Characterization of medical relevant anaerobic microorganisms by isothermal microcalorimetry.

Detection of anaerobe bacteria by culture methods requires appropriate media, special growth conditions, additional detection techniques and it typically takes several days. Therefore, anaerobes are often missed in patient specimens under routine culture conditions. Microcalorimetry may provide a simple and accurate real-time method for faster and better detection of anaerobes. An isothermal calorimeter which detect minimal changes of temperature over time was used for the calorimetric experiments. In order to find optimal growth conditions, seven reference or clinical strains of medical relevant anaerobe bacteria were tested under different circumstances. First, the strains were tested with different growth media. After determining the optimal medium for each strain, the gas phase was modified by adding 3 mL or 4 mL medium, to evaluate growth under conditions with less oxygen. Cooked Meat Medium was best supporting growth of the tested strains, including Cutibacterium acnes, Fusobacterium nucleatum, Finegoldia magna, Parvimonas micra, Bacteroides fragilis and Actinomyces odontolyticus, followed by thioglycolate. The best medium to detect Clostridioides difficile was H-Medium. All tested strains showed better growth in 4 mL medium than in 3 mL. The detection time ranged between 10 and 72 h. Our results demonstrated that the sensitivity and the detection time of anaerobe bacteria can be improved by isothermal calorimetry with optimization of growth conditions. Therefore, calorimetric detection, a practical, quick and easy-to-do method, has the potential to replace current microbiological methods.

Disruption of Cross-Feeding Inhibits Pathogen Growth in the Sputa of Patients with Cystic Fibrosis.

A critical limitation in the management of chronic polymicrobial infections is the lack of correlation between antibiotic susceptibility testing (AST) and patient responses to therapy. Underlying this disconnect is our inability to accurately recapitulate the in vivo environment and complex polymicrobial communities in vitro However, emerging evidence suggests that, if modeled and tested accurately, interspecies relationships can be exploited by conventional antibiotics predicted to be ineffective by standard AST. As an example, under conditions where Pseudomonas aeruginosa relies on cocolonizing organisms for nutrients (i.e., cross-feeding), multidrug-resistant P. aeruginosa may be indirectly targeted by inhibiting the growth of its metabolic partners. While this has been shown in vitro using synthetic bacterial communities, the efficacy of a "weakest-link" approach to controlling host-associated polymicrobial infections has not yet been demonstrated. To test whether cross-feeding inhibition can be leveraged in clinically relevant contexts, we collected sputa from cystic fibrosis (CF) subjects and used enrichment culturing to isolate both P. aeruginosa and anaerobic bacteria from each sample. Predictably, both subpopulations showed various antibiotic susceptibilities when grown independently. However, when P. aeruginosa was cultured and treated under cooperative conditions in which it was dependent on anaerobic bacteria for nutrients, the growth of both the pathogen and the anaerobe was constrained despite their intrinsic antibiotic resistance profiles. These data demonstrate that the control of complex polymicrobial infections may be achieved by exploiting obligate or facultative interspecies relationships. Toward this end, in vitro susceptibility testing should evolve to more accurately reflect in vivo growth environments and microbial interactions found within them.IMPORTANCE Antibiotic efficacy achieved in vitro correlates poorly with clinical outcomes after treatment of chronic polymicrobial diseases; if a pathogen demonstrates susceptibility to a given antibiotic in the lab, that compound is often ineffective when administered clinically. Conversely, if a pathogen is resistant in vitro, patient treatment with that same compound can elicit a positive response. This discordance suggests that the in vivo growth environment impacts pathogen antibiotic susceptibility. Indeed, here we demonstrate that interspecies relationships among microbiotas in the sputa of cystic fibrosis patients can be targeted to indirectly inhibit the growth of Pseudomonas aeruginosa The therapeutic implication is that control of chronic lung infections may be achieved by exploiting obligate or facultative relationships among airway bacterial community members. This strategy is particularly relevant for pathogens harboring intrinsic multidrug resistance and is broadly applicable to chronic polymicrobial airway, wound, and intra-abdominal infections.

Anaerobes in cystic fibrosis patients' airways.

Anaerobes are known to constitute an important part of the airway microbiota in both healthy subjects and cystic fibrosis (CF) patients. Studies on the potential role of anaerobic bacteria in CF and thus their involvement in CF pathophysiology have reported contradictory results, and the question is still not elucidated. The aim of this study was to summarize anaerobe diversity in the airway microbiota and its potential role in CF, to provide an overview of the state of knowledge on anaerobe antibiotic resistances (resistome), and to investigate the detectable metabolites produced by anaerobes in CF airways (metabolome). This review emphasizes key metabolites produced by strict anaerobic bacteria (sphingolipids, fermentation-induced metabolites and metabolites involved in quorum-sensing), which may be essential for the better understanding of lung disease pathophysiology in CF.

Anaerobic biodegradation of pharmaceutical compounds: New insights into the pharmaceutical-degrading bacteria.

Antibiotics and hormones are among the most concerning trace contaminants in the environment. Therefore, the present work aimed to identify anaerobic microorganisms with the ability to remove pharmaceutical products (PhPs) belonging to these two classes (ciprofloxacin, 17ß-estradiol and sulfamethoxazole) under different anaerobic conditions, and to elucidate the bio-removal mechanisms involved. Ciprofloxacin was efficiently biodegraded under both nitrate- and sulfate-reducing conditions reaching a PhP removal superior to 80%, whereas 17ß-estradiol was only biodegraded under nitrate-reducing conditions reaching a removal of 84%. No biodegradation of sulfamethoxazole was observed. In nitrate-reducing conditions the ciprofloxacin-degrading community was composed of Comamonas, Arcobacter, Dysgonomonas, Macellibacteroides and Actinomyces, genera while Comamonas and Castellaniella were the main bacteria present in the 17ß-estradiol-degrading community. In sulfate-reducing conditions the community was mainly composed by bacteria affiliated to Desulfovibrio, Enterococcus and Peptostreeptococcus. Interestingly, the PhP under study were biodegraded even in the absence of additional carbon source, with 85% of ciprofloxacin removed under sulfate-reducing conditions and 62% and 83% of ciprofloxacin and estradiol removed, respectively, under nitrate-reducing conditions. This work provides new insights into anaerobic bioremediation of PhP and novel PhP-degrading bacteria.

Effect of zero-valent iron and trivalent iron on UASB rapid start-up.

In order to realize the rapid start-up of upflow anaerobic sludge blanket (UASB) reactor, the iron ion in different valence state was added to UASB. The results indicated that the start-up time of R3 (FeCl3) was 48 h faster than that of R2 (zero-valent iron (ZVI)). It was because the FeCl3 could rapidly promote granulation of sludge as a flocculant. However, ZVI released Fe2+ through corrosion slowly, and then the Fe2+ increased start-up speed by enhancing enzyme activity and enriching methanogens. In addition, the ZVI and FeCl3 could promote hydrolysis acidification and strengthen the decomposition of long-chain fatty acids. The detection of iron ions showed that iron ions mainly existed in the sludge. Because the high concentration of Fe2+ could inhibit anaerobic bacteria activity, excess Fe3+ could be changed into iron hydroxide precipitation to hinder the mass transfer process of anaerobic bacteria under the alkaline condition. The FeCl3 was suitable to be added at the initial stage of UASB start-up, and the ZVI was more fitted to be used in the middle stage of reactor start-up to improve the redox ability.

Influence of Oral Anaerobic Bacteria on Hematopoietic Stem Cell Transplantation Patients: Oral Mucositis and General Condition.

OBJECTIVE: Oral mucositis (OM) caused by infection facilitated by myelosuppression and immunosuppression can be controlled through oral care. We investigated changes in oral anaerobic bacterial flora during the onset of OM with hematopoietic stem cell transplantation (HSCT). METHODS: This study included 19 patients who underwent HSCT. All received professional oral care before initiating the preparative regimen. We assessed OM, oral health and obtained microbial samples from the oral cavity during 5 assessment points: before initiating the preparative regimen; the day before HSCT (day 1); and at 7, 14, and 30 days after HSCT. Microbial species were identified by using a mass spectrometer. RESULTS: The number of patients with serious OM increased initially after HSCT and decreased thereafter. Many Streptococcus species were identified before HSCT, but these gradually decreased and were replaced by coagulase-negative staphylococci. An increase in Candida species after HSCT and the identification of Enterococcus species were significantly associated with OM. Nutritional status recovery and prognosis were significantly worse in patients who developed OM. CONCLUSIONS: To the best of our knowledge, this study is the first which shows that anaerobic bacteria were identified in patients' oral flora before and after HSCT by using a mass spectrometer. These results indicate that Enterococcus species and Candida species may have been associated with OM. OM affected the patients' improvement in nutritional status and their prognosis. We concluded that it is important to provide more complete oral care instructions and interventions to prevent these bacterial infections.

Identification of low oxygen-tolerating bacteria in prostate secretions of cancer patients and discussion of possible aetiological significance.

The microaerophylic organism Propionibacterium acnes has shown consistent association with prostate cancer (PC). Studies linking circumcision with reduced PC further support anaerobes involvement as circumcision reduces anaerobe colonisation on the glans penis. A 1988 study linked anaerobes with PC but considered them as opportunists in necrotic tumour. A hypothesis that a "Helicobacter-like" process causes PC justified this pilot study. Active surveillance patients were enrolled. Post-prostate massage urine samples were screened using the Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) technique for bacterial identification after culture in anaerobic and aerobic conditions. 8 out of 18 patients (41%) had either obligate anaerobic (n = 5) or microaerophilic (n = 4, one of whom also had anaerobes) organisms identified. None of 10 control samples contained obligate anaerobes. Although mean PSA was 63% higher in those with low oxygen tolerating bacteria, two high outliers resulted in this difference being non-significant. Given the substantially higher proportion of PC patients with organisms growing in a low concentration of oxygen when combined with previous studies compared to controls, the degree of significance was as high as smoking 5-9 cigarettes a day and needs further investigation. Translational research in trials combining Vitamin D and aspirin have begun as part of such investigation.

Commensal gut bacteria modulate phosphorylation-dependent PPARγ transcriptional activity in human intestinal epithelial cells.

In healthy subjects, the intestinal microbiota interacts with the host's epithelium, regulating gene expression to the benefit of both, host and microbiota. The underlying mechanisms remain poorly understood, however. Although many gut bacteria are not yet cultured, constantly growing culture collections have been established. We selected 57 representative commensal bacterial strains to study bacteria-host interactions, focusing on PPARγ, a key nuclear receptor in colonocytes linking metabolism and inflammation to the microbiota. Conditioned media (CM) were harvested from anaerobic cultures and assessed for their ability to modulate PPARγ using a reporter cell line. Activation of PPARγ transcriptional activity was linked to the presence of butyrate and propionate, two of the main metabolites of intestinal bacteria. Interestingly, some stimulatory CMs were devoid of these metabolites. A Prevotella and an Atopobium strain were chosen for further study, and shown to up-regulate two PPARγ-target genes, ANGPTL4 and ADRP. The molecular mechanisms of these activations involved the phosphorylation of PPARγ through ERK1/2. The responsible metabolites were shown to be heat sensitive but markedly diverged in size, emphasizing the diversity of bioactive compounds found in the intestine. Here we describe different mechanisms by which single intestinal bacteria can directly impact their host's health through transcriptional regulation.

Dissolved organic matter as a terminal electron acceptor in the microbial oxidation of steroid estrogen.

Steroid estrogen in natural waters may be biodegraded by quinone-reducing bacteria, dissolved organic matter (DOM) may serve as a terminal electron acceptor in this process. The influence of temperature, pH, dissolved oxygen and light illumination on the reduction efficiency of anthraquinone-2-disulfonate (AQS) was investigated using 17ß-estradiol (E2) as the target species. The optimum reduction conditions were found to be in the dark under anaerobic conditions at pH 8.0 and 30 °C. Quinone-reducing bacteria can use the quinone structure of DOM components as a terminal electron acceptor coupling with microbial growth to promote biodegradation. Compared with other DOM models, AQS best stimulated E2 biodegradation and the mediating effect was improved as the AQS concentration increased from 0 to 0.5 mM. However, further increase had an inhibiting effect. Natural DOM containing lake humic acid (LHA) and lake fulvic acid (LFA) had a very important accelerating effect on the degradation of E2, the action mechanism of which was consistent with that defined using DOM models. The natural DOM contained more aromatic compounds, demonstrating their greater electron-accepting capacity and generally more effective support for microorganism growth and E2 oxidation than Aldrich humic acid (HA). These results provide a more comprehensive understanding of microbial degradation of steroid estrogens in anaerobic environments and confirm DOM as an important terminal electron acceptor in pollutant transformation.

The microenvironment of injured murine gut elicits a local pro-restitutive microbiota.

The mammalian intestine houses a complex microbial community, which influences normal epithelial growth and development, and is integral to the repair of damaged intestinal mucosa(1-3). Restitution of injured mucosa involves the recruitment of immune cells, epithelial migration and proliferation(4,5). Although microenvironmental alterations have been described in wound healing(6), a role for extrinsic influences, such as members of the microbiota, has not been reported. Here, we show that a distinct subpopulation of the normal mucosal-associated gut microbiota expands and preferentially colonizes sites of damaged murine mucosa in response to local environmental cues. Our results demonstrate that formyl peptide receptor 1 (FPR1) and neutrophilic NADPH oxidase (NOX2) are required for the rapid depletion of microenvironmental oxygen and compensatory responses, resulting in a dramatic enrichment of an anaerobic bacterial consortium. Furthermore, the dominant member of this wound-mucosa-associated microbiota, Akkermansia muciniphila (an anaerobic, mucinophilic gut symbiont(7,8)), stimulated proliferation and migration of enterocytes adjacent to the colonic wounds in a process involving FPR1 and intestinal epithelial-cell-specific NOX1-dependent redox signalling. These findings thus demonstrate how wound microenvironments induce the rapid emergence of 'probiont' species that contribute to enhanced repair of mucosal wounds. Such microorganisms could be exploited as potential therapeutics.

Calorimetric studies of the growth of anaerobic microbes.

This article aims to validate the use of calorimetry to measure the growth of anaerobic microbes. It has been difficult to monitor the growth of strict anaerobes while maintaining optimal growth conditions. Traditionally, optical density and ATP concentration are usually used as measures of the growth of anaerobic microbes. However, to take these measurements it is necessary to extract an aliquot of the culture, which can be difficult while maintaining anaerobic conditions. In this study, calorimetry was used to continuously and nondestructively measure the heat generated by the growth of anaerobic microbes as a function of time. Clostridium acetobutylicum, Clostridium beijerinckii, and Clostridium cellulovorans were used as representative anaerobic microbes. Using a multiplex isothermal calorimeter, we observed that peak time (tp) of C. acetobutylicum heat evolution increased as the inoculation rate decreased. This strong correlation between the inoculation rate and tp showed that it was possible to measure the growth rate of anaerobic microbes by calorimetry. Overall, our results showed that there is a very good correlation between heat evolution and optical density/ATP concentration, validating the use of the method.

Clinical characteristics associated with mortality of patients with anaerobic bacteremia.

The presence of anaerobes in the blood stream is known to be associated with a higher rate of mortality. However, few prognostic risk factor analyses examining whether a patient's background characteristics are associated with the prognosis have been reported. We performed a retrospective case-controlled study to assess the prognostic factors associated with death from anaerobic bacteremia. Seventy-four patients with anaerobic bacteremia were treated between January 2005 and December 2014 at Aichi Medical University Hospital. The clinical information included drug susceptibility was used for analysis of prognostic factors for 30-day mortality. Multivariate logistic analyses revealed an association between the 30-day mortality rate and malignancy (OR: 3.64, 95% CI: 1.08-12.31) and clindamycin resistance (OR: 7.93, 95% CI: 2.33-27.94). The result of Kaplan-Meier analysis of mortality showed that the 30-day survival rate was 83% in clindamycin susceptible and 38.1% in clindamycin resistant anaerobes causing bacteremia. The result of log-rank test also showed that susceptibility to clindamycin affected mortality (P < 0.001). Our results indicated that malignancy and clindamycin susceptibility could be used to identify subgroups of patients with anaerobic bacteremia with a higher risk of 30-day mortality. The results of this study are important for the early and appropriate management of patients with anaerobic bacteremia.

Spectrum and treatment of anaerobic infections.

Anaerobes are the most predominant components of the normal human skin and mucous membranes bacterial flora, and are a frequent cause of endogenous bacterial infections. Anaerobic infections can occur in all body locations: the central nervous system, oral cavity, head and neck, chest, abdomen, pelvis, skin, and soft tissues. Treatment of anaerobic infection is complicated by their slow growth in culture, by their polymicrobial nature and by their growing resistance to antimicrobials. Antimicrobial therapy is frequently the only form of therapy needed, whereas in others it is an important adjunct to drainage and surgery. Because anaerobes generally are isolated mixed with aerobes, the antimicrobial chosen should provide for adequate coverage of both. The most effective antimicrobials against anaerobes are: metronidazole, the carbapenems (imipenem, meropenem, doripenem, ertapenem), chloramphenicol, the combinations of a penicillin and a beta-lactamase inhibitors (ampicillin or ticarcillin plus clavulanate, amoxicillin plus sulbactam, piperacillin plus tazobactam), tigecycline, cefoxitin and clindamycin.

Assessment of crude glycerol for Enhanced Biological Phosphorus Removal: Stability and role of long chain fatty acids.

Enhanced Biological Phosphorus Removal (EBPR) of urban wastewaters is usually limited by the available carbon source required by Polyphosphate Accumulating Organisms (PAO). External carbon sources as volatile fatty acids (VFA) or other pure organic compounds have been tested at lab scale demonstrating its ability to enhance PAO activity, but the application of this strategy at full-scale WWTPs is not cost-effective. The utilization of industrial by-products with some of these organic compounds provides lower cost, but it has the possible drawback of having inhibitory or toxic compounds to PAO. This study is focused on the utilization of crude glycerol, the industrial by-product generated in the biodiesel production, as a possible carbon source to enhance EBPR in carbon-limited urban wastewaters. Crude glycerol has non-negligible content of other organic compounds as methanol, salts, VFA and long chain fatty acids (LCFA). VFA and methanol have been demonstrated to enhance PAO activity, but there is no previous study about the effect of LCFA on PAO. This work presents the operation of an EBPR SBR system using crude glycerol as sole carbon source, studying also its long-term stability. The effect of LCFA is evaluated at short and long-term operation, demonstrating for the first time EBPR activity with LCFA as sole carbon source and its long-term failure due to the increased hydrophobicity of the sludge.

Mono- and dialkyl glycerol ether lipids in anaerobic bacteria: biosynthetic insights from the mesophilic sulfate reducer Desulfatibacillum alkenivorans PF2803T.

Bacterial glycerol ether lipids (alkylglycerols) have received increasing attention during the last decades, notably due to their potential role in cell resistance or adaptation to adverse environmental conditions. Major uncertainties remain, however, regarding the origin, biosynthesis, and modes of formation of these uncommon bacterial lipids. We report here the preponderance of monoalkyl- and dialkylglycerols (1-O-alkyl-, 2-O-alkyl-, and 1,2-O-dialkylglycerols) among the hydrolyzed lipids of the marine mesophilic sulfate-reducing proteobacterium Desulfatibacillum alkenivorans PF2803T grown on n-alkenes (pentadec-1-ene or hexadec-1-ene) as the sole carbon and energy source. Alkylglycerols account for one-third to two-thirds of the total cellular lipids (alkylglycerols plus acylglycerols), depending on the growth substrate, with dialkylglycerols contributing to one-fifth to two-fifths of the total ether lipids. The carbon chain distribution of the lipids of D. alkenivorans also depends on that of the substrate, but the chain length and methyl-branching patterns of fatty acids and monoalkyl- and dialkylglycerols are systematically congruent, supporting the idea of a biosynthetic link between the three classes of compounds. Vinyl ethers (1-alken-1'-yl-glycerols, known as plasmalogens) are not detected among the lipids of strain PF2803T. Cultures grown on different (per)deuterated n-alkene, n-alkanol, and n-fatty acid substrates further demonstrate that saturated alkylglycerols are not formed via the reduction of hypothetic alken-1'-yl intermediates. Our results support an unprecedented biosynthetic pathway to monoalkyl/monoacyl- and dialkylglycerols in anaerobic bacteria and suggest that n-alkyl compounds present in the environment can serve as the substrates for supplying the building blocks of ether phospholipids of heterotrophic bacteria.

Fusobacterium nucleatum: a commensal-turned pathogen.

Fusobacterium nucleatum is an anaerobic oral commensal and a periodontal pathogen associated with a wide spectrum of human diseases. This article reviews its implication in adverse pregnancy outcomes (chorioamnionitis, preterm birth, stillbirth, neonatal sepsis, preeclampsia), GI disorders (colorectal cancer, inflammatory bowel disease, appendicitis), cardiovascular disease, rheumatoid arthritis, respiratory tract infections, Lemierre's syndrome and Alzheimer's disease. The virulence mechanisms involved in the diseases are discussed, with emphasis on its colonization, systemic dissemination, and induction of host inflammatory and tumorigenic responses. The FadA adhesin/invasin conserved in F. nucleatum is a key virulence factor and a potential diagnostic marker for F. nucleatum-associated diseases.

Carbon monoxide. Toxic gas and fuel for anaerobes and aerobes: carbon monoxide dehydrogenases.

Carbon monoxide (CO) pollutes the atmosphere and is toxic for respiring organisms including man. But CO is also an energy and carbon source for phylogenetically diverse microbes living under aerobic and anaerobic conditions. Use of CO as metabolic fuel for microbes relies on enzymes like carbon monoxide dehydrogenase (CODH) and acetyl-CoA synthase (ACS), which catalyze conversions resembling processes that eventually initiated the dawn of life.CODHs catalyze the (reversible) oxidation of CO with water to CO2 and come in two different flavors with unprecedented active site architectures. Aerobic bacteria employ a Cu- and Mo-containing CODH in which Cu activates CO and Mo activates water and takes up the two electrons generated in the reaction. Anaerobic bacteria and archaea use a Ni- and Fe-containing CODH, where Ni activates CO and Fe provides the nucleophilic water. Ni- and Fe-containing CODHs are frequently associated with ACS, where the CODH component reduces CO2 to CO and ACS condenses CO with a methyl group and CoA to acetyl-CoA.Our current state of knowledge on how the three enzymes catalyze these reactions will be summarized and the different strategies of CODHs to achieve the same task within different active site architectures compared.

Uterine cervical ectopy during reproductive age: cytological and microbiological findings.

Cervical ectopy is common in adolescents, pregnant women, and those taking high doses of estrogen-containing contraceptives. The majority of cases have spontaneous reversion, but some cases can be persistent. Studies suggested that the adequacy of a Pap smear could be affected and there is an increased risk cervical infections. This study is a cross-sectional study conducted from December 2009 to February 2011 with 457 women with cervical ectopy and 736 without ectopy. Cervical samples were collected in vials for analysis by ThinPrep cytology (Hologic, Marlborough, MA). The Mann-Whitney test and Fisher's exact test (95% CI) were applied. The study was approved by the ethics committee of the Federal University of Ceará. The mean ages of the study group and control group were 28.7 (±14.8) and 33.6 (±7.5) years old, respectively (P < 0.0001). Negative diagnosis for malignancy and intraepithelial lesion was present in 399 (87%) cases and 705 (96%) in the study and control groups, respectively (P < 0.0001). Shift in the flora suggestive of bacterial vaginosis (BV) was observed more frequently in the study group: 74 (16.2%) than in the control group: 86(11.7%) (P = 0.017). The differences among the other morphotypes showed no significance. The smears were atypical in 12.7% (58/457) of the patients from the study group and in 4.2% (31/736) in the control group (P < 0.001; RR = 3 [2.033-4.712]). The association between ectopy and inflammatory cytology, the presence of the shift in the flora suggestive of BV and cytological atypia is evident.

Detección de microorganismos anaerobios en pacientes con pericoronaritis y sensibilidad a los antimicrobianos / Anaerobic microorganisms detection in patients with pericoronaritis and antimicrobial susceptibility

El propósito de este estudio fue la identificación de microorganismos anaerobios más frecuentemente encontrados en pericoronaritis y realizar pruebas de sensibilidad a los antimicrobianos. Se estudiaron los sacos pericoronarios del tercer molar en 20 pacientes. De las muestras recogidas en los 20 pacientes que presentaron pericoronaritis, solo en 7 (35%) hubo crecimiento de microorganismos anaerobios estrictos mientras que en los 13 restantes (65%) no se detectaron estos. En cuanto a las 12 cepas aisladas del saco pericoronario de los 7 pacientes, el microorganismo más frecuentemente encontrado fue Bifidobacterium spp en 5 casos (42%), Bifidobacterium adolescentis en 2 casos (17%), Veillonella spp en dos casos también (17%), Prevotella melaninogenica en 1 caso (8%), 1 caso Prevotella loescheii (8%) y en 1 caso a Prevotella oralis (8%). De los resultados obtenidos las bacterias anaerobias estrictas detectadas a partir de muestras de sacos pericoronarios fueron: Bifidobacterium spp., B. adolescentis, Veillonella spp, P. loeschii, P. melaninogenica y P. oralis.

Sterility testing of apheresis hematopoietic progenitor cell products using an automated blood culture system.

BACKGROUND: AABB Standards require monitoring of hematopoietic progenitor cell (HPC) products for microbial contamination. To date, there is no automated blood culture system cleared by the Food and Drug Administration for this application. Our objective was to validate the VersaTREK system (TREK Diagnostic Systems) for sterility testing of apheresis HPC products. STUDY DESIGN AND METHODS: Four aerobic bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus mitis, and Bacillus cereus), five anaerobic bacteria (Fusobacterium necrophorum, Clostridium perfringens, Bacteroides fragilis, Prevotella loescheii, and Propionibacterium acnes), and one fungus (Candida albicans) were spiked into apheresis HPC products at concentrations of 10, 10(2) , 10(3) , and 10(4) colony-forming units (CFUs)/mL. Aerobic and anaerobic bottles were incubated until positive or for up to 5 days. DNA was simultaneously extracted for polymerase chain reaction amplification of 16S ribosomal RNA (rRNA) gene. RESULTS: All aerobic bacteria grew in both bottles at all concentrations tested within 24 hours, and the time to positivity (TTP) was significantly shorter with aerobic bottles. C. albicans grew in the aerobic media at all concentrations within 30 hours. Anaerobes grew in the anaerobic bottle at all concentrations within 5 days. No bacteria were detected by using 16S rRNA gene amplification at 10(4) CFUs/mL. CONCLUSION: Compared to culture, 16S rRNA gene amplification of HPCs does not improve sensitivity or turnaround time for HPC sterility testing. The VersaTREK system is a reliable tool for detecting microbial contamination of apheresis HPC products with a limit of detection of less than or equal to 10 CFUs/mL. Inclusion of both the aerobic and the anaerobic culture bottles achieves the shortest TTP for all species tested.