Antibiotic resistance and virulence of faecal enterococci isolated from food-producing animals in Tunisia

Antimicrobial agents exert a selection pressure not only on pathogenic, but also on commensal bacteria of the intestinal tract of humans and animals. The aim of this work was to determine the occurrence of different enterococcal species and to analyse the prevalence of antimicrobial resistance and the mechanisms implicated, as well as the genetic diversity in enterococci recovered from faecal samples of food-producing animals (poultry, beef and sheep) in Tunisia. Antimicrobial resistance and the mechanisms implicated were studied in 87 enterococci recovered from 96 faecal samples from animals of Tunisian farms. Enterococcus faecium was the most prevalent species detected (46 %), followed by E. hirae (33.5 %). High percentages of resistance to erythromycin and tetracycline were found among our isolates, and lower percentages to aminoglycosides and ciprofloxacin were identified. Most of the tetracycline-resistant isolates carried the tet(M) and/or tet(L) genes. The erm(B) gene was detected in all erythromycin-resistant isolates. The ant(6)-Ia, aph(3)-Ia and aac(6)-aph(2) genes were detected in nine aminoglycoside-resistant isolates. Of our isolates, 11.5 % carried the gelE gene and exhibited gelatinase acitivity. The esp gene was detected in 10 % of our isolates and the hyl gene was not present in any isolate. The predominant species (E. faecium and E. hirae) showed a high genetic diversity by repetitive extragenic palindromic (REP)-PCR. Food animals might play a role in the spread through the food chain of enterococci with virulence and resistance traits to humans. © 2014 Springer-Verlag Berlin Heidelberg and the University of Milan

Deciphering resistance to Zymoseptoria tritici in the Tunisian durum wheat landrace accession ‘Agili39’

Septoria tritici blotch (STB), caused by Zymoseptoria tritici (Z. tritici), is an important biotic threat to durum wheat in the entire Mediterranean Basin. Although most durum wheat cultivars are susceptible to Z. tritici, research in STB resistance in durum wheat has been limited. Results: In our study, we have identified resistance to a wide array of Z. tritici isolates in the Tunisian durum wheat landrace accession ‘Agili39’. Subsequently, a recombinant inbred population was developed and tested under greenhouse conditions at the seedling stage with eight Z. tritici isolates and for five years under field conditions with three Z. tritici isolates. Mapping of quantitative trait loci (QTL) resulted in the identification of two major QTL on chromosome 2B designated as Qstb2B_1 and Qstb2B_2. The Qstb2B_1 QTL was mapped at the seedling and the adult plant stage (highest LOD 33.9, explained variance 61.6%), conferring an effective resistance against five Z. tritici isolates. The Qstb2B_2 conferred adult plant resistance (highest LOD 32.9, explained variance 42%) and has been effective at the field trials against two Z. tritici isolates. The physical positions of the flanking markers linked to Qstb2B_1 and Qstb2B_2 indicate that these two QTL are 5 Mb apart. In addition, we identified two minor QTL on chromosomes 1A (Qstb1A) and chromosome 7A (Qstb7A) (highest LODs 4.6 and 4.0, and explained variances of 16% and 9%, respectively) that were specific to three and one Z. tritici isolates, respectively. All identified QTL were derived from the landrace accession Agili39 that represents a valuable source for STB resistance in durum wheat. Conclusion: This study demonstrates that Z. tritici resistance in the ‘Agili39’ landrace accession is controlled by two minor and two major QTL acting in an additive mode. We also provide evidence that the broad efficacy of the resistance to STB in ‘Agili 39’ is due to a natural pyramiding of these QTL. A sustainable use of this Z. tritici resistance source and a positive selection of the linked markers to the identified QTL will greatly support effective breeding for Z. tritici resistance in durum wheat

Viral interference between low pathogenic avian influenza H9N2 and avian infectious bronchitis viruses in vitro and in ovo.

International audienceBACKGROUND:Low pathogenic avian influenza (LPAI) H9N2 and infectious bronchitis virus (IBV) are important pathogens of poultry, causing important economic losses for the sector. Replication interference between these two viruses was described using cell cultures (CC) and embryonated chicken eggs (ECE). Chicken embryo lung (CEL) and ECE were simultaneously or sequentially infected with IBV vaccine strain (H120) and LPAIV-H9N2 (A/Ck/TUN/145/2012) to evaluate viral interactionsin vitro and in ovo, respectively. Real-time RT-PCR was developed to specifically quantify both AIV and IBV genomes as well as viral gene copy numbers during mixed infections. The amount of IL-1 beta, in supernatants of co-infected cell cultures, was determined using an ELISA assay.RESULTS:Quantitative results of AIV and IBV co-infection showed that interferences between the two viruses yielded decreased viral growth. However, in the case of super-infection, the second virus, either AIV or IBV, induced a decrease in the growth of the first inoculated virus.CONCLUSION:It appears that either AIV or IBV has a negative impact on the other virus growth when they are inoculated simultaneously or sequentially. The ELISA results showed that higher level of secreted IL-1beta varies, depending on the viral interference conditions between both viruses, during mixed infections

A multiplex real-time RT-PCR for simultaneous detection of four most common avian respiratory viruses

International audienceA one-step multiplex real-time reverse transcription-PCR (rRT-PCR) assay was developed for simultaneous detection and quantification of four avian respiratory viruses: avian influenza virus (AIV), infectious bronchitis virus (IBV), Newcastle disease virus (NDV) and infectious laryngotracheitis virus (ILTV). In comparison with the singleplex rRT-PCR, the specificity, the sensitivity and the reproducibility of the new assay were evaluated and validated using 70 clinical samples. The optimal cutoff point, the corresponding limit of quantification (LoQ) and the limit of detection (LoD) were statistical established based on receiver operating characteristic (ROC) curve analysis. The results showed that the multiplex assay presents higher sensitivity and specificity. Correlation coefficients (R2) and amplification efficiencies (E) of all singleplex and multiplex rRT-PCR reactions are within the acceptable range. The 95% LoDs of multiplex assay were in the range [3–19] copies genomic/ µl, and its corresponding cutoff cycles were in the range [34.16–36.59]. No competitive inhibition for the detection of the four targets and no specific amplification or cross reactivity with other tested viruses was observed. Excellent results were attained in the inter-assay and intra-assay reproducibility evaluation. All identified samples by the multiplex rRT-PCR assay proved to be 100% concordant with the results of the singleplex assays. The results achieved showed that the multiplex assay is very suitable as a routine laboratory test for rapid and specific detection and quantification of co-infections in field samples

Genetic diversity and population structure of Zymoseptoria tritici on bread wheat in Tunisia using SSR markers

Septoria tritici blotch (STB) caused by Zymoseptoria tritici is the primary biotic stress for durum wheat production in Tunisia. The present study was aimed to decipher the genetic diversity and population structure of Z. tritici bread wheat isolates; in the northern regions of Tunisia this pathogen infected exclusively bread wheat. A total of 162 single-spore isolates were sampled during the 2015–2016 growing season from nine naturally infected bread wheat fields at four locations in the three main wheat-growing regions in Tunisia, previously reported as hot spots for Septoria disease (Cap Bon, Bizerte, and Beja). Collected isolates were fingerprinted using twelve polymorphic microsatellite markers to assess the genetic diversity and population structure of Z. tritici. All the microsatellite loci were polymorphic and a high genetic diversity was observed within the whole population. The highest Nei’s index value (0.42), Shannon Index (0.84) and private allele numbers (36) were found at the El Haouaria location (Cap Bon region). Furthermore, a moderate genetic differentiation within and among the surveyed locations (73% and 27%) was confirmed by analysis of molecular variance (AMOVA). On the other hand, the STRUCTURE program was shown to be less sensitive in revealing genetic structure especially at low levels of diversity. Thus, information on the genetic structure of the pathogen population collected from bread wheat is useful for designing and implementing durable and effective management strategies

Genetic diversity and population structure of Zymoseptoria tritici on bread wheat in Tunisia using SSR markers

Septoria tritici blotch (STB) caused by Zymoseptoria tritici is the primary biotic stress for durum wheat production in Tunisia. The present study was aimed to decipher the genetic diversity and population structure of Z. tritici bread wheat isolates; in the northern regions of Tunisia this pathogen infected exclusively bread wheat. A total of 162 single-spore isolates were sampled during the 2015–2016 growing season from nine naturally infected bread wheat fields at four locations in the three main wheat-growing regions in Tunisia, previously reported as hot spots for Septoria disease (Cap Bon, Bizerte, and Beja). Collected isolates were fingerprinted using twelve polymorphic microsatellite markers to assess the genetic diversity and population structure of Z. tritici. All the microsatellite loci were polymorphic and a high genetic diversity was observed within the whole population. The highest Nei’s index value (0.42), Shannon Index (0.84) and private allele numbers (36) were found at the El Haouaria location (Cap Bon region). Furthermore, a moderate genetic differentiation within and among the surveyed locations (73% and 27%) was confirmed by analysis of molecular variance (AMOVA). On the other hand, the STRUCTURE program was shown to be less sensitive in revealing genetic structure especially at low levels of diversity. Thus, information on the genetic structure of the pathogen population collected from bread wheat is useful for designing and implementing durable and effective management strategies

Antibiotic resistance and virulence of faecal enterococci isolated from food-producing animals in Tunisia

Antimicrobial agents exert a selection pressure not only on pathogenic, but also on commensal bacteria of the intestinal tract of humans and animals. The aim of this work was to determine the occurrence of different enterococcal species and to analyse the prevalence of antimicrobial resistance and the mechanisms implicated, as well as the genetic diversity in enterococci recovered from faecal samples of food-producing animals (poultry, beef and sheep) in Tunisia. Antimicrobial resistance and the mechanisms implicated were studied in 87 enterococci recovered from 96 faecal samples from animals of Tunisian farms. Enterococcus faecium was the most prevalent species detected (46 %), followed by E. hirae (33.5 %). High percentages of resistance to erythromycin and tetracycline were found among our isolates, and lower percentages to aminoglycosides and ciprofloxacin were identified. Most of the tetracycline-resistant isolates carried the tet(M) and/or tet(L) genes. The erm(B) gene was detected in all erythromycin-resistant isolates. The ant(6)-Ia, aph(3)-Ia and aac(6)-aph(2) genes were detected in nine aminoglycoside-resistant isolates. Of our isolates, 11.5 % carried the gelE gene and exhibited gelatinase acitivity. The esp gene was detected in 10 % of our isolates and the hyl gene was not present in any isolate. The predominant species (E. faecium and E. hirae) showed a high genetic diversity by repetitive extragenic palindromic (REP)-PCR. Food animals might play a role in the spread through the food chain of enterococci with virulence and resistance traits to humans. © 2014 Springer-Verlag Berlin Heidelberg and the University of Milan