Lavin A. Joseph

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.

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.

Novel Epidemic Clones of Listeria monocytogenes, United States, 2011

We identified a novel serotype 1/2a outbreak strain and 2 novel epidemic clones of Listeria monocytogenes while investigating a foodborne outbreak of listeriosis associated with consumption of cantaloupe during 2011 in the United States. Comparative analyses of strains worldwide are essential to identification of novel outbreak strains and epidemic clones.

Genetic Homogeneity of Clostridium botulinum Type A1 Strains with Unique Toxin Gene Clusters

ABSTRACT 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.

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

ABSTRACT 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.

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.

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.

Utility of Botulinum Toxin ELISA A, B, E, F kits for clinical laboratory investigations of human botulism

The Botulinum Toxin ELISA effectively provided presumptive identification of toxin in 1381 investigation samples including clinical specimens, suspect foods, and cultures. Additionally, the ELISA detected all toxins produced by a panel of stock strains representing known subtypes and was negative for non-botulinum toxin producing Clostridium and enteric pathogens. ELISA results were reproducible both within the same kit (CV < 9%) and among different production lots (CV < 23%). Fifty-five of 57 laboratories correctly identified unknown samples in a multi-laboratory study. The ELISA provides a rapid, robust in vitro screening method which will reduce animal dependence during laboratory investigations of botulism.

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

ABSTRACT 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.

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.