In vitro and in silico parameters for precise cgMLST typing of Listeria monocytogenes.

BACKGROUND: Whole genome sequencing analyzed by core genome multi-locus sequence typing (cgMLST) is widely used in surveillance of the pathogenic bacteria Listeria monocytogenes. Given the heterogeneity of available bioinformatics tools to define cgMLST alleles, our aim was to identify parameters influencing the precision of cgMLST profiles. METHODS: We used three L. monocytogenes reference genomes from different phylogenetic lineages and assessed the impact of in vitro (i.e. tested genomes, successive platings, replicates of DNA extraction and sequencing) and in silico parameters (i.e. targeted depth of coverage, depth of coverage, breadth of coverage, assembly metrics, cgMLST workflows, cgMLST completeness) on cgMLST precision made of 1748 core loci. Six cgMLST workflows were tested, comprising assembly-based (BIGSdb, INNUENDO, GENPAT, SeqSphere and BioNumerics) and assembly-free (i.e. kmer-based MentaLiST) allele callers. Principal component analyses and generalized linear models were used to identify the most impactful parameters on cgMLST precision. RESULTS: The isolate's genetic background, cgMLST workflows, cgMLST completeness, as well as depth and breadth of coverage were the parameters that impacted most on cgMLST precision (i.e. identical alleles against reference circular genomes). All workflows performed well at ≥40X of depth of coverage, with high loci detection (> 99.54% for all, except for BioNumerics with 97.78%) and showed consistent cluster definitions using the reference cut-off of ≤7 allele differences. CONCLUSIONS: This highlights that bioinformatics workflows dedicated to cgMLST allele calling are largely robust when paired-end reads are of high quality and when the sequencing depth is ≥40X.

Core Genome Multilocus Sequence Typing Scheme for Improved Characterization and Epidemiological Surveillance of Pathogenic Brucella.

Brucellosis poses a significant burden to human and animal health worldwide. Robust and harmonized molecular epidemiological approaches and population studies that include routine disease screening are needed to efficiently track the origin and spread of Brucella strains. Core genome multilocus sequence typing (cgMLST) is a powerful genotyping system commonly used to delineate pathogen transmission routes for disease surveillance and control. Except for Brucella melitensis, cgMLST schemes for Brucella species are currently not established. Here, we describe a novel cgMLST scheme that covers multiple Brucella species. We first determined the phylogenetic breadth of the genus using 612 Brucella genomes. We selected 1,764 genes that were particularly well conserved and typeable in at least 98% of these genomes. We tested the new scheme on 600 genomes and found high agreement with the whole-genome-based single nucleotide polymorphism (SNP) analysis. Next, we applied the scheme to reanalyze the genome of Brucella strains from epidemiologically linked outbreaks. We demonstrated the applicability of the new scheme for high-resolution typing required in outbreak investigations as previously reported with whole-genome SNP methods. We also used the novel scheme to define the global population structure of the genus using 1,322 Brucella genomes. Finally, we demonstrated the possibility of tracing distribution of Brucella strains by performing cluster analysis of cgMLST profiles and found nearly identical cgMLST profiles in different countries. Our results show that sequencing depth of more than 40-fold is optimal for allele calling with this scheme. In summary, this study describes a novel Brucella-wide cgMLST scheme that is applicable in Brucella molecular epidemiology and helps in accurately tracking and thus controlling the sources of infection. The scheme is publicly accessible and should represent a valuable resource for laboratories with limited computational resources and bioinformatics expertise.

Core Genome Multilocus Sequence Typing and Single Nucleotide Polymorphism Analysis in the Epidemiology of Brucella melitensis Infections.

The use of whole-genome sequencing (WGS) using next-generation sequencing (NGS) technology has become a widely accepted method for microbiology laboratories in the application of molecular typing for outbreak tracing and genomic epidemiology. Several studies demonstrated the usefulness of WGS data analysis through single-nucleotide polymorphism (SNP) calling from a reference sequence analysis for Brucella melitensis, whereas gene-by-gene comparison through core-genome multilocus sequence typing (cgMLST) has not been explored so far. The current study developed an allele-based cgMLST method and compared its performance to that of the genome-wide SNP approach and the traditional multilocus variable-number tandem repeat analysis (MLVA) on a defined sample collection. The data set was comprised of 37 epidemiologically linked animal cases of brucellosis as well as 71 isolates with unknown epidemiological status, composed of human and animal samples collected in Italy. The cgMLST scheme generated in this study contained 2,704 targets of the B. melitensis 16M reference genome. We established the potential criteria necessary for inclusion of an isolate into a brucellosis outbreak cluster to be ≤6 loci in the cgMLST and ≤7 in WGS SNP analysis. Higher phylogenetic distance resolution was achieved with cgMLST and SNP analysis than with MLVA, particularly for strains belonging to the same lineage, thereby allowing diverse and unrelated genotypes to be identified with greater confidence. The application of a cgMLST scheme to the characterization of B. melitensis strains provided insights into the epidemiology of this pathogen, and it is a candidate to be a benchmark tool for outbreak investigations in human and animal brucellosis.

Bluetongue Virus NS4 Protein Is an Interferon Antagonist and a Determinant of Virus Virulence.

UNLABELLED: Bluetongue virus (BTV) is the causative agent of bluetongue, a major infectious disease of ruminants with serious consequences to both animal health and the economy. The clinical outcome of BTV infection is highly variable and dependent on a variety of factors related to both the virus and the host. In this study, we show that the BTV nonstructural protein NS4 favors viral replication in sheep, the animal species most affected by bluetongue. In addition, NS4 confers a replication advantage on the virus in interferon (IFN)-competent primary sheep endothelial cells and immortalized cell lines. We determined that in cells infected with an NS4 deletion mutant (BTV8ΔNS4), there is increased synthesis of type I IFN compared to cells infected with wild-type BTV-8. In addition, using RNA sequencing (RNA-seq), we show that NS4 modulates the host IFN response and downregulates mRNA levels of type I IFN and interferon-stimulated genes. Moreover, using reporter assays and protein synthesis assays, we show that NS4 downregulates the activities of a variety of promoters, such as the cytomegalovirus immediate-early promoter, the IFN-ß promoter, and a promoter containing interferon-stimulated response elements (ISRE). We also show that the NS4 inhibitory activity on gene expression is related to its nucleolar localization. Furthermore, NS4 does not affect mRNA splicing or cellular translation. The data obtained in this study strongly suggest that BTV NS4 is an IFN antagonist and a key determinant of viral virulence. IMPORTANCE: Bluetongue is one of the main infectious diseases of ruminants and is caused by bluetongue virus (BTV), an arthropod-borne virus transmitted from infected to susceptible animals by Culicoides biting midges. Bluetongue has a variable clinical outcome that can be related to both virus and host factors. It is therefore critical to understand the interplay between BTV and the host immune responses. In this study, we show that a nonstructural protein of BTV (NS4) is critical to counteract the innate immune response of the host. Infection of cells with a BTV mutant lacking NS4 results in increased synthesis of IFN-ß and upregulation of interferon-stimulated genes. In addition, we show that NS4 is a virulence factor for BTV by favoring viral replication in sheep, the animal species most susceptible to bluetongue.

Mutations in the Schmallenberg Virus Gc Glycoprotein Facilitate Cellular Protein Synthesis Shutoff and Restore Pathogenicity of NSs Deletion Mutants in Mice.

UNLABELLED: Serial passage of viruses in cell culture has been traditionally used to attenuate virulence and identify determinants of viral pathogenesis. In a previous study, we found that a strain of Schmallenberg virus (SBV) serially passaged in tissue culture (termed SBVp32) unexpectedly displayed increased pathogenicity in suckling mice compared to wild-type SBV. In this study, we mapped the determinants of SBVp32 virulence to the viral genome M segment. SBVp32 virulence is associated with the capacity of this virus to reach high titers in the brains of experimentally infected suckling mice. We also found that the Gc glycoprotein, encoded by the M segment of SBVp32, facilitates host cell protein shutoff in vitro Interestingly, while the M segment of SBVp32 is a virulence factor, we found that the S segment of the same virus confers by itself an attenuated phenotype to wild-type SBV, as it has lost the ability to block the innate immune system of the host. Single mutations present in the Gc glycoprotein of SBVp32 are sufficient to compensate for both the attenuated phenotype of the SBVp32 S segment and the attenuated phenotype of NSs deletion mutants. Our data also indicate that the SBVp32 M segment does not act as an interferon (IFN) antagonist. Therefore, SBV mutants can retain pathogenicity even when they are unable to fully control the production of IFN by infected cells. Overall, this study suggests that the viral glycoprotein of orthobunyaviruses can compensate, at least in part, for the function of NSs. In addition, we also provide evidence that the induction of total cellular protein shutoff by SBV is determined by multiple viral proteins, while the ability to control the production of IFN maps to the NSs protein. IMPORTANCE: The identification of viral determinants of pathogenesis is key to the development of prophylactic and intervention measures. In this study, we found that the bunyavirus Gc glycoprotein is a virulence factor. Importantly, we show that mutations in the Gc glycoprotein can restore the pathogenicity of attenuated mutants resulting from deletions or mutations in the nonstructural protein NSs. Our findings highlight the fact that careful consideration should be taken when designing live attenuated vaccines based on deletions of nonstructural proteins since single mutations in the viral glycoproteins appear to revert attenuated mutants to virulent phenotypes.

Development of Education and Research in Anesthesia and Intensive Care Medicine at the University Teaching Hospital in Lusaka, Zambia: A Descriptive Observational Study.

BACKGROUND: Data from 2006 show that the practice of anesthesia at the University Teaching Hospital in Lusaka, Zambia was underdeveloped by international standards. Not only was there inadequate provision of resources related to environment, equipment, and drugs, but also a severe shortage of staff, with no local capability to train future physician anesthetic providers. There was also no research base on which to develop the specialty. This study aimed to evaluate patient care, education and research to determine whether conditions had changed a decade later. METHODS: A mix of qualitative data and quantitative data was gathered to inform the current state of anesthesia at the University Teaching Hospital, Lusaka, Zambia. Semistructured interviews were conducted with key staff identified by purposive sampling, including staff who had worked at the hospital throughout 2006 to 2015. Further data detailing conditions in the environment were collected by reviewing relevant departmental and hospital records spanning the study period. All data were analyzed thematically, using the framework described in the 2006 study, which described patient care, education, and research related to anesthetic practice at the hospital. RESULTS: There have been positive developments in most areas of anesthetic practice, with the most striking being implementation of a postgraduate training program for physician anesthesiologists. This has increased physician anesthesia staff in Zambia 6-fold within 4 years, and created an active research stream as part of the program. Standards of monitoring and availability of drugs have improved, and anesthetic activity has expanded out of operating theaters into the rest of the hospital. A considerable increase in the number of cesarean deliveries performed under spinal anesthetic may be a marker for safer anesthetic practice. Anesthesiologists have yet to take responsibility for the management of pain. CONCLUSIONS: The establishment of international partnerships to support postgraduate training of physician anesthetists in Zambia has created a significant increase in the number of anesthesia providers and has further developed nearly all aspects of anesthetic practice. The facilitation of the training program by a global health partnership has leveraged high-level support for the project and provided opportunities for North-South and international learning.

Multiple genome segments determine virulence of bluetongue virus serotype 8.

UNLABELLED: Bluetongue virus (BTV) causes bluetongue, a major hemorrhagic disease of ruminants. In order to investigate the molecular determinants of BTV virulence, we used a BTV8 strain minimally passaged in tissue culture (termed BTV8L in this study) and a derivative strain passaged extensively in tissue culture (BTV8H) in in vitro and in vivo studies. BTV8L was pathogenic in both IFNAR(-/-) mice and in sheep, while BTV8H was attenuated in both species. To identify genetic changes which led to BTV8H attenuation, we generated 34 reassortants between BTV8L and BTV8H. We found that partial attenuation of BTV8L in IFNAR(-/-) mice was achieved by simply replacing genomic segment 2 (Seg2, encoding VP2) or Seg10 (encoding NS3) with the BTV8H homologous segments. Fully attenuated viruses required at least two genome segments from BTV8H, including Seg2 with either Seg1 (encoding VP1), Seg6 (encoding VP6 and NS4), or Seg10 (encoding NS3). Conversely, full reversion of virulence of BTV8H required at least five genomic segments of BTV8L. We also demonstrated that BTV8H acquired an increased affinity for glycosaminoglycan receptors during passaging in cell culture due to mutations in its VP2 protein. Replication of BTV8H was relatively poor in interferon (IFN)-competent primary ovine endothelial cells compared to replication of BTV8L, and this phenotype was determined by several viral genomic segments, including Seg4 and Seg9. This study demonstrated that multiple viral proteins contribute to BTV8 virulence. VP2 and NS3 are primary determinants of BTV pathogenesis, but VP1, VP5, VP4, VP6, and VP7 also contribute to virulence. IMPORTANCE: Bluetongue is one of the major infectious diseases of ruminants, and it is listed as a notifiable disease by the World Organization for Animal Health (OIE). The clinical outcome of BTV infection varies considerably and depends on environmental and host- and virus-specific factors. Over the years, BTV serotypes/strains with various degrees of virulence (including nonpathogenic strains) have been described in different geographical locations. However, no data are available to correlate the BTV genotype to virulence. This study shows that BTV virulence is determined by different viral genomic segments. The data obtained will help to characterize thoroughly the pathogenesis of bluetongue. The possibility to determine the pathogenicity of virus isolates on the basis of their genome sequences will help in the design of control strategies that fit the risk posed by new emerging BTV strains.

Characterization of a second open reading frame in genome segment 10 of bluetongue virus.

Viruses have often evolved overlapping reading frames in order to maximize their coding capacity. Until recently, the segmented dsRNA genome of viruses of the Orbivirus genus was thought to be monocistronic, but the identification of the bluetongue virus (BTV) NS4 protein changed this assumption. A small ORF in segment 10, overlapping the NS3 ORF in the +1 position, is maintained in more than 300 strains of the 27 different BTV serotypes and in more than 200 strains of the phylogenetically related African horse sickness virus (AHSV). In BTV, this ORF (named S10-ORF2 in this study) encodes a putative protein 50-59 residues in length and appears to be under strong positive selection. HA- or GFP-tagged versions of S10-ORF2 expressed from transfected plasmids localized within the nucleoli of transfected cells, unless a putative nucleolar localization signal was mutated. S10-ORF2 inhibited gene expression, but not RNA translation, in transient transfection reporter assays. In both mammalian and insect cells, BTV S10-ORF2 deletion mutants (BTV8ΔS10-ORF2) displayed similar replication kinetics to wt virus. In vivo, S10-ORF2 deletion mutants were pathogenic in mouse models of disease. Although further evidence is required for S10-ORF2 expression during infection, the data presented provide an initial characterization of this ORF.

Virus and host factors affecting the clinical outcome of bluetongue virus infection.

UNLABELLED: Bluetongue is a major infectious disease of ruminants caused by bluetongue virus (BTV), an arbovirus transmitted by Culicoides. Here, we assessed virus and host factors influencing the clinical outcome of BTV infection using a single experimental framework. We investigated how mammalian host species, breed, age, BTV serotypes, and strains within a serotype affect the clinical course of bluetongue. Results obtained indicate that in small ruminants, there is a marked difference in the susceptibility to clinical disease induced by BTV at the host species level but less so at the breed level. No major differences in virulence were found between divergent serotypes (BTV-8 and BTV-2). However, we observed striking differences in virulence between closely related strains of the same serotype collected toward the beginning and the end of the European BTV-8 outbreak. As observed previously, differences in disease severity were also observed when animals were infected with either blood from a BTV-infected animal or from the same virus isolated in cell culture. Interestingly, with the exception of two silent mutations, full viral genome sequencing showed identical consensus sequences of the virus before and after cell culture isolation. However, deep sequencing analysis revealed a marked decrease in the genetic diversity of the viral population after passaging in mammalian cells. In contrast, passaging in Culicoides cells increased the overall number of low-frequency variants compared to virus never passaged in cell culture. Thus, Culicoides might be a source of new viral variants, and viral population diversity can be another factor influencing BTV virulence. IMPORTANCE: Bluetongue is one of the major infectious diseases of ruminants. It is caused by an arbovirus known as bluetongue virus (BTV). The clinical outcome of BTV infection is extremely variable. We show that there are clear links between the severity of bluetongue and the mammalian host species infected, while at the breed level differences were less evident. No differences were observed in the virulence of two different BTV serotypes (BTV-8 and BTV-2). In contrast, we show that the European BTV-8 strain isolated at the beginning of the bluetongue outbreak in 2006 was more virulent than a strain isolated toward the end of the outbreak. In addition, we show that there is a link between the variability of the BTV population as a whole and virulence, and our data also suggest that Culicoides cells might function as an "incubator" of viral variants.

Schmallenberg virus pathogenesis, tropism and interaction with the innate immune system of the host.

Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and "synthetic" SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV.

Genetic Diversity of Brucella melitensis Isolated from Domestic Ruminants in Iraq.

The control and eradication of brucellosis represents a critical objective for Veterinary and Health Authorities across several countries globally. Efficient surveillance programs play a pivotal role in detecting and managing outbreaks. Epidemiological investigations significantly benefit from standardized and efficient molecular typing techniques and analytical tools, enabling public health laboratories to identify the origin of outbreaks. This study aimed to sequence Brucella spp. strains isolated in Iraq from different ruminant species to verify their molecular epidemiological correlations and, above all, to shed a light on how these Iraqi isolates are positioned in the phylogenetic context of Brucella spp. The 35 isolates under study were from abortion, milk, placenta, and the fetal membranes of sheep, cattle, and buffalo. Genotyping involved various techniques: MLVA-16, Whole Genome Sequencing, MLST, and cgMLST. All the Iraqi isolates from our study clustered in MLVA-16 within the East Mediterranean clade, and all but one grouped together in the same branch of the MST tree. MST analysis showed the minimum distance of one allele between the studied isolates, except for one strain from buffalo, which was positioned farther away from the rest of the isolates. In cgMLST, the majority of strains grouped within a large cluster predominantly comprising genotypes from the Middle East. The application of different control measures in different territories based on molecular epidemiological studies would increase the chances of maximizing public health benefits and minimizing the spread of infection to disease-free or lower prevalence areas.

Multi-approach methods to predict cryptic carbapenem resistance mechanisms in Klebsiella pneumoniae detected in Central Italy.

The rapid emergence of carbapenem-resistant Klebsiella pneumoniae (Kp) strains in diverse environmental niches, even outside of the clinical setting, poses a challenge for the detection and the real-time monitoring of novel antimicrobial resistance trends using molecular and whole genome sequencing-based methods. The aim of our study was to understand cryptic resistance determinants responsible for the phenotypic carbapenem resistance observed in strains circulating in Italy by using a combined approach involving whole genome sequencing (WGS) and genome-wide association study (GWAS). In this study, we collected 303 Kp strains from inside and outside clinical settings between 2018-2022 in the Abruzzo region of Italy. The antimicrobial resistance profile of all isolates was assessed using both phenotypic and bioinformatic methods. We identified 11 strains resistant to carbapenems, which did not carry any known genetic determinants explaining their phenotype. The GWAS results showed that incongruent carbapenem-resistant phenotype was associated specifically with strains with two capsular types, KL13 and KL116 including genes involved in the capsule synthesis, encoding proteins involved in the assembly of the capsule biosynthesis apparatus, capsule-specific sugar synthesis, processing and export, polysaccharide pyruvyl transferase, and lipopolysaccharide biosynthesis protein. These preliminary results confirmed the potential of GWAS in identifying genetic variants present in KL13 and KL116 that could be associated with carbapenem resistance traits in Kp. The implementation of advanced methods, such as GWAS with increased antimicrobial resistance surveillance will potentially improve Kp infection treatment and patient outcomes.

Antibiotic resistance, plasmids, and virulence-associated markers in human strains of Campylobacter jejuni and Campylobacter coli isolated in Italy.

Campylobacteriosis, a prevalent foodborne gastrointestinal infection in Europe, is primarily caused by Campylobacter jejuni and Campylobacter coli, with rising global concerns over antimicrobial resistance in these species. This study comprehensively investigates 133 human-origin Campylobacter spp. strains (102 C. jejuni and 31 C. coli) collected in Italy from 2013 to 2021. The predominant Multilocus Sequence Typing Clonal complexes (CCs) were ST-21 CC and ST-206 CC in C. jejuni and ST-828 CC in C. coli. Ciprofloxacin and tetracycline resistance, mainly attributed to GyrA (T86I) mutation and tet(O) presence, were prevalent, while erythromycin resistance was associated with 23S rRNA gene mutation (A2075G), particularly in C. coli exhibiting multidrug-resistant pattern CipTE. Notable disparities in virulence factors among strains were observed, with C. jejuni exhibiting a higher abundance compared to C. coli. Notably, specific C. jejuni sequence types, including ST-21, ST-5018, and ST-1263, demonstrated significantly elevated counts of virulence genes. This finding underscores the significance of considering both the species and strain-level variations in virulence factor profiles, shedding light on potential differences in the pathogenicity and clinical outcomes associated with distinct C. jejuni lineages. Campylobacter spp. plasmids were classified into three groups comprising pVir-like and pTet-like plasmids families, exhibiting diversity among Campylobacter spp. The study underscores the importance of early detection through Whole Genome Sequencing to identify potential emergent virulence, resistance/virulence plasmids, and new antimicrobial resistance markers. This approach provides actionable public health data, supporting the development of robust surveillance programs in Italy.

Detection of Potential Zoonotic Agents Isolated in Italian Shelters and the Assessment of Animal Welfare Correlation with Antimicrobial Resistance in Escherichia coli Strains.

Welfare conditions in shelters, where dogs might be housed for a long period of time, may have a possible correlation with the occurrence of bacterial pathogens and their antimicrobial resistance (AMR). In this study, we assessed the occurrence of AMR in 54 strains of Escherichia coli isolated from dogs housed in 15 Italian shelters and we correlated the resistance patterns to animal welfare. We also aimed to evaluate the presence of specific pathogens with zoonotic potential in sheltered dogs. Thus, nasopharyngeal, rectal, and oral swabs were collected from a group of 20 dogs in each shelter and totaled 758 swabs. We identified 9 Staphylococcus pseudointermedius, 1 Pasteurella multocida, 9 Staphylococcus aureus, 12 Campylobacter spp., 54 Escherichia coli, 2 Salmonella enterica, and 246 Capnocytophaga spp. The antimicrobial susceptibility was assessed for the E. coli isolates using a panel of 14 antibiotics. The highest level of relative AMR was recorded for ampicillin and sulfamethoxazole. The association found between AMR and the levels of animal welfare scores in shelters was evident although not statistically significant. These results support the hypothesis that the good management of shelters can increase the level of animal welfare, thus reducing the use of antibiotics and, as a consequence, the AMR occurrence found in dogs that share their domestic environment with humans.

Genomic and Antimicrobial Surveillance of Campylobacter Population in Italian Poultry.

Campylobacter is one of the most common foodborne diseases worldwide with increasing rates of antibiotic resistance. Most cases of campylobacteriosis can be traced back to the consumption of poultry meat. Despite many efforts to reduce contamination in farms and in slaughterhouses, the persistence of this pathogen in poultry products remains a problem. This study aimed to evaluate the genetic diversity and antibiotic resistance of 542 C. jejuni and C. coli in Italian poultry, in the framework of two National Monitoring Programs. Genomes were screened for antibiotic resistance, virulence determinants and contextualized within a global collection of C. jejuni. ST2116, ST2863 and ST 832 were the most prevalent and significantly associated with Italian poultry. A worrying increase in resistance to quinolones, fluoroquinolones and tetracycline was observed in C. jejuni, while an increased occurrence of multidrug resistant (MDR) strains and strains resistant to macrolides was detected in C. coli. Low resistance rates were found for aminoglycosides. Molecular resistance determinants were consistent with the phenotypic resistance for tetracycline and quinolones. In silico analysis revealed 119 genes associated with virulence factors, with a notably higher prevalence of some genes in ST2863 genomes. This study highlights the increased resistance to macrolides and the emergence of MDR strains for C. coli, the genetic basis of AMR and the predominance of two genotypes among Campylobacter strains isolated from the Italian poultry farms.

Brucella ceti Infection in Striped Dolphins from Italian Seas: Associated Lesions and Epidemiological Data.

Brucella ceti infections have been increasingly reported in cetaceans. In this study, we analyzed all cases of B. ceti infection detected in striped dolphins stranded along the Italian coastline between 2012 and 2021 (N = 24). We focused on the pathogenic role of B. ceti through detailed pathological studies, and ad hoc microbiological, biomolecular, and serological investigations, coupled with a comparative genomic analysis of the strains. Neurobrucellosis was observed in 20 animals. The primary histopathologic features included non-suppurative meningoencephalitis (N = 9), meningitis (N = 6), and meningoencephalomyelitis (N = 5), which was also associated with typical lesions in other tissues (N = 8). Co-infections were detected in more than half of the cases, mostly involving Cetacean Morbillivirus (CeMV). The 24 B. ceti isolates were assigned primarily to sequence type 26 (ST26) (N = 21) and, in a few cases, ST49 (N = 3). The multilocus sequence typing (cgMLST) based on whole genome sequencing (WGS) data showed that strains from Italy clustered into four genetically distinct clades. Plotting these clades onto a geographic map suggests a link between their phylogeny and the topographical distribution. These results support the role of B. ceti as a primary neurotropic pathogen for striped dolphins and highlight the utility of WGS data in understanding the evolution of this emerging pathogen.

The Current Landscape of Antibiotic Resistance of Salmonella Infantis in Italy: The Expansion of Extended-Spectrum Beta-Lactamase Producers on a Local Scale.

Salmonella enterica serovar Infantis is one of the five main causes of human salmonellosis in the European Union (EU) and in recent years, has been increasingly reported to carry multiple antimicrobial resistance determinants, including extended-spectrum beta-lactamase (ESBL) genes. In our study, we used WGS-based tools to characterize S. Infantis strains circulating in the Abruzzo and Molise regions of Italy between 2017 and 2020 and compared this local dataset to the S. Infantis population present in Italy over the last two decades. Phylogenetic analyses demonstrated that the majority of strains isolated from poultry and turkeys from Abruzzo and Molise were closely related and belonged to one of the two main genetic clusters present in Italy, which were grouped predominantly as ESBL-producing strains that harbored pESI-like plasmid. We showed that 60% of the local strains carried multiple antibiotic resistance genes, including ESBL gene bla CTX-M-1 as well as aadA1, dfrA1, dfrA14, sul1, and tet(A) genes present on the pESI-like megaplasmid. The analysis of strains from Abruzzo and Molise and the publicly available Italian S. Infantis sequences revealed a dramatic increase in the number of identified AMR genes in the strains isolated after 2011. Moreover, the number of strains resistant to five or more antibiotic classes increased from 20-80% in the last decade likely due to the acquisition of the megaplasmid. The persistence of the ESBL-producing and the multidrug-resistant (MDR) clone of S. Infantis in poultry populations in Italy and in Europe requires rapid and efficient intervention strategies to prevent further expansion of the clone.

Investigating the cecal microbiota in broiler poultry farms and its potential relationships with animal welfare.

The present study assessed the modulation of cecal microbiota and correlations with Campylobacter colonization and animal welfare status. For these purposes, we conducted a cross sectional study of the cecal microbiota from 187 broilers reared in 13 batches from 10 poultry farms by performing 16S rRNA sequencing (regions V3-4). The welfare of each batch was assessed using a simplified Welfare Quality® protocol, scoring higher in organic batches, compared to both antibiotic-free and conventional batches. The bioinformatics analyses were conducted in QIIME 2 and a linear discriminant analysis determined the association between microbiota and animals with different Campylobacter carriage status and welfare levels. In the microbiota from the subjects negative for Campylobacter or with high welfare scores, Bacteroidetes was the predominant phylum with the genus Megamonas significantly increased in abundance. A greater abundance of Parabacteroides, Phascolarctobacterium, Helicobacter in poultry negative for Campylobacter was also found at the genus level. Animals with the lowest welfare scores showed an increased abundance of Proteobacteria. The results suggested a different microbial composition and diversity in the analyzed groups.

Whole Genome Sequence Analysis of Brucella abortus Isolates from Various Regions of South Africa.

The availability of whole genome sequences in public databases permits genome-wide comparative studies of various bacterial species. Whole genome sequence-single nucleotide polymorphisms (WGS-SNP) analysis has been used in recent studies and allows the discrimination of various Brucella species and strains. In the present study, 13 Brucella spp. strains from cattle of various locations in provinces of South Africa were typed and discriminated. WGS-SNP analysis indicated a maximum pairwise distance ranging from 4 to 77 single nucleotide polymorphisms (SNPs) between the South African Brucella abortus virulent field strains. Moreover, it was shown that the South African B. abortus strains grouped closely to B. abortus strains from Mozambique and Zimbabwe, as well as other Eurasian countries, such as Portugal and India. WGS-SNP analysis of South African B. abortus strains demonstrated that the same genotype circulated in one farm (Farm 1), whereas another farm (Farm 2) in the same province had two different genotypes. This indicated that brucellosis in South Africa spreads within the herd on some farms, whereas the introduction of infected animals is the mode of transmission on other farms. Three B. abortus vaccine S19 strains isolated from tissue and aborted material were identical, even though they originated from different herds and regions of South Africa. This might be due to the incorrect vaccination of animals older than the recommended age of 4-8 months or might be a problem associated with vaccine production.

A large food-borne outbreak of campylobacteriosis in kindergartens and primary schools in Pescara, Italy, May-June 2018.

Introduction. In May-June 2018, an outbreak of campylobacteriosis involved students and school staff from kindergartens and primary schools in Pescara, southern Italy.Aim. We present details of the epidemiological and microbiological investigation, and the findings of the analytical study, as well as the implemented control measures.Methodology. To identify possible risk factors associated with the observed outbreak, a case control study was conducted using a questionnaire to collect information on the date of symptoms onset, type and duration of symptoms, type of healthcare contact, school attendance, and food items consumed at school lunches during the presumed days of exposure. Attack rates were calculated for each date and school. Logistic regression models were used to estimate the odds ratios of being a case and the odds of illness by food items consumed, respectively. Moreover, we carried out a comparative genomic analysis using whole genome multilocus sequence typing (wgMLST) of Campylobacter jejuni strains isolated during the outbreak investigation to identify the source of the outbreak.Results. Overall, 222 probable cases from 21 schools were identified, and C. jejuni was successfully isolated from 60 patients. The meals in the schools involved were provided by two cooking centres managed by a joint venture between two food companies. Environmental and food sampling, epidemiological and microbiological analyses, as well as a case control study with 176 cases and 62 controls from the same schools were performed to identify the source of the outbreak. The highest attack rate was recorded among those having lunch at school on 29 May (7.8 %), and the most likely exposure was 'caciotta' cheese (odds ratio 2.40, 95 % confidence interval 1.10-5.26, P=0.028). C. jejuni was isolated from the cheese, and wgMLST showed that the human and cheese isolates belonged to the same genomic cluster, confirming that the cheese was the vehicle of the infection.Conclusion. It is plausible that a failure of the pasteurization process contributed to the contamination of the cheese batches. Timely suspension of the catering service and summer closure of the schools prevented further spread.