Eravacycline activity against clinical <Emphasis Type="Italic">S. aureus</Emphasis> isolates from China: in vitro activity,MLST profiles and heteroresistance

Background

Mortality rates for patients with Staphylococcus aureus (S. aureus) infections have improved only modestly in recent decades and S. aureus infections remain a major clinical challenge This study investigated the in vitro antimicrobial activity of erevacycline (erava) against clinical S. aureus isolates from China, as well as the heteroresistance frequency of erava and sequence types (STs) represented in the sample.

Results

A sample of 328 non-duplicate clinical S. aureus isolates, including 138 methecillin-resistant (MRSA) and 190 methecillin-sensitive (MSSA) isolates, were collected retrospectively in China. Erava exhibited excellent in vitro activity (MIC₅₀ ≤?0.25?mg/L) against MRSA and MSSA, including isolates harboring Tet specific resistance genes. The frequency of erava heteroresistance in MSSA with erava MICs?=?0.5?mg/L was 13.79% (4/29); no MRSA with erava MICs ≤0.5?mg/L exhibited heteroresistance. Heteroresistance- derived clones had no 30S ribosome subunit mutations, but their erava MICs (range, 1–4?mg/L) were suppressed dramatically in the presence of efflux protein inhibitors.

Conclusions

Conclusively, erava exhibited excellent in vitro activity against S. aureus, however hints of erava heteroresistance risk and MIC creep were detected, particularly among MSSA with MICs of 0.5?mg/L.

     

Effects of Lectins on initial attachment of cariogenic <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Oral bacteria initiate biofilm formation by attaching to tooth surfaces via an interaction of a lectin-like bacterial protein with carbohydrate chains on the pellicle. This study aimed to find naturally derived lectins that inhibit the initial attachment of a cariogenic bacterial species, Streptococcus mutans (S. mutans), to carbohydrate chains in saliva in vitro. Seventy kinds of lectins were screened for candidate motifs that inhibit the attachment of S. mutans ATCC 25175 to a saliva-coated culture plate. The inhibitory effect of the lectins on attachment of the S. mutans to the plates was quantified by crystal violet staining, and the biofilm was observed under a scanning electron microscope (SEM). Surface plasmon resonance (SPR) analysis was performed to examine the binding of S. mutans to carbohydrate chains and the binding of candidate lectins to carbohydrate chains, respectively. Moreover, binding assay between the biotinylated-lectins and the saliva components was conducted to measure the lectin binding. Lectins recognizing a salivary carbohydrate chain, Galβ1-3GalNAc, inhibited the binding of S. mutans to the plate. In particular, Agaricus bisporus agglutinin (ABA) markedly inhibited the binding. This inhibition was confirmed by SEM observation. SPR analysis indicated that S. mutans strongly binds to Galβ1-3GalNAc, and ABA binds to Galβ1-3GalNAc. Finally, the biotinylated Galβ1-3GalNAc-binding lectins including ABA demonstrated marked binding to the saliva components. These results suggest that ABA lectin inhibited the attachment of S. mutans to Galβ1-3GalNAc in saliva and ABA can be useful as a potent inhibitor for initial attachment of oral bacteria and biofilm formation.     

Enzyme-linked lectinosorbent assay of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In this study, we developed a microplate sandwich analysis of Escherichia coli and Staphylococcus aureus bacterial pathogens based on the interaction of their cell wall carbohydrates with natural receptors called lectins. An immobilized lectin-cell-biotinylated lectin complex was formed in this assay. Here, we studied the binding specificity of several plant lectins to E. coli and S. aureus cells, and pairs characterized by high-affinity interactions were selected for the assay. Wheat germ agglutinin and Ricinus communis agglutinin were used to develop enzyme-linked lectinosorbent assays for E. coli and S. aureus cells with the detection limits of 4 × 10⁶ and 5 × 10⁵ cells/mL, respectively. Comparison of the enzyme-linked immonosorbent assay and the enzyme-linked lectinosorbent assay demonstrated no significant differences in detection limit values for E. coli. Due to the accessibility and universality of lectin reagents, the proposed approach is a promising tool for the control of a wide range of bacterial pathogens.     

Madurastatin B3, a rare aziridine derivative from actinomycete <Emphasis Type="Italic">Nocardiopsis</Emphasis> sp. LS150010 with potent anti-tuberculosis activity

Since the discovery of the first antibiotic, natural products have played an important role in chemistry, biology and medicine. To explore the potential of bioactive compounds from microbes isolated from the southeast of Tibet, China, a crude extract library was constructed and screened against Staphylococcus aureus. The strain Nocardiopsis sp. LS150010 was scaled up and subjected to further chemical studies, resulting in the identification of N-salicyloyl-2-aminopropan-1,3-diol (2) and its rare aziridine derivative, madurastatin B3 (1). Their structures were determined by detailed analysis of 1D, 2D NMR and HRMS data. Compounds 1 and 2 displayed significant inhibitory activity against S. aureus and methicillin resistant S. aureus, with MIC values of 6.25 µg/mL. Compound 1 also showed potent inhibitory activity against Bacillus subtilis and Escherichia coli, as well as activity in a Mycobacterium tuberculosis Bacillus Calmette-Guérin infected THP-1 cell model.     

The influence of polyhexamethylene guanidine derivatives introduced into polyhydroxybutyrate on biofilm formation and the activity of bacterial enzymes

Escherichia coli and Staphylococcus aureus were able to produce biofilm on the surface of polyhydroxybutyrate (PHB), but their abundance depended on type and the concentrations of the polyhexamethylene guanidine (PHMG) derivatives introduced in PHB. Different types of PHMG derivatives inhibited S. aureus ATCC 6538P biofilm formation, but PHB with PHMG salt of sulfanilic acid stimulated E. coli ATCC 8739 biofilm formation. The presence of all PHMG derivatives decreased significantly the number of viable cells of the test bacteria directly proportional to the concentration of the biocidal agent. PHMG derivatives affected the activity of microbiological hydrolases with different degrees. Some of them (PHB with PHMG stearate) stimulated activity of E. coli ATCC 8739 hydrolases, other (PHB with the PHMG salt of sulfanilic acid) inhibited activity of the S. aureus ATCC 6538P hydrolases. The PHMG derivatives introduced in PHB also inhibited the activity of bacterial dehydrogenases.     

<Emphasis Type="Italic">Staphylococcus simulans</Emphasis> recombinant lysostaphin: Production,purification, and determination of antistaphylococcal activity

Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by SigmaAldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.     

Screening and molecular identification of lactic acid bacteria from <Emphasis Type="Italic">gari</Emphasis> and <Emphasis Type="Italic">fufu</Emphasis> and <Emphasis Type="Italic">gari</Emphasis> effluents

Bacterial strains were isolated from cassava-derived food products and, for the first time, from cassava by-products, with a focus on gari, a flour-like product, and the effluents from the production processes for gari and fufu (a dough also made from cassava flour). A total of 47 strains were isolated, all of which were tested to determine their resistance to acidic pH and to bile salt environments. Four of the 47 isolates tested positive in both environments, and these four isolates also showed antibacterial behaviour towards both Gram-positive and Gram-negative microbial pathogens (i.e. Methicillin-resistance Staphylococcus aureus, Listeria monocytogenes, Bacillus cereus, Salmonella enteritidis, Escherichia coli, Escherichia coli (O157), Yersinia enterocolitica). In most cases, the antibacterial activity was related to bacteriocin production. Molecular identification analysis (16S rDNA and randomly amplified polymorphic DNA-PCR) revealed that the four isolates were different strains of the same species, Lactobacillus fermentum. These results demonstrate that bacteria isolated from cassava-derived food items and cassava by-products have interesting properties and could potentially be used as probiotics.     

Site specific <Emphasis Type="Italic">N</Emphasis>-glycan profiling of NeuAc(α2-6)-Gal/GalNAc-binding bark <Emphasis Type="Italic">Sambucus nigra</Emphasis> agglutinin using LC–MS<Superscript>n</Superscript> revealed differential glycosylation

The bark of Sambucus nigra contains a complex mixture of glycoproteins that are characterized as chimeric lectins known as type II ribosome inactivating proteins and holo lectins. These type II ribosome inactivating proteins possess RNA N-glycosidase activity in subunit A and lectin activity associated with subunit B exhibiting distinct sugar specificities to NeuAc(α2-6)-Gal/GalNAc and Gal/GalNAc. In the present study we have determined the N-glycosylation pattern of type II ribosome inactivating protein specific to NeuAc(α2-6)-Gal/GalNAc (Sambucus nigra agglutinin I) by subjecting it to digestion with multiple proteases. The resulting mixture of peptides and N-glycopeptides were analyzed on liquid chromatography coupled to electro spray ionization-iontrap mass spectrometry in MSⁿ mode. MS² of precursor ions was carried out using CID which provided information on glycan sequence. In subsequent MS³ of Y₁/Y_1α ions (peptide + HexNAc)⁺ⁿ of corresponding N-glycopeptides, resulted in the fragmentation of peptide backbone confirming the site of attachment. We observed microheterogeneity in each glycan occupied site with subunit A possessing four N-glycans out of six sites with complex and paucimannose types while subunit B comprises occupancy of two sites with a paucimannose and a high mannose type. The differential N-glycosylation of subunits in SNA is discussed in the context of other type II RIPs glycans.     

Activity of novel inhibitors of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> biofilms

Staphylococcus aureus is one of the most important pathogens causing chronic biofilm infections. These are becoming more difficult to treat owing to drug resistance, particularly because S. aureus biofilms limit the efficacy of antimicrobial agents, leading to high morbidity and mortality. In the present study, we screened for inhibitors of S. aureus biofilm formation using a natural product library from the Korea Chemical Bank (KCB). Screening by crystal violet-based biomass staining assay identified hit compounds. Further examination of antibiofilm properties of these compounds was conducted and led to the identification of celastrol and telithromycin. In vitro, both celastrol and telithromycin were toxic to planktonic S. aureus and also active against a clinical methicillin-resistant S. aureus (MRSA) isolate. The effect of the compounds on preformed biofilms of clinical MRSA isolates was evaluated by confocal laser scanning microscopy (CLSM), which revealed the absence of typical biofilm architecture. In addition, celastrol and telithromycin inhibited the production of extracellular protein at selected sub-MIC concentrations, which revealed the reduced extracellular polymeric substance (EPS) secretion. Celastrol exhibited greater cytotoxicity than telithromycin. These data suggest that the hit compounds, especially telithromycin, could be considered novel inhibitors of S. aureus biofilm. Although the mechanisms of the effects on S. aureus biofilms are not fully understood, our data suggest that telithromycin could be a useful adjuvant therapeutic agent for S. aureus biofilm-related infections.     

Characterization of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates from pediatric patients with cystic fibrosis

Staphylococcus aureus is one of the major respiratory pathogens associated with cystic fibrosis (CF) patients. In this study, we collected sputum and isolated fifty S. aureus isolates from CF patients with the median age of 9.5 years old. Then we determined the profiles of these isolates by antibiotic susceptibility testing, examining their cytotoxicity and ability to internalize into an epithelial cell line (A549), as well as multiple loci sequencing typing. Predominant CF S. aureus isolates were resistant to penicillin; however, these isolates were sensitive to various antibiotics, such as vancomycin and minocycline. Different CF S. aureus isolates showed distinct cytotoxic activities, and 90 % of CF S. aureus isolates possessed the enterotoxin genes, sea and hlg. Moreover, we found that multiple different CF S. aureus isolates appeared to have the distinct capacity of invading A549 cells. ST5 (14 %), ST30 (14 %), and ST8 (10 %) were prevalent ST types in these isolates. Further analysis revealed that ST5 and ST30 isolates were less toxic than ST8 and ST15 isolates, and that the ST5, ST15, ST59, and ST87 types of CF S. aureus were less capable of invading A549 cells. Our results suggest that the ST typing method may be useful in predicting cytotoxicity and the invading capacity of S. aureus isolates from patients with CF.     

Survey of potential factors involved in the low frequency of CP5 and CP8 expression in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolates from mastitis of dairy cattle from Argentina,Chile, and Uruguay

Staphylococcus aureus produces capsular polysaccharides (CPs) both in vivo and under defined culture conditions being serotypes 5 and 8 the most prevalent. S. aureus isolates that fail to produce CP5 or CP8 are defined as non-typeable (NT). Loss of capsule expression, however, may lead to S. aureus persistence in a chronically infected host. The prevalence of NT strains of S. aureus isolated from bovine mastitis varies according to the geographic origin of the strain. The aims of this work were to detect phenotypically and genotypically the capsular profile of 144 S. aureus isolated from bovine mastitis in Argentina, Chile, and Uruguay and explore the factors that are considered to be associated with capsule expression as presence of IS257, IScap, and agr typing of non-related collection. The detection of the IS257, IScap, cap genes, and agr typing was performed using PCR. The detection and quantification of capsular polysaccharide production were performed by ELISA assays. We found that 96% of the S. aureus isolates investigated carried cap5(8) genes but over 75% of strains do not express capsule in the three countries studied. However, only 6 isolates from Argentina carried the IScap element that totally suppressed the expression of the capsule, suggesting that other factors could influence on CP expression. Moreover, the agrI/NT association was statistically significant suggesting that this profile is a phenomenon observed not only in other parts of the world but also in our region.     

Characterization of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in Goose Feces from State Parks in Northeast Ohio

Staphylococcus aureus can colonize a range of species. Although numerous studies have isolated pathogenic bacteria from wild birds, very little is known regarding S. aureus and their potential to spread methicillin-resistant (MRSA) strains. The objective of this study was to determine the presence and molecular characteristics of S. aureus in geese fecal samples collected from ten state parks across Northeast Ohio (NEO). A total of 182 fecal samples from Canada geese (Branta canadensis) were collected in April 2015. Isolates were characterized using multi-locus sequence (MLST) and spa typing, as well as PCR to detect the presence of Panton–Valentine leukocidin (PVL), mecA, and scn genes. Antibiotic susceptibility testing was done via Vitek-2 system. The overall contamination by S. aureus in fecal samples was 7.1% (13/182); 7/182 (3.8%) were MRSA and 6/182 (3.3%) were methicillin-susceptible S. aureus (MSSA). One isolate was positive for PVL. A total of eight different spa types were observed. MLST included ST5, ST8, ST291, ST298, and ST2111. One (7.7%) MSSA isolate was multi-drug resistant. The S. aureus contamination in NEO state parks ranged from 0% (park 1, 4, 8, 9) to 35% (7/20) (park 5). Parks 2, 3, 6, and 7 had 5% (1/20) positive. The results of this study indicate that the feces of geese collected at various state parks in NEO may harbor S. aureus.     

Additional Nucleotide Sequences in Precursor 16S Ribosomal RNA from <Emphasis Type="Italic">Escherichia coli</Emphasis>

MATURE 5S, 16S and 23S ribosomal RNA species present in E. coli ribosomes are the end products of complex biosyn-thetic pathways. They are formed by reduction in length, and methylation of longer RNA chains transcribed on the ribosomal RNA cistrons of E. coli DNA. While these modifications take place the ribosome structure is formed by progressive addition of ribosomal proteins and conformational changes in the resulting ribonucleoprotein precursor particles¹.     

Steroid Compounds from Far Eastern Starfishes <Emphasis Type="Italic">Henricia aspera</Emphasis> and <Emphasis Type="Italic">H. tumida</Emphasis>

Six new natural compounds were isolated from two Far Eastern starfish species, Henricia aspera and H. tumida, collected in the Sea of Okhotsk. Two new glycosylated steroid polyols were obtained from H. aspera: asperoside A and asperoside B, which were shown to be (20R,24R, 25S)-3-O-(2,3-di-O-methyl-β -D-xylopyranosyl)-24-methyl-5α-cholest-4-ene-3β, 6β,8,15α,16β,26-hexaol and (20R, 24R,25S,22E)-3-O-(2,4-di-O-methyl-β-D-xylopyranosyl)-24-methyl-5α-cholest-22-ene-3β,4β,6β,8,15α,26-hexaol, respectively. Two other glycosylated polyols, tumidoside A, with the structure elucidated as (20R, 22E)-3-O-(2,4-di-O-methyl-β -D-xylopyranosyl)-26,27-dinor-24-methyl-5α-cholest-22-ene-3β,4β,6β,8,15α,25-hexaol, and tumidoside B, whose structure was elucidated as (20R,24S)-3-O-(2,3-di-O-methyl-β-D-xylopyranosyl)-5α-cholestan-3β,4β,6β,8,15α,24-hexaol, were isolated from the two starfish species. (20R, 24S)-5α-Cholestan-3β,6β,15α,24-tetraol and (20R, 24S)-5α-cholestan-3β,6β,8,15α,24-pentaol were identified only in H. tumida. The known monoglycosides henricioside H1 and laeviuscolosides H and G were also identified in both species.     

Dynamics of antagonistic potency of <Emphasis Type="Italic">Rhodobacter capsulatus</Emphasis> PG lipopolysaccharide against endotoxin-induced effects

The dynamics of antagonistic potency of lipopolysaccharide (LPS) isolated from Rhodobacter capsulatus PG on the synthesis of proinflammatory (TNF-α, IL-1β, IL-8, IL-6, IFN-γ) and antiinflammatory (IL-10, IL-1Ra) cytokines induced by highly stimulatory endotoxins from Escherichia coli or Salmonella enterica have been studied. Using human whole blood, we have shown that R. capsulatus PG LPS inhibited most pronouncedly the endotoxin-induced synthesis of TNF-α, IL-1β, IL-8, and IL-6 during the first 6 h after endotoxin challenge. Similarly, the endotoxin-induced release of IFN-γ was abolished by R. capsulatus PG LPS as well (24 h). In contrast to the above-mentioned cytokines, the relatively weak antagonistic activity of R. capsulatus PG LPS against endotoxin-triggered production of IL-6 and IL-8 was revealed. Since R. capsulatus PG LPS displays more potent antagonistic activity against deleterious effects of S. enterica LPS than those of E. coli LPS in the cases of such cytokines as IL-1β (6 and 24 h), IL-6 and IL-8 (4 h), we conclude that the effectiveness of protective action of antagonist is mostly determined by the primary lipid A structure of the employed agonist.     

Engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for the synthesis of <Emphasis Type="Italic">N</Emphasis>-hydroxycinnamoyl tryptamine and serotonin

Plants synthesize various phenol amides. Among them, hydroxycinnamoyl (HC) tryptamines and serotonins exhibit antioxidant, anti-inflammatory, and anti-atherogenic activities. We synthesized HC–tryptamines and HC–serotonin from several HCs and either tryptamine or serotonin using Escherichia coli harboring the 4CL (4-coumaroyl CoA ligase) and CaHCTT [hydroxycinnamoyl-coenzyme A:serotonin N-(hydroxycinnamoyl)transferase] genes. E. coli was engineered to synthesize N-cinnamoyl tryptamine from glucose. TDC (tryptophan decarboxylase) and PAL (phenylalanine ammonia lyase) along with 4CL and CaHCTT were introduced into E. coli and the phenylalanine biosynthetic pathway of E. coli was engineered. Using this strategy, approximately 110.6 mg/L of N-cinnamoyl tryptamine was synthesized. By feeding 100 μM serotonin into the E. coli culture, which could induce the synthesis of cinnamic acid or p-coumaric acid, more than 99 μM of N-cinnamoyl serotonin and N-(p-coumaroyl) serotonin were synthesized.     

Engineering a <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> biosynthesis pathway into <Emphasis Type="Italic">Escherichia coli</Emphasis> for high yield triglyceride production

Background

Microbial lipid production represents a potential alternative feedstock for the biofuel and oleochemical industries. Since Escherichia coli exhibits many genetic, technical, and biotechnological advantages over native oleaginous bacteria, we aimed to construct a metabolically engineered E. coli strain capable of accumulating high levels of triacylglycerol (TAG) and evaluate its neutral lipid productivity during high cell density fed-batch fermentations.

Results

The Streptomyces coelicolor TAG biosynthesis pathway, defined by the acyl-CoA:diacylglycerol acyltransferase (DGAT) Sco0958 and the phosphatidic acid phosphatase (PAP) Lppβ, was successfully reconstructed in an E. coli diacylglycerol kinase (dgkA) mutant strain. TAG production in this genetic background was optimized by increasing the levels of the TAG precursors, diacylglycerol and long-chain acyl-CoAs. For this we carried out a series of stepwise optimizations of the chassis by 1) fine-tuning the expression of the heterologous SCO0958 and lpp β genes, 2) overexpression of the S. coelicolor acetyl-CoA carboxylase complex, and 3) mutation of fadE, the gene encoding for the acyl-CoA dehydrogenase that catalyzes the first step of the β-oxidation cycle in E. coli. The best producing strain, MPS13/pET28-0958-ACC/pBAD-LPPβ rendered a cellular content of 4.85% cell dry weight (CDW) TAG in batch cultivation. Process optimization of fed-batch fermentation in a 1-L stirred-tank bioreactor resulted in cultures with an OD_600nm of 80 and a product titer of 722.1 mg TAG L^-1 at the end of the process.

Conclusions

This study represents the highest reported fed-batch productivity of TAG reached by a model non-oleaginous bacterium. The organism used as a platform was an E. coli BL21 derivative strain containing a deletion in the dgkA gene and containing the TAG biosynthesis genes from S. coelicolor. The genetic studies carried out with this strain indicate that diacylglycerol (DAG) availability appears to be one of the main limiting factors to achieve higher yields of the storage compound. Therefore, in order to develop a competitive process for neutral lipid production in E. coli, it is still necessary to better understand the native regulation of the carbon flow metabolism of this organism, and in particular, to improve the levels of DAG biosynthesis.

     

Isolation and characterization of butanol-tolerant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Objectives

A new solvent-tolerant species, Staphylococcus aureus, was isolated and characterized during the screening of butanol-tolerant microorganisms.

Results

Three isolates of S. aureus were obtained as contaminants during improvement of butanol tolerance of E. coli K12. Their cell dry weights were 135 % that of K12 in the absence of butanol stress. S. aureus had a growth advantage over K12 when cultured with various concentrations of butanol. It can tolerate up to 3 % (v/v) butanol, while most solventogenic bacteria can tolerate only 2 % (v/v) butanol. The addition of 10–20 g glucose/l enhanced its butanol tolerance. The relative cell biomass of the S. aureus was 71–306 % that of E. coli under 5.5–10 % (v/v) ethanol stress, indicating ethanol resistance.

Conclusions

This is the first study to observe butanol-tolerant S. aureus. As this organism can be genetically manipulated, it could have a wide array of applications.

     

New approaches to modeling <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> inactivation by ultrasound

Ultrasound (US) is an effective technology to inactivate vegetative microorganisms in foods. In this study, the effect of amplitude levels (0.4, 7.5, and 37.5), duty cycles (0.3:0.7 s, 0.7:0.3 s, and 0.9: 0.1 s) and time (0, 2, 4, 6, 8, 10, 12, and 14 days) of US on inactivation of Staphylococcus aureus were investigated. In addition, genetic algorithm-artificial neural network (GA-ANN) and adaptive neuro-fuzzy inference system (ANFIS) models were used to predict inactivation of S. aureus. The GA-ANN and ANFIS were fed with three inputs of amplitude levels, duty cycles, and time. The inactivation rate of S. aureus was increased by increasing the amplitude levels, and the best inactivation was obtained at a 37.5 μm amplitude for which the S. aureus population was reduced to 2.59 CFU/mL. The high inactivation of S. aureus was achieved under a duty cycle of 0.7:0.3 s with reduction of the population to 1.49 CFU/mL. The developed GA-ANN, which included 17 hidden neurons, could predict the S. aureus population with a coefficient of determination of 0.986. The overall agreement between ANFIS predictions and experimental data was also very good (R ² ?=?0.979). Sensitivity analysis results showed that the amplitude level was the most sensitive factor for prediction of S. aureus.