Drug Repurposing of the Unithiol: Inhibition of Metallo-ß-Lactamases for the Treatment of Carbapenem-Resistant Gram-Negative Bacterial Infections.

The increasing antibiotic resistance is a clinical problem worldwide. Numerous Gram-negative bacteria have already become resistant to the most widely used class of antibacterial drugs, ß-lactams. One of the main mechanisms is inactivation of ß-lactam antibiotics by bacterial ß-lactamases. Appearance and spread of these enzymes represent a continuous challenge for the clinical treatment of infections and for the design of new antibiotics and inhibitors. Drug repurposing is a prospective approach for finding new targets for drugs already approved for use. We describe here the inhibitory potency of known detoxifying antidote 2,3-dimercaptopropane-1-sulfonate (unithiol) against metallo-ß-lactamases. Unithiol acts as a competitive inhibitor of meropenem hydrolysis by recombinant metallo-ß-lactamase NDM-1 with the KI of 16.7 µM. It is an order of magnitude lower than the KI for l-captopril, the inhibitor of angiotensin-converting enzyme approved as a drug for the treatment of hypertension. Phenotypic methods demonstrate that the unithiol inhibits natural metallo-ß-lactamases NDM-1 and VIM-2 produced by carbapenem-resistant K. pneumoniae and P. aeruginosa bacterial strains. The 3D full atom structures of unithiol complexes with NDM-1 and VIM-2 are obtained using QM/MM modeling. The thiol group is located between zinc cations of the active site occupying the same place as the catalytic hydroxide anion in the enzyme-substrate complex. The sulfate group forms both a coordination bond with a zinc cation and hydrogen bonds with the positively charged residue, lysine or arginine, responsible for proper orientation of antibiotics upon binding to the active site prior to hydrolysis. Thus, we demonstrate both experimentally and theoretically that the unithiol is a prospective competitive inhibitor of metallo-ß-lactamases and it can be utilized in complex therapy together with the known ß-lactam antibiotics.

Draft Genome Sequences of 10 Clinical K2-Type Klebsiella pneumoniae Strains Isolated in Russia.

We report here the genome sequences of 10 Klebsiella pneumoniae strains of capsular type K2 isolated in Russia from patients in an infectious clinical hospital and neurosurgical intensive care unit. The draft genome sizes range from 5.34 to 5.87 Mb and include 5,448 to 6,137 protein-coding sequences.

Comparative analysis of Klebsiella pneumoniae strains isolated in 2012-2016 that differ by antibiotic resistance genes and virulence genes profiles.

The antibacterial resistance and virulence genotypes and phenotypes of 148 non-duplicate Klebsiella pneumoniae strains collected from 112 patients in Moscow hospitals in 2012-2016 including isolates from the respiratory system (57%), urine (30%), wounds (5%), cerebrospinal fluid (4%), blood (3%), and rectal swab (1%) were determined. The majority (98%) were multidrug resistant (MDR) strains carrying blaSHV (91%), blaCTX-M (74%), blaTEM (51%), blaOXA (38%), and blaNDM (1%) beta-lactamase genes, class 1 integrons (38%), and the porin protein gene ompK36 (96%). The beta-lactamase genes blaTEM-1, blaSHV-1, blaSHV-11, blaSHV-110, blaSHV-190, blaCTX-M-15, blaCTX-M-3, blaCTX-M-55, blaOXA-48, blaOXA-244, and blaNDM-1 were detected; class 1 integron gene cassette arrays (aadA1), (dfrA7), (dfrA1-orfC), (aadB-aadA1), (dfrA17-aadA5), and (dfrA12-orfF-aadA2) were identified. Twenty-two (15%) of clinical K. pneumoniae strains had hypermucoviscous (HV) phenotype defined as string test positive. The rmpA gene associated with HV phenotype was detected in 24% of strains. The intrapersonal mutation of rmpA gene (deletion of one nucleotide at the polyG tract) was a reason for negative hypermucoviscosity phenotype and low virulence of rmpA-positive K. pneumoniae strain KPB584. Eighteen virulent for mice strains with LD50 ≤ 104 CFU were attributed to sequence types ST23, ST86, ST218, ST65, ST2174, and ST2280 and to capsular types K1, K2, and K57. This study is the first report about hypervirulent K. pneumoniae strain KPB2580-14 of ST23K1 harboring extended-spectrum beta-lactamase CTX-M-15 and carbapenemase OXA-48 genes located on pCTX-M-15-like and pOXA-48-like plasmids correspondingly.

Comparative genome analysis of novel Podoviruses lytic for hypermucoviscous Klebsiella pneumoniae of K1, K2, and K57 capsular types.

Hypermucoviscous (HV) strains of capsular types K1, K2 and K57 are the most virulent representatives of the Klebsiella pneumoniae species. Eight novel bacteriophages lytic for HV K. pneumoniae were isolated and characterized. Three bacteriophages, KpV41, KpV475, and KpV71 were found to have a lytic activity against mainly K. pneumoniae of capsular type K1. Two phages, KpV74, and KpV763 were lytic for K2 capsular type K. pneumoniae, and the phage KpV767 was specific to K57-type K. pneumoniae only. Two more phages, KpV766, and KpV48 had no capsular specificity. The phage genomes consist of a linear double-stranded DNA of 40,395-44,623bp including direct terminal repeats of 180-246 bp. The G + C contents are 52.3-54.2 % that is slightly lower than that of genomes of K. pneumoniae strains being used for phage propagation. According to the genome structures, sequence similarity and phylogenetic data, the phages are classified within the genus Kp32virus and Kp34virus of subfamily Autographivirinae, family Podoviridae. In the phage genomes, genes encoding proteins with putative motifs of polysaccharide depolymerase were identified. Depolymerase genes of phages KpV71 and KpV74 lytic for hypermucoviscous K. pneumoniae of K1 and K2 capsular type, respectively, were cloned and expressed in Escherichia coli, and the recombinant gene products were purified. The specificity and polysaccharide-degrading activity of the recombinant depolymerases were demonstrated.

Genome Sequences of Two NDM-1 Metallo-ß-Lactamase-Producing Multidrug-Resistant Strains of Klebsiella pneumoniae with a High Degree of Similarity, One of Which Contains Prophage.

We report genome sequences of two NDM-1 metallo-ß-lactamase-producing multidrug-resistant Klebsiella pneumoniae isolates of sequence type 147 (ST147) from one hospital. The genomes are highly similar and differ in prophage located in the chromosome of K. pneumoniae KPB-1470/16 and in the additional plasmid-carrying blaOXA-48 gene in K. pneumoniae KPB-417/16.

Identification of IS1R and IS10R elements inserted into ompk36 porin gene of two multidrug-resistant Klebsiella pneumoniae hospital strains.

Hospital Klebsiella pneumoniae strains (n = 196) were collected in 2012-16 from the patients of a Moscow neurosurgical intensive care unit. Klebsiella pneumoniae strains were multidrug-resistant and carried beta-lactamase genes blaSHV (97.4% of strains), blaCTX-M (84.7%), blaTEM (56.1%), blaOXA-48-like (49.0%) and blaNDM-1 (one strain), class 1 integrons (43.4% of strains) and porin protein ompK36 gene (100% of strains). The ompK36 porin protein gene disruption by insertion sequence (IS) elements and OmpK36 production loss in two strains were detected in this study. Outer membrane proteins were isolated according to Carlone et al. (Rapid microprocedure for isolating detergent-insoluble outer membrane proteins from Haemophilus species. J Clin Microbiol 1986;24:330-2). The IS10R element belonging to the IS4 family, IS10 group was detected at the position of the 41st nucleotide of the ompK36 gene in K. pneumoniae strain KPB-2304K/15 (the first report for a certain IS element in K. pneumoniae). The IS1R element belonging to the IS1 family was identified at the position of the 86th nucleotide of the ompK36 gene in the K. pneumoniae strain KPB-367K/15 (novel insertion site for IS1 element into ompK36 gene). DNA transfer of the intact ompK36 gene into the strain KPB-367K/15 by vector plasmid restored OmpK36 porin protein production and resulted in a decrease of imipenem minimal inhibitory concentration. Such data confirm the importance of IS elements in ongoing multidrug-resistant evolution in hospital Klebsiella.

Genome sequencing and comparative analysis of three hypermucoviscous Klebsiella pneumoniae strains isolated in Russia.

The prevalence and characteristics of hypermucoviscous (HV) strains among Klebsiella pneumoniae isolated in Russian hospitals were investigated. The HV strains accounted for 11% of the K. pneumoniae isolates collected in the period from 2011 to 2016, and were characterized as belonging to the K1, K2, K20 and K57 serotypes. Whole genome sequences (WGSs) of K. pneumoniae HV clinical strains KPi261 (SCPM-O-B-7850) and KPB4010 (SCPM-O-B-7846) belonging to the K1 and K2 capsular types, as well as WGSs of K. pneumoniae strain KPM9 (SCPM-O-B-7749) of the K20 capsular type isolated from freshwater, were completed. The final draft genome sequences of KPi261, KPB4010 and KPM9 strains consisted of 5 719 189, 5 431 785 and 5 427 926 bp with 57.0, 57.1 and 57.4% GC content, respectively. The chromosomal and plasmid genes associated with K. pneumoniae virulence including the capsular polysaccharide synthesis gene cluster, mucoid phenotype regulator rmpA and transcriptional activator rmpA2, the all operon associated with allantoin metabolism, the kfu operon involved in iron uptake, the aerobactin-producing system iucABCDiutA, and the iron-transport systems iroBCDN and fecIRA were detected.