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1.
Virulence ; 13(1): 77-88, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34951562

RESUMO

The extensive use of tetracycline antibiotics has led to the widespread presence of tetracycline-resistance genes in Gram-negative bacteria and this poses serious threats to human and animal health. In our previous study, we reported a method for rapid detection of Tet(X)-producers using MALDI-TOF MS. However, there have been multiple machineries involved in tetracycline resistance including efflux pump, and ribosomal protection protein. Our previous demonstrated the limitation in probing the non-Tet(X)-producing tetracycline-resistant strains. In this regard, we further developed a MALDI-TOF MS method to detect and differentiate Tet(X)-producers and non-Tet(X)-producing tetracycline-resistant strains. Test strains were incubated with tigecycline and oxytetracycline in separate tubes for 3 h and then analyzed spectral peaks of tigecycline, oxytetracycline, and their metabolite. Strains were distinguished using MS ratio for [metabolite/(metabolite+ tigecycline or oxytetracycline)]. Four control strains and 319 test strains were analyzed and the sensitivity was 98.90% and specificity was 98.34%. This was consistent with the results obtained from LC-MS/MS analysis. Interestingly, we also found that the reactive oxygen species (ROS) produced by tetracycline-susceptible strains were able to promote the degradation of oxytetracycline. Overall, the MALDITet(X)-plus test represents a rapid and reliable method to detect Tet(X)-producers, non-Tet(X)-producing tetracycline-resistant strains, and tetracycline-susceptible strains.

2.
Sci Total Environ ; 806(Pt 2): 150687, 2022 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-34597551

RESUMO

The emergence of novel plasmid-mediated high-level tigecycline resistance genes tet(X) in the Enterobacteriaceae has increased public health risk for treating severe bacterial infections. Despite growing reports of tet(X)-positive isolates detected in animal sources, the epidemiological association of animal- and environment-derived isolates with human-derived isolates remains unclear. Here, we performed a comprehensive analysis of tet(X4)-positive Escherichia coli isolates collected in a hospital in Guangdong province, China. A total of 48 tet(X4)-positive E. coli isolates were obtained from 1001 fecal samples. The tet(X4)-positive E. coli isolates were genetically diverse but certain strains that belonged to ST48, ST10, and ST877 etc. also have clonally transmitted. Most of the tet(X4) genes from these patient isolates were located on conjugative plasmids that were successfully transferred (64.6%) and generally coexisted with other antibiotic resistance genes including aadA, floR, blaTEM and qnrS. More importantly, we found the IncX1 type plasmid was a common vector for tet(X4) and was prevalent in these patient-derived strains (31.3%). This plasmid type has been detected in animal-derived strains from different species in different regions demonstrating its strong transmission ability and wide host range. Furthermore, phylogenetic analysis revealed that certain strains of patient and animal origin were closely related indicating that the tet(X4)-positive E. coli isolates were likely to have cross-sectorial clonal transmission between humans, animals, and farm environments. Our research greatly expands the limited epidemiological knowledge of tet(X4)-positive strains in clinical settings and provides definitive evidence for the epidemiological link between human-derived tet(X4)-positive isolates and animal-derived isolates.


Assuntos
Farmacorresistência Bacteriana , Escherichia coli , Animais , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Humanos , Testes de Sensibilidade Microbiana , Filogenia , Plasmídeos/genética
3.
Microbiol Spectr ; : e0116421, 2021 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-34935428

RESUMO

The emergence of tet(X) genes has compromised the clinical use of the last-line antibiotic tigecycline. We identified 322 (1.21%) tet(X) positive samples from 12,829 human microbiome samples distributed in four continents (Asia, Europe, North America, and South America) using retrospective data from worldwide. These tet(X) genes were dominated by tet(X2)-like orthologs but we also identified 12 samples carrying novel tet(X) genes, designed tet(X45), tet(X46), and tet(X47), were resistant to tigecycline. The metagenomic analysis indicated these tet(X) genes distributed in anaerobes dominated by Bacteroidaceae (78.89%) of human-gut origin. Two mobile elements ISBf11 and IS4351 were most likely to promote the transmission of these tet(X2)-like orthologs between Bacteroidaceae and Riemerella anatipestifer. tet(X2)-like orthologs was also developed during transmission by mutation to high-level tigecycline resistant genes tet(X45), tet(X46), and tet(X47). Further tracing these tet(X) in single bacterial isolate from public repository indicated tet(X) genes were present as early as 1960s in R. anatipestifer that was the primary tet(X) carrier at early stage (before 2000). The tet(X2) and non-tet(X2) orthologs were primarily distributed in humans and food animals respectively, and non-tet(X2) were dominated by tet(X3) and tet(X4). Genomic comparison indicated these tet(X) genes were likely to be generated during tet(X) transmission between Flavobacteriaceae and E. coli/Acinetobacter spp., and ISCR2 played a key role in the transmission. These results suggest R. anatipestifer was the potential ancestral source of tet(X). In addition, Bacteroidaceae of human-gut origin was an important hidden reservoir and mutational incubator for the mobile tet(X) genes that enabled spread to facultative anaerobes and aerobes. IMPORTANCE The emergence of the tigecycline resistance gene tet(X) has posed a severe threat to public health. However, reports of its origin and distribution in human remain rare. Here, we explore the origin and distribution of tet(X) from large-scale metagenomic data of human-gut origin and public repository. This study revealed the emergency of tet(X) gene in 1960s, which has refreshed a previous standpoint that the earliest presence of tet(X) was in 1980s. The metagenomic analysis from data mining covered the unculturable bacteria, which has overcome the traditional bacteria isolating and purificating technologies, and the analysis indicated that the Bacteroidaceae of human-gut origin was an important hidden reservoir for tet(X) that enabled spread to facultative anaerobes and aerobes. The continuous monitoring of mobile tigecycline resistance determinants from both culturable and unculturable microorganisms is imperative for understanding and tackling the dissemination of tet(X) genes in both the health care and agricultural sectors.

4.
Front Microbiol ; 12: 755233, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34745062

RESUMO

We determined the prevalence and transmission characteristics of mcr-1-positive Escherichia coli (MCRPEC) isolates from migratory birds Anser indicus in Guangdong, China. We identified 22 MCRPEC from 303 A. indicus fecal samples (7.3%) in Guangzhou, Zhaoqing, and Futian. The mcr-1 gene coexisted with 24 other types of antibiotic resistance genes (ARG), and 11 ARGs were highly prevalent at levels >50%. The MCRPEC displayed a diversity of sequence types (ST), and 19 distinct STs were identified with ST10, ST1146, and ST1147 as the most prevalent. In addition, these MCRPEC from birds were closely related phylogenetically to those from other sources in China. Whole-genome sequencing analysis demonstrated that mcr-1 was located on IncX4 (n=9, 40.9%), IncI2 (n=5, 22.7%) and IncP (n=1, 4.5%) plasmids and the latter shared an identical plasmid backbone with other sources. These results highlight the significance of migratory birds in the transmission of antibiotic resistance and provide powerful evidence that migratory birds are potential transmitters of antibiotic resistance.

5.
Sci Total Environ ; : 151540, 2021 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-34767892

RESUMO

Flower is an essential element in the human lifestyle but its role in disseminating antimicrobial resistance (AMR) between the environment and humans is unclear. In this study, we screened fresh flowers (Lilium spp.) collected from planting bases, market and florists in Guangzhou China aiming to investigate the prevalence of AMR genes, particularly cfr, optrA and poxtA mediating resistance to linezolid, a first-line drug for the treatment of different Gram-positive bacterial infections. We found 223 Enterococcus isolates consisting of Enterococcus faecalis, Enterococcus faecium and Enterococcus mundtii, and >50% of these isolates exhibited multiple-drug resistance. Additionally, 31 optrA-positive Enterococcus including 22 E. faecalis and 9 E. mundtii strains were recovered, however cfr and poxtA were not detected. The 22 E. faecalis strains were belonged to 7 Multilocus sequence types in which ST202 and ST376 were predominant and 9 E. mundtii strains from the same plantation bases were divided into three PFGE groups. Genetically, the majority of optrA were located on the chromosome and shared similar insertion sites and transpositions mediated by Tn554 family members. Plasmid-bearing optrA were identified in 6 E. faecalis strains where IS1216 family played key roles in horizontal transfer of optrA. These findings emphasize that the prevalence of drug resistant Enterococcus in fresh flowers is a latent danger and increases the risk of AMR dissemination to humans from the environment.

6.
Artigo em Inglês | MEDLINE | ID: mdl-34726693

RESUMO

OBJECTIVES: To determine the transmission and molecular characteristics of blaNDM-producing Escherichia coli between companion animals and their healthcare providers at veterinary clinics in Guangzhou, China. METHODS: A total of 359 samples from companion animals and their healthcare providers were collected at 14 veterinary clinics in Guangzhou, China. Genomic characteristics and clonal relationships for blaNDM-positive E. coli and complete plasmid sequences were characterized based on WGS data from combined Illumina and MinION platform reads. RESULTS: Forty-five blaNDM-positive bacteria were recovered from companion animals (n = 43) and their healthcare providers (n = 2) at 10 veterinary clinics. Overall, E. coli (73.3%, 33/45) and Klebsiella pneumoniae (13.3%, 6/45) were the most prevalent species among the seven species of blaNDM-positive bacteria. Four blaNDM variants (blaNDM-1, blaNDM-4, blaNDM-5 and blaNDM-7) were identified in 45 blaNDM-positive bacteria and blaNDM-5 was the most prevalent (77.8%, 35/45). WGS indicated that the most prevalent STs were ST405 (8/33), ST453 (6/33), ST457 (6/33) and ST410 (5/33) among the 33 blaNDM-positive E. coli isolates. Phylogenomics and PFGE analysis revealed that clonal spread of blaNDM-positive ST453 E. coli isolates between companion animals and their healthcare providers was evident. In addition, two novel IncFIB plasmids carrying blaNDM-4 (pF765_FIB and pG908_FIB) were found in this study and indicated that IS26 may promote the horizontal transmission of blaNDM between different plasmid types. CONCLUSIONS: In this study we conducted a large-scale investigation on the prevalence of blaNDM-positive E. coli isolates from companion animals and their healthcare providers and revealed the clonal spread of blaNDM-positive E. coli isolates between these two groups.

7.
Artigo em Inglês | MEDLINE | ID: mdl-34613377

RESUMO

OBJECTIVES: In this study, we developed an IS26-based CRISPR/Cas9 system as a proof-of-concept study to explore the potential of a re-engineered bacterial translocatable unit (TU) for curing and immunizing against the replication genes and antimicrobial resistance genes. METHODS: A series of pIS26-CRISPR/Cas9 suicide plasmids were constructed, and specific guide RNAs were designed to target the replication gene of IncX4, IncI2 and IncHI2 plasmids, and the antibiotic resistance genes mcr-1, blaKPC-2 and blaNDM-5. Through conjugation and induction, the transposition efficiency and plasmid-curing efficiency in each recipient were tested. In addition, we examined the efficiency of the IS26-CRISPR/Cas9 system of cell immunity against the acquisition of the exogenous resistant plasmids by introducing this system into antimicrobial-susceptible hosts. RESULTS: This study aimed to eliminate the replication genes and antimicrobial resistance genes using pIS26-CRISPR/Cas9. Three plasmids with different replicon types, including IncX4, IncI2 and IncHI2 in three isolates, two pUC19-derived plasmids, pUC19-mcr-1 and pUC19-IS26mcr-1, in two lab strains, and two plasmids bearing blaKPC-2 and blaNDM-5 in two isolates were all successfully eliminated. Moreover, the IS26-based CRISPR/Cas9 system that remained in the plasmid-cured strains could efficiently serve as an immune system against the acquisition of the exogenous resistant plasmids. CONCLUSIONS: The IS26-based CRISPR/Cas9 system can be used to efficiently sensitize clinical Escherichia coli isolates to antibiotics in vitro. The single-guide RNAs targeted resistance genes or replication genes of specific incompatible plasmids that harboured resistance genes, providing a novel means to naturally select bacteria that cannot uptake and disseminate such genes.

8.
Front Microbiol ; 12: 716393, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34497596

RESUMO

Objectives: Carbapenems, colistin, and tigecycline are critically important antibiotics in clinics. After the global appearance of bla NDM and mcr mediating the resistance to carbapenems and colistin, respectively, tigecycline becomes the last-resort drug against severe human infections caused by multidrug-resistant bacteria. Recently, a mobile tigecycline resistance gene tet(X4) has been identified in Escherichia coli, Klebsiella pneumoniae, and Acinetobacter baumannii that causes high resistance to tigecycline and other tetracyclines. In this study, the prevalence of tet(X4) in E. coli isolates from duck and goose farms in Southeast China was identified and characterized. Methods: Feces, soil, sewage, and dust samples were collected from duck and goose farms along with the southeast coast provinces of China. Antimicrobial susceptibility testing and polymerase chain reaction screening were performed to investigate the phenotype and genotype of tigecycline resistance. Conjugation, S1 pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing were used to determine the transferability, genetic location, and the genomic characteristics of tet(X4). Results: In total, 1,716 samples were collected, and 16 isolates (0.9%) recovered from Guangdong, Shandong, and Jiangsu were positive for tet(X4) gene with tigecycline minimum inhibitory concentrations ≥16 mg/L. Notably, among these tet(X4)-positive E. coil isolates, seven of them were from the environment samples (soil and sewage). PFGE and multilocus sequence typing demonstrated that ST3997 was the most prevalent sequence type (eight isolates, 50%) in Jiangsu province. By conjugation assays, 11 isolates were able to transfer tet(X4) plasmid to E. coli C600 recipient, and these plasmids belonged to IncHI1 and IncX1 detected by sequence analysis. tet(X4) was found adjacent to an insertion sequence ISCR2 downstream and a catD gene upstream for all isolates. In addition, multiple-drug resistance to tigecycline, chlortetracycline, ampicillin, florfenicol, ciprofloxacin, gentamicin, trimethoprim/sulfamethoxazole, and fosfomycin was profiled in most of the tet(X4)-positive isolates. Conclusion: The identification of tet(X4) harboring E. coli strains in duck farms and their surrounding environment enlarges our knowledge of the variety and prevalence of tigecycline resistance. The prevalence of tet(X4) raises concern for the use of tetracyclines in animal farming, and the tet(X4) gene should be listed as primary gene for resistance surveillance.

9.
Antimicrob Agents Chemother ; 65(10): e0105421, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34339270

RESUMO

The global spread of antimicrobial-resistant bacteria has been one of the most severe threats to public health. The emergence of the mcr-1 gene has posed a considerable threat to antimicrobial medication since it deactivates one last-resort antibiotic, colistin. There have been reports regarding the mobilization of the mcr-1 gene facilitated by ISApl1-formed transposon Tn6330 and mediated rapid dispersion among Enterobacteriaceae species. Here, we developed a CRISPR/Cas9 system flanked by ISApl1 in a suicide plasmid capable of exerting sequence-specific curing against the mcr-1-bearing plasmid and killing the strain with chromosome-borne mcr-1. The constructed ISApl1-carried CRISPR/Cas9 system either restored sensitivity to colistin in strains with plasmid-borne mcr-1 or directly eradicated the bacteria harboring chromosome-borne mcr-1 by introducing an exogenous CRISPR/Cas9 targeting the mcr-1 gene. This method is highly efficient in removing the mcr-1 gene from Escherichia coli, thereby resensitizing these strains to colistin. The further results demonstrated that it conferred the recipient bacteria with immunity against the acquisition of the exogenous mcr-1 containing the plasmid. The data from the current study highlighted the potential of the transposon-associated CRISPR/Cas9 system to serve as a therapeutic approach to control the dissemination of mcr-1 resistance among clinical pathogens.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Antibacterianos/farmacologia , Sistemas CRISPR-Cas/genética , Cromossomos , Colistina/farmacologia , Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Plasmídeos/genética
10.
Sci Total Environ ; 799: 149360, 2021 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-34365265

RESUMO

Tetracycline antibiotics (TCs) are massively produced and consumed in various industries resulting in large quantities of residuals in the environment. In this study, to achieve safe and efficient removal of residual TCs, a Pichia pastoris (P. pastoris) was gained to stably express glycosylated TCs degrading enzyme Tet(X) followed codon and expression parameter optimization of tet(X4). As expected, glycosylated Tet(X) still maintains efficient capacity of degrading TCs. The expressed Tet(X) maintained efficient TCs degrading ability over a pH range of 6.5 - 9.5 and temperature range of 17 - 47 °C. We tested this recombinant protein for its ability to degrade tetracycline in pond water and sewage models of tetracycline removal at starting levels of 10 mg/L substrate. 80.5 ± 3.8% and 26.2 ± 2.6% of tetracycline was degraded within 15 min in the presence of 0.2 µM Tet(X) and 50 µM NADPH, respectively. More importantly, the direct use of a Tet(X) degrading enzymes reduces the risk of gene transmission during degradation. Thus, the Tet(X) degrading enzyme expressed by P. pastoris is an effective and safe method for treating intractable TCs residues.


Assuntos
Pichia , Tetraciclinas , Antibacterianos , Pichia/genética , Saccharomycetales , Água
11.
Antibiotics (Basel) ; 10(7)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202219

RESUMO

We determined the prevalence and molecular characteristics of fosfomycin-resistant Escherichia coli from a domestic pigeon farm. A total of 79 samples collected from pigeons and their surrounding environments were screened for the presence of fosfomycin resistant isolates and these included 49 E. coli isolates that displayed high-level resistance (MIC ≥ 256 mg L-1) and carried the fosA3 gene on plasmids with sizes ranging from 80 to 370 kb. MLST analysis of these fosA3-positive E. coli isolates indicated the presence of nine sequence types (ST6856, ST8804, ST457, ST746, ST533, ST165, ST2614, ST362 and ST8805) of which ST6856 was the most prevalent (24.5%, 12/49). PFGE combined with genomic context comparative analyses indicated that the fosA3 gene was spread by horizontal transfer as well as via clonal transmission between E. coli in the pigeon farm, and IS26 played an important role in fosA3 transmission. The high prevalence of fosA3 in the pigeon farm and the high similarity of the fosA3 genomic environment between E. coli isolates from humans and pigeons indicated that the pigeon farm served as a potential reservoir for human infections. The pigeon farm was found to be an important reservoir for the fosA3 gene and this should be further monitored.

12.
Front Microbiol ; 12: 677633, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34290681

RESUMO

This study aimed to determine the prevalence and transmission characteristics of New Delhi metallo ß-lactamase (NDM)-producing Escherichia coli from ducks in Guangdong, China. In this study, a total of 28 NDM-producing E. coli isolates were recovered from 88 unduplicated diseased duck samples (31.8%) from veterinary clinics in Guangzhou, Foshan, Qingyuan, and Huizhou. Two variants, bla NDM-1 and bla NDM-5, were detected and the latter was present in 89.6% of the isolates (25/28). Multilocus sequence typing (MLST) analysis indicated that these E. coli isolates possessed six distinct STs, and ST156 was the most prevalent followed by ST648, ST746, ST354, ST10, and ST162. In addition, phylogenomic analysis found that two of the isolates that were recovered from a single sample possessed different genomes, and the bla NDM-carrying IncX3 plasmids may be horizontal transfer between E. coli isolates in the intestinal tracts of ducks. Whole-genome sequencing (WGS) analysis further revealed that bla NDM co-existed with other 25 types of antimicrobial resistance genes (ARGs), of which 16 ARGs were highly prevalent with detection rates >50%, and a high incidence of coproducing bla NDM and mcr-1 E. coli isolates (22/88, 25.0%) was detected in ducks. This study underscores the importance of surveillance for bla NDM-harboring microbes in ducks.

13.
Microb Drug Resist ; 27(12): 1624-1632, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34077284

RESUMO

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.

14.
Antibiotics (Basel) ; 10(5)2021 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-34065054

RESUMO

This study aimed to determine the global distribution and molecular characteristics of carbapenemase-producing Pseudomonas aeruginosa isolates. A total of 328 (11.1%, 328/2953) carbapenemase-producing P. aeruginosa isolates from humans were obtained from public databases as of October 2019. Of which, the blaVIM and blaIMP genes were the most prevalent carbapenemases in the P. aeruginosa isolates. These carbapenemase-producing P. aeruginosa isolates possessed 34 distinct sequence types (STs) and six predominated: ST357, ST823, ST308, ST233, ST175 and ST111. The ST357 and ST823 isolates were primarily found detected in Asia and all ST175 isolates were found in Europe. The ST308, ST233 and ST111 isolates were spread worldwide. Further, all ST823 isolates and the majority of ST111, ST233 and ST175 isolates carried blaVIM but ST357 isolates primarily carried blaIMP. ST308 isolates provide a key reservoir for the spread of blaVIM, blaIMP and blaNDM. WGS analysis revealed that ST111 carried a great diversity of ARG types (n = 23), followed by ST357 (n = 21), ST308 (n = 19), ST233 (n = 18), ST175 (n = 14) and ST823 (n = 10). The ST175 isolates carried a more diversity and frequent of aminoglycoside ARGs, and ST233 isolates harbored more tetracycline ARGs. Our findings revealed that different carbapenem resistance genes were distributed primarily in variant STs of P. aeruginosa isolates, these isolates also possessed an extensive geographical distribution that highlights the need for surveillance studies that detect carbapenemase-producing P. aeruginosa isolates in humans.

15.
mSystems ; 6(3)2021 May 18.
Artigo em Inglês | MEDLINE | ID: mdl-34006624

RESUMO

The emergence of the plasmid-mediated high-level tigecycline resistance mechanism Tet(X) threatens the role of tigecycline as the "last-resort" antibiotic in the treatment of infections caused by carbapenem-resistant Gram-negative bacteria. Compared with that of the prototypical Tet(X), the enzymatic activities of Tet(X3) and Tet(X4) were significantly enhanced, correlating with high-level tigecycline resistance, but the underlying mechanisms remain unclear. In this study, we probed the key amino acid changes leading to the enhancement of Tet(X) function and clarified the structural characteristics and evolutionary path of Tet(X) based upon the key residue changes. Through domain exchange and site-directed mutagenesis experiments, we successfully identified five candidate residues mutations (L282S, A339T, D340N, V350I, and K351E), involved in Tet(X2) activity enhancement. Importantly, these 5 residue changes were 100% conserved among all reported high-activity Tet(X) orthologs, Tet(X3) to Tet(X7), suggesting the important role of these residue changes in the molecular evolution of Tet(X). Structural analysis suggested that the mutant residues did not directly participate in the substrate and flavin adenine dinucleotide (FAD) recognition or binding, but indirectly altered the conformational dynamics of the enzyme through the interaction with adjacent residues. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and UV full-wavelength scanning experiments confirmed that each mutation led to an increase in activity without changing the biochemical properties of the Tet(X) enzyme. Further phylogenetic analysis suggested that Riemerella anatipestifer served as an important incubator and a main bridge vector for the resistance enhancement and spread of Tet(X). This study expands the knowledge of the structure and function of Tet(X) and provides insights into the evolutionary relationship between Tet(X) orthologs.IMPORTANCE The newly emerged tigecycline-inactivating enzymes Tet(X3) and Tet(X4), which are associated with high-level tigecycline resistance, demonstrated significantly higher activities in comparison to that of the prototypical Tet(X) enzyme, threatening the clinical efficacy of tigecycline as a last-resort antibiotic to treat multidrug-resistant (MDR) Gram-negative bacterial infections. However, the molecular mechanisms leading to high-level tigecycline resistance remain elusive. Here, we identified 5 key residue changes that lead to enhanced Tet(X) activity through domain swapping and site-directed mutagenesis. Instead of direct involvement with substrate binding or catalysis, these residue changes indirectly alter the conformational dynamics and allosterically affect enzyme activities. These findings further broaden the understanding of the structural characteristics and functional evolution of Tet(X) and provide a basis for the subsequent screening of specific inhibitors and the development of novel tetracycline antibiotics.

16.
Antibiotics (Basel) ; 10(4)2021 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-33923861

RESUMO

Antimicrobial resistance is recognized as one of the major global health challenges of the 21st century. Synergistic combinations for antimicrobial therapies can be a good strategy for the treatment of multidrug resistant infections. We examined the ability of a group of 29 plant essential oils as substances which enhance the antibiotic activity. We used a modified well diffusion method to establish a high-throughput screening method for easy and rapid identification of high-level enhancement combinations against bacteria. We found that 25 essential oils possessed antibacterial activity against Escherichia Coli ATCC 25922 and methicillin-resistant Staphylococcus aureus (MRSA) 43300 with MICs that ranged from 0.01% to 2.5% v/v. We examined 319 (11 × 29) combinations in a checkerboard assay with E. Coli ATCC 25922 and MRSA 43300, and the result showed that high-level enhancement combinations were 48 and 44, low-level enhancement combinations were 214 and 211, and no effects combinations were 57 and 64, respectively. For further verification we randomly chose six combinations that included orange and Petitgrain essential oils in a standard time-killing assay. The results are in great agreement with those of the well diffusion assays. Therefore, the modified diffusion method was a rapid and effective method to screen high-level enhancement combinations of antibiotics and essential oils.

17.
Appl Environ Microbiol ; 87(10)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33674440

RESUMO

We investigated the prevalence and transmission of NDM-producing Enterobacteriaceae in fecal samples of geese and environmental samples from a goose farm in southern China. The samples were cultivated on MacConkey agar plates supplemented with meropenem. Individual colonies were examined for bla NDM, and bla NDM-positive bacteria were characterized based on whole-genome sequencing (WGS) data from the Illumina and Oxford Nanopore Technologies (ONT) platforms. Of 117 samples analyzed, the carriage rates for New Delhi metallo-ß-lactamase (NDM)-positive Enterobacteriaceae were 47.1, 18, and 50% in geese, inanimate environments (sewage, soil, fodder, and dust), and mouse samples, respectively. Two variants (bla NDM-1 and bla NDM-5, in 4 and 40 isolates, respectively) were found among 44 bla NDM-positive Enterobacteriaceae; these variants belonged to eight species, and Escherichia coli was the most prevalent (50%). WGS analysis revealed that bla NDM coexisted with diverse antibiotic resistance genes (ARGs). Population structure analysis showed that most E. coli and Enterobacter sp. isolates were highly heterogeneous, while most Citrobacter sp. and P. stuartii isolates possessed extremely high genetic similarities. In addition, bla NDM-5-positive ST4358/ST48 E. coli isolates were found to be clonally spread between geese and the environment and were highly genetically similar to those reported from ducks, farm environments, and humans in China. Plasmid analysis indicated that IncX3 pHNYX644-1-like (n = 40) and untypeable pM2-1-like plasmids (n = 4) mediated bla NDM spread. pM2-1-like plasmids possessed diverse ARGs, including bla NDM-1, the arsenical and mercury resistance operons, and the maltose operon. Our findings revealed that the goose farm is a reservoir for NDM-positive Enterobacteriaceae The bla NDM contamination of wild mice and the novel pM2-1-like plasmid described here likely adds to the risk for dissemination of bla NDM and associated resistance genes.IMPORTANCE Carbapenem-resistant bacteria, in particular NDM-producing Enterobacteriaceae, have become a great threat to global public. These bacteria have been found not only in hospital and community environments but also among food animal production chains, which are recognized as reservoirs for NDM-producing Enterobacteriaceae However, the dissemination of NDM-producing bacteria in waterfowl farms has been less well explored. Our study demonstrates that the horizontal spread of bla NDM-carrying plasmids and the partial clonal spread of bla NDM-positive Enterobacteriaceae contribute to the widespread contamination of bla NDM in the goose farm ecosystem, including mice. Furthermore, we found a novel and transferable bla NDM-1-carrying multidrug resistance (MDR) plasmid that possessed multiple environmental adaptation-related genes. The outcomes of this study contribute to a better understanding of the prevalence and transmission of bla NDM-carrying Enterobacteriaceae among diverse niches in the farm ecosystem.


Assuntos
Infecções por Enterobacteriaceae/microbiologia , Enterobacteriaceae/isolamento & purificação , Gansos/microbiologia , Doenças das Aves Domésticas/microbiologia , beta-Lactamases/genética , Animais , Antibacterianos/farmacologia , China , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/genética , Infecções por Enterobacteriaceae/veterinária , Fazendas , Fezes/microbiologia , Fômites/microbiologia , Camundongos , Testes de Sensibilidade Microbiana
18.
Front Microbiol ; 12: 586504, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33613474

RESUMO

We examined the prevalence and transmission of the fosA3 gene among Citrobacter freundii isolates from flowers and the retail environments. We identified 11 fosfomycin-resistant C. freundii strains (>256 µg/mL) from 270 samples that included petals (n = 7), leaves (n = 2), dust (n = 1) and water (n = 1). These 11 isolates were multidrug-resistant and most were simultaneously resistant to fosfomycin, cefotaxime, ciprofloxacin and amikacin. Consistently, all 11 isolates also possessed bla CTX-M- 14, bla CMY- 65 / 122, aac(6')-Ib-cr, qnrS1, qnrB13/6/38 and rmtB. These fosA3-positive isolates were assigned to two distinct PFGE patterns and one (n = 9) predominated indicating clonal expansion of fosA3-positive isolates across flower markets and shops. Correspondingly, fosA3 was co-transferred with bla CTX-M- 14 via two plasmid types by conjugation possessing sizes of 110 kb (n = 9) and 260 kb (n = 2). Two representatives were fully sequenced and p12-1 and pS39-1 possessed one and two unclassified replicons, respectively. These plasmids shared a distinctive and conserved backbone in common with fosA3-carrying C. freundii and other Enterobacteriaceae from human and food animals. However, the fosA3-bla CTX-M- 14-containing multidrug resistance regions on these untypable plasmids were highly heterogeneous. To the best of our knowledge, this is the first report of fosA3 and bla CTX-M- 14 that were present in bacterial contaminants from flower shops and markets. These findings underscore a public health threat posed by untypable and transferable p12-1-like and pS39-1-like plasmids bearing fosA3-bla CTX-M- 14 that could circulate among Enterobacteriaceae species and in particular C. freundi in environmental isolates.

20.
Sci Total Environ ; 771: 144828, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33545481

RESUMO

Overuse of antibiotics in animal husbandry has led to an increase of antibiotic resistance microorganisms as well as antibiotic-resistance genes (ARGs). Duck farming in China is practiced on a large and diverse scale and the overuse of antibiotics in this field is gaining attention recently. We evaluated the diversity of ARGs from five duck farms using a functional metagenomic approach and constructed five libraries. A total of seventy-six resistant determinants were identified, of which sixty-one were gene variants or novel genes. The novel genes contained five ß-lactamase-encoding genes designated as blaDWA1, blaDWA2, blaDWA3, blaDWA4 and blaDWB1, respectively, and two genes conferring resistance to fosfomycin designated as fosA-like1 and fosA-like2. Three of the five ß-lactamase-encoding genes were further identified as extended-spectrum ß-lactamases (ESBL) that can hydrolyze both penicillins and cephalosporins. Besides, two of the five ß-lactamase-encoding genes were associated with mobile genetic elements, indicating a high potential for transfer of the genes to other bacterial hosts. The two novel fosA-like genes were able to increase the MICs of the test Escherichia coli strain from 2 µg/mL to as high as 256 µg/mL(up to 128-fold increase). Our study provides a reference for ARGs prevalence in duck farm wastes and implies that they are an important resistome reservoir, especially for novel ARGs with high spread potential.


Assuntos
Antibacterianos , Patos , Animais , Antibacterianos/farmacologia , China , Resistência Microbiana a Medicamentos/genética , Genes Bacterianos , beta-Lactamases/genética
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