GES-11-producing Acinetobacter baumannii clinical isolates from Tunisian hospitals: Long-term dissemination of GES-type carbapenemases in North Africa.

Acinetobacter baumannii is an emerging threat in healthcare facilities owing to its ability to be multidrug-resistant (MDR) and to be involved in outbreaks. GES-type extended-spectrum ß-lactamases (ESBLs) have been increasingly identified in A. baumannii. In this study, clinical A. baumannii isolates were characterised using standard biochemical methods and antibiotic susceptibility testing. Antibiotic resistance genes were sought by PCR and sequencing. Genetic support was characterised using S1 nuclease pulsed-field gel electrophoresis (PFGE) mapping, conjugation and electroporation assays. The genetic environment was investigated by PCR, and genetic relatedness was investigated by PFGE. Two MDR A. baumannii clinical isolates susceptible only to colistin and rifampicin were isolated from a tracheal aspirate of a 49-year-old woman hospitalised in 2006 at the Military Hospital of Tunis, Tunisia, and from a tracheal aspirate of a 53-year-old man hospitalised in 2010 at the Institut Orthopédique Mohamed El Kassab of Tunis, Tunisia. PCR revealed that the two isolates harboured the acquired carbapenemase blaOXA-23 and ESBL blaGES-11 genes along with chromosomally-encoded blaOXA-51 and blaADC-like genes. PFGE revealed that these A. baumannii isolates were unrelated; nevertheless, plasmid analysis revealed a similar sized plasmid following electrophoresis of the isolates. In addition, A. baumannii CIP70.10 transformants displayed similar resistance patterns. blaGES-11 was integron-borne and the ISAbaI element was identified upstream of blaOXA-23 and blaADC-like. Here we described two unrelated clinical A. baumannii isolates producing GES-11 ESBL and OXA-23 carbapenemase from two Tunisian hospitals. This work further illustrates the emergence of GES-type ß-lactamases in A. baumannii in North Africa as early as 2006.

First characterisation of plasmid-mediated quinolone resistance-qnrS1 co-expressed bla CTX-M-15 and bla DHA-1 genes in clinical strain of Morganella morganii recovered from a Tunisian Intensive Care Unit.

PURPOSE: Aim of this study was to show the emergence of the qnr genes among fluoroquinolone-resistant, AMPC and ESBL (extended-spectrum-beta-lactamase) co-producing Morganella morganii isolate. MATERIALS AND METHODS: A multi resistant Morganella morganii SM12012 isolate was recovered from pus from a patient hospitalized in the intensive care unit at the Military hospital, Tunisia. Antibiotic susceptibility was tested with the agar disk diffusion method according to Clinical and Laboratory Standards Institute guidelines. ESBLs were detected using a standard double-disk synergy test. The characterization of beta-lactamases and associated resistance genes were performed by isoelectric focusing, polymerase chain reaction and nucleotide sequencing. RESULTS: The antimicrobial susceptibility testing showed the high resistance to penicillins, cephalosporins (MICs: 64-512 µg/ml) and fluoroquinolones (MICs: 32-512 µg/ml). But M. morganii SM12012 isolate remained susceptible to carbapenems (MICs: 4-<0.25 µg/ml). The double-disk synergy test confirmed the phenotype of extended-spectrum ß-lactamases (ESBLs). Three identical ß-lactamases with pI values of 6.5, 7.8 and superior to 8.6 were detected after isoelectric focusing analysis. These ß-lactamases genes can be successfully transferred by the conjugative plasmid. Molecular analysis demonstrated the co-production of bla (DHA-1), bla (CTX-M-15) and qnrS1 genes on the same plasmid. The detection of an associated chromosomal quinolone resistance revealed the presence of a parC mutation at codon 80 (Ser80-lle80). CONCLUSION: This is the first report in Tunisia of nosocomial infection due to the production of CTX-M-15 and DHA-1 ß-lactamases in M. morganii isolate with the association of quinolone plasmid resistance. The incidence of these strains invites continuous monitoring of such multidrug-resistant strains and the further study of their epidemiologic evolution.

First report of CTX-M-9 in a clinical isolate of Enterobacter cloacae in a Tunisian hospital.

ß-lactamases are one of several mechanisms of bacterial resistance to ß-lactam antibiotics. The aim of this study was to analyze the resistance to extended-spectrum cephalosporins of a multidrug-resistant isolate of Enterobacter cloacae, BF5011. This strain was isolated from a stool culture, during the nosocomials infections occurring in the intensive care unit of the Military Hospital of Tunis in 2005. Analysis of E. cloacae BF5011 by double-disk synergy test yielded a positive result suggesting the production of extended-spectrum-ß-lactamases. Cell sonicate of this isolate is very active against cefotaxime and showed a specific activity (AS) of 7,54 U/mg for the same antibiotic. This activity was inhibited by the sulbactam and the clavulanic acid. By polymerase chain reaction and sequencing, the isolate was found to produce extended-spectrum-ß-lactamase, CTX-M-9. The bla(CTX-M-9) gene was transferred from E. cloacae by conjugation. Isoelectric focusing method revealed one band with a pI of about 8. This is the first report of CTX-M-9 in Tunisia.

[Exclusive breastfeeding and mixed feeding: knowledge, attitudes and practices of primiparous mothers]. / Allaitement maternel exclusif et allaitement mixte: connaissances, attitudes et pratiques des mères primipares.

We assessed the knowledge attitudes and practices of primiparous women with regard to exclusive breastfeeding and the use of formula milk. A total of 260 women were interviewed and the results showed that 41.5% of the women breastfed exclusively while 58.5% bottle-fed only or did so together with breastfeeding. Of those who breastfed, 43.0% did not do so soon after giving birth and did not know about colostrum. Overall, the knowledge, attitudes and practices of the mothers were unsatisfactory concerning the golden rules for successful breastfeeding, the ideal duration of exclusive breastfeeding and the food to include when introducing complementary feeding. This might be due to a low level of schooling and information, hence the need for improving strategies for maternal care during the antenatal and postnatal periods.

Anti-diabetic effect of camel milk in alloxan-induced diabetic dogs: a dose-response experiment.

This study was conducted to evaluate the effect of camel milk in alloxan-induced diabetic dogs and to follow this effect at three doses of milk. Firstly, three groups of dogs were used: two groups composed each of four diabetic dogs and receiving raw camel milk (treatment 1) or cow milk (treatment 2), and four healthy dogs getting raw camel milk (treatment 3) were used as control. Each animal was treated with 500 ml of milk daily. Secondly, we compared the effects of three amounts of camel milk: 100 ml, 250 ml and 500 ml to treat the diabetic dogs. After week 3, the dogs treated with camel milk showed a statistically significant decrease in blood glucose (from 10.88 +/- 0.55 to 6.22 +/- 0.5 mmol/l) and total protein concentrations (from 78.16 +/- 2.61 g/l to 63.63 +/- 4.43 g/l). For cholesterol levels, there was a decrease from week 2 (from 6.17 +/- 0.5 mmol/l to 4.79 +/- 0.5 mmol/l). There were no significant difference in blood glucose, cholesterol or total protein concentrations in dogs drinking 250 and 500 ml of camel milk. The dogs treated with 100 ml of camel milk did not show any significant decrease in blood glucose levels, and cholesterol and total protein concentrations. The investigation was not limited to the improvement in glycemic balance, lipids and proteins control in diabetic dogs getting camel milk, but we also noted a stability of this state after the dogs stopped to drink milk. This effect depended on the quantity of camel milk used to treat diabetic dogs.

[Exclusive breastfeeding and mixed feeding: knowledge, attitudes and practices of primiparous mothers]

We assessed the knowledge attitudes and practices of primiparous women with regard to exclusive breastfeeding and the use of formula milk. A total of 260 women were interviewed and the results showed that 41.5% of the women breastfed exclusively while 58.5% bottle-fed only or did so together with breastfeeding. Of those who breastfed, 43.0% did not do so soon after giving birth and did not know about colostrum. Overall, the knowledge, attitudes and practices of the mothers were unsatisfactory concerning the golden rules for successful breastfeeding, the ideal duration of exclusive breastfeeding and the food to include when introducing complementary feeding. This might be due to a low level of schooling and information, hence the need for improving strategies for maternal care during the antenatal and postnatal periods

First detection of CTX-M-28 in a Tunisian hospital from a cefotaxime-resistant Klebsiella pneumoniae strain.

A cefotaxime-resistant Klebsiella pneumoniae ML4313 was obtained from a patient from intensive care unit of Military hospital in Tunisia. This strain was resistant to beta-lactams, aminoglycosides, quinolones and phenicols, and tetracyclines. It was identified as producer of extended-spectrum beta-lactamases (ESBL) by double-disk synergy test between amoxicillin-clavulanate and cefotaxime, ceftriaxone, ceftazidime and aztreonam. The ESBL was identified as CTX-M-28 by sequencing of PCR products and by isoelectric focusing. The ESBL resistance was transferred by a 50kb plasmid. CTX-M-28 is closely related to CTX-M-15. This is the first description of this enzyme in Tunisia.

Identification of plasmid-encoded extended spectrum beta-lactamases produced by a clinical strain of Proteus mirabilis.

Emergence and dissemination of multiresistant strain of Proteus mirabilis have made infections treatment more difficult that this bacterium is responsible. The aim of this study is to determine the implication of the enzymatic mechanism and to describe the properties of ESBLs (extended spectrum beta-lactamases). A clinical strain of Proteus mirabilis SM514 isolated in the intensive care unit at the Military hospital in Tunisia during the period 2004 was found to be highly resistant to cephalosporins and penicilins. Cells sonicate of the isolate hydrolysed cefotaxime more efficiently than ceftriaxone and ceftazidime and had three beta-lactamases bands of approximate of isoelectric points (pI) of 5.4; 5.6 and superior to 7.6. The specific activities (AS) vary from 5.26 to 7.77U/mg of protein respectively for cefotaxime and the benzylpenicillin. These activities are inhibited by the clavulanic acid and the sulbactam. The values of the IC(50) are respectively 3.7 and 11.7muM. Only the beta-lactamases of pI 5.4 and superior to 7.6 hydrolyze the cefotaxime. Transformant produces the ESBLs of pI 5.4; 7.45 and greater than 7.6. The genes coding for this enzymes are carried by a transferable plasmids.

WITHDRAWN: [Clinical strain of Proteus mirabilis able to produce a plasmid mediated ceftazidimase.] / Identification d'une ceftazidimase plasmidique produite par une souche clinique de Proteus mirabilis.

Resistance to third generation cephalosporins due to the acquisition and expression of extended spectrum beta-lactamases (ESBLs) enzymes among Gram negative bacteria continue to pose a challenge to infection management worldwide. The aim of this study was to determine the implication of the enzymatic mechanism and to describe the properties of ESBLs from a clinical strain of Proteus mirabilis NC4150 isolated in the intensive care unit at the Military Hospital in Tunisia during the period 2004. The isolate was identified, tested for antimicrobial susceptibility and analysed for presence of ESBL genes. Two beta - lactamases which confers a multirésistance on antibiotics including the oxyimino beta-lactams were identified. Isoelectric points of these two beta-lactamases are 5.4 and 5.7. The specific activities (AS) vary from 0.2 to 36.27 U/mg of protein respectively for ampicilline and the ceftazidime. These activities are inhibited by the clavulanic acid and the sulbactam. The values of the IC 50 are respectively 16 muM and 2.4 mu M. Only the beta - lactamase of pI 5.7 hydrolyze the ceftazidime. Transformant and transconjugant produces only the ESBL (extended spectrum beta-lactamases) of pI 5.7. The gene coding for this enzyme is carried by a transferable plasmid named pNC4150.

A multiple-antibiotic resistance-independent active chloramphenicol efflux in an Escherichia coli clinical isolate.

The clinical isolate, Escherichia coli 1941, exhibits high resistance to chloramphenicol and tetracycline (minimum inhibitory concentrations of 512 micrograms/ml). Neither resistance is linked to the large conjugative plasmid present in the strain. The intracellular accumulation of radiolabeled chloramphenicol increased about 9-fold after the addition of the energy uncoupler carbonyl cyanide m-chlorophenol-hydrazone to an E. coli 1941 culture, indicating the presence of an active efflux mechanism. Sequence analysis and expression study suggested that the multiple-antibiotic resistance marRAB locus and the AcrAB drug-efflux pump were not involved in this active efflux of chloramphenicol.

Beta-lactamase-mediated resistance to extended spectrum cephalosporins among clinical isolates of Pseudomonas aeruginosa.

The role of chromosomal cephalosporinases and secondary beta-lactamases in resistance to extended spectrum cephalosporins in clinical isolates of Pseudomonas aeruginosa was investigated. Strains 687, 59, and 58 expressed an inducible chromosomal cephalosporinase, efficiently enhanced with cefoxitin and imipenem. The inducible activity in strain 802 was produced at a moderately elevated basal level and may be involved in resistance to extended spectrum cephalosporins and aztreonam. All strains produced secondary beta-lactamases inhibited by clavulanate: strains 687, 59, and 58 had carbenicillinases with pIs of 5.7 and 5.3. Strain 802 expressed a secondary beta-lactamase of pI 7.6 which may be a novel extended spectrum beta-lactamase different from known enzymes of P. aeruginosa.

[Trace metals: zinc and copper in Tunisian littoral fishes]. / Recherche de certains métaux-traces: zinc et cuivre, dans des poissons du littoral tunisien.

Investigations have been carried out in order to find out whether zinc and copper are or not accumulated in flesh and organs of some edible fish of a polluted area, namely Sfax. Tracking zinc and copper was our first aim, the second was the determination of an organotropism of the two metals. Finally, our results, would help stating whether or not Sfax-shore is polluted with zinc and copper.

Modulation of cisplatin chronotoxicity related to reduced glutathione in mice.

Intracellular reduced glutathione (GSH) concentrations were measured according to the tissue sampling-time along the 24 h scale in male B6D2F1 mice. A significant circadian rhythm in GSH content was statistically validated in liver, jejunum, colon and bone-marrow (P < or = 0.02) but not in kidney. Tissue GSH concentration increased in the dark-activity span and decreased in the light-rest span of mice. The minimum and maximum of tissue GSH content corresponded respectively to the maximum and minimum of cisplatin (CDDP) toxicity. The role of GSH rhythms with regard to CDDP toxicity was investigated, using a specific inhibitor of GSH biosynthesis, buthionine sulfoximine (BSO). Its effects were assessed on both tissue GSH levels and CDDP toxicity at three circadian times. BSO resulted in a 10-fold decrease of the 24 h-mean GSH in kidney. However a moderate GSH decrease characterized liver (-23%) and jejunum (-30%). BSO pretreatment largely enhanced CDDP toxicity which varied according to a circadian rhythm. Although BSO partly and/or totally abolished the tissue GSH rhythms, it did not modify those in CDDP toxicity. We conclude that GSH have an important influence on CDDP toxicity but not in the circadian mechanism of such platinum chronotoxicity.

Characterization of the drug resistance plasmid R2418: restriction map and role of insertion and deletion in its evolution.

Escherichia coli 2418 strain is resistant to beta-lactam antibiotics (ampicillin, carbenicillin, and cephalothin), streptomycin, tetracycline, kanamycin, and chloramphenicol. This strain contains at least two conjugative plasmids (R2418 and R2418S) encoding resistance to beta lactam antibiotics and resistance to both beta-lactam antibiotics and streptomycin, respectively. Restriction endonuclease mapping of plasmid DNAs indicates that the plasmid R2418S has evolved from R2418 DNA by the insertion of 2.5-kb DNA between BamHI and PvuII sites, and deletion of 0.5-kb DNA within the EcoRI-EcoRV region. The 2.5-kb DNA insert is responsible for streptomycin resistance. This evolution is also associated with a reduction in the efficiency of conjugal transfer for the plasmid R2418S. The conjugal transfer of streptomycin resistance occurs only through the coresidence of the conjugative plasmid R2418 or R2418S in the donor cell. In accordance with the hypothesis that the Smr determinant is due to a putative transposon, plasmid-free transconjugants resistant to streptomycin only were isolated. Southern blot analysis of HindIII chromosomal digests extracted from these transconjugants shows that the Smr determinant is inserted into different sites in chromosomal DNA.

Association of mitochondrial ADP-ribosyl transferase activity with the DNA-protein complex.

The rat liver mitochondrial DNA protein complex was purified employing a linear sucrose gradient. The ADP-ribosyl transferase activity appears to be associated with this protein DNA complex. Predominant acceptor proteins ranged between 116-30 KDa as revealed by LDS gel electrophoresis. These fractions did not contain any significant NAD glycohydrolase activity.