RESUMO
OBJECTIVES: To elucidate the mechanism of tigecycline resistance in Escherichia coli that is mediated by the tet(A) variant gene. METHODS: E. coli strain 573 carried a plasmid-borne tet(A) variant gene, tentatively designated tet(A)TIG, that conferred decreased tigecycline susceptibility (MIC 0.5â mg/L). When exposed to increasing concentrations of tigecycline (0.25-8â mg/L), mutants growing at 2, 4 and 8â mg/L were obtained and sequenced. Copies of plasmid and tet(A)TIG relative to the chromosomal DNA in the mutants were determined by WGS and quantitative PCR (qPCR). Expression of tet(A)TIG in the mutants was evaluated by RT-qPCR. The tet(A)TIG-carrying plasmids were visualized by S1-PFGE and Southern blot hybridization. PCR served for the detection of a tet(A)TIG-carrying unconventional circularizable structure (UCS). RESULTS: Tigecycline resistance with maximum MICs of 16â mg/L was seen in E. coli mutants selected in the presence of tigecycline. Compared with the parental strain, the relative copy number and transcription level of tet(A)TIG in the mutants increased significantly in the presence of 2, 4 and 8â mg/L tigecycline, respectively. With increasing tigecycline selection pressure, the tet(A)TIG-carrying plasmids in the mutants increased in size, correlating with the number of tandem amplificates of a ΔTnAs1-flanked UCS harbouring tet(A)TIG. These tandem amplificates were not stable in the absence of tigecycline. CONCLUSIONS: Tigecycline resistance is due to the tandem amplification of a ΔTnAs1-flanked tet(A)TIG-carrying plasmid-borne segment in E. coli. The gain/loss of the tandem amplificates in the presence/absence of tigecycline represents an economic way for the bacteria to survive in the presence of tigecycline.
Assuntos
Antibacterianos , Escherichia coli , Testes de Sensibilidade Microbiana , Plasmídeos , Tigeciclina , Tigeciclina/farmacologia , Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Plasmídeos/genética , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Minociclina/farmacologia , Minociclina/análogos & derivados , Amplificação de Genes , Farmacorresistência Bacteriana/genética , Sequenciamento Completo do Genoma , AntiportersRESUMO
The dissemination mechanism of the high-level tigecycline resistance gene tet(X4) in porcine Escherichia coli was investigated. tet(X4) and other antimicrobial resistance genes were located on the plasmids p1919D3-1 and p1919D62-1 and flanked by two or three copies of IS1 family elements, which can form one to three translocatable units (TUs). Using a reduced transposition model, IS1A was experimentally demonstrated to mediate the transposition of tet(X4) from a suicide plasmid into the E. coli chromosome.
Assuntos
Infecções por Escherichia coli , Escherichia coli , Animais , Antibacterianos/farmacologia , Elementos de DNA Transponíveis , Farmacorresistência Bacteriana/genética , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/genética , Humanos , Plasmídeos/genética , Suínos , Tigeciclina/farmacologiaRESUMO
OBJECTIVES: To identify the genetic context and the transferability of the multiresistance gene lsa(E) in Listeria monocytogenes. METHODS: MICs were determined by broth microdilution. Transferability of lsa(E) was investigated by conjugation, electrotransformation and natural transformation. The lsa(E)-carrying plasmid was sequenced using the Illumina MiSeq and PacBio RSII platforms. The presence of translocatable units (TUs) was examined by PCR. RESULTS: The 85 555 bp non-conjugative multiresistance plasmid pNH1 from L. monocytogenes harboured nine antimicrobial resistance genes including a multiresistance gene cluster, consisting of the genes aphA3, erm(B), aadE, spw, lsa(E) and lnu(B), and in addition the genes dfrG, tet(S) and catA8 were also located on plasmid pNH1 The multiresistance gene cluster, and each of the genes tet(S), catA8 and cadA were flanked by IS1216 elements. PCR identified four types of TUs, consisting of either the multiresistance gene cluster and one copy of IS1216, the catA8 gene and one copy of IS1216, or both, but also the tet(S) gene and one copy of IS1216, respectively. Natural transformation into Streptococcus mutans UA159 yielded transformants that harboured a novel 13 208 bp transposon, designated Tn6659. This transposon consisted of the multiresistance gene cluster bounded by IS1216 copies. All transformants displayed elevated MICs of the respective antimicrobial agents. At the integration site in the transformants, 8 bp direct target duplications (5'-ATTCAAAC-3') were found immediately up- and downstream of Tn6659. CONCLUSIONS: To the best of our knowledge, this is the first report of this novel multiresistance gene cluster and the gene catA8, flanked by IS1216 elements located on a plasmid of L. monocytogenes. Moreover, a novel functionally active multiresistance transposon was identified.
Assuntos
Listeria monocytogenes , Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla , Listeria monocytogenes/genética , Testes de Sensibilidade Microbiana , Família Multigênica , Plasmídeos/genéticaAssuntos
Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Plasmídeos/genética , beta-Lactamases/genética , Animais , Antibacterianos/farmacologia , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Carbapenêmicos/farmacologia , Escherichia coli/isolamento & purificação , Testes de Sensibilidade Microbiana , SuínosRESUMO
Biomass-based hydrogel is a promising flame-retardant material and has a high potential for applications in transportation, aerospace, building and electrical engineering, and electronics. However, rapid vat photopolymerization (VP) 3D printing of biomass-based hydrogels, especially that of all-natural ones, is still rare. Herein, a new class of VP 3D-printed hydrogels with strong covalent networks, fabricating using fully biomass materials and a commercial liquid crystal display (LCD) printer assembled with low-intensity visible light is presented. Encouragingly, the highly ordered layer-by-layer packing structures provided by VP 3D printing technology endow these hydrogels with remarkable flame retardancy, exceptional temperature resistance, advantageous combustion behaviors, and favorable mechanical strength, in particular, giving them a better limit oxygen index (83.5%) than various biomass-based hydrogels. The proposed approach enables the green design as well as the precise and efficient preparation for flame-retardant materials, paving the way for the future flame-retardant materials toward attaining green sustainability.
RESUMO
Lincomycin is widely used in respiratory and gastrointestinal infection in veterinary medicine and food animal production. Campylobacter members are vital foodborne pathogens causing campylobacteriosis, and the resistance to lincosamides is seldom reported. To date, only the rRNA methyltransferase Erm(B) has been confirmed to be associated with lincosamides resistance in Campylobacter. In this study, we identified a lnu(C) variant conferring lincomycin resistance in this pathogen of chicken origin. The Lnu(C) encoded by this gene variant showed substitution at position 8 (Asn8Lys), 11 (Phe11Leu) and 112 (Leu112Phe), when compared with the firstly reported Lnu(C) from Streptococcus agalactiae. Cloning of the lnu(C) variant into lincosamide-susceptible Campylobacter jejuni NCTC 11168 confirmed its function in conferring resistance to lincomycin with the 32-fold increased MICs. Sequencing analysis showed that the lnu(C) variant was located within a MTnSag1-like transposon together with insLNU, which is inserted between panB and cj0299 genes on the chromosome. lnu(C) gene was distributed among C. coli globally, and various STs were involved in the dissemination of lnu(C). Although transposition mediated by MTnSag1-like transposon failed to occur, the horizontal transfer mediated by natural transformation and reservoir for resistance genes may facilitate their adaptation to the antimicrobial selection pressure in chickens, which should not be ignored.
Assuntos
Infecções por Campylobacter , Campylobacter coli , Campylobacter jejuni , Campylobacter , Animais , Lincomicina/farmacologia , Antibacterianos/farmacologia , Galinhas , Campylobacter coli/genética , Farmacorresistência Bacteriana/genética , Lincosamidas/farmacologia , Campylobacter jejuni/genética , Testes de Sensibilidade MicrobianaRESUMO
OBJECTIVE: To investigate the prevalence of a tet(A) gene variant and its role in developing high-level tigecycline resistance among carbapenem-resistant Klebsiella pneumoniae (CRKP) clinical isolates. METHODS: The mechanism of high-level tigecycline resistance in CRKP mediated by a tet(A) variant was explored by induction experiments, antimicrobial susceptibility testing, whole-genome sequencing and bioinformatics analysis. The amplification and overexpression of the tet(A) variant were measured by the determination of sequencing depth, gene copy numbers, and qRT-PCR. RESULTS: A high rate (62.1%, 998/1607) of tet(A) variant carriage was observed among 1607 CRKP clinical isolates from Henan Province, China. High-level tigecycline resistance could rapidly develop by the amplification of the tet(A) variant in these isolates. The analysis of the raw sequencing data and the plasmid mapping depth revealed that the ΔtnpA homologous sequence of Tn1721 supports the amplification of the region that harbours the tet(A) variant by forming a large number of repeat arrays through translocatable units (TUs). Moreover, the epidemiological analysis of tet(A) variant-carrying structures among 1607 clinical CRKPs showed that the TU structure is widely present. CONCLUSION: The presence of a tigecycline resistance-mediating tet(A) variant in CRKP clinical isolates represents a greater health concern than initially thought and should be monitored consistently.
RESUMO
Tigecycline and carbapenems are last-resort antimicrobial agents to treat serious infections caused by multi-drug resistant bacterial pathogens. However, the co-occurrence of tigecycline and carbapenem resistance determinants challenges the clinical efficacy of these antimicrobial agents. In this study, we report the co-existence of tet(X4), bla NDM-1 and bla OXA-58 genes in the porcine Acinetobacter towneri isolate 19110F47. Sequence analysis revealed that tet(X4) gene, along with the florfenicol resistance gene floR, was flanked by three copies of IS91-like elements, which can form three different translocatable units (TUs), and were located in a 41,098-bp multidrug resistance region (MDRR) within a novel 100,354-bp genomic island (GI) region. TUs comprising floR-virD2-ISVsa3, hp-abh-tet(X4)-ISVsa3 and virD2-floR-ISVsa3-hp-abh-tet(X4)-ISVsa3 can be looped out from the chromosomal DNA and facilitate the transfer of the TU-based resistance genes into other plasmidic or chromosomal sites. In addition, the carbapenemase genes bla NDM-1 and bla OXA-58 were found on different non-conjugative multiresistance plasmids in this isolate, with the genetic contexts ISAba125-bla NDM-1-ble MBL-tnpR and ΔISAba3-bla OXA-58 -ISAba3, respectively. The simultaneous occurrence of tet(X4), bla NDM-1 and bla OXA-58 in the same porcine A. towneri isolate emphasizes the importance of antimicrobial resistance surveillance in food-producing animals.
RESUMO
The emergence of the tet(X) gene is a severe challenge to global public health security, as clinical tigecycline resistance shows a rapidly rising trend. In this research, we identified two tigecycline-resistant Acinetobacter sp. strains containing seven novel tet(X3) variants recovered from fecal samples from Chinese farms. The seven Tet(X3) variants showed 15.4% to 99.7% amino acid identity with Tet(X3). By expressing tet(X3.7) and tet(X3.9), the tigecycline MIC values for Escherichia coli JM109 increased 64-fold (from 0.13 to 8 mg/L). However, the other tet(X3) variants did not have a significant change in the MIC of tigecycline. We found that the 26th amino acid site of Tet(X3.7) changed from proline to serine, and the 25th amino acid site of Tet(X3.9) changed from glycine to alanine, which reduced the MIC of tigecycline by 2-fold [the MIC of tet(X3) to tigecycline was 16 mg/L] but did not affect its expression to tigecycline. The tet(X3) variants surrounded by mobile genetic elements appeared in the structure of gene clusters with tandem repeat sequences and were adjacent to the site-specific recombinase-encoding gene xerD. Therefore, there is a risk of horizontal transfer of resistant genes. Our study reports seven novel tet(X3) variants; the continuing emergence of tigecycline variants makes continuous monitoring of resistance to tigecycline even more critical. IMPORTANCE Although it is illegal to use tigecycline and carbapenems to treat bacterial infections in animals, we can still isolate bacteria containing both mobile resistance genes from animals, and tet(X) is currently an essential factor in degrading tigecycline. Here, we characterized two multidrug-resistant Acinetobacter sp. strains that contained vital resistance genes, such as sul2, a blaOXA-164-like gene, floR, tetM, and multiple novel tet(X3) variants with different tandem structures. It is of paramount significance that their mechanism may transfer to other Gram-negative pathogens, even if their tandem structures have no cumulative effect on tigecycline resistance.
Assuntos
Acinetobacter , Antibacterianos , Proteínas de Escherichia coli , Tigeciclina , Acinetobacter/efeitos dos fármacos , Acinetobacter/genética , Aminoácidos , Antibacterianos/farmacologia , Proteínas de Escherichia coli/genética , Integrases/genética , Testes de Sensibilidade Microbiana , Plasmídeos , Tigeciclina/farmacologiaRESUMO
To investigate the contribution of a tet(A) variant to tigecycline resistance in Enterobacter hormaechei and the recombination events that occurred during transmission of this variant. MICs were determined by broth microdilution. E. hormaechei G17 was characterized by PCR, transfer assay, S1-PFGE, Southern blot hybridization, and WGS analysis. A tet(A) variant conferring resistance to tigecycline was present in E. hormaechei G17. This strain harbored two resistance plasmids (pG17-1, 264,084 bp and pG17-2, 68,610 bp) and its E. coli transformant Tm-G17TGC one resistance plasmid (pTm-G17, 93,013 bp). The comparative analysis of pG17-1, pG17-2, and pTm-G17 showed that a tet(A) variant-carrying multiresistance gene cluster (~23 kb) originating from pG17-1 had integrated into pG17-2, forming the novel plasmid pTm-G17. In a first step, this multiresistance gene cluster was excised from pG17-1 by recombination of homologous sequences, including â³TnAs1 at both termini, thereby generating an unconventional circularizable structure (UCS). In a second step, this UCS integrated into pG17-2 via recombination between homologous sequences, including IS26 present on both, the UCS and pG17-2, thereby giving rise to the new plasmid pTm-G17. In summary, a tet(A) variant conferring resistance to tigecycline was reported in E. hormaechei. Transfer of a tet(A) variant-carrying multiresistance gene cluster between plasmids occurred in a two-step recombination process, in which homologous sequences, including either â³TnAs1 or IS26, were involved. IMPORTANCE Tigecycline is an important last-resort broad spectrum antimicrobial agent. This study describes the two-step recombination processes resulting in the transfer of the tet(A) variant gene between different plasmids in E. hormaechei, which depicts the role of recombination processes in the generation of UCSs and new plasmids, both carrying a tet(A) variant conferring resistance to tigecycline. Such processes enhance the dissemination of resistance genes, which is of particular relevance for resistance genes, such as the tet(A) variant. The presence and transmission of a tet(A) variant in E. hormaechei will compromise the efficacy of tigecycline treatment for E. hormaechei associated infection.
Assuntos
Antibacterianos , Escherichia coli , Antibacterianos/farmacologia , Enterobacter , Escherichia coli/genética , Testes de Sensibilidade Microbiana , Plasmídeos/genética , Recombinação Genética , Tigeciclina/farmacologiaRESUMO
Carbapenems and tigecycline are two important classes of antimicrobial agents to treat the infections caused by Enterobacterales. Here, we reported a plasmid carrying both bla IMP-26 and tet(A) variant in clinical Klebsiella pneumoniae KP-1572. MIC results showed that K. pneumonia KP-1572 was resistant to a wide range of antimicrobials. The bla IMP-26 and tet(A) variant were located on an identical plasmid, which was indicated by S1-PFGE and southern blotting hybridization and can be successfully transferred by electroporation. Whole-plasmid sequencing and analysis revealed that a 142,993-bp-sized plasmid, designated pIMP1572, contains an IncFIIk backbone and a variable region harboring bla IMP-26 and tet(A) variant. The plasmid pIMP1572 was apparently originated from a tet(A)-carrying IncFIIk plasmid but with a deletion length of 6,216-bp and a multiple drug resistance region (MDRR) insertion of 25,259 bp. The plasmid pIMP1572 in the present study represents the first report of the IncFIIk plasmid co-carrying bla IMP and tet(A) variant, which should be monitored.