Lowering mortality risk in CR-HvKP infection in intestinal immunohistological and microbiota restoration.

Gut damage during carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-HvKP) infection is associated with a death risk. Understanding the mechanisms by which CR-HvKP causes intestinal damage and gut microbiota alteration, and the impact on immunity, is crucial for developing therapeutic strategies. This study investigated if gastrointestinal tract damage and disruption of gut microbiota induced by CR-HvKP infection undermined host immunity and facilitated multi-organ invasion of CR-HvKP; whether the therapeutic value of the rifampicin (RIF) and zidovudine (ZDV) combination was attributed to their ability to repair damages and restore host immunity was determined. A sepsis model was utilized to assess the intestinal pathological changes. Metagenomic analysis was performed to characterize the alteration of gut microbiota. The effects of the RIF and ZDV on suppressing inflammatory responses and improving immune functions and gut microbiota were evaluated by immunopathological and transcriptomic analyses. Rapid colonic damage occurred upon activation of the inflammation signaling pathways during lethal infections. Gut inflammation compromised host innate immunity and led to a significant decrease in probiotics abundance, including Bifidobacterium and Lactobacillus. Treatment with combination drugs significantly attenuated the inflammatory response, up-regulated immune cell differentiation signaling pathways, and promoted the abundance of Bifidobacterium (33.40 %). Consistently, supplementation of Bifidobacterium alone delayed the death in sepsis model. Gut inflammation and disrupted microbiota are key disease features of CR-HvKP infection but can be reversed by the RIF and ZDV drug combination. The finding that these drugs can restore host immunity through multiple mechanisms is novel and deserves further investigation of their clinical application potential.

Deciphering mechanisms of bla NDM gene transmission between human and animals: a genomics study of bacterial isolates from various sources in China, 2015 to 2017.

BackgroundIn China, the bla NDM gene has been recovered from human bacterial isolates since 2011. After 2014, detections of this gene in animal and food bacterial isolates have increasingly been reported.AimWe aimed to understand how bla NDM-bearing bacteria could spread between humans, animals, and animal-derived food.MethodsA total of 288 non-duplicate Escherichia coli strains, including 130 bla NDM-carrying and 158 bla NDM-negative strains were collected from clinical (humans), food-producing animals (pigs) and food (retail pork) sources between 2015 and 2017. The strains were whole genome sequenced. Core-genome-multilocus-sequence-typing was conducted. To investigate if sequence types (STs) found in human, animal or food samples could have a prior origin in a clinical, animal or food-borne animal reservoir, discriminant analysis of principal components (DAPC) was used. Plasmids bearing bla NDM were characterised.ResultsThe 130 bla NDM-carrying E. coli strains comprised a total of 60 STs, with ST167 (10/51), ST77 (6/33) and ST48 (6/46) being most prevalent in clinical, animal and food sources, respectively. Some ST10 and ST167 strains were respectively found among all three sources sampled, suggesting they might enable transfer of bla NDM between sources. DAPC analysis indicated possible transmissions of ST167 from humans to animals and ST10 from animals to human. In 114 of 130 bla NDM-carrying isolates, bla NDM was located on an IncX3 plasmid.ConclusionThis study in a Chinese context suggests that cross-species transmission of certain STs of E. coli harbouring bla NDM on mobile elements, may facilitate the spread of carbapenem-resistant Enterobacteriaceae. Stringent monitoring of bla NDM-bearing E. coli in ecosystems is important.

Genomic insights into the emergence and spread of NDM-1-producing Vibrio spp. isolates in China.

BACKGROUND: Carbapenemase-producing Vibrio spp., which exhibit an XDR phenotype, have become increasingly prevalent and pose a severe threat to public health. OBJECTIVES: To investigate the genetic characteristics of NDM-1-producing Vibrio spp. isolates and the dissemination mechanisms of blaNDM-1 in Vibrio. METHODS: A total of 1363 non-duplicate Vibrio spp. isolates collected from shrimp samples in China were subjected to antimicrobial susceptibility tests and screened for blaNDM-1. The blaNDM-1-positive isolates were further characterized by PFGE, MLST, conjugation and WGS using Illumina and Nanopore platforms. Plasmid stability and fitness cost were assessed using Escherichia coli J53, Klebsiella pneumoniae Kpt80 and Salmonella spp. SA2051 as recipient strains. RESULTS: In total, 13 blaNDM-1-positive isolates were identified, all exhibiting MDR. WGS analysis revealed that the 13 blaNDM-1 genes were all associated with a derivative of Tn125. Plasmid analysis revealed that six blaNDM-1 genes were located in IncC plasmids and the other seven were carried by plasmids of two different novel types. Conjugation and plasmid stability assays showed that only the IncC plasmids could be transferred to all the recipient strains and could be stably maintained in the hosts. CONCLUSIONS: The emergence of the novel plasmids has contributed to the variable genetic contexts of blaNDM-1 in Vibrio spp. and IncC plasmids harbouring the blaNDM-1 gene could facilitate the spread of such genes between Vibrio spp. and other zoonotic pathogens, leading to a rapid dissemination of blaNDM-1 in bacterial pathogens worldwide.

Econazole as adjuvant to conventional antibiotics is able to eradicate starvation-induced tolerant bacteria by causing proton motive force dissipation.

OBJECTIVES: Bacterial antibiotic tolerance is responsible for the recalcitrance of chronic infections. This study aims to investigate a potential drug that can effectively kill antibiotic-tolerant bacteria and evaluate the ability of this drug on the eradication of tolerant cells both in vitro and in vivo. METHODS: The in vitro effect of econazole on eradicating starvation-induced tolerant bacterial populations was studied by testing the amount of survival bacteria in the presence of econazole combining conventional antibiotics. Proton motive force (PMF) was determined after econazole treatment by DiOC2(3). Finally, mouse infection models were used to detect the ability of econazole on killing the tolerant populations in vivo. RESULTS: Econazole eradicated starvation-induced tolerant cells of various bacterial species within 24 or 96 h when used in combination with conventional antibiotics. Moreover, mouse survival rate drastically increased along with the decrease of in vivo bacterial count after treatment of infected mice with the econazole and ceftazidime combination for 72 h. PMF was found to have dissipated almost completely in econazole-treated cells. CONCLUSIONS: Econazole could act in combination with conventional antibiotics to effectively eradicate bacterial tolerant cells. The combined use of econazole and ceftazidime was shown to be effective for eradicating tolerant cells in a mouse infection model. The ability of econazole to eradicate tolerant cells was due to its ability to cause dissipation of bacterial transmembrane PMF. Econazole-mediated PMF disruption is a feasible strategy for the treatment of chronic and recurrent bacterial infections.

Structural and mechanistic basis of the high catalytic activity of monooxygenase Tet(X4) on tigecycline.

BACKGROUND: Tigecycline is a tetracycline derivative that constitutes one of the last-resort antibiotics used clinically to treat infections caused by both multiple drug-resistant (MDR) Gram-negative and Gram-positive bacteria. Resistance to this drug is often caused by chromosome-encoding mechanisms including over-expression of efflux pumps and ribosome protection. However, a number of variants of the flavin adenine dinucleotide (FAD)-dependent monooxygenase TetX, such as Tet(X4), emerged in recent years as conferring resistance to tigecycline in strains of Enterobacteriaceae, Acinetobacter sp., Pseudomonas sp., and Empedobacter sp. To date, mechanistic details underlying the improvement of catalytic activities of new TetX enzymes are not available. RESULTS: In this study, we found that Tet(X4) exhibited higher affinity and catalytic efficiency toward tigecycline when compared to Tet(X2), resulting in the expression of phenotypic tigecycline resistance in E. coli strains bearing the tet(X4) gene. Comparison between the structures of Tet(X4) and Tet(X4)-tigecycline complex and those of Tet(X2) showed that they shared an identical FAD-binding site and that the FAD and tigecycline adopted similar conformation in the catalytic pocket. Although the amino acid changes in Tet(X4) are not pivotal residues for FAD binding and substrate recognition, such substitutions caused the refolding of several alpha helixes and beta sheets in the secondary structure of the substrate-binding domain of Tet(X4), resulting in the formation of a larger number of loops in the structure. These changes in turn render the substrate-binding domain of Tet(X4) more flexible and efficient in capturing substrate molecules, thereby improving catalytic efficiency. CONCLUSIONS: Our works provide a better understanding of the molecular recognition of tigecycline by the TetX enzymes; these findings can help guide the rational design of the next-generation tetracycline antibiotics that can resist inactivation of the TetX variants.

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.

Emergence of an Empedobacter falsenii strain harbouring a tet(X)-variant-bearing novel plasmid conferring resistance to tigecycline.

OBJECTIVES: To investigate the genomic and phenotypic characteristics of an MDR Empedobacter falsenii strain isolated from a Chinese patient, which was phenotypically resistant to all last-line antibiotics (carbapenems, colistin and tigecycline). METHODS: Species identity was determined by MALDI-TOF MS analysis. The complete genome sequence of the isolate was determined by WGS and the genetic elements conferring antimicrobial resistance were determined. The origin of this strain was tracked by phylogenetic analysis. RESULTS: The E. falsenii strain was genetically most closely related to an Empedobacter sp. strain isolated from the USA. Members of E. falsenii are speculated to be intrinsically resistant to colistin. The carbapenem resistance of this strain was conferred by a chromosomal blaEBR-2 variant gene. Phylogenetic analysis indicated that the gene encoding the EBR ß-lactamase was widely distributed in Empedobacter spp. Tigecycline resistance was mediated by a tet(X) variant gene encoded by a non-conjugative and non-typeable plasmid. CONCLUSIONS: The MDR phenotype of the E. falsenii isolate was conferred by different mechanisms. Findings from us and others indicate that E. falsenii may serve as a reservoir for carbapenem and tigecycline resistance determinants.

An IncR Plasmid Harbored by a Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae Strain Possesses Five Tandem Repeats of the blaKPC-2::NTEKPC-Id Fragment.

Completed sequences of three plasmids from a carbapenem-resistant hypervirulent Klebsiella pneumoniae isolate, SH9, were obtained. In addition to the pLVPK-like virulence-conferring plasmid (pVir-CR-HvKP_SH9), the two multidrug-resistant plasmids (pKPC-CR-HvKP4_SH9 and pCTX-M-CR-HvKP4_SH9) were predicted to originate from a single pKPC-CR-HvKP4-like multireplicon plasmid through homologous recombination. Interestingly, the blaKPC-2 gene was detectable in five tandem repeats exhibiting the format of an NTEKPC-Id-like transposon (IS26-ΔTn3-ISKpn8-blaKPC-2-ΔISKpn6-korC-orf-IS26). The data suggest an important role of DNA recombination in mediating active plasmid evolution.

Emergence of OXA-232 Carbapenemase-Producing Klebsiella pneumoniae That Carries a pLVPK-Like Virulence Plasmid among Elderly Patients in China.

This study reported the clonal dissemination of OXA-232-producing sequence type 15 (ST15) carbapenem-resistant Klebsiella pneumoniae among elderly patients in China. All patients were immunocompromised, suffered from multiple underlying diseases, and were hospitalized for a prolonged period; however, they slowly recovered on antimicrobial therapy. The blaOXA-232 gene was in a 6.1-kb ColKP3-type nonconjugative plasmid. The strains displayed a multidrug resistance phenotype and were not hypervirulent despite harboring a virulence plasmid. Active surveillance should be enforced to control further transmission.

Characterization of the stability and dynamics of Tn6330 in an Escherichia coli strain by nanopore long reads.

BACKGROUND: The mcr-1 gene has been widely reported in both bacterial chromosomes and plasmids, while its stability in these genetic materials is not well understood. OBJECTIVES: Our aim was to characterize the stability and dynamics of Tn6330 elements in both a plasmid and the chromosome in a single bacterial population. METHODS: Plasmid-borne and chromosomal Tn6330 were characterized by PCR, conjugation, S1-PFGE, stability assay, single-molecule long-read sequencing and bioinformatics analysis. RESULTS: Tn6330 was simultaneously detected in both a plasmid and the chromosome of a clinical Escherichia coli strain. The plasmid was found to comprise the IncFIB replicon and a phage-like replicon, as well as two integrons that harboured various mobile elements and resistance genes including mcr-1, floR, blaTEM-1b and strAB. Both plasmid-borne and chromosomal Tn6330 transposons could be re-organized into a circular intermediate that played a role in transmission of the mcr-1 gene. Tn6330 was found to be very stable in both the plasmid and chromosome after 30 passages of 12 h with or without colistin selective pressure. The decayed structure of Tn6330 in the genuine single DNA molecules of bacterial populations, although occurring at a very low frequency, could be detected for the first time, in which Tn6330 was degraded into a single ISApl1 element. CONCLUSIONS: Long-read sequencing technology is a good tool to study the evolution and stability of genetic elements in bacteria. The ultrastability of an mcr-1-encoding element in a bacterial plasmid and chromosome renders it unlikely to be eradicated quickly by the reduced use of colistin, and factors leading to the frequent demise of Tn6330 warrant further studies.

Identification and characterization of a conjugative blaVIM-1-bearing plasmid in Vibrio alginolyticus of food origin.

OBJECTIVES: To investigate the genetic features of the blaVIM-1 gene first detected in a cephalosporin-resistant Vibrio alginolyticus isolate, Vb1978. METHODS: The MICs of V. alginolyticus strain Vb1978 were determined, and the ß-lactamases produced were screened and analysed using conjugation, S1-PFGE and Southern blotting. The complete sequence of the blaVIM-1-encoding plasmid was also obtained using the Illumina and MinION sequencing platforms. RESULTS: V. alginolyticus strain Vb1978, isolated from a retail shrimp sample, was resistant to cephalosporins and exhibited reduced susceptibility to carbapenems. A novel blaVIM-1-carrying conjugative plasmid, designated pVb1978, was identified in this strain. Plasmid pVb1978 had 50 001 bp and comprised 59 predicted coding sequences (CDSs). The plasmid backbone of pVb1978 was homologous to those of IncP-type plasmids, while its replication region was structurally similar to non-IncP plasmids. The blaVIM-1 gene was found to be carried by the class 1 integron In70 and associated with a defective Tn402-like transposon. CONCLUSIONS: A novel blaVIM-1-carrying conjugative plasmid, pVb1978, was reported for the first time in V. alginolyticus, which warrants further investigation in view of its potential pathogenicity towards humans and widespread occurrence in the environment.

Selective and suppressive effects of antibiotics on donor and recipient bacterial strains in gut microbiota determine transmission efficiency of blaNDM-1-bearing plasmids.

OBJECTIVES: To test whether antibiotics of different functional categories exhibit differential potential in promoting transmission of MDR-encoding plasmids among members of the gut microbiome. METHODS: Rats inoculated with blaNDM-1-bearing Klebsiella pneumoniae were subjected to treatment with different types of antibiotics. The structural changes in the gastrointestinal (GI) tract microbiome were determined by 16S rRNA sequencing and analysis. In addition, the efficiency of transmission of blaNDM-1-bearing plasmids to different subtypes of GI tract Escherichia coli was also confirmed in vitro. RESULTS: We showed that drugs that are commonly used to treat Gram-negative bacterial infections, such as ampicillin and amoxicillin, could enrich both carbapenem-resistant Enterobacteriaceae (CRE) and antibiotic-susceptible E. coli in the GI tract, thereby promoting transmission of the blaNDM-1-bearing plasmid in the gut microbiome. In contrast, meropenem was found to minimize the population of CRE in the gut microbiome, hence treatment with this drug exhibited drastically lower potential to promote transmission of the blaNDM-1-bearing plasmid to the recipient strains. We further showed that an increased population size of Proteobacteria due to a suppressive effect on Firmicutes is a key factor in enhancing the efficiency of transmission of the blaNDM-1-bearing plasmid and hence dissemination of carbapenem-resistant strains. CONCLUSIONS: This study depicted for the first time the effect of different antibiotics on the structure of the rat GI tract microbiome, which in turn determined the pattern and rate of transmission of the blaNDM-1-bearing plasmid. Such findings can help establish new guidelines for prudent antibiotic usage to minimize the chance of dissemination of mobile resistance elements among members of the GI tract microbiome.

Increased prevalence of Escherichia coli strains from food carrying bla NDM and mcr-1-bearing plasmids that structurally resemble those of clinical strains, China, 2015 to 2017.

IntroductionEmergence of resistance determinants of bla NDM and mcr-1 has undermined the antimicrobial effectiveness of the last line drugs carbapenems and colistin.AimThis work aimed to assess the prevalence of bla NDM and mcr-1 in E. coli strains collected from food in Shenzhen, China, during the period 2015 to 2017.MethodsMultidrug-resistant E. coli strains were isolated from food samples. Plasmids encoding mcr-1 or bla NDM genes were characterised and compared with plasmids found in clinical isolates.ResultsAmong 1,166 non-repeated cephalosporin-resistant E. coli strains isolated from 2,147 food samples, 390 and 42, respectively, were resistant to colistin and meropenem, with five strains being resistant to both agents. The rate of resistance to colistin increased significantly (p < 0.01) from 26% in 2015 to 46% in 2017, and that of meropenem resistance also increased sharply from 0.3% in 2015 to 17% in 2017 (p < 0.01). All meropenem-resistant strains carried a plasmid-borne bla NDM gene. Among the colistin-resistant strains, three types of mcr-1-bearing plasmids were determined. Plasmid sequencing indicated that these mcr-1 and bla NDM-bearing plasmids were structurally similar to those commonly recovered from clinical isolates. Interestingly, both mcr-1-bearing and bla NDM-bearing plasmids were transferrable to E. coli strain J53 under selection by meropenem, yet only mcr-1-bearing plasmids were transferrable under colistin selection.ConclusionThese findings might suggest that mobile elements harbouring mcr-1 and bla NDM have been acquired by animal strains and transmitted to our food products, highlighting a need to prevent a spike in the rate of drug resistant food-borne infections.

Molecular Characterization of qnrVC Genes and Their Novel Alleles in Vibrio spp. Isolated from Food Products in China.

This study reports the prevalences of qnrVC genes in 74 ciprofloxacin-resistant Vibrio sp. isolates. Two novel functional qnrVC alleles, qnrVC8 and qnrVC9, sharing 98% and 99% nucleotide similarity with qnrVC6 and qnrVC7, respectively, were identified. Our findings suggested that carriage of qnrVC alleles, together with target mutations in gyrA and parC genes, may contribute to the development of fluoroquinolone resistance in Vibrio species, posing a serious threat to public health.

Resolution of dynamic MDR structures among the plasmidome of Salmonella using MinION single-molecule, long-read sequencing.

Background: ISCR1 is an important mobile genetic element mediating the transfer of antibiotic resistance genes. Genetic diversity regarding distribution and copy numbers of ISCR1 within a bacterial population derived from an ancestral strain, which may reflect the degree of genetic plasticity conferred by such an element, has not been studied. Objectives: To investigate the plasmid heterogeneity in Salmonella conferred by ISCR1. Methods: Nanopore long-read and other sequencing technologies were used to resolve the structures harbouring different copies of ISCR1-qnrB6 from the perspective of single molecules. Results: Salmonella London Sa128 was positive for ISCR1-qnrB6 and harboured an MDR-encoding conjugative IncF plasmid, pSa128, containing a complex class 1 integron. The plasmid pSa128T from the transconjugant was larger compared with the original plasmid pSa128, presumably due to amplification of ISCR1-qnrB6. Single-molecule, long-read analysis indicated that both plasmids in the donor and transconjugant strains were in a heterogeneous state that contains variable numbers of ISCR1-qnrB6, with four and eight copies in single plasmids being the dominant types. This type of plasmid heterogeneity in populations of one strain can be regarded as an atypical plasmidome. Conclusions: This study highlights the importance of investigation of a single plasmid structure based on long-read sequencing technologies, with a focus on analysing the complex structures of the MDR region, which is expected to exhibit genetic polymorphism or plasmid heterogeneity in various MDR-encoding elements even among members of the same strain. The availability of a single-molecule sequencing technique represents a paradigm shift in the capability of performing population genetic analysis of antibiotic-resistant organisms.

Carriage of blaKPC-2 by a virulence plasmid in hypervirulent Klebsiella pneumoniae.

Objectives: To characterize a plasmid in a K1 hypervirulent Klebsiella pneumoniae (HvKP) strain encoding both hypervirulence and carbapenem resistance phenotypes. Methods: Plasmids from HvKP strain KP70-2 were subjected to whole-plasmid sequencing using both the Illumina NextSeq 500 sequencing platform and Nanopore MinION sequencer platforms. Results: A hybrid virulence- and resistance-encoding plasmid of 240 kb, harbouring both the virulence gene rmpA2 and the carbapenemase gene blaKPC-2, was recovered from a clinical HvKP strain. Designated pKP70-2, the plasmid was found to be almost structurally identical to various known hypervirulence-encoding plasmids harboured by other HvKP strains, except for an extra MDR-encoding region located within the genetically conserved plasmid backbone. This MDR region was flanked by two copies of IS26 in the same orientation, one at each end and linked to an external 8 bp (CTAAAATT) product of target site duplications, suggesting that an insertion event was responsible for the integration of the MDR region into the virulence plasmid. The MDR region was also found to harbour mobile elements that in turn contain the antibiotic resistance genes dfrA14 and blaKPC-2. Conclusions: Based on the genetic composition of pKP70-2, we postulate that the multiple insertion elements that it harbours were responsible for mediating the plasmid recombination events that underlie continuous emergence and genetic adaptation of novel resistance- and virulence-encoding mobile elements in K. pneumoniae.

Recombination of plasmids in a carbapenem-resistant NDM-5-producing clinical Escherichia coli isolate.

Objectives: To investigate the genetic features of five plasmids recovered from an NDM-5-producing clinical Escherichia coli strain, BJ114, and to characterize the plasmid recombination event that occurred during the conjugation process. Methods: The genetic profiles of the five plasmids were determined by PCR, conjugation, S1-PFGE, Southern hybridization and WGS analysis. Plasmid sequences were analysed with various bioinformatic tools. Results: Complete sequences of five plasmids were obtained. Two small plasmids, pBJ114-141 and pBJ114-46, were speculated to have recombined into a large fusion plasmid, pBJ114T-190. When conjugated to other E. coli strains, some of the fusion plasmids were able to be resolved into the original two single plasmids. A non-conjugative plasmid, pBJ114-96, exhibited a high degree of sequence identity with the phage P7-like plasmid as well as an mcr-1-bearing plasmid. Another plasmid, pBJ114-78, was found to contain multidrug resistance genes and various mobile elements. Conclusions: The fusion plasmid recoverable from the transconjugant was found to be generated as a result of a recombination event that occurred upon interaction between a blaNDM-5-carrying plasmid and another plasmid present in the parental strain. Such recombination events presumably play a potential role in the dissemination of the blaNDM genes among different plasmids and pathogenic bacterial strains.

Functional Characterization of CTX-M-14 and CTX-M-15 ß-Lactamases by In Vitro DNA Shuffling.

This work investigated the molecular events driving the evolution of the CTX-M-type ß-lactamases by the use of DNA shuffling of fragments of the blaCTX-M-14 and blaCTX-M-15 genes. Analysis of a total of 51 hybrid enzymes showed that enzymatic activity could be maintained in most cases, yet hybrids that were active possessed fewer amino acid substitutions than those that were inactive, suggesting that point mutations in the constructs rather than reshuffling of the fragments of the two target genes would more likely cause disruption of CTX-M activity. For example, the P67L and L261P changes in a CTX-M-14 fragment could completely abolish the activity of the enzyme on all antibiotics tested. Structural analysis showed that L216 was located in the active-site ß sheet and might interact with the adjacent hydrophobic residues to stabilize the active-site ß sheet and maintain the integrity of the enzyme active site. Likewise, a single amino acid substitution, E64K, was found to exhibit a significant suppressive effect on CTX-M-15 activity. Structural analysis showed that E64 might form a salt bridge with R44, disruption of which might affect CTX-M-15 activity. Further analysis of the structure-function relationship of a range of mutant enzymes confirmed that, as can be expected, unstable enzymes lose their activity and avoid selective events. These findings suggest that the distal pockets could also contribute to the activity of the enzymes and may be regarded as alternative targets for inhibitor development.

IncFII Conjugative Plasmid-Mediated Transmission of blaNDM-1 Elements among Animal-Borne Escherichia coli Strains.

This study aims to investigate the prevalence and transmission dynamics of the blaNDM-1 gene in animal Escherichia coli strains. Two IncFII blaNDM-1-encoding plasmids with only minor structural variation in the MDR region, pHNEC46-NDM and pHNEC55-NDM, were found to be responsible for the transmission of blaNDM-1 in these strains. The blaNDM-1 gene can be incorporated into plasmids and stably inherited in animal-borne E. coli strains that can be maintained in animal gut microflora even without carbapenem selection pressure.

Prevalence and Molecular Characterization of mcr-1-Positive Salmonella Strains Recovered from Clinical Specimens in China.

The recently discovered colistin resistance element, mcr-1, adds to the list of antimicrobial resistance genes that rapidly erode the antimicrobial efficacy of not only the commonly used antibiotics but also the last-line agents of carbapenems and colistin. This study investigated the prevalence of the mobile colistin resistance determinant mcr-1 in Salmonella strains recovered from clinical settings in China and the transmission potential of mcr-1-bearing mobile elements harbored by such isolates. The mcr-1 gene was recoverable in 1.4% of clinical isolates tested, with the majority of them belonging to Salmonella enterica serotype Typhimurium. These isolates exhibited diverse pulsed-field gel electrophoresis (PFGE) profiles and high resistance to antibiotics other than colistin and particularly to cephalosporins. Plasmid analysis showed that mcr-1 was carried on a variety of plasmids with sizes ranging from ∼30 to ∼250 kb, among which there were conjugative plasmids of ∼30 kb, ∼60 kb, and ∼250 kb and nonconjugative plasmids of ∼140 kb, ∼180 kb, and ∼240 kb. Sequencing of representative mcr-1-carrying plasmids revealed that all conjugative plasmids belonged to the IncX4, IncI2, and IncHI2 types and were highly similar to the corresponding types of plasmids reported previously. Nonconjugative plasmids all belonged to the IncHI2 type, and the nontransferability of these plasmids was attributed to the loss of a region carrying partial or complete tra genes. Our data revealed that, similar to the situation in Escherichia coli, mcr-1 transmission in Salmonella was accelerated by various plasmids, suggesting that transmission of mcr-1-carrying plasmids between different species of Enterobacteriaceae may be a common event.