Clonal Spread of Colistin-Resistant Klebsiella pneumoniae Coproducing KPC and VIM Carbapenemases in Neonates at a Tunisian University Hospital.

In this study, we have attempted to report the first clonal spread of colistin-resistant Klebsiella pneumoniae coproducing KPC and VIM carbapenemases in the neonatal unit of Rabta Teaching Hospital of Tunis (Tunisia). This retrospective study was performed from January 1, 2014 to December 31, 2014 in the Microbiology Laboratory at the Rabta University Hospital of Tunis. Twenty-one nonreplicate colistin-resistant K. pneumoniae were isolated from 19 patients hospitalized in the neonatal unit and 2 patients in the adult intensive care unit (ICU). Most of the strains were isolated from invasive specimens. Pulsed-field gel electrophoresis (PFGE) and PCR analysis and nucleotide sequencing of the blaKPC and blaVIM genes were performed. Mortality was reported in 92% of cases. All the strains were resistant to colistin (minimum inhibitory concentration [MICs] ranged from 8 to 12 mg/L). The MICs for imipenem of K. pneumoniae isolates ranged from 3 to 256 mg/L for 13 strains that were characterized as intermediate or resistant. The MICs for ertapenem were higher than 32 mg/L for the 19 resistant strains. All the isolates were sensitive to tigecycline and chloramphenicol. PFGE analysis revealed two clones (I and II). Twenty of the 21 colistin-resistant, carbapenem-resistant K. pneumoniae isolates belonged to clone I. Only one strain was related to clone II. PCR analysis and nucleotide sequencing revealed that the 20 isolates belonged to clone I, coproduced the blaKPC and blaVIM genes. A single strain (clone II), which was isolated in the ICU, did not produce KPC and VIM carbapenemases. All strains did not produce OXA-48.

Multiple and mixed Helicobacter pylori infections: Comparison of two epidemiological situations in Tunisia and France.

Individuals can be infected by either a single or multiple strains of Helicobacter pylori. Multiple infection with genetically different isolates and particularly mixed infection with both antibiotic-susceptible and resistant isolates are difficult to detect and should impact the effectiveness of eradication treatment. It is largely assumed that multiple infections are more frequent in developing countries but an actual comparison developing/developed using a single methodology has never been reported. To compare the prevalence of multiple and mixed H. pylori infection in Tunisia and France, we conducted a prospective study including 42 H. pylori-culture positive infected patients (21 Tunisian and 21 French) never previously treated for H. pylori infection. One gastric biopsy was collected from antrum. Three to eleven (mean = 9) colonies were isolated from each biopsy. A total of 375 different isolates were genotyped using RAPD fingerprinting and antimicrobial susceptibility testing was performed on amoxicillin, clarithromycin, ciprofloxacin, rifampicin, tetracycline and metronidazole with E-tests. Multiple infection was defined by different RAPD fingerprintings among the different isolates from a single patient. Mixed infection was defined by different resistance profiles among the different isolates from a single patient. Multiple H. pylori infection is more prevalent in Tunisia than in France. It occurred in ten (48%) Tunisian patients and in one (5%) French patient (p < 0.001). Mixed infection is common (24%), it occurred in 4 (19%) Tunisian patients and in 6 (29%) French patients (p = 0.46) and was mainly (8/10) due to genetically related clones in single infection.

Are polymorphisms of the immunoregulatory factor CD40LG implicated in acute transfusion reactions?

The CD40 ligand (CD40L/CD154), a member of TNF superfamily, is notably expressed on activated CD4+ T-cells and stimulated platelets. CD40L is linked to a variety of pathologies and to acute transfusion reactions (ATR). Mutations in this gene (CD40LG) lead to X-linked hyper-IgM syndrome. Some CD40LG polymorphisms are associated with variable protein expression. The rationale behind this study is that CD40L protein has been observed to be involved in ATR. We wondered whether genetic polymorphisms are implicated. We investigated genetic diversity in the CD40LG using DHPLC and capillary electrophoresis for screening and genotyping (n = 485 French and Tunisian blood donors). We identified significant difference in the CD40LG linkage pattern between the two populations. Variant minor alleles were significantly over-represented in Tunisian donors (P<0.0001 to 0.0270). We found higher heterogeneity in the Tunisian, including three novel low frequency variants. As there was not a particular pattern of CD40LG in single apheresis donors whose platelet components induced an ATR, we discuss how this information may be useful for future disease association studies on CD40LG.

Genotypic and phenotypic characteristics of tunisian isoniazid-resistant Mycobacterium tuberculosis strains.

Forty three isoniazid (INH)-resistant Mycobacterium tuberculosis isolates were characterized on the basis of the most common INH associated mutations, katG315 and mabA -15C→T, and phenotypic properties (i.e. MIC of INH, resistance associated pattern, and catalase activity). Typing for resistance mutations was performed by Multiplex Allele-Specific PCR and sequencing reaction. Mutations at either codon were detected in 67.5% of isolates: katG315 in 37.2, mabA -15C→T in 27.9 and both of them in 2.4%, respectively. katG sequencing showed a G insertion at codon 325 detected in 2 strains and leading to amino acid change T326D which has not been previously reported. Distribution of each mutation, among the investigated strains, showed that katG S315T was associated with multiple-drug profile, high-level INH resistance and loss or decreased catalase activity; whereas the mabA -15C→T was more prevalent in mono-INH resistant isolates, but it was not only associated with a low-level INH resistance. It seems that determination of catalase activity aids in the detection of isolates for which MICs are high and could, in conjunction with molecular methods, provide rapid detection of most clinical INH-resistant strains.

Primary resistance to clarithromycin, metronidazole and amoxicillin of Helicobacter pylori isolated from Tunisian patients with peptic ulcers and gastritis: a prospective multicentre study.

BACKGROUND: The frequency of primary resistance to antibiotics in H. pylori isolates is increasing worldwide. In Tunisia, there are limited data regarding the pattern of H. pylori antibiotic primary resistance. AIM: To evaluate the primary resistance of H. pylori to clarithromycin, metronidazole and amoxicillin and to detect the mutations involved in clarithromycin resistance. MATERIALS AND METHODS: 273 strains isolated from adults and children were enrolled. The primary resistance to clarithromycin, metronidazole and amoxicillin was evaluated by means of E-test minimal inhibitory concentration (MIC). The real-time PCR using Scorpion primers was performed in all cases to assess clarithromycin primary resistance and point mutations involved. RESULTS: No resistance to amoxicillin was detected. For adults, resistance to clarithromycin and metronidazole was found respectively in 14.6% and 56.8%, and respectively in 18.8% and 25% in children. Overall, the rates of global primary resistance to clarithromycin and metronidazole in Tunisia were respectively determined in 15.4% and 51.3%.By the use of Scorpion PCR, the A2143G was the most frequent point mutation observed (88.1%), followed by the A2142G (11.9%); the A2142C was not found and 18 of 42 patients (42.8%) were infected by both the resistant and the susceptible genotype.The association of clarithromycin resistance with gender was not statistically significant, but metronidazole resistant strains were isolated more frequently in females (67.8%) than in males (32.2%) and the difference was significant. As for gastroduodenal diseases, the difference between strains isolated from patients with peptic ulceration and those with non peptic ulceration was not statistically significant. When about the distribution of resistant strains to clarithromycin and metronidazole between the three Tunisian cities (Tunis, Menzel Bourguiba and Mahdia), the difference was not statistically significant. CONCLUSION: Local data regarding the primary resistance of H. pylori to clarithromycin, metronidazole and amoxicillin and the main genetic mutation involved in clarithromycin resistance in vivo (A2143G) are necessary to prove a clear need for a periodic evaluation of antibiotic consumption and new therapeutic strategies in Tunisia in order to avoid the emergence of resistant strains.

Prevalence of Helicobacter pylori vacA, cagA, iceA and oipA genotypes in Tunisian patients.

BACKGROUND: Distinct virulence factors of H. pylori have been described: the vaculating cytotoxin (vacA), the cytotoxin associated gene (cagA), the induced by contact with epithelium factor Antigen (iceA gene) and the outer membrane protein oipA. In Tunisia, there are no data regarding the pattern of H. pylori genotypes; therefore, this prospective and multicentre study was the first to be done in Tunisia and aimed to investigate the prevalence of the vacA, cagA, iceA and oipA genotypes of H. pylori isolates from Tunisian patients with peptic ulceration, gastric cancer, MALT lymphoma and gastritis. METHODS: H. pylori was cultured from endoscopic biopsies obtained from 281 Tunisian patients. The vacA alleles, cagA, iceA and oipA genotypes were determined by PCR. RESULTS: The vacA s1m1, s1m2 and s2m2 were respectively found in 10.7%, 12.5% and 45.6% of strains. The s2m1 genotype was not detected in our study. The cagA was found in 61.6% of isolates. The iceA1 and the iceA2 genotypes were respectively isolated in 60.2% and in 16% of strains. The oipA genotype was detected in 90.8% of strains. Considering the vacA and iceA genotypes, the presence of multiple H. pylori strains in a single biopsy specimen was found respectively in 31.4% and 23.8%. The comparison between strains isolated from antrum and fundus showed that Tunisian patients were infected with two or more strains of different cagA, vacA, iceA and oipA genotypes and the discordance was respectively in 9.6%, 4.6%, 8.9% and 8.5% of strains. CONCLUSION: Our results showed that in 46% (131 strains among 281), the H. pylori strains were highly virulent in relation of the three or four virulent factors they could carry. These finding were described before in the literature. Tunisian patients were colonized by one or multiple strains of H. pylori in the same time in relation of presence of vacA m1/m2 and iceA1/iceA2 in the same biopsy. The discordance between strains isolated from antrum and fundus was high, and it is in favour of multicolonization.

Characterization of Tunisian Mycobacterium tuberculosis rifampin-resistant clinical isolates.

Analysis of the gene encoding the beta-subunit of Mycobacterium tuberculosis RNA polymerase (rpoB) has demonstrated a small region that harbors the mutations most frequently associated with rifampin resistance. In this study, we determined the occurrence of rifampin resistance in 544 Tunisian clinical M. tuberculosis strains isolated in a university hospital between 2004 and 2006 by using the standard-proportion agar method, the INNO-LiPA Rif.TB assay, and DNA sequencing.

Molecular epidemiology of an outbreak of multiresistant Klebsiella pneumoniae in a Tunisian neonatal ward.

During the first quarter of 1996, a major outbreak of clinical infection caused by multiresistant Klebsiella pneumoniae (MRKP) occurred in the neonatal ward of the 'Maternité Wassila Bourguiba' in Tunis, Tunisia. In total, 32 isolates of MRKP, comprising 23 clinical isolates and nine surveillance isolates, were recovered during this period and analysed for epidemiological relatedness. The isolates were compared with 17 other isolates of MRKP that were recovered during 1995. Macrorestriction profiles of total genomic DNA following XbaI restriction endonuclease digestion were analysed by PFGE; this typing classified 56% of the 32 isolates recovered in 1996 into two major clusters. Cluster A included ten isolates from 1996 and three isolates recovered in 1995, whereas cluster B included eight isolates from the outbreak of 1996. The remaining isolates were genetically unrelated to those of clusters A and B; they constituted sporadic strains. The two major clusters were also evident using other molecular typing methods, such as random amplification of polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC)-PCR, where isolates of clusters A and B could be identified on the basis of their discriminative patterns. This investigation showed the predominance of two epidemic strains, and illustrated the ease with which MRKP strains can disseminate and persist within a single ward.