Edward Wai-Chi Chan

Human Metapneumovirus Infection in the Canadian Population

ABSTRACT Human metapneumovirus (hMPV), a newly discovered paramyxovirus, has been associated with acute respiratory tract infections (ARIs) ranging from upper ARIs to severe bronchiolitis and pneumonia. Important questions remain on the contribution of hMPV to ARIs and its impact on public health. During the 2001-2002 season, we conducted a collaborative study with four provincial public health laboratories to study the prevalence of this new virus in the Canadian population. A total of 445 specimens were collected from patients of all age groups with ARIs and were tested for the presence of hMPV by reverse transcription-PCR. Of these, 66 (14.8%) tested positive for hMPV. Positive specimens were found in all age groups and in all four provinces studied. Virus activity peaked in February and March. The age range of the patients with hMPV infection was 2 months to 93 years (median age, 25 years), with similar numbers of females (35%) and males (41%). Thirty-three percent (n = 22) of hMPV-infected patients were hospitalized; of these, 27% (n = 6) had rhinitis and pneumonia, 23% (n = 5) had bronchiolitis, and 9% (n = 2) had bronchitis. The hospitalization rates were significantly higher among patients <5 years of age (P = 0.0005) and those >50 years of age (P = 0.0044) than among those 6 to 50 years of age. Phylogenetic analysis of the F gene showed that two hMPV genetic clusters were cocirculating in the 2001-2002 season, and comparison with earlier studies suggests a temporal evolutionary pattern of hMPV isolates. These results provide further evidence of the importance of hMPV in ARIs, particularly in young children and elderly individuals.

Mutations in Topoisomerase Genes of Fluoroquinolone-Resistant Salmonellae in Hong Kong

ABSTRACT A total of 88 salmonella isolates (72 clinical isolates for which the ciprofloxacin MIC was >0.06 μg/ml, 15 isolates for which the ciprofloxacin MIC was ≤0.06 μg/ml, and Salmonella enterica serotype Typhimurium ATCC 13311) were studied for the presence of genetic alterations in four quinolone resistance genes, gyrA, gyrB, parC, and parE, by multiplex PCR amplimer conformation analysis. The genetic alterations were confirmed by direct nucleotide sequencing. A considerable number of strains had a mutation in parC, the first to be reported in salmonellae. Seven of the isolates sensitive to 0.06 μg of ciprofloxacin per ml had a novel mutation at codon 57 of parC (Tyr57→Ser) which was also found in 29 isolates for which ciprofloxacin MICs were >0.06 μg/ml. Thirty-two isolates had a single gyrA mutation (Ser83→Phe, Ser83→Tyr, Asp87→Asn, Asp87→Tyr, or Asp87→Gly), 34 had both a gyrA mutation and a parC mutation (29 isolates with a parC mutation of Tyr57→Ser and 5 isolates with a parC mutation of Ser80→Arg). Six isolates which were isolated recently (from 1998 to 2001) were resistant to 4 μg of ciprofloxacin per ml. Two of these isolates had double gyrA mutations (Ser83→Phe and Asp87→Asn) and a parC mutation (Ser80→Arg) (MICs, 8 to 32 μg/ml), and four of these isolates had double gyrA mutations (Ser83→Phe and Asp87→Gly), one parC mutation (Ser80→Arg), and one parE mutation (Ser458→Pro) (MICs, 16 to 64 μg/ml). All six of these isolates and those with a Ser80→Arg parC mutation were S. enterica serotype Typhimurium. One S. enterica serotype Typhi isolate harbored a single gyrA mutation (Ser83→Phe), and an S. enterica serotype Paratyphi A isolate harbored a gyrA mutation (Ser83→Tyr) and a parC mutation (Tyr57→Ser); both of these isolates had decreased susceptibilities to the fluoroquinolones. The MICs of ciprofloxacin, levofloxacin, and sparfloxacin were in general the lowest of those of the six fluoroquinolones tested. Isolates with a single gyrA mutation were less resistant to fluoroquinolones than those with an additional parC mutation (Tyr57→Ser or Ser80→Arg), while those with double gyrA mutations were more resistant.

Multiplex PCR Amplimer Conformation Analysis for Rapid Detection of gyrA Mutations in Fluoroquinolone-Resistant Mycobacterium tuberculosis Clinical Isolates

ABSTRACT A new strategy known as multiplex PCR amplimer conformation was developed for detection of mutation in the gyrA gene of 138 clinical isolates of Mycobacterium tuberculosis. The method generated a single-stranded and heteroduplex DNA banding pattern of multiplex PCR amplimers of the region of interest that was extremely sensitive to specific mutations, thus enabling much more sensitive and reliable mutation analysis compared to the standard single-stranded conformation polymorphism technique. The genetic profiles of the gyrA gene of the 138 isolates as detected by MPAC were confirmed by nucleotide sequencing and were found to correlate strongly with the in vitro susceptibilities of the mutant strains to six fluoroquinolones (ofloxacin, levofloxacin, sparfloxacin, moxifloxacin, gatifloxacin, and sitafloxacin). All 32 isolates that contained gyrA mutations exhibited cross-resistance to the six fluoroquinolones (ofloxacin MIC for 90% of strains > 16 mg/liter), although moxifloxacin, gatifloxacin, and sitafloxacin (MIC for 90% of strains ≤ 4 mg/liter) were apparently more active than ofloxacin, levofloxacin, and sparfloxacin (MIC for 90% of strains ≥ 16 mg/liter). All gyrA mutations were clustered in codons 90, 91, and 94, and aspartic acid 94 was most frequently mutated. Twenty-three isolates without gyrA mutations were also found to exhibit reduced susceptibility to ofloxacin (MIC for 90% of strains = 4 mg/liter), but largely remained susceptible to other drugs (MIC for 90% of strains ≤ 1 mg/liter). Another 83 isolates without mutations were fully susceptible to all six fluoroquinolones (ofloxacin MIC for 90% of strains = 1 mg/liter). In conclusion, high-level phenotypic resistance to fluoroquinolones among M. tuberculosis clinical isolates, which appears to be predominantly due to gyrA mutations, may be readily detected by genotyping techniques such as multiplex PCR amplimer conformation.

Rapid Multiplex Nested PCR for Detection of Respiratory Viruses

ABSTRACT Respiratory tract infections can be caused by a heterogeneous group of viruses and bacteria that produce similar clinical presentations. Specific diagnosis therefore relies on laboratory investigation. This study developed and evaluated five groups of multiplex nested PCR assays that could simultaneously detect 21 different respiratory pathogens: influenza A virus (H1N1, H3N2, and H5N1); influenza B virus; parainfluenza virus types 1, 2, 3, 4a, and 4b; respiratory syncytial virus A and B; human rhinoviruses; human enteroviruses; human coronaviruses OC43 and 229E; severe acute respiratory syndrome coronavirus; human metapneumoviruses; Mycoplasma pneumoniae; Chlamydophila pneumoniae; Legionella pneumophila; and adenoviruses (A to F). These multiplex nested PCRs adopted fast PCR technology. The high speed of fast PCR (within 35 min) greatly improved the efficiency of these assays. The results show that these multiplex nested PCR assays are specific and more sensitive (100- to 1,000-fold) than conventional methods. Among the 303 clinical specimens tested, the multiplex nested PCR achieved an overall positive rate of 48.5% (95% confidence interval [CI], 42.9 to 54.1%), which was significantly higher than that of virus isolation (20.1% [95% CI, 15.6 to 24.6%]) and that of direct detection by immunofluorescence assay (13.5% [95% CI, 9.7 to 17.4%]). The improved sensitivity was partly due to the higher sensitivity of multiplex nested PCR than that of conventional methods in detecting cultivatable viruses. Moreover, the ability of the multiplex nested PCR to detect noncultivatable viruses, particularly rhinoviruses, coronavirus OC43, and metapneumoviruses, contributed a major gain (15.6%) in the overall positive rate. In conclusion, rapid multiplex nested PCR assays can improve the diagnostic yield for respiratory infections to allow prompt interventive actions to be taken.

Genetic and phenotypic characterization of drug-resistant Mycobacterium tuberculosis isolates in Hong Kong

Abstract 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.

Delineation of a Bacterial Starvation Stress Response Network Which Can Mediate Antibiotic Tolerance Development

ABSTRACT This study aimed at elucidating the physiological basis of bacterial antibiotic tolerance. By use of a combined phenotypic and gene knockout approach, exogenous nutrient composition was identified as a crucial environmental factor which could mediate progressive development of tolerance with markedly varied drug specificity and sustainability. Deprivation of amino acids was a prerequisite for tolerance formation, conferring condition-specific phenotypes against inhibitors of cell wall synthesis and DNA replication (ampicillin and ofloxacin, respectively), according to the relative abundances of ammonium salts, phosphate, and nucleobases. Upon further depletion of glucose, this variable phase consistently evolved into a sustainable mode, along with enhanced capacity to withstand the effect of the protein synthesis inhibitor gentamicin. Nevertheless, all phenotypes produced during spontaneous nutrient depletion lacked the sustainable, multidrug-tolerant features exhibited by the stationary-phase population and were attributed to complex interaction between starvation-mediated metabolic and stress protection responses on the basis of the following reasons: (i) the nutrition-dependent tolerance characteristics observed suggested that adaptive biosynthetic mechanisms could suppress but not fully avert tolerance under transient starvation conditions; (ii) formation of specific phenotypes could be inhibited by suppressing protein synthesis prior to nutrient depletion; (iii) bacteriostatic drugs produced only weak tolerance in the absence of starvation signals; and (iv) the attenuation of the stringent and SOS responses, as well as the functionality of other putative tolerance determinants, including rpoS, hipA, glpD, and phoU, could alter the induction requirement and drug specificity of the resultant phenotypes. These data reveal the common physiological grounds characteristic of starvation responses and the onset of antibiotic tolerance in bacteria.

Genetic characterization of mcr-1-bearing plasmids to depict molecular mechanisms underlying dissemination of the colistin resistance determinant

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 blaCTX-M, blaCMY, blaTEM, 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.

A fatal outbreak of ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae in a Chinese hospital: a molecular epidemiological study

BACKGROUND Hypervirulent Klebsiella pneumoniae strains often cause life-threatening community-acquired infections in young and healthy hosts, but are usually sensitive to antibiotics. In this study, we investigated a fatal outbreak of ventilator-associated pneumonia caused by a new emerging hypervirulent K pneumoniae strain. METHODS The outbreak occurred in the integrated intensive care unit of a new branch of the Second Affiliated Hospital of Zhejiang University (Hangzhou, China). We collected 21 carbapenem-resistant K pneumoniae strains from five patients and characterised these strains for their antimicrobial susceptibility, multilocus sequence types, and genetic relatedness using VITEK-2 compact system, multilocus sequence typing, and whole genome sequencing. We selected one representative isolate from each patient to establish the virulence potential using a human neutrophil assay and Galleria mellonella model and to establish the genetic basis of their hypervirulence phenotype. FINDINGS All five patients had undergone surgery for multiple trauma and subsequently received mechanical ventilation. The patients were aged 53-73 years and were admitted to the intensive care unit between late February and April, 2016. They all had severe pneumonia, carbapenem-resistant K pneumoniae infections, and poor responses to antibiotic treatment and died due to severe lung infection, multiorgan failure, or septic shock. All five representative carbapenem-resistant K pneumoniae strains belonged to the ST11 type, which is the most prevalent carbapenem-resistant K pneumoniae type in China, and originated from the same clone. The strains were positive on the string test, had survival of about 80% after 1 h incubation in human neutrophils, and killed 100% of wax moth larvae (G mellonella) inoculated with 1 × 106 colony-forming units of the specimens within 24 h, suggesting that they were hypervirulent K pneumoniae. Genomic analyses showed that the emergence of these ST11 carbapenem-resistant hypervirulent K pneumoniae strains was due to the acquisition of a roughly 170 kbp pLVPK-like virulence plasmid by classic ST11 carbapenem-resistant K pneumoniae strains. We also detected these strains in specimens collected in other regions of China. INTERPRETATION The ST11 carbapenem-resistant hypervirulent K pneumoniae strains pose a substantial threat to human health because they are simultaneously hypervirulent, multidrug resistant, and highly transmissible. Control measures should be implemented to prevent further dissemination of such organisms in the hospital setting and the community. FUNDING Chinese National Key Basic Research and Development Program and Collaborative Research Fund of Hong Kong Research Grant Council.

Emergence of Carbapenem-Resistant Serotype K1 Hypervirulent Klebsiella pneumoniae Strains in China

ABSTRACT We report the emergence of five carbapenem-resistant K1 hypervirulent Klebsiella pneumoniae (hvKP) strains which caused fatal infections in hospital patients in Zhejiang Province, China, upon entry through surgical wounds. Genotyping results revealed the existence of three genetically related strains which exhibited a new sequence type, ST1797, and revealed that all strains harbored the magA and wcaG virulence genes and a plasmid-borne blaKPC-2 gene. These findings indicate that K1 hvKP is simultaneously hypervirulent, multidrug resistant, and transmissible.

Emergence of Clinical Salmonella enterica Serovar Typhimurium Isolates with Concurrent Resistance to Ciprofloxacin, Ceftriaxone, and Azithromycin

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