[Show abstract][Hide abstract] ABSTRACT: Group B Streptococcus (GBS), a common bowel commensal, is a major cause of neonatal sepsis and an emerging cause of infection in immune-compromised adult populations. Because of increasing resistance to macrolides, fluoroquinolones are used to treat GBS infections in those allergic to beta-lactams, but GBS are increasingly resistant to fluoroquinolones. Fluoroquinolone resistance has been previously attributed to quinolone resistance determining regions (QRDRs) mutations. We demonstrate that some of fluoroquinolone resistance is due to efflux-mediated resistance. We tested 20 GBS strains resistant only to norfloxacin with no mutations in the QRDRs, for the efflux phenotype using norfloxacin and ethidium bromide as substrates in the presence of the efflux inhibitor reserpine. Also tested were 68 GBS strains resistant only to norfloxacin not screened for QRDRs, and 58 GBS strains resistant to ciprofloxacin, levofloxacin or moxifloxacin. Isolates were randomly selected from 221 pregnant women (35 - 37 weeks of gestation) asymptomatically carrying GBS, and 838 patients with GBS infection identified in South Korea between 2006 and 2008. The VITEK II automatic system (Biomerieux) was used to determine fluoroquinolone resistance. The reserpine associated efflux phenotype was found in more than half of GBS strains resistant only to norfloxacin with no QRDR mutations, and half where QRDR mutations were unknown. No evidence of the efflux phenotype was detected in GBS strains that were resistant to moxifloxacin or levofloxacin or both. The reserpine sensitive efflux phenotype resulted in moderate increases in norfloxacin MIC (average = 3.6 fold, range = >1-16 fold).
[Show abstract][Hide abstract] ABSTRACT: Nontypeable Haemophilus influenzae (NTHi) are Gram-negative coccobacilli that colonize the human pharynx, their only known natural reservoir. Adherence to the host epithelium facilitates NTHi colonization and marks one of the first steps in NTHi pathogenesis. Epithelial cell attachment is mediated, in part, by a pair of high molecular weight (HMW) adhesins that are highly immunogenic, antigenically diverse, and display a wide range of amino acid diversity both within and between isolates. In this study, the prevalence of hmwA, which encodes the HMW adhesin, was determined for a collection of 170 NTHi isolates recovered from the middle ears of children with otitis media (OM isolates) or throats of healthy children (commensal isolates) from Finland, Israel, and the U.S. Overall, hmwA was detected in 61% of NTHi isolates and was significantly more prevalent (P = 0.004) among OM isolates than among commensal isolates; the prevalence ratio comparing hmwA prevalence among ear isolates with that of commensal isolates was 1.47 (95% CI (1.12, 1.92)). Ninety-five percent (98/103) of the hmwA-positive NTHi isolates possessed two hmw loci. To advance our understanding of hmwA binding sequence diversity, we determined the DNA sequence of the hmwA binding region of 33 isolates from this collection. The average amino acid identity across all hmwA sequences was 62%. Phylogenetic analyses of the hmwA binding revealed four distinct sequence clusters, and the majority of hmwA sequences (83%) belonged to one of two dominant sequence clusters. hmwA sequences did not cluster by chromosomal location, geographic region, or disease status.
Infection Genetics and Evolution 10/2014; · 3.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lipooligosaccharide configurations were predicted in non-typeable Haemophilus influenzae isolates based on the presence of seven oligosaccharide-extension initiating genes (or alleles). Predicted configurations with 2-3 oligosaccharide extensions were more prevalent among middle ear than throat strains. In addition, strains with these configurations averaged higher levels of serum resistance than strains with other configurations.
Journal of clinical microbiology 04/2014; · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As a proof of principle, we used an untargeted global metabolic profiling of saliva to understand the biochemical processes associated with dental decay, dentition (primary and secondary tooth eruption) and familiality in a sample of 25 sibling pairs. Pairs were selected to represent four different combinations of dentition and tooth health: 1) both siblings with primary teeth and no decay (n=5); 2) both siblings with primary teeth and discordant for dental decay (n=6); 3) both siblings with primary teeth and dental decay (n=4); and 4) one sibling with primary teeth the other with mixed dentition and both with no dental decay (n=10). There was a strong effect of sibship on the metabolite profiles identified; this may reflect the effects of common genes, environment and behaviors, and shared oral microbial communities. Nested in the familial effects were associations of metabolite profile with dentition and with dental decay. Using three different analyses (Euclidean distance, hierarchical clustering and PCA using selected biochemicals) metabolite profiles of saliva from children with decayed teeth were more similar than the metabolite profiles of saliva from children with healthy (sound) teeth. Larger studies that include host behaviors, environmental factors, oral microbiota composition and structure, and host genetic predisposition are required to identify biomarkers for decay, and to estimate the relative contribution of host factors and oral microbes on risk of dental decay.
Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 09/2013; · 3.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are short fragments of DNA that act as an adaptive immune system protecting bacteria against invasion by phages, plasmids or other forms of foreign DNA. Bacteria without a CRISPR locus may more readily adapt to environmental changes by acquiring foreign genetic material. Uropathogenic Escherichia coli (UPEC) live in a number of environments suggesting an ability to rapidly adapt to new environments. If UPEC are more adaptive than commensal E. coli we would expect that UPEC would have fewer CRISPR loci, and -- if loci are present -- that they would harbor fewer spacers than CRISPR loci in fecal E. coli. We tested this in vivo by comparing the number of CRISPR loci and spacers, and sensitivity to antibiotics (resistance is often obtained via plasmids) among 81 pairs of UPEC and fecal E. coli isolated from women with urinary tract infection. Each pair included one uropathogen and one commensal (fecal) sample from the same female patient. Fecal isolates had more repeats (p = 0.009) and more unique spacers (p < 0.0001) at four CRISPR loci than uropathogens. By contrast, uropathogens were more likely than fecal E. coli to be resistant to ampicillin, cefazolin and trimethoprim/sulfamethoxazole. However, no consistent association between CRISPRs and antibiotic resistance was identified. To our knowledge, this is the first study to compare fecal E. coli and pathogenic E. coli from the same individuals, and to test the association of CRISPR loci with antibiotic resistance. Our results suggest that the absence of CRISPR loci may make UPEC more susceptible to infection by phages or plasmids and allow them to adapt more quickly to various environments.
Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 07/2013; · 3.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Haemophilus influenzae colonizes the human nasopharynx as a commensal, and is etiologically associated with numerous opportunistic infections of the airway; it is also less commonly associated with invasive disease. Clinical isolates of H. influenzae display extensive genomic diversity and plasticity. The development of strategies to successfully prevent, diagnose and treat H. influenzae infections depends on tools to ascertain the gene content of individual isolates.
We describe and validate a Haemophilus influenzae supragenome hybridization (SGH) array that can be used to characterize the full genic complement of any strain within the species, as well as strains from several highly related species. The array contains 31,307 probes that collectively cover essentially all alleles of the 2890 gene clusters identified from the whole genome sequencing of 24 clinical H. influenzae strains. The finite supragenome model predicts that these data include greater than 85% of all non-rare genes (where rare genes are defined as those present in less than 10% of sequenced strains). The veracity of the array was tested by comparing the whole genome sequences of eight strains with their hybridization data obtained using the supragenome array. The array predictions were correct and reproducible for ~ 98% of the gene content of all of the sequenced strains. This technology was then applied to an investigation of the gene content of 193 geographically and clinically diverse H. influenzae clinical strains. These strains came from multiple locations from five different continents and Papua New Guinea and include isolates from: the middle ears of persons with otitis media and otorrhea; lung aspirates and sputum samples from pneumonia and COPD patients, blood specimens from patients with sepsis; cerebrospinal fluid from patients with meningitis, as well as from pharyngeal specimens from healthy persons.
These analyses provided the most comprehensive and detailed genomic/phylogenetic look at this species to date, and identified a subset of highly divergent strains that form a separate lineage within the species. This array provides a cost-effective and high-throughput tool to determine the gene content of any H. influenzae isolate or lineage. Furthermore, the method for probe selection can be applied to any species, given a group of available whole genome sequences.
[Show abstract][Hide abstract] ABSTRACT: BACKGROUND: Escherichia coli is a common cause of asymptomatic and symptomatic bacteriuria in hospitalized patients. Asymptomatic bacteriuria (ASB) is frequently treated with antibiotics without a clear indication. Our goal was to determine patient and pathogen factors suggestive of ASB. METHODS: We conducted a 12-month prospective cohort study of adult inpatients with E. coli bacteriuria seen at a tertiary care hospital in St. Louis, Missouri, USA. Urine cultures were taken at the discretion of treating physicians. Bacterial isolates were tested for 14 putative virulence genes using high-throughput dot-blot hybridization. RESULTS: The median age of the 287 study patients was 65 (19--101) years; 78% were female. Seventy percent had community-acquired bacteriuria. One-hundred ten (38.3%) patients had ASB and 177 (61.7%) had symptomatic urinary tract infection (sUTI). Asymptomatic patients were more likely than symptomatic patients to have congestive heart failure (p = 0.03), a history of myocardial infarction (p = 0.01), chronic pulmonary disease (p = 0.045), peripheral vascular disease (p = 0.04), and dementia (p = 0.03). Patients with sUTI were more likely to be neutropenic at the time of bacteriuria (p = 0.046). Chronic pulmonary disease [OR 2.1 (95% CI 1.04, 4.1)] and dementia [OR 2.4 (95% CI 1.02, 5.8)] were independent predictors for asymptomatic bacteriuria. Absence of pyuria was not predictive of ASB. None of the individual virulence genes tested were associated with ASB nor was the total number of genes. CONCLUSIONS: Asymptomatic E. coli bacteriuria in hospitalized patients was frequent and more common in patients with dementia and chronic pulmonary disease. Bacterial virulence factors could not discriminate symptomatic from asymptomatic bacteriurias. Asymptomatic E. coli bacteriuria cannot be predicted by virulence screening.
[Show abstract][Hide abstract] ABSTRACT: The ure operon was significantly more prevalent in Haemophilus influenzae isolates causing otitis media and COPD associated bronchitis than those from throats of healthy individuals (97% vs. 78.1%, P < 0.001). Strains lacking the ure operon are over 8 times more likely to be throat than either otitis media or COPD isolates.
Journal of clinical microbiology 12/2012; · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Haemophilus influenzae (Hi) colonizes the human respiratory tract and is an important pathogen associated with chronic obstructive pulmonary disease (COPD). Bacterial factors that interact with the human host may be important in the pathogenesis of COPD. These factors, however, have not been well defined. The overall goal of this study was to identify bacterial genetic elements with increased prevalence among H. influenzae strains isolated from patients with COPD compared to those isolated from the pharynges of healthy individuals.
Four nontypeable H. influenzae (NTHi) strains, two isolated from the airways of patients with COPD and two from a healthy individual, were subjected to whole genome sequencing using 454 FLX Titanium technology. COPD strain-specific genetic islands greater than 500 bp in size were identified by in silico subtraction. Open reading frames residing within these islands include known Hi virulence genes such as lic2b, hgbA, iga, hmw1 and hmw2, as well as genes encoding urease and other enzymes involving metabolic pathways. The distributions of seven selected genetic islands were assessed among a panel of 421 NTHi strains of both disease and commensal origins using a Library-on-a-Slide high throughput dot blot DNA hybridization procedure. Four of the seven islands screened, containing genes that encode a methyltransferase, a dehydrogenase, a urease synthesis enzyme, and a set of unknown short ORFs, respectively, were more prevalent in COPD strains than in colonizing strains with prevalence ratios ranging from 1.21 to 2.85 (p≤0.0002). Surprisingly, none of these sequences show increased prevalence among NTHi isolated from the airways of patients with cystic fibrosis.
Our data suggest that specific bacterial genes, many involved in metabolic functions, are associated with the ability of NTHi strains to survive in the lower airways of patients with COPD.
PLoS ONE 09/2012; 7(9):e44730. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The urinary tract is the most common source for Escherichia coli bacteremia. Mortality from E. coli urinary-source bacteremia is higher than that from urinary tract infection. Predisposing factors for urinary-source E. coli bacteremia are poorly characterized.
In order to identify urinary-source bacteremia risk factors, we conducted a 12-month prospective cohort study of adult inpatients with E. coli bacteriuria that were tested for bacteremia within ±1 day of the bacteriuria. Patients with bacteremia were compared with those without bacteremia. Bacterial isolates from urine were screened for 16 putative virulence genes using high-throughput dot-blot hybridization.
Twenty-four of 156 subjects (15%) had E. coli bacteremia. Bacteremic patients were more likely to have benign prostatic hyperplasia (56% vs 19%; P = .04), a history of urogenital surgery (63% vs 28%; P = .001), and presentation with hesitancy/retention (21% vs 4%; P = .002), fever (63% vs 38%; P = .02), and pyelonephritis (67% vs 41%; P = .02). The genes kpsMT (group II capsule) (17 [71%] vs 62 [47%]; P = .03) and prf (P-fimbriae family) (13 [54%] vs 40 [30%]; P = .02) were more frequent in the urinary strains from bacteremic patients. Symptoms of hesitancy/retention (odds ratio [OR], 7.8; 95% confidence interval [CI], 1.6-37), history of a urogenital procedure (OR, 5.4; 95% CI, 2-14.7), and presence of kpsMT (OR, 2.9; 95% CI, 1-8.2) independently predicted bacteremia.
Bacteremia secondary to E. coli bacteriuria was frequent (15%) in those tested for it. Urinary stasis, surgical disruption of urogenital tissues, and a bacterial capsule characteristic contribute to systemic invasion by uropathogenic E. coli.
[Show abstract][Hide abstract] ABSTRACT: Background: Nontypeable Haemophilus influenzae (NTHi) are small gram negative bacteria that colonize the human nasopharynx, and can also cause respiratory tract infections. Since NTHi has an absolute requirement for iron from its host for aerobic growth, comparing the profile of iron acquisition genes between disease isolates from the middle ears of children with acute otitis media and commensal isolates from the throats of healthy children may elucidate their role in determining virulence.
Methods: Fluorescein-labeled probes for iron acquisition genes hxuC, hemR, and hup were prepared using strain 86-028NP as the template. Microarray slides containing NTHi DNA from middle ear (522) and throat (315) isolates were probed with a mixture of seven multilocus sequence typing gene fragments as a DNA concentration control, followed by probes for the iron acquisition genes. The bacterial DNA samples were also probed with iga and lgtC genes to differentiate NTHi from H. haemolyticus, a closely related commensal of the pharyngeal cavity.
Results: Among 315 specimens from the throat, 32 did not meet the criteria to be categorized as H. influenzae and were excluded from further analysis. Of 522 NTHi specimens from the middle ear, hxuC was present in 89.3%, hemR in 87.7%, and hup in 86.2%. Of 283 NTHi specimens from the throat, the percentages were 76%, 69.6%, and 78.8% for hxuC, hemR, and hup respectively. The prevalence ratio of middle ear specimens compared to throat specimens was 1.18 for hxuC (p < 0.001), 1.26 for hemR (p < 0.001), and 1.09 for hup (p = 0.009).
Conclusion: The higher prevalence of iron acquisition genes in NTHi isolated from infected middle ears suggests that these genes have been preserved by natural selection for survival in the middle ear space.
Infectious Diseases Society of America 2011 Annual Meeting; 10/2011
[Show abstract][Hide abstract] ABSTRACT: Extrapulmonary tuberculosis (EPTB) is an important health problem that may cause serious morbidity and diagnostic challenges. We conducted a case-control study involving 5,684, approximately 99% of bacteriologically confirmed TB patients (including 1,925 EPTB cases) diagnosed in Denmark and Greenland during 1992-2007 to gain insight to the role of host factors in EPTB pathogenesis. Among patients from Somalia and Asia, persons 25-44 and 45-64 years of age were more likely to have EPTB than persons 15-24 years of age. In contrast, among persons from Greenland, the two oldest age groups were significantly less likely to have EPTB than the youngest age group. For all the age groups, the odds for having EPTB was significantly higher among patients from Somalia and Asia and significantly lower among the patients from Greenland than among patients from Denmark. Furthermore, the occurrence of specific types of EPTB significantly varied among different age groups or origins.
The American journal of tropical medicine and hygiene 08/2011; 85(2):285-90. · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mycobacterium tuberculosis may survive for decades in the human body in a state termed latent tuberculosis infection (LTBI). We investigated the occurrence during LTBI of insertion/deletion events in a selected set of mononucleotide simple sequence repeats, DNA sequence changes in four M. tuberculosis genes, and large sequence variations in 4750 M. tuberculosis open reading frames. We studied 13 paired M. tuberculosis clinical isolates, with each pair representing a reactivation of LTBI more than three decades after primary infection. Absence of sequence variations between paired isolates in nearly all investigated loci suggests a low likelihood of bacterial replication during LTBI.
Infection, genetics and evolution: journal of molecular epidemiology and evolutionary genetics in infectious diseases 02/2011; 11(5):1164-7. · 3.22 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: It was reported previously that the major fraction of the recent decrease of tuberculosis incident cases in Arkansas had been due to a decrease in the reactivated infections. Preventing transmission of Mycobacterium tuberculosis is the key to a continued decline in tuberculosis cases. In this study, we integrated epidemiological data analysis and comparative genomics to identify host and microbial factors important to tuberculosis transmission. A significantly higher proportion of cases in large clusters (containing >10 cases) were non-Hispanic black, homeless, less than 65 years old, male sex, smear-positive sputum, excessive use of alcohol, and HIV sero-positive, compared to cases in small clusters (containing 2-5 cases) diagnosed within one year. However, being non-Hispanic black and homeless within the past year were the only two host characteristics that were identified as independent risk factors for being in large clusters. This finding suggests that social behavioral factors have a more important role in transmission of tuberculosis than does the infectiousness of the source. Comparing the genomic content of one of the large cluster strains to that of a non-clustered strain from the same community identified 25 genes that differed between the two strains, potentially contributing to the observed differences in transmission.
[Show abstract][Hide abstract] ABSTRACT: A number of observational studies and a few small or open randomized clinical trials suggest that the American cranberry may decrease incidence of recurring urinary tract infection (UTI).
We conducted a double-blind, placebo-controlled trial of the effects of cranberry on risk of recurring UTI among 319 college women presenting with an acute UTI. Participants were followed up until a second UTI or for 6 months, whichever came first. A UTI was defined on the basis of the combination of symptoms and a urine culture positive for a known uropathogen. The study was designed to detect a 2-fold difference between treated and placebo groups, as was detected in unblinded trials. We assumed 30% of participants would experience a UTI during the follow-up period.
Overall, the recurrence rate was 16.9% (95% confidence interval, 12.8%-21.0%), and the distribution of the recurrences was similar between study groups, with the active cranberry group presenting a slightly higher recurrence rate (20.0% vs 14.0%). The presence of urinary symptoms at 3 days, 1-2 weeks, and at ≥ 1 month was similar between study groups, with overall no marked differences. CONCLUSIONS.: Among otherwise healthy college women with an acute UTI, those drinking 8 oz of 27% cranberry juice twice daily did not experience a decrease in the 6-month incidence of a second UTI, compared with those drinking a placebo.
[Show abstract][Hide abstract] ABSTRACT: Our objective was to characterize 46 unique, erythromycin-sensitive, and clindamycin-resistant Streptococcus agalactiae strains from S. Korea that displayed a novel phenotype in double-disk diffusion assay. We used polymerase chain reaction to determine presence of erythromycin and clindamycin resistance genes, disc diffusion assays to determine resistance phenotype, and microbroth dilution to determine minimal inhibitory concentration. We detected a novel phenotype in the double-disk diffusion assay for inducible resistance among 46 S. agalactiae strains that were both erythromycin sensitive and clindamycin resistant. Thirty-two strains with the novel phenotype tested positive for erm(B) by DNA-DNA hybridization; sequencing of the erm(B) gene revealed mutations in the ribosomal binding site region in the erm(B) open reading frame, which is consistent with a lack of erythromycin resistance phenotype. Although identified from patients at multiple hospitals, genotyping suggested that the strains are closely related. The new phenotype shows increased sensitivity to clindamycin in the presence of erythromycin.
[Show abstract][Hide abstract] ABSTRACT: Five genetic islands (HiGI) found in Haemophilus influenzae type b strain Eagan were used as hybridization probes on type b, Haemophilus haemolyticus, and nontypeable H. influenzae (NTHi) isolates. HiGI2 and HiGI7 were significantly more prevalent in NTHi isolates from children with otitis media than in those from the throats of healthy children.
Journal of clinical microbiology 05/2010; 48(7):2565-8. · 4.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effort to develop a tuberculosis (TB) vaccine more effective than the widely used Bacille Calmette-Guérin (BCG) has led to the development of two novel fusion protein subunit vaccines: Ag85B-ESAT-6 and Ag85B-TB10.4. Studies of these vaccines in animal models have revealed their ability to generate protective immune responses. Yet, previous work on TB fusion subunit vaccine candidate, Mtb72f, has suggested that genetic diversity among M. tuberculosis strains may compromise vaccine efficacy. In this study, we sequenced the esxA, esxH, and fbpB genes of M. tuberculosis encoding ESAT-6, TB10.4, and Ag85B proteins, respectively, in a sample of 88 clinical isolates representing 57 strains from Ark, USA, and 31 strains from Turkey, to assess the genetic diversity of the two vaccine candidates. We found no DNA polymorphism in esxA and esxH genes in the study sample and only one synonymous single nucleotide change (C to A) in fbpB gene among 39 (44.3%) of the 88 strains sequenced. These data suggest that it is unlikely that the efficacy of Ag85B-ESAT-6 and Ag85B-TB10.4 vaccines will be affected by the genetic diversity of M. tuberculosis population. Future studies should include a broader pool of M. tuberculosis strains to validate the current conclusion.
BioMed Research International 01/2010; 2010:208371. · 2.71 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Persistent Escherichia coli asymptomatic bacteriuria (ASB) is common among persons with diabetes mellitus, but the duration of colonization and the rates of recolonization are unknown. We estimated the duration of colonization and the rate of recolonization among successively isolated E. coli from diabetic women with ASB and compared the virulence profiles with uropathogenic and commensal E. coli.
A total of 105 women with diabetes were enrolled in a randomized, controlled clinical trial for treatment of ASB in Manitoba, Canada, and were observed at least every 3 months for up to 3 years. We analyzed 517 isolates from 70 women with repeated E. coli ASB for genetic similarity using enterobacterial repetitive intergenic consensus polymerase chain reaction. Unique strains were screened for uropathogenic virulence characteristics using dot blot hybridization and compared with different collections of E. coli isolates.
On average, differences were found among women assigned to treatment for ASB, those treated only for symptomatic infections, and untreated women in (1) follow-up time with bacteriuria (29%, 31%, and 66%, respectively; P<.001), (2) duration of bacteriuria (2.2, 2.5, and 3.7 months, respectively; P=.04), and (3) carriage of unique isolates (2.4, 2.8, and 4 months, respectively; P=.03). Women assigned to antibiotic treatment usually had recurrent infection (76%), 64% of the time with a genetically new E. coli strain. Virulence characteristics of these isolates were comparable to those of fecal isolates from healthy women.
Treatment may reduce the overall proportion of time infected in the long term and carriage of a unique strain, but most treatment regimens were followed by subsequent recolonization. Infecting strains did not have virulence factors characteristic of uropathogenic E. coli.