Other Gram-Negative and Gram-Variable Bacilli

Actinobacillus and Aggregatibacter Species

Actinobacillus and Aggregatibacter species are coccoid to small gram-negative bacilli in the family Pasteurellaceae. These organisms are normal microbiota of the oral cavity, and less frequently the urogenital tract, in humans. They also colonize animals, which can serve as reservoirs for opportunistic human infections. The genus Aggregatibacter was created based on the phylogenetic similarity of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus, Haemophilus paraphrophilus, and Haemophilus segnis. On transfer to the new genus, Aggregatibacter aphrophilus and Aggregatibacter paraphrophilus were combined into one species, Aggregatibacter aphrophilus. The genus name reflects a propensity of these organisms to aggregate with other bacteria.

Aggregatibacter actinomycetemcomitans (formerly Actinobacillus actinomycetemcomitans ) is the best known pathogen of this group. A. actinomycetemcomitans was first described as a human pathogen in 1912 and was initially called Bacterium actinomycetem comitans. Early isolates were recovered only in conjunction with Actinomyces israelii (hence the species designation), leading to speculation that A. actinomycetemcomitans was not itself capable of causing disease. After the introduction of penicillin, it was observed that A. actinomycetemcomitans sometimes could be recovered from persistent lesions of actinomycosis after A. israelii was eradicated. By the early 1960s, recovery of this organism in pure culture from blood and other normally sterile body fluids was reported widely. The organism is best known as a cause of endocarditis but has also been isolated in pure culture from patients with meningitis, brain abscess, endophthalmitis (with and without concomitant endocarditis), soft tissue infections, parotitis, septic arthritis, osteomyelitis, spinal epidural abscess, urinary tract infection, pneumonia, empyema, and pericarditis. Soft tissue infections most commonly involve the cervicofacial area, although they can occur elsewhere, including the chest and abdomen. There are reports of A. actinomycetemcomitans mimicking actinomycosis and causing pneumonia with chest wall invasion.

Although the organism is part of the endogenous microbiota of the mouth and can be recovered from about 20% of teenagers and adults, it (along with Porphyromonas gingivalis ) is one of the major pathogens in adult and juvenile forms of periodontitis. Extraoral infections are believed to occur due to hematogenous dissemination from lesions in the oral cavity. A. actinomycetemcomitans is present in the periodontal pockets of more than 50% of adults with refractory periodontitis and 90% of patients with localized aggressive periodontitis (formerly called localized juvenile periodontitis ), a destructive form of periodontitis characterized by loss of the alveolar bone of the molars and incisors. Clonal spread of the organism within families has been demonstrated using polymerase chain reaction (PCR)–based typing systems.

A. actinomycetemcomitans is classified into seven serotypes (a through g). The prevalence of different serotypes and their association with periodontal disease varies among geographic and ethnic populations. The JP2 strain of serotype b has enhanced virulence and is associated with significantly higher prevalence of periodontitis in people of African and Mediterranean descent. Serotype c is the most prevalent subgingival type in Asian individuals as well as in Brazil and the United States. Serotypes a, b, and c are detected most frequently in German patients, and c and d are found in Korean patients.

A. actinomycetemcomitans is a successful pathogen with well-characterized virulence factors, including two exotoxins: leukotoxin and cytolethal distending toxin (Cdt). The leukotoxin selectively binds to β ₂ -integrin and destroys leukocytes by inducing apoptosis or lysis. Cdt is prevalent among certain gram-negative bacteria and acts by damaging DNA, which produces growth arrest and subsequent apoptosis of a wide variety of eukaryotic cell types. Other virulence factors include proteins Aae and ApiA, which allow the organism to adhere to epithelial cells and become internalized. Production of didanosine tetraphosphate may enhance bacterial survival within the cytoplasm. Induction of cytokines and other factors contribute to tissue destruction and resorption of alveolar bone. Intracellular survival allows the organism to evade the host immune response, penetrate the epithelial cell layer, and reach the underlying connective tissues. A. actinomycetemcomitans is further able to evade host immune responses by inducing increased expression of Toll-like receptor 2, leading to phagocytosis and apoptosis of macrophages via p38 mitogen-activated protein kinase activation and tumor necrosis factor-α production. A. actinomycetemcomitans in biofilms is strongly associated with loss of periodontal tissue attachment. The ability of this organism to adhere to abiotic surfaces and form biofilms has been attributed to type IVb–like fimbriae that are primarily composed of fimbrial lower-molecular-weight protein (Flp).

A. actinomycetemcomitans is one of the HACEK organisms, along with Haemophilus parainfluenzae, other Aggregatibacter spp. ( A. aphrophilus [formerly Haemophilus aphrophilus ] and A. segnis ), Cardiobacterium spp. ( C. hominis, C. valvarum ), Eikenella corrodens, and Kingella spp. ( K. kingae, K. denitrificans ), which have in common that they are part of the normal oral microbiota, and have slow growth in culture, the need for incubation in an atmosphere enhanced with CO ₂ for recovery in culture, and a predilection for causing endocarditis. The onset of endocarditis is usually insidious, with a mean time to diagnosis of about 3 months. In comprehensive reviews of A. actinomycetemcomitans endocarditis, almost half of patients had periodontal disease or recent dental work and over 60% had underlying native valvular disease (33%) or prosthetic valves (28%). Fever was present in fewer than 50%; peripheral manifestations and splenomegaly each occurred in about one-third. Therapy was successful in 85% to 91%, but significant embolization was common (39%) and 23% required valve replacement.

Prosthetic valve endocarditis with A. actinomycetemcomitans was usually recognized earlier than native valve endocarditis (42 vs. 106 days), which probably was attributable to a higher index of suspicion. This earlier diagnosis may account for the high cure rate achieved with antibiotics alone and a relatively low rate of embolization reported.

Culture isolation of A. actinomycetemcomitans is the usual means of diagnosis, and the fastidious, slow-growing nature of the organism makes this difficult. Cultures must be incubated in an enhanced (5%–10%) CO ₂ atmosphere. By 18 to 24 hours, a few colonies (punctate, nonhemolytic) may be apparent on blood or chocolate agar, but the organism grows slowly and incubation for at least 48 hours is needed. After further incubation, a starlike structure tends to form in the center of the mature colony. The organism grows poorly on MacConkey agar. In broth or blood cultures, the organism often grows only in small “granules” adherent to the sides of the tube or bottle, with the medium remaining clear. Although the mean duration for incubation using continuously monitored blood cultures until detection is 3 to 5 days, up to 30 days may be required, especially if the patient has received prior antibiotic therapy. This finding underscores the need to hold blood culture bottles for a prolonged time if endocarditis caused by a fastidious organism is suspected. The appearance of the organism on Gram stain is coccoid to coccobacillary, similar to Haemophilus species. A. actinomycetemcomitans is urease negative and indole negative, reduces nitrate, and usually is oxidase negative. It is catalase positive, which helps differentiate it from A. aphrophilus . Most of the HACEK organisms are included in current MALDI-TOF mass spectrometry databases, which allows for rapid and more accurate identification.

A. actinomycetemcomitans usually is susceptible to cephalosporins (especially third-generation agents), rifampin, trimethoprim-sulfamethoxazole, aminoglycosides, fluoroquinolones (including ciprofloxacin and moxifloxacin), tetracycline, azithromycin, and chloramphenicol. In vitro susceptibility to penicillin and ampicillin is variable, but test results do not necessarily correlate with the clinical outcome. In general, treatment of actinomycosis with penicillin and surgical drainage (when necessary) is sufficient, even when mixed infection is present. Vancomycin, erythromycin, and clindamycin have little activity against A. actinomycetemcomitans. The organism displays variable susceptibility to metronidazole, and in vitro synergy between metronidazole and both β-lactams and ciprofloxacin has been reported. Because of strain-to-strain variability, testing of clinical isolates is recommended. CLSI provides conditions and breakpoints for broth microdilution susceptibility testing. The bioMérieux Etest (bioMérieux, Inc., Hazelwood, MO) can be used with supplemented Mueller-Hinton agar, Haemophilus test medium, or Brucella agar supplemented with 5% sheep blood, hemin, and vitamin K incubated at 5% CO ₂ for 24 to 72 hours. In the past, penicillin or ampicillin combined with an aminoglycoside was the usual treatment for endocarditis caused by this organism. Because of the potential for β-lactamase production, reports of failures with penicillin therapy, and difficulties with susceptibility testing, third-generation cephalosporins are now considered the drugs of choice. For endocarditis caused by HACEK organisms, the American Heart Association guidelines endorsed by the Infectious Diseases Society of America recommend ceftriaxone or ampicillin-sulbactam as initial therapy. A fluoroquinolone may be used to treat patients with β-lactam allergy, but clinical data are limited.

A. actinomycetemcomitans endocarditis has developed after dental procedures despite the prophylactic use of penicillin, erythromycin, or vancomycin. Severe A. actinomycetemcomitans –associated periodontitis is usually treated with mechanical débridement in combination with oral tetracycline therapy. Tetracycline failures occur, however, and a report suggests that the combination of metronidazole and amoxicillin is effective in suppressing subgingival infection.

Five species of Actinobacillus — A. lignieresii, A. equuli, A. suis, A. hominis, and A. ureae —are rare causes of human disease. The first three are commensals and opportunistic pathogens in animals, whereas the latter two are commensals of the human upper respiratory tract. A. lignieresii, A. suis, and A. equuli rarely can cause infections after bite wounds from farm animals. These infections can be polymicrobial. One report has described a boar hunter who developed endocarditis caused by an Actinobacillus organism that resembled A. suis and A. hominis biochemically. Another report described 46 clinical A. hominis isolates acquired over a 22-year period, mostly from Copenhagen, Denmark. Before this report, there were only a few case reports of human infections caused by this organism. Most of the isolates were from the respiratory tract; 18 of 33 respiratory isolates were reported to be pure cultures of A. hominis. The remaining respiratory cultures contained at least one other common respiratory pathogen. All the patients in this series had underlying diseases, including alcoholism, cardiovascular disease, drug addiction, chronic obstructive pulmonary disease, and cancer. Most patients had fever and pulmonary infiltrates, and 9 of 36 patients for whom clinical information was available died, including 1 of the 2 patients with bacteremia. The identification of the A. hominis isolates was confirmed by ribotyping and DNA hybridization. In this and other reports, automated systems had difficulty identifying Actinobacillus species. Fatal A. hominis bacteremia has also been reported in two patients with severe underlying liver disease. A. ureae is a rare cause of bacteremia and meningitis. Nine of 14 cases of A. ureae meningitis were posttraumatic, and another occurred after neurosurgery. Several patients had underlying chronic illnesses, including alcoholism and human immunodeficiency virus (HIV) infection.

Identification of Actinobacillus species is problematic. At the genus level, these organisms are biochemically similar to Pasteurella species. Species identification can be difficult without DNA hybridization studies.

A. ureae meningitis has been treated successfully with penicillin and third-generation cephalosporins.

Aeromonas Species

Aeromonads are ubiquitous inhabitants of fresh and brackish water. They have also been recovered from chlorinated tap water, including hospital water supplies. They occasionally cause soft tissue infections and sepsis in immunocompromised hosts and increasingly have been associated with diarrheal disease and other infections in immunocompetent individuals.

Taxonomy of the aeromonads has been revised over the past few decades and continues to be in transition. Aeromonads are broadly divided into the mesophilic group, with optimal growth temperatures of 35°C to 37°C and associated with human infection, and the psychrophilic group, with optimal growth temperatures of 22°C to 25°C and associated with disease in fish. The species designations within the mesophilic group are currently largely based on DNA hybridization studies, but new information based on full-genome sequencing and microarray analysis will likely result in further taxonomic revisions. A. hydrophila, A. caviae (synonym, A. punctata ), and A. veronii biovar sobria are reported most frequently in human infections. The pathogenic potential of Aeromonas species has been attributed to several virulence factors that are very heterogeneously present among clinical isolates. The pathogenicity of A. hydrophila has been attributed to the ability of the bacterium to produce the cytotoxic enterotoxin Act and cytotonic enterotoxins Ast and Alt, as well as a variety of proteases and type III secretion systems and surface structures, including pili and S-layer, lateral, and polar flagella, which allow the organism to attach to cells and enter tissue. Carriage of multiple toxins appears to be a property of A. hydrophila but not other Aeromonas species. The aerolysin/hemolysin group of toxins, including Act, are important virulence factors in A. caviae and A. veronii biovar sobria. Alt and lateral flagella are notably significantly less prevalent in these species.

Aeromonas was first isolated more than 70 years ago, but evidence implicating this genus as a cause of gastrointestinal disease has been amassed only since the early 1980s. Reports from diverse geographic locations have associated Aeromonas species with diarrheal disease in humans; in some locales, they are recovered as commonly as Shigella or Campylobacter. Many laboratories do not routinely culture stool for Aeromonas, so the incidence of Aeromonas -associated diarrhea may be underestimated. Evidence supporting a causative role in diarrheal disease includes (1) a higher carriage rate in symptomatic compared with asymptomatic individuals; (2) an absence of other enteric pathogens in most symptomatic patients harboring Aeromonas species; (3) identification of Aeromonas enterotoxins ; (4) improvement of diarrhea with antibiotics active against Aeromonas species and clinical worsening with antibiotics ineffective against the organism; and (5) evidence of a specific secretory immune response coincident with diarrheal disease. Most of this information refers to A. hydrophila and A. cavie ; the extent of clinical information about the other species in relation to diarrheal disease is limited.

Aeromonas caviae is the predominant isolate from diarrheal stools, but in some geographic areas, A. hydrophila and A. veronii biovar sobria are frequently isolated as well. Other Aeromonas species appear to cause asymptomatic carriage only. Aeromonas -associated diarrhea usually occurs during the summer, when the concentrations of aeromonads in water are the highest. Most cases are sporadic. An epidemiologic study was unable to implicate the drinking water supply as the source of diarrheal isolates; Aeromonas isolates from diarrheal stool were genetically unrelated to those from water supplies. Aeromonas is increasingly being recognized as a cause of diarrhea in travelers returning from Asia, Africa, and Latin America. Daycare center outbreaks have been reported, although in one study, molecular typing did not suggest clonal spread. The clinical manifestations of Aeromonas -associated diarrhea are varied. Diarrhea is usually watery and self-limited, but some persons develop fever, abdominal pain, and bloody stools. Fecal leukocytes may be present. Occasionally, diarrhea may be severe or protracted, and hospitalization may be necessary. Rare cases of ischemic colitis associated with Aeromonas have been reported in healthy children and adults, and chronic colitis developing after acute Aeromonas -associated diarrhea has been reported in adults. Although no controlled trials have validated antimicrobial therapy for Aeromonas -associated diarrhea, clinical improvement has occurred with antibiotics active against the organism. Hemolytic-uremic syndrome associated with Aeromonas enterocolitis has been described in infants and adults. Aeromonas -associated diarrhea has been shown to be more prevalent in individuals with concurrent rotavirus infection. The relevance of this finding is supported by in vitro studies demonstrating that preinfection of enterocyte-like cells with rotovirus can increase the capacity of some Aeromonas strains to adhere to enterocytes.

In contrast, the evidence for pathogenic roles of aeromonads in extraintestinal infections is much more clear-cut. Most Aeromonas soft tissue infections are caused by A. hydrophila. Trauma followed by exposure to fresh or brackish water (and not salt water, even though aeromonad density in seawater is similar to that in fresh water) usually, but not invariably, precedes infection. Cellulitis develops within 8 to 48 hours, and systemic signs are common. Suppuration and necrosis around the wound are frequent, and surgical débridement is often necessary. Fasciitis, myonecrosis (occasionally associated with gas formation), and osteomyelitis may develop. In the setting of a rapidly progressive cellulitis after an injury related to water exposure, Aeromonas and Vibrio species infections should be considered in the differential diagnosis. Aeromonas soft tissue infections can develop after exposure to soil, in association with crush injuries, and as a complication of burns, typically when initial management of the burn included immersion in natural water sources. There is one reported outbreak of A. hydrophila wound infections in participants of a mud football competition in Australia. The field was “prepared” with water from an adjacent river but DNA fingerprints of the river isolates did not match those of the human isolates. Aeromonas was second only to Staphylococcus aureus in one study of bacteria causing secondary infection in untreated Buruli ulcer lesions. Aeromonas soft tissue infection is a recognized complication of the use of medicinal leeches in conjunction with reimplantation or flap surgery. A. hydrophila and other Aeromonas species are normal inhabitants of the foregut of leeches. Leeches lack the requisite proteolytic enzymes and are dependent on the symbiotic Aeromonas to digest the blood meal. Aeromonas infection has developed in about 12% of patients treated with leeches. Prophylactic antibiotics, particularly ciprofloxacin or cefotaxime, have been recommended at the time of leech application. Infections have developed despite prophylaxis, with the isolated strains determined to be resistant to ciprofloxacin. The onset of infection after the application of medicinal leeches ranges from 1 day to more than 10 days. Mild wound infection, loss of flap, myonecrosis, and sepsis may ensue.

Aeromonas bacteremia and sepsis are uncommon, but in the largest series reported to date, 143 Aeromonas bacteremias, including 104 that were monomicrobial, occurred in one institution in Taiwan over a 10-year period. A. hydrophila caused 60% of the bacteremias; most of the other isolates that were identified by species were A. veronii subtype sobria and A. caviae. Most patients in this series were immunocompromised, including 54% who were cirrhotic and 21% who had an underlying malignancy. Spontaneous bacterial peritonitis was common in cirrhotic patients with abdominal pain. There was a similar distribution of Aeromonas species in a study of 53 Aeromonas blood isolates collected from 27 medical centers in the United States over a 10-year period. Most patients were immunocompromised, and underlying malignancy was much more common than liver disease in this series. Most patients with Aeromonas sepsis do not present with diarrhea. Interestingly, about one-third of Aeromonas bacteremias are hospital acquired. Aeromonas has been recovered from hospital water supplies, and clusters of Aeromonas bacteremia have been described. In some series, the hospital-onset cases were not epidemiologically linked and endogenous gut microbiota was the presumed source. The mortality rate for Aeromonas sepsis is 33% or higher. Other species— Aeromonas jandaei, Aeromonas veronii biovar veronii, and Aeromonas schubertii —have rarely been isolated from the blood. A variety of other infections caused by Aeromonas species have been reported, including intraabdominal abscess, pancreatic abscess, hepatobiliary infection, spontaneous bacterial peritonitis in patients with cirrhosis, meningitis, endocarditis, suppurative thrombophlebitis, osteomyelitis, urinary tract infection, prostatitis, pneumonia (including near-drowning–associated pneumonia), empyema, lung abscess, tonsillitis, epiglottitis, keratitis, and otitis media. A. hydrophila epididymitis and bacteremia developed in a healthy man 24 hours after he had sexual intercourse with his wife in their swimming pool. Cultures obtained from the pool grew A. hydrophila.

Aeromonas organisms are gram-negative, nonsporulating facultative anaerobic rods that usually are β-hemolytic on blood agar and ferment carbohydrates with acid and gas production. The organisms grow well on MacConkey agar (some strains are lactose fermenters and some are not), but growth on thiosulfate citrate–bile salts–sucrose medium is variable. Selective techniques are often necessary for the isolation of Aeromonas species from mixed cultures. The organisms are more difficult to identify in stool cultures because enteric media may be inhibitory for some Aeromonas species. Either blood agar that contains ampicillin (10 or 30 µg/mL) or cefsulodin irgasan novobiocin agar can be used as a selective medium. Growth of colonies on plates usually occurs within 24 hours. Aeromonas species are oxidase positive, helping to distinguish these organisms from Enterobacteriaceae. Identification of Aeromonas to the genus level is generally not difficult, but misidentifications, particularly as Vibrio species, may occur with automated systems. Identification to species can be difficult and many clinical laboratories proceed no further, reporting an Aeromonas isolate as “ Aeromonas species” or “ Aeromonas hydrophila complex.” MALDI-TOF mass spectrometry can provide rapid and accurate identification. CLSI document M45-A2 provides interpretive criteria for disk diffusion and MIC testing for several species of Aeromonas. The clinically relevant Aeromonas species are uniformly resistant to penicillin and ampicillin, are often resistant to cefazolin and ticarcillin, and are usually but not invariably susceptible to third-generation cephalosporins, aztreonam, and carbapenems. Resistance to cefotaxime has developed on therapy. Sensitivity to piperacillin and ticarcillin-clavulanate is variable. Aeromonas species can produce serine β-lactamases, including an Ambler class D penicillinase, class C cephalosporinase, and, less frequently, Temoniera (TEM) family extended-spectrum β-lactamases. Some isolates exhibit coordinated expression of these β-lactamases after both induction and selection of derepressed mutants. Aeromonas can also harbor chromosomal CphA metallo-β-lactamases that have narrow substrate profiles and specifically hydrolyze carbapenems. Metallo-β-lactamases of the Verona integron–encoded (VIM) and imipenemase (IMP) families that confer broader β-lactam resistance have been described in strains of A. hydrophila and A. caviae, encoded on an integron and a plasmid, respectively. There are reports of increasing resistance to tetracycline and trimethoprim-sulfamethoxazole. In one report, tigecycline was active against 200 of 201 isolates. Aminoglycosides are usually active, with resistance to tobramycin being more common than resistance to gentamicin or amikacin. Fluoroquinolones are highly active against Aeromonas species, although the existence of chromosomal mutations and plasmid-mediated quinolone resistance in environmental Aeromonas strains raise concern that fluoroquinolone resistance could easily develop. Aeromonas species harboring a conjugative plasmid that confers multiple antibiotic resistance have been identified. A cephalosporin or fluoroquinolone is generally recommended for treatment of Aeromonas, with the addition of an aminoglycoside for severe infections. Because of emerging resistance, polymicrobial therapy may be considered for empirical treatment until in vitro susceptibility results are available.

Cardiobacterium Species

Cardiobacterium hominis and Cardiobacterium valvarum are the only two species in the genus Cardiobacterium. Unlike the other HACEK organisms, these organisms rarely cause disease other than endocarditis. Cardiobacterium species have been described as Pasteurella -like organisms; they are part of the microbiota in the nose, mouth, and throat and are present occasionally on other mucous membranes as well as in the gastrointestinal tract.

There are more than 80 reported cases of C. hominis infection, and all but a few have involved the heart valves. Most patients have had underlying anatomic defects (e.g., rheumatic heart disease, ventricular septal defect, congenital bicuspid valve) or prosthetic cardiac valves. Many patients with endocarditis have had severe periodontitis or prior dental procedures without antimicrobial prophylaxis. C. hominis endocarditis occurring after upper gastrointestinal endoscopy has been reported. A subacute presentation, with an insidious onset (mean of 2–5 months before diagnosis) and an absence of fever at the time of diagnosis, is common. Some of the patients have splenomegaly, anemia, immune-mediated glomerulonephritis, and hematuria, consistent with a long period between infection and diagnosis. Large vegetations, and large vessel emboli, are characteristic. The mortality rate is about 10%, and valve replacement is needed in about 30% of cases. Septic arthritis, vertebral osteomyelitis, mycotic aneurysms (intracranial and mesenteric), and neurologic involvement are reported complications of C. hominis endocarditis.

Almost all clinical isolates come from blood, although meningitis associated with endocarditis has been described. In one of the very rare cases of infection without endocarditis, a patient with adenocarcinoma of the kidney invading the cecum developed an abdominal abscess and bacteremia; abscess and blood cultures grew C. hominis and Clostridium bifermentans. There is also a case report of C. hominis pacemaker lead infection without valvular involvement. Because of phenotypic similarities, it is suspected that some clinical isolates identified as C. hominis may actually have been C. valvarum. C. valvarum has caused several endocarditis cases worldwide with a spectrum of presentations similar to C. hominis, including insidious infection, ability to cause embolism, and the need for valve replacement in the majority of cases. Most cases for which details were provided were associated with periodontitis or an antecedent dental procedure without antimicrobial prophylaxis. This species was first described as Cardiobacterium species strain B from dental plaque and has also been described among the etiologic agents in advanced lesions of children with noma.

Cardiobacterium species are pleomorphic gram-negative rods; morphology varies considerably depending on culture conditions. They often have swelling of one or both ends and retain the crystal violet dye at the poles during the Gram stain procedure. Microscopically, the organisms sometimes form rosettes, but short chains, teardrops, pairs, and clusters are also common. Supplementation of the medium with yeast extract results in a loss of the pleomorphism, and most organisms become sticklike, gram-negative rods with rounded ends. Incubation in high humidity and 3% to 5% CO ₂ maximizes recovery of the organism. Most strains grow better on sheep blood agar than chocolate agar and will grow on Mueller-Hinton agar or trypticase soy agar without blood, but grow poorly on MacConkey agar or similar selective media. Colonies of C. hominis are 1 to 2 mm in diameter on sheep blood agar, usually by 48 to 72 hours after incubation at 37°C under increased CO ₂ . However, with some systems, incubation for 5 to 7 days before growth can be confirmed is not unusual, and cultures should be held for this period or longer if C. hominis is suspected. C. valvarum is considered to be more fastidious than C. hominis, with tiny visible colonies, 0.2 to 0.8 mm in diameter, appearing on blood agar after 72 to 96 hours of incubation. Colonies of C. valvarum are nonhemolytic; however, colonies of C. hominis produce slight α-hemolysis after 3 to 4 days of incubation and develop a rough appearance, with a serpentine pattern of growth from the edge to adjacent colonies. Cardiobacterium organisms are oxidase positive and catalase negative, and they produce indole (although positivity is weak in many strains of C. hominis and absent in some oral strains of C. valvarum ). Cardiobacterium species may be misidentified as Pasteurella multocida when using the API 20NE identification strip (bioMérieux, Inc., Hazelwood, MO). The phenylphosphonate reaction can be used to separate C. hominis (positive) from C. valvarum (negative). MALDI-TOF mass spectrometry also successfully identifies Cardiobacterium and distinguishes the two species. PCR amplification of 16S ribosomal DNA from heart valve and arterioembolic tissue has detected C. hominis sequences in cases of culture-negative endocarditis.

Susceptibility tests are difficult to perform because of the slow growth of the organism and unusual nutritional requirements, although the Etest appears to be useful. When tested, the organism is usually broadly susceptible to β-lactam drugs, fluoroquinolones, chloramphenicol, rifampin, and tetracycline. Susceptibility to aminoglycosides, erythromycin, and clindamycin is variable. Isolates with the ability to produce β-lactamase have been reported. Current American Heart Association guidelines recommend treating endocarditis caused by HACEK organisms with a 4-week course of ceftriaxone, ampicillin-sulbactam, or a fluoroquinolone. In a review of cases, most patients were treated successfully with penicillin alone, ceftriaxone alone, or penicillin and aminoglycosides, with the duration of therapy ranging from 25 to 63 days. Although microbiologic cure is usually achieved, complications frequently arise during the course of therapy. Systemic embolization, mycotic aneurysm, or progressive cardiac failure has necessitated valve replacement in a number of cases.

Chromobacterium Species

Chromobacterium violaceum is a rare opportunistic human pathogen but can cause life-threatening sepsis with metastatic abscesses. The organism is a common soil and water inhabitant in tropical and subtropical areas. Most cases of human infection have come from Southeast Asia, although more than 35 cases have been reported in the United States, almost all from the Southeast (primarily Florida). Cases have also been reported from Australia and South America. Although not considered a normal inhabitant of the human gastrointestinal tract, C. violaceum was present in the feces of 3 of 65 children whose stool was cultured at the time of admission to a hospital in Atlanta.

C. violaceum infection occurs in infants, children, and adults, almost always in the summer months and usually after exposure of nonintact skin to contaminated water (often stagnant) or soil. Two cases followed near drownings. Symptoms include pain at a local site of infection, fever, nausea, vomiting, abdominal pain, and diarrhea. Local cellulitis, pustules, ulcers with necrotic bases, or lymphadenitis commonly precedes evidence of systemic infection. Septic shock develops rapidly, as can pneumonia and visceral abscesses involving the liver, spleen, and lungs. This presentation can be confused with septicemic melioidosis, which is more common than C. violaceum infection in Southeast Asia, where both diseases are endemic. The mortality rate for reported cases in the United States is about 60%. Urinary tract infection, conjunctivitis, orbital cellulitis, retropharyngeal infection with prevertebral abscess, neutropenic sepsis, osteomyelitis, brain abscess, meningitis, puerperal sepsis, and internal jugular vein thrombophlebitis have been reported. There are also a few case reports in the pediatric literature of C. violaceum –associated diarrhea. A report from Brazil of one confirmed and two suspected cases in siblings is the first cluster of suspected C. violaceum infections linked to a common source. C. violaceum infection is more common in patients with chronic granulomatous disease (CGD), but cases occur in the apparently normal host. There appears to be a higher survival rate in persons with CGD compared with patients without known neutrophil dysfunction. This may reflect a selection bias because C. violaceum infection can be the initial manifestation of CGD, with the diagnosis of CGD being established only after recovery from the infection. Deficiency of polymorphonuclear leukocyte glucose-6-phosphate dehydrogenase and neutrophil dysfunction also were present in a 3-year-old patient who died with C. violaceum sepsis. Most strains produce an antioxidant pigment, violacein, that protects the organism against oxidative stress induced by the host response to infection. Other pertinent virulence factors based on the study of only one clinical and one environmental isolate include greater endotoxicity of the outer membrane and enhanced resistance to phagocytosis in the virulent strain. Diagnosis is made by culture of blood, abscess fluid, or skin exudate.

C. violaceum organisms are long gram-negative bacilli; occasionally, the organisms are slightly curved and can be confused with vibrios. The organisms are facultatively anaerobic, with versatile and adaptable pathways for energy generation, and grow readily in 18 to 24 hours on common laboratory media containing tryptophan, which include sheep blood agar, chocolate agar, Mueller-Hinton agar, trypticase soy broth, and MacConkey agar. Incubation at 37°C usually is effective, although growth is enhanced if incubation occurs at 25°C. Most strains produce violacein, an insoluble pigment that imparts a violet-black color to the colonies on solid media under aerobic conditions, hence the species’ name ( Fig. 236.1 ). There are a few reports of infection caused by nonpigmented strains. Violacein can induce apoptosis in leukemia cell lines and is being investigated as a potential chemotherapeutic agent. The color may be lost on subculture or after therapy is begun. The organisms produce hydrogen cyanide, so a faint cyanide smell may be present. The oxidase reaction is usually positive but may be difficult to detect in pigmented strains. Demonstration of oxidase can be enhanced by incubating the culture anaerobically, which inhibits pigment formation.

Antibiotics having the greatest activity against C. violaceum generally include fluoroquinolones, chloramphenicol, tetracycline, trimethoprim-sulfamethoxazole, imipenem, and gentamicin. The ureidopenicillins are often active, but resistance to cephalosporins is common. C. violaceum is also resistant to colistin. Although aztreonam is a natural product of some strains of C. violaceum, most clinical isolates are susceptible to this agent. Because of the rarity of infection, the often fulminant course, and the high mortality rate, the optimal antibiotic therapy is unknown. Ciprofloxacin is the most active antibiotic in vitro, and there are recent case reports of successful treatment with fluoroquinolones, often in combination with other agents. Most survivors of this infection were treated with chloramphenicol or a penicillin (carboxypenicillin or a ureidopenicillin) in combination with an aminoglycoside. Relapse has occurred more than 2 weeks after the completion of therapy and apparent cure, presumably because of a residual suppurative focus. Oral trimethoprim-sulfamethoxazole, doxycycline, or ciprofloxacin has been used after 2 to 4 weeks of intravenous therapy with other antibiotics, with the oral regimen continued for several weeks to a few months to prevent relapse. Antibiotics at subinhibitory concentrations, such as occur during the postantibiotic phases of clinical therapy, have been shown to enhance quorum-sensing–related virulence factors, including violacein production, chitinase production, and biofilm formation.

Chromobacterium haemolyticum, a species newly described in 2008, has been reported to cause pediatric bacteremia, proctocolitis in children, pneumonia, and necrotizing fasciitis. These cases have been reported from the United States, Japan, and Thailand. There was known aquatic exposure in two of the cases. C. haemolyticum differs from C. violaceum in that it is nonpigmented and has strong β-hemolytic activity on sheep blood agar. Although there are also differences in several biochemical reactions, misidentifications of C. haemolyticum as nonpigmented C. violaceum occur with currently available biochemical and MALDI-TOF mass spectrometry systems; 16S rRNA sequencing is required for definitive identification. Overall, C. haemolyticum is more resistant than C. violaceum, with higher MICs for most drugs. Isolates are typically susceptible to fluoroquinolones and resistant to β-lactam antibiotics.

Dysgonomonas Species

The genus Dysgonomonas taxonomically clusters in the Bacteroides-Prevotella-Porphyromonas group and presently contains four species— D. gadei, D. capnocytophagoides, D. mossii, and D. hofstadii —that have been isolated from human sources. The type species is D. gadei but D. capnocytophagoides has been reported more frequently. D. capnocytophagoides and D. mossii were originally members of the Centers for Disease Control and Prevention (CDC) dysgonic fermenter (DF)-3 group, indicating an organism that ferments glucose and has difficulty growing on routine media. Isolates from the genus Dysgonomonas are rare but have been recovered from blood, wounds, urine, peritoneal fluid, umbilicus, stools, and gallbladder.

D. capnocytophagoides has been isolated from diarrheal stools of patients with immune deficiencies, including common variable hypogammaglobulinemia, HIV infection, diabetes with chronic renal failure, and lymphoreticular and other malignancies, and from patients receiving immunosuppressive agents, but its role as a gastrointestinal pathogen remains controversial. With the use of selective media, this organism was isolated from 11 of 690 (1.6%) stools submitted for bacterial culture at the National Cancer Institute. In another prospective study of the role of D. capnocytophagoides in diarrheal disease, the organism was recovered from 2 of 178 specimens (1.1%) submitted for Clostridioides difficile (formerly Clostridium difficile ) toxin assay and from 3 of 129 (2.3%) stool specimens from patients with HIV infection. These data suggest that the paucity of reports of recovering D. capnocytophagoides from stool specimens may not be attributable to its rarity (as a colonizer or pathogen) but rather to the inability to recover the organism on conventional media. Antibiotic therapy directed at D. capnocytophagoides produced a therapeutic response in some of these patients, including 4 of 11 in the first study. Some of the responders had diarrhea of several months’ duration, with prompt resolution after antibiotic therapy was initiated. In other patients, the clinical significance of the organism was unclear; eradication of the organism from the stool was not accompanied by resolution of diarrhea, or the diarrhea resolved without specific therapy. D. capnocytophagoides has also been isolated from the urine as a cause of biliary sepsis, from a polymicrobial thigh abscess in a patient with insulin-dependent diabetes, from liver abscesses and blood after radiofrequency ablation in a patient with hepatocellular carcinoma, and from patients with neutropenia, including the blood and stool of a patient with acute myelocytic leukemia. D. gadei and D. mossii have been isolated from the gallbladders of patients with cholecystitis. D. mossii has also been recovered repeatedly from intestinal fluid in a patient with pancreatic cancer but was not associated with obvious infection. D. hofstadii was isolated from a postoperative abdominal wound.

Organisms in the genus Dysgonomonas are coccobacillary to short gram-negative, nonmotile, facultative rods. Growth occurs on blood agar and chocolate agar after 1 to 3 days of incubation in ambient, CO ₂ -enriched, or anaerobic atmospheres, but growth is less on blood agar and no growth occurs on MacConkey agar. Routine enteric media do not support growth of D. capnocytophagoides. Selective Campylobacter media do support growth when incubated at 37°C, but not at 42°C, which is the routine incubation temperature for Campylobacter. Selective media such as cefoperazone vancomycin amphotericin B blood agar inhibit normal microbiota and allow recovery of D. capnocytophagoides from stool specimens.

Dysgonomonas colonies are gray-white and nonhemolytic with a slight sweet aromatic odor. X factor is required for growth, nitrate is not reduced, and oxidase and catalase tests are negative. Identification can be made using the API Rapid ID 32A system (bioMérieux, Durham, NC) or the VITEK 2 system (bioMérieux, Durham, NC) or by whole-cell fatty acid gas chromatography.

Despite a lack of established breakpoints, the Kirby-Bauer disk diffusion and MIC methods have been used for antimicrobial susceptibility testing. Dysgonomonas species appear to be resistant to most β-lactam drugs, fluoroquinolones, aminoglycosides, metronidazole, vancomycin, erythromycin, and gentamicin. Many strains are susceptible to chloramphenicol, trimethoprim-sulfamethoxazole, clindamycin, and tetracycline. Tetracycline or clindamycin was used in the few reported cases of diarrheal disease that responded promptly to antibiotic administration. Despite a Kirby-Bauer zone size suggesting susceptibility, imipenem failed to clear D. capnocytophagoides from the bloodstream in the one reported bacteremic patient; the bacteremia resolved after therapy with trimethoprim-sulfamethoxazole was initiated.

Kingella Species

Kingella species are members of the family Neisseriaceae. They are normal microbiota of the human oropharynx and also are occasionally found in the oral cavity in other animals. Five species— K. kingae, K. denitrificans, K. oralis, K. potus, and K. negevensis —have been described. Kingella kingae is the most frequently recognized member of the genus and has been isolated from invasive infections with the oropharynx implicated as the source. Kingella kingae is the most common cause of bone and joint infections in young children and belongs to the HACEK group of fastidious gram-negative organisms associated with endocarditis (see Chapter 213 ).

Kingella denitrificans has been implicated in cases of endocarditis, bacteremia, empyema, corneal ulcers, chorioamnionitis, granulomatous disease secondary to acquired immunodeficiency syndrome (AIDS), retropharyngeal abscess, and peritonitis in peritoneal dialysis patients. The one reported pediatric peritonitis case was polymicrobial and the source of the organisms was presumed to be the patient's dog.

The other Kingella species have been reported much less frequently. K. oralis has been isolated from subgingival plaque in patients with and without periodontitis, and its relationship to disease is unclear. The only reported infection attributed to K. potus was in a forearm wound in a zookeeper as a result of a kinkajou bite. K. negevensis is described as one of the oropharyngeal microbiota of healthy children but has not been studied in other populations. In the only reported case of invasive infection to date, PCR targeting the GroEL molecular chaperone protein gene identified K. negevensis in a culture-negative osteoarticular specimen from a child.

Kingella spp. are coccoid to medium-sized gram-negative rods that tend to resist decolorization and do not grow on MacConkey agar. The organisms are notoriously fastidious, and growth is enhanced in the presence of 5% to 10% CO _2. K. denitrificans, K. potus, and K. oralis colonies are low, convex, and 1 to 2 mm in diameter after 48 hours of incubation. They are friable and nonhemolytic and have a nondiffusible yellow pigment. K. negevensis colonies are said to resemble a small colony variant of K. kingae and are opaque, pinpoint, and faintly β-hemolytic. All species are catalase negative and oxidase positive. All species except K. potus ferment glucose. Automated biochemical identification systems can misidentify Kingella as Haemophilus spp., Gardnerella vaginalis, Neisseria spp., or Moraxella spp. Current MALDI-TOF mass spectrometry databases include K. kingae and K. denitrificans, and these organisms have been correctly identified using this technology in several reports. Amplification and sequencing of the 16S rRNA gene or species-specific nucleic acid amplification tests targeting either the rtx operon or the groEL gene have been useful for detection of Kingella in some bone and joint specimens in which cultures have not revealed the pathogen.

Approved breakpoints for broth microdilution susceptibility testing of the HACEK group have been published by the CLSI. Kingella spp. are generally susceptible to a wide range of antibiotics, including β-lactams, macrolides, tetracyclines, co-trimoxazole, and quinolones. β-Lactamase–positive isolates have been reported to be susceptible to combinations with β-lactam inhibitors.

Neisseria animaloris and Neisseria zoodegmatis

The bacteria previously known as CDC eugonic fermenter (EF)-4 have been renamed Neisseria animaloris and Neisseria zoodegmatis based on polyphasic taxonomic studies, including 16S rRNA gene sequence analysis These bacteria are nonmotile, oxidase-positive, fastidious gram-negative rods that produce acid from glucose ( eugonic fermenter ) but have relatively few other reactions and display very slow growth in anaerobic conditions. They are distinguished from Cardiobacterium and Kingella by the capacity to produce catalase and lack of indole production. Two biotypes were recognized based on the presence (EF-4a) or absence (EF-4b) of arginine hydrolase activity, DNA guanine-cytosine content, cellular fatty acid analysis, and whole-cell protein analysis. Organisms previously recognized as EF-4a are now Neisseria animaloris and EF-4b organisms are Neisseria zoodegmatis. These bacteria are normal inhabitants of the oral cavity of dogs and cats. Most human infections occur after dog bites, although infections associated with cat bites or scratches occur as well. The organism can be isolated from bite wounds that do not demonstrate signs of inflammation, but cellulitis, abscess formation, and fever may develop. Systemic infection or infection not involving skin or skin structures is extremely rare. Endophthalmitis caused by Pasteurella multocida and EF-4 occurred after a cat scratch in an 8-year-old girl. There is one report of bloodstream infection occurring in a patient with hepatic carcinoid who denied being bitten by a dog or cat. An otherwise healthy man whose dogs often licked him in the ears developed chronic EF-4 otitis media that required mastoidectomy.

These bacteria usually appear as short rods on Gram stain but can also appear as small coccoid forms or long chains. The organisms grow well on blood agar and chocolate agar within 24 hours, but grow poorly or not at all on MacConkey and similar agars. Incubation in 5% CO ₂ does not noticeably enhance growth. The colonies are small, may be slightly yellow-orange, and are smooth; some strains have a popcorn-like odor.

Penicillin G and ampicillin are active against these Neisseria species at concentrations attainable with oral administration, so amoxicillin-clavulanate, which is recommended for bite wounds, is adequate for coverage. Fluoroquinolones and tetracycline can also be used as oral therapy. Chloramphenicol and aminoglycosides have activity, whereas cephalosporins, particularly first-generation agents, are less active in vitro.

Plesiomonas shigelloides

Plesiomonas shigelloides, a ubiquitous freshwater inhabitant, has been implicated as a cause of acute diarrhea and, rarely, serious extraintestinal disease. The name Plesiomonas, from the Greek word for “neighbor,” was chosen because the organism was believed to be closely related to Aeromonas. Its classification has been a matter of some debate; it was previously classified in the family Vibrionaceae but is currently classified in the Enterobacteriaceae. P. shigelloides is the only species in the genus. The organism was originally isolated in 1947 and given the name C27. It has also been named Pseudomonas shigelloides, Aeromonas shigelloides, and Fergusonia shigelloides.

P. shigelloides is a water- and soil-associated organism that replicates at temperatures above 8°C. It is found primarily in freshwater or estuary environments within temperate and tropical climates but can exist in seawater during the warm-weather months. Asymptomatic carriage of P. shigelloides is very rare among healthy persons. The usual vehicles of transmission of plesiomonads to humans are water; food such as oysters, shrimp, or chicken ; and a variety of animals that may be colonized with the organism. The organism has been acquired during foreign travel. P. shigelloides is associated with gastroenteritis and has been identified as a cause of outbreaks, but the failure to identify an enteropathogenic mechanism, the lack of an animal model, and unsuccessful attempts to induce disease in volunteers make it impossible to firmly establish a causal relationship. Thus the clinical significance of finding the organism in a diarrheal stool is uncertain. An epidemiologic study in Ecuador found stronger evidence that Plesiomonas diarrhea was associated with rotavirus coinfection than single infection. Potential virulence factors include β-hemolysins, cytotoxins, exoenzymes, and adherence factors, but their significance is unknown.

The clinical presentation of individuals in whom P. shigelloides is isolated from diarrheal stool in the absence of detection of other pathogens varies from a mild self-limited illness to mucoid, bloody diarrhea with fecal leukocytes. A predominance of a secretory-type diarrhea has been noted, but other series have found a high percentage with a clinical illness compatible with enteroinvasive disease featuring abdominal pain, fever, bloody diarrhea, and fecal leukocytes. Most symptomatic patients have either traveled abroad or been exposed to potentially contaminated water or food. Outbreaks have been reported, particularly from Japan. The role of antibiotics for Plesiomonas -associated diarrhea is uncertain. Antimicrobial therapy did not shorten the duration of fever or diarrhea in Thai children with Plesiomonas -associated diarrhea. On the other hand, in a small nonrandomized Canadian study of patients who developed Plesiomonas -associated diarrhea after travel abroad, 8 of 9 treated patients were asymptomatic within 2 weeks, compared with 6 of 15 controls ( P < .05).

Most descriptions of extraintestinal disease come from individual case reports. These reports include cases of osteomyelitis, septic arthritis, endophthalmitis, spontaneous bacterial peritonitis, pancreatic abscess, splenic abscess, biliary disease, cholecystitis, cellulitis, pyosalpinx, epididymo-orchitis, and pneumonia. About 10 cases of neonatal sepsis with meningitis have been described. Bacteremia is rare and usually occurs in immunocompromised hosts. In a case series of Plesiomonas bacteremia from Hong Kong, all seven patients were elderly; four had biliary tract disease, and three had underlying malignancy. Bacteremia accompanying gastroenteritis has been reported in a healthy 15-year-old girl.

P. shigelloides is a motile, facultatively anaerobic, gram-negative, oxidase-positive bacillus. It is readily isolated from some enteric agars such as MacConkey agar but does not grow well on thiosulfate citrate bile salts sucrose medium. Selective techniques may be necessary for isolation of the organism from mixed cultures, such as the use of bile peptone broth or trypticase soy broth with ampicillin. The organism grows well at 35°C and produces visible colonies (nonhemolytic) within 24 hours. The organism can now be identified on gastrointestinal multiplex molecular panels, but its clinical significance is uncertain.

P. shigelloides is usually susceptible to chloramphenicol, trimethoprim-sulfamethoxazole, quinolones, cephalosporins, and imipenem. Because of β-lactamase production, most isolates are now resistant to penicillins, including ureidopenicillins, although the β-lactamase inhibitor combinations appear to be active. Susceptibilities to aminoglycosides and tetracycline are variable. Antimicrobial therapy for established enteric infections is the same as for Shigella and generally includes a fluoroquinolone or azithromycin. Systemic infections have been successfully treated with fluoroquinolones, carbapenems, or β-lactam/inhibitor combinations such as piperacillin-tazobactam.