Genetic heterogeneity and predominance of blaCTX-M -15 in cefotaxime-resistant Enterobacteriaceae isolates colonizing hospitalized children in Tunisia.

Extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae have emerged as important nosocomial pathogens. Community infections by these organisms have been also reported and were associated with previous intestinal colonization. We aimed to characterize cefotaxime-resistant Enterobacteriaceae (CTX-R-En) isolated from hospitalized children in a Tunisian paediatric ward. Seventy CTX-R-En isolates were collected from 227 rectal swabs from hospitalized children in a paediatric ward. Antimicrobial susceptibility testing was determined according to the EUCAST guidelines. Isolates were characterized by polymerase chain reaction (PCR, genes encoding: ESBLs, pAmpC, carbapenemases, plasmid-mediated quinolone resistance, virulence factors in Escherichia coli and Klebsiella pneumoniae isolates, occurrence of classes 1 and 2 integrons, phylogenetic groups of E. coli isolates, ERIC-PCR and PCR-based replicon typing) and conjugal transfer experiments. In total, 65 out of 227 (28·6%) hospitalized children were colonized with CTX-M-R-En, and 70 isolates were identified. Isolates were 59 ESBL-, 7 plasmidic-AmpC (pAmpC)-, 3 ESBL+pAmpC-, and one ESBL+carbapenemase producers. The following bla genes were identified: blaCTX-M-15 (n = 54), blaCTX-M-1 (n = 5), blaCTX-M-9 (n = 2), blaCTX-M-13 (n = 1) and blaCTX-M-14 (n = 1), blaCMY-2 (n = 5), blaCMY-4 (n = 4), blaACC-1 (n = 1) and blaOXA-48 (n = 1). Our results showed that hospitalized children were colonized with various CTX-R-En-producing several beta-lactamase enzymes.

High rates of macrolide resistance among clinical isolates of Streptococcus agalactiae in Tunisia.

One hundred sixty non duplicate erythromycin resistant Streptococcus agalactiae isolates were collected in Tunisia from January 2005 to December 2007 They were investigated to determine their resistance level to different macrolides and the mechanisms involved. Most erythromycin resistant S. agalactiae isolates were isolated from urinary specimens (38.75%, 62/160). The constitutive MLSB phenotype (cMLS) showed in 84.3% (135/160) with high MICs of macrolides and lincosamides (MIC90>256 microg/mL) and 8.2% (13/160) inducible MLSB phenotype (iMLS) with high MICs of macrolides (MIC90>256 microg/mL) and moderately increased MICs of lincosamides (MIC90=8 microg/mL). The M phenotype showed in 7.5% (12/160) with moderately increased MICs of macrolides (MIC90=32 microg/mL) and low MICs of lincosamides (MIC90=0.75 microg/mL). All strains were susceptible to quinupristun-dalfopristin association and linezolid (MIC90: 05 and 0.38 microg/mL respectively). Strains with MLSB phenotype harboured erm(B) gene with 825% (n=132), erm(TR) gene with 8.12% (n=13) and erm(B) plus mef (A) with 1.88% (n=3). All strains categorized as M phenotype carried the mef(A) gene (75%, n=12). cMLSB phenotype conferring cross resistance to macrolides, lincosamides and streptogramins B with high level of resistance was the most prevalent.

Polymorphism of ftsI gene in Haemophilus influenzae and emergence of cefotaxime resistance in two Tunisian hospitals.

The decreased affinity to ß-lactams in Haemophilus influenzae is usually caused by specific alterations in penicillin-binding protein 3 due to varieties of substitutions in ftsI gene. This study aimed to characterize the polymorphism of ftsI gene in 19 H. influenzae strains, isolated between 2014 and 2016 (different resistance phenotypes to ß-lactams (n = 9) and susceptible strains (n = 10) used for comparative purposes). All strains were characterized for capsular type by PCR and agglutination tests and for ß-lactam resistance by amplification and sequencing of ftsI. Biotyping and clonality were performed by API-NH and pulsed-field gel electrophoresis, respectively. Four strains were ß-lactamase-negative ampicillin-resistant and five were ß-lactamase-positive clavulanic-acid-resistant. One strain from each group was resistant to cefotaxime. Our isolates belonged mainly to biotype IV and I and were non-typeable and genetically unrelated. According to mutation profiles of their ftsI, strains were classified as group I (n = 3), group II (n = 4), group-III-like (n = 1) and group III (n = 1). All group II strains were further classified as subgroup IIb, except for one strain, which harboured a new mutation (N422I). Ampicillin MICs of ß-lactamase-negative ampicillin-resistant strains were 6 to 12 times the MICs of susceptible strains. Only bla TEM-1 was detected in ß-lactamase-positive clavulanic-acid-resistant strains, and was responsible for high MICs for ampicillin (>256 mg/L), whatever the ftsI mutational resistance group. The emergence of cefotaxime-resistant isolates in our country is a matter of concern and requires strict surveillance and rationalization of antibiotic use to preserve these molecules.

[Methicillin resistant Staphylococcus aureus: phenotypic and genotypic studies]. / Staphylococcus aureus résistants à la méticilline: etude phénotypique et génotypique.

BACKGROUND: Staphylococcus aureus is a human opportunistic pathogen. Its important pathogenicity and the increasingly rate of resistance to methicillin are the main causes of morbidity and mortality. AIM: In order to evaluate the epidemiologic situation of Methicillin Resistant S. aureus (MRSA) at Charles Nicolle hospital. METHODS: A four years retrospective study (January 1999-December 2002) was conducted. RESULTS: 65 non redundant MRSA isolates were collected. Identification was based on morphology, culture and biochemical characters. Antibiotic susceptibility was determined by disk diffusion method. Resistance to methicillin was confirmed by mec A PCR. Molecular typing was performed by Random Amplified Polymorphic DNA using ERIC-IR. Despite a perfect biotypic similarity between strains, ERIC-IR PCR revealed 7 genotypes. CONCLUSION: The combination of phenotypic methods and RAPD fingerprinting were easy to perform routinely for MRSA typing. However, phylogenetic relationship between strains needs more investigations.

Accessory gene regulator (agr) typing of Staphylococcus aureus isolated from human infections.

Staphylococcus aureus is a major hospital and community acquired pathogen. A total of one hundred strains were investigated. They were collected from January 2004 to July 2006 in the laboratory of microbiology at Charles Nicolle University hospital of Tunis. The isolates were identified by conventional methods. Methicillin resistance was confirmed by amplification of mecA gene by PCR. The agr groups were identified by multiplex PCR. The agr groups were distributed as follows: 19 strains belonged to group I, 16 to group II and 65 to group III. Among methicillin resistant S. aureus (MRSA), 9 (16.4%) belonged to group 1, 8 (14.5%) to group II and 38 (69.1%) to group IlI. For methicillin susceptible S. aureus (MSSA), only 10 strains (22.2%) belonged to group I, 8 (17.8%) to group II and 27 (60%) to group III. A preferential link was observed between agr group I and invasive infections (P=0.003) especially bacteremia (P=10(-4). Besides, agr groups II and III were closely related with non invasive infections (P=0.003). No association was found between other types of infections and agr groups. Likewise, no correlation was observed between agr groups, age or sex of patients and type of infections.

A comparative study of antimicrobial resistance rates and phylogenetic groups of community-acquired versus hospital-acquired invasive Escherichia coli.

OBJECTIVES: Escherichia coli is the leading cause of various infections, both in community and nosocomial settings. Our objective was to determine the antibiotic resistance rates and the phylogenetic groups of invasive E. coli and to assess the relationship between these characteristics according to the community or nosocomial origin of the strains. MATERIALS AND METHODS: One hundred non-redundant E. coli strains, causing invasive infections, were collected and investigated between 2010 and 2012. The phylogenetic groups were determined by triplex PCR. The statistical analysis was performed with Pearson χ(2) test and P-values below 0.05 were considered as statistically significant. RESULTS: Sixty-three strains were community-acquired (CA) and 37 were hospital-acquired (HA). The resistance rates among CA and HA strains were respectively: cefotaxime (11.1/37.8%), ciprofloxacin (19/43.2%), amikacin (3.2/27.2%), and cotrimoxazole (42.8/64.8%). E. coli strains caused bacteremia (CA=34.9%; HA=83.7%), peritonitis (CA=58.7%; HA=13.5%), appendicitis (CA=3.2%; HA=2.7%), and cholecystitis (CA=3.2%; HA=0%). The distribution of phylogenetic groups among CA and HA strains was: A (25.4/18.9%), B1 (9.5/16.2%), B2 (23.8/37.8%), and D group (41.3/27%). High resistance rates to cefotaxime (P=0.02), ciprofloxacin (P=0.01), amikacin (P=0.001), and cotrimoxazole (P=0.05) were statistically significantly associated with a nosocomial origin. CONCLUSION: Our results prove the diversity of phylogroups among E. coli invasive strains whatever their origin, and a higher antibiotic resistance rate in nosocomial strains. An adequate use of antibiotics and applying strict hygiene measures would limit the transmission and selection of these bacteria in hospital as well as in community settings.

Evolution of acquired resistance to third-generation cephalosporins in Enterobacteriaceae in a Tunisian hospital 1993-2001.

Between January 1993 and December 2001, the overall frequency of resistance to third-generation cephalosporins in isolates of Enterobacteriaceae from Charles Nicolle Hospital, Tunis, rose from 2.4% to 7.4%. Klebsiella pneumoniae was the most prevalent species (56%), followed by Escherichia coli (15%) and Proteus mirabilis (9%). A rate of 49% was observed among isolates from paediatric patients in 1999, caused mostly by outbreaks in the neonatal intensive care unit of K. pneumoniae and P. mirabilis isolates that produced extended-spectrum beta-lactamases.

Evaluation of a cefoxitin disk diffusion test for the routine detection of methicillin-resistant Staphylococcus aureus.

Two oxacillin disk methods were compared with a cefoxitin disk diffusion test for detection of methicillin-resistant Staphylococcus aureus (MRSA), with PCR for mecA as the reference method. When tested with 115 MRSA and 350 methicillin-susceptible S. aureus isolates, the cefoxitin disk test (specificity 100%, sensitivity 96.5%) was superior to the oxacillin disk methods (specificity 99.1%, sensitivity 90.4%). Testing with both oxacillin and cefoxitin disks would give better sensitivity (100%) than the cefoxitin test alone, but at the expense of specificity (99.1%). The cefoxitin disk test required no special test conditions and would improve the reliability of routine tests for detection of MRSA.

[Bacillus anthracis: causative agent of anthrax]. / Bacillus anthracis: agent de la maladie du charbon.

Anthrax, an acute infectious disease of historical importance, is once again regaining interest with its use as a biological weapon. It is caused by B. anthracis, a Gram positive spore forming rod usually surrounded by a capsule and producing toxin. It occurs most frequently as an epizootic or enzootic disease of herbivores that acquire spores form direct contact with contaminated soil. Spores can survive for many years in soil. Animal vaccination programs have reduced drastically the disease in developed countries. In humans, the disease is acquired following contact with anthrax infected animals or their products. 3 types of anthrax infection can occur: cutaneous, inhalational and gastro intestinal. Cutaneous anthrax is the most common observed form. When germination occurs, replicating bacteria release toxin leading to hemorrhage, edema, necrosis and death. Full virulence of B. anthracis requires the presence of both antiphagocytic capsule and 3 toxin components (protective antigen, lethal factor and edema factor). Most naturally occurring anthrax strains are sensitive to penicillin but resistant to third generation cephalosporins. Post exposure prophylaxis is indicated to prevent inhalational anthrax.

Macrolide and tetracycline resistance in clinical strains of Streptococcus agalactiae isolated in Tunisia.

Between 2007 and 2009, 226 clinical strains of Streptococcus agalactiae, recovered from female genital specimens and from gastric fluid or ear specimens from infected newborns, were isolated at the Laboratory of Microbiology of Charles Nicolle Hospital of Tunis. They were investigated to determine the prevalence of antibiotic resistance and to characterize the mechanisms of resistance to macrolide and tetracycline. All strains were susceptible to penicillin, ampicillin and quinupristin-dalfopristin. They were resistant to chloramphenicol (3.1%), rifampicin (19.1%), erythromycin (40%) and tetracycline (97.3%); 3.1% were highly resistant to streptomycin and 1.3% to gentamicin. Among the erythromycin-resistant isolates, 78.7% showed a constitutive macrolide-lincosamide-streptogramin B (MLS(B)) phenotype with high-level resistance to macrolides and clindamycin (MIC(50) >256 µg ml(-1)), 10% showed an inducible MLS(B) phenotype with high MICs of macrolides (MIC(50) >256 µg ml(-1)) and low MICs of clindamycin (MIC(50)=8 µg ml(-1)) and 2.2% showed an M phenotype with a low erythromycin-resistance level (MIC range=12-32 µg ml(-1)) and low MICs of clindamycin (MIC range: 0.75-1 µg ml(-1)). All strains were susceptible to quinupristin-dalfopristin and linezolid (MIC(90): 0.75 µg ml(-1) for each). MLS(B) phenotypes were genotypically confirmed by the presence of the erm(B) gene and the M phenotype by the mef(A) gene. Resistance to tetracycline was mainly due to the tet(M) gene (93.1%) encoding a ribosome protection mechanism. This determinant is commonly associated with the conjugative transposon Tn916 (P≤0.0002). tet(O) and tet(T) existed in a minority (2.2% and 0.4%, respectively). The efflux mechanism presented by tet(L) was less frequently present (4.5%). No significant association was found between erm(B) and tet(M) genes.

Emergence of carbapenem-resistant OXA-48 carbapenemase-producing Enterobacteriaceae in Tunisia.

We screened 21 extended spectrum ß-lactamase-producing Enterobacteriaceae with reduced susceptibility to carbapenems for carbapenemase production. Five strains (four Klebsiella pneumoniae and one Citrobacter freundii) showed carbapenemase production, which was identified as OXA-48. The bla(OXA-48) gene was detected on ~54 kb plasmids belonging to IncA/C in one case. Two isolates harboured IS1999, which is involved in bla(OXA-48) mobilization. Carbapenem resistance in enterobacteria should be regarded as an emerging clinical problem in our hospital and necessitates rigorous surveillance in order to limit its spread.

Nosocomial outbreak of imipenem-resistant Pseudomonas aeruginosa producing VIM-2 metallo-ß-lactamase in a kidney transplantation unit.

BACKGROUND: Twenty four non replicate imipenem resistant P. aeruginosa were isolated between January and November 2008, in the kidney transplantation unit of Charles Nicolle Hospital of Tunis (Tunisia). This study was conducted in order to establish epidemiological relationship among them and to identify the enzymatic mechanism involved in imipenem resistance. METHODS: Analysis included antimicrobial susceptibility profile, phenotypic (imipenem-EDTA synergy test) and genotypic detection of metallo-ß-lactamase (MBL) (PCR), O-serotyping and pulsed-field gel electrophoresis. RESULTS: All strains showed a high level of resistance to all antimicrobials tested except to colistin. The presence of MBL showed concordance between phenotypic and genotypic methods. Sixteen isolates were identified as VIM-2 MBL-producers and 13 of them were serotype O4 and belonged to a single pulsotype (A). CONCLUSIONS: This study describes an outbreak of VIM-2-producing P. aeruginosa in a kidney transplantation unit. Clinical spread of blaVIM-2 gene is a matter of great concern for carbapenem resistance in Tunisia.

Epidemiological markers of Streptococcus pyogenes strains in Tunisia.

To further understand the epidemiology of Streptococcus pyogenes or group A streptococcus (GAS) infections in Tunisia, phenotypic and genomic markers of GAS isolates, including antibiotic susceptibility, biotypes, T and emm types and toxin gene profiles, have been characterized. A total of 103 isolates, collected between 2000 and 2006, were investigated; 47 were recovered from invasive infections, and 56 from non-invasive infections. Rates of resistance to tetracycline, erythromycin, clindamycin and rifampin were 70.8%, 4.8%, 4.8% and 0.9%, respectively. High levels of resistance to streptomycin and kanamycin were observed in 1.9% and 4.8% of isolates, respectively. Biotype 3 was most common. Twenty different T patterns were observed, with a predominance of T3/13/B3264, and 38 different emm types. In both invasive and non-invasive isolates, emm118 (9.7%), emm42 (8.7%), emm1 (7.8%), st432 (6.8%), emm28 (5.8%) and emm76 (5.8%) were the most prevalent types; emm1, emm76 and emm18 were mainly observed among invasive infections, whereas emm118 (12.5%), emm42 (10.7%) and emm28 (8.9%) were predominant among non-invasive infections. The speB gene was detected in all isolates, but there were variable frequencies of speA, speC and ssa (20.3%, 32% and 25.2% respectively). Significant associations of emm1, emm18 and emm3 with speA and of emm4 and st432 with ssa were found. This first report from Tunisia revealed a unique emm distribution of GAS that differs from those of other regions. This information on the distribution of such emm types will be useful for the development of an appropriate vaccine in a country where the incidence of rheumatic fever remains high.

Phenotypic and genotypic characterization of macrolide resistant Streptococcus pneumoniae in Tunisia.

One hundred of non duplicate Streptococcus pneumoniae resistant to erythromycin collected from three teaching hospitals in Tunisia from January 1998 to December 2004 were investigated to evaluate determine their resistance level to different macrolides and the mechanisms involved. Most erythromycin resistant S. pneumoniae were isolated from respiratory tract (34%). Eighty-three percent showed constitutive MLS(B) phenotype with high MICs of macrolides and lincosamides (MIC90 >256 microg/ml), 12% M phenotype with moderately increased MICs of macrolides (MIC90: 12 microg/ml) and low MICs of lincosamides (MIC90=0.75 microg/ml) and 5% inducible MLS(B) with high MICs of macrolides (MIC90 >256 microg/ml) and moderately increased MICs of lincosamides (MIC90=8 microg/ml). All strains were susceptible to quinupristun-dafopristin association and linezolid (MIC90=1 microg/ml). Strains belonging to MLS(B) phenotype were PCR positive for the erm B gene (88%). Twelve percent categorized as M phenotype carried the mef A gene. The rates of associated resistance were 68% to penicillin G, 53% to tetracyclines, 61% to cotrimoxazole, 21% to chloramphenicol and 13% to ciprofloxacin. MLS(B) constitutive phenotype conferring cross resistance to macrolides, lincosamides and streptogramins B with high level of resistance was the most prevalent. Thus, quinupristin-dalfopristin association and linezolid remain the most active molecules.

Identification of staphylococcal cassette chromosome mec encoding methicillin resistance in Staphylococcus aureus isolates at Charles Nicolle Hospital of Tunis.

Staphylococcal cassette chromosome is a mobile element that carries the gene mecA mediating the methicillin resistance in staphylococci. In Staphylococcus aureus five types of SCCmec have been described, which differs in size and genetic composition among strains. SCCmec typing of 34 non redundant methicillin-resistant S. aureus (MRSA) recovered in 2004 at Charles Nicolle Hospital of Tunis was carried out. The isolates were identified by conventional methods. Methicillin resistance was detected by oxacillin and cefoxitin disks and confirmed by mecA PCR. The SCCmec complex types were determined by using PCR which amplify a sequence overlapping the right SCCmec chromosome junction. Strains were recovered mainly from cutaneous pus (61.7%) and blood cultures (17.64%). They were isolated from different wards: medicine (53.1%) especially from dermatology (41.2%); surgery (40.6%) and pediatrics (3.1%). Only two strains were community-acquired MRSA. Two strains (5.9%) were harboring SCCmec type I; five (14.7%) SCCmec type II and 27 (79.4%) SCCmec type III. The two community-acquired MRSA were harboring type II and III SCCmec, usually found in hospital acquired MRSA. Our findings indicate that there are only three SCCmec types at Charles Nicolle Hospital. However, the existence of SCCmec types II and III in community incite us to investigate more community-acquired MRSA.

Prevalence of agr specificity groups among methicilin resistant Staphylococcus aureus circulating at Charles Nicolle hospital of Tunis.

Methicillin resistant Staphylococcus aureus (MRSA) is a major human pathogen with many clinical aspects. In S. aureus, the accessory gene regulator (agr) globally controls the production of virulence factors. There are four agr groups. Our study was done to define the agr specificity of MRSA circulating at Charles Nicolle hospital and to investigate a possible relationship between agr groups and human disease types. From January 2004 to June 2005, a total of 57 MRSA isolated from individual hospitalized patients were collected, representing 12% of the total S. aureus isolates. The isolates were identified by conventional methods. Methicillin resistance was detected by oxacillin and cefoxitin disks and confirmed by the amplification of mecA gene by PCR. The agr groups were identified by multiplex PCR. All the strains were recovered from different wards: medicine (57.8%) especially from dermatology (56.2%), surgery (28%) and pediatrics (7%). Cutaneous pus (36.84%) and blood culture (35.08%) represented the main specimens. The agr groups were distributed as follow nine (15.7%) belonged to group I, two (3.5%) belonged to group II and 23 (40.3%) belonged to group III. For 23 strains, the agr group was not identified. A relationship between agr group and type of disease was observed: agr group III strains were associated with non invasive infections (P=0.02) and agr group I strains with invasive infections especially bacteremia (P=0.002).

[Outbreak of nosocomial urinary tract infections due to a multidrug resistant Pseudomonas aeruginosa]. / Epidémie d'infections urinaires nosocomiales à Pseudomonas aeruginosa multirésistant aux antibiotiques.

An outbreak of a multidrug resistant Pseudomonas aeruginosa including imipenem resistance occurred in the urology intensive care unit at Charles Nicolle Hospital (Tunis). All isolates presented the same antibiotic resistance pattern and were only susceptible to colistin. The epidemic strain was detected in different sites of this unit. Pulsed-field gel electrophoresis after enzymatic restriction using XbaI was performed in order to establish an epidemiologic link between these infections. Genotypic analysis showed two different patterns and the environmental source was identified in both cases. Although the same antibiotype was harbored by all the isolates, two outbreaks occurring in the same period were identified. The strengthening of hygiene measures allowed to stop the outbreak spreading. Since the hospital environment is the major source of Pseudomonas aeruginosa contamination, a continuous surveillance of the patients and the environmental sources is required for the implementation efficient control measures.