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1.  Biochemical and Computational Analysis of the Substrate Specificities of Cfr and RlmN Methyltransferases 
PLoS ONE  2015;10(12):e0145655.
Cfr and RlmN methyltransferases both modify adenine 2503 in 23S rRNA (Escherichia coli numbering). RlmN methylates position C2 of adenine while Cfr methylates position C8, and to a lesser extent C2, conferring antibiotic resistance to peptidyl transferase inhibitors. Cfr and RlmN show high sequence homology and may be evolutionarily linked to a common ancestor. To explore their individual specificity and similarity we performed two sets of experiments. We created a homology model of Cfr and explored the C2/C8 specificity using docking and binding energy calculations on the Cfr homology model and an X-ray structure of RlmN. We used a trinucleotide as target sequence and assessed its positioning at the active site for methylation. The calculations are in accordance with different poses of the trinucleotide in the two enzymes indicating major evolutionary changes to shift the C2/C8 specificities. To explore interchangeability between Cfr and RlmN we constructed various combinations of their genes. The function of the mixed genes was investigated by RNA primer extension analysis to reveal methylation at 23S rRNA position A2503 and by MIC analysis to reveal antibiotic resistance. The catalytic site is expected to be responsible for the C2/C8 specificity and most of the combinations involve interchanging segments at this site. Almost all replacements showed no function in the primer extension assay, apart from a few that had a weak effect. Thus Cfr and RlmN appear to be much less similar than expected from their sequence similarity and common target.
doi:10.1371/journal.pone.0145655
PMCID: PMC4689488  PMID: 26700482
2.  Mutations in the Bacterial Ribosomal Protein L3 and Their Association with Antibiotic Resistance 
Different groups of antibiotics bind to the peptidyl transferase center (PTC) in the large subunit of the bacterial ribosome. Resistance to these groups of antibiotics has often been linked with mutations or methylations of the 23S rRNA. In recent years, there has been a rise in the number of studies where mutations have been found in the ribosomal protein L3 in bacterial strains resistant to PTC-targeting antibiotics but there is often no evidence that these mutations actually confer antibiotic resistance. In this study, a plasmid exchange system was used to replace plasmid-carried wild-type genes with mutated L3 genes in a chromosomal L3 deletion strain. In this way, the essential L3 gene is available for the bacteria while allowing replacement of the wild type with mutated L3 genes. This enables investigation of the effect of single mutations in Escherichia coli without a wild-type L3 background. Ten plasmid-carried mutated L3 genes were constructed, and their effect on growth and antibiotic susceptibility was investigated. Additionally, computational modeling of the impact of L3 mutations in E. coli was used to assess changes in 50S structure and antibiotic binding. All mutations are placed in the loops of L3 near the PTC. Growth data show that 9 of the 10 mutations were well accepted in E. coli, although some of them came with a fitness cost. Only one of the mutants exhibited reduced susceptibility to linezolid, while five exhibited reduced susceptibility to tiamulin.
doi:10.1128/AAC.00179-15
PMCID: PMC4432171  PMID: 25845869
3.  Linezolid Dependence in Staphylococcus epidermidis Bloodstream Isolates 
Emerging Infectious Diseases  2013;19(1):129-132.
We document linezolid dependence among 5 highly linezolid-resistant (LRSE) Staphylococcus epidermidis bloodstream isolates that grew substantially faster at 32 µg/mL linezolid presence. These isolates carried the mutations T2504A and C2534T in multiple 23S rRNA copies and 2 mutations leading to relevant amino acid substitutions in L3 protein. Linezolid dependence could account for increasing LRSE emergence.
doi:10.3201/eid1901.111527
PMCID: PMC3557967  PMID: 23260390
L3 mutations; 23S rDNA mutations; growth rate; protein synthesis; ribosome; Staphylococcus epidermidis; bacteria; antibiotic; antimicrobial resistance
4.  Activity of Oxacillin versus That of Vancomycin against Oxacillin-Susceptible mecA-Positive Staphylococcus aureus Clinical Isolates Evaluated by Population Analyses, Time-Kill Assays, and a Murine Thigh Infection Model 
We compared the activity of dicloxacillin with that of vancomycin against 15 oxacillin-susceptible, methicillin-resistant Staphylococcus aureus (OS-MRSA) clinical isolates. By population analyses, we found that 6 OS-MRSA isolates were able to grow in the presence of up to 8 μg/ml dicloxacillin and 9 isolates were able to grow in 12 to >32 μg/ml dicloxacillin; all isolates grew in up to 2 μg/ml vancomycin. Both drugs exhibited similar bactericidal activities. In experimental infections, the therapeutic efficacy of dicloxacillin was significant (P < 0.05 versus untreated controls) in 10 OS-MRSA isolates and vancomycin was effective (P < 0.05) against 12 isolates; dicloxacillin had an efficacy that was comparable to that of vancomycin (P > 0.05) in 8 isolates. The favorable response to dicloxacillin treatment might suggest that antistaphylococcal penicillins could be used against OS-MRSA infections.
doi:10.1128/AAC.00103-12
PMCID: PMC3370787  PMID: 22430957
5.  Macrolide resistance determinants among Streptococcus pneumoniae isolates from carriers in Central Greece 
BMC Infectious Diseases  2012;12:255.
Background
We sought to characterize the temporal trends in nasopharyngeal carriage of macrolide-resistant pneumococci during a period with increased heptavalent pneumococcal conjugate vaccine (PCV7) coverage in Central Greece.
Methods
Streptococcus pneumoniae isolates were recovered from 2649 nasopharyngeal samples obtained from day-care center attendees in Central Greece during 2005–2009. A phenotypic and genotypic analysis of the isolates was performed, including the identification of macrolide resistance genes mef(A), subclasses mef(A) and mef(E), as well as erm(B).
Results
Of the 1105 typeable S. pneumoniae isolates, 265 (24%) were macrolide-resistant; 22% in 2005, 33.3% in 2006, 23.7% in 2007, and 20.5% in 2009 (P=0.398). Among these macrolide-resistant pneumococci, 28.5% possessed erm(B), 24.3% erm(B)+mef(E), 41.8% mef(E), and 5.3% mef(A). A mef gene as the sole resistance determinant was carried by 31% of macrolide-resistant isolates belonging to PCV7 serotypes and 75.8% of the non-PCV7 serotypes. Across the 4 annual surveillances, pneumococci carrying mef(A) gradually disappeared, whereas serotype 19F isolates carrying both erm(B) and mef(E) persisted without significant yearly fluctuations. Among isolates belonging to non-PCV7 serotypes, macrolide-resistance was observed in those of serotypes 6A, 19A, 10A, 15A, 15B/C, 35F, 35A, and 24F. In 2009, ie 5 years after the introduction of PCV7 in our country, 59% of macrolide-resistant pneumococci belonged to non-PCV7 serotypes.
Conclusions
Across the study period, the annual frequency of macrolide-resistant isolates did not change significantly, but in 2009 a marked shift to non-PCV7 serotypes occurred. Overall, more than half of the macrolide-resistant isolates possessed erm(B) either alone or in combination with mef(E). erm(B) dominated among isolates belonging to PCV7 serotypes, but not among those of non-PCV7 serotypes.
doi:10.1186/1471-2334-12-255
PMCID: PMC3484024  PMID: 23057516
7.  Hidden VIM-1 Metallo-β-Lactamase Phenotypes among Acinetobacter baumannii Clinical Isolates▿  
Journal of Clinical Microbiology  2007;46(1):346-349.
A total of 87 Acinetobacter baumannii nonrepetitive consecutive clinical isolates were tested for the presence of metallo-β-lactamases (MBLs). Results of phenotypic assays (MBL Etest, imipenem/imipenem-EDTA combined-disk test, and imipenem/EDTA double-disk synergy test) were negative in all cases, but molecular testing revealed the presence of two blaVIM-1-carrying isolates. One isolate had blaVIM-1 preceded by a weak P1 promoter, and both had inactivated P2 promoters and reduced blaVIM-1 expression, partially justifying the results revealing hidden MBL phenotypes.
doi:10.1128/JCM.01670-07
PMCID: PMC2224304  PMID: 18032624

Results 1-7 (7)