Dataset Information

Prevalence and detection of Stenotrophomonas maltophilia carrying metallo-?-lactamase blaL1 in Beijing, China.

ABSTRACT: Intrinsic ?-lactam resistance in Stenotrophomonas maltophilia is caused by bla L1 and/or bla L2, a kind of metallo-?-lactamase with a broad substrate spectrum including carbapenems. A rapid and sensitive molecular method for the detection of bla L1 in clinical samples is needed to guide therapeutic treatment. In present study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of bla L1 in clinical samples by using two methods including a chromogenic method using calcein/Mn(2+) complex and the real-time turbidity monitoring to assess the reaction. Then dissemination of L1-producing S. maltophilia was investigated from ICU patients in three top hospital in Beijing, China. The results showed that both methods detected the target DNA within 60 min under isothermal conditions (65°C). The detection limit of LAMP was 3.79 pg/?l DNA, and its sensitivity 100-fold greater than that of conventional PCR. All 21 test strains except for S. maltophilia were negative for bla L1, indicative of the high-specificity of the primers for the bla L1. A total of 22 L1-positive isolates were identified for LAMP-based surveillance of bla L1 from 105 ICU patients with clinically suspected multi-resistant infections. The sequences of these bla L1 genes were conservative with only a few sites mutated, and the strains had highly resistant to ?-lactam antibiotics. The MLST recovered that 22 strains belonged to seven different S. maltophilia sequence types (STs). Furthermore, co-occurrence of bla L1 and bla L2 genes were detected in all of isolates. Strikingly, S. maltophilia DCPS-01 was recovered to contain bla L1, bla L2, and bla NDM-1 genes, possessing an ability to hydrolyse all ?-lactams antibiotics. Our data showed the diversity types of S. maltophilia carrying bla L1 and co-occurrence of many resistant genes in the clinical strains signal an ongoing and fast evolution of S. maltophilia resulting from their wide spread in the respiratory infections, and therefore will be difficult to control.

SUBMITTER: Yang Z

PROVIDER: S-EPMC4260517 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Prevalence and detection of Stenotrophomonas maltophilia carrying metallo-β-lactamase blaL1 in Beijing, China.

Yang Zhan Z Liu Wei W Cui Qian Q Niu Wenkai W Li Huan H Zhao Xiangna X Wei Xiao X Wang Xuesong X Huang Simo S Dong Derong D Lu Sijing S Bai Changqing C Li Yan Y Huang Liuyu L Yuan Jing J

Frontiers in microbiology 20141209

Intrinsic β-lactam resistance in Stenotrophomonas maltophilia is caused by bla L1 and/or bla L2, a kind of metallo-β-lactamase with a broad substrate spectrum including carbapenems. A rapid and sensitive molecular method for the detection of bla L1 in clinical samples is needed to guide therapeutic treatment. In present study, we first described a loop-mediated isothermal amplification (LAMP) method for the rapid detection of bla L1 in clinical samples by using two methods including a chromogeni ...[more]

PMID: 25538701

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Project description:Stenotrophomonas maltophilia is an emerging nosocomial pathogen with high resistance to most clinically used antimicrobials. Tigecycline is a potential alternative antimicrobial for S. maltophilia infection treatment, but its resistance mechanism in clinical isolates is not fully elucidated. We investigated the antimicrobial susceptibility of 450 S. maltophilia isolated during 2012-2015 from three university hospitals in Beijing, China. These strains exhibited high susceptibility to minocycline (98.44%), sulfamethoxazole/trimethoprim (87.56%), tigecycline (77.78 %), doxycycline (81.33%), levofloxacin (67.56%), and ticarcillin/clavulanate (73.00%). The susceptibility of tigecycline-nonsusceptible strains (TNS) to doxycycline and levofloxacin was much lower than that of tigecycline-susceptible strains (TSS) (25.00% vs. 97.71% for doxycycline, P < 0.001; 17.00% vs. 82.00% for levofloxacin, P < 0.001). We further selected 48 TNS and TSS and compared the detection rate of eight tetracycline-specific genes by PCR and the expression level of six intrinsic multidrug resistance efflux pumps by real-time PCR. Only one tetB and two tetH genes in TNS and three tetH genes in TSS were detected, and the detection rate had no difference. The average expression level of smeD in TNS was higher than that in TSS [20.59 (11.53, 112.54) vs. 2.07 (0.80, 4.96), P < 0.001], while the average expression levels of smeA, smeI, smeO, smeV, and smrA were not significantly different, indicating that smeDEF was the predominant resistance genetic determinant in clinical S. maltophilia. Higher smeD expression was also observed in levofloxacin- and doxycycline-nonsusceptible isolates than in their corresponding susceptible isolates [16.46 (5.83, 102.24) vs. 2.72 (0.80, 6.25) for doxycycline, P < 0.001; 19.69 (8.07, 115.10) vs. 3.01(1.00, 6.03), P < 0.001], indicating that smeDEF was also the resistance genetic determinant to levofloxacin and doxycycline. The consistent resistance profile and common resistance genetic determinant highlight the importance of rational use of tigecycline for preventing the occurrence and spread of multidrug resistance.

Project description:Emergence of Enterobacteriaceae harboring metallo-β-lactamases (MBL) has raised global threats due to their broad antibiotic resistance profiles and the lack of effective inhibitors against them. We have been studied one of the emerging environmental MBL, the L1 from Stenotrophomonas maltophilia K279a. We determined several crystal structures of L1 complexes with three different classes of β-lactam antibiotics (penicillin G, moxalactam, meropenem, and imipenem), with the inhibitor captopril and different metal ions (Zn+2 , Cd+2 , and Cu+2 ). All hydrolyzed antibiotics and the inhibitor were found binding to two Zn+2 ions mainly through the opened lactam ring and some hydrophobic interactions with the binding pocket atoms. Without a metal ion, the active site is very similarly maintained as that of the native form with two Zn+2 ions, however, the protein does not bind the substrate moxalactam. When two Zn+2 ions were replaced with other metal ions, the same di-metal scaffold was maintained and the added moxalactam was found hydrolyzed in the active site. Differential scanning fluorimetry and isothermal titration calorimetry were used to study thermodynamic properties of L1 MBL compared with New Deli Metallo-β-lactamase-1 (NDM-1). Both enzymes are significantly stabilized by Zn+2 and other divalent metals but showed different dependency. These studies also suggest that moxalactam and its hydrolyzed form may bind and dissociate with different kinetic modes with or without Zn+2 for each of L1 and NDM-1. Our analysis implicates metal ions, in forming a distinct di-metal scaffold, which is central to the enzyme stability, promiscuous substrate binding and versatile catalytic activity. STATEMENT: The L1 metallo-β-lactamase from an environmental multidrug-resistant opportunistic pathogen Stenotrophomonas maltophilia K279a has been studied by determining 3D structures of L1 enzyme in the complexes with several β-lactam antibiotics and different divalent metals and characterizing its biochemical and ligand binding properties. We found that the two-metal center in the active site is critical in the enzymatic process including antibiotics recognition and binding, which explains the enzyme's activity toward diverse antibiotic substrates. This study provides the critical information for understanding the ligand recognition and for advanced drug development.

Dataset Information

Prevalence and detection of Stenotrophomonas maltophilia carrying metallo-?-lactamase blaL1 in Beijing, China.

Publications

Prevalence and detection of Stenotrophomonas maltophilia carrying metallo-β-lactamase blaL1 in Beijing, China.

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