Isolation, characterization and determination of biotechnological potential of oil-degrading bacteria from Algerian centre coast.

AIMS: The Algerian coastline is exposed to several types of pollution, including hydrocarbons. The aim of this work was to isolate oil-degrading bacteria and to explore the intrinsic bioremediation potential of part of its contaminated harbour. METHODS AND RESULTS: A collection of 119 strains, capable to grow on mineral medium supplemented with hydrocarbons, were obtained from polluted sediment and seawater collected from Sidi Fredj harbour (Algiers). Twenty-three strains were selected for further studies. Sequencing of the 16S rRNA gene showed that most isolates belong to genera of hydrocarbonoclastic bacteria (Alcanivorax), generalist hydrocarbons degraders (Marinobacter, Pseudomonas, Gordonia, Halomonas, Erythrobacter and Brevibacterium) and other bacteria not known as hydrocarbon degraders (Xanthomarina) but were able to degrade hydrocarbons. Strains related to Marinobacter and Alcanivorax were frequently isolated from our samples and resulted the most effective in degrading crude oil. Screening of catabolic genes alkB and xylA revealed the presence of alkB gene in several bacterial strains; one isolate harboured both catabolic genes while other isolates carried none of the studied genes. However, they grew in the presence of crude oil implying the existence of other biodegradation pathways. CONCLUSIONS: The samples of seawater and sediment from the Algerian coast contain high level of hydrocarbon-degrading bacteria that could be interesting and useful for future bioremediation purposes. SIGNIFICANCE AND IMPACT OF THE STUDY: This investigation demonstrates the diversity of hydrocarbon-degrading bacteria from a marine-contaminated area in Algeria, and their variable biodegradation abilities.

Influence of apixaban on commonly used coagulation assays: results from the Belgian national External Quality Assessment Scheme.

INTRODUCTION: The Belgian national External Quality Assessment Scheme performed a survey to assess the effect of the direct oral anticoagulant apixaban on the coagulation assays prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen and antithrombin as performed with a large number of reagent/instrument combinations. METHODS: Four lyophilized plasma samples spiked with apixaban (0, 41, 94 and 225 ng/mL) were sent to the 195 Belgian and Luxembourg clinical laboratories performing coagulation testing. RESULTS: PT and aPTT were barely influenced at the concentrations tested. At 225 ng/mL apixaban, PT and aPTT clotting times were only 1.15 times longer than at 0 ng/mL. Among PT reagents, RecombiPlasTin 2G® showed a slightly higher sensitivity with 225 ng/mL apixaban prolonging the PT clotting time 1.3-fold. Among aPTT reagents, there was no appreciable difference in sensitivity. Fibrinogen results were unaffected by the presence of apixaban, but antithrombin activity was considerably overestimated when measured with a FXa-based assay. At 225 ng/mL apixaban, the median percentage increase in antithrombin level was 31% when measured with the Liquid Antithrombin® reagent and 44% with the Innovance Antithrombin® reagent. CONCLUSION: Our data provide clinical laboratories with useful information on the impact of apixaban on their routine coagulation assays.

Fifteen years of Belgian experience with external quality assessment of semen analysis.

Semen analysis is difficult to standardize, quality control and quality assurance are necessary to ensure that results are accurate and precise. This Belgian EQA survey over a 15-year period, involving 121 laboratories, attempted to reduce interlaboratory variability and at the same time, encouraged participating laboratories to implement correct techniques as advised by the WHO. Over the total period, the median coefficient of variation (CV) for sperm count, irrespective of the method used was 19.2%, while using improved Neubauer chamber resulted in a significantly (p < 0.001) lower median CV (14.4%). The overall median CV for rapid progressive motility was high (37.1%), but progressive motility (15.1%) and total motility (13.8%) were acceptable. Sperm morphology revealed a large variability in 79.4% irrespective of the staining procedures or evaluation criteria used. Participation in the Belgian EQA is on voluntary basis. Both, participation and implementation of the correct techniques should be made mandatory for accreditation and benefit of patient treatment. The existing Belgian EQA program should now be harmonized with other existing EQA schemes in Europe.

Low yield by molecular detection of Chlamydophila pneumoniae in respiratory samples in Belgium questioning its etiological role in respiratory tract infections.

The actual burden of respiratory infections due to Chlamydophila pneumoniae is difficult to assess due to the major differences in positivity rates between PCR- and serology-based methods. The aim of the current study was to objectively analyse the yield of PCRs for the detection of C. pneumoniae in respiratory samples and to evaluate the additional value of performing laboratory diagnosis for C. pneumoniae in a setting of respiratory infection. The data based on routine analysis of respiratory samples with request for C. pneumoniae detection were collected from 4 large Belgian hospitals during 2 consecutive years. In total 3560 respiratory samples have been analysed and overall only 7 samples (0.2%) were found positive. Based on these observations, the critical evaluation of the actual role of C. pneumoniae in the etiology of lower respiratory infections and consequently of the extensive use of diagnostic tools for the detection of C. pneumoniae is needed.

B. anthracis in a wool-processing factory: seroprevalence and occupational risk.

In a Belgian wool-processing factory, living anthrax spores were found in raw goat hair and air dust, but confirmed anthrax cases had never been reported. Anthrax vaccines are not licensed nor recommended in Belgium. We conducted a B. anthracis seroprevalence study to investigate risk factors associated with positive serology and advise on protective measures. Overall 12·1% (8/66) employees were seropositive; 30% of persons processing raw goat hair and 20% of persons sorting raw goat hair were seropositive compared to 3% in less exposed jobs [adjusted prevalence ratio (aPR) 44·4, P=0·001; aPR 14·5, P=0·016, respectively). The number of masks used per day was protective (aPR 0·3, P=0·015). Results suggest a dose-response association for those processing raw goat hair. Host-related factors probably played a role as antibody response varied from person to person within an exposure group. Workers exposed to raw goat hair should be offered higher protection against anthrax and have access to anthrax vaccines.

First epidemiological study on exposure to Neospora caninum in different canine populations in the Algiers District (Algeria).

Neospora caninum is an important cause of abortion in cattle worldwide. Dogs act as final hosts shedding oocysts in the environment. They can also harbour the extraintestinal stage of the parasite and this may be associated with a fairly rare neuromuscular condition. The sera of 781 dogs from the Algiers District were screened by IFAT for the presence of anti-N. caninum antibodies. These dogs were distributed into four populations: local stray dogs, police dogs, dogs from breeding kennels and farm dogs. The overall seroprevalence was 21.90%. Significant differences were observed between the different populations, the highest prevalence being observed in farm (44.44%) and stray dogs (22.55%). Additionally, the highest titres were observed in farm dogs. Among studied epidemiological parameters, breed, dog origin, season and vaccination status were significantly associated with IFAT results. Additionally, a recently described real time PCR was used on the blood of 100 pound dogs and the results were compared with the serological data. A higher proportion of dogs was found to be positive by PCR when compared to the IFAT results. There was only a fairly low agreement between PCR and IFAT results which suggests that these techniques measured different aspects of the host-parasite relationship. This study indicates that the level of exposure of the canine population of Algiers area to N. caninum is very high. This would indicate a potentially high risk for N. caninum induced abortion in cattle in this region and in Algeria.

Hygiene indicator microorganisms for selected pathogens on beef, pork, and poultry meats in Belgium.

Several bacterial indicators are used to evaluate hygiene during the meat slaughtering process. The objectives of this study were to assess the Belgian baseline data on hygienic indicators and the relationship between the indicators and zoonotic agents to establish hygiene indicator criteria for cattle, pig, and chicken carcasses and meat. The study used the results from the official Belgian surveillance plan from 2000 to 2003, which included the monitoring of Escherichia coli counts (ECC), Enterobacteriaceae counts (EC), aerobic colony counts (ACC), and Pseudomonas counts (PC). The sampling method was the wet and dry swabbing technique for cattle and pig carcasses and neck skin excision for broiler and layer chicken carcasses. The 75th and 95th percentiles of ECC were -0.20 and 0.95 log CFU/cm2 for cattle carcasses, 1.20 and 2.32 log CFU/cm2 for pig carcasses, and 4.05 and 5.24 log CFU/g for chicken carcasses. The ACC were 2.1- to 4.5-log higher than the ECC for cattle, pigs, and chickens. For cattle and pig carcasses, a significant correlation between ECC, EC, and ACC was found. ECC for pork and beef samples and EC in pig carcasses were significantly higher in samples contaminated with Salmonella. In poultry samples, ECC were in general higher for samples containing Salmonella or Campylobacter. Thus, E. coli may be considered as a good indicator for enteric zoonotic agents such as Salmonella for beef, pork, and poultry samples and for Campylobacter in poultry samples.

Bifidobacteria as indicators of faecal contamination along a sheep meat production chain.

AIMS: The potential use of bifidobacteria as indicators for faecal contamination was studied along a sheep meat production and processing chain. The levels of bifidobacteria were compared with those of Escherichia coli. Total viable counts were followed along the chain (244 samples). METHODS AND RESULTS: Forty-three per cent of the samples contained bifidobacteria, of which 15% were solely detected using a PCR method based on the hsp60 gene and not by a culture-based method. Bifidobacteria were detected in only three of nine sheep faeces samples using one or the other method. However, carcasses (types C and E) were highly contaminated. These sample types (30% and 28%, respectively) were positive for bifidobacteria and negative for E. coli. The species Bifidobacterium pseudolongum and Bif. thermophilum, isolated from faecal samples, were predominant. Bifidobacterium choerinum were found in C, D, E and F sample types. CONCLUSIONS: Bifidobacteria were shown more efficient than E. coli in carcasses samples. The presence of Bif. choerinum suggested a faecal pork contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: Detection and identification of bifidobacteria, in correlation with E. coli counting, should improve hygiene quality of mutton processing chains.

A seven-year survey of Campylobacter contamination in meat at different production stages in Belgium.

The presence of Campylobacter was assessed in different samples of poultry, pork and beef meat and carcasses from slaughterhouses, production plants and retail level. An introductory study from 1997 to 1999, had the purpose of establishing the optimum dilution to detect changes in prevalence and allowed a semi-quantitative estimation of poultry and pork contamination. Following this, between 2000 and 2003, 4254 samples were taken in order to study the trends. The poultry matrixes represented the greatest number and the most highly contaminated samples, with 30.9% (in 0.01 g) positive samples, 18.7% (in 1 g), 46.9% (in 25 g) and 19.6% (in 0.01 g) for broiler carcasses, broiler fillets, prepared chicken and layer carcasses, respectively. Broiler carcasses and fillets sampled at retail level were significantly less contaminated than samples from production plants. Pork, beef and veal samples were rarely contaminated and, where contamination existed, it was at a low prevalence (maximum 5.0%). The high and unvarying prevalence of Campylobacter in poultry necessitates the implementation of intervention measures at the primary production level, in addition to methods of minimizing cross-contamination at the processing level. A survey plan in line with the present study could be used in the future to monitor the effects of the planned measures and performance objectives and to follow the evolution of Campylobacter contamination at all stages of the food chain, in accordance with European legislation.

Prevalence of enterohaemorrhagic Escherichia coli from serotype O157 and other attaching and effacing Escherichia coli on bovine carcasses in Algeria.

AIMS: Bovine meat is the principal source of human contamination of attaching and effacing Escherichia coli, including enterohaemorrhagic E. coli O157. The aim was to study the prevalence of these strains on bovine carcasses in Algeria. METHODS AND RESULTS: Two-hundred and thirty carcasses were swabbed and analysed by classical microbiological methods for total E. coli counts and for the presence of pathogenic E. coli. The E. coli counts were high, with a 75th percentile of 444.75 CFUs cm(-2). For pathogenic E. coli, more than 7% of the tested carcasses were positive for E. coli O157. Eighteen E. coli O157 strains were isolated and typed by multiplex PCR. The main isolated pathotype (78%) was eae+ stx2+ ehxA+. In addition to E. coli O157, other attaching and effacing E. coli (AEEC) were also detected from carcasses by colony hybridization after pre-enrichment and plating on sorbitol MacConkey agar using eae, stx1 and stx2 probes. Thirty carcasses (13%) on the 230 analysed harboured at least one colony positive for one of the tested probes. These positive carcasses were different from those positive for E. coli O157. Sixty-six colonies (2.9%) positive by colony hybridization were isolated. The majority (60.6%) of the positive strains harboured an enteropathogenic E. coli-like pathotype (eae+ stx-). Only three enterohaemorrhagic E. coli (EHEC)-like (eae+ stx1+) colonies were isolated from the same carcass. These strains did not belong to classical EHEC serotypes. CONCLUSIONS: In this study, the global hygiene of the slaughterhouse was low, as indicated by the high level of E. coli count. The prevalence of both E. coli O157 and other AEEC was also high, representing a real hazard for consumers. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study of this type in Algeria, which indicates that the general hygiene of the slaughterhouse must be improved.

Development of a genetic traceability test in pig based on single nucleotide polymorphism detection.

In order to assure traceability along the meat transformation process, a powerful system is required. The administrative traceability shows limits that the use of genetic markers could overcome. The individual genomes contain sequence differences, basis of the genetic polymorphism of which the genetic markers are the witnesses. Among them, two classes seem to dominate on the traceability field: the microsatellites and the single nucleotide polymorphisms (SNP). The aim of this work was to develop a genetic traceability test in pig based on SNPs mainly located in 5' and 3' untranslated regions (UTRs). A set of 21 SNP markers including new SNPs identified in this study and SNPs previously described was selected. A genotyping assay was performed on 96 individuals representing the major crossbred of the pig population in Belgium. Results showed that all individuals tested presented a different genotype. This genotyping method might help the administrative system to guarantee the traceability of pork meat along the transformation process.

Survey of the contamination of foodstuffs of animal origin by Shiga toxin producing Escherichia coli serotype O157:H7 in Belgium from 1999 to 2003.

A survey of the prevalence of Shiga toxin-producing Escherichia coli (STEC) of O157 serotype in foodstuffs of animal origin (beef, veal, pork, chicken, fish) from 1999 to 2003 in Belgium was performed. STEC strains were only isolated from beef with a prevalence of 0.73%. This percentage is low in comparison with the prevalence in other countries. Among the 76 isolated STEC O157 strains, 75% belonged to the serotype O157:H7 and 25% to the serotype O157 non H7. Moreover, the most frequent pathotype was eae stx2 ehxA (74%).

Development of bovine herpesvirus 4 as an expression vector using bacterial artificial chromosome cloning.

Several features make bovine herpesvirus 4 (BoHV-4) attractive as a backbone for use as a viral expression vector and/or as a model to study gammaherpesvirus biology. However, these developments have been impeded by the difficulty in manipulating its large genome using classical homologous recombination in eukaryotic cells. In the present study, the feasibility of exploiting bacterial artificial chromosome (BAC) cloning and prokaryotic recombination technology for production of BoHV-4 recombinants was explored. Firstly, the BoHV-4 genome was BAC cloned using two potential insertion sites. Both sites of insertion gave rise to BoHV-4 BAC clones stably maintained in bacteria and able to regenerate virions when transfected into permissive cells. Reconstituted virus replicated comparably to wild-type parental virus and the loxP-flanked BAC cassette was excised by growing them on permissive cells stably expressing Cre recombinase. Secondly, BoHV-4 recombinants expressing Ixodes ricinus anti-complement protein I or II (IRAC I/II) were produced using a two-step mutagenesis procedure in Escherichia coli. Both recombinants induced expression of high levels of functional IRAC molecules in the supernatant of infected cells. This study demonstrates that BAC cloning and prokaryotic recombination technology are powerful tools for the development of BoHV-4 as an expression vector and for further fundamental studies of this gammaherpesvirus.

A PCR method for detection of bifidobacteria in raw milk and raw milk cheese: comparison with culture-based methods.

Bifidobacteria are well known for their beneficial effects on health and are used as probiotics in food and pharmaceutical products. As they form one of the most important groups in both human and animal feces, their use as fecal indicator organisms in raw milk products has recently been proposed. Bifidobacteria species isolated in humans are different from those isolated in animals. It should therefore be possible to determine contamination origin (human or animal). A method of detecting the Bifidobacterium genus was developed by PCR targeting the hsp60 gene. The genus Bifidobacterium was identified by PCR amplification of a 217-bp hsp60 gene fragment. The degenerated primer pair specific to the Bifidobacterium genus used was tested for it specificity on 127 strains. Sensitivity was measured on artificially contaminated samples. Food can however be a difficult matrix for PCR testing since it contains PCR inhibitors. So an internal PCR control was used. An artificially created DNA fragment of 315 bp was constructed. The PCR detection method was tested on raw milk and cheese samples and compared with three culture-based methods, which comprised enrichment and isolation steps. The enrichment step used Brain Heart Infusion medium with propionic acid, iron citrate, yeast extract, supplemented with mupirocin (BHMup) or not (BH) and the isolation step used Columbia blood agar medium, supplemented with mupirocin (CMup) or not (C). The method using mupirocin at both enrichment and isolation steps and the PCR method performed from the culture in BHMup enrichment medium were shown to be the most efficient. No significant difference was observed in raw milk samples between PCR from BHMup and the culture-based method BHMup/CMup, while a significant difference was noticed between the same methods in raw milk cheese samples, which would favor using PCR. The results suggested that PCR on the hsp60 gene was convenient for a rapid detection of bifidobacteria in raw milk and raw milk cheese samples and that bifidobacteria always present throughout raw milk cheese production could be efficiently used as fecal indicators.

Comparison of four different methods for Salmonella detection in fecal samples of porcine origin.

Performances of four detection methods were evaluated for recovery of Salmonella spp. in naturally contaminated fecal specimens of porcine origin. The NMKL 71 method consisted of enrichment in Rappaport-Vassiliadis broth and plating on xylose-lysine-desoxycholate medium, whereas the SP-VG-M002 method relied on a Diasalm enrichment followed by streaking on xylose-lysine-tergitol 4 agar (XLT-4). The VIDAS SLM method was composed of double enrichment in Muller-Kauffmann tetrathionate broth and in M broths before processing in a VIDAS device. If the results were positive, the VIDAS ICS immunoenrichment was performed and the result transferred onto three different selective media. The VIDAS ICS protocol is an immunoconcentration step followed by plating on XLT-4. Seventy-eight samples were tested with all four methods simultaneously, leading to 34 positive samples with at least one method. For this assay, VIDAS SLM revealed 31 positive samples (91.2%), whereas the average positive percentage of the three other methods was 37.3% (P < 0.001). Two-paired comparisons with the VIDAS SLM method were also performed. McNemar values were systematically highly significant (P < 0.001). The proportion of agreement was significantly inferior (P < 0.05) for the comparison of VIDAS ICS and VIDAS SLM (68.7%) compared with the two other paired comparisons (average percentage, 81.5%). The conclusion reached by this trial is that VIDAS SLM significantly improves the recovery of Salmonella in naturally contaminated fecal specimens. For the paired-comparisons, NMKL 71 and SP-VG-M002 were comparable in terms of efficiency, whereas the VIDAS ICS protocol, as established by the manufacturer for food samples only, seemed less efficient than the other two.

Discrimination between Bifidobacterium species from human and animal origin by PCR-restriction fragment length polymorphism.

Bifidobacteria are normal intestinal flora in humans and animals. The genus Bifidobacterium includes 31 species of significant host specificity. Taking into account their properties, we proposed to use bifidobacteria as fecal contamination indicators. PCR-restriction fragment length polymorphism on the 16S rDNA gene was used to distinguish the different Bifidobacterium species. Sixty-four strains belonging to 13 different species were differentiated from animal or human origin using one or two restriction enzymes. Moreover, the primers used were specifics of the Bifidobacterium genus. Therefore, this method made it possible to determine both the presence of bifidobacteria in a sample and its origin of contamination.

Organisation and in vitro expression of esp genes of the LEE (locus of enterocyte effacement) of bovine enteropathogenic and enterohemorrhagic Escherichia coli.

Enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli infections are characterised by the formation of attaching and effacing (AE) lesions on intestinal epithelial cells. Secretion of extracellular proteins (EspA, EspB, and EspD) via a type III secretion apparatus is necessary for the formation of the AE lesions by human EPEC. In this study, we show that bovine EPEC and EHEC are also able to secrete polypeptides homologous to the already described Esp proteins, most probably via a type III secretion system. Bovine EPEC and EHEC strains present two different secretion profiles of Esp proteins which correlate to the pathotypes of the esp genes as determined by PCR. We also demonstrate that genes encoding secreted proteins, present in the LEE of two bovine strains, are organised in the same way as in the human EPEC strain E2348/69.

Clinical signs, reproduction of attaching/effacing lesions, and enterocyte invasion after oral inoculation of an O118 enterohaemorrhagic Escherichia coli in neonatal calves.

Attaching and effacing (AE) lesions are produced among others by enteropathogenic Escherichia coli and enterohaemorrhagic E. coli (EHEC), which differs from the former by the production of cytotoxins active on various cell cultures, the verocytotoxins, or shigacytotoxins. EHEC are associated with diarrhoea and dysentery in humans and in ruminants, mainly calves from two to eight weeks of age. Clinical signs and/or lesions have been reproduced experimentally with EHEC strains belonging to serotypes O5:K4/Nm, O26:K-:H11, O111:Nm, and O157:H7 which are isolated from cattle and/or humans. The purpose of this work was to develop an experimental model of infection in newborn calves with a bovine EHEC strain isolated from a calf which of died of diarrhoea, and belonging to the O118:H16 serotype, which is also common to both cattle and humans. The bovine O118:H16 EHEC strain was able to colonize the gut of three newborn calves, and to induce diarrhoea twenty-four hours after challenge and to produce AE lesions in the small and/or large intestines. AE lesions were detected microscopically and ultrastructurally in the small intestine of one calf and in the whole intestinal track of two calves. Internalization of bacteria and also of pedestal-bacteria complex inside of the enterocyte was observed in two of the three calves. The significance of this stage is unknown but may be related to the invasion of the calf by the bacteria. The challenge strain was isolated from the mesenteric lymph nodes of the same two calves but not from other organs or from heart blood. No blood was observed in the faeces of any of the three calves, nor were any lesions in the internal organs, which may have been related to the production of a verotoxin whose role is still unknown in cattle.

Genotypic characterization of enteropathogenic Escherichia coli (EPEC) isolated in Belgium from dogs and cats.

Enteropathogenic Escherichia coli (EPEC) are isolated from man and farm animals but also from dogs and cats. They produce typical histological lesions called 'attaching and effacing' lesions. Both plasmid and chromosomal elements are involved in the pathogenesis of EPEC infection. The presence of these genetic elements was investigated in 14 dog and three cat EPEC isolates. A bfpA-related gene was detected in five of the 17 isolates in association with high molecular weight plasmids, and a locus of enterocyte effacement (LEE) was present in all isolates. The LEE was inserted in the selC region in only 12% of the isolates. The eae, tir, espA and espB genes were analyzed by multiplex PCR. The results indicated the presence of those genes in the tested isolates with heterogeneity in the gene subtypes present: eae gamma-tir alpha-espA alpha-espB alpha (65%), eae beta-tir beta-espA beta-espB beta (29%), eae alpha-tir alpha-espA alpha-espB alpha (6%). Moreover, the espD gene was also present in dog and cat EPEC. The DEPEC and CEPEC form a heterogeneous group and five of them are closely related to human EPEC.