Investigating the Relatedness of Enteroinvasive Escherichia coli to Other E. coli and Shigella Isolates by Using Comparative Genomics.

Enteroinvasive Escherichia coli (EIEC) is a unique pathovar that has a pathogenic mechanism nearly indistinguishable from that of Shigella species. In contrast to isolates of the four Shigella species, which are widespread and can be frequent causes of human illness, EIEC causes far fewer reported illnesses each year. In this study, we analyzed the genome sequences of 20 EIEC isolates, including 14 first described in this study. Phylogenomic analysis of the EIEC genomes demonstrated that 17 of the isolates are present in three distinct lineages that contained only EIEC genomes, compared to reference genomes from each of the E. coli pathovars and Shigella species. Comparative genomic analysis identified genes that were unique to each of the three identified EIEC lineages. While many of the EIEC lineage-specific genes have unknown functions, those with predicted functions included a colicin and putative proteins involved in transcriptional regulation or carbohydrate metabolism. In silico detection of the Shigella virulence plasmid (pINV), which is essential for the invasion of host cells, demonstrated that a form of pINV was present in nearly all EIEC genomes, but the Mxi-Spa-Ipa region of the plasmid that encodes the invasion-associated proteins was absent from several of the EIEC isolates. The comparative genomic findings in this study support the hypothesis that multiple EIEC lineages have evolved independently from multiple distinct lineages of E. coli via the acquisition of the Shigella virulence plasmid and, in some cases, the Shigella pathogenicity islands.

FDA Escherichia coli Identification (FDA-ECID) Microarray: a Pangenome Molecular Toolbox for Serotyping, Virulence Profiling, Molecular Epidemiology, and Phylogeny.

UNLABELLED: Most Escherichia coli strains are nonpathogenic. However, for clinical diagnosis and food safety analysis, current identification methods for pathogenic E. coli either are time-consuming and/or provide limited information. Here, we utilized a custom DNA microarray with informative genetic features extracted from 368 sequence sets for rapid and high-throughput pathogen identification. The FDA Escherichia coli Identification (FDA-ECID) platform contains three sets of molecularly informative features that together stratify strain identification and relatedness. First, 53 known flagellin alleles, 103 alleles of wzx and wzy, and 5 alleles of wzm provide molecular serotyping utility. Second, 41,932 probe sets representing the pan-genome of E. coli provide strain-level gene content information. Third, approximately 125,000 single nucleotide polymorphisms (SNPs) of available whole-genome sequences (WGS) were distilled to 9,984 SNPs capable of recapitulating the E. coli phylogeny. We analyzed 103 diverse E. coli strains with available WGS data, including those associated with past foodborne illnesses, to determine robustness and accuracy. The array was able to accurately identify the molecular O and H serotypes, potentially correcting serological failures and providing better resolution for H-nontypeable/nonmotile phenotypes. In addition, molecular risk assessment was possible with key virulence marker identifications. Epidemiologically, each strain had a unique comparative genomic fingerprint that was extended to an additional 507 food and clinical isolates. Finally, a 99.7% phylogenetic concordance was established between microarray analysis and WGS using SNP-level data for advanced genome typing. Our study demonstrates FDA-ECID as a powerful tool for epidemiology and molecular risk assessment with the capacity to profile the global landscape and diversity of E. coli IMPORTANCE: This study describes a robust, state-of-the-art platform developed from available whole-genome sequences of E. coli and Shigella spp. by distilling useful signatures for epidemiology and molecular risk assessment into one assay. The FDA-ECID microarray contains features that enable comprehensive molecular serotyping and virulence profiling along with genome-scale genotyping and SNP analysis. Hence, it is a molecular toolbox that stratifies strain identification and pathogenic potential in the contexts of epidemiology and phylogeny. We applied this tool to strains from food, environmental, and clinical sources, resulting in significantly greater phylogenetic and strain-specific resolution than previously reported for available typing methods.

Comparative Genotypic and Phenotypic Analysis of Cronobacter Species Cultured from Four Powdered Infant Formula Production Facilities: Indication of Pathoadaptation along the Food Chain.

Cronobacter species are opportunistic pathogens commonly found in the environment. Among the seven Cronobacter species, Cronobacter sakazakii sequence type 4 (ST-4) is predominantly associated with recorded cases of infantile meningitis. This study reports on a 26-month powdered infant formula (PIF) surveillance program in four production facilities located in distinct geographic regions. The objective was to identify the ST(s) in PIF production environments and to investigate the phenotypic features that support their survival. Of all 168 Cronobacter isolates, 133 were recovered from a PIF production environment, 31 were of clinical origin, and 4 were laboratory type strains. Sequence type 1 (n = 84 isolates; 63.9%) was the dominant type in PIF production environments. The majority of these isolates clustered with an indistinguishable pulsotype and persisted for at least an 18-month period. Moreover, DNA microarray results identified two phylogenetic lineages among ST-4 strains tested. Thereafter, the ST-1 and -4 isolates were phenotypically compared. Differences were noted based on the phenotypes expressed by these isolates. The ST-1 PIF isolates produced stronger biofilms at both 28°C and 37°C, while the ST-4 clinical isolates exhibited greater swimming activity and increased binding to Congo red dye. Given the fact that PIF is a low-moisture environment and that the clinical environment provides for an interaction between the pathogen and its host, these differences may be consistent with a form of pathoadaptation. These findings help to extend our current understanding of the epidemiology and ecology of Cronobacter species in PIF production environments.

Acquisition of the lac operon by Salmonella enterica.

BACKGROUND: Classical bacteriological characteristics of Salmonella enterica indicate that the members of this species are unable to utilize lactose as a carbon source. However, lactose-fermenting (Lac+) strains of several Salmonella serovars have been isolated from different foodborne outbreaks as well as different geographical regions worldwide. In the present study, we sequenced the genomes of 13 Lac + S. enterica isolates and characterized the lac region, comparing it to the lac region in other enteric bacterial species. RESULTS: Genetic analysis of the lac operons in the S. enterica genomes revealed that they all contain intact lacI, lacZ, and lacY genes. However, lacA was truncated in all of the S. enterica subsp. enterica isolates, encoding a 56 amino acid peptide rather than the full length 220 amino acid LacA protein. Molecular analyses of the 13 isolates revealed that the lac operon resided on a plasmid in some strains and in others was integrated into the bacterial chromosome. In most cases, an insertion sequence flanked at least one end of the operon. Interestingly, the S. enterica Montevideo and S. enterica Senftenberg isolates were found to harbor a plasmid with a high degree of sequence similarity to a plasmid from Klebsiella pneumoniae strain NK29 that also harbors the lac operon. In addition, two S. enterica Tennessee isolates carried two copies of the lac operon. Phylogenetic analysis based on lacIZY gene sequences determines distinct clusters, and reveals a greater correlation between lacIZY sequence and flanking organization than with either bacterial species or genomic location. CONCLUSIONS: Our results indicate that the lac region is highly mobile among Enterobacteriaceae and demonstrate that the Lac + S. enterica subsp. enterica serovars acquired the lac region through parallel events. The acquisition of the lac operon by several S. enterica serovars may be indicative of environmental adaptation by these bacteria.

Clinical isolates of Shiga toxin 1a-producing Shigella flexneri with an epidemiological link to recent travel to Hispañiola.

Shiga toxins (Stx) are cytotoxins involved in severe human intestinal disease. These toxins are commonly found in Shigella dysenteriae serotype 1 and Shiga-toxin-producing Escherichia coli; however, the toxin genes have been found in other Shigella species. We identified 26 Shigella flexneri serotype 2 strains isolated by public health laboratories in the United States during 2001-2013, which encode the Shiga toxin 1a gene (stx1a). These strains produced and released Stx1a as measured by cytotoxicity and neutralization assays using anti-Stx/Stx1a antiserum. The release of Stx1a into culture supernatants increased ≈100-fold after treatment with mitomycin C, suggesting that stx1a is carried by a bacteriophage. Infectious phage were found in culture supernatants and increased ≈1,000-fold with mitomycin C. Whole-genome sequencing of several isolates and PCR analyses of all strains confirmed that stx1a was carried by a lambdoid bacteriophage. Furthermore, all patients who reported foreign travel had recently been to Hispañiola, suggesting that emergence of these novel strains is associated with that region.

Cronobacter: an emergent pathogen causing meningitis to neonates through their feeds.

The recognition of Cronobacter as a public health concern was raised when powdered infant formula (PIF) was linked to several neonatal meningitis outbreaks. It is an opportunistic pathogen that causes necrotising enterocolitis, infantile septicaemia, and meningitis which carries a high mortality rate among neonates. It has been also linked with cases of infection in adults and elderly. Over the past decade, much focus has been made on developing sensitive and specific characterisation, detection, and isolation methods to ascertain the quality of foods, notably contamination of PIF with Cronobacter and to understand its ability to cause disease. Whole genome sequencing has unveiled several putative virulence factors, yet the full capacity of the pathogenesis of Cronobacter has not yet been elucidated.

Comparison of chromogenic Biolog Rainbow agar Shigella/Aeromonas with xylose lysine desoxycholate agar for isolation and detection of Shigella spp. from foods.

Shigella outbreaks are widely reported throughout the world. However, it remains a challenge to isolate Shigella spp. from foods by using conventional microbiological media. The main objective of this study was to determine the effectiveness of a novel chromogenic medium, Rainbow agar Shigella/Aeromonas (Rainbow agar), for the isolation and detection of Shigella spp. in foods. All four Shigella species, S. sonnei, S. flexneri, S. dysenteriae, and S. boydii, were studied. Rainbow agar was compared with tryptic soy agar, xylose lysine desoxycholate agar (XLD), and Salmonella Shigella agar (SSA) for enumeration of Shigella spp. in pure culture. This chromogenic agar and XLD were also used to isolate Shigella spp. in artificially contaminated foods (4.8 log CFU/g of food), including lettuce, parsley, cilantro, spinach, potato salad, and shrimp. The inhibitory effect on Shigella growth by Rainbow agar was between that of XLD and SSA. All vegetables studied showed a moderately high background microflora on XLD and Rainbow agar. With artificially inoculated produce, Rainbow agar recovered about 1 to 2 log CFU more S. sonnei, S. dysenteriae, and S. boydii per g of food than did XLD. For potato salad and shrimp, which had low background microflora on Rainbow agar, Rainbow agar was slightly better in recovering Shigella spp. than XLD was in most cases. However, we found that the addition of streptomycin (6.25 mg/liter) to Rainbow agar could facilitate the isolation of Shigella in vegetables tested. In conclusion, Rainbow agar was a much more effective medium than was XLD for the isolation of Shigella spp. from foods.

Development of a multiplex real-time PCR assay with internal amplification control for the detection of Shigella species and enteroinvasive Escherichia coli.

Shigella species, particularly S. sonnei and S. flexneri, remain some of the leading bacterial etiological agents of gastrointestinal diseases in the United States and globally. The isolation and detection of these foodborne pathogens are critical for preventing the spread of disease and facilitating epidemiological investigations aimed at determining the source of a Shigella infection outbreak. A multiplex real-time PCR-based assay was developed that targets all four species of Shigella plus enteroinvasive Escherichia coli. The assay incorporates primers directed to the ipaH genes located on both the virulence plasmid and chromosome, the plasmid-encoded virulence gene mxiC, a mutated mxiC gene (mxiC::kan) that differentiates wild-type strains from a laboratory control strain, and an internal amplification control. More than 50 isolates of all four Shigella species were tested for inclusivity and specificity of the multiplex PCR assay, and more than 30 non-Shigella isolates were tested for exclusivity of the assay. The sensitivity of the assay was 1 to 3 CFU and 5 to 50 fg of target (total) DNA for the ipaH, mxiC, and mxiC::kan gene targets. The assay performed equally well and with no measurable inhibition in the Shigella target reactions when rinsates of several high-risk produce commodities (parsley, cilantro, alfalfa sprouts, and lettuce) were added to the reactions. This multiplex PCR assay is sensitive and specific and has the added dimension of discriminating all Shigella species from the positive control strain so that in any sample analysis other strains can be excluded as a source of contamination.

Development of an improved protocol for the isolation and detection of Enterobacter sakazakii (Cronobacter) from powdered infant formula.

Enterobacter sakazakii causes severe maladies and, in some cases, is fatal among infants. Powdered infant formula (PIF) contaminated with E. sakazakii has been documented as a potential cause of several outbreaks involving infants. This study describes the development of a method for the isolation and detection of E. sakazakii from PIF. It combines Taqman real-time PCR, Brilliance E. sakazakii and R&F chromogenic agars, and RAPID ID 32E biochemical tests. This method provides an expedient analysis within 1 to 2 days depending on the amount and stress status of E. sakazakii organisms and competing microorganisms in PIF. The real-time PCR has bifunctional applications, including both screening and culture confirmation of E. sakazakii.

Detection of live Salmonella sp. cells in produce by a TaqMan-based quantitative reverse transcriptase real-time PCR targeting invA mRNA.

Salmonella enterica contamination in foods is a significant concern for public health. When DNA detection methods are used for analysis of foods, one of the major concerns is false-positive results from the detection of dead cells. To circumvent this crucial issue, a TaqMan quantitative real-time RT-PCR (qRT-PCR) assay with an RNA internal control was developed. invA RNA standards were used to determine the detection limit of this assay as well as to determine invA mRNA levels in mid-exponential-, late-exponential-, and stationary-phase cells. This assay has a detection limit of 40 copies of invA mRNA per reaction. The levels of invA mRNA in mid-exponential-, late-exponential-, and stationary-phase S. enterica cells was approximately 1 copy per 3 CFU, 1 copy per CFU, and 4 copies per 10(3) CFU, respectively. Spinach, tomatoes, jalapeno peppers, and serrano peppers were artificially contaminated with four different Salmonella serovars at levels of 10(5) and less than 10 CFU. These foods were analyzed with qRT-PCR and with the FDA's Bacteriological Analytical Manual Salmonella culture method (W. A. Andrews and T. S. Hammack, in G. J. Jackson et al., ed., Bacteriological analytical manual online, http://www.cfsan.fda.gov/ approximately ebam/bam-5.html, 2007). Comparable results were obtained by both methods. Only live Salmonella cells could be detected by this qRT-PCR assay, thus avoiding the dangers of false-positive results from nonviable cells. False negatives (inhibition of the PCR) were also ruled out through the use of an RNA internal control. This assay allows for the fast and accurate detection of viable Salmonella spp. in spinach, tomatoes, and in both jalapeno and serrano peppers.

Evaluation of a revised U.S. Food and Drug Administration method for the detection and isolation of Enterobacter sakazakii in powdered infant formula: precollaborative study.

A revised U.S. Food and Drug Administration (FDA) method for the detection and isolation of Enterobacter sakazakii in powdered infant formula was developed based on real-time PCR technology complemented by culture isolation on chromogenic agars. A validation study was conducted to compare the revised FDA method to the reference FDA method. Casein and soy powdered infant formula inoculated with morphologically typical and atypical strains of E. sakazakii were analyzed. Valid results were obtained from 360 test portions and controls and showed that the revised FDA method is significantly better (P < 0.05) than the reference FDA method for the detection of typical E. sakazakii strains and the two methods are equivalent for the detection of atypical E. sakazakii strains.

Draft Genome Sequences of 12 Isolates from 3 Fusarium Species Recovered from Moldy Peanuts.

In this report, we announce the sequences of six genomes of Fusarium proliferatum (isolates MOD1-FUNGI8, -12, -13, -14, -15, and -19), four genomes of Fusarium oxysporum (MOD1-FUNGI9, -10, -11, and -16), and two genomes of the Fusarium incarnatum-Fusarium equiseti species complex (MOD1-FUNGI17 and MOD1-FUNGI18) isolated from moldy peanuts from the Washington, DC, area.

A Brief History of Shigella.

The history of Shigella, the causative agent of bacillary dysentery, is a long and fascinating one. This brief historical account starts with descriptions of the disease and its impact on human health from ancient time to the present. Our story of the bacterium starts just before the identification of the dysentery bacillus by Kiyoshi Shiga in 1898 and follows the scientific discoveries and principal scientists who contributed to the elucidation of Shigella pathogenesis in the first 100 years. Over the past century, Shigella has proved to be an outstanding model of an invasive bacterial pathogen and has served as a paradigm for the study of other bacterial pathogens. In addition to invasion of epithelial cells, some of those shared virulence traits include toxin production, multiple-antibiotic resistance, virulence genes encoded on plasmids and bacteriophages, global regulation of virulence genes, pathogenicity islands, intracellular motility, remodeling of host cytoskeleton, inflammation/polymorphonuclear leukocyte signaling, apoptosis induction/inhibition, and "black holes" and antivirulence genes. While there is still much to learn from studying Shigella pathogenesis, what we have learned so far has also contributed greatly to our broader understanding of bacterial pathogenesis.

Distribution and characterization of Shiga toxin converting temperate phages carried by Shigella flexneri in Hispaniola.

Shigella infections account for a considerable burden of acute diarrheal diseases worldwide and remain a major cause of childhood mortality in developing countries. Although, all four species of Shigella (S. dysenteriae, S. flexneri, S. boydii, and S. sonnei) cause bacillary dysentery, historically only S. dysenteriae type 1 has been recognized as carrying the genes for Shiga toxin (stx). Recent epidemiological data, however, have suggested that the emergence of stx carrying S. flexneri strains may have originated from bacteriophage-mediated inter-species horizontal gene transfer in one specific geographical area, Hispaniola. To test this hypothesis, we analyzed whole genome sequences of stx-encoding phages carried by S. flexneri strains isolated in Haiti and S. flexneri S. boydii and S. dysenteriae strains isolated from international travelers who likely acquired the infection in Haiti or the Dominican Republic. Phylogenetic analysis showed that phage sequences encoded in the Shigella strains from Hispaniola were bacteriophage φPOC-J13 and they were all closely related to a phage isolated from a USA isolate, E. coli 2009C-3133 serotype O119:H4. In addition, despite the low genetic heterogeneity of phages from different Shigella spp. circulating in the Caribbean island between 2001 and 2014, two distinct clusters emerged in Haiti and the Dominican Republic. Each cluster possibly originated from phages isolated from S. flexneri 2a, and within each cluster several instances of horizontal phage transfer from S. flexneri 2a to other species were detected. The implications of the emergence of stx-producing non-S. dysenteriae type 1 Shigella species, such as S. flexneri, spans not only the basic science behind horizontal phage spread, but also extends to medical treatment of patients infected with this pathogen.

Draft Genome Sequences of the Escherichia coli Reference (ECOR) Collection.

Here, we report the genomes of all 72 isolates belonging to the Escherichia coli reference (ECOR) collection. Strains in this collection were isolated from diverse hosts and geographic locations and have been used for more than 30 years to represent the phylogenetic diversity of E. coli.

Draft Genome Sequences of Bifidobacterium Strains Isolated from Dietary Supplements and Cultured Food Products.

Here, we present the genome sequences of 23 Bifidobacterium isolates from several commercially available dietary supplements and cultured food products. Strains of this genus are natural inhabitants of the mammalian mouth, gastrointestinal tract, and vagina. Some species are considered beneficial to human health.

Fifty-Six Draft Genome Sequences of 10 Lactobacillus Species from 22 Commercial Dietary Supplements.

Here, we present the genome sequences of 56 isolates of 10 species of the genus Lactobacillus that are considered beneficial components of the gut microbiota. The isolates examined were found in commercially available dietary supplements in the U.S. market.

Draft Genome Sequences of Escherichia albertii, Escherichia fergusonii, and Strains Belonging to Six Cryptic Lineages of Escherichia spp.

We report here the genome sequences of 55 strains belonging to the genus Escherichia from multiple animal and environmental sources. These strains include representatives of Escherichia albertii, Escherichia fergusonii, and six additional genetically distinct lineages of Escherichia spp., one of which is newly discovered and is being reported for the first time here.

Species-Wide Collection of Escherichia coli Isolates for Examination of Genomic Diversity.

Pathogenic and nonpathogenic Escherichia coli strains present a vast genomic diversity. We report the genome sequences of 2,244 E. coli isolates from multiple animal and environmental sources. Their phylogenetic relationships and potential risk to human health were examined.

Draft Genome Sequences of the Enteroinvasive Escherichia coli Strains M4163 and 4608-58.

We report here the draft genome sequences of enteroinvasive Escherichia coli (EIEC) O124:H30 strain M4163 isolated from imported French cheese and EIEC O143:H26 strain 4608-58. The assembled data determined that both strains contain multiple copies of the ipaH gene, as well as the pINV A form of the invasion plasmid.