Mutations in aarE, the ubiA Homolog of Providencia stuartii, Result in High-Level Aminoglycoside Resistance and Reduced Expression of the Chromosomal Aminoglycoside 2′-N-Acetyltransferase

The aarE1 allele was identified on the basis of the resulting phenotype of increased aminoglycoside resistance. The aarE1 mutation also resulted in a small-colony phenotype and decreased levels of aac(2′)-Ia mRNA. The deduced AarE gene product displayed 61% amino acid identity to the Escherichia coli UbiA protein, an octaprenyltransferase required for the second step of ubiquinone biosynthesis. Complementation experiments in both Providencia stuartii and E. coli demonstrated that aarE and ubiA are functionally equivalent.     

氨基糖苷类高水平耐药肠球菌的耐药及分子机制的研究

目的分析氨基糖苷类高水平耐药（HighLevelAminoglyeosideResistant,HLAR）肠球菌的耐药性与耐药基因,并研究其耐药分子机制。方法采用VITEK-2全自动鉴定仪法测定53株氨基糖苷类高水平耐药肠球菌对11种抗菌药物的最小抑菌浓度,用聚合酶链反应（PCR）检测肠球菌转座子Tn917和Tnl546／Tn916、红霉素耐药基因er—mB、毒力岛基因mefA、I类整合酶Intll、氯霉素耐药基cat、表面蛋白基因esp,并对万古霉素耐药肠球菌（Vancomy—cin—resistantEnterococc,VRE）进行基因分型（vanA、vanB、vanC）。结果53株肠球菌检出含ermB26株,占49．0％;含mefA9株,占17．0％;含Tnl546／Tn91619株,占35．8％;含Tn91711株,占20．8％;含Intll26株,占49．1％;含esp15株,占28．3％;未检出cat。53株肠球菌对复方新诺明全部耐药,对红霉素耐药高达94．3％,对高水平庆大霉素、高水平链霉素、环丙沙星、四环素和氨苄西林的耐药率分别为75．5％、73．6％、75．5％、66．0％、62．3％;对利奈唑胺和替考拉宁的敏感率高达98．1％;1株耐万古霉素,其基因和表型均为VanA。结论HLAR肠球菌可携带多种耐药基因,耐药基因与肠球菌耐药性密切相关,可能在多重耐药机制中起重要作用。     

Molecular Characterization of Acquired Enrofloxacin Resistance in Mycoplasma synoviae Field Isolates

The in vitro activity of enrofloxacin against 73 Mycoplasma synoviae field strains isolated in Israel and Europe was determined by broth microdilution. Decreased susceptibility to enrofloxacin was identified in 59% of strains, with the MICs ranging from 1 to >16 μg/ml. The estimated MIC₅₀ and MIC₉₀ values for enrofloxacin were 2 and 8 μg/ml, respectively. Moreover, this study showed that 92% of recent Israeli field isolates (2009 to 2011) of M. synoviae have MICs of ≥2 μg/ml to enrofloxacin. Comparison of the quinolone resistance-determining regions (QRDRs) in M. synoviae isolates revealed a clear correlation between the presence of one of the amino acid substitutions Asp79-Asn, Thr80-Ala/Ile, Ser81-Pro, and Asp84-Asn/Tyr/His of the ParC QRDR and decreased susceptibility to enrofloxacin (MIC, ≥1 μg/ml). Amino acid substitutions at positions GyrA 87, GyrB 401/402, and ParE 420/454 were also identified, but there was no clear-cut correlation with susceptibility to enrofloxacin. Comparison of vlhA molecular profiles revealed the presence of 9 different genotypes in the Israeli M. synoviae field isolates and 10 genotypes in the European isolates; only one vlhA genotype (type 4) was identified in both cohorts. Based on results of vlhA molecular typing, several mechanisms for emergence and dissemination of Israeli enrofloxacin-resistant M. synoviae isolates are suggested.     

Overexpression and Characterization of the Chromosomal Aminoglycoside 2′-N-Acetyltransferase of Providencia stuartii

The gene coding for aminoglycoside 2'-N-acetyltransferase Ia [AAC(2')-Ia] from Providencia stuartii was amplified by PCR and cloned. The resulting construct, pACKF2, was transferred into Escherichia coli for overexpression of AAC(2')-Ia as a fusion protein with an N-terminal hexa-His tag. The fusion protein was isolated and purified by affinity chromatography on Ni(2+)-nitrilotriacetic acid agarose and gel permeation chromatography on Superdex 75. Comparison of the specific activity of this enzyme with that of its enterokinase-digested derivative lacking the His tag indicated that the presence of the extra N-terminal peptide does not affect activity. The temperature and pH optima for activity of both forms of the 2'-N-acetyltransferase were 20 degrees C and pH 6.0, respectively, while the enzymes were most stable at 15 degrees C and pH 8.1. The Michaelis-Menten kinetic parameters for AAC(2')-Ia at 20 degrees C and pH 6.0 were determined using a series of aminoglycoside antibiotics possessing a 2'-amino group and a concentration of acetyl coenzyme A fixed at 10 times its K(m) value of 8.75 microM. Under these conditions, gentamicin was determined to be the best substrate for the enzyme in terms of both K(m) and k(cat)/K(m) values, whereas neomycin was the poorest. Comparison of the kinetic parameters obtained with the different aminoglycosides indicated that their hexopyranosyl residues provided the most important binding sites for AAC(2')-Ia activity, while the enzyme exhibits greater tolerance further from these sites. No correlation was found between these kinetic parameters and MICs determined for P. stuartii PR50 expressing the 2'-N-acetyltransferase, suggesting that its true in vivo function is not as a resistance factor.     

Cosubstrate Tolerance of the Aminoglycoside Resistance Enzyme Eis from Mycobacterium tuberculosis

We previously demonstrated that aminoglycoside acetyltransferases (AACs) display expanded cosubstrate promiscuity. The enhanced intracellular survival (Eis) protein of Mycobacterium tuberculosis is responsible for the resistance of this pathogen to kanamycin A in a large fraction of clinical isolates. Recently, we discovered that Eis is a unique AAC capable of acetylating multiple amine groups on a large pool of aminoglycoside (AG) antibiotics, an unprecedented property among AAC enzymes. Here, we report a detailed study of the acyl-coenzyme A (CoA) cosubstrate profile of Eis. We show that, in contrast to other AACs, Eis efficiently uses only 3 out of 15 tested acyl-CoA derivatives to modify a variety of AGs. We establish that for almost all acyl-CoAs, the number of sites acylated by Eis is smaller than the number of sites acetylated. We demonstrate that the order of n-propionylation of the AG neamine by Eis is the same as the order of its acetylation. We also show that the 6′ position is the first to be n-propionylated on amikacin and netilmicin. By sequential acylation reactions, we show that AGs can be acetylated after the maximum possible n-propionylation of their scaffolds by Eis. The information reported herein will advance our understanding of the multiacetylation mechanism of inactivation of AGs by Eis, which is responsible for M. tuberculosis resistance to some AGs.     

Enzymatic Modification of Aminoglycoside Antibiotics: 3-N-Acetyltransferase with Broad Specificity that Determines Resistance to the Novel Aminoglycoside Apramycin

Examination of a number of R-plasmid-containing bacterial isolates of animal origin has revealed the presence of a new aminoglycoside acetyltransferase (3-N) with a broad substrate range that includes all the disubstituted 2-deoxystreptamine antibiotics and also the novel monosubstituted antibiotic apramycin. Antibiotic derivatives acylated with hydroxyaminobutyric acid at the 1-amino position were not modified by the enzyme.     

Aminoglycoside resistance in Gram-negative blood isolates from various hospitals in Belgium and the Grand Duchy of Luxembourg. Aminoglycoside Resistance Study Group

A total of 1102 consecutive clinical blood isolates, including 897 Enterobacteriaceae and 205 non-fermenting bacilli, were obtained from 13 university and university-affiliated hospitals, which were divided into a Northern and a Southern group. Resistance to gentamicin, tobramycin, netilmicin, amikacin and isepamicin was determined using a microdilution technique according to NCCLS procedures. The overall mean resistance level was 5.9% for gentamicin, 7.7% for tobramycin, 7.5% for netilmicin, 2.8% for amikacin and 1.2% for isepamicin. Resistance to amikacin and isepamicin was significantly higher in the Northern hospitals than in the Southern hospitals. In total, 157 isolates were found not to be susceptible to aminoglycosides. By PCR, 179 aminoglycoside resistance mechanisms, i.e. 150 genes encoding modifying enzymes and 29 permeability mechanisms, were detected in 148 isolates. A resistance mechanism could not be detected in nine isolates. Moreover, in a further 14 isolates the resistance profile was not fully explained by the detected genes. The aac(6')-I genes were found to be the most predominant resistance mechanism in both the Northern and Southern isolates, followed by aac(3) genes and permeability resistance. A total of 29 non-susceptible isolates harboured a combination of genes, 72.4% of which were a combination with the aac(6')-lb gene. The majority of these combinations were broad-spectrum combinations which represented 9.0% of the resistance mechanisms in non-susceptible Enterobacteriaceae and 19.3% in the non-fermenting bacilli.     

Innate Aminoglycoside Resistance of Achromobacter xylosoxidans Is Due to AxyXY-OprZ,an RND-Type Multidrug Efflux Pump

Achromobacter xylosoxidans is an innately multidrug-resistant pathogen which is emerging in cystic fibrosis (CF) patients. We characterized a new resistance-nodulation-cell division (RND)-type multidrug efflux pump, AxyXY-OprZ. This system is responsible for the intrinsic high-level resistance of A. xylosoxidans to aminoglycosides (tobramycin, amikacin, and gentamicin). Furthermore, it can extrude cefepime, carbapenems, some fluoroquinolones, tetracyclines, and erythromycin. Some of the AxyXY-OprZ substrates are major components widely used to treat pulmonary infections in CF patients.     

Mechanism of Aminoglycoside Antibiotic Resistance in Anaerobic Bacteria: Clostridium perfringens and Bacteroides fragilis

Cell-free amino acid incorporation using ribosomes from strains of either Clostridium perfringens or Bacteroides fragilis was shown to be susceptible to inhibition by streptomycin and gentamicin. Ribosomes bound dihydrostreptomycin as effectively as ribosomes from Escherichia coli. No inactivation of streptomycin or gentamicin was detected by cell extracts of either anaerobic bacterial species. B. fragilis, grown without added hemin, menadione, and fumarate, and C. perfringens did not show any time-dependent accumulation of dihydrostreptomycin or gentamicin at concentrations tested. Decreased resistance to aminoglycosides and time-dependent uptake of dihydrostreptomycin at 500 μg/ml was observed with B. fragilis grown with hemin, menadione, and fumarate. With the last additions, cytochrome b was detected by cytochrome spectra of B. fragilis. These results demonstrate that anaerobic bacteria unable to carry out oxygen- or nitrate-dependent electron transport are resistant to streptomycin and gentamicin because of failure to transport aminoglycosides. The induction of fumarate-dependent electron transport in B. fragilis is associated with some aminoglycoside transport that is of poor efficiency relative to bacteria with electron transport to oxygen or nitrate.     

Hospital Pseudomonas aeruginosa: Surveillance of Resistance to Gentamicin and Transfer of Aminoglycoside R Factor

Most ampicillin-resistant Shigella are susceptible to cephalexin. Randomized treatment with cephalexin or ampicillin was given to 154 infants and children with acute diarrhea. Rectal swab cultures revealed Shigella in 42%, Salmonella in 6%, enteropathogenic Escherichia coli in 2%, and no pathogen in 50%. Cephalexin failed to eradicate Shigella after 5 days of treatment in 76% of patients as contrasted with 28% of ampicillin-treated patients with susceptible organisms. Shigella persisted in 78% of ampicillin-treated patients with resistant organisms. Diarrhea lasted more than 5 days in 43% of cephalexin-treated patients, in 56% of the ampicillin group with resistant organisms, but in only 9% of ampicillin-treated patients with susceptible organisms. The failure of cephalexin was due to the relatively high minimal inhibitory concentrations and minimal bacterial concentrations of 5 or 10 μg/ml and, although serum concentrations were twice the minimal bacterial concentration, they were not sufficient to demonstrate killing by the serum dilution method. In vitro susceptibility or resistance of Shigella to ampicillin correlated with clinical success or failure. Cephalexin is not a suitable drug for treatment of shigellosis in patients with ampicillin-resistant organisms.     

产ESBLs大肠埃希菌对氨基糖苷类抗生素的耐药表型与修饰酶基因研究

目的分析临床分离的产ESBLs大肠埃希菌对氨基糖苷类抗生素的耐药性,并了解其修饰酶基因携带状况及耐药机制。方法采用纸片扩散法测定临床分离的32株产ESBLs大肠埃希菌对5种氨基糖苷类抗生素的耐药性,用CLSI推荐的酶抑制剂增强纸片扩散法检测产ESBLs菌株,采用PCR法检测氨基糖苷类修饰酶基因aac（3）-Ⅰ,aac（3）-Ⅱ,8aC（6’）-Ⅰ ad,aac（6＇）-Ⅰ b,aac（6＇）-Ⅱ,ant（3＂）-Ⅰ,ant（2＂）-Ⅰ。提取携带单一修饰酶基因临床菌株的质粒并转化入宿主菌BL21中,比较临床分离菌与转化菌对5种氨基糖苷类药物敏感性及产ESBLs情况的区别。结果32株产ESBLs大肠埃希菌对庆大霉素耐药率最高（84．3％）,对阿米卡星耐药率最低（25％）。检出氯基糖苷粪修饰酶基因aac（6＇）-Ⅱ,ant（3＂）-Ⅰ,ant（2＂）-Ⅰ,阳性分别为22株（68．8％）,9株（28．1％）,5株（15．6％）,1株（3．1％）。质粒转化菌产ESBLs且同时检出原携带基因,但耐药表型有区别。结论临床分离的产ESBLs大肠埃希菌对庆大霉素耐药率最高,对阿米卡星敏感性最高,氨基糖苷类耐药与氨基糖苷类修饰酶基因表达有关,临床应对此类菌株引起重视。     

铜绿假单胞菌氨基糖苷类修饰酶基因与耐药表型的研究

目的探讨临床分离的22株铜绿假单胞菌（Pa）氨基糖苷类修饰酶（AMES）基因与耐药表型的关系。方法对22株铜绿假单胞菌采用稀释法检测庆大霉素、阿米卡星、奈替米星、妥布霉素4种抗菌药物的最低抑菌浓度（MIC），并用聚合酶链反应（PCR）法检测6种氨基糖苷类修饰酶基因。结果本组22株Pa菌庆大霉素耐药15株（68．2％）、阿米卡星耐药3株（13．6％）、奈替米星14株（63．6％）、妥布霉素15株（68．2％），检出aac（3）-Ⅰ阳性14株（63．6％）、aac（6’）-Ⅰ阳性5株（22．7％）、aac（6’）-Ⅱ阳性3株（13．6％）、ant（3”）-Ⅰ阳性1株（4．5％）和ant（2”）-Ⅰ阳性7株（31．8％），aac（3）．Ⅰ为阴性。共有18株检出氨基糖苷类修饰酶基因（81．8％）。结论分离自临床的铜绿假单胞菌氨基糖苷类修饰酶基因检出率高，且与耐药表型一致。     

Analysis of the Interactions Between Piperacillin, Ticarcillin, or Carbenicillin and Aminoglycoside Antibiotics

Enhanced activity against clinical isolates of Pseudomonas aeruginosa was demonstrated with piperacillin, ticarcillin, and carbenicillin in combination with gentamicin, tobramycin, and amikacin.     

获得性再生障碍性贫血患者外周血单个核细胞TRF1、TRF2、RAP1 mRNA表达研究

本研究检测获得性再生障碍性贫血患者外周血单个核细胞的TRF1、TRF2、RAP1基因表达水平,探讨其与获得性再生障碍性贫血发病的关系。采用实时荧光定量聚合酶链反应检测40例获得性再生障碍性贫血患者和20例正常对照组外周血单个核细胞中TRF1、TRF2、RAP1的基因表达情况。结果表明,获得性再生障碍性贫血患者的TRF1和RAP1 mRNA表达水平较正常对照组明显升高(P<0.05);TRF2 mRNA表达水平较正常对照组明显降低(P<0.01),并且TRF2与RAP1具有相关性(r=0.522,P=0.001)。结论:TRF1、TRF2、RAP1可能参与了获得性再生障碍性贫血的发病过程。     

In Vitro and In Vivo Assessment of Dermatophyte Acquired Resistance to Efinaconazole,a Novel Triazole Antifungal

Efinaconazole is a novel triazole antifungal drug for the topical treatment of onychomycosis, a nail infection caused mainly by dermatophytes. We assessed the potential of efinaconazole to induce resistance in dermatophytes by continuous exposure of Trichophyton rubrum strains to efinaconazole in vitro (12 passages) and in a guinea pig onychomycosis model (8 weeks). There was no evidence of efinaconazole resistance development in the tested strains under the experimental conditions used.