Statistical adjustment of culture-independent diagnostic tests for trend analysis in the Foodborne Diseases Active Surveillance Network (FoodNet), USA.

Background: Culture-independent diagnostic tests (CIDTs) are increasingly used to diagnose Campylobacter infection in the Foodborne Diseases Active Surveillance Network (FoodNet). Because CIDTs have different performance characteristics compared with culture, which has been used historically and is still used to diagnose campylobacteriosis, adjustment of cases diagnosed by CIDT is needed to compare with culture-confirmed cases for monitoring incidence trends. Methods: We identified the necessary parameters for CIDT adjustment using culture as the gold standard, and derived formulas to calculate positive predictive values (PPVs). We conducted a literature review and meta-analysis to examine the variability in CIDT performance and Campylobacter prevalence applicable to FoodNet sites. We then developed a Monte Carlo method to estimate test-type and site-specific PPVs with their associated uncertainties. Results: The uncertainty in our estimated PPVs was largely derived from uncertainty about the specificity of CIDTs and low prevalence of Campylobacter in tested samples. Stable CIDT-adjusted incidences of Campylobacter cases from 2012 to 2015 were observed compared with a decline in culture-confirmed incidence. Conclusions: We highlight the lack of data on the total numbers of tested samples as one of main limitations for CIDT adjustment. Our results demonstrate the importance of adjusting CIDTs for understanding trends in Campylobacter incidence in FoodNet.

Comparative Genomics of Campylobacter fetus from Reptiles and Mammals Reveals Divergent Evolution in Host-Associated Lineages.

Campylobacter fetus currently comprises three recognized subspecies, which display distinct host association. Campylobacter fetus subsp. fetus and C fetus subsp. venerealis are both associated with endothermic mammals, primarily ruminants, whereas C fetus subsp. testudinum is primarily associated with ectothermic reptiles. Both C. fetus subsp. testudinum and C. fetus subsp. fetus have been associated with severe infections, often with a systemic component, in immunocompromised humans. To study the genetic factors associated with the distinct host dichotomy in C. fetus, whole-genome sequencing and comparison of mammal- and reptile-associated C fetus was performed. The genomes of C fetus subsp. testudinum isolated from either reptiles or humans were compared with elucidate the genetic factors associated with pathogenicity in humans. Genomic comparisons showed conservation of gene content and organization among C fetus subspecies, but a clear distinction between mammal- and reptile-associated C fetus was observed. Several genomic regions appeared to be subspecies specific, including a putative tricarballylate catabolism pathway, exclusively present in C fetus subsp. testudinum strains. Within C fetus subsp. testudinum, sapA, sapB, and sapAB type strains were observed. The recombinant locus iamABC (mlaFED) was exclusively associated with invasive C fetus subsp. testudinum strains isolated from humans. A phylogenetic reconstruction was consistent with divergent evolution in host-associated strains and the existence of a barrier to lateral gene transfer between mammal- and reptile-associated C fetus Overall, this study shows that reptile-associated C fetus subsp. testudinum is genetically divergent from mammal-associated C fetus subspecies.

Multicenter Evaluation of Clinical Diagnostic Methods for Detection and Isolation of Campylobacter spp. from Stool.

The use of culture-independent diagnostic tests (CIDTs), such as stool antigen tests, as standalone tests for the detection of Campylobacter in stool is increasing. We conducted a prospective, multicenter study to evaluate the performance of stool antigen CIDTs compared to culture and PCR for Campylobacter detection. Between July and October 2010, we tested 2,767 stool specimens from patients with gastrointestinal illness with the following methods: four types of Campylobacter selective media, four commercial stool antigen assays, and a commercial PCR assay. Illnesses from which specimens were positive by one or more culture media or at least one CIDT and PCR were designated "cases." A total of 95 specimens (3.4%) met the case definition. The stool antigen CIDTs ranged from 79.6% to 87.6% in sensitivity, 95.9 to 99.5% in specificity, and 41.3 to 84.3% in positive predictive value. Culture alone detected 80/89 (89.9% sensitivity) Campylobacter jejuni/Campylobacter coli-positive cases. Of the 209 noncases that were positive by at least one CIDT, only one (0.48%) was positive by all four stool antigen tests, and 73% were positive by just one stool antigen test. The questionable relevance of unconfirmed positive stool antigen CIDT results was supported by the finding that noncases were less likely than cases to have gastrointestinal symptoms. Thus, while the tests were convenient to use, the sensitivity, specificity, and positive predictive value of Campylobacter stool antigen tests were highly variable. Given the relatively low incidence of Campylobacter disease and the generally poor diagnostic test characteristics, this study calls into question the use of commercially available stool antigen CIDTs as standalone tests for direct detection of Campylobacter in stool.

Campylobacter.

Campylobacter continues to be one of the most common bacterial causes of diarrheal illness in the United States and worldwide. Infection with Campylobacter causes a spectrum of diseases including acute enteritis, extraintestinal infections, and postinfectious complications. The most common species of Campylobacter associated with human illness is Campylobacter jejuni, but other Campylobacter species can also cause human infections. This comprehensive review includes discussion of the taxonomy, clinical manifestations of infection, epidemiology and the different methods of laboratory detection of Campylobacter.

Salmonella serotype determination utilizing high-throughput genome sequencing data.

Serotyping forms the basis of national and international surveillance networks for Salmonella, one of the most prevalent foodborne pathogens worldwide (1-3). Public health microbiology is currently being transformed by whole-genome sequencing (WGS), which opens the door to serotype determination using WGS data. SeqSero (www.denglab.info/SeqSero) is a novel Web-based tool for determining Salmonella serotypes using high-throughput genome sequencing data. SeqSero is based on curated databases of Salmonella serotype determinants (rfb gene cluster, fliC and fljB alleles) and is predicted to determine serotype rapidly and accurately for nearly the full spectrum of Salmonella serotypes (more than 2,300 serotypes), from both raw sequencing reads and genome assemblies. The performance of SeqSero was evaluated by testing (i) raw reads from genomes of 308 Salmonella isolates of known serotype; (ii) raw reads from genomes of 3,306 Salmonella isolates sequenced and made publicly available by GenomeTrakr, a U.S. national monitoring network operated by the Food and Drug Administration; and (iii) 354 other publicly available draft or complete Salmonella genomes. We also demonstrated Salmonella serotype determination from raw sequencing reads of fecal metagenomes from mice orally infected with this pathogen. SeqSero can help to maintain the well-established utility of Salmonella serotyping when integrated into a platform of WGS-based pathogen subtyping and characterization.

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.

Human infections with new subspecies of Campylobacter fetus.

Campylobacter fetus subsp. testudinum subsp. nov. is a newly proposed subspecies of C. fetus with markers of reptile origin. We summarize epidemiologic information for 9 humans infected with this bacterium. All cases were in men, most of whom were of Asian origin. Infection might have been related to exposure to Asian foods or reptiles.

An outbreak of foodborne illness among attendees of a wedding reception in Wisconsin likely caused by Arcobacter butzleri.

BACKGROUND: Arcobacter species, primarily Arcobacter butzleri, are widely distributed among animals, infrequently isolated from humans, and previously not associated with outbreaks of foodborne illness. We report results of an investigation of a foodborne outbreak that occurred among attendees of a wedding reception in Wisconsin, United States, and was likely caused by A. butzleri. METHODS: We conducted a case-control study among reception attendees and a laboratory investigation to determine the extent, source, and cause of the outbreak. A clinical case was defined as diarrhea in an attendee with illness onset ≤7 days following the wedding reception. RESULTS: The case-control study included 47 of 51 case patients and 43 non-ill attendees. Results demonstrated that consuming broasted chicken was the only factor significantly associated with illness (odds ratio 10.51; 95% confidence interval 1.28, 476.4). Five patients provided stool specimens. Comprehensive culture and polymerase chain reaction (PCR) testing did not detect common bacterial or viral pathogens. Subsequent testing with PCRs targeting 16S/23S rDNA of the three most clinically relevant Arcobacter spp. and the rpoB/C gene of A. butzleri provided products confirmed as A. butzleri (four patients) and A. cryaerophilus (one patient) by sequence analysis. CONCLUSIONS: The results of this investigation suggest that A. butzleri should be considered an agent that can cause outbreaks of foodborne illness. Rigorous investigation of outbreaks of undetermined etiology is valuable for incrementally increasing our understanding of emerging agents causing foodborne illnesses.

Clinical laboratory practices for the isolation and identification of Campylobacter in Foodborne Diseases Active Surveillance Network (FoodNet) sites: baseline information for understanding changes in surveillance data.

BACKGROUND: Campylobacter is a leading cause of foodborne illness in the United States. Understanding laboratory practices is essential to interpreting incidence and trends in reported campylobacteriosis over time and provides a baseline for evaluating the increasing use of culture-independent diagnostic methods for Campylobacter infection. METHODS: The Foodborne Diseases Active Surveillance Network (FoodNet) conducts surveillance for laboratory-confirmed Campylobacter infections. In 2005, FoodNet conducted a survey of clinical laboratories to describe routine practices used for isolation and identification of Campylobacter. A profile was assigned to laboratories based on complete responses to key survey questions that could impact the recovery and isolation of Campylobacter from stool specimens. RESULTS: Of 411 laboratories testing on-site for Campylobacter, 97% used only culture methods. Among those responding to the individual questions, nearly all used transport medium (97%) and incubated at 42°C (94%); however, most deviated from existing guidelines in other areas: 68% held specimens in transport medium at room temperature before plating, 51% used Campy blood agar plate medium, 52% read plates at <72 hours of incubation, and 14% batched plates before placing them in a microaerobic environment. In all, there were 106 testing algorithms among 214 laboratories with a complete profile; only 16 laboratories were fully adherent to existing guidelines. CONCLUSIONS: Although most laboratories used culture-based methods, procedures differed widely and most did not adhere to existing guidelines, likely resulting in underdiagnosis. Given the availability of new culture-independent testing methods, these data highlight a clear need to develop best practice recommendations for Campylobacter infection diagnostic testing.

Molecular evidence for zoonotic transmission of an emergent, highly pathogenic Campylobacter jejuni clone in the United States.

Campylobacter jejuni is a major zoonotic pathogen. A highly virulent, tetracycline-resistant C. jejuni clone (clone SA) has recently emerged in ruminant reservoirs and has become the predominant cause of sheep abortion in the United States. To determine whether clone SA is associated with human disease, we compared the clinical isolates of clone SA from sheep abortions with the human isolates of the PulseNet National Campylobacter databases at the CDC and the FDA using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST), and serotyping. The combined SmaI and KpnI PFGE pattern designations of clone SA from sheep were indistinguishable from those of 123 (9.03%) human C. jejuni isolates (total, 1,361) in the CDC database, among which 56 were associated with sporadic infections and 67 were associated with outbreaks that occurred in multiple states from 2003 to 2010. Most of the outbreaks were attributed to raw milk, while the sources for most of the sporadic cases were unknown. All clone SA isolates examined, including PFGE-matched human isolates, belong to sequence type 8 (ST-8) by MLST and serotype HS:1,8, further indicating the clonality of the related isolates from different host species. Additionally, C. jejuni clone SA was identified in raw milk, cattle feces, the feces and bile of healthy sheep, and abortion cases of cattle and goats, indicating the broad distribution of this pathogenic clone in ruminants. These results provide strong molecular and epidemiological evidence for zoonotic transmission of this emergent clone from ruminants to humans and indicate that C. jejuni clone SA is an important threat to public health.

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.

Association study between an outbreak of Guillain-Barre syndrome in Jilin, China, and preceding Campylobacter jejuni infection.

From June to July 2007, 36 cases of Guillain-Barre syndrome (GBS) occurred in a township in north China. Serological study and bacteria culture were performed to investigate the association between preceding Campylobacter jejuni infection and this GBS outbreak. Anti-C. jejuni antibodies were found in significantly higher numbers of GBS patients (IgM 84%, IgG 87.5%) than in healthy inspection cases (IgM 33%, IgG 27%). IgG anti-GM1 was the dominant anti-ganglioside antibody among the GBS patients. Seven C. jejuni isolates (four from human stool and three from poultry specimens taken from the patients' houses) were obtained. Serotyping and molecular analysis were used to investigate the genetic relatedness among these C. jejuni isolates. The four human isolates, collected from residents of the same district, were indistinguishable by both pulsed-field gel electrophoresis and multilocus sequence typing, suggesting these patients had a common source of infection. A new sequence type, sequence type-2993, was assigned to the human C. jejuni isolates, three of which belonged to Penner serotype heat-stable (HS):41. Both serotype and molecular subtype of the human C. jejuni isolates were different from those of isolates obtained from poultry specimens. Our results suggest that the antecedent C. jejuni infection triggered this GBS outbreak in China.

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.

Anti-ganglioside antibody induction by swine (A/NJ/1976/H1N1) and other influenza vaccines: insights into vaccine-associated Guillain-Barré syndrome.

BACKGROUND: Receipt of an A/NJ/1976/H1N1 "swine flu" vaccine in 1976, unlike receipt of influenza vaccines used in subsequent years, was strongly associated with the development of the neurologic disorder Guillain-Barré syndrome (GBS). Anti-ganglioside antibodies (e.g., anti-GM(1)) are associated with the development of GBS, and we hypothesized that the swine flu vaccine contained contaminating moieties (such as Campylobacter jejuni antigens that mimic human gangliosides or other vaccine components) that elicited an anti-GM(1) antibody response in susceptible recipients. METHODS: Surviving samples of monovalent and bivalent 1976 vaccine, comprising those from 3 manufacturers and 11 lot numbers, along with several contemporary vaccines were tested for hemagglutinin (HA) activity, the presence of Campylobacter DNA, and the ability to induce anti-Campylobacter and anti-GM(1) antibodies after inoculation into C3H/HeN mice. RESULTS: We found that, although C. jejuni was not detected in 1976 swine flu vaccines, these vaccines induced anti-GM(1) antibodies in mice, as did vaccines from 1991-1992 and 2004-2005. Preliminary studies suggest that the influenza HA induces anti-GM(1) antibodies. CONCLUSIONS: Influenza vaccines contain structures that can induce anti-GM(1) antibodies after inoculation into mice. Further research into influenza vaccine components that elicit anti-ganglioside responses and the role played by these antibodies (if any) in vaccine-associated GBS is warranted.

Multiplex, bead-based suspension array for molecular determination of common Salmonella serogroups.

We report the development and evaluation of a Salmonella O-group-specific Bio-Plex assay to detect the six most common serogroups in the United States (B, C(1), C(2), D, E, and O13) plus serotype Paratyphi A. The assay is based on rfb gene targets directly involved in O-antigen biosynthesis; it can be completed 45 min post-PCR amplification. The assay correctly and specifically identified 362 of 384 (94.3%) isolates tested in comparison to traditional serotyping. Seventeen isolates (4.4%) produced results consistent with what is known about the molecular basis for serotypes but different from the results of traditional serotyping, and five isolates (1.3%) generated false-negative results. Molecular determination of the serogroup for rough isolates was consistent with a common serotype in most instances, indicating that this approach has the potential to provide O-group information for isolates that do not express an O antigen. We also report the sequence of the O-antigen-encoding rfb gene cluster from Salmonella enterica serotype Poona (serogroup O13). Compared with other, previously characterized rfb regions, the O13 rfb gene cluster was most closely related to Escherichia coli O127 and O86. The O-group Bio-Plex assay described here provides an easy-to-use, high-throughput system for rapid detection of common Salmonella serogroups.

A waterborne outbreak of gastroenteritis with multiple etiologies among resort island visitors and residents: Ohio, 2004.

BACKGROUND: The implementation of treated municipal water systems in the 20th century led to a dramatic decrease in waterborne disease in the United States. However, communities with deficient water systems still experience waterborne outbreaks. In August 2004, we investigated an outbreak of gastroenteritis on South Bass Island, Ohio, an island of 900 residents that is visited by >500,000 persons each year. METHODS: To identify the source of illness, we conducted a case-control study and an environmental investigation. A case was defined as diarrhea in a person who traveled to the island during the period from May 1 through 30 September 2004 and became ill within 2 weeks after the visit. Healthy travel companions served as matched control subjects. We also performed an environmental assessment and extensive testing of island water sources. RESULTS: Among the 1450 persons reporting illness, Campylobacter jejuni, norovirus, Giardia intestinalis, and Salmonella enterica serotype Typhimurium were identified in 16, 9, 3, and 1 persons, respectively. We interviewed 100 case patients and 117 matched control subjects. Case patients were more likely to drink water on the island than control subjects (68% vs. 35%; matched odds ratio, 4.3; 95% confidence interval, 2.2-9.3). Sampling of ground water wells indicated contamination with multiple fecal microbes, including Escherichia coli, C. jejuni, Salmonella species, and Giardia species. Irregularities in sewage disposal practices that could have contaminated the underground aquifer were noted. CONCLUSIONS: The combined epidemiological and environmental investigation indicated that sewage-contaminated ground water was the likely source of this large outbreak. Long-term changes to the island's water supply and sewage management infrastructure are needed.

Sequence analysis of the rfb loci, encoding proteins involved in the biosynthesis of the Salmonella enterica O17 and O18 antigens: serogroup-specific identification by PCR.

We report sequencing of the O antigen encoded by the rfb gene cluster of Salmonella enterica serotype Jangwani (O17) and Salmonella serotype Cerro (O18). We developed serogroup O17- and O18-specific PCR assays based on rfb gene targets and found them to be sensitive and specific for rapid identification of Salmonella serogroups O17 and O18.

Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax.

Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals. It was thought to differ from Bacillus cereus, an opportunistic pathogen and cause of food poisoning, by the presence of plasmids pXO1 and pXO2, which encode the lethal toxin complex and the poly-gamma-d-glutamic acid capsule, respectively. This work describes a non-B. anthracis isolate that possesses the anthrax toxin genes and is capable of causing a severe inhalation anthrax-like illness. Although initial phenotypic and 16S rRNA analysis identified this isolate as B. cereus, the rapid generation and analysis of a high-coverage draft genome sequence revealed the presence of a circular plasmid, named pBCXO1, with 99.6% similarity with the B. anthracis toxin-encoding plasmid, pXO1. Although homologues of the pXO2 encoded capsule genes were not found, a polysaccharide capsule cluster is encoded on a second, previously unidentified plasmid, pBC218. A/J mice challenged with B. cereus G9241 confirmed the virulence of this strain. These findings represent an example of how genomics could rapidly assist public health experts responding not only to clearly identified select agents but also to novel agents with similar pathogenic potentials. In this study, we combined a public health approach with genome analysis to provide insight into the correlation of phenotypic characteristics and their genetic basis.

Molecular analysis of the rfb O antigen gene cluster of Salmonella enterica serogroup O:6,14 and development of a serogroup-specific PCR assay.

The Kauffmann-White scheme for serotyping Salmonella recognizes 46 somatic (O) antigen groups, which together with detection of the flagellar (H) antigens form the basis for serotype identification. Although serotyping has become an invaluable typing method for epidemiological investigations of Salmonella, it does have some practical limitations. We have been characterizing the genes required for O and H antigen biosynthesis with the goal of developing a DNA-based system for the determination of serotype in Salmonella. The majority of the enzymes involved in O antigen biosynthesis are encoded by the rfb gene cluster. We report the sequencing of the rfb region from S. enterica serotype Sundsvall (serogroup O:6,14). The S. enterica serotype Sundsvall rfb region is 8.4 kb in length and comprises six open reading frames. When compared with other previously characterized rfb regions, the serogroup O:6,14 sequence is most related to serogroup C(1). On the basis of DNA sequence similarity, we identified two genes from the mannose biosynthetic pathway, two mannosyl transferase genes, the O unit flippase gene and, possibly, the O antigen polymerase. The whole cluster is derived from a low-G+C-content organism. Comparative sequencing of an additional serogroup O:6,14 isolate (S. enterica serotype Carrau) revealed a highly homologous sequence, suggesting that O antigen factors O:24 and O:25 (additional O factors associated with serogroup O:6,14) are encoded outside the rfb gene cluster. We developed a serogroup O:6,14-specific PCR assay based on a region of the putative wzx (O antigen flippase) gene. This provides the basis for a sensitive and specific test for the rapid identification of Salmonella serogroup O:6,14.