Antimicrobial susceptibility of staphylococci from bovine milk samples in routine microbiological mastitis analysis in Finland

The most frequent reason for antimicrobial use in dairy herds is mastitis and knowledge about mastitis-causing pathogens and their antimicrobial susceptibility should guide treatment decisions. The overall objective of this study was to assess antimicrobial resistance (AMR) of staphylococci in mastitic milk samples in Finland. MALDI-ToF MS identified a total of 504 Staphylococcus isolates (260 S. aureus and 244 non-aureus staphylococci, NAS) originating from bovine mastitic milk samples. Phenotypic susceptibility against cefoxitin, ceftiofur, enrofloxacin, gentamycin, oxacillin, penicillin, and tetracycline was evaluated by disk diffusion method and the presence of blaZ, mecA, and mecC genes investigated by PCR. Nitrocefin test assessed these isolates’ beta-lactamase production. The most common NAS species were S. simulans, S. epidermidis, S. chromogenes, and S. haemolyticus. In total, 26.6% of the isolates (18.5% of S. aureus and 35.2% of all NAS) carried the blaZ gene. Penicillin resistance, based on disk diffusion, was lower: 18.8% of all the isolates (9.3% of S. aureus and 28.9% of all NAS) were resistant. Based on the nitrocefin test, 21.5% of the isolates produced beta-lactamase (11.6% of S. aureus and 32.0% of all NAS). Between the Staphylococcus species, the proportion of penicillin-resistant isolates varied, being lowest in S. simulans and highest in S. epidermidis. Resistance to antimicrobials other than penicillin was rare. Of the eight NAS isolates carrying the mecA gene, six were S. epidermidis. One S. aureus isolate carried the mecC gene. Agreement beyond chance, assessed by kappa coefficient, between phenotypic and genotypic resistance tests, was moderate to substantial. Some phenotypically penicillin-susceptible staphylococci carried the blaZ gene but isolates without blaZ or mec genes rarely exhibited resistance, suggesting that the more reliable treatment choice may depend upon genotypic AMR testing. Our results support earlier findings that penicillin resistance is the only significant form of antimicrobial resistance among mastitis-causing staphylococci in Finland

GATA4-targeted compound exhibits cardioprotective actions against doxorubicin-induced toxicity in vitro and in vivo : establishment of a chronic cardiotoxicity model using human iPSC-derived cardiomyocytes

Doxorubicin is a widely used anticancer drug that causes dose-related cardiotoxicity. The exact mechanisms of doxorubicin toxicity are still unclear, partly because most in vitro studies have evaluated the effects of short-term high-dose doxorubicin treatments. Here, we developed an in vitro model of long-term low-dose administration of doxorubicin utilizing human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Moreover, given that current strategies for prevention and management of doxorubicin-induced cardiotoxicity fail to prevent cancer patients developing heart failure, we also investigated whether the GATA4-targeted compound 3i-1000 has cardioprotective potential against doxorubicin toxicity both in vitro and in vivo. The final doxorubicin concentration used in the chronic toxicity model in vitro was chosen based on cell viability data evaluation. Exposure to doxorubicin at the concentrations of 1-3 mu M markedly reduced (60%) hiPSC-CM viability already within 48 h, while a 14-day treatment with 100 nM doxorubicin concentration induced only a modest 26% reduction in hiPCS-CM viability. Doxorubicin treatment also decreased DNA content in hiPSC-CMs. Interestingly, the compound 3i-1000 attenuated doxorubicin-induced increase in pro-B-type natriuretic peptide (proBNP) expression and caspase-3/7 activation in hiPSC-CMs. Moreover, treatment with 3i-1000 for 2 weeks (30 mg/kg/day, i.p.) inhibited doxorubicin cardiotoxicity by restoring left ventricular ejection fraction and fractional shortening in chronic in vivo rat model. In conclusion, the results demonstrate that long-term exposure of hiPSC-CMs can be utilized as an in vitro model of delayed doxorubicin-induced toxicity and provide in vitro and in vivo evidence that targeting GATA4 may be an effective strategy to counteract doxorubicin-induced cardiotoxicity.Peer reviewe

Nuclear Receptor-Like Structure and Interaction of Congenital Heart Disease-Associated Factors GATA4 and NKX2-5

Aims Transcription factor GATA4 is a dosage sensitive regulator of heart development and alterations in its level or activity lead to congenital heart disease (CHD). GATA4 has also been implicated in cardiac regeneration and repair. GATA4 action involves combinatorial interaction with other cofactors such as NKX2-5, another critical cardiac regulator whose mutations also cause CHD. Despite its critical importance to the heart and its evolutionary conservation across species, the structural basis of the GATA4-NKX2-5 interaction remains incompletely understood. Methods and Results A homology model was constructed and used to identify surface amino acids important for the interaction of GATA4 and NKX2-5. These residues were subjected to site-directed mutagenesis, and the mutant proteins were characterized for their ability to bind DNA and to physically and functionally interact with NKX2-5. The studies identify 5 highly conserved amino acids in the second zinc finger (N272, R283, Q274, K299) and its C-terminal extension (R319) that are critical for physical and functional interaction with the third alpha helix of NKX2-5 homeodomain. Integration of the experimental data with computational modeling suggests that the structural arrangement of the zinc finger-homeodomain resembles the architecture of the conserved DNA binding domain of nuclear receptors. Conclusions The results provide novel insight into the structural basis for protein-protein interactions between two important classes of transcription factors. The model proposed will help to elucidate the molecular basis for disease causing mutations in GATA4 and NKX2-5 and may be relevant to other members of the GATA and NK classes of transcription factors.Peer reviewe

Discovery of Small Molecules Targeting the Synergy of Cardiac Transcription Factors GATA4 and NKX2-5

Transcription factors are pivotal regulators of gene transcription, and many diseases are associated with the deregulation of transcriptional networks. In the heart, the transcription factors GATA4 and NKX2-5 are required for cardiogenesis. GATA4 and NKX2-5 interact physically, and the activation of GATA4, in cooperation with NKX2-5, is essential for stretch-induced cardiomyocyte hypertrophy. Here, we report the identification of four small molecule families that either inhibit or enhance the GATA4-NKX2-5 transcriptional synergy. A fragment-based screening, reporter gene assay, and pharmacophore search were utilized for the small molecule screening, identification, and optimization. The compounds modulated the hypertrophic agonist-induced cardiac gene expression. The most potent hit compound, N-[4-(diethylamino)phenyl]-5-methyl-3-phenylisoxazole-4-carboxamide (3, IC50 = 3 mu M), exhibited no activity on the protein kinases involved in the regulation of GATA4 phosphorylation. The identified and chemically and biologically characterized active compound, and its derivatives may provide a novel class of small molecules for modulating heart regeneration.Peer reviewe

Cardiac Actions of a Small Molecule Inhibitor Targeting GATA4–NKX2-5 Interaction

Transcription factors are fundamental regulators of gene transcription, and many diseases, such as heart diseases, are associated with deregulation of transcriptional networks. In the adult heart, zinc-finger transcription factor GATA4 is a critical regulator of cardiac repair and remodelling. Previous studies also suggest that NKX2-5 plays function role as a cofactor of GATA4. We have recently reported the identification of small molecules that either inhibit or enhance the GATA4–NKX2-5 transcriptional synergy. Here, we examined the cardiac actions of a potent inhibitor (3i-1000) of GATA4–NKX2-5 interaction in experimental models of myocardial ischemic injury and pressure overload. In mice after myocardial infarction, 3i-1000 significantly improved left ventricular ejection fraction and fractional shortening, and attenuated myocardial structural changes. The compound also improved cardiac function in an experimental model of angiotensin II -mediated hypertension in rats. Furthermore, the up-regulation of cardiac gene expression induced by myocardial infarction and ischemia reduced with treatment of 3i-1000 or when micro- and nanoparticles loaded with 3i-1000 were injected intramyocardially or intravenously, respectively. The compound inhibited stretch- and phenylephrine-induced hypertrophic response in neonatal rat cardiomyocytes. These results indicate significant potential for small molecules targeting GATA4–NKX2-5 interaction to promote myocardial repair after myocardial infarction and other cardiac injuries.Peer reviewe

Cardiac Actions of a Small Molecule Inhibitor Targeting GATA4-NKX2-5 Interaction

Transcription factors are fundamental regulators of gene transcription, and many diseases, such as heart diseases, are associated with deregulation of transcriptional networks. In the adult heart, zinc-finger transcription factor GATA4 is a critical regulator of cardiac repair and remodelling. Previous studies also suggest that NKX2-5 plays function role as a cofactor of GATA4. We have recently reported the identification of small molecules that either inhibit or enhance the GATA4-NKX2-5 transcriptional synergy. Here, we examined the cardiac actions of a potent inhibitor (3i-1000) of GATA4-NKX2-5 interaction in experimental models of myocardial ischemic injury and pressure overload. In mice after myocardial infarction, 3i-1000 significantly improved left ventricular ejection fraction and fractional shortening, and attenuated myocardial structural changes. The compound also improved cardiac function in an experimental model of angiotensin II-mediated hypertension in rats. Furthermore, the up-regulation of cardiac gene expression induced by myocardial infarction and ischemia reduced with treatment of 3i-1000 or when micro-and nanoparticles loaded with 3i-1000 were injected intramyocardially or intravenously, respectively. The compound inhibited stretch- and phenylephrine-induced hypertrophic response in neonatal rat cardiomyocytes. These results indicate significant potential for small molecules targeting GATA4-NKX2-5 interaction to promote myocardial repair after myocardial infarction and other cardiac injuries

Biological Effects of Antiprotons Are Antiprotons a Candidate for Cancer Therapy?

2009 Status Report of AD-4 Experimen

Naudan utaretulehdusta aiheuttavien stafylokokkien mikrobilääkeresistenssi

Stafylokokit ovat yleisimmät utaretulehduksen aiheuttajat naudalla. Koagulaasipositiivinen Staphylococcus aureus on perinteisesti käsitelty erikseen ja koagulaasinegatiiviset stafylokokit (KNS) yhtenä ryhmänä. Utaretulehdusta aiheuttavia KNS-lajeja on useita ja ne eroavat toisistaan taudinaiheutuskyvyltään, epidemiologialtaan ja mikrobilääkeresistenssiltään. Mikrobilääkkeitä käytetään bakteeritulehduksien hoitamiseen. Bakteerien resistenssi mikrobilääkkeille on maailmanlaajuinen ongelma ja vaikeuttaa infektioiden hoitamista eläimillä ja ihmisillä. Stafylokokeilla yleisin mikrobilääkeresistenssi on beetalaktamaasin tuottoon perustuva penisilliiniresistenssi. Penisilliini on Suomessa yleisin eläinlääkintään myytävä mikrobilääke ja sitä suositellaan penisilliiniherkkien stafylokokkien aiheuttamien utaretulehduksien hoitamiseen. Stafylokokit ovat yleisiä taudinaiheuttajia myös ihmisillä. Lähikontaktissa stafylokokit voivat siirtyä nautojen ja ihmisten välillä. Metisilliiniresistentit stafylokokit, kuten MRSA (metisilliiniresistentti Staphylococcus aureus) ja MRSE (metisilliiniresistentti Staphylococcus epidermidis), voivat naudalla esiintyessään muodostaa uhan myös ihmisten terveydelle. Stafylokokkibakteerit voivat mahdollisesti toimia resistenssigeenien reservuaarina. Monet resistenssigeenit voivat siirtyä stafylokokeilla bakteerikannasta toiseen, stafylokokkilajista toiseen sekä myös stafylokokkien ja muiden bakteerisukujen välillä. Tähän lisensiaatintutkielmaan sisältyy kirjallisuuskatsaus ja tutkimusosuus. Kirjallisuuskatsauksessa perehdytään aiempiin tutkimuksiin utaretulehdusmaidosta eristettyjen stafylokokkien mikrobilääkeresistenssistä Suomessa ja maailmalla sekä penisilliiniresistenssin mekanismeihin ja penisilliiniresistenssin toteamiseen stafylokokki-isolaateilta. Tutkimusosuudessa tavoitteena oli selvittää Suomessa vuonna 2017 otetuista utaretulehdusmaitonäytteistä eristettyjen stafylokokkien mikrobilääkeresistenssi sekä KNS-isolaattien lajijakauma. Lisäksi tavoitteena oli tilastollisten analyysien avulla arvioida kaupallisella PCR-testillä (PathoProof™ Mastitis PCR Complete-16 Assay, Thermo Fisher Scientific, Waltham, Yhdysvallat) todetun blaZ-geenin sekä kiekkodiffuusiomenetelmällä todetun ilmiasullisen penisilliiniresistenssin välistä yhteyttä. Stafylokokkilajien tunnistamiseen käytettiin massaspektrometristä MALDI-TOF MS -menetelmää (Bruker Maldi Biotyper, Bruker Daltonics Scandinavia AB, Solna, Ruotsi). Mikrobilääkeherkkyys tutkittiin kiekkodiffuusiomenetelmällä. Tilastollisissa analyyseissä beetalaktamaasigeenin ja ilmiasullisen penisilliiniresistenssin yhteydestä käytettiin McNemarin-testiä ja Kappa-arvoa. Yleisimmät maitonäytteistä eristetyt KNS-lajit olivat S. epidermidis, Staphylococcus simulans, Staphylococcus haemolyticus ja Staphylococcus chromogenes. Yleisin mikrobilääkeresistenssi oli penisilliiniresistenssi. S. aureus -isolaateista noin 11 % ja KNS-isolaateista noin 30 % oli resistenttejä penisilliinille. S. aureus -isolaateilla penisilliiniresistenssiä esiintyi vähemmän ja KNS-isolaateilla lähes saman verran kuin aiemmissa tutkimuksissa Suomessa. KNS-lajien välillä oli suuria eroja penisilliiniresistenssin yleisyydessä. Eniten penisilliiniresistenttejä isolaatteja esiintyi S. epidermidis -lajissa. S. aureus -isolaateista 3 % ja KNS-isolaateista 10 % oli resistenttejä oksasilliinille. Kefoksitiiniresistenttejä isolaatteja esiintyi vähemmän kuin oksasilliiniresistenttejä isolaatteja. Resistenssi muita tutkittuja mikrobilääkkeitä kohtaan oli vähäistä. PCR-menetelmällä blaZ-geenin kantajiksi todettujen S. aureus- ja KNS-isolaattien lukumäärät erosivat tilastollisesti merkitsevästi kiekkodiffuusiomenetelmällä penisilliiniresistenteiksi todettujen isolaattien lukumäärästä. Negatiivisten tulosten yhtenevyys oli parempi kuin positiivisten. PCR-testissä blaZ-geenin kantajiksi todetuista S. aureus -isolaateista puolet ja KNS-isolaateista kolmasosa oli penisilliiniherkkiä kiekkodiffuusiomenetelmässä

Antimicrobial susceptibility of staphylococci from bovine milk samples in routine microbiological mastitis analysis in Finland

The most frequent reason for antimicrobial use in dairy herds is mastitis and knowledge about mastitis-causing pathogens and their antimicrobial susceptibility should guide treatment decisions. The overall objective of this study was to assess antimicrobial resistance (AMR) of staphylococci in mastitic milk samples in Finland. MALDI-ToF MS identified a total of 504 Staphylococcus isolates (260 S. aureus and 244 non-aureus staphylococci, NAS) originating from bovine mastitic milk samples. Phenotypic susceptibility against cefoxitin, ceftiofur, enrofloxacin, gentamycin, oxacillin, penicillin, and tetracycline was evaluated by disk diffusion method and the presence of blaZ, mecA, and mecC genes investigated by PCR. Nitrocefin test assessed these isolates’ beta-lactamase production. The most common NAS species were S. simulans, S. epidermidis, S. chromogenes, and S. haemolyticus. In total, 26.6% of the isolates (18.5% of S. aureus and 35.2% of all NAS) carried the blaZ gene. Penicillin resistance, based on disk diffusion, was lower: 18.8% of all the isolates (9.3% of S. aureus and 28.9% of all NAS) were resistant. Based on the nitrocefin test, 21.5% of the isolates produced beta-lactamase (11.6% of S. aureus and 32.0% of all NAS). Between the Staphylococcus species, the proportion of penicillin-resistant isolates varied, being lowest in S. simulans and highest in S. epidermidis. Resistance to antimicrobials other than penicillin was rare. Of the eight NAS isolates carrying the mecA gene, six were S. epidermidis. One S. aureus isolate carried the mecC gene. Agreement beyond chance, assessed by kappa coefficient, between phenotypic and genotypic resistance tests, was moderate to substantial. Some phenotypically penicillin-susceptible staphylococci carried the blaZ gene but isolates without blaZ or mec genes rarely exhibited resistance, suggesting that the more reliable treatment choice may depend upon genotypic AMR testing. Our results support earlier findings that penicillin resistance is the only significant form of antimicrobial resistance among mastitis-causing staphylococci in Finland.Peer reviewe