The microenvironment in antibiotic susceptibility testing.

Antibiotic susceptibility testing (AST) by agar diffusion has been repeatedly standardized and, in most cases, gives results which predict clinical success when antibiotic treatment is based on such results. The formation of the inhibition zone is due to a transition from planktonic to biofilm mode of growth. The kinetics of the interaction of antibiotics with bacteria is similar during AST by agar diffusion and during administration of antibiotics to the patients. However, the Mueller-Hinton agar (MHA) recommended for AST agar diffusion test is fundamentally different from the composition of the interstitial fluid in the human body where the infections take place and human cells do not thrive in MH media. Use of RPMI 1640 medium designed for growth of eucaryotic cells for AST of Pseudomonas aeruginosa against azithromycin results in lower minimal inhibitory concentration, compared to results obtained by MHA. The reason is that the RPMI 1640 medium increases uptake and reduces efflux of azithromycin compared to MHA. During treatment of cystic fibrosis patients with azithromycin, mutational resistance occur which is not detected by AST with MHA. Whether this is the case with other antibiotics and bacteria is not known but it is of clinical importance to be studied.

Conventional methods and future trends in antimicrobial susceptibility testing.

Antimicrobial susceptibility testing is an essential task for selecting appropriate antimicrobial agents to treat infectious diseases. Constant evolution has been observed in methods used in the diagnostic microbiology laboratories. Disc diffusion or broth microdilution are classical and conventional phenotypic methods with long turnaround time and labour-intensive but still widely practiced as gold-standard. Scientists are striving to develop innovative, novel and faster methods of antimicrobial susceptibility testing to be applicable for routine microbiological laboratory practice and research. To meet the requirements, there is an increasing trend towards automation, genotypic and micro/nano technology-based innovations. Automation in detection systems and integration of computers for online data analysis and data sharing are giant leaps towards versatile nature of automated methods currently in use. Genotypic methods detect a specific genetic marker associated with resistant phenotypes using molecular amplification techniques and genome sequencing. Microfluidics and microdroplets are recent addition in the continuous advancement of methods that show great promises with regards to safety and speed and have the prospect to identify and monitor resistance mechanisms. Although genotypic and microfluidics methods have many exciting features, however, their applications into routine clinical laboratory practice warrant extensive validation. The main impetus behind the evolution of methods in antimicrobial susceptibility testing is to shorten the overall turnaround time in obtaining the results and to enhance the ease of sample processing. This comprehensive narrative review summarises major conventional phenotypic methods and automated systems currently in use, and highlights principles of some of the emerging genotypic and micro/nanotechnology-based methods in antimicrobial susceptibility testing.

Evaluating the antibacterial effect of cobalt nanoparticles against multi-drug resistant pathogens.

This study aimed to estimate the effect of cobalt nanoparticles (Co NPs) with different concentrations against multidrug-resistant (MDR) pathogenic bacteria. Three isolates of Staphylococcus aureus (gram-positive), Proteus spp. (gram-negative), and Escherichia coli (gram-negative) bacteria were extracted from various clinical examples utilizing routine methods on bacteriological culture media. The antibacterial sensitivity of commercial antibiotics such as Ciprofloxacin, Cefotaxime, Gentamycin, and Amoxicillin was broken down on a Muller Hinton agar plate and evaluated using the disk diffusion method. The study results demonstrated the antibacterial effect of the Co NPs against the bacterial isolates with three different concentrations utilized in the study. The results indicated that the Co NPs showed the highest antibacterial activity when utilizing 100 µg/ml against Escherichia coli followed by Proteus spp and Staphylococcus aureus with zones of inhibition measured as 22.2±0.1 mm, 20.3±0.15 mm, and 15.8±0.1 mm; respectively. Co NPs at a 100 µg/mL concentration showed higher inhibition zones than several common antibiotics except for Ciprofloxacin, which demonstrated better antibacterial activity against the bacterial isolates employed in this study. Scanning Electron Microscope (SEM)and X-Ray diffraction (XRD)studies confirmed that Cobalt nanoparticles (Co NPs) were synthesized from cobalt sulphate solution with a size ranging from 40 nm to 60 nm. The nanoparticles showed a crystalline structure with a round shape and smooth surface. The antibacterial resistance of Co NPs against three common bacteria such as Staphylococcus aureus, Proteus spp, and Escherichia coli was assessed in this study. The optimum concentration of the Co NPs was identified as 100 µg/ml, which could provide a similar or higher antibacterial effect.

Determination of Colistin Resistance by Simple Disk Diffusion Test Using Modified Mueller-Hinton Agar.

BACKGROUND: Colistin has become a last-resort antibiotic for the management of multidrug-resistant gram-negative bacteria. The disk diffusion test is cheap and easy to perform but may be unreliable for colistin susceptibility testing due to poor diffusion of the large colistin molecule. An improved agar diffusion test would increase the reliability of colistin susceptibility testing. This study aimed to modify Muller-Hinton agar (MHA) to improve colistin diffusion in agar. METHODS: MHA was modified by reducing the agar concentration from 100% to 30% and supplementing with protamine. We tested 60 gram-negative clinical isolates of Pseudomonas aeruginosa (N=27) and Acinetobacter calcoaceticus-baumannii complex (N=33). Disk diffusion test results were interpreted based on minimum inhibitory concentrations determined by broth microdilution. RESULTS: The modified MHA yielded the best performance metrics, including 94.7% sensitivity, 100% specificity, and an area under the curve of 0.995 (95% confidence interval, 0.982-1.000), P<0.001, at a cut-off point of 13 mm. CONCLUSIONS: A reduction of the agar concentration from 100% to 30% and the addition of protamine improved colistin diffusion in agar and allowed routine colistin susceptibility testing in a clinical microbiology laboratory, but should be handled with caution.

Determination of Colistin Resistance by Simple Disk Diffusion Test Using Modified Mueller-Hinton Agar

BACKGROUND: Colistin has become a last-resort antibiotic for the management of multidrug-resistant gram-negative bacteria. The disk diffusion test is cheap and easy to perform but may be unreliable for colistin susceptibility testing due to poor diffusion of the large colistin molecule. An improved agar diffusion test would increase the reliability of colistin susceptibility testing. This study aimed to modify Muller-Hinton agar (MHA) to improve colistin diffusion in agar.METHODS: MHA was modified by reducing the agar concentration from 100% to 30% and supplementing with protamine. We tested 60 gram-negative clinical isolates of Pseudomonas aeruginosa (N=27) and Acinetobacter calcoaceticus-baumannii complex (N=33). Disk diffusion test results were interpreted based on minimum inhibitory concentrations determined by broth microdilution.RESULTS: The modified MHA yielded the best performance metrics, including 94.7% sensitivity, 100% specificity, and an area under the curve of 0.995 (95% confidence interval, 0.982–1.000), P<0.001, at a cut-off point of 13 mm.CONCLUSIONS: A reduction of the agar concentration from 100% to 30% and the addition of protamine improved colistin diffusion in agar and allowed routine colistin susceptibility testing in a clinical microbiology laboratory, but should be handled with caution.

[Trials of the domestically produced nutrient medium «Agar Muller-Hinton II - Obolensk¼.]

The results of the comparative tests of the «Agar Muller-Hinton II - Obolensk¼ nutrient medium developed in SRCAMB, Obolensk, and the control nutrient medium imported «Mueller Hinton II Agar¼ are presented in the study. The susceptibility of bacterial clinical strains to antimicrobial agents (AMP) was determined by the disc diffusion method and the method of gradient diffusion (E-test). The carbapenemase activity of the strains carrying the carbapenemase genes was determined by CIM-test. Total 173 characterized bacterial strains of species Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis, Serratia marcescens, Enterobacter aerogenes, Escherichia coli; Photorhabdus spp., Staphylococcus aureus, Enterococcus spp. were used in the study, including producers of OXA- and NDM-types carbapenemases for gram negative bacteria. A high degree of coincidence of the results obtained on both nutrient media was shown. The consistency index of the strain sensitivity categories to AMPs (S, I, and R) was 98.2% for the disc diffusion method, and 94.4-100% - for E-test and CIM-test methods. Thus, within the framework of the Import Substitution Program, the domestic nutrient medium «MHA II-Obolensk¼ has been successfully developed. The nutrient medium meets the requirements of GOST R ISO 20776-2-2010 «Clinical laboratory testing and in vitro diagnostic test systems - Susceptibility testing of infectious agents and evaluation of performance of antimicrobial susceptibility test devices¼.

Ethno-medicinal uses and screening of plants for antibacterial activity from Similipal Biosphere Reserve, Odisha, India.

ETHNOPHARAMCOLOGICAL RELEVANCE: The present study examined the variety and the extent of medicinal plants used in the health care system of tribal inhabitants of Similipal Biosphere Reserve. In addition to this, such plants were also screened for antibacterial properties against common pathogenic bacteria. MATERIAL AND METHODS: Semi-structured interview was carried out with 42 informants (mean age 42, 86% male, 14% female) at 24 locations in and around SBR, regarding the use of plants for the treatment of various human ailments. Antibacterial screening is adopted with the documented ethnomedicinal plants using methanol and aqueous extracts against eight bacterial strains. RESULTS: A total of 187 plant species belonging to 74 families were documented for frequent medicinal uses against common ailments such as stomach problems, fever, skin diseases, diarrhea and dysentery. Although all parts of plant are used, leaves and bark are most common. Tribals used the plant parts both in form of decoction (taken orally as in internal problems) and paste (external use). Out of 187 plant species, 120 plants recorded antibacterial activity against test bacterial strain. CONCLUSIONS: This study revealed that self care using medicinal plants is a common practice by the tribes of SBR. About 64% of the used plants have scientifically proved medicinal values with respect to the antibacterial properties.

Silver/poly (lactic acid) nanocomposites: preparation, characterization, and antibacterial activity.

In this study, antibacterial characteristic of silver/poly (lactic acid) nanocomposite (Ag/PLA-NC) films was investigated, while silver nanoparticles (Ag-NPs) were synthesized into biodegradable PLA via chemical reduction method in diphase solvent. Silver nitrate and sodium borohydride were respectively used as a silver precursor and reducing agent in the PLA, which acted as a polymeric matrix and stabilizer. Meanwhile, the properties of Ag/PLA-NCs were studied as a function of the Ag-NP weight percentages (8, 16, and 32 wt% respectively), in relation to the use of PLA. The morphology of the Ag/PLA-NC films and the distribution of the Ag-NPs were also characterized. The silver ions released from the Ag/PLA-NC films and their antibacterial activities were scrutinized. The antibacterial activities of the Ag/PLA-NC films were examined against Gram-negative bacteria (Escherichia coli and Vibrio parahaemolyticus) and Gram-positive bacteria (Staphylococcus aureus) by diffusion method using Muller-Hinton agar. The results indicated that Ag/PLA-NC films possessed a strong antibacterial activity with the increase in the percentage of Ag-NPs in the PLA. Thus, Ag/PLA-NC films can be used as an antibacterial scaffold for tissue engineering and medical application.