Large Number Of Bacterial Isolates Research Articles

DropCarba – An automated magnetic digital microfluidic platform for rapid phenotypic testing of carbapenemase-producing Gram-negative bacilli

Carbapenemase-producing Gram-negative bacilli (CPGNB) is a type of antibiotic-resistant pathogens that often lead to severe clinical consequences. Phenotypic tests, such as Carba NP and blue Carba, are able to detect the resistant mechanism and provide rapid detection of carbapenemase producers to potentially guide personalized therapy. However, these tests require relatively tedious hands-on fluidic operations, and the assay format is ill-suited for automation and parallelization for improved throughput. In this study, we report an automated magnetic digital microfluidics-based platform, known as DropCarba, for parallel CPGNB detection in droplets. It automates the entire carbapenemase testing process and eliminates the need for almost all hands-on fluidic operations, which ensures high consistency and minimizes human errors with a simple “press-and-go” operation. DropCarba was validated with a large number of bacterial isolates of various Enterobacterales species (200 strains in total with 100 CPGNB and 100 non-resistant strains) in a blinded manner, and the results agree well with the benchmark Carba NP. DropCarba, with its full automation, simple operation, reduced reagent consumption, parallelization processing, and scalable manufacturing, will greatly improve CPGNB screening and make a valuable contribution to our fight against antibiotic resistance.

A scalable analytical approach from bacterial genomes to epidemiology.

Recent years have seen a remarkable increase in the practicality of sequencing whole genomes from large numbers of bacterial isolates. The availability of this data has huge potential to deliver new insights into the evolution and epidemiology of bacterial pathogens, but the scalability of the analytical methodology has been lagging behind that of the sequencing technology. Here we present a step-by-step approach for such large-scale genomic epidemiology analyses, from bacterial genomes to epidemiological interpretations. A central component of this approach is the dated phylogeny, which is a phylogenetic tree with branch lengths measured in units of time. The construction of dated phylogenies from bacterial genomic data needs to account for the disruptive effect of recombination on phylogenetic relationships, and we describe how this can be achieved. Dated phylogenies can then be used to perform fine-scale or large-scale epidemiological analyses, depending on the proportion of cases for which genomes are available. A key feature of this approach is computational scalability and in particular the ability to process hundreds or thousands of genomes within a matter of hours. This is a clear advantage of the step-by-step approach described here. We discuss other advantages and disadvantages of the approach, as well as potential improvements and avenues for future research.This article is part of a discussion meeting issue ‘Genomic population structures of microbial pathogens’.

Gplas: a comprehensive tool for plasmid analysis using short-read graphs.

SummaryPlasmids can horizontally transmit genetic traits, enabling rapid bacterial adaptation to new environments and hosts. Short-read whole-genome sequencing data are often applied to large-scale bacterial comparative genomics projects but the reconstruction of plasmids from these data is facing severe limitations, such as the inability to distinguish plasmids from each other in a bacterial genome. We developed gplas, a new approach to reliably separate plasmid contigs into discrete components using sequence composition, coverage, assembly graph information and network partitioning based on a pruned network of plasmid unitigs. Gplas facilitates the analysis of large numbers of bacterial isolates and allows a detailed analysis of plasmid epidemiology based solely on short-read sequence data.Availability and implementationGplas is written in R, Bash and uses a Snakemake pipeline as a workflow management system. Gplas is available under the GNU General Public License v3.0 at https://gitlab.com/sirarredondo/gplas.git.Supplementary information Supplementary data are available at Bioinformatics online.

16S rRNA gene sequencing and MALDI-TOF mass spectrometry based comparative assessment and bioprospection of psychrotolerant bacteria isolated from high altitudes under mountain ecosystem

The Himalayan Mountains are placed among the globally recognized biodiversity hot spots. While the Indian Himalayan Region (IHR) has been subjected to extensive studies on plant and animal biodiversity, microbial diversity is now being studied for its bioprospection. The present paper deals with the evaluation of bacterial diversity in high-altitude soil samples from IHR following polyphasic approach including comparison between the MALDI-TOF mass spectrometry and 16S rRNA gene sequencing for species-level identification. Initially, a culture collection of large number of bacterial isolates was established in the laboratory. Performing morphological and biochemical screenings, sixty-one representative isolates were selected for mass spectrometry and gene sequencing. Both the methods emerged with bacterial identification showing maximum number of Bacillus followed by Pseudomonas species. The other frequently isolated strains belonged to the genera Alcaligenes, Carnobacterium, Lysinibacillus, Microbacterium, Paenarthrobacter, Rhodococcus, Serratia and Stenotrophomonas. Although the MALDI-TOF technique appeared to be advantageous as less time-consuming in comparison with 16S rRNA-based method, the discrepancies at species level indicated the limited database of MALDI Biotyper and species complexity in the genera. The remarkable characteristics of the bacterial isolates were their tolerance to wide range of pH and temperature. Their potential to produce industrially valuable enzymes indicated their importance in bioprospection. Accessioning of these bacterial isolates in microbial culture collections is a cautious effort for their availability to conduct advanced research on these cold-adapted bacteria in future.

Novel approaches to the treatment of bacterial biofilm infections.

This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc.

Diversity and community analysis of fermenting bacteria isolated from eight major Korean fermented foods using arbitrary-primed PCR and 16S rRNA gene sequencing

Korean fermented foods are known to be beneficial for human health. Bacterial community studies have been conducted to figure out what roles the bacteria used to ferment these foods play in food fermentation. The metagenomic approach identifies both culturable and unculturable bacterial compositions, but this technique is limited in its ability to accurately determine the bacterial species from short 16S rRNA PCR products. In this study, we revisited the culture-dependent method using a relatively large number of bacterial isolates in an attempt to overcome the problem of bacterial identification, accepting that the unculturable bacterial population in each fermented food would be undetectable. Eight Korean fermented foods including kimchi, jeotgal, and meju were collected, and 1589 fermenting bacterial strains were randomly isolated. Bacteria were grouped by banding pattern using arbitrary-primed (AP) PCR prior to bacterial identification and sorted into 219 groups; 351 strains were not grouped because there was no identical AP-PCR band pattern. 16S rRNA sequence analysis identified the bacterial compositions of the fermented foods. As dominant genera, Lactobacillus and Leuconostoc strains were detected in four kimchi samples, Staphylococcus in three jeotgal samples, and Enterococcus and Bacillus in the meju sample. Interestingly, S. Equorum was most dominant in saeu-jeotgal, indicating that it is halophilic and may enhance the fermentation flavor. Further comparative analysis of this study with previous metagenomic results revealed that bacterial communities in fermented foods are highly similar at the genus level but often differ at the species level. This bacterial community study is useful for understanding the roles and functional properties of fermenting bacteria in the fermentation process of Korean fermented foods.

English

A simple and cheap method of plasmid DNA preparation from phytopathogenic Gram-negative bacteria (Xanthomonad, Erwinia stewartii) is presented here. In this method, in place of the high-priced chemicals and commercial kits, available and cheap chemicals were used for rapid isolation of small and large plasmids from different Gram negative bacteria. The time also was reduced by using this method giving a high quality plasmid production as demonstrated on the agarose gel, which make this method be used on a preparative scale to isolate sufficient quantities of plasmid DNA required for restriction analysis, cloning, or transformation experiments. A down scaled- protocol is also very useful for rapidly screening the wild plasmids in a large numbers of bacterial isolates in the experiments where hundreds of colonies should be screened for their plasmid contents such as in studying plasmid curing; antibiotic and heavy metal resistant bacteria or; xenobiotic compound degrading bacteria.   Key words: Plasmid, DNA, Erwinia, Xanthomonas, curing.

Using next generation sequencing to tackle non-typhoidal Salmonella infections

The publication of studies using next generation sequencing to analyse large numbers of bacterial isolates from global epidemics is transforming microbiology, epidemiology and public health. The emergence of multidrug resistant Salmonella Typhimurium ST313 is one example. While the epidemiology in Africa appears to be human-to-human spread and the association with invasive disease almost absolute, more needs to be done to exclude the possibility of animal reservoirs and to transfer the ability to track all Salmonella infections to the laboratories in the front line. In this mini-review we summarise what is currently known about non-typhoidal Salmonella in sub-Saharan Africa and discuss some of the issues which remain.

Comparative genotyping of Streptococcus mutans by repetitive extragenic palindromic polymerase chain reaction and multilocus sequence typing

The genetic diversity of Streptococcus mutans has been extensively studied using a variety of genotyping methods. Repetitive extragenic palindromic-polymerase chain reaction (rep-PCR) is a genotyping approach used for screening large numbers of bacterial isolates. This two-part study used multilocus sequence typing (MLST) analysis to evaluate genotypes previously identified as unique using rep-PCR. In part one, an isolate was selected from each of the 22 S.mutans rep-PCR genotype groups representing 8000 clinical isolates. For part two, four additional isolates were selected from the six most commonly occurring genotype groups (GG) for further analysis. Real-time PCR was performed using eight housekeeping S.mutans gene loci and the amplicons were sequenced. Sequence data analysis was performed using CLC DNA Workbench and alleles were compared with the PubMLST database for Oral Streptococcus using the Nakano scheme. Concatenated sequences were evaluated with MEGA using a minimum evolution method with bootstrap. All 22 rep-PCR genotypes were unique by MLST analysis. Within rep-PCR GGs, MLST matched rep-PCR in three groups demonstrating clonality; three groups exhibited more diversity with MLST. The discovery of three clonal groups is unique to this study and suggests that S.mutans genotypes are shared between unrelated subjects. Furthermore, MLST defined 19 new alleles and 26 new sequence types that have been confirmed and registered with PubMLST. Methods for processing were streamlined and a process for using MLST with rep-PCR is suggested. In conclusion, MLST verified that rep-PCR is a reliable and cost-effective method for screening large numbers of S.mutans strains for epidemiological study.

Comparison of biosurfactant detection methods reveals hydrophobic surfactants and contact‐regulated production

Biosurfactants are diverse molecules with numerous biological functions and industrial applications. A variety of environments were examined for biosurfactant-producing bacteria including soil, water and leaf surfaces. Biosurfactant production was assessed with an atomized oil assay for a large number of bacterial isolates and compared with a commonly used drop collapse assay from broth and plate cultures. The atomized oil assay detected every strain that produced a biosurfactant detectable by the drop collapse test, and also identified additional strains that were not detected with the drop collapse assay because they produced low levels of surfactant or hydrophobic (low water solubility) surfactants such as pumilacidins. Not all strains that produced a biosurfactant detectable by the drop collapse when cultured on agar surfaces produced surfactants detectable by drop collapse when cultured in broth, and vice versa. Many bacterial strains exhibited preferential production of surfactants when grown on an agar surface compared with broth cultures, and such surface enhancement of production could also be stimulated by increasing the viscosity of liquid culture media. Surface induction of surfactant production in the epiphyte Pseudomonas syringae was regulated at the transcriptional level.

A colorimetric assay of 1-aminocyclopropane-1-carboxylate (ACC) based on ninhydrin reaction for rapid screening of bacteria containing ACC deaminase

1-Aminocyclopropane-1-carboxylate (ACC) deaminase activity is an efficient marker for bacteria to promote plant growth by lowering ethylene levels in plants. We aim to develop a method for rapidly screening bacteria containing ACC deaminase, based on a colorimetric ninhydrin assay of ACC. A reliable colorimetric ninhydrin assay was developed to quantify ACC using heat-resistant polypropylene chimney-top 96-well PCR plates, having the wells evenly heated in boiling water, preventing accidental contamination from boiling water and limiting evaporation. With this method to measure bacterial consumption of ACC, 44 ACC-utilizing bacterial isolates were rapidly screened out from 311 bacterial isolates that were able to grow on minimal media containing ACC as the sole nitrogen source. The 44 ACC-utilizing bacterial isolates showed ACC deaminase activities and belonged to the genus Burkholderia, Pseudomonas or Herbaspirillum. Determination of bacterial ACC consumption by the PCR-plate ninhydrin-ACC assay is a rapid and efficient method for screening bacteria containing ACC deaminase from a large number of bacterial isolates. The PCR-plate ninhydrin-ACC assay extends the utility of the ninhydrin reaction and enables a rapid screening of bacteria containing ACC deaminase from large numbers of bacterial isolates.

Screening of lactic acid bacteria from fermented vegetables by carbohydrate profiling and PCR-ELISA.

The aim of this study was to identify potential souring agents, isolated from fermented plant material, by API 50 CHL assay and a molecular method based on polymerase chain reaction and colorimetric hybridization (PCR-ELISA). Forty-two strains of lactic acid bacteria derived from plant material were screened by taking advantage of API 50 CHL and PCR-ELISA. Oligonucleotide probes used for hybridization in PCR-ELISA were specific for lactobacilli, the Leuconostoc family, Lactobacillus pentosus/plantarum and Lactobacillus brevis. The hybrides were detected by a colour-developing reaction. Bacteria isolated from fermented cucumbers were identified as Lact. plantarum-related (Lact. plantarum and Lact. pentosus) and Leuconostoc species. Most of the strains isolated from sauerkraut were identified as Lact. pentosus/plantarum. Complementary results were obtained in the identification of bacterial strains, isolated from fermented cucumbers and sauerkraut, by API 50 CHL and PCR-ELISA. PCR-ELISA proved to be suitable for the screening of large numbers of bacterial isolates from fermented vegetables. This will be useful for the identification of strains suitable for the design of starter cultures for the fermentation of plant material.

Evaluation of genotyping large numbers of Escherichia coli isolates by enterobacterial repetitive intergenic consensus-PCR.

We analyzed a large collection of Escherichia coli isolates typed by enterobacterial repetitive intergenic consensus (ERIC)-PCR with BioNumerics gel analysis software. However, the interexperimental variation of ERIC-PCR caused the computer software to classify repeated isolates as different. ERIC-PCR should not be used as the sole determinant of genetic similarity when typing large numbers of bacterial isolates via computer software.

Detection of Microbial Growth on Polycyclic Aromatic Hydrocarbons in Microtiter Plates by Using the Respiration Indicator WST-1

We have developed a microtiter plate method for screening a large number of bacterial isolates for the ability to grow on different crystalline polycyclic aromatic hydrocarbons (PAHs). Growth on PAHs cannot easily be determined with standard growth assays because of the very low aqueous solubility and bioavailability of the PAHs. Our microtiter plate assay utilizes a new water-soluble respiration indicator, WST-1 [4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate], in combination with easily degradable carbon sources. PAH-mineralizing strains were grown on PAHs in microtiter plates for 7 to 10 days. The tetrazolium dye WST-1 was added after incubation. Dehydrogenases in growing cells reduced WST-1 to a water-soluble colored formazan, and the intensity of the color was a measure of the respiration rate. Addition of easily degradable carbon to the wells along with WST-1 resulted in a 3- to 40-fold increase in the absorbance of positive wells within 90 min, which made it possible to detect growth on fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. Addition of the electron transport blocker sodium azide unexpectedly decreased formazan formation. The method was adapted for most-probable-number enumeration of PAH degraders in soil.

Screening of bacteria for the suppression of Botrytis cinerea and Rhizoctonia solani on lettuce (Lactuca sativa) using leaf disc bioassays

Summary Two leaf disc bioassays were developed for screening bacteria as putative biological control agents of Botrytis cinerea and Rhizoctonia solani on lettuce. Aerobic spore and non-spore forming bacteria were isolated from the phylloplane, rhizoplane and rhizosphere of symptom-free lettuce plants grown in the presence and absence of chitin or composted bark soil amendments. Bacteria, previously isolated from other plants, were also included in the primary screen initially against B. cinerea. One hundred and twenty-seven of 700 isolates reduced botrytis rotting of lettuce leaves by more than 50% in the primary screen. Following a secondary screen against B. cinerea, the lead 50 isolates were also tested for suppression of R. solani infection. Four isolates significantly reduced both botrytis and rhizoctonia leaf rotting. Eleven and five isolates gave control of botrytis and rhizoctonia, respectively, equal to that given by the standard fungicides Rovral WP (iprodione) and Basilex (tolclofos methyl). The two most effective isolates against B. cinerea and R. solani were both identified as Bacillus subtilis. Use of soil amendments did not increase the proportion of efficacious isolates recovered. Effective isolates were originally recovered from roots of oilseed rape and lettuce leaves. In general, it was found that bacteria which controlled one disease effectively did not control the second disease nearly as well. The bioassay protocols developed in this study were used successfully in screening a large number of bacterial isolates in a short time.

A simple and rapid serum bactericidal assay and its evaluation in clinical isolates of Klebsiella pneumoniae

A simple and rapid assay for the determination of serum bactericidal activity was developed and evaluated in 125 clinical isolates of Klebsiella pneumoniae. The serum reactivity against these isolates was concomitantly determined by the conventional viable count technique in order to compare the efficacy of the two techniques. The rapid assay could be completed within 5–8 h and the results were recorded in terms of visible change of colour of the culture medium. Of the 125 strains tested, more than 50% were found to be resistant to 20% normal human serum. There was an excellent agreement between the two methods. The degree of discordance observed in the results obtained by the two methods was statistically not significant ( P>0.05). The conventional technique is labor intensive, cumbersome and time-consuming. The assay described here, on the other hand, is simple, easy and rapid enough to allow testing of a large number of bacterial isolates or recombinant clones in a single day. Thus, the assay can serve as an excellent alternative to the conventional technique for determining the bacterial serum susceptibility.

Phenotypic Diversity and Stability of the Intestinal Coliform Flora in Piglets During the First 3 Months of Age

The intestinal coliform bacterial populations in six piglets and their sows from two litters in the same stable were studied by the aid of an automated system for biochemical fingerprinting (The PhenePlate, or PhP system). Faecal samples from sows were collected once a week until weaning, and from piglets once a week during their first 3 mth of life. Altogether 1248 coliform isolates from 52 faecal samples were studied. The phenotypical diversity of the coliform bacteria in each faecal sample was measured as Simpson’s diversity index (D i ), and the similarities between the bacteria in different samples were measured as population similarity coefficients (S p ). The D i of the sows was initially high, but decreased in one sow during an outbreak of diarrhoea which occurred during week 3 of the study. The D i of the piglets varied between samples, and showed no tendency to increase with age. Two piglets included in the study died during the diarrhoeal outbreak, and they generally carried intestinal floras showing lower D i during their first 3 wk of life than those which survived (mean value 0.454 versus 0.744). The coliform population within each piglet was usually quite stable before the diarrhoeal outbreak, but in all piglets the population was drastically changed during and after the outbreak. Piglets within each litter shared similar coliform populations during the first week of life, but piglets from different litters had no coliforms in common. On day 75, the piglets were removed from their sows and mixed with others in different litters, and after that coliform populations of piglets from different sows showed a tendency to become more similar to each other. Samples collected during the diarrhoeal outbreak from different piglets did not contain similar coliform populations which is a strong indication that the aetiological agent of the outbreak was not any coliform bacterium. The present study shows a useful way to investigate intestinal bacteria in both man and animals. The methods are very simple to use, and fully computerised data treatment makes it possible to study large numbers of bacterial isolates. Keywords - Intestinal bacteria; Coliforms; Piglets; Diversity; Stability.

Phenotypic Diversity and Stability of the Intestinal Coliform Flora in Piglets During the First 3 Months of Age

The intestinal coliform bacterial populations in six piglets and their sows from two litters in the same stable were studied by the aid of an automated system for biochemical fingerprinting (The PhenePlate, or PhP system). Faecal samples from sows were collected once a week until weaning, and from piglets once a week during their first 3 mth of life. Altogether 1248 coliform isolates from 52 faecal samples were studied. The phenotypical diversity of the coliform bacteria in each faecal sample was measured as Simpson's diversity index (Di), and the similarities between the bacteria in different samples were measured as population similarity coefficients (Sp). The Di of the sows was initially high, but decreased in one sow during an outbreak of diarrhoea which occurred during week 3 of the study. The Di of the piglets varied between samples, and showed no tendency to increase with age. Two piglets included in the study died during the diarrhoeal outbreak, and they generally carried intestinal floras showing lower Di during their first 3 wk of life than those which survived (mean value 0.454 versus 0.744). The coliform population within each piglet was usually quite stable before the diarrhoeal outbreak, but in all piglets the population was drastically changed during and after the outbreak. Piglets within each litter shared similar coliform populations during the first week of life, but piglets from different litters had no coliforms in common. On day 75, the piglets were removed from their sows and mixed with others in different litters, and after that coliform populations of piglets from different sows showed a tendency to become more similar to each other. Samples collected during the diarrhoeal outbreak from different piglets did not contain similar coliform populations which is a strong indication that the aetiological agent of the outbreak was not any coliform bacterium. The present study shows a useful way to investigate intestinal bacteria in both man and animals. The methods are very simple to use, and fully computerised data treatment makes it possible to study large numbers of bacterial isolates.

Screening bacteria for root colonizing ability by a rapid method

A method is described for a rapid screening of a large number of bacterial isolates for their root colonizing ability based on direct microscopic observation of roots grown in water agar. Root colonizing bacteria could be seen swarming around roots and forming highly visible bright halos within 7–12 h after bacterial suspensions were placed near the root tips. To determine the relative value of the direct microscopic method, 16 randomly selected bacterial isolates were screened for their root colonizing ability by the direct observation of roots of cucumber and tomato grown in agar and by the standard dilution plating method using roots grown in soil. The rankings of the isolates for root colonizing ability obtained by the two methods were highly correlated. In 32 plant-bacterium combinations tested, the outcome of only two combinations were different in the two methods. One isolate which colonized cucumber in water agar and another isolate which colonized tomato roots in water agar did not colonize roots of these plants in the soil. Results show that the direct microscopic method can be used for a rapid qualitative screening of bacteria for their ability to colonize roots of certain plants.

Xylan-digesting Bacteria from the Rumen of Sheep Fed Maize Straw Diets

Summary: Representatives of a large number of bacterial isolates that produced clearings in xylan agar medium in an earlier study were characterized in greater detail. Butyrivibrios and ruminococci predominated. Three different biotypes of Butyrivibrio fibrisolvens, all non-cellulolytic, were identified on the basis of net acetate production or uptake, or the production of propionate. The only variable characteristic observed within the group of Ruminococcus albus isolates was the ability to solubilize cellulose. In contrast, all typical Ruminococcus flavefaciens isolates were cellulolytic. Other bacteria that occurred in smaller numbers belonged to the genera Eubacterium, Coprococcus, Fusobacterium, Bacteroides, Treponema and Clostridium.