Spatiotemporal Homogeneity of Campylobacter Subtypes from Cattle and Sheep across Northeastern and Southwestern Scotland

Source attribution using molecular subtypes has implicated cattle and sheep as sources of human Campylobacter infection. Whether the Campylobacter subtypes associated with cattle and sheep vary spatiotemporally remains poorly known, especially at national levels. Here we describe spatiotemporal patterns of prevalence, bacterial enumeration, and subtype composition in Campylobacter isolates from cattle and sheep feces from northeastern (63 farms, 414 samples) and southwestern (71 farms, 449 samples) Scotland during 2005 to 2006. Isolates (201) were categorized as sequence type (ST), as clonal complex (CC), and as Campylobacter jejuni or Campylobacter coli using multilocus sequence typing (MLST). No significant difference in average prevalence (cattle, 22%; sheep, 25%) or average enumeration (cattle, 2.7 × 10⁴ CFU/g; sheep, 2.0 × 10⁵ CFU/g) was found between hosts or regions. The four most common STs (C. jejuni ST-19, ST-42, and ST-61 and C. coli ST-827) occurred in both hosts, whereas STs of the C. coli ST-828 clonal complex were more common in sheep. Neither host yielded evidence for regional differences in ST, CC, or MLST allele composition. Isolates from the two hosts combined, categorized as ST or CC, were more similar within than between farms but showed no further spatiotemporal trends up to 330 km and 50 weeks between farm samples. In contrast, both regions yielded evidence for significant differences in ST, CC, and allele composition between hosts, such that 65% of isolates could be attributed to a known host. These results suggest that cattle and sheep within the spatiotemporal scales analyzed are each capable of contributing homogeneous Campylobacter strains to human infections.Campylobacter species are the largest cause of bacterial intestinal infection in the developed and developing world (3). Almost all reported human Campylobacter infections in the United Kingdom are caused by Campylobacter jejuni, which accounts for approximately 92% of cases, and Campylobacter coli, which accounts for most of the rest (8). Campylobacter species are carried asymptomatically in a wide range of host animals and excreted into the environment in feces (23). Humans can be infected by several routes including consumption of contaminated water (14) or food (23); indeed, case control studies indicate that consumption of poultry meat is a risk factor (11, 12, 28), but other foods including unpasteurized milk (33) and meat from cattle and sheep contaminated at the abattoir might be important (30).Cattle and sheep on farms are major hosts of Campylobacter, with up to 89% of cattle herds (31) and up to 55% of sheep flocks (26) being infected. The prevalence of C. jejuni and C. coli combined, estimated at the level of individual animals from fecal specimens, is 23 to 54% in cattle (22, 25) and up to 20% in sheep (37). Campylobacter enumeration in feces shed from individual animals ranges from <10² to 10⁷ CFU/g in both hosts (31), and the concentration shed varies with time. Meat products of cattle and sheep, by contrast, have generally lower levels of Campylobacter contamination. Prevalence values are 0.5 to 4.9% in surveys of retail beef (11a, 17, 36) and 6.9 to 12.6% in surveys of retail lamb and mutton (17, 35).Clinical Campylobacter strains can be attributed to infection sources in animals by comparing subtype frequencies in clinical cases with those in different candidate sources, including cattle, sheep, pigs, and the physical environment. Campylobacter subtype data sets are most transferable when subtypes are defined as sequence type (ST) using multilocus sequence typing (MLST). Three recent MLST-based studies based in northwestern England (34), mainland Scotland (29), northeastern Scotland (32), and New Zealand (24) have used source attribution models to infer the source of human clinical infection. The results suggest that retail chicken is the source with the highest (55 to 80%) attribution while cattle and sheep combined are ranked second (20 to 40%). These attribution models require further empirical validation but appear to be showing broadly similar results.Attribution of human Campylobacter infections to cattle and sheep raises the question of whether Campylobacter subtypes infecting farm cattle and sheep are generally homogeneous or tend to have spatiotemporal structure. Regarding spatial differences, isolates of C. jejuni from a 100-km² study of farmland area with dairy cattle and sheep in northwestern England displayed increased genetic similarity up to 1 km apart but no further trend over distances of 1 to 14 km apart (7), and isolates from three dairy cattle farms 2 or 5 km apart in the same area demonstrated differences in the frequencies of strains of clonal complexes (CCs) ST-42 and ST-61 (15). Regarding temporal differences, isolates of C. jejuni from five dairy cattle farms in the same area demonstrated differences in the frequency of strains of CC ST-61 between the spring and summer of 2003 (15). Lastly, regarding host-associated strains, STs of CCs ST-21, ST-42, and ST-61 are associated with cattle, and the more limited data for STs from sheep also show the presence of ST-21 and ST-61 (7, 15).The larger-scale spatiotemporal structure of Campylobacter strains from cattle and sheep is poorly known. The main aims of the present study were (i) to characterize C. jejuni and C. coli from cattle and sheep from two distinct geographical Scottish regions in terms of Campylobacter prevalence and enumeration and C. jejuni and C. coli ST composition and (ii) to estimate the extent of host association of C. jejuni and C. coli STs from cattle versus sheep.     

Molecular Epidemiology and Characterization of Campylobacter spp. Isolated from Wild Bird Populations in Northern England

Campylobacter infections have been reported at prevalences ranging from 2 to 50% in a range of wild bird species, although there have been few studies that have investigated the molecular epidemiology of Campylobacter spp. Consequently, whether wild birds are a source of infection in humans or domestic livestock or are mainly recipients of domestic animal strains and whether separate cycles of infection occur remain unknown. To address these questions, serial cross-sectional surveys of wild bird populations in northern England were carried out over a 2-year period. Fecal samples were collected from 2,084 wild bird individuals and screened for the presence of Campylobacter spp. A total of 56 isolates were recovered from 29 birds sampled at 15 of 167 diverse locales. Campylobacter jejuni, Campylobacter lari, and Campylobacter coli were detected by PCR, and the prevalences of different Campylobacter spp. in different avian families ranged from 0% to 33%. Characterization of 36 C. jejuni isolates by multilocus sequence typing revealed that wild birds carry both livestock-associated and unique strains of C. jejuni. However, the apparent absence of unique wild bird strains of C. jejuni in livestock suggests that the direction of infection is predominantly from livestock to wild birds. C. lari was detected mainly in wild birds sampled in an estuarine or coastal habitat. Fifteen C. lari isolates were analyzed by macrorestriction pulsed-field gel electrophoresis, which revealed genetically diverse populations of C. lari in Eurasian oystercatchers (Haematopus ostralegus) and clonal populations in magpies (Pica pica).Infection with Campylobacter spp. continues to be the leading cause of human infectious intestinal disease in the United Kingdom and has a significant economic impact (39). Consequently, there is a continuing effort to identify effective control methods. The majority of human infections (∼90%) are caused by Campylobacter jejuni subsp. jejuni (46). Other Campylobacter species, including Campylobacter coli and Campylobacter lari, can also cause enteritis in humans, but their prevalence is lower. Most C. jejuni infections are believed to result from consumption of contaminated food, including poultry meat (27, 40), red meat (52), and milk (13), which is thought to be contaminated primarily by feces. It is well established that most livestock species, including poultry, ruminants, and pigs, carry C. jejuni asymptomatically (27), making control at the farm level difficult. However, the epidemiology of C. jejuni cannot be explained solely by food-borne exposure; C. jejuni has also been isolated from a range of environmental samples, including samples of soil, water, sand, and the feces of a number of wildlife species, including wild birds (1-3). However, the role that non-food-borne exposure plays in the epidemiology of C. jejuni is currently not well defined.High prevalences of Campylobacter species infections have been found in a wide range of wild bird species, although there is great variation between taxa (2, 4, 7, 16, 35, 47, 48). Given their ability to fly long distances and their ubiquity, wild birds have the potential to play an important role in the epidemiology and evolution of Campylobacter spp. However, whether wild birds are a source of infection for humans or domestic livestock or are mainly recipients of domestic animal strains or, indeed, whether separate cycles of infection occur remain unknown. These questions remain unanswered in part because investigations of the epidemiology of Campylobacter spp. have been complicated by their high inter- and intraspecies genetic diversity (6).The methods that have been routinely used to characterize Campylobacter isolates are restricted due to genomic instability in Campylobacter populations (10, 38, 45). Multilocus sequence typing (MLST) is a method that has the advantage of being objective since it is sequence based, which allows comparison of isolates from different laboratories and accurate determination of relationships between isolates from diverse sources (11). MLST studies of C. jejuni in farm animals and the environment, including wildlife, suggest that some strains may be associated with particular host groups (6, 10, 15, 30). However, in the same studies other strains were found to occur in several host species or habitats. Few studies have investigated the molecular epidemiology of Campylobacter infection in wild bird populations using MLST, and because only a relatively small number of isolates from wild birds have been characterized by MLST, conclusions have not been drawn yet about how wild bird isolates fit into the overall phylogenetic scheme or whether wild birds act as reservoirs, amplifiers, or merely indicators of infection of domestic animals with zoonotic genotypes.In the current study a large cross-sectional survey of wild bird populations in northern England was undertaken to investigate the epidemiology of Campylobacter infection. Previous studies that have focused on the epidemiology of Campylobacter spp. solely in wild birds have investigated either a narrow range of taxonomic groups (2, 5, 17, 23, 29, 33, 43, 50) or wild birds from a limited range of habitats (18, 25, 48). Studies that have investigated a broad range of wild bird species have used Campylobacter characterization techniques that do not allow conclusions about possible host associations to be drawn or comparison of the genetic diversity of isolates between studies (21, 25, 34, 47, 53). Therefore, the aims of this study were (i) to determine the host range and prevalence of Campylobacter spp. in a wild bird population and (ii) through molecular characterization of isolates to determine whether wild birds were a likely source of infection in humans or domestic livestock and whether separate cycles of infection with host-adapted strains of Campylobacter spp. were maintained in the wild bird population.     

Antibiotic Manipulation of Intestinal Microbiota To Identify Microbes Associated with Campylobacter jejuni Exclusion in Poultry

The ability of various subsets of poultry intestinal microbiota to protect turkeys from colonization by Campylobacter jejuni was investigated. Community subsets were generated in vivo by inoculation of day-old poults with the cecal contents of a Campylobacter-free adult turkey, followed by treatment with one antimicrobial, either virginiamycin, enrofloxacin, neomycin, or vancomycin. The C. jejuni loads of the enrofloxacin-, neomycin-, and vancomycin-derived communities were decreased by 1 log, 2 logs, and 4 logs, respectively. Examination of the constituents of the derived communities via the array-based method oligonucleotide fingerprinting of rRNA genes detected a subtype of Megamonas hypermegale specific to the C. jejuni-suppressive treatments.Campylobacter jejuni, a spiral, flagellated epsilonproteobacterial commensal of poultry, is the predominant cause of bacterial food-borne illness in the United States, resulting in approximately 2 million cases per year. A role for endogenous poultry intestinal microbiota in competitive exclusion (CE) of Campylobacter was first investigated in 1982 (38). Since then, numerous studies have attempted to identify microbes associated with Campylobacter CE. Suspensions of intestinal bacteria, isolated from Campylobacter-free adult poultry and passaged under strict anaerobic conditions, were found to protect chicks from colonization by the pathogen (31). Bacteria derived from the scrapings of broiler intestinal mucosa were proven more effective than the earlier fecal culture, a result not surprising, as Campylobacter is known to preferentially colonize cecal crypts (4, 39). The CE function of the bacterial suspensions decreased with time in storage, however (39, 40). Evidence also indicates that CE may depend on the presence of strictly anaerobic bacteria (31). As an oxygen gradient likely occurs from the host epithelium into the luminal contents, a CE role for both mucosal and luminal microbes in concert is likely.Attempts have been made to identify specific microbes antagonistic to Campylobacter, and initial attempts isolated mucin-dwelling organisms with in vitro antagonistic effects against the pathogen (35, 36). Recent experiments have identified numerous bacterial groups producing anti-Campylobacter bacteriocins (29, 41, 42, 44, 45). Direct treatment of market-weight birds with the therapeutic bacteriocin Enterococcus faecium E 50-52 is effective for removal of Campylobacter spp. immediately prior to slaughter (44).Despite progress toward a solution to contamination of poultry products by Campylobacter species, incomplete or intermittent CE protection, combined with a lack of studies addressing long-term CE efficacy, indicates that the Campylobacter colonization problem is far from solved (35). In addition, risk factors for campylobacteriosis other than direct consumption of contaminated poultry include consumption of fresh vegetables and bottled water (14). Campylobacter has been found in poultry manure used to fertilize crops as well as in runoff from these farms (22, 24, 50). We believe that novel approaches for studying microbial ecology in the gut are necessary for development of intervention strategies, including competitive exclusion.The work described here takes a functional approach to identify microbes associated with protection of the intestine from Campylobacter jejuni colonization, an approach we are calling antibiotic dissection. The cecal contents from a Campylobacter-free adult turkey were inoculated into day-old poults and the microbial communities in these poults modified by treatment with therapeutic levels of antibiotics. The resulting modified microbiota were then tested for the ability to outcompete a C. jejuni challenge, and a microbe potentially associated with C. jejuni exclusion was identified.     

Thermotolerant Coliforms Are Not a Good Surrogate for Campylobacter spp. in Environmental Water

This study aimed to assess the importance of quantitatively detecting Campylobacter spp. in environmental surface water. The prevalence and the quantity of Campylobacter spp., thermotolerant coliforms, and Escherichia coli in 2,471 samples collected weekly, over a 2-year period, from 13 rivers and 12 streams in the Eastern Townships, Québec, Canada, were determined. Overall, 1,071 (43%), 1,481 (60%), and 1,463 (59%) samples were positive for Campylobacter spp., thermotolerant coliforms, and E. coli, respectively. There were weak correlations between the weekly distributions of Campylobacter spp. and thermotolerant coliforms (Spearman''s ρ coefficient = 0.27; P = 0.008) and between the quantitative levels of the two classes of organisms (Kendall tau-b correlation coefficient = 0.233; P < 0.0001). Well water samples from the Eastern Townships were also tested. Five (10%) of 53 samples from private surface wells were positive for Campylobacter jejuni, of which only 2 were positive for thermotolerant coliforms. These findings suggest that microbial monitoring of raw water by using only fecal indicator organisms is not sufficient for assessing the occurrence or the load of thermophilic Campylobacter spp. Insights into the role of environmental water as sources for sporadic Campylobacter infection will require genus-specific monitoring techniques.Campylobacter jejuni is the leading reported cause of bacterial gastroenteritis in developed countries (2). In 2004 in Canada, Campylobacter enteritis was the leading notifiable enteric food- and waterborne disease, with 9,345 reported cases (http://dsol-smed.phac-aspc.gc.ca). In Quebec province alone, nearly 3,000 cases of diarrheal illness are attributed annually to Campylobacter enteritis, more than the combined total caused by Salmonella and Shigella species, Escherichia coli O157:H7, and Yersinia enterocolitica (15). Thomas et al. recently concluded that even these numbers appear to represent a substantial underestimate of the public health burden of this enteric pathogen and that for every case of Campylobacter infection reported in Canada each year, there are an additional 23 to 49 unreported cases (47).Raw milk, untreated surface water, and poultry have all been well documented as sources of Campylobacter outbreaks (1, 8, 22, 23, 28, 32, 33, 37, 39, 42, 49). Nevertheless, most clinical cases appear as isolated, sporadic infections for which the source is rarely identified (6). Identifying the sources and routes of transmission of campylobacteriosis is essential for developing effective, targeted preventive measures.There is ample opportunity for Campylobacter spp. to contaminate environmental water, including streams, rivers, and lakes. The genus colonizes a wide variety of hosts, from domestic animals to wild birds, and thus an extensive burden of organisms is excreted via animal fecal material (2, 8). Other potential sources include discharges from wastewater treatment plants.Testing for indicator organisms (typically thermotolerant coliforms or E. coli) has generally been considered to reflect adequately the presence of enteric pathogens; consequently, campylobacters have not been explicitly monitored in water. Numerous studies (most of which were small and of short duration) have reported conflicting results regarding the value of detecting E. coli to predict Campylobacter sp. presence (4, 9, 11, 12, 16, 17, 21, 27, 29, 31, 38, 40, 43, 48). We report here a large study that analyzed 2,471 water samples from 32 different sites over 2 years to resolve this question.     

Biofilm Formation by Campylobacter jejuni Is Increased under Aerobic Conditions

The microaerophilic human pathogen Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in the developed world. During transmission through the food chain and the environment, the organism must survive stressful environmental conditions, particularly high oxygen levels. Biofilm formation has been suggested to play a role in the environmental survival of this organism. In this work we show that C. jejuni NCTC 11168 biofilms developed more rapidly under environmental and food-chain-relevant aerobic conditions (20% O₂) than under microaerobic conditions (5% O₂, 10% CO₂), although final levels of biofilms were comparable after 3 days. Staining of biofilms with Congo red gave results similar to those obtained with the commonly used crystal violet staining. The level of biofilm formation by nonmotile aflagellate strains was lower than that observed for the motile flagellated strain but nonetheless increased under aerobic conditions, suggesting the presence of flagellum-dependent and flagellum-independent mechanisms of biofilm formation in C. jejuni. Moreover, preformed biofilms shed high numbers of viable C. jejuni cells into the culture supernatant independently of the oxygen concentration, suggesting a continuous passive release of cells into the medium rather than a condition-specific active mechanism of dispersal. We conclude that under aerobic or stressful conditions, C. jejuni adapts to a biofilm lifestyle, allowing survival under detrimental conditions, and that such a biofilm can function as a reservoir of viable planktonic cells. The increased level of biofilm formation under aerobic conditions is likely to be an adaptation contributing to the zoonotic lifestyle of C. jejuni.Infection with Campylobacter jejuni is the leading cause of food-borne bacterial gastroenteritis in the developed world and is often associated with the consumption of undercooked poultry products (19). The United Kingdom Health Protection Agency reported more than 45,000 laboratory-confirmed cases for England and Wales in 2006 alone, although this is thought to be a 5- to 10-fold underestimation of the total number of community incidents (20, 43). The symptoms associated with C. jejuni infection usually last between 2 and 5 days and include diarrhea, vomiting, and stomach pains. Sequelae of C. jejuni infection include more-serious autoimmune diseases, such as Guillain-Barré syndrome, Miller-Fisher syndrome (18), and reactive arthritis (15).Poultry represents a major natural reservoir for C. jejuni, since the organism is usually considered to be a commensal and can reach densities as high as 1 × 10⁸ CFU g of cecal contents⁻¹ (35). As a result, large numbers of bacteria are shed via feces into the environment, and consequently, C. jejuni can spread rapidly through a flock of birds in a broiler house (1). While well adapted to life in the avian host, C. jejuni must survive during transit between hosts and on food products under stressful storage conditions, including high and low temperatures and atmospheric oxygen levels. The organism must therefore have mechanisms to protect itself from unfavorable conditions.Biofilm formation is a well-characterized bacterial mode of growth and survival, where the surface-attached and matrix-encased bacteria are protected from stressful environmental conditions, such as UV radiation, predation, and desiccation (7, 8, 28). Bacteria in biofilms are also known to be >1,000-fold more resistant to disinfectants and antimicrobials than their planktonic counterparts (11). Several reports have now shown that Campylobacter species are capable of forming a monospecies biofilm (21, 22) and can colonize a preexisting biofilm (14). Biofilm formation can be demonstrated under laboratory conditions, and environmental biofilms, from poultry-rearing facilities, have been shown to contain Campylobacter (5, 32, 44). Campylobacter biofilms allow the organism to survive up to twice as long under atmospheric conditions (2, 21) and in water systems (27).Molecular understanding of biofilm formation by Campylobacter is still in its infancy, although there is evidence for the role of flagella and gene regulation in biofilm formation. Indeed, a flaAB mutant shows reduced biofilm formation (34); mutants defective in flagellar modification (cj1337) and assembly (fliS) are defective in adhering to glass surfaces (21); and a proteomic study of biofilm-grown cells shows increased levels of motility-associated proteins, including FlaA, FlaB, FliD, FlgG, and FlgG2 (22). Flagella are also implicated in adhesion and in biofilm formation and development in other bacterial species, including Aeromonas, Vibrio, Yersinia, and Pseudomonas species (3, 23, 24, 31, 42).Previous studies of Campylobacter biofilms have focused mostly on biofilm formation under standard microaerobic laboratory conditions. In this work we have examined the formation of biofilms by motile and nonmotile C. jejuni strains under atmospheric conditions that are relevant to the survival of this organism in a commercial context of environmental and food-based transmission.     

Novel Clonal Complexes with an Unknown Animal Reservoir Dominate Campylobacter jejuni Isolates from River Water in New Zealand

Campylobacter jejuni is widely distributed in the environment, and river water has been shown to carry high levels of the organism. In this study, 244 C. jejuni isolates from three river catchment areas in New Zealand were characterized using multilocus sequence typing. Forty-nine of the 88 sequence types identified were new. The most common sequence types identified were ST-2381 (30 isolates), ST-45 (25 isolates), and ST-1225 (23 isolates). The majority of the sequence types identified in the river water could be attributed to wild bird fecal contamination. Two novel clonal complexes (CC) were identified, namely, CC ST-2381 (11 sequence types, 46 isolates) and CC ST-3640 (6 sequence types, 12 isolates), in which all of the sequence types were new. CC ST-2381 was the largest complex identified among the isolates and was present in two of the three rivers. None of the sequence types associated with the novel complexes has been identified among human isolates. The ST-2381 complex is not related to complexes associated with cattle, sheep, or poultry. The source of the novel complexes has yet to be identified.Contamination of the environment by bacterial pathogens is a significant health concern, as it provides a continuous source of organisms for the infection and reinfection of humans and animals. Enteric pathogens gain entry into the environment through the discharge of sewage into water and via contamination from animal feces (22). Fecal contamination is responsible for the continued presence and spread of a range of pathogenic organisms, including Campylobacter, norovirus, and Escherichia coli O157. Determining the roles of various environmental sources in human enteric disease requires an understanding of the distribution, survival, population structure, and pathogenic potential of the pathogens in the environment.Campylobacter is the most common cause of gastrointestinal illness in the industrialized world (17), imposing significant economic costs on health systems, and is associated with a number of neurological sequelae (32, 33). The majority of human campylobacter infections are caused by Campylobacter jejuni (90%), with Campylobacter coli mostly responsible for the remainder. Although Campylobacter has been isolated from a wide range of animals (41) and birds (47, 48), contaminated poultry and poultry products remain the most significant sources of human infections (10, 38, 50, 51). Campylobacter is a spiral gram-negative organism that grows best under low-oxygen conditions at 42°C. The organism is unable to grow outside an animal host, and survival in the environment is dependent on ambient temperature, oxygen levels, and sunlight.Studies worldwide examining rivers and waterways show that there is significant contamination by Campylobacter, with the sources being sewage outflow, direct fecal deposition, and pasture runoff (12, 22, 34, 37, 39). Similarly, coastal waters and estuaries can be contaminated by either sewage or bird fecal deposition (23, 35). The inability of Campylobacter to grow in the environment and its sensitivity to sunlight are thought to ensure that the organism is eventually purged from the system. However, the high levels of the organism identified in water systems have been highlighted as a risk for human infection.The characterization of campylobacter populations by multilocus sequence typing (MLST) has shown that the organism is weakly clonal and that certain clonal complexes are associated with particular animals (5, 9, 26). Isolates from human cases of infection show a wide variety of sequence types and many clonal complexes. Source attribution studies using MLST have identified poultry as causing approximately 60% of human infections (14, 38, 50). Cattle have been identified as a potential source of infection due to the high level of similarity between bovine and human strains (18, 19). There remains, however, a significant number of infections for which the source is not certain.New Zealand has one of the highest rates of campylobacteriosis in the developed world. This is due to the significant quantity of fresh chicken consumed coupled with high levels of contamination found in poultry products (1, 10, 51, 52). Campylobacter has been isolated from a range of environmental sources within New Zealand, including its rivers and streams (12, 37). Isolation rates for rivers in New Zealand range from 55 to 90%, comparable to results of studies overseas, and show the same seasonal variation as that seen elsewhere in the world (20). Pulsed-field gel electrophoresis (PFGE) analysis identified indistinguishable macrorestriction profiles for cattle, human, and river isolates, suggesting river water as a potential source of infection (8). In this study, C. jejuni isolates from three rivers in New Zealand, two on the South Island and one on the North Island, were characterized using MLST.     

Identification and Characterization of a New Ferric Enterobactin Receptor,CfrB, in Campylobacter

The ferric enterobactin (FeEnt) receptor CfrA is present in the majority of Campylobacter jejuni isolates and is responsible for high-affinity iron acquisition. Our recent work and that of others strongly suggested the existence of another FeEnt uptake system in Campylobacter. Here we have identified and characterized a new FeEnt receptor (designated CfrB) using both in vitro and in vivo systems. CfrB, a homolog of C. jejuni NCTC 11168 Cj0444, shares approximately 34% of amino acid identity with CfrA. Alignment of complete CfrB sequences showed that the CfrB is highly conserved in Campylobacter. Immunoblotting analysis using CfrB-specific antiserum demonstrated that CfrB was dramatically induced under iron-restricted conditions and was produced in the majority of Campylobacter coli (41 out of 45) and in some C. jejuni (8 out of 32) primary strains from various sources and from geographically diverse areas. All of the CfrB-producing C. coli strains also produced CfrA, which was rarely observed in the tested C. jejuni strains. Isogenic cfrB, cfrA, and cfrA cfrB double mutants were constructed in 43 diverse Campylobacter strains. Growth promotion assays using these mutants demonstrated that CfrB has a major role in FeEnt iron acquisition in C. coli. Chicken colonization experiments indicated that inactivation of the cfrB gene alone greatly reduced and even abolished Campylobacter colonization of the intestines. A growth assay using CfrB-specific antiserum strongly suggested that specific CfrB antibodies could block the function of CfrB and diminish FeEnt-mediated growth promotion under iron-restricted conditions. Together, this work reveals the complexity of FeEnt systems in the two closely related Campylobacter species and demonstrates the important role of the new FeEnt receptor CfrB in Campylobacter iron acquisition and in vivo colonization.Campylobacter species have emerged as the leading bacterial cause of food-borne human diseases in many industrialized countries since the late 1970s (25). Two major Campylobacter species, Campylobacter jejuni and Campylobacter coli, cause watery diarrhea and/or hemorrhagic colitis in humans and are also associated with Guillain-Barré syndrome, an acute flaccid paralysis that may compromise respiratory muscle function, resulting in death (24). In parallel to their increased prevalence, members of Campylobacter have become increasingly resistant to antibiotics, including fluoroquinolones and macrolides, the major drugs of choice for treating human campylobacteriosis (10). Therefore, development of new strategies to prevent and control Campylobacter infections in humans and animal reservoirs is urgently needed, which greatly relies on the better understanding of Campylobacter pathogenesis.Despite recent advances in understanding of the pathobiology of C. jejuni (9, 39), the virulence mechanisms of Campylobacter remain poorly understood. Iron is the most abundant transition metal in living organisms, with critical roles in many diverse biological systems (2); thus, iron acquisition is essential for survival and virulence of pathogenic bacteria in the host (5, 31). Examination of iron uptake in Campylobacter began in the 1980s (12), but iron uptake systems, and the associated regulatory systems, in Campylobacter species are now just beginning to be elucidated (reviewed by Miller et al. [22], Stintzi et al. [34], and Wooldridge and van Vliet [37]). Genomic data have shown a large number of genes implicated in iron scavenging, metabolism, storage, and regulation in C. jejuni (22, 34, 37). Several iron uptake systems have been identified and characterized (22, 34); among these, the ferric enterobactin (FeEnt) iron acquisition system is of particular interest because enterobactin (Ent) has the highest affinity for ferric iron of any natural siderophore compound tested (35). Furthermore, Ent is produced by a wide variety of commensal bacteria in the intestines, and this compound is likely to be produced in significant amounts by the resident microflora in the gut (37). Thus, FeEnt may be a significant source of iron for Campylobacter species during intestinal colonization even though Campylobacter species do not appear have the capacity to synthesize Ent (34).A FeEnt acquisition system in C. jejuni was identified which comprises an outer membrane receptor, CfrA, and cognate components, including a TonB-ExbB-ExbD protein complex and an ABC transporter system CeuBCDE (22, 34). The FeEnt receptor CfrA is induced under iron-restricted conditions and plays a critical role in iron acquisition and in vivo colonization by C. jejuni (27). A recent report (40) provides further molecular, antigenic, and functional evidence suggesting that CfrA is a promising subunit vaccine for preventing and controlling C. jejuni infection in humans and animal reservoirs. Interestingly, in this study one C. jejuni strain (JL11), which does not have a gene highly homologous to cfrA, could efficiently utilize FeEnt as a sole iron source for growth (40). An early study also showed that an isogenic cfrA mutant of a human C. coli strain was still fully capable of utilizing FeEnt as a sole iron source for growth (15). These studies strongly suggest that Campylobacter species possess an additional system for FeEnt-mediated iron acquisition.In this study, we demonstrate that a homolog of the C. jejuni NCTC 11168 protein Cj0444 (28) is a FeEnt receptor, designated CfrB, in Campylobacter. CfrB is highly conserved among members of Campylobacter and plays an important role in the colonization of the intestine by both C. jejuni and C. coli.     

Effects of Polyphosphate Additives on Campylobacter Survival in Processed Chicken Exudates

Campylobacter spp. are responsible for a large number of the bacterial food poisoning cases worldwide. Despite being sensitive to oxygen and nutritionally fastidious, Campylobacter spp. are able to survive in food processing environments and reach consumers in sufficient numbers to cause disease. To investigate Campylobacter persistence on processed chicken, exudates from chickens produced for consumer sale were collected and sterilized. Two types of exudates from chicken products were collected: enhanced, where a marinade was added to the chickens during processing, and nonenhanced, where no additives were added during processing. Exudates from enhanced chicken products examined in this study contained a mixture of polyphosphates. Exudate samples were inoculated with Campylobacter jejuni or Campylobacter coli strains and incubated under a range of environmental conditions, and viable bacteria present in the resultant cultures were enumerated. When incubated at 42°C in a microaerobic environment, exudates from enhanced chicken products resulted in increased survival of C. jejuni and C. coli compared with that in nonenhanced exudates in the range of <1 to >4 log CFU/ml. Under more relevant food storage conditions (4°C and normal atmosphere), the exudates from enhanced chicken products also demonstrated improved Campylobacter survival compared with that in nonenhanced exudates. Polyphosphates present in the enhanced exudates were determined to be largely responsible for the improved survival observed when the two types of exudates were compared. Therefore, polyphosphates used to enhance chicken quality aid in sustaining the numbers of Campylobacter bacteria, increasing the opportunity for disease via cross-contamination or improperly cooked poultry.Campylobacter species are the major causative agent of food-borne gastrointestinal bacterial infections in the developed world (6, 11, 21). Poultry products are a major source for the introduction of Campylobacter into the food supply (15, 16). Improperly cooked poultry and cross-contamination of other foods by raw poultry are common methods for transmission of Campylobacter to humans (5). However, Campylobacter spp. are nutritionally fastidious organisms that are sensitive to the oxygen levels present in a normal environment (O₂ = 20.9%) (21). Therefore, Campylobacter appears an unlikely candidate to persist within poultry processing and storage environments at levels sufficient to cause human disease. This conundrum directly leads to a question: what then are the elements that contribute to the ability of Campylobacter to survive through poultry processing and cold storage?To investigate this question, a food-relevant environment consisting of chicken weepage or exudate can be used to perform survival experiments on Campylobacter species. Strains of Campylobacter jejuni and Campylobacter coli were used for the survival studies since these two species are responsible for the vast majority of human cases of campylobacteriosis (20, 28). Chicken exudate is the fluid that seeps out from processed poultry carcasses and is often found to be contaminated with considerable numbers of Campylobacter bacteria. It is comprised of water, blood, fats, and other materials added to the poultry during processing. Sterilized poultry exudates make for a convenient experimental material that is also relevant to the conditions which Campylobacter will experience as a contaminant of processed poultry (2, 3). Two different types of chicken exudates were collected from commercial producers, one from chickens processed without additives (nonenhanced) and the other from chickens that were treated with a commercial marinade to increase the quality and appeal of the meat at market (enhanced). The commercial poultry marinades contain a significant amount of polyphosphate additives. Polyphosphates comprise a group of food additives that are utilized within poultry processing to enhance the moisture absorbance, color, and flavor and to reduce product shrinkage of poultry (24, 29-32). Polyphosphates have also been shown to have an antimicrobial effect on several different bacterial species (8, 10, 12). The goal of the research was to determine if polyphosphates contribute to the ability of Campylobacter to survive and persist through the supply chain, thus directly increasing the opportunity for Campylobacter-mediated food poisoning of consumers.     

Genomic Insights into the Convergence and Pathogenicity Factors of Campylobacter jejuni and Campylobacter coli Species

Whether or not bacteria form coherent evolutionary groups via means of genetic exchange and, hence, elicit distinct species boundaries remains an unsettled issue. A recent report implied that not only may the former be true but also, in fact, the clearly distinct Campylobacter jejuni and Campylobacter coli species may be converging as a consequence of increased interspecies gene flow fostered, presumably, by the recent invasion of an overlapping ecological niche (S. K. Sheppard, N. D. McCarthy, D. Falush, and M. C. Maiden, Science 320:237-239, 2008). We have reanalyzed the Campylobacter multilocus sequence typing database used in the previous study and found that the number of interspecies gene transfer events may actually be too infrequent to account, unequivocally, for species convergence. For instance, only 1 to 2% of the 4,507 Campylobacter isolates examined appeared to have imported gene alleles from another Campylobacter species. Furthermore, by analyzing the available Campylobacter genomic sequences, we show that although there seems to be a slightly higher number of exchanged genes between C. jejuni and C. coli relative to other comparable species (∼10% versus 2 to 3% of the total genes in the genome, respectively), the function and spatial distribution in the genome of the exchanged genes are far from random, and hence, inconsistent with the species convergence hypothesis. In fact, the exchanged genes appear to be limited to a few environmentally selected cellular functions. Accordingly, these genes may represent important pathogenic determinants of pathogenic Campylobacter, and convergence of (any) two bacterial species remains to be seen.High-throughput sequencing studies during the last decade have revealed that bacterial genomes are much more diverse and “fluid” than previously anticipated (14, 31). This genomic fluidity is primarily attributable to the great pervasiveness and promiscuity of horizontal gene transfer (HGT) in the bacterial world (5, 17). Nonetheless, evidence of any two distinct bacterial species or lineages merging due to directed (as opposed to promiscuous) interspecies genetic exchange was reported, probably for the first time ever, by the recent study of Sheppard et al. (26). Species convergence, if occurring, has major theoretical implications for the bacterial species concept (reviewed extensively elsewhere [9, 10, 14, 24, 30]) and important practical consequences for accurate identification of bacterial pathogens in the clinical setting.Sheppard and colleagues reported that as many as ∼18.6% of the unique alleles of housekeeping genes found in Campylobacter coli isolates may have been recently imported (through HGT) from a close relative, Campylobacter jejuni (26). The results were based on the analysis of 4,507 Campylobacter spp. isolates, which were genotyped at seven genes (loci), available though the Campylobacter multilocus sequence typing (MLST) database (4). In brief, the 4,507 genotyped isolates contained a total of 2,917 unique sequence types (STs). A unique ST represents the concatenated sequence of the seven genes present in the genome of an isolate and contains a unique sequence (allele) for at least one of the seven genes when compared against any other unique ST in the database (different isolates may be characterized by the same ST). The unique STs were assigned to either C. coli or C. jejuni species by using the program STRUCTURE (6). Neighbor-joining phylogenetic trees of all available unique alleles for each individual gene were subsequently built. Instances where the ST assignment to a species differed from the assignment of an individual gene sequence, which comprised the ST, were attributed to interspecies transfer of the gene, and the number of such instances was reported (26).Here, we have reevaluated the available Campylobacter MLST data set and show that the predominant STs, i.e., the STs characterizing >98% of the isolates, do not contain imported alleles and, hence, do not support the species convergence hypothesis. In agreement with these findings, analyses of the available Campylobacter genomic sequences indicate that the interspecies genetic exchange is limited and heavily biased toward a few genes under positive selection. In fact, housekeeping genes (such as those used in MLST) were found to be exchanged between the two species only in (rare) hitchhiking events associated with the horizontal transfer of adaptive genes. Accordingly, a clear species boundary between the C. jejuni and C. coli species is evident and it is unlikely that this boundary is being eroded.     

Correlation between Genotypic Diversity,Lipooligosaccharide Gene Locus Class Variation,and Caco-2 Cell Invasion Potential of Campylobacter jejuni Isolates from Chicken Meat and Humans: Contribution to Virulotyping

Significant interest in studying the lipooligosaccharide (LOS) of Campylobacter jejuni has stemmed from its potential role in postinfection paralytic disorders. In this study we present the results of PCR screening of five LOS locus classes (A, B, C, D, and E) for a collection of 116 C. jejuni isolates from chicken meat (n = 76) and sporadic human cases of diarrhea (n = 40). We correlated LOS classes with clonal complexes (CC) assigned by multilocus sequence typing (MLST). Finally, we evaluated the invasion potential of a panel of 52 of these C. jejuni isolates for Caco-2 cells. PCR screening showed that 87.1% (101/116) of isolates could be assigned to LOS class A, B, C, D, or E. Concordance between LOS classes and certain MLST CC was revealed. The majority (85.7% [24/28]) of C. jejuni isolates grouped in CC-21 were shown to express LOS locus class C. The invasion potential of C. jejuni isolates possessing sialylated LOS (n = 29; classes A, B, and C) for Caco-2 cells was significantly higher (P < 0.0001) than that of C. jejuni isolates with nonsialylated LOS (n = 23; classes D and E). There was no significant difference in invasiveness between chicken meat and human isolates. However, C. jejuni isolates assigned to CC-206 (correlated with LOS class B) or CC-21 (correlated with LOS class C) showed statistically significantly higher levels of invasion than isolates from other CC. Correlation between LOS classes and CC was further confirmed by pulsed-field gel electrophoresis. The present study reveals a correlation between genotypic diversity and LOS locus classes of C. jejuni. We showed that simple PCR screening for C. jejuni LOS classes could reliably predict certain MLST CC and add to the interpretation of molecular-typing results. Our study corroborates that sialylation of LOS is advantageous for C. jejuni fitness and virulence in different hosts. The modulation of cell surface carbohydrate structure could enhance the ability of C. jejuni to adapt to or survive in a host.Campylobacter jejuni is an important human enteric pathogen worldwide (3, 7, 26). Infected humans exhibit a range of clinical spectra, from mild, watery diarrhea to severe inflammatory diarrhea (28). Factors influencing the virulence of C. jejuni include motility, chemotaxis, the ability to adhere to and invade intestinal cells, intracellular survival, and toxin production (28, 30, 52). Besides its role in human enteric illnesses, C. jejuni is a predominant infectious trigger of acute postinfectious neuropathies, such as Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS) (1). Significant interest in studying the structure and biosynthesis of the core lipooligosaccharide (LOS) of C. jejuni has resulted from its potential role in these paralytic disorders. Many studies have now provided convincing evidence that molecular mimicry between C. jejuni LOS and gangliosides in human peripheral nerve tissue plays an important causal role in the pathogenesis of GBS/MFS (16, 17, 19, 21).Initial comparative studies of C. jejuni LOS structure and the corresponding DNA sequences of the LOS biosynthesis loci identified eight different LOS locus classes. Three of these classes, A, B, and C, harbor sialyltransferase genes involved in incorporating sialic acid into the LOS (42). Sialylation of the LOS core was found to be associated with ganglioside mimicry and also to affect immunogenicity and serum resistance (21). Recently, Parker et al. (43) identified 11 additional LOS classes on the basis of the sequence at the LOS biosynthesis locus. Their investigation also suggested that the LOS loci of C. jejuni strains are hot spots for genetic exchange, which can lead to mosaicism.Despite evidence on locus variation within C. jejuni LOS classes, PCR-based screening of a collection of 123 clinical and environmental strains showed that almost 60% of C. jejuni strains belong to class A, B, or C (42). Additionally, Godschalk et al. (16) found that 53% (9/17) of GBS-associated C. jejuni strains possessed LOS of class A, while 64% (35/55) of the non-GBS-associated isolates possessed LOS of class A, B, or C, and 62% (13/21) of enteritis-associated Campylobacter strains expressed LOS of class A, B, or C, as well. This relative representation of sialylated LOS classes A, B, and C was hypothesized to be advantageous for C. jejuni in the colonization and infection of various hosts (42, 49). Recently, Louwen et al. (34) demonstrated that C. jejuni strains possessing sialylated LOS (class A, B, or C) invade Caco-2 cells significantly better than nonsialylated strains (with class D or E). Knockout mutagenesis of the LOS sialyltransferase Cst-II in three C. jejuni strains revealed a significant reduction in the invasion potentials of the mutant strains (34). The possible role of LOS in adhesion and invasion was previously highlighted in the work of Perera et al. (44) and Kanipes et al. (29), where a C. jejuni waaF mutant strain showed significant reductions in levels of adherence to and invasion of INT-407 cells.LOS class diversity in C. jejuni strains isolated from chicken meat, an important source of human campylobacteriosis (6, 7, 26), has hardly been studied at all. In addition, the role of LOS class variation in the invasion potential of C. jejuni strains from chicken meat still needs to be explored. The epidemiological relevance of C. jejuni LOS gene screening can be further elaborated by correlating its results with results from other molecular-typing tools (e.g., multilocus sequence typing [MLST] and pulsed-field gel electrophoresis [PFGE]). In the present study, we screened a diverse collection of C. jejuni isolates, from consumer-packaged chicken meats and from sporadic human cases of diarrhea, by PCR for five LOS classes (A, B, C, D, and E). Then we correlated the LOS classes assigned by PCR screening with the genotypes assigned by PFGE and MLST. Finally, we tested the invasion potentials of a representative subset of C. jejuni isolates in relation to their LOS classes and genotypic diversity.     

Expanded Multilocus Sequence Typing and Comparative Genomic Hybridization of Campylobacter coli Isolates from Multiple Hosts

The purpose of this work was to evaluate the evolutionary history of Campylobacter coli isolates derived from multiple host sources and to use microarray comparative genomic hybridization to assess whether there are particular genes comprising the dispensable portion of the genome that are more commonly associated with certain host species. Genotyping and ClonalFrame analyses of an expanded 16-gene multilocus sequence typing (MLST) data set involving 85 isolates from 4 different hosts species tentatively supported the development of C. coli host-preferred groups and suggested that recombination has played various roles in their diversification; however, geography could not be excluded as a contributing factor underlying the history of some of the groups. Population genetic analyses of the C. coli pubMLST database by use of STRUCTURE suggested that isolates from swine form a relatively homogeneous genetic group, that chicken and human isolates show considerable genetic overlap, that isolates from ducks and wild birds have similarity with environmental water samples and that turkey isolates have a connection with human infection similar to that observed for chickens. Analysis of molecular variance (AMOVA) was performed on these same data and suggested that host species was a significant factor in explaining genetic variation and that macrogeography (North America, Europe, and the United Kingdom) was not. The microarray comparative genomic hybridization data suggested that there were combinations of genes more commonly associated with isolates derived from particular hosts and, combined with the results on evolutionary history, suggest that this is due to a combination of common ancestry in some cases and lateral gene transfer in others.Campylobacter species are a leading bacterial cause of gastroenteritis within the United States and throughout much of the rest of the developed world. According to the CDC, there are an estimated 2 million to 4 million cases of Campylobacter illness each year in the United States (37). Campylobacter jejuni is generally recognized as the predominant cause of campylobacteriosis, responsible for approximately 90% of reported cases, while the majority of the remainder are caused by the closely related sister species Campylobacter coli (27). Not surprisingly, therefore, the majority of research on Campylobacter has centered on C. jejuni, and C. coli is a less studied organism.A multilocus sequence typing (MLST) scheme of C. jejuni was first developed by Dingle et al. (13) on the basis of the genome sequence of C. jejuni NCTC 11168. There have also been a number of studies using the genome sequence data to develop microarrays for gene presence/absence determination across strains of C. jejuni and to identify the core genome components for the species (6, 15, 32, 33, 42, 43, 53, 57). Although C. coli is responsible for fewer food-borne illnesses than C. jejuni, the impact of C. coli is still substantial, and there is also evidence that C. coli may carry higher levels of resistance to some antibiotics (1). C. coli and C. jejuni also tend to differ in their relative prevalences in animal host species and various environmental sources (4, 48, 58), and there is some evidence that both taxa may include groups of host-specific putative ecotype strains (7, 36, 38, 39, 52, 56). At present, there is only a single draft genome sequence available for C. coli, and there are no microarray comparative genomic hybridization data for C. coli strains. Thus, there is no information on intraspecies variability in gene presence/absence in C. coli and how such variability might correlate with host species.The purpose of this work was to develop and apply an expanded 16-locus MLST genotyping scheme to evaluate the evolutionary history of Campylobacter coli isolates derived from multiple host sources and to use microarray comparative genomic hybridization to assess whether there are particular genes comprising the dispensable portion of the genome that are more commonly associated with isolates derived from different host species.     

Increase in Acid Tolerance of Campylobacter jejuni through Coincubation with Amoebae

Campylobacter jejuni is a recognized and common gastrointestinal pathogen in most parts of the world. Human infections are often food borne, and the bacterium is frequent among poultry and other food animals. However, much less is known about the epidemiology of C. jejuni in the environment and what mechanisms the bacterium depends on to tolerate low pH. The sensitive nature of C. jejuni stands in contrast to the fact that it is difficult to eradicate from poultry production, and even more contradictory is the fact that the bacterium is able to survive the acidic passage through the human stomach. Here we expand the knowledge on C. jejuni acid tolerance by looking at protozoa as a potential epidemiological pathway of infection. Our results showed that when C. jejuni cells were coincubated with Acanthamoeba polyphaga in acidified phosphate-buffered saline (PBS) or tap water, the bacteria could tolerate pHs far below those in their normal range, even surviving at pH 4 for 20 h and at pH 2 for 5 h. Interestingly, moderately acidic conditions (pH 4 and 5) were shown to trigger C. jejuni motility as well as to increase adhesion/internalization of bacteria into A. polyphaga. Taken together, the results suggest that protozoa may act as protective hosts against harsh conditions and might be a potential risk factor for C. jejuni infections. These findings may be important for our understanding of C. jejuni passage through the gastrointestinal tract and for hygiene practices used in poultry settings.Campylobacter jejuni is a major cause of human bacterial enteritis, with an incidence exceeding that of Salmonella spp. or Escherichia coli O157 (6, 28). Most infections are associated with consumption of contaminated food, primarily undercooked chicken meat, but unchlorinated water and unpasteurized milk can also be sources of Campylobacter infection (reviewed in reference 13). Apart from food-borne sources, additional risk factors include close contact with pets or farm animals and activities in recreational waters (reviewed in reference 13). C. jejuni is widely distributed in many animals and has also been reported to be isolated from surface waters (15) and, occasionally, even from groundwater (31). However, the bacterium has been shown to be relatively sensitive to environmental stress outside its hosts, including heating, disinfectants, oxygen exposure, osmotic stress, desiccation, and acidity (5, 9, 19, 35).Several hygiene practices have been implemented in broiler production facilities to reduce C. jejuni carriage in live birds. Such measures include hygiene barriers such as changing clothes before entering the broiler houses and disinfection of the interior of the building with acid between flock rotations (20). Such efforts may reduce the number of C. jejuni organisms, but the bacterium is still difficult to eradicate from contaminated farms, and subsequent outbreaks at the same farm are not rare (11). Contradictory to its fragility in different in vitro settings, C. jejuni seems to be well adapted to survive the acidic milieu of the human stomach during the passage to the lower intestinal tract, where infection is established. This is illustrated by the very low infectious dose for both broiler chickens (7) and humans (4) and indicates that the bacterium has developed strategies to avoid or withstand low pH in order to survive the transit. The gastric acid is the first line of defense against ingested pathogens. During fasting conditions in healthy humans, the luminal pH in the stomach is usually around 2.0, but it may range from 1.5 to 5.5 depending on food intake, such as a diet with a high pH, or the use of proton pump inhibitors (36). Laboratory studies have demonstrated that C. jejuni in solution survives a maximum of 30 min at pH levels below pH 2.5 and for up to 60 min at pH 3 (5, 23). When the bacterium is mixed with food, it seems to be protected, and it has been shown that C. jejuni inoculated onto ground beef survived at pH 2.5 for 2 h at 37°C (37).In the last few years, laboratory studies have identified a new potential epidemiological pathway for C. jejuni in which the bacterium colonizes unicellular eukaryotic organisms (protozoa) and thereby acquires protection from adverse environmental conditions (2, 17, 29). C. jejuni can colonize protozoa and survive longer in its protozoan host than as a free-living bacterium, and given the right temperature, the bacterium can also replicate intracellularly (1, 2). Protozoa, especially amoebae, serve as natural reservoirs or vehicles for the dissemination of several other pathogenic bacteria, including Legionella pneumophila (25), Vibrio cholerae (34), and Helicobacter pylori (38). Amoebae are abundant in virtually all natural water systems and can be found grazing on biofilms in water supply systems (14). In their trophozoite form, amoebae are naturally resistant to many environmental factors that are lethal to Campylobacter, and they can multiply at pHs ranging from 4 to 12 (16). Moreover, amoebae can enter a cyst form when challenged with unfavorable conditions. These cysts generally have a double cell wall that might explain their capability to survive chlorination, antimicrobials, and changes in pH and osmotic pressure. This resistance feature of amoebae makes them suitable hosts for other, less-resistant microorganisms (16, 32).In this study, we built on the advances gained in protozoa-Campylobacter research and investigated whether internalization of C. jejuni into Acanthamoeba affects bacterial tolerance to hydrochloric acid. Using an in vitro setup, we found that C. jejuni survived better in an acidic environment when it was coincubated with amoebae than when it was incubated as bacteria in solution. Furthermore, we show that bacterial motility and adhesion to and internalization into amoeba are trigged by moderately acidic conditions. The implications of these findings for the survival of C. jejuni in food production as well as in transit through the human stomach are discussed.     

Chickens and Cattle as Sources of Sporadic Domestically Acquired Campylobacter jejuni Infections in Finland

A substantial sampling among domestic human campylobacter cases, chicken process lots, and cattle at slaughter was performed during the seasonal peak of human infections. Campylobacter jejuni isolates (n = 419) were subtyped using pulsed-field gel electrophoresis with SmaI, and isolates representing overlapping types (n = 212) were further subtyped using KpnI for restriction. The SmaI/KpnI profiles of 55.4% (97/175) of the human isolates were indistinguishable from those of the chicken or cattle isolates. The overlapping SmaI/KpnI subtypes accounted for 69.8% (30/43) and 15.9% (32/201) of the chicken and cattle isolates, respectively. The occurrence of identical SmaI/KpnI subtypes with human C. jejuni isolates was significantly associated with animal host species (P < 0.001). A temporal association of isolates from chickens and patients was possible in 31.4% (55/175) of the human infections. Besides chickens as sources of C. jejuni in the sporadic infections, the role of cattle appears notable. New approaches to restrict the occurrence of campylobacters in other farm animals may be needed in addition to hygienic measures in chicken production. However, only about half of the human infections were attributable to these sources.The incidence of human enteric infections caused by campylobacters is highest in the summer months, showing a consistent peak at the end of July in Finland (www.ktl.fi/attachments/suomi/julkaisut/julkaisusarja_b/2008/2008b09.pdf), as well as in other Nordic countries (16, 33). Almost 70% of campylobacter infections detected in July and August in Finland are domestically acquired, whereas the annual average proportion of domestic cases is about 30%, and most of them are caused by Campylobacter jejuni (30). The prevalence of campylobacters in Finnish broiler flocks peaks simultaneously with the human cases (7), and similar sero- and genotypes have been reported among human and poultry strains isolated in Finland and in other countries (5, 8, 21-23). Several epidemiological studies have identified the handling and consumption of raw or undercooked poultry meat as a major risk factor for campylobacter enteritis (for example, see references 18, 20, and 41), whereas opposite conclusions about the significance of the consumption of chicken meat were drawn from the Swedish case-control study among young children (2) and an extensive Danish register-based study (6).Data derived from the genotyping studies of C. jejuni isolates from human infections and animals support the current suggestion that poultry is the most important single source of sporadic campylobacteriosis (12, 22, 29). However, several reports on genotype comparisons suggest that poultry may be a less significant source of campylobacters than generally thought, and other animal reservoirs should also be considered notable sources of campylobacters pathogenic to humans (3, 8, 17, 27, 31). Studies of the temporal occurrence of campylobacters in human infections and poultry flocks have revealed that the peak in prevalence, as well as some of the overlapping sero- and genotypes, is detected in humans prior to being detected in poultry (21, 28).Although cattle are well-known carriers of campylobacters, the survival of these fragile organisms in beef is poor (39, 42). In recent years, some authors (1, 4, 10) have raised the question of an indirect association between cattle and human cases. In a Finnish study combining data from the multilocus sequence typing of campylobacters isolated from production animals and from epidemiological studies of human cases, significant associations emerged between certain sequence-type complexes from human infections and contact with cattle, the consumption of unpasteurized milk, or the tasting or consumption of raw minced meat (23).The aim of this study was to investigate the contributions of poultry and cattle as sources of human C. jejuni infections in Finland by comparing over a limited time frame the macrorestriction profiles obtained from pulsed-field gel electrophoresis (PFGE) analysis of a geographically representative collection of C. jejuni isolates from domestically acquired sporadic human infections, chicken process lots, and cattle.     

Genotype and Antibiotic Resistance Analyses of Campylobacter Isolates from Ceca and Carcasses of Slaughtered Broiler Flocks

To obtain genetic information about Campylobacter jejuni and Campylobacter coli from broilers and carcasses at slaughterhouses, we analyzed and compared 340 isolates that were collected in 2008 from the cecum right after slaughter or from the neck skin after processing. We performed rpoB sequence-based identification, multilocus sequence typing (MLST), and flaB sequence-based typing; we additionally analyzed mutations within the 23S rRNA and gyrA genes that confer resistance to macrolide and quinolone antibiotics, respectively. The rpoB-based identification resulted in a distribution of 72.0% C. jejuni and 28.0% C. coli. The MLST analysis revealed that there were 59 known sequence types (STs) and 6 newly defined STs. Most of the STs were grouped into 4 clonal complexes (CC) that are typical for poultry (CC21, CC45, CC257, and CC828), and these represented 61.8% of all of the investigated isolates. The analysis of 95 isolates from the cecum and from the corresponding carcass neck skin covered 44 different STs, and 54.7% of the pairs had matching genotypes. The data indicate that cross-contamination from various sources during slaughter may occur, although the majority of Campylobacter contamination on carcasses appeared to originate from the slaughtered flock itself. Mutations in the 23S rRNA gene were found in 3.1% of C. coli isolates, although no mutations were found in C. jejuni isolates. Mutations in the gyrA gene were observed in 18.9% of C. jejuni and 26.8% of C. coli isolates, which included two C. coli strains that carried mutations conferring resistance to both classes of antibiotics. A relationship between specific genotypes and antibiotic resistance/susceptibility was observed.Campylobacteriosis is the leading food-borne bacterial gastroenteritis worldwide (12, 15). In Switzerland, the number of registered campylobacteriosis cases has rapidly increased to more than 100 per 100,000 inhabitants in the past few years (14), and this trend has also been observed in the European Union (EU) (12). However, the real number of cases is likely higher, because not all cases are reported due to the self-limiting nature of the disease and its potentially mild symptoms.Campylobacter jejuni and Campylobacter coli are commonly associated with human infection, and they can be detected in up to 85% and 15% of cases, respectively (33). Despite the important role that C. jejuni and C. coli play as zoonotic pathogens worldwide, there is little information regarding the route(s) of transmission (17). Numerous case-control and modeling studies on the infection sources of C. jejuni and C. coli have suggested that handling and consumption of contaminated poultry meat are associated with a risk of human campylobacteriosis (17, 45, 47, 49, 51). Initial meat contamination with C. jejuni or C. coli from the chicken intestine may occur during commonly used automated slaughter processing through several routes, such as the air, water, previously slaughtered flocks, or machinery (19, 36, 37).Precise genotyping and continuous comparison of the strains obtained from, e.g., the production site, flocks, slaughterhouse, retail meat, and infected humans would help to trace the source of infection and might indicate possible intervention strategies for the contaminated site.DNA sequence-based typing methods, such as multilocus sequence typing (MLST), are well suited for this purpose (28), and MLST has become the method of choice for genotyping of Campylobacter (6, 8). Moreover, extension of the classical MLST technique for C. jejuni and C. coli with sequencing of the short variable region (SVR) within the flagellin-encoding gene flaB allows a more precise differentiation among strains that have the same MLST sequence type (ST) (9, 29). An extended MLST work flow was recently developed that reduces the associated time and cost (24). In addition, the new approach allows genetic determination of antibiotic resistance to quinolones and macrolides. Resistance to these antibiotics is a worldwide issue of concern, as an increasing number of Campylobacter isolates are resistant to them. Strikingly, a number of strains are resistant to ciprofloxacin (a quinolone) and, to a lesser extent, erythromycin (a macrolide), which is problematic, because these drugs are typically used to treat campylobacteriosis. Resistance to quinolones is mainly associated with a point mutation in the DNA gyrase gene (gyrA) at position C257T, and a transition in the 23S rRNA gene at position A2075G is commonly responsible for macrolide resistance (1). Simple sequence-based analysis of these common mutational positions can therefore provide information about the antibiotic susceptibility or resistance of a strain. Besides the prudent use of antibiotics, knowledge about the genetic composition of the infectious agent can be helpful to both treat the disease and prevent the spread of resistant strains.In the current study, MLST, flaB typing, and sequence-based determination of quinolone and macrolide resistances were used to investigate the genetic background of C. jejuni and C. coli isolates collected from Swiss broilers in a spatiotemporal study in 2008. We addressed the following three aspects: (i) the diversity of Campylobacter isolates that were recovered from pooled cecum samples and the carcass neck skin, (ii) the possible impact of cross- and self-contamination during slaughter, and (iii) the antibiotic resistance of Campylobacter strains from the broiler flocks and chicken carcasses. All of the data, including the strain information and trace files, were entered into a commercial Web-based Campylobacter MLST database (SmartGene, Zug, Switzerland). This database allows users to retrieve and compare information for any analyzed strain for monitoring purposes (24).     

The Response of Campylobacter jejuni to Low Temperature Differs from That of Escherichia coli

Human infection with Campylobacter jejuni is often associated with the consumption of foods that have been exposed to both chilling and high temperatures. Despite the public health importance of this pathogen, little is known about the effects of cold exposure on its ability to survive a subsequent heat challenge. This work examined the effect of rapid exposure to chilling, as would occur in poultry processing, on the heat resistance at 56°C of two C. jejuni strains, 11168 and 2097e48, and of Escherichia coli K-12. Unlike E. coli K-12, whose cold-exposed cells showed increased sensitivity to 56°C, such exposure had only a marginal effect on subsequent heat resistance in C. jejuni. This may be explained by the finding that during rapid chilling, unlike E. coli cells, C. jejuni cells are unable to alter their fatty acid composition and do not adapt to cold exposure. However, their unaltered fatty acid composition is more suited to survival when cells are exposed to high temperatures. This hypothesis is supported by the fact that in C. jejuni, the ratio of unsaturated to saturated fatty acids was not significantly different after cold exposure, but it was in E. coli. The low-temperature response of C. jejuni is very different from that of other food-borne pathogens, and this may contribute to its tolerance to further heat stresses.Campylobacter jejuni is the leading cause of bacterial diarrheal disease worldwide and is the most common antecedent to the peripheral neuropathies Guillain-Barré syndrome and Miller Fisher syndrome (32, 35). In England and Wales, there were ca. 47,000 Campylobacter cases reported in 2006, a probable 10-fold underestimate of the true incidence, as the World Health Organization calculates that ca. 1% of the population of Europe will be infected each year.Most Campylobacter infections are food borne, although contaminated water and environmental exposure have also been implicated (5). Currently, identified high-risk factors include the consumption of chicken, especially when undercooked (15), barbecued meat (2), and raw or improperly pasteurized milk (10, 22). In all of these cases, the infecting Campylobacter population would have been exposed to both low and high temperatures before being consumed.Despite their importance as human pathogens, little is known about how campylobacters cope with hostile conditions in the transmission chain from animals to humans. Campylobacter presents an interesting conundrum. It is generally considered to be fragile compared to other food-borne pathogens (7) but is recognized as the leading cause of food-borne disease, and vehicles are frequently foods that have received a degree of heat exposure.Although Campylobacter has been shown to be able to mount an acid tolerance response (28), it lacks a σ³⁸ (RpoS) homologue (23) and cold shock proteins (17), which is thought to limit its ability to respond to hostile conditions common in the food chain. Despite lacking many of the classical bacterial stress responses, Campylobacter can survive for extended periods at low temperatures on key foods such as raw chicken (12). It is important to determine how the responses of Campylobacter to cold could affect its survival in other parts of the food chain. Does preexposure to cold make it more heat sensitive, as is the case for Escherichia coli and Salmonella spp. (20), or are its responses different, as work on one strain (27) suggests?The ability to deal with cold is inherently important to a wide range of mesophilic bacteria (29), and it is likely that some cold exposure tolerance mechanisms are highly conserved. One of the most important of these is a change in fatty acid composition, particularly in the outer membrane (36). Maintaining membrane homoviscosity is critical for continuing growth, and cellular membrane fatty acid composition is altered as the growth temperature changes (30). For example, during exposure to reduced temperatures, Salmonella spp. increase levels of unsaturated fatty acids (UFA), whereas Listeria spp. raise those of branched fatty acids in cellular membranes (33). Both responses increase membrane fluidity. Increased levels of cyclic fatty acids have been shown to have a role in raising the heat resistance of Pediococcus spp. (3) and to increase the stability of the membrane structure (9). Ulmer et al. (37) showed that as the growth temperature for Lactobacillus plantarum decreased, the proportion of UFA in cell membranes rose, leading to increased membrane fluidity and allowing the membrane to be maintained in a liquid gel state. A temperature-dependent shift in fatty acid production has been seen in E. coli. When the growth temperature is reduced, the enzyme activity of FabF increases, leading to an increase in the conversion of 16:1 fatty acids to 18:1 fatty acids (8, 11). Desaturation enhances the effects of the branched fatty acids produced during acclimation of bacterial cells to low temperatures (34).Although changes in fatty acid synthesis have been seen in Campylobacter coli upon a temperature downshift from 42 to 20°C (19), there is currently very little information on the effects of exposure to refrigeration conditions on the whole-cell fatty acid composition of C. jejuni. A study by Hazeleger et al. (17) compared changes in fatty acid composition in coccoid cells held at 4 or 12°C for 14 days with those in control spiral cells grown at 37°C for 24 h. They found that few significant changes in composition occurred at the lower temperatures, whereas at 25°C there were more marked changes in many fatty acids.The work reported here examines the effects of exposure to 6°C for 24 h on whole-cell fatty acid composition and heat resistance of C. jejuni to determine whether Campylobacter has a similar response to chill to the prototypic response of E. coli.     

Importance of Polyphosphate Kinase 1 for Campylobacter jejuni Viable-but-Nonculturable Cell Formation,Natural Transformation,and Antimicrobial Resistance

Campylobacter jejuni, a gram-negative, microaerophilic bacterium, is a predominant cause of bacterial gastroenteritis in humans. Although considered fragile and fastidious and lacking many classical stress response mechanisms, C. jejuni exhibits a remarkable capacity for survival and adaptation, successfully infecting humans and persisting in the environment. Consequently, understanding the physiological and genetic properties that allow C. jejuni to survive and adapt to various stress conditions is crucial for therapeutic interventions. Of importance is polyphosphate (poly-P) kinase 1 (PPK1), which is a key enzyme mediating the synthesis of poly-P, an essential molecule for survival, mediating stress responses, host colonization, and virulence in many bacteria. Therefore, we investigated the role of PPK1 in C. jejuni pathogenesis, stress survival, and adaptation. Our findings demonstrate that a C. jejuni Δppk1 mutant was deficient in poly-P accumulation, which was associated with a decreased ability to form viable-but-nonculturable cells under acid stress. The Δppk1 mutant also showed a decreased frequency of natural transformation and an increased susceptibility to various antimicrobials. Furthermore, the Δppk1 mutant was characterized by a dose-dependent deficiency in chicken colonization. Complementation of the Δppk1 mutant with the wild-type copy of ppk1 restored the deficient phenotypes to levels similar to those of the wild type. Our results suggest that poly-P plays an important role in stress survival and adaptation and might contribute to genome plasticity and the spread and development of antimicrobial resistance in C. jejuni. These findings highlight the potential of PPK1 as a novel target for therapeutic interventions.Campylobacter jejuni, a gram-negative, microaerophilic bacterium, occurs as a commensal among the intestinal microflora of various animals, especially chickens and cattle (6, 73). However, C. jejuni can infect human hosts, invading the intestinal mucosa and causing watery and/or bloody diarrhea (9). C. jejuni is transmitted to humans primarily through the consumption of contaminated chicken products, raw milk, or water (2, 3). Currently, C. jejuni is considered a leading bacterial cause of human food-borne gastroenteritis (3, 61) and has also been associated with a plethora of symptoms, including acute neuromuscular paralysis (Guillain-Barré syndrome) (26). Since an appropriate vaccine for human campylobacteriosis has yet to be introduced, it has been suggested that C. jejuni infections might be alternatively controlled by reducing colonization in food animals (73). Consequently, determining the physiological and genetic properties that allow the survival of C. jejuni and its colonization of animal hosts, pathogenicity, and adaptation to various stresses is of critical importance.The mechanisms underlying C. jejuni adaptation and survival under stresses imposed by its environment and host are not well understood. High variability between different C. jejuni strains and the unavailability of appropriate genetic tools and animal models have contributed to the lack of knowledge regarding its stress tolerance and pathogenicity. However, it is suggested that the capacity of C. jejuni to form viable-but-nonculturable (VBNC) cells under stress (14) and its readiness for natural transformation (68) and acquiring resistance to antibiotics (39) are among the strategies that promote stress adaptation and survival. Although little is known about the genetics underlying these processes, recent advances in C. jejuni genomics show that this bacterium carries several important genes that might play key roles in mediating stress adaptation and survival. Of particular interest are genes encoding polyphosphate (poly-P) kinases, ppk1 (CJJ81176_1361) and ppk2 (CJJ81176_0633), that were predicted to be involved in the metabolism of poly-P (22, 25, 47), an intracellular granule that impacts several physiological properties in many bacterial species, including pathogenicity, host colonization, adaptation to different environments, and survival (28, 31, 46).Poly-P kinase 1 (PPK1) is encoded by ppk1, which mediates the synthesis of all or most of the poly-P in the cell (33), while ppk2 encodes an enzyme (PPK2) that synthesizes GTP from poly-P (27). Both ppk genes have been associated with the metabolism of poly-P, which consists of phosphate residues that are linked by high-energy phosphoanhydride bonds and is widely distributed in bacterial species (60). Previous reports showed that poly-P plays important roles in bacterial survival and stress tolerance, including ATP production (8), entry of DNA through membrane channels (13, 54), capsule composition (67), maintaining nutritional requirements during starvation (34), motility, biofilm formation, and resistance to oxidative, osmotic, heat, acid and alkaline stresses, and stationary-phase survival (28, 31, 46, 48, 50, 52, 65). Because of their importance in many bacterial species, it is not surprising to assume a role for PPK and poly-P in C. jejuni survival, colonization, and stress tolerance (8).Interestingly, PPK1 has been shown to be important for C. jejuni stress responses and pathogenicity (10). However, the role of ppk1 in key metabolic and physiological responses of C. jejuni still needs further analysis. For instance, it has been proposed that during starvation, poly-P might act as a reservoir for phosphorus and energy (7). Subsequently, poly-P would be crucial for maintaining viability/metabolism in stressed cells. This has been observed in H. pylori, where the occurrence of poly-P correlated with culturability and structurally intact cells (45). Poly-P-containing nonculturable H. pylori showed a capacity for ATP and mRNA synthesis after a nutrient stimulus (45). Consequently, poly-P might be an important factor for the formation of VBNC cells by stressed bacteria, including C. jejuni. Furthermore, natural transformation is perhaps one of the most important mechanisms in the adaptation of C. jejuni, and poly-P has been reported to play a role in the entry of DNA through membrane channels (13, 54). It follows that poly-P might be important for natural transformation, adaptation, and acquisition of antibiotic resistance genes in C. jejuni. Poly-P can further impact the survival and adaptation in C. jejuni by modulating antibiotic resistance properties. For example, poly-P interacted with Escherichia coli ribosomes (42), which are known targets of several antibiotics. These observations suggest that ppk1 might be linked to important physiology and functions such as VBNC cell formation, natural transformation, and antimicrobial resistance in C. jejuni. Therefore, in the present study, we determined the contribution of PPK1 to C. jejuni stress responses and adaptation, including the ability to form VBNC cells under acid stress, natural transformation, and antimicrobial resistance. Furthermore, we assessed the impact of ppk1 deletion on in vivo chicken colonization. Our findings highlight the importance of PPK1 in C. jejuni survival, adaptation to different environmental stresses, and in vivo colonization. These findings also indicate the suitability of PPK1 as a potential target for controlling the proliferation of this pathogen.