Human coronaviruses, namely HCoV-229E and HCoV-OC43, have been identified as causal agent of upper respiratory tract infections for decades. The significance of human coronaviruses studies re-emerged after the SARS pandemic. Two novel human coronaviruses, HCoV-NL63 and HCoV-HKU1, were identified after the SARS pandemic. Up till now there are a total of five human coronaviruses being identified. After the discovery of HCoV-HKU1 in 2005, molecular epidemiology and genome studies identified the first natural recombination in human coronaviruses, which has led to the generation of different genotypes. A similar phenomenon was also observed in HCoV-OC43 in a subsequent study, resulting in the emergence of a novel genotype associated with pneumonia. Although HCoV-229E has been discovered for over forty years, information regarding its evolution and epidemiology is little and scattered. In this study, 32 HCoV-229E strains were collected from nasopharyngeal aspirates over a period of 8 years (from April 2004 to January 2012). Three genes, including RdRp (RNA-dependent-RNA polyermase), S (spike) and N (nucleocapsid), were sequenced and analyzed. Phylogenetic studies showed the existence of genetic drift among six chronological groups, including group 1 from 1979 to 1982, group 2 from 1982 to 1984, group 3 from 1990 to 1992, group 4 from 2001 to 2005, group 5 from 2005 to 2008 and group 6 from 2011 to 2012. One particular strain in 2006, HCoV-229E-HK06-24 displayed an incongruent position between the S and N gene, suggesting a possible recombination between group 4 and group 5. Additionally, five strains from 2011 to 2012 showed incongruent positions in RdRp comparing to other strains, which are suspected to be novel group 6. This study revealed the first evidence for a possible natural recombination event in HCoV-229E. The predominate group 6, which is genetically different from previous strains, may have been arisen by genetic drift. Further surveillance is required to monitor the genetic changes in HCoV-229E. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Background Methicillin resistant Staphylococcus aureus (MRSA) is endemic in healthcare settings in many countries of the world. Patients who have acquired MRSA serve as a source of transmission by contamination of their surrounding environments. Numerous studies illustrate that many different inanimate surfaces in hospitals can become a reservoir for MRSA. Objectives The objective of this study is to examine the presence of MRSA on environmental surfaces and its relationship between patients’ acquisition of MRSA by studying their molecular characteristics. Methodology The near-patient surfaces of 30 MRSA positive patients, 30 control patients and the ward environments were sampled from June 2011 to September 2011. The swabs were enriched and cultured for the presence of MRSA. The MRSA isolates obtained from environmental samples and from the clinical samples of the patients were then characterized by Spa typing. Results The MRSA found in case patients and control patients’ environmental surfaces was 97% (29/30) and 40% (12/30) respectively. Environmental surfaces that were highly contaminated by MRSA positive patients were bed sheets (70%), followed by pillows (55%), patient bed frames (52%) and patient lockers (52%). On the environmental surfaces other than the near-patient areas, ambulatory chair armrests had the highest amount of MRSA (21%), followed by fax machines which accounted for 14%. Among the 216 MRSA isolates (30 clinical isolates and 151 environmental isolates), eight spa types were found and the most predominant spa type was t1081 (63.3%) followed by t032 (17.6%) and t037 (7.4%). 27 patients were found to have the MRSA isolates with same spa type in the clinical samples and their surrounding environments. The agreement between the MRSA isolated from the clinical sample of patients and their surrounding environment was 93.1%. Conclusion Identical isolates were recovered from the patient and their environment (93.1%) which suggests possible environmental contamination of the ward cubicles, possibly contributing to endemic MRSA. More effective and rigorous use of current approaches to cleaning and decontamination is required and consideration of newer technologies to eradicate MRSA. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Background There has been an increasing awareness of the role of the hospital environment as a reservoir of Acinetobacter baumannii. A. baumannii is an important nosocomial pathogen and is difficult to control due to the increasing cases of resistance to carbapenem. Objectives The objectives of this study areto examine carbapenem-resistant Acinetobacter baumannii (CRAB) positive patients according to their environmental sample to determine how frequently the environment surrounding the patient becomes contaminated and which environmental surfaces are most commonly contaminated. Methodology During June 2011 to December 2011, data regarding 30 hospitalized patients with at least one positive CRAB clinical sample were collected from hospital X in Kowloon of Hong Kong. For 30 case patients, one patient in the ICU ward had been isolated in a single room and the other 29 patients stayed in a multi-room. Fifteen surfaces in the patient cubicle and nine surfaces in health care worker stations were evaluated for the presence of CRAB. 29 control environmental samples were obtained from the surroundings of patients without CRAB in the same cubicle and one control environmental sample was obtained from the surroundings of patients without CRAB in the other room of ICU. Pulsed-field gel electrophoresis was performed on all environmental isolates and clinical samples. Results Of the 30casepatients, 26 patients (86.7%) were found to have CRAB contamination in their surrounding environment and 6negative control patients (20%) were found to have CRAB in their environmental samples. The percentage of positive CRAB cultures in the case environment, control and health care worker stations was 28.9% (117/405), 3.4% (14/406) and 1.9% (5/265)respectively. In the surrounding case patient area, pillows (60% 18/30) and bed sheets on which the patients sleep on (60% 18/30), bed sheets covering the patients (50% 15/30) and bedside table tops (40% 12/30) were the most commonly contaminated. For 26casepatientswere found to have CRAB contamination in their surrounding environment, 23 (88.5%) of these patients were found to have the clone of isolates in the case environment related to the patients. Conclusion For patients with CRAB, the surrounding environment is frequently contaminated. Surfaces often touched by the patients are commonly contaminated. CRAB was also found on surfaces that were not closely related to the patient which are frequently touched by healthcare workers during patient care. / published_or_final_version / Microbiology / Master / Master of Medical Sciences

Dissemination of Carbapenem-Resistant Enterobacteriaceae (CRE) has raised a new challenge for health organizations all over the world. Acquisition of carbapenemase genes is the most worrisome among these CRE isolates. This study was constructed to investigate the dissemination of CRE isolates in Hong Kong and also to characterize plasmids harboring carbapenemase genes. CRE isolates were collected from public hospitals in Hong Kong from August 2006 to June 2012. Antimicrobial susceptibility of all CRE isolates was tested using disc diffusion method. Screening of carbapenemase genes (blaNDM , blaKPC, blaIMP, blaVIM and blaOXA-48) and ESBL genes (blaCTX-M and blaSHV) were also performed. Clonal relatedness was studied by multi-locus sequence typing. Characterization of plasmids was carried out by conjugation, S1-PFGE, hybridization and plasmid replicon typing. A total of 69 CRE isolates were collected including 50 K. pneumoniae, 15 E. coli, 2 E. cloacae, 1 E. aerogenes and 1 C. freundii. Eighteen carbapenemase producing Enterobacteriaceae were isolated from different patients with travel histories among these 69 isolates. Four K. pneumoniae were detected to carry blaKPC genes on different transferable plasmids as follows: 50 kb, IncX3 plasmid (ST258); 70 kb, un-typeable plasmid (ST258); 130 kb, un-typeable plasmid (ST11) and 140 kb, un-typeable plasmid (ST11). blaIMP genes were also detected in four CRE isolates to be harbored by different plasmids or located on chromosome: ST11 K. pneumoniae (50 kb, IncN), ST1 K. pneumoniae (150 kb, IncA/C), E. cloacae (130 kb, IncN-L/M) and ST899 K. pneumoniae (chromosomal located). NDM-1 (New Delhi Metallo enzyme) producing E. coli (n = 5), K. pneumoniae (n = 2), E. aerogenes (n = 1), E. cloacae (n = 1) and C. freundii (n =1) were also found in this study. Eight of them were isolated from patients travelled to different provinces of China blaNDM-1 was found to be carried by transferable plasmids in all ten isolates: IncX3 (n = 7, 50 kb), IncL/M (n = 1, 88 kb), IncA/C2 (n = 1, 140 kb) and FIIY- FIBS (n = 1, 110kb). Six of the seven IncX3 plasmids showed identical digestion profile while the other one only had two bands different from others using Restriction fragment length polymorphism (RFLP) analysis. An IncX3 plasmid pNDM-HN380 from a K. pneumoniae strain CRE380 was completely sequenced using Genome Sequencer FLX (Roche, USA). pNDM-HN380 was a 54,035 bp circular plasmid with 52 open reading frames (ORFs). The backbone of pNDM-HN380 was identical to those previous described IncX plasmids pIncX-SHV (accession number JN247852) and pEC14_35 (accession number JN935899). The blaNDM-1 gene was carried on an ISAba125 and IS26 flanked transposon-like element. And this element except IS26 and an interrupted ISAba125 was found to be identical to pNDM-BJ01 (accession number JQ001791). In conclusion, this is the first we describe a blaNDM-1 carrying IncX3 plasmid. This IncX3 plasmid was found to be predominant in the dissemination of blaNDM-1 in China. Future study of the nationwide dissemination of carbapenemase genes and also the novel IncX3 plasmids is needed. / published_or_final_version / Microbiology / Master / Master of Philosophy

The diminishing choice of effective antibiotics against resistant pathogens has forced clinicians to revive the use of old antibiotics. Hence, fosfomycin has been frequently suggested for alternative therapies given its track record of low resistance rates despite extensive use. However, there have been recent reports of plasmid-mediated fosfomycin resistance among animals and healthy humans in Asia. Accordingly, comparison of shared fosfomycin resistance mechanisms between animals and humans will shed light on the spread of resistance and guide future use of antimicrobials. This study aimed to investigate plasmid-mediated fosfomycin resistance in E. coli isolates collected from patients and animals in Hong Kong. Non-duplicate E. coli isolates were cultured from 1711 urinary isolates and 167 blood clinical samples collected from multiple centres during 1996-2008 and from fecal samples of 210 cattle, 214 pigs, 460 chickens, 398 stray cats, 368 stray dogs and 456 wild rodents during 2008-2010. A total of 2106 animal samples yielded 1693 E. coli isolates of which 831 were ESBL-producers. Fosfomycin-resistant isolates were more likely than fosfomycin-susceptible isolates to be ESBL-producers and multidrug resistant (≥3 antimicrobial classes). Of the 101 fosfomycin-resistant isolates, 97 (96.0%) were fosA3 positive and 94 (93.1%) were blaCTX-M positive. Of the 1878 clinical isolates, 18 were fosfomycin-resistant of which six were fosA3-positive and two were positive for another fosA variant (designated fosKP96). All but one fos-carrying clinical isolate was ESBL-producing. The majority of the fos-carrying E. coli strains belonged to diverse clones under two main clonal complexes CC58 accounting for 38 (36.2%) strains and CC10 for 32 (30.5%) strains. PCR mapping showed that all fosA3-containing regions were followed by a downstream IS26. In all clinical isolates and 81 (83.5%) of animal isolates, an IS26 was also found upstream. In 14 (14.4%) of animal isolates, the fosA3 gene was preceded by an upstream blaCTX-M-14-containing transposon-like structure (ΔISEcp1-blaCTX-M-14-ΔIS903 or ISEcp1-IS10-blaCTX-M-14-ΔIS903). For the remaining two animal isolates, the upstream region could not be defined. In a random subset of 18 animal isolates, fosA3 was carried on transferable plasmids with sizes of 50–200 kb and the following replicons: F2:A-:B- (n = 3), F16:A1:B- (n = 2), F24:A-B- (n = 1), IncHI2 (n = 3), IncN (n = 2), IncI1 (n = 1), B/O (n = 1) and untypeable (n = 5). Among six fosA3-carrying clinical isolates, the distributions were: F2:A-:B- (n = 2), IncN (n = 1), multi-replicon F-:A-:B1/IncN (n = 1) and untypeable (n = 2). Both fosKP96-carrying plasmids belonged to IncN. Restriction fragment length polymorphism analysis showed that the four F2:A-:B- plasmids carrying fosA3 and blaCTX-M-3 genes from a pig, dog and two patients shared an almost identical pattern. The complete plasmid sequences further demonstrated their homology. This study demonstrated the emergence of fosA3-mediated fosfomycin resistance among E. coli isolates from multiple sources. Highly similar IncFII plasmids and IS26 transposon-like structures appear to be the main vehicles for dissemination. This study also highlighted occurrence of plasmids carrying fosKP96 that may have been overlooked by others as this variant could not be detected by established PCR markers. / published_or_final_version / Microbiology / Master / Master of Philosophy

Acinetobacter baumannii is an important nosocomial pathogen worldwide because of its remarkable ability to acquire antibiotic resistance. The global emergences of multidrug-resistant A. baumannii (MDR-AB) clones are predominated by a number of widely disseminated clones, namely clonal complex (CC) 1, CC2, and CC3. In early 2010, we reported two major clones of MDR-AB, designated HKU1 and HKU2 belong to sequence types (ST) 96 and ST92, widely disseminating in our hospitals. ST92 is a predominant clone that is prevalent in more than 30 countries, whereas ST96 has been identified recently and is geographically confined to certain parts of China. Our previous study only investigated the isolates collected in the year 2005-2006. We therefore extended our investigation over a six-year period (2005-2010) to generate a more complete picture of the molecular epidemiology and resistance mechanisms in A. baumannii. Firstly, we performed the susceptibility test on various antimicrobial agents and employed molecular methods to characterize the epidemiology of the target A. baumannii isolates. For the entire study period, increased resistance rates were noted for the seven antimicrobial agents, namely imipenem, piperacillin-tazobactam, cefoperazone, ticarcillin-clavulanate, ciprofloxacin, gentamicin and amikacin (P <0.01). Worryingly, an increased trend was also observed for the pandrug-resistant rate, from 0.2% in the year 2005-2006, to 1.9-2.9% in the year 2007-2008 and up to 6.0-8.1% in the year 2009-2010 (chi square for trend, P <0.001). Pulsed-field gel electrophoresis and multilocus sequence typing (PFGE/MLST) categorized 100 out of 108 (92.6%) isolates into four clones (PFGE/MLST), namely HKU2/ST92 (n = 14), HKU3/ST254 (n = 73), HKU4/ST137 (n = 5), and HKU5/ST362 (n = 8), respectively. PCR showed that 88.9% (96/108) of the amikacin-resistant isolates were armA positive and all isolates were found to harbour at least one of the OXA-type carbapenemases with frequencies as follows: OXA-51-like (98/108, 90.7%), OXA-23-like (85/108, 78.7%), OXA-58-like (9/108, 8.3%) and OXA-24-like (8/108, 7.4%). Secondly, we compared the biological fitness of the circulating clones by performing the doubling time and adhesion experiment. The results demonstrated that HKU3/ST254 has a higher capability for replication and adherence to human bronchial epithelial cells. Together with the higher antibiotic resistance rate, the selective advantages in terms of biological fitness may facilitate the clonal expansion and wide dissemination of this lineage. Finally, whole genome sequence data showed a high amount of resistance genes intermixed with various insertion sequence (IS) elements, integrons and transponsons clustering inside the resistance islands. The presence of a second genomic resistance island conferring aminoglycoside and sulphonamide resistance, additional loci outside the resistance islands harbouring resistance genes and the high amount of antibiotic efflux pumps in various A. baumannii genomes demonstrated that resistance islands contribute a significant part to the multidrug-resistant phenotype in A. baumannii but are not the only factor. The correlation analysis further demonstrated the significance of IS elements in the dissemination of antibiotic resistance genes in the A. baumannii genomes. As a whole, whole genome sequence data may provide an informative and efficient approach to generating a more comprehensive picture to study the resistance mechanism of the epidemic strains. / published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Thesis (Ph. D.)--University of Hong Kong, 2008. / Also available in print.

Thesis (M. Med. Sc.)--University of Hong Kong, 2007.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 183-218) Also available in print.

Thesis (MSc (Production Animal Studies)--University of Pretoria, 2008. / Includes bibliographical references. Also available in print format.