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Identification of N. gonorrhoeae and Related Species

• Introduction
• Biochemical Tests
• Natural Habitat
• Pathogenicity
• Prevalence
• Taxonomy
• References

Introduction

The genus Neisseria contains a number of species which are normal flora and pathogens of humans and animals. Of these species, the species of human origin--and particularly the pathogenic species, N. gonorrhoeae and N. meningitidis--have been studied extensively in an effort to control the infections they cause.

Gonorrhea, caused by N. gonorrhoeae, is one of the most frequently reported infectious diseases in the United States and worldwide. Rapid tests have been developed to identify and distinguish N. gonorrhoeae, from the commensal Neisseria and related species which are normal flora of the oro- and nasopharynx. Because many rapid tests for the identification of N. gonorrhoeae test for a limited number of characteristics which may be shared by one or more nonpathogenic Neisseria spp., a non-gonococcal, commensal Neisseria species may be incorrectly identified as N. gonorrhoeae. Such incorrect identifications may result in serious social and medicolegal consequences for patients and their families. Thus, the primary purpose of these pages is to provide information relating to the accurate identification of N. gonorrhoeae.

Descriptions of species in these pages will, for the moment, be limited to those of human origin. Information relating to the identification species of animal origin will include a table of differential characteristics which should be consulted when a gram negative diplococcus is not readily identifiable as a human Neisseria species e.g., an isolate from a wound inflicted by an animal bite.

In addition, reference information on the taxonomy, host range, pathogenicity, natural habitat and prevalence of the Neisseria species is included.

Biochemical Tests for the Identification of N. gonorrhoeae and Related Species

Traditionally, tests used to identify strains of Neisseria species were performed as individual non-commercial tests. Although these tests have, in many cases, been superseded by commercially available products, reference laboratories may use additional individual tests to identify strains of Neisseria and related species. Table 1 provides a list of traditional tests used to identify strains of N. gonorrhoeae.

For detailed information on biochemical tests, please use the links below or in Table 1.

• Acid Detection
• Enzyme Substrate
• Nitrate Reduction
• Polysaccharide from Sucrose
• DNase

Table 1. Differential characteristics of human Neisseria spp., M. catarrhalis, and K. denitrificans

Species	Acid from					Nitrate Reduction	Polysaccharide from Sucrose	DNase	Superoxol	Pigment	Colistin Resistance
	G	M	S	F	L
N. gonorrhoeae	+	-	-	-	-	-	-	-	Strong (4+) positive ("explosive")	-	R
N. gonorrhoeae subspecies kochii	+	-	-	-	-	-	-	-	Strong (4+) positive ("explosive")	-	R
N. meningitidis	+	+	-	-	-	-	-	-	Weak (1+) to strong (4+) positive	-	R
N. lactamica	+	+	-	-	+	-	-	-	Weak (1+) to strong (3+) positive	+	R
N. polysaccharea	+	+	-	-	-	-	+	-	Weak (1+) to strong (3+) positive	-	(R)
N. cinerea	-	-	-	-	-	-	-	-	Weak (2+) positive	-	(R)
N. flavescens	-	-	-	-	-	-	+	-	Weak (2+) positive	+	S
N. mucosa	+	+	+	+	-	+	+	-	Weak (2+) positive	d	S
N. subflava biovar subflava	+	+	-	-	-	-	-	-	Weak (2+) positive	+	S
N. subflava biovar flava	+	+	-	+	-	-	-	-	Weak (2+) positive	+	S
N. subflava biovar perflava	+	+	+	+	-	-	+	-	Weak (2+) positive	+	(R)
N. sicca	+	+	+	+	-	-	+	-	Weak (2+) positive	-	S
N. elongata	-	-	-	-	-	-	-	+	-	-	S
M. catarrhalis	-	-	-	-	-	+	-	+	Weak (1+) to Strong (3+ to 4+) positive	-	(R)
K. denitrificans	+	-	-	-	-	+	-	-	-	-	R

Abbreviations: G, glucose; M, maltose; L, lactose; S, sucrose; +, most strains positive; -, most strains negative; (+), strain dependent (some strains positive; some strains negative); R, most strains resistant; (R), most strains sensitive, but some strains known to be resistant; S, all strains thought to be sensitive.

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Natural Habitat of Neisseria and Related Species

Most Neisseria and related species are normal flora in humans and animals (Table 2); their host range is shown in Table 2. However, some species such as N. gonorrhoeae are pathogens in normal hosts and those species listed as commensals may be opportunistic pathogens.

Table 2. Host Range of Neisseria species, Moraxella catarrhalis, and Kingella denitrificans

Species	Host	Pathogen/Commensal
HUMAN
N. gonorrhoeae	Man	Pathogen
N.meningitidis	Man	Occurs in carrier state Some strains cause epidemics/pandemics
N. lactamica	Man	Commensal; more frequent in children than adults
N. polysaccarea	Man	Commensal
N. cinerea	Man	Commensal
N. flavescens	Man	Isolated from outbreak of meningitis Apart from the original description, there are no reliable isolations of this species; strains isolated as N. flavescens were probably N. cinerea or N. polysaccharea
N. subflava Biovar subflava	Man	Commensal
N. subflava Biovar flava	Man	Commensal
N. subflava Biovar perflava	Man	Commensal
N. sicca	Man	Commensal
N. mucosa	Man	Commensal Similar strain isolated from dolphins
N. elongata	Man	Commensal
M. catarrhalis	Man	Possibly normal flora of the nasopharynx Causes respiratory tract infections including otitis media and sinusitis
K. denitrificans	Man	Commensal
ANIMAL
N. canis	Cat	Commensal
N. denitrificans	Guinea pig	Commensal
N. weaveri	Dog	Commensal in the upper respiratory tract of dogs; isolated from dog bites in humans
N. iguanae	Iguana	Commensal
N. ovis*	Sheep Cattle	Causes keratoconjunctivitis in sheep
N. caviae*	Guinea pig	Commensal
N. cuniculi*	Rabbit	Commensal
N. macacae	Monkey	Commensal

*Species incertae sedis, taxonomic position uncertain

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Pathogenicity of Neisseria and Related Species of Human Origin

Neisseria species

Of the Neisseria and related species, only N. gonorrhoeae is considered always to be pathogenic i.e., cause disease. N. gonorrhoeae is not considered to be normal flora under any circumstances.

N. gonorrhoeae strains may infect the mucosal surfaces of urogenital sites (cervix, urethra, rectum) and the oro- and nasopharynx (throat), causing symptomatic or asymptomatic infections. Gonococcal infections of the urogenital sites are more frequently symptomatic than asymptomatic; however, asymptomatic infections may occur. Gonococcal infections of the oro- and nasopharynx and the rectum may be asymptomatic more frequently than symptomatic. Some specific strains of N. gonorrhoeae have been associated with asymptomatic infections of urogenital sites; these include strains that require arginine, hypoxanthine, and uracil (AHU) or proline, arginine (citrulline), and uracil (PAU) to grow on chemically defined media on which gonococcal strains are characterized.

Strains of N. meningitidis may be carried as normal flora in the throat. Under some circumstances, N. meningitidis may also be pathogenic, causing sporadic cases or epidemics of meningitis. Between 3% and 30% of healthy persons in nonepidemic geographic areas may be asymptomatic carriers of N. meningitidis i.e., meningococci have colonized their throats without causing disease. The carrier state may persist for many months.

N. meningitidis causes epidemic meningitis in many parts of the world such as Sub-Saharan Africa. Certain types of N. meningitidis are usually associated with meningitis. Of a total of 13 serogroups of N. meningitidis, strains belonging to the serogroups A, B, C, and W-135 have most frequently been associated with epidemics. Group A strains have been associated with most epidemics, whereas group B, C, and W-135 strains have caused sporadic epidemics. Epidemics may occur in military recruits; in these groups, more than 95% of recruits may be asymptomatic carries of the epidemic strain while only 1% develop meningitis.

Occasionally, N. meningitidis has been isolated from the cervix or urethra of men or women. N. meningitidis is not normal flora in genitourinary sites.

Other Neisseria species are considered to be commensals i.e., they colonize the host without causing disease. Strains of these species are normal flora in the throat.

Commensal Neisseria species appear to be opportunistic pathogens i.e., these species may cause infections although are not routinely associated with specific types of infections or infections of specific sites. Most Neisseria species have been isolated occasionally from blood, cerebrospinal fluid, abscesses etc. but no consistent association between between any individual species and syndrome has been established that would warrant designating any of these species as pathogens. Some infections caused by commensal Neisseria species have occurred in persons who have deficient immune systems and who, thus, may be predisposed to infections with organisms that would not normally cause disease.

Moraxella catarrhalis

For decades, M. catarrhalis was thought to be normal flora of the oro- and nasopharynx. M. catarrhalis is now recognized to be a pathogen which causes pneumonia, systemic disease, sinusitis, otitis media and ophthalmia neonatorum. M. catarrhalis is thought to be normal flora of the nasopharynx although this species is not isolated frequently from the throats of healthy adults. It is possible that this species may colonize certain individuals in a carrier state similar to that observed with N. meningitidis.

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Prevalence of Commensal Neisseria and Related Species of Human Origin

The prevalence of gonorrhea varies geographically and is beyond the scope of this page; this topic will be discussed elsewhere.

N. meningitidis strains are carried as normal flora in the oro- and nasopharynx of adults and children. The prevalence of N. meningitidis carriage varies geographically. It has been suggested that N. meningitidis may occur more frequently in adults with gonorrhea and in homosexual men. Fewer than 1% of children are colonized by N. meningitidis during the first 4 years of life; the carriage rate increases after this age.

N. lactamica colonizes the throats of children more frequently than adults. N. lactamica colonizes the throats of as many as 4% of babies of 3 months age to a peak of 21% in children 18 to 24 months; colonization by N. lactamica then declines to 2% by age 14 to 17 years. It is estimated that 59% of children have been colonized by N. lactamica at least once by the age of 4 years. This pattern of colonization may reflect the fact that young children may drink large volumes of milk. Among Neisseria species, strains of N. lactamica are unique in their ability to use lactose; this characteristic may enhance populations of N. lactamica in the throats of younger children. Unlike N. gonorrhoeae and N. meningitidis, N. lactamica has not been implicated as a primary pathogen, although it may be an opportunistic pathogen.

The commensal Neisseria species and K. denitrificans, are normal inhabitants of the oro- or nasopharynx. These species are occasionally isolated from other sites but are not considered to be normal flora of sites other than the throat.

Because strains the commensal Neisseria species rarely grow on selective media used to isolate the gonococcus, the prevalence of these species must be determined on a medium that does not contain colistin, the antibiotic incorporated in gonococcal selective medium to inhibit the growth of the commensal species.

It is impossible to accurately determine the carriage rate of commensal Neisseria species in studies performed in the early 1900s for two reasons. Firstly, most studies of the prevalence of Neisseria spp. and N. catarrhalis (M. catarrhalis) were performed with nonselective media e.g., blood agar, which neither inhibited the growth of other bacterial species nor permitted differentiation between colonies of Neisseria and related species. Thus, it is probable that the carriage rates of some species (N. cinerea, N. polysaccharea, N. lactamica) were underestimated because these species occur in relatively small numbers and were probably overgrown by either non-neisserial species or the sucrose-positive Neisseria species (N. subflava biovar perflava, N. sicca, N. mucosa). Secondly, in these studies, strains were identified with acid detection tests that were not appropriate for the detection of the relatively small amounts of acid produced by Neisseria species, and additional differential tests now used to accurately identify commensal Neisseria species were unknown.

Some studies have been performed using selective differential media that inhibited the growth of non-neisserial species and selected for commensal Neisseria species and M. catarrhalis either by differentiating between the asaccharolytic species or between several different groups of species.

Berger and Wulf determined that the commensal Neisseria species colonzied the throats of adults as follows: N. sicca, 45%; N. perflava, 40%; N. subflava-N. flava, 11%; and "N. catarrhalis," 3%. It must be remembered that, because nitrate reduction was not used as a differential test for the identification of Neisseria species at that time, strains of N. mucosa were not recognized by these authors. Although not verifiable, it is reasonable to assume that N. mucosa strains were present in the throats of these individuals. Thus, in this study, strains of N. mucosa were identified as either N. sicca or N. perflava and the prevalences of these latter species are overestimated by the inclusion of strains of N. mucosa in their numbers. Using a selective medium at a later time, Berger found that asaccharolytic strains, i.e. strains that do not produce detectable acid from glucose, maltose, sucrose, or fructose, accounted for 15% of all neisserial isolates but, because nitrate reduction was not used to differentiate between N. cinerea and M. catarrhalis until a few years later, the relative colonization rates of these individual species were not given.

Knapp and Hook determined the prevalence and persistence of Neisseria and M. catarrhalis in the oropharynx of adults using a selective medium for commensal Neisseria species. In this study, it was not possible to differentiate between N. subflava biovar perflava (N. perflava) and N. sicca reliably because the typical colony morphology described for N. perflava (smooth, glistening, butyrous colonies) and N. sicca (dry, adherent, wrinkled colonies) was not a reproducible characteristic for the strains examined. In addition, no additional unequivocal differential tests were available to permit accurate identification of each species.

In this study, adults exhibited one of two general patterns of colonization with these species. Some adults were colonized heavily by several strains each of the sucrose-positive species, N. mucosa and N. perflava-N. sicca, whereas others were colonized sparsely by several Neisseria species. The most dramatic finding of this study not previously described was that, whereas most persons were colonized by two or three species, 5% of persons were colonized by four or five Neisseria species. These patterns of colonization were generally persistent although they were occasionally disrupted for a short period of time. For example, a person usually colonized heavily with sucrose-positive strains would occasionally exhibit an alternative pattern of colonization by many species; the original pattern of heavy colonization with sucrose-positive strains was re-established within a short period of time. It is possible that these individuals were persistently colonized by the species observed when the sucrose-positive species were suppressed, but that sucrose-positive species usually outnumbered these other species which, then, were not detected in culture. The prevalence of individual types of strains of commensal Neisseria species may vary geographically. Strains of colistin-resistant N. subflava biovar perflava have been isolated repeatedly in Chicago, yet infrequently in DeKalb County, Georgia, and Seattle, Washington. In contrast, K. denitrificans strains were isolated from 16% of patients in DeKalb County but not in Chicago or Seattle.

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References

Bovre K. Family VIII. Neisseriaceae Prevot, In NR Krieg (ed.). Manual of Systematic Bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 1984. p. 288-309.

Knapp JS. Historical perspectives and identification of Neisseria and related species. Clin Microbiol Rev 1988;1:415-431.

Knapp JS, Rice RJ. Neisseria and Branhamella. In. Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH. (ed.). Manual of Clinical Microbiology. 6th ed. American Society for Microbiology, Washington D. C, 1995.

Vedros NA. Genus I. Neisseria Trevisan 1885, 105AL, In NR Krieg (ed.). Bergey's Manual of Systematic Bacteriology, vol. 1. The Williams & Wilkins Co., Baltimore. 1984. p. 290-296.