Analysis of Antibacterial Action of Mammalian Host-Defense Cathelicidins and Induction of Resistance to Them in MßL-Producing Pseudomonas aeruginosa.

Recombinant analogs of a number of natural host-defense mammalian cathelicidins were obtained and predominant mechanism of their antibacterial action was studied. The ability of cathelicidins to suppress the growth of Pseudomonas aeruginosa producing metallo-ß-lactamases (MßL) was studied, and the possibility of appearance of cathelicidin-resistant bacteria was evaluated. Among peptides with different structures and mechanisms of action, only the strains resistant to ChMAP-28 were not obtained, which indicated minimum risk of the development of natural resistance to this cathelicidin. High antibacterial activity, wide spectrum of action, and the absence of cross-resistance effects allow considering ChMAP-28 peptide as a candidate to be developed further as a therapeutic agent against MßL-producing bacteria.

Practical Agar-Based Disk Diffusion Tests Using Sulfamoyl Heteroarylcarboxylic Acids for Identification of Subclass B1 Metallo-ß-Lactamase-Producing Enterobacterales.

The worldwide distribution of carbapenemase-producing Enterobacterales (CPE) is a serious public health concern as they exhibit carbapenem resistance, thus limiting the choice of antimicrobials for treating CPE infections. Combination treatment with a ß-lactam and one of the newly approved ß-lactamase inhibitors, such as avibactam, relebactam, or vaborbactam, provides a valuable tool to cope with CPE; however, these inhibitors are active only against serine-type carbapenemases and not against metallo-ß-lactamases (MßLs). Therefore, it is important to readily differentiate carbapenemases produced by CPE by using simple and reliable methods in order to choose an appropriate treatment. Here, we developed three practical agar-based disk diffusion tests (double-disk synergy test [DDST], disk potentiation test, and modified carbapenem inactivation method [mCIM]) to discriminate the production of subclass B1 MßLs, such as IMP-, NDM-, and VIM-type MßLs, from the other carbapenemases, especially serine-type carbapenemases. This was accomplished using B1 MßL-specific sulfamoyl heteroarylcarboxylic acid inhibitors, 2,5-dimethyl-4-sulfamoylfuran-3-carboxylic acid (SFC) and 2,5-diethyl-1-methyl-4-sulfamoylpyrrole-3-carboxylic acid (SPC), originally developed by us. The DDST and mCIM using SFC and SPC revealed high sensitivity (95.3%) and specificity (100%) in detecting B1 MßL-producing Enterobacterales. In the disk potentiation test, the sensitivities using SFC and SPC were 89.1% and 93.8%, respectively, whereas the specificities for both were 100%. These methods are simple and inexpensive and have a high accuracy rate. These methods would therefore be of immense assistance in the specific detection and discrimination of B1 MßL-producing Enterobacterales in clinical microbiology laboratories and would lead to better prevention against infection with such multidrug-resistant bacteria in clinical settings.

Tumor type classification and candidate cancer-specific biomarkers discovery via semi-supervised learning.

Identifying cancer-related differentially expressed genes provides significant information for diagnosing tumors, predicting prognoses, and effective treatments. Recently, deep learning methods have been used to perform gene differential expression analysis using microarray-based high-throughput gene profiling and have achieved good results. In this study, we proposed a new robust multiple-datasets-based semi-supervised learning model, MSSL, to perform tumor type classification and candidate cancer-specific biomarkers discovery across multiple tumor types and multiple datasets, which addressed the following long-lasting obstacles: (1) the data volume of the existing single dataset is not enough to fully exert the advantages of deep learning; (2) a large number of datasets from different research institutions cannot be effectively used due to inconsistent internal variances and low quality; (3) relatively uncommon cancers have limited effects on deep learning methods. In our article, we applied MSSL to The Cancer Genome Atlas (TCGA) and the Gene Expression Comprehensive Database (GEO) pan-cancer normalized-level3 RNA-seq data and got 97.6% final classification accuracy, which had a significant performance leap compared with previous approaches. Finally, we got the ranking of the importance of the corresponding genes for each cancer type based on classification results and validated that the top genes selected in this way were biologically meaningful for corresponding tumors and some of them had been used as biomarkers, which showed the efficacy of our method.

Origin, Diversity, and Multiple Roles of Enzymes with Metallo-ß-Lactamase Fold from Different Organisms.

ß-lactamase enzymes have generated significant interest due to their ability to confer resistance to the most commonly used family of antibiotics in human medicine. Among these enzymes, the class B ß-lactamases are members of a superfamily of metallo-ß-lactamase (MßL) fold proteins which are characterised by conserved motifs (i.e., HxHxDH) and are not only limited to bacteria. Indeed, as the result of several barriers, including low sequence similarity, default protein annotation, or untested enzymatic activity, MßL fold proteins have long been unexplored in other organisms. However, thanks to search approaches which are more sensitive compared to classical Blast analysis, such as the use of common ancestors to identify distant homologous sequences, we are now able to highlight their presence in different organisms including Bacteria, Archaea, Nanoarchaeota, Asgard, Humans, Giant viruses, and Candidate Phyla Radiation (CPR). These MßL fold proteins are multifunctional enzymes with diverse enzymatic or non-enzymatic activities of which, at least thirteen activities have been reported such as ß-lactamase, ribonuclease, nuclease, glyoxalase, lactonase, phytase, ascorbic acid degradation, anti-cancer drug degradation, or membrane transport. In this review, we (i) discuss the existence of MßL fold enzymes in the different domains of life, (ii) present more suitable approaches to better investigating their homologous sequences in unsuspected sources, and (iii) report described MßL fold enzymes with demonstrated enzymatic or non-enzymatic activities.

Phenotypic and genotypic characterization of metallo-ß-lactamase producing Pseudomonas aeruginosa isolated from burn patients.

Pseudomonas aeruginosa is an opportunistic pathogen associated with many nosocomial infections. This study aimed to detect blaIMP and blaVIM genes and their common subtypes, including bla IMP-1, bla IMP-2, bla VIM-1, and bla VIM-2, among imipenem-resistant Pseudomonas aeruginosa strains. In this study, 117 P. aeruginosa strains were isolated from clinical samples of burn wound patients in Velayat hospital, Rasht, Iran, between 2018 and 2019. These isolates were tested for antimicrobial susceptibilities by disk diffusion and Metallo-ß-Lactamase (MßL) activity. The polymerase chain reaction (PCR) test was applied to detect MßLs encoding genes in MßL-producing strains. The resistance rates were as follows: Tobramycin (59%), Gentamicin (57%), Piperacillin (52%), Ciprofloxacin (51%), Ceftazidime (32%), and Amikacin (26%). Among 27 (23%) imipenem-resistant isolates, 13 (48%) produced the MßL enzyme. PCR results of imipenem-resistant isolates showed that five and four isolates contained the blaVIM (4 blaVIM1, 2 blaVIM2) and blaIMP (4 blaIMP1, 2 blaIMP2) genes, respectively. In addition some of isolates had more than one gene. In this study, 48% of imipenem-resistant strains produced the MßL enzyme. Therefore, systematic surveillance to detect MßL-producing bacteria and rational prescription and use of carbapenems could be helpful to prevent the spread of carbapenem resistance.

The Inc FII Plasmid and its Contribution in the Transmission of blaNDM-1 and blaKPC-2 in Klebsiella pneumoniae in Egypt.

The emergence of blaKPC-2 and blaNDM-1 producing Klebsiella pneumoniae represents a great problem in many Egyptian hospitals. One hundred and twenty-six K. pneumoniae isolates from patients admitted to Assiut University Hospital were identified by an API20E kit. Carbapenemase-producing K. pneumoniae (CPKP) was detected by the modified carbapenem inactivation method (mCIM), the EDTA-modified carbapenem inactivation method (eCIM), and an E-test. Based on the polymerase chain reaction, all isolates were negative for bla-VIM-1 and bla-IMP-1, fifteen of these isolates were positive for both blaKPC-2 and blaNDM-1, two isolates were positive for blaKPC-2 only, and twenty-eight isolates were positive for bla-NDM-1 only. Although one isolate was positive for the string test, all CPKP isolates were negative for capsular genes. Only 71.1% of CPKP transferred their plasmids to their corresponding transconjugants (E. coli J53). The resistance patterns of the clinical isolates and their transconjugates were similar, except for 12 isolates, which showed differences with their transconjugates in the resistance profile of four antibiotics. Molecular typing of the plasmids based on replicon typing showed that Inc FIIK and FII plasmids predominated in isolates and their transconjugants carrying blaKPC-2 and/or blaNDM-1. Conjugative Inc FII plasmids play an important role in the spread of CPKP, and their recognition is essential to limit their spread.

Presence of high-risk clones of OXA-23-producing Acinetobacter baumannii (ST79) and SPM-1-producing Pseudomonas aeruginosa (ST277) in environmental water samples in Brazil.

This study reports the presence of hospital-associated high-risk lineages of OXA-23-producing ST79 Acinetobacter baumannii and SPM-1-producing ST277 Pseudomonas aeruginosa in urban rivers in Brazil. These findings indicate that urban rivers can act as reservoirs of clinically important multidrug-resistant bacteria, which constitute a potential risk to human and animal health.

Dilution of dipolar interactions in a spin-labeled, multimeric metalloenzyme for DEER studies.

The metallo-ß-lactamases (MßLs), which require one or two Zn(II) ions in their active sites for activity, hydrolyze the amide bond in ß-lactam-containing antibiotics, and render the antibiotics inactive. All known MßLs contain a mobile element near their active sites, and these mobile elements have been implicated in the catalytic mechanisms of these enzymes. However little is known about the dynamics of these elements. In this study, we prepared a site-specific, double spin-labeled analog of homotetrameric MßL L1 with spin labels at positions 163 and 286 and analyzed the sample with DEER (double electron electron resonance) spectroscopy. Four unique distances were observed in the DEER distance distribution, and these distances were assigned to the desired intramolecular dipolar coupling (between spin labels at positions 163 and 286 in one subunit) and to intermolecular dipolar couplings. To rid the spin-labeled analog of L1 of the intermolecular couplings, spin-labeled L1 was "diluted" by unfolding/refolding the spin-labeled enzyme in the presence of excess wild-type L1. DEER spectra of the resulting, spin-diluted enzyme revealed a single distance corresponding to the desire intramolecular dipolar coupling.

Metallo-beta-lactamase Producing Pseudomonas aeruginosa in Neonatal Septicemia.

Gram-negative bacilli are important agents causing neonatal sepsis. The organisms isolated are often resistant to multiple antimicrobials specially which are metallo-beta-lactamases (MßL) producers. Therefore, the present study was conducted with the objective to examine the incidence of MßL producing strains in multidrug resistant (MDR) Pseudomonas aeruginosa from cases of neonatal sepsis. Between January-December 2006, 1994 cases of neonatal sepsis were investigated. The isolates obtained were identified and tested for susceptibility to various antimicrobial agents. The multidrug resistant P. aeruginosa isolates were screened for the presence of MßL by imipenem-EDTA disc method. Five hundred and ninety three (29.73%) isolates were obtained from culture of neonates. Most frequent offender was P. aeruginosa (48.2%). There was an overall predominance of gram-negative organisms. MßL production was seen in 69.5% of imipenem-resistant P. aeruginosa isolates. MßL producing P. aeruginosa is an emerging threat in neonatal septicemia and a cause of concern for physicians treating such infections.