Moonsuk Kim

Cloning endangered gray wolves (Canis lupus) from somatic cells collected postmortem

The objective of the present study was to investigate whether nuclear transfer of postmortem wolf somatic cells into enucleated dog oocytes, is a feasible method to produce a cloned wolf. In vivo-matured oocytes (from domestic dogs) were enucleated and fused with somatic cells derived from culture of tissue obtained from a male gray wolf 6h after death. The reconstructed embryos were activated and transferred into the oviducts of naturally synchronous domestic bitches. Overall, 372 reconstructed embryos were transferred to 17 recipient dogs; four recipients (23.5%) were confirmed pregnant (ultrasonographically) 23-25 d after embryo transfer. One recipient spontaneously delivered two dead pups and three recipients delivered, by cesarean section, four cloned wolf pups, weighing 450, 190, 300, and 490g, respectively. The pup that weighed 190g died within 12h after birth. The six cloned wolf pups were genetically identical to the donor wolf, and their mitochondrial DNA originated from the oocyte donors. The three live wolf pups had a normal wolf karyotype (78, XY), and the amount of telomeric DNA, assessed by quantitative fluorescence in situ hybridization, was similar to, or lower than, that of the nuclear donor. In conclusion, the present study demonstrated the successful cloning of an endangered male gray wolf via interspecies transfer of somatic cells, isolated postmortem from a wolf, and transferred into enucleated dog oocytes. Therefore, somatic cell nuclear transfer has potential for preservation of canine species in extreme situations, including sudden death.

A cloned toy poodle produced from somatic cells derived from an aged female dog

To date, dogs have been cloned with somatic cell nuclear transfer (SCNT), using donor cells derived from large-breed dogs 2 months to 3 years of age. The objective of the present study was to use SCNT to produce a small-breed dog from ear fibroblasts of an aged poodle, using large-breed oocyte donors and surrogate females, and to determine the origin of its mitochondrial DNA (mtDNA) and the length of its telomeres. Oocytes were derived from large-breed donors, matured in vivo, collected by flushing oviducts, and reconstructed with somatic cells derived from an aged (14-year-old) female toy poodle. Oocytes and donor cells were fused by electric stimuli, activated chemically, and transferred into the oviducts of large-breed recipient females. Overall, 358 activated couplets were surgically transferred into the oviducts of 20 recipient dogs. Two recipients became pregnant; only one maintained pregnancy to term, and a live puppy (weighing 190 g) was delivered by Caesarean section. The cloned poodle was phenotypically and genetically identical to the nuclear donor dog; however, its mtDNA was from the oocyte donor, and its mean telomere length was not significantly different from that of the nuclear donor. In summary, we demonstrated that a small-breed dog could be cloned by transferring activated couplets produced by fusion of somatic cells from a small-breed, aged donor female with enucleated in-vivo-matured oocytes of large-breed females, and transferred into the oviduct of large-breed recipient female dogs.

Effect of Routine Sterile Gloving on Contamination Rates in Blood Culture: A Cluster Randomized Trial

BACKGROUNDnBlood culture contamination leads to inappropriate or unnecessary antibiotic use. However, practical guidelines are inconsistent about the routine use of sterile gloving in collection of blood for culture.nnnOBJECTIVEnTo determine whether the routine use of sterile gloving before venipuncture reduces blood culture contamination rates.nnnDESIGNnCluster randomized, assessor-blinded, crossover trial (ClinicalTrials.gov registration number: NCT00973063).nnnSETTINGnSingle-center trial involving medical wards and the intensive care unit.nnnPARTICIPANTSn64 interns in charge of collection of blood for culture were randomly assigned to routine-to-optional or optional-to-routine sterile gloving groups for 1854 adult patients who needed blood cultures.nnnINTERVENTIONnDuring routine sterile gloving, the interns wore sterile gloves every time before venipuncture, but during optional sterile gloving, sterile gloves were worn only if needed.nnnMEASUREMENTSnIsolates from single positive blood cultures were classified as likely contaminant, possible contaminant, or true pathogen. Contamination rates were compared by using generalized mixed models.nnnRESULTSnA total of 10 520 blood cultures were analyzed: 5265 from the routine sterile gloving period and 5255 from the optional sterile gloving period. When possible contaminants were included, the contamination rate was 0.6% in routine sterile gloving and 1.1% in optional sterile gloving (adjusted odds ratio, 0.57 [95% CI, 0.37 to 0.87]; P = 0.009). When only likely contaminants were included, the contamination rate was 0.5% in routine sterile gloving and 0.9% in optional sterile gloving (adjusted odds ratio, 0.51 [CI, 0.31 to 0.83]; P = 0.007).nnnLIMITATIONnBlood cultures from the emergency department, surgical wards, and pediatric wards were not assessed.nnnCONCLUSIONnRoutine sterile gloving before venipuncture may reduce blood culture contamination.

Improved efficiency of canine nucleus transfer using roscovitine-treated canine fibroblasts

The aim of this study was to investigate whether roscovitine (the cyclin-dependent kinase 2 inhibitor) effectively induces synchronization of the donor cell cycle at G0/G1 and to examine the effect of donor cell cycle synchronization protocols on canine somatic cell nucleus transfer. Canine fibroblasts were obtained from skin biopsy cultures taken from a 7-yr-old retriever. The donor cell cycle was synchronized either by culturing cells to reach confluency or by treating cells with 15 microg/mL roscovitine for 24h. Cell cycle stages and apoptosis were analyzed by flow cytometry. After synchronization of the donor cell cycle, cells were placed with enucleated in vivo-matured dog oocytes, fused by electric stimulation, activated, and transferred into 18 naturally estrus-synchronized surrogates. There was no significant difference in cell cycle synchronization and apoptosis rates between the confluent and roscovitine groups. After transfer of reconstructed embryos, pregnancy was detected in three of nine surrogates that received cloned embryos reconstructed with roscovitine-treated cells, whereas only one of nine surrogates was pregnant after transfer of cloned embryos reconstructed with confluent cells. One pregnant female from the confluent cell group delivered one live and one dead pup, but the live one died within 5 days after birth. Three pregnant females from the roscovitine-treated cell group delivered eight live pups and one dead pup, and one of eight live pups died within 6 days after birth. In conclusion, the current results demonstrated that reconstructing embryos with roscovitine-treated cells resulted in increased efficiency of canine somatic cell nucleus transfer.

Outbreak and control of haemorrhagic pneumonia due to Streptococcus equi subspecies zooepidemicus in dogs

This work was supported by the Brain Korea 21 Program for nVeterinary Science and the Korea Research Foundation (KRF- n2004-005-E00077).

Antituberculosis drug-induced liver injury in chronic hepatitis and cirrhosis.

OBJECTIVESnTo evaluate the incidence, risk factors and outcomes for anti-tuberculosis (TB) drug-induced liver injury (DILI) in patients with chronic liver disease including cirrhosis.nnnMETHODSnA total of 107 patients with chronic liver disease were assessed for anti-TB DILI. Anti-TB DILI was defined as elevation of alkaline phosphatase (ALP), aspartate transaminase, or alanine transaminase, or an increase in Child-Turcotte-Pugh score within 2 months of initiating anti-TB medication. The risk factors for anti-TB DILI were evaluated by multivariate logistic regression analysis.nnnRESULTSnFifty-eight (54%) patients had cirrhosis. Of 93 patients receiving one or more hepatotoxic anti-TB drugs, 18 (17%) experienced DILI: 11 (24%) among 46 patients with chronic hepatitis and 7 (15%) among 46 patients with compensated liver cirrhosis (Pxa0=xa00.271). Independent risk factors for DILI were female sex, number of hepatotoxic anti-TB drugs administered and baseline ALP levels but not cirrhosis itself. Of the 18 patients with DILI, 13 (72%) successfully completed anti-TB treatment after switching to less hepatotoxic drug regimens.nnnCONCLUSIONSnHepatotoxic anti-TB drugs may be safely used in the patients with chronic liver disease including compensated cirrhosis if number of hepatotoxic drugs used is adjusted appropriately.

Why is asymptomatic bacteriuria overtreated?: A tertiary care institutional survey of resident physicians

BackgroundAsymptomatic bacteriuria (ABU) is common and often leads to unnecessary antimicrobial use. Reducing antibiotic overuse for ABU is therefore an important issue for antimicrobial stewardship. We performed this study to investigate the appropriateness of ABU management and to evaluate physicians’ knowledge and practice regarding ABU.MethodsWe reviewed all urine cultures of ≥105xa0cfu/mL of bacteria among inpatients in a 900-bed hospital in 2011. Each episode of bacteriuria was classified into ABU or urinary tract infection (UTI). ABU was defined as a positive urine culture (≥105xa0cfu/mL) without symptoms or signs suggesting UTI. In October 2012 a cross-sectional survey of resident physicians was undertaken using an anonymous, self-administered questionnaire.ResultsWe identified 219 ABU cases among 1167 positive urine cultures, of which 70 (32.0xa0%) were inappropriately treated. Female gender, old age, pyuria, hematuria, and positive nitrite on urinalysis were associated with inappropriate ABU treatment in a multivariate analysis (Pu2009<u20090.05).The response rate to the survey was 74.2xa0% (95/128). The mean knowledge score was 37.3xa0%, and 33.7xa0% of respondents were able to distinguish ABU from UTI, but less than half knew the indications for treating ABU. Even after ABU was correctly diagnosed, concerns about postoperative infections (38.6xa0%), UTI (9.1xa0%), and abnormal urinalysis (29.5xa0%) prevented proper management. About half of the respondents reported to prescribing antibiotics for ABU despite knowing they were not indicated.ConclusionsAbout one third of ABUs were inappropriately managed. Lack of knowledge and discrepancies between knowledge and practice, contributed to antimicrobial overuse for ABU. Our findings highlight the importance of developing interventions, including education, audit and feedback, to tackle the problem of inappropriate treatment of ABU.

Impact of area under the concentration–time curve to minimum inhibitory concentration ratio on vancomycin treatment outcomes in methicillin-resistant Staphylococcus aureus bacteraemia

There have been few clinical studies on the association between the vancomycin 24-h area under the concentration-time curve (AUC24) to minimum inhibitory concentration (MIC) ratio and vancomycin treatment outcomes in methicillin-resistant Staphylococcus aureus (MRSA) infections. To examine this association and to establish a suitable cut-off value for AUC24/MIC, a multicentre prospective observational study was conducted in patients with MRSA bacteraemia. Data were collected on all patients aged ≥18 years with MRSA bacteraemia treated with vancomycin for ≥72 h without dialysis. The MIC was determined by broth microdilution (BMD) and Etest. Treatment failure was defined as (i) 30-day mortality, (ii) persistent bacteraemia (≥7 days) and (iii) recurrence (≤30 days after completion of therapy). AUC24 was estimated by a Bayesian approach based on individual vancomycin concentrations. The AUC24/MIC cut-off value for differentiating treatment success and failure was calculated by Classification and Regression Tree (CART) analysis. In total, 117 patients were enrolled, among which vancomycin treatment failure occurred in 38 (32.5%). In univariate analysis, high vancomycin MIC and low trough levels were unrelated to treatment outcomes. In the CART analysis, low vancomycin AUC24/MIC [<392.7 (BMD) and <397.2 (Etest)] was associated with treatment failure. In multivariate analysis, low AUC24/MIC was a risk factor for treatment failure [adjusted odds ratio (aOR)=3.50, 95% confidence interval (CI) 1.39-8.82 by BMD; aOR=5.61, 95% CI 2.07-15.24 by Etest]. AUC24/MIC is associated with vancomycin treatment outcomes in MRSA bacteraemia, and seeking individualised AUC24/MIC ratios above target (>400) may improve treatment outcomes.

Birth of viable puppies derived from breeding cloned female dogs with a cloned male

Since the establishment of production of viable cloned dogs by somatic cell nucleus transfer, great concern has been given to the reproductive abilities of these animals (Canis familiaris). Therefore, we investigated reproductive activity of cloned dogs by (1) performing sperm analysis using computer-assisted sperm analysis and early embryonic development, (2) assessing reproductive cycling by measuring serum progesterone (P4) levels and performing vaginal cytology, and (3) breeding cloned dogs using artificial insemination. Results showed that most parameters of sperm motility in a cloned male dog were within the reference range, and in vivo-matured oocytes from a noncloned female were successfully fertilized by spermatozoa from a cloned male dog and develop normally to the 8-cell stage. Three cloned female dogs displayed normal patterns of P4 levels and morphologic changes of the vaginal epithelium. Two cloned female dogs became pregnant using semen from a cloned male dog and successfully delivered 10 puppies by natural labor. In conclusion, these data demonstrated that both cloned male and female dogs are fertile, and their puppies are currently alive and healthy with normal growth patterns.

Impact of an Antimicrobial Stewardship Program on Unnecessary Double Anaerobic Coverage Prescription.

Background Co-administration of two or more antimicrobials with anti-anaerobic activity is not recommended except in certain circumstances. We therefore conducted an intervention to reduce unnecessary double anaerobic coverage (DAC) prescription. Materials and Methods The intervention consisted of education using an institutional intranet and prospective audits and feedback provided through collaboration between a pharmacist and an infectious diseases physician in Seoul National University Bundang Hospital, a tertiary hospital in Seongnam, Republic of Korea, in 2013. The study period was 1 year which contained 6 months of pre-intervention period and 6 months of intervention period. To estimate the overall effect of the intervention, we compared the monthly number of patients receiving unnecessary DAC for more than 3 days and the proportion of patients receiving unnecessary DAC for more than 3 days among all patients receiving DAC. Results The average monthly number of patients receiving unnecessary DAC for more than 3 days after screening decreased by 73.9% in the intervention period from 26.8 to 7.0. Wilcoxon rank sum test revealed there was a significant statistical difference in the monthly number of patients receiving unnecessary DAC for more than 3 days (P = 0.005). The proportion of patients receiving unnecessary DAC for more than 3 days after screening among all patients identified as receiving necessary or unnecessary DAC also decreased by 67.8% in the intervention period from 42.3% to 13.6% (P < 0.001). Conclusion The multidisciplinary antimicrobial stewardship program with combined methods reduced unnecessary DAC prescription successfully.