Linezolid-resistant MRSA-CC398 carrying the cfr gene, and MRSA-CC9 isolates from pigs with signs of infection in Spain.

AIMS: To perform the molecular characterization of 23 Staphylococcus aureus isolates from pigs with signs of infections recovered in Spanish farms during 2018-2019. METHODS AND RESULTS: The antimicrobial resistance pattern and virulence profile were determined. The molecular typing was performed by different molecular techniques. The transferability of the cfr gene was assessed by conjugation and its genetic environment was determined by PCR mapping. In all, 21 isolates were methicillin-resistant S. aureus (MRSA) carrying the mecA gene (SCCmecV or non-typeable SCCmec), whereas the remaining two were methicillin-susceptible (MSSA). All but one MRSA isolates (n = 20) belonged to the CC398, being the spa t011 the most prevalent (n = 11). The remaining MRSA and the two MSSA isolates were ascribed to ST9/CC9. The S. aureus isolates exhibited resistance to (number of resistant isolates): ß-lactamics (21), erythromycin and/or clindamycin (20), aminoglycosides (7), tetracycline (22), fluoroquinolones (14), chloramphenicol (5) and linezolid (1). The S. aureus isolates did not carry any of the virulence genes studied. One MRSA belonging to the CC398 showed linezolid resistance mediated by the cfr gene. The cfr gene was co-located with fexA in the Tn558 variant previously reported in the S. aureus plasmid pSCFS7. CONCLUSIONS: Two major livestock-associated genetic lineages were detected among pigs with signs of infection in Spain. The presence of the cfr gene among LA-MRSA-CC398 is of great concern not only for veterinary medicine, but also for humans in close contact. SIGNIFICANCE AND IMPACT OF THE STUDY: This work describes the molecular characterization of S. aureus isolates recovered from pigs with signs of infection and we report, as far as we know, the first description of MRSA-CC9 from pigs in Spain. Moreover, the detection of a MRSA-CC398 isolate carrying the multiresistance cfr gene highlights the need for continuous surveillance and awareness of LA-MRSA.

Diversity of Staphylococcus aureus clones in wild mammals in Aragon, Spain, with detection of MRSA ST130-mecC in wild rabbits.

AIMS: To determine the Staphylococcus aureus carriage rate in wild mammals in Aragon, northern Spain, to analyse their antimicrobial resistance phenotype/genotype and to characterize the recovered isolates. METHODS AND RESULTS: Nasal and rectal swabs of 103 mammals were collected in Aragón during the period 2012-2015. Antimicrobial susceptibility, the presence of antimicrobial resistance genes and virulence factors were investigated. Molecular characterization was carried out by spa, MLST, agr and SCCmec. Staphylococcus aureus were recovered from 23 animals (22%). Four of the 23 S. aureus were methicillin-resistant S. aureus (MRSA). Three MRSA were mecC-positive and were isolated from European rabbits and were typed as t843 (ascribed to CC130). The remaining MRSA was a mecA-carrying isolate from European hedgehog, typed as ST1-t386-SCCmecIVa-agrIII and it harboured the blaZ, erm(C), ant(6)-Ia and aph(3´)-IIIa resistance genes. A high diversity of spa-types was detected among the 19 methicillin-susceptible S. aureus isolates, which showed high susceptibility to the antimicrobials tested. The tst gene and different combinations of staphylococcal enterotoxins were found. CONCLUSIONS: Staphylococcus aureus were detected in nasal and rectal samples of wild mammals. Wild rabbits could be a reservoir of mecC-MRSA. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides information on the presence and characteristics of S. aureus from mammals in a defined geographic region in Spain.

Antibiotic resistance in Escherichia coli in husbandry animals: the African perspective.

In the last few years, different surveillances have been published in Africa, especially in northern countries, regarding antimicrobial resistance among husbandry animals. Information is still scarce, but the available data show a worrying picture. Although the highest resistance rates have been described against tetracycline, penicillins and sulphonamides, prevalence of plasmid-mediated quinolone resistance genes and extended spectrum ß-lactamase (ESBL) are being increasingly reported. Among ESBLs, the CTX-M-1 group was dominant in most African surveys. Within this group, CTX-M-15 was the main variant both in animals and humans, except in Tunisia where CTX-M-1 was more frequently detected among Escherichia coli from poultry. Certain blaCTX-M-15 -harbouring clones (ST131/B2 or ST405/D) are mainly identified in humans, but they have also been reported in livestock species from Tanzania, Nigeria or Tunisia. Moreover, several reports suggest an inter-host circulation of specific plasmids (e.g. blaCTX-M-1 -carrying IncI1/ST3 in Tunisia, IncY- and Inc-untypeable replicons co-harbouring qnrS1 and blaCTX-M-15 in Tanzania and the worldwide distributed blaCTX-M-15 -carrying IncF-type plasmids). International trade of poultry meat seems to have contributed to the spread of other ESBL variants, such as CTX-M-14, and clones. Furthermore, first descriptions of OXA-48- and OXA-181-producing E. coli have been recently documented in cattle from Egypt, and the emergent plasmid-mediated colistin resistance mcr-1 gene has been also identified in chickens from Algeria, Tunisia and South Africa. These data reflect the urgent need of a larger regulation in the use of veterinary drugs and the implementation of surveillance programmes in order to decelerate the advance of antimicrobial resistance in this continent.

Pharmacokinetics, milk penetration and PK/PD analysis by Monte Carlo simulation of marbofloxacin, after intravenous and intramuscular administration to lactating goats.

The main objectives of this study were (i) to evaluate the serum pharmacokinetic behaviour and milk penetration of marbofloxacin (MFX; 5 mg/kg), after intravenous (IV) and intramuscular (IM) administration in lactating goats and simulate a multidose regimen on steady-state conditions, (ii) to determine the minimum inhibitory concentration (MIC) and mutant prevention concentration (MPC) of coagulase negative staphylococci (CNS) isolated from caprine mastitis in Córdoba, Argentina and (iii) to make a PK/PD analysis by Monte Carlo simulation from steady-state pharmacokinetic parameters of MFX by IV and IM routes to evaluate the efficacy and risk of the emergence of resistance. The study was carried out with six healthy, female, adult Anglo Nubian lactating goats. Marbofloxacin was administered at 5 mg/kg bw by IV and IM route. Serum and milk concentrations of MFX were determined with HPLC/uv. From 106 regional strains of CNS isolated from caprine mastitis in herds from Córdoba, Argentina, MICs and MPCs were determined. MIC90 and MPC90 were 0.4 and 6.4 µg/ml, respectively. MIC and MPC-based PK/PD analysis by Monte Carlo simulation indicates that IV and IM administration of MFX in lactating goats may not be adequate to recommend it as an empirical therapy against CNS, because the most exigent endpoints were not reached. Moreover, this dose regimen could increase the probability of selecting mutants and resulting in emergence of resistance. Based on the results of Monte Carlo simulation, the optimal dose of MFX to achieve an adequate antimicrobial efficacy should be 10 mg/kg, but it is important take into account that fluoroquinolones are substrates of efflux pumps, and this fact may determine that assumption of linear pharmacokinetics at high doses of MFX may be incorrect.

Evaluation of the antiemetic efficacy of maropitant in dogs medicated with morphine and acepromazine.

OBJECTIVE: To evaluate whether maropitant (1 mg kg(-1)) injected subcutaneously (SC), administered simultaneously or 30 minutes prior to intramuscular (IM) administration of morphine (0.5 mg kg(-1)) and acepromazine (0.05 mg kg(-1)), reduces the incidence of salivation, retching and emesis in dogs. STUDY DESIGN: Randomized, controlled, prospective clinical trial. ANIMALS: Sixty dogs scheduled for an ovariohysterectomy as part of a population control program. METHODS: Dogs were randomly allocated to be administered maropitant (1 mg kg(-1)) SC simultaneously (group M0) or 30 minutes prior to (group M30) administration of morphine (0.5 mg kg(-1)) and acepromazine (0.05 mg kg(-1)) IM. A control group was administered normal saline (C) at T-30 and T0. Dogs were observed for 30 minutes after morphine-acepromazine administration. The occurrence of vomiting, retching and salivation were recorded, as well as the time to first emesis and the number of emetic events per dog. RESULTS: The occurrence of salivation was not different between the groups. Retching and vomiting occurred significantly less frequently in M30 than in the other two groups (p < 0.02). The number of emetic events was also significantly less for M30 than for the other two groups (p = 0.01). When emesis occurred, the time to the first emetic event was similar among the groups. CONCLUSIONS AND CLINICAL RELEVANCE: Maropitant (1 mg kg(-1)) SC reduced the frequency of morphine-induced emesis by as much as 70% when administered 30 minutes in advance. Simultaneous administration of maropitant and morphine-acepromazine produced no measurable effect on the frequency of retching or vomiting.

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.

Context-sensitive recovery of neuromuscular function from vecuronium in dogs: Effects of dose and dosing protocol.

Recovery of neuromuscular function is a gradual phenomenon whereby function progresses from absent to normal. The speed of spontaneous recovery can be used to predict the time when neuromuscular function is expected to be restored. However, the speed of recovery might be affected by the dose of the neuromuscular blocker administered, and by the dosing regimen of that dose. The effects of both factors on the speed of spontaneous recovery from vecuronium were evaluated. Seven dogs were anesthetized three times and the train-of-four (TOF) ratio was measured with acceleromyography. Vecuronium was administered at 0.1 mg/kg, 0.2 mg/kg, or 0.1 mg/kg followed by two doses of 0.05 mg/kg was administered each time. In the divided-dose treatment group, aliquots were administered on return of the first twitch (T1) of the TOF from the previous dose. The duration of surgical block, from injection to return of T1, was longest for the divided-dose protocol, intermediate for 0.2 mg/kg single bolus, and shortest for 0.1 mg/kg (P < 0.0001). The recovery period, from return of T1 to a TOF ratio ≥0.9, was longer for 0.2 mg/kg administered as a single bolus than for the other two groups (P = 0.007). Doubling the dose of a single bolus of vecuronium extended the time of surgical block and prolonged the duration of the recovery period. However, dividing that dose into smaller aliquots extended the period of surgical block while shortening the recovery period. Hence, the spontaneous reappearance of T1 should not be used in isolation to predict the time to complete recovery of neuromuscular function.

Antibiogramj: A tool for analysing images from disk diffusion tests.

BACKGROUND AND OBJECTIVES: Disk diffusion testing, known as antibiogram, is widely applied in microbiology to determine the antimicrobial susceptibility of microorganisms. The measurement of the diameter of the zone of growth inhibition of microorganisms around the antimicrobial disks in the antibiogram is frequently performed manually by specialists using a ruler. This is a time-consuming and error-prone task that might be simplified using automated or semi-automated inhibition zone readers. However, most readers are usually expensive instruments with embedded software that require significant changes in laboratory design and workflow. METHODS: Based on the workflow employed by specialists to determine the antimicrobial susceptibility of microorganisms, we have designed a software tool that, from images of disk diffusion tests, semi-automatises the process. Standard computer vision techniques are employed to achieve such an automatisation. RESULTS: We present AntibiogramJ, a user-friendly and open-source software tool to semi-automatically determine, measure and categorise inhibition zones of images from disk diffusion tests. AntibiogramJ is implemented in Java and deals with images captured with any device that incorporates a camera, including digital cameras and mobile phones. The fully automatic procedure of AntibiogramJ for measuring inhibition zones achieves an overall agreement of 87% with an expert microbiologist; moreover, AntibiogramJ includes features to easily detect when the automatic reading is not correct and fix it manually to obtain the correct result. CONCLUSIONS: AntibiogramJ is a user-friendly, platform-independent, open-source, and free tool that, up to the best of our knowledge, is the most complete software tool for antibiogram analysis without requiring any investment in new equipment or changes in the laboratory.

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.

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.

Antibiotic resistance in Escherichia coli isolates obtained from animals, foods and humans in Spain.

Antibiotic resistance was investigated in 474 Escherichia coli isolates recovered from animal faeces (broilers, pigs, pets, bulls and horses), human faeces (patients and healthy volunteers) and food products of animal origin. E. coli isolates (3260) recovered from human significant infectious samples were also included. There was a high frequency of nalidixic acid, ciprofloxacin and gentamicin resistance in E. coli isolates from broilers (88, 38 and 40%, respectively), and from foods (53, 13 and 17%). High levels of resistance to trimethoprim-sulphamethoxazole and tetracycline have been found in E. coli isolates from broilers, pigs and foods. These data raise important questions about the potential impact of antibiotic use in animals and the possible entry of resistant pathogens into the food chain.

First detection of methicillin-resistant Staphylococcus aureus ST398 and Staphylococcus pseudintermedius ST68 from hospitalized equines in Spain.

Eight coagulase-positive staphylococci from equines with different pathologies obtained between 2005 and 2011 were investigated. Isolates were characterized by different molecular techniques (spa-, agr-, MLST), and clonal relatedness of strains was investigated by ApaI and SmaI PFGE. Anti-microbial resistance and virulence profiles were determined. Six isolates were identified as Staphylococcus aureus, and two as Staphylococcus pseudintermedius. Of these, four isolates were methicillin-resistant S. aureus (MRSA) ST398 and one S. pseudintermedius was mecA positive and typed as ST68. One MRSA ST398 strain was isolated in 2005 and might be one of the earliest MRSA ST398 descriptions in Spain. All 5 mecA-positive strains were multidrug resistant and were isolated from hospitalized equines. Three MRSA ST398 strains carried the recently described transposon Tn559 within the chromosomal radC gene. The mecA-positive S. pseudintermedius ST68 strain was also multidrug resistant and harboured the erm(B)-Tn5405-like element. This ST68 strain presented a clear susceptible phenotype to oxacillin and cefoxitin regardless of the presence of an integral and conserved mecA gene and mecA promoter, which enhances the need for testing the presence of this gene in routine analysis to avoid treatment failures. These data reflect the extended anti-microbial resistance gene acquisition capacities of both bacterial species and evidence their pathogenic properties. The first detection of MRSA ST398 and S. pseudintermedius ST68 in horses in Spain is reported.

Chromosomal integration of the novel plasmid pUR3912 from methicillin-susceptible Staphylococcus aureus ST398 of human origin.

The novel erm(T)-cadDX-carrying plasmid pUR3912 has recently been described in the methicillin-susceptible Staphylococcus aureus ST398-t571 strain C3912 from a healthy human in Spain. Structural analysis revealed that pUR3912 belongs to the pC194 replicon family, replicates via a rolling circle mechanism and harbours putative double-strand (dso) and single-strand (sso) origins of replication. Besides its plasmid location, a copy of pUR3912 was also found in the chromosomal DNA of strain C3912. Two IS431 copies, which flank the plasmid, most probably mediated its chromosomal integration. Its ability to not only exist extrachromosomally, but also to integrate into the chromosomal DNA ensures persistence and dissemination of pUR3912.

A description of preimaginal stages of Pseudaspidapion botanicum Alonso-Zarazaga & Wang, 2011 (Apionidae, Curculionoidea).

The preimaginal stages including egg, mature larva and pupa of Pseudaspidapion botanicum Alonso-Zarazaga & Wang, 2011 were described and figured, diagnostic characters of larva and pupa were discussed, and corresponding biological information was supplied. The nomenclature of frontal setae in the larva compared with curculionid weevils, the absence of the hypopharyngeal bracon in the larva, and the metafemoral setae in the pupa were discussed. Common and different characters among the larvae of Pseudaspidapion botanicum, Aspidapion radiolus (Marsham, 1802) and Aspidapion aeneum (Fabricius, 1775) were also provided.

Methicillin-resistant Staphylococcus aureus (MRSA) ST398 in a farmer with skin lesions and in pigs of his farm: clonal relationship and detection of lnu(A) gene.

Skin infection associated with methicillin-resistant Staphylococcus aureus (MRSA)-ST398 was detected in a pig-farmer, and MRSA-ST398 isolates were also detected in nasal samples of the patient and of 11/12 pigs on his farm. Twelve MRSA isolates were obtained from skin lesions (n = 6) and nasal samples (n = 6) of the patient in two sampling moments and 11 MRSA isolates from nasal samples of pigs. They were typed as t011-SCCmecIVa-agrI and t108-SCCmecV-agrI (patient and pigs) and t588-SCCmecV-agrI (patient). The following resistance genes were detected (number isolates): tet(K) (1), tet(L) (23), tet(M) (13), erm(A) (13), erm(C) (13), msr(A) (11), lnu(A) (21), aph(2'')-acc(6') (3), ant(4') (13), aph(3') (12), dfrS1 (15) and dfrK (22). Seventeen human and animal MRSA-ST398 isolates showed indistinguishable PFGE patterns (A1-spa-t011 or B2-spa-t108) and similar phenotypic-genotypic characteristics, including the presence of the lnu(A) gene, associated with lincomycin resistance. Potential pig-to-human transference of ST398 is suggested in this study. The first detection of the lnu(A) gene in MRSA-ST398 is reported.

Characterization of the mechanisms of fluoroquinolone resistance in vancomycin-resistant enterococci of different origins.

The mutations in gyrA and parC genes were analyzed in 22 vancomycin-resistant enterococci of different origins and species, which had varying susceptibility to ciprofloxacin (minimum inhibitory concentration, MIC: 0.5- >256 mg/l). All vanA or vanb2-containing strains with ciprofloxacin MIC of >32 mg/l presented amino acid changes in GyrA protein (S83I, S83Y, S83R or S83I-E87G) with/without changes in ParC protein (S80I or S80R or S80l). Strains with lower ciprofloxacin MICs presented the GyrA and parC wild type. One vanA-containing Enterococcus durans strain with a ciprofloxacin MIC of 64 mg/l presented the S83I and S80I changes in GyrA and ParC proteins, respectively. Two vanB2 Enterococcus faecium strains were typed by multi-locus-sequencetyping and both were ascribed to the CC17 clonal complex with two sequence-types (ST78 and ST17-like). All seven vancomycin-resistant and ciprofloxacin-resistant E. faecium strains showed ampicillin resistance (MIC 32-256 mg/l), identifying the following amino acid changes in PBP5 protein: Q461K, V462K, H470Q, M485A, N496K, A499T, E525D, N546T, A558T, G582S, K632Q, P642L, E629V and P667S, together with a serine insertion at position 466´. The 12 Enterococcus gallinarum and Enterococcus casseliflavus isolates included in the study exhibited an MIC for ciprofloxacin in the range 0.5-16 mg/l and no amino acid changes were identified in GyrA or ParC proteins. Specific mutations in gyrA and parC genes are associated with fluoroquinolone resistance in E. faecium and E. durans of different origins.

A new species of genus Pseudaspidapion Wanat, 1990 (Coleoptera, Apionidae) from China.

Pseudaspidapion botanicumsp. n. from China is described and figured. Its host plant is Grewia biloba G. Don var. parviflora (Bunge) Hand.-Mazz (Malvaceae: Grewioideae). The genus Harpapion Voss, 1966 is recorded as new for China and Vietnam and two comb. n. are proposed: Harpapion vietnamense (Korotyaev, 1985) (from Aspidapion) and Harpapion coelebs (Korotyaev, 1987) (from Pseudaspidapion). A key to the known species of the genus Pseudaspidapion from China is presented.

Class 1 integrons lacking qacEDelta1 and sul1 genes in Escherichia coli isolates of food, animal and human origins.

OBJECTIVES: To study the frequency and diversity of class 1 integrons lacking the 3'-conserved segment (CS) in intI1-positive Escherichia coli isolates of different origins. METHODS: The presence of intI1 was previously detected in 84 E. coli isolates of food (21 isolates), animal (32) and healthy-human volunteer (31) origins. The qacEDelta1-sul1 genes were analyzed by PCR and those isolates that lacked these genes were included in this work. The genetic structure of class 1 integrons was determined, using the PCR and sequencing primer-walking strategy. Isolates and plasmids were typed. RESULTS: Class 1 integrons lacking the 3'-CS were found in 13 of the 84 intI1-positive E. coli isolates (15.5%) of food, animal, and human origins. All 13 isolates showed unrelated patterns by REP-PCR. The following gene cassette arrangements were identified inside the class 1 integrons of these 13 strains: dfrA1; dfrA5; dfrA12-orfF-aadA2-cmlA1-aadA1-qacH-IS440-sul3; dfrA12-orfF-aadA2-cmlA1-aadA1-IS440-sul3; estX-psp-aadA2-cmlA1-aadA1-qacH-IS440-sul3; and a new arrangement estX-psp-aadA2-cmlA1Delta-IS1294-DeltacmlA1-aadA1-qacH-IS440-sul3 that contain the IS1294 into the cmlA1 gene (included in GenBank, number EU704128). Complete or truncated mef(B) gene was detected upstream of sul3 gene in this type of integrons. Plasmids were identified in four of the studied strains by PCR-replicon-typing, detecting different combinations of IncY, I1, FIC, FII, FIB plasmids. Non-classic integrons were located into plasmids of 100-150 kb in four studied strains. CONCLUSIONS: Occurrence and diversity of class 1 integrons lacking 3'-CS among the studied intI1-positive E. coli isolates of different origins were relatively high. The sul3 gene was detected in most of class 1 integrons lacking 3'-CS.

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.