The hidden 'mycobacteriome' of the human healthy oral cavity and upper respiratory tract.

The incidence of opportunistic non-tuberculous mycobacteria (NTM) infections has increased considerably in the past decades causing an array of infections, including respiratory and soft-tissue infections. NTM are ubiquitous and can be found in numerous environments, including households and water plants. However, NTM have not been reported to be associated with the healthy human oral microbiome. Since the oral cavity and upper respiratory track are the main ports of entry of microorganisms into the human body, elucidating NTM diversity and prevalence will assist in the assessment of the potential risks of infection elicited by these opportunistic pathogens. Here, we report the identification of a 'non-tuberculous mycobacteriome' in healthy individuals. We employed a modified DNA extraction procedure in conjunction with mycobacterial-specific primers to screen niches in the oral cavity (buccal mucosa and dental plaque) and upper respiratory tract (nostrils and oropharynx) of 10 healthy subjects. A total of 50 prevalent operational taxonomic units sequenced on MiSeq (Illumina) using 16S rRNA V3-V4 region were detected across all screened niches, showing the presence of diverse NTM communities. NTM DNA was detected in the nostrils of all 10 subjects, in buccal mucosa of 8 subjects, in the oropharynx of 7 subjects, and in the dental plaques of 5 subjects. Results from quantitative PCR showed each individual harbored 10(3)-10(4) predicted NTM per each screened niche. The modification of standard DNA isolation methods to increase sensitivity toward mycobacterial species represents an important step to advance the knowledge of the oral as well as the overall human microbiome. These findings clearly reveal for the first time that healthy individuals harbor a 'non-tuberculous mycobacteriome' in their oral cavity and upper respiratory tract and may have important implications in our understanding of infections caused by NTM.

Validation of Mycobacterium tuberculosis Rv1681 protein as a diagnostic marker of active pulmonary tuberculosis.

The development of an accurate antigen detection assay for the diagnosis of active tuberculosis (TB) would represent a major clinical advance. Here, we demonstrate that the Mycobacterium tuberculosis Rv1681 protein is a biomarker for active TB with potential diagnostic utility. We initially identified, by mass spectroscopy, peptides from the Rv1681 protein in urine specimens from 4 patients with untreated active TB. Rabbit IgG anti-recombinant Rv1681 detected Rv1681 protein in lysates and culture filtrates of M. tuberculosis and immunoprecipitated it from pooled urine specimens from two TB patients. An enzyme-linked immunosorbent assay formatted with these antibodies detected Rv1681 protein in unconcentrated urine specimens from 11/25 (44%) TB patients and 1/21 (4.8%) subjects in whom TB was initially clinically suspected but then ruled out by conventional methods. Rv1681 protein was not detected in urine specimens from 10 subjects with Escherichia coli-positive urine cultures, 26 subjects with confirmed non-TB tropical diseases (11 with schistosomiasis, 5 with Chagas' disease, and 10 with cutaneous leishmaniasis), and 14 healthy subjects. These results provide strong validation of Rv1681 protein as a promising biomarker for TB diagnosis.

Current and novel approaches to vaccine development against tuberculosis.

Antibiotics and vaccines are the two most successful medical countermeasures that humans have created against a number of pathogens. However a select few e.g., Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB) have evaded eradication by vaccines and therapeutic approaches. TB is a global public health problem that kills 1.4 million people per year. The past decade has seen significant progress in developing new vaccine candidates, but the most fundamental questions in understanding disease progression and protective host responses that are responsible for controlling Mtb infection still remain poorly resolved. Current TB treatment requires intense chemotherapy with several antimicrobials, while the only approved vaccine is the classical viable whole-cell based Bacille-Calmette-Guerin (BCG) that protects children from severe forms of TB, but fails to protect adults. Taken together, there is a growing need to conduct basic and applied research to develop novel vaccine strategies against TB. This review is focused on the discussion surrounding current strategies and innovations being explored to discover new protective antigens, adjuvants, and delivery systems in the hopes of creating an efficacious TB vaccine.

Detection and identification of species-specific bacteria associated with synanthropic mites.

Internal bacterial communities of synanthropic mites Acarus siro, Dermatophagoides farinae, Lepidoglyphus destructor, and Tyrophagus putrescentiae (Acari: Astigmata) were analyzed by culturing and culture-independent approaches from specimens obtained from laboratory colonies. Homogenates of surface-sterilized mites were used for cultivation on non-selective agar and DNA extraction. Isolated bacteria were identified by sequencing of the 16S rRNA gene. PCR amplified 16S rRNA genes were analyzed by terminal restriction fragment length polymorphism analysis (T-RFLP) and cloning sequencing. Fluorescence in situ hybridization using universal bacterial probes was used for direct bacterial localization. T-RFLP analysis of 16S rRNA gene revealed distinct species-specific bacterial communities. The results were further confirmed by cloning and sequencing (284 clones). L. destructor and D. farinae showed more diverse communities then A. siro and T. putrescentiae. In the cultivated part of the community, the mean CFUs from four mite species ranged from 5.2 × 10(2) to 1.4 × 10(3) per mite. D. farinae had significantly higher CFUs than the other species. Bacteria were located in the digestive and reproductive tract, parenchymatical tissue, and in bacteriocytes. Among the clones, Bartonella-like bacteria occurring in A. siro and T. putresecentiae represented a distinct group related to Bartonellaceae and to Bartonella-like symbionts of ants. The clones of high similarity to Xenorhabdus cabanillasii were found in L. destructor and D. farinae, and one clone related to Photorhabdus temperata in A. siro. Members of Sphingobacteriales cloned from D. farinae and A. siro clustered with the sequences of "Candidatus Cardinium hertigii" and as a separate novel cluster.

Enterococcus faecalis with the gelatinase phenotype regulated by the fsr operon and with biofilm-forming capacity are common in the agricultural environment.

The prevalence of gelatinase activity and biofilm formation among environmental enterococci was assessed. In total, 396 enterococcal isolates from swine and cattle faeces and house flies from a cattle farm were screened for gelatinase activity. The most prevalent phenotype on Todd-Hewitt agar with 1.5% skim milk was the weak protease (WP) (72.2% of isolates), followed by the strong protease (SP) 18.7%, and no protease (NP) (9.1%). The majority of WP isolates was represented by Enterococcus hirae (56.9%), followed by Enterococcus faecium (25.9%), Enterococcus casseliflavus (10.4%), Enterococcus gallinarum (5.2%) and Enterococcus saccharolyticus (1.7%). All WP isolates were negative for gelE (gelatinase) and sprE (serine protease) as well as the fsrABDC operon that regulates the two proteases, and only four isolates (7.0%) formed biofilms in vitro. All SP isolates were Enterococcus faecalis positive for the fsrABDC, gelE, sprE genes and the majority (91.2%) formed a biofilm. Diversity of NP isolates was relatively evenly distributed among E. hirae, E. faecium, E. casseliflavus, E. gallinarum, Enterococcus durans, E. saccharolyticus and Enterococcus mundtii. All NP isolates were negative for the fsr operon and only four E. hirae (11.1%) formed a biofilm. Of further interest was the loss of the gelatinase phenotype (18.9% of isolates) from SP isolates after 4 month storage at 4-8 degrees C and several passages of subculture. Results of reverse transcription PCR analysis indicated that mRNA was produced for all the genes in the frs operon and sequencing of the gelE gene did not reveal any significant mutations. However, gelatinase was not detectable by Western blot analysis. Our study shows that E. faecalis with the complete fsr operon and the potential to form a biofilm are relatively common in the agricultural environment and may represent a source/reservoir of clinically relevant strains. In addition, many environmental enterococci, especially E. hirae, produce an unknown WP that can hydrolyse casein but does not contribute to biofilm formation. The stability of the gelatinase phenotype in E. faecalis and its regulation will require additional studies.

Potential of houseflies to contaminate ready-to-eat food with antibiotic-resistant enterococci.

It was shown previously that houseflies in fast-food restaurants commonly carry antibiotic-resistant and potentially virulent enterococci. In this study, the potential of field-collected houseflies to contaminate ready-to-eat (RTE) food with enterococci was assessed by laboratory bioassays. Houseflies were collected with a sweep net in a cattle feedlot and exposed in groups of 5, 10, 20, and 40 to a beef patty (from an RTE hamburger) for 0.5, 1.0, 3.0, and 24 h. The exposure of RTE food to flies resulted in 100% contamination with enterococci in all bioassays, regardless of the number of houseflies and the length of exposure time. In addition, with the increasing number of houseflies as well as with the increasing time exposure, the concentration of enterococci in RTE food increased. Even a short time exposure (0.5 h) resulted in food contamination, ranging from 3.1 x 10(3) CFU/g (5 houseflies) to 8.4 x 10(4) CFU/g (40 houseflies). The analysis of 23 randomly selected enterococcal isolates from RTE food after the fly exposure revealed a single species, Enterococcus faecalis. In contrast, four Enterococcus species, including E. faecalis (57.1%), E. gallinarum (19.1%), E. hirae (14.3%), and E. faecium (9.5%), represented 21 randomly selected and identified isolates from houseflies. Phenotypic screening showed that E. faecalis isolates from RTE food were resistant to ciprofloxacin (17.4%), tetracycline (13.0%), erythromycin (13.0%), and chloramphenicol (4.3%). This study demonstrates a great potential of houseflies from a cattle feedlot to contaminate RTE food with enterococci in a short time.

Influx of enterococci and associated antibiotic resistance and virulence genes from ready-to-eat food to the human digestive tract.

The influx of enterococcal antibiotic resistance (AR) and virulence genes from ready-to-eat food (RTEF) to the human digestive tract was assessed. Three RTEFs (chicken salad, chicken burger, and carrot cake) were sampled from five fast-food restaurants five times in summer (SU) and winter (WI). The prevalence of enterococci was significantly higher in SU (92.0% of salad samples and 64.0% of burger samples) than in WI (64.0% of salad samples and 24.0% of burger samples). The overall concentrations of enterococci during the two seasons were similar ( approximately 10(3) CFU/g); the most prevalent were Enterococcus casseliflavus (41.5% of isolates) and Enterococcus hirae (41.5%) in WI and Enterococcus faecium (36.8%), E. casseliflavus (27.6%), and Enterococcus faecalis (22.4%) in SU. Resistance in WI was detected primarily to tetracycline (50.8%), ciprofloxacin (13.8%), and erythromycin (4.6%). SU isolates were resistant mainly to tetracycline (22.8%), erythromycin (22.1%), and kanamycin (13.0%). The most common tet gene was tet(M) (35.4% of WI isolates and 11.9% of SU isolates). The prevalence of virulence genes (gelE, asa1, cylA, and esp) and marker genes for clinical isolates (EF_0573, EF_0592, EF_0605, EF_1420, EF_2144, and pathogenicity island EF_0050) was low (< or =12.3%). Genotyping of E. faecalis and E. faecium using pulsed-field gel electrophoresis revealed that the food contamination likely originated from various sources and that it was not clonal. Our conservative estimate (single AR gene copy per cell) for the influx of tet genes alone to the human digestive tract is 3.8 x 10(5) per meal (chicken salad). This AR gene influx is frequent because RTEFs are commonly consumed and that may play a role in the acquisition of AR determinants in the human digestive tract.

Ecology of antibiotic resistance genes: characterization of enterococci from houseflies collected in food settings.

In this project, enterococci from the digestive tracts of 260 houseflies (Musca domestica L.) collected from five restaurants were characterized. Houseflies frequently (97% of the flies were positive) carried enterococci (mean, 3.1 x 10(3) CFU/fly). Using multiplex PCR, 205 of 355 randomly selected enterococcal isolates were identified and characterized. The majority of these isolates were Enterococcus faecalis (88.2%); in addition, 6.8% were E. faecium, and 4.9% were E. casseliflavus. E. faecalis isolates were phenotypically resistant to tetracycline (66.3%), erythromycin (23.8%), streptomycin (11.6%), ciprofloxacin (9.9%), and kanamycin (8.3%). Tetracycline resistance in E. faecalis was encoded by tet(M) (65.8%), tet(O) (1.7%), and tet(W) (0.8%). The majority (78.3%) of the erythromycin-resistant E. faecalis isolates carried erm(B). The conjugative transposon Tn916 and members of the Tn916/Tn1545 family were detected in 30.2% and 34.6% of the identified isolates, respectively. E. faecalis carried virulence genes, including a gelatinase gene (gelE; 70.7%), an aggregation substance gene (asa1; 33.2%), an enterococcus surface protein gene (esp; 8.8%), and a cytolysin gene (cylA; 8.8%). Phenotypic assays showed that 91.4% of the isolates with the gelE gene were gelatinolytic and that 46.7% of the isolates with the asa1 gene aggregated. All isolates with the cylA gene were hemolytic on human blood. This study showed that houseflies in food-handling and -serving facilities carry antibiotic-resistant and potentially virulent enterococci that have the capacity for horizontal transfer of antibiotic resistance genes to other bacteria.