Evaluation of core genome and whole genome multilocus sequence typing schemes for Campylobacter jejuni and Campylobacter coli outbreak detection in the USA.

Campylobacter is a leading causing of bacterial foodborne and zoonotic illnesses in the USA. Pulsed-field gene electrophoresis (PFGE) and 7-gene multilocus sequence typing (MLST) have been historically used to differentiate sporadic from outbreak Campylobacter isolates. Whole genome sequencing (WGS) has been shown to provide superior resolution and concordance with epidemiological data when compared with PFGE and 7-gene MLST during outbreak investigations. In this study, we evaluated epidemiological concordance for high-quality SNP (hqSNP), core genome (cg)MLST and whole genome (wg)MLST to cluster or differentiate outbreak-associated and sporadic Campylobacter jejuni and Campylobacter coli isolates. Phylogenetic hqSNP, cgMLST and wgMLST analyses were also compared using Baker's gamma index (BGI) and cophenetic correlation coefficients. Pairwise distances comparing all three analysis methods were compared using linear regression models. Our results showed that 68/73 sporadic C. jejuni and C. coli isolates were differentiated from outbreak-associated isolates using all three methods. There was a high correlation between cgMLST and wgMLST analyses of the isolates; the BGI, cophenetic correlation coefficient, linear regression model R 2 and Pearson correlation coefficients were >0.90. The correlation was sometimes lower comparing hqSNP analysis to the MLST-based methods; the linear regression model R 2 and Pearson correlation coefficients were between 0.60 and 0.86, and the BGI and cophenetic correlation coefficient were between 0.63 and 0.86 for some outbreak isolates. We demonstrated that C. jejuni and C. coli isolates clustered in concordance with epidemiological data using WGS-based analysis methods. Discrepancies between allele and SNP-based approaches may reflect the differences between how genomic variation (SNPs and indels) are captured between the two methods. Since cgMLST examines allele differences in genes that are common in most isolates being compared, it is well suited to surveillance: searching large genomic databases for similar isolates is easily and efficiently done using allelic profiles. On the other hand, use of an hqSNP approach is much more computer intensive and not scalable to large sets of genomes. If further resolution between potential outbreak isolates is needed, wgMLST or hqSNP analysis can be used.

Cronobacter sakazakii Infections in Two Infants Linked to Powdered Infant Formula and Breast Pump Equipment - United States, 2021 and 2022.

Cronobacter sakazakii, a species of gram-negative bacteria belonging to the Enterobacteriaceae family, is known to cause severe and often fatal meningitis and sepsis in young infants. C. sakazakii is ubiquitous in the environment, and most reported infant cases have been attributed to contaminated powdered infant formula (powdered formula) or breast milk that was expressed using contaminated breast pump equipment (1-3). Previous investigations of cases and outbreaks have identified C. sakazakii in opened powdered formula, breast pump parts, environmental surfaces in the home, and, rarely, in unopened powdered formula and formula manufacturing facilities (2,4-6). This report describes two infants with C. sakazakii meningitis reported to CDC in September 2021 and February 2022. CDC used whole genome sequencing (WGS) analysis to link one case to contaminated opened powdered formula from the patient's home and the other to contaminated breast pump equipment. These cases highlight the importance of expanding awareness about C. sakazakii infections in infants, safe preparation and storage of powdered formula, proper cleaning and sanitizing of breast pump equipment, and using WGS as a tool for C. sakazakii investigations.

Ongoing Outbreak of Extensively Drug-Resistant Campylobacter jejuni Infections Associated With US Pet Store Puppies, 2016-2020.

Importance: Extensively drug-resistant Campylobacter jejuni infections cannot be treated with any commonly recommended antibiotics and pose an increasing public health threat. Objectives: To investigate cases of extensively drug-resistant C jejuni associated with pet store puppies and describe the epidemiologic and laboratory characteristics of these infections. Design, Setting, and Participants: In August 2017, health officials identified, via survey, patients with C jejuni infections who reported contact with puppies sold by pet stores. In conjunction with state and federal partners, the Centers for Disease Control and Prevention investigated cases of culture-confirmed C jejuni infections in US patients with an epidemiologic or molecular association with pet store puppies between January 1, 2016, and February 29, 2020. Available records from cases occurring before 2016 with genetically related isolates were also obtained. Main Outcomes and Measures: Patients were interviewed about demographic characteristics, health outcomes, and dog exposure during the 7 days before illness onset. Core genome multilocus sequence typing was used to assess isolate relatedness, and genomes were screened for resistance determinants to predict antibiotic resistance. Isolates resistant to fluoroquinolones, macrolides, and 3 or more additional antibiotic classes were considered to be extensively drug resistant. Cases before 2016 were identified by screening all sequenced isolates submitted for surveillance using core genome multilocus sequence typing. Results: A total of 168 patients (median [interquartile range] age, 37 [19.5-51.0] years; 105 of 163 female [64%]) with an epidemiologic or molecular association with pet store puppies were studied. A total of 137 cases occurred from January 1, 2016, to February 29, 2020, with 31 additional cases dating back to 2011. Overall, 117 of 121 patients (97%) reported contact with a dog in the week before symptom onset, of whom 69 of 78 (88%) with additional information reported contact with a pet store puppy; 168 isolates (88%) were extensively drug resistant. Traceback investigation did not implicate any particular breeder, transporter, distributer, store, or chain. Conclusions and Relevance: Strains of extensively drug-resistant C jejuni have been circulating since at least 2011 and are associated with illness among pet store customers, employees, and others who come into contact with pet store puppies. The results of this study suggest that practitioners should ask about puppy exposure when treating patients with Campylobacter infection, especially when they do not improve with routine antibiotics, and that the commercial dog industry should take action to help prevent the spread of extensively drug-resistant C jejuni from pet store puppies to people.

Comparison of Molecular Subtyping and Antimicrobial Resistance Detection Methods Used in a Large Multistate Outbreak of Extensively Drug-Resistant Campylobacter jejuni Infections Linked to Pet Store Puppies.

Campylobacter jejuni is a leading cause of enteric bacterial illness in the United States. Traditional molecular subtyping methods, such as pulsed-field gel electrophoresis (PFGE) and 7-gene multilocus sequence typing (MLST), provided limited resolution to adequately identify C. jejuni outbreaks and separate out sporadic isolates during outbreak investigations. Whole-genome sequencing (WGS) has emerged as a powerful tool for C. jejuni outbreak detection. In this investigation, 45 human and 11 puppy isolates obtained during a 2016-2018 outbreak linked to pet store puppies were sequenced. Core genome multilocus sequence typing (cgMLST) and high-quality single nucleotide polymorphism (hqSNP) analysis of the sequence data separated the isolates into the same two clades containing minor within-clade differences; however, cgMLST analysis does not require selection of an appropriate reference genome, making the method preferable to hqSNP analysis for Campylobacter surveillance and cluster detection. The isolates were classified as sequence type 2109 (ST2109)-a rarely seen MLST sequence type. PFGE was performed on 38 human and 10 puppy isolates; PFGE patterns did not reliably predict clustering by cgMLST analysis. Genetic detection of antimicrobial resistance determinants predicted that all outbreak-associated isolates would be resistant to six drug classes. Traditional antimicrobial susceptibility testing (AST) confirmed a high correlation between genotypic and phenotypic antimicrobial resistance determinations. WGS analysis linked C. jejuni isolates in humans and pet store puppies even when canine exposure information was unknown, aiding the epidemiological investigation during the outbreak. WGS data were also used to quickly identify the highly drug-resistant profile of these outbreak-associated C. jejuni isolates.

Corrigendum: Whole Genome Sequencing: Bridging One-Health Surveillance of Foodborne Diseases.

[This corrects the article DOI: 10.3389/fpubh.2019.00172.].

Whole Genome Sequencing: Bridging One-Health Surveillance of Foodborne Diseases.

Infections caused by pathogens commonly acquired from consumption of food are not always transmitted by that route. They may also be transmitted through contact to animals, other humans or the environment. Additionally, many outbreaks are associated with food contaminated from these non-food sources. For this reason, such presumed foodborne outbreaks are best investigated through a One Health approach working across human, animal and environmental sectors and disciplines. Outbreak strains or clones that have propagated and continue to evolve in non-human sources and environments often show more sequence variation than observed in typical monoclonal point-source outbreaks. This represents a challenge when using whole genome sequencing (WGS), the new gold standard for molecular surveillance of foodborne pathogens, for outbreak detection and investigation. In this review, using recent examples from outbreaks investigated in the United States (US) some aspects of One Health approaches that have been used successfully to solve such outbreaks are presented. These include using different combinations of flexible WGS based case definition, efficient epidemiological follow-up, traceback, surveillance, and testing of potential food and environmental sources and animal hosts.

An Overview of PulseNet USA Databases.

PulseNet USA is the molecular surveillance network for foodborne disease in the United States. The network consists of state and local public health laboratories, as well as food regulatory agencies, that follow PulseNet's standardized protocols to perform pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) and analyze the results using standardized software. The raw sequences are uploaded to the GenomeTrakr or PulseNet bioprojects at the National Center for Biotechnology Information. The PFGE patterns and analyzed sequence data are uploaded in real time with associated demographic data to the PulseNet national databases managed at the Centers for Disease Control and Prevention. The PulseNet databases are organism specific and provide a central storage location for molecular and demographic data related to an isolate. Sequences are compared in the databases, thereby facilitating the rapid detection of clusters of foodborne diseases that may represent widespread outbreaks. WGS genotyping data, for example, antibiotic resistance and virulence profiles, are also uploaded in real time to the PulseNet databases to improve food safety surveillance activities.

Multidrug-Resistant Campylobacter jejuni Outbreak Linked to Puppy Exposure - United States, 2016-2018.

Campylobacter causes an estimated 1.3 million diarrheal illnesses in the United States annually (1). In August 2017, the Florida Department of Health notified CDC of six Campylobacter jejuni infections linked to company A, a national pet store chain based in Ohio. CDC examined whole-genome sequencing (WGS) data and identified six isolates from company A puppies in Florida that were highly related to an isolate from a company A customer in Ohio. This information prompted a multistate investigation by local and state health and agriculture departments and CDC to identify the outbreak source and prevent additional illness. Health officials from six states visited pet stores to collect puppy fecal samples, antibiotic records, and traceback information. Nationally, 118 persons, including 29 pet store employees, in 18 states were identified with illness onset during January 5, 2016-February 4, 2018. In total, six pet store companies were linked to the outbreak. Outbreak isolates were resistant by antibiotic susceptibility testing to all antibiotics commonly used to treat Campylobacter infections, including macrolides and quinolones. Store record reviews revealed that among 149 investigated puppies, 142 (95%) received one or more courses of antibiotics, raising concern that antibiotic use might have led to development of resistance. Public health authorities issued infection prevention recommendations to affected pet stores and recommendations for testing puppies to veterinarians. This outbreak demonstrates that puppies can be a source of multidrug-resistant Campylobacter infections in humans, warranting a closer look at antimicrobial use in the commercial dog industry.

Determination of Evolutionary Relationships of Outbreak-Associated Listeria monocytogenes Strains of Serotypes 1/2a and 1/2b by Whole-Genome Sequencing.

We used whole-genome sequencing to determine evolutionary relationships among 20 outbreak-associated clinical isolates of Listeria monocytogenes serotypes 1/2a and 1/2b. Isolates from 6 of 11 outbreaks fell outside the clonal groups or "epidemic clones" that have been previously associated with outbreaks, suggesting that epidemic potential may be widespread in L. monocytogenes and is not limited to the recognized epidemic clones. Pairwise comparisons between epidemiologically related isolates within clonal complexes showed that genome-level variation differed by 2 orders of magnitude between different comparisons, and the distribution of point mutations (core versus accessory genome) also varied. In addition, genetic divergence between one closely related pair of isolates from a single outbreak was driven primarily by changes in phage regions. The evolutionary analysis showed that the changes could be attributed to horizontal gene transfer; members of the diverse bacterial community found in the production facility could have served as the source of novel genetic material at some point in the production chain. The results raise the question of how to best utilize information contained within the accessory genome in outbreak investigations. The full magnitude and complexity of genetic changes revealed by genome sequencing could not be discerned from traditional subtyping methods, and the results demonstrate the challenges of interpreting genetic variation among isolates recovered from a single outbreak. Epidemiological information remains critical for proper interpretation of nucleotide and structural diversity among isolates recovered during outbreaks and will remain so until we understand more about how various population histories influence genetic variation.

Outbreaks of Salmonellosis From Small Turtles.

OBJECTIVE: Turtle-associated salmonellosis (TAS), especially in children, is a reemerging public health issue. In 1975, small pet turtles (shell length <4 inches) sales were banned by federal law; reductions in pediatric TAS followed. Since 2006, the number of multistate TAS outbreaks has increased. We describe 8 multistate outbreaks with illness-onset dates occurring in 2011-2013. METHODS: We conducted epidemiologic, environmental, and traceback investigations. Cases were defined as infection with ≥ 1 of 10 molecular subtypes of Salmonella Sandiego, Pomona, Poona, Typhimurium, and I 4,[5],12:i:-. Water samples from turtle habitats linked to human illnesses were cultured for Salmonella. RESULTS: We identified 8 outbreaks totaling 473 cases from 41 states, Washington DC, and Puerto Rico with illness onsets during May 2011-September 2013. The median patient age was 4 years (range: 1 month-94 years); 45% percent were Hispanic; and 28% were hospitalized. In the week preceding illness, 68% (187 of 273) of case-patients reported turtle exposure; among these, 88% (124 of 141) described small turtles. Outbreak strains were isolated from turtle habitats linked to human illnesses in seven outbreaks. Traceback investigations identified 2 Louisiana turtle farms as the source of small turtles linked to 1 outbreak; 1 outbreak strain was isolated from turtle pond water from 1 turtle farm. CONCLUSIONS: Eight multistate outbreaks associated with small turtles were investigated during 2011-2013. Children <5 years and Hispanics were disproportionately affected. Prevention efforts should focus on patient education targeting families with young children and Hispanics and enactment of state and local regulations to complement federal sales restrictions.

Molecular subtyping of Clostridium botulinum by pulsed-field gel electrophoresis.

Pulsed-field gel electrophoresis (PFGE) has been extensively used to estimate the genetic diversity of Clostridium botulinum. In addition, PFGE is the standard method for investigating foodborne outbreaks associated with various enteric pathogens, including C. botulinum. PFGE can be used to exclude a suspected but not confirmed food source when the patterns of the food and clinical isolates are different. Indistinguishable PFGE patterns may also be useful for linking isolates between patients or to a food source, but results must be interpreted within an epidemiological context to ensure isolates are truly related. Here, we describe a standardized laboratory protocol for molecular subtyping of C. botulinum by PFGE.

Identification and characterization of multidrug-resistant Salmonella enterica serotype Albert isolates in the United States.

Salmonella enterica is one of the most common causes of bacterial foodborne illness in the United States. Although most Salmonella infections are self-limiting, antimicrobial treatment of invasive salmonellosis is critical. The primary antimicrobial treatment options include fluoroquinolones or extended-spectrum cephalosporins, and resistance to these antimicrobial drugs may complicate treatment. At present, S. enterica is composed of more than 2,600 unique serotypes, which vary greatly in geographic prevalence, ecological niche, and the ability to cause human disease, and it is important to understand and mitigate the source of human infection, particularly when antimicrobial resistance is found. In this study, we identified and characterized 19 S. enterica serotype Albert isolates collected from food animals, retail meat, and humans in the United States during 2005 to 2013. All five isolates from nonhuman sources were obtained from turkeys or ground turkey, and epidemiologic data suggest poultry consumption or live-poultry exposure as the probable source of infection. S. enterica serotype Albert also appears to be geographically localized to the midwestern United States. All 19 isolates displayed multidrug resistance, including decreased susceptibility to fluoroquinolones and resistance to extended-spectrum cephalosporins. Turkeys are a likely source of multidrug-resistant S. enterica serotype Albert, and circulation of resistance plasmids, as opposed to the expansion of a single resistant strain, is playing a role. More work is needed to understand why these resistance plasmids spread and how their presence and the serotype they reside in contribute to human disease.

Clostridium botulinum strains producing BoNT/F4 or BoNT/F5.

Botulinum neurotoxin type F (BoNT/F) may be produced by Clostridium botulinum alone or in combination with another toxin type such as BoNT/A or BoNT/B. Type F neurotoxin gene sequences have been further classified into seven toxin subtypes. Recently, the genome sequence of one strain of C. botulinum (Af84) was shown to contain three neurotoxin genes (bont/F4, bont/F5, and bont/A2). In this study, eight strains containing bont/F4 and seven strains containing bont/F5 were examined. Culture supernatants produced by these strains were incubated with BoNT/F-specific peptide substrates. Cleavage products of these peptides were subjected to mass spectral analysis, allowing detection of the BoNT/F subtypes present in the culture supernatants. PCR analysis demonstrated that a plasmid-specific marker (PL-6) was observed only among strains containing bont/F5. Among these strains, Southern hybridization revealed the presence of an approximately 242-kb plasmid harboring bont/F5. Genome sequencing of four of these strains revealed that the genomic backgrounds of strains harboring either bont/F4 or bont/F5 are diverse. None of the strains analyzed in this study were shown to produce BoNT/F4 and BoNT/F5 simultaneously, suggesting that strain Af84 is unusual. Finally, these data support a role for the mobility of a bont/F5-carrying plasmid among strains of diverse genomic backgrounds.

Outbreak of Salmonella enterica serotype Infantis infection in humans linked to dry dog food in the United States and Canada, 2012.

CASE DESCRIPTION--In April 2012, Salmonella enterica serotype Infantis was detected in an unopened bag of dry dog food collected during routine retail surveillance. PulseNet, a national bacterial subtyping network, identified humans with Salmonella Infantis infection with the same genetic fingerprint as the dog food sample. CLINICAL FINDINGS--An outbreak investigation identified 53 ill humans infected with the outbreak strain during January 1 to July 5, 2012, in 21 states and 2 provinces in Canada; 20 (38%) were children ≤ 2 years old, and 12 of 37 (32%) were hospitalized. Of 21 ill people who remembered the dog food brand, 12 (57%) reported a brand produced at a plant in Gaston, SC. Traceback investigations also identified that plant. The outbreak strain was isolated from bags of dry dog food and fecal specimens obtained from dogs that lived with ill people and that ate the implicated dry dog food. TREATMENT AND OUTCOME--The plant was closed temporarily for cleaning and disinfection. Sixteen brands involving > 27,000 metric tons (> 30,000 tons) of dry dog and cat food were recalled. Thirty-one ill dogs linked to recalled products were reported through the FDA consumer complaint system. CLINICAL RELEVANCE-- A one-health collaborative effort on epidemiological, laboratory, and traceback investigations linked dry dog foods produced at a plant to illnesses in dogs and humans. More efforts are needed to increase awareness among pet owners, health-care professionals, and the pet food industry on the risk of illness in pets and their owners associated with dry pet foods and treats.

Multistate outbreak of listeriosis associated with cantaloupe.

BACKGROUND: Although new pathogen-vehicle combinations are increasingly being identified in produce-related disease outbreaks, fresh produce is a rarely recognized vehicle for listeriosis. We investigated a nationwide listeriosis outbreak that occurred in the United States during 2011. METHODS: We defined an outbreak-related case as a laboratory-confirmed infection with any of five outbreak-related subtypes of Listeria monocytogenes isolated during the period from August 1 through October 31, 2011. Multistate epidemiologic, trace-back, and environmental investigations were conducted, and outbreak-related cases were compared with sporadic cases reported previously to the Listeria Initiative, an enhanced surveillance system that routinely collects detailed information about U.S. cases of listeriosis. RESULTS: We identified 147 outbreak-related cases in 28 states. The majority of patients (127 of 147, 86%) were 60 years of age or older. Seven infections among pregnant women and newborns and one related miscarriage were reported. Of 145 patients for whom information about hospitalization was available, 143 (99%) were hospitalized. Thirty-three of the 147 patients (22%) died. Patients with outbreak-related illness were significantly more likely to have eaten cantaloupe than were patients 60 years of age or older with sporadic illness (odds ratio, 8.5; 95% confidence interval, 1.3 to ∞). Cantaloupe and environmental samples collected during the investigation yielded isolates matching all five outbreak-related subtypes, confirming that whole cantaloupe produced by a single Colorado farm was the outbreak source. Unsanitary conditions identified in the processing facility operated by the farm probably resulted in contamination of cantaloupes with L. monocytogenes. CONCLUSIONS: Raw produce, including cantaloupe, can serve as a vehicle for listeriosis. This outbreak highlights the importance of preventing produce contamination within farm and processing environments.

Genetic diversity among Clostridium botulinum strains harboring bont/A2 and bont/A3 genes.

Clostridium botulinum type A strains are known to be genetically diverse and widespread throughout the world. Genetic diversity studies have focused mainly on strains harboring one type A botulinum toxin gene, bont/A1, although all reported bont/A gene variants have been associated with botulism cases. Our study provides insight into the genetic diversity of C. botulinum type A strains, which contain bont/A2 (n = 42) and bont/A3 (n = 4) genes, isolated from diverse samples and geographic origins. Genetic diversity was assessed by using bont nucleotide sequencing, content analysis of the bont gene clusters, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Sequences of bont genes obtained in this study showed 99.9 to 100% identity with other bont/A2 or bont/A3 gene sequences available in public databases. The neurotoxin gene clusters of the subtype A2 and A3 strains analyzed in this study were similar in gene content. C. botulinum strains harboring bont/A2 and bont/A3 genes were divided into six and two MLST profiles, respectively. Four groups of strains shared a similarity of at least 95% by PFGE; the largest group included 21 out of 46 strains. The strains analyzed in this study showed relatively limited genetic diversity using either MLST or PFGE.

Genomic characterization of Listeria monocytogenes strains involved in a multistate listeriosis outbreak associated with cantaloupe in US.

A multistate listeriosis outbreak associated with cantaloupe consumption was reported in the United States in September, 2011. The outbreak investigation recorded a total of 146 invasive illnesses, 30 deaths and one miscarriage. Subtyping of the outbreak associated clinical, food and environmental isolates revealed two serotypes (1/2a and 1/2b) and four pulsed-field gel electrophoresis two-enzyme pattern combinations I, II, III, and IV, including one rarely seen before this outbreak. A DNA-microarray, Listeria GeneChip®, developed by FDA from 24 Listeria monocytogenes genome sequences, was used to further characterize a representative sample of the outbreak isolates. The microarray data (in the form of present or absent calls of specific DNA sequences) separated the isolates into two distinct groups as per their serotypes. The gene content of the outbreak-associated isolates was distinct from that of the previously-reported outbreak strains belonging to the same serotypes. Although the 1/2b outbreak associated isolates are closely related to each other, the 1/2a isolates could be further divided into two distinct genomic groups, one represented by pattern combination I strains and the other represented by highly similar pattern combinations III and IV strains. Gene content analysis of these groups revealed unique genomic sequences associated with these two 1/2a genovars. This work underscores the utility of multiple approaches, such as serotyping, PFGE and DNA microarray analysis to characterize the composition of complex polyclonal listeriosis outbreaks.

Detection and differentiation of Clostridium botulinum type A strains using a focused DNA microarray.

A focused oligonucleotide microarray featuring 62 probes targeting strain variable regions of the Clostridium botulinum strain ATCC 3502 genome sequence was developed to differentiate C. botulinum type A strains. The strain variable regions were selected from deletions identified among a panel of 10 type A strains compared to the strain ATCC 3502 genome sequence using high density comparative genomic hybridization microarrays. The focused microarray also featured specific probes for the detection of the neurotoxin genes of various serotypes (A-G), toxin gene cluster components (ha70 and orfX1), and fldB as a marker for proteolytic clostridia (Group I). Eight pairs of strains selected from separate type A botulism outbreaks were included in the 27 subtype A1-A4 strains examined in this study. Each outbreak related strain pair consisted of strains isolated from different sources (stool and food). Of the eight outbreak related strain pairs, six groups of strains with indistinguishable hybridization patterns were identified. Outbreak related strains were shown to have identical hybridization patterns. Strain pairs from three separate outbreaks involving strains harboring both the type A neurotoxin gene (bont/A) and an unexpressed type B neurotoxin gene (bont/B) shared the same probe hybridization profile. The focused microarray format provides a rapid approach for neurotoxin gene detection and preliminary determination of the relatedness of strains isolated from different sources.

Genetic homogeneity of Clostridium botulinum type A1 strains with unique toxin gene clusters.

A group of five clonally related Clostridium botulinum type A strains isolated from different sources over a period of nearly 40 years harbored several conserved genetic properties. These strains contained a variant bont/A1 with five nucleotide polymorphisms compared to the gene in C. botulinum strain ATCC 3502. The strains also had a common toxin gene cluster composition (ha-/orfX+) similar to that associated with bont/A in type A strains containing an unexpressed bont/B [termed A(B) strains]. However, bont/B was not identified in the strains examined. Comparative genomic hybridization demonstrated identical genomic content among the strains relative to C. botulinum strain ATCC 3502. In addition, microarray data demonstrated the absence of several genes flanking the toxin gene cluster among the ha-/orfX+ A1 strains, suggesting the presence of genomic rearrangements with respect to this region compared to the C. botulinum ATCC 3502 strain. All five strains were shown to have identical flaA variable region nucleotide sequences. The pulsed-field gel electrophoresis patterns of the strains were indistinguishable when digested with SmaI, and a shift in the size of at least one band was observed in a single strain when digested with XhoI. These results demonstrate surprising genomic homogeneity among a cluster of unique C. botulinum type A strains of diverse origin.

A real-time multiplexed PCR assay for rapid detection and differentiation of Campylobacter jejuni and Campylobacter coli.

Campylobacter species are the leading agents of bacterial gastroenteritis worldwide. C. jejuni and C. coli together are responsible for more than 95% of all cases of Campylobacter-induced diarrheal disease in the United States. Detection of campylobacteria in clinical samples by conventional culture is problematic and slow due to their complex taxonomy, fastidious growth requirements, and biochemical inertness. The current study describes a rapid, sensitive, and specific real-time polymerase chain reaction (PCR) assay capable of detecting and differentiating C. jejuni (hippuricase gene, hipO) and C. coli (serine hydroxymethyltransferase gene, glyA) in a single reaction, directly from clinical isolates and human feces. The analytical specificity of the assay was demonstrated with a diverse range of Campylobacter species, related organisms, and other diarrhea-inducing bacterial pathogens. The analytical sensitivity of the multiplexed, PCR assay was 10 genome copies for both C. jejuni and C. coli. Following a rapid DNA extraction method (QIAGEN QIAamp DNA stool Mini Kit), the multiplexed PCR identified C. jejuni or C. coli in 100% of fecal samples containing 10(3) colony-forming units (CFU) per gram of feces. This assay represents the first real-time PCR method capable of detecting and differentiating C. jejuni and C. coli in a single reaction.