A comparative phenotypic and genomic analysis of methicillin-resistant Staphylococcus aureus ST45 isolates from cellulitis and from osteomyelitis in Taiwan.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 45 is a globally disseminated MRSA lineage. Herein, we investigated whether MRSA ST45 isolates from cellulitis and from osteomyelitis display distinctive phenotypic and genomic characteristics. METHODS: A total of 15 MRSA ST45 isolates from cellulitis (CL-MRSAs; n = 6) or osteomyelitis (OM-MRSAs; n = 9) were collected in a Taiwan hospital. These MRSA ST45 isolates were characterized for their antimicrobial susceptibility, biofilm-forming ability, cellular infectivity in vitro, and pathogenicity in vivo. Four CL-MRSA and six OM-MRSA ST45 isolates were selected for whole-genome sequencing (WGS). RESULTS: Antibiotic resistance tests showed that all OM-MRSA ST45 strains, but not CL-MRSA ST45 strains, were resistant to ciprofloxacin, levofloxacin, gentamicin and doxycycline. Compared to the CL-MRSA ST45 isolates, the OM-MRSA ST45 isolates had stronger biofilm-forming ability and cellular infectivity, and caused more severe disease in mice. WGS analysis revealed that these OM-MRSA ST45 isolates carry multiple common mutations or polymorphisms in genes associated with antibiotic resistance and virulence. Moreover, the transposable elements IS256 and IS257R2 were found only in the OM-MRSA ST45 isolates. CONCLUSIONS: The emergence and spread of the highly pathogenic and multidrug-resistant ST45 MRSAs identified from osteomyelitis may pose a serious threat on public health.

Influence of IS256 on Genome Variability and Formation of Small-Colony Variants in Staphylococcus aureus.

Staphylococcus aureus has acquired resistance to nearly all antibiotics used in clinical practice. Whereas some resistance mechanisms are conferred by uptake of resistance genes, others evolve by mutation. In this study, IS256 has been shown to play a role, e.g., in S. aureus strains displaying intermediate resistance to vancomycin (VISA). To characterize the IS256 insertion sites in the genomes of two closely related sequence type 247 (ST247) VISA strains, all insertions were mapped in both VISA and a susceptible control strain. The results showed that the three ST247 strains contained the highest number so far of IS256 insertions for all sequenced S. aureus strains. Furthermore, in contrast to the case with the other IS elements in these genomes, the IS256 insertion sites were not identical in the closely related strains, indicating a high transposition frequency of IS256 When IS256 was introduced into a laboratory strain which was then cultured in the presence of antibiotics, it was possible to isolate small-colony variants (SCVs) that possessed IS256 insertions in guaA and hemY that displayed increased resistance to vancomycin and aminoglycosides, respectively. For these clones, a very rapid reversion to the wild type that resembled the fast reversion of clinical SCVs was observed. The reversion was caused by excision of IS256 in a small number of fast-growing clones that quickly outcompeted the SCVs in broth cultures. In conclusion, the presence of IS256 confers a strong genomic plasticity that is useful for adaptation to antibiotic stress.

Characterization of the IncA/C plasmid pSCEC2 from Escherichia coli of swine origin that harbours the multiresistance gene cfr.

OBJECTIVES: To determine the complete nucleotide sequence of the multidrug resistance plasmid pSCEC2, isolated from a porcine Escherichia coli strain, and to analyse it with particular reference to the cfr gene region. METHODS: Plasmid pSCEC2 was purified from its E. coli J53 transconjugant and then sequenced using the 454 GS-FLX System. After draft assembly, predicted gaps were closed by PCR with subsequent sequencing of the amplicons. RESULTS: Plasmid pSCEC2 is 135 615 bp in size and contains 200 open reading frames for proteins of ≥100 amino acids. Analysis of the sequence of pSCEC2 revealed two resistance gene segments. The 4.4 kb cfr-containing segment is flanked by two IS256 elements in the same orientation, which are believed to be involved in the dissemination of the rRNA methylase gene cfr. The other segment harbours the resistance genes floR, tet(A)-tetR, strA/strB and sul2, which have previously been found on other IncA/C plasmids. Except for these two resistance gene regions, the pSCEC2 backbone displayed >99% nucleotide sequence identity to that of other IncA/C family plasmids isolated in France, Chile and the USA. CONCLUSIONS: The cfr gene was identified on an IncA/C plasmid, which is well known for its broad host range and transfer and maintenance properties. The location on such a plasmid will further accelerate the dissemination of cfr and co-located resistance genes among different Gram-negative bacteria. The genetic context of cfr on plasmid pSCEC2 underlines the complexity of cfr transfer events and confirms the role that insertion sequences play in the spread of cfr.

Genetic environment of the multi-resistance gene cfr in methicillin-resistant coagulase-negative staphylococci from chickens, ducks, and pigs in China.

The present study focussed on the analysis of the genetic environment of the multi-resistance gene cfr detected among 21, mostly methicillin-resistant, coagulase-negative Staphylococcus (CoNS) isolates obtained from chickens, ducks and pigs in China. It included sequencing of the regions up- and downstream of the cfr gene on various plasmid types in 13 isolates, such as pSS-02 and pSS-02-like (n=7), pSS-03-like (n=1), pJP1-like (n=3), pSS-04 (n=1) and pJP2 (n=1). This analysis revealed that insertion sequences (IS21-558, IS256, IS257, or IS1216E) and other resistance genes (aacA-aphD and aadD for aminoglycoside resistance, ble for bleomycin resistance, fosD for fosfomycin resistance, erm(B) and erm(C) for macrolide-lincosamide-streptogramin B resistance, or fexA for phenicol resistance) coexisted on the respective plasmids. In the chromosomal copies of cfr identified in eight S. lentus isolates, the cfr gene was found to be bracketed by insertion sequences, such as IS256 or ISEnfa5. Stability tests confirmed that all chromosomal cfr-containing regions could be looped out via IS-mediated recombination. The observations made in this study extend the rather rudimentary knowledge about the genetic environment of cfr in staphylococci from chickens and ducks and confirmed that insertion sequences play an important role in the dissemination of cfr, not only among different types of plasmids, but also for the integration in the chromosomal DNA.

Molecular characterization of lugdunin inactivation mechanisms and their association with Staphylococcus lugdunensis genetic types.

BACKGROUND AND PURPOSE: Our previous studies showed that lugdunin activities are associated with Staphylococcus lugdunensis genotypes, and most isolates do not exhibit lugdunin activity. As a continuation of our previous analysis, we focused on the reasons for defects in lugdunin production in S. lugdunensis clinical isolates. METHODS: A comparative analysis of 36 S. lugdunensis whole genome sequencing data revealed three major mutation types, unknown deletion mechanism that caused most of lug operon genes lost, mobile genetic element (MGE) insertion, and nonsense mutations, which potentially damaged lugdunin production. A total of 152 S. lugdunensis clinical isolates belonging to lugdunin nonproducers were further examined for the above three mutation types. PCR products were sequenced to examine these variations. RESULTS: Forty-six of the 152 isolates were CRISPR-Cas IIC isolates, including 26 ST27, 14 ST4, and 6 ST29 isolates; further investigation confirmed that all of their lug operons had lost almost all lug operon genes except lugM. An IS256 insertion in lugA was identified in 16 isolates, and most isolates (15 over 16) belonged to ST3. In addition, three nonsense mutations caused by single nucleotide substitutions (an adenine deletion in lugB at the 361th and 1219th nucleotides and an adenine deletion in lugC at the 1612nd nucleotide) that were frequently observed among 36 S. lugdunensis whole genome sequencing data were further observed in our clinical isolates. These three nonsense mutations were frequently found in most of CRISPR-Cas IIIA strains, especially in ST6 isolates. CONCLUSION: Our findings suggest that the mechanisms affecting lugdunin production are associated with S. lugdunensis molecular types.

Comparative Genomics Identifies Novel Genetic Changes Associated with Oxacillin, Vancomycin and Daptomycin Susceptibility in ST100 Methicillin-Resistant Staphylococcus aureus.

Infections due to vancomycin-intermediate S. aureus (VISA) and heterogeneous VISA (hVISA) represent a serious concern due to their association with vancomycin treatment failure. However, the underlying molecular mechanism responsible for the hVISA/VISA phenotype is complex and not yet fully understood. We have previously characterized two ST100-MRSA-hVISA clinical isolates recovered before and after 40 days of vancomycin treatment (D1 and D2, respectively) and two in vitro VISA derivatives (D23C9 and D2P11), selected independently from D2 in the presence of vancomycin. This follow-up study was aimed at further characterizing these isogenic strains and obtaining their whole genome sequences to unravel changes associated with antibiotic resistance. It is interesting to note that none of these isogenic strains carry SNPs in the regulatory operons vraUTSR, walKR and/or graXRS. Nonetheless, genetic changes including SNPs, INDELs and IS256 genomic insertions/rearrangements were found both in in vivo and in vitro vancomycin-selected strains. Some were found in the downstream target genes of the aforementioned regulatory operons, which are involved in cell wall and phosphate metabolism, staphylococcal growth and biofilm formation. Some of the genetic changes reported herein have not been previously associated with vancomycin, daptomycin and/or oxacillin resistance in S. aureus.

Molecular Analysis of Pathogenicity, Adhesive Matrix Molecules (MSCRAMMs) and Biofilm Genes of Coagulase-Negative Staphylococci Isolated from Ready-to-Eat Food.

This paper provides a snapshot on the pathogenic traits within CoNS isolated from ready-to-eat (RTE) food. Eighty-five strains were subjected to biofilm and slime production, as well as biofilm-associated genes (icaA, icaD, icaB, icaC, eno, bap, bhp, aap, fbe, embP and atlE), the insertion sequence elements IS256 and IS257 and hemolytic genes. The results showed that the most prevalent determinants responsible for the primary adherence were eno (57.6%) and aap (56.5%) genes. The icaADBC operon was detected in 45.9% of the tested strains and was correlated to slime production. Moreover, most strains carrying the icaADBC operon simultaneously carried the IS257 insertion sequence element. Among the genes encoding for surface proteins involved in the adhesion to abiotic surfaces process, atlE was the most commonly (31.8%) followed by bap (4.7%) and bhp (1.2%). The MSCRAMMs, including fbe and embp were detected in the 11.8% and 28.2% of strains, respectively. A high occurrence of genes involved in the hemolytic toxin production were detected, such as hla_yiD (50.6%), hlb (48.2%), hld (41.2%) and hla_haem (34.1%). The results of the present study revealed an unexpected occurrence of the genes involved in biofilm production and the high hemolytic activity among the CoNS strains, isolated from RTE food, highlighting that this group seems to be acquiring pathogenic traits similar to those of S. aureus, suggesting the need to be included in the routine microbiological analyses of food.

First report of the multiresistance gene cfr in Pasteurella multocida strains of avian origin from China.

OBJECTIVES: The aim of this study was to investigate the presence and genetic environment of the multiresistance gene cfr gene in Pasteurella multocida of avian origin from China. METHODS: A total of 113 P. multocida isolates were collected from sick poultries (ducks, chickens and geese) from 2003 to 2016 in Southern China and were screened for the presence of the cfr gene by PCR. The cfr-carrying P. multocida strains were subjected to antimicrobial susceptibility testing, S1 nuclease PFGE and Southern blot hybridisation, conjugative transfer and analysis of genetic environment of the cfr gene. RESULTS: Among 113 P. multocida isolates, strains FJ6671 and FJ6683 from Muscovy duck harboured the cfr gene and presented a multiresistant phenotype. The cfr gene in the two strains was located on an ∼40-kb conjugative plasmid in different genetic environments, including ISApl12-cfr-IS26 and IS26-cfr-IS256. CONCLUSIONS: These results demonstrate plasmid-carried cfr in P. multocida and suggest that transposition and homologous recombination mediated by IS26, ISApl1 and IS256 might have played an important role in transfer of the cfr gene in P. multocida. To the best of our knowledge, this is the first report of the cfr gene in P. multocida. Active and ongoing surveillance of cfr in P. multocida is urgently warranted.

Daptomycin resistance in methicillin-resistant Staphylococcus aureus is conferred by IS256 insertion in the promoter of mprF along with mutations in mprF and walK.

Mechanisms underlying the emergence of daptomycin resistance in Staphylococcus aureus remain unclear. In this study, Staphylococcus aureus strain 3d0, isolated from a patient with bloodstream infection and belonging to the predominant Chinese hospital-associated methicillin-resistant S. aureus (MRSA) clone ST239, was serially passaged on gradient broth containing daptomycin for 34 days. The whole genomes of 3d0 and its serial passage strains were sequenced and compared. Five single nucleotide polymorphisms, four IS256 insertions, and one 39-bp insert occurred in the progress of daptomycin resistance acquisition. IS256 insertion in the mprF promoter region resulted in mprF overexpression. Two novel point mutations in mprF and walK, leading to amino acid substitutions in MprF (G299V and L473I) and WalK (L7Q and Y225N), were shown by allelic replacement experiments to increase the minimum inhibitory concentration (MIC) of daptomycin by 2-4 times. Allelic replacement of both mprF and walK in strain 3d0 increased the daptomycin MIC by 4-8-fold, indicating that mprF and walK mutations synergistically contribute to daptomycin non-susceptibility. Notably, these mutants acquired resistance without losing fitness and exhibited decreased expression of cell wall degradation-related genes. In conclusion, this study revealed novel mutations of MRSA daptomycin resistance acquisition in vitro as well as several novel mutations in walK and mprF, and includes the first in-depth analysis of the mprF promoter. This study sheds light on how MRSA may acquire daptomycin resistance during daptomycin treatment.

IS256-Mediated Overexpression of the WalKR Two-Component System Regulon Contributes to Reduced Vancomycin Susceptibility in a Staphylococcus aureus Clinical Isolate.

Vancomycin (VAN)-intermediate-resistant Staphylococcus aureus (VISA) is continually isolated globally, with a systematic review suggesting a prevalence of 2% in all blood culture samples. Most VISA strains exhibit common characteristics, such as a thickened cell wall, reduced autolysis, and attenuated virulence. Here, based on multi-omics approaches, we have characterized clinical VISA isolates obtained through prolonged antimicrobial treatment in a single patient. All VISA isolates were isogenic, based on multi-locus sequence typing (MLST) ST5, SCCmec type II (2A), and spa type t17639. Core-genome single nucleotide variations (SNVs) found among thirteen isolates during the patient's hospitalization, indicated clonality, but not notable genetic features of the VISA phenotype. We determined the complete genome sequence of VAN-susceptible strain KG-03 (minimum inhibitory concentration [MIC] 0.5 µg/mL) and two VISA strains, KG-18 and KG-22 (MIC 8.0 and 4.0 µg/mL, respectively). Comparative genome analysis showed remarkable strain-specific IS256 insertions. RNA-Seq transcriptome analysis revealed IS256-mediated overexpression of the walKR two-component system in VISA KG-18, possibly leading to modulation of cell wall integrity (lytM and sceD) and surface charge (mprF and dltABCD). In addition, secretome analysis indicated that cell wall-anchored proteins (Protein A, SasG, and SdrD) were significantly decreased. KG-18 and KG-22 exhibit thickened cell wall, and are relatively resistant to lysostaphin, which cleaves a staphylococcus-unique pentaglycine chain in the peptidoglycan. We conclude that KG-18 achieved reduced susceptibility to VAN by IS256-mediated WalKR overexpression, leading to a markedly thickened cell wall for trapping free VAN molecules with redundant D-Ala-D-Ala targets. In addition, a positively charged surface with lysyl-phosphatidylglycerol and depolarization of wall teichoic acid could contribute to inhibiting cationic daptomycin and VAN antimicrobial activity. Comparative omics approaches in this study strongly suggest that fully complete and annotated genome sequences will be indispensable for characterizing overall VISA phenotype.

Invasive Methicillin-Resistant Staphylococcus aureus USA500 Strains from the U.S. Emerging Infections Program Constitute Three Geographically Distinct Lineages.

USA500 isolates are clonal complex 8 (CC8) Staphylococcus aureus strains closely related to the prominent community- and hospital-associated USA300 group. Despite being relatively understudied, USA500 strains cause a significant burden of disease and are the third most common methicillin-resistant S. aureus (MRSA) strains identified in the U.S. Emerging Infections Program (EIP) invasive S. aureus surveillance. To better understand the genetic relationships of the strains, we sequenced the genomes of 539 USA500 MRSA isolates from sterile site infections collected through the EIP between 2005 and 2013 in the United States. USA500 isolates fell into three major clades principally separated by their distribution across different U.S. regions. Clade C1 strains, found principally in the Northeast, were associated with multiple IS256 insertion elements in their genomes and higher levels of antibiotic resistance. C2 was associated with Southern states, and E1 was associated with Western states. C1 and C2 strains all shared a frameshift in the gene encoding AdsA surface-attached surface protein. We propose that the term "USA500" should be used for CC8 strains sharing a recent common ancestor with the C1, C2, and E1 strains but not in the USA300 group.IMPORTANCE In this work, we have removed some of the confusion surrounding the use of the name "USA500," placed USA500 strains in the context of the CC8 group, and developed a strategy for assignment to subclades based on genome sequence. Our new phylogeny of USA300/USA500 will be a reference point for understanding the genetic adaptations that have allowed multiple highly virulent clonal strains to emerge from within CC8 over the past 50 years.

Role of insertion sequence element is256 as a virulence marker and its association with biofilm formation among methicillin-resistant Staphylococcus epidermidis from hospital and community settings in Chennai, South India.

The objective of this study was to detect the association of biofilm formation with IS256 among clinical and carrier isolates of methicillin-resistant Staphylococcus epidermidis (MRSE). A total of 71 MRSE isolates were included in this study. Phenotypic detection of biofilm formation was done by Congo red agar method. Detection of genes associated with biofilm formation (icaAD, aap and atlE) and insertion sequence IS256 was done by polymerase chain reaction. Of the 71 MRSE isolates,19/40 (47.5%) clinical isolates from hospital settings and 11/31 (35.5%) carrier isolates from community settings respectively were found to be positive for all the three genes tested, namely, icaAD+, aap+ and atlE+ genes. Nearly 80% of clinical isolates were found to harbour IS256, whereas only 13% of community isolates harboured IS256.

Increase in IS256 transposition in invasive vancomycin heteroresistant Staphylococcus aureus isolate belonging to ST100 and its derived VISA mutants.

In Staphylococcus aureus, transposition of IS256 has been described to play an important role in biofilm formation and antibiotic resistance. This study describes the molecular characterization of two clinical heterogeneous vancomycin-intermediate S. aureus (hVISA) isolates recovered from the same patient (before and after antibiotic treatment) and two VISA derivatives obtained by serial passages in the presence of vancomycin. Our results showed that antibiotic treatment (in vivo and in vitro) could enhance IS256 transposition, being responsible for the eventual loss of agr function. As far as we know this is the first study that reports the increase of IS256 transposition in isogenic strains after antibiotic treatment in a clinical setting.

Studies on coexistence of mec gene, IS256 and novel sasX gene among human clinical coagulase-negative staphylococci.

Methicillin-resistant coagulase-negative staphylococci (MRCoNS) are major nosocomial pathogens which cause device-related infections. Presence of various virulence factors along with methicillin resistance favor improved CoNS pathogenicity and their dissemination among population. In the present study, mec gene prevalence was analyzed along with SCC mec diversity among 55 human clinical CoNS isolates. PCR screening of insertion sequence (IS256) was also conducted to elucidate their association with methicillin resistance among selected CoNS species. In addition to this, PCR screening and sequence analysis of novel colonization-associated gene sasX was also carried out. High incidences of mec gene, IS256 and their association have been noted among all of the CoNS species tested. Interestingly, eight CoNS isolates were found to harbor sasX gene including S. epidermidis, S. hemolyticus and S. saprophtyicus species. Remarkably they were also found to have the coexistence of mec gene and IS256 in their genome. Increased SCC mec diversity with non-typeable elements was also observed among CoNS isolates. Presence of sasX gene in CoNS with mec gene and insertion sequence 256 and also the identification of non-typeable SCC mec element make the study novel and interesting.

Complete Genome Sequence of the MRSA Isolate HC1335 from ST239 Lineage Displaying a Truncated AgrC Histidine Kinase Receptor.

Methicillin-resistant Staphylococcus aureus (MRSA) is still one of the most important hospital pathogen globally. The multiresistant isolates of the ST239-SCCmecIII lineage are spread over large geographic regions, colonizing and infecting hospital patients in virtually all continents. The balance between fitness (adaptability) and virulence potential is likely to represent an important issue in the clonal shift dynamics leading the success of some specific MRSA clones over another. The accessory gene regulator (agr) is the master quorum sensing system of staphylococci playing a role in the global regulation of key virulence factors. Consequently, agr inactivation in S. aureus may represent a significant mechanism of genetic variability in the adaptation of this healthcare-associated pathogen. We report here the complete genome sequence of the methicillin-resistant S. aureus, isolate HC1335, a variant of the ST239 lineage, which presents a natural insertion of an IS256 transposase element in the agrC gene encoding AgrC histidine kinase receptor.

Biofilm formation of ica operon-positive Staphylococcus epidermidis from different sources.

Information on the prevalence of biofilm-related factors (PIA, Bhp, Aap, Embp) in Staphylococcus epidermidis of animal origin is scarce. In this study, 263 S. epidermidis isolates of diverse origin (animal, farmers, patients, and laboratory staff) were investigated for the presence of the ica operon (icaRADBC). The icaRADBC-positive isolates were further characterized by means of biofilm formation, presence of other biofilm-related genes, antimicrobial resistance, and population structure. Of all isolates, 28.5% (n = 75) were icaRADBC-positive, including 16.5% of animal origin, 29.1% farmer isolates, and 44.6% hospital-associated isolates (including patients and laboratory staff isolates). Most icaRADBC-positive isolates carried embp (n = 73), aap (n = 57), bhp (n = 22), and IS256 (n = 29). Statistical differences were found between animal and patient isolates for the presence of icaRADBC, bhp, and aap. No statistically significant relation was found between the presence of one or more genes and the level of biofilm formation. Most icaRADBC-positive isolates belonged to the clonal complex 5 (formerly 2) and most sequence types corresponded to types previously observed in community and nosocomial S. epidermidis populations. Although the prevalence of S. epidermidis in the nasal cavity of bovines and poultry is low, some isolates belong to STs related to ica-positive clinical strains.

Genetic determinants and biofilm formation of clinical Staphylococcus epidermidis isolates from blood cultures and indwelling devises.

For a long time, Staphylococcus epidermidis, as a member of the coagulase-negative staphylococci, was considered as part of the physiological skin flora of the human being with no pathogenic significance. Today, we know that S. epidermidis is one of the most prevalent causes for implant-associated and nosocomial infections. We performed pheno- and genotypic analysis (ica, IS256, SCCmec types, agr groups) of biofilm formation in 200 isolates. Fifty percent were genetically ica-positive and produced biofilm. Among all studied isolates, agr II and III and SCCmec type I were the most prevalent, whereas within the selected multi-resistant isolates (29%), agr I and III and SCCmec type II dominated. SCCmec type I and mecA-negative S. epidermidis isolates were associated with agr II. The majority of the blood culture and biopsy isolates were assigned to agr III and SCCmec type I, whereas agr II was predominantly detected in mecA-negative S. epidermidis isolated from catheter and implant materials. MLST analysis revealed the major clonal lineages of ST2, ST5, ST10, and ST242 (total 13 STs). ST2 isolates from blood cultures were icaA/D-positive and harbored SCCmec types II and III and IS256, whereas the icaA/D- and IS256-positive ST23 isolates were assigned to SCCmec types I and IV.

IS-mediated loss of virulence by Aeromonas salmonicida: A tangible piece of an evolutionary puzzle.

Insertion sequences (IS) are abundant in the bacterial fish pathogen Aeromonas salmonicida genome. IS are involved in rearrangement events that lead to the loss of virulence. In previous work, we studied a plasmid rearrangement that causes the deletion of the type three secretion system in A. salmonicida, resulting in a loss of virulence. We showed that the rearrangement is caused by the recombination of two IS (ISAS11) on an unstable plasmid (pAsa5). However, many rearrangements cannot be explained by our experimental approach and are thought to be the result of more complex or incomplete rearrangement events, as suggested by other plasmid loss profiles observed in various A. salmonicida strains. In this commentary, we examine the genetic instability of A. salmonicida indicating that its genome is rapidly evolving.