Diagnosis of Arboleda-Tham syndrome by whole genome sequencing in an Asian boy with severe developmental delay.

Diagnosis of a 9-month-old boy brought to our genetics clinic with chief complaints of developmental delay (DD), failure to thrive, microcephaly, trunk hypotonia and hypertonia of the extremities. Multiple congenital defects but no significant syndromes or diseases were impressed. The chromosomal analysis and array comparative genomic hybridization (aCGH) revealed no significant pathogenic changes. Whole Genome Sequencing (WGS) identified a p.Glu1139fs de novo mutation of the KAT6A gene. The patient's phenotype was consistent clinically with Arboleda-Tham syndrome (ARTHS). Reviewing the literature showed that this is the first patient in Taiwan detected by WGS and that it involves a novel mutation. Comparing the highly variable clinical presentations of this syndrome with our patient, this boy's features and severe developmental defects seem to be due to a late-truncating mutation at the carboxyl end of the KAT6A protein. Our study demonstrates the power of WGS to confirm a diagnosis within 4 weeks for this rare condition.

Increased mcr-1 in pathogenic Escherichia coli from diseased swine, Taiwan.

PURPOSE: To investigate the increasing prevalence of colistin resistance in animal Escherichia coli isolates and their mcr-1-carrying plasmids, especially those shared by isolates from human and retail meats. METHODS: E. coli from diseased swine and poultry recovered between 2012 and 2016 were studied. Susceptibility was determined using broth microdilution method or Vitek II system. Fifty-eight mcr-1-positive isolates were randomly selected for further testing, including pulsed-field gel electrophoresis (PFGE) for clonality determination, S1- or I-CeuI-PFGE and Southern blotting for localization of mcr-1, and conjugation for transmissibility. Whole genome sequencing (WGS) was performed for the genetic structure of plasmids. RESULTS: Among the 1234 E. coli isolates from diseased swine, colistin resistance increased from 14.6% (14/96) in 2012 to 43.8% (63/144) in 2016 with a paralleled increase in mcr-1-positivity from 12.5% (12/96) to 33.3% (48/144) in 2016 (P < 0.001), but no such significant increase was observed in the 489 diseased poultry isolates. The 58 clonally diverse isolates were resistant to multiple antibiotics commonly used in humans. PFGE and Southern blotting revealed one chromosome-located mcr-1 and various mcr-1-borne plasmids, all of which were transferable to E. coli J53. WGS revealed that the prevalent 60-kb and 30-kb plasmid was the same as pHNSHP45 in China and pESTMCR in Estonia, respectively, which were both present in human isolates in Taiwan. CONCLUSION: Increased colistin resistance and mcr-1-positivity in diseased swine isolates and detection of mcr-1 carried on similar plasmids in isolates from animals and humans stress the need to monitor resistance in both sectors.

Lineage-specific SNPs for genotyping of Mycobacterium tuberculosis clinical isolates.

Tuberculosis (TB) is a severe infectious disease worldwide. Genetic variation of the causative agent, Mycobacterium tuberculosis (MTB), determines the outcomes of infection and anti-TB treatment. Until recently, there has been no effective and convenient way for classifying clinical isolates based on the DNA sequences of the divergent lineages of MTB infecting human populations. Here, we identified single nucleotide polymorphisms (SNPs) of six representative strains from Taiwan by whole-genome sequencing and comparing the results to the sequence of the H37Rv reference strain. One hundred and ten SNPs, each unique to one of the six strains, were used to genotype 150 additional isolates by applying DNA mass spectrometry. Lineage-specific SNPs were identified that could distinguish the major lineages of the clinical isolates. A subset including 32 SNPs was found to be sufficient to type four major groups of MTB isolates in Taiwan (ancient Beijing, modern Beijing, East African-Indian, and Latin-American Mediterranean). However, there was high genetic homozygosity within the Euro-American lineage, which included spoligotype-classified Haarlem and T strains. By whole-genome sequencing of 12 representative Euro-American isolates, we identified multiple subtype-specific SNPs which allowed us to distinguish two major branches within the Euro-American lineage.

Copy Number Change of the NDM-1 sequence in a multidrug-resistant Klebsiella pneumoniae clinical isolate.

The genetic features of the antimicrobial resistance of a multidrug resistant Klebsiella pneumoniae strain harboring bla NDM-1 were investigated to increase our understanding of the evolution of NDM-1. The strain, KPX, came from a Taiwanese patient with a hospitalization history in New Delhi. Complete DNA sequencing was performed; and the genes responsible for antimicrobial resistance were systematically examined and isolated by library screening. KPX harbored two resistance plasmids, pKPX-1 and pKPX-2, which are 250-kb and 141-kb in size, respectively, with bla NDM-1 present on pKPX-1. The plasmid pKPX-1 contained genes associated with the IncR and IncF groups, while pKPX-2 belonged to the IncF family. Each plasmid carried multiple antimicrobial resistance genetic determinants. The gene responsible for resistance to carbapenems was found on pKPX-1 and that for resistance to aztreonam was found on pKPX-2. To our surprise, we discovered that bla NDM-1 exists on pKPX-1 as multiple copies in the form of tandem repeats. Amplification of bla NDM-1 was found to occur by duplication of an 8.6-kb unit, with the copy number of the repeat varying from colony to colony. This repeat sequence is identical to that of the pNDM-MAR except for two base substitutions. The copy number of bla NDM-1 of colonies under different conditions was assessed by Southern blotting and quantitative PCR. The bla NDM-1 sequence was maintained in the presence of the antimicrobial selection; however, removal of antimicrobial selection led to the emergence of susceptible bacterial populations with a reduced copy number or even the complete loss of the bla NDM-1 sequence. The dynamic nature of the NDM-1 sequence provides a strong argument for judicious use of the broad-spectrum antimicrobials in order to reduce the development and spread of antimicrobial resistance among pathogens.

Complete genome sequence of Klebsiella oxytoca E718, a New Delhi metallo-ß-lactamase-1-producing nosocomial strain.

We report the complete genome sequence of Klebsiella oxytoca E718, a New Delhi metallo-ß-lactamase-1 (NDM-1)-producing strain isolated from a renal transplant patient. The genome contains a 6,097,032-bp chromosome and two multidrug resistance plasmids with sizes of 324,906 bp and 110,781 bp.

Complexity of the Mycoplasma fermentans M64 genome and metabolic essentiality and diversity among mycoplasmas.

Recently, the genomes of two Mycoplasma fermentans strains, namely M64 and JER, have been completely sequenced. Gross comparison indicated that the genome of M64 is significantly bigger than the other strain and the difference is mainly contributed by the repetitive sequences including seven families of simple and complex transposable elements ranging from 973 to 23,778 bps. Analysis of these repeats resulted in the identification of a new distinct family of Integrative Conjugal Elements of M. fermentans, designated as ICEF-III. Using the concept of "reaction connectivity", the metabolic capabilities in M. fermentans manifested by the complete and partial connected biomodules were revealed. A comparison of the reported M. pulmonis, M. arthritidis, M. genitalium, B. subtilis, and E. coli essential genes and the genes predicted from the M64 genome indicated that more than 73% of the Mycoplasmas essential genes are preserved in M. fermentans. Further examination of the highly and partly connected reactions by a novel combinatorial phylogenetic tree, metabolic network, and essential gene analysis indicated that some of the pathways (e.g. purine and pyrimidine metabolisms) with partial connected reactions may be important for the conversions of intermediate metabolites. Taken together, in light of systems and network analyses, the diversity among the Mycoplasma species was manifested on the variations of their limited metabolic abilities during evolution.

Sequencing and comparative genome analysis of two pathogenic Streptococcus gallolyticus subspecies: genome plasticity, adaptation and virulence.

Streptococcus gallolyticus infections in humans are often associated with bacteremia, infective endocarditis and colon cancers. The disease manifestations are different depending on the subspecies of S. gallolyticus causing the infection. Here, we present the complete genomes of S. gallolyticus ATCC 43143 (biotype I) and S. pasteurianus ATCC 43144 (biotype II.2). The genomic differences between the two biotypes were characterized with comparative genomic analyses. The chromosome of ATCC 43143 and ATCC 43144 are 2,36 and 2,10 Mb in length and encode 2246 and 1869 CDS respectively. The organization and genomic contents of both genomes were most similar to the recently published S. gallolyticus UCN34, where 2073 (92%) and 1607 (86%) of the ATCC 43143 and ATCC 43144 CDS were conserved in UCN34 respectively. There are around 600 CDS conserved in all Streptococcus genomes, indicating the Streptococcus genus has a small core-genome (constitute around 30% of total CDS) and substantial evolutionary plasticity. We identified eight and five regions of genome plasticity in ATCC 43143 and ATCC 43144 respectively. Within these regions, several proteins were recognized to contribute to the fitness and virulence of each of the two subspecies. We have also predicted putative cell-surface associated proteins that could play a role in adherence to host tissues, leading to persistent infections causing sub-acute and chronic diseases in humans. This study showed evidence that the S. gallolyticus still possesses genes making it suitable in a rumen environment, whereas the ability for S. pasteurianus to live in rumen is reduced. The genome heterogeneity and genetic diversity among the two biotypes, especially membrane and lipoproteins, most likely contribute to the differences in the pathogenesis of the two S. gallolyticus biotypes and the type of disease an infected patient eventually develops.

Genome sequence of the repetitive-sequence-rich Mycoplasma fermentans strain M64.

Mycoplasma fermentans is a microorganism commonly found in the genitourinary and respiratory tracts of healthy individuals and AIDS patients. The complete genome of the repetitive-sequence-rich M. fermentans strain M64 is reported here. Comparative genomics analysis revealed dramatic differences in genome size between this strain and the recently completely sequenced JER strain.

Genetic diversity of capsular polysaccharide biosynthesis in Klebsiella pneumoniae clinical isolates.

Klebsiella pneumoniae is an enteric pathogen causing community-acquired and hospital-acquired infections in humans. Epidemiological studies have revealed significant diversity in capsular polysaccharide (CPS) type and clinical manifestation of K. pneumoniae infection in different geographical areas of the world. We have sequenced the capsular polysaccharide synthesis (cps) region of seven clinical isolates and compared the sequences with the publicly available cps sequence data of five strains: NTUH-K2044 (K1 serotype), Chedid (K2 serotype), MGH78578 (K52 serotype), A1142 (K57 serotype) and A1517. Among all strains, six genes at the 5' end of the cps clusters that encode proteins for CPS transportation and processing at the bacterial surface are highly similar to each other. The central region of the cps gene clusters, which encodes proteins for polymerization and assembly of the CPS subunits, is highly divergent. Based on the collected sequence, we found that either the wbaP gene or the wcaJ gene exists in a given K. pneumoniae strain, suggesting that there is a major difference in the CPS biosynthesis pathway and that the K. pneumoniae strains can be classified into at least two distinct groups. All isolates contain gnd, encoding gluconate-6-phosphate dehydrogenase, at the 3' end of the cps gene clusters. The rmlBADC genes were found in CPS K9-positive, K14-positive and K52-positive strains, while manC and manB were found in K1, K2, K5, K14, K62 and two undefined strains. Our data indicate that, while overall genomic organization is similar between different pathogenic K. pneumoniae strains, the genetic variation of the sugar moiety and polysaccharide linkage generate the diversity in CPS molecules that could help evade host immune attack.

Genomic diversity of citrate fermentation in Klebsiella pneumoniae.

BACKGROUND: It has long been recognized that Klebsiella pneumoniae can grow anaerobically on citrate. Genes responsible for citrate fermentation of K. pneumoniae were known to be located in a 13-kb gene cluster on the chromosome. By whole genome comparison of the available K. pneumoniae sequences (MGH 78578, 342, and NTUH-K2044), however, we discovered that the fermentation gene cluster was present in MGH 78578 and 342, but absent in NTUH-K2044. In the present study, the previously unknown genome diversity of citrate fermentation among K. pneumoniae clinical isolates was investigated. RESULTS: Using a genomic microarray containing probe sequences from multiple K. pneumoniae strains, we investigated genetic diversity among K. pneumoniae clinical isolates and found that a genomic region containing the citrate fermentation genes was not universally present in all strains. We confirmed by PCR analysis that the gene cluster was detectable in about half of the strains tested. To demonstrate the metabolic function of the genomic region, anaerobic growth of K. pneumoniae in artificial urine medium (AUM) was examined for ten strains with different clinical histories and genomic backgrounds, and the citrate fermentation potential was found correlated with the genomic region. PCR detection of the genomic region yielded high positive rates among a variety of clinical isolates collected from urine, blood, wound infection, and pneumonia. Conserved genetic organizations in the vicinity of the citrate fermentation gene clusters among K. pneumoniae, Salmonella enterica, and Escherichia coli suggest that the 13-kb genomic region were not independently acquired. CONCLUSION: Not all, but nearly half of the K. pneumoniae clinical isolates carry the genes responsible for anaerobic growth on citrate. Genomic variation of citrate fermentation genes in K. pneumoniae may contribute to metabolic diversity and adaptation to variable nutrient conditions in different environments.

Genome sequencing and comparative analysis of Klebsiella pneumoniae NTUH-K2044, a strain causing liver abscess and meningitis.

Nosocomial infections caused by antibiotic-resistant Klebsiella pneumoniae are emerging as a major health problem worldwide, while community-acquired K. pneumoniae infections present with a range of diverse clinical pictures in different geographic areas. In particular, an invasive form of K. pneumoniae that causes liver abscesses was first observed in Asia and then was found worldwide. We are interested in how differences in gene content of the same species result in different diseases. Thus, we sequenced the whole genome of K. pneumoniae NTUH-K2044, which was isolated from a patient with liver abscess and meningitis, and analyzed differences compared to strain MGH 78578, which was isolated from a patient with pneumonia. Six major types of differences were found in gene clusters that included an integrative and conjugative element, clusters involved in citrate fermentation, lipopolysaccharide synthesis, and capsular polysaccharide synthesis, phage-related insertions, and a cluster containing fimbria-related genes. We also conducted comparative genomic hybridization with 15 K. pneumoniae isolates obtained from community-acquired or nosocomial infections using tiling probes for the NTUH-K2044 genome. Hierarchical clustering revealed three major groups of genomic insertion-deletion patterns that correlate with the strains' clinical features, antimicrobial susceptibilities, and virulence phenotypes with mice. Here we report the whole-genome sequence of K. pneumoniae NTUH-K2044 and describe evidence showing significant genomic diversity and sequence acquisition among K. pneumoniae pathogenic strains. Our findings support the hypothesis that these factors are responsible for the changes that have occurred in the disease profile over time.

Mobilization of qnrB2 and ISCR1 in plasmids.

The DNA sequences of two IncHI2 plasmids, pEC-IMP and pEC-IMPQ, from metallo-beta-lactamase-producing Enterobacter cloacae clinical isolates were determined. The two conjugative plasmids are almost identical, but pEC-IMPQ carries an additional segment containing an orf513 (ISCR1), a truncated 3' conserved sequence, and a qnrB2. Comparative analyses provide support for the proposed ISCR1-mediated gene mobilization.

Dysregulation of GIMAP genes in non-small cell lung cancer.

The GIMAP (GTPase of the immunity-associated protein) gene family includes seven functional members residing on human chromosome 7. GIMAP genes encode GTP-binding proteins that share a unique primary structure and whose function is largely unknown. However, gene ablation studies reveal that Gimap4 plays an important role in regulating the apoptosis of T cells. In a pilot microarray analysis on six cases of non-small cell lung cancer (NSCLC), we discovered that the expression of GIMAP family members, but not the neighboring non-GIMAP genes, was uniformly lower in the tumor tissues, compared to that in the adjacent nontumor tissues. This finding was subsequently confirmed by quantitative PCR assays in a total of twenty NSCLCs, and we found that GIMAP6 and GIMAP8 showed striking reduction of gene expression in the tumors. In contrast, GIMAP8 mRNA level was abnormally elevated in the adjacent nontumor tissues as compared to that in the control lung tissues. Such reciprocal expression of GIMAPs suggests that this unique gene family might contribute to the pathogenesis of and immune reactions to NSCLC.

Comparative genome analysis of Vibrio vulnificus, a marine pathogen.

The halophile Vibrio vulnificus is an etiologic agent of human mortality from seafood-borne infections. We applied whole-genome sequencing and comparative analysis to investigate the evolution of this pathogen. The genome of biotype 1 strain, V. vulnificus YJ016, was sequenced and includes two chromosomes of estimated 3377 kbp and 1857 kbp in size, and a plasmid of 48,508 bp. A super-integron (SI) was identified, and the SI region spans 139 kbp and contains 188 gene cassettes. In contrast to non-SI sequences, the captured gene cassettes are unique for any given Vibrio species and are highly variable among V. vulnificus strains. Multiple rearrangements were found when comparing the 5.3-Mbp V. vulnificus YJ016 genome and the 4.0-Mbp V. cholerae El Tor N16961 genome. The organization of gene clusters of capsular polysaccharide, iron metabolism, and RTX toxin showed distinct genetic features of V. vulnificus and V. cholerae. The content of the V. vulnificus genome contained gene duplications and evidence of horizontal transfer, allowing for genetic diversity and function in the marine environment. The genomic information obtained in this study can be applied to monitoring vibrio infections and identifying virulence genes in V. vulnificus.

Potential pool of non-heart-beating kidney donors in Taiwan: chart review in a regional hospital.

The annual number of renal transplantations performed in Taiwan is strictly limited by the availability of donor organs. One way to expand the donor pool is the use of non-heart-beating (NHB) donors. This study evaluated the potential number of NHB kidney-donors in Taiwan using a retrospective death chart review from a regional hospital in patients who died between January 1 and December 31, 1999. Exclusion criteria were extremities of age, systemic infection, malignancies other than primary brain tumor, uncontrolled diabetes mellitus, hypertension, kidney disease, or deaths with incomplete records. Detailed biomedical data of potential donors were collected. Of the 840 in-hospital deaths, 258 were in patients aged 3 to 65 years. Among these patients, 52 (6%) did not meet any exclusion criteria and were classified as potential kidney donors. Of these 52 patients, eight (1%) were classified as very suitable donors, defined as no diabetes mellitus, no hypertension, good renal function (serum creatinine < 1.3 mg/dL), age less than 50 years, and death in the intensive care unit or emergency service. Twenty-five of the remaining 44 sub-optimal potential donors who did not meet the criteria of very suitable were classified as having high potential to be donors by a combined scoring system (sum score = 5-6). The results suggest that there is a considerable number of potential NHB kidney-donors in Taiwan. Programs to advocate use of NHB kidney donors and education of the public and transplant professionals might significantly increase the number of kidneys available for transplantation in Taiwan.