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Objectives While an increase in the levels of MDR in Salmonella enterica sevorar Choleraesuis has been reported in Europe, little is known about the situation in Spain. Therefore, we first aimed to assess the phenotypic resistance profile and to determine the presence of genetic determinants of resistance of S. Choleraesuis isolates collected in animal and human. Our second objective was to identify and characterize clusters of highly related isolates. Methods We analysed 50 human and 45 animal isolates retrieved from 2006 to 2021 using the disc diffusion method and performed WGS followed by analyses of genetic determinants and phylogenetic analysis. Results All isolates were of ST145 and corresponded to the variant Kunzendorf. Swine isolates harboured a significantly higher number of antimicrobial resistance genes than human isolates, and often carried plasmid replicons of the IncHI2/IncHI2A type (42% of all animal isolates). In addition, we identified several MDR S. Choleraesuis strains circulating in humans and swine between 2006 and 2021. The phylogenetic analyses identified four clades associated with specific patterns of resistance genes and plasmid replicons. The clades also included isolates that differed in terms of year and region of isolation as well as host of origin. Conclusions This One Health approach highlights that reducing human MDR S. Choleraesuis infections may require the adoption of strategies that not only seek to prevent cases in humans but also to characterize and reduce the infection burden in swine. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Salmonella enterica subspecies enterica is one of the most important foodborne pathogens and the causative agent of salmonellosis, which affects both humans and animals producing numerous infections every year. The study and understanding of its epidemiology are key to monitoring and controlling these bacteria. With the development of whole-genome sequencing (WGS) technologies, surveillance based on traditional serotyping and phenotypic tests of resistance is being replaced by genomic surveillance. To introduce WGS as a routine methodology for the surveillance of food-borne Salmonella in the region, we applied this technology to analyze a set of 141 S. enterica isolates obtained from various food sources between 2010 and 2017 in the Comunitat Valenciana (Spain). For this, we performed an evaluation of the most relevant Salmonella typing methods, serotyping and sequence typing, using both traditional and in silico approaches. We extended the use of WGS to detect antimicrobial resistance determinants and predicted minimum inhibitory concentrations (MICs). Finally, to understand possible contaminant sources in this region and their relationship to antimicrobial resistance (AMR), we performed cluster detection combining single-nucleotide polymorphism (SNP) pairwise distances and phylogenetic and epidemiological data. The results of in silico serotyping with WGS data were highly congruent with those of serological analyses (98.5% concordance). Multi-locus sequence typing (MLST) profiles obtained with WGS information were also highly congruent with the sequence type (ST) assignment based on Sanger sequencing (91.9% coincidence). In silico identification of antimicrobial resistance determinants and minimum inhibitory concentrations revealed a high number of resistance genes and possible resistant isolates. A combined phylogenetic and epidemiological analysis with complete genome sequences revealed relationships among isolates indicative of possible common sources for isolates with separate sampling in time and space that had not been detected from epidemiological information. As a result, we demonstrate the usefulness of WGS and in silico methods to obtain an improved characterization of S. enterica enterica isolates, allowing better surveillance of the pathogen in food products and in potential environmental and clinical samples of related interest. [ABSTRACT FROM AUTHOR]

Background: Swine are considered a major source of foodborne salmonellosis, a public health issue further complicated by the circulation of multidrug-resistant Salmonella strains that threaten the safety of the food chain. The current study aimed to identify patterns that can help to understand the epidemiology of antimicrobial resistance (AMR) in Salmonella in pigs in Spain through the application of several multivariate statistical methods to data from the AMR national surveillance programs from 2001 to 2017. Results: A total of 1,318 pig Salmonella isolates belonging to 63 different serotypes were isolated and their AMR profiles were determined. Tetracycline resistance across provinces in Spain was the highest among all antimicrobials and ranged from 66.7% to 95.8%, followed by sulfamethoxazole resistance (range: 42.5% − 77.8%), streptomycin resistance (range: 45.7% − 76.7%), ampicillin resistance (range: 24.3% − 66.7%, with a lower percentage of resistance in the South-East of Spain), and chloramphenicol resistance (range: 8.5% − 41.1%). A significant increase in the percentage of resistant isolates to chloramphenicol, sulfamethoxazole, ampicillin and trimethoprim from 2013 to 2017 was observed. Bayesian network analysis showed the existence of dependencies between resistance to antimicrobials of the same but also different families, with chloramphenicol and sulfamethoxazole in the centre of the networks. In the networks, the conditional probability for an isolate susceptible to ciprofloxacin that was also susceptible to nalidixic acid was 0.999 but for an isolate resistant to ciprofloxacin that was also resistant to nalidixic acid was only 0.779. An isolate susceptible to florfenicol would be expected to be susceptible to chloramphenicol, whereas an isolate resistant to chloramphenicol had a conditional probability of being resistant to florfenicol at only 0.221. Hierarchical clustering further demonstrated the linkage between certain resistances (and serotypes). For example, a higher likelihood of multidrug-resistance in isolates belonging to 1,4,[5],12:i:- serotype was found, and in the cluster where all isolates were resistant to tetracycline, chloramphenicol and florfenicol, 86.9% (n = 53) of the isolates were Typhimurium. Conclusion: Our study demonstrated the power of multivariate statistical methods in discovering trends and patterns of AMR and found the existence of serotype-specific AMR patterns for serotypes of public health concern in Salmonella isolates in pigs in Spain. [ABSTRACT FROM AUTHOR]

Salmonella enterica, a Gram-negative zoonotic bacterium, is mainly a food-borne pathogen and the main cause of diarrhea in humans worldwide. The main reservoirs are found in poultry farms, but they are also found in wild birds. The development of antibiotic resistance in S. enterica species raises concerns about the future of efficient therapies against this pathogen and revives the interest in bacteriophages as a useful therapy against bacterial infections. Here, we aimed to decipher and functionally annotate 10 new Salmonella phage genomes isolated in Spain in the light of phage therapy. We designed a bioinformatic pipeline using available building blocks to de novo assemble genomes and perform syntaxic annotation. We then used genome-wide analyses for taxonomic annotation enabled by vContact2 and VICTOR. We were also particularly interested in improving functional annotation using remote homologies detection and comparisons with the recently published phage-specific PHROG protein database. Finally, we searched for useful functions for phage therapy, such as systems encoded by the phage to circumvent cellular defenses with a particular focus on anti-CRISPR proteins. We, thus, were able to genetically characterize nine virulent phages and one temperate phage and identify putative functions relevant to the formulation of phage cocktails for Salmonella biocontrol. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar Infantis (S. Infantis) is a broiler-associated pathogen which ranks in the fourth position as a cause of human salmonellosis in the European Union. Here, we report a comparative genomic analysis of two clinical S. Infantis isolates recovered in Spain from children who just returned from Peru. The isolates were selected on the basis of resistance to cefotaxime, one of the antibiotics of choice for treatment of S. enterica infections. Antimicrobial susceptibility testing demonstrated that they were resistant to eight classes of antimicrobial agents: penicillins, cephalosporins, phenicols, aminoglycosides, tetracyclines, inhibitors of folate synthesis, (fluoro)quinolones and nitrofurans, and one of them was also resistant to fosfomycin. As shown by whole-genome sequence analysis, each isolate carried a pESI-like megaplasmid of ca. 300 kb harboring multiple resistance genes [blaCTX-M-65, aph(4)-Ia, aac(3)-IVa, aph(3′)-Ia, floR, dfrA14, sul1, tet(A), aadA1 ± fosA3], as well as genes for resistance to heavy metals and disinfectants (mer, ars and qacEΔ1). These genes were distributed in two complex regions, separated by DNA belonging to the plasmid backbone, and associated with a wealth of transposable elements. The two isolates had a D87Y amino acid substitution in the GyrA protein, and truncated variants of the nitroreductase genes nfsA and nsfB, accounting for chromosomally encoded resistances to nalidixic acid and nitrofurantoin, respectively. The two S. Infantis isolates were assigned to sequence type ST32 by in silico multilocus sequence typing (MLST). Phylogenetic analysis revealed that they were closely related, differing only by 12 SNPs, although they were recovered from different children two years apart. They were also genetically similar to blaCTX-M-65-positive ± fosA3 isolates obtained from humans and along the poultry production chain in the USA, South America, as well as from humans in several European countries, usually associated with a travel history to America. However, this is the first time that the S. Infantis blaCTX-M-65 ± fosA3 MDR clone has been reported in Spain. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Salmonella enterica is a well-adapted zoonotic bacterium associated to cases of gastroenteritis and bacteremia with increased morbidity and mortality. In this study, three isolates of Salmonella Typhimurium obtained from human clinical samples, showing colistin resistance and low-level resistance to quinolones, have been genetically characterized. We detected the co-occurrence of mcr-1 and qnrS1 on a single IncHI2 plasmid in isolates of Salmonella Typhimurium obtained from Spanish children without a travel history. The multiresistant region contained numerous resistance genes. Isolates were clonally related, which suggests the presence of these clones in the community and the potential to cause outbreaks affecting the most susceptible population. It is necessary to monitor the presence of these plasmid-mediated resistance genes in human European strains of Salmonella spp. because of the risk of producing outbreaks of community-acquired infections. [ABSTRACT FROM AUTHOR]

Salmonella enterica serovar Choleraesuis is the aetiological agent of swine paratyphoid being a highly invasive zoonotic pathogen. Wild boar natural populations are experiencing a demographical expansion as well as some farms are breeding this species to release for hunting with management sometimes identical to that of domestic pigs, including supplementation, grouping, and antibiotic treatments. This situation increases the chance of contact between wild boars and livestock, and potentially induces stress, with different sanitary consequences. The present work aims to describe the clinical features of recent outbreaks caused by S. Choleraesuis in wild boar from central‐western Spain, as well as the antimicrobial resistance and phylogenetic relationships of isolates involved. 28 strains of S. Choleraesuis were isolated from 28 different wild boars belonging to 10 different game states located in central western Spain and submitted to the Clinical Veterinary Hospital (CVH) of the University of Extremadura. Samples were taken from different organs and cultured according to the ISO 6579:2002 procedure. Suspicious colonies were identified by PCR and antimicrobial resistance was evaluated by disc diffusion susceptibility test and the presence of the main resistance genes as well as 18 plasmid replicons frequently found among the Enterobacteriaceae was verified by PCR. Pulsed field gel electrophoresis was applied to determine the genetic relationship between isolates. The outbreaks under study were characterized by high mortality (35%–84%) and a septicaemic presentation. S. Choleraesuis was isolated from all the wild boars analysed, and 26 of the 28 isolates presented resistance to at least one antibiotic. The predominant resistances found were against sulphonamide, streptomycin, tetracycline, and doxicicline and sul1, strA‐strB, and tetA were the most prevalent resistance genes among isolates. 10 strains carried FIIA, FIB+H/1 or FIIA+H/1 plasmids. PFGE classified the isolates into four different profiles, grouped into two clusters. This results show that prevention against S. Choleraesuis must be considered in the sanitary programs of the wild boar breeders. [ABSTRACT FROM AUTHOR]

A screening of antimicrobial resistance and its genetic determinants has been performed on 300 Salmonella enterica isolates collected during 2004-2008 from human infections in Spain. Salmonella Typhimurium and Salmonella Enteritidis were the major serotypes, which were found with similar frequencies covering 80% of the bacterial collection. Salmonella Typhimurium isolates frequently shared low susceptibility to antimicrobials of the penta-resistance phenotype (ACSSuT) and/or cephalosporin resistance. The ACSSuT profile was found closely linked to int1-associated gene cassettes, with major elements carrying DNA fragments of 1.0 Kb (aadA2 gene) plus 1.2 Kb (blaPSE-1 gene) or 2.0 Kb (aadA1 and blaOXA-1 genes). Among these, ACSSuT and cephalosporin resistances were associated in Salmonella Typhimurium isolates expressing the blaOXA gene. β-lactamase activities were also detected from isolates carrying blaTEM, blaCMY, or blaSHV, although only the two last genes expressed extended-spectrum β-lactamases. The clonal analysis of S. enterica strains suggests that both horizontal and vertical transfer mechanisms are involved in the wide dissemination of their antimicrobial resistance. [ABSTRACT FROM AUTHOR]

Abstract: Plasmids implicated in the mobilisation of β-lactamase genes in extended-spectrum β-lactamase (ESBL)- and AmpC-producing Salmonella enterica isolates recovered from three Spanish hospitals were characterised. The temporal stability of these plasmids and of the resistance phenotype without antimicrobial pressure was also assessed in the laboratory setting. The resistance determinants and their genetic environments were characterised by PCR sequencing, and their genomic location was analysed by S1 nuclease pulsed-field gel electrophoresis (PFGE) and I-CeuI PFGE, followed by Southern blot hybridisation. The 11 S. enterica studied strains carried bla CTX-M-9 (serovar Virchow, 2 isolates), bla CTX-M-10 (Virchow, 2), bla CTX-M-14 (Enteritidis, 1), bla CTX-M-15 (Gnesta and S. enterica group C, 2), bla SHV-2 (Livingstone, 1), bla SHV-12 (Enteritidis, 1) and bla CMY-2 (Bredeney, 2). The ISEcp1–bla CTX-M-14–IS903 and ISEcp1–bla CTX-M-15–orf477 genetic structures were detected. IncI1 and IncA/C plasmids carried bla CTX-M-14, bla CTX-M-15, bla SHV-2, bla SHV-12 and bla CMY-2 genes. bla CTX-M-9 included in an In60 complex integron and bla CTX-M-10 linked to a phage-related element were found in non-typeable plasmids. Conjugation and temporal stability experiments were performed in vitro through daily passages (100 days) in the absence of antimicrobial pressure. In the stability experiments, 5 of the 11 tested isolates lost the ESBL or AmpC plasmidic genes and this was associated with concomitant loss of the whole or partial plasmid. In conclusion, successful plasmids belonging to different Inc groups mobilise ESBL- and AmpC-encoding genes in S. enterica. Loss of ESBL/AmpC genes in the absence of antimicrobial pressure might explain the low prevalence of these β-lactamases among Salmonella isolates. [Copyright &y& Elsevier]

Summary: A chronic proliferative rhinitis in sheep associated with Salmonella enterica subspecies diarizonae serovar 61:k:1, 5, (7) is described. Ten adult sheep from eight traditionally managed Rasa Aragonesa flocks located in Aragon (Spain) were studied. Clinical signs began with bilateral thick mucus nasal discharge, wheezing and snoring and progressed to partial or complete obstruction of the nostrils. Necropsy examination revealed swollen ventral nasal turbinates with a roughened mucosal surface partially covered by small polyps. Histopathology revealed chronic proliferative rhinitis with a predominant population of neutrophils that infiltrated the mucosal epithelium. Plasma cells and macrophages were present in the lamina propria. Organisms expressing Salmonella antigen immunohistochemically were detected within epithelial cells. Salmonella enterica subspecies diarizonae serovar 61:k:1, 5, (7) was isolated as a sole microorganism from nasal swabs taken from five animals. The implication of finding this bacterium in various diseases in sheep and its role as a potential zoonosis are discussed. [Copyright &y& Elsevier]

Abstract: The effect of high hydrostatic pressure treatments on a cocktail of four Salmonella Enteritidis strains inoculated in commercial Spanish potato omelette (tortilla de patatas) was studied. Inactivation of Salmonella was influenced by treatment time and pressure. Treatments at 600MPa for 5 or 8min reduced the counts of Salmonella on selective medium (XLD agar) by 5.9 and 6.5 log cycles, respectively. Investigation of sublethally injured cells by growth on triple agar layer (TAL) plates or in non-selective medium (TSA) revealed that the population of sublethally injured cells able to grow on TSA could be 2.5 to 3.0 log cycles higher (depending on treatment) compared to non-injured cells. The TAL method only allowed recovery of a fraction of sublethally injured cells, suggesting different degrees of cell damage. In addition, tailings were observed in survival curves for treatments above 500MPa both for selective and non-selective determinations. Omelette samples inoculated with Salmonella and treated at 500MPa (5min) or 680MPa (8min) were stored at 6°C for 15days. Counts of survivors on selective medium increased at day 3 of storage for the less intense HHP treatment (suggesting repair of sublethally injured cells), but not until day 10 for the 680MPa treatment. Temperature abuse of the treated omelettes (6h at 22°C) applied at day 3 of storage increased the concentrations of cells able to grow on non-selective as well as on selective media, an effect that was more pronounced in the samples treated at 500MPa for 5min. The results suggest that temperature abuse may compromise the efficacy of HHP treatments against Salmonella in omelettes. Industrial relevance: The efficacy of HHP treatments depends greatly on the food substrate and microorganisms. Therefore, the degree of protection afforded by HHP treatments for each foodborne pathogen and food system need to be determined experimentally. So far, there are no previous studies addressing the efficacy of HHP treatments on inactivation of Salmonella in Spanish omelettes. This type of RTE food is widely consumed not only in Spain, but also in many other countries, especially as an appetizer. Industrial application of HHP treatments on omelettes could improve their safety while at the same time enhancing the perception of this food product as a safer food. [Copyright &y& Elsevier]

Abstract: The purpose of this study was to investigate the prevalence of Salmonella contamination and main serovars in pig slaughterhouses in Spain including carcasses, live animals and the environment. A total of 896 pig carcasses were randomly selected and swabbed before chilling in 3–5 visits to four pig slaughterhouses (A, B, C and D). Salmonella contamination was detected in 39.7% of the carcasses. The prevalence of positive carcasses was similar amongst slaughterhouses but significant differences were observed when taking sampling day into consideration within each of the slaughterhouses. Furthermore, a significant reduction in the prevalence of Salmonella contaminated carcasses (10.8%) was demonstrated in slaughterhouses C and D after chilling and cooling procedures. Sixteen batches of 10 animals were tracked from farm-to-slaughterhouse in slaughterhouses A and B to investigate the relationship between carcass contamination and contamination in live animals entering the slaughterhouse. No difference was found between infected and uninfected animals with respect to Salmonella contamination of the carcass although an increase in Salmonella contamination during the processing of live pigs into pork carcasses was evident. Regarding contamination in the slaughterhouse environment, Salmonella was isolated from most of the evaluated points in the slaughter line of the four studied slaughterhouses. Holding pens were identified as highly contaminated and what is more the ineffectiveness of the routinely cleaning protocols at this level was demonstrated in slaughterhouses C and D. The predominant Salmonella serovars found in carcasses, live pigs entering the slaughterhouse and the environment of the slaughterhouse were S. Typhimurium, S. Rissen, S. Derby and S. 4,[5],12:i:-. The same serovars were found in all the stages supporting the hypothesis that infected pigs are the main source of Salmonella contamination within slaughterhouses. [Copyright &y& Elsevier]

Abstract: A total of 226 chicken samples (carcasses, legs, wings, necks and breasts) were obtained (73 in 1993 and 153 in 2006) from 10 retail outlets in North-Western Spain and screened for the presence of Salmonella. Isolates were subjected to serotyping, phage typing (Salmonella Enteritidis and Salmonella Typhimurium) and antimicrobial susceptibility testing (15 antimicrobials; disk diffusion method). Salmonella was detected in 40 (55%) samples in 1993 and 19 (12.4%) in 2006 (P <0.001). The serotypes (S. Enteritidis, Salmonella Poona, Salmonella Infantis, Salmonella Newport and S. Typhimurium) and phage types (1, 4, 14b and 35 in the case of S. Enteritidis and 193 for S. Typhimurium) detected are among the main types responsible for human salmonellosis in Spain. All strains were multi-resistant (resistant to 3–13 antimicrobials). The average number of resistances per strain increased (P <0.05) from 3.98 in 1993 to 5.00 in 2006. An increase in the incidence of resistance was observed between 1993 and 2006 for cephalothin (P <0.01), enrofloxacin (P <0.001) and tetracycline (P <0.01). The decreases in the prevalence of Salmonella between 1993 and 2006 suggest that the mandatory measures introduced over the last decade in the European Union to reduce the incidence of Salmonella in poultry have apparently been successful. However, the increase in antibiotic resistance rates is of concern and constitutes a threat to public health. Because the data in this study demonstrated that chicken in North-Western Spain is a potential source of antibiotic-resistant Salmonella strains, the need of consumer education on good sanitary practices is highlighted. [Copyright &y& Elsevier]

The mechanisms of antimicrobial resistance were characterized in 90 Salmonella enterica isolates either resistant or with intermediate resistance to amoxicillin/clavulanate (AMCR/I) or resistant to third-generation cephalosporins (C3GR). These isolates were recovered in three Spanish hospitals during 2007-2009. The C3GR phenotype was expressed by three isolates that carried the following extended-spectrum β-lactamase genes: phage-associated blaCTX M-10 in S. Virchow, blaCTX-M-14a surrounded by ISEcp1 and IS903 in S. Enteritidis, and blaCTX-M-15 linked to ISEcp1 and orf477 in S. Gnesta (first description in this serotype). The AMCR/I phenotype was found in 87 isolates (79 S. Typhimurim, 7 S. Enteritidis, and one S. Thompson). The blaPSE-1 gene, followed by blaOXA-1 was mostly found among S. Typhimurim, and the blaTEM-1 gene among S. Enteritidis. Three different gene combinations [blaPSE-1+floR+aadA2+sul+tet(G); blaOXA-1+catA+aadA1/strA-strB+sul+tet(B) and blaTEM-1+ cmlA1+aadA/strA-strB+sul+tet(A)/tet(B) genes] were associated with the ampicillin-chloramphenicol-streptomycin-sulfon- amides-tetracycline phenotype in 68 AMCR/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 blaPSE-1 gene cassettes, respectively, were detected. The blaOXA-1+aadA1 arrangement was detected in 23 isolates, and the aac(6')-Ib-cr+blaOXA-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. [ABSTRACT FROM AUTHOR]

Among the 1233 Salmonella enterica isolates obtained in two Spanish hospitals, five isolates (0.4%) (serovars: Virchow, four; Livingstone, one) had the phenotype of an extended-spectrum β-lactamase (ESBL) producer. The genetic characterization of the ESBL of S. enterica Livingstone revealed a blaSHV-2 gene. The blaCTX-M-10 gene in a phage-related genetic environment was found in one S. enterica Virchow isolate, and the blaCTX-M-9 gene within the In60 integron was found in the three remaining Virchow isolates. These three isolates presented indistinguishable or closely related pulsed-field gel electrophoresis patterns among themselves and also as compared with the two other blaCTX-M-9-containing isolates previously obtained from animals. ESBL production is an emerging mechanism of resistance in S. enterica in the two studied hospitals. [ABSTRACT FROM AUTHOR]

Abstract: In this study, 119 multidrug-resistant isolates of non-typhoidal Salmonella enterica serovars collected in Spain (2002–2004) were screened for integrons. Among the isolates, 73.1% contained class 1 integrons, however classes 2 and 3 were not detected. Integrons containing gene cassettes were found in S. Enteritidis (16/32), S. Typhimurium biphasic (18/32) and monophasic [4,5,12:i:–] (11/19), S. Virchow (17/18) and S. Brandenburg (8/8), but not in S. Hadar (0/10). Ten complete and four incomplete gene cassettes, combined in 10 variable regions, were identified, one of which (2100bp/dfrA1–597bp-aadA24) was a new description. Most integrons mapped on plasmids of ca. 40–340kb. Exceptions were 1000bp/aadA2 and 1200bp/bla PSE-1 found on the chromosome of biphasic S. Typhimurium, probably as part of SGI1-like structures. [Copyright &y& Elsevier]

This study was the first conducted in Spain to evaluate the occurrence of antimicrobial resistance and multi-resistance in Salmonella isolates recovered from finishing pigs from Spanish swine farms distributed over the whole country. For this purpose, 290 Salmonella isolates recovered from apparently healthy finishing pigs in a farm-based cross-sectional study and 192 Salmonella isolates recovered from faecal samples of finishing pigs suffering from diarrhoea were investigated. Resistance to a panel of 17 antimicrobials was determined using a broth microdilution technique. Resistance was a common finding and was detected in 90.3% of the Salmonella isolates from apparently healthy finishing pigs and 95.3% of the Salmonella isolates from clinically diseased finishing pigs. Resistance was particularly high among isolates of serogroup B and serovars Typhimurium and its monophasic variant S. 4,5,12:i:-. Higher frequencies of resistance were found to tetracycline, sulphamethoxazole, streptomycin, spectinomycin, ampicillin, chloramphenicol and trimethoprim-sulphamethoxazole. Less than 10% of the isolates were resistant to amoxicillin/clavulanic acid, neomycin, cephalotin, apramycin and gentamicin. Resistance to ciprofloxacin, colistin and ceftiofur was rare (under 1%). Multi-resistance, defined as resistance to four or more drugs, was detected in more than 50% of the isolates. Although multi-resistance was particularly frequent among isolates of S. Typhimurium, it was also high among other serovars as Bredeney and the S. Typhimurium monophasic variant. 4,5,12:i:-. [ABSTRACT FROM AUTHOR]

The present study is the first conducted in Spain to estimate the bacteriological herd prevalence of Salmonella enterica in fattening units and to describe the Salmonella serovar diversity on these farms using a sample representative of the entire swine population. For this purpose, 10 faecal samples were collected from 10 different pens containing pigs close to market weight in a total of 232 fattening units. Total sample size was proportionally distributed according to the fattener census in each of the regions of the country and all the samples were examined by culture of 25 g of faecal material. One hundred (43.1%) farms had at least one Salmonella-positive sample (95% CI: 37–49.1%). Salmonella enterica was detected in 290 (12.5%) pooled faecal floor samples (95% CI: 11.2–13.8%). The apparent herd prevalence of salmonellosis was similar among multi-site, finishing and farrow to finish farms. Overall, 24 different serovars were identified, with S. Typhimurium, S. Rissen and S. Derby being the most common both at herd and sample level. Results of phage typing were available for the 91 isolates of S. Typhimurium. A total number of 10 different phage types were identified, with DT 193 being the most frequent. Phage types DT 104, DT 104b and DT U302, which have been associated with several multi-resistant patterns, accounted for 23% and 29% of the Typhimurium total isolates or Typhimurium infected farms respectively. [ABSTRACT FROM AUTHOR]

After Salmonella Enteritidis and S. Typhimurium, S. 4,[5],12:i:- is the most reported serovar in human clinical cases. During the past 20 years, many tools have been used for its typing and second-phase flagellar deletion characterization. Currently, whole genome sequencing (WGS) and different bioinformatic programs have shown the potential to be more accurate than earlier tools. To assess this potential, we analyzed by WGS and in silico typing a selection of 42 isolates of S. 4,[5],12:i:- and S. Typhimurium with different in vitro characteristics. Comparative analysis showed that SeqSero2 does not differentiate fljB-positive S. 4,[5],12:i:- strains from those of serovar Typhimurium. Our results proved that the strains selected for this work were non-clonal S. 4,[5],12:i:- strains circulating in Spain. Using WGS data, we identified 13 different deletion types of the second-phase flagellar genomic region. Most of the deletions were generated by IS26 insertions, showing orientation-dependent conserved deletion ends. In addition, we detected S. 4,[5],12:i:- strains of the American clonal line that would give rise to the Southern European clone in Spain. Our results suggest that new S. 4,[5],12:i:- strains are continuously emerging from different S. Typhimurium strains via different genetic events, at least in swine products. [ABSTRACT FROM AUTHOR]

Simple Summary: There is a lack of knowledge about the impact that phages present in the environment may have against certain Salmonella serovars. Thus, an improved understanding of Salmonella phage diversity will provide a better insight into the role of phages in Salmonella ecology and diversity. The results of this study showed that the poultry farm environment could represent a valuable source of Salmonella phages, which are more varied in broiler than in layer farms. The exploration of novel nonantibiotic interventions in the field, such as the use of bacteriophages, is necessary to avoid the presence of Salmonella. Bacteriophages are a group of viruses widely distributed in nature, strictly associated with the prokaryotic cell. Researchers have demonstrated the success of phage therapy in reducing Salmonella counts in poultry products. However, the impact that phage concentration in the environment may have against certain Salmonella serovars is not well understood. Therefore, the aim of this study was to assess Salmonella phage prevalence in commercial poultry farms in terms of the production type: layers or broilers. The most prevalent Salmonella serovars isolated in poultry production were used for phage isolation. Salmonella specific phages were isolated from 141 layer and broiler farms located in the Valencia region during 2019. Analysis of the samples revealed that 100% presented Salmonella phages, the most prevalent being against serovar S. Enteritidis (93%), followed by S. Virchow (59%), S. Typhimurium (55%), S. Infantis (52%) and S. Ohio (51%). These results indicate that poultry farms could represent an important source of Salmonella phages. Nevertheless, further studies are needed to assess the epidemiology of phages against other serovars present in other countries and their diversity from the point of view of molecular studies. [ABSTRACT FROM AUTHOR]

• A prevalence of 45.3 % of isolations of Salmonella enterica subsp. diarizonae was found. • The number of positive samples in nostrils was higher than in faeces. • Ninety per cent of the analyzed farms had positive animals. • Farms with a previous diagnosis of CPR were more likely to have a positive animal. Salmonella enterica subsp. diarizonae serotype 61: k: 1,5, (7) (SED) is a microorganism well adapted to sheep; however, it has also been described producing chronic proliferative rhinitis (CPR) in ovine. CPR causes a proliferative inflammation of the ventral nasal turbinates that may totally obstruct the nasal cavity. The main objective of the present study was to investigate the prevalence of SED in nostrils and stool of sheep without CPR clinical signs in commercial sheep farms of Spain with and without previous clinical cases of CPR. Five samplings were performed in 10 commercial sheep farms for one year. Samples from nostrils and faeces were taken from four animals without CPR visible clinical signs that belonged to four different age ranges at each farm visit. The prevalence of positive animals was 45.3 %, and the number of positive samples in nostrils was higher than in faeces (38.5 % vs 22.5 %). Only on one farm was no positive result obtained in the entire study. In almost all positive farms, sheep belonging to the youngest age ranges accounted for more than 50 % of positive isolates. Finally, farms with a previous diagnosis of CPR were 1.784 times more likely to have an animal with positive isolation than farms without a previous diagnosis. This could suggest that the infection pressure in the farm might favour the occurrence of clinical cases of the disease. However, further studies will be necessary to unravel why this saprophytic bacterium is able to cross the epithelial barrier causing severe rhinitis in certain animals. [ABSTRACT FROM AUTHOR]

The resistance plasmid pUO-StVR2, derived from virulence plasmid pSLT, is widespread in clinical isolates of Salmonella enterica serovar Typhimurium recovered in Spain and other European countries. pUO-StVR2 carries several genes encoding a FetMP-Fls system, which could be involved in iron uptake. We therefore analyzed S. Typhimurium LSP 146/02, a clinical strain selected as representative of the isolates carrying the plasmid, and an otherwise isogenic mutant lacking four genes (fetMP-flsDA) of the fetMP-fls region. Growth curves and determination of the intracellular iron content under iron-restricted conditions demonstrated that deletion of these genes impairs iron acquisition. Thus, under these conditions, the mutant grew significantly worse than the wild-type strain, its iron content was significantly lower, and it was outcompeted by the wild-type strain in competition assays. Importantly, the strain lacking the fetMP-flsDA genes was less invasive in cultured epithelial HeLa cells and replicated poorly upon infection of RAW264.7 macrophages. The genes were introduced into S. Typhimurium ATCC 14028, which lacks the FetMP-Fls system, and this resulted in increased growth under iron limitation as well as an increased ability to multiply inside macrophages. These findings indicate that the FetMP-Fls iron acquisition system exceeds the benefits conferred by the other high-affinity iron uptake systems carried by ATCC 14028 and LSP 146/02. We proposed that effective iron acquisition by this system in conjunction with antimicrobial resistance encoded from the same plasmid have greatly contributed to the epidemic success of S. Typhimurium isolates harboring pUO-StVR2. [ABSTRACT FROM AUTHOR]

Resistance to the quinolones nalidixic acid (NAL) and ciprofloxacin (CIP) and the occurrence of quinolone resistance determinants have been investigated in 300 non-typhoidal Salmonella from human origin, isolated in the years between 2004 and 2008, in 6 hospitals within Extremadura (Spain). Salmonella Enteritidis was the major serotype found among quinolone-resistant isolates, most of which were clustered by clonal analysis to a single clone, which presented D87 or S83 substitutions in GyrA. Eleven isolates presented the non-classical quinolone resistance phenotype (resistance to CIP and susceptibility to NAL), lacking mutations in the quinolone resistance determinant region of topoisomerase genes. Among them, one Salmonella Typhimurium isolate carried a qnrS1 gene in a low-molecular-weight plasmid, pQnrS1-HLR25, identical to plasmids previously found in the UK, Taiwan, and USA. The occurrence of this genetic element could represent a risk for the horizontal transmission of quinolone resistance among Enterobacteriaceae in the Iberian Peninsula., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Introduction: Salmonellosis is an important public health problem. Turtles are increasingly involved in the role of transmitters of this infection to humans., Methods: Salmonella cases are reported to the local Surveillance Agency where interviews are carried out to address possible exposures. Molecular epidemiology techniques were used to identify species., Results: In this article we report two examples of this type of infection in two places, 300 km apart in Spain. In Barcelona a turtle transmitted the disease to a small baby via her mother, and in Castellón 5 related cases of Salmonella infections were detected, and all were transmitted by imported turtles. Molecular epidemiology techniques confirmed the turtle-person transmissions and showed strong relationships between cases in Castellón and Barcelona., Discussion: These examples represent the tip of the iceberg of what is happening with pet reptiles as regards transmission of this infection. We believe that it is important to assess the impact of this type of infection in each country, in order to subsequently promote prevention strategies such as: regulations for pet shops, and educating/informing families who buy reptiles as pets., (Copyright © 2012 Elsevier España, S.L. All rights reserved.)

We analyzed a collection of 60 Salmonella enterica 4,5,12:i:- phage type U302 multidrug-resistant monophasic variant strains, isolated in Spain between 2000 and 2007. Most strains showed resistance to ampicillin (A), chloramphenicol (C), sulfamethoxazole (Su), gentamicin (G), streptomycin (S), tetracycline (T), and co-trimoxazole (SxT) (an ACSuGSTSxT resistance pattern). Only one pulsed-field gel electrophoresis (PFGE) type was detected, with 19 subtypes (Simpson's index of diversity [SID]=0.89). Multiple-locus variable-number tandem-repeat analysis (MLVA) showed more variability, with 32 profiles (SID=0.97), but only showed diversity at the STTR5 and STTR6 loci. PCR and sequencing demonstrated all strains contained the same allantoin-glyoxylate pathway deletion. Four types of deletions were detected in the fljAB operon, all starting at the same position, at the STM2758 gene, and followed by an IS26 insertion. Furthermore, a representative set of strains of the four deletion types harbored plasmids with IS26. We propose that a Salmonella enterica serotype Typhimurium U302 multidrug-resistant (ACSuGSTSxT) strain, defective for the allantoin-glyoxylate pathway and containing IS26 at plasmid pU302L, could be the ancestor of the variant in Spain.

Background: There are few studies about the effect of alcoholic beverages in food-borne disease outbreaks., Methods: We studied this effect during an outbreak of Salmonella ohio infection that occurred in Castellón (Spain) in May 2000. S. ohio was isolated in stools of patients and also in implicated foods., Results: Analysis was restricted to the group that was exposed to contaminated food (51 persons). We compared attack rates by level of alcohol intake (none, 1-40 gm, and >40 gm). The adjusted relative risks in the two alcohol categories were 0.73 [95% confidence interval (CI) = 0.46-1.15] and 0.54 (95% CI = 0.31-0.96), respectively., Conclusions: We discuss the possible protective effect of alcoholic beverages in some food-borne infectious outbreaks.