Gram-Negative Cocci

For a general discussion on Gram-negative bacteria, readers are referred to the last part of this chapter. With reference to Gram-negative cocci, members of the follow- ing three genera are considered important pathogens:

• Neisseria spp.

 N. gonorrhoeae

 N. meningitidis

• Moraxella spp.

 M. catarrhalis

• Haemophilus (It is a coccobacillus and sometimes listed among the Gram- negative bacilli)

 H. influenzae

NEISSERIA SPECIES

Neisseria spp. are aerobic, Gram-negative, nonmotile, oxidase positive cocci that do not produce endospores. Members of several species of Neisseria, including N. sicca, N. mucosa, N. lactamica, and N. flavescens, commonly colonize mucous membranes of mouth and nasopharynx. Two species are important human pathogens. These include N. gonorrhoeae and N. meningitidis.

Neisseria gonorrhoeae

Neisseria gonorrhoeae, often referred to as gonococcus, causes gonorrhea, a disease that was quite rampant during the pre-antibiotics era. This bacterium infects only humans; no animal cases and no other natural reservoirs are known.

Disease

Gonorrhea is a sexually transmitted disease. Isolation of N. gonorrhoeae from inanimate objects is occasionally reported but their role in the causation of gonorrhea is equivocal. The infection initially involves the urethra in men and cervix in women. Incubation period may range from less than 2 days to more than 1 week, and the symptoms include purulent discharges and dysuria. Disseminated gonorrhea may involve the joints, heart, meninges, eyes, and the pharynx. In women, it may spread to the fallopian tube and cause pelvic inflammatory disease (PID), which may result in ectopic pregnancy and infertility. More than 400,000 cases of PID occur annually in the United States. Gonorrhea is more common among women than men perhaps due to the fact that many female cases remain asymptomatic hence do not receive timely medical attention. Since infected females may not present any symptom, they usually play a major role in the dissemination of gonorrhea in general population. Gonorrhea in males is mostly symptomatic and easily detected.

Virulence Factors

Only a few virulence factors are known. Certain proteins, namely pilin, protein I and II, and an IgA specific proteinase, appear to play an important role in the pathogenesis of this bacterium. In order to infect, the pathogen must attach to the mucosal cells of the epithelial walls by means of fimbriae. The bacterium invades the spaces separating columnar epithelial cells, which are found in the oral pharynx, eyes, rectum, urethra, cervical opening, and external genitals of prepubescent females.

Laboratory Diagnosis

Blood agar is an excellent isolation medium, but some bacteria and yeasts are known to have inhibitory effect on N. gonorrhoeae. Therefore, New York City agar is preferred for the selective isolation of N. gonorrhoeae. The inoculated plates should be incubated at 35°C, aerobically (anaerobic incubation is also acceptable), for 24–72 hours. Microscopic examination of pus demonstrating presence of Gram-negative diplococci can be helpful (Fig. 8.1). This bacterium produces acid from glucose, but not from maltose, lactose sucrose, or fructose.

Antimicrobial Sensitivity

Initially, penicillin was the wonder drug for the treatment of gonorrhea and is credited with the eradication of this disease from most of the industrialized countries. However, during the past few decades, a large number of multiple drug resistant cases have emerged. Currently, the preferred agents include azithromycin, doxycy- cline, and ceftriaxone. The CDC recommends a single dose of ceftriaxone. Prevention is best achieved by the use of condoms.

Figure 8.1.    Gram-negative diplococci seen in a smear prepared from Neisseria gonorrhoeae

isolated from a case of gonorrhea.

Neisseria meningitidis (Meningococcus) 

Neisseria meningitidis is strictly a human pathogen and is frequently isolated from throat and nasal swabs of apparently healthy individuals. Its isolation from vaginal and labial swabs has also been occasionally noted. 

Disease 

Neisseria meningitidis can be isolated from the upper respiratory tract of about 10% of the healthy population. The primary site of infection is the nasopharynx from where it may spread to the blood stream and cause septicemia and meningitis. Clinical symptoms may also include headache and fever. Children younger than 3 years of age are most susceptible, but cases involving adults are not uncommon. The infection is mostly acquired through inhalation of bioaerosol. Living in close quar- ters with infected persons is a major risk factor. The disease in untreated patients with meningococcal meningitis is almost always fatal.

Virulence Factors 

Virulence factors include pili that facilitate colonization, lipooligosaccharide which contains the endotoxin moiety that is responsible for fever and other symptoms, opacity proteins, and capsular polysaccharides. Host factors such as splenectomy and terminal complement deficiency play a role in susceptibility to severe infection in a minority of patients.

Laboratory Diagnosis 

Gram staining of cerebrospinal fluid (CSF) can provide clues to a presumptive diagnosis. Suitable clinical specimens, such as throat swab and CSF, can be cultured on blood agar and grown aerobically (growth is better with 3%–5% carbon dioxide) at 35°C for 1–2 days. Neisseria meningitidis is fastidious and sensitive to low temperature; therefore, the specimens should not be stored in a refrigerator. Colonies are very small, round, smooth, and whitish. Specially designed commercial kits for blood culture are useful. 

Antibiotic Sensitivity 

Penicillin and cephalosporins are effective antibiotics. Chloramphenicol is indicated in some cases but is rarely used. Rifampin and ciprofloxacin are used for prophylaxis, however, increasing resistance to ciprofloxacin is being reported in some areas. Vaccination with a tetravalent vaccine from the polysaccharide capsular antigen is very effective in preventing meningococcal meningitis in military recruits. Its effect on children is, however, questionable.

MORAXELLA CATARRHALIS

Disease 

Moraxella catarrhalis, occasionally referred to as Branhamella catarrhalis, is an important causal agent of otitis media in children. It can also cause meningitis, endocarditis, bronchopulmonary infections, and neonatal conjunctivitis. This bacterium can be isolated from the throat swabs of apparently healthy individuals.

Laboratory Diagnosis 

Blood agar is a useful medium for the isolation of M. catarrhalis. Incubation should be done aerobically at 35°C for 24 hours. The colonies are convex, nonpigmented, and smooth. This is a fast-growing bacterium. It is oxidase positive and β- galactosidase negative. Occasionally, Moraxella spp. are mistaken for Neisseria spp. Important differences between the two taxa are summarized in Table 8.1. 

Antibiotic Sensitivity 

Moraxella catarrhalis infections are often hard to treat. Most strains produce β- lactamase. The clavulanate-supplemented amoxicillin can be useful. Other effective antibiotics include tetracycline, cephalosporins, and fluoroquinolones.

Table 8.1    Some Delineating Properties of Neisseria and Moraxella spp.

Species	NY city agar	Chocolate agar	Acid from maltose	Acid from lactose
N. gonorrhoeae	Growth	No growth	Negative	Negative
N. meningitidis	Growth	No growth	Positive	Negative
N. mucosa	No growth	Growth	Positive	Negative
N. sicca	No growth	Growth	Positive	Negative
N. lactamica	Growth	Variable	Positive	Positive
N. flavescens	No growth	Growth	Negative	Negative
M. catarrhalis	Variable	Variable	Negative	Negative

Table 8.2    Some Physiological Differences between Some Clinically Significant Species of the Genus Haemophilus

Glucose

Species	Hemolysis	fermentation	Mannose fermentation	Catalase test
H. influenzae H. haemolyticus H. parahaemolyticus H. parainfluenzae	None Present Present None	Positive Positive Positive Positive	Negative Negative Negative Positive	Positive Positive Positive Variable

HAEMOPHILUS INFLUENZAE 

Members of the genus Haemophilus are commonly found in the mouth and upper respiratory tract of humans. Ten species including (but not limited to) H. influenzae, H. parainfluenzae, H. haemolyticus, and H. parahaemolyticus are commonly isolated from human sources. These are Gram-negative coccobacilli that are often grouped together with other Gram-negative bacilli. Only H. influenza is of main clinical significance. It must be stated here that the term “Haemophilus influenzae” is a classic example of misnomer in microbiology. It is neither hemophilic nor a causal agent of influenza. It can be isolated from the respiratory tract of approximately 5% of apparently healthy persons. Some physiological differences among commonly encountered Haemophilus spp. are noted in Table 8.2.

Disease 

Haemophilus influenzae causes acute or chronic severely invasive infection of the respiratory tract and may involve meninges, especially strains belonging to the serotype b, generally referred to as “Hib.” It is a common cause of infection in children younger than 3 years of age. Prior to the introduction of vaccine, Hib accounted for nearly 20,000 cases of invasive infections in the United States annu- ally. Infection is contracted via inhalation of H. influenzae-laden bioaerosol. In adults, infections involving sinuses and ears are also seen. 

Virulence Factors 

Haemophilus influenzae serotype b (Hib) has polyribitol phosphate capsule that helps the bacterium resist phagocytosis. In addition, pili help it with the initial attachment and colonization.

Laboratory Diagnosis 

Most strains of H. influenzae do not grow well on blood agar without a feeder bacterium, which is usually S. aureus. Staphylococcus aureus strains produce a growth factor called “V factor” that helps H. influenza grow (Fig. 8.2). Chocolate agar, which does not need a feeder bacterium is, therefore, a preferred medium for the isolation of H. influenza. On chocolate agar, the colonies appear as small dewdrops. The growth conditions include incubation at 35°C and moist atmosphere with 5%– 10% CO2. 

Taxonomy 

As stated above, genus Haemophilus has many species, which are normally associated with the mouth and upper respiratory tract. A delineating scheme for some of the closely related species is depicted in Table 8.2.

Figure 8.2.    A demonstration of the use of feeder bacterium S. aureus for the isolation of H. influenzae. See color insert.

Antibiotic Sensitivity 

About 20%–30% strains produce β-lactamase, hence are resistant to penicillin (ampicillin). Effective agents include cephalosporins, erythromycin, and fluoroquinolones. Vaccination is the most effective prophylaxis for children younger than 18 months of age. 

AN OVERVIEW OF GRAM-NEGATIVE BACTERIA

Gram-negative bacteria are the most abundant in nature. While some are found in humans as commensals, only a few are pathogenic, and a great majority of them are

Figure 8.3.    Schema for the grouping of some clinically significant Gram-negative bacteria. *Some species are lactose fermenters. **Some taxa present curved rods and some are coccobacilli (see text)

harmless to humans. Because of their abundance in nature and their metabolic and structural diversity, there has been a considerable amount of controversy in their taxonomic classification. Traditionally, Gram-negative bacteria have been placed under the division Gracillicutes in the Kingdom Monera. Prompted by recent advances in molecular genetics, the newer generation of microbiologists believes that Gram-negative bacteria are genetically too diverse to be placed under one division and resect the classic concept of high-level division of bacteria into four classes that were initially based on cell wall composition and Gram staining. There may be some strength in this argument, but molecular taxonomy is still far from being fully developed and practical. We believe that until and unless a workable taxonomic scheme based on molecular genetics is at least partially if not fully developed, the traditional system should remain in place. 

The single most important component in the cell wall of Gram-negative bacteria is the abundance of lipopolysaccharide, also called LPS or just endotoxin, which plays a critical role in their pathogenicity. LPS triggers the innate immune response and production of cytokines that play a critical role in inflammation and cellular immunity. As for the taxonomy, readers are referred to Bergey’s Manual of Determinative Bacteriology for a scholarly discussion and an in-depth classification. Figure 8.3 presents a simple plan for grouping clinically significant Gram-negative bacteria.