Genetičke i genomske (molekularne) dijagnostičke metode u identifikaciji uzročnika infekcija mokraćnog sustava i sastavnica urinarne mikrobiote

Standard urine culture (SUC) is a widespread diagnostic test. According to various guidelines, SUC is not a recommended first-line diagnostic tool for uncomplicated urinary tract infections (UTIs). However, its results are valuable in patients with complicated UTIs, hospitalized patients, and those who failed empirical antibiotic therapy. The emergence of antibiotic resistance has reaffirmed the urine culture as the gold standard in UTI workup. Nevertheless, turn-around-time and cost-effectiveness of SUC are the main incentives for continuous exploration of new, faster, and more sensitive procedures for evaluating the count and species of microorganisms and their susceptibility to antibiotics. Therefore, we considered it important to write a review that analyzes the advantages and disadvantages of state-of-the-art UTI diagnostics. We aim to compare standard cultivation methods with diagnostic modalities based on multiplex PCR, 16S RNA genes sequencing, and next-generation sequencing suitable for analyzing whole urinary microbiomes.Standardna urinokultura česta je dijagnostička pretraga, no prema smjernicama se ne preporučuje u obradi nekompliciraih infekcija mokraćnog sustava, već je indicirana za bolesnike s kompliciranim infekcijama mokraćnog sustava, hospitalizirane bolesnike i one kod kojih empirijska antibiotska terapija nije polučila klinički odgovor. Pojava bakterijske rezistencije na antibiotike naglašava važnost urinokulture s antibiogramom kao zlatnog standarda u obradi infekcija mokraćnog sustava. Molekularne dijagnostičke metode kao što su multipleks PCR, sekvenciranje 16S RNA gena i sekvenciranje nove generacije, prikladne su za detekciju urinarnih patogena i analizu urinarnog mikrobioma, a zbog brzine i osjetljivosti nalaze svoje mjesto u kliničkoj obradi urinarnih infekcija. Cilj ovog rada je usporediti prednosti i nedostatke suvremenih metoda dijagnosticiranja infekcija mokraćnog sustava s klasičnim kultivacijskim metodama

Treatment of soft tissue infection and osteomyelitis with multi-drug resistant Acinetobacter baumannii following a tibial fracture surgery: case report

Cilj: Cilj je prikazati kirurško i antimikrobno liječenje komplicirane kirurške infekcije uzrokovane Acinetobacter baumannii. Prikaz slučaja: 57-godišnja pacijentica bila je premještena u našu ustanovu tri tjedna nakon osteosinteze proksimalnog multifragmentarnog prijeloma tibije s postoperacijskom infekcijom kosti i mekog tkiva uzrokovanom A. baumannii. Prema nalazu iz vanjske ustanove, osim na karbapeneme, fluorokinolone i aminoglikozide, soj je bio rezistentan i na sulbaktam i kolistin. U trenutku prijema pacijentica je bila febrilna, povišenih upalnih parametara i na terapiji ciprofloksacinom i rifampicinom. U našoj ustanovi ponovno su uzeti mikrobiološki uzorci iz kojih je bio izoliran multirezistentan A. baumannii, ali osjetljiv na sulbaktam i kolistin. Uz nekrektomiju mekog tkiva i sekvestrektomiju zahvaćenog dijela kosti te rekonstrukcijsko kirurško liječenje, provedena je i kombinirana terapija kolistinom i fosfomicinom tijekom 14 dana. Nakon 23 dana hospitalizacije pacijentica je dobrog općeg stanja i lokalnog nalaza otpuštena na fizikalnu terapiju. Zaključak: A. baumannii uzrokuje manje od 3% kirurških infekcija, a osobito su rizični bolesnici s prethodnim ozljedama kosti i mekog tkiva te prethodno provedenom antibiotskom terapijom. Kolistin je terapija izbora za liječenje infekcija uzrokovanih multirezistentnim A. baumannii. Iako je A. baumannii intrinzično rezistentan na fosfomicin, smatra se kako se u kombinaciji s kolistinom postiže sinergističko djelovanje. Budući da je fosfomicin mala molekula, dobro prodire i u biofilm. Kod naše pacijentice kombinacijom antimikrobne terapije i kirurškog liječenja postigao se odličan klinički ishod.Aim: The aim was to present surgical and antimicrobial treatment of complicated surgical site infection (SSI) caused by A. baumannii. Case report: 57-year-old female patient was admitted 3 weeks after plate osteosynthesis for proximal tibial multifragmented fracture. The surgery was performed in another institution. She presented with SSI and osteomyelitis due to A. baumannii. According to microbiology results from another institution, the strain was resistant to carbapenems, fluoroquinolones, aminoglycosides, and to sulbactam and colistin. At presentation she was already being treated with ciprofloxacin and rifampicin but was nevertheless febrile with elevated inflammation markers. New sets of microbiology samples were taken, and the cultures yielded a multi-drug resistant A. baumannii, although sensitive to sulbactam and colistin. In addition to soft tissue necrectomy and sequestrectomy of devitalized bone with subsequent reconstructive surgery, the patient was treated with 14-day long parenteral combination therapy – colistin and fosfomycin. After 23-day-long hospitalization, the patient was transferred to physical rehabilitation clinic in good general health and with satisfactory wound healing. Conclusion: Acinetobacter baumannii causes less than 3% of SSI-s, and patients with previous traumatic injuries and antibiotic therapy are most at risk. Colistin is preferred therapy for multi-drug resistant A. baumannii. Although A. baumannii is intrinsically resistant to fosfomycin, it is thought that combination therapy with colistin yields synergy. Since fosfomycin is a small molecule, it penetrates biofilm well. Due to extensive surgical and antimicrobial treatment, our patient had a good clinical outcome

Direct Identification of Urinary Tract Pathogens by MALDI-TOF/TOF Analysis and De Novo Peptide Sequencing

For mass spectrometry-based diagnostics of microorganisms, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is currently routinely used to identify urinary tract pathogens. However, it requires a lengthy culture step for accurate pathogen identification, and is limited by a relatively small number of available species in peptide spectral libraries (≤3329). Here, we propose a method for pathogen identification that overcomes the above limitations, and utilizes the MALDI-TOF/TOF MS instrument. Tandem mass spectra of the analyzed peptides were obtained by chemically activated fragmentation, which allowed mass spectrometry analysis in negative and positive ion modes. Peptide sequences were elucidated de novo, and aligned with the non- redundant National Center for Biotechnology Information Reference Sequence Database (NCBInr). For data analysis, we developed a custom program package that predicted peptide sequences from the negative and positive MS/MS spectra. The main advantage of this method over a conventional MALDI-TOF MS peptide analysis is identification in less than 24 h without a cultivation step. Compared to the limited identification with peptide spectra libraries, the NCBI database derived from genome sequencing currently contains 20, 917 bacterial species, and is constantly expanding. This paper presents an accurate method that is used to identify pathogens grown on agar plates, and those isolated directly from urine samples, with high accuracy

Campylobacter fetus Bacteremia Related to Vascular Prosthesis and Pseudoaneurysm Infection: A Case Report and Review

Background. Campylobacter fetus rarely causes gastrointestinal diseases but shows an affinity for the endovascular epithelium. Methods. We describe a case of C. fetus bacteremia related to vascular prosthesis and pseudoaneurysm infection, with a review of the literature. Results. A 67-year-old male was admitted with a history of fever, weakness and painful swelling of the groin. After unsuccessful treatment with ciprofloxacin, the patient was transferred to our hospital, where he had been previously treated for aortoiliac occlusive disease including a prosthetic aortobifemoral and popliteal bypass with polyester graft placement. An angiography showed a pseudoaneurysm in the groin and, therefore, repair of the pseudoaneurysm, removal of the prosthesis and biologic graft placement were performed. Blood cultures and tissue samples of the vascular prosthesis and pseudoaneurysm yielded C. fetus resistant to ciprofloxacin. The patient was treated with meropenem for four weeks, followed by amoxicillin-clavulanate for another two weeks after discharge. Eight previously published cases of C. fetus bacteremia due to infected cardiovascular prosthetic devices (prosthetic heart valves, implantable cardioverter-defibrillators and a permanent pacemaker) were summarized in the review. Conclusions. To our knowledge, this is the first report of a C. fetus bacteremia related to post-surgical infection of a vascular prosthesis causing a pseudoaneurysm

N-Terminal chemical derivatization of peptides with 4-formyl-benzenesulfonic acid and electrospray positive and negative tandem mass spectrometry with high abundance of b-ion series

Rationale: Selective derivatization of peptide N-terminus with 4-formyl-benzenesulfonic acid (FBSA) enables chemically activated fragmentation in positive and negative ion modes (ESI+/−) under charge reduction conditions. Overlapped positive and negative tandem mass spectra show b-ions making the assignment of b-ion series fragments easy and accurate. Methods: We developed an FBSA–peptide microwave-assisted derivatization procedure. Derivatized and nonderivatized bovine serum albumin tryptic peptides and insulin non-tryptic peptide were compared after tandem mass spectrometry (MS/MS) analysis in positive and negative ion modes. A high-quality data set of sulfonated b-ions obtained in negative tandem mass spectra of singly charged FBSA–peptides were matched to detected b-ions in positive MS/MS spectra. Moreover, negative spectra signals were converted and matched against y-ions in positive tandem mass spectra to identify complete peptide sequences. Results: The FBSA derivatization procedure produced a significantly improved MS/MS data set (populated by high-intensity signals of b- and y-ions) compared to commonly used N-terminal sulfonation reagents. Undesired side reactions almost do not occur, and the procedure reduces the derivatization time. It was found that b-ion intensities comprise 15% and 13% compared to combined ion intensities generated in positive- and negative ion modes, respectively. High visibility of b-ion series in negative ion mode can be attributed to N-terminal sulfonation that had no negative effect on the production of b- and y-ion series in positive ion mode. Conclusions: The FBSA derivatization and de novo sequencing approach outlined here is a reliable method for accurate peptide sequence assignment. Increased production of b-ions in positive- and negative ion modes greatly improves peak assignment and thus enables accurate sequence reconstruction. Implementation of the named methodology would improve the quality of de novo sequencing data and reduce the number of misinterpreted spectra.</p

Outbreak of healthcare-associated bacteremia caused by Burkholderia gladioli due to contaminated multidose vials with saline solutions in three Croatian hospitals

Objectives: Burkholderia gladioli has been associated with infections in patients with cystic fibrosis, chronic granulomatous disease, and other immunocompromising conditions. The aim of this study was to better depict the outbreak of healthcare-associated bacteremia caused by B. gladioli due to exposure to contaminated multidose vials with saline solutions. ----- Methods: An environmental and epidemiologic investigation was conducted by the Infection Prevention and Control Team (IPCT) to identify the source of the outbreak in three Croatian hospitals. ----- Results: During a 3-month period, 13 B. gladioli bacteremia episodes were identified in 10 patients in three Croatian hospitals. At the time of the outbreak, all three hospitals used saline products from the same manufacturer. Two 100-ml multidose vials with saline solutions and needleless dispensing pins were positive for B. gladioli. All 13 bacteremia isolates and two isolates from the saline showed the same antimicrobial susceptibility patterns and pulsed-field gel electrophoresis profile, demonstrating clonal relatedness. ----- Conclusion: When an environmental pathogen causes an outbreak, contamination of intravenous products must be considered. Close communication between the local IPCT and the National Hospital Infection Control Advisory Committee is essential to conduct a prompt and thorough investigation and find the source of the outbreak