Devitalization of Bacteria in Composted Cattle Manure with Natural Additives and Risk for Environment.

Nowadays, there is an effort to improve the effectiveness of the composting process, supported by the addition of various supplements to reduce soil nutrition losses and increase soil remediation. The aim of this study was to examine the devitalization effect of natural additives like zeolite-clinoptilolite and its combination with hydrated lime in composted cattle manure on indicator and pathogen bacteria. The composting process was running in three static piles of cattle manure mixed with wheat straw (control, zeolite-lime, and zeolite) for 126 days. Composted manure substrates were determined for physicochemical (temperature, pH, nitrogen and phosphorus content, C/N, organic matter, and moisture) and microbiological analyses (Salmonella spp., indicator bacteria). The effects of additives were reflected in changes in physicochemical factors, e.g., an increase in temperature (<53 °C) or pH (<9.3). According to Pearson correlation, these changes (pH, Nt, Pt) resulted in a significant decrease (p < 0.001) of indicator bacteria (two or three orders) in zeolite pile or zeolite-lime pile. Die-off of Salmonella spp. in the zeolite-lime pile was indicated within 41 days; in other piles, this occurred on day 63. Our results can aid in further optimizing the composting of cattle manure in order to lower environmental pollution and the risk of human infection.

Effect of Wastewater Treatment on Bacterial Community, Antibiotic-Resistant Bacteria and Endoparasites.

Wastewater and wastewater treatment plants serve as urban reservoirs of pathogenic microorganisms. Wastewaters frequently contain bacteria, antibiotic-resistant bacteria, and developmental stages of parasites with significant zoonotic potential. Five wastewater treatment plants in the central part of Slovakia were investigated to determine the effect of treatment on bacterial community, antibiotic-resistant bacteria, and the occurrence of helminth eggs. Although all monitored chemical factors (chemical oxygen demand, biochemical oxygen demand, N-NH4, total nitrogen, and total phosphorus) in the effluent were in line with the legislative standards for discharge into public waterways, the results of minimal inhibitory concentrations show that reclaimed water harbors E. coli resistant to several commonly used antibiotics (ampicillin, piperacillin, and tazobactam, combine ampicillin and sulbactam, cefotaxime, tetracycline). The presence of endoparasite developmental stages in wastewater and sludge (Ascaris spp., Hymenolepis nana, eggs from the Ancylostomatidae family, Giardia duodenalis) indicates potential health risks for humans and workers at these sites. Treatment such as composting before applying sludge to land is necessary to reduce human pathogens.

New Insight on Antibiotic Resistance and Virulence of Escherichia coli from Municipal and Animal Wastewater.

Antibiotic resistance of the indicator microorganism Escherichia coli was investigated in isolates from samples collected during the course of one year from two wastewater treatment plants treating municipal and animal wastes in Slovakia, respectively. The genes of antibiotic resistance and virulence factors in selected resistant E. coli isolates were described. A high percentage of the isolates from municipal and animal wastewater were resistant to ampicillin, streptomycin, tetracycline, ceftiofur, ceftriaxone, and enrofloxacin. In the selected E. coli isolates, we detected the following phenotypes: ESBL (20.4% in animal wastewater; 7.7% in municipal wastewater), multidrug-resistant (17% of animal and 32% of municipal isolates), high resistance to quinolones (25% of animal and 48% of municipal samples), and CTX-M (7.9% of animal and 17.3% of municipal isolates). We confirmed an integro-mediated antibiotic resistance in 13 E. coli strains from municipal and animal wastewater samples, of which the Tn3 gene and virulence genes cvaC, iutA, iss, ibeA, kps, and papC were detected in six isolates. One of the strains of pathogenic E. coli from the animal wastewater contained genes ibeA with papC, iss, kpsII, Int1, Tn3, and Cit. In addition, one blaIMP gene was found in the municipal wastewater sample. This emphasises the importance of using the appropriate treatment methods to reduce the counts of antibiotic-resistant microorganisms in wastewater effluent.

Detection of Antimicrobial Resistance of Bacteria Staphylococcus chromogenes Isolated from Sheep's Milk and Cheese.

Antimicrobial and multidrug resistance is detected in nonaureus staphylococci, including Staphylococcus chromogenes, which commonly causes intramammary infections. Recent clinical studies point to the presence of methicillin-resistant S. chromogenes. Therefore, this study aims to determine the prevalence of this species in samples of sheep's milk and cheeses made from them. Isolates were identified by polymerase chain reaction and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF). A total of 208 staphylococcal isolates were identified. Of these, 18% were identified as S. chromogenes. The antimicrobial resistance of the identified isolates was determined using the agar dilution method against penicillin, ceftaroline, teicoplanin, gentamicin, erythromycin, tetracycline, and ofloxacin. The highest resistance was found to penicillin (95%), tetracycline (86%), and oxacillin (81%). The highest sensitivity was confirmed for gentamicin (55%). The study also confirmed the presence of methicillin resistant staphylococcal isolates (30%) based on the phenotypic manifestation of antimicrobial resistance and detection of the presence of the mecA gene. The study shows that the tested isolates (62%) were multidrug resistant. Resistance to two antibiotics was most often found (39%).

Antimicrobial Resistance of Staphylococcus sp. Isolated from Cheeses.

S. aureus and some species of coagulase-negative staphylococci, including S. chromogenes and S. simulans, commonly cause intramammary infections. However, little attention was paid to the antimicrobial resistance of these species with respect to their occurrence in dairy products, for example, popular sheep and goat cheeses made from unpasteurized milk. The aim of this study was to investigate such sheep and goat cheeses for the occurrence and antimicrobial resistance of the relevant staphylococci species. The staphylococcal isolates were identified by polymerase chain reaction (130 isolates) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry. The most common species of S. aureus (56 isolates) were identified, as well as S. chromogenes (16 isolates) and S. simulans (10 isolates). Antimicrobial resistance to penicillin, oxacilin, ceftaroline, teicoplanin, gentamicin, erythromycin, tetracycline and ofloxacin was subsequently determined in these species using the agar dilution method. The highest resistance was confirmed in all species, especially to penicillin (91%) and erythromycin (67%). The highest sensitivity was confirmed to ofloxacin (83%). Due to the high incidence of penicillin and oxacilin-resistant staphylococci, the mecA gene was detected by polymerase chain reaction, which was confirmed only in S. aureus isolates (19%). Our study shows that the tested strains (77%) were resistant to more than one antibiotic at a time.

Comparison of Campylobacter spp. and flock health indicators of broilers in Iceland.

INTRODUCTION: The safety of poultry meat products and contamination with microorganisms is based on appropriate reduction of the presence of pathogens during poultry rearing and is closely related to the level of rearing hygiene, including the type of housing, stocking density, microclimate, sanitation and ventilation. OBJECTIVE: The aim of the study is to evaluate the prevalence of Campylobacter-positive samples in Iceland during 2016-2018, and to compare the potential influence of individual parameters of welfare on the prevalence of Campylobacter spp. MATERIAL AND METHODS: Positivity of excrement and caecum samples for Campylobacter spp. was determined according to ISO 10272-1: 2006 and 2017. Data of welfare indicators were collected during the rearing period and in the slaughterhouse. RESULTS: Considerable seasonality was observed in the prevalence of Campylobacter spp. The prevalence of campylobacteriosis on the investigated broiler farms was significantly higher (p ˂0.05) during the summer. Comparison of welfare parameters on Campylobacter-positive and Campylobacter-negative farms failed to indicate a significantly higher level of observed welfare indicators in birds from Campylobacter-positive farms (p˃0.05). In comparing small, medium and big farms, a significantly higher occurrence was observed (p<0.05) of the FPD score over 40, stocking density, and the average slaughter weight, and percentage of mortality over 2% in small farms. CONCLUSIONS: Intensive management and the environment affect the welfare of poultry and its resistance to infections (Campylobacteriosis) and thus increase the health risk. Checking the welfare parameters in a slaughter house provides delayed improvement of the environment on farms, but it can also lead to changes in the following production cycles (decreasing of stocking density).

Antibiotic resistance and virulence of Escherichia coli strains isolated from animal rendering plant.

Processing of animal carcasses and other animal wastes in rendering plants is a significant source of antibiotic resistant microorganisms. The main goal of this study was to investigate the resistance to 18 antibacterial agents including ß-lactams, fluoroquinolones, colistin and virulence factors (iss, tsh, cvaC, iutA, papC, kps and ibeA genes) in 88 Escherichia coli strains isolated from a rendering plant over 1 year period. ESBL (Extended-spectrum beta-lactamases) and plasmid-mediated Amp were screened by interpretative reading of MIC. ESBL phenotype was detected in 20.4% of samples and high level of resistance to fluoroquinolone was found in 27.2% of strains. Cephalosporinase CTX-M1, cephamycinase CMY-2, integrase 1 and transposon 3 genes were detected by PCR. Furthermore, there were found three CMY-2 producing E. coli with O25b-ST131, resistant to the high level of enrofloxacin and containing the gene encoding the ferric aerobactin receptor (iutA). One enrofloxacin resistant E. coli strain possessed iss, ibeA, kps and papC virulence genes also with CMY-2, integrase1 and Tn3. ST131 E. coli with CMY-2 has a zoonotic potential and presents a serious health risk to humans.

Devitalisation of pathogens in stored pig slurry and potential risk related to its application to agricultural soil.

The study investigated the risks arising from application of pig slurry to soil in relation to viability of Salmonella typhimurium, Escherichia coli, total coliforms, faecal enterococci and eggs of Ascaris suum at different temperatures. Potential effect of changes in physico-chemical parameters, particularly dry matter (DM), pH and ammonia, were also investigated. Examination showed that S. typhimurium was devitalised after storage in the slurry for 115 days at 4 °C and after 90 days at 20 and 42 °C. Devitalization of E. coli and faecal entrerococci required more than 115 at temperature of 4 °C and faecal enterococci were recovered from slurry after 115 days of storage even at temperature of 20 °C. Total coliforms survived for 115 days at all investigated temperatures. Complete devitalization of A. suum eggs was not achieved even after 115 days at 42 °C. Our investigations indicated potential microbial and parasitic risk related to application of pig slurry to soil even after 115 days of storage.

Extended spectrum beta-lactamases in Escherichia coli from municipal wastewater.

INTRODUCTION AND OBJECTIVE: Over the past decades, awareness of the environmental load of resistant organisms has increased. The presented paper focuses on antibiotic resistance and detection of resistance genes in environmental E. coli and on the evaluation of biofilm formation in ESBLs (extended spectrum beta-lactamase) producing E. coli isolated from an urban wastewater treatment plant. MATERIALS AND METHOD: Wastewater samples and artificially added polystyrene pellets were used as the source for E. coli isolation. Minimal inhibitory concentrations of 19 antibiotics were determined according to CLSI (2013). Biofilm formation was investigated by crystal violet or resazurin methods. CTX-M, carbapenemases, qnrS, mobile elements and virulence factors were determined by PCR. Clonal relatedness of strains was detected by principal component analysis by a Maldi biotyper. RESULTS: ESBL phenotype was detected in 26% of environmental strains. CTX-M, CMY-2 and qnrS genes of antibiotic resistance were detected. IMP gene together with integron 1 in one ertapenem resistant E. coli was also recorded. There was no evident correlation between antibiotic resistance, virulence and biofilm production. CONCLUSIONS: The results showed that the wastewater is a source of ESBLs, carbapenemases and plasmid fluoroquinolone resistance. Strains with biofilm production, antibiotic resistance of CTX-M group, CMY-2, qnrS genes and virulence factors present a potential environmental health risk.

Antibiotic resistance of Escherichia coli isolated from a poultry slaughterhouse.

The aim of the study was to investigate the antibiotic resistant E. coli strains isolated from bioaerosols and surface swabs in a slaughterhouse as a possible source of poultry meat contamination. The highest air coliforms contamination was during shackling, killing and evisceration of poultry. The strains showed resistance to ampicillin (89%), ceftiofur (62%) and cefquinome (22%), while resistance to ampicillin with sulbactam was only 6%. Resistance to streptomycin and gentamicin was detected in 43% vs. 14% isolates; to tetracycline 33%; to chloramphenicol and florfenicol in 10% vs. 18% isolates; to cotrimoxazol in 35% isolates; to enrofloxacin in 43% isolates. The higher MIC of ceftazidime (3.6 mg x l(-1)) and ceftriaxon (5.2 mg x l(-1)) revealed the presence of ESBLs in 43% of isolates. From 19 selected phenotypically ESBL positive strains, 16 consisted of CMY-2 genes, while CTX-M genes were not detected by PCR. Maldi tof analysis of selected E. coli showed a clear clonal relatedness of environmental strains from various withdrawals.