Effect of Local Administration of Meglumine Antimoniate and Polyhexamethylene Biguanide Alone or in Combination with a Toll-like Receptor 4 Agonist for the Treatment of Papular Dermatitis due to Leishmania infantum in Dogs.

Papular dermatitis is a cutaneous manifestation of canine Leishmania infantum infection associated with mild disease. Although it is a typical presentation, nowadays, there is still no established treatment. This study evaluated the safety and clinical efficacy of local meglumine antimoniate, locally administered polyhexamethylene biguanide (PHMB) alone or PHMB in combination with a Toll-like receptor 4 agonist (TLR4a) for the treatment of papular dermatitis due to L. infantum and assessed parasitological and immunological markers in this disease. Twenty-eight dogs with papular dermatitis were divided randomly into four different groups; three of them were considered treatment groups: PHMB (n = 5), PHMB + TLR4a (n = 4), and meglumine antimoniate (n = 10)), and the remaining were considered the placebo group (n = 9), which was further subdivided into two sub-groups: diluent (n = 5) and TLR4a (n = 4). Dogs were treated locally every 12 h for four weeks. Compared to placebo, local administration of PHMB (alone or with TLR4a) showed a higher tendency towards resolution of papular dermatitis due to L. infantum infection at day 15 (χ2 = 5.78; df = 2, p = 0.06) and day 30 (χ2 = 4.; df = 2, p = 0.12), while local meglumine antimoniate administration demonstrated the fastest clinical resolution after 15 (χ2 = 12.58; df = 2, p = 0.002) and 30 days post-treatment (χ2 = 9.47; df = 2, p = 0.009). Meglumine antimoniate showed a higher tendency towards resolution at day 30 when compared with PHMB (alone or with TLR4a) (χ2 = 4.74; df = 2, p = 0.09). In conclusion, the local administration of meglumine antimoniate appears to be safe and clinically efficient for the treatment of canine papular dermatitis due to L. infantum infection.

FtsE, the Nucleotide Binding Domain of the ABC Transporter Homolog FtsEX, Regulates Septal PG Synthesis in E. coli.

The peptidoglycan (PG) layer, a crucial component of the tripartite E.coli envelope, is required to maintain cellular integrity, protecting the cells from mechanical stress resulting from intracellular turgor pressure. Thus, coordinating synthesis and hydrolysis of PG during cell division (septal PG) is crucial for bacteria. The FtsEX complex directs septal PG hydrolysis through the activation of amidases; however, the mechanism and regulation of septal PG synthesis are unclear. In addition, how septal PG synthesis and hydrolysis are coordinated has remained unclear. Here, we have shown that overexpression of FtsE leads to a mid-cell bulging phenotype in E.coli, which is different from the filamentous phenotype observed during overexpression of other cell division proteins. Silencing of the common PG synthesis genes murA and murB reduced bulging, confirming that this phenotype is due to excess PG synthesis. We further demonstrated that septal PG synthesis is independent of FtsE ATPase activity and FtsX. These observations and previous results suggest that FtsEX plays a role during septal PG hydrolysis, whereas FtsE alone coordinates septal PG synthesis. Overall, our study findings support a model in which FtsE plays a role in coordinating septal PG synthesis with bacterial cell division. IMPORTANCE The peptidoglycan (PG) layer is an essential component of the E.coli envelope that is required to maintain cellular shape and integrity. Thus, coordinating PG synthesis and hydrolysis at the mid-cell (septal PG) is crucial during bacterial division. The FtsEX complex directs septal PG hydrolysis through the activation of amidases; however, its role in regulation of septal PG synthesis is unclear. Here, we demonstrate that overexpression of FtsE in E.coli leads to a mid-cell bulging phenotype due to excess PG synthesis. This phenotype was reduced upon silencing of common PG synthesis genes murA and murB. We further demonstrated that septal PG synthesis is independent of FtsE ATPase activity and FtsX. These observations suggest that the FtsEX complex plays a role during septal PG hydrolysis, whereas FtsE alone coordinates septal PG synthesis. Our study indicates that FtsE plays a role in coordinating septal PG synthesis with bacterial cell division.

Fungal cell barriers and organelles are disrupted by polyhexamethylene biguanide (PHMB).

The similarities between fungal and mammalian cells pose inherent challenges for the development of treatments for fungal infections, due to drug crossover recognition of host drug targets by antifungal agents. Thus, there are a limited number of drug classes available for treatment. Treatment is further limited by the acquisition and dissemination of antifungal resistance which contributes to the urgent need of new therapies. Polyhexamethylene biguanide (PHMB) is a cationic antimicrobial polymer with bactericidal, parasiticidal and fungicidal activities. The antifungal mechanism of action appears to involve preferential mechanical disruption of microbial cell structures, offering an alternative to conventional antifungals. However, the antifungal mechanisms have been little studied. The aim of this study was to characterise PHMB's activities on selected yeast (Saccharomyces cerevisiae, Candida albicans) and filamentous fungal species (Fusarium oxysporum, Penicillium glabrum). Fungal membrane disruption, cell entry and intracellular localisation activities of PHMB were evaluated using viability probe entry and polymer localisation studies. We observed that PHMB initially permeabilises fungal cell membranes and then accumulates within the cytosol. Once in the cytosol, it disrupts the nuclear membrane, leading to DNA binding and fragmentation. The electrostatic interaction of PHMB with membranes suggests other intracellular organelles could be potential targets of its action. Overall, the results indicate multiple antifungal mechanisms, which may help to explain its broad-spectrum efficacy. A better understanding of PHMB's mechanism(s) of action may aid the development of improved antifungal treatment strategies.

Antimicrobial activities of polyhexamethylene biguanide against biofilm-producing Prototheca bovis causing bovine mastitis.

Prototheca spp. is a frequent cause of bovine mastitis and is highly resistant to commonly used disinfectants. This study aimed to: (1) evaluate the antimicrobial activity of polyhexamethylene biguanide (PHMB) against mastitis-causing Prototheca spp., and (2) evaluate the biofilm production ability of Prototheca spp. A total of 85 Prototheca bovis and 2 Prototheca blaschkeae isolates from bovine mastitis cases were submitted to biofilm production assays and antimicrobial susceptibility tests against PHMB and disinfectants commonly used in dairy herds (chlorhexidine digluconate, povidone-iodine, sodium dichloroisocyanurate, and sodium hypochlorite). The minimal inhibitory concentration (MIC) and minimal algicidal concentration (MAC) were determined by microdilution assays. We observed that PHMB (MIC90: ≥2 µg/mL and MAC90: ≥4 µg/mL) and chlorhexidine gluconate (MIC90 and MAC90: ≥2 µg/mL) presented the highest antimicrobial activity against P. bovis isolates, followed by sodium dichloroisocyanurate (MIC90 and MAC90: ≥1,400 µg/mL), sodium hypochlorite (MIC90 and MAC90: ≥2,800 µg/mL), and povidone-iodine (MIC90 and MAC90: ≥3,200 µg/mL). Concerning P. blaschkeae isolates, PHMB (MIC and MAC ≥1 µg/mL) and chlorhexidine gluconate (MIC and MAC ≥1 µg/mL) were the disinfectants that presented the lowest concentration values required to inhibit the isolates. Regarding biofilms formation, 63.5% (n = 54/85) of the P. bovis isolates were classified as strong, 28.3% (n = 24/85) moderate, and 8.2% (n = 7/85) weak biofilm producers. In contrast, the P. blaschkeae isolates were classified as weak and moderate biofilm producers. These findings suggest that PHMB has the potential to be used for teat and milking-equipment disinfection for the prevention of mastitis-causing Prototheca spp. in dairy herds.

The importance of inflammation control for the treatment of chronic diabetic wounds.

Diabetic chronic wounds cause massive levels of patient suffering and economic problems worldwide. The state of chronic inflammation arises in response to a complex combination of diabetes mellitus-related pathophysiologies. Advanced treatment options are available; however, many wounds still fail to heal, exacerbating morbidity and mortality. This review describes the chronic inflammation pathophysiologies in diabetic ulcers and treatment options that may help address this dysfunction either directly or indirectly. We suggest that treatments to reduce inflammation within these complex wounds may help trigger healing.

Efficacy of natural antimicrobial peptides versus peptidomimetic analogues: a systematic review.

Aims: This systematic review was carried out to determine whether synthetic peptidomimetics exhibit significant advantages over antimicrobial peptides in terms of in vitro potency. Structural features - molecular weight, charge and length - were examined for correlations with activity. Methods: Original research articles reporting minimum inhibitory concentration  values against Escherichia coli, indexed until 31 December 2020, were searched in PubMed/ScienceDirect/Google Scholar and evaluated using mixed-effects models. Results: In vitro antimicrobial activity of peptidomimetics resembled that of antimicrobial peptides. Net charge significantly affected minimum inhibitory concentration values (p < 0.001) with a trend of 4.6% decrease for increments in charge by +1. Conclusion: AMPs and antibacterial peptidomimetics exhibit similar potencies, providing an opportunity to exploit the advantageous stability and bioavailability typically associated with peptidomimetics.

Antifungal Polymeric Materials and Nanocomposites.

Rising global populations due to medicinal advancements increases the patient population susceptible to superficial and severe fungal infections. Fungi often implicated in these diseases includes the dermatophytes (Microsporum spp., Epidermophtyon spp., Trichophyton spp.) as well as species of the Candida spp., Aspergillosis spp. and Cryptococcus spp. genera. In addition, increasing global populations leads to increasing agricultural demands. Thus, fungal infections of preharvested crops and stored food by plant pathogens such as Magnaporthe oryzae and Fusarium oxysporum can have detrimental socioeconomic effects due to food insecurity. Current antifungal strategies are based mainly on small molecule antifungal drugs. However, these drugs are limited by poor solubility and bioavailability. Furthermore, antifungal resistance against these drugs are on the rise. Thus, antimicrobial polymers offer an alternative antifungal strategy. Antifungal polymers are characterised by cationic and hydrophobic regions where the cationic regions have been shown to interact with microbial phospholipids and membranes. These polymers can be synthetic or natural and demonstrate distinct antifungal mechanisms ranging from fungal cell membrane permeabilisation, cell membrane depolarisation or cell entry. Although the relative importance of such mechanisms is difficult to decipher. Due to the chemical properties of these polymers, they can be combined with other antimicrobial compounds including existing antifungal drugs, charcoals, lipids and metal ions to elicit synergistic effects. In some cases, antifungal polymers and nanocomposites show better antifungal effects or reduced toxicity compared to the widely used small molecule antifungal drugs. This review provides an overview of antimicrobial polymers and nanocomposites with antifungal activity and the current understanding of their antifungal mechanisms.

Cellular gene delivery via poly(hexamethylene biguanide)/pDNA self-assembled nanoparticles.

Cellular gene delivery via polycations has wide implications for the potential of gene therapy, but it has remained a challenge due to the plethora of pre- and post-uptake barriers that must be overcome to reach desired efficiency. Herein we report poly(hexamethylene biguanide) (PHMB) as a nano-vector for intracellular delivery of plasmid DNA (pDNA) and oligodeoxynucleotides (ODNs). PHMB and pDNA or ODNs self-assembled into complex nanoparticles at different pH values (7.4 and 12). Their size, charge, cellular uptake, and gene-expression efficiency are assessed and compared to PEI analogues. The systematic results show that the nanoparticles are effective in delivering plasmid DNA and ODNs to model cell lines in culture (HepG2, HEK293T, HeLa), with measurable changes in gene expression levels, comparable to and, in some conditions, even higher than PEI. The well-accepted safety profile of PHMB makes it a valuable candidate for consideration as an effective intracellular DNA vector for further study and potential clinical translation.

Development of a fixation-free fluorescence in situ hybridization for the detection of Salmonella species.

Salmonella is one of the most important infectious bacteria causing severe gastroenteritis and deaths in humans and animals, and the prompt diagnosis is crucial for effective control and treatment. The detection of Salmonella still depends principally on culture-based methods, which is time-consuming and laborious. Recently, polyhexamethylene biguanide (PHMB) was discovered to have cellular delivery properties and its combination with the fluorescence in situ hybridization (FISH) method was exploited for oligomer delivery and the rapid detection of Salmonella spps in this study. Cell-associated fluorescence was quantified using Volocity® 3-D image analysis software (Volocity 6.3, PerkinElmer, Inc.). PHMB complexed with fluorophore-labelled species-specific oligomers permeabilized freshly grown viable strains of Salmonella cells and mediated strong cell-associated fluorescence signals. This strategy further enabled a fixation-free protocol and hybridization in a single reaction. Using the modified FISH method, monoculture Salmonella strains were validated as well as detected in artificially contaminated water and milk within a turnaround period of 5 h. The method was observed to be comparable with the standard FISH technique (sFISH; P > 0.05). The findings suggest that fixation-free delivery and hybridization of oligomers using PHMB can provide a simplified and prompt strategy for Salmonella detection at the species level, and promote early management responses to the disease and appropriate antimicrobial therapy.

The Effects of Polyhexamethylene Biguanide (PHMB) and TLR Agonists Alone or as Polyplex Nanoparticles against Leishmania infantum Promastigotes and Amastigotes.

Dogs are the main reservoir for Leishmania infantum, manifesting from a subclinical to a fatal disease. Limited treatments are available, although new antiparasitics and immunomodulators are pursued. Polyhexamethylene biguanide (PHMB) has a broad antimicrobial spectrum, including antiparasitic activity. Here, we evaluated the potential for Toll-like receptor agonists (TLRa) and PHMB alone, and as polyplex nanoparticles containing PHMB and TLR4 or TLR9 agonists, to selectively kill L. infantum. Susceptibility of L. infantum promastigotes to PHMB, miltefosine, and allopurinol was performed, and the half-maximum inhibitory concentrations (IC50) were determined. Then, DH-82 cells were infected and treated with PHMB alone or combined with TLR4a (MPLA-SM) or TLR9a (CpG ODNs) and allopurinol alone. The IC50 values of L. infantum promastigotes were PHMB (1.495 µM), miltefosine (9.455 µM), and allopurinol (0.124 µM). After infection, treated DH-82 cells displayed a lower percentage (p = 0.0316), intensity (p = 0.0002), and index of infection (p = 0.0022) when compared to non-treated cells. PHMB induced lower percentage of infection alone (p = 0.043), in combination with TLR9a (p = 0.043), and with TLR4a (p = 0.0213). Supernatants were collected and used to measure TNF-α and IL-6 levels. Increased TNF-α was observed after PHMB plus TLR4a, relative to uninfected and infected untreated macrophages (p = 0.043). PHMB combined with TLR4a shows promise as a potential anti-L. infantum drug combination, as well as inducer of proinflammatory response, as demonstrated by decreased infection and increased TNF-α production.

Genetic diversity and risk factors for the transmission of antimicrobial resistance across human, animals and environmental compartments in East Africa: a review.

BACKGROUND: The emergence and spread of antimicrobial resistance (AMR) present a challenge to disease control in East Africa. Resistance to beta-lactams, which are by far the most used antibiotics worldwide and include the penicillins, cephalosporins, monobactams and carbapenems, is reducing options for effective control of both Gram-positive and Gram-negative bacteria. The World Health Organization, Food and Agricultural Organization and the World Organization for Animal Health have all advocated surveillance of AMR using an integrated One Health approach. Regional consortia also have strengthened collaboration to address the AMR problem through surveillance, training and research in a holistic and multisectoral approach. This review paper contains collective information on risk factors for transmission, clinical relevance and diversity of resistance genes relating to extended-spectrum beta-lactamase-producing (ESBL) and carbapenemase-producing Enterobacteriaceae, and Methicillin-resistant Staphylococcus aureus (MRSA) across the human, animal and environmental compartments in East Africa. MAIN BODY: The review of the AMR literature (years 2001 to 2019) was performed using search engines such as PubMed, Scopus, Science Direct, Google and Web of Science. The search terms included 'antimicrobial resistance and human-animal-environment', 'antimicrobial resistance, risk factors, genetic diversity, and human-animal-environment' combined with respective countries of East Africa. In general, the risk factors identified were associated with the transmission of AMR. The marked genetic diversity due to multiple sequence types among drug-resistant bacteria and their replicon plasmid types sourced from the animal, human and environment were reported. The main ESBL, MRSA and carbapenem related genes/plasmids were the blaCTX-Ms (45.7%), SCCmec type III (27.3%) and IMP types (23.8%), respectively. CONCLUSION: The high diversity of the AMR genes suggests there may be multiple sources of resistance bacteria, or the possible exchange of strains or a flow of genes amongst different strains due to transfer by mobile genetic elements. Therefore, there should be harmonized One Health guidelines for the use of antibiotics, as well as regulations governing their importation and sale. Moreover, the trend of ESBLs, MRSA and carbapenem resistant (CAR) carriage rates is dynamic and are on rise over time period, posing a public health concern in East Africa. Collaborative surveillance of AMR in partnership with regional and external institutions using an integrated One Health approach is required for expert knowledge and technology transfer to facilitate information sharing for informed decision-making.

Doxycycline induces Hok toxin killing in host E. coli.

The antibacterial efficacy of the tetracycline antibiotics has been greatly reduced by the development of resistance, hence a decline in their clinical use. The hok/sok locus is a type I toxin/antitoxin plasmid stability element, often associated with multi-drug resistance plasmids, especially ESBL-encoding plasmids. It enhances host cell survivability and pathogenicity in stressful growth conditions, and increases bacterial tolerance to ß-lactam antibiotics. The hok/sok locus forms dsRNA by RNA:RNA interactions between the toxin encoding mRNA and antitoxin non-coding RNA, and doxycycline has been reported to bind dsRNA structures and inhibit their cleavage/processing by the dsRNase, RNase III. This study investigated the antibacterial activities of doxycycline in hok/sok host bacteria cells, the effects on hok/sok-induced changes in growth and the mechanism(s) involved. Diverse strains of E. coli were transformed with hok/sok plasmids and assessed for doxycycline susceptibility and growth changes. The results show that the hok/sok locus increases bacterial susceptibility to doxycycline, which is more apparent in strains with more pronounced hok/sok-induced growth effects. The increased doxycycline susceptibility occurs despite ß-lactam resistance imparted by hok/sok. Doxycycline was found to induce bacterial death in a manner phenotypically characteristic of Hok toxin expression, suggesting that it inhibits the toxin/antitoxin dsRNA degradation, leading to Hok toxin expression and cell death. In this way, doxycycline could counteract the multi-drug resistance plasmid maintenance/propagation, persistence and pathogenicity mechanisms associated with the hok/sok locus, which could potentially help in efforts to mitigate the rise of antimicrobial resistance.

The development of species-specific antisense peptide nucleic acid method for the treatment and detection of viable Salmonella.

Genotypic based detection methods using specific target sites in the pathogen genome can complement phenotypic identification. We report the development of species-specific antisense peptide nucleic acid (PNA) combined with selective and differential enrichment growth conditions for Salmonella treatment and detection. An antisense PNA oligomer targeting the Salmonella ftsZ gene and conjugated with a cell-penetrating peptide ((KFF)3K) was exploited to probe bacteria cultured in three different growth media (Muller Hinton broth (MHB), Rappaport-Vassiliadis Soya Peptone Broth (RVS, Oxoid), and in-house modified Rappaport-Vassiliadis Soya Peptone Broths (mRVSs). Also, water and milk artificially contaminated with bacteria were probed. Antisense PNA provided detectable changes in Salmonella growth and morphology in all media and artificially contaminated matrices except RVS. Salmonella was detected as elongated cells. On the contrary, treated Escherichia coli did not elongate, providing evidence of differentiation and selectivity for Salmonella. Similarly, Salmonella probed with mismatched PNAs did not elongate. Antisense oligomers targeted ftsZ mRNA in combination with selective growth conditions can provide a detection strategy for viable Salmonella in a single reaction, and act as a potential tool for bacteria detection in real food and environmental samples.

Identification of Escherichia coli from broiler chickens in Jordan, their antimicrobial resistance, gene characterization and the associated risk factors.

BACKGROUND: Avian pathogenic Escherichia coli (APEC) is the principle cause of colibacillosis affecting poultry. The main challenge to the poultry industry is antimicrobial resistance and the emergence of multidrug resistant bacteria that threaten the safety of the food chain. Risk factors associated with emergence of antimicrobial resistance among avian pathogenic E. coli were correlated with the inappropriate use of antimicrobials along with inadequate hygienic practices, which encourages the selection pressure of antimicrobial resistant APEC. The aim of this study was to isolate, identify, serogroup and genotype APEC from broilers, assess their antibiotic resistance profile, expressed genes and the associated risk factors. RESULTS: APEC was isolated from the visceral organs of sick chickens with a prevalence of 53.4%. The most prevalent serotypes were O1, O2, O25 and O78, in percentage of 14.8, 12.6, 4.4 and 23.7%, respectively. Virulence Associated Genes; SitA, iss, iucD, iucC, astA, tsh cvi and irp2 were detected in rate of 97.4, 93.3, 75, 74, 71, 46.5, 39 and 34%, respectively and 186 (69.2%) isolates possess > 5-10 genes. The highest resistance was found against sulphamethoxazole-trimethoprim, florfenicol, amoxicillin, doxycycline and spectinomycin in percentage; 95.5, 93.7, 93.3, 92.2 and 92.2%, respectively. Sixty-eight percent of APEC isolates were found to have at least 5 out of 8 antimicrobial resistant genes. The most predominant genes were Int1 97%, tetA 78.4%, bla TEM 72.9%, Sul1 72.4%, Sul2 70.2%. Two risk factors were found to be associated with the presence of multi-drug resistant APEC in broiler chickens, with a P value ≤0.05; the use of ground water as source of drinking water and farms located in proximity to other farms. CONCLUSIONS: This study characterized the VAGs of avian pathogenic E. coli and establish their antimicrobial resistance patterns. The widespread of antimicrobial resistance of APEC isolates and detection of ARGs highlighted the need to monitor the spread of ARGs in poultry farms and the environment in Jordan. Use of ground water and closely located farms were significant risk factors associated with the presence of MDR APEC in broiler chickens in Jordan.

Polyhexamethylene Biguanide:Polyurethane Blend Nanofibrous Membranes for Wound Infection Control.

Polyhexamethylene biguanide (PHMB) is a broad-spectrum antiseptic which avoids many efficacy and toxicity problems associated with antimicrobials, in particular, it has a low risk of loss of susceptibility due to acquired antimicrobial resistance. Despite such advantages, PHMB is not widely used in wound care, suggesting more research is required to take full advantage of PHMB's properties. We hypothesised that a nanofibre morphology would provide a gradual release of PHMB, prolonging the antimicrobial effects within the therapeutic window. PHMB:polyurethane (PU) electrospun nanofibre membranes were prepared with increasing PHMB concentrations, and the effects on antimicrobial activities, mechanical properties and host cell toxicity were compared. Overall, PHMB:PU membranes displayed a burst release of PHMB during the first hour following PBS immersion (50.5-95.9% of total released), followed by a gradual release over 120 h (≤25 wt % PHMB). The membranes were hydrophilic (83.7-53.3°), gradually gaining hydrophobicity as PHMB was released. They displayed superior antimicrobial activity, which extended past the initial release period, retained PU hyperelasticity regardless of PHMB concentration (collective tensile modulus of 5-35% PHMB:PU membranes, 3.56 ± 0.97 MPa; ultimate strain, >200%) and displayed minimal human cell toxicity (<25 wt % PHMB). With further development, PHMB:PU electrospun membranes may provide improved wound dressings.

Doxycycline inhibits pre-rRNA processing and mature rRNA formation in E. coli.

In bacteria, RNase III cleaves the initial long primary ribosomal RNA transcripts/precursors (pre-rRNAs), thereby releasing the pre-16S and pre-23S rRNAs for maturation. This cleavage is specified by the double-stranded secondary structures flanking the mature rRNAs, and not necessarily by the nucleotide sequences. Inhibition of this cleavage would lead to a build-up of pre-rRNA molecules. Doxycycline has earlier been shown to bind synthetic double-stranded RNAs and inhibit their cleavage by RNase III. Since bacterial rRNA processing is primarily dependent on RNase III cleavage (which is inhibited by doxycycline), doxycycline could therefore inhibit the normal processing of bacterial rRNA. In this study, the effect of doxycycline on bacterial rRNA processing was investigated by analyzing the amounts of various rRNAs in growing Escherichia coli cells treated with doxycycline. The results showed a doxycycline dose-dependent decrease in mature 16S and 23S rRNAs, concurrent with an accumulation of the initial rRNA transcripts and long precursors. Morphologically, treated cells were elongated at low drug concentrations, while nucleoid degeneration indicative of cell death occurred at higher drug concentrations. These observations suggest that doxycycline inhibits the cleavage and processing of bacterial rRNA transcripts/precursors, leading to impaired formation of mature rRNAs, and the consequent inhibition of protein synthesis for which the tetracycline group of antibiotics are renowned. Since rRNA structure and processing pathway is conserved among bacterial species, this mechanism may account for the broad spectrum of antibiotic activity and selective microbial protein synthesis inhibition of doxycycline and the tetracyclines.

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.

Phenotypic indications of FtsZ inhibition in hok/sok-induced bacterial growth changes and stress response.

The hok/sok locus has been shown to enhance the growth of bacteria in adverse growth conditions such as high temperature, low starting-culture densities and antibiotic treatment. This is in addition to their well-established plasmid-stabilization effect via post-segregational killing of plasmid-free daughter cells. It delays the onset of growth by prolonging the lag phase of bacterial culture, and increases the rate of exponential growth when growth eventually begins. This enables the cells adapt to the prevailing growth conditions and enhance their survival in stressful conditions. These effects functionally complement defective SOS response mechanism, and appear analogous to the growth effects of FtsZ in the SOS pathway. In this study, the role of FtsZ in the hok/sok-induced changes in bacterial growth and cell division was investigated. Morphologic studies of early growth-phase cultures and cells growing under temperature stress showed elongated cells typical of FtsZ inhibition/deficiency. Both ftsZ silencing and over-expression produced comparable growth effects in control cells, and altered the growth changes observed otherwise in the hok/sok+ cells. These changes were diminished in SOS-deficient strain containing mutant FtsZ. The involvement of FtsZ in the hok/sok-induced growth changes may be exploited as drug target in host bacteria, which often propagate antibiotic resistance elements.

Bactericidal and Anti-biofilm Effects of Polyhexamethylene Biguanide in Models of Intracellular and Biofilm of Staphylococcus aureus Isolated from Bovine Mastitis.

Staphylococcus aureus infection is a common cause of mastitis, reducing milk yield, affecting animal welfare and causing huge economic losses within the dairy industry. In addition to the problem of acquired drug resistance, bacterial invasion into udder cells and the formation of surface biofilms are believed to reduce antibiotic efficacy, leading to treatment failure. Here, we investigated the antimicrobial activities of enrofloxacin, an antibiotic that is commonly used in mastitis therapy and polyhexamethylene biguanide (PHMB), an antimicrobial polymer. The antimicrobial activities were tested against intracellular S. aureus in infected Mac-T cells (host cells). Also, fluorescein-tagged PHMB was used to study PHMB uptake and localization with S. aureus within the infected Mac-T cells. Anti-biofilm activities were tested by treating S. aureus biofilms and measuring effects on biofilm mass in vitro. Enrofloxacin and PHMB at 15 mg/L killed between 42 to 92 and 99.9% of intracellular S. aureus, respectively. PHMB-FITC entered and colocalized with the intracellular S. aureus, suggesting direct interaction of the drug with the bacteria inside the host cells. Enrofloxacin and PHMB at 15 mg/L reduced between 10 to 27% and 28 to 37% of biofilms' mass, respectively. The half-maximal inhibitory concentrations (IC50) obtained from a cytotoxicity assay were 345 ± 91 and 21 ± 2 mg/L for enrofloxacin and PHMB, respectively; therefore, both compounds were tolerated by the host cells at high concentrations. These findings suggest that both antimicrobials are effective against intracellular S. aureus and can disrupt biofilm structures, with PHMB being more potent against intracellular S. aureus, highlighting the potential application of PHMB in mastitis therapy.

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.