Multidrug-resistant Clinical Strains Research Articles

Green Synthesis, Characterization and Antimicrobial Activity of Copper Oxide Nanomaterial Derived from Momordica charantia.

BackgroundIn the emerging field of nanotechnology, copper oxide (CuO) nanomaterials are considered to be one of the most important transition metal oxides owing to its fascinating properties. Its synthesis from green chemistry principles is gaining importance as next-generation antibiotics due to its simplicity, eco-friendliness, and cost-effectiveness. In the present study, CuO nanorods (CuO NRs) were synthesized from the aqueous fruit extract of Momordica charantia and characterized using different analytical techniques. Further, the biomedical therapeutic potential was evaluated against multi-drug resistant microbial strains.Materials and MethodsTo synthesize CuO NRs, 0.1M of CuSO4.5H2O solution was added to aqueous extract of Momordica charantia in a 1:3 (v/v) ratio (pH=11) and heated at 50°C followed by washing and drying. The synthesized CuO NRs were subjected to characterization using different analytical techniques such as UV visible spectroscopy, zeta sizer equipped with zeta potential, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), scanning electron microscopy (SEM) equipped with energy-dispersive X-ray spectroscopy (EDS) and transmission electron microscopy (TEM). Further, the application as a biomedical therapeutic potential was evaluated in vitro using well diffusion method against eleven multidrug-resistant clinical bacterial strains, a fungus- Trichophyton rubrum and in ovo against the R2B virus using haemagglutination (HA) test.ResultsCharacterization was preliminarily done by the spectral study that confirms the absorbance band at 245nm. FTIR analysis at 628 cm−1 peak identified copper oxide vibration. SEM analysis revealed agglomerated particle clusters. However, with TEM clear nanorods of average diameter of 61.48 ± 2 nm were observed. EDAX confirmed CuO formation while XRD showed a typical monoclinic structure with 6 nm crystallite size. Biological screening of CuO NRs showed significant results against both in vitro and in ovo methods. Significant inhibitory activity (p<0.0001) was noted against most of the resistant human pathogenic strains including both Gram-positive and Gram-negative bacteria. The highest efficacy was observed against Bacillus cereus with a 31.66 mm zone of inhibition. Besides, the therapeutic potential of CuO NRs against Corynebacterium xerosis, Streptococcus viridians and R2B strain of Newcastle disease is reported for the first time.ConclusionBased on the present results, it could be expected that green synthesized CuO NRs would find potential applications in the field of nanomedicine.

Environmentally Benign Nanoantibiotics with a Built-in Deactivation Switch Responsive to Natural Habitats.

The massive use of antibiotics in healthcare and agriculture has led to their artificial accumulation in natural habitats, which risks the structure and function of the microbial communities in ecosystems, threatens food and water security, and accelerates the development of resistome. Ideally, antibiotics should remain fully active in clinical services while becoming deactivated rapidly once released into the environment, but none of the current antibiotics meet this criterion. Here, we show a nanoantibiotic design that epitomizes the concept of carrying a built-in "OFF" switch responsive to natural stimuli. The environmentally benign nanoantibiotics consist of cellulose backbones covalently grafted with hydrophilic polymer brushes that by themselves are antimicrobially inactive. In their nanostructured forms in services, these cellulose-based polymer molecular brushes are potent killers for both Gram-positive and Gram-negative bacteria, including clinical multidrug-resistant strains; after services and being discharged into the environment, they are shredded into antimicrobially inactive pieces by cellulases that do not exist in the human body but are abundant in natural habitats. This study illuminates a new concept of mitigating the environmental footprints of antibiotics with rationally designed nanoantibiotics that can be dismantled and disabled by bioorthogonal chemistry occurring exclusively in natural habitats.

Anti-oxidant, antibacterial, anti-biofilm, and anti-quorum sensing activities of four essential oils against multidrug-resistant bacterial clinical isolates

Purpose of the studyOutbreaks of multidrug-resistant bacteria are increasingly reported at the clinical setting. The antimicrobial, anti-biofilm, anti-quorum sensing, and anti-oxidant activities of four essential oils extracted from Cinnamomum verum, Origanum majorana, Thymus vulgaris, and Eugenia caryophyllata against Gram-positive and Gram-negative multidrug-resistant bacteria were evaluated in vitro. Materials and methodsThis study was conducted on 105 multidrug resistant clinical strains. Inhibition diameter zone, minimum inhibitory concentration, and minimum bactericide concentration of the oils were determined using agar disc diffusion method and microdilution. The ability of the 4 essential oils to inhibit the production of bacterial biofilms was tested on polystyrene plates, as well as their inhibitory effect on the production of violacein by Chromobacterium violaceum CV026. The anti-oxidant activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl scavenging method. ResultsEssential oils of Cinnamomum verum, Thymus vulgaris and Eugenia caryophyllata showed an important antibacterial activity. The inhibition diameter zone was higher than 20 mm for 90.24 %, 85.71 % and 60.95 % of strains respectively.These essential oils have a remarkable anti-biofilm and anti-quorum sensing activities against almost all the species studied. Clove extract revealed the highest anti-oxidant activity (Pourcentage of inhibtion of DPPH = 90.3 %). ConclusionThese results supported the use of the 4 essential oils as alternative or complementary agents to treat infections caused by multidrug-resistant bacteria, and to prevent biofilm formation and quorum sensing signaling. They might be used as a safe anti-oxidants instead of harmful artificial ones.

Discovery of a novel and selective fungicide that targets fungal cell wall to treat dermatomycoses: 1,3-bis(3,4-dichlorophenoxy)propan-2-aminium chloride.

Fungal infections are highly prevalent and are responsible for high rates of morbidity and mortality. In this context, the search for new treatment alternatives is very relevant. Analyse chemical compounds for antifungal potential against dermatomycosis fungi. The antifungal activity of 121 compounds, intermediates or derivatives of 1,3-bis(aryloxy)propane substituted at C-2 (111 compounds) and isothiouronium derivatives (10 compounds) was investigated through susceptibility tests, mechanism of action, toxicity and hydrogel incorporation. The compound 1,3-bis(3,4-dichlorophenoxy)propan-2-aminium chloride (2j) was the most active fungicide against dermatophytes and Candida spp., at very low concentrations (0.39-3.12µg/mL), including action on resistant and multidrug-resistant clinical strains. Compound 2j has presented a promising toxicity profile, showing selectivity index >10, relative to human lymphocytes. The compound was classified as non-irritant by the HET-CAM test and did not cause histopathological alterations in pig ear skin, thus presenting an excellent perspective for topical application. 2j targets the fungal cell wall, which was confirmed by scanning electron microscopy, which also indicated the additional ability of 2j to inhibit the Candida albicans pseudohyphae formation and biofilm of Microsporum canis. Compound 2j was incorporated in a hydrogel with bioadhesive potential. The results of the human skin permeation showed that 2j remained significantly in the epidermis, ideally for the dermatomycosis treatment. Therefore, the compound 2j demonstrated the potential for antifungal drug development, with a action mechanism elucidated and already applied in a semisolid formulation as a new therapeutic option for fungal skin infections.

Tween 80 induces a carbon flux rerouting in Mycobacterium tuberculosis

As a means to increase the growth rate and reduce aggregation, Tween 80 is routinely added to growth media during mycobacterial culturing. This detergent has, however, been associated with causing alterations to the morphology, pathogenicity and virulence of these bacteria. In an attempt to better understand the underlying mechanism of these alterations, we investigated the effect of Tween 80 on the metabolomes of a M. tuberculosis lab strain (H37Rv) and multidrug-resistant clinical strain (R179), using GC-GCxTOF-MS metabolomics. The metabolite markers identified indicated Tween 80-induced disparities in the central carbon metabolism of both strains, with an upregulation in the glyoxylate cycle, glucogenogenesis and the pentose phosphate pathway. The results also signified an increased production of mycobacterial biosynthetic precursors such as triacylglycerols, proteinogenic amino acids and nucleotide precursors, in the presence of the detergent. Collectively, these metabolome variations mimic the phenotypic changes observed when M. tuberculosis is grown in vivo, in a lipid rich environment. However, in addition to the increased availability of oleic acid as a carbon source from Tween 80, the observed variations, and the morphological changes associated with the detergent, could also be a result of an overall stress response in these bacteria. This study is the first to identify specific metabolome variations related to the addition of Tween 80 to the growth media during M. tuberculosis culturing. The consideration of these results during the method development and data interpretation phases of future metabolomics investigations will improve the quality of the analyses as well as the credibility of potential research outcomes. These results will also assist in the interpretation of research questions specifically aimed at aspects of mycobacterial metabolism, even when using other methodologies such as transcriptomics or fluxomics.

Short tryptophan- and arginine-rich peptide shows efficacy against clinical methicillin-resistant Staphylococcus aureus strains isolated from skin and soft tissue infections

In recent years methicillin-resistant Staphylococcus aureus has posed a challenge in treating skin and soft tissue infections. Finding new antimicrobial agents has therefore become imperative. We evaluated the in vitro antimicrobial activity of a synthetic peptide, P6, against multidrug resistant clinical strains of Staphylococcus aureus isolated from skin and soft tissue infections. The P6 antimicrobial effect was evaluated in vitro by determining MIC/MBC, the ratio of live/dead cells and the effects induced at membrane level. The therapeutic efficiency was determined against human skin cells. P6 inhibited growth for all strains between 8 and 16 mg/L and killed all bacterial strains at 16 mg/L. The therapeutic potential was found to be 30 and 15 in the presence of BSA. We showed that P6 localizes at membrane level, where it acts slowly, by depolarizing it and affecting its integrity. P6 can be considered a good candidate for use as an antimicrobial agent in topical applications.

Design, synthesis, and evaluation of novel 2-substituted 1,4-benzenediol library as antimicrobial agents against clinically relevant pathogens

Development of new antimicrobial agents, capable of combating resistant and multidrug-resistant fungal and bacterial clinical strains, is necessary. This study presents the synthesis and antimicrobial screening of 42 2-substituted-1,4-benzenediols, being 10 novel compounds. In total, 23 compounds showed activity against fungi and/or bacteria. Benzenediol compounds 2, 5, 6, 8, 11, and 12 demonstrated broad spectrum antimicrobial actions, including resistant and multidrug-resistant species of dermatophytes (Trichophyton mentagrophytes), Candida spp. and the ESKAPE panel of bacteria. Minimum inhibitory concentrations of these compounds for fungi and bacterial strains ranged from 25 to 50 µg/ml and 8–128 µg/ml, respectively. The antifungal mechanism of action is related to the fungal cell wall of dermatophytes and membrane disruption to dermatophytes and yeasts, in the presence of compound 8. Specific structural changes, such as widespread thinning along the hyphae and yeast lysis, were observed by scanning electron microscopy. The effects of compound 8 on cell viability are dose-dependent; however they did not cause genotoxicity and mutagenicity in human leukocyte cells nor haemolysis. Moreover, the compounds were identified as nonirritant by the ex-vivo Hen’s egg test-chorioallantoic membrane (HET-CAM). The furan-1,4-benzenediol compound 5 showed in vivo efficacy to combat S. aureus infection using embryonated chicken eggs. Therefore, the compounds 8, and 5 are promising as hits for the development of new antimicrobial drugs with reduced toxicity.

Genomic analysis of a multidrug-resistant clinical Providencia rettgeri (PR002) strain with the novel integron ln1483 and an A/C plasmid replicon.

Whole-genome sequence analysis was performed on a multidrug-resistant Providencia rettgeri PR002 clinical strain isolated from the urine of a hospitalized patient in Pretoria, South Africa, in 2013. The resistome, mobilome, pathogenicity island(s), as well as virulence and heavy-metal resistance genes of the isolate, were characterized using whole-genome sequencing and bioinformatic analysis. PR002 had a genome assembly size of 4,832,624bp with a GC content of 40.7%, an A/C2 plasmid replicase gene, four integrons/gene cassettes, 17 resistance genes, and several virulence and heavy metal resistance genes, confirming PR002 as a human pathogen. A novel integron, In1483, harboring the gene blaOXA-2 , was identified, with other uncharacterized class 1 integrons harboring aacA4cr and dfrA1. Aac(3')-IIa and blaSCO-1 , as well as blaPER-7 , sul2, and tet(B), were found bracketed by composite Tn3 transposons, and IS91, IS91, and IS4 family insertion sequences, respectively. PR002 was resistant to all antibiotics tested except amikacin, carbapenems, cefotaxime-clavulanate, ceftazidime-clavulanate, cefoxitin, and fosfomycin. PR002 was closely related to PR1 (USA), PRET_2032 (SPAIN), DSM_1131, and NCTC7477 clinical P. rettgeri strains, but not close enough to suggest it was imported into South Africa from other countries. Multidrug resistance in P. rettgeri is rare, particularly in clinical settings, making this case an important incident requiring urgent attention. This is also the first report of an A/C plasmid in P. rettgeri. The array, multiplicity, and diversity of resistance and virulence genes in this strain are concerning, necessitating stringent infection control, antibiotic stewardship, and periodic resistance surveillance/monitoring policies to preempt further horizontal and vertical spread of these resistance genes.

Multidrug-resistant Acinetobacter baumannii strains with NDM-1: Molecular characterization and in vitro efficacy of meropenem-based combinations.

Acinetobacter baumannii is an important cause of hospital-acquired, multidrug-resistant (MDR) infections occurring worldwide. Anti-microbial combination regimens may be the only feasible treatment option for affected patients. In the present study, the efficacy of the combined therapy of meropenem with colistin, ampicillin-sulbactam, tazobactam and vancomycin against clinical strains of MDR A. baumannii was determined. Anti-microbial susceptibility testing was performed and resistance genes were characterized by a multiplex polymerase chain reaction (PCR)-reverse line blot assay. The genetic background of New Delhi metallo-β-lactamase 1 (NDM-1) was analysed by primer walking. The presence of NDM-1 was detected using the modified Hodge test and the EDTA-combined disk test. To screen for synergistic drug effects, the fractional inhibitory concentration index was calculated using a checkerboard assay. The results of the PCR as well as the sequence analyses suggested that NDM-1 was located downstream of the ISAba125 element. In addition, a synergistic effect was determined for meropenem + vancomycin, meropenem + tazobactam and meropenem + ampicillin + sulbactam in two strains each, and in four strains for meropenem + colistin. A total of five A. baumannii strains with resistance to numerous antibiotics and carrying numerous resistance genes were identified. In the strains of A. baumannii, the NDM-1 gene was integrated in a transposon structure with a copy of the ISAba125 insertion sequence. However, the genetic background was not identical among the different species and strains. The genetic variability of NDM-1 may facilitate the rapid dissemination of this gene. In conclusion, meropenem may enhance the efficacy of antibiotics in A. baumannii strains with NDM-1-associated MDR.

Restoring the activity of the antibiotic aztreonam using the polyphenol epigallocatechin gallate (EGCG) against multidrug-resistant clinical isolates of Pseudomonas aeruginosa.

Introductio n. Pseudomonas aeruginosa is an important Gram-negative pathogen that is intrinsically multidrug-resistant (MDR) and frequently associated with healthcare-associated outbreaks. With increasing resistance to antibiotics and with very few novel drugs under development, clinicians often use combinations to treat critically ill patients.Aim. The aim of this study was to evaluate the ability of epigallocatechin (EGCG) to restore the activity of aztreonam against clinical MDR strains of P. aeruginosa.Methodology. Checkerboard and time-kill kinetic assays were performed to assess synergy in vitro and the Galleria mellonella model of infection was used to test the efficacy of the combination in vivo. Accumulation assays were performed to gain insight into the mechanism of action.Results. The results demonstrate that synergy between aztreonam and EGCG exists [fractional inhibitory concentration indices (FICIs) 0.02-0.5], with the combination affording significantly (P=<0.05) enhanced bacterial killing, with a >3 log10 reduction in colony-forming units ml-1 at 24 h. EGCG was able to restore susceptibility to aztreonam to a level equal to or below the breakpoint set by the European Committee for Antimicrobial Susceptibility Testing. In G. mellonella, the combination was superior to monotherapy, with increased larval survival observed (94 % vs ≤63 %). We also demonstrated the relatively low toxicity of EGCG to human keratinocytes and G. mellonella larvae. Accumulation assay data suggest that the mechanism of synergy may be due to EGCG increasing the uptake of aztreonam.Conclusion. EGCG was able to restore the activity of aztreonam against MDR P. aeruginosa. The data presented support further evaluation of the aztreonam-EGCG combination and highlight its potential for use in clinical medicine.

Comparative metabolomics shows the metabolic profiles fluctuate in multi-drug resistant Escherichia coli strains

In this study, two susceptible strains and two multi-drug resistant clinical Escherichia coli strains were obtained by Kirby-Bauer method, and then a GC–MS-based metabolomics method was used to compare the differential expression of metabolites between two drug sensitive (CK1 and CK2) and two multidrug-resistant (MDR1 and MDR2) clinical strains of E. coli. We characterized a total of 273 metabolites, including 77 commonly altered metabolites, between MDR vs. antibiotic sensitive strains. Interestingly, the PCA score plot clearly discriminated drug sensitive and MDR strains. The following bioinformatics analysis showed that biosynthesis of amino acids, biosynthesis of phenylpropanoids and purine metabolism were commonly enriched in MDR strains. Moreover, microbial metabolism in diverse environments, carbon metabolism，and pyrimidine metabolism pathways were more likely to be enriched MDR1 strain, while ABC transporters, and cysteine and methionine metabolism pathways were enriched in MDR2 strains. The enzyme activities in several involved metabolic pathways were further measured and metabolite candidates were validated by GC–MS-SIM method. These results indicated that antibiotic resistance affects the metabolite profiles of bacteria. In general, our study provides evidence on the study and prediction of MDR characteristics and mechanisms in bacteria at the metabolite level. Biological significanceOveruse and abuse of antibiotics has led to the emergence of antibiotic-resistant strains of bacteria; however, relatively little is known about their resistance mechanisms. In this study, metabolomics method was used to compare the differential expression of metabolites between sensitive and multidrug-resistant clinical strains of E. coli. Results show that the PCA score plot clearly discriminated sensitive and MDR strains, indicating that they had different metabolic profiles. Further, bioinformatics analysis showed that biosynthesis of amino acids, biosynthesis of phenylpropanoids and purine metabolism may be related to resistance. Finally, the enzyme activities in several involved metabolic pathways were further measured and metabolite candidates were validated by GC–MS-SIM method. In general, our study provides evidence on the study and prediction of MDR characteristics and mechanisms in bacteria at the metabolite level.

Ts2631 Endolysin from the Extremophilic Thermus scotoductus Bacteriophage vB_Tsc2631 as an Antimicrobial Agent against Gram-Negative Multidrug-Resistant Bacteria.

Bacteria that thrive in extreme conditions and the bacteriophages that infect them are sources of valuable enzymes resistant to denaturation at high temperatures. Many of these heat-stable proteins are useful for biotechnological applications; nevertheless, none have been utilized as antibacterial agents. Here, we demonstrate the bactericidal potential of Ts2631 endolysin from the extremophilic bacteriophage vB_Tsc2631, which infects Thermus scotoductus, against the alarming multidrug-resistant clinical strains of Acinetobacter baumannii, Pseudomonas aeruginosa and pathogens from the Enterobacteriaceae family. A 2–3.7 log reduction in the bacterial load was observed in antibacterial tests against A. baumannii and P. aeruginosa after 1.5 h. The Ts2631 activity was further enhanced by ethylenediaminetetraacetic acid (EDTA), a metal ion chelator (4.2 log reduction in carbapenem-resistant A. baumannii) and, to a lesser extent, by malic acid and citric acid (2.9 and 3.3 log reductions, respectively). The EDTA/Ts2631 combination reduced all pathogens of the Enterobacteriaceae family, particularly multidrug-resistant Citrobacter braakii, to levels below the detection limit (>6 log); these results indicate that Ts2631 endolysin could be useful to combat Gram-negative pathogens. The investigation of A. baumannii cells treated with Ts2631 endolysin variants under transmission electron and fluorescence microscopy demonstrates that the intrinsic antibacterial activity of Ts2631 endolysin is dependent on the presence of its N-terminal tail.

Whole-genome analysis of EC129, an NDM-5-, CTX-M-14-, OXA-10- and MCR-1-co-producing Escherichia coli ST167 strain isolated from Japan.

The emergence and spread of carbapenemase-producing Enterobacteriaceae is a worldwide concern. This study reports the whole genome sequence of an NDM-5-, CTX-M-14-, OXA-10- and MCR-1-co-producing Escherichia coli sequence type 167 (ST167) multidrug-resistant clinical strain (EC129) isolated from a sputum sample of a hospitalised patient diagnosed with pneumonia. The genome of E. coli EC129 was subjected to next-generation sequencing and reads were assembled. The draft genome was annotated using DDBJ Read Annotation Pipeline DFAST server, followed by subsequent in silico analysis. The genome of E. coli ST167 strain EC129 is 5319159 bp in length and contains 5022 protein-coding sequences. The blaNDM-5, blaCTX-M-14, blaOXA-10 and mcr-1 genes were detected along with other antimicrobial resistance genes conferring resistance to aminoglycosides, fluoroquinolones, sulfonamides, trimethoprim and tetracyclines. Antimicrobial susceptibility testing revealed that the isolate was resistant to all antimicrobial agents except colistin. To our knowledge, this study is the first to report anE. coli ST167 strain co-producing NDM-5, CTX-M-14, OXA-10 and MCR-1 isolated from a sputum sample of an individual with pneumonia in Japan, thus elucidating the molecular characteristics and resistance gene diversity of this strain.

Circulation of imipenem-resistant Acinetobacter baumannii ST10, ST2 and ST3 in a university teaching hospital from Tehran, Iran.

Multi-drug resistant (MDR) Acinetobacter baumannii has introduced a worldwide health crisis. The purposes of this study were to characterize the clonal relatedness among MDR clinical strains and to introduce a new two-locus typing method confirmed by multi-locus sequence typing (MLST). In this study, we determined antimicrobial resistance, detected genes associated with carbapenem resistance and characterized clonal relatedness among 99 clinical isolates extracted from 82 hospitalized inpatients in a university hospital. Of the 99 A. baumannii isolates, 92.9% (92/99) were resistant to imipenem and 97.9% (97/99) had an MDR profile. We found that the high prevalence of blaVIM [94.9% (94/99)] and blaOXA-23-like [93.93% (93/99)] is the main mechanism of carbapenem resistance. This study proposes a new two-locus typing (blaOXA-51-like and ampC) method for the rapid identification of clonal complexes (CCs). The results of this method and confirmation by MLST show that clinical isolates carry blaOXA-68 as well as ampC-10 or ampC-20 genes belonging to CC10 (ST10); blaOXA-66 and ampC-2 belonging to CC2 (ST2); and blaOXA-71 and ampC-3 belonging to CC3 (ST3). One isolate had blaOXA-90 with an undetermined allele number of ampC belonging to ST513. The high prevalence of MDR strains and the circulation of four limited clones, including ST10 (45/99), ST2 (41/99), ST3 (12/99) and ST513 (1/99), in the clinical setting highlights the importance of a rigorous infection control programme. The two-locus typing method has more discrimination than the application of each method separately and it could be applied for the rapid determination of the CC without performing MLST.

Characterization of myophage AM24 infecting Acinetobacter baumannii of the K9 capsular type.

In the present study, we investigate the biological properties and genomic organization of virulent bacteriophage AM24, which specifically infects multidrug-resistant clinical Acinetobacterbaumannii strains with a K9 capsular polysaccharide structure. The phage was identified as a member of the family Myoviridae by transmission electron microscopy. The AM24 linear double-stranded DNA genome of 97,177bp contains 167 open reading frames. Putative functions were assigned for products of 40 predicted genes, including proteins involved in nucleotide metabolism and DNA replication, packaging of DNA into the capsid, phage assembly and structural proteins, and bacterial cell lysis. The gene encoding the tailspike, which possesses depolymerase activity towards the corresponding capsular polysaccharides, is situated in the phage genome outside of the structural module, upstream of the genes responsible for packaging of DNA into the capsid. The data on characterization of depolymerase-carrying phage AM24 contributes to our knowledge of the diversity of viruses infecting different capsular types of A.baumannii.

In vitro activities of ceftazidime/avibactam alone or in combination with antibiotics against multidrug-resistant Acinetobacter baumannii isolates.

Infections caused by multidrug-resistant (MDR) Acinetobacter baumannii (A. baumannii) are a growing problem because of the limited options for treatment. The number of antimicrobials that are currently being developed is still insufficient to control this global threat. Combination therapies of antibiotics with different antimicrobial mechanisms have been proposed as the best options for treating MDR A. baumannii infections. The objective of this study was to investigate the in-vitro effectiveness of ceftazidime/avibactam alone or in combination with antibiotics against MDR A. baumannii isolates using time-kill assays. Forty clinical MDR strains were screened, and minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of ceftazidime/avibactam, colistin, levofloxacin, meropenem, tigecycline, and tobramycin were determined by microbroth dilution method. The in-vitro synergistic activities of ceftazidime/avibactam with antibiotic combinations were determined by time-kill assays at 1× MIC and 4× MIC against five MDR A. baumannii isolates. Based on MIC results, all isolates of A. baumannii were resistant to ceftazidime/avibactam, except for AB-5. All isolates were found to be resistant to meropenem and levofloxacin. At 4× MIC, all of the tested antibiotics showed bactericidal effect (≥3log10killing). The synergistic activities of ceftazidime/avibactam+colistin, ceftazidime/avibactam+tobramycin and ceftazidime/avibactam+tigecycline combinations at 1× MIC were observed against studied 5/5, 4/5 and 4/5 strains, respectively. Furthermore, all of the tested combinations at 4× MIC were additive at 24h. No antagonism was observed. The findings of this study suggest that a significant bactericidal effect was seen with all tested combinations. These findings present significant implications for antibiotic choice for the treatment of infections caused by MDR A. baumannii.

Studies on the mechanism of multidrug resistance of Acinetobacter baumannii by proteomic analysis of the outer membrane vesicles of the bacterium

Acinetobacter baumannii, an opportunistic pathogen is responsible for pneumonia, meningitis and a wide range of other infections. It is typically involved in nosocomial infections. Outer membrane vesicles (OMVs) released from the bacteria play an important role in the bacterial physiology including pathogenesis. In this study, OMVs were isolated and characterized form sensitive and multidrug-resistant clinical strains of A. baumannii. A comparative proteomics analysis of the OMVs from the A. baumannii sensitive and MDR strain revealed the increased content of carbapenemase and a host of other related antibiotic resistant enzymes in the OMVs of the A. baumannii MDR strain. The OMVs of antibiotic-sensitive strain contained only 8 antibiotic resistance-conferring proteins, whereas the OMVs of multidrug resistance contained 24 proteins. Growth studies on the sensitive and MDR strain in the presence of polymyxin B and OMVs prepared from the respective strains revealed that the OMVs protected the bacteria against the antibiotic. Bacteria were grown in the presence of antibiotic and an indole derivative has shown that the minimum inhibitory concentration was reduced in the presence of derivative indicating that the derivative regulated the production of OMVs. Interestingly, it was also found that the degradosome complex involved in RNA processing was associated with the OMVs. These studies reveal that OMVs may play an important role in antibiotic resistance/persistence.

Anti-mycobacterial activity of some medicinal plants used traditionally by tribes from Madhya Pradesh, India for treating tuberculosis related symptoms

Ethnopharmacological relevanceTuberculosis (TB) is one of the highly infectious disease caused by the bacterium Mycobacterium tuberculosis (Mtb). Several medicinal plants are traditionally used by the tribal healers of some tribal pockets of Madhya Pradesh (M.P.), India in the treatment of various ailments including TB related symptoms. The information of traditional knowledge of plants is empirical lacking systematic scientific investigation. Aim of the studyTo provide a scientific rationale for the traditional uses of some medicinal plants in treating prolonged cough, chest complaints and TB by ethnic groups in four districts of the state of M.P., India. The anti-mycobacterial activity of these plants was also investigated against multidrug-resistant (MDR) strains of Mtb. The active plant extracts were also assessed for general cytotoxicity by human monocytic leukemia cell line, THP-1 macrophages. Materials and methodsSemi structured interviews and guided field-walk methods were used to gather information on medicinal plants used by the tribal traditional healers. The study was carried out in the tribal pockets of Anuppur, Mandla, Umariya and Dindori districts of M.P., India. A total of 35 plant species distributed in 22 families were selected based on folklore uses suggested by the tribal traditional healer. The ethanol extracts of plants were evaluated for anti-mycobacterial activity against Mtb H37Rv and six MDR clinical isolates of Mtb. The anti-mycobacterial activity of plants was determined in terms of minimum inhibitory concentration (MIC) using standard resazurin microtitre plate assay (REMA). The plant extracts found to be active against Mtb were further evaluated for general cytotoxicity against human THP-1 macrophages using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. The selectivity index (SI) of active plant extracts were also calculated on the basis of MIC and cytotoxicity. ResultsOut of the 35 plants, 11 plant species showed anti-mycobacterial activity with MIC ranging from 500 to 31.25 µg/mL against Mtb H37Rv. The plant extracts also exhibited anti-mycobacterial activity against six multi-drug resistant (MDR) clinical strains of Mtb isolated from sputum samples of pulmonary TB patients. In vitro cytotoxicity of active plant extracts was also assessed in human THP-1 macrophages. The IC50 (50% inhibitory concentration) values of most of the plant extracts on THP-1 was found to be higher than MIC values against Mtb, indicating that the THP-1 cells are not adversely affected at concentrations that are effective against Mtb. Significant anti-mycobacterial activity was observed for eleven plants viz., Alstonia scholaris (L.) R. Br., Glycyrrhiza glabra L., Holorrhena antidysentrica (Roth) Wall. exA.DC., Mallotus philippensis (Lam.) Müll. Arg., Eulophia nuda Lindl., Cocculus hirsutus (L.) Diels, Pueraria tuberosa (Willd.) DC., Cyperus rotundus L., Curcuma caesia Roxb., Sphaeranthus indicus L. and Plumbago zeylanica L. which lends support to their traditional uses. ConclusionThe present investigation supports the potential role of plants used by tribal healers as our results have shown that these plants exhibit anti-mycobacterial activity in the acceptable range against Mtb. Our study clearly lends support to the traditional uses of some plants in TB related symptoms as we have found them to exhibit significant in vitro anti-TB activity. To the best of our knowledge, anti-mycobacterial activity in plants against Mtb is being reported for the first time from tribal areas of M.P., India by the current study.

Draft genome sequence of an NDM-5, CTX-M-15 and OXA-1 co-producing Escherichia coli ST167 clinical strain isolated from a urine sample.

Escherichia coli is the leading cause of urinary tract infections worldwide. Carbapenemase-producing clinical isolates leave few therapeutic options. Here we report the draft genome sequence of an NDM-5, CTX-M-15, and OXA-1 co-producing E. coli sequence type 167 (ST167) multidrug-resistant clinical strain (ECWJ1) isolated from a urine sample of a hospitalised male patient diagnosed with intracranial injury from a road traffic accident. The genome of E. coli ECWJ1 was sequenced using an Illumina HiSeq™ 4000 platform. The generated sequence reads were assembled using CLC Genomics Workbench. The draft genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) server, and further bioinformatics analysis was performed. Escherichia coli ECWJ1 belongs to ST167. The total genome length of the isolate is 4992753bp, with 4890 protein-coding sequences. The presence of blaNDM-5, blaCTX-M-15 and blaOXA-1 genes was detected in addition to other antimicrobial resistance genes conferring resistance to aminoglycosides, fluoroquinolones, sulphonamides, trimethoprim and tetracyclines. Antimicrobial susceptibility testing revealed, that besides tigecycline and colistin, this isolate is resistant to all antimicrobials tested. To our knowledge, this is the first report of an E. coli ST167 strain co-producing NDM-5, CTX-M-15 and OXA-1 isolated from urine sample in China. It may help us to understand the genomic features, molecular characteristics and resistance gene diversity of this bacterial pathogen. The genome sequence of E. coli ECWJ1 can be used as a reference sequence for comparative analysis of NDM-5-producing E. coli, aiming to reveal the mechanism of NDM-5 dissemination in this pathogen.

Isolation, characterization and efficacy of phage MJ2 against biofilm forming multi-drug resistant Enterobacter cloacae.

Biofilm is involved in a variety of infections, playing a critical role in the chronicity of infections. Enterobacter cloacae is a biofilm-forming and multi-drug-resistant (MDR) nosocomial pathogen leading to significant morbidity and mortality. This study aimed at isolation of a bacteriophage against MDR clinical strain of E. cloacae and its efficacy against bacterial planktonic cells and biofilm. A bacteriophage MJ2 was successfully isolated from wastewater and was characterized. The phage exhibited a wide range of thermal and pH stability and demonstrated considerable adsorption to host bacteria in the presence of CaCl2 or MgCl2. Transmission electron microscopy (TEM) showed MJ2 head as approximately 62 and 54nm width and length, respectively. It had a short non-contractile tail and was characterized as a member of the family Podoviridae [order Caudovirales]. The phage MJ2 was found to possess 11 structural proteins (12-150kDa) and a double-stranded DNA genome with an approximate size of 40kb. The log-phase growth of E. cloacae both in biofilm and suspension was significantly reduced by the phage. The E. cloacae biofilm was formed under different conditions to evaluate the efficacy of MJ2 phage. Variable reduction pattern of E. cloacae biofilm was observed while treating it for 4h with MJ2, i.e., biofilm under static conditions. The renewed media with intervals of 24, 72, and 120h showed biomass decline of 2.8-, 3-, and 3.5-log, respectively. Whereas, the bacterial biofilm formed with dynamic conditions with refreshing media after 24, 72, and 120h demonstrated decline in growth at 2.5-, 2.6-, and 3.3-log, respectively. It was, therefore, concluded that phage MJ2 possessed considerable inhibitory effects on MDR E. cloacae both in planktonic and biofilm forms.