## 3 Results and Discussion  
Sixty milk samples from three species, namely cows, sheep, and goats, were tested for a number of pathogens by both conventional culture-based methods as well as by the real-time PCR method. The isolated bacteria from the milk samples are shown in Table 2. Overall, _K. pneumoniae_ strains were detected in all analyzed unpasteurized ovine milk samples (\(n=20\)), whereas _Enterococcus_ strains were detected in all of the examined bovine (\(n=20\)) and coprime (\(n=20\)) milk samples, therefore comprising 66.6% of the total analyzed milk samples (Table 2). _Staphylococcus saprophvticus_ was identified only in the analyzed ovine milk samples (\(n=20\); 33.3%). In regard to the rest of the examined pathogens (_Staphylococcus aureus, Staphylococcus epidermidis_, and _Escherichia coli_), they were not identified in any of the examined samples, independently of the species. The presence of _K. pneumoniae_, which belongs to the pathogens that cause mastitis, has been reported mostly in dairy cows [30; 31] and it is regulated by good hygiene practices as its presence has been associated with feces and manure treatment, wood, and inorganic bedding. _K. pneumoniae_ can act opportunistically as a pathogen in patients under immunosuppression and as such has been extensively studied in human infection through the incidence of severe hospital outbreaks. It is a current issue for public health since many _K. pneumoniae_ strains are resistant to antibiotics of beta-lactam, which are hydrolyzed by the enzyme beta-lactamases produced by the bacteria [31]. The isolates that were confirmed as _K. pneumoniae_ were then further analyzed for antibiotic resistance with the NG-Test Carba 5 kit, which detects the presence of five carbapenemases. Strains of _Klebsiella pneumoniae_ that are resistant to the antibiotics carcinogenesis were first identified in 1996 and are considered important nosocomial pathogens causing serious infections associated with increased mortality [32]. The Carbapenemresistant _Klebsiella pneumoniae_ have genes most commonly carried in plasmids that encode for the production of enzymes called carbapenemases that have the ability to hydrolyze the carbapenems. Carbapenemases are enzymes that belong to the family of \(\beta\)-lactamases but are extremely versatile and can recognize almost all hydrolyzable \(\beta\)-lactams, and most importantly, they are resistant to inhibition by \(\beta\)-lactamase inhibitors [33]. In this study, the detection of the most common carbapenemases was performed such as: KPC (_\"Klebsiella pneumoniae_ carbapenemase\"), OXA (\"oxacillin-hydrolyzing \(\beta\)-lactamases\"), IMP (\"active \begin{table}
\begin{tabular}{l c c c c} \hline \hline
**Strains** & **Total** & **Bovine** & **Ovine** & **Caprine** \\ \hline _Klebsiella pneumoniae_ & 20 (33.3\% *) & 0/20 & 20/20 & 0/20 \\ _Enterococcus_ spp. & 40 (66.7\% *) & 20/20 & 0/20 & 20/20 \\ _Staphylococcus_ & & & & \\ _saprophvticus_ & 20 (33.3\% *) & 0/20 & 20/20 & 0/20 \\ _Staphylococcus aureus_ & 0 & 0/20 & 0/20 & 0/20 \\ _Staphylococcus epidermidis_ & 0 & 0/20 & 0/20 & 0/20 \\ _Escherichia coli_ & 0 & 0/20 & 0/20 & 0/20 \\ \hline \hline \end{tabular}  
* Percentage of the total milk samples, independently of the species.  
\end{table} 
Table 2: Isolated bacteria in animal milk samples verified by chromogen agar and conventional PCR. on impienem\"), VIM (\"Verona integron-encoded metallo-lactamase\"), and NDM (\"New Delhi Metallo-\(\beta\)-Lactamase\"). As presented in Figure 1, the results of the in vitro multiplex immunoassay showed that at least one carbapenemase was produced by four (20.0%) isolates. In particular, NDM carbapenemase production was observed in two _K. pneumoniae_ isolates (10%) while an equal number of isolates produced OXA-48 carbapenemase. This is a significant finding because as of the year 2005, there were no cases of non-susceptible _Klebsiella_ in carbapenem at a European level but in the following years, carbapenem-resistant _K.