Exploring Antibiotic Resistance Diversity in Leuconostoc spp. by a Genome-Based Approach: Focus on the {lsaA} Gene

Leuconostoc spp. are environmental microorganisms commonly associated with fermented foods. Absence of antibiotic resistance (AR) in bacteria is a critical issue for global food safety. Herein, we updated the occurrence of AR genes in the Leuconostoc genus through in silico analyses of the genomes of 17 type strains. A total of 131 putative AR traits associated with the main clinically relevant antibiotics were detected. We found, for the first time, the lsaA gene in L. fallax ATCC 700006T and L. pseudomesenteroides NCDO 768T. Their amino acid sequences displayed high similarities (59.07% and 52.21%) with LsaA of Enterococcusfaecalis V583, involved in clindamycin (CLI) and quinupristin-dalfopristin (QUD) resistance. This trait has different distribution patterns in Leuconostoc nontype strains-i.e., L. pseudomesenteroides, L. lactis and L. falkenbergense isolates from fermented vegetables, cheeses, and starters. To better explore the role of lsaA, MIC for CLI and QUD were assessed in ATCC 700006T and NCDO 768T; both strains were resistant towards CLI, potentially linking lsaA to their resistant phenotype. Contrarily, NCDO 768T was sensitive towards QUD; however, expression of lsaA increased in presence of this antibiotic, indicating an active involvement of this trait and thus suggesting a revision of the QUD thresholds for this species

Pleural tuberculosis: medical thoracoscopy greatly increases the diagnostic accuracy

Our objective was to evaluate the efficacy of a standardised work-up in the diagnosis of pleural tuberculosis (TB) that included fibreoptic bronchoscopy and medical thoracoscopy. A consecutive series of 52 pleural TB patients observed during the period 2001-2015 was evaluated retrospectively. 20 females, mean (range) age 39.7 (18-74) years, and 32 males, mean (range) age 45.75 (21-83) years, were included (28 non-EU citizens (53.8%)). The diagnosis of TB infections was established by identification (using stains, culture or molecular tests) of Mycobacterium tuberculosis in the pleura, sputum and/or bronchial specimens, or by evidence of caseous granulomas on pleural biopsies. Patients with and without lung lesions were considered separately. The diagnostic yield of the microbiological tests on pleural fluid was 17.3% (nine out of 52 patients). Among the 18 patients with lung lesions, bronchial samples (washing, lavage or biopsy) were positive in 50% of cases (nine patients). Cultures of pleural biopsies were positive in 63% of cases (29 out of 46 patients); pleural histology was relevant in all patients. Without pleural biopsy, a diagnosis would have been reached in 15 out of 52 patients (28.6%) and in four of them only following culture at 30-40 days. An integrated diagnostic work-up that includes all the diagnostic methods of interventional pulmonology is required for a diagnosis of pleural TB. In the majority of patients, a diagnosis can be reached only with pleural biopsy

Active surveillance for carbapenemaseproducing Klebsiella pneumoniae and correlation with infection in subjects attending an Italian tertiary-care hospital: a 7-year retrospective study

Objectives The distribution of carbapenemase-producing Klebsiella pneumoniae (CPKP) phenotypes and genotypes in samples collected during 2011–2018 was evaluated. The association between patients with CPKP-positive rectal swab and those with CPKP infection, as well as the overall analysis of CPKP-infected patients, was performed. Setting The study was performed in a tertiary-care hospital located in Northern Italy. Participants Two groups were considered: 22 939 ‘atrisk’ patients submitted to active surveillance for CPKP detection in rectal swabs/stools and 1094 CPKP-infected patients in which CPKP was detected in samples other than rectal swabs. Results CPKP-positive rectal swabs were detected in 5% (1150/22 939). A CPKP infection was revealed in 3.1% (719/22 939) of patients: 582 with CPKP-positive rectal swab (50.6% of the 1150 CPKP-positive rectal swabs) and 137 with CPKP-negative rectal swab. The 49.4% (568/1150) of the patients with CPKP-positive rectal swab were carriers. The overall frequency of CPKP-positive patients (carriers and infected) was almost constant from 2012 to 2016 (excluding the 2015 peak) and then increased in 2017–2018. blaKPC was predominant followed by blaVIM. No difference was observed in the frequency of CPKP-positive rectal swab patients among the different material groups. Among the targeted carbapenemase genes, blaVIM was more significantly detected from urine than from other samples. Conclusions The high prevalence of carriers without evidence of infection, representing a potential reservoir of CPKP, suggests to maintain the guard about this problem, emphasising the importance of active surveillance for timely detection and separation of carriers, activation of contact precautions and antibiotic treatment guidance on suspicion of infection

Finding a correct species assignment for a Metschnikowia strain: insights from the genome sequencing of strain DBT012

: Metschnikowia pulcherrima is an important yeast species that is attracting increased interest thanks to its biotechnological potential, especially in agri-food applications. Phylogenetically related species of the so-called 'pulcherrima clade' were first described and then reclassified in one single species, which makes the identification an intriguing issue. Starting from the whole-genome sequencing of the protechnological strain Metschnikowia sp. DBT012, this study applied comparative genomics to calculate similarity with the M. pulcherrima clade publicly available genomes with the aim to verify if novel single-copy putative phylogenetic markers could be selected, in comparison with the commonly used primary and secondary barcodes. The genome-based bioinformatic analysis allowed the identification of 85 consensus single-copy orthologs, which were reduced to three after split decomposition analysis. However, wet-lab amplification of these three genes in nonsequenced type strains revealed the presence of multiple copies, which made them unsuitable as phylogenetic markers. Finally, average nucleotide identity (ANI) was calculated between strain DBT012 and available genome sequences of the M. pulcherrima clade, although the genome dataset is still rather limited. Presence of multiple copies of phylogenetic markers as well as ANI values were compatible with the recent reclassification of the clade, allowing the identification of strain DBT012 as M. pulcherrima

Higher recovery rate of microorganisms from cerebrospinal fluid samples by the BACTEC culture system in comparison with agar culture

The aim of this study was to assess the diagnostic value of the BACTEC FX blood culture (BC) system as compared to the agar culture (AC) of cerebrospinal fluid samples (CSF), evaluating the recovery rate and the time to detection of microorganisms in a 3.5-year period. From December 2011 to May 2015, 1326 CSF samples (694 patients) were submitted to both AC and BC. Among the 150 positive samples (96 patients), 165 microorganisms were detected: 81 by both the protocols, 77 by BC alone, and 7 by AC alone, demonstrating a higher detection rate of BC (95.8%) than AC (53.3%). Although BC presents some disadvantages, it is able to improve the yield of clinically significant microorganisms, and it could potentially reduce the reporting time as compared to AC. The results obtained highlighted the necessity of a combined approach for the successful detection of central nervous system microbial infections

Comparison of the BACTEC™ blood culture system with conventional culture of cerebrospinal fluid samples.

Objective – Many invasive andlife-threatening infections are diagnosed by microbial culture of sterile bodyfluid specimens. The standard procedures used for conventional cultivation ofbacteria and yeasts from sterile body fluids other than blood involveinoculation onto a solid medium. The aim of this study was to evaluate theBactec blood culture system (Becton Dickinson,Sparks, MD) in comparison with the conventional culturemethod for microbial isolation from cerebrospinal fluid (CSF) samples. Methods - From January 2012 to November2013, 788 samples of CSF collected from 390 patients with the clinical suspicion of centralnervous system infection (meningitis, encephalitis) or polytrauma involvingcentral nervous system were routinely sent to the Bacteriology laboratory of the Unit ofClinical Microbiology at the University Hospital of Parma for the diagnosisof infections by bacteria and fungi. Conventional cultures were performed on chocolate agar,blood agar, MacConkey agar plates for aerobic bacteria, and when possibledepending on the volume of sample available on bile-esculine agar and Schaedleragar plates for anaerobic bacteria (KIMA, Piove di Sacco-PD, Italy) accordingto standard procedures; in parallel, an aliquot (1ml) of each sample wasinoculated directly into an aerobic Bactec Ped Plus/F bottle added withFastidious Organism Supplement (Becton Dickinson) which improves theopportunity for growth of fastidious organisms. When a bottle signaled apositive result by the Bactec FX instrument a Gram stain was performed and analiquot was subcultured onto conventional media. Results – Among the 87 positive samplesbelonging to 51 patients analyzed, clinically significant microorganisms(mainly bacteria and in three cases fungi) were identified from 45 samples(51.72%) by both methods. For the remaining 42 specimens (48.28%), growth wasdetected by the Bactec system, while there was no growth onto solid mediainoculated directly with the samples. No microorganism which went undetected bythe Bactec system was detected by conventional cultures. The most frequentlymicroorganisms recovered only by the Bactec system were gram-positive cocci (21coagulase-negative staphylococci, 5 Streptococcus spp., 1 Kocuriakristinae and 1 Rothia mucilaginosa) while theGram-negative bacilli and Gram-positive bacteria were equally recovered by bothculture methods. In two samples belonging to different patients the Bactecsystem alone allowed the isolation of Staphylococcus aureus and in eightsamples (belonging 4 patients) of Gram-negative bacilli. Conclusion – The Bactec system was shownto enhance the detection of microorganisms in CSF versus conventionalmethods of 48.28%. Although part of these additional positive results could belikely referred as due to contamination during sample collection, we canconclude that when applied and evaluated on a wider group of samples, theBactec blood culture system might be in the future routinely used to improvethe yield of clinically significant microorganisms from cerebrospinal-fluid

Seven-year active surveillance on carbapenemase-producing Klebsiella pneumoniae colonisation in subjects attending an Italian University hospital

Background: Carbapenem-resistantEnterobacteriaceae (CRE),especially Klebsiella pneumoniae, havespread globally and representa serious and growing threat to public health.Theaim of this study is theevaluation of the situation about the distribution of the phenotypes and genotypes of thecarbapenemase-producing (CP) Klebsiella pneumoniae strains circulating in the University-Hospital of Parma as a result of theapplication of theactive surveillance plan during thelast seven years. Materials/methods: A total of 34.043 rectal swabs from 22.944 high-risk patients (e.g. discharges from, or readmissions to,acutecarefacilities, long-term carefacilities and intensivecare units) from November 2011 to October 2018.Therectal swabs wereinoculated onto chromogenicagar and the bluecolonies referring to presumptive CRE weresubcultured on MacConkey agar with a carbapenem disk.Thecarbapenem nonsusceptible strains wereidentified by MALDI-TOF MS and, when K. pneumoniae was revealed, thestrains weresubmitted to CR confirmation by antimicrobial susceptibility testing and to phenotypical analysis by modified Hodgeand disk diffusion inhibition tests,and to genotypical characterization. Results: Carbapenem-resistant K. pneumoniae strains were detected in 1178 cases and the production of carbapenemase was revealed in 1150 cases (5%).With regard to the genes detected by the molecular genotyping assay,all types of carbapenemase genes were detected among theanalyzed samples.The KPC gene was predominant followed by the VIM target, while OXA-48 and NDM genes were morerarely observed. In about 1% of the CP K. pneumoniae strains none of thetargeted genes was revealed. Conclusions:Since 2013, the Centers for Disease Control and Prevention assigned the highest threat level to CRE and declared that CRE require urgent public health attention.The detailed data regarding a correlation between the CP K. pneumoniaecolonized subjects and thoseinfected in thesame period will beextensively presented. In our experience, theresults of theapplication of activesurveillance on high risk patient categories suggest to maintain the guard about this problem

Epidemiologia delle enteriti nella popolazione adulta e pediatrica nell’area di Parma

Introduzione. L’enterite acuta è una delle principali cause di morbilità e mortalità nel mondo, soprattutto nei bambini; tuttavia, i metodi diagnostici convenzionali per il rilevamento di patogeni enterici richiedono tempo, sono laboriosi, spesso mancano di sensibilità e specificità lasciando casi non diagnosticati. Scopo. Questo studio riporta un quadro epidemiologico degli enteropatogeni rilevati in tutti i pazienti con enterite, di sospetta origine batterica e/o virale, pediatrici ed in una selezione di pazienti adulti in un periodo di 2,5 anni. Materiali e metodi. 2532 campioni di feci (2128 pediatrici, 1651 italiani 477 stranieri, 1420 ricoverati 708 ambulatoriali, età media 4 anni, e 404 adulti, 363 italiani 44 stranieri, 236 ricoverati e 168 ambulatoriali, età media 37 anni), sono stati esaminati mediante FilmArray® Gastrointestinal Panel (FA-GP) e, quando possibile, microscopia elettronica (1590) per la rilevazione di virus e un saggio di real-time PCR per enterovirus (2508). I campioni risultati positivi per batteri e parassiti sono stati sottoposti anche a metodi diagnostici convenzionali. Risultati. FA-GP è risultato positivo in 1332 campioni (52,6%): in 919 (69%) erano presenti singoli agenti mentre in 413 (31%) agenti multipli, per un totale di 1890 agenti. Quelli più comunemente rilevati sono stati E. coli enteropatogeno, rotavirus, norovirus e sapovirus. FA-GP ha rilevato 1101 agenti (488 dei quali identificati come singolo patogeno) non inclusi nel sospetto clinico. La percentuale di recupero dei metodi convenzionali è stata 40,8% per i batteri, 34.2% per i virus e 68,2% per i parassiti. Enterovirus è stato rivelato nel 7,9% (198/2508) dei campioni, come unico agente in 75 casi. Conclusioni. Questo studio, che evidenzia anche l'utilità di saggi a rilevazione multipla, conferma il ruolo prevalente degli agenti virali, quali rotavirus e norovirus, nelle enteriti pediatriche e di Campylobacter in quelle degli adulti; inoltre, dimostra la crescente prevalenza dell'infezione da E. coli enteropatogeno e sapovirus

A new laboratory workflow for the diagnosis of gastroenteritis in pediatric patients by using FilmArray® Gastrointestinal Panel

Introduction. Pathogen-induced acute gastroenteritis is one of the leading cause of childhood morbidity and mortality worldwide. Conventional diagnostic methods for routine detection of enteric pathogens are time consuming, labor-intensive, often lack sensitivity and specificity, and leave undiagnosed cases. More recently, the introduction of the syndromic multiplex PCR systems has allowed to simultaneously detecting a wider range of enteric pathogens. This study represents a 2-year picture of the epidemiology of enteric pathogens in children suffering from gastroenteritis using the FilmArray® Gastrointestinal Panel (FA-GP), a multiplex molecular assay that allows to simultaneously detecting a large panel of pathogens independently of the etiological suspicion. Materials and Methods. A total of 2128 stool samples, collected from children with clinical suspicion of bacterial and/or viral gastroenteritis attending the University Hospital of Parma, was submitted to the FA-GP and, when an adequate aliquot was available, to electron microscopy (n = 1494) for virus detection and to an enterovirus-targeting real-time PCR (n = 2110). Specimens with positive results for Salmonella, Yersinia enterocolitica, Vibrio, diarrheagenic Escherichia coli/Shigella, Campylobacter, Plesiomonas shigelloides by the FA-GP were also submitted to conventional diagnostic methods. When a parasite was detected by the FA-GP, the same sample and/or additional samples up to three per patient were submitted to conventional assays as confirmed. Results. The FA-GP gave positive results in 1171 (55%) cases: 801 (68.4%) contained a single agent and 370 (31.6%) multiple agents, for a total of 1660 pathogens. Enteropathogenic E. coli, rotavirus, norovirus, toxigenic Clostridioides difficile, and sapovirus were the most commonly detected pathogens. A total of 970 additional agents (424 of which as single pathogen) was detected by the FA-GP and not included in the clinical suspicion. The overall recovery rate of the conventional methods from stools that resulted positive by the FA-GP was 39% for bacteria, 35.7% and 82.9% for Giardia intestinalis and Cryptosporidium, respectively, and ranged from 3.2% to 62.8% for viruses, if excluding all electron microscopy-negative astroviruses. Enterovirus, an agent not targeted by the FA-GP, was revealed in 9.2% (194/2110) of the examined samples, and in 73 cases it was the only agent detected. Discussion and Conclusions. The results of this study allowed to extend the range of detectable pathogens independently of the clinical suspicion, to detect co-infections in almost one third of children positive for at least one agent and to show that conventional methods would have missed more than half of the enteric agents detected by the FA-GP