Antibiotic prophylaxis and hospitalization of horses subjected to median laparotomy: gut microbiota trajectories and abundance increase of Escherichia

Introduction: Horse clinics are hotspots for the accumulation and spread of clinically relevant and zoonotic multidrug-resistant bacteria, including extended-spectrum β-lactamase producing (ESBL) Enterobacterales. Although median laparotomy in cases of acute equine colic is a frequently performed surgical intervention, knowledge about the effects of peri-operative antibiotic prophylaxis (PAP) based on a combination of penicillin and gentamicin on the gut microbiota is limited. Methods: We collected fecal samples of horses from a non-hospitalized control group (CG) and from horses receiving either a pre-surgical single-shot (SSG) or a peri-operative 5-day (5DG) course of PAP. To assess differences between the two PAP regimens and the CG, all samples obtained at hospital admission (t0), on days three (t1) and 10 (t2) after surgery, were screened for ESBL-producing Enterobacterales and subjected to 16S rRNA V1–V2 gene sequencing. Results: We included 48 samples in the SSG (n = 16 horses), 45 in the 5DG (n = 15), and 20 in the CG (for t0 and t1, n = 10). Two samples of equine patients receiving antibiotic prophylaxis (6.5%) were positive for ESBL-producing Enterobacterales at t0, while this rate increased to 67% at t1 and decreased only slightly at t2 (61%). Shannon diversity index (SDI) was used to evaluate alpha-diversity changes, revealing there was no significant difference between horses suffering from acute colic (5DG, SDImean of 5.90, SSG, SDImean of 6.17) when compared to the CG (SDImean of 6.53) at t0. Alpha-diversity decreased significantly in both PAP groups at t1, while at t2 the onset of microbiome recovery was noticed. Although we did not identify a significant SDImean difference with respect to PAP duration, the community structure (beta-diversity) was considerably restricted in samples of the 5DG at t1, most likely due to the ongoing administration of antibiotics. An increased abundance of Enterobacteriaceae, especially Escherichia, was noted for both study groups at t1. Conclusion: Colic surgery and PAP drive the equine gut microbiome towards dysbiosis and reduced biodiversity that is accompanied by an increase of samples positive for ESBL-producing Enterobacterales. Further studies are needed to reveal important factors promoting the increase and residency of ESBL-producing Enterobacterales among hospitalized horses

Frequency, Local Dynamics, and Genomic Characteristics of ESBL-Producing Escherichia coli Isolated From Specimens of Hospitalized Horses

Previous research identified veterinary clinics as hotspots with respect to accumulation and spread of multidrug resistant extended-spectrum β-lactamase (ESBL)-producing Escherichia coli (EC). Therefore, promoting the prudent use of antibiotics to decrease selective pressure in that particular clinical environment is preferable to enhance biosecurity for animal patients and hospital staff. Accordingly, this study comparatively investigated the impact of two distinct perioperative antibiotic prophylaxis (PAP) regimens (short-term versus prolonged) on ESBL-EC carriage of horses subjected to colic surgery. While all horses received a combination of penicillin/gentamicin (P/G) as PAP, they were assigned to either the “single-shot group” (SSG) or the conventional “5-day group” (5DG). Fecal samples collected on arrival (t0), on the 3rd (t1) and on the 10th day after surgery (t2) were screened for ESBL-EC. All isolates were further investigated using whole genome sequences. In total, 81 of 98 horses met the inclusion criteria for this study. ESBL-EC identified in samples available at t0, t1 and t2 were 4.8% (SSG) and 9.7% (5DG), 37% (SSG) and 47.2% (5DG) as well as 55.6% (SSG) and 56.8% (5DG), respectively. Regardless of the P/G PAP regimen, horses were 9.12 times (95% CI 2.79–29.7) more likely to carry ESBL-EC at t1 compared to t0 (p < 0.001) and 15.64 times (95% CI 4.57–53.55) more likely to carry ESBL-EC at t2 compared to t0 (p < 0.001). ESBL-EC belonging to sequence type (ST) 10, ST86, ST641, and ST410 were the most prevalent lineages, with blaCTX–M–1 (60%) being the dominant ESBL gene. A close spatio-temporal relationship between isolates sharing a particular ST was revealed by genome analysis, strongly indicating local spread. Consequently, hospitalization itself has a strong impact on ESBL-EC isolation rates in horses, possibly masking differences between distinct PAP regimens. The results of this study reveal accumulation and spread of multi-drug resistant ESBL-EC among horses subjected to colic surgery with different P/G PAP regimens, challenging the local hygiene management system and work-place safety of veterinary staff. Moreover, the predominance of particular ESBL-EC lineages in clinics providing health care for horses needs further investigation.Peer Reviewe

Antibiotic prophylaxis and hospitalization of horses subjected to median laparotomy: gut microbiota trajectories and abundance increase of Escherichia

IntroductionHorse clinics are hotspots for the accumulation and spread of clinically relevant and zoonotic multidrug-resistant bacteria, including extended-spectrum β-lactamase producing (ESBL) Enterobacterales. Although median laparotomy in cases of acute equine colic is a frequently performed surgical intervention, knowledge about the effects of peri-operative antibiotic prophylaxis (PAP) based on a combination of penicillin and gentamicin on the gut microbiota is limited.MethodsWe collected fecal samples of horses from a non-hospitalized control group (CG) and from horses receiving either a pre-surgical single-shot (SSG) or a peri-operative 5-day (5DG) course of PAP. To assess differences between the two PAP regimens and the CG, all samples obtained at hospital admission (t0), on days three (t1) and 10 (t2) after surgery, were screened for ESBL-producing Enterobacterales and subjected to 16S rRNA V1–V2 gene sequencing.ResultsWe included 48 samples in the SSG (n = 16 horses), 45 in the 5DG (n = 15), and 20 in the CG (for t0 and t1, n = 10). Two samples of equine patients receiving antibiotic prophylaxis (6.5%) were positive for ESBL-producing Enterobacterales at t0, while this rate increased to 67% at t1 and decreased only slightly at t2 (61%). Shannon diversity index (SDI) was used to evaluate alpha-diversity changes, revealing there was no significant difference between horses suffering from acute colic (5DG, SDImean of 5.90, SSG, SDImean of 6.17) when compared to the CG (SDImean of 6.53) at t0. Alpha-diversity decreased significantly in both PAP groups at t1, while at t2 the onset of microbiome recovery was noticed. Although we did not identify a significant SDImean difference with respect to PAP duration, the community structure (beta-diversity) was considerably restricted in samples of the 5DG at t1, most likely due to the ongoing administration of antibiotics. An increased abundance of Enterobacteriaceae, especially Escherichia, was noted for both study groups at t1.ConclusionColic surgery and PAP drive the equine gut microbiome towards dysbiosis and reduced biodiversity that is accompanied by an increase of samples positive for ESBL-producing Enterobacterales. Further studies are needed to reveal important factors promoting the increase and residency of ESBL-producing Enterobacterales among hospitalized horses

Studies on the importance of the neurotrophic factor GDNF for the interaction between enteric glial cells and enterocytes for the regulation of the intestinal barrier

In der vorliegenden Dissertation wurde das Zusammenspiel von enterischen Gliazellen (EGC) und Darmepithelzellen (Caco-2) thematisiert, wobei der Fokus auf der Bedeu-tung des neurotrophen Faktors GDNF für die Interaktion zwischen den beiden genann-ten Zelltypen lag. Weiterhin wurde evaluiert, ob die Tyrosinkinase RET auch in Darme-pithelzellen für die GDNF-Signaltransduktion unter Ruhebedingungen und bei Entzün-dungen verantwortlich ist. Als Grundlage diente ein Ko-Kultur-Modell mit Caco-2 und EGC. Durch Permeabili-täts- und Widerstandsmessungen wurden die Auswirkungen von GDNF auf Zell-Monolayer ermittelt. Effekte auf die Barrieredifferenzierung wurden anhand subkon-fluenter Zell-Monolayer charakterisiert, wohingegen die Auswirkungen auf Entzün-dungsstimuli an konfluenten Zellen untersucht wurden. Veränderungen von Junktions-proteinen wurden mit Immunfluoreszenzfärbungen und Western-Blot-Analysen aufge-zeigt. Abschließend erfolgte eine Analyse humaner Gewebeproben von Patienten mit und ohne chronisch-entzündlichen Darmerkrankungen (CED) in Bezug auf deren GDNF-Expression. Die verwendeten intestinalen Epithelzellen exprimieren die GDNF-Rezeptoren GFRα1, GFRα2, GFRα3 und RET. Nach Etablierung des Kultursystems zeigten Permeabilitäts-messungen, Messungen des Epithelwiderstandes sowie Immunfluoreszenz-Färbungen, dass die Differenzierung der Darmepithelzellen in der Ko-Kultur mit EGC durch GDNF vermittelt wird. Zudem war eine GDNF-abhängige, barrierestabilisierende Wirkung in einem Inflammationsmodell zu beobachten. Weiterhin wurde nachgewiesen, dass GDNF-Effekte auf Enterozyten auch im Darmepithel über die RET-Tyrosinkinase mit nachfolgender Hemmung des p38-MAPK-Signalwegs bedingt werden. Eine Stimulation der EGC mit Zytokinen bestätigte eine Hochregulation der GDNF-Expression und Sek-retion. In humanen Proben war intestinales GDNF bei schwerer Entzündung reduziert. Zusammenfassend wurde erstmalig der Nachweis erbracht, dass von EGC sezerniertes GDNF die Differenzierung der Barriere in Darmepithelzellen induziert und diese gegen einen Zytokin-vermittelten Zusammenbruch schützt. Dies wird über eine RET-abhängige Regulation der p38-MAPK vermittelt. Die Reduktion der GDNF-Konzentration in transmuralen Gewebeproben von Patienten mit CED trägt möglicher-weise zur Pathogenese der CED bei.The present thesis adresses the interaction of enteric glial cells (EGC) and intestinal epithelial cells (Caco-2), focusing on the importance of the neurotrophic factor GDNF for the interaction between the two cell types. Furthermore, it was evaluated whether the tyrosine kinase RET is also responsible for GDNF signal transduction in intestinal epithelial cells under resting conditions and during inflammation. A co-culture model with Caco-2 and EGC served as the base for further investigations. Permeability and resistance measurements were used to determine the effects of GDNF on cell monolayers. Effects on barrier differentiation were characterized using subconfluent cell monolayers, whereas effects on inflammatory stimuli were investigated in confluent cells. Changes in junctional proteins were revealed by immunofluorescence staining and Western blot analysis. Finally, human tissue samples from patients with and without chronic inflammatory bowel disease (IBD) were analyzed with regard to their GDNF expression. The intestinal epithelial cells used, express the GDNF receptors GFRα1, GFRα2, GFRα3 and RET. After establishment of the culture system, permeability measurements, epithelial resistance measurements and immunofluorescence staining showed that the differentiation of intestinal epithelial cells in co-culture with EGC is mediated by GDNF. Additionally, a GDNF-dependent, barrier-stabilizing effect was observed in an inflammation model. Furthermore, it was shown that GDNF effects on enterocytes are also caused in the intestinal epithelium via RET tyrosine kinase with subsequent inhibition of the p38 MAPK signaling pathway. Stimulation of EGC with cytokines confirmed an upregulation of GDNF expression and secretion. In human samples, intestinal GDNF was reduced in severe inflammation. In summary, it was demonstrated for the first time that GDNF secreted by EGC induces barrier differentiation in intestinal epithelial cells and protects them against cytokine-mediated breakdown. This is mediated via RET-dependent regulation of p38 MAPK. The reduction of GDNF levels in transmural tissue samples from patients with IBD may contribute to the pathogenesis of IBD

Perioperative antibiotic prophylaxis in horses undergoing aseptic colic surgery

Im Rahmen dieser Promotion wurde eine prospektive, randomisierte Studie zu postoperativen Komplikationen bei Pferden nach chirurgischen Eingriffen bei kolikoperierten Patienten erstellt. Ziel dieser Studie bestand darin, Unterschiede hinsichtlich postoperativer Komplikationen zwischen zwei verschiedenen Regimen von Antibiotika (Single-Shot vs. konventionelle Antibiotikagabe) zu evaluieren und eine Single-Shot Gabe als antibiotische Prophylaxe bei diesen Eingriffen zukünftig in Erwägung zu ziehen. Für die prospektive Studie konnten 67 Pferde ausgewertet werden, die in der Klinik für Pferde, FU Berlin einer Kolikoperation unterzogen wurden. Die Pferde wurden randomisiert einer der beiden Gruppen zugeordnet. Alle Pferde wurden intensiv über zehn Tage überwacht. In den Wundbeurteilungen an Tag drei, fünf und zehn wurden mittels eines Scores die Kriterien Exsudation, Schwellung und Nahtdehiszenz evaluiert. Weiterhin wurden die Blutwerte Leukozyten, Hämatokrit, Totalprotein, Serumamyloid A, Fibrinogen und Laktat prä- und postoperativ in den ersten fünf Tagen und am zehnten Tag ermittelt. 1. Hauptfrage: Ist eine Single-Shot Antibiotikagabe gleichwertig zu dem konventionellen perioperativen Antibiotikamanagement hinsichtlich möglicher, postoperativer Komplikationen? Mittels eines linearen Modells mit negativer Binomialverteilung wurden die Wundscores beurteilt. Es gab keinen signifikanten Unterschied hinsichtlich des Gesamtscores aller Tage zwischen den zu vergleichenden Gruppen (p = 0,985, lineare Regression). Sowohl bei der Entstehung von Wundinfektionen in den ersten zehn Tagen postoperativ (p = 0,2, exakter Fischer-Test) als auch bei der Entstehung von Wundinfektionen innerhalb dreißig Tagen postoperativ (p = 0,07, exakter Fischer-Test) gab es keinen signifikanten Unterschied. Auch hinsichtlich der Entstehung von postoperativer Colitis (p = 0,2, exakter Fischer-Test) als auch der Entstehung postoperativer hämolytischer Anämie (p = 0,317, exakter Fischer-Test) gab es keine signifikanten Unterschiede zwischen den Gruppen. Bei einer Studienpower von 60% und einem gesetzten Limit von 15%, entstand eine Differenz von 7,7 % zwischen den zu vergleichenden Gruppen in der Beurteilung des Auftretens von postoperativ auftretenden Komplikationen. Es existiert somit keine Unterlegenheit der Single-Shot Gruppe gegenüber der konventionellen Gruppe hinsichtlich des Auftretens von unerwünschten Komplikationen postoperativ. 1. Detailfragen: Welchen Einfluss hat das perioperative Antibiotikamanagement (SingleShot versus konventionell) auf die Entzündungsparameter im Blut des Pferdes? Welchen Einfluss hat das perioperative Management weiterhin auf die Entwicklung resistenter Erreger in der Nase und dem Kot der Pferde? Mithilfe eines linearen gemischten Regressionsmodells wurden die Blutwerte ausgewertet. Es existierte kein signifikanter Unterschied zwischen den SAA-Werten (p = 0,799, lineares Regressionsmodell), den Leukozyten (p = 0,998, lineares Regressionsmodell), den Fibrinogenwerten (p = 0,545, lineares Regressionsmodell) und den Totalproteinwerten (p = 0,722, lineares Regressionsmodell) zwischen den beiden zu vergleichenden Gruppen. Eine Auswertung der erhobenen Nasentupfer- und Kotproben wurde mit dem Chi-QuadratTest und dem exakten Fisher-Test erhoben. Auch hier fand sich kein signifikanter Unterschied von MRSA (p = 0,288, Chi-Quadrat-Test), MDR-Enterobacterales (p = 0,286, Chi-Quadrat– Test) und Acinetobacter baumannii (p = 0,493, exakter Fisher-Test) in den gewonnenen Nasentupferproben am dritten Tag postoperativ zwischen den zu vergleichenden Gruppen. Hinsichtlich der Kotproben an diesem Tag war kein signifikanter Unterschied bei dem Auftreten von MDR-Enterobacterales (p = 0,099, Chi-Quadrat-Test) und Acinetobacter baumannii (p = 0,503, Chi-Quadrat-Test) zu verzeichnen. Auch am zehnten Tag postoperativ gab es keinen signifikanten Unterschied hinsichtlich des Auftretens von MRSA (p = 0,920, Chi-QuadratTest), MDR-Enterobacterales (p = 0,481, exakter Fisher-Test) und Acinetobacter baumannii (p = 0,494, exakter Fisher-Test) in der Nase der Studienteilnehmer. Die Kotprobenergebnisse wiesen ebenfalls keine signifikanten Unterschiede zwischen den Gruppen mit MDR-Enterobacterales (p = 0,938, Chi-QuadratTest) und Acinetobacter baumannii (p = 0,240, exakter Fisher-Test) auf. Auffällig war jedoch, dass das Auftreten von multiresistenten E.coli in der Kotprobe und Acinetobacter baumannii in Nasentupfer- und Kotprobe ausschließlich in der konventionellen Gruppe nachzuweisen war. Es konnte jedoch keine Signifikanz nachgewiesen werden. Zusammenfassend ist der Einsatz eines perioperativen Managements in Form eines SingleShots bei sauberen und sauber-kontaminierten Operationen im Bereich der Kolikchirurgie zu empfehlen. Auch diese Studie verdeutlicht, dass ein vermehrter Einsatz von Antibiotika das Auftreten von resistenten Bakterien begünstigt. Um postoperative Komplikationen im Rahmen einer antimikrobiellen Single-Shot Gabe noch sicherer zu beurteilen, empfehlen sich weitere Studien mit höheren Fallzahlen.For this dissertation a randomised prospective study was carried out to assess postoperative complications when using two types of perioperative antibiotic treatments in horses undergoing emergency laparotomies. The aim of this study was to identify differences in postoperative complications between the two types of perioperative antibiotic treatments (Single-Shot versus 120 hour treatment). Sixtyseven horses who were undergoing a surgical emergency celiotomy in the clinic for horses at the FU Berlin were included in the prospective study. All patients were randomised to one of the treatment groups. All horses were monitored for ten days after surgery. A scoring system for exsudation, swelling and incisional dehiscence was established. A wound assesment on day three, five and ten after surgery was carried out. The blood parameters leucocytes, packed cell volume, total protein, serum amyloid a, fibrinogen and lactat were measured on the first to the fifth day and on day ten after surgery. 1. Main aim of the study: Is a use of single shot antibiotic treatment perioperative equivalent in regard to surgical side infections and other postoperative complications in horses to the common 120 hours antibiotic treatment? For the evaluation of the wound scores a linear regressionmodel with negative binomial distributation was used. There were no significant differences in the total scores between both antibiotic groups (p = 0.985, linear regression). In the development of surgical side infections in the first ten days after surgery (p = 0.2, exact fisher-test) as well as the development within thirty days postoperative (p = 0.07, fisher-exact test) there was no significant difference. Regarding the appearance of postsurgical colitis in horses (p = 0.2, exact fisher-test) and the development of postoperative hemolytic anemia (p = 0.317, exact fisher-test) there were no significant differences between both groups. With a study power of 60% and a staid limit of 15% there was a difference of 7,7% between the comparative groups in the evaluation of the occurence of postoperative complications. There was no inferiority of the single shot group adverse the conventional group with the regard to the occurence of postoperative complications. 1. Detail questions: How does the postoperative antimicrobial treatment (single shot versus conventional) influence the inflammation parameters in the blood of the horses? Is there an influence of the antibiotic treatment on the appearance of resistant bacteria in the nose and feces of the horses? With the use of a linear mixed regression modell the blood parameters of the horses were evaluated. There were no significant differences between the serum amyloid a parameters (p = 0.799, linear regressionmodel), the leucocytes (p = 0.988, linear regressionmodel), the fibrinogen parameters (p = 0.545, linear regressionmodel), and the total protein parameters (p = 0.722, linear regressionmodel), between both groups. To evaluate the samples from the noses and faeces the chi square test and the fisher exact test was utilized. There were no significant differences in the colonization with MRSA (p = 0.288, chi square test), MDR-Enterobacterales (p = 0.286, chi square test) and Acinetobacter baumannii (p = 0.493, fisher exact test) in the nose samples between the groups on the third day after surgery. The faecal samples on this day showed no significant differences in the colonization of MDR-Enterobacterales (p = 0.099, chi square test) and Acinetobacter baumannii (p = 0.503, chi square test). On the tenth day after surgery there were no significant difference on the establishment of MRSA (p = 0.920, chi square test), MDR-Enterobacterales (p = 0.481, fisher exact test) and Acinetobacter baumannii (p = 0.494, fisher exact test) in the nose samples between the groups. No significant differences were found in the establishment of MDR-Enterobacterales (p = 0.938, chi square test) and Acinetobacter baumannii (p = 0.240, fisher exact test) in the faecal samples on this day. It was noticable that the colonization with multidrug resistant E.coli and Acinetobacter baumannii in the nose and the faecal samples was only found in the conventional group. The resulat was not significant. In summary the use of a single shot as a perioperative prophylaxis in horses undergoing an emergency laparotomy in clean and clean-contamined surgerys is advisable. This prospective study shows that an increased use of antibiotic prophylaxis and therapy promote the occurence of resistant bacteria. To evaluate postoperative complications after a single shot use more safely additional studys with a higher study population would be recommendable

Measurements of transepithelial electrical resistance (TEER) are affected by junctional length in immature epithelial monolayers

The measurement of transepithelial electrical resistance (TEER) is a common technique to determine the barrier integrity of epithelial cell monolayers. However, it is remarkable that absolute TEER values of similar cell types cultured under comparable conditions show an immense heterogeneity. Based on previous observations, we hypothesized that the heterogeneity of absolute TEER measurements can not only be explained by maturation of junctional proteins but rather by dynamics in the absolute length of cell junctions within monolayers. Therefore, we analyzed TEER in epithelial cell monolayers of Caco2 cells during their differentiation, with special emphasis on both changes in the junctional complex and overall cell morphology within monolayers. We found that in epithelial Caco2 monolayers TEER increased until confluency, then decreased for some time, which was then followed by an additional increase during junctional differentiation. In contrast, permeability of macromolecules measured at different time points as 4 kDA fluorescein isothiocyanate (FITC)-dextran flux across monolayers steadily decreased during this time. Detailed analysis suggested that this observation could be explained by alterations of junctional length along the cell borders within monolayers during differentiation. In conclusion, these observations confirmed that changes in cell numbers and consecutive increase of junctional length have a critical impact on TEER values, especially at stages of early confluency when junctions are immature

Measurements of transepithelial electrical resistance (TEER) are affected by junctional length in immature epithelial monolayers

The measurement of transepithelial electrical resistance (TEER) is a common technique to determine the barrier integrity of epithelial cell monolayers. However, it is remarkable that absolute TEER values of similar cell types cultured under comparable conditions show an immense heterogeneity. Based on previous observations, we hypothesized that the heterogeneity of absolute TEER measurements can not only be explained by maturation of junctional proteins but rather by dynamics in the absolute length of cell junctions within monolayers. Therefore, we analyzed TEER in epithelial cell monolayers of Caco2 cells during their differentiation, with special emphasis on both changes in the junctional complex and overall cell morphology within monolayers. We found that in epithelial Caco2 monolayers TEER increased until confluency, then decreased for some time, which was then followed by an additional increase during junctional differentiation. In contrast, permeability of macromolecules measured at different time points as 4 kDA fluorescein isothiocyanate (FITC)-dextran flux across monolayers steadily decreased during this time. Detailed analysis suggested that this observation could be explained by alterations of junctional length along the cell borders within monolayers during differentiation. In conclusion, these observations confirmed that changes in cell numbers and consecutive increase of junctional length have a critical impact on TEER values, especially at stages of early confluency when junctions are immature

Wesen, Erscheinungsformen und Entwicklungsrichtungen des imperialistischen Ruestungsexports in die Entwicklungslaender

SIGLEMilitaerbibliothek Dresden - 90 14 0607 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman