Cartilage Regeneration in Humans with Adipose Tissue-Derived Stem Cells and Adipose Stromal Vascular Fraction Cells: Updated Status.

Adipose tissue-derived stem cells (ASCs) in the form of stromal vascular fraction (SVF) and cultured expansion have been applied in clinical settings in some countries to treat osteoarthritis (OA) of knees, one of the most common debilitating, incurable disorders. Since the first report of successful cartilage-like tissue regeneration with autologous adipose SVF containing ASCs, there has been a gradual increase in the number of publications confirming such results. Thus far, most of the reports have been limited to treatments of OA of knees. Recently, successful applications of adipose SVF in treating OA of ankles and hips have been reported. In addition, several groups have reported modified methods of applying adipose SVF, such as combining bone marrow stimulation with adipose SVF or adding additional extracellular matrix (ECM) in treating OA. Here, we present an updated, systematic review of clinical effectiveness and safety in treating OA of knees, ankles, and one hip since 2016 using ASCs in the form of adipose SVF or in cultured expansion, along with a description and suggestion of potential biological mechanisms of cartilage regeneration.

Current use of autologous adipose tissue-derived stromal vascular fraction cells for orthopedic applications.

Autologous adipose stromal vascular fractions (SVFs) containing adipose tissue-derived stem cells (ASCs) are currently being used in clinical settings for various orthopedic applications for human patients. Due to its potential capability of regenerating cartilage, bone, and tendons, autologous adipose SVFs are being tried in treating patients with osteoarthritis (OA), chondromalacia, meniscus tear, osteonecrosis of the femoral head, and tendon injuries. Here, we have reviewed available human clinical studies with regard to patient applications of autologous adipose SVF containing ASCs, specifically assessing effectiveness and safety in the field of orthopedic disorders. All studies reviewed in this article presents potential benefits of autologous adipose SVF in various orthopedic applications without any serious side effects.

Complex Class 1 Integron Carrying qnrB62 and blaVIM-2 in a Citrobacter freundii Clinical Isolate.

The coexistence of qnrB62 and blaVIM-2 was detected in a Citrobacter clinical isolate. The reduced fluoroquinolone susceptibility is attributable to qnrB62, mutations of quinolone-resistance-determining regions, and an efflux pump or pumps. The genetic context surrounding chromosomal qnrB62 was a novel complex class 1 integron (In1184::ISCR1::qnrB62) containing a unique gene array (blaVIM-2-aacA4'-8-gucD). An 18-nucleotide deletion at the 3' end of the pspA gene [pspA(Δ18)], upstream of qnrB62, and an inverted repeat region (IRR2) were detected in In1184::ISCR1::qnrB62, indicating past transposition events.

Why cannot a ß-lactamase gene be detected using an efficient molecular diagnostic method?

OBJECTIVE: Fast detection of ß-lactamase (bla) genes can minimize the spread of antibiotic resistance. Although several molecular diagnostic methods have been developed to detect limited bla gene types, these methods have significant limitations, such as their failure to detect almost all clinically available bla genes. We have evaluated a further refinement of our fast and accurate molecular method, developed to overcome these limitations, using clinical isolates. METHODS: We have recently developed the efficient large-scale bla detection method (large-scaleblaFinder) that can detect bla gene types including almost all clinically available 1,352 bla genes with perfect specificity and sensitivity. Using this method, we have evaluated a further refinement of this method using clinical isolates provided by International Health Management Associates, Inc. (Schaumburg, Illinois, USA). Results were interpreted in a blinded manner by researchers who did not know any information on bla genes harbored by these isolates. RESULTS: With only one exception, the large-scaleblaFinder detected all bla genes identified by the provider using microarray and multiplex PCR. In one of the Escherichia coli test isolates, a blaDHA-1 gene was detected using the multiplex PCR assay but it was not detected using the large-scaleblaFinder. CONCLUSION: The truncation of a blaDHA-1 gene is an important reason for an efficient molecular diagnostic method (large-scaleblaFinder) not to detect the bla gene.

Fast and Accurate Large-Scale Detection of ß-Lactamase Genes Conferring Antibiotic Resistance.

Fast detection of ß-lactamase (bla) genes allows improved surveillance studies and infection control measures, which can minimize the spread of antibiotic resistance. Although several molecular diagnostic methods have been developed to detect limited bla gene types, these methods have significant limitations, such as their failure to detect almost all clinically available bla genes. We developed a fast and accurate molecular method to overcome these limitations using 62 primer pairs, which were designed through elaborate optimization processes. To verify the ability of this large-scale bla detection method (large-scaleblaFinder), assays were performed on previously reported bacterial control isolates/strains. To confirm the applicability of the large-scaleblaFinder, the assays were performed on unreported clinical isolates. With perfect specificity and sensitivity in 189 control isolates/strains and 403 clinical isolates, the large-scaleblaFinder detected almost all clinically available bla genes. Notably, the large-scaleblaFinder detected 24 additional unreported bla genes in the isolates/strains that were previously studied, suggesting that previous methods detecting only limited types of bla genes can miss unexpected bla genes existing in pathogenic bacteria, and our method has the ability to detect almost all bla genes existing in a clinical isolate. The ability of large-scaleblaFinder to detect bla genes on a large scale enables prompt application to the detection of almost all bla genes present in bacterial pathogens. The widespread use of the large-scaleblaFinder in the future will provide an important aid for monitoring the emergence and dissemination of bla genes and minimizing the spread of resistant bacteria.

Structural basis for carbapenem-hydrolyzing mechanisms of carbapenemases conferring antibiotic resistance.

Carbapenems (imipenem, meropenem, biapenem, ertapenem, and doripenem) are ß-lactam antimicrobial agents. Because carbapenems have the broadest spectra among all ß-lactams and are primarily used to treat infections by multi-resistant Gram-negative bacteria, the emergence and spread of carbapenemases became a major public health concern. Carbapenemases are the most versatile family of ß-lactamases that are able to hydrolyze carbapenems and many other ß-lactams. According to the dependency of divalent cations for enzyme activation, carbapenemases can be divided into metallo-carbapenemases (zinc-dependent class B) and non-metallo-carbapenemases (zinc-independent classes A, C, and D). Many studies have provided various carbapenemase structures. Here we present a comprehensive and systematic review of three-dimensional structures of carbapenemase-carbapenem complexes as well as those of carbapenemases. We update recent studies in understanding the enzymatic mechanism of each class of carbapenemase, and summarize structural insights about regions and residues that are important in acquiring the carbapenemase activity.

Structure of ADC-68, a novel carbapenem-hydrolyzing class C extended-spectrum ß-lactamase isolated from Acinetobacter baumannii.

Outbreaks of multidrug-resistant bacterial infections have become more frequent worldwide owing to the emergence of several different classes of ß-lactamases. In this study, the molecular, biochemical and structural characteristics of an Acinetobacter-derived cephalosporinase (ADC)-type class C ß-lactamase, ADC-68, isolated from the carbapenem-resistant A. baumannii D015 were investigated. The blaADC-68 gene which encodes ADC-68 was confirmed to exist on the chromosome via Southern blot analysis and draft genome sequencing. The catalytic kinetics of ß-lactams and their MICs (minimum inhibitory concentrations) for A. baumannii D015 and purified ADC-68 (a carbapenemase obtained from this strain) were assessed: the strain was resistant to penicillins, narrow-spectrum and extended-spectrum cephalosporins, and carbapenems, which were hydrolyzed by ADC-68. The crystal structure of ADC-68 was determined at a resolution of 1.8 Å. The structure of ADC-68 was compared with that of ADC-1 (a non-carbapenemase); differences were found in the central part of the Ω-loop and the C-loop constituting the edge of the R1 and R2 subsites and are close to the catalytic serine residue Ser66. The ADC-68 C-loop was stabilized in the open conformation of the upper R2 subsite and could better accommodate carbapenems with larger R2 side chains. Furthermore, a wide-open conformation of the R2-loop allowed ADC-68 to bind to and hydrolyze extended-spectrum cephalosporins. Therefore, ADC-68 had enhanced catalytic efficiency against these clinically important ß-lactams (extended-spectrum cephalosporins and carbapenems). ADC-68 is the first reported enzyme among the chromosomal class C ß-lactamases to possess class C extended-spectrum ß-lactamase and carbapenemase activities.

Phosphorylation-dependent interaction between a serine/threonine kinase PknA and a putative cell division protein Wag31 in Mycobacterium tuberculosis.

Mycobacterium tuberculosis genome contains eleven serine/threonine protein kinases (STPKs). Among these ST- PKs, PknA is a key component of signal transduction pathway that regulates cell shape and possibly cell division in M. tuberculosis via reversible phosphorylation of intracellular proteins. The in vitro peptide library screen showed that Wag31, a putative cell division protein, was a new substrate phosphorylated by PknA. The signal transduction pathway involving Wag31 and PknA plays a unique role in M. tuberculosis growth regulation that may participate in the pathogenesis of tuberculosis. In this study, genes of PknA, wild-type Wag31 (Wag31WT), phosphoablative Wag31T73A, and phosphomimetic Wag31T73E were cloned and expressed. Far-western analyses were performed using partial purified PknA and completely purified Wag31 proteins (Wag31WT, Wag31T73A, and Wag31T73E). Far-western analysis data revealed that the direct interaction between PknA and Wag31 is dependent on the phos- phorylation state of Wag31, which can represent a novel target for the development of new anti-tuberculosis drugs.

Determination of pentapeptide repeat units in Qnr proteins by the structure-based alignment approach.

The Qnr proteins belong to the pentapeptide repeat protein family. Despite each pentapeptide repeat unit being an important structural determinant, accurately determining pentapeptide repeat units in the Qnr proteins through sequence-based alignments has been challenging. In the present study, the pentapeptide repeat units of the nine representative Qnr proteins have been precisely determined via the structure-based alignment approach using homology modeling and superposition of their best models.

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.

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.

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.

PNGM-1, a novel subclass B3 metallo-ß-lactamase from a deep-sea sediment metagenome.

OBJECTIVES: In order to find antimicrobial resistance gene(s) pre-dating the use of antibiotics through metagenomics, functional screening of a metagenomic library from the deep-seep sediments of Edison Seamount (ca. 10000 years old) was performed. METHODS: Among 60 antimicrobial-resistant clones, a single clone with the highest minimum inhibitory concentration (MIC) for ampicillin was selected. Sequence analysis revealed a new metallo-ß-lactamase (MBL) gene, designated as blaPNGM-1. PNGM-1 retains a zinc ion-binding motif (H116XH118XD120H121, H196 and H263), conserved in subclass B3 MBLs. The catalytic parameters of purified PNGM-1 and the MICs of ß-lactams for Escherichia coli TOP10 transformants harbouring the blaPNGM-1 gene were assessed. RESULTS: Antimicrobial susceptibility testing indicated reduced susceptibility to penicillins, narrow- and extended-spectrum cephalosporins, and carbapenems in E. coli TOP10 transformants harbouring the blaPNGM-1 gene. In addition, kinetic analyses revealed that PNGM-1 hydrolysed almost all ß-lactams. CONCLUSIONS: The PNGM-1 enzyme is the first case of a subclass B3 MBL derived from a functional metagenomic library of a deep-sea sediment that pre-dates the antibiotic era.

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.

Potential use of mesenchymal stem cells in human meniscal repair: current insights.

The menisci of the human knee play an important role in maintaining normal functions to provide stability and nutrition to the articular cartilage, and to absorb shock. Once injured, these important structures have very limited natural healing potential. Unfortunately, the traditional arthroscopic meniscectomy performed on these damaged menisci may predispose the joint toward early development of osteoarthritis. Although a very limited number of studies are available, mesenchymal stem cells (MSCs) have been investigated as an alternative therapeutic modality to repair human knee meniscal tears. This review summarizes the results of published applications of MSCs in human patients, which showed that the patients who received MSCs (autologous adipose tissue-derived stem cells or culture-expanded bone marrow-derived stem cells) presented symptomatic improvements, along with magnetic resonance imaging evidences of the meniscal repair.

Antimicrobial Resistance of Hypervirulent Klebsiella pneumoniae: Epidemiology, Hypervirulence-Associated Determinants, and Resistance Mechanisms.

Klebsiella pneumoniae is one of the most clinically relevant species in immunocompromised individuals responsible for community-acquired and nosocomial infections, including pneumonias, urinary tract infections, bacteremias, and liver abscesses. Since the mid-1980s, hypervirulent K. pneumoniae, generally associated with the hypermucoviscosity phenotype, has emerged as a clinically significant pathogen responsible for serious disseminated infections, such as pyogenic liver abscesses, osteomyelitis, and endophthalmitis, in a generally younger and healthier population. Hypervirulent K. pneumoniae infections were primarily found in East Asia and now are increasingly being reported worldwide. Although most hypervirulent K. pneumoniae isolates are antibiotic-susceptible, some isolates with combined virulence and resistance, such as the carbapenem-resistant hypervirulent K. pneumoniae isolates, are increasingly being detected. The combination of multidrug resistance and enhanced virulence has the potential to cause the next clinical crisis. To better understand the basic biology of hypervirulent K. pneumoniae, this review will provide a summarization and discussion focused on epidemiology, hypervirulence-associated factors, and antibiotic resistance mechanisms of such hypervirulent strains. Epidemiological analysis of recent clinical isolates in China warns the global dissemination of hypervirulent K. pneumoniae strains with extensive antibiotic resistance in the near future. Therefore, an immediate response to recognize the global dissemination of this hypervirulent strain with resistance determinants is an urgent priority.

Biology of Acinetobacter baumannii: Pathogenesis, Antibiotic Resistance Mechanisms, and Prospective Treatment Options.

Acinetobacter baumannii is undoubtedly one of the most successful pathogens responsible for hospital-acquired nosocomial infections in the modern healthcare system. Due to the prevalence of infections and outbreaks caused by multi-drug resistant A. baumannii, few antibiotics are effective for treating infections caused by this pathogen. To overcome this problem, knowledge of the pathogenesis and antibiotic resistance mechanisms of A. baumannii is important. In this review, we summarize current studies on the virulence factors that contribute to A. baumannii pathogenesis, including porins, capsular polysaccharides, lipopolysaccharides, phospholipases, outer membrane vesicles, metal acquisition systems, and protein secretion systems. Mechanisms of antibiotic resistance of this organism, including acquirement of ß-lactamases, up-regulation of multidrug efflux pumps, modification of aminoglycosides, permeability defects, and alteration of target sites, are also discussed. Lastly, novel prospective treatment options for infections caused by multi-drug resistant A. baumannii are summarized.

Regeneration of Cartilage in Human Knee Osteoarthritis with Autologous Adipose Tissue-Derived Stem Cells and Autologous Extracellular Matrix.

This clinical case series demonstrates that percutaneous injections of autologous adipose tissue-derived stem cells (ADSCs) and homogenized extracellular matrix (ECM) in the form of adipose stromal vascular fraction (SVF), along with hyaluronic acid (HA) and platelet-rich plasma (PRP) activated by calcium chloride, could regenerate cartilage-like tissue in human knee osteoarthritis (OA) patients. Autologous lipoaspirates were obtained from adipose tissue of the abdominal origin. Afterward, the lipoaspirates were minced to homogenize the ECM. These homogenized lipoaspirates were then mixed with collagenase and incubated. The resulting mixture of ADSCs and ECM in the form of SVF was injected, along with HA and PRP activated by calcium chloride, into knees of three Korean patients with OA. The same affected knees were reinjected weekly with additional PRP activated by calcium chloride for 3 weeks. Pretreatment and post-treatment magnetic resonance imaging (MRI) data, functional rating index, range of motion (ROM), and pain score data were then analyzed. All patients' MRI data showed cartilage-like tissue regeneration. Along with MRI evidence, the measured physical therapy outcomes in terms of ROM, subjective pain, and functional status were all improved. This study demonstrates that percutaneous injection of ADSCs with ECM contained in autologous adipose SVF, in conjunction with HA and PRP activated by calcium chloride, is a safe and potentially effective minimally invasive therapy for OA of human knees.