Emergence of a multiresistant KPC-3 and VIM-1 carbapenemase-producing Escherichia coli strain in Spain.

OBJECTIVES: To characterize the mechanisms involved in carbapenem resistance, as well as the genetic elements supporting their mobilization, in a multidrug-resistant Escherichia coli isolate. METHODS: The E. coli isolate was obtained from a patient with fatal urinary sepsis. Antimicrobial susceptibility testing was performed by the disc diffusion and agar dilution methods. The E. coli molecular type and phylogroup were determined using multilocus sequence typing and the triple PCR technique, respectively. PCR and sequencing were used for virulence and resistance genotype characterization. Plasmid content and gene location were analysed by S1-PFGE, I-Ceu1-PFGE and hybridization experiments. Transformation assays were performed. RESULTS: The E. coli strain, typed as ST448 and phylogroup B1, was resistant to all tested antibiotics except fosfomycin, tigecycline and tetracycline. The following resistance and virulence genetic structures were obtained: ISKpn7 + bla(KPC-3) + ISKpn6 linked to Tn4401; tnpR + aac(6')-Ib'-9 + aadA1 + bla(OXA-9) + tnpR + bla(TEM-1a) + tnpB + strB + strA + sul2; intI1 + bla(VIM-1) + aac(6')-Ib' + aphA15 + aadA1 + catB2 + qacEΔ1-sul1 + orf5; ISEcp1 + bla(CMY-2); IS26 + bla(SHV-12); aph(3')-I; aac(3)-IV; floR; catA; and fimA. Mutations in the ampC promoter (-18, -1 and +58) and substitutions in the GyrA (Ser-83→Leu and Asp-87→Asn) and ParC (Ser-80→Ile) proteins were observed. IncFII (ST2), IncA/C and ColE(TP) plasmids of 145.5, 87 and <2 kb, respectively, were found. The bla(VIM-1) gene was located in a non-typeable plasmid of >300 kb, and the bla(KPC-3) gene in the 145.5 kb IncFII plasmid. Transformant strains carried the IncFII and ColE(TP) plasmids, and the bla(KPC-3), bla(TEM-1a), bla(OXA-9), aadA1, aac(6')-Ib'-9, aac(3)-IV and floR genes. CONCLUSIONS: This is the first report of the co-production of KPC-3, VIM-1, SHV-12, OXA-9 and CMY-2 in a unique clinical multiresistant E. coli isolate. The dissemination of these genes on mobile genetic elements is alarming and complicates antimicrobial therapies.

High clonality and diversity of virulence determinants among bla(PSE)-positive Salmonella Typhimurim isolates recovered in three geographically distant Spanish hospitals.

Molecular typing, the presence of Salmonella genomic island 1 (SGI1), and virulence factors were studied in 45 bla(PSE)-positive S. Typhimurium isolates from 3 hospitals. All isolates belonged to sequence type ST19, presented low clonal diversity, and harbored SGI1. The wide diversity of virulence factors was classified into 3 major virulotype groups.

Genetic characterization of the mechanisms of resistance to amoxicillin/clavulanate and third-generation cephalosporins in Salmonella enterica from three Spanish hospitals.

The mechanisms of antimicrobial resistance were characterized in 90 Salmonella enterica isolates either resistant or with intermediate resistance to amoxicillin/clavulanate (AMC(R/I)) or resistant to third-generation cephalosporins (C3G(R)). These isolates were recovered in three Spanish hospitals during 2007-2009. The C3G(R) phenotype was expressed by three isolates that carried the following extended-spectrum ß-lactamase genes: phage-associated bla(CTX M-10) in S. Virchow, bla(CTX-M-14a) surrounded by ISEcp1 and IS903 in S. Enteritidis, and bla(CTX-M-15) linked to ISEcp1 and orf477 in S. Gnesta (first description in this serotype). The AMC(R/I) phenotype was found in 87 isolates (79 S. Typhimurim, 7 S. Enteritidis, and one S. Thompson). The bla(PSE-1) gene, followed by bla(OXA-1) was mostly found among S. Typhimurim, and the bla(TEM-1) gene among S. Enteritidis. Three different gene combinations [bla(PSE-1) +floR+aadA2+sul+tet(G); bla(OXA-1) +catA+aadA1/strA-strB+sul+tet(B) and bla(TEM-1) + cmlA1+aadA/strA-strB+sul+tet(A)/tet(B) genes] were associated with the ampicillin-chloramphenicol-streptomycin-sulfonamides-tetracycline phenotype in 68 AMC(R/I) S. enterica isolates. Class 1 integrons were observed in 79% of the isolates and in most of them (45 isolates) two integrons including the aadA2 and bla(PSE-1) gene cassettes, respectively, were detected. The bla(OXA-1) +aadA1 arrangement was detected in 23 isolates, and the aac(6')-Ib-cr+bla(OXA-1) +catB3+arr3 in another one. Non-classic class 1 integrons were found in three isolates: dfrA12+orfF+aadA2+cmlA1+aadA1 (1 isolate), dfrA12+orfF+aadA2+ cmlA1+aadA1+qacH+IS440+sul3 (1 isolate) and dfrA12+orfF+aadA2+cmlA1+aadA1+qacH+IS440+ sul3+orf1+mef(B)Δ-IS26 (1 isolate). Taken together, these results underline the need for clinical concern regarding ß-lactam resistance in Salmonella and thus for continuous monitoring.

Genetic characterization of the mechanisms of resistance to amoxicillin/clavulanate and third-generation cephalosporins in Salmonella enterica from three Spanish hospitals

The mechanisms of antimicrobial resistance were characterized in 90 Salmonella enterica isolates either resistant or with intermediate resistance to amoxicillin/clavulanate (AMC(R/I)) or resistant to third-generation cephalosporins (C3G(R)). These isolates were recovered in three Spanish hospitals during 2007-2009. The C3G(R) phenotype was expressed by three isolates that carried the following extended-spectrum β-lactamase genes: phage-associated bla(CTX M-10) in S. Virchow, bla(CTX-M-14a) surrounded by ISEcp1 and IS903 in S. Enteritidis, and bla(CTX-M-15) linked to ISEcp1 and orf477 in S. Gnesta (first description in this serotype). The AMC(R/I) phenotype was found in 87 isolates (79 S. Typhimurim, 7 S. Enteritidis, and one S. Thompson). The bla(PSE-1) gene, followed by bla(OXA-1) was mostly found among S. Typhimurim, and the bla(TEM-1) gene among S. Enteritidis. Three different gene combinations [bla(PSE-1) +floR+aadA2+sul+tet(G); bla(OXA-1) +catA+aadA1/strA-strB+sul+tet(B) and bla(TEM-1) + cmlA1+aadA/strA-strB+sul+tet(A)/tet(B) genes] were associated with the ampicillin-chloramphenicol-streptomycin-sulfonamides-tetracycline phenotype in 68 AMC(R/I) S. enterica isolates. Class 1 integrons were observed in 79% of the isolates and in most of them (45 isolates) two integrons including the aadA2 and bla(PSE-1) gene cassettes, respectively, were detected. The bla(OXA-1) +aadA1 arrangement was detected in 23 isolates, and the aac(6’)-Ib-cr+bla(OXA-1) +catB3+arr3 in another one. Non-classic class 1 integrons were found in three isolates: dfrA12+orfF+aadA2+cmlA1+aadA1 (1 isolate), dfrA12+orfF+aadA2+ cmlA1+aadA1+qacH+IS440+sul3 (1 isolate) and dfrA12+orfF+aadA2+cmlA1+aadA1+qacH+IS440+ sul3+orf1+mef(B)Δ-IS26 (1 isolate). Taken together, these results underline the need for clinical concern regarding β-lactam resistance in Salmonella and thus for continuous monitoring (AU)

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In vivo selection of aac(6')-Ib-cr and mutations in the gyrA gene in a clinical qnrS1-positive Salmonella enterica serovar Typhimurium DT104B strain recovered after fluoroquinolone treatment.

OBJECTIVES: To characterize the mechanisms implicated in the in vivo selection of quinolone and aminoglycoside resistance in a faecal Salmonella enterica serovar Typhimurium DT104B strain recovered after ciprofloxacin treatment of a hospitalized elderly patient with acute gastroenteritis. METHODS: Two Salmonella Typhimurium isolates were obtained before (Se6) and after (Se20) treatment and they were typed by PFGE and multilocus sequence typing (MLST). Antimicrobial susceptibility was determined by disc diffusion and agar dilution methods. Class 1, 2 and 3 integrons and resistance mechanisms were studied by PCR and sequencing. Plasmids were typed. RESULTS: Both Salmonella Typhimurium strains were resistant to tetracycline, streptomycin and sulphonamides, while Se20 was also resistant to nalidixic acid, ciprofloxacin, norfloxacin, levofloxacin, ofloxacin, amikacin, tobramycin, kanamycin and trimethoprim. PFGE and MLST showed a clonal relationship between the strains, which belonged to the sequence type ST36. Both strains contained the repC-sul2-strA-strB structure and tet(A) and qnrS1 genes, and strain Se20 also contained the aac(6')-Ib-cr gene, the Ser83-->Tyr substitution in GyrA and one class 1 integron with the dfrA17 + aadA5 gene cassette arrangement lacking qacEDelta1 + sul1. Two different transconjugants from Salmonella Se20 (TCSe20B and TCSe20L) harboured qnrS1 and sul2 genes and the class 1 integron. The TCSe20B strain also acquired the aac(6')-Ib-cr gene located on a non-typeable plasmid. qnrS1 was identified on a ColE-type plasmid and the class 1 integron on an IncI1-type plasmid. CONCLUSIONS: This is the first report of in vivo selection of the aac(6')-Ib-cr gene and the Ser83-->Tyr change in GyrA in a qnrS1-positive Salmonella Typhimurium strain after ciprofloxacin treatment; the in vitro transfer of both plasmid-mediated quinolone resistance genes was also demonstrated.

Genetic environment of sul genes and characterisation of integrons in Escherichia coli isolates of blood origin in a Spanish hospital.

The prevalence and characterisation of integrons and the genetic environment of sulphonamide resistance genes were studied in 135 Escherichia coli isolates recovered from blood cultures in a Spanish hospital during 2007. Class 1 and 2 integrons were identified in 54 isolates (intI1, 52 isolates; intI2, 1 isolate; and intI1+intI2, 1 isolate). Of the 53 intI1-positive isolates, 36 (67.9%) contained the classic class 1 integron including the qacEDelta1-sul1 region, and 11 different gene cassette arrangements were demonstrated in 33 of these isolates. Seventeen intI1-positive isolates lacked the qacEDelta1-sul1 region, and 8 gene cassette arrangements were demonstrated in 12 of these isolates. Seventy-one isolates showed a sulphonamide-resistant phenotype, 63 of which contained sul genes. The sul1 gene was associated with intI1 in 36 of 42 sul1-positive isolates, and the sul3 gene was associated with non-classic class 1 integrons in 5 of 7 sul3-positive isolates. Finally, sul2 was found associated with strA-strB genes in 32 of 35 sul2-positive isolates, identifying 11 genetic structures, 1 of them presenting the IS150 element disrupting the strB gene; this structure was included in GenBank with accession no. FJ705354. Almost one-half of the E. coli isolates from blood cultures contained integrons and sul genes. Moreover, sul genes were detected in different structures, one of them new, and could be important determinants in antibiotic resistance dissemination.