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

Objective: To find out the spatial autocorrelation of antibiotic resistance of S. pneumonia and test the significance of distance as a risk factor. Methods: Descriptions of penicillin and macrolide resistance in EARS-Net countries from 2006 to 2010 were given. Global moran’s I and Anselin moran’s I were used to assess the spatial autocorrelation and gravity model was used to test the significance of distance and other socio – economic factors. Results: The trend of resistance in Europe was stable. Positive spatial autocorrelation existed from 2006 to 2010 for penicillin (Z(I): 0.16-0.2) and 2009 to 2010 for macrolide (Z(I): 0.11 -0.13). Some clusters (hotspots) were identified; they were Cyprus (2006-2010 for penicillin and 2009 to 2010 for macrolide), Spain (2006 for penicillin), France (2006 for penicillin), Romania (2009 for penicillin and macrolide) and Bulgaria (2009 for penicillin and macrolide). The result of gravity model showed that only parameters of population in 2007 for penicillin (p<0.05) and parameter of distance in 2009 for penicillin (p<0.05) in Cyprus were statistically significant. Conclusion: Distance was not a risk factor of high prevalence of antibiotic resistance of S. pneumoniae although there was a positive spatial autocorrelation. Improvement in surveillance system and appropriate public action were recommended for controlling the spread of resistant strain of S. pneumoniae. / published_or_final_version / Public Health / Master / Master of Public Health

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

Background A pandemic clone, Escherichia coli sequence type 131 (ST131), has been disseminated worldwide and represents an important cause of antimicrobial resistant infections. The spread of this resistant clone has become a great public health concern. Objectives The aims of this study were to investigate the prevalence of ST131 in Escherichia coli isolates from human urinary cultures in Hong Kong and study the antimicrobial phenotypes of ST131. Methodology This study included 340 E. coli clinical urinary isolates obtained from patients in four district hospitals between May 2013 and July 2013 in Hong Kong. Antimicrobial susceptibilities were assessed by disk diffusion method with reference to CLSI. The isolates were investigated by phylogroup-specific and ST131-specific PCR assays. ST131 strains were further assessed for subclone distribution, antimicrobial resistance and extended-spectrum β-lactamase (ESBL) type. Results A total of 18.5% (63/340) of the E. coli population was identified as ST131. ST131 isolates were significantly more likely than non-ST131 isolates to be ciprofloxacin resistant (69.8%, 44/63 versus 31.0%, 86/277; P <0.001), gentamicin resistant (38.1%, 24/63 versus 24.9%, 69/277; P=0.03) and ESBL producers (41.3%, 26/63 versus 18.8%, 52/277; P <0.001). Among the ST131 E. coli isolates, 68.3% (43/63) belonged to the H30 subclone. Most H30 isolates were ST131-O25b (97.7%, 42/43). Also, the ST131-H30 E. coli subclone was statistically associated with ciprofloxacin resistance compared with the non-H30 ST131 isolates (P <0.001). Additionally, strains which were co-resistant to ciprofloxacin, co-trimoxazole and gentamicin were overwhelmingly associated with the H30 subclone than non-H30 (23.3%, 10/43 versus 0%, 0/20; P=0.02). Conclusion This study showed that ST131 isolates were widespread among human E. coli urinary isolates in Hong Kong. The increase in antimicrobial resistance phenotypes are highlighted with ST131, especially the H30 subclone isolates. The dissemination of the ST131 resistant clonal group has aroused clinical attention and limited the choice of empirical treatment. / published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

No description available.

No description available.

The characteristics and expression of laboratory derived chloramphenicol (CM) resistance in P. aeruginosa were examined. Resistant strains exhibiting single cell resistance of 1.5 to 2 mg/mL were readily isolated following one passage in CM at 150 to 1000 (mu)g/mL. Isogenic strains, selected on CM at 150 and 500 (mu)g/mL were chosen for detailed study. Resistance was not a consequence of drug detoxification or altered sensitivity of the target site. The resistant strains exhibited unusual phenotypic properties including pronounced variations in growth rate, CM susceptibility and cell morphology as a function of the composition of the growth medium. Growth in CM also resulted in significant alterations in amino acid transport and respiratory capacity, the extent of which varied with the strain, the growth medium and the concentration of CM. These drug and medium-dependent alterations were determined to reside in an increased and highly specific requirement for Ca('2+), Mg('2+), Mn('2+) or Sr('2+). Manipulation of the divalent cation concentration of a variety of growth media resulted in dramatic alterations in growth rate, resistance and amino acid transport. Ca('2+) was significantly more effective than the latter three ions. The expression of native and plasmid-mediated CM resistance was also modified by the external concentration of divalent cations. In view of the nature and specificity of the cation requirement, it was concluded that (1) divalent cation-mediated alterations of outer membrane permeability are fundamental to the expression of native and acquired CM resistance in P. aeruginosa; (2) laboratory-derived CM resistance involves envelope changes, such that interaction with divalent cations promotes more effective exclusion of CM. The latter conclusion is supported by other divalent cation-dependent alterations in envelope function in the resistant strains.

This research tested the hypotheses that (a) suboptimal root-zone temperatures (RZT) limit the soybean-Bradyrhizobium N$ sb2$-fixing symbiosis primarily through an inhibition of symbiosis establishment and (b) this inhibition is modified by the genotype of micro- or macrosymbiont. Controlled environment and field experiments were conducted utilizing two soybean genotypes and six B. japonicum strains. At 19$ sp circ$C RZT fixed nitrogen levels decreased by 30-40%, predominantly due to a restriction in the latter stages of nodule development. Reductions of 10% and 30% in specific nodule activity rates at 19$ sp circ$C and 15$ sp circ$C RZT respectively, indicated nodule function to be comparatively insensitive to low RZT. Soybean genotypes did not differ in seedling nodulation or N$ sb2$-fixation under cool-soil, field or controlled environment, conditions. At all temperatures, commercial B. japonicum strain 532C was more efficient, but not effective, than strains obtained from the cool-soils of Northern Japan. Under cool-soil field conditions, two of the latter strains increased seedling nodulation and N$ sb2$-fixation.

The characteristics of the effects of chloramphenicol on Pseudomonas aeruginosa were examined. Resistant strains were easily isolated following a single passage in chloramphenicol at 150 $ mu$g/ml to 500 $ mu$g/ml. Drug detoxification or altered sensitivity of the target site could not be the mechanism of resistance. This resistance to chloramphenicol was correlated with the addition of an outer membrane protein with a molecular weight of 49 kDa and the loss of two outer membrane proteins, one with the molecular weight of 19 kDa and the other of about 10 kDa. The highly specific requirement of the resistant strains for Ca$ sp{2+}$, Mg$ sp{2+}$, Mn$ sp{2+}$ or Sr$ sp{2+}$ described by Irvin and Ingram (1982) was confirmed by the observation that the outer membrane of the resistant cells contained twice as much Mg$ sp{2+}$ cation as the sensitive cells. Many other experiments designed to observe the effects of chloramphenicol on the outer membrane of P. aeruginosa failed. It was concluded that the observations made in this study strongly suggested a "re-structuring" of the outer membrane of P. aeruginosa, rendering the resistant cells more impermeable to chloramphenicol.