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September 22, 2019  |  

Comparative genomics of Pseudomonas syringae reveals convergent gene gain and loss associated with specialization onto cherry (Prunus avium).

Genome-wide analyses of the effector- and toxin-encoding genes were used to examine the phylogenetics and evolution of pathogenicity amongst diverse strains of Pseudomonas syringae causing bacterial canker of cherry (Prunus avium), including pathovars P. syringae pv morsprunorum (Psm) races 1 and 2, P. syringae pv syringae (Pss) and P. syringae pv avii. Phylogenetic analyses revealed Psm races and P. syringae pv avii clades were distinct and were each monophyletic, whereas cherry-pathogenic strains of Pss were interspersed amongst strains from other host species. A maximum likelihood approach was used to predict effectors associated with pathogenicity on cherry. Pss possesses a smaller repertoire of type III effectors but has more toxin biosynthesis clusters than Psm and P. syringae pv avii. Evolution of cherry pathogenicity was correlated with gain of genes such as hopAR1 and hopBB1 through putative phage transfer and horizontal transfer respectively. By contrast, loss of the avrPto/hopAB redundant effector group was observed in cherry-pathogenic clades. Ectopic expression of hopAB and hopC1 triggered the hypersensitive reaction in cherry leaves, confirming computational predictions. Cherry canker provides a fascinating example of convergent evolution of pathogenicity that is explained by the mix of effector and toxin repertoires acting on a common host.© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

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September 22, 2019  |  

Comparative genomics of Spiraeoideae-infecting Erwinia amylovora strains provides novel insight to genetic diversity and identifies the genetic basis of a low-virulence strain.

Erwinia amylovora is the causal agent of fire blight, one of the most devastating diseases of apple and pear. Erwinia amylovora is thought to have originated in North America and has now spread to at least 50 countries worldwide. An understanding of the diversity of the pathogen population and the transmission to different geographical regions is important for the future mitigation of this disease. In this research, we performed an expanded comparative genomic study of the Spiraeoideae-infecting (SI) E. amylovora population in North America and Europe. We discovered that, although still highly homogeneous, the genetic diversity of 30 E. amylovora genomes examined was about 30 times higher than previously determined. These isolates belong to four distinct clades, three of which display geographical clustering and one of which contains strains from various geographical locations (‘Widely Prevalent’ clade). Furthermore, we revealed that strains from the Widely Prevalent clade displayed a higher level of recombination with strains from a clade strictly from the eastern USA, which suggests that the Widely Prevalent clade probably originated from the eastern USA before it spread to other locations. Finally, we detected variations in virulence in the SI E. amylovora strains on immature pear, and identified the genetic basis of one of the low-virulence strains as being caused by a single nucleotide polymorphism in hfq, a gene encoding an important virulence regulator. Our results provide insights into the population structure, distribution and evolution of SI E. amylovora in North America and Europe.© 2017 BSPP AND JOHN WILEY & SONS LTD.

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September 22, 2019  |  

Whole-genome comparison of high and low virulent Staphylococcus aureus isolates inducing implant-associated bone infections.

Staphylococcus aureus can cause wide range of infections from simple soft skin infections to severe endocarditis, bacteremia, osteomyelitis and implant associated bone infections (IABI). The focus of the present investigation was to study virulence properties of S. aureus isolates from acute and chronic IABI by means of their in vivo lethality, in vitro osteoblasts invasion, biofilm formation and subsequently whole genome comparison between high and low virulent strains. Application of insect infection model Galleria mellonella revealed high, intermediate and low virulence phenotypes of these clinical isolates, which showed good correlation with osteoblast invasion and biofilm formation assays. Comparative genomics of selected high (EDCC 5458) and low (EDCC 5464) virulent strains enabled the identification of molecular factors responsible for the development of acute and chronic IABI. Accordingly, the low virulent strain EDCC 5464 harbored point mutations resulting in frame shift mutations in agrC (histidine kinase in agr system), graS (histidine kinase in graSR, a two component system) and efeB (peroxidase in efeOBU operon, an iron acquisition system) genes. Additionally, we found a mobile element (present 11 copies in EDCC 5464) inserted at the end of ß-hemolysin (hlb) and sarU genes, which are involved in the pathogenesis and regulation of virulence gene expression in coordination with quorum sensing system. All these results are in good support with the low virulence behavior of EDCC 5464. From the previous literature, it is well known that agr defective S. aureus clinical strains are isolated from the chronic infections. Similarly, low virulent EDCC 5464 was isolated from chronic implant-associated bone infections infection whereas EDCC 5458 was obtained from acute implant-associated bone infections. Laboratory based in vitro and in vivo results and insights from comparative genomic analysis could be correlated with the clinical conclusion of IABIs and allows evidence-based treatment strategies based on the pathogenesis of the strain to cure life devastating implant-associated infections. Copyright © 2018 Elsevier GmbH. All rights reserved.

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September 22, 2019  |  

Genomic comparison of highly virulent, moderately virulent, and avirulent strains from a genetically closely-related MRSA ST239 sub-lineage provides insights into pathogenesis.

The genomic comparison of virulent (TW20), moderately virulent (CMRSA6/CMRSA3), and avirulent (M92) strains from a genetically closely-related MRSA ST239 sub-lineage revealed striking similarities in their genomes and antibiotic resistance profiles, despite differences in virulence and pathogenicity. The main differences were in the spa gene (coding for staphylococcal protein A), lpl genes (coding for lipoprotein-like membrane proteins), cta genes (genes involved in heme synthesis), and the dfrG gene (coding for a trimethoprim-resistant dihydrofolate reductase), as well as variations in the presence or content of some prophages and plasmids, which could explain the virulence differences of these strains. TW20 was positive for all genetic traits tested, compared to CMRSA6, CMRSA3, and M92. The major components differing among these strains included spa and lpl with TW20 carrying both whereas CMRSA6/CMRSA3 carry spa identical to TW20 but have a disrupted lpl. M92 is devoid of both these traits. Considering the role played by these components in innate immunity and virulence, it is predicted that since TW20 has both the components intact and functional, these traits contribute to its pathogenesis. However, CMRSA6/CMRSA3 are missing one of these components, hence their intermediately virulent nature. On the contrary, M92 is completely devoid of both the spa and lpl genes and is avirulent. Mobile genetic elements play a potential role in virulence. TW20 carries three prophages (?Sa6, ?Sa3, and ?SPß-like), a pathogenicity island and two plasmids. CMRSA6, CMRSA3, and M92 contain variations in one or more of these components. The virulence associated genes in these components include staphylokinase, entertoxins, antibiotic/antiseptic/heavy metal resistance and bacterial persistence. Additionally, there are many hypothetical proteins (present with variations among strains) with unknown function in these mobile elements which could be making an important contribution in the virulence of these strains. The above mentioned repertoire of virulence components in TW20 likely contributes to its increased virulence, while the absence and/or modification of one or more of these components in CMRSA6/CMRSA3 and M92 likely affects the virulence of the strains.

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September 22, 2019  |  

Genetic adaptation of a mevalonate pathway deficient mutant in Staphylococcus aureus.

In this study we addressed the question how a mevalonate (MVA)-auxotrophic Staphylococcus aureus?mvaS mutant can revert to prototrophy. This mutant couldn’t grow in the absence of MVA. However, after a long lag-phase of 4-6 days the mutant adapted from auxotrophic to prototrophic phenotype. During that time, it acquired two point mutations: One mutation in the coding region of the regulator gene spx, which resulted in an amino acid exchange that decreased Spx function. The other mutation in the upstream-element within the core-promoter of the mevalonolactone lactonase gene drp35. This mutation led to an increased expression of drp35. In repeated experiments the mutations always occurred in spx and drp35 and in the same order. The first detectable mutation appeared in spx and allowed slight growth; the second mutation, in drp35, increased growth further. Phenotypical characterizations of the mutant showed that small amounts of the lipid-carrier undecaprenol are synthesized, despite the lack of mvaS. The growth of the adapted clone, ?mvaSad, indicates that the mutations reawake a rescue bypass. We think that this bypass enters the MVA pathway at the stage of MVA, because blocking the pathway downstream of MVA led to growth arrest of the mutant. In addition, the lactonase Drp35 is able to convert mevalonolactone to MVA. Summarized, we describe here a mutation-based two-step adaptation process that allows resuscitation of growth of the ?mvaS mutant.

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September 22, 2019  |  

Comparative genomics of Pseudomonas sp. strain SI-3 associated with macroalga Ulva prolifera, the causative species for green tide in the Yellow Sea.

Algae-bacteria associations occurred widely in marine habitats, however, contributions of bacteria to macroalgal blooming were almost unknown. In this study, a potential endophytic strain SI-3 was isolated from Ulva prolifera, the causative species for the world’s largest green tide in the Yellow Sea, following a strict bleaching treatment to eliminate epiphytes. The genomic sequence of SI-3 was determined in size of 4.8 Mb and SI-3 was found to be mostly closed to Pseudomonas stutzeri. To evaluate the characteristics of SI-3 as a potential endophyte, the genomes of SI-3 and other 20 P. stutzeri strains were compared. We found that SI-3 had more strain-specific genes than most of the 20 P. stutzeri strains. Clusters of Orthologous Groups (COGs) analysis revealed that SI-3 had a higher proportion of genes assigned to transcriptional regulation and signal transduction compared with the 20 P. stutzeri strains, including four rhizosphere bacteria, indicating a complicated interaction network between SI-3 and its host. P. stutzeri is renowned for its metabolic versatility in aromatic compounds degradation. However, significant gene loss was observed in several aromatic compounds degradation pathways in SI-3, which may be an evolutional adaptation that developed upon association with its host. KEGG analysis revealed that dissimilatory nitrate reduction to ammonium (DNRA) and denitrification, two competing dissimilatory nitrate reduction pathways, co-occurred in the genome of SI-3, like most of the other 20 P. stutzeri strains. We speculated that DNRA of SI-3 may contribute a competitive advantage in nitrogen acquisition of U. prolifera by conserving nitrogen in NH4+ form, as in the case of microalgae bloom. Collectively, these data suggest that Pseudomonas sp. strain SI-3 was a suitable candidate for investigation of the algae-bacteria interaction with U. prolifera and the ecological impacts on algal blooming.

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September 22, 2019  |  

Potential survival and pathogenesis of a novel strain, Vibrio parahaemolyticus FORC_022, isolated from a soy sauce marinated crab by genome and transcriptome analyses.

Vibrio parahaemolyticus can cause gastrointestinal illness through consumption of seafood. Despite frequent food-borne outbreaks of V. parahaemolyticus, only 19 strains have subjected to complete whole-genome analysis. In this study, a novel strain of V. parahaemolyticus, designated FORC_022 (Food-borne pathogen Omics Research Center_022), was isolated from soy sauce marinated crabs, and its genome and transcriptome were analyzed to elucidate the pathogenic mechanisms. FORC_022 did not include major virulence factors of thermostable direct hemolysin (tdh) and TDH-related hemolysin (trh). However, FORC_022 showed high cytotoxicity and had several V. parahaemolyticus islands (VPaIs) and other virulence factors, such as various secretion systems (types I, II, III, IV, and VI), in comparative genome analysis with CDC_K4557 (the most similar strain) and RIMD2210633 (genome island marker strain). FORC_022 harbored additional virulence genes, including accessory cholera enterotoxin, zona occludens toxin, and tight adhesion (tad) locus, compared with CDC_K4557. In addition, O3 serotype specific gene and the marker gene of pandemic O3:K6 serotype (toxRS) were detected in FORC_022. The expressions levels of genes involved in adherence and carbohydrate transporter were high, whereas those of genes involved in motility, arginine biosynthesis, and proline metabolism were low after exposure to crabs. Moreover, the virulence factors of the type III secretion system, tad locus, and thermolabile hemolysin were overexpressed. Therefore, the risk of foodborne-illness may be high following consumption of FORC_022 contaminated crab. These results provided molecular information regarding the survival and pathogenesis of V. parahaemolyticus FORC_022 strain in contaminated crab and may have applications in food safety.

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September 22, 2019  |  

Emergence of an XDR and carbapenemase-producing hypervirulent Klebsiella pneumoniae strain in Taiwan.

Carbapenemase-producing Klebsiella pneumoniae causes high mortality owing to the limited therapeutic options available. Here, we investigated an emergent carbapenem-resistant K. pneumoniae strain with hypervirulence found among KPC-2-producing strains in Taiwan.KPC-producing K. pneumoniae strains were collected consecutively from clinical specimens at the Taipei Veterans General Hospital between January 2012 and December 2014. Capsular types and the presence of rmpA/rmpA2 were analysed, and PFGE and MLST performed using these strains. The strain positive for rmpA/rmpA2 was tested in an in vivo mouse lethality study to verify its virulence and subjected to WGS to delineate its genomic features.A total of 62 KPC-2-producing K. pneumoniae strains were identified; all of these belonged to ST11 and capsular genotype K47. One strain isolated from a fatal case with intra-abdominal abscess (TVGHCRE225) harboured rmpA and rmpA2 genes. This strain was resistant to tigecycline and colistin, in addition to carbapenems, and did not belong to the major cluster in PFGE. TVGHCRE225 exhibited high in vivo virulence in the mouse lethality experiment. WGS showed that TVGHCRE225 acquired a novel hybrid virulence plasmid harbouring a set of virulence genes (iroBCDN, iucABCD, rmpA and rmpA2, and iutA) compared with the classic ST11 KPC-2-producing strain.We identified an XDR ST11 KPC-2-producing K. pneumoniae strain carrying a hybrid virulent plasmid in Taiwan. Active surveillance focusing on carbapenem-resistant hypervirulent K. pneumoniae strains is necessary, as the threat to human health is imminent.

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September 22, 2019  |  

Extensive genomic diversity among Mycobacterium marinum strains revealed by whole genome sequencing.

Mycobacterium marinum is the causative agent for the tuberculosis-like disease mycobacteriosis in fish and skin lesions in humans. Ubiquitous in its geographical distribution, M. marinum is known to occupy diverse fish as hosts. However, information about its genomic diversity is limited. Here, we provide the genome sequences for 15 M. marinum strains isolated from infected humans and fish. Comparative genomic analysis of these and four available genomes of the M. marinum strains M, E11, MB2 and Europe reveal high genomic diversity among the strains, leading to the conclusion that M. marinum should be divided into two different clusters, the “M”- and the “Aronson”-type. We suggest that these two clusters should be considered to represent two M. marinum subspecies. Our data also show that the M. marinum pan-genome for both groups is open and expanding and we provide data showing high number of mutational hotspots in M. marinum relative to other mycobacteria such as Mycobacterium tuberculosis. This high genomic diversity might be related to the ability of M. marinum to occupy different ecological niches.

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September 22, 2019  |  

Co-location of the blaKPC-2, blaCTX-M-65, rmtB and virulence relevant factors in an IncFII plasmid from a hypermucoviscous Klebsiella pneumoniae isolate.

Hypervirulent variants of klebsiella pneumoniae (hvKP), which cause serious infections not only healthy individuals, but also the immunocompromised patients, have been increasingly reported recently. One conjugation of a hypermucoviscous strian SWU01 co-carried the resistance gene blaKPC-2 and virulence gene iroN by the PCR detection from three carbapenem-resistance hvKP. To know the genetic context of this plasmid. The whole genome of this strain was sequenced. We got a 162,552-bp plasmid (pSWU01) which co-carried the resistance gene blaKPC-2 and virulence gene iroN. It is composed of a typical IncFII-type backbone, five resistance genes including blaCTX-M-65, blaKPC-2, blaSHV-12, blaTEM-1 and rmtB, and several virulence relevant factors including iroN, traT and toxin-antitoxin systems. The plasmid pSWU01 co-carrying the multidrug resistance determinants and virulence relevant factors from the hypermucoviscous K. pneumoniae represents a novel therapeutic challenge. Copyright © 2018 Elsevier Ltd. All rights reserved.

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September 22, 2019  |  

Comparative genomics of Salmonella enterica serovar Montevideo reveals lineage-specific gene differences that may influence ecological niche association.

Salmonella enterica serovar Montevideo has been linked to recent foodborne illness outbreaks resulting from contamination of products such as fruits, vegetables, seeds and spices. Studies have shown that Montevideo also is frequently associated with healthy cattle and can be isolated from ground beef, yet human salmonellosis outbreaks of Montevideo associated with ground beef contamination are rare. This disparity fuelled our interest in characterizing the genomic differences between Montevideo strains isolated from healthy cattle and beef products, and those isolated from human patients and outbreak sources. To that end, we sequenced 13 Montevideo strains to completion, producing high-quality genome assemblies of isolates from human patients (n=8) or from healthy cattle at slaughter (n=5). Comparative analysis of sequence data from this study and publicly available sequences (n=72) shows that Montevideo falls into four previously established clades, differentially occupied by cattle and human strains. The results of these analyses reveal differences in metabolic islands, environmental adhesion determinants and virulence factors within each clade, and suggest explanations for the infrequent association between bovine isolates and human illnesses.

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September 22, 2019  |  

The energy-coupling factor transporter module EcfAA’T, a novel candidate for the genetic basis of fatty acid-auxotrophic small-colony variants of Staphylococcus aureus.

Staphylococcal small-colony variants (SCVs) are invasive and persistent due to their ability to thrive intracellularly and to evade the host immune response. Thus, the course of infections due to this phenotype is often chronic, relapsing, and therapy-refractory. In order to improve treatment of patients suffering from SCV-associated infections, it is of major interest to understand triggers for the development of this phenotype, in particular for strains naturally occurring in clinical settings. Within this study, we comprehensively characterized two different Staphylococcus aureus triplets each consisting of isogenic strains comprising (i) clinically derived SCV phenotypes with auxotrophy for unsaturated fatty acids, (ii) the corresponding wild-types (WTs), and (iii) spontaneous in vitro revertants displaying the normal phenotype (REVs). Comparison of whole genomes revealed that clinical SCV isolates were closely related to their corresponding WTs and REVs showing only seven to eight alterations per genome triplet. However, both SCVs carried a mutation within the energy-coupling factor (ECF) transporter-encoding ecf module (EcfAA’T) resulting in truncated genes. In both cases, these mutations were shown to be naturally restored in the respective REVs. Since ECF transporters are supposed to be essential for optimal bacterial growth, their dysfunction might constitute another mechanism for the formation of naturally occurring SCVs. Another three triplets analyzed revealed neither mutations in the EcfAA’T nor in other FASII-related genes underlining the high diversity of mechanisms leading to the fatty acid-dependent phenotype. This is the first report on the ECF transporter as genetic basis of fatty acid-auxotrophic staphylococcal SCVs.

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September 22, 2019  |  

Comparison of highly and weakly virulent Dickeya solani strains, with a view on the pangenome and panregulon of this species.

Bacteria belonging to the genera Dickeya and Pectobacterium are responsible for significant economic losses in a wide variety of crops and ornamentals. During last years, increasing losses in potato production have been attributed to the appearance of Dickeya solani. The D. solani strains investigated so far share genetic homogeneity, although different virulence levels were observed among strains of various origins. The purpose of this study was to investigate the genetic traits possibly related to the diverse virulence levels by means of comparative genomics. First, we developed a new genome assembly pipeline which allowed us to complete the D. solani genomes. Four de novo sequenced and ten publicly available genomes were used to identify the structure of the D. solani pangenome, in which 74.8 and 25.2% of genes were grouped into the core and dispensable genome, respectively. For D. solani panregulon analysis, we performed a binding site prediction for four transcription factors, namely CRP, KdgR, PecS and Fur, to detect the regulons of these virulence regulators. Most of the D. solani potential virulence factors were predicted to belong to the accessory regulons of CRP, KdgR, and PecS. Thus, some differences in gene expression could exist between D. solani strains. The comparison between a highly and a low virulent strain, IFB0099 and IFB0223, respectively, disclosed only small differences between their genomes but significant differences in the production of virulence factors like pectinases, cellulases and proteases, and in their mobility. The D. solani strains also diverge in the number and size of prophages present in their genomes. Another relevant difference is the disruption of the adhesin gene fhaB2 in the highly virulent strain. Strain IFB0223, which has a complete adhesin gene, is less mobile and less aggressive than IFB0099. This suggests that in this case, mobility rather than adherence is needed in order to trigger disease symptoms. This study highlights the utility of comparative genomics in predicting D. solani traits involved in the aggressiveness of this emerging plant pathogen.

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September 22, 2019  |  

Variant O89 O-antigen of E. coli is associated with group 1 capsule loci and multidrug resistance.

Bacterial surface polysaccharides play significant roles in fitness and virulence. In Gram-negative bacteria such as Escherichia coli, major surface polysaccharides are lipopolysaccharide (LPS) and capsule, representing O- and K-antigens, respectively. There are multiple combinations of O:K types, many of which are well-characterized and can be related to ecotype or pathotype. In this investigation, we have identified a novel O:K permutation resulting through a process of major genome reorganization in a clade of E. coli. A multidrug-resistant, extended-spectrum ß-lactamase (ESBL)-producing strain – E. coli 26561 – represented a prototype of strains combining a locus variant of O89 and group 1 capsular polysaccharide. Specifically, the variant O89 locus in this strain was truncated at gnd, flanked by insertion sequences and located between nfsB and ybdK and we apply the term O89m for this variant. The prototype lacked colanic acid and O-antigen loci between yegH and hisI with this tandem polysaccharide locus being replaced with a group 1 capsule (G1C) which, rather than being a recognized E. coli capsule type, this locus matched to Klebsiella K10 capsule type. A genomic survey identified more than 200 E. coli strains which possessed the O89m locus variant with one of a variety of G1C types. Isolates from our collection with the combination of O89m and G1C all displayed a mucoid phenotype and E. coli 26561 was unusual in exhibiting a mucoviscous phenotype more recognized as a characteristic among Klebsiella strains. Despite the locus truncation and novel location, all O89m:G1C strains examined showed a ladder pattern typifying smooth LPS and also showed high molecular weight, alcian blue-staining polysaccharide in cellular and/or extra-cellular fractions. Expression of both O-antigen and capsule biosynthesis loci were confirmed in prototype strain 26561 through quantitative proteome analysis. Further in silico exploration of more than 200 E. coli strains possessing the O89m:G1C combination identified a very high prevalence of multidrug resistance (MDR) – 85% possessed resistance to three or more antibiotic classes and a high proportion (58%) of these carried ESBL and/or carbapenemase. The increasing isolation of O89m:G1C isolates from extra-intestinal infection sites suggests that these represents an emergent clade of invasive, MDR E. coli.

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September 22, 2019  |  

Prevalence and genomic structure of bacteriophage phi3 in human derived livestock-associated MRSA from 2000 to 2015.

Whereas the emergence of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) clonal complex 398 (CC398) in animal husbandry and its transmission to humans are well documented, less is known about factors driving the epidemic spread of this zoonotic lineage within the human population. One factor could be the bacteriophage phi3, which is rarely detected in S. aureus isolates from animals but commonly found among isolates from humans, including those of the human-adapted methicillin-susceptible S. aureus (MSSA) CC398 clade. The proportion of phi3-carrying MRSA spa-CC011 isolates, which constitute presumptively LA-MRSA within the multilocus sequence type (MLST) clonal complex 398, was systematically assessed for a period of 16 years to investigate the role of phi3 in the adaptation process of LA-MRSA to the human host. For this purpose, 632 MRSA spa-CC011 isolates from patients of a university hospital located in a pig farming-dense area in Germany were analyzed. Livestock-associated acquisition of MRSA spa-CC011 was previously reported as having increased from 1.8% in 2000 to 29.4% in 2014 in MRSA-positive patients admitted to this hospital. However, in this study, the proportion of phi3-carrying isolates rose only from 1.1% (2000 to 2006) to 3.9% (2007 to 2015). Characterization of the phi3 genomes revealed 12 different phage types ranging in size from 40,712 kb up to 44,003 kb, with four hitherto unknown integration sites (genes or intergenic regions) and several modified bacterial attachment (attB) sites. In contrast to the MSSA CC398 clade, phi3 acquisition seems to be no major driver for the readaptation of MRSA spa-CC011 to the human host. Copyright © 2018 American Society for Microbiology.

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