Biomedical subjects

Fanrong Kong

Publications and source records attributed to Fanrong Kong.

At least 19 recordsLinked to original sources

PubMed2026

Genome-sequencing-based benchmarking of antimicrobial resistance, treatment outcomes and healthcare transmission events for Clostridioides difficile infection in Australian hospitals.

BACKGROUND: Clostridioides difficile infection (CDI) remains a priority for infection prevention and control in health care, particularly with the emergence of hypervirulent strains and antimicrobial resistance (AMR). AIM: To characterize the genomic epidemiology and AMR profiles of culture-confirmed CDI cases within tertiary hospitals in Australia. METHODS: A total of 155 C. difficile isolates from 142 patients with CDI diagnosed in four hospitals between 2023 and 2025 were studied. Data collected included patient demographics, severity of infection, antibiotic treatment and clinical outcomes at 8 weeks. Phenotypic susceptibility to vancomycin, fidaxomicin, metronidazole, moxifloxacin, meropenem, tetracycline and rifaximin were determined by agar dilution. Isolates underwent whole-genome sequencing (WGS) for genotyping and resistome assessment. FINDINGS: WGS differentiated 39 distinct sequence types among CDI isolates across different healthcare services. In total, 100 isolates were singletons and 55 (35% clustering rate) isolates were considered to be genomically related (difference of two or fewer single-nucleotide polymorphisms). Of these, 12 patients (8.5%) with close hospital contact formed six epidemiologically linked clusters. Phenotypic susceptibility results were obtained for 134 (86.4%) CDI isolates. There was no phenotypic resistance to vancomycin [minimum inhibitory concentration required to inhibit the growth of 90% of isolates (MIC90) 1 mg/L], metronidazole (MIC90 0.5 mg/L) or fidaxomicin (MIC90 0.5 mg/L). There was no association in the study cohort between the presence of resistance genes or reduced phenotypic susceptibility and CDI recurrence. CONCLUSION: Genomic analysis of C. difficile isolates did not identify any outbreaks or an association between the sequence type or presence of a resistance gene and clinical outcomes. High-resolution characterization and identification of antibiotic resistance, CDI clinical relapse and recent transmission offered by genome sequencing can provide important benchmarks for hospital infection control.

Antibiotic resistance↗

PubMed2006

Identification of a Streptococcus agalactiae serotype III subtype 4 clone in association with adult invasive disease in Hong Kong.

Characterization of Streptococcus agalactiae serotype III isolates revealed a subtype 4 clone that has an indistinguishable pulsed-field gel electrophoresis pattern and possesses a C-alpha protein, IS1381, and a novel sequence type (ST), ST 283, by multilocus sequence tagging. This clone was significantly associated with diseases caused by invasive strains from nonpregnant adults (P <or= 0.01, chi-square test) and was not present in the genital tracts of pregnant mothers.

DNA Transposable Elements↗

PubMed2006

Influence of disk separation distance on accuracy of the disk approximation test for detection of inducible clindamycin resistance in Staphylococcus spp.

We undertook this study to assess the accuracy of the clindamycin-erythromycin disk approximation test (D-test) for detection of inducible clindamycin resistance in Staphylococcus spp. One hundred sixty-three Staphylococcus aureus and 68 coagulase-negative Staphylococcus (CoNS) spp. which were erythromycin nonsusceptible but clindamycin susceptible were tested using the D-test performed at both 15-mm and 22-mm disk separations and compared with genotyping as the "gold standard." The rate of inducible clindamycin resistance was 96.3% for S. aureus and 33.8% for CoNS spp. The sensitivities of the D-tests performed at 15 mm and 22 mm were 100% and 87.7%, respectively, and specificities were 100% for both. The use of 22-mm disk separation for the D-test to detect inducible clindamycin resistance results in an unacceptably high very major error rate (12.3%). All isolates with false-negative results harbored the ermA gene, and the majority were methicillin-resistant Staphylococcus aureus. False-negative results were associated with smaller clindamycin zone sizes and double-edged zones. We recommend using a disk separation distance of </=15 mm. There is wide geographic variation in the rates of inducible clindamycin resistance, and each laboratory should determine the local rate before deciding whether to either perform the D-test routinely or else report that all erythromycin-resistant S. aureus isolates are also clindamycin resistant.

Anti-Bacterial Agents↗

PubMed2006

Development of a DNA microarray to identify the Streptococcus pneumoniae serotypes contained in the 23-valent pneumococcal polysaccharide vaccine and closely related serotypes.

Streptococcus pneumoniae is a major worldwide human pathogen. This investigation has developed a reliable and accurate DNA microarray method for inter-species differentiation of S. pneumoniae and intra-species differentiation of the 23 groups of S. pneumoniae including serotypes represented in the 23-valent pneumococcal vaccine and the other 20 closely related serotypes. In addition to 16S rDNA probes, serotype- or serogroup-specific probes targeting the capsular polysaccharide synthesis (cps) genes, wzy or capA were generated. We adopted a two-step multiplex PCR to improve the sensitivity of detection to a level of 10(5) cfu/ml in pure culture or 50 ng DNA. A total of 169 isolates (from China, Australia, Canada and New Zealand) including 147 belonging to 23-valent vaccine and closely related serotypes of S. pneumoniae, 11 belonging to other serotypes and 11 of different species commonly isolated from respiratory tract were tested to verify the method. The DNA microarray method developed provides a sensitive means to rapidly identify the members of the most common S. pneumoniae serotypes in patients and to monitor their distribution in different patient groups and geographic locations. Such information is needed for disease surveillance and to monitor vaccine efficacy.

Bacterial Proteins↗

PubMed2006

Streptococcus agalactiae Cbeta protein gene (bac) sequence types, based on the repeated region of the cell-wall-spanning domain: relationship to virulence and a proposed standardized nomenclature.

The Cbeta protein (Bac) of Streptococcus agalactiae (group B streptococcus; GBS) is an IgA binding protein encoded by bac, of which at least 39 sequence types have been described, based on polymorphisms in the repeated region of the cell-wall-spanning domain ('bac sequence types'). Cbeta is usually found in serotype Ib, less commonly in serotype II, and rarely in other serotypes. The aim of this study was to examine the prevalence, variety and distribution, among GBS serotypes and between invasive and superficial isolates, of bac sequence types. A total of 1101 GBS isolates were tested, from 10 countries, with a bac-specific PCR, and amplicons from all 255 (23 %) with positive results were sequenced. Ninety-seven percent (184/190) of serotype Ib and 37 % of serotype II isolates were bac positive. The Calpha protein gene (bca) was present in 98 % (251/255), and insertion sequences IS1381 and IS861 in 94 % (239/255), of bac-positive isolates. The authors identified 59 bac sequence types belonging to 19 groups, based on length, from 496 to 946 bp, with up to six sequence variants (a-f) in each group. The median bac sequence length of invasive isolates was significantly shorter than that of superficial isolates overall (640 versus 586 bp; P < 0.001) and specifically for serotype Ib (541 versus 676 bp; P < 0.001), and invasive isolates were significantly (P < 0.001) more likely to have one or more 18 bp deletions relative to the original published bac sequence (X59771). bac sequence typing is a useful addition to the previously described genotyping system, and will help to predict relative virulence among S. agalactiae serotype Ib strains.

Antigens, Bacterial↗

PubMed2006

Molecular serotype identification of Streptococcus agalactiae of bovine origin by multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay.

We used a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay and sequencing of a variable region of the cps cluster to identify serotypes of 140 Streptococcus agalactiae (group B Streptococcus; GBS) isolates from cattle. Only 71 (51%) isolates were typeable using antisera, but molecular serotypes (MS) were assigned to 133 (95%) and 139 (99%) isolates by partial cpsE-cpsF-cpsG sequencing and mPCR/RLB, respectively. Ninety-four isolates (67%) belonged to MS III and most belonged to a molecular serosubtype (msst) III-3, which is uncommon among GBS isolates from humans. Our results demonstrate that cps clusters of bovine GBS differ significantly from those of GBS isolates from humans.

Animals↗

PubMed2006

Simultaneous detection of nine antibiotic resistance-related genes in Streptococcus agalactiae using multiplex PCR and reverse line blot hybridization assay.

Streptococcus agalactiae (group B streptococcus [GBS]) is the leading cause of neonatal and maternal sepsis. Penicillin is recommended for intrapartum prophylaxis, but erythromycin or clindamycin is used for penicillin-allergic carriers. Antibiotic resistance (AR) has increased recently and needs to be monitored. We have developed a multiplex PCR-based reverse line blot (mPCR/RLB) hybridization assay to detect, simultaneously, seven genes encoding AR--erm(A/TR), erm(B), mef(A/E), tet(M), tet(O), aphA-3, and aad-6--and two AR-related genes, int-Tn and mreA. We tested 512 GBS isolates from Asia and Australasia and compared mPCR/RLB with antibiotic susceptibility phenotype or single-gene PCR. Phenotypic resistance to tetracycline was identified in 450 (88%) isolates, of which 442 had tet(M) (93%) and/or tet(O) (6%). Of 67 (13%) erythromycin-resistant isolates, 18 were susceptible to clindamycin, i.e., had the M phenotype, encoded by mef(A/E); 39 had constitutive (cMLS(B)) and 10 inducible clindamycin resistance, and of these, 34 contained erm(B) and 12 erm(A/TR). Of four additional isolates with mef(A/E), three contained erm(B) with cMLS(B) and one was erythromycin susceptible. Of 61 (12%) clindamycin-resistant isolates, 20 were susceptible to erythromycin and two had intermediate resistance. Based on sequencing, 21 of 22 isolates with mef had mef(E), and 8 of 353 with int-Tn had an atypical sequence. Several AR genes, erm(B), tet(O), aphA-3, aad-6, and mef(A/E), were significantly more common among Asian than Australasian isolates, and there were significant differences in distribution of AR genes between GBS serotypes. Our mPCR/RLB assay is simple, rapid, and suitable for surveillance of antibiotic resistance in GBS.

Anti-Bacterial Agents↗

PubMed2006

Reverse line blot assay for direct identification of seven Streptococcus agalactiae major surface protein antigen genes.

We developed a multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB) to detect the genes encoding members of the family of variable surface-localized proteins of Streptococcus agalactiae (group B streptococcus [GBS]), namely, Bca (Calpha), Rib, Epsilon (Epsilon/Alp1/Alp5), Alp2, Alp3, and Alp4, and the immunoglobulin A binding protein, Bac (Cbeta). We used the assay to identify these genes in a collection of well-characterized GBS isolates and reference strains. The results showed that mPCR/RLB avoids the common problems of cross-reaction and nontypability associated with protein typing using antisera. It is as sensitive as, but more practical than, separate gene-specific PCRs and would be suitable for large molecular epidemiological studies of GBS.

Antigens, Bacterial↗

PubMed2006

Use of PCR and reverse line blot hybridization macroarray based on 16S-23S rRNA gene internal transcribed spacer sequences for rapid identification of 34 mycobacterium species.

The aim of this study was to develop a PCR and reverse line blot hybridization (PCR-RLB) macroarray assay based on 16S-23S rRNA gene internal transcribed spacer sequences for the identification and differentiation of 34 mycobacterial species or subspecies. The performance of the PCR-RLB assay was assessed and validated by using 78 reference strains belonging to 55 Mycobacterium species, 219 clinical isolates which had been identified as mycobacteria by high-performance liquid chromatography or gas chromatography, three skin biopsy specimens from patients with suspected leprosy which had been shown to contain acid-fast bacilli, and isolates of 14 nonmycobacterial species. All mycobacteria were amplified in the PCR and hybridized with a genus-specific probe (probe MYC). The 34 species-specific probes designed in this study hybridized only with the corresponding Mycobacterium species. The mycobacterial PCR-RLB assay is an efficient tool for the identification of clinical isolates of mycobacteria; it can reliably identify mixed mycobacterial cultures and M. leprae in skin biopsy specimens.

DNA, Ribosomal Spacer↗

PubMed2006

Evaluation of a multiplex PCR-based reverse line blot-hybridization assay for identification of serotype and surface protein antigens of Streptococcus agalactiae.

A 33-primer multiplex PCR-based reverse line blot (mPCR/RLB) assay was developed to identify Streptococcus agalactiae serotypes and surface protein antigen genes simultaneously. It was evaluated by using 551 clinical isolates. The mPCR/RLB assay was more sensitive than conventional serotyping, especially for protein antigen typing, but otherwise the results correlated well.

Antigens, Bacterial↗

PubMed2006

Simultaneous detection and identification of Candida, Aspergillus, and Cryptococcus species by reverse line blot hybridization.

We report on a reverse line blot (RLB) assay, utilizing fungal species-specific oligonucleotide probes to hybridize with internal transcribed spacer 2 region sequences amplified using a nested panfungal PCR. Reference and clinical strains of 16 Candida species (116 strains), Cryptococcus neoformans (five strains of Cryptococcus neoformans var. neoformans, five strains of Cryptococcus neoformans var. grubii, and six strains of Cryptococcus gatti), and five Aspergillus species (68 strains) were all correctly identified by the RLB assay. Additional fungal species (16 species and 26 strains) not represented on the assay did not exhibit cross-hybridization with the oligonucleotide probes. In simulated clinical specimens, the sensitivity of the assay for Candida spp. and Aspergillus spp. was 10(0.5) cells/ml and 10(2) conidia/ml, respectively. This assay allows sensitive and specific simultaneous detection and identification of a broad range of fungal pathogens.

Aspergillus↗

PubMed2006

Use of PCR and reverse line blot hybridization assay for rapid simultaneous detection and serovar identification of Chlamydia trachomatis.

The aim of this study was to develop and evaluate multiplex and nested PCR-reverse line blot (RLB) hybridization assays for detection and serovar identification of Chlamydia trachomatis. Two sets of primers targeting the VD2 region of the omp1 gene and one set targeting the cryptic plasmid were designed for use in multiplex (both targets) and nested PCR (omp1 only). For the RLB assay, labeled omp1 amplicons were hybridized to a membrane containing probes specific for 15 C. trachomatis serovars. The assays were used to test 429 clinical specimens, which had been previously tested for C. trachomatis using the COBAS AMPLICOR system. Specimens were tested without knowledge of the COBAS AMPLICOR result. Of 205 specimens that were positive by COBAS AMPLICOR, 201 (98%) were positive by multiplex PCR-RLB and 188 (92%) were also positive by omp1 nested PCR-RLB. In addition, three of 224 COBAS AMPLICOR-negative specimens were positive by omp1 nested PCR-RLB. One hundred sixty-six of 191 (87%) specimens in which C. trachomatis serovars were identified contained only one serovar and 25 (13%) contained two or three serovars. Serovars D, E, and F were found in 31 (16%), 83 (43%), and 51 (27%) specimens, respectively. Serovar E (41%) was the most commonly identified single serovar. Serovars J and K were found alone uncommonly (<2% each), but 18 of 25 (72%) specimens with multiple C. trachomatis serovars contained one or both (10 specimens) of these serovars. The nested (ompI) PCR-RLB is a specific and sensitive method for simultaneous detection and serovar identification of C. trachomatis, which can reliably identify mixed C. trachomatis serovars. It is suitable for use in epidemiological studies.

Bacterial Typing Techniques↗

PubMed2006

Use of a serotype-specific DNA microarray for identification of group B Streptococcus (Streptococcus agalactiae).

Group B Streptococcus (GBS; Streptococcus agalactiae) is an important cause of sepsis and meningitis. Nine GBS serotypes, based on capsular polysaccharide (CPS) antigens, have been described. Their distribution varies worldwide and needs to be monitored to understand the epidemiology of GBS disease and inform the development of vaccines. In this study, we sequenced cpsH of GBS serotype II (cpsHII) and compared it with that of the other eight serotypes to identify serotype-specific regions. We then developed a DNA microarray based on the cpsH gene and used it to test 88 GBS isolates-9 serotype reference strains and 79 clinical isolates-and 7 other bacterial and fungal species which are commonly present in the vagina flora. The microarray was shown to be specific and reproducible. This is the first report of a microarray which can identify the nine GBS serotypes. The use of a microarray has advantages over traditional serotyping methods and will be of practical value in both reference and diagnostic laboratories.

Bacterial Capsules↗

PubMed2006

Multiplex PCR-based reverse line blot hybridization assay to identify 23 Streptococcus pneumoniae polysaccharide vaccine serotypes.

We developed a multiplex PCR-based reverse line blot assay to identify 23 pneumococcal serotypes represented in the polysaccharide vaccine, using 334 well-characterized isolates, representing all 90 serotypes, and 268 "unknowns." The assay identified all target serotypes, but 11, which cross-react with 1 to 4 nonvaccine serotypes, could be distinguished using serotype-specific antisera.

Antibodies, Bacterial↗

PubMed2005

A molecular-capsular-type prediction system for 90 Streptococcus pneumoniae serotypes using partial cpsA-cpsB sequencing and wzy- or wzx-specific PCR.

In a previous study, a molecular capsular type (MCT) prediction system for 51 Streptococcus pneumoniae serotypes was developed based on a combination of partial cpsA-cpsB sequencing and serotype(s)/group(s)-specific PCR. In this study, another 169 S. pneumoniae isolates were added to the existing database of 427 isolates, including representatives of all 39 serotypes not previously studied. In addition to the authors' own limited sequence data for all 90 serotypes, cpsA-cpsB sequence data published by the S. pneumoniae capsular loci-sequencing group (http://www.sanger.ac.uk/Projects/S_pneumoniae/CPS/) at the Sanger Institute or available from GenBank were incorporated into the database. All serotypes, except 25A, were represented by at least two isolates. The number of sequence types identified was 138, of which 110 corresponded to single conventional serotypes (CSs); of these, 57 were represented by two or more isolates. Twenty-six sequence types were shared by between two and four CSs. To resolve these shared cpsA-cpsB sequence types and increase the discriminatory power of our system, the genes encoding the capsular polysaccharide flippase (wzx) and polymerase (wzy) were annotated and 24 new serotype(s)/group(s)-specific PCRs targeting wzy and two targeting wzx were designed. Using both cpsA-cpsB sequencing and wzx/wzy PCR, MCT correctly predicted the CSs of 516 (73 %) and the serogroup of an additional 155 (22 %) of the 708 isolates evaluated. For 5 % of isolates, MCT could not distinguish between members of five serotype pairs (37 isolates) containing members of different serogroups. Although further study of the relationship between MCT and CS is needed, this system now allows serotype or serogroup identification of 95 % of S. pneumoniae isolates.

Bacterial Capsules↗

PubMed2005

Simultaneous detection and serotype identification of Streptococcus agalactiae using multiplex PCR and reverse line blot hybridization.

Streptococcus agalactiae (group B streptococcus, GBS) is an important cause of sepsis in neonates and their mothers, and the elderly and immunocompromised patients. Ongoing surveillance to monitor GBS serotype distribution is needed to guide the development and assess the feasibility of GBS conjugate vaccines. The authors previously developed a molecular serotype identification method based on serotype-specific PCR and partial sequencing of cps genes. In this study, a novel 10-primer pair multiplex PCR and reverse line blot (mPCR/RLB) hybridization assay was developed for simultaneous detection and serotype identification of all nine GBS serotypes. For all 316 GBS isolates tested the mPCR/RLB results corresponded with those of conventional serotyping and individual serotype-specific PCR, and the method was more convenient and practical than either alternative.

Base Sequence↗

PubMed2005

Comparison of a 3-set genotyping system with multilocus sequence typing for Streptococcus agalactiae (Group B Streptococcus).

Group B streptococcus (GBS; Streptococcus agalactiae) is the most common cause of neonatal and obstetric sepsis and is an increasingly important cause of septicemia in elderly individuals and immunocompromised patients. Epidemiological studies of GBS infections require comprehensive typing systems that provide information about variable characteristics, such as antigenic type, virulence, or antibiotic resistance, as well as the "backbone" structure or the genetic lineage of isolates. We have previously described a 3-set genotyping system that identifies the molecular serotype (MS) or molecular serosubtype (msst), the protein gene profile, and the presence of several mobile genetic elements (F. Kong, D. Martin, G. James, and G. L. Gilbert, J. Med. Microbiol. 52:337-344, 2003). In this study, 83 clinical GBS isolates which had been previously studied by multilocus sequence typing (MLST) (N. Jones, J. F. Bohnsack, S. Takahashi, K. A. Oliver, M. S. Chan, F. Kunst, P. Glaser, C. Rusniok, D. W. Crook, R. M. Harding, N. Bisharat, and B. G. Spratt, J. Clin. Microbiol. 41:2530-2536, 2003) were examined by using the 3-set genotyping system. Genotypes were assigned to five isolates that were nontypeable by conventional serotyping. There were 27 "3-set" genotypes, 24 multilocus sequence types (STs), and 35 unique combinations (or strains), of which the 4 most common, msst III-2 (ST-17), msst III-1 (ST-19), Ia-1 (ST-23), and V-1 (ST-1), accounted for more than 60% of isolates. The 83 isolates were grouped into seven clusters, with a good correlation between the multilocus STs and the genotypes. The combination of 3-set genotyping and MLST adds discriminatory power to strain typing of GBS, which will be useful for future studies of the epidemiology and pathogenesis of GBS disease.

Bacterial Proteins↗

PubMed2004

Postgenomic taxonomy of human ureaplasmas -- a case study based on multiple gene sequences.

In 2000, the full genome sequence of Ureaplasma parvum (previously known as Ureaplasma urealyticum) serovar 3 was released. In 2002, after prolonged debate, it was agreed that the former U. urealyticum should be divided into two species -- U. parvum and U. urealyticum. To provide additional support for this decision and improve our understanding of the relationship between these two species, the authors studied four 'core' genes or gene clusters in ATCC reference strains of all 14 serovars of U. parvum and U. urealyticum. These 'core' regions were the rRNA gene clusters, the EF-Tu genes (tuf), urease gene clusters and multiple-banded antigen genes (mba). The known U. parvum genome sequences (GenBank accession no. NC_002162) were used as reference. DNA insertions and deletions (indels) were found in all of the gene regions studied, except tuf, but they were found only between, not within, the two species. An incidental finding was that there was inter-copy heterogeneity for rRNA gene cluster sequences. Sequence analysis (sequence heterogeneity and especially indels) of all four selected targets consistently supported the separation of human ureaplasmas into two species. Except for multiple-banded antigen, there was less heterogeneity in amino acid sequences of proteins, between species, than in the nucleic acid sequences of the corresponding genes. The degrees of heterogeneity at the 5' end of the species-specific regions of multiple-banded antigen were almost identical for both amino acid and nucleotide sequences. Analysis of the authors' results provided an interesting case study to help resolve some common problems in the use of sequence data to infer phylogenetic relationships and support taxonomic changes. It is recommended that, to avoid confusion, the new nomenclature be used for human ureaplasmas in future publications.

Bacterial Proteins↗