Inoculum Size Influences Bacterial Cross Contamination between Surfaces

Many factors have been shown to influence bacterial transfer between surfaces, including surface type, bacterial species, moisture level, pressure, and friction, but the effect of inoculum size on bacterial transfer has not yet been established. Bacterial cross contamination rates during performance of common food service tasks were previously determined in our laboratory using nalidixic acid-resistant Enterobacter aerogenes. Eight different transfer rates were determined, each involving a minimum of 30 volunteers. The influence of source inoculum level on the percentage of bacteria transferred (percent transfer rates) and log₁₀ CFU per recipient surface was determined using statistical analysis. The effect of inoculum size on transfer rate was highly statistically significant (P < 0.0001) for all transfer rate data combined (352 observations) and for each individual cross contamination rate, except for data on contamination via transfer from chicken to hand through a glove barrier (P = 0.1643). Where inoculum size on the source was greater, transfer rates were lower, and where inoculum size on the source was less, transfer rates were higher. The negative linear trend was more obvious for activities that had a larger range of inoculum sizes on the source surface. This phenomenon has serious implications for research seeking to determine bacterial cross contamination rates, since the different transfer efficiencies that were previously shown to be associated with certain activities may actually be the result of differing initial inoculum levels. The initial inoculum size on the source and the amount of bacteria transferred must both be considered to accurately determine bacterial transfer rates.     

Regulation of growth of Acinetobacter calcoaceticus NCIB8250 on L-mandelate in batch culture.

Batch culture of Acinetobacter calcoaceticus in L-mandelate- or phenylglyoxylate-salts medium showed an unusual non-exponential pattern unless the inoculum had been grown on benzyl alcohol. There were transient accumulations of benzaldehyde and benzyl alcohol caused by the limitation of L-mandelate oxidation by low activities of benzaldehyde dehydrogenase and the diversion of reducing power to the formation of benzyl alcohol. In vivo enzymic activities were estimated from patterns of substrate utilization in batch cultures containing pairs of substrates. When bacteria previously grown in L-mandelate-salts medium were inoculated into media containing L-mandelate and a second carbon source, metabolism of L-mandelate was arithmetical in the presence of benzoate, catechol or succinate, but accelerated on exhaustion of the second substrate. This indicated repression of the enzymes involved in L-mandelate oxidation. Inoculation of bacteria grown in benzoate-salts medium into medium containing L-mandelate and benzoate gave diauxie with initial utilization of benzoate. Similar experiments showed that benzoate oxidation was not repressed by catechol and only partially repressed by succinate. Measurement of L-mandelate dehydrogenase, phenylglyoxylate carboxy-lyase and benzaldehyde dehydrogenase I in bacterial extracts showed no evidence for feedback inhibition by intermediates of the pathway. The rates of L-mandelate and benzoate utilization by bacterial suspensions were inhibited by succinate and catechol but not by other intermediates of the pathway.     

Genetic transfer of the mcd gene in soil

AIMS: To investigate the role of horizontal gene transfer of mcd (methylcarbamate-degrading) gene in high genetic diversity of carbofuran-degrading bacteria. METHODS AND RESULTS: The actuality of genetic transfer from degraders to an Agrobacterium tumefaciens strain was determined in liquid medium. The mcd gene was chosen for transfer experiments. Transconjugants were obtained irrespective of the type of the donor strain (Gram-positive or Gram-negative), size of the inoculum, or nature and concentration of the pesticide in the medium. Soil microcosms, inoculated with or without the donor and/or recipient strains were used. The size of the initial degrading population (treated or untreated soil) and the nature of the inoculated donor strains were considered. More transconjugants were isolated in the previously treated soil than in the untreated soil. Agrobacterium transconjugants were isolated even when the donor strain was not inoculated, probably as a result of gene transfer from indigenous degrading population to the recipient strain. Moreover, potential transconjugants belonging to the Pseudomonas genus were isolated. CONCLUSIONS: Our results seem to demonstrate that the mcd gene is transferable in soil among bacterial populations. SIGNIFICANCE AND IMPACTS OF THE STUDY: The transfer of the mcd gene is partly responsible for the high genetic diversity of micro-organisms able to catabolize carbofuran.     

Significance of inoculum size in biofilm formation by staphylococci

In the present study the effect of inoculum size, ranging from 10(6) to 1 cell, in biofilm formation by staphylococci was determined by microtiter plate test. The initial inoculum size had a dramatic effect on the quantity of biofilm formed. A decreased number of bacteria in initial inoculum always resulted in a decreased production of biofilm.     

Microbial Characterization during the Early Habitation of the International Space Station

An evaluation of the microbiota from air, water, and surface samples provided a baseline of microbial characterization onboard the International Space Station (ISS) to gain insight into bacterial and fungal contamination during the initial stages of construction and habitation. Using 16S genetic sequencing and rep-PCR, 63 bacterial strains were isolated for identification and fingerprinted for microbial tracking. Of the bacterial strains that were isolated and fingerprinted, 19 displayed similarity to each other. The use of these molecular tools allowed for the identification of bacteria not previously identified using automated biochemical analysis and provided a clear indication of the source of several ISS contaminants. Strains of Bradyrhizobium and Sphingomonas unable to be identified using sequencing were identified by comparison of rep-PCR DNA fingerprints. Distinct DNA fingerprints for several strains of Methylobacterium provided a clear indication of the source of an ISS water supply contaminant. Fungal and bacterial data acquired during monitoring do not suggest there is a current microbial hazard to the spacecraft, nor does any trend indicate a potential health risk. Previous spacecraft environmental analysis indicated that microbial contamination will increase with time and will require continued surveillance.     

Enterococcus faecalis Gene Transfer under Natural Conditions in Municipal Sewage Water Treatment Plants

The ability of Enterococcus faecalis to transfer various genetic elements under natural conditions was tested in two municipal sewage water treatment plants. Experiments in activated sludge basins of the plants were performed in a microcosm which allowed us to work under sterile conditions; experiments in anoxic sludge digestors were performed in dialysis bags. We used the following naturally occurring genetic elements: pAD1 and pIP1017 (two so-called sex pheromone plasmids with restricted host ranges, which are transferred at high rates under laboratory conditions); pIP501 (a resistance plasmid possessing a broad host range for gram-positive bacteria, which is transferred at low rates under laboratory conditions); and Tn916 (a conjugative transposon which is transferred under laboratory conditions at low rates to gram-positive bacteria and at very low rates to gram-negative bacteria). The transfer rate between different strains of E. faecalis under natural conditions was, compared to that under laboratory conditions, at least 10⁵-fold lower for the sex pheromone plasmids, at least 100-fold lower for pIP501, and at least 10-fold lower for Tn916. In no case was transfer from E. faecalis to another bacterial species detected. By determining the dependence of transfer rates for pIP1017 on bacterial concentration and extrapolating to actual concentrations in the sewage water treatment plant, we calculated that the maximum number of transfer events for the sex pheromone plasmids between different strains of E. faecalis in the municipal sewage water treatment plant of the city of Regensburg ranged from 10⁵ to 10⁸ events per 4 h, indicating that gene transfer should take place under natural conditions.     

Aerobic and anaerobic degradation and mineralization of 14C-chitin by water column and sediment inocula of the York River estuary, Virginia.

Potential rates of chitin degradation (Cd) and mineralization (Cm) by estuarine water and sediment bacteria were measured as a function of inoculum source, temperature, and oxygen condition. In the water column inoculum, 88 to 93% of the particulate chitin was mineralized to CO2 with no apparent lag between degradation and mineralization. No measurable dissolved pool of radiolabel was found in the water column. For the sediment inocula, 70 to 90% of the chitin was degraded while only 55 to 65% was mineralized to CO2. 14C label recoveries in the dissolved pool were 19 to 21% for sand, 17 to 24% in aerobic mud, and 12 to 21% for the anaerobic mud. This uncoupling between degradation and mineralization occurred in all sediment inocula. More than 98% of the initial 14C-chitin was recovered in the three measured fractions. The highest Cd and Cm values, 30 and 27% day-1, occurred in the water column inoculum at 25 degrees C. The lowest Cd and Cm values were found in the aerobic and anaerobic mud inocula incubated at 15 degrees C. Significant differences in Cd and Cm values among water column and sediment inocula as well as between temperature treatments were evident. An increased incubation temperature resulted in shorter lag times before the onset of chitinoclastic bacterial growth, degradation, and mineralization and resulted in apparent Q10 values of 1.1 for water and 1.3 to 2.1 for sediment inocula. It is clear that chitin degradation and mineralization occur rapidly in the estuary and that water column bacteria may be more important in this process than previously acknowledged.     

Conjugational transfer of recombinant DNA in cultures and in soils: host range of Pseudomonas putida TOL plasmids.

Recombinant TOL plasmid pWWO-EB62 allows Pseudomonas putida to grow on p-ethylbenzoate. This plasmid can be transferred to other microorganisms, and its catabolic functions for the metabolism of alkylbenzoates are expressed in a limited number of gram-negative bacteria, including members of pseudomonad rRNA group I and Escherichia coli. Transfer of the recombinant plasmid to Erwinia chrysanthemi was observed, but transconjugants failed to grow on alkylbenzoates because they lost catabolic functions. Pseudomonads belonging to rRNA groups II, III, and IV, Acinetobacter calcoaceticus, and Alcaligenes sp. could not act as recipients for TOL, either because the plasmid was not transferred or because it was not stably maintained. The frequency of transfer of pWWO-EB62 from P. putida as a donor to pseudomonads belonging to rRNA group I was on the order of 1 to 10(-2) transconjugant per recipient, while the frequency of intergeneric transfer ranged from 10(-3) to 10(-7) transconjugant per recipient. The profile of potential hosts was conserved when the donor bacterium was Escherichia coli or Erwinia chrysanthemi instead of P. putida. No intergeneric gene transfer of the recombinant TOL plasmid was observed in soils; however, intraspecies transfer did take place. Intraspecies transfer of TOL in soils was affected by the type of soil used, the initial inoculum size, and the presence of chemicals that could affect the survival of the donor or recipient bacteria.     

Conjugational transfer of recombinant DNA in cultures and in soils: host range of Pseudomonas putida TOL plasmids.

Recombinant TOL plasmid pWWO-EB62 allows Pseudomonas putida to grow on p-ethylbenzoate. This plasmid can be transferred to other microorganisms, and its catabolic functions for the metabolism of alkylbenzoates are expressed in a limited number of gram-negative bacteria, including members of pseudomonad rRNA group I and Escherichia coli. Transfer of the recombinant plasmid to Erwinia chrysanthemi was observed, but transconjugants failed to grow on alkylbenzoates because they lost catabolic functions. Pseudomonads belonging to rRNA groups II, III, and IV, Acinetobacter calcoaceticus, and Alcaligenes sp. could not act as recipients for TOL, either because the plasmid was not transferred or because it was not stably maintained. The frequency of transfer of pWWO-EB62 from P. putida as a donor to pseudomonads belonging to rRNA group I was on the order of 1 to 10(-2) transconjugant per recipient, while the frequency of intergeneric transfer ranged from 10(-3) to 10(-7) transconjugant per recipient. The profile of potential hosts was conserved when the donor bacterium was Escherichia coli or Erwinia chrysanthemi instead of P. putida. No intergeneric gene transfer of the recombinant TOL plasmid was observed in soils; however, intraspecies transfer did take place. Intraspecies transfer of TOL in soils was affected by the type of soil used, the initial inoculum size, and the presence of chemicals that could affect the survival of the donor or recipient bacteria.     

Bioprocess intensification in flow-through monolithic microbioreactors with immobilized bacteria

Microporous polymers (with porosity up to 90%) with a well-prescribed internal microstructure were prepared in monolithic form to construct a flow-through microbioreactor in which phenol-degrading bacteria, Pseudomonas syringae, was immobilized. Initially, bacteria was forced seeded within the pores and subsequently allowed to proliferate followed by acclimatization and phenol degradation at various initial substrate concentrations and flow rates. Two types of microporous polymer were used as the monolithic support. These polymers differ with respect to their pore and interconnect sizes, macroscopic surface area for bacterial support, and phase volume. Polymer with a nominal pore size of 100 microm with phase volume of 90% (with highly open pore structure) yielded reduced bacterial proliferation, while the polymer with nominal pore size of 25 microm with phase volume of 85% (with small interconnect size and large pore area for bacterial adhesion) yielded monolayer bacterial proliferation. Bacteria within the 25 microm polymer support remained monolayered, without any apparent production of extracellular matrix during the 30-day continuous experimental period. The microbioreactor performance was characterized in terms of volumetric utilization rate and compared with the published data, including the case where the same bacteria was immobilized on the surface of microporous polymer beads and used in a packed bed during continuous degradation of phenol. It is shown that at similar initial substrate concentration, the volumetric utilization in the microreactor is at least 20-fold more efficient than the packed bed, depending on the flow rate of the substrate solution. The concentration of the bacteria within the pores of the microreactor decreases from 2.25 cells per microm2 on the top surface to about 0.4 cells per microm2 within 3 mm reactor depth. If the bacteria-depleted part of the microreactor is disregarded, the volumetric utilization increases by a factor of 30-fold compared with the packed bed. This efficiency increase is attributed to the reduction of diffusion path for the substrate and nutrients and enhanced availability of the bacteria for bioconversion in the absence of biofilm formation as well as the presence of flow over the surface of the monolayer bacteria.     

Biofilm formation and the food industry,a focus on the bacterial outer surface

The ability of many bacteria to adhere to surfaces and to form biofilms has major implications in a variety of industries including the food industry, where biofilms create a persistent source of contamination. The formation of a biofilm is determined not only by the nature of the attachment surface, but also by the characteristics of the bacterial cell and by environmental factors. This review focuses on the features of the bacterial cell surface such as flagella, surface appendages and polysaccharides that play a role in this process, in particular for bacteria linked to food‐processing environments. In addition, some aspects of the attachment surface, biofilm control and eradication will be highlighted.     

Influence of Culture Parameters on Biological Hydrogen Production by Clostridium saccharoperbutylacetonicum ATCC 27021

Summary Various medium components (carbon and nitrogen sources, iron, inoculum size) and environmental factors (initial pH and the agitation speed) were evaluated for their effects on the rate and the yield of hydrogen production by Clostridium saccharoperbutylacetonicum. Among the carbon sources assessed, cells grown on disaccharides (lactose, sucrose and maltose) produced on the average more than twice (2.81 mol-H2/mol sugar) as much hydrogen as monosaccharides (1.29 mol-H2/mol sugar), but there was no correlation between the carbon source and the production rate. The highest yield (2.83 mol/mol) was obtained in lactose and sucrose but the highest production rate (1.75 mmol/h) in sucrose. Using glucose as carbon source, yeast extract was the best nitrogen source. A parallel increase between the production rate and the yield was obtained by increasing glucose concentration up to 40 g/l (1.76 mol-H2/mol, 3.39 mmol/h), total nitrogen as yeast extract up to 0.1% (1.41 mol/mol, 1.91 mmol/h) and agitation up to 100 rev/min (1.66 mol-H2/mol, 1.86 mmol/h). On the other hand, higher production rates were favoured in preference to the yield at a neutral initial pH 7 (2.27 mmol/h), 1000 mg iron/l or more (1.99 mmol/h), and a larger inoculum size, 10%, (2.36 mmol/h) whereas an initial alkaline pH of 8.5 (1.72 mol/mol), a lower iron concentration of 25 mg/l (1.74 mol/mol) and smaller inoculum size, 1%, (1.85 mol/mol) promoted higher yield over production rate.     

木质素降解菌BYL-7的筛选及降解条件优化

【背景】微生物降解木质素因其具有降解效率高和环保等特点而备受关注。【目的】筛选高效木质素降解真菌,并对其降解条件进行优化。【方法】通过愈创木酚-马铃薯葡萄糖琼脂(potato dextroseagar,PDA)和苯胺蓝平板法筛选高效木质素降解菌株,利用单因素筛选及响应面试验对培养条件进行优化。【结果】筛选到一株高效木质素降解菌BYL-7,经形态和多序列分析初步确定为Trametes versicolor。单因素试验证明初始pH、温度和接种量为降解木质素显著影响因子,响应面试验确定降解木质素最优条件为:初始pH 6.7,温度25°C,接种量8%。在此条件下,碱性木质素降解率为36.5%,比未优化前提高54.0%;水稻秸秆木质素、半纤维素和纤维素降解率分别为32.8%、21.5%、13.2%,其中木质素降解率比未优化前提高36.1%;漆酶活性在第6天达到峰值120.0 U/L,比未优化前提高25.0%;木质素过氧化物酶活性在第6天达到峰值1343.8U/L,比未优化前提高36.0%;锰过氧化物酶活性在第5天达到峰值463.8U/L,比未优化前提高31.7%。【结论】研究结果为木质素的降解提...     

Production of acid proteinases byHumicola lutea immobilized in polyacrylamide gel

Summary Fungal spores ofHumicola lutea 120–5 were entrapped in 5% polyacrylamide gel and were cultivated for 44–48 h to form a mycelial network inside the beads. A dense mycelial growth also occurred on the surface of the beads. It was possible to reuse the immobilized mycelium for production of acid proteinases in 12 different batches without loss of mechanical stability. The inoculum size should be controlled prior to its transfer into fresh production medium. Maximal enzyme production exceeding the level of free cell fermentation was registered in the fourth to seventh cycles. According to the size of the inoculum, half of the initial production rate was reached after 7–14 batches.     

Biodegradation of phenol by bacterial strains from petroleum-refining wastewater purification plant

The ability of four strains of bacteria derived from a biological petroleum-refining wastewater purification plant to carry out the biodegradation of phenol was studied. Two of the strains belonging to the genus Pseudomonas were found to be characterised by high effectiveness of the removal of phenol which was used as sole carbon and energy source (the strains were designated P1 and P2). In turn the effect of inoculum size, initial concentration of substrate (500 and 1,000 mg phenol/L) and temperature (10, 20 and 30 degrees C) on the rate of phenol degradation by strains P1, P2 and mixture of both was investigated. It was found that strain P1 which was identified as Pseudomonas fluorescens degraded phenol better than strain P2--Pseudomonas cepacia. The rate of phenol biodegradation was significantly affected by size of inoculum and temperature of incubation. Phenol was removed the fastest with the highest inoculum used. The optimal temperature was about 20 degrees C. At 10 and 30 degrees C the process of biodegradation was visibly inhibited. The rate of phenol utilisation was also found to decrease with increased concentration of substrate.     

Influence of soil pH on the nitrate-reducing microbial populations and their potential to reduce nitrate to NO and N2O

Abstract After the introduction of Rhizobium leguminosarum biovar trifolii into natural loamy sand and silt loam, bacterial numbers increased only directly after inoculation. Thereafter, bacterial numbers decreased until an equilibrium was reached. This decrease was exponential on a log scale and could be described by the function Y = A + B − R ', where Y is the log number of rhizobial cells at time: T ; A represents the lgo of the final population size; B is the difference between the log (initial number of bacteria) and A ; R is the daily reduction factor of Y−A and t is time in days after inoculation. The final population sizes increased with increasing inoculum densities (10⁴−10⁸ bacteria/g soil). In sterilized soil, however, the populations increased up to an equilibrium, which was not affected by the inoculum density.
The final population sizes were higher in silt loam than in loamy sand in natural, as well as in sterilized soil. The final population size was reached earlier in natural silt loam than in loamy sand. Also the growth rate in sterilized soil was higher in silt loam than in loamy sand. The growth rate of low inoculum densities in silt loam was exponential and approximately the same as in yeast extract mannitol broth. The growth rate in loamy sand could be improved by incresing the bulk density of the soil from 1.0 to 1.4 g/cm³.     

Bacterial hand contamination and transfer after use of contaminated bulk-soap-refillable dispensers

Bulk-soap-refillable dispensers are prone to extrinsic bacterial contamination, and recent studies demonstrated that approximately one in four dispensers in public restrooms are contaminated. The purpose of this study was to quantify bacterial hand contamination and transfer after use of contaminated soap under controlled laboratory and in-use conditions in a community setting. Under laboratory conditions using liquid soap experimentally contaminated with 7.51 log(10) CFU/ml of Serratia marcescens, an average of 5.28 log(10) CFU remained on each hand after washing, and 2.23 log(10) CFU was transferred to an agar surface. In an elementary-school-based field study, Gram-negative bacteria on the hands of students and staff increased by 1.42 log(10) CFU per hand (26-fold) after washing with soap from contaminated bulk-soap-refillable dispensers. In contrast, washing with soap from dispensers with sealed refills significantly reduced bacteria on hands by 0.30 log(10) CFU per hand (2-fold). Additionally, the mean number of Gram-negative bacteria transferred to surfaces after washing with soap from dispensers with sealed-soap refills (0.06 log(10) CFU) was significantly lower than the mean number after washing with contaminated bulk-soap-refillable dispensers (0.74 log(10) CFU; P < 0.01). Finally, significantly higher levels of Gram-negative bacteria were recovered from students (2.82 log(10) CFU per hand) than were recovered from staff (2.22 log(10) CFU per hand) after washing with contaminated bulk soap (P < 0.01). These results demonstrate that washing with contaminated soap from bulk-soap-refillable dispensers can increase the number of opportunistic pathogens on the hands and may play a role in the transmission of bacteria in public settings.     

Use of Experimental Factorial Design for Optimization of Hexavalent Chromium Removal by a Bacterial Consortium: Soil Microcosm Bioremediation

Hexavalent chromium Cr(VI) is regularly introduced into the environment through diverse anthropogenic activities. It is highly toxic, mutagenic and carcinogenic, and because of its solubility in water, chromate contamination can be difficult to contain. Bacteria can reduce chromate to insoluble and less toxic trivalent chromium Cr(III), and thus increasing attention is paid to chromate bioremediation to reduce its ecotoxicological impacts. In this study, the factorial design 2³ was employed to optimize critical parameters responsible for higher Cr(VI) removal by a bacterial consortium. The factors considered were pH, temperature, and inoculum size at two markedly different levels. All three dependent variables have significant effect on Cr(VI) reduction. Optimal Cr(VI) removal by the bacterial consortium occurred at pH 9, temperature 37°C, and inoculum size OD = 3. Analysis of variance (ANOVA) showed a high coefficient of determination (R²) value of 0.984, thus ensuring a satisfactory adjustment of the second-order regression model with the experimental data. In addition, the effect of bioaugmentation of Cr(VI)-polluted soil microcosms with the bacterial consortium was investigated using the best factor levels. Contaminated soil by 20 and 60 mg/Kg of Cr(VI) showed reductions of 83% and 65% of initial Cr(VI) by the bacterial consortium, suggesting that this bacterial consortium might diminish phytoavailable Cr(VI) in soil and be useful for cleaning up chromium-contaminated sites.     

Effect of operating variables on biofilm formation and performance of an anaerobic fluidized-bed bioreactor

The effect of various operating variables such as initial inoculum circulation, dilution rate, chemical oxygen demand (COD) loading rate, and quantity and quality of inoculum on the process of film formation on sand surface and reactor performance were studied using synthetic glucose based wastewater. It was found that the film formation process is favored by a high dilution rate, a large quantity of inoculum, and an inoculum having high methane producing capacity. Experimental observations indicate that the biofilm formation process is initiated by methanogenic bacteria.