Pongolini, S. | bioRxiv Science

bioRxiv2025

Inactivation of Influenza A Viruses (H1N1, H5N1) During Grana-Type Raw Milk Cheesemaking: Implications for Foodborne Transmission Risk

BackgroundThe detection of H5N1 highly pathogenic avian influenza virus (HPAIV) in lactating dairy cattle in the United States, with high viral titers in raw milk, has raised concerns about potential zoonotic transmission through the consumption of unpasteurized milk and raw-milk dairy products. While inactivation studies exist for pasteurized milk, data on virus persistence during the manufacture of raw-milk cheeses remain scarce. AimTo evaluate the survival and inactivation of avian influenza viruses (AIV), including both low pathogenic (LPAIV, H1N1) and highly pathogenic (HPAIV, H5N1) strains, during the production and ripening of Grana-type hard cheeses made from raw cows milk. MethodsExperimental cheesemaking was conducted using raw milk artificially contaminated with A/duck/Italy/281904-2/06 (H1N1; 107.75 EID50/mL) or A/duck/Italy/326224-2/22 (H5N1 clade 2.3.4.4b; 106.75 EID50/mL). Cheeses were produced in accordance with Parmigiano Reggiano production standards and ripened for 30 days at 5-6 {degrees}C. Viral presence was assessed in finished cheeses by inoculation on SPF embryonated chicken eggs (ECE), hemagglutination (HA) assay, and monoclonal antibody-based ELISA. ResultsNo infectious virus was detected in any cheese sample produced from contaminated milk following two blind passages in SPF-ECE. Both HA and ELISA tests yielded negative results, indicating complete inactivation of the virus. ConclusionThis study demonstrates that the traditional Grana-type cheese production process-- including curd cooking, acidification, and ripening--effectively inactivates both LPAIV and HPAIV, even at high contamination levels. These findings support the microbiological safety of hard cheeses made from raw milk with regard to AIV, contributing to risk assessment and food safety policies during avian influenza outbreaks.

microbiology↗

bioRxiv2018

Gene composition as a potential barrier to large recombinations in the bacterial pathogen Klebsiella pneumoniae

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

evolutionary biology↗