Antimicrobial resistance and prevalence of extended-spectrum beta-lactamase-producing Klebsiella species in East Tennessee dairy farms.

Klebsiella species commonly reside in dairy cattle guts and are consistently exposed to beta-lactam antibiotics, including ceftiofur, which are frequently used on the U.S. dairy farms. This may impose selection pressure and result in the emergence of extended-spectrum beta-lactamase (ESBL)-producing strains. However, information on the status and antimicrobial resistance (AMR) profile of ESBL-Klebsiella spp. in the U.S. dairy farms is largely unknown. This study aimed to determine the prevalence and AMR profile of ESBL-Klebsiella spp. and the factors affecting their occurrence in dairy cattle farms. Rectal fecal samples (n = 508) and manure, feed, and water samples (n = 64) were collected from 14 dairy farms in Tennessee. Samples were directly plated on CHROMagar ESBL, and presumptive Klebsiella spp. were confirmed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Antimicrobial susceptibility testing was performed on the isolates against panels of 14 antimicrobial agents from 10 classes using minimum inhibitory concentration. Of 572 samples, 57 (10%) were positive for ESBL-Klebsiella spp. The fecal prevalence of ESBL-Klebsiella spp. was 7.2% (95% CI: 6.5-8.0). The herd-level fecal prevalence of ESBL-Klebsiella spp. was 35.7% (95% CI: 12.7-64.8). The fecal prevalence of ESBL-Klebsiella spp. was significantly higher in calves than in cows and higher in cows with higher parity (≥3) as compared to cows with low parity (P < 0.001). Most (96.5%, n = 57) ESBL-Klebsiella spp. were resistant to ceftriaxone. The highest level of acquired co-resistance to ceftriaxone in ESBL-Klebsiella spp. was to sulfisoxazole (66.7%; 38/57). About 19% of ESBL-Klebsiella spp. were multidrug resistant. The presence of ESBL-producing Klebsiella spp. in dairy cattle, feed, and water obtained from troughs could play a crucial epidemiological role in maintaining and spreading the bacteria on farms and serving as a point source of transmission. IMPORTANCE: We collected 572 samples from dairy farms, including rectal feces, manure, feed, and water. We isolated and identified extended-spectrum beta-lactamase (ESBL)-Klebsiella spp. and conducted an antimicrobial susceptibility test and analyzed different variables that may be associated with ESBL-Klebsiella spp. in dairy farms. The results of our study shed light on how ESBL-Klebsiella spp. are maintained through fecal-oral routes in dairy farms and possibly exit from the farm into the environment. We determine the prevalence of ESBL-Klebsiella spp. and their antimicrobial susceptibility profiles, underscoring their potential as a vehicle for multiple resistance gene dissemination within dairy farm settings. We also collected data on variables affecting their occurrence and spread in dairy farms. These findings have significant implications in determining sources of community-acquired ESBL-Enterobacteriaceae infections and designing appropriate control measures to prevent their spread from food animal production systems to humans, animals, and environments.

Interaction between microalgae and phycosphere bacteria in a binary cultivation system-based dairy farm wastewater treatment.

The combination of microalgal culture and wastewater treatment is an emerging topic. This study investigated the use of different microalgae to treat different types of dairy farm wastewater. The results showed that the removal of ammonia nitrogen and total phosphorus by mixed microalgae was over 99% and 80%, respectively. The highest production of protein in biomass and extracellular polymeric substances was observed in high-concentration wastewater. In the phycosphere, the abundance of Proteobacteria and Cyanobacteria increased, while that of Bacteroidota decreased. Phycosphere bacteria were strongly correlated with microalgal growth and the composition of extracellular polymeric substances, especially with bound extracellular polymeric substances relative to soluble extracellular polymeric substances. Genes associated with photosynthesis and respiration in phycosphere bacteria were upregulated, contributing to the material exchange capacity in the microalgal-bacterial systems. The interaction between microalgae and phycosphere bacteria thus represents the core of the binary cultivation system-based wastewater treatment and requires further investigation.

Metabolites and physical scores as possible predictors for postpartum culling in dairy cows.

The purpose of the study was to explore the associations of serum non-esterified fatty acids (NEFA) and ß-hydroxybutyrate (BHBA) concentrations with the body condition score (BCS) and rumen fill score (RFS) in order to predict the risk of postpartum culling, and to further investigate effective monitoring stages during the dry period. From October 2012 to March 2014, clinically healthy Holstein heifers and cows were sampled once before calving, and the occurrence of culling within 60 days in milk (DIM) was investigated. The discriminatory ability of each parameter was evaluated using receiver operating characteristic (ROC) analysis. Of the 720 cows sampled between 14 and 2 days before the actual day of calving in the study, 42 cull cows (mean DIM ± SE: 22.0 ± 2.6) were confirmed. The areas under the curve (AUC) of the ROC for predicting culling using serum NEFA concentrations were 0.6 and 0.7 at 14 to 2 and 7 to 2 days before calving, respectively. The AUC for the RFS was 0.7 for both periods, indicating the same diagnostic level as the serum NEFA concentration. Both the serum NEFA concentration and RFS were possible predictors in cows with ≥ 2 parities, but not in cows with 0-1 parity sampled even at 7 to 2 days before calving. The serum BHBA concentration and BCS were not suitable predictors of culling for any period or parity. These results indicate that RFS has a discriminatory ability comparable to the serum NEFA concentration for predicting culling within 60 DIM.

Bacterial Diversity and Antimicrobial Resistance of Microorganisms Isolated from Teat Cup Liners in Dairy Farms in Shandong Province, China.

Global milk consumption exceeds 800 million tons a year and is still growing. Milk quality and its products are critical to human health. A teat cup makes direct contact with the cow's teats during milking and its cleanliness is very important for the quality of raw milk. In this study, the microorganism from post-milking teat cup liners were collected from six dairy farms in Shandong Province of China, the bacterial species were identified using microbial mass spectrometry, the minimum inhibitory concentrations of the isolated strains against ten antimicrobial agents were determined using the broth microdilution method, and the antimicrobial resistance genes were detected by PCR. The results indicated that the most frequently isolated bacteria in this study were Bacillus licheniformis (39/276, 14.13%), followed by Bacillus pumilus (20/276, 7.25%), Bacillus cereus (17/276, 6.16%), and Bacillus subtili (16/276, 5.80%). The isolates exhibited the highest average resistance to lincomycin (87.37%), followed by sulfadiazine (61.05%) and streptomycin (42.63%); the highest detection rate of resistance genes was Sul1 (55.43%), followed by ant(4') (51.09%), tet(M) (25.36%), blaKPC (3.62%) and qnrS (3.62%). These findings imply the necessity for enhanced measures in disinfecting cow udders and milking equipment, highlighting the persistently challenging issue of antimicrobial resistance in Shandong Province.

Identification of Enterococcus spp. by MALDI-TOF mass spectrometry isolated from clinical mastitis and bulk tank milk samples.

BACKGROUND: Throughout a three-year study period, 1,577 bovine clinical mastitis samples and 302 bulk tank samples were analyzed from ten Brazilian dairy herds. Enterococcus spp. was isolated and identified in 93 (5.9%) clinical mastitis samples. In addition, 258 Enterococcus spp. were isolated from the bulk tank samples of the same herds. The identification of Enterococcus spp. isolated from bulk tanks and milk samples of clinical mastitis were accomplished by phenotypic characteristics and confirmed by MALDI-TOF Mass Spectrometry (MS). Fisher test was performed to verify the difference between bulk tanks and mastitis samples. RESULTS: The following species were identified from clinical mastitis: E. saccharolyticus (62.4%), E. faecalis (19.4%), E. faecium (15.1%), E. hirae (1.1%), E. mundtii (1.1%), E. durans (1.1%). Furthermore, from 258 bulk tank milk samples, eight enterococci species were isolated: E. faecalis (67.8%), E. hirae (15.1%), E. faecium (4.6%), E. saccharolyticus (4.6%), E. mundtii (3.1%), E. caseliflavus ( 2.7%), E. durans (1.2%), E. galinarum (0.8%). CONCLUSIONS: The difference in species predominance in bulk tank samples (67.8% of E. faecalis) and clinical mastitis (62.4% of E. saccharolyticus) was unexpected and caught our attention. Although Enterococcus spp. are traditionally classified as an environmental mastitis agent, in the present study, E. saccharolyticus behaved as a contagious agent of mastitis, which consequently changed the control patterns to be implemented.

Comparative Antimicrobial Resistance and Prevalence of Methicillin Resistance in Coagulase-Positive Staphylococci from Conventional and Organic Dairy Farms in South Korea.

Bovine mastitis (BM) has caused huge economic and financial losses in the dairy industry worldwide, with Staphylococcus aureus as one of its major pathogens. BM treatment still relies on antibiotics and its extensive use often generates methicillin-resistant S. aureus (MRSA) and mupirocin-resistant S. aureus (MuRSA). This study compared the antimicrobial resistance trend in coagulase-positive Stapholococci (CoPS) isolated from BM milk in conventional and organic dairy farms and checked prevalence of MRSA and MuRSA. A total of 163 presumptive Staphylococci were isolated, wherein 11 out of 74 from 4 conventional farms (CF1, CF2, CF3, CF4) and 17 out of 89 from 3 organic farms (OF1, OF2, OF3) exhibited coagulase activity. Multiplex-PCR amplification confirmed at least one coagulase-positive isolate from CF1, CF2, CF3, CF4, and OF1 as S. aureus, denoted by the presence of the nuc gene. Three isolates from CF2 contained the mecA gene, indicating MRSA prevalence, while the MuRSA gene marker, mupA, was not detected in any of the isolates. Antimicrobial testing showed that conventional farm isolates were more resistant to antibiotics, especially ampicillin and tetracycline. This suggests a risk of developing multidrug resistance in dairy farms if antibiotic use is not properly and strictly monitored and regulated.

Assessment of microbial communities in a dairy farm from a food safety perspective.

Microbial communities associated with dairy farm operations have a significant influence on food safety, dairy product quality, and animal health. This study aimed to create a microbial mapping at a dairy farm to learn about their bacterial diversity, distribution, and potential dissemination pathways. The investigation included the detection of key zoonotic pathogens, enumeration of Staphylococcus aureus and Escherichia coli as indicators of typical bacterial loads in a dairy production environment, and a microbiome analysis using metagenomics. A total of 160 samples (environmental, udder swabs, feed, feces, raw milk, and water) were collected during winter (N = 80) and spring (N = 80). In winter, Cronobacter spp. were detected in four feed and two water samples; L. monocytogenes was identified in two samples, one from feces and one from a cattle mat; E. coli O157:H7 was found in two feed samples. On the other hand, during spring, Cronobacter spp. were present in four feed samples and one hallway drain, with only one feed sample testing positive for E. coli O157:H7, while L. monocytogenes was absent during the spring season. Regarding microbial counts, there was no significant difference between the two seasons (p = 0.068) for S. aureus; however, a significant difference (p = 0.025) was observed for E. coli. Environmental microbiome analysis showed the presence of Proteobacteria (46.0 %) and Firmicutes (27.2 %) as the dominant phyla during both seasons. Moraxellaceae (11.8 %) and Pseudomonadaceae (10.62 %) were notable during winter, while Lactobacillaceae (13.0 %) and Enterobacteriaceae (12.6 %) were prominent during spring. These findings offer valuable insights into microbial distribution within a dairy farm and potential risks to animal and human health through environmental cross-contamination.

Attitudes and professional values of veterinarians and veterinary students toward positive welfare states for dairy cattle.

Research that involves agricultural animal welfare has typically aimed to improve welfare by decreasing disease, distress, and pain. Positive welfare does not necessarily occur with the absence of suffering but in combination with opportunities for behaviors or affective states desired by animals. Our objectives were to describe Canadian bovine veterinarians' and veterinary students' attitudes, professional normative values, and perceived ability to promote positive welfare for dairy cows, and to explore participants' provided rationale. With an online cross-sectional survey, Canadian veterinary practitioners (n = 78) and veterinary students (n = 148) were asked, on a 7-point Likert scale, about their attitudes, perceived professional normative values, and perceived ability of veterinarians to promote positive welfare for dairy cows. We used an applied thematic analysis approach within the qualitative description methodology to analyze participants' open-ended text responses. Quantitatively, participants had very favorable attitudes (mean ± SE; 6.3 ± 0.04) and perceived favorable values (5.7 ± 0.06) in the veterinary community toward positive welfare opportunities for dairy cows. Three themes were identified to explain the professional normative values: influences from within the veterinary profession, influences from outside the veterinary profession, and personal views of participants. Participants were confident that veterinarians could suggest positive welfare opportunities for dairy cows (6.1 ± 0.06) but were uncertain that the decision to suggest these opportunities to producers was within a veterinarian's control (4.3 ± 0.11) and were not confident that implementation of positive welfare opportunities was under a veterinarian's control (2.1 ± 0.07). Three themes were identified to explain the barriers to veterinarians promoting positive welfare opportunities for dairy cows: not practical to implement, resistance to change, and concern for the animal. Participants stated that many positive welfare opportunities were impractical or expensive to implement. We conclude that positive attitudes and positive professional values exist in the veterinary community toward positive welfare for dairy cows but that much uncertainty exists regarding a veterinarian's ability to influence change to current practices.

Knowledge, Attitudes, Practices, and Risk Perception of Antimicrobial Use and Antimicrobial Resistance Among Dairy Farm Owners/Workers in Addis Ababa, Ethiopia.

Introduction: Antimicrobial resistance (AMR) is one of the most significant global health threats to the public, animals, and the ecosystem. Inappropriate use of antibiotics in food animals is considered a major driver of AMR in humans. This study was conducted to assess the knowledge, attitude, practices, and risk perception (KAPP) of dairy farm owners/workers in Addis Ababa about antibiotic use and resistance. Methods: A face-to-face interview using a structured questionnaire was conducted with 281 respondents in four selected subcities of Addis Ababa. The responses provided by each participant were recoded into a binary scale based on the mean score of each domain. Pearson chi-square was used to check the association between the KAPP and sociodemographic characteristics of the respondents and logistic regression analysis was done to explore the factors associated with KAPP. Results: Overall, more than half of the surveyed dairy farm owners/workers had good knowledge (57.7%) and appropriate practice (53.0%), while less than half of the respondents showed desirable attitudes (47.7%) and positive risk perceptions (42.7%). The findings revealed a strong association between the respondents' KAPP and education and between knowledge and risk perception and farming experience. Conclusion: This study found that continuous education of dairy farm owners/workers regarding antimicrobial usage and antimicrobial resistance in dairy farms will increase their awareness and perception of risk as well as motivate them to adopt desirable attitudes and appropriate practices, and consequently limit inappropriate use of antimicrobials leading to mitigating emergence of AMR.

Isolation, Identification, and Characterization of Bacillus cereus Group Bacteria Isolated from the Dairy Farm Environment and Raw Milk in Tunisia.

Members of the Bacillus cereus group are well-known opportunistic foodborne pathogens. In this study, the prevalence, hemolytic activity, antimicrobial resistance profile, virulence factor genes, genetic diversity by enterobacterial repetitive intergenic consensus (ERIC)-polymerase chain reaction (PCR) genotyping, and adhesion potential were investigated in isolates from a Tunisian dairy farm environment and raw milk. A total of 200 samples, including bedding, feces, feed, liquid manure, and raw bovine milk, were examined. Based on PCR test targeting sspE gene, 59 isolates were detected. The prevalence of B. cereus group isolates in bedding, feces, liquid manure, feed, and raw milk was 48%, 37.8%, 20%, 17.1%, and 12.5%, respectively. Out of the tested strains, 81.4% showed ß-hemolytic on blood agar plates. An antimicrobial resistance test against 11 antibiotics showed that more than 50% of the isolates were resistant to ampicillin and novobiocin, while a high sensitivity to other antibiotics tested was observed in most isolates. The distribution of enterotoxigenic genes showed that 8.5% and 67.8% of isolates carried hblABCD and nheABC, respectively. In addition, the detection rate of cytotoxin K (cytk), enterotoxin T (bceT), and ces genes was 72.9%, 64.4%, and 5.1%, respectively. ERIC-PCR fingerprinting genotype analysis allowed discriminating 40 different profiles. The adhesion potential of B. cereus group on stainless steel showed that all isolates were able to adhere at various levels, from 1.5 ± 0.3 to 5.1 ± 0.1 log colony-forming unit (CFU)/cm2 for vegetative cells and from 2.6 ± 0.4 to 5.7 ± 0.3 log CFU/cm2 for spores. An important finding of the study is useful for updating the knowledge of the contamination status of B. cereus group in Tunisia, at the dairy farm level.

An observational study demonstrates human-adapted Staphylococcus aureus strains have a higher frequency of antibiotic resistance compared to cattle-adapted strains isolated from dairy farms making farmstead cheese.

BACKGROUND: Staphylococcus aureus is a multi-host zoonotic pathogen causing human and livestock diseases. Dairy farms that make artisan cheese have distinctive concerns for S. aureus control. Antimicrobial-resistant (AMR) S. aureus is a public and animal health concern. There is a need to study the population structure of AMR S. aureus at the human-animal interface and understand the path of zoonotic transmission. This cross-sectional observational study aimed to assess the genetic diversity and AMR patterns of S. aureus isolated from cattle and humans on conventional and organic Vermont dairy farms that produce and sell farmstead cheese. RESULTS: A convenience sample of 19 dairy farms in Vermont was enrolled, and 160 S. aureus isolates were collected from cow quarter milk (CQM), bulk tank milk (BTM), human-hand and -nasal swabs. After deduplication, 89 isolates were used for the analysis. Sequence types (STs) were determined by multilocus sequence typing and cataloged to the PubMLST database. Nine defined and five novel STs were identified. For BTM and CQM samples, six STs were identified within cow-adapted CC97 and CC151. Two human-adapted STs were isolated from BTM and CQM. Seven human-adapted clonal complexes with eight STs were identified from human samples. One cow-adapted ST was isolated from a human. Antimicrobial susceptibility of the isolates was tested using disc diffusion and broth microdilution methods. Approximately 27% of the isolates were beta-lactam resistant and blaZ gene-positive. S. aureus isolates from human swabs were more likely to carry blaZ compared to isolates from CQM or BTM. S. aureus isolated from cows and humans on the same farm belonged to different STs. CONCLUSION: Humans were more likely to carry beta-lactam-resistant S. aureus compared to cows, and on organic farms only human-adapted blaZ positive STs were isolated from BTM. Moreover, we identified potential spillover events of S. aureus sequence types between host species. The presence of penicillin-resistant-human-adapted S. aureus on both organic and conventional dairy farms highlights a "One Health" concern at the junction of public and animal health requiring further surveillance.

Fifty years of environmental progress for United States dairy farms.

Dairy farms in the United States have changed in many ways over the past 50 yr. Milk production efficiency has increased greatly, with ∼30% fewer cows producing about twice the amount of milk today. Other improvements include increases in crop yields, fuel efficiency of farm equipment, and efficiency in producing most resources used on farms (e.g. electricity, fuel, fertilizer). These improvements have led to changes in the environmental impact of farms. Through simulation of representative dairy farms in 1971 and 2020, changes in nutrient losses and farmgate life cycle assessments of greenhouse gas (GHG) emissions, fossil energy use, and blue (ground and surface) water use were determined for 6 regions and the United States. For all environmental metrics studied, intensities expressed per unit of fat- and protein-corrected milk produced were reduced, but the total effects over all farms or milk produced increased for 5 of the 13 environmental metrics. Reductions in the impacts of dairy farms in the eastern United States were offset by large increases in western regions because of a major increase in cow numbers in the West. The national average intensity of GHG emissions decreased by 42%, which gave just a 14% increase in the total GHG emissions of all dairy farms over the 50-yr period. The intensity of fossil energy use decreased by 54%, with the total for all farms decreasing by 9%. Water use related to milk production decreased in intensity by 28%, but due to the large increase in dairy production in the dry western regions that have a greater dependence on irrigated feed crops, total blue water use increased by 42%. Major pathways of nitrogen loss included ammonia volatilization, leaching, and denitrification, where total ammonia emissions related to US dairy farms increased by 29%, while leaching losses decreased by 39%, with little change in nitrous oxide emissions. Simulated nitrogen and phosphorus runoff losses totaled for all dairy farms decreased by 27% to 51% through more efficient fertilizer use, reduced tillage, and greater use of cover crops. Emissions of methane and reactive non-methane volatile organic compounds increased by 32% and 53%, respectively, due to greater use of long-term manure storage and silage stored in bunkers and piles. Although much progress has been made in improving production efficiency, continued improvements with new strategies and technologies are needed to meet the demand for dairy products and mitigate total environmental impacts, particularly in view of projected climate variability.

Tracking the extracellular and intracellular antibiotic resistance genes across whole year in wastewater of intensive dairy farm.

Monitoring the annual variation of antibiotic resistance genes (ARGs) in livestock wastewater is important for determining the high-risk period of transfer and spread of animal-derived antibiotic resistance into the environment. However, the knowledge regarding the variation patterns of ARGs, especially intracellular ARGs (iARGs) and extracellular ARGs (eARGs), over time in livestock wastewater is still unclear. Herein, we conducted a year-round study to trace the profiles of ARGs at a Chinese-intensive dairy farm, focusing on the shifts observed in different months. The results showed significant differences in the composition and variation between iARGs and eARGs. Tetracycline, sulfonamide, and macrolide resistance genes were the major types of iARGs, while cfr was the major type of eARG. The environmental adaptations of the host bacteria determine whether ARGs appear as intracellular or extracellular forms. The total abundance of ARGs was higher from April to September, which can be attributed to the favorable climatic conditions for bacterial colonization and increased antibiotic administration during this period. Integron was found to be highly correlated with most iARGs, potentially playing a role in the presence of these genes within cells and their similar transmission patterns in wastewater. The intracellular and extracellular bacterial communities were significantly different, primarily because of variations in bacterial adaptability to the high salt and anaerobic environment. The intracellular co-occurrence network indicated that some dominant genera in wastewater, such as Turicibacter, Clostridium IV, Cloacibacillus, Subdivision5_genera_incertae_sedis, Saccharibacteria_genera_incertae_sedis and Halomonas, were potential hosts for many ARGs. To the best of our knowledge, this study demonstrates, for the first time, the annual variation of ARGs at critical points in the reuse of dairy farm wastewater. It also offers valuable insights into the prevention and control of ARGs derived from animals.

Biological Control of Hyalomma Ticks in Cattle by Fungal Isolates.

Ticks pose a major threat to cattle health and production in Pakistan because they transmit pathogens of diseases like Babesiosis and Theileriosis. Hyalomma spp., found across Africa, Asia, and Europe, are especially problematic. This study explored biocontrol of Hyalomma spp. using spore-free fungal culture filtrates collected from dairy farm soil in Kohat, Pakistan. Three fungal species of the genera Alternaria, Aspergillus, and Penicillium were isolated, and their filtrates were tested against tick adults and larvae. Filtrate concentrations were prepared at different strengths. Data were taken after the exposure of adults and larvae ticks to various concentrations of the fungal filtrates. Results indicated that at 100% concentration, all fungal filtrates induced 100% mortality in adults and larvae. Decreasing filtrate concentration lowered tick mortality. The lowest concentration caused the least mortality. The effect was time- and dose-dependent. In conclusion, spore-free fungal culture filtrates can provide biocontrol of Hyalomma spp. in a time- and concentration-dependent manner. Further research should explore the active compounds causing mortality and optimal application methods. The process outlined here provides a natural biocontrol alternative to chemical pesticides to reduce tick infestations and associated cattle diseases in Pakistan.

Phylogenetic variation in raw cow milk microbiota and the impact of forage combinations and use of silage inoculants.

Introduction: The microbiota of bulk tank raw milk is known to be closely related to that of microbial niches of the on-farm environment. Preserved forage types are partof this ecosystem and previous studies have shown variations in their microbial ecology. However, little is known of the microbiota of forage ration combinations and the transfer rates of associated species to milk. Methods: We identified raw milk bacteria that may originate from forage rations encompassing either hay (H) or grass/legume silage uninoculated (GL) as the only forage type, or a combination of GL and corn silage uninoculated (GLC), or grass/legume and corn silage both inoculated (GLICI). Forage and milk samples collected in the fall and spring from 24 dairy farms were analyzed using 16S rRNA gene high-throughput sequencing following a treatment with propidium monoazide to account for viable cells. Results and discussion: Three community types separating H, GL, and GLICI forage were identified. While the H community was co-dominated by Enterobacteriaceae, Microbacteriaceae, Beijerinckiaceae, and Sphingomonadaceae, the GL and GLICI communities showed high proportions of Leuconostocaceae and Acetobacteraceae, respectively. Most of the GLC and GLICI rations were similar, suggesting that in the mixed forage rations involving grass/legume and corn silage, the addition of inoculant in one or both types of feed does not considerably change the microbiota. Raw milk samples were not grouped in the same way, as the GLC milk was phylogenetically different from that of GLICI across sampling periods. Raw milk communities, including the GLICI group for which cows were fed inoculated forage, were differentiated by Enterobacteriaceae and other Proteobacteria, instead of by lactic acid bacteria. Of the 113 amplicon sequence variants (ASVs) shared between forage rations and corresponding raw milk, bacterial transfer rates were estimated at 18 to 31%. Silage-based forage rations, particularly those including corn, share more ASVs with raw milk produced on corresponding farms compared to that observed in the milk from cows fed hay. These results show the relevance of cow forage rations as sources of bacteria that contaminate milk and serve to advance our knowledge of on-farm raw milk contamination.

Antimicrobial Resistance Profiles of Staphylococcus Isolated from Cows with Subclinical Mastitis: Do Strains from the Environment and from Humans Contribute to the Dissemination of Resistance among Bacteria on Dairy Farms in Colombia?

Staphylococcus is a very prevalent etiologic agent of bovine mastitis, and antibiotic resistance contributes to the successful colonization and dissemination of these bacteria in different environments and hosts on dairy farms. This study aimed to identify the antimicrobial resistance (AMR) genotypes and phenotypes of Staphylococcus spp. isolates from different sources on dairy farms and their relationship with the use of antibiotics. An antimicrobial susceptibility test was performed on 349 Staphylococcus strains (S. aureus, n = 152; non-aureus staphylococci (NAS), n = 197) isolated from quarter milk samples (QMSs) from cows with subclinical mastitis (176), the teats of cows (116), the milking parlor environment (32), and the nasal cavities of milk workers (25). Resistance and multidrug resistance percentages found for S. aureus and NAS were (S. aureus = 63.2%, NAS = 55.8%) and (S. aureus = 4.6%, NAS = 11.7%), respectively. S. aureus and NAS isolates showed resistance mainly to penicillin (10 IU) (54.1% and 32.4%) and ampicillin (10 mg) (50.3% and 27.0%) drugs. The prevalence of AMR Staphylococcus was higher in environmental samples (81.3%) compared to other sources (52.6-76.0%). In S. aureus isolates, the identification of the blaZ (83.9%), aacAaphD (48.6%), ermC (23.5%), tetK (12.9%), and mecA (12.1%) genes did not entirely agree with the AMR phenotype. We conclude that the use of ß-lactam antibiotics influences the expression of AMR in Staphylococcus circulating on dairy farms and that S. aureus isolates from the environment and humans may be reservoirs of AMR for other bacteria on dairy farms.

Clonal spread of non-O157 Shiga toxigenic Escherichia coli O21:H25 in raw water buffalo milks.

AIMS: This study was conducted to investigate the presence of Shiga toxin-producing O157 and non-O157 E. coli in raw water buffalo milk, as well as to determine the virulence gene profiles, phylogroups, sequence types, and serotypes of the isolated strains. METHODS AND RESULTS: A total of 200 hand-milked raw water buffalo milk samples were collected from 200 different water buffaloes over a period of three months from 20 different farms. Isolation of STEC was performed using CHROMagar STEC. Presence of stx1, stx2, and eaeA genes were investigated by mPCR. Phylogroups and sequence types of E. coli strains were determined by Clermont phylotyping and MLST. Serotyping was performed using PCR or WGS. According to the results, two milk samples obtained from two different farms were found as STEC-positive. All Stx-positive E. coli isolates belonged to phylogenetic group A and were assigned to ST10. WGS results indicated that serotype of two isolates was O21:H25 and average nucleotide identity was detected at 99.99%. Thirteen additional registered E. coli O21:H25 assembled WGS data were obtained from EnteroBase and a phylogenetic tree was constructed. CONCLUSIONS: With this study, the presence of stx2 harboring E. coli O21:H25 in milk was identified for the first time. Although the identified serotype is considered a non-pathogen seropathotype, we conclude it could play an important role in the environmental circulation of Stx-phages and consequently contribute to the emergence of new STEC-related outbreaks.

Death-and-culling rates of calves and associated economic losses in the first month of life on dairy farms in eastern Hokkaido, Japan.

The objective of this study was to analyze death and culling (DC) of calves during the first month of life and associated economic losses on dairy farms in eastern Hokkaido, Japan. The DC in the first month of life of 4411 Holstein and Wagyu crossbred calves born in the year 2019-2020 on 39 dairy farms milking Holsteins was investigated. Based on a target DC rate of 6.75%, farms were classified into two groups, those with high DC rates (HDC, 11.68%, n = 18) and those with low DC rates (LDC, 2.67%, n = 21), and analyzed for DC factors (breed, sex, parity of dams, and housing type of dams) and diseases causing DC, their loss estimates, and replenishment of DC calves (birth rate, purchase of heifers, and housing type of dams). Comparisons between groups were made using the Kaplan-Meier method, the Mann-Whitney U test, and chi-square test. The DC rate of Holsteins was significantly higher (P < 0.001) for HDC farms than for LDC ones. But, the DC rate of Wagyu crossbreds was not different between the groups, which suggested hybrid vigor. The DC rates for digestive diseases were significantly higher (P < 0.01) in the HDC farms, except for congenital diseases and deaths of unknown cause. The overall loss estimates of DC per calf-month for all farms was 8892, JPY/calf-months, 14,726 for HDC farms, and 4065 for LDC farms. The loss estimates of items with significant differences in DC rates were significantly higher (P < 0.05) in the HDC farms, with higher loss estimates for Holsteins and digestive diseases being the most common characteristics of HDC farms. In binomial logistic regression analysis with "HDC farms or not" as the response variable and replenishment of DC calves as the explanatory variable, HDC was significantly more likely (OR: 1.10, P < 0.05) on farms with a higher birth proportion of Holsteins, and HDC farms supplemented the DC calves by increasing the birth proportion of Holsteins.

Molecular identification of blaTEM and blaCTX-M genes in multidrug-resistant Escherichia coli found in milk samples from dairy cattle farms in Tulungagung, Indonesia.

Introduction: Escherichia coli is an opportunistic bacteria that can grow easily, produce toxins, and resist antibiotics. The phenomenon of E. coli developing multidrug resistance is currently the subject of extensive research. The objective of this study was to molecularly identify blaTEM and blaCTX-M genes in multidrug-resistant E. coli found in milk samples from dairy cattle farms in Tulungagung, Indonesia. Material and Methods: One hundred and ten milk samples were collected from 45 dairy cattle farms in Tulungagung, Indonesia. Indole, methyl red, Voges-Proskauer and in citrate tests and triple iron sugar agar tests were used to identify E. coli. Multidrug resistance was determined in isolates through antibiotic sensitivity tests using tetracycline, streptomycin, trimethoprim, chloramphenicol and aztreonam. Extended-spectrum beta lactamase enzyme production was confirmed by double-disc synergy test (DDST). Molecular identification was performed to confirm the blaTEM and blaCTX-M genes. Results: One hundred and one (91.82%) E. coli strains were isolated from the samples. The antibiotic sensitivity test showed four (3.96%) multidrug-resistant (MDR) and one (0.99%) ESBL-positive E. coli by DDST confirmation. There were three (77.78%) blaTEM genes and one (0.99%) blaCTX-M gene discovered in the MDR E. coli isolates using PCR for molecular identification. Conclusion: The findings of the blaTEM and blaCTX-M genes encoding ESBL E. coli in dairy cattle milk in Tulungagung, Indonesia is concerning and argues for prompt action to stop the emergence of antibiotic resistance which has an impact on public health.

Corynebacterium suedekumii sp. nov and Corynebacterium breve sp. nov., isolated from raw cow's milk.

Four Gram-positive, rod-shaped, none-sporeforming, non-motile isolates were obtained from various raw milk samples taken from the cooling tank on a research farm in Königswinter, Germany. Based on phylogenetic analysis of the 16S rRNA genes and whole genome sequences, all isolates were assigned to the genus Corynebacterium, but were divided in two different groups. All isolates contained C18 : 1 cis 9 and C16 : 0 as predominant fatty acids, as well as traces of C18 : 0. They all contained menaquinones MK-8 (H2) and MK-9 (H2) and produced mycolic acids characteristic for the majority of species belonging to the genus Corynebacterium. 16S rRNA gene sequence similarity values to the closest related type strains Corynebacterium humireducens DSM 45392T and Corynebacterium pilosum DSM 20521T were below 98.7 %, average nucleotide identity values were below 86 % and digital DNA-DNA-hybridization values were below 25 %, indicating that the isolates represent two novel species. The names Corynebacterium suedekumii sp. nov. and Corynebacterium breve sp. nov. are proposed, represented by the type strains LM112T (=DSM 116216T=HAMBI 3782T) and R4T (=DSM 116183T=HAMBI 3785T), respectively.