Emerging Strategies to Combat ESKAPE Pathogens in the Era of Antimicrobial Resistance: A Review

The acronym ESKAPE includes six nosocomial pathogens that exhibit multidrug resistance and virulence: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. Persistent use of antibiotics has provoked the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) bacteria, which render even the most effective drugs ineffective. Extended spectrum β-lactamase (ESBL) and carbapenemase producing Gram negative bacteria have emerged as an important therapeutic challenge. Development of novel therapeutics to treat drug resistant infections, especially those caused by ESKAPE pathogens is the need of the hour. Alternative therapies such as use of antibiotics in combination or with adjuvants, bacteriophages, antimicrobial peptides, nanoparticles, and photodynamic light therapy are widely reported. Many reviews published till date describe these therapies with respect to the various agents used, their dosage details and mechanism of action against MDR pathogens but very few have focused specifically on ESKAPE. The objective of this review is to describe the alternative therapies reported to treat ESKAPE infections, their advantages and limitations, potential application in vivo, and status in clinical trials. The review further highlights the importance of a combinatorial approach, wherein two or more therapies are used in combination in order to overcome their individual limitations, additional studies on which are warranted, before translating them into clinical practice. These advances could possibly give an alternate solution or extend the lifetime of current antimicrobials

Antibiofilm and antipersister activity of acetic acid against extensively drug resistant Pseudomonas aeruginosa PAW1.

Pseudomonas aeruginosa is an ESKAPE pathogen associated with difficult-to-treat burn wound and surgical-site infections. This study aimed to characterise an extensively drug resistant (XDR) P. aeruginosa isolate (designated PAW1) and to investigate the antibiofilm and antipersister effect of acetic acid on PAW1. PAW1 was identified using biotypic (VITEK) and genotypic (16S rDNA) analysis. Minimum inhibitory concentration (MIC) and disc susceptibility testing showed high level resistance against all antibiotics from classes including beta lactams, cephems, carbapenems and fluoroquinolones. It was therefore identified as extensively drug resistant (XDR), showing resistance to all antibiotics except for, aminoglycoside (gentamicin and netilmicin) and lipopeptides (polymyxin B). Time kill assays showed antibiotic tolerant, persister cell formation in presence of 100X MICs of gentamicin and polymyxin B. Other virulence traits such as ability to produce lipase, protease, haemolysin, and siderophores and to form biofilms were additional factors which may contribute to its pathogenicity. PAW1 showed promising susceptibility against acetic acid with MIC and minimum biofilm inhibitory concentration of 0.156% (v/v). Percent viability of PAW1 was dependent on dose and treatment time of acetic acid. 0.625% acetic acid treatment of 5 minutes was effective in killing >90% planktonic cells showing lesser toxicity to L929 cells (IC50 = 0.625%). Biofilm disruption caused due to acetic acid was also dose dependent, showing 40.57% disruption after treatment with 0.625% acetic acid for 5 minutes. FESEM imaging and live dead staining of planktonic and biofilm forms of PAW1 confirmed that acetic acid treatment caused 19.04% of cell shrinkage and disruption of extracellular matrix resulting in killing of cells. Antipersister activity of acetic acid was demonstrated by showing complete killing of PAW1 at 4X MIC. Overall, this study characterised an XDR isolate P. aeruginosa showing resistance and tolerance to various antibiotics. Antipersister and antibiofilm effect of acetic acid demonstrates the importance of forgotten topical agents as an effective strategy to treat XDR pathogens

Understanding the role of miRNAs in governing the drought sensitive response of a rice mega variety, Swarna at reproductive stage

To combat drought stress, the major abiotic stress in rice, major genomic regions governing stress tolerance under field conditions have been identified and exploited, though their molecular basis remains elusive till date. We have recently reported the miRNAs/mRNA modules important for drought stress response in these genomic regions. To further understand this secondary regulation by miRNA/mRNA modules in a mega variety, Swarna, which is sensitive to drought stress at reproductive stage, we generated sRNA-seq data. Our analysis identified 9 putative novel miRNAs and 27 differentially expressed known miRNAs at booting stage under drought stress. The major miRNA/transcript modules, identified through degradome analysis and transcript abundance studies, that had an impact on drought stress response of plant and yield included osa-miR169a/LOC_Os07g41720, Osa-miR171b/f, Osa-miR172d-3p/5p, Osa-miR1876/ LOC_Os11g38330, Osa-miR397a and Osa-miR530–3p. The results indicated a basis for the low spikelet fertility and high grain chalkiness of the mega variety Swarna under drought stress through the modulation of expression of Osa-miR397a/ LOC_Os03g03510 and LOC_Os03g51220 and Osa-miR530–3p/ LOC_Os10g40510 modules. This study provides the potential target genes for improving Swarna, a globally important variety, so as to ensure food security under climate change scenario

Table_2_Understanding plant–microbe interaction of rice and soybean with two contrasting diazotrophic bacteria through comparative transcriptome analysis.xlsx

Understanding the beneficial plant–microbe interactions is becoming extremely critical for deploying microbes imparting plant fitness and achieving sustainability in agriculture. Diazotrophic bacteria have the unique ability to survive without external sources of nitrogen and simultaneously promote host plant growth, but the mechanisms of endophytic interaction in cereals and legumes have not been studied extensively. We have studied the early interaction of two diazotrophic bacteria, Gluconacetobacter diazotrophicus (GAB) and Bradyrhizobium japonicum (BRH), in 15-day-old seedlings of rice and soybean up to 120 h after inoculation (hai) under low-nitrogen medium. Root colonization of GAB in rice was higher than that of BRH, and BRH colonization was higher in soybean roots as observed from the scanning electron microscopy at 120 hai. Peroxidase enzyme was significantly higher at 24 hai but thereafter was reduced sharply in soybean and gradually in rice. The roots of rice and soybean inoculated with GAB and BRH harvested from five time points were pooled, and transcriptome analysis was executed along with control. Two pathways, “Plant pathogen interaction” and “MAPK signaling,” were specific to Rice-Gluconacetobacter (RG), whereas the pathways related to nitrogen metabolism and plant hormone signaling were specific to Rice-Bradyrhizobium (RB) in rice. Comparative transcriptome analysis of the root tissues revealed that several plant–diazotroph-specific differentially expressed genes (DEGs) and metabolic pathways of plant–diazotroph-specific transcripts, viz., chitinase, brassinosteroid, auxin, Myeloblastosis (MYB), nodulin, and nitrate transporter (NRT), were common in all plant–diazotroph combinations; three transcripts, viz., nitrate transport accessory protein (NAR), thaumatin, and thionin, were exclusive in rice and another three transcripts, viz., NAC (NAM: no apical meristem, ATAF: Arabidopsis thaliana activating factor, and CUC: cup-shaped cotyledon), ABA (abscisic acid), and ammonium transporter, were exclusive in soybean. Differential expression of these transcripts and reduction in pathogenesis-related (PR) protein expression show the early interaction. Based on the interaction, it can be inferred that the compatibility of rice and soybean is more with GAB and BRH, respectively. We propose that rice is unable to identify the diazotroph as a beneficial microorganism or a pathogen from an early response. So, it expressed the hypersensitivity-related transcripts along with PR proteins. The molecular mechanism of diazotrophic associations of GAB and BRH with rice vis-à-vis soybean will shed light on the basic understanding of host responses to beneficial microorganisms.</p

Table_5_Understanding plant–microbe interaction of rice and soybean with two contrasting diazotrophic bacteria through comparative transcriptome analysis.xlsx

Understanding the beneficial plant–microbe interactions is becoming extremely critical for deploying microbes imparting plant fitness and achieving sustainability in agriculture. Diazotrophic bacteria have the unique ability to survive without external sources of nitrogen and simultaneously promote host plant growth, but the mechanisms of endophytic interaction in cereals and legumes have not been studied extensively. We have studied the early interaction of two diazotrophic bacteria, Gluconacetobacter diazotrophicus (GAB) and Bradyrhizobium japonicum (BRH), in 15-day-old seedlings of rice and soybean up to 120 h after inoculation (hai) under low-nitrogen medium. Root colonization of GAB in rice was higher than that of BRH, and BRH colonization was higher in soybean roots as observed from the scanning electron microscopy at 120 hai. Peroxidase enzyme was significantly higher at 24 hai but thereafter was reduced sharply in soybean and gradually in rice. The roots of rice and soybean inoculated with GAB and BRH harvested from five time points were pooled, and transcriptome analysis was executed along with control. Two pathways, “Plant pathogen interaction” and “MAPK signaling,” were specific to Rice-Gluconacetobacter (RG), whereas the pathways related to nitrogen metabolism and plant hormone signaling were specific to Rice-Bradyrhizobium (RB) in rice. Comparative transcriptome analysis of the root tissues revealed that several plant–diazotroph-specific differentially expressed genes (DEGs) and metabolic pathways of plant–diazotroph-specific transcripts, viz., chitinase, brassinosteroid, auxin, Myeloblastosis (MYB), nodulin, and nitrate transporter (NRT), were common in all plant–diazotroph combinations; three transcripts, viz., nitrate transport accessory protein (NAR), thaumatin, and thionin, were exclusive in rice and another three transcripts, viz., NAC (NAM: no apical meristem, ATAF: Arabidopsis thaliana activating factor, and CUC: cup-shaped cotyledon), ABA (abscisic acid), and ammonium transporter, were exclusive in soybean. Differential expression of these transcripts and reduction in pathogenesis-related (PR) protein expression show the early interaction. Based on the interaction, it can be inferred that the compatibility of rice and soybean is more with GAB and BRH, respectively. We propose that rice is unable to identify the diazotroph as a beneficial microorganism or a pathogen from an early response. So, it expressed the hypersensitivity-related transcripts along with PR proteins. The molecular mechanism of diazotrophic associations of GAB and BRH with rice vis-à-vis soybean will shed light on the basic understanding of host responses to beneficial microorganisms.</p

Table_8_Understanding plant–microbe interaction of rice and soybean with two contrasting diazotrophic bacteria through comparative transcriptome analysis.docx

Understanding the beneficial plant–microbe interactions is becoming extremely critical for deploying microbes imparting plant fitness and achieving sustainability in agriculture. Diazotrophic bacteria have the unique ability to survive without external sources of nitrogen and simultaneously promote host plant growth, but the mechanisms of endophytic interaction in cereals and legumes have not been studied extensively. We have studied the early interaction of two diazotrophic bacteria, Gluconacetobacter diazotrophicus (GAB) and Bradyrhizobium japonicum (BRH), in 15-day-old seedlings of rice and soybean up to 120 h after inoculation (hai) under low-nitrogen medium. Root colonization of GAB in rice was higher than that of BRH, and BRH colonization was higher in soybean roots as observed from the scanning electron microscopy at 120 hai. Peroxidase enzyme was significantly higher at 24 hai but thereafter was reduced sharply in soybean and gradually in rice. The roots of rice and soybean inoculated with GAB and BRH harvested from five time points were pooled, and transcriptome analysis was executed along with control. Two pathways, “Plant pathogen interaction” and “MAPK signaling,” were specific to Rice-Gluconacetobacter (RG), whereas the pathways related to nitrogen metabolism and plant hormone signaling were specific to Rice-Bradyrhizobium (RB) in rice. Comparative transcriptome analysis of the root tissues revealed that several plant–diazotroph-specific differentially expressed genes (DEGs) and metabolic pathways of plant–diazotroph-specific transcripts, viz., chitinase, brassinosteroid, auxin, Myeloblastosis (MYB), nodulin, and nitrate transporter (NRT), were common in all plant–diazotroph combinations; three transcripts, viz., nitrate transport accessory protein (NAR), thaumatin, and thionin, were exclusive in rice and another three transcripts, viz., NAC (NAM: no apical meristem, ATAF: Arabidopsis thaliana activating factor, and CUC: cup-shaped cotyledon), ABA (abscisic acid), and ammonium transporter, were exclusive in soybean. Differential expression of these transcripts and reduction in pathogenesis-related (PR) protein expression show the early interaction. Based on the interaction, it can be inferred that the compatibility of rice and soybean is more with GAB and BRH, respectively. We propose that rice is unable to identify the diazotroph as a beneficial microorganism or a pathogen from an early response. So, it expressed the hypersensitivity-related transcripts along with PR proteins. The molecular mechanism of diazotrophic associations of GAB and BRH with rice vis-à-vis soybean will shed light on the basic understanding of host responses to beneficial microorganisms.</p

Table_4_Understanding plant–microbe interaction of rice and soybean with two contrasting diazotrophic bacteria through comparative transcriptome analysis.docx

Understanding the beneficial plant–microbe interactions is becoming extremely critical for deploying microbes imparting plant fitness and achieving sustainability in agriculture. Diazotrophic bacteria have the unique ability to survive without external sources of nitrogen and simultaneously promote host plant growth, but the mechanisms of endophytic interaction in cereals and legumes have not been studied extensively. We have studied the early interaction of two diazotrophic bacteria, Gluconacetobacter diazotrophicus (GAB) and Bradyrhizobium japonicum (BRH), in 15-day-old seedlings of rice and soybean up to 120 h after inoculation (hai) under low-nitrogen medium. Root colonization of GAB in rice was higher than that of BRH, and BRH colonization was higher in soybean roots as observed from the scanning electron microscopy at 120 hai. Peroxidase enzyme was significantly higher at 24 hai but thereafter was reduced sharply in soybean and gradually in rice. The roots of rice and soybean inoculated with GAB and BRH harvested from five time points were pooled, and transcriptome analysis was executed along with control. Two pathways, “Plant pathogen interaction” and “MAPK signaling,” were specific to Rice-Gluconacetobacter (RG), whereas the pathways related to nitrogen metabolism and plant hormone signaling were specific to Rice-Bradyrhizobium (RB) in rice. Comparative transcriptome analysis of the root tissues revealed that several plant–diazotroph-specific differentially expressed genes (DEGs) and metabolic pathways of plant–diazotroph-specific transcripts, viz., chitinase, brassinosteroid, auxin, Myeloblastosis (MYB), nodulin, and nitrate transporter (NRT), were common in all plant–diazotroph combinations; three transcripts, viz., nitrate transport accessory protein (NAR), thaumatin, and thionin, were exclusive in rice and another three transcripts, viz., NAC (NAM: no apical meristem, ATAF: Arabidopsis thaliana activating factor, and CUC: cup-shaped cotyledon), ABA (abscisic acid), and ammonium transporter, were exclusive in soybean. Differential expression of these transcripts and reduction in pathogenesis-related (PR) protein expression show the early interaction. Based on the interaction, it can be inferred that the compatibility of rice and soybean is more with GAB and BRH, respectively. We propose that rice is unable to identify the diazotroph as a beneficial microorganism or a pathogen from an early response. So, it expressed the hypersensitivity-related transcripts along with PR proteins. The molecular mechanism of diazotrophic associations of GAB and BRH with rice vis-à-vis soybean will shed light on the basic understanding of host responses to beneficial microorganisms.</p

Table_11_Understanding plant–microbe interaction of rice and soybean with two contrasting diazotrophic bacteria through comparative transcriptome analysis.xlsx

Understanding the beneficial plant–microbe interactions is becoming extremely critical for deploying microbes imparting plant fitness and achieving sustainability in agriculture. Diazotrophic bacteria have the unique ability to survive without external sources of nitrogen and simultaneously promote host plant growth, but the mechanisms of endophytic interaction in cereals and legumes have not been studied extensively. We have studied the early interaction of two diazotrophic bacteria, Gluconacetobacter diazotrophicus (GAB) and Bradyrhizobium japonicum (BRH), in 15-day-old seedlings of rice and soybean up to 120 h after inoculation (hai) under low-nitrogen medium. Root colonization of GAB in rice was higher than that of BRH, and BRH colonization was higher in soybean roots as observed from the scanning electron microscopy at 120 hai. Peroxidase enzyme was significantly higher at 24 hai but thereafter was reduced sharply in soybean and gradually in rice. The roots of rice and soybean inoculated with GAB and BRH harvested from five time points were pooled, and transcriptome analysis was executed along with control. Two pathways, “Plant pathogen interaction” and “MAPK signaling,” were specific to Rice-Gluconacetobacter (RG), whereas the pathways related to nitrogen metabolism and plant hormone signaling were specific to Rice-Bradyrhizobium (RB) in rice. Comparative transcriptome analysis of the root tissues revealed that several plant–diazotroph-specific differentially expressed genes (DEGs) and metabolic pathways of plant–diazotroph-specific transcripts, viz., chitinase, brassinosteroid, auxin, Myeloblastosis (MYB), nodulin, and nitrate transporter (NRT), were common in all plant–diazotroph combinations; three transcripts, viz., nitrate transport accessory protein (NAR), thaumatin, and thionin, were exclusive in rice and another three transcripts, viz., NAC (NAM: no apical meristem, ATAF: Arabidopsis thaliana activating factor, and CUC: cup-shaped cotyledon), ABA (abscisic acid), and ammonium transporter, were exclusive in soybean. Differential expression of these transcripts and reduction in pathogenesis-related (PR) protein expression show the early interaction. Based on the interaction, it can be inferred that the compatibility of rice and soybean is more with GAB and BRH, respectively. We propose that rice is unable to identify the diazotroph as a beneficial microorganism or a pathogen from an early response. So, it expressed the hypersensitivity-related transcripts along with PR proteins. The molecular mechanism of diazotrophic associations of GAB and BRH with rice vis-à-vis soybean will shed light on the basic understanding of host responses to beneficial microorganisms.</p

Table_9_Understanding plant–microbe interaction of rice and soybean with two contrasting diazotrophic bacteria through comparative transcriptome analysis.xlsx

Understanding the beneficial plant–microbe interactions is becoming extremely critical for deploying microbes imparting plant fitness and achieving sustainability in agriculture. Diazotrophic bacteria have the unique ability to survive without external sources of nitrogen and simultaneously promote host plant growth, but the mechanisms of endophytic interaction in cereals and legumes have not been studied extensively. We have studied the early interaction of two diazotrophic bacteria, Gluconacetobacter diazotrophicus (GAB) and Bradyrhizobium japonicum (BRH), in 15-day-old seedlings of rice and soybean up to 120 h after inoculation (hai) under low-nitrogen medium. Root colonization of GAB in rice was higher than that of BRH, and BRH colonization was higher in soybean roots as observed from the scanning electron microscopy at 120 hai. Peroxidase enzyme was significantly higher at 24 hai but thereafter was reduced sharply in soybean and gradually in rice. The roots of rice and soybean inoculated with GAB and BRH harvested from five time points were pooled, and transcriptome analysis was executed along with control. Two pathways, “Plant pathogen interaction” and “MAPK signaling,” were specific to Rice-Gluconacetobacter (RG), whereas the pathways related to nitrogen metabolism and plant hormone signaling were specific to Rice-Bradyrhizobium (RB) in rice. Comparative transcriptome analysis of the root tissues revealed that several plant–diazotroph-specific differentially expressed genes (DEGs) and metabolic pathways of plant–diazotroph-specific transcripts, viz., chitinase, brassinosteroid, auxin, Myeloblastosis (MYB), nodulin, and nitrate transporter (NRT), were common in all plant–diazotroph combinations; three transcripts, viz., nitrate transport accessory protein (NAR), thaumatin, and thionin, were exclusive in rice and another three transcripts, viz., NAC (NAM: no apical meristem, ATAF: Arabidopsis thaliana activating factor, and CUC: cup-shaped cotyledon), ABA (abscisic acid), and ammonium transporter, were exclusive in soybean. Differential expression of these transcripts and reduction in pathogenesis-related (PR) protein expression show the early interaction. Based on the interaction, it can be inferred that the compatibility of rice and soybean is more with GAB and BRH, respectively. We propose that rice is unable to identify the diazotroph as a beneficial microorganism or a pathogen from an early response. So, it expressed the hypersensitivity-related transcripts along with PR proteins. The molecular mechanism of diazotrophic associations of GAB and BRH with rice vis-à-vis soybean will shed light on the basic understanding of host responses to beneficial microorganisms.</p

Table_6_Understanding plant–microbe interaction of rice and soybean with two contrasting diazotrophic bacteria through comparative transcriptome analysis.xlsx

Understanding the beneficial plant–microbe interactions is becoming extremely critical for deploying microbes imparting plant fitness and achieving sustainability in agriculture. Diazotrophic bacteria have the unique ability to survive without external sources of nitrogen and simultaneously promote host plant growth, but the mechanisms of endophytic interaction in cereals and legumes have not been studied extensively. We have studied the early interaction of two diazotrophic bacteria, Gluconacetobacter diazotrophicus (GAB) and Bradyrhizobium japonicum (BRH), in 15-day-old seedlings of rice and soybean up to 120 h after inoculation (hai) under low-nitrogen medium. Root colonization of GAB in rice was higher than that of BRH, and BRH colonization was higher in soybean roots as observed from the scanning electron microscopy at 120 hai. Peroxidase enzyme was significantly higher at 24 hai but thereafter was reduced sharply in soybean and gradually in rice. The roots of rice and soybean inoculated with GAB and BRH harvested from five time points were pooled, and transcriptome analysis was executed along with control. Two pathways, “Plant pathogen interaction” and “MAPK signaling,” were specific to Rice-Gluconacetobacter (RG), whereas the pathways related to nitrogen metabolism and plant hormone signaling were specific to Rice-Bradyrhizobium (RB) in rice. Comparative transcriptome analysis of the root tissues revealed that several plant–diazotroph-specific differentially expressed genes (DEGs) and metabolic pathways of plant–diazotroph-specific transcripts, viz., chitinase, brassinosteroid, auxin, Myeloblastosis (MYB), nodulin, and nitrate transporter (NRT), were common in all plant–diazotroph combinations; three transcripts, viz., nitrate transport accessory protein (NAR), thaumatin, and thionin, were exclusive in rice and another three transcripts, viz., NAC (NAM: no apical meristem, ATAF: Arabidopsis thaliana activating factor, and CUC: cup-shaped cotyledon), ABA (abscisic acid), and ammonium transporter, were exclusive in soybean. Differential expression of these transcripts and reduction in pathogenesis-related (PR) protein expression show the early interaction. Based on the interaction, it can be inferred that the compatibility of rice and soybean is more with GAB and BRH, respectively. We propose that rice is unable to identify the diazotroph as a beneficial microorganism or a pathogen from an early response. So, it expressed the hypersensitivity-related transcripts along with PR proteins. The molecular mechanism of diazotrophic associations of GAB and BRH with rice vis-à-vis soybean will shed light on the basic understanding of host responses to beneficial microorganisms.</p