Methicillin Resistant Staphylococcus aureus (MRSA) in raw buffalo milk and its fate along the human gastrointestinal tract: an in vitro study

Methicillin resistant Staphylococcus aureus (MRSA) has become one of the major public health challenges worldwide. MRSA strains are capable of causing from mild non-life-threatening to severe infections of the skin and soft tissues, and even death. Skin infections caused by MRSA include primary pyodermas such as folliculitis, furuncles, carbuncles, and impetigo. Infections involving the soft tissues include cellulitis and pyo-myositis, which are less common but can cause serious morbidity. The contamination of raw food, especially meat and milk, by MRSA is well documented as well as its transmission to humans via animal contact. In addition, humans can act as reservoirs of MRSA without showing any clinical signs, thus they can contaminate foods by handling. Its presence in several kinds of food has suggested the possibility of MRSA to act as a foodborne pathogen. Although the hypothesis is suggestive, there are insufficient evidence to consider the foodstuff a vehicle of MRSA infection. For instance, nothing is known about its ability to survive under human gastroenteric conditions. Despite of the potential hazard for human health there is a lack of date on prevalence of MRSA in some foods, such as buffalo milk and buffalo dairy products. The high consumptions of buffalo drinking milk and dairy products worldwide involve an elevated number of consumers of all ages, and this is crucial considering their potential role in the transmission of foodborne pathogens. To address these issues, the aims of the thesis are: i) to assess the occurrence of MRSA in new ecological niches such as buffalo dairy farms and buffalo tank milk from Italy in order to better understand the epidemiology of MRSA ii) to study the fate of MRSA strains isolated from foods and from humans along the human gastrointestinal tract and its inter-species interaction with the human gut microbiota. Regarding the occurrence of MRSA in raw buffalo milk, seventy-five bulk tank milk (BTM) samples from farms and 24 nasal swabs from farm workers were collected, respectively. Three (4%) out of 75 BTM samples and 1 (4%) out of 24 nasal swabs were MRSA-positive. The milk isolates showed the following genotypes: ST1/t127/Va and ST72/t3092/V, while the human isolate was characterized as ST1/t127/IVa. No ST398 were found. All the isolates were multidrug resistant but vancomycin susceptible, carrying the icaA gene, while they tested negative for pvl and ses genes. This study demonstrates for the first time in Europe that MRSA might be present in dairy buffalo farms and in raw buffalo milk. For what concern the second aim, a MRSA ST398/t011/V strain, previously isolated from raw cow milk, and a human origin MRSA strain were inoculated into two foods of animal origin respectively. The pH of the matrices was gradually decreased to 2.0 in 2 hours, during which time they were kept at 37°C and periodically homogenized. The same MRSA strains levels were inoculated within an intestinal in vitro simulator and it was periodically analyzed their fate along the whole transit. Mucin agar carriers replaced the intestinal mucus layer and a basic feed medium represented the intestinal lumen contents. A three-day in vitro study was performed using microbiota from the pooled faeces of healthy individuals that were stabilized simulating colon conditions. The MRSA population survived the decreasing gastric pH levels unharmed, but it was affected by the organic acids produced by the enteric microbiota along the transit into the simulator. It was, in fact, no longer viable after 24 h of incubation with luminal colon microbiota, whereas counts of 4 log cfu/g were still obtained in the mucin agar carriers after 72 h of incubation. Despite the ability of MRSA to overcome human stomach acidic conditions, these results confirm the hypothesis that competitive microbiota may control MRSA intestinal colonization.