Subtyping of Salmonella enterica serovar Newport outbreak isolates by CRISPR-MVLST and determination of the relationship between CRISPR-MVLST and PFGE results.

Salmonella enterica subsp. enterica serovar Newport (S. Newport) is the third most prevalent cause of food-borne salmonellosis. Rapid, efficient, and accurate methods for identification are required to track specific strains of S. Newport during outbreaks. By exploiting the hypervariable nature of virulence genes and clustered regularly interspaced short palindromic repeats (CRISPRs), we previously developed a sequence-based subtyping approach, designated CRISPR-multi-virulence-locus sequence typing (CRISPR-MVLST). To demonstrate the applicability of this approach, we analyzed a broad set of S. Newport isolates collected over a 5-year period by using CRISPR-MVLST and pulsed-field gel electrophoresis (PFGE). Among 84 isolates, we defined 38 S. Newport sequence types (NSTs), all of which were novel compared to our previous analyses, and 62 different PFGE patterns. Our data suggest that both subtyping approaches have high discriminatory abilities (>0.95) with a potential for clustering cases with common exposures. Importantly, we found that isolates from closely related NSTs were often similar by PFGE profile as well, further corroborating the applicability of CRISPR-MVLST. In the first full application of CRISPR-MVLST, we analyzed isolates from a recent S. Newport outbreak. In this blinded study, we confirmed the utility of CRISPR-MVLST and were able to distinguish the 10 outbreak isolates, as defined by PFGE and epidemiological data, from a collection of 20 S. Newport isolates. Together, our data show that CRISPR-MVLST could be a complementary approach to PFGE subtyping for S. Newport.

CRISPR-MVLST subtyping of Salmonella enterica subsp. enterica serovars Typhimurium and Heidelberg and application in identifying outbreak isolates.

BACKGROUND: Salmonella enterica subsp. enterica serovars Typhimurium (S. Typhimurium) and Heidelberg (S. Heidelberg) are major causes of foodborne salmonellosis, accounting for a fifth of all annual salmonellosis cases in the United States. Rapid, efficient and accurate methods for identification are required for routine surveillance and to track specific strains during outbreaks. We used Pulsed-field Gel Electrophoresis (PFGE) and a recently developed molecular subtyping approach termed CRISPR-MVLST that exploits the hypervariable nature of virulence genes and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) to subtype clinical S. Typhimurium and S. Heidelberg isolates. RESULTS: We analyzed a broad set of 175 S. Heidelberg and S. Typhimurium isolates collected over a five-year period. We identified 21 Heidelberg Sequence Types (HSTs) and 37 Typhimurium STs (TSTs) that were represented by 27 and 45 PFGE pulsotypes, respectively, and determined the discriminatory power of each method. CONCLUSIONS: For S. Heidelberg, our data shows that combined typing by both CRISPR-MVLST and PFGE provided a discriminatory power of 0.9213. Importantly, CRISPR-MVLST was able to separate common PFGE patterns such as JF6X01.0022 into distinct STs, thus providing significantly greater discriminatory power. Conversely, we show that subtyping by either CRISPR-MVLST or PFGE independently provides a sufficient discriminatory power (0.9345 and 0.9456, respectively) for S. Typhimurium. Additionally, using isolates from two S. Typhimurium outbreaks, we demonstrate that CRISPR-MVLST provides excellent epidemiologic concordance.

The combination of CRISPR-MVLST and PFGE provides increased discriminatory power for differentiating human clinical isolates of Salmonella enterica subsp. enterica serovar Enteritidis.

Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) is a major cause of foodborne salmonellosis. Rapid, efficient and accurate methods for identification are required to track specific strains of S. Enteritidis during outbreaks of human salmonellosis. By exploiting the hypervariable nature of virulence genes and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs), we previously developed a powerful sequence-based subtyping approach, designated CRISPR-MVLST. To substantiate the applicability of CRISPR-MVLST, we analyzed a broad set of S. Enteritidis isolates collected over a six-year period. Among 141 isolates we defined 22 Enteritidis Sequence Types (ESTs), the majority of which were novel. Notably, strains exhibiting the common PFGE pattern, JEGX01.0004 (characteristic of ∼40% of S. Enteritidis isolates in the United States), were separated into twelve distinct sequence types. Conversely, isolates of EST4, the most predominant EST we observed, comprised eight different PFGE patterns. Importantly, we showed that some genotypes that were previously associated with the food supply chain at the farm level have now been identified in clinical samples. CRISPR sequence data shows subtle but distinct differences among different alleles of S. Enteritidis, suggesting that evolution of these loci occurs vertically, as opposed to previously reported evolution by spacer acquisition in other bacteria.

Prevalence of Salmonella cerro in laboratory-based submissions of cattle and comparison with human infections in Pennsylvania, 2005-2010.

The aim of this study was to identify Salmonella serotypes infecting cattle in Pennsylvania, to compare infection rates for the predominant serotype, Salmonella enterica serotype Cerro, with the infection rates for the same serotype in humans, and to study the clonal diversity and antimicrobial resistance for this serotype in cattle from 2005 to 2010. Clonal diversity among the selected isolates was studied using pulsed-field gel electrophoresis (PFGE) and repetitive (rep)-polymerase chain reaction (PCR). Salmonella Cerro showed the single largest increase as a cause of cattle infections over the study period. The proportional distribution of Salmonella Cerro serotype among laboratory-submitted Salmonella positive cases in cattle was 36.1% in the year 2010 compared to 14.3% in 2005. A simultaneous decrease in serotype Newport infections was also observed in cattle (25% in 2005, to 10.1% in 2010). Studies of clonal diversity for cattle and human isolates revealed a predominant PFGE type but showed some variability. All tested isolates (n = 60) were susceptible to sulfamethoxazole-trimethoprim, but 2% of cattle isolates (n = 1/50) and 20% of human isolates (n = 2/10) showed resistance to tetracycline and sulfisoxazole. One human isolate showed additional resistance to ampicillin and gentamicin. This study suggests an increase in Salmonella Cerro infections in the cattle population and a decrease in Salmonella Newport infections. The increase in Cerro infections appears to be restricted to the cattle population, but occasional human infections occur.

Multidrug-resistant Salmonella isolates from retail chicken meat compared with human clinical isolates.

AIM: To examine the prevalence of antimicrobial-resistant Salmonella in chicken meat and correlate with isolates from ill humans. METHODS: We isolated Salmonella from raw chicken purchased from a randomly selected sample of retail outlets in central Pennsylvania during 2006-2007. Salmonella isolates from meat were compared, using pulsed-field gel electrophoresis, to isolates in the PulseNet database of Salmonella recovered from humans. RESULTS: Of 378 chicken meat samples, 84 (22%) contained Salmonella. Twenty-six (31%) of the Salmonella isolates were resistant to > or = 3 antimicrobials and 18 (21%) were resistant to ceftiofur. All ceftiofur-resistant isolates exhibited reduced susceptibility (minimum inhibitory concentration >2 microg/mL) to ceftriaxone and carried a bla(CMY) gene, as detected by polymerase chain reaction. Among the 28 Salmonella serovar Typhimurium isolates, 20 (71.4%) were resistant to > or = 3 antimicrobials and 12 (42.9%) were resistant to ceftiofur. One ceftiofur-resistant Salmonella serovar Typhimurium poultry isolate exhibited a rare pulsed-field gel electrophoresis pattern indistinguishable from a human isolate in PulseNet; both isolates carried the bla(CMY-2) gene. CONCLUSIONS: These data demonstrate the presence of multidrug-resistant Salmonella in poultry meat, including bla(CMY) plasmid-mediated genes that confer resistance to both ceftiofur, used in poultry, and ceftriaxone, used for treating salmonellosis in humans. This study illustrates the potential for molecular subtyping databases to identify related Salmonella isolates from meat and ill humans, and suggests that chicken could be a source for multidrug-resistant salmonellosis in humans.

Escherichia coli O157:H7 Outbreak Associated with Restaurant Beef Grinding.

Escherichia coli O157:H7 is a common cause of foodborne illness in the United States. Beef ground at establishments regulated by the U.S. Department of Agriculture, Food Safety and Inspection Service is routinely tested for E. coli O157:H7. Prior to December 2013, boxed beef product (wholesale cuts of beef, such as beef loin, packaged into bags and boxed for shipping) was not always tested for this pathogen. Downstream processors or retailers may grind the product; and, if the ground beef is not cooked to the recommended temperature, pathogens on the exterior of the beef introduced to the interior through grinding may survive. On 18 October 2013, the Allegheny County Health Department identified two E. coli O157:H7 cases, both of whom were food handlers at restaurant A, a restaurant that ground locally produced boxed beef for hamburgers on site. Case finding was conducted through public messaging, employee surveys, and disease surveillance. All potential cases were interviewed using a standard questionnaire. A confirmed case was defined as laboratory-confirmed E. coli O157:H7 with exposure to restaurant A. A probable case was defined as a patient with compatible symptoms and exposure to restaurant A but without laboratory confirmation. All human and food isolates were characterized by pulsed-field gel electrophoresis and multilocus variable-number tandem repeat analysis. The analysis identified 14 confirmed and 10 probable cases of E. coli; 18 nonintact ground beef samples tested positive for E. coli O157:H7. Nine confirmed cases were restaurant A employees. All confirmed cases recalled eating a restaurant A hamburger in the 10 days before illness onset; most cases reported consuming medium to rare hamburgers. Multiple pulsed-field gel electrophoresis and multilocus variable-number tandem repeat analysis patterns were identified among both the human and ground beef isolates, and the patient isolates matched those found in ground beef samples. Restaurant A voluntarily closed for 1.5 days, changed beef suppliers, ceased grinding beef in-house, and has had no new cases since reopening.

A comparison of non-typhoidal Salmonella from humans and food animals using pulsed-field gel electrophoresis and antimicrobial susceptibility patterns.

Salmonellosis is one of the most important foodborne diseases affecting humans. To characterize the relationship between Salmonella causing human infections and their food animal reservoirs, we compared pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility patterns of non-typhoidal Salmonella isolated from ill humans in Pennsylvania and from food animals before retail. Human clinical isolates were received from 2005 through 2011 during routine public health operations in Pennsylvania. Isolates from cattle, chickens, swine and turkeys were recovered during the same period from federally inspected slaughter and processing facilities in the northeastern United States. We found that subtyping Salmonella isolates by PFGE revealed differences in antimicrobial susceptibility patterns and, for human Salmonella, differences in sources and invasiveness that were not evident from serotyping alone. Sixteen of the 20 most common human Salmonella PFGE patterns were identified in Salmonella recovered from food animals. The most common human Salmonella PFGE pattern, Enteritidis pattern JEGX01.0004 (JEGX01.0003ARS), was associated with more cases of invasive salmonellosis than all other patterns. In food animals, this pattern was almost exclusively (99%) found in Salmonella recovered from chickens and was present in poultry meat in every year of the study. Enteritidis pattern JEGX01.0004 (JEGX01.0003ARS) was associated with susceptibility to all antimicrobial agents tested in 94.7% of human and 97.2% of food animal Salmonella isolates. In contrast, multidrug resistance (resistance to three or more classes of antimicrobial agents) was observed in five PFGE patterns. Typhimurium patterns JPXX01.0003 (JPXX01.0003 ARS) and JPXX01.0018 (JPXX01.0002 ARS), considered together, were associated with resistance to five or more classes of antimicrobial agents: ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline (ACSSuT), in 92% of human and 80% of food animal Salmonella isolates. The information from our study can assist in source attribution, outbreak investigations, and tailoring of interventions to maximize their impact on prevention.

Outbreak of human Salmonella Typhimurium infections linked to contact with baby poultry from a single agricultural feed store chain and mail-order hatchery, 2009.

BACKGROUND: Over 30 outbreaks of human salmonellosis linked to contact with live poultry from mail-order hatcheries were reported to Centers for Disease Control and Prevention between 1990 and 2010. In May 2009, we investigated an outbreak of human Salmonella Typhimurium infections, primarily affecting children. METHODS: A case was defined as a person with the outbreak strain of Salmonella Typhimurium, as determined by pulsed-field gel electrophoresis and multiple-locus variable-number tandem repeat analysis, in a Pennsylvania or New York resident with illness onset between May 1 and September 1, 2009. We conducted a case-control study to examine the relationship between illness and live poultry contact. Controls were age-matched and geographically-matched. Traceback and environmental investigations were conducted. RESULTS: We identified 36 case-patients in Pennsylvania and New York; 36% were children aged ≤5 years. Case-patients were more likely than controls to report live baby poultry contact (matched odds ratio [mOR]: 17.0; 95% confidence interval [CI]: 2.7-710.5), contact with chicks (mOR: 14.0; 95% CI: 2.1-592.0), ducklings (mOR: 8.0; 95% CI: 1.1-355.0) and visiting agricultural feed stores (mOR: 6.0; 95% CI: 1.3-55.2). Most (83%) visited agricultural Feed Store Chain Y, a national agricultural feed store chain, which received poultry from Hatchery C, which is supplied by multiple egg sources. Salmonella Typhimurium was isolated from a source duck flock, but had a different pulsed-field gel electrophoresis pattern than the outbreak strain. CONCLUSIONS: Live baby poultry remain an important source of human salmonellosis, particularly among children. Preventing these infections requires comprehensive interventions at hatcheries and agricultural feed stores; pediatricians should inform patients of risks associated with live poultry contact.

Human Salmonella infections linked to contaminated dry dog and cat food, 2006-2008.

OBJECTIVE: Human Salmonella infections associated with dry pet food have not been previously reported. We investigated such an outbreak of Salmonella Schwarzengrund and primarily affecting young children. PATIENTS AND METHODS: Two multistate case-control studies were conducted to determine the source and mode of infections among case-patients with the outbreak strain. Study 1 evaluated household exposures to animals and pet foods, and study 2 examined risk factors for transmission among infant case-patients. Environmental investigations were conducted. RESULTS: Seventy-nine case-patients in 21 states were identified; 48% were children aged 2 years or younger. Case-households were significantly more likely than control households to report dog contact (matched odds ratio [mOR]: 3.6) and to have recently purchased manufacturer X brands of dry pet food (mOR: 6.9). Illness among infant case-patients was significantly associated with feeding pets in the kitchen (OR: 4.4). The outbreak strain was isolated from opened bags of dry dog food produced at plant X, fecal specimens from dogs that ate manufacturer X dry dog food, and an environmental sample and unopened bags of dog and cat foods from plant X. More than 23 000 tons of pet foods were recalled. After additional outbreak-linked illnesses were identified during 2008, the company recalled 105 brands of dry pet food and permanently closed plant X. CONCLUSIONS: Dry dog and cat foods manufactured at plant X were linked to human illness for a 3-year period. This outbreak highlights the importance of proper handling and storage of pet foods in the home to prevent human illness, especially among young children.

The key role of pulsed-field gel electrophoresis in investigation of a large multiserotype and multistate food-borne outbreak of Salmonella infections centered in Pennsylvania.

Five different serotypes of Salmonella enterica were implicated in a large outbreak linked to fresh Roma tomatoes served at gas station deli counters in Pennsylvania and nearby states during July 2004: S. enterica serotypes Javiana, Anatum, Thompson, Typhimurium, and Muenchen. One of these serotypes, Anatum, was isolated from both tomatoes and patients. Pulsed-field gel electrophoresis (PFGE) played a key role in identifying the outbreak-associated isolates and distinguishing them from unrelated sporadic isolates. It also demonstrated that the genetic fingerprints of serotype Anatum isolates derived from patients were indistinguishable from those derived from tomatoes. Rapid communication of PFGE fingerprints with other public health laboratories through the Centers for Disease Control and Prevention's PulseNet USA national molecular surveillance network for bacterial food-borne pathogens facilitated the tracking of this outbreak in other states. The work described in this report emphasizes the laboratory's role in core public health functions and services, thereby providing a highly visible example of public health in action.

Activation of prophage eib genes for immunoglobulin-binding proteins by genes from the IbrAB genetic island of Escherichia coli ECOR-9.

Four distinct Escherichia coli immunoglobulin-binding (eib) genes, each of which encodes a surface-exposed protein that binds immunoglobulins in a nonimmune manner, are carried by separate prophages in E. coli reference (ECOR) strain ECOR-9. Each eib gene was transferred to test E. coli strains, both in the form of multicopy recombinant plasmids and as lysogenized prophage. The derived lysogens express little or no Eib protein, in sharp contrast to the parental lysogen, suggesting that ECOR-9 has an expression-enhancing activity that the derived lysogens lack. Supporting this hypothesis, we cloned from ECOR-9 overlapping genes, ibrA and ibrB (designation is derived from "immunoglobulin-binding regulator"), which together activated eib expression in the derived lysogens. The proteins encoded by ibrA and ibrB are very similar to uncharacterized proteins encoded by genes of Salmonella enterica serovar Typhi and E. coli O157:H7 (in a prophage-like element of the Sakai strain and in two O islands of strain EDL933). The genomic segment containing ibrA and ibrB has been designated the IbrAB island. It contains regions of homology to the Shiga toxin-converting prophage, Stx2, as well as genes homologous to phage antirepressor genes. The left boundary between the IbrAB island and the chromosomal framework is located near min 35.8 of the E. coli K-12 genome. Homology to IbrAB was found in certain other ECOR strains, including the other five eib-positive strains and most strains of the phylogenetic group B2. Sequencing of a 1.1-kb portion of ibrAB revealed that the other eib-positive strains diverge by