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Abstract: This study investigated four methods for the recovery of enteroviruses from sterilized raw wastewater, activated sludge, thickened sludge and treated wastewater, inoculated with Echovirus 11, Gregory prototype. The adsorption–elution method recommended by the US Environmental Protection Agency (EPA) was better for Echovirus 11 recovery than a sonication method, a modified EPA method and a membrane adsorption elution method since it resulted in the highest detection levels by cell culture and RT-PCR (Friedman’s test, p <0.00041 and p =0.041, respectively). [Copyright &y& Elsevier]

The radiation field for an annular-type photo-catalytic reactor, used for wastewater disinfection by UV radiation, has been modelled. The spatial domain was described in terms of two cylindrical coordinates and the field of direction with two angular variables. The UV radiation entering through the inner reactor wall was obtained from the extended source with volumetric emission (a UV lamp). The system parameters for the radiative-transfer equation were taken from experimental measurements. Resorting to computational experiments, the effects produced by different concentrations of radiation absorbing particles (bacteria) were investigated. We have succeeded in determining the incident radiation profiles and local volumetric rate of energy absorption profiles, both as a function of the spatial position inside the reactor. The experimental values were compared with the theoretical predictions, good agreement was obtained. The errors were never higher than 10%. [Copyright &y& Elsevier]

Abstract The agricultural reuse of urban sewage sludge in degraded croplands has seen a wide acceptance for biowaste recycling and for the improvement of soil fertility. However, soil degradation and health issues may arise from the fact that sludge addition causes chemical and biological contamination if mismanaged. To closely monitor the long term soil-sludge interactions, a field study was conducted over a three-year period under a semi-arid climate. More precisely, changes in topsoil properties were assessed in 4-m2 uncropped plots amended with sludge rates equivalent to 0, 40, 80 and 120 t ha−1 year−1 for two soil textures: soil A (sandy loam) and soil S (sandy). Results showed that all studied parameters changed significantly in a dose-dependent manner for both soils as compared to untreated controls. As such, sludge addition improved consistently TOC, N, P and K content up to soils treated with 120 t ha−1 year−1. The enhancement of soil nutrient status impacted positively on biological properties, including microbial biomass and soil enzyme activities (dehydrogenase, protease and phosphatase). However, the variation of soil properties was more important in soil A characterized by a higher fine fraction thus stronger retention capacity. Accordingly, XRD analysis of soil A revealed several identifiable peaks representing kaolinite and illite clays in contrast to soil S. Unexpectedly, high to excessive sludge doses of 80 and 120 t ha−1 year−1 did not provoke soil degradation after three repeated annual amendments. In fact, pH values in both soils remained within neutral to alkaline range (7.76–8.63); total heavy metals (Cu, Zn, Ni and Pb), soil salinity and fecal coliforms were all below threshold values for contaminated soils. Highlights • Tunisian croplands have low organic matter content due to overexploitation. • Three successive annual sewage sludge amendments improved soil fertility. • Soil biological activities were positively correlated with the sludge dose. • Soil degradation was not observed even at excessive sludge doses. • A finer texture had more impact on soil parameters after sludge addition. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus is a versatile bacterium, which can infect or colonize a variety of host species. The objective of this study was to characterize S. aureus isolates recovered from nasal swabs of 167 healthy ewes sampled from 12 farms in different areas of Tunisia during the period of 2014–2015. Genetic lineages, virulence factors and antibiotic resistance mechanisms were determined for recovered isolates. S. aureus was detected in 45 out of 167 tested samples (26.9%). All isolates were methicillin-susceptible (MSSA) and the majority of them were susceptible to tested antibiotics with few exceptions (% of resistance): penicillin (8.8), ciprofloxacin (4.4), and tobramycin or tetracycline (2.2, each). Twelve different spa types were detected (t15098, t15099, t1773, t3576, t1534, t5428, t3750, t5970 t254, t2883, t127 and t933), two of them were new (t15098 and t15099). S. aureus isolates were ascribed to agr I (n = 23), agr II (n = 1) and agr III (n = 20), and one was non-typeable. According to the sequence-type (ST) determined and/or the spa- type detected, the 45 S. aureus isolates were assigned to six clonal complexes, with CC522 (44.4%) and CC130 (37.7%) being the most common lineages. Twenty-one (46.6%) and two (4.2%) isolates harbored the tst and eta genes encoding TSST-1 and ETA, respectively. In conclusion, nares of healthy ewes could be a reservoir of MSSA CC522 and CC130, lineages associated with TSST-1 and ETA that might represent a risk to human health. [ABSTRACT FROM AUTHOR]

Pentachlorophenol (PCP) is quite persistent in the environment and severely affects different ecosystems including forest soil. The main objective of this work was to study different bioremediation processes of artificially PCP (100 mg kg−1) contaminated forest soil (Sc). In fact, we used bioaugmentation by adding two different bacterial consortia B1 and B2, biostimulation procedures by amendments based on forest compost (FC), municipal solid waste compost (MC), sewage sludge (SS), and phosphate, and their combined treatments. Soil physical and chemical properties, residual PCP, soil microbial biomass carbon, soil respiration and some enzymatic activities at zero time and after 30 d of incubation, were evaluated. A net reduction of PCP, 71% of the initial concentration, after 30 d-incubation occurred in the sample Sc+B1+FC, as the best performance among all treatments, due to natural attenuation, immobilization of PCP molecules in the forest soil through organic amendments, and the action of the exogenous microbial consortium B1. The single application of FC or B1 led to a depletion of PCP concentration of 52% and 41%, respectively. Soil microbial biomass carbon decreased in PCP contaminated soil but it increased when organic amendment also in combination with microbial consortia was carried out as bioremediation action. Soil respiration underwent no changes in contaminated soil and increased under FC based bioremediation treatment. These results demonstrate that the combined treatments of biostimulation and bioaugmentation might be a promising process for remediation of PCP contaminated soil. [Display omitted] • PCP reduction by 32% in soil after 30 days occurred due to natural attenuation. • PCP contamination depressed soil microbial biomass and enzymatic activities. • Microbial consortia and organic amendments further reduced the PCP content. • Combined biostimulation with FC and bioaugmentation with B1 achieved the best result. [ABSTRACT FROM AUTHOR]

Abstract: Salmonella serovars are increasing in importance as significant pathogens of both human and animals. Although water and wastewater are treated to eliminate pathogenic microorganisms, they still play an important role in the transmission of Salmonella spp. In this study, bacteriophages infecting Salmonella spp. were isolated from wastewater and evaluated; for their potential to lyse environmental Salmonella strains in vitro at different MOIs and temperatures; and to control the wastewater bacterial community. Three distinct phages designated sww65, sww275, and sww297; as defined by plaque morphology, electron microscopy and host range; were obtained from wastewater. Challenge tests were performed at 37, and 30°C with the infection of the Salmonella cultures with individual phage, a mixture of two phages, and cocktail of three phages at MOIs of 100, 102, and 104 PFU/CFU. At 30, and 37°C, a cocktail of three phages reduced all of the Salmonella cultures tested. These results required a high multiplicity of infection. However, when infected with only one phage or a mixture of two phages at MOIs of 100 or 10 2 PFU/CFU, an emergence of bacterial resistance was observed. The dynamic monitoring of wastewater enterobacterial community was conducted using Enterobacterial Repetitive Intergenic Consensus-PCR (ERIC-PCR). The number of bands decreased gradually with the use of individual phage or phage cocktails. Moreover, the dynamic monitoring of Salmonella community during wastewater treatment was performed using PCR detection of virulence gene invA. The results correlated with the ERIC-PCR fingerprints, and suggested that Salmonella community was affected by the phage treatment. Indeed, in wastewater, bacteriophages are reducing Salmonella and other members of the Enterobacteriaceae. These results indicated that dynamic changes are closely related with the process of treatment. The introduction of wide host range bacteriophages in wastewater can have a potential impact on the dynamics of the microbial communities, manifested by the reduction or the elimination of microbial species. [Copyright &y& Elsevier]

Abstract: Food-borne diseases caused by Salmonella enterica are a significant public health concern around the world. Since 2002, S. enterica serovar Kentucky has shown an increase in several countries with the concurrent emergence of multidrug-resistant isolates. The spread of such strains in the environment poses a major public health problem. A total of 57 Salmonella Kentucky strains isolated from different sources during the period 2005 to 2008 in Tunisia, were characterized by their antimicrobial and mercury resistance profiles; ability to form a biofilm; virulence invA/spvC genes and quorum sensing sdiA gene. A total of 10.6% of the isolates demonstrated multidrug-resistance against 3 to 13 antibiotics with ciprofloxacin resistance occurring in 33% of human isolates. In addition, 37% of the isolates exhibited minimum inhibitory concentrations value to mercuric chloride, ranging from 8 to 32μgml−1 and were considered as resistant strains. The majority of strains tested were able to form a biofilm, especially for environmental and animal derived isolates. Therefore, the biofilm seems to comprise a normal and favorable capability in the life of Salmonella Kentucky in the environment. Interestingly, all the isolates possessed the sdiA gene, 87.7% of isolates possessed the invA gene, and no isolate harbored the spvC gene. The emergence of resistance to ciprofloxacin in human Salmonella Kentucky isolates, added to the presence of invA and sdiA genes, and the production of biofilm could be the decisive factors in the dissemination of S. Kentucky strains on a large scale. [Copyright &y& Elsevier]

Abstract: Since 2002, the emergence of multidrug resistant Salmonella enterica serovar Kentucky (S. Kentucky) and the associated salmonellosis with treatment failure were declared in different parts of the world and were in most of the case contracted during travels to Northeast and Eastern Africa. In the present work, we reported an epidemiological study of S. Kentucky isolated from different environmental and clinical origins in Tunisia, using Pulsed-field gel electrophoresis (PFGE); Enterobacterial Repetitive Intergenic Consensus (ERIC-2) fingerprinting; Plasmid profiling; and antibiotic resistance profiles. ERIC-2 fingerprinting allowed the differentiation of 14 different patterns versus only 4 pulsotypes. Besides, a high proportion of strains were found to be nontypeable by XbaI-PFGE and/or by plasmid profiling (plasmid-free strains). The antibiotic resistance was mainly detected against streptomycin (80.7%), sulfonamides (42.1%) and tetracycline (15.7%). Furthermore, two avian strains were shown to be resistant to trimethoprim–sulfamethoxazole and three clinical strains have demonstrated multidrug-resistant phenotypes (against 5 to 10 antibiotics) and all of them exhibited resistance against nalidixic acid, ciprofloxacin, ofloxacin and tetracycline. ERIC-2 PCR was found to be the most discriminative. However, combination of the three typing methods offer a better mean for differentiating S. Kentucky isolates, monitoring the multiresistant types and determination of their origin. [Copyright &y& Elsevier]

Abstract: In this study, the performance of a combined subsurface vertical and horizontal flow constructed wetlands system, designed for rural domestic wastewaters treatment and with theoretical hydraulic retention time of 2 d and 3.6 d, respectively, was investigated. Several water quality parameters including pH, BOD5, COD, TSS, TKN and TP, and faecal bacteria’s number in both raw and treated wastewaters were monitored during a macrophytes life cycle. At the same time, the growth of the rooted plants, reeds and cattails was investigated through the measurement of the height and leaves number. The average influent characteristics were as follows: pH (7.98 ± 0.27), BOD5 (420 ± 144 mg/L), COD (1339 ± 352 mg/L), TSS (798 ± 302 mg/L), TKN (205 ± 70 mg/L), TP (30.7 ± 12.4 mg/L), faecal coliforms ([2.69 ± 4.14] × 107 CFU/100 mL) and faecal streptococci ([9.35 ± 1.81] × 106 CFU/100 mL). The main treatment performance results showed the following average removal rates: BOD5 (93 ± 2%), COD (89 ± 3%), TSS (98 ± 1.5%), TKN (38 ± 19%), TP (72 ± 16%). The average bacterial reduction between the inlet and the outlet was of the order of 4 ± 1 logarithmic units for faecal coliforms and 3 ± 1 logarithmic units for faecal streptococci. Analysis of the results reveals a temporal variation in the system’s performance depending on the primary treatment operation and on the macrophytes growth rate. Reeds and cattails start their life cycle at the beginning of the spring and continue their development during summer. During the autumn season, the plants reach their optimum growth rate, and after that, they enter the dormant phase. The quality of the treated wastewaters was evaluated according to Tunisian standards. The average effluent pH, BOD5, TSS and faecal bacteria were in agreement with the standards, but COD, nitrogen and phosphorus residual loads were still above the values required by the quality criteria. [Copyright &y& Elsevier]

Abstract: The current work tackles the problem of soil microbial growth and biofilm expansion assessment in infiltration percolation process in order to optimize the technique design and monitoring. Experiments were conducted on a sand filled PVC column fed at a pulsed rhythm of 8 sequences per day in order to maximize the soil aeration that generates a better oxygen renewal in the soil gas phase, enhances the oxidation of the oxidisable matters and improves sanitary performances. As we target the biofilm expansion study, we assessed the microbial biomass at different depths of the soil matrix (10, 25, 50 and 75 cm), by the chloroform fumigation–extraction and DNA extraction methods. Results showed that the microbial biomass increases with operating time especially between 10 and 25 cm depths until the 96th operating day (OPD) and decreases notably after this time. A highly significant positively correlation (r = 0.97) is registered between results of microbial biomass quantification by fumigation–extraction method (FEM) expressed as carbon material (biomass C) and those obtained by DNA extraction method (DNA-EM). In addition, we performed an enumeration of the biofilm total microbial flora using the solid medium cultivation method which corroborates the microbial biomass quantification results. In order to illustrate the biofilm shape generated by bacterial cells and extracellular polymeric substances (EPS)in the vicinity of the sand grains, we performed different scanning electron microscope (SEM) micrographs at different elapsed operation times. [Copyright &y& Elsevier]

Abstract: For small settlements sanitation, constructed wetland systems offer a suitable social economic alternative to the classic wastewater treatment plants. In order to study the treatment mechanisms occurring in the soil–plant system, we set up a small-scale pilot wastewater treatment plant composed by a settler/digester followed by two trains combining two different types of subsurface flow constructed wetlands called ‘hybrid systems’. The first train represents a combination of a vertical flow reed (Phragmites australis) bed followed by a horizontal bed, planted with Typha, whereas in the second one, the combination is reversed. The hybrid systems are fed intermittently with a hydraulic load of 0.2 m per day given in two sequences of one hour separated by a six-hour rest period. We followed the quality of the effluent at the outlet of the beds. The two hybrid systems present satisfactory performances: yields higher than 90% in organic matter removal are registered. For the first system we could obtain a substantial nitrification but uncompleted denitrification whereas for the second system we observed that the total nitrogen (NTK) reduction was achieved by denitrification and nitrification remains almost complete. The pathogenic bacteria abatement is satisfactory for the two systems but more remarkable for the second (2 u log). [Copyright &y& Elsevier]

Abstract: The use of municipal solid waste compost (MSWC) as soil organic amendment is of an economic and environmental interest. However, little is known about the effectiveness of MSWC application on agricultural soil in northern Africa arid climate. We assessed the impact of five years'' applications of different organic and mineral fertilizers on wheat grain yields and soil chemical and microbial characteristics. Soils were treated with MSWC at rates of 40 (C1) and 80 (C2) Mgha−1, farmyard manure at a rate of 40Mgha−1 (M), chemical fertilizers (Cf) and the combinations (C1Cf, C2Cf, MCf). Wheat grain yield was enhanced with all amendments. Parallel increases of heavy metal levels and faecal coliform were also recorded except for Cf treatments. Based on wheat grain yield, heavy metal and faecal coliform data, we determined the treatment effectiveness index (Exx ), calculated by dividing the pollutant increase ratio by the grain yield increase ratio. The treatment effectiveness index E C1 indicated lower faecal and heavy metal pollution with positive gains in wheat yields. Despite polluting effects on soil determined by the different treatments, no significant differences between treatments were observed in total bacterial count and soil bacterial community structure, as shown by 16S rRNA gene PCR-denaturing gradient gel electrophoresis banding patterns and 16S rRNA gene Length Heterogeneity-PCR analysis. According to the collected data, the use of MSWC at a rate of 40Mgha−1 might be recommended. [Copyright &y& Elsevier]

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 (tet A/ tet B), sulfonamide (sul 1/ sul 3), and chloramphenicol (cml A), 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 mcr 1-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 tet A, sul1 / sul3 and cml A 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. • 94 CTX-resistant E. coli isolates recovered from poultry faeces (22.4%) and meat samples (63.8%). • The majority of isolates harboured bla CTX-M-1 and bla CTX-M-55 genes. • mcr -1 gene was detected in 52 out of 54 colistin-resistant isolates. • MLST and PFGE revealed 13 sequence types (STs) and 28 profiles. [ABSTRACT FROM AUTHOR]

• 120 bovine faecal samples and 103 raw bovine/caprine milk samples analysed. • Eight samples (3.6%) contained ESBL-producing E. coli (ESBL-EC) (5: raw milk; 3: cattle faeces). • Four colistin-resistant ESBL-EC isolates of bovine origin (mcr -1-positive). • ESBL-EC strains: mcr -1/CTX-M-1/D-ST1642, mcr -1/CTX-M-1/A-ST10, CTX-M-15/B1-ST394, and CTX-M-15/A-ST46. • Most of bovine ESBL-EC isolates were multidrug-resistant. 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. [ABSTRACT FROM AUTHOR]