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.

Campylobacter fetus subsp. testudinum subsp. nov., isolated from humans and reptiles.

A polyphasic study was undertaken to determine the taxonomic position of 13 Campylobacter fetus-like strains from humans (n = 8) and reptiles (n = 5). The results of matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MS and genomic data from sap analysis, 16S rRNA gene and hsp60 sequence comparison, pulsed-field gel electrophoresis, amplified fragment length polymorphism analysis, DNA-DNA hybridization and whole genome sequencing demonstrated that these strains are closely related to C. fetus but clearly differentiated from recognized subspecies of C. fetus. Therefore, this unique cluster of 13 strains represents a novel subspecies within the species C. fetus, for which the name Campylobacter fetus subsp. testudinum subsp. nov. is proposed, with strain 03-427(T) ( = ATCC BAA-2539(T) = LMG 27499(T)) as the type strain. Although this novel taxon could not be differentiated from C. fetus subsp. fetus and C. fetus subsp. venerealis using conventional phenotypic tests, MALDI-TOF MS revealed the presence of multiple phenotypic biomarkers which distinguish Campylobacter fetus subsp. testudinum subsp. nov. from recognized subspecies of C. fetus.

Multilocus sequence typing confirms wild birds as the source of a Campylobacter outbreak associated with the consumption of raw peas.

From August to September 2008, the Centers for Disease Control and Prevention (CDC) assisted the Alaska Division of Public Health with an outbreak investigation of campylobacteriosis occurring among the residents of Southcentral Alaska. During the investigation, pulsed-field gel electrophoresis (PFGE) of Campylobacter jejuni isolates from human, raw pea, and wild bird fecal samples confirmed the epidemiologic link between illness and the consumption of raw peas contaminated by sandhill cranes for 15 of 43 epidemiologically linked human isolates. However, an association between the remaining epidemiologically linked human infections and the pea and wild bird isolates was not established. To better understand the molecular epidemiology of the outbreak, C. jejuni isolates (n=130; 59 from humans, 40 from peas, and 31 from wild birds) were further characterized by multilocus sequence typing (MLST). Here we present the molecular evidence to demonstrate the association of many more human C.jejuni infections associated with the outbreak with raw peas and wild bird feces. Among all sequence types (STs) identified, 26 of 39 (67%) were novel and exclusive to the outbreak. Five clusters of overlapping STs (n=32 isolates; 17 from humans, 2 from peas, and 13 from wild birds) were identified. In particular, cluster E (n=7 isolates; ST-5049) consisted of isolates from humans,peas, and wild birds. Novel STs clustered closely with isolates typically associated with wild birds and the environment but distinct from lineages commonly seen in human infections. Novel STs and alleles recovered from human outbreak isolates allowed additional infections caused by these rare genotypes to be attributed to the contaminated raw peas.

Outbreak of campylobacteriosis associated with consumption of raw peas.

BACKGROUND: Campylobacter jejuni is a leading cause of acute gastroenteritis worldwide, and most cases are identified as sporadic events rather than as parts of recognized outbreaks. We report findings from a substantial 2008 campylobacteriosis outbreak with general implications for fresh produce safety. METHODS: We conducted a matched case-control study to determine the source of the outbreak and enhanced surveillance to identify additional cases. Clinical and environmental specimens were tested for Campylobacter, and isolates were subtyped by pulsed-field gel electrophoresis (PFGE). RESULTS: By routine surveillance, we identified 63 cases of laboratory-confirmed infection. Only raw peas, consumed by 30 (67%) of 45 case-patients and by 15 (17%) of 90 control participants, were associated with illness (adjusted odds ratio: 8.2; P<.001). An additional 69 patients (26 laboratory-confirmed) who reported eating raw peas within 10 days of illness onset were identified through enhanced surveillance. In all, 5 cases were hospitalized, and Guillain-Barré syndrome developed in 1 case; none died. The implicated pea farm was located near a Sandhill crane (Grus canadensis) stopover and breeding site. Of 36 environmental samples collected, 16 were positive for C. jejuni-14 crane-feces samples and 2 pea samples. We identified 25 unique combined SmaI-KpnI PFGE patterns among clinical isolates; 4 of these combined PFGE patterns identified in 15 of 55 human isolates were indistinguishable from PFGE patterns identified in environmental samples. CONCLUSIONS: This investigation established a rare laboratory-confirmed link between a campylobacterosis outbreak and an environmental source and identified wild birds as an underrecognized source of produce contamination.

Genetic relationships among reptilian and mammalian Campylobacter fetus strains determined by multilocus sequence typing.

Reptile Campylobacter fetus isolates and closely related strains causing human disease were characterized by multilocus sequence typing. They shared approximately 90% nucleotide sequence identity with classical mammalian C. fetus, and there was evidence of recombination among members of these two groups. The reptile group represents a possible separate genomospecies capable of infecting humans.

Antimicrobial resistance incidence and risk factors among Helicobacter pylori-infected persons, United States.

Helicobacter pylori is the primary cause of peptic ulcer disease and an etiologic agent in the development of gastric cancer. H. pylori infection is curable with regimens of multiple antimicrobial agents, and antimicrobial resistance is a leading cause of treatment failure. The Helicobacter pylori Antimicrobial Resistance Monitoring Program (HARP) is a prospective, multicenter U.S. network that tracks national incidence rates of H. pylori antimicrobial resistance. Of 347 clinical H. pylori isolates collected from December 1998 through 2002, 101 (29.1%) were resistant to one antimicrobial agent, and 17 (5%) were resistant to two or more antimicrobial agents. Eighty-seven (25.1%) isolates were resistant to metronidazole, 45 (12.9%) to clarithromycin, and 3 (0.9%) to amoxicillin. On multivariate analysis, black race was the only significant risk factor (p < 0.01, hazard ratio 2.04) for infection with a resistant H. pylori strain. Formulating pretreatment screening strategies or providing alternative therapeutic regimens for high-risk populations may be important for future clinical practice.

Specific, sensitive, and quantitative enzyme-linked immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen.

The bioterrorism-associated human anthrax epidemic in the fall of 2001 highlighted the need for a sensitive, reproducible, and specific laboratory test for the confirmatory diagnosis of human anthrax. The Centers for Disease Control and Prevention developed, optimized, and rapidly qualified an enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum. The qualified ELISA had a minimum detection limit of 0.06 micro g/mL, a reliable lower limit of detection of 0.09 micro g/mL, and a lower limit of quantification in undiluted serum specimens of 3.0 micro g/mL anti-PA IgG. The diagnostic sensitivity of the assay was 97.8%, and the diagnostic specificity was 97.6%. A competitive inhibition anti-PA IgG ELISA was also developed to enhance diagnostic specificity to 100%. The anti-PA ELISAs proved valuable for the confirmation of cases of cutaneous and inhalational anthrax and evaluation of patients in whom the diagnosis of anthrax was being considered.