High-throughput microfluidic system for long-term bacterial colony monitoring and antibiotic testing in zero-flow environments

In this study, a high-throughput microfluidic system is presented. The system is comprised of seven parallel channels. Each channel contains 32 square-shaped microchambers. After simulation studies on samples loaded into the microchambers, and the solute exchange between the microchambers and channels, the long-term culture of Escherichia coli (E. coli) HB101 in the microchambers is realized. Using the principle that L-arabinose (L-Ara) can induce recombinant E. coli HB101 pGLO to synthesize green fluorescent protein (GFP), the real-time analysis of GFP expression in different initial bacterial densities is performed. The results demonstrate that higher initial loading densities of the bacterial colony cause bacterial cell to enter log-phase proliferation sooner. High or low initial loading densities of the bacterial cell suspension induce the same maximum growth rates during the log-phase. Quantitative on-chip analysis of tetracycline and erythromycin inhibition on bacterial cell growth is also conducted. Bacterial morphology changes during antibiotic treatment are observed. The results show that tetracycline and erythromycin exhibit different inhibition activities in E. coli cells. Concentrations of 3 μg/mL tetracycline can facilitate the formation of long filamentous bacteria with the average length of more than 50 μm. This study provides an on-chip framework for bacteriological research in a high-throughput manner and the development of recombinant bacteria-based biosensors for the detection of specific substances.     

Screening of different contaminated environments for polyhydroxyalkanoates-producing bacterial strains

Total sixteen bacterial strains were isolated and purified from the samples collected from sugarcane molasses soil, sewage water and long-chain-hydrocarbon-contaminated area of the Punjab University, Lahore, Pakistan. Tolerance to different antibiotics was studied and strains showed multiple antibiotic resistance. All strains were characterized for Gram stain, biochemical reactions and polyhydroxyalkanoate (PHA) production. Total fourteen strains were Gram negative and two were Gram positive, while biochemically nine PHA producers showed affiliation to Pseudomonas, Enterobacter, Citrobacter, Bacillus and Escherichia. Screening for PHA production was done by Sudan black staining and nine out of sixteen strains exhibited PHA producing ability. PHA production was optimized for different growth parameters, like nitrogen concentration, pH and temperature. PHA extraction was done by solvent extraction method. Bacterial strains US1 and M1 accumulated up to 30% PHA of their cell dry weight on PHA extraction by solvent extraction method. Bacterial strain US1 was identified by 16S rRNA gene analysis as P. aeruginosa (DQ455691). PHA production was confirmed by PCR amplification of 500 bp fragment from PHA polymerase (Pha C) gene; five strains from nine PHA producers gave positive results on PCR. Pha C gene fragment of US1 was sequenced and submitted to Gene Bank under the accession number DQ455690. The amino acid sequence showed homology using the protein BLAST at 129–132 sites with different PHA synthases of the Pseudomonas sp.     

Alternatives to antibiotics for the control of bacterial disease in aquaculture

The wide and frequent use of antibiotics in aquaculture has resulted in the development and spread of antibiotic resistance. Because of the health risks associated with the use of antibiotics in animal production, there is a growing awareness that antibiotics should be used with more care. This is reflected in the recent implementation of more strict regulations on the prophylactic use of antibiotics and the presence of antibiotic residues in aquaculture products. For a sustainable further development of the aquaculture industry, novel strategies to control bacterial infections are needed. This review evaluates several alternative biocontrol measures that have emerged recently. Most of these methods are still in research phase; few have been tested in real aquaculture settings. It is important to further develop different strategies that could be combined or used in rotation in order to maximise the chance of successfully protecting the animals and to prevent resistance development.     

Interaction of aminoglycosides and the other antibiotic on selected bacterial strains

The aim of this study was to analyse the activity interaction of aminoglycosides (gentamicin, kanamycin, streptomycin and dihydrostreptomycin) when combined with other antibiotics (lincomycin, benzylpenicillin, amoxicillin, cephalexin, spectinomycin and erythromycin), on selected clinical bacterial strains. The checkerboard method has been selected from the traditional assays for the measurement of antibiotic interaction. Checkerboard results for all strains demonstrated synergism for nine cases (9/112--8%). Additive effects were predominant--73/112--65.2%. In 12.5% neutral effects were shown, but in 11.6% of combinations FIC indexes were not possible to calculate, because of the resistance of clinical strains to the highest concentration of at least one antibiotic. The best results were achieved for combinations of dihydrostreptomycin with procaine penicillin because of higher number of cases synergy effect was observed. Antagonism of aminoglycosides and beta-lactams in case of gentamicin and amoxicillin for E. coli and E. cloacea strains were shown. Potential activity for combination of streptomycin and erythromycin was shown.     

Determination of biological and physicochemical parameters of Artemia franciscana strains in hypersaline environments for aquaculture in the Colombian Caribbean

Background

Artemia (Crustacea, Anostraca), also known as brine shrimp, are typical inhabitants of extreme environments. These hypersaline environments vary considerably in their physicochemical composition, and even their climatic conditions and elevation. Several thalassohaline (marine) environments along the Colombian Caribbean coast were surveyed in order to contribute to the knowledge of brine shrimp biotopes in South America by determining some vital biological and physicochemical parameters for Artemia survival. Additionally, cyst quality tests, biometrical and essential fatty acids analysis were performed to evaluate the economic viability of some of these strains for the aquaculture industry.

Results

In addition to the three locations (Galerazamba, Manaure, and Pozos Colorados) reported in the literature three decades ago in the Colombian Caribbean, six new locations were registered (Salina Cero, Kangaru, Tayrona, Bahía Hondita, Warrego and Pusheo). All habitats sampled showed that chloride was the prevailing anion, as expected, because of their thalassohaline origin. There were significant differences in cyst diameter grouping strains in the following manner according to this parameter: 1) San Francisco Bay (SFB-Control, USA), 2) Galerazamba and Tayrona, 3) Kangarú, 4) Manaure, and 5) Salina Cero and Pozos Colorados. Chorion thickness values were smaller in Tayrona, followed by Salina Cero, Galerazamba, Manaure, SFB, Kangarú and Pozos Colorados. There were significant differences in naupliar size, grouping strains as follows (smallest to largest): 1) Galerazamba, 2) Manaure, 3) SFB, Kangarú, and Salina Cero, 4) Pozos Colorados, and 5) Tayrona. Overall, cyst quality analysis conducted on samples from Manaure, Galerazamba, and Salina Cero revealed that all sites exhibited a relatively high number of cysts.g^-1. Essential fatty acids (EFA) analysis performed on nauplii from cyst samples from Manaure, Galerazamba, Salina Cero and Tayrona revealed that cysts from all sites exhibited high arachidonic acid:20:4(n-6) (ArA) and eicosapentaenoic acid: 20:5(n-3) (EPA) levels comparable to the control sample (SFB). In contrast, most cysts collected (including SFB) at different locations, and during different months, presented low docosahexaenoic acid: 22:6(n-3) (DHA) levels (Manaure was the only exception with high DHA levels). Some variations in EPA and ArA levels were observed in all sites, contrasting with the much lower DHA levels which remained constant for all locations, except for Manaure which exhibited variable DHA levels. DHA/EPA ratio was overall very low for all sites compared to SFB cysts. All strains had a low DHA/ArA, but a high EPA/ArA ratio, including the control.

Conclusion

The Colombian A. franciscana habitats analyzed were determined to be thalassohaline, and suitable for A. franciscana development. EFA profiles demonstrated that Tayrona, Galerazamba, Manaure and Salina Cero strains are suitable food for marine fish and crustacean culture because of their high EPA/ArA ratio, but might have to be fortified with DHA rich emulsions depending on the nutritional requirements of the species to be cultured, because of their overall low DHA content. The relatively small nauplii are appropriate for marine larvaeculture. In contrast, the strains from Tayrona, Kangarú, Salina Cero, and Pozos Colorados may be of use but limited to Artemia small biomass production quantities, because of the small surface area of their respective locations; Artemia could be exploited at these locations for local aquaculture applications. In general, cyst quality evaluation for Manaure, Salina Cero and Galerazamba cysts revealed that cysts from these three locations could improve their quality by concentrating efforts on cyst processing techniques. Finally, most locations had great A. franciscana production potential and require different degrees of water quality and/or infrastructure management.     

Isolation of antibiotic resistance bacterial strains from Eastern Siberia permafrost sediments

A collection of bacterial antibiotic resistance strains isolated from arctic permafrost subsoil sediments of various age and genesis was created. The collection included approximately 100 strains of Gram-positive (Firmicutes, Arthrobacter) and Gram-negative bacteria (Bacteroidetes, γ-Proteobacteria, and α-Proteobacteria) resistant to aminoglycoside antibiotics (gentamicin, kanamycin, and streptomycin), chloramphenicol and tetracycline. Antibiotic resistance spectra were shown to differ in Gram-positive and Gram-negative bacteria. Multidrug resistance strains were found for the first time in ancient bacteria. In studies of the molecular nature of determinants for streptomycin resistance, determinants of the two types were detected: strA-strB genes coding for aminoglycoside phosphotransferases and genes aadA encoding aminoglycoside adenylyltransferases. These genes proved to be highly homologous to those of contemporary bacteria.     

Isolation of antibiotic resistance bacterial strains from East Siberia permafrost sediments

A collection of bacterial antibiotic resistance strains isolated from arctic permafrost subsoil sediments of various age and genesis was created. The collection included approximately 100 strains of Gram-positive (Firmicutes, Arthrobacter) and Gram-negative bacteria (Bacteroidetes, gamma-Proteobacteria, and alpha-Proteobacteria) resistant to aminoglycoside antibiotics (gentamycin, kanamycin, and streptomycin), chloramphenicol and tetracycline. Antibiotic resistance spectra were shown to differ in Gram-positive and Gram-negative bacteria. Multidrug resistance strains were found for the first time in ancient bacteria. In studies of the molecular nature of determinants for streptomycin resistance, determinants of the two types were detected: strA-strB genes coding for aminoglycoside phosphotransferases and genes aadA encoding aminoglycoside adenylyltransferases. These genes proved to be highly homologous to those of contemporary bacteria.     

Cerebellar protein expression in three different mouse strains and their relevance for motor performance

The present study uses a proteomic approach to link motor function to cerebellar protein expression in 129X1/SvJ, C57BL/6J and nNOS WT mice. Poor performance on the Rota rod, the standard test for motor coordination, was detected in 129X1/SvJ mice. No gross impairments of neurological, cognitive and behavioural functions were observed. Identification and quantification of 48 proteins revealed reduced expression of calbindin, septin 5 and syntaxin binding protein 1 in 129X1/SvJ. In nNos WT glucose-6-phosphate 1 dehydrogenase X was decreased whereas dihydropyrimidinase-related protein-4 was increased. In C57BL/6J stress-70 protein, alpha enolase, NAD-dependent deacetylase sirtuin 2, septin 2, dihydropyrimidinase-related protein-2 and brain derived neurotrophic factor showed elevated levels. Neurological examination, Rota rod test, Morris Water Maze, Multiple-T-Maze, Open field and Elevated plus-maze were employed to study motor, cognitive and behavioural function. Mice were sacrificed and cerebellar tissue was homogenized. Proteins were extracted and separated on two-dimensional gel electrophoresis with subsequent in-gel digestion followed by mass spectrometrical analysis of peptides (MALDI-TOF/TOF-TOF). Quantification of spots was carried out by specific software. A strong association of impaired motor function with altered cerebellar protein expression of calbindin, septin 5 and syntaxin binding protein 1in 129X1/SvJ was observed and is in agreement with previous observations of motor deficiencies in a calbindin knock-out mouse. These results have to be taken into account when using 129X1/SvJ for biochemical, toxicological or gene targeting experiments as well as when studying the above-mentioned proteins or corresponding pathways and cascades in this mouse strain.     

Development of a vital fluorescent staining method for monitoring bacterial transport in subsurface environments

Previous bacterial transport studies have utilized fluorophores which have been shown to adversely affect the physiology of stained cells. This research was undertaken to identify alternative fluorescent stains that do not adversely affect the transport or viability of bacteria. Initial work was performed with a groundwater isolate, Comamonas sp. strain DA001. Potential compounds were first screened to determine staining efficiencies and adverse side effects. 5-(And 6-)-carboxyfluorescein diacetate, succinimidyl ester (CFDA/SE) efficiently stained DA001 without causing undesirable effects on cell adhesion or viability. Members of many other gram-negative and gram-positive bacterial genera were also effectively stained with CFDA/SE. More than 95% of CFDA/SE-stained Comamonas sp. strain DA001 cells incubated in artificial groundwater (under no-growth conditions) remained fluorescent for at least 28 days as determined by epifluorescent microscopy and flow cytometry. No differences in the survival and culturability of CFDA/SE-stained and unstained DA001 cells in groundwater or saturated sediment microcosms were detected. The bright, yellow-green cells were readily distinguished from autofluorescing sediment particles by epifluorescence microscopy. A high throughput method using microplate spectrofluorometry was developed, which had a detection limit of mid-10(5) CFDA-stained cells/ml; the detection limit for flow cytometry was on the order of 1,000 cells/ml. The results of laboratory-scale bacterial transport experiments performed with intact sediment cores and nondividing DA001 cells revealed good agreement between the aqueous cell concentrations determined by the microplate assay and those determined by other enumeration methods. This research indicates that CFDA/SE is very efficient for labeling cells for bacterial transport experiments and that it may be useful for other microbial ecology research as well.     

Prevalence of genetically similar Flavobacterium columnare phages across aquaculture environments reveals a strong potential for pathogen control

Intensive aquaculture conditions expose fish to bacterial infections, leading to significant financial losses, extensive antibiotic use and risk of antibiotic resistance in target bacteria. Flavobacterium columnare causes columnaris disease in aquaculture worldwide. To develop a bacteriophage-based control of columnaris disease, we isolated and characterized 126 F. columnare strains and 63 phages against F. columnare from Finland and Sweden in 2017. Bacterial isolates were virulent on rainbow trout (Oncorhynchus mykiss) and fell into four previously described genetic groups A, C, E and G, with genetic groups C and E being the most virulent. Phage host range studied against a collection of 227 bacterial isolates (from 2013 to 2017) demonstrated modular infection patterns based on host genetic group. Phages infected contemporary and previously isolated bacterial hosts, but bacteria isolated most recently were generally resistant to previously isolated phages. Despite large differences in geographical origin, isolation year or host range of the phages, whole-genome sequencing of 56 phages showed high level of genetic similarity to previously isolated F. columnare phages (Ficleduovirus, Myoviridae). Altogether, this phage collection demonstrates a potential for use in phage therapy.     

62株阴沟肠杆菌的生化特性和药敏结果

目的探讨临床分离阴沟肠杆菌的实用鉴定方法.方法对VITEK-32微生物分析仪鉴定所得的62株阴沟肠杆菌的生化反应和药敏结果作回顾性分析.结果 62株阴沟肠杆菌用鉴定卡鉴定的可信度在94%～99%,其中51株为99%、占82.3%,4株为95%、占6.5%,其他7株、占11.2%.阴沟肠杆菌对除外亚胺培南的20种抗生素均有不同程度的耐药.结论脲酶、侧金盏花醇、精氨酸水解和赖氨酸脱羧等试验对肠杆菌属中5个常见菌种(阴沟、坂崎、产气、聚团和格高菲肠杆菌)的鉴别有重要意义.     

A suite of recombinant luminescent bacterial strains for the quantification of bioavailable heavy metals and toxicity testing

Background  

Recombinant whole-cell sensors have already proven useful in the assessment of the bioavailability of environmental pollutants like heavy metals and organic compounds. In this work 19 recombinant bacterial strains representing various Gram-positive (Staphylococcus aureus and Bacillus subtilis) and Gram-negative (Escherichia coli, Pseudomonas fluorescens) bacteria were constructed to express the luminescence encoding genes luxCDABE (from Photorhabdus luminescens) as a response to bioavailable heavy metals ("lights-on" metal sensors containing metal-response elements, 13 strains) or in a constitutive manner ("lights-off" constructs, 6 strains).     

High performance liquid chromatography detection of phototrophic bacterial pigments in aquatic environments

Pigment extracts of phototrophic bacteria isolated from Lake Kinneret (Rhodopseudomonas palustris, Thiocapsa roseopersicina, Prosthecochloris aestuaris andChlorobium phaeobacteroides) were studied by means of high performance liquid chromatography (HPLC). An absorption wavelength of 360 nm provided the best resolution among the pigments of the species tested and between them and chlorophylla. Signature pigments were identified for each of these species, and their presence was thereby monitored in lake water samples.C. phaeobacteroides, which was observed in the anaerobic hypolimnion and predominated in the metalimnion, was recognized by a characteristic cluster of major chlorophyllous pigment peaks. The spectral qualities of these pigments were close but not identical to published data on bacteriochlorophylle, presumably due to the use of different solvents for extraction. The intensity of these pigment peaks was employed to determine the depth of the greatest phototrophic bacterial biomass, which was not related to that of algae.     

Power and performance management of virtualized computing environments via lookahead control

There is growing incentive to reduce the power consumed by large-scale data centers that host online services such as banking, retail commerce, and gaming. Virtualization is a promising approach to consolidating multiple online services onto a smaller number of computing resources. A virtualized server environment allows computing resources to be shared among multiple performance-isolated platforms called virtual machines. By dynamically provisioning virtual machines, consolidating the workload, and turning servers on and off as needed, data center operators can maintain the desired quality-of-service (QoS) while achieving higher server utilization and energy efficiency. We implement and validate a dynamic resource provisioning framework for virtualized server environments wherein the provisioning problem is posed as one of sequential optimization under uncertainty and solved using a lookahead control scheme. The proposed approach accounts for the switching costs incurred while provisioning virtual machines and explicitly encodes the corresponding risk in the optimization problem. Experiments using the Trade6 enterprise application show that a server cluster managed by the controller conserves, on average, 22% of the power required by a system without dynamic control while still maintaining QoS goals. Finally, we use trace-based simulations to analyze controller performance on server clusters larger than our testbed, and show how concepts from approximation theory can be used to further reduce the computational burden of controlling large systems.

An azido-oxazolidinone antibiotic for live bacterial cell imaging and generation of antibiotic variants

An azide-functionalised analogue of the oxazolidinone antibiotic linezolid was synthesised and shown to retain antimicrobial activity. Using facile ‘click’ chemistry, this versatile intermediate can be further functionalised to explore antimicrobial structure–activity relationships or conjugated to fluorophores to generate fluorescent probes. Such probes can report bacteria and their location in a sample in real time. Modelling of the structures bound to the cognate 50S ribosome target demonstrates binding to the same site as linezolid is possible. The fluorescent probes were successfully used to image Gram-positive bacteria using confocal microscopy.     

Molecular identification and antibiotic control of endophytic bacterial contaminants from micropropagated Aglaonema cultures

Endophytic bacterial contamination is a major constraint to the establishment and maintenance of aseptic Aglaonema cultures. The objectives of the present study included the identification of endophytic bacterial contaminants from micropropagated Aglaonema cultures and the investigation of effective antibiotic treatment for their control. Bacterial contaminants isolated from 181 infected stem nodal explants of six Aglaonema cultivars were identified following the amplification of 16S ribosomal RNA gene and partial sequence analysis. A total of thirteen different bacterial species were identified and these were found to be mostly associated with soil and water. The bacteria were subsequently subjected to antimicrobial susceptibility and minimum inhibitory concentration tests. Three antibiotics, including gentamicin, tetracycline and chloramphenicol, were selected for their effectiveness at low concentrations of 4?C32?mg?l^?1 to inhibit bacterial growth in most of the bacterial species found in the present study. The incorporation of these antibiotics into the culture medium was found to effectively reduce the incidence of bacterial contamination in three of the four Aglaonema cultivars tested. Therefore, sanitation of the irrigation water and growth substrate while raising the stock plants, as well as the appropriate use of antibiotics during the in vitro culture stage will be important factors governing the success of Aglaonema micropropagation in the future.     

Accurate assessment of antibiotic susceptibility and screening resistant strains of a bacterial population by linear gradient plate

The dynamics of a bacterial population exposed to the minimum inhibitory concentration (MIC) of an antibiotic is an important issue in pharmacological research. Therefore, a novel antibiotic susceptibility test is urgently needed that can both precisely determine the MIC and accurately select antibiotic-resistant strains from clinical bacterial populations. For this purpose, we developed a method based on Fick's laws of diffusion using agar plates containing a linear gradient of antibiotic. The gradient plate contained two layers. The bottom layer consisted of 15 mL agar containing the appropriate concentration of enrofloxacin and allowed to harden in the form of a wedge with the plate slanted such that the entire bottom was just covered. The upper layer consisted of 15 mL plain nutrient agar added with the plate held in the horizontal position. After allowing vertical diffusion of the drug from the bottom agar layer for 12 h, the enrofloxacin concentration was diluted in proportion to the ratio of the agar layer thicknesses. The uniform linear concentration gradient was verified by measuring the enrofloxacin concentration on the agar surface. When heavy bacterial suspensions were spread on the agar surface and incubated for more than 12 h, only resistant cells were able to form colonies beyond the boundary of confluent growth of susceptible cells. In this way, the true MIC of enrofloxacin was determined. The MICs obtained using this linear gradient plate were consistent with those obtained using conventional antibiotic susceptibility tests. Discrete colonies were then spread onto a gradient plate with higher antibiotic concentrations; the boundary line increased significantly, and gene mutations conferring resistance were identified. This new method enables the rapid identification of resistant strains in the bacterial population. Use of the linear gradient plate can easily identify the precise MIC and reveal the dynamic differentiation of bacteria near the MIC. This method allows the study of genetic and physiological characteristics of individual strains, and may be useful for early warning of antibiotic resistance that may occur after use of certain antimicrobial agents, and guide clinical treatment.