Rapid Microarray-Based Identification of DifferentmecAAlleles in Staphylococci

ABSTRACTTo screen isolates and to identifymecAalleles, publishedmecAsequences were analyzed, and a microarray for the rapid discrimination ofmecAalleles was designed. A GenBank analysis yielded 135 full-length gene sequences annotated asmecA. These sequences clustered into 32 different alleles corresponding to 28 unique amino acid sequences and to 15 distinct hybridization patterns on this microarray. A collection of 78 clinical and veterinary isolates ofStaphylococcusspp. was characterized using this assay. Nine of the 15 expected patterns, as well as one as-yet-unknown pattern, were identified. These patterns were detected in various epidemic methicillin-resistantStaphylococcus aureusstrains, inS. pseudintermedius, and in coagulase-negative species such asS. epidermidis,S. fleurettii, orS. haemolyticus. There was no correlation between the differentmecAhybridization patterns and the SCCmectype. Determination of MICs showed thatmecAalleles corresponding to only four of these nine patterns were associated with β-lactam resistance. ThemecAalleles that did not confer β-lactam resistance were largely restricted to coagulase-negative staphylococci of animal origin, such asS. sciuriandS. vitulinus. Because of the diversity of sequences and the different impact on β-lactam susceptibility, the existence of differentmecAalleles needs to be taken into account when designing diagnostic assays for the detection ofmecA.

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Activity of Debio1452, a FabI Inhibitor with Potent Activity against Staphylococcus aureus and Coagulase-Negative Staphylococcus spp., Including Multidrug-Resistant Strains

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05119-14 ◽

2015 ◽

Vol 59 (5) ◽

pp. 2583-2587 ◽

Cited By ~ 20

Author(s):

Robert K. Flamm ◽

Paul R. Rhomberg ◽

Nachum Kaplan ◽

Ronald N. Jones ◽

David J. Farrell

Keyword(s):

Staphylococcus Aureus ◽

Antimicrobial Agents ◽

Fatty Acid Biosynthesis ◽

Multidrug Resistant ◽

Coagulase Negative Staphylococcus ◽

Significant Activity ◽

Coagulase Negative Staphylococci ◽

Content Type ◽

Mrsa Strains ◽

Human Infections

ABSTRACTStaphylococcus aureusand coagulase-negative staphylococci (CoNS) are responsible for a wide variety of human infections. The investigational antibacterial Debio1450 (previously AFN-1720), a prodrug of Debio1452 (previously AFN-1252), specifically targets staphylococci without significant activity against other Gram-positive or Gram-negative species. Debio1452 inhibits FabI, an enzyme critical to fatty acid biosynthesis in staphylococci. The activity of Debio1452 against CoNS, methicillin-susceptibleS. aureus(MSSA), and methicillin-resistantS. aureus(MRSA), including significant clones, was determined. A globally diverse collection of 574 patient isolates from 35 countries was tested that included CoNS (6 species, 103 strains), MSSA (154 strains), MRSA (163 strains), and molecularly characterized strains (includingspa-typed MRSA clones; 154 strains). The isolates were tested for susceptibility by CLSI broth microdilution methods against Debio1452 and 10 comparators. The susceptibility rates for the comparators were determined using CLSI and EUCAST breakpoint criteria. AllS. aureusand CoNS strains were inhibited by Debio1452 concentrations of ≤0.12 and ≤0.5 μg/ml, respectively. The MIC50s for MSSA, MRSA, and molecularly characterized MRSA strains were 0.004 μg/ml, and the MIC90s ranged from 0.008 to 0.03 μg/ml. The MICs were higher for the CoNS isolates (MIC50/90, 0.015/0.12 μg/ml). AmongS. aureusstrains, resistance was common for erythromycin (61.6%), levofloxacin (49.0%), clindamycin (27.6%), tetracycline (15.7%), and trimethoprim-sulfamethoxazole (7.0%). Debio1452 demonstrated potent activity against MSSA, MRSA, and CoNS. Debio1452 showed significantly greater activity overall (MIC50, 0.004 μg/ml) than the other agents tested against these staphylococcal species, which included dominant MRSA clones and strains resistant to currently utilized antimicrobial agents.

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Biosynthesis of the Unique Wall Teichoic Acid of Staphylococcus aureus Lineage ST395

mBio ◽

10.1128/mbio.00869-14 ◽

2014 ◽

Vol 5 (2) ◽

Cited By ~ 25

Author(s):

Volker Winstel ◽

Patricia Sanchez-Carballo ◽

Otto Holst ◽

Guoqing Xia ◽

Andreas Peschel

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Gene Transfer ◽

Horizontal Gene Transfer ◽

Biosynthesis Pathway ◽

Glycerol Phosphate ◽

Coagulase Negative Staphylococci ◽

Content Type ◽

Gram Positive Bacteria ◽

Wall Teichoic Acids

ABSTRACT The major clonal lineages of the human pathogen Staphylococcus aureus produce cell wall-anchored anionic poly-ribitol-phosphate (RboP) wall teichoic acids (WTA) substituted with d-Alanine and N-acetyl-d-glucosamine. The phylogenetically isolated S. aureus ST395 lineage has recently been found to produce a unique poly-glycerol-phosphate (GroP) WTA glycosylated with N-acetyl-d-galactosamine (GalNAc). ST395 clones bear putative WTA biosynthesis genes on a novel genetic element probably acquired from coagulase-negative staphylococci (CoNS). We elucidated the ST395 WTA biosynthesis pathway and identified three novel WTA biosynthetic genes, including those encoding an α-O-GalNAc transferase TagN, a nucleotide sugar epimerase TagV probably required for generation of the activated sugar donor substrate for TagN, and an unusually short GroP WTA polymerase TagF. By using a panel of mutants derived from ST395, the GalNAc residues carried by GroP WTA were found to be required for infection by the ST395-specific bacteriophage Φ187 and to play a crucial role in horizontal gene transfer of S. aureus pathogenicity islands (SaPIs). Notably, ectopic expression of ST395 WTA biosynthesis genes rendered normal S. aureus susceptible to Φ187 and enabled Φ187-mediated SaPI transfer from ST395 to regular S. aureus. We provide evidence that exchange of WTA genes and their combination in variable, mosaic-like gene clusters have shaped the evolution of staphylococci and their capacities to undergo horizontal gene transfer events. IMPORTANCE The structural highly diverse wall teichoic acids (WTA) are cell wall-anchored glycopolymers produced by most Gram-positive bacteria. While most of the dominant Staphylococcus aureus lineages produce poly-ribitol-phosphate WTA, the recently described ST395 lineage produces a distinct poly-glycerol-phosphate WTA type resembling the WTA backbone of coagulase-negative staphylococci (CoNS). Here, we analyzed the ST395 WTA biosynthesis pathway and found new types of WTA biosynthesis genes along with an evolutionary link between ST395 and CoNS, from which the ST395 WTA genes probably originate. The elucidation of ST395 WTA biosynthesis will help to understand how Gram-positive bacteria produce highly variable WTA types and elucidate functional consequences of WTA variation.

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Novel Type XII Staphylococcal Cassette ChromosomemecHarboring a New Cassette Chromosome Recombinase, CcrC2

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01692-15 ◽

2015 ◽

Vol 59 (12) ◽

pp. 7597-7601 ◽

Cited By ~ 58

Author(s):

Zhaowei Wu ◽

Fan Li ◽

Dongliang Liu ◽

Huping Xue ◽

Xin Zhao

Keyword(s):

United States ◽

Staphylococcus Aureus ◽

Methicillin Resistance ◽

The United States ◽

Gene Complex ◽

Coagulase Negative Staphylococci ◽

Typing Method ◽

Content Type ◽

Staphylococcal Cassette Chromosome ◽

Sequence Identities

ABSTRACTExcision and integration of staphylococcal cassette chromosomemec(SCCmec) are mediated by cassette chromosome recombinases (Ccr), which play a crucial role in the worldwide spread of methicillin resistance in staphylococci. We report a novelccrgene,ccrC2, in the SCCmecof aStaphylococcus aureusisolate, BA01611, which showed 62.6% to 69.4% sequence identities to all publishedccrC1sequences. A further survey found that theccrC2gene was mainly located among coagulase-negative staphylococci (CoNS) and could be found in staphylococcal isolates from China, the United States, France, and Germany. Theccrgene complex harboring theccrC2gene was designated a type 9 complex, and the SCCmecof BA01611 was considered a novel type and was designated type XII (9C2). This novel SCCmecelement in BA01611 was flanked by a pseudo-SCC element (ΨSCCBA01611) carrying a truncatedccrA1gene. Both individual SCC elements and a composite SCC were excised from the chromosome based on detection of extrachromosomal circular intermediates. We advocate inclusion of the ccrC2gene and type 9ccrgene complex during revision of the SCCmectyping method.

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In VitroActivity against Staphylococcus aureus of a Novel Antimicrobial Agent, PRF-119, a Recombinant Chimeric Bacteriophage Endolysin

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00217-11 ◽

2011 ◽

Vol 55 (9) ◽

pp. 4416-4419 ◽

Cited By ~ 29

Author(s):

Evgeny A. Idelevich ◽

Christof von Eiff ◽

Alexander W. Friedrich ◽

Domenico Iannelli ◽

Guoqing Xia ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Antimicrobial Agent ◽

The Novel ◽

Good Activity ◽

Coagulase Negative Staphylococci ◽

Methicillin Resistant ◽

Content Type ◽

Microdilution Method ◽

Antistaphylococcal Activity ◽

Bacteriophage Endolysin

ABSTRACTAntistaphylococcal activity of the novel chimeric endolysin PRF-119 was evaluated with the microdilution method. The MIC50and MIC90of 398 methicillin-susceptibleStaphylococcus aureusisolates were 0.098 μg/ml and 0.391 μg/ml, respectively (range, 0.024 to 0.780 μg/ml). Both the MIC50and MIC90values of 776 methicillin-resistantS. aureusisolates were 0.391 μg/ml (range, 0.024 to 1.563 μg/ml). All 192 clinical isolates of coagulase-negative staphylococci exhibited MIC values of >50 μg/ml. In conclusion, PRF-119 exhibited very good activity specifically againstS. aureus.

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Expansion of a Plasmid Classification System for Gram-Positive Bacteria and Determination of the Diversity of Plasmids in Staphylococcus aureus Strains of Human, Animal, and Food Origins

Applied and Environmental Microbiology ◽

10.1128/aem.00870-12 ◽

2012 ◽

Vol 78 (16) ◽

pp. 5948-5955 ◽

Cited By ~ 29

Author(s):

Carmen Lozano ◽

Lourdes García-Migura ◽

Carmen Aspiroz ◽

Myriam Zarazaga ◽

Carmen Torres ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Classification System ◽

Gel Electrophoresis ◽

Pulsed Field Gel Electrophoresis ◽

Content Type ◽

Plasmid Content ◽

Gram Positive Bacteria ◽

Human Animal ◽

Different Origins

ABSTRACTAn expansion of a previously described plasmid classification was performed and used to reveal the plasmid content of a collection of 92Staphylococcus aureusstrains of different origins.repgenes of other genera were detected inStaphylococcus. S1 pulsed-field gel electrophoresis (PFGE) hybridizations were performed with 18 representativeS. aureusstrains, and a high number of plasmids of different sizes and organizations were detected.

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Dissemination of the Samecfr-Carrying Plasmid among Methicillin-Resistant Staphylococcus aureus and Coagulase-Negative Staphylococcal Isolates in China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04580-14 ◽

2015 ◽

Vol 59 (6) ◽

pp. 3669-3671 ◽

Cited By ~ 17

Author(s):

Jia Chang Cai ◽

Yan Yan Hu ◽

Hong Wei Zhou ◽

Gong-Xiang Chen ◽

Rong Zhang

Keyword(s):

Staphylococcus Aureus ◽

Methicillin Resistant Staphylococcus Aureus ◽

Mobile Element ◽

Sequence Type ◽

Coagulase Negative Staphylococci ◽

Methicillin Resistant ◽

Content Type ◽

Staphylococcal Species ◽

Complete Sequencing ◽

Staphylococcal Cassette Chromosome

ABSTRACTSixcfr-harboring methicillin-resistantStaphylococcus aureus(MRSA) isolates, which belonged to the same clone of sequence type 5 (ST5)-staphylococcal cassette chromosomemecelement II (SCCmecII)-spat311, were investigated in this study. Complete sequencing of acfr-carrying plasmid, pLRSA417, revealed an 8,487-bp fragment containing a Tn4001-like transposon,cfr,orf1, and ISEnfa4. This segment, first identified in an animal plasmid, pSS-01, was observed in several plasmids from clinical coagulase-negative staphylococci in China, suggesting that thecfrgene, which might originate from livestock, was located in the same mobile element and disseminated among different clinical staphylococcal species.

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Evolution and Single-Nucleotide Polymorphisms in Methicillin-Resistant Staphylococcus aureus Strains with Reduced Susceptibility to Vancomycin and Daptomycin, Based on Determination of the Complete Genome

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05159-14 ◽

2015 ◽

Vol 59 (6) ◽

pp. 3585-3587 ◽

Cited By ~ 10

Author(s):

Tetsuo Yamaguchi ◽

Shingo Suzuki ◽

Sakiko Okamura ◽

Yuri Miura ◽

Ayaka Tsukimori ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Single Nucleotide Polymorphisms ◽

Complete Genome ◽

Nucleotide Polymorphisms ◽

Single Nucleotide ◽

Methicillin Resistant ◽

Content Type ◽

Rifampin Resistance ◽

Generation Sequencing

ABSTRACTWe obtained a series of methicillin-resistantStaphylococcus aureusisolates, including both daptomycin-susceptible strain TD1 and daptomycin-resistant strain TD4, from a patient. We determined the complete genome sequences of TD1 and TD4 using next-generation sequencing, and only four single-nucleotide polymorphisms (SNPs) were identified, one each incapB(E58K),rpoB(H481Y),lytN(I16V), andmprF(V351E). We determined that these four SNPs were sufficient to cause the strains to develop daptomycin, vancomycin, and rifampin resistance.

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Heparin Mimics Extracellular DNA in Binding to Cell Surface-Localized Proteins and Promoting Staphylococcus aureus Biofilm Formation

mSphere ◽

10.1128/msphere.00135-17 ◽

2017 ◽

Vol 2 (3) ◽

Cited By ~ 15

Author(s):

Surabhi Mishra ◽

Alexander R. Horswill

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Binding Proteins ◽

Extracellular Dna ◽

Enhancement Effect ◽

Heparin Binding ◽

Coagulase Negative Staphylococci ◽

Content Type ◽

Catheter Lock ◽

Main Component

ABSTRACT Staphylococcus aureus and coagulase-negative staphylococci (CoNS) are the leading causes of catheter implant infections. Identifying the factors that stimulate catheter infection and the mechanism involved is important for preventing such infections. Heparin, the main component of catheter lock solutions, has been shown previously to stimulate S. aureus biofilm formation through an unknown pathway. This work identifies multiple heparin-binding proteins in S. aureus, and it reveals a potential mechanism through which heparin enhances biofilm capacity. Understanding the details of the heparin enhancement effect could guide future use of appropriate lock solutions for catheter implants. Staphylococcus aureus is a leading cause of catheter-related bloodstream infections. Biofilms form on these implants and are held together by a matrix composed of proteins, polysaccharides, and extracellular DNA (eDNA). Heparin is a sulfated glycosaminoglycan that is routinely used in central venous catheters to prevent thrombosis, but it has been shown to stimulate S. aureus biofilm formation through an unknown mechanism. Data presented here reveal that heparin enhances biofilm capacity in many S. aureus and coagulase-negative staphylococcal strains, and it is incorporated into the USA300 methicillin-resistant S. aureus (MRSA) biofilm matrix. The S. aureus USA300 biofilms containing heparin are sensitive to proteinase K treatment, which suggests that proteins have an important structural role during heparin incorporation. Multiple heparin-binding proteins were identified by proteomics of the secreted and cell wall fractions. Proteins known to contribute to biofilm were identified, and some proteins were reported to have the ability to bind eDNA, such as the major autolysin (Atl) and the immunodominant surface protein B (IsaB). Mutants defective in IsaB showed a moderate decrease in biofilm capacity in the presence of heparin. Our findings suggested that heparin is substituting for eDNA during S. aureus biofilm development. To test this model, eDNA content was increased in biofilms through inactivation of nuclease activity, and the heparin enhancement effect was attenuated. Collectively, these data support the hypothesis that S. aureus can incorporate heparin into the matrix and enhance biofilm capacity by taking advantage of existing eDNA-binding proteins. IMPORTANCE Staphylococcus aureus and coagulase-negative staphylococci (CoNS) are the leading causes of catheter implant infections. Identifying the factors that stimulate catheter infection and the mechanism involved is important for preventing such infections. Heparin, the main component of catheter lock solutions, has been shown previously to stimulate S. aureus biofilm formation through an unknown pathway. This work identifies multiple heparin-binding proteins in S. aureus, and it reveals a potential mechanism through which heparin enhances biofilm capacity. Understanding the details of the heparin enhancement effect could guide future use of appropriate lock solutions for catheter implants.

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Linezolid Surveillance Results for the United States (LEADER Surveillance Program 2014)

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02803-15 ◽

2016 ◽

Vol 60 (4) ◽

pp. 2273-2280 ◽

Cited By ~ 51

Author(s):

Robert K. Flamm ◽

Rodrigo E. Mendes ◽

Patricia A. Hogan ◽

Jennifer M. Streit ◽

James E. Ross ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Streptococcus Pneumoniae ◽

Ribosomal Proteins ◽

The United States ◽

23S Rrna ◽

Surveillance Program ◽

Coagulase Negative Staphylococci ◽

Content Type ◽

Mrsa Strains ◽

Viridans Group Streptococci

ABSTRACTThelinezolidexperience andaccuratedetermination ofresistance (LEADER) surveillance program has monitored linezolid activity, spectrum, and resistance since 2004. In 2014, a total of 6,865 Gram-positive pathogens from 60 medical centers from 36 states were submitted. The organism groups evaluated wereStaphylococcus aureus(3,106), coagulase-negative staphylococci (CoNS; 797), enterococci (855),Streptococcus pneumoniae(874), viridans group streptococci (359), and beta-hemolytic streptococci (874). Susceptibility testing was performed by reference broth microdilution at the monitoring laboratory. Linezolid-resistant isolates were confirmed by repeat testing. PCR and sequencing were performed to detect mutations in 23S rRNA, L3, L4, and L22 proteins and acquired genes (cfrandoptrA). The MIC50/90forStaphylococcus aureuswas 1/1 μg/ml, with 47.2% of isolates being methicillin-resistantStaphylococcus aureus. Linezolid was active against allStreptococcus pneumoniaestrains and beta-hemolytic streptococci with a MIC50/90of 1/1 μg/ml and against viridans group streptococci with a MIC50/90of 0.5/1 μg/ml. Among the linezolid-nonsusceptible MRSA strains, one strain harboredcfronly (MIC, 4 μg/ml), one harbored G2576T (MIC, 8 μg/ml), and one containedcfrand G2576T with L3 changes (MIC, ≥8 μg/ml). Among CoNS, 0.75% (six isolates) of all strains demonstrated linezolid MIC results of ≥4 μg/ml. Five of these were identified asStaphylococcus epidermidis, four of which containedcfrin addition to the presence of mutations in the ribosomal proteins L3 and L4, alone or in combination with 23S rRNA (G2576T) mutations. Six enterococci (0.7%) were linezolid nonsusceptible (≥4 μg/ml; five with G2576T mutations, including one with an additionalcfrgene, and one strain withoptrAonly). Linezolid demonstrated excellent activity and a sustained susceptibility rate of 99.78% overall.

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Characterization of a Novel Arginine Catabolic Mobile Element (ACME) and Staphylococcal Chromosomal CassettemecComposite Island with Significant Homology to Staphylococcus epidermidis ACME Type II in Methicillin-Resistant Staphylococcus aureus Genotype ST22-MRSA-IV

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01756-10 ◽

2011 ◽

Vol 55 (5) ◽

pp. 1896-1905 ◽

Cited By ~ 54

Author(s):

Anna C. Shore ◽

Angela S. Rossney ◽

Orla M. Brennan ◽

Peter M. Kinnevey ◽

Hilary Humphreys ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Staphylococcus Epidermidis ◽

Mobile Element ◽

Type I ◽

Type Ii ◽

Coagulase Negative Staphylococci ◽

Methicillin Resistant ◽

Content Type ◽

Arginine Catabolic Mobile Element

ABSTRACTThe arginine catabolic mobile element (ACME) is prevalent among methicillin-resistantStaphylococcus aureus(MRSA) isolates of sequence type 8 (ST8) and staphylococcal chromosomal cassettemec(SCCmec) type IVa (USA300) (ST8-MRSA-IVa isolates), and evidence suggests that ACME enhances the ability of ST8-MRSA-IVa to grow and survive on its host. ACME has been identified in a small number of isolates belonging to other MRSA clones but is widespread among coagulase-negative staphylococci (CoNS). This study reports the first description of ACME in two distinct strains of the pandemic ST22-MRSA-IV clone. A total of 238 MRSA isolates recovered in Ireland between 1971 and 2008 were investigated for ACME using a DNA microarray. Twenty-three isolates (9.7%) were ACME positive, and all were either MRSA genotype ST8-MRSA-IVa (7/23, 30%) or MRSA genotype ST22-MRSA-IV (16/23, 70%). Whole-genome sequencing and comprehensive molecular characterization revealed the presence of a novel 46-kb ACME and staphylococcal chromosomal cassettemec(SCCmec) composite island (ACME/SCCmec-CI) in ST22-MRSA-IVh isolates (n= 15). This ACME/SCCmec-CI consists of a 12-kb DNA region previously identified in ACME type II inS. epidermidisATCC 12228, a truncated copy of the J1 region of SCCmectype I, and a complete SCCmectype IVh element. The composite island has a novel genetic organization, with ACME located withinorfXand SCCmeclocated downstream of ACME. One PVL locus-positive ST22-MRSA-IVa isolate carried ACME located downstream of SCCmectype IVa, as previously described in ST8-MRSA-IVa. These results suggest that ACME has been acquired by ST22-MRSA-IV on two independent occasions. At least one of these instances may have involved horizontal transfer and recombination events between MRSA and CoNS. The presence of ACME may enhance dissemination of ST22-MRSA-IV, an already successful MRSA clone.

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