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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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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 β-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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.

Objectives: To characterize a novel virulence-resistance plasmid pSE380T carried by a Salmonella enterica serotype Enteritidis clinical strain SE380., Methods: The plasmid pSE380T was conjugated to Escherichia coli strain J53 and sequenced by PacBio RSII, followed by subsequent annotation and genetic analysis., Results: Sequence analysis of this plasmid revealed that the entire Salmonella Enteritidis-specific virulence plasmid, pSEN, had been incorporated into an IncHI2 MDR plasmid, which comprises the cephalosporin and fosfomycin resistance determinants blaCTX-M-14 and fosA3. Based on BLAST analysis and scrutiny of insertion footprints, the insertion event was found to involve a replicative transposition process mediated by IS26, an IS element frequently detected in various resistance plasmids. The resulting pSE380T plasmid also comprises backbone elements of IncHI2 and IncFIA plasmids, producing a rare fusion product that simultaneously encodes functional features of both, i.e. virulence, resistance and high transmissibility., Conclusions: This is a novel hybrid plasmid mediating MDR and virulence from a clinical Salmonella Enteritidis strain. This plasmid is likely to be transmissible amongst various serotypes of Salmonella and other Enterobacteriaceae species, rendering a wide range of bacterial pathogens resistant to cephalosporins and fosfomycin, and further enhancing their virulence potential. It will be important to monitor the spread and further evolution of this plasmid among the Enterobacteriaceae strains., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Objectives: To analyse and compare mcr-1-bearing plasmids from animal Escherichia coli isolates, and to investigate potential mechanisms underlying dissemination of mcr-1., Methods: Ninety-seven ESBL-producing E. coli strains isolated from pig farms in China were screened for the mcr-1 gene. Fifteen mcr-1-positive strains were subjected to molecular characterization and bioinformatic analysis of the mcr-1-bearing plasmids that they harboured., Results: Three major types of mcr-1-bearing plasmids were recovered: IncX4 (∼33 kb), IncI2 (∼60 kb) and IncHI2 (∼216-280 kb), among which the IncX4 and IncI2 plasmids were found to harbour the mcr-1 gene only, whereas multiple resistance elements including bla CTX-M , bla CMY , bla TEM , fosA, qnrS, floR and oqxAB were detected, in various combinations, alongside mcr-1 in the IncHI2 plasmids. The profiles of mcr-1-bearing plasmids in the test strains were highly variable, with coexistence of two mcr-1-bearing plasmids being common. However, the MIC of colistin was not affected by the number of mcr-1-carrying plasmids harboured. Comparative analysis of the plasmids showed that they contained an mcr-1 gene cassette with varied structures (mcr-1-orf, ISApl1-mcr-1-orf and Tn6330), with the IncHI2 type being the most active in acquiring foreign resistance genes. A novel transposon, Tn6330, with the structure ISApl1-mcr-1-orf-ISApl1 was found to be the key element mediating translocation of mcr-1 into various plasmid backbones through formation of a circular intermediate., Conclusions: The mcr-1 gene can be disseminated via multiple mobile elements including Tn6330, its circular intermediate and plasmids harbouring such elements. It is often co-transmitted with other resistance determinants through IncHI2 plasmids. The functional mechanism of Tn6330, a typical composite transposon harbouring mcr-1, should be further investigated., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Several observational studies have been published investigating the association between oral fluoroquinolone use and the development of retinal detachment; however, the findings are not concordant. This study is a meta-analysis of the existing literature and estimates the overall absolute risk of such an event., Methods: Electronic databases were searched for observational studies on the association between oral fluoroquinolone and retinal detachment up to August 2014. Studies that did not meet the criteria for meta-analysis were narratively reviewed. Cases of retinal detachment during current fluoroquinolone use were also extracted for absolute risk calculation., Results: Seven observational studies were included. Three (case-control and self-controlled case series studies) were eligible for meta-analysis and four (cohort studies) were narratively reviewed. The rate ratio of the case-control studies was 1.82 (95% CI 0.67-4.93), I(2) =96% and the incidence rate ratio of the self-controlled case series was 1.03 (95% CI 0.84-1.27), I(2) =36%. Three of the four cohort studies found no significant association between oral fluoroquinolone use and the development of retinal detachment. The pooled absolute risk of retinal detachment whilst on current oral fluoroquinolone treatment is estimated to be 4.85 per 1000000 prescriptions (95% CI 0.78-8.91)., Conclusions: The findings of this systematic review and meta-analysis do not support an association between oral fluoroquinolone use and the development of retinal detachment. Given the low absolute risk, such an event would be rare if there were an association. The current prescribing practice for fluoroquinolones should not be altered because of a previously suggested potential risk of retinal detachment., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: A study reported a significant association between oral fluoroquinolones and the development of retinal detachment among current users of oral fluoroquinolones (Etminan M, Forooghian F, Brophy JM et al. JAMA 2012; 307: 1414-9). However, other published studies have discordant results. This study aimed to investigate this association and to estimate the absolute risk of developing retinal detachment in patients exposed to oral fluoroquinolones., Methods: A self-controlled case series study was conducted with data retrieved from the Hong Kong Clinical Data Analysis and Reporting System database and the Taiwan National Health Insurance Research Database. Hong Kong and Taiwanese patients who had prescriptions for oral fluoroquinolones and a procedure for retinal detachment between 2001 and 2012 and between 2000 and 2010, respectively, were defined as cases and included in the analysis., Results: A total of 9 events were found during the fluoroquinolone-exposed period and 1407 events were found during the non-exposed period. The adjusted incidence rate ratio in the combined model was 1.26 (0.65-2.47). The crude absolute risk of experiencing retinal detachment whilst on oral fluoroquinolones was ∼1.3 per 200 000 prescriptions., Conclusions: Our study does not support the association between the use of fluoroquinolones and the development of retinal detachment and our findings are strikingly similar to that of the study conducted in Denmark. Doubt is cast on the association between the use of fluoroquinolones and the development of retinal detachment. Therefore, the use of fluoroquinolones should not be precluded based on the current evidence on the risk of retinal detachment. The impact of different ethnicities on the response to fluoroquinolones should also be investigated., (© The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Objectives: Since the emergence of multidrug-resistant and extensively drug-resistant tuberculosis, there has been a call for a rapid assay to detect rifampicin-resistant strains that can be implemented into a routine service to analyse all strains in a specific geographical location. Denaturing HPLC (dHPLC) is a rapid screening test that can detect mutations in PCR amplicons. The aim of this study was to evaluate the dHPLC analysis of rifampicin-resistant Mycobacterium tuberculosis isolates using an extensive strain collection from Hong Kong and the UK and a collection of 84 consecutive clinical isolates., Methods: DNA from 51 rifampicin-resistant M. tuberculosis strains from the UK and Hong Kong identified from 1996 to 2005 was extracted and each mutation was defined by capillary electrophoresis. A 400 bp PCR product was amplified from each strain, heteroduplexed with a known susceptible control (H37Rv) and analysed by dHPLC at 67.0 degrees C., Results: Forty-five out of 51 (88.2%) rifampicin-resistant strains with known DNA mutations were detected by dHPLC. Two out of 84 clinical isolates were phenotypically rifampicin-resistant and dHPLC detected a mutation in the rpoB amplicon for both these isolates. dHPLC detected a mutation in 1 out of 82 phenotypically rifampicin-susceptible isolates (M482T, a non-cluster I/II mutation). In a combined analysis of all strains and isolates, mutation detection by dHPLC analysis exhibited 88.2% sensitivity and 98.8% specificity., Conclusions: This study shows that dHPLC analysis is sensitive and specific and could be implemented in a routine clinical service.

Objectives: To characterize 250 drug-resistant Mycobacterium tuberculosis (MTB) isolates in Hong Kong with respect to their drug susceptibility phenotypes to five common anti-tuberculosis drugs (ofloxacin, rifampicin, ethambutol, isoniazid and pyrazinamide) and the relationship between such phenotypes and the patterns of genetic mutations in the corresponding resistance genes (gyrA, rpoB, embB, katG, inhA, ahpC and pncA)., Methods: The MIC values of the aforementioned anti-tuberculosis drugs were determined for each of the 250 drug-resistant MTB clinical isolates by the absolute concentration method. Genetic mutations in the corresponding resistance genes in these MTB isolates were identified by PCR-single-stranded conformation polymorphism/multiplex PCR amplimer conformation analysis (SSCP/MPAC), followed by DNA sequencing of the purified PCR products., Results: Resistance to four or five drugs was commonly observed in these MTB isolates; such phenotypes accounted for over 34% of the 250 isolates. The most frequently observed phenotypes were those involving both rifampicin and isoniazid, with or without additional resistance to the other drugs. A total of 102 novel mutations, which accounted for 80% of all mutation types detected in the 7 resistance genes, were recovered. Correlation between phenotypic and mutational data showed that genetic changes in the gyrA, rpoB and katG genes were more consistently associated with a significant resistance phenotype. Despite this, however, a considerable proportion of resistant MTB isolates were found to harbour no detectable mutations in the corresponding gene loci., Conclusions: These findings expand the spectrum of potential resistance-related mutations in MTB clinical isolates and help consolidate the framework for the development of molecular methods for delineating the drug susceptibility profiles of MTB isolates in clinical laboratories.

This study was initiated throughout Hong Kong, to reveal the characteristics of community-acquired infections. All specimens collected by general practitioners from infected patients were followed prospectively, and those that were culture-positive were analysed. Four thousand seven hundred and forty-one specimens were collected from 3977 patients by 89 doctors from July 2000 to October 2001. The most common specimens were throat swabs (33%), urine (26%) and sputa (16%). The average culture-positive rate was 28%. The most common organisms were Escherichia coli (18%), beta-haemolytic streptococci (15%) and Staphylococcus aureus (12%). Fluoroquinolone resistance was relatively high (up to 35%) in organisms commonly causing urinary tract infection (E. coli, Proteus and Morganella). Although none of the pneumococci was resistant to penicillin 1 mg/L, the proportion with intermediate resistance (0.1-1 mg/L) was alarming (81%). There were three strains of methicillin-resistant S. aureus. A decrease in ampicillin resistance but a high prevalence of macrolide resistance were noted in Haemophilus influenzae. All Neisseria gonorrhoeae isolates were resistant to penicillin, up to 79% to the fluoroquinolones, 15% to spectinomycin, but all were susceptible to ceftriaxone. Respiratory pathogens (Streptococcus pneumoniae, beta-haemolytic streptococci and H. influenzae) were relatively susceptible to the newer fluoroquinolones (0-2%, 0.5-6% and 2% resistant, respectively) or third-generation cephalosporins (0-2% resistant). The distribution of organisms and their antibiotic resistance varied over time. Thus frequent surveillance is needed to provide information on the drugs of choice for different infections.