An in vitro study into the antimicrobial and cytotoxic effect of Acticoat™ dressings supplemented with chlorhexidine.

Burn injuries can cause traumatic and debilitating physical trauma, with burn wounds prone to bacterial infection. This study examined in vitro the effectiveness of the silver nanoparticle based antimicrobial dressing, Acticoat™, in combination with a range of antimicrobial compounds against Staphylococcus aureus and Pseudomonas aeruginosa and investigated potential cytotoxic effects in multi-layered differentiated keratinocyte models. Acticoat™ with chlorhexidine was found to be highly effective against S. aureus and P. aeruginosa across a 3 day incubation period on pig skin models. MTT assays and histological staining of keratinocyte models revealed Acticoat™ had a cytotoxic effect following initial contact with the cells and cytotoxicity was exacerbated when dressings were coated with chlorhexidine and antimicrobial peptide formulations. Spectrophotometric analysis suggested that the silver nanoparticles may mobilise from the dressing as nanoclusters or silver salts, which may relate to the observed cytotoxicity. The bacterial strains used in this study showed a substantial tolerance to Acticoat™ with biofilm-like communities observed on the dressing surfaces. This could be mitigated with chlorhexidine, albeit with an increase in cytotoxicity. The clinical significance of these findings in terms of infection control and wound healing remain to be determined; the potential benefit of bactericidal activity must be balanced against cytotoxicity, and the prevalence and potential transmission of the silver tolerant phenotype must also be assessed.

Transcriptional profiling of biofilms formed on chilled beef by psychrotrophic meat spoilage bacterium, Pseudomonas fragi 1793.

Pseudomonas fragi is the predominant bacterial species associated with spoiled aerobically stored chilled meat worldwide. It readily forms biofilms on meat under refrigerated temperature conditions used in the meat industry. Biofilm growth leads to slime development on meat which in turn becomes a major quality defect. To understand the genetic regulation that aids P. fragi to survive under chilled conditions used in the meat industry, as well to obtain an overview of the transcriptomic behavior of this organism when grown as biofilms, RNA sequencing was carried out for the main stages of the P. fragi 1793 biofilm. RNA was extracted at different stages of the biofilm cycle namely initiation, maturation and dispersal. At the same time, the biofilm growth was assessed by fluorescent staining and imaging using confocal laser scanning microscope. The results of RNA sequencing were verified by qRT-PCR using twelve genes that were most significantly up and down regulated at each stage. Differential expression analysis at biofilm maturation revealed 332 significantly upregulated genes and 37 downregulated genes relative to initiation. Differential expression analysis at biofilm dispersal reveled 658 upregulated and 275 downregulated genes relative to initiation. During biofilm maturation and dispersal, genes coding for flp family type IVb pilin, ribosome modulation factor, creatininase were the most upregulated genes while genes encoding for iron uptake systems including TonB-dependent siderophore receptor and taurine transport were significantly down regulated. The results show that protein synthesis and cellular multiplication cease after the biofilm population maximum has reached.

Survival of Salmonella Under Heat Stress is Associated with the Presence/Absence of CRISPR Cas Genes and Iron Levels.

Clustered regularly interspaced short palindromic repeats (CRISPR) cas genes have been linked to stress response in Salmonella. Our aim was to identify the presence of CRISPR cas in Salmonella and its response to heat in the presence of iron. Whole genomes of Salmonella (n = 50) of seven serovars were compared to identify the presence of CRISPR cas genes, direct-repeats and spacers. All Salmonella genomes had all cas genes present except S. Newport 2393 which lacked these genes. Gene-specific primers were used to confirm the absence of these genes in S. Newport 2393. The presence/absence of CRISPR cas genes was further investigated among 469 S. Newport genomes from PATRIC with 283 genomes selected for pan-genome analysis. The response of eleven Salmonella strains of various serovars to gradual heat in ferrous and ferric forms of iron was investigated. A total of 32/283 S. Newport genomes that lacked all CRISPR cas genes clustered together. S. Newport 2393 was the most heat-sensitive strain at higher iron levels (200 and 220 pm) in ferrous and ferric forms of iron. The absence of CRISPR cas genes in S. Newport 2393 may contribute to its increase in heat sensitivity and iron may play a role in this. The high reduction in numbers of most Salmonella strains exposed to heat makes it unfeasible to extract RNA and conduct transcription studies. Further studies should be conducted to validate the survival of Salmonella when exposed to heat in the presence/absence of CRISPR cas genes and different iron levels.

Effect of chitosan and gum Arabic with natamycin on the aroma profile and bacterial community of Australian grown black Périgord truffles (Tuber melansoporum) during storage.

This study aimed to assess the effect of chitosan or gum Arabic edible coatings, with natamycin (200, 300, 400 mg/L) on the aroma profiles of Western Australian grown truffles at five storage intervals: 0, 7, 14, 21, and 28 days using solid-phase microextraction (SPME)-followed by gas chromatography-mass spectrometry (GC-MS). The population structure of the bacterial community of both untreated and chitosan-natamycin (400 mg/L) coated truffles were assessed using metagenomic sequencing analysis alongside GC-MS. The results demonstrated that all the coating treatments were able to have a positive impact in halting or delaying the changes of truffle aroma throughout the storage period, with chitosan-natamycin (400 mg/L) coating having the best preservation results compared to the other coatings. Only 9 volatile organic compounds (VOCs) were found to have significant changes in chitosan-natamycin (400 mg/L) coated truffles throughout the storage period compared to 11 VOCs in untreated controls. The result also demonstrated the gradual change of fresh truffle's bacteria communities over the storage period. Over 4 weeks of storage, the dominant bacterial classes of the truffles (α-Proteobacteria, Bacteroidia or Actinobacteria classes) were replaced by Bacteroidia, Actinobacteria, Deltaprotobacteria and γ-Proteobacteria classes. The preliminary results from this study show that edible coatings can affect the VOC and bacterial communities of the truffles which may have implications for future research into truffle preservation techniques.

Changes in STEC and bacterial communities during enrichment of manufacturing beef in selective and non-selective media.

Detection and isolation of Shiga toxin-producing Escherichia coli (STEC) from manufacturing beef is challenging and it may be affected by microbial changes during enrichment. This study was designed to understand population changes during enrichment of beef from an integrated (Samples A and B) and a fragmented (Samples C and D) abattoir. The samples were enriched in buffered peptone water (BPW), Assurance GDS MPX top 7 STEC mEHEC®, BAX® E. coli O157:H7 MP and PDX-STEC media then were processed for 16 S rRNA sequencing. Escherichia dominated Sample B enrichment broths regardless of the media used (71.6-97.9%) but only in mEHEC broth (79.6%) of Sample A. Escherichia was dominant in Sample C in mEHEC (95.2%) and PDX-STEC (99.2%) broths but less in BPW (58.5%) and MP (64.9%) broths. In Sample D, Clostridium dominated in mEHEC (65.5%), MP (80.2%) and PDX-STEC (90.6%) broths. O157 STEC was isolated from Sample C only. The study suggested that MP may not be as effective as mEHEC and PDX-STEC and that Clostridium could interfere with enrichment of Escherichia. Understanding the ecological changes during enrichment provides meaningful insight to optimising the enrichment protocol for STEC and subsequently enhance the efficiency of STEC detection.

Antimicrobial efficacy of nisin-loaded bacterial cellulose nanocrystals against selected meat spoilage lactic acid bacteria.

This study aimed to explore the potential of bacterial cellulose nanocrystals (BCNs) loaded with nisin against selected meat spoilage lactic acid bacteria (LAB) in vitro. BCNs were produced by H2SO4 hydrolysis, and nisin-loaded BCNs were produced through the complexation method. All nanocrystals were assessed for their zeta-potential, encapsulation efficiency and antimicrobial activity. Different nisin concentrations were tested and the most effective nanocrystals were further characterised. BCNs had an average zeta-potential of - 43 mV and all nisin-loaded BCNs produced with 5 mg/ml BCNs suspension had zeta-potential values ≥- 30 mV. The encapsulation efficiency of nisin varied from 80.5 to 93.3 % and crystallinity of BCNs was not influenced by nisin encapsulation. Microbial inactivation was achieved by BCN loaded with 2.0 and 2.5 mg/ml nisin. Therefore, nisin-loaded BCNs may be used as antimicrobial agents in active food packaging.

Salmonella survival after exposure to heat in a model meat juice system.

The effect of heat against eleven Salmonella strains in model meat juices was examined. Juices from beef, lamb and goat were made from either the fatty layer (FL), muscle (M) or a mixture of both (FLM). The pH of each FLM sample was altered to match the pH of PBS and vice versa to determine the pH effect on the survival of Salmonella against the effect of heat. Salmonella were exposed to either gradual heating to 70 °C in FLM, M and FL or heat shock at 70 °C for 5 min in FLM. Fat, fatty acid profile and iron content of the juices were determined. Gradual heat treatment significantly (p ≤ 0.05) reduced Salmonella as compared to the untreated controls (~1.92-7.61 log CFU ml-1) while heat shock significantly (p ≤ 0.05) reduced Salmonella as compared to the untreated controls (~5.80-7.36 log CFU ml-1). Survival of Salmonella was higher in lamb juices than other juices. The fat content in lamb FL (3.25%) was significantly higher (p ≤ 0.05) than beef (1.30%) and goat FL (1.42%). Iron content in lamb FLM (~127 mg kg-1) was significantly (p ≤ 0.05) lower than beef (~233 mg kg-1) and goat FLM (~210 mg kg-1). The omega 6 and linoleic acid content in goat FLM (~36.0% and ~34.4%) was significantly higher (p ≤ 0.05) than beef (~29.1% and ~27.1%). Fat, fatty acids and iron may differentially protect Salmonella against the effect of heat in these juices.

Survival of Salmonella on Red Meat in Response to Dry Heat.

ABSTRACT: Red meat is associated with Salmonella outbreaks, resulting in negative impacts for the processing industry. Little work has been reported on the use of dry heat as opposed to moist heat against Salmonella on red meat. We determined the effect of drying at 25°C and dry heat at 70°C with ∼10% relative humidity for 1 h against 11 Salmonella strains of multiple serovars on beef, lamb, and goat and rubber as an inert surface. Each strain at ∼108 CFU/mL was inoculated (100 µL) onto ±1 g (1 cm2) of each surface and allowed to attach for 15 min in a microcentrifuge tube. Samples were then exposed to 70 and 25°C with ∼10% relative humidity in a heating block. Surviving Salmonella numbers on surfaces were enumerated on a thin agar layer medium. If numbers were below the limit of detection (2.01 log CFU/cm2), Salmonella cells were enriched before plating to determine the presence of viable cells. Water loss (percent) from meat after at 25 and 70°C was determined. Whole genomes of Salmonella were interrogated to identify the presence-absence of stress response genes (n = 30) related to dry heat that may contribute to the survival of Salmonella. The survival of Salmonella at 25°C was significantly higher across all surfaces (∼6.09 to 7.91 log CFU/cm2) than at 70°C (∼3.66 to 6.33 log CFU/cm2). On rubber, numbers of Salmonella were less than the limit of detection at 70°C. Water loss at 70°C (∼17.72 to 19.89%) was significantly higher than at 25°C (∼2.98 to 4.11%). Salmonella cells were not detected on rubber, whereas survival occurred on all red meat at 70°C, suggesting its protective effect against the effect of heat. All Salmonella strains carried 30 stress response genes that likely contributed to survival. A multi-antibiotic-resistant Salmonella Typhimurium 2470 exhibited an increase in heat resistance at 70°C on beef and lamb compared with other strains. Our work shows that dry heat at 70°C for 1 h against Salmonella on red meat is not a practical approach for effectively reducing or eliminating them from red meat.

Characterization of the biofilm matrix composition of psychrotrophic, meat spoilage pseudomonads.

Psychrotrophic Pseudomonas species are the key spoilage bacteria of aerobically stored chilled meat. These organisms readily form biofilms on meat under refrigerated conditions leading to consumer rejection and associated economic losses. Limited information is available on the matrix composition of the biofilms formed by these bacteria. We quantified and characterized the main components of the matrix of mono-species biofilms of selected Pseudomonas fragi and Pseudomonas lundensis strains using chemical analysis and Raman spectroscopy. The biofilms were grown at 10 °C and 25 °C on nitro-cellulose membranes placed on surface sterilized beef cuts. Extra-cellular polymeric substances of the matrix were extracted in soluble and bound forms and were chemically assessed for total carbohydrates, proteins and extra-cellular DNA. Both Pseudomonas species showed a significant increase in total carbohydrates and total proteins when grown at 10 °C as compared to 25 °C. Extra-cellular DNA did not show a strong correlation with growth temperature. Raman spectra were obtained from planktonic bacteria and membrane grown biofilms at 10 °C and 25 °C. Higher levels of guanine were detected in planktonic cells as compared to biofilm cells. This study suggests that psychrotrophic Pseudomonas species may respond to cold stress by increasing extra-cellular polymer secretions.

Bacterial community analysis using 16S rRNA amplicon sequencing in the boning room of Australian beef export abattoirs.

Microbial contamination associated with beef slaughter and boning has been investigated using traditional culture dependent approaches. However, conventional counting methods have disadvantages of detecting only cultivable bacterial groups that may be a small subset of the true microbial population. This study investigated the microbiology in the boning room of an integrated (abattoir A) and a fragmented (abattoir B) Australian beef export abattoirs using culture independent 16S rRNA gene amplicon sequencing coupled with total viable count (TVC). Transmission of microbial populations during processing of carcases onto beef trim was monitored and compared between the two abattoirs. The results showed that the abattoirs produced beef trim with a mean TVC of 2.64-2.70 log10 CFU/cm2. Initial counts of microbes on the chilled carcases entering the boning room were <1.5 log10 CFU/cm2 and the environmental surfaces had ≤2.0 log10 CFU/cm2 throughout the boning room. Profiling of 16S gene sequences demonstrated that the contamination of boned products (beef trim) may be a result of contamination accumulating from environmental surfaces that are regularly in contact with beef trim. The 16S data also showed that the bacterial communities on the carcases and trim shared similar community composition with microbiota on environmental surfaces at varying proportions depending on the day of processing. Bacteroidales, Clostridiales, Enterobacteriales, Lactobacillales and Pseudomonadales were predominantly present in the bacterial communities in both abattoirs. However, the changes in relative abundance of these bacteria through the boning process varied between the abattoirs. The findings from this study suggested that the transfer of bacterial contaminants in the beef cattle boning room can be dynamic, and a 16 s rRNA gene sequencing-based approach can improve our understanding of the sources of contamination in the boning environment.

Effectiveness of bacterial cellulose in controlling purge accumulation and improving physicochemical, microbiological, and sensorial properties of vacuum-packaged beef.

The application of bacterial cellulose (BC) as a wrapping material for vacuum-packaged beef was studied and compared against unwrapped beef for up to 3 weeks. The impact of BC wrap on the weight loss, purge accumulation, and drip loss were assessed along with low-field nuclear magnetic resonance, physicochemical, microbiological, and sensorial evaluations. The BC wrap significantly (P < 0.05) reduced purge accumulation in vacuum packages which was confirmed by an increased swelling ratio and scanning electron microscopy images. Colorimetric measurements showed significantly (P < 0.05) increased redness and yellowness values in wrapped samples compared to unwrapped samples. BC wrap did not affect pH, tenderness, and odor of meat, but significantly (P < 0.05) increased lipid oxidation, and numbers of lactic acid bacteria and Brochothrix thermosphacta counts. This study shows that BC wrap has potential as a purge absorbent in vacuum packaged meat. PRACTICAL APPLICATION: Bacteria cellulose has good water holding capacity that can be utilized to absorb purge exudate from beef. It helps to improve the appearance and consequently consumer acceptance of vacuum packed beef.

Analysis of Bacterial Diversity in Relation to the Presence of the Top 7 Shiga Toxin-Producing Escherichia coli throughout Australian Beef Abattoirs.

ABSTRACT: There is increasing evidence that diversity changes in bacterial communities of beef cattle correlate to the presence of Shiga toxin-producing Escherichia coli (STEC). However, studies that found an association between STEC and bacterial diversity have been focused on preslaughter stages in the beef supply chain. This study was designed to test a hypothesis that there are no differences in bacterial diversity between samples with and those without the presence of the top 7 STEC (O26, O45, O103, O111, O121, O145, and O157) throughout processing in an integrated (abattoir A) and a fragmented (abattoir B) Australian beef abattoir. Slaughter and boning room surface samples from each abattoir were analyzed using 16S rRNA amplicon sequencing and tested for the top 7 STEC following the Food Safety and Inspection Service protocol. Potential positives through slaughter were similar between the abattoirs (64 to 81%). However, abattoir B had substantially reduced potential positives in the boning room compared with abattoir A (abattoir A: 23 and 48%; abattoir B: 2 and 7%). Alpha diversity between the sample groups was not significantly different (P > 0.05) regardless of different STEC markers. Nonmetric multidimensional scaling of slaughter samples showed that the bacterial composition in fecal and hide samples shared the least similarity with the communities in carcass and environmental samples. Surface samples from slaughter (carcass and environmental) and boning (carcass, beef trim, and environmental) all appeared randomly plotted on the scale. This indicated that the STEC presence also did not have a significant effect (P > 0.05) on beta diversity. Although presence of STEC appeared to correlate with changes in diversity of fecal and hide bacterial communities in previous studies, it did not appear to have the same effect on other samples throughout processing.

A Comparison of 16S rRNA Profiles Through Slaughter in Australian Export Beef Abattoirs.

Microbial contamination of beef cattle carcases and subsequent cross-contamination during processing is inevitable and virtually impossible to prevent. The understanding of microbial contamination in the beef industry is currently limited to hypotheses based on traditional microbiological tools. Additionally, the complex structural and functional responses of beef cattle microbial communities to the fragmentation in the supply chain remain unknown. This study used 16S rRNA gene sequencing in combination with traditional microbiology to monitor and compare changes in the microbiota throughout slaughter in an integrated (abattoir A) and a fragmented (abattoir B) beef abattoir in Australia. Briefly, the primary difference between an integrated and a fragmented abattoir is that fragmented abattoirs receive cattle from multiple sources, whereas integrated abattoirs typically receive cattle that has been produced using the same production system and from a limited number of sources. The composition in the bacterial communities varied between the abattoirs, though the presence of the most predominant bacterial species within the microbiota at each abattoir was similar. Lactobacillales (2.4-56.2%) and Pseudomonadales (2.4-59.4%) most notably dominated hides, carcases, and the environment in abattoir B. In abattoir A, Bacteroidales (3.9-43.8%), Lactobacillales (0.0-61.9%), and Pseudomonadales (0.5-72.1%) fluctuated but generally shared the dominance over the rest. Combined results of total viable count (TVC) and 16S rRNA gene profiling indicated that an upward hide pulling system adopted by abattoir B may lead to increased transmission of hide contaminants to post-hide pull carcases. Abattoir B had 3.2 log10CFU/cm2 reduction from hide to carcase, where abattoir A had 4.5 log10CFU/cm2 reduction. The findings from this study indicated that common beef-associated microbiota exist in varying composition in Australian abattoirs, and 16S rRNA amplicon sequencing is a powerful tool to understand in-depth movement of microbial contaminants.

In situ characterisation of biofilms formed by psychrotrophic meat spoilage pseudomonads.

Psychrotrophic Pseudomonas species form biofilms on meat during refrigerated and temperature abuse conditions. Biofilm growth leads to slime formation on meat which is a key organoleptic degradation characteristic. Limited research has been undertaken characterising biofilms grown on meat during chilled aerobic storage. In this work, biofilms formed by two key meat spoilage organisms, Pseudomonas fragi and Pseudomonas lundensis were studied in situ using five strains from each species. Biofilm structures were studied using confocal microscope images, cellular arrangement, cell counts and biomass quantifications. This work demonstrated that highly dense, compact biofilms are a characteristic of P. fragi strains. P. lundensis formed biofilms with loosely arranged cells. The cells in P. fragi biofilm appear to be vertically oriented whereas this characteristic was absent in P. lundensis biofilms formed under identical conditions. Despite the continued access to nutrients, biofilms formed on meat by proteolytic Pseudomonas species dispersed after a population maximum was reached.

Draft Genome Sequences of Four Antibiotic-Resistant Salmonella Strains Isolated from Australian Red Meat Animal Species.

The genome sequences of four antibiotic-resistant Salmonella strains isolated from red meat animals in Australia are presented. Multidrug-resistant Salmonella enterica serovar Heidelberg 329 and Salmonella enterica serovar Typhimurium 2470 harbored an IncHI2 plasmid similar to the multidrug-resistant S. Heidelberg strain N13-01290 plasmid pN13-01290_23 previously isolated in Canada.

The Predominance of Psychrotrophic Pseudomonads on Aerobically Stored Chilled Red Meat.

Microbial spoilage of meat during chilled aerobic storage causes significant financial losses to the industry. Even with modern day preservation techniques, spoilage remains an unsolved problem. Spoilage of meat is a complex process that involves the activity of endogenous enzymes and microorganisms. Psychrotrophic Pseudomonas species are the key microorganisms that cause spoilage in aerobically stored chilled meat. Spoilage pseudomonads are highly robust and able to withstand stressful environmental conditions that would otherwise inhibit the growth of other spoilage organisms. In order to implement efficient control measures, and to minimize spoilage, a thorough understanding of the characteristics of spoilage pseudomonads is essential. This review focuses on the spoilage process and the key metabolic attributes of the main psychrotrophic spoilage Pseudomonas species to explain their predominance on meat over other psychrotrophic bacteria. This review also highlights less studied, but important, characteristics of psychrotrophic pseudomonads such as biofilm formation and quorum sensing in the context of meat spoilage. The importance of the use of model systems that are closely applicable to the food industry is also discussed in detail.

PCR screening of antimicrobial resistance genes in faecal samples from Australian and Chinese children.

OBJECTIVES: Recent public awareness campaigns on the risk of antibiotic resistance in pathogenic microbes has placed pressure on governments to enforce stricter antimicrobial stewardship policies on hospitals and the agricultural industry. In this study, faecal samples from Australian and Chinese children were screened for the presence of antimicrobial resistance genes (ARGs) in order to identify demographics at risk of carriage of these genes and to examine antimicrobial stewardship policies from the two countries that may influence carriage. METHODS: Faecal samples from 46 Australian and 53 Chinese children were screened by PCR for the presence of six clinically relevant ARGs. Clinical and demographic data were also collected from each patient. RESULTS: More than 90% of faecal samples from Chinese children tested positive for ß-lactam, macrolide, tetracycline and aminoglycoside resistance genes, which was substantially higher than Australian samples. Besides country of origin, no clear trend could be seen to predict carriage of ARGs. The exception to this was Chinese-born children who immigrated to Australia having higher rates of carriage of blaTEM and tetM genes than children born and still living in Australia. CONCLUSIONS: These data indicate that Chinese children are more likely to carry certain ARGs than Australian children. The Chinese government has recently implemented strict policies to control the overuse of antibiotics in hospitals. However, many of these policies do not extend to the agricultural industry, which could explain the differences seen in this study.

Integration of Emerging Biomedical Technologies in Meat Processing to Improve Meat Safety and Quality.

Modern-day processing of meat products involves a series of complex procedures designed to ensure the quality and safety of the meat for consumers. As the size of abattoirs increases, the logistical problems associated with large-capacity animal processing can affect the sanitation of the facility and the meat products, potentially increasing transmission of infectious diseases. Additionally, spoilage of food from improper processing and storage increases the global economic and ecological burden of meat production. Advances in biomedical and materials science have allowed for the development of innovative new antibacterial technologies that have broad applications in the medical industry. Additionally, new approaches in tissue engineering and nondestructive cooling of biological specimens could significantly improve organ transplantation and tissue grafting. These same strategies may be even more effective in the preservation and protection of meat as animal carcasses are easier to manipulate and do not have the same stringent requirements of care as living patients. This review presents potential applications of emerging biomedical technologies in the food industry to improve meat safety and quality. Future research directions investigating these new technologies and their usefulness in the meat processing chain along with regulatory, logistical, and consumer perception issues will also be discussed.

In Vitro Antimicrobial Efficacy of Tobramycin Against Staphylococcus aureus Biofilms in Combination With or Without DNase I and/or Dispersin B: A Preliminary Investigation.

Staphylococcus aureus in biofilms is highly resistant to the treatment with antibiotics, to which the planktonic cells are susceptible. This is likely to be due to the biofilm creating a protective barrier that prevents antibiotics from accessing the live pathogens buried in the biofilm. S. aureus biofilms consist of an extracellular matrix comprising, but not limited to, extracellular bacterial DNA (eDNA) and poly-ß-1, 6-N-acetyl-d-glucosamine (PNAG). Our study revealed that despite inferiority of dispersin B (an enzyme that degrades PNAG) to DNase I that cleaves eDNA, in dispersing the biofilm of S. aureus, both enzymes were equally efficient in enhancing the antibacterial efficiency of tobramycin, a relatively narrow-spectrum antibiotic against infections caused by gram-positive and gram-negative pathogens, including S. aureus, used in this investigation. However, a combination of these two biofilm-degrading enzymes was found to be significantly less effective in enhancing the antimicrobial efficacy of tobramycin than the individual application of the enzymes. These findings indicate that combinations of different biofilm-degrading enzymes may compromise the antimicrobial efficacy of antibiotics and need to be carefully assessed in vitro before being used for treating medical devices or in pharmaceutical formulations for use in the treatment of chronic ear or respiratory infections.

Efficacy of Antibacterial Peptides Against Peptide-Resistant MRSA Is Restored by Permeabilization of Bacteria Membranes.

Clinical application of antimicrobial peptides (AMPs), as with conventional antibiotics, may be compromised by the development of bacterial resistance. This study investigated AMP resistance in methicillin resistant Staphylococcus aureus, including aspects related to the resilience of the resistant bacteria toward the peptides, the stability of resistance when selection pressures are removed, and whether resistance can be overcome by using the peptides with other membrane-permeabilising agents. Genotypically variant strains of S. aureus became equally resistant to the antibacterial peptides melittin and bac8c when grown in sub-lethal concentrations. Subculture of a melittin-resistant strain without melittin for 8 days lowered the minimal lethal concentration of the peptide from 170 µg ml-1 to 30 µg ml-1. Growth for 24 h in 12 µg ml-1 melittin restored the MLC to 100 µg ml-1. Flow cytometry analysis of cationic fluorophore binding to melittin-naïve and melittin-resistant bacteria revealed that resistance coincided with decreased binding of cationic molecules, suggesting a reduction in nett negative charge on the membrane. Melittin was haemolytic at low concentrations but the truncated analog of melittin, mel12-26, was confirmed to lack haemolytic activity. Although a previous report found that mel12-26 retained full bactericidal activity, we found it to lack significant activity when added to culture medium. However, electroporation in the presence of 50 µg ml-1 of mel12-26, killed 99.3% of the bacteria. Similarly, using a low concentration of the non-ionic detergent Triton X-100 to permeabilize bacteria to mel12-26 markedly increased its bactericidal activity. The observation that bactericidal activity of the non-membranolytic peptide mel12-26 was enhanced when the bacterial membrane was permeablized by detergents or electroporation, suggests that its principal mechanism in reducing bacterial survival may be through interaction with intracellular organelles or processes. Additionally, our results showed that the haemolytic peptide bac8c, had increased antibacterial activity at non-haemolytic concentrations when used with membrane-permeabilizing surfactants.