Therapeutic potential of second degree's skin burns by topical dressing of Teucrium ramosissimum that promotes re-epithelialization.

The aim of the report is to assess the protective effect of powder aerial part of Teucrium ramosissimum (TS) on the in vivo wound-healing of second-degree burn injuries. Teucrium phytocompounds were characterized by FTIR, HPLC, and GC/MS spectra. Burn wound models were employed to evaluate the in vivo wound-healing activity. Thirty six wistar rats with burn wounds were divided into six groups and treated daily with TS, the mixture of Teucrium and honey (TS-HY), thymol and Dermosalic® (0.05%) (DS) creams. Skin epithelialization was monitored on the 4th, 13th, and 21st days. Proteins and the level of malondialdehyde in the burned skin were assessed. Microscopic and macroscopic investigations of skin wound tissues showed significant wound closure rate via complete epidermal reepithelization and regeneration, higher protein content, collagen synthesis and deposition, hair follicles growth post wounding that were promoted in TS-, thymol-, TS-HY- and DS-treated wound tissues compared to the untreated burned wound tissues that was characterized by the absence of the epithelialization, vascularization and the formation of the epidermis layer. Additionally, the skin healing potential of TS and TS-HY was validated by markedly decreased of lipid peroxidation. Overall, TS was found to possess complete wound closure and improves the healing process.

Detection of AmpC and ESBL-producing Enterobacterales isolated from urinary tract infections in Tunisia.

Urinary tract infections (UTIs) are the most frequent human infections in community and hospitals. This study aimed to determine the distribution of bacterial uropathogens among urinary tract infections diagnosed within the regional hospital Houcine Bouzaiene (Gafsa, South West Tunisia) during a survey of 54 days from the 8th of November to the 31st of December 2017. Enterobacterales strains were tested for antimicrobial resistance by disk diffusion method and extended-spectrum ß-lactamase (ESBL) production was tested by double-disc synergy test. Strains were further subjected to a molecular assessment of ESBL and AmpC ß-lactamase production by PCR. Overall, 173 bacterial isolates were studied, out of which 91.3% were Enterobacterales. Escherichia coli was the dominant pathogen, followed by Klebsiella pneumoniae. High to moderate resistance rates were observed, ranging from 66% to 90.7% for penicillins, from 6.7% to 18.6% for cephalosporins and from 16.2% to 25.4% for fluoroquinolones. Enterobacterales with decreased susceptibility to third-generation cephalosporins (3rd GC) carried several resistance genes: blaCTX-M group 1 and group 9, and ACC and FOX AmpC ß-lactamase genes. Overall, ESBLs and AmpC ß-lactamases were detected in 57% and 14% of the 3rd GC-resistant isolates, respectively. This study proved the high potential of K. pneumaniae species to develop resistance against commonly used antibiotics. Thus, rigorous monitoring of the antibiotic resistance of clinical pathogens have to be implemented in Tunisia. Our results are very relevant to evaluate efficiency of the Tunisian therapeutic strategies against UTIs and adapt them to the emerging problem of antimicrobial resistance.

Escherichia coli colonizing healthy children in Tunisia: High prevalence of extra-intestinal pathovar and occurrence of non-extended-spectrum-ß-lactamase-producing ST131 clone.

This study was performed to investigate the distribution of antimicrobial resistance genes and extra-intestinal virulence determinants in a collection of 98 Escherichia coli strains isolated from rectal swabs of healthy children. Forty-six isolated strains were resistant to at least one of the tested antibiotics (usually active against enterobacteria). They were mainly resistant to ampicillin and ticarcillin (42.97%), tetracyclin (26.5%), and trimethoprim/sulfamethoxazole (18.4%). No resistance to the third generation of cephalosporins, carbapenems, aminoglycosides and colistin was found. Resistance to penicillins was encoded by blaTEM-1 (n=34) and blaSHV-1 genes (n=4). Tetracyclin resistance was encoded by tetB (n=12), tetA (n= 5), and tetC (n=1) genes. Amongst resistant quinolones isolated (n=5), chromosomal mutations in gyrA and parC genes were detected in four isolates and qnrS1 gene in two strains. Nine plasmid replicon types were detected; IncFIB (n=36) and IncI1 (n=7) were the most frequent ones. Isolates frequently belonged to phylogenetic groups A (51.1%) and D (27.5%). Extra-intestinal pathovar (n=38) occurred mainly in B2 phylogroup (P=0.0002). Amongst them, two isolates (non-extended-spectrum-ß-lactamase (ESBL)-producers) belonged to the pandemic clone ST131. A significant distribution of virulence determinants and pathogenicity island marker was observed within strains belonging to B2 and D phylogroups. Interestingly, our results showed that ExPEC strains, including ST131 pandemic clone, are present within fecal isolates in healthy children. These findings highlight the importance of intestinal microbiota as a reservoir for virulent and resistant strains. Thus, reinforcing hand hygiene and antibiotic rational use is imperative to avoid the diffusion of these pathogens in the community.

First report of extensively-drug-resistant Proteus mirabilis isolate carrying plasmid-mediated blaNDM-1 in a Tunisian intensive care unit.

Emergence of the New Delhi metallo-ß-lactamase (NDM-1), an Ambler class B metallo-ß-lactamase able to hydrolyse all ß-lactams except monobactams, constitutes a critical and increasingly important medical issue. The acquisition of blaNDM-1 is of particular concern for Proteus mirabilis, which is intrinsically resistant to tetracycline, tigecycline and colistin, as this will make clinical treatment extremely difficult. To the authors' knowledge, this is the first report of the blaNDM-1 gene in an extensively-drug-resistant P. mirabilis clinical isolate carrying plasmid-mediated resistance to carbapenems (blaNDM-1), cephalosporins (blaCMY-4), aminoglycosides (aph3 VIa and aph3 Ia) and fluoroquinolones (qnrA6).

Role of association of OmpK35 and OmpK36 alteration and blaESBL and/or blaAmpC genes in conferring carbapenem resistance among non-carbapenemase-producing Klebsiella pneumoniae.

In Klebsiella pneumoniae, loss of the two major outer membrane porins (OMPs) OmpK35 and OmpK36 confers resistance to carbapenems in strains producing extended-spectrum ß-lactamases (ESBLs) or plasmid-mediated AmpC-type ß-lactamases. This study investigated mechanisms responsible for carbapenem resistance in non-carbapenemase-producing K. pneumoniae (NCPK). All carbapenem-resistant Enterobacteriaceae (CRE) at Charles Nicolle Hospital (Tunis, Tunisia) were collected over a 6-year period (2010-2015). Among the 334 CRE strains collected, 44 (13.2%) were NCPK. MIC ranges for ertapenem, imipenem and meropenem were 1 to >32 mg/L, 0.125-8 mg/L and 0.125-32 mg/L, respectively. All strains showed a multidrug-resistant (MDR) phenotype and were negative for carbapenemase activity. None of the carbapenemase genes searched for were found. ESBL production was confirmed in all isolates except one [CTX-M-15 (n = 39) and SHV-5 (n = 4)]. Three isolates produce DHA-1 (associated with CTX-M-15 in two strains). Molecular fingerprints grouped the 44 NCPK isolates into seven clusters. In seven representative strains of these clusters, SDS-PAGE results showed that four isolates lacked the OmpK35 porin, one isolate lacked OmpK36 and two isolates lacked both OmpK35 and OmpK36. Sequencing of the corresponding porin genes showed amino acid insertions and deletions leading to early termination of translation, point mutations in the promoter region, or insertion sequences disrupting the gene coding sequence. Loss or deficiency of OMPs, coupled with ESBL and/or AmpC production, plays an important role in conferring carbapenem resistance in K. pneumoniae. Dissemination of these MDR bacteria in our hospital may create serious therapeutic problems in the future.

NDM-1- and OXA-23-producing Acinetobacter baumannii isolated from intensive care unit patients in Tunisia.

Gastrointestinal colonisation by carbapenem-resistant Acinetobacter baumannii (CRAB) is a critical step before nosocomial infection. This study evaluated CRAB intestinal carriage in patients admitted to a Tunisian ICU and determined the antimicrobial resistance mechanisms involved. From December 2014 to February 2015, all 63 patients admitted to the ICU were screened for rectal CRAB colonisation upon admission and once weekly thereafter. ICU patients who acquired a CRAB nosocomial infection were also included. ß-Lactamases and associated resistance genes were screened by PCR sequencing, and molecular typing was performed by PFGE and MLST. The CRAB faecal carriage rate at admission was 4.8% (3/63). The CRAB acquisition rate during ICU stay was analysed in 39 of the remaining 60 patients and the rate of acquired CRAB faecal carriage was 15.4% (6/39); 4 patients also showed an ICU-acquired CRAB infection (one patient was a faecal carrier and suffered infection). Overall, 13 CRAB isolates were collected from 12 patients, of which 11 isolates showed resistance to all antibiotics tested except colistin. blaOXA-23 and blaNDM-1 were detected in 11 and 2 isolates, respectively. All OXA-23-producing strains carried armA, tetB, sul1 and catB, and some of them carried aph(3')-VIa, blaTEM-1, aph(3')-Ia and ant(2'')-Ia. The blaNDM-1-positive isolates harboured aph(3')-VIa and catB. Three PFGE patterns and two STs were identified [ST195 (n = 11), ST1089 (n = 2, NDM-1-positive)]. Whether imported or acquired during ICU stay, CRAB colonisation is a major risk factor for the occurrence of serious nosocomial infection. Systematic screening of faecal carriage is mandatory to prevent their spread.

Emergence of plasmid-mediated colistin-resistance in CMY-2-producing Escherichia coli of lineage ST2197 in a Tunisian poultry farm.

Our study aimed to investigate colistin resistance and the mechanisms involved in a collection of 35 extended-spectrum beta-lactamase (ESBL) and 13 CMY-2-producing E. coli strains which were previously recovered from chicken gut microbiota in Tunisia, as well as to determine the genetic location of mcr genes. Forty-eight ESBL and CMY-2-producing E. coli strains were obtained from 137 fecal samples of healthy chickens during 2013. These strains were tested for colistin resistance by the broth microdilution method, and screened for mcr-1 and mcr-2 genes by PCR. Two of these strains were colistin-resistant (MIC = 8 mg/L). Both harbored the mcr-1 gene, were CMY-2 producers, and were additionally resistant to tetracycline, ciprofloxacin, chloramphenicol, gentamicin, tobramycin and trimethoprim-sulfamethoxazole. They shared phylogroup A, the same pulsed-field gel electrophoresis (PFGE)-pattern, and were typed as ST2197. In both strains, ISApl1 and pap2 were detected upstream and downstream of mcr-1 gene, respectively. The analysis of the two mcr-1-positive strains and their transconjugants by PCR-based replicon typing and S1-PFGE, demonstrated that mcr-1 gene is linked to an IncP plasmid (~242 kb), and blaCMY-2 to an IncI1 plasmid (97 kb). The occurrence of E. coli harboring mcr-1 gene among intestinal microbiota in poultry and its location on a conjugative plasmid could represent a risk for public health. The evolution of this type of resistant microorganisms should be evaluated in the future.

An Outbreak of NDM-1-Producing Klebsiella pneumoniae, Associated with OmpK35 and OmpK36 Porin Loss in Tunisia.

OBJECTIVES: To describe clinical and molecular characteristics of an outbreak due to metallo-ß-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 blaOXA-48. All strains lacked at least one OMP, and carbapenem resistance levels were remarkably elevated in strains lacking OmpK35 and OmpK36. blaNDM-1 was located in IncFIA-type conjugative plasmid, with the same genetic context in all strains. The epidemiological diffusion of blaNDM-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 ß-lactamase expression, in conferring high carbapenem resistance.

Antimicrobial susceptibility and MLVA analysis of S. Typhimurium strains isolated from human and poultry samples in Tunisia.

INTRODUCTION: Salmonella enterica infections are a significant public health concern worldwide, being Salmonella Typhimurium one of the most prevalent serovars. Human salmonellosis is typically associated with the consumption of contaminated foods, such as poultry, eggs and processed meat. The extensive use of antimicrobials in humans and animals has led to an increase in multidrug resistance among Salmonella strains, becoming multidrug-resistant (MDR) strains a major public health concern. METHODOLOGY: This study was designed to investigate the antimicrobial susceptibility and the genotypic diversity of Salmonella Typhimurium strains isolated in Tunisia from human and poultry sources from 2009 to 2015. Fortyfive strains were analyzed by disk-diffusion test to determine the antimicrobial susceptibility. The presence of antimicrobial resistance genes was tested by PCR, and genotyping was performed using multiple-locus variable-number tandem repeats analysis (MLVA). RESULTS: About 50% of the strains were resistant to at least 3 antibiotics (multidrug-resistant strains, MDR). The most frequent resistance profile in clinical strains was AMP-TIC-TET-MIN-SXT (n = 7) and TET-MIN in poultry origin strains (n = 7). The MLVA typing grouped the strains in 2 main clusters. Cluster I was mostly formed by human isolates, whereas in cluster II both human and poultry isolates were grouped. Simpson's diversity index was 0.870 and 0.989 for antimicrobial resistance profiles and MLVA, respectively. CONCLUSIONS: Multiresistance is common in Salmonella Typhimurium isolated from human and poultry sources in Tunisia. The genotyping results suggest that some strains isolated from both sources may descend from a common subtype.

Rectal Carriage of Extended-Spectrum Beta-Lactamase and Carbapenemase Producing Gram-Negative Bacilli in Intensive Care Units in Tunisia.

This study was conducted to evaluate the rate of fecal carriage of Gram-negative bacilli (GNB) resistant to third-generation cephalosporins (third GC) in patients hospitalized in the intensive care unit (ICU) of Charles Nicolle Hospital of Tunis and to identify the enzymatic mechanisms involved. From February to April 2014, rectal swabs were collected from all patients (n = 38) at admission and once weekly thereafter to identify acquisition. They were cultured on desoxycholate-lactose-agar plates supplemented with cefotaxime (2 mg/L). The rate of fecal carriage of GNB resistant to third GC was 0% (0/38) at admission and the acquisition rate was 45.16% (14/31). Nineteen GNB resistant to C3G were collected from 14 patients. The major species collected were Acinetobacter baumannii (n = 5), Klebsiella pneumoniae (n = 5), and Enterobacter cloacae (n = 5). Thirteen extended-spectrum ß-lactamase (ESBL) producing GNB were found; CTX-M-15 (n = 10) and CTX-M-14 (n = 1) among Enterobacteriacae and GES-12 (n = 2) among A. baumannii. Ten strains were carbapenem resistant. OXA-48 (n = 4) and NDM-1 (n = 1) were detected among Enterobacteriacae and OXA-23 (n = 5), and GES-11 (n = 1) were detected in A. baumannii. Gene encoding the ACT-16 AmpC-type-ß-lactamase was detected in two isolates. All Escherichia coli isolates were assigned to group B2. Among virulence genes, prevalence of fimH, fuyA, ompT, pai, and usp were highest observed in all E. coli isolates. Among K. pneumoniae mrkD and entB were the most frequent (n = 5) followed by ybtS (n = 4) and kfu (n = 2). This study revealed a high prevalence of fecal carriage of multidrug-resistant GNB, including ESBLs, carbapenemases, and cephalosporinases producing bacteria in patients hospitalized in ICU.

Community fecal carriage of broad-spectrum cephalosporin-resistant Escherichia coli in Tunisian children.

The spread of extended spectrum ß-lactamases (ESBL) and plasmid mediated AmpC ß-lactamases (pAmpC) was evaluated in Escherichia coli strains collected from the intestinal microbiota of healthy children in Tunisia. The carriage rate of CTXRE. coli was 6.6% (7 of 105 samples) and one strain/sample was further characterized (7 isolates). These isolates harbored blaCTX-M-1 (n = 4), blaCTX-M-15 (n = 2), and blaCMY-2 gene (n = 1), which were usually located on FIB replicon type and carried class 1 integrons. The acc(6')-Ib-cr variant was identified in one isolate that harbored blaCTX-M-15. CTXRE. coli isolates were genetically unrelated and belonged to B1 (n = 3/ST155/ST398/ST58), D (n = 2/ST117/ST493), B2 (n = 1/ST127), and A (n = 1/ST746) phylogroups. Strain virulence scores varied from 3 to 12, and frequently harbored the pathogenicity island PAI IV536. The intestinal tract of healthy children constitute an important reservoir of ESBL producing E. coli. Thus, improvement of hygiene measures mainly in the school environment and rational use of antibiotics would be of great help in preventing selection and diffusion of resistant strains from intestinal microbiota.

High Prevalence of Gut Microbiota Colonization with Broad-Spectrum Cephalosporin Resistant Enterobacteriaceae in a Tunisian Intensive Care Unit.

Healthcare-associated infections due to cefotaxime-resistant (CTX-R) Enterobacteriaceae have become a major public health threat, especially in intensive care units (ICUs). Often acquired nosocomially, CTX-R Enterobacteriaceae can be introduced initially by patients at admission. This study aimed to determine the prevalence and genetic characteristics of CTX-R Enterobacteriaceae-intestinal carriage in ICU patients, to evaluate the rate of acquisition of these organisms during hospitalization, and to explore some of the associated risk factors for both carriage and acquisition. Between December 2014 and February 2015, the 63 patients admitted in the ICU of Charles Nicolle hospital were screened for rectal CTX-R Enterobacteriaceae colonization at admission and once weekly thereafter to identify acquisition. CTX-R Enterobacteriaceae fecal carriage rate was 20.63% (13/63) at admission. Among the 50 non-carriers, 35 were resampled during their hospitalization and the acquisition rate was 42.85% (15/35). Overall, 35 CTX-R Enterobacteriaceae isolates were collected from 28 patients (25 Klebsiella pneumoniae, seven Escherichia coli, and three Enterobacter cloacae strains). Seven patients were simultaneously colonized with two CTX-R Enterobacteriaceae isolates. CTX-M-15 was detected in most of the CTX-R Enterobacteriaceae isolates (30/35, 88.23%). Three strains co-produced CMY-4 and 22 strains were carbapenem-resistant and co-produced a carbapenemase [OXA-48 (n = 13) or NDM-1 (n = 6)]. Molecular typing of K. pneumoniae strains, revealed eight Pulsed field gel electrophoresis (PFGE) patterns and four sequence types (ST) [ST101, ST147, ST429, and ST336]. However, E. coli isolates were genetically unrelated and belonged to A (n = 2), B1 (n = 2) and B2 (n = 3) phylogenetic groups and to ST131 (two strains), ST572 (two strains), ST615 (one strain) and ST617 (one strain). Five colonized patients were infected by CTX-R Enterobacteriaceae (four with the same strain identified from their rectal swab and one with a different strain). Whether imported or acquired during the stay in the ICU, colonization by CTX-R Enterobacteriaceae is a major risk factor for the occurrence of serious nosocomial infections. Their systematic screening in fecal carriage is mandatory to prevent the spread of these multidrug resistant bacteria.

High prevalence of extended-spectrum and plasmidic AmpC beta-lactamase-producing Escherichia coli from poultry in Tunisia.

This study was conducted to detect extended spectrum beta-lactamases (ESBLs) and plasmidic AmpC beta-lactamase (pAmpC-BL)-producing Escherichia coli isolates in industrial poultry samples were collected from healthy chickens of the three farms. Samples were inoculated onto desoxycholate-lactose-agar plates supplemented with cefotaxime (2mg/L). E. coli was identified by biochemical and molecular methods and antibiotic susceptibility testing by the disk diffusion method. Genes encoding ESBLs and pAmpC-BL were detected by PCR and sequencing. Phylogenetic groups were determined by triplex PCR. The molecular typing of strains was done by pulsed field gel electrophoresis (PFGE) and Multilocus Sequence Typing (MLST) in those isolates showing different PFGE patterns. Cefotaxime-resistant E. coli isolates were recovered in 48 of 137 fecal samples (35%), and one isolate/sample was further studied. The following beta-lactamase genes were detected: blaCTX-M-1 (29 isolates, isolated in all three farms), blaCTX-M-15 (5 isolates, confined in farm II), blaCTX-M-14 and blaCMY-2 (one isolate and 13 isolates, respectively, in farm III). The 48 cefotaxime-resistant isolates were distributed into phylogroups: B1 (n=21), A (n=15) and D (n=12). PFGE analysis revealed 19 unrelated patterns: 15 different profiles among ESBL-positive strains and 4 among the CMY-2-positive isolates. The following sequence types-associated phylogroups were detected: a) CTX-M-1-positive strains: lineages ST542-B1, ST212-B1, ST58-B1, ST155-B1 and ST349-D; b) CTX-M-15-positive strain: lineage ST405-D; c) CTX-M-14-positive strain: lineage ST1056-B1; d) CMY-2-positive strains: lineages ST117-D, ST2197-A, and ST155-B1. Healthy chickens constitute an important reservoir of ESBL- and pAmpC-BL-producing E. coli isolates that potentially could be transmitted to humans via the food chain or by direct contact.

Cooccurrence of Multiple AmpC ß-Lactamases in Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis in Tunisia.

Over a period of 40 months, plasmid-mediated AmpC ß-lactamases were detected in Tunis, Tunisia, in 78 isolates (0.59%) of Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis. In 67 isolates, only one ampC gene was detected, i.e., blaCMY-2-type (n = 33), blaACC (n = 23), blaDHA (n = 6) or blaEBC (n = 5). Multiple ampC genes were detected in 11 isolates, with the following distribution: blaMOX-2, blaFOX-3, and blaCMY-4/16 (n = 6), blaFOX-3 and blaMOX-2 (n = 3), and blaCMY-4 and blaMOX-2 (n = 2). A great variety of plasmids carrying these genes was found, independently of the species and the bla gene. If the genetic context of blaCMY-2-type is variable, that of blaMOX-2, reported in part previously, is unique and that of blaFOX-3 is unique and new.

Prevalence and characterization of plasmid-mediated quinolone resistance genes in extended-spectrum ß-lactamase-producing Enterobacteriaceae in a Tunisian hospital.

The objective of the study was to assess the prevalence of plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrB, qnrC, qnrD, qnrS, aac(6')-Ib-cr, qepA, and oqxAB) in a collection of 120 extended-spectrum ß-lactamases (ESBLs)-producing enterobacteria and to characterize them. Overall, PMQR determinants were detected in 72 (60%) isolates (20 Escherichia coli, 32 Klebsiella pneumoniae, and 20 Enterobacter cloacae). PMQR frequencies were as follows: qnr genes (25.8%), oqxAB (21.6%), and aac(6')-Ib-cr variant (19.2%). Four qnr alleles were identified as qnrB1 (83.8%), qnrB4 (6.4%), qnrB2 (3.2%), and qnrS1 (6.4%). qnr genes were mainly detected in E. cloacae (50%), aac(6')-Ib-cr in E. coli (47.5%), and oqxAB in K. pneumoniae (65%). Overall, blaCTX-M-15 (90.3%) was the most prevalent blaESBL type followed by blaSHV-12 (6.4%) and blaSHV-27 (2.7%). Rates of mutations in gyrA and parC genes were 75% for E. coli, 72.8% for K. pneumoniae, and 50% for E. cloacae. Isolates with mutations in their quinolone resistance-determining regions exhibited high fluoroquinolones resistance levels compared to those with wild ones. Genetic study of PMQR-harboring isolates revealed a great genomic diversity among each Enterobacteriaceae species. Our findings indicate the high prevalence of PMQR determinants among ESBL-producing Enterobacteriaceae isolates from our hospital and their diffusion in various unrelated CTX-M-15-producing clones.

Multidrug resistance and high virulence genotype in uropathogenic Escherichia coli due to diffusion of ST131 clonal group producing CTX-M-15: an emerging problem in a Tunisian hospital.

A collection of 201 Escherichia coli strains isolated from urine of patients in a Tunisian hospital between January 2006 and July 2008 was studied. Microbial identification was done by conventional methods, and antibiotic susceptibility with disk diffusion method was performed according to the Clinical Laboratory and Standards Institute guidelines. Detection of extended-spectrum beta-lactamase (ESBL) was performed by double-disk synergy test (DDST) and identification was done by PCR and sequencing. ESBL-producing isolates were subjected to molecular typing by random amplified polymorphic DNA (RAPD) and ST131 detection by PCR. Four phylogenetic groups (A, B1, B2 and D), 18 virulence genes and CTX-M group were individualized using PCR. Statistical analysis was done by Pearson χ2 test and Mann-Whitney U test. The strains were recovered primarily from urology (28%), maternity (19%) and medicine (16%) wards. Antibiotic resistance rates were ampicilin (72.1%), nalidixic acid (41.8%), ciprofloxacin (38.8%), gentamicin (23.9%) and cefotaxime (17.4%). Thirty-one of cefotaxime-resistant isolates (n = 35) had a positive DDST and harboured bla CTX-M-15 gene. Twenty of them (64.5%) belonged to the ST131 clone and showed the same RAPD DNA profile. Ciprofloxacin- and cotrimoxazole-susceptible isolates were significantly associated with phylogenetic group B2, whereas isolates that were resistant to these molecules were associated with B1 and D phylogenetic groups, respectively. Virulence genes were significantly more frequent among ciprofloxacin- and cotrimoxazole-susceptible strains than those resistant to these antibiotics. However, CXT-M-15-producing isolates were associated with many virulence genes. Isolates concomitantly susceptible to the three antimicrobials agents (ciprofloxacin, cefotaxime and cotrimoxazole) were significantly associated with group B2 and high virulence score, whereas isolates with resistance patterns especially those including resistance to ciprofloxacin belonged predominantly to B1 phylogroup and haboured few virulence genes. The emergence of virulent and multidrug-resistant E. coli is a concerning development that deserves close attention in our institution.

Characterization of extended spectrum ß-lactamase-producing Escherichia coli in community-acquired urinary tract infections in Tunisia.

This study was conducted to investigate the molecular epidemiology of extended spectrum ß-lactamase (ESBL)-producing Escherichia coli in community-acquired (urinary tract) infections (CA-UTI) in Tunisia. Between January 2007 and December 2009, 15 E. coli isolates were collected at the laboratory of microbiology of Charles Nicolle Hospital of Tunis. Microbial identification was done with conventional methods. Antibiotic susceptibility was determined by disk diffusion method and ESBL detection was done with double-disk synergy test. ESBL typing was performed by polymerase chain reaction (PCR) and sequencing. Phylogenetic groups, virulence factors, and sequence type (ST)131 were determined by PCR. Genetic relatedness between strains was examined by pulsed-field gel electrophoresis (PFGE) after restriction with XbaI. The prevalence of ESBL-producing E. coli in CA-UTI was 0.046%. The majority of isolates were multidrug resistant. ESBL types were CTX-M-15 (n=13) and SHV-12 (n=2). The most common phylogenetic group was B2 (n=11) and virulence score was greater than or equal to 9 in nine strains. PFGE revealed 12 clusters. The majority of isolates (n=14) belonged to ST131 clone and 11 of them were CTX-M producers. In conclusion, this is the first detailed documentation of CA-ESBLs producing E. coli in Tunisia. Of particular concern is the emergence in our community of the highly diffusing CTX-M-15-B2-ST131 E. coli clone, which requires strengthening surveillance measures to countervail this emergent public health problem.

Characterization and molecular epidemiology of extended spectrum beta-lactamase producing Enterobacter cloacae isolated from a Tunisian hospital.

In 2009, out of the 66 nonrepetitive Enterobacter cloacae collected at Charles Nicolle hospital in Tunisia, 44 were extended spectrum ß-lactamase (ESBL) producers. The aim of the current study was to detect and characterize the genes encoding the ESBLs including blaTEM, blaSHV, and blaCTX-M groups by polymerase chain reaction and sequencing. Pulsed-field gel electrophoresis (PFGE) analysis was used to determine the genetic relatedness between isolates. All strains were susceptible to carbapenems. They were resistant to fluoroquinolones, gentamicin, tobramycin, and trimethoprim+sulfamethoxazole but variably resistant to netilmicin, amikacin, and tetracyclines. Sequence analysis of the polymerase chain reaction products revealed the presence of blaCTX-M-15 (39 strains), blaSHV-12 (6 strains), and blaSHV-27 (1 strain). The coexistence of two ESBLs was observed in two isolates harboring, respectively, SHV-12+CTX-M-15 and SHV-27+CTX-M-15. PFGE revealed 36 unrelated profiles. Diffusion of E. cloacae producing CTX-M-15 ESBL in our hospital is the consequence of dissemination of identical or related plasmids harboring the CTX-M-15 gene.

Carbapenem-resistant Acinetobacter baumannii producing the carbapenemase OXA-23 in Tunisia.

AIM: To analyze the mechanisms of resistance to carbapenems among imipenem resistant A. baumannii recovered from different wards at Charles Nicolle Hospital. METHODS: From January to December 2007, 50 carbapenem-resistant A. baumannii isolates were recovered from hospitalized patients. MICs were performed by agar dilution method and interpreted according to CLSI guidelines. Metallo-ß-lactamase production was evaluated using imipenem-EDTA disk synergy test. PCR and DNA sequencing targeting blaOXA genes were performed and pulsed field gel electrophoresis was used for epidemiologic study. RESULTS: Most of the isolates were obtained from patients hospitalized in surgery (62%) and Intensive Care Units (22%). All strains showed high level of resistance to ticarcillin (MIC50 > 2048µg/ml), ticarcillin-clavulanic acid (MIC50 >1024µg/ml), aztreonam (MIC50 = 512µg/ml), ceftazidim (MIC50 = 512µg/ml), imipenem (MIC50 = 512µg/ml), meropenem (MIC50 =128µg/ml) and cefepime (MIC50 = 256µg/ml). Metallo-ß-lactamase production was negative for all isolates. The co-existence of blaOXA-51-like/ blaOXA-23-like was detected in 82% (n= 41). The genes blaOXA- 24-like and blaOXA-58-like were not found in any isolate. All isolates harboured a blaOXA-51-like gene. Sequencing confirmed the presence of blaOXA-23 and blaOXA-69 genes. Eight distinct patterns were observed (A: 41 isolates, B: 1 isolate, C: 1 isolate, D: 1 isolate, E: 1 isolate, F: 2 isolates, G: 1 isolate, H: 2 isolates). CONCLUSION: Production of OXA-23 was the important mechanism of resistance to carbapenem among A. baumannii. Strengthening of prevention measures are required to control further spread of carbapenemases in Tunisia.