Activated sludge and UV-C254 for Sapovirus, Aichivirus, Astrovirus, and Adenovirus processing.

In this study, the detection rates of four enteric viruses, Human Astrovirus (HAstVs), Aichivirus (AiVs), Human Adenovirus (HAdVs), and Sapovirus (SaVs) are carried out to assess the virological quality of the treated wastewater. A total of 140 samples was collected from wastewater treatment plant WWTP of Tunis-City. Real-time RT-PCR and conventional RT-PCR results showed high frequencies of detection of the four enteric viruses investigated at the entry and exit of the biological activated sludge procedure and a significant reduction in viral titers after tertiary treatment with UV-C254 irradiation. These results revealed the ineffectiveness of the biological activated sludge treatment in removing viruses and the poor quality of the treated wastewater intended for recycling, agricultural reuse, and safe discharge into the natural environment. The UV-C254 irradiation, selected while considering the non-release of known disinfection by-products because of eventual reactions with the large organic and mineral load commonly present in the wastewater.

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.

Effectiveness of combined tools: adsorption, bioaugmentation and phytoremediation for pesticides removal from wastewater.

The aim of this study was the comparison of two process in pentachlorophenol (PCP: 100 mg L-1) removal by combined process bioaugmentation-adsorption and bioaugmentation-phytoremediation in secondary treated wastewater (STWW). The phytoremediation procedure was conducted by using two plants such as Typha angustifolia and Schoenoplectus acutus, and the bioaugmentation procedure was operated by Pseudomonas putida HM 627618 as a plant growth promoting bacteria (PGPR). The adsorption process was performed by palm date activated carbon. The PCP monitoring was assessed by high performance liquid chromatography (HPLC) and the optical density determination at 600 nm (OD600). The performance of the two processes was observed by the determination of total bacteria, chlorophylls and physical and chemical analysis (COD, pH, conductivity, chloride, and organic carbon). The alfalfa seed germination test was conducted to assess the two operational performance procedures. According to the results obtained from the physical and chemical analysis of the treated STWW, there was no significant differences in the pH and in the EC content of the bioaugmentation-phytoremediation treatment, while a significant increase of the EC content was observed in the bioaugmentation-adsorption to 5.08 mS cm-1. The COD value significantly decreased up to 1320 mg L-1 in bioaugmentation-adsorption treatment (control value 2400 mg L-1) and 98 mg L-1 in bioaugmentation-phytoremediation treatment (control value 98 mg L-1). Microbial biomass monitoring of P. putida shows significant greater in both processes in the order of 9.18 and 7.01 Log CFU mL-1 for bioaugmentation-adsorption and bioaugmentation-phytoremediation, respectively. The chlorophyll content in Typha angustifolia and Schoenoplectus acutus significantly decreased after 144 h with the exception of the chlorophyll a content of Schoenoplectus acutus in which the content increased up to 3.31 mg mL-1. Comparing the performance of these two treatments, it was found according to HPLC analysis that the bioaugmentation-adsorption process was more efficient in removing about 97% of PCP after 48 h, against around 90% of PCP after 72 h for the phytoremediation-bioaugmentation. The alfalfa seeds showed a germination rate after the 5th day of incubation of 100% and 95%, respectively for the PCP-non-contaminated and treated STWW, while for wastewater containing PCP the germination was totally inhibited.

This paper describing sensitive methods of combined bioaugmentation-phytoremediation and bioaugmentation-adsorption for pentachlorophenol (PCP) depletion in wastewater. The novelty is the choice of a macrophyte Typha angustifolia and Schoenoplectus acutus in constructed wetland fixed in clay matrix. The two-selected plants are still used for the elimination of heavy metals but not for pesticide in wastewater. Also, the combined process bioaugmentation-adsorption was not tested in other researches. On the other side, in this study, the phytoremediation technique combined with bacteria positively affected the plants activity in order to promote pollutant remediation. Hence, the Pseudomonas putida HM 627618 in wastewater with the macrophyte presence or date stone adsorbent have a great capacity to reduce this pollutant (PCP) by improving the bioremediation process.

Effects of heavy metals on growth and biofilm-producing abilities of Salmonella enterica isolated from Tunisia.

This study aims to test the toxicity of some metallic elements on Salmonella enterica strains and their power to grow and to develop a biofilm to overcome this environmental stress. From 50 selected strains of Salmonella, 70% belong to the Kentucky serotypes that is the most frequent one, followed by the other serotypes such as Amsterdam 6%, anatum 4%, derby 4% Enteritidis 4%, Zanzibar 4%, typhyrimium 2%, gallinaruim 2%, inbondaka 2% and Newport 2%. All the strains have presented the invA invasion gene involved in the virulence and Salmonella infection. Genotypic BOX-PCR analysis of these strains showed 18 profiles, with a discrimination index of 0.93. The Salmonella growth has mainly revealed that the variation of the rates of different metallic elements showed a significant influence on the Salmonella growth. The qualitative, quantitative study and biofilm tubes showed that 40% of the strains have a strong capacity to form biofilm, and the wild-type phenotypes (RDAR; rigid film; Strong), This phenotype varies according to the nature and the concentration of the metal (0.1 mM-1 mM) considered. In the presence of copper, zinc, cobalt, and chromium, the Salmonella strains showed a potent capacity to form a biofilm with a slight variation in the wild-type phenotype. However, when chromium rates increased, Salmonella loses the RDAR morphotype. Addition of mercury and cadmium in the growth medium reduced the production of Salmonella biofilm by around 14 and 15%, respectively, if compared with the control free of metals.

Macrophyte and indigenous bacterial co-remediation process for pentachlorophenol removal from wastewater.

This study has contributed in the description of bioaugmentation-phytoremediation efficiency process using Typha angustifolia concerning PCP tolerance and removal from wastewater. Samples of wastewater were collected from industrial wastewater plants, namely row wastewater effluent "E.WW", primary wastewater "P.WW", secondary wastewater "S.WW", clarified wastewater "AC.WW". These effluents were spiked with PCP at different rate (100, 500, and 1000 mg.L-1), physical, chemical and biological properties were monitored. A second experiment was set up in order to check the efficiency of phytoremediation treatments of the different effluents artificially contaminated with 200 mg.L-1 PCP after 20 days lab scale experiment. An important PCP removal by indigenous bacteria was showed in S. WW with values from 1000 to 72.2 mg.L-1 from T0 (start of the experiment) to TF (end of the experiment), respectively. Phytoremediation process allowed a decrease of PCP rate from 200 to 6.4 mg.L-1, a decrease of chloride content from 14.0 to 4.0 mg.L-1 in S.WW samples was observed. Furthermore, a significant increase of bacterial number in S.WW and AC.WW to 1.700 × 106 and 1.450 × 106 CFU.mL-1, respectively was observed. In addition, the DGGE analysis showed that after bioaugmentation-phytoremediation treatments, the highest species richness and relative abundance in wastewater effluent was observed. Novelty statement Pentachlorophenol (PCP) is one of highly toxic of polychlorophenols and required to continuously monitor in environment. This paper presenting a sensitive method phytoremediation and bioaugmentation for PCP biotransformation in wastewater. The novelty is the choice of a macrophyte Typha angustifolia, which is still used for the elimination of heavy metals but it not used for pesticide and pollutant removal in wastewater. Also, there are different analysis that was performed in order to check phyto-technique process (DGGE and HPLC). On the other side, in this study, the phyto-techniques with Typha angustifolia positively affected intrinsic microorganisms in order to promote pollutant remediation. So, the intrinsic microorganisms in wastewater with the macrophyte presence have a great capacity to reduce this pollutant and improve the bioremediation process.

High rates of antibiotic resistance and biofilm production in Escherichia coli isolates from food products of animal and vegetable origins in Tunisia: a real threat to human health.

The aim of this study was to compare the antibiotic susceptibility of eighty Escherichia coli isolates from vegetables and food products of animal origin in Tunisia, and to study their genes encoding antibiotic resistance and in vitro biofilm forming capacity. Antimicrobial susceptibilities were determined, as well as PCR investigation of genes associated with antibiotic resistance. Biofilm formation was tested using four different methods: the microtiter plate-, MTT-staining-, XTT-staining-, and the Congo Red Agar assays. High antibiotic resistance rates were observed for amoxicillin (68.7%), amoxicillin/clavulanic acid (73.7%), gentamicin (68.7%), kanamycin (66.2%), nalidixic acid (36.2%), streptomycin (68.7%) and tetracycline (35%). The majority of isolates was multidrug resistant and biofilm producer. MTT testing showed that vegetables isolates were significantly higher biofilm producers compared to foods of animal origins. This study showed that E. coli isolates from food products were reservoirs of genes encoding antibiotic-resistance and have a high propensity to produce biofilm.

Exhaustion of pentachlorophenol in soil microcosms with three Pseudomonas species as detoxification agents.

Pentachlorophenol (PCP) is a toxic compound, which is widely used as a wood preservative product and general biocide. It is persistent in the environment and has been classified as a persistent organic pollutant to be reclaimed in many countries. Bioremediation is an emerging approach to rehabilitating areas polluted by recalcitrant xenobiotics. In the present study, we evaluated the potential of three strains of Pseudomonas (P. putida S121, P. rhizophila S211, and P. fuscovagiceae S115) as bioremediation agents in depletion and detoxification of PCP in soil microcosms. PCP removal was effectively optimized using a central-composite experimental design and response surface methodology (RSM). The optimum conditions for maximum PCP removal yield (85 ± 5%) were: 500 mg/kg PCP concentration, 108 UFC/g soil inoculum size of each strain and 55 days incubation period. The bacterial strains, P. putida, P. rhizophila, and P. fuscovagiceae, showed good capability to tolerate and degrade PCP so that they could be successfully used in synergistic effect to treat PCP polluted soils.

Bacterial consortium biotransformation of pentachlorophenol contaminated wastewater.

The aims of this study were (i) to compare PCP removal (100 mg L-1) by two bacterial consortia B1 and B2 in sterile wastewater (STWW) and liquid mineral medium (MSM), (ii) PCP effect in biofilm formation and antimicrobial susceptibility. PCP removal was measured by high-performance liquid chromatography (HPLC) during 168 h at 30 °C. Biofilm formation was assessed with two approaches: Congo Red Agar and Microtiter-plate. Antimicrobial susceptibility was determined by the agar disc diffusion technique. The results showed that the PCP removal for consortium B1 and B2 after 168 h was 70 and 97.5% in STWW; 62.2 and 85.5% in MSM, respectively. In addition, PCP addition showed an increase in biofilm development especially for B2 consortium around 3.5 nm in 100 mg L-1 PCP. PCP added in the Muller Hinton (MH) medium and Gentamicin disc showed a clear increase in diameter of cell lysis around 2 to 4.5 cm.

Surfactant efficiency on pentachlorophenol-contaminated wastewater enhanced by Pseudomonas putida AJ 785569.

This study aims to evaluate the effect of three surfactants on the removal of PCP (800 mg L-1) from Secondary Treated Wastewater (STWW) by Pseudomonas putida AJ 785569. The effect of surfactants [sodium lauryl sulfate (SDS) as anionic, Tween 80 (TW80) as non-anionic and cetyltrimethylammonium bromide (CTAB) as cationic] is tested about the three following aspects: (1) bacterial growth, (2) bacterial biofilm formation or development and (3) PCP rate removal. The results showed that strain P. putida AJ 785569 could adsorb around 30 mg L-1 and remove 600 mg L-1 of PCP within 168 h of incubation. The SDS developed the growth of bacteria and the removal of PCP. This PCP removal in mineral salt medium (MSM) is around 760 mg L-1 (95% degradation) higher than the ones registered with CTAB and TW80 with a value 506.75 (63% degradation) and 364.1 mg L-1 (45% degradation), respectively. The obtained results of chloride concentration showed an important relation with PCP removal during incubation with an important value. Monitoring the development of bacterial biofilm, in MSM medium added with PCP (100 mg L-1) by strain P. putida AJ 785569, showed a significant increase in the optical density value from 0.9 to 4 at λ = 595 nm, a modification of strain P. putida AJ 785569's morphotype, density and color colonies.

Study of the diversity of 16S-23S rDNA internal transcribed spacer (ITS) typing of Escherichia coli strains isolated from various biotopes in Tunisia.

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.

Removal of pentachlorophenol from contaminated wastewater using phytoremediation and bioaugmentation processes.

The phytoremediation procedure was conducted by Lemna gibba (L) and Typha angustifolia (T) and the bioaugmentation procedure used P. putida HM627618. The ability of the selected P. putida HM627618 to tolerate and remove PCP (200 mg L-1) was measured by high performance liquid chromatography analysis and optical density at 600 nm. Five different experiments were conducted in secondary treated wastewater for PCP testing removal (100 mg L-1) including two phytoremediation assays (T + PCP; L + PCP), three bioaugmentation-phytoremediation assays (T + B + PCP; L + B + PCP; L + T + B + PCP) and a negative control assay with PCP. Various analytical parameters were determined in this study such as bacterial count, chlorophylls a and b, COD, pH and PCP content. The main results showed that the average PCP removal by P. putida HM627618 was around 87.5% after 7 days of incubation, and 88% of PCP removal was achieved by treatment (T + B) after 9 days. During these experiments, pH, COD and chloride content showed a net increase in all treatments. The chlorophylls a and b in case of (T) and (L) Chlorophylls a and b for T and L phytoremediation showed a decrease with a value less than 10 µg/mg of fresh material after 20 days of cultivation.

From farm to fork: identical clones and Tn6674-like elements in linezolid-resistant Enterococcus faecalis from food-producing animals and retail meat.

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.

Linezolid-resistant (Tn6246::fexB-poxtA) Enterococcus faecium strains colonizing humans and bovines on different continents: similarity without epidemiological link.

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.

Community-level genetic profiles of actinomycetales in long-term biowaste-amended soils.

Actinomycetales is an order of actinobacteria that have an important role in the decomposition of organic matter. Their abundance and distribution can reflect a good level of soil fertility as well as biological activity. In this research study, actinomycetal diversity in soil was investigated under various field treatments with biowastes. Initially, unvegetated agricultural soil plots of 4 m2 had been annually amended with increasing rates of municipal solid waste compost (MSWC at 40, 80 and 120 t ha-1 year-1) and farmyard manure (FM at 40 and 120 t ha-1 year-1) for eight consecutive years. Control consisted of unamended soil and all treatments were distributed in four randomized complete blocks. At the end of the experimental period, total DNA was extracted from fresh topsoil samples (0-20 cm) then nested PCR-DGGE sequencing method was applied to assess the long-term effect of treatments on the diversity of actinomycetes. Analytical outcomes revealed the presence of ten actinomycetal families with Streptomycetaceae, Pseudonocardiaceae and Nocardioidaceae being the most dominant regardless to changes in experimental conditions. Besides, the long-term accumulation of both biowastes in soil affected the diversity of actinomycetal communities in different ways including contribution, stimulation or inhibition. Interestingly, soil treated with MSWC at an equivalent rate of 40 t ha-1 year-1 was likely to provide optimal growth conditions for major identified genera because it showed the highest actinomycetal diversity as compared to the rest of the treatments.

Characterization of Pseudomonas aeruginosa isolated from various environmental niches: New STs and occurrence of antibiotic susceptible "high-risk clones".

The aim of this study was to investigate the antimicrobial phenotypes, major virulence factors, and the molecular typing of 66 P. aeruginosa isolates collected from various sources: human patients and hospital environment, raw milk, poultry meat, chicken/sheep fecal samples, wastewater, thermal water, and seawater. All isolates, except one, were susceptible to all tested antibiotics. exoA, lasB, rhlR, and lasR genes were harbored by 60 isolates. Forty-six, 18, and 2 isolates amplified exoS, exoU, and exoS+exoU genes, respectively. Twenty-one isolates showed high elastase and pigment production. The PFGE typing identified 26 pulsotypes. Some pulsotypes included isolates from different environmental niches and areas. Twelve selected isolates were typed by MLST and eight different STs were found, three of them were new. Our results highlighted the dissemination of some clones amongst different settings and the occurrence of antibiotic susceptible 'high-risk clones' that might be very harmful when acquiring genes encoding antibiotic resistance.

Dispersal of linezolid-resistant enterococci carrying poxtA or optrA in retail meat and food-producing animals from Tunisia.

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.

Detection of Sapoviruses in two biological lines of Tunisian hospital wastewater treatment.

The efficiency of rotating biodisks and natural oxidizing lagoon procedures is investigated at a Tunisian semi-industrial pilot plant, El Menzeh I, where the wastewater is mainly provided by three different neighbouring hospital clinics. Throughout 2011, 102 wastewater samples were collected from the two mentioned wastewater treatment procedures. Results showed that the Sapovirus (SaV) frequency was approximately 29.4% using the real-time reverse transcription polymerase chain reaction (RT-PCR) technique, and about 16.6% using the conventional RT-PCR. Also, the SaV genogroups and genotypes were identified and genotyping revealed that all of the four Tunisian SaV strains obtained belonged to the two genogroups GIV.1 and GGI.3. In addition, two new genotypes, D and C, were detected. A moderate decrease in the SaV frequencies was observed at the exit of the two treatment processes and the SaV removal rate was around 90% in the natural oxidizing lagoons and 94% in the rotating biodisks procedure showing the temperate sensitivity of these viruses to the implemented biological wastewater. Therefore, an urgent disinfection process should be implemented downstream of the two biological treatment procedures for safe release of treated effluent in the different natural environments. Abbreviations: NoV: Noroviruses; SaV: Sapoviruses; EC: Electrical Conductivity; COD: Chemical Oxygen Demand; BOD5: Biological Oxygen Demand; SS: Suspended Solids; NH4-N: Ammonium Nitrogen; P-PO4: Ortho-Phosphate; AlCl3: aluminum chloride.

Detection of optrA in the African continent (Tunisia) within a mosaic Enterococcus faecalis plasmid from urban wastewaters.

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.

Detection of Aichi virus genotype B in two lines of wastewater treatment processes.

Enteric viruses are released in important quantities into the environment where they can persist for a very long time. At very low doses, they can cause human gastroenteritis, and are responsible for a substantial number of waterborne diseases. The aims of this study were multiple: firstly, to study the circulation of Aichi viruses (AiV) in wastewater sampled at the scale of a pilot wastewater treatment plant; secondly, to evaluate the performance of two wastewater treatment procedures, as natural oxidizing lagoons and rotating Biodisks, concerning the AiV removal; and finally, to determine the different type of AiV genotype found during this study. Hence, the pilot wastewater treatment plant is principally irrigated by the wastewater of three neighbouring clinics. Wastewater samples were collected during 2011 from the two lines of biological treatment procedures. AiV detection in wastewater were achieved using the Reverse Transcription Polymerase Chain Reaction (RT-PCR) technique, and the identification of AiV genotype was realized by the direct sequencing of PCR products. The result revealed that AiV strains were identified in 50% (n = 51) of the wastewater samples. A significant increase of the AiV detection frequency was registered from upstream to downstream of the five ponds constituting the natural oxidizing lagoon process, and at the exit of the rotating Biodisks procedure. All detected AiV strains showed the highest nucleotide sequence identity to genotype B that has been recently observed in patients in Asia. This finding represented the first Tunisian survey that revealed and mentioned the first detection of AiV genotype B in sewage and by the same argued for a noticeable resistance or survival of this type of virus in the two lines of treatment considered.

Staphylococcus aureus isolated from wastewater treatment plants in Tunisia: occurrence of human and animal associated lineages.

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.