Increased levels of soluble HLA-G molecules in Tunisian patients with chronic hepatitis B infection.

Hepatitis B virus (HBV) infection is a global health problem. The mechanisms of immune tolerance in HBV infection are still unclear. The host immune response plays a critical role in determining the outcome of HBV infection. Human leucocyte antigen-G (HLA-G) is involved in immunotolerogenic process and infectious diseases. This study aimed to explore the implication of soluble HLA-G (sHLA-G) and its isoforms in HBV infection. Total sHLA-G (including shedding HLA-G1 and HLA-G5) was analysed by ELISA in 95 chronic HBV patients, 83 spontaneously resolvers and 100 healthy controls (HC). To explore the presence of sHLA-G dimers, we performed an immunoprecipitation and a Western blot analysis on positive samples for sHLA-G in ELISA. The serum levels of sHLA-G were significantly increased in patients with chronic HBV patients compared to spontaneously resolvers and HC (P<.0001). Interestingly, we found an increased level of sHLA-G1 in chronic HBV patients than in spontaneously resolvers and HC (P<.001). In addition, the expression of HLA-G5 seems to be higher in the sera of chronic HBV patients than spontaneously resolvers (P=.026). The analysis of HLA-G dimers showed the presence of homodimers in 93% of chronic HBV patients, 67% in spontaneously resolvers and 60% in HC. These results provide evidence that sHLA-G may have a crucial role in the outcome of HBV infection and could be proposed as a biomarker for infection outcome. Based on its tolerogenic function, HLA-G might be considered as a new promising immunotherapeutic approach to treat the chronic infection with HBV.

Association of an HLA-G 14-bp Insertion/Deletion polymorphism with high HBV replication in chronic hepatitis.

Identification of an HLA-G 14-bp Insertion/Deletion (Ins/Del) polymorphism at the 3' untranslated region of HLA-G revealed its importance in HLA-G mRNA stability and HLA-G protein level variation. We evaluated the association between the HLA-G 14-bp Ins/Del polymorphism in patients with chronic Hepatitis B virus (HBV) infection in a case-control study. Genomic DNA was extracted from 263 patients with chronic HBV hepatitis and 246 control subjects and was examined for the HLA-G 14-bp Ins/Del polymorphism by PCR. The polymorphic variants were genotyped in chronic HBV seropositive cases stratified according to HBV DNA levels, fibrosis stages and in a control population. There was no statistical significant association between the 14-bp Ins/Del polymorphism and increased susceptibility to HBV infection neither for alleles (P = 0.09) nor for genotypes (P = 0.18). The stratification of HBV patients based on HBV DNA levels revealed an association between the 14-bp Ins/Del polymorphism and an enhanced HBV activity with high HBV DNA levels. In particular, the Ins allele was significantly associated with high HBV DNA levels (P = 0.0024, OR = 1.71, 95% CI 1.2-2.4). The genotype Ins/Ins was associated with a 2.5-fold (95% CI, 1.29-4.88) increased risk of susceptibility to high HBV replication compared with the Del/Del and Ins/Del genotypes. This susceptibility is linked to the presence of two Ins alleles. No association was observed between the 14-bp Ins/Del polymorphism and fibrosis stage of HBV infection. We observed an association between the 14-bp Ins/Del polymorphism and high HBV replication characterized by high HBV DNA levels in chronic HBV patients. These results suggest a potential prognostic value for disease outcome evaluation.

Prevalence, identification by a DNA microarray-based assay of human and food isolates Listeria spp. from Tunisia.

OBJECTIVES: We aimed at evaluating the prevalence of Listeria species isolated from food samples and characterizing food and human cases isolates. MATERIAL AND METHODS: Between 2005 and 2007, one hundred food samples collected in the markets of Tunis were analysed in our study. Five strains of Listeria monocytogenes responsible for human listeriosis isolated in hospital of Tunis were included. Multiplex PCR serogrouping and pulsed field gel electrophoresis (PFGE) applying the enzyme AscI and ApaI were used for the characterization of isolates of L. monocytogenes. We have developed a rapid microarray-based assay to a reliable discrimination of species within the Listeria genus. RESULTS: The prevalence of Listeria spp. in food samples was estimated at 14% by using classical biochemical identification. Two samples were assigned to L. monocytogenes and 12 to L. innocua. DNA microarray allowed unambiguous identification of Listeria species. Our results obtained by microarray-based assay were in accordance with the biochemical identification. The two food L. monocytogenes isolates were assigned to the PCR serogroup IIa (serovar 1/2a). Whereas human L. monocytogenes isolates were of PCR serogroup IVb, (serovars 4b). These isolates present a high similarity in PFGE. Food L. monocytogenes isolates were classified into two different pulsotypes. These pulsotypes were different from that of the five strains responsible for the human cases. CONCLUSION: We confirmed the presence of Listeria spp. in variety of food samples in Tunis. Increased food and clinical surveillance must be taken into consideration in Tunisia to identify putative infections sources.

Prevalence and characterisation of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates in healthy volunteers in Tunisia.

The objective of this investigation was to analyse the carriage rate of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in faecal samples of healthy humans in Tunisia and to characterise the recovered isolates. One hundred and fifty samples were inoculated on MacConkey agar plates supplemented with cefotaxime (2 µg/ml) for ESBL-positive E. coli recovery. The characterisation of ESBL genes and their genetic environments, detection of associated resistance genes, multilocus sequence typing (MLST) and phylogroup typing were performed by polymerase chain reaction (PCR) and sequencing. The presence and characterisation of integrons and virulence factors were studied by PCR and sequencing. ESBL-positive E. coli isolates were detected in 11 of 150 faecal samples (7.3%) and one isolate/sample was further characterised. These isolates contained the blaCTX-M-1 (ten isolates) and blaTEM-52c genes (one isolate). The ISEcp1 (truncated by IS10 in four strains) and orf477 sequences were found upstream and downstream, respectively, of all bla (CTX-M-1) genes. Seven different sequence types (STs) and three phylogroups were identified among CTX-M-1-producing isolates [ST/phylogroup (number of isolates)]: ST58/B1 (3), ST57/D (2), ST165/A (1), ST155/B1 (1), ST10/A (1), ST398/A (1) and ST48/B1 (1). The TEM-52-producing isolate was typed as ST219 and phylogroup B2. Six ESBL isolates contained class 1 integrons with the gene cassettes dfrA17-aadA5 (five isolates) and dfrA1-aadA1 (one). Healthy humans in the studied country could be a reservoir of CTX-M-1-producing E. coli.

Molecular analysis and antimicrobial resistance of Salmonella isolates recovered from raw meat marketed in the area of "Grand Tunis", Tunisia.

A total of 315 samples of chicken (60), beef (144), minced meat (56), lamb meat (33), merguez (10) and fish (12) were collected from various local outlet stores in the area of "Grand Tunis", Tunisia between 2006 and 2008. Salmonella was recovered from 80 samples with the highest occurrence in chicken (48.3%) followed by beef (29.8%), minced meat (10.7%) and lamb (6.0%). No Salmonella were isolated from 12 fish and 10 merguez samples (typical Tunisian sausages). Nine serovars were identified among the isolates with the predominance of Salmonella Typhimurium (n=25) followed by Salmonella Kentucky (n=14), Salmonella Suberu (n=12) and Salmonella Zanzibar (n=11). Isolated Salmonella were characterized by serotyping, pulsed-field gel electrophoresis (PFGE) analysis, plasmid content and antimicrobial resistance profiling. Sixteen (20.0%) Salmonella isolates displayed resistance to ampicillin (13 isolates), streptomycin (five isolates), cefoperazone (two isolates), furazolodine (two isolates), with seven of these isolates displaying multiple resistance to at least two of these antimicriobal agents. PFGE analysis showed homogenous restriction patterns in each serovar. Compiled serotyping, PFGE analysis, plasmid profiling and antimicrobial resistance data provided additional discrimination.

Nasal carriage of Staphylococcus aureus in healthy humans with different levels of contact with animals in Tunisia: genetic lineages, methicillin resistance, and virulence factors.

Nasal swabs of 423 healthy humans who showed different levels of contact with animals (frequent, 168; sporadic, 94; no contact, 161) were obtained in Tunisia (2008-2009), and 99 of them presented other associated risk factors. Methicillin-resistant Staphylococcus aureus (MRSA) was detected in one of these 423 samples (0.24%), retrieved from a veterinarian. The MRSA isolate was mecA-positive, typed as ST80-t203-SCCmecIVc-agrIII, and contained tet(K), ant(6)-Ia, and aph(3')-IIIa genes encoding tetracycline, streptomycin, and kanamycin resistance, respectively. This MRSA isolate also contained the lukF/lukS virulence gene encoding Panton-Valentine leukocidin. Fifty-four (12.8%) additional nasal samples contained methicillin-susceptible S. aureus (MSSA) and one isolate/sample was characterized. A high diversity of spa types (n = 43; 4 new) and pulsed-field gel electrophoresis (PFGE) types (n = 37) was detected among the 55 recovered S. aureus strains. The percentages of antimicrobial resistance/detected resistance genes were as follows: tetracycline [22%/tet(K)-tet(L)-tet(M)], erythromycin [5%/msrA], ciprofloxacin [14.5%], trimethoprim-sulfamethoxazole [2%/dfrA], streptomycin [11%/ant(6)-Ia], kanamycin [7%/aph(3')-IIIa], amikacin [5%], and chloramphenicol [2%]. Four and two isolates carried the lukF/lukS and eta and/or etb genes, respectively, and always in individuals with contact with animals. Eleven isolates carried the tst gene and were recovered from individuals with different levels of contact with animals.

Antimicrobial resistance and molecular analysis of non-typhoidal Salmonella isolates from human in Tunisia.

During the period from 2006 to 2007, a total of 32 clinical isolates of Salmonella enterica were isolated from diarrheagenic stool samples and further examined for their susceptibility to various antibiotics. Sixteen of the human isolates were from the capital Tunis, 11 were from Sousse, four were from Nabeul and one was from Mahdia, Tunisia. The isolates were serotyped and identified at the National Centre of Enteropathogenic Bacteria, Pasteur Institute, Tunis (Centre National de Salmonella, Shigella et Vibrio - Institut pasteur de Tunis); nine distinct serovars were identified: Enteritidis (n=20), Typhimurium (n=4), Zanzibar (n=2), Manhattan (n=1), Bovismorbificans (n=1), Amsterdam (n=1), Saint Paul (n=1), Kentucky (n=1) and Muenster (n=1). Our results showed that 25 Salmonella isolates (78.1 %) were resistant to antibiotics with 20 isolates (62.5 %) displayed resistance to ampicillin. Isolates sharing invA gene, as shown by PCR amplification, were further characterized by the techniques of pulsed-field gel electrophoresis (PFGE) using the restriction enzyme XbaI and plasmid analysis to determine possible genetic relationships among Salmonella enterica clinical isolates and to assess genetic diversity. Plasmid profiling identified seven plasmid profiles (with 1-5 plasmids) among the isolates; four isolates (Salmonella Kentucky, Salmonella Muenster, Salmonella Bovismorbificans and Salmonella Zanzibar) did not carry any plasmid. The isolates were differentiated into 10 distinct XbaI-pulsotypes. Our findings show genetic diversity among the different serovars and cluster analysis of compiled serotyping, PFGE, plasmid profiling and antibiotic resistance data provided additional discrimination.

Survival study of enterotoxigenic Escherichia colistrain in seawater and wastewater microcosms.

In order to survey osmotic and oligotrophic stress consequence on pathogenic enterobacteria discharged in marine areas, we examined enterotoxigenic Escherichia coli (ETEC) and a reference (Ecoli O126:B16) strains during their survival (47 days) in wastewater microcosms, submerged in natural seawater and maintained in laboratory conditions. The results revealed that the survival time for the two strains was prolonged when bacterial cells were previously incubated in wastewater, with less cellular membrane damage. In addition, the wild clinical E. coli strain showed a better survival capacity than the reference E. coli strain one. For both, we noted some modifications in biochemical profiles relatively to the initial state, notably when they were previously incubated in wastewater microcosm.

Evidence for the involvement of galectin-3 in mesenchymal stem cell suppression of allogeneic T-cell proliferation.

Human bone marrow-derived mesenchymal stem cells (MSC) are multipotent non-hematopoietic progenitors that have regulatory activity on immune cells. NOD- and Toll-like receptors (NLR, TLR) have several roles in immunity, including those relevant to pathogen recognition and shaping the course of immune responses by controlling gene expression. We have shown that these innate immune receptors are expressed by hematopoietic CD34+ progenitors and MSC. To uncover genes critical in MSC function, first we have used microarray to screen for potential transcripts whose levels are altered in response to NOD-1 and TLR-2 activation, and second we validated some candidate genes using real-time RT-PCR, Western blots and cellular assays. Amongst the altered genes, galectin-3 was upregulated at both mRNA and protein levels in response to TLR-2 activation. Interestingly, MSC secreted galectin-3, a protein known to modulate T-cell proliferation, gene expression, cell adhesion and migration. Knockdown of galectin-3 in MSC using small interfering RNA (siRNA) reduced the immunosuppressive effect of MSC on mixed lymphocyte cultures when compared to cells treated with an irrelevant siRNA (P < 0.05). Collectively, the data emphasize a new role of galectin-3 in the immunomodulatory function of MSC and indicate that NOD signalling pathway is also functional in these cells.

Esterase as an enzymatic signature of Geodermatophilaceae adaptability to Sahara desert stones and monuments.

AIM: To assess esterase profiling of members of Geodermatophilaceae isolated from desert stones and monuments in Tunisia and Egypt. METHODS AND RESULTS: Members of Geodermatophilaceae family isolated from desert stones and monuments in Tunisia and Egypt were characterized by partial 16S rRNA sequences. Twenty-five strains were clustered in three dissimilar groups of the genera Geodermatophilus (12 strains), Blastococcus (5 strains) and Modestobacter (3 strains). Isolates were also screened and typed based on major groups of esterase hydrolytic activity. Their esterase patterns were determined and compared to those of ten reference strains belonging to Geodermatophilaceae family. Strains exhibited a diverse and complex pattern of electrophoretic esterase bands, and 31 haplotypes were obtained for the 35 investigated strains. Esterases produced by members of Geodermatophilaceae family have an optimal activity around 40 degrees C and at pH 8. Esterases from Geodermatophilus strains display a high resistance to thermal inactivation and alkaline pH and retaining 30 and 20% of activity after heating for 20 min at 120 degrees C and at pH 12, respectively, and were completely inactivated after 30 min at 120 degrees C. Enzyme activity has been strongly activated in the presence of Ca(2+)and Mg(2+) ions and moderately by Zn(2+) and was markedly inhibited by Cu(2+) and Co(2+) ions. CONCLUSIONS: Geodermatophilaceae isolates share a rich and particular pool of esterase activities that could be directly linked to harsh conditions characterizing their ecological habitat including high level of aridity, temperature, ionic strength and low nutrient availability. SIGNIFICANCE AND IMPACT OF THE STUDY: Esterase could be considered as enzymatic signature that outlines adaptability of Geodermatophilaceae in arid area.

Survival of Escherichia coli strains in Mediterranean brackish water in the Bizerte lagoon in northern Tunisia.

This study investigated survival and virulence of Escherichia coli strains exposed to natural conditions in brackish water. Two E. coli strains (O126:B16 and O55:B5) were incubated in water microcosms in the Bizerte lagoon in northern Tunisia and exposed for 12 days to natural sunlight in June (231 to 386 W/m2, 26 +/- 1 degrees C, 30 g/L) and in April (227 to 330 W/m2, 17 +/- 1 degrees C, 27 g/L) or maintained in darkness for 21 days (17 +/- 1 degrees C, 27 g/L). The results revealed that sunlight was the most significant inactivating factor (decrease of 3 Ulog within 48 hours for the two strains) compared to salinity and temperature (in darkness). Survival time of the strains was prolonged as they were maintained in darkness. Local strain (E. coli O55:B5) showed better survival capacity (T90 = 52 hours) than E. coli O126:B16 (T90 = 11 h). For both, modifications were noted only for some metabolic activities of carbohydrates hydrolysis. Cytotoxicity of the two strains, tested on Vero cell, was maintained during the period of survival.

Biological control of grey mould in strawberry fruits by halophilic bacteria.

AIMS: Grey mould caused by Botrytis cinerea is an economically important disease of strawberries in Tunisia and worldwide. The aim of this study was to select effective halophilic bacteria from hypersaline ecosystems and evaluate the abilities of antifungal bacteria to secrete extracellular hydrolytic enzymes, anti-Botrytis metabolites and volatiles. METHODS AND RESULTS: Grey mould was reduced in strawberry fruits treated with halophilic antagonists and artificially inoculated with B. cinerea. Thirty strains (20.2%) were active against the pathogen and reduced the percentage of fruits infected after 3 days of storage at 20 degrees C, from 50% to 91.66%. The antagonists were characterized by phenotypic tests and 16S rDNA sequencing. They were identified as belonging to one of the species: Virgibacillus marismortui, B. subtilis, B. pumilus, B. licheniformis, Terribacillus halophilus, Halomonas elongata, Planococcus rifietoensis, Staphylococcus equorum and Staphylococcus sp. The effective isolates were tested for antifungal secondary metabolites. CONCLUSIONS: Moderately halophilic bacteria may be useful in biological control against this pathogen during postharvest storage of strawberries. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of such bacteria may constitute an important alternative to synthetic fungicides. These moderate halophiles can be exploited in commercial production and application of the effective strains under storage and greenhouse conditions.

[Chemical synthesis of lactococcin B and functional evaluation of the N-terminal domain using a truncated synthetic analogue]. / Synthèse chimique de la lactococcine B et évaluation fonctionnelle de son extrémité N-terminale grâce à un analogue synthétique tronqué.

The lactococcin B (LnB) is a hydrophobic, positively charged bacteriocin, produced by Lactococcus lactis ssp. cremoris 9B4. It consists of a peptidic chain made up of 47 amino acid residues, and inhibits Lactococcus exclusively. In order to study its biological activity a synthetic lactococcin B (LnBs) was obtained by solid-phase chemical synthesis using a Fmoc strategy. LnBs was shown to be indistinguishable from the natural peptide. In addition, a synthetic (7-47) LnBst analogue was obtained by withdrawal of peptidyl-resin after the 41 cycle of LnBs peptide chain assembly. The synthetic N-terminal truncated (7-47) LnBst analogue was found to be inactive on indicator strains. Our results strongly suggest that the first six N-terminal amino acid residues are involved in the bactericidal activity of LnB.

Treatment of chickpea with Rhizobium isolates enhances the expression of phenylpropanoid defense-related genes in response to infection by Fusarium oxysporum f. sp. ciceris.

Differential expression of phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS) and isoflavone reductase (IFR) genes involved in phenylpropanoids metabolism was investigated using Northern blot analyses in chickpea seedlings bacterized with Rhizobium isolates (PchDMS and Pch43) and further challenged with Fusarium oxysporum f. sp. ciceris (Foc) race 0. Gene activation patterns in the moderately resistant accession INRAT87/1 were compared with those exhibited by the susceptible accession ILC482 at various time intervals after inoculation with Foc, to determine whether differences in levels or timing of transcript accumulation could be correlated with differences in the susceptibility of chickpea accessions to Foc. Gene activation was higher in the moderately resistant accession INRAT87/1 than in the susceptible ILC482. Pre-treatment of chickpea seedlings with Rhizobium isolates before inoculation with the pathogen enhanced the accumulation of the three genes' mRNA transcripts. In parallel, changes in the soluble phenolic pool produced through pathways involving these enzymes were analyzed in chickpea roots. A strong accumulation of these compounds was revealed at 72 hpi in both accessions. After that time, these high levels of phenolic compounds were maintained until the end of the experiment in the moderately resistant accession, while they have significantly declined in the susceptible accession. HPLC analyses revealed a very high accumulation of the constitutive isoflavones, formononetin and biochanin A and their glycoside conjugates in chickpea roots inoculated with Rhizobium isolates and/or challenged with Foc, as compared to the controls. Our results suggest that the increased accumulation of phenolic compounds, observed in chickpea seedlings inoculated with Foc, can be attributed to increased expression of genes in the phenylpropanoid pathway and that such gene expression is enhanced by pre-treatment with Rhizobium isolates.

Diversity of structures carrying the aac(6')-aph(2") gene in clinical Enterococcus faecalis and Enterococcus faecium strains isolated in Tunisia.

The diversity of structures carrying the aac(6')-aph(2") gene was studied in 46 high-level gentamicin-resistant Enterococcus faecalis and Enterococcus faecium clinical strains recovered in a Tunisian hospital during the period 2000-2003. The inclusion of the aac(6')-aph(2") gene within the Tn4001 composite element or in its truncated forms (lacking the IS256 at the right, the left or at both sides of the aac(6')-aph(2") gene) was investigated by PCR and sequencing. The aac(6')-aph(2") gene was included in the composite Tn4001 element in 19 of 34 high-level gentamicin-resistant E. faecalis strains (56%) and in 1 of 12 E. faecium strains (12%). A truncated form of Tn4001 lacking IS256 at the left-hand (in 10 E. faecalis and 8 E. faecium), at the right-hand (3 E. faecalis and 2 E. faecium) or at both sides of the aac(6')-aph(2") gene (in 2 E. faecalis and 1 E. faecium) was also detected in 26 of our enterococci. The transference by conjugation of the aac(6')-aph(2") gene, associated with other resistance genes, was demonstrated in seven of the high-level gentamicin-resistant E. faecalis strains.

Antibiotic resistance and mechanisms implicated in clinical enterococci in a Tunisian hospital.

Susceptibility testing for 15 antibiotics was performed in a series of 191 clinical enterococci recovered in a Tunisian Hospital during 2000-2003. Species detected were the following ones (number of isolates): E. faecalis (139), E. faecium (41), E. casseliflavus (5), E. gallinarum (3), E. avium (2) and E. hirae (1). The percentages of antibiotic resistance detected were as follows (E. faecalis/ E. faecium/ other species) : penicillin (0/ 73/ 9%), tetracycline (78/ 44/ 54%), chloramphenicol (52/ 29/ 27%), erythromycin (66/ 100/ 82%), spiramycin (84/ 83/ 64%), pristinamycin (100/ 0/ 73%), trimethoprim-sulfamethoxazole (88/ 78/ 91%), rifampicin (72/ 41/ 0%), vancomycin (0/ 0/ 36%), teicoplanin (0/ 0/ 0%), high-level-resistance for gentamicin (24/ 29/ 45%), streptomycin (34/ 56/ 55%) and kanamycin (41/ 68/ 55%). Increased vancomycin minimum inhibitory concentrations (MICs) were only detected in E. casseliflavus and E. gallinarum isolates (MIC range 8-24 microg/ml). The erm(B), catA, tet(M), aac(6')-aph(2''), aph(3')-IIIa, and ant(6)-Ia genes were detected in 91%, 32%, 86%, 98%, 100%, and 72% of the E. faecium and E. faecalis isolates resistant to erythromycin, chloramphenicol, tetracycline and high-level-resistant to gentamicin, kanamycin and streptomycin, respectively. A total of 20 unrelated pulsed-field-gel-electrophoresis patterns were found in the series of 46 high-level gentamicin-resistant E. faecalis and E. faecium isolates of this study.

Combined effect of alkali pretreatment and sodium chloride addition on the olive fermentation process.

Green olives of the Tunisian variety "Meski" were treated according to a Spanish-style green olive preservation process by using an alkaline treatment (1.5, 2 and 2.5% (w/v) NaOH) to eliminate bitterness, combined with different brine concentrations (6, 9 and 12% (w/v) NaCl). A spontaneous fermentation by the environmental microflora took place. Results showed that 2% NaOH solution and 9% sodium chloride brine was an optimal combination inducing the best growth of Lactobacillus species (10(8) CFU/ml) and acidity of 0.726 g lactic acid/100 ml brine. In all trials and independently of the treatment, Lb. plantarum was the most dominant strain of Lactobacillus. Moreover, pretreatment with lye and lactic fermentation of olives contributed to coliform elimination.

Characterisation of nasal Staphylococcus delphini and Staphylococcus pseudintermedius isolates from healthy donkeys in Tunisia.

REASONS FOR PERFORMING STUDY: Staphylococcus intermedius group (SIG) bacteria can colonise the nares of some animals but are also emerging pathogens in humans and animals. OBJECTIVES: To analyse SIG nasal carriage in healthy donkeys destined for food consumption in Tunisia and to characterise recovered isolates. METHODS: Nasal swabs from 100 healthy donkeys were tested for SIG recovery, and isolates were identified by biochemical and molecular methods. Antimicrobial susceptibility of isolates was tested and detection of antimicrobial resistance and virulence genes was performed. Isolates were typed at the clonal level by multilocus sequence typing and SmaI pulsed-field gel electrophoresis. RESULTS: Staphylococcus delphini and Staphylococcus pseudintermedius (included in SIG) were obtained in 19% and 2% of the tested samples, respectively, and one isolate per sample was characterised. All isolates were meticillin susceptible and mecA negative. Most S. delphini and S. pseudintermedius isolates showed susceptibility to all antimicrobials tested, with the exception of 2 isolates resistant to tetracycline (tet(M) gene) or fusidic acid. The following toxin genes were identified (percentage of isolates): lukS-I (100%), lukF-I (9.5%), siet (100%), se-int (90%), seccanine (19%) and expA (9.5%). Thirteen different pulsed-field gel electrophoresis profiles were identified among the 21 SIG isolates. Additionally, the following 9 different sequence types (STs) were detected by multilocus sequence typing, 6 of them new: ST219 (6 isolates), ST12 (5 isolates), ST220 (3 isolates), ST13, ST50, ST193, ST196, ST218 and ST221 (one isolate each). CONCLUSIONS: Staphylococcus delphini and S. pseudintermedius are common nasal colonisers of donkeys, generally susceptible to the antimicrobials tested; nevertheless, these SIG isolates contain virulence genes, including the recently described exfoliative gene (expA) and several enterotoxin genes, with potential implications for public health. This is the first description of S. delphini in Tunisia. The Summary is available in Chinese - see Supporting information.

Heterogeneity among infecting strains of Pseudomonas aeruginosa in diverse departments of a large Tunisian hospital.

Clinical isolates of Pseudomonas aeruginosa were obtained during a half-year screening period of five different wards of the La Rabta Hospital (Tunis). Distinct clinical isolates (N= 82) were obtained from patients, 40 (48%) of which originated from the Department of Otolaryngology. In order to define the local epidemiology of this opportunistic organism, all strains were serotyped, analysed for pyocin production and genetically characterized with the help of pulsed-field gel electrophoresis (PFGE). The data show that, despite the frequent occurrence of identical serotypes, most of the isolates represent unique pyocin types (N= 53) and genotypes (N= 64). A combination of the pyocin and PFGE data showed that nearly all strains were of unique types, except for two pairs of strains. A limited number of strain clusters was observed on the basis of DNA typing data alone. This involved eight genotypes, some of which were clustered with respect to clinical environment or time. Genotype 22 occurred most frequently (6/83, 7%) and independently of time and locale, indicating that it may represent either a clonal type constituting a major fraction of all P. aeruginosa isolates in the region or a more prevalent organism. Despite a relatively high incidence of P. aeruginosa infections, the polyclonality of these strains shows that, in La Rabta Hospital, pseudomonal infections are not primarily due to excessive spread of a single bacterial genotype.

Impact of Heavy Metals on the Selective Phenotypical Markers of Pseudomonas aeruginosa.

The aim of this study was to characterize the impact of heavy metals on phenotypical markers of Pseudomonas aeruginosa. Twenty-two isolates of P. aeruginosa, either clinical (20) or secondary treated wastewater (2), were used to inoculate micro-ecosystems of sterile distilled water or secondary waste effluent in the presence of subminimal inhibitory concentrations of a variety of heavy metals commonly encountered in the aquatic naturally habitat (Ca2+, Co2+, Cr3+, Cu2+, Hg2+, Ni2+, Zn2+). Micro-ecosystems were exposed to visible light at laboratory temperature and individual strains were reisolated after a 1-, 3-, or 6-month period. The re-isolates (129) were characterized using hierarchical classification analysis in order to define affinities among variants of P. aeruginosa. Subsequently, discriminant analysis was used to detect eventual relationships among the different phenotypical markers studied. Results of the hierarchical classification, based on qualitative or quantitative approaches, showed clearly that incubation of P. aeruginosa in the presence of heavy metals altered the studied phenotypical markers, namely serotype, phage type, MIC of metals, and pyocin type. Discriminant analysis showed that the studied phenotypical markers could be classified into four clusters: C1 (L1 and L2 phage types, Hg tolerance and/or resistance, S2 serotype), C2 (P2 pyocin type, Cd tolerance and/or resistance, S1 serotype), C3 (Co and Cr tolerance and/or resistance) and C4 (P1 pyocin type, Ni, Zn, and Cu tolerance and/or resistance).