Campylobacter John Moore, Deborah Corcoran, James Dooley, Séamus Fanning, Brigid Lucey, Motoo Matsuda, David Mcdowell, Francis Mégraud, B. Cherie Millar, Rebecca O’Mahony, et al.

To cite this version:

John Moore, Deborah Corcoran, James Dooley, Séamus Fanning, Brigid Lucey, et al.. Campylobac- ter. Veterinary Research, BioMed Central, 2005, 36 (3), pp.351-382. ￿10.1051/vetres:2005012￿. ￿hal- 00902984￿

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HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Vet. Res. 36 (2005) 351–382 351 © INRA, EDP Sciences, 2005 DOI: 10.1051/vetres:2005012 Review article

Campylobacter

John E. MOOREa*, Deborah CORCORANb, James S.G. DOOLEYc, Séamus FANNINGd, Brigid LUCEYe, Motoo MATSUDAf, David A. MCDOWELLg, Francis MÉGRAUDh, B. Cherie MILLARa, Rebecca O’MAHONYd, Lisa O’RIORDANa, Michele O’ROURKEd, Juluri R. RAOi, Paul J. ROONEYa, Andrew SAILSj, Paul WHYTEd

a Northern Ireland Public Health Laboratory, Department of Bacteriology, Belfast City Hospital, Belfast BT9 7AD, Northern Ireland, United Kingdom b Molecular Diagnostics Unit, Cork Institute of Technology, Bishopstown, Cork, Ireland c School of Biomedical Sciences, University of Ulster, Coleraine, Co. Londonderry, BT52 1SA, Northern Ireland, United Kingdom d Centre for Food Safety, Faculties of Agriculture, Medicine & Veterinary Medicine, University College, Belfield, Dublin 4, Ireland e Department of Medical Microbiology, University Hospital, Wilton, Cork, Ireland f Laboratory of Molecular Biology, School of Environmental Health Sciences, Azabu University, Sagamihara, 229-8501, Japan g Department of Food Studies, University of Ulster, Jordanstown, Newtownabbey, Co. Antrim, Northern Ireland, United Kingdom h Laboratoire de Bactériologie, CHU Pellegrin, Place Amélie Raba-Léon, 33076 Bordeaux, France i Department of Applied Plant Science, Queen’s University, The Agriculture and Food Science Centre, Newforge Lane, Belfast, BT9 5PX, Northern Ireland, United Kingdom j Health Protection Agency, Institute of Pathology, Newcastle General Hospital, Newcastle upon tyne NE4 6BE, United Kingdom

(Received 6 December 2004; accepted 1 February 2005)

Abstract – Species within the genus, Campylobacter, have emerged over the last three decades as significant clinical pathogens, particularly of human public health concern, where the majority of acute bacterial enteritis in the Western world is due to these organisms. Of particular concern are the species, C. jejuni and C. coli, which are responsible for most of these gastrointestinal-related infections. Although these organisms have already emerged as causative agents of zoonoses, several aspects of their epidemiology and pathophysiology are only beginning to emerge. Trends in increasing antibiotic resistance are beginning to emerge with oral antibiotics, which may be the drug of choice for when it is necessary to intervene chemotherapeutically. This review wishes to examine (i) emerging clinical aspects of the disease, such as Guillain Barré syndrome (GBS), (ii) the association between these organisms and poultry as a natural host, (iii) environmental aspects of Campylobacter epidemiology, (iv) the emergence of atypical campylobacters (v) emerging trends in antibiotic resistance, (vi) adoption of modern methods for the detection of campylobacters. epidemiology / poultry / PCR / zoonosis / antibiotic resistance

* Corresponding author: [email protected] 352 J.E. Moore et al.

Table of contents

1. Introduction ...... 352 2. Historical emergence of Campylobacter ...... 352 3. Clinical aspects of Campylobacter infections ...... 353 3.1. Enteric infection...... 353 3.2. Systemic infection...... 354 3.3. Post-infectious manifestations ...... 354 4. Human epidemiology and foods of animal origin...... 355 4.1. Campylobacters and poultry ...... 355 4.2. Campylobacters and other food animals...... 356 4.3. Control in foods of animal origin ...... 356 5. Environmental campylobacters ...... 357 5.1. Water...... 357 5.2. Sewage and water treatment plants...... 357 5.3. Farms ...... 358 5.4. Food related environments...... 359 6. Atypical campylobacters ...... 360 7. Antibiotic resistance ...... 362 7.1. Antimicrobial susceptibility testing in Campylobacter spp...... 363 7.2. Surveillance of antimicrobial resistance in Campylobacter spp...... 364 7.3. Genetic mechanisms associated with antimicrobial resistance in Campylobacter spp...... 365 7.4. Gene cassettes and class 1 integrons in Campylobacter spp...... 368 7.5. MDR-mediated by antimicrobial efflux systems...... 369 8. Campylobacter detection...... 370 9. Conclusions ...... 373

1. INTRODUCTION resistant Campylobacter strains cause more prolonged or more severe illness than do Campylobacter jejuni is a major cause of antimicrobial-susceptible strains. foodborne illness causing human acute bac- terial enteritis worldwide [8, 164]. Overall the high incidence of clinical disease asso- 2. HISTORICAL EMERGENCE ciated with this organism, its low infective OF CAMPYLOBACTER dose in humans [137], and its potentially serious sequelae, confirms its importance Campylobacter spp. have long been as a significant public health hazard [8, associated with the cause of veterinary dis- 164]. eases, such as diarrhoea in cattle, and septic Numbers of infections have declined abortions in cattle and sheep. Their associ- slightly in some parts of the world during ation with human blood cultures in the late recent years, but the overall disease burden 1950’s was rare and hence Campylobacter spp. is still significant, thus there remains an was deemed to be an opportunistic human urgent need to better understand how this pathogen. It is only in the last 30 years that disease is transmitted into and within the these organisms have been recognised as a human food chain. Such challenges are major cause of human illness. Campylo- increased by the observation that an increas- bacters may have been observed in the ing number of Campylobacter isolates from stools of diarrhoeic infants in Germany as humans and the human food chain exhibit early as 1880. The first recognised identifi- antibiotic resistance and that antimicrobial- cation was made by McFadyen and Stockman Campylobacter 353 in 1913 (cited in [102]) in association with of epidemiological research and conse- abortions in sheep. Confirmatory tests were quently to the realization that campylo- carried out by Smith in 1918 (cited in [102]) bacters have now emerged as a significant when similar organisms were isolated from public health problem for both developed aborted bovine foetuses. The organisms and underdeveloped countries [44, 147, were originally assigned to the Vibrio genus, 153–156, 165]. due to their spiral appearance and hence Smith named the organism Vibrio fetus. However, it was not until 1947 that the 3. CLINICAL ASPECTS OF human infection was first associated with CAMPYLOBACTER INFECTIONS the microaerophilic vibrios, which was associated with a pregnancy-related infec- tion, where the fœtus died. In 1957, the 3.1. Enteric infection work of Elizabeth King (cited in [102]) pro- Thermotolerant campylobacters (Campy- posed two different types of vibrios associ- lobacter jejuni/Campylobacter coli) consti- ated with enteric diseases, the first being V. tute the most frequent cause of intestinal fetus and the second was found to be ther- mophilic in nature. It was not until 1963 that infections worldwide. The main symptom the genus Campylobacter (meaning “a curved observed is diarrhea which can vary from rod”) was proposed as it realized that the limited to voluminous stools which may be organism could not utilize sugars and had a watery or bloody. Another frequent digestive different G+C content to that of Vibrio spp. tract symptom is abdominal pain, whereas The work of King was later corroborated vomiting is uncommon. Fever, headache, with the work of Dekeyser and Butzler in asthenia, and anorexia are also present and 1972 (cited in [102]), when isolation pro- may precede diarrhea [97]. Campylobacters cedures for thermophilic campylobacters are enteroinvasive bacteria which lead to were developed. This method involved the colitis and, in some instances, resemble filtering of stools samples through 0.64 µ inflammatory bowel disease. When pain is membrane filters and inoculating the filters the major feature of the infection, differen- onto agar. This method proved too cumber- tiation from appendicitis may be difficult. some and in 1977, Martin Skirrow from Normally the disease develops two to three Worcester Public Health Laboratory, days after ingestion of contaminated food described a simple direct technique, involv- and the symptoms resolve themselves ing the direct culturing of faeces onto blood within a week. In comparison to Salmonella agar containing vancomycin, polymyxin or Shigella infections, Campylobacter infec- and trimethoprim [153–156]. Plates were tions are usually less acute (less fever and incubated at 43 °C in an microaerophilic general symptoms) with a higher tendency atmosphere containing 5% (v/v) O2, 10% toward recurrence if no treatment is given; (v/v) CO2 and 85% (v/v) N2. Since then, however, they are not distinguishable with- several methodological modifications have out performing a coproculture. Stools remain been made, thereby allowing the universal positive for several weeks. Treatment adoption of such methods and variants of appears to be beneficial if it is administered standard methods, which allow routine early enough in the course of the disease diagnostic clinical microbiology laborato- [146]. The recommended drugs are eryth- ries to attempt the isolation of campylo- romycin, or amoxicillin or a fluoroquinolone bacters from faecal specimens. or tetracycline, provided the bacterium has The improved isolation methods led to not acquired a resistance. the publication of the first report of the fre- Campylobacter enteritis may occur in all quency of campylobacters in association age groups but clinical presentation can with humans, thereby leading to an avalanche vary according to age. In infants, the risk of 354 J.E. Moore et al. dehydration or convulsion exists. Breast flow. Nevertheless, the frequency of septi- feeding protects against the clinical expres- cemia detected in the case of Campylo- sion of the infection [105]. Symptoms appear bacter enteric infections remains very low during the weaning period. (0.1%), especially when compared to those In hyperexposed subjects, immunity associated with Salmonella [156]. develops and the infection becomes sub- There is one Campylobacter species, C. clinical. It occurs in developing countries in fetus, which is rarely found as a cause of children who are repeatedly infected but enteritis but is quite often isolated in sys- also in certain populations in Western coun- temic infections. The number of systemic tries, e.g. raw milk drinkers and workers in infections observed with C. fetus indeed poultry abattoirs. exceeds the number due to thermotolerant In contrast, a decreased immune response, campylobacters. However, more than half as may occur in elderly people or in people of the patients harbour an underlying dis- whose immunity is impaired by an underlying ease, as indicated previously. This bacter- disease (diabetes, cirrhosis, cancer, immu- iemia induces fever and leads to metastatic nosuppression, HIV infection), increases localization. A number of tissues can be the risk of developing a severe infection. In involved, especially the vascular endothelium a study, the risk of Campylobacter infection (aneurism, thrombophlebitis, endocarditis), was multiplied by 40 when subjects were bones, joints, meninges, etc. Despite its HIV positive compared to controls [160]. name, C. fetus does not appear to induce fre- quent abortions in humans, only a few cases For unknown reasons, the male gender is have been reported. These infections must also an important risk factor for Campylo- be treated vigorously because of a bad prog- bacter infection. Furthermore, a decreased nosis. In a survey of more than 100 cases, gastric acidity, for example following pro- death occurred in 15% of the cases, one- ton pump inhibitor consumption, has been third being attributable to the infection, and shown to be a risk factor [106]. A few local a relapse occurred in 10%. The proposed complications have been documented such treatment includes gentamicin and a second as appendicitis, peritonitis, cholecystitis, antibiotic, e.g. amoxicillin or Augmentin® hepatitis or pancreatitis but are extremely or ciprofloxacin or imipenem, according to rare. the location and the susceptibility profile. The main Campylobacter species involved is C. jejuni which is responsible for 80 to 3.3. Post-infectious manifestations 85% of all enteric Campylobacter infec- tions. C. coli ranks second (10 to 15%). As with other enteropathogenic bacteria, Although the latter’s source may be differ- C. jejuni can cause post-infectious manifes- ent, pigs being the main reservoir, this does tations, e.g. reactive arthritis, urticaria, ery- not seem to lead to a different type of dis- thema nodosum. Interestingly, a case of ease. The other campylobacters such as immunoproliferative small intestinal dis- Campylobacter lari, Campylobacter upsa- ease associated with C. jejuni has also been liensis, and Campylobacter fetus are more described recently [74]. These complica- seldomly found, but vary depending on dif- tions seldomly occur (< 1%). The most ferent regions of the world. C. upsaliensis, important post-infectious manifestation to for example, is frequently isolated in South be considered is Guillain-Barré syndrome Africa [69]. [157]. This syndrome is an acute demyeli- nating disease affecting the peripheral neurons 3.2. Systemic infection and is characterized by an ascending paraly- sis. Three clinical forms can be distinguished, Campylobacters are invasive bacteria the last one being the Miller Fisher syn- which may translocate and reach the blood drome, where ataxia and ophthalmoplegia Campylobacter 355 are observed. C. jejuni enteritis is the infec- The handling and consumption of poultry tion most frequently observed before Guil- meat has been previously linked to human lain Barré syndrome and occurs in 30 to illness [8, 9], especially when eaten raw and 50% of all cases. It is estimated to occur in undercooked or recontaminated following 1 in 3 000 C. jejuni infections. The patho- cooking. genic mechanism relies on antigen mimicry between oligosaccharides from the C. jejuni 4.1. Campylobacters and poultry lipopolysaccharides and the GM1 ganglio- side of the peripheral neuron membrane The role of poultry in the epidemiology [195]. The serogroup first described in of human Campylobacteriosis was clearly Japan is C. jejuni PEN19 but other sero- demonstrated in Belgium during the dioxin groups have been described in Europe [37]. crisis in 1999. As a result detecting feeds This syndrome is very severe, leading to a contaminated with abnormally high levels 2 to 3% mortality and major neurological of dioxins in feeds, domestically produced sequelae in 20% of the cases. The other chicken and eggs were withdrawn from patients experience a partial or total recov- retail outlets in Belgium. The resulting tem- ery [22]. The most severe cases are induced porary deficit in supply of poultry and eggs by C. jejuni [77, 183]. over subsequent months resulted in an esti- Recently, Helms et al. [59] in Denmark mated 40% reduction in the numbers of evaluated the global mortality rate of patients human Campylobacter cases reported. The in the year following a bacterial enteric incidence of Campylobacter cases rose to infection, after an adjustment on comorbid- similar levels to those observed prior to the ity, and surprisingly found an excess mor- crisis when the ban on poultry meat was tality after Campylobacter infection (OR = lifted [182]. 1.35, 95% CI = 1.02–1.80). Intestinal colonisation in broiler chicks is rarely detected until at least 7 days of age. In summary, Campylobacter infections Once colonised, chicks normally remain are very common self-limited diseases. asymptomatic carriers until they reach Their frequency generates numerous health slaughter age [51]. Wide variations in flock care expenses. Furthermore, life-threaten- infection prevalences up to 100% have been ing systemic diseases are diagnosed more previously reported in surveillance studies and more readily and the most severe Guil- [64]. The most significant routes of trans- lain Barré syndromes are the post infectious mission by Campylobacter to commercial consequence of this infection, making poultry flocks at the pre-harvest level Campylobacter infection a major public remain unclear. However, a number of epi- health issue. demiological studies have suggested that inadequate disinfection between chick place- ments, age disposition, the use of multi-unit 4. HUMAN EPIDEMIOLOGY sites, the proximity of other livestock, sea- AND FOODS OF ANIMAL ORIGIN son and lapses in biosecurity are significant risk factors [21]. The role of other vectors Campylobacter jejuni is now recognised such as litter beetles, house flies and wild as one of the main causes of bacterial food- birds have also been identified as potential borne disease in many developed countries transmission risks [27]. Conflicting reports with Campylobacter coli less frequently have emerged on the ability of campylo- implicated [43]. Foods of animal origin, in bacters to infect successive generations either particular poultry, have been identified as by direct vertical transmission from hen to significant sources of this enteropathogen chick via the egg or by horizontal transmis- as a result of infection and contamination at sion within the hatchery environment [125, the pre-harvest and harvest levels [126]. 162]. Difficulties in the identification of 356 J.E. Moore et al. significant infection routes to commercial sheep have been shown to be generally flocks at farm level have been further com- lower with approximately 20% of animals pounded by the strain diversity observed in intestinal carriers [197]. The high preva- both flock and environmental isolates from lences of campylobacters in pigs have been various studies and the frequently observed reported previously in numerous studies co-infection of birds with multiple strains and dressed pig carcases have been shown of Campylobacter jejuni [108, 125]. The to be more frequently contaminated than use of contaminated water for drinking in either beef or sheep [107]. This is most poultry houses has also been recognised as likely attributable to the fact that pig car- a significant risk factor for colonisation by cases undergo a communal scalding stage Campylobacter and may in fact be under- early in the slaughter process combined estimated due to the existence of viable- with the fact that the skin remains on the non-culturable or highly stressed forms of carcase following all of the dressing proce- the organism in environmental samples, dures. including farm water supplies [109]. Contaminated shellfish have also been The high prevalence of campylobacters implicated as a vehicle in the dissemination in poultry flocks at the pre-harvest level is of Campylobacteriosis. Harvesting shellfish further exacerbated due to multiple oppor- from Campylobacter-contaminated waters tunities for cross-contamination to occur would appear to be the most likely cause of during slaughter and processing. The high infection [193]. Campylobacters have also throughputs of modern poultry slaughter been isolated frequently from asympto- plants has necessitated the development of matic companion animals, with symptoms automated equipment in, for example, the of enteritis frequently reported in younger stages of scalding, plucking and eviscera- animals [56]. Transmission of campylobacters tion. The net effect of processing large num- from pets to humans has been confirmed in bers of carcases from different sources very previous case studies and identified as a often leads to the dissemination of enteric potential risk factor in epidemiological inves- pathogens, including Campylobacter from tigations, particularly young children in the early stages of the slaughtering process. contact with puppies exhibiting enteritic Also, as skin is normally not removed from symptoms [158]. dressed carcases, large numbers of campy- lobacters can remain in situ on the finished 4.3. Control in foods of animal origin raw product thus increasing the likelihood of exposure to the consumer. A longitudinally integrated approach to controlling campylobacters along the entire 4.2. Campylobacters and other food food chain should be adopted for foods of animals animal origin, in particular, poultry. Con- trol should be directed primarily at the pre- The gastrointestinal tracts of other food vention of colonisation in food animals animal species have also been shown to be through the implemention of Good Hygi- frequently colonised with campylobacters, enic Practices (GHP), biosecurity measures particularly, C. jejuni and C. coli [99]. and husbandry practices which should be Reported rates of intestinal Campylobacter incorporated in Hazard Analysis Critical carriage in food animals have varied widely Control Point (HACCP) based risk man- between studies [23]. The digestive tract of agement systems [189]. Efforts at harvest clinically normal cattle has been demon- level should be concentrated on practices strated to be a significant reservoir for a number designed to control and reduce levels of faecal of Campylobacter spp. [12], with prevalences contamination during live bird transporta- of the enteropathogen in cattle ranging from tion, slaughter and carcase dressing [9, 191]. 0–80%. Prevalences of Campylobacter in In addition consumers and food handlers Campylobacter 357 should be made aware of the role that they 3.7% of all DALY’s), mainly through play in reducing the incidence of Campylo- infectious diarrhoea. Gastrointestinal dis- bacter infection by preventing cross-con- eases are often severe due to under-nutrition tamination in kitchens or food preparation and lack of intervention strategies in the areas [61]. developing nations and virtually 9/10 account Other potential options currently availa- for infant deaths alone [11]. Major enteric ble to reduce the levels of this enteropath- pathogens in the infant’s mortality include ogen on food animal carcases include, irra- rotavirus, Campylobacter jejuni, enterotoxi- diation [100], chemical decontamination genic bacteria (Escherichia coli, Shigella [190], steam pasteurization and hot water spp. and Vibrio cholerae 01) and possibly immersion [192]. enteropathogens (E. coli, Aeromonas spp. V. cholerae O139) enterotoxigenic Bacte- More recently, l’Agence française de roides fragilis, Clostridium difficile and sécurité sanitaire des aliments (AFSSA) Cryptosporidium parvum. All except the has published an extensive review article C. parvum are easily controlled by chlorin- entitled “Appréciation des risques alimen- ation of water, but re-contamination of taires liés aux Campylobacters: Application treated water is a huge problem. Emerging au couple poulet/Campylobacter jejuni”, environmental pathogens, such as Helico- which should be consulted for further infor- bacter pylori and Burkholderia pseudomallei, mation. This may be obtained on-line at may well be of significance in some http://www.afssa.fr. regions. In adults, much less is understood of various sequelae such as myocarditis, diabetes, reactive arthritis and cancers some 5. ENVIRONMENTAL months-years after initial infections. Also, CAMPYLOBACTERS besides the traditional pathogens (helminths, Entamoeba histolytica, Giardia lamblia 5.1. Water hepatitis A and E) various enteroviruses, C. jejuni and H. pylori are emerging issues Waterborne outbreaks associated with in adults. contamination of drinking water by Campy- lobacter jejuni are rather common in the 5.2. Sewage and water treatment plants Nordic countries Sweden, Norway or Fin- land, where in sparsely populated districts The presence of bacterial pathogens groundwater is commonly used without (Listeria monocytogenes, Campylobacter disinfection. Campylobacters, Escherichia coli and jejuni, Escherichia coli O157 and coli, or other coliforms have rarely been Salmonella spp.) in eight Swedish sewage detected in potential sources. Using a com- treatment plants (STP), with four different bination of Penner serotyping and pulsed- treatment methods, focusing on detection field gel electrophoresis (digestion with of zoonotic bacteria in raw and treated SmaI and KpnI), Hanninen et al. [57] stud- sludge were investigated [142]. Restriction ied three waterborne outbreaks in Finland enzyme analysis and pulsed field gel elec- caused by C. jejuni and used sample vol- trophoresis of Salmonella serotypes indi- umes of 4 000 to 20 000 mL for analysis of cated that Salmonella persists in STP and Campylobacters and sample volumes of 1 that there is a continuous supply of new to 5 000 mL for analysis of coliforms and strains. There are differences in treatment E. coli, depending on the sampling site, methods concerning the reduction of path- confirming the likely reservoir of an out- ogens and indicator bacteria. If spread on break. Poor water quality, sanitation and arable land, sludge increases the environ- hygiene account for some 1.7 million deaths mental load of pathogens and thereby a year world-wide (3.1% of all deaths and increase the risk for spreading diseases to 358 J.E. Moore et al. people and animals. Said et al. [143] water sources investigated. The use of these reported outbreaks of infectious diseases water sources for drinking and domestic over the last 30 years associated with pri- purposes poses a serious threat to the health vate water supplies (PWS). The majority and well-being of the users and calls for (16 outbreaks) were reported after the intro- urgent South African government interven- duction of enhanced surveillance. Although tion [112]. PWS only serve 0.5% of the population, 36% of drinking water outbreaks are asso- 5.3. Farms ciated with PWS. The main pathogen, Campylobacter, was implicated in 13 (52%) Campylobacter is the most commonly outbreaks. Most reported outbreaks (88%) reported notifiable disease in New Zealand. occurred in commercial or Category Two Savill et al. [150] investigated the reservoirs supplies, which potentially affect larger of Campylobacter in a defined geographi- populations. The main factors implicated in cal area within New Zealand and compared these outbreaks are temporary or transient strains isolated from humans and environ- populations, treatment (lack or failure), the mental sources within this area as a prelude presence of animals and heavy rains. The to investigating the likely transmission public health problem associated with PWS routes to humans. Campylobacter jejuni was could be prevented by the identification and commonly found in faeces from dairy cows, understanding of risk factors, by the proper beef cattle, sheep and ducks, chicken car- protection of water sources and adequate casses, sheep offal and surface waters and treatment and maintenance. This could be C. coli was commonly found in sheep fae- facilitated through the introduction of a risk ces. Minihan et al. [99] reported that the assessment as part of a scheme for the water prevalence of Campylobacter spp. faecal supplies. Ottoson [120] investigated the shedding within pens was positively corre- prevalence of pathogens (e.g. rotavirus, lated to the pen, the month of sampling and Salmonella typhimurium, Campylobacter the Campylobacter spp. contamination sta- jejuni, Giardia lamblia and Cryptosporidium tus of the pen dividing bars and the water parvum) in greywater in a local treatment trough surface. They suggested that Campy- system at Vibyasen (north of Stockholm, lobacter spp. should be considered as a Sweden) and the faecal load of these path- pathogen shed in the faeces of a substantial ogens and were used to form the basis of a proportion of feedlot cattle. Guan and Holley screening-level quantitative microbial risk [55] reviewed available international data assessment (QMRA) using faecal indicator and the developing situation in Western bacteria and chemical biomarkers. Growth Canada upon the survival of major patho- conditions for Salmonella in greywater sed- gens including Escherichia coli O157:H7, iments were also investigated and risk mod- Salmonella, and found significant variabil- elling based on replication in the system ity in resistance to environmental challenge increased the probability of infection from that are characteristic of the organisms them- Salmonella 1000-fold, but it was still lower selves. The survival of pathogens were than the risk of a rotavirus infection. The longer in environmental samples at cool microbial quality of several, usually untreated, temperatures but their abilities differed surface domestic water sources, used by when exposed in liquid and solid manure. rural communities in the Venda Region of Theoretical extrapolations from cattle manure South Africa, was assessed to gauge their environments, indicated that holding manure fitness for human consumption and to high- at 25 °C for 90 days would appear to render light the possible impact of waterborne dis- the cattle manure pathogen-free. However, eases. Salmonella, Shigella, Vibrio cholerae, with good hygienic practice during harvest, Campylobacter, Aeromonas and Plesio- a very low level of this pathogen can be monas were isolated from several of the achieved on dressed carcasses. Poultry, Campylobacter 359 particularly chickens, account for the major- Millar et al. [98]. Enterobacteriaceae and ity of human infections caused by Campy- Campylobacter jejuni, were determined by lobacter. Reduction or elimination of this quantitative real-time PCR (qPCR) than by pathogen in the poultry reservoir is an essen- cultivation [73], as some of these bacteria tial step in minimizing the public health may have been in a potentially hazardous problem. However, farm-based interven- active but non-cultivable state and this tion measures are still not available because method provides a viable alternative for of the lack of understanding of the ecolog- biosafety and hygiene monitoring reasons. ical aspects of C. jejuni on poultry farms Yang et al. [194] reported that retail chicken and Sahin et al. [141] have elaborately dis- meat, raw milk and environmental water are cussed the horizontal and vertical transmis- commonly contaminated with C. jejuni and sions of Campylobacter infections affected could serve as a potential risk for consumers by immune status of the poultry host and the in eastern China, especially if proper hygi- environmental conditions in the production enic and cooking conditions are not main- system. Eifert et al. [36] compared various tained. The rapid and sensitive detection of sampling techniques (cloacal swabs, faecal C. jejuni is necessary for the maintenance samples and environmental surface “drag” of a safe food/water supply in China and the swabs) on 3, 5 and 7 weeks old poultry birds real-time PCR assay provides a specific, for presence of Arcobacter butzleri, a sensitive and rapid method for quantitative causal agent of human enteritis and found detection of C. jejuni. Pearce et al. [124] that environmental swabs recorded the reported that although Campylobacter is highest percentage recovery, while intesti- highly prevalent in the intestinal tracts of nal tracts had none. Gaynor et al. [47] dem- swine arriving at the slaughter facility, this onstrated via gene expression studies the microorganism does not progress through capacity of C. jejuni to adapt to multiple the slaughtering operation and is not detect- environmental niches. The genetic evolu- able on carcasses after overnight chilling. tionary mechanisms of adaption provides Sharma et al. [152] critically examined the the first whole-genome molecular explora- potential of emerging water-borne patho- tion of the effect of laboratory culture and gens in both developed and developing storage on colonization and virulence prop- nations and the global epidemiology of a erties of this pathogen. In this respect, it is number of cases involving hepatitis viruses interesting to note that Campylobacter (including hepatitis E virus), Campylo- cases occurring in rural populations of bacter jejuni, microsporidia, cyclospora, Michigan, USA, attributable to poultry hus- Yersinia enterocolitica, calciviruses and bandry and of some cases occurred in indi- environmental bacteria like Mycobacterium viduals who were not poultry farmers by spp., Aeromonads, Legionella pneumophila occupation, is highlighted by the studies of and multidrug-resistant Pseudomonas aer- Potter et al. [131]. uginosa that have been associated with water-borne illnesses. It also examines the possible reasons, such as an increase in the 5.4. Food related environments number of immunocompromised individu- Novel employment of lactate dehydroge- als, urbanization and horizontal gene trans- nase release from porcine aortic endothelial fer, that may underlie their emergence. The cells (PAEC) as a quantitative marker of isothermal amplification method nucleic cytotoxic activity in thermophilic Campylo- acid sequence-based amplification (NASBA), bacter spp. including Campylobacter jejuni, which amplifies RNA, has been reported as C. coli, C. lari and urease-positive ther- useful for the detection of microbial patho- mophilic campylobacters (UPTC), from gens in food and environmental samples [29]. human faecal isolates, poultry and environ- More recently Birk et al. [18] have devel- mental sources has been demonstrated by oped a food-based model system which is 360 J.E. Moore et al. a suitable model system for the study of sur- tions of UPTC appeared, isolates of UPTC vival of C. jejuni in food systems. This have been reported in several European model employs chicken juice as the test countries (The Netherlands in 1997 [39], matrix and may be useful in anticipating the Northern Ireland in 1996, 1999 and 2003 survival of C. jejuni in foods, thereby lead- [66, 93, 193], England in 1998 [42]) and one ing to the development of new preservation Asian country (Japan in 1996 and 2002 [92, systems. 94]). Consequently, about 200 UPTC isolates have been found from the natural environ- ment, river water, sea water and shellfish, 6. ATYPICAL CAMPYLOBACTERS including wild birds, but not from any domestic or wild animals. Therefore, the In relation to human Campylobacteriosis, natural environment is an important reser- C. jejuni, C. coli, C. upsaliensis, C. hyoin- voir of the UPTC organisms. However, in testinalis, C. lari, C. fetus, C. sputorum bio- a study by Waldenstrom et al. [184] were var sputorum have been demonstrated to be unable to detect any UPTC organisms in a implicated as gastrointestinal pathogens, wild bird population in Sweden. though some are rare [71]. C. concisus, C. When On and Harrington studied the curvus, C. gracilis, C. rectus and C. showae taxonomic and epidemiological relationship are detected in association with the oral cav- among Campylobacter species by numeri- ity [82]. Alternatively, C. mucosalis, C. hel- cal analysis of amplified fragment length veticus C. sputorum and biovar faecalis are polymorphism (AFLP), a high level of isolated from animals [114, 161]. genetic diversity in C. lari, particularly Moreover, some other atypical and amongst UPTC isolates, was identified [117]. emerging Campylobacter organisms than Multilocus enzyme electrophoresis analy- those described above have interestingly sis, which were shown to be the most suc- been identified to occur for these ten years. cessful at discriminating UPTC organisms Therefore, the aim of the present article is at the subspecies level, whereas serotyping, to review atypical and emerging examples phage-typing, antibiogram typing and flag- among the genus. Five years after, follow- ellin typing were unsuccessful [101], also ing the original description of C. lari organ- demonstrated that the UPTC isolates (n = 31) isms by Skirrow and Benjamin in 1980 isolated from several countries and sources [155], Bolton et al. [20] isolated the first 10 examined are genetically hypervariable and atypical isolates of C. lari, urease-positive form a cluster separate from the C. lari thermophilic Campylobacter (UPTC) from (n = 3) cluster [94]. the natural environment in England in 1985 [20]. This was the first example of urease- In relation to the pathogenesis, Sekizuka producing bacteria among the genus et al. [151] has found short flaA-like Campylobacter. Then, four UPTC isolates sequences, containing internal termination were found for the first time from humans, codons (TAG), incomplete genes or pseu- two from the faeces with two diarrheal dis- dogenes of flaA in two Japanese UPTC iso- eases, one from the appendix with one lates [151]. Furthermore, shorter flaA genes appendicitis and one from the urine with without any internal termination codons than one urinary tract infection in 1986–1989 those of C. jejuni and C. coli were demon- [17, 96]. Until now, only these four clinical strated in the isolates of UPTC from the nat- cases of UPTC isolates have been pub- ural environment in England [151] and in lished. However, any association of UPTC Northern Ireland (T. Gondo, unpublished with human disease still remains unclear. data). The reason(s) why any of UPTC UPTC organisms were demonstrated to organisms have not been identified as a belong within C. lari possibly as a biovar cause of gastrointestinal disease for humans, [122] or a variant [96]. After these descrip- may partly be due to the shorter and/or Campylobacter 361 shorter pseudogene structure of flaA. No and for the genus as a whole was performed phenotypic and genotypic characteristics of [70]. Three unidentified 16S rDNA Campy- urease from UPTC have yet been described. lobacter genus-specific amplicons of faecal In 1983–1985, C. hyointestinalis organ- origin were sequenced and demonstrated to isms, distinguished from previously described be 99% similar [70]. These were previously catalase-positive Campylobacter species undescribed and uncultivated Campylo- by colony morphology, ability to produce bacter species. The organism from faeces, specific PCR assay, was detected in 10 of H2S in triple sugar iron agar, glycine toler- ant, intolerant to 3.0% NaCl, ability to grow the 20 faecal samples but not in any saliva 25 °C, sensitive to cephalothin and resistant samples. Then, the authors proposed to to nalidixic acid, were first isolated from the term “Candidatus Campylobacter hominis” intestines of pigs with proliferative enteritis [70]. Nextly, they developed an isolation and other animals (faeces from cattle and strategy employing initial non-selective intestine of a hamster) [49, 50]. About ten membrane filtration onto fastidious anaer- years after from the first description of C. obe agar for the uncultivated C. hominis intestinalis, seven isolates resembled but organisms [72]. The unique species status distinct from the type strain and other refer- of the isolates, whose nearest phylogenetic ence strains of C. intestinalis were obtained neighbours were C. gracilis and C. sputo- from porcine stomachs [116]. Based on the rum, was further confirmed by taxonomic numerical analysis of 38 phenotypic char- study of 47 phenotypic characteristics [72]. acters, DNA-DNA hybridization studies C. lanienae is a new catalase-positive and DNA base compositions, On et al. [116] species that was first described from the proposed the name C. hyointestinalis subsp. faeces of healthy abattoir workers in Swit- lawsonii. Alternatively, C. hyointestinalis zerland in 2000 [81]. Nucleotide sequence subsp. hyointestinalis was accordingly given of the 16S rDNA, DNA-DNA homology [116]. When Harrington and On examined test and G+C content of genome DNA dem- phylogenetic relationships of C. hyointesti- onstrated that the new organism constituted nalis subspecies by means of 16S rDNA a previously undescribed species, whose near- sequences, they found that the sequence est phylogenetic neighbours were C. hyoin- identities among C. hyointestinalis subsp. testinalis subsp. hyointestinalis, C. fetus lawsonii isolates exceeded 99.9% and among and C. mucosalis [81]. Rapid PCR-biprobe C. intestinalis subsp. hyointestinalis iso- identification scheme based on the real- lates ranged from 96.4 to 100% [58]. time PCR was developed for the Campylo- Sequence identities between isolates repre- bacter taxa pathogenic for humans, including senting the two subspecies ranged from C. lanienae [82]. This new organism has 95.7 to 99.0%. Surprisingly, an interventing also been isolated from the faeces of six sequence was identified in the C. hyointes- healthy pigs in Japan [148] and from the tinalis subsp. lawsonii strains [58]. AFLP fin- faeces of bovine and beef cattle, in the beef gerprinting method was also demonstrated cattle, C. lanienae was the most frequently to allow the classification of the C. hyoin- detected species (49%), in Canada [62, 63, testinalis at the subspecies level [35]. 115, 118]. In addition, an intervening When, in 1998, Lawson et al. [70] exam- sequence of 226 bp in the 16S rDNA was ined saliva and faeces from 20 healthy indi- found in four isolates of the six of C. lan- viduals in order to know the variety of ienae in Japan [148]. campylobacters in their gastrointestinal In 1998, On et al. first identified the tract, PCR assays specific for nine species 15 strains isolated from faeces of 14 cattle of Campylobacter (C. sputorum, C. concisus, in United Kingdom and one human diar- C. upsaliensis, C. helveticus, C. lari, C. fetus, rhoea in Canada among 44 catalase nega- C. hyointestinalis, C. jejuni and C. coli), tive and urease-positive Campylobacter 362 J.E. Moore et al. group as a new C. sputorum biovar parau- tic animals (pigs, bovines and cattle) in reolyticus, by a phylogenetic study based Europe and North America (Canada), More- on phenotypic characterization, numerical over, seven isolates of C. hyointestinalis analysis of whole-cell protein profiles, DNA- subsp. lawsonii have been isolated from the DNA hybridization and sequence analysis porcine stomach in UK. Thus, healthy of 16S rDNA [115]. They demonstrated the domestic animals including wild animals clonality of C. sputorum bv. paraureolytics could potentially be important reservoirs of determined by macrorestricion profiling these new atypical and emerging organisms and biotyping by using the 18 isolates iso- of Campylobacter in humans. lated over a 12-month period from seven dairy cows contained in a single herd [181], Their study also indicated that the organism 7. ANTIBIOTIC RESISTANCE can persist in cattle for a long-term, at least 12 months. Campylobacter enteritis is considered to Alderton et al. in 1995 [5] reported the be a zoonotic disease, and domestic animals 11 isolates from intestinal lesions of pigs such as poultry, cattle and pigs can act as with proliferative enteritis including an sources of infection [59, 95]. Transmission organism formerly described as strain RMIT to man usually results in sporadic infection, 32AT as a new name C. hyoilei sp. nov. [33]. and is often associated with improper han- The phenotypic characteristics of these dling or cooking of food. The majority of organisms indicated that they are closely cases of clinical Campylobacter enteritis related to each other and are not isolates of are sufficiently mild or self-limiting not to other Campylobacter spp. commonly iso- require antimicrobial chemotherapy [6]. lated from pigs. They also suggested that Nevertheless, in severe or recurrent cases this organism is more closely related to C. where antibiotics are required, susceptibil- jejuni than C. coli based on the sequence ity testing is important to ensure appropri- differences of 16S rDNA. However, it was ate and timely treatment [13, 134, 179]. confirmed that C. coli strains and C. hyoilei Serious systemic infection may also be strains were indistinguishable based on treated with an aminoglycoside such as gentamicin [153]. Tetracyclines have been examining a variety of phenotypic and gen- suggested as an alternative choice in the otypic criteria and both represent the same treatment of clinical Campylobacter enteri- species [180]. Although differentiation tis, but in practice are rarely used. However, between C. hyoilei and C. coli using geno- macrolides remain the agents of choice, and typic and phenotypic analyses were dem- resistance rates to erythromycin remain onstrated, the taxonomic subcommittee of comparatively low [104]. Fluoroquinolo- the International Committee on Systematic nes, offer an effective therapy, against most Prokaryotes finally concluded that the epi- enteric pathogens, to treat acute bacterial thet ‘hyoilei’could in principle be revised as diarrhoea; with ciprofloxacin being used an infrasubspecific designation. Therefore, extensively as prophylaxis for travellers the subcommittee discouraged the use of [52]. Emergence of resistance to these the name C. hyoilei. agents however, has since made their effi- In conclusion, for the last ten years, cacy less certain. Resistance was reported about 200 UPTC isolates have found only to develop among patients after treatment in the natural environment in Europe and with fluoroquinolones [14], and was also Japan, whereas several atypical and emerg- found to coincide with the introduction of ing Campylobacter taxons (C. hominis, these agents in veterinary medicine [1, 2, C. lanienae and C. sputorum subsp. parau- 38]. However, an increasing number of reolyticus) have been newly found mainly Campylobacter isolates resistant to these in the faeces of healthy humans and domes- drugs are now being cultured from both Campylobacter 363 clinical and food samples in several Euro- method are lower when compared to similar pean countries, Canada and the United values obtained by the agar dilution method States. Since the 1990s, a significant increase regardless of the organism tested [41, 48, 60]. in the prevalence of resistance to macrolides However when E-test and the agar dilution and fluoroquinolones among Campylo- method are used on a small number of iso- bacter spp. have been reported and this is lates from a single geographic location, accept- recognised as an emerging public health able agreement between both approaches problem in many European countries [40]. for susceptibility categorisation is achieved. Entry of these isolates into the food chain For larger collections, microdilution is the could represent a significant threat to public preferred protocol especially when suscep- health. tibility to nalidixic acid and trimethoprim- sulfamethoxazole are being considered [84]. 7.1. Antimicrobial susceptibility testing Molecular techniques offer an alternative in Campylobacter spp. means of assessing antimicrobial resistance Several laboratory methods, including among bacterial isolates. In a study of qui- disc diffusion, broth microdilution, agar nolone-resistant Campylobacter a majority dilution and the Epsilometer-test (E-test) of isolates analysed were shown to possess have been applied to determine in vitro sus- a common mutation [128]. The predominant ceptibility profile(s) of Campylobacter to a genetic alteration responsible for confer- range of antimicrobial agents [15, 41, 45, ring resistance to ciprofloxacin in C. jejuni 46, 48, 84, 119, 149]. Despite the availabil- and C. coli is the result of a mutation in the ity of comparable standardised procedures gyrA gene, whereby many isolates tested for many organisms, based on, the approved demonstrated a Thr-86-Ile substitution in the guidelines defined by the National Com- A-subunit of DNA gyrase [185]. A Mismatch mittee for Clinical Laboratory Standards Amplification Mutation Assay (MAMA)- (NCCLS), no internationally accepted cri- PCR has been successfully applied to the teria for susceptibility testing of Campylo- detection of ciprofloxacin resistance in bacter spp. are available and breakpoints do C. jejuni and C. coli, and this protocol is a not exist. Consequently it is not possible to convenient screening tool among these iso- directly compare the resistance profiles of lates [196]. This method used a conserved isolates cultured from various origins. forward primer and a reverse diagnostic More, recently however, the NCCLS Sub- primer, which together generate a 264-bp Committee on Veterinary Antimicrobial Susceptibility Testing approved an agar product that is a positive indication of the dilution protocol as a valid method. presence of the Thr-86-Ile amino acid substi- tution, consistent with resistance to cipro- Several authors have compared the per- floxacin. A “real-time” PCR-based approach formance of the methods above and was recently developed to detect the C-to-T reported a correlation between E-test and agar dilution methods. Values determined nucleotide polymorphism associated with however can vary depending on the antimi- the latter amino acid substitution in the crobial agent(s) being considered [48]. This gyrA-encoding gene [34]. In this case, fluores- observation was particularly evident with cence resonance energy transfer technology respect to C. jejuni [84]. Comparing MIC (FRET) can be applied to the analysis of values obtained by E-test and the agar dilu- melting curves when a specific probe tion protocols, Ge et al. [48] reported values hybridises to a DNA template. This proto- ranging from 21.4 to 62% for gentamicin col can be adapted to include additional and nalidixic acid respectively. Whilst the mutations providing rapid and reproducible E-test is convenient, relatively simple to screening methods for ciprofloxacin resist- perform, MIC values determined by this ant Campylobacter isolates. 364 J.E. Moore et al.

Undoubtedly, one of the advantages of The use of antimicrobial agents on farm using these methods includes the possibil- animals, both to treat infection and as ity of direct detection from a sample obvi- growth promoters is a cause of concern, and ating the need for culture [28]. Molecular the increasing rates of resistance among methods can facilitate analysis of organ- Campylobacter spp. to these agents appear isms that may be sub-lethally damaged and to make a conservative policy on the use of difficult to grow, and these strategies also antibiotics in farm animals advisable [1, offer the possibility of screening large numbers 127]. Antibiotics of the macrolide-lincosa- of isolates for a specific mutation within a mide group have been used in treating food single assay. The disadvantages of using animals worldwide for several decades. molecular detection methods include the Their uses have included the control of dys- failure to detect resistance if a new, unexpected entery and Mycoplasma infections in or rare resistance mechanism is present swine, and for treating mastitis in cattle [104], and the necessity to perform a sepa- [40]. The use of macrolides and other com- rate assay for each antimicrobial agent tested. pounds for growth promotion has been Furthermore, no standards exist for per- banned, in all European Union countries forming genetic testing methods [28]. For with effect from July 1999. Fluoroquinolo- these reasons, it may be more useful to com- nes are available for treating food animals bine phenotypic and genotypic methods of in many countries, and Table I shows the veterinary licensing “time-line” of this group susceptibility testing. of antibiotics in a number of European countries. It is difficult to evaluate the actual 7.2. Surveillance of antimicrobial usage of these agents in food animals, but it resistance in Campylobacter spp. is noteworthy that fluoroquinolone treat- ment of Campylobacter-colonised broiler Transmission of antimicrobial resist- chickens has induced fluoroquinolone resist- ance from food animals to humans can ance under experimental conditions [65]. occur via the food chain [126, 133]. It is dif- Supplementing animal feed with antibi- ficult to determine the precise extend of the otics is estimated to constitute more than risk posed to human health [127]. Neverthe- half the total antimicrobial use worldwide less, food animals are a significant reservoir [188]. It has been reported that in Denmark of antibiotic resistant zoonotic pathogens. the consumption (per animal) of antibiotics Continuous monitoring of susceptibility pro- such as macrolides and tetracyclines in files of Campylobacter spp. to a panel of agriculture was 2–4 times higher than con- antimicrobial agents is necessary for a number sumption (per patient) in human medicine of reasons. Firstly, there are increasing rates [1]. Emergence of antimicrobial resistance of resistance to the agents of choice used in among zoonotic pathogens has led to the the treatment of clinical enteric infection development of a continuous surveillance [1]. This suggests a need to supply alterna- system of antimicrobial resistance among tive antimicrobials which remain therapeu- bacteria isolated from pigs, cattle and broil- tically effective. Secondly, the emergence ers in Denmark. The Danish Integrated of multidrug-resistant (MDR) organisms Antimicrobial Monitoring Programme (DAN- must be monitored carefully [86]. Finally, MAP) has set out to establish a baseline for mechanisms for the transfer of resistance comparison with future prospective studies both within Campylobacter spp. and between to enable the determination of trends over different genera of enteric organisms by time [1]. Monitoring strategies such as this means of mobile genetic elements may may have a positive impact on the effective present a significant threat to the continued treatment of human enteric Campylobacter efficacy of antimicrobial chemotherapy infection. To date, with the exception of [76, 113]. ciprofloxacin resistance, there is a scarcity Campylobacter 365

Table I. Veterinary licensing of fluoroquinolones in selected European countries.

Country Antimicrobial Licensing year Animal species

Ireland Enrofloxacin Prior to 1987 Cattle, pigs, poultry United Kingdom Enrofloxacin 1993 Cattle, pigs, poultry Danofloxacin 1993 Poultry Marbofloxacin 1995 Cattle Difloxacin 1998 Poultry Denmark Enrofloxacin 1991 Cattle, pigs, poultry Danofloxacin 1993 Poultry Difloxacin 1998 Poultry, turkey Marbofloxacin 1998 Cattle, pigs, dogs, cats Spain Enrofloxacin 1986 Cattle, pigs, poultry Difloxacin 1998 Poultry The Netherlands Enrofloxacin 1987 Cattle, pigs, poultry Difloxacin 1998 Poultry France Enrofloxacin 1991 Cattle, poultry Danofloxacin 1996 Cattle Marbofloxacin 1993 Cattle Difloxacin 1998 Poultry of scientific evidence for the transmission transfer of such resistance determinants of antimicrobial resistance as a direct result (acquisition) together with any genetic mod- of the use of antimicrobial agents in veter- ification of pre-existing genes through point inary medicine [127]. mutations (intrinsic) or some other genetic event, are thought to be the main mecha- 7.3. Genetic mechanisms associated nisms contributing to bacterial resistance [2, 10, 140]. Self-transmissible elements with antimicrobial resistance including plasmids, transposons and bacte- in Campylobacter spp. riophage all facilitate the acquisition and Bacterial populations can respond to the subsequent dissemination of resistance threat of an antimicrobial agent by evolving determinants. In addition, integrons, when some type of resistance mechanism(s) [138, associated with plasmids and/or bacteri- 159]. The imposed selective pressure ophage are now considered efficient vehi- results in the development of a correspond- cles for the transfer of resistance markers ing resistance determinant, either through among unrelated bacterial populations [24]. direct acquistition or intrinsically by mod- The isolation rate of plasmids from ification of a host gene target, designed to Campylobacter spp. has been shown to vary facilitate evasion of the inhibitory substance. considerably between 44 and 91% for clin- For example environmental selection follow- ical and poultry isolates respectively in one ing enrofloxacin treatment of chickens infected study [75], compared with a plasmid isola- with fluoroquinolone-sensitive Campylo- tion rate of 19% reported in a separate study bacter spp. resulted in the emergence of the [14]. As shown in Table II, tetracycline, corresponding resistant isogenic strains kanamycin and chloramphenicol resist- suggesting that this organism is hypermutat- ances are primarily plasmid-mediated. His- able under these conditions [87]. Horizontal torically, tetracycline resistance has been 366 J.E. Moore et al.

Table II. Genetic mechanisms responsible for antimicrobial resistance detected to date in C. jejuni and C. coli.

Antibiotic Mechanism of resistance Reference

Aminoglycosides (with the Chromosomal: enzymatic modification of antibiotics. [113, 175] exception of kanamycin) Integron-mediated resistance Kanamycin Majority plasmid-borne, remainder chromosomal; [140] resistance through enzymatic modification of kanamycin Chloramphenicol Plasmid-borne, resistance through modification of [186] the target site (ribosome) or alteration of the antibiotic Ciprofloxacin Chromosomal: modification of gyrA and parC confers [3, 4, 16] resistance Erythromycin Chromosomally mediated, resistance through [166] modification of the target site (ribosome) β-Lactams Chromosomal; three mechanisms, decreased uptake [129] through modification of a porin, alteration of a penicillin binding protein, or production of a β- lactamase Tetracycline tetO gene, plasmid-borne in the majority of cases, [7, 78, 167] resistance mediated through ribosomal protection

Trimethoprim dfr1 gene, chromosomal, located to the remnants of an [53] integron dfr9 gene, chromosomal, located to the remnants of a transposon Resistance arising through modification of the trimethoprim target Multidrug-resistance Efflux pump with a broad specificity; preventing [25] (MDR) accumulation of antibiotics particularly well researched and docu- only 15.9% of the isolates analysed for mented [173] The tetO gene conferring tet- plasmids contained these mobile elements racycline resistance has a G+C content of and none of the tetracycline resistant strains 40% [90], which is close to that of the tetM were found to harbour plasmid DNA [10]. gene of Streptococcus pneumoniae, with Chloramphenicol resistance, although rare which it shares 75% homology [89]. It is in campylobacters [10], has also been significantly higher than that for C. jejuni reported to be plasmid mediated [140]. A chlo- and C. coli chromosomal (32.5 mol%) and ramphenicol resistance determinant cloned plasmid (33 mol%) DNAs [90, 169]. Based from a C. coli plasmid was sequenced and on this evidence, Taylor and Courvalin found to have 43 and 57% homology with [168] have suggested that the tetO gene was chloramphenicol acetyltransferase (CAT) pro- acquired by Campylobacter spp. from a teins from other Gram-positive and -negative Gram-positive coccus, and that divergence origins [186]. A kanamycin resistance deter- occurred over time. More recently, in Brazil, minant, aphA-3 was found located distal to Campylobacter 367 this cat gene and Sagara et al. [140] reported and from chickens [40] and detection of a link between kanamycin and chloram- erythromycin resistance may be determined phenicol resistance in Campylobacter fol- by PCR methods [163]. Nevertheless, lowing a number of cloning experiments erythromycin is considered to be one of the involving the cat and kan resistance genes safest drugs effective against Campylobacter. of a plasmid from a multiple-antibiotic- Resistance may also develop during the resistant C. coli isolate. course of human treatment. Similar to Kanamycin resistance in Campylobacter tetracycline (outlined above), erythromycin is more commonly associated with C. coli is a potent inhibitor of protein synthesis, than with C. jejuni [140]. Like tetracycline binding reversibly to several ribosomal resistance, kanamycin resistance determinants targets including the Domain V-located on were located both on the chromosome and the 23S rRNA gene locus, in addition to the on self-transmissible plasmids [168]. These ribosomal structural proteins, L2, L4, L15 determinants are frequently found on the and L22. Three major point mutations occuring within the former locus, and same plasmids as tetracycline-resistance responsible for erythromycin resistance, determinants [67, 170]. Kotarski et al. [67] were defined [177]. The MIC in each case also observed that the Kmr determinant were 128 µg/mL, as defined by the agar could translocate between plasmid and dilution method. A combined PCR-RFLP chromosomal DNA, suggesting that the Kmr assay was evaluated as a direct means of determinant in campylobacters may be located detection. Similarly, a PCR-based line-probe on a transposable element of approximately assay focusing on the 23S rRNA target only, 4 kb. A number of genes responsible for r was developed as a simple means to detect Km in campylobacters have been identified isolates resistant to erythromycin [111]. including aphA-1, aphA-2, aphA-3 and aphA- Whilst useful, this approach detected only 7, with a plasmid location reported for both 50% of mutations arising in resistant Japanese aphA-3 and aphA-7 [168, 172]. The aphA- isolates analysed. It is consieveable that not 3 gene is often found on large plasmids that all of the mechanisms contributing to also encode tetO. aphA-3 like tetO is macrolide resistance in Campylobacter have thought to have originated from a Gram- been described to date. The possibility arises positive source and is commonly found in that at least some of these will be MIC- staphylococcal and streptococcal species dependent. Vacher et al. [177] did not consider [68]. aphA-7 determinants on the other the possible involvement of the ribosomal hand have been reported to be found on structural proteins, whereas Corcoran and small plasmids that do not encode any other Fanning (unpublished) sequenced several resistance determinants [171]. Although L4 and L22 genes and defined associated aphA-7 has been shown to have a broad host polymorphisms in resistant isolates. None range, in that it can be expressed in both of these isolates possesed any of the E. coli and S. gordonii, its low G+C ratio corresponding nucleotide polymorphisms (32.8%), matching that of C. jejuni, suggests in the 23S rRNA gene. Similarly, the this gene may be indigenous to campylobacters involvement of an efflux system (below) [172]. A chromosomal location for aphA-1 cannot be ruled out [88]. Inhibition of this has been reported for a Campylobacter-like pump system with phenylalanine-arginene organism [121]. Table II lists some of the b-naphthylamide (PAβN) was shown to potential means by which Campylobacter restore susceptibility to Campylobacter in acquires antimicrobial resistance markers. a dose dependent manner. Macrolides are the agents of choice for Fluoroquinolones are important drugs treating Campylobacter infections. Resistance used in human and animal medicine, and to erythromycin is mainly found in strains are often the agents of choice used to treat of animal origin, especially C. coli in pigs campylobacteriosis in humans. Currently, 368 J.E. Moore et al. in the European Union, fluoroquinolones more gene cassettes and convert them into are licenced for use in a number of food functionally expressed genes [135]. It is animals, however emerging resistance to this these gene cassettes that encode the resist- important class of antimicrobial is recognised ance determinants to several antimicrobial as a significant public health problem [128]. agents [24]. This observation may in turn, lead to Campylo- Although several classes of integrons bacter-associated deaths among vulnerable have been described to date, class 1 are clin- members in the community. Resistance to ically significant. A typical class 1 integron fluoroquinolones arises following a point includes two conserved segments (CS), mutation [ACAATA], which produces a Thr-86-Ile amino acid substitution in the known as the 5’- and 3’-CS segments, Quinolone Resistance Determining Region flanking the central gene cassette. An intI (QRDR) of the gyrA subunit-encoding gene gene encoding an integrase enzyme is [185]. Genetic alterations in this region are located within the 5’-CS, which is respon- often associated with high-level resistance sible for the recombination of an incoming to nalidixic acid (MIC > 64–128 µg/mL) gene cassette at a specific att1 attachment and ciprofloxacin (MIC > 16–64 µg/mL). site. Also, within this region is a promoter Similarly, resistance to fluoroquinolnes may which facilitates the efficient expression of also be associated with the activation of a any integrated gene cassette. The 3’-CS multi-gene efflux pump system (see below contains two open reading frames (ORFs) and [25]). encoding resistance to quaternary ammo- The presence of chromosomally-located nium compounds (qac) and sulphonamide transposons among Campylobacter spp. (sul1), respectively. Integrons can incorpo- had not been reported prior to 1998. The rate and express more than one gene cas- dfr9 gene-encoding trimethoprim resist- sette, provided that its location is flanked by ance was located to the remnant of a trans- the 5’- and 3’-CS domains. Thus integrons poson inserted in the genome of a number may contain a number of recombined gene of clinical C. jejuni isolates, [53]. The G+C cassettes, oriented in a classical “head-to- content of dfr9 was found to be 40%, (sim- tail” arrangement, conferring a multi-drug ilar to the tetO gene described earlier), resistant (MDR) phenotype on any isolate which is considerably higher than that of in which these genetic elements are located. Campylobacter spp, and was previously Previously, integron-like structures were detected in a transposon located in the reported in Campylobacter isolates raising genome of porcine isolates of E. coli [26]. A study by Richardson and Park [136] iden- the possibility that these elements may tified an insertion sequence that was encode antimicrobial resistance and possi- flanked by direct repeat sequences in the bly function as a potential vehicle for dis- chromosome of certain isolates of C. jejuni, semination of resistance among Campylo- which appeared to be a non-functional bacter spp. [85]. Gibreel and Sköld [53], transposable element and the spread of anti- reported the existence of chromosomally biotic resistance under natural conditions located integrons carrying a dfr1 containing imay be due to a combination of gene trans- gene cassette (Tab. II) in C. jejuni. In a fer systems acting in parallel or in series [30]. recent study investigating of a large collection of unrelated Campylobacter spp., isolates 7.4. Gene cassettes and class 1 integrons of both human and animal origin, complete in Campylobacter spp. class 1 integrons were identified [113]. In this case, the gene cassettes contained two Integron structures are naturally occur- ORFs, one of which conferred resistance to ring gene expression systems that can the aminoglycoside antibiotics, streptomy- potentially capture and integrate one or cin/spectinomycin. Campylobacter 369

As the use of aminoglycoside therapy may Campylobacter-mediated efflux system be considered as an appropriate treatment referred to as the cmeABC-operon. This option for some Campylobacter-related three gene operon efflux pump system con- infections, the recent identification of integrons tributes to multidrug resistance (MDR) in containing aminoglycoside-encoding genes C. jejuni and probably in C. coli also (Cor- (aadA2 and aac4), suggests that the coran and Fanning, unpublished) and con- possibility now exists for treatment failure sists of an inner-membrane transporter to occur, due to the presence of these genetic (encoded by the cmeC gene), a periplasmic elements in Campylobacter spp. [76, 113]. fusion protein (cmeB) and an outer-mem- Furthermore, the presence of class 1 integrons brane channel protein (cmeA). Susceptibil- in several Campylobacter isolates may in ity studies by Lin et al. [79, 80] using wild part offer an explanation for the high levels type and isogenic mutants in C. jejuni dem- of resistance to sulphonamides, frequently onstrated that inactivation of the CmeABC reported among these organisms. Increasing pump by insertional mutagenesis substan- prevalence of macrolide- and quinolone- tially increased the susceptibility of C. jejuni resistance is more usually attributed to specific to several classes of antimicrobial agent(s) mutations in chromosomally located genes, and also to heavy metals and bile salts [79]. though the future involvement of plasmid Resistance to bile salts may be a necessary encoded integrons cannot be ruled out. step for successful colonization of animal O’Halloran et al. [113] suggest that integrons intestines, contributing to bacterial patho- may be partly responsible for horizontal gensis [87]. gene transfer as a potential vehicle for dissemination of MDR phenotypes among Efflux mechanisms have an important Campylobacter spp. These findings may impact on antimicrobial resistance [187]. have further implications for future therapeutic Resistance by efflux can be easily dissem- strategies, leading to reduced drug efficacy inated [91]. In several cases the genetic ele- and/or treatment failures in the case of ments encoding efflux pumps and their reg- MDR organisms, whose transmission through ulators are located on plasmids, or on the food chain poses a real threat to public conjugative or transformable transposons health. located on plasmids or in the chromosome. More importantly, efflux mediated resist- ance mechanisms can spread between phy- 7.5. MDR-mediated by antimicrobial logenically very different species. This has efflux systems been exemplified by the macrolide-medi- Active efflux pumps are known to con- ated efflux, not only among streptococci, tribute to intrinsic and acquired resistance but also to other Gram-negative bacteria to a range of antimicrobial agents [79, 87, [83]. Co-transfer with genes for other resist- 91]. These pumps reduce the intracellular ance classes may also take place if these are accumilation of antimicrobial agents and present together on large mobile genetic other compounds and this feature is now elements. recognised as a major mechanism of resist- In conclusion, thermophilic Campylo- ance in pathogenic organisms [110, 130, bacter spp. are among the commonest bac- 178]. Comparitive genomics has identified terial cause of gastroenteritis in developed a number of efflux transporters and some of countries. Our knowledge of this organisms these are classified as H+-antiporters. In epidemiology is limited. Campylobacterio- Campylobacter spp., the resistance to nod- sis is a zoonosis, and farm and companion- ulation and cell division (RND) family animals are significant reservoirs of the [176] is associated with high-level fluoro- organism with the potential for transmis- quinolone resistance [79, 132, 187]. This sion to humans. There is evidence that fresh resistance is linked to the activation of the meat, especially poultry, is a major source 370 J.E. Moore et al. of infection [164]. In addition, antibiotic 8. CAMPYLOBACTER DETECTION resistant C. jejuni and C. coli are now being reported with increased frequency [86, Campylobacter species and in particular 113]. Erythromycin and less commonly, Campylobacter jejuni and Campylobacter ciprofloxacin, remain the agents of choice coli are the most common cause of gastro- for the treatment of severe or recurrent enteritis in humans in the developed world. Campylobacter enteritis in humans. How- Ever since its recognition as a cause of dis- ever, it has been suggested by some inves- ease in humans, detection of this zoonosis tigators that fluoroquinolone and macrolide has relied on culture-based methods. In use in animals (for treatment and preven- fact, the original development of a culture tion) leads to the development of resistance media for the isolation of Campylobacter among human isolates [38], whilst others from human faeces by Martin Skirrow in suggested that resistance in C. jejuni and 1977 [154] helped to firmly establish its C. coli can be accounted for, at least in part, role in human disease. Prior to this work, by use of antimicrobials to treat human Campylobacter detection was reliant on infection. The association between the use membrane filtration of faecal samples onto of valuable drugs in veterinary medicine non-selective media, a laborious and cum- and the emergence of resistance in human bersome method. Although Skirrow’s isolates and visa versa requires a more com- medium was effective for isolating campy- plete understanding. lobacters from human faeces, it was less suitable for animal and environmental Treatment with antimicrobials is a risk specimens, owing to the presence of con- factor for infection with organisms that are taminating species. This led to the develop- simultaneously resistant to several drugs ment of the more selective Preston medium and this may contribute to mortality [59]. by Bolton and Robertson [19] suitable for Horizontal gene transfer is a significant the isolation of Campylobacter from foods mechanism for disseminating antimicrobial and environmental samples. In subsequent resistance among unrelated bacterial years following these publications, further populations [10, 126]. Integron structures improvements have led to more sensitive play a pivotal role and have been identified and selective media for the improved detec- in several Gram-negative bacterial species tion of Campylobacter in faecal samples. including food-borne pathogens, such as However, even with these improvements Salmonella spp., E. coli and Shigella spp. culture-based methods have a number of [31, 138, 159]. Studies are now reporting limitations. The methods are slow and in the the existence of these structures in Campy- case of human faecal samples require up to lobacter and therefore their role and contri- 48 h to yield a presumptive isolate, which bution to antimicrobial resistance must be then requires confirmation using pheno- assessed [76, 113]. typic tests. In the case of food samples, Overall, amplifying the reservoir of where cell numbers can be low in a back- resistance by whatever means is inherently ground of high numbers of other competing problematic, making transmission to humans flora, enrichment culture in broth media is via food or other means more likely. Quan- required to recover small numbers of cells titative evaluation of associated risks will prior to plating on selective media. This can lead to the development and effective lengthen the detection process with up to implementation of rational guidelines for five days bring required to achieve a result. antimicrobial use [59, 127]. Eliminating Culture-based methods may also select against Campylobacter transmission via the food less common species, such as Campylobacter chain must remain a veterinary and public upsalienesis and Campylobacter lari, leading health priority. to possible misdiagnosis and underestimation Campylobacter 371 of the true burden of infection with these sample preparation methods and the use of other species. gel electrophoresis end-point detection The limitations of culture-based proce- methods, requiring manipulation of ampli- dures led to the development of alternative fication products following PCR cycling, methods for the detection of campylo- hampered the transition of these methods bacters in foods and faecal samples. The from research to routine microbiology labora- development of both poly and monoclonal tories. Adaptation of PCR assays into a micro- antibodies specific for campylobacters has plate hybridisation format or PCR-ELISA facilitated the development of a number of increased the specificity and sensitivity of antibody-based tests. Latex agglutination detection. Lawson et al. [71] developed a tests for the identification of presumptive panel of PCR-ELISA assays which they Campylobacter isolates have been devel- used in a large-scale survey of the detection oped, which can provide rapid more rapid of Campylobacter species in human gastroen- species confirmation than conventional teritis. The assays could detect and differen- tiate between C. jejuni, C. coli, C. upsaliensis, phenotypic tests [103]. A commercial enzyme- C. helveticus, C. fetus, C. hyointestinalis and linked immunosorbent assay (ProSpecT C. lari with the PCR-ELISA results being Microplate assay; Alexon-Trend, USA) compared with conventional culture meth- was developed for the detection of C. jejuni ods. The PCR-ELISA assays detected and C. coli directly in faecal samples, from campylobacters in culture-negative faecal humans with gastroenteritis [174]. This samples and also more importantly, detected assay was demonstrated to have a sensitiv- mixed infections with more than one Campy- ity of 96% and a specificity of 99% when lobacter species. The assays did provide applied to 50 Campylobacter culture-posi- information on the identity and occurrence tive and 114 culture-negative faecal speci- of species that are not detected by culture, mens [174]. A second prospective study of however the authors did note that PCR was 1205 faecal samples demonstrated similar more expensive and labour-intensive than results with a sensitivity of detection of culture. The use of such assays may prove 97.7% [32]. The assay also provided more useful in further large-scale epidemiologi- rapid results when compared to conven- cal surveys of Campylobacter infection and tional culture, with results being available provide evidence of the role of Campylo- within hours rather than days. Such rapid bacter species, other than C. jejuni and methods may prove useful in cases where C. coli, in human disease. early diagnosis may alter patient manage- The first report of a PCR assay for the ment or treatment. detection of campylobacters in foods was Since its discovery PCR has impacted on made by Giesendorf et al. [54] who described virtually all areas of microbiology and in a PCR assay for the rapid and sensitive particular has been used to detect microbial detection of Campylobacter species in pathogens in a wide range of sample types. chicken products. The assay was applied to The first application of PCR for the specific the detection of Campylobacter species in detection of C. jejuni and C. coli was both naturally contaminated and artificially described in 1992 [123]. The assay targeted inoculated samples of chicken skin follow- the flagellin A gene of C. jejuni and C. coli ing enrichment culture of the samples for and was demonstrated to be specific for 18 h in Preston enrichment broth. The assay these two species and successfully detected demonstrated a limit of detection of 25 CFU 30–60 bacteria per PCR reaction in seeded of Campylobacter species per gram of human faecal samples. This report also tissue following the 18 h enrichment. The demonstrated the potential of the PCR- use of PCR for the detection of Campylo- based methods to detect very low numbers bacter in foods is hampered by the rela- of Campylobacter cells. However, complex tively large sample size (25 g in most 372 J.E. Moore et al. routine test procedures) compared to the relating closely with culture. The quantita- final template volume in the PCR assay tive detection of Campylobacter directly in (often 1–5 µL). For a PCR assay to replace raw-meat rinse fluid samples was also dem- conventional culture methods, it must have onstrated however the limit of detection a limit of detection sufficiently sensitive to was compromised by the presence of PCR be able to detect a single Campylobacter inhibitors and the low numbers of cells cell in 25 g food. In order to biologically present (Sails et al., unpublished data). The amplify the numbers of cells present, many use of sensitive, quantitative PCR methods PCR-based studies have utilised enrich- for the detection of Campylobacter during ment culture prior to application of the PCR food processing could be used to determine assay. There have been many further points in the food production process where reports of PCR assays for the detection of contamination occurs and where controls campylobacters in a range of sample types could be introduced to reduce or eliminate including foods, environmental waters and Campylobacter from retail food products, other environmental samples. Although thereby reducing the risk to the consumer. these assays may be useful as an adjunct to enrichment culture, by reducing the total The ultimate goal of nucleic acid based time of detection by two or more days, they detection methods is to facilitate direct are still limited by inefficient sample prep- detection of pathogens in food samples with- aration methods. Many PCR assays have a out the need for enrichment culture. This limit of detection of a single cell per reac- would permit more rapid detection of path- tion, however the inability to separate low ogens, thereby reducing the time of detec- numbers of cells away from the sample tion to hours, rather than days. In order to matrix remains the “bottleneck” for the adop- determine the viability of the detected path- tion of PCR-based methodologies in routine ogen, the assay must target a cellular proc- food microbiology testing laboratories. ess or molecule, which has been shown to Specific and sensitive methods are required be associated with bacterial viability under to separate the target cells away from the all conditions tested. Conventional PCR sample matrix in a form amenable for PCR- methods detect chromosomal gene sequences, based detection. In a recent study, paramag- which can be present in non-viable cells. netic beads were utilised as a method for Therefore, direct detection by conventional isolating Campylobacter from chicken cecal PCR cannot determine the viability of the contents and faecal samples, prior to PCR detected cells somewhat limiting the use- [139]. The beads initially bound to the cells fulness of such methods in food microbiol- in the liquid sample matrix and then lysis ogy testing. Detection of viable cells using buffer was added to lyse the cells releasing messenger RNA (mRNA) as the target for the DNA, which then also bound to the reverse transcriptase PCR (RT-PCR) has beads. The DNA was then washed and used been investigated for several microbial as template in the PCR assay. This proce- pathogens including C. jejuni [144]. The dure may prove useful in food testing labo- RT-PCR assay was demonstrated to differ- ratories, however further studies are required entiate between viable and heat-killed cells to validate this approach for food sample of C. jejuni, however the method of killing testing. and post-treatment holding conditions did The introduction of real-time PCR meth- influence the rate of mRNA degradation in ods have facilitated the development of the cells. Further studies are required to quantitative PCR assays for the detection of investigate the effect of different killing Campylobacter in foods [145], milk and methods and post–treatment holding condi- environmental waters [194]. The assays tions to determine the factors which influ- demonstrated a range of quantitation over ence the rate of mRNA degradation in dead 6 orders of magnitude with the results cor- cells. This will allow the above factors to be Campylobacter 373 investigated and the results related to food has become routine from both clinical as processing methods. well as from environmental specimens and Campylobacter detection methods have although relatively complicated to perform, improved significantly since the initial iso- routine isolation has been carried out with lation of campylobacters using membrane success for this past 20 years or so. filtration and non-selective media in the Campylobacter spp. are the most com- early 1970s. Improvements in molecular mon cause of acute gastroenteritis in the methods have facilitated the development developed world. Thermophilic campylo- of nucleic acid-based detection methods bacters, i.e. those Campylobacter spp. which which are more rapid, sensitive and spe- are able to proliferate at 42 °C, particularly cific. In the future, improvements in sample C. jejuni, C. coli and C. lari, are of partic- extraction methods allowing more sensitive ular interest to the food industry, as these detection of cells by PCR will facilitate the campylobacters form the natural microflora uptake of these methods by microbiology of the gastrointestinal tract of several laboratories. Eventually, biological growth domestic and pet animals including poultry. or amplification in vitro may be replaced Although campylobacters are the most com- with DNA amplification, with culture media mon cause of acute human enteritis, their being replaced by PCR reagents and the routes of infection and transmission to man incubator being replaced by the thermal are still not fully understood. Further work cycler. is still required to find the source(s) of these organisms of major public health concern. 9. CONCLUSIONS ACKNOWLEDGEMENTS The past three decades have witnessed the rise of Campylobacter enteritis in man All authors are listed in alphabetical order, from virtual obscurity to notoriety, with with the exception of the corresponding author, present isolation rates superseding those of as each author played an equal role in the prep- other enteric pathogens such as Salmonella aration of this manuscript. J.E. Moore and D.A. spp. and Shigella spp. in most developed McDowell are funded by the Research & Devel- countries. Unlike the salmonellae and other opment Office, Department of Health & Public enteric pathogens, the majority (ca. 99%) of Safety, Northern Ireland (Infectious Disease – clinical reports concerning Campylobacter Recognised Research Group (RRG) 9.9). are sporadic and Campylobacter enteritis outbreaks are rare. 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