Genomics Insight into cfr-Mediated Linezolid-Resistant LA-MRSA in Italian Pig Holdings.

The cfr genes encode for a 23S rRNA methyltransferase, conferring a multiresistance phenotype to phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A antibiotics. These genes have been described in staphylococci, including methicillin-resistant Staphylococcus aureus (MRSA). In this study, we retrospectively performed an in-depth genomic characterisation of three cfr-positive, multidrug-resistant (MDR) livestock-associated (LA) MRSA clonal complexes (CCs) 1 and 398 detected in different Italian pig holdings (2008-2011) during population studies on Italian livestock (2008-2014). We used a combined Illumina and Oxford Nanopore Technologies (ONT) whole genome sequencing (WGS) approach on two isolates (the 2008 CC1 and the 2010 CC398 isolates, but not the 2011 CC1 isolate). Interestingly, the three isolates presented different cfr variants, with only one displaying a linezolid-resistant phenotype. In isolate 2008 CC1, the cfr gene was identified within a Tn558 composite transposon-like structure flanked by IS elements located on a novel 44,826 bp plasmid. This represents the first report of CC1 LA-MRSA harbouring the cfr gene in its functional variant. Differently, cfr was chromosomally located in isolate 2010 CC398. Our findings have significant public health implications, confirm the need for the continuous genomic surveillance of cfr-positive zoonotic LA-MRSA, and backdate cfr presence in LA-MRSA from Italian pigs to at least 2008.

Evidence of structural rearrangements in ESBL-positive pESI(like) megaplasmids of S.Infantis.

The increasing prevalence of pESI(like)-positive, multidrug-resistant (MDR) S. Infantis in Europe is a cause of major concern. As previously demonstrated, the pESI(like) megaplasmid is not only a carrier of antimicrobial resistant (AMR) genes (at least tet, dfr, and sul genes), but also harbours several virulence and fitness genes, and toxin/antitoxin systems that enhance its persistence in the S. Infantis host. In this study, five prototype pESI(like) plasmids, of either CTX-M-1 or CTX-M-65 ESBL-producing strains, were long-read sequenced using Oxford Nanopore Technology (ONT), and their complete sequences were resolved. Comparison of the structure and gene content of the five sequenced plasmids, and further comparison with previously published pESI(like) sequences, indicated that although the sequence of such pESI(like) 'mosaic' plasmids remains almost identical, their structures appear different and composed of regions inserted or transposed after different events. The results obtained in this study are essential to better understand the plasticity and the evolution of the pESI(like) megaplasmid, and therefore to better address risk management options and policy decisions to fight against AMR and MDR in Salmonella and other food-borne pathogens. Graphical representation of the pESI-like plasmid complete sequence (ID 12037823/11). Block colours indicate the function of the genes: red: repB gene; pink: class I integrons (IntI); yellow; mobile elements; blue: resistance genes; green: toxin/anti-toxin systems; grey: mer operon; light green: genes involve in conjugation.

The hazard of carbapenemase (OXA-181)-producing Escherichia coli spreading in pig and veal calf holdings in Italy in the genomics era: Risk of spill over and spill back between humans and animals.

Carbapenemase-producing Enterobacterales (CPE) are considered a major public health issue. In the frame of the EU Harmonized AMR Monitoring program conducted in Italy in 2021, 21 epidemiological units of fattening pigs (6.98%; 95% CI 4.37-10.47%; 21/301) and four epidemiological units of bovines <12 months (1.29%; 95% CI 0.35-3.27%, 4/310) resulted positive to OXA-48-like-producing E. coli (n = 24 OXA-181, n = 1 OXA-48). Whole Genome Sequencing (WGS) for in-depth characterization, genomics and cluster analysis of OXA-181-(and one OXA-48) producing E. coli isolated, was performed. Tracing-back activities at: (a) the fattening holding of origin of one positive slaughter batch, (b) the breeding holding, and (c) one epidemiologically related dairy cattle holding, allowed detection of OXA-48-like-producing E. coli in different units and comparison of further human isolates from fecal samples of farm workers. The OXA-181-producing isolates were multidrug resistant (MDR), belonged to different Sequence Types (STs), harbored the IncX and IncF plasmid replicons and multiple virulence genes. Bioinformatics analysis of combined Oxford Nanopore Technologies (ONT) long reads and Illumina short reads identified bla OXA-181 as part of a transposon in IncX1, IncX3, and IncFII fully resolved plasmids from 16 selected E. coli, mostly belonging to ST5229, isolated during the survey at slaughter and tracing-back activities. Although human source could be the most likely cause for the introduction of the bla OXA-181-carrying IncX1 plasmid in the breeding holding, concerns arise from carbapenemase OXA-48-like-producing E. coli spreading in 2021 in Italian fattening pigs and, to a lesser extent, in veal calf holdings.

First report of the zoonotic nematode Baylisascaris procyonis in non-native raccoons (Procyon lotor) from Italy.

Baylisascaris procyonis is a nematode parasite of the raccoon (Procyon lotor), and it can be responsible for a severe form of larva migrans in humans. This parasite has been reported from many countries all over the world, after translocation of its natural host outside its native geographic range, North America. In the period between January and August 2021, 21 raccoons were cage-trapped and euthanized in Tuscany (Central Italy), in the context of a plan aimed at eradicating a reproductive population of this non-native species. All the animals were submitted for necroscopic examination. Adult ascariids were found in the small intestine of seven raccoons (prevalence 33.3%). Parasites have been identified as B. procyonis based on both morphometric and molecular approaches. The aim of the present article is to report the first finding of this zoonotic parasite from Italy, highlighting the sanitary risks linked to the introduction of alien vertebrate species in new areas.

Emergence of IncHI2 Plasmids With Mobilized Colistin Resistance (mcr)-9 Gene in ESBL-Producing, Multidrug-Resistant Salmonella Typhimurium and Its Monophasic Variant ST34 From Food-Producing Animals in Italy.

A collection of 177 genomes of Salmonella Typhimurium and its monophasic variant isolated in 2014-2019 from Italian poultry/livestock (n = 165) and foodstuff (n = 12), previously screened for antimicrobial susceptibility and assigned to ST34 and single-locus variants, were studied in-depth to check the presence of the novel mcr-9 gene and to investigate their genetic relatedness by whole genome sequencing (WGS). The study of accessory resistance genes revealed the presence of mcr-9.1 in 11 ST34 isolates, displaying elevated colistin minimum inhibitory concentration values up to 2 mg/L and also a multidrug-resistant (MDR) profile toward up to seven antimicrobial classes. Five of them were also extended-spectrum beta-lactamases producers (bla SHV - 12 type), mediated by the corresponding antimicrobial resistance (AMR) accessory genes. All mcr-9-positive isolates harbored IncHI2-ST1 plasmids. From the results of the Mash analysis performed on all 177 genomes, the 11 mcr-9-positive isolates fell together in the same subcluster and were all closely related. This subcluster included also two mcr-9-negative isolates, and other eight mcr-9-negative ST34 isolates were present within the same parental branch. All the 21 isolates within this branch presented an IncHI2/2A plasmid and a similar MDR gene pattern. In three representative mcr-9-positive isolates, mcr-9 was demonstrated to be located on different IncHI2/IncHI2A large-size (∼277-297 kb) plasmids, using a combined Illumina-Oxford Nanopore WGS approach. These plasmids were also compared by BLAST analysis with publicly available IncHI2 plasmid sequences harboring mcr-9. In our plasmids, mcr-9 was located in a ∼30-kb region lacking different genetic elements of the typical core structure of mcr-9 cassettes. In this region were also identified different genes involved in heavy metal metabolism. Our results underline how genomics and WGS-based surveillance are increasingly indispensable to achieve better insights into the genetic environment and features of plasmid-mediated AMR, as in the case of such IncHI2 plasmids harboring other MDR genes beside mcr-9, that can be transferred horizontally also to other major Salmonella serovars spreading along the food chain.

Plasmodium matutinum Transmitted by Culex pipiens as a Cause of Avian Malaria in Captive African Penguins (Spheniscus demersus) in Italy.

Avian malaria is a parasitic disease of birds caused by protozoa belonging to the genus Plasmodium, within the order Haemosporida. Penguins are considered particularly susceptible, and outbreaks in captive populations can lead to high mortality. We used a multidisciplinary approach to investigate the death due to avian malaria, occurred between 2015 and 2019, in eight African penguins (Spheniscus demersus) kept in two Italian zoos located in central Italy, and situated about 30 km apart. We also provided information about the presence and circulation of Plasmodium spp. in mosquitoes in central Italy by sampling mosquitoes in both zoos where penguin mortalities occurred. In the eight dead penguins, gross and histopathological lesions were consistent with those previously observed by other authors in avian malaria outbreaks. Organs from dead penguins and mosquitoes collected in both zoos were tested for avian malaria parasites by using a PCR assay targeting the partial mitochondrial conserved region of the cytochrome b gene. Identification at species level was performed by sequencing analysis. Plasmodium matutinum was detected in both dead penguins and in mosquitoes (Culex pipiens), while Plasmodium vaughani in Culex pipiens only. Parasites were not found in any of the PCR tested Aedes albopictus samples. Based on our phylogenetic analysis, we detected three previously characterized lineages: Plasmodium matutinum LINN1 and AFTRU5, P. vaughani SYAT05. In Culex pipiens we also identified two novel lineages, CXPIP32 (inferred morphospecies Plasmodium matutinum) and CXPIP33 (inferred morphospecies P. vaughani). Significantly, LINN1 and AFTRU5 were found to be associated to penguin deaths, although only LINN1 was detected both in penguins (along the years of the study) and in Culex pipiens, while AFTRU5 was detected in a single penguin dead in 2017. In conclusion, in our study Plasmodium matutinum was found to cause avian malaria in captive penguins kept in Europe, with Culex pipiens being its most probable vector. Our results are in agreement with previous studies suggesting that Culex pipiens is one of the main vectors of Plasmodium spp. in Europe and the Northern Hemisphere. Zoos maintaining captive penguins in temperate areas where Culex pipiens is abundant should be well aware of the risks of avian malaria, and should put every effort to prevent outbreaks, in particular during the periods when the number of vectors is higher.

Carbapenemase IncF-borne blaNDM-5 gene in the E. coli ST167 high-risk clone from canine clinical infection, Italy.

The blaNDM-5-producing E. coli Sequence Type (ST)167 high-risk clone is emerging worldwide in human clinical cases, while its presence in companion animals is sporadic and has never been described in Italy. Using a combined Oxford Nanopore (ONT) long-reads and Illumina short-reads sequencing approach, an E. coli ST167 isolated from a hospitalized dog, was in-depth characterized by WGS and the plasmid containing blaNDM-5 was fully reconstructed. The complete sequence of the pMOL008 mosaic plasmid (F36:F31:A4:B1; pMOL008) harbouring blaNDM-5, was resolved and characterized. Moreover, a (pro)phage and IncFII, containing blaCMY-2 and ermB, and IncI2 plasmid types were also identified. pMOL008 was almost identical to blaNDM-5-containing plasmids from E. coli ST167 isolated from Italian human clinical cases and from a Swiss dog and colonized humans. blaNDM-5 was located in a class 1 integron together with aadA2, aac(3)-IIa, mph(A), sul1, tet(A) and dfrA12. The risk of spill-over and spill-back transmission of carbapenem-resistance genes, related plasmids and strains between humans and dogs, represents a Public Health threat and highlights the importance of the One Health approach for the AMR surveillance.

Livestock-Associated Methicillin-Resistant Staphylococcus aureus and Related Risk Factors in Holdings of Veal Calves in Northwest Italy.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) have emerged worldwide as zoonotic pathogens. Data on LA-MRSA in veal calf production in Italy are lacking; the aim of this survey was to fill current knowledge gaps in its prevalence and characteristics. Between February 2012 and January 2013 nasal swabs were taken from 1650 three- to six-month-old veal calves on 55 farms in Piedmont (northwest Italy), including gathering-related epidemiological data. S. aureus were screened for methicillin resistance by phenotypic and molecular (mecA gene detection) methods. MRSA were further genotyped by multilocus sequence typing. About 30% of the herds tested positive for MRSA: three different clonal complexes (CC398, CC97, and CC1) and staphylococcal cassette chromosome mec types (IVa, IVb, and V) were detected. Multilevel logistic regression model indicated poor cleaning, importation from Austria, and animal age as risk factors and coagulase-negative staphylococci colonization as a predictive factor for the occurrence of MRSA. The detection of CCs circulating in pigs and dairy cattle in Italy underscores the ability of the LA-MRSA clones to spread among animal production systems. In addition to maintaining preventive control measures for human health, better cleaning procedures need to be implemented, especially after new calves have been introduced into the herd.

Novel IncFII plasmid harbouring blaNDM-4 in a carbapenem-resistant Escherichia coli of pig origin, Italy.

OBJECTIVES: To the best of our knowledge, we describe the first evidence in Europe of an MDR, blaNDM-4-positive Escherichia coli isolated from a food-producing animal, harboured by a novel IncFII plasmid of which we report the complete sequence. METHODS: One blaNDM-4-positive E. coli isolated in 2019 from the caecal contents of a fattening pig in Italy was in-depth characterized by combined bioinformatic analysis of Oxford Nanopore long reads and Illumina short reads, for in silico typing, determination of the blaNDM-4 genetic context and full reconstruction of the blaNDM-4-carrying plasmid. RESULTS: The isolate belonged to ST641 and to the genoserotype O108:H23 and tested positive for different virulence genes and plasmid replicons. The MDR phenotype of resistance to all ß-lactams, carbapenems, sulfamethoxazole and trimethoprim was mediated by blaTEM-1B, blaNDM-4, sul1/sul3 and dfrA12, respectively. The blaNDM-4 gene was harboured by a novel 53 043 bp IncFII plasmid (pMOL412_FII) composed of four main genetic regions, including an MDR region (MRR-NDM-4) of 16 kb carrying blaNDM-4 and several antimicrobial resistance genes located in a class 1 integron. pMOL412_FII was closely related to another ∼90.3 kb plasmid (pM109_FII) harbouring blaNDM-4 in an E. coli isolated from a human patient in Myanmar. CONCLUSIONS: To the best of our knowledge, we have identified for the first time in Europe an NDM-producing Enterobacterales in livestock and resolved the complete sequence of the novel pMOL412_FII plasmid harbouring blaNDM-4 in an MRR. A global One Health approach, comparing genomic data from different sources and geographical areas, may help to trace back and control possible plasmid-borne carbapenemase gene transmission between animals and humans and along the food chain at international level.

Molecular epidemiology of Salmonella Infantis in Europe: insights into the success of the bacterial host and its parasitic pESI-like megaplasmid.

Salmonella Infantis is one of the five serovars most frequently causing human salmonellosis in Europe, mainly associated with poultry. A clone harbouring a conjugative plasmid of emerging S. Infantis (pESI)-like megaplasmid, carrying multidrug resistant (MDR) and extended-spectrum beta-lactamases (ESBL) genes, has spread in the Italian broiler chicken industry also causing human illness. This work is aimed at elucidating the molecular epidemiology of S. Infantis and pESI-like in Europe using whole-genome sequencing and bioinformatics analysis, and to investigate the genetic relatedness of S. Infantis clones and pESI-like from animals, meat, feed and humans provided by institutions of nine European countries. Two genotyping approaches were used: chromosome or plasmid SNP-based analysis and the minimum spanning tree (MST) algorithm based on core-genome multilocus sequence typing (cgMLST). The European S. Infantis population appeared heterogeneous, with different genetic clusters defined at core-genome level. However, pESI-like variants present in 64.1 % of the isolates were more genetically homogeneous and capable of infecting different clonal lineages in most of the countries. Two different pESI-like with ESBL genes (n=82) were observed: blaCTX-M-1-positive in European isolates and blaCTX-M-65-positive in American isolates (study outgroup). Both variants had toxin-antitoxin systems, resistance genes towards tetracyclines, trimethoprim, sulphonamides and aminoglycosides, heavy metals (merA) and disinfectants (qacEΔ). Worryingly, 66 % of the total isolates studied presented different gyrA chromosomal point mutations associated with (fluoro)quinolone resistance (MIC range 0.125-0.5 mg/L), while 18 % displayed transferable macrolide resistance mediated by mph, mef and erm(B) genes. Proper intervention strategies are needed to prevent further dissemination/transmission of MDR S. Infantis and pESI-like along the food chain in Europe.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) spa type t127, Sequence Type (ST)1, quickly spreads and persists among young pigs.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) clones other than Clonal Complex (CC)398, as CC1, have been isolated in pigs in some countries, and appeared to be prevalent in Italy. The aim of this study was to evaluate the capability of Sequence Type (ST)1, CC1, LA-MRSA clone to colonize and to be transmitted among piglets. Eighteen caesarean-derived/colostrum-deprived piglets of 35 days of age were assigned randomly to three groups: four seeder piglets were contaminated with a spa type t127, ST1, SCCmec V, MRSA (Group A), 10 MRSA-negative piglets were exposed to Group A after 2 days post-contamination, dpc (Group B) and 4 piglets were used as control group (Group C). Piglets were evaluated until 44 dpc (Group A) or at 42 days post-exposure, dpe (Group B) and then euthanized and necropsied. All nasal and skin cultures of Group A resulted MRSA-positive throughout the experiment starting from two dpc, while Group C tested always MRSA-negative. At first sampling, all Group B piglets became positive and remained positive throughout the experiment. This is the first colonization/transmission study with a CC1 LA-MRSA in pigs. The results add further knowledge on the ability of CC1 LA-MRSA to colonize pigs, and on colonization/transmission patterns, both suggesting good host adaptation.

Corrigendum: Colistin Resistance Mediated by mcr-1 in ESBL-Producing, Multidrug Resistant Salmonella Infantis in Broiler Chicken Industry, Italy (2016-2017).

[This corrects the article DOI: 10.3389/fmicb.2018.01880.].

Colistin Resistance Mediated by mcr-1 in ESBL-Producing, Multidrug Resistant Salmonella Infantis in Broiler Chicken Industry, Italy (2016-2017).

Colistin-resistance mediated by mobilisable and plasmid-borne mcr genes has emerged worldwide, threatening the efficacy of colistin, a last resort antibiotic increasingly used for treating human invasive infections by multidrug-resistant or extensively drug-resistant Enterobacteriaceae. In this study, we report the first evidence of mcr-1-mediated colistin resistance in four multidrug resistant (MDR) out of 324 Salmonella infantis from the Italian antimicrobial resistance (AMR) monitoring (2001-2017) in broilers and broiler meat. Two were also Extended Spectrum Beta-Lactamases (ESBL)-producing isolates. Characterization by whole genome sequencing (WGS), located mcr-1.1 on an incX4 plasmid. Phylogenetic analysis of these isolates with selected Italian S. Infantis previously isolated from animals, meat and human clinical cases with unknown epidemiological relationship, demonstrated that ESBL-producing, mcr-1-positive isolates belonged to the emerging pESI-like-positive-ESBL-producing clone described in Italy in 2015.

Non-toxigenic Corynebacterium ulcerans sequence types 325 and 339 isolated from two dogs with ulcerative lesions in Italy.

Corynebacterium ulcerans, an emerging pathogen related to C. diphtheriae and C. pseudotuberculosis, is able to cause disease in both human and animal hosts. C. ulcerans may harbor acquired virulence factors such as dermonecrotic exotoxin phospholipase D (PLD) and the prophage-encoded diphtheria toxin (DT). Infections typically occur in persons reporting close contact with animals. In pets, C. ulcerans has been isolated from both asymptomatic carriers and clinically affected dogs and cats. We describe the isolation and characterization of C. ulcerans strains from 2 pet dogs with ulcerative lesions in Italy. The 2 isolates tested negative for both DT genes, but were PLD-producers and belonged to sequence types (STs) 325 and 339. These 2 cases highlight that C. ulcerans cutaneous infections might be underestimated in pets, given that many veterinary laboratories do not routinely consider and/or identify Corynebacterium species from cutaneous samples. Early detection and molecular typing of C. ulcerans is essential in order to implement effective treatment and to prevent diffusion and possible zoonotic transmission of certain STs.

Evidence of a Prototheca Zopfii Genotype 2 Disseminated Infection in a Dog with Cutaneous Lesions.

Protothecosis is a disease caused by saprophyte aerobic unicellular algae belonging to the genus Prototheca. In dogs, it mainly occurs as a disseminated form, with initial clinical manifestations often referable to the gastrointestinal tract, followed by typical ocular and neurological signs. So far, Prototheca zopfii genotype 2 infection has been reported in severe forms of disseminated protothecosis, while in dogs has never been associated with cutaneous forms. In this study, we describe a case of Prototheca zopfii genotype 2 infection in a dog characterized by nodular and ulcerative dermatitis and with evidence of dissemination. In December 2015, a 5-year-old unneutered male English Setter dog was presented with a 4-month history of footpads ulcerations and multifocal nodular lesions (3-5 cm diameter) on both front limbs. Cytological examination of the aspirated fluid collected from all nodules revealed the presence of sporangic forms compatible with Prototheca spp. organisms. Suspected Prototheca spp. colonies were isolated from the aspirated fluid and identified as Prototheca zopfii genotype 2 by molecular methods. Few days after the visit, the patient developed serious neurological and ocular signs, and the owners elected humane euthanasia. To the authors' knowledge, this case could represent the first report of a disseminated Prototheca zopfii genotype 2 infection associated with cutaneous lesions in a dog. This study underlines the importance of considering Prototheca zopfii genotype 2 infection in the differential etiological diagnosis of nodular and ulcerative dermatitis in dogs.

A methicillin-resistant Staphylococcus aureus (MRSA) Sequence Type 8, spa type t11469 causing infection and colonizing horses in Italy.

A Methicillin-resistantStaphylococcus aureus(MRSA) was isolated in Italy from a pathological sample of a mare presenting chronic purulent sinusitis and that had undergone frontal-sinus surgery three months before. Humans, horses, dogs and environmental samples were subsequently collected at the mare's stable and at the Veterinary Hospital, where the mare was operated/hospitalized, and screened for the presence of MRSA that was detected from other horses and from the environment at both sites. All the MRSA isolates belonged to clonal complex (CC)8, ST8-t11469-SCCmec-IVa, and showed similar phenotypic and genetic multidrug resistance patterns and macrorestriction-pulsed-field gel electrophoresis profiles. The only MRSA detected from humans was a CC1, ST1-t127-SCCmec-IVa. This paper represents the first report of a clinical MRSA infection in a horse in Italy. This study also supports the opinion that improper use of antibiotics and hospitalization/surgery can represent risk factors for MRSA colonization/infection in horses, and that the environment is among important sources for exposure.

Antimicrobial Activity of Essential Oils Against Staphylococcus aureus in Fresh Sheep Cheese.

Essential oils (EOs) are aromatic oily liquids extracted from different parts of specific plants, well known especially for their aromatic and antibacterial properties. Nowadays, EOs are exploited in the food sector mainly for their aromatic properties. Thanks to their antimicrobial activity, however, they could also be used as additives to increase the safety and the shelf-life of food products. Aim of this study was to assess the antimicrobial activity of Thymus vulgaris L. oil and of Origanum vulgare L. oil against Staphylococcus aureus both in vitro and on fresh cheese, and to determine whether the use of EOs can modify the microbiological and/or chemical-physical properties of the products. The antimicrobial activity against S. aureus in vitro was assessed by preparation of the aromatogram (diffusion in agar test), minimum inhibitory concentration test and minimum bactericidal concentration assessment. Raw sheep milk was experimentally contaminated with a strain of S. aureus ATCC 25922 and was used to produce three types of fresh cheese: without EOs, with thyme and oregano EOs (both EOs at a concentration of 1:1000). The samples were analysed on the day of production, after three and seven days. The results obtained from the tests showed that the concentration of S. aureus and the counts of lactic flora remained unchanged for all types of cheese. Even the chemical-physical parameters were constant. The results of inhibition tests on the cheese disagree with those relating to the in vitro tests. Most likely this is due to the ability of EOs to disperse in the lipids the food: the higher the fat content is, the lower the oil fraction will be able to exert the antimicrobial activity.

Sheep Milk Yogurt from a Short Food Supply Chain: Study of the Microbiological, Chemico-Physical and Organoleptic Parameters in Relation to Shelf-Life.

Aim of this work was to analyse some microbiological, chemico-physical and organoleptic parameters of sheep milk yogurt during and after its declared shelf-life. Five samples of a sheep's milk yogurt of the same lot, collected from a short supply chain ovine dairy farm of the Roman province, were analysed. Declared shelf-life of the product was 30 days. The products were examined at 2, 14, 30, 35 and 40 days from the production date, performing the following microbiological analyses: enumeration of i) colony-forming units characteristic of the yogurt, ii) Enterobacteriaceae, iii) yeasts and/or moulds at 25°C. Microbiological identification was performed by miniature biochemical tests and for the lactic acid bacteria also by PCR. At every test interval, evaluation of organoleptic parameters and pH was also performed. The analysed product maintained an almost constant amount of lactic acid bacteria until the end of the declared shelf-life. Concerning lactic acid bacteria, a 100% concordance of the results observed by using biochemical identification methods and PCR assays was obtained. After 14 days from the production, the presence of yeasts (Candida famata) was revealed, while the presence of moulds was detected after 30 days. Ralstonia picketii, an environmental microorganism, was also isolated. The results obtained in this study indicate that yogurt spoilage is mainly due to the growth of specific microorganisms of spoilage, such as yeasts and moulds.