Antibacterial mechanism of Pseudomonas aeruginosa UKMP14T rhamnolipids against multidrug resistant Acinetobacter baumannii.

Rhamnolipids, a major category of glycolipid biosurfactant, have recently gained enormous attention in medical field because of their relevance as effective antibacterial agents against a wide variety of pathogenic bacteria. Our previous studies have shown that rhamnolipids from an environmental isolate of Pseudomonas aeruginosa UKMP14T possess antibacterial, anti-adhesive and anti-biofilm activity against multidrug-resistant ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter sp.) pathogens. However, the mechanism of their antibacterial action remains unclear. Thus, this study aimed to elucidate the mechanism of the antibacterial action of P. aeruginosa UKMP14T rhamnolipids by studying the changes in cells of one of the ESKAPE pathogens, Acinetobacter baumannii, which is the most difficult strain to kill. Results revealed that rhamnolipid treatment rendered A. baumannii cells more hydrophobic as evaluated through contact angle measurements. It also induced the release of cellular proteins measuring 510 µg/mL at a rhamnolipid concentration of 1000 µg/mL. In addition, rhamnolipids were found to be bactericidal in their action as they could permeate the inner membranes, leading to a leak-out of nucleotides. More than 50 % of the cells were found to be killed upon 1000 µg/mL rhamnolipid treatment as observed through fluorescence microscopy. Other cellular changes such as irregular shape and size, membrane perturbations, clumping, shrinkage and physical damage were clearly visible in SEM, FESEM and laser micrographs. Furthermore, rhamnolipid treatment inhibited the levels of acyl-homoserine lactones (AHLs) in A. baumannii, which are vital for their biofilm formation and virulence. The obtained results indicate that P. aeruginosa UKMP14T rhamnolipids target outer and inner bacterial membranes through permeation, including physical damage to the cells, leading to cell leakage. Furthermore, AHL inhibition appears to be the mechanism behind their anti-biofilm action. All these observations can be correlated to rhamnolipids' antibacterial effect against A. baumannii.

Improved complete genome sequence of Bdellovibrio bacteriovorus 109J, a widely studied laboratory strain of predatory bacteria.

The complete genome sequence of Bdellovibrio bacteriovorus 109J, a well-studied laboratory strain of predatory bacteria, first determined in 2014. Here we report an improved complete genome sequence of B. bacteriovorus 109J, incorporating 16 assembly and 87 nucleotide corrections. This revised genome will be helpful to studies on the predatory bacteria.

Whole genome sequencing of methicillin-resistant Staphylococcus aureus clinical isolates from Terengganu, Malaysia, indicates the predominance of the EMRSA-15 (ST22-SCCmec IV) clone.

Despite the importance of methicillin-resistant Staphylococcus aureus (MRSA) as a priority nosocomial pathogen, the genome sequences of Malaysian MRSA isolates are currently limited to a small pool of samples. Here, we present the genome sequence analyses of 88 clinical MRSA isolates obtained from the main tertiary hospital in Terengganu, Malaysia in 2016-2020, to obtain in-depth insights into their characteristics. The EMRSA-15 (ST22-SCCmec IV) clone of the clonal complex 22 (CC22) lineage was predominant with a total of 61 (69.3%) isolates. Earlier reports from other Malaysian hospitals indicated the predominance of the ST239 clone, but only two (2.3%) isolates were identified in this study. Two Indian-origin clones, the Bengal Bay clone ST772-SCCmec V (n = 2) and ST672 (n = 10) were also detected, with most of the ST672 isolates obtained in 2020 (n = 7). Two new STs were found, with one isolate each, and were designated ST7879 and ST7883. From the core genome phylogenetic tree, the HSNZ MRSA isolates could be grouped into seven clades. Antimicrobial phenotype-genotype concordance was high (> 95%), indicating the accuracy of WGS in predicting most resistances. Majority of the MRSA isolates were found to harbor more than 10 virulence genes, demonstrating their pathogenic nature.

Complete genome sequence of Paenibacillus sp. VCA1 isolated from crater lake of the El Chichón Volcano.

We report the complete genome of Paenibacillus sp. strain VCA1, which was isolated from sediment from El Chichón Volcano. This genome consists of 6,690,819 bp and 6,312 coding sequences, with 51.8% G+C content. Whole-genome sequencing was performed to explore the strain's biocontrol and plant growth promotion properties.

Insights into bacterial community metatranscriptome and metabolome in river water influenced by palm oil mill effluent final discharge.

AIMS: This study aimed to investigate the effect of palm oil mill effluent (POME) final discharge on the active bacterial composition, gene expression, and metabolite profiles in the receiving rivers to establish a foundation for identifying potential biomarkers for monitoring POME pollution in rivers. METHODS AND RESULTS: The POME final discharge, upstream (unpolluted by POME), and downstream (effluent receiving point) parts of the rivers from two sites were physicochemically characterized. The taxonomic and gene profiles were then evaluated using de novo metatranscriptomics, while the metabolites were detected using qualitative metabolomics. A similar bacterial community structure in the POME final discharge samples from both sites was recorded, but their composition varied. Redundancy analysis showed that several families, particularly Comamonadaceae and Burkholderiaceae [Pr(>F) = 0.028], were positively correlated with biochemical oxygen demand (BOD5) and chemical oxygen demand (COD). The results also showed significant enrichment of genes regulating various metabolisms in the POME-receiving rivers, with methane, carbon fixation pathway, and amino acids among the predominant metabolisms identified (FDR < 0.05, PostFC > 4, and PPDE > 0.95). This was further validated through qualitative metabolomics, whereby amino acids were detected as the predominant metabolites. CONCLUSIONS: The results suggest that genes regulating amino acid metabolism have significant potential for developing effective biomonitoring and bioremediation strategies in river water influenced by POME final discharge, fostering a sustainable palm oil industry.

Exoprotease exploitation and social cheating in a Pseudomonas aeruginosa environmental lysogenic strain with a noncanonical quorum sensing system.

Social cheating is the exploitation of public goods that are costly metabolites, like exoproteases. Exoprotease exploitation in Pseudomonas aeruginosa has been studied in reference strains. Experimental evolution with reference strains during continuous growth in casein has demonstrated that nonexoprotease producers that are lasR mutants are selected while they behave as social cheaters. However, noncanonical quorum-sensing systems exist in P. aeruginosa strains, which are diverse. In this work, the exploitation of exoproteases in the environmental strain ID4365 was evaluated; ID4365 has a nonsense mutation that precludes expression of LasR. ID4365 produces exoproteases under the control of RhlR, and harbors an inducible prophage. As expected, rhlR mutants of ID4365 behave as social cheaters, and exoprotease-deficient individuals accumulate upon continuous growth in casein. Moreover, in all continuous cultures, population collapses occur. However, this also sometimes happens before cheaters dominate. Interestingly, during growth in casein, ID4565's native prophage is induced, suggesting that the metabolic costs imposed by social cheating may increase its induction, promoting population collapses. Accordingly, lysogenization of the PAO1 lasR mutant with this prophage accelerated its collapse. These findings highlight the influence of temperate phages in social cheating.

Simultaneous detection of various pathogenic Escherichia coli in water by sequencing multiplex PCR amplicons.

Waterborne diseases due to pathogen contamination in water are a serious problem all over the world. Accurate and simultaneous detection of pathogens in water is important to protect public health. In this study, we developed a method to simultaneously detect various pathogenic Escherichia coli by sequencing the amplicons of multiplex PCR. Our newly designed multiplex PCR amplified five genes for pathogenic E. coli (uidA, stx1, stx2, STh gene, and LT gene). Additional two PCR assays (for aggR and eae) were also designed and included in the amplicon sequencing analysis. The same assays were also used for digital PCR (dPCR). Strong positive correlations were observed between the sequence read count and the dPCR results for most of the genes targeted, suggesting that our multiplex PCR-amplicon sequencing approach could provide quantitative information. The method was also successfully applied to monitor the level of pathogenic E. coli in river water and wastewater samples. The approach shown here could be expanded by targeting genes for other pathogens.

Resistance against two lytic phage variants attenuates virulence and antibiotic resistance in Pseudomonas aeruginosa.

Background: Bacteriophage therapy is becoming part of mainstream Western medicine since antibiotics of clinical use tend to fail. It involves applying lytic bacteriophages that self-replicate and induce cell lysis, thus killing their hosts. Nevertheless, bacterial killing promotes the selection of resistant clones which sometimes may exhibit a decrease in bacterial virulence or antibiotic resistance. Methods: In this work, we studied the Pseudomonas aeruginosa lytic phage φDCL-PA6 and its variant φDCL-PA6α. Additionally, we characterized and evaluated the production of virulence factors and the virulence in a Galleria mellonella model of resistant mutants against each phage for PA14 and two clinical strains. Results: Phage φDCL-PA6α differs from the original by only two amino acids: one in the baseplate wedge subunit and another in the tail fiber protein. According to genomic data and cross-resistance experiments, these changes may promote the change of the phage receptor from the O-antigen to the core lipopolysaccharide. Interestingly, the host range of the two phages differs as determined against the Pseudomonas aeruginosa reference strains PA14 and PAO1 and against nine multidrug-resistant isolates from ventilator associated pneumonia. Conclusions: We show as well that phage resistance impacts virulence factor production. Specifically, phage resistance led to decreased biofilm formation, swarming, and type III secretion; therefore, the virulence towards Galleria mellonella was dramatically attenuated. Furthermore, antibiotic resistance decreased for one clinical strain. Our study highlights important potential advantages of phage therapy's evolutionary impact that may be exploited to generate robust therapy schemes.

CRISPR-Cas Controls Cryptic Prophages.

The bacterial archetypal adaptive immune system, CRISPR-Cas, is thought to be repressed in the best-studied bacterium, Escherichia coli K-12. We show here that the E. coli CRISPR-Cas system is active and serves to inhibit its nine defective (i.e., cryptic) prophages. Specifically, compared to the wild-type strain, reducing the amounts of specific interfering RNAs (crRNA) decreases growth by 40%, increases cell death by 700%, and prevents persister cell resuscitation. Similar results were obtained by inactivating CRISPR-Cas by deleting the entire 13 spacer region (CRISPR array); hence, CRISPR-Cas serves to inhibit the remaining deleterious effects of these cryptic prophages, most likely through CRISPR array-derived crRNA binding to cryptic prophage mRNA rather than through cleavage of cryptic prophage DNA, i.e., self-targeting. Consistently, four of the 13 E. coli spacers contain complementary regions to the mRNA sequences of seven cryptic prophages, and inactivation of CRISPR-Cas increases the level of mRNA for lysis protein YdfD of cryptic prophage Qin and lysis protein RzoD of cryptic prophage DLP-12. In addition, lysis is clearly seen via transmission electron microscopy when the whole CRISPR-Cas array is deleted, and eliminating spacer #12, which encodes crRNA with complementary regions for DLP-12 (including rzoD), Rac, Qin (including ydfD), and CP4-57 cryptic prophages, also results in growth inhibition and cell lysis. Therefore, we report the novel results that (i) CRISPR-Cas is active in E. coli and (ii) CRISPR-Cas is used to tame cryptic prophages, likely through RNAi, i.e., unlike with active lysogens, active CRISPR-Cas and cryptic prophages may stably co-exist.

Impaired glucose metabolism by deleting the operon of hydrogenase 2 in Escherichia coli.

Although Escherichia coli has four hydrogenases, their definite roles in fermentation are still not clear. In this study, all the operon deletion mutants of E.coli hydrogenases (∆hya, ∆hyb, ∆hyc, or ∆hyf) were constructed to evaluate the hydrogen metabolism in comparison to their respective single-gene deletion mutants of large subunits (∆hyaB, ∆hybC, ∆hycE, and ∆hyfG). Besides the hyc operon mutant that expectedly showed no hydrogen synthesis, the hyb operon mutant showed low hydrogen production and demonstrated significantly reduced growth under anaerobic conditions. The present work also provided first-hand data where deleterious effects of operon deletion were compared with single-gene deletion mutations and the results showed that the former type of deletion was found to cause more prominent phenotypic effects than the latter one. Interestingly, hyb operon mutant was remarkably distinct from other operon mutants, specifically in its inability to utilize glucose under both aerobic and anaerobic conditions. Further studies on this mutant revealed a significant reduction of the total intracellular ATP and NADH concentrations, which could explain its impaired glucose metabolism. In this way, Hyd-2 was verified as crucial not only in glucose metabolism but also in energy balance and redox homeostasis of the cells. Furthermore, a decreased expression of glucose metabolism-associated genes, particularly ppc and pykA, indicated their regulation by hyb operon, and thereby, glucose consumption. Moreover, the transcriptional changes in this mutant indicated the wide genomic connectivity of hyb operon to other metabolisms.

Cultivation of gastrointestinal microbiota in a new growth system revealed dysbiosis and metabolic disruptions in carcinoma-bearing rats.

A challenge in the study of gastrointestinal microbiota (GITm) is the validation of the genomic data with metabolic studies of the microbial communities to understand how the microbial networks work during health and sickness. To gain insights into the metabolism of the GITm, feces from healthy and sick rats with cancer were inoculated in a defined synthetic medium directed for anaerobic prokaryote growth (INC-07 medium). Significant differences between cultures of healthy and sick individuals were found: 1) the consumption of the carbon source and the enzyme activity involved in their catabolism (e.g., sucrase, lactase, lipases, aminotransferases, and dehydrogenases); 2) higher excretion of acetic, propionic, isobutyric, butyric, valeric, and isovaleric acids; 3) methane production; 4) ability to form biofilms; and 5) up to 500 amplicon sequencing variants (ASVs) identified showed different diversity and abundance. Moreover, the bowel inflammation induced by cancer triggered oxidative stress, which correlated with deficient antioxidant machinery (e.g., NADPH-producing enzymes) determined in the GITm cultures from sick individuals in comparison with those from control individuals. Altogether, the data suggested that to preserve the microbial network between bacteria and methanogenic archaea, a complete oxidation of the carbon source may be essential for healthy microbiota. The correlation of 16S rRNA gene metabarcoding between cultures and feces, as well as metabolomic data found in cultures, suggest that INC-07 medium may be a useful tool to understand the metabolism of microbiota under gut conditions.

Microbial Community Structures and Methanogenic Functions in Wetland Peat Soils.

Methane metabolism in wetlands involves diverse groups of bacteria and archaea, which are responsible for the biological decomposition of organic matter under certain anoxic conditions. Recent advances in environmental omics revealed the phylogenetic diversity of novel microbial lineages, which have not been previously placed in the traditional tree of life. The present study aimed to verify the key players in methane production, either well-known archaeal members or recently identified lineages, in peat soils collected from wetland areas in Japan. Based on an ana-lysis of microbial communities using 16S rRNA gene sequencing and the mole-cular cloning of the functional gene, mcrA, a marker gene for methanogenesis, methanogenic archaea belonging to Methanomicrobiales, Methanosarcinales, Methanobacteriales, and Methanomassiliicoccales were detected in anoxic peat soils, suggesting the potential of CH4 production in this natural wetland. "Candidatus Bathyarchaeia", archaea with vast metabolic capabilities that is widespread in anoxic environments, was abundant in subsurface peat soils (up to 96% of the archaeal community) based on microbial gene quantification by qPCR. These results emphasize the importance of discovering archaea members outside of traditional methanogenic lineages that may have significant functions in the wetland biogeochemical cycle.

A simple approach for random genomic insertion-deletions using ambiguous sequences in Escherichia coli.

Escherichia coli K-12, being one of the best understood and thoroughly analyzed organisms, is the preferred platform for genetic and biochemical research. Among all genetic engineering approaches applied on E. coli, the homologous recombination approach is versatile and precise, which allows engineering genes or large segments of the chromosome directly by using polymerase chain reaction (PCR) products or synthetic oligonucleotides. The previously explained approaches for random insertion and deletions were reported as technically not easy and laborious. This study, first, finds the minimum length of homology extension that is efficient and accurate for homologous recombination, as 30 nt. Second, proposes an approach utilizing PCR products flanking ambiguous NNN-sequence (30-nt) extensions, which facilitate the homologous recombination to recombine them at multiple regions on the genome and generate insertion-deletion mutations. Further analysis found that these mutations were varying in number, that is, multiple genomic regions were deleted. Moreover, evaluation of the phenotype of all the multiple random insertion-deletion mutants demonstrated no significant changes in the normal metabolism of bacteria. This study not only presents the efficiency of ambiguous sequences in making random deletion mutations, but also demonstrates their further applicability in genomics.

Quorum quenching of autoinducer 2 increases methane production in anaerobic digestion of waste activated sludge.

The ubiquitous signaling molecule autoinducer 2 (AI-2) is involved in intra- and interspecies communication, most notably between Gram-negative and Gram-positive bacteria. AI-2 accumulates during the exponential phase of the Escherichia coli (E. coli) monoculture and then rapidly decreases upon entry into the stationary phase. However, deleting both the genes encoding AI-2 synthase (LuxS) and the lsr operon regulator (LsrR) in the E. coli genome causes impaired AI-2 production and continuous AI-2 scavenging from the environment. This genetically-engineered E. coli mutant capable of quenching AI-2 quorum sensing (QS) system was utilized to evaluate the effect of AI-2 quenching on the anaerobic digestion of waste activated sludge (WAS) because the role of QS system via AI-2 in the process remains obscure. In this study, E. coli ∆luxS lsrR mutant cells were microencapsulated in sodium alginate beads and incubated with WAS anaerobically. After 15 days of anaerobic fermentation, the WAS containing double mutant cells produced significantly more methane than that of the parent E. coli cells. AI-2 quenching occurred concurrently with a shift of microbial communities that contribute to increasing acetate consumption by the Methanosarcina spp. resulting in an increase in methane production. KEY POINTS: • Impact of autoinducer 2 quenching in complex bacterial populations were determined. • Key microorganisms contributing to the increase of methane in WAS anaerobic digestion were found. • The AI-2 quenching is a potential regulatory in wastewater treatment and bioenergy research.

Advancement of Metatranscriptomics towards Productive Agriculture and Sustainable Environment: A Review.

While chemical fertilisers and pesticides indeed enhance agricultural productivity, their excessive usage has been detrimental to environmental health. In addressing this matter, the use of environmental microbiomes has been greatly favoured as a 'greener' alternative to these inorganic chemicals' application. Challenged by a significant proportion of unidentified microbiomes with unknown ecological functions, advanced high throughput metatranscriptomics is prudent to overcome the technological limitations in unfolding the previously undiscovered functional profiles of the beneficial microbiomes. Under this context, this review begins by summarising (1) the evolution of next-generation sequencing and metatranscriptomics in leveraging the microbiome transcriptome profiles through whole gene expression profiling. Next, the current environmental metatranscriptomics studies are reviewed, with the discussion centred on (2) the emerging application of the beneficial microbiomes in developing fertile soils and (3) the development of disease-suppressive soils as greener alternatives against biotic stress. As sustainable agriculture focuses not only on crop productivity but also long-term environmental sustainability, the second half of the review highlights the metatranscriptomics' contribution in (4) revolutionising the pollution monitoring systems via specific bioindicators. Overall, growing knowledge on the complex microbiome functional profiles is imperative to unlock the unlimited potential of agricultural microbiome-based practices, which we believe hold the key to productive agriculture and sustainable environment.

Impact of 5-fluorouracil on anaerobic digestion using sewage sludge.

The role of bacterial interaction is vital to control bacterial functions; however, it has not been fully understood in microbial consortia (including anaerobic digestion). In this study, fluorouracil (FU), which is an anticancer agent and a quorum sensing (QS) inhibitor to some of the Gram-negative bacteria was found to inhibit methane production from sewage sludge under anaerobic conditions, as shown in a result where methane production in the presence of FU was eight times lower than the control (sewage sludge without FU). Whereas FU did not influence the hydrolysis process, in the acidogenesis/acetogenesis process, butyrate, and acetate accumulated in samples with FU. Also, in the methanogenesis process, FU remarkably inhibited methane production by acetoclastic methanogens rather than by the hydrogenotrophic ones. This result agreed with the result that growth and methane production of Methanosarcina acetivorans C2A was inhibited in the presence of FU. However, the inhibitory effect of FU was high in the condition that both bacteria and archaea were active. It indicates that FU influences methanogens and bacteria in the process of methane fermentation. The analyses of microbial communities (bacteria and archaea) showed that the abundance ratio of the Firmicutes phyla is high, and hydrogenotrophic methanogens become dominant in the presence of FU. Conversely, the abundance of Spirochaetes significantly decreased under FU. The inhibition of methane production by FU was due to the growth inhibition of methanogenic archaea and the changes in the composition of the bacterial population.

Cu2O/TiO2 decorated on cellulose nanofiber/reduced graphene hydrogel for enhanced photocatalytic activity and its antibacterial applications.

Photocatalysis has gained attention as a viable wastewater remediation technique. However, the difficulty of recovering powder-based photocatalyst has often become a major limitation for their on-site practical application. Herein, we report on the successful in-situ preparation of a novel three-dimensional (3D) photocatalyst consisting of Cu2O/TiO2 loaded on a cellulose nanofiber (CNF)/reduced graphene hydrogel (rGH) via facile hydrothermal treatment and freeze-drying. The 3D macrostructure not only provides a template for the anchoring of Cu2O and TiO2 but also provides an efficient electron transport pathway for enhanced photocatalytic activity. The results showed that the Cu2O and TiO2 were uniformly loaded onto the aerogel framework resulting in the composites with large surface area with exposed actives sites. As compared to bare rGH, CNF/rGH, Cu2O/CNF/rGH and TiO2/CNF/rGH, the Cu2O/TiO2/CNF/rGH showed improved photocatalytic activity for methyl orange (MO) degradation. MO degradation pathway is proposed based on GC-MS analysis. The enhanced photoactivity can be attributed to the charge transfer and electron-hole separation from the synergistic effect of Cu2O/TiO2 anchored on CNF/rGH. In terms of their anti-bacterial activity towards Staphylococcus aureus and Escherichia coli, the synergistic effect of the Cu2O/TiO2 anchored on the CNF/rGH framework showed excellent activity towards the bacteria.

A Novel Archaeal Lineage in Boiling Hot Springs around Oyasukyo Gorge (Akita, Japan).

A novel deep-branching archaeal lineage was discovered at high-temperature hot springs around Oyasukyo Gorge in Akita Prefecture, Japan. Actively boiling hot spring water contained >1×104 microbes mL-1. The microbial community composition assessed by analyzing 16S rRNA gene amplicons revealed that the dominant bacterial phyla were Proteobacteria and Aquificae (>50% of the microbial composition) in samples collected in 2016 and 2019, respectively. Approximately 10% of the reads obtained in both years were not assigned to any taxonomy. The more detailed phylogenetic positions of the unassigned sequences identified using a clone library and phylogenetic tree showed that they formed a clade that was independent, distantly related to known phyla, and had low similarity (<82%) to all other sequences in available databases. The present results suggest that this novel archaeal phylum-level lineage thrives in boiling hot springs in Japan.

Engineering anaerobic digestion via optimizing microbial community: effects of bactericidal agents, quorum sensing inhibitors, and inorganic materials.

Anaerobic digestion of sewage sludge (SS) is one of the effective ways to reduce the waste generated from human life activities. To date, there are many reports to improve or repress methane production during the anaerobic digestion of SS. In the anaerobic digestion process, many microorganisms work positively or negatively, and as a result of their microbe-to-microbe interaction and regulation, methane production increases or decreases. In other words, understanding the complex control mechanism among the microorganisms and identifying the strains that are key to increase or decrease methane production are important for promoting the advanced production of bioenergy and beneficial compounds. In this mini-review, the literature on methane production in anaerobic digestion has been summarized based on the results of antibiotic addition, quorum sensing control, and inorganic substance addition. By optimizing the activity of microbial groups in SS, methane or acetate can be highly produced. KEY POINTS: • Bactericidal agents such as an antibiotic alter microbial community for enhanced CH4 production. • Bacterial interaction via quorum sensing is one of the key points for biofilm and methane production. • Anaerobic digestion can be altered in the presence of several inorganic materials.