Rubén González-Sanz

Emergence of extended-spectrum β-lactamases and AmpC-type β-lactamases in human Salmonella isolated in Spain from 2001 to 2005

OBJECTIVES To study the resistance to third-generation cephalosporins in Salmonella strains isolated from humans in a 5 year period in Spain, and to identify the responsible genes and their dissemination. METHODS Twenty-seven isolates were analysed by PCR and sequencing to identify the genes responsible for the beta-lactamase resistance phenotypes. The transferability of the phenotypes was tested by conjugation to Escherichia coli K12J53, plasmid detection with S1-PFGE, hybridization and PCRs of the transconjugants. The genetic relationship was determined by PFGE. RESULTS We found bla(CTX-M-9) and bla(CTX-M-10) in Salmonella Virchow PT19. bla(CTX-M-14) was detected in Salmonella (IV) 44:z(4),z(23):-, Salmonella Enteritidis PT6a, Salmonella Typhimurium DT193 and Salmonella Typhimurium DT104B. bla(CTX-M-1) was found in Salmonella Litchfield. bla(CTX-M-15) and bla(CTX-M-32) were found in Salmonella Enteritidis PT1. bla(SHV-12) was found in Salmonella Blockley, Salmonella Hadar PT2, Salmonella Enteritidis PT21, Salmonella Enteritidis PT1 and Salmonella Bredeney. bla(SHV-2) was found in Salmonella Livingstone. bla(CMY-2) was detected in Salmonella Bredeney, Salmonella Newport, Salmonella Enteritidis PT5b and Salmonella Heidelberg. bla(DHA-1) was detected for the first time in Spain in Salmonella Newport. One strain of Salmonella Senftenberg harboured two extended-spectrum beta-lactamases, bla(SHV-12) and bla(CTX-M-9). We have found a large variety of beta-lactamase families as well as several members of major relevance, such as CTX-M-15, CTX-M-32, CMY-2 and DHA-1. XbaI-PFGE, conjugation assays and S1-PFGE hybridization showed that all these beta-lactamases were mediated by plasmids. CONCLUSIONS This study demonstrates the emergence of a public health risk related to resistance to beta-lactams in Salmonella. The resistance trends need to be monitored carefully.

Surveillance of antibiotic resistance evolution and detection of class 1 and 2 integrons in human isolates of multi-resistant Salmonella Typhimurium obtained in Uruguay between 1976 and 2000

OBJECTIVES To study the evolution of antibiotic resistance in isolates of Salmonella enterica subspecies enterica serovar Typhimurium (Salmonella Typhimurium) obtained in Uruguay between the years 1976 and 2000, and to determine the incidence of class 1 and 2 integrons in the multi-resistant isolates. METHODS We studied 258 strains of Salmonella Typhimurium from various sources, isolated between 1976 and 2000. We determined the evolution of antibiotic resistance and the distribution of class 1 and 2 integrons in all isolates by means of disk diffusion assays and PCR. RESULTS During the period 1989-2000 resistance to streptomycin was 56.8%, tetracycline 13.6%, sulfonamides 11.2%, and ampicillin 7.2%. Resistance to gentamicin, kanamycin, chloramphenicol, and nalidixic acid were lower than 5%; no resistance was detected to fluoroquinolones, oxyiminocephalosporins, and amikacin. These results show a dramatic decrease with respect to values found in the period 1976-1988. In this period, resistance to streptomycin was 63.2%, tetracycline 36.8%, sulfonamides 32.3%, and ampicillin 27.8%. Throughout the two periods, 29 multi-resistant Salmonella Typhimurium strains were isolated harboring some class of integron: 15 strains had only intI2, 11 strains presented both intI1 and intI2, and three isolates only intI1. CONCLUSIONS Our results show a marked decrease in resistance throughout these years, along with a correlation between resistance to different antibiotics and the presence of integrons.

Characterization of multidrug-resistant Enterobacteriaceae carrying plasmid-mediated quinolone resistance mechanisms in Spain

OBJECTIVES The aim of this study was to detect and characterize plasmid-mediated quinolone resistance determinants as well as genes responsible for additional resistances in Enterobacteriaceae isolates in Spain. METHODS The resistance genes were identified by PCR and sequencing. Plasmid analysis was carried out by S1-PFGE and PCR-based replicon typing. Conjugation assays were performed to link resistance genes to plasmids. The genetic relationships among the strains were determined by XbaI-PFGE. RESULTS One hundred and twenty-three isolates carried qnr as the only quinolone resistance determinant. One Salmonella Bredeney was positive for qnrB2 harboured on a 320 kb conjugative IncHI2 plasmid. One Salmonella Newport was positive for qnrB4 harboured on a 70 kb conjugative IncFIIs plasmid. Twenty-five Salmonella Thompson were positive for qnrA1. Twenty-two harboured a 220 kb non-conjugative and non-typeable plasmid, two a 220 kb conjugative IncHI2 plasmid and one a 120 kb non-conjugative IncA/C plasmid. qnrS1 was always detected on non-conjugative ColE(TP) plasmids of various sizes. Thus, two Salmonella Montevideo strains carried a 20 kb plasmid while Salmonella Typhimurium strains carried plasmids of 10 kb (n = 91) or 30 kb (n = 2). One Escherichia coli was positive for qnrA1 detected on a 220 kb conjugative IncHI2 plasmid. qnr alleles and β-lactamases were associated in Salmonella Bredeney (harbouring bla(SHV-12)), Salmonella Newport (harbouring bla(DHA-1)) and E. coli (harbouring bla(CTX-M-9)). CONCLUSIONS This is the first epidemiological study of qnr genes in Enterobacteriaceae isolates from Spain. Salmonella plasmids bearing qnr alleles are not a localized phenomenon in Spain and wide variation in plasmids and co-resistance was detected. The presence of qnr determinants in Salmonella serotypes commonly reported in human disease is concerning.

Antimicrobial resistance and phage and molecular typing of Salmonella strains isolated from food for human consumption in Spain.

The aims of this study were to ascertain the population structure and antimicrobial susceptibility of Salmonella enterica serovars isolated in 2002 from food in 16 Spanish regions. Serovars were characterized by serotyping, phage typing, antimicrobial susceptibility, and pulsed-field gel electrophoresis (PFGE) typing, and 264 nonrelated strains were selected for further analysis. The main sources were eggs and their derivatives (21.6% of strains), poultry and related products (16.6%), and seafood (16.3%). High serotype diversity was detected (51 serotypes); the most common were Enteritidis (n = 96, 36.3%) and Typhimurium (n = 53, 20.1%), followed by a miscellaneous group of 49 different serotypes (n = 115, 43.5%). A 15% increase in Salmonella Enteritidis isolation was observed. Common phage types for Salmonella Enteritidis were PT1 (41.6% of isolates), PT4 (9.4%), PT6 (9.4%), and PT6a (9.4%), and common types for Salmonella Typhimurium were DTU302 (18.8%), DT104 (15.1%), and DT104B (13.2%). Salmonella Enteritidis strains were categorized into eight PFGE types with a similarity of 81 to 96%, and 73.9% of the strains were grouped into just one cluster. Salmonella Typhimurium isolates were divided into 13 PFGE types with a similarity of 64 to 86%, and one predominant clone contained 41.5% of the strains. Resistance rates for Salmonella Enteritidis, Salmonella Typhimurium, and the miscellaneous group were, respectively, 8.3, 69.8, and 13.9% for ampicillin, 3.1, 52.8, and 59% for streptomycin, 40.6, 22.6, and 10.4% for nalidixic acid, 15.6, 71.7, and 31.1% for tetracycline, 7.3, 18.8, and 9.5% for trimethoprim-sulfamethoxazole, 0, 50.9, and 4.3% for chloramphenicol, and 6.2, 71.7, and 17.4% for multiple (at least four) antimicrobials. All the strains remained susceptible to other beta-lactams and fluoroquinolones. Surveillance of S. enterica isolated from food is strongly recommended to reduce community exposure to antimicrobial resistant strains.

ISG15 Is Upregulated in Respiratory Syncytial Virus Infection and Reduces Virus Growth through Protein ISGylation

ABSTRACT Human respiratory syncytial virus (RSV), for which neither a vaccine nor an effective therapeutic treatment is currently available, is the leading cause of severe lower respiratory tract infections in children. Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like protein that is highly increased during viral infections and has been reported to have an antiviral or a proviral activity, depending on the virus. Previous studies from our laboratory demonstrated strong ISG15 upregulation during RSV infection in vitro. In this study, an in-depth analysis of the role of ISG15 in RSV infection is presented. ISG15 overexpression and small interfering RNA (siRNA)-silencing experiments, along with ISG15 knockout (ISG15−/−) cells, revealed an anti-RSV effect of the molecule. Conjugation inhibition assays demonstrated that ISG15 exerts its antiviral activity via protein ISGylation. This antiviral activity requires high levels of ISG15 to be present in the cells before RSV infection. Finally, ISG15 is also upregulated in human respiratory pseudostratified epithelia and in nasopharyngeal washes from infants infected with RSV, pointing to a possible antiviral role of the molecule in vivo. These results advance our understanding of the innate immune response elicited by RSV and open new possibilities to control infections by the virus. IMPORTANCE At present, no vaccine or effective treatment for human respiratory syncytial virus (RSV) is available. This study shows that interferon-stimulated gene 15 (ISG15) lowers RSV growth through protein ISGylation. In addition, ISG15 accumulation highly correlates with the RSV load in nasopharyngeal washes from children, indicating that ISG15 may also have an antiviral role in vivo. These results improve our understanding of the innate immune response to RSV and identify ISG15 as a potential target for virus control.

Distribución de los serotipos y fagotipos de Salmonella de origen humano aislados en España en 1997-20

Introduccion. La salmonelosis continua siendo una de las causas principales de gastroenteritis en Espana, siendo la serotipificacion el marcador epidemiologico universalmente utilizado para la caracterizacion de los aislamientos de Salmonella spp. Algunos serotipos se identifican muy frecuentemente, reduciendo el poder de discriminacion de esta tecnica. Por ello, para el estudio epidemiologico de las salmonelosis producidas por estos serotipos es necesario utilizar marcadores complementarios como la fagotipificacion. Metodos. Se serotipificaron, por aglutinacion directa, las cepas de Salmonella spp. de origen humano recibidas en el Laboratorio Nacional de Referencia de Salmonella y Shigella (LNRSSE) entre los anos 1997 y 2001 y se fagotipificaron, segun esquemas internacionales, las cepas de los serotipos Enteritidis, Typhimurium, Hadar, Virchow y Typhi. Resultados. Se analizaron 30.856 cepas de Salmonella spp. procedentes de la mayoria de las Comunidades Autonomas. Los serotipos Enteritidis (51%) y Typhimurium (24%) fueron los mayoritarios. Las combinaciones serotipo/fagotipo mas frecuentes fueron: Enteritidis/FT1 (18%), Enteritidis/FT4 (15%), Enteritidis/FT6a (5%), Typhimurium/FT104 (5%) y Enteritidis/FT6 (3%). Las cepas del serotipo Enteritidis/FT1 tuvieron el mayor aumento en este periodo de tiempo, pasando del 11,61% en 1997 al 24,74% en 2001. Conclusiones. La utilizacion jerarquica de la serotipificacion y posteriormente de la fagotipificacion en cepas de Salmonella spp. de los serotipos mas frecuentes aumento enormemente el poder de discriminacion de la serotipificacion. Su aplicacion en estudios epidemiologicos es de gran utilidad en la caracterizacion temprana de cepas relacionadas.

Novel genetic environment of qnrB2 associated with TEM-1 and SHV-12 on pB1004, an IncHI2 plasmid, in Salmonella Bredeney BB1047 from Spain.

The presence of the qnrB2 gene in animal isolates and zoo-notic pathogens opens the possibility that genetic exchange and plasmid acquisition of the qnrB2 gene could occur in the faecal flora of the animals. Interestingly, p137.25 belongs to the IncN plasmid family that is able to replicate in different enterobacter-ial strains, but also seems prevalent in faecal flora from animals. In fact, a study performed on a large collection of E. coli from the USA demonstrated that the prevalence of IncN plasmids is high in avian E. coli (10% – 16%) but negative in E. coli from faeces of healthy humans. 15 This evidence supports the hypothesis that the Salmonella 137.25 strain acquired the qnrB2 gene on an IncN plasmid circulating in avian bacterial flora. This strain could cause infections in humans through the food chain and the resistance plasmid contributes to the dissemination of the qnrB2 gene in other Enterobacteriaceae. None to declare.

Spread of a multiresistant CTX-M-9-producing Salmonella enterica serotype Virchow phage type 19 in Spain.

The purpose of this study was to survey Salmonella enterica serotype Virchow phage type 19 (S. Virchow PT19) strains submitted to the Spanish National Reference Laboratory for Salmonella (SNRLS) from 2002 to 2006 in order to determine the rate type and genetic background of β-lactam resistance and to further identify the associated resistances. Ninety-nine S. Virchow PT19 strains were analysed. Antimicrobial susceptibility was determined by the disk diffusion method using Mueller–Hinton agar medium. Polymerase chain reaction (PCR) assays and, later, sequencing of the obtained fragments were performed for the molecular characterisation of the resistances. Pulsed-field gel electrophoresis (PFGE) and plasmid analysis (using conjugation, Southern blot hybridisation and replicon typing) were used for characterisation. The characterisation of S. Virchow PT19 strains allowed the identification of a clonal multiresistant S. Virchow PT19 harbouring an IncH12 plasmid with the blaCTX-M-9 gene within the complex integron In60 distributed across Spain. An IncH12 plasmid widely reported and studied in Enterobacteria is described in a clonal multiresistant S. Virchow PT19 which has successfully spread throughout Spain