Evaluation of a Bead-Based Salmonella Molecular Serotyping Method for Salmonella Isolated from Food and Environmental Samples.

Salmonella is a leading cause of foodborne illness worldwide, and foods containing Salmonella (except raw meat and poultry products) are considered adulterated. Serotyping of Salmonella is an essential part of surveillance and investigation of outbreaks. This study evaluated a bead-based Salmonella molecular serotyping (SMS) method, which included the O-group 1, H-antigen, alternate target, and O-group 2 assays, compared with traditional serotyping. Salmonella was isolated from food, pet food, and environmental samples or were reference strains. A total of 572 isolates were analyzed by using two formats of the SMS method in comparison with traditional methods: 485 were analyzed by using Radix SMS (a custom user-mixed format), 218 were analyzed by using Luminex SMS (a commercial kit format), and 131 of the total isolates were analyzed by both formats for comparison. The SMS method was evaluated on the basis of the successful identification of antigens by the probes included in the method. The method identified 550 (96.2%) isolates as expected, 6 (1.0%) isolates were not identified as initially expected but were shown to be correctly identified by SMS after reanalysis by traditional serotyping, and 16 (2.8%) isolates not identified as expected possessed an antigen that should have been detected by the method but was not. Among the isolates considered correctly identified, 255 (44.6%) were identified to a single serovar, 44 (7.7%) required additional biochemical testing to differentiate variants or subspecies, and 251 (43.9%) were partially serotyped because probes for some antigens were not in the assay or had allelic variation for known serovars. Whole genome sequencing, SeqSero, and the Salmonella In Silico Typing Resource gave added confirmation for three isolates. Addition of the O-group 2 assay enabled the identification of 55 (9.6%) of 572 isolates. The SMS method could fully or partially serotype most isolates within a day. The SMS method should be a valuable tool when faster screening methods are needed, such as outbreaks and screening large numbers of environmental isolates.

Epidemiologic patterns of human Salmonella serotype diversity in the USA, 1996-2016.

Although researchers have described numerous risk factors for salmonellosis and for infection with specific common serotypes, the drivers of Salmonella serotype diversity among human populations remain poorly understood. In this retrospective observational study, we partition records of serotyped non-typhoidal Salmonella isolates from human clinical specimens reported to CDC national surveillance by demographic, geographic and seasonal characteristics and adapt sample-based rarefaction methods from the field of community ecology to study how Salmonella serotype diversity varied within and among these populations in the USA during 1996-2016. We observed substantially higher serotype richness in children <2 years old than in older children and adults and steadily increasing richness with age among older adults. Whereas seasonal and regional variation in serotype diversity was highest among infants and young children, variation by specimen source was highest in adults. Our findings suggest that the risk for infection from uncommon serotypes is associated with host and environmental factors, particularly among infants, young children and older adults. These populations may have a higher proportion of illness acquired through environmental transmission pathways than published source attribution models estimate.

Variable expression of O:61 in Salmonella group C2.

According to the Kauffmann-White scheme, 39 pairs of serovars in Salmonella serogroup C2 differ only by the minor antigen O:6(1). We characterized strains from 10 serovars representing five Salmonella serogroup C2 pairs. All strains demonstrated variable expression of O:6(1). These results indicate that these pairs are not distinct serovars.

Molecular phylogeny of the salmonellae: relationships among Salmonella species and subspecies determined from four housekeeping genes and evidence of lateral gene transfer events.

The salmonellae are a diverse group of bacteria within the family Enterobacteriaceae that includes two species, Salmonella enterica and Salmonella bongori. In order to characterize the phylogenetic relationships of the species and subspecies of Salmonella, we analyzed four housekeeping genes, gapA, phoP, mdh and recA, comprising 3,459 bp of nucleotide sequence data for each isolate sequenced. Sixty-one isolates representing the most common serotypes of the seven subspecies of Salmonella enterica and six isolates of Salmonella bongori were included in this study. We present a robust phylogeny of the Salmonella species and subspecies that clearly defines the lineages comprising diphasic and monophasic subspecies. Evidence of intersubspecies lateral gene transfer of the housekeeping gene recA, which has not previously been reported, was obtained.

Salmonella bacteriuria: an increasing entity in elderly women in the United States.

Salmonellosis is a major cause of gastroenteritis in the United States and can lead to septicaemia, and other extra-intestinal illness including urinary tract infections (UTIs). To examine trends in Salmonella bacteriuria in the United States, surveillance data from the National Salmonella Surveillance System from 1980 to the end of 1999 were reviewed. Overall, 17442 urinary Salmonella isolates were reported, representing 2% of all Salmonella isolates from a known source. This proportion increased from 2% during 1980--1984 to 4% during 1995--1999. The median age of persons from whom these isolates came was 51 years; 12,176 (70 %) were women. Compared to the last national survey conducted between 1968 and 1979, the rate of Salmonella bacteriuria increased among women, from 2.0 per million persons in 1980 to 3.7 in 1999; the highest rate occurring in women > or = 70 years. National reporting of Salmonella bacteriuria increased in absolute incidence and as a proportion of all Salmonella, especially in elderly women and may represent an increase in the incidence of Salmonella UTIs. Better understanding of the uropathogenicity of Salmonella serotypes may further clarify the mechanisms of Salmonella UTIs.

Sequencing and comparative analysis of flagellin genes fliC, fljB, and flpA from Salmonella.

Salmonella isolates have traditionally been classified by serotyping, the serologic identification of two surface antigens, O-polysaccharide and flagellin protein. Serotyping has been of great value in understanding the epidemiology of Salmonella and investigating disease outbreaks; however, production and quality control of the hundreds of antisera required for serotyping is difficult and time-consuming. To circumvent the problems associated with antiserum production, we began the development of a system for determination of serotype in Salmonella based on DNA markers. To identify flagellar antigen-specific sequences, we sequenced 280 alleles of the three genes that are known to encode flagellin in Salmonella, fliC, fljB, and flpA, representing 67 flagellar antigen types. Analysis of the data indicated that the sequences from fliC, fljB, and flpA clustered by the antigen(s) they encode not by locus. The sequences grouped into four clusters based on their conserved regions. Three of the four clusters included multiple flagellar antigen types and were designated the G complex, the Z4 complex, and the alpha cluster. The fourth cluster contained a single antigen type, H:z(29). The amino acid sequences of the conserved regions within each cluster have greater than 95% amino acid identity, whereas the conserved regions differ substantially between clusters (75 to 85% identity). Substantial sequence heterogeneity existed between alleles encoding different flagellar antigens while alleles encoding the same flagellar antigen were homologous, suggesting that flagellin genes may be useful targets for the molecular determination of flagellar antigen type.

Evaluation of methods for subtyping Campylobacter jejuni during an outbreak involving a food handler.

In October 1998, the Centers for Disease Control and Prevention (CDC) assisted in an investigation of an outbreak of campylobacteriosis at a school in Salina, Kansas. Twenty-two isolates were submitted from the Kansas state public health laboratory to CDC, 9 associated with the outbreak and 13 epidemiologically unrelated sporadic isolates. Pulsed-field gel electrophoresis (PFGE) using SmaI and SalI was initially used to validate the epidemiologic data. We then tested the ability of other subtyping techniques to distinguish the outbreak-associated isolates from unrelated sporadic isolates. The methods employed were somatic O serotyping, PCR-restriction fragment length polymorphism (RFLP) analysis of flaA, DNA sequence analysis of 582 bp of flaA that included the short variable region (SVR), and sequencing of the entire flaA gene. PFGE was the most discriminatory technique, yielding 11 SmaI and 10 SalI restriction profiles. All outbreak isolates were indistinguishable by PFGE, somatic O serotyping, and sequencing of the 582-bp region of the flaA gene. fla typing by PCR-RFLP grouped one sporadic isolate with the outbreak strain. Analysis of the DNA sequence of a 582-bp segment of flaA produced strain groupings similar to that generated by PCR-RFLP but further differentiated two flaA PCR-RFLP types (with a 1-bp difference in the 582-bp region). Two sporadic strains were distinct by flaA PCR-RFLP but differed only by a single base substitution in the 582-bp region. The entire flaA gene was sequenced from strains differing by a single base pair in the 582-bp region, and the data revealed that additional discrimination may in some cases be obtained by sequencing outside the SVR. PFGE was superior to all other typing methods tested for strain discrimination; it was crucial for understanding the Kansas outbreak and, when SmaI was used, provided adequate discrimination between unrelated isolates.

Campylobacter jejuni.

Campylobacter jejuni is the most frequently diagnosed bacterial cause of human gastroenteritis in the United States. The emergence of antimicrobial-resistant and, in particular, of fluoroquinolone-resistant C. jejuni infections in Europe and the United States, temporally associated with the approval of use of fluoroquinolones in veterinary medicine, is an important public health concern. Recent research has provided strong evidence for an association between Campylobacter infection and Guillain-Barr Syndrome (GBS), and Campylobacter is the most frequent antecedent infection in GBS. The consumption of undercooked poultry and cross-contamination of other foods with uncooked meat products are leading risk factors for human campylobacteriosis. Reinforcing hygienic practices at each link in the food chain, from producer to consumer, is critical in preventing the disease.

Campylobacter jejuni--an emerging foodborne pathogen.

Campylobacter jejuni is the most commonly reported bacterial cause of foodborne infection in the United States. Adding to the human and economic costs are chronic sequelae associated with C. jejuni infection--Guillian-Barré syndrome and reactive arthritis. In addition, an increasing proportion of human infections caused by C. jejuni are resistant to antimicrobial therapy. Mishandling of raw poultry and consumption of undercooked poultry are the major risk factors for human campylobacteriosis. Efforts to prevent human illness are needed throughout each link in the food chain.

Use caution with serologic testing for Helicobacter pylori infection in children.

Commercial serologic assays accurately detect adult Helicobacter pylori infection. Their use in children remains controversial. An ELISA to detect H. pylori IgG in children was developed and compared with three commercial assays. ELISA standardization was done with sera from all ages and validation was done with another cohort of sera with known H. pylori status. Three commercial serologic assays were subsequently compared against this pediatric ELISA at independent sites, at which 142 pediatric serum samples from different countries were evaluated. The pediatric ELISA was 91.4% sensitive. Assay 3 demonstrated a sensitivity of 78%. Less sensitivity was observed for assay 1 (70%) and assay 2 (63%). Accuracy of commercial assays was greatly reduced when sera from developing countries and younger ages were evaluated. Results of serologic tests used to diagnose H. pylori should be interpreted with caution when evaluating children with abdominal pain. Accurate serologic assays in children may be more important for epidemiologic research than for clinical decision making.

Resistance to antimicrobial chemotherapy: a prescription for research and action.

The growing problem of resistance to antimicrobial chemotherapy was discussed by participants at the February 1995 workshop at Emory University on population biology, evolution, and control of infectious diseases. They discussed the nature and source of this problem and identified areas of research in which information is lacking for the development of programs to control of the emergence and spread of resistant bacteria. Particular attention was given to theoretical (mathematical modeling) and empirical studies of the within and between-host population biology (epidemiology) and the evolution of microbial resistance to chemotherapeutic agents. Suggestions were made about the kinds of models and data needed, and the procedures that could be employed to stem the ascent and dissemination of resistant bacteria. This article summarizes the observations and recommendations made at the 1995 meeting and in the correspondence between participants that followed. It concludes with an update on the theoretical and empirical research on the between- and within-host population biology and evolution of resistance to antimicrobial chemotherapy most of which has been done since that meeting.

Discrimination of Campylobacter jejuni isolates by fla gene sequencing.

Comparison of the entire coding sequence of flaA (1,764 nucleotides) from 15 isolates of Campylobacter jejuni showed two regions of high variability, one region approximately from base positions 700 to 1,450 and a short variable region (SVR) from base positions 450 to 600. Parsimony analysis of the SVR sequences yielded a dendrogram similar to that which was derived by analysis of the entire gene. PCR was used to generate templates, and the SVR was sequenced with primers constructed to hybridize to conserved flanking sequences. The SVRs of 22 isolates of C. jejuni from four outbreaks that have been well characterized and a larger panel of isolates from three additional outbreaks were sequenced. Analysis of the nucleotide sequences produced results that grouped the isolates very similarly to other subtyping techniques. Sequence data were also generated for isolates from three additional outbreaks. Categorizing the isolates by fla SVR DNA sequence placed them in epidemiologically relevant groups. Sequence analysis of the C. jejuni flaA SVR may be a useful tool for epidemiologic investigations and could complement or replace serotyping and other subtyping methods.

Molecular characterization of the gene encoding H antigen in Escherichia coli and development of a PCR-restriction fragment length polymorphism test for identification of E. coli O157:H7 and O157:NM.

Recent outbreaks of disease caused by Escherichia coli O157:H7 have focused much attention on this newly emerged pathogen. Identification of the H7 flagellar antigen is critical for the confirmation of E. coli O157:H7; however, clinical isolates are frequently nonmotile and do not produce detectable H antigen. To further characterize nonmotile isolates (designated NM), we developed a PCR-restriction fragment length polymorphism (PCR-RFLP) test to identify and characterize the gene encoding the H antigen (fliC) in E. coli. The entire coding sequence of fliC was amplified by PCR, the amplicon was restricted with RsaI, and the restriction fragment pattern was examined after gel electrophoresis. Two hundred eighty E. coli isolates representing serotypes O157:H7 and O157:NM, flagellar antigen H7 groups associated with other O serogroups, and all other flagellar antigen groups were analyzed. A single restriction pattern (pattern A) was identified for O157:H7 isolates, O157:NM isolates that produced Shiga toxin (formerly Shiga-like toxin or verotoxin), and 16 of 18 O55:H7 isolates. Flagellar antigen group H7 isolates of non-O157 serotypes had one of three banding patterns distinct from pattern A. A wide variety of patterns were found among isolates of the other 52 flagellar antigen groups; however, none was identical to the O157:H7 pattern. Thirteen of 15 nonmotile strains that did not produce the A pattern had patterns that matched those of other known H groups. The PCR-RFLP in conjunction with O serogroup determination will be useful in identifying E. coli O157:H7 and related strains that do not express immunoreactive H antigen and could be expanded to include other clinically important E. coli strains.

Characterization of nonmotile variants of Escherichia coli O157 and other serotypes by using an antiflagellin monoclonal antibody.

An antiflagellin monoclonal antibody (15D8) was used to detect the presence of flagella in nonmotile variants of several pathogenic Escherichia coli serotypes. Of the 48 isolates examined, 15 reacted with monoclonal antibody 15D8 and were culturally confirmed to be motile. Of the 38 clinical strains designated O157:NM or O157:H-, 7 were antibody reactive and motile and agglutinated with anti-H7 sera.

Molecular subtyping of toxigenic Vibrio cholerae O139 causing epidemic cholera in India and Bangladesh, 1992-1993.

Since October 1992, > 150,000 cases of cholera have been reported from India and Bangladesh; the great majority of Vibrio cholerae isolates belong to the newly established serogroup O139. To better understand the interaction of genetic and epidemiologic factors responsible for their sudden appearance and rapid spread, representative toxigenic V. cholerae O139 isolates were molecularly characterized and compared with a set of toxigenic V. cholerae O1 and non-O1/non-O139 strains. DNA sequences of the cholera toxin B subunit gene and multilocus enzyme electrophoresis markers of V. cholerae O139 strains were identical to those of V. cholerae O1 isolates of the seventh pandemic. Two distinct ribotypes and four pulsed-field gel electrophoretic patterns were observed for O139 strains. V. cholerae O139 strains were very similar to V. cholerae O1 strains of the seventh pandemic but clearly different from the toxigenic V. cholerae strains of serogroups other than O1 and O139.

Detection of Chlamydia pneumoniae in clinical specimens by polymerase chain reaction using nested primers.

A nested primers strategy was used to develop a two-step PCR test for the direct species-specific detection of the 16s rRNA gene of Chlamydia pneumoniae. This test was applied to 58 nasopharyngeal or oropharyngeal swab specimens collected from patients in studies of community-acquired pneumonia and in a local outbreak of respiratory disease. Twelve patients (21%) showed evidence of Chlamydia pneumoniae infection in serological tests (7/56; 13%), culture (8/58; 14%) or PCR (10/58; 17%). Nested PCR but not single-step PCR was found to be as sensitive as culture or serology for detection of infection with this organism. In summary, nested PCR can be useful in direct testing of clinical specimens for Chlamydia pneumoniae, making additional DNA purification steps unnecessary.

The molecular epidemiology of cholera in Latin America.

To explain the sudden appearance and rapid spread of cholera in Latin America in January 1991, molecular techniques were used to define Vibrio cholerae O1 isolates from around the world. Restriction fragment length polymorphisms of rRNA and ctxA genes, DNA sequence of cholera toxin B subunit gene ctxB, and multilocus enzyme electrophoresis data were used to characterize 197 isolates. Worldwide, there are at least four distinct toxigenic El Tor V. cholerae O1 clones: the seventh pandemic (Eastern Hemisphere), US Gulf Coast, Australian, and Latin American. Nontoxigenic V. cholerae O1 previously isolated in Brazil, Mexico, and Peru are unlike current toxigenic isolates. The Latin American clone probably represents an extension of the seventh pandemic into the Western Hemisphere, while the US Gulf Coast clone most likely evolved separately. These data will be useful in monitoring the spread of cholera, determining the origin of outbreaks in both hemispheres, and implicating specific vehicles of transmission.

Use of automated sequencing of polymerase chain reaction-generated amplicons to identify three types of cholera toxin subunit B in Vibrio cholerae O1 strains.

Cholera toxin is the principal factor causing the profuse intestinal fluid secretion that is characteristic of cholera. The DNA sequences of the cholera toxin subunit B structural genes from 45 Vibrio cholerae O1 strains isolated in 29 countries over a period of 70 years were determined by automated DNA sequencing of polymerase chain reaction-generated amplicons. Three types of cholera toxin B subunit gene (ctxB) were identified. Genotype 1 was found in strains of classical biotype worldwide and El Tor biotype strains associated with the U.S. Gulf Coast, genotype 2 was found in El Tor biotype strains from Australia, and genotype 3 was found in El Tor biotype strains from the seventh pandemic and the recent Latin American epidemic. All base changes correspond to an amino acid substitution in the B subunit of the cholera toxin. Heterogenicity in the B subunit could have implications for vaccine development and diagnostic tests for cholera toxin and antitoxin. We conclude that this technology provides timely and potentially useful epidemiological information.

Use of polymerase chain reaction for detection of toxigenic Vibrio cholerae O1 strains from the Latin American cholera epidemic.

In January 1991, an outbreak of cholera started in Peru and spread throughout most of Latin America within 8 months. As of March 1992, over 450,000 cases and approximately 4,000 deaths have been reported to the Pan American Health Organization. The causative organism is toxigenic Vibrio cholerae O1 of the El Tor biotype and is distinct from the U.S. Gulf Coast strains. A polymerase chain reaction (PCR) that amplifies a 564-bp fragment of the cholera toxin A subunit gene (ctxA) was used to identify toxigenic V. cholerae O1 strains. A total of 150 V. cholerae O1 isolates were tested. They were of unknown toxin status, were associated with recent outbreaks, and were isolated from patients, food, and water. One hundred forty isolates were found to be toxigenic both by PCR and the routine diagnostic enzyme-linked immunosorbent assay. Thirty-eight known toxigenic strains isolated worldwide from 1921 to 1991 were also positive in the PCR. A collection of 18 nontoxigenic V. cholerae O1 strains, 35 Escherichia coli heat-labile-enterotoxin-I-producing strains, 26 Campylobacter strains, and 8 strains of Aeromonas hydrophila, previously reported to produce cholera toxin-like toxin, were all negative in the ctxA PCR. We conclude that this PCR is a diagnostic method that specifically detects toxin genes in V. cholerae O1 strains in a reference laboratory. It is more rapid and less cumbersome than other diagnostic methods for detection of toxicity in these strains.