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Herein, we sequenced and assembled the genome of a Shigella sonneiisolate carrying several small plasmids using a hybrid approach that combined Oxford Nanopore Technologies and Illumina platforms.

AbstractObjective:To assess the incidence of colonization and infection with carbapenemase-producing Enterobacteriaceae (CPE) and carbapenem-resistant Acinetobacter baumannii(CR-Ab) in the ICUs of our city hospitals before and during the coronavirus disease 2019 (COVID-19) pandemic.Methods:We conducted a multicenter, before-and-after, cross-sectional study to compare the rates of colonization and infection with CPE and/or CR-Ab in 2 study periods, period 1 (January–April 2019) and period 2 (January–April 2020). Incidence rate ratios (IRRs) and 95% confidence intervals (CIs) of weekly colonization and infection rates for each period were compared for the 2 study periods using Poisson regression. Weekly trends in the incidence of colonization or infection for each study period were summarized using local weighted (Loess) regression.Results:We detected no significant change in either IRR and weekly trend in CPE colonization and infection during the 2 study periods. A shift from KPC to other CPE mechanisms (OXA-48 and VIM) was observed during period 2. Compared to period 1, during period 2 the IRR of colonization and infection with CR-Ab increased 7.5- and 5.5-fold, respectively. Genome sequencing showed that all CR-Ab strains belonged to the CC92/IC2 clonal lineage. Clinical strains clustered closely into a single monophyletic group in 1 of the 3 centers, whereas they segregated in 2 different clusters in the other 2 centers, which strongly indicates horizontal transmission.Conclusions:Our findings indicate the need to conduct infection control activities targeted against the spread of antimicrobial resistance between and within hospitals during the COVID-19 pandemic, and if necessary, remodulating them according to the new organizational structures imposed by the pandemic.

•Cefiderocol showed a high failure rate for XDR-AB infections in the CREDIBLE-CR trial.•A case series of ICU patients receiving cefiderocol for XDR-AB infections was analysed.•Microbiological failure occurred in 54% of patients.•Microbiological failure rate was higher in patients achieving suboptimal fCmin/MIC.•Attainment of suboptimal PK/PD target could explain the low eradication rate.•Higher PK/PD targets could be desirable in VAP given the low penetration into ELF.

: Carbapenemase-producing Enterobacterales (CPE) represent a public health concern. The limited antimicrobial options against CPE have led to the development of novel antimicrobial molecules. In the present study, we characterized the genetic determinants associated with the resistance to ceftazidime/avibactam (CAZ-AVI), meropenem/vaborbactam (MER-VAB), imipenem/relebactam (IMI-REL) and cefiderocol (CFD) in a carbapenemase-producing Klebsiella pneumoniaestrain isolated from a critically ill patient.

•We propose a culture-based protocol combining rectal CPE detection and carbapenemase identification.•Workflow integrates automatic digital analysis of chromogenic media (WASPLab®) with rapid tests for carbapenemases.•WASPLab showed a sensitivity/specificity of 100%/79.4% to correctly segregate plates with/without Enterobacteriaceae.•Workflow proved to be fast and reliable, being particularly suitable for areas endemic for KPC-producing K. pneumoniae.

To explore pharmacokinetic/pharmacodynamic (PK/PD) profile of continuous infusion (CI) ceftazidime-avibactam for treating difficult-to-treat resistant Gram-negative (DTR-GN) infections in critical patients undergoing continuous venovenous haemodiafiltration (CVVHDF).

ABSTRACTWe evaluated a real-time single-peak (11.109-Da) detection assay based on matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) for the identification of Klebsiella pneumoniaecarbapenemase (KPC)-producing K. pneumoniae. Our results demonstrated that the 11.109-Da peak was detected in 88.2% of the KPC producers. Analysis of blaKPC-producing K. pneumoniaeshowed that the gene encoding the 11.109-Da protein was commonly (97.8%) associated with the Tn4401a isoform.

ABSTRACTAcute rheumatic fever (ARF) is a postsuppurative sequela caused by Streptococcus pyogenesinfections affecting school-age children. We describe here the occurrence of an ARF outbreak that occurred in Bologna province, northeastern Italy, between November 2012 and May 2013. Molecular analysis revealed that ARF-related group A Streptococcus(GAS) strains belonged to the M-18 serotype, including subtypes emm18.29and emm18.32. All M-18 GAS strains shared the same antigenic profile, including SpeA, SpeB, SpeC, SpeL, SpeM, and SmeZ. Matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) analysis revealed that M-18 GAS strains grouped separately from other serotypes, suggesting a different S. pyogeneslineage. Single nucleotide polymorphisms and phylogenetic analysis based on whole-genome sequencing showed that emm18.29and emm18.32GAS strains clustered in two distinct groups, highlighting genetic variations between these subtypes. Comparative analysis revealed a similar genome architecture between emm18.29and emm18.32strains that differed from noninvasive emm18.0strains. The major sources of differences between M-18 genomes were attributable to the prophage elements. Prophage regions contained several virulence factors that could have contributed to the pathogenic potential of emm18.29and emm18.32strains. Notably, phage FSPBO.1 carried erythrogenic toxin A gene (speA1) in six ARF-related M-18 GAS strains but not in emm18.0strains. In addition, a phage-encoded hyaluronidase gene (hylP.2) presented different variants among M-18 GAS strains by showing internal deletions located in the a-helical and TSßH regions. In conclusion, our study yielded insights into the genome structure of M-18 GAS strains responsible for the ARF outbreak in Italy, thus expanding our knowledge of this serotype.

ABSTRACTKlebsiella pneumoniaeis at the forefront of antimicrobial resistance for Gram-negative pathogenic bacteria, as strains resistant to third-generation cephalosporins and carbapenems are widely reported. The worldwide diffusion of these strains is of great concern due to the high morbidity and mortality often associated with K. pneumoniaeinfections in nosocomial environments. We sequenced the genomes of 89 K. pneumoniaestrains isolated in six Italian hospitals. Strains were selected based on antibiotypes, regardless of multilocus sequence type, to obtain a picture of the epidemiology of K. pneumoniaein Italy. Thirty-one strains were carbapenem-resistant K. pneumoniaecarbapenemase producers, 29 were resistant to third-generation cephalosporins, and 29 were susceptible to the aforementioned antibiotics. The genomes were compared to all of the sequences available in the databases, obtaining a data set of 319 genomes spanning the known diversity of K. pneumoniaeworldwide. Bioinformatic analyses of this global data set allowed us to construct a whole-species phylogeny, to detect patterns of antibiotic resistance distribution, and to date the differentiation between specific clades of interest. Finally, we detected an ∼1.3-Mb recombination that characterizes all of the isolates of clonal complex 258, the most widespread carbapenem-resistant group of K. pneumoniae. The evolution of this complex was modeled, dating the newly detected and the previously reported recombination events. The present study contributes to the understanding of K. pneumoniaeevolution, providing novel insights into its global genomic characteristics and drawing a dated epidemiological scenario for this pathogen in Italy.

Multidrug resistance associated with extended-spectrum beta-lactamase (ESBL) and Klebsiella pneumoniaecarbapenemase (KPC) among K. pneumoniaeis endemic in southern Europe. We retrospectively analyzed the impact of resistance on the appropriateness of empirical therapy and treatment outcomes of K. pneumoniaebloodstream infections (BSIs) during a 2-year period at a 1420-bed tertiary-care teaching hospital in northern Italy. We identified 217 unique patient BSIs, including 92 (42) KPC-positive, 49 (23) ESBL-positive, and 1 (0.5) metallo-beta-lactamase-positive isolates. Adequate empirical therapy was administered in 74 of infections caused by non-ESBL non-KPC strains, versus 33 of ESBL and 23 of KPC cases (p < 0.0001). To clarify the impact of resistance on BSI treatment outcomes, we compared several different models comprised of non-antibiotic treatment-related factors predictive of patients’ 30-day survival status. Acute Physiology and Chronic Health Evaluation (APACHE) II score determined at the time of positive blood culture was superior to other investigated models, correctly predicting survival status in 83 of the study cohort. In multivariate analysis accounting for APACHE II, receipt of inadequate empirical therapy was associated with nearly a twofold higher rate of death (adjusted hazard ratio 1.9, 95 confidence interval 1.1–3.4; p = 0.02). Multidrug-resistant K. pneumoniaeaccounted for two-thirds of all K. pneumoniaeBSIs, high rates of inappropriate empirical therapy, and twofold higher rates of patient death irrespective of underlying illness.

ABSTRACTColistin is a key drug for the treatment of infections caused by extensively drug-resistant strains of Enterobacteriaceaeproducing carbapenemases. However, the emergence of colistin resistance is being increasingly reported, especially among Klebsiella pneumoniaestrains producing KPC-type carbapenemases (KPC-KP). In this work, we investigated colistin-susceptible (KPB-1) and colistin-resistant (KPB-2) sequential isolates obtained from a patient with a KPC-KP infection before and after low-dosage colistin treatment, respectively. By using a next-generation sequencing approach and comparative genomic analysis of the two isolates, we detected in KPB-2 a nonsynonymous nucleotide substitution in the gene encoding the PmrB sensor kinase, resulting in a leucine-to-arginine substitution at amino acid position 82. Compared with KPB-1, KPB-2 exhibited upregulated transcription of pmrAand of pmrK, which is part of the pmrHFIJKLMoperon responsible for modification of the colistin lipopolysaccharide target. Complementation with wild-type pmrBin KPB-2 restored colistin susceptibility and reduced the transcription of pmrAand pmrKto basal levels, while expression of PmrBL82Rin KPB-1 did not alter colistin susceptibility or upregulate pmrAand pmrKexpression, confirming the dominance of wild-type PmrB versus the PmrBL82Rmutant. The present results indicated that PmrB mutations mediating colistin resistance may be selected during low-dosage colistin treatment. The colistin-resistant phenotype of KPB-2 was stable for up to 50 generations in the absence of selective pressure and was not associated with a significant fitness cost in a competition experiment.

ABSTRACTColistin is one of the few agents that retain activity against extensively drug-resistant strains of Klebsiella pneumoniaeproducing KPC-type carbapenemases (KPC-KP). However, resistance to colistin is increasingly reported among KPC-KP. Comparative genomic analysis of a pair of sequential KPC-KP isolates from the same patient including a colistin-susceptible isolate (KKBO-1) and a colistin-resistant isolate (KKBO-4) selected after colistin exposure revealed that insertional inactivation of the mgrBgene, encoding a negative regulator of the PhoQ/PhoP signaling system, is a genetic mechanism for acquired colistin resistance. The role of mgrBinactivation in acquired colistin resistance was confirmed by complementation experiments with wild-type mgrB, which restored colistin susceptibility in KKBO-4, and by construction of an mgrBdeletion mutant from KKBO-1, which exhibited a colistin-resistant phenotype. Insertional mgrBinactivation was also detected in 60% of colistin-resistant mutants selected from KKBO-1 in vitro, following plating on colistin-containing medium, confirming the role (although not unique) of this mechanism in the emergence of acquired colistin resistance. In colistin-resistant mutants carrying insertional inactivation or deletion of the mgrBgene, upregulated transcription of phoP, phoQ, and pmrK(which is part of the pmrHFIJKLMoperon) was detected. These findings confirmed the MgrB regulatory role in K. pneumoniaeand were in agreement with the known association between upregulation of the PhoQ/PhoP system and activation of the pmrHFIJKLMoperon, which eventually leads to resistance to polymyxins by modification of the lipopolysaccharide target.

The choice of foraging areas by large carnivores can be driven both by prey abundance and landscape attributes and it is likely that the relative importance of these two components changes on different spatial scales. In the Bohemian Forest (southwestern Bohemia, Czech Republic), we focused on the effect of microhabitat. We tested if Eurasian lynx Lynx lynxhunted merely in areas where its main prey, roe deer Capreolus capreolusand red deer Cervus elaphus, occurred (‘prey-occurrence hypothesis’) or if there were fine-scale habitat features that increased prey catchability (‘landscape hypothesis’). Fine-scale habitat features were recorded at sites where an ungulate had been killed and located using telemetry or by chance (in winter: N = 29 roe deer, N = 18 red deer; in summer: N = 33 roe deer, N = 5 red deer). We compared these features with those recorded at locations where live red or roe deer were recorded using telemetry (N = 100 per species per six-month period). In winter, lynx killed both roe and red deer at sites where there was a greater heterogeneity in terms of visibility than at sites where live ungulates were recorded, i.e. at kill sites there were both good stalking cover and good visibility. In addition, the risk of predation for red deer was negatively correlated with tree density. In summer, the risk of predation for roe deer was not associated with any of the habitat variables measured. Thus, the presence of a kill was associated with particular fine-scale habitat features in winter, while in summer it was simply associated with where prey occurred. A deeper understanding of the type of habitat favoured by lynx is fundamental to the management and conservation of this species. Based on our results, forest management should ensure that the level of habitat heterogeneity is favourable for lynx.

ABSTRACTDetection of West Nile virus (WNV) by nucleic acid amplification technology (NAAT) is used widely to screen blood and organ donations in areas where WNV is endemic. We report a false-positive result of a WNV transcription-mediated amplification assay (TMA) in a patient with viremia that was caused by Usutu virus, a mosquito-borne flavivirus.

Most seabird species display colonial behavior during the breeding period which implies that food resources around breeding sites can easily go depleted. Seabirds need to both reach profitable areas, which can be located far from the colony, and return to the colony regularly. In this context, flexibility in movement behavior may be crucial for breeding success. During chick-rearing, Procellariformes species can alternate short trips lasting 1–4 days for chick provisioning with longer trips for self-provisioning in what has been called a dual-foraging strategy. We analyzed foraging trips from 136 Scopoli’s shearwaters from three Mediterranean colonies tracked with GPS during 6 chick-rearing seasons to assess whether the adoption of a dual foraging strategy depends on the quality of habitat surrounding the colony. We found a marked dual-foraging strategy only in birds from the Linosa colony which was the largest colony in terms of breeding pairs and was characterized by having a lower marine habitat quality. Birds from this colony performed foraging trips that extended up to 369 km from the nest and lasted more than 10 days. In general, the decision to perform long lasting trips was triggered by lower values of primary production and higher offspring weight. Contrary to expectation, the decision to feed far from the colony was not related to the parents’ weight. At the same time, despite the higher productivity offered by distant areas, the higher proportion of long trips performed by birds breeding in poor areas was not sufficient to maintain the same body mass as the ones breeding in richer areas.