SeptiFast Real-Time PCR for Detection of Bloodborne Pathogens in Patients with Severe Sepsis or Septic Shock

Several studies have been performed investigating the role of a real-time multiplex polymerase chain reaction assay LightCycler® SeptiFast® with inconsistent results. In prospective evaluation of adult patients with severe sepsis or septic shock SeptiFast assay and blood culture results were compared regarding concordance, the impact of SeptiFast assay on antimicrobial therapy adjustment, time to results and the role of SeptiFast assay as a marker of disease severity. 63 blood sample sets were collected from 57 patients. 51 (80.9%) results were concordant negative and 7 (11.1%) concordant positive. In one (1.6%) sample set blood culture was positive and SeptiFast assay negative, in three (4.8%) sample sets with negative blood cultures pathogens were detected by SeptiFast assay and in one (1.6%) patient an additional pathogen was detected by SeptiFast assay. If blood culture is considered as „gold standard“, 1 (1.6%) SeptiFast false negative and 4 (6.3%) false positive results were identified (sensitivity 87.5%, specificity 92.6%, negative predictive value 97.8%). Antibiotic treatment was adjusted according to SeptiFast assay in 4 (6.3%) cases. Time to final results was significantly shorter with SeptiFast assay (32 ± 23 h vs. 97 ± 28 h, p<0.0001). Positive SeptiFast assay was not associated with higher mortality, C-reactive protein or procalcitonin (p=0.74, p=0.44 and p=0.12, respectively). According to our results SeptiFast assay can be used as a valuable add-on to blood culture in diagnostic workup of patients with severe sepsis and septic shock but it cannot replace the blood culture

Potential value of a rapid syndromic multiplex PCR for the diagnosis of native and prosthetic joint infections: a real-world evidence study

Introduction: The BIOFIRE Joint Infection (JI) Panel is a diagnostic tool that uses multiplex-PCR testing to detect microorganisms in synovial fluid specimens from patients suspected of having septic arthritis (SA) on native joints or prosthetic joint infections (PJIs). Methods: A study was conducted across 34 clinical sites in 19 European and Middle Eastern countries from March 2021 to June 2022 to assess the effectiveness of the BIOFIRE JI Panel. Results: A total of 1527 samples were collected from patients suspected of SA or PJI, with an overall agreement of 88.4 % and 85 % respectively between the JI Panel and synovial fluid cultures (SFCs). The JI Panel detected more positive samples and microorganisms than SFC, with a notable difference on Staphylococcus aureus, Streptococcus species, Enterococcus faecalis, Kingella kingae, Neisseria gonorrhoeae, and anaerobic bacteria. The study found that the BIOFIRE JI Panel has a high utility in the real-world clinical setting for suspected SA and PJI, providing diagnostic results in approximately 1 h. The user experience was positive, implying a potential benefit of rapidity of results' turnover in optimising patient management strategies. Conclusion: The study suggests that the BIOFIRE JI Panel could potentially optimise patient management and antimicrobial therapy, thus highlighting its importance in the clinical setting

Molecular characterisation of carbapenem-resistant Pseudomonas aeruginosa isolated from patients and the environment

Pseudomonas aeruginosa je vseprisotna okoljska oportunistična bakterija. Občasno jo osamimo kot del človeške mikrobiote. Lahko povzroča okužbe povezane z zdravljenjem in je intrinzično odporna proti številnim antibiotikom. Med maloštevilnimi zdravili, ki so na voljo za zdravljenje psevdomonasnih okužb so karbapenemi, tudi odpornost proti njim narašča. P. aeruginosa iz bolnišnic lahko prehaja v komunalni sistem odpadnih vod in v vode čistilnih naprav. Primerjali smo klinično pomembne proti karbapenemom odporne P. aeruginosa (CRPA) in CRPA iz vod dveh čistilnih naprav, ter ugotavljali prekrivanje genotipov in razlike v genih za odpornost in virulenco. V laboratoriju, kamor pošiljajo na preiskave vzorce iz več zdravstvenih ustanov, smo zbrali seve CRPA iz urina in dihal v obdobju enega leta. V istem obdobju smo mesečno vzorčili vodo iz iztoka dveh čistilnih naprav in na selektivnem gojišču osamili seve CRPA. Občutljivost za protimikrobna zdravila smo določali z disk difuzijsko metodo po EUCASTu. Seve smo tipizirali s PFGE. Za vsak pulzotip smo izbrali enega do devet predstavnikov, pri katerih smo nato določali sekvenco celotnega genoma (WGS). Na osnovi WGS smo izvedli tipizacijo MLST in analizo rezistenčnih ter virulenčnih determinant. Skupaj smo osamili 213 CRPA sevov (65 pulzotipov), 130 iz kliničnih vzorcev (38 poulzotipov) in 83 iz okoljskih vzorcev (31 pulzotipov). Večino kliničnih CRPA sevov (125 od 130 sevov in 37 od 38 pulzotipov) smo izolirali iz kužnin iz večje učne bolnišnice. Ostale klinične seve (5 od 130) smo razvrstili v 3 pulzotipe. Najpogostejšemu kliničnemu pulzotip (Pt1) je pripadalo 57 sevov (45,6 %), našli pa smo ga samo v večji učni bolnišnici. Samo dva pulzotipa (Pt17 in Pt63) sta bila tako v večji učni bolnišnici kot tudi v manjših zdravstvenih organizacijah. Okoljske CRPA seve smo porazdelili v 31 pulzotipov (26 pulzotipov iz večje čistilne naprave, 11 pulzotipov iz manjše čistilne naprave). Podobno kot med kliničnimi sevi je tudi med sevi iz čistilnih naprav prevladoval posamični pulzotip (Pt10 – 21,7 %). Prekrivanje med kliničnimi in okoljskimi pulzotipi in MLST tipi CRPA, je bilo nizko. Le 9 pulzotipov se je pojavljalo v več kot eni inštituciji (zdravstveni inštituciji/čistilni napravi). Z MLST smo med 112 analiziranimi sevi določili 49 ST. Le 10 ST je bilo tako iz kliničnih kot iz okoljskih vzorcev, med njimi po vsem svetu razširjena ST111 in ST235. Klinični in okoljski sevi CRPA so se razlikovali glede odpornosti proti antibiotikom. Večina izolatov je bila odporna samo proti imipenemu in/ali meropenemu. Pri sevih, ki so bili dodatno odporni, smo opisali devet različnih različnih vzorcev odpornosti. Klinične seve smo našli pri vseh vzorcih odpornosti, medtem ko smo okoljske seve našli le pri 4 dodatnih vzorcih odpornosti. Analiza rezistoma je potrdila gene za intrinzične mehanizme odpornosti. Našli smo tudi ene povezane s horizontalnim genskim prenosom, kot so geni za karbapenemaze (16 % sevov) in različne encime, ki modificirajo aminoglikozide. Pri 33 % sevov smo potrdili mutacije v girazi. Vsi sevi z geni za karbapenemaze so bili večkratno odporni. Z WGS smo določili tudi prisotnost virulenčnih genov med analiziranimi sevi. Glede teh se sevi iz okolja in klinični sevi večinoma niso razlikovali. Izjema so bili geni za eksotoksine Y, U, T in S. Pri večini sevov smo našli gene za Y in T, gene za U in S smo našli le pri pri 14 oz. 36 sekvenčnih tipih. Geni za S in U so bili bolj pogosti pri kliničnih sevih, medtem ko so bili geni za T bolj pogosti med okoljskimi sevi. Nizka stopnja prekrivanja med kliničnimi in okoljskimi CRPA sevi kaže, da gre za dva neodvisna rezervoarja. Klinični CRPA sevi imajo nekoliko drugačen set virulenčnih determinant in bolj raznolike vzorce odpornosti.P. aeruginosa is opportunistic, ubiquitous bacterium, occasionally isolated as a part of human microbiota. It can cause severe hospital acquired infections. It is intrinsically resistant to many antibiotics. Carbapenems are among the few available options for treatment of P. aeruginosa infections, but the carbapenem resistance is increasing worldwide. P. aeruginosa from hospitals could potentially be transmitted to the wastewater systems. The aim of our study was t to compare clinically relevant carbapenem resistant P. aeruginosa (CRPA) with CRPA isolates from two wastewater treatment plants (WWTP) and to detect possible overlap in genotypes and presence of resistance and virulence genes. During the one year period CRPA cultivated from urine and respiratory diagnostic samples in the laboratory serving several health care facilities were collected. Two WWTPs were sampled monthly and CRPA were isolated on selective media. Susceptibility was determined by disk diffusion method according to EUCAST standards. Strains were PFGE typed. For each pulsotype from one to nine representatives were selected and subjected to whole genome sequencing (WGS). WGS was used for MLST and for analysis of resistance and virulence determinants. Altogether 213 CRPA strains (65 pulsotypes) were collected, of which 130 were from clinical samples (38 pulsotypes) and 83 were from environmental (31 pulsotypes) samples. Majority of clinical CRPA strains (125 of 130 strains and 37 of 38 pulsotypes) were from larger teaching hospital. Other clinical strains (5 of 130) belonged to 3 pulsotypes. The most common clinical pulsotype (Pt1) was encountered in 57 strains (45,6 %), but was present only in larger teaching hospital. Only two pulsotypes (Pt17 and Pt63) were encountered in larger teaching hospital as well as in smaller health institutions. Environmental CRPA strains belonged to 31 pulsotypes (26 in larger WWTP and 11 in smaller WWTP). Similar as among clinical strains also here a single pulsotype was prevalent (Pt10 – 21,7 %). Only 9 pulsotypes were shared between two or more settings (hospital or WWTP). Overlap between CRPA genotypes from patients and WWTP according to PFGE and MLST typing was low. Only 9 pulsotypes were shared between two or more settings (hospital or WWTP). Similar results were obtained with MLST typing where 49 STs were determined in 112 analysed strains, but only 10 STs overlapped between environment and patients, among them the worldwide distributed types ST111 and ST235. Clinical and environmental CRPA differed in antibiotic resistance. The majority of isolates was resistant only to imipenem and/or meropenem. Strains with additional resistances were distributed into nine different patterns. All of them included clinically relevant CRPA, while environmental CRPA showed only four additional resistance patterns. Resistome analysis demonstrated different intrinsic resistance mechanisms, but also genes associated with horizontal gene transfer (HGT) such as genes for carbapenemases - VIM family (16 % of strains) and genes for aminoglycoside modifying enzymes. Mutations in gyrase was found in 33 % of strains. All strains with carbapenemases were multiresistant. Prevalence of virulence genes assessed from WGS was mostly uniform across the analysed strains. Exemptions were genes for type exotoxins Y, U, T and S. Genes for Y and T were present in majority of isolates, while genes for U and S were present only in 14 and 36 ST, respectively. It seems that genes associated with exotoxins S and U are relatively more frequent in clinical strains, while are genes associated with exotoxin T more frequent in environmental strains. In summary, we have shown a low overlap between clinically relevant and environmental CRPA genotypes, indicating that these two reservoirs are independent. CRPA in hospital environment have slightly different set of virulence determinants and higher variety of resistance patterns

Molekularna identifikacija klinično pomembnih bakterij, vključno enterobakterij s primerjavo delnega nukleotidnega zaporedja gena za 16S ribosomsko RNK

Background: The correct identification of bacterial isolates is paramount for the correct diagnosis and treatment of patients. The standard approach for bacterial identification relies on phenotypic tests, which are often time-consuming and unreliable when identifying atypical strains. We instead hypothesized a molecular approachfast and reliable identification could be achieved by comparing a 16S ribosomal RNA gene sequence with publicly available sequences. Methods: In the present study, DNA from seventeen bacterial species was extracted and amplified by PCR, and then a partial 16S ribosomal RNA gene sequence was obtained and compared with sequences deposited in public DNA sequence databasesEMBL/GeneBank/DDBJ and RDP-II. Results: Bacterial species, belonging to several familiesCoriobacteriaceae, Bacteroidaceae, Clostri- diaceae, Staphylococcaceae, as well as three out of nine Enterobacteriaceae were specifically identified by partial 16S ribosomal RNA gene sequence to the species level. For six other species of enteric bacteria specific identification of species failed, since the results of genotypic identification yielded several species. Conclusions: We conclude identification of bacteria with partial 16S ribosomal RNA gene sequence is an efficient complement to phenotypic identification, except for enteric bacteria.Za učinkovito diagnostiko bakterijskih okužb in zdravljenje je potrebna hitra in natančna identifikacija bakterije. Običajen postopek je fenotipska identifikacija, ki je pogosto zahtevna in nenatančna za atipične izolate. V naši študiji smo predpostavili, da je možno identificirati bakterije s primerjavo delnih nukleotidnih zaporedij genov, ki kodirajo 16S ribosomsko RNKs primerjavo z nukleotidnimi zaporedji v javno dostopnih podatkovnih bazah za zaporedja DNK: EMBL, GenBank, DDBJ in RDP-II. Še posebej nas je zanimala možnost identifikacije enterobakterij, saj mnogi avtorji navajajo, da te skupine bakterij ni moč ločiti na osnovi zaporedij genov 16S ribosomske RNK. Za izvedbo študije smo pridobili 15 tipskih sevov in dva klinična izolata iz zbirke Zavoda za zdravstveno varstvo Maribor, katerih rutinska fenotipska identifikacija je težavna. Iz bakterij smo izolirali DNK ter z verižno reakcijo s polimerazo (PCR) pomnožili variabilen odsek gena za 16S ribosomsko RNK. Pridelke PCR smo očistili in poslali na sekveniranje s prednjim začetnim oligonukleotidom v podjetje Macrogen, Koreja (www.macrogen.com). Pridobljena nukleotidna zaporedja DNK smo nato z dvema iskalnima algoritmomaBLAST, v bazah GeneBank/EMBL/DDBJ, ter z algoritmom Sequence match v bazi RDP-II primerjali z vloženimi nukleotidnimi zaporedji, za morebitno identifikacijo vrste. Specifično smo uspeli identificirati vrste: Staphylococcus epidermidis,Bacteroides fragilis, Clostridium perfringens, Peptostreptococcus anaerobius, Eggerthella lenta, Enterobacter cloacae, Yersinia enterocolitica, Proteus mirabilis, Streptococcus pyogenes, Enterococcus faecalis. Medtem ko Escherichia coli, Serratia marcescens, Shigella flexneri, Klebsiella pneumoniae, Citrobacter freundii nismo uspeli nedvoumno identificirati, čeprav je klinično nujno. Vendar menimo, da je opisan genotipski pristop za identifikacijo vrste bakterije učinkovito dopolnilo fenotipski identifikaciji, še posebno pri identifikaciji atipičnih izolatov

Arnica Montana L. supercritical extraction optimization for antibiotic and anticancer activity

Arnica montana L. flower heads are known for their antioxidant, antimicrobial, and anticancer activity. The aim of this work was to optimize the process of supercritical CO(2) extraction, to achieve high extraction yield and high content of biologically active components, and to confirm the antimicrobial and anticancer activity of the extract. The influence of pressure and temperature on the total phenolic content, antioxidant activity, and proanthocyanidin content was evaluated. The pressure and temperature were found to be interdependent. A temperature of 60°C and a pressure of 30 MPa resulted in a high extraction yield, antioxidant activity and phenolic content. The content of proanthocyanidins was highest at a pressure between 18 and 24 MPa. The extracts inhibited three different microorganisms successfully; Staphylococcus aureus, Escherichia coli and Candida albicans, at concentrations ranging from 0.1 to 5.16 mg/ml and showed anticancer activity decrease up to 85% at a concentration of 0.5 mg/ml

Low overlap between carbapenem resistant <i>Pseudomonas aeruginosa</i> genotypes isolated from hospitalized patients and wastewater treatment plants

<div><p>The variability of carbapenem-resistant <i>Pseudomonas aeruginosa</i> strains (CRPA) isolated from urine and respiratory samples in a large microbiological laboratory, serving several health care settings, and from effluents of two wastewater treatment plants (WWTP) from the same region was assessed by PFGE typing and by resistance to 10 antibiotics. During the 12-month period altogether 213 carbapenem-resistant <i>P</i>. <i>aeruginosa</i> isolates were cultured and distributed into 65 pulsotypes and ten resistance profiles. For representatives of all 65 pulsotypes 49 different MLSTs were determined. Variability of clinical and environmental strains was comparable, 130 carbapenem-resistant <i>P</i>. <i>aeruginosa</i> obtained from 109 patients were distributed into 38 pulsotypes, while 83 isolates from WWTPs were classified into 31 pulsotypes. Only 9 pulsotypes were shared between two or more settings (hospital or WWTP). Ten MLST were determined for those prevalent pulsotypes, two of them (ST111 and ST235) are among most successful CRPA types worldwide. Clinical and environmental carbapenem-resistant <i>P</i>. <i>aeruginosa</i> strains differed in antibiotic resistance. The highest proportion of clinical isolates was resistant to piperacillin/tazobactam (52.3%) and ceftazidime (42.3%). The highest proportion of environmental isolates was resistant to ceftazidime (37.1%) and ciprofloxacin (35.5%). The majority of isolates was resistant only to imipenem and/or meropenem. Strains with additional resistances were distributed into nine different patterns. All of them included clinically relevant strains, while environmental strains showed only four additional different patterns.</p></div

PFGE dendrogram depicting the genetic relatedness of the 66 pulsotypes found among the carbapenem-resistant <i>P</i>. <i>aeruginosa</i> isolates.

<p>Only one representative of a single pulsotype was included. Locations where the pulsotype was found are marked with the sign X. Other—other clinical settings.</p

Distribution of different carbapenem-resistant <i>P</i>. <i>aeruginosa</i> pulsotypes in clinical and environmental isolates.

<p>Distribution of different carbapenem-resistant <i>P</i>. <i>aeruginosa</i> pulsotypes in clinical and environmental isolates.</p

Venn diagram showing overleap of pulsotypes between clinical and environmental settings.

<p>For clearer picture and because of low number of isolates we grouped together Hospital B and smaller clinical settings. N = number of typeable isolates; PFGE = number of pulsotypes.</p

Resistance patterns of clinical and environmental carbapenem-resistant <i>P</i>. <i>aeruginosa</i> isolates and their corresponding pulsotypes.

<p>Pulsotypes in bold share the resistance pattern and were found in both environments (patients and water).</p