Co-occurrence of genes encoding carbapenemase, ESBL, pAmpC and non-ß-Lactam resistance among Klebsiella pneumonia and E. coli clinical isolates in Tunisia.

This study aimed to investigate the molecular mechanisms of carbapenem and colistin resistance in K. pneumoniae and E. coli isolates obtained from hospitalized patients in Carthagene International Hospital of Tunis. A total of 25 K. pneumoniae and 2 E. coli clinical isolates with reduced susceptibility to carbapenems were recovered. Susceptibility testing and phenotypic screening tests were carried out. ESBL, AmpC, carbapenemase and other antibiotic resistance genes were sought by PCR-sequencing. The presence of plasmid-mediated colistin resistance genes (mcr-1-8) was examined by PCR and the nucleotide sequence of the mgrB gene was determined. The analysis of plasmid content was performed by PCR-Based Replicon Typing (PBRT). The clonality of isolates was assessed by PFGE and multilocus sequence typing (MLST). All of the isolates produced carbapenemase activity. They showed a great variation in the distribution of ESBL, AmpC, carbapenemase and other plasmid-mediated resistance determinants. K. pneumoniae isolates carried blaNDM-1 (n = 11), blaOXA-48 (n = 11), blaNDM-1 + blaOXA-48 (n = 1), blaNDM-1 + blaVIM-1 (n = 1), blaOXA-204 (n = 1), along with blaCTX-M , blaOXA , blaTEM , blaCMY , blaDHA and blaSHV genes variants on conjugative plasmid of IncL/M, IncR, IncFIIK , IncFIB, and IncHI1 types. Three sequence types ST101, ST307 and ST15 were identified. The mgrB alteration g109a (G37S) was detected in a single colistin-resistant, NDM-1 and OXA-48-coproducing K. pneumoniae isolate. The two E. coli isolates belonged to ST95, co-produced NDM-1 and CTX-M-15, and harboured plasmid of IncFII and IncFIB types. To our knowledge, this is the first report in Tunisia of NDM-1, OXA-48, and CTX-M-15 coexistence in colistin-resistant K. pneumoniae ST15.

Characterisation of nasal Staphylococcus delphini and Staphylococcus pseudintermedius isolates from healthy donkeys in Tunisia.

REASONS FOR PERFORMING STUDY: Staphylococcus intermedius group (SIG) bacteria can colonise the nares of some animals but are also emerging pathogens in humans and animals. OBJECTIVES: To analyse SIG nasal carriage in healthy donkeys destined for food consumption in Tunisia and to characterise recovered isolates. METHODS: Nasal swabs from 100 healthy donkeys were tested for SIG recovery, and isolates were identified by biochemical and molecular methods. Antimicrobial susceptibility of isolates was tested and detection of antimicrobial resistance and virulence genes was performed. Isolates were typed at the clonal level by multilocus sequence typing and SmaI pulsed-field gel electrophoresis. RESULTS: Staphylococcus delphini and Staphylococcus pseudintermedius (included in SIG) were obtained in 19% and 2% of the tested samples, respectively, and one isolate per sample was characterised. All isolates were meticillin susceptible and mecA negative. Most S. delphini and S. pseudintermedius isolates showed susceptibility to all antimicrobials tested, with the exception of 2 isolates resistant to tetracycline (tet(M) gene) or fusidic acid. The following toxin genes were identified (percentage of isolates): lukS-I (100%), lukF-I (9.5%), siet (100%), se-int (90%), seccanine (19%) and expA (9.5%). Thirteen different pulsed-field gel electrophoresis profiles were identified among the 21 SIG isolates. Additionally, the following 9 different sequence types (STs) were detected by multilocus sequence typing, 6 of them new: ST219 (6 isolates), ST12 (5 isolates), ST220 (3 isolates), ST13, ST50, ST193, ST196, ST218 and ST221 (one isolate each). CONCLUSIONS: Staphylococcus delphini and S. pseudintermedius are common nasal colonisers of donkeys, generally susceptible to the antimicrobials tested; nevertheless, these SIG isolates contain virulence genes, including the recently described exfoliative gene (expA) and several enterotoxin genes, with potential implications for public health. This is the first description of S. delphini in Tunisia. The Summary is available in Chinese - see Supporting information.

Prevalence and characterisation of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli isolates in healthy volunteers in Tunisia.

The objective of this investigation was to analyse the carriage rate of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in faecal samples of healthy humans in Tunisia and to characterise the recovered isolates. One hundred and fifty samples were inoculated on MacConkey agar plates supplemented with cefotaxime (2 µg/ml) for ESBL-positive E. coli recovery. The characterisation of ESBL genes and their genetic environments, detection of associated resistance genes, multilocus sequence typing (MLST) and phylogroup typing were performed by polymerase chain reaction (PCR) and sequencing. The presence and characterisation of integrons and virulence factors were studied by PCR and sequencing. ESBL-positive E. coli isolates were detected in 11 of 150 faecal samples (7.3%) and one isolate/sample was further characterised. These isolates contained the blaCTX-M-1 (ten isolates) and blaTEM-52c genes (one isolate). The ISEcp1 (truncated by IS10 in four strains) and orf477 sequences were found upstream and downstream, respectively, of all bla (CTX-M-1) genes. Seven different sequence types (STs) and three phylogroups were identified among CTX-M-1-producing isolates [ST/phylogroup (number of isolates)]: ST58/B1 (3), ST57/D (2), ST165/A (1), ST155/B1 (1), ST10/A (1), ST398/A (1) and ST48/B1 (1). The TEM-52-producing isolate was typed as ST219 and phylogroup B2. Six ESBL isolates contained class 1 integrons with the gene cassettes dfrA17-aadA5 (five isolates) and dfrA1-aadA1 (one). Healthy humans in the studied country could be a reservoir of CTX-M-1-producing E. coli.

Nasal carriage of Staphylococcus aureus in healthy humans with different levels of contact with animals in Tunisia: genetic lineages, methicillin resistance, and virulence factors.

Nasal swabs of 423 healthy humans who showed different levels of contact with animals (frequent, 168; sporadic, 94; no contact, 161) were obtained in Tunisia (2008-2009), and 99 of them presented other associated risk factors. Methicillin-resistant Staphylococcus aureus (MRSA) was detected in one of these 423 samples (0.24%), retrieved from a veterinarian. The MRSA isolate was mecA-positive, typed as ST80-t203-SCCmecIVc-agrIII, and contained tet(K), ant(6)-Ia, and aph(3')-IIIa genes encoding tetracycline, streptomycin, and kanamycin resistance, respectively. This MRSA isolate also contained the lukF/lukS virulence gene encoding Panton-Valentine leukocidin. Fifty-four (12.8%) additional nasal samples contained methicillin-susceptible S. aureus (MSSA) and one isolate/sample was characterized. A high diversity of spa types (n = 43; 4 new) and pulsed-field gel electrophoresis (PFGE) types (n = 37) was detected among the 55 recovered S. aureus strains. The percentages of antimicrobial resistance/detected resistance genes were as follows: tetracycline [22%/tet(K)-tet(L)-tet(M)], erythromycin [5%/msrA], ciprofloxacin [14.5%], trimethoprim-sulfamethoxazole [2%/dfrA], streptomycin [11%/ant(6)-Ia], kanamycin [7%/aph(3')-IIIa], amikacin [5%], and chloramphenicol [2%]. Four and two isolates carried the lukF/lukS and eta and/or etb genes, respectively, and always in individuals with contact with animals. Eleven isolates carried the tst gene and were recovered from individuals with different levels of contact with animals.

Detection of unrelated Escherichia coli strains harboring genes of CTX-M-15, OXA-1, and AAC(6')-Ib-cr enzymes in a Tunisian hospital and characterization of their integrons and virulence factors.

Beta-lactamase characterization was carried out in a collection of 18 extended-spectrum beta-lactamase (ESBL)-positive Escherichia coli isolates from blood (n=8) and urine (n=10) obtained in 2007 in a tunisian Hospital. All isolates were clonally unrelated according to PFGE analysis. Seventeen strains presented the bla(CTX-M-)15 gene associated with bla (OXA-)1 and four of these strains with the (TEM-)1(b) gene. The remaining ESBL-positive strain contained the bla (CTX-M-)9 gene associated with the bla (OXA-)1 and bla (TEM-)1(b) genes. The orf477 sequence was identified downstream of the bla(CTX-M-)15 gene in all 17 bla(CTX-M-)15-positive strains, and ISEcp1 upstream in 15 of them (in eight cases truncated by IS26). The presence of a class 1 integron was demonstrated in 4 of the 18 ESBL-positive strains (22.2%), with dfrA17 + aadA5 (3 strains) and dfrA12 + orfF + aadA2 (1 strain) being the gene cassettes identified. The variant aac(6´)-Ib-cr was found in 15 bla(CTX-M-)15-containing strains. All 18 ESBL-positive strains were typed as phylogroup B2 and contained at least three of the eight tested virulence genes (fimA, papGIII, hlyA, cnf1, papC, aer, eae and bfp). Six bla(CTX-M-)15-positive strains were included in the serotype O25b and one of them was typed as ST131. Another bla(CTX-M-)15-positive strain serotype-O25 was typed as ST638. The bla(CTX-M-)15, aac(6')- Ib-cr, and aac(3)-II genes were co-transferred by conjugation from 7 donor strains to E. coli CSH26 recipient strain. The bla(CTXM-)15 gene is prevalent among ESBL-positive E. coli strains in the studied hospital, that is frequently found together with aac(6')- Ib-cr, and aac(3)-II genes. The detection of the clone O25b-St131 in a bla(CTX-M-)15 strain corroborates its worldwide dissemination.

Polymorphism in pbp5 gene detected in clinical Enterococcus faecium strains with different ampicillin MICs from a Tunisian hospital.

The polymorphism in pbp5 gene was investigated in nine unrelated clinical gentamicin-resistant Enterococcus faecium strains with different minimal-inhibitory-concentration values for ampicillin (six ampicillin-resistant and three ampicillin-susceptible). Five alleles were detected when the pbp5 C-terminal region was analysed, two of them in the ampicillin-resistant strains showed a new allele characterised by the Thr416Ala and Val462Ala substitutions. Two different alleles were identified when the pbp5 N-terminal region was studied; one of them in the unique strain (E. faecium 83) that presented very low ampicillin MIC (<0.125 microg/ml) and a nucleotidic mutation implicating a stop codon at 451 position. RT-PCR experiments carried out on five E.faecium positive results indication the expression of this gene. Specific mutations in pbp5 gene could be responsible of the high MIC values of some of the E. faecium strains.

Detection of virulence factors in high-level gentamicin-resistant Enterococcus faecalis and Enterococcus faecium isolates from a Tunisian hospital.

Phenotypic and genotypic determination of virulence factors were carried out in 46 high-level gentamicin-resistant (HLGR) clinical Enterococcus faecalis (n=34) and Enterococcus faecium (n=12) isolates recovered from different patients in La Rabta Hospital in Tunis, Tunisia, between 2000 and 2003 (all these isolates harboured the aac(6')-aph(2") gene). The genes encoding virulence factors (agg, gelE, ace, cylLLS, esp, cpd, and fsrB) were analysed by PCR and sequencing. The production of gelatinase and hemolysin, the adherence to caco-2 and hep-2 cells, and the capacity for biofilm formation were investigated in all 46 HLGR enterococci. The percentages of E. faecalis isolates harbouring virulence genes were as follows: gelE, cpd, and ace (100%); fsrB (62%); agg (56%); cylLLS (41.2%); and esp (26.5%). The only virulence gene detected among the 12 HLGR E. faecium isolates was esp (58%). Gelatinase activity was detected in 22 of the 34 E. faecalis isolates (65%, most of them with the gelE+-fsrB+ genotype); the remaining 12 isolates were gelatinase-negative (with the gelE+-fsrB- genotype and the deletion of a 23.9 kb fragment of the fsr locus). Overall, 64% of the cylLLS-containing E. faecalis isolates showed beta-hemolysis. A high proportion of our HLGR E. faecalis isolates, in contrast to E. faecium, showed moderate or strong biofilm formation or adherence to caco-2 and hep-2 cells.

Diversity of structures carrying the aac(6')-aph(2") gene in clinical Enterococcus faecalis and Enterococcus faecium strains isolated in Tunisia.

The diversity of structures carrying the aac(6')-aph(2") gene was studied in 46 high-level gentamicin-resistant Enterococcus faecalis and Enterococcus faecium clinical strains recovered in a Tunisian hospital during the period 2000-2003. The inclusion of the aac(6')-aph(2") gene within the Tn4001 composite element or in its truncated forms (lacking the IS256 at the right, the left or at both sides of the aac(6')-aph(2") gene) was investigated by PCR and sequencing. The aac(6')-aph(2") gene was included in the composite Tn4001 element in 19 of 34 high-level gentamicin-resistant E. faecalis strains (56%) and in 1 of 12 E. faecium strains (12%). A truncated form of Tn4001 lacking IS256 at the left-hand (in 10 E. faecalis and 8 E. faecium), at the right-hand (3 E. faecalis and 2 E. faecium) or at both sides of the aac(6')-aph(2") gene (in 2 E. faecalis and 1 E. faecium) was also detected in 26 of our enterococci. The transference by conjugation of the aac(6')-aph(2") gene, associated with other resistance genes, was demonstrated in seven of the high-level gentamicin-resistant E. faecalis strains.

Phenotypic and genotypic study of gelatinase and beta-haemolysis activities in faecal enterococci of poultry in Portugal.

The detection of gelatinase and beta-haemolysis activities was carried out in 83 faecal enterococci (43 Enterococcus faecalis, 33 E. faecium, five E. durans and two E. hirae) of poultry origin. In addition, the presence of genes of the gelE-fsrABC locus and of the cyl operon (cylL(L), cylL(S), cylA, cylB and cylM) were studied by polymerase chain reaction and correlated with gelatinase and beta-haemolysis production, respectively. Most of our E. faecalis isolates were gelatinase-positive (88%), being this activity not frequent in the other enterococcal species (2.5%). Only one of the 33 E. faecium isolates showed a positive gelatinase reaction. All enterococci that showed gelatinase activity harboured the gelE and fsrABC genes, although these genes were also detected in four E. faecalis and one E. durans gelatinase-negative isolates. Most of our non-E. faecalis gelatinase-negative isolates did not harbour gelE-fsrABC genes. A high proportion of faecal enterococci of poultry origin harboured genes of the cyl operon (71%), although only 7% contained the five cyl tested genes (all of them E. faecalis). Only one isolate of our series could express beta-haemolysis, harbouring the whole cyl operon. The cylL(S) genotype was the most prevalent in our enterococci (39%) and also the most prevalent among our E. faecalis isolates (60%). Other genotypes detected were the following ones (% of enterococci): cylA + cylB + cylM (13%), cylL(L) + cylA (4%), cylL(L) (4%), cylL(L) + cylA + cylB + cylM (2%), cylL(L) + cylA + cylM (1%) and cylA + cylM (1%). Both phenotypic and genotypic assays are important to evaluate the virulence potential of enterococci.

Antibiotic resistance and mechanisms implicated in clinical enterococci in a Tunisian hospital.

Susceptibility testing for 15 antibiotics was performed in a series of 191 clinical enterococci recovered in a Tunisian Hospital during 2000-2003. Species detected were the following ones (number of isolates): E. faecalis (139), E. faecium (41), E. casseliflavus (5), E. gallinarum (3), E. avium (2) and E. hirae (1). The percentages of antibiotic resistance detected were as follows (E. faecalis/ E. faecium/ other species) : penicillin (0/ 73/ 9%), tetracycline (78/ 44/ 54%), chloramphenicol (52/ 29/ 27%), erythromycin (66/ 100/ 82%), spiramycin (84/ 83/ 64%), pristinamycin (100/ 0/ 73%), trimethoprim-sulfamethoxazole (88/ 78/ 91%), rifampicin (72/ 41/ 0%), vancomycin (0/ 0/ 36%), teicoplanin (0/ 0/ 0%), high-level-resistance for gentamicin (24/ 29/ 45%), streptomycin (34/ 56/ 55%) and kanamycin (41/ 68/ 55%). Increased vancomycin minimum inhibitory concentrations (MICs) were only detected in E. casseliflavus and E. gallinarum isolates (MIC range 8-24 microg/ml). The erm(B), catA, tet(M), aac(6')-aph(2''), aph(3')-IIIa, and ant(6)-Ia genes were detected in 91%, 32%, 86%, 98%, 100%, and 72% of the E. faecium and E. faecalis isolates resistant to erythromycin, chloramphenicol, tetracycline and high-level-resistant to gentamicin, kanamycin and streptomycin, respectively. A total of 20 unrelated pulsed-field-gel-electrophoresis patterns were found in the series of 46 high-level gentamicin-resistant E. faecalis and E. faecium isolates of this study.

Characterization of antibiotic resistance genes and virulence factors in faecal enterococci of wild animals in Portugal.

Antibiotic susceptibility was tested in 140 non-selected enterococci (73 Enterococcus faecalis, 45 E. faecium and 22 of other species) recovered from faecal samples of 77 wild animals in Portugal. Susceptibility testing for 11 antibiotics (vancomycin, teicoplanin, ampicillin, streptomycin, gentamicin, kanamycin, chloramphenicol, tetracycline, erythromycin, quinupristin-dalfopristin and ciprofloxacin) was determined by disk diffusion and agar dilution methods. Forty-four isolates (31.4%) showed susceptibility to all the antibiotics tested (5.5% of E. faecalis; 62.2% of E. faecium; and 78.6% of E. hirae). Neither ampicillin-resistance nor acquired-vancomycin-resistance was detected and 1.4% of the isolates showed high-level-resistance for gentamicin or streptomycin. Tetracycline and erythromycin resistances were shown in 28.6% and 20.1% of the isolates, respectively. Antibiotic resistance genes were studied by polymerase chain reaction (PCR) and sequencing and tet(M) + tet(L), erm(B) or aac(6')-aph(2'') genes were detected in most of tetracycline-, erythromycin- or gentamicin-resistant enterococci respectively. Genes encoding virulence factors were studied by PCR and a wide variety of virulence genes were detected in most of E. faecalis isolates but were rarely found in E. faecium and not detected in the other species. The prevalence of genes encoding virulence factors in E. faecalis was as follows: cpd (98.6%), gelE (75.3%), agg (30.1%), fsr (17.8%), ace (9.6%) and esp (4.1%). Low percentages of antibiotic resistance was found in the faecal enterococci of wild animals but a wide variety of virulence genes were detected among E. faecalis isolates although were rare in the other species.