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Methicillin-resistant Staphylococcus aureus (MRSA) is an important pathogen causing health care-infections in the world, especially in burns. The aim of this study was to assess the extent of dissemination of MRSA isolated from burn patients in Burn Intensive Care Unit in Tunisia and to evaluate the frequency of virulence and antibiotics resistance genes. Among the 72 S. aureus isolates analyzed in the study, 54% were MRSA. The majority of MRSA (94.8%) were multidrug resistant and they had a high resistance rates to kanamycin (94.8%), tobramycin (90%), tetracycline (94.8%) and ciprofloxacin and rifampicin (87%, each). The gene aac(6′)-Ie-aph(2″)-Ia conferring resistance to kanamycine and tobtamycin were detected in all isolates and the aph(3′)-Ia gene conferring resistance to gentamicin were detected in 2.8% of resistant isolates. Tetracycline resistance genes tet(M), tet(K) and tet(L) were detected in 100%, 10.8% and 2.8% of the isolates, respectively. The SCCmec type III and the agr type I were the most predominant (69.2% and 90%, respectively). The 27 SCCmecIII-agrI isolates were clustered into two PFGE types A and B. The two representative isolates of PFGE clusters A and B belonged to ST239-t037 and ST241-t037 respectively. As conclusion, our results showed a high prevalence of MRSA in trauma burn intensive care unit belonging to two multidrug resistant clones ST239/ST241-agrI-t037-SCCmecIII MRSA. We also demonstrated that MRSA was disseminated between burn patients.

Antimicrobial-resistant Escherichia coli isolates have emerged in various ecologic compartments and evolved to spread globally. We sought to (1.) investigate the occurrence of ESBL-producing E. coli (ESBL-Ec) in feces from free-range chickens in a rural region and (2.) characterize the genetic background of antimicrobial resistance and the genetic relatedness of collected isolates. Ninety-five feces swabs from free-range chickens associated with two households (House 1/House 2) in a rural region in northern Tunisia were collected. Samples were screened to recover ESBL-Ec, and collected isolates were characterized for phenotype/genotype of antimicrobial resistance, integrons, and molecular typing (pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST)). Overall, 47 ESBL-Ec were identified, with the following genes detected: 35 blaCTX-M-1, 5 blaCTX-M-55, 5 blaCTX-M-15, 1 blaSHV-2, and 1 blaSHV-12. Resistance to fluoroquinolones, tetracycline, sulfonamides, and colistin was encoded by aac(6′)-Ib-cr (n = 21), qnrB (n = 1), and qnrS (n = 2); tetA (n = 17)/tetB (n = 26); sul1 (n = 29)/sul2 (n = 18); and mcr-2 (n = 2) genes, respectively. PFGE and MLST identified genetic homogeneity of isolates in House 1; however, isolates from House 2 were heterogeneous. Notably, among nine identified sequence types, ST58, ST69, ST224, and ST410 belong to pandemic high-risk clonal lineages associated with extrapathogenic E. coli. Minor clones belonging to ST410 and ST471 were shared by chickens from both households. The virulence genes fyuA, fimH, papGIII, and iutA were detected in 35, 47, 17, and 23 isolates, respectively. Findings indicate a high occurrence of ESBL-Ec in free-range chickens and highlight the occurrence of pandemic zoonotic clones.

Tuberculous Spondylodiscitis is the most common form of musculoskeletal tuberculosis. Molecular techniques on fresh tissues are proved to improve the diagnosis of tuberculous spondylodiscitis and to allow a rapid diagnosis to initiate the treatment and prevent neurological complications.The objective of the present study was to assess the diagnostic performances of single tube nested PCR and GeneXpert ultra in the diagnosis of tuberculous spondylodiscitis on formalin fixed paraffin embedded tissues.This study included 63 tuberculous spondylodiscitis cases collected from June 2014 to January 2020 and corresponding to 27 definite tuberculous spondylodiscitis with positive microbiology, and 36 probable tuberculous spondylodiscitis, with histopathological, clinical and radiological findings consistent with tuberculous spondylodiscitis but with negative microbiology. The sensitivity, specificity, positive predictive value and negative predictive value of nested PCR and GeneXpert ultra were determined with reference to microbiology.Nested PCR was positive in 47 (75%) cases: 26/27 definite tuberculous spondylodiscitis and 21/36 probable tuberculous spondylodiscitis. GeneXpert ultra was positive in only 6 (10%) cases corresponding to definite tuberculous spondylodiscitis. The sensitivity, specificity, positive predictive value and negative predictive value of nested PCR on formalin fixed paraffin embedded tissues were 96%, 100%, 100%, 83% respectively. For GeneXpert ultra, these rates were 22%, 100%, 100% and 25% respectively.Nested PCR and GeneXpert ultra on formalin fixed paraffin embedded tissues are useful tools for the diagnosis of tuberculous spondylodiscitis, especially for cases where microbiological investigations were not carried out. Both techniques have excellent specificity but single tube nested PCR is more sensitive. Key Points • Molecular techniques are routinely performed on fresh tissues • GeneXpert and nested PCR on formalin fixed paraffin embedded tissues are reliable for the diagnosis of tuberculous spondylodiscitis • Nested PCR is more sensitive than Genexpert for diagnosing tuberculous spondylodiscitis.

Healthcare-associated infections (HAIs) are with high rates of mortality and an additional cost, in onco-hematology patients.To assess the prevalence trends of HAIs in the onco-hematology ward of the Tunisian National Bone Marrow Transplant Center (NBMTC), and to determine the principal associated risk factors.Six repeated point prevalence surveys were conducted, from May 2018 to March 2019, using a two months interval. All patients hospitalized in the day of the survey were included. Risk factors of HAIs were expressed as odds ratios (ORs) with 95% confidence intervals (CIs). They were assessed using a logistic regression model.Nineteen patients out of a total of 74 patients have been diagnosed with 19 HAIs, representing a prevalence of 25.7%. No significant downward or upward trend of prevalence was revealed over time (p=0.3). The most common HAI was respiratory tract infection (57.9%) with a prevalence of 14.9%. Multiple logistic regression analysis revealed that HAI was significantly associated with neutropenia (Adjusted OR: 14; 95% CI: 1.5-127; p=0.01) and duration of central venous catheter (Adjusted OR: 1.1; 95% CI: 1-1.2; p=0.005).High prevalence of HAIs in our center with a high rate of mortality, requiring identifying potential problems in infection control practices.

Microbial contamination of hematopoietic stem cells (HSC), used for autologous and allogenic transplantations, is rare but could cause serious blood stream infection in transplanted patients. These infections occur immediately, or later following the formation of biofilm on the catheter lumen. The present study describes an intermittent B. cepacia HSC contamination associated with nosocomial bacteremia: from October 2011 to April 2015, 17 B. cepacia strains were isolated in HSC bags ( n = 14) and blood cultures ( n = 3) in patients hospitalized in the National Bone Marrow Transplant Center. Two epidemiologic investigations in the National Blood Transfusion Center, allowing the isolation of three strains in hygiene samples, and four interventions in this institution were done. To identify the source of this contamination, a molecular investigation was done on 23 B. cepacia strains isolated in our center from 2007 to 2015. PFGE analysis revealed five clusters. The major cluster included 18 strains isolated from HSC bags ( n = 14), blood culture ( n = 1), and water cans and bath ( n = 3). The second cluster (B) including only two and the remaining clusters (C, D, and E) contained single strains isolated before the epidemic period. These findings confirmed that the origin of the outbreak was the contaminated water used in the water bath during the thawing step of HSC bags. Based on this result, new sterile water was used for every defrosting, but HSC bags contamination persisted. In May 2015, the water bath was replaced with a dry bath and no B. cepacia strain was isolated from that date to April 2020.

Objective: To report a series of Granulomatous Spondylodiscitis (GS) with focus on the histopathological features of the different forms of GS. Design: Case series. Setting: Pathology department of Charles Nicolle’s Hospital of Tunisia Participants: This study included 57 patients diagnosed with GS. There were 44 (77.2%) female patients and 13 (22.8%) male patients (sex ratio = 0.28). Intervention: Not applicable. Outcome measures: Clinical, microbiological and histopathological features were assessed in this study. Results: Fifty-seven patients with GS were enrolled: 51 tuberculous spondylodiscitis (TS), 2 fungal spondylodiscitis (FS), 3 brucellar spondylodiscitis (BS) and 1 case of sarcoidosis. Granulomas with necrosis were seen in 38 (66.6%) cases: 36 TS and 2 FS, while granulomas without necrosis were observed in the remaining 19 cases: 15 TS, 3 BS and 1 sarcoidosis. In all cases of TS, granulomas were epithelioid type, associated with histiocytic type granulomas in 7 cases. Caseous necrosis was seen in 35 cases of TS and suppurative granuloma in one case. The 3 cases of BS exhibited non-necrotizing and histiocytic type granulomas. The 2 cases of FS showed histiocytic, epithelioid and necrotizing granulomas. Necrosis was mixed: suppurative and caseous in both cases of FS. Sarcoidosis was characterized with epithelioid type granulomas without necrosis. Conclusion: Granuloma with caseous necrosis is highly suggestive of TS but does not rule out FS. Certain fungi can exhibit this type of necrosis as do tuberculosis species. Suppurative inflammation, although rare in TS, does exist. Histiocytic type granuloma without necrosis is suggestive of brucellosis.

Objectives: We conducted a survey of extended spectrum β-lactamase (ESBL) among infectious Escherichia coli and Klebsiella pneumoniae clinical isolates in the Bone Marrow Transplant Centre of Tunisia during a period of 10 years (from 2002 to 2011). Methods: Bacterial identification was carried on the basis of biochemical characteristics and API identification systems. Antibiotic susceptibility was tested by disc diffusion method on Muller-Hinton agar. Results: During the study period, 34.5% of 142 K. pneumoniae strains and 11.46% of 218 E. coli strains were ESBL producing. An increasing trend was observed for ESBL production in the selected bacteria. Conclusion: Screening for ESBL poduction among infectious K. pneumoniae and E. coli strains is paramount to guide the use of antimicrobial agents for the treatment of these pathogens in the hematopoietic stem cell transplant recipients population.

Bacteremia become fearsome in hematopoietic stem cell transplant (HSCT) recipients with the emergence of multidrug-resistant (MDR) strains.Our purpose was to investigate the prevalence of MDR bacteremia in HSCT recipients at the Tunisian National Bone Marrow Transplant Center, associated factors and attributable mortality rate.Our retrospective study (January 2010-December 2017) included all MDR bacteremia in the Hematology department. MDR rods were: extended spectrum beta-lactamase producing Enterobacterales (ESBL-E), P. aeruginosa and A. baumannii resistant to at least three families of antibiotics, methicillin-resistant S. aureus (MRSA) and vancomycin resistant E. faecium (VRE).The prevalence of MDR bacteremia among HSCT recipients was 5.9% (48/816) with a stable trend over time (rs=0.18). Neutropenia, prior hospitalization, prior antibiotherapy and prior colonization with MDR pathogens were observed in 59%, 58%, 48% and 31% of cases, respectively. Imipenem was the most prescribed first-line antibiotic (50%). The attributable mortality rate was 13%. MDR bacteria (n=48) belonged to ESBL-E (60%), P. aeruginosa (19%), A. baumannii (13%), MRSA (4%) and VRE (4%). For ESBL-E and P. aeruginosa, the rates of antibiotic resistance were respectively, 17% and 44% to imipenem, 31% and 56% to amikacin and 15% and 0% to colistin. Strains of A. baumannii were susceptible only to colistin. The MRSA (n=2) were resistant to ciprofloxacin and gentamicin and susceptible to glycopeptides. The VRE (n=2) were susceptible to linezolid and tigecycline.Low prevalence of MDR bacteremia in HSCT recipients but high attributable mortality rate, requiring reinforcement of hygiene measures.

As part of its pedagogical reform, the Faculty of Medicine of Tunis integrated since 1998 the objective clinical evaluation in multiple stations (ECOSM), as sanctioning evaluation at the end of the internships. However, this method has never been evaluated in nutrition.Carry out a docimological analysis of the ECOSM in nutrition intended for students of the 2nd year of the Second Cycle of Medical Studies (DCEM 2).This was a transversal descriptive prospective study on the ECOSM for sanctioning purposes, students of DCEM 2 having carried out an internship at the National Institute of Nutrition in Tunis during the academic year 2017- 2018.The general average of the ECOSM for the population studied was 13.92 ± 1.45 out of 20. Half of the stations were of acceptable difficulty. More than a third of the questions (37%) had good or excellent discrimination. For all the stations combined, the average Cronbach's alpha coefficient was 0.62, indicating an unacceptable internal homogeneity with great heterogeneity. Likewise, the Cronbach's alpha coefficients calculated for all stations and all questions were strictly less than 0.7. The flattening coefficient was negative, equal to -0.946, and the distribution curve was platykurtic.At the end of this work, we recommend making students and teachers particularly aware of the importance of evaluating the various objectives during the internship before the ECOSM test in order to fill in the gaps and ensure that the internship objectives are met.

Multi-station objective clinical evaluation (MSOCE) is an equitable and genuine formative and normative assessment tool that is deemed relevant to its effectiveness, reliability and validity in clinical evaluation. However, no experience with its application in pathological anatomy has been published.To carry out a critical analysis and a perceptual study of the MSOCE in pathological anatomy in medical students.This was a cross-sectional, multicenter prospective study in which we developed and tested a MSOCE on 32 medical students of the 1st year of the second cycle of medical studies. Docimological analysis of the 12 stations and the 28 questions of the different stations was done by the file "AnItem.xls".The mean total score obtained at the MSOCE was 36.2/50 (range : 29 to 41). The majority of stations were of acceptable difficulty (92%) and of poor discrimination (92%). The majority of questions were easy (57%) with poor discrimination (75% of the total questions). One question on 28 was "ideal", 4%. The MSOCE had a large heterogeneity (average Cronbach alpha of 0.29), and discriminated well between the average students (platykurtic, left-spread, negatively asymmetric distribution curve). The perception analysis showed that 27 students (84%) were satisfied with the OMSCE and thought that it was close to the reality of the clinical training.The OMSCE tested was easy and discriminated mostly between average students. It was well perceived by the students and close to the reality of the clinical training. Ongoing analysis will further improve the quality of MSOCE.

Little is known about the role of forestland and non-fertilized agriculture soils as reservoirs of extended-spectrum beta-lactamase (ESBL) and plasmid-borne AmpC (pAmpC)-producing Escherichia coli isolates. Thus, in the present study, 210 soil samples from various origins (forest of Oued Zen (Ain Drahem), non-agriculture soils from different park gardens in Tunis City, cereal culture soils and home gardens) were investigated to characterize cefotaxime-resistant E. coli isolates. A total of 22 ESBL/pAmpC-producing E. coli were collected, and all harbored variants of the blaCTX-M gene (15 blaCTX-M-1, 5 blaCTX-M-55 and 2 blaCTX-M-15). A total of seven and two isolates harbored also blaEBC and blaDHA-like genes, respectively. Resistances to tetracycline, sulfonamides and fluoroquinolones were encoded by tetA (n = 4)/tetB (n = 12), sul1 (n = 17)/sul2 (n = 19) and aac(6’)-Ib-cr (n = 2)/qnrA (n = 1)/qnrS (n = 1) genes, respectively. A total of seven isolates were able to transfer by conjugation cefotaxime-resistance in association or not with other resistance markers. PFGE showed that ten and two isolates were clonally related (pulsotypes P1 and P2). The 10 P1 isolates had been collected from forestland, cereal culture soils and an urban park garden in Tunis City, arguing for a large spread of clonal strains. Our findings highlight the occurrence of ESBL/pAmpC-E. coli isolates in soils under limited anthropogenic activities and the predominance of CTX-M enzymes that are largely disseminated in E. coli from humans and animals in Tunisia.

Brucellosis is a systemic infection caused by facultative intra-cellular bacteria of the Brucella genus that may involve multiple organs and tissues. The disease is a zoonosis of worldwide distribution, occurring in all continents, but is more frequent in many countries of the Middle East, the Mediterranean region, and Latin America. The spine is the most common site of musculoskeletal involvement, followed by the sacroiliac joints. This chapter aims to assess the clinical, biological, and imaging features of spinal brucellosis.

Percutaneous spine biopsy is an effective modality for the diagnosis and management of spinal infection when a microbiological diagnosis for a known associated organism has not been established by blood cultures or serological tests. It allows obtaining of specimens for microbiological and histopathological diagnosis. This safe and cost- and time-saving procedure is less painful and less invasive than open surgical biopsy. Several imaging-guidance methods are available, including fluoroscopy, computed tomography (CT), ultrasonography, and magnetic resonance imaging, but CT and fluoroscopy remain the most frequently used ones. A good knowledge of spinal anatomy, meticulous biopsy technique, and close collaboration among the interventional radiologist, pathologist, microbiologist, and clinician is essential to obtaining good results and avoiding complications.

The aim of this retrospective study was to investigate the resistance determinants and genetic relatedness of colistin-resistant Klebsiella pneumoniae (ColR-Kp) isolates in the National Bone Marrow Transplant Center of Tunis (NBMTC) between 2002 and 2013. Minimum inhibitory concentrations (MICs) for colistin of ColR-Kp isolates were assessed using a UMIC broth microdilution kit. All isolates were screened by PCR for the resistance genes mcr-1, blaCTX-M-1, blaSHV, blaTEM, blaOXA-1, blaOXA-48, blaKPC, blaIMP, blaVIM, blaNDM, blaGES, blaIMI and blaAmpC as well as class 1, 2 and 3 integrons. The clonality of ColR-Kp isolates was investigated by pulsed-field gel electrophoresis (PFGE). A total of 24 non-duplicate ColR-Kp isolates were collected with MICs ranging from 16 mg/L to >64 mg/L, representing 3.4% (24/709) of the total K. pneumoniae isolates. The colistin resistance gene mcr-1 was not found. The blaSHV gene was present in all of the isolates, blaTEM in 18 isolates, blaCTX-M-1 and blaOXA-1 in 15 isolates each and blaCMY-16 in two isolates. The blaOXA-48 gene was found in five carbapenem-resistant isolates and was associated with blaCTX-M-1, blaSHV, blaTEM and blaOXA-1. All isolates were negative for the remaining carbapenemases. Class 1 integron was found in 19 isolates. PFGE showed non-clonal spread of ColR-Kp isolates in this center. The increasing rate of colistin resistance in K. pneumoniae isolates in NBMTC was neither associated to clonal diffusion nor to the plasmid-mediated colistin resistance gene mcr-1. Its association with the use of colistin in total digestive decolonization in transplant patients at NBMTC must be investigated.

BACKGROUND Tuberculous spondylodiscitis (TS) is the most common form of musculoskeletal tuberculosis. Currently, histology is widely used to distinguish tuberculous from nontuberculous disease. OBJECTIVES The aim of the present study was to assess the accuracy of histology compared with bacteriology in the diagnosis of TS. METHODS This is a single-center case series carried out from January 2014 to February 2018 in a pathology department. It included 121 discovertebral biopsies of infective spondylodiscitis. The measures of diagnostic accuracy of histology were determined taking bacteriology as criterion standard. RESULTS Among the 121 cases, 55 (45.4%) were diagnosed as TS by histological and/or bacteriological findings, 17 (30.9%) were classified as definite TS by bacteriology, and the remaining 38 (69.1%) had positive histology and negative bacteriology. There were 2 false-negatives, which histologically displayed suppuration without granuloma, and 3 false-positives; in one case, histology displayed granulomas without necrosis and culture isolated Brucella. In the 2 others, histology revealed granulomas with caseous-like necrosis and microbiology isolated fungal species. The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of histology in the diagnosis of TS were 88.2%, 93.4%, 83.3%, 95.5%, and 92%, respectively. CONCLUSIONS Histology is proved to be an accurate diagnostic tool in TS. Suppurative forms of TS without granuloma are rare and represent the main cause of false-negative histology. Suggestive histology of TS does not rule out fungal and brucellar spondylodiscitis. Caseous necrosis is not pathognomonic of tuberculosis. Fungal infection can also exhibit such type of necrosis.

There is a lack of information concerning commensal Neisseria, as most studies focus on pathogenic Neisseria. To evaluate the use of Multilocus Sequence Typing (MLST) as a molecular identification tool, we determined the sequences of 700 bp fragments in seven housekeeping genes (abcZ, adk, aroE, fumC, gdh, pdhC and pgm) of 24 commensal Neisseria isolates collected from neutropenic patients in the Bone Marrow Transplant Center of Tunisia. Results were then compared to those obtained by conventional biochemical testing. In 79% (19/24), more than one possibility was given by MLST and in 46% (11/24), one of the possibilities offered by MLST, agreed with the result given by conventional biochemical testing.

Objectives This study investigated the prevalence of extended-spectrum β-lactamase-producing Klebsiella pneumoniae (ESBL-KP) strains in the National Bone Marrow Transplant Center of Tunis between 2002 and 2011 as well as their associated antimicrobial resistance patterns and molecular features. Methods Antimicrobial susceptibility was determined by the disk diffusion method according to CA-SFM guidelines. All of the strains were screened for β-lactamase genes, plasmid-encoded AmpC genes and integrons. Carbapenemase genes were analysed by PCR and sequencing for strains showing reduced susceptibility to ertapenem. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE) and multilocus sequencing typing (MLST). Results A total of 128 non-repetitive ESBL-KP strains (23.4%) responsible for infection or colonisation were recovered among 548 K. pneumoniae strains. The isolates were also multidrug-resistant. Molecular analysis revealed the prevalence of blaSHV-type (92.2%), followed by blaOXA-1 (81.3%) and blaCTX-M-1 group (73.4%). Four ertapenem-resistant ESBL-KP strains (3.1%) carried the blaOXA-48 gene associated with the blaCTX-M-15 gene. Class 1 integrons were the most prevalent among the isolates (85.2%). High diversity was demonstrated by PFGE with limited clonal dissemination of 1 major (n = 13 strains) and 11 minor clusters (each comprising 2–3 strains). MLST of representative strains also showed high diversity with two main epidemic clones: ST15, associated with the major cluster; and ST101, associated with five minor clusters (n = 11 strains). Conclusions This study provides relevant information on the epidemiology of ESBL-KP strains in oncohaematology patients, of which 18.8% belonged to the specific CTX-M–15 K. pneumoniae clones ST15 and ST101.

The aim of this study was to investigate the molecular features among Klebsiella pneumoniae and Escherichia coli strains showing a resistant/intermediate-resistant phenotype to ertapenem (R/IR-ERT), implicated in colonization/infection in patients of the Hematology and Graft Units of the National Bone Marrow Transplant Center of Tunisia (3-year period, 2011–2014). The major carbapenemase, extended-spectrum beta-lactamase, and plasmidic AmpC beta-lactamase genes were analyzed and characterized by PCR and sequencing. Genetic relatedness was determined by pulsed-field gel electrophoresis (PFGE) using XbaI and multilocus sequencing typing. The blaOXA-48 and blaKPC carbapenemase genes were detected among R/IR-ERT isolates. All R/IR-ERT K. pneumoniae strains (n = 19) had blaOXA-48 gene, and 14/19 strains also harbored the blaCTX-M-15 gene. Eight different PFGE patterns were detected among these K. pneumoniae isolates, and they showed eight different sequences types, ST11 and ST15 being the most prevalent ones. ...

Background Extended spectrum β-lactamase-producing Enterobacteriaceae (ESBL-E) create a therapeutic challenge and have high potential for dissemination. The purpose of our study was to investigate the epidemiology of these infections in hematopoietic stem cell transplant (HSCT) recipients and to determine the genes encoding ESBL. Material/methods This retrospective study comprised adult patients hospitalized at the National Bone Marrow Transplant Center (NBMTC) and infected with ESBL-E post-HSCT between January 2006 and December 2016. The search for the ESBL and carbapenemase genes was performed by polymerase chain reaction (PCR) amplification. Molecular typing was performed by pulsed field gel electrophoresis (PFGE) after digestion with XbaI. Results Forty ESBL-E were responsible for infections in 34 HSCT recipients (3.3% of total HSCT recipients). Prior hospital stay, prior antibiotic therapy and prior colonization with ESBL-E were reported in 62.5%, 70% and 50% of the infectious episodes, respectively. The initial antibiotic treatment was appropriate in 67.7% of cases. Imipenem was the most prescribed antibiotic (64.5%). The mortality rate due to ESBL-E infection was 8.8%. The ESBL-E, isolated mainly from blood cultures (40%), belonged mostly to K. pneumoniae (n=19) and E. coli (n=17). Associated antibiotic resistance rates were 17.5% for ertapenem, 85% for ciprofloxacin and 30% for amikacin. The predominant gene encoding ESBL was blaCTX-M (55%). Among the seven carbapenem-resistant strains, four had the blaOXA-48 gene and two the blaKPC gene. There was no clonal relationship between the strains. Conclusion There was low prevalence of ESBL-E infections in HSCT recipients in our center, with no epidemic distribution but non-negligible mortality rate.

We analyzed 85 Neisseria spp. strains collected by swabbing from neutropenic patients to determine the prevalence of reduced susceptibility to penicillin and to ascertain the clonal relationship between these strains. High genetic diversity and an elevated level of penicillin resistance were found among commensal Neisseria clinical isolates.

Identification of Streptococcus pneumoniae among other α-haemolytic streptococci is based on phenotypic or genotypic characteristics such as colony morphology, bile solubility and optochin susceptibility. This study reports three optochin-resistant S. pneumoniae strains isolated from immunocompromised patients in Tunisia. The three isolates were positive for the bile solubility test. Biochemical identification with API® 20 Strep was not discriminatory for two strains. The three strains had different serotypes (6C, 19F and 23F) and three different sequence types (ST386, ST320 and ST326). Sequencing of the atpA and atpC genes for each strain showed only modification in atpC. The mutations Met13→Val or Val48→Ile were observed in two strains. However, in the third strain a novel type of mutation (Val15→Ile) was identified.

Objectives: A retrospective study was conducted in the Bone Marrow Transplant Center of Tunisia during a period of 10 years (from 2002 to 2011) in order to report the prevalence of infectious multi-drug resistant bacteria. Methods: Bacterial identification was carried on the basis of biochemical characteristics and API identification systems. Antibiotic susceptibility was tested by disc diffusion method on Muller-Hinton agar. Results: During the study period, 34.5% of 142 Klebsiella pneumoniae strains and 11.46% of 218 Escherichia coli strains were extended-spectrum beta-lactamase (ESBL) producers. Also, 32.8% of 210 strains of Pseudomonas aeruginosa were imipenem and/or ceftazidime resistant and 20.75% of 106 strains of Staphylococcus aureus were methicillin resistant. A rising trend was observed for the prevalence of the selected multidrug resistant bacteria. Conclusion: These findings may have important clinical implications in prophylaxis and selection of antibiotic treatment. Continuous surveillance is needed, especially for onco-hematological patients. Keywords: Infectious multi-drug resistant bacteria, immunocompromised patients, Tunisia.

In the present work, nearly the entire 16S rRNA gene sequences of 46 clinical samples of Neisseria spp. were determined, and the aligned sequences were analyzed to investigate the diversity of 16S rRNA genes in each commensal Neisseria species. Two 16S rRNA types were identified in two Neisseria sicca strains, three 16S rRNA types in five Neisseria macacae strains, fourteen 16S rRNA types in twenty Neisseria flavescens isolates, and fourteen 16S rRNA types in nineteen Neisseria mucosa isolates. The number of nucleotides that were different between 16S rRNA sequences within specie ranged from 1 to 15. We found high intraspecific sequence variation in 16S rRNA genes of Neisseria spp. strains.

Background: An important observation during quantification experiments of Staphylococcus epidermidis biofilm is that there is a great difference in the biofilm biomass of different strains despite the same experimental conditions. Objectives: This study aimed to study the genotypic background beyond differential rates of Polysaccharide Intercellular Adhesion (PIA) production in S. epidermidis biofilm forming strains. Methods: A number of 126 strains were isolated from blood cultures (n = 40), catheter cultures (n = 50), and other specimens (n = 36). The strains were obtained from patients hospitalized at the bone marrow transplant center of Tunis. Biofilm micro-plate assay, hemagglutination, and susceptibility to proteinase K methods were used to assess biofilm characteristics in the studied strains. Conventional and real time PCR were used to assess genotypic background of biofilm formation. Results: Using PCR method, we demonstrated that there is a significant difference in ica genes (P < 0.01) and not in adhesion rsb and sar genes distribution between biofilm forming and non-biofilm forming strains. Almost all strains harbored agr type I. None of studied strains harbored IS256 inside ica operon. Ica-independent biofilm formation was detected in 11 strains that were confirmed to have proteinaceous matrix. Using Kernel density estimation, we established that biofilm biomass was higher in ica-dependent than ica-independent biofilm forming population. Using qRT-PCR, we found a significant correlation between biofilm biomass and RNAIII expression level (r2 = 0.95); but no correlation was found for biofilm biomass neither with icaA nor with ccpA genes. Conclusions: Data reported here indicated that there is no specific genetic combination beyond the quantity of biofilm biomass in S. epidermidis. Biofilm biomass seemed to be controlled by RNAIII expression level. Further interest should be directed to biofilm dispersal since it seems that the key difference in biofilm biomass ability of S. epidermidis strains relates to factors regulating this stage.

Introduction Item analysis is the process of collecting, summarizing and using information from students' responses to assess test items' quality. This study used this approach to evaluate the quality of items and examinations given in the Faculty of Medicine of Tunis (FMT). Methods This study concerned the examinations of 2012-2013 (principal session). It analyzed 3138 items from 66 examinations, of which, 46 were multidisciplinary (187 disciplines). A total of 2515 students took the examinations. "AnItem.xls" file was used for the analysis that focused on difficulty, discrimination and internal consistency. Results Mean difficulty for all examinations was optimum (mean difficulty index: 0.59). Majority of items (89.17%) were either easy or of acceptable difficulty. Mean discrimination for all examinations was moderate (mean item discrimination coefficient: 0.28) with poor discrimination in 23.62% of items. Maximal discrimination occurred with disciplines of difficulty index between 0.4-0.6. « Ideal » items represented 27.02%. Mean internal consistency for all examinations was acceptable (Cronbach's alpha: 0.79). Disciplines with nonacceptable internal consistency (68.45%) contained a maximum of 33 items (each one) and a positive correlation between their alpha and the number of their questions. Distributions were mostly (72.73%) platykurtic and negatively asymmetric (89.39%). First year of studies had the best parameters. Conclusion Our examinations had an acceptable internal consistency, and a good level of difficulty and discrimination. They tended to facility and discriminated basically students of medium level. Item analysis is useful as a guide to item writers to improve the overall quality of questions in the future.

Based on difficulty and discrimination indices and cognitive levels of Bloom, we assessed in this study the quality of Microbiology exam questions (main session 2012-2013, Faculty of Medicine of Tunis).We analyzed 70 questions: 16 (exam"A") given to 533 students (1st year), 28 and 26 (exams"B1" and "B2") given respectively to 285 and 292 students (3rd year). For every question, we determined difficulty and discrimination indices and the highest cognitive level required to resolve it. We calculated mean difficulty and discrimination indices for each exam and cognitive level, and mean indices of discrimination for every difficulty degree.The 70 questions were of optimum difficulty (0.58), good discrimination (0.31) and explored mainly (58.57%) the lowest cognitive level. For both years, mean indices of difficulty were acceptable, while those of discrimination were good (0.33) and marginal (0.27) for respectively 3rd and 1st year. "A" explored Lower Orders of Cognitive Skills (LOCS), "B2" both Lower and High Orders and "B1" all orders. Mean difficulty indices of every cognitive level were acceptable except for the median one (0.83). Mean discrimination indices were good for all cognitive levels except for LOCS of the 1st year (0.27). Mean indices of discrimination were marginal (0.29) for difficult questions and good for others. Compared to B2, B1 was more attainable and discriminative, free of poor discrimination questions, and explored all cognitive orders.Our study remains specific to particular questions and generalizations seem difficult. However, it can serve as a guideline to other similar studies.

Staphylococcus epidermidis is a leading cause of hospital-acquired infections, mostly associated with the use of medical devices in immunocompromised patients. It originates from the patient's own skin flora, which is subject to severe changes as a result of selective pressure exerted by the hospital environment. This notion led us to compare S. epidermidis isolates from catheter related infections (CRI), non-catheter related bacteremia (NCRB) and catheter hub cultures (commensal isolates). The collection comprised 47 CRI strains from the Bone Marrow Transplant Centre of Tunis, 25 NCRB strains and 25 commensal isolates from patients hospitalized in the same center. Antimicrobial resistance and virulence-associated genes (icaABC, aap, atlE, bhp, fbe, embp, and IS256), polysaccharide intercellular adhesin synthesis, and biofilm formation were investigated. The clonal relationship of strains was investigated by pulsed field gel electrophoresis. Whereas bhp, atlE, fbe, embp, and aap were almost ubiquitously amplified, resistance to oxacillin, kanamycin, tobramycin, gentamicin, cotrimoxazole, and fosfomycin, biofilm production, ica genes, and IS256 were significantly more frequent in invasive (CRI and NCRB strains) than in commensal strains. Moreover, strong biofilm production was significantly more frequent among CRI strains than in NCRB strains. In conclusion, when S. epidermidis is isolated from blood cultures, the detection of strong biofilm production may be significant with regard to judging whether the detected strain is an etiologic agent of CRI.

To investigate the prevalence of resistance to macrolide, lincosamide and streptogramin (MLS) antibiotics in Gram-positive cocci isolated in a Bone Marrow Transplant Center of Tunisia, we tested the antibiotic susceptibility of 172 clinical isolates of Staphylococcus epidermidis, Streptococcus mitis and Enterococcus faecium to macrolide erythromycin and spiramycin, the lincosamide clindamycin and the streptogramin pristinamycin. These three groups of organisms were mostly resistant to macrolides and lincosamide, but were commonly susceptible to pristinamycin. The resistance phenotypes of erythromycin-resistant isolates were determined by the five-disc test with erythromycin, spiramycin, lincomycin, clindamycin and pristinamycin, which showed that most exhibited constitutive MLS resistance. In order to determine the prevalence of the resistance genotypes and the resistance mechanisms, the prevalence of the erythromycin resistance methylase (erm) (A), erm(B), erm(C), msr(A) and macrolide efflux (mef) (A) genes in the erythromycin-resistant isolates was identified by polymerase chain reaction (PCR) analysis. The resistance was due mainly to the presence of ermB in E. faecium (80%), ermC in S. epidermidis (53%) and mefA in S. mitis (65%).

This study was undertaken to determine the species distribution and antibiotic resistance patterns of coagulase-negative Staphylococci (CoNS) other than Staphylococcus epidermidis. A total of 142 CoNS (except S. epidermidis) strains were isolated from a variety of clinical specimens in neutropenic patients at the Bone Marrow Transplant Centre of Tunisia between 2002 and 2004. All CoNS isolates were further identified by Api ID32 STAPH and ITS-PCR and antibiotic sensitivity was performed by disc diffusion method. Staphylococcus haemolyticus was the commonest species (38%) followed by Staphylococcus hominis (36%). All isolates were sensitive to vancomycin and 8 (6%) strains showed a reduced sensitivity to teicoplanin. Resistance to penicillin G and methicillin was 84 and 60%, respectively. Methicillin-resistant CoNS strains were determined to be more resistant to antibiotics than methicillin-susceptible CoNS strains. The mecA gene was detected by PCR in 65% (92/142) CoNS isolates. Out of 92 mecA-positive isolates, 90 were phenotypically methicillin-resistant and two were methicillin-susceptible. Phylogenetic analysis, carried out to study the evolution of mecA genes between different Staphylococcal species, revealed a high homology for such genes among Staphylococci.

Characterisation of extended-spectrum beta-lactamase (ESBL) genes and their genetic environments as well as the presence of integrons were analysed in nine Klebsiella pneumoniae and two Escherichia coli ESBL-positive isolates recovered in the Centre of Bone Marrow Transplantation of Tunisia. All strains harboured the bla(CTX-M-15) gene and presented minimum inhibitory concentrations for cefotaxime and ceftazidime of 256-1024 mg L(-1) and 16-512 mg L(-1), respectively, and eight of them showed different pulsed-field gel electrophoresis patterns. The bla(OXA-1) and bla(TEM-1) genes were detected in eight and ten strains, respectively. In addition, bla(SHV-1), bla(SHV-11) and bla(SHV-27) were found in six, one and one K. pneumoniae strains, respectively. The new variant bla(SHV-103) was characterised in one K. pneumoniae strain. The intI1 gene was detected in eight K. pneumoniae strains and the dfrA5+ereA2 and aadA gene cassettes were found in one and five strains, respectively. All strains harboured a 70 kb plasmid, and its transference in addition to bla(CTX-M-15), bla(TEM-1b) and bla(OXA-1) genes was demonstrated from three K. pneumoniae to E. coli. ISEcp1 and orf477 were located upstream and downstream, respectively, of the bla(CTX-M-15) gene in 10 strains. The occurrence of the bla(CTX-M-15) gene in unrelated strains might have originated from the dissemination of mobile genetic elements in which ISEcp1 may have played an important role.

Thirty methicillin-resistant Staphylococcus epidermidis strains isolated from catheters and blood cultures from neutropenic patients were studied. They were classified into 17 multidrug-resistance patterns. Polymerase cahin reaction analysis revealed that methicillin resistance was encoded by the mecA gene in all strains, and aminoglycosides resistance was due to aac(6')-Ie-aph(2'')-Ia (23 strains), ant(4')-Ia (13), and aph(3')-IIIa (1) genes. The aac(6')-Ie-aph(2'')-Ia gene was detected concomitantly with aph(3')-IIIa, and ant(4')-Ia genes in one and nine strains, respectively. Erythromycin resistance was encoded by the ermC (11 strains), ermA (6), and msrA (2) genes. The ermC gene was inducibly expressed in five strains, whereas the ermA was exclusively constitutively expressed. The icaA and icaC genes were detected in 19 strains; however, biofilm production was observed in only 16 strains. Most strains harbored multiple plasmids of variable sizes ranging from 2.2 to 70 kb, and two strains were plasmid-free. PFGE identified 15 distinct PFGE types, and five predominant genotypes were found. Our study showed the occurrence of complex genetic phenomenons. In unrelated strains, evidence of horizontal transfer of antibiotic-encoding genes and/or ica operon, and in indistinguishable strains, there is a quite good likelihood of independent steps of loss and/or gain of these genes. This genome dynamicity might have enhanced the invasiveness power of these methicillin-resistant S epidermidis strains.

Escherchia coli is the most common etiological agent of urinary tract infections. In this study we had two goals: First of all, to find out if urine stains isolated from our patients--having the particularity of being immunocompromised--would have a virulence genes distribution different from the one observed in strains isolated from ordinary patients. Second, we wanted to identify a common virulence profile associated to these particular strains. The prevalence of virulence factors (VF)-encoding genes was analyzed by PCR. Of the tested VF-encoding genes, malX (80%), ompT (79%), fyuA (74%), usp (67%), chuA (66%), iroN (59%), iutA (56%), papC (36%), pap AH (30%), papEF (28%), hlyA (28%), papG allele II (25%), cnf1 (21%), focG (20%),cvaC (20%) and papG allele III (7%) were significantly associated to urinary strains. Virulence genes distribution of urinary strains isolated from onco-hematology patients and the one observed in strains isolated from ordinary patients are almost the same. The virulence profiles containing adhesins type 1, S and F1C fimbriae, siderophore genes and three individual genes ompT, usp and malX were present in half of the urinary strains and were significantly associated to them. Two virulence signatures occurred significantly in UTI-causing strains (12%). These findings provide first insight into the virulence of UTI-causing E. coli strains isolated in onco-hematology patients.