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Objectives To characterize the relationship of tet(X4)-positive isolates from different hosts and environments. Methods PCR and MALDI-TOF MS were used to identify the tet(X4)-positive isolates. The MICs of 13 antimicrobial agents were determined by broth microdilution. Illumina technology was used to sequence all of the isolates. One isolate was randomly selected from Escherichia coli ST761 clones for long-read sequencing to obtain plasmid sequences. Bioinformatics analysis was used to determine the phylogeny of 46 tet(X4)-positive E. coli ST761 strains. Results A total of 12 tet(X4)-positive isolates, 8 E. coli and 4 Aeromonas simiae, were obtained from six lairages of a slaughterhouse. These isolates exhibited resistance to at least three classes of antimicrobials, including tigecycline. The majority of them, seven E. coli and three A. simiae, represent separate clonal groups. Notably, the seven E. coli isolates belonged to ST761, a common ST carrying the tet(X4) gene that has been identified in 39 isolates from animals, meat, wastewater and humans from seven Chinese provinces. All 46 tet(X4)-positive E. coli ST761 strains from various sources have a close phylogenetic relationship (0–72 SNPs), with a high nucleotide sequence similarity of resistance genes and the tet(X4)-carrying IncX1-IncFIA(HI1)-IncFIB(K) hybrid plasmid, indicating a clonal relationship of tet(X4)-positive E. coli ST761 among animals, food, the environment and humans. Conclusions The clonal relationship of tet(X4)-positive E. coli ST761 between humans and animals poses a previously underestimated threat to public health. To the best of our knowledge, this is the first description of tet(X4)-positive A. simiae.

Objectives To characterize a clinical Klebsiella pneumoniae isolate from China co-harbouring tet(X4), blaOXA-181 and the aerobactin operon on an IncFIBk-FII-X3-ColKP3 hybrid plasmid. Methods A tigecycline-resistant strain was recovered from the intestinal sample of a patient. It was subjected to antimicrobial susceptibility testing, conjugation assay, virulence testing, WGS, bioinformatics analysis, plasmid stability testing and fitness cost testing. Results The strain K. pneumoniae T877 was resistant to tigecycline, intermediate to piperacillin/tazobactam and ertapenem, and positive for tet(X), blaOXA-181 and the virulence-associated operon iutAiucABCD, which were located on the same plasmid, named pKPT877-hybrid. It was 99.96% identical to the IncFIBk-FII plasmid pSCH6109-Vir (accession number CP050860) from K. pneumoniae strain SCH6109 at 96% coverage with the absence of a 50 kb region on pKPT877-hybrid; this region was highly homologous to the 51 kb IncX3-ColKP3-type, blaOXA-181-carrying plasmid pOXA181-191773 (accession number CP080367). Plasmid pKPT877-hybrid was conjugatively transferable to the ST11 K. pneumoniae strains FJ8 and KP04. pKPT877-hybrid did not have a significant impact on the fitness cost and could be maintained stably in T877. Conclusions We report for the first time (to the best of our knowledge) the co-transfer of last-line antibiotic resistance determinants [tet(X4) and blaOXA-181] and the aerobactin operon (iutAiucABCD) by a mobile IncFIBk-FII-X3-ColKP3 hybrid plasmid, which can be stably maintained in K. pneumoniae strains, even in the absence of antibiotic selective pressure. Once the plasmid transfers to a K. pneumoniae with porin deficiency, the strain might have high levels of resistance to carbapenems and tigecycline, which are the last line of defence against infections. Heightened and continuous efforts are needed to control its dissemination.

ObjectivesTo characterize a novel plasmid-mediated tigecycline resistance-related gene, tet(Y), in a clinical Acinetobacter baumannii isolate from China.MethodsThe tet(Y)-encoded tigecycline-resistant A. baumannii 2016GDAB1 was screened through antimicrobial susceptibility testing and WGS. The function of tet(Y) was verified by complementation of tet(Y). The plasmid transferability and stability were detected via plasmid conjugation and in vitro bacterial passaging. The 3D structure of Tet(Y) was predicted and docked using tFold and AutoDock Vina.ResultsThe tigecycline-resistant A. baumannii 2016GDAB1 was isolated from bronchoalveolar lavage fluid of a patient with hospital-acquired pneumonia. However, this strain did not harbour any common tigecycline resistance genes, determinants or mutations. 2016GDAB1 belongs to the non-epidemic clone ST355 (Oxford scheme), which has been mainly reported in animals. The tet(Y) gene was located on a 72 156 bp plasmid and genomic environment analysis revealed that Tn5393 may play a role in tet(Y) transmission, whereas phylogenetic analysis indicated the origin of tet(Y) as from Aeromonas. Overexpression of tet(Y) resulted in a 2- to 4-fold increase in tigecycline MIC. Introduction of the tet(Y)-harbouring plasmid p2016GDAB1 via electroporation resulted in a 16-fold increase in tigecycline MIC but failed to transfer into the tigecycline-susceptible A. baumannii recipient via conjugation. Isolates carrying the tet(Y) gene were vulnerable to tigecycline pressure and exhibited decreased susceptibility to tigecycline. A tet(Y)-carrying plasmid was stably maintained in the host strains.ConclusionsThis study identified the tigecycline resistance-related gene tet(Y) in A. baumannii. This gene conferred an increased tigecycline MIC and the transposable element Tn5393 may play a role in its transmission across isolates.

Objectives Carbapenem-resistant Klebsiella pneumoniae (CR-KP) infections represent severe threats to public health worldwide. The aim of this study was to assess potential synergistic interaction between tigecycline and aminoglycosides via in vitro and in vivo studies. Methods Antibiotic resistance profiles and molecular characteristics of 168 CR-KP clinical isolates were investigated by susceptibility testing, PCR and MLST. Chequerboard tests and time–kill assays were performed for 20 CR-KP isolates to evaluate in vitro synergistic effects of tigecycline combined with aminoglycosides. A tissue-cage infection model of rats was established to evaluate in vivo synergistic effects. Different doses of tigecycline and aminoglycosides alone or in combination were administered for 7 days via tail vein injection. Antibiotic efficacy was evaluated in tissue-cage fluid and emergence of resistance was screened. Results The chequerboard tests showed that this combination displayed synergistic or partial synergistic activity against CR-KP. The time–kill assays further demonstrated that strong synergistic effects of such a combination existed against isolates that were susceptible to both drugs but for resistant isolates no synergy was observed if clinical pharmacokinetics were taken into consideration. The in vivo study showed that the therapeutic effectiveness of combination therapies was better than that of monotherapy for susceptible isolates, suggesting in vivo synergistic effects. Furthermore, combinations of tigecycline with an aminoglycoside showed significant activity in reducing the occurrence of tigecycline-resistant mutants. Conclusions Compared with single drugs, tigecycline combined with aminoglycosides could exert synergistic effects and reduce the emergence of tigecycline resistance. Such a combination might be an effective alternative when treating CR-KP infections in clinical practice.

Objectives To determine the most common tigecycline resistance mechanisms in carbapenem-resistant Acinetobacter baumannii isolates obtained during the global Tigecycline Evaluation Surveillance Trial (TEST). Methods Tigecycline MICs were determined by broth microdilution. WGS was used to screen for the previously identified tigecycline resistance mechanisms, as well as mutations in resistance-nodulation-cell division (RND)-type efflux pump regulators. Results From a total 313 isolates, 113 genetically unique tigecycline-resistant isolates were analysed. The most frequent and worldwide distributed mechanism associated with tigecycline resistance was disruption of adeN, which encodes the repressor of the RND efflux pump AdeIJK, either by IS elements or nucleotide deletions causing premature stop codons. However, mutations leading to amino acid substitutions and disruption by IS elements within the two-component regulatory system adeRS, which regulates expression of the AdeABC efflux pump, correlate with higher tigecycline MICs, but these were found less frequently and were mainly restricted to Southern European countries. Furthermore, an altered version of tviB was identified in several tigecycline-resistant isolates that did not have putative resistance mutations within RND-type regulators. The resistance determinants tet(A) and tet(X), as well as resistance mutations in putative resistance determinants trm, plsC, rrf, msbA and genes encoding 30S ribosomal proteins, were not identified in any isolate. Conclusions The most prevalent tigecycline resistance mechanisms were caused by alterations in the regulators of RND-type efflux pumps. These data provide the basis for further characterization of regulator alterations and their contribution to increased efflux and tigecycline resistance, and also should be taken into account in drug discovery programmes to overcome the contribution of efflux pumps.

Background Tigecycline and carbapenems are regarded as vital antimicrobials to treat serious bacterial infections. Co-occurrence of resistance genes conferring resistance to both tigecycline and carbapenems in Pseudomonas spp. was not investigated. Objectives To characterize a megaplasmid co-harbouring tmexCD1-toprJ1 and blaVIM-2 in Pseudomonas putida of migratory bird origin. Methods One tigecycline- and carbapenem-resistant strain was isolated and characterized by antimicrobial susceptibility testing, conjugation assay, WGS and bioinformatics analysis. Results The strain P. putida ZXPA-20 resistant to meropenem and tigecycline was positive for blaVIM-2 and tmexCD1-toprJ1 genes. The gene blaVIM-2 was inserted into the backbone of the megaplasmid pZXPA-20 within a Tn5090-like structure. The genetic context of tmexCD1-toprJ1 in the megaplasmid was identical to many chromosomal tmexCD1-toprJ1 of Pseudomonas species. Plasmid-mediated tmexCD1-toprJ1 gene cluster in Pseudomonas spp. was more common than that in Klebsiella pneumoniae. To the best of our knowledge, this is the first report of co-occurrence of blaVIM-2 and tmexCD1-toprJ1 in one plasmid. Conclusions Emergence of plasmid-mediated carbapenem and tigecycline resistance genes in P. putida from migratory birds highlighted the importance of surveillance of novel mobile resistance genes in migratory birds, which may play a vital role in global transmission of novel resistance genes.

ObjectivesTo determine the in vitro susceptibility of members of the Mycobacterium abscessus complex to routinely tested antibiotics and to an extended antibiotic panel.MethodsNon-duplicate isolates for which susceptibility testing results were available were included in this study. Retrospective laboratory records were reviewed, including tigecycline susceptibility results, and testing was performed with additional drugs, including vancomycin, dalbavancin, telavancin, oritavancin, rifabutin, delafloxacin, eravacycline, clofazimine and bedaquiline using broth microdilution (Sensititre, Thermo Fisher).ResultsA total of 218 M. abscessus complex isolates were included for retrospective review, of which 151 were respiratory isolates. Of these 218 isolates, 211 were available for additional testing with the extended antibiotic panel. Of these, 146 were respiratory isolates. One isolate had a vancomycin MIC of 2 mg/L and MICs of all other isolates were >8 mg/L. All isolates had MICs of >8 mg/L for oritavancin, dalbavancin and telavancin. One isolate had a delafloxacin MIC of 4 mg/L and MICs of all other isolates were >8 mg/L. The MIC50/MIC90s of rifabutin, tigecycline, eravacycline, clofazimine and bedaquiline were 16/32, 0.5/1, 0.12/0.25, 0.12/0.25 and 0.06/0.12 mg/L, respectively.ConclusionsIn vitro activity was demonstrated for clofazimine, bedaquiline and eravacycline, indicating potential for inclusion as standardized therapy for M. abscessus complex infections.

Objectives To reconstruct the evolutionary history of the clinical Acinetobacter baumannii XH1056, which lacks the Oxford scheme allele gdhB. Methods Susceptibility testing was performed using broth microdilution and agar dilution. The whole-genome sequence of XH1056 was determined using the Illumina and Oxford Nanopore platforms. MLST was performed using the Pasteur scheme and the Oxford scheme. Antibiotic resistance genes were identified using ABRicate. Results XH1056 was resistant to all antibiotics tested, apart from colistin, tigecycline and eravacycline. MLST using the Pasteur scheme assigned XH1056 to ST256. However, XH1056 could not be typed with the Oxford MLST scheme as gdhB is not present. Comparative analyses revealed that XH1056 contains a 52 933 bp region acquired from a global clone 2 (GC2) isolate, but is otherwise closely related to the ST23 A. baumannii XH858. The acquired region in XH1056 also contains a 34 932 bp resistance island that resembles AbGRI3 and contains the armA, msrE-mphE, sul1, blaPER-1, aadA1, cmlA1, aadA2, blaCARB-2 and ere(B) resistance genes. Comparison of the XH1056 chromosome to that of GC2 isolate XH859 revealed that the island in XH1056 is in the same chromosomal region as that in XH859. As this island is not in the standard AbGRI3 position, it was named AbGRI5. Conclusions XH1056 is a hybrid isolate generated by the acquisition of a chromosomal segment from a GC2 isolate that contains a resistance island in a new location—AbGRI5. As well as generating ST256, it appears likely that a single recombination event is also responsible for the acquisition of AbGRI5 and its associated antibiotic resistance genes.

Objectives The recently discovered tigecycline-inactivating enzyme Tet(X4) can confer high-level tigecycline resistance on its hosts, which makes it a public health concern. This study focused on isolation and screening of Tet(X4)-positive Enterobacteriaceae from the gut microbiota of a cohort of healthy individuals in Singapore. Methods MinION and Illumina sequencing was performed to obtain the complete genome sequences of Escherichia coli 2EC1-1 and 94EC. Subsequently, 109 human faecal samples were screened retrospectively for eravacycline-resistant Enterobacteriaceae strains, which were further tested for tet(X4) by PCR. The taxonomy of the isolated strains was determined by 16S rRNA gene PCR and Sanger sequencing. Results Comparative genomic analysis of E. coli 2EC1-1 and 94EC revealed that both carry tet(X4), which is encoded by IncI1-type plasmids p2EC1-1 and p94EC-2, respectively. Retrospective screening of faecal samples collected from 109 healthy individuals showed that the faecal carriage rate of Tet(X4)-producing Enterobacteriaceae is 10.1% (95% CI = 5.1%–17.3%), suggesting that tet(X4) is widely distributed in the gut microbiota of healthy individuals in Singapore. Conclusions To the best of our knowledge, this is the first report on the prevalence of tet(X4) in the gut microbiota of a healthy human cohort, as well as the first description of this resistance mechanism outside of China. Our findings suggest that surveillance of tet(X4) in community settings is vital to monitor the spread of this resistance mechanism.

Background The emergence of plasmid-mediated tet(X3)/tet(X4) genes is threatening the role of tigecycline as a last-resort antibiotic to treat clinical infections caused by XDR bacteria. Considering the possible public health threat posed by tet(X) and its variants [which we collectively call ‘tet(X) genes’ in this study], global monitoring and surveillance are urgently required. Objectives Here we conducted a worldwide survey of the global distribution and spread of tet(X) genes. Methods We analysed a comprehensive dataset of bacterial genomes in conjunction with surveillance data from our laboratory and the NCBI database, as well as sufficient metadata to characterize the results. Results The global distribution features of tet(X) genes were revealed. We clustered three types of genetic backbones of tet(X) genes embedded or transferred in bacterial genomes. Our pan-genome analyses revealed a large genetic pool composed of tet(X)-carrying sequences. Moreover, phylogenetic trees of tet(X) genes and tet(X)-like proteins were built. Conclusions To the best of our knowledge, our results provide the first view of the global distribution of tet(X) genes, demonstrate the features of tet(X)-carrying fragments and highlight the possible evolution of tigecycline-inactivation enzymes in diverse bacterial species and habitats.

BackgroundSPR206 is a novel polymyxin analogue. Activity against clinical isolates is little documented.MethodsA collection of 200 MDR, carbapenem-resistant, tigecycline-resistant, colistin-resistant and non-MDR clinical isolates of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Stenotrophomonas maltophilia was obtained from 50 centres across China (2016–17). All isolates were derived from respiratory tract, urine and blood samples. Strains were purposely selected on the basis of phenotypes, genotypes and specimen origins. MICs of SPR206 and other antimicrobials were determined.ResultsSPR206 was active against all bacteria tested except colistin-resistant isolates. The MIC50/90 values of SPR206 for colistin-resistant strains were comparable to known polymyxins (16/128 versus 8/128 mg/L). SPR206 exhibited potent activity against colistin-susceptible OXA-producing A. baumannii (MIC50/90 = 0.064/0.125 mg/L), NDM-producing Enterobacteriaceae (MIC50/90 = 0.125/0.25 mg/L) and KPC-2-producing Enterobacteriaceae (MIC50/90 = 0.125/0.5 mg/L). In fact, SPR206 was the most potent agent tested, with 2- to 4-fold lower MICs than colistin and polymyxin B for A. baumannii, P. aeruginosa and Enterobacteriaceae. Additionally, MIC values of SPR206 (MIC50/90 = 0.064/0.125 mg/L) were 16- to 32-fold lower than those of tigecycline (MIC50/90 = 2/2 mg/L) for tigecycline-susceptible carbapenem-resistant A. baumannii.ConclusionsSPR206 showed good in vitro activity against MDR, tigecycline-resistant and non-MDR clinical isolates of Gram-negative pathogens. SPR206 also exhibited superior potency to colistin and polymyxin B, with 2- to 4-fold lower MIC50/90 values.

Background Mycobacterium abscessus causes chronic pulmonary infections. Owing to its resistance to most classes of antibiotics, treatment is complex and cure rates are only 45%. Tigecycline is active against M. abscessus, but severe toxicity and the need for IV administration limit its use. Objectives To assess the potential of inhaled tigecycline as a treatment for M. abscessus pulmonary disease, by measuring its efficacy in a mouse model of chronic M. abscessus pulmonary disease, establishing the intracellular activity of tigecycline against M. abscessus in human macrophages and measuring the activity of tigecycline in the sputum of cystic fibrosis patients. Methods We infected GM-CSF knockout mice with M. abscessus by intrapulmonary aerosol. Infected mice were treated with tigecycline in 0.25, 1.25 and 2.5 mg doses, by inhalation, or untreated, for 28 days. Tigecycline was added to human peripheral blood-derived macrophages infected with M. abscessus to assess its intracellular activity. We performed a time–kill kinetics experiment of tigecycline against M. abscessus with and without sputum of cystic fibrosis patients. Results Inhaled tigecycline proved highly effective against M. abscessus in GM-CSF knockout mice. The effect was dose dependent. Tigecycline showed potent activity against M. abscessus in macrophages and retained most of its activity in the presence of sputum of cystic fibrosis patients. Conclusions Inhaled tigecycline may represent a viable treatment option for M. abscessus pulmonary disease, where treatment outcomes are currently very poor. A stable and safe formulation is required to proceed to further pharmacodynamic studies and ultimately clinical trials.

Objectives To report a novel tigecycline resistance gene, tet(X6), and its variants in four bacterial species isolated from chickens and pigs in China. Methods WGS was conducted to identify the suspected resistance genes in the tigecycline-resistant Myroides phaeus 18QD1AZ29W. Functional cloning, homology modelling and molecular docking were performed to compare the function with other Tet(X) variants. Retrospective screening for tet(X6) was conducted for 80 isolates in our WGS data collection, and all genomic environments of tet(X6)-positive isolates were analysed. Results The tigecycline-resistant M. phaeus 18QD1AZ29W isolated from a pig farm in Shandong in 2018 was positive for tet(X2) and a novel tet(X) gene, designated tet(X6). Tet(X6) could increase the MICs of all tested tetracyclines/glycylcyclines for Escherichia coli only 2- to 4-fold, which was possibly due to a lower tetracycline binding capacity of Tet(X6) compared with that of other Tet(X) variants. Retrospective screening showed that seven other isolates (7/80, 8.8%), comprising four Proteus spp. and three Acinetobacter spp. from chickens and pigs in Shandong and Guangdong, were positive for three different variants of tet(X6). The analysis of the genomic environment revealed that two tet(X6)-positive isolates from M. phaeus and Proteus cibarius, respectively, contained ISCR2, which may play a role in tet(X6) transmission. Conclusions This study identified a novel type of tigecycline resistance gene, tet(X6), in Myroides, Acinetobacter and Proteus from chickens and swine. Tet(X6) conferred lower tetracycline/glycylcycline MICs than other Tet(X) variants, and ISCR2 may play a role in the transmission of tet(X6).

Objectives To investigate the role of Major Facilitator Superfamily (MFS)-type transporters from Acinetobacter baumannii AYE in tigecycline efflux. Methods Two putative tetracycline transporter genes of A. baumannii AYE (tetA and tetG) were heterologously expressed in Escherichia coli and drug susceptibility assays were conducted with tigecycline and three other tetracycline derivatives. The importance of TetA in tigecycline transport in A. baumannii was determined by complementation of tetA in WT and Resistance Nodulation cell Division (RND) gene knockout strains of A. baumannii ATCC 19606. Gene expression of the MFS-type tetA gene and RND efflux pump genes adeB, adeG and adeJ in A. baumannii AYE in the presence of tigecycline was analysed by quantitative real-time RT–PCR. Results Overproduction of TetA or TetG conferred resistance to doxycycline, minocycline and tetracycline in E. coli. Cells expressing tetA, but not those expressing tetG, conferred resistance to tigecycline, implying that TetA is a determinant for tigecycline transport. A. baumannii WT and RND-knockout strains complemented with plasmid-encoded tetA are significantly less susceptible to tigecycline compared with non-complemented strains. Efflux pump genes tetA and adeG are up-regulated in A. baumannii AYE in the presence of subinhibitory tigecycline concentrations. Conclusions TetA plays an important role in tigecycline efflux of A. baumannii by removing the drug from cytoplasm to periplasm and, subsequently, the RND-type transporters AdeABC and AdeIJK extrude tigecycline across the outer membrane. When challenged with tigecycline, tetA is up-regulated in A. baumannii AYE. Synergy between TetA and the RND-type transporters AdeABC and/or AdeIJK appears necessary for A. baumannii to confer higher tigecycline resistance via drug efflux.

Objectives To characterize a novel tigecycline resistance gene, tet(X6), and a novel SXT-related integrative and conjugative element (ICE), ICEPgs6Chn1, found in a tigecycline-resistant Proteus genomospecies 6 strain, T60. Methods Strain T60 was identified by the VITEK 2 system, biochemical reactions and an SNP-based approach. The genetic profile of strain T60 was determined by WGS analysis. ICEPgs6Chn1 was analysed by PCR, conjugation experiments and bioinformatics tools. tet(X6) was characterized by cloning and protein structure prediction. Results Strain T60 was resistant to ampicillin, tetracycline, tigecycline, florfenicol, colistin and kanamycin, but susceptible to cefotaxime; it also exhibited high MICs of eravacycline (32 mg/L) and omadacycline (>64 mg/L). Only one chromosome was identified and tet(X6) was located in chromosomal ICEPgs6Chn1, a member of the SXT/R391 ICE family, of 114 368 bp and encoding the antimicrobial resistance genes floR, strB, strA, aph(3′)-Ia, aac(3)-IV, aph(4)-Ia, tet(X6) and sul2. The circular intermediate of ICEPgs6Chn1 was detected by PCR and sequencing, but conjugation experiments showed that it was not self-transmissible. Cloning of the novel gene tet(X6) and protein structure prediction revealed that Tet(X6) confers tigecycline resistance. Conclusions To our knowledge, this is the first report of a novel SXT/R391 ICE in a Proteus genomospecies 6 strain. Importantly, a novel high-level tigecycline resistance gene, tet(X6), emerged for the first time in the SXT/R391 element of Proteus genomospecies 6, revealing that ICEs may serve as an important platform for the accumulation of antibiotic resistance genes.

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 β-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.

Objectives To report data for ceftazidime/avibactam and comparators against meropenem-non-susceptible Enterobacteriaceae collected globally (excluding centres in the USA) from 2015 to 2017 as part of the International Network For Optimal Resistance Monitoring (INFORM) surveillance programme. Methods MICs and susceptibility were determined using EUCAST broth microdilution methodology and EUCAST breakpoints. Isolates were screened to detect genes encoding β-lactamases using multiplex PCR assays. MBL-positive isolates were those in which one or more of the IMP, VIM and/or NDM genes were detected. Results A total of 1460 meropenem-non-susceptible isolates were collected and, of the agents on the panel, susceptibility was highest to ceftazidime/avibactam, colistin and tigecycline [73.0%, 77.0% (1081/1403) and 78.1%, respectively]. Ceftazidime/avibactam was not active against MBL-positive isolates (n=367); these isolates showed the highest rates of susceptibility to colistin (92.1%, 303/329), tigecycline (71.9%) and amikacin (46.6%). A total of 394 isolates were resistant to ceftazidime/avibactam and, of the 369 isolates that were screened, 98.4% were found to carry a gene encoding an MBL enzyme. Among isolates that were identified as carbapenemase positive and MBL negative (n=910), susceptibility was highest to ceftazidime/avibactam (99.8%). Susceptibility was also highest to ceftazidime/avibactam among isolates that were carbapenemase negative and MBL negative (94/98, 95.9%). Conclusions These data highlight the need for continued surveillance of antimicrobial activity as well as the need for new antimicrobials to treat infections caused by meropenem-non-susceptible Enterobacteriaceae, for which the options are extremely limited.

Objectives To assess the prevalence, antimicrobial susceptibilities and molecular characteristics of ESBL-producing Escherichia coli and Klebsiella pneumoniae infecting patients receiving care in Canadian hospitals from January 2007 to December 2016. Methods Clinical isolates of E. coli (n = 8387) and K. pneumoniae (n = 2623) submitted to CANWARD, an ongoing Canadian national surveillance study, were tested using the CLSI reference broth microdilution method to determine their susceptibility to 15 antimicrobial agents. ESBL-producing E. coli and K. pneumoniae confirmed by the CLSI phenotypic method and putative AmpC-producing E. coli underwent PCR testing and DNA sequencing to identify resistance genes. Annual proportions of isolates harbouring ESBL and AmpC genes were assessed by the Cochran–Armitage test of trend. Results The annual proportion of isolates of E. coli that were ESBL producing increased from 3.4% in 2007 to 11.1% in 2016 (P 95% of ESBL-producing E. coli were susceptible to amikacin, colistin, ertapenem, meropenem and tigecycline. The proportion of isolates of K. pneumoniae that were ESBL producing increased from 1.3% in 2007 to 9.7% in 2016 (P 95% of ESBL-producing K. pneumoniae were susceptible to amikacin and meropenem. CTX-M-15 was the predominant genotype in both ESBL-producing E. coli (64.2% of isolates) and ESBL-producing K. pneumoniae (51.0%). The annual proportion of isolates of E. coli that were AmpC producing [annual proportion mean 1.9% (range 0.3%–3.1%)] was unchanged from 2007 to 2016 (P > 0.5). Conclusions The prevalence of both ESBL-producing E. coli and K. pneumoniae increased significantly in Canada during the study period while the prevalence of AmpC-producing E. coli remained low and stable.

Objectives The CANWARD surveillance study was established in 2007 to annually assess the in vitro susceptibilities of a variety of antimicrobial agents against bacterial pathogens isolated from patients receiving care in Canadian hospitals. Methods 42 936 pathogens were received and CLSI broth microdilution testing was performed on 37 355 bacterial isolates. Limited patient demographic data submitted with each isolate were collated and analysed. Results Of the isolates tested, 43.5%, 33.1%, 13.2% and 10.2% were from blood, respiratory, urine and wound specimens, respectively; 29.9%, 24.8%, 19.0%, 18.1% and 8.2% of isolates were from patients in medical wards, emergency rooms, ICUs, hospital clinics and surgical wards. Patient demographics associated with the isolates were: 54.6% male/45.4% female; 13.1% patients aged ≤17 years, 44.3% 18–64 years and 42.7% ≥65 years. The three most common pathogens were Staphylococcus aureus (21.2%, both methicillin-susceptible and MRSA), Escherichia coli (19.6%) and Pseudomonas aeruginosa (9.0%). E. coli were most susceptible to meropenem and tigecycline (99.9%), ertapenem and colistin (99.8%), amikacin (99.7%) and ceftolozane/tazobactam and plazomicin (99.6%). Twenty-three percent of S. aureus were MRSA. MRSA were most susceptible to ceftobiprole, linezolid and telavancin (100%), daptomycin (99.9%), vancomycin (99.8%) and tigecycline (99.2%). P. aeruginosa were most susceptible to ceftolozane/tazobactam (98.3%) and colistin (95.0%). Conclusions The CANWARD surveillance study has provided 10 years of reference antimicrobial susceptibility testing data on pathogens commonly causing infections in patients attending Canadian hospitals.

[Objectives]: To elucidate the potential mutation-driven mechanisms involved in the acquisition of tigecycline resistance by the opportunistic pathogen Stenotrophomonas maltophilia. The mutational trajectories and their effects on bacterial fitness, as well as cross-resistance and/or collateral susceptibility to other antibiotics, were also addressed., [Methods]: S. maltophilia populations were submitted to experimental evolution in the presence of increasing concentrations of tigecycline for 30 days. The genetic mechanisms involved in the acquisition of tigecycline resistance were determined by WGS. Resistance was evaluated by performing MIC assays. Fitness of the evolved populations and individual clones was assessed by measurement of the maximum growth rates., [Results]: All the tigecycline-evolved populations attained high-level resistance to tigecycline following different mutational trajectories, yet with some common elements. Among the mechanisms involved in low susceptibility to tigecycline, mutations in the SmeDEF efflux pump negative regulator smeT, changes in proteins involved in the biogenesis of the ribosome and modifications in the LPS biosynthesis pathway seem to play a major role. Besides tigecycline resistance, the evolved populations presented cross-resistance to other antibiotics, such as aztreonam and quinolones, and they were hypersusceptible to fosfomycin, suggesting a possible combination treatment. Further, we found that the selected resistance mechanisms impose a relevant fitness cost when bacteria grow in the absence of antibiotic., [Conclusions]: Mutational resistance to tigecycline was easily selected during exposure to this antibiotic. However, the fitness cost may compromise the maintenance of S. maltophilia tigecycline-resistant populations in the absence of antibiotic., Work in our laboratory is supported by grants from the Instituto de Salud Carlos III (Spanish Network for Research on Infectious Diseases, grant RD16/0016/0011)—cofinanced by the European Development Regional Fund ‘A Way to Achieve Europe’, by grant S2017/BMD-3691 InGEMICS-CM, funded by Comunidad de Madrid (Spain) and European Structural and Investment Funds and by the Spanish Ministry of Economy and Competitivity (BIO2017-83128-R).

ObjectivesIn a new experimental model of carbapenemase-producing Klebsiella pneumoniae osteomyelitis we evaluated the efficacy of colistin alone and in various combinations and examined the emergence of colistin-resistant strains and cross-resistance to host defence peptides (HDPs).MethodsKPC-99YC is a clinical strain with intermediate susceptibility to meropenem (MIC = 4 mg/L) and full susceptibility to gentamicin, colistin and tigecycline (MICs = 1 mg/L) and fosfomycin (MIC = 32 mg/L). Time–kill curves were performed at 4× MIC. Osteomyelitis was induced in rabbits by tibial injection of 2 × 108 cfu. Treatment started 14 days later for 7 days in seven groups: (i) control; (ii) colistin; (iii) colistin + gentamicin; (iv) colistin + tigecycline; (v) colistin + meropenem; (vi) colistin + meropenem + gentamicin; and (vii) colistin + fosfomycin.ResultsIn vitro, colistin was rapidly bactericidal, but regrowth occurred after 9 h. Combinations of colistin with meropenem or fosfomycin were synergistic, whereas combination with tigecycline was antagonistic. In vivo, colistin alone was not effective. Combinations of colistin with meropenem or fosfomycin were bactericidal (P ConclusionsIn this model, combinations of colistin plus meropenem, with or without gentamicin, or colistin plus fosfomycin were the only effective therapies. The combination of colistin and tigecycline should be administered with caution, as it may be antagonistic in vitro and in vivo.

Background MDR-TB and XDR-TB have poor outcomes. Objectives To examine the efficacy of tigecycline monotherapy in the hollow fibre system model of TB. Methods We performed pharmacokinetic/pharmacodynamic studies using tigecycline human-like concentration-time profiles in the hollow fibre system model of TB in five separate experiments using Mycobacterium tuberculosis in log-phase growth or as semi-dormant or intracellular bacilli, as monotherapy. We also compared efficacy with the isoniazid/rifampicin/pyrazinamide combination (standard therapy). We then applied extinction mathematics, morphisms and Latin hypercube sampling to identify duration of therapy with tigecycline monotherapy. Results The median tigecycline MIC for 30 M. tuberculosis clinical and laboratory isolates (67% MDR/XDR) was 2 mg/L. Tigecycline monotherapy was highly effective in killing M. tuberculosis in log-phase-growth and semi-dormant and intracellular M. tuberculosis. Once-a-week dosing had the same efficacy as daily therapy for the same cumulative dose; thus, tigecycline efficacy was linked to the AUC0-24/MIC ratio. Tigecycline replacement by daily minocycline after 4 weeks of therapy was effective in sterilizing bacilli. The AUC0-24/MIC ratio associated with optimal kill was 42.3. Tigecycline monotherapy had a maximum sterilizing effect (day 0 minus day 28) of 3.06 ± 0.20 log10 cfu/mL (r2 = 0.92) compared with 3.92 ± 0.45 log10 cfu/mL (r2 = 0.80) with optimized standard therapy. In our modelling, at a tigecycline monotherapy duration of 12 months, the proportion of patients with XDR-TB who reached bacterial population extinction was 64.51%. Conclusions Tigecycline could cure patients with XDR-TB or MDR-TB who have failed recommended therapy. Once-a-week tigecycline could also replace second-line injectables in MDR-TB regimens.

OBJECTIVES To describe the microbiology and antimicrobial resistance patterns of cultured samples acquired from Canadian ICUs. METHODS From 2007 to 2016, tertiary care centres from across Canada submitted 42938 bacterial/fungal isolates as part of the CANWARD surveillance study. Of these, 8130 (18.9%) were from patients on ICUs. Susceptibility testing guidelines and MIC interpretive criteria were defined by CLSI. RESULTS Of the 8130 pathogens collected in this study, 58.2%, 36.3%, 3.1% and 2.4% were from respiratory, blood, wound and urine specimens, respectively. The top five organisms collected from Canadian ICUs accounted for 55.4% of all isolates and included Staphylococcus aureus (21.5%), Pseudomonas aeruginosa (10.6%), Escherichia coli (10.4%), Streptococcus pneumoniae (6.5%) and Klebsiella pneumoniae (6.4%). MRSA accounted for 20.7% of S. aureus collected, with community-associated (CA) MRSA genotypes increasing in prevalence over time (P

Objectives We investigated the in vitro activities of cefiderocol, ceftazidime/avibactam, ceftolozane/tazobactam and other related drugs against imipenem-resistant Pseudomonas aeruginosa, imipenem-resistant Acinetobacter baumannii and Stenotrophomonas maltophilia isolates. Methods Non-duplicated bacteraemia isolates (n = 300) of imipenem-resistant P. aeruginosa (n = 100), imipenem-resistant A. baumannii (n = 100) and S. maltophilia (n = 100) were evaluated. Imipenem-resistant P. aeruginosa and imipenem-resistant A. baumannii isolates were defined as isolates exhibiting imipenem MIC ≥8 mg/L, as determined using the VITEK 2 system. The MICs of 11 other antimicrobial agents for the isolates were determined by the broth microdilution method. Iron-depleted CAMHB was used to determine the MICs of cefiderocol. Results The rates of colistin resistance of imipenem-resistant P. aeruginosa and imipenem-resistant A. baumannii were 5% and 10%, respectively. The MIC90 values of cefiderocol, ceftolozane/tazobactam, ceftazidime/avibactam, tigecycline and colistin were as follows: imipenem-resistant P. aeruginosa: 1, 4, 16, >4 and 2 mg/L; imipenem-resistant A. baumannii: 8, >64, >64, 4 and 2 mg/L; and S. maltophilia: 0.25, >64, >64, 2 and >8 mg/L, respectively. For imipenem-resistant A. baumannii isolates, the MICs of cefiderocol, ceftolozane/tazobactam and ceftazidime/avibactam were ≤4 mg/L for 88%, 8% and 1% of the isolates, respectively. Cefiderocol MICs were ≤4 mg/L for the five colistin-resistant imipenem-resistant P. aeruginosa isolates and 70% of the 10 colistin-resistant imipenem-resistant A. baumannii isolates. Conclusions Cefiderocol exhibited more potent in vitro activity than ceftolozane/tazobactam and ceftazidime/avibactam against imipenem-resistant P. aeruginosa, imipenem-resistant A. baumannii and S. maltophilia isolates.

OBJECTIVES: We aimed to establish a novel murine intra-abdominal foreign body infection model to study the activity of anidulafungin and tigecycline against dual species Candida albicans/Staphylococcus aureus biofilms. METHODS: In vitro and in vivo single and dual species biofilms were developed inside serum-coated triple-lumen catheters placed in 24-well plates or implanted intraperitoneally in BALB/c mice. The effect of tigecycline and anidulafungin alone and in combination was tested using clinically relevant concentrations. Scanning electron microscopy was used to visualize the mature biofilm structure developed intraperitoneally. Flow cytometry was used to determine the immunological response upon infection. Immunoblot analysis allowed us to determine the effect of anidulafungin on poly-β-(1,6)-N-acetylglucosamine in in vitro-grown S. aureus biofilms. RESULTS: We determined the MIC, MBC and in vitro susceptibility profile for anidulafungin and tigecycline against C. albicans and S. aureus in mixed and single species biofilms. We demonstrated that anidulafungin acts synergistically when combined with tigecycline against in vivo intra-abdominal biofilms. Moreover, we reveal that anidulafungin reduces the abundance of S. aureus poly-β-(1,6)-N-acetylglucosamine. The influx of neutrophils is much increased when infected with mixed biofilms compared with single species biofilms. CONCLUSIONS: Currently, treatment of intra-abdominal infections, in particular polymicrobial catheter-associated peritonitis, is ineffective. To the best of our knowledge, this is the first study that provides insight into new possible options for treatment of C. albicans/S. aureus biofilms present in the abdominal cavity. ispartof: JOURNAL OF ANTIMICROBIAL CHEMOTHERAPY vol:73 issue:10 pages:2806-2814 ispartof: location:England status: published

Background Carbapenemase-producing Klebsiella pneumoniae causes high mortality owing to the limited therapeutic options available. Here, we investigated an emergent carbapenem-resistant K. pneumoniae strain with hypervirulence found among KPC-2-producing strains in Taiwan. Methods KPC-producing K. pneumoniae strains were collected consecutively from clinical specimens at the Taipei Veterans General Hospital between January 2012 and December 2014. Capsular types and the presence of rmpA/rmpA2 were analysed, and PFGE and MLST performed using these strains. The strain positive for rmpA/rmpA2 was tested in an in vivo mouse lethality study to verify its virulence and subjected to WGS to delineate its genomic features. Results A total of 62 KPC-2-producing K. pneumoniae strains were identified; all of these belonged to ST11 and capsular genotype K47. One strain isolated from a fatal case with intra-abdominal abscess (TVGHCRE225) harboured rmpA and rmpA2 genes. This strain was resistant to tigecycline and colistin, in addition to carbapenems, and did not belong to the major cluster in PFGE. TVGHCRE225 exhibited high in vivo virulence in the mouse lethality experiment. WGS showed that TVGHCRE225 acquired a novel hybrid virulence plasmid harbouring a set of virulence genes (iroBCDN, iucABCD, rmpA and rmpA2, and iutA) compared with the classic ST11 KPC-2-producing strain. Conclusions We identified an XDR ST11 KPC-2-producing K. pneumoniae strain carrying a hybrid virulent plasmid in Taiwan. Active surveillance focusing on carbapenem-resistant hypervirulent K. pneumoniae strains is necessary, as the threat to human health is imminent.

Background Mycobacterium abscessus complex (MABC) is the most common non-tuberculous mycobacterium that causes complicated skin and soft tissue infections (cSSTIs). The selection of antimycobacterial agents for successful treatment of such infections is a critical issue. Objectives To investigate the antimicrobial susceptibility patterns of MABC isolates from skin and soft tissue to a variety of antimycobacterial agents. Methods Sixty-seven MABC isolates were collected and partial gene sequencing of secA1, rpoB and hsp65 was used to classify them into three subspecies: M. abscessus subsp. abscessus (MAB), M. abscessus subsp. massiliense (MMA) and M. abscessus subsp. bolletii (MBO). The MICs of 11 antimycobacterial agents for these 67 isolates were determined using a broth microdilution method and commercial Sensititre RAPMYCOI MIC plates, as recommended by CLSI. Results In total, 28 MAB, 38 MMA and 1 MBO were isolated from patients with cSSTIs at our hospital. Most MABC strains were resistant to ciprofloxacin, doxycycline, imipenem, linezolid, minocycline, moxifloxacin and trimethoprim/sulfamethoxazole. In addition, most MABC strains were intermediately susceptible or resistant to cefoxitin. Eighteen of the 28 MABs and 1 MBO isolate harboured the T28 polymorphism in the erm(41) gene. Two of the 38 MMA isolates had an rrl A2059G point mutation. Most of the MABC strains were susceptible to amikacin and tigecycline. Conclusions In Taiwan, amikacin, clarithromycin and tigecycline have good activity against MMA and MAB erm(41) C28 sequevar isolates, whereas amikacin and tigecycline, rather than clarithromycin, have good activity against both MBO and MAB erm(41) T28 sequevar isolates. Clinical trials are warranted to correlate these data with clinical outcomes.

Objectives To estimate UK prevalence and incidence of clinically significant carbapenemase-producing Enterobacteriaceae (CPE), and to determine epidemiological characteristics, laboratory methods and infection prevention and control (IPC) measures in acute care facilities. Methods A 6 month survey was undertaken in November 2013-April 2014 in 21 sentinel UK laboratories as part of the European Survey on Carbapenemase-Producing Enterobacteriaceae (EuSCAPE) project. Up to 10 consecutive, non-duplicate, clinically significant and carbapenem-non-susceptible isolates of Escherichia coli or Klebsiella pneumoniae were submitted to a reference laboratory. Participants answered a questionnaire on relevant laboratory methods and IPC measures. Results Of 102 isolates submitted, 89 (87%) were non-susceptible to ≥1 carbapenem, and 32 (36%) were confirmed as CPE. CPE were resistant to most antibiotics, except colistin (94% susceptible), gentamicin (63%), tigecycline (56%) and amikacin (53%). The prevalence of CPE was 0.02% (95% CI = 0.01%-0.03%). The incidence of CPE was 0.007 per 1000 patient-days (95% CI = 0.005-0.010), with north-west England the most affected region at 0.033 per 1000 patient-days (95% CI = 0.012-0.072). Recommended IPC measures were not universally followed, notably screening high-risk patients on admission (applied by 86%), using a CPE 'flag' on patients' records (70%) and alerting neighbouring hospitals when transferring affected patients (only 30%). Most sites (86%) had a laboratory protocol for CPE screening, most frequently using chromogenic agar (52%) or MacConkey/CLED agars with carbapenem discs (38%). Conclusions The UK prevalence and incidence of clinically significant CPE is currently low, but these MDR bacteria affect most UK regions. Improved IPC measures, vigilance and monitoring are required.

Objectives The objective of this study was to summarize available data on polymyxin-based combination therapy or monotherapy for carbapenem-resistant Gram-negative bacteria. Methods This is a systematic review. We included observational studies and randomized controlled trials (RCTs) comparing polymyxin monotherapy versus polymyxin-based combination therapy in adult patients with infections caused by carbapenem-resistant or carbapenemase-producing Gram-negative bacteria. Only named antibiotic regimens were included. The primary outcome was 30 day mortality. Unadjusted OR (uOR) and adjusted OR where available with 95% CI were pooled in random-effects meta-analyses. Results Twenty-two studies including 28 comparisons were included. Polymyxin monotherapy was associated with a uOR of 1.58 (95% CI = 1.03-2.42) for mortality compared with polymyxin/carbapenem combination therapy (seven observational studies, 537 patients), without heterogeneity. Subgrouping studies to serious and critical risk of bias resulted in uORs of 0.94 (95% CI = 0.42-2.09) and 1.94 (95% CI = 1.17-3.23), respectively. Mortality was significantly higher with polymyxin monotherapy compared with combination therapy with tigecycline, aminoglycosides or fosfomycin (potentially double-coverage regimens): uOR of 1.57 (95% CI = 1.06-2.32) overall (10 observational studies and 1 RCT, 585 patients, no heterogeneity) and uOR of 2.09 (95% CI = 1.21-3.6) for Klebsiella pneumoniae bacteraemia (7 observational studies, 285 patients, no heterogeneity); very low quality evidence. Two RCTs and one observational study assessing rifampicin/colistin combination therapy for Acinetobacter baumannii infections showed no difference in mortality compared with colistin monotherapy; moderate quality evidence. Conclusions The significant association observed in observational studies between polymyxin monotherapy and mortality cannot be taken as proof of combination therapy effects due to the low quality of the evidence. The only three RCTs to date show no effect of rifampicin/colistin or fosfomycin/colistin on mortality for Acinetobacter infections.

Molecular resistance markers for pan-resistant isolates of KPC-producing Klebsiella pneumoniae were identified and showed core-genome MLST to be a promising tool for tracking outbreaks and transmission pathways.KPC-2-producing Klebsiella pneumoniae (KPC-KP) ST258 has been rapidly expanding and is often associated with serious nosocomial infections. Last-line antibiotics such as colistin and tigecycline often remain the only treatment option. We describe here the evolving genetic background of KPC-KP isolates in Crete, Greece.We tested the antibiotic susceptibility of 34 clinical isolates from patients hospitalized in 2010 and 2013-14. Whole-genome sequences of these isolates were analysed for acquired resistance genes and gene mutations.All KPC-KP isolates belonged to ST258 with the exception of one ST147 isolate. From 2014, 26% of isolates were non-susceptible to all antibiotics, compared with 0 of 11 isolates from 2010. Colistin resistance was associated with mutations in mgrB, which was present in 61% of isolates from 2014. Core-genome MLST analysis showed that pan-resistant isolates were closely related and appeared in two separate clusters.KPC-KP is rapidly evolving to pan-resistance in Crete. We identified molecular resistance markers for pan-resistant isolates and showed that core-genome MLST is a promising tool for molecular fingerprinting of KPC-KP ST258.

Antibiotic resistance is an ongoing global medical crisis and we are in great need of new antibacterial agents to combat rapidly emerging resistant pathogens. Tigecycline is one of few drugs that have been introduced into medicine during the last two decades. It is a broad-spectrum third generation tetracycline that is active against multidrug-resistant bacteria that cause complicated infections.In this thesis I examined the development of tigecycline resistance in Escherichia coli and associated in vitro and in vivo fitness effects. Selections of spontaneous E. coli mutants revealed relatively high accumulation rates of changes in the multidrug efflux system AcrAB-TolC regulation network and in heptose biosynthesis and transport pathways important for lipopolysaccharide (LPS) synthesis. Both groups of mutations led to reduced susceptibility to tigecycline and slower growth compared to the wild-type bacteria. Additional in vitro fitness assays and in vivo competitions showed that LPS mutants were less fit than efflux mutants, providing a possible explanation for why up-regulation of multidrug efflux pumps is the main tigecycline resistance mechanism reported in clinical isolates.Tigecycline was designed to evade the two most common tetracycline resistance mechanisms conferred by Tet proteins, efflux and ribosomal protection. However, tigecycline is a substrate for the tetracycline modifying enzyme Tet(X). Screening of Tet protein mutant libraries showed that it is possible to select Tet mutants with minimal inhibitory concentrations of tigecycline that reach clinically relevant levels. Mutations in Tet proteins that permitted a better protection from tigecycline frequently exhibited reduced activity against earlier generations of tetracyclines, except for the Tet(X) enzyme mutants, which were better at inactivating all tested tetracyclines. This is particularly worrisome because different variants of Tet(X) have recently spread to multidrug-resistant pathogens through horizontal gene transfer. Therefore, Tet(X) mutants with improved activity threaten the medical future of tetracyclines.Multidrug resistance is easily disseminated through horizontally spreading conjugative plasmids. pUUH239.2 is an example of a successful conjugative plasmid that caused the first clonal outbreak of extended spectrum β-lactamase-producing Klebsiella pneumoniae in Scandinavia. This plasmid was formed after rearrangements between two different plasmid backbones and it carries resistance genes to multiple antibiotic classes, heavy metals, and detergents.

Objectives Tigecycline is a broad-spectrum antibiotic acting at the level of the 30S ribosomal subunit to inhibit translation. While Streptococcus pneumoniae remains susceptible to tigecycline, resistance is beginning to emerge in some species and mainly involves efflux or mutations in ribosome constituents. We describe here the characterization of S. pneumoniae mutants selected for resistance to tigecycline. Methods Molecular determinants of resistance to tigecycline in S. pneumoniae were studied through WGS of two series of mutants made resistant to tigecycline in vitro in a stepwise fashion and by reconstructing tigecycline resistance using DNA transformation. Results The tigecycline-resistant S. pneumoniae M1TGC-6 and M2TGC-6 mutants were cross-resistant to tetracycline and minocycline. A role in tigecycline resistance could be attributed to 4 of the 12 genes that were mutated in both mutants. Mutations in ribosomal proteins S10 and S3, acquired early and late during selection, respectively, were implicated in resistance in both mutants. Similarly, mutations were detected in the four alleles of the 16S ribosomal RNA at sites involved in tigecycline binding and the number of mutated alleles correlated with the level of resistance. Finally, the gene spr1784 encodes an RsmD-like 16S rRNA methyltransferase for which inactivating mutations selected in the S. pneumoniae tigecycline-resistant mutants were found to decrease susceptibility to tigecycline. Conclusions This first report about tigecycline resistance mechanisms in S. pneumoniae revealed that, in contrast to Gram-negative species, for which efflux appears central for tigecycline resistance, resistance in the pneumococcus occurs through mutations related to ribosomes.

Objectives Antimicrobial therapy for sepsis caused by carbapenem- and colistin-resistant Klebsiella pneumoniae is not well established. We hypothesized that the early use of gentamicin in cases due to susceptible organisms would decrease the crude mortality rate of this infection. Methods This retrospective cohort study examined 50 cases of sepsis caused by carbapenem-resistant K. pneumoniae occurring between June 2012 and February 2013 during an outbreak of K. pneumoniae ST512 producing KPC-3, SHV-11 and TEM-1. Survival curves categorized by the use of gentamicin were constructed using the Kaplan-Meier method and compared using the log-rank test. Eight multivariate models using Cox regression were designed to study the risk factors for mortality and test the hypothesis. Results The 30 day crude mortality rate was 38%. The use of targeted gentamicin was associated with reduced mortality (20.7% versus 61.9%, P = 0.02). In all multivariate regression models, the use of gentamicin was independently associated with lower mortality until Day 30 (HR 0.17-0.29, P = 0.03-0.002 depending on the model) after controlling for other potential confounding variables such as age, optimal treatment, renal function, severity of infection, underlying disease, use of tigecycline and previous hospitalization. Conclusions Gentamicin reduced the mortality from sepsis caused by this K. pneumoniae ST512 clone producing KPC-3, SHV-11 and TEM-1.

BACKGROUND Clostridium difficile infection (CDI) is mediated by potent extracellular toxins and is spread largely via bacterial spores. We and others have shown that some antibiotics stimulate C. difficile toxin production in a strain-specific manner; however, the effects of newer anti-C. difficile antibiotics on this process remain to be investigated. METHODS The effects of the protein synthesis inhibitor tigecycline on sporulation and toxin A and toxin B production were compared in historical (strain 9689) and hypervirulent BI/NAP1/027 (strain 5325) isolates of C. difficile in vitro. RESULTS Tigecycline at 1/4× MIC stimulated an increased and earlier toxin A and/or B gene expression in both the historical and the hypervirulent strains, although a commensurate increase in toxin protein production was observed only in the 9689 strain. In fact, in the hypervirulent 5325 strain, toxin production was dramatically suppressed. By comparison, subinhibitory concentrations of vancomycin and metronidazole also stimulated increased protein toxin production by the historical, but not the hypervirulent, strain. In addition, tigecycline dose-dependently reduced viable spore production by both the 9689 and 5325 strains. Vancomycin treatment also suppressed spore formation in both C. difficile strains; however, metronidazole, while reducing spore formation in the 9689 strain, stimulated a near 2 log increase in spore production by the 5325 isolate. CONCLUSIONS In summary, these findings suggest that the treatment of CDI patients with tigecycline could effectively both control disease progression and limit its spread by disrupting sporulation.

Results: One hundred and one patients were included in the analysis; 68 (67.3%) received MT and 33 (32.7%) received CT. Pneumonia was the most common infection (50.5%), 68.6% of cases being associated with mechanical ventilation. Colistin (67.6%) and carbapenems (14.7%) were the most common drugs used in MT; colistin plus tigecycline (27.3%) and carbapenem plus tigecycline (12.1%) were the most frequent combinations. Crude 30 day mortality was 23.5% and 24.2% for the MT and CT groups, respectively (RR ¼ 1.03; 95% CI 0.49 – 2.16; P ¼ 0.94). Multivariate analysis of 30 day survival showed no trend towards reduced 30 day mortality with CT (HR ¼1.35; 95% CI 0.53–3.44; P ¼ 0.53). Subgroup analysis showed similar results. Conclusions: Our data do not support an association of CT with reduced mortality in MDRAB infections. More data for specific types of infection and combinations are needed.

Objectives: Combination therapy is recommended for the treatment of KPC-producing Klebsiella pneumoniae (KPC-Kp), but the optimal regimen for colistin-resistant strains is unknown. We compared the synergistic activity and post-antibiotic effect (PAE) of colistin in combination with other antimicrobials against colistin-susceptible and -resistant KPC-Kp bloodstream isolates. Methods: The genotypes of nine colistin-susceptible and eight colistin-resistant KPC-Kp bloodstream isolates were analysed using PCR and amplicon sequencing. Combinations of colistin, meropenem, tigecycline, rifampicin and teicoplanin were then screened using the Etest, a chequerboard assay and time – kill studies. Synergistic combinations were also analysed with respect to the PAE in time –kill curves and the PAE at clinically achievable concentrations. Results: Insertional inactivation of the PhoQ/PhoB two-component regulatory system bymgrB-IS5 was identified in 6/8 (75%) colistin-resistant KPC-Kp. Colistin/rifampicin combinations resulted in no interactions [fractional inhibitory concentration (FIC) indices 1.5 –2] for colistin-susceptible strains, but were uniformly synergistic (FIC indices 0.1 –0.4) against colistin-resistant KPC-Kp. Time –kill kinetic analysis, at clinically achievable fixed concentrations of rifampicin and colistin, confirmed synergy and produced persistent growth inhibition (3 h) of colistin-resistant KPC-Kp strains exposed to colistin/rifampicin or colistin/rifampicin/tigecycline combinations. Conclusions: Combinations of colistin plus rifampicin, and less frequently tigecycline, exhibited synergistic activity in vitro against colistin-resistant KPC-Kp strains.

Objectives To examine the incidence of pancreatitis among subjects enrolled in the tigecycline clinical trial programme, summarize cases and examine concomitant use of other pancreatitis-causing medications. Methods Subject data from Phase 3 and 4 comparative tigecycline studies were included in the analysis; investigator-reported adverse events of ‘pancreatitis’, ‘necrotizing pancreatitis’ or ‘pancreas disorder’ were reviewed. Data were summarized and cases were reported. No statistical comparisons were made. The incidence of overall pancreatitis with 95% CIs was calculated. The Wilson score method was used to calculate CIs. Results Nineteen subjects with investigator-determined pancreatitis were identified from the programme database, which included 3788 subjects treated with tigecycline and 3646 subjects treated with a comparator. There were 9 cases identified among the tigecycline-treated subjects [9 of 3788 (0.24%; 95% CI, 0.11–0.45)] and 10 cases among the comparator-treated subjects [10 of 3646 (0.27%; 95% CI, 0.13–0.50)]. The demographic characteristics of the subjects with pancreatitis were similar between treatment groups. The median duration of tigecycline therapy was 8.0 days compared with 11.0 days of comparator treatment. Concomitant or prior exposure to a Badalov class I medication was evident in the majority of subjects who developed pancreatitis. A numerically higher number of tigecycline-treated subjects were exposed to furosemide prior to the onset of pancreatitis than comparator-treated subjects. Conclusions Pancreatitis was uncommon in subjects treated with tigecycline, with an occurrence of

OBJECTIVES Acinetobacter baumannii is an important opportunistic pathogen and multidrug-resistant isolate. Although tigecycline is a potent antibiotic for treating infections with multidrug-resistant isolates, resistance is becoming a problem. This study aimed to explore the mechanism of tigecycline resistance in A. baumannii. METHODS A serial passage experiment was performed to collect isolates selected by tigecycline. The expression of efflux pumps was quantified in the final selected isolate, 19606-T8. The whole genome of 19606-T8 was sequenced and the putative mutations were confirmed using PCR and Sanger sequencing. A complementation experiment was performed to evaluate the contribution of the mutations to decreased susceptibility to tigecycline. The significance of a deletion mutation was further investigated in terms of growth rate and antibiotic susceptibilities. RESULTS We collected serial isolates by selective pressure of tigecycline, and designated them 19606-T1 to 19606-T8. The efflux pumps AdeABC, AdeFGH and AdeIJK were not overexpressed in 19606-T8, which had decreased susceptibility to tigecycline. Isolate 19606-T8 carried one deletion mutation in trm and three non-synonymous substitutions in msbA (A84V), lolA (P91L) and filC (N168K). The deletion mutation in trm (encoding S-adenosyl-L-methionine-dependent methyltransferase) resulted in decreased susceptibility to tigecycline as well as to minocycline and doxycycline. In complementation experiments, the MICs of tigecycline, minocycline and doxycycline in a tigecycline-resistant isolate were restored by complementation with wild-type trm. CONCLUSIONS Given that the deletion mutation in trm was associated with decreased susceptibility to tigecycline and that a wild-type trm plasmid could restore the susceptibility, trm is considered to play an important role in decreased susceptibility to tigecycline in A. baumannii.

We report the case of a kidney-transplant patient, suffering an intra-abdominal abscess at the surgical site caused by a carbapenem-resistant ST258 Klebsiella pneumoniae clone, producing the KPC-3 carbapenemase. Under tigecycline treatment, the patient developed a sepsis caused by a carbapenem-susceptible ST258 K. pneumoniae strain. Complete DNA sequences of the plasmids carried by the resistant and susceptible strains from this patient were determined.The complete DNA sequences of plasmids were obtained by applying the 454 Genome Sequencer FLX-PLUS procedure on a library constructed of total plasmid DNA purified from the carbapenem-resistant and -susceptible strains.In the carbapenem-resistant strain, four plasmids encoding 24 resistance genes, including blaKPC-3, and two putative virulence clusters were detected. In the susceptible strain, large rearrangements occurred in the KPC-carrying plasmid, causing the deletion of the entire Tn4401::blaKPC-3 transposon, with the consequent reversion of the strain to carbapenem susceptibility. The patient was successfully treated with carbapenems and fully recovered.The description of the plasmid content in these two strains gives interesting insights into the plasticity of KPC-carrying plasmids in K. pneumoniae.

Tigecycline is an approved treatment for complicated skin and soft-tissue infections (cSSTIs). The efficacy of tigecycline as monotherapy or in combination with other antibacterials in the treatment of cSSTI in routine practice is described.Individual patient-level data were pooled from five European observational studies (July 2006 to October 2011).A total of 254 cSSTI patients who received tigecycline were included (mean age 63.2 ± 14.9 years). Of these, 34.4% were in intensive care units, 54.5% acquired their infection in hospital and 90.9% had at least one comorbidity. Infection most commonly affected the limbs (62.4%) and 43.8% of infections were classified as necrotizing. The mean Acute Physiology and Chronic Health Evaluation (APACHE) II and Sequential Organ Failure Assessment (SOFA) scores at the beginning of treatment were 15.0 ± 7.9 (n = 205) and 5.8 ± 3.9 (n = 32), respectively, indicating high disease severity. Staphylococcus aureus (52.7%), Escherichia coli (18.0%) and Enterococcus faecium (12.0%) were the most frequently isolated pathogens; 32.9% of infections were polymicrobial and 30.5% were due to resistant pathogens. Overall, 71.8% received tigecycline as monotherapy and 28.2% as combination therapy for a mean duration of 12 days. Clinical response rates at the end of treatment were 79.6% for all patients who received the standard dosage (183/230), 86.7% for patients who received tigecycline as monotherapy (143/165), 75.0% for patients with a nosocomial infection (96/128), 75.3% for patients with an APACHE II score15 (61/81) and 58.3% for patients with a SOFA score ≥ 7 (7/12).In these real-life studies, tigecycline, alone and in combination, achieved favourable clinical response rates in patients with cSSTI with a high severity of illness.

Tigecycline is approved for the treatment of complicated skin and soft-tissue infections (cSSTIs) and complicated intra-abdominal infections (cIAIs) in adults. In this analysis the safety and tolerability profile of tigecycline (used alone or in combination) for the treatment of patients with approved indications of cSSTI and cIAI were examined under real-life clinical conditions.Individual patient-level data were pooled from five European observational studies (July 2006 to October 2011). A total of 254 cSSTI and 785 cIAI patients were included. The mean age was 63 years; 34.4% and 56.6% were in intensive care units, 90.9% and 88.1% had at least one comorbidity and mean Acute Physiology and Chronic Health Evaluation (APACHE) II scores at the beginning of treatment were 15.0 ± 7.9 and 16.9 ± 7.6, respectively.Data on adverse events (AEs) were available for 198 cSSTI and 590 cIAI patients in three studies. Nausea and vomiting were reported in ≤ 2% of patients. The most common serious AEs were multi-organ failure (4.0% and 10.0% in cSSTI and cIAI patients, respectively) and sepsis (4.0% and 6.1%, respectively). Death was recorded for 24/254 (9.4%) cSSTI and 147/785 (18.7%) cIAI patients. Mortality rates were higher in the group with a baseline APACHE II score of15 compared with those with a score of ≤ 15 (18.7% versus 3.5% for cSSTI patients and 23.8% versus 16.0% for cIAI patients). A similar trend was seen when cIAI patients were stratified by Sequential Organ Failure Assessment (SOFA) score.The safety and tolerability of tigecycline, alone and in combination, are consistent with the level of critical illness among patients in these real-life studies.

Objectives Vancomycin-resistant enterococci (VRE) can be associated with serious bacteraemia. The focus of this study was to characterize the molecular epidemiology of VRE from bacteraemia cases that were isolated from 1999 to 2009 as part of Canadian Nosocomial Infection Surveillance Program (CNISP) surveillance activities. Methods From 1999 to 2009, enterococci were collected from across Canada in accordance with the CNISP VRE surveillance protocol. MICs were determined using broth microdilution. PCR was used to identify vanA, B, C, D, E, G and L genes. Genetic relatedness was examined using multilocus sequence typing (MLST). Results A total of 128 cases of bacteraemia were reported to CNISP from 1999 to 2009. In 2007, a significant increase in bacteraemia rates was observed in western and central Canada. Eighty-one of the 128 bacteraemia isolates were received for further characterization and were identified as Enterococcus faecium. The majority of isolates were from western Canada (60.5%), followed by central (37.0%) and eastern (2.5%) Canada. Susceptibilities were as follows: daptomycin, linezolid, tigecycline and chloramphenicol, 100%; quinupristin/dalfopristin, 96.3%; high-level gentamicin, 71.6%; tetracycline, 50.6%; high-level streptomycin, 44.4%; rifampicin, 21.0%; nitrofurantoin, 11.1%; clindamycin, 8.6%; ciprofloxacin, levofloxacin and moxifloxacin, 1.2%; and ampicillin, 0.0%. vanA contributed to vancomycin resistance in 90.1% of isolates and vanB in 9.9%. A total of 17 sequence types (STs) were observed. Beginning in 2006 there was a shift in ST from ST16, ST17, ST154 and ST80 to ST18, ST412, ST203 and ST584. Conclusions The increase in bacteraemia observed since 2007 in western and central Canada appears to coincide with the shift of MLST STs. All VRE isolates remained susceptible to daptomycin, linezolid, chloramphenicol and tigecycline.