Multi-locus sequence typing and in vitro antimicrobial resistance of equine Streptococcus equi subspecies zooepidemicus strains.

Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) is one of the most important pathogens frequently associated with the main causes of equine infertility. In this study, we surveyed 22 strains of S. zooepidemicus collected during 2021 from cervico-uterine swabs of mares with endometritis. The genetic variability of the isolated strains was studied by multi-locus sequence typing (MLST) from whole-genome sequencing (WGS) data. The average length of reconstructed genomes was 2,088,286 bp (95% CI: 2,061,569 bp-2,114,967 bp), which was expected for S. zooepidemicus genomes. The assembled genomes were assigned to sequence types (STs) using the S. zooepidemicus scheme targeting seven loci (arcC, nrdE, proS, spi, tdk, tpi, yqiL) available in PubMLST database. MLST revealed a wide variability of STs with two (9.1%) novel STs identified in this study, precisely ST521 with two isolates and ST522 with one isolate. Furthermore, 4/22 (18.2%) isolates were assigned to ST92, 3/22 (13.6%) to ST205, 2/22 (9.1%) to ST475, and one strain (4.5%) for each of the following STs: ST10, ST30, ST39, ST49, ST101, ST132, ST147, ST314, ST369, ST467. Isolates were also tested for antimicrobial resistance using Kirby-Bauer disk diffusion method. Resistance to amoxicillin-clavulanate, ampicillin, amikacin, gentamicin, streptomycin, enrofloxacin, sulfamethoxazole-trimethoprim, tetracycline, oxytetracycline represented the most common resistance profile (13/22, 59.1%). No correlation between specific ST and antimicrobial resistance profile was found. Our study provides a comprehensive insight into the epidemiology, ST diversity and antimicrobial resistance profile of S. zooepidemicus strains, isolated in Italy, causing subfertility problems in mares.

SARS-CoV-2 in Animal Companions: A Serosurvey in Three Regions of Southern Italy.

Several animal species have been found to be susceptible to SARS-CoV-2 infection. The occurrence of infection in dogs and cats living in close contact with owners deserves particular attention from public health authorities in a One Health approach. In this study, we conducted serological screening to identify SARS-CoV-2 exposure in the sera from dogs and cats in three regions of southern Italy sampled during the years 2021 and 2022. We collected 100 serum samples in 2021 (89 from dogs and 11 from cats) and 640 in 2022 (577 from dogs and 63 from cats). Overall, the ELISA positivity rate was found to be 2.7% (20/740), with higher seroprevalence in dogs. Serum neutralization tests confirmed positivity only in two samples collected from dogs, and the assays, performed with serologically distinct SARS-CoV-2 variants, showed variant-specific positivity. This paper shows that monitoring SARS-CoV-2 exposure in animals might be affected by the viral antigenic evolution, which requires continuous updates to the serological tests used. Serological surveys are useful in understanding the true extent of exposure occurring in specific animal populations, not suffering the same limitations as molecular tests, and could help in identifying the infecting virus if tests able to characterize the immune response are used. The use of variant-specific validated serological methods should always be considered in serosurvey studies in order to determine the real impact of emerging variants on animal populations and its implications for veterinary and human health, as well as to identify potential reservoirs of the virus and its evolutionary changes.

High Genetic Diversity and Virulence Potential in Bacillus cereus sensu lato Isolated from Milk and Cheeses in Apulia Region, Southern Italy.

The Bacillus cereus group includes species that act as food-borne pathogens causing diarrheal and emetic symptoms. They are widely distributed and can be found in various foods. In this study, out of 550 samples of milk and cheeses, 139 (25.3%) were found to be contaminated by B. cereus sensu lato (s.l.). One isolate per positive sample was characterized by Multilocus Sequence Typing (MLST) and for the presence of ten virulence genes. Based on MLST, all isolates were classified into 73 different sequence types (STs), of which 12 isolates were assigned to new STs. Virulence genes detection revealed that 90% and 61% of the isolates harboured the nheABC and the hblCDA gene cluster, respectively. Ninety-four percent of the isolates harboured the enterotoxin genes entS and entFM; 8% of the isolates possessed the ces gene. Thirty-eight different genetic profiles were identified, suggesting a high genetic diversity. Our study clearly shows the widespread diffusion of potentially toxigenic isolates of B. cereus s.l. in milk and cheeses in the Apulia region highlighting the need to adopt GMP and HACCP procedures along every step of the milk and cheese production chain in order to reduce the public health risk linked to the consumption of foods contaminated by B. cereus s.l.

Pilot Study on the Action of Thymus vulgaris Essential Oil in Treating the Most Common Bacterial Contaminants and Salmonella enterica subsp. enterica Serovar Derby in Poultry Litter.

The indiscriminate use of antimicrobials in poultry farms is linked to the increase in multi-resistant bacteria. Accordingly, based on the antimicrobial properties of Thyme Essential Oil (TEO), the present study evaluated the effects of TEO on the reduction of common microbial contaminants and Salmonella on poultry litter. A litter bulk sample was collected in a broiler farm and qualitative/quantitative investigations identified Escherichia coli and Mammaliicoccus lentus. The experimental contamination with Salmonella Derby wild strain was also performed. All pathogens showed phenotypic and genotypic resistance to different classes of antibiotics. The litter, split in different units, was treated with aqueous solutions of TEO at different concentrations (5% to 1.25%), demonstrating its effectiveness in reducing the total number of bacteria. The strongest antibacterial action was observed at the lowest concentration against Enterobacteriaceae, with a growth reduction compared to the positive control of 73.3% and 77.8% against E. coli and Salmonella Derby, respectively, while towards M. lentus the reduction was 50%. Our data confirm the antimicrobial activity of TEO and suggest its possible application for the treatment of poultry litter as an effective and natural approach for the prevention of diseases caused by the most common bacteria that colonize poultry farms, counteracting the onset of antibiotic resistance.

How Well Do We Handle the Sample Preparation, FT-ICR Mass Spectrometry Analysis, and Data Treatment of Atmospheric Waters?

FT-ICR MS (Fourier-transform ion cyclotron resonance mass spectrometry) analysis has shown great potential to aid in the understanding of the extremely high molecular diversity of cloud water samples. The main goal of this work was to determine the differences in terms of formula assignment for analytical (i.e., measurement replicates) and experimental replicates of a given cloud water sample. The experimental replicates, obtained by solid phase extraction, were also compared to the results obtained for freeze-dried samples to evaluate whether the presence of salts interferes with the analysis. Two S/N ratios, generally adopted for atmospheric samples, were evaluated, and three different algorithms were used for assignment: DataAnalysis 5.3 (Bruker), Composer (Sierra Analytics), and MFAssignR (Chemical Advanced Resolution Methods Lab). In contrast to other works, we wanted to treat this comparison from the point of view of users, who usually must deal with a simple list of m/z ratios and intensity with limited access to the mass spectrum characteristics. The aim of this study was to establish a methodology for the treatment of atmospheric aqueous samples in light of the comparison of three different software programs, to enhance the possibility of data comparison within samples.

Analysis of Gene Single Nucleotide Polymorphisms in COVID-19 Disease Highlighting the Susceptibility and the Severity towards the Infection.

Many factors may influence the risk of being infected by SARS-CoV-2, the coronavirus responsible for coronavirus disease 2019 (COVID-19). Exposure to the virus cannot explain the variety of an individual's responses to the virus and the high differences of effect that the virus may cause to some. While a person's preexisting condition and their immune defenses have been confirmed to play a major role in the disease progression, there is still much to learn about hosts' genetic makeup towards COVID-19 susceptibility and risk. The host genetic makeup may have direct influence on the grade of predisposition and outcomes of COVID-19. In this study, we aimed to investigate the presence of relevant genetic single nucleotide polymorphisms (SNPs), the peripheral blood level of IL6, vitamin D and arterial blood gas (ABG) markers (pH, oxygen-SpO2 and carbon dioxide-SpCO2) on two groups, COVID-19 (n = 41, study), and the healthy (n = 43, control). We analyzed cytokine and interleukin genes in charge of both pro-inflammatory and immune-modulating responses and those genes that are considered involved in the COVID-19 progression and complications. Thus, we selected major genes, such as IL1ß, IL1RN (IL-1 ß and α receptor) IL6, IL6R (IL-6 receptor), IL10, IFNγ (interferon gamma), TNFα (tumor necrosis factor alpha), ACE2 (angiotensin converting enzyme), SERPINA3 (Alpha-1-Antiproteinase, Antitrypsin member of Serpin 3 family), VDR (vitamin D receptor Tak1, Bsm1 and Fok1), and CRP (c-reactive protein). Though more research is needed, these findings may give a better representation of virus pleiotropic activity and its relation to the immune system.

Diagnosis of SARS-CoV-2 during the Pandemic by Multiplex RT-rPCR hCoV Test: Future Perspectives.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has rapidly become a significant threat to public health. However, among the Coronaviridae family members, there are other viruses that can also cause infections in humans. Among these, severe acute respiratory syndrome (SARS-CoV) and Middle East respiratory syndrome (MERS-CoV) have posed significant threats to human health in the past. Other human pathogenic coronaviruses have been identified, and they are known to cause respiratory diseases with manifestations ranging from mild to severe. In this study, we evaluated the performance of a multiplex RT-rPCR specific to seven human pathogenic coronaviruses in mainly detecting SARS-CoV-2 directly from nasopharyngeal swabs obtained from suspected COVID-19 infected patients, while simultaneously detecting different human pathogenic coronaviruses in case these were also present. We tested 1195 clinical samples suspected of COVID-19 infection. The assay identified that 69% of the samples tested positive for SARS-CoV-2 (1195), which was confirmed using another SARS-CoV-2 RT-PCR kit available in our laboratory. None of these clinical samples were positive for SARS-CoV, MERS-CoV or HCoV. This means that during the endemic phase of COVID-19, infection with other human pathogenic coronaviruses, even the common cold coronavirus (HCoV), is very uncommon. Our study also confirmed that the multiplex RT-rPCR is a sensitive assay for detecting SARS-CoV-2 regardless of differences among the variants. This multiplex RT-rPCR is also time- and cost-saving and very easy to apply in the diagnostic laboratory due to its simple procedure and its stability in storage after preparation. These features make the assay a valuable approach in screening procedures for the rapid detection of SARS-CoV-2 and other human pathogenic coronaviruses that could affect public health.

Toxigenic Genes, Pathogenic Potential and Antimicrobial Resistance of Bacillus cereus Group Isolated from Ice Cream and Characterized by Whole Genome Sequencing.

Bacillus cereus is isolated from a variety of foods where it may cause food spoilage and/or food poisoning due to its toxigenic and pathogenic nature. In this study, we identified members of B. cereus groups in 65% of the ice cream samples analyzed, which were characterized based on multi locus variable number tandem repeats analysis (MLVA) and whole genome sequencing (WGS). The MLVA revealed that 36 strains showed different allelic profiles. Analyses of WGS data enabled the identification of three members of the B. cereus group: B. cereus sensu stricto, B. mosaicus and B. thuringiensis. Based on the multi locus sequence typing (MLST) scheme, the strains were classified in 27 sequence types (STs), including ST26 that causes food poisoning. Toxin genes' detection revealed the presence of the genes encoding nonhemolytic enterotoxin (NHE), hemolysin BL (HBL), cytotoxin K (cytK) and cereulide (ces) in 100%, 44%, 42% and 8% of the strains, respectively. The identification of eleven antimicrobial resistance (AMR) genes predicted the resistance to five different antimicrobials, and the resistance to beta-lactam antibiotics was confirmed with a phenotypic antimicrobial test. Taken together, the results showed that the B. cereus strains isolated from ice cream were a potential hazard for consumer safety.

Isolation and characterization of Yersinia enterocolitica from foods in Apulia and Basilicata regions (Italy) by conventional and modern methods.

Yersiniosis is the third most reported food-borne zoonosis in Europe. The aim of the present study was to perform the search for Yersinia enterocolitica in food samples collected from Apulia and Basilicata regions (Southern Italy) and to characterize any isolates by classical and modern analytical methods. A total of 130 samples were analyzed between July 2018 and July 2019: most of them were raw milk and dairy products made from it. Furthermore, 8 out of 130 samples were individual milk samples collected from bovines reared in a Brucella-free farm which showed false positive serological reaction for brucellosis due to the presence of pathogenic Y. enterocolitica O:9 biotype 2 in faeces. The Real Time PCR targeting the ail gene and the culture method were performed to detect pathogenic Y. enterocolitica. Isolates were subjected to API 20E (Biomerieux) and MALDI-TOF MS (Matrix Assisted Laser Desorption Ionization Time-of-Flight) for species identification. All samples were negative for the ail gene. The culture method allowed to isolate suspicious colonies from 28 samples. The API 20E system and the MALDI-TOF MS technique identified 20 Y. enterocolitica and 1 Y. intermedia in a concordant way. The remaining 7 strains were all identified as Y. enterocolitica by the API 20E system, while the MALDI-TOF MS recognized 4 Y. intermedia, 1 Y. bercovieri and 2 Y. massiliensis. Genotypic characterization of the discordant strains was performed by rMLST and it confirmed the MALDI-TOF MS' results. Only non-pathogenic Y. enterocolitica biotype 1A strains were found, although with a non-negligible prevalence (P = 0.15 with CI 95% = ± 0.06). This study indicates a poor circulation of pathogenic Y. enterocolitica in food products made and marketed in the investigated areas. However, the small number of samples, insufficient for some food categories such as meat and vegetable, does not allow to exclude the presence of pathogenic strains at all.

Influence of strong iron-binding ligands on cloud water oxidant capacity.

Iron (Fe) plays a dual role in atmospheric chemistry: it is involved in chemical and photochemical reactivity and serves as a micronutrient for microorganisms that have recently been shown to produce strong organic ligands. These ligands control the reactivity, mobility, solubility and speciation of Fe, which have a potential impact on Fe bioavailability and cloud water oxidant capacity. In this work, the concentrations of Fe-binding ligands and the conditional stability constants were experimentally measured for the first time by Competitive Ligand Exchange-Adsorptive Cathodic Stripping Voltammetry (CLE-ACSV) technique in cloud water samples collected at puy de Dôme (France). The conditional stability constants, which indicate the strength of the Fe-ligand complexes, are higher than those considered until now in cloud chemistry (mainly Fe-oxalate). To understand the effect of Fe complexation on cloud water reactivity, we used the CLEPS cloud chemistry model. According to the model results, we found that Fe complexation impacts the hydroxyl radical formation rate: contrary to our expectations, Fe complexation by natural organic ligands led to an increase in hydroxyl radical production. These findings have important impacts on cloud chemistry and the global iron cycle.

Laniakea@ReCaS: exploring the potential of customisable Galaxy on-demand instances as a cloud-based service.

BACKGROUND: Improving the availability and usability of data and analytical tools is a critical precondition for further advancing modern biological and biomedical research. For instance, one of the many ramifications of the COVID-19 global pandemic has been to make even more evident the importance of having bioinformatics tools and data readily actionable by researchers through convenient access points and supported by adequate IT infrastructures. One of the most successful efforts in improving the availability and usability of bioinformatics tools and data is represented by the Galaxy workflow manager and its thriving community. In 2020 we introduced Laniakea, a software platform conceived to streamline the configuration and deployment of "on-demand" Galaxy instances over the cloud. By facilitating the set-up and configuration of Galaxy web servers, Laniakea provides researchers with a powerful and highly customisable platform for executing complex bioinformatics analyses. The system can be accessed through a dedicated and user-friendly web interface that allows the Galaxy web server's initial configuration and deployment. RESULTS: "Laniakea@ReCaS", the first instance of a Laniakea-based service, is managed by ELIXIR-IT and was officially launched in February 2020, after about one year of development and testing that involved several users. Researchers can request access to Laniakea@ReCaS through an open-ended call for use-cases. Ten project proposals have been accepted since then, totalling 18 Galaxy on-demand virtual servers that employ ~ 100 CPUs, ~ 250 GB of RAM and ~ 5 TB of storage and serve several different communities and purposes. Herein, we present eight use cases demonstrating the versatility of the platform. CONCLUSIONS: During this first year of activity, the Laniakea-based service emerged as a flexible platform that facilitated the rapid development of bioinformatics tools, the efficient delivery of training activities, and the provision of public bioinformatics services in different settings, including food safety and clinical research. Laniakea@ReCaS provides a proof of concept of how enabling access to appropriate, reliable IT resources and ready-to-use bioinformatics tools can considerably streamline researchers' work.

Emerging Mutations Potentially Related to SARS-CoV-2 Immune Escape: The Case of a Long-Term Patient.

SARS-CoV-2 isolates from long-term COVID-19 patients play a significant role in understanding the mechanisms of infection and virus persistence. This study describes a SARS-CoV-2 isolate from a 53-year-old woman from Apulia (Italy), who was COVID-19 positive for approximately four months. In this paper we aimed to investigate any potential correlation between genetic mutations and clinical features of this case of infection. The viral isolate was assigned to lineage B.1.177.51 through whole-genome sequencing (WGS) and harbored a novel set of mutations on the Spike protein (V143D, del144/145 and E484K); furthermore, seroneutralization assays showed impaired response of the surveyed strain to BNT162b2 (Comirnaty) Pfizer/BioNTech vaccine-induced (average reduction of 70%) and convalescent sera (average reduction of 19.04%), when compared to VOC P.1. This study highlights the importance of genomic surveillance for the management of the COVID-19 pandemic, the relevance of monitoring of emerging SARS-CoV-2 mutations in all lineages, and the necessity of testing the response of emerging variants to available therapies and vaccines.

Tracking the international spread of SARS-CoV-2 lineages B.1.1.7 and B.1.351/501Y-V2 with grinch.

Late in 2020, two genetically-distinct clusters of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with mutations of biological concern were reported, one in the United Kingdom and one in South Africa. Using a combination of data from routine surveillance, genomic sequencing and international travel we track the international dispersal of lineages B.1.1.7 and B.1.351 (variant 501Y-V2). We account for potential biases in genomic surveillance efforts by including passenger volumes from location of where the lineage was first reported, London and South Africa respectively. Using the software tool grinch (global report investigating novel coronavirus haplotypes), we track the international spread of lineages of concern with automated daily reports, Further, we have built a custom tracking website (cov-lineages.org/global_report.html) which hosts this daily report and will continue to include novel SARS-CoV-2 lineages of concern as they are detected.

Accurate detection and quantification of SARS-CoV-2 genomic and subgenomic mRNAs by ddPCR and meta-transcriptomics analysis.

SARS-CoV-2 replication requires the synthesis of a set of structural proteins expressed through discontinuous transcription of ten subgenomic mRNAs (sgmRNAs). Here, we have fine-tuned droplet digital PCR (ddPCR) assays to accurately detect and quantify SARS-CoV-2 genomic ORF1ab and sgmRNAs for the nucleocapsid (N) and spike (S) proteins. We analyzed 166 RNA samples from anonymized SARS-CoV-2 positive subjects and we observed a recurrent and characteristic pattern of sgmRNAs expression in relation to the total viral RNA content. Additionally, expression profiles of sgmRNAs, as determined by meta-transcriptomics sequencing of a subset of 110 RNA samples, were highly correlated with those obtained by ddPCR. By providing a comprehensive and dynamic snapshot of the levels of SARS-CoV-2 sgmRNAs in infected individuals, our results may contribute a better understanding of the dynamics of transcription and expression of the genome of SARS-CoV-2 and facilitate the development of more accurate molecular diagnostic tools for the stratification of COVID-19 patients.

Genomic Surveillance of Circulating SARS-CoV-2 in South East Italy: A One-Year Retrospective Genetic Study.

In order to provide insights into the evolutionary and epidemiological viral dynamics during the current COVID-19 pandemic in South Eastern Italy, a total of 298 genomes of SARS-CoV-2 strains collected in the Apulia and Basilicata regions, between March 2020 and January 2021, were sequenced. The genomic analysis performed on the draft genomes allowed us to assign the genetic clades and lineages of belonging to each sample and provide an overview of the main circulating viral variants. Our data showed the spread in Apulia and Basilicata of SARS-CoV-2 variants which have emerged during the second wave of infections and are being currently monitored worldwide for their increased transmission rate and their possible impact on vaccines and therapies. These results emphasize the importance of genome sequencing for the epidemiological surveillance of the new SARS-CoV-2 variants' spread.

Degradation of nanoplastics in the environment: Reactivity and impact on atmospheric and surface waters.

Microplastics (MPs) and nanoplastics (NPs) are ubiquitous and contaminate soil, surface waters, atmospheric aerosol, precipitations, indoor and outdoor environments. However, the occurrence, transformation and fate of NPs in the environment are still unclear. In this work, polystyrene nanoparticles (PS-NPs) are used as a proxy of NPs to study their reactivity and potential impact on atmospheric and surface waters. In particular, the reactivity with hydroxyl radicals (OH) in the aqueous phase is investigated. For the first time, a reactivity constant for the reaction of NPs with OH is measured, strongly dependent on the exposed particle surface area of NPs. Degradation products (short chain carboxylic acids and aromatic compounds), obtained by direct and OH-mediated photolysis of PS-NPs suspensions, are identified by mass spectrometry. Irradiation of a PS-NPs suspension under natural sunlight for 1 year has shown the formation of formic acid and organic compounds similar to those found in riverine and cloud dissolved organic matter, which could contribute significantly to the dissolved organic matter in the aqueous phase.

Emerging high-risk ST101 and ST307 carbapenem-resistant Klebsiella pneumoniae clones from bloodstream infections in Southern Italy.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CR-KP) is an urgent public health issue in Italy. This pattern of resistance is due mainly to dissemination of carbapenemase genes. Molecular characterization of carbapenem-resistant Klebsiella pneumoniae (CR-KP) strains was performed over a three-year period. In-depth analysis was performed on a subset of emerging CR-KP ST101 and ST307 clones. METHODS: A prospective study was performed on 691 patients with CR-KP bloodstream infections hospitalized in 19 hospitals located in three large provinces in Southern Italy. Carbapenemase genes were identified via genotyping methods. Multi-locus sequence typing (MLST) and Whole Genome Sequencing (WGS) were carried out on ST101 and ST307 isolates. RESULTS: Among the CR-KP isolates, blaKPC was found in 95.6%, blaVIM was found in 3.5%, blaNDM was found in 0.1% and blaOXA-48 was found in 0.1%. The blaKPC-3 variant was identified in all 104 characterized KPC-KP isolates. MLST of 231 representative isolates revealed ST512 in 45.5%, ST101 in 20.3% and ST307 in 18.2% of the isolates. cgMLST of ST307 and ST101 isolates revealed presence of more than one beta-lactam resistance gene. Amino acid substitution in the chromosomal colistin-resistance gene pmrB was found in two ST101 isolates. CONCLUSIONS: ST512 is widespread in Southern Italy, but ST101 and ST307 are emerging since they were found in a significant proportion of cases. Aggressive infection control measures and a continuous monitoring of these high-risk clones are necessary to avoid rapid spread of CR-KP, especially in hospital settings.

Detection of a novel clone of Acinetobacter baumannii isolated from a dog with otitis externa.

In this study, the isolation of Acinetobacter baumannii in a dog with clinical bilateral otitis externa is described. Moreover, to investigate the zoonotic potential of the isolate, microbiological examinations on the family members were performed. An A. baumanniistrain was isolated from nasal swab in one of the dog owners. The identity of bacterial strains, either from dog and owner, was confirmed by phenotypic and molecular typing (wgMLST). Furthermore, to assess the pathogenic potential of the isolates a deep characterization of virulence and antibiotic resistance genes was done by Whole Genome Sequencing (WGS). Finally, the susceptibility towards a wide panel of antimicrobials was investigated. In our knowledge, this is the first recorded case of A. baumanniiisolation from canine auricular swabs in Italy. And interestingly, this study underlines the possible spread of this microorganism from human to animal.

Evaluation of Antibiotic Resistance of Helicobacter pylori Strains Isolated in Bari, Southern Italy, in 2017-2018 by Phenotypic and Genotyping Methods.

Objective: Helicobacter pylori antibiotic resistance is a constantly evolving process and local surveillance is warranted to guide clinicians in the choice of therapy. Materials and Methods: Antibiotic susceptibility testing was performed by E-test on 92 H. pylori strains, and resistance to clarithromycin and levofloxacin was also evaluated using a commercially available genotyping method. Results: In naïve patients the resistance to clarithromycin, levofloxacin, and metronidazole was 37.7%, 26.2%, and 16.4%, respectively, significantly lower than the percentage found in treated patients. Concomitant resistance to ≥2 antibiotics was also observed in naïve patients. The A2143G mutation of the 23S-rRNA gene was the most frequently detected, also in naïve patients. The highest minimum inhibitory concentration (MIC)50 value (256 mg/L) was associated with A2142 mutations in all the patients carrying them. For levofloxacin resistance a mutation in codon 87 was detected in 63.9% and in codon 91 in 36.1% of the H. pylori strains, without significant differences in the patients groups. A mutation in codon 87 was associated with the highest MIC50 value (32 mg/L). Conclusions: In our area, a high prevalence of H. pylori primary resistance was detected; these rates were higher in patients who had experienced failure of several courses of therapy. A better knowledge of the local epidemiology of resistance, and the genotypes responsible, will improve the H. pylori eradication rates.