Detection and quantification of microplastics in Posidonia oceanica banquettes in the Gulf of Gabes, Tunisia.

Plastic pollution and microplastic (MP) debris are some of the most significant solid waste pollutants, threatening the marine environment and causing sediment accumulation. Coastal seagrass areas are usually important habitats that support multiple living species and provide several ecosystem services. This study aimed to determine the abundance, characteristics, and composition of microplastics on the southern side of the Tunisian Mediterranean Sea by using Posidonia oceanica (P. oceanica) as a crucial trap for microplastics. Samples of Posidonia leaves were collected from the Tunisian coastal area of Gabes-City. The characterization of microplastic detritus was carried out by stereomicroscopy, and acid digestion of Posidonia tissue leaves was performed for qualitative and quantitative analysis of MPs using NMR spectroscopy. The study revealed pellets, threads, and fragments of polymers as the frequent forms found in MPs. Polyethylene, polystyrene, and bis(2-ethyl-hexyl) phthalates were the most abundant materials detected. P. oceanica leaves contributed notably to microplastic subsidence, seafloor horizontal migration, and sediment burial. Thus, marine flora appeared to be a good tool to detect and monitor plasticizers, and further studies of the P. oceanica seagrass areas will help in developing a more comprehensive knowledge of chemicals spreading over a geographical zone. The results obtained will be used for developing baseline data on plasticizer contamination on the wide-ranging marine coast.

Widespread of the Vienna/Hungarian/Brazilian CC8-ST239-SCCmec III MRSA clone in patients hospitalized in the Tunisian Burn and Traumatology Center.

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-ß-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.

Genetic characterization of ESBL/pAmpC-producing Escherichia coli isolated from forest, urban park and cereal culture soils.

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.

Genetic characterization of extended-spectrum ß-lactamase-producing Enterobacteriaceae from a biological industrial wastewater treatment plant in Tunisia with detection of the colistin-resistance mcr-1 gene.

This study evaluated the occurrence of extended-spectrum ß-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-ß-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.

Genetic characterization of ESBL-producing Escherichia coli and Klebsiella pneumoniae isolated from wastewater and river water in Tunisia: predominance of CTX-M-15 and high genetic diversity.

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 (CTXR) 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.

Detection of Extended-Spectrum ß-Lactamases (ESBL) Producing Enterobacteriaceae from Fish Trapped in the Lagoon Area of Bizerte, Tunisia.

Extended-spectrum ß-lactamase and their molecular mechanism in Enterobacteriaceae were analyzed in 126 fish samples of 9 various wild species, living in the lagoon of Bizerte in Tunisia. Fifty-nine (59) Gram-negative strains were isolated and identified as Escherichia coli (n = 24), Klebsiella pneumonia (n = 21), Citrobacter freundii (n = 8), and Shigella boydii (n = 6). Forty-seven ESBL producers were identified using the synergic test. ß-Lactamase genes detected were bla CTX-M-1 (E. coli/15; K. pneumonia/8; C. freundii/1; Sh. boydii/1), bla CTX-M-1+ bla OXA-1 (E. coli/4; K. pneumonia/3), bla CTX-M-1+ bla TEM-1-a (K. pneumonia/2), bla CTX-M-15+ bla TEM-1-a (K. pneumonia/1; Sh. boydii/1), bla CTX-M-15+ bla OXA-1 (K. pneumonia/1), bla CTX-M-15 (E. coli/3; K. pneumonia/1; Sh. boydii/3), and bla CTX-M-9 (C. freundii/3). Most strains (84.7%) showed a multiresistant phenotype. qnrA and qnrB genes were identified in six E. coli and in ten E. coli+one K. pneumonia isolates, respectively. The resistance to tetracycline and sulfonamide was conferred by the tet and sul genes. Characterization of phylogenic groups in E. coli isolates revealed phylogroups D (n = 20 strains), B2 (n = 2), and A (n = 2). The studied virulence factor showed prevalence of fimA genes in 9 E. coli isolates (37.5%). Similarly, no strain revealed the three other virulence factors tested (eae, aer, and cnf1). Our findings confirmed that the lagoons of Bizerte may be a reservoir of multidrug resistance/ESBL-producing Enterobacteriaceae. This could lead to indisputable impacts on human and animal health, through the food chain.

Co-occurrence of mcr-1 mediated colistin resistance and ß-lactamase-encoding genes in multidrug-resistant Escherichia coli from broiler chickens with colibacillosis in Tunisia.

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 ß-lactamases and minimum inhibitory concentration for colistin were determined according to Clinical and Laboratory Standards Institute guidelines. ß-Lactam and non-ß-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 blaCTX-M-1, blaTEM, and blaSHV ß-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.

High prevalence of mcr-1 encoding colistin resistance and first identification of blaCTX-M-55 in ESBL/CMY-2-producing Escherichia coli isolated from chicken faeces and retail meat in Tunisia.

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. blaTEM, blaSHV, blaCTX-M, and blaCMY 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: blaCTX-M-1 +/- blaTEM-1 (n = 65), blaCTX-M-55 +/- blaTEM-1 (n = 21), blaCTX-M-14 (n = 1), and blaSHV-12 (n = 3). The blaCMY-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 blaCTX-M-55 in poultry in Tunisia.

mcr-1 encoding colistin resistance in CTX-M-1/CTX-M-15- producing Escherichia coli isolates of bovine and caprine origins in Tunisia. First report of CTX-M-15-ST394/D E. coli from goats.

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 (CTXR). 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.

Extended-spectrum ß-lactamase-producing Enterobacteriaceae from animal origin and wastewater in Tunisia: first detection of O25b-B23-CTX-M-27-ST131 Escherichia coli and CTX-M-15/OXA-204-producing Citrobacter freundii from wastewater.

OBJECTIVES: This study aimed to isolate and characterise extended-spectrum ß-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 blaTEM, blaSHV, blaCTX-M and blaOXA-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 blaCTX-M-1 and blaCTX-M-15 genes were predominant in E. coli and K. pneumoniae isolates. E. cloacae and C. braakii isolates harboured the blaSHV-12 gene. The C. freundii isolated from wastewater carried blaCTX-M-15, blaTEM-1 and blaOXA-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-B23-CTX-M-27-ST131 E. coli isolates and of C. freundii carrying blaCTX-M-15, blaTEM-1 and blaOXA-204 in Tunisia.

Multidrug Resistance and the Predominance of blaCTX-M in Extended Spectrum Beta-Lactamase-Producing Enterobacteriaceae of Animal and Water Origin.

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.