Transmission of antibiotic resistance genes through mobile genetic elements in Acinetobacter baumannii and gene-transfer prevention.

Antibiotic resistance is a major global public health concern. Acinetobacter baumannii is a nosocomial pathogen that has emerged as a global threat because of its high levels of resistance to many antibiotics, particularly those considered as last-resort antibiotics, such as carbapenems. Mobile genetic elements (MGEs) play an important role in the dissemination and expression of antibiotic resistance genes (ARGs), including the mobilization of ARGs within and between species. We conducted an in-depth, systematic investigation of the occurrence and dissemination of ARGs associated with MGEs in A. baumannii. We focused on a cross-sectoral approach that integrates humans, animals, and environments. Four strategies for the prevention of ARG dissemination through MGEs have been discussed: prevention of airborne transmission of ARGs using semi-permeable membrane-covered thermophilic composting; application of nanomaterials for the removal of emerging pollutants (antibiotics) and pathogens; tertiary treatment technologies for controlling ARGs and MGEs in wastewater treatment plants; and the removal of ARGs by advanced oxidation techniques. This review contemplates and evaluates the major drivers involved in the transmission of ARGs from the cross-sectoral perspective and ARG-transfer prevention processes.

Designing Short Peptides to Block the Interaction of SARS-CoV-2 and Human ACE2 for COVID-19 Therapeutics.

To date, the current COVID-19 pandemic caused by SARS-CoV-2 has infected 99.2 million while killed 2.2 million people throughout the world and is still spreading widely. The unavailability of potential therapeutics against this virus urges to search and develop new drugs. SARS-CoV-2 enters human cells by interacting with human angiotensin-converting enzyme 2 (ACE2) receptor expressed on human cell surface through utilizing receptor-binding domain (RBD) of its spike glycoprotein. The RBD is highly conserved and is also a potential target for blocking its interaction with human cell surface receptor. We designed short peptides on the basis of our previously reported truncated ACE2 (tACE2) for increasing the binding affinity as well as the binding interaction network with RBD. These peptides can selectively bind to RBD with much higher affinities than the cell surface receptor. Thus, these can block all the binding residues required for binding to cell surface receptor. We used selected amino acid regions (21-40 and 65-75) of ACE2 as scaffold for the de novo peptide design. Our designed peptide Pep1 showed interactions with RBD covering almost all of its binding residues with significantly higher binding affinity (-13.2 kcal mol-1) than the cell surface receptor. The molecular dynamics (MD) simulation results showed that designed peptides form a stabilized complex with RBD. We suggest that blocking the RBD through de novo designed peptides can serve as a potential candidate for COVID-19 treatment after further clinical investigations.

Mutation-Based Antibiotic Resistance Mechanism in Methicillin-Resistant Staphylococcus aureus Clinical Isolates.

ß-Lactam antibiotics target penicillin-binding proteins and inhibit the synthesis of peptidoglycan, a crucial step in cell wall biosynthesis. Staphylococcus aureus acquires resistance against ß-lactam antibiotics by producing a penicillin-binding protein 2a (PBP2a), encoded by the mecA gene. PBP2a participates in peptidoglycan biosynthesis and exhibits a poor affinity towards ß-lactam antibiotics. The current study was performed to determine the diversity and the role of missense mutations of PBP2a in the antibiotic resistance mechanism. The methicillin-resistant Staphylococcus aureus (MRSA) isolates from clinical samples were identified using phenotypic and genotypic techniques. The highest frequency (60%, 18 out of 30) of MRSA was observed in wound specimens. Sequence variation analysis of the mecA gene showed four amino acid substitutions (i.e., E239K, E239R, G246E, and E447K). The E239R mutation was found to be novel. The protein-ligand docking results showed that the E239R mutation in the allosteric site of PBP2a induces conformational changes in the active site and, thus, hinders its interaction with cefoxitin. Therefore, the present report indicates that mutation in the allosteric site of PBP2a provides a more closed active site conformation than wide-type PBP2a and then causes the high-level resistance to cefoxitin.

Purple corn extract (PCE) alleviates cigarette smoke (CS)-induced DNA damage in rodent blood cells by activation of AMPK/Foxo3a/MnSOD pathway.

Purple corn extract (PCE) is a nutraceutical, an activator of AMPK, and it has antioxidants and anticancer properties. Therefore, PCE could be a candidate for alleviating cigarette smoke (CS)-induced oxidative DNA damage. This study examined whether PCE can have a protective effect on blood cells in an animal model of cigarette smoke (CS)-induced DNA damage. PCE was orally administered to CS-inhaled Spraque-Dawley (SD) rats, followed by the target cells being examined for markers of DNA damage. The study also sought to elucidate the mechanism of PCE action in the PCE treated animals. SD rat inhalation of CS was for once a day for 30 min, repeated for 7 days. PCE was administered orally before CS inhalation. Pretreatment of the animals with oral PCE kept the numbers of white blood cells (WBC) as well as neutrophils (NE), lymphocytes (LY), monocytes (Mo), eosinophils (EO), abd jasophils (BA) from increasing as those were increased in the CS-inhaling SD rats. The amount of phosphorylated γ-H2AX, a DNA damage marker, was assayed in the circulating blood cells collected from the animals and western blot analysis with anti-Foxo3a, p-Foxo3a, p-AMPK, MnSOD antibodies were performed on those cells. PCE protected the circulating blood cells from CS inhalation-induced DNA damage by 44% as assayed by increases in γ-H2AX. PCE also increased the nuclear localization of Foxo3a by 52% over control cells. Mechanistically, PCE appears to efficiently protect various blood cell types from CS-induced DNA damage through removal of ROS via activation of the AMPK/Foxo3a/MnSOD pathway.

Structural Insights for Core Scaffold and Substrate Specificity of B1, B2, and B3 Metallo-ß-Lactamases.

Metallo-ß-lactamases (MBLs) hydrolyze almost all ß-lactam antibiotics, including penicillins, cephalosporins, and carbapenems; however, no effective inhibitors are currently clinically available. MBLs are classified into three subclasses: B1, B2, and B3. Although the amino acid sequences of MBLs are varied, their overall scaffold is well conserved. In this study, we systematically studied the primary sequences and crystal structures of all subclasses of MBLs, especially the core scaffold, the zinc-coordinating residues in the active site, and the substrate-binding pocket. We presented the conserved structural features of MBLs in the same subclass and the characteristics of MBLs of each subclass. The catalytic zinc ions are bound with four loops from the two central ß-sheets in the conserved αß/ßα sandwich fold of MBLs. The three external loops cover the zinc site(s) from the outside and simultaneously form a substrate-binding pocket. In the overall structure, B1 and B2 MBLs are more closely related to each other than they are to B3 MBLs. However, B1 and B3 MBLs have two zinc ions in the active site, while B2 MBLs have one. The substrate-binding pocket is different among all three subclasses, which is especially important for substrate specificity and drug resistance. Thus far, various classes of ß-lactam antibiotics have been developed to have modified ring structures and substituted R groups. Currently available structures of ß-lactam-bound MBLs show that the binding of ß-lactams is well conserved according to the overall chemical structure in the substrate-binding pocket. Besides ß-lactam substrates, B1 and cross-class MBL inhibitors also have distinguished differences in the chemical structure, which fit well to the substrate-binding pocket of MBLs within their inhibitory spectrum. The systematic structural comparison among B1, B2, and B3 MBLs provides in-depth insight into their substrate specificity, which will be useful for developing a clinical inhibitor targeting MBLs.

Potential Benefits of Allogeneic Haploidentical Adipose Tissue-Derived Stromal Vascular Fraction in a Hutchinson-Gilford Progeria Syndrome Patient.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare, fatal, and genetic disorder in the LMNA gene encoding for prelamin A. Normally, prelamin A is processed to become lamin A protein. In HGPS patients, there is a heterozygous mutation in LMNA gene, in which there is a deletion of genetic codes responsible for 50 amino acids at the C-terminus of prelamin A. The processing of the abnormal prelamin A results in abnormal lamin A protein, called progerin, causing symptoms of accelerated early aging, probably due to the inflammaging process. It is well known that adipose tissue-derived mesenchymal stem cells (MSCs) have anti-inflammatory effects by modulating inflammatory cytokines and by extracellular vesicles. Here, we present a case of an HGPS patient who responded positively to injections of allogeneic haploidentical adipose tissue-derived stromal vascular fractions containing MSCs by showing rapid height and weight growth along with increased blood level of insulin-like growth factor 1.

Freshwater viral metagenome reveals novel and functional phage-borne antibiotic resistance genes.

BACKGROUND: Antibiotic resistance developed by bacteria is a significant threat to global health. Antibiotic resistance genes (ARGs) spread across different bacterial populations through multiple dissemination routes, including horizontal gene transfer mediated by bacteriophages. ARGs carried by bacteriophages are considered especially threatening due to their prolonged persistence in the environment, fast replication rates, and ability to infect diverse bacterial hosts. Several studies employing qPCR and viral metagenomics have shown that viral fraction and viral sequence reads in clinical and environmental samples carry many ARGs. However, only a few ARGs have been found in viral contigs assembled from metagenome reads, with most of these genes lacking effective antibiotic resistance phenotypes. Owing to the wide application of viral metagenomics, nevertheless, different classes of ARGs are being continuously found in viral metagenomes acquired from diverse environments. As such, the presence and functionality of ARGs encoded by bacteriophages remain up for debate. RESULTS: We evaluated ARGs excavated from viral contigs recovered from urban surface water viral metagenome data. In virome reads and contigs, diverse ARGs, including polymyxin resistance genes, multidrug efflux proteins, and ß-lactamases, were identified. In particular, when a lenient threshold of e value of ≤ 1 × e-5 and query coverage of ≥ 60% were employed in the Resfams database, the novel ß-lactamases blaHRV-1 and blaHRVM-1 were found. These genes had unique sequences, forming distinct clades of class A and subclass B3 ß-lactamases, respectively. Minimum inhibitory concentration analyses for E. coli strains harboring blaHRV-1 and blaHRVM-1 and catalytic kinetics of purified HRV-1 and HRVM-1 showed reduced susceptibility to penicillin, narrow- and extended-spectrum cephalosporins, and carbapenems. These genes were also found in bacterial metagenomes, indicating that they were harbored by actively infecting phages. CONCLUSION: Our results showed that viruses in the environment carry as-yet-unreported functional ARGs, albeit in small quantities. We thereby suggest that environmental bacteriophages could be reservoirs of widely variable, unknown ARGs that could be disseminated via virus-host interactions. Video abstract.

SHV Hyperproduction as a Mechanism for Piperacillin-Tazobactam Resistance in Extended-Spectrum Cephalosporin-Susceptible Klebsiella pneumoniae.

This study aimed to determine the mechanism of resistance to piperacillin-tazobactam (TZP) in Klebsiella pneumoniae bloodstream isolates that are susceptible to extended-spectrum cephalosporins (ESCs). Antibiotic susceptibility was determined for K. pneumoniae isolated from children with bacteremia. The ß-lactamase genes were detected using a large-scale bla detection method (LARGE-SCALEblaFinder) and confirmed by sequencing analysis. The isolates were further characterized by ß-lactamase activity assays and multilocus sequence typing. Among the 300 bloodstream isolates of K. pneumoniae, 11 (3.7%) were TZP resistant but ESC susceptible. The TZP minimum inhibitory concentrations (MICs) of the isolates ranged from 128/4 to >2,048/4 mg/L. Avibactam markedly inhibited piperacillin resistance, reducing the MICs to the range of ≤1 to 8 mg/L. Among the 11 isolates, four hyperproduced SHV-1 and two hyperproduced SHV-11, exhibiting 77- to 496-fold higher ß-lactamase activity compared with the SHV-1- and SHV-11-producing reference strains that are susceptible to TZP. OXA-1 was coproduced in three isolates, and the remaining two isolates produced TEM-30. Transformants with recombinant plasmids carrying the ß-lactamase genes demonstrated an increase in MICs of TZP. The TZP-resistant and ESC-susceptible isolates were not epidemiologically related. Hyperproduction of SHV-1 and SHV-11 represents a novel mechanism for reducing TZP activity in K. pneumoniae isolates resistant to ESCs. Continuous monitoring and investigation of TZP-resistant isolates are needed in the current era of high TZP consumption.

The Occurrence and Characterization of Extended-Spectrum-Beta-Lactamase-Producing Escherichia coli Isolated from Clinical Diagnostic Specimens of Equine Origin.

Escherichia coli isolates were recovered from clinical specimens of equine patients admitted to the Texas A&M Veterinary Medical Teaching Hospital over a five-year period. Ceftiofur resistance was used as a marker for potential extended-spectrum beta-lactamase (ESBL)-activity, and of the 48 ceftiofur-resistant E. coli isolates, 27.08% (n = 13) were phenotypically ESBL-positive. Conventional PCR analysis followed by the large-scalebla Finder multiplex PCR detected the ESBL genes, CTX-M-1 and SHV, in seven out of the 13 isolates. Moreover, beta-lactamase genes of TEM-1-type, BER-type (AmpC), and OXA-type were also identified. Sequencing of these genes resulted in identification of a novel TEM-1-type gene, called blaTEM-233, and a study is currently underway to determine if this gene confers the ESBL phenotype. Furthermore, this report is the first to have found E. coli ST1308 in horses. This subtype, which has been reported in other herbivores, harbored the SHV-type ESBL gene. Finally, one out of 13 E. coli isolates was PCR-positive for the carbapenemase gene, blaIMP-1 despite the lack of phenotypically proven resistance to imipenem. With the identification of novel ESBL gene variant and the demonstrated expansion of E. coli sequence types in equine patients, this study underscores the need for more investigation of equines as reservoirs for ESBL-producing pathogens.

Dual activity of PNGM-1 pinpoints the evolutionary origin of subclass B3 metallo-ß-lactamases: a molecular and evolutionary study.

Resistance to ß-lactams is one of the most serious problems associated with Gram-negative infections. ß-Lactamases are able to hydrolyze ß-lactams such as cephalosporins and/or carbapenems. Evolutionary origin of metallo-ß-lactamases (MBLs), conferring critical antibiotic resistance threats, remains unknown. We discovered PNGM-1, the novel subclass B3 MBL, in deep-sea sediments that predate the antibiotic era. Here, our phylogenetic analysis suggests that PNGM-1 yields insights into the evolutionary origin of subclass B3 MBLs. We reveal the structural similarities between tRNase Zs and PNGM-1, and demonstrate that PNGM-1 has both MBL and tRNase Z activities, suggesting that PNGM-1 is thought to have evolved from a tRNase Z. We also show kinetic and structural comparisons between PNGM-1 and other proteins including subclass B3 MBLs and tRNase Zs. These comparisons revealed that the B3 MBL activity of PNGM-1 is a promiscuous activity and subclass B3 MBLs are thought to have evolved through PNGM-1 activity.

The novel metallo-ß-lactamase PNGM-1 from a deep-sea sediment metagenome: crystallization and X-ray crystallographic analysis.

Metallo-ß-lactamases (MBLs) are present in major Gram-negative pathogens and environmental species, and pose great health risks because of their ability to hydrolyze the ß-lactam rings of antibiotics such as carbapenems. PNGM-1 was the first reported case of a subclass B3 MBL protein that was identified from a metagenomic library from deep-sea sediments that predate the antibiotic era. In this study, PNGM-1 was overexpressed, purified and crystallized. Crystals of native and selenomethionine-substituted PNGM-1 diffracted to 2.10 and 2.30 Šresolution, respectively. Both the native and the selenomethionine-labelled PNGM-1 crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 122, b = 83, c = 163 Å, ß = 110°. Matthews coefficient (VM) calculations suggested the presence of 6-10 molecules in the asymmetric unit, corresponding to a solvent content of ∼31-58%. Structure determination is currently in progress.

Clinical Protocol of Producing Adipose Tissue-Derived Stromal Vascular Fraction for Potential Cartilage Regeneration.

Osteoarthritis (OA) is one of the most common debilitating disorders. Recently, numerous attempts have been made to improve the functions of the knees by using different forms of mesenchymal stem cells (MSCs). In Korea, bone marrow concentrates and cord blood-derived stem cells have been approved by the Korean Food and Drug Administration (KFDA) for cartilage regeneration. In addition, an adipose tissue-derived stromal vascular fraction (SVF) has been allowed by the KFDA for joint injections in human patients. Autologous adipose tissue-derived SVF contains extracellular matrix (ECM) in addition to mesenchymal stem cells. ECM excretes various cytokines that, along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, may help MSCs to regenerate cartilage and improve knee functions. In this article, we presented a protocol to improve knee functions by regenerating cartilage-like tissue in human patients with OA. The result of the protocol was first reported in 2011 followed by a few additional publications. The protocol involves liposuction to obtain autologous lipoaspirates that are mixed with collagenase. This lipoaspirates-collagenase mixture is then cut and homogenized to remove large fibrous tissue that may clog up the needle during the injection. Afterwards, the mixture is incubated to obtain adipose tissue-derived SVF. The resulting adipose tissue-derived SVF, containing both adipose tissue-derived MSCs and remnants of ECM, is injected into knees of patients, combined with HA and calcium chloride activated PRP. Included are three cases of patients who were treated with our protocol resulting in improvement of knee pain, swelling, and range of motion along with MRI evidence of hyaline cartilage-like tissue.