Identification of Specific Gene Sequences Conserved in Contemporary Epidemic Strains of Salmonella enterica

Genetic elements specific to recent and contemporary epidemic strains of Salmonella enterica were identified using comparative genomic analysis. Two epidemic multidrug-resistant (MDR) strains, MDR Salmonella enterica serovar Typhimurium definitive phage type 104 (DT104) and cephalosporin-resistant MDR Salmonella enterica serovar Newport, and an epidemic pansusceptible strain, Salmonella serovar Typhimurium DT160, were subjected to Salmonella gene microarray and suppression subtractive hybridization analyses. Their genome contents were compared with those of coexisting sporadic strains matched by serotype, geographic and temporal distribution, and host species origin. These paired comparisons revealed that epidemic strains of S. enterica had specific genes and gene regions that were shared by isolates of the same subtype. Most of these gene sequences are related to mobile genetic elements, including phages, plasmids, and plasmid-like and transposable elements, and some genes may encode proteins conferring growth or survival advantages. The emergence of epidemic MDR strains may therefore be associated with the presence of fitness-associated genetic factors in addition to their antimicrobial resistance genes.     

Comprehensive analysis of <Emphasis Type="BoldItalic">Salmonella</Emphasis> sequence polymorphisms and development of a LDR-UA assay for the detection and characterization of selected serotypes

Salmonella is a major cause of food-borne disease, and Salmonella enterica subspecies I includes the most clinically relevant serotypes. Salmonella serotype determination is important for the disease etiology assessment and contamination source tracking. This task will be facilitated by the disclosure of Salmonella serotype sequence polymorphisms, here annotated in seven genes (sefA, safA, safC, bigA, invA, fimA, and phsB) from 139 S. enterica strains, of which 109 belonging to 44 serotypes of subsp. I. One hundred nineteen polymorphic sites were scored and associated to single serotypes or to serotype groups belonging to S. enterica subsp. I. A diagnostic tool was constructed based on the Ligation Detection Reaction—Universal Array (LDR-UA) for the detection of polymorphic sites uniquely associated to serotypes of primary interest (Salmonella Hadar, Salmonella Infantis, Salmonella Enteritidis, Salmonella Typhimurium, Salmonella Gallinarum, Salmonella Virchow, and Salmonella Paratyphi B). The implementation of promiscuous probes allowed the diagnosis of ten further serotypes that could be associated to a unique hybridization pattern. Finally, the sensitivity and applicability of the tool was tested on target DNA dilutions and with controlled meat contamination, allowing the detection of one Salmonella CFU in 25 g of meat.     

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.     

Comparison of Salmonella enterica Serovar Typhimurium LT2 and Non-LT2 Salmonella Genomic Sequences, and Genotyping of Salmonellae by Using PCR

Genes of Salmonella enterica serovar Typhimurium LT2 expected to be specifically present in Salmonella were selected using the Basic Local Alignment Search Tool (BLAST) program. The 152 selected genes were compared with 11 genomic sequences of Salmonella serovars, including Salmonella enterica subsp. I and IIIb and Salmonella bongori (V), and were clustered into 17 groups by their comparison patterns. A total of 38 primer pairs were constructed to represent each of the 17 groups, and PCR was performed with various Salmonella subspecies including Salmonella enterica subsp. I, II, IIIa, IIIb, IV, VI, and V to evaluate a comprehensive DNA-based scheme for identification of Salmonella subspecies and the major disease-causing Salmonella serovars. Analysis of PCR results showed that Salmonella enterica subsp. I was critically divided from other subspecies, and Salmonella strains belonging to S. enterica subsp. I were clustered based on their serovars. In addition, genotypic relationships within S. enterica subsp. I by PCR results were investigated. Also, Salmonella signature genes, Salmonella enterica serovar Typhimurium signature genes, and Salmonella enterica subsp. I signature genes were demonstrated based on their PCR results. The described PCR method suggests a rapid and convenient method for identification of Salmonella serovars that can be used by nonspecialized laboratories. Genome sequence comparison can be a useful tool in epidemiologic and taxonomic studies of Salmonella.     

Bacteriophage Therapy To Reduce Salmonella Colonization of Broiler Chickens

Acute enteric infections caused by salmonellas remain a major public health burden worldwide. Poultry, particularly chickens, are known to be the main reservoir for this zoonotic pathogen. Although some progress has been made in reducing Salmonella colonization of broiler chickens by using biosecurity and antimicrobials, it still remains a considerable problem. The use of host-specific bacteriophages as a biocontrol is one possible intervention by which Salmonella colonization could be reduced. A total of 232 Salmonella bacteriophages were isolated from poultry farms, abattoirs, and wastewater in 2004 and 2005. Three phages exhibiting the broadest host ranges against Salmonella enterica serotypes Enteritidis, Hadar, and Typhimurium were characterized further by determining their morphology and lytic activity in vitro. These phages were then administered in antacid suspension to birds experimentally colonized with specific Salmonella host strains. The first phage reduced S. enterica serotype Enteritidis cecal colonization by ≥4.2 log₁₀ CFU within 24 h compared with controls. Administration of the second phage reduced S. enterica serotype Typhimurium by ≥2.19 log₁₀ CFU within 24 h. The third bacteriophage was ineffective at reducing S. enterica serotype Hadar colonization. Bacteriophage resistance occurred at a frequency commensurate with the titer of phage being administered, with larger phage titers resulting in a greater proportion of resistant salmonellas. The selection of appropriate bacteriophages and optimization of both the timing and method of phage delivery are key factors in the successful phage-mediated control of salmonellas in broiler chickens.     

Development of an oligonucleotide microarray method for Salmonella serotyping

Adequate identification of Salmonella enterica serovars is a prerequisite for any epidemiological investigation. This is traditionally obtained via a combination of biochemical and serological typing. However, primary strain isolation and traditional serotyping is time‐consuming and faster methods would be desirable. A microarray, based on two housekeeping and two virulence marker genes (atpD, gyrB, fliC and fljB), has been developed for the detection and identification of the two species of Salmonella (S. enterica and S. bongori), the five subspecies of S. enterica (II, IIIa, IIIb, IV, VI) and 43 S. enterica ssp. enterica serovars (covering the most prevalent ones in Austria and the UK). A comprehensive set of probes (n = 240), forming 119 probe units, was developed based on the corresponding sequences of 148 Salmonella strains, successfully validated with 57 Salmonella strains and subsequently evaluated with 35 blind samples including isolated serotypes and mixtures of different serotypes. Results demonstrated a strong discriminatory ability of the microarray among Salmonella serovars. Threshold for detection was 1 colony forming unit per 25 g of food sample following overnight (14 h) enrichment.     

Sequence Analysis of the rfb Loci, Encoding Proteins Involved in the Biosynthesis of the Salmonella enterica O17 and O18 Antigens: Serogroup-Specific Identification by PCR

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

PCR Detection of Salmonella enterica Serotype Montevideo in and on Raw Tomatoes Using Primers Derived from hilA

Salmonellae have been some of the most frequently reported etiological agents in fresh-produce-associated outbreaks of human infections in recent years. PCR assays using four innovative pairs of primers derived from hilA and sirA, positive regulators of Salmonella invasive genes, were developed to identify Salmonella enterica serotype Montevideo on and in tomatoes. Based on examination of 83 Salmonella strains and 22 non-Salmonella strains, we concluded that a pair of hilA primers detects Salmonella specifically. The detection limits of the PCR assay were 10¹ and 10⁰ CFU/ml after enrichment at 37°C for 6 and 9 h, respectively. When the assay was validated by detecting S. enterica serotype Montevideo in and on artificially inoculated tomatoes, 10² and 10¹ CFU/g were detected, respectively, after enrichment for 6 h at 37°C. Our results suggest that the hilA-based PCR assay is sensitive and specific, and can be used for rapid detection of Salmonellae in or on fresh produce.     

Plasmid Profile and Pulsed–Field Gel Electrophoresis Analysis of Salmonella enterica Isolates from Humans in Turkey

This study was conducted for typing Salmonella enterica subspecies enterica strains in Turkey using pulsed–field gel electrophoresis (PFGE) and plasmid DNA profile analysis. Fourty-two strains were isolated from clinical samples obtained from unrelated patients with acute diarrhea. The samples were collected from state hospitals and public health laboratories located at seven provinces in different regions of Turkey at different times between 2004 and 2010. The strains were determined to belong to 4 different serovars. The Salmonella enterica strains belonged to the serovars Salmonella Enteritidis (n = 23), Salmonella Infantis (n = 14), Salmonella Munchen (n = 2), and Salmonella Typhi (n = 3). Forty-two Salmonella enterica strains were typed with PFGE methods using XbaI restriction enzyme and plasmid analysis. At the end of typing, 11 different PFGE band profiles were obtained. Four different PFGE profiles (type 1, 4, 9, and 10) were found among serotype S. Enteritidis species, 3 different PFGE profiles (type 3, 5, 6) were found among S. Infantis species, 2 different PFGE profiles were found among S. Typhi species (type 2 and 11), and 2 different PFGE profiles were found among S. Munchen species (type 7, 8). The UPGMA dendrogram was built on the PFGE profiles. In this study, it was determined that 4 strains of 42 Salmonella enterica strains possess no plasmid, while the isolates have 1–3 plasmids ranging from 5.0 to 150 kb and making 12 different plasmid profiles (P1–P12). In this study, we have applied the analysis of the PFGE patterns and used bioinformatics methods to identify both inter and intra serotype relationships of 4 frequently encountered serotypes for the first time in Turkey.     

Microarray-Based Detection of Salmonella enterica Serovar Enteritidis Genes Involved in Chicken Reproductive Tract Colonization

Salmonella enterica serovar Enteritidis has developed the potential to contaminate table eggs internally, by colonization of the chicken reproductive tract and internalization in the forming egg. The serotype Enteritidis has developed mechanisms to colonize the chicken oviduct more successfully than other serotypes. Until now, the strategies exploited by Salmonella Enteritidis to do so have remained largely unknown. For that reason, a microarray-based transposon library screen was used to identify genes that are essential for the persistence of Salmonella Enteritidis inside primary chicken oviduct gland cells in vitro and inside the reproductive tract in vivo. A total of 81 genes with a potential role in persistence in both the oviduct cells and the oviduct tissue were identified. Major groups of importance include the Salmonella pathogenicity islands 1 and 2, genes involved in stress responses, cell wall, and lipopolysaccharide structure, and the region-of-difference genomic islands 9, 21, and 40.     

Molecular Analysis of the rfb O Antigen Gene Cluster of Salmonella enterica Serogroup O:6,14 and Development of a Serogroup-Specific PCR Assay

The Kauffmann-White scheme for serotyping Salmonella recognizes 46 somatic (O) antigen groups, which together with detection of the flagellar (H) antigens form the basis for serotype identification. Although serotyping has become an invaluable typing method for epidemiological investigations of Salmonella, it does have some practical limitations. We have been characterizing the genes required for O and H antigen biosynthesis with the goal of developing a DNA-based system for the determination of serotype in Salmonella. The majority of the enzymes involved in O antigen biosynthesis are encoded by the rfb gene cluster. We report the sequencing of the rfb region from S. enterica serotype Sundsvall (serogroup O:6,14). The S. enterica serotype Sundsvall rfb region is 8.4 kb in length and comprises six open reading frames. When compared with other previously characterized rfb regions, the serogroup O:6,14 sequence is most related to serogroup C₁. On the basis of DNA sequence similarity, we identified two genes from the mannose biosynthetic pathway, two mannosyl transferase genes, the O unit flippase gene and, possibly, the O antigen polymerase. The whole cluster is derived from a low-G+C-content organism. Comparative sequencing of an additional serogroup O:6,14 isolate (S. enterica serotype Carrau) revealed a highly homologous sequence, suggesting that O antigen factors O:24 and O:25 (additional O factors associated with serogroup O:6,14) are encoded outside the rfb gene cluster. We developed a serogroup O:6,14-specific PCR assay based on a region of the putative wzx (O antigen flippase) gene. This provides the basis for a sensitive and specific test for the rapid identification of Salmonella serogroup O:6,14.     

Detection of Salmonella enterica Subpopulations by Phenotype Microarray Antibiotic Resistance Patterns

Three strains of Salmonella enterica serotype Enteritidis were compared to Salmonella enterica serotype Heidelberg, Salmonella enterica serotype Newport, and Salmonella enterica serovar Typhimurium for growth in the presence of 240 antibiotics arranged within a commercial high-throughput phenotype microarray. The results show that antibiotic resistances were different for subpopulations of serotype Enteritidis separated only by genetic drift.     

Characterization of the RpoS Status of Clinical Isolates of Salmonella enterica

The stationary-phase-inducible sigma factor, σ^S (RpoS), is the master regulator of the general stress response in Salmonella and is required for virulence in mice. rpoS mutants can frequently be isolated from highly passaged laboratory strains of Salmonella. We examined the rpoS status of 116 human clinical isolates of Salmonella, including 41 Salmonella enterica serotype Typhi strains isolated from blood, 38 S. enterica serotype Typhimurium strains isolated from blood, and 37 Salmonella serotype Typhimurium strains isolated from feces. We examined the abilities of these strains to produce the σ^S protein, to express RpoS-dependent catalase activity, and to resist to oxidative stress in the stationary phase of growth. We also carried out complementation experiments with a cloned wild-type rpoS gene. Our results showed that 15 of the 41 Salmonella serotype Typhi isolates were defective in RpoS. We sequenced the rpoS allele of 12 strains. This led to identification of small insertions, deletions, and point mutations resulting in premature stop codons or affecting regions 1 and 2 of σ^S, showing that the rpoS mutations are not clonal. Thus, mutant rpoS alleles can be found in freshly isolated clinical strains of Salmonella serotype Typhi, and they may affect virulence properties. Interestingly however, no rpoS mutants were found among the 75 Salmonella serotype Typhimurium isolates. Strains that differed in catalase activity and resistance to hydrogen peroxide were found, but the differences were not linked to the rpoS status. This suggests that Salmonella serotype Typhimurium rpoS mutants are counterselected because rpoS plays a role in the pathogenesis of Salmonella serotype Typhimurium in humans or in the transmission cycle of the disease.     

Inhibition of Salmonella enterica Cells in Deli-Type Salad by Enterocin AS-48 in Combination with Other Antimicrobials

The cyclic antibacterial peptide enterocin AS-48 acted synergistically with p-hydroxybenzoic acid methyl ester (PHBME) and with 2-nitropropanol against Salmonella enterica serovar Enteritidis CECT 4300 in Russian salad. In challenge tests on a cocktail of Salmonella strains (S. enterica ssp. enterica serotype Typhi CECT 409, S. enterica ssp. enterica serovar Choleraesuis CECT 915, S. enterica ssp. enterica serovar Enteritidis CECT 4300, S. enterica ssp. arizonae serovar Arizonae CECT 4395, and S. enterica ssp. salamae CECT 4000) in salad at 10°C, the combinations of PHBME and AS-48 (80 μg/g) or 2-nitropropanol and AS-48 (40 μg/g) reduced the concentrations of viable Salmonella from 4.27 to 4.75 log CFU/g down to the limit of detection for 7 days. Salmonella populations did not increase in control samples (without any antimicrobials or in the presence of AS-48 alone) probably due to the low pH of this type of salad and low temperature of incubation, but retained over 85% viability after 1 week. This work opens new possibilities to expand the spectrum of inhibition of enterocin AS-48 against Salmonella enterica.     

An Allelotyping PCR for Identifying Salmonella enterica serovars Enteritidis, Hadar, Heidelberg, and Typhimurium

Current commercial PCRs tests for identifying Salmonella target genes unique to this genus. However, there are two species, six subspecies, and over 2,500 different Salmonella serovars, and not all are equal in their significance to public health. For example, finding S. enterica subspecies IIIa Arizona on a table egg layer farm is insignificant compared to the isolation of S. enterica subspecies I serovar Enteritidis, the leading cause of salmonellosis linked to the consumption of table eggs. Serovars are identified based on antigenic differences in lipopolysaccharide (LPS)(O antigen) and flagellin (H1 and H2 antigens). These antigenic differences are the outward appearance of the diversity of genes and gene alleles associated with this phenotype.We have developed an allelotyping, multiplex PCR that keys on genetic differences between four major S. enterica subspecies I serovars found in poultry and associated with significant human disease in the US. The PCR primer pairs were targeted to key genes or sequences unique to a specific Salmonella serovar and designed to produce an amplicon with size specific for that gene or allele. Salmonella serovar is assigned to an isolate based on the combination of PCR test results for specific LPS and flagellin gene alleles. The multiplex PCRs described in this article are specific for the detection of S. enterica subspecies I serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.Here we demonstrate how to use the multiplex PCRs to identify serovar for a Salmonella isolate.     

Pet Snakes as a Reservoir for Salmonella enterica subsp. diarizonae (Serogroup IIIb): a Prospective Study

Reptile-associated Salmonella infections are an increasing problem for humans. We have prospectively screened two breeding groups of 16 pet snakes for colonization with Salmonella species. Various serovars of S. enterica subsp. diarizonae were found in 81% of the snakes. To avoid transmission, strict hygienic precautions should be applied when reptiles are handled.     

Fast DNA Serotyping and Antimicrobial Resistance Gene Determination of Salmonella enterica with an Oligonucleotide Microarray-Based Assay

Salmonellosis caused by Salmonella (S.) belongs to the most prevalent food-borne zoonotic diseases throughout the world. Therefore, serotype identification for all culture-confirmed cases of Salmonella infection is important for epidemiological purposes. As a standard, the traditional culture method (ISO 6579:2002) is used to identify Salmonella. Classical serotyping takes 4–5 days to be completed, it is labor-intensive, expensive and more than 250 non-standardized sera are necessary to characterize more than 2,500 Salmonella serovars currently known. These technical difficulties could be overcome with modern molecular methods. We developed a microarray based serogenotyping assay for the most prevalent Salmonella serovars in Europe and North America. The current assay version could theoretically discriminate 28 O-antigens and 86 H-antigens. Additionally, we included 77 targets analyzing antimicrobial resistance genes. The Salmonella assay was evaluated with a set of 168 reference strains representing 132 serovars previously serotyped by conventional agglutination through various reference centers. 117 of 132 (81%) tested serovars showed an unique microarray pattern. 15 of 132 serovars generated a pattern which was shared by multiple serovars (e.g., S. ser. Enteritidis and S. ser. Nitra). These shared patterns mainly resulted from the high similarity of the genotypes of serogroup A and D1. Using patterns of the known reference strains, a database was build which represents the basis of a new PatternMatch software that can serotype unknown Salmonella isolates automatically. After assay verification, the Salmonella serogenotyping assay was used to identify a field panel of 105 Salmonella isolates. All were identified as Salmonella and 93 of 105 isolates (88.6%) were typed in full concordance with conventional serotyping. This microarray based assay is a powerful tool for serogenotyping.     

Effects of Norspermidine and Spermidine on Biofilm Formation by Potentially Pathogenic Escherichia coli and Salmonella enterica Wild-Type Strains

Polyamines are present in all living cells. In bacteria, polyamines are involved in a variety of functions, including biofilm formation, thus indicating that polyamines may have potential in the control of unwanted biofilm. In the present study, the effects of the polyamines norspermidine and spermidine on biofilms of 10 potentially pathogenic wild-type strains of Escherichia coli serotype O103:H2, Salmonella enterica subsp. enterica serovar Typhimurium, and S. enterica serovar Agona were investigated. We found that exogenously supplied norspermidine and spermidine did not mediate disassembly of preformed biofilm of any of the E. coli and S. enterica strains. However, the polyamines did affect biofilm production. Interestingly, the two species reacted differently to the polyamines. Both polyamines reduced the amount of biofilm formed by E. coli but tended to increase biofilm formation by S. enterica. Whether the effects observed were due to the polyamines specifically targeting biofilm formation, being toxic for the cells, or maybe a combination of the two, is not known. However, there were no indications that the effect was mediated through binding to exopolysaccharides, as earlier suggested for E. coli. Our results indicate that norspermidine and spermidine do not have potential as inhibitors of S. enterica biofilm. Furthermore, we found that the commercial polyamines used contributed to the higher pH of the test medium. Failure to acknowledge and control this important phenomenon may lead to misinterpretation of the results.     

Extracellular DNA Inhibits Salmonella enterica Serovar Typhimurium and S. enterica Serovar Typhi Biofilm Development on Abiotic Surfaces

Extracellular DNA (eDNA) was identified and characterized in a 2-day-old biofilms developed by Salmonella enterica ser. Typhimurium SR-11 and S. enterica ser. Typhi ST6 using confocal laser scanning microscopy (CLSM) and enzymatic extraction methods. Results of microtitre plate assay and CLSM analysis showed both Salmonella strains formed significantly more biofilms in the presence of DNase I; Furthermore, a remarkable decrease of biofilm formation was observed when eDNA was added in the inoculation. However, for the pre-established biofilms on polystyrene and glass, no significant difference was observed between the DNase I treated biofilm and the corresponding non-treated controls. In conclusion, these results demonstrate that eDNA is a novel matrix component of Salmonella biofilms. This is the first evidence for the presence of eDNA and its inhibitive and destabilizing effect during biofilm development of S. enterica ser. Typhimurium and S. enterica ser. Typhi on abiotic surfaces.     

Multilocus Sequence Typing Supports the Hypothesis that Cow- and Human-Associated Salmonella Isolates Represent Distinct and Overlapping Populations

A collection of 179 human and 156 bovine clinical Salmonella isolates obtained from across New York state over the course of 1 year was characterized using serotyping and a multilocus sequence typing (MLST) scheme based on the sequencing of three genes (fimA, manB, and mdh). The 335 isolates were differentiated into 52 serotypes and 72 sequence types (STs). Analyses of bovine isolates collected on different farms over time indicated that specific subtypes can persist over time on a given farm; in particular, a number of farms showed evidence for the persistence of a specific Salmonella enterica serotype Newport sequence type. Serotypes and STs were not randomly distributed among human and bovine isolates, and selected serotypes and STs were associated exclusively with either human or bovine sources. A number of common STs were geographically widespread. For example, ST6, which includes isolates representing serotype Typhimurium as well as the emerging serotype 4,5,12:i:-, was found among human and bovine isolates in a number of counties in New York state. Phylogenetic analyses supported the possibility that serotype 4,5,12:i:- is closely related to Salmonella serotype Typhimurium. Salmonella serotype Newport was found to represent two distinct evolutionary lineages that differ in their frequencies among human and bovine isolates. A number of Salmonella isolates carried two copies of manB (33 isolates) or showed small deletion events in fimA (nine isolates); these duplication and deletion events may provide mechanisms for the rapid diversification of Salmonella surface molecules. We conclude that the combined use of an economical three-gene MLST scheme and serotyping can provide considerable new insights into the evolution and transmission of Salmonella.