Gram-Negative Bacilli

Asporogenous Gram-negative bacilli of clinical importance can be divided into two major groups. Glucose-fermenting, oxidase-negative, and catalase-positive members constitute one group, called Enterobacteriaceae. Several members of this group are normally present in human intestines and others are causal agents of serious infec- tions. The second group, somewhat more heterogeneous, usually called nonfermentative Gram-negative bacilli, are glucose nonfermenters. They are widely distributed in nature and prefer aquatic habitats. However, several members of this group are frequently isolated from human sources and known to cause serious infections. A simple and practical scheme for the grouping of important pathogenic Gram-negative bacteria is depicted at the end of the previous chapter.

Members of the family Enterobacteriaceae are further divided into two groups; lactose fermenters, also called fecal coliforms or just coliforms, include Escherichia coli, Klebsiella, and Enterobacter. Lactose nonfermenters include many pathogenic species, such as Salmonella typhi, Shigella dysenteriae, and Yersinia pestis.

SPECIMEN COLLECTION

Clinical specimens, such as fecal matter, urine, blood, and other body fluids, should be collected aseptically and processed immediately. Otherwise, transport media should be used.

MEDIA AND LABORATORY DIAGNOSIS

Most Gram-negative rods grow on a wide range of media; some are selective, and others are nonselective (also called all-purpose media). Specific media for different groups will be described under the relevant section. Some of the media commonly used for their isolation are listed in Table 9.1.

As is evident from the information summarized in Table 9.1, MacConkey, Eosin methylene blue, and Hektoen agars have low selectivity, but Bismuth sulfite and

Table 9.1 Growth and Colony Characteristics of Some Members of the Family Enterobacteriaceae on Some Selective Media (Modified after Howard et al., Clinical and Pathogenic Microbiology, Mosby, St. Louis)

			Media
Species	MacConkey agar	Eosin-methylene blue agar	Hektoen agar	Bismuth sulfite agar	Brilliant green agar
Escherichia coli	Flat, red or pink	Metallic sheen	Yellow-orange	No growth	No growth
Klebsiella pneumoniae	Dark pink, mucoid	Colorless	Yellow or green	No growth	No growth
Enterobacter spp.	Pink, slightly mucoid	Purple	Yellow-orange	No growth	No growth
Serratia spp.	Colorless to slightly pink	Lavender or colorless	Color-less	No growth	No growth
Proteus mirabilis	Colorless	Colorless	Color-less	No growth	No growth
Salmonella spp.	Colorless	Colorless	Green or blue	Green-black	Pink-white
Shigella spp.	Colorless	Colorless	Green or blue	No growth	No growth
Yersinia pestis	Colorless or peach	Colorless or purple	Salmon	No growth	No growth

brilliant green agars are highly selective. In addition, Salmonella spp. and Escherichia coli can be differentiated from others by their ability to produce “black butt” on triple sugar iron agar (not included in Table 9.1).

Incubation is done for 18–24 hours at 35°C under aerobic or anaerobic condi- tions. Serratia marcescens can grow at 1–5°C and Escherichia coli does well at 45°C. In general, members of Enterobacteriaceae are easy to isolate and identify. Diagnosis of certain infections may require serologic test.

Occasionally, it is better to use nonselective media, especially for isolation from blood and sterile body fluid (e.g., cerebrospinal fluid). Sometimes use of enrichment broth, such as Gram-negative (GN) broth and Salenite-F (SF) broth, is desirable, especially for Salmonella and Shigella spp.

ENTEROBACTERIACEAE

As stated above, members of the family Enterobacteriaceae are Gram-negative, nonspore-forming bacilli that are oxidase negative, catalase positive, and ferment glucose with acid production. They reduce nitrate to nitrite. Important members of Enterobacteriaceae, commonly known as enteric bacteria, are included in the schema depicted at the end of the preceding chapter. Escherichia coli and Klebsiella pneumoniae constitute normal intestinal microbiota. Species of Salmonella, Shigella and Yersinia are also called enteric bacteria, but they do not represent normal intestinal microbiota, although they can survive in the intestines and that is where they lodge in the case of infection. Clinically significant members of the family enterobacteriaceae and some of their physiological differences are depicted in Table 9.2.

Clinical Significance

Escherichia coli

Escherichia coli and most other fecal coliforms are most abundant in sewage and sewage-contaminated water. Therefore, the infections are often acquired by ingestion of food and beverages contaminated with untreated water. Since this is the most abundant bacterium in feces, poor personal hygiene also plays important role in the dissemination of E. coli related infections. Escherichia coli are the only clinically significant species of the genus Escherichia. It has been incriminated in the cases of diarrhea, gastroenteritis, septicemia, and neonatal meningitis. It is also responsible for nearly 70% of all the cases of urinary tract infections (UTI).

Virulence Factors Many strains of E. coli produce endotoxins, heat labile enterotoxins (LT) and heat stable enterotoxins (ST). These toxins are usually produced by strains in O groups, including 06, 08, 015, 025, 063, 091, 0111, 0145, 0147, 0152, 0157, and 0159. Other virulence factors include pili and fimbriae which help with attachment, hemolysin, and a Shiga toxin-like substance. In addition to O antigens, two other antigens described as H and K antigens are produced by some strains.

Enterobacter cloacae 

Enterobacter cloacae is an opportunistic pathogen in immune-compromised patients. It accounts for 4%–10% of all cases of bacteremia caused by Gram-negative bacteria. The infection is characterized by fever and may be fatal if not controlled in a timely manner. Other clinical conditions may include endocarditis, ventriculitis, meningitis, and occasionally urinary tract infections. Enterobacter cloacae along with Klebsiella pneumoniae and Serratia marcescens are often associated with the contamination of intravenous fluids, and are thus a major source of hospital-acquired infection. Another Enterobacter spp., E. aerogenes, is also often isolated from clinical specimens. Members of the genus Enterobacter generally yield a positive result in ornithine decarboxylase tests and are capable of growing in potassium cyanide. In addition, strains belonging to most species of this genus are motile.

Klebsiella pneumoniae 

Klebsiella pneumoniae is the only pathogenic species of the genus Klebsiella. Like E. coli, it is a common fecal bacterium and abundantly present in sewage and untreated water. Infection can be endogenous or exogenous, and is a major cause of hospital-acquired infections. It is an important causal agent of pneumonia, lung abscess, septicemia, meningitis, and otitis externa, and a major cause of UTI. Klebsiella pneumoniae strains are nonmotile and yield a negative result in ornithine decarboxylase and indole tests. Most strains of K. pneumoniae can grow at 45°C. 

Therapeutic measures may depend on the clinical manifestation. For example, oral antibiotics are frequently used for the management of UTI and intravenous antibiotics are often used to treat pneumonia. Resistance factors carried by some organisms, such as extended spectrum beta lactamases, make the use of antimicro- bial resistance testing crucial in all serious infections. 

Virulence Factors Virulence factors include cell wall receptors, polysaccharide capsules, a large plasmid, enterotoxins, and K antigens.

Proteus mirabilis 

Proteus mirabilis (more frequently) and P. vulgaris (less commonly) are important pathogenic species of the genus Proteus. This bacterium is often associated with UTI. Proteus is also occasionally incriminated in pneumonia and septicemia. The virulence factors include production of urease, which breaks down urea into ammonia, the latter being highly toxic to the cells. This also contributes to struvite deposition in the upper urinary tract. Many strains produce bacteriocin. Two other species of the genus Proteus are commonly encountered in clinical specimens. These include P. penneri and P. vulgaris. They can be differentiated from each other by indole, ornithine decarboxylase, esculin hydrolysis, and salicin fermentation tests. Only P. vulgaris strains yield a positive indole test and only P. mirabilis are ornithine decarboxylase positive. Most strains (but not all) of P. penneri, especially biotype 2, are positive in salicin fermentation and esculin hydrolysis tests.

Salmonella Species 

Salmonella typhi has been reported mostly from humans. It is the well-known causal agent of typhoid, the deadly disease that affects millions worldwide. In the United States, approximately 400 cases are reported each year. The symptoms of typhoid may include fever, headache, gastrointestinal discomfort, loss of electrolytes, and dehydration. The disease is often fatal if not treated in a timely manner. The infection is usually food and waterborne. Chlorination of water has greatly reduced incidence of typhoid in industrialized countries, but the disease still remains a major public health concern in poor countries. Asymptomatic carriers of S. typhi are not uncommon. 

Salmonella paratyphi and S. choleraesuis, the other two pathogenic species of Salmonella, are commonly associated with poultry, cattle, pig, sheep, lizards, and so on. Human infections are usually acquired due to ingestion of contaminated meat, poultry, or other food products. The symptoms may include diarrhea, dysentery, bronchopneumonia, pyelonephritis, and meningitis. 

Virulence Factors The virulence factors include ability of the bacterium to adhere to and penetrate epithelial cells and its ability to multiply within the phago- cytic cells. O antigens and LPS are also important virulence-related properties. 

Serratia Species 

Genus Serratia has several species of which seven are generally encountered in clinical specimens. These include S. marcescens, S. ficaria, S. liquefaciens, S. odor- ifera, S. plymuthica, S. rubidaea, and S. fonticola. Of these, S. marcescens is perhaps the most important. This species is characterized by positive DNAse, lipase, gelatin hydrolysis, and sorbitol fermentation, and negative arabinose fermentation tests. An interesting feature of certain Serratia spp. is their ability to produce characteristic red pigment at ambient temperature (20–25°C), but not at 35°C. Serratia marcescens is associated, although less commonly than other organisms, with UTI, bacteremia (especially in the presence of an IV catheter), and neonatal meningitis (often nosocomially acquired infections).

Shigella Species 

Of all Shigella spp., S. dysenteriae is the most important pathogen. It causes shigellosis or bacillary dysentery. Symptoms may include fever, cramps, and diarrhea. The diarrhea may be accompanied by mucous and may eventually become bloody. Death may occur due to electrolyte imbalance. Fecal contamination of food and water is a common source of infection. Globally, shigellosis is responsible for a high degree of mortality and morbidity each year. Occasionally, Shigella strains are mistaken for E. coli. Normally, E. coli strains are motile, produce gas from glucose, and ferment lactose and mucate. In contrast, Shigella spp. are nonmotile and negative in gas production from glucose, and lactose and mucate fermentation tests. 

Virulence Factors 

The bacterium penetrates the intestine and invades intestinal cells, resulting in the lysis of the cells. Virulence factors include production of the deadly Shiga toxin, which is cytotoxic, enterotoxic, and neurotoxic.

Yersinia Species

Yersinia pestis causes plague, the deadliest of all infectious diseases, which has left a vivid mark in the history of medieval Europe. During the recent years, authentic cases of plague have been reported from many parts of the world including the United States and India. Plague can be divided into two distinct clinical forms:

1. Bubonic plague is acquired via fleas that infect rodents, such as rats. The symptoms include fever and lymphadenitis.

2. Pneumonic plague is transmitted from infected persons to healthy ones. The symptoms include pneumonia. This stage is a highly contagious.

Virulence Factors 

Virulence factors include the ability of the bacterium to invade tissue, its ability to survive in the phagosomes, and virulence plasmids. Several strains are known to produce pesticin, which is a bacteriocin. Bacteriocins are toxic proteins that inhibit growth of closely related bacteria, and with certain reservations, they can be called narrow spectrum antibiotics.

Laboratory Diagnosis 

 Clinically significant species of Yersinia grow on most media that have been discussed earlier. Storage at 4°C over an extended period can help with the enrichment and enhance isolation in culture. But this technique of cold storage enrichment has very little diagnostic value. Gram negative rods can be easily seen in the stained smears (Fig. 9.1). Serological test for Y. enterolytica O8 also has only a limited value. The diagnosis of plague is generally made on the basis of clinical findings with limited support from the laboratory observations. Of the several species of genus Yersinia (10 species by some counts), Y. enterocolitica, Y. pseudotuberculosis, Y. kristensensii, Y. intermedia, and Y. frederiksenii can be also encountered in clinical specimens. Therefore, species differentiation is required. Yersinia pestis strains are generally nonmotile, indole negative, do not hydrolyze urea, and yield a positive ornithine decarboxylase test.

Antibiotic Sensitivity 

 Susceptibility of enteric bacteria to antimicrobial agents is highly variable and it may differ from strain to strain. Resistance to antibiotics that were previously considered effective is quite common these days. A sensitivity test is, therefore, highly desirable. Usually, the local health care institutions keep a track of their susceptibility pattern. The antibiotics that may be effective include ticaracillin, clavulanic acid, second-generation cephalosporins, fluoroquinolones, kanamycin, tetracycline, and trimethoprim sulfamethoxazole.

Figure 9.1.    A Gram-stained smear prepared from a Y. pestis strain isolated from a case of plague

(courtesy: CDC).

Citrobacter Species

Citrobacter spp. are motile bacilli that may or may not always ferment lactose. Another distinguishing feature is their negative reaction in lysine decarboxylase tests. In addition to differences shown in the Table 9.3, the three species discussed here can be differentiated from each other on the basis of indole test and hydrogen sulfide production on triple sugar agar. Infections caused by Citrobacter spp. are often nosocomial and usually noted in immunocompromised persons. Species fre- quently isolated from human cases include C. freundii, C. diversus, and C. amalo- naticus. These often occur in the urinary tract, the respiratory tract, wounds, and cutaneous infections. Cases of septicemia, brain abscess, and meningitis have been occasionally noted in association with C. freundii and C. diversus. Strains of Citrobacter spp. have traditionally been sensitive to ampicillin and cephalothin, but instances of resistance to these drugs is on rise.

Edwardsiella Species

Two species of the genus Edwardsiella, E. tarda and E. hoshinae, are clinically significant. These species are not uncommon in human environments and are occasionally isolated from reptiles, fishes, and other animals. These two species are associated with gastrointestinal disorders mostly in the tropics. Edwardsiella tarda has occasionally been held as the causal agent in cases of septic shock, hepatic abscess, meningitis,

Table 9.3    Some Physiological Differences among Certain Members of the Family

Enterobacteriaceae That Are Relatively Uncommon Pathogens

to be the causal agent are limited to urinary tract infections and peritonitis. Strains of K. ascorbata can be mistaken for E. coli. They generally yield positive results in citrate utilization, lysine decarboxylation, and sorbitol and raffinose fermentation tests while E. coli are mostly negative. Kluyvera ascorbata strains are sensitive to ampicillin and cephalothin.

Providencia Species

From clinical perspective, the most important species of Providencia are P. stuartii and P. rettgeri. The infections are mostly noted in elderly persons with urinary tract disorders. These species are also known to colonize skin damaged by severe burns. Urinary tract infections are more common in patients with catheters. Strains of Providencia spp. are generally negative in hydrogen sulfide tests. They can be mistaken for Shigella spp., but can be differentiated on the basis of motility and citrate utilization tests, which are generally positive for Providencia spp. and negative for Shigella spp. Most strains of P. rettgeri and P. stuartii are sensitive to nitrofurantoin and beta lactam antibiotics. Important physiological differences among the species of Providencia and other uncommon pathogens belonging to the family Enterobacteriaceae are summarized in Table 9.3.

GLUCOSE NONFERMENTERS

Glucose nonfermenters, also known as NFB (nonfermenting bacteria), mostly include common environmental bacteria that seem to prefer wet conditions. Some NFB are also important opportunistic pathogens. These include the following:

• Acinetobacter
• Alcaligenes
• Flavobacterium
• Moraxella
• Pseudomonas
• Xanthomonas
• Burkholderia

Growth Requirements

NFB grow well on blood agar and on some of the other media used for Enterobacteriaceae, especially MacConkey agar. Selective media for Pseudomonas aeruginosa include acitamide agar, cetrimide agar, and OFPBL medium for Burkholderia (Pseudomonas) cepacia.

Except for a few exceptions, a majority of NFBs are strict aerobes, fast growers, and prefer incubation at 35°C. Distinguishing colony characteristics of some NFBs on blood agar are described below, along with other relevant features.

Acinetobacter Species 

On blood agar, colonies are raised, creamy, circular, and opaque, with hemolysis. It also grows well on MacConkey agar. Members of this genus are commonly present in soil and water. They can also be isolated from skin. This bacterium is not known to infect humans with unimpaired immunity. However, its association with nosocomial infections, pneumonia, meningitis, UTIs, septicemia, and wound infection has been reported. Acinetobacter baumannii is considered pathogenic to humans. Lately, this bacterium has become a more common cause of infections associated with stays in the intensive care unit and prolonged hospitalizations.

Alcaligenes Species 

A species of clinical significance is A. faecalis. These are oxidase positive and grow well on MacConkey agar. On blood agar, the colonies are nonpigmented, glistening, convex, and produce a fruity odor. Like Acinetobacter baumannii, Alcaligenes faecalis is mostly associated with infections in immune-compromised patients. Its isolation from sputum and urine has been occasionally reported. Also, like A. baumannii, it is known to cause nosocomial septicemia in some cases.

Flavobacterium Species 

Flavobacterium meningosepticum is the only species of clinical significance. It is oxidase positive and it may or may not grow on MacConkey agar. On blood agar, the colonies are circular, convex, smooth, and yellowish orange. This bacterium has been incriminated in a few cases of neonatal meningitis. Cases of meningitis, pneumonia, and septicemia in adults have been reported on rare occasions. Other notable species are F. indologenes, F. odoratum, and F. thalpohilum, which have been noted in association with the rare cases of meningitis, septicemia, urogenital tract disorders, and wounds. But their role in the causation of the disease is uncertain.

Moraxella Species 

Important species of the genus Moraxella include M. atlantae, M. lacunata, M. nonliquefaciens, and M. osloensis. These species are oxidase positive and they may or may not grow on MacConkey agar. On blood agar, the colonies are tiny and translucent. Moraxella spp. are normally present on mucous membrane, but they are generally considered harmless. However, strains identified as M. lacunata are occasionally associated with conjunctivitis and corneal infections. Moraxella osloensis are generally present in the genitourinary tract. 

Xanthomonas Species

Xanthomonas maltophilia, formerly known as Pseudomonas maltophilia, is a clinically significant species of the genus Xanthomonas. Most strains are oxidase negative and grow well on MacConkey agar. On blood agar, the colonies are lavender colored at first and turn grayish green with age. Some strains produce an ammonialike odor. Xanthomonas maltophilia strains are sometimes isolated from clinical specimens in hospitalized patients and involved in cases of pneumonia, bacteremia, endocarditis, meningitis, and UTI. Most infections are nosocomial and often noted in immunocompromised subjects. In a majority of such cases, infections are associated with the instrumentation or manipulative procedures.

Burkholderia Species

An important species of the genus Burkholderia, from the clinical perspective, is B. cepacia, formerly known as Pseudomonas cepacia. Like P. aeruginosa, B. cepacia is also widely distributed in nature. Most strains are oxidase negative and grow on a wide range of media including MacConkey agar and blood agar. Selective media have been developed for the isolation of this bacterium. It is one of the most important causal agents of respiratory tract infections in cystic fibrosis patients and an occasional cause of nosocomial infections. Urinary tract infections are often associated with the use of the catheter. This bacterium has also been isolated from intravenous catheters. Septicemia associated with the use of contaminated catheters has been reported. Burkholderia cepacia has also been associated with the nosocomial outbreaks of pneumonia and septicemia. Like P. aeruginosa, B. cepacia infections are difficult to treat. Antimicrobial resistance testing is important in determining the most ideal therapy.

Pseudomonas Species

Genus Pseudomonas has many species, but the most important member is P. aeruginosa. A majority of strains are oxidase positive and they grow well on MacConkey agar. On blood agar, the colonies are usually large, irregular, round, and have a ground glass appearance with grape-like odor. Pseudomonas spp. are dynamic bacteria, abundantly present in aquatic systems, and play an extremely important role in recycling the biomass in ecosystem.

In this era of indiscriminate use of antibiotics, corticosteroids, and immune suppressants, it is hard to draw a sharp line between pathogenic and nonpathogenic microorganisms. However, of all the bacteria listed as glucose nonfermenters, P. aeruginosa is of immense clinical importance. It is an important opportunistic pathogen and is also frequently incriminated in nosocomial infections such as pneumonia and bacteremia. Of the other notable species of this genus, P. fluorescens and P. putida are normally present in the upper respiratory tract and are occasionally isolated from blood, cerebrospinal fluid, pleural fluid, urine, and wounds, but their actual role in the causation of the diseases is uncertain. Another species, P. mallei, has been noted in association with glanders, a disease often noted in horses. Pseudomonas pseudomallei, a closely related species, is generally associated with glander-like disease in humans, mostly in Southeast Asia. But this bacterium is normally present in soil and water in many parts of the world. Perhaps next to P. aeruginosa, P. stutzeri is of some clinical significance. It has been isolated from blood, cerebrospinal fluid, discharges from the middle ear, sputum, and urine and is believed to be the causative agent at least in some of the cases. Among the other notables are P. alcaligenes, P. gladioli, and P. pickettii, which have been isolated from blood, urine, cerebrospinal fluid, and the respiratory tract of patients suffering from empyema, endocarditis, bacteremia, meningitis, and cystic fibrosis.

Disease 

Pseudomonas aeruginosa is known to cause a wide range of clinical complications including burn and wound infection, pneumonia, chronic pulmonary disorders, sep- ticemia, otitis externa, and UTI. Pseudomonas aeruginosa is also known to cause major complications in cystic fibrosis cases. Diseases caused by P. aeruginosa are generally rare in immunocompetent persons. Infections are mostly noted in patients with underlying conditions, such as intravenous drug use, serious burn injuries, granulocytopenia, long-term antibiotic therapy, and indwelling catheter or other devices. 

Virulence Factors 

 Virulence factors include endotoxin (LPS), exotoxins A and S, cytotoxins (leukocidins), proteinases, phospholipases (responsible for the destruc- tion of pulmonary surfactants), and pili that help with adherence. Extracellular proteinases are believed to be responsible for the degradation of a wide range of proteins associated with structural components and homeostasis. 

Antibiotic Sensitivity 

 Pseudomonas aeruginosa is resistant to many of the commonly used antimicrobial agents. Therefore, sensitivity testing is a necessity. Some effective agents include aminoglycosides, ceftazidine, piperacillin, carbapenems, and ciprofloxacin.

UNCOMMON NONFERMENTATIVE TAXA

Chryseomonas luteola 

Strains of C. luteola are characterized by the presence of polar flagella. They are motile, oxidase negative, and grow well on MacConkey agar. Isolates identified as C. luteola have been occasionally noted in the cases of wounds and abscesses, and have also been associated with a few cases of peritonitis and bacteremia, mostly in critically ill persons. 

Eikenella corrodens 

Eikenella corrodens strains are oxidase positive and nonmotile. They do not grow on MacConkey agar. This bacterium is normally present on the mucous membrane, mostly in the nasopharynx and gastrointestinal tract. Strains have been also isolated from cases of meningitis, and infections involving the neck and head, as well as bite wounds, mostly in immunocompromised patients. 

Kingella kingae 

Kingella spp. are facultative anaerobic, coccobacilli that are nonmotile, and oxidase negative, and may or may not grow on MacConkey agar. Strains of K. kingae have been occasionally isolated from blood, urine, throat, and wound samples. It is an opportunistic pathogen, and has been occasionally incriminated in the cases of endocarditis, arthritis, osteomyelitis, and septicemia. A majority of patients in such cases were children.

Weeksella virosa 

Weeksella virosa strains are also nonmotile and oxidase positive. Their growth on MacConkey agar is variable, mostly poor. They inhabit the urogenital tract of approximately 2% of women and are frequently isolated from the vaginal swabs of women who have had multiple sexual partners. There seems to be a relationship between the number of sexual partners and frequency of isolation of this bacterium from vaginal swabs. It has been occasionally incriminated in the cases of urethritis, peritonitis, pneumonia, sepsis, and vaginal infection.

Antibiotic Sensitivity 

Antibiotic sensitivity of these abovementioned nonfermentative Gram-negative bacilli can be hard to predict. Therefore, it is important to perform sensitivity tests, if their role in the causation of disease is suspected.