Mohamed Salah Abbassi

Prevalence and Diversity of Integrons and Associated Resistance Genes in Escherichia coli Isolates from Poultry Meat in Tunisia

Fifty-five Escherichia coli isolates were acquired from chicken and turkey meat obtained from two slaughterhouses in Tunis. Eighty-nine percent, 80%, 78%, 67%, 45%, 27%, 7%, 4%, and 2% of these isolates showed resistance to tetracycline, trimethoprim/sulfamethoxazole, streptomycin, nalidixic acid, ampicillin, chloramphenicol, ciprofloxacin, colistine, and gentamicin, respectively. No resistance was detected to cefotaxime, ceftazidime, or amikacin. bla(TEM) gene was found in 22 of 25 ampicillin-resistant isolates, and 1 isolate harbored bla(OXA-1) gene. Tetracycline resistance was predominately mediated by the tetA gene. The sul1, sul2, and sul3 genes, alone or combined, were detected in 46 of 48 sulfonamide-resistant isolates, and sul1 and sul3 were included in class 1 integrons in some cases. Sixty percent of isolates harbored integrons (class 1, 30 isolates; class 2, 5 isolates). Class 2 integrons contained in all cases the dfrA1-sat1-aadA1-orfX gene cassette arrangement. Nine gene cassette arrangements have been detected among class 1 integrons, containing different alleles of dfrA (five alleles) and aadA (2 alleles) genes, which encode trimethoprim and streptomycin resistance, respectively. An uncommon gene cassette array (sat-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3) has been identified in three class 1 integron-positive isolates, and one additional isolate had this same structure with the insertion of IS26 inside the aadA1 gene (included in GenBank with accession no. FJ160769). The 55 studied isolates belong to the four phylogenic groups of E. coli, and phylogroups A and D were the most prevalent ones. At least one virulence-associated gene (fimA, papC, or aer) was detected in 44 of the 55 (80%) studied isolates. E. coli isolates of poultry origin could be a reservoir of antimicrobial-resistance genes and of integrons, and its evolution should be tracked in the future.

Genetic characterisation of CTX-M-15-producing Klebsiella pneumoniae and Escherichia coli strains isolated from stem cell transplant patients in Tunisia

Characterisation of extended-spectrum beta-lactamase (ESBL) genes and their genetic environments as well as the presence of integrons were analysed in nine Klebsiella pneumoniae and two Escherichia coli ESBL-positive isolates recovered in the Centre of Bone Marrow Transplantation of Tunisia. All strains harboured the bla(CTX-M-15) gene and presented minimum inhibitory concentrations for cefotaxime and ceftazidime of 256-1024 mg L(-1) and 16-512 mg L(-1), respectively, and eight of them showed different pulsed-field gel electrophoresis patterns. The bla(OXA-1) and bla(TEM-1) genes were detected in eight and ten strains, respectively. In addition, bla(SHV-1), bla(SHV-11) and bla(SHV-27) were found in six, one and one K. pneumoniae strains, respectively. The new variant bla(SHV-103) was characterised in one K. pneumoniae strain. The intI1 gene was detected in eight K. pneumoniae strains and the dfrA5+ereA2 and aadA gene cassettes were found in one and five strains, respectively. All strains harboured a 70 kb plasmid, and its transference in addition to bla(CTX-M-15), bla(TEM-1b) and bla(OXA-1) genes was demonstrated from three K. pneumoniae to E. coli. ISEcp1 and orf477 were located upstream and downstream, respectively, of the bla(CTX-M-15) gene in 10 strains. The occurrence of the bla(CTX-M-15) gene in unrelated strains might have originated from the dissemination of mobile genetic elements in which ISEcp1 may have played an important role.

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.

High prevalence of extended-spectrum and plasmidic AmpC beta-lactamase-producing Escherichia coli from poultry in Tunisia

This study was conducted to detect extended spectrum beta-lactamases (ESBLs) and plasmidic AmpC beta-lactamase (pAmpC-BL)-producing Escherichia coli isolates in industrial poultry samples were collected from healthy chickens of the three farms. Samples were inoculated onto desoxycholate-lactose-agar plates supplemented with cefotaxime (2mg/L). E. coli was identified by biochemical and molecular methods and antibiotic susceptibility testing by the disk diffusion method. Genes encoding ESBLs and pAmpC-BL were detected by PCR and sequencing. Phylogenetic groups were determined by triplex PCR. The molecular typing of strains was done by pulsed field gel electrophoresis (PFGE) and Multilocus Sequence Typing (MLST) in those isolates showing different PFGE patterns. Cefotaxime-resistant E. coli isolates were recovered in 48 of 137 fecal samples (35%), and one isolate/sample was further studied. The following beta-lactamase genes were detected: blaCTX-M-1 (29 isolates, isolated in all three farms), blaCTX-M-15 (5 isolates, confined in farm II), blaCTX-M-14 and blaCMY-2 (one isolate and 13 isolates, respectively, in farm III). The 48 cefotaxime-resistant isolates were distributed into phylogroups: B1 (n=21), A (n=15) and D (n=12). PFGE analysis revealed 19 unrelated patterns: 15 different profiles among ESBL-positive strains and 4 among the CMY-2-positive isolates. The following sequence types-associated phylogroups were detected: a) CTX-M-1-positive strains: lineages ST542-B1, ST212-B1, ST58-B1, ST155-B1 and ST349-D; b) CTX-M-15-positive strain: lineage ST405-D; c) CTX-M-14-positive strain: lineage ST1056-B1; d) CMY-2-positive strains: lineages ST117-D, ST2197-A, and ST155-B1. Healthy chickens constitute an important reservoir of ESBL- and pAmpC-BL-producing E. coli isolates that potentially could be transmitted to humans via the food chain or by direct contact.

Occurrence of blaCTX-M-1,qnrB1 and virulence genes in avian ESBL-producing Escherichia coli isolates from Tunisia

Avian ESBL-producing Escherichia coli isolates have been increasingly reported worldwide. Animal to human dissemination, via food chain or direct contact, of these resistant bacteria has been reported. In Tunisia, little is known about avian ESBL- producing E. coli and further studies are needed. Seventeen ESBL-producing Escherichia coli isolates from poultry feces from two farms (Farm 1 and farm 2) in the North of Tunisia have been used in this study. Eleven of these isolates (from farm 1) have the same resistance profile to nalidixic acid, sulfonamides, streptomycin, tetracycline, and norfloxacine (intermediately resistant). Out of the six isolates recovered from farm 2, only one was co-resistant to tetracycline. All isolates, except one, harbored blaCTX-M-1 gene, and one strain co-harbored the blaTEM-1 gene. The genes tetA and tetB were carried, respectively, by 11 and 1 amongst the 12 tetracycline-resistant isolates. Sulfonamides resistance was encoded by sul1, sul2, and sul3 genes in 3, 17, and 5 isolates, respectively. The qnrB1 was detected in nine strains, one of which co-harbored qnrS1 gene. The search for the class 1 and 2 integrons by PCR showed that in farm 1, class 1 and 2 integrons were found in one and ten isolates, respectively. In farm 2, class 1 integron was found in only one isolate, class 2 was not detected. Only one gene cassette arrangement was demonstrated in the variable regions (VR) of the 10 int2-positive isolates: dfrA1- sat2-aadA1. The size of the VR of the class 1 integron was approximately 250 bp in one int1-positive isolate, whereas in the second isolate, no amplification was observed. All isolates of farm 1 belong to the phylogroup A (sub-group A0). However, different types of phylogroups in farm 2 were detected. Each of the phylogroups A1, B22, B23 was detected in one strain, while the D2 phylogroup was found in 3 isolates. The virulence genes iutA, fimH, and traT were detected in 3, 7, and 3 isolates, respectively. Two types of gene combination were detected: iutA+fimH+traT in 3 isolates and iutA+fimH in one isolate. The isolates recovered in farm 1 showed the same profile of PFGE macro-restriction, while isolates of farm 2 presented unrelated PFGE patterns. We conclude that these avian ESBL-producing E. coli isolates show homo- and heterogenic genetic background and that plasmids harboring ESBL genes could be involved in the dissemination of this resistance phenotype.

Phenotypic and Genotypic Characterization of Salmonella enterica Recovered from Poultry Meat in Tunisia and Identification of New Genetic Traits

Thirty-seven Salmonella enterica isolates obtained from poultry meat in Tunisia were included in this study for characterization of antibiotic resistance mechanisms. High percentages of resistance were detected to ampicillin, sulfonamides, tetracycline, nalidixic acid, and streptomycin (32.4%-89.2%), and lower percentages to amoxicillin-clavulanic acid, kanamycin, amikacin, trimethoprim-sulfamethoxazol, and chloramphenicol (2.7%-18.9%). All strains showed susceptibility to ceftazidime, cefotaxime, gentamicin, and ciprofloxacin. Class 1 integrons were detected in 30% of Salmonella isolates, and four different gene cassette arrangements were detected, including genes implicated in resistance to aminoglycosides (aadA1 and aadA2) and trimethoprim (dfrA1). Four different Pc variants (PcW, PcH1, PcH1(TTN-10), PcW(TGN-10)) with inactive P2 have been found among these isolates. Integron-positive isolates were ascribed to eight different serotypes. A Salmonella Schwarzengrund isolate harbored a new class 1 integron containing the qacH-dfrA1b-aadA1b-catB2 gene cassette arrangement, with the very unusual PcH1(TTN-10) promoter, which has been registered in GenBank (accession no. HQ874651). Different plasmid replicon types were demonstrated among integron-positive isolates: IncI1 (8 isolates), IncN (8), IncP (2), IncFIB (2), and IncFII (2). Ten different pulsed-field gel electrophoresis profiles were detected among the 11 integron-positive isolates and 8 different sequence types were identified by multilocus sequence typing, one of them (registered as ST867) was new, detected in 3 Salmonella Zanzibar isolates. A high diversity of clones is observed among poultry Salmonella isolates and a high proportion of them show a multiresistant phenotype with very diverse mobile genetic structures that could be implicated in bacterial dissemination in different environments.

Molecular typing of staphylococcal communities isolated during municipal solid waste composting process

This study investigates the molecular typing occurrence of staphylococcal communities during composting process of municipal solid waste. One hundred staphylococcal strains were isolated during the composting process and analysed either by phenotypic and genotypic methods. Amplified ribosomal DNA restriction analysis (ARDRA), internal transcripted spacer-polymerase chain reaction (ITS-PCR) and pulsed-field gel electrophoresis (PFGE) were used as molecular tools. During the digestion phase of composting process three staphylococcal species were recovered:Staphylococcus xylosus (48%),Staphylococcus lentus (40%), andStaphylococcus hominis (12%). In the maturation phase in addition to the latter species four species were also recovered at low frequencies:Staphylococcus lugdunensis, Staphylococcus haemolyticus, Staphylococcus warneri andStaphylococcus sciuri. Therefore, the composition of staphylococcal communities was temperature-, and phase dependant. The molecular methods showed that the ARDRA was not able to differentiate between strains of the same species. However, the ITS-PCR and the PFGE methods allowed interspecies and intraspecies discrimination, respectively.

Detection of optrA in the African continent (Tunisia) within a mosaic Enterococcus faecalis plasmid from urban wastewaters

Objectives Oxazolidinone resistance is a serious limitation in the treatment of MDR Enterococcus infections. Plasmid-mediated oxazolidinone resistance has been strongly linked to animals where the use of phenicols might co-select resistance to both antibiotic families. Our goal was to assess the diversity of genes conferring phenicol/oxazolidinone resistance among diverse enterococci and to characterize the optrA genetic environment. Methods Chloramphenicol-resistant isolates (>16 mg/L, n = 245) from different sources (hospitals/healthy humans/wastewaters/animals) in Portugal, Angola and Tunisia (1996-2016) were selected. Phenicol (eight cat variants, fexA, fexB) or phenicol + oxazolidinone [cfr, cfr(B), optrA] resistance genes were searched for by PCR. Susceptibility (disc diffusion/microdilution), filter mating, stability of antibiotic resistance (500 bacterial generations), plasmid typing (S1-PFGE/hybridization), MLST and WGS (Illumina-HiSeq) were performed for optrA-positive isolates. Results Resistance to phenicols (n = 181, 74%) and phenicols + oxazolidinones (n = 2, 1%) was associated with the presence of cat(A-8) (40%, predominant in hospitals and swine), cat(A-7) (29%, predominant in poultry and healthy humans), cat(A-9) (2%), fexB (2%) and fexA + optrA (1%). fexA and optrA genes were co-located in a transferable plasmid (pAF379, 72 918 bp) of two ST86 MDR Tunisian Enterococcus faecalis (wastewaters) carrying several putative virulence genes. MICs of chloramphenicol, linezolid and tedizolid were stably maintained at 64, 4 and 1 mg/L, respectively. The chimeric pAF379 comprised relics of genetic elements from different Gram-positive bacteria and origins (human/porcine). Conclusions To the best of our knowledge, we report the first detection of optrA in an African country (Tunisia) within a transferable mosaic plasmid of different origins. Its identification in isolates from environmental sources is worrisome and alerts for the need of a concerted global surveillance on the occurrence and spread of optrA.

Characterization of Pc Promoter Variants of Class 1 Integrons in Escherichia coli Isolates from Poultry Meat

Integrons are important genetic elements implicated in acquisition and expression of antimicrobial resistance genes. Gene cassettes of class 1 integrons may be differently expressed depending on the Pc promoter variant. Thirty-four Escherichia coli isolates (carrying 38 class 1 integrons), recovered from poultry meat in previous studies in Tunisia and selected by their specific traits, were further characterized in this study. Integron promoter variants and the pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) of isolates were determined. Three types of promoter variants were identified among the 38 class 1 integrons (PcW, PcH1, and PcS); the weak promoters were the most predominant. A high clonal diversity of the E. coli strains was demonstrated by PFGE or by MLST. Fifteen PFGE profiles were detected among 19 integron-positive isolates of phylogroup B2, and 12 different sequence types were identified by MLST among the remaining 15 isolates (ST48, ST88, ST101, ST117, ST155, ST189, ST351, ST359, ST410, ST641, ST665, and one new ST). These data reflect that the presence of integrons in these isolates is not due to the clonal dispersion but to dissemination of genetic structures carrying integrons in different clones. To the best of our knowledge, this is the first report examining the gene cassette promoter variants in class 1 integrons of E. coli isolates from poultry meat origin. The predominance of promoters implicated in weak expression/high excision activity of gene cassette arrays is of interest because they could theoretically enhance the capacity of integrons to adapt to antibiotic pressure.

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.