Bacteriemia caused by Actinomyces sp. (Microbacterium hydrocarbonoxydans) in a ten-year-old boy with carious dental

Cilj : Prikaz slučaja bakterijemije, uzrokovane bakterijom Actinomyces sp., kod desetogodišnjeg dječaka s karioznim zubalom. Prikaz bolesnika: Prikazali smo dječaka koji je višekratno hospitaliziran zbog ponavljajućih febriliteta i povišenih upalnih parametara. Tijekom pregleda uočeni su povećani limfni čvorovi smješteni uz stražnji rub sternokleidomastoideusa, palpatorno bezbolni i pomični. Unatrag mjesec dana, višekratno je dobivao peroralne antibiotike radi tonzilofaringitisa praćenog povišenom temperaturom. U uzorcima krvi za hemokulturu porasli su gram pozitivni štapići koji su, Api-Coryne sustavom za identifikaciju, identificirani kao Arcanobacterium haemolyticum. Zbog rijetkosti takvog izolata, soj smo poslali na dodatnu identifikaciju 16S rDNA metodom sekvencioniranja genoma kojom se izolira Actinomyces sp., a Microbacterium hydrocarbonoxydans naknadnom identifikacijom pomoću MALDI-TOF metode. Daljnjom hematološkom obradom pacijenta dokazan je Hodgkinov limfom. Zaključak: Obzirom da API sustavi identifikacije mogu biti nepouzdani, rijetke i neuobičajene izolate kod sistemskih infekcija i(li) bakterijemija potrebno je identificirati pouzdanijim i modernijim sustavima ili molekularnim metodama. Dostupnost molekularnih metoda i masene spektrometrije u rutinskoj laboratorijskoj dijagnostici omogućit će točniju i češću identifikaciju ovakvih izolata. Ako, kod sistemske aktinomikoze i(li) bakterijemije uzrokovane vrstama roda Actinomyces, nema dobrog odgovora na ciljanu terapiju, trebalo bi posumnjati na malignu bolest u pozadini.

Engineering the Enantioselectivity and Thermostability of a (+)-γ-Lactamase from Microbacterium hydrocarbonoxydans for Kinetic Resolution of Vince Lactam (2-Azabicyclo[2.2.1]hept-5-en-3-one)

ABSTRACT To produce promising biocatalysts, natural enzymes often need to be engineered to increase their catalytic performance. In this study, the enantioselectivity and thermostability of a (+)-γ-lactamase from Microbacterium hydrocarbonoxydans as the catalyst in the kinetic resolution of Vince lactam (2-azabicyclo[2.2.1]hept-5-en-3-one) were improved. Enantiomerically pure (−)-Vince lactam is the key synthon in the synthesis of antiviral drugs, such as carbovir and abacavir, which are used to fight against HIV and hepatitis B virus. The work was initialized by using the combinatorial active-site saturation test strategy to engineer the enantioselectivity of the enzyme. The approach resulted in two mutants, Val54Ser and Val54Leu, which catalyzed the hydrolysis of Vince lactam to give (−)-Vince lactam, with 99.2% (enantiomeric ratio [E] > 200) enantiomeric excess (ee) and 99.5% ee (E > 200), respectively. To improve the thermostability of the enzyme, 11 residues with high temperature factors (B-factors) calculated by B-FITTER or high root mean square fluctuation (RMSF) values from the molecular dynamics simulation were selected. Six mutants with increased thermostability were obtained. Finally, the mutants generated with improved enantioselectivity and mutants evolved for enhanced thermostability were combined. Several variants showing (+)-selectivity (E value > 200) and improved thermostability were observed. These engineered enzymes are good candidates to serve as enantioselective catalysts for the preparation of enantiomerically pure Vince lactam. IMPORTANCE Enzymatic kinetic resolution of the racemic Vince lactam using (+)-γ-lactamase is the most often utilized means of resolving the enantiomers for the preparation of carbocyclic nucleoside compounds. The efficiency of the native enzymes could be improved by using protein engineering methods, such as directed evolution and rational design. In our study, two properties (enantioselectivity and thermostability) of a γ-lactamase identified from Microbacterium hydrocarbonoxydans were tackled using a semirational design. The protein engineering was initialized by combinatorial active-site saturation test to improve the enantioselectivity. At the same time, two strategies were applied to identify mutation candidates to enhance the thermostability based on calculations from both a static (B-FITTER based on the crystal structure) and a dynamic (root mean square fluctuation [RMSF] values based on molecular dynamics simulations) way. After combining the mutants, we successfully obtained the final mutants showing better properties in both properties. The engineered (+)-lactamase could be a candidate for the preparation of (−)-Vince lactam.