Caracterización de mecanismos de resistencia a carbapenémicos en aislados clínicos de Pseudomonas aeruginosa en un hospital español / Characterisation of carbapenem-resistance mechanisms in clinical Pseudomonas aeruginosa isolates recovered in a Spanish hospital

INTRODUCCIÓN: Los carbapenémicos son los antibióticos betalactámicos con mayor espectro de actividad en el tratamiento de infecciones por Pseudomonas aeruginosa. El objetivo de este trabajo fue caracterizar molecularmente una colección de aislados de P. aeruginosa resistentes a carbapenémicos (PARC). MÉTODOS: Se recogieron 85 aislados PARC de 60 pacientes en el Hospital San Pedro, Logroño (período 2008-2011). La relación clonal se determinó por electroforesis en gel de campo pulsado (PFGE), la sensibilidad a 15 antipseudomónicos por método de difusión en disco y las alteraciones en oprD, la caracterización de integrones y la tipificación molecular (MLST) por PCR y secuenciación. RESULTADOS: Las 85 PARC se clasificaron en 35 perfiles diferentes de PFGE. Se seleccionaron 61 cepas de los 60 pacientes y se observó que eran multirresistentes, aunque ninguna mostró fenotipo carbapenemasa. Se detectó un gran polimorfismo de OprD, destacando que el 59% de las cepas presentaban un codón de finalización prematuro. ISPa1328 e ISPsp4 truncaban el gen oprD en 2 cepas (GenBank KF517097 y KF517098). El 67% de las cepas presentó integrones de clase 1 con genes codificantes de enzimas modificantes de aminoglucósidos, 2 de las cuales portaban un nuevo integrón: aac(3)-Ia+aadA1h (nombrado In272, GenBank GQ144317). Se detectaron 4 secuencias tipo (ST) (número de cepas): ST175 (35), ST308 (3), ST235 (2) y ST639 (1). CONCLUSIÓN: La multirresistencia, el alto polimorfismo de oprD, el alto porcentaje de integrones, la moderada relación clonal de las cepas y la elevada diseminación epidémica de clones de alto riesgo son aspectos de gran preocupación clínica para erradicar la diseminación de PARC

INTRODUCTION: Carbapenems are the beta-lactam antibiotics with the best spectrum of activity in the treatment of Pseudomonas aeruginosa infections. The objective of this study was to molecularly characterise a collection of carbapenem-resistant P. aeruginosa isolates (PARC). METHODS: A total of 85 PARC isolates were recovered from 60 patients in the Hospital San Pedro, Logroño (period 2008-2011). Clonal relationship was determined using pulsed-field gel electrophoresis (PFGE), susceptibility testing to 15 anti-pseudomonal agents was performed using the disk diffusion method, and alterations in oprD, characterisation of integrons and molecular typing (MLST) using PCR and sequencing. RESULTS: The 85 PARC were classified into 35 different PFGE profiles. Of the 61 selected strains from 60 patients all of them were multiresistant, although none of them showed a carbapenemase phenotype. High polymorphism was detected in OprD, emphasising that 59% of the strains had a premature stop codon. ISPa1328 and ISPsp4 insertion sequences truncated oprD gene into 2 strains (GenBank KF517097 and KF517098). Two-thirds (67%) of the strains showed class 1 integrons with genes encoding aminoglycoside modifying enzymes, and 2 of them carried a new integron: aac(3)-Ia+aadA1h, named In272, GenBank GQ144317. Four sequence types were detected (Strain Nos.): ST175 (35), ST308 (3), ST235 (2), and ST639 (1). CONCLUSION: Multidrug resistance, high polymorphism in oprD, a high percentage of integrons, moderate clonal relationship of strains, and the high epidemic dissemination of high-risk clones are clinical aspects of great concern in order to eradicate the spread of PARC

Characterisation of carbapenem-resistance mechanisms in clinical Pseudomonas aeruginosa isolates recovered in a Spanish hospital. / Caracterización de mecanismos de resistencia a carbapenémicos en aislados clínicos de Pseudomonas aeruginosa en un hospital español.

INTRODUCTION: Carbapenems are the beta-lactam antibiotics with the best spectrum of activity in the treatment of Pseudomonas aeruginosa infections. The objective of this study was to molecularly characterise a collection of carbapenem-resistant P. aeruginosa isolates (PARC). METHODS: A total of 85 PARC isolates were recovered from 60patients in the Hospital San Pedro, Logroño (period 2008-2011). Clonal relationship was determined using pulsed-field gel electrophoresis (PFGE), susceptibility testing to 15anti-pseudomonal agents was performed using the disk diffusion method, and alterations in oprD, characterisation of integrons and molecular typing (MLST) using PCR and sequencing. RESULTS: The 85 PARC were classified into 35 different PFGE profiles. Of the 61selected strains from 60patients all of them were multiresistant, although none of them showed a carbapenemase phenotype. High polymorphism was detected in OprD, emphasising that 59% of the strains had a premature stop codon. ISPa1328 and ISPsp4 insertion sequences truncated oprD gene into 2 strains (GenBank KF517097 and KF517098). Two-thirds (67%) of the strains showed class 1 integrons with genes encoding aminoglycoside modifying enzymes, and 2 of them carried a new integron: aac(3)-Ia+aadA1h, named In272, GenBank GQ144317. Four sequence types were detected (Strain Nos.): ST175 (35), ST308 (3), ST235 (2), and ST639 (1). CONCLUSION: Multidrug resistance, high polymorphism in oprD, a high percentage of integrons, moderate clonal relationship of strains, and the high epidemic dissemination of high-risk clones are clinical aspects of great concern in order to eradicate the spread of PARC.

Changes in genetic lineages, resistance, and virulence in clinical methicillin-resistant Staphylococcus aureus in a Spanish hospital.

A total of 204 methicillin-resistant Staphylococcus aureus (MRSA) isolates were isolated in a Spanish hospital in two different periods (2001 and 2009). The percentages of MRSA isolates detected in 2001 and 2009 were 29 and 27 %, respectively. Genetic lineages, resistance mechanisms, and virulence traits were determined in these isolates. The most frequent detected lineage in both periods was S. aureus protein A (spa)-type t067, assigned to clonal complex (CC) 5 (CC5-t067), being more prevalent in 2001 (93 %) than in 2009 (71 %). The remaining CCs and spa-types detected were (%2001/%2009): CC5-t002 (0/5), CC8-t008 (1/16), CC8-t024 (0/1), CC8-t190 (0/3), CC8-t2849 (0/2), CC22-t032 (0/2), CC30-t012 (1/0), CC228-t109 (1/0), CC228-t1318 (2/0), and CC247-t051 (2/0). Most of the MRSA were isolated from wounds, representing 39 % in 2001 and 63 % in 2009. The emergence of MRSA CC8 isolates, mainly from wounds, seemed to occur in the second period. Resistance to (%2001/%2009) quinolones (99/87), aminoglycosides (98/88), macrolides (32/30), lincosamides (30/17), and tetracycline (2/1) was found in isolates in both periods. Trimethoprim-sulfamethoxazole resistance was detected only in 2001 (1 %), and chloramphenicol (1 %) and mupirocin resistance (11 %) were detected only in 2009. An association between staphylococcal enterotoxin gene profiles and CCs was detected in most of the cases. The egc-cluster was related to CC5, CC22, CC30, and CC228 and most of the CC8 isolates presented the sed, sej, and ser genes. Four tst-1-positive (CC5 and CC30) isolates were detected in 2001 and two lukS/F-PV-positive isolates were detected in 2009. Therefore, there is still a predominance of CC5-t067 in our region, although an increase of lineage CC8 was observed.

Outbreak caused by a multi-resistant Klebsiella pneumoniae strain of new sequence type ST341 carrying new genetic environments of aac(6')-Ib-cr and qnrS1 genes in a neonatal intensive care unit in Spain.

An outbreak due to a Klebsiella pneumoniae clone occurred in a neonatal intensive care unit of a Spanish Hospital in which three newborns were infected (all with gestational age ≤29 weeks; two of them died) and seven were colonized (gestational age >32 weeks; none died). One K. pneumoniae strain per patient was further characterized. The 10 strains showed an indistinguishable pulsed-field-gel-electrophoresis pattern, were typed in the phylogenetic group KpI and were ascribed into a new sequence type registered as ST341. All 10 strains presented the same multiple-antibiotic-resistant phenotype, showed extended-spectrum-beta-lactamase production, and harbored the bla(CTX-M-15), bla(SHV-11), bla(OXA-1,)aac(6')-Ib-cr, qnrS1, aac(3)-II, aph(3')-Ia and aadA5 resistance genes. No class 1 or class 2 integrons were detected. The bla(CTX-M-15) gene presented the following genetic environment: ISEcp1-bla(CTX-M-15)-orf477. These strains contained two copies of the aac(6')-Ib-cr gene included in the following new genetic environments: aac(3)-II-IS26-aac(6')-Ib-cr-bla(OXA-1) and aac(3)-II-IS26-ΔcatB3-bla(OXA-1)-aac(6')-Ib-cr (registered at GenBank with accession numbers GQ438247 and GQ438248, respectively). The genetic environment of the qnrS1 gene (IS26-ΔISEcl2-qnrS1) (GenBank accession number GQ438249) was also not described previously. The aac(6')-Ib-cr, qnrS1, bla(CTX-M-15), aac(3)-II, and bla(OXA-1) genes, located in a plasmid of 33.5 kb, could be transferred to Escherichia coli by transformation.

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.