Dairy Cows' Udder Pathogens and Occurrence of Virulence Factors in Staphylococci

Abstract

This study investigated 960 Slovak and Czech spotted cattle from four different conventional (non-organic) dairy herds located in Eastern Slovakia and Czechia during early lactation (14-100 days after calving). Dairy cows were examined clinically; milk from fore-stripping of each udder quarter was subjected to sensory examination and assessed by the California mastitis test (CMT), and laboratory analyses of bacterial pathogens in milk, including virulence factors, were conducted. Positive CMT scores (1-3) for one or more quarters were detected in 271 (28.2%) of the examined animals. Out of 230 infected milk samples, representing 24.0% of all dairy cows, staphylococci (59.1% of positive findings) were the most commonly isolated organisms, followed by E. coli (11.3%), streptococci Str. uberis (9.1%) and Str. agalactiae (3.4%), and enterococci (6.1%). From 136 isolates of S. aureus (38 isolates) and non-aureus staphylococci (NAS; 98 isolates), virulence factors and their resistance to 14 antimicrobials were detected using the disk diffusion method, with PCR detection of the methicillin resistance gene, mecA. An increased incidence of clinical and chronic forms of mastitis has been reported in mastitic cows in which staphylococci, especially S. aureus and NAS (S. chromogenes, S. warneri, and S. xylosus), have been detected and compared to other isolated udder pathogens. From those species, S. aureus and isolates of NAS mentioned above showed multiple virulence factors that are more likely to hydrolyze DNA, hemolysis, produce gelatinase and biofilm, and have multi-drug resistance as compared to other less virulent staphylococci. Generally, the isolated staphylococci showed 77.2% resistance to one or more antimicrobials, in particular to aminoglycosides, β-lactams, macrolides, or cephalosporins. Isolates that showed the ability to form a biofilm were more resistant to more than one antimicrobial than isolates without biofilm production. Multi-drug resistance to three or more antimicrobial classes was recorded in 16 isolates (11.7%), and the presence of the mecA gene was also confirmed in two isolates of S. aureus and two species of NAS.

Keywords: antibiotics; biofilm; early lactation; mastitis; methicillin resistance gene; non-aureus staphylococci.

Figure 1

DNase test and biofilm production on Congo Red agar for staphylococcal detection. Source: Zigo et al. [6].

Figure 2

Prevalence of mastitis in monitored dairy herds during early lactation. Note: CZ 1–2: dairy farms situated in Czechia; SK 1–2: dairy farms situated in Slovakia. Subclinical mastitis: no signs are observed, the udder and milk appear normal, but an infection is still present with a positive CMT score and an increased SCC. Clinical mastitis: signs that are mild, moderate, or severe. Chronic mastitis: detected based on history (previous treatment) of clinical examination of the udder and positive CMT score.

Figure 3

Comparison of resistance to more than one antimicrobial in isolates forming or non-forming biofilm at a time. Note: n—number of resistant isolates to more than one antimicrobial.

Figure 4

Detection of mecA gene in Staphylococcus spp. isolates isolated from infected raw milk samples using the PCR method (527 bp). L: 100 bp ladder; Line 1: isolate S. xylosus without mecA gene; Line 2: isolate S. capitis without mecA gene; Line 3: isolate S. chromogenes with mecA gene; Line 4: isolate S. warneri with mecA gene; Lines 5 and 6: isolate S. aureus with mecA gene; Line 7: isolate S. haemolyticus without mecA gene; Line 8: reference strain CCM 4750 S. aureus (positive control); Line 9: water (negative control).