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Abstract: Bleomycin is a highly potent cytotoxic and genotoxic agent used in the chemotherapy of various types of tumors. It is a radiomimetic anticancer drug that produces single- and double-stranded [...]

Purpose: Broomrape produces serious damage to many legume crops and, particularly, becomes a limiting factor for faba bean (Vicia faba L.) production in the Mediterranean basin. Currently, several traditional methods of control have been developed, but none has proved to be effective for this parasite. However, breeding for resistance to this pest remains as one of the most feasible and environment-friendly methods for managing broomrape, but the mechanisms governing the interaction between the parasite and the host are not yet well understood. Therefore, we studied the behavior and molecular and enzymatic changes associated with the resistance to Orobanche foetida in faba bean mutants, which were obtained through radiation mutagenesis. Materials and methods: Three faba bean genotypes were used in this study, the variety ‘Badï’, characterized by high productivity in Orobanche-free soils and susceptibility to O. foetida, and two mutant lines P2M3 and P7M3 (derived from radio mutagenesis program), selected for their higher resistance to O. foetida in a field evaluation. The infection progress and the relative changes in the co-culture response, the enzymatic activities changes and the efficiency of the root extract stimulants from the host plant were followed and evaluated in all the genotypes. Results: Experiments showed that low induction of seed germination is a major component of resistance in these lines against O. foetida. This is confirmed by the in vitro experiments with root exudates. The parallel reduction in infection was accompanied by the continuous enhancement of the peroxidase activity, the polyphenol oxidase activity and the phenylalanine ammonia lyase activity in faba bean roots. Conclusion: These data suggest the contribution of these enzymes in faba bean resistance to O. foetida broomrape induced by the use of gamma rays. Management of Orobanche by way of crop selection, based on these enzyme systems is a possible option. [ABSTRACT FROM AUTHOR]

BACKGROUND Some radiation-mutagenised chickpea mutants potentially resistant to the broomrape, Orobanche foetida Poir., were selected through field trials. The objectives of this work were to confirm resistance under artificial infestation, in pots and mini-rhizotron systems, and to determine the developmental stages of broomrape affected by resistance and the relevant resistance mechanisms induced by radiation mutagenesis. RESULTS Among 30 mutants tested for resistance to O. foetida, five shared strong resistance in both pot experiments and mini-rhizotron systems. Resistance was not complete, but the few individuals that escaped resistance displayed high disorders of shoot development. Results demonstrated a 2-3-fold decrease in stimulatory activity of root exudates towards broomrape seed germination in resistant mutants in comparison with non-irradiated control plants and susceptible mutants. Resistance was associated with an induction of broomrape necrosis early during infection. When infested, most of the resistant mutants shared enhanced levels of soluble phenolic contents, phenylalanine ammonia lyase activity, guaiacol peroxidase activity and polyphenol oxidase activity, in addition to glutathione and notably ascorbate peroxidase gene expression in roots. CONCLUSION Results confirmed enhanced resistance in chickpea radiation-mutagenised mutants, and demonstrated that resistance is based on alteration of root exudation, presumed cell-wall reinforcement and change in root oxidative status in response to infection. © 2016 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Bacillus licheniformis RBS 5 was isolated from thermal spring in Tunisia. The isolate coproduce α-amylase and protease enzymes. The a-amylase activity showed an optimal activity at approximately 65°C and in wide pH interval ranging from 4 to 9. This enzyme was stable over the range of 45 to 70°C after 30 min of incubation and in the pH range of 8 to 10. Protease activity was optimal; at 80°C, pH 12. This enzyme was stable until 60°C over the pH range of 10 to 12. EDTA at concentration of 5 mM reduces slightly both activities evoking the serine alkaline protease. Cationic ions (Ca2+, Cu2+, Zn2+, and Mg2+) have an inhibition effect on a-amylase. However, protease activity was enhanced by Ca2+, Cu2+ and Mg2+); the other cations reduce slightly the proteolytic activity. SDS and H2O2 were found as inhibitors for both activities whereas Triton X-100 and perfume have no effect. Taken together, these traits make protease activity of B. licheniformis RBS 5 as efficient for use in detergent industry. [ABSTRACT FROM AUTHOR]

A new thermostable, haloalkaline, solvent stable SDS-induced serine protease was purified and characterized from a thermophilic Geobacillus toebii LBT 77 newly isolated from a Tunisian hot spring. This study reveals the potential of the protease from Geobacillus toebii LBT 77 as an additive to detergent with spectacular proprieties described for the first time. The protease was purified to homogeneity by ammonium sulfate precipitation followed by Sephadex G-75 and DEAE-Cellulose chromatography. It was a monomeric enzyme with molecular weight of 30 kDa. The optimum pH, temperature, and NaCl for maximum protease activity were 13.0, 95°C, and 30%, respectively. Activity was stimulated by Ca2+, Mg2+, DTNB, β-mercaptoethanol, and SDS. The protease was extremely stable even at pH 13.25, 90°C, and 30% NaCl and in the presence of hydrophilic, hydrophobic solvents at high concentrations. The high compatibility with ionic, nonionic, and commercial detergents confirms the utility as an additive to cleaning products. Kinetic and thermodynamic characterization of protease revealed Km=1 mg mL−1,  Vmax=217.5 U mL−1, Kcat/Km=99 mg mL−1 S−1, Ea=51.5 kJ mol−1, and ΔG⁎=56.5 kJ mol−1. [ABSTRACT FROM AUTHOR]

Background: ESBL-producing bacteria are a clinical problem in the management of diseases caused by these pathogens. Worldwide, systemic infections with BL enzymes are evolving by mutations from classical bla genes in an intensified manner and they continue to be transferred across species. Results: E.cloacae BF1417 isolate and its transconjugants gave positive results with the DDST, suggesting the presence of ESBL. Sequence analysis revealed a blaSHV-ESBL-type gene that differs from the gene encoding SHV-1 by five point mutations resulting in three amino acid substitutions in the coding region: C123R, I282T and L286P. This novel SHV-type enzyme was designated SHV-128. The conjugation tests and plasmid characterization showed that the blaSHV-128 is located on a conjugative plasmid IncFII type. Expression studies demonstrated that the above mutations participated in drug resistance, hydrolysis of extended spectrum β-lactam and the change of the isoelectric point of the protein. Conclusion: These findings underscore the diversity by which antibiotic resistance can arise and the evolutionary potential of the clinically important ESBL enzymes. In addition, this study highlights the need for systematic surveillance of ESBL-mediated resistance as well as in clinical areas and communities. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae ML2011, a multiresistant isolate, was isolated from the Military Hospital of Tunis (Tunisia). The determination of the minimal inhibitory concentrations exhibited by K. pneumoniae ML2011 was performed by Etest. The crude extract of the isolates contains four different β-lactamases with pI 5.5,7.3,7.6, and 8.6. Only the β-lactamases with pI 7.3 andpI 8.6 were transferred by transformation and conjugation experiment. Molecular characterization of these genes was performed by PCR and sequencing. The chromosomal β-lactamases are TEM (pI 5.5) and SHV-1 (7.6). CTX-M-28 (pI 8.6) and the novel variant of SHV named SHV- 104 (pI 7.3) were encoded by bla gene located on a 50 kb highly conjugative plasmid. The SHV-104 β-lactamase was produced in E. coli and purified. Its profile of activity was determined. Compared to SHV-1, SHV-104 contains one mutation, R202S. Their kinetic parameters were similar except for cefotaxime. The analysis of the predicted structure of SHV-104 indicated that the R202S mutation suppresses a salt bridge present in SHV-1. Therefore, the overall flexibility of the protein increased and might improve the hydrolysis of cefotaxime. We can conclude that the multiresistant phenotype of K. pneumoniae ML2011 strain is mainly linked to the production of CTX-M-28 since SHV-104 possesses a narrow spectrum of activity. [ABSTRACT FROM AUTHOR]

Objectives: The aim of this study was to investigate the prevalence and the emergence of plasmid-mediated quinolone resistance among broad-spectrum beta-lactam-resistant Proteus mirabilis and Morganella morganii clinical isolates recovered in the Military Hospital in Tunisia. Methods: Of 200 strains examined, 50 exhibited resistance to quinolones. Quinolone resistance determinants ( qnr and aac(6′)-Ib-cr) were characterized by multiplex PCR and sequencing. Chromosomal quinolone resistance mutations in the quinolone resistance-determining region (QRDR) and class 1 integron characterization were analysed by PCR and sequencing. The clonal relationship between the isolates was studied by pulsed-field gel electrophoresis (PFGE). Results: Fourteen isolates harboured qnrA6 and among them 8 (57%) were extended-spectrum beta-lactamase (ESBL) producers, whilst 12 (85%) isolates harboured blaDHA-1. Mutations in the QRDR were detected in gyrA (Ser83Ile, Glu87Lys), gyrB (Ser464Phe), and parC (Ser80Ile). qnrA6 and blaDHA-1 genes were found embedded in complex sul1-type class 1 integrons. A gene cassette carrying aac(6′)-Ib-cr was found located in the class 1 integron upstream of the qacEΔ1 gene. According to the PFGE analysis, the isolates were clonally unrelated. Conclusions: This is the first description in North Africa of class 1 integrons carrying blaDHA-1, qnrA6 gene, and aac(6′)-Ib-cr determinants in clinical strains of Proteus mirabilis and Morganella morganii. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae ML2508 was isolated from a patient at the surgery unit of the Military Hospital (Hôpital Militaire de Tunis), Tunisia. It was identified as a producer of extended-spectrum β-lactamases (ESBLs) by the double-disk synergy test. The β-lactamases produced by the strain were characterized by isoelectric focusing, determination of the specific activities against penicillins and cephalosporins, determination of the inhibitory concentration required to inhibit 50% of enzyme activity (IC), and the inhibition effect of EDTA on putative metallo-β-lactamases. The crude extract of K. pneumoniae ML2508 contains five different β-lactamases with pI 5.5, 7.3, 7.6, 8.1, and 8.6. Only the β-lactamase with pI 8.1 was transferred by transformation and conjugation experiments. Molecular characterization of these genes was performed by PCR and sequencing. The four chromosomal β-lactamases are TEM (pI 5.5), 2 SHV (pI 7.3 and 7.6), and CTX-M-28 (pI 8.6). The β-lactamase with pI 8.1 was encoded by bla gene located on a 50-kb highly conjugative plasmid. The study of the genetic context of bla was realized by PCR-mapping and DNA sequencing. A novel variant of tnp designated IS Ecp1C was detected upstream of the gene. [ABSTRACT FROM AUTHOR]

Klebsiella pneumoniae ML0306 was isolated from a patient in the intensive care unit of the Military hospital of Tunis in Tunisia. The strain was resistant to β-lactams, aminoglycosides, quinolones, phenicols and tetracyclines. Crude extract from the isolate showed a broad-substrate profile by hydrolyzing benzylpenicillin, ampicillin, cloxacilline, cephalothin, cefotaxime and ceftriaxone but not ceftazidime, cefoxitin, cefpirome, imipinem and aztreonam. The isolate was identified as a producer of pI 7.6 β-lactamase. Sequencing of the β-lactamase gene showed that it coded for a penicillinase SHV-11. This is the first description of this enzyme in Tunisia. The gene encoding this enzyme was located on the chromosome and a 50 kb conjugative plasmid. The overproduction of SHV-11 was involved in the wading of the hydrolytic spectrum of the strain. [ABSTRACT FROM AUTHOR]

Activation of 2-deoxyglucose transport in isolated rat fat cells by insulin is dependent upon the presence of Ca2+ in the external medium. When calcium concentration is kept below 100 μM, insulin acts like a partial agonist, giving only half of the maximal activation obtained normally with a millimolar concentration of this ion. Ocytocin, whose insulin-like action on adipocytes activates glucose oxidation by these cells was found to be unable to affect the rate of 2-deoxy- glucose transport. This, together with previous observations, suggests that calcium ions play a role in the mechanism of insulin action possibly by binding selectively to membrane sites involved in the transmission of the hormonal message to the glucose carrier. Ocytocin seems to trigger only intracellular glucose metabolism and it appears that there is an absolute requirement for calcium ions in the activation of a still unknown membrane signal. [ABSTRACT FROM AUTHOR]

Abstract: Escherichia coli CA0210 was identified in a stool culture of a 03-mounth-newonate in Tunisia. This strain was resistant to β-lactams, including ureidopenicillins, ticarcillin–clavulanic acid, cefpirome, ceftazidime, and cefotaxime, but it remained susceptible to imipenem and cefoxitin. The β-lactam–hydrolyzing β-lactamase gene of E. coli CA0210 and the upstream and downstream regions were cloned, sequenced, and expressed in E. coli DH5α. These resistances were carried by a 1080-bp chromosomal gene that encoded a β-lactamase with a pI of 6.3. Cloning and sequencing experiments showed that the corresponding bla TEM-15 gene was part of a chromosomally located Tn801 transposon. [Copyright &y& Elsevier]

Abstract: A clinical isolate of Escherichia coli LBT04 was found to have a high-level resistance to broad-spectrum β-lactams. Analysis of this strain by the disk diffusion test revealed synergies between clavulanic acid and ceftazidime, cefotaxime. Clavulanic acid decreased the MICs of ceftazidime, cefotaxime, and ceftriaxone, which suggested that LBT04 produced an extended-spectrum β-lactamase. These resistances were carried by a 1080-bp chromosomal gene that encoded a β-lactamase with a pI of 6.3. Cloning and sequencing experiments showed that this β-lactamase revealed identity with the bla TEM-1 gene encoding the TEM-1 β-lactamase, except for a replacement of the Glu residue at position 104 by Lys, and of the Gly residue at position 238 by Ser. These two mutations were encountered in TEM-15 β-lactamase, but this is the first description of this enzyme in the E. coli species in Tunisian hospitals. To cite this article: C. Chouchani et al., C. R. Biologies 330 (2007). [Copyright &y& Elsevier]

Klebsiella pneumoniae ML2011, a multiresistant isolate, was isolated from the Military Hospital of Tunis (Tunisia). The determination of the minimal inhibitory concentrations exhibited by K. pneumoniae ML2011 was performed by Etest. The crude extract of the isolates contains four different β -lactamases with pI 5.5, 7.3, 7.6, and 8.6. Only the β -lactamases with pI 7.3 and pI 8.6 were transferred by transformation and conjugation experiment. Molecular characterization of these genes was performed by PCR and sequencing. The chromosomal β -lactamases are TEM (pI 5.5) and SHV-1 (7.6). CTX-M-28 (pI 8.6) and the novel variant of SHV named SHV-104 (pI 7.3) were encoded by bla gene located on a 50 kb highly conjugative plasmid. The SHV-104 β -lactamase was produced in E. coli and purified. Its profile of activity was determined. Compared to SHV-1, SHV-104 contains one mutation, R202S. Their kinetic parameters were similar except for cefotaxime. The analysis of the predicted structure of SHV-104 indicated that the R202S mutation suppresses a salt bridge present in SHV-1. Therefore, the overall flexibility of the protein increased and might improve the hydrolysis of cefotaxime. We can conclude that the multiresistant phenotype of K. pneumoniae ML2011 strain is mainly linked to the production of CTX-M-28 since SHV-104 possesses a narrow spectrum of activity.

This study investigated the genetic environment of bla(CTX-M) genes and associated resistance genes in seven Proteus mirabilis and six Morganella morganii extended spectrum β-lactamase (ESBL)-positive isolates. The isolates were recovered from hospitalized patients with respiratory or urinary tract infections at the Military Hospital of Tunis, Tunisia. Twenty-one of the 200 strains exhibited non-susceptibility to third generation cephalosporins and, among these strains, the double-disk synergy test confirmed the ESBL phenotype in 13 isolates. These ESBL producers were co-resistant to chloramphenicol, tetracycline and oflaxacin but remained susceptible to ertapenem (MIC<0.25 mg l(-1)). The presence and nature of bla(CTX-M-15), bla(CTX-M-8), bla(TEM-24), bla(TEM-1) and bla(TEM-2) genes was determined by PCR and sequencing. Chromosomal localization of the bla(CTX-M-15) gene was confirmed in all strains, with the exception of M. morganii isolate M-17991, by Southern-blot analysis performed either on chromosomal or plasmid DNA. M. morganii M-12012 and M. morganii M-6019 showed the same PFGE pattern, whereas the remaining CTX-M-15-producing isolates were unrelated. The presence of ISEcp1 was ascertained in CTX-M-15-producing isolates. A class 1 integron with different gene cassettes (dfrA1, orfC and aadB) was found in five P. mirabilis and six M. morganii isolates.

Klebsiella pneumoniae ML1708 exhibited a multiresistance phenotype, including resistance to all beta-lactams tested, chloramphenicol, ciprofloxacin, nalidixic acid, tetracycline, and streptomycin. The double-disk synergy test was positive. ML1708 harbored a 50 kb conjugative plasmid that encoded a beta-lactamase of pI 5.5. The corresponding bla gene was identified by polymerase chain reaction and sequencing as a bla(TEM) gene. The deduced protein sequence revealed a new variant of TEM-1 beta-lactamase designated TEM-164. TEM-164 contains the unusual following mutations: L40V and I279T. These modifications may result in a change of the pI to 5.5 and hydrolyze cefotaxime and ceftazidime.

Escherichia coli CA0210 was identified in a stool culture of a 03-month-neonate in Tunisia. This strain was resistant to beta-lactams, including ureidopenicillins, ticarcillin-clavulanic acid, cefpirome, ceftazidime, and cefotaxime, but it remained susceptible to imipenem and cefoxitin. The beta-lactam-hydrolyzing beta-lactamase gene of E. coli CA0210 and the upstream and downstream regions were cloned, sequenced, and expressed in E. coli DH5alpha. These resistances were carried by a 1080-bp chromosomal gene that encoded a beta-lactamase with a pI of 6.3. Cloning and sequencing experiments showed that the corresponding blaTEM-15 gene was part of a chromosomally located Tn801 transposon.

Klebsiella pneumoniae CH0905 strain exhibiting high-level cefotaxime resistance was isolated from a stool culture in the intensive care unit. The resistance gene responsible was shown to be located on a conjugative 60-kb plasmid designated pCH0905. The minimum inhibitory concentration (MIC) values for cefotaxime and ceftazidime of the original isolate and the transconjugates were 256 mug/ml. Isoelectric focusing of a protein preparation from the K. pneumoniae strain showed beta-lactamases with the pI values of 7.6 and 6.3. A 1,080-bp fragment amplified with PCR was cloned into the pGEM-T Easy vector. The nucleotide sequence of the complete 1,080 bp was determined. Sequence analysis revealed that the bla(TEM) gene of pCH0905 differed from bla(TEM-1) by two mutations, leading to the following amino acid substitutions: the glutamic acid residue at position 104 by lysine and the glycine residue at position 238 by serine (Ambler numbering). The association of these two mutations was described previously in TEM-15 beta-lactamase, but this is the first detection of this enzyme in Tunisia.

A novel natural TEM beta-lactamase with extended-spectrum activity, TEM-138, was identified in a ceftazidime-resistant clinical isolate of Salmonella enterica serovar Infantis. Compared to TEM-1, TEM-138 contains the following mutations: E104K, N175I, and G238S. The bla(TEM-138) gene was located on a 50-kb transferable plasmid. Expression studies with Escherichia coli revealed efficient ceftazidimase and cefotaximase activities for TEM-138.

Using reversible electropermeabilization of cells and spheroplasts, we show that the cell wall and plasma membrane partly account for bleomycin resistance by acting as two independent barriers. We also report on the presence of a membrane protein that may be responsible for bleomycin internalization and toxicity in Saccharomyces cerevisiae.

The resistance phenotype of the clinical isolate of Escherichia coli 1941 was characterized by high-level resistance to penicillins and to combinations amoxicillin-ticarcillin/clavulanate and ampicillin/sulbactam. This resistance was carried by the conjugative plasmid pEC1941 that encoded a beta-lactamase activity. The purified enzyme focused at pI 5.4 and was strongly inhibited in vitro by clavulanic acid (IC50 = 0.09 microM). Nucleotide sequence analysis revealed identity between the plasmid borne blaTEM gene of E. coli 1941 and the blaTEM-1B gene, except for a single C-to-T substitution at position 32 in the promoter region leading to the overlapping promoters Pa and Pb. No alterations in the expression of outer membrane porins OmpC and OmpF have been detected. These findings show that the resistance of E. coli 1941 to the combinations of beta-lactams with beta-lactamase inhibitors is related to high-level production of TEM-1 enzyme expressed from the strong promoters Pa and Pb.

Pseudomonas aeruginosa 802 was isolated at Rabta hospital in Tunis and was resistant to extended-spectrum cephalosporins and aztreonam. It produced a pI 7.6 extended-spectrum beta-lactamase (ESBL). The ESBL, named LBT 802, was purified to homogeneity by filtration on Sephadex G-75 followed by CM-Sepharose chromatography and high-performance liquid chromatography (HPLC) on a TSK-gel SP-5PW column. The LBT 802 enzyme had a molecular mass of 30 kDa. It showed a broad-substrate profile by hydrolyzing benzylpenicillin, ampicillin, cephalothin, cephaloridine, cefotaxime, ceftriaxone, and cefpirome but not ceftazidime, cefoxitin, imipenem, or aztreonam. The highest hydrolytic efficiency (Vmax/Km) was obtained for ampicillin, cephalothin, cephaloridine, and benzylpenicillin. Among extended-spectrum cephalosporins the best substrate was ceftriaxone followed by cefotaxime and cefpirome. LBT 802 activity was inhibited by clavulanic acid, sulbactam, imipenem, cefoxitin, and aztreonam. It showed its lowest Ki values for clavulanic acid, imipenem and sulbactam.