Successful Transplantation of Primary Hepatocytes from DsRed Mice into Fah-/-;Scid/Scid Mice; A Future In Vivo Model System for Receiving Human Hepatocytes

We have developed a system for studying hepatocellular growth potential in which liver cells are introduced into the diseased livers of Fah-/-; Scid/Scid double knockout mice. To use this system to study cell transplantation, DsRed  liver cells were introduced into severe immunodeficient Fah-/-; Scid/Scid double knockout  mice. In regenerated recipient livers, up to 20% of the mouse liver is repopulated by DsRed mouse hepatocytes demonstrating the creation of a functional mouse liver in which parenchyma is derived from DsRed mouse hepatocytes. The severe immunodeficient Fah-/-;Scid/Scid double knockout mice provide a tool for studying hepatocellular biology. Keywords: Human Hepatocytes, Transplantation, DsRed and Fah-/-;Scid/Scid Mice

Occurrence and genetic characteristics of mcr-1-positive colistin-resistant E. coli from poultry environments in Bangladesh

ObjectivesColistin is one of the last-resort antibiotics for treatment of multi-drug resistant (MDR) Gram negative bacterial infections. We determined occurrence and characteristics of mcr-1-producing E. coli obtained from live bird markets (LBM), rural poultry farms (RPF) and rural household backyard poultry (HBP) in Bangladesh.MethodsWe tested 104 extended-spectrum β-lactamase (ESBL)-producing E. coli isolated during 2017-2018 from poultry sources for colistin resistance. We analyzed the resistant isolates for mcr gene and characterized mcr positive isolates for antibiotic susceptibility, antibiotic resistance genes, transmissible plasmids and clonal diversity.ResultsOf 104 isolates, 98 (94%) had MICcolistin ≥4 μg/mL and 14 (13.5%) were positive for mcr-1 of which 10 were from LBM (n = 10), 3 from RFP and 1 from HBP. All 14 mcr-1 E. coli were resistant to third generation cephalosporin and tetracycline, while 12 were resistant to fluoroquinolone and sulphamethoxazole, 10 were to aminoglycosides and 3 were to nitrofurantoin. Four isolates carried conjugative mcr-1 plasmid of 23 to 55 MDa in size. The 55 MDa plasmid found in 2 isolates carried additional resistant genes including blaCTX-M-group-1 and blaTEM-1 (ESBL), qnrB (fluoroquinolone) and rmtB (aminoglycoside). These plasmids belong to IncF family with additional replicons: HI1 and N. ERIC-PCR revealed a heterogeneous banding pattern of mcr-1 positive isolates.ConclusionWe report a 13.5% prevalence of mcr-1 positive MDR E. coli in poultry fecal samples predominantly from LBMs in Bangladesh accentuating the need for safe disposal of poultry feces and hygiene practices among people exposed to poultry.</div

Impact of contrasting poultry exposures on human, poultry, and wastewater antibiotic resistomes in Bangladesh

Interactions between humans, animals, and the environment are considered critical foci for addressing antimicrobial resistance (AMR). However, One Health data on AMR in low- and middle-income countries are presently scarce. Using metagenomics, we investigated whether and how the fecal antibiotic resistomes of humans are influenced by exposure to intensive and non-intensively reared poultry within contrasting settings of urban wet markets (n = 13) and rural households (n = 7) in Bangladesh. We also considered poultry (n = 10) and wastewater (n = 10) resistomes in these settings. We found that occupational poultry exposures did not significantly alter the human fecal resistome. In contrast, macrolide-lincosamide-streptogramin and streptothricin antibiotic resistance genes (ARGs) were enriched in poultry from urban wet markets relative to rural household chickens. Wastewater had the highest ARG richness, though this was only partially explained by poultry cecal and human fecal sources. Wastewater also contained clinically significant carbapenem ARGs. This study therefore provides critical insight into the distribution of ARGs in Bangladesh

Fecal Colonization With Multidrug-Resistant E. coli Among Healthy Infants in Rural Bangladesh

Third generation cephalosporins (3GC) are one of the main choices for treatment of infections caused by multidrug-resistant (MDR) Gram-negative bacteria. Due to their overuse, an increasing trend of resistance to 3GC has been observed in developing countries. Here, we describe fecal colonization of 3GC-resistant (3GCr) Escherichia coli in healthy infants (1–12 months old) living in rural areas of Bangladesh. We found that stool samples of 82% of infants (n = 100) were positive for 3GCr E. coli with a mean ± standard deviation of 6.21 ± 1.32 log10 CFU/g wet weight of stool. 3GCr E. coli encompasses an average one third (33%) of the total E. coli of stool. Almost 77% (n = 63) of these 3GCr E. coli were MDR (or resistant to ≥3 classes of antibiotics). Around 90% (n = 74) of 3GCr E. coli were extended spectrum beta-lactamase (ESBL)-producing in which blaCTX–M–group–1 was the predominant (96%, n = 71) ESBL-gene followed by blaTEM (41%, n = 30) and blaOXA–1 (11%, n = 8). A significant proportion (26.5%, n = 22) of 3GCr E. coli was pathogenic, comprising two types, enteroaggregative (EAEC, n = 19) and enteropathogenic (EPEC, n = 3). Colonization of 3GCr E. coli in infant guts was not associated with demographic characteristics such as age, sex, mode of delivery, maternal and infant antibiotic use, disease morbidity, and feeding practices. The high rate of colonization of 3GCr E. coli in infants’ guts is a serious public health concern which needs immediate attention and warrants further studies to explore the cause

Impact of opioid-free analgesia on pain severity and patient satisfaction after discharge from surgery: multispecialty, prospective cohort study in 25 countries

Background: Balancing opioid stewardship and the need for adequate analgesia following discharge after surgery is challenging. This study aimed to compare the outcomes for patients discharged with opioid versus opioid-free analgesia after common surgical procedures.Methods: This international, multicentre, prospective cohort study collected data from patients undergoing common acute and elective general surgical, urological, gynaecological, and orthopaedic procedures. The primary outcomes were patient-reported time in severe pain measured on a numerical analogue scale from 0 to 100% and patient-reported satisfaction with pain relief during the first week following discharge. Data were collected by in-hospital chart review and patient telephone interview 1 week after discharge.Results: The study recruited 4273 patients from 144 centres in 25 countries; 1311 patients (30.7%) were prescribed opioid analgesia at discharge. Patients reported being in severe pain for 10 (i.q.r. 1-30)% of the first week after discharge and rated satisfaction with analgesia as 90 (i.q.r. 80-100) of 100. After adjustment for confounders, opioid analgesia on discharge was independently associated with increased pain severity (risk ratio 1.52, 95% c.i. 1.31 to 1.76; P &lt; 0.001) and re-presentation to healthcare providers owing to side-effects of medication (OR 2.38, 95% c.i. 1.36 to 4.17; P = 0.004), but not with satisfaction with analgesia (beta coefficient 0.92, 95% c.i. -1.52 to 3.36; P = 0.468) compared with opioid-free analgesia. Although opioid prescribing varied greatly between high-income and low- and middle-income countries, patient-reported outcomes did not.Conclusion: Opioid analgesia prescription on surgical discharge is associated with a higher risk of re-presentation owing to side-effects of medication and increased patient-reported pain, but not with changes in patient-reported satisfaction. Opioid-free discharge analgesia should be adopted routinely

Foxc2CreERT2 knock-in mice mark stage-specific Foxc2-expressing cells during mouse organogenesis

浜松医科大学学位論文　医博第759号（平成29年3月13日

Co-exposure of chromium or cadmium and a low concentration of amoxicillin are responsible to emerge amoxicillin resistant Staphylococcus aureus

ABSTRACT: Background: Heavy metals and antimicrobials co-exist in many environmental settings. The co-exposure of heavy metals and antimicrobials can drive emergence of antimicrobial resistant (AMR) Enterobacteriaceae. We hypothesized that co-exposure to heavy metals and a low concentration of antibiotic might alter antimicrobial susceptibility patterns, which facilitate emergence of AMR Staphylococcus aureus. Methods: The growth kinetics of antimicrobial susceptible S. aureus was carried out in the presence of chromium or cadmium salt and a low concentration of antibiotics. Subsequently, the antimicrobial susceptibility pattern was determined by the Kirby-Bauer disc diffusion method. Moreover, the mRNA copy number was determined by reverse transcription polymerase chain reaction. Results: The antimicrobial susceptibility profile revealed that the zone of inhibition (ZOI) for ampicillin, amoxicillin, ciprofloxacin and doxycycline was significantly decreased in chromium pre-exposed S. aureus compared to unexposed bacteria, whereas cadmium pre-exposed bacteria only showed significant decreased in ZOI for amoxicillin. Moreover, the MIC of amoxicillin for S. aureus was increased by 8-fold in chromium and 32-fold in cadmium when bacteria were co-exposed with low concentrations of amoxicillin. The mRNA expression of femX, mepA and norA also significantly increased in S. aureus after exposure to chromium and a low concentration of amoxicillin. Conclusion: Cultivation of S. aureus at the minimum levels of chromium or cadmium and a low concentration of amoxicillin increased the inhibitory concentration of amoxicillin through inducing bacterial efflux pumps and antibiotic resistant genes. However, it is warranted to assess the whole transcriptome to find out all responsible factors behind this de novo amoxicillin resistant S. aureus

Characterization of a serologically atypical Shigella flexneri Z isolated from diarrheal patients in Bangladesh and a proposed serological scheme for Shigella flexneri.

BACKGROUND:Atypical Shigella flexneri Z variant, that agglutinate with E1037 group factor specific monoclonal antisera against Shigella flexneri IV-I but not with other group or type specific antisera, has continuously being isolated in Bangladesh since 1997. Later this serotype has been reported in Indonesia, China and Argentina. Despite being a provisional serotype, continuous isolation of these strains in diverse geographical regions implicated a great necessity to study the overall characteristics of these strains. Therefore, we extensively characterized S. flexneri Z strains using various phenotypic and molecular tools. METHOD:Of 3569 S. flexneri isolated between 1997 and 2015, 95 strains were identified as S. flexneri Z using a panel of polyvalent absorbed antisera and monoclonal antisera of S. flexneri (MASF). Of them, randomly selected 65 strains were molecular O-serotyped using multiplex PCR and characterized using different phenotypic and molecular techniques (i.e.biotyping, plasmid profile, virulence marker and PFGE) to determine relationship with other subserotypes of S. flexneri. RESULTS:All these atypical S. flexneri Z strains were agglutinated with MASF B and IV-I antisera. Concordantly, these strains were positive to opt-gene, responsible for MASF IV-I sero-positive phenotype. However, molecular O-serotyping of all 65 strains could not differentiate between Z and Yb giving similar amplification products (wzx1-5 and opt). Contrarily, MASF based serotypic scheme distinguished among Z and Yb as well as Ya. All these S. flexneri Z showed typical biochemical reaction of S. flexneri, harboured a 140 MDa virulence plasmid and virulence markers namely ipaH, ial, sen, sigA and sepA genes. Along with the virulence plasmid, small plasmids (2.6, 1.8 and 1.6 MDa) were present as core plasmid. Moreover, a middle ranged plasmid and a 4.0 MDa sized plasmid were observed in 65% and 20% strains, respectively. Analysis of PFGE on XbaI-digested chromosomal DNA of Bangladeshi strains showed that S. flexneri Z had a close relatedness with Ya and Yb but completely different than the strains of Xa, Xb, 2a and 2b. This observation was found to be unequivocal while the overall result of biotyping, plasmid profile, and virulence factors was compared. Therefore, we conclude that these atypical serotype Z isolated in Bangladesh had a clonal relationship with Ya and Yb of Bangladesh and the opt gene played an important role in serotypic switching among them. Current serotyping scheme of S. flexneri strains fails to place many such atypical strains (1c, 1c+6, 1d, type 4, and 4c) including S. flexneri Z isolated from different parts of the world. Therefore, an updated serotyping scheme for identification of subserotypes of S. flexneri has been proposed to avoid multiple naming of the same subserotype having similar agglutination pattern

Supplementary material files: Impact of contrasting poultry exposures on human, poultry, and wastewater antibiotic resistomes in Bangladesh

Supplementary material files for 'Impact of Contrasting Poultry Exposures on Human, Poultry, and Wastewater Antibiotic Resistomes in Bangladesh'. Using metagenomics, we investigated whether and how the faecal antibiotic resistomes of humans are influenced by exposure to intensive and non-intensively reared poultry within contrasting settings of urban wet markets (n=13) and rural households (n=7) in Bangladesh. We also considered poultry (n=10) and wastewater (n=10) resistomes in these settings.This record contains nine files: one file containing supplementary tables S1-S1 (Williams Supplementary tables.xlsx), and eight files containing Supplementary figures from S1 to S8 (Williams Supplementary figure S1.docx, etc).</p

Human Colonization with Extended-Spectrum Beta-Lactamase-Producing E. coli in Relation to Animal and Environmental Exposures in Bangladesh: An Observational One Health Study

Background: Human exposure to intensively farmed livestock is a potential risk for transmission of antibiotic-resistant bacteria (ARB) but few studies have assessed the relative role of animal vs. environmental sources of ARB in low-resource community settings. Objectives: We conducted an observational study to compare ARB colonization and antibiotic-resistant gene prevalence and abundance in humans with high or low exposure to poultry in rural households, commercial poultry farms, and urban markets in Bangladesh. Methods: Extended-spectrum β-lactamase (ESBL)-producing and carbapenem-resistant E. coli were quantified in feces from adults with high or low poultry exposure (n=100, respectively), poultry (n=200), drinking water (n=120), and wastewater (n=120) from 40 rural households, 40 poultry farms, and 40 urban markets. Results: ESBL-producing E. coli (ESBL-EC) prevalence was 67.5% (95% CI: 61.0, 74.0) in samples from adults, 68.0% (95% CI: 61.5, 74.5) in samples from poultry, and 92.5% (95% CI: 87.7, 97.3) in wastewater samples. Carbapenem-resistant E. coli prevalence was high in market wastewaters [30% (95% CI: 15.0, 45.0)] but low in humans (1%) and poultry (1%). Human, poultry, and wastewater isolates shared common resistance genes: blaCTX-M-1, qnr, and blaTEM. Human colonization was not significantly associated with exposure to poultry or setting (rural, farm, or market). Ninety-five percent of commercial poultry farms routinely administered antibiotics. Susceptibility tests were significantly different in household vs. farm and market poultry isolates for four of seven antibiotic classes. In human isolates, there were no differences except aminoglycoside resistance (16.4% high vs. 4.4% low exposure, p=0.02). Urban market wastewaters and poultry samples had significantly higher concentrations of ESBL-EC (p<0.001) and blaCTX-M-1 (p<0.001) compared with samples from farms and rural households. Discussion: ESBL-EC colonization was high in humans but not significantly associated with exposure to poultry. Bidirectional transmission of antibiotic resistance is likely between humans, poultry, and the environment in these community settings, underlining the importance of One Health mitigation strategies