In vitro model systems for exploring oral biofilms: From single-species populations to complex multi-species communities.

Numerous in vitro biofilm model systems are available to study oral biofilms. Over the past several decades, increased understanding of oral biology and advances in technology have facilitated more accurate simulation of intraoral conditions and have allowed for the increased generalizability of in vitro oral biofilm studies. The integration of contemporary systems with confocal microscopy and 16S rRNA community profiling has enhanced the capabilities of in vitro biofilm model systems to quantify biofilm architecture and analyse microbial community composition. In this review, we describe several model systems relevant to modern in vitro oral biofilm studies: the constant depth film fermenter, Sorbarod perfusion system, drip-flow reactor, modified Robbins device, flowcells and microfluidic systems. We highlight how combining these systems with confocal microscopy and community composition analysis tools aids exploration of oral biofilm development under different conditions and in response to antimicrobial/anti-biofilm agents. The review closes with a discussion of future directions for the field of in vitro oral biofilm imaging and analysis.

Co-occurrence of yeast, streptococci, dental decay, and gingivitis in the post-partum period: results of a longitudinal study.

Objective: The interactions between yeast and streptococci species that lead to dental decay and gingivitis are poorly understood. Our study describes these associations among a cohort of 101 post-partum women enrolled in the Center for Oral Health Research in Appalachia, 2012-2013. Methods: All eligible women without dental caries were included (n = 21) and the remainder were randomly sampled to represent the total number of decayed, missing, and filled teeth (DMFT) at enrollment. We used amplicon sequencing and qPCR of saliva from 2, 6, 12 and 24 visits to determine microbiome composition. Results: Active decay and generalized gingivitis were strongly predictive of each other. Using adjusted marginal models, Candida albicans and Streptococcus mutans combined were associated with active decay (OR = 3.13; 95% CI 1.26, 7.75). However, C. albicans alone (OR = 2.33; 95% CI: 0.81, 6.75) was associated with generalized gingivitis, but S. mutans alone was not (OR = 0.55; 95% CI: 0.21, 1.44). Models including microbiome community state types (CSTs) showed CSTs positively associated with active decay were negatively associated with generalized gingivitis. Discussion: C. albicans is associated with active decay and generalized gingivitis, but whether one or both are present depends on the structure of the co-existing microbial community.

Introducing BAIT (Biofilm Architecture Inference Tool): a software program to evaluate the architecture of oral multi-species biofilms.

Biofilm model systems are used to study biofilm growth and predict the effects of anti-biofilm interventions within the human oral cavity. Many in vitro biofilm model systems use a confocal laser scanning microscope (CLSM) in conjunction with image analysis tools to study biofilms. The aim of this study was to evaluate an in-house developed image analysis software program that we call BAIT (Biofilm Architecture Inference Tool) to quantify the architecture of oral multi-species biofilms following anti-biofilm interventions using a microfluidic biofilm system. Differences in architecture were compared between untreated biofilms and those treated with water (negative control), sodium gluconate ('placebo') or stannous fluoride (SnF2). The microfluidic system was inoculated with pooled human saliva and biofilms were developed over 22 h in filter-sterilized 25 % pooled human saliva. During this period, biofilms were treated with water, sodium gluconate, or SnF2 (1000, 3439 or 10 000 p.p.m. Sn2+) 8 and 18 h post-inoculation. After 22 h of growth, biofilms were stained with LIVE/DEAD stain, and imaged by CLSM. BAIT was used to calculate biofilm biovolume, total number of objects, surface area, fluffiness, connectivity, convex hull porosity and viability. Image analysis showed oral biofilm architecture was significantly altered by 3439 and 10 000 p.p.m. Sn2+ treatment regimens, resulting in decreased biovolume, surface area, number of objects and connectivity, while fluffiness increased (P<0.01). In conclusion, BAIT was shown to be able to measure the changes in biofilm architecture and detects possible antimicrobial and anti-biofilm effects of candidate agents.

A Sensitive Thresholding Method for Confocal Laser Scanning Microscope Image Stacks of Microbial Biofilms.

Biofilms are surface-attached microbial communities whose architecture can be captured with confocal microscopy. Manual or automatic thresholding of acquired images is often needed to help distinguish biofilm biomass from background noise. However, manual thresholding is subjective and current automatic thresholding methods can lead to loss of meaningful data. Here, we describe an automatic thresholding method designed for confocal fluorescent signal, termed the biovolume elasticity method (BEM). We evaluated BEM using confocal image stacks of oral biofilms grown in pooled human saliva. Image stacks were thresholded manually and automatically with three different methods; Otsu, iterative selection (IS), and BEM. Effects on biovolume, surface area, and number of objects detected indicated that the BEM was the least aggressive at removing signal, and provided the greatest visual and quantitative acuity of single cells. Thus, thresholding with BEM offers a sensitive, automatic, and tunable method to maintain biofilm architectural properties for subsequent analysis.

The Role of Mobile Genetic Elements in the Spread of Antimicrobial-Resistant Escherichia coli From Chickens to Humans in Small-Scale Production Poultry Operations in Rural Ecuador.

Small-scale production poultry operations are increasingly common worldwide. To investigate how these operations influence antimicrobial resistance and mobile genetic elements (MGEs), Escherichia coli isolates were sampled from small-scale production birds (raised in confined spaces with antibiotics in feed), household birds (no movement constraints; fed on scraps), and humans associated with these birds in rural Ecuador (2010-2012). Isolates were screened for genes associated with MGEs as well as phenotypic resistance to 12 antibiotics. Isolates from small-scale production birds had significantly elevated odds of resistance to 7 antibiotics and presence of MGE genes compared with household birds (adjusted odds ratio (OR) range = 2.2-87.9). Isolates from humans associated with small-scale production birds had elevated odds of carrying an integron (adjusted OR = 2.0; 95% confidence interval (CI): 1.06, 3.83) compared with humans associated with household birds, as well as resistance to sulfisoxazole (adjusted OR = 1.9; 95% CI: 1.01, 3.60) and trimethoprim/sulfamethoxazole (adjusted OR = 2.1; 95% CI: 1.13, 3.95). Stratifying by the presence of MGEs revealed antibiotic groups that are explained by biological links to MGEs; in particular, resistance to sulfisoxazole, trimethoprim/sulfamethoxazole, or tetracycline was highest among birds and humans when MGE exposures were present. Small-scale production poultry operations might select for isolates carrying MGEs, contributing to elevated levels of resistance in this setting.

Modeling the Emergence of Antibiotic Resistance in the Environment: an Analytical Solution for the Minimum Selection Concentration.

Environmental antibiotic risk management requires an understanding of how subinhibitory antibiotic concentrations contribute to the spread of resistance. We develop a simple model of competition between sensitive and resistant bacterial strains to predict the minimum selection concentration (MSC), the lowest level of antibiotic at which resistant bacteria are selected. We present an analytical solution for the MSC based on the routinely measured MIC, the selection coefficient (sc) that expresses fitness differences between strains, the intrinsic net growth rate, and the shape of the bacterial growth dose-response curve with antibiotic or metal exposure (the Hill coefficient [κ]). We calibrated the model by optimizing the Hill coefficient to fit previously reported experimental growth rate difference data. The model fit varied among nine compound-taxon combinations examined but predicted the experimentally observed MSC/MIC ratio well (R2 ≥ 0.95). The shape of the antibiotic response curve varied among compounds (0.7 ≤ κ ≤ 10.5), with the steepest curve being found for the aminoglycosides streptomycin and kanamycin. The model was sensitive to this antibiotic response curve shape and to the sc, indicating the importance of fitness differences between strains for determining the MSC. The MSC can be >1 order of magnitude lower than the MIC, typically by the factor scκ This study provides an initial quantitative depiction and a framework for a research agenda to examine the growing evidence of selection for resistant bacterial communities at low environmental antibiotic concentrations.

Association of Escherichia coli ST131 lineage with risk of urinary tract infection recurrence among young women.

OBJECTIVES: The aim of this study was to test the hypothesis that urinary tract infections (UTIs) caused by Escherichia coli of the sequence type 131 (ST131) lineage are more likely to recur than UTIs caused by other E. coli lineages. METHODS: Isolates from 221 young women with UTI caused by E. coli participating in a randomised controlled trial were used. Participants were followed for 6 months or until UTI recurrence. RESULTS: Sequence type was not associated with risk of recurrence. Isolates in the ST131 lineage were more resistant than other STs to quinolones (6.2% vs. 1.3%) but not trimethoprim/sulfamethoxazole (15.4% vs. 15.0%). CONCLUSIONS: These results do not support an increased risk of recurrent UTI among otherwise healthy women with UTI caused by E. coli ST131.

Conservation in gene encoding Mycobacterium tuberculosis antigen Rv2660 and a high predicted population coverage of H56 multistage vaccine in South Africa.

H56/AERAS-456+IC31 (H56), composed of two early secretion proteins, Ag85B and ESAT-6, and a latency associated protein, Rv2660, and the IC31 Intercell adjuvant, is a new fusion subunit vaccine candidate designed to induce immunity against both new infection and reactivation of latent tuberculosis infection. Efficacy of subunit vaccines may be affected by the diversity of vaccine antigens among clinical strains and the extent of recognition by the diverse HLA molecules in the recipient population. Although a previous study showed the conservative nature of Ag85B- and ESAT-6-encoding genes, genetic diversity of Rv2660c that encodes RV2660 is largely unknown. The population coverage of H56 as a whole yet remains to be assessed. The present study was conducted to address these important knowledge gaps. DNA sequence analysis of Rv2660c found no variation among 83 of the 84 investigated clinical strains belonging to four genetic lineages. H56 was predicted to have as high as 99.6% population coverage in the South Africa population using the Immune Epitope Database (IEDB) Population Coverage Tool. Further comparison of H56 population coverage between South African Blacks and Caucasians based on the phenotypic frequencies of binding MHC Class I and Class II supertype alleles found that all of the nine MHC-I and six of eight MHC-II human leukocyte antigen (HLA) supertype alleles analyzed were significantly differentially expressed between the two subpopulations. This finding suggests the presence of race-specific functional binding motifs of MHC-I and MHC-II HLA alleles, which, in turn, highlights the importance of including diverse populations in vaccine clinical evaluation. In conclusion, H56 vaccine is predicted to have a promising population coverage in South Africa; this study demonstrates the utility of integrating comparative genomics and bioinformatics in bridging animal and clinical studies of novel TB vaccines.

Degradation of Extracellular Antibiotic Resistance Genes with UV254 Treatment.

Disinfected wastewater effluent contains a complex mixture of biomolecules including DNA. If intact genes conveying antibiotic resistance survive the disinfection process, environmental bacteria may take them up. We treated plasmid pWH1266, which contains ampicillin resistance gene blaTEM-1 and tetracycline resistance gene tetA, with UV254 doses up to 430 mJ/cm2 and studied the ability of those genes to be acquired by Acinetobacter baylyi. The plasmids required approximately 20-25 mJ/cm2 per log10 loss of transformation efficiency. We monitored plasmid DNA degradation using gel electrophoresis and qPCR with both short amplicons (∼200 bps, representative of ARG amplicon lengths commonly used for environmental monitoring) and long amplicons (800-1200 bps, designed to cover the entire resistance genes). The rate of transformability loss due to UV254 treatment was approximately 20× and 2× larger than the rate of gene degradation measured with the short and long amplicons qPCR, respectively. When extrapolated to account for the length of the entire pWH1266 plasmid, the qPCR rate constants were 2-7× larger than the rate constants measured with transformation assays. Gel electrophoresis results confirmed that DNA cleavage was not a major inactivating mechanism. Overall, our results demonstrate that qPCR conservatively measures the potential for a gene to be transformed by environmental bacteria following UV254 treatment.

Antibiotic Resistance in Animal and Environmental Samples Associated with Small-Scale Poultry Farming in Northwestern Ecuador.

The effects of animal agriculture on the spread of antibiotic resistance (AR) are cross-cutting and thus require a multidisciplinary perspective. Here we use ecological, epidemiological, and ethnographic methods to examine populations of Escherichia coli circulating in the production poultry farming environment versus the domestic environment in rural Ecuador, where small-scale poultry production employing nontherapeutic antibiotics is increasingly common. We sampled 262 "production birds" (commercially raised broiler chickens and laying hens) and 455 "household birds" (raised for domestic use) and household and coop environmental samples from 17 villages between 2010 and 2013. We analyzed data on zones of inhibition from Kirby-Bauer tests, rather than established clinical breakpoints for AR, to distinguish between populations of organisms. We saw significantly higher levels of AR in bacteria from production versus household birds; resistance to either amoxicillin-clavulanate, cephalothin, cefotaxime, and gentamicin was found in 52.8% of production bird isolates and 16% of household ones. A strain jointly resistant to the 4 drugs was exclusive to a subset of isolates from production birds (7.6%) and coop surfaces (6.5%) and was associated with a particular purchase site. The prevalence of AR in production birds declined with bird age (P < 0.01 for all antibiotics tested except tetracycline, sulfisoxazole, and trimethoprim-sulfamethoxazole). Farming status did not impact AR in domestic environments at the household or village level. Our results suggest that AR associated with small-scale poultry farming is present in the immediate production environment and likely originates from sources outside the study area. These outside sources might be a better place to target control efforts than local management practices. IMPORTANCE In developing countries, small-scale poultry farming employing antibiotics as growth promoters is being advanced as an inexpensive source of protein and income. Here, we present the results of a large ecoepidemiological study examining patterns of antibiotic resistance (AR) in E. coli isolates from small-scale poultry production environments versus domestic environments in rural Ecuador, where such backyard poultry operations have become established over the past decade. Our previous research in the region suggests that introduction of AR bacteria through travel and commerce may be an important source of AR in villages of this region. This report extends the prior analysis by examining small-scale production chicken farming as a potential source of resistant strains. Our results suggest that AR strains associated with poultry production likely originate from sources outside the study area and that these outside sources might be a better place to target control efforts than local management practices.

Distribution of Enteroinvasive and Enterotoxigenic Escherichia coli Across Space and Time in Northwestern Ecuador.

Although Escherichia coli infections are common throughout the developing world, their prevalence patterns in space and over time are not well characterized. We used serial case control data collected from 16 communities in northwestern Ecuador between 2004 and 2010, to examine the prevalence of enteroinvasive E. coli (EIEC) and enterotoxigenic E. coli (ETEC). At its peak, the regional prevalence of EIEC was 8.3 infections/100 persons but this decreased to 1 infection/1,000 persons. The regional prevalence of ETEC ranged from 8 infections/1,000 persons to 3.7 infections/100 persons. The prevalence pattern of EIEC resembled that of a large epidemic whereas the prevalence of ETEC was more stable over time. Here, we provide community-based evidence for temporal shifts in the dominant E. coli pathotype from EIEC to ETEC over a multi-year time period. Furthermore, genotype analysis suggests that a given strain of EIEC and ETEC can persist in this region for long periods, up to 24 and 55 months, respectively.

Unexpected distribution of the fluoroquinolone-resistance gene qnrB in Escherichia coli isolates from different human and poultry origins in Ecuador.

Fluoroquinolone resistance can be conferred through chromosomal mutations or by the acquisition of plasmids carrying genes such as the quinolone resistance gene (qnr). In this study, 3,309 strains of commensal Escherichia coli were isolated in Ecuador from: (i) humans and chickens in a rural northern coastal area (n = 2368, 71.5%) and (ii) chickens from an industrial poultry operation (n = 827, 25%). In addition, 114 fluoroquinolone-resistant strains from patients with urinary tract infections who were treated at three urban hospitals in Quito, Ecuador were analyzed. All of the isolates were subjected to antibiotic susceptibility screening. Fluoroquinolone-resistant isolates (FRIs) were then screened for the presence of qnrB genes. A significantly higher phenotypic resistance to fluoroquinolones was determined in E. coli strains from chickens in both the rural area (22%) and the industrial operation (10%) than in strains isolated from humans in the rural communities (3%). However, the rates of qnrB genes in E. coli isolates from healthy humans in the rural communities (11 of 35 isolates, 31%) was higher than in chickens from either the industrial operations (3 of 81 isolates, 6%) or the rural communities (7 of 251 isolates, 2.8%). The occurrence of qnrB genes in human FRIs obtained from urban hospitals was low (1 of 114 isolates, 0.9%). These results suggested that the qnrB gene is more widely distributed in rural settings, where antibiotic usage is low, than in urban hospitals and industrial poultry operations. The role of qnrB in clinical resistance to fluoroquinolones is thus far unknown.

Coaggregation occurs between microorganisms isolated from different environments.

Coaggregation, the specific recognition and adherence of different microbial species, is thought to enhance biofilm formation. To date, no studies have focused on the ability of microorganisms isolated from a broad range of environments to coaggregate with each other and it is unclear whether coaggregation promotes the transmission of microorganisms between environmental niches. We aimed to evaluate the coaggregation ability of 29 bacteria and one fungus, isolated from a range of different environments, and to characterize the cell-surface polymers that mediate coaggregation between selected pairs. Strains were categorized as belonging to one of the four microbial archetypes: aquatic, broad environment, human opportunistic pathogen or human oral. A total of 23 of the 30 strains (77%) coaggregated with at least one other and 21/30 (70%) coaggregated with strains belonging to other archetypes. Nasopharyngeal bacteria belonging to the human opportunistic pathogen archetype showed the least number of coaggregations, and five Haemophilus influenzae strains did not coaggregate. Protease and sugar treatments indicated that coaggregation between strains of different archetypes was often mediated by lectin-saccharide interactions (9 of 15 evaluated pairs). In conclusion, coaggregation can occur between taxonomically disparate species isolated from discrete environments. We propose that these organisms be labeled as 'cross-environment coaggregating organisms'. The ability to coaggregate may aid species to colonize non-indigenous biofilms.

Effects of selection pressure and genetic association on the relationship between antibiotic resistance and virulence in Escherichia coli.

Antibiotic selection pressure and genetic associations may lead to the cooccurrence of resistance and virulence in individual pathogens. However, there is a lack of rigorous epidemiological evidence that demonstrates the cooccurrence of resistance and virulence at the population level. Using samples from a population-based case-control study in 25 villages in rural Ecuador, we characterized resistance to 12 antibiotics among pathogenic (n = 86) and commensal (n = 761) Escherichia coli isolates, classified by the presence or absence of known diarrheagenic virulence factor genes. The prevalences of resistance to single and multiple antibiotics were significantly higher for pathogenic isolates than for commensal isolates. Using a generalized estimating equation, antibiotic resistance was independently associated with virulence factor carriage, case status, and antibiotic use (for these respective factors: odds ratio [OR] = 3.0, with a 95% confidence interval [CI] of 1.7 to 5.1; OR = 2.0, with a 95% CI of 1.3 to 3.0; and OR = 1.5, with a 95% CI of 0.9 to 2.5). Virulence factor carriage was more strongly related to antibiotic resistance than antibiotic use for all antibiotics examined, with the exception of fluoroquinolones, gentamicin, and cefotaxime. This study provides epidemiological evidence that antibiotic resistance and virulence factor carriage are linked in E. coli populations in a community setting. Further, these data suggest that while the cooccurrence of resistance and virulence in E. coli is partially due to antibiotic selection pressure, it is also genetically determined. These findings should be considered in developing strategies for treating infections and controlling for antibiotic resistance.

Unexpected distribution of the fluoroquinolone resistance gene qnrB in Escherichia coli isolates from different human and poultry origins in Ecuador

Fluoroquinolone resistance can be conferred through chromosomal mutations or by the acquisition of plasmids carrying genes such as the quinolone resistance gene (qnr). In this study, 3,309 strains of commensal Escherichia coli were isolated in Ecuador from: (i) humans and chickens in a rural northern coastal area (n = 2368, 71.5%) and (ii) chickens from an industrial poultry operation (n = 827, 25%). In addition, 114 fluoroquinolone-resistant strains from patients with urinary tract infections who were treated at three urban hospitals in Quito, Ecuador were analyzed. All of the isolates were subjected to antibiotic susceptibility screening. Fluoroquinolone-resistant isolates (FRIs) were then screened for the presence of qnrB genes. A significantly higher phenotypic resistance to fluoroquinolones was determined in E. coli strains from chickens in both the rural area (22%) and the industrial operation (10%) than in strains isolated from humans in the rural communities (3%). However, the rates of qnrB genes in E. coli isolates from healthy humans in the rural communities (11 of 35 isolates, 31%) was higher than in chickens from either the industrial operations (3 of 81 isolates, 6%) or the rural communities (7 of 251 isolates, 2.8%). The occurrence of qnrB genes in human FRIs obtained from urban hospitals was low (1 of 114 isolates, 0.9%). These results suggested that the qnrB gene is more widely distributed in rural settings, where antibiotic usage is low, than in urban hospitals and industrial poultry operations. The role of qnrB in clinical resistance to fluoroquinolones is thus far unknown (AU)

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Comparative Profile of Heme Acquisition Genes in Disease-Causing and Colonizing Nontypeable Haemophilus influenzae and Haemophilus haemolyticus.

Nontypeable Haemophilus influenzae (NTHI) are Gram-negative bacteria that colonize the human pharynx and can cause respiratory tract infections, such as acute otitis media (AOM). Since NTHI require iron from their hosts for aerobic growth, the heme acquisition genes may play a significant role in avoiding host nutritional immunity and determining virulence. Therefore, we employed a hybridization-based technique to compare the prevalence of five heme acquisition genes (hxuA, hxuB, hxuC, hemR, and hup) between 514 middle ear strains from children with AOM and 235 throat strains from healthy children. We also investigated their prevalences in 148 Haemophilus haemolyticus strains, a closely related species that colonizes the human pharynx and is considered to be nonpathogenic. Four out of five genes (hxuA, hxuB, hxuC, and hemR) were significantly more prevalent in the middle ear strains (96%, 100%, 100%, and 97%, respectively) than in throat strains (80%, 92%, 93%, and 85%, respectively) of NTHI, suggesting that strains possessing these genes have a virulence advantage over those lacking them. All five genes were dramatically more prevalent in NTHI strains than in H. haemolyticus, with 91% versus 9% hxuA, 98% versus 11% hxuB, 98% versus 11% hxuC, 93% versus 20% hemR, and 97% versus 34% hup, supporting their potential role in virulence and highlighting their possibility to serve as biomarkers to distinguish H. influenzae from H. haemolyticus. In summary, this study demonstrates that heme acquisition genes are more prevalent in disease-causing NTHI strains isolated from the middle ear than in colonizing NTHI strains and H. haemolyticus isolated from the pharynx.

Efflux-mediated resistance identified among norfloxacin resistant clinical strains of group B Streptococcus from South Korea.

OBJECTIVES: 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. 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. METHODS: 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, Durham, NC, USA) was used to determine fluoroquinolone resistance. RESULTS: 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 minimum inhibitory concentration (average=3.6 fold, range=>1-16 fold). CONCLUSIONS: A substantial portion of GBS strains resistant to norfloxacin have an efflux phenotype.

Prevalence, distribution, and sequence diversity of hmwA among commensal and otitis media non-typeable Haemophilus influenzae.

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 or nasopharynges 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.

Otitis media associated polymorphisms in the hemin receptor HemR of nontypeable Haemophilus influenzae.

Nontypeable Haemophilus influenzae (NTHi) colonize the human pharynx asymptomatically, and are also an important cause of otitis media (OM). Previous studies have demonstrated that some genes are more prevalent in OM-causing NTHi strains than in commensal strains, suggesting a role in virulence. These studies, however, are unable to investigate the possible associations between gene polymorphisms and disease. This study examined amino acid polymorphisms and sequence diversity in a potential virulence gene, the hemin receptor hemR, from a previously characterized NTHi strain collection containing both commensal and OM organisms to identify possible associations between the polymorphisms and otitis media. The full open reading frame of hemR was sequenced from a total of 146 NTHi isolates, yielding a total of 47 unique HemR amino acid sequences. The predicted structure of HemR showed substantial similarity to a class of monomeric TonB dependent, ligand-gated channels involved in iron acquisition in other gram negative bacteria. Fifteen amino acid polymorphisms were significantly more prevalent at the 90% confidence level among commensal compared to OM isolates. Upon controlling for the confounding effect of population structure, over half of the polymorphism-otitis media relationships lost statistical significance, emphasizing the importance of assessing the effect of population structure in association studies. The seven polymorphisms that retained significance were dispersed throughout the protein in various functional and structural domains, including the signal peptide, N-terminal plug domain, and intra- and extracellular loops. The alternate amino acid of only one of these seven polymorphisms was more common among OM isolates, demonstrating a strong trend toward the consensus sequence among disease causing NTHi. We hypothesize that variability at these positions in HemR may result in a reduced ability to acquire iron, rendering NTHi with such versions of the gene less fit for survival in the middle ear environment.

Phase variation and host immunity against high molecular weight (HMW) adhesins shape population dynamics of nontypeable Haemophilus influenzae within human hosts.

Nontypeable Haemophilus influenzae (NTHi) is a bacterium that resides within the human pharynx. Because NTHi is human-restricted, its long-term survival is dependent upon its ability to successfully colonize new hosts. Adherence to host epithelium, mediated by bacterial adhesins, is one of the first steps in NTHi colonization. NTHi express several adhesins, including the high molecular weight (HMW) adhesins that mediate attachment to the respiratory epithelium where they interact with the host immune system to elicit a strong humoral response. hmwA, which encodes the HMW adhesin, undergoes phase variation mediated by 7-base pair tandem repeats located within its promoter region. Repeat number affects both hmwA transcription and HMW-adhesin production such that as the number of repeats increases, adhesin production decreases. Cells expressing large amounts of HMW adhesins may be critical for the establishment and maintenance of NTHi colonization, but they might also incur greater fitness costs when faced with an adhesin-specific antibody-mediated immune response. We hypothesized that the occurrence of large deletion events within the hmwA repeat region allows NTHi cells to maintain adherence in the presence of antibody-mediated immunity. To study this, we developed a mathematical model, incorporating hmwA phase variation and antibody-mediated immunity, to explore the trade-off between bacterial adherence and immune evasion. The model predicts that antibody levels and avidity, catastrophic loss rates, and population carrying capacity all significantly affected numbers of adherent NTHi cells within a host. These results suggest that the occurrence of large, yet rare, deletion events allows for stable maintenance of a small population of adherent cells in spite of HMW adhesin specific antibody-mediated immunity. These adherent subpopulations may be important for sustaining colonization and/or maintaining transmission.