Effect of Gamma Irradiation on Pathogenic Staphylococcus aureus in Packaged Ready-to-Eat Salads Treated with Biological Extracts.

Providing pathogen-free ready-to-eat (RTE) salads is critical for all consumers, especially individuals with weakened immunity. In this study, the efficacy of γ-irradiation on Staphylococcus aureus (S. aureus) in freshly packaged salads (4.24 log CFU/g) treated with essential oil (EO) and myrtle juice during 10 days of storage and their impact on organoleptic properties were investigated. EO was extracted by hydrodistillation and the chemical composition was analyzed by gas chromatography with Flame Ionization Detector (GC/FID) and gas chromatography/mass spectrometry (GC/MS). Myrtle juice was prepared from fresh fruits. The cytotoxic effects of Thymus capitatus (T. capitatus) EO against a normal human umbilical vein endothelial cell (HUVEC) were assessed. GC/FID and GC-MS analysis of the thyme EO revealed the presence of 13 compounds, including carvacrol (79.55%) and p-cymene (7.93%) as major components. The EO was found to be noncytotoxic, with concentrations lower than 0.16 µL/mL. A reduction of more than 3 log CFU/g and a total inactivation of S. aureus were achieved with the combination of gamma irradiation at 0.5 kGy with myrtle juice at 6 µL/mL and EO at 0.08 µL/mL, respectively. The treatment of fresh RTE salads with thyme and myrtle juice was evaluated as acceptable by the sensory panel. The combined effect showed a synergistic potential on the inactivation of S. aureus.

Inactivation of biohazards in healthcare wastewater by E-Beam and Gamma irradiation: a comparative study.

The main objective of this study is to evaluate the effect of irradiation by Gamma rays and Electron Beam (E-Beam) on naturally occurring microorganisms shed in healthcare wastewater issued from multi-specialties hospital. We examined the susceptibility of naturally occurring total indicator bacteriophages towards Gamma rays and E-Beam irradiation to evaluate their appropriateness as viral indicators for healthcare wastewater quality control. Results showed that healthcare wastewater is a rich matrix containing bacteriophages surrogates of pathogenic waterborne viruses (4.5 Log10 PFU/100 mL for SOMCPH and 2.3 Log10 PFU/100 mL for FRNAPH), antibiotic resistant bacteria (Mean concentrations from 2.3 to 5.5 Log10 CFU/100 mL), molds and yeasts (2.7 Log10 CFU/100 mL), and spores of Clostridium perfringens (Mean concentration of 3.3 Log10 CFU/100 mL). After E-Beam irradiation, naturally occurring bacteria in healthcare wastewater showed lower resistance patterns (D10 values ranging between 0.21 ± 0.005 and 0.59 ± 0.005) compared to those obtained after Gamma irradiation (D10 values ranging between 0.25 ± 0.015 and 0.70 ± 0.0001). Spores of Clostridium perfringens were the most resistant assayed microbes either after E-Beam (D10 values of 3.74 ± 0.005) or Gamma irradiation (D10 values of 4.77 ± 0.025) of collected samples. According to inactivation patterns, a dose of 10 kGy was sufficient for a complete inactivation of spores. Bacteriophages isolated from healthcare wastewater showed the same resistance patterns as those previously obtained in urban treated sewage and were inactivated using higher doses than waterborne bacteria (D10 values of SOMCPH 1.46 ± 0.057; D10 values of FRNAPH 1.03 ± 0.057). Their resistance to irradiation treatment in such complex matrix corroborates their use to survey the viral quality of healthcare wastewater before their discharge in the urban sanitation network. D10 value analysis showed that bacteria and bacteriophages inactivation by E-Beam irradiation required lower doses than those required for their inactivation using Gamma rays. According to inactivation patterns, a dose of 7 kGy was sufficient for total inactivation of both pathogenic bacteria and viruses. Thus, E-Beam irradiation seems to be an efficient physical pre-treatment process for healthcare wastewater treatment prior to its discharge in urban sanitation system to ensure compliance with environmental standards and protect public health.

Occurrence of Staphylococcus spp. and investigation of fecal and animal viral contaminations in livestock, river water, and sewage from Tunisia and Romania.

The objective of this study was to determine the occurrence of Staphylococcus spp., Escherichia coli, somatic coliphages, F-specific RNA bacteriophages, hepatitis E virus (HEV), and bovine enterovirus (BEV) in fecal and water samples. The occurrence of Staphylococcus spp. was investigated in treated wastewater samples collected from slaughterhouse of Tunisia. Results showed that Staphylococcus spp. were detected in the totality of collected samples with an average mean of 5.44 Log10 (CFU/100 ml). Regarding fecal indicator bacteria, E. coli was detected in the totality of water samples and was more abundant in Tunisian samples than in samples collected from Romania (P < 0.05). Concerning somatic coliphages and F-specific RNA bacteriophages used as viral indicators, they were detected in all raw and treated wastewaters. Bovine enterovirus (BEV) was detected in 20.1% and 28% of bovine stool samples collected from Tunisia and Romania, respectively. BEV was also detected in 60% of porcine stool samples from Romania. BEV was absent in all treated sewage samples. HEV was detected in raw sewage and bovine fecal sample from Romania with low occurrence and none sample from Tunisia was positive. This study may give us an insight into the monitoring of water quality in Tunisia and Romania.

Comparison of the Performance of Different Microbial Source Tracking Markers among European and North African Regions.

Microbial source tracking (MST) has been extensively used to detect the sources of fecal pollution in water. The inclusion of MST in water management strategies improves the ecological status of the ecosystem and human and animal health under interdisciplinary analysis in all aspects of health care for humans, animals, and the environment (One Health approach). In this study, the performance of MST markers targeting host-specific Bacteroidales (HF183 and Rum-2-Bac) and species (HMBif and CWBif) were evaluated in raw sewage collected from human, ruminant, swine, and poultry sources in Tunisia, Cyprus, Ireland, and Spain. In addition, the ratio between somatic coliphages and bacteriophages infecting GA17 (SOMCPH/GA17PH) was measured in Tunisia and Spain. The obtained results showed variability of the bacterial markers between the four countries, suggesting that their usefulness could be affected by several conditions (dietary habits, agricultural practices, and climatic conditions) that differ between countries. The Rum-2-Bac marker stood out as a valid MST tool, particularly in Ireland, whereas CWBif was the best option in Tunisia, Spain, and Cyprus. The human-specific HMBif marker showed good sensitivity and specificity in Tunisia, Spain, and Ireland, whereas HF183 showed a low specificity. However, HF183 was suggested as a good human marker in Ireland and Cyprus because of its higher concentration than HMBif. Regarding viral markers, the ratio of SOMCPH/GA17PH showed a clear discrimination between human and nonhuman samples. The combined use of molecular bacterial markers and the ratio of SOMCPH/GA17PH may improve the success of MST.

Total coliphages removal by activated sludge process and their morphological diversity by transmission electron microscopy.

This study was conducted to isolate phages in treated sewage collected from wastewater treatment plant, and explore their morphological diversity by transmission electron microscopy (TEM). Fates of total bacteriophages and their reduction by biological treatment were also assayed. Phages were isolated using the plaque assay then negatively stained and observed by electron microscope. Electron micrographs showed different types of phages with different shapes and sizes. The majority of viruses found in treated sewage ranged from 30 to 100 nm in capsid diameter. Many of them were tailed, belonging to Siphoviridae, Myoviridae and Podoviridae families. Non-tailed phage particles were also found at a low rate, presumably belonging to Leviviridae or Microviridae families. This study shows the diversity and the abundance of bacteriophages in wastewater after biological treatment. Their persistence in wastewater reused in agriculture should raise concerns about their potential role in controlling bacterial populations in the environment. They should be also included in water treatment quality controlling guidelines as fecal and viral indicators.

Antibiotic resistance genes in bacterial and bacteriophage fractions of Tunisian and Spanish wastewaters as markers to compare the antibiotic resistance patterns in each population.

The emergence and increased prevalence of antibiotic resistance genes (ARGs) in the environment may pose a serious global health concern. This study evaluates the abundance of several ARGs in bacterial and bacteriophage DNA via real-time qPCR in samples from five different sampling points in Tunisia; three wastewater treatment plants (WWTP 1, 2 and 3) and wastewater from two abattoirs slaughtering different animals. Results are compared with those obtained in the Barcelona area, in northeast Spain. Eight ARGs were quantified by qPCR from total and phage DNA fraction from the samples. Three ß-lactamases (bla(TEM), bla(CTX-M) cluster 1 and bla(CTX-M) cluster 9), two quinolone resistance genes (qnrA and qnrS), the mecA gene that confers resistance to methicillin in Staphylococcus aureus, the emerging armA gene, conferring resistance to aminoglycosides and sul1, the most extended gene conferring resistance to sulfonamides, were evaluated. Sul1 and bla(TEM) were the most prevalent ARGs detected at all five Tunisian sampling points, similarly with the observations in Barcelona. bla(CTX-M-9) was more prevalent than bla(CTX-M-1) both in bacterial and DNA within phage particles in all samples analysed. mecA and armA were almost absent in Tunisian waters from human or animal origin in contrast with Barcelona that showed a medium prevalence. qnrA was more prevalent than qnrS in bacterial and phage DNA from all sampling points. In conclusion, our study shows that ARGs are found in the bacterial and is reflected in the phage DNA fraction of human and animal wastewaters. The densities of each ARGs vary depending on the ARGs shed by each population and is determined by the characteristics of each area. Thus, the evaluation of ARGs in wastewaters seems to be suitable as marker reflecting the antibiotic resistance patterns of a population.

A comparison of two extraction methods for the detection of Enteroviruses in raw sludge.

The aim of this study was to compare two viral extraction methods for the detection of naturally occurring Enteroviruses in raw sludge. The first method (M1) is based on an ultracentrifugation step. In the second one (M2), viral RNA was extracted directly after viral elution from suspended solids. Genomes of enteroviruses were quantified by a quantitative real time PCR (qRT-PCR) in sludge samples. Somatic coliphages and F-specific RNA phages, considered as viral indicators of enteric viruses in sludge, were enumerated by the double layer agar technique. Results showed that direct assay of RNA extraction yielded higher genomic copies of enteric viruses (with an average of 5.07Log10 genomic copies/100 mL). After the ultracentrifugation assay in the second method, genomic copies number decreases (with an average of 4.39Log10 genomic copies/100 mL). This can be explained by an eventual concentration of inhibitors existing in sludge samples. Phages enumeration results showed their presence in all sludge samples with an average of (5.69Log10 PFU/100 mL) for somatic coliphages and (4Log10 PFU/100 mL) for F-specific RNA phages. This emphasizes the use of somatic coliphages as viral indicators for enteroviruses in environmental samples and especially in raw sludge samples in wastewater treatment plants prior to agricultural use.

Effect of gamma irradiation on bacteriophages used as viral indicators.

This study aimed to examine the susceptibility of indicator bacteriophages towards γ-radiation to evaluate their appropriateness as viral indicators for water quality control. The effects of γ-radiation on naturally occurring somatic coliphages, F-specific coliphages and Escherichia coli were examined in raw sewage and sewage sludge. As well, the effects of radiation on bacteriophages ΦX174 and MS2, and E. coli all grown in the laboratory and seeded in distilled water, autoclaved raw sewage and a 1% peptone solution were evaluated. The inactivation of E. coli was fairly similar in all matrices. In contrast, inactivation of bacteriophages was significantly greater in distilled water than in the other matrices. These results showed the great influence of the matrix characteristics on virus inactivation. Somatic coliphages in raw sewage and sewage sludge and ΦX174 in autoclaved sewage were inactivated similarly and were far more resistant than F-specific coliphages, MS2 and E. coli. As well, F-specific RNA bacteriophages in raw sewage and sewage sludge and MS2 in autoclaved sewage were inactivated similarly and were more resistant than E. coli. In contrast, MS2 was more susceptible to γ-radiation than E. coli in distilled water. Our results showed that ΦX174 is a suitable indicator for estimating virus inactivation by γ-irradiation and corroborate the use of somatic coliphages to survey the viral quality of treated water and sludges.

Presence and fate of coliphages and enteric viruses in three wastewater treatment plants effluents and activated sludge from Tunisia.

PURPOSE: The role of water in the transmission of infectious diseases is well defined; it may act as a reservoir of different types of pathogens. Enteric viruses can survive and persist for a long time in water, maintaining infectivity in many instances. This suggests the need to include virus detection in the evaluation of the microbiological quality of waters. METHODS: In this study, enteric viruses (enteroviruses and hepatitis A virus (HAV)) were investigated by RT-PCR and coliphages (known as indicators of viral contamination) were enumerated with the double-layer technique agar in effluents and sewage sludge from three Tunisian wastewater treatment plants. RESULTS AND DISCUSSION: The molecular detection of enteric viruses revealed 7.7% of positive activated sludge samples for enteroviruses. None of the samples was positive for HAV. Molecular virus detection threshold was estimated to be 10(3) PFU/100 ml. All samples contained high concentrations of coliphages except those of dry sludge. Reductions in the concentrations of bacteriophages attained by the wastewater treatment plants are of the order of magnitude as reductions described elsewhere. Peak concentrations in raw wastewater were associated with winter rains and suspended materials rate in analysed samples. Our data which is the first in North Africa showed that similar trends of coliphages distribution to other studies in other countries. CONCLUSION: No clear correlation between studied enteric viruses and coliphages concentration was proved. Coliphages abundance in collected samples should raise concerns about human enteric viruses transmission as these residues are reused in agricultural fields.

Performance of a submerged membrane bioreactor for the aerobic treatment of abattoir wastewater.

The performance of a submerged membrane bioreactor (SMBR) has been investigated for abattoir wastewater (AW) treatment. The chemical oxygen demand (COD) of permeate has not exceeded 25 mg L(-1) providing an average COD removal of 98%. Microbiological analysis showed that the SMBR has allowed a complete removal of fecal coliforms, Listeria and Salmonella. A significant reduction in the excess biomass production was also observed. In fact, the yield of biomass production (Yobs) ranged between 0 and 0.106 g suspended solids/g COD removed. The study of the dynamic of bacterial communities using the single strand conformation polymorphism (SSCP) method showed a significant change in the population structure and revealed a correlation between the sludge production yield and the bacterial communities.

Full-length genome sequences of hepatitis C virus subtype 4f.

Hepatitis C virus genotype 4 (HCV-4) is very common in central Africa, prevalent in the Middle East, and is becoming increasingly frequent among southern Europeans. We have determined the complete nucleotide sequences of HCV-4f strains and investigated their phylogenetic relationships with other genotypes. We amplified the complete genome of two HCV subtype 4f strains, IFBT84 and IFBT88. The HCV-4f strains have a total of 9181 and 9304 nt, respectively, including the 5' untranslated region followed by a single open reading frame. There was no evidence of genomic recombination in the IFBT84 and IFBT88 strains. The sequences of the HCV-4f strain genomes are closer to those of HCV-1b than to genotypes 2, 3, 5 and 6. It is important to know the full-length sequences of HCV-4 subtypes in order to classify them correctly and to obtain more detailed knowledge about HCV epidemiology and sensitivity to interferon.

Determining the source of nosocomial transmission in hemodialysis units in Tunisia by sequencing NS5B and E2 sequences of HCV.

Hepatitis C virus infection is a significant problem in hemodialysis units. HCV is very variable genetically with six genotypes. Clinical and epidemiological investigation of a new infection requires the determination of both the genotype and the strain of the HCV involved. A prospective, epidemiologic study of 395 dialysis patients in Tunisia was conducted from November 2001 to November 2003 to identify the source of nosocomial transmission using phylogenetic analysis of NS5b and E2 sequences. Hepatitis C infection was diagnosed by screening for anti-HCV antibodies and HCV RNA in sera using third generation ELISA and a qualitative RT-PCR assay. HCV strains were genotyped by sequencing the NS5b region. The genetic relatedness of the HCV strains was studied by sequencing the NS5b and the HVR-1 regions of the HCV genome. Two de novo cases of HCV infection were detected during the follow-up. One of them has been described previously. The case described in this study occurred in a center in which 12 patients were already infected with HCV strains belonging to genotypes 1b (n = 8) and 1a (n = 4). Phylogenetic analysis of the NS5b region from the HCV strains circulating in this center disclosed four clusters, confirmed by analysis of the HVR-1 region, providing strong evidence for nosocomial infection. Epidemiological data showed that these patients were dialyzed during the same shift and in the same area. Phylogenetic analysis of NS5b sequences is useful for determining the HCV genotype and providing evidence of nosocomial transmission.

Hepatitis C virus infection among dialysis patients in Tunisia: incidence and molecular evidence for nosocomial transmission.

In order to study the incidence of hepatitis C virus (HCV) infection in Tunisian haemodialysis patients and detect its nosocomial transmission, 395 patients were enrolled in a prospective study (November 2001-2003). HCV serological and virological status was determined initially using, respectively a third generation ELISA and an RT-PCR qualitative assay. The genotype of the HCV isolates was determined by sequencing NS5B region. The issue of nosocomial transmission was addressed by sequencing the HVR-1 region of the E2 gene. About 20% of the patients had anti-HCV antibodies and HCV-RNA was detected in 73% of the anti-HCV positive patients. Two cases of de novo HCV infection were identified in two dialysis centers, during virological follow-up of patients susceptible to HCV infection. The incidence of de novo HCV infection was 0.5%. Determining the genotypes in the first center disclosed that all HCV-positive patients were infected with genotype 1b; sequencing of the HVR-1 region of the E2 gene provided strong evidence that the isolate from the newly infected patient and another infected dialysis patient were closely related, confirming nosocomial contamination. The investigation of the second center is pending. Besides, one patient with negative HCV serology had detectable HCV-RNA at the beginning of the study. This case had HCV genotype 1b, two other infected dialysis patients in the same unit had HCV genotypes 4k and 3a; thus precluding nosocomial transmission. Thanks to molecular and phylogenetic methods, one case of nosocomial HCV transmission in haemodialysis was confirmed. Epidemiological investigation suggested nosocomial transmission via the medical and/or nursing staff.