Changes in mammary infection status in dairy cows during the dry period using dry cow therapy approaches on three farms.

This case study evaluated the mammary infection status of dairy cows during the dry periods and explored the associated problems in their quarters with dry cow therapy (DCT). This study assessed intramammary infections, antibiotic efficacy, and antimicrobial resistance of pathogens in 464-quarter milk samples from 59 dairy cows during the dry periods after applying blanket DCT, non-DCT, and selective DCT approaches on three farms. The recovery rates of intramammary infections were 95% (19/20 quarters) with blanket DCT on farm A, 70% (14/20) with non-DCT on farm B, and 19% (4/21) with selective DCT on farm C. Analysis of mammary infections in cows with DCT revealed that mammary infections were controlled by blanket DCT, well controlled by non-DCT, and substantial problems remained in selective DCT. Lower intramammary infection prevalence in the quarters at postpartum appeared to be associated with higher recovery of mammary infections, fewer new infections, and lower uncured mammary infections within the herds. Antibacterial resistance in 14 coagulase-negative staphylococci isolated to six antimicrobial drugs was suggested to be linked to antibiotic use on the farm. Follow-up studies on the quarter-based infection status with DCT will assist in improving mastitis control in cows during the dry period.

Occurrence, genetic diversity, and antimicrobial resistance of methicillin-resistant Staphylococcus spp. in hospitalized and non-hospitalized cats in Brazil.

Methicillin-resistant Staphylococci (MRS) cause infections at various sites and exhibit multidrug resistance. Despite their importance in veterinary medicine, only little is known about Staphylococcus spp. colonizing and infecting cats. Therefore, in this study, we aimed to isolate and identify Staphylococcus spp. colonizing hospitalized and non-hospitalized domestic cats and analyze their antimicrobial resistance profiles, genetic diversity, and risk factors associated with MRS colonization. A total of 218 oral and axillary swabs were obtained from 109 cats, including 77 non-hospitalized and 32 hospitalized cats. After plating on selective media, the isolates were identified via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and rpoB and 16S rRNA gene sequencing. Subsequently, antimicrobial sensitivity of the strains was assessed, and they were screened for mecA gene. Methicillin-resistant S. haemolyticus (MRSH) isolates were subjected to multilocus sequence typing, whereas methicillin-resistant S. pseudintermedius (MRSP) and S. felis isolates were subjected to whole genome sequencing. S. felis was most commonly isolated from non-hospitalized cats (28.1%), whereas S. pseudintermedius and MRS were commonly isolated from hospitalized cats (25%). MRSH isolates from hospitalized animals were classified as ST3. The identified MRSP strains belonged to two well-known sequence types, ST551 and ST71. Moreover, antimicrobial use (p = 0.0001), hospitalization (p = 0.0141), and comorbidities (p = 0.002) were associated with increased MRS prevalence in cats.

Staphylococcus pseudintermedius ST2660 isolated from a cat has strong biofilm-forming ability and increases biofilm formation at cat's normal body temperature.

Staphylococcus pseudintermedius has been isolated from dogs, cats, and horses and is also known as an emergent zoonotic agent. We administered orbifloxacin, a fluoroquinolone, to treat bacterial infections of cutaneous wounds caused by excessive grooming of the skin in contact with the subcutaneous port of the subcutaneous ureteral bypass (SUB) system in a cat. However, after 80 days of treatment, a severe abscess was observed in the wound and fluoroquinolone-resistant S. pseudintermedius was isolated from the abscess. The isolate was identified as a novel sequence type (ST) 2660 and contained genes for leukocidins (lukS and lukF), exfoliative toxin (siet), and biofilm regulation (icaA and icaD). The isolate was resistant to macrolide, lincosamide, fluoroquinolone, and tetracycline classes. In addition, the isolate had strong biofilm-forming ability which significantly increased with culturing at 39 °C compared with that at 37 °C, suggesting that the isolate prefers a cats' body temperature as the optimal biofilm growth condition. Notably, the biofilms were increased in the presence of doxycycline with culturing at 39 °C. This study is the first report in Japan on the new sequence type of S. pseudintermedius isolated from a companion animal and clarifies the distinctive virulence of S. pseudintermedius.

Prevalence and molecular characterization of multidrug-resistant coagulase negative staphylococci from urban wastewater in Delhi-NCR, India.

Antimicrobial resistance (AMR) is global health concern escalating rapidly in both clinical settings and environment. The effluent from pharmaceuticals and hospitals may contain diverse antibiotics, exerting selective pressure to develop AMR. To study the aquatic prevalence of drug-resistant staphylococci, sampling was done from river Yamuna (3 sites) and wastewater (7 sites) near pharmaceutical industries in Delhi-NCR, India. 59.25% (224/378) were considered presumptive staphylococci while, methicillin resistance was noted in 25% (56/224) isolates. Further, 23 methicillin-resistant coagulase negative staphylococci (MR-CoNS) of 8 different species were identified via 16S rRNA gene sequencing. Multidrug resistance (MDR) was noted in 60.87% (14/23) isolates. PCR based detection of antibiotic resistance genes revealed the number of isolates containing mecA (7/23), blaZ (6/23), msrA (10/23), aac(6')aph (2") (2/23), aph(3')-IIIa (2/23), ant(4')-Ia (1/23), dfrG (4/23), dfrA(drfS1) (3/23), tetK (1/23) and tetM (1/23). The current research highlights the concerning prevalence of MDR-CoNS in aquatic environment in Delhi.

Unravelling the complex mechanisms of multidrug resistance in bovine mastitis pathogens: Insights into antimicrobial resistance genes, biofilm dynamics, and efflux systems.

Mastitis remains a paramount economic threat to dairy livestock, with antibiotic resistance severely compromising treatment efficacy. This study provides an in-depth investigation into the multidrug resistance (MDR) mechanisms in bacterial isolates from bovine mastitis, emphasizing the roles of antimicrobial resistance genes (ARGs), biofilm formation, and active efflux systems. A total of 162 Staphylococci, eight Escherichia coli, and seven Klebsiella spp. isolates were obtained from 215 milk samples of clinical and subclinical mastitis cases. Antibiotic susceptibility testing identified Twenty Staphylococci (12.35 %), six E. coli (75 %) and seven Klebsiella (100 %) identified as MDR displaying significant resistance to ß-lactams and tetracyclines The Multiple Antibiotic Resistance (MAR) index of these isolates ranged from 0.375 to 1.0, highlighting extensive resistance. Notably, 29 of the 33 MDR isolates produced biofilms on Congo red agar, while all exhibited biofilm formation in the Microtitre Plate assay. Critical ARGs (blaZ, blaTEM, blaCTX-M, tetM, tetA, tetB, tetC, strA/B, aadA) and efflux pump genes (acrB, acrE, acrF, emrB, norB) regulating active efflux were identified. This pioneering study elucidates the synergistic contribution of ARGs, biofilm production, and efflux pump activity to MDR in bovine mastitis pathogens. To our knowledge, this comprehensive study is the first of its kind, offering novel insights into the complex resistance mechanisms. The findings underscore the imperative need for advanced antibiotic stewardship and strategic interventions in dairy farming to curb the rise of antibiotic-resistant infections, thereby protecting both animal and public health.

Synergistic effects of oxidative and acid stress on bacterial membranes of Escherichia coli and Staphylococcus simulans.

Oxidative stress in combination with acid stress has been shown to inactivate a wide spectrum of microorganisms, including multi-resistant bacteria. This occurs e.g. in phagolysosomes or during treatment by cold atmospheric pressure plasmas (CAP) and possibly depends on the cell membrane. We therefore explored the effects of CAP-generated reactive oxygen and nitrogen species (RONS) on bacterial growth inhibition and membranes in neutral and acidic suspensions. We observed that growth inhibition was most efficient when bacteria were treated by a mix of short and long-lived RONS in an acidic environment. Membrane packing was affected mainly upon contact with short-lived RONS, while also acidity strongly modulated packing. Under these conditions, Gram-negative bacteria displayed large potassium release while SYTOX Green influx remained marginal. Growth inhibition of Gram-negative bacteria correlated well with outer membrane (OM) permeabilization that occurred upon contact with short and/or long-lived RONS in synergy with acidity. In Gram-positive bacteria, CAP impaired membrane potential possibly through pore formation upon contact with short-lived RONS while formation of membrane protein hydroperoxides was probably involved in these effects. In summary, our study provides a wide perspective on understanding inactivation mechanisms of bacteria by RONS in combination with acidity.

Green synthesized silver nanoparticles from Ocimum sanctum: A potent inhibitor of biofilm forming ability and efflux pumps in bacteria causing bovine mastitis.

Therapeutic management of mastitis faces significant challenges due to multidrug resistance. In the present study, multi-drug-resistant (MDR) Staphylococcus spp, Klebsiella pneumoniae, and Escherichia coli were isolated from bovine clinical mastitis cases and the phenotypic and genotypic multidrug resistance profiling was carried out. Silver nanoparticles (AgNPs) were biosynthesized using Ocimum sanctum leaf extracts and characterized via UV Vis absorption, Fourier Transform Infrared Spectroscopy, X-ray diffraction studies, Energy dispersive spectroscopy and Electron Microscopy. The determined minimum inhibitory concentration and minimum bactericidal concentration of the AgNPs against the recovered MDR isolates were 62.5 µg/ml and 125 µg/ml respectively. At a concentration of 50 µg/ml, the AgNPs demonstrated biofilm inhibitory activities of 80.35 % for MDR E. coli, 71.29 % for S. aureus and 60.18 % for MDR K. pneumoniae. Post-treatment observations revealed notable differences in biofilm formation across bacterial isolates. Furthermore, AgNP treatment led to significant downregulation of expression of the efflux pump genes acrB, acrE, acrF, and emrB in Gram-negative isolates and norB in Staphylococci isolates. This research underscores the potential for the development of an eco-friendly antimicrobial alternative in the form of green synthesized silver nanoparticles to combat drug resistance offering potential antibiofilm and efflux pump inhibitory activities.

Molecular identification, virulence, and antibiotic-resistant genes characteristics of Staphylococcus spp., isolated from milk samples.

Background: Food safety and food-borne infections are major subjects of global interest. Dairy products are considered as important source for these infections. Aim: The present study was conducted to identify the occurrence and to genotype isolates of Staphylococcus spp. recovered from milk samples in Al-Diwaniyah City, Iraq. Methods: The current study included the collection of 50 milk samples purchased from local stores in the current city. These samples were subjected to bacterial cultivation and biochemical tests. Later, the growth was used to extract the genomic DNA that was exposed to PCR and partial gene sequencing both targeted the 16S rRNA gene at a specific genetic piece. Results: The PCR results demonstrated the amplification of the genetic fragment of five genetic clusters for each of Staphylococcus aureus (SAD11, SAD12, SAD13, SAD14, and SAD15), Staphylococcus epidermidis (SED1, SED2, SED3, SED4, and SED5), and Staphylococcus intermedius (SID1, SID2, SID3, SID4, and SID5). The PCR products were sent out to sequencing and reported that the current isolates were similar in their genetic content with global isolates at 95.34% to 97.59%, 96.21% to 97.57%, and 96.09% to 97.88%, respectively, of identity. Conclusion: The present findings show high genetic variations among isolates of S. aureus, S. epidermidis, and S. intermedius recovered from milk samples, and these genotypes are found in different infection settings related to humans and animals, which may pose high risks to humans and animals.

Emergence of Staphylococcus argenteus in pediatrics: Molecular insights from a hospital in East China.

Staphylococcus argenteus is a novel species within the Staphylococcus aureus complex and can cause serious bloodstream infections (BSIs) in humans, which have been mainly reported in adults, especially the elderly. In this study, we analyzed the molecular characterization of a strain of S. argenteus (22WJ8192) isolated from the peripheral vein blood sample of a seven-month-old female infant in Eastern China. The 22WJ8192 belonged to sequence type (ST)2250 and harbored six antibiotic-resistance genes and 53 virulence genes and was resistant to penicillin. Additionally, we conducted a comparative analysis of the molecular characteristics of S. argenteus sourced from various origins within the dataset, predominantly from the National Center for Biotechnology Information Collection (NCBI) genome database. Antibiotic-resistance genes blaR1, blaI_of_Z, blaZ, fosB-Saur, tet(L), aph(3")-IIIa, mecA, and dfrG were more prevalent among the strains of human origin. Virulence genes lukF-PV, sak, sdrE, scn, sdrC, and sdrD were more prevalent among strains of human origin. The presence of antibiotic-resistance genes blaR1, blaI_of_Z, blaZ, fosB-Saur, and aph(3")-IIIa in strain 22WJ8192 was also more common among strains of human origin in the dataset. Conversely, the antibiotic-resistance genes tet(L), mecA, and dfrG, typically found in strains of human origin, were not detected in 22WJ8192. Additionally, virulence genes lukF-PV, sak, sdrE, scn, sdrC, and sdrD present in 22WJ8192 exhibited a higher prevalence among strains of human origin in the dataset. In conclusion, this study emphasizes the potential of S. argenteus ST2250 to induce severe bloodstream infections in infants, shedding light on the molecular characteristics of this strain.

Epidemiology and antibiotic resistance of staphylococci on commercial pig farms in Cape Town, South Africa.

Staphylococci are responsible for a wide range of infections in animals. The most common species infecting animals include Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus intermedius. Recent increases in antibiotic use and antibiotic resistance in animals highlight the need to understand the potential role of commercial livestock as a reservoir of staphylococci and antibiotic resistance genes. Nasal swabs were collected from 143 apparently healthy pigs and 21 pig farm workers, and 45 environmental swabs of feed and water troughs, from two commercial pig farms in the Western Cape, South Africa. Staphylococci were isolated, identified using mass-spectrometry, and antimicrobial susceptibility testing and Illumina whole genome sequencing were performed. One hundred and eighty-five (185) Staphylococcus spp. isolates were obtained, with Mammalicoccus sciuri (n = 57; 31%) being the most common, followed by S. hyicus (n = 40; 22%) and S. aureus (n = 29; 16%). S. epidermidis was predominantly identified in the farm workers (n = 18; 86%). Tetracycline resistance was observed across all species, with rates ranging from 67 to 100%. Majority of M. sciuri isolates (n = 40; 70%) were methicillin resistant, with 78% (n = 31) harbouring mecA. M. sciuri isolates had genes/elements which were associated with SCCmec_type_III (3A) and SCCmec_type_VIII(4A) and were mostly observed in ST61 strains. ST239 strains were associated with SCCmec_type_III(3A). High rates of tetracycline resistance were identified among staphylococci in the pig farms in Western Cape, South Africa. This highlights the need for policy makers to regulate the use of this antibiotic in pig farming.

Prevalence and antimicrobial resistance trends among vaginal bacteria isolates from pregnant bitches.

Many of the reproductive tract infections in the bitches are caused by bacteria that can normally be present on the vaginal mucosa. These bacteria also might have an important role as the cause responsible for pregnancy loss and fetal deaths. The choice of antibiotic therapy for the pregnant animal is narrow and represents a severe problem in veterinary practice, especially due to increased antimicrobial resistance. Due to incorrect antimicrobials use in breeding kennels, the aim of the present study was to assess the occurrence of the bacterial flora isolated from the pregnant bitches and their antibiotic sensitivity. The study was carried out at the private Veterinary clinic in Novi Sad, Serbia. The vaginal swabs were taken from 60 bitches diagnosed with pregnancy and were sent to be laboratory tested. Based on the results, the most common isolated pathogens were Staphylococcus pseudintermedius (20%) and beta-hemolytic streptococci (18.33%). Furthermore, significant resistance to antibiotics from beta-lactams group was detected. It is of particular importance that antimicrobial treatment be evidence based in order to reduce the overuse of antimicrobials due to increased concern regarding antimicrobial resistance.

Antibacterial and antibiofilm effect of essential oils on staphylococci isolated from cheese - application of the oil mixture in a cheese model.

The aim of the research was to examine the antimicrobial and antibiofilm effects of angelica, immortelle, laurel, hyssop, and sage plant dust essential oils (EOs) against isolated strains of Staphylococcus spp. from cheeses, in vitro and in the model of white cheese. MALDI-TOF MS analysis confirmed two Staphylococcus aureus strains and two coagulase-negative, identified as S. saprophyticus and S. warneri. All isolates produce biofilm, where the strains of S. aureus showed slightly better adherence. The main component of angelica EO was ß-phellandrene (48.19 %), while α-pinene (20.33 %) were dominant in immortelle EO, in hyssop EO cis-pinocamphone (37.25 %), in laurel EO 1,8-cineole (43.15 %) and in sage EO epirosmanol (26.25 %). The sage EO exhibited the strongest antistaphylococcal activity against all isolates. Synergism was also detected in combination of sage with hyssop or laurel EO. Better antibiofilm activity was confirmed for sage EO compared to hyssop EO. The mixture of sage/laurel EOs reduced the total number of staphylococci in the cheese after 4 days. Results indicate that in vitro applied EOs showed significant antistaphylococcal and antibiofilm activity, while the oil mixture reduced the initial total number of staphylococci.

The relationship between resistance evolution and carbon metabolism in Staphylococcus xylosus under ceftiofur sodium stress.

Staphylococcus xylosus has emerged as a bovine mastitis pathogen with increasing drug resistance, resulting in substantial economic impacts. This study utilized iTRAQ analysis to investigate the mechanisms driving resistance evolution in S. xylosus under ceftiofur sodium stress. Findings revealed notable variations in the expression of 143 proteins, particularly glycolysis-related proteins (TpiA, Eno, GlpD, Ldh) and peptidoglycan (PG) hydrolase Atl. Following the induction of ceftiofur sodium resistance in S. xylosus, the emergence of resistant strains displaying characteristics of small colony variants (SCVs) was observed. The transcript levels of TpiA, Eno, GlpD and Ldh were up-regulated, TCA cycle proteins (ICDH, MDH) and Atl were down-regulated, lactate content was increased, and NADH concentration was decreased in SCV compared to the wild strain. That indicates a potential role of carbon metabolism, specifically PG hydrolysis, glycolysis, and the TCA cycle, in the development of resistance to ceftiofur sodium in S. xylosus.

Staphylococcus spp. as part of the microbiota and as opportunistic pathogen in free-ranging black-tuffed marmosets (Callithrix penicillata) from urban areas: Epidemiology, antimicrobial resistance, and pathology.

BACKGROUND: Marmosets (Callithrix sp.), including black-tuffed marmosets (C. penicillata), are neotropical primates that can be highly adapted to urban environments, especially parks and forested areas near cities. Staphylococcus spp. are part of the microbiota of many different hosts and lead to opportunistic severe infection. Isolates from wild animals can be resistant to antimicrobial drugs. However, there are a few studies that evaluated Staphylococcus spp. in neotropical primates. The goal of this study was to evaluate Staphylococcus spp. isolated from free-ranging black-tuffed marmosets. METHODS: Marmosets were captured in six urban parks. After sedation, skin and rectal swabs and feces were sampled. Staphylococcus spp. isolates were identified by MALDI-ToF and their antimicrobial susceptibility was determined. RESULTS: Over 30% of captured individuals were positive for Staphylococcus spp., and S. aureus was the most isolated species followed by Mammaliicoccus (Staphylococcus) sciuri. With the exception of the marmoset subjected to necropsy, none of the other had lesions, which supports that notion that Staphylococcus spp. are members of the microbiota, but also opportunistic pathogens. Most isolates were susceptible to all antimicrobials tested; however, one isolate of S. epidermidis was resistant to multiple antimicrobials (penicillin, cefoxitin, ciprofloxacin, clindamycin, and erythromycin). We considered S. aureus as the main staphylococci to colonize black-tuffed marmosets. CONCLUSIONS: Black-tuffed marmosets can be colonized by several Staphylococcus species, most frequently by S. aureus, and the majority of isolates were sensible to the antimicrobials tested. One S. epidermidis isolate was considered multidrug resistant.

Integrating multi-wet laboratory diagnostics to study staphylococci in animals in Uganda.

BACKGROUND: Several diagnostic environments in Uganda lack real-time, robust and high-throughput technologies for comprehensive typing of microbes, which is a setback to infectious disease surveillance. This study combined various wet laboratory diagnostics to understand the epidemiology of pathogenic staphylococci isolated from animals in Uganda and the implications for global health security priorities. METHODS: A retrospective study was conducted employing records and pathogenic staphylococci (from animals) archived at the Central Diagnostic Laboratory (CDL), Makerere University, Uganda, between January 2012 and December 2019. The bacteria were speciated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and tested for virulence factors [beta lactamases, lecithinase, deoxyribonuclease (DNase), haemolysins] and resistance to ten antimicrobials of clinical and veterinary relevance. Tetracycline and methicillin resistance genes were also tested. RESULTS: The prevalent diseases were mastitis in cattle and skin infections in dogs. Of the 111 staphylococci tested by MALDI-TOF MS, 79 (71.2%) were Staphylococcus aureus, 27 (24.3%) were Staphylococcus pseudintermedius and 5 (4.5%) were Staphylococcus schleiferi. All these strains expressed haemolysins. The prevalence of strains with lecithinase, penicillinase, cephalosporinase and DNase was 35.9% (14/39), 89.7% (35/39), 0.0% (0/39) and 87.2% (34/39), respectively. Staphylococci were primarily resistant to early penicillins (over 80%), tetracycline (57.7%), and chloramphenicol (46.2%). Minimal resistance was noted with cloxacillin (0.0%), ciprofloxacin (9.6%), and cefoxitin (3.8%). The prevalence of multidrug resistance (MDR) was 78.8% for general staphylococci, 82.2% for S. aureus, 73.1% for S. pseudintermedius, and 60.0% for S. schleiferi. Multidrug resistant staphylococci were significantly more prevalent in the cattle isolates than in the dog isolates (P < 0.05). The prevalence of methicillin-resistant staphylococci (MRS) tested by resistance to cefoxitin and mecA carriage was 3.8%. These four strains were all isolated from dog skin infections. The tetK gene was the most predominant (35.4%), followed by tetM (25.0%). CONCLUSION: In resource-constrained settings, the approach of integrated diagnostics promises sustainable disease surveillance and the addressing of current capacity gaps. The emergence of MRS (zoonotic bacteria) in companion animals creates a likelihood of reduced treatment options for related human infections, a threat to global health.

Oxacillin-resistant Staphylococcus spp.: Impacts on fatality in a NICU in Brazil - confronting the perfect storm.

OBJECTIVE: Analyse the incidence, risk factors, antimicrobial susceptibility profile, and fatality in neonates infected with oxacillin-resistant Staphylococcus spp. (ORS). METHODS: In this retrospective observational descriptive cohort study, the medical records of neonates admitted to the Neonatal Intensive Care Unit (NICU) from January 2015 to June 2022 were analysed. Participants were monitored daily through the National Healthcare Safety Network. RESULTS: Among the 1610 neonates, 193 (12 %) developed ORS infections, primarily in the bloodstream (96.8 %). The incidence of these infections/patient-days decreased by 51.8 % between 2016 (8.3) and 2022 (4). The median age of affected neonates was 17.5 days (IQR:12-28.7). Pre-emptive prescription of fourth-generation cephalosporins (OR=14.36; P<0.01) emerged as a risk factor in the multivariate analysis. Staphylococcus epidermidis was the most prevalent species (60.1 %), with one isolate showing a "susceptible, increased exposure" profile to vancomycin. Additionally, 2 % of pathogens were extensively drug-resistant (XDR). ORS infections were associated with prolonged hospital stays (from 10 to 46 days) and increased mortality (from 10.2 % to 19.2 %). The median time between infection and the fatal outcome was 15 days (IQR:8-40), and Staphylococcus capitis was the most lethal species (26.7 %). CONCLUSIONS: The high incidence of ORS infections was linked to extended hospitalisation and increased mortality, highlighting the complexity of this situation - a "perfect storm." This underscores the urgency of implementing effective interventions for managing and preventing ORS infections in the NICU.

Identification and antimicrobial susceptibility profiles of Staphylococcus species isolated from raw cow milk, and swabs in smallholder dairy farms in Meta district, Eastern Ethiopia.

BACKGROUND: The safety of milk production in terms of foodborne infections is a worldwide issue, particularly in developing countries where production is often unhygienic. A cross-sectional study was conducted from December 2018 to August 2019 in the Meta District of Eastern Hararghe Zone, Oromia Regional State, Ethiopia. We aim to assess milk hygiene practices among smallholder dairy farmers, estimate the prevalence of Staphylococcus aureus in raw cow milk and swabs, assess associated risk factors, and the antimicrobial susceptibility test of S. aureus isolates. Face-to-face interviews with 30 respondents randomly selected from smallholder dairy farmers were used to assess the potential risk factors for S. aureus contaminations in milk. A total of 177 samples were examined using standard microbiological testing. The disc diffusion technique was also employed to assess the antibiotic susceptibility of the isolates. The data was analyzed using STATA® version 14.0 statistical software. RESULTS: According to the milk hygiene assessment, 80% of respondents did not wash cow udder before milking, did not use detergent to clean milk containers, and did not keep milk refrigerated before consumption or sale, while 63.3% of milk consumers ingested raw milk. They had never heard of staphylococci foodborne disease. Likewise, the overall prevalence of S. aureus was 12.42% (95%CI: 8.32-18.98). The prevalence of S. aureus in udder milk, equipment swabs, and milkers' hands was 18.8%, 26.7%, and 30%, respectively. The prevalence of S. aureus in milk is significantly associated with age, and mastitis history (p < 0.05). Moreover, old and mastitis positive animals were eight (OR: 8.40; 95%CI: 1.68-41.89) and four (OR: 4.33; 95%CI: 1.37-13.66) times more likely to be infected by S. aureus than adult, and mastitis negative animal. The isolates were resistant to penicillin G (97.4%) and tetracycline (69.2%) whereas susceptible to kanamycin, streptomycin, vancomycin, and cefotaxime, at 84.6%, 71.8%, 64%, and 58.8%, respectively. CONCLUSION: This study revealed the presence of antimicrobial-resistant patterns of S. aureus on commonly used antibiotics, as well as inadequate milk handling practices in the study area. Thus, awareness should be created on proper milk handling and hygiene as well as appropriate uses of antibiotics should be encouraged.

Coagulase-negative staphylococci from bovine milk: Antibiogram profiles and virulent gene detection.

BACKGROUND: Coagulase-negative Staphylococcus species are an emerging cause of intramammary infection, posing a significant economic and public health threat. The aim of this study was to assess the occurrence of coagulase-negative Staphylococcus species in bovine milk and dairy farms in Northwestern Ethiopia and to provide information about their antibiotic susceptibility and virulence gene profiles. METHODS: The cross-sectional study was conducted from February to August 2022. Coagulase-negative Staphylococcus species were isolated from 290 milk samples. Species isolation and identification were performed by plate culturing and biochemical tests and the antimicrobial susceptibility pattern of each isolate was determined by the Kirby-Bauer disc diffusion test. The single-plex PCR was used to detect the presence of virulent genes. The STATA software version 16 was used for data analysis. The prevalence, proportion of antimicrobial resistance and the number of virulent genes detected from coagulase-negative Staphylococcus species were analyzed using descriptive statistics. RESULTS: Coagulase-negative Staphylococcus species were isolated in 28.6%, (95% CI: 23.5-34.2) of the samples. Of these, the S. epidermidis, S. sciuri, S. warneri, S. haemolyticus, S. simulans, S. chromogens, S. cohnii, and S. captis species were isolated at the rates of 11, 5.2, 3.4, 3.1, 3.1, 1, 1, and 0.7% respectively. All the isolates showed a high percentage (100%) of resistance to Amoxicillin, Ampicillin, and Cefotetan and 37.5% of resistance to Oxacillin. The majority (54.2%) of coagulase-negative isolates also showed multidrug resistance. Coagulase-negative Staphylococcus species carried the icaD, pvl, mecA, hlb, sec, and hla virulent genes at the rates of 26.5%, 22.1%, 21.7%, 9.6%, 9.6% and 8.4% respectively. CONCLUSION: The present study revealed that the majority of the isolates (54.2%) were found multidrug-resistant and carriage of one or more virulent and enterotoxin genes responsible for intramammary and food poisoning infections. Thus, urgent disease control and prevention measures are warranted to reduce the deleterious impact of coagulase-negative species. To the best of our knowledge, this is the first study in Ethiopia to detect coagulase-negative Staphylococcus species with their associated virulent and food poisoning genes from bovine milk.

Light-activated conjugated polymer nanoparticles to defeat pathogens associated with bovine mastitis.

Bovine mastitis (BM) represents a significant challenge in the dairy industry. Limitations of conventional treatments have prompted the exploration of alternative approaches, such as photodynamic inactivation (PDI). In this study, we developed a PDI protocol to eliminate BM-associated pathogens using porphyrin-doped conjugated polymer nanoparticles (CPN). The PDI-CPN protocol was evaluated in four mastitis isolates of Staphylococcus and in a hyper-biofilm-forming reference strain. The results in planktonic cultures demonstrated that PDI-CPN exhibited a bactericidal profile upon relatively low light doses (∼9.6 J/cm2). Furthermore, following a seven-hour incubation period, no evidence of cellular reactivation was observed, indicating a highly efficient post-photodynamic inactivation effect. The successful elimination of bacterial suspensions encouraged us to test the PDI-CPN protocol on mature biofilms. Treatment using moderate light dose (∼64.8 J/cm2) reduced biofilm biomass and metabolic activity by up to 74% and 88%, respectively. The impact of PDI-CPN therapy on biofilms was investigated using scanning electron microscopy (SEM), which revealed nearly complete removal of the extracellular matrix and cocci. Moreover, ex vivo studies conducted on bovine udder skin demonstrated the efficacy of the therapy in eliminating bacteria from these scaffolds and its potential as a prophylactic method. Notably, the histological analysis of skin revealed no signs of cellular degeneration, suggesting that the protocol is safe and effective for BM treatment. Overall, this study demonstrates the potential of PDI-CPN in treating and preventing BM pathogens. It also provides insights into the effects of PDI-CPN on bacterial growth, metabolism, and survival over extended periods, aiding the development of effective control strategies and the optimization of future treatments.

Emergence of novel methicillin resistant Staphylococcus pseudintermedius lineages revealed by whole genome sequencing of isolates from companion animals and humans in Scotland.

Staphylococcus pseudintermedius is an opportunistic pathogen in dogs, and infection in humans is increasingly found, often linked to contact with dogs. We conducted a retrospective genotyping and antimicrobial susceptibility testing study of 406 S. pseudintermedius isolates cultured from animals (dogs, cats and an otter) and humans across Scotland, from 2007 to 2020. Seventy-five sequence types (STs) were identified, among the 130 isolates genotyped, with 59 seen only once. We observed the emergence of two methicillin resistant Staphylococcus pseudintermedius (MRSP) clones in Scotland: ST726, a novel locally-evolving clone, and ST551, first reported in 2015 in Poland, possibly linked to animal importation to Scotland from Central Europe. While ST71 was the most frequent S. pseudintermedius strain detected, other lineages that have been replacing ST71 in other countries, in addition to ST551, were detected. Multidrug resistance (MDR) was detected in 96.4% of MRSP and 8.4% of MSSP. A single MRSP isolate was resistant to mupirocin. Continuous surveillance for the emergence and dissemination of novel MDR MRSP in animals and humans and changes in antimicrobial susceptibility in S. pseudintermedius is warranted to minimise the threat to animal and human health.