Total Colony-forming Unit Counts Research Articles

Towards a new biological control approach for Photorhabdus temperata bioinsecticide production through the bioconversion of Tunisian industrial wastewater

A novel bioconversion approach of Tunisian wastewater to low-cost Photorhabdus temperata bioinsecticide is presented in this study. Our results showed that when cultured on the food industry wastewater (WS4), P. temperata cells exhibited oral toxicity of about 42%, which is the same as those cultured in complex medium (CM), used as control. Moreover, variants small colony polymorphism (Vsm) of the strain K122 was completely avoided after a prolonged incubation. However, viable but non-culturable (VBNC) state was enhanced with the maximum colony-forming units (CFU) count of 9 × 106 cells/mL obtained after 48 h of incubation in the WS4. According to flow cytometry analysis, almost 100% of P. temperata cells were viable until 48 h of incubation. The appearance of propidium iodide (PI) positively stained cells was observed after a prolonged incubation with a maximum of 17% of damaged cells in WS1. In order to follow the progress of P. temperata fermentation process carried out in industrial wastewater, we established for the first time, the mathematical relationship between total cell counts, CFU counts and oral toxicity of P. temperata strain K122. Indeed, irrespective of the medium used, the relationship between CFU count and total cell count followed a power law. Additionally, when plotting CFU count, or total cell count against toxicity, a semi-log linear relationship was obtained. Our results proved the efficiency of this bioconversion approach to produce bioinsecticide based on the entomopathogenic bacterium P. temperata, with practical benefits in terms of cost production and wastewater management.

Prevalence and quantification of contamination of knitted cotton outer gloves during hip and knee arthroplasty surgery

Knitted cotton outer gloves offer protection against surgical glove perforation and provide improved grip on instruments. These gloves absorb blood and other fluids during surgery, and may therefore also accumulate contaminating bacteria. To date, there is no published data on microbial contamination of such gloves during surgery. Knitted cotton outer gloves used in primary and revision hip and knee arthroplasty from two Swiss hospitals were analysed by quantitative bacteriology. Samples were subjected to sonication and vortexing, followed by membrane filtration of the sonicate. Membranes were incubated under aerobic and anaerobic culture conditions, respectively, for 21 days. Total microbial load for each pair of gloves was determined by colony-forming units (CFU) count. Strain identification was performed with MALDI-TOF. A total of 43 pairs of gloves were collected from continuous series of surgeries. Under aerobic culture conditions, total CFU counts ranged 0-1103, 25 (58%) samples remaining sterile, and 4 (9%) yielding > 100CFU. Under anaerobic culture conditions, total CFU counts ranged 0-3579, 22 (51%) samples remaining sterile, 6 (14%) yielding > 100CFU. The only covariate significantly associated with the level of contamination was the provider hospital (p < 0.0001 for aerobic and p = 0.007 for anaerobic cultures). Strain identification revealed only skin commensals, mainly coagulase-negative staphylococci and Propionibacterium spp. While contamination of surgical latex gloves is a well-known issue, no study has examined so far contamination of knitted cotton outer gloves. No or very low microbial contamination could be identified in the majority of the knitted cotton outer gloves assayed. However, a relevant proportion showed contamination far higher than estimated minimal thresholds for implant-associated infection. Clinical relevance of these findings remains to be established.

Aldehyde dehydrogenase activity in cryopreserved cord blood cells for quality assessment prior to transplantation.

In umbilical cord blood transplantation (UCBT), the number of cluster of differentiation (CD)34+ cells and colony‑forming units (CFUs) in the cord blood (CB) graft positively correlate with patient survival. Therefore, these parameters are currently used for quality assessment of the cryopreserved CB cells in the attached segment that is considered representative of the CB in the main bag prior to UCBT. Since aldehyde dehydrogenase (ALDH) activity is high in hematopoietic stem cells, the number of ALDH‑bright (ALDHbr) cells was examined in comparison with the number of CD34+ cells and CFUs for the quality assessment of CB units. In the cryopreserved main bag, the number of ALDHbr cells in the CB unit exhibited positive correlation with the number of CD34+ cells, and with CFU‑granulocytes/macrophages and total CFU counts. Furthermore, the concentration of ALDHbr cells in the cryopreserved attached segment was not significantly different compared to that of the main bag, suggesting that the attached segment is representative of the main bag. In conclusion, the present study suggested that ALDHbr cell counts in the cryopreserved attached segments may serve as a quality assessment indicator for CB units prior to UCBT.

A simple model describes development of early peaks in oomycete zoospore inoculum detected in southern UK outdoors horticultural reservoirs

The numbers of water-borne oomycete propagules in outdoor reservoirs used in horticultural nurseries within the UK are investigated in this study. Water samples were recovered from 11 different horticultural nurseries in the southern UK during Jan–May in 2 “cool” years (2010 and 2013; winter temperatures 2.0 and 0.4°C below UK Met Office 30 year winter average, respectively) and 2 “warm” years (2008 and 2012; winter temperatures 1.2 and 0.9°C above UK Met Office 30 year winter average, respectively). Samples were analyzed for total number of oomycete colony forming units (CFU), predominantly members of the families Saprolegniaceae and Pythiaceae, and these were combined to give monthly mean counts. The numbers of CFU were investigated with respect to prevailing climate in the region: mean monthly air temperatures calculated by using daily observations from the nearest climatological station. The investigations show that the number of CFU during spring can be explained by a linear first-order equation and a statistically significant &lt;em&gt;r&lt;/em&gt;&lt;sup&gt;2&lt;/sup&gt; value of 0.66 with the simple relationship: [&lt;em&gt;CFU&lt;/em&gt;] = &lt;em&gt;a&lt;/em&gt;(&lt;em&gt;T&lt;/em&gt; − &lt;em&gt;T&lt;/em&gt;&lt;span&gt;&lt;em&gt;&lt;sub&gt;b&lt;/sub&gt;&lt;/em&gt;&lt;/span&gt;) − &lt;em&gt;b&lt;/em&gt;, where &lt;em&gt;a&lt;/em&gt; is the rate of inoculum development with temperature &lt;em&gt;T&lt;/em&gt;, and &lt;em&gt;b&lt;/em&gt; is the baseload population at temperatures below &lt;em&gt;T&lt;/em&gt;&lt;span&gt;&lt;em&gt;&lt;sub&gt;b&lt;/sub&gt;&lt;/em&gt;&lt;/span&gt;. Despite the majority of oomycete CFU detected being non-phytopathogenic members of the Saprolegniaceae, total oomycete CFU counts are still of considerable value as indicators of irrigation water treatment efficacy and cleanliness of storage tanks. The presence/absence of &lt;em&gt;Pythium&lt;/em&gt; spp. was also determined for all samples tested, and &lt;em&gt;Pythium&lt;/em&gt; CFU were found to be present in the majority, the exceptions all being particularly cold months (January and February 2010, and January 2008). A simple scenario study (+2 deg C) suggests that abundance of water-borne oomycetes during spring could be affected by increased temperatures due to climate change.

Contamination of Stethoscopes and Physicians' Hands After a Physical Examination

To compare the contamination level of physicians' hands and stethoscopes and to explore the risk of cross-transmission of microorganisms through the use of stethoscopes. We conducted a structured prospective study between January 1, 2009, and May 31, 2009, involving 83 inpatients at a Swiss university teaching hospital. After a standardized physical examination, 4 regions of the physician's gloved or ungloved dominant hand and 2 sections of the stethoscopes were pressed onto selective and nonselective media; 489 surfaces were sampled. Total aerobic colony counts (ACCs) and total methicillin-resistant Staphylococcus aureus (MRSA) colony-forming unit (CFU) counts were assessed. Median total ACCs (interquartile range) for fingertips, thenar eminence, hypothenar eminence, hand dorsum, stethoscope diaphragm, and tube were 467, 37, 34, 8, 89, and 18, respectively. The contamination level of the diaphragm was lower than the contamination level of the fingertips (P<.001) but higher than the contamination level of the thenar eminence (P=.004). The MRSA contamination level of the diaphragm was higher than the MRSA contamination level of the thenar eminence (7 CFUs/25 cm(2) vs 4 CFUs/25 cm(2); P=.004). The correlation analysis for both total ACCs and MRSA CFU counts revealed that the contamination level of the diaphragm was associated with the contamination level of the fingertips (Spearman's rank correlation coefficient, ρ=0.80; P<.001 and ρ=0.76; P<.001, respectively). Similarly, the contamination level of the stethoscope tube increased with the increase in the contamination level of the fingertips for both total ACCs and MRSA CFU counts (ρ=0.56; P<.001 and ρ=.59; P<.001, respectively). These results suggest that the contamination level of the stethoscope is substantial after a single physical examination and comparable to the contamination of parts of the physician's dominant hand.

Lncrna Hematlas Defines Blood Lineage-Specific RNA Expression Signatures and Novel Lincrna Biomarkers

Long non-coding RNAs (lncRNAs) recently emerged as central regulators of chromatin and gene expression. We created a comprehensive lncRNA HemAtlas in human and murine blood cells. We sampled RNA from differentiated granulocytes, monocytes, erythroid precursors, in vitro maturated megakaryocytes, CD4-T and CD8-T cells, NK cells, B cells and stem cells (human CD34+ cord blood hematopoietic stem and progenitor cells [CB-HSPCs]) and subjected them to microarray analysis of mRNA and lncRNA expression. Moreover, the human LncRNA HemAtlas was complemented with human hematopoietic stem cells (HSCs; CD34+/CD38-), megakaryocytic/erythroid progenitors (MEPs; CD34+/CD38+/CD45RA-/CD123-), common myeloid progenitors (CMPs; CD34+/CD38+/CD45RA-/CD123+) and granulocytic/monocytic progenitors (GMPs; CD34+/CD38+/CD45RA+/CD123+) from fetal liver (FL), CB and peripheral blood (PB) HSPCs.The complete microarray profiling of the differentiated cells yielded a total of 1588 (on Arraystar® platform) and 1439 lncRNAs (on NCode® platform), which were more than 20-fold differentially expressed between the blood lineages. Thus, a core fraction of lncRNAs is modulated during differentiation. LncRNA subtype comparison for each lineage, schematics of mRNA:lncRNA lineage coexpression and genomic loci correlation revealed a complex genetic interplay regulating hematopoiesis.Integrated bioinformatic analyses determined the top 50 lineage-specific lncRNAs for each blood cell lineage in both species, while gene set enrichment analysis (GSEA) confirmed lineage identity. The megakaryocytic/erythroid expression program was already evident in MEPs, while monocytoc/granulocytic signatures were found in GMPs.Amongst all significantly associated genes, 46% were lncRNAs, while 5% belonged to the subgroup of long intervening non-coding RNAs (lincRNA).For human megakaryocytes, erythroid cells, monocytes, granulocytes and HSPCs we validated four lincRNA candidates, respectively, to be specifically expressed by qRT-PCR. RNAi knock-down studies using two shRNA constructs per candidate demonstrated an impact on proliferation, survival or lineage specification for at least one specific lincRNA per lineage. We detected a 3 to 4.5-fold increased colony-forming capacity upon knockdown of the HSPC-specific PTMAP6 lincRNA in methylcellulose colony-forming unit (CFU) assays. Inversely, knockdown of monocyte-specific DB519945 resulted in 3.5 to 5.5-fold reduction of the total number of CFUs. Likewise, the total CFU counts was 4.3-fold reduced upon knockdown of megakaryocyte-specific AK093872. Kockdown of the granulocyte-specific LINC00173 perturbed granulocytic in vitro differentiation as assessed by the percentage of CD66b+/CD13+ granulocytes (2-fold reduction) and nuclear lobulation (MGG-stained cytospins). The erythroid-specific transcript AY034471 showed 25 to 50% reduction in burst-forming units in collagen-based assays.Thus, our study provides a global human hematopoietic lncRNA expression resource and defines blood-lineage specific lncRNA marker and regulator genes. Disclosures:No relevant conflicts of interest to declare.

Quantitative Analysis of Fecal Flora in Goat Kids with and without Floppy Kid Syndrome

Floppy kid syndrome (FKS) affects goat kids in the first month of life and is associated with high morbidity and mortality rates. The condition is characterized by neurological signs that can be ascribed to increased plasma D-lactate concentrations. The source of D-lactate has not been identified conclusively, but D-lactate-producing bacteria in the large intestine are thought to be involved. To determine the number of colony-forming unit (CFUs) of certain groups of bacteria in the feces of kids with and without FKS. Nineteen goat kids with clinical signs of FKS, acidemia (pH ≤ 7.2), and plasma D-lactate concentration >7 mM and 15 healthy goat kids without acidemia (pH >7.2) and D-lactate concentration <1 mM. In this case-control study, the goat kids were examined clinically and blood was collected to measure D-lactate concentration, blood gases, and acid-base parameters. Fecal samples were collected and the total aerobic bacterial count and CFU counts of coliforms, enterococci, staphylococci, streptococci, lactobacilli, and clostridia were determined using the surface plating method. Goat kids with FKS had a mean plasma D-lactate concentration of 10.9 ± 3.7 mM compared with 0.3 ± 0.9 mM in healthy kids, and significantly greater CFU counts for enterococci, streptococci, staphylococci, and lactobacilli than healthy kids. The groups of bacteria present in greater numbers in the feces of goat kids with FKS include several D-lactate-producing species, which makes dysbacteriosis a likely cause of the increased plasma D-lactate concentration in FKS.

Epiphytic microbial communities on foliage of fungicide treated and non-treated flowering dogwoods

Microbial populations on leaves of Cornus florida (flowering dogwood) in two state parks, a commercial nursery (CN) and at Tennessee State University (TSU) were assessed monthly from May through September. Trees at the TSU location were treated with propiconazole or thiophanate-methyl, at the CN with thiophanate-methyl in rotation with propiconazole at 12–14 days intervals. The highest numbers of colony forming units (CFU) were detected in Rock Island State Park (RISP) and in the CN while the lowest numbers were in Fall Creek Falls State Park (FCFSP) and TSU. Incidence of powdery mildew caused by Erysiphe pulchra was severe in non-treated flowering dogwood at CN and TSU, not in the state parks. Fungicides treatments were highly effective in controlling powdery mildew, but did not significantly affect the microbial populations as indicated by total CFU counts. Because E. pulchra is a biotroph and does not grow on artificial media, it was not included in the tabulation. Experimental data suggest that fungicide treatments did not kill all epiphytes; if they did, there was a rapid re-colonization and replacement with new organisms. Bacteria and yeasts CFU counts were higher than for filamentous fungi. No difference in microbial diversity was detected between fungicide-treated and non-treated trees. In addition to Cladosporium spp. isolated from fungicide treated plots at CN and TSU, several epiphytes known to suppress foliar diseases were isolated from the two state parks where fungicides were not applied.

NO TITLE AVAILABLE

Polishing of dental prostheses can cause a dangerous cycle of cross-contamination involving dentists, laboratory technicians, patients and auxiliary personnel. The aim of this study was to show the microbial contamination in the dental laboratory during the polishing procedure of complete dentures. For this purpose, 4 experiments were conducted. Experiment I -- Determination of the total colony-forming units (CFU) counts contaminating complete maxillary dentures. During the polishing procedure, determination of the CFU counts transferred to the operator (Experiment II) and of the total CFU counts transferred to previously sterilized complete dentures (Experiment III). Experiment IV -- The total counts of remaining CFU in the lathe spindle after Experiments II and III. Complete dentures were highly contaminated (mean = 1.4 x 10(7) CFU/mL). There was a elevated level of contamination by splatter and aerosols. There was high microbial transfer from the contaminated lathe spindle to the sterile prostheses (mean = 1.7 x 10(7) CFU/mL). The spindles were highly contaminated after polishing procedures (mean = 3.5 x 10(8) CFU/mL). The polishing of dental prostheses is a possible source of transmission of communicable diseases in the laboratory and requires improved techniques for infection control.

Enumeration of Viable Fungi in Occupational Environments: A Comparison of Samplers and Media

The performance of sampling for fungi using four widely used microbiological air sampling devices and three broad spectrum media was compared in seven occupational settings. Total colony forming units counts ranged from 10 to 3700 CFU/m3. The sampling efficiencies of the Andersen N-6 sampler, slit sampler, and Reuter centrifugal air sampler (RCS) were comparable, whereas the SAS sampler underestimated CFU counts by approximately 50%. The correlation between the RCS results and the other three samplers used was lower compared to their mutual correlations. There were no large differences in performance of the three media tested: malt extract agar, dichloran glycerol (18) agar, and dichloran rose bengal chloramphenicol agar. Coefficients of variation of duplicate samples were large compared to commonly used methods for sampling dust and chemicals. It is concluded that the N-6 and slit sampler perform best, although RCS is useful in probes because it is easy to handle. The three media tested yielded similar results. Further testing of DG-18 is recommended, as it inhibits fast growing genera and, thus, counting colonies is easier.

Microbiota and crevicular fluid collagenase activity in the osseointegrated dental implant sulcus: A comparison of sites in edentulous and partially edentulous patients

The soft tissues adjacent to osseointegrated dental implants (OII) were investigated using clinical, biochemical and microbiological methods. Tooth and implant crevices were compared in 15 partially edentulous patients, examining 28 peri-implant and 19 periodontal sites, and in 6 edentulous patients, examining 13 implant sites. Sites were classified by standard periodontal indices; the crevicular fluid flow determined; crevicular fluid was collected for collagenase assays; and the subgingival bacterial flora was examined and cultured. Differences in clinical parameters were noted in that implants had significantly less keratinized gingiva and deeper probing depths. Crevicular fluid was present in the OII sulcus but the crevicular fluid flow did not differ from that observed from tooth sites either in the partially edentulous or edentulous patients. Tissue collagenase activity and collagenase inhibitor were detected in the implant crevicular fluid and, as in periodontal sites, a strong inverse relationship was found between the levels of active collagenase and collagenase inhibitor. Microbiology included darkfield microscopy, anaerobic culturing for total colony forming unit counts and identification of black pigmented Bacteroides (BPB). Few differences were observed between implants and teeth in partially edentulous patients, indicating that crevices around teeth may act as reservoirs of bacteria which can colonize implant sites. A higher percentage of BPBs and wet spreaders (Capnocytophaga) was noted at partially edentulous implant sites when compared with edentulous implant sites, perhaps reflecting the lower numbers of periodontal pathogens present in edentulous mouths. Overall, the characteristics of implant sulci appear to be similar to periodontal sulci with respect to crevicular fluid flow and microflora.