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This study aimed to characterize the first vancomycin-resistant Enterococcus faecalis (VREfs) isolate from patient with neutropenic in Tunisia by whole-genome sequencing (WGS). This strain was detected from routine rectal swab from an 8-year-old child with bone marrow aplasia, residing in a rural area, on September 20, 2021. The strain was isolated after 12 days of hospitalization at the National Bone Marrow Transplant Center. Minimum Inhibitory Concentrations of vancomycin and teicoplanin were >256 and 16 mg/L, respectively. WGS revealed that the strain belonged to the ST249 clone, exclusively reported in avian (poultry and ducks) vancomycin-susceptible E. faecalis isolates in six studies from four countries, primarily Denmark. The vanA gene was carried by the Tn 1546 transposon mobilized by a pTW9-like plasmid. The ardA gene, a CRISPR-Cas system neutralization factor, was detected in this strain. In summary, this is the first report of avian-associated E. faecalis ST249 in clinical samples. Initially vancomycin susceptible, the strain acquired a pTW9-like plasmid carrying the classical vanA -Tn 1546 transposon. This acquisition was facilitated by the sex pheromone-response mechanisms and the ard A gene and CRISPR-Cas system neutralization.

Aims: In Tunisia, limited research has focused on characterizing clinical vancomycin-resistant Enterococcus faecium (VREfm). This study aimed to bridge this knowledge gap by molecular characterization of antimicrobial resistance, determining the genetic elements mediating vancomycin-resistance, and whole-genome sequencing of one representative VREfm isolate., Methods and Results: Over 6 years (2011-2016), a total of eighty VREfm isolates responsible for infection or colonization were identified from hospitalized patients, with the incidence rate increasing from 2% in 2011 to 27% in 2016. All of these strains harbored the vanA gene. The screening for antimicrobial resistance genes revealed the predominance of ermB, tetM, and aac(6')-Ie-aph(2'')-Ia genes and 81.2% of strains harbored the Tn1545. Pulsed-field gel electrophoresis identified seven clusters, with two major clusters (belonging to ST117 and ST80) persisting throughout the study period. Seven Tn1546 types were detected, with type VI (truncated transposon) being the most prevalent (57.5%). Whole-genome sequencing revealed a 3 028 373 bp chromosome and five plasmids. Mobile genetic elements and a type I CRISPR-cas locus were identified. Notably, the vanA gene was carried by the classic Tn1546 transposon with ISL3 insertion on a rep17pRUM plasmid., Conclusion: A concerning trend in the prevalence of VREfm essentially attributed to CC17 persistence and to horizontal transfer of multiple genetic variants of truncated vanA-Tn1546., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Aims: This study was conducted to evaluate the in vitro activity of clinically relevant aminoglycosides and to determine the prevalence of genes encoding aminoglycoside modifying enzymes (AMEs) and 16S ribosomal RNA (rRNA) methyltransferases among aminoglycoside-resistant E. coli (n = 61) and K. pneumoniae (n = 44) clinical isolates. Associated resistances to beta-lactams and their bla genes as well as the genetic relatedness of isolates were also investigated., Materials and Methods: A total of 105 aminoglycoside-resistant E. coli (n = 61) and K. pneumoniae (n = 44) isolates recovered between March and May 2017 from 100 patients hospitalized in different wards of Charles Nicolle Hospital of Tunis, Tunisia, were studied. Minimal inhibitory concentrations of aminoglycoside compounds were determined by broth microdilution method. Aminoglycosides resistance encoding genes [aph(3´)-Ia, aph(3') IIa, aph(3´)-VIa, ant(2″)-Ia, aac(3)-IIa, aac(3)-IVa, aac(6')-Ib, rmtA, rmtB, rmtC, armA, and npmA] and bla genes were investigated by PCR and sequencing. Genetic relatedness was examined by multilocus sequence typing (MLST) for representative isolates., Results: High rates of aminoglycoside resistance were found: gentamicin (85.7%), tobramycin (87.6%), kanamycin (78.0%), netilmincin (74.3%), and amikcin (18.0%). Most common AME gene was aac(3)-IIa (42%), followed by aac(6')-Ib (36.2%) and aph(3')-VIa (32.4%). The majority of isolates were resistant to beta-lactams and blaCTX-M-15 was the most common ESBL. The blaNDM-1 and blaOXA-48 were also produced by 1 and 23 isolates, respectively. Novel sequence types have been reported among our isolates and high-risk clonal lineages have been detected, such as E. coli ST43 (ST131 in Achtman MLST scheme) and K. pneumoniae (ST11/ST13)., Conclusions: The high prevalence of aminoglycoside resistance rates and the diversity of corresponding genes, with diverse β-lactamase enzymes among genetically heterogeneous clinical isolates present a matter of concern., (© The Author(s) 2024. Published by Oxford University Press on behalf of Applied Microbiology International.)

Acinetobacter baumannii has emerged as a main nosocomial pathogen exhibiting high rates of resistance to clinically relevant antibiotics. Six pandrug-resistant A. baumannii (PDR-A. baumannii) were recovered from three patients in a Tunisian Intensive Care Unit (ICU) between 10th and 16th of May 2018 resulting in one fatal case and raising the possibility of an outbreak. On 18th of May environmental screening of ICU surfaces was carried out. On 22nd of May a fourth patient was infected with PDR-A. baumannii and died. A second investigation was carried out for environmental screening and PDR-A. baumannii was isolated from the respirator. Antimicrobial susceptibility testing was performed according to EUCAST (2019) guidelines. MIC of colistin was determined by broth microdilution method. PCR was used to detect 14 beta-lactamases/carbapenemases and mcr (mcr-1 to mcr-5) genes. The genetic relatedness of PDR-A. baumannii isolates was determined by PFGE and MLST. Seven PDR-A. baumannii isolates were recovered from four patients, one MDR strain from wash basin, a PDR strain from hand sanitizer bottle and another PDR strain from respirator. All PDR-A. baumannii (n = 9) harbored blaOXA-69 gene and none carried mcr. Moreover, seven carried blaGES and blaOXA-23 genes. PFGE identified four pulsotypes (A, B, C, and D) with the pulsotype A gathering seven PDR-A. baumannii isolates: six from three patients and one from hygiene sample. MLST revealed that all PDR-A. baumannii isolates of pulsotype A belonged to the pandemic clone ST2. Systematic screening of MDR and PDR-A. baumannii is highly recommended to limit dissemination of such strains in ICUs.

This study sought to investigate the occurrence and subsequently to characterize extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae from urban and rural stagnant water samples during the wet season (December to February) in several regions of northern Tunisia. From 56 stagnant water samples, 14 ESBL-producing Enterobacteriaceae were recovered, including 9 Escherichia coli, 3 Klebsiella pneumoniae, and 2 K. oxytoca. Most isolates were multidrug-resistant, with ESBL production primarily encoded by blaCTX-M-15 (n = 8) and blaCTX-M-1 (n = 4) followed by blaCTX-M-55 (n = 1) and blaTEM-26 (n = 1). One K. pneumoniae isolate co-harbored blaKPC and blaCTX-M-15 genes. Class 1 integrons were detected in 4 isolates, however, sul1, sul2, and aac(6')-Ib-cr genes were detected in eleven, two, and four isolates, respectively. The nine E. coli isolates belonged to seven sequence types namely, B2/ST131 (3 isolates), A/ST164, A/ST10, A/ST224, A/ST38, A/ST155, and A/ST69 (each of them one isolate). The three K. pneumoniae isolates were assigned to three sequence types: ST101, ST405 (harboring CTX-M-15 and KPC), and ST1564. Overall, the phenotypic and genotypic traits of collected isolates mirror the molecular epidemiology of ESBL-producing enterobacteria in Tunisia and highlight the potential role of stagnant water in both urban and rural areas as a reservoir of ESBL-producing Enterobacteriaceae.

This study sought to evaluate the probiotic properties and the food preservation ability of lactic acid bacteria isolates collected from the intestines of wild marine fishes (gilthead seabream ( Sparus aurata ) ( n = 60) and whiting fish ( Merlangius merlangus ) ( n = 40)) from the Mediterranean sea in the area of Mostaganem city, Algeria. Forty-two isolates were identified as: Enterococcus durans ( n = 19), Enterococcus faecium ( n = 15), Enterococcus faecalis ( n = 4), Lactococcus lactis subp. lactis ( n = 3), and Lactobacillus plantarum ( n = 1). All isolates showed inhibition to at least one indicator strain, especially against Listeria monocytogenes , Staphylococcus aureus , Paenibacillus larvae , Vibrio alginolyticus , Enterococcus faecalis , Bacillus cereus , and Bacillus subtilis . In all collected isolates, PCR analysis of enterocin-encoding genes showed the following genes: entP ( n = 21), ent 1071A/B ( n = 11), entB ( n = 8), ent L50A/B ( n = 7), ent AS48 ( n = 5), and entX ( n = 1). Interestingly, 15 isolates harbored more than one ent gene. Antimicrobial susceptibility, phenotypic virulence, and genes encoding virulence factors were investigated by PCR. Resistance to tetracycline ( n = 8: tetL + tetK) , erythromycin ( n = 7: 5 ermA , 2 msrA , and 1 mef (A/E)), ciprofloxacin ( n = 1), gentamicin ( n = 1: aac (6')- aph (2″)), and linezolid ( n = 1) were observed. Three isolates were gelatinase producers and eight were α-hemolytic. Three E. durans and one E. faecium harbored the hyl gene. Eight isolates showing safety properties (susceptible to clinically relevant antibiotics, free of genes encoding virulence factors) were tested to select probiotic candidates. They showed high tolerance to low pH and bile salt, hydrophobicity power, and co-culture ability. The eight isolates showed important phenotypic and genotypic traits enabling them to be promising probiotic candidates or food bio-conservers and starter cultures.

Pseudomonas aeruginosa is one of the major infectious agents in burn patients. Globally, high rates of antimicrobial resistance in P. aeruginosa have been reported, which is a cause of concern. The objective of this study was to determine the rate of resistance to carbapenems in P. aeruginosa isolates recovered from burn patients in Tunisia, to search genes encoding for carbapenemases and to determine their epidemiological markers (serotypes). A retrospective study was conducted in the Burn Intensive Care Unit (BICU) of the Trauma and Burn Centre of Ben Arous, Tunisia, and P. aeruginosa isolates collected from burn patients, from January to December 2018 were investigated. Carbapenemase screening was performed by Carbapenem Inactivation Method (CIM) and by EDTA-disk test for all carbapenem resistant isolates. Genes encoding carbapenemases (blaVIM, blaIMP, blaGES, blaNDM, and blaKPC) were investigated by PCR and selected carbapenemase genes were sequenced. During the study period, 104 non duplicated P. aeruginosa isolates were recovered. Most of them were isolated from skin samples (45.1%) and blood culture (22.1%) and belonged to O:11 (19.2%), O:12, and O:5 (12.5%, each) serotypes. High rates of resistance were observed for carbapenems (64.4%). Among the 67 carbapenem resistant isolates, 58 (86.5%) harbored blaVIM gene and 55 (82%) blaGES gene; in addition, 48 (71.6%) co-harbored blaVIM and blaGES genes. After sequencing, the blaVIM-2 and blaGES-5 gene variants were identified in seven randomly selected isolates. To the best of our knowledge, this is the first description of P. aeruginosa simultaneously harboring blaVIM-2 and blaGES-5 genes.

Antimicrobial-resistant Escherichia coli isolates have emerged in various ecologic compartments and evolved to spread globally. We sought to (1.) investigate the occurrence of ESBL-producing E. coli (ESBL-Ec) in feces from free-range chickens in a rural region and (2.) characterize the genetic background of antimicrobial resistance and the genetic relatedness of collected isolates. Ninety-five feces swabs from free-range chickens associated with two households (House 1/House 2) in a rural region in northern Tunisia were collected. Samples were screened to recover ESBL-Ec, and collected isolates were characterized for phenotype/genotype of antimicrobial resistance, integrons, and molecular typing (pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)). Overall, 47 ESBL-Ec were identified, with the following genes detected: 35 bla CTX-M-1, 5 bla CTX-M-55 , 5 bla CTX-M-15 , 1 bla SHV-2, and 1 bla SHV-12 . Resistance to fluoroquinolones, tetracycline, sulfonamides, and colistin was encoded by aac(6') -Ib- cr ( n = 21), qnr B ( n = 1), and qnr S ( n = 2); tet A ( n = 17)/ tet B ( n = 26); sul 1 ( n = 29)/ sul 2 ( n = 18); and mcr -2 ( n = 2) genes, respectively. PFGE and MLST identified genetic homogeneity of isolates in House 1; however, isolates from House 2 were heterogeneous. Notably, among nine identified sequence types, ST58, ST69, ST224, and ST410 belong to pandemic high-risk clonal lineages associated with extrapathogenic E. coli . Minor clones belonging to ST410 and ST471 were shared by chickens from both households. The virulence genes fyuA, fimH, papGIII, and iutA were detected in 35, 47, 17, and 23 isolates, respectively. Findings indicate a high occurrence of ESBL-Ec in free-range chickens and highlight the occurrence of pandemic zoonotic clones.

Mastitis remains the most frequent and the most expensive disease of dairy breeding. The objective of the study was to study S. aureus isolates collected from subclinical bovine mastitis in the Tiaret region, Algeria, by determining their antimicrobial susceptibility and their virulence traits. Sixty-two S. aureus isolates collected from subclinical bovine mastitis were studied by determining their antimicrobial susceptibility according to CLSI guidelines, and nine genes encoding virulence factors and resistance to methicillin and penicillin were determined by PCR. Multi-drug resistance was observed in 19 (30.64%) isolates and five (8%) were methicillin-resistant S. aureus (MRSA), four of them harbored the mecA gene; however, the mecC gene was not detected. Out of 59 penicillin-resistant isolates, 14 harbored the blaZ gene; one of them co-harbored the mecA gene. The following virulence genes were detected: eta (n = 23; 37%), icaA (20; 32.2%), icaD (18; 29%), etb (16; 25.8%), luk E-D (14; 22.5%), and sea (6; 9.6%). The tsst-1, lukF/lukS, and luk-M genes were not detected. The occurrence of MRSA and multi-drug resistant (MDR) S. aureus isolates as well as genes encoding virulence factors playing an important role in the pathogenesis of subclinical bovine mastitis and of harmful potential to human is a cause for concern., (© The Author(s) 2023. Published by Oxford University Press on behalf of Applied Microbiology International.)

The emergence and spread of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals is a major global public health concern. The current study sought to characterize 25 MRSA clinical isolates collected in a Tunisian hospital from December 2015 to September 2016, with the genetic lineages, virulence factors, and antibiotic resistance mechanisms determined for these isolates. Three spa-types were detected: t037 (23 isolates), t932, and t2235 (one isolate each). Isolates were ascribed to agr I (n = 20), agr II (n = 1), with four nontypeable isolates. Depending on sequence type (ST), the 25 MRSA isolates were assigned to two clonal complexes (CC8 and CC5), with a predominance of the lineage ST239-CC8 (n = 24; 96%). All isolates belonging to CC8 had the SCCmec type III, while the unique CC5 isolate had SCCmec type IV. Antimicrobial susceptibility testing revealed high levels of resistance to aminoglycosides, tetracycline, ciprofloxacin and rifampicin for the majority of isolates belonging to the ST239-CC8 lineage. The ST149-CC5 isolate was susceptible to non-β-lactam antibiotics. One isolate harbored the tsst-1 gene (4%); however, lukS/LukF-PV, eta and etb genes were not detected. The MDR ST239-CC8 clone would seem to be widespread in this hospital. Therefore, a rigorous hygienic control system is urgently required., (© The Author(s) 2022. Published by Oxford University Press on behalf of Applied Microbiology International.)

Background: Escherichia coli ( E. coli ) is one of the main etiological agents responsible for bovine mastitis (BM), neonatal calf diarrhea (NCD), and avian colibacillosis (AC). This study aimed to assess resistance and virulence genes content, biofilm-forming ability, phylogenetic groups, and genetic relatedness in E. coli isolates recovered from clinical cases of BM, NCD, and AC., Materials/methods: A total of 120 samples including samples of milk ( n = 70) and feces ( n = 50) from cows with BM and calves with NCD, respectively, were collected from different farms in Northern Tunisia. Bacterial isolation and identification were performed. Then, E. coli isolates were examined by disk diffusion and broth microdilution method for their antimicrobial susceptibility and biofilm-forming ability. PCR was used to detect antimicrobial resistance genes (ARGs), virulence genes (VGs), phylogenetic groups, and Enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) for their clonal relationship., Results: Among the 120 samples, 67 E. coli isolates (25 from BM, 22 from AC, and 20 from NCD) were collected. Overall, 83.6% of isolates were multidrug resistant. Thirty-six (53.73%) isolates were phenotypically colistin-resistant (CREC), 28.3% (19/67) were ESBL producers (ESBL-EC), and forty-nine (73.1%) formed biofilm. The bla TEM gene was found in 73.7% (14/19) of isolates from the three diseases, whilst the bla CTXM-g-1 gene was detected in 47.3% (9/19) of isolates, all from AC. The most common VG was the fim A gene (26/36, 72.2%), followed by aer (12/36, 33.3%) , cnf1 (6/36, 16.6%) , pap C (4/36, 11.1%), and stx 1 and stx 2 genes (2/36; 5.5% for each). Phylogenetic analysis showed that isolates belonged to three groups: A (20/36; 55.5%), B2 (7/36; 19.4%), and D (6/36; 16.6%). Molecular typing by ERIC-PCR showed high genetic diversity of CREC and ESBL E. coli isolates from the three animal diseases and gave evidence of their clonal dissemination within farms in Tunisia., Conclusion: The present study sheds new light on the biofilm-forming ability and clonality within CREC and ESBL-EC isolated from three different animal diseases in Tunisian farm animals.

Extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae have emerged as important nosocomial pathogens. Community infections by these organisms have been also reported and were associated with previous intestinal colonization. We aimed to characterize cefotaxime-resistant Enterobacteriaceae (CTX-R-En) isolated from hospitalized children in a Tunisian paediatric ward. Seventy CTX-R-En isolates were collected from 227 rectal swabs from hospitalized children in a paediatric ward. Antimicrobial susceptibility testing was determined according to the EUCAST guidelines. Isolates were characterized by polymerase chain reaction (PCR, genes encoding: ESBLs, pAmpC, carbapenemases, plasmid-mediated quinolone resistance, virulence factors in Escherichia coli and Klebsiella pneumoniae isolates, occurrence of classes 1 and 2 integrons, phylogenetic groups of E. coli isolates, ERIC-PCR and PCR-based replicon typing) and conjugal transfer experiments. In total, 65 out of 227 (28·6%) hospitalized children were colonized with CTX-M-R-En, and 70 isolates were identified. Isolates were 59 ESBL-, 7 plasmidic-AmpC (pAmpC)-, 3 ESBL+pAmpC-, and one ESBL+carbapenemase producers. The following bla genes were identified: bla CTX-M-15 (n = 54), bla CTX-M-1 (n = 5), bla CTX-M-9 (n = 2), bla CTX-M-13 (n = 1) and bla CTX-M-14 (n = 1), bla CMY-2 (n = 5), bla CMY-4 (n = 4), bla ACC-1 (n = 1) and bla OXA-48 (n = 1). Our results showed that hospitalized children were colonized with various CTX-R-En-producing several beta-lactamase enzymes., (© 2022 The Society for Applied Microbiology.)

Colistin (also known as polymyxin E), a polymyxin antibiotic discovered in the late 1940s, has recently reemerged as a last-line treatment option for multidrug-resistant infections. However, in recent years, colistin-resistant pathogenic bacteria have been increasingly reported worldwide. Accordingly, the presented review was undertaken to identify, integrate and synthesize current information regarding the detection and transmission of colistin-resistant bacteria across the African continent, in addition to elucidating their molecular mechanisms of resistance. PubMed, Google Scholar and Science Direct were employed for study identification, screening and extraction. Overall, based on the developed literature review protocol and associated inclusion/exclusion criteria, 80 studies published between 2000 and 2021 were included comprising varying bacterial species and hosts. Numerous mechanisms of colistin resistance were reported, including chromosomal mutation(s) and transferable plasmid-mediated colistin resistance (encoded by mcr genes). Perhaps unexpectedly, mcr-variants have exhibited rapid emergence and spread across most African regions. The genetic variant mcr-1 is predominant in humans, animals and the natural environment, and is primarily carried by IncHI2- type plasmid. The highest number of studies reporting the dissemination of colistin-resistant Gram-negative bacteria were conducted in the North African region., (© 2022 Society for Applied Microbiology.)

Antimicrobial resistance represents a global health problem, with infections due to pathogenic antimicrobial resistant bacteria (ARB) predicted to be the most frequent cause of human mortality by 2050. The phenomenon of antimicrobial resistance has spread to and across all ecological niches, and particularly in livestock used for food production with antimicrobials consumed in high volumes. Similarly, hospitals and other healthcare facilities are recognized as significant 'hotspots' of ARB and antimicrobial resistance genes (ARGs); however, over the past decade, new and previously overlooked ecological niches are emerging as hidden reservoirs of ARB/ARGs. Increasingly extensive and intensive industrial activities, degradation of natural environments, burgeoning food requirements, urbanization, and global climatic change have all dramatically affected the evolution and proliferation of ARB/ARGs, which now stand at extremely concerning ecological levels. While antimicrobial resistant bacteria and genes as they originate and emanate from livestock and human hosts have been extensively studied over the past 30 years, numerous ecological niches have received considerably less attention. In the current descriptive review, the authors have sought to highlight the importance of wildlife as sources/reservoirs, pathways and receptors of ARB/ARGs in the environment, thus paving the way for future primary research in these areas., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Aims: Worldwide, studies regarding antimicrobial resistance in rabbits are scarce. In addition, it seems that rearing conditions have important impact on emergence and spread of antimicrobial-resistant bacteria. Thus, the authors sought to (1) assess the role of rabbits residing across diverse ecosystems as potential reservoirs of antimicrobial-resistant enterococci and (2) investigate the genetic background of detected resistances., Methods and Results: Faecal samples from 60 healthy farmed rabbits (one farm), 35 laboratory rabbits and 31 wild rabbits were analysed. Overall, 97 enterococci isolates were accumulated, as follows: 44 E. faecium, 37 E. faecalis, 7 E. gallinarum, 5 E. durans and 4 E. avium. E. faecalis isolates were statistically associated with farm rabbits and wild rabbits (p < 0.05). High rates of resistance were observed for tetracycline (60.8%; tetM [n = 48; 81.3%], tetO [n = 7; 11.8%] and tetL [n = 1; 1.7%]), erythromycin (43.3%; msr(A) [n = 14; 33.3%] and ermB [n = 13; 31%]), ampicillin (29.9%), streptomycin (26.8%; ant(6)-Ia [n = 3, 11.5%]) and vancomycin (21.6%; vanA [one E. faecium + one E. faecalis; 9.5%]). Low frequencies of resistance were observed for teicoplanin (9.2%), linezolid (8.2%), ciprofloxacin (7.2%) and gentamicin (1%; aac(6')-Ie-aph(2″)-Ia). Resistance to ampicillin and vancomycin was associated with laboratory rabbits (p < 0.05). Int-Tn (Tn916/1545) was detected in 27 (27.8%) isolates, of which 10 isolates co-harboured tetM and ermB genes, while 16 comprised tetM., Conclusion: Findings indicate that clinically relevant enterococci species isolated from rabbits are frequently resistant to antimicrobials and harbour a range of genes associated with the Tn916/1545 family., Significance and Impact of the Study: This study highlights the high rates of antimicrobial-resistant enterococci from rabbits and the occurrence of both vancomycin- and linezolid-resistant isolates, potentially representing a very serious threat to human and animal health., (© 2022 Society for Applied Microbiology.)

Multidrug-resistant (MDR) Enterococcus faecium ( Efm ) infections continue to increase worldwide, although epidemiological studies remain scarce in lower middle-income countries. We aimed to explore which strains circulate in E. faecium causing human infections in Tunisian healthcare institutions in order to compare them with strains from non-human sources of the same country and finally to position them within the global E. faecium epidemiology by genomic analysis. Antibiotic susceptibility testing was performed and transfer of vancomycin- vanA and ampicillin- pbp5 resistance was performed by conjugation. WGS-Illumina was performed on Tunisian strains, and these genomes were compared with Efm genomes from other regions present in the GenBank/NCBI database ( n = 10,701 Efm genomes available May 2021). A comparison of phenotypes with those predicted by the recent ResFinder 4.1-CGE webtool unveiled a concordance of 88%, with discordant cases being discussed. cgMLST revealed three clusters [ST18/CT222 ( n = 13), ST17/CT948 strains ( n = 6), and ST203/CT184 ( n = 3)], including isolates from clinical, healthy-human, retail meat, and/or environmental sources in different countries over large time spans (10-12 years). Isolates within each cluster showed similar antibiotic resistance, bacteriocin, and virulence genetic patterns. pbp5 -AmpR was transferred by VanA-AmpR-ST80 (clinical) and AmpR-ST17- Efm (bovine meat). Identical chromosomal pbp5 -platforms carrying metabolic/virulence genes were identified between ST17/ST18 strains of clinical, farm animal, and retail meat sources. The overall results emphasize the role of high-resolution genotyping as provided by WGS in depicting the dispersal of MDR- Efm strains carrying relevant adaptive traits across different hosts/regions and the need of a One Health task force to curtail their spread.

Aims: Molecular characterization of extended-spectrum β-lactamases (ESBLs) among Salmonella Kentucky and Typhimurium isolates: partial sequence analysis of the types of β-lactamases found in these isolates, clonality, resistance and supposed emergence of ESBL-producing strains., Methods and Results: A retrospective study surveyed the ESBLs occurring in a total of 1404 Salmonella Kentucky and Typhimurium isolates collected over a 5-year period in Tunisia. Antimicrobial susceptibility tests, ESBL phenotype determination (double-disc synergy) were performed. Polymerase chain reaction assays were used for the detection of β-lactamase genes (bla TEM , bla SHV , bla OXA-1 and bla CTX-M ), class 1 and class 2 integrases (intI1 and intI2) and the 3' conserved segment (3'-CS) of class 1 integron (qacEΔ1+sul1). Sequencing of amplicons of β-lactamase genes was performed. Percentage of 9.8 of the isolates (S. Kentucky = 117, S. Typhimurium = 20) were either resistant to penicillin and had decreased susceptibility to cefotaxime or had a positive double-disc synergy test result. Polymerase chain reaction detected that these isolates harboured one or more β-lactamase genes (bla TEM , bla SHV , bla OXA-1 or bla CTX-M ). TEM-1, TEM-34, CTX-M15, CTX-M9 and CTX-M61 type ESBLs were identified through sequencing. The novel Salmonella cefotaxime-hydrolysing β-lactamase, CTX-M61/TEM-34, detected in this study showed the emergence of new CTX-M-type ESBLs in Tunisia. There were found 33 different multidrug resistance (MDR) patterns., Conclusion: These findings highlighted the proliferation of ESBLs and MDR in Salmonella Kentucky and Typhimurium isolates from numerous regions and sources in Tunisia, indicating an emerging public health concern., Significance and Impact of the Study: For the first time CTX-M-61/TEM-34, a novel cefotaxime-hydrolysing β-lactamase of Salmonella had been detected., (© 2021 The Society for Applied Microbiology.)

We investigated the 16S-23S rRNA intergenic spacer region (ISR)-PCR and the phylogenetic PCR analyses of 150 Escherichia coli isolates as tools to explore their diversity, according to their sampling origins, and their relative dominance in these sampling sources. These genetic markers are used to explore phylogenetic and genetic relationships of these 150 E. coli isolates recovered from different environmental sources (water, food, animal, human and vegetables). These isolates are tested for their biochemical pattern and later genotyped through the 16S-23S rRNA intergenic spacer PCR amplification and their polymorphism investigation of PCR-amplified 16S-23S rDNA ITS. The main results of the pattern band profile revealed one to four DNA fragments. Distributing 150 E. coli isolates according to their ITS and using RS-PCR, revealed four genotypes and four subtypes. The DNA fragment size ranged from 450 to 550 bp. DNA band patterns analysis revealed considerable genetic diversity in interspecies. Thus, the 450 and 550 bp sizes of the common bands in all E. coli isolates are highly diversified. Genotype I appeared as the most frequent with 77.3% (116 isolates), genotype II with 12% (18 isolates); genotype III with 9.7% (14 isolates), and the IV rarely occurred with 4% (2 isolates). Distributing the E. coli phylogroups showed 84 isolates (56%) of group A, 35 isolates (23.3%) of group B1, 28 isolates (18.7%) of group B2 and only three isolates (2%) of group D., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

We aimed to study the antibiotic susceptibility and possible occurrence of extended-spectrum beta-lactamases (ESBL)/carbapenemase-producing Escherichia coli isolates collected from rabbits in Tunisia. In all, 35 faecal samples from healthy rabbits were collected from one farm and E. coli were isolated from three media: antibiotic-free TBX agar, TBX+2 mg l -1 cefotaxime and TBX+1 mg l -1 imipenem. In total, 39 E. coli isolates were recovered; the majority showed resistance to at least one antibiotic and none was ESBL producer. Carbapenem resistance was detected in 16 isolates from either selective or un-selective media. Phenotypic methods used to detect carbapenemase production showed two positive isolates by Modified Hodge Test, six metallo-carbapenemase producers (Imipenem disc+EDTA) and all were temocillin resistant (possible OXA-48 carbapenemase). bla VIM and bla IMP type genes were detected in two and one isolates, respectively; one of them harboured both genes. Isolates contained common genes encoding resistance to sulphonamides (sul1, sul2), tetracycline (tetA, tetB, tetC) and fluoroquinolones (qnrS, aac(6')-Ib-cr). Class 1 and 2 integrons were detected in five and four isolates, respectively. These findings highlight the importance of rabbit production as reservoir of carbapenem-resistant E. coli and argument the first report of bla VIM and bla IMP genes in livestock in Tunisia., (© 2021 The Society for Applied Microbiology.)

Little is known about the role of forestland and non-fertilized agriculture soils as reservoirs of extended-spectrum beta-lactamase (ESBL) and plasmid-borne AmpC (pAmpC)-producing Escherichia coli isolates. Thus, in the present study, 210 soil samples from various origins (forest of Oued Zen (Ain Drahem), non-agriculture soils from different park gardens in Tunis City, cereal culture soils and home gardens) were investigated to characterize cefotaxime-resistant E. coli isolates. A total of 22 ESBL/pAmpC-producing E. coli were collected, and all harbored variants of the blaCTX-M gene (15 blaCTX-M-1, 5 blaCTX-M-55 and 2 blaCTX-M-15). A total of seven and two isolates harbored also blaEBC and blaDHA-like genes, respectively. Resistances to tetracycline, sulfonamides and fluoroquinolones were encoded by tetA (n = 4)/tetB (n = 12), sul1 (n = 17)/sul2 (n = 19) and aac(6')-Ib-cr (n = 2)/qnrA (n = 1)/qnrS (n = 1) genes, respectively. A total of seven isolates were able to transfer by conjugation cefotaxime-resistance in association or not with other resistance markers. PFGE showed that ten and two isolates were clonally related (pulsotypes P1 and P2). The 10 P1 isolates had been collected from forestland, cereal culture soils and an urban park garden in Tunis City, arguing for a large spread of clonal strains. Our findings highlight the occurrence of ESBL/pAmpC-E. coli isolates in soils under limited anthropogenic activities and the predominance of CTX-M enzymes that are largely disseminated in E. coli from humans and animals in Tunisia., (© The Author(s) 2021. Published by Oxford University Press on behalf of FEMS. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Multiantimicrobial-resistant Escherichia coli isolates are a global human health problem causing increasing morbidity and mortality. Genes encoding antimicrobial resistance are mainly harbored on mobile genetic elements (MGEs) such as transposons and plasmids as well as integrons, which enhance their rapid spread. The aim of this study was to characterize 83 multiantimicrobial-resistant E. coli isolates recovered from healthy broiler chickens. Among 78 tetracycline-resistant isolates, the tetA , tetB , and tetC genes were detected in 59 (75.6%), 14 (17.9%), and one (1.2%) isolates, respectively. The sul1 , sul2 , and sul3 genes were detected 31 (46.2%), 16 (23.8%), and 6 (8.9%) isolates, respectively, among 67 sulfonamide-resistant isolates. The PCR-based replicon typing method showed plasmids in 29 isolates, IncFIB (19), IncI1-I γ (17), IncF (14), IncK (14), IncFIC (10), IncP (8), IncY (3), IncHI2 (1), and IncX (1). The class 1 and 2 integrons were detected in 57 and 2 isolates, respectively; one isolate harbored both integrons. Seven and one gene cassette arrays were identified in class 1 and class 2 integrons, respectively. Our findings show that multiantimicrobial-resistant E. coli isolates from chickens serve as reservoirs of highly diverse and abundant tet and sul genes and plasmid replicons. Such isolates and MGEs pose a potential health threat to the public and animal farming., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Mohamed Salah Abbassi et al.)

Aim: The objective of this study was to characterize lactic acid bacteria (LAB) from rabbits to be used as potential autochthonous probiotic., Methods and Results: Fifteen faecal samples were collected from wild and laboratory rabbits. One hundred and eight isolates were collected and tested for their inhibitory power against eight pathogenic bacteria. Among them, 43 Enterococcus isolates were able to inhibit at least one pathogen. Enterocine genes entA, entB and entP were detected in 14, 17 and 22 isolates, respectively. These isolates were tested for their antibiotic susceptibility and genes encoding virulence factors. Relevant phenotypes of antibiotic resistance were observed especially for ampicillin, vancomycin and linezolid. The following virulence genes were detected (number of positive isolates): hyl (5), esp (8), gelE (30), agg (2), ace (21), efa (6), CylL L/s (5), cob (26), cpd (32) and ccf (33). Five isolates were considered as safe and showed tolerance to both acid and bile salt., Conclusion: Bacteriocinogenic enterococci isolates from rabbits may show relevant resistance phenotypes and virulence factors. In addition, one Enterococcus durans isolate presents promising autochthonous probiotic candidate., Significance and Impact of the Study: This study reveals interesting properties for E. durans isolate and supports their utilization as autochthonous probiotic in rabbit husbandry., (© 2021 The Society for Applied Microbiology.)

The prevalence of Salmonella in dogs and cats was investigated and analysed for serotyping, susceptibility to antimicrobial drugs and risk factors assessment. In total, 151 faecal samples from 103 and 48 healthy and nonhealthy (diarrheic) cats and dogs, respectively were examinated. Salmonellae were confirmed by laboratory and biomedical characteristics and further serotyped then subjected to antimicrobial susceptibility tests. Risk factors that are typically associated with the shedding of salmonellae were assessed using Fisher's exact tests. Salmonella was detected in 18% (n=27/151) of pets. Most of the positive samples 85%(n=23/27) were from healthy cats and 7.4% (n=2/27) from healthy dogs and 7.4% (n = 2/27) from a diarrhoeic cat and diarrhoeic dog. Only one sample from each diarrhoeic cat and diarrhoeic dog were positive for Salmonella. total, 25 salmonellae (93% of strains) were serotyped as S. Thompson mostly originated form healthy cats (n = 23/25). All were resistant to tetracycline and trimethoprim-sulfamethoxazole and expressed ed only resisted an overall intermediate susceptibility patterns to ciprofloxacin. Also, multidrug resistant S. Kentucky and S. Minnesota were identified from a diarrhoeic and an healthy dog, respectively. This is the first isolation report of Salmonella from cats and dogs in Libya. It indeed represents a public health concern wich requires further monitoring.

The present study investigated the colonization rates and antimicrobial susceptibility of Staphylococcus species isolated from the nostrils of healthy horses. A nonselective laboratory approach was applied, followed by confirmation using a Phoenix automated microbiological system. Among the 92 horses included in the study, 48.9% (45/92) carried Staphylococcus species of mostly the coagulase-negative staphylococci (CoNS) type yielding 70 Staphylococcus strains. Of these strains, 37.1% (26/70; 24 CoNS and 2 coagulase-positive staphylococci; CoPS) were identified as methicillin-resistant staphylococci (MRS) expressing significant resistance to important antimicrobial classes represented mainly by subspecies of CoNS. This is the first study reporting a high prevalence of various Staphylococcus species, particularly strains of CoNS expressing multidrug resistance patterns of public health concern, colonizing healthy horses in Libya., (©2021 The Japanese Society of Equine Science.)

This study evaluated the occurrence of extended-spectrum β-lactamases (ESBL) and associated resistance genes, integrons, and plasmid types, as well as the genetic relatedness of enterobacterial isolates in the wastewater treatment plant (WWTP) of La Charguia, Tunis City (Tunisia). A total of 100 water samples were collected at different points of the sewage treatment process during 2017-2019. Antimicrobial susceptibility was conducted by the disc-diffusion method. blaCTX-M, blaTEM and blaSHV genes as well as those encoding non-β-lactam resistance, the plasmid types, occurrence of class1 integrons and phylogenetic groups of Escherichia coli isolates were determined by PCR/sequencing. Genomic relatedness was determined by multi-locus sequence typing (MLST) for selected isolates. In total, 57 ESBL-producer isolates were recovered (47 E. coli, eight Klebsiella pneumoniae, 1 of the Citrobacter freundii complex and 1 of the Enterobacter cloacae complex). The CTX-M-15 enzyme was the most frequently detected ESBL, followed by CTX-M-27, CTX-M-55 and SHV-12. One E. coli isolate harboured the mcr-1 gene. The following phylogroups/sequence types (STs) were identified among ESBL-producing E. coli isolates: B2/ST131 (subclade-C1), A/ST3221, A/ST8900, D/ST69, D/ST2142, D/ST38, B1/ST2460 and B1/ST6448. High numbers of isolates harboured the class 1 integrons with various gene cassette arrays as well as IncP-1 and IncFIB plasmids. Our findings confirm the importance of WWTPs as hotspot collectors of ESBL-producing Enterobacteriaceae with a high likelihood of spread to human and natural environments., (© The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.)

Aims: Characterization of quinolone-resistant Salmonella Kentucky and Typhimurium isolates in Tunisia from various sources, detection of some plasmid-mediated quinolone resistance genes and the genetic relatedness., Methods: A total of 1404 isolates of S. Kentucky (n = 1059)/S. Typhimurium (n = 345) from various sources from all over Tunisia were tested for quinolone resistance by disk diffusion method. Minimum inhibitory concentrations of nalidixic acid, ciprofloxacin and ofloxacin were determined. Quinolone-resistant isolates were screened for plasmid-mediated quinolone-resistance genes (qnrA,qnrB,qnrS, aac(6')-Ib-cr and qepA) by polymerase chain reaction (PCR). Mutations in the quinolone-resistance-determining regions of the gyrA and parC genes were detected by PCR and DNA sequencing. Pulsed-field gel electrophoresis and multilocus sequence typing were accomplished for isolates harbouring plasmid-mediated quinolone-resistance genes., Results: According to our selection criteria (NAL = resistance phenotype; CIP = resistant with diameter 0, or intermediate), only 63 S. Kentucky/41 S. Typhimurium isolates were investigated: 49% (5/104) were multidrug resistant. Two S. Typhimurium isolates harboured qnrB19 with different PFGE profiles. A mutation was detected in the gyrA gene for each of these two isolates. MLST revealed the presence of ST313 and ST34, an endemic sequence type., Conclusion: Our study highlights the presence of quinolone multidrug-resistant Salmonella in humans and animals in Tunisia. This is the first report of S. Typhimurium ST34 in Africa and qnrB19 in Tunisia., Significance and Impact of the Study: This is the first report that describes not only the current epidemiological situation of the quinolone resistance in S. Kentucky and Typhimurium isolated from various sources and regions in Tunisia, but also, the genetic resistance determinants associated with phenotypic antibiotic resistance and the molecular mechanisms of their quinolone-resistance. Also, we provide the first report of S. Typhimurium ST34 in Africa, and the first report of qnrB19 in Salmonella in Tunisia., (© 2020 The Society for Applied Microbiology.)

We sought to determine the relative value of conventional molecular methods and whole-genome sequencing (WGS) for subtyping Salmonella enterica serovar Enteritidis recovered from 2000 to 2015 in Tunisia and to investigate the genetic diversity of this serotype. A total of 175 Salmonella Enteritidis isolates were recovered from human, animal, and foodborne outbreak samples. Pulsed-field gel electrophoresis (PFGE), multiple locus variable-number tandem repeat analysis (MLVA), and whole-genome sequencing were performed. Eight pulsotypes were detected for all isolates with PFGE (DI = 0.518). Forty-five Salmonella Enteritidis isolates were selected for the MLVA and WGS techniques. Eighteen MLVA profiles were identified and classified into two major clusters (DI = 0.889). Core genome multilocus typing (cgMLST) analysis revealed 16 profiles (DI = 0.785). Whole-genome analysis indicated 660 single-nucleotide polymorphism (SNP) divergences dividing these isolates into 43 haplotypes (DI = 0.997). The phylogenetic tree supported the classification of Salmonella Enteritidis isolates into two distinct lineages subdivided into five clades and seven subclades. Pairwise SNP differences between the isolates ranged between 302 and 350. We observed about 311 SNP differences between the two foodborne outbreaks, while only less or equal to 4 SNP differences within each outbreak. SNP-based WGS typing showed an excellent discriminatory power comparing with the conventional methods such as PFGE and MLVA. Besides, we demonstrate the added value of WGS as a complementary subtyping method to discriminate outbreak from non-outbreak isolates belonging to common subtypes. It is important to continue the survey of Salmonella Enteritidis lineages in Tunisia using WGS.

Tetracycline resistance is still considered one of the most abundant antibiotic resistances among pathogenic and commensal microorganisms. The aim of this study was to evaluate the prevalence of tetracycline resistance ( tet ) genes in broiler chickens in Tunisia, and this was done by PCR. Individual cloacal swabs from 195 broiler chickens were collected at two slaughterhouses in the governorate of Ben Arous (Grand Tunis, Tunisia). Chickens were from 7 farms and belonged to 13 lots consisting of 15 animals randomly selected. DNA was extracted and tested for 14 tet genes. All the lots examined were positive for at least 9 tet genes, with an average number of 11 tet genes per lot. Of the 195 animals tested, 194 (99%) were positive for one or more tet genes. Tet (L), tet (M) and tet (O) genes were found in 98% of the samples, followed by tet (A) in 90.2%, tet (K) in 88.7% and tet (Q) in 80%. These results confirm the antimicrobial resistance impact in the Tunisian poultry sector and suggest the urgent need to establish a robust national antimicrobial resistance monitoring plan. Furthermore, the molecular detection of antibiotic resistance genes directly in biological samples seems to be a useful means for epidemiological investigations of the spread of resistance determinants.

Background: Methicillin-resistant staphylococci (MRS) are an emerging global problem with serious public health concern., Aims: This study investigated the prevalence and antimicrobial susceptibility of commensal Staphylococcus species isolated from healthy and clinical cats and dogs., Methods: Nasal swab samples were collected from animals and processed using selective and semi-selective mediums. Presumptive isolates were subjected to biochemical testing and analyzed using the Phoenix automated identification and susceptibility testing system. PCRs protocols were used to screen for mecA and pvl genes., Results: In total, 151 pets (103 cats and 48 dogs) were enrolled, of which 14 dogs (29%) and 24 cats (23%) were colonized with various Staphylococcus species mainly originated from healthy animals. A total of 38 staphylococci isolates were collected and distributed between 24 coagulase-negative and 14 coagulase-positive staphylococci. Only 13 staphylococci strains were identified as MRS, out of which only five isolates expressed that the mecA gene exclusively originated from healthy pets., Conclusion: This is the first study reporting the prevalence and colonization status of staphylococci species and MRS strains isolated from cats and dogs in Libya. The study reports important information of medical and clinical importance on antimicrobial and multidrug resistance of different staphylococci strains, particularly the coagulase negative species., Competing Interests: The authors declare that there is no conflict of interest.

Aquatic environments are crucial hotspots for the dissemination of antibiotic resistant microorganisms and resistance genes. Thus, the purpose of this study was to investigate the occurrence and the genetic characterization of cefotaxime-resistant (CTX R ) Enterobacteriaceae at a Tunisian semi-industrial pilot plant with biological treatment (WWPP) and its receiving river (Rouriche River, downstream from WWPP) located in Tunis City, during 2017-2018. We collected 105 and 15 water samples from the WWPP and the Rouriche River, respectively. Samples were screened to recover ESBL-producing Enterobacteriaceae (ESBL-E) and isolates were characterized for phenotype/genotype of antimicrobial resistance, integrons, plasmid types and molecular typing (multilocus sequence typing, MLST). Among 120 water samples, 33 and 4 contained ESBL-producing E. coli and K. pneumoniae isolates, respectively. Most isolates were multidrug resistant and produced CTX-M-15 (28 isolates), CTX-M-1 (4 isolates), CTX-M-55 (2 isolates), CTX-M-27 (one isolate), SHV-12 (one isolate) and VEB beta-lactamases (one isolate). All K. pneumoniae were CTX-M-15-positive. Four colistin-resistant isolates were found (MIC 4-8 μg/ml), but they were negative for the mcr genes tested. Class 1 integrons were detected in 21/25 trimethoprim/sulfamethoxazole-resistant isolates, and nine of them carried the gene cassette arrays: aadA2 + dfrA12 (n = 4), aadA1 + dfrA15 (n = 2), aadA5 + dfrA17 (n = 2) and aadA1/2 (n = 1). The IncP and IncFIB plasmids were found in 30 and 16 isolates, respectively. Genetic lineages detected were as follows: E. coli (ST48-ST10 Cplx, ST2499, ST906, ST2973 and ST2142); K. pneumoniae: (ST1540 and ST661). Our findings show a high rate of CTX-M-15 and high genetic diversity of ESBL-E isolates from WWPP and receiving river water.

Objectives: Colibacillosis caused by avian pathogenic Escherichia coli (APEC) is considered a major hindrance in poultry farming worldwide. This study aimed to characterize the genetic content and the relatedness between multidrug-resistant E. coli isolates from broiler chickens died due to colibacillosis from three farms from Tunisia., Methods: One hundred samples were collected from chickens' fresh carcasses from three poultry farms in Tunisia. E. coli isolation and identification were performed. Then, antimicrobial susceptibility regarding antibiotics, the ability to produce β-lactamases and minimum inhibitory concentration for colistin were determined according to Clinical and Laboratory Standards Institute guidelines. β-Lactam and non-β-lactam antimicrobial resistance genes, integrons, virulence genes, and phylogenetic groups were investigated using polymerase chain reaction. The genetic relatedness of the E. coli isolates was analysed by pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)., Results: A high infection rate of E. coli (50%) in infected organs of chickens was observed. The majority of E. coli isolates were multidrug resistant (96%); among them, 24% were colistin resistant and 30% were ESBL producing. Seven of 12 colistin-resistant isolates harboured the mcr-1 gene; among them, 10 were ESBL producing and carried bla CTX-M-1 , bla TEM , and bla SHV β-lactamase-encoding genes. E. coli isolates were assigned to different phylogroups but most of them (74%) belonged to the pathogenic phylogroup B2. Molecular typing by PFGE showed that some E. coli isolates harbouring ESBL-mcr-1 genes were clonally related. MLST revealed the presence of four different ST lineages among ESBL- and mcr-1-carrying E. coli: ST4187, ST3882; ST5693, and ST8932 with clonal dissemination of E. coli ST4187 between two of the farms., Conclusion: This is the first report of ESBL-mcr-1-carrying E. coli isolates of a clinically relevant phylogenetic group (B2) from chickens that died due to colibacillosis in Tunisian poultry farms., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objectives: poxtA is the most recently described gene conferring acquired resistance to linezolid, a relevant antibiotic for treating enterococcal infections. We retrospectively screened for poxtA in diverse enterococci and aimed to characterize its genetic/genomic contexts., Methods: poxtA was screened by PCR in 812 enterococci from 458 samples (hospitals/healthy humans/wastewater/animals/retail food) obtained in Portugal/Angola/Tunisia (1996-2019). Antimicrobial susceptibility testing was performed for 13 antibiotics (EUCAST/CLSI). poxtA stability (∼500 generations), transfer (filter mating), clonality (SmaI-PFGE) and location (S1-PFGE/hybridization) were tested. WGS (Illumina-HiSeq) was performed for clonal representatives., Results: poxtA was detected in Enterococcus faecium from six samples (1.3%): a healthy human (rectal swab) in Porto, Portugal (ST32/2001); four farm cows (milk) in Mateur, Tunisia (ST1058/2015); and a hospitalized patient (faeces) in Matosinhos, Portugal (ST1058/2015). All expressed resistance to linezolid (MIC = 8 mg/L), chloramphenicol, tetracycline and erythromycin, with variable resistance to ciprofloxacin and streptomycin. ST1058-poxtA-carrying isolates from Tunisia and Portugal differed by two SNPs and had similar plasmid content. poxtA, located in an IS1216-flanked Tn6246-like element, co-hybridized with fexB on one or more plasmids per isolate (one to three plasmids of 30-100 kb), was stable after several generations and transferred only from ST1058. ST1058 strains carried resistance/virulence genes (Efmqnr/acm) possibly induced under selective quinolone treatment., Conclusions: poxtA has been circulating in Portugal since at least 2001, corresponding to the oldest description worldwide to date. We also extend the reservoir of poxtA to bovines. The similar linezolid-resistant poxtA-carrying strains colonizing humans and livestock on different continents, and without a noticeable relationship, suggests a recent transmission event or convergent evolution of E. faecium populations in different hosts and geographic regions., (© The Author(s) 2020. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Avian industries have been reported as an important contributor in the worldwide spread of antibiotic resistance owing to some particular practices especially the overuse of antibiotics. Thus in this study, we aimed to characterize extended-spectrum-beta-lactamase (ESBL) and acquired-AmpC-beta-lactamase (aAmpC)-producing Escherichia coli isolates from chicken faeces and raw meat in Tunisia. During the year 2018, 286 faecal chicken swabs and 47 raw chicken meat samples were collected and processed to recover cefotaxime-resistant E. coli. Antimicrobial susceptibility was performed by disk-diffusion and/or broth-microdilution. bla TEM , bla SHV , bla CTX-M , and bla CMY genes were investigated by PCR/sequencing. Genes encoding resistance to colistin (mcr-1 to mcr-8), tetracycline (tetA/tetB), sulfonamide (sul1/sul3), and chloramphenicol (cmlA), were analysed by PCR. Class 1 integrons were investigated by PCR/sequencing. Phylogenetic groups of all isolates were determined. PFGE and MLST were performed for representative isolates. PCR-based replicon typing was performed in mcr1-harbouring isolates. Cefotaxime-resistant E. coli was detected in 22.4% (64/286) and 63.8% (30/47) of faeces and meat samples, respectively. Ninety isolates were ESBL-producers and harboured the genes: bla CTX-M-1 +/- bla TEM-1 (n = 65), bla CTX-M-55 +/- bla TEM-1 (n = 21), bla CTX-M-14 (n = 1), and bla SHV-12 (n = 3). The bla CMY-2 gene was detected in four ESBL-negative isolates. Isolates belonged to phylogroups D (50%), A (36.2%), B1 (9.6%), and B2 (4.3%). Fifty-four were colistin-resistant and 52 carried the mcr-1 gene. The tetA, sul1/sul3 and cmlA genes were detected among resistant isolates and 76 harboured class 1 integrons. MLST analysis revealed 13 sequence types (STs). The isolates were classified into 28 PFGE types. The IncP, IncFIB, and IncI1 replicons were detected among mcr-1-positive strains. We report a high frequency of ESBL-producers and colistin-resistant E. coli in chicken and derived food and the detection for the first time of bla CTX-M-55 in poultry in Tunisia., Competing Interests: Declaration of competing interest The authors declare that there are no conflicts of interest., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objectives: The aim of this study was to characterise Escherichia coli strains harbouring plasmid-mediated quinolone resistance (PMQR) genes recovered from various samples (n = 116) from healthy and diarrhoeic animals in Tunisia., Methods: All nalidixic acid-resistant E. coli isolates were screened for the presence of PMQR genes. Isolates positive for PMQR genes were investigated by PCR for chromosomal mutations in the quinolone resistance-determining regions (QRDRs) of GyrA and ParC, the presence of class 1 and class 2 integrons, genes encoding tetracycline and sulfonamide resistance, genes encoding virulence factors, and phylogenetic group. Genetic relationships was determined by pulsed-field gel electrophoresis (PFGE)., Results: Amongst 51 nalidixic acid-resistant isolates, 9 harboured PMQR genes (5 co-harbouredqnrS1 and qnrB1, 3 harboured qnrS1 and 1 harboured qnrB1). Two types of mutation in the QRDR of GyrA were observed: S83L and D87N (eight isolates) and S83L (one isolate). For the QRDR of ParC, the substitution S80I was observed in four isolates. A class 1 integron was found in six isolates. The tetA or tetB gene was observed in six isolates and both tetA and tetB were co-harboured by two isolates. The sul1, sul2 and sul3 genes were detected in six, four and one isolates, respectively. According to the presence of specific virulence genes, the nine strains were classified as UPEC (5), EAEC (3) and EPEC (1). Three isolates from turkey faeces were clonally related by PFGE., Conclusion: These findings highlight the plausible role of the avian industry as a reservoir of human pathogenic E. coli strains., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: Increasing numbers of linezolid-resistant Enterococcus carrying optrA are being reported across different niches worldwide. We aimed to characterize the first optrA-carrying Enterococcus faecalis obtained from food-producing animals and retail meat samples in Tunisia., Methods: Seven optrA-carrying E. faecalis obtained from chicken faeces (n=3, August 2017) and retail chicken meat (n=4, August 2017) in Tunisia were analysed. Antimicrobial susceptibility was determined by disc diffusion, broth microdilution and Etest against 13 antibiotics, linezolid and tedizolid, respectively (EUCAST/CLSI). optrA stability (∼600 bacterial generations), transfer (filter mating) and location (S1-PFGE/hybridization) were characterized. WGS (Illumina-HiSeq) was done for four representatives that were analysed through in silico and genomic mapping tools., Results: Four MDR clones carrying different virulence genes were identified in chicken faeces (ST476) and retail meat (the same ST476 clone plus ST21 and ST859) samples. MICs of linezolid and tedizolid were stably maintained at 8 and 1-2 mg/L, respectively. optrA was located in the same transferable chromosomal Tn6674-like element in ST476 and ST21 clones, similar to isolates from pigs in Malaysia and humans in China. ST859 carried a non-conjugative plasmid of ∼40 kb with an impB-fexA-optrA segment, similar to plasmids from pigs and humans in China., Conclusions: The same chromosomal and transferable Tn6674-like element was identified in different E. faecalis clones from humans and animals. The finding of retail meat contaminated with the same linezolid-resistant E. faecalis strain obtained from a food-producing animal highlights the potential role of the food chain in the worrisome dissemination of optrA that can be stably maintained without selective pressure over generations., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The objective of this study was to isolate and characterize ESBL-producing Escherichia coli (ESBL-EC) from raw bovine and caprine milk samples, as well as from bovine faeces in Tunisia. Therefore, 120 bovine faecal samples and 9 caprine raw milk samples were collected from 2 extensive dairy-cow-farms and 5 ovine farms, respectively. In addition, 94 raw bovine milk samples, from containers and holding tanks from 50 small public-markets in the North of Tunisia, were processed for the isolation of cefotaxime-resistant E. coli (CTX R ). Antimicrobial susceptibility testing was carried out by disc-diffusion/broth-microdilution methods. The presence of genes encoding ESBL, as well as those encoding colistin (mcr-1 to 5 genes)- sulfonamide-, tetracycline-, gentamicin-, quinolone and chloramphenicol-resistance and class 1 integrons were tested by PCR (and sequencing in some cases). ESBL-EC isolates were further characterized by phylogrouping and MLST/PFGE typing. Eight samples (3.6%) contained ESBL-EC isolates (3/2 from raw bovine/goat milk and 3 from cattle faeces) and one isolate/sample was characterized. Four ESBL-EC isolates, all of bovine origin (3 faeces/1 milk), were resistant to colistin (MIC: 8-16 μg/ml), harboured the mcr-1 gene and carried IncP- and IncFIB-type plasmids. The 8 ESBL-EC strains had the following characteristics: a) bovine faeces: mcr-1/CTX-M-1/D-ST1642 (3 strains); b) raw milk: mcr-1/CTX-M-1/A-ST10 (1 strain); CTX-M-15/B1-ST394 (3 strains), and CTX-M-15/A-ST46 (1 strain). Most of bovine ESBL-EC isolates were multidrug-resistant (4/5). Our results showed that ESBL-EC were detected in bovine and caprine samples (CTX-M-1/CTX-M-15 producers), being some of them colistin-resistant (associated with mcr-1 gene), and they belonged to international clonal lineages., (Copyright © 2019. Published by Elsevier Ltd.)

Objectives: The epidemiology of Enterococcus resistant to priority antibiotics including linezolid has mainly been investigated in developed countries and especially in hospitals. We aimed to evaluate the contribution of different non-human reservoirs for the burden of MDR enterococci in Tunisia, where scarce data are available., Methods: Samples (n = 287) were collected from urban wastewater (n = 57), retail meat (n = 29; poultry/bovine/ovine), milk (n = 89; bovine/ovine), farm animal faeces (n = 80; poultry/bovine/ovine) and pets (n = 32; rabbit/dogs/cats/birds) in different Tunisian regions (2014-17). They were plated onto Slanetz-Bartley agar after pre-enrichment without antibiotics. Standard methods were used for bacterial identification and characterization of antibiotic resistance and virulence genes (PCR), antibiotic susceptibility testing (disc diffusion/broth microdilution; EUCAST/CLSI) and clonality (SmaI-PFGE/MLST)., Results: All samples carried Enterococcus (n = 377 isolates) resistant to antibiotics considered to be critical or highly important by WHO. Even without antibiotic selection, 38% of Enterococcus faecalis (Efs) and 22% of Enterococcus faecium (Efm) were identified as MDR. Linezolid-resistant isolates (5%; MIC = 8 mg/L) comprised six poxtA-carrying Efm (cow milk), seven optrA-carrying Efs (chicken faeces/meat) and five Efm lacking cfr/optrA/poxtA (poultry/bovine/ovine/wastewater). Clinically relevant Efm clones (clade A1) were identified in animal/meat sources. Ampicillin resistance (1%) was confined to ST18/ST78-like MDR Efm clones from bovine meat/milk samples carrying relevant virulence markers (e.g. ptsD/IS16)., Conclusions: This study provides evidence of the contribution of livestock and foodstuffs to the dispersal of acquired linezolid resistance genes including poxtA and optrA. We report the first poxtA-carrying Efm in Tunisia, and for the first time in bovine samples, stressing the urgent need for alternative measures to counteract the spread of linezolid-resistant enterococci globally., (© The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

This study aimed to analyse the frequency of genes encoding virulence factors and to characterize resistance profiles of Staphylococcus aureus isolated from raw milk. In total, 47 and 9 S. aureus isolates were recovered from 150 and 100 raw bovine and ovine milk samples, respectively, in Tunisia. The majority of isolates was resistant to penicillin, and no methicillin-resistant S. aureus was detected. Eighteen and two isolates harboured etd and eta genes respectively. Sixteen enterotoxin-encoding genes were detected (n, %): sed (25, 44·6%), sec (16, 28·6%), sei (16, 28·6%), seh (13, 23·2%), seln (13, 23·2%), sell (10, 17·8%), seg (9, 16%), selu (8, 14·3%), selq (7, 12·5%), selo (7, 12·5%), selm (7, 12·5%), seb (7, 12·5%), sea (6, 10·7%), selk (3, 5·4%), ser (1, 1·8%) and selp (1, 1·8%). Ten isolates carried the tsst1 gene. All isolates carried the haemolysin toxin (hla, hld and hlg). The immune evasion cluster system-type B was predominant (20 isolates) followed by C (3 isolates), A and E (1 isolate each). The occurrence of enterotoxigenic S. aureus in raw milk constitutes a potential risk for human health. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper describes the characteristics of Staphylococcus aureus isolated from raw milk samples from healthy cows and ewes collected from small family farms in Tunisia. Fifty-six strains were analysed by determining their antibiotic susceptibility and genes encoding antibiotic resistance and virulence factors. Methicillin-resistant strains were not detected, and overall low level of antimicrobial resistance was reported. However, our strains harboured several genes encoding virulence factors and 87·5% of them carried at least one gene encoding for enterotoxins showing a high risk of spread of food-borne diseases., (© 2019 The Society for Applied Microbiology.)

Objectives: This study aimed to isolate and characterise extended-spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) isolates from animals and wastewater in Tunisia., Methods: ESBL-E from wastewater (n=123 samples), faeces of healthy animals (poultry, sheep, goats and calves) (n=140) and raw milk from healthy cows (n=42) and goats (n=20) were investigated. Antimicrobial susceptibility was determined according to CLSI recommendations. The bla TEM , bla SHV , bla CTX-M and bla OXA-48 genes were analysed by PCR and sequencing. Phylogenetic groups were determined by PCR for Escherichia coli isolates. The clonality of E. coli and Klebsiella pneumoniae isolates was determined by XbaI-PFGE and MLST., Results: A total of 81 E. coli, 20 K. pneumoniae, 4 Enterobacter cloacae, 1 Citrobacter freundii and 1 Citrobacter braakii were isolated. The bla CTX-M-1 and bla CTX-M-15 genes were predominant in E. coli and K. pneumoniae isolates. E. cloacae and C. braakii isolates harboured the bla SHV-12 gene. The C. freundii isolated from wastewater carried bla CTX-M-15 , bla TEM-1 and bla OXA-204 . E. coli isolates belonged to phylogroups A (37), B1 (25), B2 (7) and D (12). Seventy-eight E. coli isolates were typeable by PFGE and were classified into 34 pulsotypes. The K. pneumoniae isolates belonged to 11 pulsotypes. The E. coli isolates belonged to sequence types ST131, ST224, ST162, ST845, ST5204, ST69, ST141 and ST10. The K. pneumoniae isolates belonged to ST405, ST147, ST564, ST307, ST152, ST45, ST661 and ST1564., Conclusion: This is the first report of O25b-B2 3 -CTX-M-27-ST131 E. coli isolates and of C. freundii carrying bla CTX-M-15 , bla TEM-1 and bla OXA-204 in Tunisia., (Copyright © 2019 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Objectives: Methicillin-resistant staphylococci, including methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP), pose a threat to animal and human health worldwide. Veterinary staff and pets may play a role in the spread of resistant clones., Methods: A total of 125 samples from veterinary staff (n=50), dogs (n=49) and cats (n=26) were investigated. Obtained isolates were tested for the methicillin resistance gene mecA and were subjected to multiplex PCR to differentiate coagulase-positive species. Following SCCmec and spa typing, isolates were tested for the presence of various toxin and virulence genes and phenotypic resistance to common antimicrobials., Results: Overall, 4 MRSA were isolated from two veterinarians and two dogs and 19 MRSP were found in eleven dogs (12 isolates) and five cats (7 isolates). The MRSA isolates possessed sea (2) and eta (3) virulence genes and the MRSP isolates possessed sea (6), expA (15), expB (1) and siet (19) genes. SCCmec type II and three spa types (t186, t1816 and t10897) were identified in the MRSA isolates. Most of the MRSP isolates belonged to SCCmec types II (2 isolates) and V (10 isolates); however, the remaining 7 isolates were untypeable and contained class C1 mec. The majority of isolates were multidrug-resistant (MDR)., Conclusion: These findings show that pets and veterinarians could be potential sources of MDR-MRSA and MDR-MRSP in Iran. Taken together, these findings warrant future investigations on the epidemiology and public-health significance of MDR-MRSA and MDR-MRSP both in veterinarians and companion animals in Iran., (Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Objectives: This study aimed to screen for and characterise methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) in nasal swabs and milk from healthy cows from different regions in Tunisia., Methods: A total of 141 Staphylococcus spp. isolates were recovered from milk and nasal samples of cows. S. aureus isolates were further characterised by determining their antimicrobial susceptibilities, genes encoding antimicrobial resistance and virulence factors, biofilm production, agr type and PFGE. spa and SCCmec typing and MLST were also performed for the MRSA isolate., Results: Twenty-seven isolates (19.1%) were identified as S. aureus, of which 26 were MSSA and 1 was MRSA. The MSSA isolates were resistant to penicillin (73.1%), fusidic acid (61.5%), clindamycin (34.6%) and erythromycin (34.6%). The MRSA isolate, from a milk sample, was resistant to cefoxitin, penicillin, fusidic acid, amikacin and clindamycin. Twenty-five isolates (92.6%) had at least one enterotoxin gene. Only four isolates (14.8%) were positive for the tsst-1 gene. Genes encoding the exfoliative toxins D and A were detected in 9 (33.3%) and 6 (22.2%) isolates, respectively. The single MRSA isolate and 22 MSSA isolates were biofilm-producers on Congo red agar plates. Twelve pulsotypes were identified amongst 25 MSSA isolates revealing the clonal diversity of these isolates; however, one MSSA isolate was identified as CC398. The MRSA isolate was PVL-negative and was typed as ST97-t267-agrI-SCCmecV., Conclusion: Contamination of milk with S. aureus, especially enterotoxin- and TSST-1-positive strains, poses a potential public-health threat. This is the first report of MRSA of bovine origin in Tunisia., (Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

Staphylococcus aureus is recognized worldwide as one of the major agents of dairy cow intra-mammary infections. This microorganism can express a wide spectrum of pathogenic factors used to attach, colonize, invade and infect the host. The present study evaluated 120 isolates from eight different countries that were genotyped by RS-PCR and investigated for 26 different virulence factors to increase the knowledge on the circulating genetic lineages among the cow population with mastitis. New genotypes were observed for South African strains while for all the other countries new variants of existing genotypes were detected. For each country, a specific genotypic pattern was found. Among the virulence factors, fmtB , cna , clfA and leucocidins genes were the most frequent. The sea and sei genes were present in seven out of eight countries; seh showed high frequency in South American countries (Brazil, Colombia, Argentina), while sel was harboured especially in one Mediterranean country (Tunisia). The etb , seb and see genes were not detected in any of the isolates, while only two isolates were MRSA (Germany and Italy) confirming the low diffusion of methicillin resistance microorganism among bovine mastitis isolates. This work demonstrated the wide variety of S. aureus genotypes found in dairy cattle worldwide. This condition suggests that considering the region of interest might help to formulate strategies for reducing the infection spreading.

Livestock and food products of animal origin constitute important reservoirs of intestinal and extraintestinal pathogenic Escherichia coli including antibiotic-resistant E. coli isolates. To assess potential risks to public health related to E. coli strains of animal origin in Tunisia, 65 E. coli isolates recovered from healthy animals and food products of animal origin were studied. Antimicrobial susceptibility was determined according to CLSI guidelines and genes encoding antibiotic resistance as well as virulence factors were investigated by PCR. High rates of antibiotic resistance were observed to kanamycin (78.4%), gentamicin (75.3%) and streptomycin (75.3%, encoded by strA-strB (7 isolates)), amoxicillin (64.6%), amoxicillin/clavulanic acid (60%), tetracycline (44.6%; tetA (8 isolates) and tetB (7 isolates)), nalidixic acid (27.6%, qnrS (3 isolates), qnrB (2 isolates) and qnrA (one isolate)) and sulfonamides (36.9%; sul1 (1 isolate), sul2 (4 isolates), and sul3 (1 isolate)). Virulotypes classified some isolates as STEC (3%), MNEC (1.5%) and atypical EPEC (1.5%). This study demonstrated high rates of antimicrobial resistance and the presence of some pathogenic pathovars from animal origins that are a cause of concern for public health.

Our study aimed to investigate colistin resistance and the mechanisms involved in a collection of 35 extended-spectrum beta-lactamase (ESBL) and 13 CMY-2-producing E. coli strains which were previously recovered from chicken gut microbiota in Tunisia, as well as to determine the genetic location of mcr genes. Forty-eight ESBL and CMY-2-producing E. coli strains were obtained from 137 fecal samples of healthy chickens during 2013. These strains were tested for colistin resistance by the broth microdilution method, and screened for mcr-1 and mcr-2 genes by PCR. Two of these strains were colistin-resistant (MIC = 8 mg/L). Both harbored the mcr-1 gene, were CMY-2 producers, and were additionally resistant to tetracycline, ciprofloxacin, chloramphenicol, gentamicin, tobramycin and trimethoprim-sulfamethoxazole. They shared phylogroup A, the same pulsed-field gel electrophoresis (PFGE)-pattern, and were typed as ST2197. In both strains, ISApl1 and pap2 were detected upstream and downstream of mcr-1 gene, respectively. The analysis of the two mcr-1-positive strains and their transconjugants by PCR-based replicon typing and S1-PFGE, demonstrated that mcr-1 gene is linked to an IncP plasmid (~242 kb), and bla CMY-2 to an IncI1 plasmid (97 kb). The occurrence of E. coli harboring mcr-1 gene among intestinal microbiota in poultry and its location on a conjugative plasmid could represent a risk for public health. The evolution of this type of resistant microorganisms should be evaluated in the future., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The aim of this work was the genetic characterization of cefotaxime-resistant enterobacteria from animals (53 samples), the surface water of rivers (17 samples), and wastewater treatment plants (43 samples) in Tunisia. A total of 48 (42.4%) cefotaxime-resistant isolates were recovered. An extended spectrum beta-lactamase (ESBL) phenotype with a positive double-disk synergy test (DDST) was exhibited by 34 (70.8%) and 14 (29.1%) isolates from water and animal origins, respectively. Isolates from water were identified as: Escherichia coli (n = 17), Hafnia spp. (n = 13), Citrobacter spp. (n = 1), Enterobacter cloacae (n = 1), Klebsiella pneumoniae (n = 1), and K. oxytoca (n = 1). Animal isolates were identified as: E. coli (n = 11), E. cloacae (n = 1), Hafnia spp. (n = 1), and K. pneumoniae (n = 1). PCR investigation of blaCTX-M, blaTEM, and blaSHV genes showed that amongst the 48 isolates with a positive DDST, 41 (87.5%) carried the blaCTX-M gene, 1 isolate harbored the blaSHV gene, and 1 isolate coharbored blaCTX-M with blaSHV genes. The class 1 and 2 integrons were detected in 27 (56.2%) and 1 (2%) isolates, respectively. Our study showed a significant occurrence of ESBL-producing enterobacteria in animals and aquatic environments with a predominance of blaCTX-M genes., (© 2019 S. Karger AG, Basel.)

Objectives: Oxazolidinone resistance is a serious limitation in the treatment of MDR Enterococcus infections. Plasmid-mediated oxazolidinone resistance has been strongly linked to animals where the use of phenicols might co-select resistance to both antibiotic families. Our goal was to assess the diversity of genes conferring phenicol/oxazolidinone resistance among diverse enterococci and to characterize the optrA genetic environment., Methods: Chloramphenicol-resistant isolates (>16 mg/L, n = 245) from different sources (hospitals/healthy humans/wastewaters/animals) in Portugal, Angola and Tunisia (1996-2016) were selected. Phenicol (eight cat variants, fexA, fexB) or phenicol + oxazolidinone [cfr, cfr(B), optrA] resistance genes were searched for by PCR. Susceptibility (disc diffusion/microdilution), filter mating, stability of antibiotic resistance (500 bacterial generations), plasmid typing (S1-PFGE/hybridization), MLST and WGS (Illumina-HiSeq) were performed for optrA-positive isolates., Results: Resistance to phenicols (n = 181, 74%) and phenicols + oxazolidinones (n = 2, 1%) was associated with the presence of cat(A-8) (40%, predominant in hospitals and swine), cat(A-7) (29%, predominant in poultry and healthy humans), cat(A-9) (2%), fexB (2%) and fexA + optrA (1%). fexA and optrA genes were co-located in a transferable plasmid (pAF379, 72 918 bp) of two ST86 MDR Tunisian Enterococcus faecalis (wastewaters) carrying several putative virulence genes. MICs of chloramphenicol, linezolid and tedizolid were stably maintained at 64, 4 and 1 mg/L, respectively. The chimeric pAF379 comprised relics of genetic elements from different Gram-positive bacteria and origins (human/porcine)., Conclusions: To the best of our knowledge, we report the first detection of optrA in an African country (Tunisia) within a transferable mosaic plasmid of different origins. Its identification in isolates from environmental sources is worrisome and alerts for the need of a concerted global surveillance on the occurrence and spread of optrA., (© The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

This study focused on 77 isolates of Salmonella enterica serotype Enteritidis collected during 2009 to 2013 from healthy and sick chickens and environmental farm samples in Tunisia. Resistance to 14 antimicrobials and the encoding genes were analyzed. 66, 26, 6.5, 3.9 and 1.3% were pan-susceptible or showed resistance to nalidixic acid (Asp87 to Tyr and Asp87 to Asn substitutions in GyrA), ampicillin (bla TEM-1-like and bla SHV ), sulfonamides (sul1and sul3) and streptomycin (strB), respectively. A single isolate with intermediate susceptibility to ciprofloxacin was positive for qnrB, whereas qnrA, qnrS or aac(6')-Ib-cr were not detected. The virulotype of the isolates was established by testing ten virulence genes. The orgA, ssaQ, mgtC, siiD, sopB genes, located on Salmonella pathogenicity islands, and spvC of the serotype-specific virulence plasmid, were common to all isolates. In contrast, the prophage-associated sopE-1, sodC1 and gipA genes and the fimbrial bcfC gene were variably represented. All isolates except one contained the virulence plasmid, which appeared either alone or together with one or more additional plasmids. One isolate carried a single plasmid of ca. 90Kb which may be derived from the virulence plasmid (60Kb). Overall, seven resistotypes, six virulotypes and six plasmid profiles were identified. XbaI-PFGE revealed four related pulsotypes (X1-X4), with 80% of the isolates sharing the X1 pattern. The latter isolates exhibited different resistance, virulence and plasmid profiles, suggesting that mobile genetic elements, particularly prophages and plasmids, are of central importance for the evolution and adaptation of S. Enteritidis circulating in chicken farms in Tunisia., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: The aim of this study was to characterize 32 MRSA isolates recovered from wound specimens of patients in a Hospital in Tripoli, Libya, during 2013., Methods: MRSA isolates were characterized by determining their antibiotic susceptibilities, genes encoding antibiotic resistance and virulence factors, the SCCmec class, agr type, spa typing, PFGE and MLST., Results: PFGE and MLST revealed that all isolates were clonal and belonged to the Clonal Complex 5 (CC5). They harboured the SCCmecV and the agrII and the spa type was t6065. The majority of isolates were resistant to cefoxitin (32, 100%), penicillin (32, 100%), ampicillin (32, 100%), enrofloxacin (32, 100%), ciprofloxacin (32, 100%), fusidic acid (32, 100%), gentamicin (32, 100%), kanamycin (32, 100%), trimethoprim (32, 100%), and erythromycin (30, 93.7%). The main genes encoding antibiotic resistance were: blaZ (31, 96.8%), ermC (30, 93.7%), aph(3')-III a (3, 9.4%), aac6-aph2 (32, 100%), InuA (3, 9.4%), tetM (3, 9.4%), tetL (3, 9.4%), dfrG (28, 87.5%), fusC (32, 100%). All isolates were PVL negative; however, exfoliative-encoding genes (eta: 25) and enterotxin genes (seb: 32, seo: 32, sei: 32, ser: 32, seu: 32, seg: 32, sej: 32, sed: 31, sen: 29, seh: 26, sec: 26, sea: 6, sek: 5), haemolysin (hla (32), hld (32), hlg (32)) and immune evasion cluster proteins (scn: 32, sak: 32) were relevant., Conclusion: To the best of our knowledge this is the first report of a specific clonal spread of a multi-drug resistant MRSA-CC5- SCCmecV in a Libyan Hospital., (Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.)

The objective was to characterize Staphylococcus aureus isolated from two wastewater treatment plants (WWTPs) located in Tunis City (Tunisia), during the period 2014-2015. Genetic lineages, antibiotic resistance mechanisms and virulence factors were determined for the recovered isolates. S. aureus isolates were recovered from 12 of the 62 wastewater samples tested (19.35%), and one isolate/sample was characterized, all of them being methicillin-susceptible (MSSA). Six spa types (t587, t674, t224, t127, t701 and t1534) were found among the 12 isolates, and the spa-t587, associated with the new sequence type ST3245, was the most predominant one (7 isolates). The remaining isolates were assigned to five clonal complexes (CC5, CC97, CC1, CC6 and CC522) according to the sequence-type determined and/or the spa-type detected. S. aureus isolates were ascribed to agrI (n = 3), agrII (n = 7) and agrIII (n = 1); however, one isolate was non-typeable. S. aureus showed resistance to (number of isolates): penicillin (12), erythromycin (7), tetracycline (one) and clindamycin (one). Among the virulence factors investigated, only one isolate harboured the tst gene, encoding the TSST-1 (toxic shock syndrome toxin 1). Despite the low number of studied isolates, the present study reports the occurrence of both human- and animal-associated S. aureus clonal complexes in WWTPs in Tunisia.