Modestas Ruzauskas

Reflection paper on MRSA in food-producing and companion animals: epidemiology and control options for human and animal health

The scope of this reflection paper was to review the latest research on the risk of MRSA infection and colonization in animals. Attention focused on occurrence, risk factors for colonization and infection, and human contact hazard for livestock, horses, and companion animals. Whereas the clonal relationship between MRSA strains of CC398 is straightforward in livestock this is less obvious in horses. Small companion animals typically share MRSA strains that seem to exchange with a human reservoir. Management and therapeutic options have been suggested for livestock, horses, companion animals, as well as instructions on safety measures for persons in contact with animals. Conclusions were drawn with emphasis on future research activities, especially to confirm the apparent evolution of the organism and to demonstrate efficiency of control strategies.

Prevalence of trimethoprim resistance genes in Escherichia coli isolates of human and animal origin in Lithuania

A total of 456 non-repetitive Escherichia coli isolates from human clinical specimens (urinary, n=134; cervix, vagina and prostate, n=52; blood, pus and wounds, n=45), healthy animals (cattle, n=45; poultry, n=20) and diseased animals (cattle, n=53; swine, n=64; poultry, n=43) obtained in Lithuania during the period 2005-2008 were studied for trimethoprim (TMP) resistance and the prevalence of dfr genes. A TMP resistance rate in the range of 18-26 % respective to the origin was found in clinical isolates, 23-40 % in isolates from diseased animals and 9-20 % in isolates from healthy animals. Of 112 TMP-resistant isolates, 103 carried at least one of the six dfrA genes (dfrA1, dfrA5, dfrA8, dfrA12, dfrA14 and dfrA17) as determined by multiplex PCR and RFLP. The dfrA1 and dfrA17 genes were found most frequently in clinical isolates (17 and 19 isolates, respectively), whilst dfrA1 and dfrA14 genes dominated in isolates of animal origin (25 and 13 isolates, respectively). The dfrA5, dfrA12 and dfrA8 genes were detected at lower frequencies. The association with class 1/class 2 integrons was confirmed for 73-100 % of dfr genes found in most groups of isolates, except for the isolates from diseased swine. In this group, the majority of dfr-positive isolates (67 %, 8/12) carried dfrA8 (6/12) or dfrA14 genes (2/12) that were not associated with integrons. Non-integron location was also confirmed for the remaining dfrA8 genes (six clinical isolates and one isolate from diseased cattle) and for dfrA14 genes (two isolates from diseased cattle and swine each). All cassette-independent dfrA14 genes were found to be located within the strA gene. This study on the prevalence and distribution of TMP resistance genes among E. coli isolates of human and animal origin in Lithuania demonstrates that dfr genes are carried most frequently as gene cassettes within class 1 and/or class 2 integrons. However, TMP resistance in some of the isolates was found to be mediated by non-integron-associated dfrA8 and dfrA14 genes, indicating the existence of alternative sources for the spread of resistance.

Detection of the mcr-1 gene in Escherichia coli prevalent in the migratory bird species Larus argentatus

Sir, To the best of our knowledge, we describe the first known occurrence of the mcr-1 gene in Escherichia coli carried by a wild migratory bird—the European herring gull (Larus argentatus). Recently, the first plasmid-mediated colistin resistance determinant, mcr-1, has been identified in Enterobacteriaceae prevalent in humans, domestic animals and food. Colistin is a polymyxin antibiotic widely used in animal production and currently increasingly prescribed for therapeutic usage in human medicine, as a consequence of the spread of MDR Gram-negatives. The mcr-1 gene, encoding a phosphoethanolamine transferase, was first identified among Chinese enterobacterial isolates and later on it was detected in some European countries including Denmark, the UK, Germany, Belgium and France. In the present case, the mcr-1 gene was detected in non-ESBLproducing E. coli. The study involved 22 ESBL-positive and 95 ESBL-negative E. coli isolates obtained from 160 European herring gulls tested for the presence of MDR. The study protocol was approved by the Lithuanian Environmental Protection Agency (protocol number 15.10-A4-8844). Among 117 isolates tested, 8 of them had elevated colistin MICs of 4–8 mg/L and a single isolate harboured the mcr-1 gene. The gene was detected by PCR following the protocol described previously. Faeces of birds were

Prevalence of methicillin-resistant Staphylococcus haemolyticus in companion animals: a cross-sectional study

BackgroundAmong coagulase-negative staphylococci, Staphylococcus haemolyticus is the second most frequently isolated species from human blood cultures and has the highest level of antimicrobial resistance. This species has zoonotic character and is prevalent both in humans and animals. Recent studies have indicated that methicillin-resistant S. haemolyticus (MRSH) is one of the most frequent isolated Staphylococcus species among neonates in intensive care units. The aim of this study was to determine the presence of MRSH in different groups of companion animals and to characterize isolates according their antimicrobial resistance.MethodsSamples (n = 754) were collected from healthy and diseased dogs and cats, female dogs in pure-breed kennels, healthy horses, and kennel owners. Classical microbiological tests along with molecular testing including PCR and 16S rRNA sequencing were performed to identify MRSH. Clonality of the isolates was assessed by Pulsed Field Gel Electrophoresis using the Sma I restriction enzyme. Antimicrobial susceptibility testing was performed using the broth micro-dilution method. Detection of genes encoding antimicrobial resistance was performed by PCR. Statistical analysis was performed using the R Project of Statistical Computing, “R 1.8.1” package.ResultsFrom a total of 754 samples tested, 12 MRSH isolates were obtained. No MRSH were found in horses and cats. Eleven isolates were obtained from dogs and one from a kennel owner. Ten of the dog isolates were detected in pure-breed kennels. The isolates demonstrated the same clonality only within separate kennels.The most frequent resistances of MRSH isolates was demonstrated to benzylpenicillin (91.7%), erythromycin (91.7%), gentamicin (75.0%), tetracycline (66.7%), fluoroquinolones (41.7%) and co-trimoxazole (41.7%). One isolate was resistant to streptogramins. All isolates were susceptible to daptomycin, rifampin, linezolid and vancomycin. The clone isolated from the kennel owner and one of the dogs was resistant to beta-lactams, macrolides, gentamicin and tetracycline.ConclusionsPure-breed kennels keeping 6 or more females were determined to be a risk factor for the presence of MRSH strains. MRSH isolated from companion animals were frequently resistant to some classes of critically important antimicrobials, although they remain susceptible to antibiotics used exclusively in human medicine.

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.

Microbiome and antimicrobial resistance genes in microbiota of cloacal samples from European herring gulls (Larus argentatus)

Abstract Introduction: The aim of the study was to determine microbiota in the cloacal samples of European herring gulls (Larus argentatus) and to compare a variety of genes encoding antimicrobial resistance in cultivable and non-cultivable bacteria. Material and Methods: Cloacal samples from European herring gulls were collected from a Kaunas city dump. Cultivable microbiota were isolated, their microbial susceptibility was tested, and genes encoding antimicrobial resistance were detected. Additionally, a metagenomic study was performed using Next-Generation Sequencing (NGS). Results: In total, 697 different operational taxonomic units at genus level were detected; however, only 63 taxonomic units were detected at the amount of ≥0.1% of the total number of DNA copies. Catellicoccus marimammalium was found to have the highest prevalence. The bacterial amount of other genera was up to 5% with the most highly prevalent being Psychrobacter (4.7%), Helicobacter (4.5%), unclassified Enterococcaceae (3.2%), Pseudomonas (2.9%), and Brachyspira (2.6%). Conclusions: C. marimammalium are predominant microbiota in the cloacal samples of Larus argentatus. This species of gulls is a reservoir of bacteria carrying a wide-spectrum of genes encoding antimicrobial resistance. The same genes were detected in both cultivable microbiota and in the total DNA of the samples.

Development and characterization of the gummy–supplements, enriched with probiotics and prebiotics

ABSTRACT The aim of this study was to develop gummy–supplements (G-S) based on probiotics (Lactobacillus plantarum LUHS135 and L. paracasei LUHS244), prebiotics (psyllium husk), and apple pomace as an pectin source, and to evaluate viable lactic acid bacteria (LAB) count, total phenolic compounds (TPC) content, antioxidant activity, colour coordinates, texture parameters, and overall acceptability of the developed G-S. Antimicrobial properties of the used LAB strains against Pseudomonas aeruginosa, Proteus mirabilis, Escherichia coli, Salmonella enterica, Staphylococcus aureus, Enterococcus faecalis, and Streptococcus mutans were investigated. Higher lightness, yellowness, and acceptability of the G-S with gelatin were found. G-S with agar showed harder texture, and agar/gelatin selection has a significant influence on TPC content in G-S. The antioxidant activity of G-S was depended on the strain of LAB and the use of psyllium husk. LUHS244 inhibited all the tested pathogenic strains. The developed G-S formula simply allowed to produce higher value products.

Sea Buckthorn (Hippophae rhamnoides L.) and Quince (Cydonia oblonga L.) Juices and Their By-Products as Ingredients Showing Antimicrobial and Antioxidant Properties for Chewing Candy: Nutraceutical Formulations

Sustainable and environmentally friendly approaches to the production of health foods have become very popular. The concept of this study was to develop chewing candy (CC)—nutraceutical formulations based on sea buckthorn (Hippophae rhamnoides L.) and quince (Cydonia oblonga L.) juice and juice by-products (BuJ, QuJ, BuBP, and QuBP, resp.), as ingredients showing antimicrobial properties against Klebsiella pneumoniae, Salmonella enterica, Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis, methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecalis, E. faecium, and Bacillus cereus. Two texture-forming agents (agar and gelatin) were tested for CC formulation. BuJ, QuJ, BuBP, and QuBP showed antimicrobial activity against all the pathogens tested, and the largest inhibition zones against Bacillus and Proteus mirabilis were observed for BuJ and QuJ, respectively. Agar and/or gelatin selection has a significant influence on CC texture ( ), and interactions of agar and/or gelatin selection × juice or juice by-products and sea buckthorn or quince × juice or juice by-products were also significant ( ). The best acceptability was shown for CC prepared with agar and BuBP (131.7) and with gelatin and QuJ (132.0). The addition of BuJ, QuJ, BuBP, and QuBP increases the antioxidant activity of CC by five times. Finally, not just juice, but also juice by-products, have great potential as desirable antimicrobial ingredients for the food industry.

The effects of ultrasonication, fermentation with Lactobacillus sp., and dehydration on the chemical composition and microbial contamination of bovine colostrum

The aim of this study was to evaluate the influence of ultrasonication, fermentation with Lactobacillus plantarum LUHS135 and Lactobacillus paracasei LUHS244, and different methods of dehydration on the chemical composition of bovine colostrum (BC), including the fatty acid and free amino acid profile and the content of micro- and macroelements. In addition, we analyzed the changes in lactic acid bacteria count, microbial contamination (aerobic mesophilic spore-forming bacteria, enterobacteria including Escherichia coli, and fungi/yeasts), the abundance of biogenic amines, and the concentration of nucleotide monophosphates. Significant effects of different treatments on the free amino acid profile were established, and an increase of lysine concentration by 1.2 to 95.9% was observed in treated BC. All of the treatments reduced the concentration of cadaverine, histamine, and tyramine in BC. The concentrations of macro- and microelements in BC followed the following order Ca > Na > K > Mg and Zn > Fe > Sr > Ba > Mn > Cu > Al > Se > Mo > Cr > Ni > Sn > Co > Pb > Cd. By combining the fermentation with Lactobacillus plantarum strain LUHS135 and vacuum drying, it was possible to increase the abundance of nucleotide monophosphates by more than 100%. All of the treatments reduced the microbial contamination of BC. Thus, the combination of ultrasonication, fermentation, and dehydration can be used for improving the properties and safety of BC.

Nutraceuticals in gummy candies form prepared from lacto‐fermented lupine protein concentrates, as high‐quality protein source, incorporated with Citrus paradise L. essential oil and xylitol

1 Lithuanian University of Health Sciences, Tilzes g. 18, Kaunas LT-47181, Lithuania 2 University of Latvia, Jelgavas iela 1, Riga LV-1004, Latvia 3 Institute of Food Safety, Animal Health and Environment, Lejupes iela 3, Riga LV-1076, Latvia 4 Institute of Food Hygiene, Universit€at Leipzig, An den Tierkliniken 1, Leipzig 04103, Germany 5 Kaunas University of Technology, Radvilenu pl. 19, Kaunas LT-50254, Lithuania