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1.  Novel mutations in penicillin-binding protein genes in clinical Staphylococcus aureus isolates that are methicillin resistant on susceptibility testing, but lack the mec gene 
Objectives
Methicillin-resistant Staphylococcus aureus (MRSA) is an important global health problem. MRSA resistance to β-lactam antibiotics is mediated by the mecA or mecC genes, which encode an alternative penicillin-binding protein (PBP) 2a that has a low affinity to β-lactam antibiotics. Detection of mec genes or PBP2a is regarded as the gold standard for the diagnosis of MRSA. We identified four MRSA isolates that lacked mecA or mecC genes, but were still phenotypically resistant to pencillinase-resistant β-lactam antibiotics.
Methods
The four human S. aureus isolates were investigated by whole genome sequencing and a range of phenotypic assays.
Results
We identified a number of amino acid substitutions present in the endogenous PBPs 1, 2 and 3 that were found in the resistant isolates but were absent in closely related susceptible isolates and which may be the basis of resistance. Of particular interest are three identical amino acid substitutions in PBPs 1, 2 and 3, occurring independently in isolates from at least two separate multilocus sequence types. Two different non-conservative substitutions were also present in the same amino acid of PBP1 in two isolates from two different sequence types.
Conclusions
This work suggests that phenotypically resistant MRSA could be misdiagnosed using molecular methods alone and provides evidence of alternative mechanisms for β-lactam resistance in MRSA that may need to be considered by diagnostic laboratories.
doi:10.1093/jac/dkt418
PMCID: PMC3922151  PMID: 24216768
β-lactams; MRSA; mecA; mecC
2.  A Staphylococcus xylosus Isolate with a New mecC Allotype 
Recently, a novel variant of mecA known as mecC (mecALGA251) was identified in Staphylococcus aureus isolates from both humans and animals. In this study, we identified a Staphylococcus xylosus isolate that harbors a new allotype of the mecC gene, mecC1. Whole-genome sequencing revealed that mecC1 forms part of a class E mec complex (mecI-mecR1-mecC1-blaZ) located at the orfX locus as part of a likely staphylococcal cassette chromosome mec element (SCCmec) remnant, which also contains a number of other genes present on the type XI SCCmec.
doi:10.1128/AAC.01882-12
PMCID: PMC3591899  PMID: 23274660
3.  Whole genome sequencing identifies zoonotic transmission of MRSA isolates with the novel mecA homologue mecC 
EMBO Molecular Medicine  2013;5(4):509-515.
Several methicillin-resistant Staphylococcus aureus (MRSA) lineages that carry a novel mecA homologue (mecC) have recently been described in livestock and humans. In Denmark, two independent human cases of mecC-MRSA infection have been linked to a livestock reservoir. We investigated the molecular epidemiology of the associated MRSA isolates using whole genome sequencing (WGS). Single nucleotide polymorphisms (SNP) were defined and compared to a reference genome to place the isolates into a phylogenetic context. Phylogenetic analysis revealed two distinct farm-specific clusters comprising isolates from the human case and their own livestock, whereas human and animal isolates from the same farm only differed by a small number of SNPs, which supports the likelihood of zoonotic transmission. Further analyses identified a number of genes and mutations that may be associated with host interaction and virulence. This study demonstrates that mecC-MRSA ST130 isolates are capable of transmission between animals and humans, and underscores the potential of WGS in epidemiological investigations and source tracking of bacterial infections.
doi:10.1002/emmm.201202413
PMCID: PMC3628104  PMID: 23526809
cattle; mecC; MRSA; sheep; zoonosis
4.  Genetic Variability in Beta-Defensins Is Not Associated with Susceptibility to Staphylococcus aureus Bacteremia 
PLoS ONE  2012;7(2):e32315.
Introduction
Human beta-defensins are key components of human innate immunity to a variety of pathogens, including Staphylococcus aureus. The aim of the present study was to investigate a potential association between gene variations in DEFB1 and DEFB103/DEFB4 and the development of S. aureus bacteremia (SAB) employing a case-control design.
Methods
Cases were unique patients with documented SAB, identified with the National S. aureus Bacteremia Register, a comprehensive dataset of all episodes of community associated-SABs (CA-SAB) occurring in children (≤20 yrs) in Denmark from 1990 to 2006. Controls were age-matched healthy individuals with no history of SAB. DNA obtained from cases and controls using the Danish Newborn Screening Biobank were genotyped for functional polymorphisms of DEFB1 by Sanger sequencing and copy number variation of the DEFB103 and DEFB4 genes using Pyrosequencing-based Paralogue Ratio Test (P-PRT).
Results
193 ethnic Danish SAB cases with 382 age-matched controls were used for this study. S. aureus isolates represented a variety of bacterial (i.e., different spa types) types similar to SAB isolates in general. DEFB1 minor allele frequencies of rs11362 (cases vs. controls 0.47/0.44), rs1800972 (0.21/0.24), and rs1799946 (0.32/0.33) were not significantly different in cases compared with controls. Also, DEFB4/DEFB103 gene copy numbers (means 4.83/4.92) were not significantly different in cases compared with controls.
Conclusions
Using a large, unique cohort of pediatric CA-SAB, we found no significant association between DEFB1 genetic variation or DEFB4/DEFB103 gene copy number and susceptibility for SAB.
doi:10.1371/journal.pone.0032315
PMCID: PMC3285211  PMID: 22384213
5.  Novel Types of Staphylococcal Cassette Chromosome mec Elements Identified in Clonal Complex 398 Methicillin-Resistant Staphylococcus aureus Strains▿‡ 
The structures of staphylococcal cassette chromosome mec (SCCmec) elements carried by 31 clonal complex 398 (CC398) methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from the participants at a conference were analyzed. The SCCmecs were classified into novel types, namely, IX, X, V(5C2&5) subtype c, and IVa. Type V(5C2&5) subtype c, IX, and X SCCmecs carried genes conferring resistance to metals. The structures of SCCmecs from CC398 strains were distinct from those normally found in humans, adding to the evidence that humans are not the original host for CC398.
doi:10.1128/AAC.01475-10
PMCID: PMC3101438  PMID: 21422209
6.  Molecular Epidemiology of Panton-Valentine Leukocidin-Positive Staphylococcus aureus in Spain: Emergence of the USA300 Clone in an Autochthonous Population ▿  
Journal of Clinical Microbiology  2010;49(1):433-436.
We characterized all of the Panton-Valentine leukocidin (PVL)-positive Staphylococcus aureus isolates collected between 2005 and 2008 in the Bilbao, Spain, area. For the first time, the USA300 clone is reported as predominant among PVL-positive clones in a European autochthonous population, requiring active monitoring of the incidence of USA300 in Spain and throughout Europe.
doi:10.1128/JCM.02201-10
PMCID: PMC3020469  PMID: 21068288
7.  Two Distinct Clones of Methicillin-Resistant Staphylococcus aureus (MRSA) with the Same USA300 Pulsed-Field Gel Electrophoresis Profile: a Potential Pitfall for Identification of USA300 Community-Associated MRSA▿  
Journal of Clinical Microbiology  2009;47(11):3765-3768.
Analysis of methicillin-resistant Staphylococcus aureus (MRSA) characterized as USA300 by pulsed-field gel electrophoresis identified two distinct clones. One was similar to community-associated USA300 MRSA (ST8-IVa, t008, and Panton-Valentine leukocidin positive). The second (ST8-IVa, t024, and PVL negative) had different molecular characteristics and epidemiology, suggesting independent evolution. We recommend spa typing and/or PCR to discriminate between the two clones.
doi:10.1128/JCM.00934-09
PMCID: PMC2772603  PMID: 19759225
8.  A Common Variant of Staphylococcal Cassette Chromosome mec Type IVa in Isolates from Copenhagen, Denmark, Is Not Detected by the BD GeneOhm Methicillin-Resistant Staphylococcus aureus Assay ▿  
Journal of Clinical Microbiology  2009;47(5):1524-1527.
Rapid tests for detection of methicillin-resistant Staphylococcus aureus (MRSA) carriage are important to limit the transmission of MRSA in the health care setting. We evaluated the performance of the BD GeneOhm MRSA real-time PCR assay using a diverse collection of MRSA isolates, mainly from Copenhagen, Denmark, but also including international isolates, e.g., USA100-1100. Pure cultures of 349 MRSA isolates representing variants of staphylococcal cassette chromosome mec (SCCmec) types I to V and 103 different staphylococcal protein A (spa) types were tested. In addition, 53 methicillin-susceptible Staphylococcus aureus isolates were included as negative controls. Forty-four MRSA isolates were undetectable; of these, 95% harbored SCCmec type IVa, and these included the most-common clone in Copenhagen, spa t024-sequence type 8-IVa. The false-negative MRSA isolates were tested with new primers (analyte-specific reagent [ASR] BD GeneOhm MRSA assay) supplied by Becton Dickinson (BD). The ASR BD GeneOhm MRSA assay detected 42 of the 44 isolates that were false negative in the BD GeneOhm MRSA assay. Combining the BD GeneOhm MRSA assay with the ASR BD GeneOhm MRSA assay greatly improved the results, with only two MRSA isolates being false negative. The BD GeneOhm MRSA assay alone is not adequate for MRSA detection in Copenhagen, Denmark, as more than one-third of our MRSA isolates would not be detected. We recommend that the BD GeneOhm MRSA assay be evaluated against the local MRSA diversity before being established as a standard assay, and due to the constant evolution of SCCmec cassettes, a continuous global surveillance is advisable in order to update the assay as necessary.
doi:10.1128/JCM.02153-08
PMCID: PMC2681823  PMID: 19297600
9.  Rapid Increase of Genetically Diverse Methicillin-Resistant Staphylococcus aureus, Copenhagen, Denmark 
Emerging Infectious Diseases  2007;13(10):1533-1540.
Community-onset MRSA with diverse genetic backgrounds is rapidly emerging in this previously low MRSA prevalence area.
In Copenhagen, methicillin-resistant Staphylococcus aureus (MRSA) accounted for <15 isolates per year during 1980–2002. However, since 2003 an epidemic increase has been observed, with 33 MRSA cases in 2003 and 110 in 2004. We analyzed these 143 cases epidemiologically and characterized isolates by pulsed-field gel electrophoresis, Staphylococcus protein A (spa) typing, multilocus sequence typing, staphylococcal chromosome cassette (SCC) mec typing, and detection of Panton-Valentine leukocidin (PVL) genes. Seventy-one percent of cases were community-onset MRSA (CO-MRSA); of these, 36% had no identified risk factors. We identified 29 spa types (t) and 16 sequence types (STs) belonging to 8 clonal complexes and 3 ST singletons. The most common clonal types were t024/ST8-IV, t019/ST30-IV, t044/ST80-IV, and t008/ST8-IV (USA300). A total of 86% of isolates harbored SCCmec IV, and 44% had PVL. Skin and soft tissue infections dominated. CO-MRSA with diverse genetic backgrounds is rapidly emerging in a low MRSA prevalence area.
doi:10.3201/eid1310.070503
PMCID: PMC2851516  PMID: 18258003
MRSA; Panton-Valentine leukocidin; MLST; spa typing; Staphylococcus aureus; Community-onset MRSA; SCCmec; USA300; PFGE; Nursing home; research
10.  Molecular Epidemiology and Antimicrobial Susceptibility of Clinical Staphylococcus aureus from Healthcare Institutions in Ghana 
PLoS ONE  2014;9(2):e89716.
The objective of this study was to determine the antimicrobial susceptibility patterns and clonal diversity of clinical Staphylococcus aureus isolates from Ghana. A total of 308 S. aureus isolates from six healthcare institutions located across Northern, Central and Southern Ghana were characterized by antibiotyping, spa typing and PCR detection of Panton Valentine leukocin (PVL) genes. Methicillin-resistant S. aureus (MRSA) were confirmed by PCR detection of mecA gene and further characterized by SCCmec and multi-locus sequence typing (MLST). The prevalence of antimicrobial resistance was below 5% for all agents tested except for penicillin (97%), tetracycline (42%) and erythromycin (6%). Ninety-one spa types were found, with t355 (ST152, 19%), t084 (ST15, 12%) and t314 (ST121, 6%) being the most frequent types. Based on established associations between spa and MLST types, isolates were assigned to 16 clonal complexes (CCs): CC152 (n = 78), CC15 (n = 57), CC121 (n = 39), CC8 (n = 36), CC5 (n = 33), CC1 (n = 29), CC45 (n = 9), CC88 (n = 8), CC30 (n = 4), CC9 (n = 3), CC25 (n = 2), CC97 (n = 2) CC20 (n = 2), CC707 (n = 2), CC7 (n = 3) and CC522 (n = 1). Most isolates (60%) were PVL-positive, especially those belonging to ST152, ST121, ST5, ST15, ST1, ST8, and ST88. Nine (3%) isolates were MRSA belonging to seven distinct clones: ST88-IV (n = 2), ST250-I (n = 2), ST8-IV (n = 1), ST72-V (n = 1), ST789-IV (n = 1), ST2021-V (n = 1), and ST239-III (n = 1). The study confirmed a high frequency of PVL-positive S. aureus in Africa, low prevalence of antimicrobial resistance and high diversity of MRSA lineages in Ghana compared to developed countries and other African countries. The detection of known pandemic MRSA clones in the absence of routine MRSA identification in most Ghanaian clinical microbiology laboratories calls for capacity building to strengthen surveillance and prevent spread of these clones.
doi:10.1371/journal.pone.0089716
PMCID: PMC3934920  PMID: 24586981
11.  Methicillin-Resistant Staphylococcus aureus Colonization: A Three-Year Prospective Study in a Neonatal Intensive Care Unit in Italy 
PLoS ONE  2014;9(2):e87760.
Background
Methicillin resistant Staphylococcus aureus (MRSA) is a major etiological agent of infection in neonatal intensive care units (NICUs). Routes of entry of this organism can be different and the transmission pathway complex. Colonized neonates are the main endogenous reservoir.
Methods and Results
We conducted a prospective three-year study on MRSA colonization recruiting 722 neonates admitted between 2009 and 2012. Nasal swabs were cultured weekly and MRSA isolates were submitted to molecular typing. The annual incidence density of acquisition of MRSA ranged from a maximum of 20.2 cases for 1000 patient-days during the first year to a minimum of 8.8 cases in the second one to raise again up to 13.1 cases during the third year. The mean weekly colonization pressure fluctuated from 19.1% in the first year to 13.4% in the second year and 16.8% in the third year. It significantly correlated with the number of MRSA acquisitions in the following week. Overall, 187 (25.9%) subjects tested positive for MRSA. A non multiresistant, tst positive, ST22-MRSA-IVa spa t223 strain proved to be endemic in the NICU, being identified in 166 (88.8%) out of 187 colonized neonates. Sporadic or epidemic occurrence of other strains was detected.
Conclusions
An MRSA strain belonging to the tst1 positive, UK-EMRSA-15/ “Middle Eastern Variant” appeared to be endemic in the NICU under investigation. During the three-year period, substantial changes occurred in case-mix of patients moving towards a higher susceptibility to MRSA colonization. The infection control procedures were able to decrease the colonization rate from more than 40% to approximately 10%, except for an outbreak due to a CA-MRSA strain, ST1-MRSA-IVa, and a transient increase in the colonization prevalence rate coincident with a period of substantial overcrowding of the ward. Active surveillance and molecular typing contributed to obtain a reliable picture of the MRSA dissemination in NICU.
doi:10.1371/journal.pone.0087760
PMCID: PMC3914835  PMID: 24505312
12.  Incidence and Characterisation of Methicillin-Resistant Staphylococcus aureus (MRSA) from Nasal Colonisation in Participants Attending a Cattle Veterinary Conference in the UK 
PLoS ONE  2013;8(7):e68463.
We sought to determine the prevalence of nasal colonisation with methicillin-resistant Staphylococcus aureus among cattle veterinarians in the UK. There was particular interest in examining the frequency of colonisation with MRSA harbouring mecC, as strains with this mecA homologue were originally identified in bovine milk and may represent a zoonotic risk to those in contact with dairy livestock. Three hundred and seven delegates at the British Cattle Veterinarian Association (BCVA) Congress 2011 in Southport, UK were screening for nasal colonisation with MRSA. Isolates were characterised by whole genome sequencing and antimicrobial susceptibility testing. Eight out of three hundred and seven delegates (2.6%) were positive for nasal colonisation with MRSA. All strains were positive for mecA and none possessed mecC. The time since a delegate’s last visit to a farm was significantly shorter in the MRSA-positive group than in MRSA-negative counterparts. BCVA delegates have an increased risk of MRSA colonisation compared to the general population but their frequency of colonisation is lower than that reported from other types of veterinarian conference, and from that seen in human healthcare workers. The results indicate that recent visitation to a farm is a risk factor for MRSA colonisation and that mecC-MRSA are rare among BCVA delegates (<1% based on sample size). Contact with livestock, including dairy cattle, may still be a risk factor for human colonisation with mecC-MRSA but occurs at a rate below the lower limit of detection available in this study.
doi:10.1371/journal.pone.0068463
PMCID: PMC3711812  PMID: 23869220
13.  Meticillin-resistant Staphylococcus aureus with a novel mecA homologue in human and bovine populations in the UK and Denmark: a descriptive study 
The Lancet Infectious Diseases  2011;11(8):595-603.
Summary
Background
Animals can act as a reservoir and source for the emergence of novel meticillin-resistant Staphylococcus aureus (MRSA) clones in human beings. Here, we report the discovery of a strain of S aureus (LGA251) isolated from bulk milk that was phenotypically resistant to meticillin but tested negative for the mecA gene and a preliminary investigation of the extent to which such strains are present in bovine and human populations.
Methods
Isolates of bovine MRSA were obtained from the Veterinary Laboratories Agency in the UK, and isolates of human MRSA were obtained from diagnostic or reference laboratories (two in the UK and one in Denmark). From these collections, we searched for mecA PCR-negative bovine and human S aureus isolates showing phenotypic meticillin resistance. We used whole-genome sequencing to establish the genetic basis for the observed antibiotic resistance.
Findings
A divergent mecA homologue (mecALGA251) was discovered in the LGA251 genome located in a novel staphylococcal cassette chromosome mec element, designated type-XI SCCmec. The mecALGA251 was 70% identical to S aureus mecA homologues and was initially detected in 15 S aureus isolates from dairy cattle in England. These isolates were from three different multilocus sequence type lineages (CC130, CC705, and ST425); spa type t843 (associated with CC130) was identified in 60% of bovine isolates. When human mecA-negative MRSA isolates were tested, the mecALGA251 homologue was identified in 12 of 16 isolates from Scotland, 15 of 26 from England, and 24 of 32 from Denmark. As in cows, t843 was the most common spa type detected in human beings.
Interpretation
Although routine culture and antimicrobial susceptibility testing will identify S aureus isolates with this novel mecA homologue as meticillin resistant, present confirmatory methods will not identify them as MRSA. New diagnostic guidelines for the detection of MRSA should consider the inclusion of tests for mecALGA251.
Funding
Department for Environment, Food and Rural Affairs, Higher Education Funding Council for England, Isaac Newton Trust (University of Cambridge), and the Wellcome Trust.
doi:10.1016/S1473-3099(11)70126-8
PMCID: PMC3829197  PMID: 21641281

Results 1-13 (13)