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PubMed2002

Evaluation of the 3D BacT/ALERT automated culture system for the detection of microbial contamination of platelet concentrates.

Bacterial transmission remains the major component of morbidity and mortality associated with transfusion-transmitted infections. Platelet concentrates are the most common cause of bacterial transmission. The BacT/ALERT 3D automated blood culture system has the potential to screen platelet concentrates for the presence of bacteria. Evaluation of this system was performed by spiking day 2 apheresis platelet units with individual bacterial isolates at final concentrations of 10 and 100 colony-forming units (cfu) mL-1. Fifteen organisms were used which had been cited in platelet transmission and monitoring studies. BacT/ALERT times to detection were compared with thioglycollate broth cultures, and the performance of five types of BacT/ALERT culture bottles was evaluated. Sampling was performed immediately after the inoculation of the units, and 10 replicates were performed per organism concentration for each of the five types of BacT/ALERT bottles. The mean times for the detection of these 15 organisms by BacT/ALERT, with the exception of Propionibacterium acnes, ranged from 9.1 to 48.1 h (all 10 replicates were positive). In comparison, the time range found using thioglycollate was 12.0-32.3 h (all 10 replicates were positive). P. acnes' BacT/ALERT mean detection times ranged from 89.0 to 177.6 h compared with 75.6-86.4 h for the thioglycollate broth. BacT/ALERT, with the exception of P. acnes, which has dubious clinical significance, gave equivalent or shorter detection times when compared with the thioglycollate broth system. The BacT/ALERT system detected a range of organisms at levels of 10 and 100 cfu mL-1. This study validates the BacT/ALERT microbial detection system for screening platelets. Currently, the system is the only practically viable option available for routinely screening platelet concentrates to prevent bacterial transmission.

Aerobiosis↗

PubMed2003

Lack of interleukin-10 leads to intestinal inflammation, independent of the time at which luminal microbial colonization occurs.

Previous studies have demonstrated that the resident bacteria harbored by interleukin (IL)-10 gene-deficient mice initiate an enterocolitis in the neonatal period. The associated intestinal injury is characterized by an increase in the secretion of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, and by a systemic response to endogenous bacterial antigens, supporting the hypothesis that a lack of tolerance may be the initiating cause. Whether bacterial initiation of this enterocolitis would occur in the adult intestine or whether it is only seen in the developing neonatal intestine was not known. Adult (9 weeks of age), axenic, luminally sterile IL-10 gene-deficient mice, which do not spontaneously develop enterocolitis, were inoculated with intestinal microbial flora. These mice rapidly developed intestinal injury and demonstrated elevated levels of IFN-gamma in cecal and colonic tissue. This response precedes a systemic spleen cell response to stimulation by bacterial antigens. Similarly, axenic, IL-10 gene-deficient mice exposed to microflora as neonates experience a comparable intestinal injury and IFN-gamma release before the appearance of IFN-gamma-producing cells in the spleen. Microbial colonization in control mice leads to systemic IL-10 production, but not systemic IFN-gamma production, suggesting that an IL-10-mediated suppression regulates the response in normal control mice but is absent in IL-10 gene-deficient mice. Our results suggest that the point at which intestinal microbial colonization occurs does not significantly influence the severity or specificity of the inflammatory response in IL-10 gene-deficient mice. The lack of tolerance to bacterial antigens appears to result from the absence of IL-10 during bacterial exposure.

Animals↗

PubMed2005

Use of chitosan to prolong mozzarella cheese shelf life.

This study was undertaken to evaluate the feasibility of using chitosan, a natural antimicrobial substance, to improve the preservation of a very perishable cheese. The effectiveness of chitosan to inhibit the growth of spoilage microorganisms in Mozzarella cheese was studied during refrigerated storage. A lactic acid/chitosan solution was added directly to the starter used for Mozzarella cheese manufacturing. Mozzarella cheese samples were stored at 4 degrees C for about 10 d and microbial populations as well as the pH were monitored. Results demonstrated that chitosan inhibited the growth of some spoilage microorganisms such as coliforms, whereas it did not influence the growth of other microorganisms, such as Micrococcaceae, and lightly stimulated lactic acid bacteria.

Anti-Infective Agents↗

PubMed2005

Development of improved method for isolation of Mycobacterium avium subsp. paratuberculosis from bulk tank milk: effect of age of milk, centrifugation, and decontamination.

Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne's disease in cattle and it has been suggested that this organism may be associated with Crohn's disease in humans. Cows at the advanced stage of the disease shed this organism into both their milk and feces. The objective of this study was to develop a more efficient procedure for isolating MAP from bulk tank raw milk. Bulk tank raw milk (50 mL) samples 3 to 13 d old after collection without spiking were investigated to evaluate the effects of milk age on the efficacy of decontamination. Milk samples, 2 to 3 d old, were seeded with MAP at levels of 50 to 200 colony forming units/mL in experiments involving factorial design to evaluate 1) the effects of different decontaminating reagents and decontamination procedures on recovery of MAP, and 2) partition MAP in milk fractions after centrifugation in raw milk. Decontamination in 20 mL of 0.75% hexadecylpyridinium chloride (HPC) at room temperature (22 degrees C) for 2 to 5 h, with shaking, at intervals was found to be the most effective procedure for decontaminating milk 2 to 3 d old. Prolonged exposure to decontaminants, additional incubations in antibiotics, or at higher temperature (37 degrees C) significantly reduced recovery of live MAP. Enhanced growth of microbial contaminants was noticed in samples decontaminated overnight at room temperature compared to those decontaminated for 2 to 5 h. Decontamination of 6 d old milk samples required extra incubation in antibiotic brew. Decontamination of milk samples that are 8 d and older was not effective in removing microbial contaminants. The MAP cells preferentially partitioned into the cream fraction after centrifugation, and combining the milk cream and pellet fractions enhanced recovery of MAP. A recovery rate of 16.6% was estimated with the use of our improved protocol.

Animals↗

PubMed1991

[Microbial biocenosis in normal newborn babies and in septic diseases].

A study was made of the symbion microflora of different biotopes in 185 neonates with sepsis and 200 healthy neonates on natural feeding. Appreciable differences were revealed in the formation of the microbial ecosystems in children with sepsis as compared to the healthy neonates. In the septic process the microbiological profile for different biotopes is poorly differentiated and is characterized by the similarity of opportunistic bacteria isolated. The children who suffered pyoseptic pathology need additional bacterial therapy aimed at the recovery of the normal microflora.

Bacteria↗

PubMedDate not supplied

[Bacteriological quality of spring and well water in Yaounde (Cameroon)].

The drinking water supply in Yaounde, Cameroon, often fails to meet demand and this leads many people to seek water from springs and wells the microbiological quality of which is unknown. We carried out a microbiological survey of five spring water points and ten wells. The fifteen study sites were located in various residential districts and were selected according to geographical location and number of users. Microorganisms were isolated on specific culture media using the membrane filter technique. We found that these water supplies contained many types of bacteria including Pseudomonas aeruginosa, Aeromonas hydrophila and indicators of fecal contamination. Maximum annual concentrations differed at the various sites and over time, from 750 to 98,000 CFU/l for P. aeruginosa, 40 to 78,000 CFU/l for A. hydrophila and 300 to 58,000 CFU/l for fecal indicator bacteria. The density of fecal bacteria was strongly correlated (p < 0.01) with those of P. aeruginosa in spring water and A. hydrophila in well water. The degree of correlation between microbial concentrations differed between sites. The source of fecal contamination differed between sites and was animal, human or both. The densities of bacteria recorded were high. Therefore, consumers of water drawn from these sources are at short-term risk of contracting waterborne diseases.

Cameroon↗

PubMed2001

Bacteria and Archaea physically associated with Gulf of Mexico gas hydrates.

Although there is significant interest in the potential interactions of microbes with gas hydrate, no direct physical association between them has been demonstrated. We examined several intact samples of naturally occurring gas hydrate from the Gulf of Mexico for evidence of microbes. All samples were collected from anaerobic hemipelagic mud within the gas hydrate stability zone, at water depths in the ca. 540- to 2,000-m range. The delta(13)C of hydrate-bound methane varied from -45.1 per thousand Peedee belemnite (PDB) to -74.7 per thousand PDB, reflecting different gas origins. Stable isotope composition data indicated microbial consumption of methane or propane in some of the samples. Evidence of the presence of microbes was initially determined by 4,6-diamidino 2-phenylindole dihydrochloride (DAPI) total direct counts of hydrate-associated sediments (mean = 1.5 x 10(9) cells g(-1)) and gas hydrate (mean = 1.0 x 10(6) cells ml(-1)). Small-subunit rRNA phylogenetic characterization was performed to assess the composition of the microbial community in one gas hydrate sample (AT425) that had no detectable associated sediment and showed evidence of microbial methane consumption. Bacteria were moderately diverse within AT425 and were dominated by gene sequences related to several groups of Proteobacteria, as well as Actinobacteria and low-G + C Firmicutes. In contrast, there was low diversity of Archaea, nearly all of which were related to methanogenic Archaea, with the majority specifically related to Methanosaeta spp. The results of this study suggest that there is a direct association between microbes and gas hydrate, a finding that may have significance for hydrocarbon flux into the Gulf of Mexico and for life in extreme environments.

Archaea↗

PubMed2002

Calorimetric determination of inactivation parameters of micro-organisms.

AIMS: This study aimed to apply differential scanning calorimetry (DSC) to evaluate the thermal inactivation kinetics of bacteria. METHODS AND RESULTS: The apparent enthalpy (DeltaH) of Escherichia coli cells was evaluated by a temperature scan in a DSC after thermal pretreatment in the calorimeter to various temperatures between 56 and 80 degrees C. Conventional semilogarithmic survival curve analysis was combined with a linearly increasing temperature protocol. Calorimetrically determined D and z values were compared to those obtained from plate count data collected under isothermal conditions to validate the new approach. CONCLUSIONS: The calculated D values using both apparent enthalpy and viability data for cells heat treated in the DSC were similar to the D values obtained from isothermal treatment. Temperatures for 1 through 10-log microbial population reductions, calculated from plate count and enthalpy data, were in agreement within 0.5-2.4 degrees C at a 4 degrees C min-1 heating rate. SIGNIFICANCE AND IMPACT OF THE STUDY: This novel calorimetric method provides an approach to obtain accurate and reproducible kinetic parameters for inactivation. The calorimetric method here described is time efficient and is conducted under conditions similar to food processing conditions.

Calorimetry, Differential Scanning↗

PubMed2006

Comparison of bacterial plaque samples from titanium implant and tooth surfaces by different methods.

Studies have shown similarities in the microflora between titanium implants or tooth sites when samples are taken by gingival crevicular fluid (GCF) sampling methods. The purpose of the present study was to study the microflora from curette and GCF samples using the checkerboard DNA-DNA hybridization method to assess the microflora of patients who had at least one oral osseo-integrated implant and who were otherwise dentate. Plaque samples were taken from tooth/implant surfaces and from sulcular gingival surfaces with curettes, and from gingival fluid using filter papers. A total of 28 subjects (11 females) were enrolled in the study. The mean age of the subjects was 64.1 years (SD+/-4.7). On average, the implants studied had been in function for 3.7 years (SD+/-2.9). The proportion of Streptococcus oralis (P<0.02) and Fusobacterium periodonticum (P<0.02) was significantly higher at tooth sites (curette samples). The GCF samples yielded higher proportions for 28/40 species studies (P-values varying between 0.05 and 0.001). The proportions of Tannerella forsythia (T. forsythensis), and Treponema denticola were both higher in GCF samples (P<0.02 and P<0.05, respectively) than in curette samples (implant sites). The microbial composition in gingival fluid from samples taken at implant sites differed partly from that of curette samples taken from implant surfaces or from sulcular soft tissues, providing higher counts for most bacteria studied at implant surfaces, but with the exception of Porphyromonas gingivalis. A combination of GCF and curette sampling methods might be the most representative sample method.

Bacteroides↗

PubMed1993

Microbiological predictors of caries risk.

The prediction of caries risk has been of long-standing interest. Generally, few of the tests involving oral bacteria or their products have become accepted. Presently, the main focus is on counts of lactobacilli (L) and mutans streptococci (MS). Due to their positive numerical association with human caries and the linkage of this association to carbohydrate consumption, counts of L and MS may, potentially, serve not only as a caries risk predictor but also as an indicator of carbohydrate consumption, another caries-risk factor. The value of counts of L and MS as caries-risk predictors has been evaluated by means of studies providing data on test sensitivity, specificity, and predictive values. These and other studies indicate that their use for the prediction of caries risk of individuals is not possible but is more promising for that of the caries risk of groups (e.g., identification of high-caries-risk subjects); further, the prediction of low caries risk may be more reliable than that of high caries risk. The influence of test variables on the test results has been discussed. These include the level of caries increment, subject age, methods of caries evaluation, use of saliva or dental plaque as test sample, sampling frequency, type of bacterial growth medium, and the use of simplified methods rather than conventional laboratory procedures for microbial enumeration. An approach to optimize the use of microbiological caries-risk predictors in different populations as well as their use in conjunction with other caries-risk predictors has been discussed. The latter include the incipient caries lesion or past caries experience and salivary buffering capacity and flow rate. Due to the multifactorial nature of caries etiology, it is expected that multivariate approaches rather than the use of single parameters may improve caries risk prediction for individuals as well as groups of subjects.

Colony Count, Microbial↗

PubMed2000

An archaeal iron-oxidizing extreme acidophile important in acid mine drainage.

A new species of Archaea grows at pH approximately 0.5 and approximately 40 degrees C in slime streamers and attached to pyrite surfaces at a sulfide ore body, Iron Mountain, California. This iron-oxidizing Archaeon is capable of growth at pH 0. This species represents a dominant prokaryote in the environment studied (slimes and sediments) and constituted up to 85% of the microbial community when solution concentrations were high (conductivity of 100 to 160 millisiemens per centimeter). The presence of this and other closely related Thermoplasmales suggests that these acidophiles are important contributors to acid mine drainage and may substantially impact iron and sulfur cycles.

Biofilms↗

PubMed1994

DNA probe analyses of the survival of selected periodontal pathogens following scaling, root planing, and intra-pocket irrigation.

This clinical study evaluated the survival rates of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and Prevotella intermedia in periodontal pockets following scaling and root planing and intra-pocket irrigation with antimicrobial agents in patients with moderate and severe periodontitis. The number of target organisms was determined utilizing DNA probes. Adult periodontitis patients were selected on the basis that the subgingival flora contained at least one of the target organisms. Forty-eight (48) inflamed pockets > or = 5 mm in depth with probing attachment loss and containing at least one of the target species were then selected in 7 adult patients who harbored these bacteria. Following baseline clinical and bacterial examination, all patients received thorough scaling and root planing. In addition, 1 or 2 teeth in each patient which harbored the target flora at baseline were randomly assigned to each of the following 4 treatment modalities: 1) control group, no irrigation; 2) saline group, irrigation with 2 cc of physiologic saline; 3) tetracycline group, irrigation with 2 cc of aqueous tetracycline hydrochloride, 50 mg/ml (5%); and 4) chlorhexidine group, irrigation with 2 cc 0.12% chlorhexidine. All selected sites (5 to 8 per patient) were nonadjacent teeth. Clinical parameters and microbial analysis were recorded again at one week, and one month post-irrigation. The survival rate of the target microorganisms was determined and the effect of irrigation with antimicrobial agents on this microflora was compared with the control groups (1 and 2).(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed2006

Pathogen reduction in unpasteurized apple cider: adding cranberry juice to enhance the lethality of warm hold and freeze-thaw steps.

U.S. Food and Drug Administration (FDA) regulations require processors of apple cider sold wholesale to use processing steps that ensure a 5-log reduction in numbers of the pertinent pathogen, generally considered to be Escherichia coli O157:H7. Current widely used validated pathogen-reduction steps are thermal pasteurization and UV light treatment. These techniques may be unaffordable or undesirable for some processors. This study investigated the cran-cider process, which is the addition of cranberry juice at a 15% (vol/vol) level, followed by warm hold (45 degrees C for 2 h) and freeze-thaw steps (-20 degrees C for 24 h, 5 degrees C for 24 h). When enumeration procedures did not include injury repair, the cran-cider process achieved a > or = 5-log reduction in numbers of E. coli O157:H7, Salmonella serovars, and Listeria monocytogenes. However, an injury-repair step was included in the pathogen enumeration procedure in confirmatory trials, and the resulting E. coli O157:H7 reductions of 3.5 to 4.2 log did not meet the FDA requirement. Consumer evaluation of apple cider subjected to the cran-cider process was favorable with a mean (n = 197) score of 5.8 on a seven-point hedonic scale (where 6 equals "like moderately") and 89% of panelists giving the product a positive score of 5, 6, or 7. The cran-cider process provides a novel way to improve microbial safety of unpasteurized apple cider, but it does not meet FDA-mandated pathogen reductions for wholesalers. However, cider makers selling apple cider only at retail could use the process to improve the safety of their product, provided containers were labeled with the FDA-mandated consumer warning.

Beverages↗

PubMed2006

Artisanal and experimental Pecorino Siciliano cheese: microbial dynamics during manufacture assessed by culturing and PCR-DGGE analyses.

Traditional artisanal Pecorino Siciliano (PS) cheeses, and two experimental PS cheeses were manufactured using either raw or pasteurised ewes' milk with the addition of starter cultures. The bacterial diversity and dynamics of the different cheese types were evaluated both by culturing and characterisation of isolates, and a culture-independent approach based on the 16S ribosomal RNA (rRNA) gene. Following cultivation, artisanal and experimental cheese types showed similar microbial counts, and isolates belonging to Lactococcus lactis, Streptococcus thermophilus, Enterococcus faecalis and Leuconostoc mesenteroides were identified by phenotypic characterisation and comparison of the restriction fragment length polymorphism (RFLP) of the 16S rRNA gene to that of reference species. The culture-independent fingerprinting technique PCR and denaturing gradient gel electrophoresis (DGGE) of V6 to V8 regions of the 16S rRNA gene of samples taken during artisanal PS cheese manufacture, from raw milk to the ripened cheese, indicated relevant shifts in the microbial community structure. The dominance of Streptococcus bovis and Lactococcus lactis species in the traditional artisanal PS was revealed by 16S rRNA gene sequencing. Comparison of DGGE profiles of samples from milk to ripened cheese, derived from artisanal procedure and the two experimental PS cheeses during production showed similar trends with the presence of intense bands in common. Nevertheless, the profiles of several artisanal cheeses from different farms appeared more diverse, and these additional species are probably responsible for the generally superior flavour and aroma development of traditional PS cheese.

Animals↗

PubMed2001

Protegrin-1 enhances bacterial killing in thermally injured skin.

OBJECTIVE: Septic complications and the emergence of drug-resistant microbes represent serious risks to patients. Recently, naturally occurring peptides have been discovered that possess potent and broad-spectrum antimicrobial activity. Protegrin-1 is particularly attractive for clinical use in human wounds because, unlike defensins, protegrin-1 retains broad antimicrobial and antifungal activity at physiologic salt concentration and in the presence of serum. The objective of this study was to examine the efficacy of protegrin-1 in killing multiple drug-resistant microbes isolated from human burn patients. DESIGN: For thein vitroexperiment, bilayer radial diffusion was performed comparing standard antibiotics with protegrin-1 on multiple-drug-resistant microbial organisms isolated from infected burn wounds. In vivo, rats received a 20% total body surface area partial-thickness burn by immersion in 60 degrees C water for 20 secs followed by wound seeding with 106 colony forming units of Silvadene-resistant Pseudomonas aeruginosa. SETTING: University of Michigan research laboratory. SUBJECTS: Adult, male Sprague-Dawley rats. INTERVENTIONS: Rats were randomized into three groups: those receiving synthetic protegrin-1, acetic acid (carrier), or gentamicin (positive control). Protegrin-1 was administered by topical application or intradermal injection. Wound tissues were harvested aseptically at different time points for quantitative bacterial counts. MEASUREMENTS AND MAIN RESULTS: In vivo and in vitro experiments revealed rapid and significant decreases in bacterial counts for protegrin-1-treated groups compared with controls. CONCLUSIONS: This study shows that protegrin-1 potentially may be used as an alternative or adjunct therapy to standard agents used to treat wound infections.

Administration, Topical↗

PubMed2005

Modeling the survival of Salmonella spp. in chorizos.

The survival of Salmonella spp. in chorizos has been studied under the effect of storage conditions; namely temperature (T=6, 25, 30 degrees C), air inflow velocity (F=0, 28.4 m/min), and initial water activity (a(w0)=0.85, 0.90, 0.93, 0.95, 0.97). The pH was held at 5.0. A total of 20 survival curves were experimentally obtained at various combinations of operating conditions. The chorizos were stored under four conditions: in the refrigerator (Ref: T=6 degrees C, F=0 m/min), at room temperature (RT: T=25 degrees C, F=0 m/min), in the hood (Hd: T=25 degrees C, F=28.4 m/min), and in the incubator (Inc: T=30 degrees C, F=0 m/min). Semi-logarithmic plots of counts vs. time revealed nonlinear trends for all the survival curves, indicating that the first-order kinetics model (exponential distribution function) was not suitable. The Weibull cumulative distribution function, for which the exponential function is only a special case, was selected and used to model the survival curves. The Weibull model was fitted to the 20 curves and the model parameters (alpha and beta) were determined. The fitted survival curves agreed with the experimental data with R(2)=0.951, 0.969, 0.908, and 0.871 for the Ref, RT, Hd, and Inc curves, respectively. Regression models relating alpha and beta to T, F, and a(w0) resulted in R(2) values of 0.975 for alpha and 0.988 for beta. The alpha and beta models can be used to generate a survival curve for Salmonella in chorizos for a given set of operating conditions. Additionally, alpha and beta can be used to determine the times needed to reduce the count by 1 or 2 logs t(1D) and t(2D). It is concluded that the Weibull cumulative distribution function offers a powerful model for describing microbial survival data. A comparison with the pathogen modeling program (PMP) revealed that the survival kinetics of Salmonella spp. in chorizos could not be adequately predicted using PMP which underestimated the t(1D) and t(2D). The mean of the Weibull probability density function correlated strongly with t(1D) and t(2D), and can serve as an alternative to the D-values normally used with first-order kinetic models. Parametric studies were conducted and sensitivity of survival to operating conditions was evaluated and discussed in the paper. The models derived herein provide a means for the development of a reliable risk assessment system for controlling Salmonella spp. in chorizos.

Animals↗

PubMed2002

A geostatistical analysis of small-scale spatial variability in bacterial abundance and community structure in salt marsh creek bank sediments.

Small-scale variations in bacterial abundance and community structure were examined in salt marsh sediments from Virginia's eastern shore. Samples were collected at 5 cm intervals (horizontally) along a 50 cm elevation gradient, over a 215 cm horizontal transect. For each sample, bacterial abundance was determined using acridine orange direct counts and community structure was analyzed using randomly amplified polymorphic DNA fingerprinting of whole-community DNA extracts. A geostatistical analysis was used to determine the degree of spatial autocorrelation among the samples, for each variable and each direction (horizontal and vertical). The proportion of variance in bacterial abundance that could be accounted for by the spatial model was quite high (vertical: 60%, horizontal: 73%); significant autocorrelation was found among samples separated by 25 cm in the vertical direction and up to 115 cm horizontally. In contrast, most of the variability in community structure was not accounted for by simply considering the spatial separation of samples (vertical: 11%, horizontal: 22%), and must reflect variability from other parameters (e.g., variation at other spatial scales, experimental error, or environmental heterogeneity). Microbial community patch size based upon overall similarity in community structure varied between 17 cm (vertical) and 35 cm (horizontal). Overall, variability due to horizontal position (distance from the creek bank) was much smaller than that due to vertical position (elevation) for both community properties assayed. This suggests that processes more correlated with elevation (e.g., drainage and redox potential) vary at a smaller scale (therefore producing smaller patch sizes) than processes controlled by distance from the creek bank.

Bacteria↗

PubMed1992

High cell density and high-productivity microbial fermentation.

Several interesting approaches to high cell density systems, molecular strategies coupled with fermentation technology, and updated traditional strategies have been used in the past year to obtain high productivity in the formation of important products. The most significant advances include new strategies for controlling high cell density fermentation reactors and expression of heterologous proteins in different strains of yeasts.

Bacteria↗