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The COVID-19 pandemic, especially during the first wave, posed a great challenge to the hospital management and generally promoted nosocomial pathogen dissemination. MDR A. baumannii can easily spread and persist for a long time on surfaces, causing outbreaks in health care settings.

Consumption of raw food, especially smoked fish, meat, soft cheeses, and vegetables, contaminated with Listeria monocytogenes can cause listeriosis, which can be invasive in pregnant women, elderly...

Acinetobacter baumanniiis a nosocomial pathogen that poses a serious threat due to the rise of incidence of multidrug resistant (MDR) strains. During the COVID-19 pandemic, MDRA.baumanniiclones have caused several outbreaks worldwide. Here we describe a detailed investigation of an MDRA. baumanniioutbreak that occurred at Fondazione IRCCS Policlinico San Matteo (Pavia, Italy). A total of 96A. baumanniistrains, isolated between January and July 2020 from 41 inpatients (both SARS-CoV-2 positive and negative) in different wards, were characterized by phenotypic and genomic analyses combining Illumina and Nanopore sequencing. Antibiotic susceptibility testing revealed that all isolates were resistant to carbapenems and the sequence analysis attributed this to the carbapenemase geneblaOXA-23. Screening of virulence factors unveiled that all strains carried determinants for biofilm formation, while plasmid analysis revealed the presence of two plasmids, one of which was a ⍰100kbp long and encoded a phage sequence.A core genome-based phylogeny was inferred to integrate outbreak strain genomes with background genomes from public databases and from the local surveillance program. All strains belonged to the globally disseminated ST2 clone and were mainly divided into two clades. Isolates from the outbreak clustered with surveillance isolates from 2019, suggesting that the outbreak was caused by two strains that were already circulating in the hospital before the start of the pandemic. The intensive spread ofA. baumanniiin the hospital was enhanced by the extreme emergency situation of the first COVID-19 pandemic wave that resulted in minor attention to infection prevention and control practices.ImportanceThe COVID-19 pandemic, especially during the first wave, posed a great challenge to the hospital management and generally promoted nosocomial pathogen dissemination. Multidrug resistant (MDR)A. baumanniican easily spread and persist for a long time on surfaces, causing outbreaks in healthcare settings. Infection prevention and control practices, epidemiological surveillance and microbiological screening are fundamental in order to control such outbreaks.Here, we sequenced the genomes of 96 isolates from an outbreak of MDRA. baumanniistrains using both short- and long-read technology in order to reconstruct the outbreak events in fine detail. The sequence data demonstrated that two endemic clones of MDRA. baumanniiwere the source of this large hospital outbreak during the first COVID-19 pandemic wave, confirming the effect of COVID-19 emergency disrupting the protection provided by the use of the standard prevention procedures.

Studies are needed to better understand the genomic evolution of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This study aimed to describe viral quasispecies population of upper and lower respiratory tract by next-generation sequencing in patients admitted to intensive care unit. A deep sequencing of the S gene of SARS-CoV-2 from 109 clinical specimens, sampled from the upper respiratory tract (URT) and lower respiratory tract (LRT) of 77 patients was performed. A higher incidence of non-synonymous mutations and indels was observed in the LRT among minority variants. This might be explained by the ability of the virus to invade cells without interacting with ACE2 (e.g. exploiting macrophage phagocytosis). Minority variants are highly concentrated around the gene portion encoding for the Spike cleavage site, with a higher incidence in the URT; four mutations are highly recurring among samples and were found associated with the URT. Interestingly, 55.8% of minority variants detected in this locus were TG and GT transversions. Results from this study evidenced the presence of selective pressure and suggest that an evolutionary process is still ongoing in one of the crucial sites of spike protein associated with the spillover to humans.

Here, we describe the isolation of a strain of the genus Pantoea encoding a VIM carbapenemase, the first to our knowledge. The strain, isolated from a rectal swab of a 10-day-old newborn admitted to a neonatal intensive care unit (NICU), was identified through whole-genome sequencing analyses as Pantoea brenneri. The strain harbored the carbapenemases gene blaVIM-1. The prompt application of contact measures and the isolation of the newborn prevented the dissemination of VIM-producing P. brenneri and of the plasmid carrying the VIM-1 gene to other newborns.

Klebsiella pneumoniae (Kp) is one of the most important nosocomial pathogens world-wide, being responsible for frequent hospital outbreaks and causing sepsis and multi-organ infections with a high mortality rate and frequent hospital outbreaks. The most prevalent and widely disseminated lineage of K. pneumoniae is clonal group 258 (CG258), which includes the highly resistant “high-risk” genotypes ST258 and ST11. Recent studies revealed that very large recombination events have occurred during the recent emergence of Kp lineages. A striking example is provided by ST258, which has undergone a recombination event that replaced over 1 Mb of the genome with DNA from an unrelated Kp donor. Although several examples of this phenomenon have been documented in Kp and other bacterial species, the significance of these very large recombination events for the emergence of either hyper-virulent or resistant clones remains unclear. Here we present an analysis of 834 Kp genomes that provides data on the frequency of these very large recombination events (defined as those involving >100Kb), their distribution within the genome, and the dynamics of gene flow within the Kp population. We note that very large recombination events occur frequently, and in multiple lineages, and that the majority of recombinational exchanges are clustered within two overlapping genomic regions, which result to be involved by recombination events with different frequencies. Our results also indicate that certain non-CG258 lineages are more likely to act as donors to CG258 recipients than others. Furthermore, comparison of gene content in CG258 and non-CG258 strains agrees with this pattern, suggesting that the success of a large recombination depends on gene composition in the exchanged genomic portion.Author SummaryKlebsiella pneumoniae (Kp) is an opportunistic bacterial pathogen, a major cause of deadly infections and outbreaks in hospitals worldwide. This bacterium is able to exchange large genomic portions (up to a fourth of the entire genome) within a single recombination event. Indeed, the most epidemiologically important Kp clone, is actually a hybrid which emerged after a > 1Mb recombination event. In this work, we investigated how recombinations affected the evolution of the most studied Kp Clonal Group, CG258. We found that large recombinations occurred frequently during Kp evolution, and occurred preferentially in a well-delimited genomic region. Furthermore, we found that four epidemiologically important clones emerged after large recombinations. We identified the donors of several large recombinations: despite many Kp lineages acted as donors during CG258 evolution, two of them have been involved more frequently. We hypothesize that the observed pattern of donors-recipients in recombinations, and the presence of a large recombinogenic region in Kp genome, could be related to gene composition. Indeed, genomic analyses showed a pattern compatible with this hypothesis, suggesting that gene content can represent a main factor in the success of a large recombination.

Consumption of raw food, especially smoked fish, meat, soft cheeses, and vegetables, contaminated with

Neutralizing antibodies that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein are among the most promising approaches against COVID-191,2. A bispecific IgG1-like molecule (CoV-X2) has been developed on the basis of C121 and C135, two antibodies derived from donors who had recovered from COVID-193. Here we show that CoV-X2 simultaneously binds two independent sites on the RBD and, unlike its parental antibodies, prevents detectable spike binding to the cellular receptor of the virus, angiotensin-converting enzyme 2 (ACE2). Furthermore, CoV-X2 neutralizes wild-type SARS-CoV-2 and its variants of concern, as well as escape mutants generated by the parental monoclonal antibodies. We also found that in a mouse model of SARS-CoV-2 infection with lung inflammation, CoV-X2 protects mice from disease and suppresses viral escape. Thus, the simultaneous targeting of non-overlapping RBD epitopes by IgG-like bispecific antibodies is feasible and effective, and combines the advantages of antibody cocktails with those of single-molecule approaches. The bispecific IgG1-like CoV-X2 prevents SARS-CoV-2 spike binding to ACE2, neutralizes SARS-CoV-2 and its variants of concern, protects against disease in a mouse model, whereas the parental monoclonal antibodies generate viral escape.

From February to April 2020, Lombardy (Italy) reported the highest numbers of SARS-CoV-2 cases worldwide. By analyzing 346 whole SARS-CoV-2 genomes, we demonstrate the presence of seven viral lineages in Lombardy, frequently sustained by local transmission chains and at least two likely to have originated in Italy. Six single nucleotide polymorphisms (five of them non-synonymous) characterized the SARS-CoV-2 sequences, none of them affecting N-glycosylation sites. The seven lineages, and the presence of local transmission clusters within three of them, revealed that sustained community transmission was underway before the first COVID-19 case had been detected in Lombardy., The Lombardy region of Italy was heavily affected early in the SARS-CoV-2 pandemic. Here, the authors use whole genome sequencing and show that there were multiple introductions into the region, with transmission occurring before the first case was detected.

The SARS-CoV-2 pandemic that we are currently experiencing is exerting a massive toll both in human lives and economic impact. One of the challenges we must face is to try to understand if and how different variants of the virus emerge and change their frequency in time. Such information can be extremely valuable as it may indicate shifts in aggressiveness, and it could provide useful information to trace the spread of the virus in the population. In this work we identified and traced over time 7 amino acid variants that are present with high frequency in Italy and Europe, but that were absent or present at very low frequencies during the first stages of the epidemic in China and the initial reports in Europe. The analysis of these variants helps defining 6 phylogenetic clades that are currently spreading throughout the world with changes in frequency that are sometimes very fast and dramatic. In the absence of conclusive data at the time of writing, we discuss whether the spread of the variants may be due to a prominent founder effect or if it indicates an adaptive advantage.

The circulation of carbapenem-resistant Klebsiella pneumoniae (CRKP) is a significant problem worldwide. In this work we characterize the isolates and reconstruct the spread of a multi-clone epidemic event that occurred in an Intensive Care Unit in a hospital in Northern Italy. The event took place from August 2015 to May 2016 and involved 23 patients. Twelve of these patients were colonized by CRKP at the gastrointestinal level, while the other 11 were infected in various body districts. We retrospectively collected data on the inpatients and characterized a subset of the CRKP isolates using antibiotic resistance profiling and whole genome sequencing. A SNP-based phylogenetic approach was used to depict the evolutionary context of the obtained genomes, showing that 26 of the 32 isolates belong to three genome clusters, while the remaining six were classified as sporadic. The first genome cluster was composed of multi-resistant isolates of sequence type (ST) 512. Among those, two were resistant to colistin, one of which indicating the insurgence of resistance during an infection. One patient hospitalized in this period was colonized by two strains of CRKP, both carrying the blaKPC gene (variant KPC-3). The analysis of the genome contig containing the blaKPC locus indicates that the gene was not transmitted between the two isolates. The second infection cluster comprised four other genomes of ST512, while the third one (ST258) colonized 12 patients, causing five clinical infections and resulting in seven deaths. This cluster presented the highest level of antibiotic resistance, including colistin resistance in all 17 analyzed isolates. The three outbreaking clones did not present more virulence genes than the sporadic isolates and had different patterns of antibiotic resistance, however, were clearly distinct from the sporadic ones in terms of infection status, being the only ones causing overt infections.

Acinetobacter baumannii is a known opportunistic pathogen, dangerous for public health mostly due to its ability to rapidly acquire antibiotic-resistance traits. Its genome was described as characterized by remarkable plasticity, with a high frequency of homologous recombinations and proliferation of Insertion Sequences (IS). The SMAL pulsotype is an A. baumannii strain currently isolated only in Italy, characterized by a low incidence and a high persistence over the years. In this present work, we have conducted a comparative genomic analysis on this clone. The genome of 15 SMAL isolates was obtained and characterized in comparison with 24 other assemblies of evolutionary related isolates. The phylogeny highlighted the presence of a monophyletic clade (named ST78A), which includes the SMAL isolates. ST78A isolates have a low rate of homologous recombination and low gene content variability when compared to two related clades (ST78B and ST49) and to the most common A. baumannii variants worldwide (International Clones I and II). Remarkably, genomes in the ST78A clade present a high number of IS, including classes mostly absent in the other related genomes. Among these IS, one copy of IS66 was found to interrupt the gene comEC/rec2, involved in the acquisition of exogenous DNA. The genomic characterization of SMAL isolates shed light on the surprisingly low genomic plasticity and the high IS proliferation present in this strain. The interruption of the gene comEC/rec2 by an IS in the SMAL genomes brought us to formulate an evolutionary hypothesis according to which the proliferation of IS is slowing the acquisition of exogenous DNA, thus limiting genome plasticity. Such genomic architecture could explain the epidemiological behavior of high persistence and low incidence of the clone and provides an interesting framework to compare ST78 with the highly epidemic International Clones, characterized by high genomic plasticity.

Vertically-transmitted bacterial symbionts are widespread in ticks and have manifold impacts on the epidemiology of tick-borne diseases. For instance, they may provide essential nutrients to ticks, affect vector competence, induce immune responses in vertebrate hosts, or even evolve to become vertebrate pathogens. The deer or blacklegged tick Ixodes scapularis harbours the symbiont Rickettsia buchneri in its ovarian tissues. Here we show by molecular, proteomic and imaging methods that R. buchneri is also capable of colonising the salivary glands of wild I. scapularis. This finding has important implications for the diagnosis of rickettsial infections and for pathogen-symbiont interactions in this notorious vector of Lyme borreliosis.

The circulation of carbapenem-resistant Klebsiella pneumoniae (CRKP) is a significant problem worldwide. In this work we characterize the isolates and reconstruct the spread of a multi-clone epidemic event that occurred in an Intensive Care Unit in a hospital in Northern Italy. The event took place from August 2015 to May 2016 and involved 23 patients. Twelve of these patients were colonized by CRKP at the gastrointestinal level, while the other 11 were infected in various body districts. We retrospectively collected data on the inpatients and characterized a subset of the CRKP isolates using antibiotic resistance profiling and whole genome sequencing. A SNP-based phylogenetic approach was used to depict the evolutionary context of the obtained genomes, showing that 26 of the 32 isolates belong to three genome clusters, while the remaining six were classified as sporadic. The first genome cluster was composed of multi-resistant isolates of sequence type (ST) 512. Among those, two were resistant to colistin, one of which indicating the insurgence of resistance during an infection. One patient hospitalized in this period was colonized by two strains of CRKP, both carrying the blaKPC gene (variant KPC-3). The analysis of the genome contig containing the blaKPC locus indicates that the gene was not transmitted between the two isolates. The second infection cluster comprised four other genomes of ST512, while the third one (ST258) colonized 12 patients, causing five clinical infections and resulting in seven deaths. This cluster presented the highest level of antibiotic resistance, including colistin resistance in all 17 analyzed isolates. The three outbreaking clones did not present more virulence genes than the sporadic isolates and had different patterns of antibiotic resistance, however, were clearly distinct from the sporadic ones in terms of infection status, being the only ones causing overt infections.

Rickettsiales are a lineage of obligate intracellular Alphaproteobacteria, encompassing important human pathogens, manipulators of host reproduction, and mutualists. Here we report the discovery of a novel Rickettsiales bacterium associated with Paramecium, displaying a unique extracellular lifestyle, including the ability to replicate outside host cells. Genomic analyses show that the bacterium possesses a higher capability to synthesise amino acids, compared to all investigated Rickettsiales. Considering these observations, phylogenetic and phylogenomic reconstructions, and re-evaluating the different means of interaction of Rickettsiales bacteria with eukaryotic cells, we propose an alternative scenario for the evolution of intracellularity in Rickettsiales. According to our reconstruction, the Rickettsiales ancestor would have been an extracellular and metabolically versatile bacterium, while obligate intracellularity would have evolved later, in parallel and independently, in different sub-lineages. The proposed new scenario could impact on the open debate on the lifestyle of the last common ancestor of mitochondria within Alphaproteobacteria.

International audience; Acinetobacter species assigned to the Acinetobacter calcoaceticus-baumannii (Acb) complex, are Gram-negative bacteria responsible for a large number of human infections. The population structure of Acb has been studied using two 7-gene MLST schemes, introduced by Bartual and coworkers (Oxford scheme) and by Diancourt and coworkers (Pasteur scheme). The schemes have three genes in common but underlie two coexisting nomenclatures of sequence types and clonal complexes, which complicates communication on A. baumannii genotypes. The aim of this study was to compare the characteristics of the two schemes to make a recommendation about their usage. Using genome sequences of 730 strains of the Acb complex, we evaluated the phylogenetic congruence of MLST schemes, the correspondence between sequence types, their discriminative power and genotyping reliability from genomic sequences. In silico ST re-assignments highlighted the presence of a second copy of the Oxford gdhB locus, present in 553/730 genomes that has led to the creation of artefactual profiles and STs. The reliability of the two MLST schemes was tested statistically comparing MLST-based phylogenies to two reference phylogenies (core-genome genes and genome-wide SNPs) using topology-based and likelihood-based tests. Additionally, each MLST gene fragment was evaluated by correlating the pairwise nucleotide distances between each pair of genomes calculated on the core-genome and on each single gene fragment. The Pasteur scheme appears to be less discriminant among closely related isolates, but less affected by homologous recombination and more appropriate for precise strain classification in clonal groups, which within this scheme are more often correctly monophyletic. Statistical tests evaluate the tree deriving from the Oxford scheme as more similar to the reference genome trees. Our results, together with previous work, indicate that the Oxford scheme has important issues: gdhB paralogy, recombination, primers sequences, position of the genes on the genome. While there is no complete agreement in all analyses, when considered as a whole the above results indicate that the Pasteur scheme is more appropriate for population biology and epidemiological studies of A. baumannii and related species and we propose that it should be the scheme of choice during the transition toward, and in parallel with, core genome MLST.

Rickettsialesare a lineage of obligatorily intracellularAlphaproteobacteria, encompassing important human pathogens, manipulators of host reproduction, and mutualists. Here we report the discovery of a novelRickettsialesbacterium associated withParamecium, displaying a unique extracellular lifestyle, including the ability to replicate outside host cells. Genomic analyses show that the bacterium possesses a higher capability to synthesize amino acids, compared to all investigatedRickettsiales. Considering these observations, phylogenetic and phylogenomic reconstructions, and re-evaluating the different means of interaction ofRickettsialesbacteria with eukaryotic cells, we propose an alternative scenario for the evolution of intracellularity inRickettsiales. According to our reconstruction, theRickettsialesancestor would have been an extracellular and metabolically versatile bacterium, while obligate intracellularity and genome reduction would have evolved later in parallel and independently in different sub-lineages. The proposed new scenario could impact on the open debate on the lifestyle of the last common ancestor of mitochondria withinAlphaproteobacteria.

Here we report an outbreak of extended spectrum β-lactamase-producing Klebsiella pneumoniae that occurred in a neonatal intensive care unit in Northern Italy and involved 97 patients. Progressively tightened sets of containment measures were implemented but the epidemic event was stopped only 9 months later. The final, effective, containment strategy consisted of the application of strict geographic cohorting of colonized infants and their nursing staff, the suspension of any new admission and a rigorous daily sterilization protocol for all surfaces and fomites in the ward. A posteriori characterization of the outbreak strain was performed using both traditional microbiology and molecular biology techniques, and whole genome sequencing, allowing to compare outbreak isolates with other strains collected in the previous two years. The results allowed to determine that the outbreak strain had been circulating inside the ward since the year before. Genomic characterization revealed that the strain carried a wide array of virulence and antibiotic resistance determinants, including gene bla(TEM-206), which had never been reported in a clinical isolate of K. pneumoniae before. The presence of such a high number of determinants for antibiotic resistance imposes significant therapeutic limitations on the treatment of infections, thus, further epidemiological investigations are needed to evaluate the prevalence of the newly described variant.

Gut microbiota has been implicated in the etiopathogenesis of colorectal cancer. The development of colorectal cancer is a multistep process by which healthy epithelium slowly develops into preneoplastic lesions, which in turn progress into malignant carcinomas over time. In particular, sporadic colorectal cancers can arise from adenomas (about 85% of cases) or serrated polyps through the “adenoma-carcinoma” or the “serrated polyp-carcinoma” sequences, respectively. In this study, we performed 16 S rRNA gene sequencing of bacterial DNA extracted from faecal samples to compare the microbiota of healthy subjects and patients with different preneoplastic and neoplastic lesions. We identified putative microbial biomarkers associated with stage-specific progression of colorectal cancer. In particular, bacteria belonging to the Firmicutes and Actinobacteria phyla, as well as members of the Lachnospiraceae family, proved to be specific of the faecal microbiota of patients with preneoplastic lesions, including adenomas and hyperplastic polyps. On the other hand, two families of the Proteobacteria phylum, Alcaligeneaceae and Enterobacteriaceae, with Sutterella and Escherichia/Shigella being the most representative genera, appeared to be associated with malignancy. These findings, once confirmed on larger cohorts of patients, can represent an important step towards the development of more effective diagnostic strategies.

We retrospectively analyzed a rare Salmonella enterica serovar Manhattan outbreak that occurred in Italy in 2009 to evaluate the potential of new genomic tools based on differential single nucleotide polymorphism (SNP) analysis in comparison with the gold standard genotyping method, pulsed-field gel electrophoresis. A total of 39 isolates were analyzed from patients ( n = 15) and food, feed, animal, and environmental sources ( n = 24), resulting in five different pulsed-field gel electrophoresis (PFGE) profiles. Isolates epidemiologically related to the outbreak clustered within the same pulsotype, SXB_BS.0003, without any further differentiation. Thirty-three isolates were considered for genomic analysis based on different sets of SNPs, core, synonymous, nonsynonymous, as well as SNPs in different codon positions, by Bayesian and maximum likelihood algorithms. Trees generated from core and nonsynonymous SNPs, as well as SNPs at the second and first plus second codon positions detailed four distinct groups of isolates within the outbreak pulsotype, discriminating outbreak-related isolates of human and food origins. Conversely, the trees derived from synonymous and third-codon-position SNPs clustered food and human isolates together, indicating that all outbreak-related isolates constituted a single clone, which was in line with the epidemiological evidence. Further experiments are in place to extend this approach within our regional enteropathogen surveillance system.

Klebsiella pneumoniae is 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. pneumoniae infections in nosocomial environments. We sequenced the genomes of 89 K. pneumoniae strains 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. pneumoniae in Italy. Thirty-one strains were carbapenem-resistant K. pneumoniae carbapenemase 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. pneumoniae worldwide. 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. pneumoniae evolution, providing novel insights into its global genomic characteristics and drawing a dated epidemiological scenario for this pathogen in Italy.

The emergence of carbapenemase producing Enterobacteriaceae has raised major public health concern. The aim of this study was to investigate the molecular epidemiology and the mechanism of carbapenem resistance acquisition of multidrug-resistant Klebsiella pneumoniae isolates from 20 neonates in the neonatal intensive care unit (NICU) of the V. Monaldi Hospital in Naples, Italy, from April 2015 to March 2016. Genotype analysis by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) identified PFGE type A and subtypes A1 and A2 in 17, 2, and 1 isolates, respectively, and assigned all isolates to sequence type (ST) 104. K. pneumoniae isolates were resistant to all classes of β-lactams including carbapenems, fosfomycin, gentamicin, and trimethoprim-sulfamethoxazole, but susceptible to quinolones, amikacin, and colistin. Conjugation experiments demonstrated that resistance to third-generation cephems and imipenem could be transferred along with an IncA/C plasmid containing the extended spectrum β-lactamase blaSHV -12 and carbapenem-hydrolyzing metallo-β-lactamase blaV IM-1 genes. The plasmid that we called pIncAC_KP4898 was 156,252 bp in size and included a typical IncA/C backbone, which was assigned to ST12 and core genome (cg) ST12.1 using the IncA/C plasmid MLST (PMLST) scheme. pIncAC_KP4898 showed a mosaic structure with blaV IM-1 into a class I integron, blaSHV -12 flanked by IS6 elements, a mercury resistance and a macrolide 2'-phosphotransferase clusters, ant(3″), aph(3″), aacA4, qnrA1, sul1, and dfrA14 conferring resistance to aminoglycosides, quinolones, sulfonamides, and trimethoprim, respectively, several genes predicted to encode transfer functions and proteins involved in DNA transposition. The acquisition of pIncAC_KP4898 carrying blaV IM-1 and blaSHV -12 contributed to the spread of ST104 K. pneumoniae in the NICU of V. Monaldi Hospital in Naples.

Introduction Listeria monocytogenes (Lm) is a bacterium widely distributed in nature and able to contaminate food processing environments, including those of dairy products. Lm is a primary public health issue, due to the very low infectious dose and the ability to produce severe outcomes, in particular in elderly, newborns, pregnant women and immunocompromised patients. Methods In the period between April and July 2015, an increased number of cases of listeriosis was observed in the area of Pavia, Northern Italy. An epidemiological investigation identified a cheesemaking small organic farm as the possible origin of the outbreak. In this work we present the results of the retrospective epidemiological study that we performed using molecular biology and genomic epidemiology methods. The strains sampled from patients and those from the target farm's cheese were analyzed using PFGE and whole genome sequencing (WGS) based methods. The performed WGS based analyses included: a) in-silico MLST typing; b) SNPs calling and genetic distance evaluation; c) determination of the resistance and virulence genes profiles; d) SNPs based phylogenetic reconstruction. Results Three of the patient strains and all the cheese strains resulted to belong to the same phylogenetic cluster, in Sequence Type 29. A further accurate SNPs analysis revealed that two of the three patient strains and all the cheese strains were highly similar (0.8 SNPs of average distance) and exhibited a higer distance from the third patient isolate (9.4 SNPs of average distance). Discussion Despite the global agreement among the results of the PFGE and WGS epidemiological studies, the latter approach agree with epidemiological data in indicating that one the patient strains could have originated from a different source. This result highlights that WGS methods can allow to better.

Bacteriology has embraced the next-generation sequencing revolution, swiftly moving from the time of single genome sequencing to the age of genomic epidemiology. Hundreds and now even thousands of genomes are being sequenced for single bacterial species, allowing unprecedented levels of resolution and insight in the evolution and epidemic diffusion of the main bacterial pathogens. Here, we present a review of some of the most recent and groundbreaking studies in this field.

Multidrug-resistant (MDR) Klebsiella pneumoniae is one of the most important causes of nosocomial infections worldwide. After the spread of strains resistant to beta-lactams at the end of the previous century, the diffusion of isolates resistant to carbapenems and colistin is now reducing treatment options and the containment of infections. Carbapenem-resistant K. pneumoniae strains have spread rapidly among Italian hospitals, with four subclades of pandemic clonal group 258 (CG258). Here we show that a single Italian hospital has been invaded by three of these subclades within 27 months, thus replicating on a small scale the “Italian scenario.” We identified a single clone responsible for an epidemic outbreak involving seven patients, and we reconstructed its star-like pattern of diffusion within the intensive care unit. This epidemiological picture was obtained through phylogenomic analysis of 16 carbapenem-resistant K. pneumoniae isolates collected in the hospital during a 27-month period, which were added to a database of 319 genomes representing the available global diversity of K. pneumoniae strains. Phenotypic and molecular assays did not reveal virulence or resistance determinants specific for the outbreak isolates. Other factors, rather than selective advantages, might have caused the outbreak. Finally, analyses allowed us to identify a major subclade of CG258 composed of strains bearing the yersiniabactin virulence factor. Our work demonstrates how the use of combined phenotypic, molecular, and whole-genome sequencing techniques can help to identify quickly and to characterize accurately the spread of MDR pathogens.

We announce the draft genome sequence of Clostridium tyrobutyricum strain DIVETGP. This strain was isolated from cow's milk used for Grana Padano cheese production. The genome was obtained using Illumina HiSeq technology and comprises 45 contigs for 3,018,999 bp, with a G+C content of 30.8%.

Sawflies are important pests of various plant species. Diprion pini (L.) and Neodiprion sertifer (Geoffroy) (Hymenoptera: Diprionidae) are two of the most important sawfly pests in Italy, and both species are parasitized by the hymenopteran parasitoid Dahlbominus fuscipennis (Zetterstedt). Bacterial endosymbionts are currently studied for their high potential in strategies of biocontrol in a number of insect species. In this study, we investigated the presence of symbiotic bacteria ( Wolbachia and Cardinium ) in the three species of hymenoptera mentioned earlier, both in wild and laboratory populations. Although all samples were negative for the presence of Cardinium , 100% prevalence for Wolbachia was detected, as all examined individuals resulted to be PCR positive. Furthermore, 16S rDNA and ftsZ gene sequencing indicated that all individuals from the three hymenopteran species are infected by a single Wolbachia strain. Additionally, we report the presence of gynandromorphic individuals in D. pini , both in wild and laboratory-reared populations. Heat treatments on D. pini colonies removed the Wolbachia symbionts, but they also prevented the development of adults.

Acetic acid bacteria (AAB) live in sugar rich environments, including food matrices, plant tissues, and the gut of sugar-feeding insects. By comparing the newly sequenced genomes of Asaia platycodi and Saccharibacter sp., symbionts of Anopheles stephensi and Apis mellifera, respectively, with those of 14 other AAB, we provide a genomic view of the evolutionary pattern of this bacterial group and clues on traits that explain the success of AAB as insect symbionts. A specific pre-adaptive trait, cytochrome bo3 ubiquinol oxidase, appears ancestral in AAB and shows a phylogeny that is congruent with that of the genomes. The functional properties of this terminal oxidase might have allowed AAB to adapt to the diverse oxygen levels of arthropod guts.

We announce the draft genome sequence of Salmonella enterica subsp. enterica serovar Manhattan strain 111113, isolated from a patient during an outbreak in northern Italy. The genome, which was obtained with Illumina MiSeq technology, is composed of 21 contigs for a total of 4,684,342 bp, with a G+C content of 52.17%.

We report the draft genome sequence of the Stenotrophomonas maltophilia strain EPM1, found in association with a culture of Giardia duodenalis . The draft genome sequence of S. maltophilia strain EPM1, obtained with Roche 454 GS-FLX Titanium technology, is composed of 19 contigs totaling 4,785,869 bp, with a G+C content of 66.37%.

International audience

Background Global climate change can seriously impact on the epidemiological dynamics of vector-borne diseases. In this study we investigated how future climatic changes could affect the climatic niche of Ixodes ricinus (Acari, Ixodida), among the most important vectors of pathogens of medical and veterinary concern in Europe. Methods Species Distribution Modelling (SDM) was used to reconstruct the climatic niche of I. ricinus, and to project it into the future conditions for 2050 and 2080, under two scenarios: a continuous human demographic growth and a severe increase of gas emissions (scenario A2), and a scenario that proposes lower human demographic growth than A2, and a more sustainable gas emissions (scenario B2). Models were reconstructed using the algorithm of “maximum entropy”, as implemented in the software Maxent 3.3.3e; 4,544 occurrence points and 15 bioclimatic variables were used. Results In both scenarios an increase of climatic niche of about two times greater than the current area was predicted as well as a higher climatic suitability under the scenario B2 than A2. Such an increase occurred both in a latitudinal and longitudinal way, including northern Eurasian regions (e.g. Sweden and Russia), that were previously unsuitable for the species. Conclusions Our models are congruent with the predictions of range expansion already observed in I. ricinus at a regional scale and provide a qualitative and quantitative assessment of the future climatically suitable areas for I. ricinus at a continental scale. Although the use of SDM at a higher resolution should be integrated by a more refined analysis of further abiotic and biotic data, the results presented here suggest that under future climatic scenarios most of the current distribution area of I. ricinus could remain suitable and significantly increase at a continental geographic scale. Therefore disease outbreaks of pathogens transmitted by this tick species could emerge in previous non-endemic geographic areas. Further studies will implement and refine present data toward a better understanding of the risk represented by I. ricinus to human health.