Clonal groups of high-level gentamicin-resistant Enterococcus faecium isolated from municipal wastewater and clinical samples in Tehran, Iran

Aims:  Clonality among high-level gentamicin-resistant Enterococcus faecium (HLGR-EF) isolates obtained from clinical and sewage treatment plants (STP) were investigated using PhePlate system (PhP), ribotyping and pulsed-field gel electrophoresis (PFGE).
Methods and Results:  During 1 year study (September 2005–2006), a total of 106 HLGR-EF isolates were collected from clinical ( n  = 48) and STP ( n  = 58) samples in Tehran, Iran. Biochemical fingerprinting of these isolates using the PhP showed the presence of 21 PhP types (diversity index, Di  = 0·97) among the clinical and 21 PhP types ( Di  = 0·91) among the STP isolates. Representative isolates of each PhP type ( n  = 42) were further characterized by the ribotyping method. Sixteen ribotypes were identified among the isolates with five types shared between the clinical and STP isolates. PFGE recognized 24 clonal types among these isolates with three pulsotypes shared between the clinical and STP isolates. Combination of the two techniques (PFGE and ribotyping) resulted in 24 ( Di  = 0·96) and 16 ( Di  = 0·93) types among the strains isolated from clinical and STP samples, respectively.
Conclusions:  We concluded that the combination of PhP typing, ribotyping and PFGE could be extremely discriminatory when examining HLGR-EF isolates.
Significance and Impact of the Study:  The emergence of highly diverse HLGR-EF population in Iran is of serious concern especially because of their multi-resistances.     

MITOL-dependent ubiquitylation negatively regulates the entry of PolγA into mitochondria

Mutations in mitochondrial replicative polymerase PolγA lead to progressive external ophthalmoplegia (PEO). While PolγA is the known central player in mitochondrial DNA (mtDNA) replication, it is unknown whether a regulatory process exists on the mitochondrial outer membrane which controlled its entry into the mitochondria. We now demonstrate that PolγA is ubiquitylated by mitochondrial E3 ligase, MITOL (or MARCH5, RNF153). Ubiquitylation in wild-type (WT) PolγA occurs at Lysine 1060 residue via K6 linkage. Ubiquitylation of PolγA negatively regulates its binding to Tom20 and thereby its mitochondrial entry. While screening different PEO patients for mitochondrial entry, we found that a subset of the PolγA mutants is hyperubiquitylated by MITOL and interact less with Tom20. These PolγA variants cannot enter into mitochondria, instead becomes enriched in the insoluble fraction and undergo enhanced degradation. Hence, mtDNA replication, as observed via BrdU incorporation into the mtDNA, was compromised in these PEO mutants. However, by manipulating their ubiquitylation status by 2 independent techniques, these PEO mutants were reactivated, which allowed the incorporation of BrdU into mtDNA. Thus, regulated entry of non-ubiquitylated PolγA may have beneficial consequences for certain PEO patients.

This study shows that mitochondrial entry of the replicative polymerase PolgA is regulated by ubiquitylation by the E3 ligase MITOL; however, by manipulating their ubiquitylation status, some progressive external ophthalmoplegia mutants whose PolgA is polyubiquitylated and cannot enter the mitochondrion can be reactivated and hence become functionally active.