Confronting the complexities of antimicrobial management for Staphyloccous aureus causing bovine mastitis: an innovative paradigm.

Globally, Mastitis is a disease commonly affecting dairy cattle which leads to the use of antimicrobials. The majority of mastitis etiological agents are bacterial pathogens and Staphylococcus aureus is the predominant causative agent. Antimicrobial treatment is administered mainly via intramammary and intramuscular routes. Due to increasing antimicrobial resistance (AMR) often associated with antimicrobial misuse, the treatment of mastitis is becoming challenging with less alternative treatment options. Besides, biofilms formation and ability of mastitis-causing bacteria to enter and adhere within the cells of the mammary epithelium complicate the treatment of bovine mastitis. In this review article, we address the challenges in treating mastitis through conventional antibiotic treatment because of the rising AMR, biofilms formation, and the intracellular survival of bacteria. This review article describes different alternative treatments including phytochemical compounds, antimicrobial peptides (AMPs), phage therapy, and Graphene Nanomaterial-Based Therapy that can potentially be further developed to complement existing antimicrobial therapy and overcome the growing threat of AMR in etiologies of mastitis.

Phenotypic and genotypic characterization of colistin-resistant Escherichia Coli with mcr-4, mcr-5, mcr-6, and mcr-9 genes from broiler chicken and farm environment.

BACKGROUND: Colistin is an antibiotic used as a last-resort to treat multidrug-resistant Gram-negative bacterial infections. Colistin had been used for a long time in veterinary medicine for disease control and as a growth promoter in food-producing animals. This excessive use of colistin in food animals causes an increase in colistin resistance. This study aimed to determine molecular characteristics of colistin-resistant Escherichia coli in broiler chicken and chicken farm environments. RESULTS: Four hundred fifty-three cloacal and farm environment samples were collected from six different commercial chicken farms in Kelantan, Malaysia. E. coli was isolated using standard bacteriological methods, and the isolates were tested for antimicrobial susceptibility using disc diffusion and colistin minimum inhibitory concentration (MIC) by broth microdilution. Multiplex PCR was used to detect mcr genes, and DNA sequencing was used to confirm the resistance genes. Virulence gene detection, phylogroup, and multilocus sequence typing (MLST) were done to further characterize the E. coli isolates. Out of the 425 (94%; 425/453) E. coli isolated from the chicken and farm environment samples, 10.8% (48/425) isolates were carrying one or more colistin-resistance encoding genes. Of the 48 colistin-resistant isolates, 54.2% (26/48) of the mcr positive isolates were genotypically and phenotypically resistant to colistin with MIC of colistin ≥ 4 µg/ml. The most prominent mcr gene detected was mcr-1 (47.9%; 23/48), followed by mcr-8 (18.8%; 9/48), mcr-7 (14.5%; 7/48), mcr-6 (12.5%; 6/48), mcr-4 (2.1%; 1/48), mcr-5 (2.1%; 1/48), and mcr-9 (2.1%; 1/48) genes. One E. coli isolate originating from the fecal sample was found to harbor both mcr-4 and mcr-6 genes and another isolate from the drinking water sample was carrying mcr-1 and mcr-8 genes. The majority of the mcr positive isolates were categorized under phylogroup A followed by phylogroup B1. The most prevalent sequence typing (ST) was ST1771 (n = 4) followed by ST206 (n = 3). 100% of the mcr positive E. coli isolates were multidrug resistant. The most frequently detected virulence genes among mcr positive E. coli isolates were ast (38%; 18/48) followed by iss (23%; 11/48). This is the first research to report the prevalence of mcr-4, mcr-5, mcr-6, mcr-7, and mcr-8 genes in E. coli from broiler chickens and farm environments in Malaysia. CONCLUSION: Our findings suggest that broiler chickens and broiler farm environments could be reservoirs of colistin-resistant E. coli, posing a risk to public health and food safety.

Antimicrobial activities of graphene oxide against biofilm and intracellular Staphylococcus aureus isolated from bovine mastitis.

BACKGROUND: S. aureus is one of the causative agents of bovine mastitis. The treatment using conventional antimicrobials has been hampered due to the development of antimicrobial resistance and the ability of the bacteria to form biofilms and localize inside the host cells. OBJECTIVES: Here, the efficacy of graphene oxide (GO), a carbon-based nanomaterial, was tested against the biofilms and intracellular S. aureus invitro. Following that, the mechanism for the intracellular antimicrobial activities and GO toxicities was elucidated. METHODS: GO antibiofilm properties were evaluated based on the disruption of biofilm structure, and the intracellular antimicrobial activities were determined by the survival of S. aureus in infected bovine mammary cells following GO exposure. The mechanism for GO intracellular antimicrobial activities was investigated using endocytosis inhibitors. GO toxicity towards the host cells was assessed using a resazurin assay. RESULTS: At 100 ug/mL, GO reduced between 30 and 70% of S. aureus biofilm mass, suggesting GO's ability to disrupt the biofilm structure. At 200 ug/mL, GO killed almost 80% of intracellular S. aureus, and the antimicrobial activities were inhibited when cells were pre-treated with cytochalasin D, suggesting GO intracellular antimicrobial activities were dependent on the actin-polymerization of the cell membrane. At < 250 ug/mL, GO enhanced the viability of the Mac-T cell, and cells were only affected at higher dosages. CONCLUSION: The in vitro efficacy of GO against S. aureus in vitro suggested the compound could be further tested in Vivo to zrecognize its potential as one of the components of bovine mastitis therapy.

Prevalence, Antimicrobial Resistance, and Characterization of Staphylococcus aureus Isolated from Subclinical Bovine Mastitis in East Coast Malaysia.

S. aureus is the pathogen that is commonly associated with subclinical mastitis, causing significant economic losses to dairy farms. This infection responds poorly to antimicrobial treatment, which could be due to the development of AMR, biofilm formation, and the intracellular invasion of S. aureus into bovine mammary cells leading to treatment failure. Thus, it is important to understand the challenge of this problem. Therefore, the present study aims to determine the prevalence, antimicrobial resistance, and characterization of S. aureus that was isolated from subclinical bovine mastitis in East Coast Malaysia. A total of 235 milk samples from dairy cows were collected from selected farms in Kelantan and Pahang. The samples were subjected to a somatic cell analysis to identify subclinical mastitis, followed by bacteria isolation and antimicrobial susceptibility testing. The isolated S. aureus were further analyzed for their ability to form biofilms and invade the bovine mammary epithelial cells (MAC-T cells) in in vitro infections modeling using a gentamicin protection assay. The overall total of 74/235 (31.4%; 95% CI = 0.31; 0.32) of the milk samples demonstrated >200,000 somatic cells/mL, suggesting the presence of subclinical mastitis in the animals. A total of 39/235 (16.5%; 95% CI = 0.16, 0.17) of the milk samples harbored S. aureus which demonstrated resistance towards the following antimicrobials: penicillin (18/39, 46%), ampicillin (17/39, 43.6%), oxacillin (12/39, 31%), tetracycline (10/39, 26%), and erythromycin (7/39, 18%). AMR was recorded for a total of (17/39, 43.6%) of S. aureus isolates. All isolates formed biofilms, with (8/30, 27%) strongly biofilm-forming, (18/30, 60%) moderately biofilm-forming, and the remaining (4/30, 13%) of isolates weakly biofilm-forming. Interestingly, the AMR isolates appear to produce weak and moderate biofilm. Moreover, (6/20, 30%) of the S. aureus isolates were invasive towards MAC-T cells, as indicated by their ability to evade gentamicin treatment. The study demonstrated the presence of AMR, invasiveness, and biofilm formation in S. aureus that was isolated from subclinical mastitis. This characteristic presents additional challenges to existing antimicrobial therapy.

Phylogenetically Diverse Escherichia coli Strains from Chicken Coharbor Multiple Carbapenemase-Encoding Genes (bla NDM -bla OXA-blaIMP).

Carbapenem-resistant Enterobacteriaceae (CRE) has been a public health risk in several countries, and recent reports indicate the emergence of CRE in food animals. This study was conducted to investigate the occurrence, resistance patterns, and phylogenetic diversity of carbapenem-resistant E. coli (CREC) from chicken. Routine bacteriology, PCR detection of E. coli species, multiplex PCR to detect carbapenemase-encoding genes, and phylogeny of CRE E. coli were conducted. The results show that 24.36% (19/78) were identified as CREC based on the phenotypic identifications of which 17 were positive for the tested carbapenemases genes. The majority, 57.99% (11/19), of the isolates harbored multiple carbapenemase genes. Four isolates harbored all bla NDM, bla OXA, and bla IMP, and five and two different isolates harbored bla NDM and bla OXA and bla OXA and bla IMP, respectively. The meropenem, imipenem, and ertapenem MIC values for the isolates ranged from 2 µg/mL to ≥256 µg/mL. Phylogenetic grouping showed that the CREC isolates belonged to five different groups: groups A, B1, C, D, and unknown. The detection of CREC in this study shows that it has become an emerging problem in farm animals, particularly, in poultry farms. This also implies the potential public health risks posed by CRE from chicken to the consumers.

Antibacterial Activity of Ikarugamycin against Intracellular Staphylococcus aureus in Bovine Mammary Epithelial Cells In Vitro Infection Model.

Staphylococcus aureus is an ubiquitous and versatile pathogen associated with a wide range of diseases. In animals, this bacterium is one of the causative agents of bovine mastitis, responsible for huge economic losses in the dairy industry. Besides the development of antibiotic resistance, the intracellular survival of S. aureus within udder cells has rendered many antibiotics ineffective, leading to therapeutic failure. Our study therefore aims to investigate the in vitro bactericidal activity of ikarugamycin (IKA) against intracellular S. aureus using a bovine mammary epithelial cells (Mac-T cells) infection model and determine the cytotoxic effect. Minimum inhibitory concentration (MIC) was used to determine the antibacterial activity of IKA, and Mac-T cells were infected with S. aureus using gentamicin protection assay. IKA intracellular antibacterial activity assays were used to determine the bactericidal activity of IKA against intracellular S. aureus. The cytotoxicity of IKA against Mac-T cells was evaluated using the resazurin assay. We showed that, S. aureus is susceptible to IKA with a MIC value of 0.6 µg/mL. IKA at 4 × MIC and 8 × MIC have bactericidal activity by reducing 3 and 5 logs10 CFU/mL of S. aureus in the first six-hour of treatment respectively. In addition, IKA demonstrated intracellular killing activity by killing 90% of intracellular S. aureus at 5 µg/mL. This level is comparatively lower than 9.2 µg/mL determined as the half-maximal inhibitory concentration (IC50) of IKA required to kill 50% of Mac-T cells, highlighting a lower concentration required for bactericidal effect compared to the cytotoxic effect. The study highlighted that importance of IKA as a potential antibiotic candidate to be explored for the in vivo efficacy in treating S. aureus mastitis.

Prevalence of Antimicrobial Resistance (AMR) Salmonella spp. and Escherichia coli Isolated from Broilers in the East Coast of Peninsular Malaysia.

Salmonella species (spp.) and Escherichia coli (E. coli) are the most common infectious pathogens in poultry. Antimicrobials are given either as growth promoters or as treatment, thereby increasing the possibility of the emergence of antimicrobial resistance (AMR). We determined the prevalence of AMR for both pathogens isolated from broiler farms in the East Coast of Peninsular Malaysia from 2018-2019. A total of 384 cloacal swabs were collected, followed by bacterial isolation, confirmation, and antimicrobial susceptibility tests. The overall prevalence of Salmonella spp. and E. coli were 6.5% and 51.8%, respectively. Salmonella spp. and E. coli displayed resistance towards the following antimicrobials: erythromycin (100% for both pathogens), chloramphenicol (76.2% and 84.5%, respectively), tetracycline (62% and 94.6%, respectively), ampicillin (47.7% and 87%, respectively), sulfamethoxazole/trimethoprim (42.9% and 83.3%, respectively), ciprofloxacin (4.8% and 23.8%, respectively), nalidixic acid (9.6% and 60.7%, respectively), streptomycin (19% and 66%, respectively), kanamycin (28.6% and 57%, respectively), cephalothin (0% and 11%, respectively), and gentamicin (0% and 20.2%, respectively). Multidrug resistance (MDR) was recorded in 82% of Salmonella spp. and 100% of E. coli. These findings demonstrate the high prevalence of AMR in both pathogens in broiler farms on the East Coast of Peninsular Malaysia. These findings could be attributed to the excessive use of antimicrobial agents by poultry farm owners. Enhanced control measures and a strong monitoring system should be urgently implemented in order to reduce the emergence of antimicrobial resistance.

Antimicrobial Polymers: The Potential Replacement of Existing Antibiotics?

Antimicrobial resistance is now considered a major global challenge; compromising medical advancements and our ability to treat infectious disease. Increased antimicrobial resistance has resulted in increased morbidity and mortality due to infectious diseases worldwide. The lack of discovery of novel compounds from natural products or new classes of antimicrobials, encouraged us to recycle discontinued antimicrobials that were previously removed from routine use due to their toxicity, e.g., colistin. Since the discovery of new classes of compounds is extremely expensive and has very little success, one strategy to overcome this issue could be the application of synthetic compounds that possess antimicrobial activities. Polymers with innate antimicrobial properties or that have the ability to be conjugated with other antimicrobial compounds create the possibility for replacement of antimicrobials either for the direct application as medicine or implanted on medical devices to control infection. Here, we provide the latest update on research related to antimicrobial polymers in the context of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) pathogens. We summarise polymer subgroups: compounds containing natural peptides, halogens, phosphor and sulfo derivatives and phenol and benzoic derivatives, organometalic polymers, metal nanoparticles incorporated into polymeric carriers, dendrimers and polymer-based guanidine. We intend to enhance understanding in the field and promote further work on the development of polymer based antimicrobial compounds.

Targeting the Bacterial Protective Armour; Challenges and Novel Strategies in the Treatment of Microbial Biofilm.

Infectious disease caused by pathogenic bacteria continues to be the primary challenge to humanity. Antimicrobial resistance and microbial biofilm formation in part, lead to treatment failures. The formation of biofilms by nosocomial pathogens such as Staphylococcus aureus (S. aureus), Pseudomonas aeruginosa (P. aeruginosa), and Klebsiella pneumoniae (K. pneumoniae) on medical devices and on the surfaces of infected sites bring additional hurdles to existing therapies. In this review, we discuss the challenges encountered by conventional treatment strategies in the clinic. We also provide updates on current on-going research related to the development of novel anti-biofilm technologies. We intend for this review to provide understanding to readers on the current problem in health-care settings and propose new ideas for new intervention strategies to reduce the burden related to microbial infections.

Polyhexamethylene Biguanide and Nadifloxacin Self-Assembled Nanoparticles: Antimicrobial Effects against Intracellular Methicillin-Resistant Staphylococcus aureus.

The treatment of skin and soft tissue infections caused by methicillin-resistant Staphylococcus aureus (MRSA) remains a challenge, partly due to localization of the bacteria inside the host's cells, where antimicrobial penetration and efficacy is limited. We formulated the cationic polymer polyhexamethylene biguanide (PHMB) with the topical antibiotic nadifloxacin and tested the activities against intracellular MRSA in infected keratinocytes. The PHMB/nadifloxacin nanoparticles displayed a size of 291.3 ± 89.6 nm, polydispersity index of 0.35 ± 0.04, zeta potential of +20.2 ± 4.8 mV, and drug encapsulation efficiency of 58.25 ± 3.4%. The nanoparticles killed intracellular MRSA, and relative to free polymer or drugs used separately or together, the nanoparticles displayed reduced toxicity and improved host cell recovery. Together, these findings show that PHMB/nadifloxacin nanoparticles are effective against intracellular bacteria and could be further developed for the treatment of skin and soft tissue infections.

Targeting the hard to reach: challenges and novel strategies in the treatment of intracellular bacterial infections.

Infectious diseases continue to threaten human and animal health and welfare globally, impacting millions of lives and causing substantial economic loss. The use of antibacterials has been only partially successful in reducing disease impact. Bacterial cells are inherently resilient, and the therapy challenge is increased by the development of antibacterial resistance, the formation of biofilms and the ability of certain clinically important pathogens to invade and localize within host cells. Invasion into host cells provides protection from both antibacterials and the host immune system. Poor delivery of antibacterials into host cells causes inadequate bacterial clearance, resulting in chronic and unresolved infections. In this review, we discuss the challenges associated with existing antibacterial therapies with a focus on intracellular pathogens. We consider the requirements for successful treatment of intracellular infections and novel platforms currently under development. Finally, we discuss novel strategies to improve drug penetration into host cells. As an example, we discuss our recent demonstration that the cell penetrating cationic polymer polyhexamethylene biguanide has antibacterial activity against intracellular Staphylococcus aureus. LINKED ARTICLES: This article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro-organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc.

The antimicrobial polymer PHMB enters cells and selectively condenses bacterial chromosomes.

To combat infection and antimicrobial resistance, it is helpful to elucidate drug mechanism(s) of action. Here we examined how the widely used antimicrobial polyhexamethylene biguanide (PHMB) kills bacteria selectively over host cells. Contrary to the accepted model of microbial membrane disruption by PHMB, we observed cell entry into a range of bacterial species, and treated bacteria displayed cell division arrest and chromosome condensation, suggesting DNA binding as an alternative antimicrobial mechanism. A DNA-level mechanism was confirmed by observations that PHMB formed nanoparticles when mixed with isolated bacterial chromosomal DNA and its effects on growth were suppressed by pairwise combination with the DNA binding ligand Hoechst 33258. PHMB also entered mammalian cells, but was trapped within endosomes and excluded from nuclei. Therefore, PHMB displays differential access to bacterial and mammalian cellular DNA and selectively binds and condenses bacterial chromosomes. Because acquired resistance to PHMB has not been reported, selective chromosome condensation provides an unanticipated paradigm for antimicrobial action that may not succumb to resistance.

Bactericidal effects of polyhexamethylene biguanide against intracellular Staphylococcus aureus EMRSA-15 and USA 300.

OBJECTIVES: The treatment of skin infections caused by Staphylococcus aureus is limited by acquired antibiotic resistance and poor drug delivery into pathogen and host cells. Here, we investigated the antibacterial activities of six topically used antimicrobials and a cationic polymer, polyhexamethylene biguanide (PHMB), against intracellular MSSA strain RN4420 and MRSA strains EMRSA-15 and USA 300. METHODS: The MICs of antimicrobials were determined for MSSA and MRSA strains, and the bactericidal activities of nadifloxacin and PHMB against intracellular MRSA were determined using infected keratinocytes. Fluorescein-tagged PHMB (PHMB-FITC) was used to study PHMB uptake, co-localization with intracellular EMRSA-15 and retention in keratinocytes. The mechanism(s) of PHMB uptake into keratinocytes were studied using a dynamin inhibitor, dynasore. RESULTS: Gentamicin, nadifloxacin and PHMB showed the lowest MICs for MRSA. Nadifloxacin at 10 mg/L killed 80% of intracellular EMRSA-15, but was not effective against USA 300. PHMB at 4 mg/L killed almost 100% of intracellular EMRSA-15 and USA 300. PHMB entered keratinocytes, co-localized with intracellular EMRSA-15 and was retained by the cells for over 5 h. PHMB uptake and its intracellular antibacterial activities were inhibited by the dynamin inhibitor, dynasore. CONCLUSIONS: PHMB kills intracellular MRSA via direct interaction with pathogens inside keratinocytes and host cell entry is dynamin dependent.

Pathogen- and Host-Directed Antileishmanial Effects Mediated by Polyhexanide (PHMB).

BACKGROUND: Cutaneous leishmaniasis (CL) is a neglected tropical disease caused by protozoan parasites of the genus Leishmania. CL causes enormous suffering in many countries worldwide. There is no licensed vaccine against CL, and the chemotherapy options show limited efficacy and high toxicity. Localization of the parasites inside host cells is a barrier to most standard chemo- and immune-based interventions. Hence, novel drugs, which are safe, effective and readily accessible to third-world countries and/or drug delivery technologies for effective CL treatments are desperately needed. METHODOLOGY/PRINCIPAL FINDINGS: Here we evaluated the antileishmanial properties and delivery potential of polyhexamethylene biguanide (PHMB; polyhexanide), a widely used antimicrobial and wound antiseptic, in the Leishmania model. PHMB showed an inherent antileishmanial activity at submicromolar concentrations. Our data revealed that PHMB kills Leishmania major (L. major) via a dual mechanism involving disruption of membrane integrity and selective chromosome condensation and damage. PHMB's DNA binding and host cell entry properties were further exploited to improve the delivery and immunomodulatory activities of unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG ODN). PHMB spontaneously bound CpG ODN, forming stable nanopolyplexes that enhanced uptake of CpG ODN, potentiated antimicrobial killing and reduced host cell toxicity of PHMB. CONCLUSIONS: Given its low cost and long history of safe topical use, PHMB holds promise as a drug for CL therapy and delivery vehicle for nucleic acid immunomodulators.