Rate of Beta-Lactam Resistance and Epidemiological Features of S. Aureus-Associated Bovine Mastitis in Cross-Bred Ethiopian Cows: Systematic Review

Introduction

Bovine mastitis, a grave and substantial ailment, poses a considerable challenge to dairy production.^1,2 Particularly in low-income countries like Ethiopia, it demands special attention from farmers. Numerous factors have been linked to mastitis, including lower milk production, milk that is judged unsafe for human consumption due to antibiotic residues, veterinary expenses, and the eventual culling of long-term sick cows.³ The categorization of bovine mastitis primarily revolves around the distinction between subclinical and clinical cases, as determined by the severity level.

Over 90% of Ethiopia’s total milk production loss is attributed to subclinical mastitis, according to study made by scholars.^4,5 In addition, cross-breed dairy cows are predicted to cost 38 US dollars per lactation. Furthermore, it has an effect on public health since dairy products can shed zoonotic pathogens and their toxin, which poses a major risk to human health.⁶ Mastitis, can result from a variety of illnesses caused by bacteria, fungi, and viruses. In domestic animals, mastitis can be caused by at least 137 etiological agents, with bacteria being the most common cause.⁷ One of the most infectious bacteria that cause mastitis in cows and spoiling of milk is S. aureus.⁸

There is proof of the careless use of antimicrobial agents by medical professionals, unskilled practitioners, and others engaged in drug usage and animal husbandry in Ethiopia. Antibiotic resistance is also greatly influenced by elements related to the delivery of medication itself, such as an unfavorable length of treatment and mode of administration. Both in humans and animals, the incidence of staphylococcal infections has been steadily rising. The reason behind this trend is the increment in multidrug-resistant types of these illnesses, which has made treating them more difficult. There are several different processes underlying the emergence of antimicrobial resistance (AMR) in S. aureus. The most well-known resistance mechanisms include: antibiotics being inactivated by enzymes, changing the targets of antibiotics to reduce their affinity, antibiotics being trapped, efflux, pumps, and limited drug uptake through biofilm formation.^7–11

It is essential to comprehend the complex interactions among these multifactorial and toxin-specific virulence variables in order to create successful treatment plans to combat S. aureus infections. By focusing on these elements, it may be possible to design new intervention strategies and preventative measures to counteract the pathogenicity of these adaptable bacteria. Future studies in this area will probably provide more information about the mechanisms behind S. aureus virulence and help develop mitigation methods for its harmful effects on human health.^10,11

The continuous expression of several virulence factors in response to environmental stimuli during infections suggests the presence of global regulators, in which the expression of numerous unrelated target genes is controlled by a single regulatory determinant.¹² She and Sek genes are detected in isolates and cause subclinical mastitis(SCM), while the CC479 strains of S. aureus are connected to severe clinical mastitis in cattle.¹³ Mean while, sed and sej isolates are mainly associated with continual mastitis.¹⁴ These regulators produce chemicals that aid in the survival of bacteria, allowing them to adapt to harsh settings. Drug resistance is a major public health risk associated with S. aureus. The death rate for people with drug-sensitive S. aureus infections is 64% lower than that of people with methicillin-resistant infections.

Methicillin resistance¹⁵ is caused by the acquisition of the mecA gene and is typified by the synthesis of PBP2A, an alternative penicillin-binding protein. There is a limited affinity of this protein for beta-lactam antibiotics.¹⁶ When it comes to dairy animals, Staphylococcus aureus usually affects the integument, nasal mucosa, and skull. But during milking time, an infected udder quarter serves as the main source of infection for non-infected animals. Depending on the developed standard state of the nation, this can happen through contaminated milkers hands or milking machines¹⁷ separating S. aureus from other species by coagulase formation, mannitol fermentation, and trehalose usage is part of the identification and isolation procedures.¹⁸ Staphylococcus aureus is the most common infectious and zoonotic pathogen in Ethiopia that causes mastitis and significantly lowers output. Numerous studies, undertaken in different places and times, have examined the prevalence of S. aureus infection in bovine mastitis, accounting for prevalence at the cow, herd, and quarter levels. Recent studies show that between 66.07% and 10% of both asymptomatic and symptomatic instances of bovine mastitis have S. aureus present.^19–21

The purpose of this systematic review is aimed to offer useful information about S. aureus, a pathogen spread through milk, to experts in the fields of animal, human, and environmental health. It will function as a thorough information source for putting preventative and control measures, such as infection prevention, resistance reduction, and surveillance design, into practice on dairy farms. In both human and veterinary medicine, systematic reviews are frequently used to compile the results of several research studies. The combined proportion of the epidemiological distribution and the beta-lactam resistance profile of the S. aureus infection among Ethiopian dairy cows was, thus, the purpose of this review.

Methodology

The STROBIE (Preferred Reporting Items for Systematic Reviews and Meta-analysis) checklist (Supplementary file-1 STROBIE checklist for included cross-sectional studies) served as the basis for the systematic review. Based on the highlighted protocols, the checklists were utilized to verify that pertinent data from the chosen publications had been included.

Search Engine/Strategy

The systematic review was based on the STROBIE (Preferred Reporting Items for Systematic Reviews and Meta-analysis) criteria (Supplementary file-1 STROBIE checklist for included cross-sectional studies). The checklists used to confirm that the relevant data from the selected publications was incorporated in compliance with the protocols that were indicated. The literature search was conducted between November 20, 2021, and December 30, 2022. A comprehensive search strategy was independently devised by two authors (M.B. and B.M.) to locate included studies. Both manual methods and databases, such as PubMed, Web of Science, Google Scholar, and HINARI, were used for the literature searches. What is the prevalence or percentage of S. aureus infection among the bacteria that cause mastitis in Ethiopian dairy cows that are milked? How does this affect both clinical and subclinical mastitis? The CoCoPop (Condition, Context, and Population) paradigm was applied in order to locate relevant articles. The context was Ethiopia (Co), the condition was staphylococcus infection (Co), and the population was cows (Pop).

The search approach used a range of crucial keywords and Medical Subject Heading (MeSH) terms. At this point, epidemiology, mastitis/infection of the mammary gland, bovine/lactating cows, cross-sectional study, staphylococcus aureus, and staphylococcus infection are the MeSH terms used. Subsequently, pertinent terms and expressions were integrated, and the Boolean operator “AND /OR” was employed to do an online search. The search procedures that were used included Staphylococcus OR Staphylococcus infection OR Staphylococcus aureus AND (occurrence OR prevalence OR infection rate) AND (cows OR dairy cows OR) AND mastitis OR AND Ethiopia. The only language that could be published in was English. All identified studies were imported into End-Note 20 to remove duplicates.

Criteria for Selection of Articles

The following points were important considerations when selecting relevant studies such as all studies should have observational study designs, there should be precise estimations at the quarter- and cow-levels of the percentage of S. aureus infection in mastitis among bacterial pathogens; and studies was selected based on the occurrence of S. aureus in bovine species. All positive cases are chosen for SCM based on the primary clinical indicators and CMT. In the subsequent selection of all positive cases, the specimens that were cultivated, papers dictating S. aureus isolation and identification using conventional bacteriology techniques, the research was published in English, and the study was carried out in Ethiopia, were considered in their review work. Indexed and published papers that met the aforementioned inclusion requirements were chosen. Exclusion criteria included cohort and case control studies, case series, conventional reviews, experimental (clinical) research, and outbreak reports. Lastly, the data extraction process employed these inclusion and exclusion criteria (study screening strategy and reasons for exclusion). (Figure 1).

Figure 1 PRISMA flow chart for selection of included and excluded of studies. Notes: PRISMA figure adapted from Page MJ, McKenzie JE, Bossuyt PM et al The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. BMJ. 2021;372:n71. doi: 10.1136/bmj.n71. Creative Commons.22

Data Extraction Tools

The form contained studies from which M.D. and B.M. separately extracted the following data: the first author’s name, the year the study was conducted, the study design, the study regions, the geographic allocation, the number of animals, the number of CMT positive and culture positive quarters, and the actual prevalence of Staphylococcus aureus infection in cattle mastitis.

Quality Assessment and Evaluation of Study

The AMSTAR-2 (Supplementary file-2) quality evaluation was used in this review to evaluate the research protocols methodological quality and evidence strength in relation to the review question. The quality evaluation of the included studies in this systematic review and meta-analysis is what determines the outcomes (https://drive.google.com/file/d/186it7bH)

Data Synthesis and Statistical Analysis

Based on the papers that were examined, an estimate of the average prevalence of S. aureus in both clinical and subclinical instances of bovine mastitis was made using the SPSS software version 25. To ascertain the average prevalence of S. aureus, a subgroup calculation was carried out, taking into account the degree of mastitis, the “study year” and the study location. Three groups (earlier, middle, and latest) were created for the research year in order to track the evolution of S. aureus prevalence in cows during the course of the study. Furthermore, depending on the pooled papers, research year categories were created, including groups like (≤2012), (2013–2017), and (≥2018).

Results

Search results for Quarter Wise Prevalence Studies

Figure 1 illustrates how 458 articles were read using a variety of extra techniques and electronic resources. Using duplicate removal and eligibility tagging, a total of fifty-six articles were eliminated. After the titles and abstracts were screened, a total of 252 papers were disqualified. 49 of the 150 objects that were reported were retrieved once eligibility was established. In the end, only 26 full-text publications for the synthesis of S. aureus could be found.

Characteristics of Included Studies

Publicly available reports of staphylococcal infection in bovine mastitis in cross-breed, nursing cows from Ethiopia are included in this review. The complete study consists of twenty-six (26) relevant studies that address S. aureus infection in both clinical and subclinical bovine mastitis. All the studies included in this review, with one exception, used a cross-sectional methodology. This review includes studies conducted in different regions of Ethiopia between 2008 and 2022. For every study included in this evaluation, the minimum sample size was 4119, and the largest was 1502.²³

All of the included investigations used standard microbiological techniques to isolate S. aureus. These techniques are detailed by NMC.¹ Furthermore, the usual procedures for bacterial isolation and identification, bacteriological culture, and CMT for subclinical mastitis were used. In order to assess the pooled proportion of S. aureus infection in Ethiopia, 8441 cows total and 10,346 milk samples at the quarter level were used. The apparent prevalence of S. aureus in bovine mastitis ranged from 10 to 66 point six percent. The detailed elements of the investigation are presented in Table 1 and Figure 2. According to Hailemelekot et al (2018), 66% of S. aureus cases were detected in the Amhara area.²⁴ However, according to Balemi et al,¹⁹ the Oromia regional state had the lowest frequency (10%) in 2018.

Figure 2 Prevalence of S. aureus in the respect of the study year. Notes: The horizontal -axis represents the publication year listed while the y-axis is average prevalence of S. aureus.

Table 1 Characteristics of Included Studies (n=26)

The extracted data pertaining to the primary components of the Materials and Methods sections of the reviewed papers is compiled in Table 2. The aspects that were most thoroughly explained were sample size calculations, study designs, ethical issues, and suitable explanations of sampling, environments, and correct laboratory practices (isolation techniques). On the other hand, the farm’s management system, housing arrangement, hygienic methods, and statistical management of the results were all either incompletely explained. In relation to the study animals descriptions (eg g. Risk factors), it was determined that this element lacked clarity.

Table 2 Studies Distributed According to Clarity Scores of the Elements of the Materials and Methods Sections

Geographical Distribution of Bovine Mastitis Associated S. Aureus in Ethiopia

According to Figure 3 and Table 3, the Amhara and Tigray regions had the highest average prevalence of S. aureus (40%), followed by AA (39 point 5%), Oromia (34 point 3%), and SNNPRS (21%). Twelve of the 26 investigations were conducted in the region of Tigray, three in Amhara, four in AA, four in SNNPRS, and three in Oromia.

Figure 3 Geographical distribution of S. aureus in Ethiopia.

Table 3 Mean Prevalence of the S. aureus with Number of Studies in Regions of Ethiopia

Prevalence of S. aureus in Different Study Year Categories

>2018 (43%) was the study year category with the greatest average proportion of S. aureus in mastitis, followed by <2012 (32%), and 2013–2017 (34%). Figure 4 illustrates how S. aureus infections in mastitis became more common over time.

Figure 4 Prevalence of S. aureus in different study year categories in Ethiopia.

The Average Prevalence of S. Aureus in Clinical and Subclinical Mastitis

As depicted in Figure 5, the staphylococcal infection rate in subclinical mastitis was 84%, but it was only 16% in clinical mastitis. Table 4 demonstrates that most research employed the California Mastitis Test to identify subclinical mastitis and clinical observation to identify clinical mastitis. Abebe et al,²¹ discovered that the highest percentage / prevalence (48%) of S. aureus in subclinical mastitis, and Zeryehun and Abera,³⁰ discovered that the prevalence of clinical mastitis was the lowest at 1%.

Figure 5 Average prevalence of S. aureus in clinical and subclinical mastitis in Ethiopia.

Table 4 Average Prevalence of Bovine Mastitis Associated S. Aureus in the Level of Mastitis

Beta Lactam Antimicrobial Resistance Rate of S. aureus

Antibiotics are the sole known treatment for bovine mastitis in Ethiopia. The most often used class of antibiotics is specifically beta-lactams, which include ceftriaxone, cefotaxime, ampicillin, penicillin, and amoxicillin. The percentage of glands that experience bacteriological cure following antimicrobial therapy for clinical mastitis has been observed to vary widely.⁴⁴

The pace of recovery is affected by bacterial factors, such as antibiotic resistance and strain, management variables, and cow-specific factors. These variables also affect the proportion of treatments that are successful.^25,45–47 The existence of plasmids of various sizes has been connected to a number of antimicrobial resistances. It has been discovered that S. aureus possesses two main plasmid types, which may be involved in the pathogenicity and/or antibiotic resistance of the bacteria. Agar disc-diffusion studies, resistance-encoding gene identification, and broth dilution testing are just a few of the methods that can be used to assess antimicrobial resistance.

The beta-lactam antibiotic resistance rate of S. aureus linked to bovine mastitis is the subject of 14 investigations (Table 5) in total (containing ampicillin, cephalosporin resistance, penicillin, and amoxicillin). Most of the included papers offer a comprehensive account of the whole pathogen isolation procedure in cases of both clinical and subclinical bovine mastitis, together with the results of many antibiotic susceptibility testing. A minimum of one S. aureus isolate has been reported from the selected publications, and they ought to have included one beta-lactam antibiotic. There is only one paper that appears more than once in this systematic review.

Table 5 Summary of Various Beta-Lactam Resistance for S. aureus in the Treatment of Bovine Mastitis (n=14)

Among the 14 included trials, the resistance rate of S. aureus to various beta lactam antibiotics ranged from 0% to 100%.

Of the total studies (14), two of those^48,57 found that 100% of the patients had penicillin resistance when treating S. aureus mastitis in cattle. Nonetheless, a study of 52 found that penicillin was 100% successful in getting rid of S. aureus. This study includes cefoxitin, ceftriaxone, and cefotaxime among other cephalosporins. Cefotaxime and cefoxitin showed the highest resistance rates (80%) among the three cephalosporins. When treated with penicillin, S. aureus had the highest average resistance rate (75%) to that of amoxicillin (67%), cephalosporin (57%), and ampicillin (50%), in that order (Figure 6).

Figure 6 Beta lactam antimicrobial resistance rate of S. aureus in the treatment of bovine mastitis.

Discussion

These 26 studies provide an average prevalence of 35.4% (95% confidence interval: 0.31–0.41) among bacterial pathogens, with an emphasis on S. aureus in Ethiopia. Girma et al²⁸ in Ethiopia’s Tigray regional state reported similar results. The overall percentage of S. aureus linked to bovine mastitis was lower than the results of several separate investigations carried out in Ethiopia, including 51% by Abebe et al,²¹ 47% by Fessha.³³ 66% by Hailemelekot,²⁴ and 45% by Birhanu et al.⁵ The total percentage of the pathogen in mastitis in domestic cows, however, was higher than in several of the included studies, among which Tesfaye et al (24%),^27,28 Zerihun et al (21%), and Abebe et al³⁰ reported 21%.²⁰

In cases of both clinical and subclinical mastitis, the sub-total occurrence of S. aureus infection varied depending on the study year: 32% (>2012), 34% (2013–2017), and 43% (> 2018) for each study year group. This systematic review found that the rate of S. aureus infection in bovine mastitis increased when the included publications were categorized based on the study year. It is estimated that approximately 50% of S. aureus-induced mastitis generate β-lactamase, which may be due to increased antibiotic resistance via a variety of mechanisms.⁴⁷ The proliferation of resistant strains in people, animals, and the environment may be related to this. Contributing causes could also include Ethiopia’s restricted access to therapeutic treatment and the underuse of antibiotics in veterinary medicine. The development of S. aureus biofilms in cow mastitis is likewise associated with a reduction in antibiotic sensitivity. This is explained by a decrease in the metabolic activity of the bacteria within biofilms and a reduction in the amount of antibiotics that pass through the biofilm matrix.⁵⁸ There are other circumstances as well, like sub-inhibitory antibiotic concentrations. Moreover, lactose and proteases found in milk promote the growth of S. aureus biofilms inside the mammary gland of cows.^59,60

The Amhara Region had the highest percentage of S. aureus infections (40.2%) among the study areas, while the SNNPRS region had the lowest (21%). This may have to do with the average annual relative humidity, the cleanliness of the farming system, and the prudent use of antibiotics.

Moreover, the production and consumption of raw milk and other dairy products often take place in unhygienic conditions in Ethiopia.⁶¹ Consequently, there is an extremely high chance of coming into contact with S. aureus as a result of consuming dairy products.⁶² Research conducted in Ethiopia has demonstrated that S. aureus is present in milk at several points throughout the milk value chain. This phenomenon may be explained by contamination from mastitis-affected cows, cross-infection of milk at collection sites from contaminated milk from infected farms, improper handling practices, and use of unclean equipment.⁶³

Within the global context, this particular study found that the mean incidence of S. aureus-associated bovine mastitis (35.4%) was almost identical to the percentage found in a study conducted in China (36.23%) by Wang et al⁶⁴ The estimated percentages for the US, Kenya, and Canada are as follows: 20.8–23.3% in the US, 20–22% in Canada, and 15.7% in Kenya.⁶⁵ However, the result was lower than Elhaig and Selim’s claimed 56.7%, with⁶⁶ in Nigeria.

During milking, S. aureus spreads among cows; thus, it is necessary to work together to minimize the spread of the infection to healthy animals. S. aureus isolates from cows are also a major source of foodborne illnesses, and raw milk and bulk tank products are crucial conduits for the spread of infection to people. Researchers in Ethiopia have shown that adult milkers between the ages of 30 and 40 had higher prevalence of S. aureus on their hands. These factors included inadequate udder cleaning, not washing hands before milking, using a shared towel, not dipping the teat after milking, routinely disinfecting the milking area, and lacking knowledge about the contagious nature of S. aureus and MRSA. Additionally, farms with semi-intensive management systems, inadequate barn drainage systems, and cows who had previously been exposed to mastitis showed greater prevalence of the organism.⁶⁷

This systematic study revealed that, in relation to the degree of mastitis, the average occurrence of S. aureus infection was higher in subclinical mastitis (26%) than in clinical mastitis in cows (5%). As a result, our finding demonstrated that S. aureus infection has a significant economic impact on Ethiopia’s dairy industry, as subclinical mastitis is the main obstacle facing the industry in poor nations.⁶⁸ Due to decreased milk quality and production, S. aureus is a very opportunistic infection that usually co-occurs with subclinical mastitis and results in large economic losses.⁶⁹ It has been demonstrated that intramuscular infection by CC97 strains in nursing cows can lead to asymptomatic, subclinical, or chronic infections, making pathogen control in dairy herds more challenging.⁷⁰

Despite significant variance in the overall rate of resistance among different types of antimicrobial drugs, S. aureus resistance to these agents poses a significant concern. According to the epidemiological estimate, 68% of Ethiopians were resistant to beta-lactam antibiotics overall. This rate is quite high, and may be caused by the production of beta-lactamase by S. aureus, which degrades the beta-lactam ring in the drug. Another possible reason could be a changed PBP with reduced affinity for most beta-lactam antibiotics.

S. aureus had a 75% beta-lactam resistance rate to penicillin, a 67% rate to amoxicillin, a 57% rate to cephalosporin, and a 50% rate to ampicillin. The penicillin resistance to S. aureus was the highest. This might be because penicillin is widely available and utilized. Even non-professionals (farmers, drug dealers, and animal owners) possess penicillin, which they self-administer without understanding the proper dosage or timing.⁷¹ Compared to other beta lactam medicines, S. aureus had the highest resistance to penicillin in the current investigation. As reported by Gianneechini et al,⁷² André et al,⁷³ Turutoglu et al,⁷⁴ Kalmus et al,⁷⁵ Sakwinska et al,⁷⁶ Peles et al,⁷⁷ Jørgensen et al,⁷⁸ Anderson et al,⁷⁸ and Tenhagen et al,⁷⁹ respectively, this percentage was higher than the values reported in Argentina (47.6%), Brazil (69.9%), Turkey (62%), Estonia (61%), France (30%), Hungary (30%), Germany (17%), the United States (10%), and Norway (6%), respectively. However, the percentage of resistance exhibited a lesser magnitude in comparison to the values that have been previously documented in, Tigray (90%),⁸⁰ Addis Ababa (95.3%),⁸¹ Hawassa (100%),⁸² Iran (100%),⁸³ India (74.7%)⁸⁴ and 80% in Sweden.⁸⁵

The results of the systematic review show that S. aureus had a 50% mean rate of ampicillin resistance. Our results, which are somewhat inferior to those of prior Ethiopian research on ampicillin resistance in Tigray (100%) and Hawassa (70.9%),⁸⁶ it was shown that S. aureus had the lowest level of ampicillin resistance (50%), in this particular investigation. However, it has been noted that ampicillin resistance may increase to 76.1–89.7% in Japan.⁸⁷ The majority of S. aureus isolates from bovine mastitis in Nepal showed total resistance (100%) to ampicillin (73.2%), whereas in Uganda (73.2%), India (74.42%), and Nepal (20.5%) showed resistance to ampicillin.⁸⁸

In order to prevent antibiotic resistance and minimize the use of antibiotics, society must be empowered through education. The aforementioned therapies are required under the current standard guidelines for the use of antibiotics. The objective areas also include continuing professional development and pre- and in-service education for health professionals. The public and society have been empowered through the dissemination of mass media information regarding the usage of antibiotics and resistance. The system’s ability to manage the threats that AMR has introduced is still lacking.

Thus, continuous intervention for scaling up is necessary for both AMR containment and prevention. Empowerment and education remain crucial strategies for stopping and containing MRSA. MRSA containment and prevention require a coordinated and integrated approach at the global, institutional, national, and individual levels. A comprehensive, evidence-based response approach is necessary due to the multidimensional character of MRSA, which encompasses biological, behavioral, technical, economic, regulatory, and educational components. Preventive and control measures must be put into place in healthcare facilities as well as the general public in order to lessen the danger of infection and the need for antibiotics. This will help to stop the emergence of resistant strains, which need to be quickly found and contained to stop them from spreading to other areas. In order to effectively address the challenges posed by MRSA, foster stewardship practices, and drive innovation, collaboration among all sectors is important.

Conclusion

The thorough investigation’s findings demonstrate the widespread distribution of S. aureus, with particularly high sub-group average prevalence in the Amhara and Tigray regions. Furthermore, a rise in S. aureus prevalence in recent years has been shown by sub-group calculation depending on study year. The incidence of S. aureus in subclinical mastitis in dairy cows is five times higher than in clinical mastitis. In general, S. aureus exhibits a high rate of beta lactamase resistance; however, in cases of mastitis in local breed cattle, the rate of resistance to Penicillin is highest. The relevance for the economy and public health is shown by the prevalence of S. aureus in subclinical mastitis. Veterinarians, medical professionals, veterinary pharmacists, farm laborers, milkers, and consumers of raw milk must thus make it a top priority to control and prevent S. aureus in bovine mastitis while also lowering the incidence of beta-lactam resistance.