Infections caused by KPC-producing Klebsiella pneumoniae isolates: clinical, laboratory and epidemiological investigation

The aim of the study was the clinical, laboratory and epidemiological investigation of infections caused by KPC-producing Klebsiella pneumoniae (KPC-KP).A prospective observational cohort study was conducted (5/08-10/10) in a tertiary care hospital. All patients with at least one clinical sample positive for KPC-KP were identified and followed up. Patients’ clinical and epidemiological characteristics were studied. Rectal screening was performed and a simple phenotypic algorithm for surveillance cultures was evaluated. The study isolates were screened for carbapenemase production and their susceptibility profile was determined. The presence of blaKPC was detected by PCR and sequencing. Genetic relatedness of the isolates was determined by MLST. During the study period, a total of 414 KPC-KP isolates were recovered from clinical samples of 185 patients. Medical records were available for evaluation for 175 of them. Susceptibility testing showed higher in vitro activity for tigecycline (95.4%), fosfomycin (93.8%), gentamicin (86.7%) and colistin (79.5%). Comparative evaluation of tigecycline susceptibility testing methods revealed significant discrepancies between Vitek2 and reference broth microdilution, with Vitek2 producing higher MICs, resulting in false resistance. PCR detected the concomitant presence of blaVIM-1 and blaKPC-2 gene in 21 isolates. MLST classified the isolates into 5 STs with predominance of ST258 (82.3%). The culture-based phenotypic algorithm for rectal screening was proved highly sensitive and specific. This method accurately detected the type of carbapenemase for all KPC-producing isolates. The study population consisted of 175 patients either infected (95) or colonized (80) with KPC-KP isolates. The most common infection type was BSI (62 patients). Prior rectal colonization was the only independent predictor of clinical infection. Overall mortality was 36% being significantly higher for clinically infected patients. Infection mortality was 32.6%. Microbiological response was noted for 43.4% of the patients. In the cohort of 62 patients with BSI, age, APACHE II score at infection onset and inappropriate antimicrobial treatment were identified as independent predictors of infection mortality. All patients that received combination schemes had favourable infection outcome; in contrast, infection mortality was significantly higher among patients treated with appropriate monotherapy. Investigation of risk factors for CR KPC-KP acquisition showed that colistin resistance was not associated with colistin administration. The only independent predictor was prior rectal colonization with CR KPC-KP isolate. Molecular typing showed that both CR and CS isolates belonged to the same clone. This study investigated laboratory, clinical and therapeutic issues; the importance of appropriate antimicrobial therapy and the superiority of combination regimens in the treatment of KPC-infections was demonstrated. The study also revealed the shortcomings of Vitek2 for tigecycline susceptibility testing, the inaccuracies of which may falsely restrict treatment options. In addition, a new low-cost phenotypic algorithm for direct and specific detection of carbapenemase producers from surveillance cultures has been proposed.Σκοπός της μελέτης ήταν η εργαστηριακή, κλινική και επιδημιολογική διερεύνηση των λοιμώξεων από στελέχη Klebsiella pneumoniae που παράγουν KPC καρβαπενεμάση (KPC-KP). Μελετήθηκαν προοπτικά, για διάστημα 2,5 ετών (5/08-10/10), όλοι οι ασθενείς Τριτοβάθμιου Νοσοκομείου από τους οποίους απομονώθηκε στέλεχος KPC-KP. Αναλύθηκαν τα κλινικά και επιδημιολογικά τους χαρακτηριστικά και διερευνήθηκαν οι παράγοντες κινδύνου για εμφάνιση κλινικής λοίμωξης, για δυσμενή έκβαση στους ασθενείς με βακτηριαιμία και για λοίμωξη ή αποικισμό από ανθεκτικό στην κολιστίνη στέλεχος (CR). Πραγματοποιήθηκε έλεγχος ορθικής φορείας και αξιολογήθηκε και ένας νέος φαινοτυπικός αλγόριθμος για τις καλλιέργειες επιτήρησης. Ο έλεγχος των απομονωθέντων στελεχών περιελάμβανε τον καθορισμό του προφίλ ευαισθησίας, τον φαινοτυπικό και μοριακό έλεγχο και τη μοριακή τυποποίηση. Κατά το χρονικό διάστημα της μελέτης απομονώθηκαν 414 στελέχη KPC-KP από 185 ασθενείς. Ο έλεγχος της ευαισθησίας έδειξε ότι υψηλότερη δραστικότητα εμφάνισαν η τιγεκυκλίνη (95,4%), η φωσφομυκίνη (93,8%), η γενταμικίνη (86,7%) και η κολιστίνη (79,5%). Σημαντικός βαθμός ασυμφωνίας προέκυψε στον έλεγχο ευαισθησίας της τιγεκυκλίνης μεταξύ του Vitek2 και της μεθόδου αναφοράς, με τάση για ψευδή αντοχή. Ο έλεγχος παρουσίας γονιδίων άλλων καρβαπενεμασών έδειξε ότι 21 στελέχη έφεραν και το γονίδιο blaVIM-1. Η μοριακή τυποποίηση με τη μέθοδο MLST ανέδειξε 5 ST τύπους με επικρατούντα τον ST258 (82,3%). O νέος φαινοτυπικός αλγόριθμος στις καλλιέργειες επιτήρησης παρουσίασε υψηλή ευαισθησία και ειδικότητα, ενώ η ταυτοποίηση του είδους της καρβαπενεμάσης ήταν ακριβής για όλα τα KPC-θετικά στελέχη. Κλινική λοίμωξη εμφάνισαν 95 ασθενείς, με συχνότερη τη βακτηριαιμία. Μοναδικός ανεξάρτητος παράγοντας κινδύνου για την εμφάνιση λοίμωξης ήταν η προηγηθείσα ορθική φορεία. Η ολική θνητότητα ήταν 36% στο σύνολο των ασθενών και βρέθηκε σημαντικά υψηλότερη στους ασθενείς με λοίμωξη. Η θνητότητα λοίμωξης ήταν 32,6%. Στη μελέτη των παραγόντων που σχετίζονται με την έκβαση στους ασθενείς με βακτηριαιμία η ηλικία, το APACHE II score κατά την έναρξη της λοίμωξης και η μη χορήγηση κατάλληλης αντιμικροβιακής αγωγής αναδείχτηκαν ως ανεξάρτητοι προγνωστικοί παράγοντες για δυσμενή έκβαση. Η χορήγηση συνδυασμών δραστικών αντιμικροβιακών οδήγησε σε κλινική επιτυχία για το σύνολο των ασθενών, ενώ η θνητότητα της λοίμωξης στην ομάδα της μονοθεραπείας ήταν σημαντικά υψηλότερη. Η διερεύνηση των παραγόντων κινδύνου για λοίμωξη ή αποικισμό από στέλεχος CR KPC-KP δεν ανέδειξε συσχέτιση με την προηγηθείσα χορήγηση κολιστίνης. Μοναδικός ανεξάρτητος παράγοντας κινδύνου ήταν η προηγηθείσα ορθική φορεία CR KPC-KP. Από τον έλεγχο της κλωνικότητας προέκυψε ότι όλα τα στελέχη ανήκαν στον ίδιο κλώνο. Οι λοιμώξεις από στελέχη KPC-KP παρουσιάζουν υψηλή επίπτωση και συνοδεύονται από υψηλή θνητότητα. Στην παρούσα μελέτη επιχειρήθηκε να απαντηθούν ερωτήματα που τίθενται στην κλινική και εργαστηριακή πράξη. Στο πλαίσιο αυτό, αναδείχτηκε η υπεροχή της συνδυαστικής αντιμικροβιακής αγωγής αλλά και η αδυναμία του αυτοματοποιημένου συστήματος Vitek2 να ανιχνεύσει αξιόπιστα την ευαισθησία στην τιγεκυκλίνη. Επίσης αναπτύχθηκε ένας νέος φαινοτυπικός αλγόριθμος που επιτρέπει την ταχεία και ειδική για τον τύπο της καρβαπενεμάσης ανίχνευση στις καλλιέργειες ορθικών επιχρισμάτων

Necrotizing Skin and Soft Tissue Infection Due to <i>Syncephalastrum</i> Species and <i>Fusarium solani</i> Species Complex Following Open Tibia Fracture

Fungal necrotizing skin and soft tissue infection (NSSTI) represents a rare clinical entity. An extremely rare case of NSSTI, following an open tibia fracture in a 36-year-old male caused by both Syncephalastrum spp. and Fusarium solani species complex (SC) is presented. The infection was diagnosed through direct microscopy, cultures and histology. The disease had a long course. The patient underwent a total of seven consecutive surgical debridements, while proper and timely antifungal treatment was initiated and included liposomal amphotericin B and voriconazole. He gradually recovered and 4 years later he is completely functioning and healthy. Invasive fungal infections are well-documented causes of high morbidity and mortality in immunocompromised individuals, whereas in immunocompetent hosts, trauma-related fungal infections have also been reported. It is of note that Syncephalastrum spp. has very rarely been identified to cause infection in immunocompromised or immunocompetent hosts, whereas Fusarium spp. has rarely been involved in skin necrotic lesions in non-immunocompromised individuals. A high suspicion index, especially in necrotic lesions in trauma patients, is pivotal for early diagnosis, which may lead to lower mortality as well as lower amputation rates. Definite diagnosis through microscopy, histology and/or cultures are of paramount importance, whereas PCR testing may also be extremely useful

Chronic prostatic infection : microbiological findings in two Mediterranean populations

Introduction/Aim: Despite accumulated knowledge, several microbiological aspects of chronic bacterial prostatitis (CBP) remain uncertain. The aim of our study was to determine microbiological characteristics on our CBP population. Materials: The material of this retrospective study consisted in bacterial isolates from urine and/or prostatic secretions or sperm cultures (total ejaculate) obtained from individuals with prostatitis symptoms and from patients with febrile relapses of CBP visiting our department, from 03/2009 to 03/2015. Retrospective data from an Italian single-center database (years 2009-2015) were also collected for a tentative comparison of pathogen prevalence between chronic bacterial prostatitis cases assessed in Greece and Italy. Results: A total of 389 bacterial isolates obtained from eligible Greek patients constituted the material of the study. While E coli was the most frequent individual pathogen, Gram-positive species were overly more frequent than Gram-negative. Besides the high frequency of E. coli and E. faecalis isolates the most remarkable similarity between Greek and Italian databases was the wide array of different Gram-positive and Gram-negative species isolated from CBP patients. Conclusions: In Greece, the incidence of CBP is possibly higher than that reported in international surveys. Similarities between Greek and Italian databases suggest geographical trends in CBP epidemiology

A Combined Disk Test for Direct Differentiation of Carbapenemase-Producing Enterobacteriaceae in Surveillance Rectal Swabs

Carbapenemase-producing Enterobacteriaceae (CPE) are rapidly spreading worldwide. Early detection of fecal CPE carriers is essential for effective infection control. Here, we evaluated the performance of a meropenem combined disk test (CDT) for rapidly differentiating CPE isolates directly from rectal swabs. The screening method was applied for 189 rectal swabs from hospitalized patients at high risk for CPE carriage. Swabs were suspended in 1 ml saline and cultured for confluent growth onto a MacConkey agar plate with a meropenem (MER) disk alone, a MER disk plus phenyl boronic acid (PBA), a MER disk plus EDTA, and a MER disk plus PBA and EDTA. An inhibition zone of &lt;= 25 mm around the MER disk alone indicated carriage of carbapenem-resistant organisms. Furthermore, &gt;= 5-mm differences in the inhibition zone between MER disks without and with the inhibitors (PBA, EDTA, or both) were considered positive results for detecting Klebsiella pneumoniae carbapenemase (KPC), metallo-beta-lactamase (MBL), or both carbapenemases, respectively. For comparison, rectal suspensions were tested using MacConkey plates with ertapenem (MacERT) disks and PCR (PCR-S) for carbapenemase genes. Of the 189 samples, 97 were genotypically confirmed as CPE positive by one of the three protocols tested. The CDT, MacERT disks, and PCR-S assays exhibited sensitivities of 94.8%, 96.9%, and 94.8% and specificities of 100%, 98.9%, and 100%, respectively, for detecting CPE-positive swabs. Moreover, the CDT correctly differentiated the production of KPC, MBL, or both carbapenemases in 78 of the 97 (80.4%) CPE-positive rectal swabs. Our results demonstrate that the CDT may provide a simple and inexpensive method for detecting and differentiating the carbapenemase type within a single day without requiring further testing and additional delay, supporting the timely implementation of infection control measures

Risk Factors and Outcomes Associated with Acquisition of Colistin-Resistant KPC-Producing Klebsiella pneumoniae: a Matched Case-Control Study▿

A matched 1:3 case-control study investigated factors predicting colistin-resistant versus colistin-susceptible KPC-producing Klebsiella pneumoniae acquisition and its impact on patient outcomes. Case patients were more often admitted from other institutions (P = 0.019) and had longer therapy with β-lactam/β-lactamase inhibitors (P = 0.002) and higher overall mortality (P = 0.05). All 52 study isolates were clonally related, suggesting horizontal dissemination. None of these parameters independently predicted colistin resistance, which probably occurred in a susceptible KPC-KP strain that was subsequently disseminated horizontally

Predominance of international clone 2 OXA-23-producing-Acinetobacter baumannii clinical isolates in Greece, 2015: results of a nationwide study

In a previous nationwide study in Greece, OXA-58 was the sole carbapenemase present among carbapenem-resistant Acinetobacter baumannii (CRAB) isolated between 2000 and 2009. In this study, the antibiotic resistances, carbapenemase gene content and clonal relatedness of 194 single-patient CRAB clinical isolates collected randomly during 2015 from 11 tertiary hospitals located throughout Greece were investigated. Antimicrobial susceptibility was determined using commercial and dilution methods. PCR assays for carbapenemase genes were performed. Clonality was tested by a scheme based on two multiplex PCRs and single-locus blaOXA-51-like sequence-based typing. Furthermore, Pasteur's multilocus sequence typing (MLST) scheme and pulsed-field gel electrophoresis (PFGE) were applied to 31 selected representative isolates. The most active antibiotics were trimethoprim/sulfamethoxazole (SXT) (34.6% of isolates susceptible), minocycline (71.6%), colistin (72.7%) and tigecycline (MIC50/90 values, 1/2 mg/L). The blaOXA-23-like gene was identified in 188 isolates (96.9%), blaOXA-23-like together with blaOXA-58-like in 3 isolates (1.5%), blaOXA-58-like in 2 isolates (1.0%) and blaOXA-40-like in 1 isolate (0.5%). ISAba1 was found upstream of the blaOXA-23-like gene in all isolates. International clone (IC) 2 comprised 157 isolates (80.9%), IC1 comprised 36 isolates (18.6%) and ST78 comprised 1 isolate (0.5%). All IC2 and IC1 isolates tested by MLST were ST2 and ST1, respectively. Seven PFGE types were detected. IC2 isolates were resistant to more antibiotics than IC1, except for SXT. This nationwide study showed that CRAB isolates in Greek hospitals currently produce almost uniformly the OXA-23 carbapenemase and belong mainly to IC2 and, to a lesser extent, IC1. Of particular concern, colistin susceptibility is recently severely reduced

Linezolid Dependence in Staphylococcus epidermidis Bloodstream Isolates

We document linezolid dependence among 5 highly linezolid-resistant (LRSE) Staphylococcus epidermidis bloodstream isolates that grew substantially faster at 32 mu g/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