Thioredoxin 1 Participates in the Activity of the Salmonella enterica Serovar Typhimurium Pathogenicity Island 2 Type III Secretion System

The facultative intracellular pathogen Salmonella enterica serovar Typhimurium relies on its Salmonella pathogenicity island 2 (SPI2) type III secretion system (T3SS) for intracellular replication and virulence. We report that the oxidoreductase thioredoxin 1 (TrxA) and SPI2 are coinduced for expression under in vitro conditions that mimic an intravacuolar environment, that TrxA is needed for proper SPI2 activity under these conditions, and that TrxA is indispensable for SPI2 activity in both phagocytic and epithelial cells. Infection experiments in mice demonstrated that SPI2 strongly contributed to virulence in a TrxA-proficient background whereas SPI2 did not affect virulence in a trxA mutant. Complementation analyses using wild-type trxA or a genetically engineered trxA coding for noncatalytic TrxA showed that the catalytic activity of TrxA is essential for SPI2 activity in phagocytic cells whereas a noncatalytic variant of TrxA partially sustained SPI2 activity in epithelial cells and virulence in mice. These results show that TrxA is needed for the intracellular induction of SPI2 and provide new insights into the functional integration between catalytic and noncatalytic activities of TrxA and a bacterial T3SS in different settings of intracellular infections.In Escherichia coli, thioredoxin 1 (TrxA, encoded by trxA) is an evolutionary conserved 11-kDa cytosolic highly potent reductase that supports the activities of various oxidoreductases and ribonucleotide reductases (1, 29) and interacts with a number of additional cytoplasmic proteins through the formation of temporary covalent intermolecular disulphide bonds (32). Consequently, as trxA mutants of E. coli (51), Helicobacter pylori (13), and Rhodobacter sphaeroides (34) show increased sensitivity to hydrogen peroxide, TrxA has been defined as a significant oxidoprotectant. In addition, TrxA possess a protein chaperone function that is disconnected from cysteine interactions (30, 32).Salmonella enterica serovar Typhimurium is closely related to E. coli. During divergent evolution, the Salmonella genome acquired a number of virulence-associated genes (20). Many of these genes are clustered on genetic regions termed Salmonella pathogenicity islands (or SPIs). Of these, SPI1 and SPI2 code for separate type III secretion systems (T3SSs). T3SSs are supramolecular virulence-associated machineries that, in several pathogenic gram-negative bacterial species, enable injection of effector proteins from the bacteria into host cells (22, 57). The effector proteins, in turn, manipulate intrinsic host cell functions to facilitate the infection.The SPI1 T3SS of S. serovar Typhimurium is activated for expression in the intestine in response to increased osmolarity and decreased oxygen tension (22, 57). SPI1 effector proteins are primarily secreted into cells that constitute the epithelial layer and interfere with host cell Cdc42 and Rac-1 signaling and actin polymerization. This enables the bacteria to orchestrate their own actin-dependent uptake into nonphagocytic cells (57). SPI1 effector proteins also induce inflammatory signaling and release of interleukin-1β from infected cells (25, 26).Subsequent systemic progression of S. serovar Typhimurium from the intestinal tissue relies heavily on an ability to survive and replicate in phagocytic cells (18, 46, 53, 54). S. serovar Typhimurium uses an additional set of effector proteins secreted by the SPI2 T3SS for replication inside host cells and for coping with phagocyte innate responses to the infection (10, 11, 54). The functions of SPI2 effectors include diversion of vesicular trafficking, induction of apoptotic responses, and manipulation of ubiquitination of host proteins (28, 40, 45, 53). Hence, SPI2 effector proteins create a vacuolar environment that sustains intracellular replication of S. serovar Typhimurium (28).In addition to pathogenicity islands, the in vivo fitness of Salmonella spp. relies on selected functions shared with other enterobacteria. Thus, many virulence genes are integrated into “housekeeping” gene regulatory networks, coded for by a core genome, which steer bacterial stress responses (12, 17, 27, 55). Selected anabolic pathways also contribute to virulence of S. serovar Typhimurium (18, 27), evidently by providing biochemical building blocks for bacterial replication (36).In S. serovar Typhimurium, TrxA is a housekeeping protein that strongly contributes to virulence in cell culture and mouse infection models (8). However, the mechanism by which TrxA activity adds to virulence has not been defined. Here we show that the contribution of TrxA to virulence of S. serovar Typhimurium associates with its functional integration with the SPI2 T3SS under conditions that prevail in the intracellular vacuolar compartment of the host cell. These findings ascribe a novel role to TrxA in bridging environmental adaptations with virulence gene expression and illuminate a new aspect of the interaction between evolutionary conserved and horizontally acquired gene functions in bacteria.     

Application of Fourier Transform Infrared Spectroscopy and Chemometrics for Differentiation of Salmonella enterica Serovar Enteritidis Phage Types

Fourier transform infrared (FT-IR) spectroscopy and chemometric techniques were used to discriminate five closely related Salmonella enterica serotype Enteritidis phage types, phage type 1 (PT1), PT1b, PT4b, PT6, and PT6a. Intact cells and outer membrane protein (OMP) extracts from bacterial cell membranes were subjected to FT-IR analysis in transmittance mode. Spectra were collected over a wavenumber range from 4,000 to 600 cm⁻¹. Partial least-squares discriminant analysis (PLS-DA) was used to develop calibration models based on preprocessed FT-IR spectra. The analysis based on OMP extracts provided greater separation between the Salmonella Enteritidis PT1-PT1b, PT4b, and PT6-PT6a groups than the intact cell analysis. When these three phage type groups were considered, the method based on OMP extract FT-IR spectra was 100% accurate. Moreover, complementary local models that considered only the PT1-PT1b and PT6-PT6a groups were developed, and the level of discrimination increased. PT1 and PT1b isolates were differentiated successfully with the local model using the entire OMP extract spectrum (98.3% correct predictions), whereas the accuracy of discrimination between PT6 and PT6a isolates was 86.0%. Isolates belonging to different phage types (PT19, PT20, and PT21) were used with the model to test its robustness. For the first time it was demonstrated that FT-IR analysis of OMP extracts can be used for construction of robust models that allow fast and accurate discrimination of different Salmonella Enteritidis phage types.Over the past 10 years there has been an increase in the incidence of gastrointestinal infections caused by Salmonella enterica serovar Enteritidis, which is now one of the leading S. enterica serotypes worldwide (21, 27). Poultry, poultry products, cattle, and dairy products are the predominant sources of Salmonella-contaminated food products that cause human salmonellosis (28). Large-scale infections continue to occur in developed countries (8). Unrestricted international movement of commercially prepared food and food ingredients and dissimilarities in government and industry food safety controls during the processing, distribution, and marketing of products have surely contributed to the increase in food-borne outbreaks. Salmonella is a tremendous challenge for the agricultural and food processing industries because of its ability to survive under adverse conditions, such as low levels of nutrients and suboptimal temperatures (4, 13).Salmonella Enteritidis isolates can be categorized for epidemiological purposes by using a variety of typing tools (13). These tools include typing techniques such as serological and phage typing (29) and antibiotic resistance patterns (25). These methods are now supplemented by molecular genetics techniques, such as DNA fingerprinting (23), plasmid profiling (16), and pulsed-field gel electrophoresis (26). Phage typing has been used to diagnose Salmonella outbreaks, including S. enterica serovar Typhi and S. enterica serovar Typhimurium outbreaks (29). It is useful to evaluate whether isolates obtained from different sources at different times are similar or distinct in terms of their reactions with a specific collection of bacteriophages used for typing. The correlation between phage type and the source of an epidemic is high (22). Although very effective, existing classification methods are time-consuming, laborious, and expensive, and they often require special training of personnel and expertise, which can prevent a rapid response to the presence of pathogenic bacterial species.Fourier transform infrared (FT-IR) spectroscopy has been successfully used for differentiation and classification of microorganisms at the species and subspecies levels (7, 9, 12, 15, 18, 19, 20). This technique has been shown to have high discriminatory power and allows identification of bacteria at distinct taxonomic levels based on differences in the infrared absorption patterns of microbial cells. FT-IR spectroscopy has been used to differentiate and characterize intact microbial cells based on outer membrane cell components, including lipopolysaccharides (LPS), lipoproteins, and phospholipids (24). Several studies in which S. enterica serotypes have been discriminated using multivariate data analysis and FT-IR spectroscopy have been performed (1, 2, 10, 11). Kim et al. (11) compared the FT-IR spectra of intact cells and the FT-IR spectra of outer membrane protein (OMP) extracts from S. enterica serotypes to discriminate serotypes. Analysis of spectra of OMP extracts in the 1,800- to 1,500-cm⁻¹ region resulted in 100% correct classification of the serotypes investigated.Previously, there have been no reports of differentiation of Salmonella Enteritidis phage types by FT-IR spectroscopy and chemometric methods. To discriminate closely related phage types of Salmonella Enteritidis in this study, intact cells and OMP extracts of bacterial cell membranes were subjected to FT-IR analysis. The isolates analyzed included isolates belonging to five of the phage types of Salmonella Enteritidis found most frequently in Portuguese hospitals in the period from 2004 to 2006, phage type 1 (PT1), PT1b, PT4b, PT6, and PT6a (5, 14). Chemometric models were used to discriminate between phage types based on infrared spectra.     

Short-Term Signatures of Evolutionary Change in the Salmonella enterica Serovar Typhimurium 14028 Genome

Salmonella enterica serovar Typhimurium is a Gram-negative pathogen that causes gastroenteritis in humans and a typhoid-like disease in mice and is often used as a model for the disease promoted by the human-adapted S. enterica serovar Typhi. Despite its health importance, the only S. Typhimurium strain for which the complete genomic sequence has been determined is the avirulent LT2 strain, which is extensively used in genetic and physiologic studies. Here, we report the complete genomic sequence of the S. Typhimurium strain 14028s, as well as those of its progenitor and two additional derivatives. Comparison of these S. Typhimurium genomes revealed differences in the patterns of sequence evolution and the complete inventory of genetic alterations incurred in virulent and avirulent strains, as well as the sequence changes accumulated during laboratory passage of pathogenic organisms.The genomes of related bacteria can differ in three ways: (i) gene content, where one bacterial species or strain harbors genes absent from the other organism; (ii) nucleotide substitutions within largely conserved DNA sequences, which can result in amino acid changes in orthologous proteins, form pseudogenes, and promote distinct expression patterns of genes present in the two organisms; and (iii) changes in gene arrangement, caused by inversions and translocations. These differences have been observed not only across bacterial species but also among strains belonging to the same species. Recent genomic analyses have revealed that many bacterial pathogens of humans are virtually monomorphic (1) and exhibit very limited sequence diversity, raising questions about the nature of the genetic changes governing distinct behaviors. Furthermore, several bacterial pathogens that have been subjected to extensive passage in the laboratory display altered virulence characteristics, but the genetic basis for these alterations remains largely unknown. Here, we address both of these questions by determining and analyzing the genome sequences of closely related isolates of Salmonella enterica serovar Typhimurium, a Gram-negative pathogen that has been used as a preeminent model to investigate basic genetic mechanisms (2, 8, 46, 59), as well as the interaction between bacterial pathogens and mammalian hosts (11, 41).The genus Salmonella is divided into two species: Salmonella bongori and Salmonella enterica, which together comprise over 2,300 serovars differing in host specificity and the disease conditions they promote in various hosts. For example, S. enterica serovar Typhi is human restricted and causes typhoid fever, whereas serovar Typhimurium is a broad-host-range organism that causes gastroenteritis in humans and a typhoid-like disease in mice. Although the complete genome sequences of 15 Salmonella enterica strains are available, there is only a single representative of S. Typhimurium—strain LT2 (31). Despite its wide application in genetic analysis, strain LT2 is highly attenuated for virulence in both in vitro and in vivo assays (52, 56), leading many investigators to use other S. Typhimurium isolates to examine the genetic basis for bacterial pathogenesis (11, 14, 16).Over 300 virulence genes (3, 5, 47) have already been identified in Salmonella enterica serovar Typhimurium 14028 (now termed S. enterica subsp. enterica serovar Typhimurium ATCC 14028), which is a descendant of CDC 60-6516, a strain isolated in 1960 from pools of hearts and livers of 4-week-old chickens (P. Fields, personal communication). Whereas strain 14028 has been typed as LT2, a designation based on phage sensitivity (27), the two strains were isolated from distinct sources decades apart, which makes their genealogy and exact relationship obscure. A derivative of the original 14028 strain with a rough colony morphology (due to changes in O-antigen expression) was designated 14028r to distinguish it from the original smooth strain, renamed 14028s, and was used in a genetic screen for Salmonella virulence genes because it retained lethality for mice and the ability to survive within murine macrophages. Strain 14028 was also used for the identification of Salmonella genes that were specifically expressed during infection of a mammalian host (30). Both 14028 and LT2 possess a 90-kb virulence plasmid promoting intracellular replication and systemic disease (14), but they differ in their prophage contents, as is often the case among S. Typhimurium strains (12, 13).To identify the individual changes that differentiate S. Typhimurium strains and to assess the nature of variation that arises during laboratory storage and passage, we determined the genome sequence of strain 14028s. This genome was then used as a reference for sequencing its progenitors, including the original source strain CDC 60-6516 and the earliest smooth and rough variants. Our analysis uncovered the genomic differences that arose during the past decades of laboratory cultivation and showed that derivatives with different virulence potentials can follow distinct patterns of sequence evolution.     

Spontaneous Excision of the Salmonella enterica Serovar Enteritidis-Specific Defective Prophage-Like Element φSE14

Salmonella enterica serovar Enteritidis has emerged as a major health problem worldwide in the last few decades. DNA loci unique to S. Enteritidis can provide markers for detection of this pathogen and may reveal pathogenic mechanisms restricted to this serovar. An in silico comparison of 16 Salmonella genomic sequences revealed the presence of an ∼12.5-kb genomic island (GEI) specific to the sequenced S. Enteritidis strain NCTC13349. The GEI is inserted at the 5′ end of gene ydaO (SEN1377), is flanked by 308-bp imperfect direct repeats (attL and attR), and includes 21 open reading frames (SEN1378 to SEN1398), encoding primarily phage-related proteins. Accordingly, this GEI has been annotated as the defective prophage SE14 in the genome of strain NCTC13349. The genetic structure and location of φSE14 are conserved in 99 of 103 wild-type strains of S. Enteritidis studied here, including reference strains NCTC13349 and LK5. Notably, an extrachromosomal circular form of φSE14 was detected in every strain carrying this island. The presence of attP sites in the circular forms detected in NCTC13349 and LK5 was confirmed. In addition, we observed spontaneous loss of a tetRA-tagged version of φSE14, leaving an empty attB site in the genome of strain NCTC13349. Collectively, these results demonstrate that φSE14 is an unstable genetic element that undergoes spontaneous excision under standard growth conditions. An internal fragment of φSE14 designated Sdf I has been used as a serovar-specific genetic marker in PCR-based detection systems and as a tool to determine S. Enteritidis levels in experimental infections. The instability of this region may require a reassessment of its suitability for such applications.The genus Salmonella comprises a heterogeneous group of Gram-negative bacteria, differentiable by biochemical and serological properties. More than 2,500 Salmonella serovars have been identified according to the serospecificities of the somatic and flagellar antigens. Some serovars, exemplified by Salmonella enterica serovar Typhimurium and S. Enteritidis, can infect a broad range of hosts. However, a subset of serovars, such as S. Typhi, a human-specific pathogen, show a high degree of adaptation to a specific host.In the last few decades, S. Enteritidis has emerged as a major health problem worldwide (31). This pathogen colonizes the reproductive organs of infected birds without causing discernible illness and survives host defenses during the formation of the egg (25, 27). The production of a capsule-like O antigen structure by certain wild-type strains of S. Enteritidis (30, 46) has been associated with reproductive tract tropism and improved survival within eggs (26, 27, 45). Egg contamination can originate before oviposition by direct contamination of the yolk, albumen, or eggshell membranes with bacteria from the infected reproductive organs of the birds or after or during oviposition by penetration of bacteria from contaminated feces through the eggshell (8, 14, 25). Transmission of the bacterium to humans occurs mainly through the consumption of contaminated eggs or egg products (8, 14, 25). Upon infection of a human host, S. Enteritidis causes self-limiting gastroenteritis similar to that caused by other nontyphoidal Salmonella serovars.According to information gathered from 84 countries responding to a global survey conducted by the World Health Organization (WHO), S. Enteritidis and S. Typhimurium accounted for ∼70% of all human and nonhuman isolates of Salmonella reported worldwide between 1995 and 2008. In fact, S. Enteritidis alone accounted for 61.4% of the ∼1.5 million human isolates of Salmonella reported during this period, according to the WHO Global Foodborne Infections Network Country Databank (http://www.who.int/salmsurv). Remarkably, S. Enteritidis is the second most prevalent cause of Salmonella infection in humans, after S. Typhimurium, in the United States (10).The high global prevalence of S. Enteritidis makes the development of a rapid, sensitive, and highly specific detection system critical to collect accurate epidemiologic data. The identification of loci that serve as specific markers for DNA-based identification of this pathogen may also provide insights into pathogenic mechanisms restricted to this serovar. Genomic regions that are unique to given serovars are especially suitable for such epidemiologic detection (3). For instance, Agron and colleagues identified an S. Enteritidis-specific genomic region of ∼4,060 bp adjacent to the ydaO gene, carrying six open reading frames (ORFs) that they designated lygA to lygF (1). A PCR-based assay successfully detected the presence of an internal fragment of this serovar-specific region in most strains in a diverse collection of clinical and environmental S. Enteritidis isolates and not in 73 non-Enteritidis isolates of S. enterica representing 34 different serovars (1). Since then, this region has been widely used as an S. Enteritidis-specific molecular marker in the development of several PCR-based assays for detection and epidemiological typing of Salmonella serovars in clinical and environmental samples (2, 11, 32, 37, 44, 53). Recently, an S. Enteritidis-specific real-time quantitative PCR (qPCR) assay based on the detection of this region was developed (15). This qPCR assay has been used in a series of studies of the distribution and replication kinetics of S. Enteritidis in experimentally infected animals (16-21).We performed a bioinformatic study to identify genomic regions specific to S. Enteritidis and found a genomic island (GEI) that includes the S. Enteritidis-specific locus lyg (1). This island has been annotated as the defective prophage SE14 in the genome of S. Enteritidis strain NCTC13349 (52). Although we demonstrate that the location in the genome and the overall genetic structure of the island are conserved in wild-type isolates of S. Enteritidis from different origins, we detected strains that do not carry the island in their genomes. Finally, we demonstrate here that the island corresponds to an unstable element that undergoes spontaneous excision from the genome of S. Enteritidis under standard growth conditions.     

Development of a Cell Culture Method To Isolate and Enrich Salmonella enterica Serotype Enteritidis from Shell Eggs for Subsequent Detection by Real-Time PCR

Salmonella enterica serotype Enteritidis is a major cause of nontyphoidal salmonellosis from ingestion of contaminated raw or undercooked shell eggs. Current techniques used to identify Salmonella serotype Enteritidis in eggs are extremely laborious and time-consuming. In this study, a novel eukaryotic cell culture system was combined with real-time PCR analysis to rapidly identify Salmonella serotype Enteritidis in raw shell eggs. The system was compared to the standard microbiological method of the International Organization for Standardization (Anonymous, Microbiology of food and animal feeding stuffs—horizontal method for the detection of Salmonella, 2002). The novel technique utilizes a mouse macrophage cell line (RAW 264.7) as the host for the isolation and intracellular replication of Salmonella serotype Enteritidis. Exposure of macrophages to Salmonella serotype Enteritidis-contaminated eggs results in uptake and intracellular replication of the bacterium, which can subsequently be detected by real-time PCR analysis of the DNA released after disruption of infected macrophages. Macrophage monolayers were exposed to eggs contaminated with various quantities of Salmonella serotype Enteritidis. As few as 10 CFU/ml was detected in cell lysates from infected macrophages after 10 h by real-time PCR using primer and probe sets specific for DNA segments located on the Salmonella serotype Enteritidis genes sefA and orgC. Salmonella serotype Enteritidis could also be distinguished from other non-serogroup D Salmonella serotypes by using the sefA- and orgC-specific primer and probe sets. Confirmatory identification of Salmonella serotype Enteritidis in eggs was also achieved by isolation of intracellular bacteria from lysates of infected macrophages on xylose lysine deoxycholate medium. This method identifies Salmonella serotype Enteritidis from eggs in less than 10 h compared to the more than 5 days required for the standard reference microbiological method of the International Organization for Standardization (Microbiology of food and animal feeding stuffs—horizontal method for the detection of Salmonella, 2002).Nontyphoidal salmonellosis is an invasive intestinal disease contracted predominately by ingestion of food contaminated with serotypes of the gram-negative bacterial species Salmonella enterica. Gastroenteritis caused by Salmonella spp. represents a large portion of the natural food-borne illnesses that occur worldwide each year. Bacterial virulence is established in part by the bacterium''s ability to invade and survive within host cells (20). S. enterica is capable of survival within a wide array of host cells, including epithelial cells, dendritic cells, and macrophages in both animal and cell culture models (16, 17, 18, 19). However, survival in macrophages is required for initiation of systemic infection (24). Two chromosomal pathogenicity islands, SPI-1 and SPI-2, which are present in all Salmonella enterica serotypes, are essential for the invasion of epithelial cells and intracellular replication in macrophages, respectively (13, 14).There are currently over 2,500 distinct serotypes of S. enterica (http://www.pasteur.fr/sante/clre/cadrecnr/salmoms/WKLM_2007.pdf). Of these, Salmonella enterica serovar Enteritidis and Salmonella enterica serovar Typhimurium are most commonly associated with food-borne illness in humans (4). Raw and undercooked shell eggs have been implicated as vehicles for the transmission of both of these serotypes of Salmonella enterica (9, 38). However, Salmonella serotype Enteritidis infection has been more frequently linked to shell egg consumption, whereas Salmonella serotype Typhimurium infection is more often associated with the consumption of contaminated chicken meat (8). Of the 309 documented outbreaks of Salmonella serotype Enteritidis in the United States from 1990 to 2001, 241 were attributed to the consumption of raw or undercooked eggs (6). Salmonella serotype Enteritidis phage types 4, 8, and 13 have been implicated in the majority of salmonellosis cases from the consumption of egg products (5). In addition, Salmonella serotype Enteritidis is able to colonize laying hen reproductive organs and developing eggs and has been shown to persist in eggs after they have been laid (23).A variety of methods have been developed in order to expedite the detection of salmonellae in eggs, including GeneQuence DNA hybridization, PCR analysis, and enzyme-linked immunosorbent assay (3, 27, 37). However, these methods require lengthy enrichment steps prior to the application of the respective methods. Real-time PCR (RT-PCR) is a promising new method currently used for detection of a wide variety of bacterial pathogens in food matrices (12, 15, 22, 34, 40). However, this technique can be ineffective for the detection of Salmonella serotype Enteritidis in foods such as eggs due to the presence of PCR-inhibitory components (41).In this study, we developed a novel detection system to allow for the specific identification of viable Salmonella serotype Enteritidis in raw shell eggs. The method developed is based on the ability of Salmonella to invade and replicate within macrophages as part of its life cycle within a host. In theory, cultured eukaryotic cell lines exposed to Salmonella-contaminated foods will allow the penetration and replication of Salmonella while confining food particles and noninvasive bacteria to the extracellular environment, allowing the isolation and enrichment of intracellular Salmonella for subsequent detection by commercially available techniques, such as RT-PCR. In practice, a suitable mammalian cell monolayer is exposed to a particular food matrix suspected of harboring salmonellae. The exposure is promoted for sufficient time to allow cell contact and engulfment of salmonellae. The mammalian cell monolayer is then washed sufficiently to remove the food matrix and extracellular microorganisms. The infected cell monolayer is reconstituted with fresh medium and further incubated to allow for intracellular multiplication of Salmonella (postinfection). After the infection is terminated, the culture medium is discarded, the infected cells are disrupted, and the DNA present in the resultant lysates is analyzed by RT-PCR using primers and probes specific for unique Salmonella DNA sequences. We utilized this method for the presumptive and confirmatory identification of Salmonella serotype Enteritidis in raw shell eggs.     

Inhibition of the Early Stage of Salmonella enterica Serovar Enteritidis Biofilm Development on Stainless Steel by Cell-Free Supernatant of a Hafnia alvei Culture

Compounds present in Hafnia alvei cell-free culture supernatant cumulatively negatively influence the early stage of biofilm development by Salmonella enterica serovar Enteritidis on stainless steel while they also reduce the overall metabolic activity of S. Enteritidis planktonic cells. Although acylhomoserine lactones (AHLs) were detected among these compounds, the use of several synthetic AHLs was not able to affect the initial stage of biofilm formation by this pathogen.Biofilms are groups of bacteria encased in a self-produced extracellular matrix (5, 6). Biofilms formed on stainless steel (SS) surfaces in food-processing areas are of great importance since they may lead to food spoilage and transmission of diseases (2, 16). This sessile mode of life allows bacteria to enjoy a number of advantages, such as increased resistance to antimicrobial agents (9, 12). Notably, it is widely accepted that bacteria (both planktonic and biofilm cells) communicate by releasing and sensing signaling compounds in a process commonly known as quorum sensing (13, 18, 24).Salmonella enterica serovar Enteritidis is one of the most important bacterial pathogens worldwide (7, 17). Hafnia alvei are frequent psychrotrophic members of the Enterobacteriaceae community in meat products, playing a role in their spoilage, while they have been shown to be capable of producing signaling compounds (3). In this study, in order to determine any possible influence of compounds produced by H. alvei on the biofilm-forming ability of S. Enteritidis, the latter was left to develop biofilm on SS surfaces in the presence of conditioned medium obtained after the growth of the former. Biofilm formation was assessed directly by detaching cells and enumerating them and, also, indirectly by automated conductance measurements.     

Flagellated but Not Hyperfimbriated Salmonella enterica Serovar Typhimurium Attaches to and Forms Biofilms on Cholesterol-Coated Surfaces

The asymptomatic, chronic carrier state of Salmonella enterica serovar Typhi occurs in the bile-rich gallbladder and is frequently associated with the presence of cholesterol gallstones. We have previously demonstrated that salmonellae form biofilms on human gallstones and cholesterol-coated surfaces in vitro and that bile-induced biofilm formation on cholesterol gallstones promotes gallbladder colonization and maintenance of the carrier state. Random transposon mutants of S. enterica serovar Typhimurium were screened for impaired adherence to and biofilm formation on cholesterol-coated Eppendorf tubes but not on glass and plastic surfaces. We identified 49 mutants with this phenotype. The results indicate that genes involved in flagellum biosynthesis and structure primarily mediated attachment to cholesterol. Subsequent analysis suggested that the presence of the flagellar filament enhanced binding and biofilm formation in the presence of bile, while flagellar motility and expression of type 1 fimbriae were unimportant. Purified Salmonella flagellar proteins used in a modified enzyme-linked immunosorbent assay (ELISA) showed that FliC was the critical subunit mediating binding to cholesterol. These studies provide a better understanding of early events during biofilm development, specifically how salmonellae bind to cholesterol, and suggest a target for therapies that may alleviate biofilm formation on cholesterol gallstones and the chronic carrier state.The serovars of Salmonella enterica are diverse, infect a broad array of hosts, and cause significant morbidity and mortality in impoverished and industrialized nations worldwide. S. enterica serovar Typhi is the etiologic agent of typhoid fever, a severe illness characterized by sustained bacteremia and a delayed onset of symptoms that afflicts approximately 20 million people each year (14, 19). Serovar Typhi can establish a chronic infection of the human gallbladder, suggesting that this bacterium utilizes novel mechanisms to mediate enhanced colonization and persistence in a bile-rich environment.There is a strong correlation between gallbladder abnormalities, particularly gallstones, and development of the asymptomatic Salmonella carrier state (47). Antibiotic regimens are typically ineffective in carriers with gallstones (47), and these patients have an 8.47-fold-higher risk of developing hepatobiliary carcinomas (28, 46, 91). Elimination of chronic infections usually requires gallbladder removal (47), but surgical intervention is cost-prohibitive in developing countries where serovar Typhi is prevalent. Thus, understanding the progression of infection to the carrier state and developing alternative treatment options are of critical importance to human health.The formation of biofilms on gallstones has been hypothesized to facilitate enhanced colonization of and persistence in the gallbladder. Over the past 2 decades, bacterial biofilms have been increasingly implicated as burdens for food and public safety worldwide, and they are broadly defined as heterogeneous communities of microorganisms that adhere to each other and to inert or live surfaces (17, 22, 67, 89, 102). A sessile environment provides selective advantages in natural, medical, and industrial ecosystems for diverse species of commensal and pathogenic bacteria, including Streptococcus mutans (40, 92, 104), Staphylococcus aureus (15, 35, 100), Escherichia coli (21, 74), Vibrio cholerae (39, 52, 107), and Pseudomonas aeruginosa (23, 58, 73, 105). Bacterial biofilms are increasingly associated with many chronic infections in humans and exhibit heightened resistance to commonly administered antibiotics and to engulfment by professional phagocytes (54, 55, 59). The bacterial gene expression profiles for planktonic and biofilm phenotypes differ (42, 90), and the changes are likely regulated by external stimuli, including nutrient availability, the presence of antimicrobials, and the composition of the binding substrate.Biofilm formation occurs in sequential, highly ordered stages and begins with attachment of free-swimming, planktonic bacteria to a surface. Subsequent biofilm maturation is characterized by the production of a self-initiated extracellular matrix (ECM) composed of nucleic acid, proteins, or exopolysaccharides (EPS) that encase the community of microorganisms. Planktonic cells are continuously shed from the sessile, matrix-bound population, which can result in reattachment and fortification of the biofilm or systemic infection and release of the organism into the environment. Shedding of serovar Typhi by asymptomatic carriers can contaminate food and water and account for much of the person-to-person transmission in underdeveloped countries.Our laboratory has previously reported that bile is required for formation of mature biofilms with characteristic EPS production by S. enterica serovars Typhimurium, Enteritidis, and Typhi on human gallstones and cholesterol-coated Eppendorf tubes (18, 78). Cholesterol is the primary constituent of human cholesterol gallstones, and use of cholesterol-coated tubes creates an in vitro uniform surface that mimics human gallstones (18). It was also demonstrated that Salmonella biofilms that formed on different surfaces had unique phenotypes and required expression of specific EPS (18, 77), yet the factors mediating Salmonella binding to gallstones and cholesterol-coated surfaces during the initiation of biofilm formation remain unknown. Here, we show that the presence of serovar Typhimurium flagella promotes binding specifically to cholesterol in the early stages of biofilm development and that the FliC subunit is a critical component. Bound salmonellae expressing intact flagella provided a scaffold for other cells to bind to during later stages of biofilm growth. Elucidation of key mechanisms that mediate adherence to cholesterol during Salmonella bile-induced biofilm formation on gallstone surfaces promises to reveal novel drug targets for alleviating biofilm formation in chronic cases.     

Genome-Wide Transposon Mutagenesis Identifies a Role for Host Neuroendocrine Stress Hormones in Regulating the Expression of Virulence Genes in Salmonella

Bacterial sensing of environmental signals plays a key role in regulating virulence and mediating bacterium-host interactions. The sensing of the neuroendocrine stress hormones epinephrine (adrenaline) and norepinephrine (noradrenaline) plays an important role in modulating bacterial virulence. We used MudJ transposon mutagenesis to globally screen for genes regulated by neuroendocrine stress hormones in Salmonella enterica serovar Typhimurium. We identified eight hormone-regulated genes, including yhaK, iroC, nrdF, accC, yedP, STM3081, and the virulence-related genes virK and mig14. The mammalian α-adrenergic receptor antagonist phentolamine reversed the hormone-mediated effects on yhaK, virK, and mig14 but did not affect the other genes. The β-adrenergic receptor antagonist propranolol had no activity in these assays. The virK and mig14 genes are involved in antimicrobial peptide resistance, and phenotypic screens revealed that exposure to neuroendocrine hormones increased the sensitivity of S. Typhimurium to the antimicrobial peptide LL-37. A virK mutant and a virK mig14 double mutant also displayed increased sensitivity to LL-37. In contrast to enterohemorrhagic Escherichia coli (EHEC), we have found no role for the two-component systems QseBC and QseEF in the adrenergic regulation of any of the identified genes. Furthermore, hormone-regulated gene expression could not be blocked by the QseC inhibitor LED209, suggesting that sensing of hormones is mediated through alternative signaling pathways in S. Typhimurium. This study has identified a role for host-derived neuroendocrine stress hormones in downregulating S. Typhimurium virulence gene expression to the benefit of the host, thus providing further insights into the field of host-pathogen communication.Bacterial sensing of environmental signals plays a key role in regulating virulence gene expression and bacterium-host interactions. It is increasingly recognized that detection of host-derived molecules, such as the neuroendocrine stress hormones (catecholamines) epinephrine (adrenaline) and norepinephrine (noradrenaline), plays an important role in modulating bacterial virulence (29, 42).Physical and psychological stress has been linked to increased severity and susceptibility to infection in humans and other animals (23, 42), and epinephrine/norepinephrine levels are an important factor in this. Stress triggers an increase in plasma epinephrine levels (31), and plasma levels of epinephrine and norepinephrine have been reported to increase with patients suffering from postoperative sepsis compared to patients with no complications (32). Administration of norepinephrine and epinephrine to otherwise healthy subjects increases the severity of bacterial infections, including Clostridium perfringens in humans and enterohemorrhagic Escherichia coli (EHEC) in calves (42, 63, 65). Treatment with norepinephrine also increases the virulence of Salmonella enterica serovar Enteritidis in chicks and Salmonella enterica serovar Typhimurium in mice, with a substantial increase in bacterial numbers recovered from the cecum and liver in both cases (47, 65).Norepinephrine is found in large concentrations in the gut due to release by gastrointestinal neurones; indeed up to half the norepinephrine in the body may be produced in the enteric nervous system (ENS) (3). Epinephrine, while not normally found in the gut, is present in the bloodstream and is also produced by macrophages in response to bacteria-derived lipopolysaccharide (LPS) (12, 26). S. Typhimurium is an enteropathogen, can also cross the epithelial barrier to cause systemic infection, and will therefore encounter both these molecules in the normal infection cycle.Phenotypes induced by stress hormones in bacteria include increased adherence of EHEC to bovine intestinal mucosa (63), upregulation of type III secretion and Shiga toxin production in EHEC (22, 60), upregulation of type III secretion in Vibrio parahaemolyticus (51), increase in invasion of epithelial cells and breakdown of epithelial tight junctions by Campylobacter jejuni (15), affected motility and expression of iron uptake genes in S. Typhimurium (8, 9, 36), and modulated virulence in Borrelia burgdorferi (59). Epinephrine and norepinephrine can overcome the growth inhibition of many bacteria, including Salmonella, in serum-containing media (13, 43), due to the ability to act as a siderophore to facilitate iron uptake (13, 28, 47).Norepinephrine and epinephrine also interact with bacterial quorum-sensing (QS) systems. QS is a process of bacterial cell-cell communication in which each cell produces small signal molecules termed “autoinducers” (AIs), which regulate gene expression when a critical threshold concentration and therefore population density have been reached. QS affects diverse processes, including motility, virulence, biofilm formation, type III secretion, and luminescence (6, 64).The EHEC AI-3 QS system is important for motility and expression of the type III secretion system encoded by the locus of enterocyte effacement (LEE) (60). AI-3 sensing and signal transduction are mediated via the QseBC and QseEF two-component systems, respectively. Epinephrine and norepinephrine can substitute for AI-3, causing cross talk between the two signaling systems and induction of type III secretion and motility (57, 60). The sensor kinase QseC is autophosphorylated upon binding either epinephrine or norepinephrine (14), demonstrating the presence of adrenergic receptors in bacteria. These adrenergic phenotypes can also be blocked by the mammalian α- and β-adrenergic antagonists phentolamine and propranolol, although it should be noted that QseC is blocked only by the former (14, 60). This suggests the occurrence of cross talk between bacterial and mammalian cell signaling systems and the existence of multiple bacterial adrenergic sensors.To elucidate the role of host-derived stress hormones in the physiology and pathogenicity of S. Typhimurium, we used MudJ transposon mutagenesis to screen globally for epinephrine- and norepinephrine-regulated genes in S. Typhimurium.     

Changes in Ultrastructure and Fourier Transform Infrared Spectrum of Salmonella enterica Serovar Typhimurium Cells after Exposure to Stress Conditions

The effect of exposure to acid (pH 2.5), alkaline (pH 11.0), heat (55°C), and oxidative (40 mM H₂O₂) lethal conditions on the ultrastructure and global chemical composition of Salmonella enterica serovar Typhimurium CECT 443 cells was studied using transmission electron microscopy and Fourier transform infrared spectroscopy (FT-IR) combined with multivariate statistical methods (hierarchical cluster analysis and factor analysis). Infrared spectra exhibited marked differences in the five spectral regions for all conditions tested compared to those of nontreated control cells, which suggests the existence of a complex bacterial stress response in which modifications in a wide variety of cellular compounds are involved. The visible spectral changes observed in all of the spectral regions, together with ultrastructural changes observed by transmission electron microscopy and data obtained from membrane integrity tests, indicate the existence of membrane damage or alterations in membrane composition after heat, acid, alkaline, and oxidative treatments. Results obtained in this study indicate the potential of FT-IR spectroscopy to discriminate between intact and injured bacterial cells and between treatment technologies, and they show the adequacy of this technique to study the molecular aspects of bacterial stress response.Salmonella spp. are an important cause of bacterial food-borne disease all over the world, causing a diversity of illnesses that include typhoid fever, gastroenteritis, and septicemia (11). According to epidemiological data from the European Union, a total of 131,468 laboratory-confirmed salmonellosis cases were reported in 2008, with two serovars, Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis, being responsible for 79.9% of all cases (13). The detection and identification of pathogens in foods are a basic cornerstone of food safety, because they make it possible to identify sources of contamination, provide data on the evaluation of risk reduction measures, and identify the food chain operations, processes, batches, or products representing a threat to public health. Furthermore, they also are fundamental in the epidemiological investigation of food-borne diseases. The presence of stress-injured bacterial cells in foods represents a challenge to those involved in food quality assurance, as routine microbiological procedures may yield negative results for sublethally injured cells. Thus, food could be presumed to be safe and free from pathogenic cells but during storage become dangerous due to the recovery and growth of previously injured cells.Given the fact that bacterial cells react to the different environmental stress conditions by inducing structural and physiological changes, Fourier transform infrared (FT-IR) spectroscopy, which reflects the biochemical composition of the cellular constituents of bacteria that include water, fatty acids, proteins, polysaccharides, and nucleic acids (26), should be able to monitor the changes occurring in bacterial cells in response to several food-related stress conditions. The potential of this methodology to detect and differentiate sublethally heat-injured and dead Listeria monocytogenes and S. Typhimurium cells and to discriminate between diverse heat treatment intensities has been highlighted (2, 20). FT-IR spectroscopy also has been successfully applied to the identification and classification of bacteria such as Acinetobacter (35), Brucella (23), Campylobacter (24, 25), Escherichia coli (1), Lactobacillus (10, 27), Listeria (28, 29), Salmonella (9, 17), Staphylococcus (8, 19), and Yersinia (18).The main aim of this study was to assess ultrastructural modifications and infrared (IR) spectral changes at different degrees of stress exposure and to discriminate between injured and noninjured S. Typhimurium cells after their exposure to heat, acid, alkaline, and oxidative lethal conditions. Results obtained also could help us improve our knowledge of S. Typhimurium cell damage and strategies of response to these adverse conditions.     

Respective Roles of Culturable and Viable-but-Nonculturable Cells in the Heterogeneity of Salmonella enterica Serovar Typhimurium Invasiveness

The existence of Salmonella enterica serovar Typhimurium viable-but-nonculturable (VBNC) cells is a public health concern since they could constitute unrecognized sources of infection if they retain their pathogenicity. To date, many studies have addressed the ability of S. Typhimurium VBNC cells to remain infectious, but their conclusions are conflicting. An assumption could explain these conflicting results. It has been proposed that infectivity could be retained only temporarily after entry into the VBNC state and that most VBNC cells generated under intense stress could exceed the stage where they are still infectious. Using a Radioselectan density gradient centrifugation technique makes it possible to increase the VBNC-cell/culturable-cell ratio without increasing the exposure to stress and, consequently, to work with a larger proportion of newly VBNC cells. Here, we observed that (i) in the stationary phase, the S. Typhimurium population comprised three distinct subpopulations at 10, 24, or 48 h of culture; (ii) the VBNC cells were detected at 24 and 48 h; (iii) measurement of invasion gene (hilA, invF, and orgA) expression demonstrated that cells are highly heterogeneous within a culturable population; and (iv) invasion assays of HeLa cells showed that culturable cells from the different subpopulations do not display the same invasiveness. The results also suggest that newly formed VBNC cells are either weakly able or not able to successfully initiate epithelial cell invasion. Finally, we propose that at entry into the stationary phase, invasiveness may be one way for populations of S. Typhimurium to escape stochastic alteration leading to cell death.Like several readily culturable pathogenic bacterial species, Salmonella enterica has been shown to enter into a viable-but-nonculturable (VBNC) state in response to environmental stresses (25, 33). In this state, cells display integrity and activities but escape detection by conventional culture-based monitoring (24). The physiological significance of this phenotype is unclear: some authors have proposed that it is part of an adaptive response aimed at long-term survival under adverse conditions (22, 32); others argue that it is a consequence of stochastic cellular deterioration and that VBNC cells are on their way to death (4, 10, 12, 23). In any case, the existence of VBNC pathogens is a public health concern since they may constitute unrecognized sources of infection if they retain their pathogenicity.To date, many studies have addressed the ability of VBNC pathogens to remain infectious, but the conclusions of some investigators are conflicting (15, 36). In vitro experiments have shown that VBNC cells of Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Oranienburg can recover their culturability (13, 27, 30, 31). This phenomenon, called resuscitation, confirms that at least some VBNC cells ultimately remain able to multiply and are therefore potentially infectious. On the other hand, most in vivo studies ruled out the ability of S. Typhimurium VBNC cells to initiate infection in mice and chicken or to resuscitate during their passage in the animal gut (6, 17, 34, 35). However, one study reported evidence of the maintenance of pathogenicity by VBNC cells of S. Oranienburg in a model of morphine-immunosuppressed mice (1). An assumption could explain these apparently opposite results. It has been proposed that infectivity could be retained only temporarily after entry into the VBNC state (8, 19, 26). Experiments intended for testing the ability of VBNC cells to retain their pathogenicity cannot be fully conclusive if the inocula still contain culturable cells. Therefore, all previously published animal experiments with S. Typhimurium were conducted on populations with VBNC-cell/culturable-cell ratios around 10,000:1. Such populations were obtained after strong exposure to stress, either under intense stressing factors for a short period (e.g., germicidal UV-C for 2 min [6]) or under mild stressing factors for a long period (e.g., starvation for a minimum of 1 week [35]). In such populations, most VBNC cells could exceed the stage where they are still infectious, and the negative outcomes of infection studies could actually reflect their inability to specifically address the fraction of recent VBNC cells.A Radioselectan density gradient centrifugation technique was shown to fractionate stationary-phase populations of Escherichia coli into two subpopulations (10, 12, 18). Interestingly, the VBNC cells formed during a 48-h E. coli culture were specifically recovered in the high-density (HD) subpopulation (12). This technique thus gives the opportunity to increase the VBNC-cell/culturable-cell ratio without increasing exposure to stress and, consequently, to work with a larger proportion of cells having recently entered the VBNC state.Here, this technique was used to discriminate different stationary-phase S. Typhimurium subpopulations. We further investigated the invasiveness of these cell subpopulations by using both gene expression assays of invasion genes and in vitro invasion tests. Thus, the aim of this study was to assess the invasiveness of the cell subpopulations in accordance with their cellular states.     

Interaction of Candida albicans with an Intestinal Pathogen,Salmonella enterica Serovar Typhimurium

Candida albicans is an opportunistic human fungal pathogen that normally resides in the gastrointestinal tract and on the skin as a commensal but can cause life-threatening invasive disease. Salmonella enterica serovar Typhimurium is a gram-negative bacterial pathogen that causes a significant amount of gastrointestinal infection in humans. Both of these organisms are also pathogenic to the nematode Caenorhabditis elegans, causing a persistent gut infection leading to worm death. In the present study, we used a previously developed C. elegans polymicrobial infection model to assess the interactions between S. Typhimurium and C. albicans. We observed that when C. elegans is infected with C. albicans and serovar Typhimurium, C. albicans filamentation is inhibited. The inhibition of C. albicans filamentation by S. Typhimurium in C. elegans appeared to be mediated by a secretary molecule, since filter-sterilized bacterial supernatant was able to inhibit C. albicans filamentation. In vitro coculture assays under planktonic conditions showed that S. Typhimurium reduces the viability of C. albicans, with greater effects seen at 37°C than at 30°C. Interestingly, S. Typhimurium reduces the viability of both yeast and filamentous forms of C. albicans, but the killing appeared more rapid for the filamentous cells. The antagonistic interaction was also observed in a C. albicans biofilm environment. This study describes the interaction between two diverse human pathogens that reside within the gastrointestinal tract and shows that the prokaryote, S. Typhimurium, reduces the viability of the eukaryote, C. albicans. Identifying the molecular mechanisms of this interaction may provide important insights into microbial pathogenesis.Candida albicans, the most common human fungal pathogen, is a prototypical opportunistic organism that lives harmlessly in the human gastrointestinal tract but has the ability to cause life-threatening invasive disease. Bloodstream infection with C. albicans remains the most lethal form (10), with translocation of the gastrointestinal mucosa being an important pathogenic mechanism, especially in hemato-oncology patients and those who have undergone abdominal surgery. A key virulence determinant of C. albicans is its ability to transition from yeast to a filamentous form (16, 17, 19, 22). This morphogenesis appears important for tissue adherence and invasion (22). Furthermore, C. albicans has the ability to form complex biofilms on medical devices (13) and on human mucosal surfaces, such as the gastrointestinal and bronchial mucosa. C. albicans biofilm formation has immense clinical and economic consequences (13).Recently the interactions between this important fungal pathogen and bacteria were described (11, 12, 18). These studies focus on the interaction between C. albicans and nonfermenting, gram-negative bacteria, such as Pseudomonas aeruginosa and Acinetobacter baumannii, whose interactions are likely found in the clinical environment, especially in the respiratory tracts of critically ill patients and on wounds of patients with burn injuries (7, 20). Of interest, these bacteria show antagonistic properties toward C. albicans, with a predilection toward reducing the viability of C. albicans filaments. In order to study these prokaryote-eukaryote interactions, our laboratory developed a polymicrobial infection model system using Caenorhabditis elegans as a substitute host (18). Previously, we showed that C. albicans causes a persistent lethal infection of the C. elegans intestinal tract (6). This leads to overwhelming C. albicans intestinal proliferation with subsequent filamentation through the worm cuticle (6). Given these characteristics, we decided to use this model to study the interaction of C. albicans with another intestinal pathogen, Salmonella enterica serovar Typhimurium.S. Typhimurium is a gram-negative organism that belongs to the Enterobacteriaceae family. It is a gastrointestinal tract pathogen of humans, being responsible for approximately 2 million to 4 million cases of enterocolitis each year in the United States (4, 8, 21, 23). During infection, S. Typhimurium competes with normal intestinal flora (23). Its virulence pathways are well described, and it has been shown to cause a persistent and lethal gut infection of the nematode C. elegans, similar to infection seen with C. albicans (1, 14). Given this and the fact that C. albicans is a common inhabitant of the human gastrointestinal tract, we used the C. elegans polymicrobial infection model (18) to study the interactions between S. Typhimurium and C. albicans. Understanding the interactions between these diverse organisms within the complex milieu of an intestinal tract may provide important pathogenic and therapeutic insights.     

Comparison of Genotypes of Salmonella enterica Serovar Enteritidis Phage Type 30 and 9c Strains Isolated during Three Outbreaks Associated with Raw Almonds

In 2000 to 2001, 2003 to 2004, and 2005 to 2006, three outbreaks of Salmonella enterica serovar Enteritidis were linked with the consumption of raw almonds. The S. Enteritidis strains from these outbreaks had rare phage types (PT), PT30 and PT9c. Clinical and environmental S. Enteritidis strains were subjected to pulsed-field gel electrophoresis (PFGE), multilocus variable-number tandem repeat analysis (MLVA), and DNA microarray-based comparative genomic indexing (CGI) to evaluate their genetic relatedness. All three methods differentiated these S. Enteritidis strains in a manner that correlated with PT. The CGI analysis confirmed that the majority of the differences between the S. Enteritidis PT9c and PT30 strains corresponded to bacteriophage-related genes present in the sequenced genomes of S. Enteritidis PT4 and S. enterica serovar Typhimurium LT2. However, PFGE, MLVA, and CGI failed to discriminate between S. Enteritidis PT30 strains related to outbreaks from unrelated clinical strains or between strains separated by up to 5 years. However, metabolic fingerprinting demonstrated that S. Enteritidis PT4, PT8, PT13a, and clinical PT30 strains metabolized l-aspartic acid, l-glutamic acid, l-proline, l-alanine, and d-alanine amino acids more efficiently than S. Enteritidis PT30 strains isolated from orchards. These data indicate that S. Enteritidis PT9c and 30 strains are highly related genetically and that PT30 orchard strains differ from clinical PT30 strains metabolically, possibly due to fitness adaptations.Salmonella enterica is one of the major causes of bacterial food-borne illness worldwide. Many serovars of S. enterica serovar Enteritidis emerged as serious problems in the human food supply during the 1980s, and these cases were associated mostly with undercooked eggs and poultry (26). The phage typing of S. Enteritidis strains associated with egg-associated outbreaks had indicated that phage types 8 (PT8) and PT13a were the most common PTs in the United States (12), and PT4 was the most common in Europe (22). Through education and quality improvements, the incidence of S. Enteritidis due to egg products has decreased in the United States (18). However, several recent outbreaks have identified new sources for S. Enteritidis, specifically mung bean sprouts, tomatoes, and raw whole almonds (3, 13, 31).At the time of the 2001 outbreak, almonds and other low-moisture foods were considered an unlikely source of food-borne illness. Almonds are California''s major tree nut crop and have ranked first in California agricultural exports for many years, accounting for 60% of world production in 2000 (14) and 80% in 2008 (http://www.almondboard.com/AboutTheAlmondBoard/Documents/2008-Almond-Board-Almanac.pdf). However, no outbreaks associated with almonds had been reported before 2001. In the spring of 2001, Canadian health officials identified a link between illnesses caused by S. Enteritidis and the consumption of raw almonds (6). Outbreak-related cases were identified from November 2001 to July 2001 in several provinces across Canada and in several regions in the United States (13). During the traceback investigation, almond retailers, processors, and growers were identified, and S. Enteritidis PT30 was cultured from almond samples, a huller/sheller facility, and environmental samples from the orchards (30). The ability to identify the contaminated food source for this outbreak was aided significantly by the previously rare occurrence of S. Enteritidis PT30. S. Enteritidis PT30 continued to be isolated from one of the outbreak-associated orchards during a 5-year period, suggesting that this organism was highly fit for persistence in this environment (30).In 2004, another rare S. Enteritidis PT (PT9c) was linked to a second outbreak associated with raw almonds. Similarly to the first outbreak, both phage typing and pulsed-field gel electrophoresis (PFGE) aided the identification of related cases caused by S. Enteritidis PT9c that occurred over a large geographical region of the United States and Canada (3). A third S. Enteritidis PT30 outbreak associated with raw almonds was reported in Sweden in 2005 to 2006 (15).We have characterized, by molecular methods, S. Enteritidis strains recovered from clinical, almond, and orchard samples related to these three outbreaks to determine whether they were related genotypically. Additional S. Enteritidis strains representing some common phage types also were examined for comparison. Strains were genotyped by PFGE profiling, multilocus variable-number tandem repeat analysis (MLVA), and comparative genomic indexing (CGI) with a S. enterica serovar Typhimurium LT2/Enteritidis PT4 microarray to determine relatedness and whether an association with the source could be determined.     

Population Structure of the Lyme Borreliosis Spirochete Borrelia burgdorferi in the Western Black-Legged Tick (Ixodes pacificus) in Northern California

Factors potentially contributing to the lower incidence of Lyme borreliosis (LB) in the far-western than in the northeastern United States include tick host-seeking behavior resulting in fewer human tick encounters, lower densities of Borrelia burgdorferi-infected vector ticks in peridomestic environments, and genetic variation among B. burgdorferi spirochetes to which humans are exposed. We determined the population structure of B. burgdorferi in over 200 infected nymphs of the primary bridging vector to humans, Ixodes pacificus, collected in Mendocino County, CA. This was accomplished by sequence typing the spirochete lipoprotein ospC and the 16S-23S rRNA intergenic spacer (IGS). Thirteen ospC alleles belonging to 12 genotypes were found in California, and the two most abundant, ospC genotypes H3 and E3, have not been detected in ticks in the Northeast. The most prevalent ospC and IGS biallelic profile in the population, found in about 22% of ticks, was a new B. burgdorferi strain defined by ospC genotype H3. Eight of the most common ospC genotypes in the northeastern United States, including genotypes I and K that are associated with disseminated human infections, were absent in Mendocino County nymphs. ospC H3 was associated with hardwood-dominated habitats where western gray squirrels, the reservoir host, are commonly infected with LB spirochetes. The differences in B. burgdorferi population structure in California ticks compared to the Northeast emphasize the need for a greater understanding of the genetic diversity of spirochetes infecting California LB patients.In the United States, Lyme borreliosis (LB) is the most commonly reported vector-borne illness and is caused by infection with the spirochete Borrelia burgdorferi (3, 9, 52). The signs and symptoms of LB can include a rash, erythema migrans, fever, fatigue, arthritis, carditis, and neurological manifestations (50, 51). The black-legged tick, Ixodes scapularis, and the western black-legged tick, Ixodes pacificus, are the primary vectors of B. burgdorferi to humans in the United States, with the former in the northeastern and north-central parts of the country and the latter in the Far West (9, 10). These ticks perpetuate enzootic transmission cycles together with a vertebrate reservoir host such as the white-footed mouse, Peromyscus leucopus, in the Northeast and Midwest (24, 35), or the western gray squirrel, Sciurus griseus, in California (31, 46).B. burgdorferi is a spirochete species with a largely clonal population structure (14, 16) comprising several different strains or lineages (8). The polymorphic ospC gene of B. burgdorferi encodes a surface lipoprotein that increases expression within the tick during blood feeding (47) and is required for initial infection of mammalian hosts (25, 55). To date, approximately 20 North American ospC genotypes have been described (40, 45, 49, 56). At least four, and possibly up to nine, of these genotypes are associated with B. burgdorferi invasiveness in humans (1, 15, 17, 49, 57). Restriction fragment length polymorphism (RFLP) and, subsequently, sequence analysis of the 16S-23S rRNA intergenic spacer (IGS) are used as molecular typing tools to investigate genotypic variation in B. burgdorferi (2, 36, 38, 44, 44, 57). The locus maintains a high level of variation between related species, and this variation reflects the heterogeneity found at the genomic level of the organism (37). The IGS and ospC loci appear to be linked (2, 8, 26, 45, 57), but the studies to date have not been representative of the full range of diversity of B. burgdorferi in North America.Previous studies in the northeastern and midwestern United States have utilized IGS and ospC genotyping to elucidate B. burgdorferi evolution, host strain specificity, vector-reservoir associations, and disease risk to humans. In California, only six ospC and five IGS genotypes have been described heretofore in samples from LB patients or I. pacificus ticks (40, 49, 56) compared to approximately 20 ospC and IGS genotypes identified in ticks, vertebrate hosts, or humans from the Northeast and Midwest (8, 40, 45, 49, 56). Here, we employ sequence analysis of both the ospC gene and IGS region to describe the population structure of B. burgdorferi in more than 200 infected I. pacificus nymphs from Mendocino County, CA, where the incidence of LB is among the highest in the state (11). Further, we compare the Mendocino County spirochete population to populations found in the Northeast.