Antibiotic Potentiation as a Promising Strategy to Combat Macrolide Resistance in Bacterial Pathogens.

Antibiotics, which hit the market with astounding impact, were once called miracle drugs, as these were considered the ultimate cure for infectious diseases in the mid-20th century. However, today, nearly all bacteria that afflict humankind have become resistant to these wonder drugs once developed to stop them, imperiling the foundation of modern medicine. During the COVID-19 pandemic, there was a surge in macrolide use to treat secondary infections and this persistent use of macrolide antibiotics has provoked the emergence of macrolide resistance. In view of the current dearth of new antibiotics in the pipeline, it is essential to find an alternative way to combat drug resistance. Antibiotic potentiators or adjuvants are non-antibacterial active molecules that, when combined with antibiotics, increase their activity. Thus, potentiating the existing antibiotics is one of the promising approaches to tackle and minimize the impact of antimicrobial resistance (AMR). Several natural and synthetic compounds have demonstrated effectiveness in potentiating macrolide antibiotics against multidrug-resistant (MDR) pathogens. The present review summarizes the different resistance mechanisms adapted by bacteria to resist macrolides and further emphasizes the major macrolide potentiators identified which could serve to revive the antibiotic and can be used for the reversal of macrolide resistance.

Antimicrobial resistance heterogeneity among multidrug-resistant Gram-negative pathogens: Phenotypic, genotypic, and proteomic analysis.

Microbes evolve rapidly by modifying their genomes through mutations or through the horizontal acquisition of mobile genetic elements (MGEs) linked with fitness traits such as antimicrobial resistance (AMR), virulence, and metabolic functions. We conducted a multicentric study in India and collected different clinical samples for decoding the genome sequences of bacterial pathogens associated with sepsis, urinary tract infections, and respiratory infections to understand the functional potency associated with AMR and its dynamics. Genomic analysis identified several acquired AMR genes (ARGs) that have a pathogen-specific signature. We observed that blaCTX-M-15, blaCMY-42, blaNDM-5, and aadA(2) were prevalent in Escherichia coli, and blaTEM-1B, blaOXA-232, blaNDM-1, rmtB, and rmtC were dominant in Klebsiella pneumoniae. In contrast, Pseudomonas aeruginosa and Acinetobacter baumannii harbored blaVEB, blaVIM-2, aph(3'), strA/B, blaOXA-23, aph(3') variants, and amrA, respectively. Regardless of the type of ARG, the MGEs linked with ARGs were also pathogen-specific. The sequence type of these pathogens was identified as high-risk international clones, with only a few lineages being predominant and region-specific. Whole-cell proteome analysis of extensively drug-resistant K. pneumoniae, A. baumannii, E. coli, and P. aeruginosa strains revealed differential abundances of resistance-associated proteins in the presence and absence of different classes of antibiotics. The pathogen-specific resistance signatures and differential abundance of AMR-associated proteins identified in this study should add value to AMR diagnostics and the choice of appropriate drug combinations for successful antimicrobial therapy.

Hybrid genome assembly and annotation of multidrug-resistant Staphylococcus aureus genotype ST672-SCCmec type IVd (2B).

OBJECTIVES: The emergence of multidrug-resistant Staphylococcus aureus strains is mainly mediated by mobile genetic elements, such as Staphylococcal Cassette Chromosome mec (SCCmec). Currently, SCCmec elements in S. aureus are classified into 15 types, with type IV being the most common in hospital and community-associated methicillin-resistant S. aureus. Among different subtypes of SCCmec type IV strains (IVa-IVn), the complete genome sequence of the SCCmec IVd (2B) subtype is still lacking. Here, we report the complete genome sequence of multidrug-resistant S. aureus SCCmec typeIVd (2B) isolate, S. aureus S145. METHODS: Staphylococcus aureus S145 was subjected to phenotypic and genotypic characterization. The whole-genome sequencing of S145 was performed using a hybrid-genome approach. The antibiotic-resistance genes were detected and compared with 112 publicly available S. aureus genomes. RESULTS: We obtained a complete genome of S145 with 2.7 Mbp length, Guanine-Cytosine (GC) content of 32.8%, and 2,548 protein-coding regions with 79 virulence factors and 90 antibiotic resistance genes. The S145 has ∼17-kb SCCmec, which encodes genes such as mecA, mecR1, ccrA2B2, and SCCmec IVd (2B) subtype gene CG002. We detected a ∼30-kb multidrug-resistant plasmid with eight antibiotic-resistant genes forming three clusters. Cluster1 encoded for penicillin (blaI-blaZ-blaR1), Cluster2 for aminoglycoside-streptothricin (aph(3')-IIIa-sat4-ΔANT(6)-Ia), and Cluster3 for macrolides (msr(A)-mph(C)) resistance genes. Comparative analysis of Cluster1-Cluster3 revealed that the genetic organization of these clusters resembles resistance genes present in plasmids of USA300 S. aureus SCCmec type IVa strains. CONCLUSION: Here, we report the complete genome sequence of S. aureus SCCmec IVd (2B) that can be used as a reference genome for further comparative genomic analysis.

Respiratory tract microbiome and pneumonia.

The respiratory system, like the gut, harbors a vast variety of microorganisms which include bacteria, viruses and fungi. The advent of next generation sequencing and multi-omic approaches has revealed the diversity and functional significances of microorganisms in the respiratory health. It has been identified that there has been a co-evolution of indigenous respiratory microbiota and the human immune system. However, an immune response is usually generated when the homeostasis of the microbiota is disturbed. The respiratory microbiome has been identified to be important in shaping the respiratory immunity. Gut microbiota and oral microbiota are also known to be pivotal in shaping the immune system of the respiratory tract and influence its microbial dynamics. Proteobacteria, Firmicutes, and Bacteroidetes have been identified to be predominant in the respiratory system. While, Streptococcus, Prevotella, Fusobacteria, and Veillonella forms the major part, potential pathogens, such as Haemophilus and Neisseria, also form a small fraction of the healthy lung microbiome. Dysbiosis of respiratory microbiome can lead to increased colonization of opportunistic pathogens that can lead to respiratory infections such as pneumonia. This chapter describes the microbial diversity of respiratory system and the role of respiratory microbiome during respiratory infections like pneumonia. The chapter also discusses few strategies that have been proved effective in preventing pneumonia.

ß-Lactam potentiators to re-sensitize resistant pathogens: Discovery, development, clinical use and the way forward.

ß-lactam antibiotics are one of the most widely used and diverse classes of antimicrobial agents for treating both Gram-negative and Gram-positive bacterial infections. The ß-lactam antibiotics, which include penicillins, cephalosporins, monobactams and carbapenems, exert their antibacterial activity by inhibiting the bacterial cell wall synthesis and have a global positive impact in treating serious bacterial infections. Today, ß-lactam antibiotics are the most frequently prescribed antimicrobial across the globe. However, due to the widespread use and misapplication of ß-lactam antibiotics in fields such as human medicine and animal agriculture, resistance to this superlative drug class has emerged in the majority of clinically important bacterial pathogens. This heightened antibiotic resistance prompted researchers to explore novel strategies to restore the activity of ß-lactam antibiotics, which led to the discovery of ß-lactamase inhibitors (BLIs) and other ß-lactam potentiators. Although there are several successful ß-lactam-ß-lactamase inhibitor combinations in use, the emergence of novel resistance mechanisms and variants of ß-lactamases have put the quest of new ß-lactam potentiators beyond precedence. This review summarizes the success stories of ß-lactamase inhibitors in use, prospective ß-lactam potentiators in various phases of clinical trials and the different strategies used to identify novel ß-lactam potentiators. Furthermore, this review discusses the various challenges in taking these ß-lactam potentiators from bench to bedside and expounds other mechanisms that could be investigated to reduce the global antimicrobial resistance (AMR) burden.

Co-infection of Lactococcus garvieae and Tilapia lake virus (TiLV) in Nile tilapia Oreochromis niloticus cultured in India.

Tilapia lake virus (TiLV) and Lactococcus garvieae are 2 major pathogens of cultured Nile tilapia Oreochromis niloticus. In June-July 2018, a disease outbreak was reported in Nile tilapia cultured in brackish water floating cages in Kerala, India. Affected fish died gradually, and cumulative mortality reached ~75% within 1 mo. In the present study, TiLV and L. garvieae were isolated from the infected fish and confirmed. Nucleotide analysis of the partial sequence of segment 3 revealed that the present TiLV isolate showed 100% similarity with TiLV MF574205 and 97.65% similarity with TiLV KU552135 isolated in Israel. The partial 16S rDNA nucleotide sequence of L. garvieae shared 99% similarity with the 16S rDNA nucleotide sequence of L. garvieae isolated from Nile tilapia in Brazil. Eight virulence genes (hly1, hly2, hly3, NADH oxidase, adhPav, LPxTG-1, LPxTG-4, adhC1) were amplified in the present isolate. In the experimental challenge study, the onset of mortality started earlier in fish co-infected with TiLV and L. garvieae (3 d post-infection [dpi]) compared to other groups. Cumulative mortality (90% at 12 dpi) was significantly higher in the co-infected group than in fish infected with TiLV (60% at 12 dpi) and L. garvieae (40% at 12 dpi) alone. This study reveals that synergistic co-infection with TiLV and other bacteria may increase mortality in disease outbreaks. To the best of our knowledge, this is the first reported co-infection of L. garvieae with TiLV associated with mass mortality in Nile tilapia in India.

The bane of coastal marine environment: A fatal case of Vibrio vulnificus associated cellulitis and septicaemia.

Vibrio vulnificus is a Gram negative motile bacterium known to cause fatal septicaemia and wound infection. It is commonly associated with the consumption of under-cooked seafood or exposure to marine environment. We report a case of a 55 year old male patient, who was presented with right lower limb cellulitis and septicaemia due to V. vulnificus. V. vulnificus infection in India are rare. However, increasing reports of V. vulnificus from India recommends considering the pathogen while dealing necrotising fasciitis especially in the proximity of marine environment.

Potential effectiveness and adverse implications of repurposing doxycycline in COVID-19 treatment.

Introduction: COVID-19 infection with no known-specific drugs or vaccines has impacted mankind and has become beyond precedence. Currently, re-purposing of existing drugs is the only therapeutic option for managing COVID-19 symptoms and associated co-infections to reduce mortality. Antimicrobials as varied as antiparasitic, antiviral, and antibiotics are under various stages of evaluation.Areas covered: Recently, doxycycline, a broad-spectrum antibiotic that has also reported antiviral and anti-inflammatory properties was widely investigated in clinical trials, either alone or in combination with other drugs, and repurposed for COVID-19 treatment. In the review, the potential therapeutic applications of doxycycline in COVID-19 treatment and its potential adverse implications with respect to antimicrobial resistance bestowed by repurposing the antibiotic have been expounded.Expert opinion: 'Fighting disease with already existing antibiotics' and 'antimicrobial resistance progression' are like two arms of a balance that has to be carefully equilibrated. Any imbalance by the inappropriate or indiscriminate use of the repurposed drugs would cause a disastrous increase in antimicrobial resistance (AMR). Hence, cautious parallel assessment of potential long-term consequences of AMR is of great importance to mankind as its impacts would prevail even after the current pandemic.

First report of Vibrio cholerae O9, novel st520, isolated from a child with bacteraemia-associated sepsis.

Vibrios have been identified to cause extra-intestinal complications apart from the occasional cholera-like diarrhoeal outbreaks. The non-O1/O139 Vibrio cholerae strains are ubiquitous in environmental water bodies and hence pose a threat to people even without obvious risk factors. We describe a case of sepsis in a child with spinal dysraphism caused by a V. cholerae O9 strain belonging to a novel sequence type (ST520). The present case highlights the need of considering V. cholerae non-O1/O139 as one of the pathogens while dealing with sepsis cases, and also, the study expounds the importance of proper characterisation of the pathogen for an effective treatment.

Epidemiological and pathogenic characteristics of Haitian variant V. cholerae circulating in India over a decade (2000-2018).

Vibrio cholerae, causative agent of the water-borne disease cholera still threatens a large proportion of world's population. The major biotypes of the pathogen are classical and El Tor. There have been recent reports of variant V. cholerae strains circulating around the world. In the present study, the epidemiological status of V. cholerae strains circulating in the country over a decade was assessed. Also, a comprehensive analysis of the difference in pathogenicity between the different biotypes of V. cholerae strains was evaluated both in-vitro and in-vivo. The amount of CT produced by different biotypes of V. cholerae strains were analyzed by GM1 ELISA and the probable reasons for the difference in toxin production was discussed. MLST analysis grouped the isolates into a single Sequence Type (ST 69) whereas PFGE analysis clustered the isolates into ten different pulsotypes revealing molecular diversity. The circulating strains were identified to produce cholera toxin and CT mRNA intermediate to the classical and prototype El Tor strains. Also, the circulating strains were identified to possess four ToxR binding sequences. In-vivo pathogenicity analysis by rabbit ileal loop fluid accumulation assay revealed the Haitian variant strains to be more hyperemic than the prototype strains.

Adaptive laboratory evolution of Vibrio cholerae to doxycycline associated with spontaneous mutation.

Cholera, caused by the Gram-negative bacterium Vibrio cholerae, remains a serious threat in underdeveloped countries. Although rehydration therapy has been the mainstay of disease management, antibiotics are also being used as an adjunct treatment, resulting in an increase in the circulation of antimicrobial-resistant V. cholerae strains. In the present study, adaptive laboratory evolution, whole-genome sequencing and molecular docking studies were performed to identify putative mutations related to doxycycline resistance in V. cholerae isolates. The V57L mutation in the RpsJ protein was identified to be important in conferring doxycycline resistance. As revealed by molecular docking studies, the mutation was identified to alter the ribosome structure near the doxycycline binding site. Doxycycline stress also induced co-resistance to colistin, a last-resort antibiotic to treat extensively drug-resistant bacteria. This study illustrates for the first time a possible mechanism of doxycycline-selected resistance in V. cholerae as well as doxycycline-selected co-resistance, warranting strict restrictions on the indiscriminate use of antibiotics.

Tryptanthrin, a potential biofilm inhibitor against toxigenic Vibrio cholerae, modulating the global quorum sensing regulator, LuxO.

Cholera caused by the Gram-negative bacterium Vibrio cholerae still remains a major health burden in developing countries due to its high transmissibility and multidrug resistance. Alternative strategies are in quest to curtail the disease focusing on antivirulent approaches, such as biofilm inhibition, which make bacteria more susceptible to antibiotic therapies. The biofilm state is important for V. cholerae pathogenesis and its persistence in the environment. In the present study, tryptanthrin, a phytochemical, has been identified as possessing strong anti-biofilm activity at sub MIC (2 µg ml-1) against V. cholerae. LuxO was identified as the putative target of tryptanthrin by molecular docking and real time analysis. The phytochemical was identified as safe and possessed synergistic action with ciprofloxacin, a commonly used quinolone antibiotic to treat cholera. Collectively, the study establishes the first report on the anti-biofilm property of tryptanthrin by targeting LuxO, which could serve as a potential antivirulent therapy to combat V. cholerae infections.

Decoding the proteomic changes involved in the biofilm formation of Enterococcus faecalis SK460 to elucidate potential biofilm determinants.

BACKGROUND: Enterococcus faecalis is a major clinically relevant nosocomial bacterial pathogen frequently isolated from polymicrobial infections. The biofilm forming ability of E. faecalis attributes a key role in its virulence and drug resistance. Biofilm cells are phenotypically and metabolically different from their planktonic counterparts and many aspects involved in E. faecalis biofilm formation are yet to be elucidated. The strain E. faecalis SK460 used in the present study is esp (Enterococcal surface protein) and fsr (two-component signal transduction system) negative non-gelatinase producing strong biofilm former isolated from a chronic diabetic foot ulcer patient. We executed a label-free quantitative proteomic approach to elucidate the differential protein expression pattern at planktonic and biofilm stages of SK460 to come up with potential determinants associated with Enterococcal biofilm formation. RESULTS: The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses of proteomic data revealed that biofilm cells expressed higher levels of proteins which are associated with glycolysis, amino acid biosynthesis, biosynthesis of secondary metabolites, microbial metabolism in diverse environments and stress response factors. Besides these basic survival pathways, LuxS-mediated quorum sensing, arginine metabolism, rhamnose biosynthesis, pheromone and adhesion associated proteins were found to be upregulated during the biofilm transit from planktonic stages. The selected subsets were validated by quantitative real-time PCR. In silico functional interaction analysis revealed that the genes involved in upregulated pathways pose a close molecular interaction thereby coordinating the regulatory network to thrive as a biofilm community. CONCLUSIONS: The present study describes the first report of the quantitative proteome analysis of an esp and fsr negative non gelatinase producing E. faecalis. Proteome analysis evidenced enhanced expression of glycolytic pathways, stress response factors, LuxS quorum signaling system, rhamnopolysaccharide synthesis and pheromone associated proteins in biofilm phenotype. We also pointed out the relevance of LuxS quorum sensing and pheromone associated proteins in the biofilm development of E. faecalis which lacks the Fsr quorum signaling system. These validated biofilm determinants can act as potential inhibiting targets in Enterococcal infections.

Molecular Adaptations and Antibiotic Resistance in Vibrio cholerae: A Communal Challenge.

Cholera, caused by the Gram-negative bacterium Vibrio cholerae, remains a major problem in developing countries. Although the disease can be managed by oral rehydration therapy, antibiotics are widely used nowadays to treat the disease. However, chemoprophylaxis has been proven to have no effect on the spread of the disease, but acts as a major driver for antimicrobial resistance (AMR). V. cholerae has evolved different ways to combat antibiotics used against them. This review comprehends the different molecular mechanisms of antibiotic resistance in V. cholerae, gaps in the development of new antibiotics, and the alternative strategies that can be used to treat the disease. The review advocates the use of antivirulence compounds rather than antibacterial compounds as a strategy to limit the increasing AMR. Also, the review expounds the role of community in preventing the diseases and tackling the global burden of AMR.

Genome sequence of a multidrug-resistant Klebsiella pneumoniae ST78 with high colistin resistance isolated from a patient in India.

OBJECTIVES: Multidrug-resistant (MDR) Klebsiella pneumoniae isolates with colistin resistance are a major concern in healthcare settings. This study aimed to evaluate the genome-wide distribution of antimicrobial resistance genes in K. pneumoniae CRKP I with high colistin resistance isolated from a patient in India. METHODS: The whole genome of K. pneumoniae CRKP I was sequenced on an Illumina MiSeq platform. De novo genome assembly was performed using SPAdes v.3.0.0, and the genome sequence was analysed using bioinformatics tools available from the Center for Genomic Epidemiology. RESULTS: The genome of K. pneumoniae CRKP I is 5.1 Mb in size and contains different classes of antimicrobial resistance genes. The isolate is highly resistant to colistin owing to a point mutation in mgrB gene, encoding a negative regulator of the PhoP/PhoQ two-component system. Multilocus sequence typing (MLST) showed that K. pneumoniae CRKP I belongs to ST78. CONCLUSION: These data provide useful information for comparative genomic analysis regarding the dissemination of antimicrobial resistance genes in K. pneumoniae. To our knowledge, this is the first report of a MDR K. pneumoniae with high colistin resistance belonging to ST78 causing infection in a human.

Genomic Insights into Biofilm-Forming Enterococcus faecalis SK460 Isolated from a Chronic Diabetic Ulcer Patient.

Enterococcus faecalis is recognized as one of the leading pathogens causing nosocomial infections. Here we report a draft genome sequence of Enterococcus faecalis SK460, isolated from a chronic diabetic foot ulcer patient. This strain exhibits various biofilm-associated genes, virulence genes, and antibiotic-resistance genes related to aminoglycoside, macrolide, and tetracycline resistance.

Insights into the Draft Genome Sequence of a Haitian Variant Vibrio cholerae Strain Isolated from a Clinical Setting in Kerala, South India.

We report here the draft genome sequence of a Haitian variant Vibrio cholerae strain, W4-13, isolated from Kerala, South India, possessing cholera toxin gene in chromosomes I and II. The sequence will be useful to achieve a profound understanding on its evolution, with emphasis on its pathogenesis and antibiotic resistance.