Agar Dilution Method Research Articles

SPECIATION AND ANTIMICROBIAL SUSCEPTIBILITY OF ENTEROCOCCI FROM PATIENTS IN A TERTIARY CARE HOSPITAL

Introduction: Enterococci have evolved from normal commensals to multidrug resistant pathogen. Enterococci are often reported in association with hospital-acquired infections. Management of Enterococcal infections has become challenging due to the emergence of multidrug resistant strains. Objectives: To speciate and detect the antibiotic susceptibility of Enterococci isolated from different clinical specimens.Materials and Methods: Atotal of 100 Enterococci strains isolated from clinical samples were tested using standard microbiological procedures. The minimum inhibitory concentration (MIC) for vancomycin was tested by agar dilution and E-strip methods. Results: Enterococci infections were more common in age groups, 21-40 years and 41-60 years with male preponderance. Isolates of Enterococci were more frequently associated with post operative wound infection (26%). E. faecalis (55%) was the predominant species, followed by E. faecium (45%). E. faecium showed 80% resistance to penicillin and ampicillin, while E. faecalis showed 38% resistance. All Enterococci isolates were 100% sensitive to vancomycin, linezolid and teicoplanin by the disc diffusion method; however, MIC for vancomycin by agar dilution and E-strip methods showed one strain of E. faecium resistant to vancomycin. Conclusion: Enterococci identication to the species level should be stressed in routine laboratory practice. Vancomycin resistance detection in Enterococci by the MIC method should be preferred as it helps reduce the morbidity and mortality due to Vancomycin resistant Enterococci.

Actividad antifúngica In Vitro de extractos etanólicos de Cnidoscolus urens L. en el control de Colletotrichum spp. en Lycopersicum esculentum: una perspectiva agrícola sostenible

Anthracnose, caused by the pathogenic fungi Colletotrichum spp., poses a significant threat to table tomato (Lycopersicum esculentum) cultivation. This study delves into the potential of plant extracts from Cnidoscolus urens L. as an alternative biocontrol strategy to combat this disease. Rich in secondary metabolites like terpenes, which are instrumental in plant defense, these extracts also comprise esters and fatty acids. Although the latter are not classified as secondary metabolites, they contribute significantly to the plant's biochemical makeup. Our objective was to gauge the in vitro inhibitory efficacy of ethanolic extracts derived from the leaves and stems of Cnidoscolus urens L. against Colletotrichum spp. To achieve this, an agar dilution method with varying extract concentrations was employed. The results showed that concentrations ranging from treatment 3 to treatment 8 effectively inhibited fungal mycelial growth. Interestingly, the extracts' origin, whether from leaves or stems, did not show any significant differential impact on their inhibitory activity. These insights emphasize the consistent effect of Cnidoscolus urens L. extracts in stalling Colletotrichum spp. growth, underscoring their potential as biological antifungal agents in agriculture. Given the pronounced in vitro effectiveness of both leaf and stem extracts, they beckon further exploration as part of sustainable agricultural strategies to combat prominent diseases like anthracnose.

EVALUATION OF ANTIBACTERIAL AND ANTIOXIDANT PROPERTIES OF LEAF EXTRACT OF CHRYSOPHYLLUM ALBIDUM AGAINST SELECTED ENTERIC BACTERIAL PATHOGENS

This study investigated the antibacterial and antioxidant potentials of Chrysophyllum albidum leaf extract against selected enteric bacterial pathogens (Escherichia coli, Klebsiella pneumoniae, Shigella dysenteriae, Salmonella typhi, Proteus mirabilis and Proteus vulgaris). Fresh leaves of Chrysophyllum albidum were shade air-dried and ground into fine powder. Thereafter, the leaves powder was cold extracted using methanol and sterile distilled water in ratio 3:2(v/v). The mixture obtained was concentrated in vacuo using a rotary evaporator and lyophilized. The crude extract was screened for antibacterial, phytochemicals and antioxidant properties. The antibacterial properties were determined using agar well diffusion and agar dilution methods while the antioxidant and phytochemical assay were analyzed using standard methods. The phytochemical screening of the extract revealed the presence of tannins, alkaloids, flavonoids, saponins, steroids, terpenoids, reducing sugar and cardiac glycosides. The zones of inhibition shown by the extract at 10 mg/mL against the bacterial isolates ranged between 10 mm and 22 mm. The highest zone of inhibition (22 mm) was expressed against Escherichia coli at a concentration of 10 mg/mL. The MICs ranged between 1.25 mg/mL and 5mg/mL while MBCs ranged between 2.5 mg/mL and 10 mg/mL. The antioxidant assay of leaf extract showed appreciable antioxidant potential when compared with ascorbic acid used as standard. The leaf extract exhibited percentage of 92.03% at a concentration of 500 µg/mL while ascorbic acid exhibited percentage of 96.54% at the same concentration. This study, therefore showed that leaf extract of Chrysophyllum albidum exhibited significant antibacterial and antioxidant activities against the test isolates.

Epidemiological investigation and drug resistance characteristics of Riemerella anatipestifer strains from large-scale duck farms in Shandong Province, China from March 2020 to March 2022

Infectious serositis is a common disease caused by Riemerella anatipestifer (R. anatipestifer) in ducks, characterized by respiratory distress, septicemia, and neurological symptoms. In this study, 1,020 samples (brain and liver) were collected from ducks with suspected R. anatipestifer infection from March 2020 to March 2022 in Shandong Province, of which 171 R. anatipestifer strains were identified by PCR and isolation culture. The serotype of all strains was analyzed, and 74 strains were subjected to drug sensitivity tests and drug resistance genes detection. The results showed that the overall prevalence rate of R. anatipestifer in Shandong Province was 16.7% (171/1,020), with most strains coming from brain samples of ducklings under 3-mo old collected from September to December each year. Histopathological examination showed that heart vessels of the diseased duck were highly dilated and filled with red blood cells, with obvious fibrin exudates outside the pericardium, and fatty degeneration of liver cells. There were 45 strains of serotype 1, 45 strains of serotype 2, 2 strains of serotype 4, 33 strains of serotype 6, 44 strains of serotype 7, and 2 strains of serotype 10. The minimum inhibitory concentration (MIC) of 10 common antibiotics against 74 representative strains was determined by the agar dilution method. It was found that 74 strains had the most severe resistance to gentamicin (77%) and fully susceptible to ceftriaxone, but the 81.1% isolated strains were multidrug resistant. Resistance genes testing of 74 R. anatipestifers showed that tetracycline resistance gene tet X had the highest detection rate of 95.9%, followed by macrolide resistance gene ermF with 77%, and the rate of β-lactam resistance gene blaTEM is the lowest (10.8%). The animal experiment of 4 R. anatipestifer strains with different serotypes showed that they had strong pathogenicity to 7-day-old ducklings, which could cause nervous symptoms, and the mortality rate was 58% to 70%. The autopsy showed obvious pathological changes. These findings of this study on R. anatipestifer will help us to understand the latest prevalence, drug resistance characteristics, and pathogenicity of R. anatipestifer in Shandong, China, and provide a scientific guide for the treatment and control of the disease.

Antibacterial and anti-biofilm activity of radezolid against Staphylococcus aureus clinical isolates from China.

Although the potent antibacterial ability of radezolid against Staphylococcus aureus has been widely reported worldwide, its antibacterial and anti-biofilm activity against the S. aureus clinical isolates from China remains elusive. In this study, the minimum inhibitory concentration (MIC) of radezolid was determined in S. aureus clinical isolates from China using the agar dilution method, and the relationship between radezolid susceptibility and ST distribution was also investigated. The anti-biofilm activity of radezolid against S. aureus was determined by a crystal violet assay and compared with that of linezolid and contezolid. The quantitative proteomics of S. aureus treated with radezolid was analyzed, and the genetic mutations in radezolid-induced resistant S. aureus were determined by whole-genome sequencing. The dynamic changes in transcriptional expression levels of several biofilm-related genes were analyzed by quantitative RT-PCR. Our data showed that radezolid MIC ranged from ≤0.125 to 0.5 mg/L, which was almost 1/4 × MIC of linezolid against S. aureus, indicating the greater antibacterial activity of radezolid than linezolid. The S. aureus clinical isolates with radezolid MICs of 0.5 mg/L were most widely distributed in ST239 of MRSA and ST7 of MSSA. Moreover, the more robust anti-biofilm activity of radezolid with subinhibitory concentrations (1/8 × MIC and 1/16 × MIC) was demonstrated against S. aureus when compared with that of contezolid and linezolid. Genetic mutations were found in glmS, 23S rRNA, and DUF1542 domain-containing protein in radezolid-induced resistant S. aureus selected by in vitro induction of drug exposure. Quantitative proteomic analysis of S. aureus indicated that the global expression of some biofilm-related and virulence-related proteins was downregulated. Quantitative RT-PCR further confirmed that the expressions of some downregulated biofilm-related proteins, including sdrD, carA, sraP, hlgC, sasG, spa, sspP, fnbA, and oatA, were decreased after 12 h and 24 h of exposure to radezolid. Conclusively, radezolid shows robust antibacterial and anti-biofilm activity against S. aureus clinical isolates from China when compared with contezolid and linezolid.

Polyclonal Spread of Fosfomycin Resistance among Carbapenemase-Producing Members of the Enterobacterales in the Czech Republic.

Fosfomycin (FOS) has been recently reintroduced into clinical practice, but its effectiveness against multidrug-resistant (MDR) Enterobacterales is reduced due to the emergence of FOS resistance. The copresence of carbapenemases and FOS resistance could drastically limit antibiotic treatment. The aims of this study were (i) to investigate fosfomycin susceptibility profiles among carbapenem-resistant Enterobacterales (CRE) in the Czech Republic, (ii) to characterize the genetic environment of fosA genes among the collection, and (iii) to evaluate the presence of amino acid mutations in proteins involved in FOS resistance mechanisms. During the period from December 2018 to February 2022, 293 CRE isolates were collected from different hospitals in the Czech Republic. FOS MICs were assessed by the agar dilution method (ADM), FosA and FosC2 production was detected by the sodium phosphonoformate (PPF) test, and the presence of fosA-like genes was confirmed by PCR. Whole-genome sequencing was conducted with an Illumina NovaSeq 6000 system on selected strains, and the effect of point mutations in the FOS pathway was predicted using PROVEAN. Of these strains, 29% showed low susceptibility to fosfomycin (MIC, ≥16 μg/mL) by ADM. An NDM-producing Escherichia coli sequence type 648 (ST648) strain harbored a fosA10 gene on an IncK plasmid, while a VIM-producing Citrobacter freundii ST673 strain harbored a new fosA7 variant, designated fosA7.9. Analysis of mutations in the FOS pathway revealed several deleterious mutations occurring in GlpT, UhpT, UhpC, CyaA, and GlpR. Results regarding single substitutions in amino acid sequences highlighted a relationship between ST and specific mutations and an enhanced predisposition for certain STs to develop resistance. This study highlights the occurrence of several FOS resistance mechanisms in different clones spreading in the Czech Republic. IMPORTANCE Antimicrobial resistance (AMR) currently represents a concern for human health, and the reintroduction of antibiotics such as fosfomycin into clinical practice can provide further option in treatment of multidrug-resistant (MDR) bacterial infections. However, there is a global increase of fosfomycin-resistant bacteria, reducing its effectiveness. Considering this increase, it is crucial to monitor the spread of fosfomycin resistance in MDR bacteria in clinical settings and to investigate the resistance mechanism at the molecular level. Our study reports a large variety of fosfomycin resistance mechanisms among carbapenemase-producing Enterobacterales (CRE) in the Czech Republic. Our study summarizes the main achievements of our research on the use of molecular technologies, such as next-generation sequencing (NGS), to describe the heterogeneous mechanisms that reduce fosfomycin effectiveness in CRE. The results suggest that a program for widespread monitoring of fosfomycin resistance and epidemiology fosfomycin-resistant organisms can aide timely implementation of countermeasures to maintain the effectiveness of fosfomycin.

Ceftazidime/avibactam-resistant meropenem-susceptible KPC-producing Klebsiella pneumoniae: Analysis of cases and evaluation of in vitro activity of fosfomycin-containing combinations

Little is known regarding outcomes and optimal therapeutic regimens of infections caused by Klebsiella pneumoniaecarbapenemase-producing Klebsiella pneumoniae (KPC-Kp) resistant to ceftazidime/avibactam (CZA) and susceptible to meropenem (MEM). Although susceptible to MEM in vitro, the possibility of developing MEM resistance overtime is a concern. We describe the clinical characteristics of patients with colonization/infection due to KPC variants with a focus on the in vitro activity of fosfomycin (FOS)-containing combinations. Patients with colonization/infection due to a KPC variant were included. Fosfomycin susceptibility was performed by agar dilution method. Synergistic activity of FOS-based combinations was evaluated by gradient strip-agar diffusion method. The emergence of in vitro MEM resistance was also tested. Eleven patients were included: eight with infection [four with ventilator-associated pneumonia and four with bloodstream infections] and three with colonization. Previous therapy with CZA was administered to all patients (with a mean cumulative duration of 23 days). All subjects with infection received meropenem, in monotherapy (n=4) or with amikacin (n=2) or fosfomycin (n=2), and achieved clinical cure. A new CZA-susceptible and MEM-resistant KPC-Kp strain was subsequently isolated in three patients (27.3%). Meropenem/vaborbactam (MVB) showed high in vitro activity, while FOS+MEM combination was synergistic in 40% of cases. In vitro resistance to MEM was observed with maintenance of CZA resistance. Detection of KPC variants may occur within the same patient, especially if CZA has been previously administered. Although clinical success has been obtained with carbapenems, the emergence of MEM resistance is a concern. Fosfomycin plus meropenem is synergistic and may be a valuable combination option for KPC variants, while MVB may be considered in monotherapy. The detection of KPC variants in an endemic setting for KPC-Kp represents a worryingly emerging condition. The optimal therapeutic approach is still unknown and the development of meropenem resistance is of concern, which may lead to therapeutic failure in clinical practice. In these cases, the addition of fosfomycin to meropenem, or a more potent antibiotic, such as meropenem/vaborbactam, may be valuable therapeutic options.

Geographic patterns of antimicrobial susceptibilities for Bacteroides spp. worldwide: Results from the Antimicrobial Testing Leadership and Surveillance (ATLAS) programme, 2007–2020

Antimicrobial susceptibilities of 4,973 Bacteroides species isolates recovered from various sources of patients from 12 countries (99.6% from European countries) in the Antimicrobial Testing Leadership and Surveillance (ATLAS) program, 2007-2020, were investigated. The minimum inhibitory concentrations (MICs) of the isolates with six commonly used agents were determined using the agar dilution method. Among the isolates, ten Bacteroides species were included: B. fragilis (n = 3,180, 64.0%) was most frequently encountered, followed by B. thetaiotaomicron (n = 675) and B. ovatus (n = 409). During the 14 years, the proportion of B. fragilis declined, but the proportion of non-fragilis Bacteroides species increased. More than 90% of the isolates tested were susceptible to piperacillin-tazobactam, meropenem, and tigecycline. Significantly lower susceptibility rates to cefoxitin (p<0.001), clindamycin (p<0.001), piperacillin/tazobactam (p<0.001), and tigecycline (p = 0.006) were observed among non-fragilis Bacteroides species isolates than among B. fragilis isolates. Moreover, the susceptibility rates to clindamycin (p = 0.003) and tigecycline (p = 0.044) decreased significantly among non-fragilis Bacteroides species over time. Clindamycin susceptibility rates higher than 80% were found in Greece (100%), Sweden (86.3%), and the United Kingdom (80.7%), and the lowest susceptibility rates were found in the United States (42.9%) and Japan (53.9%). In conclusion, the susceptibility of Bacteroides species to commonly used antibiotics varied geographically. Empirical antibiotic therapy for suspected anaerobic infections with clindamycin and cefoxitin should be avoided due to high resistance rates. Piperacillin-tazobactam, meropenem, metronidazole, and tigecycline could be considered favourable options for the treatment of infections caused by Bacteroides species.

Molecular epidemiology and comparative genomics of carbapenemase-producing Escherichia coli isolates from 19 tertiary hospitals in China from 2019 to 2020.

The clinical use of carbapenems is facing challenges due to increased carbapenemase-producing Escherichia coli (CP-EC) infections over the past decade. Meanwhile, whole-genome sequencing (WGS) is an important method for bacterial epidemiological research. We aim to provide more gene-based surveys to explore the genomics and occurrence of CP-EC in China. A total of 780 Escherichia coli isolates were collected by the China Antimicrobial Resistance Surveillance Trial (CARST) from 2019 to 2020. An antibacterial susceptibility test was performed by using the agar dilution method. CP-EC were detected by the modified carbapenem inactivation method (mCIM), EDTA-modified carbapenem inactivation method (eCIM), and polymerase chain reaction (PCR). Homology analysis was performed by multilocus sequence typing (MLST). A conjugation experiment was performed to verify the transferability of plasmids carrying carbapenemase genes. WGS was conducted to explore the gene-environment of the carbapenemase gene. Of the 780 Escherichia coli isolates, 31 isolates were insensitive to carbapenem with a rate of 4%. Among them, 13 CP-EC isolates had transferability of the bla NDM gene. These isolates belonged to nine distinct sequence types (STs), with some correlation. We found that two (2/13, 15.4%) of the CP-EC isolates that were collected from blood specimens were highly pathogenic and also showed high transferability of the bla NDM gene. In addition, eight (8/13, 61.5%) of the CP-EC isolates were found to be multidrug-resistant. With the increasing use of carbapenem, CP-EC isolates accounted for nearly half of the total carbapenem-insensitive Escherichia coli isolates. Our findings highlight the urgent need to pay attention to CP-EC isolates in bloodstream infections and ESBL-producing CP-EC isolates. Based on the One Health concept, we suggest various measures, including the development of bacterial vaccines, antibiotic management, and establishment of better medical environments, to avoid the outbreak of CP-EC.

Antibacterial Activity of Ginkgo biloba Extracts against Clavibacter michiganensis subsp. michiganensis, Pseudomonas spp., and Xanthomonas vesicatoria

Phytopathogenic bacteria represent a risk to global food production by impacting a variety of crops. The aim of this study was to characterize the contents of bioactive constituents in extracts from Ginkgo biloba L. leaves and fruits and test their activity against six phytopathogenic bacteria that affect horticultural crops. Gas chromatography–mass spectrometry (GC−MS) was used for the chemical profiling of the aqueous methanol extracts, and their bacteriostatic activity against Clavibacter michiganensis subsp. michiganensis, Pseudomonas cichorii, Pseudomonas syringae pv. pisi, Pseudomonas syringae pv. syringae, Pseudomonas syringae pv. tomato, and Xanthomonas vesicatoria (formerly Xanthomonas campestris pv. vesicatoria) was determined in vitro through the agar dilution method. The protective effect of the leaf extract was tested in vivo against the most relevant bacterial pathogens based on their economic/scientific importance, i.e., C. michiganensis subsp. michiganensis and P. syringae pv. pisi, in tomato (Solanum lycopersicum L.) and pea (Pisum sativum L.) plants, respectively, under greenhouse conditions. The GC−MS characterization of G. biloba extracts revealed the presence of dihydro-4-hydroxy-2(3H)-furanone, 2,4-dimethyl-3-hexanol, catechol, 3-O-methyl-D-fructose, 4,6-di-O-methyl-α-D-galactose, methyl 2-O-methyl-α-D-xylofuranoside, and 3-methyl mannoside. In vitro growth inhibition tests showed that, while the fruit extract had no activity, the leaf extract exhibited minimum inhibitory concentrations between 500 and 1000 μg mL−1, which may be attributed to the presence of 2,4-dimethyl-3-hexanol and catechol. In vivo tests of the leaf extract demonstrated full protection in tomato and pea plants at 1000 and 1500 μg∙mL−1, respectively. The results indicate that G. biloba leaves may be employed as a biorational source for integrated pest management in horticulture.

Thin Layer Chromatography Bio-autography guided identification of Antibacterial constituents of leaf extract of Stereospermum kunthianum Cham. (Bignoniaceae)

Background: There is an incessant quest for the discovery of novel antibacterial agents.&nbsp;Stereospermum kunthianum Cham.&nbsp; Bignoniaceae) is used in traditional medicine in Nigeria and other&nbsp;parts of Africa in the treatment of venereal diseases e.g., syphilis,&nbsp; wounds, refractory cough, malaria,&nbsp;tooth ache leprosy etc. The present work investigates the antimicrobial activity and phytochemical&nbsp;constituents of the ethanolic leaf extract of the plant.&nbsp; Methods: Ethanolic leaf extract of S.Kunthianum was obtained by macerations method. Agar well&nbsp;diffffusion method was used to carry&nbsp; out preliminary antimicrobial susceptibility testing. The test was&nbsp;carried out at extract concentration of 100, 200 and 400 mgmL-1 . The&nbsp; agar dilution method was used to&nbsp;determine the Minimum Inhibitory Concentration (MIC) of the extracts. Phytochemical screening&nbsp; was&nbsp;conducted using standard procedures. Thin layer chromatography bioautography analysis was carried&nbsp;out using the agar overlay&nbsp; method. Six micro-organisms including two gram positive- Bacillus subtilis&nbsp;and Staphylococcus aureus, two gram negative Salmonella&nbsp; typhi , and Pseudomonas aeruginosa, two&nbsp;fungi, Aspergillus niger and Candida albicans were used as test organisms.&nbsp; Results: The ethanol extracts of S.kunthianum leaves showed antimicrobial activity against B.subtilis,&nbsp;S.aureus and S.typhi with zones of&nbsp; Inhibition ranging from 13±1.41 to 29.5 ± 1.00 mm and MIC values&nbsp;ranging from 16 -64 mgmL-1 . Phytochemical screening revealed the&nbsp; presence of saponins, flflavonoids&nbsp;and terpenoid compounds&nbsp; Conclusion: The study showed that the antibacterial activities of the leaf extract are probably due to&nbsp;polyphenolic and steroidal&nbsp; compounds.&nbsp; &nbsp

Can beta-lactamase resistance genes in anaerobic Gram-negative gut bacteria transfer to gut aerobes?

The study was conceived with the hypothesis that human aerobic gut flora could act as a reservoir of ß-lactamases and contribute to the emergence of ß-lactam resistance by transferring ß-lactamase genes to resident anaerobes. Thus, we studied the repertoire of ß-lactam resistance determinants (ß-lactamases associated with aerobes and anaerobes) in Gram-negative anaerobes. The phenotypic resistance against ß-lactams and the presence of aerobic and anaerobic ß-lactamases were tested in Gram-negative anaerobic isolates (n = 200) by agar dilution method and targeted PCR, respectively. In addition, whole-genome sequencing (WGS) was used to study the ß-lactam resistance determinants in 4/200 multi-drug resistant (MDR) strains. The resistance to ß-lactams was as follows: imipenem (0.5%), cefoxitin (26.5%), and piperacillin-tazobactam (27.5%). None of the isolates showed the presence of ß-lactamases found in aerobic microorganisms. The presence of anaerobic ß-lactamase genes viz. cfiA, cepA, cfxA, cfiAIS [the intact segment containing cfiA gene (350 bp) and upstream IS elements (1.6-1.7 kb)] was detected in 10%, 9.5%, 21.5%, and 0% isolates, respectively. The WGS data showed the presence of cfiA, cfiA4, cfxA, cfxA2, cfxA3, cfxA4, cfxA5 in MDR strains. The study showed a distinct dichotomy in repertoires of ß-lactamases between aerobes and anaerobes.

Effect of selected botanical compounds on Ascosphaera apis and Apis mellifera

Ascosphaera apis is a worldwide spread fungal pathogen that invades the honey bee brood and causes chalkbrood disease. In many countries, its control with fungicides is banned due to residue contamination of bee products and health risk. Since then, there is no alternative treatment. In this study, an antifungal activity of 10 essential oils or their components has been investigated. The agar dilution method showed considerable differences between its antifungal activity with the best results exhibited by thyme oil (MIC50 = 57.7 µL/L) followed by cedarwood oil (MIC50 = 64.9 µL/L), thyme (MIC50 = 82.5 µL/L), clove bud oil (MIC50 = 114.2 µL/L), and cinnamon oil (MIC50 = 136.6 µL/L). These botanical compounds have also been tested using a honey bee tolerance by complete exposure method. The oil tolerated the most by honey bees was from cedarwood (LC50 = 33.4 µL). The composition and quantity of main components of the oils mentioned above were determined by GC-MS/MS. Thymol is responsible for the antifungal activity of thyme oil, whereas, in clove bud and cinnamon oil, the main component is eugenol. Cedarwood oils contain primarily gama and alpha cendrene. The results suggest the analyzed compounds can be used to control multiple honey bee pathogens simultaneously due to their broad spectrum of antimicrobial activity and enhanced efficacy by synergistic effect.

Prevalence and molecular characterization of methicillin resistant Staphylococcus aureus (MRSA) and vancomycin resistant Staphylococcus aureus (VRSA) in a tertiary care hospital

Resistance shown by Staphylococcus aureus to methicillin; mediated by mecA, and vancomycin; mediated by vanA, has led to difficulty in treatment of related infections. Despite reports showing methicillin resistant S. aureus (MRSA) and vancomycin resistant S. aureus (VRSA) in Nepal, and need for their regular surveillance, no study has been conducted on it in our hospital. So, this study is aimed to determine prevalence of MRSA, VRSA and their molecular characterization along with antibiogram. A descriptive cross-sectional study was conducted from August to December, 2022 in Clinical Microbiology Laboratory of NMCTH among S. aureus (n=160) isolated from various clinical specimens after receiving ethical approval from NMC-IRC. AST was done by modified Kirby-Bauer’s disc diffusion method. MRSA and VRSA were detected by cefoxitin disc method and agar dilution method respectively. Inducible clindamycin resistance was detected by D-test. Resistant genes (mecA, PVL, and vanA) were detected using conventional PCR. Prevalence of MRSA was found to be 31.2% (50/160) but none of the isolates were resistant to vancomycin. Total 7 (46.6%) mecA and 7 (46.6%) PVL genes were detected among 15 selected MRSA isolates but vanA was not found. All the MRSA isolates were susceptibile to co-trimoxazole, tigecycline, chloramphenicol, vancomycin, teicoplanin and linezolid. The resistance rate against ciprofloxacin, ofloxacin, and clindamycin was 52.0%, 44.0%, and 68.0% (20.0% iMLSB, 28.0% cMLSB and 16.0% MS-phenotypes) respectively. Prompt implementation of hospital antibiotic policy and AMR Act by government along with regular surveillance of MRSA and VRSA seems essential to contain MRSA infections. Co-trimoxazole could be treatment option against MRSA in our setting, keeping vancomycin in reserve. However, large scale studies are required to establish this conclusion.

Evaluation of disk diffusion method for testing the rifampicin, erythromycin, and tetracycline susceptibility of Clostridioides (prev. Clostridium) difficile

IntroductionAntimicrobial resistance (AMR) is one of the greatest threats to animal and public health. Clostridioides (prev. Clostridium) difficile is a major burden to healthcare and a relevant AMR gene reservoir. Despite the known importance of AMR in C. difficile epidemiology and treatment, antimicrobial susceptibility testing for this pathogen is still based on the determination of the minimal inhibitory concentration (MIC) by the agar dilution method, which is technically demanding and labor-intensive. In this study, the disk diffusion method was used to evaluate the susceptibility of C. difficile to erythromycin, rifampicin, and tetracycline. Material and methodsA total of 155 isolates isolated between 2011 and 2022 from humans and animals in Brazil were simultaneously tested using the disk diffusion method and the epsilometer test (Etest) for these three antimicrobials on Brucella blood agar supplemented with vitamin K and hemin. ResultsThe results suggest that disk diffusion can be an interesting routine tool to identify erythromycin- and rifampicin-resistant C. difficile isolates (≥20 mm cut-off) and wild type (WT) strains (≥28 mm). However, the disk diffusion protocol tested in this study does not seem suitable for tetracycline because of the common misclassification of resistant strains.

Phenotypic and Genotypic Correlation of Antimicrobial Susceptibility of Bacteroides fragilis: Lessons Learnt.

Background Bacteroides fragilis is an opportunistic pathogen causing severe infections, including bacteremia. There have been increased reports of antimicrobial resistance in B. fragilis. However, phenotypic testing of susceptibility is time consuming and not cost effective for anaerobes. The present study investigates the correlation of phenotypic susceptibility with genotypic markers; to determine if these could be considered for deciding empirical therapy for B. fragilis. Material and methods Bacteroides fragilis isolates from various clinical samples including exudates, tissue, and body fluids were collected between November 2018 and January 2020 in the Department of Clinical Microbiology, Christian Medical College (CMC) Vellore. Species identification was done by MatrixAssistedLaserDesorptionIonization time of flight mass spectrometry (MALDI TOF) according to the manufacturer's instructions. A total number of 51 B. fragilis isolates were tested against metronidazole, clindamycin, piperacillin/tazobactam, and meropenem phenotypically by agar dilution method using Clinical & Laboratory Standards Institute (CLSI) 2019 guidelines and minimum inhibitory concentrations (MIC) were interpretated. The genotypic markers for antimicrobial resistance genes (nim, emrF, and cfiA) were studied by polymerase chain reaction (PCR) assay as per the standard protocol on all isolates to detect resistance genes. Results B. fragilis isolates in this study expressed 45%, 41%, and 16% phenotypic resistance to clindamycin, metronidazole, and meropenem, respectively, with least resistance topiperacillin/tazobactam (6%). Among the metronidazole resistant isolates, 52% harbored nim gene. Nim gene was also present in 76% (23/30) of the metronidazole susceptible isolates. Similarly, cfiA was present in all eight meropenem resistant isolates in addition to 22% (9/41) of the susceptible isolates. All cfiA negative isolates were phenotypically susceptible. Interestingly, 74% (17/23) of the clindamycin resistant isolates were positive for ermF. Conclusions Detection of a limited set of genes does not always correlate with phenotypic resistance to metronidazole and clindamycindue to the reported influence of insertion sequence (IS) elements, efflux, and other genetic determinants. Certainly, the absence of the cfiA gene can be employed to rule out meropenem resistance. However, redundant use of antibiotics such as meropenem along with metronidazole could be avoided for B. fragilis, which might otherwise elevate meropenem resistance. Recommendation of metronidazole requires prior phenotypic testing due to the reported 41% resistance.

Multicentre Clinical Evaluation of a Molecular Diagnostic Assay to Identify Neisseria gonorrhoeae Infection and Detect Antimicrobial Resistance

ObjectivesAntimicrobial resistance (AMR) in Neisseria gonorrhoeae (N. gonorrhoeae) is an urgent threat to public health, with the emergence of highly resistant strains such as the FC428 clone. This study aimed to evaluate the high-resolution melting assay of N. gonorrhoeae AMR (HRM-NG-AMR) for diagnosing N. gonorrhoeae infection and detecting extended-spectrum cephalosporins and azithromycin resistance. MethodsA multicentre collection of 1488 samples, including 770 isolates and 718 urogenital swabs, was used to evaluate the performance of the HRM-NG-AMR assay. The presence of N. gonorrhoeae was confirmed by culture. Minimum inhibitory concentrations of antibiotics against the tested isolates were determined using the agar dilution method. ResultsRegarding N. gonorrhoeae identification, HRM-NG-AMR had a sensitivity of 95.15% (95% CI 91.65–97.28) and a specificity of 96.44% (95% CI 94.17–97.89) using culture as standard. Regarding AMR detection, the specificity ranged from 96.29% (95% CI 94.57–97.50) for cefixime to 99.52% (95% CI 98.68–99.85) for azithromycin. Additionally, the sensitivity ranged from 31.34% (95% CI 20.87–43.97) for azithromycin to 79.10% (95% CI 63.52–89.42) for ceftriaxone. It was determined that 664 of 672 (98.81%) and 615 of 672 (91.52%) N. gonorrhoeae isolates were susceptible to ceftriaxone and cefixime, respectively, by detecting non-mosaic penA. Lastly, 40 genotypic FC428-related strains with the penA-60.001 allele were accurately identified. ConclusionsThe HRM-NG-AMR assay showed promising diagnostic performance for detecting N. gonorrhoeae infection and predicting AMR. This study aimed to evaluate the application of this assay in the clinical setting to enhance AMR surveillance and treatment intervention.

Antimicrobial Peptides Designed against the Ω-Loop of Class A β-Lactamases to Potentiate the Efficacy of β-Lactam Antibiotics.

Class A serine β-lactamases (SBLs) have a conserved non-active site structural domain called the omega loop (Ω-loop), in which a glutamic acid residue is believed to be directly involved in the hydrolysis of β-lactam antibiotics by providing a water molecule during catalysis. We aimed to design and characterise potential pentapeptides to mask the function of the Ω-loop of β-lactamases and reduce their efficacy, along with potentiating the β-lactam antibiotics and eventually decreasing β-lactam resistance. Considering the Ω-loop sequence as a template, a group of pentapeptide models were designed, validated through docking, and synthesised using solid-phase peptide synthesis (SPPS). To check whether the β-lactamases (BLAs) were inhibited, we expressed specific BLAs (TEM-1 and SHV-14) and evaluated the trans-expression through a broth dilution method and an agar dilution method (HT-SPOTi). To further support our claim, we conducted a kinetic analysis of BLAs with the peptides and employed molecular dynamics (MD) simulations of peptides. The individual presence of six histidine-based peptides (TSHLH, ETHIH, ESRLH, ESHIH, ESRIH, and TYHLH) reduced β-lactam resistance in the strains harbouring BLAs. Subsequently, we found that the combinational effect of these peptides and β-lactams sensitised the bacteria towards the β-lactam drugs. We hypothesize that the antimicrobial peptides obtained might be considered among the novel inhibitors that can be used specifically against the Ω-loop of the β-lactamases.

Toxic heavy metal/oxyanion tolerance in haloarchaea from some saline and hypersaline ecosystems.

Toxic heavy metal/oxyanion contamination has increased severely through the last decades. In this study, 169 native haloarchaeal strains were isolated from different saline and hypersaline econiches of Iran. After providing pure culture and performing morphological, physiological, and biochemical tests, haloarchaea resistance toward arsenate, selenite, chromate, cadmium, zinc, lead, copper, and mercury were surveyed using an agar dilution method. On the basis of minimum inhibitory concentrations (MICs), the least toxicities were found with selenite and arsenate, while the haloarchaeal strains revealed the highest sensitivity for mercury. On the other hand, the majority of haloarchaeal strains exhibited similar responses to chromate and zinc, whereas the resistance level of the isolates to lead, cadmium, and copper was very heterogeneous. 16 S ribosomal RNA (rRNA) gene sequence analysis revealed that most haloarchaeal strains belong to the Halorubrum and Natrinema genera. The obtained results from this study showed that among the identified isolates, Halococcus morrhuae strain 498 had an exceptional resistance toward selenite and cadmium (64 and 16 mM, respectively). Also, Halovarius luteus strain DA5 exhibited a remarkable tolerance against copper (32 mM). Moreover, strain Salt5, identified as Haloarcula sp., was the only strain that could tolerate all eight tested heavy metals/oxyanions and had a significant tolerance of mercury (1.5 mM).

Genome Analysis of Pseudomonas aeruginosa Strains from Chronically Infected Patients with High Levels of Persister Formation

The appearance of persister cells with low metabolic rates are key factors leading to antibiotic treatment failure. Such persisters are multidrug tolerant and play a key role in the recalcitrance of biofilm-based chronic infections. Here, we present the genomic analyses of three distinct Pseudomonas aeruginosa Egyptian persister-isolates recovered from chronic human infections. To calculate the persister frequencies, viable counts were determined before and after treatment with levofloxacin. The susceptibilities of isolates to different antibiotics were determined using the agar-dilution method. To determine their recalcitrance, the levofloxacin persisters were further challenged with lethal concentrations of meropenem, tobramycin, or colistin. Furthermore, the biofilm formation of the persister strains was estimated phenotypically, and they were reported to be strong biofilm-forming strains. The genotypic characterization of the persisters was performed using whole genome sequencing (WGS) followed by phylogenetic analysis and resistome profiling. Interestingly, out of the thirty-eight clinical isolates, three isolates (8%) demonstrated a persister phenotype. The three levofloxacin-persister isolates were tested for their susceptibility to selected antibiotics; all of the tested isolates were multidrug resistant (MDR). Additionally, the P. aeruginosa persisters were capable of surviving over 24 h and were not eradicated after exposure to 100X-MIC of levofloxacin. WGS for the three persisters revealed a smaller genome size compared to PAO1-genome. Resistome profiling indicated the presence of a broad collection of antibiotic-resistance genes, including genes encoding for antibiotic-modifying enzymes and efflux pump. Phylogenetic analysis indicated that the persister isolates belong to a distinct clade rather than the deposited P. aeruginosa strains in the GenBank. Conclusively, the persister isolates in our study are MDR and form a highly strong biofilm. WGS revealed a smaller genome that belongs to a distinct clade.