Current Name	Previous Name
Moraxella catarrhalis	Branhamella catarrhalis, Neisseria catarrhalis
Neisseria gonorrhoeae
Neisseria meningitidis
*Other Neisseria* spp.**
N. animaloris	CDC group EF-4a
N. cinerea
N. lactamica
N. polysaccharea
N. subflava	N. subflava, N. flava, and N. perflava
N. sicca
N. mucosa
N. flavescens

General Characteristics

Species of the family Neisseriaceae, genus Neisseria, are discussed in this chapter, along with family Moraxellaceae, species Moraxella catarrhalis, because of their biochemical and morphologic similarities. The organisms are all oxidase-positive, gram-negative diplococci that do not elongate when exposed to subinhibitory concentrations of penicillin. The rodlike Neisseria spp. are described in Chapter 28.

Except for Neisseria gonorrhoeae, the organisms considered in Table 40-1 are normal inhabitants of the upper respiratory tract of humans. Humans are the only natural host for N. gonorrhoeae, primarily a clinically significant pathogen found in the urogenital tract and never considered normal flora. Asymptomatic carriers of gonorrhea are the primary reservoir for dissemination in the human population. The number of reported and identified cases of infection with N. gonorrhoeae is probably significantly higher than statistical data indicate because of the number of unreported cases.

TABLE 40-1

Epidemiology

Organism	Habitat (Reservoir)	Mode of Transmission
Moraxella catarrhalis	Normal human flora of upper respiratory tract; occasionally colonizes female genital tract	Spread of patient’s endogenous strain to normally sterile sites. Person-to-person nosocomial spread by contaminated respiratory droplets also can occur
Neisseria gonorrhoeae	Not part of normal human flora. Only found on mucous membranes of genitalia, anorectal area, oropharynx, or conjunctiva at time of infection	Person-to-person spread by sexual contact, including rectal intercourse and orogenital sex. May also be spread from infected mother to newborn during birth. Asymptomatic carriers are a significant reservoir for increased disease transmission.
Neisseria meningitidis	Colonizes oropharyngeal and nasopharyngeal mucous membranes of humans. Humans commonly carry the organism without symptoms	Person-to-person spread by contaminated respiratory droplets, usually in settings of close contact
Other Neisseria spp.	Normal human flora of the upper respiratory tract	Spread of patient’s endogenous strain to normally sterile sites. Person-to-person spread may also be possible, but these species are not common causes of human infections
Neisseria animaloris	Not part of normal human flora. Animal oral and respiratory commensal organism	Animal contact, particularly bites or scratches from dogs and cats

The two pathogenic species of Neisseria, N. gonorrhoeae and N. meningitidis, are transmitted person to person. N. gonorrhoeae is sexually transmitted, and N. meningitidis is spread via contaminated respiratory droplets. Infections caused by M. catarrhalis and the other Neisseria spp. usually involve a patient’s endogenous strain.

Pathogenesis and Spectrum of Disease

As noted in Table 40-2, infections caused by M. catarrhalis are usually localized to the respiratory tract and rarely disseminate.

TABLE 40-2

Pathogenesis and Spectrum of Disease

Organism	Virulence Factors	Spectrum of Disease and Infections
Moraxella catarrhalis	Uncertain; factors associated with cell envelope probably facilitate attachment to respiratory epithelial cells	Most infections are localized to sites associated with the respiratory tract and include otitis media, sinusitis, and pneumonia. Lower respiratory tract infections often target elderly patients and those with chronic obstructive pulmonary disease. Rarely causes disseminated infections such as bacteremia or meningitis.
Neisseria gonorrhoeae	Several surface factors, such as pili (types T1-T2 virulent and T3-T5 avirulent), mediate the exchange of genetic material between strains and attachment to human mucosal cell surface, invasion of host cells, and survival through the inhibition of phagocytosis in the presence neutrophils. Genetic-phase variation of pilus structure between types T1 through T5 allows the organism to vary its antigenic structure, preventing recognition by host immune cells. Capsule, lipooligosaccharide (endotoxin), and outer cell membrane proteins I-III are important in antigenic variation and for eliciting an inflammatory response. Protein II (Opa) facilitates adherence to phagocytic and epithelial cells. Protein II (RMP) blocks the bactericidal effect of host IgG. Outer membrane porin (PorB) provides protection from the host’s immune response, including serum complement–mediated cell death.	A leading cause of sexually transmitted diseases. Genital infections include acute purulent urethritis, prostatitis, and epididymitis in males and acute cervicitis in females. These infections also may be asymptomatic in females. Other localized infections include pharyngitis, anorectal infections, and conjunctivitis (e.g., ophthalmia neonatorum of newborns acquired during birth from an infected mother). Disseminated infections result when the organism spreads from a local infection to cause pelvic inflammatory disease or disseminated gonococcal infection that includes bacteremia, arthritis, and metastatic infection at other body sites. Pelvic inflammatory disease (PID) may cause sterility, ectopic pregnancy or perihepatitis also referred to as Fitz-Hugh–Curtis syndrome.
Neisseria meningitidis	Surface structures, perhaps pili, facilitate attachment to mucosal epithelial cells and invasion to the submucosa. Once in the blood, survival is mediated by production of a polysaccharide capsule. Endotoxin release mediates many of the systemic manifestations of infection, such as shock. Cellular proteins are similar to those described for N. gonorrhoeae, including Por and Opa. Two porin proteins are produced (PorA and PorB). IgA protease degrades membrane-associated IgA, increasing the host’s susceptibility to invasion.	Life-threatening, acute, purulent meningitis. Meningitis may be accompanied by appearance of petechiae (i.e., rash) that is associated with meningococcal bacteremia (i.e., meningococcemia). Bacteremia leads to thrombocytopenia, disseminated intravascular coagulation, and shock. Disseminated disease is often fatal. Less common infections include conjunctivitis, pneumonia, and sinusitis.
Other Neisseria spp.	Unknown; probably of low virulence	Rarely involved in human infections. When infections occur, they can include bacteremia, endocarditis, and meningitis.
Neisseria animaloris	Unknown	Cellulitis or abscess formation secondary to infected bite wounds; systemic infection (rare).

N. gonorrhoeae is a leading cause of sexually transmitted disease, and infections caused by this organism usually are localized to the mucosal surfaces where the host is initially exposed to the organism (e.g., cervix, conjunctiva, pharyngeal surface, anorectal area, or urethra of males). Localized infections may be asymptomatic or acute with a pronounced purulent response. Not all infections remain localized, and dissemination from the initial infection site can lead to severe disseminated disease (see Table 40-2). Isolates with nutritional requirements for arginine, hypoxanthine, and uracil (AHU strains) are often isolated from disseminated infections, most often from women although also from asymptomatic males.

N. meningitidis is a leading cause of fatal bacterial meningitis. However, the virulence factors responsible for the spread of this organism from a patient’s upper respiratory tract to the bloodstream and meninges, causing life-threatening infections, are not fully understood (see Table 40-2).

The other Neisseria spp. are not considered pathogens and are often referred to as the saprophytic Neisseria. Although they are most commonly encountered as contaminants in clinical specimens, they may occasionally be identified in bacteremia and endocarditis.

Laboratory Diagnosis

Specimen Collection and Transport

The pathogenic Neisseria spp. described in this chapter are very sensitive to drying and temperature extremes. In addition to general information on specimen collection and transport provided in Table 5-1, there are some special requirements for isolation of N. gonorrhoeae and N. meningitidis.

Swabs are acceptable for N. gonorrhoeae testing if the specimen will be plated within 6 hours; however, reduced recovery may result within 30 minutes of collection. If cotton swabs are used, the transport medium should contain charcoal (Ames medium) to inhibit toxic fatty acids present in the fibers. Calcium alginate has also been found to be inhibitory. Dacron or rayon fibers are recommended. N. gonorrhoeae should be inoculated to growth media immediately after specimen collection. The sample should then be placed in a container able to sustain an atmosphere of increased carbon dioxide (CO₂) during transport. Specially packaged media consisting of selective agar in plastic trays that contain a CO₂-generating system are commercially available (JEMBEC plates). The JEMBEC system (Figure 40-1) is transported to the laboratory at room temperature. Upon receipt in the laboratory, the agar surface is cross-streaked to obtain isolated colonies, and the plate is incubated at 35°C in 3% to 5% CO₂. Additional commercial transport systems that may be useful, particularly when the collection site is separate from the diagnostic laboratory, are the Bio-Bag, Gono-Pak, and Transgrow.

Figure 40-1 JEMBEC system. Plate contains modified Thayer-Martin medium. The CO₂-generating tablet is composed of sodium bicarbonate and citric acid. After inoculation the tablet is placed in the well, and the plate is closed and placed in the zip-lock plastic pouch. The moisture in the agar activates the tablet, generating a CO₂ atmosphere in the pouch.

Recovery of N. gonorrhoeae or N. meningitidis from normally sterile body fluids requires no special methods, except for blood cultures. Both organisms are sensitive to sodium polyanethol sulfonate (SPS), the preservative typically found in blood culture broths. If a blood culture broth is inoculated, the SPS content should not exceed 0.025%. In addition, if blood is first collected in Vacutainer tubes containing SPS (Becton Dickinson, Sparks, Maryland), the specimen must be transferred to the broth culture system within 1 hour of collection.

Nasopharyngeal swabs collected to detect N. meningitidis carriers should be plated immediately to the JEMBEC system, or they should be submitted on swabs placed in charcoal transport media.

Specimen Processing

The JEMBEC system should be incubated at 35° to 37°C as soon as the plate is received in the laboratory. Body fluids (e.g., joint or cerebrospinal fluid [CSF]) should be stored until cultured at 37°C, because both gonococci and meningococci are sensitive to cold.

Any volume of clear fluid greater than 1 mL suspected of containing either of these pathogens should be centrifuged at room temperature at 1500× g for 15 minutes. The supernatant fluid should then be removed and the sediment should be vortexed and inoculated onto the appropriate media (described later).

Any specimens or cultures in which N. meningitidis is a consideration should be handled in a biological safety cabinet to avoid laboratory-acquired infections.

Direct Detection Methods

Gram Stain

The members of the genus Neisseria discussed in this chapter and M. catarrhalis appear as gram-negative diplococci (Figure 40-2) with adjacent sides flattened. They are often referred to as “kidney bean”–shaped diplococci. Direct Gram staining of urethral discharge from symptomatic males with urethritis is an important test for gonococcal disease. The appearance of gram-negative diplococci inside polymorphonuclear leukocytes is diagnostic in this situation. However, because the normal vaginal and rectal flora are composed of gram-negative coccobacilli, which can resemble Neisseria spp., direct examination of endocervical secretions in symptomatic women is only presumptive evidence of gonorrhea, and the diagnosis must be confirmed by culture. In addition, avirulent strains (i.e., pili types 3 to 5) may be present as extracellular diplococci; these are not pathogenic. Pharyngeal specimens should not be Gram stained, because nonpathogenic, commensal Neisseria spp. may be present, and these are not diagnostic of infection.

The direct Gram stain of body fluids for either N. gonorrhoeae or N. meningitidis is best accomplished using a cytocentrifuge, which can concentrate small numbers of organisms 100-fold.

Commercial Molecular Assays

Molecular assays have replaced old enzyme-linked immunosorbent assay systems for rapid diagnosis of N. gonorrhoeae. The U.S. Food and Drug Administration (FDA) has cleared a number of amplified and nonamplified tests. (For a discussion of molecular technology, see Chapter 8.) The nonamplified DNA probe assay, PACE 2 (Hologic, Inc., Bedford, MA), has a chemiluminescent detection system for direct detection of gonococcal ribosomal RNA (rRNA) in genital and conjunctival specimens. This test performs well in high-risk patients, is rapid (results are available in 2 hours), and is suitable for screening many patients simultaneously. The Gen-Probe Accuprobe test targets rRNA after lysis of bacteria; the rRNA is detected using a single-strand chemiluminescent DNA probe. The hybrids are then detected in a luminometer. In addition, the Digene CT/GC Dual ID HC2 (HC2; Qiagen) detects RNA-DNA hybrids using antibody-mediated recognition of the hybrids and visualization of a chemiluminescent substrate.

Amplified assays, which are more sensitive than the nonamplified assay, are commercially available from Roche Diagnostic Systems. They include the AMPLICOR and COBAS AMPLICOR PCR (Branchburg, New Jersey) and the Hologic Gen-Probe Aptima Combo 2 transcription-mediated amplification (Bedford, MA) and other tests produced by other manufacturers. The ProbeTec ET (Becton Dickinson, Sparks, Maryland) also is available. These tests are suitable for large-scale screening programs, but none are admissible as evidence in medicolegal cases. An advantage of all molecular assays is the ability to test for Chlamydia trachomatis from the same specimen at the same time. N. gonorrhoeae DNA can be found in a specimen for up to 3 weeks after successful treatment, so amplified molecular assays should not be used to assess cure.

Molecular assays have also been developed to detect N. meningitidis. Sequence-based typing methods combined with serologic typing are currently recommended. Molecular targets for identification include PCR and sequencing of a variety of genes, including PorA, PorB, FetA (associated with PorA), global housekeeping genes, penA (penicillin susceptibility) and Factor H binding protein.

Following the manufacturer’s recommendations is important in evaluating molecular diagnostic tests to identify Neisseria spp. Some of the assays have limitations with regard to the type of specimen that may be used, cross-reactivity with nonpathogenic species, and assay inhibition and false-negative results caused by substances present in patient samples.

Antigen Detection

The detection of Neisseria meningitidis capsular polysaccharide antigen in body fluids (e.g., urine, serum, CSF) is no longer recommended in the United States.

Cultivation

Media of Choice

N. meningitidis, M. catarrhalis, and saprophytic Neisseria spp. grow well on 5% sheep blood and chocolate agars. N. gonorrhoeae is more fastidious and requires an enriched chocolate agar for growth on primary culture. Because gonococci and sometimes meningococci must be isolated from sites that contain large numbers of normal flora (e.g., genital tract or upper respiratory tract), selective media have been developed to facilitate their recovery. The first of these was Thayer-Martin medium, a chocolate agar with an enrichment supplement (IsoVitaleX) and the antimicrobials colistin (to inhibit gram-negative bacilli), nystatin (to inhibit yeast), and vancomycin (to inhibit gram-positive bacteria). This original medium was subsequently modified to include trimethoprim (to inhibit swarming Proteus spp.), and its name was changed to modified Thayer-Martin medium (MTM). Martin-Lewis (ML) medium is similar to MTM except that anisomycin, an antifungal agent, is substituted for nystatin and the concentration of vancomycin is increased. GC-LECT agar is a selective medium that contains additional antimicrobials to inhibit bacteria found in oropharyngeal specimens; it includes vancomycin and lincomycin (to inhibit gram-positive bacteria), colistin (to inhibit gram-negative bacteria), amphotericin B (to inhibit yeast), and trimethoprim (to inhibit swarming Proteus spp. and Capnocytophaga spp.).

New York City (NYC) medium, a transparent medium containing lysed horse blood, horse plasma, yeast dialysate, and the same antibiotics as MTM, also has been used. The advantage of NYC medium is that genital mycoplasmas (Mycoplasma hominis and Ureaplasma urealyticum; see Chapter 45) also grow on this agar. Some strains of N. gonorrhoeae are inhibited by the concentration of vancomycin in the selective media, so the addition of nonselective chocolate agar is recommended, especially in suspect cases that are culture negative or for sterile specimens (e.g., joint fluid).

Unlike the pathogenic species, some of the saprophytic Neisseria spp. (N. flavescens, N. mucosa, N. sicca, and N. subflava) may grow on MacConkey agar, although poorly. N. gonorrhoeae and N. meningitidis will grow in most broth blood culture media but grow poorly in common nutrient broths such as thioglycollate and brain-heart infusion. M. catarrhalis and the other Neisseria spp. grow well in almost any broth medium.

Incubation Conditions and Duration

Agar plates should be incubated at 35° to 37°C for 72 hours in a CO₂-enriched, humid atmosphere. N. gonorrhoeae, N. meningitidis, and M. catarrhalis grow best under conditions of increased CO₂ (3% to 7%). This atmosphere can be achieved using a candle jar, CO₂-generating pouch, or CO₂ incubator. Only white, unscented candles should be used in candle jars, because other types may be toxic to N. gonorrhoeae and N. meningitidis.

Humidity can be provided by placing a pan with water in the bottom of a CO₂ incubator or by placing a sterile gauze pad soaked with sterile water in the bottom of a candle jar (Figure 40-3).

Colonial Appearance

Table 40-3 describes the colonial appearance and other distinguishing characteristics (e.g., pigment) of M. catarrhalis and the Neisseria spp. on chocolate agar.

TABLE 40-3

Colonial Appearance and Other Characteristics on Chocolate Agar *

Organism	Appearance
Moraxella catarrhalis	Large, nonpigmented or gray, opaque, smooth; friable “hockey puck” consistency; colony may be moved intact over surface of agar
Neisseria gonorrhoeae	Small, grayish white, convex, translucent, shiny colonies with either smooth or irregular margins; may be up to five different colony types on primary plates
N. meningitidis	Medium, smooth, round, moist, gray to white; encapsulated strains are mucoid; may be greenish cast in agar underneath colonies
N. animaloris	Some strains exhibit yellow to tan pigment; odor resembles popcorn
N. cinerea	Small, grayish white; translucent; slightly granular
N. flavescens	Medium, yellow, opaque, smooth
N. lactamica	Small, nonpigmented or yellowish, smooth, transparent
N. mucosa	Large, grayish white to light yellow, translucent; mucoid because of capsule
N. polysaccharea	Small, grayish white to light yellow, translucent, raised
N. sicca	Large, nonpigmented, wrinkled, coarse and dry, adherent
N. subflava	Medium, greenish yellow to yellow, smooth, entire edge

^*Appearance on blood agar is the same as on chocolate agar except for pigmentation; colonies are less opaque on blood agar.

Approach to Identification

Various commercial systems are available for the rapid identification of the coccoid Neisseria spp. and M. catarrhalis. Some of these systems are described briefly in Table 13-1. These systems employ biochemical or enzymatic substrates and work very well for the pathogenic species (N. gonorrhoeae, N. meningitidis, and M. catarrhalis). A heavy inoculum of the organism is required, but because these systems detect the activity of preformed enzymes, viability of the organisms in the inoculum is not essential. Manufacturers’ instructions should be followed exactly; several systems have been developed only for strains isolated on selective media and should not be used to test other gram-negative diplococci.

Biochemical Identification

Table 40-4 presents some conventional biochemical tests that traditionally have been used to identify these organisms definitively. The extent to which identification of isolates is carried out depends on the source of the specimen and the suspected species of the organism involved.

TABLE 40-4

Biochemical and Physiologic Characteristics of Moraxella catarrhalis and Coccoid Neisseria spp.

	GROWTH ON:			RAPID FERMENTATION SUGARS
Organism	Modified Thayer-Martin*	Nutrient Agar at 35°C	Blood or Chocolate Agar at 25°C	Glucose	Maltose	Lactose	Nitrate Reduction	Gas from Nitrate Reduction	0.1% Nitrite Reduction
Moraxella catarrhalis^†	v	+	+	−	−	−	+	−	v
Neisseria cinerea^‡	v	+	−	−^§	−	−	−	−	+
N. flavescens	−	+	+	−	−	−	−	−	+^\|\|
N. gonorrhoeae^¶	+	−	−	+	−	−	−	−	−
N. lactamica	+	v	v	+	+	+	−	−	+
N. meningitidis	+	−	−	+	+	−	−	−	v
N. mucosa	−	+	+	+ or (+)	+	−	+	+	+
N. sicca^#	−	+	+	+ or (+)	+	−	−	−	+
N. subflava^#	−	+	+	v	+	−	−	−	+

+, >90% of strains positive; (+), >90% of strains positive but reaction may be delayed (i.e., 2 to 7 days); −, >90% of strains negative; v, variable.

^*Growth defined as >10 colonies.

^†Butyrate and DNase positive.

^‡Neisseria cinerea may be differentiated from N. flavescens by a positive reaction with the amylosucrase test.

^§Some strains of N. cinerea may appear glucose-positive in some rapid systems and be mistaken for N. gonorrhoeae. However, N. cinerea grows on nutrient agar at 35°C and reduces nitrite, unlike the gonococcus.

^||Only 2 of 10 strains were tested.

^¶Kingella denitrificans may grow on modified Thayer-Martin agar and be mistaken for N. gonorrhoeae on microscopic examination. However, K. denitrificans can reduce nitrate and is catalase-negative, unlike the gonococcus.

^#Neisseria subflava produces a yellow pigment on Loeffler’s agar; N. sicca does not.

Data compiled from Janda WM, Knapp JS: Neisseria and Moraxella catarrhalis. In Murray PR, Baron EJ, Jorgensen JH et al, editors: Manual of clinical microbiology, ed 8, Washington, DC, 2003, ASM Press; and Weyant RS, Moss CW, Weaver RE et al, editors: Identification of unusual pathogenic gram-negative aerobic and facultatively anaerobic bacteria, ed 2, Baltimore, 1996, Williams & Wilkins.

An isolate from a child or a person involved in a case of sexual abuse must be identified unequivocally because of the medicolegal ramifications of these results. It is recommended that these organisms be identified using at least two different types of tests; that is, biochemical, immunologic, enzymatic, or the nonamplified DNA probe previously discussed. Isolates from normally sterile body fluids should also be completely identified. However, isolates from genital sites in adults at risk of sexually transmitted disease (STD) can be identified presumptively; that is, oxidase-positive, gram-negative diplococci that grow on gonococcal selective agar. Likewise, an oxidase-positive, gram-negative diplococcus that hydrolyzes tributyrin from an eye or ear culture can be identified as M. catarrhalis (see Figure 13-8).

Comments About Specific Organisms

Determination of carbohydrate utilization patterns historically has been performed in cysteine trypticase soy agar (CTA) with 1% dextrose, maltose, lactose, and sucrose (see Procedure 40-1 on the Evolve site). This medium is no longer widely used, because it does not work well for oxidative Neisseria spp., specifically N. gonorrhoeae and N. meningitidis. Therefore, carbohydrate utilization patterns are currently determined by inoculating an extremely heavy suspension of the organism to be tested in a small volume of buffered, low-peptone substrate with the appropriate carbohydrate. These methods do not require subculture or growth, and results are available in approximately 4 hours. Commercially available methods include the Rim-Neisseria Test (Rapid Identification Method–Neisseria) (Remel Laboratories), the Neisseria Kwik Test (Micro-Biologics) and the Gonobio Test (I.A.F Production).

Procedure 40-1 Carbohydrate Utilization Method—CTA

Purpose

The carbohydrate utilization method is the traditional method used to identify Neisseria spp. based on carbohydrate utilization in cysteine trypticase soy agar (CTA) with the addition of 1% of a specific carbohydrate (glucose, maltose, lactose, or sucrose) and phenol red as a pH indicator.

Method

1. Using an inoculating loop, prepare a heavy inoculum in saline of a pure isolate from a subculture no more than 24 hours old obtained on nonselective media.

2. Using an inoculating needle, transfer the sample from the prepared tube and stab the top half of the CTA carbohydrate tube. Inoculating one of each of the CTA media: glucose, maltose, lactose, and sucrose.

3. Tightly cap the tubes and incubate at 35°-37°C in ambient air. Incubate an uninoculated control tube simultaneously.

4. After incubation, examine the tubes within 24 to 72 hours for a yellow color at the top of the media; this indicates acid production and a positive result for the carbohydrate utilization test.

Expected Results and Quality Control

Positive: Yellow color at the top of the tube only, indicating carbohydrate utilization.

Negative: No color change compared to control uninoculated tube.

Organism	Glucose	Maltose	Lactose	Sucrose
N gonorrhoeae ATCC 43069

Pos Neg Neg Neg

Pos Pos Neg Neg

Pos Pos Pos Neg

Pos Pos Neg Pos

Neg Neg Neg Neg

Avoid incubation in carbon dioxide (CO₂), which may alter the pH of the media, resulting in a color change to yellow throughout the entire tube and a false-positive reaction. A yellow color change throughout the tube also may indicate the presence of contaminating organisms. Caution should be used in interpreting the test result, and other confirmatory tests also should be performed.

The saprophytic Neisseria spp. are not routinely identified in the clinical laboratory. N. cinerea may be misidentified as N. gonorrhoeae if the isolate produces a weak positive glucose reaction. However, it grows on nutrient agar at 35°C, whereas the gonococcus does not. Moreover, N. cinerea is inhibited by colistin, whereas N. gonorrhoeae is not.

M. catarrhalis can be differentiated from the gonococci and meningococci based on its growth on blood agar at 22°C and on nutrient agar at 35°C, the reduction of nitrate to nitrite, its inability to utilize carbohydrates, and its production of DNase. M. catarrhalis is the only member of this group of organisms that hydrolyzes DNA.

Chromogenic substrate enzyme tests for beta-galactosidase, gamma-glutamyl aminopeptidase, and prolyl-hydroxylprolyl aminopeptidase are available for the differentiation of N. gonorrhoeae, N. meningitidis, N. lactamica, and M. catarrhalis. M. catarrhalis lacks all three of these enzymes. The presence of prolyl-hydroxylprolyl aminopeptidase alone identifies an organism as N. gonorrhoeae. The presence of beta-galactosidase and gamma-glutamyl aminopeptidase indicates N. meningitidis. Two commercial chromogenic substrate kits are the Gonocheck II (EY Laboratories, San Mateo, California) and BactiCard Neisseria (Remel Laboratories, Lenexa, Kansas). A limitation of these methods is misidentification of various nonpathogenic strains of Neisseria spp. In addition, isolate colonies on selective media should be used to avoid misidentification of contaminants as a Neisseria spp. Modified chromogenic substrate kits, such as the BactiCard Neisseria, can be used to identify and speciate Neisseria and Haemophilus organisms from selective and nonselective media. These modified tests use a combination of enzyme substrate tests and additional biochemical tests.

N. lactamica may grow on selective media and may be confused with N. meningitidis. The ONPG test (Procedure 13-33) is used to determine an organism’s ability to produce beta-galactosidase, which is an indicator of lactose utilization. N. lactamica is ONPG positive, and N. gonorrhoeae is ONPG negative.

The eugonic fermenter N. animaloris propagates well on routine laboratory media and ferments glucose; this distinguishes it from dysgonic fermenters that grow poorly on blood and chocolate agars (see Chapter 31). N. animaloris ferments no carbohydrates other than glucose and is indole negative and arginine dihydrolase positive.

Antimicrobial Susceptibility Testing and Therapy

Although beta-lactamase production is common among M. catarrhalis isolates, many beta-lactam antibiotics maintain activity. Because several other agents are also effective, susceptibility testing to guide therapy is not routinely required (Table 40-5).

TABLE 40-5

Antimicrobial Therapy and Susceptibility Testing

Species	Therapeutic Options	Potential Resistance to Therapeutic Options	Validated Testing Methods*	Comments
Moraxella catarrhalis	Several beta-lactams are effective, including beta-lactam/beta-lactamase–inhibitor combinations, cephalosporins, macrolides, quinolones, and trimethoprim- sulfamethoxazole	Commonly produce beta-lactamases that mediate resistance to ampicillin. Although not common, resistance to erythromycin and trimethoprim-sulfamethoxazole may occur	See CLSI document M45 methods	Testing to guide therapy is not routinely needed
Neisseria gonorrhoeae	Recommended therapy includes ceftriaxone and other broad-spectrum cephalosporins. Macrolides also may be used	Penicillin resistance by beta- lactamase production is common	As documented in Chapter 12: disk diffusion, agar dilution, limited commercial methods	Testing by disk diffusion may not detect decrease in quinolone activity. No ceftriaxone resistance has been documented
Neisseria meningitidis	Supportive therapy for shock and antimicrobial therapy using penicillin, ceftriaxone, cefotaxime, or chloramphenicol	Subtle increases in beta-lactam resistance have been described, but clinical relevance is uncertain. Beta-lactamase production is extremely rare Reduced fluoroquinolone susceptibility possible	As documented in Chapter 12: broth dilution, agar dilution and CLSI guidelines	Testing to guide therapy is not routinely needed
Other Neisseria spp.	Usually susceptible to penicillin and other beta-lactams	Uncertain; potential for beta-lactamase production	Not available
Neisseria animaloris	Not well characterized; purported susceptibility to penicillin, ampicillin, ciprofloxacin, and ofloxacin	Unknown; first-generation cephems appear less active than penicillins	Not available

^*Validated testing methods include standard methods recommended by the Clinical and Laboratory Standards Institute (CLSI) and commercial methods approved by the U.S. Food and Drug Administration (FDA).

Standard methods have been established for performing in vitro susceptibility testing with N. gonorrhoeae and N. meningitidis (see Chapter 12). The Clinical and Laboratory Standards Institute (CLSI) recommends the use of agar dilution for minimum inhibitory concentration (MIC) measurements and GC agar containing 1% growth supplement for N. gonorrhoeae disk diffusion methods.

In addition, various agents can be considered for testing and therapeutic use. Quinolones were widely used to treat gonorrhea; however, resistance to these agents has emerged (i.e., quinolone-resistant N. gonorrhoeae [QRNG]), and they are no longer recommended for treatment of gonorrhea.

The Gonococcal Isolate Surveillance Project (GISP) has identified six categories of antibiotic susceptibility patterns for the characterization of isolates: PPNG (plasmid-mediated beta-lactamase positive); TRNG (plasmid-mediated tetracycline resistance, MIC ≥ 16 µg/mL); PPNG-TRNG; Penr; Tetr (MIC = 2-8 µg/mL) (chromosomal-mediated resistance patterns); and CMRNG (Penr combined with Tetr).

Because of the increase in QRNG and PPNG isolates, broad-spectrum cephalosporins have become the treatment of choice for N. gonorrhoeae. However, treatment failures have been associated with the use of broad-spectrum cephalosporins. Oral and intravenous regimens are recommended, depending on the severity and location of the infection.

Beta-lactamase production in N. meningitidis is extremely rare, although decreased susceptibility to penicillin, mediated by altered penicillin-binding proteins, is emerging. Optimum laboratory methods for detecting this relatively low level of resistance have not yet been established, and the impact of this resistance on the clinical efficacy of penicillin is not known. The CLSI recommends that susceptibility testing be performed by disk diffusion on Mueller-Hinton agar or using cation-adjusted Mueller-Hinton broth in microdilution.