Virulence determinants, phylogenetic groups and fluoroquinolone resistance in Escherichia coli isolated from cystitis and pyelonephritis.

AIM OF THE STUDY: The aim of this study is to assess the relation between virulence genotype, phylogenetic group and susceptibility to fluoroquinolones and the urinary tract infection type including pyelonephritis and cystitis due to Escherichia coli. MATERIALS AND METHODS: Between 2006 and 2007, 129 non-duplicate E. coli isolates from pyelonephritis (n=56) and cystitis (n=73) were prospectively collected. The antibiotic susceptibility was done by disk diffusion method. The phylogenetic groups, A, B1, B2 and D and 18 virulence genes were determined by multiplex PCR. Statistical analysis was done with the Pearson χ2 test, Mann-Whitney U-test, Kruskal-Wallis test and stepwise multivariable logistic regression analysis, P values below 0.05 were considered statistically significant. RESULTS: For the pyelonephritis group, sex ratio was 0.3, the median age for women was 30 years and for men it was 54 years. For the cystitis group, sex ratio was 0.4, the median age for women was 41.5 years and for men it was 67.8 years. Significant statistical correlations were found between pyelonephritis isolates and susceptibility to ciprofloxacin (P=4 10(-5)), papG allele II (P=2 10(-6)), hlyA (P=10(-03)), iroN (P=0.04), iha (P=0.03) and ompT (P=0.03) virulence genes, high virulence score (P=0.008) and B2 phylogenetic group (P=0.03). In multivariate logistic regression analysis, papG II as predictor of pyelonephritis, no correlation could be established for the cystitis group. CONCLUSION: Our findings argue for a direct link between pyelonephritis, virulence factors, susceptibility to fluroquinolones and B2 phylogenetic group among uropthogenic E. coli.

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.

Correlation between meticillin-resistant Staphylococcus aureus prevalence and infection control initiatives within southern and eastern Mediterranean hospitals.

The Mediterranean region has been identified as an area of hyper-endemicity for multi-resistant hospital pathogens. To better understand potential drivers behind this situation, we attempted to correlate already published meticillin-resistant Staphylococcus aureus (MRSA) data from 27 hospitals, participants in the Antibiotic Resistance Surveillance & Control in the Mediterranean Region (ARMed) project, with responses received from the same institutions to questionnaires which dealt with various aspects of infection control and antibiotic stewardship. No difference could be ascertained between high and low prevalence hospitals in terms of scores from replies to structured questions regarding infection control set-up, hand hygiene facilities and antibiotic stewardship practices. However, we did identify differences in terms of bed occupancy and isolation facilities. Hospitals reporting frequent episodes of overcrowding, particularly involving several departments, and which found regular difficulties sourcing isolation beds, had significantly higher MRSA proportions. This suggests that infrastructural deficits related to insufficient bed availability and compounded by inadequate isolation facilities could potentiate MRSA hyper-endemicity in south-eastern Mediterranean hospitals.

Infection control and antibiotic stewardship practices reported by south-eastern Mediterranean hospitals collaborating in the ARMed project.

The prevalence of multiply resistant organisms (MROs) reported from south-eastern Mediterranean hospitals highlights the need to identify possible contributory factors to help design control interventions. This was investigated through a structured questionnaire, which examined infection control and antibiotic stewardship practices in hospitals participating or collaborating with the Antibiotic Resistance SurveilLance & Control in the Mediterranean Region (ARMed) project. A total of 45 hospitals (78.9% of invited institutions) responded to the questionnaire; 60% indicated that they faced periods of overcrowding when available bed complement was insufficient to cope with hospital admissions and 62% reported difficulties in isolating patients with MROs due to lack of available beds. Most hospitals relied mainly on washing to achieve hand hygiene, whether by non-medicated or disinfectant soaps. Dependence on solid bars of soap (28.9%) and cloth towels (37.8%) were among the problems identified as well as inconvenient distances of sinks from patient beds (66.6%). Alcohol hand rub was the predominant hand hygiene product in only 7% of hospitals. Programmes for better antibiotic use were mostly limited in scope; 33.3% reported having antibiotic prescribing guidelines and 53.3% of hospitals fed back resistance rates to prescribers. Auditing of antibiotic consumption, whether institution- or unit-based, was carried out in 37.8% of responding hospitals. Multi-faceted approaches aimed at improving isolation of patients with MROs, increasing the emphasis on hand hygiene by encouraging greater use of alcohol hand rubs and introducing effective antibiotic stewardship programmes should be encouraged in south-eastern Mediterranean hospitals.

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.

Antibiotic resistance in the southeastern Mediterranean--preliminary results from the ARMed project.

Sporadic reports from centres in the south and east of the Mediterranean have suggested that the prevalence of antibiotic resistance in this region appears to be considerable, yet pan-regional studies using comparable methodology have been lacking in the past. Susceptibility test results from invasive isolates of Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Enterococcus faecium and faecalis routinely recovered from clinical samples of blood and cerebrospinal fluid within participating laboratories situated in Algeria, Cyprus, Egypt, Jordan, Lebanon, Malta, Morocco, Tunisia and Turkey were collected as part of the ARMed project. Preliminary data from the first two years of the project showed the prevalence of penicillin non-susceptibility in S. pneumoniae to range from 0% (Malta) to 36% (Algeria) [median: 29%] whilst methicillin resistance in Staphylococcus aureus varied from 10% in Lebanon to 65% in Jordan [median: 43%]. Significant country specific resistance in E. coli was also seen, with 72% of isolates from Egyptian hospitals reported to be resistant to third generation cephalosporins and 40% non-susceptible to fluoroquinolones in Turkey. Vancomycin non-susceptibility was only reported in 0.9% of E. faecalis isolates from Turkey and in 3.8% of E. faecium isolates from Cyprus. The preliminary results from the ARMed project appear to support previous sporadic reports suggesting high antibiotic resistance in the Mediterranean region. They suggest that this is particularly the case in the eastern Mediterranean region where resistance in S. aureus and E. coli seems to be higher than that reported in the other countries of the Mediterranean.

Antibiotic resistance in the southeastern Mediterranean - preliminary results from the ARMed project.

Sporadic reports from centres in the south and east of the Mediterranean have suggested that the prevalence of antibiotic resistance in this region appears to be considerable, yet pan-regional studies using comparable methodology have been lacking in the past. Susceptibility test results from invasive isolates of Staphylococcus aureus, Streptococcus pneumoniae, Escherichia coli, Enterococcus faecium and faecalis routinely recovered from clinical samples of blood and cerebrospinal fluid within participating laboratories situated in Algeria, Cyprus, Egypt, Jordan, Lebanon, Malta, Morocco, Tunisia and Turkey were collected as part of the ARMed project. Preliminary data from the first two years of the project showed the prevalence of penicillin non-susceptibility in S. pneumoniae to range from 0% (Malta) to 36% (Algeria) [median: 29%] whilst methicillin resistance in Staphylococcus aureus varied from 10% in Lebanon to 65% in Jordan [median: 43%]. Significant country specific resistance in E. coli was also seen, with 72% of isolates from Egyptian hospitals reported to be resistant to third generation cephalosporins and 40% non-susceptible to fluoroquinolones in Turkey. Vancomycin non-susceptibility was only reported in 0.9% of E. faecalis isolates from Turkey and in 3.8% of E. faecium isolates from Cyprus. The preliminary results from the ARMed project appear to support previous sporadic reports suggesting high antibiotic resistance in the Mediterranean region. They suggest that this is particularly the case in the eastern Mediterranean region where resistance in S. aureus and E. coli seems to be higher than that reported in the other countries of the Mediterranean.

[Bacteriologic profile of bacteremia due to multi-drug resistant bacteria at Charles-Nicolle Hospital of Tunis]. / Profil bactériologique des bactériémies à germes multirésistants à l'hôpital Charles-Nicolle de Tunis.

OBJECTIVE: The authors had for aim to evaluate the place of multi-drug resistant bacteria (MDR) in nosocomial bacteremia. MATERIALS AND METHODS: A retrospective study was carried out at the Microbiology laboratory of Charles Nicolle hospital of Tunis (2001-2003). One hundred and ninety-five isolated MDR [third generation cephalosporin resistant enterobacteria, methicillin-resistant Staphylococcus aureus (MRSA), Acinetobacter baumannii and Pseudomonas aeruginosa resistant to ceftazidime and imipenem]. An automated system was used to detect bloodstream infections. Microorganism identification was performed by conventional methods and antibiotic susceptibilities were determined by the disk diffusion method. RESULTS: MDR bacteria were resistant to third generation cephalosporins (29%), A. baumannii (24%), P. aeruginosa (24%), and MRSA (10%). ERC3G were resistant to aminosides and fluorquinolones. A. baumannii and P. aeruginosa had high resistance rates. Associated resistance rates in MRSA were moderate. CONCLUSION: MDR bacteria are of great concern in our hospital. This situation emphasizes the importance to maintain rigorous measures of hygiene as well as adapted antibiotic prescriptions.

Characterisation of CMY-4, an AmpC-type plasmid-mediated beta-lactamase in a Tunisian clinical isolate of Proteus mirabilis.

A strain of Proteus mirabilis resistant to beta-lactams, including cefoxitin, was isolated from the urine of a woman from Tunisia. Its antibiotic susceptibility pattern and that of the Escherichia coli transconjugant suggested the presence of an AmpC-type beta-lactamase. Two bands of beta-lactamase activity (pI 5.4 and 9.2) were detected by isoelectric focusing. The nucleotide sequence of the gene encoding the AmpC-type enzyme was determined. The deduced amino acid sequence was 98-99% identical to CMY-3 and to those of the plasmid-mediated AmpC-type beta-lactamases originated from Citrobacter freundii and 97% identical to the chromosome-encoded beta-lactamase of a Tunisian clinical isolate of C. freundii. This enzyme differs from CMY-2 by one substitution (Arg for Trp at position 221) and from CMY-3 by two substitutions (Glu for Gly at position 42 and Ser for Asn at position 363) and we propose the denomination CMY-4.

Two novel transferable extended-spectrum beta-lactamases from Klebsiella pneumoniae in Tunisia.

Two novel beta-lactamases conferring multiresistance to antibiotics including oxyimino beta-lactams have been identified in two nosocomial K. pneumoniae strains isolated in Tunis in 1986 and 1988. Both enzymes were encoded by ca. 150-kilobase plasmids. Donor and transconjugant strains producing these enzymes exhibited highly similar pattern of resistance (CTX phenotype) to beta-lactams including penicillins and oxyimino beta-lactams e.g. cefotaxime, ceftriaxone, ceftazidime, and aztreonam. High and variable synergy (16 to 1066-fold) was obtained when combined to 0.1 microgram/ml of clavulanate (beta-lactamase inhibitor). The isoelectric points of these two enzymes were 5.4 and 6.4. These beta-lactamases differed from TEM types by hydrolysis for cefotaxime or ceftriaxone but were inhibited by clavulanate and cloxacillin. DNA hybridization studies suggested that that the genes of these enzymes may be derived from genes encoding TEM-type enzymes.

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.

Prevalence of methicillin-resistant Staphylococcus aureus in eight African hospitals and Malta.

Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious therapeutic problem worldwide, and its frequency in most African countries has not been reported. This study was aimed at determining the prevalence and antibiotic susceptibility patterns of MRSA in eight large hospitals (>500 beds) in Africa and Malta, from 1996 to 1997. Susceptibility to methicillin (oxacillin) and to other drugs was determined by E test (AB Biodisk, Solna, Sweden) on a total of 1440 clinical isolates of S. aureus. Methicillin resistance was detected in 213 (15%) of the 1440 isolates tested. The rate of MRSA was relatively high in Nigeria, Kenya, and Cameroon (21-30%), and below 10% in Tunisia, Malta, and Algeria. All MRSA isolates were sensitive to vancomycin, with MICs 60%) of MRSA strains were multiresistant. There is a need to maintain surveillance and control of MRSA infections in Africa.

Species incidence and antimicrobial-agent resistance patterns of Enterobacteriaceae in Charles Nicolle Hospital, Tunis, from 1983 to 1987.

Between July 1983 and December 1987, 13,108 strains of enterobacteriaceae were isolated at Charles Nicolle Hospital in Tunis. This study reports the prevalence of different species isolated, their resistance and the evolution of bacterial resistance during that period. There appeared to be a great stability in the distribution of bacterial groups. Among the commonly sensitive species, Proteus mirabilis showed a high proportion of strains resistant to ampicillin (79.3%), carbenicillin (75.9%), cefalotin (73.8%) and gentamicin (46%). The proportions of resistant strains in P. mirabilis were much the same for each successive year from 1983 to 1987, and the percentages of resistant strains in the majority of the bacterial species were similarly stable. Amikacin and cefotaxime remained the most active antibiotics against enterobacteriaceae.

[Non-typhoid Salmonella in pediatric patients in Tunis (Hospital Charles-Nicolle) from 1980 to 1991]. / Salmonelles non typhoïdiques en milieu pédiatrique a Tunis (Hôpital Charles-Nicolle) de 1980 a 1991.

252 nontyphoidic salmonellae strains isolated from pediatrics were identified by using standard laboratory procedures and were serotyped with commercial antisera. Antibiotic susceptibility tests were performed by the agar diffusion method. Mostly isolates obtained from feces (182) and blood (67) and 89% of them from newborns and nursings. S. sér. Wien represented 75% of the isolates. The other serovars were essentially S. sér Typhimurium, S. sér Infantis and S. sér Enteritidis. All the isolates of S. sér Wien and S. sér Typhimurium were resistant to amoxicillin, carbenicillin, cephalothin, streptomycin, chloramphenicol and tetracycline from 1980. From 1987 S. sér Wien isolates developed resistance to cefotaxime and amikacin. Distribution nontyphoidic salmonellae strains according to the year was variable. Maximal isolates were in September and October. Two major epidemics were observed in this study, in 1982 and 1989. 94% of the strains determined nosocomial infections. The source of infection was established during the epidemic of 1989. The application of hygiene measures led to decline the number of isolates in 1990 and their disappearance in 1991, any strains of S. sér Wien was isolated.

Characteristics of the antibiotic resistance plasmid in Salmonella typhi isolated in Tunis in 1990.

A multiresistant Salmonella typhi (S typhi, strain 302) was isolated from a blood culture of a patient in the Infectious Diseases department of Rabta Hospital in Tunis. The following tests were carried out: antibiotic susceptibility testing by the agar diffusion method; determination of the minimum inhibitory concentration against four beta-lactam antibiotics (amoxicillin, ceftriaxone, ceftazidime, imipenem), chloramphenicol, gentamicin, and amikacin by the agar dilution method; conjugation with E coli K12 for study of transferability of resistance markers; and electrophoresis of plasmid DNA extracts on agarose gel. S typhi 302 was resistant to amoxicillin, streptomycin, tetracycline, chloramphenicol, and sulfamide-trimethoprim, and this resistance was transferable in toto with a frequency of 10(-4). The MICs of amoxicillin and chloramphenicol were, respectively, 1024 (due to the production of TEM-1 beta-lactamase) and 256 mg/l. These resistance markers were carried by a plasmid of about 40 kb, similar to the Salmonella wien plasmid. The easy acquisition of a multiresistance plasmid by S typhi suggests that epidemiological monitoring of this serovar should be carried out.

[Beta-lactam and aminoglycoside resistant phenotypes in enterobacteria isolated in pediatrics]. / Phénotypes de résistance aux beta-lactamines et aux aminosides des entérobactéries isolées en milieu pédiatrique.

Beta-lactams and aminoglycosides are widely used in the therapy of serious Gram negative infections in pediatrics. In order to survey acquired resistance, the phenotype of resistance to beta-lactams and aminoglycosides were characterized for 260 strains of enterobacteriaceae among the mainly isolated species in pediatrics. These phenotypes were established according to the results of the disk susceptibility tests for beta-lactams respectively ampicillin, ticarcillin, cefaloridin and cefotaxim, and aminoglycosides, streptomycin (S), gentamicin (G), tobryamycin (T), amikacin (A), and netilmicin (N). 49% of E. coli and 73% of P. mirabilis strains were resistant to 3 beta-lactams (RRRS). 72% of Salmonella spp. strains were resistant to 4 beta-lactams (RRRR). For the majority of strains, the mainly observed resistance phenotype to aminoglycosides was streptomycin resistance excepted for Salmonella spp., which was S + KGTNA. The high level of acquired resistance to beta-lactams and aminoglycosides among Enterobacteriaceae with the selection of multiresistant strains would be explained by the wild use of cefotaxim and amikacin in pediatrics.

[Methicillin resistant Staphylococcus aureus. Decrease of incidence in a medical resuscitation unit at CHU Charles-Nicolle in Tunis]. / Staphylococcus aureus méticilline résistant. Diminution de son incidence en réanimation médicale au CHU Charles-Nicolle de Tunis.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major pathogen in hospitals. Between 1985 to 1988, there has been an increase in the number of MRSA (92 %) caused by an epidemic emergency in medical care unit of our hospital. Infections control measures allowed to stop these outbreaks (20 % MRSA in 1990). But incidence of MRSA persist to decrease without supplement control measures and in 1994, only 7 % of Staphylococcus aureus strains are MRSA.

[Staphylococcus aureus nosocomial infections in an intensive care milieu (1985-1989) in Tunis]. / Infections nosocomiales à Staphylococcus aureus en milieu de réanimation médicale (1985-1989) à Tunis.

221 strains of Staphylococcus aureus oxacillin resistant (MetiR) caused nosocomial infections were isolated from 1985 to 1989 in a medical intensive care unit. The survey of susceptibility to antibiotics was established according to the computerized data of disk susceptibility test. The resistance phenotypes to beta-lactams, aminoglycosides and macrolides were established for epidemiological study. S. aureus infections were mainly bacteraemia (31%) and peritonitis (12%). These isolates were resistant to oxacillin with a high level (mean MIC 386 micrograms/ml). Their resistance phenotypes were MLSBc (constitutive resistance to macrolides, lincosamine and streptogramines B) in 53% and S + KGT (resistance to streptomycin, kanamycin, gentamicin and tobramycin) in 61%. All the isolates were susceptible to pristinamycin and vancomycin (MIC 0.1 and 2 micrograms/ml). These phenotypes related to the spread of multiply drug resistant strains were responsible of nosocomial outbreaks. Strains with the same pattern of resistance were isolated among the medical staff and in the environment. Infection control measures allowed to stop these outbreaks.

Epidemiological analysis of Acinetobacter baumannii strains isolated from a surgical intensive care unit.

A serious epidemic of Acinetobacter baumannii resistant to imipenem occurred in the surgical intensive care unit of the hospital Charles-Nicolle in Tunis during February 1994, causing two deaths among three patients. The Acinetobacter strains were isolated from various samples of the intensive care unit. The techniques used for typing were biotyping, antibiogram, plasmid profiles and chromosomal DNA by random amplified polymorphic DNA (RAPD). The A baumannii strains isolated from patients exhibited an identical pattern with all the epidemiological markers utilized; the strains from the surrounding areas showed four and six different patterns respectively for phenotypic and genotypic characters. The strain isolated from a care table had the same phenotypic and genotypic pattern as that of the patients' strains.