A. Ben Hassen

Nosocomial outbreak of acute gastroenteritis in a neonatal intensive care unit in tunisia caused by multiply drug resistantSalmonella wien producing SHV-2 beta-lactamase

In a Tunisian hospital 27 babies, including 12 who were premature, in a single intensive care unit suffered acute gastroenteritis in the period from January to May 1988. The mean age at the onset of gastroenteritis was 8.4 days; nine babies died.Salmonella wien was isolated from stools (all babies) and blood (4 babies). It was also isolated from the stools of one nurse and from a mattress. Twelve of the babies had received cefotaxime, which was successfully replaced by oral colimycin. The outbreak was stopped by the implementation of infection control measures. All isolates ofSalmonella wien were of the same biotype, and had the same antibiotic resistance pattern (third generation cephalosporins, monobactams, aminoglycosides, chloramphenicol, trimethoprim and sulphonamides) and plasmid DNA restriction pattern. The isolates were all susceptible to a combination of cefotaxime and clavulanic acid (a β-lactamase inhibitor), which displayed synergy, suggesting the presence of a β-lactamase (geometric mean MICs 11.24 µg/ml for cefotaxime alone and 0.24 µg/ml in combination with 0.1 µg/ml potassium clavulanate). All isolates produced TEM-1 and SHV-2 β-lactamase which was not transferable toEscherichia coli by conjugation. The presence of the SHV-2 enzyme inSalmonella wien may allow it to adapt to newer β-lactams which is a cause for concern in this hospital.

Multiplex PCR detection of the antibiotic resistance genes in Staphylococcus aureus strains isolated from auricular infections

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.

High prevalence of mec complex C and ccrC is independent of SCCmec type V in Staphylococcus haemolyticus

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.

Antibiotic resistance and molecular characterization of clinical isolates of methicillin-resistant coagulase-negative staphylococci isolated from bacteremic patients in oncohematology

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.

Stenotrophomonas maltophilia responsible for respiratory infections in neonatal intensive care unit: antibiotic susceptibility and molecular typing.

OBJECTIVE 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). MATERIALS AND METHODS 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. RESULTS 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. CONCLUSION Identification of two clonal strains of S. maltophilia responsible of respiratory infection. Epidemic strains are hardly eradicated when colonization is established.

Analysis of cytomegalovirus (CMV) viremia using the pp65 antigenemia assay, the amplicor CMV test, and a semi-quantitative polymerase chain reaction test after allogeneic marrow transplantation

Abstract: A pp65 antigenemia assay for polymorphonuclear leukocytes (PMNLs) (CINAkit Rapid Antigenemia), and a qualitative polymerase chain reaction (PCR) test for plasma ‘PCR‐P qual’ (Amplicor cytomegalovirus [CMV] test) were performed for 126 samples (blood and plasma) obtained from 18 bone marrow transplant patients, over a 9‐month surveillance period. Among those samples, 92 were assayed with a semi‐quantitative PCR test for PMNLs ‘PCR‐L quant.’ The number of samples with a positive CMV test for antigenemia and PCR‐P qual assays was 20.63% and 12.7%, respectively, whereas the PCR‐L quant assay was positive in 48 of the 92 samples assayed (52.17%). The rates of concordance of the results of PCR‐P qual and antigenemia, PCR‐P qual and PCR‐L quant, antigenemia and PCR‐L quant were 92%, 65.2% and 66.8%, respectively. The analysis of the results for the 92 specimens tested by all 3 methods showed a rate of concordance of 63% among all methods. Good agreement (κ=0.72) was found only between pp65 Ag and PCR‐P qual assays. Clinical disease correlates with an antigenemia high viral load. Three patients had CMV disease despite preemptive therapy, and all of them had graft‐versus‐host‐disease (GVHD). PMNLs‐based assays are more efficient in monitoring CMV reactivation, but for high‐risk patients with GVHD, more sensitive assays (real‐time PCR) must be done.

Species distribution and antibiotic sensitivity pattern of coagulase-negative Staphylococci other than Staphylococcus epidermidis isolated from various clinical specimens

This study was undertaken to determine the species distribution and antibiotic resistance patterns of coagulase-negative Staphylococci (CoNS) other than Staphylococcus epidermidis. A total of 142 CoNS (except S. epidermidis) strains were isolated from a variety of clinical specimens in neutropenic patients at the Bone Marrow Transplant Centre of Tunisia between 2002 and 2004. All CoNS isolates were further identified by Api ID32 STAPH and ITS-PCR and antibiotic sensitivity was performed by disc diffusion method. Staphylococcus haemolyticus was the commonest species (38%) followed by Staphylococcus hominis (36%). All isolates were sensitive to vancomycin and 8 (6%) strains showed a reduced sensitivity to teicoplanin. Resistance to penicillin G and methicillin was 84 and 60%, respectively. Methicillin-resistant CoNS strains were determined to be more resistant to antibiotics than methicillin-susceptible CoNS strains. The mecA gene was detected by PCR in 65% (92/142) CoNS isolates. Out of 92 mecA-positive isolates, 90 were phenotypically methicillin-resistant and two were methicillin-susceptible. Phylogenetic analysis, carried out to study the evolution of mecA genes between different Staphylococcal species, revealed a high homology for such genes among Staphylococci.

Enterococcus faecium isolated from bone marrow transplant patients in Tunisia: High prevalence of antimicrobial resistance and low pathogenic power

OBJECTIVES 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. MATERIALS AND METHODS 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. RESULTS 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. CONCLUSION 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.

Detection of SHV-1 β-lactamase in Pseudomonas aeruginosa strains by genetic methods

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.

Identification des gènes de β-lactamase à spectre étendu de type SHV chez Pseudomonas aeruginosa par PCR-restriction fragment length polymorphism et insertion site restriction-PCR

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.