Search

Objectives: To describe clinical and molecular characteristics of an outbreak due to metallo-β-lactamases (MBLs) producing Klebsiella pneumoniae collected at Charles Nicolle Hospital of Tunis and to analyze the impact of outer membrane porin (OMP) loss on carbapenem resistance levels., Methods: Between 2010 and 2015, 178 carbapenem-resistant Enterobacteriaceae were isolated. Screening for MBL production was performed using combined disk diffusion method, with imipenem and ethylene diamine tetraacetic acid (EDTA) as inhibitors. Resistance genes and virulence factors were identified by polymerase chain reaction (PCR) and sequencing. Genotyping was performed by pulsed-field gel electrophoresis and multilocus sequence typing. Genetic environment of carbapenemase genes was determined by PCR mapping. Conjugation assays were performed, and plasmids were assigned to incompatibility groups by PCR-based replicon typing. OMPs were profiled by sodium dodecyl sulfate-polyacrilamide gel electrophoresis, and porin genes were sequenced., Results: Nineteen K. pneumoniae (10.6%) showing MBL activity were isolated from patients hospitalized on four different wards. NDM-1 was the only MBL identified, in association with bla OXA-48 . All strains lacked at least one OMP, and carbapenem resistance levels were remarkably elevated in strains lacking OmpK35 and OmpK36. bla NDM-1 was located in IncFIA-type conjugative plasmid, with the same genetic context in all strains. The epidemiological diffusion of bla NDM-1 was due to two clones, one major clone belonging to sequence type (ST) 147 (n = 16) and the other clone belonging to ST307 (n = 3)., Conclusions: This study describes an outbreak of NDM-1-producing K. pneumoniae strains, isolated from a Tunisian hospital, caused by two clones belonging to ST147 and ST307; and highlights the role of OMPs loss, in combination with β-lactamase expression, in conferring high carbapenem resistance.

Purpose: Since their emergence at the beginning of the century, OXA-48 carbapenemases have spread in the community and in hospitals. To assess the diversity of OXA-48-producing bacterial strains and plasmids in the hospital setting, we studied the strains isolated from patients in three hospitals in the Paris area., Materials and Methods: All possible OXA-48-like strains were included in the study. OXA-48-like and extended-spectrum beta-lactamase-encoding genes were identified, and fingerprinting analysis was performed for all Escherichia coli and Klebsiella pneumoniae strains. The backbones and close genetic environments of bla OXA-48 were assessed by amplifying genes that were specific to the pOXA-48a plasmid and PCR, encompassing the junctions between bla OXA-48 and its direct genetic environment., Results: Overall, 68 strains from 30 patients were studied. These strains belonged to seven different enterobacterial species. OXA-48, OXA-204, and OXA-401 were identified in 62, 3, and 3 isolates, respectively. Additional broad-spectrum beta-lactamases were identified in 34% (23/68) of the strains. The strain diversity was high between and within patients. Identical patterns were observed only within individual patients or among epidemiologically related patients. Plasmid mapping was performed in the 62 OXA-48-producing strains and the 3 OXA-405-producing strains, resulting in the identification of 5 different patterns., Conclusion: Because of their ability to transfer between strains, OXA-48 carbapenemases have a high risk of dissemination and may become endemic in France.

Six multidrug-resistant Klebsiella pneumoniae isolates were recovered from injured Libyan combatants. Production of carbapenemase was screened by using commercial combination tablets from Rosco combined with a temocillin disk. Polymerase chain reaction (PCR) and sequencing were used to detect several carbapenemase genes and to characterize their genetic environment. Genetic support was studied by mating-out assays. Plasmid size was identified by the KADO method. PCR and sequencing allowed characterization of plasmid scaffold. Genotyping was performed by pulse-field gel electrophoresis (PFGE) and multilocus sequence typing. PCR was used to check for the presence of nine genes linked to virulence in K. pneumoniae. No carbapenemase was identified by Rosco disks, but all isolates showed high-level temocillin resistance. All of them harbored blaOXA-48 in the transposon Tn1999.2, on a self-conjugative plasmid of about 60 kb, similar to pOXA-48. PFGE revealed three clusters in which isolates were genetically related: The first comprised FM9 and FM10, and the second comprised FM1, FM4, and FM5. FM2 formed a third distinct clone. Sequence types ST101, ST11, and ST147 were identified in keeping with PFGE results. The entB, ycfM, ybtS, and mrkD genes were detected in all isolates, and kfu gene was present in the three ST101 strains. This work confirms the current and successful spread of blaOXA-48 by horizontal dissemination of a single IncL/M plasmid through different genetic backbones with strong epidemic potential. It also highlights the need for rapid and reliable phenotypic detection methods. Attempts to link virulence factors and the production of this carbapenemase deserve further studies.

Seventy-one isolates of Enterobacteriaceae (17 Escherichia coli, 50 Klebsiella pneumoniae, and 4 Enterobacter cloacae) producing extended-spectrum-β-lactamases (ESBLs) were collected between April 2008 and March 2010 in an intensive care unit and surgical ward of Tlemcen Hospital (West of Algeria). Sequencing identified the bla(CTX-M-15) determinant in 69 isolates and bla(CTX-M-3) in 2 isolates. None of the studied strains produced the class D carbapenemase OXA-48. Repetitive Extragenic palindromic polymerase chain reaction showed a high degree of genotypic diversity among E. coli strains and two major clonal populations of K. pneumoniae (CKp1 n=11 and CKp5 n=25) which were further identified as members of the multilocus sequence typing types (ST931) and (ST15), respectively. The ST15 isolates harbored more resistance genes and virulence factors than the ST931 isolates. The characterization of the spacer region between ISEcp1 and bla(CTX-M) for CTX-M-15 producers individualized two populations. One that derived from the CTX-M-3 under Algerian clinical context and one that is universally found. The dissemination of ESBLs in the studied Enterobacteriaceae isolates was mainly due to the epidemic clones of K. pneumoniae and to genetic transit of plasmids among unrelated strains.

This study was conducted to identify the molecular mechanisms of imipenem resistance in a Klebsiella pneumoniae (Kp16137) isolate recovered in August 2008 at the University Hospital Sahloul, Sousse, Tunisia. The strain was identified with the API 20E system; antibiotic-containing disks were used for detection of antibiotic susceptibility by a disk diffusion assay. We investigated the presence of β-lactamases by PCR, using specific primers for bla(TEM), bla(SHV), bla(CTX-M), bla(OXA), bla(CMY), bla(ACC), bla(FOX), bla(IMP), bla(KPC), bla(VIM), and by sequencing. Extraction of plasmid DNA from Kp16137 and the transconjugant was performed by the method of Kado. Southern transfer was performed on nylon. The membrane was hybridized with a specific probe for the bla(CMY-2) gene. Outer membrane proteins were isolated and were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 12% polyacrylamide gel. K. pneumoniae Kp16137 was resistant to all available β-lactams, including third generation cephalosporins and carbapenems. The screening of β-lactamases showed the presence of three β-lactamases: TEM-1, SHV-61, and CMY-4. The CMY-4 β-lactamase was located on an 80-kb plasmid. An analysis of the outer membrane proteins of this isolate revealed that it lacked a porin of 42 kDa. The loss of this outer membrane protein band correlated with imipenem resistance in this strain. In K. pneumoniae 16137, synthesis of a plasmid-mediated β-lactamase: AmpC CMY-4, together with alteration in permeability led to resistance to all available β-lactams and carbapenems.

Two mutants of Escherichia coli (V1 and V2) with acquired mechanisms of resistance to fluoroquinolones were isolated sequentially from blood cultures of a patient with cholangiocarcinoma treated repeatedly with ofloxacin; a third mutant (V3) was isolated under ciprofloxacin therapy. All mutants were related clonally. V1 was susceptible to quinolones but with diminished susceptibility to ofloxacin. V2 was hypersusceptible to nalidixic acid but had high-level resistance to ofloxacin. V3 was resistant to all quinolones. Ofloxacin selected for original gyrA and parC mutations, leading to the unusual and misleading resistance phenotypes of V1 and V2, whereas efflux played a major role in the increased resistance of V3.

One hundred extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae were recovered from the intensive care unit and the urology ward of the University Hospital of Sahloul in Tunisia between May 2005 and May 2006. The majority of strains showed a high level of resistance to cefotaxime and ceftazidime. Double-disk synergy test and E-test strips were used to confirm production of ESBLs. The molecular analysis revealed that the majority of strains (91%) carried genes encoding CTX-M-15. SHV-12 and SHV-2a were produced, respectively, by 9% and 3% of the strains. Pulsed-field gel electrophoresis of ESBL-producing Klebsiella pneumoniae isolates revealed four different clonal groups and three for Escherichia coli, showing the absence of spread of any epidemic clone. The CTX-M-15 ESBL-producing E. coli of the major clonal group belong to the B2 phylogenetic group, to the sequence type 131, and has a high virulence potential. In conclusion, CTX-M-15 ESBLs accounted for the overwhelming majority of ESBL types among Enterobacteriaceae from our hospital. This study confirms the high rate of ESBLs in Tunisia and further demonstrates the worldwide spread of genes coding for CTX-M-15 enzymes in clinical isolates.

The aim of this study was to describe the distribution of the trimethoprim-sulfamethoxazole resistance genes and their association with class 1 integrons in a collection of clinical isolates of Enterobacteriaceae recovered at the University Hospital Sahloul in Tunisia. A total of 80 isolates of Enterobacteriaceae were studied, including six different species. There were 35 extended-spectrum beta-lactamases (ESBL)-producing isolates. Resistance to trimethoprim-sulfamethoxazole was assessed by the disk diffusion method. Polymerase chain reaction (PCR) with primers specific for sul1, sul2, and sul3 was used to detect the three known sulphonamide resistance genes. The presence of class 1 integrons in the studied isolates was detected using PCR and the resistance gene cassettes were characterized by directly sequencing the PCR products obtained with 5'conserved segment (5'CS) and 3'conserved segment (3'CS) primers. The int1 gene was found in 68 out of 80 enterobacterial isolates. The sul1 gene was found in 22 isolates (27.5%), sul2 gene in 5 isolates (6.25%), and both genes in 49 isolates (61.25%). Eight of the studied isolates had no dfr alleles, and in the remaining 72 isolates, 7 dfr genes were identified. The most prevalent were dfrA7 (40%) and dfrA17 (33%). Class 1 integrons were found to be an important genetic element of resistance to trimethoprim-sulfamethoxazole among the clinical isolates of Enterobacteriaceae. The types, combinations, and frequency of the gene cassettes in integrons provide useful data for the surveillance of antimicrobial resistance in our hospital and for the prescription practice of cotrimoxazole.

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.