Campylobacter: Virulence Factors and Pathogenesis

Abstract

The species from the genus Campylobacter are the common causes of foodborne bacterial pathogens found worldwide. The diseases that arise from the infection of this bacterial agent are sometimes self-limiting or can range from mild symptoms to fatal illnesses. The disease is reported in more than 500 million cases of diarrhea annually. The taxonomy, pathogenesis and treatment of Campylobacter is been discussed here. Several virulence factors of Campylobacter are involved in playing a crucial role in pathogenesis, e.g., the chemotactically controlled cellular motility, the bacterial adhesion, the invasion into the host cell, and toxin formation. When a specific diagnosis is made, antibiotic therapy is advocated for use to reduce symptoms. The random use of antibiotics in the treatment of infectious diseases has brought about the emergence of many antibiotic-resistant bacteria, which have become a public health problem and a menace to society.

1. Introduction

Campylobacter was first described in 1913 [1] but was initially classified in the genus Vibrio. With its similarities to the Helicobacter genus, it was finally grouped into the genus Campylobacter (family Campylobacteraceae, order Campylobacterales, class Epsilonproteobacteria, phylum Proteobacteria), which consisted of 33 species and subspecies, showing a broad ecological distribution [2].

Campylobacter is one of the most common causes of bacterial gastroenteritis worldwide [3]. Currently, there are several species within the genus Campylobacter divided into 43 child taxa with a validly published species (http://www.bacterio.net/campylobacter.html) [4] as shown in Table 1. The most common sources of transmission of its infection are contaminated water, raw or contaminated milk, and food especially undercooked meat or meat products [5].

S/No	Campylobacter specie
1	Campylobacter sputorum
2	Campylobacter jejuni
3	Campylobacter fetus
4	Campylobacter coli
5	Campylobacter concisus
6	Campylobacter laridis
7	Campylobacter mucosalis
8	Campylobacter hyointestinalis
9	Campylobacter curvus
10	Campylobacter upsaliensis
11	Campylobacter rectus
12	Campylobacter helveticus
13	Campylobacter showae
14	Campylobacter gracilis
15	Campylobacter lanienae
16	Campylobacter hominis
17	Campylobacter insulaenigrae
18	Campylobacter canadensis
19	Campylobacter peloridis
20	Campylobacter cuniculorum
21	Campylobacter avium
22	Campylobacter ureolyticus
23	Campylobacter subantarcticus
24	Campylobacter volucris
25	Campylobacter corcagiensis
26	Campylobacter iguaniorum
27	Campylobacter hepaticus
28	Campylobacter geochelonis
29	Campylobacter pinnipediorum
30	Campylobacter ornithocola
31	Campylobacter blaseri
32	Campylobacter armoricus
33	Campylobacter novaezeelandiae
34	Campylobacter vulpis
35	Campylobacter taeniopygiae
36	Campylobacter portucalensis
37	Campylobacter massiliensis
38	Campylobacter estrildidarum
39	Campylobacter aviculae
40	Campylobacter bilis
41	Campylobacter anatolicus
42	Campylobacter suis
42	Campylobacter majalis

Table 1.

Currently described Campylobacter species.

http://www.bacterio.net/campylobacter.html.

The taxonomic classification of Campylobacter species has been long characterized by the phenotype of bacterial isolates, where molecular characterization played a minute role in the description of bacteria [6]. There is a drastic change involving all the branches of taxonomy with the advent of DNA sequencing technologies, the standards for the description of Campylobacter species have been updated to a biphasic approach with genotype and phenotype descriptions both being important [7].

DNA-based classifications also have no universal standard and different methodologies (eg. multi-locus sequence typing vs. whole genome sequencing) that allow different resolving capacities to distinguish between strain variants as well as dealing with complex evolutionary phenomena such as recombination in different ways. Nonetheless, taxonomic updates based on DNA sequences are essential and have led to the inclusion of species previously not classified as Campylobacter [8] and the exclusion and reclassification of others [9].

The members of the Campylobacter genus share so many common features. Morphologically, Campylobacter is a slightly curved or spiral, rod-shaped bacteria with single, bipolar, or entire absence flagellum depending on the specie, motile and non-spore-forming, obligate microaerophilic (requires minimum 5% O2 level, Nitrogen 85%, 10% CO2) heat labile, thermophilic, Gram-negative bacteria and exhibits optimum growth at 42 ̊ C [10, 11, 12, 13]. An atmosphere containing increased hydrogen is required by some species for microaerobic growth [10]. Campylobacter species measure approximately 0.2 to 0.8 by 0.5 to 5 m in size and are chemoorganotrophs which acquire their energy sources from amino acids or tricarboxylic acid cycle intermediates [14].

This bacterium takes residence in different places, commonly the gastrointestinal tracts of many animal species where it serves as commensal or pathogenic [15]. Most infections in humans are attributed to Campylobacter jejuni and Campylobacter coli, additional species are isolated from humans too [13]. C. jejuni is the major causative agent of human foodborne gastroenteritis globally resulting in more than 500 million cases of diarrhea annually [16, 17]. In severe cases of infection incriminated by C. jejuni, there is a development of post-infection complications such as Guillain Barré Syndrome [18].

The genome size of C. jejuni and C. coli is approximately 1600 ± 1700 kilobases (kb). This is comparatively small in relation to the enteropathogens such as Escherichia coli with a genome size of approximately 4500 kb [19, 20]. Since the genome of C. jejuni was sequenced, it revealed the presence of hypervariable sequences that consist of homopolymeric tracts [21].

Among the species of Campylobacter, C. jejuni show a high level of variability within many sequences, helping it to adapt to different harsh environments [22]. Within these hypervariable genes, several phenotypic variations and vast diversity within Campylobacter populations occur, especially after transmission from animals or humans. This is shown in different experiments performed in vivo and in vitro, resulting in several genetic changes and mutations that contribute to a high genetic diversity [23, 24]. When samples collected from animals and humans are sequenced and typed, only a few genotypes are similar bringing about strain-dependent pathogenicity and specific colonization ability [25, 26].

2. Pathogenesis

Campylobacter is very infectious as low infective doses of 500 to 800 CFU can cause a problem in humans [27, 28]. Thermotolerant campylobacters, such as C. Jejuni and C. coli, are the most frequent cause of bacterial infection of the lower intestine worldwide [29]. The mechanism of pathogenesis of C. jejuni comprises of four main stages: adhesion to intestinal cells, colonization of the digestive tract, invasion of targeted cells, and toxin production [30].

Several virulence factors of Campylobacter are involved in playing a crucial role in pathogenesis, e.g., the chemotactically controlled cellular motility, the bacterial adhesion, the invasion into the host cell, and toxin formation. In addition to the roles of virulence factors in host colonization, additional factors are involved in successful colonization, such as various genes, antigens, mechanisms of iron utilization, and the response to oxidative and environmental stress. The poor knowledge in understanding, which bacterial and cellular factors are, involved in pathogenicity is not only due to the genetic inter- and intrastrain variability but also to differences between the laboratory strains and the different host cell lines and protocols used in the different laboratories [31]. However, even if the exact mechanism of infection in humans is not yet known, three basic steps can be identified [32].

First, the colonization of the intestinum, especially the crypts of the gut mucosa, a specific adhesion occurs to proteins of the host epithelium, followed by the invasion of the intestinal cells and the translocation of the bacterium, either trans- or paracellularly. At this point, Campylobacter multiplies in the intestinal mucosa releasing toxins that necrotize the intestinal villi. The damage to the intestinal epithelium results in a loss of function, opening of the shielding barrier and the tight junctions, induction of inflammation, release of electrolytes from the systemic compartment of the host to the lumen of the gut, and finally to strong and bloody diarrhea. Furthermore, the adhesion of the bacteria to the epithelial cells is accompanied by a strong pro-inflammatory immune response [33].

2.1 The mechanism of pathogenesis

Humans get infected primarily by contact with live animals or through consumption of contaminated foodstuffs, contact with live poultry, consumption of poultry meat [34, 35], Pork meat [36], beef [37], drinking water from untreated water sources, and raw milk [38].

For adequate attachment to a host, microorganisms require adherence factors which are usually surface appendages such as the pili that are located on the surface of many Gram-negative and Gram-positive species. Genome annotations of several C. jejuni strains do not include obvious pilus or pilus-like open reading frames [21, 38]. A multi-protein type II-like secretion system of a type that is associated with pilus assembly in Vibrio cholerae and Neisseria gonorrhoeae was identified as part of the competence machinery, but an actual pilus-like structure has not been identified [39].

Campylobacter colonization of the host mechanism involves primary intestinal cells – Islets of cobblestone cells. The intestinal mucus attenuates C. jejuniinvasion in-vitro in chicken. A specific avian intestinal factor rather than tissue tropism underlies Campylobacter commensalism in Chickens [40]. Upon infection, Campylobacter elicits the secretion of Interlukin-8, IL-8 and Cytokine. The bacteria adhere preferentially to mucus overlying the intestinal tissue. There is also an interaction of C. jejuni with tissue or mucus via the flagella [41]. Several factors have been identified as influencing the binding of Campylobacter to epithelial cells of the host in-vitro [30].

Other proteins involved in campylobacter virulence and adhesions to the host call are the CadF (Campylobacter adhesin to fibronectin) and Peb1 proteins. Inactivation of the CadF gene seemed to render C. jejuni capable of colonizing the cecum of chicks. Also, CadF protein is required for optimal bacterial adhesion to the extracellular matrix and the colonization of newly hatched Leghorn chicken [42].

CadF binds specifically to fibronectin, which is located basolaterally on epithelial cells in situ [43, 44]. CadF is required for maximal binding and invasion by C. jejuni in vitro, and cadF mutants are greatly reduced in chick colonization compared with the wild type [44].

Another characterized adhesion is the JlpA, a surface exposed lipoprotein that is highly required for HEp-2 cell binding [45]. JlpA binds to Hsp90α, some of which is surface localized in these cells [46]. The process of JlpA binding to Hsp90α activates NF-κB and p38 mitogen-activated protein (MAP) kinase, both of them contribute to proinflammatory responses [46]. This is an indication that some of the inflammatory processes that are observed during the pathogenesis of C. jejuni might be related to JlpA-dependent adherence. Another lipoprotein, CapA, was implicated as a possible adhesion. CapA is an autotransporter that is homologous to an autotransporter adhesin, and CapA-deficient mutants have decreased adherence to Caco-2 cells and decreased colonization and persistence in a chick model [47].

Some putative adhesins of C. jejuni are located in the periplasm such as the Peb1 adhesin, also known as CBF1 required for adherence to HeLa cells [48, 49]. Peb1 shares homology with the periplasmic-binding proteins of amino acid ATP-binding cassette (ABC) transporters [50, 51]. Peb1 binds to both aspartate and glutamate with high affinity, and peb1-deficient mutants cannot grow if these amino acids are the major carbon source [51]. Although Peb1 has not been localized to the inner or outer membrane, some has been observed in culture supernatants [51]. Furthermore, Peb1 contains a predicted signal peptidase II recognition site, a common motif in surface-localized lipoproteins, and so there is a possibility that some Peb1 is surface accessible, despite the failure of fractionation techniques to demonstrate this [50, 51]. Mutants that lack peb1 colonize mice poorly, but this could be attributed to the loss of either the adhesion or the amino-acid-transport functions, or both [49, 51].

The cytotoxic activity in C. jejuni is associated with a cytolethal-distending toxin (CDT) [52]. CDT induces DNA double-strand breaks leading to cell cycle arrest in the G2 phase and provokes cell distension and eventually cell death [52, 53, 54]. CDT also seems to play a role in the invasion and/or survival of C. Jejuni in HeLa cells [55].

The growth temperature of the bacteria significantly affects the ability of C. jejuni to bind to epithelial cell lines in-vitro. Its maximum adhesion to INT-407 is 37°C [56]. Generally, the binding of C. jejuni to cultured cells is not affected by temperature or the Phylogenetic origin of the target cell. The number of bacteria in the inoculum or multiplicity of infection regulates the ability of Campylobacter to invade [57].

The Cytotoxic effect is characterized by remarkable cell distension that is obvious 48 to 72 h after the addition of bacteria-free supernatant and results in cell death. This cell distension is evident in the appearance of HeLa cells, which are star-shaped [53].

Adhesion, invasion, and cytotoxic assay indicate that the ability to invade and induce IL-8 production, to produce CDT, and to resist bile salt is widespread among C. jejuni isolates [58], nevertheless, a higher degree of bile salt resistance and more. Pronounced CDT productions are associated with strains causing enteritis in humans. Furthermore, the CheY appears to be a modulator of C. jejuni virulence.

Molecular studies revealed a high rate of variation of homopolymeric runs – lipooligosaccharides, capsules, or flagellin that are responsible for virulence, are also important for the survival strategy of C. jejuni [21]. The flagellar apparatus is more important for the invasion and translocation of C. jejuni in contact with the host cell and for Chicken gut colonization. The Lipooligosaccharide (LOS) of C. jejuni is highly variable but their structures resemble human neuronal gangliosides. It is thought that this phenomenon results in autoimmune disorders, including Guillain–Barré syndrome (GBS), a paralytic neuropathy that occurs following approximately 1 in every 1000 cases of campylobacterosis, and Miller–Fisher syndrome, a variant of GBS. Much research works have been done to advance the understanding of the mechanism by which C. Jejuni infections result in the conditions above [59, 60, 61, 62].

The presence of variation in the capsule structure observed has been linked to multiple mechanisms that include phase variation of structural genes and an O-methyl phosphoramidate modification [63, 64, 65, 66]. Many strains of C. jejuni are thought to produce both LOS and a high molecular weight lipopolysaccharide (HMW LPS) that is a highly variable capsular polysaccharide. The structural capsules of several C. jejuni strains have been determined including strain 11,168 and strain RM1221 showing their similarities and differences in structure. The former presented with 6-methyl-d-glycero-α-l-glucoheptose, β-d-glucouronic acid modified with 2-amino-2-deoxyglycerol, β-d-GalfNAc and β-d-ribose [64], and contains a novel modification on the GalfNAc [65], the latter having 6-deoxy-d-manno-heptose and d-xylose [67], which are two sugars that are not often detected in bacterial polysaccharides while some strains possess teichoic acid-like or hyaluronic acid-like capsules [68, 69]. The C. jejuni capsule is responsible for serum resistance, the adherence and invasion of epithelial cells, chick colonization and virulence in a ferret model [70, 71, 72].

Other important genes -CiaB (Campylobacter invasion antigen B) is involved in mutagenesis and is required for the secretion process and effective entry of the bacterium into the host cell [73, 74, 75]; while the Cjl121c is essential for host colonization and virulence [76].

Mutation experiments showed that many genes involved in Campylobacter virulence are conserved across species (e.g., CadF, Peb1, jlpA, cdt operon, CiaB, and flagellin genes). Among the same strains of the same origin, there are differences in virulence characteristics [77].

Campylobacteriosis in humans presents symptoms including diarrhea (often bloody), cramping, abdominal pain, nausea, and headaches [78] most commonly caused by the species C. jejuni and C. coli [79, 80]. It has also been tagged as an ‘emerging Campylobacter spp.’ [13], including Campylobacter concisus [81]. Campylobacter sputorum [82], Campylobacter upsaliensis [82], Campylobacter ureolyticus [83] and Campylobacter hyointestinalis [84]. C. jejuni, C. coli, Campylobacter lari, and C. upsaliensis form a genetically close group referred to as the thermotolerant campylobacters because they grow optimally at 42°C [85] and the remaining Campylobacter species are classified into other general groups [86]. The increasing availability of genetic and genomic data on the species’ characteristics and ecological associations due to the improved diagnostic technologies has transformed our perception of the clinical importance of “emerging Campylobacter spp.” [84].

Generally, Campylobacter causes a self-limiting clinical illness that lasts 5 to 7 days; the infection resolves without the use of antimicrobials in the majority of cases but 5–10% of patients relapse after their initial illness [87]. However, the infection can take a much more severe course, especially in infants, elderly people, and immunosuppressed patients (e.g., HIV), so intensified antibiotic treatment is necessary [88].

3. Treatment

The culture-independent diagnostic tests (CIDT) detect the presence of specific antigens or DNA sequences and are recently more used for the detection of bacterial enteric infections such as Campylobacter, Salmonella, Shigella, Shigatoxin–producing E. coli, Vibrio, and Yersinia [89].

Various PCR methods are used for the specific identification of Campylobacter e.g. single locus sequencing of the flagellar flaA and flaB genes and Multilocus sequence typing. Campylobacter-specific genome sections can also be detected by multiplex PCR [90].

Standardized molecular typing methods such as pulsed-field gel electrophoresis [91] and flagellin typing (fla typing) by restriction fragment length polymorphism analysis of a PCR product [92] are in use worldwide.

The random use of antibiotics in the treatment of infectious diseases has brought about the emergence of many antibiotic-resistant bacteria, which have become a public health problem and a menace to society. In recent years, several studies have reported the problem of antibiotic resistance in the various strains of bacteria [93, 94].

In self-limiting infection, no treatment is required but uncomplicated enterocolitis is managed symptomatically with fluid therapy consisting of electrolytes and volume substitution [95, 96, 97].

In cases where a specific diagnosis is made and a severe progression to a fatal illness, immunosuppression, or lack of improvement of symptoms antibiotic therapy with macrolides (azithromycin), fluoroquinolones (ciprofloxacin), and tetracyclines is recommended. Resistance testing should be performed routinely for these cases. The use of cephalosporins should be highly avoided due to high resistance rates. Macrolides, fluoroquinolones and aminoglycosides are classified as critically important antimicrobials, while tetracycline is considered a highly important antimicrobial [98]. Resistance to (fluoro)quinolones and tetracyclines is highly prevalent in Campylobacter spp. isolates, while resistance to erythromycin is typically low to moderate [99, 100, 101].

Macrolides are the first-line antibiotic for the treatment of enteric gastroenteritis, while fluoroquinolones and tetracyclines remain as alternatives [102, 103, 104]. Systemic infections are routinely treated with aminoglycosides [104, 105] with low resistance [100].

Fluoroquinolones act by primarily targeting the DNA gyrase [106]. DNA gyrase is a heterotetrameric type IIA topoisomerase, consisting of two polypeptide subunits (GyrA and GyrB, encoded by gyrA and gyrB, respectively), catalyzing ATP-dependent negative supercoiling of DNA to regulate replication, repair and gene expression [107, 108, 109]. Resistance to (fluoro)quinolones among Campylobacter spp. is largely mediated by chromosomal mutations in the quinolone resistance-determining region (QRDR) of gyrA, typically conferred by the C257T nucleotide mutation (Thr-86-Ile) [110].

The prevalence of Campylobacter gastroenteritis can be significantly reduced by risk-based vaccination, although, there is no commercial vaccine available at the moment, which is also due to the great antigenic diversity of the bacterium. A capsule polysaccharide-based vaccine has proven successful against diarrhea in primates [111]. In order to increase its immunogenicity in humans, it has been coupled to liposomes [112].

4. Conclusion

It is established that Infections caused by Campylobacter are one of the major food-borne causes of gastroenteritis, which can range from mild symptoms to fatal illness worldwide. With the advancement in molecular technology, the epidemiology of some Campylobacter infections remains a mystery.

Care must be taken in severe forms, especially in infants, elderly people, and immunosuppressed patients (e.g., HIV), and must be placed on antibiotic therapy bearing in mind that random use of antibiotics has brought about antibiotic-resistant strains of the bacteria. Further research work is needed to unveil more information.

Conflict of interest

The authors declare no conflict of interest.