinical Laboratory Standards; values for other strains were obtained at CDC on GC II base medium (Becton Dickinson, Cockeysville, MD) supplemented with 1% IsoVitaleX. **Very slight inhibition seen occasionally with colonies growing to the edge of the disk. Note: N. gonorrhoeae strains CDC 10,328 and CDC 10,329 are currently used at CDC for quality assurance of susceptibilities to fluoroquinolones (ciprofloxacin and ofloxacin) recommended for the primary treatment of uncomplicated gonorrhea.

Table 2. Criteria for the Interpretation of Susceptibilities of Strains of Neisseria gonorrhoeae

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