Lipopeptides from Bacillus amyloliquefaciens strain 32a as promising biocontrol compounds against the plant pathogen Agrobacterium tumefaciens.

Despite the potential biological importance of lipopeptides from Bacillus amyloliquefaciens as antimicrobial compounds, their effects on Agrobacterium tumefaciens biofilms have not been previously studied. These latter are important virulence factors for the development and re-occurrence of crown gall disease. As part of the development of a new biopesticide acting as anti-biofilm and biocontrol agent, we investigated for the first time the ability of a mixture of lipopeptides produced by B. amyloliquefaciens strain 32a to inhibit the tumor formation on plants and to reduce the formation of biofilms by the phytopathogenic A. tumefaciens strains C58 and B6. The mixture was found to display a strong biosurfactant activity as well as bactericidal activity against planktonic Agrobacterium cells. Moreover, the lipopeptide treatment inhibited biofilm formation in the range of 79.58 ± 0.60-100.00 ± 0.00% and dislodged 43.42 ± 0.91-93.89 ± 2.70% of preformed biofilm. For these assays, fluorescence microscopy did not show any adherent cell in the anti-adhesive assay and only few ones in the cell-dislodging assay. More importantly, lipopeptide-enriched extract inhibits tumor formation on tomato stem when treatments were applied after pathogen adhesion to wounded tissues. By virtue of its ability to inhibit biofilms formed on biotic and abiotic surfaces and to control efficiently tumor development, the 32a lipopeptide mixture may represent an excellent new tool for an efficient biocontrol of crown gall disease.

A broad-range PCR technique for the diagnosis of infective endocarditis

Abstract Infective endocarditis (IE) remains a severe and potentially fatal disease demanding sophisticated diagnostic strategies for detection of the causative microorganisms. The aim of the present study was to develop a broad-range 16S ribosomal RNA gene polymerase chain reaction in the routine diagnostic of IE for the early diagnosis of fatal disease. A broad-range PCR technique was selected and evaluated in terms of its efficiency in the diagnosis of endocarditis using 19 heart valves from patients undergoing cardiovascular surgeries at the Habib Bourguiba Hospital of Sfax, Tunisia, on the grounds of suspected IE. The results demonstrated the efficiency of this technique particularly in cases involving a limited number of bacteria since it helped to increase detection sensitivity. The technique proved to be efficient, particularly, in the bacteriological diagnosis of IE in contexts involving negative results from conventional culture methods and other contexts involving bacterial species that were not amenable to identification by phenotypic investigations. Indeed, the sequencing of the partial 16S ribosomal RNA gene revealed the presence of Bartonella henselae, Enterobacter sp., and Streptococcus pyogenes in three heart valves with the negative culture. It should be noted that the results obtained from the polymerase chain reaction-sequencing identification applied to the heart valve and the strain isolated from the same tissue were not consistent with the ones found by the conventional microbiological methods in the case of IE caused by Gemella morbillorum. In fact, the results from the molecular identification revealed the presence of Lactobacillus jensenii. Overall, the results have revealed that the proposed method is sensitive, reliable and might open promising opportunities for the early diagnosis of IE.

A broad-range PCR technique for the diagnosis of infective endocarditis.

Infective endocarditis (IE) remains a severe and potentially fatal disease demanding sophisticated diagnostic strategies for detection of the causative microorganisms. The aim of the present study was to develop a broad-range 16S ribosomal RNA gene polymerase chain reaction in the routine diagnostic of IE for the early diagnosis of fatal disease. A broad-range PCR technique was selected and evaluated in terms of its efficiency in the diagnosis of endocarditis using 19 heart valves from patients undergoing cardiovascular surgeries at the Habib Bourguiba Hospital of Sfax, Tunisia, on the grounds of suspected IE. The results demonstrated the efficiency of this technique particularly in cases involving a limited number of bacteria since it helped to increase detection sensitivity. The technique proved to be efficient, particularly, in the bacteriological diagnosis of IE in contexts involving negative results from conventional culture methods and other contexts involving bacterial species that were not amenable to identification by phenotypic investigations. Indeed, the sequencing of the partial 16S ribosomal RNA gene revealed the presence of Bartonella henselae, Enterobacter sp., and Streptococcus pyogenes in three heart valves with the negative culture. It should be noted that the results obtained from the polymerase chain reaction-sequencing identification applied to the heart valve and the strain isolated from the same tissue were not consistent with the ones found by the conventional microbiological methods in the case of IE caused by Gemella morbillorum. In fact, the results from the molecular identification revealed the presence of Lactobacillus jensenii. Overall, the results have revealed that the proposed method is sensitive, reliable and might open promising opportunities for the early diagnosis of IE.

Multicentric study in five African countries of antibiotic susceptibility for three main pathogens: Streptococcus pneumoniae, Staphylococcus aureus, and Pseudomonas aeruginosa.

Antibiotic resistance is a growing clinical and epidemiological problem. We report on the antibiotic susceptibility of three pathogens isolated from patients in Algeria, Egypt, Morocco, Senegal, and Tunisia during 2010-2011. In total, 218 Streptococcus pneumoniae, 428 Staphylococcus aureus, and 414 Pseudomonas aeruginosa strains were collected. S. pneumoniae resistance was noted against penicillin (30.2%), erythromycin (27.4%), cefpodoxime (19.1%), amoxicillin (12.0%), cefotaxime (7.4%), and levofloxacin (3.2%). All the strains were teicoplanin susceptible. Staphylococcus aureus methicillin resistance differed between countries, from 5.0% in Senegal to 62.7% in Egypt. Levofloxacin resistance was low in all countries, and the highest rate (in Egypt) was still only 13.6% for intermediate and resistant strains combined. Most strains were susceptible to fosfomycin (99.3%) and pristinamycin (94.2%). P. aeruginosa resistance was found against levofloxacin (30.4%), ciprofloxacin (29.9%), tobramycin (19.7%), ceftazidime (19.2%), and imipenem (17.9%), but not colistin. Antibiotic susceptibility varied widely between countries, with resistance typically most prevalent in Egypt.

Spinal brucellosis in South of Tunisia: review of 32 cases.

BACKGROUND CONTEXT: Brucellosis remains an important economic and public health problem in some parts of the world. The spine is the most common site of musculoskeletal involvement of brucellosis. PURPOSE: Assess the clinical, laboratory, radiological findings, and outcomes of vertebral involvement in brucellosis. STUDY DESIGN: A retrospective study. PATIENT SAMPLE: Thirty-two patients with spinal brucellosis during a period of 21 years (1990-2010) were included. OUTCOME MEASURES: Clinical and radiological improvement. METHODS: Diagnosis made on clinical presentation, laboratory findings, radiographic evidence, and the Brucellar etiology was considered when seroagglutination tests were positive at a titer of 1/160 or higher, and/or Brucella spp were isolated in the blood or sample cultures. RESULTS: The mean age of patients was 51±15.85 years (23 males, 9 females; age range, 19-74 years). The median diagnostic delay was 3 months. Back or neck pain (100% of patients), fever (78%), and sweats (68.6%) were the most common symptoms. Cultures of blood specimens from five patients (15.6%) were positive for Brucella melitensis. Four patients (12.5%) had motor weakness or paralysis. Magnetic resonance imaging was performed in 24 (75%) cases. Paravertebral masses, epidural masses, and psoas abscesses were detected in 65.6%, 59.4%, and 28.1% of patients, respectively. The lumbar vertebra was the most frequently involved region with the rate of 68.7%, followed by thoracal (18.7%), cervical (6.3%), lumbosacral (6.3%), and thoracolumbar (3.1%) segments. The duration of antimicrobial therapy of brucellosis (median, 6 months; range, 3-13 months) varied according to clinical response and the presence of epidural and paravertebral masses. There were no deaths or severe sequelae in this study. CONCLUSIONS: Brucellar spondylitis should be considered in patients with back pain and fever in endemic areas. A high index of suspicion and clinical, laboratory, and radiological examinations help to confirm the diagnosis of vertebral involvement.

Nosocomial dissemination of Providencia stuartii isolates carrying bla OXA-48, bla PER-1, bla CMY-4 and qnrA6 in a Tunisian hospital.

OBJECTIVES: To report an outbreak due to Providencia stuartii isolates carrying bla(OXA-48), bla(PER-1), bla(CMY-4) and qnrA6 in a Tunisian hospital in 2011. METHODS: Eight intensive care unit (ICU) patients infected/colonized by extended-spectrum ß-lactamase (ESBL)-producing P. stuartii between March and May 2011 were included. Molecular epidemiology was studied by PFGE. Antibiotic resistance genes were analysed by PCR and sequencing and the plasmid incompatibility group by a PCR-based replicon typing scheme. RESULTS: Eight patients were colonized with ESBL-producing P. stuartii isolates. All these isolates were clonally related and found to carry bla(OXA-48), bla(PER-1), bla(CMY-4), qnrA6 and aac-6'-Ib genes on the same self-conjugative IncA/C plasmid. The same strain was also cultured from environmental samples in the ICU. All these isolates were susceptible to carbapenems. Only one colonized patient developed P. stuartii pleurisy and was effectively treated with imipenem alone. CONCLUSIONS: This is the first report of an outbreak due to P. stuartii isolates carrying bla(OXA-48) in Tunisia. The simultaneous expression of various resistance genes (bla(OXA-48), bla(CMY-4), bla(PER-1), qnrA and aac-6'-Ib) by P. stuartii isolates is alarming.

Antibacterial, anti-chlamydial, and cytotoxic activities of a marine snail (Hexaplex trunculus) phospholipase A2: an in vitro study.

In order to report pharmacological characterization of marine snail (Hexaplex trunculus) hepatopancreatic phospholipase A(2) (mSDPLA(2)), we have talked for the first time the antimicrobial activity against different pathogenic bacterial strains, anti-chlamydial activity as well as its cytotoxic activity against McCoy cell lines. mSDPLA(2), showed a high level of activity towards Gram-positive bacteria as Staphylococcus aureus and Staphylococcus epidermidis. Whereas Gram-negative bacteria, unfortunately, exhibited a higher resistance, mSDPLA(2) was also found to have a strong cytotoxic activity, causing significant morphological alterations of the McCoy cell lines surfaces and to be a hinder to the proliferation. Moreover, mSDPLA(2) proved to have a very potent anti-chlamydial activity. Over 95 % inhibition of chlamydial inclusions were obtained at a concentration of 10 µg/ml of mSDPLA(2) after 24 h postinfection. Interestingly, at a concentration of 10 µg/ml of mSDPLA(2), the proliferation of McCoy cells was not affected. Approximately 50 % inhibition of cell growth was obtained with a concentration of 37 µg/mL of mSDPLA(2). mSDPLA(2) could be considered as an excellent candidate for the development of a new anti-infective agent. This enzyme showed significant antimicrobial activities.

A proposed mouse model to study male infertility provoked by genital serovar E, Chlamydia trachomatis.

Chlamydia trachomatis is a common sexually transmitted pathogen. The impact of chlamydial infection on male infertility is controversial. The aim of this study was to assess the role of C trachomatis human genital serovar E on sperm function, induction of apoptosis in spermatozoa, and reproductive performance, using the Swiss male mice model. Fertile mice were inoculated in the meatus urethra with 10(6) C trachomatis inclusion-forming units at day 0. The studied parameters were evaluated 7, 15, 21, and 30 days postinoculation (pi) in infected and sham-infected controls. Semen parameters of the infected mice groups were significantly lower than those of the control groups at the different days pi. DNA fragmentation study indicated that the mean percentages of apoptotic spermatozoa in the infected mice groups were significantly higher than those in the control groups 7 and 15 days pi, whereas the mean percentages of necrotic spermatozoa in the infected mice groups were significantly higher than those in the control group on the 30th day pi. A decrease in reproductive performance was observed at different days pi in infected male mice groups when compared to the control groups. Furthermore, a statistically significant decrease in the mean number of infant mice was observed at 21 and 30 days pi. In conclusion, our data showed that inoculation of fertile male Swiss mice in the meatus urethra with C trachomatis could lead to alteration of semen parameters, induction of apoptosis in spermatozoa, and decrease of the reproductive performance of male mice.

Diversity of ß-lactamases in Pseudomonas aeruginosa isolates producing metallo-ß-lactamase in two Tunisian hospitals.

This study was conducted to identify the ß-lactamase content of 30 metallo-ß-lactamase-producing Pseudomonas aeruginosa isolated in 2007 from two Tunisian hospitals and to investigate their genetic relatedness. All these isolates produced VIM-2. bla(PER-1), bla(PSE-1), bla(OXA-2), and bla(OXA-10) were identified in 17, 5, 21, and 1 isolates, respectively. These enzymes were often associated in the same isolate: 26 isolates had at least two ß-lactamases. The predominant serotype was O12. Pulsed-field gel electrophoresis revealed genetic diversity among the metallo-ß-lactamase-producing P. aeruginosa isolates. This is the first report on the existence of bla(PER-1), bla(PSE-1), bla(OXA-2), and bla(OXA-10) in Tunisia.

[Investigation of three nosocomial outbreaks of Serratia marcescens in an intensive care unit in Sfax-Tunisia]. / Investigation de trois épidémies d'infections nosocomiales à Serratia marcescens survenues dans le service de réanimation-CHU de Sfax, Tunisie.

UNLABELLED: THE AIM of the study was to type Serratia marcescens responsible for nosocomial outbreaks in an intensive care unit in Sfax -Tunisia. METHODS: The relatedness between S. marcescens isolates was studied by Pulsed field gel electrophoresis (PFGE). We included 56 strains of Serratia marcescens isolated from patients hospitalized in the intensive care unit during 2003 and 2004. Seven epidemiological unrelated strains of Serratia marcescens were also tested. Samples from environment and hands of the nursing and medical staff were collected and cultured to identify the source of contamination. RESULTS: All strains showed a wild type of antimicrobial susceptibility. PFGE typing revealed that three different clones were present. None of the cultures taken from hands of unit staff and from environmental samples yielded positive results for S. marcescens. CONCLUSION: We have confirmed the presence of three consecutive outbreaks caused by three genetically unrelated bacterial clones of Serratia marcescens in the intensive care unit ward. These outbreaks are closely related to the frequent use of colistin and the lack of measures of hygiene in this ward.

Molecular epidemiology and genetic environment of acquired bla ACC-1 in Salmonella enterica serotype Livingstone causing a large nosocomial outbreak in Tunisia.

Eighty-four isolates of Salmonella enterica serovar Livingstone were collected from patients hospitalized in a pediatric ward in Sfax Hospital (South Tunisia). These isolates were responsible for two nosocomial outbreaks in 2000 and 2002. Twenty-eight clinical isolates of S. enterica serovar Livingstone were also obtained in two other Tunisian hospitals in Monastir (Central Tunisia) and Tunis (North Tunisia), respectively, in 2002 and 2003. Pulsed-field gel electrophoresis yielded that these isolates were closely related. Antimicrobial susceptibility testing showed a particular beta-lactam resistance phenotype, suggestive of the presence of an AmpC-type enzyme in 111 of the 112 clinical isolates. bla(ACC-1) was characterized by polymerase chain reaction (PCR) and sequence analysis in the 111 isolates. TEM-1 was characterized in all strains and SHV-2a in only two strains. The genetic organization of bla(ACC-1) was determined by PCR mapping and sequencing. The plasmid-borne bla(ACC-1) gene mapped immediately downstream from ISEcp1. This ISEcp1 insertion sequence was itself disrupted by IS26 insertion sequences. A supplementary deletion of 13 bp was observed in ISEcp1 upstream IS26, in all isolates from Tunis, except one. PCR analysis and sequencing also revealed the presence of tnpR, bla(SCO-1), gdha, IS1353, and TniB Delta 1.

Broad-range PCR, cloning and sequencing of the full 16S rRNA gene for detection of bacterial DNA in synovial fluid samples of Tunisian patients with reactive and undifferentiated arthritis.

INTRODUCTION: Broad-range rDNA PCR provides an alternative, cultivation-independent approach for identifying bacterial DNA in reactive and other form of arthritis. The aim of this study was to use broad-range rDNA PCR targeting the 16S rRNA gene in patients with reactive and other forms of arthritis and to screen for the presence of DNA from any given bacterial species in synovial fluid (SF) samples. METHODS: We examined the SF samples from a total of 27 patients consisting of patients with reactive arthritis (ReA) (n = 5), undifferentiated arthritis (UA) (n = 9), rheumatoid arthritis (n = 7), and osteoarthritis (n = 6) of which the latter two were used as controls. Using broad-range bacterial PCR amplifying a 1400 bp fragment from the 16S rRNA gene, we identified and sequenced at least 24 clones from each SF sample. To identify the corresponding bacteria, DNA sequences were compared to the EMBL (European Molecular Biology Laboratory) database. RESULTS: Bacterial DNA was identified in 20 of the 27 SF samples (74, 10%). Analysis of a large number of sequences revealed the presence of DNA from more than one single bacterial species in the SF of all patients studied. The nearly complete sequences of the 1400 bp were obtained for most of the detected species. DNA of bacterial species including Shigella species, Escherichia species, and other coli-form bacteria as well as opportunistic pathogens such as Stenotrophomonas maltophilia and Achromobacter xylosoxidans were shared in all arthritis patients. Among pathogens described to trigger ReA, DNA from Shigella sonnei was found in ReA and UA patients. We also detected DNA from rarely occurring human pathogens such as Aranicola species and Pantoea ananatis. We also found DNA from bacteria so far not described in human infections such as Bacillus niacini, Paenibacillus humicus, Diaphorobacter species and uncultured bacterium genera incertae sedis OP10. CONCLUSIONS: Broad-range PCR followed by cloning and sequencing the entire 16S rDNA, allowed the identification of the bacterial DNA environment in the SF samples of arthritic patients. We found a wide spectrum of bacteria including those known to be involved in ReA and others not previously associated with arthritis.

IgA antibodies against the Epstein-Barr nuclear antigen1 as a valuable biomarker for the diagnosis of nasopharyngeal carcinoma in Tunisian patients.

Serological tests for Epstein-Barr virus (EBV) have been used for many years as diagnostic predictors of nasopharyngeal carcinoma. It has been shown previously that the conventional immunofluorescence assay has a limited diagnostic value, especially in young patients from North African area. In the search for more reliable immunoglobulin (Ig) G or IgA antibody markers for the diagnosis of nasopharyngeal carcinoma, immunoblot analysis was performed using a full spectrum of EBV proteins. Sera were collected from 108 patients with nasopharyngeal carcinoma and three control groups composed of 18 patients with lymphoma, 18 other patients with autoimmune diseases and 55 healthy EBV carriers. It was observed that the IgA Epstein-Barr nuclear antigen 1 (EBNA1), IgA early antigen (EA)-p138 and IgG EA-p138 antibodies represent the most specific anti-EBV responses in either young or older patients with nasopharyngeal carcinoma which yield higher positive rates compared to the three control groups. Since the IgA EBNA1 response showed the highest sensitivity value for the detection of nasopharyngeal carcinoma, a novel enzyme-linked immunosorbent assay (ELISA) was established using a GST-EBNA1 protein expressed in bacteria, containing the P-threonine EBNA1 subtype cloned from DNA EBV sequence of C15 xenograft cells. Detection rates were 85.7% and 94.9% in young and older patients with nasopharyngeal carcinoma respectively, while only 3.6%, 11.1%, and 16.6% in healthy EBV carriers, patients with lymphoma and patients with autoimmune diseases, respectively. Thus, IgA EBNA1 ELISA may be useful for early diagnosis and mass screening of nasopharyngeal carcinoma in Tunisia even in young patients.

Short- and long-term outcomes of surgery for active infective endocarditis: a Tunisian experience.

From January 1997 to December 2006, all patients with a Duke criteria-based definite diagnosis of infective endocarditis (IE) operated on during the active phase in a Tunisian high volume tertiary-care centre were included. Among the 186 patients with IE identified during the study period, 88 (48.35%) required surgery in the active phase. Mean age was 34.9 years, 54 (61.4%) were men. The infected valve was native in 70 cases (79.5%) and prosthetic in 18 (20.5%). Streptococcus sp. were the most common causative microorganisms. The most frequent indication for operation was congestive heart failure. There were 24 in-hospital deaths (27.27% early mortality). By multivariate analysis, severe congestive heart failure (HR=13.82, 95% CI [3.38-38.15], P<0.001) and large >15 mm vegetations (HR=6.02, 95% CI [1.48-18.52], P=0.03) were predictive of in-hospital mortality. Survivors were followed-up from 3 to 120 months, mean of 28.6. Actuarial 5- and 10-year survivals free from the combined endpoint of recurrent IE, cardiovascular death and late surgery in survivors were 69+/-5% and 63+/-7%, respectively. In conclusion, despite medical progress, surgery for endocarditis in Tunisia remains challenging and yields high mortality rates. Severe heart failure is the most powerful predictor of mortality. Long-term outcome is, however, satisfactory.

Aberrant methylation of p16, DLEC1, BLU and E-cadherin gene promoters in nasopharyngeal carcinoma biopsies from Tunisian patients.

BACKGROUND: Aberrant methylation of tumor suppressor gene (TSG) promoters has been extensively investigated in nasopharyngeal carcinomas (NPC) from South East Asia but not from North Africa. PATIENTS AND METHODS: The methylation status of p16, deleted in lung and esophageal cancer (DLEC1), zinc finger, MYND-type containing 10 (BLU) and E-cadherin gene promoters was investigated in 44 Tunisian NPC biopsies and three NPC xenografts, by methylation-specific PCR (MSP) combined with a quantitative assessment of some of the samples. RESULTS: The frequencies of aberrant promoter methylation were similar to previous figures reported for Asian series: p16 27/44 (65%), DLEC1 38/44 (86.3%), BLU 15/44 (34.1%) and E-cadherin 35/44 (79.5%). Although in other malignancies, aberrant promoter hypermethylation increases with patient age, it was at the same high frequency in the juvenile and adult forms of Tunisian NPCs. However, there was a strong association between aberrant methylation of E-cadherin promoter and lymph node invasion (p < 0.01). In addition, aberrant methylation of the BLU promoter was significantly correlated with an undifferentiated histological type (p = 0.03). CONCLUSION: Aberrant methylation of tumor suppressor genes occurs with the same high frequency in NPCs from North Africa as in South East Asia, regardless of patient age.

Analysis of bacterial DNA in synovial tissue of Tunisian patients with reactive and undifferentiated arthritis by broad-range PCR, cloning and sequencing.

INTRODUCTION: Bacteria and/or their antigens have been implicated in the pathogenesis of reactive arthritis (ReA). Several studies have reported the presence of bacterial antigens and nucleic acids of bacteria other than those specified by diagnostic criteria for ReA in joint specimens from patients with ReA and various arthritides. The present study was conducted to detect any bacterial DNA and identify bacterial species that are present in the synovial tissue of Tunisian patients with reactive arthritis and undifferentiated arthritis (UA) using PCR, cloning and sequencing. METHODS: We examined synovial tissue samples from 28 patients: six patients with ReA and nine with UA, and a control group consisting of seven patients with rheumatoid arthritis and six with osteoarthritis (OA). Using broad-range bacterial PCR producing a 1,400-base-pair fragment from the 16S rRNA gene, at least 24 clones were sequenced for each synovial tissue sample. To identify the corresponding bacteria, DNA sequences were compared with sequences from the EMBL (European Molecular Biology Laboratory) database. RESULTS: Bacterial DNA was detected in 75% of the 28 synovial tissue samples. DNA from 68 various bacterial species were found in ReA and UA samples, whereas DNA from 12 bacteria were detected in control group samples. Most of the bacterial DNAs detected were from skin or intestinal bacteria. DNA from bacteria known to trigger ReA, such as Shigella flexneri and Shigella sonnei, were detected in ReA and UA samples of synovial tissue and not in control samples. DNA from various bacterial species detected in this study have not previously been found in synovial samples. CONCLUSION: This study is the first to use broad-range PCR targeting the full 16S rRNA gene for detection of bacterial DNA in synovial tissue. We detected DNA from a wide spectrum of bacterial species, including those known to be involved in ReA and others not previously associated with ReA or related arthritis. The pathogenic significance of some of these intrasynovial bacterial DNAs remains unclear.

Ureaplasma urealyticum, Ureaplasma parvum, Mycoplasma hominis and Mycoplasma genitalium infections and semen quality of infertile men.

BACKGROUND: Genital ureaplasmas (Ureaplasma urealyticum and Ureaplasma parvum) and mycoplasmas (Mycoplasma genitalium and Mycoplasma hominis) are potentially pathogenic species playing an etiologic role in both genital infections and male infertility. Reports are, however, controversial regarding the effects of these microorganisms infections in the sperm seminological variables. This study aimed at determining the frequency of genital ureplasmas and mycoplasmas in semen specimens collected from infertile men, and at comparing the seminological variables of semen from infected and non-infected men with these microorganisms. METHODS: A total of 120 semen samples collected from infertile men were investigated. Semen specimens were examined by in-house PCR-microtiter plate hybridization assay for the presence of genital ureaplasmas and mycoplasmas DNA. Semen analysis was assessed according to the guidelines of the World Health Organization. Standard parametric techniques (t-tests) and nonparametric techniques (Wilcoxon tests) were used for statistical analysis. RESULTS: The frequency of genital ureaplasmas and mycoplasmas detected in semen samples of infertile men was respectively 19.2% (23/120) and 15.8% (19/120). The frequency of Ureaplasma urealyticum (15%) was higher than that of Mycoplasma hominis (10.8%), Ureaplasma parvum (4.2%) and Mycoplasma genitalium (5%). Mixed species of mycoplasmas and ureaplasmas were detected in 6.7% of semen samples. Comparison of the parameters of the standard semen analysis between the male partners of the infertile couples with and without genital ureaplasmas and mycoplasmas infection showed that the presence of Mycoplasma hominis DNA in semen samples is associated with low sperm concentration (p = 0.007) and abnormal sperm morphology (p = 0.03) and a negative correlation between sperm concentration and the detection of Mycoplasma genitalium in semen samples of infertile men (p = 0.05). The mean values of seminal volume, pH, vitality, motility and leukocyte count were not significantly related either to the detection of genital mycoplasmas DNA or to the detection of ureaplasmas DNA in semen specimens. CONCLUSION: Our results demonstrate that genital mycoplasmas and ureaplasmas seem to be widespread among the male partners of infertile couples in Tunisia. Genital mycoplasmas infections of the male genital tract could negatively influence semen quality. Our results also indicate that PCR-microtiter plate hybridization assay method provides a rapid and effective technique to detect human genital mycoplasmas and ureaplasmas which is useful for etiological and epidemiological studies of these pathogens.

Polymorphism analysis of Epstein-Barr virus isolates of nasopharyngeal carcinoma biopsies from Tunisian patients.

Many studies suggest that the focal distribution of nasopharyngeal carcinoma (NPC) may be influenced not only by host genetics, diet and environments but also by interplay with Epstein-Barr virus (EBV) genetics. Specific EBV gene variants (the A and C types, the BamHI f configuration, a C terminal 30 bp deletion and a N terminal loss of an XhoI site in the BNLF1 gene) have been explored in high incidence areas in southern Asian NPC patients. In contrast, in Tunisia where NPC represents the most frequent type of Head and Neck cancer the distribution of these polymorphisms remains poorly investigated. In order to characterize the epidemiology of EBV variants in Tunisian NPC patients, we have investigated the A or B type of the EBV nuclear antigen (EBNA)2 gene, the C or D type of the BamHI W1/I1 region, the F/f variants of the BamHI F region and the presence or the absence of the XhoI site, 30 bp deletion and Taq1 site in the BNLF1 gene in 47 NPC biopsies, 12 being younger than 30 and 35 older than 30. Our results show a unique genetic profile of the tumor EBV strains regarding the A and D types, the prototype F and retention of the XhoI restriction site in the N terminal region of BNLF1 gene. With regard to the C terminal region of this gene, four genetic profiles were detected: (1) the occurrence of the 30 bp deletion in association with the Taq1 site in 39 cases (83%), (2) the presence of the Taq1 site by itself in 5 cases, (3) the occurrence of the 30 bp deletion by itself in 2 cases and (4) the occurrence of a new deletion of 81 bp covering the 30 bp deletion in association with the Taq1 site in one case. With the exception of the 81 bp deletion, which has not been previously described in the literature, the summarized results have shown the same genetic profile in Tunisian NPC tumor isolates as tumor isolates from other North African and Mediterranean countries. Hence, the observed EBV polymorphisms are not fully specific of to the Tunisian NPCs. Nevertheless, the notion of a divergence between North African and Asian tumor EBV isolates is reinforced by this study.

Emergence of multidrug-resistant Klebsiella pneumoniae isolates producing VIM-4 metallo-beta-lactamase, CTX-M-15 extended-spectrum beta-lactamase, and CMY-4 AmpC beta-lactamase in a Tunisian university hospital.

Klebsiella pneumoniae clinical isolates resistant to carbapenems were recovered from 11 patients in the hospital of Sfax, Tunisia. The isolates were closely related as shown by pulsed-field gel electrophoresis, and they produced VIM-4 metallo-enzyme, CTX-M-15 extended-spectrum beta-lactamase, and CMY-4 AmpC enzyme. The bla(VIM-4) gene is part of a class 1 integron.