Revolutionizing orthopedic healthcare: a systematic review unveiling recombinant antimicrobial peptides.

The increasing demand for orthopedic surgeries, including joint replacements, is driven by an aging population and improved diagnosis of joint conditions. Orthopedic surgeries carry a risk of infection, especially in patients with comorbidities. The rise of antibiotic resistance exacerbates this issue, necessitating alternatives like in vitro bioengineered antimicrobial peptides (AMPs), offering broad-spectrum activity and multiple action mechanisms. This review aimed to assess the prevalence of antimicrobial potential and the yield after purification among recombinant AMP families. The antimicrobial potential was evaluated using the Minimum Inhibitory Concentration (MIC) values against the most common bacteria involved in clinical infections. This systematic review adhered to PRISMA guidelines, focusing on in vitro studies of recombinant AMPs. The search strategy was run on PubMed, Scopus and Embase up to 30th March 2023. The Population, Exposure and Outcome model was used to extract the data from studies and ToxRTool for the risk of bias analysis. This review included studies providing peptide production yield data and MIC values against pathogenic bacteria. Non-English texts, reviews, conference abstracts, books, studies focusing solely on chemical synthesis, those reporting incomplete data sets, using non-standard MIC assessment methods, or presenting MIC values as ranges rather than precise concentrations, were excluded. From 370 publications, 34 studies on AMPs were analyzed. These covered 46 AMPs across 18 families, with Defensins and Hepcidins being most common. Yields varied from 0.5 to 2,700 mg/L. AMPs were tested against 23 bacterial genera, with MIC values ranging from 0.125 to >1,152 µg/mL. Arenicins showed the highest antimicrobial activity, particularly against common orthopedic infection pathogens. However, AMP production yields varied and some AMPs demonstrated limited effectiveness against certain bacterial strains. This systematic review emphasizes the critical role of bioengineered AMPs to cope infections and antibiotic resistance. It meticulously evaluates recombinant AMPs, focusing on their antimicrobial efficacy and production yields. The review highlights that, despite the variability in AMP yields and effectiveness, Arenicins and Defensins are promising candidates for future research and clinical applications in treating antibiotic-resistant orthopedic infections. This study contributes significantly to the understanding of AMPs in healthcare, underscoring their potential in addressing the growing challenge of antibiotic resistance. Systematic review registration:https://osf.io/2uq4c/.

Association between the Presence of Resistance Genes and Sanitiser Resistance of Listeria monocytogenes Isolates Recovered from Different Food-Processing Facilities.

Sanitisers are widely used in cleaning food-processing facilities, but their continued use may cause an increased resistance of pathogenic bacteria. Several genes have been attributed to the increased sanitiser resistance ability of L. monocytogenes. This study determined the presence of sanitiser resistance genes in Irish-sourced L. monocytogenes isolates and explored the association with phenotypic sanitiser resistance. The presence of three genes associated with sanitiser resistance and a three-gene cassette (mdrL, qacH, emrE, bcrABC) were determined in 150 L. monocytogenes isolates collected from Irish food-processing facilities. A total of 23 isolates contained bcrABC, 42 isolates contained qacH, one isolate contained emrE, and all isolates contained mdrL. Additionally, 47 isolates were selected and grouped according to the number and type of resistance genes, and the minimal inhibitory concentration (MIC) of these isolates for benzalkonium chloride (BAC) was determined experimentally using the broth microdilution method. The BAC resistance of the strain carrying the bcrABC gene cassette was significantly higher than that of strains lacking the gene cassette, and the BAC resistance of the strain carrying the qacH gene was significantly higher than that of strains lacking the qacH gene (p < 0.05). Isolates harbouring both the qacH and bcrABC genes did not show higher BAC resistance. With respect to environmental factors, there was no significant difference in MIC values for isolates recovered from different processing facilities. In summary, this investigation highlights the prevalence of specific sanitiser resistance genes in L. monocytogenes isolates from Irish food-processing settings. While certain genes correlated with increased resistance to benzalkonium chloride, the combination of multiple genes did not necessarily amplify this resistance.

Diversity of the Microbiota of Traditional Izmir Tulum and Izmir Brined Tulum Cheeses and Selection of Potential Probiotics.

High-throughput DNA sequencing (HTS) was used to study the microbial diversity of commercial traditional Izmir Tulum (IT) and Izmir Brined Tulum (IBT) cheeses from Izmir, Türkiye. Simultaneously, cultivation-dependent methods were used to isolate, identify and characterize bacterial strains displaying probiotic potential. At the phylum level, Firmicutes dominated the microbiota of both cheese types comprising >98% of the population. Thirty genera were observed, with Streptococcus being the most abundant genus and with Streptococcus thermophilus and S. infantarius subsp. infantarius being the most abundant species. Genera, including Bifidobacterium and Chryseobacterium, not previously associated with IT and IBT, were detected. IT cheeses displayed higher operational taxonomic units (OTUs; Richness) and diversity index (Simpson) than IBT cheeses; however, the difference between the diversity of the microbiota of IT and IBT cheese samples was not significant. Three Lacticaseibacillus paracasei strains isolated from IBT cheeses exhibited probiotic characteristics, which included capacity to survive under in vitro simulated gastrointestinal conditions, resistance to bile salts and potential to adhere to HT-29 human intestinal cells. These findings demonstrate that Tulum cheeses harbor bacterial genera not previously reported in this cheese and that some strains display probiotic characteristics.

Association of Virulence, Biofilm, and Antimicrobial Resistance Genes with Specific Clonal Complex Types of Listeria monocytogenes.

Precise classification of foodborne pathogen Listeria monocytogenes is a necessity in efficient foodborne disease surveillance, outbreak detection, and source tracking throughout the food chain. In this study, a total of 150 L. monocytogenes isolates from various food products, food processing environments, and clinical sources were investigated for variations in virulence, biofilm formation, and the presence of antimicrobial resistance genes based on their Whole-Genome Sequences. Clonal complex (CC) determination based on Multi-Locus Sequence Typing (MLST) revealed twenty-eight CC-types including eight isolates representing novel CC-types. The eight isolates comprising the novel CC-types share the majority of the known (cold and acid) stress tolerance genes and are all genetic lineage II, serogroup 1/2a-3a. Pan-genome-wide association analysis by Scoary using Fisher's exact test identified eleven genes specifically associated with clinical isolates. Screening for the presence of antimicrobial and virulence genes using the ABRicate tool uncovered variations in the presence of Listeria Pathogenicity Islands (LIPIs) and other known virulence genes. Specifically, the distributions of actA, ecbA, inlF, inlJ, lapB, LIPI-3, and vip genes across isolates were found to be significantly CC-dependent while the presence of ami, inlF, inlJ, and LIPI-3 was associated with clinical isolates specifically. In addition, Roary-derived phylogenetic grouping based on Antimicrobial-Resistant Genes (AMRs) revealed that the thiol transferase (FosX) gene was present in all lineage I isolates, and the presence of the lincomycin resistance ABC-F-type ribosomal protection protein (lmo0919_fam) was also genetic-lineage-dependent. More importantly, the genes found to be specific to CC-type were consistent when a validation analysis was performed with fully assembled, high-quality complete L. monocytogenes genome sequences (n = 247) extracted from the National Centre for Biotechnology Information (NCBI) microbial genomes database. This work highlights the usefulness of MLST-based CC typing using the Whole-Genome Sequence as a tool in classifying isolates.

Antimicrobial Resistance Genes Analysis of Publicly Available Staphylococcus aureus Genomes.

Staphylococcus aureus is a pathogen that can cause severe illness and express resistance to multiple antimicrobial agents. It is part of the ESKAPE organisms and it has been included by the Centers for Disease Control and Prevention (CDC) of USA in the list of serious threats to humans. Many antimicrobial mechanisms have been identified, and, in particular, antimicrobial resistance genes (ARGs) can be determined by whole genome sequencing. Mobile genetic elements (MGEs) can determine the spread of these ARGs between strains and species and can be identified with bioinformatic analyses. The scope of this work was to analyse publicly available genomes of S. aureus to characterise the occurrence of ARGs present in chromosomes and plasmids in relation to their geographical distribution, isolation sources, clonal complexes, and changes over time. The results showed that from a total of 29,679 S. aureus genomes, 24,765 chromosomes containing 73 different ARGs, and 21,006 plasmidic contigs containing 47 different ARGs were identified. The most abundant ARG in chromosomes was mecA (84%), while blaZ was the most abundant in plasmidic contigs (30%), although it was also abundant in chromosomes (42%). A total of 13 clonal complexes were assigned and differences in ARGs and CC distribution were highlighted among continents. Temporal changes during the past 20 years (from 2001 to 2020) showed that, in plasmids, MRSA and macrolide resistance occurrence decreased, while the occurrence of ARGs associated with aminoglycosides resistance increased. Despite the lack of metadata information in around half of the genomes analysed, the results obtained enable an in-depth analysis of the distribution of ARGs and MGEs throughout different categories to be undertaken through the design and implementation of a relatively simple pipeline, which can be also applied in future works with other pathogens, for surveillance and screening purposes.

Integration of genomics in surveillance and risk assessment for outbreak investigation.

Keeping food safe is a challenge that needs continuous surveillance for the sake of consumers' health. The main issue when a food-borne pathogen outbreak occurs is represented by the identification of the source(s) of contamination. Delivering this information in a timely manner helps to control the problem, with positive outcomes for everyone, especially for the consumers, whose health is in this way preserved, and for the stakeholders involved in food production and distribution, who could face enormous economic losses if recalls or legal issues occur. Whole genome sequencing (WGS) is a tool recently implemented for the characterisation of isolates and the study of outbreaks because of its higher efficiency and faster results, when compared to traditional typing methods. Lower sequencing costs and the development of many bioinformatic tools helped its spread, and much more attention has been given to its use for outbreak investigation. It is important to reach a certain level of standardisation, though, for ensuring result reproducibility and interoperability. Moreover, nowadays it is possible, if not mandatory for Open Science Practices, to share WGS data in publicly available databases, where raw reads, assembled genomes and their corresponding metadata can be easily found and downloaded. The scope of this Fellowship was to provide the Fellow all the training necessary for successfully integrating genomics to surveillance and risk assessment of food-borne pathogens from farm to fork.

Correlation of organic acid tolerance and genotypic characteristics of Listeria monocytogenes food and clinical isolates.

A collection of Listeria monocytogenes isolates from various food products, food processing environments and clinical sources (n = 153) were evaluated for their tolerance to acetic, lactic and propionic acids. A large variation in tolerance was observed amongst isolates under mildly acidic conditions (pH 5.3) for acetic (5-20 mM undissociated acid) and propionic acid (2-10 mM undissociated acid) but there was less variation for lactic acid (3-6 mM undissociated acid). Analysis of the isolate genome sequences for a complement of genes previously shown to have a role in acid tolerance revealed that thiT, gadT2, gadD2 and gadD3 genes were linked to higher acetic acid tolerance (P < 0.05) while lisRK was linked to higher tolerance to propionic acid (P = 1 × 10-11). An absence of plasmid genes was also linked with isolates showing higher tolerance for all acids. Scoary GWAS analysis revealed that a total of 333, 207, and 333 genes were associated with acid tolerance for acetic, lactic, and propionic acid, respectively (P < 0.05). However, the p-value adjusted with Bonferroni's method for multiple comparisons did not reveal any significant associations. Isolates were grouped into clonal complexes (CC) using Multi Locus Sequence Typing (MLST) and MIC values for the three acids were determined for representative strains. One complex, CC18, showed significantly higher (P ≤ 0.05) acetic and propionic acid MIC values than other groups, whereas only CC7 type isolates revealed significantly higher (P ≤ 0.001) lactic acid MIC values. The results demonstrate that MLST typing could be linked to acid tolerance phenotypic traits which is important in predicting the behaviour of L. monocytogenes in food products.

Inhibition of Listeria monocytogenes by Phage Lytic Enzymes Displayed on Tailored Bionanoparticles.

The high mortality rate associated with Listeria monocytogenes and its ability to adapt to the harsh conditions employed in food processing has ensured that this pathogen remains a serious problem in the ready-to-eat food sector. Bacteriophage-derived enzymes can be applied as biocontrol agents to target specific foodborne pathogens. We investigated the ability of a listeriophage endolysin and derivatives thereof, fused to polyhydroxyalkanoate bionanoparticles (PHA_BNPs), to lyse and inhibit the growth of L. monocytogenes. Turbidity reduction assays confirmed the lysis of L. monocytogenes cells at 37 °C upon addition of the tailored BNPs. The application of BNPs also resulted in the growth inhibition of L. monocytogenes. BNPs displaying only the amidase domain of the phage endolysin were more effective at inhibiting growth under laboratory conditions (37 °C, 3 × 107 CFU/mL) than BNPs displaying the full-length endolysin (89% vs. 83% inhibition). Under conditions that better represent those found in food processing environments (22 °C, 1 × 103 CFU/mL), BNPs displaying the full-length endolysin demonstrated a greater inhibitory effect compared to BNPs displaying only the amidase domain (61% vs. 54% inhibition). Our results demonstrate proof-of-concept that tailored BNPs displaying recombinant listeriophage enzymes are active inhibitors of L. monocytogenes.

Variability in Cold Tolerance of Food and Clinical Listeria monocytogenes Isolates.

The aim of this study was to investigate the level of strain variability amongst food and clinical Listeria monocytogenes isolates growing at low temperatures (4 and 7 °C) in both laboratory media and real food matrices. Isolates (n = 150) grown in laboratory media demonstrated a large variation in growth profiles measured using optical density. Overall, it was noted that clinical isolates exhibited a significantly higher growth rate (p ≤ 0.05) at 7 °C than the other isolates. Analysis of variance (ANOVA) tests of isolates grouped using Multi Locus Sequence Typing (MLST) revealed that clonal complex 18 (CC18) isolates were significantly (p ≤ 0.05) faster growing at 4 °C than other CC-type isolates while CC101, CC18, CC8, CC37 and CC14 were faster growing than other CC types at 7 °C. Euclidean distance and Ward method-based hierarchical clustering of mean growth rates classified 33.33% of isolates as faster growing. Fast and slow growing representative isolates were selected from the cluster analysis and growth rates were determined using plate count data in laboratory media and model food matrices. In agreement with the optical density experiments, CC18 isolates were faster and CC121 isolates were slower than other CC types in laboratory media, UHT milk and fish pie. The same trend was observed in chocolate milk but the differences were not statistically significant. Moreover, pan-genome analysis (Scoary) of isolate genome sequences only identified six genes of unknown function associated with increased cold tolerance while failing to identify any known cold tolerance genes. Overall, an association that was consistent in laboratory media and real food matrices was demonstrated between isolate CC type and increased cold tolerance.

Omnibus Modeling of Listeria monocytogenes Growth Rates at Low Temperatures.

Listeria monocytogenes is a pathogen of considerable public health importance with a high case fatality. L. monocytogenes can grow at refrigeration temperatures and is of particular concern for ready-to-eat foods that require refrigeration. There is substantial interest in conducting and modeling shelf-life studies on L. monocytogenes, especially relating to storage temperature. Growth model parameters are generally estimated from constant-temperature growth experiments. Traditionally, first-order and second-order modeling (or primary and secondary) of growth data has been done sequentially. However, omnibus modeling, using a mixed-effects nonlinear regression approach, can model a full dataset covering all experimental conditions in one step. This study compared omnibus modeling to conventional sequential first-order/second-order modeling of growth data for five strains of L. monocytogenes. The omnibus model coupled a Huang primary model for growth with secondary models for growth rate and lag phase duration. First-order modeling indicated there were small significant differences in growth rate depending on the strain at all temperatures. Omnibus modeling indicated smaller differences. Overall, there was broad agreement between the estimates of growth rate obtained by the first-order and omnibus modeling. Through an appropriate choice of fixed and random effects incorporated in the omnibus model, potential errors in a dataset from one environmental condition can be identified and explored.

Evaluation of lactic acid bacteria strains isolated from fructose-rich environments for their mannitol-production and milk-gelation abilities.

Mannitol is a sugar alcohol, or polyol, widely used in the food industry because of its low-calorie properties. Industrial production of mannitol is difficult and expensive. However, certain bacterial species are known to produce mannitol naturally, including certain lactic acid bacteria and fructophilic lactic acid bacteria (LAB). In this study, bacterial strains isolated from fructose-rich sources, including flowers, leaves, and honey, were identified by 16S rRNA sequence analysis as Leuconostoc, Fructobacillus, Lactococcus, and Lactobacillus species and 4 non-LAB species. DNA profiles generated by pulsed-field gel electrophoresis discriminated 32 strains of Leuconostoc mesenteroides and 6 Fructobacillus strains. Out of 41 LAB strains isolated, 32 were shown to harbor the mdh gene, which encodes the mannitol dehydrogenase enzyme, and several showed remarkable fructose tolerance even at 50% fructose concentrations, indicating their fructophilic nature. Several of the strains isolated, including Leuconostoc mesenteroides strains DPC 7232 and DPC 7261, Fructobacillus fructosus DPC 7237, and Fructobacillus fructosus DPC 7238, produced higher mannitol concentrations than did the positive control strain Limosilactobacillus reuteri DSM 20016 during an enzymatic screening assay. Mannitol concentrations were also examined via HPLC in 1% fructose de Man, Rogosa, and Sharpe medium (FMRS) or 1% fructose milk (FM). Among the strains, Fructobacillus fructosus DPC 7238 displayed high fructose utilization (9.27 g/L), high mannitol yield (0.99 g of mannitol/g of fructose), and greatest volumetric productivities (0.46 g/L per h) in FMRS. However, Leuconostoc mesenteroides DPC 7261 demonstrated the highest fructose utilization (8.99 g/L), mannitol yield (0.72 g of mannitol/g of fructose), and volumetric productivities (0.04 g/L per h) in FM. Storage modulus G' (>0.1 Pa) indicated a shorter gelation time for Limosilactobacillus reuteri DSM 20016 (8.73 h), followed by F. fructosus DPC 7238 (11.57 h) and L. mesenteroides DPC 7261 (14.52 h). Our results show that fructose-rich niches can be considered important sources of fructophilic LAB strains, with the potential to be used as starter cultures or adjunct cultures for the manufacture of mannitol-enriched fermented dairy products and beverages.

Effectiveness of current hygiene practices on minimization of Listeria monocytogenes in different mushroom production-related environments.

BACKGROUND: The commercial production of Agaricus bisporus is a three stage process: 1) production of compost, also called "substrate"; 2) production of casing soil; and 3) production of the mushrooms. Hygiene practices are undertaken at each stage: pasteurization of the substrate, hygiene practices applied during the production of casing soil, postharvest steam cookout, and disinfection at the mushroom production facilities. However, despite these measures, foodborne pathogens, including Listeria monocytogenes, are reported in the mushroom production environment. In this work, the presence of L. monocytogenes was evaluated before and after the application of hygiene practices at each stage of mushroom production with swabs, samples of substrate, casing, and spent mushroom growing substrates. RESULTS: L. monocytogenes was not detected in any casing or substrate sample by enumeration according to BS EN ISO 11290-2:1998. Analysis of the substrate showed that L. monocytogenes was absent in 10 Phase II samples following pasteurization, but was then present in 40% of 10 Phase III samples. At the casing production facility, 31% of 59 samples were positive. Hygiene improvements were applied, and after four sampling occasions, 22% of 37 samples were positive, but no statistically significant difference was observed (p > .05). At mushroom production facilities, the steam cookout process inactivated L. monocytogenes in the spent growth substrate, but 13% of 15 floor swabs at Company 1 and 19% of 16 floor swabs at Company 2, taken after disinfection, were positive. CONCLUSION: These results showed the possibility of L. monocytogenes recontamination of Phase III substrate, cross-contamination at the casing production stage and possible survival after postharvest hygiene practices at the mushroom growing facilities. This information will support the development of targeted measures to minimize L. monocytogenes in the mushroom industry.

Prevalence, virulence characterization, and genetic relatedness of Listeria monocytogenes isolated from chicken retail points and poultry slaughterhouses in Turkey.

Listeria monocytogenes is one of the most important foodborne pathogens and is a causal agent of listeriosis in humans and animals. The aim of this study was to determine the prevalence, serogroups, antibiotic susceptibility, virulence factor genes, and genetic relatedness of L. monocytogenes strains isolated from 500 poultry samples in Turkey. The isolation sources of 103 L. monocytogenes strains were retail markets (n = 100) and slaughterhouses (n = 3). L. monocytogenes strains were identified as serogroups 1/2a-3a (75.7%, lineage I), 1/2c-3c (14.56%, lineage I), 1/2b-3b-7 (5.82%, lineage II), 4a-4c (2.91%, lineage III), and 4b-4d-4e (0.97%, lineage III). Most of the L. monocytogenes strains (93.2%) were susceptible to the antibiotics tested. PCR analysis indicated that the majority of the strains (95% to 100%) contained most of the virulence genes (hylA, plcA, plcB, prfA, mpl, actA, dltA, fri, flaA inlA, inlC, and inlJ). Pulsed-field gel electrophoresis (PFGE) demonstrated that there were 18 pulsotypes grouped at a similarity of > 90% among the strains. These results indicate that it is necessary to prevent the presence of L. monocytogenes in the poultry-processing environments to help prevent outbreaks of listeriosis and protect public health.

Inhibition of L. monocytogenes Biofilm Formation by the Amidase Domain of the Phage vB_LmoS_293 Endolysin.

Listeria monocytogenes is a ubiquitous Gram-positive bacterium that is a major concern for food business operators because of its pathogenicity and ability to form biofilms in food production environments. Bacteriophages (phages) have been evaluated as biocontrol agents for L. monocytogenes in a number of studies and, indeed, certain phages have been approved for use as anti-listerial agents in food processing environments (ListShield and PhageGuard Listex). Endolysins are proteins produced by phages in the host cell. They cleave the peptidoglycan cell wall, thus allowing release of progeny phage into the environment. In this study, the amidase domain of the phage vB_LmoS_293 endolysin (293-amidase) was cloned and expressed in Escherichia. coli(E. coli). Muralytic activity at different concentrations, pH and temperature values, lytic spectrum and activity against biofilms was determined for the purified 293-amidase protein. The results showed activity on autoclaved cells at three different temperatures (20 °C, 37 °C and 50 °C), with a wider specificity (L. monocytogenes 473 and 3099, a serotype 4b and serogroup 1/2b-3b-7, respectively) compared to the phage itself, which targets only L. monocytogenes serotypes 4b and 4e. The protein also inhibits biofilm formation on abiotic surfaces. These results show the potential of using recombinant antimicrobial proteins against pathogens in the food production environment.