Salah Hammami

Prevalence and Diversity of Integrons and Associated Resistance Genes in Escherichia coli Isolates from Poultry Meat in Tunisia

Fifty-five Escherichia coli isolates were acquired from chicken and turkey meat obtained from two slaughterhouses in Tunis. Eighty-nine percent, 80%, 78%, 67%, 45%, 27%, 7%, 4%, and 2% of these isolates showed resistance to tetracycline, trimethoprim/sulfamethoxazole, streptomycin, nalidixic acid, ampicillin, chloramphenicol, ciprofloxacin, colistine, and gentamicin, respectively. No resistance was detected to cefotaxime, ceftazidime, or amikacin. bla(TEM) gene was found in 22 of 25 ampicillin-resistant isolates, and 1 isolate harbored bla(OXA-1) gene. Tetracycline resistance was predominately mediated by the tetA gene. The sul1, sul2, and sul3 genes, alone or combined, were detected in 46 of 48 sulfonamide-resistant isolates, and sul1 and sul3 were included in class 1 integrons in some cases. Sixty percent of isolates harbored integrons (class 1, 30 isolates; class 2, 5 isolates). Class 2 integrons contained in all cases the dfrA1-sat1-aadA1-orfX gene cassette arrangement. Nine gene cassette arrangements have been detected among class 1 integrons, containing different alleles of dfrA (five alleles) and aadA (2 alleles) genes, which encode trimethoprim and streptomycin resistance, respectively. An uncommon gene cassette array (sat-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3) has been identified in three class 1 integron-positive isolates, and one additional isolate had this same structure with the insertion of IS26 inside the aadA1 gene (included in GenBank with accession no. FJ160769). The 55 studied isolates belong to the four phylogenic groups of E. coli, and phylogroups A and D were the most prevalent ones. At least one virulence-associated gene (fimA, papC, or aer) was detected in 44 of the 55 (80%) studied isolates. E. coli isolates of poultry origin could be a reservoir of antimicrobial-resistance genes and of integrons, and its evolution should be tracked in the future.

Occurrence of blaCTX-M-1,qnrB1 and virulence genes in avian ESBL-producing Escherichia coli isolates from Tunisia

Avian ESBL-producing Escherichia coli isolates have been increasingly reported worldwide. Animal to human dissemination, via food chain or direct contact, of these resistant bacteria has been reported. In Tunisia, little is known about avian ESBL- producing E. coli and further studies are needed. Seventeen ESBL-producing Escherichia coli isolates from poultry feces from two farms (Farm 1 and farm 2) in the North of Tunisia have been used in this study. Eleven of these isolates (from farm 1) have the same resistance profile to nalidixic acid, sulfonamides, streptomycin, tetracycline, and norfloxacine (intermediately resistant). Out of the six isolates recovered from farm 2, only one was co-resistant to tetracycline. All isolates, except one, harbored blaCTX-M-1 gene, and one strain co-harbored the blaTEM-1 gene. The genes tetA and tetB were carried, respectively, by 11 and 1 amongst the 12 tetracycline-resistant isolates. Sulfonamides resistance was encoded by sul1, sul2, and sul3 genes in 3, 17, and 5 isolates, respectively. The qnrB1 was detected in nine strains, one of which co-harbored qnrS1 gene. The search for the class 1 and 2 integrons by PCR showed that in farm 1, class 1 and 2 integrons were found in one and ten isolates, respectively. In farm 2, class 1 integron was found in only one isolate, class 2 was not detected. Only one gene cassette arrangement was demonstrated in the variable regions (VR) of the 10 int2-positive isolates: dfrA1- sat2-aadA1. The size of the VR of the class 1 integron was approximately 250 bp in one int1-positive isolate, whereas in the second isolate, no amplification was observed. All isolates of farm 1 belong to the phylogroup A (sub-group A0). However, different types of phylogroups in farm 2 were detected. Each of the phylogroups A1, B22, B23 was detected in one strain, while the D2 phylogroup was found in 3 isolates. The virulence genes iutA, fimH, and traT were detected in 3, 7, and 3 isolates, respectively. Two types of gene combination were detected: iutA+fimH+traT in 3 isolates and iutA+fimH in one isolate. The isolates recovered in farm 1 showed the same profile of PFGE macro-restriction, while isolates of farm 2 presented unrelated PFGE patterns. We conclude that these avian ESBL-producing E. coli isolates show homo- and heterogenic genetic background and that plasmids harboring ESBL genes could be involved in the dissemination of this resistance phenotype.

Spatio-Temporal Identification of Areas Suitable for West Nile Disease in the Mediterranean Basin and Central Europe

West Nile virus (WNV) is a mosquito-transmitted Flavivirus belonging to the Japanese encephalitis antigenic complex of the Flaviviridae family. Its spread in the Mediterranean basin and the Balkans poses a significant risk to human health and forces public health officials to constantly monitor the virus transmission to ensure prompt application of preventive measures. In this context, predictive tools indicating the areas and periods at major risk of WNV transmission are of paramount importance. Spatial analysis approaches, which use environmental and climatic variables to find suitable habitats for WNV spread, can enhance predictive techniques. Using the Mahalanobis Distance statistic, areas ecologically most suitable for sustaining WNV transmission were identified in the Mediterranean basin and Central Europe. About 270 human and equine clinical cases notified in Italy, Greece, Portugal, Morocco, and Tunisia, between 2008 and 2012, have been considered. The environmental variables included in the model were altitude, slope, night time Land Surface Temperature, Normalized Difference Vegetation Index, Enhanced Vegetation Index, and daily temperature range. Seasonality of mosquito population has been modelled and included in the analyses to produce monthly maps of suitable areas for West Nile Disease. Between May and July, the most suitable areas are located in Tunisia, Libya, Egypt, and North Cyprus. Summer/Autumn months, particularly between August and October, characterize the suitability in Italy, France, Spain, the Balkan countries, Morocco, North Tunisia, the Mediterranean coast of Africa, and the Middle East. The persistence of suitable conditions in December is confined to the coastal areas of Morocco, Tunisia, Libya, Egypt, and Israel.

Towards the PCR-based identification of Palaearctic Culicoides biting midges (Diptera: Ceratopogonidae): Results from an international ring trial targeting four species of the subgenus Avaritia

BackgroundBiting midges of the genus Culicoides (Diptera: Ceratopogonidae) are biological vectors of internationally important arboviruses. To understand the role of Culicoides in the transmission of these viruses, it is essential to correctly identify the species involved. Within the western Palaearctic region, the main suspected vector species, C. obsoletus, C. scoticus, C. dewulfi and C. chiopterus, have similar wing patterns, which makes it difficult to separate and identify them correctly.MethodsIn this study, designed as an inter-laboratory ring trial with twelve partners from Europe and North Africa, we assess four PCR-based assays which are used routinely to differentiate the four species of Culicoides listed above. The assays based on mitochondrial or ribosomal DNA or microarray hybridisation were tested using aliquots of Culicoides DNA (extracted using commercial kits), crude lysates of ground specimens and whole Culicoides (265 individuals), and non-Culicoides Ceratopogonidae (13 individuals) collected from across Europe.ResultsA total of 800 molecular assays were implemented. The in-house assays functioned effectively, although specificity and sensitivity varied according to the molecular marker and DNA extraction method used. The Obsoletus group specificity was overall high (95-99%) while the sensitivity varied greatly (59.6-100%). DNA extraction methods impacted the sensitivity of the assays as well as the type of sample used as template for the DNA extraction.ConclusionsThe results are discussed in terms of current use of species diagnostic assays and the future development of molecular tools for the rapid differentiation of cryptic Culicoides species.

Phenotypic and Genotypic Characterization of Salmonella enterica Recovered from Poultry Meat in Tunisia and Identification of New Genetic Traits

Thirty-seven Salmonella enterica isolates obtained from poultry meat in Tunisia were included in this study for characterization of antibiotic resistance mechanisms. High percentages of resistance were detected to ampicillin, sulfonamides, tetracycline, nalidixic acid, and streptomycin (32.4%-89.2%), and lower percentages to amoxicillin-clavulanic acid, kanamycin, amikacin, trimethoprim-sulfamethoxazol, and chloramphenicol (2.7%-18.9%). All strains showed susceptibility to ceftazidime, cefotaxime, gentamicin, and ciprofloxacin. Class 1 integrons were detected in 30% of Salmonella isolates, and four different gene cassette arrangements were detected, including genes implicated in resistance to aminoglycosides (aadA1 and aadA2) and trimethoprim (dfrA1). Four different Pc variants (PcW, PcH1, PcH1(TTN-10), PcW(TGN-10)) with inactive P2 have been found among these isolates. Integron-positive isolates were ascribed to eight different serotypes. A Salmonella Schwarzengrund isolate harbored a new class 1 integron containing the qacH-dfrA1b-aadA1b-catB2 gene cassette arrangement, with the very unusual PcH1(TTN-10) promoter, which has been registered in GenBank (accession no. HQ874651). Different plasmid replicon types were demonstrated among integron-positive isolates: IncI1 (8 isolates), IncN (8), IncP (2), IncFIB (2), and IncFII (2). Ten different pulsed-field gel electrophoresis profiles were detected among the 11 integron-positive isolates and 8 different sequence types were identified by multilocus sequence typing, one of them (registered as ST867) was new, detected in 3 Salmonella Zanzibar isolates. A high diversity of clones is observed among poultry Salmonella isolates and a high proportion of them show a multiresistant phenotype with very diverse mobile genetic structures that could be implicated in bacterial dissemination in different environments.

Detection of circulation of West Nile virus in equine in the north-west of Tunisia

Two outbreaks of West Nile Fever (FWN) were observed in the Sahel of Tunisia in 1997 and 2003. Several cases of meningitis and meningoencephalitis have been described in humans during these two outbreaks. However, no animal or clinical findings or seroconversion have been detected despite a high seroprevalence in human beings found around the affected areas. Few data are available regarding the spreading of this virus in other parts of the country. The purpose of this study was to detect a possible WNV spread in horses in some areas of Tunisia considered to be at risk for WNV but which had not been affected by previous outbreaks. A total of 133 equine blood samples were collected in six delegations from three governorates in the north-west of Tunisia. A second blood sampling was taken from animals that were tested negative after the first sampling for IgG to identify possible seroconversion. Detection of IgG was done using competitive ELISA. A significant viral spread was detected in the study area. Out of 133 samples tested for IgG during the first sampling, 36 samples were tested positive (27.1%). Two seroconversions were detected between September and October 2008 out of 84 samples tested. Statistical analysis showed a significant association between the presence of a wetland within 10 km and seroconversion. The presence of cattle seems to be a protective factor.

Molecular epidemiology of bluetongue virus serotype 1 circulating in Italy and its connection with northern Africa

Western BTV-1 emerged in the Mediterranean basin in 2006 and it has since been isolated in southern and northern European countries. Six BTV-1 strains isolated from infected sheep in Italy between 2006 and 2013 and a BTV-1 strain isolated from an infected sheep in Tunisia in 2011 were fully sequenced. The seven strains were shown to be nearly identical in each gene segment. The Seg-2 sequences of the BTV-1 strains group according to the year of isolation reflecting the time of BTV incursions in Italy. Combined results suggest that BTV-1 strains isolated in Sardinia, Sicily and mainland Italy in 2012 and 2013 have a direct northern African origin. The Italian strains originated from a strain closely related to a BTV-1 strain isolated in Tunisia in 2011. Better surveillance programs with northern and sub-Saharan African countries should be implemented making the control of spread of BTV easier and effective.

Identifying Areas Suitable for the Occurrence of Rift Valley Fever in North Africa: Implications for Surveillance.

Rift Valley fever (RVF) is a vector-borne zoonotic disease that has caused widespread outbreaks throughout Africa and the Arabian Peninsula, with serious consequences for livestock-based economies and public health. Although there have never been any reports of RVF in Morocco, Algeria, Tunisia and Libya, it is a priority disease in the Maghreb, due to the threat of introduction of the virus through transboundary livestock movements or infected mosquito vectors. However, the implementation of surveillance activities and early warning contingency plans requires better knowledge of the epidemiological situation. We conducted a multicriteria decision analysis, integrating host distribution with a combination of important ecological factors that drive mosquito abundance, to identify hotspots and suitable time periods for RVF enzootic circulation (i.e. stable transmission at a low to moderate level for an extended period of time) and an RVF epizootic event (i.e. a sudden occurrence of a large number of infected animals over a large geographic area) in the Maghreb. We also modelled vector species distribution using available information on vector presence and habitat preference. We found that the northern regions of the Maghreb were moderately suitable for RVF enzootics, but highly suitable for RVF epizootics. The vector species distribution model identified these regions as the most favourable mosquito habitats. Due to the low density of animal hosts and arid conditions, the desert region showed low RVF suitability, except in oases. However, the presence of competent vectors in putative unsuitable areas underlines the need for further assessments of mosquito habitat preference. This study produced monthly RVF suitability maps useful for animal health managers and veterinary services involved in designing risk-based surveillance programmes. The suitability maps can be further enhanced using existing country-specific sources of information and by incorporating knowledge - as it becomes available - on the epidemiology of the disease and distribution of vectors in the Maghreb.

Détection de la circulation de virus West Nile chez les Équidés dans le nord-ouest de la Tunisie

Two outbreaks of West Nile Fever (FWN) were observed in the Sahel of Tunisia in 1997 and 2003. Several cases of meningitis and meningoencephalitis have been described in humans during these two outbreaks. However, no animal or clinical findings or seroconversion have been detected despite a high seroprevalence in human beings found around the affected areas. Few data are available regarding the spreading of this virus in other parts of the country. The purpose of this study was to detect a possible WNV spread in horses in some areas of Tunisia considered to be at risk for WNV but which had not been affected by previous outbreaks. A total of 133 equine blood samples were collected in six delegations from three governorates in the north-west of Tunisia. A second blood sampling was taken from animals that were tested negative after the first sampling for IgG to identify possible seroconversion. Detection of IgG was done using competitive ELISA. A significant viral spread was detected in the study area. Out of 133 samples tested for IgG during the first sampling, 36 samples were tested positive (27.1%). Two seroconversions were detected between September and October 2008 out of 84 samples tested. Statistical analysis showed a significant association between the presence of a wetland within 10 km and seroconversion. The presence of cattle seems to be a protective factor.

Strengthening cooperation on vectorborne diseases in Europe

In 2007 a new European network was established to improve the coordination of research on three midgeborne viral diseases that are expanding their range in Europe. Writing on behalf of the members of Med_Reo_Net, Catherine Cêtre-Sossah, the coordinator of this project, explains the aims of the initiative.