A first insight into genetic diversity of Mycobacterium bovis isolated from extrapulmonary tuberculosis patients in South Tunisia assessed by spoligotyping and MIRU VNTR.

INTRODUCTION: In Tunisia, almost 77% of clinically and bacteriologically diagnosed cases of extrapulmonary tuberculosis (EPTB) are zoonotic TB, caused by M. bovis. Although several studies have analyzed bovine TB in cattle in Tunisia, no study has evaluated the risk of transmission to humans in such an endemic country. We aimed to study the genetic diversity of M. bovis human isolates, to ascertain the causes of human EPTB infection by M. bovis and to investigate the distribution and population structure of this species in Tunisia. MATERIALS AND METHODS: A total of 110 M. bovis isolates taken from patients with confirmed EPTB were characterized by spoligotyping and MIRU-VNTR typing methods. RESULTS: Among the 15 spoligotypes detected in our study, 6 (SB0120, SB0121, SB2025, SB1200, SB1003 and SB0134) were the most prevalent (83.5%) of which SB0120, SB0121 and SB2025 were the most prevailing. MIRU-VNTR typing method showed a high genotypic and genetic diversity. The genetic differentiation based on MIRU-VNTR was significant between populations from South East (Tataouine, Medenine) and Central West (Gafsa, Sidi Bouzid, Kasserine) regions. Of note, 13/15 (86.7%) spoligotypes detected in our study were previously identified in cattle in Tunisia with different frequencies suggesting a peculiar ability of some genotypes to infect humans. Using combined spoligotyping and MIRU-VNTR method, a high clustering rate of 43.9% was obtained. Our results underlined that human EPTB due to M. bovis was more commonly found in female gender and in young patients. Most of our patients, 66.4% (73/110) were raw milk or derivatives consumers, whereas 30.9% (34/110) patients would have contracted EPTB through contact with livestock. The findings suggest that the transmission of Zoonotic TB caused by M. bovis to humans mainly occurred by oral route through raw milk or derivatives. CONCLUSION: Our study showed the urgent need of a better veterinary control with the implementation of effective and comprehensive strategies in order to reach a good protection of animals as well as human health.

Screening and Detecting Salmonella in Different Food Matrices in Southern Tunisia Using a Combined Enrichment/Real-Time PCR Method: Correlation with Conventional Culture Method.

A combined enrichment/ newly developed invA TaqMan® real-time PCR (qPCR) method as a screening assay to detect Salmonella spp. in 500 naturally food matrices is evaluated. DNA template for qPCR was extracted from an overnight pre-enriched sample in buffered peptone water using lysis-guanidine isothiocyanate method. Heterologous internal amplification control (IAC) was incorporated during qPCR assays and co-amplified with the invA gene of the target pathogen. InvA qPCR exhibited 100% specificity when testing 94 Salmonella strains (inclusivity) and 32 non-Salmonella strains (exclusivity). The qPCR showed a consistent detection of two copies of the invA gene/PCR reaction, a good intra- and inter-run reproducibility with a good PCR efficiency (89.6%). QPCR was sensitive and showed Salmonella detection at 8.5 × 100 CFU mL-1 of artificially spiked poultry meat -BWP solution in less than 40 cycles. When analyzing 500 different food matrices and comparing the results with the ISO 6579:2002 conventional culture method, the sensitivity and specificity were 100 and 76.6%, respectively. QPCR showed Salmonella spp. DNA in raw poultry meat 27/45 (60%), milk 31/93 (33.3%), raw red meat 5/13 (38.5%), and fish 11/46 (23.9%) samples. The prevalence of Salmonella spp. in cakes, dairy, cooked meals, charcuterie products using qPCR was 11/14 (26.8%), 5/22 (22.7%), 32/150 (21.3%), and 5/20 (25%), respectively, compared to 0% as demonstrated by culture. S. Anatum was the most common serovar found associated with red meat compared to S. kentucky isolated from fish and poultry meat. In conclusion, our study is the first to use a combined enrichment/invA qPCR method as a screening assay to detect Salmonella DNA in different types of commercialized food in Southern Tunisia. QPCR results indicate that Salmonella contamination is common in milk and in other types of food samples.

Isolation and Identification of Campylobacter spp. from Poultry and Poultry By-Products in Tunisia by Conventional Culture Method and Multiplex Real-Time PCR.

Thermophilic Campylobacter spp. are one of the primary causes of bacterial human diarrhea. The consumption of poultry meats, by-products, or both is suspected to be a major cause of human campylobacteriosis. The aims of this study were to determine the prevalence of thermophilic Campylobacter spp. in fresh poultry meat and poultry by-products by conventional culture methods and to confirm Campylobacter jejuni and Campylobacter coli isolates by using the multiplex PCR assay. Two hundred fifty fresh poultry samples were collected from a variety of supermarkets and slaughterhouses located in Sfax, Tunisia, including chicken (n =149) and turkey (n =101). The samples were analyzed using conventional microbiological examinations according to the 2006 International Organization for Standardization method (ISO 10272-1) for Campylobacter spp. Concurrently, a real-time PCR was used for identification of C. jejuni and C. coli . Of the 250 samples of poultry meat and poultry by-products, 25.6% (n = 64) were contaminated with Campylobacter spp. The highest prevalence of Campylobacter spp. was found in chicken meat (26.8%) followed by turkey meat (23.7%). Among the different products, poultry breasts showed the highest contamination (36.6%) followed by poultry by-products (30%), poultry wings (28%) and poultry legs (26%) showed the lowest contamination, and no contamination was found on neck skin. Of the 64 thermophilic Campylobacter isolates, C. jejuni (59.7%) was the most frequently isolated species and 10.9% of the isolates were identified as C. coli . All of the 64 Campylobacter isolates identified by the conventional culture methods were further confirmed by PCR. The seasonal peak of Campylobacter spp. contamination was in the warm seasons (spring and summer). The study concluded that high proportions of poultry meat and poultry by-products marketed in Tunisia are contaminated by Campylobacter spp. Furthermore, to ensure food safety, poultry meats must be properly cooked before consuming.

First-time detection and identification of the Mycobacterium tuberculosis Complex members in extrapulmonary tuberculosis clinical samples in south Tunisia by a single tube tetraplex real-time PCR assay.

INTRODUCTION: Tunisia has one of the highest burdens of extrapulmonary tuberculosis (EPTB) among tuberculosis (TB) cases but the contribution of MTBC-mediated human EPTB is unknown. EPTB diagnosis is challenging due to the paucibacillary nature of clinical samples. Therefore, a need of a simplified molecular method for sensitive and specific TB detection and differentiation of MTBC members caused EPTB remains a priority to an early diagnosis, optimize successful anti-TB treatment and minimize transmission. We evaluated the performance of a single tube tetraplex Taq Man real time PCR for EPTB detection and differentiation between MTBC members directly on extrapulmonary samples. MATERIALS AND METHODS: Extrapulmonary samples obtained from clinically suspected EPTB patients from 2013 to April 2015 were tested by Ziehl Neelsen Staining, mycobacterial culture and qPCR assay for RD1, RD9, RD12 and ext-RD9 targets (MTBC-RD qPCR). The performance of qPCR was compared to a reference standard based on MTBC culture and/or at least two criteria of a composite reference standard (CRS) including clinical, radiological, histopathological and therapeutic findings. RESULTS: EPTB was identified in 157/170 (92.4%) of included patients of whom 99 (63%) were confirmed by culture and 58 (36.9%) by CRS criteria. The sensitivity and specificity of qPCR, in comparison to the reference standard were 100% (157/157) and 92.3% (12/13), respectively. The sensitivity of qPCR was statistically significant as compared to culture and smear microscopy (P< 0.001). QPCR results showed M. bovis identification in 77.1% of extrapulmonary samples in occurrence to lymphadenitis infection. M. tuberculosis and M.bovis BCG were detected in 21.6% and 1.3% of cases, respectively. CONCLUSIONS: MTBC-RD qPCR proved to be a rapid and sensitive assay for simultaneously TB detection and MTBC members identification on extrapulmonary samples within 1.5 days after sample receipt. Its high sensitivity could make this method a useful tool in diagnosing TB in addition to routine conventional methods and TB clinical parameters.

Molecular characterization of Mycobacterium tuberculosis strains resistant to isoniazid.

OBJECTIVE/BACKGROUND: Tuberculosis is a major public health problem and the emergence of drug resistance complicates the situation even more. It is therefore crucial to implement all conclusions from the studies that aim at a better understanding of the molecular mechanisms which govern the emergence and the evolution of drug resistance. The aim of this study is to assess the degree of involvement of the inhA and katG genes in the acquisition of isoniazid resistance in clinical strains of Mycobacterium tuberculosis. METHODS: The inhA and katG genes were sequenced in 21 strains of M. tuberculosis with different resistance profiles and from different regions. RESULTS: Analysis of the sequences obtained by comparison to those of the reference strain H37Rv showed that 95.2% had mutations. KatG S315T was the most common mutation (85.7%). The mutation katG T275A was revealed in two strains (9.5%). Two different point mutations in the inhA gene and its promoter region were identified as C-15T and G56A at a frequency equal to 14% and 10%, respectively. The G56A mutation is a new silent mutation. Our study showed no correlation between found mutations and multidrug resistance. Among the 21 strains studied, only one strain showed no mutations. CONCLUSION: In terms of this study, we characterized the mutations involved in resistance to isoniazid. katG S315T was by far the most frequent mutation, followed by C-15T. The frequency of these mutations was concordant with those reported in literature including those in intermediate tuberculosis endemic countries.

Co-composting of spent coffee ground with olive mill wastewater sludge and poultry manure and effect of Trametes versicolor inoculation on the compost maturity.

The co-composting of spent coffee grounds, olive mill wastewater sludge and poultry manure was investigated on a semi-industrial scale. In order to reduce the toxicity of the phenolic fraction and to improve the degree of composting humification, composts were inoculated with the white-rot fungus Trametes versicolor in the early stages of the maturation phase. During composting, a range of physico-chemical parameters (temperature and both organic matter and C/N reduction), total organic carbon, total nitrogen, elemental composition, lignin degradation and spectroscopic characteristics of the humic acids (HAs) were determined; impacts of the composting process on germination index of Hordeum vulgare and Lactuca sativa were assessed. The coffee waste proved to be a highly compostable feedstock, resulting in mature final compost with a germination index of 120% in less than 5 months composting. In addition, inoculation with T. versicolor led to a greater degree of aromatization of HA than in the control pile. Moreover, in the inoculated mixture, lignin degradation was three times greater and HA increased by 30% (P<0.05), compared to the control pile. In the T. versicolor inoculated mixture, the averages of C and N were significantly enhanced in the HA molecules (P<0.05), by 26% and 22%, respectively. This improvement in the degree of humification was confirmed by the ratio of optical densities of HA solutions at 465 and 665 nm which was lower for HA from the treated mixture (4.5) than that from the control pile (5.4).

[Duodenal duplication revealed by acute pancreatitis. A case report]. / Duplication duodenale revelee par une Pancreatite aigue. A propos d'un cas.

BACKGROUND: Duodenum duplications are uncommon congenital anomalies. Most symptomatic cases are diagnosed in childhood and usually present with obstructive or bleeding symptoms. Acute pancreatitis is rarely attributed to these cysts. AIM: To report a new case of duodenum duplication revealed by acute pancreatitis. CASE REPORT: This 3 year old child presented with an acute pancreatitis. Abdominal ultrasonography and Computer tomography were performed showing a cystic mass depending of the 2nd duodenum. Diagnosis of duodenal duplication is made in laparotomy. A surgical resection of the duplication was performed respecting the papilla. Microscopic examination of the specimen confirmed the duodenal duplication. The patient was asymptomatic after the intervention. CONCLUSION: Duodenum duplications are uncommon congenital anomalies. Acute pancreatitis might be revealing presentation.

Evolution of the fatty fraction during co-composting of olive oil industry wastes with animal manure: maturity assessment of the end product.

Olive mill wastewater sludge, resulting from the natural evaporation of olive oil processing effluent, was co-composted with poultry manure and changes in the lipid fraction investigated. Composting was achieved after approximately 9 months, leading to a compost with high stability and maturity (C/N ratio: 11.9; cation exchange capacity (CEC): 85.9 meq 100 g(-1) organic matter, CEC/total organic carbon: 4.2 meq g(-1); humic acids carbon/fulvic acids carbon: 2.2) useable directly in agriculture and having the same fertilizing capacity as farmyard manure. Composting led to a reduction in the lipid fraction by at least 95%. Unsaturated fatty acids, particularly polyunsaturated acids, were the most degraded (reduction of 55%) leading to an increase in saturated fatty acids. This change was confirmed by the relative increase in the peroxide index from 5 to 32.5 meq O(2)kg(-1) fats, and a decrease in the C(18:2)/C(16:0) ratio from 0.9 to 0.3. In addition, this study demonstrated that 1.2% of the humic acids component of the compost comprised fatty acids.

Biological activity during co-composting of sludge issued from the OMW evaporation ponds with poultry manure-Physico-chemical characterization of the processed organic matter.

Olive mill sludge (OMS), a by-product resulting from natural evaporation of olive oil processing effluent, poses a major environmental threat. A current cost-effective practice of OMS management is composting. A mixture of OMS (60%) with poultry manure (PM) was successfully composted for 210 days. During the process, effluents of olive oil mill and confectionary were used to keep moisture at optimal level (40-60%). Biological indicators reflecting stability of the compost (microbial biota respiration and enumeration, and germination index) were analysed for the assessment of the product quality. The composted mixture showed a high microbial activity with a succession of microbial populations depending on the temperature reached during the biodegradation. The pathogen content from PM decreased with composting as did phytotoxic compounds. Phenols and lipids were reduced, respectively, by 40% and 84% while germination index increased with composting progress. Fourier transform infrared (FTIR) spectroscopic analysis revealed that the final compost improved the aromatic content compared to the starting materials, with a decrease in aliphatic groups and a reduction in the easily assimilated components by the microflora acting during the biological process. The final compost was characterized by relatively high organic matter content (26.21%), a low C/N ratio (16.21), an alkaline pH (8.32), a relatively high electrical conductivity (9.21mS/cm) and a high level of nutrients. The germination index for Lepidium sativum L. was 87.71% after 210 days of composting, showing that the final compost was not phytotoxic.

Elimination of polyphenols toxicity from olive mill wastewater sludge by its co-composting with sesame bark.

Olive mill wastes represent a significant environmental problem in Mediterranean areas where they are generated in huge quantities in a short period of time. Their high phenol, lipid and organic acid concentrations turn them into phytotoxic materials, but these wastes also contain valuable resources such as a large proportion of organic matter and a wide range of nutrients that could be recycled. Composting is one of the technologies used for the valorization of this effluent, producing a fertilizer useful for poor soils.The present work deals with the changes that occur in the content of phenolic compounds and the biotoxicity of the oxidized substrate which result from the composting of olive mill wastewater (OMW) sludge with sesame bark. The total organic matter decreased 52.72% while water-soluble phenol degradation decreased 72% after 7 months of processing. Gas chromatography coupled with mass spectroscopy was used to confirm the elimination of polyphenols during composting. Initially, the analysis showed three abundant polyphenolic compounds, one of which was identified as the 4-hydroxyphenyl-ethanol (tyrosol), a well-known antioxidant in OMW. After 7 months of composting, all of the phenolic compounds disappeared. The phytotoxic effects of OMW sludge, assessed by the plant index germination, increased during the composting to reach 80% after 210 days. This trend was confirmed by the correlation between physico-chemical and toxicity parameters. The results obtained confirmed the stability of the compost prepared from OMW sludge with sesame bark and indicated a gradual detoxification as the compost matured.

Maturity assessment of composted olive mill wastes using UV spectra and humification parameters.

In this study, two olive mill wastes - exhausted olive cake (EOC) and paste of olive mill wastewater naturally dehydrated (POMW) - were co-composted and mixed with 25% sesame bark (SB). The humification process was evidenced by quantifying the humic substances and the generally accepted humification indices: (i) the ratio of humic acid (HA) carbon to fulvic acid (FA) carbon (CHA/CFA), (ii) the ratio of water soluble organic carbon (CW) to total organic nitrogen (Cw/Norg), (iii) and the ratio of humic acid carbon to total organic carbon CHA/Corg and by determining the absorbance ratios: E2/E4, E2/E6 and E4/E6. The results showed that the time required to reach maturity was dependant on the chemical properties of the initial raw materials used. The compost including EOC had more nitrogen and synthesised more polymerised HA, the POMW compost also had acceptable degrees of stability and maturity at the end of the process. Maturation was confirmed by a decline in Cw below 1.7, an increase in nitrogen, in HA, in CHA/CFA and an elimination of phytotoxicity. Composts produced with olive mill wastes, experimented on potato culture in the field, can be considered beneficial to soils because of their humification indexes and no toxicity.

Quality assessment of composts prepared with olive mill wastewater and agricultural wastes.

The co-composting of solid residue from olive oil production process, exhausted olive cake (EOC), with poultry manure (PM) watered with olive mill wastewater (OMW) was considered as an efficient method for the treatment of olive oil extraction effluent having high organic content including phenolic polluting compounds. The process was carried out by using three aerated windrows of variable compositions. OMW was used continuously during the bio-oxidative period, which lasted three months, to replace water for windrow moistening. The main process parameters (temperature, pH, humidity and C/N) were monitored over four months to ascertain the maturity of the compost. The composting process lasted four months during which 26 moistenings of the mixtures were performed with OMW or water to keep moisture within the ideal range of 45-60% (w/w). At the maturity stage, the C/N ratios were less than 16, pH of the resulting products were slightly alkaline (pH=8) and electrical conductivity was relatively high in the OMW mixtures (5.46-5.48 Sm(-1)) when compared with water application. Nitrates increased (0.16-0.42%) and phenol contents were reduced by more than 49%. Mature composts were then used as an amendment for potato production in a field where no inhibitory effect was observed. Potato productivity increased 10-23% as a result of compost application. No noticeable negative impact of OMW on the soil system was observed. Phenolic compound concentrations in the stabilised composts were comparable in the three studied mixtures (different sites) and averaged 0.24%. Considering previous results and this three year study, it has been observed that the benefit of these composts demonstrated the potential sustainable agronomic production of potato while using locally available recycled organic materials.