Gram-Positive Bacilli

Important pathogens in this group of bacteria are restricted to three genera that include aerobes as well as anaerobes:

• Clostridium spp. (anaerobic)

• Bacillus spp. (aerobic)

• Listeria spp. (aerobic to facultative anaerobic)

Most Gram-positive rods grow well on blood agar. However, selective media are available in some cases, for example, cycloserine–cefoxitin–fructose–egg yolk agar (CCFA) for C. difficile.

CLOSTRIDIUM SPECIES

Clostridia are anaerobic, spore-forming (sporogenous), Gram-positive rods. They are widely distributed in nature and many are common soil-borne bacteria. Their ability to produce spores enables them to survive under adverse environmental conditions and also affords them greater protection from chemical and physical agents. Recent genetic studies seem to challenge the conventional classification of the members of this genus. Depending on the school of thought, number of species assigned to genus Clostridium varies from 20 to more than 100. Of these, four species, namely C. tetani, C. botulinum, C. perfringens and C. difficile, are most important pathogens. Some of the physiological differences among the clinically significant Clostridium spp. are summarized in Table 6.1.

Clostridium tetani

Clostridium tetani is anaerobic, Gram-positive, endospore-forming bacillus that is normally present in soil and dust. Its isolation from fecal matter is not uncommon.

Table 6.1    Some Differentiating Characteristics of Clostridium spp.

Species	Mannose fermentation	Indole Test	Lacithinase test	Lipase
C. botulinum C. difficile C. perfringens C. tetani	Negative Positive Positive Negative	Negative Negative Negative Variable	Negative Negative Positive Negative	Positive Negative Negative Negative

Disease 

Clostridium tetani causes tetanus, a dreaded, painful, and often fatal disease. The incubation period may range from 1 to 54 days (generally 6–15 days). Widespread use of antitetanus vaccines in United States and other industrialized countries has greatly reduced number of cases, but tetanus is still fairly common in rest of the world. There are several clinical variations of tetanus but the generalized form of the disease is most common. The organs involved include masseter and back muscles and the autonomic nervous system. The most important symptoms are persistent spasm of back muscles, cardiac arrhythmia, irregular blood pressure, sweating, dehydration, and a condition called Risus sardonicus, or twisted facial muscles.

Virulence Factor 

Clostridium tetani produces two types of toxins: tetanospasmin, a spasmogenic neurotoxin, and tetanolysin, a hemolytic toxin. In addition, it produces endospores, which resist most antimicrobial substances produced by components of the immune system.

Laboratory Diagnosis 

Tetanus is usually diagnosed on the basis of clinical symptoms and patient’s history of exposure to soil, dust, or rusted metal. Cultures are seldom attempted and not always successful. Gram staining can provide useful information in early stages (Fig. 6.1).

Antibiotic Sensitivity 

Tetanus toxin is irreversibly bound to tissue so supportive care is of primary impor- tance. However, tetanus immune globulin combined with metronidazole should be administered. Active toxin production is limited by wound management. Penicillin is also an acceptable alternative to metronidazole. The best protection against tetanus is obtained by vaccination with three doses of tetanus toxoid, followed by booster doses every 10 years.

Figure 6.1.    A Gram-stained smear of Clostridium tetani showing Gram-positive vegetative rods, some with a terminal endospore.

Clostridium botulinum 

Like C. tetani, C. botulinum is an ubiquitous bacterium that is commonly isolated from soil and stagnant water. Eight serotypes (A, B, Cα, Cβ, D, E, F, and G) have been reported.

Disease 

Clostridium botulinum causes botulism, which is a paralytic disease. Three clinical forms of botulism are known: food borne infection, caused by ingestion of toxin- infested food, infant infection, caused by ingestion of food contaminated with bacterium, and wound infection, resulting from bacterial contamination. Symptoms of food-borne infection include blurred vision, constipation, abdominal discomfort, and muscle weakness. Death occurs due to respiratory failure.

Virulence Factor 

Most strains produce botulinol (botulinum toxin), an extremely potent toxin that acts on the neuromuscular junction. The bacterium has an additional advantage due to its ability to produce endospores.

Laboratory Diagnosis 

In the cases of botulinum food poisoning, fecal material is cultured on enriched media. Since such materials are heavily contaminated with a wide range of

Figure 6.2.    A Gram-stained smear prepared from a culture of Clostridium botulinum, showing

Gram-positive rods with endospores.

fast-growing bacteria, it is better to first prepare a saline suspension of the fecal matter and then leave it for 10 minutes in a water bath held at 80°C. This process kills vegetative forms of most contaminating bacteria. The sample is then inoculated on an enriched medium such as egg yolk agar. The incubation is done anaerobically at 35°C. Gram-positive rods with endospores are easily seen in Gram-stained smears (Fig. 6.2).

Antibiotic Sensitivity 

A trivalent botulinum antitoxin has been used with success. Penicillin should also be administered to those with wound botulism. Metronidazole is also believed to be an effective agent and is an acceptable alternative in those allergic to penicillin.

Clostridium perfringens 

Like other Clostridia, C. perfringens is ubiquitous and commonly present in soil and water. It is also frequently isolated from human feces. The endospores enable this bacterium to survive under unfavorable environmental conditions.

Disease 

Clostridium perfringens causes gas gangrene, involving invasion of healthy muscles surrounding the infected site. The condition can be life-threatening. Infections occur as a result of contamination of fresh wounds. Bullae and necrosis may develop in the infected wound, which gradually becomes dark colored.

Virulence Factors 

Strains of C. perfringens are known to produce several toxins. These are usually characterized as α, β, and λ toxins. In addition, phospholipases and proteinases produced by some strains probably play a role in tissue destruction and gas production.

Antibiotic Sensitivity 

Proper wound care is required and the wounds can heal normally in due course. Medication with penicillin may be required in serious cases. 

Clostridium difficile 

A small number of C. difficile bacteria are normally present in the human intestinal tract. Disease develops when the normal bacterial population is destroyed by the prolonged use of broad-spectrum antibiotics. 

Disease 

Clostridium difficile causes diarrhea, mostly following long-term therapy with broad-spectrum antibiotics. The clinical condition is called pseudo membranous colitis (PMC) and may lead to dehydration, electrolyte imbalance, toxic megacolon, and colon perforation. These bacteria are frequently isolated from hospital floors, bedpans, and equipment, and found in the intestine of 10%–20% of healthy persons. A hypervirulent epidemic strain, referred to as NAP-1, has become more prevalent over the recent years. It is believed to be responsible for much of the increasing virulence and mortality being seen with this infection. It effects the expression of toxins A and B, as noted in the virulence factors section.

Virulence Factors 

Most strains of C. difficile produces two toxins (A and B). Toxin A is enterotoxin and B is cytopathic. Toxin B is believed to be more significant in causing the clinical disease. 

Laboratory Diagnosis 

Tissue culture, polymerase chain reaction (PCR), latex agglutination and ELISA are helpful diagnostic tools. Bacterial culture can be made on cycloserine–cefoxitin– fructose–egg yolk agar. Toxin formation needs to be demonstrated in order to dis- tinguish active infection from colonization. In the clinical setting, the most useful tests are the ELISA for toxins A and B and PCR for the gene for toxin B.

Antibiotic Sensitivity 

Effective agents include metronidazole and oral vancomycin, which is not absorbed. Replenishing some of the bacteria (e.g., Lactobacillus) normally found in intestinal tract can be helpful. A new agent, fidaxomicin, was approved by the FDA in 2011 for the treatment of C. difficile–related medical conditions.

LACTOBACILLUS SPECIES 

Lactobacilli are not pathogenic bacteria, but a section has been added here because they are also Gram-positive, anaerobic, asporogenous bacilli. They play a positive role in human health, especially in the health and well being of women by protecting vaginal surfaces from colonization by harmful bacteria. Genus Lactobacillus has several species. Many are found in the gastrointestinal tract and vagina. Lactobacillus acidophilus ferments glycogen into lactic acid, which causes an acidic environ- ment in the vagina, thus excluding other bacteria that prefer neutral pH. Surprisingly, L. acidophilus is mostly absent in the vagina of prepubescent and postmenopausal women, suggesting a possible connection with estrogen.

BACILLUS SPECIES 

Member of the genus Bacillus are aerobic, sporogenous, rod-shaped bacteria. The term “bacillus” refers to a specific bacterial genus, and also refers to a specific bacterial shape, that is, the rod shape. The genus Bacillus has more than 40 species, but only one is an important pathogen, B. anthracis. Another member of this genus, B. cereus, is also of clinical significance. Physiological differences between B. anthracis and B. cereus are summarized in Table 6.2.

Table 6.2    Growth-Related Differences between B. anthracis B. cereus and B. thuringiensis

Characteristic	B. anthracis	B. cereus and B. thuringiensis
Thiamine requirement	Positive	Negative
Hemolysis on sheep blood agar	Negative	Positive
Poly D-glutamic acid capsule	Positive	Negative
Lysis by gamma phage	Positive	Negative
Motility	Negative	Positive
Growth on chloral hydrate agar	Negative	Positive
String-of-pearls test	Positive	Negative

Bacillus anthrasis 

Bacillus anthracis, also known as anthrax bacillus, causes the deadly disease anthrax in humans. The bacterium can be found in soil. Cattles in developing countries often suffer from skin anthrax. The infections are acquired either through inhalation or through direct contact with the skin of infected animals or their hides. Anthrax is almost nonexistent in the United States and other industrialized countries. Increased awareness in recent years is primarily due to its potential use as a biological weapon by terrorists. The bacterium produces highly resistant endospores and it can be easily grown in culture.

Disease 

Three clinical types of anthrax—pulmonary, cutaneous, and gastrointestinal—are generally recognized. The infection may spread via blood to meninges. The pulmo- nary form, relatively less common than the other two, is an airborne infection. The symptoms include fever, cough, headache, and vomoting. In advanced cases, lymph nodes are enlarged and symptoms of meningeal infection may develop. This form of infection can take a rapid course, leading to death in a very short time, often within 3 days. The cutaneous form involves necrotizing ulcers on skin which may or may not take a systemic course. The lesions become increasingly dark with time. Mortality is uncommon in the cases of skin anthrax. Gastrointestinal anthrax is aquired through ingestion of contaminated food. The bacterium may invade the upper intestinal tract, cecum, and ileum. Its symptoms include nausea and malaise. Hematogenous dissemination leads to systemic infection which is usually fatal.

Virulence Factors 

Bacillus anthracis posses a capsule made of poly D-glutamic acid that helps it evade host defenses. Most B. anthracis strains produce tripartite toxins which comprise three proteins, named LF, PA, and EF. 

Laboratory Diagnosis 

Specimens (blood, pus, and other body fluids) must be collected aseptically. The bacterium grows well on blood agar at 35°C. Phenylethyl alcohol agar is used for isolation from heavily contaminated specimens. Bacillus anthracis is a fast-growing bacterium. Class III or Class IV safety cabinets should be used when handling sus- pected specimens or cultures. Fresh isolates are strongly Gram-positive and endospores can be readily seen. The Gram reaction weakens as the cultures age.

Taxonomy 

Due to the presence of a capsule, B. anthracis is easily differentiated from other members of the genus Bacillus. Important delineating characteristics of main species of the genus Bacillus are summarized in Table 6.3.

Table 6.3 Physiological Differences between Some Common Species of the Genus Bacillus

Species hydrolysis       Penicillin         Lecithinase        β-hemolysis           Starch         Capsule

B. anthracis	Sensitive	Variable	None	None	Present
B. cereus	Resistant	Positive	Present	Positive	Absent
B. subtilis	Sensitive	Negative	Variable	Positive	Absent
B. sphaericus	Sensitive	Negative	Negative	Negative	Absent

Antibiotic Sensitivity 

Effective agents include doxycycline, ciprofloxacin, and chloramphenicol.

Bacillus cereus 

Bacillus cereus is a common soil-borne bacterium and is easily isolated from the air. Contamination of cereals, vegetables, meat, and cooked food commonly occurs. 

Disease 

Food poisoning is the most common form of B. cereus infection. The disease is acquired via the ingestion of contaminated food. Symptoms include nausea, vomiting, diarrhea, and abdominal pain. Cases of ocular infection are occasionally reported. Such infections often follow a traumatic injury and simultaneous contamination with the soil.

Virulence Factors

Two enterotoxins, one heat stable and the other heat labile, have been reported. Cereolysin and a phospholipase are believed to cause tissue damage.

Laboratory Diagnosis 

Bacillus cereus is easily grown on most media, especially on blood agar. Incubation is done at 35°C under aerobic conditions. Bacillus cereus strains show peritrichous flagella, which can be visualized by the use of Leifson flagella stain (Fig. 6.3).

Antibiotic Sensitivity 

Antibiotic therapy is not indicated in most cases. Serious conditions can be treated with clindamycin, vancomycin, gentamycin, or ciprofloxacin.

Figure 6.3.    A smear from the culture of a B. cereus strain showing peritrichous flagella (Leifson flagella stain). See color insert.

LISTERIA SPECIES 

Listeria monocytogenes 

Listeria are Gram-positive, facultative anaerobes, nonsporogenous bacilli. The only species of medical importance is L. monocytogenes. 

Disease 

Listeria monocytogenes causes meningitis and septicemia and accounts for 12%– 15% of all cases of meningitis in infants less than one month old or elderly persons aged more than 70 years. The bacterium is commonly present in the environment and it can grow at 4°C. Because of its ability to grow at low temperatures, the practice of storing leftover baby food including the feeding bottle in the refrigerator may play important role in infants contracting this disease. Infections occur due to consumption of contaminated food or animal product. Of the human population, 1%–5% harbors this bacterium in intestine and vagina. The possibility that some newborns acquire this infection while passing through the birth canal cannot be ruled out.

Virulence Factors 

Most pathogenic strains are β-hemolytic and also produce phospholipases.

Table 6.4    Some Differentiating Characteristics of Listeria spp.

Species	Hemolysis	D-Xylose fermentation	L-Rhamnose fermentation
L. monocytogenes	Positive	Negative	Positive
L. innocua	Negative	Negative	Variable
L. ivanovii	Positive	Positive	Negative
L. welshimeri	Negative	Positive	Variable
L. seeligeri	Negative	Positive	Negative
L. grayi	Negative	Negative	Negative

Laboratory Diagnosis

Blood agar, Columbia agar, and colistin-nalidixic acid (CNA) agar are good isolation media. Incubation is done at 35°C. Small smooth, whitish, translucent, and moist colonies approximately 1 mm in diameter develop within 24 hours. The β-hemolysis can be seen under the colonies. Fermentation tests are positive with L-rhamnose and negative with xylose.

Genus Listeria has six species that include L. monocytogenes, L. innocua, L. ivanovii, L. welshimeri, L. seeligeri, and L. grayi. Except for L. monocytogenes, all other Listeria spp. have been isolated from nonhuman as well as human sources. It is, therefore, important to differentiate L. monocytogenes from other five closely related species (Table 6.4).

Antibiotic Sensitivity

The therapeutic agents generally used for clinical management include ampicillin, gentamicin, and trimethoprim-sulfa.

Erysipelothrix rhusiopathiae

Erysipelothrix rhusiopathiae is a Gram-positive, asporogenous, catalase negative bacillus. In humans, it mostly causes localized inflammation of skin characterized by painful, red zones. The infection may spread to the blood and the vascular system, resulting in septicemia and endocarditis. The bacterium is widely distributed in nature and can be isolated from cattle, horses, pigs, mice, and turkeys, which may either be carriers or infected with E. rhusiopathiae. Most infections are due to occupational exposure in those such as butchers, fisherman, slaughterhouse workers, and farmers who come into close contact with these animals.

Virulence Factors

No clearly defined virulence factor has been identified but its ability to attach to heart valves and produce enzymes such as neuraminidase and hyaluronidase could possible play a role in the pathogenesis.

Laboratory Diagnosis 

Erysipelothrix rhusiopathiae grows well on blood agar. Better results are obtained if the biopsied tissue is homogenized and then inoculated on the media. The incuba- tion is done at 35°C. This bacterium is aerobic, but the growth is generally better if the incubation is done in a carbon dioxide incubator. The colonies grow slowly and may take up to 7 days to fully develop. They may present variable size and texture from rough and large to small and clear. This bacterium differs from other common Gram-positive pathogenic bacilli by its catalase negative properties. 

Antibiotic Sensitivity 

Erysipelothrix rhusiopathiae strains are mostly sensitive to ampicillin, clindamycin, and cephalosporins.