[Show abstract][Hide abstract] ABSTRACT: This study was designed to isolate Shigella spp. strains from food and stool samples by a combination of PCR and culture methods and characterize their serotypes, antibiotic resistance profiles, virulence genes and pulsed-field gel electrophoresis (PFGE) patterns to investigate possible clonal relationships amongst strains circulating.
Six Shigella spp. strains were isolated from 280 food samples against 16 Shigella isolates from 236 stool samples of symptomatic patients and asymptomatic food handlers during the period from January 2007 to December 2009 in Public Health Regional Laboratory of Nabeul. The detection of ipaH, ipaBCD, ial, ShET-1 and ShET-2 was performed by a PCR technique with specific primers.
The use of PCR technique improved the rate of detecting Shigella in stool samples from 6·7 to 14% and in food samples from 2·1 to 8·6%. Percentage of Shigella isolates and ipaH-specific PCR demonstrated a marked pattern of seasonality, increasing in summer and fall seasons for human and food isolates. Amongst the environmental strains, 50% of isolates were invasive. However, for the 16 clinical strains isolated, nine were found to be positive for both ial and ipaBCD gene and 11 were found to produce ShET-1 and/or ShET-2. XbaI PFGE analysis revealed the presence of a predominant clone amongst Shigella sonnei strains recovered from different sources circulating in Nabeul, Tunisia, throughout the years 2007-2009.
This study demonstrated the existence of Shigella in food samples and dispersion of different virulence genes amongst these isolates, which appear to constitute an environmental source of epidemic spread. The clonal relationships amongst strains isolated from food elements and human stools indicate the incrimination of different kinds of foods as vehicle of transmission of Shigella, which are usually escaped from detection by traditional culture methods.
[Show abstract][Hide abstract] ABSTRACT: In 2009, out of the 66 nonrepetitive Enterobacter cloacae collected at Charles Nicolle hospital in Tunisia, 44 were extended spectrum β-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.
[Show abstract][Hide abstract] ABSTRACT: Staphylococcus haemolyticus is one of the most clinically relevant coagulase-negative staphylococci (CoNS), particularly in immunocompromised patients; however, little is known regarding its molecular epidemiology. In this work, we characterized the genetic background and the SCCmec region of 36 methicillin-resistant S. haemolyticus (MRSHae) and 10 methicillin-susceptible S. haemolyticus (MSSHae) collected from neutropenic patients in Tunisia between 2002 and 2004. The molecular characterization of MRSHae by pulsed-field gel electrophoresis (PFGE) showed that the great majority of the isolates (77.8%) belonged to only four types. SCCmec typing by polymerase chain reaction (PCR) and Southern hybridization showed that isolates belonging to each PFGE type could carry either one or two SCCmec types. SCCmec V was the most common, but mec complex C was frequently associated to ccr allotypes other than ccrC. The mec complex class C was predominant in MRSHae (47%) and ccrC was predominant among both methicillin-resistant and -susceptible isolates (31 and 50%, respectively). Interestingly, one half (50%) of the MRSHae isolates analyzed lacked the known ccr complexes (ccrAB and ccrC), although they carried the mecA. Conversely, all MSSHae carrying a ccrC complex were multidrug-resistant, although they lack the mecA. The results suggest that ccrC and mec complex C are frequent and may exist autonomously and independently of SCCmec type V in S. haemolyticus. Moreover, the data obtained suggest that small chromosomal rearrangements promoting the loss or structural variation of mec and ccr complex appear to occur frequently, which probably provide S. haemolyticus with a specialized means for SCCmec trapping and/or diversification.
European Journal of Clinical Microbiology 08/2011; 31(4):605-14. DOI:10.1007/s10096-011-1354-3 · 2.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Polymerase chain reaction (PCR) amplification of antibiotic resistance genes as well as staphylococcal cassette chromosome mec (SCCmec) typing and pulsed-field gel electrophoresis (PFGE) of SmaI macrorestriction fragments of genomic DNA were used to characterize 45 methicillin-resistant coagulase-negative staphylococci (MRCoNS) isolates responsible of bacteremia recovered in patients at the Bone Marrow Transplant Centre of Tunisia in 1998-2007. Among the 45 MRCoNS isolates, Staphylococcus epidermidis was the most prevalent species (75.6%) followed by Staphylococcus haemolyticus (22.2%) and Staphylococcus hominis (2.2%). Extended susceptibility profiles were generated for MRCoNS against 16 antimicrobial agents. Out of 45 mecA-positive strains, 43 (95.6%) were phenotypically methicillin-resistant and two (4.4%) were methicillin-susceptible. The msr(A) was the most prevalent gene (13 isolates; 48.1%) among erythromycin-resistant isolates. The erm(C) was found alone in seven (25.9%) or in combination with both erm(A) and erm(B) in two (7.4%) isolates. The aac(6')-Ie-aph(2″)-Ia was the most prevalent gene among aminoglycoside-resistant isolates, detected alone in 14 isolates (33.3%) isolates, in combination with ant(4')-Ia in 18 (42.8%) isolates, in combination with aph(3')-IIIa in four (9.5%) or with both ant(4')-Ia and aph(3')-IIIa in two (4.7%) isolates. The ant(4')-Ia was detected in three (7.1%) isolates and the aph(3')-IIIa in one (2.4%) isolate. Among tetracycline-resistant isolates, six (85.7%) strains harbored the tet(K) gene and one (14.3%) strain carried tet(K) and tet(M) genes. SCCmec types IV (31%) and III (24.5%), the most prevalent types detected, were found to be more resistant to non-β-lactam antibiotics. A wide diversity of isolates was observed by PFGE among MRCoNS.
[Show abstract][Hide abstract] ABSTRACT: Objectif
Le but de cette étude était de comparer des isolats cliniques de Candida tropicalis afin de déterminer leur polymorphisme génétique.
Les isolats de C. tropicalis ont été collectés pendant une période de sept mois (août 2007–avril 2008) à partir de patients hospitalisés dans une unité de réanimation de l’hôpital La Rabta en Tunisie.
Matériels et méthodes
Tous les isolats de C. tropicalis collectés des patients hospitalisés dans cette unité ont été typés par la technique d’électrophorèse en champ pulsé (PFGE).
Durand cette période, 15 patients se sont avérés positifs pour C. tropicalis et 34 isolats ont été collectés. Parmi ces 34 isolats, 29 appartenaient au même pulsotype A (avec trois sous-types A1, A2, A3), alors que deux isolats appartenaient au pulsotype B et trois au pulsotype C.
La majorité des isolats appartenaient au même pulsotype (85 %) mais le sous-type A1 était le plus représenté par rapport aux sous-types A2 et A3.
[Show abstract][Hide abstract] ABSTRACT: To investigate the prevalence of resistance to macrolide, lincosamide and streptogramin (MLS) antibiotics in Gram-positive cocci isolated in a Bone Marrow Transplant Center of Tunisia, we tested the antibiotic susceptibility of 172 clinical isolates of Staphylococcus epidermidis, Streptococcus mitis and Enterococcus faecium to macrolide erythromycin and spiramycin, the lincosamide clindamycin and the streptogramin pristinamycin. These three groups of organisms were mostly resistant to macrolides and lincosamide, but were commonly susceptible to pristinamycin. The resistance phenotypes of erythromycin-resistant isolates were determined by the five-disc test with erythromycin, spiramycin, lincomycin, clindamycin and pristinamycin, which showed that most exhibited constitutive MLS resistance. In order to determine the prevalence of the resistance genotypes and the resistance mechanisms, the prevalence of the erythromycin resistance methylase (erm) (A), erm(B), erm(C), msr(A) and macrolide efflux (mef) (A) genes in the erythromycin-resistant isolates was identified by polymerase chain reaction (PCR) analysis. The resistance was due mainly to the presence of ermB in E. faecium (80%), ermC in S. epidermidis (53%) and mefA in S. mitis (65%).
[Show abstract][Hide abstract] ABSTRACT: The performances of five commercial TaqMan real-time PCR assays for the detection of Mycoplasma pneumoniae in respiratory tract specimens were evaluated in comparison with an in-house real-time PCR. All kits allowed prompt and specific results, validated by the use of an internal control. The Nanogen kit showed the best clinical sensitivity.
[Show abstract][Hide abstract] ABSTRACT: We propose a simple and rapid method to discriminate SHV-type extended spectrum beta-lactamase (ESBL) genes in P. aeruginosa based on PCR techniques (PCR-RFLP and RSI-PCR). We studied 22 producing ESBL P. aeruginosa strains isolated from seven immunocompromised patients (19 isolates) and from environmental swabs (three isolates) at the Bone Marrow Transplantation Center of Tunis. Screening PCR with primer pairs designed to detect gene encoding TEM, SHV, OXA group I, OXA group II, OXA-18 and PER-1 ESBL was positive for bla(OXA18) and bla(SHV) genes in all isolates. Pulsed field gel electrophoresis using SpeI endonuclease defined five genotypic groups. For at least one isolate corresponding to each genotype observed, restriction of PCR products by DdeI and BsrI revealed the same restriction pattern that the bla(SHV-1) negative control; in the same way, RSI-PCR products digestion by NruI, thus excluding 35, 238 and 240 mutations characterizing reported ESBL in P. aeruginosa (SHV-2a, SHV5 et SHV12), and suggesting that studied bla(SHV) genes were not ESBL ones. Genomic DNA hybridization by southern blot with probe consisting in bla(SHV-1) gene was positive in these isolates. Sequencing the full-length open reading frame revealed nucleotide sequence of the bla(SHV-1). PCR-RFLP and RSI-PCR results were then confirmed. This approach is effective for screening P. aeruginosa for ESBL genes carriage in epidemiological studies and for detecting new variants.
[Show abstract][Hide abstract] ABSTRACT: The influence of protoplasting and protoplast regeneration on antibiotic activity, transfer of biosynthesis encoding genes in local Streptomyces spp. CN207 was studied. The frequency of regenerated protoplasts in the lag phase was 1.7x10 3 CFU/ml, in the beginning of the exponential phase 0.4x10 2 CFU/ml, in the exponential growth phase 2.5x10 3 CFU/ml, and 1.0x10 5 CFU/ml in stationary phase. The protoplast formation and regeneration technique resulted in a new isolate strain of Streptomyces spp.PR01 that produced approximately 5 fold more Streptomyces spp. CN207 antibiotic. The protoplast fusion resulted in increased isolation of variants with higher antibiotic activity. Recombinant Streptomyces coelicolor PF04 was increased 10 times more than the wild strain. The processes also affected on the strain resistance to some antibiotics but had no effect on the components of the antibiotic. The characteristics of this recombinant product were similar considerably to Streptomyces spp. CN207 product. Our data, in principal, indicate the possibility of transferring antibiotics cluster genes by fusion and provide a starting point for genetic and biochemical investigations of CN207 biosynthesis.
AFRICAN JOURNAL OF BIOTECHNOLOGY 10/2008; 7:3155-3161. · 0.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Twelve multidrug-resistant Pseudomonas aeruginosa (MDRPA) isolates were recovered over a period of two years in the National Bone Marrow Transplant Centre of Tunisia. MDRPA isolates were isolated from seven patients and from three environmental samples. Isoelectric focusing revealed pIs of 8.2, 5.5 and 7.6 in all MDRPA isolates. These strains produced the OXA-18 extended spectrum beta-lactamase and an SHV type beta-lactamase as shown by screening PCR analysis. DNA hybridization confirmed this inference, detecting bla(SHV) gene in these isolates. Pulsed-field gel electrophoresis (PFGE) defined one predominant genomic group; group A (seven isolates) and four different genotypes containing one to two isolates. Clonally related isolates were recovered from three patients and from two washbasins. Sequencing DNA of cluster representative strains identified the classical bla(SHV-1) gene. For these strains, the nucleotide sequence of the structural bla(SHV-1) gene was nearly identical to those previously described. Such enzyme has not been reported from P. aeruginosa. This is the first report of the SHV-1 penicillinase in epidemic P. aeruginosa strain.
[Show abstract][Hide abstract] ABSTRACT: Investigation of the occurrence and antibiotic susceptibility of Enterococcus faecium isolates, collected during four years from neutropenic patients at the Tunisian bone marrow transplantation centre.
E. faecium strains were identified by conventional methods and by the Api20 Strep (Bio-Mérieux, France). Antibiotic susceptibility was determined by the disk diffusion method on Mueller-Hinton agar and interpreted as recommended by CA-SFM. MICs of ampicillin, vancomycin, and teicoplanin were determined by E-test method.
Two hundred and thirty five E. faecium isolates were recovered from stool cultures or rectal swabs (229), throat (three), urine (two), and pus of wound (one). None was responsible for bacteraemia. Ampicillin resistance, without production of beta-lactamase, was observed in 43.8% of isolates. All the isolates were susceptible to glycopeptides. High rates of resistance were observed: high-level resistance (HLR) to gentamicin (33.6%), HLR to kanamycin (55.7%), HLR to streptomycin (47.6%), erythromycin (86.4%), ciprofloxacin (78.7%), rifampicin (85%), and tetracycline (43%). Strains with HLR to gentamicin were significantly more resistant to ampicillin and streptomycin. Multiple drug resistance was observed in most isolates.
These findings demonstrated the low pathogenic power of E. faecium in our patients, and the high frequencies of resistance to ampicillin and aminoglycosides. In the absence of glycopeptide-resistance, vancomycin remains an alternative treatment against multidrug resistant strains.
[Show abstract][Hide abstract] ABSTRACT: We report the analysis of genetic determinants conferring resistance to pristinamycin in Staphylococcus epidermidis strains and epidemiology typing of these strains by pulsed-field gel electrophoresis.
Staphylococcus epidermidis (346 isolates) were searched for strains with pristinamycin resistance. Pristinamycin-resistant strains (seven isolates) were isolated in five patients with haematological cancer in the Bone Marrow Transplant Centre of Tunisia in 2002. Resistance to pristinamycin was observed in 2% of isolates. The seven pristinamycin-resistant strains shared resistance to oxacillin (MIC = 8-512 microg ml(-1)), gentamicin (MIC = 16-512 microg ml(-1)), erythromycin (MIC > 1024 microg ml(-1)), lincomycin (MIC > 1024 microg ml(-1)), pristinamycin (MIC = 4-16 microg ml(-1)) and rifampin (MIC = 128-256 microg ml(-1)). erm genes were amplified: ermA from six strains and ermC from one. vga gene encoding streptogramins A resistance (pristinamycin résistance) was amplified from all strains and typed as vgaA by analysis after electrophoresis of restriction profiles of vga amplicons (two fragments with Sau3A of 164 and 378 bp; one fragment with EcoRI). Pulsed-field gel electrophoresis (PFGE) of SmaI chromosomal DNA digests of the seven S. epidermidis isolates divided them into two distinct pattern types: pulsed-field type A (classified from A1 to A6 subtypes) and type B. The six strains harbouring ermA genes belonged to the PFGE type A while the strain harbouring ermC genes belonged to the PFGE type B. We characterized an epidemic strain carrying the vgaA and ermA genes responsible for the outbreak.
Two clones of pristinamycin-resistant S. epidermidis were isolated in our patients. One of them, isolated in all patients, had expanded over six months suggesting acquisition by cross-contamination.
Increasing isolation of pristinamycin resistant S. epidermidis strains is an alarming indicator of nosocomial dissemination. The vector will be determined to establish a system of epidemiological surveillance.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to investigate the molecular epidemiology of Stenotrophomonas maltophilia strains responsible for respiratory infection in a neonatal intensive care unit (NICU) in Tunis City, isolated during 22 months (December 2003-September 2005).
Twelve strains of S. maltophilia isolated from tracheal aspirates of distinct infants and two environmental strains were tested for antibiotic susceptibility and genotyped by pulsed-field gel electrophoresis (PFGE) method.
Unlike a large heterogeneity demonstrated by the antibiotyping method, PFGE identified two concomitant outbreaks consisting of nine, including an environmental strain (clone A), and four strains (clone B), respectively; a distinguishable strain was classified in a unique pattern (PFGE type C). The long-term dissemination of these strains is a characteristic feature of these outbreaks. Improvement of hygienic conditions attributed to a markedly decrease in their isolation frequencies. Concomitant outbreaks and long period persistence of S. maltophilia in NICU is an important finding of this study.
Identification of two clonal strains of S. maltophilia responsible of respiratory infection. Epidemic strains are hardly eradicated when colonization is established.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to determine the overall percentage of beta-lactams susceptibility, beta-lactamase production, penicillin binding protein (PBP) modification and serotypes of colonizing Haemophilus influenzae strains.
A total of 50 isolates of colonized H. influenzae, isolated from neutropenic patients. The prevalence of beta-lactams resistance and beta-lactamase production were recorded for each strains using E-test strips and chromogenic cephalosporin test, then were determined their resistance genes (bla(TEM) and bla(ROB)) by PCR as well as their capsular types by standard slide agglutination serotyping (SAST) and capsular genes amplification.
Thirty-two percent of the 50 strains were amoxicillin resistant, among these, 20% were resistant by beta-lactamase production, and they produced all type TEM beta-lactamase. Four percent of the isolates had PBP modification and three strains (6%) associated the two resistance mechanisms. Slide agglutination serotyping showed that 95.8% of the strains were unencapsulated, and 4.1% were of serogroup b. The result was confirmed by PCR capsular typing.
By the light of these results, our findings suggest that it becomes important to follow the evolution of the resistance background of our strains, and that the majority of colonizing H. influenzae strains isolated in our center are unencapsulated.
[Show abstract][Hide abstract] ABSTRACT: Thirty-five Staphylococcus aureus strains from auricular infections were isolated. The identification of strains was confirmed by Api ID 32 Staph strips, the antibiotic susceptibility test was performed using ATB Staph kit. PCR assay was used to detect the oxacillin resistance gene (mecA) and the erythromycin genes (ermA, ermB, ermC, msrA and mef). The susceptibility profile of all strains revealed a low resistance level to oxacillin and erythromycin. The PCR results show that 60 % of the strains are mecA positive. The frequency of erythromycin genes was: ermA (+) 22.8 %, ermB (+) 45.7, ermC (+) 17.1, msrA (+) 28.6. The mef gene was not detected in any strain. No correlations between genotypic and phenotypic methods for the determination of oxacillin and erythromycin resistance was found. However, multiplex PCR technique was shown to be a fast, practical and economic technique for the detection of methicillin-and erythromycin-resistant staphylococci.
[Show abstract][Hide abstract] ABSTRACT: Appreciation of the frequency, the level and the genetic support of methicillin resistance.
Seventy-three strains of coagulase negative staphylococci isolated from various specimens, from January to June 2004, were studied. The phenotypic detection was carried out by disk diffusion test using oxacillin and cefoxitin disks, by the determination of oxacillin Minimal Inhibitor Concentration (E-test), by the oxacillin screening test at a concentration of 4 mug/ml and by the search of the penicillin binding protein PBP2a using the slide latex agglutination test. The results of these methods were compared to PCR of mecA gene.
Forty-eight strains carried mecA gene whose 30 were detected by the oxacillin disk, the cefoxitin disk, the oxacillin screening test, the slide latex agglutination test and had a MIC from 24 to 256 mug/ml. Seventeen strains were not detected by oxacillin disk but by cefoxitin disk and the slide latex agglutination test. Among these strains, 13 (76%) had oxacillin MIC from 0.5 to 1,5 mug/ml and not grew on oxacillin agar screening, while 4 (24%) had oxacillin MIC from 6 to 16 mug/ml and grew on this agar. One strain had oxacillin MIC of 0,19 mug/ml and was not detected with any phenotypic method.
The determination of oxacillin MIC, the search of the PBP2a or more simply the cefoxitin disk had permitted to detect the strains mecA gene (+) with resistant and pre-resistant phenotype but not the strain with sensible phenotype (2.1%).
[Show abstract][Hide abstract] ABSTRACT: Following systematic screening for ceftazidime-resistant (CAZ-R) Pseudomonas aeruginosa, 24 isolates producing extended-spectrum beta-lactamase (ESBL) were recovered during a 24-month period at the National Bone Marrow Transplant Centre of Tunisia. These isolates were from seven immunocompromised patients and from environmental swabs. ESBLs inhibited by clavulanic acid were detected by double-disk diffusion tests. Isoelectric focusing revealed that these isolates produced two to four beta-lactamases with pIs of 5.5, 6.1, 6.4, 7.6 or 8.2, and PCR detected the presence of bla(OXA-18), bla(SHV) and bla(TEM) genes in 24, 21 and two isolates, respectively. Pulsed-field gel electrophoresis defined two dominant genotypic groups: group A (16 isolates) and group B (four isolates). Sequencing of PCR products from representative isolates identified the bla(OXA-18) gene and revealed nucleotide sequences belonging to the bla(SHV-1) and bla(TEM-1) genes. Isolates producing OXA-18 belonged to genomic group A and were isolated from four immunocompromised patients in the haematology and graft units, and from two wash-basins in the graft unit. No immunocompromised patient harboured the clonal epidemic strain upon admission. This is the first report of the OXA-18-type ESBL in P. aeruginosa in Tunisia, and the first description of an outbreak caused by an OXA-18-producing strain of P. aeruginosa.
[Show abstract][Hide abstract] ABSTRACT: Slime production, principal virulence factor of Staphylococcus epidermidis associated with catheter-related infections is mediated by icaADBC operon wich expression is subject to phase variation. Reversible transposition of IS256 element into this operon is one of the most important mechanisms of biofilm phenotypic variation. Our study compared 28 S. epidermidis strains from catheter-related infection to 28 strains from nasal carriage concerning slime production on Congo red agar plate and ica genes and IS256 presence by PCR. ica operon was present among all slime-producing strains, and was absent among slime-negative strains. Only 79% of ica-positive strains were slime producers and no insertion of IS256 element was detected inside ica genes. A significative difference was found between catheter-related infections strains and commensal ones in terms of oxacillin (67,8 versus 35,7%) and ofloxacin resistance (75 versus 35,7%), slime production (64,2 versus 28,5%), phase variability (46,4 versus 7,1%) and ica genes presence (82,1 versus 35,7%). Our study demonstrates the role of ica genes, of phenotypic variability of slime production and antibiotic multiresistance as virulence factors of S. epidermidis associated with catheter-related infections; it confirms also the complexity and the diversity of regulation mechanisms implicated in biofilm formation.