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1.  Hybrid Shiga Toxin-Producing and Enterotoxigenic Escherichia sp. Cryptic Lineage 1 Strain 7v Harbors a Hybrid Plasmid 
Applied and Environmental Microbiology  2016;82(14):4309-4319.
Hybrid isolates of Shiga toxin-producing Escherichia coli (STEC) and enterotoxigenic E. coli (ETEC) encoding heat-stable enterotoxin (ST) are being reported with increasing frequency from a variety of sources. However, information regarding the plasmids that these strains harbor is scarce. In this study, we sequence and characterize a plasmid, p7v, from the STEC/ETEC hybrid strain 7v. Whole-genome phylogenetic analyses of STEC/ETEC hybrid strains and prototype E. coli isolates of other pathotypes placed 7v in the Escherichia sp. cryptic lineage 1 (CL1) clade. The complete plasmid, p7v, was determined to be 229,275 bp and encodes putative virulence factors that are typically carried on STEC plasmids as well as those often carried on ETEC plasmids, indicating that the hybrid nature of the strain extends beyond merely encoding the two toxins. Plasmid p7v carries two copies of sta with identical sequences, which were discovered to be divergent from the sta sequences found in the prototype human ETEC strains. Using a nomenclature scheme based on a phylogeny constructed from sta and stb sequences, the sta encoded on p7v is designated STa4. In silico analysis determined that p7v also encodes the K88 fimbria, a colonization factor usually associated with porcine ETEC plasmids. The p7v sequence and the presence of plasmid-encoded virulence factors are compared to those of other STEC/ETEC CL1 hybrid genomes and reveal gene acquisition/loss at the strain level. In addition, the interrogation of 24 STEC/ETEC hybrid genomes for identification of plasmid replicons, colonization factors, Stx and ST subtypes, and other plasmid-encoded virulence genes highlights the diversity of these hybrid strains.
IMPORTANCE Hybrid Shiga toxin-producing Escherichia coli/enterotoxigenic Escherichia coli (STEC/ETEC) strains, which have been isolated from environmental, animal, and human clinical samples, may represent an emerging threat as food-borne pathogens. Characterization of these strains is important for assessing virulence potential, aiding in the development of pathogen detection methods, and understanding how the hybrid strains evolve to potentially have a greater impact on public health. This study represents, to our knowledge, both the first characterization of a closed plasmid sequence from a STEC/ETEC hybrid strain and the most comprehensive phylogenetic analysis of available STEC/ETEC hybrid genomes to date. The results demonstrate how the mobility of plasmid-associated virulence genes has resulted in the creation of a diverse plasmid repertoire within the STEC/ETEC hybrid strains.
PMCID: PMC4959189  PMID: 27208138
2.  Strain-Level Discrimination of Shiga Toxin-Producing Escherichia coli in Spinach Using Metagenomic Sequencing 
PLoS ONE  2016;11(12):e0167870.
Consumption of fresh bagged spinach contaminated with Shiga toxin-producing Escherichia coli (STEC) has led to severe illness and death; however current culture-based methods to detect foodborne STEC are time consuming. Since not all STEC strains are considered pathogenic to humans, it is crucial to incorporate virulence characterization of STEC in the detection method. In this study, we assess the comprehensiveness of utilizing a shotgun metagenomics approach for detection and strain-level identification by spiking spinach with a variety of genomically disparate STEC strains at a low contamination level of 0.1 CFU/g. Molecular serotyping, virulence gene characterization, microbial community analysis, and E. coli core gene single nucleotide polymorphism (SNP) analysis were performed on metagenomic sequence data from enriched samples. It was determined from bacterial community analysis that E. coli, which was classified at the phylogroup level, was a major component of the population in most samples. However, in over half the samples, molecular serotyping revealed the presence of indigenous E. coli which also contributed to the percent abundance of E. coli. Despite the presence of additional E. coli strains, the serotype and virulence genes of the spiked STEC, including correct Shiga toxin subtype, were detected in 94% of the samples with a total number of reads per sample averaging 2.4 million. Variation in STEC abundance and/or detection was observed in replicate spiked samples, indicating an effect from the indigenous microbiota during enrichment. SNP analysis of the metagenomic data correctly placed the spiked STEC in a phylogeny of related strains in cases where the indigenous E. coli did not predominate in the enriched sample. Also, for these samples, our analysis demonstrates that strain-level phylogenetic resolution is possible using shotgun metagenomic data for determining the genomic relatedness of a contaminating STEC strain to other closely related E. coli.
PMCID: PMC5145215  PMID: 27930729
3.  Prevalence of Stx-producing Shigella species isolated from French Travelers Returning from the Caribbean: An Emerging Pathogen with International Implications 
Shiga toxins are potent cytotoxins that inhibit host cell protein synthesis, leading to cell death. Classically, these toxins are associated with intestinal infections due to Shiga toxin-producing Escherichia coli or Shigella dysenteriae serotype 1 and infections with these strains can lead to hemolytic uremic syndrome. Over the past decade there is increasing recognition that Shiga toxin is produced by additional Shigella species. We recently reported the presence and expression of stx genes in Shigella flexneri 2a clinical isolates. The toxin genes were carried by a new stx-encoding bacteriophage and infection with these strains correlated with recent travel to Haiti or the Dominican Republic. In this study we further explored the epidemiological link to this region by utilizing the French National Reference Center for Escherichia coli, Shigella and Salmonella collection to survey the frequency of Stx-producing Shigella species isolated from French travelers returning from the Caribbean. About 21% of the isolates tested were found to encode and produce Stx. These isolates included strains of S. flexneri 2a, S. flexneri Y, and S. dysenteriae 4. All of the travelers whom were infected with Stx-producing Shigella had recently traveled to Haiti, the Dominican Republic, or French Guiana. Furthermore, whole genome sequencing found that the toxin genes were encoded by a prophage that was highly identical to the phage we identified in our previous study. These findings demonstrate that this new stx-encoding prophage is circulating within that geographical area, has spread to other continents, and is capable of spreading to multiple Shigella serogroups.
PMCID: PMC4497849  PMID: 25980352
Shigella; Shiga toxin; Haiti; Dominican Republic
4.  Updates on feline aelurostrongylosis and research priorities for the next decade 
Parasites & Vectors  2016;9:389.
Feline aelurostrongylosis, caused by the metastrongyloid nematode Aelurostrongylus abstrusus, is an important gastropod-borne parasitic lung disease in cats. Infection with A. abstrusus is widespread globally, but the increasing awareness of this parasite and the advent of more sensitive diagnostics have contributed to the apparent increase in its prevalence and geographic expansion. Clinical features may range in severity from subclinical to life-threatening respiratory disease. Parasitological standard techniques, such as visualization of the nematode first larval stage in faecal and respiratory (bronchial mucus or pleural fluid) samples, remain the mainstays of diagnosis. However, diagnosis is evolving with recent advances in serological and molecular testing, which can improve the time to initiation of effective anthelmintic therapy. Despite numerous anthelmintics that are now available as treatment options, the role of host immunity and lifestyle factors in selecting cats that may benefit from more targeted anthelmintic prophylaxis or treatment practice remains unclear and is likely to guide therapeutic choices as newer data become available. This review summarizes the biology, epidemiology, pathophysiology, diagnosis and treatment options currently available for feline aelurostrongylosis.
PMCID: PMC4936016  PMID: 27387914
Aelurostrongylus abstrusus; Aelurostrongylosis; Cat; Lungworm
5.  Folate status and concentrations of serum folate forms in the US population: National Health and Nutrition Examination Survey 2011–2 
The British journal of nutrition  2015;113(12):1965-1977.
Serum and red blood cell (RBC) total folate are indicators of folate status. No nationally representative population data exist for folate forms. We measured serum folate forms [5-methyltetrahydrofolate (5-methylTHF), unmetabolized folic acid (UMFA), non-methyl folate (sum of THF, 5-formylTHF, 5,10-methenylTHF), and MeFox (5-methylTHF oxidation product)] by HPLC-MS/MS and RBC total folate by microbiologic assay in US persons ≥1 year (n ~7500) participating in the National Health and Nutrition Examination Survey 2011–2. Data analysis for serum total folate was conducted including and excluding MeFox. Concentrations (geometric mean; detection rate) of 5-methylTHF (37.5 nmol/L; 100%), UMFA (1.21 nmol/L; 99.9%), MeFox (1.53 nmol/L; 98.8%), and THF (1.01 nmol/L; 85.2%) were mostly detectable. 5-FormylTHF (3.6%) and 5,10-methenylTHF (4.4%) were rarely detected. The biggest contributor to serum total folate was 5-methylTHF (86.7%); UMFA (4.0%), non-methyl folate (4.7%), and MeFox (4.5%) contributed smaller amounts. Age was positively related to MeFox but showed a U-shaped pattern for other folates. We generally noted sex and race-ethnic biomarker differences and weak (Spearman r <0.4) but significant (P <0.05) correlations with physiologic and lifestyle variables. Fasting, kidney function, smoking, and alcohol intake showed negative associations. BMI and body surface area showed positive associations with MeFox but negative associations with other folates. All biomarkers showed significantly higher concentrations with recent folic acid-containing dietary supplement use. These first-time population data for serum folate forms generally show similar associations with demographic, physiologic, and lifestyle variables as serum total folate. Patterns observed for MeFox may suggest altered folate metabolism dependent on biological characteristics.
PMCID: PMC4804191  PMID: 25917925
NHANES; folate vitamers; 5-methyltetrahydrofolate; unmetabolized folic acid; non-methyl folate; folate oxidation product; LC-MS/MS
6.  Application of Metagenomic Sequencing to Food Safety: Detection of Shiga Toxin-Producing Escherichia coli on Fresh Bagged Spinach 
Applied and Environmental Microbiology  2015;81(23):8183-8191.
Culture-independent diagnostics reduce the reliance on traditional (and slower) culture-based methodologies. Here we capitalize on advances in next-generation sequencing (NGS) to apply this approach to food pathogen detection utilizing NGS as an analytical tool. In this study, spiking spinach with Shiga toxin-producing Escherichia coli (STEC) following an established FDA culture-based protocol was used in conjunction with shotgun metagenomic sequencing to determine the limits of detection, sensitivity, and specificity levels and to obtain information on the microbiology of the protocol. We show that an expected level of contamination (∼10 CFU/100 g) could be adequately detected (including key virulence determinants and strain-level specificity) within 8 h of enrichment at a sequencing depth of 10,000,000 reads. We also rationalize the relative benefit of static versus shaking culture conditions and the addition of selected antimicrobial agents, thereby validating the long-standing culture-based parameters behind such protocols. Moreover, the shotgun metagenomic approach was informative regarding the dynamics of microbial communities during the enrichment process, including initial surveys of the microbial loads associated with bagged spinach; the microbes found included key genera such as Pseudomonas, Pantoea, and Exiguobacterium. Collectively, our metagenomic study highlights and considers various parameters required for transitioning to such sequencing-based diagnostics for food safety and the potential to develop better enrichment processes in a high-throughput manner not previously possible. Future studies will investigate new species-specific DNA signature target regimens, rational design of medium components in concert with judicious use of additives, such as antibiotics, and alterations in the sample processing protocol to enhance detection.
PMCID: PMC4651076  PMID: 26386062
7.  FDA Escherichia coli Identification (FDA-ECID) Microarray: a Pangenome Molecular Toolbox for Serotyping, Virulence Profiling, Molecular Epidemiology, and Phylogeny 
Applied and Environmental Microbiology  2016;82(11):3384-3394.
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.
PMCID: PMC4959244  PMID: 27037122
8.  Urine sodium excretion increased slightly among U.S. adults between 1988–2010123 
The Journal of nutrition  2014;144(5):698-705.
Little information is available on temporal trends in sodium intake in the U.S. population using urine sodium excretion as a biomarker. Our aim was to assess 1988–2010 trends in estimated 24-h urine sodium (24hUNa) excretion among U.S. adults (20–59 y) participating in the cross-sectional National Health and Nutrition Examination Survey (NHANES). We used subsamples from a 1988–1994 convenience sample, a 2003–2006 1/3 random sample, and a 2010 1/3 random sample to comply with resource constraints. We estimated 24hUNa excretion from measured sodium concentrations in spot urine samples by use of calibration equations (for men and women) derived from the INTERSALT study. Estimated 24hUNa excretion increased over the 20-y period (1988–1994, 2003–2006, and 2010) [mean ± SEM (n)]: 3160 ± 38.4 mg/d (1249), 3290 ± 29.4 mg/d (1235), and 3290 ± 44.4 mg/d (525), respectively (Ptrend = 0.022). We observed significantly higher mean estimated 24hUNa excretion in each survey period (P <0.001) for men compared to women (31–33%) and for persons with higher body mass index (BMI) (32–35% for obese vs. normal weight) or blood pressure (–26% for hypertensive vs. normal blood pressure). After adjusting for age, sex, and race-ethnicity, temporal trends in mean estimated 24hUNa excretion remained statistically significant (Ptrend = 0.004). We observed no temporal trends in mean estimated 24hUNa excretion among BMI subgroups, nor after adjusting for BMI. While several limitations apply to this analysis (the use of a convenience sample in 1988–1994 and using estimated 24hUNa excretion as a biomarker of sodium intake), these first NHANES data suggest that mean estimated 24hUNa excretion increased slightly in U.S. adults over the last 2 decades and this increase may be explained by a shift in the distribution of BMI.
PMCID: PMC4806532  PMID: 24623847
9.  Stx-Producing Shigella Species From Patients in Haiti: An Emerging Pathogen With the Potential for Global Spread 
Open Forum Infectious Diseases  2015;2(4):ofv134.
Shiga toxins (Stx) are commonly produced by Shigella dysenteriae serotype 1 and Stx-producing Escherichia coli. However, the toxin genes have been detected in additional Shigella species. We recently reported the emergence of Stx-producing Shigella in travelers in the United States and France who had recently visited Hispaniola (Haiti and the Dominican Republic). In this study, we confirm this epidemiological link by identifying Stx-producing Shigella from Haitian patients attending clinics near Port-au-Prince. We also demonstrate that the bacteriophage encoding Stx is capable of dissemination to stx-negative Shigella species found in Haiti, suggesting that Stx-producing Shigella may become more widespread within that region.
PMCID: PMC4606844  PMID: 26484357
bacteriophage; Haiti; Shiga toxin; Shigella
10.  Albuminuria Prevalence in First Morning Void Compared with Previous Random Urine from Adults in the National Health and Nutrition Examination Survey, 2009–2010 
Clinical chemistry  2013;59(4):675-683.
Albuminuria, defined as urine albumin/creatinine ratio (ACR) ≥30 mg/g, is a diagnostic component of chronic kidney disease (CKD). National estimates of ACR and CKD prevalence have been based on single random urine samples. Although 2 urine samples or a first morning void are known to produce different estimates of ACR, the impact of differing urine sampling schemes on nationally estimated rates of CKD is unknown.
In 2009–2010, the National Health and Nutrition Examination Survey (NHANES) participants provided 2 untimed urine samples for sequential ACR measurement: an initial random urine collected in the NHANES mobile examination center and a subsequent first morning void collected at home. Rates of albuminuria were calculated in the overall population and broken down by demographics, diagnosed diabetes and hypertension status, and estimated glomerular filtration rate (eGFR).
Overall, 43.5% of adults with increased ACR (≥30 mg/g) in a random urine also had increased ACR in a first morning urine. This percentage was higher among individuals ≥50 years old (48.9%), males (53.3%), participants with diagnosed diabetes (56.3%) and hypertension (51.5%), and eGFR <60 mL/min/1.72m2 (56.9%). The use of confirmed increased ACR (defined as the presence of ACR ≥30 mg/g in both samples taken within 10 days) to define CKD resulted in a lower overall prevalence (11.6%) than first morning urine (12.7%) or random spot urine only (15.2%).
ACR measured on random urine samples appears to overestimate the prevalence of albuminuria compared to first morning urine collections.
PMCID: PMC4562457  PMID: 23315482
11.  Acquisition of the lac operon by Salmonella enterica 
BMC Microbiology  2015;15:173.
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.
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.
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.
Electronic supplementary material
The online version of this article (doi:10.1186/s12866-015-0511-8) contains supplementary material, which is available to authorized users.
PMCID: PMC4549013  PMID: 26303940
12.  Draft Genome Sequences of the Enteroinvasive Escherichia coli Strains M4163 and 4608-58 
Genome Announcements  2015;3(1):e01395-14.
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.
PMCID: PMC4319576  PMID: 25657266
13.  Novel Microarray Design for Molecular Serotyping of Shiga Toxin-Producing Escherichia coli Strains Isolated from Fresh Produce 
Applied and Environmental Microbiology  2014;80(15):4677-4682.
Serotyping Escherichia coli is a cumbersome and complex procedure due to the existence of large numbers of O- and H-antigen types. It can also be unreliable, as many Shiga toxin-producing E. coli (STEC) strains isolated from fresh produce cannot be typed by serology or have only partial serotypes. The FDA E. coli identification (FDA-ECID) microarray, designed for characterizing pathogenic E. coli, contains a molecular serotyping component, which was evaluated here for its efficacy. Analysis of a panel of 75 reference E. coli strains showed that the array correctly identified the O and H types in 97% and 98% of the strains, respectively. Comparative analysis of 73 produce STEC strains showed that serology and the array identified 37% and 50% of the O types, respectively, and that the array was able to identify 16 strains that could not be O serotyped. Furthermore, the array identified the H types of 97% of the produce STEC strains compared to 65% by serology, including six strains that were mistyped by serology. These results show that the array is an effective alternative to serology in serotyping environmental E. coli isolates.
PMCID: PMC4148803  PMID: 24837388
14.  Genetic Diversity and Virulence Potential of Shiga Toxin-Producing Escherichia coli O113:H21 Strains Isolated from Clinical, Environmental, and Food Sources 
Applied and Environmental Microbiology  2014;80(15):4757-4763.
Shiga toxin-producing Escherichia coli strains of serotype O113:H21 have caused severe human diseases, but they are unusual in that they do not produce adherence factors coded by the locus of enterocyte effacement. Here, a PCR microarray was used to characterize 65 O113:H21 strains isolated from the environment, food, and clinical infections from various countries. In comparison to the pathogenic strains that were implicated in hemolytic-uremic syndrome in Australia, there were no clear differences between the pathogens and the environmental strains with respect to the 41 genetic markers tested. Furthermore, all of the strains carried only Shiga toxin subtypes associated with human infections, suggesting that the environmental strains have the potential to cause disease. Most of the O113:H21 strains were closely related and belonged in the same clonal group (ST-223), but CRISPR analysis showed a great degree of genetic diversity among the O113:H21 strains.
PMCID: PMC4148811  PMID: 24858089
15.  Characterization of enteropathogenic and Shiga toxin-producing Escherichia coli in cattle and deer in a shared agroecosystem 
Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogen. Cattle are suggested to be an important reservoir for STEC; however, these pathogens have also been isolated from other livestock and wildlife. In this study we sought to investigate transmission of STEC, enterohemorrhagic E. coli (EHEC) and enteropathogenic E. coli (EPEC) between cattle and white-tailed deer in a shared agroecosystem. Cattle feces were collected from 100 animals in a Michigan dairy farm in July 2012, while 163 deer fecal samples were collected during two sampling periods (March and June). The locations of deer fecal pellets were recorded via geographic information system mapping and microsatellite multi-locus genotyping was used to link the fecal samples to individual deer at both time points. Following subculture to sorbitol MacConkey agar and STEC CHROMagar, the pathogens were characterized by serotyping, stx profiling, and PCR-based fingerprinting; multilocus sequence typing (MLST) was performed on a subset. STEC and EHEC were cultured from 12 to 16% of cattle, respectively, and EPEC was found in 36%. Deer were significantly less likely to have a pathogen in March vs. June where the frequency of STEC, EHEC, and EPEC was 1, 6, and 22%, respectively. PCR fingerprinting and MLST clustered the cattle- and deer-derived strains together in a phylogenetic tree. Two STEC strains recovered from both animal species shared MLST and fingerprinting profiles, thereby providing evidence of interspecies transmission and highlighting the importance of wildlife species in pathogen shedding dynamics and persistence in the environment and cattle herds.
PMCID: PMC4381715  PMID: 25883908
STEC; EPEC; EHEC; cattle; deer; transmission; MLST; fingerprinting
16.  Recent advances in the epidemiology, clinical and diagnostic features, and control of canine cardio-pulmonary angiostrongylosis 
Veterinary Research  2014;45(1):92.
The aim of this review is to provide a comprehensive update on the biology, epidemiology, clinical features, diagnosis, treatment, and prevention of canine cardio-pulmonary angiostrongylosis. This cardiopulmonary disease is caused by infection by the metastrongyloid nematode Angiostrongylus vasorum. The parasite has an indirect life cycle that involves at least two different hosts, gastropod molluscs (intermediate host) and canids (definitive host). A. vasorum represents a common and serious problem for dogs in areas of endemicity, and because of the expansion of its geographical boundaries to many areas where it was absent or uncommon; its global burden is escalating. A. vasorum infection in dogs can result in serious disorders with potentially fatal consequences. Diagnosis in the live patient depends on faecal analysis, PCR or blood testing for parasite antigens or anti-parasite antibodies. Identification of parasites in fluids and tissues is rarely possible except post mortem, while diagnostic imaging and clinical examinations do not lead to a definitive diagnosis. Treatment normally requires the administration of anthelmintic drugs, and sometimes supportive therapy for complications resulting from infection.
PMCID: PMC4189173  PMID: 25283220
17.  Pilus distribution among lineages of group b streptococcus: an evolutionary and clinical perspective 
BMC Microbiology  2014;14:159.
Group B Streptococcus (GBS) is an opportunistic pathogen in both humans and bovines. Epidemiological and phylogenetic analyses have found strains belonging to certain phylogenetic lineages to be more frequently associated with invasive newborn disease, asymptomatic maternal colonization, and subclinical bovine mastitis. Pilus structures in GBS facilitate colonization and invasion of host tissues and play a role in biofilm formation, though few large-scale studies have estimated the frequency and diversity of the three pilus islands (PIs) across diverse genotypes. Here, we examined the distribution of pilus islands (PI) 1, 2a and 2b among 295 GBS strains representing 73 multilocus sequence types (STs) belonging to eight clonal complexes. PCR-based RFLP was also used to evaluate variation in the genes encoding pilus backbone proteins of PI-2a and PI-2b.
All 295 strains harbored one of the PI-2 variants and most human-derived strains contained PI-1. Bovine-derived strains lacked PI-1 and possessed a unique PI-2b backbone protein allele. Neonatal strains more frequently had PI-1 and a PI-2 variant than maternal colonizing strains, and most CC-17 strains had PI-1 and PI-2b with a distinct backbone protein allele. Furthermore, we present evidence for the frequent gain and loss of genes encoding certain pilus types.
These data suggest that pilus combinations impact host specificity and disease presentation and that diversification often involves the loss or acquisition of PIs. Such findings have implications for the development of GBS vaccines that target the three pilus islands.
PMCID: PMC4074840  PMID: 24943359
Streptococcus agalactiae; Pilus; MLST; Molecular epidemiology
18.  Development of a Tiered Multilocus Sequence Typing Scheme for Members of the Lactobacillus acidophilus Complex 
Applied and Environmental Microbiology  2013;79(23):7220-7228.
Members of the Lactobacillus acidophilus complex are associated with functional foods and dietary supplements because of purported health benefits they impart to the consumer. Many characteristics of these microorganisms are reported to be strain specific. Therefore, proper strain typing is essential for safety assessment and product labeling, and also for monitoring strain integrity for industrial production purposes. Fifty-two strains of the L. acidophilus complex (L. acidophilus, L. amylovorus, L. crispatus, L. gallinarum, L. gasseri, and L. johnsonii) were genotyped using two established methods and compared to a novel multilocus sequence typing (MLST) scheme. PCR restriction fragment length polymorphism (PCR-RFLP) analysis of the hsp60 gene with AluI and TaqI successfully clustered 51 of the 52 strains into the six species examined, but it lacked strain-level discrimination. Random amplified polymorphic DNA PCR (RAPD-PCR) targeting the M13 sequence resulted in highly discriminatory profiles but lacked reproducibility. In this study, an MLST scheme was developed using the conserved housekeeping genes fusA, gpmA, gyrA, gyrB, lepA, pyrG, and recA, which identified 40 sequence types that successfully clustered all of the strains into the six species. Analysis of the observed alleles suggests that nucleotide substitutions within five of the seven MLST loci have reached saturation, a finding that emphasizes the highly diverse nature of the L. acidophilus complex and our unconventional application of a typically intraspecies molecular typing tool. Our MLST results indicate that this method could be useful for characterization and strain discrimination of a multispecies complex, with the potential for taxonomic expansion to a broader collection of Lactobacillus species.
PMCID: PMC3837765  PMID: 24038697
19.  Draft Genome Sequence of the Multidrug-Resistant Escherichia coli Strain LR09, Isolated from a Wastewater Treatment Plant 
Genome Announcements  2014;2(2):e00272-14.
We report the draft genome sequence of Escherichia coli O1:H6 strain LR09, which was isolated from a wastewater treatment plant and displays high resistance to five fluoroquinolone antimicrobials. The assembled data determine that the strain clusters with E. coli phylogroup F and harbors a plasmid conferring resistance to a broad spectrum of antibiotics.
PMCID: PMC3974944  PMID: 24699962
20.  Shiga toxin 2 overexpression in Escherichia coli O157:H7 strains associated with severe human disease 
Microbial pathogenesis  2011;51(6):466-470.
Variation in disease severity among E. coli O157:H7 infections may result from differential expression of Shiga toxin 2 (Stx2). Eleven strains belonging to four prominent phylogenetic clades, including clade 8 strains representative of the 2006 U.S. spinach outbreak, were examined for stx2 expression by real-time PCR and western blot analysis. Clade 8 strains were shown to overexpress stx2 basally, and following induction with ciprofloxacin when compared to strains from clades 1-3. Differences in stx2 expression generally correlated with Stx2 protein levels. Single-nucleotide polymorphisms identified in regions upstream of stx2AB in clade 8 strains were largely absent in non-clade 8 strains. This study concludes that stx2 overexpression is common to strains from clade 8 associated with hemolytic uremic syndrome, and describes SNPs which may affect stx2 expression and which could be useful in the genetic differentiation of highly-virulent strains.
PMCID: PMC3205445  PMID: 21864671
Shiga toxin; E. coli O157:H7; Clade 8; Stx2; hemolytic uremic syndrome
21.  Rapid Genomic-Scale Analysis of Escherichia coli O104:H4 by Using High-Resolution Alternative Methods to Next-Generation Sequencing 
Two technologies, involving DNA microarray and optical mapping, were used to quickly assess gene content and genomic architecture of recent emergent Escherichia coli O104:H4 and related strains. In real-time outbreak investigations, these technologies can provide congruent perspectives on strain, serotype, and pathotype relationships. Our data demonstrated clear discrimination between clinically, temporally, and geographically distinct O104:H4 isolates and rapid characterization of strain differences.
PMCID: PMC3294476  PMID: 22210216
22.  Biomarkers of folate status in NHANES: a roundtable summary123456 
A roundtable to discuss the measurement of folate status biomarkers in NHANES took place in July 2010. NHANES has measured serum folate since 1974 and red blood cell (RBC) folate since 1978 with the use of several different measurement procedures. Data on serum 5-methyltetrahydrofolate (5MTHF) and folic acid (FA) concentrations in persons aged ≥60 y are available in NHANES 1999–2002. The roundtable reviewed data that showed that folate concentrations from the Bio-Rad Quantaphase II procedure (Bio-Rad Laboratories, Hercules, CA; used in NHANES 1991–1994 and NHANES 1999–2006) were, on average, 29% lower for serum and 45% lower for RBC than were those from the microbiological assay (MA), which was used in NHANES 2007–2010. Roundtable experts agreed that these differences required a data adjustment for time-trend analyses. The roundtable reviewed the possible use of an isotope-dilution liquid chromatography–tandem mass spectrometry (LC-MS/MS) measurement procedure for future NHANES and agreed that the close agreement between the MA and LC-MS/MS results for serum folate supported conversion to the LC-MS/MS procedure. However, for RBC folate, the MA gave 25% higher concentrations than did the LC-MS/MS procedure. The roundtable agreed that the use of the LC-MS/MS procedure to measure RBC folate is premature at this time. The roundtable reviewed the reference materials available or under development at the National Institute of Standards and Technology and recognized the challenges related to, and the scientific need for, these materials. They noted the need for a commutability study for the available reference materials for serum 5MTHF and FA.
PMCID: PMC3127517  PMID: 21593502
23.  Biomarkers of vitamin B-12 status in NHANES: a roundtable summary123456 
A roundtable to discuss the measurement of vitamin B-12 (cobalamin) status biomarkers in NHANES took place in July 2010. NHANES stopped measuring vitamin B-12–related biomarkers after 2006. The roundtable reviewed 3 biomarkers of vitamin B-12 status used in past NHANES—serum vitamin B-12, methylmalonic acid (MMA), and total homocysteine (tHcy)—and discussed the potential utility of measuring holotranscobalamin (holoTC) for future NHANES. The roundtable focused on public health considerations and the quality of the measurement procedures and reference methods and materials that past NHANES used or that are available for future NHANES. Roundtable members supported reinstating vitamin B-12 status measures in NHANES. They noted evolving concerns and uncertainties regarding whether subclinical (mild, asymptomatic) vitamin B-12 deficiency is a public health concern. They identified the need for evidence from clinical trials to address causal relations between subclinical vitamin B-12 deficiency and adverse health outcomes as well as appropriate cutoffs for interpreting vitamin B-12–related biomarkers. They agreed that problems with sensitivity and specificity of individual biomarkers underscore the need for including at least one biomarker of circulating vitamin B-12 (serum vitamin B-12 or holoTC) and one functional biomarker (MMA or tHcy) in NHANES. The inclusion of both serum vitamin B-12 and plasma MMA, which have been associated with cognitive dysfunction and anemia in NHANES and in other population-based studies, was preferable to provide continuity with past NHANES. Reliable measurement procedures are available, and National Institute of Standards and Technology reference materials are available or in development for serum vitamin B-12 and MMA.
PMCID: PMC3127527  PMID: 21593512
24.  Ancestral Lineages of Human Enterotoxigenic Escherichia coli▿ †  
Journal of Clinical Microbiology  2010;48(8):2916-2924.
Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhea among children living in and among travelers visiting developing countries. Human ETEC strains represent an epidemiologically and phenotypically diverse group of pathogens, and there is a need to identify natural groupings of these organisms that may help to explain this diversity. Here, we sought to identify most of the important human ETEC lineages that exist in the E. coli population, because strains that originate from the same lineage may also have inherited many of the same epidemiological and phenotypic traits. We performed multilocus sequence typing (MLST) on 1,019 ETEC isolates obtained from humans in different countries and analyzed the data against a backdrop of MLST data from 1,250 non-ETEC E. coli and eight ETEC isolates from pigs. A total of 42 different lineages were identified, 15 of which, representing 792 (78%) of the strains, were estimated to have emerged >900 years ago. Twenty of the lineages were represented in more than one country. There was evidence of extensive exchange of enterotoxin and colonization factor genes between different lineages. Human and porcine ETEC have probably emerged from the same ancestral ETEC lineage on at least three occasions. Our findings suggest that most ETEC strains circulating in the human population today originate from well-established, globally widespread ETEC lineages. Some of the more important lineages identified here may represent a smaller and more manageable target for the ongoing efforts to develop effective ETEC vaccines.
PMCID: PMC2916599  PMID: 20534806
25.  Single Nucleotide Polymorphism Analysis of the Major Tripartite Multidrug Efflux Pump of Escherichia coli: Functional Conservation in Disparate Animal Reservoirs despite Exposure to Antimicrobial Chemotherapy ▿ †  
AcrAB-TolC imparts a strong intrinsic resistance phenotype to many clinically significant molecules in Escherichia coli. This complex is composed of a pump, AcrB, and a periplasmic protein, AcrA, that exports substrates through a common outer membrane porin, TolC. A sequence survey of the pump-specific components, acrA and acrB, was conducted on three discrete animal reservoirs: rodents, bovines, and catfish. Although two of the reservoirs (bovine and catfish) were agrarian, and antibiotic use (ceftiofur and oxytetracycline/Romet 30, respectively) was reported for them, the vast majority of structural polymorphisms were silent except for T104A (AcrA) and Q733R (AcrB), found in certain bovine-derived strains. Overall, the genes were well conserved, with high ratios of synonymous to nonsynonymous substitutions (dS/dN ratios), consistent with or, in the case of acrB, better than those of standard multilocus sequence typing (MLST) loci. Furthermore, predicted recombination points from single nucleotide polymorphism (SNP) patterns in acrB support a modular evolution of transporter proteins, consistent with an ancient origin. However, functional studies with clones representing the major silent SNPs and the nonsilent mutation in acrB failed to generate significant differences in resistance to a range of common efflux pump substrates. Interestingly, a comparison between log-phase acrA and acrB expression profiles yielded inconsistent trends, with acrB expression increasing modestly (<1.8-fold) in many strains from the antibiotic-enriched pools. Our results suggest that structural polymorphisms in this major efflux pump system may not contribute significantly to adaptive resistance by altering function or substrate specificity but may have a potential use in improving phylogenetic relationships and/or source tracking.
PMCID: PMC2826003  PMID: 20038628

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