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Genome-wide high-throughput screening to investigate essential genes involved in methicillin-resistant Staphylococcus aureus Sequence Type 398 survival.

ABSTRACT: Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) Sequence Type 398 (ST398) is an opportunistic pathogen that is able to colonize and cause disease in several animal species including humans. To better understand the adaptation, evolution, transmission and pathogenic capacity, further investigations into the importance of the different genes harboured by LA-MRSA ST398 are required. In this study we generated a genome-wide transposon mutant library in an LA-MRSA ST398 isolate to evaluate genes important for bacterial survival in laboratory and host-specific environments. The transposon mutant library consisted of approximately 1 million mutants with around 140,000 unique insertion sites and an average number of unique inserts per gene of 44.8. We identified LA-MRSA ST398 essential genes comparable to other high-throughput S. aureus essential gene studies. As ST398 is the most common MRSA isolated from pigs, the transposon mutant library was screened in whole porcine blood. Twenty-four genes were specifically identified as important for bacterial survival in porcine blood. Mutations in 23 of these genes resulted in attenuated bacterial fitness. Seven of the 23 genes were of unknown function, whereas 16 genes were annotated with functions predominantly related to carbon metabolism, pH shock and a variety of regulations and only indirectly to virulence factors. Mutations in one gene of unknown function resulted in a hypercompetitive mutant. Further evaluation of these genes is required to determine their specific relevance in blood survival.

SUBMITTER: Christiansen MT

PROVIDER: S-EPMC3923074 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Genome-wide high-throughput screening to investigate essential genes involved in methicillin-resistant Staphylococcus aureus Sequence Type 398 survival.

Christiansen Mette T MT Kaas Rolf S RS Chaudhuri Roy R RR Holmes Mark A MA Hasman Henrik H Aarestrup Frank M FM

PloS one 20140212 2

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) Sequence Type 398 (ST398) is an opportunistic pathogen that is able to colonize and cause disease in several animal species including humans. To better understand the adaptation, evolution, transmission and pathogenic capacity, further investigations into the importance of the different genes harboured by LA-MRSA ST398 are required. In this study we generated a genome-wide transposon mutant library in an LA-MRSA ST398 iso ...[more]

PMID: 24563689

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Project description:Livestock-associated methicillin-resistant Staphylococcus aureus sequence type (ST) 398 (LA-MRSA ST398) is a genetic lineage for which pigs are regarded as the main reservoir. An increasing prevalence of LA-MRSA ST398 has been reported in areas with high livestock density throughout Europe. In this study, we investigated the drivers contributing to the introduction and spread of LA-MRSA ST398 through the pig farming system in southern Italy. Whole-genome sequencing (WGS) of LA-MRSA ST398 isolates collected in 2018 from pigs (n = 53) and employees (n = 14) from 10 farms in the Calabria region of Italy were comparatively analyzed with previously published WGS data from Italian ST398 isolates (n = 45), an international ST398 reference collection (n = 89), and isolates from Danish pig farms (n = 283), which are the main suppliers of pigs imported to Italy. Single-nucleotide polymorphisms (SNP) were used to infer isolate relatedness, and these data were used together with data from animal trading to identify factors contributing to LA-MRSA ST398 dissemination. The analyses support the existence of two concurrent pathways for the spread of LA-MRSA ST398 in southern Italy: (i) multiple introductions of LA-MRSA ST398 through the import of colonized pigs from other European countries, including Denmark and France, and (ii) the spread of distinct clones dependent on local trading of pigs between farms. Phylogenetically related Italian and Danish LA-MRSA ST398 isolates shared extensive similarities, including carriage of antimicrobial resistance genes. Our findings highlight the potential risk of transboundary transmission of antimicrobial-resistant bacterial clones with a high zoonotic potential during import of pigs from countries with high LA-MRSA prevalence.IMPORTANCE Over the past decade, livestock-associated methicillin-resistant Staphylococcus aureus sequence type 398 (LA-MRSA ST398) has spread among pig holdings throughout Europe, in parallel with the increased incidence of infections among humans, especially in intensive pig farming regions. Despite the growing prevalence of LA-MRSA ST398 in Italian pig farms, the transmission dynamics of this clone in Italy remains unclear. This work provides genome-based evidence to suggest transboundary LA-MRSA ST398 transmission through trading of colonized pigs between European countries and Italy, as well as between farms in the same Italian region. Our findings show that both international trading and local trading of colonized pigs are important factors contributing to the global spread of LA-MRSA ST398 and underscore the need for control measures on and off the farm to reduce the dissemination of this zoonotic pathogen.

Project description:BackgroundSevere infections with highly virulent community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) are a global problem. However, the molecular events defining the evolution of CA-MRSA are still poorly understood. MRSA of sequence type (ST) 398 is known to frequently infect livestock, while ST398 isolates infecting humans are commonly methicillin-susceptible or represent MRSA originating from livestock-associated (LA)-MRSA.MethodsWe used whole genome sequencing of newly detected CA-MRSA ST398 isolates, in comparison to geographically matched LA-MRSA and methicillin-sensitive ST398, to determine their evolutionary history. Furthermore, we used phenotypic analyses including animal infection models to gain insight into the evolution of virulence in these CA-MRSA isolates. Finally, we determined methicillin resistance and expression of the methicillin resistance-conferring gene mecA and its penicillin-binding protein product, PBP2a, in a large series of CA-MRSA strains of divergent STs.ResultsWe report several cases of severe and fatal infections due to ST398 CA-MRSA. The responsible isolates showed the typical genetic characteristics reported for human-adapted methicillin-sensitive ST398. Whole genome sequencing demonstrated that they evolved from human-adapted, methicillin-susceptible clones on several different occasions. Importantly, the isolates had not undergone consistent genetic alterations or changes in virulence as compared to their methicillin-susceptible predecessors. Finally, we observed dramatically and consistently lower methicillin resistance and expression of the resistance gene mecA, as compared to hospital-associated MRSA strains, in a diverse selection of CA-MRSA strains.ConclusionsOur study presents evidence for the development of highly virulent human-adapted ST398 CA-MRSA isolates from methicillin-susceptible predecessors. Notably, our investigation indicates that, in contrast to widespread notions, the development of CA-MRSA is not necessarily associated with the acquisition of specific virulence genes or other virulence-increasing changes. Rather, our findings emphasize the importance of the CA-MRSA-characteristic staphylococcal cassette chromosome mec types, which provide only low-level methicillin resistance, for that process. Our findings are of particular importance for the diagnosis of CA-MRSA, inasmuch as they indicate that the presence of specific virulence genes cannot generally be used for that purpose.

Project description:Continuing introduction of multi-drug resistant, zoonotic pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) in horse clinics challenges the biosafety of employees and animal patients. This study was aimed to determine the occurrence of mobile genetic elements facilitating survival in the early stages of invasive infection in different host species, including humans and horses, in MRSA carried by equine patients admitted to a large horse clinic. A total of 341 equine patients were investigated for carriage of MRSA by hygiene screening directly at hospital admission. MRSA were further investigated by antimicrobial susceptibility testing, whole-genome sequencing and genomic composition, including virulence factors involved in immune evasion and host adaption. From a total of 340 validated specimens from equine nostrils, 3.5% yielded positive results for MRSA. All MRSA were found to be closely related belonging to sequence type (ST) 398_t011 with up to four additional antimicrobial resistances. All MRSA harbored a specific Staphylococcal Pathogenicity Island (SaPIbov5) involved in facilitating survival in ruminant and equine plasma. Moreover, a β-hemolysin (hlb) converting ΦSa3 phage encoding the human-specific Immune Evasion Cluster (IEC) was present in 72% of the isolates. An equid-specific leukotoxin encoded by a further temperate phage (Saeq1) was only rarely detected (22%). Despite the absence of β-hemolysin production for all IEC-positive ST398, a prominent hemolysis zone was demonstrable on sheep blood agar. Thus, IEC might remain undetected among the ST398 lineage, since the presence of IEC is commonly associated with reduction of hemolysis in S. aureus belonging to other genetic backgrounds. Here we describe MRSA-ST398 harboring different mobile genetic elements encoding variants of immune evasion factors and toxins previously shown to contribute to S. aureus invasive diseases in specific host species or ecologic niches. We suggest these combinations contribute to the adaptation of MRSA belonging to ST398 with respect to epidemic spread across different habitats and hosts, and may therefore confer a host "generalist" phenotype.

Dataset Information

Genome-wide high-throughput screening to investigate essential genes involved in methicillin-resistant Staphylococcus aureus Sequence Type 398 survival.

Publications

Genome-wide high-throughput screening to investigate essential genes involved in methicillin-resistant Staphylococcus aureus Sequence Type 398 survival.

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