Assessment of bacteriocin production by clinical Pseudomonas aeruginosa isolates and their potential as therapeutic agents.

INTRODUCTION: Bacterial infections and the rising antimicrobial resistance pose a significant threat to public health. Pseudomonas aeruginosa produces bacteriocins like pyocins, especially S-type pyocins, which are promising for biological applications. This research focuses on clinical P. aeruginosa isolates to assess their bacteriocin production, inhibitory spectrum, chemical structure, antibacterial agents, and preservative potential. METHODS: The identification of P. aeruginosa was conducted through both phenotypic and molecular approaches. The inhibitory spectrum and antibacterial potential of the isolates were assessed. The kinetics of antibacterial peptide production were investigated, and the activity of bacteriocin was quantified in arbitrary units (AU ml-1). Physico-chemical characterization of the antibacterial peptides was performed. Molecular weight estimation was carried out using SDS-PAGE. qRT-PCR analysis was employed to validate the expression of the selected candidate gene. RESULT: The antibacterial activity of P. aeruginosa was attributed to the secretion of bacteriocin compounds, which belong to the S-type pyocin family. The use of mitomycin C led to a significant 65.74% increase in pyocin production by these isolates. These S-type pyocins exhibited the ability to inhibit the growth of both Gram-negative (P. mirabilis and P. vulgaris) and Gram-positive (S. aureus, S. epidermidis, E. hirae, S. pyogenes, and S. mutans) bacteria. The molecular weight of S-type pyocin was 66 kDa, and its gene expression was confirmed through qRT-PCR. CONCLUSION: These findings suggest that S-type pyocin hold significant potential as therapeutic agents against pathogenic strains. The Physico-chemical resistance of S-type pyocin underscores its potential for broad applications in the pharmaceutical, hygiene, and food industries.

In vitro assessment of the effect of magnetic fields on efficacy of biosynthesized selenium nanoparticles by Alborzia kermanshahica.

Cyanobacteria represent a rich resource of a wide array of unique bioactive compounds that are proving to be potent sources of anticancer drugs. Selenium nanoparticles (SeNPs) have shown an increasing potential as major therapeutic platforms and led to the production of higher levels of ROS that can present desirable anticancer properties. Chitosan-SeNPs have also presented antitumor properties against hepatic cancer cell lines, especially the Cht-NP (Chitosan-NPs), promoting ROS generation and mitochondria dysfunction. It is proposed that magnetic fields can add new dimensions to nanoparticle applications. Hence, in this study, the biosynthesis of SeNPs using Alborzia kermanshahica and chitosan (CS) as stabilizers has been developed. The SeNPs synthesis was performed at different cyanobacterial cultivation conditions, including control (without magnetic field) and magnetic fields of 30 mT and 60 mT. The SeNPs were characterized by uv-visible spectroscopy, Fourier-transform infrared spectroscopy (FT-IR), Dynamic light scattering (DLS), zeta potential, and TEM. In addition, the antibacterial activity, inhibition of bacterial growth, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC), as well as the antifungal activity and cytotoxicity of SeNPs, were performed. The results of uv-visible spectrometry, DLS, and zeta potential showed that 60 mT had the highest value regarding the adsorption, size, and stabilization in compared to the control. FTIR spectroscopy results showed consistent spectra, but the increased intensity of peaks indicates an increase in bond number after exposure to 30 mT and 60 mT. The results of the antibacterial activity and the inhibition zone diameter of synthesized nanoparticles showed that Staphylococcus aureus was more sensitive to nanoparticles produced under 60 mT. Se-NPs produced by Alborzia kermanshahica cultured under a 60 mT magnetic field exhibit potent antimicrobial and anticancer properties, making them a promising natural agent for use in the pharmaceutical and biomedical industries.

Silver nanoparticle biosynthesis utilizing Ocimum kilimandscharicum leaf extract and assessment of its antibacterial activity against certain chosen bacteria.

The use of plants in the biological production of silver nanoparticles for antibacterial applications is a growing field of research. In the current work, we formulated Ocimum kilimandscharicum extracts using silver nanoparticles, and evaluated its potential antibacterial activity. Aqueous and methanol plant extracts were used to reduce silver nitrate at different time intervals (30 to 150 minutes) and pH (2 to 11). The UV-visible absorption spectrum recorded for methanol and aqueous extracts revealed a successful synthesis of AgNPs for methanol and aqueous extracts. The antimicrobial activity of the AgNPs was evaluated against Escherichia coli ATCC 25922, Salmonella choleraesuius ATCC 10708, and Staphylococcus aureus ATCC 25923 The best inhibition zone for the methanol and aqueous-mediated AgNPs, ranging from 12 ± 1 to 16 ± 1mm. Additionally, the methanol and aqueous extract silver nanoparticles had the same Minimum Inhibitory Concentration (6.25 ± 0.00 mg/ml), whereas the Minimum Bactericidal Concentrations were 12.5 ± 0.00 and 25 ± 0.00 mg/ml, respectively. The highest inhibition zone of 16 ± 1 mm was observed against Salmonella choleraesuius with 50 ± 0.00 mg/ml aqueous silver nanoparticles. The results show that the silver nanoparticles made with Ocimum kilimandscharicum have antibacterial action against those microorganisms.

Curation, inference, and assessment of a globally reconstructed gene regulatory network for Streptomyces coelicolor.

Streptomyces coelicolor A3(2) is a model microorganism for the study of Streptomycetes, antibiotic production, and secondary metabolism in general. Even though S. coelicolor has an outstanding variety of regulators among bacteria, little effort to globally study its transcription has been made. We manually curated 29 years of literature and databases to assemble a meta-curated experimentally-validated gene regulatory network (GRN) with 5386 genes and 9707 regulatory interactions (~ 41% of the total expected interactions). This provides the most extensive and up-to-date reconstruction available for the regulatory circuitry of this organism. Only ~ 6% (534/9707) are supported by experiments confirming the binding of the transcription factor to the upstream region of the target gene, the so-called "strong" evidence. While for the remaining interactions there is no confirmation of direct binding. To tackle network incompleteness, we performed network inference using several methods (including two proposed here) for motif identification in DNA sequences and GRN inference from transcriptomics. Further, we contrasted the structural properties and functional architecture of the networks to assess the reliability of the predictions, finding the inference from DNA sequence data to be the most trustworthy approach. Finally, we show two applications of the inferred and the curated networks. The inference allowed us to propose novel transcription factors for the key Streptomyces antibiotic regulatory proteins (SARPs). The curated network allowed us to study the conservation of the system-level components between S. coelicolor and Corynebacterium glutamicum. There we identified the basal machinery as the common signature between the two organisms. The curated networks were deposited in Abasy Atlas ( https://abasy.ccg.unam.mx/ ) while the inferences are available as Supplementary Material.

Recombinant production of Trx-Ib-AMP4 and Trx-E50-52 antimicrobial peptides and antimicrobial synergistic assessment on the treatment of methicillin-resistant Staphylococcus aureus under in vitro and in vivo situations.

PURPOSE: The production of alternative novel antimicrobial agents is considered an efficient way to cope with multidrug resistance among pathogenic bacteria. E50-52 and Ib-AMP4 antimicrobial peptides (AMPs) have illustrated great proven antibacterial effects. The aim of this study was recombinant production of these AMPs and investigation of their synergistic effects on methicillin-resistant Staphylococcus aureus (MRSA). METHOD: At first, the codon optimized sequences of the Ib-AMP4 (UniProt: 024006 (PRO_0000020721), and E50-52 (UniProtKB: P85148) were individually ligated into the pET-32α vector and transformed into E. coli. After the optimization of production and purification steps, the MIC (Minimum inhibitory concentration), time kill and growth kinetic tests of recombinant proteins were determined against MRSA. Finally, the in vivo wound healing efficiency was tested. RESULTS AND CONCLUSION: The recorded MIC of recombinant Trx-Ib-AMP4, Trx-E50-52 against MRSA bacterium were 0.375 and 0.0875 mg/mL respectively. The combination application of the produced AMPs by the checkerboard method confirmed their synergic activity. The results of the time-kill showed sharply decrease of the number of viable cells with over five time reductions in log10 CFU/mL by the combination of Trx-E50-52 and Trx-IbAMP4 at 2 × MIC within 240 min. The growth kinetic results confirmed the combination of Trx-E50-52 and Trx-IbAMP4 had much greater success in the reduction of over 50 % of MRSA suspensions' turbidity within the first hour. Wound healing assay and histological analysis of infected mice treated with Trx-Ib-AMP4 or Trx-E50-52 compared with those treated with a combination of Trx-Ib-AMP4 and Trx-E50-52 showed significant synergic effects.

Utilization of Crab Waste for Cost-Effective Bioproduction of Prodigiosin.

This study aimed to establish the culture process for the cost-effective production of prodigiosin (PG) from demineralized crab shell powder (de-CSP), a fishery processing byproduct created via fermentation. Among the tested PG-producing strains, Serratia marcescens TNU02 was demonstrated to be the most active strain. Various ratios of protein/de-CSP were used as the sources of C/N for PG biosynthesis. The PG yield was significantly enhanced when the casein/de-CSP ratio was controlled in the range of 3/7 to 4/6. TNU02 produced PG with a high yield (5100 mg/L) in a 15 L bioreactor system containing 4.5 L of a newly-designed liquid medium containing 1.6% C/N source (protein/de-CSP ratio of 3/7), 0.02% (NH4)2SO4, 0.1% K2HPO4, and an initial pH of 6.15, at 27 °C for 8 h in dark conditions. The red pigment was purified from the culture broth and then quantified as being PG by specific Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry (MALDI-TOF MS) and UV spectra analysis. The purified PG demonstrated moderate antioxidant and effective inhibition against four cancerous cell lines. Notably, this study was the first to report on using crab wastes for PG bioproduction with high-level productivity (5100 mg/L) in a large scale (4.5 L per pilot) in a short period of fermentation time (8 h). The salt compositions, including (NH4)2SO4 and K2HPO4, were also a novel finding for the enhancement of PG yield by S. marcescens in this report.

Inactivating pathogenic bacteria in greywater by biosynthesized Cu/Zn nanoparticles from secondary metabolite of Aspergillus iizukae; optimization, mechanism and techno economic analysis.

The inactivation of antibiotic resistant Escherichia coli (Gram negative) and Staphylococcus aureus (Gram positive) seeded in greywater by bimetallic bio-nanoparticles was optimized by using response surface methodology (RSM). The bimetallic nanoparticles (Cu/Zn NPs) were synthesized in secondary metabolite of a novel fungal strain identified as Aspergillus iizukae EAN605 grown in pumpkin medium. Cu/Zn NPs were very effective for inhibiting growth of E. coli and S. aureus. The maximum inactivation was optimized with 0.028 mg mL-1 of Cu/Zn NPs, at pH 6 and after 60 min, at which the reduction of E. coli and S. aureus was 5.6 vs. 5.3 and 5.2 vs. 5.4 log reduction for actual and predicted values, respectively. The inactivation mechanism was described based on the analysis of untreated and treated bacterial cells by Field emission scanning electron microscopy (FESEM), Energy Dispersive X-Ray Spectroscopy (EDS), Atomic Force Microscopy (AFM) revealed a damage in the cell wall structure due to the effect of Cu/Zn NPs. Moreover, the Raman Spectroscopy showed that the Cu/Zn NPs led to degradation of carbohydrates and amino structures on the bacteria cell wall. The Fourier transform infrared spectroscopy (FTIR) analysis confirmed that the destruction take place in the C-C bond of the functional groups available in the bacterial cell wall. The techno economic analysis revealed that the biosynthesis Cu/Zn NPs is economically feasible. These findings demonstrated that Cu/Zn NPs can effectively inhibit pathogenic bacteria in the greywater.

Green synthesis, characterization and antibacterial activity of silver nanoparticles by Malus domestica and its cytotoxic effect on (MCF-7) cell line.

This article reports the utilization of Malus domestica for the synthesis of silver nanoparticles (AgNPs) with cytotoxic activity against the Michigan Cancer Foundation-7 (MCF-7) cell line as well as their antibacterial and radical scavenging potential. The biosynthesized AgNPs were confirmed using various analytical characterization techniques. The cytotoxic effect of Malus domestica-AgNPs (M.d-AgNPs) was studied by MTT assay and scavenging efficacy was assessed by DPPH, nitric oxide radical and phosphomolybdate assays. Furthermore, green synthesized nanoparticles were evaluated for their antibacterial activity against multidrug resistant-clinical isolates. M.d-AgNPs were observed to be almost spherical in shape with an average diameter from 50 to 107.3 nm as assessed by TEM and DLS. M.d-AgNPs revealed the dose-dependent antioxidant activity and antimicrobial activity against multidrug-resistant bacterial strain viz. Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Escherichia coli. Also, in vitro studies revealed dose-dependent cytotoxic effects of M.d-AgNPs treated MCF-7 cell line. The data strongly suggest that M.d-AgNPs had a potential antioxidant, antimicrobial and cytotoxicity activity.

Bio-engineering and cellular imaging of silver nanoparticles as weaponry against multidrug resistant human pathogens.

'Go green' has also been implied to nanotechnology by harbouring eco-benign principle for a cleaner production of silver nanoparticles (AgNPs). This was achieved using a nitrate reducing Bacillus subtilis L1 (KT266579.1) inhabiting rhizosphere soil under optimized laboratory conditions, highlighting on its antibacterial modus operandi. Nano-characteristics and antimicrobial mechanism were investigated using spectroscopic and electron microscopic studies. Spectroscopic and microscopic characterization revealed typical surface plasmon resonance (SPR) with λmax 420 nm showing mean particle size of ~28.30 nm and spherical shaped nanoparticles. Antimicrobial susceptibility pattern of clinically important pathogens (n = 15) exposed to AgNPs at 10 µg, 15 µg and 20 µg/mL for 18 h was found significant in a dose dependent fashion. Electron and atomic force microscopic (AFM) studies have demonstrated the typical bactericidal effect of AgNPs (<25 µg/mL) associated with 'pitting effect', cell shrinkage and increase in surface roughness. The EDX spectrum of the control and treated bacteria showed the intrusion of AgNPs inside the bacterial cells endorsing the event of bacterial paralysis. DNA fragmentation assay demonstrated significant DNA damage in the form of smear, indicative of genotoxicity at ≤32 µg and ≤16 µg/mL of AgNPs respectively for Gram positive and negative strains in <12 h. These results suggest that AgNPs possess excellent antimicrobial activity, providing a potential lead for developing a broad spectrum antibacterial agent and extending its therapeutic modalities targeting antibiotic resistant strains at gene level.

Assessment of Microbiological Safety of Water in Public Swimming Pools in Guangzhou, China.

This study assessed microbiological safety of water from public swimming pools in Guangzhou, China. Water samples from 39 outdoor municipal swimming pools were collected from late June to early September, 2013 and subjected to detection of protozoa (Giardia and Cryptosporidium) and bacteria (Pseudomonas aeruginos, total coliforms, E. coli, E. coli O157, Shigella, and Salmonella). Cryptosporidium and Giardia were both detected in 5 (12.8%) swimming pools. Total coliforms were detected in 4 (10.3%) samples with concentrations ranging from 1.3 to 154.0 MPN/100 mL while E. coli was detected in 4 (10.3%) samples with concentrations ranging from 0.5 to 5.3 MPN/100 mL. P. aeruginosa was detected in 27 (69.2%) samples but E. coli O157, Shigella and Salmonella were not detected. Among these swimming pools, 9 (23%) met the Chinese National Standard of residual chlorine levels and 24 (62%) were tested free of residual chlorine at least once. The multi-locus sequence typing (MLST) analysis showed that all P. aeruginosa isolates belonged to new sequence types (STs) with dominant ST-1764 and ST-D distributed in different locations within the area. Some P. aeruginosa strains were resistant to medically important antibiotics. Results indicate potential public health risks due to the presence of microbiological pathogens in public swimming pools in this area.

Bioprocessing strategies for cost-effective large-scale biogenic synthesis of nano-MgO from endophytic Streptomyces coelicolor strain E72 as an anti-multidrug-resistant pathogens agent.

In this report, the local nano-MgO synthesizer strain has been isolated from Ocimum sanctum plant and deposited in GenBank as endophytic Streptomyces coelicolor strain E72. Its intracellular metabolic fraction that contains 7.2 µg/µl of carbohydrate, 6.3 g/l of protein and 5.2 nmol/hr/ml of nitrate reductase used to produce multi-surface shaped nano-MgO with diameter ~25 nm. To the best of our knowledge, this is the first report using statistical nanobiotechnological strategies (Plackett -Burman, Box-Behnken and Taguchi experimental designs) to study and evaluate the endophytic S. coelicolor biomass production (123.3 g/l) and extract the highest bioactive metabolites that used for biogenic synthesis of nano-MgO (320 g/l) through exponential sucrose pulses feeding fermentation strategy after 192 hr in semi industrial scale bioreactor (7 L). Purified nano-MgO applied in vitro against multi-drug resistant human pathogens and the large inhibition zone recorded against Shigella flexneri (108 ± 10.53 mm). The average of MICs was recorded as 25 µg/ml that inhibited 90% of the pathogenic living cells and compared with 100 mg/ml ampicilin/sulbactam solution that killed 40% of the same pathogen. These results are expected to gather sufficient knowledge to discover and develop a new cheap and eco-friendly nano-MgO as an extremely strong antimicrobial agent used in biomedical applications.

Toward the Drug Factory Microbiome: Microbial Community Variations in Antibiotic-Producing Clean Rooms.

Microbiome projects are currently booming around the globe, enabled by advances in culture-independent microbial community analysis and high-throughput sequencing. One emerging application of microbiome science involves exploring microbial diversity in built environments, and one unexplored built environment is the pharmaceutical factory, notably factories producing antibiotics, as they could be enriched in antibiotic-resistant microbes. To examine the drug factory microbiome, we launched this interdisciplinary hypothesis-generating study to benchmark culture-independent microbiome analysis in drug manufacturing units producing antibiotics and nonantibiotic drugs, against traditional microbial identification and quantification techniques. Over a course of 4 months, we prospectively collected 234 samples from antibiotic (kanamycin and amoxicillin) and nonantibiotic (acetaminophen) production clean rooms within a pharmaceutical factory in Egypt. All samples were analyzed by traditional culture-based methods, and microbial communities of representative samples were profiled by16S rRNA gene sequencing. In addition, antibiotic resistance profiles of some samples were determined, and representative resistance genes were screened. The 16S rRNA analysis revealed a typical predominance of Proteobacteria (36%), Firmicutes (31%), and Bacteroidetes (16%). The microbial composition of the samples was highly affected by the use of water, environmental conditions during the production process, the presence of personnel, and the type of the product. The effect of these factors was confirmed by total aerobic microbial counts and identification of biomarker microbes. In conclusion, these observations can aid in the future for optimal design and management of pharmaceutical manufacturing units, and speak to a greater need for implementing microbiome research in the quality assurance of built environments.

Sakacin-A antimicrobial packaging for decreasing Listeria contamination in thin-cut meat: preliminary assessment.

BACKGROUND: Minimally processed ready-to-eat products are considered a high-risk food because of the possibility of contamination with pathogenic bacteria, including Listeria monocytogenes from the animal reservoir, and the minimal processing they undergo. In this study, a sakacin-A anti-Listeria active package was developed and tested on thin-cut veal meat slices (carpaccio). RESULTS: Enriched food-grade sakacin-A was obtained from a cell-free supernatant of a Lactobacillus sakei culture and applied (0.63 mg cm-2 ) onto the surface of polyethylene-coated paper sheets to obtain an active antimicrobial package. The coating retained antimicrobial features, indicating that the process did not affect sakacin-A functionality, as evidenced in tests carried out in vitro. Thin-cut veal meat slices inoculated with Listeria innocua (a surrogate of pathogenic L. monocytogenes) were laid on active paper sheets. After 48 h incubation at 4 °C, the Listeria population was found to be 1.5 log units lower with respect to controls (3.05 vs 4.46 log colony-forming units (CFU) g-1 ). CONCLUSION: This study demonstrates the possibility of using an antimicrobial coating containing sakacin-A to inhibit or decrease the Listeria population in ready-to-eat products, thus lowering the risk of food-related diseases. © 2016 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The next generation of antimicrobial peptides (AMPs) as molecular therapeutic tools for the treatment of diseases with social and economic impacts.

Anti-infective drugs have had a key role in the contemporary world, contributing to dramatically decrease mortality rates caused by infectious diseases worldwide. Antimicrobial peptides (AMPs) are multifunctional effectors of the innate immune system of mucosal surfaces and present antimicrobial activity against a range of pathogenic viruses, bacteria, and fungi. However, the discovery and development of new antibacterial drugs is a crucial step to overcome the great challenge posed by the emergence of antibiotic resistance. In this review, we outline recent advances in the development of novel AMPs with improved antimicrobial activities that were achieved through characteristic structural design. In addition, we describe recent progress made to overcome some of the major limitations that have hindered peptide biosynthesis.

Production of a polar fish antimicrobial peptide in Escherichia coli using an ELP-intein tag.

An important aspect related to infectious pathogens is their exceptional adaptability in developing resistance, which leads to a perpetual challenge in the discovery of antimicrobial drugs with novel mechanisms of action. Among them, antimicrobial peptides (AMPs) stand out as promising anti-infective molecules. In order to overcome the high costs associated with isolation from natural sources or chemical synthesis of AMPs we propose the expression of Pa-MAP 2, a polyalanine AMP. Pa-MAP 2 was fused to an ELP-intein tag where the ELP (Elastin-like polypeptide) was used to promote aggregation and fast and cost-effective isolation after expression, and the intein was used to stimulate a controlled AMP release. For these, the vector pET21a was used to produce Pa-MAP 2 fused to the N-termini region of a modified Mxe GyrA intein followed by 60 repetitions of ELP. Purified Pa-MAP 2 showed a MIC of 25µM against E. coli ATCC 8739. Batch fermentation demonstrated that Pa-MAP-2 can be produced in both rich and defined media at yields 50-fold higher than reported for other AMPs produced by the ELP-intein system, and in comparable yields to expression systems with protease or chemical cleavage.

Lanthipeptides: chemical synthesis versus in vivo biosynthesis as tools for pharmaceutical production.

Lanthipeptides (also called lantibiotics for those with antibacterial activities) are ribosomally synthesized post-translationally modified peptides having thioether cross-linked amino acids, lanthionines, as a structural element. Lanthipeptides have conceivable potentials to be used as therapeutics, however, the lack of stable, high-yield, well-characterized processes for their sustainable production limit their availability for clinical studies and further pharmaceutical commercialization. Though many reviews have discussed the various techniques that are currently employed to produce lanthipeptides, a direct comparison between these methods to assess industrial applicability has not yet been described. In this review we provide a synoptic comparison of research efforts on total synthesis and in vivo biosynthesis aimed at fostering lanthipeptides production. We further examine current applications and propose measures to enhance product yields. Owing to their elaborate chemical structures, chemical synthesis of these biomolecules is economically less feasible for large-scale applications, and hence biological production seems to be the only realistic alternative.

Microbial degradation of chitin waste for production of chitosanase and food related bioactive compounds.

Ecological samples rich in microbial diversity like cow dung, legume rhizosphere, fish waste and garden soil were used for isolation of chitosan-degrading microorganisms. Selected isolates were used for production of chitosanase and food related bioactive compounds by conversion of biowaste. Production of glucosamine (Gln), N-acetylglucosamine (NAG), chitooligosaccharides (COS), antioxidants, antibacterial compounds and prebiotics was carried out by microbial fermentation of biowaste. The highest chitosanase activity (8 U/mL) was observed in Aspergillus sp. isolated from fish market waste and it could produce Gln and NAG while Streptomyces sp. isolated from garden soil was able to produce COS along with Gln and NAG. Radical scavenging activity was observed in culture supernatants of 35% of studied isolates, and 20% isolates secreted compounds which showed positive effect on growth of Bifidobacterium. Antibacterial compounds were produced by 40% of selected isolates and culture supernatants of two microbial isolates, Streptomyces zaomyceticus C6 and one of garden soil isolates, were effective against both gram positive and negative bacteria.

Association between vancomycin trough concentration and area under the concentration-time curve in neonates.

National treatment guidelines for invasive methicillin-resistant Staphylococcus aureus (MRSA) infections recommend targeting a vancomycin 24-h area under the concentration-time curve (AUC0-24)-to-MIC ratio of >400. The range of vancomycin trough concentrations that best predicts an AUC0-24 of >400 in neonates is not known. This understanding would help clarify target trough concentrations in neonates when treating MRSA. A retrospective chart review from a level III neonatal intensive care unit was performed to identify neonates treated with vancomycin over a 5-year period. Vancomycin concentrations and clinical covariates were utilized to develop a one-compartment population pharmacokinetic model and examine the relationships between trough and AUC0-24 in the study neonates. Monte Carlo simulations were performed to examine the effect of dose, postmenstrual age (PMA), and serum creatinine level on trough and AUC0-24 achievement. A total of 1,702 vancomycin concentrations from 249 neonates were available for analysis. The median (interquartile range) PMA was 39 weeks (32 to 42 weeks) and weight was 2.9 kg (1.6 to 3.7 kg). Vancomycin clearance was predicted by weight, PMA, and serum creatinine level. At a trough of 10 mg/liter, 89% of the study neonates had an AUC0-24 of >400. Monte Carlo simulations demonstrated that troughs ranging from 7 to 11 mg/liter were highly predictive of an AUC0-24 of >400 across a range of PMA, serum creatinine levels, and vancomycin doses. However, a trough of ≥10 mg/liter was not readily achieved in most simulated subgroups using routine starting doses. Higher starting doses frequently resulted in troughs of >20 mg/liter. A vancomycin trough of ∼10 mg/liter is likely adequate for most neonates with invasive MRSA infections based on considerations of the AUC0-24. Due to pharmacokinetic and clinical heterogeneity in neonates, consistently achieving this target vancomycin exposure with routine starting doses is difficult. More robust clinical dosing support tools are needed to help clinicians with dose individualization.