Effects of chitin and its derivatives on human cancer cells lines.

The present study is focused on the effect of chitin derivatives against human cancer cell lines RD and Hep2. As an outcome from this research, chitin was cytotoxic at IC50 = 400 µg/ml and 200 µg/ml against Hep2 cells and RD cells lines, respectively. Irradiated chitin had an IC50 value of 450 µg/ml for Hep2 and an IC50 of 200 µg/ml for RD. The lowest IC50 is attributed to chitosan, 300 µg/ml in Hep2 and 190 µg/ml in RD.

Virulence profiles and antibiotic susceptibility patterns of Klebsiella pneumoniae strains isolated from different clinical specimens.

AIM OF THE STUDY: To detect virulence factors in 54 Klebsiella pneumoniae isolates from different clinical specimens: urine (26), blood (11), pus (11), lung (four), cerebrospinal fluid (one) and ascitic fluid (one). MATERIAL AND METHODS: PCR was used to investigate virulence genes encoding adhesins (fimH-1, mrkD, kpn, ycfM), siderophores (entB: enterobactin, iutA: aerobactin, irp-1, irp-2, ybtS, fyuA: yersiniabactin, iroN: catechols receptor), protectines or invasins (rmpA, magA, traT) and toxins (hlyA, cnf-1). The serum resistance, capsule and hypermucoviscosity, and ability to form biofilm and produce siderophores were sought by phenotypic assays. The in vivo virulence was assessed in mice infected by intraperitoneal way. Antimicrobial susceptibility was tested by diffusion method. RESULTS: The most common virulence genes were fimH-1 (100%), mrkD (96.3%), ycfM (96.3%), and entB (100%). kpn and yersiniabactin genes were found at medium rates of 63% and 46.3% and at lower prevalence, were genes traT (1.8%), iroN (3.7%), iutA (5.5%) and rmpA (3.7%). magA, hlyA and cnf-1 genes were not detected. The capsule, serum resistance, biofilm formation, mannose-sensitive or -resistant haemagglutination and hypermucoviscosity were observed in 100%, 92.6%, 88.8%, 94.4%, 68.5% and 9.2% of isolates, respectively. The prevalence of siderophores was consistent with that of genotypic detection. The LD50 in mice was very low (<10(2) CFUs) for isolates with the most virulence factors. A rate of 74.1% of isolates showed a multidrug resistance (MDR) pattern. CONCLUSIONS: The distribution of virulence profiles according to the clinical origin suggests a role of enterobactin in urinary infections and yersiniabactin in the invasiveness. The fimbriae F1 and F3, capsule, enterobactin, serum resistance and biofilm formation, were commonly found in isolates, they seem to be at the basis of classic pathogenicity of K. pneumoniae. The invasiveness enhancers, aerobactin, yersiniabactin, catechols receptor, mucoid factor and hypermucoviscosity, detected concomitantly in some isolates, constitute a threat for vulnerable populations, even more if they are in combination with antibiotic resistance.

CTX-M-15 extended-spectrum beta-lactamases in Enterobacteriaceae in the intensive care unit of Tlemcen Hospital, Algeria.

The aim of this study was to detect extended-spectrum beta-lactamases (ESBL) in Enterobacteriaceae isolates in the intensive care unit (ICU) of Tlemcen hospital in north-western Algeria. Antimicrobial susceptibility testing, molecular typing, characterization of ESBL-encoding genes and the genetic environment, conjugation experiments and plasmid analysis were carried out. In all, 28 Enterobacteriaceae isolates were isolated from specimens recovered from patients in the ICU and 2 from surfaces of the unit. Of these, 11 isolates (4 Escherichia coli, 5 Klebsiella pneumoniae and 2 Enterobacter cloacae) produced ESBL of the CT-X-M-15 type. Molecular typing of the isolates showed the clonal nature of 4 K. pneumoniae isolates. The bla(CTXM-15) gene was genetically linked to insertion sequence lSEcp1B and was transferable by conjugation from 3 isolates. Regular monitoring of resistance mechanisms, the establishment of a prevention strategy, and more rational and appropriate use of antibiotics are needed.

CTX-M-15 extended-spectrum beta-lactamases in Enterobacteriaceae in the intensive care unit of Tlemcen hospital, Algeria

The aim of this study was to detect extended-spectrum beta-lactamases [ESBL] in Enterobacteriaceae isolates in the intensive care unit [ICU] of Tlemcen hospital in north-western Algeria. Antimicrobial susceptibility testing, molecular typing, characterization of ESBL-encoding genes and the genetic environment, conjugation experiments and plasmid analysis were carried out. In all, 28 Enterobacteriaceae isolates were isolated from specimens recovered from patients in the ICU and 2 from surfaces of the unit. Of these, 11 isolates [4 Escherichia coti, 5 Klebsiellapneumoniae and 2 Enterobacter cloacae] produced ESBL of the CT-X-M-15 type. Molecular typing of the isolates showed the clonal nature of 4 K, pneumonlae isolates. The bla[CTMX-15] gene was genetically linked to insertion sequence ISEcp1B and was transferable by conjugation from 3 isolates. Regular monitoring of resistance mechanisms, the establishment of a prevention strategy, and more rational and appropriate use of antibiotics are needed

Prevalence and characterization of extended-spectrum beta-lactamases in Klebsiella pneumoniae in Algiers hospitals (Algeria).

AIM OF THE STUDY: To determine the prevalence and the diversity of extended-spectrum beta-lactamases (ESBLs) in 196 Klebsiella pneumoniae clinical isolates collected from three hospitals in Algiers. MATERIALS AND METHODS: Antibiograms were done on Mueller-Hinton agar plates with the disc-diffusion method and MICs were determined by agar-dilution method. Mating experiments were performed in agar medium. Plasmid DNA was extracted by the alcalin-lysis method. Total DNA was extracted with a Qiagen mini kit and screened for bla(TEM) and bla(CTX-M) genes by PCR. Linkage of bla(CTX-M) genes with insertion sequence ISEcp1B and class 1 integrons was investigated by PCR. PCR products were sequenced by the Sanger method. The epidemiological relationships between ESBL-producing K. pneumoniae isolates were analyzed by ERIC-PCR. RESULTS: Thirty-nine (19.9%) isolates were found to produce ESBLs belonging to CTX-M-1 group and TEM penicillinases (CTX-M-3, CTX-M-15 and TEM-1). ERIC-PCR analysis showed that the isolates are genetically unrelated. The bla(TEM) and bla(CTX-M) genes as well as aminoglycosides and sulfonamides resistance determinants were found located in self-transferable plasmids of approximately 85 kb. The class 1 integrons and the insertion sequence ISEcp1B were present in the isolates and in their transconjugants. ISEcp1B was found genetically linked to the bla(CTX-M) genes and located 127bp upstream, with the presence of the V and W sequences. CONCLUSION: The study revealed a high rate of ESBL-producing K. pneumoniae in Algerian hospitals, resulting from horizontal dissemination of mobile bla(CTX-M) genes.

Antibiotic susceptibility and mechanisms of beta-lactam resistance among clinical strains of Pseudomonas aeruginosa: first report in Algeria.

OBJECTIVE: Pseudomonas aeruginosa is a major causative agent of hospital infections. Studies on this subject being rare in Algeria, we determined the antibiotic susceptibility of P. aeruginosa and investigated the mechanisms of beta-lactam resistance and the spread of multidrug resistant strains in the university affiliated Hospital of Tlemcen (Algeria). DESIGN: One hundred and ninety-nine consecutive strains of P. aeruginosa were collected between November 2005 and February 2007. MICs of antibiotics were measured by the agar dilution method. The resistance mechanisms to beta-lactams were identified phenotypically or by molecular methods (isoelectrofocusing, PCR and sequencing). Strains expressing a secondary beta-lactamase were serotyped and genotyped (Random Amplified Polymorphic DNA). RESULTS: The proportion of susceptible isolates were: ticarcillin (56%), piperacillin-tazobactam (81%), ceftazidime (88%), cefepime (80%), aztreonam (64%), imipenem (65%), amikacin (83%), tobramycin (81%) and ciprofloxacin (97%) according to the French CASFM breakpoints. Resistance to beta-lactams was linked to the production of transferable beta-lactamases (16%), overproduction of cephalosporinase AmpC (12%) and/or non-enzymatic mechanisms such as the loss of porin OprD (35%) and overproduction of the active efflux system MexAB-OprM (24%). High level resistance to ticarcillin was due to the expression of beta- lactamase OXA-10 alone or associated with TEM-110. A genotypic analysis revealed the spread of a multidrug resistant epidemic clone expressing these two acquired beta-lactamases in the surgical ICU. CONCLUSIONS: This study shows that resistance to antibiotics, in particular to imipenem of P. aeruginosa, is becoming a cause of concern in the Hospital of Tlemcen.

Prevalence of beta-lactams resistance among Escherichia coli clinical isolates from a hospital in Algiers.

A high prevalence of beta-lactams resistance among Enterobacteriaceae have been reported worldwide; however, there are not sufficient data on this issue in Algeria. beta-Lactams susceptibility of 203 Escherichia coli clinical isolates was determined by agar diffusion method, and production of extended-spectrum beta-lactamases (ESBL) was screened by double-disk synergy test. This analysis showed five well-defined phenotypes: 1) 62 isolates (30.5%) were susceptible to all beta-lactams; 2) 135 isolates (66.5%) presented a broad-spectrum beta-lactamases phenotype (BSBL); 3) three isolates (1.5%) were defined as producing ESBLs; 4) two isolates (1%) were AmpC cephalosporinase producers; and 5) one isolate (0.5%) presented a phenotype of cell-decreased permeability to beta-lactams. Isoelectric focusing revealed beta-lactamases with isolectric points of 5.4 or 7.6 for isolates with BSBL phenotype; approximately 9.0 for two ESBL isolates; 5.4, 7.6 and approximately 9.0 for the remaining ESBL isolate; and 5.4 and approximately 9.0 for the AmpC isolates. The cefotaxime hydrolysis corresponds to the basic bands with an isoelectric point of approximately 9.0. Conjugation assay showed transfer of penicillinase and AmpC resistance phenotypes and their corresponding beta-lactamases to recipient E. coli BM21 in association with plasmids of 71.4 kb for the AmpC isolates and from 40-56 kb for penicillinase isolates. This result showed that the AmpC phenotype is plasmid mediated. ESBL isolates were found not to transfer their resistance through conjugation experiment. Polymerase chain reaction (PCR) experiments using primers specific to blaTEM, blaAmpC and blaCTX-M genes showed specific amplification with blaCTX-M primer for two ESBL isolates; blaTEM and blaCTX-M for the remaining ESBL isolate; and blaTEM and blaAmpC for the AmpC isolates and their corresponding transconjugants. The study showed a high rate of isolates producing penicillinase, and low frequencies of AmpC and ESBL phenotypes. AmpC beta-lactamases were plasmid mediated, and ESBLs belong to the CTM-M type.

Prevalence of β - lactams resistance among Escherichia coli clinical isolates from a hospital in Algiers

La prevalencia de la resistencia a los betalactámicos entre las enterobacterias es alta en todo el mundo, pero en Argelia no se dispone desuficientes datos. Se determinó la sensibilidad a los betalactámicos de 203 cepas clínicas de Escherichia coli mediante difusión en agar y seanalizó la producción de betalactamasas de espectro extendido (BLEE) mediante la técnica de la sinergia del doble disco. Este análisis mostrócinco fenotipos bien definidos: 1) 62 cepas (30,5%) fueron sensibles a todos los betalactámicos; 2) 135 cepas (66,5%) presentaron unfenotipo caracterizado por betalactamasas de amplio espectro (BLEA); 3) 3 cepas (1,5%) se definieron como productoras de BLEE; 4) 2 cepas(1%) fueron productoras de cefalosporinasa tipo AmpC; y 5) una (0,5%) presentó un fenotipo de disminución de la permeabilidad celulara los betalactámicos. La determinación del punto isoeléctrico mostró betalactamasas con puntos isoeléctricos de 5,4 o 7,6 para las cepascon fenotipo BLEA; ∼9,0 para 2 cepas productoras de BLEE; 5,4, 7,6 y ∼9,0 para la tercera cepa productora de BLEE; y 5,4 y ∼9,0 para lascepas productoras de cefalosporinasa AmpC. La hidrólisis de cefotaxima se corresponde con las bandas básicas con un punto isoeléctricode ∼9,0. El ensayo de conjugación mostró una transferencia de los fenotipos de resistencia de penicilinasas y cefalosporinasa AmpC y suscorrespondientes betalactamasas a E. coli BM21 en asociación con plásmidos de 71,4 kb para las cepas productoras de cefalosporinasaAmpC y de 40-56 kb para las productoras de penicilinasas. Este resultado mostró que el fenotipo productor de cefalosporinasa AmpC estámediado por plásmidos. Las cepas productoras de BLEE no transfirieron su resistencia mediante el ensayo de conjugación. La reacción encadena de la polimerasa utilizando primers específicos para los genes blaTEM, blaAmpC y blaCTX-M mostró una amplificación específica con elprimer para blaCTX-M en dos cepas productoras de BLEE; los primers para blaTEM y blaCTX-M para la tercera cepa productora de BLEE; y losprimers para blaTEM y blaAmpC para las cepas productoras de cefalosporinasa AmpC y sus cepas transconjugantes correspondientes. El estudiomostró una alta tasa de cepas productoras de penicilinasas y una baja frecuencia de fenotipos productores de AmpC y BLEE. Las betalactamasasproductoras de cefalosporinasa AmpC estaban mediadas por plásmidos y las BLEE pertenecieron al tipo CTM-M

A high prevalence of β-lactams resistance among Enterobacteriaceae have been reported worldwide; however, there are not sufficient dataon this issue in Algeria. β-Lactams susceptibility of 203 Escherichia coli clinical isolates was determined by agar diffusion method, and productionof extended-spectrum β-lactamases (ESBL) was screened by double-disk synergy test. This analysis showed five well-defined phenotypes:1) 62 isolates (30.5%) were susceptible to all β-lactams; 2) 135 isolates (66.5%) presented a broad-spectrum β-lactamases phenotype(BSBL); 3) three isolates (1.5%) were defined as producing ESBLs; 4) two isolates (1%) were AmpC cephalosporinase producers; and 5) one isolate(0.5%) presented a phenotype of cell-decreased permeability to β-lactams. Isoelectric focusing revealed β-lactamases with isolectric pointsof 5.4 or 7.6 for isolates with BSBL phenotype; ∼9.0 for two ESBL isolates; 5.4, 7.6 and ∼9.0 for the remaining ESBL isolate; and 5.4 and ∼9.0for the AmpC isolates. The cefotaxime hydrolysis corresponds to the basic bands with an isoelectric point of ∼9.0. Conjugation assay showedtransfer of penicillinase and AmpC resistance phenotypes and their corresponding β-lactamases to recipient E. coli BM21 in association withplasmids of 71.4 kb for the AmpC isolates and from 40–56 kb for penicillinase isolates. This result showed that the AmpC phenotype is plasmidmediated. ESBL isolates were found not to transfer their resistance through conjugation experiment. Polymerase chain reaction (PCR) experimentsusing primers specific to blaTEM , blaAmpC and blaCTX-M genes showed specific amplification with blaCTX-M primer for two ESBL isolates;blaTEM and blaCTX-M for the remaining ESBL isolate; and blaTEM and blaAmpC for the AmpC isolates and their corresponding transconjugants. Thestudy showed a high rate of isolates producing penicillinase, and low frequencies of AmpC and ESBL phenotypes. AmpC β-lactamases wereplasmid mediated, and ESBLs belong to the CTM-M type

Genotypic and phenotypic characterization of potential virulence of intestinal avian Escherichia coli strains isolated in Algeria.

In order to characterize potential pathogenic Escherichia coli strains isolated from diarrheic hens and chickens originating from intensive battery rearing in North Algeria, the presence of a large range of virulence factors and markers was studied in 50 strains by DNA-DNA hybridization on colonies and phenotypic tests. The sequences we focused on were those coding for adhesins F5, F41, F17, Pap, Afa, and Sfa; intimin Eae; and toxins STa, STb, LT1, Stx1, Stx2, CNF1, and CNF2. The phenotypes explored were the colicins, aerobactin, hemolysins, and hemagglutinin production and serum resistance. The genotypic and phenotypic tests enabled us to categorize the isolates into two distinct groups: those with a potential to invade the host (27 strains were serum resistant and/or produced aerobactin), among which three strains were also potentially diarrheagenic, one strain was LT1 + F17+ Afa+ Pap+ (enterotoxigenic E. coli) and the two others were Stx1 (verotoxigenic E. coli). Twenty-three strains were colicinogenic, including 19 strains producing colicin V. This latter factor was also detected in isolates negative for the other virulence factors. On the basis of the type of erythrocytes agglutinated, we established 14 mannose-resistant hemagglutination patterns among the 37 strains tested, including 22 serum-resistant and/or aerobactin producing strains and 15 strains negative for these two characters. None of the strains produced alpha hemolysin, whereas two strains produced beta hemolysin and enterohemolysin, respectively. Congo red fixation was observed in 25 strains. No relationship could be detected between Congo red fixation and the presence of other virulence markers, such as serum resistance and aerobactin production. This study shows that among isolates originating from the feces of diarrheic chickens, the proportion of potentially diarrheagenic E. coli strains is low.

An Algerian child homozygous for the M470V polymorphism and for a deletion of two nucleotides in exon 10 of the CFTR gene, shows severe cystic fibrosis symptoms.

When screening for the presence of major cystic fibrosis mutations in Algerian cystic fibrosis families by heteroduplex formation, aberrant heteroduplexes were observed for exon 10 in one family. Here we describe the clinical and molecular findings in a severely affected child of this family, homozygous for the 1609delCA and for the M470V polymorphism.

[Optimization of culture conditions for the production of Salmonella typhi O and H antigenic suspensions]. / Optimisation des conditions de culture pour la production de suspensions antigéniques O et H de Salmonella typhi.

A Behring's production protocol of antigenic suspensions O and H of Salmonella typhi has been studied. The object of this study has been the Search of optimal conditions of cultivation and their exploitation on fermentor. A fermentation using the I.P.A culture medium has been realized to compare its results with that obtained using Behring's culture medium (B.C.M). The optimal cultivation conditions obtained are a waving rate of 400 tr/mn, a pH of 7.6 and an important air flow. Relating to nutritious constituents, the adequate glucose concentration is about 8g/1 and it seems better to replace meat extract by yeast extract. Comparatively to I.P.A culture medium, one of the most important advantage of Behring's culture medium modified reside in the conservation of the antigenicity of Salmonella typhi.

A simple adult-mouse test for tissue invasiveness in Yersinia enterocolitica strains of low experimental virulence.

The virulence of Yersinia enterocolitica depends on the presence of a 70-kilobase plasmid, called the Vwa plasmid. This situation is particularly favourable for studies of the mechanism of pathogenicity, but these are hindered by the lack of a suitable animal test to monitor the virulence of the human-pathogenic strains isolated outside the USA which belong to serogroups O:3, O:9 and O:5,27. We observed that, after oral administration to the mouse, the Vwa-positive strains of these serogroups produce a discrete systemic infection while the Vwa-negative strains do not. We present here a simple mouse-virulence test based on this observation.

Study of the incompatibility and replication of the 70-kb virulence plasmid of Yersinia.

The 70-kb virulence plasmid, vir, from four Yersinia enterocolitica and one Y. pseudotuberculosis strains are incompatible with IncFI plasmids F'Lac and R386 while they are compatible with plasmids representing nine other incompatibility groups. Hybridization experiments carried out on one of these virulence plasmids showed that it contains the F incompatibility determinant D, incD. This determinant was cloned onto pACYC184 and the recombinant clone expressed incompatibility with F'Lac. We conclude that the incompatibility observed between F or R386 and the 70-kb virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis is mediated by incD. Replication genes (rep) from the same plasmid were cloned independently in Escherichia coli. Rep and incD map on two different BamHI fragments. Surprisingly, the replicon isolated is not sensitive to inc D incompatibility. Apart from incD, vir and F share extremely little homology. In particular, there is no evidence for the presence of an F-like transfer operon on vir.

[Pathogenesis of post-irradiation infection. 2. Role of polynucleated neutrophils in the defense of irradiated rats against Yersinia enterocolitica]. / Pathogénie des infections après irradiation. 2. Rôle des polynucléaires neutrophiles dans la défense des rats irradiés contre Yersinia enterocolitica.

Wistar R inbred rats showed a substantial mortality when they were given Yersinia enterocolitica eight days after a 6.5 Gy total body irradiation. The possibility to abolish the high susceptibility of these irradiated rats to Yersinia enterocolitica by intravenous injections of isogenic neutrophils is presented: irradiated rats injected with 7 to 10.10(7) isogenic neutrophils, by the intravenous route, just before or after the administration of Yersinia enterocolitica, were not susceptible. On the contrary, control irradiated rats, not transfused, were killed by the same bacterial challenge.