Isolation and Identification of Campylobacter spp. from Food and Food-Related Environment

Abstract

Campylobacter species are among the most common causes of bacterial gastroenteritis in humans worldwide. The genus Campylobacter consists of at least 39 validly published species with wide distribution in various hosts and environments, which are either pathogens for humans or animals, or not pathogenic as identified so far. Various methods have been used for detecting campylobacters including conventional culture methods, molecular (such as polymerase chain reaction), immunological methods and genome sequencing. Currently, isolation and subsequent identification of the target campylobacters are required by most of the regulatory bodies globally. The multiple Campylobacter species exhibit diverse physiological and metabolic characteristics and growth requirements, which can interfere with the sensitivity and specificity of culture-dependent methods. Furthermore, strains among each species may behavior differently in various culture media and under various culture conditions. Therefore, it is important to apply appropriate isolation and identification methods for different types of species and samples based on specific purposes. This chapter will review the development and the current status of culture-dependent methods for the isolation and detection of various Campylobacter species from food and food-related environments during the next generation sequencing era.

1. Introduction

1.1 Organisms and brief history

For the last three decades, Campylobacter species have been the focus of growing attention because of the increasing frequency with which they have been isolated from various sources including man, animals, food and water [1]. Campylobacter species are among the most common causes of bacterial gastroenteritis in human worldwide [2, 3, 4], which account annually for approximately 166 million foodborne illnesses around the world [5].

The name “Campylobacter” is an ancient Greek word meaning “curved rod”. Campylobacter species are Gram-negative, spiral, rod-shaped, or curved bacteria with a single polar flagellum, bipolar flagella, or no flagellum depending on the species, non-spore-forming, and approximately 0.2 to 0.8 by 0.5 to 5 μm [6, 7, 8]. The typical shape of Campylobacter looks more like a spiral or helical one rather than a curved rod shape, which can change its shape into filamentous or coccoid to adapt to the stressful conditions [9, 10]. When two or more bacterial cells are grouped together, an “S” or a “V” gull-wing shape is formed [11]. The majority of Campylobacter species have a characteristic corkscrew-like motion due to a single polar flagellum at one or both ends of the cell [11], with the exceptions that C. gracilis is non-motile and C. showae contains multiple flagella [12].

Escherich observed the Campylobacter-like non-culturable spiral-shaped organism in infants’ stool samples in 1886 [1, 13]. These bacteria, called related Vibrio by then, were first isolated from the uterine mucus of a pregnant sheep from a flock of 150 ewes that were experiencing an abortion rate of 33% in 1906 by McFadyean and Stockmanin in the United Kingdom [14]. A few years later, an apparently identical organism, firstly named as a spirillum and then as Vibrio fetus, was isolated from the fetal membranes of aborting cattle by Smith and Taylor in the United States [15, 16]. In 1949, Stegenga and Terpstra demonstrated the pathogenic role of V. fetus venerealis in enzootic sterility in cows [1, 17]. In 1931, winter dysentery in calves was attributed to infection with a “vibrio” that they called Vibrio jejuni [18], and a similar organism associated with swine dysentery was identified by Doyle [19].

Campylobacter-like organisms, were first isolated from humans in 1938 from the blood of patients suffering from diarrhea in a milk-borne outbreak affecting 355 people in the United States, and the causative bacterium for this outbreak was named “V. jejuni” [20]. This has been regarded as the first well-documented instance of human Campylobacter infection [1]. Subsequently, V. fetus was isolated from the blood of three pregnant women admitted to hospital because of fever of unknown origin in 1947 [21], and King described the isolation of “related Vibrio” from blood samples of children with diarrhea in 1957 [22]. Up to 1972, only 12 cases of ‘related vibrio’infections were reported in the literature [1]. The reason for these small numbers of the reports was that the optimal or specific selective culture techniques for the isolation of ‘related vibrio’, later called as Campylobacter [23, 24], from feces were not developed at that time.

The first successful isolation of Campylobacter from feces or stool samples of patients with diarrhea was accomplished in Belgium using the technique of direct membrane filtration onto agar medium containing several antibiotics in 1968 and published in 1972 [25]. This study also found that C. jejuni was highly susceptible to erythromycin [26]. Consequently, in 1977, a selective culture procedure was recommended using selective medium containing antibiotics with incubation at 43°C in a microaerobic atmosphere (5% oxygen, 10% carbon dioxide, and 85% hydrogen) [27]. This facilitated the isolation of campylobacters, C. jejuni and C. coli (the two species known at the time to cause gastroenteritis), with greater ease [27]. The successful isolation of campylobacters from human feces based on the above selective media led to the recognition that Campylobacter is a leading cause of human diarrheal illness in many countries [5, 28].

Since the establishment of genus Campylobacter in 1963 with Campylobacter fetus as the species type [23], the taxonomy of the family Campylobacteraceae has transformed extensively [12]. The genus Campylobacter belongs to the family Campylobacteraceae proposed in 1991, the order Campylobacterales, the class Epsilonproteobacteria, and the phylum Proteobacteria. The class Epsilonproteobacteria presently comprises four closely related genera, Campylobacter, Arcobacter, Dehalospirillum and Sulfurospirillum [http://www.bacterio.net/index.html]. By 2021, the genus Campylobacter consists of 39 validly published species, 11 subspecies and 4 biovars [https://lpsn.dsmz.de/genus/campylobacter] (Table 1).

Taxon	Host	Disease		Foodborne	References
		In human	In animal
C. armoricus	Surface water, human stool	humans displaying enteric infection	Unknown		[29]
C. aviculae	Lab Zebra Finches	Unknown	Unknown		[30]
C. avium	Poultry	Unknown	Unknown	Yes	[31, 32]!!!
C. blaseri sp. nov.	Common seals	Unknown	Unknown
C. canadensis	Whooping cranes	Unknown	Unknown	Yes
C. coli	Pigs, poultry, ostriches, cattle, sheep, penguin	Gastroenteritis meningitis, acute cholecystitis	Gastroenteritis, infectious Hepatitis	Yes
C. concisus	Humans, domestic pets	Gastroenteritis, periodontitis, IBD	Unknown	Yes
C. corcagiensis	lion-tailed macaques	Unknown
C. cuniculorum	Rabbits	Unknown	Unknown
C. curvus	Humans	Periodontitis, gastroenteritis	Unknown
C. estrildidarum	Lab Zebra Finches	Unknown	Unknown		[30]
C. fetus subsp. fetus	Cattle, sheep, reptiles	Gastroenteritis, septicemia	Spontaneous abortion
C. fetus subsp. venerealis	Cattle, sheep	Septicemia	Infectious infertility
C. fetus subsp. venerealis bv. intermedius	Cattle	Unknown	Genital campylobacteriosis
C. fetus subsp. testudinum	Human, reptile	Unknown	Unknown
C. geochelonis sp. nov	Western Hermann’s tortoise	Not known	Unknown
C gracilis	Humans	Periodontitis	Unknown
C. helveticus	Dogs, cats	Periodontitis	Gastroenteritis
C. hepaticus sp. nov	Poultry	Unknown	Sporty liver disease		[33]
C. hominis	Humans	Unknown	Unknown
C. hyointestinalis subsp. hyointestinalis	Cattle, deer, pigs, hamsters	Gastroenteritis	Gastroenteritis	Yes
C. hyointestinalis subsp. lawsonii	Pigs	Unknown	Unknown	Yes
C. insulaenigrae	Seals, porpoises	Unknown	Unknown	Yes
C. iguaniorum sp. nov	Reptile (lizards)	Unknown	Unknown
C. jejuni subsp. doylei	Humans	Septicemia, gastroenteritis	Unknown	Yes
C. jejuni subsp. jejuni	Poultry, cattle, pigs, ostriches, wild birds, penguin	Gastroenteritis, Guillain-Barré syndrome	Spontaneous abortion, avian hepatitis	Yes
C. lanienae	Cattle	Unknown	Unknown	Yes
C. lari subsp. concheus	Shellfish	Gastroenteritis	Unknown	Yes
C. lari subsp. lari	Wild birds, dogs, poultry, shellfish, horses	Gastroenteritis, septicemia	Avian gastroenteritis	Yes
C. lari subsp. ornithocola	Wild bird	Unknown
C. mucosalis	Pigs	Unknown	Unknown	Yes
C. novaezeelandiae sp. nov.	Birds and water	Unknown	Unknown
C. peloridis	Shellfish	Unknown	Unknown	Yes
C. portucalensis	Preputial mucosa of bulls	Unknown	Unknown		[34]
C. rectus	Humans	Periodontitis	Unknown
C. showae	Humans	Periodontitis	Unknown
C. sputorum bv. sputorum	Humans, cattle, pigs, sheep	Gastroenteritis, abscesses	Spontaneous abortion	Yes
C. sputorum bv. fecalis	Sheep, cattle	Unknown	Unknown
C. sputorum bv. paraureolyticus	Human, cattle	Unknown	Unknown
C. subantarcticus	Birds in the subantarctic	Unknown	Unknown	Yes
C. taeniopygiae	Lab zebra finch	Unknown	Unknown		[30]
C. troglodytes sp. nov	Wild chimpanzees	Unknown	Unknown
C. upsaliensis	Dogs, cats	Gastroenteritis	Gastroenteritis
C. ureolyticus	Humans	Gastroenteritis, Crohn’s disease	Unknown
C. volucris	Black-headed gulls	Unknown	Unknown
C. vulpis	wild red foxes fox	Unknown	Unknown

Table 1.

List of validly published! And newly proposed!! Species, subspecies and biovars in the genus campylobacter and their common hosts and disease associations in humans and animals.

!: Valid species (39 species, 11 subspecies (subsp), and 4 biovars (bv)) as included on the website: https://lpsn.dsmz.de/genus/campylobacter

!!: Indicated as “sp. nov” in the table.

!!!: All the reference is referred to [31] and [32], except those indicated differently.

In addition, species within the Campylobacter genus can be grouped according to association with different host environments (e.g. animal intestinal tracts and human oral cavity) [35] and propensity to cause disease in animal and human hosts [13, 36] and (Table 1). Zoonotic Campylobacter species are commensal organisms found in the intestinal tract of a variety of mammals, birds and reptiles as well as in related environments, including water and soil [8, 11, 13, 37]. Among the zoonotic species, C. jejuni is responsible for approximately 81% of human gastrointestinal-related Campylobacter infections with C. coli, C. fetus, C. lari and C. upsaliensis being responsible for 8.4%, 0.2%, 0.1% and 0.09%, respectively. The remainder of human Campylobacter infections are from other species or undifferentiated “campylobacters” [38]. Other zoonotic species including C. fetus, C. hyointestinalis, C. upsaliensis, C. sputorum, C. concisus and C. ureolyticus have also been recognized as causal agents of human gastroenteritis [2, 37]. Of these, C. concisus, C. upsaliensis and C. ureolyticus are considered as emerging or under-recognized disease-associated species, where due to the advances in molecular biology and culture methodologies these species are becoming increasingly recognized as pathogens [7, 11]. In addition to causing gastroenteritis in humans, a number of Campylobacter species are potential oral pathogens and have been commonly isolated from the human oral cavity. These include C. concisus, C. showae, C. gracilis, C. curvus, C. rectus, and C. ureolyticus [35]. While campylobacters do not typically cause disease in animals, C. fetus subspecies fetus and C. fetus subspecies venerealis are important causes of reproductive system disorders and abortions in ruminants, and particularly C. fetus subsp. venerealis is restricted to cattle and causes bovine genital campylobacteriosis, and C. hepaticus causes Spotty Liver Disease in layer chickens [13, 33, 36] (Table 1). Based on the route of transmission to humans, campylobacters can also be classified as foodborne or non-foodborne groups (Table 1) [31]. This chapter will focus on the foodborne campylobacters potentially from food and food-related environments.

King [22] observed that the incubation at 42°C enhanced the growth of campylobacters, which led to the concept of “thermophilic” campylobacters. Traditionally, based on the tolerance to the temperature 42°C - 43°C (able to grow at this temperature), campylobacters are also referred as “thermophilic” and “non-thermophilic” groups, with C. jejuni, C. coli, C. lari and C. upsaliensis as the four human pathogenic species of Campylobacter and are often referred to as the thermophilic Campylobacter species. However, these species do not exhibit true thermophily (growth at 55°C or above) [39]. For example, the D value (the time it takes to reduce a microbial population by 1 logarithm) at 50°C for C. jejuni strains in skimmed milk was demonstrated to be between 1.3–4.5 min, and inoculation of a heat tolerant strain of C. jejuni into roast beef at a level of $5.9\times {10}^6$ /g resulted in no survivors by the time the internal temperature had risen to 55°C [40]. Similarly, a low D value between 0.7–1.4 at 60°C for C. jejuni and C. coli strains was found [41]. Therefore, it was suggested that the organisms which could grow at 41–43°C, should be referred to as “thermotolerant” Campylobacter species [42]. Therefore, the term “thermotolerant” is used in this chapter.

1.2 Distribution and diseases

Campylobacter species are commensal microorganisms of the gastrointestinal tract of many wild animals (birds such as ducks and gulls), farm animals (cattle and pigs), and companion animals (such as dogs and cats) (Table 1) [8, 11, 13]. The organisms can also be found in the internal organs of animals [43, 44]. The main route of transmission to humans is generally believed to be foodborne, via undercooked meat and meat products, particularly the poultry products, as well as raw or contaminated milk [11, 13]. Despite it is well-known fastidious nature, Campylobacter is also isolated from environmental sources, such as lake, river, soil, sea, and sewage, suggesting that environmental water is a possible vehicle that transmits Campylobacter to humans [8]. Different species of the Campylobacter genus naturally colonize a wide range of hosts (including pets, farm animals and wild animals) and are frequently detected in contaminated food products, indicating that these organisms are potentially transmissible to humans [7].

Many Campylobacter species are known pathogens in either humans or animals, or both [7] (Table 1). The main disease in humans caused by campylobacters is gastroenteritis worldwide, which is mainly due to C. jejuni [2, 7] and accounts annually for approximately 166 million foodborne illnesses around the world [5]. Meanwhile, C. jejuni infection may lead to autoimmune conditions known as Guillain-Barré syndrome (GBS) and Miller Fisher syndrome. Campylobacter species have also been associated with a range of gastrointestinal conditions, including inflammatory bowel diseases (IBD), Barrett’s esophagus, and colorectal cancer [7]. In addition, they have been reported to be involved in extragastrointestinal manifestations, including bacteremia, lung infections, brain abscesses, meningitis, and reactive arthritis, in individual cases and small cohorts of patients [2, 7]. The precise role of Campylobacter species in the development of these clinical conditions is largely unknown. A growing number of Campylobacter species other than C. jejuni and C. coli have been recognized as emerging human and animal pathogens. The development of new molecular and innovative culture methodologies enhanced the detection and isolation of a range of under-recognized or emerging, and nutritionally fastidious Campylobacter species, including C. concisus, C. upsaliensis and C. ureolyticus. It has been found that these emerging Campylobacter species are associated with a range of gastrointestinal diseases, particularly gastroenteritis, IBD and periodontitis. Some cases of the gastrointestinal tract infection by these bacteria can lead to life-threatening extragastrointestinal diseases [2, 7]. Based on the high number of recent Campylobacter infections reported in the region, campylobacteriosis ranks as the third cause of death behind listeriosis and salmonellosis in the EU [38].

1.3 Objectives

Various methods have been used for detecting campylobacters including conventional culture methods, molecular (such as polymerase chain reaction or PCR) and immunological methods, and genome sequencing analysis. Currently, isolation and subsequent identification of the target campylobacters are required by most of the regulatory bodies globally. The multiple species of Campylobacter exhibit diverse physiological and metabolic characteristics, and growth requirements, which can interfere with the sensitivity and specificity of culture-dependent methods. Furthermore, strains among each species may behave differently in various culture media and under various culture conditions. Therefore, it is important to apply appropriate isolation and identification methods for different types of species and samples based on specific purposes. The subsequent detection and characterization can also be challenging for comprehensive and accurate identification, particularly for source attribution and epidemiological investigations. This chapter will firstly reviews the physiology of the organism in order to better understand the isolation procedure, followed by a review of the culture-dependent detection methods in combination with technologies newly developed for various Campylobacter species from food and food-related environment that may contaminate the food chain.

2. Physiology of campylobacters and growth requirements of different species

It is important to understand the unusual physiology of campylobacters in vivo and in vitro for the development of optimal culture procedures in vitro. A review by Park [45] indicated that compared with most foodborne bacterial pathogens which are considered to have stronger ability to survive the harmful conditions imposed by food processing and preservation, Campylobacter species require uniquely fastidious growth conditions and are usually more sensitive to environmental stress. It is generally believed that campylobacters may also lack many of the well-characterized adaptive responses that support the resistance to stress in other bacteria [45]. These factors may lead to the difficulty to recover the campylobacters from food and food-related environment. However, several reports indicated that campylobacters have peculiar mechanisms such as their inherent genome plasticity and gene regulators that respond to changing environments which enhance their survival in hostile conditions [46, 47, 48].

2.1 Microaerobic requirement

As microaerophilic and capnophilic microorganisms, Campylobacter species generally require a microaerophilic atmosphere with reduced oxygen (approximately 5–10% O₂) and elevated carbon dioxide (5–10% CO₂) concentrations for its optimal growth in vitro [2, 11]. They have a respiratory type of metabolism. Several Campylobacter species including C. concisus, C. curvus, C. gracilis, C. mucosalis, C. rectus, C. showae and some strains of C. hyointestinalis require extra hydrogen (3–7% H₂) or formate acting as an electron donor for microaerobic growth and successful recovery. Although most of the Campylobacter species require microaerobic conditions for growth, however certain species such as C. concisus can grow under or prefer anaerobic conditions for growth [2, 49]. There are also some strains being aerotolerant [50, 51, 52].

2.2 Optimal growth and survival temperature, pH and water content

The minimum, optimum and maximum growth temperatures (°C) of campylobacters are 32, 42–43 and 45 respectively, and minimum, optimum and maximum growth pH values are pH 4.9, 6.5–7.5 and 9.5 [11, 45, 53]. Campylobacter species will not survive below a pH of 4.9 and above pH 9.0 and grow optimally at pH 6.5–7.5 [11]. The organisms will grow with water activity (a_w) at 0.987 and 0.997 [11, 45, 53]. Growth does not occur in environments with aw lower than 0.987 (sensitive to concentrations of sodium chloride (NaCl) greater than 2%w/v), while optimal growth occurs at $a_{\mathrm{w}}=0.997$ (approximately 0.5% w/v NaCl) [11].

Generally, some Campylobacter species (e.g. C. jejuni, C. coli, C. lari, C. upsaliensis, C. helveticus and C. insulaenigrae) are referred to thermotolerant with an optimal growth temperature of 37–42°C and a maximum temperature of ∼46°C. The remaining Campylobacter species are considered not thermotolerant in the literature, with an optimal growth temperature of 37°C [54, 55]. However, based on the official publications for the validated species and subspecies in Campylobacter genus classification up to date (https://lpsn.dsmz.de/genus/campylobacter; accessed on 2021-12-21), among 39 validly published species, 28 species with 2 subspecies are indicated as thermotolerant (able to grow at 42°C) with the majority of the strains (90–100% strains) able to grow at 42°C [Table 1 with relevant references]. These thermotolerant species/subspecies include C. armoricus, C. aviculae sp. nov., C. avium, C. blaseri, C. canadensis, C. coli, C. corcagiensis, C. estrildidarum sp. nov., C. helveticus, C. hepaticus, C. hyointestinalis subsp. hyointestinalis, C. hyointestinalis subsp. lawsonii, C. jejuni subsp. jejuni, C. lanienae, C. lari subsp. concheus, C. lari subsp. lari, C. mucosalis, C. novaezeelandiae, C. ornithocola, C. peloridis, C. portucalensis, C. sputorum, C. subantarcticus, C. taeniopygiae sp. nov., C. upsaliensis, C. volucris and C. vulpis. Seven species with three subspecies are non-thermotolerant or poorly thermotolerant (only 0–10% strains thermotolerant), including C. fetus subsp. venerealis, C. geochelonis, C. iguaniorum, C. insulaenigrae, C. jejuni subsp. doylei, C. pinnipediorum subsp. caledonicus and C. pinnipediorum subsp. pinnipediorum. Others are partially thermotolerant (26–89% strains), which can be divided into three groups, namely, (−) (11–25% strains are thermotolerant) including C. hominis and C. rectus; (V) (26–74%) including C. curvus, C. fetus subsp. testudinum, C. gracilis, C. showae and C. ureolyticus; and (+) (75–89%) including C. concisus, C. cuniculorum and C. fetus subsp. fetus. It is worthwhile to note that there are different degrees of tolerance to growth temperatures among the strains of the same species, which should be taken into consideration when the incubation temperatures are chosen for the diagnostic testing.

Generally speaking, campylobacters are unable to grow below 30°C [45, 56]. It was suggested that the absence of cold-shock proteins might be responsible for the inability of this pathogen to grow at lower temperatures [57]. However, although not growing, C. jejuni was found to survive for more than 4 h at 27°C and 60–62% relative humidity on food contact surfaces [58]. These physiological characteristics reduce the ability of campylobacters to multiply outside of an animal host, and in food during their processing and long term storage [45]. Due to the fluctuation of body temperatures in reptiles, Campylobacter species in reptiles have adapted to larger temperature ranges and are more tolerant to lower temperatures than those found in mammals and birds. For example, the proposed Campylobacter geochelonis sp. nov., isolated from western Hermann’s tortoise grows at 25°C and not at 42°C [54].

In pure cultures, Campylobacter spp. are normally inactivated by frozen storage at −15°C in as few as 3 days [59], although freezing does not eliminate the pathogen from contaminated foods [60]. Hazeleger [57] revealed that aged C. jejuni cells survived the longest at 4°C. Under the cold and other stress conditions, campylobacters may enter viable but non-culturable (VBNC) state [11]. The campylobacters at VBNC state may affect the sensitivity of culture-dependent detection procedures.

2.2.1 Antibiotics resistance

Most campylobacters are resistant to a few antibiotics including amphotericin B, cefoperazone, colistin, cycloheximide, polymyxin B, rifampin and trimethoprim at different concentrations, which have been used as supplements in selective media [61]. In addition, C. jejuni, C. coli, C hyointestinalis and C, fetus, but not C. upsaliensis, have also been shown to be resistant to Aztreonam [62]. There is evidence that some strains of C. coli and even a few strains of C. jejuni are likely to have been missed due to their sensitivity to cephalothin. Several species, including C. hyointestinalis, C. upsaliensis and C. fetus are inhibited by the high amount of cefoperazone contained in the selective medium [61, 63, 64]. Generally speaking, C. upsaliensis, a commonly believed to be an important human pathogen, is sensitive to the antibiotics routinely used in Campylobacter selective media. The recovery of this organism will rely on the development of widely applicable, effective techniques for its isolation, such as membrane filtration based techniques [63]. The presence of these antibiotics in the selective media generally used for the isolation of Campylobacter species (e.g., Skirrow’s medium) may well lead to the suboptimal identification of C. upsaliensis in clinical specimens at most centers. It has been shown that the procedure (Cape Town Protocol) using membrane filtration and antibiotic-free agar isolate more Campylobacter species than the agar alone with antibiotics [65]. Therefore, the usage of antibiotics must be carefully selected for the culture media to avoid the false negative results.

2.2.2 Essential nutrient requirements for growth in vitro

Campylobacters obtain their energy sources from amino acids or tricarboxylic acid cycle intermediates [6, 66].Campylobacter is generally considered a non-saccharolytic bacterium because of inability to ferment or use glucose and other carbohydrates as growth substrates, which has been supported by genome sequence analysis [67, 68, 69] and recent growth-independent phenotype microarray analyses that allows monitoring the respiratory activity of metabolically active cells [70]. Furhter studies confirmed that pentoses and hexoses like glucose, fructose, galactose, rhamnose and the disaccharides lactose, maltose, trehalose and sucrose do not enhance the respiratory activity of C. jejuni [66, 71, 72, 73]. The Campylobacter culture media contains mainly the peptamin or peptone that provides amino acids (source of carbon), sulfide, and nitrogen required for making their energies, yeast extract provides B vitamins (coenzymes), and sodium chloride to maintain osmotic equilibrium [74]. Campylobacter enrichment broth must contain sources of iron, such as blood, hemin, and ferrous sulfate [61].

2.2.3 Considerations for developing a new media or culture procedure or strategies

Taken together, the studies of metabolic activities have been mainly focused on C. jejuni, which showed an intriguing metabolic diversity among different strains. The diverse growth properties of C. jejuni isolates result from the presence or absence of various metabolic genes involved in the strain-specific utilization of particular substrates such as fucose, asparagine or glutamine and peptides. In addition, C. jejuni isolates contain different sets of group A chemoreceptor tlp genes that respond differently to potential nutrients [75, 76]. The presence of various chemosensory receptor genes in C. jejuni suggests that different strains may not respond equivalently to certain nutrients and consequently cannot utilize and benefit from the same growth substrates. These may affect the isolation of all the strains from the same Campylobacter species using the same medium.

3. Isolation and detection

Traditionally, campylobacters have been detected using culture-dependent procedures followed by the identification, confirmation and typing of the isolates using biochemical, immunological and molecular methods [11, 13, 77]. Cultural isolation remains the gold standard for confirming the presence of live bacteria in a sample. Molecular and immunological methods, particularly PCR [13], and recently the whole genome sequencing tools [78, 79, 80, 81, 82] have also been used for detection of Campylobacter species in different sources. The following will review the methods of culture, identification and confirmation, and, characterization of the isolates in detail.

3.1 Culture methods

3.1.1 History of methods development

Campylobacter-like organisms were first isolated from the blood of humans in 1938 using tryptose phosphate beef broth or brain broth at 37°C, but cultures using media including Endo medium, blood agar, plain agar, Herrold’s, Loeffler’s, liver agar, and milk agar for fecal samples failed [1, 20]. King in 1957 [22] demonstrated that campylobacters referred to as the V. fetus group and the “related vibrios” by then, grew in thioglycollate medium under microaerophilic condition in a Brewer anaerobic jar. Some strains grew on MacConkey’s agar, but failed to grow on several solid media such as SS (Shigella-Salmonella) agar, Simmons’ citrate agar or Christensen’s urea agar. In addition, it was found that the V. fetus strains preferred a temperature between 25°C and 37°C with very little or no growth occurring at 42°C, while “related vibrios” (now C. jejuni) strains failed to grow at 25°C, but grew optimally at 42°C, with no growth occurring at 52°C. The first successful isolation of Campylobacter from feces of patients was accomplished in 1968 and published in 1972, by using the technique of direct membrane filtration (0.65 μm pore size) onto fluid thioglycolate-agar medium containing antibiotics (bacitracin, polymyxin B sulfate, novobiocin and actidione [1, 25]. This was followed by the development of a selective medium in 1977, which enabled the isolation of C. jejuni and C. coli from human feces [27]. In Skirrow’s study, initially the filtrates of fecal suspensions prepared by passing samples through Millipore filters based on that described by [25] were cultured on plain blood agar. Then this study also developed a procedure using medium containing vancomycin, polymyxin B, and trimethoprim, and incubation condition at 43°C in an atmosphere of 5% oxygen, 10% carbon dioxide, and 85% nitrogen [27]. The successful isolation of campylobacters from human feces based on the above selective media led to the recognition that Campylobacter is a leading cause of human diarrheal illness in many countries [5, 28]. Since then, extensive efforts have been made to develop, evaluate and validate the existing and new media, and procedures for the isolation and identification of campylobacters from food and food-related environments.

The main media and procedures currently in use were developed during 1980s [61]. The characteristics of these organisms allowing growth and selection have been largely understood [83]. The prescence of Campylobacter cells in larger numbers from clinical (or animal fecal) sample makes their recovery reasonably straightforward. The ability of thermotolerant campylobacters particularly C. jejuni and C. coli, to grow at 42°C, has enhanced the selective isolation of thermotolerant campylobacters by inhibiting the growth of other bacterial species. The applications of antibiotic cocktails in selective media have become refined in their combinations of the types and concentrations over time with continuously improved bacterial recovery rates. For food and water samples, and perhaps clinical samples requiring lengthy periods of transportation before analysis, the recovery of stressed cells can also be challenging [11, 83]. The procedures for the isolation of Campylobacter spp. from foods were adapted originally from clinical microbiology protocols since the 1970s [61, 84]. So far more than 20 of each selective enrichment broth and selective agar media have been developed (Table 2) with different scales of evaluation and validation [13, 37, 61, 77, 83, 85]. As campylobacters do not ferment carbohydrates, peptones are included in all media as a nutrient source. Some such as Preston broth, Bolton broth, Exeter broth and Campylobacter enrichment broth contain meat or yeast extract [61]. Most media are developed based on several commonly used media such as nutrient broth or agar, Brucella, Columbia, thioglycollate media with the addition of various antibiotics and with or without blood [61]. Most Campylobacter media contain antibiotics and blood which neutralizes trimethoprim antagonists. Oxygen quenching agents are also used to overcome the adverse effects of toxic oxygen derivatives that can form when media are exposed to light (e.g. hydrogen peroxide and superoxide) [61]. Selective or non-selective blood agar media were successful in isolating new Campylobacter species [61]. Depending on the purposes, quantitative and qualitative procedures have been developed. Currently there are a few agar plates available for detection and enumeration purposes. In addition to those developed in the early days including the most used currently, modified charcoal cefoperazonedeoxycholate agar (mCCDA), Skirrow, Campy BAP, Karmali, and Abeyta-Hunt-Bark, several new agar plates have been developed, including Campy-Line, Campy-Cefex and several commercially available chromogenic agars during the last two decades (Table 2) [96]. Currently, the most commonly used enrichment media include Bolton broth, Preston broth and Exeter broth [85].

Enrichment broth media		Agar media
Name	Reference	Name	Reference
Thioglycollate broth	[22, 61]	Thioglycollate	[61, 85]
Preston broth	[61]	Dekeyser	[85]
VTP Brucella-FBP broth (VTP) or VTP FBP broth	[61]	Skirrow blood	[27, 61, 85]
Modified charcoal cefoperazone deoxycholate (mCCD) broth	[61]	Blaser A	[85]
Doyle and Roman enrichment broth	[61]	Blaser-Wang	[85]
Park and Sanders broth	[61]	Butzler and BU40 (modified Butzler) (contains bacitracin, novobiocin, cycloheximide, colistin, and cefazolin)	[61]
Exeter broth	[61]	Butzler (Virion) (BV) (cefoperazone, rifampin, colistin, and amphotericin B)	[61]
Christopher broth	[85]	Butzler (Oxoid)	[61]
Lander broth	[85]	Butzler selective medium or Campy BAP	[61]
Waterman broth	[85]	Preston agar (Campylobacter Agar)	[61]
Hunt and Radle broth	[61]	Waterman agar	[85]
Bolton broth (Campylobacter enrichment broth – Bolton formula)	[61]	Modified Butzler agar (MBA)	[61, 86]
Campylobacter enrichment broth (CEB) (a commercial version of Bolton broth with A commercially available enrichment broth varies only in the substitution of natamycin for cycloheximide	[87]	Blaser medium or Campy BAP (with vancomycin, trimethoprim, polymyxin B, cephalothin, and amphotericin)	[61]
Rosef and Kapperud Campylobacter enrichment broth (RKCEB),	[61, 86]	Charcoal cefoperazone deoxycholate (CCDA) (Campylobacter Blood-Free Selective Agar)	[61]
Rosef	[61]	mCCD agar	[61]
Lovett’s broth (Brucella broth with FBP, vancomycin, polymyxin B)	[88]	Campylobacter Selective medium (CAT)	[61]
Blood-free Campylobacter medium (BFCM)	[86]	Karmali agar	[61]
Mueller and Hinton broth without or with antibiotics (MHBH)	[61]	Campy Brucella agar (CBAP),	[61, 86]
Fennell’s medium	[61]	Mueller and Hinton agar with antibiotics (MHBA) or without antibiotics	[61]
Semi-solid medium or selective semisolid Brucella medium (SSBM),	[61, 86]	Columbia Blood Agar	[61]
Buffered Peptone Water (transportation and carcass rinse and pre-enrichment)	[61, 89, 90]	Abeyta–Hunt–Bark (A-H-B) agar (Heart infusion agar with yeast extract and antibiotics)	[91]
Weybridge’s (Transportation media)	[92]	Campy-Cefex	[93]
Wang’s semi-solid Transportation Medium	[89]	Campy Line agar with sulfamethoxazole (CLA-S),	[94]
Wang’s Freezing/storage Medium	[89]	CampyFood agar Internationally validated method for detection & enumeration (ISO 16140/AOAC)	BioMérieux, France
Cary-Blair transportation medium	[91]	CASA Chromogenic Medium for enteric Campylobacter species	AES Chemunex, France
A-H slant	[91]	RAPID’ Campylobacter agar (chromogenic)	Bio-Rad (Certified NF VALIDATION according to the ISO 16140 standard)
Brain Heart Infusion broth (motility testing)	[95]	CHROMagar™ Campylobacter CAC (chromogenic for thermotolerant Campylobacter)	CHROMagar, Paris, France
The aztreonam amphotericin vancomycin (AAV) experimental campylobacter selective medium	[61, 62]	Brilliance CampyCount AGAR (chromogenic for Campylobacter jejuni and Campylobacter coli)	ThermoFisher Scientific
CampyFood broth (Internationally validated method for detection & enumeration (ISO 16140/AOAC))	BioMérieux, France	Campylosel	Bio-Mérieux, France

Table 2.

List of culture media used for isolation, transportation and maintenance of campylobacters.

3.1.2 Current culture procedures

So far, three types of basic culture procedures have been commonly employed for the isolation of campylobacters. These methods include 1) membrane filtration onto non-selective or selective agar media; 2) direct plating on selective agar, either blood-based or charcoal-based; and 3) selective enrichment in broth followed by streaking onto selective agar [61, 77]. Various culture supplements and procedures have been examined or standardized to improve selective isolation of Campylobacter species [11, 13, 37, 61, 77, 83]. The above three approaches have been used for qualitative detection or enumeration (semi-quantitative and quantitative) using the most probable number (MPN) procedure or direct plating method [97].

Although many culture-independent detection methods have emerged over time, isolation by culture is still the “gold” standard procedure for the detection of campylobacters for regulatory bodies. Despite the continued improvements in the isolation procedures of Campylobacter, however, challenges remain, which reduce the efficiency of these methodologies [98]. As discussed in the section related to the growth requirements, Campylobacter exhibits dynamic and malleable physiological and metabolic characteristics that have impacts on the sensitivity and specificity of culture-dependent methods. So far, there is no single or “standard” accepted method of isolating and detecting all Campylobacter species from all kinds of sample types, due to different requirements of temperature, microaerobic conditions, nutrients and susceptibility to selective antibiotics. However, there are some generally agreed procedures for common types of Campylobacter species and some types of food samples [13, 37, 61, 77, 83, 85]. Several protocols have been published or recommended by recognized authorities, such as 1) the International Standards Organization [99, 100]; 2) US Food and Drug Administration (FDA) [91]; 3) the U.S. Department of Agriculture (USDA) - Food Safety and Inspection Service (FSIS) [89]; 4) the Public Health England [95]; 5) World Organization for Animal Health (OIE) [92]; 6) Health Canada (HC) [101]; 7) Australia and New Zealand [102]. These methods use the most effective protocols to isolate thermotolerant Campylobacter spp. (mainly C. jejuni and C. coli) from food, primarily poultry products [77]. Cape Town protocol is also a well-known protocol used in South Africa, which employs membrane filtration and antibiotic-free agar plating for a broad spectrum of Campylobacter species [65].

The method of choice to isolate low numbers of Campylobacter from contaminated food samples is the combination of enrichment broth with selective plating or direct plating on selective agars. For analysis of fecal samples or certain types of food samples, direct plating is often preferred due to the presence of large numbers of non-stressed campylobacters in feces. Several selective agars have been used for various purposes including regulatory requirement in US [65, 89, 95]. However, due to the slow growth of Campylobacter species, many are lost to competition by contaminant bacteria naturally present in foods. As mentioned earlier, certain antibiotics in the selective media may inhibit the growth of certain Campylobacter species and strains. By taking advantage of the unique motility of campylobacteria, the membrane filtration method known as “Cape Town protocol” allows these organisms in samples to penetrate the cellulose filters of 0.45-mm or 0.65-mm pore sizes to antibiotic-free blood or other agar. This method has shown a great advantage in isolating a wider spectrum of Campylobacter species, including C. upsaliensis, C. concisus, C. curvus, C. rectus, C. sputorum biovar sputorum and C. hyointestinalis as well as the standard C. jejuni and C. coli from human stools [65]. However, this method takes longer time, i.e., a couple of days more than conventional direct plating [103] and also depends on the presence of a large number of Campylobacter cells with cell motility [104]. The membrane filtration method has been evaluated for food and water samples [104, 105, 106, 107]. One study [104] showed that the minimum numbers of motile bacteria required for this method were 2.2 and 2.1 log colony forming unit (CFU) for 24-h cultures and centrifuged cells, respectively, and 4.1 and 3.4 log CFU of coccoid and nonmotile mutant cells, respectively. Broiler meat samples after enrichment in Bolton’s broth showed that approximately 1.7 log CFU of Campylobacter can be detected with pure colonies on agar plates using this filtration method. The results from the studies [104, 105, 106, 107] demonstrate that the motility of the bacteria influences passage through cellulose filters and that 0.65-mm-pore-size filters on agar plates help obtain pure Campylobacter colonies from enriched food samples [104, 105]. A novel and simple filtration procedure after enrichment in Rosef’s enrichment broth was developed using a hydrophobic grid membrane filter (HGMF) on antibiotic-free semisolid medium (SSM). The HGMF-SSM method showed higher recovery rates using turkey samples and pig fecal samples compared with Rosef’s broth enrichment procedure [105]. A study using selective enrichment combined with membrane filtration has shown a similar recovery rate but with 20-fold fewer false-positive for campylobacters in water compared with the enrichment procedure [106]. Another study [107] showed that the filtration method, and real-time PCR and digital PCR were more sensitive than enrichment culture method using inoculated sprouts samples. The filtration method showed a similar detection ability to PCR in all samples.

For the enrichment procedures, particularly for the samples with low numbers of cells under stress, a preliminary (resuscitation) period of incubation at reduced temperature (37°C) for about 4 h prior to increasing the temperature to 42°C (for thermotolerant species) for the remainder of the 48 h of incubation time has been used commonly. However, procedures using staged periodic increases in temperature to aid adaptation and recovery can be time-consuming and overly complex [61]. Several factors have been considered during the development or application of the media for campylobacters from food, including incubation at 37°C instead of 42 or 43°C and changes in the types and concentrations of antibiotics or various combinations of selective enrichment broth with selective agars, in order not to inhibit a wider spectrum of the organisms such as C. upsaliensis, C. jejuni subsp. doylei and some strains of C. coli and C. lari [108, 109, 110, 111]. The use of immunomagnetic separation methods to concentrate all cells and to remove competitive microorganisms could be used as an option. However, this method is problematic due to low capture efficacy because of the unique movement of campylobacters and with the few studies undertaken on campylobacters showing limited efficacy when applied to naturally contaminated samples [112], and possible surface antigenic variations of the campylobacters if a single antibody is used for capture [83]. Considerably less studied are the prevalence and importance of species other than C. jejuni and C. coli, especially as related to food as a source of illness. Most Campylobacter species have different growth requirements to C. jejuni and C. coli, and until recently, specific methods for isolation have not been applied. In order to enhance the detection sensitivity and accuracy, and shorten the turnaround time of culture process, the screening of the presence of campylobacters in the enrichment broth during or after enrichment could be conducted using various methods, such as PCR [89].

Microaerobic systems are also important to support Campylobacter growth in vitro. Different methods have been developed to generate microaerobic atmospheres for routine use during the enrichment of food samples or during the incubation of inoculated plate media. The microaerobic atmosphere is usually generated in a gassed jar system, either by continuous flow of the mixed gas through the containers or by evacuation and gas replacement. If a large number of samples are processed, the evacuation-replacement is a more economical and practical way, for which, the air in the jar or gas tank is removed by a vacuum pump, and then replaced with a desired microaerobic gas mix [61, 91, 113]. Other commercial sachets that generate microaerobic conditions can also be used, particularly for a small number of samples or when other evacuation-replacement system(s) not available [114]. Plastic bags utilized to freeze food products with a “ziplock” type closing to prevent air leaks have been successfully used with gas-generating sachets and manual evacuation-replacement systems to be flushed with a desired microaerophilic gas mixture [115, 116]. Due to specific growth requirements, certain species or stains require H₂ content [6]. For a large number of samples, or to create unique microaerobic gas mixes with increased H₂ content, more sophisticated microaerobic workstations can be used [44]. In addition to generating the microaerobic conditions, there have been several attempts to use O₂-quenching agents added to enrichment broths and agar plates for the isolation of Campylobacter species to reduce the toxic effects of oxygen radicals. These O₂-quenching agents include blood or alkaline hematin, charcoal, iron salts, norepinephrine, ferrous sulfate, sodium metabisulfite and sodium pyruvate (known as FBP supplement). In general, if blood or charcoal is added to agar plates, no other O₂ quenching compounds are required [61, 108]. Optimum growth can also be maintained in a tri-gas incubator [117] or a continued culture bioreactors [118]. Possibly even further neglected is the requirement for gaseous hydrogen in the cultivation atmosphere, without which many species cannot grow. From that perspective, the lack of commercially available”gas packs”to generate a microaerobic atmosphere that includes H₂ is unfortunate, especially since the growth of C. jejuni and C. coli are also enhanced in the presence of H₂. The inclusion of oxygen quenching supplements in pre-enrichment media seems to be a widely adopted practice to allow broth cultures to be incubated in air [61, 91, 101].

3.1.3 Regulatory use for risk assessment and control

The prevention of transmission to humans is paramount in reducing the incidence and burden of Campylobacter disease in humans. In the last two decades, extensive risk assessment and baseline studies on the distribution of the organisms in the food chain have been conducted in several countries [8, 31, 119]. The ubiquity of Campylobacter in the environment and poultry products presents difficulties in investigating the complex pathways for infection with no single specific point for effective prevention and control. Regardless of the high levels of contamination by this pathogen, particularly in poultry carcasses and its products, raw meat contaminated with Campylobacter is still allowed to be sold at retail in most countries. Since raw poultry is the main source of infection, poultry has been the focus for reduction with various success using different approaches in several countries including Iceland, New Zealand and UK [8, 31, 119]. Recently, the United States of America has introduced a standard and a compliance guide for poultry industries to reduce the campylobacters in raw poultry [120]. In Europe, a Process Hygiene Criterion (PHC) (Commission Regulation (EU) 2017/1495 of 23 August 2017 amending Regulation (EC) No 2073/2005) for Campylobacter spp. came into effect in January 2018. This PHC set a limit of the Campylobacter load (less than 1000 CFU/g) on broiler carcasses to control contamination of carcasses during the slaughtering process through monitoring and taking corrective actions when the mandated targets are breached [121].

3.1.4 Impacts of sampling and culture procedures on the recovery sensitivity and isolation of strain types

3.1.4.1 Detection sensitivity

According to the US Centers for Disease Control and Prevention (CDC), in approximately 80 and 56% of the cases of foodborne illness and death respectively in the USA, causal agents were not identified [122, 123]. In Canada, among 115 foodborne outbreaks reported from 2008 to 2014, 7.8% of outbreaks did not identify the etiologic agent [124]. Campylobacterosis is ranked as one of the major foodborne illnesses [123]. Campylobacter species. may account for some of the illnesses for which etiological agents were not identified. In fact, a study [125] in Canada indicated that current methods for isolation of Campylobacter species from clinical samples might fail to recover isolates from positive samples, particularly those in cryptic taxa of Campylobacter. Furthermore, patients may also be colonized with more than one genotype of Campylobacter [126]. Existing isolation methods have technical limitations in isolating this fastidious bacterium, such as a growth competition with indigenous bacteria in food samples. When compared with PCR methods, it was demonstrated that Campylobacter culture failed to correctly detect Campylobacter in 30% of positive patient stool specimens [127] or detected fewer species [128]. The studies comparing the culture and PCR methods for food have demonstrated that PCR methods identified more positive samples than culture methods [107, 129].

Several reasons could attribute to the relatively low sensitivity of culture methods compared with molecular methods. Sample collection and preparation for the sensitive recovery of live campylobacters may be important [77, 130]. For food and water samples, and perhaps clinical samples requiring lengthy periods of transportation before analysis, the recovery of stressed cells can be challenging [83]. Another interfering factor is the influence of microbiologically diverse and complex food or other sample matrices. A study [111] compared the effects of Bolton and Preston selective media on the microbiota compositions and isolation frequencies using next-generation sequencing (NGS) analysis of 16S rRNA. The results showed that Bolton and Preston-selective enrichments generated different microbiota communities and that the sequence of combining the selective media also critically affects the isolation frequency by altering microbiota compositions. For example, the highly prevalent Escherichia coli in Bolton media negatively affected the efficacy of Campylobacter isolation. The study [78] compared five different commercialized selective Campylobacter media for the ability to isolate Campylobacter from broiler fecal samples using 16S rRNA tagged-pyrosequencing of the isolated colonies. Sequencing results indicated that 0.04% of the total fecal microbial community was Campylobacter, and 88–97% of the putative colonies were in fact Campylobacter. The study also revealed that incubation atmosphere had little effect on recovery, but a significant difference in media specificity was found at 42 vs. 37°C. Different culture media showed different non-Campylobacter sequence types. Therefore, there are significant challenges in culturing Campylobacter on selective and/or differential media due to the presence of other competitive microorganisms, which can likely influence the metabolism of Campylobacter. In addition, the diversity of various host or environment matrices, such as poultry- or bovine-specific matrices, may also induce biochemical changes in Campylobacter, which further obscure isolation and identification. In addition, exposure to environmental stresses such as temperature, pH, aw, and starvation triggers a response that often results in the so-called “viable but non-culturable” (VBNC) form that appears to be capable of surviving as an intact and potentially infectious agent yet resistant to conventional culture [48, 83].

3.1.4.2 Impact on the isolation of different strain types

Most official protocols including that of the ISO, US FDA and HC require 25 g of meat for testing. However, retail packages typically contain multiple meat pieces with weights exceeding the required amount. This raises a concern that the testing of the required amount taken from only one of multiple pieces of meat from the same package may not be representative of the whole package. In addition, several studies have shown multiple strains could be present in a single chicken flock [131, 132, 133, 134], chickens from a positive flock could contaminate the raw chicken meat at the slaughterhouse [134, 135] and internal organs and raw meat samples had multiple Campylobacter strains [43, 44]. Therefore, slaughter and packaging processes could lead to the contamination of chicken carcasses/raw meat from the same or different flock(s) with multiple strains. The selection of samples from the same package may affect the isolation of different strains in contaminated samples. Furthermore, the effects of various culture procedures on the isolation of different genotypes of campylobacters are not well understood. Overall, the culture conditions including temperature and types of media affected the genotypes of campylobacters recovered from raw chicken [136, 137, 138].

Taken together, specific isolation procedures and culture media influence the diversity of Campylobacter species recovered from samples. Temperature, media, time, and enrichment all influence the ability to isolate Campylobacter. The infective dose of the pathogen is low (approximately 500 CFU) [139, 140]. These studies highlight the importance of method sensitivity, and the need to collect multiple isolates from both clinical samples and potential sources of infection to support source attribution and epidemiological investigations.

3.2 Identification, confirmation and typing methods

The next step for the successful isolation of a strain is the identification and confirmation of the presumptive isolate. Traditionally, identification and confirmation of an isolate consisted of the examination of colony morphology, phase-contrast microscopic examination of morphology and corkscrew-like motility of the suspect, followed by confirmation using immunological assays (e.g., agglutination tests), biochemical and phenotypical testing, and molecular approaches [91, 101]. More recently, microbial identification based on proteomic profiling using Bruker ® MALDI Biotyper and genomic sequencing approaches have been recommended [82, 89, 98]. However, different published official protocols use different tests screening, confirmation and detection using agar colonies or enrichment broth. The identification and selection of suspect colonies from the selective agar are the first and crucial steps. To assist the accurate identification and selection of the Campylobacter-like colonies on the agar plate, particularly when the colonies are atypical or mixed with other floral microorganisms, specific, rapid and quantitative colony blot immunoassay [137, 141] and molecular tests, such as PCR, can be used [107].

The final stage in strain characterization is subtyping to allow rigorous assessment for epidemiology and source attribution purposes. Various strain subtyping approaches, including phenotyping and genotyping, have been developed and applied. The classic techniques for differentiating isolates phenotypically are based on the presence or absence of biological or metabolic activities expressed by the organism. Since 1980s, a few phenotyping schemes have been developed, including Skirrow-Benjamin and Preston biotyping schemes [142, 143], Penner (haemagglutination) and Lior slide agglutination serotyping schemes [144, 145], plasmid typing [146, 147], bacteriophage typing [148], and multilocus enzyme electrophoresis [149]. The most popularly used phenotypic methods to differentiate thermotolerant Campylobacter (mainly C. jejuni and C. coli) isolates include Skirrow-Benjamin biotyping scheme, Penner and Lior serotyping schemes, and multilocus enzyme electrophoresis [91, 101, 119, 149]. Although most of these methods lack discriminatory power, they are still used and are efficient to characterize bacterial food-borne pathogens [149]. All of the tests described above can be used alone or in combination to isolate and identify Campylobacter species, particularly C. jejuni and C. coli.

The need to enhance the limited discriminatory power of the traditional phenotyping methods in epidemiological investigations has led to the development of molecular typing methodologies. These improved technologies have been instrumental in reporting source attributions of sporadic infections and outbreaks with Campylobacter by providing information on the genetic subtypes. Many molecular subtyping methods have been developed to characterize Campylobacter species, but only a few are commonly used in molecular epidemiology studies [83, 138, 150, 151]. The commonly used methods include: pulsed-field gel electrophoresis (PFGE), flaA short variable region sequence typing (flaA-SVR), flaA restriction fragment length polymorphism analysis (flaA-RFLP), multi-locus sequence typing (MLST), extended MLST (eMLST), ribotyping, random amplification of polymorphic DNA (RAPD), microarray comparative genomic hybridization (MCGH), comparative genomic fingerprinting, single nucleotide polymorphisms, high-resolution melting analysis (HRM), Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), and nucleotide and whole genome sequencing [150, 152]. The molecular typing methods have played a significant role in tracking sources of Campylobacter spp. infection [150]. However, most of the above-mentioned technologies are based only on a small fraction of the genome [150, 152]. In recent years, various emerging next generation sequencing (NGS) platforms and various pipelines for different purposes have been developed [153]. The NGS has been applied in the development of molecular tests, and in recent years various platforms have been used for whole strain genome sequencing for campylobacters for various purposes including strain differentiation with more discrimination power [82, 152, 154]. The method has been recommended for use with the detection and typing of campylobacters by several regulatory organizations such as USDA [89].

Overall, typing methods play an instrumental role in the identification, monitoring, and prevention of Campylobacter infections. The use of multiple phenotypic and genotypic or molecular typing methods can improve species and subspecies discrimination and is appropriate when trying to identify pathogenic organisms like C. jejuni, C. coli, and C. laridis [151]. Serotyping and biotyping methods have been commonly used for identifying bacterial isolates and for epideomiological purposes in the past and currently in some countries [155]. These phenotypic methods, however, cannot provide as much discriminatory power as genotyping methods. The MLST, PFGE and AFLP have been found to have greater discriminatory powers when compared with techniques like ribotyping and flagellin typing. It is not yet possible to identify a perfect typing method for all non-pathogenic and pathogenic Campylobacter species. However, currently available techniques including NGS, when used in concert, would fulfill the requirements for epidemiological and source attribution purposes. The development of a validated and practical typing method or methods could make routine subtyping of Campylobacter species feasible.

4. Discussion and conclusion

Currently, the genus Campylobacter contains 39 validly published species, 11 subspecies and 4 biovars, according to the list of prokaryotic names with standing in nomenclature. These diversified species of the genus Campylobacter transit through various animal and environmental compartments to humans and animals, which emphasize the need to adopt an integrated One-Health approach in Campylobacter epidemiology, risk assessment and prevention. Thermotolerant campylobacters, such as C. jejuni, C. coli and C. lari, are the most implicated species in Campylobacter infections in humans. However, there are many other emerging and unusual species, which cannot be detected using the currently available culture methods. Problems with recovering different strains within the same species or specific relevant strains using the same media formulation are often encountered because of the multiple resistance of campylobacters to antibiotics. Many studies highlight the importance of method sensitivity and the need to collect multiple isolates from both clinical samples and potential sources of infection to support epidemiological investigations. All the above-mentioned testing limitations may have contributed to missed or inadequate source attribution and a limited understanding of the epidemiology of Campylobacter gastroenteritis. Therefore, the development of the new strategies, employing comprehensive procedures to isolate and detect all the strains and species of campylobacters would be ideal. In order to prevent and control the transmission of foodborne campylobacters to humans, the baseline studies and risk assessments at larger scales with more systemic and collaborative approaches have been strengthened in recent years. The measures to reduce the burden of campylobacters in poultry products have been implemented in several countries with success, including the regulatory requirements to meet the limit of Campylobacter load on the carcasses of young chicken and turkeys in the US and Europe. To overcome difficulties in preserving most fresh foods with short shelf-life, the food industry urgently demands novel rapid tests at reasonable cost that employ improved culture and culture-independent methods able to accurately detect low numbers of viable Campylobacter cells. These innovative methods will reduce significant economic loss and the health risks to public.

Acknowledgments

The authors thank Beverley Phipps-Todd for providing laboratory support throughout many years in developing procedures for the isolation and detection of campylobacters, and Sohail Naushad, Ruimin Gao and Isaac Firth, for providing critical review of this manuscript.

Conflict of interest

The authors declare no conflict of interest.