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.     

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.     

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.     

A New Borrelia Species Defined by Multilocus Sequence Analysis of Housekeeping Genes

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.Multilocus sequence typing (MLST) and multilocus sequence analysis (MLSA) have been shown to be powerful and pragmatic molecular methods for typing large numbers of microbial strains for population genetics studies, delineation of species, and assignment of strains to defined bacterial species (4, 13, 27, 40, 44). To date, MLST/MLSA schemes have been applied only to a few vector-borne microbial populations (1, 6, 30, 37, 40, 41, 47).Lyme borreliosis (LB) spirochetes comprise a diverse group of zoonotic bacteria which are transmitted among vertebrate hosts by ixodid (hard) ticks. The most common agents of human LB are Borrelia burgdorferi (sensu stricto), Borrelia afzelii, Borrelia garinii, Borrelia lusitaniae, and Borrelia spielmanii (7, 8, 12, 35). To date, 15 species have been named within the group of LB spirochetes (6, 31, 32, 37, 38, 41). While several of these LB species have been delineated using whole DNA-DNA hybridization (3, 20, 33), most ecological or epidemiological studies have been using single loci (5, 9-11, 29, 34, 36, 38, 42, 51, 53). Although some of these loci have been convenient for species assignment of strains or to address particular epidemiological questions, they may be unsuitable to resolve evolutionary relationships among LB species, because it is not possible to define any outgroup. For example, both the 5S-23S intergenic spacer (5S-23S IGS) and the gene encoding the outer surface protein A (ospA) are present only in LB spirochete genomes (36, 43). The advantage of using appropriate housekeeping genes of LB group spirochetes is that phylogenetic trees can be rooted with sequences of relapsing fever spirochetes. This renders the data amenable to detailed evolutionary studies of LB spirochetes.LB group spirochetes differ remarkably in their patterns and levels of host association, which are likely to affect their population structures (22, 24, 46, 48). Of the three main Eurasian Borrelia species, B. afzelii is adapted to rodents, whereas B. valaisiana and most strains of B. garinii are maintained by birds (12, 15, 16, 23, 26, 45). However, B. garinii OspA serotype 4 strains in Europe have been shown to be transmitted by rodents (17, 18) and, therefore, constitute a distinct ecotype within B. garinii. These strains have also been associated with high pathogenicity in humans, and their finer-scale geographical distribution seems highly focal (10, 34, 52, 53).In this study, we analyzed the intra- and interspecific phylogenetic relationships of B. burgdorferi, B. afzelii, B. garinii, B. valaisiana, B. lusitaniae, B. bissettii, and B. spielmanii by means of a novel MLSA scheme based on chromosomal housekeeping genes (30, 48).     

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.     

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.     

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.     

Development of a Two-Color Fluorescence In Situ Hybridization Technique for Species-Level Identification of Human-Infectious Cryptosporidium spp.

A two-color fluorescence in situ hybridization assay that allows for the simultaneous identification of Cryptosporidium parvum and C. hominis was developed. The assay is a simple, rapid, and cost-effective tool for the detection of the major Cryptosporidium species of concern to public health.Cryptosporidium (Apicomplexa) is a genus of protozoan parasites with species and genotypes that infect humans, domesticated livestock, companion animals, and wildlife worldwide (5, 6, 14, 15, 20, 23). The majority of cases of cryptosporidiosis in humans are caused by Cryptosporidium parvum or C. hominis (8, 10, 19, 24), although rare cases due to species such as C. meleagridis, C. felis, or C. canis have been reported (8, 9, 11-13, 17, 18, 22). The specific identification and characterization of Cryptosporidium species are central to the control of this disease in humans and a wide range of animals.One of the most widely adopted techniques for the identification of microorganisms in complex microbial communities is fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes (2-4). This method relies on the hybridization of synthetic oligonucleotide probes to specific regions within the rRNA of the organism. While FISH has been applied for the detection of Cryptosporidium oocysts in water samples (21), no FISH probes that successfully differentiate C. hominis from C. parvum have been reported.We have reported previously on the design of a species-specific probe, Cpar677, that detects C. parvum (1). In this study, we report on the design and validation of a C. hominis species-specific probe, Chom253. Together, the two probes were used here for the development of a two-color, microscopy-based FISH assay for the simultaneous detection of C. parvum and C. hominis.     

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.     

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.     

Alignment of the c-Type Cytochrome OmcS along Pili of Geobacter sulfurreducens

Immunogold localization revealed that OmcS, a cytochrome that is required for Fe(III) oxide reduction by Geobacter sulfurreducens, was localized along the pili. The apparent spacing between OmcS molecules suggests that OmcS facilitates electron transfer from pili to Fe(III) oxides rather than promoting electron conduction along the length of the pili.There are multiple competing/complementary models for extracellular electron transfer in Fe(III)- and electrode-reducing microorganisms (8, 18, 20, 44). Which mechanisms prevail in different microorganisms or environmental conditions may greatly influence which microorganisms compete most successfully in sedimentary environments or on the surfaces of electrodes and can impact practical decisions on the best strategies to promote Fe(III) reduction for bioremediation applications (18, 19) or to enhance the power output of microbial fuel cells (18, 21).The three most commonly considered mechanisms for electron transfer to extracellular electron acceptors are (i) direct contact between redox-active proteins on the outer surfaces of the cells and the electron acceptor, (ii) electron transfer via soluble electron shuttling molecules, and (iii) the conduction of electrons along pili or other filamentous structures. Evidence for the first mechanism includes the necessity for direct cell-Fe(III) oxide contact in Geobacter species (34) and the finding that intensively studied Fe(III)- and electrode-reducing microorganisms, such as Geobacter sulfurreducens and Shewanella oneidensis MR-1, display redox-active proteins on their outer cell surfaces that could have access to extracellular electron acceptors (1, 2, 12, 15, 27, 28, 31-33). Deletion of the genes for these proteins often inhibits Fe(III) reduction (1, 4, 7, 15, 17, 28, 40) and electron transfer to electrodes (5, 7, 11, 33). In some instances, these proteins have been purified and shown to have the capacity to reduce Fe(III) and other potential electron acceptors in vitro (10, 13, 29, 38, 42, 43, 48, 49).Evidence for the second mechanism includes the ability of some microorganisms to reduce Fe(III) that they cannot directly contact, which can be associated with the accumulation of soluble substances that can promote electron shuttling (17, 22, 26, 35, 36, 47). In microbial fuel cell studies, an abundance of planktonic cells and/or the loss of current-producing capacity when the medium is replaced is consistent with the presence of an electron shuttle (3, 14, 26). Furthermore, a soluble electron shuttle is the most likely explanation for the electrochemical signatures of some microorganisms growing on an electrode surface (26, 46).Evidence for the third mechanism is more circumstantial (19). Filaments that have conductive properties have been identified in Shewanella (7) and Geobacter (41) species. To date, conductance has been measured only across the diameter of the filaments, not along the length. The evidence that the conductive filaments were involved in extracellular electron transfer in Shewanella was the finding that deletion of the genes for the c-type cytochromes OmcA and MtrC, which are necessary for extracellular electron transfer, resulted in nonconductive filaments, suggesting that the cytochromes were associated with the filaments (7). However, subsequent studies specifically designed to localize these cytochromes revealed that, although the cytochromes were extracellular, they were attached to the cells or in the exopolymeric matrix and not aligned along the pili (24, 25, 30, 40, 43). Subsequent reviews of electron transfer to Fe(III) in Shewanella oneidensis (44, 45) appear to have dropped the nanowire concept and focused on the first and second mechanisms.Geobacter sulfurreducens has a number of c-type cytochromes (15, 28) and multicopper proteins (12, 27) that have been demonstrated or proposed to be on the outer cell surface and are essential for extracellular electron transfer. Immunolocalization and proteolysis studies demonstrated that the cytochrome OmcB, which is essential for optimal Fe(III) reduction (15) and highly expressed during growth on electrodes (33), is embedded in the outer membrane (39), whereas the multicopper protein OmpB, which is also required for Fe(III) oxide reduction (27), is exposed on the outer cell surface (39).OmcS is one of the most abundant cytochromes that can readily be sheared from the outer surfaces of G. sulfurreducens cells (28). It is essential for the reduction of Fe(III) oxide (28) and for electron transfer to electrodes under some conditions (11). Therefore, the localization of this important protein was further investigated.     

Cultivation of Fastidious Bacteria by Viability Staining and Micromanipulation in a Soil Substrate Membrane System

Soil substrate membrane systems allow for microcultivation of fastidious soil bacteria as mixed microbial communities. We isolated established microcolonies from these membranes by using fluorescence viability staining and micromanipulation. This approach facilitated the recovery of diverse, novel isolates, including the recalcitrant bacterium Leifsonia xyli, a plant pathogen that has never been isolated outside the host.The majority of bacterial species have never been recovered in the laboratory (1, 14, 19, 24). In the last decade, novel cultivation approaches have successfully been used to recover “unculturables” from a diverse range of divisions (23, 25, 29). Most strategies have targeted marine environments (4, 23, 25, 32), but soil offers the potential for the investigation of vast numbers of undescribed species (20, 29). Rapid advances have been made toward culturing soil bacteria by reformulating and diluting traditional media, extending incubation times, and using alternative gelling agents (8, 21, 29).The soil substrate membrane system (SSMS) is a diffusion chamber approach that uses extracts from the soil of interest as the growth substrate, thereby mimicking the environment under investigation (12). The SSMS enriches for slow-growing oligophiles, a proportion of which are subsequently capable of growing on complex media (23, 25, 27, 30, 32). However, the SSMS results in mixed microbial communities, with the consequent difficulty in isolation of individual microcolonies for further characterization (10).Micromanipulation has been widely used for the isolation of specific cell morphotypes for downstream applications in molecular diagnostics or proteomics (5, 15). This simple technology offers the opportunity to select established microcolonies of a specific morphotype from the SSMS when combined with fluorescence visualization (3, 11). Here, we have combined the SSMS, fluorescence viability staining, and advanced micromanipulation for targeted isolation of viable, microcolony-forming soil bacteria.     

Selection of a Bacillus pumilus Strain Highly Active against Ceratitis capitata (Wiedemann) Larvae

Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), the Mediterranean fruit fly (medfly), is one of the most important fruit pests worldwide. The medfly is a polyphagous species that causes losses in many crops, which leads to huge economic losses. Entomopathogenic bacteria belonging to the genus Bacillus have been proven to be safe, environmentally friendly, and cost-effective tools to control pest populations. As no control method for C. capitata based on these bacteria has been developed, isolation of novel strains is needed. Here, we report the isolation of 115 bacterial strains and the results of toxicity screening with adults and larvae of C. capitata. As a result of this analysis, we obtained a novel Bacillus pumilus strain, strain 15.1, that is highly toxic to C. capitata larvae. The toxicity of this strain for C. capitata was related to the sporulation process and was observed only when cultures were incubated at low temperatures before they were used in a bioassay. The mortality rate for C. capitata larvae ranged from 68 to 94% depending on the conditions under which the culture was kept before the bioassay. Toxicity was proven to be a special characteristic of the newly isolated strain, since other B. pumilus strains did not have a toxic effect on C. capitata larvae. The results of the present study suggest that B. pumilus 15.1 could be considered a strong candidate for developing strategies for biological control of C. capitata.The Mediterranean fruit fly (medfly), Ceratitis capitata, is considered a highly invasive agricultural and economically important pest throughout the world. In less than 200 years the range of this species has expanded from its native habitat in sub-Saharan Africa, and it has become a cosmopolitan species (26) that is present on five continents (14, 46). The wide distribution of the medfly is attributed, among other things, to its remarkably polyphagous behavior (more than 300 host plants have been reported) (43), to its resistance to cold climates (65), and to successful establishment after multiple introductions (30, 49) as a result of the increasing frequency of global trade (46).Medfly infestations cause serious economic losses and sometimes result in complete loss of crops (76). Numerous methods have been tried to control medfly populations, including chemical products, such as malathion and other organophosphate insecticides (4, 8), classic biological control programs based on the release of some of parasitoids and predators (38, 41, 44), toxic baits (2, 13, 31, 32, 35, 56), mass trapping systems (24, 51), the sterile insect technique (7, 34, 61, 63, 72, 73), and development of integrated strategies of management (71). In spite of all of these attempts, control of Mediterranean fruit fly populations has been ineffective, and losses associated with this pest worldwide are constantly increasing (21, 46).Insecticides based on microbial agents (bacteria, fungi, and viruses) are a promising alternative that has received a great deal of attention for control of C. capitata (5, 13, 18, 40, 55), but so far no such insecticide has reached a commercial stage. Among the microbial insecticides, bacteria are very successful agents in biological control programs (17, 29). The entomopathogenic bacteria belonging to the genus Bacillus are natural agents used for biological control of invertebrate pests and are the basis of many commercial insecticides. Three species of the genus Bacillus have been mass produced and commercialized: Bacillus sphaericus, Bacillus thuringiensis, and Paenibacillus popilliae (formerly Bacillus popilliae) (29, 54). These organisms have different spectra and levels of activity that are correlated with the nature of the toxins, which are very frequently produced during sporulation (16, 17). B. thuringiensis was the first Bacillus species used in biological control programs for pests and human vector disease insects (17, 62). During its stationary phase, this Gram-positive, aerobic, ubiquitous, endospore-forming bacterium produces parasporal crystalline inclusions composed mainly of two types of insecticidal proteins (Cry and Cyt toxins) (62) that are toxic to a variety of insects, in some cases at the species level.There have been some reports of B. thuringiensis strains active against other fruit flies (3, 37, 58, 59, 67), but there has been no report of any Bacillus strain with activity against C. capitata.The aim of this study was to search for novel bacteria belonging to the genus Bacillus, specifically B. thuringiensis, with activity against adults and larvae of C. capitata that could be used as biological control agents. Isolation of 115 bacterial strains, evaluation of the insecticidal activities of these strains, and identification of a novel strain of Bacillus pumilus that is highly toxic to C. capitata larvae are reported here. In addition, we found that toxicity was observed only when cultures of B. pumilus strain 15.1 were exposed to low temperatures. The isolation of this novel pathogenic strain could be important for future development of biotechnological strategies aimed at reducing the economic losses caused by C. capitata.