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Backflow of pathogens and endotoxins from the renal pelvis to the bloodstream is one supposed mechanism for infectious complications development after endourological stones surgery. The aim of this study is detecting to prove bacterial spread into irrigation fluid and bloodstream during percutaneous nephrolithotomy (PCNL) and to correlate these findings with clinical and microbiological parameters and infectious complications (IC).Bladder urine culture (BUC) was retrieved before PCNL; during the procedure, 2 blood samples (BC) and an irrigation fluid sample during fragmentation (SFUC) were collected for culture. Stone culture (SC) was also obtained. Patients were evaluated post-operatively for IC.Sixty-one patients were prospectively included. IC occurred in 15 patients (24.6%). SFUC was positive in 7/61 (11.5%); BC in 10/61 (16.4%). Among patients with positive BC; BUC, SFUC and SC were positive in 9 (90%), 6 (60%), and 8 (80%) cases, respectively. Out of 10 patients with positive BC, 4 developed post-operative IC. Pre-operative renal impairment (p = 0.04), intraoperative-evaluated stone residual (p = 0.02), BUC (p = 0.004), and SC (p = 0.008) were associated with IC. No correlation was found between bacterial spread in the irrigation fluid and blood and IC.We proved that bacteria can be detected into the irrigation fluid and blood during PCNL. This transient bacteremia appears to be unrelated to IC development.

Infection prevention and control (IPC) practices are of paramount importance for preventing the spread of multidrug-resistant organisms (MDROs) in hospitals, especially in the intensive care unit (ICU). Whole-genome sequencing (WGS) is seen as a promising tool for IPC, but its employment is currently still limited.

Background and aims. Scedosporium species and Lomentospora prolificans are the filamentous fungi isolated more frequently from the cystic fibrosis (CF) lower airways, after Aspergillus fumigatus. Previous studies showed that, in CF patients, Scedosporium species and Lomentospora prolificans are responsible for airways colonization/ infection, enhancing pulmonary deterioration, and for severe invasive infections in CF patients undergoing lung transplantation. Aims of our project were to evaluate the prevalence of Scedosporium and Lomentospora species in Italian patients with CF, and to evaluate the interest of the Scedosporium-selective colture medium SceSel compared to routinely used agar-based culture media, in order to improve laboratory diagnostic performances.Materials and methods. A total of 1977 sputum samples from 1154 CF patients were collected and processed according to CF Italian recommendations in the year 2017 (January 1st to December 31st). The SceSel agar was used in addition to the routine culture procedures to burst growth and isolation of Scedosporium/ Lomentospora species. The fungal isolates were identified by classical mycological methods and polymerase chain reaction-based DNA sequencing of ITS1 and ITS4 regions. Susceptibility of the isolates to antifungal drugs was investigated by E-test method. Results. Among the 1154 enrolled patients, 62 (5.3%) were positive for Scedosporium/Lomentospora species (median age: 27y; range 8-64y; 33 male). Out of 1977 samples, 93 were positive for Scedosporium/Lomentospora species, and 13 (13.9%) were recovered only on SceSel agar. According to molecular analyses, isolation rates of each species were: S. apiospermum 53.1%, S. boydii 37.5%, L. prolificans 6.3% and S. aurantiacum 3.1%. Amphotericin B minimum inhibitory concentration (MIC) values were above or equal to 2 mg/L in all strains. Voriconazole MIC values were below or equal to 0.38 mg/L in almost all strains, proving this drug to be the most effective antimycotic for members of Scedosporium species. Otherwise, Lomentospora prolificans showed to be resistant to all considered antifungal drugs, only Voriconazole seems to be active on some Lomentospora prolificans isolates. Conclusions. Our results suggest that SceSel agar should be used in combination with routine media as standard microbiological protocol and procedures are not always adequate to isolate Scedosporium and Lomentospora species in CF respiratory samples. Molecular identification and susceptibility tests are needed, especially for strains isolated from critical patients and those considered for transplantation, as they might develop invasive scedosporiosis.

Mycobacterium abscessus (MABS) infection represents significant management challenge in cystic fibrosis (CF) patients. This retrospective study (2005-2016) aims to determine the prevalence of the subspecies of MABS isolated from CF patients, to evaluate the persistence over the years of a single subspecies of MABS and to correlate mutations responsible for macrolides and amikacin resistance with MIC values. We investigated 314 strains (1 isolate/patient/year) isolated from the lower respiratory tract of 51 chronically infected CF patients. Sequencing of rpoB gene was performed to identify the MABS subspecies. The erm(41) gene was sequenced to differentiate the strains with and without inducible macrolide resistance. Regions of 23S and 16S rRNA were sequenced to investigate mutations responsible for constitutive resistance to macrolides and aminoglycosides, respectively. Antibiotic susceptibility, using commercial microdilution plates, was evaluated according to CLSI. M. abscessus subsp. abscessus accounted for 64% of the isolates, bolletii subspecies for 16% and massiliense subspecies for 20%. All the massiliense strains presented truncated erm(41) gene while 12 abscessus strains presented the mutation T28-C in the erm(41) gene, which makes it inactive. The 23S rRNA analysis did not show constitutive resistance to macrolides in any strain. Mutation of the 16S rRNA gene was highlighted in 2 strains out of 314, in agreement with high MIC values. The correct identification at the subspecies level and the molecular analysis of 23S rRNA, 16S rRNA and erm gene is useful to guide the treatment strategy in patients with M. abscessus lung infection.

Background The analysis of microbiome in respiratory samples is a topic of great interest in chronic respiratory diseases. The method used to prepare sputum samples for microbiome analysis is very heterogeneous. The selection of the most suitable methodology for DNA extraction is fundamental to have the most representative data. The objective of this study was to compare different conditions for DNA extraction from sputum in adult patients with bronchiectasis. Methods Five sputum samples from bronchiectasis patients were collected at the Policlinico Hospital in Milan, Italy. Eighteen conditions for DNA extraction were compared, including two enzyme-based (Roche and Zymo) and one beads-based (Mobio) technique. These techniques were tested with/without Dithiothreitol (DTT) and with/without lysostaphin (0.18 and 0.36 mg/mL) step. DNA was quantified, tested using Real-time PCR for 16S rDNA and S. aureus and, then, microbiome was evaluated. Results Although 16S rDNA was similarly detected across all the different techniques, Roche kit gave the highest DNA yield. The lowest Ct values for Real-time PCR for S. aureus was identified when lysostaphin was added. Considering genera from microbiome, alpha diversity indices did not show any significant differences between techniques, while relative abundances were more similar in presence of DTT. Conclusions None of the conditions emerged to be superior to the others even if enzyme-based kits seem to be needed in order to have a higher extraction yield. Electronic supplementary material The online version of this article (10.1186/s40248-018-0166-z) contains supplementary material, which is available to authorized users.

Introduction: There is no consensus on which is the best method to extract bacterial DNA from sputum. We aimed at comparing 20 different DNA extraction techniques on both mock community and sputum samples. Methods: One mock was built and 5 sputa from cystic fibrosis and bronchiectasis patients were evaluated at the Policlinico Hospital in Milan, Italy. 20 techniques for sputum bacterial DNA extraction were compared, including two enzyme-based (Roche and Zymo) and two beads-based (Mobio and QIAGEN) techniques which were tested with/without Dithiothreitol (DTT) and with/without lysostaphin (0.18 and 0.36 mg/mL) steps. Extractions were performed in duplicate to evaluate the variability among samples. DNA was quantified and, then, tested using Real time PCR for 16S rDNA and S. aureus. Results: Although 16S rDNA was similarly detected across all the different techniques in 20 ng of extract (range 19-21 Ct), Roche kit gave the highest DNA yield in both mock and sputa (see figure). The mean standard deviation between replicates was lower in DTT-treated vs non-treated samples (0.17 vs 0.47 Ct, respectively). In both enzymatic-based techniques, the lowest Ct values for Real time PCR for S. aureus in both mock and sputa was identified when lysostaphin was added. Conclusion: An enzyme-based kit with the addition of both DTT treatment and lysostaphin steps seems to be the preferred technique for DNA bacterial extraction on sputum.

Bacteria from the Burkholderia cepacia complex (Bcc) are capable of causing severe infections in patients with cystic fibrosis (CF). Bcc infection is often extremely difficult to treat due to its intrinsic resistance to multiple antibiotics. In addition, it seems to speed up the decline of lung function and is considered a contraindication for lung transplantation in CF. This study investigates the species of the Bcc strains recovered from chronically infected CF subjects by means of: isolation, identification methods and complete recA nucleotide sequences of 151 samples. Molecular typing showed that B. cenocepacia III is the dominant strain found in the group of subjects being treated at the Milan CF Centre (Italy) and that the infection is chronically maintained by the same species. Defining species by means of molecular analysis yields important information for the clinician in order to establish the most appropriate therapy and implement correct measures for prevention of transmission among CF subjects.

Burkholderia cepacia complex (Bcc) includes several phenotypically similar but genotypically distinct gram-negative bacteria (GNB) that can colonize the respiratory tract of Cystic Fibrosis (CF) patients. Pathogens are difficult to treat due to intrinsic resistance to multiple antibiotics and are associated to a more rapid decline in lung function and to increased mortality, particularly after lung transplantation. For all these reasons, chronic infection by Burkholderia (B) cenocepacia is presently considered a relative or absolute contraindication in almost all lung transplant centres. We report the case of a young adult CF patient chronically colonized by B multivorans genomovar II, with diabetes and end-stage renal disease treated with renal replacement therapy: a few months after lung transplantation, she developed post-surgery B multivorans bacteremia and multiple brain abscesses. This severe infection did not improve despite multiple standard antibiotic regimen. The introduction of ceftazidime-avibactam, a new β-lactam/ β-lactamase inhibitor combination resulted in clinical recovery and in radiological and biochemical improvement.

The taxonomic position of members of the Mycobacterium abscessus complex has been the subject of intensive investigation and, in some aspects confusion, in recent years as a result of varying approaches to genetic data interpretation. Currently, the former species Mycobacterium massiliense and Mycobacterium bolletii are grouped together as Mycobacterium abscessus subsp. bolletii. They differ greatly, however, as the former M. bolletii has a functional erm(41) gene that confers inducible resistance to macrolides, the primary therapeutic antimicrobials for M. abscessus, while in the former M. massiliense the erm(41) gene is non-functional. Furthermore, previous whole genome studies of the M. abscessus group support the separation of M. bolletii and M. massiliense. To shed further light on the population structure of Mycobacterium abscessus, 43 strains and three genomes retrieved from GenBank were subjected to pairwise comparisons using three computational approaches: verage ucleotide dentity, enome to enome istance and single nucleotide polymorphism analysis. The three methods produced overlapping results, each demonstrating three clusters of strains corresponding to the same number of taxonomic entities. The distances were insufficient to warrant distinction at the species level, but met the criteria for differentiation at the subspecies level. Based on prior erm(41)-related phenotypic data and current genomic data, we conclude that the species M. abscessus encompasses, in adjunct to the presently recognized subspecies M. abscessus subsp. abscessus and M. abscessus subsp. bolletii, a third subspecies for which we suggest the name M. abscessus subsp. massiliense comb. nov. (type strain CCUG 48898T=CIP 108297T=DSM 45103T=KCTC 19086T).

Different steps and conditions for DNA extraction for microbiota analysis in sputum have been reported in the literature. We aimed at testing both dithiothreitol (DTT) and enzymatic treatments of sputum samples and identifying the most suitable DNA extraction technique for the microbiota analysis of sputum. Sputum treatments with and without DTT were compared in terms of their median levels and the coefficient of variation between replicates of both DNA extraction yield and real-time PCR for the 16S rRNA gene. Treatments with and without lysozyme and lysostaphin were compared in terms of their median levels of real-time PCR for S. aureus. Two enzyme-based and three beads-based techniques for DNA extraction were compared in terms of their DNA extraction yield, real-time PCR for the 16S rRNA gene and microbiota analysis. DTT treatment decreased the coefficient of variation between replicates of both DNA extraction yield and real-time PCR. Lysostaphin (either 0.18 or 0.36 mg/mL) and lysozyme treatments increased S. aureus detection. One enzyme-based kit offered the highest DNA yield and 16S rRNA gene real-time PCR with no significant differences in terms of alpha-diversity indexes. A condition using both DTT and lysostaphin/lysozyme treatments along with an enzymatic kit seems to be preferred for the microbiota analysis of sputum samples. © 2018 by the authors. Licensee MDPI, Basel, Switzerland.

Objective Rasamsonia is a new genus which comprises both thermotolerant Talaromyces and thermophilic Geosmithia species. Rasamsonia argillacea (RA) has recently been reported as the cause of invasive mycosis in human chronic granulomatous disease and also as airway colonizer in patients with cystic fibrosis (CF). The aim of this work is to follow patients colonized by emerging fungus Rasamsonia and study their clinical characteristics and conditions. Methods Over a two years period (2013–2014), seven out of the 798 CF patients, followed at Milan centre, showed at least one positive culture for Rasamsonia . RA was identified macroscopically and microscopically by lactic blue stain. Molecular biology analysis on RA strains will be carried out. Results No correlation between RA colonization and patient sex (4F, 3M) was found. Six patients carried at least one copy of mutated allele DF508. Five patients received antimycotic oral therapy (4 itraconazole and 1 voriconazole) before RA recovery. Two out of the patients (both treated with itraconazole) were intermittently colonized with RA. One of patients was also chronically colonized with Scedosporium apiospermum. Pulmonary function (FEV1) remained substantially unvaried. Conclusion All patients were chronically colonized also by various bacteria species: probably the presence of bacteria and the resulting inflammatory response can predispose CF airways to RA acquisition. Further studies are needed to asses the possible association between genotype of patients and colonization by RA, whether antimycotic administration could be a risk factor for RA acquisition and to investigate the RA impact on pulmonary function.

Objectives To evaluate the molecular profile of MRSA strains isolated from the low respiratory tract of CF patients chronically colonized, in order to investigate its origin (HA-MRSA or CA-MRSA) and to examine, in a subgroup of patients, the acquisition of strains with a different molecular profile in the course of time. Methods Forty-four patients (9%) out of the 793 followed at the CF Centre of Milan suffer from MRSA chronic infection. Seventy-eight MRSA strains isolated from these patients have been characterized by: Multiplex PCR ( mec A gene and the SCC mec -type), spa -typing and a specific PCR for Panton Valentine (PVL) gene. Thirty-four out of 44 patients have been monitored over time (T1 = 2012; T2=2014) in order to investigate the molecular profile of MRSA strains isolated after the first infection. Results All 78 MRSA strains have the mecA gene; 76% present SCC mec type IV (CA-MRSA), 21% belong to spa-type t032 (UK-EMRSA-15) and 20% to spa-type t008. Among 4 strains with spa-type t008 and SCC mec type IVa (USA-300), only two show the PVL gene. The remaining 24% present SCC mec type I (HA-MRSA). The molecolar profile of the strains isolated in T2 was the same of the one isolated at T1 in 29/34 patient while 5/34 had different profile. Conclusion The molecular characterization of MRSA strains isolated at Milan CF Centre has shown a high prevalence of CA-MRSA (SCC mec type IV). The distribution of spa-type shows to be similar to that described in other European countries. The different MRSA molecular profiles suggest to carry out genotyping analyses as well, in order to highlight the presence of clones in our CF population.

Objectives To perform molecular typing of clinical strains of RGM isolated from the lower respiratory tract of CF patients with persistent, transient and intermittent infections. Secondary, to assess whether chronic infections are sustained by the same bacterial species, over time. Methods Twenty-three patients, out of the 793 followed at the Regional Reference Centre for Cystic Fibrosis of Milan, have a persistent RGM infection, and 19 show an intermittent infection. From October 2013 to October 2014, 65 strains were isolated and typed by gene sequencing of the rpo B gene. The gene sequences obtained were compared to those already present in the GenBank database to discriminate subspecies of RGM. Results Molecular typing of 46 strains (at two different follow-up times: T1, T2), isolated from 23 patients with chronic RGM colonization, reveals that chronic infection are sustained, in 100% of cases, by MAC (61% M. abscessus sensu stricto, 26% M. bolletii and 13% M. massiliense ), and species identified at T1 is the same identified at T2. In 19 patients, the intermittent infection is sustained by different species of RGM: 37% M. chelonae, 26% M. abscessus sensu stricto, 26% M. bolletii, 5.5% M. fortuitum and 5.5% M. massiliense. Conclusion The chronic infections are sustained by MAC, with a higher prevalence of M. abscessus sensu stricto, and that species acquired at T1 persists over time. Intermittent infections are sustained by M. chelonae, M. fortuitum and MAC. Further correlation studies between the clinical course of the pulmonary disease and the presence of MAC different subspecies are recommended in order to create appropriate therapeutic schemes.