Cooperative antibiotic resistance facilitates horizontal gene transfer.

The rise of ß-lactam resistance among pathogenic bacteria, due to the horizontal transfer of plasmid-encoded ß-lactamases, is a current global health crisis. Importantly, ß-lactam hydrolyzation by ß-lactamases, not only protects the producing cells but also sensitive neighboring cells cooperatively. Yet, how such cooperative traits affect plasmid transmission and maintenance is currently poorly understood. Here we experimentally show that KPC-2 ß-lactamase expression and extracellular activity were higher when encoded on plasmids compared with the chromosome, resulting in the elevated rescue of sensitive non-producers. This facilitated efficient plasmid transfer to the rescued non-producers and expanded the potential plasmid recipient pool and the probability of plasmid transfer to new genotypes. Social conversion of non-producers by conjugation was efficient yet not absolute. Non-cooperative plasmids, not encoding KPC-2, were moderately more competitive than cooperative plasmids when ß-lactam antibiotics were absent. However, in the presence of a ß-lactam antibiotic, strains with non-cooperative plasmids were efficiently outcompeted. Moreover, plasmid-free non-producers were more competitive than non-producers imposed with the metabolic burden of a plasmid. Our results suggest that cooperative antibiotic resistance especially promotes the fitness of replicons that transfer horizontally such as conjugative plasmids.

Impact of target site mutations and plasmid associated resistance genes acquisition on resistance of Acinetobacter baumannii to fluoroquinolones.

Among bacterial species implicated in hospital-acquired infections are the emerging Pan-Drug Resistant (PDR) and Extensively Drug-Resistant (XDR) Acinetobacter (A.) baumannii strains as they are difficult to eradicate. From 1600 clinical specimens, only 100 A. baumannii isolates could be recovered. A high prevalence of ≥ 78% resistant isolates was recorded for the recovered isolates against a total of 19 tested antimicrobial agents. These isolates could be divided into 12 profiles according to the number of antimicrobial agents to which they were resistant. The isolates were assorted as XDR (68; 68%), Multi-Drug Resistant (MDR: 30; 30%), and PDR (2; 2%). Genotypically, the isolates showed three major clusters with similarities ranging from 10.5 to 97.8% as revealed by ERIC-PCR technique. As a resistance mechanism to fluoroquinolones (FQs), target site mutation analyses in gyrA and parC genes amplified from twelve selected A. baumannii isolates and subjected to sequencing showed 12 profiles. The selected isolates included two CIP-susceptible ones, these showed the wild-type profile of being have no mutations. For the ten selected CIP-resistant isolates, 9 of them (9/10; 90%) had 1 gyrA/1 parC mutations (Ser 81 → Leu mutation for gyrA gene and Ser 84 → Leu mutation for parC gene). The remaining CIP-resistant isolate (1/10; 10%) had 0 gyrA/1 parC mutation (Ser 84 → Leu mutation for parC gene). Detection of plasmid-associated resistance genes revealed that the 86 ciprofloxacin-resistant isolates carry qnrA (66.27%; 57/86), qnrS (70.93%; 61/86), aac (6')-Ib-cr (52.32%; 45/86), oqxA (73.25%; 63/86) and oqxB (39.53%; 34/86), while qepA and qnrB were undetected in these isolates. Different isolates were selected from profiles 1, 2, and 3 and qnrS, acc(6,)-ib-cr, oqxA, and oqxB genes harbored by these isolates were amplified and sequenced. The BLAST results revealed that the oqxA and oqxB sequences were not identified previously in A. baumannii but they were identified in Klebsiella aerogenes strain NCTC9793 and Klebsiella pneumoniae, respectively. On the other hand, the sequence of qnrS, and acc(6,)-ib-cr showed homology to those of A. baumannii. MDR, XDR, and PDR A. baumannii isolates are becoming prevalent in certain hospitals. Chromosomal mutations in the sequences of GyrA and ParC encoding genes and acquisition of PAFQR encoding genes (up to five genes per isolate) are demonstrated to be resistance mechanisms exhibited by fluoroquinolones resistant A. baumannii isolates. It is advisable to monitor the antimicrobial resistance profiles of pathogens causing nosocomial infections and properly apply and update antibiotic stewardship in hospitals and outpatients to control infectious diseases and prevent development of the microbial resistance to antimicrobial agents.

Plasma Damage Control: From Biomolecules to Cells and Skin.

The antibacterial and cell-proliferative character of atmospheric pressure plasma jets (APPJs) helps in the healing process of chronic wounds. However, control of the plasma-biological target interface remains an open issue. High vacuum ultraviolet/ultraviolet (VUV/UV) radiation and RONS flux from plasma may cause damage of a treated tissue; therefore, controlled interaction is essential. VUV/UV emission from argon APPJs and radiation control with aerosol injection in plasma effluent is the focus of this research. The aerosol effect on radiation is studied by a fluorescent target capable of resolving the plasma oxidation footprint. In addition, DNA damage is evaluated by plasmid DNA radiation assay and cell proliferation assay to assess safety aspects of the plasma jet, the effect of VUV/UV radiation, and its control with aerosol injection. Inevitable emission of VUV/UV radiation from plasmas during treatment is demonstrated in this work. Plasma has no antiproliferative effect on fibroblasts in short treatments (t < 60 s), while long exposure has a cytotoxic effect, resulting in decreased cell survival. Radiation has no effect on cell survival in the medium due to absorption. However, a strong cytotoxic effect on the attached fibroblasts without the medium is apparent. VUV/UV radiation contributes 70% of the integral plasma effect in induction of single- and double-strand DNA breaks and cytotoxicity of the attached cells without the medium. Survival of the attached cells increases by 10% when aerosol is introduced between plasma and the cells. Injection of aerosol in the plasma effluent can help to control the plasma-cell/tissue interaction. Aerosol droplets in the effluent partially absorb UV emission from the plasma, limiting photon flux in the direction of the biological target. Herein, cold and safe plasma-aerosol treatment and a safe operational mode of treatment are demonstrated in a murine model.

Antimicrobial susceptibilities and comparative whole genome analysis of two isolates of the probiotic bacterium Lactiplantibacillus plantarum, strain ATCC 202195.

A synbiotic containing Lactiplantibacillus plantarum [American Type Culture Collection (ATCC) strain identifier 202195] and fructooligosaccharide was reported to reduce the risk of sepsis in young infants in rural India. Here, the whole genome of two isolates of L. plantarum ATCC 202195, which were deposited to the ATCC approximately 20 years apart, were sequenced and analyzed to verify their taxonomic and strain-level identities, identify potential antimicrobial resistant genes and virulence factors, and identify genetic characteristics that may explain the observed clinical effects of L. plantarum ATCC 202195. Minimum inhibitory concentrations for selected antimicrobial agents were determined using broth dilution and gradient strip diffusion techniques. The two L. plantarum ATCC 202195 isolates were genetically identical with only three high-quality single nucleotides polymorphisms identified, and with an average nucleotide identity of 99.99%. In contrast to previously published reports, this study determined that each isolate contained two putative plasmids. No concerning acquired or transferable antimicrobial resistance genes or virulence factors were identified. Both isolates were sensitive to several clinically important antibiotics including penicillin, ampicillin and gentamicin, but resistant to vancomycin. Genes involved in stress response, cellular adhesion, carbohydrate metabolism and vitamin biosynthesis are consistent with features of probiotic organisms.

Colistin Resistance in Aeromonas spp.

The increase in the use of antimicrobials such as colistin for the treatment of infectious diseases has led to the appearance of Aeromonas strains resistant to this drug. However, resistance to colistin not only occurs in the clinical area but has also been determined in Aeromonas isolates from the environment or animals, which has been determined by the detection of mcr genes that confer a resistance mechanism to colistin. The variants mcr-1, mcr-3, and mcr-5 have been detected in the genus Aeromonas in animal, environmental, and human fluids samples. In this article, an overview of the resistance to colistin in Aeromonas is shown, as well as the generalities of this molecule and the recommended methods to determine colistin resistance to be used in some of the genus Aeromonas.

Multidrug resistance IncC plasmid carrying blaCMY-97 in Shiga toxin-producing Escherichia coli ST215-H54 of ovine origin.

CMY-type ß-lactamases are the most reported plasmid-mediated AmpC (pAmpC), with the CMY-2-like group being the most clinically relevant described in Escherichia coli at human-animal-environment interface. Shiga toxin-producing E. coli (STEC) lineages are zoonotic pathogens commonly reported causing serious clinical conditions in humans, including severe diarrheagenic diseases. Therefore, this study aimed to investigate a multidrug-resistant (MDR) STEC isolate (A313) recovered from a healthy sheep and carrying mobile blaCMY-97, that encodes a pAmpC belonging to the CMY-2-like group. The A313 isolate exhibited a MDR profile to clinically relevant antimicrobials (i.e., cephalosporins, aminoglycosides, and fluoroquinolones), but reduced susceptibility to extended-spectrum cephalosporins and aztreonam. Besides, virulence genes (stx2, gad and iutA) were detected in A313, which belonged to ST215/CC10 and phylogenetic group A, whereas the fimH54 was identified. The blaCMY-97 gene and other antimicrobial resistance determinants [aph(6)-Id, aph(3″)-Ib, aac(3)-IId, aadA5, floR, tetA, sul1, and sul2], as well as genes encoding tolerance to mercury (merRTPCADE), were harbored by an IncC plasmid (named pA313-CMY-97, ~ 176 kb). A novel genetic context of blaCMY-2-like, in which a 208-bp ISEcp1 was truncated by an IS26 in the opposite orientation upstream of the blaCMY-97 gene (IS26-∆ISEcp1-blaCMY-97-blc-sugE-encR), was also identified in pA313-CMY-97. To the best of our knowledge, this is the first report on the acquisition of blaCMY-97 into a plasmid. Therefore, we reported ovine as reservoir of clinically relevant MDR bacteria carrying mobile blaCMY-97 with potential for zoonotic transmission.

High-risk clones of extended-spectrum ß-lactamase-producing Klebsiella pneumoniae isolated from the University Hospital Establishment of Oran, Algeria (2011-2012).

The purpose of the study was to characterize the resistome, virulome, mobilome and Clustered Regularly Interspaced Short Palindromic Repeats-associated (CRISPR-Cas) system of extended-spectrum ß-lactamase producing Klebsiella pneumoniae (ESBL-KP) clinical isolates and to determine their phylogenetic relatedness. The isolates were from Algeria, isolated at the University Hospital Establishment of Oran, between 2011 and 2012. ESBL-KP isolates (n = 193) were screened for several antibiotic resistance genes (ARGs) using qPCR followed by Pulsed-Field Gel Electrophoresis (PFGE). Representative isolates were selected from PFGE clusters and subjected to whole-genome sequencing (WGS). Genomic characterization of the WGS data by studying prophages, CRISPR-Cas systems, Multi-Locus Sequence Typing (MLST), serotype, ARGs, virulence genes, plasmid replicons, and their pMLST. Phylogenetic and comparative genomic were done using core genome MLST and SNP-Based analysis. Generally, the ESBL-KP isolates were polyclonal. The whole genome sequences of nineteen isolates were taken of main PFGE clusters. Sixteen sequence types (ST) were found including high-risk clones ST14, ST23, ST37, and ST147. Serotypes K1 (n = 1), K2 (n = 2), K3 (n = 1), K31 (n = 1), K62 (n = 1), and K151 (n = 1) are associated with hyper-virulence. CRISPR-Cas system was found in 47.4%, typed I-E and I-E*. About ARGs, from 193 ESBL-KP, the majority of strains were multidrug-resistant, the CTX-M-1 enzyme was predominant (99%) and the prevalence of plasmid-mediated quinolone resistance (PMQR) genes was high with aac(6')-lb-cr (72.5%) and qnr's (65.8%). From 19 sequenced isolates we identified ESBL, AmpC, and carbapenemase genes: blaCTX-M-15 (n = 19), blaOXA-48 (n = 1), blaCMY-2 (n = 2), and blaCMY-16 (n = 2), as well as non-ESBL genes: qnrB1 (n = 12), qnrS1 (n = 1) and armA (n = 2). We found IncF, IncN, IncL/M, IncA/C2, and Col replicon types, at least once per isolate. This study is the first to report qnrS in ESBL-KP in Algeria. Our analysis shows the concerning co-existence of virulence and resistance genes and would support that genomic surveillance should be a high priority in the hospital environment.

Tripodal scaffolds with three appended imidazole thiones for Cu(I) chelation and protection from Cu-mediated oxidative stress.

Imidazole thiones appear as interesting building blocks for Cu(I) chelation and protection against Cu-mediated oxidative stress. Therefore, a series of tripodal molecules derived from nitrilotriacetic acid appended with three imidazole thiones belonging either to histamine-like or histidine-like moieties were synthesized. These tripods demonstrate intermediate affinity between that previously measured for tripodal analogues bearing three thiol moieties such as cysteine and those grafted with three thioethers, like methionines, consistently with the thione group in the imidazole thione moiety existing as a tautomer between a thiol and a thione. The two non-alkylated tripods derived from thioimidazole, TH and TH* demonstrated three orders of magnitude larger affinity for Cu(I) (logKpH 7.4 = 14.3) than their analogues derived from N,N'-dialkylated thioimidazole TMe and TEt (logKpH 7.4 = 11-11.6). Their efficiency to inhibit Cu-mediated oxidative stress is demonstrated by several assays involving ascorbate consumption or biomolecule damages and correlates with their ability to chelate Cu(I), related to their conditional complexation constants at pH 7.4. The two non-alkylated tripods derived from thioimidazole, TH and TH* are significantly more powerful in reducing Cu-mediated oxidative stress than their analogues derived from N,N'-dialkylated thioimidazole TMe and TEt.

Novel Plasmid-Borne Fimbriae-Associated Gene Cluster Participates in Biofilm Formation in Escherichia coli.

This study reported the involvement of a gene cluster from a conjugative plasmid in the biofilm formation of Escherichia coli. We used a novel EZ-Tn5 transposon technique to generate a transposon library and used arbitrarily primed PCR to detect the insertion sites in biofilm formation-deficient mutants. To validate the function of candidate biofilm formation genes, the genes were cloned into plasmid pBluescript II SK (+) and transformed into E. coil DH5α. Biofilm production from the transformants was then assessed by phenotypic biofilm formation using Crystal Violet staining and microscopy. A total of 3,000 transposon mutants of E. coli DH5α-p253 were screened, of which 28 were found to be deficient in biofilm formation. Further characterization revealed that 24/28 mutations were detected with their insertions in chromosome, while the remaining 4 mutations were evidenced that the functional genes for biofilm formation were harbored in the plasmid. Interestingly, the plasmid sequencing showed that these four transposon mutations were all inserted into a fimbriae-associated gene cluster (fim-cluster). This fim-cluster is a hybrid segment spanning a 7,949 bp sequence, with a terminal inverted repeat sequence and two coding regions. In summary, we performed a high-efficiency screening to a library constructed with the EZ-Tn5-based transposon approach and identified the gene clusters responsible for the biofilm production of E. coli, especially the genes harbored in the plasmid. Further studies are needed to understand the spread of this novel plasmid-mediated biofilm formation gene in clinical E. coli isolates and the clinical impacts.

Development of multiplexing gene silencing system using conditionally induced polycistronic synthetic antisense RNAs in Escherichia coli.

Although efficient methods of gene silencing have been established in eukaryotes, many different techniques are still used in bacteria due to the lack of a standardized tool. Here, we developed a convenient and efficient method to downregulate the expression of a specific gene using ∼140 nucleotide RNA with a 24-nucleotide antisense region from an arabinose-inducible expression plasmid by taking Escherichia coli lacZ and phoA genes encoding ß-galactosidase and alkaline phosphatase, respectively, as target genes to evaluate the model. We examined the antisense RNA (asRNA) design, including targeting position, uORF stability elements at the 5'-end, and Hfq-binding module at the 3'-end, and inducer amount required to obtain effective experimental conditions for gene silencing. Furthermore, we constructed multiplexed dual-acting asRNA genes in the plasmid, which were transcribed as polycistronic RNA and were able to knockdown multiple target genes simultaneously. We observed the highest inhibition level of 98.6% when lacZ was targeted using the pMKN104 asRNA expression plasmid, containing a five times stronger PBAD -10 promoter sequence with no requirement of the Hfq protein for repression. These features allow the system to be utilized as an asRNA expression platform in many bacteria, besides E. coli, for gene regulation.

Expansion and persistence of antibiotic-specific resistance genes following antibiotic treatment.

Oral antibiotics are commonly prescribed to non-hospitalized adults. However, antibiotic-induced changes in the human gut microbiome are often investigated in cohorts with preexisting health conditions and/or concomitant medication, leaving the effects of antibiotics not completely understood. We used a combination of omic approaches to comprehensively assess the effects of antibiotics on the gut microbiota and particularly the gut resistome of a small cohort of healthy adults. We observed that 3 to 19 species per individual proliferated during antibiotic treatment and Gram-negative species expanded significantly in relative abundance. While the overall relative abundance of antibiotic resistance gene homologs did not significantly change, antibiotic-specific gene homologs with presumed resistance toward the administered antibiotics were common in proliferating species and significantly increased in relative abundance. Virome sequencing and plasmid analysis showed an expansion of antibiotic-specific resistance gene homologs even 3 months after antibiotic administration, while paired-end read analysis suggested their dissemination among different species. These results suggest that antibiotic treatment can lead to a persistent expansion of antibiotic resistance genes in the human gut microbiota and provide further data in support of good antibiotic stewardship.Abbreviation: ARG - Antibiotic resistance gene homolog; AsRG - Antibiotic-specific resistance gene homolog; AZY - Azithromycin; CFX - Cefuroxime; CIP - Ciprofloxacin; DOX - Doxycycline; FDR - False discovery rate; GRiD - Growth rate index value; HGT - Horizontal gene transfer; NMDS - Non-metric multidimensional scaling; qPCR - Quantitative polymerase chain reaction; RPM - Reads per million mapped reads; TA - Transcriptional activity; TE - Transposable element; TPM - Transcripts per million mapped reads.

Biomimetic Hydrogels Loaded with Nanofibers Mediate Sustained Release of pDNA and Promote In Situ Bone Regeneration.

Polymer hydrogels are generally insufficient biomechanics, strong resistance to cell adhesion, and weak bioactivity which limits their application in bone tissue engineering considerably. In order to develop a bone tissue engineering material with both good mechanical properties, osteogenic and angiogenic activity. Nanofibers carrying DNA plasmid (pNF) are introduced to gelatin methacryloyl (GelMA) and thiolated chitosan (TCS) system for preparing a novel GelMA/TCS/pNF composite hydrogel with dual network structure. By characterization of the compressive measurements, the resulting composite scaffold shows greatly enhanced mechanical strength (0.53 MPa) and is not damaged after 20 cycles of compression. And the fabricated composite scaffold displays sustained release of bone morphogenetic protein-2 that can induce osteogenic differentiation and angiopoietin-1 that promotes vascularization. The cell experiment shows that this system can significantly promote MC3T3-E1 cell attachment, proliferation, as well as osteogenic-related and angiogenic-related genes expression of MC3T3-E1 cells. Moreover, the in vivo results show that the composite scaffold with activated gene fibers can significantly promote osteogenesis and vascularization leading to favorable capacity of bone regeneration, meaning that the resulting biomimetic composite hydrogel scaffolds are excellent candidates for bone repair materials.

Collateral sensitivity associated with antibiotic resistance plasmids.

Collateral sensitivity (CS) is a promising alternative approach to counteract the rising problem of antibiotic resistance (ABR). CS occurs when the acquisition of resistance to one antibiotic produces increased susceptibility to a second antibiotic. Recent studies have focused on CS strategies designed against ABR mediated by chromosomal mutations. However, one of the main drivers of ABR in clinically relevant bacteria is the horizontal transfer of ABR genes mediated by plasmids. Here, we report the first analysis of CS associated with the acquisition of complete ABR plasmids, including the clinically important carbapenem-resistance conjugative plasmid pOXA-48. In addition, we describe the conservation of CS in clinical E. coli isolates and its application to selectively kill plasmid-carrying bacteria. Our results provide new insights that establish the basis for developing CS-informed treatment strategies to combat plasmid-mediated ABR.

Prevalence and Molecular Characterization of Extended Spectrum ß-Lactamase, Plasmid-Mediated Quinolone Resistance, and Carbapenemase-Producing Gram-Negative Bacilli in Burkina Faso.

The spreading of carbapenemase-producing gram-negative bacilli (GNB) must be considered as an "urgent" threat. The aim of this study was to determine the prevalence of extended spectrum ß-lactamase (ESBL), plasmid-mediated quinolone resistance (PMQR), and carbapenemase-producing GNB and to characterize the supporting genes in GNB specimens isolated from patients and healthy volunteers in Burkina Faso. From April to June 2016, carbapenemase-producing GNB screening was performed in 1,230 consecutive clinical specimens, and 158 fecal samples from inpatients and healthy volunteers without digestive pathology at Souro Sanou University Hospital, Bobo Dioulasso. Strains were identified by matrix-assisted laser desorption ionization-time of flight and antimicrobial susceptibility was tested with the disk diffusion method on Müller-Hinton agar. The presence of carbapenemase, ESBL, and PMQR genes was assessed by multiplex PCR. The molecular epidemiological study was performed using multilocus sequence typing analysis. From the 1,230 clinical samples, 443 GNB strains were isolated among which 4 (0.9%) were carbapenemase-producing isolates (Escherichia coli, n = 1; Acinetobacter baumannii, n = 3). Among the 158 fecal samples tested for carbapenemase-producing Enterobacteriaceae carriage, 13 (8.2%) were carbapenemase-producing isolates (E. coli, n = 4; Klebsiella pneumoniae, n = 6; A. baumannii, n = 2; Acinetobacter nosocomialis, n = 1; Acinetobacter bereziniae, n = 1). The strains from the two groups were resistant to broad-spectrum cephalosporins (100% for both), gentamicin (100% and 64.3%), levofloxacin (100% and 85.7%), and to amikacin (0% and 7.1%). The carbapenemase-encoding genes blaNDM-1, blaOxa-58, blaOxa-181, and blaVIM-2 were detected in clinical and in fecal samples. The majority (10/11) of the enterobacterial strains carried also blaCTX-M-15. The majority of the strains belonged to ST692 for E. coli, to ST147 for K. pneumoniae and to ST2 for A. baumannii. This study confirms the presence of carbapenemase-producing GNB in samples from patients and healthy volunteers. More effective active surveillance activities are needed.

The Gut Microbiome as a Reservoir for Antimicrobial Resistance.

This review will consider the gut as a reservoir for antimicrobial resistance, colonization resistance, and how disruption of the microbiome can lead to colonization by pathogenic organisms. There is a focus on the gut as a reservoir for ß-lactam and plasmid-mediated quinolone resistance. Finally, the role of functional metagenomics and long-read sequencing technologies to detect and understand antimicrobial resistance genes within the gut microbiome is discussed, along with the potential for future microbiome-directed methods to detect and prevent infection.

Whole-genome sequence analysis of Salmonella Infantis isolated from raw chicken meat samples and insights into pESI-like megaplasmid.

There has been an increase in the number of reports on Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) isolated from animals and humans. Recent studies using whole genome sequencing (WGS) have provided evidence on the likely contribution of a unique conjugative megaplasmid (pESI; ~280 kb) to the dissemination of this serovar worldwide. In the present study, twenty-two unrelated Salmonella strains [S. Infantis (n = 20) and Salmonella 6,7:r:- (n = 2)] and their plasmids were investigated using next generation sequencing technologies (MiSeq and MinION) to unravel the significant expansion of this bacteria in Turkey. Multi-locus sequence typing, plasmid replicons, resistance gene contents as well as phylogenetic relations between strains were determined. According to the WGS data, all S. Infantis possessed the relevant megaplasmid backbone genes and belonged to sequence type 32 (ST32) with the exception of a single novel ST7091. Tetracycline and trimethoprim/sulfamethoxazole resistance were found to be widespread in S. Infantis strains and the resistant strains exclusively carried the tetA, sul1, sul2 and dfrA14 genes. One S. Infantis isolate was also a carrier of the plasmid-mediated ampC via blaCMY-2, gene. Moreover, full genomes of four S. Infantis isolates were reconstructed based on hybrid assembly. All four strains contained large plasmids (240-290 kb) similar to previously published megaplasmid (pESI) and accompanied by several small plasmids. The megaplasmid backbone contained a toxin-antitoxin system, two virulence cassettes and segments associated with heavy metals resistance, while variable regions possessed several antibiotic resistance genes flanked by mobile elements. This study indicated that pESI-like megaplasmid is widely disseminated within the tested S. Infantis strains of chicken meat, warranting further genomic studies on clinical strains from humans and animals to uncover the overall emergence and spread of this serovar.

Synthesis and evaluation of etoposide and podophyllotoxin analogs against topoisomerase IIα and HCT-116 cells.

Etoposide is a widely-used anticancer agent that targets human type II topoisomerases. Evidence suggests that metabolism of etoposide in myeloid progenitor cells is associated with translocations involved in leukemia development. Previous studies suggest halogenation at the C-2' position of etoposide reduces metabolism. Halogens were introduced into the C-2' position by electrophilic aromatic halogenation onto etoposide (ETOP, 1), podophyllotoxin (PPT, 2), and 4-dimethylepipodophyllotoxin (DMEP, 3), and to bridge the gap of knowledge regarding the activity of these metabolically stable analogs. Five halogenated analogs (6-10) were synthesized. Analogs 8-10 displayed variable ability to inhibit DNA relaxation. Analog 9 was the only analog to show concentration-dependent enhancement of Top2-mediated DNA cleavage. Dose response assay results indicated that 8 and 10 were most effective at decreasing the viability of HCT-116 and A549 cancer cell lines in culture. Flow cytometry with 8 and 10 in HCT-116 cells provide evidence of sub-G1 cell populations indicative of apoptosis. Taken together, these results indicate C-2' halogenation of etoposide and its precursors, although metabolically stable, decreases overall activity relative to etoposide.

Hormetic dose-dependent response about typical antibiotics and their mixtures on plasmid conjugative transfer of Escherichia coli and its relationship with toxic effects on growth.

Bacterial resistance caused by the abuse of antibiotics has attracted worldwide attention. However, there are few studies exploring bacterial resistance under the environmental exposure condition of antibiotics that is featured by low-dose and mixture. In this study, sulfonamides (SAs), sulfonamide potentiators (SAPs) and tetracyclines (TCs) were used to determine the effects of antibiotics on plasmid RP4 conjugative transfer of Escherichia coli (E. coli) under single or combined exposure, and the relationship between the effects of antibiotics on conjugative transfer and growth was investigated. The results show that the effects of single or binary antibiotics on plasmid RP4 conjugative transfer all exhibit a hormetic phenomenon. The linear regression reveals that the concentrations of the three antibiotics promoting conjugative transfer are correlated with the concentrations promoting growth and the physicochemical properties of the compounds. The combined effects of SAs-SAPs and SAs-TCs on plasmid conjugative transfer are mainly synergistic and antagonistic. While SAPs provide more effective concentrations for the promotion of conjugative transfer in SAs-SAPs mixtures, SAs play a more important role in promoting conjugative transfer in SAs-TCs mixtures. Mechanism explanation shows that SAs, SAPs and TCs inhibit bacterial growth by acting on their target proteins DHPS, DHFR and 30S ribosomal subunit, respectively. This study indicates that toxic stress stimulates the occurrence of conjugative transfer and promotes the development of bacterial resistance, which will provide a reference for resistance risk assessment of antibiotic exposure.

Preliminary view of the global distribution and spread of the tet(X) family of tigecycline resistance genes.

BACKGROUND: The emergence of plasmid-mediated tet(X3)/tet(X4) genes is threatening the role of tigecycline as a last-resort antibiotic to treat clinical infections caused by XDR bacteria. Considering the possible public health threat posed by tet(X) and its variants [which we collectively call 'tet(X) genes' in this study], global monitoring and surveillance are urgently required. OBJECTIVES: Here we conducted a worldwide survey of the global distribution and spread of tet(X) genes. METHODS: We analysed a comprehensive dataset of bacterial genomes in conjunction with surveillance data from our laboratory and the NCBI database, as well as sufficient metadata to characterize the results. RESULTS: The global distribution features of tet(X) genes were revealed. We clustered three types of genetic backbones of tet(X) genes embedded or transferred in bacterial genomes. Our pan-genome analyses revealed a large genetic pool composed of tet(X)-carrying sequences. Moreover, phylogenetic trees of tet(X) genes and tet(X)-like proteins were built. CONCLUSIONS: To the best of our knowledge, our results provide the first view of the global distribution of tet(X) genes, demonstrate the features of tet(X)-carrying fragments and highlight the possible evolution of tigecycline-inactivation enzymes in diverse bacterial species and habitats.

Antibiotics Interfere with the Evolution of Plasmid Stability.

Extra-chromosomal genetic elements are important drivers of bacterial evolution, and their evolutionary success depends on positive selection for the genes they encode. Examples are plasmids encoding antibiotic resistance genes that are maintained in the presence of antibiotics (e.g., [1-3]). Plasmid maintenance is considered a metabolic burden to the host [4]; hence, when the cost of plasmid carriage outweighs its benefit, plasmid-free segregants are expected to outcompete plasmid-carrying cells, eventually leading to plasmid loss [5-7]. Thus, in the absence of positive selection, plasmid survival hinges upon stable persistence in the population. The ubiquity of plasmids in nature suggests that plasmids having a negligible effect on host fitness may evolve stable inheritance and thus gain a long-term persistence in the population, also in the absence of positive selection [8]. Nonetheless, the transition of plasmids into stably inherited genetic elements remains understudied. Here, we show that positive selection for a plasmid-encoded gene interferes with the evolution of plasmid stability. Evolving plasmids under different selection regimes in Escherichia coli, we find that antibiotics led to plasmid amplification, resulting in plasmid instability. Thus, under positive selection, suboptimal solutions for plasmid stability were maintained in the population hindering long-term plasmid persistence. Indeed, a survey of Escherichia plasmids confirms that antibiotic resistance genes are rarely found on small plasmids. Our results show that a plasmid-mediated advantage for the host may manifest in reduced plasmid evolutionary success. Considering plasmids as autonomously evolving entities holds promise for understanding the factors that govern their evolution.