DNA Nanoflower Eye Drops with Antibiotic-Resistant Gene Regulation Ability for MRSA Keratitis Target Treatment.

Methicillin-resistant Staphylococcus aureus (MRSA) biofilm-associated bacterial keratitis is highly intractable, with strong resistance to ß-lactam antibiotics. Inhibiting the MRSA resistance gene mecR1 to downregulate penicillin-binding protein PBP2a has been implicated in the sensitization of ß-lactam antibiotics to MRSA. However, oligonucleotide gene regulators struggle to penetrate dense biofilms, let alone achieve efficient gene regulation inside bacteria cells. Herein, an eye-drop system capable of penetrating biofilms and targeting bacteria for chemo-gene therapy in MRSA-caused bacterial keratitis is developed. This system employed rolling circle amplification to prepare DNA nanoflowers (DNFs) encoding MRSA-specific aptamers and mecR1 deoxyribozymes (DNAzymes). Subsequently, ß-lactam antibiotic ampicillin (Amp) and zinc oxide (ZnO) nanoparticles are sequentially loaded into the DNFs (ZnO/Amp@DNFs). Upon application, ZnO on the surface of the nanosystem disrupts the dense structure of biofilm and fully exposes free bacteria. Later, bearing encoded aptamer, the nanoflower system is intensively endocytosed by bacteria, and releases DNAzyme under acidic conditions to cleave the mecR1 gene for PBP2a down-regulation, and ampicillin for efficient MRSA elimination. In vivo tests showed that the system effectively cleared bacterial and biofilm in the cornea, suppressed proinflammatory cytokines interleukin 1ß ï¼ˆIL-1ß） and tumor neocrosis factor-alpha （TNF-α）, and is safe for corneal epithelial cells. Overall, this design offers a promising approach for treating MRSA-induced keratitis.

In-Silico molecular screening of natural compounds as a potential therapeutic inhibitor for Methicillin-resistant Staphylococcus aureus inhibition.

Methicillin-resistant Staphylococcus aureus (MRSA) is a life-threatening superbug causing infectious diseases such as pneumonia, endocarditis, osteomyelitis, etc. Conventional antibiotics are ineffective against MRSA infections due to their resistance mechanism against the antibiotics. The Penicillin Binding Protein (PBP2a) inhibits the activity of antibiotics by hydrolyzing the ß-lactam ring. Thus, alternate treatment methods are needed for the treatment of MRSA infections. Natural bioactive compounds exhibit good inhibition efficiency against MRSA infections by hindering its enzymatic mechanism, efflux pump system, etc. The present work deals with identifying potential and non-toxic natural bioactive compounds (ligands) through molecular docking studies through StarDrop software. Various natural bioactive compounds which are effective against MRSA infections were docked with the protein (6VVA). The ligands having good binding energy values and pharmacokinetic and drug-likeness properties have been illustrated as potential ligands for treating MRSA infections. From this exploration, Luteolin, Kaempferol, Chlorogenic acid, Sinigrin, Zingiberene, 1-Methyl-4-(6-methylhepta-1,5-dien-2-yl)cyclohex-1-ene, and Curcumin have found with good binding energies of -8.6 kcal/mol, -8.4 kcal/mol, -8.2 kcal/mol, -7.5 kcal/mol, -7.4 kcal/mol, -7.3 kcal/mol, and -7.2 kcal/mol, respectively.

In vitro and computational studies of the ß-lactamase inhibition and ß-lactam potentiating properties of plant secondary metabolites.

ß-lactam resistance in bacteria is primarily mediated through the production of ß-lactamases. Among the several strategies explored to mitigate the issue of ß-lactam resistance, the use of plant secondary metabolites in combination with existing ß-lactams seem promising. The present study aims to identify possible ß-lactam potentiating plant secondary metabolites following in vitro and in silico approaches. Among 180 extracts from selected 30 medicinal plants, acetone extract of Ficus religiosa (FRAE) bark recorded the least IC50 value of 3.9 mg/ml. Under in vitro conditions, FRAE potentiated the activity of ampicillin, which was evidenced by the significant reduction in IC50 values of ampicillin against multidrug resistant bacteria. Metabolic profiling following HR-LCMS analysis revealed the presence of diverse metabolites viz. flavonoids, alkaloids, terpenoids, etc. in FRAE. Further, ensemble docking of the FRAE metabolites against four Class A ß-lactamase (SHV1, TEM1, KPC2 and CTX-M-27) showed quercetin, taxifolin, myricetin, luteolin, and miquelianin as potential inhibitors with the least average binding energy. In molecular dynamic simulation studies, myricetin formed the most stable complex with SHV1 and KPC-2 while miquelianin with TEM1 and CTX-M-27. Further, all five metabolites interacted with amino acid residue Glu166 in Ω loop of ß-lactamase, interfering with the deacylation step, thereby disrupting the enzyme activity. The pharmacokinetics and ADMET profile indicate their drug-likeness and non-toxic nature, making them ideal ß-lactam potentiators. This study highlights the ability of metabolites present in FRAE to act as ß-lactamase inhibitors.Communicated by Ramaswamy H. Sarma.

Identification of natural inhibitor against L1 ß-lactamase present in Stenotrophomonas maltophilia.

Antibiotic resistance is threatening the medical industry in treating microbial infections. Many organisms are acquiring antibiotic resistance because of the continuous use of the same drug. Gram-negative organisms are developing multi-drug resistance properties (MDR) due to chromosomal level changes that occurred as a part of evolution or some intrinsic factors already present in the organism. Stenotrophomonas maltophilia falls under the category of multidrug-resistant organism. WHO has also urged to evaluate the scenario and develop new strategies for making this organism susceptible to otherwise resistant antibiotics. Using novel compounds as drugs can ameliorate the issue to some extent. The ß-lactamase enzyme in the bacteria is responsible for inhibiting several drugs currently being used for treatment. This enzyme can be targeted to find an inhibitor that can inhibit the enzyme activity and make the organism susceptible to ß-lactam antibiotics. Plants produce several secondary metabolites for their survival in adverse environments. Several phytoconstituents have antimicrobial properties and have been used in traditional medicine for a long time. The computational technologies can be exploited to find the best compound from many compounds. Virtual screening, molecular docking, and dynamic simulation methods are followed to get the best inhibitor for L1 ß-lactamase. IMPPAT database is screened, and the top hit compounds are studied for ADMET properties. Finally, four compounds are selected to set for molecular dynamics simulation. After all the computational calculations, withanolide R is found to have a better binding and forms a stable complex with the protein. This compound can act as a potent natural inhibitor for L1 ß-lactamase.

Whole genome sequencing snapshot of multi-drug resistant Klebsiella pneumoniae strains from hospitals and receiving wastewater treatment plants in Southern Romania.

We report on the genomic characterization of 47 multi-drug resistant, carbapenem resistant and ESBL-producing K. pneumoniae isolates from the influent (I) and effluent (E) of three wastewater treatment plants (WWTPs) and from Romanian hospital units which are discharging the wastewater in the sampled WWTPs. The K. pneumoniae whole genome sequences were analyzed for antibiotic resistance genes (ARGs), virulence genes and sequence types (STs) in order to compare their distribution in C, I and E samples. Both clinical and environmental samples harbored prevalent and widely distributed ESBL genes, i.e. blaSHV, blaOXA, blaTEM and blaCTX M. The most prevalent carbapenemase genes were blaNDM-1, blaOXA-48 and blaKPC-2. They were found in all types of isolates, while blaOXA-162, a rare blaOXA-48 variant, was found exclusively in water samples. A higher diversity of carbapenemases genes was seen in wastewater isolates. The aminoglycoside modifying enzymes (AME) genes found in all types of samples were aac(6'), ant(2'')Ia, aph(3'), aaD, aac(3) and aph(6). Quinolone resistance gene qnrS1 and the multi-drug resistance oqxA/B pump gene were found in all samples, while qnrD and qnrB were associated to aquatic isolates. The antiseptics resistance gene qacEdelta1 was found in all samples, while qacE was detected exclusively in the clinical ones. Trimethroprim-sulfamethoxazole (dfrA, sul1 and sul2), tetracyclines (tetA and tetD) and fosfomycin (fosA6, known to be located on a transpozon) resistance genes were found in all samples, while for choramphenicol and macrolides some ARGs were detected in all samples (catA1 and catB3 / mphA), while other (catA2, cmIA5 and aac(6')Ib / mphE and msrE) only in wastewater samples. The rifampin resistance genes arr2 and 3 (both carried by class I integrons) were detected only in water samples. The highly prevalent ARGs preferentially associating with aquatic versus clinical samples could ascribe potential markers for the aquatic (blaSHV-145, qacEdelta1, sul1, aadA1, aadA2) and clinical (blaOXA-1, blaSHV-106,blaTEM-150, aac(3)Iia, dfrA14, oqxA10; oqxB17,catB3, tetD) reservoirs of AR. Moreover, some ARGs (oqxA10; blaSHV-145; blaSHV-100, aac(6')Il, aph(3')VI, armA, arr2, cmlA5, blaCMY-4, mphE, msrE, oqxB13, blaOXA-10) showing decreased prevalence in influent versus effluent wastewater samples could be used as markers for the efficiency of the WWTPs in eliminating AR bacteria and ARGs. The highest number of virulence genes (75) was recorded for the I samples, while for E and C samples it was reduced to half. The most prevalent belong to three functional groups: adherence (fim genes), iron acquisition (ent, fep, fyu, irp and ybt genes) and the secretion system (omp genes). However, none of the genes associated with hypervirulent K. pneumoniae have been found. A total of 14 STs were identified. The most prevalent clones were ST101, ST219 in clinical samples and ST258, ST395 in aquatic isolates. These STs were also the most frequently associated with integrons. ST45 and ST485 were exclusively associated with I samples, ST11, ST35, ST364 with E and ST1564 with C samples. The less frequent ST17 and ST307 aquatic isolates harbored blaOXA-162, which was co-expressed in our strains with blaCTX-M-15 and blaOXA-1.

Genomic Characterization of a Multidrug-Resistant and Hypermucoviscous/Hypervirulent Klebsiella quasipneumoniae subsp. similipneumoniae ST4417 Isolated from a Sewage Treatment Plant.

Aim: Clinical strains of Klebsiella quasipneumoniae subsp. similipneumoniae have been reported worldwide. Multidrug-resistant (MDR) hypermucoviscous (hm)/hypervirulent (hv) lineages have become a global problem for public health worldwide. Therefore, this study aimed to characterize by whole-genome sequencing a MDR-hm/hv K. quasipneumoniae subsp. similipneumoniae SWT10 strain belonging to the new sequence type ST4417 isolated from a sewage treatment plant. Results: The SWT10 strain was recovered from a sewage treatment plant in Brazil and presented the hm and MDR phenotypes. Resistome analysis showed antimicrobial resistance genes associated with resistance to fluoroquinolones, ß-lactams, tetracyclines, trimethoprim, aminoglycosides, sulfonamides, macrolides, and fosfomycin as well as several heavy metal resistance genes. Virulome analysis showed virulence factors related to hv lineages. Multilocus sequence typing analysis revealed the new ST4417, which was grouped in CC1584 by the minimum-spanning tree. Besides, five plasmid incompatibility groups, two prophage-related sequences, and 66 genomic islands were detected. Conclusion: This study reports for the first time the genome sequence of a MDR-hm/hv K. quasipneumoniae subsp. similipneumoniae recovered from the environment, which contributes to a better understanding about these lineages as well as for surveillance studies worldwide.

Ten-Year Trends and Clinical Relevance of the Antimicrobial Resistance Genotype in Respiratory Isolates of Streptococcus pneumoniae.

BACKGROUND: Antimicrobial resistance of Streptococcus pneumoniae, especially against ß-lactam antibiotics, is a global concern. We aimed to analyze a 10-year trend in the antimicrobial resistance genotype of respiratory isolates of S. pneumoniae and to clarify whether resistance genotypes were correlated with phenotypic drug susceptibility, pathogenicity, and host clinical background. METHODS: Respiratory isolates of S. pneumoniae from 2003 to 2012 were analyzed with polymerase chain reaction for the presence of ß-lactam resistance gene mutations on pbp1a, pbp2x, and pbp2b. Sixty-eight strains isolated from different patients in 2012 were particularly analyzed for the association between genotypes and clinical data. RESULTS: The 10-year trend analysis showed a recent increase in gPRSP (genotypic penicillin-resistant S. pneumoniae) with all 3 ß-lactam resistance genes (from 21.7 to 35.3% in 3 years) and a steady level of gPSSP (genotypic penicillin-susceptible S. pneumoniae) without any ß-lactam resistance genes (13.2% in 2012). This resistance trend in genotypes was more prominent than resistance phenotypes determined with a drug susceptibility test. The probability of being a causative pathogen did not differ in gPSSP (55.6%), gPISP (genotypic penicillin-intermediate resistant S. pneumoniae; 54.3%), and gPRSP (54.2%). There was no significant difference in the ratio of patients who presented with respiratory failure in respiratory infection caused by gPSSP, gPISP, or gPRSP. Host clinical characteristics including age and gender were not different among resistance genotypes. CONCLUSIONS: There was no difference in pathogenicity or clinical background between gPSSP, gPISP, and gPRSP. Antimicrobial resistance in respiratory isolates of S. pneumoniae was more prevalent in genotypes than in phenotypes.

Identification of Mycobacterial Genes Involved in Antibiotic Sensitivity: Implications for the Treatment of Tuberculosis with ß-Lactam-Containing Regimens.

In a Mycobacterium smegmatis mutant library screen, transposon mutants with insertions in fhaA, dprE2, rpsT, and parA displayed hypersusceptibility to antibiotics, including the ß-lactams meropenem, ampicillin, amoxicillin, and cefotaxime. Sub-MIC levels of octoclothepin, a psychotic drug inhibiting ParA, phenocopied the parA insertion and enhanced the bactericidal activity of meropenem against Mycobacterium tuberculosis in combination with clavulanate. Our study identifies novel factors associated with antibiotic resistance, with implications in repurposing ß-lactams for tuberculosis treatment.

Discovery of potent wall teichoic acid early stage inhibitors.

The widespread emergence of methicillin-resistant Staphylococcus aureus (MRSA) has dramatically eroded the efficacy of current ß-lactam antibiotics and created an urgent need for novel treatment options. Using an S. aureus phenotypic screening strategy, we have identified small molecule early stage wall teichoic acid (WTA) pathway-specific inhibitors predicted to be chemically synergistic with ß-lactams. These previously disclosed inhibitors, termed tarocins, demonstrate by genetic and biochemical means inhibition of TarO, the first step in WTA biosynthesis. Tarocins demonstrate potent bactericidal synergy in combination with broad spectrum ß-lactam antibiotics across diverse clinical isolates of methicillin-resistant Staphylococci. The synthesis and structure-activity relationships (SAR) of a tarocin series will be detailed. Tarocins and other WTA inhibitors may provide a rational strategy to develop Gram-positive bactericidal ß-lactam combination agents active against methicillin-resistant Staphylococci.

Fate and behavior of extended-spectrum ß-lactamase-producing genes in municipal sewage treatment plants.

Extended-spectrum ß-lactamases (ESBLs) have the capability of hydrolyzing a variety of the newer ß-lactam antibiotics, including the third-generation cephalosporins and monobactams known as a rapidly evolving group of ESBLs. The purpose of this study was to investigate the occurrence and fate of ß-lactamase producing genes (CTX-M type 1, type2, CTX-M probe for all groups except CTX-M-1, and TEM, SHV, OXA) through wastewater treatment utilities. ß-lactamase producing genes in influent, digested sludge, activated sludge, and disinfected effluent were monitored. The results showed that influent contained high level of all target genes, and all CTX-M types, SHV, and OXA gene decreased significantly in biological treatment process such as activated sludge process and anaerobic digestion, however, TEM type was not effectively eliminated. Possibly, host microbes of TEM could be most resistant in target genes or to some extent gene transfer occurred in wastewater treatment processes. All target genes were significantly reduced during disinfection. Consequently, wastewater treatment process apparently reduced host microbes carrying ß-lactamase producing genes effectively, although they are selectively removed in biological processes. In addition, the significant reduction during disinfection was shown, although slightly differences of removal efficiency were observed in resistance.

Discovery of wall teichoic acid inhibitors as potential anti-MRSA ß-lactam combination agents.

Innovative strategies are needed to combat drug resistance associated with methicillin-resistant Staphylococcus aureus (MRSA). Here, we investigate the potential of wall teichoic acid (WTA) biosynthesis inhibitors as combination agents to restore ß-lactam efficacy against MRSA. Performing a whole-cell pathway-based screen, we identified a series of WTA inhibitors (WTAIs) targeting the WTA transporter protein, TarG. Whole-genome sequencing of WTAI-resistant isolates across two methicillin-resistant Staphylococci spp. revealed TarG as their common target, as well as a broad assortment of drug-resistant bypass mutants mapping to earlier steps of WTA biosynthesis. Extensive in vitro microbiological analysis and animal infection studies provide strong genetic and pharmacological evidence of the potential effectiveness of WTAIs as anti-MRSA ß-lactam combination agents. This work also highlights the emerging role of whole-genome sequencing in antibiotic mode-of-action and resistance studies.

Engineered Amp C ß-lactamase as a fluorescent screening tool for class C ß-lactamase inhibitors.

Class C ß-lactamases mediate antibiotic resistance in bacteria by efficiently hydrolyzing a broad range of ß-lactam antibiotics. With their clinical significance and the lack of commercially available effective inhibitors, development of class C ß-lactamase inhibitors has become one of the recent hot issues in the pharmaceutical industry. In this paper, we report the protein engineering of a fluorescent Amp C ß-lactamase mutant designated as V211Cf for the in vitro screening of class C ß-lactamase inhibitors. When a fluorescein (f) was incorporated at the entrance of the enzyme's active site (position 211), Amp C ß-lactamase from Enterobacter cloacae P99 was tailor-made into a novel fluorescent biosensing protein that could display a fluorescence enhancement upon binding with its ß-lactam substrates/inhibitors. With its catalytic activity close to the wild-type level, V211Cf can act as a "natural" fluorescent drug target for screening small binding molecules. In addition, V211Cf can allow specific detection for its active-site binding molecules and discriminate them from nondruglike molecules in the screen. Furthermore, V211Cf is amenable to a high throughput format. Taken together, V211Cf demonstrates the potential as an efficient tool for screening class C ß-lactamase inhibitors and facilitates the discovery of therapeutics that can combat the clinically important class C ß-lactamases.

Positively cooperative binding of zinc ions to Bacillus cereus 569/H/9 beta-lactamase II suggests that the binuclear enzyme is the only relevant form for catalysis.

Metallo-beta-lactamases catalyze the hydrolysis of most beta-lactam antibiotics and hence represent a major clinical concern. While enzymes belonging to subclass B1 have been shown to display maximum activity as dizinc species, the actual metal-to-protein stoichiometry and the affinity for zinc are not clear. We have further investigated the process of metal binding to the beta-lactamase II from Bacillus cereus 569/H/9 (known as BcII). Zinc binding was monitored using complementary biophysical techniques, including circular dichroism in the far-UV, enzymatic activity measurements, competition with a chromophoric chelator, mass spectrometry, and nuclear magnetic resonance. Most noticeably, mass spectrometry and nuclear magnetic resonance experiments, together with catalytic activity measurements, demonstrate that two zinc ions bind cooperatively to the enzyme active site (with K(1)/K(2)> or =5) and, hence, that catalysis is associated with the dizinc enzyme species only. Furthermore, competitive experiments with the chromophoric chelator Mag-Fura-2 indicates K(2)<80 nM. This contrasts with cadmium binding, which is clearly a noncooperative process with the mono form being the only species significantly populated in the presence of 1 molar equivalent of Cd(II). Interestingly, optical measurements reveal that although the apo and dizinc species exhibit undistinguishable tertiary structural organizations, the metal-depleted enzyme shows a significant decrease in its alpha-helical content, presumably associated with enhanced flexibility.

NADPH-dependent glutamate dehydrogenase in Penicillium chrysogenum is involved in regulation of beta-lactam production.

The interactions between the ammonium assimilatory pathways and beta-lactam production were investigated by disruption of the NADPH-dependent glutamate dehydrogenase gene (gdhA) in two industrial beta-lactam-producing strains of Penicillium chrysogenum. The strains used were an adipoyl-7-ADCA- and a penicillin-producing strain. The gdhA gene disruption caused a decrease in maximum specific growth rate of 26 % and 35 % for the adipoyl-7-ADCA-producing strain and the penicillin-producing strain, respectively, compared to the corresponding reference strains. Interestingly, no beta-lactam production was detected in either of the DeltagdhA strains. Supplementation with glutamate restored growth but no beta-lactam production was detected for the constructed strains. Cultures with high ammonium concentrations (repressing conditions) and with proline as nitrogen source (de-repressed conditions) showed continued beta-lactam production for the reference strains whereas the DeltagdhA strains remained non-productive under all conditions. By overexpressing the NAD-dependent glutamate dehydrogenase, the specific growth rate could be restored, but still no beta-lactam production was detected. The results indicate that the NADPH-dependent glutamate dehydrogenase may be directly or indirectly involved in the regulation of beta-lactam production in industrial strains of P. chrysogenum.

Redefining penems.

The antimicrobial class of penems has the potential to address most of the relevant resistance issues associated with beta-lactam antibiotics because of their exceptionally broad spectrum of antibacterial activity and their intrinsic stability against hydrolytic attack by many beta-lactamases including ESBL and AmpC enzymes. The subclass of carbapenems covers the spectrum of hospital pathogens whereas the subclass of penems covers community pathogens. The only currently available penem, faropenem, has a low propensity for resistance development, beta-lactamase induction and selection of carbapenem-resistant Pseudomonas aeruginosa. This makes it attractive for the treatment of community-acquired infections and for step-down or sequential therapy following carbapenem treatment without jeopardizing the activity of carbapenems or the entire beta-lactam class in the hospital environment.

In vitro activity of levofloxacin against recent Gram-negative nosocomial pathogens.

The objective of our study was to evaluate the in vitro activity of levofloxacin in comparison with other antibiotics (coamoxiclav, ceftriaxone, ceftazidime, meropenem, aztreonam, ciprofloxacin and netilmicin) against about 500 Gram-negative nosocomial microorganisms, isolated during 2003. The bactericidal activities (minimal bactericidal activity and killing curve) of levofloxacin and the other antibiotics were also evaluated. The results confirm the excellent in vitro activity of levofloxacin against multiresistant nosocomial Gram-negative pathogens, including the 73 extended spectrum beta-lactamase producer strains (90% of Escherichia coli, Enterobacter cloacae and Klebsiella pneumoniae were inhibited at 0.5 mg/l). Of particular interest is its susceptibility and bactericidal activity against Stenotrophomonas maltophilia strains. In conclusion, due to the favorable pharmacokinetics and pharmacodynamics of the molecule, associated with its safety, levofloxacin may represent a valid therapeutic option in the treatment of severe Gram-negative nosocomial infections.

Quantitation of transcription and clonal selection of single living cells with beta-lactamase as reporter.

Gene expression was visualized in single living mammalian cells with beta-lactamase as a reporter that hydrolyzes a substrate loaded intracellularly as a membrane-permeant ester. Each enzyme molecule changed the fluorescence of many substrate molecules from green to blue by disrupting resonance energy transfer. This wavelength shift was detectable by eye or color film in individual cells containing less than 100 beta-lactamase molecules. The robust change in emission ratio reveals quantitative heterogeneity in real-time gene expression, enables clonal selection by flow cytometry, and forms a basis for high-throughput screening of pharmaceutical candidate drugs in living mammalian cells.