Identification of the protein coding capability of coronavirus defective viral genomes by mass spectrometry.

During coronavirus infection, in addition to the well-known coronavirus genomes and subgenomic mRNAs, an abundance of defective viral genomes (DVGs) can also be synthesized. In this study, we aimed to examine whether DVGs can encode proteins in infected cells. Nanopore direct RNA sequencing and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis were employed. With the protein databases generated by nanopore direct RNA sequencing and the cell lysates derived from the RNA-protein pull-down assay, six DVG-encoded proteins were identified by LC-MS/MS based on the featured fusion peptides caused by recombination during DVG synthesis. The results suggest that the coronavirus DVGs have the capability to encode proteins. Consequently, future studies determining the biological function of DVG-encoded proteins may contribute to the understanding of their roles in coronavirus pathogenesis and the development of antiviral strategies.

Biological characterization of coronavirus noncanonical transcripts in vitro and in vivo.

BACKGROUND: In addition to the well-known coronavirus genomes and subgenomic mRNAs, the existence of other coronavirus RNA species, which are collectively referred to as noncanonical transcripts, has been suggested; however, their biological characteristics have not yet been experimentally validated in vitro and in vivo. METHODS: To comprehensively determine the amounts, species and structures of noncanonical transcripts for bovine coronavirus in HRT-18 cells and mouse hepatitis virus A59, a mouse coronavirus, in mouse L cells and mice, nanopore direct RNA sequencing was employed. To experimentally validate the synthesis of noncanonical transcripts under regular infection, Northern blotting was performed. Both Northern blotting and nanopore direct RNA sequencing were also applied to examine the reproducibility of noncanonical transcripts. In addition, Northern blotting was also employed to determine the regulatory features of noncanonical transcripts under different infection conditions, including different cells, multiplicities of infection (MOIs) and coronavirus strains. RESULTS: In the current study, we (i) experimentally determined that coronavirus noncanonical transcripts were abundantly synthesized, (ii) classified the noncanonical transcripts into seven populations based on their structures and potential synthesis mechanisms, (iii) showed that the species and amounts of the noncanonical transcripts were reproducible during regular infection but regulated in altered infection environments, (iv) revealed that coronaviruses may employ various mechanisms to synthesize noncanonical transcripts, and (v) found that the biological characteristics of coronavirus noncanonical transcripts were similar between in vitro and in vivo conditions. CONCLUSIONS: The biological characteristics of noncanonical coronavirus transcripts were experimentally validated for the first time. The identified features of noncanonical transcripts in terms of abundance, reproducibility and variety extend the current model for coronavirus gene expression. The capability of coronaviruses to regulate the species and amounts of noncanonical transcripts may contribute to the pathogenesis of coronaviruses during infection, posing potential challenges in disease control. Thus, the biology of noncanonical transcripts both in vitro and in vivo revealed here can provide a database for biological research, contributing to the development of antiviral strategies.

The identification of soy sauce adulterated with bean species and the origin using headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry.

Soy sauce is one of the significant seasonings in Asia but is often mislabeled in ingredients or substituted with geographical information. With no adequate methods to distinguish the bean sources and the origins of soy sauce, our study designed a seamless headspace solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME/GC-MS) for analyzing unique volatile components of different soy sauces. Over 400 volatile flavor compounds were identified and the assistance of chemometric analysis successfully discriminated different bean sources (black bean and soybean) and producing regions (Taiwan and Japan). The chemometric models can also perfectly evaluate real samples together with adulterated samples. In brief, these soy sauce volatile signatures can solve the problem of authentication and assist the whole industry in preventing adulteration and producing countries' counterfeit.

Determination of adulteration, geographical origins, and species of food by mass spectrometry.

Food adulteration, mislabeling, and fraud, are rising global issues. Therefore, a number of precise and reliable analytical instruments and approaches have been proposed to ensure the authenticity and accurate labeling of food and food products by confirming that the constituents of foodstuffs are of the kind and quality claimed by the seller and manufacturer. Traditional techniques (e.g., genomics-based methods) are still in use; however, emerging approaches like mass spectrometry (MS)-based technologies are being actively developed to supplement or supersede current methods for authentication of a variety of food commodities and products. This review provides a critical assessment of recent advances in food authentication, including MS-based metabolomics, proteomics and other approaches.

Hierarchical Composite Nanoarchitectonics with a Graphitic Core, Dendrimer and Fluorocarbon Domains, and a Poly(ethylene glycol) Shell as O2 Reservoirs for Reactive Oxygen Species Production.

Graphene oxide (GO), single-walled carbon nanohorn (CNHox), and nitrogen-doped CNH (N-CNH) were functionalized with fluorinated poly(ethylene glycol) (F-PEG) and/or with a fluorinated dendrimer (F-DEN) to prepare a series of assembled nanocomposites (GO/F-PEG, CNHox/F-PEG, N-CNH/F-PEG, N-CNH/F-DEN, and N-CNH/F-DEN/F-PEG) that provide effective multisite O2 reservoirs. In all cases, the O2 uptake increased with time and saturated after 10-20 min. When graphitic carbons (GO and CNHox) were coated with F-PEG, the O2 uptake doubled. The O2 loading was slightly higher in N-CNH compared to CNHox. Notably, coating N-CNH with F-DEN or F-PEG, or with both F-DEN and F-PEG, was more effective. The best performance was obtained with the N-CNH/F-DEN/F-PEG nanocomposite. The O2 uptake kinetics and mechanisms were analyzed in terms of the Langmuir adsorption equation based on a multibinding site assumption. This allowed the precise determination of multiple oxygen binding sites, including on the graphitic structure and in the dendrimer, F-DEN, and F-PEG. After an initial rapid, relatively limited release, the amount of O2 trapped in the nanomaterials remained high (>95%). This amount was marginally lower for the functionalized composites, but the oxygen stored was reserved for longer times. Finally, it is shown that these systems can generate singlet oxygen after irradiation by a light-emitting diode, and this production correlates with the amount of O2 loaded. Thus, it was anticipated that the present nanocomposites hierarchically assembled from components with different characters and complementary affinities for oxygen can be useful as O2 reservoirs for singlet oxygen generation to kill bacteria and viruses and to perform photodynamic therapy.

Chemoenzymatic Synthesis of Asymmetrically Branched Human Milk Oligosaccharide Lacto-N-Hexaose.

We herein reported the first chemoenzymatic synthesis of lacto-N-hexaose (LNH) by combining chemical carbohydrate synthesis with a selectively enzymatic glycosylation strategy. A tetrasaccharide core structure GlcNH2ß1→3 (GlcNAcß1→6) Galß1→4Glc, a key precursor for subsequent enzymatic glycan extension toward asymmetrically branched human milk oligosaccharides, was synthesized in this work. When the order of galactosyltransferase-catalyzed reactions was appropriately arranged, the ß1,4-galactosyl and ß1,3-galactosyl moieties could be sequentially assembled on the C6-arm and C3-arm of the tetrasaccharide, respectively, to achieve an efficient LNH synthesis. Lacto-N-neotetraose (LNnH), another common human milk oligosaccharide, was also synthesized en route to the target LNH.

Clinical characteristics and treatment outcomes of SARS-CoV-2 delta variant outbreak, Pingtung, Taiwan, June 2021.

BACKGROUND: An outbreak of SARS-CoV-2 Delta variant infection occurred in Pingtung, Taiwan, in June 2021. In this study, we aimed to elucidate the clinical characteristics of the Delta-variant SARS-CoV-2 infection and the treatment outcome of antiviral agents in patients from Pingtung County in Southern Taiwan. METHODS: A total of 11 patients with Delta-variant COVID-19 were consecutively admitted to a governmental hospital in June 2021. Baseline characteristics and treatment outcome were evaluated. RESULTS: All patients were symptomatic. The most common symptoms were cough (72.7%), followed by fever (54.5%), headache (18.2%) and dysosmia/dysgeusia (18.2%). Two patients developed pneumonia without mechanical ventilation requirement. Compared to patients without pneumonia, those with pneumonia had higher aspartate aminotransferase (AST) (21.0 vs. 126.0 IU/L, P = 0.03) and lactate dehydrogenase (LDH) (143.1 vs. 409.0 IU/mL, P = 0.03), and ferritin (0.2 vs. 2.0 mg/L, P = 0.046) levels. Pneumonia improved after 2-week treatment, and no mortality occurred after 30 days of diagnosis. The median duration of viral shedding duration of viral shedding was 16.5 days (range 11-42 days) (defined by time to repeated negative real-time reverse transcriptase polymerase chain reaction (RT-PCR) or a cycle threshold (CT) value ≥ 30). CONCLUSION: We demonstrated the clinical characteristics of Delta-variant COVID-19 and treatment outcome of antiviral agents. The risk factors attributed to pneumonia were higher serum AST, ferritin, and LDH levels.

Proteomics for species authentication of cod and corresponding fishery products.

Seafood substitutions is a global problem and come under the spotlight in recent years. In Taiwan, Greenland halibut is usually substituted for the cod because of its lower price. Nowadays, DNA technology is widely used for fish species identifications; however, it still has concern about the DNA of processed fishery products might be destroyed. This study was designed to develop a proteomic-based method for fish and fishery product authentication by using ultra performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS) with Sequential window acquisition of all theoretical fragment ion spectra (SWATH). The protein biomarkers from the meat of Alaska pollock, Atlantic cod, and Greenland halibut were identified and validated for species authentication of cod and corresponding fishery products, which might prevent consumer substitutions and fish product mislabeling. Besides, the E. coli proteins can be measured from existing SWATH-MS data though retrospective analysis successfully, it might present the quality of fish meat.

Mortality outcomes with hydroxychloroquine and chloroquine in COVID-19 from an international collaborative meta-analysis of randomized trials.

Substantial COVID-19 research investment has been allocated to randomized clinical trials (RCTs) on hydroxychloroquine/chloroquine, which currently face recruitment challenges or early discontinuation. We aim to estimate the effects of hydroxychloroquine and chloroquine on survival in COVID-19 from all currently available RCT evidence, published and unpublished. We present a rapid meta-analysis of ongoing, completed, or discontinued RCTs on hydroxychloroquine or chloroquine treatment for any COVID-19 patients (protocol: https://osf.io/QESV4/ ). We systematically identified unpublished RCTs (ClinicalTrials.gov, WHO International Clinical Trials Registry Platform, Cochrane COVID-registry up to June 11, 2020), and published RCTs (PubMed, medRxiv and bioRxiv up to October 16, 2020). All-cause mortality has been extracted (publications/preprints) or requested from investigators and combined in random-effects meta-analyses, calculating odds ratios (ORs) with 95% confidence intervals (CIs), separately for hydroxychloroquine and chloroquine. Prespecified subgroup analyses include patient setting, diagnostic confirmation, control type, and publication status. Sixty-three trials were potentially eligible. We included 14 unpublished trials (1308 patients) and 14 publications/preprints (9011 patients). Results for hydroxychloroquine are dominated by RECOVERY and WHO SOLIDARITY, two highly pragmatic trials, which employed relatively high doses and included 4716 and 1853 patients, respectively (67% of the total sample size). The combined OR on all-cause mortality for hydroxychloroquine is 1.11 (95% CI: 1.02, 1.20; I² = 0%; 26 trials; 10,012 patients) and for chloroquine 1.77 (95%CI: 0.15, 21.13, I² = 0%; 4 trials; 307 patients). We identified no subgroup effects. We found that treatment with hydroxychloroquine is associated with increased mortality in COVID-19 patients, and there is no benefit of chloroquine. Findings have unclear generalizability to outpatients, children, pregnant women, and people with comorbidities.

Honey proteomic signatures for the identification of honey adulterated with syrup, producing country, and nectar source using SWATH-MS approach.

Honey is widely consumed by humans, due to its multiple applications as a food constituent and its therapeutic effects. This study reports on the discrimination of honey products from different geographical and botanical sources, as well as honey products containing distinct forms of syrup used in honey adulteration. Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS)-based proteomic analysis combined with chemometrics was successfully applied in identifying characteristic proteins that can be used as biomarkers of the original source of honey. Honey samples from different producing regions (Tainan, Changhua, and Taichung), countries (Taiwan and Thailand), and distinct botanical sources (longan and litchi) were clearly distinguished by the developed orthogonal projections to latent structures discriminant analysis (OPLS-DA) model with good fitness and prediction ability. Furthermore, we successfully discriminated the adulteration of honey with syrup in different proportions (even with honey content as low as 20%) with this proteomic SWATH-MS platform.

Effects of Substituting Polyisoprenyl Carbanions for Ethoxyl Groups of Bis-[3-(triethoxysilyl)propyl] Tetrasulfide on the Properties of Carbon Black and Silica-Reinforced NR/SSBR.

The coupling agent TESPT (bis-[3-(triethoxysilyl)propyl] tetrasulfide) was modified by substituting polyisoprenyl (PI) carbanions for the ethoxyl groups on silicon for increasing the interaction of rubber with its fillers. The modification was carried out by the reaction of TESPT with polyisoprenyllithium, which had been previously prepared by anionic polymerization of isoprene using butyllithium. The success of the substitution was confirmed by Fourier-transform infrared spectroscopy, and the molecular weight of the modified TESPT (PI-TESPT) was determined from gel permeation chromatography measurements. The effects of tethered PI, as well as of its chain length, on the mechanical and dynamic properties of rubber compounds were examined using a universal testing machine and dynamic mechanical analysis (DMA). In rubber sample preparation, the amount of PI3-TESPT (PI of 2900 g/mol) used in rubber compounding is equal to that of the reference sample with TESPT (S TESPT). For S PI-TESPT samples, the amounts of PI6-TESPT (PI of 5500 g/mol) and PI14-TESPT (PI of 13,700 g/mol) used were calculated as molar ethoxyl groups which are nearly equivalent to those of PI3-TESPT. At the same wt % (parts per hundred, phr) of elemental sulfur in rubber compounds, despite the order of cross-linking density being S TESPT (sample prepared with TESPT) > S PI3-TESPT > S PI6-TESPT > S PI14-TESPT, the exhibited tensile strength is of the order of S PI3-TESPT > S PI6-TESPT > S TESPT ≈ S PI14-TESPT. The better mechanical properties of S PI3-TESPT, as opposed to those of S TESPT, could be attributed to the extra reinforcement from the PI-rubber chain linkage and better silica dispersion, as suggested by the mixing torque and Payne effect (ΔG') measurements. While the dynamic properties of S PI3-TESPT are inferior to those of S TESPT, these properties can be improved by adding more elemental sulfur to increase the cross-linking density.

Variations in bovine milk oligosaccharides after calving using p-aminobenzoic ethyl ester closed-ring labeling and negative ion electrospray LC/MS/MS.

A strategy was proposed to analyze bovine milk oligosaccharides using p-aminobenzoic ethyl ester (ABEE) closed-ring labeling and C18 capillary liquid chromatography negative ion electrospray tandem mass spectrometry. Linkage specific fragment ions were used to identify oligosaccharide isomers. By constructing the mass chromatograms using linkage specific fragment ions, isomers were differentiated based on m/z values as well as temporal separation provided by liquid chromatography. In addition to disialyllactose and the single isomer lacto-N-neohexaose, four pairs of linkage isomers including 3'/6'-sialyllactose (3'/6'-SL), 3'/6'-sialyllactosamine (3'/6'-SLN), 3'/6'-sialylgalactosyl-lactose (3'/6'-SGL), and lacto-N-tetraose/lacto-N-neotetraose (LNT/LNnT) in bovine milk were investigated. Variations of bovine milk oligosaccharides in a lactation period of 72 h after calving were studied. Sialylated oligosaccharide was found to be distinctively more abundant in milk of the first 24 h, decreasing in successive milkings. For the first time, the variation of lacto-N-tetraose in bovine milk was reported.

A multicenter, randomized, open-label, controlled trial to evaluate the efficacy and tolerability of hydroxychloroquine and a retrospective study in adult patients with mild to moderate coronavirus disease 2019 (COVID-19).

OBJECTIVE: In this study, we evaluated the efficacy of hydroxychloroquine (HCQ) against coronavirus disease 2019 (COVID-19) via a randomized controlled trial (RCT) and a retrospective study. METHODS: Subjects admitted to 11 designated public hospitals in Taiwan between April 1 and May 31, 2020, with COVID-19 diagnosis confirmed by pharyngeal real-time RT-PCR for SARS-CoV-2, were randomized at a 2:1 ratio and stratified by mild or moderate illness. HCQ (400 mg twice for 1 d or HCQ 200 mg twice daily for 6 days) was administered. Both the study and control group received standard of care (SOC). Pharyngeal swabs and sputum were collected every other day. The proportion and time to negative viral PCR were assessed on day 14. In the retrospective study, medical records were reviewed for patients admitted before March 31, 2020. RESULTS: There were 33 and 37 cases in the RCT and retrospective study, respectively. In the RCT, the median times to negative rRT-PCR from randomization to hospital day 14 were 5 days (95% CI; 1, 9 days) and 10 days (95% CI; 2, 12 days) for the HCQ and SOC groups, respectively (p = 0.40). On day 14, 81.0% (17/21) and 75.0% (9/12) of the subjects in the HCQ and SOC groups, respectively, had undetected virus (p = 0.36). In the retrospective study, 12 (42.9%) in the HCQ group and 5 (55.6%) in the control group had negative rRT-PCR results on hospital day 14 (p = 0.70). CONCLUSIONS: Neither study demonstrated that HCQ shortened viral shedding in mild to moderate COVID-19 subjects.

Leucocin C-607, a Novel Bacteriocin from the Multiple-Bacteriocin-Producing Leuconostoc pseudomesenteroides 607 Isolated from Persimmon.

Leuconostoc pseudomesenteroides 607, isolated from persimmon fruit, was found to have high inhibitory activity against Listeria monocytogenes and several other Gram-positive bacteria. Inhibitory substances were purified from culture supernatant by ion-exchange chromatography, Sep-Pak C18 cartridge, and reverse-phase high-performance liquid chromatography (RP-HPLC). Two antibacterial peptides were observed during the purification procedures. One of these peptides had a molecular size of 4623.05 Da and a partial N-terminal amino acid sequence of NH2-KNYGNGVHxTKKGxS, in which the YGNGV motif is specific for class IIa bacteriocins. A BLAST search revealed that this bacteriocin was similar to leucocin C from Leuconostoc mesenteroides. Leucocin C-specific primers were designed and a single PCR product was amplified. Analysis of the nucleotide sequence has revealed a putative peptide differing by only one amino acid residue from the sequence of leucocin C. No identical peptide or protein has been reported in the literature, and this peptide, termed leucocin C-607, was therefore considered to be a new variant of leucocin C produced by Leuc. pseudomesenteroides 607. Another antibacterial peptide purified from the same culture supernatant had a molecular size of 3007.7 or 3121.97 Da. However, detailed information regarding this second peptide remains to be determined. Distinct characteristics, such as heat stability and inhibitory spectrum, were observed for the two bacteriocins produced by Leuc. pseudomesenteroides 607. These results suggested that Leuc. pseudomesenteroides 607 produces leucocin C-607 along with another unknown bacteriocin.

Resistance to first- and second-line antituberculosis drugs in Southern Taiwan: Implications for empirical treatment.

BACKGROUND: Multidrug-resistant and extensively drug-resistant tuberculosis infections cause public health concerns worldwide. Local epidemiologic data about the drug resistance of Mycobacterium tuberculosis isolate (Mtb) is critical to guide appropriate empirical therapy to cure patients and restrain the spread of tuberculosis. METHODS: Antituberculosis susceptibility testing was performed for 287 Mtbs, including 63 MDR-Mtbs collected in southern Taiwan from 2011 to 2015. Tuberculosis patients were classified into newly diagnosed cases and previously treated cases based on patients' medical history. RESULTS: Almost no resistance was found to the tested second-line antituberculosis drugs in non-MDR-Mtbs. Higher resistance rates to ethambutol, ofloxacin, and streptomycin were observed in MDR-Mtbs compared to non-MDR-Mtbs. Among 63 MDR-Mtbs, 61.9% of patients were newly diagnosed and 38.1% were previously treated cases. For MDR-Mtb, the drug-resistance rates in previously treated cases were significantly higher for ethambutol, pyrazinamide, ofloxacin, moxifloxacin, streptomycin, and p-aminosalicylic acid. When MDR-Mtbs are identified in previously treated cases, empirical administration of ethambutol, pyrazinamide, ofloxacin, or moxifloxacin may not provide effective treatment. The resistance rates to these drugs were all more than 50%. Furthermore, 25% of MDR-Mtbs from previously treated patients were resistant to p-aminosalicylic acid. CONCLUSION: We observed almost no resistance to the tested second-line antituberculosis drugs among non-MDR-Mtbs. Anti-tuberculosis regimen with pyrazinamide, ethambutol, fluoroquinolone, kanamycin, cycloserine and p-aminosalicylic acid can be empirically used for newly diagnosed MDR-TB cases. For previously treated MDR-TB patients, empirical ethambutol, pyrazinamide, ofloxacin, or moxifloxacin may not provide effective treatment because the resistance rates to these drugs were all >50%.

The Effects of PM2.5 from Asian Dust Storms on Emergency Room Visits for Cardiovascular and Respiratory Diseases.

A case-crossover study examined how PM2.5 from Asian Dust Storms (ADS) affects the number of emergency room (ER) admissions for cardiovascular diseases (CVDs) and respiratory diseases (RDs). Our data indicated that PM2.5 concentration from ADS was highly correlated with ER visits for CVDs and RDs. The odds ratios (OR) increased by 2.92 (95% CI: 1.22-5.08) and 1.86 (95% CI: 1.30-2.91) per 10 µg/m³ increase in PM2.5 levels, for CVDs and RDs, respectively. A 10 µg/m³ increase in PM2.5 from ADSs was significantly associated with an increase in ER visits for CVDs among those 65 years of age and older (an increase of 2.77 in OR) and for females (an increase of 3.09 in OR). In contrast, PM2.5 levels had a significant impact on RD ER visits among those under 65 years of age (OR = 1.77). The risk of ER visits for CVDs increased on the day when the ADS occurred in Taiwan and the day after (lag 0 and lag 1); the corresponding risk increase for RDs only increased on the fifth day after the ADS (lag 5). In Taiwan's late winter and spring, the severity of ER visits for CVDs and RDs increases. Environmental protection agencies should employ an early warning system for ADS to reduce high-risk groups' exposure to PM2.5.

Dense magnetized plasma associated with a fast radio burst.

Fast radio bursts are bright, unresolved, non-repeating, broadband, millisecond flashes, found primarily at high Galactic latitudes, with dispersion measures much larger than expected for a Galactic source. The inferred all-sky burst rate is comparable to the core-collapse supernova rate out to redshift 0.5. If the observed dispersion measures are assumed to be dominated by the intergalactic medium, the sources are at cosmological distances with redshifts of 0.2 to 1 (refs 10 and 11). These parameters are consistent with a wide range of source models. One fast burst revealed circular polarization of the radio emission, but no linear polarization was detected, and hence no Faraday rotation measure could be determined. Here we report the examination of archival data revealing Faraday rotation in the fast radio burst FRB 110523. Its radio flux and dispersion measure are consistent with values from previously reported bursts and, accounting for a Galactic contribution to the dispersion and using a model of intergalactic electron density, we place the source at a maximum redshift of 0.5. The burst has a much higher rotation measure than expected for this line of sight through the Milky Way and the intergalactic medium, indicating magnetization in the vicinity of the source itself or within a host galaxy. The pulse was scattered by two distinct plasma screens during propagation, which requires either a dense nebula associated with the source or a location within the central region of its host galaxy. The detection in this instance of magnetization and scattering that are both local to the source favours models involving young stellar populations such as magnetars over models involving the mergers of older neutron stars, which are more likely to be located in low-density regions of the host galaxy.

Characterization of Fosfomycin Resistant Extended-Spectrum ß-Lactamase-Producing Escherichia coli Isolates from Human and Pig in Taiwan.

To investigate the efficacy of fosfomycin against extended-spectrum ß-lactamases (ESBL) producing Escherichia coli in Taiwan and the resistance mechanisms and characterization of human and pig isolates, we analyzed 145 ESBL-producing isolates collected from two hospitals (n = 123) and five farms (n = 22) in Taiwan from February to May, 2013. Antimicrobial susceptibilities were determined. Clonal relatedness was determined by PFGE and multi-locus sequence typing. ESBLs, ampC, and fosfomycin resistant genes were detected by PCR, and their flanking regions were determined by PCR mapping and sequencing. The fosfomycin resistant mechanisms, including modification of the antibiotic target (MurA), functionless transporters (GlpT and UhpT) and their regulating genes such as uhpA, cyaA, and ptsI, and antibiotic inactivation by enzymes (FosA and FosC), were examined. The size and replicon type of plasmids carrying fosfomycin resistant genes were analyzed. Our results revealed the susceptibility rates of fosfomycin were 94% for human ESBL-producing E. coli isolates and 77% for pig isolates. The PFGE analysis revealed 79 pulsotypes. No pulsotype was found existing in both human and pig isolates. Three pulsotypes were distributed among isolates from two hospitals. ISEcp1 carrying blaCTX-M-group 9 was the predominant transposable elements of the ESBL genes. Among the thirteen fosfomycin resistant isolates, functionless transporters were identified in 9 isolates. Three isolates contained novel amino acid substitutions (Asn67Ile, Phe151Ser and Trp164Ser, Val146Ala and His159Tyr, respectively) in MurA (the target of fosfomycin). Four isolates had fosfomycin modified enzyme (fosA3) in their plasmids. The fosA3 gene was harboured in an IncN-type plasmid (101 kbp) in the three pig isolates and an IncB/O-type plasmid (113 kbp) in the human isolate. In conclusion, we identified that 6% and 23% of the ESBL-producing E. coli from human and pigs were resistant to fosfomycin, respectively, in Taiwan. No clonal spread was found between human and pig isolates. Functionless transporters were the major cause of fosfomycin resistance, and the fosA3-transferring plasmid between isolates warrants further monitoring.