Deciphering the Coevolutionary Dynamics of L2 ß-Lactamases via Deep Learning.

L2 ß-lactamases, serine-based class A ß-lactamases expressed by Stenotrophomonas maltophilia, play a pivotal role in antimicrobial resistance (AMR). However, limited studies have been conducted on these important enzymes. To understand the coevolutionary dynamics of L2 ß-lactamase, innovative computational methodologies, including adaptive sampling molecular dynamics simulations, and deep learning methods (convolutional variational autoencoders and BindSiteS-CNN) explored conformational changes and correlations within the L2 ß-lactamase family together with other representative class A enzymes including SME-1 and KPC-2. This work also investigated the potential role of hydrophobic nodes and binding site residues in facilitating the functional mechanisms. The convergence of analytical approaches utilized in this effort yielded comprehensive insights into the dynamic behavior of the ß-lactamases, specifically from an evolutionary standpoint. In addition, this analysis presents a promising approach for understanding how the class A ß-lactamases evolve in response to environmental pressure and establishes a theoretical foundation for forthcoming endeavors in drug development aimed at combating AMR.

Colistin-degrading proteases confer collective resistance to microbial communities during polymicrobial infections.

BACKGROUND: The increasing prevalence of resistance against the last-resort antibiotic colistin is a significant threat to global public health. Here, we discovered a novel colistin resistance mechanism via enzymatic inactivation of the drug and proposed its clinical importance in microbial communities during polymicrobial infections. RESULTS: A bacterial strain of the Gram-negative opportunistic pathogen Stenotrophomonas maltophilia capable of degrading colistin and exhibiting a high-level colistin resistance was isolated from the soil environment. A colistin-degrading protease (Cdp) was identified in this strain, and its contribution to colistin resistance was demonstrated by growth inhibition experiments using knock-out (Δcdp) and complemented (Δcdp::cdp) mutants. Coculture and coinfection experiments revealed that S. maltophilia carrying the cdp gene could inactivate colistin and protect otherwise susceptible Pseudomonas aeruginosa, which may seriously affect the clinical efficacy of the drug for the treatment of cystic fibrosis patients with polymicrobial infection. CONCLUSIONS: Our results suggest that Cdp should be recognized as a colistin resistance determinant that confers collective resistance at the microbial community level. Our study will provide vital information for successful clinical outcomes during the treatment of complex polymicrobial infections, particularly including S. maltophilia and other colistin-susceptible Gram-negative pathogens such as P. aeruginosa. Video abstract.

Dual Activity BLEG-1 from Bacillus lehensis G1 Revealed Structural Resemblance to B3 Metallo-ß-Lactamase and Glyoxalase II: An Insight into Its Enzyme Promiscuity and Evolutionary Divergence.

Metallo-ß-lactamases (MBLs) are class B ß-lactamases from the metallo-hydrolase-like MBL-fold superfamily which act on a broad range of ß-lactam antibiotics. A previous study on BLEG-1 (formerly called Bleg1_2437), a hypothetical protein from Bacillus lehensis G1, revealed sequence similarity and activity to B3 subclass MBLs, despite its evolutionary divergence from these enzymes. Its relatedness to glyoxalase II (GLXII) raises the possibility of its enzymatic promiscuity and unique structural features compared to other MBLs and GLXIIs. This present study highlights that BLEG-1 possessed both MBL and GLXII activities with similar catalytic efficiencies. Its crystal structure revealed highly similar active site configuration to YcbL and GloB GLXIIs from Salmonella enterica, and L1 B3 MBL from Stenotrophomonas maltophilia. However, different from GLXIIs, BLEG-1 has an insertion of an active-site loop, forming a binding cavity similar to B3 MBL at the N-terminal region. We propose that BLEG-1 could possibly have evolved from GLXII and adopted MBL activity through this insertion.

Structural insights into the mechanism of pH-selective substrate specificity of the polysaccharide lyase Smlt1473.

Polysaccharide lyases (PLs) are a broad class of microbial enzymes that degrade anionic polysaccharides. Equally broad diversity in their polysaccharide substrates has attracted interest in biotechnological applications such as biomass conversion to value-added chemicals and microbial biofilm removal. Unlike other PLs, Smlt1473 present in the clinically relevant Stenotrophomonas maltophilia strain K279a demonstrates a wide range of pH-dependent substrate specificities toward multiple, diverse polysaccharides: hyaluronic acid (pH 5.0), poly-ß-D-glucuronic (celluronic) acid (pH 7.0), poly-ß-D-mannuronic acid, and poly-α-L-guluronate (pH 9.0). To decode the pH-driven multiple substrate specificities and selectivity in this single enzyme, we present the X-ray structures of Smlt1473 determined at multiple pH values in apo and mannuronate-bound states as well as the tetra-hyaluronate-docked structure. Our results indicate that structural flexibility in the binding site and N-terminal loop coupled with specific substrate stereochemistry facilitates distinct modes of entry for substrates having diverse charge densities and chemical structures. Our structural analyses of wild-type apo structures solved at different pH values (5.0-9.0) and pH-trapped (5.0 and 7.0) catalytically relevant wild-type mannuronate complexes (1) indicate that pH modulates the catalytic microenvironment for guiding structurally and chemically diverse polysaccharide substrates, (2) further establish that molecular-level fluctuation in the enzyme catalytic tunnel is preconfigured, and (3) suggest that pH modulates fluctuations resulting in optimal substrate binding and cleavage. Furthermore, our results provide key insight into how strategies to reengineer both flexible loop and regions distal to the active site could be developed to target new and diverse substrates in a wide range of applications.

Structural basis for an exceptionally strong preference for asparagine residue at the S2 subsite of Stenotrophomonas maltophilia dipeptidyl peptidase 7.

The emergence of drug-resistant bacteria has become a major problem worldwide. Bacterial dipeptidyl peptidases 7 and 11 (DPP7s and DPP11s), belonging to the family-S46 peptidases, are important enzymes for bacterial growth and are not present in mammals. Therefore, specific inhibitors for these peptidases are promising as potential antibiotics. While the molecular mechanisms underlining strict specificity at the S1 subsite of S46 peptidases have been well studied, those of relatively broad preference at the S2 subsite of these peptidases are unknown. In this study, we performed structural and biochemical analyses on DPP7 from Stenotrophomonas maltophilia (SmDPP7). SmDPP7 showed preference for the accommodation of hydrophobic amino acids at the S2 subsite in general, but as an exception, also for asparagine, a hydrophilic amino acid. Structural analyses of SmDPP7 revealed that this exceptional preference to asparagine is caused by a hydrogen bonding network at the bottom of the S2 subsite. The residues in the S2 subsite are well conserved among S46 peptidases as compared with those in the S1 subsite. We expect that our findings will contribute toward the development of a universal inhibitor of S46 peptidases.

Faropenem reacts with serine and metallo-ß-lactamases to give multiple products.

Penems have demonstrated potential as antibacterials and ß-lactamase inhibitors; however, their clinical use has been limited, especially in comparison with the structurally related carbapenems. Faropenem is an orally active antibiotic with a C-2 tetrahydrofuran (THF) ring, which is resistant to hydrolysis by some ß-lactamases. We report studies on the reactions of faropenem with carbapenem-hydrolysing ß-lactamases, focusing on the class A serine ß-lactamase KPC-2 and the metallo ß-lactamases (MBLs) VIM-2 (a subclass B1 MBL) and L1 (a B3 MBL). Kinetic studies show that faropenem is a substrate for all three ß-lactamases, though it is less efficiently hydrolysed by KPC-2. Crystallographic analyses on faropenem-derived complexes reveal opening of the ß-lactam ring with formation of an imine with KPC-2, VIM-2, and L1. In the cases of the KPC-2 and VIM-2 structures, the THF ring is opened to give an alkene, but with L1 the THF ring remains intact. Solution state studies, employing NMR, were performed on L1, KPC-2, VIM-2, VIM-1, NDM-1, OXA-23, OXA-10, and OXA-48. The solution results reveal, in all cases, formation of imine products in which the THF ring is opened; formation of a THF ring-closed imine product was only observed with VIM-1 and VIM-2. An enamine product with a closed THF ring was also observed in all cases, at varying levels. Combined with previous reports, the results exemplify the potential for different outcomes in the reactions of penems with MBLs and SBLs and imply further structure-activity relationship studies are worthwhile to optimise the interactions of penems with ß-lactamases. They also exemplify how crystal structures of ß-lactamase substrate/inhibitor complexes do not always reflect reaction outcomes in solution.

Structural characterization of cystathionine γ-lyase smCSE enables aqueous metal quantum dot biosynthesis.

The development and utilization of inorganic material biosynthesis have evolved into single macromolecular systems. A putative cystathionine γ-lyase of bacteria Stenotrophomonas maltophilia (smCSE) is a newly identified biomolecule that enables the synthesis of nanomaterials. Due to the lack of structural information, the mechanism of smCSE biosynthesis remains unclear. Herein, we obtain two atomic-resolution smCSE-form X-ray structures and confirm that the conformational changes of Tyr108 and Lys206 within the enzyme active sites are critical for the protein-driven synthesis of metal sulfide quantum dots (QDs). The structural stability of tetramer and the specificity of surface amino acids are the basis for smCSE to synthesize quantum dots. The size of QD products can be regulated by predesigned amino acids and the morphology can be controlled through proteolytic treatments. The growth rate is enhanced by the stabilization of a flexible loop in the active site, as shown by the X-ray structure of the engineered protein which fused with a dodecapeptide. We further prove that the smCSE-driven route can be applied to the general synthesis of other metal sulfide nanoparticles. These results provide a better understanding of the mechanism of QD biosynthesis and a new perspective on the control of this biosynthesis by protein modification.

Flavonoids from Woodfordia fruticosa as potential SmltD inhibitors in the alternative biosynthetic pathway of peptidoglycan.

SmltD is an ATP-dependent ligase that catalyzes the condensation of UDP-MurNAc-l-Ala and l-Glu to form UDP-MurNAc-l-Ala-l-Glu, in the newly discovered peptidoglycan biosynthesis pathway of a Gram-negative multiple-drug-resistant pathogen, Stenotrophomonas maltophilia. Phytochemical investigation of the 70% ethanol extract from Woodfordia fruticosa flowers collected in Myanmar led to the identification of anti-SmltD active flavonoids, kaempferol 3-O-(6''-galloyl)-ß-d-glucopyranoside (1), astragalin (2), and juglalin (3). Among them, 1 showed the most potent SmltD inhibitory activity. An enzyme steady-state kinetic study revealed that 1 exerted competitive inhibition with respect to ATP. The results of this study provided an attractive foundation for the further development of novel inhibitors of SmltD.

Polypyridine ligands as potential metallo-ß-lactamase inhibitors.

Bacteria have developed multiple resistance mechanisms against the most used antibiotics. In particular, zinc-dependent metallo-ß-lactamase producing bacteria are a growing threat, and therapeutic options are limited. Zinc chelators have recently been investigated as metallo-ß-lactamase inhibitors, as they are often able to restore carbapenem susceptibility. We synthesized polypyridyl ligands, N,N'-bis(2-pyridylmethyl)-ethylenediamine, N,N,N'-tris(2-pyridylmethyl)-ethylenediamine, N,N'-bis(2-pyridylmethyl)-ethylenediamine-N-acetic acid (N,N,N'-tris(2-pyridylmethyl)-ethylenediamine-N'-acetic acid, which can form zinc(II) complexes. We tested their ability to restore the antibiotic activity of meropenem against three clinical strains isolated from blood and metallo-ß-lactamase producers (Klebsiella pneumoniae, Enterobacter cloacae, and Stenotrophomonas maltophilia). We functionalized N,N,N'-tris(2-pyridylmethyl)-ethylenediamine with D-alanyl-D-alanyl-D-alanine methyl ester with the aim to increase bacterial uptake. We observed synergistic activity of four polypyridyl ligands with meropenem against all tested isolates, while the combination N,N'-bis(2-pyridylmethyl)-ethylenediamine and meropenem was synergistic only against New Delhi and Verona integron-encoded metallo-ß-lactamase-producing bacteria. All synergistic interactions restored the antimicrobial activity of meropenem, providing a significant decrease of minimal inhibitory concentration value (by 8- to 128-fold). We also studied toxicity of the ligands in two normal peripheral blood lymphocytes.

Effects of resveratrol on the growth and enzyme production of Stenotrophomonas maltophilia: a burn wound pathogen.

OBJECTIVE: The purpose of this study was to identify the potential of resveratrol in inhibiting the growth and production of two enzymes, hyaluronidase and protease, in Stenotrophomonas maltophilia, which has become a burn wound pathogen of great significance. METHOD: Stenotrophomonas maltophilia (ATCC 17666) was cultured in nutrient broth and the microbial load was standardised to 0.5 McFarland standard at 600nm. The study included antimicrobial assays (well diffusion and resazurin dye binding method), hyaluronidase expression regulation assay (hyaluronic acid hydrolysis assay and turbidity assay) and protease expression regulation assay (casein hydrolysis assay and determination of specific activity of protease using tyrosine standard). RESULTS: The minimum inhibitory concentration (MIC) of resveratrol against Stenotrophomonas maltophilia was found to be 125µg/ml. Hyaluronidase production in the organism treated with resveratrol was found to be half that in the untreated organism. The specific activity of protease produced by the organism treated with resveratrol was found to be one-quarter that in the untreated organism, as analysed by the tyrosine standard estimation protocol. CONCLUSION: Resveratrol was found to be a potent compound to treat Stenotrophomonas maltophilia infections. In addition to the antimicrobial and enzyme-regulatory properties of resveratrol, it also shows anti-oxidant and anti-inflammatory properties. This finding has great scope clinically as resveratrol may prove to be an ideal drug to treat burn wound infections.

Characterization and rational design for substrate specificity of a prolyl endopeptidase from Stenotrophomonas maltophilia.

A novel prolyl endopeptidase from Stenotrophomonas maltophilia, SmPEP, was discovered and characterized. The specific activity of the recombinant SmPEP expressed by Escherichia coli BL21 (DE3), was 68.3 U/mg at pH 8.0 and 37 °C. In order to improve the substrate specificity for long-chain peptide, rational design was applied based on the structure constructed by homology modeling. Inter-domain sites within the ß-propeller domain were chosen for the mutation to weaken the inter-domain interaction and form an open conformation for long-chain substrate entering into the active site. The substrate specificity on a designed long-chain substrate, PQPQLPYPQPQLP, of the mutants F263A and E184 G increased 8.77 and 5.75 times respectively versus wild-type. After the saturated mutation of the both sites, the reactive rate of mutant F263 V on 13-mer peptide was 10.2 times higher than that of the wild-type. Then the mutant F263 V was used in the hydrolysis of casein, and the ACE inhibitory activity of the hydrolysate was significantly improved compared with wild type enzyme, which verified the efficiency of the design strategy.

Efficient synthesis of L-phosphinothricin using a novel aminoacylase mined from Stenotrophomonas maltophilia.

L-phosphinothricin (L-PPT) is a competitive and environmentally friendly herbicide. To develop an efficient approach for synthesis of l-PPT, a kinetic resolution route with a novel aminoacylase (SmAcy) mined from Stenotrophomonas maltophilia using N-acetyl-PPT as a substrate was first constructed. This SmAcy exhibited high hydrolytic activity and excellent enantioselectivity (E > 200) toward N-acetyl-PPT. Optically pure l-PPT (> 99.9 % eep) was acquired with high conversion (> 49 %) within 4 h by the whole cells. On the basis of the docking analysis, a main reason for high enantioselectivity (E > 200) of SmAcy towards l-enantiomer would be that the D-N-acetyl-PPT cannot interact with the key general acid-base residue and the metal ions. A low-cost and simple preparation process of the substrate from commercially available racemic PPT for production of L-PPT was provided. A chemical racemization method of the unreacted D-enantiomer of substrate was also provided to recycle the unwanted substrate enantiomer. This study provides a potential route for the industrial production of L-PPT.

Impaired Airway Epithelial Barrier Integrity in Response to Stenotrophomonas maltophilia Proteases, Novel Insights Using Cystic Fibrosis Bronchial Epithelial Cell Secretomics.

Stenotrophomonas maltophilia is a Gram-negative opportunistic pathogen that can chronically colonize the lungs of people with cystic fibrosis (CF) and is associated with lethal pulmonary hemorrhage in immunocompromised patients. Its secreted virulence factors include the extracellular serine proteases StmPR1, StmPR2, and StmPR3. To explore the impact of secreted virulence determinants on pulmonary mucosal defenses in CF, we examined the secretome of human CFBE41o- bronchial epithelial cells in response to treatment with S. maltophilia K279a cell culture supernatant (CS) using a liquid-chromatography-tandem mass spectrometry (LC-MS/MS) based label-free quantitative (LFQ) shotgun proteomics approach for global profiling of the cell secretome. Secretome analysis identified upregulated pathways mainly relating to biological adhesion and epithelial cell signaling in infection, whereas no specific pathways relating to the immune response were enriched. Further exploration of the potentially harmful effects of K279a CS on CF bronchial epithelial cells, demonstrated that K279a CS caused CFBE41o- cell condensation and detachment, reversible by the serine protease inhibitor PMSF. K279a CS also decreased trans-epithelial electrical resistance in CFBE41o- cell monolayers suggestive of disruption of tight junction complexes (TJC). This finding was corroborated by an observed increase in fluorescein isothiocyanate (FITC) dextran permeability and by demonstrating PMSF-sensitive degradation of the tight junction proteins ZO-1 and occludin, but not JAM-A or claudin-1. These observations demonstrating destruction of the CFBE41o- TJC provide a novel insight regarding the virulence of S. maltophilia and may explain the possible injurious effects of this bacterium on the CF bronchial epithelium and the pathogenic mechanism leading to lethal pulmonary hemorrhage.

Phosphoglycerate mutase affects Stenotrophomonas maltophilia attachment to biotic and abiotic surfaces.

Stenotrophomonas maltophilia biofilm formation is of increasing medical concern, particularly for lung infections. However, the molecular mechanisms facilitating the biofilm lifestyle in S. maltophilia are poorly understood. We generated and screened a transposon mutant library for mutations that lead to altered biofilm formation compared to wild type. One of these mutations, in the gene for glycolytic enzyme phosphoglycerate mutase (gpmA), resulted in impaired attachment on abiotic and biotic surfaces. As adherence to a surface is the initial step in biofilm developmental processes, our results reveal a unique factor that could affect S. maltophilia biofilm initiation and, possibly, subsequent development.

Characterization of an efficient estrogen-degrading bacterium Stenotrophomonas maltophilia SJTH1 in saline-, alkaline-, heavy metal-contained environments or solid soil and identification of four 17ß-estradiol-oxidizing dehydrogenases.

The efficient bioremediation of estrogen contamination in complex environments is of great concern. Here the strain Stenotrophomonas maltophilia SJTH1 was found with great and stable estrogen-degradation efficiency even under stress environments. The strain could utilize 17ß-estradiol (E2) as a carbon source and degrade 90% of 10 mg/L E2 in a week; estrone (E1) was the first degrading intermediate of E2. Notably, diverse pH conditions (3.0-11.0) and supplements of 4% salinity, 6.25 mg/L of heavy metal (Cd2+ or Cu2+), or 1 CMC of surfactant (Tween 80/ Triton X-100) had little effect on its cell growth and estrogen degradation. The addition of low concentrations of copper and Tween 80 even promoted its E2 degradation. Bioaugmentation of strain SJTH1 into solid clay soil achieved over 80% removal of E2 contamination (10 mg/kg) within two weeks. Further, the whole genome sequence of S. maltophilia SJTH1 was obtained, and a series of potential genes participating in stress-tolerance and estrogen-degradation were predicted. Four dehydrogenases similar to 17ß-hydroxysteroid dehydrogenases (17ß-HSDs) were found to be induced by E2, and the four heterogenous-expressed enzymes could oxidize E2 into E1 efficiently. This work could promote bioremediation appliance potential with microorganisms and biodegradation mechanism study of estrogens in complex real environments.

Structural and biochemical analysis of the metallo-ß-lactamase L1 from emerging pathogen Stenotrophomonas maltophilia revealed the subtle but distinct di-metal scaffold for catalytic activity.

Emergence of Enterobacteriaceae harboring metallo-ß-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 ß-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-ß-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-ß-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 ß-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.

Molecular Basis of Class A ß-Lactamase Inhibition by Relebactam.

ß-Lactamase production is the major ß-lactam resistance mechanism in Gram-negative bacteria. ß-Lactamase inhibitors (BLIs) efficacious against serine ß-lactamase (SBL) producers, especially strains carrying the widely disseminated class A enzymes, are required. Relebactam, a diazabicyclooctane (DBO) BLI, is in phase 3 clinical trials in combination with imipenem for the treatment of infections by multidrug-resistant Enterobacteriaceae We show that relebactam inhibits five clinically important class A SBLs (despite their differing spectra of activity), representing both chromosomal and plasmid-borne enzymes, i.e., the extended-spectrum ß-lactamases L2 (inhibition constant 3 µM) and CTX-M-15 (21 µM) and the carbapenemases KPC-2, -3, and -4 (1 to 5 µM). Against purified class A SBLs, relebactam is an inferior inhibitor compared with the clinically approved DBO avibactam (9- to 120-fold differences in half maximal inhibitory concentration [IC50]). MIC assays indicate relebactam potentiates ß-lactam (imipenem) activity against KPC-producing Klebsiella pneumoniae, with similar potency to avibactam (with ceftazidime). Relebactam is less effective than avibactam in combination with aztreonam against Stenotrophomonas maltophilia K279a. X-ray crystal structures of relebactam bound to CTX-M-15, L2, KPC-2, KPC-3, and KPC-4 reveal its C2-linked piperidine ring can sterically clash with Asn104 (CTX-M-15) or His/Trp105 (L2 and KPCs), rationalizing its poorer inhibition activity than that of avibactam, which has a smaller C2 carboxyamide group. Mass spectrometry and crystallographic data show slow, pH-dependent relebactam desulfation by KPC-2, -3, and -4. This comprehensive comparison of relebactam binding across five clinically important class A SBLs will inform the design of future DBOs, with the aim of improving clinical efficacy of BLI-ß-lactam combinations.

In Vitro Activity of Minocycline against U.S. Isolates of Acinetobacter baumannii-Acinetobacter calcoaceticus Species Complex, Stenotrophomonas maltophilia, and Burkholderia cepacia Complex: Results from the SENTRY Antimicrobial Surveillance Program, 2014 to 2018.

We evaluated the activity of minocycline and comparator agents against a large number of Stenotrophomonas maltophilia (n = 1,289), Acinetobacter baumannii-Acinetobacter calcoaceticus species complex (n = 1,081), and Burkholderia cepacia complex (n = 101) isolates collected from 2014 to 2018 from 87 U.S. medical centers spanning all 9 census divisions. The isolates were collected primarily from hospitalized patients with pneumonia (1,632 isolates; 66.0% overall), skin and skin structure infections (354 isolates; 14.3% overall), bloodstream infections (266 isolates; 10.8% overall), urinary tract infections (126 isolates; 5.1% overall), intra-abdominal infections (61 isolates; 2.5% overall), and other infections (32 isolates; 1.3% overall). Against the A. baumannii-A. calcoaceticus species complex, colistin was the most active agent, exhibiting MIC50/90 values at ≤0.5/2 µg/ml and 92.4% susceptibility. Minocycline ranked second in activity, with MIC50/90 values at 0.25/8 µg/ml and susceptibility at 85.7%. Activity for these two agents was reduced against extensively drug-resistant and multidrug-resistant isolates of the Acinetobacter baumannii-Acinetobacter calcoaceticus species complex. Only two agents showed high levels of activity (susceptibility, >90%) against S. maltophilia, minocycline (MIC50/90, 0.5/2 µg/ml; 99.5% susceptible) and trimethoprim-sulfamethoxazole (MIC50/90, ≤0.5/1 µg/ml; 94.6% susceptible). Minocycline was active against 92.8% (MIC90, 4 µg/ml) of trimethoprim-sulfamethoxazole-resistant S. maltophilia isolates. Various agents exhibited susceptibility rates of nearly 90% against the B. cepacia complex isolates; these were trimethoprim-sulfamethoxazole (MIC50/90, ≤0.5/2 µg/ml; 93.1% susceptible), ceftazidime (MIC50/90, 2/8 µg/ml; 91.0% susceptible), meropenem (MIC50/90, 2/8 µg/ml; 89.1% susceptible), and minocycline (MIC50/90, 2/8 µg/ml; 88.1% susceptible). These results indicate that minocycline is among the most active agents for these three problematic potential pathogen groups when tested against U.S. isolates.

Population Structure, Molecular Epidemiology, and ß-Lactamase Diversity among Stenotrophomonas maltophilia Isolates in the United States.

Stenotrophomonas maltophilia is a Gram-negative, nonfermenting, environmental bacillus that is an important cause of nosocomial infections, primarily associated with the respiratory tract in the immunocompromised population. Aiming to understand the population structure, microbiological characteristics and impact of allelic variation on ß-lactamase structure and function, we collected 130 clinical isolates from across the United States. Identification of 90 different sequence types (STs), of which 63 are new allelic combinations, demonstrates the high diversity of this species. The majority of the isolates (45%) belong to genomic group 6. We also report excellent activity of the ceftazidime-avibactam and aztreonam combination, especially against strains recovered from blood and respiratory infections for which the susceptibility is higher than the susceptibility to trimethoprim-sulfamethoxazole, considered the "first-line" antibiotic to treat S. maltophilia Analysis of 73 blaL1 and 116 blaL2 genes identified 35 and 43 novel variants of L1 and L2 ß-lactamases, respectively. Investigation of the derived amino acid sequences showed that substitutions are mostly conservative and scattered throughout the protein, preferentially affecting positions that do not compromise enzyme function but that may have an impact on substrate and inhibitor binding. Interestingly, we detected a probable association between a specific type of L1 and L2 and genomic group 6. Taken together, our results provide an overview of the molecular epidemiology of S. maltophilia clinical strains from the United States. In particular, the discovery of new L1 and L2 variants warrants further study to fully understand the relationship between them and the ß-lactam resistance phenotype in this pathogen.IMPORTANCE Multiple antibiotic resistance mechanisms, including two ß-lactamases, L1, a metallo-ß-lactamase, and L2, a class A cephalosporinase, make S. maltophilia naturally multidrug resistant. Thus, infections caused by S. maltophilia pose a big therapeutic challenge. Our study aims to understand the microbiological and molecular characteristics of S. maltophilia isolates recovered from human sources. A highlight of the resistance profile of this collection is the excellent activity of the ceftazidime-avibactam and aztreonam combination. We hope this result prompts controlled and observational studies to add clinical data on the utility and safety of this therapy. We also identify 35 and 43 novel variants of L1 and L2, respectively, some of which harbor novel substitutions that could potentially affect substrate and/or inhibitor binding. We believe our results provide valuable knowledge to understand the epidemiology of this species and to advance mechanism-based inhibitor design to add to the limited arsenal of antibiotics active against this pathogen.

Roles of the Two-MnSOD System of Stenotrophomonas maltophilia in the Alleviation of Superoxide Stress.

Manganese-dependent superoxide dismutase (MnSOD, SodA) and iron-dependent SOD (FeSOD, SodB) are critical cytosolic enzymes for alleviating superoxide stress. Distinct from the singular sodA gene in most bacteria, Stenotrophomonas maltophilia harbors two sodA genes, sodA1 and sodA2. The roles of SodA1, SodA2, and SodB of S. maltophilia in alleviating superoxide stress were investigated. The expression of sod genes was determined by promoter-xylE transcriptional fusion assay and qRT-PCR. SodA2 and sodB expressions were proportional to the bacterial logarithmic growth, but unaffected by menadione (MD), iron, or manganese challenges. SodA1 was intrinsically unexpressed and inducibly expressed by MD. Complementary expression of sodA1 was observed when sodA2 was inactivated. The individual or combined sod deletion mutants were constructed using the gene replacement strategy. The functions of SODs were assessed by evaluating cell viabilities of different sod mutants in MD, low iron-stressed, and/or low manganese-stressed conditions. Inactivation of SodA1 or SodA2 alone did not affect bacterial viability; however, simultaneously inactivating sodA1 and sodA2 significantly compromised bacterial viability in both aerobic growth and stressed conditions. SodA1 can either rescue or support SodA2 when SodA2 is defective or insufficiently potent. The presence of two MnSODs gives S. maltophilia an advantage against superoxide stress.