Combined effects of spray-drying conditions and postdrying storage time and temperature on Salmonella choleraesuis and Salmonella typhimurium survival when inoculated in liquid porcine plasma

Altres ajuts: GC/2014DI066The objective of this study was to determine the effectiveness of the spray-drying process on the inactivation of Salmonella choleraesuis and Salmonella typhimurium spiked in liquid porcine plasma and to test the additive effect of immediate postdrying storage. Commercial spray-dried porcine plasma was sterilized by irradiation and then reconstituted (1:9) with sterile water. Aliquots of reconstituted plasma were inoculated with either S. choleraesuis or S. typhimurium, subjected to spray-drying at an inlet temperature of 200°C and an outlet temperature of either 71 or 80°C, and each spray-drying temperature combinations were subjected to either 0, 30 or 60 s of residence time () as a simulation of residence time typical of commercial dryers. Spray-dried samples were stored at either 4·0 ± 3·0°C or 23·0 ± 0·3°C for 15 days. Bacterial counts of each Salmonella spp., were completed for all samples. For both Salmonella spp., spray-drying at both outlet temperatures reduced bacterial counts about 3 logs at 0 s, while there was about a 5·5 log reduction at 60 s. Storage of all dried samples at either 4·0 ± 3·0°C or 23·0 ± 0·3°C for 15 days eliminate all detectable bacterial counts of both Salmonella spp. Safety of raw materials from animal origin like spray-dried porcine plasma (SDPP) may be a concern for the swine industry. Spray-drying process and postdrying storage are good inactivation steps to reduce the bacterial load of Salmonella choleraesuis and Salmonella typhimurium. For both Salmonella spp., spray-drying at 71°C or 80°C outlet temperatures reduced bacterial counts about 3 log at residence time (RT) 0 s, while there was about a 5.5 log reduction at RT 60 s. Storage of all dried samples at either 4.0 ± 3.0°C or 23.0 ± 0.3°C for 15 days was effective for eliminating detectable bacterial counts of both Salmonella spp. Significance and Impact of the Study: Safety of raw materials from animal origin like spray-dried porcine plasma (SDPP) may be a concern for the swine industry. Spray-drying process and postdrying storage are good inactivation steps to reduce the bacterial load of Salmonella choleraesuis and Salmonella typhimurium. For both Salmonella spp., spray-drying at 71°C or 80°C outlet temperatures reduced bacterial counts about 3 log at residence time (RT) 0 s, while there was about a 5.5 log reduction at RT 60 s. Storage of all dried samples at either 4.0 ± 3.0°C or 23.0 ± 0.3°C for 15 days was effective for eliminating detectable bacterial counts of both Salmonella spp

Diversity of Multi-Drug Resistant Avian Pathogenic Escherichia coli (APEC) Causing Outbreaks of Colibacillosis in Broilers during 2012 in Spain

Avian pathogenic Escherichia coli (APEC) are the major cause of colibacillosis in poultry production. In this study, a total of 22 E. coli isolated from colibacillosis field cases and 10 avian faecal E. coli (AFEC) were analysed. All strains were characterised phenotypically by susceptibility testing and molecular typing methods such as pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST). The presence of 29 virulence genes associated to APEC and human extraintestinal pathogenic E. coli (ExPEC) was also evaluated. For cephalosporin resistant isolates, cephalosporin resistance genes, plasmid location and replicon typing was assessed. Avian isolates belonged to 26 O:H serotypes and 24 sequence types. Out of 22 APEC isolates, 91% contained the virulence genes predictors of APEC; iutA, hlyF, iss, iroN and ompT. Of all strains, 34% were considered ExPEC. PFGE analysis demonstrated a high degree of genetic polymorphism. All strains were multi-resistant, including those isolated from healthy animals. Eleven strains were resistant to cephalosporins; six contained blaCTX-M-14, two blaSHV-12, two blaCMY-2 and one blaSHV-2. Two strains harboured qnrA, and two qnrA together with aac(6’)-Ib-cr. Additionally, the emergent clone O25b:H4-B2-ST131 was isolated from a healthy animal which harboured blaCMY-2 and qnrS genes. Cephalosporin resistant genes were mainly associated to the presence of IncK replicons. This study demonstrates a very diverse population of multi-drug resistant E. coli containing a high number of virulent genes. The E. coli population among broilers is a reservoir of resistance and virulence-associated genes that could be transmitted into the community through the food chain. More epidemiological studies are necessary to identify clonal groups and resistance mechanisms with potential relevance to public health.This work was partially supported by the grants AGL2011- 28836 and AGL2013-47852-R from the Ministerio de Economía y Competitividad (España) and grants CN2012/303 and EM2014/001 (Consellería de Cultura, Educación e Ordenación Universitaria, Xunta de Galicia and the European Regional Development Fund, ERDF). Work from LMG is supported by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) and the European Social FundS

Molecular epidemiology and mechanisms of cephalosporin resistance in escherichia coli of different origins; broilers, flies and white storks

Las bacterias resistentes a antibióticos, y en concreto a betalactamasas de espectro extendido (BLEEs), se han convertido en un problema grave. Su prevalencia ha incrementado rápidamente a nivel mundial debido a una diseminación pandémica de plásmidos y a la introducción de genes de resistencia a BLEEs en clones exitosos (Coque, Baquero et al. 2008, Brolund 2014). Según la Organización Mundial de la Salud (OMS), los antimicrobianos más críticos en sanidad humana y animal son las cefalosporinas de tercera generación, las fluoroquinolonas, los macrólidos y los aminoglicósidos (Collignon, Powers et al. 2009, Collignon, Conly et al. 2016). Por lo tanto, esta tesis se ha centrado en las resistencias a cefalosporinas, y en particular resistencias a BLEEs y betalactamasas tipo AmpC. La mayoría de las investigaciones se han centrado en la epidemiología de las resistencias a antibióticos en medicina humana y veterinaria; sin embargo, en los últimos años ha habido un aumento de los estudios dirigidos a bacterias multiresistentes que circulan en ambientes naturales actuando como reservorios (Allen, Donato et al. 2010). Una vez las bacterias están presentes en el ambiente, pueden transmitirse a los humanos. A través de los estudios incluidos en la actual tesis, hemos investigado la aparición y los mecanismos de diseminación de Escherichia coli resistentes a cefalosporinas de diferentes nichos ecológicos; pollos de engorde como ejemplo de animales de producción, moscas (Musca domestica) como vectores mecánicos y reservorios de genes de resistencia que contribuyen a la diseminación de resistencias en el ambiente de granja; y finalmente, cigüeñas blancas (Ciconia ciconia) como ejemplo de vectores de larga distancia y centinelas de la presión humana. Con este enfoque, intentamos entender la transmisibilidad de las resistencias entre nichos diferentes; e identificar los clones, los genes de resistencia y los plásmidos involucrados.Bacteria resistant to antimicrobials, and specifically to extended-spectrum beta-lactamases (ESBLs), have become of increasing concern. Its prevalence has increased fast worldwide due to a pandemic dissemination of plasmids and the introduction of ESBL resistance genes into successful clones (Coque, Baquero et al. 2008, Brolund 2014). According to the World Health Organization (WHO), the most critical antimicrobials in human and animal health are third-generation cephalosporins, fluoroquinolones, macrolides and aminoglycosides (Collignon, Powers et al. 2009, Collignon, Conly et al. 2016). Therefore, this thesis has focused in resistance to cephalosporins and particularly to ESBLs and AmpC type of resistance. Due to the overuse of antimicrobials in humans and veterinary medicine, the emergence of multi-drug resistance has increased in the last decades. Most of the research has focused on the epidemiology of antibiotic resistance in human and veterinary medicine; however, in the last years it has been an increasing attention on how multiresistant bacteria circulates in natural environments and how these niches can act as reservoirs of resistant traits (Allen, Donato et al. 2010). Once the resistant bacteria are present in the environment, it can easily get in contact with humans. Through the studies compiled in this thesis, we have investigated the occurrence and mechanisms of dissemination of cephalosporin resistance (CR) Escherichia coli from different ecological niches; broilers as an example of food-producing animals, houseflies (Musca domestica) as mechanical vectors and reservoir of resistance genes contributing to the spread of resistance in the farm environment; and finally white storks (Ciconia ciconia) as an example of long-distance vectors and sentinels of human pressure. With this approach, we intend to understand the transmissibility of these resistances among different niches; and identify clones, resistance genes and plasmids involved

Molecular epidemiology and mechanisms of cephalosporin resistance in escherichia coli of different origins; broilers, flies and white storks

Las bacterias resistentes a antibióticos, y en concreto a betalactamasas de espectro extendido (BLEEs), se han convertido en un problema grave. Su prevalencia ha incrementado rápidamente a nivel mundial debido a una diseminación pandémica de plásmidos y a la introducción de genes de resistencia a BLEEs en clones exitosos (Coque, Baquero et al. 2008, Brolund 2014). Según la Organización Mundial de la Salud (OMS), los antimicrobianos más críticos en sanidad humana y animal son las cefalosporinas de tercera generación, las fluoroquinolonas, los macrólidos y los aminoglicósidos (Collignon, Powers et al. 2009, Collignon, Conly et al. 2016). Por lo tanto, esta tesis se ha centrado en las resistencias a cefalosporinas, y en particular resistencias a BLEEs y betalactamasas tipo AmpC. La mayoría de las investigaciones se han centrado en la epidemiología de las resistencias a antibióticos en medicina humana y veterinaria; sin embargo, en los últimos años ha habido un aumento de los estudios dirigidos a bacterias multiresistentes que circulan en ambientes naturales actuando como reservorios (Allen, Donato et al. 2010). Una vez las bacterias están presentes en el ambiente, pueden transmitirse a los humanos. A través de los estudios incluidos en la actual tesis, hemos investigado la aparición y los mecanismos de diseminación de Escherichia coli resistentes a cefalosporinas de diferentes nichos ecológicos; pollos de engorde como ejemplo de animales de producción, moscas (Musca domestica) como vectores mecánicos y reservorios de genes de resistencia que contribuyen a la diseminación de resistencias en el ambiente de granja; y finalmente, cigüeñas blancas (Ciconia ciconia) como ejemplo de vectores de larga distancia y centinelas de la presión humana. Con este enfoque, intentamos entender la transmisibilidad de las resistencias entre nichos diferentes; e identificar los clones, los genes de resistencia y los plásmidos involucrados.Bacteria resistant to antimicrobials, and specifically to extended-spectrum beta-lactamases (ESBLs), have become of increasing concern. Its prevalence has increased fast worldwide due to a pandemic dissemination of plasmids and the introduction of ESBL resistance genes into successful clones (Coque, Baquero et al. 2008, Brolund 2014). According to the World Health Organization (WHO), the most critical antimicrobials in human and animal health are third-generation cephalosporins, fluoroquinolones, macrolides and aminoglycosides (Collignon, Powers et al. 2009, Collignon, Conly et al. 2016). Therefore, this thesis has focused in resistance to cephalosporins and particularly to ESBLs and AmpC type of resistance. Due to the overuse of antimicrobials in humans and veterinary medicine, the emergence of multi-drug resistance has increased in the last decades. Most of the research has focused on the epidemiology of antibiotic resistance in human and veterinary medicine; however, in the last years it has been an increasing attention on how multiresistant bacteria circulates in natural environments and how these niches can act as reservoirs of resistant traits (Allen, Donato et al. 2010). Once the resistant bacteria are present in the environment, it can easily get in contact with humans. Through the studies compiled in this thesis, we have investigated the occurrence and mechanisms of dissemination of cephalosporin resistance (CR) Escherichia coli from different ecological niches; broilers as an example of food-producing animals, houseflies (Musca domestica) as mechanical vectors and reservoir of resistance genes contributing to the spread of resistance in the farm environment; and finally white storks (Ciconia ciconia) as an example of long-distance vectors and sentinels of human pressure. With this approach, we intend to understand the transmissibility of these resistances among different niches; and identify clones, resistance genes and plasmids involved

Molecular epidemiology and mechanisms of cephalosporin resistance in Escherichia coli of different origins : broilers, flies and white storks = Epidemiología molecular y mecanismos de resistencia a cefalosporinas en Escherichia coli de diferentes orígenes : pollos de engorde, moscas y cigüeñas blancas /

Departament responsable de la tesi: Departament de Medicina i Cirurgia Animals.A la portada: CReSA, IRTA.Las bacterias resistentes a antibióticos, y en concreto a betalactamasas de espectro extendido (BLEEs), se han convertido en un problema grave. Su prevalencia ha incrementado rápidamente a nivel mundial debido a una diseminación pandémica de plásmidos y a la introducción de genes de resistencia a BLEEs en clones exitosos (Coque, Baquero et al. 2008, Brolund 2014). Según la Organización Mundial de la Salud (OMS), los antimicrobianos más críticos en sanidad humana y animal son las cefalosporinas de tercera generación, las fluoroquinolonas, los macrólidos y los aminoglicósidos (Collignon, Powers et al. 2009, Collignon, Conly et al. 2016). Por lo tanto, esta tesis se ha centrado en las resistencias a cefalosporinas, y en particular resistencias a BLEEs y betalactamasas tipo AmpC. La mayoría de las investigaciones se han centrado en la epidemiología de las resistencias a antibióticos en medicina humana y veterinaria; sin embargo, en los últimos años ha habido un aumento de los estudios dirigidos a bacterias multiresistentes que circulan en ambientes naturales actuando como reservorios (Allen, Donato et al. 2010). Una vez las bacterias están presentes en el ambiente, pueden transmitirse a los humanos. A través de los estudios incluidos en la actual tesis, hemos investigado la aparición y los mecanismos de diseminación de Escherichia coli resistentes a cefalosporinas de diferentes nichos ecológicos; pollos de engorde como ejemplo de animales de producción, moscas (Musca domestica) como vectores mecánicos y reservorios de genes de resistencia que contribuyen a la diseminación de resistencias en el ambiente de granja; y finalmente, cigüeñas blancas (Ciconia ciconia) como ejemplo de vectores de larga distancia y centinelas de la presión humana. Con este enfoque, intentamos entender la transmisibilidad de las resistencias entre nichos diferentes; e identificar los clones, los genes de resistencia y los plásmidos involucrados.Bacteria resistant to antimicrobials, and specifically to extended-spectrum beta-lactamases (ESBLs), have become of increasing concern. Its prevalence has increased fast worldwide due to a pandemic dissemination of plasmids and the introduction of ESBL resistance genes into successful clones (Coque, Baquero et al. 2008, Brolund 2014). According to the World Health Organization (WHO), the most critical antimicrobials in human and animal health are third-generation cephalosporins, fluoroquinolones, macrolides and aminoglycosides (Collignon, Powers et al. 2009, Collignon, Conly et al. 2016). Therefore, this thesis has focused in resistance to cephalosporins and particularly to ESBLs and AmpC type of resistance. Due to the overuse of antimicrobials in humans and veterinary medicine, the emergence of multi-drug resistance has increased in the last decades. Most of the research has focused on the epidemiology of antibiotic resistance in human and veterinary medicine; however, in the last years it has been an increasing attention on how multiresistant bacteria circulates in natural environments and how these niches can act as reservoirs of resistant traits (Allen, Donato et al. 2010). Once the resistant bacteria are present in the environment, it can easily get in contact with humans. Through the studies compiled in this thesis, we have investigated the occurrence and mechanisms of dissemination of cephalosporin resistance (CR) Escherichia coli from different ecological niches; broilers as an example of food-producing animals, houseflies (Musca domestica) as mechanical vectors and reservoir of resistance genes contributing to the spread of resistance in the farm environment; and finally white storks (Ciconia ciconia) as an example of long-distance vectors and sentinels of human pressure. With this approach, we intend to understand the transmissibility of these resistances among different niches; and identify clones, resistance genes and plasmids involved

Impact of the use of β-lactam antimicrobials on the emergence of Escherichia coli isolates resistant to cephalosporins under standard pig-rearing conditions

The aim of this study was to evaluate if the treatments with ceftiofur and amoxicillin are risk factors for the emergence of cephalosporin resistant (CR) E. coli in a pig farm during the rearing period. One hundred 7-day-old piglets were divided into two groups, a control (n = 50) group and a group parenterally treated with ceftiofur (n = 50). During the fattening period, both groups were subdivided in two. A second treatment with amoxicillin was administered in feed to two of the four groups, as follows: group 1 (untreated, n = 20), group 2 (treated with amoxicillin, n = 26), group 3 (treated with ceftiofur, n = 20), and group 4 (treated with ceftiofur and amoxicillin, n = 26). During treatment with ceftiofur, fecal samples were collected before treatment (day 0) and at days 2, 7, 14, 21, and 42 posttreatment, whereas with amoxicillin, the sampling was extended 73 days posttreatment. CR E. coli bacteria were selected on MacConkey agar with ceftriaxone (1 mg/liter). Pulsed-field gel electrophoresis (PFGE), MICs of 14 antimicrobials, the presence of cephalosporin resistance genes, and replicon typing of plasmids were analyzed. Both treatments generated an increase in the prevalence of CR E. coli, which was statistically significant in the treated groups. Resistance diminished after treatment. A total of 47 CR E. coli isolates were recovered during the study period; of these, 15 contained blaCTX-M-1, 10 contained blaCTX-M-14, 4 contained blaCTX-M-9, 2 contained blaCTX-M-15, and 5 contained blaSHV-12. The treatment with ceftiofur and amoxicillin was associated with the emergence of CR E. coli during the course of the treatment. However, by the time of finishing, CR E. coli bacteria were not recovered from the animals

Foraging at solid urban waste disposal sites as risk factor for cephalosporin and colistin resistant Escherichia coli carriage in white storks (Ciconia ciconia)

White stork (Ciconia ciconia) may act as a reservoir and vehicle of cephalosporin resistant (CR) Escherichia coli. Between 2011 and 2014, we sampled white storks from colonies exposed to different degrees of anthropic pressure across the major areas of natural distribution of white storks in Spain. Cloacal swab samples (n = 467) were obtained from individuals belonging to 12 different colonies from six different regions. Additionally, 70 samples were collected from recently deposited droppings at the base of nesting platforms. We phenotypically characterized E. coli isolates, confirmed presence of CR genes and classified plasmids. Risk factors for acquiring these genes were assessed. Overall, 8.8% (41 out of 467) storks carried CR E. coli in their cloaca and five (7.1%) were identified from recently deposited droppings; therefore, 46 isolates were further characterized. Of them, 20 contained blaCTX–M–1, nine blaCMY–2, six blaCTX–M–14, four blaSHV–12, three blaCTX–M–15, two blaCTX–M–32, one blaCTX–M–1 together with blaCMY–2, and one blaCTX–M–1 together with blaSHV–12. All were multidrug-resistant, and four harbored the plasmid-mediated colistin resistance mcr-1 gene. CR genes were associated with the presence of IncI1, IncFIB, and IncN replicon families. XbaI-macrorestriction analysis revealed a great diversity among most of the XbaI-PFGE types, but indistinguishable types were also seen with isolates obtained from different locations. Clonal complex 10 was the most common among CR E. coli and two blaCTX–M–15 positive isolates were identified as B2-ST131. Carriage of CR E. coli was significantly higher in colonies located close to solid urban waste disposal sites in which foraging on human waste was more likely and in one case to cattle grazing. The co-occurrence of blaCMY–2 and mcr-1 on plasmids of E. coli isolated from wild birds as early as 2011 is of note, as the earliest previous report of mcr-1 in wild birds is from 2016. Our study shows that foraging at landfills and in association with cattle grazing are important risk factors for the acquisition of CR E. coli in white storks.This work was partially supported by the grants AGL2013-47852-R and RTI2018-095586-B-C22 from the Ministerio de Economía y Competitividad (MINECO), project RTA2011-00111-C03 funded by the Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA) and by the CERCA program from Generalitat de Catalunya. The VHIR-HUVH was supported by Plan Nacional de I+D+i 2013–2016, Instituto de Salud Carlos III and the Spanish Ministry of Health (FIS PI15/00604) and the Subdirección General de Redes y Centros de Investigación Cooperativa, MINECO, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0003) – co-financed by European Development Regional Fund “A way to achieve Europe. AM-M is a Ph.D. student registered with the Universidad Autònoma de Barcelona. The contract of LM-G was supported by INIA and the European Social Fund. JP-P was funded by a grant from the Complutense University of Madrid (CT45/15-CT46/15). FT-M was supported by the Isabel Maria Lopez Martinez Memorial Scholarship at the University of Saskatchewan (Canada). Trapping activities of adult birds were partially supported in the framework of SEO/BirdLife Migra program and financed by Fundación Iberdrola España and Storch Schweiz, by grant CGL2012-32544 from the MINECO, and by grant 511/2012 from the Organismo Autónomo de Parques Nacionales.Peer reviewe

Distribution of virulence-associated gene profiles, phylogeny, serotyping and MLST results among all 32 strains.

<p>Phylo, phylogroup; ST, sequence type; Cplx, clonal complex. Adhesins <i>fimH</i> (D-mannose-specific adhesin of type I fimbriae), <i>fimAvMT78</i> (FimA variant MT78 of type 1 fimbriae), <i>papEF</i> and <i>papG</i> (P fimbria subunits), and <i>sfa/focDE</i> (S fimbrial adhesin/putative F1C fimbrial adhesin); toxins <i>cdtB</i> (cytolethal distending toxin), <i>hlyF</i> (hemolysin F), and <i>astA</i> (EAST1, enteroaggregative E. coli heat-stable toxin); siderophores fyuA (yersiniabactin), <i>iutA</i> (aerobactin), <i>iroN</i> (novel catecholate siderophore receptor), and <i>irp-2</i> (iron repressible associated with yersiniabactin synthesis); protectins <i>kpsM</i> (groups II and III, specifically targeting the K1, K2 and K5 genes of group II capsules), <i>cvaC</i> (ColV, colicin V from serum resistance-associated plasmids), <i>iss</i> (surface exclusion serum survival protein), and <i>traT</i> (serum resistance); miscellaneous virulence genes <i>ompT</i> (protease), <i>ibeA</i> (invasion of brain endothelium), <i>malX</i> (PAI, pathogenicity island marker), and <i>usp</i> (uropathogenic-specific protein, bacteriocin).</p><p>^a Virulence-associated genes shown in boldface are the five genes characteristics of APEC strains.</p><p>Distribution of virulence-associated gene profiles, phylogeny, serotyping and MLST results among all 32 strains.</p