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1.
Citrus tristeza virus (CTV) is one of the most devastating pathogens of citrus. Its genome is organized into 12 open reading frames (ORFs), of which ten ORFs located at the 3′-terminus of the genome have multiple biological functions. The ten genes at the 3′-terminus of the genome of a severe isolate (CTV-S4) and three ORFs (CP, CPm and p20) of three other isolates (N4, S45 and HB1) were cloned into pGBKT7 and pGADT7 yeast shuttle vectors. Yeast two-hybridization (Y2H) assays results revealed a strong self-interaction for CP and p20, and a unique interaction between the CPm of CTV-S4 (severe) and CP of CTV-N4 (mild) isolates. Bimolecular fluorescence complementation also confirmed these interactions. Analysis of the deletion mutants delineated the domains of CP and p20 self-interaction. Furthermore, the domains responsible for CP and p20 self-interactions were mapped at the CP amino acids sites 41–84 and p20 amino acids sites 1–21 by Y2H. This study provided new information on CTV protein interactions which will help for further understanding the biological functions.  相似文献   

2.
ObjectivesTo characterize Alcaligenes faecalis metallo-β-lactamase (MBL) AFM-2 and AFM-3 from clinical Pseudomonas aeruginosa isolates NDTH10366, NDTH9845 and WTJH17.MethodsClinical isolates were whole-genome sequenced using the Illumina and Oxford Nanopore platforms. MICs of clinical isolates and transformants containing MBL genes were determined using broth microdilution methods. Kinetic parameters of purified AFM and NDM-1 were measured using a spectrophotometer. The AFM structure was modelled with SWISS-MODEL.ResultsNDTH10366 and NDTH9845 were extensively drug-resistant (XDR) isolates carrying blaAFM-2 and multiple copies of blaKPC-2, whereas WTJH17 was an XDR isolate carrying blaAFM-3. The plasmid-borne blaAFM-2 and blaAFM-3 genes are associated with a novel ISCR element, ISCR29. AFM-2 and AFM-3, differing from AFM-1 by one amino acid substitution each, shared 86.2% and 86.6% amino acid sequence identity with NDM-1, respectively. Phylogenetic analysis confirmed the close relationship between AFM and NDM. Expression of AFM and NDM-1 under their native promoters in DH5α and PAO1 led to elevated MICs for all tested β-lactams except aztreonam. Comparable catalytic abilities were observed for AFM and NDM-1 when hydrolysing nitrocefin, cefepime, imipenem and biapenem, whereas for other tested β-lactams AFM displayed weaker enzymatic activities. Modelling AFM structure revealed a characteristic αβ/βα fold with two zinc-binding active sites.ConclusionsAFM from clinical P. aeruginosa isolates demonstrated β-lactamase activity comparable to NDM-1. Co-carriage of blaAFM and blaKPC renders clinical P. aeruginosa isolates non-susceptible to all antipseudomonal β-lactams. The association of blaAFM genes with translocatable genetic elements and plasmids highlights their concerning potential for dissemination.  相似文献   

3.
The in vitro activity of doripenem was evaluated against a recent collection of extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae and Pseudomonas aeruginosa isolates (201 ESBL-producing Enterobacteriaceae [153 Escherichia coli and 48 Klebsiella pneumoniae] and 201 P. aeruginosa). Comparator agents included amikacin, tobramycin, ciprofloxacin, cefepime, cefotaxime, ceftazidime piperacillin-tazobactam, imipenem, and meropenem. Both doripenem and meropenem inhibited 100% of the ESBL-producing Enterobacteriaceae at ≤0.5 μg/mL. For these isolates, the MIC90 of doripenem (0.12 μg/mL) was 4-fold lower than that of imipenem (0.5 μg/mL). Against P. aeruginosa, the MIC90 of doripenem and meropenem was 2 μg/mL, 4-fold lower than that of imipenem (8 μg/mL). At an MIC of ≤2 μg/mL, doripenem, meropenem, and imipenem inhibited 90.5%, 89.6%, and 82.1% of P. aeruginosa isolates, respectively. Doripenem maintained activity against imipenem-nonsusceptible isolates of P. aeruginosa; at an MIC of ≤4 μg/mL, it inhibited 15 of the 25 isolates with MICs for imipenem of >4 μg/mL. Doripenem is active against ESBL-producing Enterobacteriaceae and P. aeruginosa isolates. Its activity is similar to that of meropenem and slightly better than that of imipenem. The results of this study suggest that doripenem could be an alternative therapeutic agent for infections caused by these organisms.  相似文献   

4.
5.
Carbapenems have been considered as last line antibiotics for treatment of multidrug-resistant (MDR) Acinetobacter baumannii but carbapenem resistant A. baumannii has been increased during the last decade in many parts of the world. OXA-type β-lactamase enzymes are the most common cause of carbapenem resistance in A. baumannii and presence of ISAba1 in upstream of these genes may increase the expression of these OXA genes. The aim of this study was to determine, for the first time, the antibiotic resistance pattern and prevalence of OXA type β-lactamases among nosocomial A. baumannii isolates from northwest of Iran. A total of 100 A. baumannii isolates were recovered from hospitalized patients in a university hospital in northwest of Iran. Sixty-two percent of isolates were resistant to imipenem. All isolates carried bla(OXA-51)-like gene. Among imipenem resistant isolates, 88.7% carried bla(OXA-23)-like, 1.6% carried bla(OXA-40)-like, and 3.2% had bla(OXA-58)-like resistance genes. Ninety percent of isolates contained ISAba1 element and in 74.2% of imipenem resistant isolates, ISAba1 was located in upstream of bla(OXA-23)-like. The results of this study demonstrated high prevalence of OXA-type carbapenemase among MDR A. bumanii in the Northwest of Iran.  相似文献   

6.
We characterized 34 methicillin-resistant Staphylococcus aureus strains isolated in Paraguay in 2005. The strains belonged to two clones. The major clone (sequence type 5 [ST5] or ST221, spa type t149, staphylococcal cassette chromosome mec [SCCmec] type I) was similar to the Cordobes/Chilean clone spreading through South America, and the minor clone (ST239 or ST889, spa type t037, SCCmec type IIIA) was related to the Brazilian clone.  相似文献   

7.
Nontuberculous mycobacteria (NTM) that cannot be identified to the species level by reverse line blot hybridization assays and sequencing of the 16S rRNA gene comprise a challenge for reference laboratories. However, the number of 16S rRNA gene sequences added to online public databases is growing rapidly, as is the number of Mycobacterium species. Therefore, we re-analysed 178 Mycobacterium isolates with 53 previously unmatched 16S rRNA gene sequences, submitted to our national reference laboratory in 1999–2007. All sequences were again compared with the GenBank database sequences and the isolates were re-identified using two commercially available identification kits, targeting separate genetic loci. Ninety-three out of 178 isolates (52%) with 20 different 16S rRNA gene sequences could be assigned to validly published species. The two reverse line blot assays provided false identifications for three recently described species and 22 discrepancies were recorded in the identification results between the two reverse line blot assays. Identification by reverse line blot assays underestimates the genetic heterogeneity among NTM. This heterogeneity can be clinically relevant because particular sub-groupings of species can cause specific disease types. Therefore, sequence-based identification is preferable, at least at the reference laboratory level, although the exact targets needed for clinically useful results remain to be established. The number of NTM species in the environment is probably so high that unidentifiable clinical isolates should be given a separate species status only if this is clinically meaningful.  相似文献   

8.
A total of 3160 clinical isolates of Escherichia coli from intra-abdominal infections were collected during 2008–2009 from 13 European countries. The frequency of extended-spectrum β-lactamase (ESBL)-producing isolates in Europe was 11%. The most active antibiotics tested were typically imipenem, ertapenem, and amikacin, although the activity of all non-carbapenem antibiotics was lower when tested against ESBL-positive isolates than when tested against ESBL-negative isolates. Ertapenem exhibited 99.3% susceptibility with all isolates, and 96.8% susceptibility with ESBL-positive isolates. With application of the ertapenem CLSI clinical breakpoint for resistance (MIC ≥1 mg/L), only six isolates (0.2%) were ertapenem-resistant, and only three of these were available for molecular characterization. Of those three, only one was ESBL-positive (CTX-M-14), and two were carbapenemase-positive (OXA-48). All three were negative for, VIM, NDM and KPC carbapenemases. Although the level of ertapenem resistance in E. coli is very low, further monitoring of ertapenem susceptibility and molecular characterization of ertapenem-resistant isolates is needed.  相似文献   

9.
The study reports for the first time the identification of CTX-M-14-like and CTX-M-27-like extended-spectrum β-lactamases (ESBLs) belonging to the CTX-M-9 group in Klebsiella pneumoniae and Escherichia coli isolated from the neonatal stool in India. The plasmid carrying the blaCTX-M-9 group in both the isolates was transferable. Till date, no other CTX-M group, except the CTX-M-1 group, has been reported from India. A total of 77% of the neonates had ESBL-producing K. pneumoniae or E. coli in their stool, and blaCTX-M-15 was the predominant ESBL gene. Although the CTX-M-9 group was found in the stool and did not cause infection, the detection of the CTX-M-9 group might be a prelude to future infections.  相似文献   

10.
Four phenotypic methods (three dimensional test, AmpC test, cloxacillin synergy test and cefotetan/cefotetan-cloxacillin E-test) to detect plasmid-mediated AmpC β-lactamases (pAmpC) were compared in 125 clinical Enterobacteriaceae isolates with AmpC profile: 74 E. coli (bla (CMY-2): 70; bla (DHA-1): 4), five K. pneumoniae (bla (CMY-2): 2; bla (DHA-1): 3), six P. mirabilis (bla (CMY-2): 6) and 40 negative isolates for pAmpC β-lactamases. All evaluated methods showed a good sensitivity (>95%) but low values of specificity (<60%) in E. coli, explained by an increase of AmpC expression caused by chromosomal ampC promoter/attenuator mutations (-42, -18, -1, +58, predominantly). The cefotetan/cefotetan-cloxacillin or cloxacillin synergy test may be advocated as phenotypic screening test, and the AmpC test as confirmatory test for detection of pAmpC in isolates that lack or minimally express chromosomally encoded AmpC β-lactamases. In the case of E. coli, the phenotypic evaluated tests were not able to differentiate between chromosomal ampC overexpression or acquisition of plasmid-encoded ampC genes.  相似文献   

11.
Purpose: Klebsiella pneumoniae is considered an important pathogen causing nosocomial and community-acquired infections and is often associated with the production of extended-spectrum β-lactamases (ESBL) belonging to SHV and CTX-M families, which are frequently described as a part of complex integrons, facilitate their horizontal transfer to other related as well as unrelated microbes. The present study was undertaken to investigate the occurrence and characterization of integrons among K pneumoniae isolates producing ESBL in a tertiary referral hospital. Materials and Methods: A total of 136 clinical isolates of K pneumoniae were investigated for the presence of ESBL. Their ESBL genes were characterized by multiplex polymerase chain reaction (PCR). Integrase gene PCR was performed to detect the presence of integron. The isolates were further typed by random amplification of polymorphic DNA (RAPD). Result: Out of 136 K pneumoniae isolates, 63 (46%) were confirmed to be ESBL producers. SHV (68%) and CTX–M (67%) ESBL genes were the most common in our study. Of the 63 ESBL-positive isolates, 58 (92%) strains carried integrons; 52 strains (82%) carried only class 1 integron, whereas 6 (9%) isolates harboured both class 2 integrons and the class 1 gene. However, in ESBL negatives, only 29 (40%) strains were positive for class 1 integron and none for class 2 integron. Conclusion: The presence of class 2 integron amongst ESBL-producing K pneumoniae is being described for the first time in this part of the world. The findings of this study strongly suggest that integrons have a role in the dissemination of ESBL-mediated resistance among the nosocomial isolates of K pneumonia.  相似文献   

12.
Two carbapenem-resistant Klebsiella pneumoniae isolates producing the plasmid-encoded carbapenem-hydrolyzing OXA-48 were identified. These isolates, recovered from two patients hospitalized in two different hospitals in Tunisia in December 2010, were not clonally related. Molecular investigations showed that both isolates co-produced the narrow-spectrum β-lactamases TEM-1 and SHV-1, together with the extended-spectrum β-lactamase CTX-—15.  相似文献   

13.
Campylobacter jejuni O:41 strains are found in association with Guillain-Barré syndrome in South Africa. Strains of this serotype collected over 17 years were characterized by amplified fragment length polymorphism and flagellin typing to determine their clonal nature. Despite minor variation in GM1 expression, all of the strains were genetically indistinguishable, indicating that they are representative of a genetically stable clone.  相似文献   

14.
The objective of this study was to investigate the observation of daptomycin resistance in Corynebacterium striatum, both in vivo and in vitro. We describe a case of C. striatum bacteremia in a patient with a left ventricular assist device (LVAD); the initial isolate recovered was daptomycin susceptible with a minimum inhibitory concentration (MIC) of 0.125 μg/ml. Two months later, and after daptomycin therapy, the individual became bacteremic with an isolate of C. striatum with a daptomycin MIC of >256 μg/ml. To study the prevalence of daptomycin resistance in C. striatum, clinical isolates of C. striatum were grown in broth culture containing daptomycin to investigate the emergence of resistance to this antimicrobial. Molecular typing was used to evaluate serial isolates from the index patient and the clinical isolates of C. striatum we assayed. In vitro analysis of isolates from the index patient and 7 of 11 additional C. striatum isolates exhibited the emergence of high-level daptomycin resistance, despite initially demonstrating low MICs to this antimicrobial agent. This phenotype was persistent even after serial subculture in the absence of daptomycin. Together, these data demonstrate that caution should be taken when using daptomycin to treat high-inoculum infections and/or infections of indwelling medical devices with C. striatum. To our knowledge, this is the first report characterizing the emergence of daptomycin resistance in C. striatum.  相似文献   

15.
In order to assess the prevalence of carbapenem-hydrolyzing class D β-lactamase genes in Acinetobacter spp. isolates in China, we conducted a polymerase chain reaction (PCR)-based surveillance of OXA-type β-lactamase gene clusters for a total of 2,880 Acinetobacter spp. isolates collected from 23 Chinese provinces. All isolates were tested for susceptibility to 12 antimicrobial agents and showed high rates of resistance to all these agents except minocycline. We also found that the vast majority of carbapenem-resistant Acinetobacter spp. were OXA-23-like-producing isolates, predominantly Acinetobacter baumannii isolates. Besides, bla OXA-58-like and bla OXA-24-like genes were detected in 32 and 11 isolates, respectively, involving many provinces throughout China. Furthermore, these two carbapenem-resistance determinants were located on transferable plasmids in most cases, indicating an emerging threat for both OXA-58-like- and OXA-24-like-producing Acinetobacter spp. isolates in China. Interestingly, a novel homologue of the bla OXA-143 gene was identified in a susceptible Acinetobacter pittii isolate. Overall, these observations suggest that the bla OXA-23-harboring A. baumannii isolates are the most frequent carbapenem-resistant Acinetobacter spp. in China, and the bla OXA-24-like and bla OXA-58-like genes have emerged as potential threats of hospital outbreaks of multidrug-resistant Acinetobacter spp.  相似文献   

16.
Genetic analysis of group A rotavirus isolates detected in the feces of children admitted to hospitals in Novosibirsk and Omsk within four epidemic seasons—2007, 2007–2008, 2009–2010, and 2010–2011—was carried out. 1416 Group A rotavirus isolates were genotyped by the multiplex PCR method. It is shown that, in 2007–2011 in Western Siberia, isolates of the widespread genotypes G1P[8], G4P[8], G2P[4], and G3P[8] cocirculated. In a few cases, genotypes G9P[8], G2P[8], G3P[9], and G4P[6] have been determined. In 2008 in Omsk and in 2009 in Novosibirsk, dominant genotype G1P[8] was replaced by genotype G4P[8]. The occurrence and spectrum of circulating genotypes differed and changed every epidemic season in both cities. Phylogenetic analysis of nucleotide sequences of three genome fragments that encode proteins VP4 (VP8*), VP7, and VP6 showed that most of the Novosibirsk and Omsk isolates clustered together and exhibited a high degree of homology with isolates identified in other regions of Eurasia. In addition, for the first time in Novosibirsk, 14 rotavirus isolates (genotypes G9, G1 and G4) related to the rare subline P[8]b (OP354-like) of the gene encoding the protein VP4 were detected, while in Omsk only the isolate Omsk08-381/G9P[8]b was found. The results obtained in this study indicate the need for long-term monitoring of rotavirus isolates circulating in Western Siberia, which is important when choosing a rotavirus vaccine to immunize young children and to improve the diagnostic kits for the infection, as well as to understand the epidemiology and evolution of group A rotaviruses.  相似文献   

17.
Equine infectious anaemia virus (EIAV) is classified within the Retroviridae and, like other lentivirus, has the propensity for considerable antigenic variation. An extensive phylogenetic analysis in Bayesian fashion, with significant amounts of new EIAV gag sequence information, revealed a strong geographic compartmentalization clearly related to the phylogeographic history of modern horses, pointing out that New World EIAV strains form a distinct group with a potentially common origin. This evidence suggests that a single founder event may have occurred during the reintroduction of horses to the Americas by European colonists in the 15th century, a possibility that raises many interesting scenarios with implications for all evolutionary and ecological studies.  相似文献   

18.
19.
Antimicrobial resistance (AMR) in Neisseria gonorrhoeae is a major health threat compromising the gonorrhoea treatment globally. AMR surveillance including whole genome sequencing (WGS)-based epidemiology provides ideal resolution to identify and describe AMR gonococcal clones, AMR determinants and populations, which can inform management guidelines and antimicrobial stewardship policies. Our aims were to, for the first time, elucidate the WGS-based epidemiology and characterize AMR determinants of gonococcal strains spreading in Ukraine, 2013–2018. Gonococcal isolates (n = 150) from Ternopil and Dnipro, Ukraine (2013–2018), were subjected to AMR testing (Etest) for eight antimicrobials and WGS. Overall, 11.3% of isolates were resistant to ciprofloxacin, 6.0% to tetracycline, and 0.7% to benzylpenicillin. No isolates were resistant to azithromycin, spectinomycin, ceftriaxone, or cefixime, but one isolate was bordering resistance to both cephalosporins. Twenty-five MLST STs, 50 NG-MAST STs, and 34 NG-STAR types were identified. The phylogenomic analysis revealed six main clusters, mostly associated with the internationally described multidrug-susceptible gonococcal lineage. Resistance to ciprofloxacin was associated with GyrA S91F and ParC S87R mutations; tetracyclines with rpsJ V57M and tetM; penicillins with mosaic penA-34.001 and β-lactamase; mtrR; PorB1b G101D, and PBP1 L421P mutations. One isolate of the multidrug-resistant NG-MAST ST1407, MLST ST1901 was found, which was bordering resistance to ceftriaxone and cefixime. The antimicrobial susceptibility of gonococcal strains spreading in Ternopil and Dnipro, Ukraine, in 2013–2018 was surprisingly high. Continued and expanded gonococcal AMR surveillance, ideally including WGS, in Ukraine is essential. This could inform action plans and public health policies to control the spread of AMR gonococcal strains in Ukraine.  相似文献   

20.
We investigated the prevalence of metallo-β-lactamases (MBLs) among 1,827 Enterobacteriaceae isolates collected in 2006 and evaluated the VITEK 2 microbiology system, modified Hodge test, and 2 combined disk tests as the screening tools for MBLs by using these isolates and 77 previously characterized IMP-8 producers. The IMP-8 MBL was identified in 18 isolates of 2006, and the IMP-8-positive isolates represented 0.2%, 1.1%, and 5.0% of all Escherichia coli, Klebsiella pneumoniae, and Enterobacter cloacae isolates, respectively. Only one-third of all MBL producers could be recognized by either VITEK 2 or the Hodge test. MBL production could be identified in 38 (40%) of the 95 IMP-8-producing isolates by the combined disk test using meropenem disks supplemented by phenylboronic acid and EDTA, and only 2 (2.1%) isolates gave positive results in the combined disk test using meropenem disks supplemented with dipicolinic acid. Of all IMP-8 producers, 37.9%, 50.5%, and 32.6% were nonsusceptible to tigecycline, fluoroquinolones, and both, respectively. In conclusion, this study demonstrated the lack of distinct phenotypes that could be easily identified among the IMP-8-producing Enterobacteriaceae isolates at a Taiwanese hospital. Continuous surveillance and monitoring are needed because the widespread of tigecycline- and fluoroquinolone-coresistant MBL producers may become a serious therapeutic problem.  相似文献   

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