María Concepción Porrero

Staphylococcus aureus CC398: Host Adaptation and Emergence of Methicillin Resistance in Livestock

ABSTRACT Since its discovery in the early 2000s, methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 398 (CC398) has become a rapidly emerging cause of human infections, most often associated with livestock exposure. We applied whole-genome sequence typing to characterize a diverse collection of CC398 isolates (n = 89), including MRSA and methicillin-susceptible S. aureus (MSSA) from animals and humans spanning 19 countries and four continents. We identified 4,238 single nucleotide polymorphisms (SNPs) among the 89 core genomes. Minimal homoplasy (consistency index = 0.9591) was detected among parsimony-informative SNPs, allowing for the generation of a highly accurate phylogenetic reconstruction of the CC398 clonal lineage. Phylogenetic analyses revealed that MSSA from humans formed the most ancestral clades. The most derived lineages were composed predominantly of livestock-associated MRSA possessing three different staphylococcal cassette chromosome mec element (SCCmec) types (IV, V, and VII-like) including nine subtypes. The human-associated isolates from the basal clades carried phages encoding human innate immune modulators that were largely missing among the livestock-associated isolates. Our results strongly suggest that livestock-associated MRSA CC398 originated in humans as MSSA. The lineage appears to have undergone a rapid radiation in conjunction with the jump from humans to livestock, where it subsequently acquired tetracycline and methicillin resistance. Further analyses are required to estimate the number of independent genetic events leading to the methicillin-resistant sublineages, but the diversity of SCCmec subtypes is suggestive of strong and diverse antimicrobial selection associated with food animal production. IMPORTANCE Modern food animal production is characterized by densely concentrated animals and routine antibiotic use, which may facilitate the emergence of novel antibiotic-resistant zoonotic pathogens. Our findings strongly support the idea that livestock-associated MRSA CC398 originated as MSSA in humans. The jump of CC398 from humans to livestock was accompanied by the loss of phage-carried human virulence genes, which likely attenuated its zoonotic potential, but it was also accompanied by the acquisition of tetracycline and methicillin resistance. Our findings exemplify a bidirectional zoonotic exchange and underscore the potential public health risks of widespread antibiotic use in food animal production. Modern food animal production is characterized by densely concentrated animals and routine antibiotic use, which may facilitate the emergence of novel antibiotic-resistant zoonotic pathogens. Our findings strongly support the idea that livestock-associated MRSA CC398 originated as MSSA in humans. The jump of CC398 from humans to livestock was accompanied by the loss of phage-carried human virulence genes, which likely attenuated its zoonotic potential, but it was also accompanied by the acquisition of tetracycline and methicillin resistance. Our findings exemplify a bidirectional zoonotic exchange and underscore the potential public health risks of widespread antibiotic use in food animal production.

Monitoring and Characterization of Extended-Spectrum β-Lactamases in Escherichia coli Strains from Healthy and Sick Animals in Spain in 2003

ABSTRACT Genes encoding CTX-M-14, CTX-M-9, CTX-M-1, CTX-M-32, SHV-12, TEM-52, or CMY-2 β-lactamases were detected in 21 Escherichia coli strains recovered during 2003 from sick animals (11 of 459 [2.4%] strains) and healthy animals (10 of 158 [6.3%] strains) in Spain. Twelve of these strains harbored blaCTX-M genes and showed unrelated pulsed-field gel electrophoresis patterns.

armA and aminoglycoside resistance in Escherichia coli.

We report armA in an Escherichia coli pig isolate from Spain. The resistance gene was borne by self-transferable IncN plasmid pMUR050. Molecular analysis of the plasmid and of the armA locus confirmed the spread of this resistance determinant.

Molecular characterization of spa type t127, sequence type 1 methicillin-resistant Staphylococcus aureus from pigs

OBJECTIVES The aim of this study was to provide molecular characterization of methicillin-resistant Staphylococcus aureus (MRSA) spa type t127, sequence type (ST) 1 isolates, detected in a European baseline survey in holdings of breeding pigs, to determine phenotypic and genotypic drug resistance and to compare the results with those obtained from a collection of t127, ST1 MRSA and methicillin-susceptible S. aureus (MSSA) clinical isolates. METHODS Twenty-four t127, ST1 MRSA from dust sampled in different breeding holdings in Italy, Spain and Cyprus were studied, along with 2 t127, ST1 MRSA from fattening pigs and 11 human t127, ST1 MRSA and MSSA. Genotyping was performed using multilocus sequence typing (MLST), spa typing and PFGE. SCCmec elements were characterized by multiplex-PCR and resistance and pathogenicity genes by PCR and microarray. RESULTS PFGE patterns separated a porcine cluster (PC) from a human cluster (HC), with 75% similarity. The PC carried SCCmec cassette type V, while all isolates of the HC carried SCCmec cassette type IVa. Kanamycin resistance mediated by aadD, fluoroquinolone and erm(A)-mediated macrolide resistance and the absence of the sakA gene were features of the PC only. All isolates of both clusters were positive for LukE-LukD and LuF-LukS-HlgA leukotoxin genes and one human MSSA harboured Panton-Valentine leucocidin genes. CONCLUSIONS Despite differences in the host-specific genetic features, the possibility of PC transmission to humans cannot be excluded. MRSA spa type t127, ST1 from pigs possesses several virulence and resistance genes towards major classes of antimicrobials and may represent a serious therapeutic challenge in case of invasive infections in humans.

Evaluation of four protocols for the detection and isolation of thermophilic Campylobacter from different matrices

Aims:  To identify the optimal method for detection of thermophilic Campylobacter at various stages in the food chain, three culture‐dependent (direct plating, Bolton and Preston enrichment) and one molecular method (qPCR) were compared for three matrices: poultry faeces (n = 38), neck skin (n = 38) and packed fresh meat (n = 38).

Livestock-Associated Methicillin Resistant and Methicillin Susceptible Staphylococcus aureus Sequence Type (CC)1 in European Farmed Animals: High Genetic Relatedness of Isolates from Italian Cattle Herds and Humans

Methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type (ST)1, Clonal Complex(CC)1, SCCmec V is one of the major Livestock-Associated (LA-) lineages in pig farming industry in Italy and is associated with pigs in other European countries. Recently, it has been increasingly detected in Italian dairy cattle herds. The aim of this study was to analyse the differences between ST1 MRSA and methicillin-susceptible S. aureus (MSSA) from cattle and pig herds in Italy and Europe and human isolates. Sixty-tree animal isolates from different holdings and 20 human isolates were characterized by pulsed-field gel electrophoresis (PFGE), spa-typing, SCCmec typing, and by micro-array analysis for several virulence, antimicrobial resistance, and strain/host-specific marker genes. Three major PFGE clusters were detected. The bovine isolates shared a high (≥90% to 100%) similarity with human isolates and carried the same SCCmec type IVa. They often showed genetic features typical of human adaptation or present in human-associated CC1: Immune evasion cluster (IEC) genes sak and scn, or sea; sat and aphA3-mediated aminoglycoside resistance. Contrary, typical markers of porcine origin in Italy and Spain, like erm(A) mediated macrolide-lincosamide-streptograminB, and of vga(A)-mediated pleuromutilin resistance were always absent in human and bovine isolates. Most of ST(CC)1 MRSA from dairy cattle were multidrug-resistant and contained virulence and immunomodulatory genes associated with full capability of colonizing humans. As such, these strains may represent a greater human hazard than the porcine strains. The zoonotic capacity of CC1 LA-MRSA from livestock must be taken seriously and measures should be implemented at farm-level to prevent spill-over.

Antimicrobial resistance in indicator Escherichia coli isolates from free-ranging livestock and sympatric wild ungulates in a natural environment (Northeastern Spain)

ABSTRACT Antimicrobial resistance was assessed in indicator Escherichia coli isolates from free-ranging livestock and sympatric wild boar (Sus scrofa) and Iberian ibex (Capra pyrenaica) in a National Game Reserve in northeastern Spain. The frequency of antimicrobial resistance was low (0% to 7.9%). However, resistance to an extended-spectrum cephalosporin and fluoroquinolones was detected.

A Livestock-Associated, Multidrug-Resistant, Methicillin-Resistant Staphylococcus aureus Clonal Complex 97 Lineage Spreading in Dairy Cattle and Pigs in Italy

ABSTRACT Pandemic methicillin-resistant Staphylococcus aureus (MRSA) clonal complex 97 (CC97) lineages originated from livestock-to-human host jumps. In recent years, CC97 has become one of the major MRSA lineages detected in Italian farmed animals. The aim of this study was to characterize and analyze differences in MRSA and methicillin-susceptible S. aureus (MSSA) mainly of swine and bovine origins. Forty-seven CC97 isolates, 35 MRSA isolates, and 6 MSSA isolates from different Italian pig and cattle holdings; 5 pig MRSA isolates from Germany; and 1 human MSSA isolate from Spain were characterized by macrorestriction pulsed-field gel electrophoresis (PFGE) analysis, multilocus sequence typing (MLST), spa typing, staphylococcal cassette chromosome mec (SCCmec) typing, and antimicrobial resistance pattern analysis. Virulence and resistance genes were investigated by PCR and microarray analysis. Most of the isolates were of SCCmec type V (SCCmec V), except for two German MRSA isolates (SCCmec III). Five main clusters were identified by PFGE, with the German isolates (clusters I and II) showing 60.5% similarity with the Italian isolates, most of which (68.1%) grouped into cluster V. All CC97 isolates were Panton-Valentine leukocidin (PVL) negative, and a few (n = 7) tested positive for sak or scn. All MRSA isolates were multidrug resistant (MDR), and the main features were erm(B)- or erm(C)-mediated (n = 18) macrolide-lincosamide-streptogramin B resistance, vga(A)-mediated (n = 37) pleuromutilin resistance, fluoroquinolone resistance (n = 33), tet(K) in 32/37 tet(M)-positive isolates, and blaZ in almost all MRSA isolates. Few host-associated differences were detected among CC97 MRSA isolates: their extensive MDR nature in both pigs and dairy cattle may be a consequence of a spillback from pigs of a MRSA lineage that originated in cattle as MSSA and needs further investigation. Measures should be implemented at the farm level to prevent spillover to humans in intensive farming areas.

Detection of methicillin‐resistant Staphylococcus aureus in Iberian pigs

Aims:  Iberian pigs are bred in Spain for the production of high‐value dry‐cured products, whose export volumes are increasing. Animals are typically reared outdoors, although indoor farming is becoming popular. We compared carriage of methicillin‐resistant Staphylococcus aureus (MRSA) in Iberian pigs, raised indoors and outdoors, with intensively farmed Standard White pigs.

Cattle Drive Salmonella Infection in the Wildlife–Livestock Interface

The genus Salmonella is found throughout the world and is a potential pathogen for most vertebrates. It is also the most common cause of food‐borne illness in humans, and wildlife is an emerging source of food‐borne disease in humans due to the consumption of game meat. Wild boar is one of the most abundant European game species and these wild swine are known to be carriers of zoonotic and food‐borne pathogens such as Salmonella. Isolation of the pathogen, serotyping and molecular biology are necessary for elucidating epidemiological connections in multi‐host populations. Although disease management at population level can be addressed using a number of different strategies, such management is difficult in free‐living wildlife populations due to the lack of experience with the wildlife–livestock interface. Herein, we provide the results of a 4‐year Salmonella survey in sympatric populations of wild boar and cattle in the Ports de Tortosa i Beseit National Game Reserve (NE Spain). We also evaluated the effects of two management strategies, cattle removal and increased wild boar harvesting (i.e. by hunting and trapping), on the prevalence of the Salmonella serovar community. The serovars Meleagridis and Anatum were found to be shared by cattle and wild boar, a finding that was confirmed by 100% DNA similarity patterns using pulse field gel electrophoresis. Cattle removal was more efficient than the culling of wild boar as a means of reducing the prevalence of shared serotypes, which underlines the role of cattle as a reservoir of Salmonella for wild boar. To our knowledge, this is the first attempt to manage Salmonella in the wild, and the results have implications for management.