Search

Since April 2022, waves of Omicron cases have surfaced in Taiwan and spread throughout the island. Using high-throughput sequencing of the SARS-CoV-2 genome, we have analyzed 2405 PCR-positive swab samples from 2339 individuals and identified the Omicron BA.2.3.7 variant as a major lineage within the recent community outbreak in Taiwan.

Aeromonas dhakensis is an emerging human pathogen which causes fast and severe infections worldwide. Under the gradual pressure of lacking useful antibiotics, finding a new strategy against A. dhakensis infection is urgent. To understand its pathogenesis, we created an A. dhakensis AAK1 mini-Tn10 transposon library to study the mechanism of A. dhakensis infection. By using a Caenorhabditis elegans model, we established a screening platform for the purpose of identifying attenuated mutants. The uvrY mutant, which conferred the most attenuated toxicity toward C. elegans, was identified. The uvrY mutant was also less virulent in C2C12 fibroblast and mice models, in line with in vitro results. To further elucidate the mechanism of UvrY in controlling the toxicity in A. dhakensis, we conducted a transcriptomic analysis. The RNAseq results showed that the expression of a unique hemolysin ahh1 and other virulence factors were regulated by UvrY. Complementation of Ahh1, one of the most important virulence factors, rescued the pore-formation phenotype of uvrY mutant in C. elegans; however, complementation of ahh1 endogenous promoter-driven ahh1 could not produce Ahh1 and rescue the virulence in the uvrY mutant. These findings suggest that UvrY is required for the expression of Ahh1 in A. dhakensis. Taken together, our results suggested that UvrY controls several different virulence factors and is required for the full virulence of A. dhakensis. The two-component regulator UvrY therefore a potential therapeutic target which is worthy of further study.

Most bacteria possess alcohol dehydrogenase (ADH) genes (Adh genes) to mitigate alcohol toxicity, but these genes have functions beyond alcohol degradation. Previous research has shown that ADH can modulate quorum sensing in Acinetobacter baumannii, a rising opportunistic pathogen. However, the number and nature of Adh genes in A. baumannii have not yet been fully characterized. We identified seven alcohol dehydrogenases (NAD+-ADHs) from A. baumannii ATCC 19606, and examined the roles of three iron-containing ADHs, ADH3, ADH4, and ADH6. Marker-less mutation was used to generate Adh3, Adh4, and Adh6 single, double, and triple mutants. Disrupted Adh4 mutants failed to grow in ethanol-, 1-butanol-, or 1-propanol-containing mediums, and recombinant ADH4 exhibited strongest activity against ethanol. Stress resistance assays with inorganic and organic hydroperoxides showed that Adh3 and Adh6 were key to oxidative stress resistance. Virulence assays performed on the Galleria mellonella model organism revealed that Adh4 mutants had comparable virulence to wild-type, while Adh3 and Adh6 mutants had reduced virulence. The results suggest that ADH4 is primarily involved in alcohol metabolism, while ADH3 and ADH6 are key to stress resistance and virulence. Further investigation into the roles of other ADHs in A. baumannii is warranted.

In this study, we show that Acinetobacter baumannii ATCC 19606 harbors two sets of ohrR-ohr genes, respectively encoded in chromosomal DNA and a pMAC plasmid. We found no significant difference in organic hydroperoxide (OHP) resistance between strains with or without pMAC. However, a disk diffusion assay conducted by exposing wild-type, ∆ohrR-C, C represented gene on chromosome, or ∆ohr-C single mutants, or ∆ohrR-C∆ohr-C double mutants to tert-butyl hydroperoxide (tBHP) found that the ohrR-p-ohr-p genes, p represented genes on pMAC plasmid, may be able to complement the function of their chromosomal counterparts. Interestingly, ∆ohr-C single mutants generated in A. baumannii ATCC 17978, which does not harbor pMAC, demonstrated delayed exponential growth and loss of viability following exposure to 135 μg of tBHP. In a survival assay conducted with Galleria mellonella larvae, these mutants demonstrated almost complete loss of virulence. Via an electrophoretic mobility shift assay (EMSA), we found that OhrR-C was able to bind to the promoter regions of both chromosomal and pMAC ohr-p genes, but with varying affinity. A gain-of-function assay conducted in Escherichia coli showed that OhrR-C was not only capable of suppressing transformed ohr-C genes but may also repress endogenous enzymes. Taken together, our findings suggest that chromosomal ohrR-C-ohr-C genes act as the major system in protecting A. baumannii ATCC 19606 from OHP stresses, but the ohrR-p-ohr-p genes on pMAC can provide a supplementary protective effect, and the interaction between these genes may affect other aspects of bacterial viability, such as growth and virulence.

This paper reports a comparative study on characterization and thermal shock behavior of air plasma sprayed Al2O3-13wt.%TiO2 coatings using two kinds of raw materials, i.e. nanostructural and micro-structural (traditional) feedstock powders. The characterization, before and after thermal shock test, was carried out using micro-Vickers hardness tester, XRD and SEM. The thermal shock test was carried out using a water quenching method by employing cyclic heat treatment between ambient temperature and 650°C in air. The results showed that in spite of having denser structure, the nanostructural coating showed hardness a little lower than the traditional one at both conditions of before and after thermal shock tests. However, the nanostructural coating showed very good thermal shock behaviour.

This study aimed to understand the effect of a different binder on fundamental properties and corrosion behavior of WC based HVOF coatings. Three kinds of coatings, each with a different binder, were prepared using commercial feedstock powders, i.e. WC-12Co, WC-10Co4Cr and WC-12Ni. Micro Vickers hardness tester, XRD and SEM were used. The corrosion performance was evaluated using a salt spray tester. It was found that the WC-10Co4Cr coating showed the highest corrosion resistance and superior hardness comparable to the WC-12Co coating.

A 269-kilobase conjugative plasmid, pK29, from a Klebsiella pneumoniae strain was sequenced. The plasmid harbors multiple antimicrobial resistance genes, including those encoding CMY-8 AmpC-type and CTX-M-3 extended-spectrum β-lactamases in the common backbone of IncHI2 plasmids. Mechanisms for dissemination of the resistance genes are highlighted in comparative genomic analyses.

The bacterium Xanthomonas campestris pathovar campestris (XCC) 17 is a local isolate that causes crucifer black rot disease in Taiwan. In this study, its proteome was separated using 2-DE and the well-resolved proteins were excised, trypsin digested, and analyzed by MS. Over 400 protein spots were analyzed and 281 proteins were identified by searching the MS or MS/MS spectra against the proteome database of the closely related XCC ATCC 33913. Functional categorization of the identified proteins matched 141 (50%) proteins to 81 metabolic pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. In addition, we performed a comparative proteome analysis of the pathogenic strain 17 and an avirulent strain 11A to reveal the virulence-related proteins. We detected 22 up-regulated proteins in strain 17 including the degrading enzymes EngXCA, HtrA, and PepA, which had been shown to have a role in pathogenesis in other bacteria, and an anti-host defense protein, Ohr. Thus, further functional studies of these up-regulated proteins with respect to their roles in XCC pathogenicity are suggested.

Fengycin, a lipopeptidic antibiotic, is synthesized nonribosomally by five fengycin synthetases (FenC, FenD, FenE, FenA, and FenB) in Bacillus subtilis F29-3. This work demonstrates that these fengycin synthetases interlock to form a chain, which coils into a 14.5-nm structure. In this chain, fengycin synthetases are linked in the order FenC-FenD-FenE-FenA-FenB by interactions between the C-terminal region of an upstream enzyme and the N-terminal region of its downstream partner enzyme, with their amino acid activation modules arranged colinearly with the amino acids in fengycin. This work also reveals that fengycin is synthesized on this fengycin synthetase chain, explaining how fengycin is synthesized efficiently and accurately. The results from this investigation demonstrate that forming a peptide synthetase complex is crucial to nonribosomal peptide synthesis.

Most of the published complete genome sequences of Helicobacter pylori strains are limited to clinical isolates associated with gastritis, peptic ulcers, or gastric cancer. The genome sequences of three H. pylori strains isolated from patients with gastric mucosa-associated lymphoid tissue (MALT) lymphoma are presented here to facilitate studies of H. pylori -associated MALT lymphomagenesis.

A plasmid containing the qnrS quinolone resistance determinant and the gene encoding the SHV-2 β-lactamase has been discovered from a clinical Klebsiella pneumoniae strain isolated in Taiwan. The complete 98-kb sequence of this plasmid, designated pK245, was determined by using a whole-genome shotgun approach. Transfer of pK245 conferred low-level resistance to fluoroquinolones in electroporant Escherichia coli epi300. The sequence of the immediate region surrounding qnrS in pK245 is nearly identical (>99% identity) to those of pAH0376 from Shigella flexneri and pINF5 from Salmonella enterica serovar Infantis, the two other qnr S-carrying plasmids reported to date, indicating a potential common origin. Other genes conferring resistance to aminoglycosides ( aacC2 , strA , and strB ), chloramphenicol ( catA2 ), sulfonamides ( sul 2), tetracycline ( tetD ), and trimethoprim ( dfrA14 ) were also detected in pK245. The dfrA14 gene is carried on a class I integron. Several features of this plasmid, including three separate regions containing putative replicons, a partitioning-control system, and a type II restriction modification system, suggest that it may be able to replicate and adapt in a variety of hosts. Although no critical conjugative genes were detected, multiple insertion sequence elements were found scattered throughout pK245, and these may facilitate the dissemination of the antimicrobial resistance determinants. We conclude that pK245 is a chimera which acquired its multiple antimicrobial resistance determinants horizontally from different sources. The identification of pK245 plasmid expands the repertoire of the coexistence of quinolone and extended-spectrum-β-lactam resistance determinants in plasmids carried by various species of the family Enterobacteriaceae in different countries.

The thermophilic bacterium Thermus aquaticus NTU103 harbors a 1965-bp plasmid, pTA103. Sequencing analysis revealed that pTA103 contains two open reading frames. One of the open reading frames (orf2) shares no significant homology with protein in the data bank. The other one has 50% similarity and 34% identity with RepA-like protein of pRm1132f, which is a rolling-circle replication (RCR) plasmid isolated from Sinorhizobium meliloti. S1 nuclease analysis demonstrated that pTA103 contains a single-stranded intermediate, confirming that pTA103 replicates via RCR mechanism. Sequence data also revealed putative double-stranded origin and single-stranded origin sites, indicating the importance of these cis elements in pTA103 replication.

The iac locus is involved in indole-3-acetic acid (IAA) catabolism in Acinetobacter baumannii. Nine structural genes of iac are transcribed in the same direction, whereas iacR, which encodes a MarR-type transcriptional regulator, is transcribed in the opposite direction. The IacA protein, which is encoded by the second structural gene of the iac locus, is expressed in an IAA-dependent manner. Here, we characterized gene expression from this locus in wild type A. baumannii and in an iacR mutant; this revealed that the iacH promoter is negatively regulated by IacR. The transcriptional site of iacH was determined by using 5′ rapid amplification of cDNA ends; one IacR-binding site was identified between positions −35 and +28 of the iacH promoter. Sequence analysis and an electrophoretic mobility shift assay indicated that recombinant IacR binds specifically to a sequence with dyad symmetry in the iacR-iacH overlapping promoters in the absence of IAA. In addition, a two-plasmid expression system in Escherichia coli showed that IAA probably serves as a ligand that binds to IacR and releases it from the iacH promoter, thereby allowing RNA polymerase to transcribe iac. Thus, iac is expressed in order to promote IAA degradation, whereas free IacR is required for iac repression. We conclude that IacR serves as a key regulator of IAA degradation in A. baumannii in the rhizosphere. These results provide new insights into the possible role of A. baumannii in the environment.

Aeromonas taiwanensis was first described in 2010 on the basis of one clinical wound isolate (strain LMG 24683 T = A2-50 T = CECT 7403 T ) from Taiwan. We present here the genome sequence of A. taiwanensis LMG 24683 T , which carries several genes encoding virulence determinants and Ambler class C and D β-lactamases.

Vibrio vulnificus, a gram-negative marine bacterium, has been recognized as an important pathogen of both humans and eels for decades. V. vulnificus infection is characterized by the rapid spread of this organism from intestine or skin into deeper tissue and even the bloodstream to result in septicemia and/or necrotic skin lesions. One of gene regions, region XII, was found to be associated with one of the two lineages of V. vulnificus divided based on the multilocus sequence typing (MLST) data of six housekeeping genes. The superintegron is unique to each Vibrio species, containing different kinds of gene cassettes. The functions of gene cassettes and open reading frames (ORFs) in superintegrons remain largely unknown. The publication of nucleotide sequences of two BT1 V. vulnificus genomes and the BT2 virulence plasmid has opened up opportunities for mining new virulence genes for mice and eels as well as developing new diagnostic methods. Moreover, the complete genome information can be applied to epidemiology study and food safety monitoring. Because of the high mortality rate of systemic infection with V. vulnificus, an effective vaccine against this organism is desired, particularly for individuals at high risk. Contaminated bivalve molluscan shellfish, including oysters, clams, and mussels, are major sources of per os infection by V. vulnificus. The vast information generated from the genomes of major representative Vibrio species has enabled comparative analysis and provided an opportunity to investigate the biology of this group of marine bacteria.

Acinetobacter baumannii harbours a gene cluster similar to the iac locus of Pseudomonas putida 1290, which can catabolize the plant hormone indole 3-acetic acid (IAA) as an energy source. However, there has been no evidence showing that IAA can be utilized by A. baumannii. This study showed that A. baumannii can grow in M9 minimal medium containing IAA as the sole carbon source. A mutagenesis study indicated that iacA, encoded in the iac locus of A. baumannii, is involved in the catabolism of IAA. As shown by western blotting analysis, the IacA protein was detected in A. baumannii grown in M9 minimal medium with IAA but not with pyruvate, suggesting that the expression of iacA is regulated by the presence of IAA. In vitro studies have shown that IacA can oxidize indole, an IAA-like molecule, converting it to indoxyl, which spontaneously dimerises to form indigo. In this study, we show that the crude extracts from either wild-type A. baumannii or Escherichia coli overexpressing IacA can oxidize IAA. These results imply that the iac gene cluster of A. baumannii is involved in IAA degradation and that the iacA gene is upregulated when cells encounter IAA in their native environments.

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.

Thermus thermophilus and Thermus aquaticus are thermophilic bacteria that are frequently found to attach to solid surfaces in hot springs to form biofilms. Uridine diphosphate (UDP)-galactose-4'-epimerase (GalE) is an enzyme that catalyzes the conversion of UDP-galactose to UDP-glucose, an important biochemical step in exopolysaccharide synthesis. We expressed GalE obtained from T. thermophilus HB8 in Escherichia coli and found that the enzyme is stable at 80 degrees C and can epimerize UDP-galactose to UDP-glucose and UDP-N-acetylgalactosamine (UDP-GalNAc) to UDP-N-acetylglucosamine (UDP-GlcNAc). Enzyme overexpression in T. thermophilus HB27 led to an increased capacity of biofilm production. Therefore, the galE gene is important to biofilm formation because of its involvement in epimerizing UDP-galactose and UDP-N-acetylgalactosamine for exopolysaccharide biosynthesis.

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.

Fengycin is a lipopeptidic antibiotic produced nonribosomally by Bacillus subtilis F29-3. Synthesis of this antibiotic requires five fengycin synthetases encoded by fenC, fenD, fenE, fenA, and fenB. In this study, we analyze the functions of the enzyme encoded by fenE, which contains two amino acid activation modules, FenE1 and FenE2. ATP-PP(i) exchange assay revealed that FenE1 activates l-Glu and FenE2 activates l-Ala, l-Val, and l-2-aminobutyric acid, indicating that FenE activates the fifth and the sixth amino acids in fengycin. Furthermore, l-Val is a better substrate than l-Ala for FenE2 in vitro, explaining why B. subtilis F29-3 normally produces twice as much of fengycin B than fengycin A, which contains d-Val and d-Ala at the sixth amino acid position, respectively. Results presented herein suggest that fengycin synthetase genes and amino acids in fengycin are colinear.

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.

Background The black tiger shrimp (Penaeus monodon) is one of the most important aquaculture species in the world, representing the crustacean lineage which possesses the greatest species diversity among marine invertebrates. Yet, we barely know anything about their genomic structure. To understand the organization and evolution of the P. monodon genome, a fosmid library consisting of 288,000 colonies and was constructed, equivalent to 5.3-fold coverage of the 2.17 Gb genome. Approximately 11.1 Mb of fosmid end sequences (FESs) from 20,926 non-redundant reads representing 0.45% of the P. monodon genome were obtained for repetitive and protein-coding sequence analyses. Results We found that microsatellite sequences were highly abundant in the P. monodon genome, comprising 8.3% of the total length. The density and the average length of microsatellites were evidently higher in comparison to those of other taxa. AT-rich microsatellite motifs, especially poly (AT) and poly (AAT), were the most abundant. High abundance of microsatellite sequences were also found in the transcribed regions. Furthermore, via self-BlastN analysis we identified 103 novel repetitive element families which were categorized into four groups, i.e., 33 WSSV-like repeats, 14 retrotransposons, 5 gene-like repeats, and 51 unannotated repeats. Overall, various types of repeats comprise 51.18% of the P. monodon genome in length. Approximately 7.4% of the FESs contained protein-coding sequences, and the Inhibitor of Apoptosis Protein (IAP) gene and the Innexin 3 gene homologues appear to be present in high abundance in the P. monodon genome. Conclusions The redundancy of various repeat types in the P. monodon genome illustrates its highly repetitive nature. In particular, long and dense microsatellite sequences as well as abundant WSSV-like sequences highlight the uniqueness of genome organization of penaeid shrimp from those of other taxa. These results provide substantial improvement to our current knowledge not only for shrimp but also for marine crustaceans of large genome size.

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 the13-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.