Genomic insights into in-ICU emergence of last-resort antimicrobial resistance in a rare, carbapenem resistant, ST16 Klebsiella pneumoniae strain from Jodhpur, India.

OBJECTIVES: To investigate the genomic differences between two extensively drug resistant, ST16 strains of Klebsiella pneumoniae recovered from patients in the same ICU, one of which was colistin resistant. METHODS: Antimicrobial susceptibilities of the isolates were determined using VITEK-2. Hybrid assemblies for both strains were generated using Oxford Nanopore and Illumina technologies. The sequence type, capsule type, O-locus type, antimicrobial resistance determinants and plasmids carried by the isolates were inferred from the genome sequence. The phylogenetic placement, antimicrobial resistance, and virulence determinants of the isolates relative to a collection (n = 871) of ST16 isolates were assessed. RESULTS: Both BC16, a colistin-resistant blood stream isolate and U23, a colistin-sensitive urinary isolate displayed near-identical antimicrobial resistance profiles and genome sequences with varying plasmid profiles. The BC16 genome only had 21 SNPs relative to U23 and belonged to the same capsule, O-antigen locus and multi-locus sequence types. The mgrB locus in BC16 was disrupted by an IS5 element. Phylogenetically, U23 and BC16 were placed on a clade with 4 strains belonging to K-type K48 and O-type O2a as opposed to majority (n = 807) of the strains (K-type K51 and O-type O3b). CONCLUSIONS: BC16 was a colistin resistant derivative of U23, which evolved colistin resistance by an IS5-mediated disruption of the mgrB locus, likely during treatment of the index patient with colistin in the ICU. The strains belong to a rare subtype of ST16 with unique capsular and O-antigen types underscoring the utility of genomic surveillance networks and open-access genomic surveillance data in tracking problem clones.

Unusual Hypermucoviscous Clinical Isolate of Klebsiella pneumoniae with No Known Determinants of Hypermucoviscosity.

Klebsiella pneumoniae can be broadly classified into classical strains that cause drug-resistant, hospital-associated infections and hypervirulent strains that cause invasive, community-acquired, drug-susceptible infections. Hypermucoviscosity in Klebsiella pneumoniae has been associated with immune evasion and hypervirulence. A string-test-positive, hypermucoviscous strain of Klebsiella pneumoniae, P34, was isolated from the cystic lesion of a patient who reported to a tertiary care hospital in Jodhpur, Rajasthan, India. Given the antibiotic-susceptible and hypermucoviscous nature of the isolate, it was suspected to belong to the hypervirulent lineage of Klebsiella pneumoniae. However, P34 did not overproduce capsular polysaccharides and also remained susceptible to the antimicrobial effects of human serum when tested alongside strains that were non-hypermucoviscous. Sequencing of the genome of P34 revealed the absence of any large virulence plasmids or integrative conjugative elements that usually carry hypermucoviscosity- and hypervirulence-associated genes. P34 also lacked key virulence determinants such as aerobactin, yersiniabactin, and salmochelin biosynthesis clusters. In addition, P34 lacked homologs for genes associated with enhanced capsule synthesis and hypermucoviscosity, such as rmpA, rmpA2, rmpC, and rmpD (regulator of mucoid phenotype). These observations suggest that P34 may harbor novel genetic determinants of hypermucoviscosity independent of the indirectly acting rmpA and the recently described rmpD. IMPORTANCE Hypermucoviscosity is a characteristic of hypervirulent Klebsiella pneumoniae strains, which are capable of causing invasive disease in community settings. This study reports phenotyping and genomic analysis of an unusual clinical isolate of Klebsiella pneumoniae, P34, which exhibits hypermucoviscosity and yet does not harbor rmp (regulator of mucoid phenotype) genes, which are known determinants of hypermucoviscosity (rmpA and rmpD). Similar clinical isolates belonging to the K. pneumoniae complex that are hypermucoviscous but do not harbor the rmp loci have been reported from India and abroad, indicating the prevalence of unknown determinants contributing to hypermucoviscosity. Therefore, strains like P34 will serve as model systems to mechanistically study potentially novel determinants of hypermucoviscosity in the K. pneumoniae complex.

Vancomycin-Induced Modulation of Gram-Positive Gut Bacteria and Metabolites Remediates Insulin Resistance in iNOS Knockout Mice.

The role of oxidative and nitrosative stress has been implied in both physiology and pathophysiology of metabolic disorders. Inducible nitric oxide synthase (iNOS) has emerged as a crucial regulator of host metabolism and gut microbiota activity. The present study examines the role of the gut microbiome in determining host metabolic functions in the absence of iNOS. Insulin-resistant and dyslipidemic iNOS-/- mice displayed reduced microbial diversity, with a higher relative abundance of Allobaculum and Bifidobacterium, gram-positive bacteria, and altered serum metabolites along with metabolic dysregulation. Vancomycin, which largely depletes gram-positive bacteria, reversed the insulin resistance (IR), dyslipidemia, and related metabolic anomalies in iNOS-/- mice. Such improvements in metabolic markers were accompanied by alterations in the expression of genes involved in fatty acid synthesis in the liver and adipose tissue, lipid uptake in adipose tissue, and lipid efflux in the liver and intestine tissue. The rescue of IR in vancomycin-treated iNOS-/- mice was accompanied with the changes in select serum metabolites such as 10-hydroxydecanoate, indole-3-ethanol, allantoin, hippurate, sebacic acid, aminoadipate, and ophthalmate, along with improvement in phosphatidylethanolamine to phosphatidylcholine (PE/PC) ratio. In the present study, we demonstrate that vancomycin-mediated depletion of gram-positive bacteria in iNOS-/- mice reversed the metabolic perturbations, dyslipidemia, and insulin resistance.

Comparative data analsysis of two multi-drug resistant homoserine lactone and rhamnolipid producing Pseudomonas aeruginosa from diabetic foot infected patient.

Pseudomonas aeruginosa generally forms strong biofilm during chronic condition of wound. The whole mechanism of biofilm formation works in tandem with quorum sensing circuit of the organism in order to produce virulence. Here we report the draft genome sequence of two diabetic foot ulcer Pseudomonas aeruginosa isolates (VIT PC 7 and VIT PC 9) displaying homoserine lactone, rhamnolipid producing, biofilm phenotype and antibiotic resistance genes related to carbapenem, aminoglycoside, beta- lactamase and tetracycline resistance. The whole genome sequencing library was prepared according to the Oxford Nanopore's SQK-LSK108 kit protocol on Oxford Nanopore's Minion platform. The 7.1 Mb and 6.3-Mb draft genome sequence with GC content of 65.8% and 66.4% respectively provides insight into their resistance mechanism and virulence factors.

Genome Sequence of Bacillus vallismortis TD3, a Salt-Tolerant Strain Isolated from the Sediments of a Solar Saltern in Tamil Nadu, India.

Various Bacillus spp. capable of producing enzymes with industrially desirable properties have been isolated from adverse environments. Here, we announce the 3.91-Mbp draft genome sequence of a moderately salt-resistant Bacillus vallismortis strain, TD3, capable of producing several industrially relevant enzymes.

Genome Sequence of a Moderately Halophilic Bacillus cereus Strain, TS2, Isolated from Saltern Sediments.

We report the 5.3-Mbp genome sequence of Bacillus cereus strain TS2, which was isolated from the sediments of a solar saltern in southern India. Genome analysis of B. cereus TS2, a salt-resistant strain, will improve our understanding of how B. cereus, a food pathogen, responds to hyperosmotic stress.

Comparison of the long term efficacy of radiofrequency ablation and surgical turbinoplasty in inferior turbinate hypertrophy: a randomized clinical study.

OBJECTIVE: To compare the long term effectiveness of radiofrequency thermal ablation with that of surgical turbinoplasty, a prospective, randomized, blinded clinical study was conducted. METHODS: Eighty six patients suffering from nasal obstruction due to bilateral inferior turbinate hypertrophy were enrolled. Forty four patients were operated by radiofrequency ablation, while 42 underwent surgical turbinoplasty. The outcomes of both techniques were compared in terms of symptoms using visual analogue score (VAS) and endoscopic scores. RESULTS: A statistically significant difference in the VAS scores existed between the two groups by the 3rd month in terms of nasal obstruction (p=.0002) and headache (p=.0001), by the 6th month in terms of nasal secretions (p=.007), by the end of 1 year in terms of sneezing (p=.023) and no statistically significant difference even till the end of year with respect to hyposmia. CONCLUSIONS: Radiofrequency thermal ablation is more effective than surgical turbinoplasty in terms of treating nasal obstruction and equally effective in terms of managing sneezing. Radiofrequency ablation has the advantage of being a day care procedure and minimal complications.

Pleiotropic Regulation of Virulence Genes in Streptococcus mutans by the Conserved Small Protein SprV.

Streptococcus mutans, an oral pathogen associated with dental caries, colonizes tooth surfaces as polymicrobial biofilms known as dental plaque. S. mutans expresses several virulence factors that allow the organism to tolerate environmental fluctuations and compete with other microorganisms. We recently identified a small hypothetical protein (90 amino acids) essential for the normal growth of the bacterium. Inactivation of the gene, SMU.2137, encoding this protein caused a significant growth defect and loss of various virulence-associated functions. An S. mutans strain lacking this gene was more sensitive to acid, temperature, osmotic, oxidative, and DNA damage-inducing stresses. In addition, we observed an altered protein profile and defects in biofilm formation, bacteriocin production, and natural competence development, possibly due to the fitness defect associated with SMU.2137 deletion. Transcriptome sequencing revealed that nearly 20% of the S. mutans genes were differentially expressed upon SMU.2137 deletion, thereby suggesting a pleiotropic effect. Therefore, we have renamed this hitherto uncharacterized gene as sprV (streptococcal pleiotropic regulator of virulence). The transcript levels of several relevant genes in the sprV mutant corroborated the phenotypes observed upon sprV deletion. Owing to its highly conserved nature, inactivation of the sprV ortholog in Streptococcus gordonii also resulted in poor growth and defective UV tolerance and competence development as in the case of S. mutans Our experiments suggest that SprV is functionally distinct from its homologs identified by structure and sequence homology. Nonetheless, our current work is aimed at understanding the importance of SprV in the S. mutans biology.IMPORTANCEStreptococcus mutans employs several virulence factors and stress resistance mechanisms to colonize tooth surfaces and cause dental caries. Bacterial pathogenesis is generally controlled by regulators of fitness that are critical for successful disease establishment. Sometimes these regulators, which are potential targets for antimicrobials, are lost in the genomic context due to the lack of annotated homologs. This work outlines the regulatory impact of a small, highly conserved hypothetical protein, SprV, encoded by S. mutans We show that SprV affects the transcript levels of various virulence factors required for normal growth, biofilm formation, stress tolerance, genetic competence, and bacteriocin production.

Gene Regulation by the LiaSR Two-Component System in Streptococcus mutans.

The LiaSR two-component signal transduction system regulates cellular responses to several environmental stresses, including those that induce cell envelope damages. Downstream regulons of the LiaSR system have been implicated in tolerance to acid, antibiotics and detergents. In the dental pathogen Streptococcus mutans, the LiaSR system is necessary for tolerance against acid, antibiotics, and cell wall damaging stresses during growth in the oral cavity. To understand the molecular mechanisms by which LiaSR regulates gene expression, we created a mutant LiaR in which the conserved aspartic acid residue (the phosphorylation site), was changed to alanine residue (D58A). As expected, the LiaR-D58A variant was unable to acquire the phosphate group and bind to target promoters. We also noted that the predicted LiaR-binding motif upstream of the lia operon does not appear to be well conserved. Consistent with this observation, we found that LiaR was unable to bind to the promoter region of lia; however, we showed that LiaR was able to bind to the promoters of SMU.753, SMU.2084 and SMU.1727. Based on sequence analysis and DNA binding studies we proposed a new 25-bp conserved motif essential for LiaR binding. Introducing alterations at fully conserved positions in the 25-bp motif affected LiaR binding, and the binding was dependent on the combination of positions that were altered. By scanning the S. mutans genome for the occurrence of the newly defined LiaR binding motif, we identified the promoter of hrcA (encoding a key regulator of the heat shock response) that contains a LiaR binding motif, and we showed that hrcA is negatively regulated by the LiaSR system. Taken together our results suggest a putative role of the LiaSR system in heat shock responses of S. mutans.

Genome Sequencing of a Mung Bean Plant Growth Promoting Strain of P. aeruginosa with Biocontrol Ability.

Pseudomonas aeruginosa PGPR2 is a mung bean rhizosphere strain that produces secondary metabolites and hydrolytic enzymes contributing to excellent antifungal activity against Macrophomina phaseolina, one of the prevalent fungal pathogens of mung bean. Genome sequencing was performed using the Ion Torrent Personal Genome Machine generating 1,354,732 reads (6,772,433 sequenced bases) achieving ~25-fold coverage of the genome. Reference genome assembly using MIRA 3.4.0 yielded 198 contigs. The draft genome of PGPR2 encoded 6803 open reading frames, of which 5314 were genes with predicted functions, 1489 were genes of known functions, and 80 were RNA-coding genes. Strain specific and core genes of P. aeruginosa PGPR2 that are relevant to rhizospheric habitat were identified by pangenome analysis. Genes involved in plant growth promoting function such as synthesis of ACC deaminase, indole-3-acetic acid, trehalose, mineral scavenging siderophores, hydrogen cyanide, chitinases, acyl homoserine lactones, acetoin, 2,3-butanediol, and phytases were identified. In addition, niche-specific genes such as phosphate solubilising 3-phytase, adhesins, pathway-specific transcriptional regulators, a diguanylate cyclase involved in cellulose synthesis, a receptor for ferrienterochelin, a DEAD/DEAH-box helicase involved in stress tolerance, chemotaxis/motility determinants, an HtpX protease, and enzymes involved in the production of a chromanone derivative with potent antifungal activity were identified.

Identification and structure elucidation of a novel antifungal compound produced by Pseudomonas aeruginosa PGPR2 against Macrophomina phaseolina.

Pseudomonas aeruginosa PGPR2 was found to protect mungbean plants from charcoal rot disease caused by Macrophomina phaseolina. Secondary metabolites from the culture supernatant of P. aeruginosa PGPR2 were extracted with ethyl acetate and the antifungal compound was purified by preparative HPLC using reverse phase chromatography. The purified compound showed antifungal activity against M. phaseolina and other phytopathogenic fungi (Fusarium sp., Rhizoctonia sp. Alternaria sp., and Aspergillus sp.). The structure of the purified compound was determined using (1)H, (13)C, 2D NMR spectra and liquid chromatography-mass spectrometry (LC-MS). Spectral data suggest that the antifungal compound is 3,4-dihydroxy-N-methyl-4-(4-oxochroman-2-yl)butanamide, with the chemical formula C14H17NO5 and a molecular mass of 279. Though chemically synthesized chromanone derivatives have been shown to have antifungal activity, we report for the first time, the microbial production of a chromanone derivative with antifungal activity. This ability of P. aeruginosa PGPR2 makes it a suitable strain for biocontrol.

Influence of periplasmic oxidation of glucose on pyoverdine synthesis in Pseudomonas putida S11.

Fluorescent pseudomonads catabolize glucose simultaneously by two different pathways, namely, the oxidative pathway in periplasm and the phosphorylative pathway in cytoplasm. This study provides evidence for the role of glucose metabolism in the regulation of pyoverdine synthesis in Pseudomonas putida S11. We have characterized the influence of direct oxidation of glucose in periplasm on pyoverdine synthesis in P. putida S11. We identified a Tn5 transposon mutant of P. putida S11 showing increased pyoverdine production in minimal glucose medium (MGM). This mutant designated as IST1 had Tn5 insertion in glucose dehydrogenase (gcd) gene. To verify the role of periplasmic oxidation of glucose on pyoverdine synthesis, we constructed mutants S11 Gcd(-) and S11 PqqF(-) by antibiotic cassette mutagenesis. These mutants of P. putida S11 with loss of glucose dehydrogenase gene (gcd) or cofactor pyrroloquinoline quinone biosynthesis gene (pqqF) showed increased pyoverdine synthesis and impaired acid production in MGM. In minimal gluconate medium, the pyoverdine production of wild-type strain S11 and mutants S11 Gcd(-) and S11 PqqF(-) was higher than in MGM indicating that gluconate did not affect pyoverdine synthesis. In MGM containing PIPES-NaOH (pH 7.5) buffer which prevent pH changes due to gluconic acid production, strain S11 produced higher amount of pyoverdine similar to mutants S11 Gcd(-) and S11 PqqF(-). Therefore, it is proposed that periplasmic oxidation of glucose to gluconic acid decreases the pH of MGM and thereby influences pyoverdine synthesis of strain S11. The increased pyoverdine synthesis enhanced biotic and abiotic surface colonization of the strain S11.

Inactivation of the transcriptional regulator-encoding gene sdiA enhances rice root colonization and biofilm formation in Enterobacter cloacae GS1.

Enterobacter cloacae GS1 is a plant growth-promoting bacterium which colonizes rice roots. In the rhizosphere environment, N-acyl homoserine lactone (NAHL)-like quorum-sensing signals are known to be produced by host plants and other microbial inhabitants. E. cloacae GS1 was unable to synthesize NAHL quorum-sensing signals but had the NAHL-dependent transcriptional regulator-encoding gene sdiA. This study was aimed at understanding the effects of SdiA and NAHL-dependent cross talk in rice root colonization by E. cloacae GS1. Pleiotropic effects of sdiA inactivation included substantial increases in root colonization and biofilm formation, suggesting a negative role for SdiA in bacterial adhesion. We provide evidence that sdiA inactivation leads to elevated levels of biosynthesis of curli, which is involved in cellular adhesion. Extraneous addition of NAHLs had a negative effect on root colonization and biofilm formation. However, the sdiA mutant of E. cloacae GS1 was insensitive to NAHLs, suggesting that this NAHL-induced inhibition of root colonization and biofilm formation is SdiA dependent. Therefore, it is proposed that NAHLs produced by both plant and microbes in the rice rhizosphere act as cross-kingdom and interspecies signals to negatively impact cellular adhesion and, thereby, root colonization in E. cloacae GS1.

Proteolytic enzyme mediated antagonistic potential of Pseudomonas aeruginosa against Macrophomina phaseolina.

A new antagonistic bacterial strain PGPR2 was isolated from the mungbean rhizosphere and documented for the production of hydrolytic enzymes with antifungal activity. Based on the phylogenetic analysis of the 16S rRNA gene sequence and phenotyping, this strain was identified as Pseudomonas aeruginosa. Maximum protease activity (235 U/mL) was obtained at 24 h of fermentation. The protease was purified to homogeneity in three steps: ammonium sulphate precipitation, anion exchange chromatography on DEAE- cellulose resin and gel filtration chromatography using P6 column. The purified enzyme had a molecular weight of -33 kDa. The purified protease exhibited maximum activity at pH 6.0 and retained 80% of activity in a pH range of 5.0 - 9.0. Proteolytic activity was maximum in a temperature range of 40-70 degrees C. However, the enzyme was stable at 40 degrees C for 60 min. Among the metals tested, Mg2+ enhanced the protease activity. Internal amino acid sequence of the protease obtained by MALDI -ToF and subsequent Mascot database search showed maximum similarity to the HtpX protease of P. aeruginosa strain PA7. Thus, by virtue of its early production time, thermostability and effective antifungal ability, the protease purified and characterized from P. aeruginosa PGPR2 has several potential applications as fungicidal agents in agriculture.

Genome sequence of Staphylococcus arlettae strain CVD059, isolated from the blood of a cardiovascular disease patient.

We have isolated a Staphylococcus arlettae strain, strain CVD059, from the blood of a rheumatic mitral stenosis patient. Here, we report the genome sequence and potential virulence factors of this clinical isolate. The draft genome of S. arlettae CVD059 is 2,565,675 bp long with a G+C content of 33.5%.

Genome sequence of the plant growth-promoting rhizobacterium Pseudomonas putida S11.

Here we report the genome sequence of a plant growth-promoting rhizobacterium, Pseudomonas putida S11. The length of the draft genome sequence is approximately 5,970,799 bp, with a G+C content of 62.4%. The genome contains 6,076 protein-coding sequences.

Genome sequence of the plant growth-promoting bacterium Enterobacter cloacae GS1.

Here, we present the genome sequence of Enterobacter cloacae GS1. This strain proficiently colonizes rice roots and promotes plant growth by improving plant nutrition. Analyses of the E. cloacae GS1 genome will throw light on the genetic factors involved in root colonization, growth promotion, and ecological success of this rhizobacterium.

Influence of siderophore pyoverdine synthesis and iron-uptake on abiotic and biotic surface colonization of Pseudomonas putida S11.

Fluorescent pseudomonads produce a characteristic fluorescent pigment, pyoverdines as their primary siderophore for iron acquisition under iron-limiting conditions. Here, we report the identification of a random transposon mutant IST3 of Pseudomonas putida S11 showing tolerance to iron starvation stress condition and increased pyoverdine production. The insertion of the Tn5 transposon was found to be in pstS gene of pstSR operon encoding sensor histidine kinase protein of the two-component signal transduction system. A pyoverdine negative derivative of IST3 mutant constructed was sensitive to iron stress condition. It indicated that increased survival of IST3 under iron-limiting condition was due to higher pyoverdine production. The iron starvation tolerant mutant (IST3) exhibited enhanced pyoverdine-mediated iron uptake in minimal medium which significantly improved its biofilm formation, seed adhesion and competitive root colonization.

Root colonization of a rice growth promoting strain of Enterobacter cloacae.

Enterobacter cloacae GS1 was isolated by in-planta enrichment of a rice rhizoplane bacterial community. It displayed strong seed adherence ability (2.5 × 10(5) cfu/seed) and colonized rice roots reaching up to 1.65 × 10(9) cfu/g of fresh root weight in a gnotobiotic root colonization system. E. cloacae GS1 was motile, able to solubilize tricalcium phosphate, and produced indole acetic acid like substances (15 µg/ml). As an introduced bioinoculant in non-sterile soil, E. cloacae GS1 colonized rice roots and significantly improved the fresh weight, root length, shoot length, and nitrogen content in inoculated rice seedlings as compared to uninoculated controls. This isolate was tagged with green fluorescent protein and various stages of root colonization in gnotobiotic hydroponic environment and non-sterile soil environment were followed by fluorescence microscopy. Owing to its effective root colonizing ability and growth promoting potential, Enterobacter cloacae GS1 is a promising symbiotic bioinoculant for rice.