Small Inocula Research Articles

Autotrophic hydrogen-producing biofilm growth sustained by a cathode as the sole electron and energy source

It is still unclear whether autotrophic microbial biocathode biofilms are able to self-regenerate under purely cathodic conditions without any external electron or organic carbon sources. Here we report on the successful development and long-term operation of an autotrophic biocathode whereby an electroactive biofilm was able to grow and sustain itself with CO2 as a sole carbon source and using the cathode as electron source, with H2 as sole product. From a small inoculum of 15mgCOD (in 250mL), containing 30.3% Archaea, the bioelectrochemical system operating at −0.5V vs. SHE enabled an estimated biofilm growth of 300mg as COD over a period of 276days. A dramatic change in the microbial population was observed during this period with Archaea disappearing completely (<0.1% of population). The predominant phyla enriched were Proteobacteria (57.3%), Firmicutes (12.4%), Bacteroidetes (11.6%) and Actinobacteria (1.1%). Up to 9.2LH2m−2day−1 (1.88Am−2) was achieved when the cathode potential was decreased to −0.75V vs. SHE. This study demonstrates that purely autotrophic biofilm growth coupled to proton reduction to hydrogen alone can be sustained with a cathode as the sole electron source, while avoiding the development of H2-consuming microorganisms such as methanogens and acetogens.

β-Adrenergic blockade protects BALB/c mice against infection with a small inoculum of Leishmania mexicana mexicana (LV4).

In order to test the influence of the sympathetic nervous system on Leishmania mexicana infection, groups of female BALB/c mice were treated (i.p.) with the non-selective β-adrenergic receptor (β-AR) antagonist (S)-propranolol (5mg/kg thrice a day), the β2-AR agonist clenbuterol (1mg/kg once a day) or the α2-AR antagonist yohimbine (2mg/kg twice a day) during 5days. During the second day of treatments, mice were inoculated in the footpad with 1×10(6) or 1×10(3) metacyclic promastigotes of L. mexicana mexicana (LV4). The lesion size was measured weekly, and parasite burden on week 12. In mice treated with (S)-propranolol, the percentage of splenic T lymphocytes producing IFN-γ after antigen challenge was determined by flow cytometry. In mice infected with 1×10(6) parasites, only (S)-propranolol caused a reduction of footpad swelling (p<0.05, weeks 11-12), without effects on parasite burden, or in the percentage of IFN-γ-immunopositive CD4(+) or CD8(+) T lymphocytes. In mice infected with 1×10(3) parasites, the effects of treatments vs. control group were as follows: (a) inhibition of footpad swelling by (S)-propranolol (p<0.01, weeks 3-12), clenbuterol (p<0.05, weeks 7-10), and yohimbine (p<0.01, week 7); (b) a decrease of the parasite burden by (S)-propranolol (p<0.01) and yohimbine (p<0.05); (c) in control mice the percentage of CD4(+) T-cells producing IFN-γ was 6.2±0.5%, while in those treated with (S)-propranolol it increased to 8.7±0.6% (p<0.01); (d) in control mice the percentage of CD8(+) T-cells producing IFN-γ was 3.1±0.4%, while in those treated with (S)-propranolol it increased to 10.4±0.2% (p<0.01). These results indicate that the blockade of β-ARs during infection of BALB/c mice with an inoculum of L. mexicana mexicana similar to that delivered by the bite of a sand fly produces a Th1 bias in the immune response, favoring an increment of T lymphocytes secreting IFN-γ, which correlated with a reduced parasite burden (p<0.05, Spearman's test). We suggest that β-AR antagonists could be of therapeutic value, either as treatment or as adjuvant of vaccines for L. mexicana.

DNA Extraction Methods for Panbacterial and Panfungal PCR Detection in Intraocular Fluids

Purpose: Three different methods of DNA extraction from intraocular fluids were compared with subsequent detection for bacterial and fungal DNA by universal PCR amplification.Material and Methods: Three DNA extraction methods, from aqueous and vitreous humors, were evaluated to compare their relative efficiency. Bacterial (Gram positive and negative) and fungal strains were used in this study: Escherichia coli, Staphylococcus epidermidis and Candida albicans. The quality, quantification, and detection limit for DNA extraction and PCR amplification were analyzed. Validation procedures for 13 aqueous humor and 14 vitreous samples, from 20 patients with clinically suspected endophthalmitis were carried out.Results: The column-based extraction method was the most time-effective, achieving DNA detection limits ≥102 and 103 CFU/100 µL for bacteria and fungi, respectively. PCR amplification detected 100 fg, 1 pg and 10 pg of genomic DNA of E. coli, S. epidermidis and C. albicans respectively. PCR detected 90.0% of the causative agents from 27 intraocular samples collected from 20 patients with clinically suspected endophthalmitis, while standard microbiological techniques could detect only 60.0%. The most frequently found organisms were Streptococcus spp. in 38.9% (n = 7) of patients and Staphylococcus spp. found in 22.2% (n = 4).Conclusions: The column-based extraction method for very small inocula in small volume samples (50–100 µL) of aqueous and/or vitreous humors allowed PCR amplification in all samples with sufficient quality for subsequent sequencing and identification of the microorganism in the majority of them.

Host-pathogen interactions and immune evasion strategies in Francisella tularensis pathogenicity.

Francisella tularensis is an intracellular Gram-negative bacterium that causes life-threatening tularemia. Although the prevalence of natural infection is low, F. tularensis remains a tier I priority pathogen due to its extreme virulence and ease of aerosol dissemination. F. tularensis can infect a host through multiple routes, including the intradermal and respiratory routes. Respiratory infection can result from a very small inoculum (ten organisms or fewer) and is the most lethal form of infection. Following infection, F. tularensis employs strategies for immune evasion that delay the immune response, permitting systemic distribution and induction of sepsis. In this review we summarize the current knowledge of F. tularensis in an immunological context, with emphasis on the host response and bacterial evasion of that response.

An Outpatient, Ambulant-Design, Controlled Human Infection Model Using Escalating Doses of Salmonella Typhi Challenge Delivered in Sodium Bicarbonate Solution

Typhoid fever is a major global health problem, the control of which is hindered by lack of a suitable animal model in which to study Salmonella Typhi infection. Until 1974, a human challenge model advanced understanding of typhoid and was used in vaccine development. We set out to establish a new human challenge model and ascertain the S. Typhi (Quailes strain) inoculum required for an attack rate of 60%-75% in typhoid-naive volunteers when ingested with sodium bicarbonate solution. Groups of healthy consenting adults ingested escalating dose levels of S. Typhi and were closely monitored in an outpatient setting for 2 weeks. Antibiotic treatment was initiated if typhoid diagnosis occurred (temperature ≥38°C sustained ≥12 hours or bacteremia) or at day 14 in those remaining untreated. Two dose levels (10(3) or 10(4) colony-forming units) were required to achieve the primary objective, resulting in attack rates of 55% (11/20) or 65% (13/20), respectively. Challenge was well tolerated; 4 of 40 participants fulfilled prespecified criteria for severe infection. Most diagnoses (87.5%) were confirmed by blood culture, and asymptomatic bacteremia and stool shedding of S. Typhi was also observed. Participants who developed typhoid infection demonstrated serological responses to flagellin and lipopolysaccharide antigens by day 14; however, no anti-Vi antibody responses were detected. Human challenge with a small inoculum of virulent S. Typhi administered in bicarbonate solution can be performed safely using an ambulant-model design to advance understanding of host-pathogen interactions and immunity. This model should expedite development of diagnostics, vaccines, and therapeutics for typhoid control.

A novel knee prosthesis model of implant-related osteo- myelitis in rats

Background and purpose There have been numerous reports of animal models of osteomyelitis. Very few of these have been prosthesis models that imitate human conditions. We have developed a new rat model of implant-related osteomyelitis that mimics human osteomyelitis, to investigate the pathology of infection after orthop edic implant surgery.Methods 2 wild-type strains of Staphylococcus aureus, MN8 and UAMS-1, and their corresponding mutants that are unable to produce poly-N-acetyl glucosamine (PNAG) (ica::tet) were injected into the medullary canals of the femur and tibia at 3 different doses: 102, 103, and > 104 CFU/rat. We measured clinical signs, inflammatory markers, radiographic signs, histopathology, and bacteriology in the infected animals.Results An inoculum of at least 104 cfu of either wild-type bacterial strain resulted in histological, bacteriological, and radiographic signs of osteomyelitis with loosening of the prosthesis. An inoculum of 103 CFU gave signs of osteomyelitis but the prosthesis remained in situ. Bacterial inocula of 102 cfu gave no signs of osteolysis.Interpretation We have established a new knee prosthesis model that is suitable for reliable induction of experimental implant-associated osteomyelitis with the prosthesis in situ, using a small inoculum of S. aureus. At a dose of 103 CFU/rat, bacteria unable to produce PNAG (ica::tet) had only minor defects in their virulence.

Test of colonisation scenarios reveals complex invasion history of the red tomato spider mite Tetranychus evansi.

The spider mite Tetranychus evansi is an emerging pest of solanaceous crops worldwide. Like many other emerging pests, its small size, confusing taxonomy, complex history of associations with humans, and propensity to start new populations from small inocula, make the study of its invasion biology difficult. Here, we use recent developments in Approximate Bayesian Computation (ABC) and variation in multi-locus genetic markers to reconstruct the complex historical demography of this cryptic invasive pest. By distinguishing among multiple pathways and timing of introductions, we find evidence for the “bridgehead effect”, in which one invasion serves as source for subsequent invasions. Tetranychus evansi populations in Europe and Africa resulted from at least three independent introductions from South America and involved mites from two distinct sources in Brazil, corresponding to highly divergent mitochondrial DNA lineages. Mites from southwest Brazil (BR-SW) colonized the African continent, and from there Europe through two pathways in a “bridgehead” type pattern. One pathway resulted in a widespread invasion, not only to Europe, but also to other regions in Africa, southern Europe and eastern Asia. The second pathway involved the mixture with a second introduction from BR-SW leading to an admixed population in southern Spain. Admixture was also detected between invasive populations in Portugal. A third introduction from the Brazilian Atlantic region resulted in only a limited invasion in Europe. This study illustrates that ABC methods can provide insights into, and distinguish among, complex invasion scenarios. These processes are critical not only in understanding the biology of invasions, but also in refining management strategies for invasive species. For example, while reported observations of the mite and outbreaks in the invaded areas were largely consistent with estimates of geographical expansion from the ABC approach, historical observations failed to recognize the complex pathways involved and the corresponding effects on genetic diversity.

Comparative Study of Microcalorimetric Behavior of Escherichia coli, Proteus mirabilis and Klebsiella pneumoniae

To analyze the microcalorimetric behaviour of Escherichia coli, Proteus mirabilis and Klebsiella pneumoniae, and to test the prospective use of microcalorimetry in the early diagnosis of infections by enterobacteria. For our experiments we used cultures of E. coli, P. mirabilis and K. pneumoniae. For the experimental setup, we used a Calvet microcalorimeter with the constant temperature of 309 K inside. Using the measured difference of heat power generated by the cells over time, we obtained growth graphs of the three bacterial species grown at different densities. The generated curves have characteristic plot that repeats for each bacterial species at all the concentrations studied. We observed an inversely proportional relationship between inoculum size and the time until signal detection; the smaller inoculum, the longer time for the signal detection. In all conducted experiments, even with low number of bacteria (10 CFU/ml) as inoculum, we could identify bacterial growth in the sample within 8 hours. Microcalorimetry could be an efficient technique used for the early detection and identification of enterobacteria in a culture medium.

Colonization of Caenorhabditis elegans by Bacillus nematocida B16, a Bacterial Opportunistic Pathogen

Background: Soil-dwelling Bacillus nematocida B16 can kill Caenorhabditis elegans via a Trojan horse-like mechanism. However, colonization is a key problem that must be solved during the infection process. Aims: To study the molecular mechanism involved in the colonization of B. nematocida B16 against the host C. elegans. Methods: GFP-expressing strain B16g was constructed and its nematocidal activity was assayed. ‘Feeding transfer’ experiments were carried out separately using of B16 and B16g strains to explore the colonization mode of the bacteria. Fluorescence microscopy was used to observe the interactions between fluorescent signal and the quantity of bacteria in the intestine. A mariner-based transposon called TnYLB-1 was also applied in the random mutagenesis of B16 to screen the mutants with impaired colonization of nematode worms and identify potential localization-related genes. Results and Conclusion: A small inoculum of the bacteria resulted in its proliferation in the C. elegans intestine. The fluorescence signal was enhanced with increasing bacterial density in the intestine. Several candidate genes with possibly important roles in colonization were found. These results provide a solid foundation for further elucidation of the infection process at the molecular level and enrichment of our knowledge of bacterial pathogenesis.

Growth of Salmonella Typhimurium DT104 at 30°C Is Not Affected by Anatomical Location on the Chicken Carcass†

Development of models for growth of Salmonella in the chicken food matrix is time-consuming and expensive. The current study was undertaken to examine growth of Salmonella on different anatomical locations of the chicken carcass. The purpose was to determine whether anatomical location should be included as an independent variable in predictive models for chicken. Eleven anatomical locations were studied: skin (wing, breast, drumstick, and thigh), meat surface (wing, breast, drumstick, and thigh), and meat interior (breast, drumstick, and thigh). Background microflora, pH, and growth (lag time, λ; growth rate, μ; and time for a 3-log increase, t(3)) at 30°C for a small inoculum size (0.92 ± 0.30 log per portion) of Salmonella Typhimurium DT104 were examined. Four or six replicate storage trials were conducted per anatomical location (n = 46 growth curves). Portion sizes were 1.12 ± 0.17 g (mean ± standard deviation) for meat and 0.25 ± 0.08 g for skin. A two-phase linear model was used to determine λ and μ. The effect of anatomical location on dependent variables was assessed by one-way analysis of variance. pH values differed (P < 0.001) among anatomical locations, with skin (6.86 ± 0.20). dark meat (6.39 ± 0.20) . white meat (5.97 ± 0.20). Background microflora (4.32 ± 1.66 log per portion) was variable and not affected (P > 0.05) by anatomical location. Likewise, λ (1.90 ± 0.75 h), μ (0.648 ± 0.120 log/h), and t(3) (6.71 ± 0.82 h) at 30°C were not affected (P > 0.05) by anatomical location. Although there were differences in pH among anatomical locations, these differences were not sufficient to affect growth of Salmonella Typhimurium DT104 at 30°C. If this observation holds for other storage conditions and strains, then anatomical location does not need to be included as an independent variable in predictive models for chicken. This would save significant time and money for the predictive microbiologist.

The Influence of The Degree of Grinding to Obtain of Some Fermented Mashes Based on Whole Wheat and Husked Millet

The purpose of this article was to study the influence of degree of milling whole wheat (was ground in 4 variants of grain) and husked millet (was finely ground) to obtain fermented mashes type Braga. There have been 4 mashes of grist of whole wheat, grist husked millet, hop pellets and water and were boiled under continuous stirring and then cooled. Their content has diluted with water and then strainer by pressed through a sieve. Each mash-filtrate has divided in two and added white sugar 7%, resulting two series of sweet mashes. One has inoculated with a mixed culture of yeast and lactic bacteria and the other only with yeast culture. After fermentation were analyzed fermented mashes (pH, volatile and total acidity, total and soluble dry substances, dynamic viscosity, alcohol concentration, number of yeast and bacteria cells (UFC/mL). All determinations have made in triplicate. For both series of fermented mashes observed an increase in viscosity for increasing grain particles of grist. Increased viscosity was higher when using only inoculums of yeast culture. pH values for both series of mashes varied in opposite directions. Towards lower pH's initial pH was significant for the series of mashes fermented with mixed cultivation of microorganisms. Are balanced values of alcoholic strength of fermented mashes at 25°C for 40 h. Very small inoculums of yeast ensures appropriate alcohol concentration, but not a total acidity that provides drinking fermented sour-sweet character type specific Braga. According to the ingredients used and method of obtaining of fermented mashes recommended wheat grain milling at 0.3 and/or 0.4 mm for a positive sensory evaluation.

Contribution of the Lipopolysaccharide to Resistance of Shigella flexneri 2a to Extreme Acidity

Shigella flexneri is endemic in most underdeveloped countries, causing diarrheal disease and dysentery among young children. In order to reach its target site, the colon, Shigella must overcome the acid environment of the stomach. Shigella is able to persist in this stressful environment and, because of this ability it can initiate infection following the ingestion of very small inocula. Thus, acid resistance is considered an important virulence trait of this bacterium. It has been reported that moderate acid conditions regulate the expression of numerous components of the bacterial envelope. Because the lipopolysaccharide (LPS) is the major component of the bacterial surface, here we have addressed the role of LPS in acid resistance of S. flexneri 2a. Defined deletion mutants in genes encoding proteins involved in the synthesis, assembly and length regulation of the LPS O antigen were constructed and assayed for resistance to pH 2.5 after adaptation to pH 5.5. The results showed that a mutant lacking O antigen was significantly more sensitive to extreme acid conditions than the wild type. Not only the presence of polymerized O antigen, but also a particular polymer length (S-OAg) was required for acid resistance. Glucosylation of the O antigen also contributed to this property. In addition, a moderate acidic pH induced changes in the composition of the lipid A domain of LPS. The main modification was the addition of phosphoethanolamine to the 1′ phosphate of lipid A. This modification increased resistance of S. flexneri to extreme acid conditions, provide that O antigen was produced. Overall, the results of this work point out to an important role of LPS in resistance of Shigella flexneri to acid stress.

English

Three strains,&nbsp;Bacillus subtilis&nbsp;B1,&nbsp;B. subtilis&nbsp;B18 and&nbsp;Bacillus&nbsp;thuringiensis&nbsp;B12,were&nbsp;screened&nbsp;from wheat bran&nbsp;to produce&nbsp;milk-clotting enzyme. Among them,&nbsp;B. subtilis&nbsp;B1 exhibited considerable milk-clotting activity with low proteolytic activity. After&nbsp;response surface methodology&nbsp;optimization,&nbsp;milk-clotting activity&nbsp;wasimproved from 782 SU ml-1&nbsp;to 1129.05 &plusmn; 74.55&nbsp;SU mL-1&nbsp;with a small inoculum size (0.130%, v/v).&nbsp;The&nbsp;optimized&nbsp;medium contained glucose (16.2 gL-1), wheat&nbsp;bran (30 gL-1), NaCl (5 gL-1), MgSO4&middot;7H2O (5 gL-1), KH2PO4&nbsp;(2 gL-1) and CaCO3&nbsp;(3 gL-1). The&nbsp;milk-clotting enzyme&nbsp;from&nbsp;B. subtilis&nbsp;B1 has the optimum pH 5.5 and CaCl2concentration 50 mmol L-1. It is completely inactivated after 5 min at 70&deg;C. The result showed that&nbsp;B. subtilis&nbsp;B1 was a promising strain for industrial milk-clotting enzyme production. &nbsp; Key words:&nbsp;Milk-clotting enzyme,&nbsp;Bacillus subtilis, submerged fermentation, wheat bran.

A Substantial Transmission Bottleneck among Newly and Recently HIV‐1–Infected Injection Drug Users in St Petersburg, Russia

There are limited data on the genetic complexity of human immunodeficiency virus type 1 (HIV-1) after transmission among a cohort of injection drug users (IDUs). We used single-genome amplification of HIV-1 env to determine the genotypic characteristics of virus among IDUs with acute infection in St Petersburg, Russia. Our results indicate that a single variant was transmitted in a majority of cases (9 of 13 participants), which is analogous to what is observed in sexual transmission. These data are most consistent with a genetic bottleneck during transmission by injection drug use that is due to a small inoculum, which most often results in the transmission of a low-complexity viral population.

A SEMI-STOCHASTIC MODEL FOR HIV POPULATION DYNAMICS

A semi-stochastic model is developed and investigated for human immunodeficiency virus type-1 (HIV-1) population dynamics. The model includes both stochastic parts (changes of CD4+ T cells) and deterministic parts (changes of free virions). Using the best currently available parameter values, we estimate the distributions of the time of occurrence and the magnitude of the early peak in virions. We investigated the effects of varying parameter values on mean solutions in order to assess the stochastic effects of between-patient variability. Numerical simulation shows that the lower the infection rate, the higher the death rate of the infected cells, more rapid clearance of virions and lower rate of virion emission by the infected cells result in lower speed of infection progression and magnitude but a greater variability in response. We also examine the probability that a small viral inoculum fails to establish an infection. Further, we theoretically quantify the expected variability around the infected equilibrium for each population by using diffusion approximation.

Estimating Risk from Small Inocula by Using Population Growth Parameters

Risk from an uncertain small inoculum depends on variability of single-cell lag times. However, quantifying single-cell variability is technically challenging. It is possible to estimate this variability using population growth parameters. We demonstrate this possibility using data from literature and show a Bayesian scheme for performing this task.

Diagnóstico microbiológico de las infecciones oculares

The recent increase in the practice of ocular surgery and widespread use of contact lenses have led to an increase in the incidence of severe ocular infections. Inadequate management of these infections can result in irreversible loss of vision. Microbiological diagnosis is essential when the lesions are non-specific, recurrent, or unresponsive to antibiotic therapy, but is hampered by the difficulty of analyzing limited sample volumes containing small inocula. This document presents a review of the most important ocular infections according to the structure affected, and describes the most common causes of these infections and the situations in which a microbiological diagnosis is recommended. Information is included on the sample type and sampling methods, sample transport to the laboratory, and laboratory management and processing techniques, with special attention to liquid culture media. The yield of smear examination and culture of each type of ocular specimen is specified. Lastly, the molecular methods recently developed for the diagnosis of ocular fungal infections and Acanthamoeba infections are described.

Estimating establishment probabilities of Cladocera introduced at low density: an evaluation of the proposed ballast water discharge standards

The International Maritime Organization has proposed ballast water discharge standards to reduce densities of taxa transported in ballast water. While reduced propagule pressure will almost certainly lower the risk of species introductions, we ask if the proposed standards will be effective against taxa capable of rapid population growth from small inocula, such as parthenogenetic taxa. We modified a diffusion approximation to calculate establishment probabilities as the probability of reaching a critical threshold density necessary to initiate sexual reproduction (and production of diapausing eggs for long-term persistence) before the onset of adverse environmental conditions. We parameterize our model for six Cladocera using growth rates measured during enclosure experiments conducted under favourable environmental conditions (i.e., using a precautionary approach). We then examine predicted establishment probabilities at different inoculum densities to determine the efficacy of the proposed ballast water discharge standards. Our results indicate that future technologies in compliance with the proposed standards could reduce establishment probabilities for some parthenogenetic zooplankton threefold; however, there is still a risk of establishment for some taxa.

Cultivation and Laboratory Maintenance of Chlamydia pneumoniae

Chlamydiae are Gram-negative obligate intracellular parasites. Chlamydia pneumoniae is a human respiratory pathogen that causes pneumonia, bronchitis, sinusitis, and pharyngitis. C. pneumoniae has also been associated with cardiovascular disease. C. pneumoniae can only be grown in cell culture and is more difficult to isolate from specimens than Chlamydia trachomatis. Commonly used cell lines for isolation of C. trachomatis are not sensitive for C. pneumoniae. The most sensitive cell lines for isolation of C. pneumoniae are HL and HEp-2. Centrifugation of the inoculum onto the monolayer and inclusion of cycloheximide in the medium enhance isolation. Inclusions are smaller than those of other chlamydiae and are visualized by staining with FITC-conjugated genus- or C. pneumoniae-specific monoclonal antibodies. Slow expansion and use of a small inoculum are key to successful culture. Infectious organisms can be purified by use of Hypaque-76 gradients to titers >1 x 10(8)/ml.

The Fatty Acid Biosynthesis Enzyme FabI Plays a Key Role in the Development of Liver-Stage Malarial Parasites

The fatty acid synthesis type II pathway has received considerable interest as a candidate therapeutic target in Plasmodium falciparum asexual blood-stage infections. This apicoplast-resident pathway, distinct from the mammalian type I process, includes FabI. Here, we report synthetic chemistry and transfection studies concluding that Plasmodium FabI is not the target of the antimalarial activity of triclosan, an inhibitor of bacterial FabI. Disruption of fabI in P. falciparum or the rodent parasite P. berghei does not impede blood-stage growth. In contrast, mosquito-derived, FabI-deficient P. berghei sporozoites are markedly less infective for mice and typically fail to complete liver-stage development in vitro. This defect is characterized by an inability to form intrahepatic merosomes that normally initiate blood-stage infections. These data illuminate key differences between liver- and blood-stage parasites in their requirements for host versus de novo synthesized fatty acids, and create new prospects for stage-specific antimalarial interventions.