Laboratory automation reduces time to report of positive blood cultures and improves management of patients with bloodstream infection.

The impact on time to results (TTR) and clinical decisions was evaluated for mono-microbial positive blood cultures (BC) processed using the BD Kiestra Work Cell Automation (WCA) system. Positive BC were processed by the WCA system by full-automatic subculture on solid media and digital imaging after 8 h of incubation (8-h method) followed by identification (ID) and antimicrobial susceptibility testing (AST). To evaluate the accuracy of the 8-h method, ID and AST from 8-h and overnight incubated colonies were compared for the same organisms. To evaluate its clinical impact, results from 102 BC processed by the 8-h method (cases) were compared with those from 100 BC processed by overnight incubation method (controls) in a comparable period. Identification after 8-h and overnight incubation gave concordant results in 101/102 (99.0%) isolates. Among a total of 1379 microorganism-antimicrobial combinations, categorical agreement was 99.4% (1371/1379); no very major error, 7 major errors, and one minor error were observed. TTR in cases (32.8 h ± 8.3 h) was significantly (p < 0.001) shorter than in controls (55.4 h ± 13.3 h). A significant reduction was observed for duration of empirical therapy (cases 54.8 h ± 23.3 h vs controls 86.9 h ± 34.1 h, p < 0.001) and 30-day crude mortality rate (cases 16.7% vs controls 29.0%, p < 0.037). Automation and 8-h digital reading of plates from positive BC, followed by ID and AST, greatly reduce TTR and shorten the duration of antimicrobial empiric therapy, possibly improving outcome in patients with mono-microbial bloodstream infections.

Carbapenemase-producing Enterobacteriaceae isolates resistant to last-line antibiotics in an Italian general hospital.

The global dissemination of carbapenemase-producing Enterobacteriaceae (CPE) is of great concern for public health. These bacteria have the potential for rapid dissemination in healthcare settings and cause infections associated with high rates of morbidity and mortality. A total of 221 carbapenem non-susceptible Enterobacteriaceae isolates were collected from patients admitted to an Italian general hospital from January 2016 to March 2017. Among these isolates, 78.3% were carbapenemase producers: 96% were positive for the blaKPC gene and the remainder for the blaVIM gene (allelic variant VIM-1). CPE isolates were mainly Klebsiella pneumoniae, but we also detected carbapenemase enzymes in Citrobacter freundii, Enterobacter cloacae and Escherichia coli. Among CPE isolates, 79.2% exhibited co-resistance to two or more non-b-lactam agents and 38% of these isolates (all KPC-positive) were resistant to colistin. This percentage reached 55% among CPE isolated from the bloodstream. All patients with colistin-resistant CPE isolates recovered from blood samples showed an unfavorable outcome within 7 days from the first positive blood culture. Our data show the dissemination of a high percentage of CPE isolates co-resistant to last-line antibiotics. In addition, we report the first identification in our hospital of CPE isolates harboring the blaVIM gene and Escherichia coli harboring the blaKPC gene. These results underline the need to implement antibiotic stewardship and infection control programs, and emphasize the need for novel antimicrobial agents active against CPE.

Carbapenem-Resistant Klebsiella pneumoniae: Results of a Laboratory Surveillance Program in an Italian General Hospital (August 2014-January 2015) : Surveillance of Carbapenem-resistant Klebsiella pneumoniae.

In this study we report the analysis of 131 Klebsiella pneumoniae (K. pneumoniae) clinical isolates from patients hospitalized in various wards, of Perugia General Hospital, from August 2014 to January 2015. Forty two isolates (32.1 %), were resistant to at least one carbapenem antibiotic and, among these isolates, 14 (33.3 %) exhibited resistance to colistin. All isolates were carbapenemases producers and 41 (97.6 %) harboured the bla KPC gene. Carbapenem-resistant K. pneumoniae isolates (CRKPs) were, also, typed for the genotypic diversity and the results revealed the circulation of two major clusters.This surveillance study evidences the spread of CRKP isolates in Perugia General Hospital and confirms that carbapenem-resistant K. pneumoniae isolates have reached epidemic dissemination in Italy. In addition the percentage of resistance to colistin resulted to be less than that observed in other hospital laboratories across Italy. In conclusion the circulation of these isolates should be monitored and appropriate policy of surveillance must be used, in a target manner, in order to reduce the spread of carbapenem-resistant isolates.

Worrisome trend of new multiple mechanisms of linezolid resistance in staphylococcal clones diffused in Italy.

In order to assess the frequency of clinically relevant linezolid-resistant staphylococcal isolates, and the role of linezolid in maintaining and coselecting multiple resistance mechanisms (cfr, 23S rRNA, L3/L4 mutations), a prospective Italian study was performed from 2010 to 2011 to confirm the diffusion of three major multidrug-resistant clones (ST2, ST5, ST23).

Hyper-Virulent Listeria monocytogenes Strains Associated With Respiratory Infections in Central Italy.

Listeria monocytogenes (Lm) is a foodborne pathogen causing listeriosis. Invasive forms of the disease mainly manifest as septicaemia, meningitis and maternal-neonatal infections. Lm-associated respiratory infections are very rare and little known. We reported two Lm respiratory infection cases occurred in Central Italy during the summer of 2020, in the midst of the SARS-CoV2 pandemic. In addition to collect the epidemiological and clinical characteristics of the patients, we used Whole Genome Sequencing to study the genomes of the Lm isolates investigating their virulence and antimicrobial profiles and the presence of genetic mobile elements. Both the strains belonged to hypervirulent MLST clonal complexes (CC). In addition to the Listeria Pathogenicity Island 1 (LIPI-1), the CC1 strain also carried LIPI-3 and the CC4 both LIPI-3 and LIPI-4. Genetic determinants for antimicrobial and disinfectants resistance were found. The CC1 genome presented prophage sequences but they did not interrupt the comK gene, involved in the phagosomal escape of Lm. None of the strains carried plasmids. Lm is an important, although rare, opportunistic pathogen for respiratory tract and lung infections. To avoid dangerous diagnostic delays of these severe clinical forms, it is important to sensitize hospital laboratories to this rare manifestation of listeriosis considering Lm in the differential diagnosis of respiratory infections.

Analysis of meticillin-susceptible and meticillin-resistant biofilm-forming Staphylococcus aureus from catheter infections isolated in a large Italian hospital.

Several characteristics were analysed in 37 Staphylococcus aureus isolates from nosocomial catheter infections: the PFGE profile after SmaI digestion of chromosomal DNA, the ability to form a biofilm on a polystyrene surface, antibiotic susceptibility patterns (penicillin, oxacillin, erythromycin, tetracycline, clindamycin, telithromycin, gentamicin, ciprofloxacin, quinupristin/dalfopristin, rifampicin, vancomycin and linezolid), and the presence of genetic determinants of antibiotic resistance and biofilm formation. All strains but three (92 %) were able to grow on a plastic surface as a biofilm. An almost complete association was found between phenotypes and genotypic traits of antibiotic resistance, whilst PFGE profiling showed the highly polyclonal composition of the set of strains under study. Sixteen isolates (43 %) were meticillin-resistant and were subjected to staphylococcal cassette chromosome mec (SCCmec) and cassette chromosome recombinase (ccr) complex type determination by multiplex PCR. Only a subgroup of six strains belonged to the archaic clone PFGE type and bore the SCCmec/ccrAB type I structure. Among the remaining strains some presented small rearrangements of the SCCmec/ccrAB genetic locus, whilst others could barely be traced back to a known structural type. These observations suggest that, at the local level and at a particular site of infection, S. aureus may show great genetic variability and escape the general rule of expansion of the S. aureus pandemic clones.

Absidia Corymbifera in an immune competent accident victim with multiple abdominal injuries: case report.

BACKGROUND: We report a case of mucormycosis in a healthy 17-year-old accident victim with multiple abdominal injuries which was caused by infection with Absidia Corymbifera, a ubiquitous saphrophyte in the ground. CASE PRESENTATION: The patient was admitted to hospital with massive abdominal trauma. During an 8-hour emergency operation he received transfusions of compacted red blood cells, plasma, platelets and hemagel. He developed a crush syndrome with acute renal failure, resolved with extra-corporeal dialysis and had to undergo splenectomy because of spleen hematoma. As wound secretion and central venous catheter (CVC) blood cultures and drainage fluid were positive for Enterococcus Faecium, Providentia Rettgeri, Hafnia Alvei and Candida Albicans, tecoplanin, metronidazole, imipenem, and flucanozole were administered. Although the CVC was changed high fever persisted and discharge continued from the large abdominal wound. Repeated tampons in different sections and wound secretion smears were positive for A. corymbifera. Flucanozole was stopped and liposomal amphotericin (Ambisome; 5 mg/Kg i.v.) given for over 3 months. The patient improved; fever gradually disappeared. After 8 days, tampons and wound secretion smears were negative for A. corymbifera. No other fungal infections developed. Drainage fluid was later positive for tecoplanin-resistant E. faecium and Pseudomonas Aeroginosa responding only to meropenem and ciprofloxacin. Abdominal computerized tomography visualized fluid accumulation around the iliac-femoral bypass. Abcess was ruled out when scintigraphy showed no tracer uptake. The lesion was drained. Drainage fluid cultures were negative for bacteria and fungi. Fluid accumulation gradually disappeared with prolonged antibiotic and antifungal therapy. One year after the accident the patient is in good health, with normal quality of life. CONCLUSION: Successful outcome was due to early, specific antifungal therapy, at sufficiently high dosage which was prolonged for an adequate period of time. Early diagnosis of mucormycosis is essential for efficacious anti-fungal treatment and prevention of irreversible spread of mucormycosis to vital organs. It presupposes awareness that A. corymbifera infection can develop in healthy individuals who are stressed and traumatized through skin-ground contact in accidents.

[Clinico-microbiological comments on various cases of candidemia]. / Osservazioni clinico-microbiologiche su alcuni casi di candidemia.

During a 3 year period 48 patients with one or more blood cultures positive for Candida spp. were enrolled in the study. One patient presented 3 consecutives episodes of candidemia with infection of the port a cath. Fifty cases of candidemia were diagnosed. Candidemia with infection of the central venous catheter was the most frequent diagnosis (52%); in 20% of cases a tissue localization was also present (disseminated candidiasis). Thirty-four out of 50 episodes occurred in the Surgical Department. Among risk factors the most frequent resulted: prolonged antibiotic treatment (100%), intravascular catheter (86%), parenteral nutrition (74%), abdominal surgery (46%). C. albicans was identified more frequently than others Candida spp., resistance to fluconazole was detected in 20% of strains tested. 34/45 episodes of fungemia were treated with fluconazole, none reported side effects. In 5 cases fluconazole was discontinued for clinical failure. Clinical outcome in patients with Candida infection depends on other factors beside in vitro drug susceptibility tests.

A multidrug-resistant Pseudomonas aeruginosa isolate from a lethal case of sepsis induces necrosis of human neutrophils.

A multidrug-resistant Pseudomonas aeruginosa (r-Pa) was isolated from a lethal case of sepsis in a bone marrow transplant recipient. Genotypic analysis of P. aeruginosa isolates demonstrated that sepsis was secondary to gut colonization. The interactions between r-Pa and patient's neutrophils were studied. The results indicate that: (1) the patient's neutrophil killing activity and nitric oxide production against r-Pa or drug sensitive P. aeruginosa (s-Pa) were profoundly impaired; (2) r-Pa cells, but not s-Pa cells or their filtered culture supernatants, induced necrosis of healthy donor neutrophils. Neutrophil necrosis emerges as a remarkable event in the pathogenesis of P. aeruginosa sepsis.

Susceptibility of Streptococcus pyogenes from throat cultures to macrolide antibiotics and influence of collection criteria.

OBJECTIVE: To assess the incidence of resistance to erythromycin and to the three other macrolide antibiotics most extensively used in Italy (azithromycin, clarithromycin and roxithromycin) among clinical strains of Streptococcus pyogenes freshly isolated from throat cultures of pediatric patients in an area of Central Italy. METHODS: Two sets of isolates were examined. The strains of the first set (n=100) were collected according to a protocol admitting only throat swabs from untreated patients with symptoms of acute pharyngotonsillitis. The second set (n=180) consisted of strains isolated from throat cultures during the routine activity of diagnostic laboratories, no particular protocol being applied. RESULTS: A trimodal distribution of strains was observed in relation to their macrolide susceptibility levels: two clusters were constituted by highly susceptible and highly resistant strains, respectively; a third, middle cluster consisted of strains displaying low-level resistance (or even intermediate susceptibility, in a minority of isolates, to clarithromycin). The distribution of individual isolates in the three modal clusters was the same with all four drugs. Both MIC ranges and MIC50s almost overlapped in the isolates of the two sets, whereas MIC90s were far higher in the strains of the second set (4 micro g/mL for clarithromycin, 8 micro g/mL for erythromycin and azythromycin, and 16 micro g/mL for roxithromycin) than in those of the first (0.125 micro g/mL for all four drugs). Resistant strains were 5% among the isolates of the first set and three times as many among those of the second. CONCLUSIONS: The lower incidence of macrolide resistance recorded in the first set is probably more reliable: the threefold incidence observed in the second set may be overestimated due to the lower frequency of strains involved in drug-responsive infections and to the increased occurrence of strains from unsuccessfully treated patients.