Gram‐Negative Anaerobes

Gram‐Negative Anaerobes

John F. Prescott


Gram‐negative anaerobes represent an extremely diverse group of bacteria, so that generalization about their pathogenesis is difficult. Their study has been hindered by their requirement for anaerobic growth conditions, and often for specialized media; indeed, many species have never been successfully cultured. Modern metagenomic analysis of clinical specimens is potentially clarifying (Yip et al. 2021) but sometimes gives challenging discrepancies about the role of different species in animal diseases, for example in the role of mixed aerobic‐anaerobic bacteria in bovine metritis (Jeon and Galvão 2018). Most of the anaerobic species encountered in veterinary clinical practice form part of the normal microbiota found in the mouth and other parts of the intestinal, upper respiratory, and female genitourinary tracts of various animals. In these sites, they are normally present in often dense polymicrobial consortia, for example forming biofilms at the gingival margin. A small number of well‐defined species of these bacteria are recognized as important pathogens of animals; these species primarily belong to the genera Bacteroides, Dichelobacter, Fusobacterium, Porphyromonas, Prevotella and Treponema, and are discussed individually in the chapter. These infections are often associated with the common aerobic opportunist pathogens of different animal species, sometimes with facultative species such as Escherichia coli and frequently with mixtures of anaerobic species (“mixed aerobic‐anaerobic infections”). In vivo, these often polymicrobial mixtures, commonly containing five to eight or more bacterial species, may act synergistically in enhancing disease, particularly in the presence of predisposing conditions such as local trauma. Cooperative mechanisms involved in these synergistic interactions include the supply of energy substrates, provision of essential growth factors, production of a lower redox potential, the additive effect of virulence factors in protection against host defenses or in enhancing host damage, and a combination of these mechanisms.

General Bacterial Pathogenesis Aspects

In the process of establishing infection, and depending on the site involved, pathogenic anaerobic bacteria either need to generate a local anaerobic microenvironment or be able to tolerate oxygen exposure. Anaerobic microenvironments may be created by bacterial damage to the host or because of synergy with facultatively anaerobic bacteria that consume oxygen, or both. Similarly, oxygen tolerance allows anaerobes to survive in infected tissues until conditions are more conducive for their multiplication and invasion. Many pathogenic anaerobes can grow at low oxygen tensions by using superoxide dismutase, catalase or nicotinamide adenine dinucleotide oxidase to protect themselves against the toxic effects of oxygen.

In many cases the mechanisms by which Gram‐negative pathogenic anaerobes cause disease are often ill defined. Until recently, progress has been hampered by difficulties in growing the bacteria, by a lack of genomic information, by the large number of different opportunist pathogens involved in these often polymicrobial infections, and by the inability to genetically manipulate many of the species involved. Tissue destruction may arise from host immune responses and inflammation that are triggered by specific bacterial products causing the local release of a wide variety of inflammatory cytokines, chemokines and other mediators. Fermentation products such as lactic acid, butyric acid, and ammonia, which create the characteristic putrid odor of anaerobic infections, may also have inflammatory and cytotoxic effects.

A summary of common anaerobic infections in selected important animal species is given in Table 35.1. A description of the main pathogenic genera and what is known about their pathogenesis follows. To conserve space, some older key references have not been included but can be found in previous editions of this chapter.

Table 35.1 Common examples of mixed aerobic‐Gram‐negative and anaerobic bacterial infections in selected animal species.

Species Infection Common aerobic pathogen(s) Anaerobes
Cattle Metritis Trueperella pyogenes, Escherichia coli Fusobacterium necrophorum, Porphyromonas melaninogenica

Interdigital necrobacillosis ? F. necrophorum, Porphyromonas melaninogenica

Liver abscesses T. pyogenes F. necrophorum
Cats Cat bite abscesses and infections Pasteurella multocida, Streptococcus spp. Fusobacterium spp., Porphyromonas spp., Bacteroides spp., Prevotella spp., Propionibacterium spp.
Dogs Bite infections Pasteurella canis, other Pasteurella spp., Streptococcus spp., Staphylococcus spp. Fusobacterium spp., Porphyromonas spp., Prevotella spp., Propionibacterium spp., Bacteroides spp.

Periodontal disease Aggregatibacter actimomycetemacomitans, Campylobacter rectus, Eikenella corrodens, Streptococcus spp. Actinomyces spp., Porphyromonas gingivalis, Porphyromonas gulae, Prevotella intermedia, Prevotella nigrans, Tannerella forsythia
Horses Pleuritis, pleuropneumonia Streptococcus equi subsp. zooepidemicus, Pasteurella caballi Fusobacterium equinum, Bacteroides spp., Prevotella spp.
Kangaroos, wallabies Macropod progressive periodontal disease (“lumpy jaw”) Mannheimia spp. (?) Porphyromonas spp., including P. loveana, Fusobacterium spp., Bacteroides spp., Fretibacterium spp., Desulfomicrobium spp.
Sheep Footrot Dichelobacter is aerotolerant Dichelobacter nodosus, F. necrophorum
Swine Injection site abscess T. pyogenes F. necrophorum, Bacteroides spp., Porphyromonas spp.


Characteristics of the Organisms and Pathogenic Species

The genus Fusobacterium are obligate anaerobic, non‐motile, Gram‐negative often spindle‐shaped (fusiform) or long filamentous, bacilli. There are 17 species currently recognized in the genus, including both pathogenic and non‐pathogenic species; all produce large amounts of butyric acid as a fermentation product.

Fusobacterium necrophorum is a major animal pathogen (Nagaraja et al. 2005). Fusobacterium canifelinum, Fusobacterium nucleatum, and Fusobacterium russi are members of the normal oral microbiota of dogs and cats and are frequently isolated from their bite wounds in humans (Citron 2002; Conrads et al. 2004). Fusobacterium equinum, part of the normal oral microbiota of horses, is isolated from oral‐associated and respiratory diseases of horses (Racklyeft and Love 2000). F. nucleatum, which has three subspecies, is also found in the mouth of humans and is an important cause of gingivitis and periodontal disease in humans and is also linked to the severity and progression of colonic cancer (Umana et al. 2019).

Fusobacterium necrophorum

F. necrophorum contains two subspecies, necrophorum and funduliforme, which differ in cellular morphology, colonial characteristics, growth patterns in broth, extracellular enzymes, virulence factors and virulence, as well as in frequency of occurrence in infections (Nagaraja et al. 2005). Subspecies necrophorum, which is more common in animal disease, is more virulent for mice (Zhang et al. 2006) and possesses a haemagglutinin (haem) gene (Narongwanichgarn et al. 2003). F. necrophorum is also a human pathogen, with most isolates from human infections being subsp. funduliforme (Riordan 2007; Tadepalli et al. 2008c).

Source of Infection: Ecology, Evolution and Epidemiology

F. necrophorum is a normal inhabitant of the mouth, gastrointestinal and urogenital tracts of animals, and survives well in soil, especially in well‐manured soil.

Types of Disease and Pathologic Changes

F. necrophorum is a major bovine pathogen, associated with numerous necrotic disease conditions often termed “necrobacillosis.” These occur in many domestic and wild animals and may involve any part of the body (Nagaraja 1998; Nagaraja et al. 2005). It causes a variety of necrotic infections including necrotic stomatitis of calves, lambs, and pigs, footrot in cattle and sheep, lung and liver abscesses in cattle and pigs, and jaw abscesses in wild ruminants and marsupials. Among F. necrophorum infections in cattle, hepatic necrobacillosis (liver abscesses), necrotic laryngitis (calf diphtheria), and interdigital necrobacillosis (footrot) have the most severe economic impacts but mixed aerobic–anaerobic uterine infections involving Trueperella pyogenes and F. necrophorum are also a significant cause of loss.

In humans, it is a common cause of Lemierre syndrome (also called necrobacillosis or post‐anginal sepsis), a severe infection of young adults spreading from the throat by septic thrombophlebitis of the jugular vein (Riordan 2007).

Virulence Factors and Pathogenomics

Several structural features, toxins, and enzymes play a role in the pathogenesis of F. necrophorum infections. Hemagglutinin, endotoxic lipopolysaccharides (LPS), and leukotoxin are proven virulence factors that participate in the pathogenesis of fusobacterial infections (Table 35.2). Suggested more minor virulence‐associated factors include hemolysin, polysaccharide capsule, adhesins or pili, platelet aggregation factor, dermonecrotic toxin, and extracellular enzymes including proteases, lipases and DNases. A 42 kDa outer‐membrane protein (OMP) may be involved in adhesion to cells (Menon et al. 2018). As discussed below, a role in virulence for type Vc secretion system (T5cSS) trimeric autotransporter adhesins has been suggested (Umana et al. 2019).

Table 35.2 Major virulence factors of Fusobacterium necrophorum.

Factor Characteristics Mechanism of action Role in pathogenesis
Haemagglutinin Outer membrane protein (19 kDa) Agglutinates erythrocytes of various animals Mediates attachment to rumen epithelial cells and hepatocytes
Leukotoxin Extracellular protein (molecular weight 336 kDa) Cytotoxic to neutrophils, macrophages, hepatocytes, and rumen epithelial cells Protects against phagocytosis by neutrophils and Kupffer cells, damages hepatic parenchyma by the release of cytolytic products
Endotoxin Cell‐wall (lipid A) component of lipopolysaccharide Cytotoxic and necrotic effects and induces disseminated intravascular coagulation. Creates anaerobic microenvironment conducive for anaerobic growth

Hemagglutinin is a low molecular weight OMP (19 kDa) that may play a significant role in adherence to epithelial cells, an initial step in pathogenesis (Kanoe et al. 1998; Narongwanichgarn et al. 2003). The concentration and chemical composition of endotoxic LPS varies depending on the subspecies, with the biological activity of LPS of subspecies necrophorum greater than that of subsp. funduliforme (Garcia et al. 1999).

Leukotoxin is a secreted protein that is toxic to ruminant leukocytes, macrophages, hepatocytes, and possibly rumen epithelial cells (Narayanan et al. 2002; Nagaraja et al. 2005). Bovine neutrophils are more susceptible to toxin than peripheral blood mononuclear cells, whereas leukotoxin is less toxic to horse neutrophils and is non‐toxic to swine and rabbit neutrophils. This specificity may be a function of the presence of high‐affinity receptors, or increased numbers of receptors on the surface of ruminant leukocytes. At high concentrations leukotoxin causes primary cell necrosis.

Leukotoxin mediates several potentially important pathogenic mechanisms, including modulation of the host immune system by its toxicity, cellular activation of leukocytes, and apoptosis‐mediated killing of phagocytes and immune effector cells (Narayanan et al. 2002). The importance of leukotoxin as a virulence factor in F. necrophorum infections is indicated by a correlation between toxin production and ability to induce abscesses in laboratory animals, an inability of leukotoxin‐negative strains to induce foot abscesses in cattle following intradermal inoculation, and a relationship between anti‐leukotoxin antibody titers and protection against infection in experimental challenge studies (Nagaraja et al. 2005). Carriage of the leukotoxin structural gene lktA is almost universal in invasive bovine strains of F. necrophorum, although it is less common in non‐bovine invasive animal and human strains (Ludlam et al. 2009).

The leukotoxin operon consists of three genes, lktB, lktA, and lktC. The 335 kDa LktA protein is considerably larger than leukotoxins produced by other bacteria (Narayanan et al. 2001). The protein may represent a new class of bacterial leukotoxins since it has no sequence similarity to other leukotoxins and is also unusual in that it lacks cysteine. The 62 kDa LktB protein has sequence similarity to HxuB, the heme–hemopexin utilization protein of Haemophilus influenzae. The LktB protein contains a putative polypeptide‐transport‐associated domain, suggesting that it is involved in the secretion of the LktA leukotoxin. The function of the LktC protein is currently unknown. The subspecies funduliforme lkt operon is organized identically to the subsp. necrophorum operon. Although the overall sequence similarity of the Lkt proteins is high between the two subspecies, the LktB and LktA proteins have significant differences in their N‐terminal sequences (Tadepalli et al. 2008b). The toxic activity of the LktA protein when expressed in E. coli indicates that the other proteins encoded in the leukotoxin operon are not required to produce biologically active toxin. They likely play a role in secretion of the toxin across the cytoplasmic and outer membranes of F. necrophorum.

All three lkt genes appear to be co‐transcribed from a promoter located in the intergenic region upstream of the lktB gene (Zhang et al. 2006). The sequence and the size of the intergenic region differs in the two subspecies, being 548 bp in subsp. necrophorum and 337 bp in subsp. funduliforme. The subsp. funduliforme promoter activity is weaker than that of subsp. necrophorum (Tadepalli et al. 2008b) and the differences in toxicity may relate to enhanced transcription in subsp. Fusobacterium.

Bioinformatic analyses indicate that Fusobacterium species may be unique among pathogens in possessing only the major protein type V secretion system (T5SS) and lacking types I–IV and VI (Umana et al. 2019). The T5SS system is further uniquely divided into five distinct categories (T5aSS–T5eSS) of autotransporters, with the majority of those characterized, including the F. necrophorum system, having adhesin properties. F. necrophorum, however, lacks the T5SS‐secreted FadA protein family and most strains lack the otherwise numerous surface‐associated proteins with MORN (membrane occupation and recognition nexus) 2 domains associated with the adhesiveness and invasiveness of other fusobacteria, including F. nucleatum (Manson McGuire et al. 2014; Umana et al. 2019).


Although the general process is clear, a detailed understanding of the molecular basis of F. necrophorum pathogenesis is still lacking. The basic processes of the pathogenesis of bovine liver abscessation is illustrated in Figure 35.1.

In respect to host association, F. necrophorum lacks the genes associated with the actively invasive characteristics of species such as F. nucleatum and has been characterized as a “passively invading” Fusobacterium species (Manson McGuire et al. 2014; Umana et al. 2019). Although the organism can attach to different cell lines, possibly through the action of hemagglutin and a 42 kDa OMP, the precise mechanisms have not been fully determined. Bioinformatic analysis supports earlier understanding that F. necrophorum infection in animals generally requires local epithelial damage of some type. For example, liver abscesses in cattle occur secondary to infection of the rumen wall (the “rumenitis–liver abscess complex”) as a result of rapid fermentation of grain by rumen microbes and the consequent accumulation of organic acids resulting in rumen acidosis (Nagaraja and Lechtenberg 2007). This acid‐induced rumenitis, sometimes together with damage to the protective surface by coarse objects (e.g. sharp feed particles, hair), predispose the damaged rumen wall to colonization by F. necrophorum. The organism then causes local infection and small rumen wall abscesses. Bacterial emboli are subsequently shed into the portal circulation where they are filtered by the liver, resulting in infection and abscess formation.

Evasion of host defenses is primarily by leukotoxin‐mediated cytotoxicity, preventing neutrophil phagocytosis and clearance of organisms from the rumen wall and liver. In addition, neutrophil death likely leads to parenchymal cell damage and abscess formation, thus maintaining an anaerobic environment in the aerobic hepatic tissue. A decrease in the local neutrophil response due to leukotoxin‐mediated death of neutrophils could also enhance colonization by the opportunistic pathogens with which this organism is very commonly associated, such as T. pyogenes in the rumen wall, liver, and feet, and Prevotella melaninogenica in the feet (Nagaraja 1998; Amachawadi et al. 2017). Host damage is likely a result of both phagocyte destruction and of LPS‐induced release of inflammatory mediators. Metagenomic analysis of cattle liver abscesses suggests an additional opportunistic role for Pseudomonas species, among other genera (Amachawadi et al. 2021).

Schematic illustration of basic pathogenesis of liver abscessation of cattle caused by Fusobacterium necrophorum.

Figure 35.1 Basic pathogenesis of liver abscessation of cattle caused by Fusobacterium necrophorum. Many details of the pathogenesis of this and other important diseases caused by this organism remain to be determined. LPS, lipopolysaccharide; OMP, outer‐membrane protein; T5cSS, type Vc secretion system.

Calf diphtheria is a necrotic laryngitis occurring in cattle up to three years of age characterized by necrosis of the mucosa and underlying tissues of the larynx and adjacent structures. The organism is normally present in the upper respiratory tract, but does not penetrate healthy mucosa; hence, a breach in the mucosal integrity is required for the onset of infection. The infection can be acute or chronic and is non‐contagious. In severe cases, cattle may die from aspiration pneumonia.

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Nov 13, 2022 | Posted by in GENERAL | Comments Off on Gram‐Negative Anaerobes

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