Showing papers by "Alessandra Carattoli published in 2007"

Extended-Spectrum β-Lactamase CTX-M-1 in Escherichia coli Isolates from Healthy Poultry in France

Abstract: Genes encoding extended-spectrum beta-lactamase CTX-M-1 were detected in 12 Escherichia coli isolates recovered over a 7-month period from the ceca of healthy poultry in seven districts in France in 2005. Eleven of those strains were not clonally related and had a bla(CTX-M-1) gene located on transferable plasmids of different sizes and structures.

Dissemination of an Extended-Spectrum-β-Lactamase blaTEM-52 Gene-Carrying IncI1 Plasmid in Various Salmonella enterica Serovars Isolated from Poultry and Humans in Belgium and France between 2001 and 2005

Abstract: Food-producing animals are the primary reservoir of zoonotic pathogens, and the rate of detection of extended-spectrum β-lactamase (ESBL)-producing Escherichia coli and Salmonella strains has increased in recent years. ESBLs are widely detected in various human medical institutions, but they are not so frequently reported in bacterial populations circulating in animals. In Belgium and France the emergence of resistance to extended-spectrum cephalosporins, such as ceftriaxone and ceftiofur, has been recently reported in Salmonella enterica serovar Virchow isolates from poultry and humans (1, 15). Resistance was due to the ESBL genes blaCTX-M-2 or blaCTX-M-9 carried on large conjugative plasmids. Since 2001 a large number of strains have been isolated from poultry (more than 150 in Belgium) and a more limited number from humans (n = 15) in Belgium and France showing resistance to extended-spectrum cephalosporins by production of an ESBL not belonging to the CTX-M family and with various additional resistances to other antibiotic families. The serovars concerned were Agona, Derby, Infantis, Paratyphi B dT+, and Typhimurium. In particular, the emergence of extended-spectrum cephalosporin-resistant serovar Infantis with more than 80 strains isolated from poultry and 4 strains from humans caused some concern. The purpose of the present study was to identify the ESBL gene and its location in these strains. Strains studied are shown in Table ​Table1.1. Antibiotic susceptibility testing was done by the disk diffusion method, and the MICs of ceftriaxone and ceftiofur were determined as described previously (1, 14, 15). Resistance to extended-spectrum cephalosporins from all Salmonella strains was transferred to an E. coli recipient strain by conjugation as previously described (1, 14, 15), and all E. coli transconjugant strains showed the same antibiotic resistance profile (Table ​(Table1).1). Other resistances from multidrug-resistant strains were not transferred by conjugation. According to the MICs, the levels of resistance to ceftiofur and ceftriaxone were lower in the transconjugant strains than in the parental strains, but this was also observed in a previous study (14). PCR assays to detect ESBL genes (TEM, SHV, and CTX-M) were performed on parental and transconjugant strains using previously described primers (1, 14, 15), and nucleotide sequencing of the amplicons identified the blaTEM-52 resistance gene in all strains. Plasmids extracted from the transconjugants were further characterized by PstI restriction analysis showing that they were all identical and greater than 100 kb in size (Fig. ​(Fig.1).1). Southern blot hybridization experiment with a blaTEM-52 gene probe was performed as described previously (12). It revealed two PstI fragments of 2.9 and 2.75 kb. In fact, this PstI restriction profile corresponded exactly to that of blaTEM-52-carrying plasmids isolated in 2002 and 2003 from four isolates of S. enterica serovars Typhimurium, Enteritidis, and Panama from French patients with gastroenteritis (14). A study performed in 2001 and 2002 on Salmonella isolated from poultry, poultry products, and human patients in The Netherlands revealed that the TEM-52 variant was the most common ESBL detected in this bacterial collection (6). In particular, TEM-52-producing salmonellae of the serovars Blockley, Virchow, Typhimurium, and Paratyphi B were identified from poultry, and strains of the serovars Thompson, London, Enteritidis, and Blockley were identified from human patients (6). Several sporadic cases of E. coli TEM-52 producers were reported in animals: dogs in Portugal, rabbits in Spain, and beef meat in Denmark (2, 4, 8). These findings suggest a wide dissemination of this ESBLs in Europe in animals and humans. The presence of the blaTEM-52 gene in E. coli, as well as in different Salmonella serovars, strongly indicated that it is not due to the spread of a single clone but to the horizontal transmission of this resistance trait. To better identify the molecular mechanism of dissemination of this ESBL, the blaTEM-52-positive plasmids were typed by the PCR-based replicon typing as previously described (3), demonstrating that they all belong to the IncI1 incompatibility group. IncI1 plasmids were recently described in E. coli and Salmonella strains of different serovars isolated in the United Kingdom associated with relevant β-lactamases such as CMY-2, CMY-7, and CTX-M-15, suggesting a large prevalence of IncI1 plasmids in Europe (7). FIG. 1. Restriction analysis (PstI) (upper panel) and Southern blot hybridization with a blaTEM-52 probe (lower panel) of plasmid DNAs isolated from E. coli transconjugants. Lane 1, E. coli transconjugant 988SA01TC1; lane 2, E. coli transconjugant 2004/10101TC1; ... TABLE 1. Characteristics of the Salmonella strains and their transconjugants producing TEM-52 used in this study To identify the mobile genetic element carrying the blaTEM-52 gene, the plasmid DNA of E. coli transconjugant 04-3486TC1 extracted with a QIAfilter Midi kit (QIAGEN, Courtaboeuf, France) was digested with ClaI and ligated into the ClaI-restricted phagemid pBK-CMV (Stratagene). Recombinant plasmids were introduced into E. coli DH10B by electroporation (Bio-Rad Gene Pulser II; Bio-Rad, Marnes-La-Coquette, France) and selected on Mueller-Hinton (MH) agar (Bio-Rad) containing kanamycin (30 μg/ml) and ceftazidime (2 μg/ml). Recombinant plasmids that possessed a 4.7-kb insert were selected. Nucleotide sequencing of the insert indicated that the blaTEM-52 gene was located on a Tn3 transposon. To complete the transposon sequence, nucleotide sequencing was further performed by genome walking on the native plasmid. This Tn3 transposon structure is shown in Fig. ​Fig.2.2. Its nucleotide sequence is deposited in GenBank under accession number {"type":"entrez-nucleotide","attrs":{"text":"EF141186","term_id":"119632822","term_text":"EF141186"}}EF141186. Very few sequences of complete Tn3 elements from Salmonella are currently available (10). Complete plasmid-borne Tn3 elements, however, specifying non-ESBLs have recently been described in serovar Typhimurium from a rabbit and in serovar Infantis from poultry and shown to be linked to either the tetracycline resistance gene tet(A) or the quinolone resistance gene qnrS (9, 13). FIG. 2. Genetic organization of the blaTEM-52 carrying transposon on a conjugative IncI1 plasmid from serovar Typhimurium strain 04-3486. The position and orientation of the genes are indicated by arrows. Gray arrows correspond to plasmid genes flanking the transposon. ... Among the extended-spectrum cephalosporin-resistant Salmonella strains studied, five isolates belonging to serovars Agona, Paratyphi B dT+, and Typhimurium, showed an additional multidrug resistance profile with resistances to chloramphenicol, florfenicol, streptomycin, spectinomycin, sulfonamide, tetracycline, and trimethoprim (Table ​(Table1).1). This multidrug resistance profile is characteristic of SGI1 antibiotic resistance gene clusters, which were previously identified in these serovars (11). Identification of SGI1 and mapping of its antibiotic resistance gene cluster performed as described previously (5) showed that two of the five isolates possessed SGI1 and the SGI1-A variant in serovar Typhimurium strain 04-3486 and serovar Agona strain 777SA01, respectively (Table ​(Table1).1). Serovar Agona strains with SGI1-A are frequently isolated from poultry in Belgium (5). The serovar Typhimurium isolate carrying SGI1 was further shown to be of phage type DT104, a dominant multidrug-resistant clone that has spread all over the world (11). To our knowledge, this is the first time that multidrug-resistant strains carrying SGI1 together with a plasmid-borne ESBL gene have been reported, and further surveillance of such strains is thus warranted. Since most of the strains showing extended-spectrum cephalosporin resistance were of serovar Infantis, these were further investigated for clonality by XbaI and BlnI macrorestriction pulsed-field gel electrophoresis analysis. The Infantis isolates showing the same resistance profile and whatever their origin, poultry or human, showed identical XbaI and BlnI macrorestriction profiles, indicating that these were clonal (data not shown). In conclusion, the present study showed the spread of an IncI1 plasmid carrying the blaTEM-52 gene among S. enterica serovars Agona, Derby, Infantis, Paratyphi B dT+, and Typhimurium, as well as the spread of a single Infantis clone carrying this plasmid mainly in poultry. It is thus likely that humans infected with these strains were contaminated by ingestion of undercooked poultry products. The further spread of such plasmids in multidrug-resistant strains carrying SGI1 is of concern.

Population structure and resistance genes in antibiotic-resistant bacteria from a remote community with minimal antibiotic exposure.

Abstract: In a previous study, we detected unexpectedly high levels of acquired antibiotic resistance in commensal Escherichia coli isolates from a remote Guarani Indian (Bolivia) community with very low levels of antibiotic exposure and limited exchanges with the exterior. Here we analyzed the structure of the resistant E. coli population from that community and the resistance mechanisms. The E. coli population (113 isolates from 72 inhabitants) showed a high degree of genetic heterogeneity, as evidenced by phylogenetic grouping (77% group A, 10% group B1, 8% group D, 5% group B2) and genotyping by randomly amplified polymorphic DNA (RAPD) analysis (44 different RAPD types). The acquired resistance genes were always of the same types as those found in antibiotic-exposed settings [ bla TEM , bla PSE-1 , catI , cmlA6 , tet (A), tet (B), dfrA1 , dfrA7 , dfrA8 , dfrA17 , sul1 , sul2 , aphA1 , aadA1 , aadA2 , aadA5 , aadB , and sat-1 ]. Class 1 and class 2 integrons were found in 12% and 4% of the isolates, respectively, and harbored arrays of gene cassettes similar to those already described. The cotransferability of multiple-resistance traits was observed from selected isolates and was found to be associated with resistance conjugative plasmids of the F, P, and N types. Overall, these data suggest that the resistance observed in this remote community is likely the consequence of the dissemination of resistant bacteria and resistance genes from antibiotic-exposed settings (rather than of an independent in situ selection) which involved both the clonal expansion of resistant strains and the horizontal transfer/recombination of mobile genetic elements harboring resistance genes.

Multicopy blaOXA-58 Gene as a Source of High-Level Resistance to Carbapenems in Acinetobacter baumannii

Abstract: The mechanisms at the origin of heterogeneous carbapenem resistance levels observed among Acinetobacter baumannii isolates collected in 2005 in a large University Hospital of Rome, Italy, were investigated. These isolates were related and possessed similar plasmids carrying the carbapenem-hydrolyzing oxacillinase gene blaOXA-58 but showed variable levels of resistance to carbapenems. Analysis of sequences surrounding the blaOXA-58 gene showed genetic variability, with the presence in several isolates of multiple copies of the blaOXA-58 gene; this extra copy number was likely related to an IS26-mediated transposition or recombination process.

Comparative analysis of IncHI2 plasmids carrying blaCTX- M- 2 or blaCTX- M- 9 from Escherichia coli and Salmonella enterica strains isolated from poultry and humans

Abstract: Salmonella enterica bla(CTX-M-2) and bla(CTX-M-9) plasmid backbones from isolates from Belgium and France were analyzed. The bla(CTX-M-2-)plasmids from both human and poultry isolates were related to the IncHI2 pAPEC-O1-R plasmid, previously identified in the United States in avian Escherichia coli strains; the bla(CTX-M-9) plasmids were closely related to the IncHI2 R478 plasmid.

Acquisition and diffusion of blaCTX‐M‐9 gene by R478‐IncHI2 derivative plasmids

Abstract: Escherichia coli and Salmonella enterica isolates carrying the blaCTX-M-9 gene located on plasmids prevailed at the Hospital de la Santa Creu i Sant Pau, Barcelona, Spain in the 1996–1999 period. The blaCTX-M-9-carrying plasmids showed a great variability in size, suggesting the mobilization of the gene among different plasmid scaffolds. The aim of the present work was to identify and better characterize the plasmids involved in the spread of the blaCTX-M-9 gene. Results showed that the majority of these strains carried plasmids belonging to the IncHI2 incompatibility group. The IncHI2 plasmids were further characterized, and found to be related to the reference IncHI2 plasmid R478.

Expanded-spectrum β-Lactamase and Plasmid-mediated Quinolone Resistance

Abstract: To the Editor: The emergence of plasmid-mediated, and thus transferable, quinolone resistance determinants has been recently discovered (1) and shown to involve the pentapeptide repeat protein Qnr, which interacts with DNA gyrase and topoisomerase IV to prevent quinolone inhibition (2,3). Qnr determinants confer resistance to nalidixic acid and reduced susceptibility to fluoroquinolones (3). They have been identified worldwide in a variety of enterobacterial species and were often associated to expanded-spectrum β-lactamases (ESBLs) (2). The association between the ESBL VEB-1 and the QnrA1 determinants was reported (4). Because plasmid co-localization of QnrA and VEB-1 encoding genes has been reported repeatedly from scattered clonally-unrelated enterobacterial isolates, our objective was to use replicon typing to trace a possible dissemination of a common plasmid worldwide. The blaVEB-1 and/or qnrA-positive plasmids that have been included in the study were from 17 isolates previously described in detail (3–8) (Table). Escherichia coli transconjugants (Tc) were obtained for 14 of 17 clinical isolates, allowing an accurate replicon typing since original clinical isolates might harbor several plasmids. They were collected from 1999 to 2005, from patients hospitalized in different parts of the world (Table). The 13 blaVEB-1-positive isolates were from 5 countries (France, Turkey, Algeria, Thailand, and Canada), scattered on 4 continents. Among them, the Providencia stuartii and Proteus mirabilis isolates from Algeria were negative for qnrA1. In addition, 4 blaVEB-1-negative but qnrA1-positive isolates recovered from France and Australia were also included in the study. Table Features of the VEB-1– or QnrA-positive isolates used in this study* PCR-based replicon typing (PBRT), which recognizes FIA, FIB, FIC, HI1, HI2, I1-Ig, L/M, N, P, W, T, A/C, K, B/O, X, Y, and FII replicons (9), was applied to type the resistance plasmids from all the strains. Amplicons were confirmed by DNA sequencing and used as probes in hybridization experiments on purified plasmids (data not shown). PBRT results showed that the 13 blaVEB-1-positive plasmids (including 11 qnrA1-positive) belonged to the IncA/C incompatibility group. DNA sequencing identified the A/C2 replicon variant (European Molecular Biology Laboratory no. {"type":"entrez-nucleotide","attrs":{"text":"AM087198","term_id":"90855305","term_text":"AM087198"}}AM087198) in all these plasmids (Table). Plasmids of this type were recently identified in the United States and in Italy carrying the AmpC-type cephalosporinase CMY-4–encoding gene (10). In 2 strains (E. coli TcGOC and Citrobacter freundii LUT), the IncA/C2 plasmids were associated with additional replicons, which suggests the presence of multiple plasmids or fusions between plasmids of different backbones. By contrast, all the 4 blaVEB-1-negative isolates but qnrA1-positive were negative for the A/C replicon, except transconjugant TcK147; however, sequencing identified an A/C1-type replicon in that strain. These results indicated that the genes encoding QnrA1 and VEB-1, when identified concomitantly in a given isolate, were always located on plasmids belonging to the same IncA/C2-incompatibility group that may vary in size and digestion pattern (Table; unpub. data). In addition, we showed that plasmids carrying the blaVEB-1 gene but lacking qnrA1 were also of the IncA/C2 type (Table). Plasmids that were blaVEB-1-negative but qnrA1-positive were of distinct replicon types, thus suggesting independent acquisition of the qnrA1 gene on different plasmids. It is remarkable that since VEB-1 is apparently always encoded by IncA/C2 plasmids, when genes for QnrA1 and VEB-1 are found together, they also occur on IncA/C2 plasmids. Thus, evidence here shows that the IncA/C2 plasmid is the main vehicle of the blaVEB-1 gene worldwide, on which the qnrA1 gene may be added. The possibility that both blaVEB-1 and qnrA1 genes may be identified on a single genetic structure in several isolates has been recently shown with their identification within the same sul1-type integron (6). Since results of these experiments provided a good marker for tracing blaVEB-1-positive plasmids, and taking in account the property of A/C-type plasmids to have a broad range of hosts (note: this has not been demonstrated for the specific A/C2 subgroup), we tried to amplify the A/C2 replicon in a collection of 15 blaVEB-1-positive and clonally unrelated Pseudomonas aeruginosa isolates from France, Thailand, India, and Kuwait. The blaVEB-1 gene was supposed to be chromosome-encoded in those isolates. PCR failed to give any positive results, confirming the absence of an IncA/C-type plasmid and also ruling out the hypothesis of IncA/C2-type plasmid co-integration at the origin of blaVEB-1 acquisition in P. aeruginosa. The spread of plasmids carrying a large array of resistance genes among Enterobacteriaceae is of concern since this provides a convenient genetic mechanism for a given strain to become panresistant to antimicrobial drugs. In particular, the recent identification of the Qnr determinants have shown that plasmids may provide resistance (or at least reduced susceptibility) to quinolones and fluoroquinolones, whereas they are already known to carry resistance to β-lactams, aminoglycosides, chloramphenicol, tetracycline, rifampin, sulfonamides, and disinfectants. pQR1 (4) or p1 (6) are examples of well-characterized plasmids that mediate multidrug resistance by carrying blaVEB-1 and qnrA1, together with aminoglycoside resistance genes aadB, aacA1, and aadA1, chloramphenicol resistance gene cmlA, rifampin resistance gene arr2, disinfectant resistance gene qacI, and sulfonamides resistance gene sul1. Our study showed that the IncA/C2-type plasmids may be the source of such worldwide dissemination. It means that 1 plasmid scaffold has brought the same (or at least very similar) multidrug resistance to multiple enterobacterial species in different continents.

Optimization of High-Resolution Melting Analysis for Low-Cost and Rapid Screening of Allelic Variants of Bacillus anthracis by Multiple-Locus Variable-Number Tandem Repeat Analysis

Abstract: Background: Molecular genotyping of Bacillus anthracis , the etiologic agent of anthrax, is important for differentiating and identifying strains from different geographic areas and for tracing strains deliberately released in a bioterrorism attack. We previously described a multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA) based on 25 marker loci. Although the method has great differentiating power and reproducibility, faster genotyping at low cost may be requested to accurately identify B. anthracis strains in the field. Methods: We used the High Resolution Melter-1 (Idaho Technology) and a saturating dye of double-stranded DNA (LCGreen I) to identify alleles via PCR and melting-curve analysis of the amplicons. We applied high-resolution melting analysis (HRMA) to a collection of 19 B. anthracis strains. Results: HRMA produced reproducible results for 6 of the 25 B. anthracis loci tested. These easily interpretable and distinguishable melting curve results were consistent with MLVA results obtained for the same alleles. The feasibility of this method was demonstrated in testing of different allelic variants for the 6 selected loci. Conclusions: The described HRMA application for screening B. anthracis VNTR loci is fast and widely accessible and may prove particularly useful under field conditions.

Characterization of the IncA/C plasmid pCC416 encoding VIM-4 and CMY-4 β-lactamases

Abstract: Objectives: To characterize the antibiotic resistance regions of pCC416, a VIM-4- and CMY-4-encoding plasmid from clinical enterobacteria, and to elucidate its relation with the CMY-encoding plasmids widely diffused in Salmonella. Methods: The enterobacterial multiresistant plasmid pCC416 was derived from an Escherichia coli transconjugant and characterized. Conventional and long-range PCR assays were performed using primers specific for VIM-4- and CMY-4-encoding segments of pCC416. Amplicons were characterized by sequencing. blaVIM-4, blaMY-4 and IntI1-specific probes were prepared from PCR products and used for the identification of various pCC416 clones. VIM- and CMY-positive Bam HI and Sau 3AI fragments of pCC416 were cloned into pACYC184 and their sequences were determined by gene walking. Results: The pCC416 plasmid contained two distinct resistant loci carrying β-lactamase genes. The blaVIM-4 gene was part of an integron located in a complex, multidrug-resistant region of novel structure, interspersed with mobile elements or remnants thereof and being similar to various regions of other resistance plasmids. Nevertheless, a region in the 3′ end of this structure resembled the respective region found in a CMY-2-encoding plasmid from Salmonella. The blaCMY-4 gene was identified within an 11.3 kb region also related to the CMY-2-encoding plasmids. Conclusions: pCC416 probably evolved from an IncA/C2, CMY-encoding plasmid through acquisition of a VIM-encoding In4-type integron providing an example of accretion of resistance determinants in a single replicon. © The Author 2007.

Outbreak of Acinetobacter baumannii producing the carbapenem-hydrolyzing oxacillinase OXA-58 in Rome, Italy.

Abstract: In this study 45 epidemic and sporadic isolates of Acinetobacter baumannii were investigated by antimicrobial resistance, integron identifications and genotyping. Isolates were genotyped by random amplified polymorphism (RAPD) DNA and pulsed-field gel electrophoresis (PFGE). Four different RAPD patterns were observed among the isolates of our collection, further discerned in six PFGE types. Two prevalent genotypes were identified, one corresponding to a carbapenem resistant epidemic clone, causing an outbreak at the intensive care unit of a hospital of Rome. Two class 1 integrons, carrying different gene cassette arrays, were identified among the two prevalent genotypes. Nucleotide analysis of the integron-variable regions revealed the presence of the aacA4, orfO, bla(OXA-20), and aacC1, orfX, orfX', aadA1 gene cassette arrays, respectively. All the carbapenem resistant strains analyzed in this study carried the bla (OXA-58) gene located on plasmids.