Orientin Enhances Colistin-Mediated Bacterial Lethality through Oxidative Stress Involvement.

Bacterial resistance to colistin has prompted the search for alternative strategies to enhance antibacterial potential. Combination therapy remains one of the viable strategies in antibacterial therapy and has been proven to be effective in reducing the risk of resistance. In this study, the potential of orientin for enhancing the antibacterial activity of colistin was assessed against Klebsiella pneumoniae and Pseudomonas aeruginosa in vitro. The involvement of oxidative stress in such enhancement was also assessed. The minimum inhibitory concentrations (MICs) of colistin and orientin were 16 µg/mL and 64 µg/mL against K. pneumoniae and 64 µg/mL and 256 µg/mL against P. aeruginosa respectively. For the combination therapy, orientin potentiates the antibacterial effect of colistin with a friction inhibitory concentration index (FICI) of 0.37 and 0.31 against K. pneumoniae and P. aeruginosa, respectively. This observation suggests a synergistic interaction, with the MIC of colistin being reduced by 3- and 4-fold in the presence of orientin against K. pneumoniae and P. aeruginosa, respectively. Additionally, treatment with the combination of colistin and orientin induced oxidative stress against both organisms through increased cellular levels of superoxide anion radicals with concomitant increase in NAD+/NADH and ADP/ATP ratios. These findings suggest that orientin enhanced colistin in the killing of the test bacteria and the cotreatment of colistin and orientin induced oxidative stress, through reactive oxygen species generation, which consequently facilitated bacterial lethality without causing drug-drug interactions. Although, the data presented in this study has supported the capability of orientin for strengthening antibacterial activity of colistin toward the fight against drug-resistant Gram-negative bacteria, studies focusing on the exact target and mechanism of action of orientin are underway.

Draft Genome Sequences of Three Identified Rhizobacteria Associated with Maize Rhizosphere.

In this study, we sequenced the entire genomes of three identified rhizobacterial strains associated with maize plantation. Genome annotation of the sequenced data revealed several putative growth-promoting proteins associated with the production of indoleacetic acids and siderophore, the assimilation of nitrogen, and phosphorus solubilization.

Metagenome-Assembled Genomes Isolated from a Human Fecal Diarrhea Sample.

Diarrheal infection is the second leading infectious disease that is killing children under the age of 5 years. This study investigates the microbial community within a fecal sample from a diarrhea-affected child through shotgun metagenomic sequencing.

Metagenomes and Assembled Genomes from Diarrhea-Affected Cattle (Bos taurus).

The de novo metagenome assembly for C1-TPA is 68,577,389 bp long spread over 10,108 contigs, while that of C3-TPA is 55,517,929 bp distributed over 9,415 contigs. A total of 8 metagenome-assembled genomes (MAGs) were extracted from C1-TPA, and 10 were extracted from C3-TPA. Both samples have a Flavobacterium sp. and a Pseudomonas sp. in common among their bacterial communities.

Distribution of Important Probiotic Genes and Identification of the Biogenic Amines Produced by Lactobacillus acidophilus PNW3.

The genome of Lactobacillus acidophilus PNW3 was assessed for probiotic and safety potentials. The genome was completely sequenced, assembled using SPAdes, and thereafter annotated with NCBI prokaryotic genome annotation pipeline (PGAP) and rapid annotation using subsystem technology (RAST). Further downstream assessment was determined using appropriate bioinformatics tools. The production of biogenic amines was confirmed through HPLC analysis and the evolutionary trend of the strain was determined through the Codon Tree pipeline. The strain was predicted as a non-human pathogen. A plethora of encoding genes for lactic acids and bioactive peptides production, adhesion molecules, resistance to the harsh gut environmental conditions, and improvement of the host metabolism, which are putative for important probiotic functionalities, were located at different loci within the genome. A bacteriocin predicted to be helveticin J was identified as one of the secondary metabolites. The maximum zone of inhibition exhibited by the crude bacteriocin against STEC E. coli O177 was 21.7 ± 0.58 mm and 24.3 ± 1.15 mm after partial purification (250 µg/mL). Three coding sequences were identified for insertion sequences and one for the CRISPR-Cas fragment. The protein-encoding sequence for Ornithine decarboxylase was found within the genome. L. acidophilus PNW3 presents important features categorizing it as a viable and safe probiotic candidate, though further safety investigations are necessary. The application of probiotics in livestock-keeping would ensure improved public health and food security.

Feces Metagenomes and Metagenome-Assembled Genome Sequences from Two Separate Dogs (Canis lupus familiaris) with Multiple Diarrheal Episodes.

This study reports two feces metagenomes (D84 and D85) and six metagenome-assembled genomes (MAGs). The assembled MAGs include Pseudomonas sp. strain NID84 and Acinetobacter sp. strain N2D84 from D84 and Enterococcus sp. strain N4D85, Enterococcus sp. strain N5D85, Lactobacillus sp. strain N6D85, and Leuconostoc sp. strain N7D85 from D85. Acinetobacter sp. N2D84 was identified as a human pathogen with a probability of 92%.

Whole-Genome Sequencing of Multidrug-Resistant Campylobacter fetus subsp. fetus NWU_ED24, Isolated from a Bovine Sheath Wash Sample.

Campylobacter fetus subsp. fetus is an opportunistic human pathogen that is frequently identified as a cause of intestinal infections as well as bloodstream infections. This bacterium is well known to cause spontaneous abortions in sheep and cows. The strain reported in this study was isolated from a preputial wash sample from a bull in South Africa.

Whole-genome sequence of multi-drug resistant Pseudomonas aeruginosa strains UY1PSABAL and UY1PSABAL2 isolated from human broncho-alveolar lavage, Yaoundé, Cameroon.

Pseudomonas aeruginosa has been implicated in a wide range of post-operation wound and lung infections. A wide range of acquired resistance and virulence markers indicate surviving strategy of P. aeruginosa. Complete-genome analysis has been identified as efficient approach towards understanding the pathogenicity of this organism. This study was designed to sequence the entire genome of P. aeruginosa UY1PSABAL and UY1PSABAL2; determine drug-resistance profiles and virulence factors of the isolates; assess factors that contribute toward stability of the genomes; and thereafter determine evolutionary relationships between the strains and other isolates from similar sources. The genomes of the MDR P. aeruginosa UY1PSABAL and UY1PSABAL2 were sequenced on the Illumina Miseq platform. The raw sequenced reads were assessed for quality using FastQC v.0.11.5 and filtered for low quality reads and adapter regions using Trimmomatic v.0.36. The de novo genome assembly was made with SPAdes v.3.13 and annotated using Prokka v.2.1.1 annotation pipeline; Rapid Annotation using Subsytems Technology (RAST) server v.2.0; and PATRIC annotation tool v.3.6.2. Antimicrobial resistance genes and virulence determinants were searched through the functional annotation data generated from Prokka, RAST and PATRIC annotation pipelines; In addition to ResFinder and Comprehensive Antibiotic Resistance Database (CARD) which were employed to determine resistance genes. The PHAge Search Tool Enhanced Release (PHASTER) web server was used for the rapid identification and annotation of prophage sequences within bacterial genome. Predictive secondary metabolites were identified with AntiSMASH v.5.0. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and cas genes regions were also investigated with the CRISPRone and CRISPRFinder server. The genome sizes of 7.0 and 6.4 Mb were determined for UY1PSABAL and UY1PSABAL2 strains with G+C contents of 66.1% and 66.48% respectively. ß-lactamines resistance genes blaPAO, aminoglycoside phosphorylating enzymes genes aph(3')-IIb, fosfomycine resistance gene fosA, vancomycin vanW and tetracycline tetA were among identified resistance genes harboured in both isolates. UY1PSABAL bore additional aph(6)-Id, aph(3'')-Ib, ciprofloxacin-modifying enzyme crpP and ribosomal methylation enzyme rmtB. Both isolates were found harbouring virulence markers such as flagella and type IV pili; and also present various type III secretion systems such as exoA, exoS, exoU, exoT. Secondary metabolites such as pyochelin and pyoverdine with iron uptake activity were found within the genomes as well as quorum-sensing systems, and various fragments for prophages and insertion sequences. Only the UY1PSABAL2 contains CRISPR-Cas system. The phylogeny revealed a very close evolutionary relationship between UY1PSABAL and the similar strain isolated from Malaysia; the same trend was observed between UY1PSABAL2 and the strain from Chinese origin. Complete analyses of the entire genomes provide a wide range of information towards understanding pathogenicity of the pathogens in question.

Integrated genome-based probiotic relevance and safety evaluation of Lactobacillus reuteri PNW1.

This study evaluates whole-genome sequence of Lactobacillus reuteri PNW1 and identifies its safety genes that may qualify it as a putative probiotic. It further extracted the bacteriocin produced by the strain and tested its effectiveness against pathogenic STEC E. coli O177. The genomic DNA was sequenced on illuminal Miseq instrument and the sequenced data was assessed for quality reads before assembled with SPAdes. The draft assembly was annotated with Prokaryotic Genome Annotation Pipeline (PGAP) and Rapid Annotations using Subsystems Technology (RAST). Further downstream analyses were carried out using appropriate bioinformatic tools. Production of biogenic amines was biochemically confirmed through HPLC analysis. The assembled genome was 2,430,215 bp long in 420 contigs with 39% G+C content. Among all known genes, putatively responsible for the production of toxic biochemicals, only arginine deiminase (EC3.5.3.6) was spotted. Coding sequences (CDS) putative for D-lactate dehydrogenase (EC1.1.1.28), L-lactate dehydrogenase (EC1.1.1.27) and bacteriocin helveticin J were found within the genome together with plethora of other probiotic important genes. The strain harbours only resistant genes putative for Lincosamide (lnuC) and Tetracycline resistant genes (tetW). There was no hit found for virulence factors and probability of the strain being a human pathogen was zero. Two intact prophage regions were detected within the genome of L. reuteri PNW1 and nine CDS were identified for insertion sequence by OASIS which are belong to seven different families. Five putative CDS were identified for the CRISPR, each associated with Cas genes. Maximum zone of inhibition exhibited by the bacteriocin produced L. reuteri PNW1 is 20.0±1.00 mm (crude) and 23.3±1.15 mm (at 0.25 mg/ml) after being partially purified. With the strain predicted as non-human pathogen, coupled with many other identified desired features, L. reuteri PNW1 stands a chance of making good and safe candidates for probiotic, though further in-vivo investigations are still necessary.

Time-kill kinetics and biocidal effect of Euclea crispa leaf extracts against microbial membrane.

OBJECTIVE: To evaluate antimicrobial potential of the fractions partitioned from Euclea crispa leaf extract and determination of their impact on cell membrane disruption. METHODS: Antimicrobial potentials were evaluated via susceptibility test, determination of minimum inhibitory concentrations (MICs) and time-kill kinetics of the potent fractions. Degree of membrane disruption was determined by the amount of proteins and nucleotides released from within the cells and SEM images of the membrane after 120 min of treatment. RESULTS: The largest inhibition zone (25.5 ± 0.50 mm) was obtained by ethylacetate fraction against Aeromonas hydrophilla at 10 mg/mL. The lowest MIC (0.16 mg/mL) was exhibited by n-butanol and ethylacetate fractions against test bacteria while all fractions exhibited MIC values between 0.31 and 1.25 mg/mL against susceptible yeast. n-Butanol fraction achieved absolute mortality against Bacillus pumulis (B. pumulis) and Klebsiella pneumoniae (K. pneumoniae) after 90 and 120 min contact time respectively at 1 × MIC. Total mortality also achieved by n-hexane fraction against B. pumulis and K. pneumoniae after 90 and 120 min respectively at 2 × MIC. Ethylacetate fraction achieved absolute mortality against both bacteria after 120 min at 2 × MIC. n-Hexane fraction achieved total mortality against Candida albicans after 120 min at 1 × MIC. Maximum amount of proteins (0.566 µg/mL) was released from K. pneumoniae by n-butanol fraction at 2 × MIC after 120 min of treatment while the maximum amount of nucleotides released (4.575 µg) was from B. pumulis by n-hexane fraction under similar condition. CONCLUSION: This study suggests the leaf of Euclea crispa a source of bioactive compound with membrane attack as one of the mechanisms of its biocidal action.