Nano silver combines hydrogen peroxide against multi-resistant pathogens in Egyptian dairy cows

Purpose: The past few decades have seen a rapid and global increase in the emergence of multidrug-resistant bacteria (MDR).
Methods: The purpose of the current study is to isolate the most common MDR bacteria from dairy farms and cattle slaughterhouses, then evaluate their resistance patterns and evaluate resistance activity. bacteria of nano silver-hydrogen peroxide (AgNPs-H2O2) as an alternative to conventional antibiotics. In this regard, 200 samples were collected from two dairy farms and a beef slaughterhouse in Dakhliya province, Egypt.

Results: Interestingly, out of 120 samples collected from dairy farms, the incidence of isolates were 26.7, 23.3, 21.7, 16.7 and 11.7. % for S. typhimurium, E. coli O157: H7, L. monocytogenes, K. pneumoniae and P. aeruginosa, respectively. Meanwhile, the overall prevalence rates were 30, 25, 22.5, 17.5 and 5% for E. coli O157: H7, L. monocytogenes, S. typhimurium, P. aeruginosa and K. pneumoniae, respectively. Correspondingly, for 80 samples collected from a beef slaughterhouse. The antibiotic susceptibility pattern revealed that all isolates exhibited resistance to at least four of the antibiotics tested, with resistance index values ​​for many antibiotics. (MAR) ranged from 0.44 to 0.88. Furthermore, the commercial AgNPs-H2O2 product is characterized by a transmission electron microscope (TEM) and a zeta potential that shows spherical particles with a surface charge – 0.192 mV. Antibacterial activity of synthetic nano silver (AgNP) with H2O2 products against MDR strains was evaluated by measuring the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time curve. time annihilated.

Conclusion: Current data report high rates of MDR infection in dairy farms and slaughterhouses. More importantly, AgNPs-H2O2 silver nanoparticles exhibited broad-spectrum bactericidal activity against MDR strains, demonstrating their promising use as a safe, environmentally friendly, secretive antimicrobial agent. cost savings. To our knowledge, this study is a pioneer in investigating the potential alternative antimicrobial role of silver nanoparticles to control many drug-resistant pathogens in Egypt.

Nano silver combines hydrogen peroxide against multi-resistant pathogens in Egyptian dairy cows

(Bản quyền NanoCMM Technology)


Antibiotic resistance (AMR) has been considered a serious global threat to animal and human health, food security and development. This global threat is mainly the result of indiscriminate use of antimicrobial agents, thereby significantly reducing or even losing their effectiveness.  It should be emphasized that antibiotic-resistant pathogens endanger the human and veterinary ability to treat infectious diseases. Obviously, the application of antimicrobial agents on food-producing animals, for prophylactic and / or therapeutic purposes, creates a significant selection pressure that contributes to the occurrence, and transmission of antibiotic resistance through food supplies. Food of animal origin may be contaminated with various zoonotic pathogens that may result from improper production, processing methods on livestock farms and / or processing lines. feed, which in turn leads to the transmission of these pathogens to the consumer.

Among other diseases, mastitis is a multifactorial disease affecting milk yield and quality in dairy farms.  It should be noted that the main sources of milk contamination in dairy farms include handling, the type of management and hygiene practices on the farm, and therefore, many microorganisms can Dairy and its surroundings at the farm level, representing important sources of foodborne illness.  This problem poses a major public health hazard, while the pathogens that frequently cause and isolates are Salmonella enterica, Escherichia coli O157: H7, Pseudomonas aeruginosa, Listeria monocytogenes and Klebsiella pneumoniae.  It should be noted that foodborne pathogens such as Staphylococcus aureus, Streptococcus agalactiae, Escherichia coli, Pseudomonas aeruginosa, Corynebacterium bovis, and Bacillus cereus are considered to be the main causes of mastitis, thus leading to damage. loss of production and human illness resulting from consumption of contaminated food. dairy products. In addition, L. monocytogenes is also considered a foodborne pathogen, transmitted by meat, poultry, milk and plant products, while E. coli O157: H7 is widely known as a Major pathogens associated with foodborne diseases are observed in dairy products. When examined, beef cattle also contain pathogenic E. coli. 20

Furthermore, Klebsiella pneumoniae is considered to be one of the environmental agents causing clinical, subclinical mastitis and milk quality degradation. This pathogen causes severe mastitis due to its antibiotic resistance, rapid development of toxic shock and death of the animal.  Furthermore, most human infections caused by Salmonella originate from foods of animal origin such as dairy and meat, or from contact with contaminated animals and environments. Clearly, inappropriate farm practices in the production, handling and marketing of meat facilitate the transfer of foodborne pathogens to meat and meat products.

To combat this problem, antibiotic use has been considered the first choice in the treatment of bacterial infections in dairy cows, especially mastitis, which leads to a widespread and latent antibiotic residue in milk. risk of resistance to microorganisms in the environment. Furthermore, cattle are exposed to a wide variety of contaminants, including faecal bacteria, feed or the environment, resulting in the transmission of these organisms during slaughter on the body. meat, posing a high risk of food safety. Furthermore, the existence of live and environmental bacteria in the farm environment represents a major reservoir for the transfer of antibiotic resistance genes to pathogenic bacteria. In addition, some actions during slaughter at the slaughterhouse, such as removing and slaughtering meat, can contribute to carcass contamination. This challenge is often exacerbated by asymptomatic livestock carriers that pose a major public health hazard along the food chain.

Consequently, more stringent hygiene measures combined with law enforcement at slaughterhouses and during slaughter are key factors in reducing the risk of meat contamination. Likewise, there is an urgent need to find eco-friendly alternatives to conventional antibiotics to combat the widespread multidrug resistance (MDR) problem and the emergence of its fast.

It should be noted that recent years have seen remarkable progress in discovering the role of nanotechnology in providing the large growth and transformation of nanoparticles (NPs) with properties. Proprietary chemistry as a promising tool used in medicine and agriculture to overcome the limitations caused by conventional antibiotics. The application of nanomaterials, mainly Silver nanoparticles (AgNPs), has spurred attention in many different aspects; nanomedicine, academic, industry and field aspects. Silver nanoparticles exhibit amazing bactericidal activity on a wide variety of Gram-positive and Gram-negative bacteria, including food-borne pathogens such as Escherichia coli O157: H7, Salmonella, Listeria monocytogenes, Campylobacter jejuni and Staphylococcus aureus .Mechanisms behind AgNPs actions include their induction of bacterial cell destruction through the generation of reactive oxygen species (ROS) in a number of bacteria including E. coli, K . p neumoniae, and Pseudomonas aeruginosa. 36 , 44 , 45

Importantly, there are fewer reports of bacterial resistance to nano silver than for conventional antibiotics. The antimicrobial activity of NPs is protected by various factors, such as the size, shape, stability and usage concentration of NPs. Taking into account, the most common and useful determinants for the relative bactericidal activity of the various synthetic nanomaterials are the minimum inhibitory concentration (MIC) and concentration Minimal bactericidal (MBC).

With the above information, this study was initially performed to show the incidence and survival of food-borne MDR pathogens, mainly five main species, including Salmonella enterica, Escherichia coli O157: H7. , Pseudomonas aeruginosa, Listeria monocytogenes, and Klebsiella pneumoniae have been isolated from various sources. These sources include milk, bulk milk, milking equipment, beef carcasses, walls, workers’ knives and hands (cotton swabs) from dairy farms and slaughterhouses in Dakahliya Governorate, Egypt. . We also aim to evaluate the antibacterial activity of commercially synthesized AgNPs-H2O2 silver nanoparticles on representative isolated MDR pathogens combined with the discovery of the bactericidal mechanism of AgNPs using assays. test of different cells.

Materials and methods

Ethical considerations

Ethical consent to this study was obtained from the Research, Publication and Ethics guidelines of the Faculty of Veterinary Medicine, Mansoura University, Egypt, where all relevant legal regulations were complied with. of Egypt for research and publishing. The participating dairy workers provided informed consent in the cotton swab collection process and the study followed the guidelines outlined in the Helsinki Declaration.

Research area, sample collection and preparation

In this study, a total of 200 samples were collected from two dairy farms and one beef slaughterhouse in the province of El-Dakahlia, Egypt, between September and November 2018. Regarding feces The total number of samples collected from farms dairy cows was 120 at a rate of 20 samples from each source / farm, while these samples were collected and included samples from large tank milk (BTMs). ) (100 mL), milking equipment and cotton swabs of dairy workers. For the slaughter establishment, total samples collected were 80 samples, at a rate of 20 samples from each source, samples including samples of beef (25 gm), walls, knives and cotton swabs were collected. full. sterile conditions. All samples are then transported into an ice box to the laboratory of the Department of Hygiene and Animal Diseases, Mansoura University, and are under further processing. Then, five main types of bacteria were isolated and characterized from the samples collected; including E. c oli O157: H7, Listeria monocytogenes, Salmonella typhimurium, Pseudomonas aeruginosa, and Klebsiella pneumonia, following the procedure described elsewhere. 51 Isolation and characterization steps are performed using suitable selective culture media, and different incubation conditions followed by colonization, morphology and biochemical profiles. . Furthermore, the validation of identified strains is performed through serological and molecular characterization as described elsewhere. 21 , – 52 – 55

Antibiotic susceptibility test and isolation of MDR bacteria

Antibiotic susceptibility models to E were confirmed. Cells O157: H7, Listeria monocytogenes, Salmonella typhimurium, Pseudomonas aeruginosa, and strains of pneumonia Klebsiella for selected antibiotics (Oxoid, Hampshire, UK) are shown in Table 1. These samples were performed by a Kirby-Bauer disc diffusion test using Mueller-Hinton Agar (MHA) (BioMeriéux, Vienna, Austria) according to the guidance of the Institute of Clinical and Laboratory Standards (CLSI). Isolates were classified as resistant, intermediate or sensitive by measuring the diameter of the inhibitory zone around each plate, as described by CLSI. Then, the Multiple Resistance Index (MDR) for each type of resistance was then calculated from the amount of resistance to each strain, divided by the total number of antibiotics tested. 57 Isolates that show resistance to three or more antibiotics are considered multi-resistant strain. 58

Table 1 Antibiotics were used to test the antibiotic susceptibility of five types of bacteria

Table 1 Antibiotics were used to test the antibiotic susceptibility of five types of bacteria

Culture and preparation conditions of bacteria

Bacterial strains (N = 10) expressing MDR with three or more antibiotics were prepared according to the methods mentioned in the other section. In short, all bacterial samples were inoculated on Mueller Hinton (MHB) medium (BioMeriéux, Vienna, Austria) and were cultured in aerobic medium at 37ºC / 24 h, after which a loop was obtained. streak on MHA plates and sub-cultures for purification on the same medium. Pure colonies are harvested and kept at -80ºC. Thereafter, 0.5 McFarland microbiological preparations were prepared by direct colony suspension as recommended by CLSI guidelines. 56 Bacterial turbidity was spectrally adjusted by UV / Visible 6715 scanning spectrometer (Jenway, Canada) to 0.08–0.12 at optical density (OD) 625 nm, creating a microbial suspension have (1–2) x10^8 colony forming units (CFU) / mL.

Properties of the commercial nano silver AgNPs-H2O2

AgNPs-H2O2 (Top Superpower Vision) was purchased and supplied by the El-Delta Center as a commercial product for the silver nanotechnology company, Mansoura, Egypt. Product stock solution consists of 45 nm (0.00004467 mL / liter) nano silver with Hydrogen Peroxide (50% liter) and natural herbs, mint (1 mL / liter) at a concentration of 5 mL / liter of water then product is diluted in Mueller Hinton broth (MHB). The mean morphology and mean size of the commercial nano product were characterized through the TEM and zeta potentials as described elsewhere,  using the Zeta Potential Ver by Malvern Instruments Ltd. 2.3 at Central Laboratory, Electron Microscope Unit, Department of Agriculture, Mansoura University, Mansoura, Egypt

In vitro antimicrobial activity of nano silver AgNPs-H2O2 against MDR strains
Determination of minimum inhibitor concentration (MIC) value

The inhibitory ability of AgNPs-H2O2 products against strains of MDR bacteria (N = 5) was evaluated using MIC. MIC is determined by a broth dilution method according to CLSI guidelines. 60 Specifically, MIC was performed in 96-well microtiter plates by a double micro-dilution method. The AgNPs-H2O2 mixture was prepared to the desired commercial concentration by dilution in sterile distilled water and then the mixture was diluted by 1/10 in sterile Mueller Hinton Broth (MHB). In this regard, a 50 µL MHB amount was sown into each well, starting from the 2nd well to the 11th well, followed by a 2-fold dilution of the commercial nano product in MHB. Then, 100 µL of this mixture was inoculated in the first well, then 50 µL was transferred to subsequent wells except the 12th well which acted as a negative control (drug-free well). Then, dilute 50 µL of bacterial suspension to 1/150 in sterile MHB with microbiological 10^6 distributed in all wells then mix the plates, and plates are incubated at 35 ° C for 24 hours. MIC is defined as the point where bacterial growth is completely inhibited in microbial dilution wells that can be detected visually under transmitted light without turbidity. Therefore, the first well without microbial growth is considered to be the MIC, expressed in μg / mL. To determine the MIC of nano silver of all strains tested, they were exposed to 0–100 μg / mL AgNPs. Furthermore, the MIC of each isolate was identified three times for data verification. The test includes only the medium and media containing silver nanoparticles as reference controls. As mentioned above,

Determination of minimum bactericidal concentration (MBC)

The lowest concentration of AgNPs-H2O2 mixture required to destroy the last 99.9% of bacterial inoculum called MBC, was determined after dilution in broth by subculture 50 µL from sock. both wells had no visible turbidity on MHA plates and incubated at 35 ± 1 ° C for 16–18 hours. The plates were then studied for the presence or absence of bacterial colonies and MBC was determined to be the lowest NP concentration that could completely inhibit bacterial growth. The NPs product mode of action is assessed by the MBC / MIC ratio, where scores 1, 2 and 4 are considered bactericidal and bacteriostatic if scores> 4.  Likewise, the lowest dilution without growth of visible macroscopic bacteria was identified as MBC. This was done according to the CLSI method for antibacterial drugs, described in document M7-A9. 63

Check kill time (kill time curve)

Interactions between nano silver bacteria strains AgNPs-H2O2 and MDR were determined using a time lethal test under AgNPs concentrations of 0.25xMIC and 1xMIC as described elsewhere. 56 In summary, after reading the MIC for each AgNPs-H2O2 and MDR strain, three tubes with 10 mL MHB of 5 × 10^5 CFU / mL bacterial suspension were tested at 0.25 × MIC and 1 × MIC. , and a third tube of one type was used as the growth control, while the nano silver AgNPs-H2O2 were substituted for MHB as the negative control. All tubes were then incubated at 35 ± 1 ° C for different intervals (2, 4, 6, 8, 12 and 24 hours). 64 Live / dead bacterial cells (CFU / mL) of each tube were quantified on MHA plates by time-related agar plate method. These numbers are plotted on the chart depicting the kill time curve versus the positive and negative control curves. 48

Statistical analysis

Statistical analysis was performed using SPSS statistical package, The frequency of inhibited samples was compared between different concentrations of the drug. Furthermore, the kappa test was used to check for consistency between the results of MIC and MBC. The result is considered significant at P <0.05, then the mean CFU concentration is compared at different times using the variance analysis (ANOVA) test to detect the difference. difference between the mean values. Finally, the Duncan multi-range test was used to make different comparisons between vehicles. Data are presented as means and standard error, and the results are considered significant when P <0.05.


Prevalence of MDR species of bacteria among inspected dairy farms and beef slaughterhouses

The prevalence and frequency distribution of bacterial species from dairy farms and slaughterhouses are shown in Table 2. Among other species, S. typhimurium was the most dominant bacterial species (26.7%) isolated from samples collected from tested dairy farms, where milking equipment had spectral ratios. The highest variable was 35%, followed by a worker swab (25%), while the lowest prevalence rate was from BTMs (20%). Following S. typhimurium, E. coli O157: H7 had the second occurrence level of 23.3%, while the isolation frequency was 30%, 25% and 15% respectively for BTM, milking device. and a worker’s cotton swab. The overall prevalence of L. monocytogenes was 21.7% in dairy farm samples while the recovery rates between sources analyzed were 25%, 20%, and 20% for cotton swabs, BTMs and instruments. milking workers, respectively. Among 120 samples collected from dairy farms, K. pneumoniae was recovered at a rate of 16.7%, while BTM had a 25% higher isolation rate, followed by cotton swabs of workers (15 %) and milking equipment (10%). On the other hand, P. aeruginosa showed the lowest rate of detected bacteria among all isolates (11.7%), while the isolate rate sequentially from farm source was 20%, 10 % and 5% from workers’ hand gauze. , milking equipment, and BTM, respectively.

Table 2 Frequency distribution of bacterial species recovered from dairy farms and slaughterhouses

Table 2 Frequency distribution of bacterial species recovered from dairy farms and slaughterhouses

According to samples collected from beef slaughterhouses, of the 80 samples collected, the overall incidence rates were 30%, 25%, 22.5%, 17.5%, and 5% for E. coli O157: H7, L. monocytogenes, S. typhimurium, P. aeruginosa, and K. pneumoniae, respectively. Furthermore, workers’ knives, gauze hands and carcasses are major sources of E. coli O157: H7, with recovery rates of 40%, 40% and 30%, respectively. Meanwhile, hot spots for L. monocytogenes contamination in slaughterhouses were found to be high on wall gauze (40%), followed by carcasses (30%) and 20% on knife gauze. Most of the S. typhimurium in the slaughterhouse was isolated from wall gauze and carcasses (both 30%), followed by workers’ hand gauze (20%), while at least detected from gauze (10 %). There is no major difference between different sources for P. aeruginosa isolate. K. pneumoniae is mainly isolated from workers’ knives and gauze at the rate of 10%, and cannot be detected from other sources.

Antibiotic resistance patterns of bacterial species isolated from dairy farms and slaughterhouses

Out of all isolates, 40 confirmed strains were isolated from dairy farms and slaughterhouses (five per strain) were tested for antibiotic sensitivity by diffusion. plate-Bauer dispersion to assess their resistance patterns (Table 3).

Table 3 Multi-drug resistance (MDR) models of recovered bacterial species (N = 40) from dairy farms and cattle slaughterhouses

Table 3 Multi-drug resistance (MDR) models of recovered bacterial species (N = 40) from dairy farms and cattle slaughterhouses

As shown in Table 3, the percentages of overall resistance patterns of the strains isolated from dairy farms with their respective bacteria were as follows: 37.5%, 46.2%, 25 %, 28.6% and 70%, for E. coli O157: H 7, L. monocytogenes, S. typhimurium, P. aeruginosa, and K. pneumoniae, respectively. These results clearly indicate that K. pneumoniae showed the highest level of resistance among bacteria species isolated from dairy farms. Meanwhile, the percentage resistance levels of strains isolated from beef slaughter samples compared to their respective bacteria species are as follows: 85.7%, 50%, 44.4%, 25 % and 20% for P. aeruginosa, K. pneumoniae, S. typhimurium, E. coli O157: H 7, and L. monocytogenes, respectively. According to these results, a multiple antibiotic resistance index (MAR) was identified and it was shown that all isolates exhibited resistance to at least four antibiotics.

In general, MAR index values ​​ranged from 0.44–0.88, while K. pneumoniae had the highest MAR index of 0.88, with resistance to six types of antibiotics demonstrated ( neomycin, kanamycin, tetracycline, nalidixic acid, amoxicillin and gentamycin). The recovered S. typhimurium strains had a MAR index of 0.63 with resistance to 5 antibiotics; norfloxacin, amoxicillin, ciprofloxacin, Chloramphenicol and Trimethoprim / sulphamethoxazole. Furthermore, strains of E. coli O157: H 7 were confirmed to exhibit resistance to erythromycin, amoxicillin, tetracycline and streptomycin, with a MAR index of 0.5. Regarding L. monocytogenes, they demonstrated resistance to five antibiotics; Rifampicin, Cefotaxime, tetracycline, gentamycin and chloramphenicol, with a MAR index value of 0.45. On the other hand, P. aeruginosa had the lowest MAR index value of 0.44 with a model resistant to ciprofloxacin, norfloxacin, streptomycin and levofloxacin.

Characteristics of the silver nanoparticle AgNP-H2O2

Figures 1 and 2 show TEM images and zeta potentials of used AgNPs-H 2 O 2 nano silver. Stock solution of 100 nm nano silver product was created in culture medium and further dilutions were performed in Luria-Bertani broth. NP morphology, shape and size were measured using TEM and Malvern Nano Zeta Sizer (Malvern Zetasize Nano-zs90). Spherical nanoparticles with well-defined particle size range (30,17–67.92 nm) have an estimated zeta potential of -0,192 mV.

Figure 1 Characteristics of the product AgNPs-H2O2, TEM microscope image and size distribution of nano-silver

Figure 1 Characteristics of the product AgNPs-H2O2, TEM microscope image and size distribution of nano-silver

Figure 2 Zeta potential of AgNPs-H2O2 product

Figure 2 Zeta potential of AgNPs-H2O2 product

Antibacterial activity of nano silver AgNPs-H2O2 on MDR bacteria

The microbial broth dilution method was used to evaluate the bactericidal activity of AgNPs-H2O2 against MDR bacteria (Table 4). MIC values ​​of products to inhibit bacterial growth were 6,25, 12,5, 3,125, 6,25 and 25 µg / mL for E. coli O157: H7, L. monocytogenes, S, respectively. typhimurium, P. aeruginosa and K. pneumonia e. . The MBC of AgNPs-H2O2 ranged from 6.25 to 50 µg / mL for all strains tested. The MBC / MIC ratio is a measure that indicates the bactericidal ability of a compound under investigation. Relationships between the MIC and MBC assays were measured with the Kappa test. A high degree of association between both assays (83, 82 and 80%) was detected for S. typhimurium, E. coli O157: H7 and K. pneumoniae, respectively. However, a low association (30%) was noted for both L. monocytogenes and P. aeruginosa (Table 4).

Table 4 The MIC values, MBC and the MBC / MIC ratio of the AgNPs-H2O2 Product

Table 4 The MIC values, MBC and the MBC / MIC ratio of the AgNPs-H2O2 Product

In the current study, nano silver AgNPs-H2O2 showed a bactericidal effect against all strains tested except for P. aeruginosa that exhibited bacteriostatic effect. As mentioned before, the bactericidal effect of AgNPs-H2O2 on MDR bacteria, namely; E. coli O157: H7, L. monocytogenes, S. typhimurium, P. aeruginosa, and K. pneumoniae; Isolation from dairy farms and a beef slaughterhouse were evaluated using different assays (MIC, MBC, and slaughter test). The time-dependent bactericidal action of the nano silver product is shown in Figure 3 and this assay is performed against the MIC value of each selected strain. The bactericidal activity was assessed by a practical decrease in cell viability (CFU / mL) at 2, 4, 6, 8, 12 and 24 hours for each isolate. All strains tested had high growth rates at 2 and 4 hours after exposure to AgNPs-H2O2 nano silver. Importantly, growth decreased after 6 hours and reached full inhibition after 24 hours, with the exception of P. aeruginosa, which showed immediate bacterial growth even after 24 hours.

Figure 3 AgNPs-H2O2 time-killing assay against multidrug-resistant bacteria.

Figure 3 AgNPs-H2O2 lethality trials against multidrug-resistant bacteria. (A) L. monocytogenes, (B) P. aeruginosa, (C) S. typhimurium, (D) E. coli O157: H7, (E) K. pneumoniae, and (F) comparison kill time assay of AgNPs -H2O2 against all tested MDR bacteria. Measurements are made at 0, 2, 4, 6, 8, 12 and 24 hours post-treatment, microbiological viability is expressed in CFU / mL, data points are expressed as averages. ± SEM for three tests.

Discussion of nano silver – H2O2 efficiency

Cattle are considered the main reservoir for a number of zoonotic pathogens. Existing research has reported interesting new data on the relatively high prevalence of zoonotic MDR bacteria isolated from dairy farms and associated beef slaughterhouses. evaluation of their drug resistance models and antibacterial activity of nano silver AgNPs-H2O2 as an alternative to conventional antibiotics. To our knowledge, current work is the first involving the discovery of the role of silver nanoparticles in the control of many drug-resistant pathogens isolated from dairy farms and slaughterhouses. beef in Egypt.

Some previous studies have shown that some animal pathogens, including E. coli O157: H7, reside in the intestines of animals and are excreted in feces and are not expelled in milk; however, contaminated milk during lactation has been reported. Notably, as depicted in Table 2, our study reported a relatively high incidence of 23.3% and 22.5% for E. coli O157: H7 isolated from the sites. Dairy farms and beef slaughterhouses, respectively. Corresponding spawn rates were 30% in both BTM and carcasses from dairy and slaughterhouses. The prevalence is higher than reported in some previous studies.  This difference could be due to poor hygiene practices on both farm and slaughterhouses, in addition to skin / manure-carcass contamination at processing plants playing an important role. in carcass contamination. In contrast, prevalence of E. coli O157: H7 (2.7%, 3.2% and 8%) from beef samples in slaughterhouses has been reported in previous studies. In addition, a low 10% isolation rate from BTMs was observed elsewhere. Consistent with L. monocytogenes, it is classified as the third major foodborne pathogen and its existence in milk and dairy products, in addition to having adverse effects on the industry. dairy industry and public health. L. monocytogenes can be transmitted to humans through contaminated milk and meat. In this study, the prevalence of L. monocytogen es infection was 21.7% and 25%, respectively, in dairy farms and slaughterhouses. Taking this into account, milking equipment had the highest isolation rate, at 25%, followed by 20% for both the BTM and the worker swab. These results are consistent with some previous findings that isolated L. monocytogenes at the rate of 21-26% from raw fresh milk. Additionally, another earlier study reported that 20% BTM samples containing L. monocytogenes on a dairy farm. 83 All of these findings refer to infected animals, poor silage quality, and inadequate hygiene practices are possible sources of L. monocytogenes contamination in dairy farms. Our findings also suggest the prevalence of L. monocytogenes in slaughterhouses, which is supported by some previous studies indicating that animal transport stress leads to increased rates of shedding of animals. bacteria.86

In fact, S. typhimurium is one of the most frequent animal-to-human pathogens that cause a number of human illnesses from consuming contaminated food, including raw meat and milk.  Interestingly, our data obtained from dairy farms showed a high incidence of S. typhimurium (26.7%), where the frequency of isolation levels increased gradually to 35%. , 25% and 20% from milking equipment, workers. ‘swab, and BTM, respectively. These results are strongly correlated with those reported in a previous study where Salmonella spp. Can be isolated from bulk tank milk and dairy cow manure samples. On the other hand, 9% and 8% lower isolation rates of Salmonella from worker milk and gauze were reported in a previous study in Sharkia, Egypt. Another previous study failed to identify Salmonella in BTM from dairy farms. Deviation of current results from previous results can be attributed to a number of factors, including differences in hygiene and hygiene practices applied during milking. Notably, a high incidence of S. typhimurium (26.7%) was found in the slaughterhouse, while the highest isolation frequency (30%) was reported from carcass and wall gauze samples. . These data are consistent with some previous reports. Contaminated carcasses during butcher or skin removal, repeated use of slaughtering equipment for different animals, improper disinfection of utensils, and sharpening of knives on unclean objects will all contribute to a high chance of Salmonella infection in slaughterhouses.

As for P. aeruginosa, it has been considered a broad host pathogen. In cattle, P. aeruginosa has been implicated in many diseases, particularly mastitis. Previous studies have shown that the presence of P. aeruginosa in milk samples can be caused by a variety of unhygienic maintenance errors, including inadequate cleaning of the refrigerator or refrigerator in bulk, and This could be caused by contaminated milk entering contaminated water at the farm level. Notably, data obtained from our current study show that the P aeruginosa distribution between dairy farms and slaughterhouses was 11.7% and 17.5%, respectively. An almost similar isolation rate of P. aeruginosa of 9.4% has been reported in previous works. On the other hand, lower isolation rates of 3.6 and 5.4% have been indicated in some previous reports. This difference may be due to previously mentioned factors related to unhygienic practices that lead to milk contamination with polluted water at the farm level. In the same strain, Klebsiella spp. are Gram-negative bacteria that have a significant effect on milk production and the survival rate of animals. It has been linked to mastitis outbreaks. The prevalence rates of K. pneumoniae in our current results from dairy farms and one slaughterhouse were 16.7% and 5%, respectively. These findings suggest the existence of this bacterium in the animal environment from a variety of sources. An 8.6% lower prevalence rate was reported in a previous report from dairy farms. Klebsiella spp. frequent excretion in the feces of healthy cows leads to higher levels of contamination in litter, and manure in barns and alleys together leads to higher rates of Klebsiella infection on the scalp and hence Contaminate milk.

As shown in Table 3, the antibiotic histogram of the five bacterial species isolated from dairy farms and beef slaughterhouses shows the presence of multidrug resistance among all isolates. . As illustrated in Table 3, the reported resistance rates of strains isolated from dairy farms were 70%, 46.2%, 35.7%, 28.6% and 25% for K. pneumoniae, L. monocytogenes, E. coli O157: H7, P. aeruginosa, and S. typhimurium, respectively. Meanwhile, among slaughterhouses, the drug resistance model was 85.7% for P. aeruginosa strains, 50% for K. pneumoniae, followed by 44.4%, 25% and 20% for strains. with S. typhimurium, E. coli O157: H7, and L. monocytogenes, respectively. Clearly, current data suggest that the predominant MDR bacteria among food-borne isolates has increased over the past decades. Resistance patterns of strains evaluated against antibiotics commonly used in the veterinary sector showed that all strains had MAR for at least four antibiotics. Our findings concur with findings reported elsewhere. The high resistance of E. coli O157: H7, L. monocytogenes and K. pneumoniae to tetracycline correlates with the widespread use of tetracycline in dairy farms for the treatment of infectious diseases. In the current study, all strains had a MAR index ranging from 0.44–0.88, as it was well known that isolates with a MAR index less than 0.2 were given. are from rarely used sources of antibiotics. These data confirm that the use of antimicrobial agents is strongly associated with the risk of growth of MDR bacteria. It was previously noted that resistant strains carefully selected during antibiotic treatment persisted for a long time in the intestinal tract when this treatment was completed. Furthermore, these resistant strains can affect animal health and may be spread to other animals, especially to their offspring and companion animals.

Interestingly, the testing of nano silver AgNPs-H2O2 using the TEM and zeta potentials represents a major potential tool for determining the morphology, size and surface charge of nanoparticles, and thus understand the physical stability of the nanoparticles. 111 Nanoparticles are known to have good physical activity when their nanocrystals have positive or negative zeta potential values ​​greater than -30 mV to +30 mV due to the electrostatic repulsion of the individual particles. Used products have a zeta potential value of -0,192 mV, which means sufficient repulsion results in better physical glue stability with a high degree of confidence. Whereas the agglutination and flocculation of nanoparticles are associated with small zeta potential values ​​due to the gravitational forces of van der Waals acting on them, aimed at physical instability.

Over the past decades, global attention has turned to MDR bacteria as a major challenge to human and animal health. 114 Control strategies for such bacteria are largely based on the use of conventional antibiotics. However, their inappropriate use can lead to a global catastrophe and spur us to look for alternatives to these drugs. Nanotechnology has been considered a biological nano weapon that helps re-investigate the biological properties of previously known antimicrobial compounds by controlling their size to modify their potential effects. . 115 In this study, we aimed to evaluate the antibacterial activity of nano silver AgNPs-H 2 O 2 against MDR bacteria through several in vitro assays (MIC, MBC, and kill time test). , and therefore, to evaluate the effectiveness of the different concentrations and times on the cell viability of the bacteria were tested. The MIC test is used to determine the lowest concentration of tested NPs needed to completely inhibit bacterial growth with increasing concentration of the product. Our data showed that all strains were sensitive to the NPs tested, where the MIC values ​​were as follows: 3,125 µg / mL for S. typhimurium, 6.25 µg / mL for both E. coli O157: H7 and P. aeruginosa, 12.5 µg / mL for L. monocytogenes, and 25 µg / mL for K. pneumoniae.

The MBC value of AgNPs-H2O2 indicated that S. typhimurium had the highest sensitivity to NP product at 6.25 µg / mL, while E. coli O157: H7 needed a higher concentration to inhibit growth. they were at 12.5 µg / mL, while the growth of P. aeruginosa, L. monocytogenes, and K .pneumoniae decreased completely at 50 µg / mL. These findings are consistent with the results of Zarei et al.  who reported antibacterial activity values ​​of nano-silver against S. typhimurium as MIC of 3.12 µg / mL and MBC of 6.25 µg. / mL. On the other hand, 2 µg / mL AgNPs with a mean size of 30 nm had bacteriostatic effect on P. aeruginosa. 117 For all MDR strains, except P. aeruginosa, MBC / MIC ratio indicates that AgNPs-H2O2 has a bactericidal rather than bacteriostatic effect, and is clinically preferred because dead bacteria lead to rapid termination of infection, and better clinical results. and less likely to develop resistance. and the infection spreads. Thus, these actions in turn reduce the occurrence of resistant mutations.  It should be emphasized that silver nanoparticles have broad spectrum activity against Gram-negative bacteria including members of the genera Escherichia, Pseudomonas and Salmonella, in addition to Gram-positive bacteria including Clostridium, Listeria, Staphylococcus and Streptococcus, through binding to the bacterial cell membrane leads to disruption of its function, invading bacteria and releasing silver ions. Post-treatment microbial viability can be assessed with kill time tests to describe the least time required to induce bacteriostatic or bactericidal effects. Rapid bacterial spawning times are one of the key essentials of the ability to infect bacteria, a trait that can be a good target for preventing a viable infection. The association level analysis using the Kappa test between MIC and MBC results could confirm the compatibility between the two assessment methods in addition to revealing the higher degree of association and the reliability of the assays. experience. Hence, the kappa test provides more information than a simple calculation of the deal amount. AgNPs-H2O2 silver NPs evaluation of a product’s bactericidal power relative to the MIC values ​​and time of its claims of bactericidal power in a dose-dependent manner and time. Our results are consistent with results reported in an earlier study, people who found that E. coli counts decreased from 10^7 to 10^1 CFU / mL at 14 hours after treatment with silver ions, and therefore, the behavior of the nanoparticles can be the same as that of silver ions.  As observed in our study, the nano product used is nano silver combined with H2O2. This combination rapidly reduced the viability of bacteria over time with complete death of MDR strains after 12–24 hours. This proposed action may be thought to have a higher bactericidal effect since more than 100 times is achieved when such a combination is achieved due to the Fenton-like reaction between two agents that produce a Hydroxy (OH) group,  are considered. one of the most potent bioactive ROS. 42


With the above information, our study highlights a worrying trend for higher rates of MDR bacteria in dairy farms and beef slaughterhouses in Egypt, suggesting that antibiotic use has excreta. discrimination in the treatment of infectious diseases. Obviously, an urgent call is needed to find an alternative to these antibacterial agents. More importantly, AgNPs-H2O2 demonstrated a promising strong bactericidal activity against MDR bacteria regardless of the level of resistance against the strains tested. Our current data also conclude that AgNPs-H2O2 silver nanoparticles could be suggested as an environmentally friendly broad spectrum bactericidal agent, and our research suggests further future research on these similar lines against other bacteria and the same nanoparticles. Furthermore, the monitoring of sanitary and biosafety measures as well as the handling of antibiotics must be carefully examined.


Reference source:

Enhanced antibacterial effect of antibiotics in combination with silver nanoparticles against animal pathogens