Structural and Functional Characterization of OXA-48: Insight into Mechanism and Structural Basis of Substrate Recognition and Specificity.

Class D ß-lactamase OXA-48 is widely distributed among Gram-negative bacteria and is an important determinant of resistance to the last-resort carbapenems. Nevertheless, the detailed mechanism by which this ß-lactamase hydrolyzes its substrates remains poorly understood. In this study, the complex structures of OXA-48 and various ß-lactams were modeled and the potential active site residues that may interact with various ß-lactams were identified and characterized to elucidate their roles in OXA-48 substrate recognition. Four residues, namely S70, K73, S118, and K208 were found to be essential for OXA-48 to undergo catalytic hydrolysis of various penicillins and carbapenems both in vivo and in vitro. T209 was found to be important for hydrolysis of imipenem, whereas R250 played a major role in hydrolyzing ampicillin, imipenem, and meropenem most likely by forming a H-bond or salt-bridge between the side chain of these two residues and the carboxylate oxygen ions of the substrates. Analysis of the effect of substitution of alanine in two residues, W105 and L158, revealed their roles in mediating the activity of OXA-48. Our data show that these residues most likely undergo hydrophobic interaction with the R groups and the core structure of the ß-lactam ring in penicillins and the carbapenems, respectively. Unlike OXA-58, mass spectrometry suggested a loss of the C6-hydroxyethyl group during hydrolysis of meropenem by OXA-48, which has never been demonstrated in Class D carbapenemases. Findings in this study provide comprehensive knowledge of the mechanism of the substrate recognition and catalysis of OXA-type ß-lactamases.

IS26-mediated formation of a virulence and resistance plasmid in Salmonella Enteritidis.

Objectives: To characterize a novel virulence-resistance plasmid pSE380T carried by a Salmonella enterica serotype Enteritidis clinical strain SE380. Methods: The plasmid pSE380T was conjugated to Escherichia coli strain J53 and sequenced by PacBio RSII, followed by subsequent annotation and genetic analysis. Results: Sequence analysis of this plasmid revealed that the entire Salmonella Enteritidis-specific virulence plasmid, pSEN, had been incorporated into an IncHI2 MDR plasmid, which comprises the cephalosporin and fosfomycin resistance determinants blaCTX-M-14 and fosA3. Based on BLAST analysis and scrutiny of insertion footprints, the insertion event was found to involve a replicative transposition process mediated by IS26, an IS element frequently detected in various resistance plasmids. The resulting pSE380T plasmid also comprises backbone elements of IncHI2 and IncFIA plasmids, producing a rare fusion product that simultaneously encodes functional features of both, i.e. virulence, resistance and high transmissibility. Conclusions: This is a novel hybrid plasmid mediating MDR and virulence from a clinical Salmonella Enteritidis strain. This plasmid is likely to be transmissible amongst various serotypes of Salmonella and other Enterobacteriaceae species, rendering a wide range of bacterial pathogens resistant to cephalosporins and fosfomycin, and further enhancing their virulence potential. It will be important to monitor the spread and further evolution of this plasmid among the Enterobacteriaceae strains.

Evolution and comparative genomics of pAQU-like conjugative plasmids in Vibrio species.

Objectives: To investigate a set of MDR conjugative plasmids found in Vibrio species and characterize the underlying evolution process. Methods: pAQU-type plasmids from Vibrio species were sequenced using both Illumina and PacBio platforms. Bioinformatics tools were utilized to analyse the typical MDR regions and core genes in the plasmids. Results: The nine pAQU-type plasmids ranged from ∼160 to 206 kb in size and were found to harbour as many as 111 core genes encoding conjugative, replication and maintenance functions. Eight plasmids were found to carry a typical MDR region, which contained various accessory and resistance genes, including ISCR1-blaPER-1-bearing complex class 1 integrons, ISCR2-floR, ISCR2-tet(D)-tetR-ISCR2, qnrVC6, a Tn10-like structure and others associated with mobile elements. Comparison between a plasmid without resistance genes and different MDR plasmids showed that integration of different mobile elements, such as IS26, ISCR1, ISCR2, IS10 and IS6100, into the plasmid backbone was the key mechanism by which foreign resistance genes were acquired during the evolution process. Conclusions: This study identified pAQU-type plasmids as emerging MDR conjugative plasmids among important pathogens from different origins in Asia. These findings suggest that aquatic bacteria constitute a major reservoir of resistance genes, which may be transmissible to other human pathogens during food production and processing.

IncI1 Plasmids Carrying Various blaCTX-M Genes Contribute to Ceftriaxone Resistance in Salmonella enterica Serovar Enteritidis in China.

Resistance to extended-spectrum ß-lactams in Salmonella, in particular, in serotypes such as Salmonella enterica serovar Enteritidis that are frequently associated with clinical infections, is a serious public health concern. In this study, phenotypic characterization of 433 clinical S. Enteritidis strains obtained from a nationwide collection of the Chinese Center for Disease Control and Prevention during the period from 2005 to 2010 depicted a trend of increasing resistance to ceftriaxone from 2008 onwards. Seventeen (4%) of the strains were found to be resistant to ceftriaxone, 7% were found to be resistant to ciprofloxacin, and 0.7% were found to be resistant to both ciprofloxacin and ceftriaxone. Most of the ceftriaxone-resistant S. Enteritidis strains (15/17) were genetically unrelated and originated from Henan Province. The complete sequence of an IncI1 plasmid, pSE115, which belonged to a novel sequence type, was obtained. This 87,255-bp IncI1 plasmid was found to harbor a blaCTX-M-14 gene in a novel multidrug resistance region (MRR) within the tra locus. Although the majority of strains were also found to contain conjugative IncI1 plasmids with a size similar to that of pSE115 (∼90 kb) and harbor a variety of blaCTX-M group 1 and group 9 elements, the novel MRR site at the tra locus in pSE115 was not detectable in the other IncI1 plasmids. The findings from this study show that cephalosporin resistance in S. Enteritidis strains collected in China was mainly due to the dissemination of IncI1 plasmids carrying blaCTX-M, resembling the situation in which IncI1 plasmids serve as major vectors of blaCTX-M variants in other members of the Enterobacteriaceae.

IncHI2 Plasmids Are the Key Vectors Responsible for oqxAB Transmission among Salmonella Species.

This study reported and analyzed the complete sequences of two oqxAB-bearing IncHI2 plasmids harbored by a clinical Salmonella enterica serovar Typhimurium strain and an S Indiana strain of animal origin, respectively. In particular, pA3T recovered from S Indiana comprised the resistance determinants oqxAB, aac(6')Ib-cr, fosA3, and blaCTX-M-14 Further genetic screening of 63 oqxAB-positive Salmonella isolates revealed that the majority carried IncHI2 plasmids, confirming that such plasmids play a pivotal role in dissemination of oqxAB in Salmonella spp.

Evolution and dissemination of OqxAB-like efflux pumps, an emerging quinolone resistance determinant among members of Enterobacteriaceae.

The OqxAB efflux pump, a plasmid-mediated quinolone resistance (PMQR) determinant, has become increasingly prevalent among members of Enterobacteriaceae over the past decade. To investigate the evolution and dissemination routes of the oqxAB operon, we assessed the prevalence of oqxAB-like elements among various Gram-negative bacterial species and analyzed the genotypic and phenotypic characteristics of organisms harboring such elements. With a comprehensive genotyping approach, a chromosome-based oqxAB operon was detectable in all Klebsiella pneumoniae strains tested, including organisms isolated before the year 1984. Sequence and phylogenetic analyses confirmed that the oqxAB operon in K. pneumoniae isolates was genetically closest to their plasmid-borne counterparts recoverable only from Escherichia coli and Salmonella isolates collected from the year 2003 onward. Chromosomal elements with much lower sequence homology were also found among the Enterobacter spp. but not other Gram-negative species. Contrary to the quinolone resistance phenotypes which were consistently observable among organisms with oqxAB-harboring plasmids, chromosomal oqxAB elements generally did not confer quinolone resistance, except for K. pneumoniae strains, which exhibited a typical oqxAB-mediated phenotype characterized by cross-resistance to olaquindox, chloramphenicol, and the quinolones. Gene expression analysis illustrated that such phenotypes were due to elevated expression of the chromosomal oqxAB operon. Furthermore, transposition of the oqxAB operon from the bacterial chromosome to plasmids was found to result in a >80-fold increase in the level of expression of the OqxAB pump, confirming its status as the first constitutively expressed efflux system located in bacterial mobile elements.

First detection of AmpC ß-lactamase bla(CMY-2) on a conjugative IncA/C plasmid in a Vibrio parahaemolyticus isolate of food origin.

Vibrio parahaemolyticus is an important causative agent of gastroenteritis, with the consumption of contaminated seafood being the major transmission route. Resistance to penicillin is common among V. parahaemolyticus strains, whereas cephalosporin resistance remains rare. In an attempt to assess the current prevalence and characteristics of antibiotic resistance of this pathogen in common food samples, a total of 54 (17% of the total samples) V. parahaemolyticus strains were isolated from 318 meat and seafood samples purchased from supermarkets and wet markets in Shenzhen, China, in 2013. These isolates exhibited high-level resistance to ampicillin, yet they were mostly susceptible to other antimicrobials, except for two that were resistant to extended-spectrum cephalosporins. The ß-lactamase gene blaPER-1 was detectable in one strain, V. parahaemolyticus V43, which was resistant to both third- and fourth-generation cephalosporins. Compared to other blaPER-1-positive V. parahaemolyticus strains reported in our previous studies, strain V43 was found to harbor an ∼200-kb conjugative plasmid carrying genes that were different from the antimicrobial resistance genes reported from the previous studies. The ß-lactamase gene blaCMY-2 was detectable for the first time in another V. parahaemolyticus isolate, V4, which was resistant to third-generation cephalosporins. This blaCMY-2 gene was shown to be located in an ∼150-kb IncA/C-type conjugative plasmid with a genetic structure consisting of traB-traV-traA-ISEcp1-blaCMY-2-blc-sugE-encR-orf1-orf2-orf3-orf4-dsbC-traC, which is identical to that of other IncA/C conjugative plasmids in Enterobacteriaceae, albeit with a different size. These findings indicate that the transmission of extended-spectrum-ß-lactamase (ESBL) and AmpC ß-lactamase genes via conjugative plasmids can mediate the development of extended-spectrum cephalosporin resistance in V. parahaemolyticus, thereby posing a potential threat to public health.

Dissemination of IncI2 Plasmids That Harbor the blaCTX-M Element among Clinical Salmonella Isolates.

The extended-spectrum-ß-lactamase (ESBL) determinant CTX-M-55 is increasingly prevalent in Escherichia coli but remains extremely rare in Salmonella. This study reports the isolation of a plasmid harboring the blaCTX-M-55 element in a clinical Salmonella enterica serotype Typhimurium strain resistant to multiple antibiotics. This plasmid is genetically identical to several known IncI2-type elements harbored by E. coli strains recovered from animals. This finding indicates that IncI2 plasmids harboring the blaCTX-M genes may undergo cross-species migration among potential bacterial pathogens, with E. coli as the major source of such elements.

Complete nucleotide sequence of a conjugative plasmid carrying bla(PER-1).

The nucleotide sequence of a self-transmissible plasmid pVPH1 harboring bla(PER-1) from Vibrio parahaemolyticus was determined. pVPH1 was 183,730 bp in size and shared a backbone similar to pAQU1 and pAQU2, differing mainly in an ∼40-kb multidrug resistance (MDR) region. A complex class 1 integron was identified together with ISCR1 and bla(PER-1) (ISCR1-bla(PER-1)-gst-abct-qacEΔ1-sul1), which was shown to form a circular intermediate playing an important role in the dissemination of bla(PER-1).

Emergence of clinical Salmonella enterica serovar Typhimurium isolates with concurrent resistance to ciprofloxacin, ceftriaxone, and azithromycin.

Salmonella infection is an important public health issue for which the needs of antimicrobial treatment are increasing. A total of 546 human clinical S. enterica serovar Typhimurium isolates were recovered from patients in hospitals in China during the period of 2005 to ∼ 2011. Twenty percent of the isolates exhibited resistance to ciprofloxacin, and 4% were resistant to ceftriaxone. Importantly, for the first time, 12 (2%) S. Typhimurium isolates resistant to both ciprofloxacin and ceftriaxone were recovered; among these 12 isolates, two were also resistant to azithromycin, and one was resistant to all other drugs tested. The combined effects of various transferrable extended-spectrum ß-lactamase determinants and a novel efflux-based ciprofloxacin resistance mechanism encoded by the mobile efflux gene oqxAB were responsible for the emergence of these extremely (highly) drug-resistant (XDR) S. Typhimurium isolates. The dissemination of resistance genes, such as those encoding ESBLs and the OqxAB pump, among Salmonella organisms will speed up the selection of XDR Salmonella, posing a huge threat to public health and Salmonella infection control.

First detection of oqxAB in Salmonella spp. isolated from food.

Food-borne salmonellosis is an important public health problem worldwide and the second leading cause of food-borne illnesses in Hong Kong. In this study, the prevalence and antimicrobial resistance of Salmonella in meat products in Hong Kong were determined. Interestingly, a plasmid-mediated quinolone resistance (PMQR) gene combination, oqxAB, which mediates resistance to nalidixic acid, chloramphenicol, and olaquindox, was for the first time detectable on the chromosomes of two Salmonella enterica serovar Derby isolates. Further surveillance of oqxAB in Salmonella will be needed.

Chlorogenic acid exhibits cholesterol lowering and fatty liver attenuating properties by up-regulating the gene expression of PPAR-α in hypercholesterolemic rats induced with a high-cholesterol diet.

Hypercholesterolemia is a major risk factor for the development of cardiovascular disease and nonalcoholic fatty liver disease. Natural compounds have been proved to be useful in lowering serum cholesterol to slow down the progression of cardiovascular disease and nonalcoholic fatty liver disease. In the present study, the hypocholesterolemic and hepatoprotective effects of the dietary consumption of chlorogenic acid were investigated by monitoring plasma lipid profile (total cholesterol, triglycerides, high-density lipoprotein and low-density lipoprotein) in Sprague-Dawley rats fed with a normal diet, a high-cholesterol diet or a high-cholesterol diet supplemented with chlorogenic acid (1 or 10 mg/kg/day p.o.) for 28 days. Chlorogenic acid markedly altered the increased plasma total cholesterol and low-density lipoprotein but decreased high-density lipoprotein induced by a hypercholesterolemic diet with a dose-dependent improvement on both atherogenic index and cardiac risk factor. Lipid depositions in liver were attenuated significantly in hypercholesterolemic animals supplemented with chlorogenic acid. It is postulated that hypocholesterolemic effect is the primary beneficial effect given by chlorogenic acid, which leads to other secondary beneficial effects such as atheroscleroprotective, cardioprotective and hepatoprotective functions. The hypocholesterolemic functions of chlorogenic acid are probably due to the increase in fatty acids unitization in liver via the up-regulation of peroxisome proliferation-activated receptor α mRNA.

Characterization of multidrug-resistant Proteus mirabilis isolated from chicken carcasses.

This study investigated genotypic and phenotypic features of antimicrobial resistance of Proteus mirabilis isolated from chicken products. Resistance to a broad spectrum of antimicrobial agents was commonly observed in the test isolates: tetracycline (100%), sulfamethoxazole (80%), chloramphenicol (66%), nalidixic acid (66%), ampicillin (60%), streptomycin (56%), ciprofloxacin (52%), kanamycin (46%), gentamicin (38%), ceftriaxone (36%), cefotaxime (34%), ceftiofur (22%), and amoxicillin-clavulanic acid (16%). The ß-lactamases TEM-1 and OXA-1, and extended-spectrum ß-lactamases CTX-M-9 and CMY-2 were detected in ß-lactam-resistant isolates. Single mutations in gyrA and parC were found to be contributing factors for fluoroquinolone resistance. Plasmid-mediated quinolone resistance (PMQR) genes qnrA and qnrD were detected in six fluoroquinolone-resistant isolates and a superintegron element, SXT, was detected in 14 out of 50 isolates. The high-level of antimicrobial resistance of P. mirabilis isolated from food products may pose a potential threat to public health.

Characterization of Salmonella food isolates with concurrent resistance to ceftriaxone and ciprofloxacin.

Foodborne salmonellosis is an important public health problem worldwide. Most human Salmonella infections occur through the consumption of contaminated food of animal origin. The study reported the first isolation of two Salmonella enterica serovar Oranienburg strains from pork in China with concurrent resistance to ciprofloxacin and ceftriaxone. Both isolates also showed resistance to norfloxacin, trimethoprim-sulfamethoxazole, and chloramphenicol, and an elevated minimal inhibitory concentraton of azithromycin; one strain was also resistant to amikacin, gentamicin, tetracycline, and amoxicillin-clavulanic acid. Salmonella ceftriaxone resistance was due to the production of IncN plasmidborne CTX-M-14 ESBL, and their ciprofloxacin resistance was mediated by target mutations and efflux pump activity. This is the first time that ceftriaxone- and ciprofloxacin-resistant Salmonella was reported in meat products, which may be due to the uses of antibiotics in animal production. The study warrants the continuous surveillance of multidrug-resistant Salmonella in meat products and cautious use of antibiotics in food animals.

Transcriptional Regulation and Functional Characterization of the Plasmid-Borne oqxAB Genes in Salmonella Typhimurium.

Coexistence of oqxAB and aac(6')-Ib-cr is often associated with the expression of fluoroquinolone resistance in Salmonella. The actual role of the plasmid-borne oqxAB gene and its regulatory mechanism compared to its chromosomally encoded counterpart in Klebsiella pneumoniae remain unclear We found that cloning of oqxAB gene only or chromosomally encoded oqxABR (ABRc) locus did not lead to an increase of ciprofloxacin (CIP) minimum inhibitory concentration (MIC) in S. Typhimurium, while cloning of the plasmid-encoded oqxABR (ABRp) locus led to a 4-fold increase in CIP MIC, reaching 0.0065 µg/mL. The co-carriage of these constructs with aac(6')-Ib-cr further increased the CIP MIC to 0.25 µg/mL in S. Typhimurium carrying aac(6')-Ib-cr and ABRp. Analysis of the transcription start site sequences showed that the expression level of suppressor protein gene, oqxR, in strains carrying ABRp was lower than that of its chromosomal counterpart due to the truncated promoter region in ABRp. The lower expression of OqxR in ABRp led to the overexpression of OqxAB, which elevated CIP MIC and exhibited a synergistic antimicrobial effect with the aac(6')-Ib-cr gene product to confer intermediate CIP (MIC = 0.25 µg/mL) in S. Typhimurium. Global transcriptional regulators in S. Typhimurium did not seem to play a role in regulating the plasmid-borne oqxAB genes. In conclusion, findings in this work showed that neither aac(6')-Ib-cr nor oqxABRp, but the combination of both genes, could mediate intermediated resistance to fluoroquinolone in Salmonella. The truncated promoter region in the oqxR gene of the plasmid-encoded locus led to the constituted expression of oqxAB genes. IMPORTANCE The transferable mechanisms of quinolone resistance (TMQR) gene, oqxAB, has been widely detected in Salmonella and is commonly associated with aac(6')-Ib-cr. It is thought to be associated with fluoroquinolone resistance, while its ancestor gene from K. pneumoniae is not. This study evaluated the actual role of the plasmid-borne oqxAB genes in Salmonella and showed that it was not able to mediate intermediated resistance to fluoroquinolone and only did so when it coexisted with aac(6')-Ib-cr. Chromosomally encoded oqxABRc from K. pneumoniae was not able to mediate enhanced CIP MIC due to tight regulation by the suppressor oqxR. However, plasmid-encoded oqxABRp enabled oqxAB to be expressed constitutionally due to the truncated promoter region of oqxR, leading to lower expression of the suppressor oqxR. This study clarified the roles of oqxAB and aac(6')-Ib-cr in mediating fluoroquinolone resistance in Salmonella and provides insights into the regulation of plasmid-encoded TMQR determinant, oqxAB.

Active maintenance of proton motive force mediates starvation-induced bacterial antibiotic tolerance in Escherichia coli.

Recent evidence suggests that metabolic shutdown alone does not fully explain how bacteria exhibit phenotypic antibiotic tolerance. In an attempt to investigate the range of starvation-induced physiological responses underlying tolerance development, we found that active maintenance of the transmembrane proton motive force (PMF) is essential for prolonged expression of antibiotic tolerance in bacteria. Eradication of tolerant sub-population could be achieved by disruption of PMF using the ionophore CCCP, or through suppression of PMF maintenance mechanisms by simultaneous inhibition of the phage shock protein (Psp) response and electron transport chain (ETC) complex activities. We consider disruption of bacterial PMF a feasible strategy for treatment of chronic and recurrent bacterial infections.

Over-Expression of ISAba1-Linked Intrinsic and Exogenously Acquired OXA Type Carbapenem-Hydrolyzing-Class D-ß-Lactamase-Encoding Genes Is Key Mechanism Underlying Carbapenem Resistance in Acinetobacter baumannii.

Acinetobacter baumannii is an important clinical pathogen which often causes fatal infections among seriously ill patients. Treatment options for managing infections caused by this organism have become limited as a result of emergence of carbapenem resistant strains. In the current study, whole genome sequencing, gene expression studies and enzyme kinetics analyses were performed to investigate the underlying carbapenem resistance mechanisms in fourteen clinical A. baumannii strains isolated from two hospitals, one each in Hong Kong and Henan Province, People's Republic of China. A large majority of the A. baumannii strains (11/14) were found to belong to the International Clone II (IC-II), among which six were ST208. Twelve of these strains were carbapenem resistant and found to either harbor bla OXA- 23/bla OXA- 72, or exhibit over-expression of the bla OXA- 51 gene upon ISAba1 insertion. Enzymatic assay confirmed that the OXA variants, including those of bla OXA - 51, exhibited strong carbapenem-degrading activities. In terms of other intrinsic mechanisms, a weak correlation was observed between reduced production of outer membrane porin CarO/expression resistance-nodulation-division (RND) efflux AdeB and phenotypic resistance. This finding implied that over-production of carbapenem-hydrolyzing-class D-ß-lactamases (CHDLs), including the intrinsic bla OXA- 51 gene and the acquired bla OXA- 23 and bla OXA- 24 elements, is the key mechanism of carbapenem resistance in A. baumannii. This view is confirmed by testing the effect of NaCl, a known bla OXA inhibitor, which was found to cause reduction in carbapenem MIC by twofolds to eightfolds, suggesting that inhibiting OXA type carbapenemases represents the most effective strategy to control phenotypic carbapenem resistance in A. baumannii.

Efficient generation of complete sequences of MDR-encoding plasmids by rapid assembly of MinION barcoding sequencing data.

Background: Multidrug resistance (MDR)-encoding plasmids are considered major molecular vehicles responsible for transmission of antibiotic resistance genes among bacteria of the same or different species. Delineating the complete sequences of such plasmids could provide valuable insight into the evolution and transmission mechanisms underlying bacterial antibiotic resistance development. However, due to the presence of multiple repeats of mobile elements, complete sequencing of MDR plasmids remains technically complicated, expensive, and time-consuming. Results: Here, we demonstrate a rapid and efficient approach to obtaining multiple MDR plasmid sequences through the use of the MinION nanopore sequencing platform, which is incorporated in a portable device. By assembling the long sequencing reads generated by a single MinION run according to a rapid barcoding sequencing protocol, we obtained the complete sequences of 20 plasmids harbored by multiple bacterial strains. Importantly, single long reads covering a plasmid end-to-end were recorded, indicating that de novo assembly may be unnecessary if the single reads exhibit high accuracy. Conclusions: This workflow represents a convenient and cost-effective approach for systematic assessment of MDR plasmids responsible for treatment failure of bacterial infections, offering the opportunity to perform detailed molecular epidemiological studies to probe the evolutionary and transmission mechanisms of MDR-encoding elements.