Phenotypical and Genotypical Antimicrobial Resistance of Coagulase-negative staphylococci Isolated from Cow Mastitis.

The objectives of this study were to determine the prevalence and antimicrobial resistance of coagulase-negative staphylococci (CNS) isolated from dairy cows with subclinical mastitis. Antimicrobial resistance in staphylococci were evaluated by breakpoint values specific to the species (EU-CAST). The presence of resistance-encoding genes was detected by multiplex PCR. A total of 191 CNS isolates were obtained. The CNS isolates were typically resistant to penicillin (67.4%), tetracyc-line (18.9%), and erythromycin (13.7%). CNS isolates (78.0%) were resistant to at least one antimicrobial compound, and 22.0% were multiresistant. The multiresistant isolates were predominantly Staphylococcus chromogenes (28.6%), Staphylococcus warneri (19%) and Staphylococcus haemolyticus (14.3%). According to MIC pattern data, multiresistant isolates showed the highest resistance (p<0.05) rates to penicillin (85.7%), tetracycline (66.7%), and erythromycin (48.2%), but all of them were sensitive to daptomycin, oxacillin, qiunupristin/dalfopristin, and vancomycin. S. chromogenes (9.5%), S. haemolyticus (4.8%), and S. capitis ss capitis (2.4%) strains were resistant to methicillin; their resistance to oxacillin and penicillin was more than 8 mg/l. A high rate of resistance to penicillin was linked to a blaZ gene found in 66.6% of the isolated multiresistant CNS strains. Resistance to tetracycline via the tetK (38.1%) gene and penicillin via the mecA (23.8%) gene were detected less frequently. Gene msrAB was responsible for macrolides and lincosamides resistance and detected in 28.6% of the CNS isolates. Antimicrobial resistance genes were identified more frequently in S. epidermidis, S. chromogenes, and S. warneri.

Characterization of Staphylococcus pseudintermedius isolated from diseased dogs in Lithuania.

The aim of this study was to characterize Staphylococcus pseudintermedius for its antimicrobial resistance and virulence factors with a special focus on methicillin-resistant (MRSP) strains isolated from sick dogs in Lithuania. Clinically sick adult dogs suffering from infections (n=214) and bitches with reproductive disorders (n=36) from kennels were selected for the study. Samples (n=192) from the 250 tested (76.8%) dogs were positive for Staphylococcus spp. Molecular profiling using the species-specific nuc gene identified 51 isolates as S. pseudintermedius (26.6% from a total number of isolated staphylococci) of which 15 isolates were identified as MRSP. Ten MRSP isolates were isolated from bitches with reproductive disorders from two large breeding kennels. Data on susceptibility of S. pseudintermedius to different antimicrobials revealed that all isolates were susceptible to vancomycin, daptomycin and linezolid. Two isolates (3.9%) were resistant to rifampicin. A high resistance was seen towards penicillin G (94.1%), tetracycline (64.7%) and macrolides (68.7%). Resistance to fluoroquinolones ranged from 25.5% (gatifloxacin) to 31.4% (ciprofloxacin). The most prevalent genes encoding resistance included blaZ, aac(6')-Ie-aph(2'')-Ia, mecA, and tet(M). The Luk-I gene encoding a leukotoxin was detected in 29% of the isolates, whereas the siet gene encoding exfoliative toxin was detected in 69% of the S. pseudintermedius isolates. This report of MRSP in companion animals represents a major challenge for veterinarians in terms of antibiotic therapy and is a concern for both animal and public health.

Gut microbiota isolated from the European pond turtle (Emys orbicularis) and its antimicrobial resistance.

The aim of the study was to isolate cultivable gut microbiota from European pond turtles kept at the Lithuanian Zoo and to determine antimicrobial resistance of the isolates. The study subjects included 8 elderly turtles living at the Lithuanian Zoo for about 50 years as well as their offspring - 24 young individuals (1-2 years old) that were hatched at the same zoo. Animals were not exposed by treatment with antimicrobials during the last 3 years. Gut samples were taken from the cloaca and inoculated onto universal media. Isolates then were identified using sequence analysis of 16S rRNA. The antimicrobial susceptibility testing was performed using the agar diffusion method according to Kirby-Bauer. Clinical breakpoints according to CLSI whenever possible, were used for interpretation of susceptibility. Bacterial isolates resistant to at least three antimicrobials of different classes were treated as multi-resistant. Fifty-two bacterial isolates were obtained and identified from turtle gut samples. The most prevalent genera included Aeromonas, Chryseobacterium and Citrobacter. Fifty percent of the isolates obtained from elderly turtles (CI 95% - 19.01-80.99) and 54.8% (CI 95% - 39.75-69.85) of the isolates from young animals were identified as multi-resistant. The most common resistance rates of the isolates from both groups of the turtles were observed toward ampicillin (86.6%), ciprofloxacin (61.5%) and gentamicin (40.4%). The lowest number of resistant isolates were detected toward combination of sulfamethoxazole-trimethoprim (26.9%). The study revealed that European pond turtles kept in captivity are carriers of multi-resistant bacteria however, further studies need to be performed to investigate whether the resistant microorganisms are natural microbiota for this species or they were acquired in the zoo.

First report of swine-associated methicillin-resistant Staphylococcus aureus ST398 in Lithuania.

During 2011, 160 nasal samples were taken from pigs on 8 different farms in Lithuania. Four methicillin-resistant Staphylococcus aureus (MRSA) isolates were obtained. The isolates were ST398, spa type t011 and SCCmec V and none carried the lukF/lukS genes. Strains were resistant to tetracycline, attributed to tetK and tetM genes, and to erythromycin owing to the ermB gene. One MRSA strain was resistant to trimethoprim/sulfamethoxazole and carried the dfrK gene. This is the first report on the presence and characteristics of livestock-associated MRSA isolated from pigs in Lithuania.

Antimicrobial resistance patterns to beta-lactams of gram-positive cocci isolated from bovine mastitis in Lithuania.

The aim of the study was to isolate gram-positive cocci from cows with mastitis and to determine their resistance to beta-lactamic antibiotics. Eight hundred and nine strains were isolated and identified as staphylococci (n=516), streptococci (n=199) and enterococci (n=94) from sub-clinical and clinical cases of bovine mastitis in Lithuania. The most common causative agents of udder disease included: S. epidermidis (n=176), S. aureus (n=176), S. agalactiae (n=134), S. hyicus (136) and E. hirae (n=68). Isolates were analysed for antimicrobial resistance to penicillin, ampicillin, amoxicillin, cephalothin, cephalexin, amoxicillin + clavulanate. The susceptibility patterns were analysed using the agar disk diffusion method. S. aureus showed the highest level of resistance to amoxicillin (81.3%), penicillin (76.7%) and ampicillin (78.4%). The corresponding values for CNS strains were 59.7%, 59.7% and 50.6% against penicillin, ampicillin and amoxicillin respectively. Streptococci were the most frequently resistant to amoxicillin (29.3%), and enterococci to penicillin (27%), amoxicillin (27.5%) and amoxicillin/clavulanic acid (23.8%). The resistance of all tested mastitis pathogens to aminopenicillins and penicillin highly correlated (r=0.83). Compared with other antibiotics, amoxicillin and clavulanic acid combination tended to be more effective (p<0.05) against all tested bacteria in vitro. However, S. aureus, in 38.1% of cases, was resistant to this combination of antimicrobials. This study demonstrates that S. epidermidis, S. aureus, S. hyicus, S. agalactiae and E. hirae remain the most frequent mastitis causative agents on Lithuanian cattle farms. The highest resistance in vitro to penicillins was demonstrated by S. aureus, S. hyicus and S. intermedius. Resistance to cephalosporins remains low, irrespective of bacterial species of gram-positive cocci.

Correlation of different biochemical parameters in blood sera of healthy and sick cows.

Metabolic diseases of cows represent the leading internal pathology in Lithuania in terms of incidence and economic impact. This paper summarizes the mineral metabolic state of milk cows, and details the influence of feeding on serum levels of calcium, nonorganic phosphorus, magnesium, potassium, sodium, glucose, total protein, and urea quantity, and correlationd among them, in healthy dry or post-calving cows as well as in cows with osteomalacia and milk fever after calving. There was less pronounced hypocalcaemia and there were only minor changes in phosphorus, magnesium, potassium and sodium in the serum of healthy dry and post-calving cows that had silage and mineral-vitamin supplements, compared to cows that did not have supplements and silage. There was a fall in calcium and phosphorus (to 1.89 +/- 0.12 mmol/L and 0.71 +/- 0.06 mmol/L, respectively) in the blood of cows sick with milk fever after calving, while levels of magnesium and potassium were increased. The correlation between calcium and phosphorus was r = 0.6993, p < 0.001 in the serum of sick cows. There was a fall in calcium, phosphorus and magnesium (to 1.86 +/- 0.46 mmol/L, 0.75 +/- 0.37 mmol/L, and 0.60 +/- 0.19 mmol/L, respectively) and an increase in sodium level (to 158.90 +/- 19.30 mmol/L) in the blood of cows with osteomalacia in comparison with healthy cows.

Interaction between the levels of hormones and minerals in sera of healthy and sick cows.

An interaction between blood levels of parathyroid hormone, calcitonin, 1.25-dihydroxycholecalciferol and levels of calcium, phosphorus and magnesium was examined in 85 cows, which included healthy cows and cows with ostemalacia, mastitis and paresis. Levels of parathyroid hormone (PTH) and calcitonin were determined in vitro using IMMULITE analyser (Diagnostic Products Corporation, USA), by means of immunometric assay. Levels of vitamin D were measured using the enzyme linked immunosorbent assay (ELISA). Levels of calcium, phosphorus and magnesium were determined using the automated Eos-Bravo analyser (Hospitex Diagnostics, Italy) with HOSPITEX reagents. The lowest blood levels of calcium (1.38 +/- 0.18 mmol/L) and phosphorus (0.65 +/- 0.12 mmol/L) were found in cows with parturient paresis. Decreased blood levels of phosphorus and magnesium were also determined in cows with osteomalacia. For cows with parturient paresis, which received a mineral supplement, the average serum level of calcium was by 20.7% higher than the level found in those which did not receive a supplement, and the level of phosphorus was by 23.6% higher, however, these levels remained low. The blood level of parathyroid hormone ranged from 3.47 to 5.20 pmol/L in healthy cows and from 3.95 to 15.21 pmol/L in sick cows. The highest and statistically significant increase in blood PTH level (up to 18.31 +/- 1.88 pmol/L) was found in cows with parturient paresis. The blood level of PTH correlated inversely with the level of calcium in cows with osteomaliacia (r = -0.89) and in cows with parturient paresis (r = -0.49 and r = -0.61, respectively). The serum level of calcitonin ranged from 1.46 pmol/L to 2.40 pmol/L in healthy and sick cows and the difference was not statistically significant. Lower serum levels of vitamin D were found in heifers-in-calf and in cows with mastitis. A clear correlation between levels of calcitonin, vitamin D and macronutrients was not found.