Nurmohammad Shaikh

Escherichia coli O157:H7 Shiga Toxin-Encoding Bacteriophages: Integrations, Excisions, Truncations, and Evolutionary Implications

As it descended from Escherichia coli O55:H7, Shiga toxin (Stx)-producing E. coli (STEC) O157:H7 is believed to have acquired, in sequence, a bacteriophage encoding Stx2 and another encoding Stx1. Between these events, sorbitol-fermenting E. coli O157:H(-) presumably diverged from this clade. We employed PCR and sequence analyses to investigate sites of bacteriophage integration into the chromosome, using evolutionarily informative STEC to trace the sequence of acquisition of elements encoding Stx. Contrary to expectations from the two currently sequenced strains, truncated bacteriophages occupy yehV in almost all E. coli O157:H7 strains that lack stx(1) (stx(1)-negative strains). Two truncated variants were determined to contain either GTT or TGACTGTT sequence, in lieu of 20,214 or 18,895 bp, respectively, of the bacteriophage central region. A single-nucleotide polymorphism in the latter variant suggests that recombination in that element extended beyond the inserted octamer. An stx(2) bacteriophage usually occupies wrbA in stx(1)(+)/stx(2)(+) E. coli O157:H7, but wrbA is unexpectedly unoccupied in most stx(1)-negative/stx(2)(+) E. coli O157:H7 strains, the presumed progenitors of stx(1)(+)/stx(2)(+) E. coli O157:H7. Trimethoprim-sulfamethoxazole promotes the excision of all, and ciprofloxacin and fosfomycin significantly promote the excision of a subset of complete and truncated stx bacteriophages from the E. coli O157:H7 strains tested; bile salts usually attenuate excision. These data demonstrate the unexpected diversity of the chromosomal architecture of E. coli O157:H7 (with novel truncated bacteriophages and multiple stx(2) bacteriophage insertion sites), suggest that stx(1) acquisition might be a multistep process, and compel the consideration of multiple exogenous factors, including antibiotics and bile, when chromosome stability is examined.

Patterned progression of bacterial populations in the premature infant gut

Significance It is increasingly apparent that bacteria in the gut are important determinants of health and disease in humans. However, we know remarkably little about how this organ transitions from a sterile/near-sterile state at birth to one that soon harbors a highly diverse biomass. We show in premature infants a patterned progression of the gut bacterial community that is only minimally influenced by mode of delivery, antibiotics, or feeds. The pace of this progression is most strongly influenced by gestational age, with the microbial population assembling slowest for infants born most prematurely. These data raise the possibility that host biology, more than exogenous factors such as antibiotics, feeds, and route of delivery, drives bacterial populations in the premature newborn infant gut. In the weeks after birth, the gut acquires a nascent microbiome, and starts its transition to bacterial population equilibrium. This early-in-life microbial population quite likely influences later-in-life host biology. However, we know little about the governance of community development: does the gut serve as a passive incubator where the first organisms randomly encountered gain entry and predominate, or is there an orderly progression of members joining the community of bacteria? We used fine interval enumeration of microbes in stools from multiple subjects to answer this question. We demonstrate via 16S rRNA gene pyrosequencing of 922 specimens from 58 subjects that the gut microbiota of premature infants residing in a tightly controlled microbial environment progresses through a choreographed succession of bacterial classes from Bacilli to Gammaproteobacteria to Clostridia, interrupted by abrupt population changes. As infants approach 33–36 wk postconceptional age (corresponding to the third to the twelfth weeks of life depending on gestational age at birth), the gut is well colonized by anaerobes. Antibiotics, vaginal vs. Caesarian birth, diet, and age of the infants when sampled influence the pace, but not the sequence, of progression. Our results suggest that in infants in a microbiologically constrained ecosphere of a neonatal intensive care unit, gut bacterial communities have an overall nonrandom assembly that is punctuated by microbial population abruptions. The possibility that the pace of this assembly depends more on host biology (chiefly gestational age at birth) than identifiable exogenous factors warrants further consideration.

Development of an enhanced human gastrointestinal epithelial culture system to facilitate patient-based assays

Objective The technology for the growth of human intestinal epithelial cells is rapidly progressing. An exciting possibility is that this system could serve as a platform for individualised medicine and research. However, to achieve this goal, human epithelial culture must be enhanced so that biopsies from individuals can be used to reproducibly generate cell lines in a short time frame so that multiple, functional assays can be performed (ie, barrier function and host–microbial interactions). Design We created a large panel of human gastrointestinal epithelial cell lines (n=65) from patient biopsies taken during routine upper and lower endoscopy procedures. Proliferative stem/progenitor cells were rapidly expanded using a high concentration of conditioned media containing the factors critical for growth (Wnt3a, R-spondin and Noggin). A combination of lower conditioned media concentration and Notch inhibition was used to differentiate these cells for additional assays. Results We obtained epithelial lines from all accessible tissue sites within 2 weeks of culture. The intestinal cell lines were enriched for stem cell markers and rapidly grew as spheroids that required passage at 1:3–1:4 every 3 days. Under differentiation conditions, intestinal epithelial spheroids showed region-specific development of mature epithelial lineages. These cells formed functional, polarised monolayers covered by a secreted mucus layer when grown on Transwell membranes. Using two-dimensional culture, these cells also demonstrated novel adherence phenotypes with various strains of pathogenic Escherichia coli. Conclusions This culture system will facilitate the study of interindividual, functional studies of human intestinal epithelial cells, including host–microbial interactions.

A precise reconstruction of the emergence and constrained radiations of Escherichia coli O157 portrayed by backbone concatenomic analysis

Single nucleotide polymorphisms (SNPs) in stable genome regions provide durable measurements of species evolution. We systematically identified each SNP in concatenations of all backbone ORFs in 7 newly or previously sequenced evolutionarily instructive pathogenic Escherichia coli O157:H7, O157:H−, and O55:H7. The 1,113 synonymous SNPs demonstrate emergence of the largest cluster of this pathogen only in the last millennium. Unexpectedly, shared SNPs within circumscribed clusters of organisms suggest severely restricted survival and limited effective population sizes of pathogenic O157:H7, tenuous survival of these organisms in nature, source-sink evolutionary dynamics, or, possibly, a limited number of mutations that confer selective advantage. A single large segment spanning the rfb-gnd gene cluster is the only backbone region convincingly acquired by recombination as O157 emerged from O55. This concatenomic analysis also supports using SNPs to differentiate closely related pathogens for infection control and forensic purposes. However, constrained radiations raise the possibility of making false associations between isolates.

The IrgA Homologue Adhesin Iha Is an Escherichia coli Virulence Factor in Murine Urinary Tract Infection

ABSTRACT The role of the Escherichia coli iron-regulated gene homologue adhesin (Iha) in the pathogenesis of urinary tract infections (UTIs) is unknown. We performed a series of complementary analyses to confirm or refute the hypothesis that Iha is a virulence factor in uropathogenic E. coli. Fecal E. coli isolates exhibited significantly lower prevalences of iha (range, 14 to 22%) than did clinical isolates from cases of pediatric cystitis or pyelonephritis, adult pyelonephritis or urosepsis, or bacteremia (range, 38 to 74%). Recombinant Iha from E. coli pyelonephritis isolate CFT073 conferred upon nonadherent E. coli ORN172 the ability to adhere to cultured T-24 human uroepithelial cells. In a well-established mouse model of ascending UTI, CFT073 and its derivative UPEC76 (a pap [P fimbriae] mutant version of strain CFT073) each significantly outcompeted their respective iha deletion mutants in CBA/J mice 48 h after bladder challenge (P < 0.03 for urine, both kidneys, and bladders of both constructs, except for bladders of mice challenged with UPEC76 and its deletion mutant, where P = 0.11). These data suggest that IhaCFT073 is a virulence factor and might be a target for anti-UTI interventions.

Diarrhea Etiology in a Pediatric Emergency Department: A Case Control Study

BACKGROUND The etiology of childhood diarrhea is frequently unknown. METHODS We sought Aeromonas, Campylobacter, Escherichia coli O157:H7, Pleisiomonas shigelloides, Salmonella, Shigella, Vibrio, and Yersinia (by culture), adenoviruses, astroviruses, noroviruses, rotavirus, and Shiga toxin-producing E. coli (STEC; by enzyme immunoassay), Clostridium difficile (by cytotoxicity), parasites (by microscopy), and enteroaggregative E. coli (EAEC; by polymerase chain reaction [PCR] analysis) in the stools of 254 children with diarrhea presenting to a pediatric emergency facility. Age- and geographic-matched community controls without diarrhea (n = 452) were similarly studied, except bacterial cultures of the stool were limited only to cases. RESULTS Twenty-nine (11.4%) case stools contained 13 Salmonella, 10 STEC (6 O157:H7 and 4 non-O157:H7 serotypes), 5 Campylobacter, and 2 Shigella. PCR-defined EAEC were present more often in case (3.2%) specimens than in control (0.9%) specimens (adjusted odds ratio [OR], 3.9; 95% confidence interval [CI], 1.1-13.7), and their adherence phenotypes were variable. Rotavirus, astrovirus, and adenovirus were more common among cases than controls, but both groups contained noroviruses and C. difficile at similar rates. PCR evidence of hypervirulent C. difficile was found in case and control stools; parasites were much more common in control specimens. CONCLUSIONS EAEC are associated with childhood diarrhea in Seattle, but the optimal way to identify these agents warrants determination. Children without diarrhea harbor diarrheagenic pathogens, including hypervirulent C. difficile. Our data support the importance of taking into account host susceptibility, microbial density, and organism virulence traits in future case-control studies, not merely categorizing candidate pathogens as being present or absent.

Sepsis From the Gut: The Enteric Habitat of Bacteria That Cause Late-Onset Neonatal Bloodstream Infections

BACKGROUND Late-onset sepsis is a major problem in neonatology, but the habitat of the pathogens before bloodstream invasion occurs is not well established. METHODS We examined prospectively collected stools from premature infants with sepsis to find pathogens that subsequently invaded their bloodstreams, and sought the same organisms in stools of infants without sepsis. Culture-based techniques were used to isolate stool bacteria that provisionally matched the bloodstream organisms, which were then genome sequenced to confirm or refute commonality. RESULTS Of 11 children with late-onset neonatal bloodstream infections, 7 produced at least 1 stool that contained group B Streptococcus (GBS), Serratia marcescens, or Escherichia coli before their sepsis episode with provisionally matching organisms. Of 96 overlap comparison subjects without sepsis temporally associated with these cases, 4 were colonized with provisionally matching GBS or S. marcescens. Of 175 comparisons of stools from randomly selected infants without sepsis, 1 contained a GBS (this infant had also served as an overlap comparison subject and both specimens contained provisionally matching GBS). Genome sequencing confirmed common origin of provisionally matching fecal and blood isolates. The invasive E. coli were present in all presepticemic stools since birth, but gut colonization with GBS and S. marcescens occurred closer to time of bloodstream infection. CONCLUSIONS The neonatal gut harbors sepsis-causing pathogens, but such organisms are not inevitable members of the normal microbiota. Surveillance microbiology, decolonization, and augmented hygiene might prevent dissemination of invasive bacteria between and within premature infants.

Precise Dissection of an Escherichia coli O157:H7 outbreak by Single Nucleotide Polymorphism Analysis

ABSTRACT The current pathogen-typing methods have suboptimal sensitivities and specificities. DNA sequencing offers an opportunity to type pathogens with greater degrees of discrimination using single nucleotide polymorphisms (SNPs) than with pulsed-field gel electrophoresis (PFGE) and other methodologies. In a recent cluster of Escherichia coli O157:H7 infections attributed to salad bar exposures and romaine lettuce, a subset of cases denied exposure to either source, although PFGE and multiple-locus variable-number tandem-repeat analysis (MLVA) suggested that all isolates had the same recent progenitor. Interrogation of a preselected set of 3,442,673 nucleotides in backbone open reading frames (ORFs) identified only 1 or 2 single nucleotide differences in 3 of 12 isolates from the cases who denied exposure. The backbone DNAs of 9 of 9 and 3 of 3 cases who reported or were unsure about exposure, respectively, were isogenic. Backbone ORF SNP set sequencing offers pathogen differentiation capabilities that exceed those of PFGE and MLVA.

Environmental Enteric Dysfunction Includes a Broad Spectrum of Inflammatory Responses and Epithelial Repair Processes

Background & Aims Environmental enteric dysfunction (EED), a chronic diffuse inflammation of the small intestine, is associated with stunting in children in the developing world. The pathobiology of EED is poorly understood because of the lack of a method to elucidate the host response. This study tested a novel microarray method to overcome limitation of RNA sequencing to interrogate the host transcriptome in feces in Malawian children with EED. Methods In 259 children, EED was measured by lactulose permeability (%L). After isolating low copy numbers of host messenger RNA, the transcriptome was reliably and reproducibly profiled, validated by polymerase chain reaction. Messenger RNA copy number then was correlated with %L and differential expression in EED. The transcripts identified were mapped to biological pathways and processes. The children studied had a range of %L values, consistent with a spectrum of EED from none to severe. Results We identified 12 transcripts associated with the severity of EED, including chemokines that stimulate T-cell proliferation, Fc fragments of multiple immunoglobulin families, interferon-induced proteins, activators of neutrophils and B cells, and mediators that dampen cellular responses to hormones. EED-associated transcripts mapped to pathways related to cell adhesion, and responses to a broad spectrum of viral, bacterial, and parasitic microbes. Several mucins, regulatory factors, and protein kinases associated with the maintenance of the mucous layer were expressed less in children with EED than in normal children. Conclusions EED represents the activation of diverse elements of the immune system and is associated with widespread intestinal barrier disruption. Differentially expressed transcripts, appropriately enumerated, should be explored as potential biomarkers.

New insights into environmental enteric dysfunction

Environmental enteric dysfunction (EED) has been recognised as an important contributing factor to physical and cognitive stunting, poor response to oral vaccines, limited resilience to acute infections and ultimately global childhood mortality. The aetiology of EED remains poorly defined but the epidemiology suggests a multifactorial combination of prenatal and early-life undernutrition and repeated infectious and/or toxic environmental insults due to unsanitary and unhygienic environments. Previous attempts at medical interventions to ameliorate EED have been unsatisfying. However, a new generation of imaging and ‘-omics’ technologies hold promise for developing a new understanding of the pathophysiology of EED. A series of trials designed to decrease EED and stunting are taking novel approaches, including improvements in sanitation, hygiene and nutritional interventions. Although many challenges remain in defeating EED, the global child health community must redouble their efforts to reduce EED in order to make substantive improvements in morbidity and mortality worldwide.