Incidence and Genomic Background of Antibiotic Resistance in Food-Borne and Clinical Isolates of Salmonella enterica Serovar Derby from Spain.

Salmonella enterica serovar Derby (S. Derby) ranks fifth among nontyphoidal Salmonella serovars causing human infections in the European Union. S. Derby isolates (36) collected between 2006 and 2018 in a Spanish region (Asturias) from human clinical samples (20) as well as from pig carcasses, pork- or pork and beef-derived products, or wild boar (16) were phenotypically characterized with regard to resistance, and 22 (12 derived from humans and 10 from food-related samples) were also subjected to whole genome sequence analysis. The sequenced isolates belonged to ST40, a common S. Derby sequence type, and were positive for SPI-23, a Salmonella pathogenicity island involved in adherence and invasion of the porcine jejune enterocytes. Isolates were either susceptible (30.6%), or resistant to one or more of the 19 antibiotics tested for (69.4%). Resistances to tetracycline [tet(A), tet(B) and tet(C)], streptomycin (aadA2), sulfonamides (sul1), nalidixic acid [gyrA (Asp87 to Asn)] and ampicillin (blaTEM-1-like) were detected, with frequencies ranging from 8.3% to 66.7%, and were higher in clinical than in food-borne isolates. The fosA7.3 gene was present in all sequenced isolates. The most common phenotype was that conferred by the tet(A), aadA2 and sul1 genes, located within identical or closely related variants of Salmonella Genomic Island 1 (SGI1), where mercury resistance genes were also present. Diverse IncI1-I(α) plasmids belonging to distinct STs provided antibiotic [blaTEM-1, tet(A) and/or tet(B)] and heavy metal resistance genes (copper and silver), while small pSC101-like plasmids carried tet(C). Regardless of their location, most resistance genes were associated with genetic elements involved in DNA mobility, including a class one integron, multiple insertion sequences and several intact or truncated transposons. By phylogenetic analysis, the isolates were distributed into two distinct clades, both including food-borne and clinical isolates. One of these clades included all SGI1-like positive isolates, which were found in both kinds of samples throughout the entire period of study. Although the frequency of S. Derby in Asturias was very low (0.5% and 3.1% of the total clinical and food isolates of S. enterica recovered along the period of study), it still represents a burden to human health linked to transmission across the food chain. The information generated in the present study can support further epidemiological surveillance aimed to control this zoonotic pathogen.

Genomic Analysis of Ciprofloxacin-Resistant Salmonella enterica Serovar Kentucky ST198 From Spanish Hospitals.

Salmonella enterica serovar Kentucky (S. Kentucky) with sequence type (ST) 198 and highly resistant to ciprofloxacin (ST198-Cip R ) has emerged as a global MDR clone, posing a threat to public health. In the present study, whole genome sequencing (WGS) was applied to characterize all Cip R S. Kentucky detected in five Spanish hospitals during 2009-2018. All Cip R isolates (n = 13) were ST198 and carried point mutations in the quinolone resistance-determining regions (QRDRs) of both gyrA (resulting in Ser83Phe and Asp87Gly, Asp87Asn, or Asp87Tyr substitutions in GyrA) and parC (with Thr57Ser and Ser80Ile substitutions in ParC). Resistances to other antibiotics (ampicillin, chloramphenicol, gentamicin, streptomycin, sulfonamides, and tetracycline), mediated by the bla TEM- 1 B , catA1, aacA5, aadA7, strA, strB, sul1, and tet(A) genes, and arranged in different combinations, were also observed. Analysis of the genetic environment of the latter resistance genes revealed the presence of multiple variants of SGI1 (Salmonella genomic island 1)-K and SGI1-P, where all these resistance genes except catA1 were placed. IS26 elements, found at multiple locations within the SGI1 variants, have probably played a crucial role in their generation. Despite the wide diversity of SGI1-K- and SGI1-P-like structures, phylogenetic analysis revealed a close relationship between isolates from different hospitals, which were separated by a minimum of two and a maximum of 160 single nucleotide polymorphisms. Considering that S. enterica isolates resistant to fluoroquinolones belong to the high priority list of antibiotic-resistant bacteria compiled by the World Health Organization, continuous surveillance of the S. Kentucky ST198-CIP R clone is required.

Colistin Resistance in Monophasic Isolates of Salmonella enterica ST34 Collected From Meat-Derived Products in Spain, With or Without CMY-2 Co-production.

Colistin is a last-resort antibiotic in fighting severe infections caused by multidrug resistant Gram negative pathogens in hospitals. Zoonotic bacteria acquire colistin resistance in animal reservoirs and mediate its spread along the food chain. This is the case of non-typhoid serovars of Salmonella enterica. Colistin-resistant S. enterica in foods represents a threat to human health. Here, we assessed the prevalence of colistin-resistance in food-borne isolates of S. enterica (2014-2019; Asturias, Spain), and established the genetic basis and transferability of this resistance. Five out of 231 isolates tested (2.2%) were resistant to colistin. Four of them, belonging to the European monophasic ST34 clone of S. Typhimurium, were characterized in the present study. They were collected from pork or pork and beef meat-derived products, either in 2015 (three isolates) or 2019 (one isolate). Molecular typing with XbaI-PFGE and plasmid profiling revealed distinct patterns for each isolate, even though two of the 2015 isolates derived from the same sample. The MICs of colistin ranged from 8 to 16 mg/L. All isolates carried the mcr-1.1 gene located on conjugative plasmids of the incompatibility groups IncX4 (2015 isolates) or IncHI2 (2019 isolate). Apart from colistin resistance, the four isolates carried chromosomal genes conferring resistance to ampicillin, streptomycin, sulfonamides and tetracycline [bla TEM-1, strA-strB, sul2, and tet(B)] and heavy metals, including copper and silver (silESRCFBAGP and pcoGE1ABCDRSE2), arsenic (arsRSD2A2BCA1D1) ± mercury (merEDACPTR), which are characteristically associated with the European ST34 monophasic clone. The 2019 isolate was also resistant to other antibiotics, comprising third generation cephalosporins and cephamycins. The latter phenotype was conferred by the bla CMY-2 gene located on an IncI1-I(α)-ST2 plasmid. Results in the present study identified meat-derived products as a reservoir of a highly successful clone harboring transferable plasmids which confer resistance to colistin and other clinically important antibiotics. An important reduction in the number of food-borne S. enterica detected during the period of the study, together with the low frequency of colistin resistance, underlines the success of One Health initiatives, such as those implemented at the UE, to control zoonotic bacteria along the food chain and to halt the spread of antimicrobial resistance.

Molecular Characterization of Salmonella enterica Serovar Enteritidis, Genetic Basis of Antimicrobial Drug Resistance and Plasmid Diversity in Ampicillin-Resistant Isolates.

Salmonella enterica serovar Enteritidis is the most common cause of human salmonellosis worldwide. In this study, all clinical isolates of Salmonella Enteritidis recovered between January 2008 and June 2014 in a Spanish region (491) were screened for antimicrobial drug resistance and the phage type (PT) was determined for a significant number (265). PT1, PT14b, PT56, PT6, PT4, and PT8 were the predominant PTs, accounting together for 82% of the isolates. A total of 38.3% of the isolates were susceptible to all antimicrobials tested, 46.4% and 6.1% isolates were resistant to nalidixic acid and ampicillin, respectively, and single isolates were resistant to two (ampicillin and nalidixic acid) or six (ampicillin, chloramphenicol, streptomycin, sulfonamides, tetracycline, and trimethoprim) agents. Nalidixic acid resistance was statistically associated with PT1 and PT14b (p < 0.05, 95% CI), and ampicillin resistance with PT6/PT6a (p < 0.05, 95% CI). All ampicillin-resistant isolates (30) carried a plasmid-encoded blaTEM-1. All except one harbored the virulence plasmid specific of Salmonella Enteritidis (IncFIIA + IncFIB; 28 isolates) or a blaTEM-1-positive variant herein (IncFIIA + IncFIB; 1 isolate). Five additional blaTEM-1 plasmids, of the ColE1, IncX, IncF, and IncI incompatibility groups, were identified. The IncI plasmid, found in the single multidrug-resistant isolate, carried the strAB and sul2 genes together with genes of the virulence plasmid, including the spv operon. The obtained results highlight the high diversity of blaTEM-1 plasmids conferring ampicillin resistance in Salmonella Enteritidis, and support clonal expansion as the main cause of nalidixic acid resistance in this serovar.

Incidence and Genetic Bases of Nitrofurantoin Resistance in Clinical Isolates of Two Successful Multidrug-Resistant Clones of Salmonella enterica Serovar Typhimurium: Pandemic "DT 104" and pUO-StVR2.

In this study, the incidence and genetic bases of nitrofurantoin resistance were established for clinical isolates of two successful clones of Salmonella enterica serovar Typhimurium, the pandemic "DT 104" and the pUO-StVR2 clone. A total of 61 "DT 104" and 40 pUO-StVR2 isolates recovered from clinical samples during 2008-2014 and assigned to different phage types, were tested for nitrofurantoin susceptibility. As previously shown for older isolates, all newly tested pUO-StVR2 isolates were highly resistant to nitrofurantoin (minimal inhibitory concentration [MIC] of 128 µg/ml), while 42.6%, 24.6%, and 32.8% of the "DT 104" isolates were susceptible, showed intermediate resistance or were highly resistant, with MICs of 8, 64, and 128 µg/ml, respectively. The genetic bases of nitrofurantoin resistance were established by PCR amplification and sequencing of the nfsA and nfsB genes encoding oxygen-insensitive nitroreductases. pUO-StVR2 isolates shared identical alterations in both nfsA (IS1 inserted into the coding region) and nfsB (in frame duplication of two codons). "DT 104" isolates with intermediate or high resistance had a missense mutation affecting the start codon of nfsA, while a single resistant isolate carried an additional frameshift mutation affecting nfsB. Complementation studies, performed with wild-type nfsA and nfsB, cloned independently and together into low and high copy-number vectors, confirmed NfsA and NfsB as responsible for nitrofurantoin toxicity. The same alterations persisted along time in isolates of each clone belonging to different phage types. Accordingly, changes leading to nitrofurantoin resistance have probably occurred before phage type diversification.

Antimicrobial Drug Resistance and Molecular Typing of Salmonella enterica Serovar Rissen from Different Sources.

Salmonella enterica serovar Rissen is one of the most common serovars found in pigs and pork products in different countries, including Spain. However, information on the molecular bases of antimicrobial drug resistance and the population structure of Salmonella Rissen from different sources in Spain is limited. The present study focused on 84 isolates collected in Spain from pig and beef carcasses, foods and clinical samples associated with sporadic cases of gastroenteritis, and one outbreak. The majority of the isolates were resistant to tetracycline (73.8%), mainly conferred by tet(A). Resistances to streptomycin (aadA1-like, aadA2, and strAB), sulfonamides (sul1, sul2, and sul3), trimethoprim (dfrA1-like and dfrA12), ampicillin (blaTEM-1-like), and chloramphenicol (cmlA1-like) were also detected, with frequencies ranging from 12% to 20.2%. Most of the identified genes were carried by integrons, including three class 1 integrons of the sul1 type, a class 1 integron of the sul3 type, and the class 2 integron of Tn7. Two sul1 integrons, the sul3 integron, and the class 2 integron are first reported in Salmonella Rissen. Typing of the isolates with XbaI pulsed-field gel electrophoresis detected a major clone, which was circulating in humans and animals during the past decade, and was responsible for the outbreak. The obtained results are relevant for food safety and public health.

IncA/C plasmids mediate antimicrobial resistance linked to virulence genes in the Spanish clone of the emerging Salmonella enterica serotype 4,[5],12:i:-.

OBJECTIVES: To broaden knowledge of the molecular bases and genetics of multidrug resistance in clinical isolates of Salmonella enterica serotype 4,5,12:i:- belonging to the Spanish clone. METHODS: The relatedness of the isolates was determined by phage typing and XbaI-PFGE. Resistance genes, integrons and transposable elements were identified by PCR amplification and sequencing. Plasmids were characterized by alkaline lysis, S1-PFGE, conjugation, replicon typing and Southern blot hybridization. RESULTS: The isolates were closely related and resistant to five to seven antimicrobials (ampicillin, chloramphenicol, gentamicin, streptomycin/spectinomycin, sulphonamides, trimethoprim and tetracycline, arranged in different combinations). Most of the responsible genes were provided by a conventional class 1 integron with the dfrA12-orfF-aadA2 variable region, an atypical class 1 integron containing sul3 next to the estX-psp-aadA2-cmlA1-aadA1 variable region and a truncated Tn1721 transposon carrying tet(A). A defective Tn21 with the mer operon and ISVsa3 associated with sul2 were also detected. All resistance genes and mobile genetic elements were located on large, non-conjugative and highly variable plasmids carrying one (A/C) or two (A/C and N) replicons, as well as virulence genes of pSLT. CONCLUSIONS: IncA/C plasmids are responsible for multidrug resistance in an increasing number of relevant human and animal bacterial pathogens, and hence are regarded as an important threat to public health. Those found in the Spanish clone of Salmonella 4,5,12:i:- constitute a relevant example of short-term evolution, and could have been involved in the successful adaptation of this pathogen.

Detailed structure of integrons and transposons carried by large conjugative plasmids responsible for multidrug resistance in diverse genomic types of Salmonella enterica serovar Brandenburg.

OBJECTIVES: To evaluate the incidence, molecular basis and distribution among genomic types of antimicrobial drug resistance in Salmonella enterica (S.) serovar Brandenburg isolates recorded in the Principality of Asturias, Spain. METHODS: Thirty-seven S. Brandenburg isolates were tested for susceptibility to antimicrobial agents and typed by random amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE). PCR amplifications, together with DNA cloning and sequencing, were used to identify resistance genes, integrons and transposons and to establish the structure and physical associations between them. Conjugation experiments were applied to establish the location of the identified elements. RESULTS: Twenty-one isolates were resistant to one or more unrelated drugs. Resistances to streptomycin, tetracycline, kanamycin, chloramphenicol, ampicillin and trimethoprim-sulfamethoxazole, encoded by aadA1, tet(A) or tet(B), aphA1, catA1, bla(TEM) and dfrA1-sul1-sul3, respectively, were most frequently observed. Multidrug resistance (32.4%) was mainly mediated by mobile genetic elements. These included: (i) class 1 integrons (with dfrA1-aadA1 gene cassettes in their variable region), which were part of Tn21-related transposons associated with Tn9; (ii) a Tn1721-derivative containing tet(A); (iii) a defective Tn10 that carried tet(B), and was linked to an integron; and (iv) large conjugative plasmids carrying a class 1 integron-Tn21-Tn9-like structure, together with the Tn1721- or the Tn10-related element. Two-way-RAPD and XbaI-PFGE discriminated the isolates into 15 and 12 profiles, respectively. CONCLUSIONS: Complex genetic elements have apparently been responsible for the recruitment, assembly and dispersion of resistance genes among the highly diverse genomic types of S. Brandenburg, identified as causal agents of human salmonellosis in the Principality of Asturias, over recent years.

[Outbreak of gastroenteritis in a nursery school caused by a strain of Salmonella enterica serovar Typhimurium carrying the hybrid virulence-resistance plasmid pUO-StVR2]. / Brote de gastroenteritis en una guardería causado por una cepa de Salmonella enterica serovar Typhimurium portadora del plásmido híbrido de resistencia-virulencia pUO-StVR2.

OBJECTIVE: Epidemiological and microbiological study of a salmonellosis outbreak, affecting 22 children in a nursery school in Oviedo (Spain). METHODS: Attack rates and epidemic curves were determined, and bacterial typing methods were applied. RESULTS: The outbreak was attributed to a Salmonella enterica serovar Typhimurium strain, belonging to an emergent type characterized by the presence of a hybrid virulence-resistance plasmid of 125-130 kb, named pUO-StVR2. The attack rate of confirmed cases vs. possible cases was 27.2% vs. 23.5% for the children and 0 vs. 26.5% for the staff of the affected center. The source of the infection could not be identified. Nevertheless, according to the evolution of the cases over time, the transmission route was likely to be personal contact between the staff and children, which facilitates fecal-oral dissemination. All but one of the 27 isolates analyzed (from 22 patients) showed identical features: R-profile, plasmid-profile, RAPD-type, PFGE-type; all were non-phage-typeable, with the exception of a DT104b isolate. pUO-StVR2 is probably a derivative of the virulence plasmid pSLT from the LT2 type strain that acquired an R-region complex (ACSSuT/blaOXA-catA1-strA/ B-aadA1-sul1-sul2-tet[B]), in which the blaOXA-aadA1 genes are part of the variable region of a class 1 integron. CONCLUSION: This outbreak is an example of how a Salmonella enterica serovar Typhimurium strain belonging to a type that is probably endemic in Spain can be transferred to the community and affect a susceptible population.

Brote de gastroenteritis en una guardería causado por una cepa de Salmonella enterica serovar Typhimurium portadora del plásmido híbrido de resistencia-virulencia pUO-StVR2 / Outbreak of gastroenteritis in a nursery school caused by a strain of Salmonella enterica serovar Typhimurium carrying the hybrid virulence-resistance plasmid pUO-StVR2

Objetivo. El estudio epidemiológico y microbiológico de un brote de salmonelosis ocurrido en 2004, que afectó a 22 niños de una guardería en Oviedo. Métodos. Se determinaron las tasas de ataque y curvas epidémicas y se aplicaron técnicas de tipificación bacteriana. Resultados. El brote pudo atribuirse a una cepa de Salmonella enterica serovar Typhimurium perteneciente a un grupo emergente que alberga un plásmido híbrido de virulencia-resistencia de 125-130 kb, denominado pUO-StVR2. Las tasas de ataque para los casos confirmados frente a los posibles fue 27,2% frente a 23,5% para los niños y 0 frente a 26,5% para el personal del centro. La fuente de infección no pudo ser identificada, aunque según la evolución temporal de los casos se podría considerar que el modo de transmisión fue el contacto cuidadoras-niños, facilitando la diseminación fecal-oral. Todos menos uno de los 27 aislamientos analizados (de 22 pacientes) presentaban características idénticas: perfil-R, perfil de plásmidos, RAPD-tipo, PFGE-tipo y eran no fagotipificables (la excepción fue un aislamiento con fagotipo DT104b). pUO-StVR2 es un derivado del plásmido de virulencia, pSLT, de la cepa tipo LT2 que ha ganado una región-R compleja (ACSSuT/blaOXA-catA1-strA/ B-aadA1-sul1-sul2-tet[B]) en la que los genes blaOXA-aadA1 forman parte de la región variable de un integrón de clase 1. Conclusión. Este brote constituye un ejemplo de cómo una cepa de S. enterica serovar Typhimurium, perteneciente a un tipo probablemente ya endémico en la península Ibérica, puede transmitirse a la comunidad y afectar a un colectivo susceptible (AU)

Objective. Epidemiological and microbiological study of a salmonellosis outbreak, affecting 22 children in a nursery school in Oviedo (Spain). Methods. Attack rates and epidemic curves were determined, and bacterial typing methods were applied. Results. The outbreak was attributed to a Salmonella enterica serovar Typhimurium strain, belonging to an emergent type characterized by the presence of a hybrid virulence-resistance plasmid of 125-130 kb, named pUO-StVR2. The attack rate of confirmed cases vs. possible cases was 27.2% vs. 23.5% for the children and 0 vs. 26.5% for the staff of the affected center. The source of the infection could not be identified. Nevertheless, according to the evolution of the cases over time, the transmission route was likely to be personal contact between the staff and children, which facilitates fecal-oral dissemination. All but one of the 27 isolates analyzed (from 22 patients) showed identical features: R-profile, plasmid-profile, RAPD-type, PFGE-type; all were non-phage-typeable, with the exception of a DT104b isolate. pUO-StVR2 is probably a derivative of the virulence plasmid pSLT from the LT2 type strain that acquired an R-region complex (ACSSuT/blaOXA-catA1-strA/ B-aadA1-sul1-sul2-tet[B]), in which the blaOXA-aadA1 genes are part of the variable region of a class 1 integron. Conclusion. This outbreak is an example of how a Salmonella enterica serovar Typhimurium strain belonging to a type that is probably endemic in Spain can be transferred to the community and affect a susceptible population (AU)