The Mla system and its role in maintaining outer membrane barrier function in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia are ubiquitous Gram-negative bacteria found in both natural and clinical environments. It is a remarkably adaptable species capable of thriving in various environments, thanks to the plasticity of its genome and a diverse array of genes that encode a wide range of functions. Among these functions, one notable trait is its remarkable ability to resist various antimicrobial agents, primarily through mechanisms that regulate the diffusion across cell membranes. We have investigated the Mla ABC transport system of S. maltophilia, which in other Gram-negative bacteria is known to transport phospholipids across the periplasm and is involved in maintaining outer membrane homeostasis. First, we structurally and functionally characterized the periplasmic substrate-binding protein MlaC, which determines the specificity of this system. The predicted structure of the S. maltophilia MlaC protein revealed a hydrophobic cavity of sufficient size to accommodate the phospholipids commonly found in this species. Moreover, recombinant MlaC produced heterologously demonstrated the ability to bind phospholipids. Gene knockout experiments in S. maltophilia K279a revealed that the Mla system is involved in baseline resistance to antimicrobial and antibiofilm agents, especially those with divalent-cation chelating activity. Co-culture experiments with Pseudomonas aeruginosa also showed a significant contribution of this system to the cooperation between both species in the formation of polymicrobial biofilms. As suggested for other Gram-negative pathogenic microorganisms, this system emerges as an appealing target for potential combined antimicrobial therapies.

Study of the biosynthesis and functionality of polyphosphate in Bifidobacterium longum KABP042.

Polyphosphate (poly-P) biosynthesis in bacteria has been linked to many physiological processes and has been characterized as an interesting functional molecule involved in intestinal homeostasis. We determined the capacity for poly-P production of 18 probiotic strains mainly belonging to Bifidobacterium and former Lactobacillus genera, showing that poly-P synthesis varied widely between strains and is dependent on the availability of phosphate and the growth phase. Bifidobacteria were especially capable of poly-P synthesis and poly-P kinase (ppk) genes were identified in their genomes together with a repertoire of genes involved in phosphate transport and metabolism. In Bifidobacterium longum KABP042, the strain we found with highest poly-P production, variations in ppk expression were linked to growth conditions and presence of phosphate in the medium. Moreover, the strain produced poly-P in presence of breast milk and lacto-N-tetraose increased the amount of poly-P synthesized. Compared to KABP042 supernatants low in poly-P, exposure of Caco-2 cells to KABP042 supernatants rich in poly-P resulted in decreased epithelial permeability and increased barrier resistance, induction of epithelial protecting factors such as HSP27 and enhanced expression of tight junction protein genes. These results highlight the role of bifidobacteria-derived poly-P as a strain-dependent functional factor acting on epithelial integrity.

A Stenotrophomonas maltophilia TetR-Like Transcriptional Regulator Involved in Fatty Acid Metabolism Is Controlled by Quorum Sensing Signals.

Stenotrophomonas maltophilia is an environmental bacterium as well as an emerging opportunistic multidrug-resistant pathogen. They use the endogenous diffusible signal factor (DSF) quorum sensing (QS) system to coordinate population behavior and regulate virulence processes but can also respond to exogenous N-acyl-homoserine lactone (AHL) signals produced by neighboring bacteria. The effect of these QS signals on the global gene expression of this species remains, however, unknown. Whole-transcriptome sequencing analyses were performed for exponential cultures of S. maltophilia K279a treated with exogenous DSF or AHLs. Addition of DSF and AHLs signals resulted in changes in expression of at least 2-fold for 28 and 82 genes, respectively. Interestingly, 22 of these genes were found upregulated by both QS signals, 14 of which were shown to also be induced during the stationary phase. Gene functions regulated by all conditions included lipid and amino acid metabolism, stress response and signal transduction, nitrogen and iron metabolism, and adaptation to microoxic conditions. Among the common top upregulated QS core genes, a putative TetR-like regulator (locus tag SMLT2053) was selected for functional characterization. This regulator controls its own ß-oxidation operon (Smlt2053-Smlt2051), and it is found to sense long-chain fatty acids (FAs), including the QS signal DSF. Gene knockout experiments reveal that operon Smlt2053-Smlt2051 is involved in biofilm formation. Overall, our findings provide clues on the effect that QS signals have in S. maltophilia QS-related phenotypes and the transition from the exponential to the stationary phase and bacterial fitness under high-density growth. IMPORTANCE The quorum sensing system in Stenotrophomonas maltophilia, in addition to coordinating the bacterial population, controls virulence-associated phenotypes, such as biofilm formation, motility, protease production, and antibiotic resistance mechanisms. Biofilm formation is frequently associated with the persistence and chronic nature of nosocomial infections. In addition, biofilms exhibit high resistance to antibiotics, making treatment of these infections extremely difficult. The importance of studying the metabolic and regulatory systems controlled by quorum sensing autoinducers will make it possible to discover new targets to control pathogenicity mechanisms in S. maltophilia.

Improved mini-Tn7 Delivery Plasmids for Fluorescent Labeling of Stenotrophomonas maltophilia.

Fluorescently labeled bacterial cells have become indispensable for many aspects of microbiological research, including studies on biofilm formation as an important virulence factor of various opportunistic bacteria of environmental origin such as Stenotrophomonas maltophilia. Using a Tn7-based genomic integration system, we report the construction of improved mini-Tn7 delivery plasmids for labeling of S. maltophilia with sfGFP, mCherry, tdTomato and mKate2 by expressing their codon-optimized genes from a strong, constitutive promoter and an optimized ribosomal binding site. Transposition of the mini-Tn7 transposons into single neutral sites located on average 25 nucleotides downstream of the 3'-end of the conserved glmS gene of different S. maltophilia wild-type strains did not have any adverse effects on the fitness of their fluorescently labeled derivatives. This was demonstrated by comparative analyses of growth, resistance profiles against 18 antibiotics of different classes, the ability to form biofilms on abiotic and biotic surfaces, also independent of the fluorescent protein expressed, and virulence in Galleria mellonella. It is also shown that the mini-Tn7 elements remained stably integrated in the genome of S. maltophilia over a prolonged period of time in the absence of antibiotic selection pressure. Overall, we provide evidence that the new improved mini-Tn7 delivery plasmids are valuable tools for generating fluorescently labeled S. maltophilia strains that are indistinguishable in their properties from their parental wild-type strains. IMPORTANCE The bacterium S. maltophilia is an important opportunistic nosocomial pathogen that can cause bacteremia and pneumonia in immunocompromised patients with a high rate of mortality. It is now considered as a clinically relevant and notorious pathogen in cystic fibrosis patients but has also been isolated from lung specimen of healthy donors. The high intrinsic resistance to a wide range of antibiotics complicates treatment and most likely contributes to the increasing incidence of S. maltophilia infections worldwide. One important virulence-related trait of S. maltophilia is the ability to form biofilms on any surface, which may result in the development of increased transient phenotypic resistance to antimicrobials. The significance of our work is to provide a mini-Tn7-based labeling system for S. maltophilia to study the mechanisms of biofilm formation or host-pathogen interactions with live bacteria under non-destructive conditions.

Complete genome sequences of Lacticaseibacillus paracasei INIA P272 (CECT 8315) and Lacticaseibacillus rhamnosus INIA P344 (CECT 8316) isolated from breast-fed infants reveal probiotic determinants.

Lacticaseibacillus paracasei INIA P272 and Lacticaseibacillus rhamnosus INIA P344, isolated from breast-fed infants, are two promising bacterial strains for their use in functional foods according to their demonstrated probiotic and technological characteristics. To better understand their probiotic characteristics and evaluate their safety, here we report the draft genome sequences of both strains as well as the analysis of their genetical content. The draft genomes of L. paracasei INIA P272 and L. rhamnosus INIA P344 comprise 3.01 and 3.26 Mb, a total of 2994 and 3166 genes and a GC content of 46.27 % and 46.56 %, respectively. Genomic safety was assessed following the EFSA guidelines: the identification of both strains was confirmed through Average Nucleotide Identity, and the absence of virulence, pathogenic and antibiotic resistance genes was demonstrated. The genome stability analysis revealed the presence of plasmids and phage regions in both genomes, however, CRISPR sequences and other mechanisms to fight against phage infections were encoded. The probiotic abilities of both strains were supported by the presence of genes for the synthesis of SCFA, genes involved in resistance to acid and bile salts or a thiamine production cluster. Moreover, the encoded exopolysaccharide biosynthesis genes could provide additional protection against the deleterious gastrointestinal conditions, besides which, playing a key role in adherence and coaggregation of pathogenic bacteria together with the high number of adhesion proteins and domains encoded by both genomes. Additionally, the bacteriocin cluster genes found in both strains, could provide an advantageous ability to compete against pathogenic bacteria. This genomic study supports the probiotic characteristics described previously for these two strains and satisfies the safety requirements to be used in food products.

Strain-specific interspecies interactions between co-isolated pairs of Staphylococcus aureus and Pseudomonas aeruginosa from patients with tracheobronchitis or bronchial colonization.

Dual species interactions in co-isolated pairs of Staphylococcus aureus and Pseudomonas aeruginosa from patients with tracheobronchitis or bronchial colonization were examined. The genetic and phenotypic diversity between the isolates was high making the interactions detected strain-specific. Despite this, and the clinical origin of the strains, some interactions were common between some co-isolated pairs. For most pairs, P. aeruginosa exoproducts affected biofilm formation and reduced growth in vitro in its S. aureus counterpart. Conversely, S. aureus did not impair biofilm formation and stimulated swarming motility in P. aeruginosa. Co-culture in a medium that mimics respiratory mucus promoted coexistence and favored mixed microcolony formation within biofilms. Under these conditions, key genes controlled by quorum sensing were differentially regulated in both species in an isolate-dependent manner. Finally, co-infection in the acute infection model in Galleria mellonella larvae showed an additive effect only in the co-isolated pair in which P. aeruginosa affected less S. aureus growth. This work contributes to understanding the complex interspecies interactions between P. aeruginosa and S. aureus by studying strains isolated during acute infection.

Genome Sequence of the Reuterin-Producing Strain Limosilactobacillus reuteri INIA P572.

Limosilactobacillus reuteri is a beneficial bacterium that inhabits the gastrointestinal tract of different mammals. Diverse beneficial effects have been attributed to specific strains, in part mediated by the production of reuterin. Here, we report the draft genome sequence of L. reuteri INIA P572, a reuterin-producing strain isolated from pig feces.

Oral colonization by Levilactobacillus brevis KABPTM-052 and Lactiplantibacillus plantarum KABPTM-051: A Randomized, Double-Blinded, Placebo-Controlled Trial (Pilot Study).

BACKGROUND: To determine the oral colonization capacity of the strains Levilactobacillus brevis KABPTM-052 (CECT 7480) and Lactiplantibacillus plantarum KABPTM-051 (CECT 7481) in healthy subjects. MATERIAL AND METHODS: This randomized, double-blinded, placebo-controlled study included 40 volunteers (22 females, 18 males; age range 18-55 years) with healthy gingiva or mild gingivitis, allocated to receiving probiotic chewing gum (n=20) or placebo (n=20) b.i.d for 6 weeks. At baseline and after 6 weeks of treatment, a periodontics specialist collected saliva samples to assess probiotic colonization by qPCR, and analysed dental plaque, gingival index and dental probing pocket depth in Community Periodontal Index (CPI) teeth subset. Protocol was registered as NCT03540498. RESULTS: Treatment compliance was high (99%). Both L. brevis and L. plantarum were detected in the oral microbiota at baseline. After 6 weeks, volunteers receiving probiotic showed a significant increase of both L. brevis (p = 0.017) and L. plantarum (p = 0.004) versus placebo. This effect remained significant after adjusting for gender and gingival index at baseline. In the probiotic group, reduction in plaque index significantly correlated to higher levels of L. brevis (rho = 0.57, p = 0.022) but not of L. plantarum at study endpoint, and the number of subjects with dental plaque was reduced during intervention (7 of 17, p = 0.016). No such effects were observed in the placebo group. No adverse drug reactions were reported. CONCLUSIONS: Levilactobacillus brevis KABPTM-052 and Lactiplantibacillus plantarum KABPTM-051 colonize the buccal microbiota of healthy volunteers, and higher colonization by L. brevis positively correlated to reduction in dental plaque. Key words:Probiotic, Levilactobacillus brevis, Lactiplantibacillus plantarum, oral colonization, oral microbiota, dental plaque.

Probiotic Properties of Bifidobacterium longum KABP042 and Pediococcus pentosaceus KABP041 Show Potential to Counteract Functional Gastrointestinal Disorders in an Observational Pilot Trial in Infants.

Functional gastrointestinal disorders (FGIDs) are a common concern during the first year of life. Recognized as gut-brain axis disorders by Rome IV criteria, FGIDs etiology is linked to altered gut-brain interaction, intestinal physiology, and microbiota. In this regard, probiotics have emerged as a promising therapy for infant FGIDs. In this study, we have investigated the probiotic potential of the strains Bifidobacterium longum KABP042 and Pediococcus pentosaceus KABP041-isolated from healthy children's feces-in the treatment of FGIDs. To this scope, genome sequences of both strains were obtained and subjected to in silico analyses. No virulence factors were detected for any strain and only the non-transferable erm(49) gene, which confers resistance to erythromycin and clindamycin, was identified in the genome of B. longum KABP042. Safety of both strains was confirmed by acute oral toxicity in rats. In vitro characterization revealed that the strains tolerate gastric and bile challenges and display a great adhesion capacity to human intestinal cells. The two strains mediate adhesion by different mechanisms and, when combined, synergically induce the expression of Caco-2 tight junction proteins. Moreover, growth inhibition experiments demonstrated the ability of the two strains alone and in combination to antagonize diverse Gram-negative and Gram-positive bacterial pathogens during sessile and planktonic growth. Pathogens' inhibition was mostly mediated by the production of organic acids, but neutralization experiments strongly suggested the presence of additional antimicrobial compounds in probiotic culture supernatants such as the bacteriocin Lantibiotic B, whose gene was detected in the genome of B. longum KABP042. Finally, an exploratory, observational, pilot study involving 36 infants diagnosed with at least one FGID (infant colic and/or functional constipation) showed the probiotic formula was well tolerated and FGID severity was significantly reduced after 14 days of treatment with the 2 strains. Overall, this work provides evidence of the probiotic and synergic properties of strains B. longum KABP042 and P. pentosaceus KABP041, and of their potential to treat pediatric FGIDs. Clinical Trial Registration: [www.ClinicalTrials.gov], [identifier NCT04944628].

Genetic Variants of the DSF Quorum Sensing System in Stenotrophomonas maltophilia Influence Virulence and Resistance Phenotypes Among Genotypically Diverse Clinical Isolates.

The pathogenicity of Stenotrophomonas maltophilia is regulated in part by its quorum sensing (QS) system. The main QS signaling molecule in S. maltophilia is known as diffusible signal factor (DSF), and the rpf gene cluster is responsible for its synthesis and perception. Two cluster variants have been previously described, rpf-1 and rpf-2, which differ basically in the conditions under which DSF is produced. Here, correlations between the rpf variant and antibiotic susceptibility, LPS electrophoretic profiles and virulence-related phenotypes were evaluated for a collection of 78 geographically and genetically diverse clinical strains of S. maltophilia. In general there were associations between previously established genogroups and the genetic variant of the rpf cluster. However, only few genotype-phenotype correlations could be observed. Resistance to the ß-lactam antibiotics ceftazidime and ticarcillin was associated with strains carrying the rpf-1 variant, whereas strains of variant rpf-2, particularly those of genogroup C, showed higher resistance levels to colistin. Strains of variant rpf-2 were also significantly more virulent to Galleria mellonella larvae than those of rpf-1, most likely due to an increased ability of rpf-2 strains to form biofilms. A comparative genomic analysis revealed the presence of proteins unique to individual genogroups. In particular, the strains of genogroup C share an operon that encodes for a new virulence determinant in S. maltophilia related to the synthesis of an alternative Flp/Tad pilus. Overall, this study establishes a link between the DSF-based QS system and the virulence and resistance phenotypes in this species, and identifies potential high-risk clones circulating in European hospitals.

Sulfonamide-based diffusible signal factor analogs interfere with quorum sensing in Stenotrophomonas maltophilia and Burkholderia cepacia.

Aim:Stenotrophomonas maltophilia (Sm) and Burkholderia cepacia complex (BCC) are Gram-negative bacterial pathogens, which are typically multidrug resistant and excellent biofilm producers. These phenotypes are controlled by quorum sensing (QS) systems from the diffusible signal factor (DSF) family. We aim to interfere with this QS system as an alternative approach in combatting such difficult-to-treat infections. Materials & methods: A library of sulfonamide-based DSF bioisosteres was synthesized and tested against the major phenotypes regulated by QS. Results & conclusion: Several analogs display significant antibiofilm activity while the majority increase the action of the last-resort antibiotic colistin against Sm and BCC. Most compounds inhibit DSF synthesis in the Sm K279a strain. Our results support the strategy of interfering with QS communications to combat multidrug resistance.

Gene duplications in the E. coli genome: common themes among pathotypes.

BACKGROUND: Gene duplication underlies a significant proportion of gene functional diversity and genome complexity in both eukaryotes and prokaryotes. Although several reports in the literature described the duplication of specific genes in E. coli, a detailed analysis of the extent of gene duplications in this microorganism is needed. RESULTS: The genomes of the E. coli enteroaggregative strain 042 and other pathogenic strains contain duplications of the gene that codes for the global regulator Hha. To determine whether the presence of additional copies of the hha gene correlates with the presence of other genes, we performed a comparative genomic analysis between E. coli strains with and without hha duplications. The results showed that strains harboring additional copies of the hha gene also encode the yeeR irmA (aec69) gene cluster, which, in turn, is also duplicated in strain 042 and several other strains. The identification of these duplications prompted us to obtain a global map of gene duplications, first in strain 042 and later in other E. coli genomes. Duplications in the genomes of the enteroaggregative strain 042, the uropathogenic strain CFT073 and the enterohemorrhagic strain O145:H28 have been identified by a BLASTp protein similarity search. This algorithm was also used to evaluate the distribution of the identified duplicates among the genomes of a set of 28 representative E. coli strains. Despite the high genomic diversity of E. coli strains, we identified several duplicates in the genomes of almost all studied pathogenic strains. Most duplicated genes have no known function. Transcriptomic analysis also showed that most of these duplications are regulated by the H-NS/Hha proteins. CONCLUSIONS: Several duplicated genes are widely distributed among pathogenic E. coli strains. In addition, some duplicated genes are present only in specific pathotypes, and others are strain specific. This gene duplication analysis shows novel relationships between E. coli pathotypes and suggests that newly identified genes that are duplicated in a high percentage of pathogenic E. coli isolates may play a role in virulence. Our study also shows a relationship between the duplication of genes encoding regulators and genes encoding their targets.

Heterogeneous Colistin-Resistance Phenotypes Coexisting in Stenotrophomonas maltophilia Isolates Influence Colistin Susceptibility Testing.

The polymyxin antibiotic colistin shows in vitro activity against Stenotrophomonas maltophilia. However, an increased incidence of colistin-resistant isolates has been recently observed. In addition, in vitro evaluation of colistin susceptibility for this organism has been problematic. The aims of this study were to investigate the colistin-resistance phenotypes displayed by S. maltophilia and their potential association with the challenging determination of colistin susceptibilities for this organism by even the recommended method. Colistin-resistance phenotypes were inferred by use of the recommended broth microdilution method in different clinical isolates of S. maltophilia. Most of the strains showed non-interpretable minimum inhibitory concentrations (MICs) for colistin due to an incomplete growth inhibition in wells of the microdilution plate. In addition, the subpopulation of bacteria resistant to colistin showed an increased ability to form biofilms on the plastic surface of MIC plates. The observed incomplete growth inhibition in the microdilution plates is compatible with a progressive adaptation to colistin or a heterogeneous susceptibility to this antibiotic. Therefore, to determine the existence of heteroresistance or adaptive resistance, four colistin-resistant clinical isolates were subjected to serial Etest assays, growth rate analyses, and the population analysis profile test. The experiments indicated that these S. maltophilia isolates display a colistin-resistant sub-population that survives and multiplies in the presence of the antibiotic. Interestingly, this phenomenon might not be explainable by the natural background mutation rate alone since the development of a resistant sub-population occurred upon the contact with the antibiotic and it was reversible. This complex colistin-resistance phenotype is exhibited differently by the different isolates and significantly affected colistin susceptibility testing. Furthermore, it can coexist with adaptive resistance to colistin as response to pre-incubation with sub-inhibitory concentrations of the antibiotic. Overall, the combined action of heterogeneous colistin-resistance mechanisms in S. maltophilia isolates, including colistin-induced biofilm formation, may hamper the correct interpretation of colistin susceptibility tests, thus having potentially serious implications on antimicrobial-therapy decision making.

High-resolution diffusion pattern of human infections by Salmonella enterica serovar Napoli in Northern Italy explained through phylogeography.

Salmonella enterica serovar Napoli (serovar Napoli) is an emerging cause of human salmonellosis in Northern Italy. No specific reservoirs of serovar Napoli have been identified in Italy, so far. However, the environment, especially surface waters, has been hypothesized as an important source of infection based on the observation that genotypically different clusters of serovar Napoli are detected in different geographical macro-areas. To further support the hypothesis of a spatially-restricted pattern of serovar Napoli diffusion, a spatial segregation of serovar Napoli lineages should be observed also at smaller geographical scale. However, classical genotyping techniques used for Salmonella, such as pulsed-field gel electrophoresis (PFGE), did not possess enough discriminatory power to highlight spatial clustering of serovar Napoli within the macro-areas. To this purpose, we performed phylogeographical analyses based on genome-wide single nucleotide polymorphisms to test whether spatio-temporal evolution patterns of serovar Napoli in Northern Italy could be recognized with high geographical resolution, i.e. at local level. Specifically, we analyzed the local spread of the main PFGE clonal group, responsible for more than 60% of human infections in the study area, that did not show any geographical differentiation by PFGE within Northern Italy, i.e. the macro-area considered in the study. Both discrete and continuous phylogeography highlighted the existence of two main geographically-restricted clades: a Southern clade corresponding to the Po Valley and a Northern clade corresponding to the Pre-Alps area. Furthermore, the phylogeographical analyses suggested that the most probable site of origin of the clone was in an area of the Po Valley at the confluence of the Po and Ticino rivers, one of the most important Italian wetlands. These findings provide further support to the hypothesis that environmental transmission may play an important role in the ecology of serovar Napoli.

Quorum Sensing Signaling and Quenching in the Multidrug-Resistant Pathogen Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is an opportunistic Gram-negative pathogen with increasing incidence in clinical settings. The most critical aspect of S. maltophilia is its frequent resistance to a majority of the antibiotics of clinical use. Quorum Sensing (QS) systems coordinate bacterial populations and act as major regulatory mechanisms of pathogenesis in both pure cultures and poly-microbial communities. Disruption of QS systems, a phenomenon known as Quorum Quenching (QQ), represents a new promising paradigm for the design of novel antimicrobial strategies. In this context, we review the main advances in the field of QS in S. maltophilia by paying special attention to Diffusible Signal Factor (DSF) signaling, Acyl Homoserine Lactone (AHL) responses and the controversial Ax21 system. Advances in the DSF system include regulatory aspects of DSF synthesis and perception by both rpf-1 and rpf-2 variant systems, as well as their reciprocal communication. Interaction via DSF of S. maltophilia with unrelated organisms including bacteria, yeast and plants is also considered. Finally, an overview of the different QQ mechanisms involving S. maltophilia as quencher and as object of quenching is presented, revealing the potential of this species for use in QQ applications. This review provides a comprehensive snapshot of the interconnected QS network that S. maltophilia uses to sense and respond to its surrounding biotic or abiotic environment. Understanding such cooperative and competitive communication mechanisms is essential for the design of effective anti QS strategies.

Identification of a major Listeria monocytogenes outbreak clone linked to soft cheese in Northern Italy - 2009-2011.

BACKGROUND: Molecular subtyping and enhanced surveillance in Lombardy region identified a cluster of possibly related listeriosis cases from 2006 to 2010. This cluster grouped 31 isolates that belonged to serotype 1/2a and Sequence Type 38 (ST38) as defined by Multilocus Sequence Typing (MLST). METHODS: Our study expanded the previous investigation to include cases from 2011 to 2014 and used Multi-Virulence-Locus Sequence Typing (MVLST) on all ST38 isolates to better understand their epidemiology and possibly identify a common source outbreak. RESULTS: Out of 306 L. monocytogenes clinical isolates collected, 43 (14.1%) belonged to ST38 with cases occurring in nine out of twelve Lombardy provinces. The ST38 isolates were split by MVLST into two Virulence Types (VTs): VT80 (n = 12) and VT104 (n = 31). VT104 cases were concentrated between 2009 and 2011 in two provinces, Bergamo and Milan. An epidemiologic investigation was performed and in one case, a matching VT104 isolate was retrieved from a soft cheese sample from a patient's refrigerator. CONCLUSIONS: Our findings revealed a major listeriosis outbreak in Northern Italy linked to soft cheese in 2009-2011, which went undetected by local health authorities. Our study shows that integrating subtyping methods with conventional epidemiology can help identify the source of L. monocytogenes outbreak clones.

Epidemiology and Molecular Typing of Pregnancy-Associated Listeriosis Cases in Lombardy, Italy, over a 10-Year Period (2005-2014).

In developed countries, pregnancy-related listeriosis accounts for 20-43% of total invasive listeriosis. This work describes the first pregnancy-related listeriosis survey in Italy based on two data sources, that is, mandatory notification system and regional laboratory-based network. Out of 610 listeriosis cases reported over a 10-year period, 40 were pregnancy-related (6.6%). Among these, 29 pregnancy-related isolates were available and have been analysed with serotyping, Pulsed-Field Gel Electrophoresis, and Multi-Virulence-Locus Sequence Typing. No maternal fatality was recorded, but 11 (29.7%) pregnancies resulted in a foetal death, a miscarriage, or a birth of a foetus dying immediately after birth. The average incidence of pregnancy-related listeriosis was 4.3 cases per 100000 births, and the proportion of pregnancy-associated listeriosis among ethnic minorities was significantly higher compared to the general population (30.0% versus 3.5%, P < 0.01). L. monocytogenes isolates belonged to serotypes 1/2a, 1/2b, and 4b, with the latter significantly more prevalent among pregnancy-related isolates. Twenty different pulsotypes were distinguished and 16 out of the 29 isolates were classified into seven clusters. A total of 16 virulence types (VTs) were identified. Five VTs accounted for 45% of the total cases and coincided with those of previously described Epidemic Clones (ECs) of L. monocytogenes.

Salmonella enterica Serotype Napoli is the First Cause of Invasive Nontyphoidal Salmonellosis in Lombardy, Italy (2010-2014), and Belongs to Typhi Subclade.

Salmonella enterica serotype Napoli (S. Napoli) is currently emerging in Europe and particularly in Italy, where in 2014 it caused a large outbreak associated with elevated rates of bacteremia. However, no study has yet investigated its invasive ability and phylogenetic classification. Here, we show that between 2010 and 2014, S. Napoli was the first cause of invasive salmonellosis affecting 40 cases out of 687 (invasive index: 5.8%), which is significantly higher than the invasive index of all the other nontyphoidal serotypes (2.0%, p < 0.05). Genomic and phylogenetic analyses of an invasive isolate revealed that S. Napoli belongs to Typhi subclade in clade A, Paratyphi A being the most related serotype and carrying almost identical pattern of typhoid-associated genes. This work presents evidence of invasive capacity of S. Napoli and argues for reconsideration of its nontyphoidal category.

A Multischool Outbreak Due to Salmonella enterica serovar Napoli Associated with Elevated Rates of Hospitalizations and Bacteremia, Milan, Italy, 2014.

A multischool outbreak of salmonellosis caused by Salmonella enterica serovar Napoli was investigated in the province of Milan from October to November 2014, following an increase in school absenteeism coinciding with two positive cases. Epidemiological studies detected 47 cases in four primary schools: 46 children and 1 adult woman (51.4% males and 48.6% females, median age 8.9). From these, 14 cases (29.8%) were severe and resulted in hospitalization, including 6 children (12.8%) who developed an invasive salmonellosis. The epidemic curve revealed an abnormally long incubation period, peaking 1 week after the first confirmed case. Twenty-five available isolates were typed by pulsed-field gel electrophoresis showing an identical pattern. The isolate belongs to ST474, an ST composed exclusively of Salmonella Napoli human strains isolated in France and Italy. Antibiotic resistance analysis showed resistance to aminoglycosides, correlating with the presence of the aminoglycoside resistance gene aadA25 in its genome. Trace-back investigations strongly suggested contaminated ham as the most likely food vehicle, which was delivered by a common food center on 21 October. Nevertheless, this ingredient could not be retrospectively investigated since it was no longer available at the repository. This represents the largest Salmonella Napoli outbreak ever reported in Italy and provides a unique scenario for studying the outcome of salmonellosis caused by this emerging and potentially invasive nontyphoidal serotype.

Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Napoli Strain SN310, Cause of a Multischool Outbreak in Milan, Italy, in 2014.

We report the draft genome sequence of Salmonella enterica subsp. enterica serovar Napoli strain SN310, isolated from a stool sample of an affected pupil during a multischool outbreak in 2014 in Milan, Italy. This represents the first reported draft genome sequence of the emerging serovar Napoli.