Molecular characterisation of antimicrobial resistance and virulence genes in Escherichia coli strains isolated from diarrhoeic and healthy rabbits in Tunisia

[EN] The purpose of this study was to identify Escherichia coli isolates in diarrhoeic and healthy rabbits in Tunisia and characterise their virulence and antibiotic resistance genes. In the 2014-2015 period, 60 faecal samples from diarrhoeic and healthy rabbits were collected from different breeding farms in Tunisia. Susceptibility to 14 antimicrobial agents was tested by disc diffusion method and the mechanisms of gene resistance were evaluated using polymerase chain reaction and sequencing methods. Forty E. coli isolates were recovered in selective media. High frequency of resistance to tetracycline (95%) was detected, followed by different levels of resistance to sulphonamide (72.5%), streptomycin (62.5%), trimethoprim-sulfamethoxazole (60%), nalidixic acid (32.5%), ampicillin (37.5%) and ticarcillin (35%). E. coli strains were susceptible to cefotaxime, ceftazidime and imipenem. Different variants of blaTEM, tet, sul genes were detected in most of the strains resistant to ampicillin, tetracycline and sulphonamide, respectively. The presence of class 1 integron was studied in 29 sulphonamide-resistant E. coli strains from which 15 harboured class 1 integron with four different arrangements of gene cassettes, dfrA17+aadA5 (n=9), dfrA1 + aadA1 (n=4), dfrA12 + addA2 (n=1), dfrA12+orf+addA2 (n=1). The qnrB gene was detected in six strains out of 13 quinolone-resistant E. coli strains. Seventeen E. coli isolates from diarrhoeic rabbits harboured the enteropathogenic eae genes associated with different virulence genes tested (fimA, cnf1, aer), and affiliated to B2 (n=8) and D (n=9) phylogroups. Isolated E. coli strains from healthy rabbit were harbouring fim A and/or cnf1 genes and affiliated to A and B1 phylogroups. This study showed that E. coli strains from the intestinal tract of rabbits are resistant to the widely prescribed antibiotics in medicine. Therefore, they constitute a reservoir of antimicrobial-resistant genes, which may play a significant role in the spread of antimicrobial resistance. In addition, the eae virulence gene seemed to be implicated in diarrhoea in breeder rabbits in Tunisia.The work was supported by Tunisian Ministry of Higher Education, Scientific Research and Technology (LR16IP03). Many thanks go to the members of the Department of Animal Production, National Institute of Agronomy of Tunisia for their help in collecting the samples.Ben Rhouma, R.; Jouini, A.; Klibi, A.; Hamrouni, S.; Boubaker, A.; Kmiha, S.; Maaroufi, A. (2020). Molecular characterisation of antimicrobial resistance and virulence genes in Escherichia coli strains isolated from diarrhoeic and healthy rabbits in Tunisia. World Rabbit Science. 28(2):81-91. https://doi.org/10.4995/wrs.2020.10879OJS8191282Allen H.K., Donato J., Wang H.H, Cloud-Hansen K.A., Davies J. 2010. Call of the wild: antibiotic resistance genes in natural environments. 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Methicillin-Resistant <i>Staphylococcus</i> <i>aureus</i> Strains Isolated from Burned Patients in a Tunisian Hospital: Molecular Typing, Virulence Genes, and Antimicrobial Resistance

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the major causes of a variety of infections in hospitals and the community. Their spread poses a serious public health problem worldwide. Nevertheless, in Tunisia and other African countries, very little molecular typing data on MRSA strains is currently available. In our study, a total of 64 MRSA isolates were isolated from clinical samples collected from burned patients hospitalized in the Traumatology and Burns Center of Ben Arous in Tunisia. The identification of the collection was based on conventional methods (phenotypic and molecular characterization). The characterization of the genetic support for methicillin resistance was performed by amplification of the mecA gene by polymerase chain reaction (PCR), which revealed that 78.12% of S. aureus harbors the gene. The resistance of all the collection to different antibiotic families was studied. Indeed, the analysis of strain antibiotic susceptibility confirmed their multi-resistant phenotype, with high resistance to ciprofloxacin, gentamicin, penicillin, erythromycin, and tetracycline. The resistance to the last three antibiotics was conferred by the blaZ gene (73.43%), the erm(C) gene (1.56%), the msr(A) gene (6.25%), and tet(M) gene (7.81%), respectively. The clonal diversity of these strains was studied by molecular typing of the accessory gene regulator (agr) system, characterization of the SCCmec type, and spa-typing. The results revealed the prevalence of agr types II and III groups, the SCCmec type III and II cassettes, and the dominance of spa type t233. The characterization of the eight enterotoxins genes, the Panton-Valentine leukocidin and the toxic shock syndrome toxin, was determined by PCR. The percentage of virulence genes detected was for enterotoxins (55%), tst (71.88%), leukocidin E/D (79.69%), and pvl (1.56%) factors. Furthermore, our results revealed that the majority of the strains harbor IEC complex genes (94%) with different types. Our findings highlighted the emergence of MRSA strains with a wide variety of toxins, leukocidin associated with resistance genes, and specific genetic determinants, which could constitute a risk of their spread in hospitals and the environment and complicate infection treatment

Lineages, Virulence Gene Associated and Integrons among Extended Spectrum &beta;-Lactamase (ESBL) and CMY-2 Producing Enterobacteriaceae from Bovine Mastitis, in Tunisia

Extended Spectrum Beta-Lactamase (ESBL) Enterobacteriaceae are becoming widespread enzymes in food-producing animals worldwide. Escherichia coli and Klebseilla pneumoniae are two of the most significant pathogens causing mastitis. Our study focused on the characterization of the genetic support of ESBL/pAmpC and antibiotic resistance mechanisms in cefotaxime-resistant (CTXR) and susceptible (CTXS) Enterobacteriaceae isolates, recovered from bovine mastitis in Tunisia, as well as the analyses of their clonal lineage and virulence-associated genes. The study was carried out on 17 ESBL/pAmpC E. coli and K. pneumoniae and 50 CTXS&nbsp;E. coli. Detection of resistance genes and clonal diversity was performed by PCR amplification and sequencing. The following &beta;-lactamase genes were detected: blaCTX-M-15 (n = 6), blaCTX-M-15 + blaOXA-1 (2), bla CTX-M-15 + blaOXA-1 + blaTEM-1b (2), blaCTX-M-15 + blaTEM-1b (4), blaCMY-2 (3). The MLST showed the following STs: ST405 (n = 4 strains); ST58 (n = 3); ST155 (n = 3); ST471 (n = 2); and ST101 (n = 2). ST399 (n = 1) and ST617 (n = 1) were identified in p(AmpC) E. coli producer strains. The phylogroups A and B1 were the most detected ones, followed by the pathogenic phylogroup B2 that harbored the shigatoxin genes stx1/stx2, associated with the cnf, fimA, and aer virulence factors. The qnrA/qnrB, aac(6&prime;)-Ib-cr genes and integrons class 1 with different gene cassettes were detected amongst these CTXR/S isolated strains. The presence of different genetic lineages, associated with resistance and virulence genes in pathogenic bacteria in dairy farms, may complicate antibiotic therapies and pose a potential risk to public health

Inhibitory effect of high hydrostatic pressure, nisin, and moderate heating on the inactivation of Paenibacillus sp. and Terribacillus aidingensis spores isolated from UHT milk

International audienceThe sensitivity of Paenibacillus sp. and Terribacillus aidingensis spores to combined action of HP, nisin and moderate heating were investigated for the first time. The results showed that the effect of HP on spores varies with pressure level, treatment temperature, spore species and presence of nisin. T. aidingensis spores were more resistant than Paenibacillus sp., whatever the treatment applied. The inactivation rates of both spore species did not exceed 2.7log (CFU/mL) after HP treatment at 600 MPa/50°C/10 min. However, a high synergistic effect of nisin and HP was observed after a treatment at 500 MPa/10 min/50°C with addition of nisin during HP and into the recovery medium, leading to 6log (CFU/mL) and 4log (CFU/mL) reduction for Paenibacillus and Terribacillus spores, respectively. Finally, the fraction of germinated spores by HP was measured and the results showed that germination induction is not the only underlying mechanisms of nisin sensitization by HP

The Synergistic Effect of High Pressure and Nisin on the Inactivation of Heat-Resistant and Pathogenic Spores in Food Matrices

International audience[Technical Session 7 – Food Processing Technologies]Introduction: The high demand for minimally processed food makes high pressure processing (HPP) one of the most promising non-thermal technologies for food application. While HPP efficiently inactivates vegetative bacteria, bacterial spores show strong resistance, especially at mild temperatures. To ensure spore inactivation in food matrices, the addition of other hurdle(s), such as mild heat or antimicrobial agents, is necessary.Purpose: HPP in combination with nisin was investigated as a non-thermal method for the inactivation of pathogenic and thermoresistant bacterial spores in food matrices.Methods: Six bacterial strains were studied with regard to the diversity of their origins and properties: Bacillus pumilus, Bacillus sporothermodurans, Bacillus licheniformis, Bacillus weihenstephanensis, Bacillus subtilis and Clostridium sp. (botulinum type E-like). Spores were treated in a buffer, skim milk or a liquid medium simulating cooked ham. Pressure levels ranging from 200 MPa to 600 MPa were applied for 10 min at 20°C or 50°C. Nisin was added during and/or after HPP to a final concentrations of 50 or 20 UI/mL.Results: While no significant reduction of spore cultivability was observed at any pressure at 20 °C, the addition of nisin at low concentration (ten times lower than the legal concentration) during and after HPP treatment induced a highly synergistic effect on Bacillus spp. inactivation, with spore count below the detection limit (inactivation > 6 log/mL). Moreover, spores remained sensitive to nisin up to 6h after HPP.Significance: The food industry usually pressurizes foods at room temperature, resulting in, only, inactivation of vegetative cells. The present work shows that combining HPP with nisin can lead to a synergistic Bacillus spp. spore inactivation, even after treatments at 20°C. The addition of nisin in foods before their pressurization can, therefore, be an efficient way to ensure the inactivation of bacterial spores

High pressure sensitization of heat-resistant and pathogenic foodborne spores to nisin

International audienceToday, there is no effective non-thermal method to inactivate unwanted bacterial spores in foods. High-Pressure (HP) process has been shown to act synergistically with moderate heating and the bacteriocin nisin to inactivate spores but the mechanisms have not been elucidated. The purpose of the present work was to investigate in depth the synergy of HP and nisin on various foodborne spore species and to bring new elements of understandings.For this purpose, spores of Bacillus pumilus, B. sporothermodurans, B. licheniformis, B. weihenstephanensis, and Clostridium sp. were suspended in MES buffer, in skim milk or in a liquid medium simulating cooked ham brine and treated by HP at 500 MPa for 10 min at 50 °C or 20 °C. Nisin (20 or 50 IU/mL) was added at three different points during treatment: during HP, during and or in the plating medium of enumeration. In the latter two cases, a high synergy was observed with the inhibition of the spores of Bacillus spp. The evaluation of the germinated fraction of Bacillus spp. spores after HP revealed that this synergy was likely due to the action of nisin on HP-sensitized spores, rather than on HP-germinated spores. Thus, the combination of nisin and HP can lead to Bacillus spp. spore inhibition at 20 °C. And Nisin can act on HP-treated spores, even if they are not germinated.This paper provides new information about the inhibition of spores by the combination of HP and nisin. The high synergy observed at low temperature has not been reported yet and could allow food preservation without the use of any thermal process