Bradyrhizobium japonicum FN1 produces an inhibitory substance that affects competition for nodule occupancy

Bacteriocins are narrow spectrum antibiotics of bacterial origin that can affect competition in resource-limited environments such as the rhizosphere. Therefore, bacteriocins may be good candidates for manipulation in order to generate more competitive inocula for soybean. In this study,<i> B. japonicum</i> FN1 along with other Bradyrhizobia in our culture collection were screened for bacteriocin-like activity. A total of five distinct inhibitory activities could be observed. FN1 genes putatively involved in bacteriocin production were computationally identified. These genes were mutagenized and the subsequent strains were screened for loss of inhibitory activity. Mutant strain BRJ-48, with an insert in<i> bjfn1_01204</i>, displayed a loss of the ability to inhibit an indicator strain. This loss could be complemented by the introduction of a plasmid expressing <i>bjfn1_01204 </i>in trans. The strain carrying the mutation did not affect competition in broth cultures, but was shown to be less competitive for nodule occupancy. Annotation suggests that <i>bjfn1_01204</i> encodes a carboxymuconolactone decarboxylase, however the direct contribution of how this enzyme contributes to inhibiting the tester strain remains unknown.

A phenotypic screen using splitCas9 identifies essential genes required for actin regulation during host cell egress and invasion by Toxoplasma gondii

Apicomplexan parasites, such as Toxoplasma gondii, possess unique organelles, cytoskeletal structures, signalling cascades, replicate by internal budding within a specialised compartment and actively invade and exit the host cell, to name a few aspects of the unique biology that characterise this phylum. Due to their huge phylogenetic distance from well established model organisms, such as opisthokonts, comparative genomics has a limited capacity to infer gene functions and conserved proteins can fulfil different roles in apicomplexans. Indeed, approximately 30% of all genes are annotated as hypothetical and many had a crucial role during the asexual life cycle in genome-wide screens. While the current CRISPR/Cas9-based screens allow the identification of fitness conferring genes, only little information about the respective functions can be obtained. To overcome this limitation, and to group genes of interest into functional groups, we established a conditional Cas9-system in T. gondii that allows phenotypic screens. Using an indicator strain for F-actin dynamics and apicoplast segregation, we identified critical genes required for defined steps during the asexual life cycle. The detailed characterisation of two of these candidates revealed them to be critical for host cell egress and invasion and to act at different time points in the disassembly of the intravacuolar F-actin network. While the signalling linking factor (SLF) is an integral part of a signalling complex required for early induction of egress, a novel conoid protein (conoid gliding protein, CGP) acts late during egress and is required for the activation of gliding motility.

Antibiotic Spectrum and Stability of Crude Extract of Extracellular Metabolites from Bacillus mageterium LB01-17

Crop pathogens including fungi and bacteria seriously affect the quality and yields of agricultural products. The purpose of this study was to detect the antibiotic spectrum of the crude extract of extracellular metabolites produced by Bacillus megaterium LB01-17 and its stability to heat, acid, alkali and ultraviolet light. The methods of mycelium growth rate and inhibitory zones were used to test the antimicrobial activity of the crude extract on 13 kinds of crop pathogenic fungi and 6 kinds of bacteria. The activity stability of the crude extract was determined under different pH values, acid-base environment and UV radiation time with Colletotrichum gloeosporioides in postharvest mango as the indicator strain. The crude extract displayed broad spectrum activity against all tested fungi with the inhibition rate on 5 kinds of fungi more than 83 %. Inhibitory effects of the crude extract on the growth of 6 kinds of bacteria were also observed and strongest inhibition to Xanthomonas oryzae pv.oryzae in rice was showed (inhibitory zone diameter 22.37 mm). By the detection of stability of the crude extract to acid, alkali, heat and UV, the results demonstrated that the inhibition rate of the crude extract against Colletotrichum gloeosporioides was stable at pH 2-8 and 77.13 % of inhibition rate was still kept after treated at 140 ºC for 20 min and the crude extract had a stable activity to UV, and the inhibition rate was almost unchanged after 6 h.

The preliminary optimization of culture components for improving antifungal production by a wild-type soil bacteria Bacillus subtilis

Antifungal compounds are produced by Bacillus species under various growth conditions. While optimizing the antifungal production by using the one-factor-at-a-time (OFAT) and response surface methodology (RSM) approaches mannose and malt extract were identified as the most suitable carbon and nitrogen sources respectively. The RSM was applied to determine the optimum conditions of the three parameters such as pH, carbon and nitrogen sources for improved production. Optimum concentrations of carbon and nitrogen sources were 0.3% and 0.6% respectively with optimum media pH of 7.0 which showed optimum value of 40 AU/ml of antifungal compound against the Candida albicans SC5314 used as an indicator strain. In the present study, the F-value was determined as 0.0034 to imply that the model is significant. The goodness of the fit of the model was tested using coefficient of determination, R2 value, that tantamounts to 0.8562. The identification of antifungal compounds with their molecular masses was accomplished by MALDI-TOF mass spectrometry after n- butanol extraction. The present study thus has provided a platform to upgrade the yield of antifungal compounds which have got immense clinical potential to tread Candidosis.

Enterococcus mundtii Isolated from Slovak Raw Goat Milk and Its Bacteriocinogenic Potential

Enterococci are lactic acid bacteria. Most of them can adapt well to the food system due to their salt and acid-tolerance. Moreover, many enterococcal species have been found to produce antimicrobial substances of proteinaceous character, i.e., bacteriocins/enterocins. In this study, Enterococcus mundtii EM ML2/2 with bacteriocinogenic potential was identified in Slovak raw goat milk. This strain demonstrated inhibition activity against up to 36% of Gram-positive indicator bacteria, and in concentrated form the bacteriocin substance (pH 6.3) showed the highest inhibition activity (1600 AU/mL) against the principal indicator strain E. avium EA5. Semi-purified substance (SPS) EM ML2/2 produced inhibition activity up to 3200 AU/mL. Concentrated bacteriocin substance and SPS maintained active (inhibition activity up to 100 AU/mL) for three months under −20 °C storage conditions. The strain showed susceptible antibiotic profile, and it did not form biofilm. No production of damaging enzymes was noted. It was nonhemolytic, as well as DNase, and gelatinase-negative. It grew well in skim milk, and it was salt and acid-tolerant. The bacteriocin potential of E. mundtii species isolated from Slovak raw goat milk has not previously been detected, so this is an original contribution which may stimulate addtitional research and application studies.

Antimicrobial Resistance Genes and Bacteria Detected in Hospital Sewage May Provide Valuable Information in Clinical Antimicrobial Resistance

Aim: Clinical antimicrobial resistance (AMR) is a significant threat to public health, which is often unclear due to representative data from human populations that are challenging to obtain. Study the associations between consumption of antibiotics and antimicrobial resistance bacteria or genes (ARB or ARGs) that will benefit from elucidating the AMR. Methods: The details of antibiotics usage were calculated based on the actual consumption in the target hospital, ARB was detected by culture method, and ARGs were evaluated by metagenomics. Results: Our study revealed that culture-based single-indicator-strain approaches only capture the AMR in 16.17% infectious samples. 1573 bacterial species and 885 types of ARGs were found in the hospital sewage. The consumption of antibiotics influences the resistance profiles that were significant in E.coli, but the strength varies among bacteria. In all ARGs group, ARGs of aminoglycosides was the most common, followed by sulfonamide, tetracycline, phenicol, macrolides, and quinolones, comprising to 82.6% of all ARGs. Five hundred nineteen pairs of ARGs and bacterial species showed a significant correlation (r > 0.8). The co-occurrence patterns of bacteria- ARGs mirrors the AMR of the clinic. Antibiotic usage will affect the abundance of ARG in sewage, with a hysteresis effect. Conclusion: The ARGs- bacteria co-occurrence patterns from wastewater could be a valuable bio-indicator to reflect the emergence of ARB in the future. Developing a predictive risk model of AMR on this basis will facilitate the rational use of antibiotics.

New indicator Escherichia coli strain for rapid and accurate detection of supF mutations

Background The supF gene of Escherichia coli is useful for forward mutation analysis in bacterial and mammalian cells used in mutagenesis and DNA repair studies. Indicator E. coli strains, such as KS40/pOF105, have been used to analyze supF mutations. However, KS40/pOF105 is not enough to select supF mutants on nutrient-rich agar plates. Therefore, in this study, a new indicator E. coli strain for rapid and accurate detection of supF mutations was developed. Results The gyrA and rpsL genes with an amber mutation were integrated into the chromosomal DNA of E. coli KS40 to produce a new indicator strain, RF01. RF01 cells transformed by the wild-type supF gene were sensitive to nalidixic acid and streptomycin on LB agar plates. supF mutant frequencies and mutation spectra in RF01 were similar to those in KS40/pOF105. In addition, some mutations in supF were only detected in RF01. Conclusion RF01 is a new and useful indicator E. coli strain for analyzing supF mutations.

Identification and Characterization of the Bacteriocin Carocin S3 from the Multiple Bacteriocin Producing Strain of Pectobacterium carotovorum subsp. carotovorum

Background: Pectobacterium carotovorum subsp. carotovorum belongs to the Enterobacteriaceae family, which causes soft-rot disease in numerous plants worldwide resulting in significant economic losses. Results from our previous studies showed that the strain H-rif-8-6 produces low-molecular-weight bacteriocin (LMWB) Carocin S1. Interestingly, TH22-10, the caroS1K:Tn5 insertional mutant in H-rif-8-6, loses Carocin S1 producing ability, but still produces other LMWBs which the indicator strain SP33 can detect. The SP33 is one of the many strains that are sensitive toward the cytotoxic effects of Carocin S3K, but not Carocin S1. The result revealed that H-rif-8-6 is a multiple-bacteriocin producing strain.Results: In this study, a 4.1-kb DNA fragment was isolated from the chromosomal DNA of Pcc strain, H-rif-8-6, by a DNA probe using the caroS1K gene as the template. DNA sequencing and analysis by GenBank revealed two complete open reading frames (ORFs), designated ORF1 and ORF2, which were identified within the sequence fragment. ORF1 and ORF2, similar to the identified carocin S2 genes, encode the killer (Carocin S3K) and the immunity (Carocin S3I) proteins, respectively, which were homologous to the colicin E3 gene. Carocin S3K and Carocin S3I were expressed, isolated, and purified in Escherichia coli BL21 after subcloning of the expression plasmid pGS3KI or pGSK3I. SDS-PAGE analysis showed that the relative masses of Carocin S3K and Carocin S3I were 95.6 kDa and 10.2 kDa, respectively. The results reveal that Carocin S3K has higher antimicrobial and specific antimicrobial activities for Pcc along with a nuclease activity than Carocin S3I. However, Carocin S3I inhibits the activity of Carocin S3K. Interestingly, a high concentration of Carocin S3I protein is also a DNA nuclease, and Carocin S3K also inhibits its activity.Conclusion: This study showed that another type of bacteriocin was found in Pectobacterium carotovorum. This new type of bacteriocin, Carocin S3, has the killer protein, Carocin S3K, and the immunity protein, Carocin S3I.

Identification and Characterization of the Bacteriocin Carocin S3 from the Multiple Bacteriocin Producing Strain of Pectobacterium carotovorum subsp. carotovorum

Background: Pectobacterium carotovorum subsp. carotovorum belongs to the Enterobacteriaceae family, which causes soft-rot disease in numerous plants worldwide resulting in significant economic losses. Results from our previous studies showed that the strain H-rif-8-6 produces low-molecular-weight bacteriocin (LMWB) Carocin S1. Interestingly, TH22-10, the caroS1K:Tn5 insertional mutant in H-rif-8-6, loses Carocin S1 producing ability, but still produces other LMWBs which the indicator strain SP33 can detect. The SP33 is one of the many strains that are sensitive toward the cytotoxic effects of Carocin S3K, but not Carocin S1. The result revealed that H-rif-8-6 is a multiple-bacteriocin producing strain.Results: In this study, a 4.1-kb DNA fragment was isolated from the chromosomal DNA of Pcc strain, H-rif-8-6, by a DNA probe using the caroS1K gene as the template. DNA sequencing and analysis by GenBank revealed two complete open reading frames (ORFs), designated ORF1 and ORF2, which were identified within the sequence fragment. ORF1 and ORF2, similar to the identified carocin S2 genes, encode the killer (Carocin S3K) and the immunity (Carocin S3I) proteins, respectively, which were homologous to the colicin E3 gene. Carocin S3K and Carocin S3I were expressed, isolated, and purified in Escherichia coli BL21 after subcloning of the expression plasmid pGS3KI or pGSK3I. SDS-PAGE analysis showed that the relative masses of Carocin S3K and Carocin S3I were 95.6 kDa and 10.2 kDa, respectively. The results reveal that Carocin S3K has higher antimicrobial and specific antimicrobial activities for Pcc along with a nuclease activity than Carocin S3I. However, Carocin S3I inhibits the activity of Carocin S3K. Interestingly, a high concentration of Carocin S3I protein is also a DNA nuclease, and Carocin S3K also inhibits its activity.Conclusion: This study showed that another type of bacteriocin was found in Pectobacterium carotovorum. This new type of bacteriocin, Carocin S3, has the killer protein, Carocin S3K, and the immunity protein, Carocin S3I.

Rapid Carbapenem Inactivation Method (rCIM): A New Phenotypic Method for Rapidly Detecting Carbapenemase-Producing Enterobacterales

Background: Rapid and accurate methods for detecting carbapenemase-producing Enterobacterales (CPE) are essential for improving patient prognosis and preventing the spread of these microbes. In this study, 103 carbapenem-resistant Enterobacterales (CRE) isolates were collected from clinical specimens; of these, 55 CRE isolates were included in the retrospective analysis, and 48 CRE isolates were included in the prospective evaluation. Using sequencing results as the gold standard, we evaluated the performance of the rapid carbapenem inactivation method (rCIM) for detecting carbapenemases in comparison with the modified carbapenem inactivation method (mCIM) and CNPt-Direct test. In rCIM, the test isolate was incubated with meropenem (MEM) disks, and the supernatant obtained via centrifugation was incubated with the indicator strain Escherichia coli ATCC 25922. Growth of the indicator strain was monitored using a nephelometer. Results: The cut-off value of rCIM was 0.50 McFarland units. In retrospective analysis, the percent positive agreement and percent negative agreement of rCIM for detecting carbapenemase were 97.1% and 100%, respectively, and these values were higher than those for mCIM and the CNPt-Direct test. In the prospective evaluation, rCIM was linked to a sensitivity and specificity of 96.4% and 95%, respectively. Conclusion: rCIM may be a rapid (<3 h), economical, simple, and reliable method for screening CPE isolates, and it is expected to be routinely implemented in clinical microbiology laboratories.