E-test Method Research Articles

Candida auris MIC testing by EUCAST and clinical and laboratory standards institute broth microdilution, and gradient diffusion strips; to be or not to be amphotericin B resistant?

ObjectivesReported amphotericin B resistance rates for Candida auris vary considerably. This may reflect clinically relevant differences in susceptibility, technical issues with testing, or adoption of a clinical breakpoint that bisects the wild-type population. We compared reference methods and two gradient diffusion strips using a shared C. auris strain collection. MethodsForty C. auris strains from nine U.S. states and ≥3 clades were included. Fourteen MIC data sets were generated using EUCAST E.Def 7.4, Clinical and Laboratory Standards Institute (CLSI) M27Ed4, Etest, and MTS (Liofilchem) strip MICs. MICs ≤1 mg/L were classified as susceptible. ResultsEUCAST and CLSI amphotericin B MIC testing were robust across the included method variables. The modal MIC was 1 mg/L, distributions unimodal and narrow with similar GM-MICs (0.745–1.072); however, susceptibility classification varied (0–28% resistance). Gradient diffusion strip testing resulted in wider and bimodal distributions for 8/9 data sets. If adopting, per manufacturer's protocol, double inoculation for the Etest method, the modal MIC increased to 2–4 mg/L and resistance rates to 45–63% versus 25–30% with the single inoculation. The EUCAST, CLSI, Etest, and MTS strip MICs correlated to the optical density of drug-free control EUCAST wells, suggesting that some isolates grew better than others and that this was associated with MIC. Discussion: The EUCAST and CLSI MIC results were in close agreement, whereas the strip test showed wider and bimodal distributions with reader to reader and centre to centre variation. Our study adds to the concern for commercial MIC testing of amphotericin B against C. auris and suggests the current breakpoint leads to random susceptibility classification.

Multicentre evaluation of in vitro activity of contezolid against drug-resistant Staphylococcus and Enterococcus.

To investigate susceptibility to contezolid, a novel oxazolidinone, multicentre surveillance was conducted involving 2449 strains of Staphylococcus and Enterococcus collected from 65 hospitals across China. The MICs of contezolid, linezolid and other clinically significant antibiotics were determined by the broth microdilution method. Consistency with the broth microdilution method for contezolid was assessed using agar dilution method, as well as disc diffusion and ETEST for linezolid, respectively. WGS was conducted on all 20 linezolid-resistant and 30 randomly non-resistant strains to analyse linezolid resistance genes (optrA, poxtA, cfr) and 23S rRNA mutation sites. All strains exhibited WT susceptibility to contezolid, while resistance proportions to daptomycin, vancomycin, teicoplanin, tigecycline and eravacycline ranged from 0% to 5.2% in Staphylococcus, and from 0% to 7.8% in Enterococcus. Linezolid resistance was higher in Enterococcus faecalis (4.4%) compared with Enterococcus faecium (0.2%). Contezolid showed a lower MIC50 (0.5 mg/L) than linezolid (2 mg/L) for methicillin-resistant Staphylococcus. Against Enterococcus, contezolid demonstrated a cumulative MIC percentage of 70% for VRE and 39.1% for E. faecalis (at MIC = 1 mg/L), whereas linezolid showed 0% and 1.1%, respectively. Among the 20 linezolid-resistant Enterococcus strains, all carried the optrA gene without 23S rRNA mutations. For contezolid, MICs were 4 mg/L for 19 strains and 2 mg/L for 1 strain. The ETEST, agar dilution and disc diffusion methods showed essential and categorical agreements of >90% for linezolid, with no major errors or very major errors. Contezolid demonstrated significant in vitro antibacterial activity against methicillin-resistant Staphylococcus, VRE and linezolid-resistant E. faecalis.

Associations between biofilm formation and virulence factors among clinical Helicobacter pylori isolates

IntroductionHelicobacter pylori (H. pylori) causes several gastrointestinal diseases. Its virulence factors contributing to disease development include biofilm formation, cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA) proteins that induce host tissue damage. In addition, urease activity enables H. pylori growth in the gastric acidic environment. This work aimed to characterize bacterial factors associated with biofilm production among 89 clinical H. pylori isolates, collected from patient gastric biopsies. MethodsBiofilm production was detected using the crystal violet method. PCR was performed to determine vacA genotype (s1m1, s1m2, s2m1 and s2m2) and cagA gene presence. Urease activity was measured via the phenol red method. Susceptibility to six antibiotics was assessed by the Etest method. ResultsMost H. pylori isolates produced biofilm. No association was found between biofilm-formation capacity and cagA presence or vacA genotype. Urease activity levels varied across isolates; no association was found between biofilm-formation and urease activity. Clarithromycin resistance was measured in 49 % of the isolates. Isolates susceptible to tetracycline were more commonly strong biofilm producers. In contrast, a significantly higher rate of strong biofilm producers was observed among resistant isolates to amoxicillin, levofloxacin and rifampicin, compared to susceptible isolates. Non-biofilm producers were more common among isolates sensitive to rifampicin and metronidazole, compared to resistant isolates. ConclusionsFurther studies are needed to understand the factors that regulate biofilm production in order to search for treatments for H. pylori biofilm destruction.

Pheno- and Genotypic Epidemiological Characterization of Vancomycin-Resistant Enterococcus faecium Isolates from Intensive Care Unit Patients in Central Türkiye.

Vancomycin-resistant Enterococcus faecium (VRE) has been detected in Türkiye. Only limited information is available on its dissemination in the central regions of the country. This study describes the first epidemiological characterization of VRE clinical isolates detected in patients in a hospital in the province of Aksaray. In this one-year study conducted between 2021 and 2022, stool samples from intensive care unit patients were screened for VRE using the phenotypic E-test method, and the antibiotic sensitivity test was analyzed by using the VITEK® 2 system. A molecular assay for confirmation of species level was carried out by 16S rRNA gene-based sequencing and testing for antibiotic resistance (vanA or vanB) and virulence factor-encoding genes (esp, asa1, and hyl). Further, genotypic characterization was determined by macro-restriction fragment pattern analysis (MRFPA) of genomic DNA digested with SmaI restriction enzyme. Of the total 350 Enterococcus positive patients from different hospital intensive care units, 22 (6.3%) were positive for VRE using the phenotypic E-test method. All isolates showed resistance to ampicillin, ciprofloxacin, vancomycin, and teicoplanin and positive amplification for the vanA gene. However, none of the isolates was positive for the vanB gene. The most prevalent virulence gene was esp. The results indicate that the isolates are persistent in the hospital environment and subsequently transmitted to hospitalized patients, thus representing challenges to an outbreak and infection control. These study results would also help formulate more effective strategies to reduce the transmission and propagation of VRE contamination in various hospital settings.

Investigation of gyrA and parC mutations and the prevalence of plasmid-mediated quinolone resistance genes in Klebsiella pneumoniae clinical isolates

BackgroundThe emergence of fluoroquinolone resistance in clinical isolates of Klebsiella pneumoniae is a growing concern. To investigate the mechanisms behind this resistance, we studied a total of 215 K. pneumoniae isolates from hospitals in Bushehr province, Iran, collected between 2017 and 2019. Antimicrobial susceptibility test for fluoroquinolones was determined. The presence of plasmid mediated quinolone resistance (PMQR) and mutations in quinolone resistance-determining region (QRDR) of gyrA and parC genes in ciprofloxacin-resistant K. pneumoniae isolates were identified by PCR and sequencing.ResultsOut of 215 K. pneumoniae isolates, 40 were resistant to ciprofloxacin as determined by E-test method. PCR analysis revealed that among these ciprofloxacin-resistant isolates, 13 (32.5%), 7 (17.5%), 40 (100%), and 25 (62.5%) isolates harbored qnrB, qnrS, oqxA and aac(6’)-Ib-cr genes, respectively. Mutation analysis of gyrA and parC genes showed that 35 (87.5%) and 34 (85%) of the ciprofloxacin-resistant isolates had mutations in these genes, respectively. The most frequent mutations were observed in codon 83 of gyrA and codon 80 of parC gene. Single gyrA substitution, Ser83→ Ile and Asp87→Gly, and double substitutions, Ser83→Phe plus Asp87→Ala, Ser83→Tyr plus Asp87→Ala, Ser83→Ile plus Asp87→Tyr, Ser83→Phe plus Asp87→Asn and Ser83→Ile plus Asp87→Gly were detected. In addition, Ser80→Ile and Glu84→Lys single substitution were found in parC gene.ConclusionsOur results indicated that 90% of isolates have at least one mutation in QRDR of gyrA orparC genes, thus the frequency of mutations was very significant and alarming in our region.

Comparison of Different Methods for Assaying the In Vitro Activity of Cefiderocol against Carbapenem-Resistant Pseudomonas aeruginosa Strains: Influence of Bacterial Inoculum.

Carbapenem-resistant Pseudomonas aeruginosa infections represent a critical public health concern, highlighting the need for the development of effective antibiotics. Cefiderocol demonstrated potent in vitro activity against Pseudomonas aeruginosa, particularly in strains that are resistant to other drugs. However, concerns regarding the emergence of drug-resistant strains persist. This study, conducted with 109 carbapenem-resistant Pseudomonas aeruginosa strains from the Spanish Hospital (Dr. Balmis, Alicante). The study evaluated susceptibility to cefiderocol in comparison to alternative antibiotics and including their susceptibility to bacterial inoculum, while assessing various testing methods. Our findings revealed high susceptibility to cefiderocol against carbapenem-resistant strains, with only 2 of 109 strains exhibiting resistance. Comparative analysis demonstrated superiority of cefiderocol towards alternative antibiotics. Both the E-test and disk-diffusion methods showed 100% concordance with the microdilution method in classifying strains as susceptible or resistant. However, 4.6% (5/109) of disc zone diameters fell within the technical uncertainty zone, so the E-test technique was found to be more useful in routine clinical practice. Additionally, escalating bacterial inoculum correlated with decreases in vitro activity, so this parameter should be adjusted very carefully in in vivo studies. This study underscores cefiderocol's potential as a therapeutic option for carbapenem-resistant Pseudomonas aeruginosa infections. However, the emergence of drug-resistant strains emphasizes the critical need for a wise use of antibiotics and a continuous monitoring of resistance to antibiotics. Based on our in vitro data, further investigation concerning the impact of bacterial inoculum on drug efficacy is warranted in order to detect resistance mechanisms and optimize treatment strategies, thereby mitigating the risk of resistance.

Antibiotic Resistance in Acetic Acid Bacteria Originating from Vinegar.

Acetic acid bacteria (AAB) are major contributors to the production of fermented vinegar, offering various cultural, culinary, and health benefits. Although the residual unpasteurized AAB after vinegar production are not pathogens, these are necessary and require safety evaluations, including antibiotic resistance, before use as a starter. In this research, we investigated the antibiotic resistance profiles of 26 AAB strains, including various species of Komagataeibacter and Acetobacter, against 10 different antibiotics using the E-test method. All strains exhibited resistance to aztreonam and clindamycin. Komagataeibacter species demonstrated a 50% resistance rate to ciprofloxacin, analogous to Acetobacter species, but showed twice the resistance rates to chloramphenicol and erythromycin. Genomic analysis of K. saccharivorans CV1 identified intrinsic resistance mechanisms, such as multidrug efflux pumps, thereby enhancing our understanding of antibiotic resistance in acetic acid-producing bacteria. These findings enhance understanding of antibiotic resistance in AAB for food safety and new antimicrobial strategies, suggesting the need for standardized testing methods and molecular genetic study.

Comparative study of antifungal susceptibility testing methods for clinical Candida albicans isolates

PurposeCandida albicans is the second most common cause of candidemia in Malaysia. The Clinical and Laboratory Standards Institute (CLSI) broth microdilution method is the gold standard for determining its minimum inhibitory concentration (MIC); however, it is laborious and time-consuming. This study was conducted to evaluate the usefulness of alternative methods, namely Sensititre YeastOne (SYO), VITEK 2 system, and E-test for determining the MIC of clinical C. albicans isolates. Materials and methodsThe susceptibilities of 95 C. albicans isolates were compared between SYO, VITEK 2 system, and E-test with CLSI broth microdilution method. The categorical agreement (CA), essential agreement (EA), very major errors (VME), major errors (ME) and minor errors (MiE) were calculated. ResultsOur finding showed the CA varied for SYO from 96.8% to 100%, while the EA ranged from 91.6% to 100%. The SYO method showed 1.1% of VME and ME, and up to 3.2% of MiE. Next, the CA and EA ranges for the VITEK 2 system were 97.8%–100% and 23.2%–100%, respectively. In the VITEK 2 technique, 1.1% of VME were found. For the E-test, the CA varied from 83.2% to 100% while the EA ranged from 64.2% to 98.9%. The E-test method showed 1.1% of VME and up to 16.8% of MiE. ConclusionsIn conclusion, SYO and VITEK 2 (except flucytosine) could be potential alternatives to the CLSI broth microdilution method in determining the MIC of C. albicans.

Effect and mechanism of Qingre Huashi decoction on drug-resistant Helicobacter pylori.

Helicobacter pylori (HP), the most common pathogenic microorganism in the stomach, can induce inflammatory reactions in the gastric mucosa, causing chronic gastritis and even gastric cancer. HP infection affects over 4.4 billion people globally, with a worldwide infection rate of up to 50%. The multidrug resistance of HP poses a serious challenge to eradication. It has been de-monstrated that compared to bismuth quadruple therapy, Qingre Huashi decoction (QHD) combined with triple therapy exhibits comparable eradication rates but with a lower incidence of adverse reactions; in addition, QHD can directly inhibit and kill HP in vitro. To explore the effect and mechanism of QHD on clinically multidrug-resistant and strong biofilm-forming HP. In this study, 12 HP strains were isolated in vitro after biopsy during gastroscopy of HP-infected patients. In vitro, the minimum inhibitory concentration (MIC) values for clinical HP strains and biofilm quantification were determined through the E-test method and crystal violet staining, respectively. The most robust biofilm-forming strain of HP was selected, and QHD was evaluated for its inhibitory and bactericidal effects on the strain with strong biofilm formation. This assessment was performed using agar dilution, E-test, killing dynamics, and transmission electron microscopy (TEM). The study also explored the impact of QHD on antibiotic resistance in these HP strains with strong biofilm formation. Crystalline violet method, scanning electron microscopy, laser confocal scanning microscopy, and (p)ppGpp chromatographic identification were employed to evaluate the effect of QHD on biofilm in strong biofilm-forming HP strains. The effect of QHD on biofilm and efflux pump-related gene expression was evaluated by quantitative polymerase chain reaction. Non-targeted metabolomics with UHPLC-MS/MS was used to identify potential metabolic pathways and biomarkers which were different between the NC and QHD groups. HP could form biofilms of different degrees in vitro, and the intensity of formation was associated with the drug resistance of the strain. QHD had strong bacteriostatic and bactericidal effects on HP, with MICs of 32-64 mg/mL. QHD could inhibit the biofilm formation of the strong biofilm-forming HP strains, disrupt the biofilm structure, lower the accumulation of (p)ppGpp, decrease the expression of biofilm-related genes including LuxS, Spot, glup (HP1174), NapA, and CagE, and reduce the expression of efflux pump-related genes such as HP0605, HP0971, HP1327, and HP1489. Based on metabolomic analysis, QHD induced oxidative stress in HP, enhanced metabolism, and potentially inhibited relevant signaling pathways by upregulating adenosine monophosphate (AMP), thereby affecting HP growth, metabolism, and protein synthesis. QHD exerts bacteriostatic and bactericidal effects on HP, and reduces HP drug resistance by inhibiting HP biofilm formation, destroying its biofilm structure, inhibiting the expression of biofilm-related genes and efflux pump-related genes, enhancing HP metabolism, and activating AMP in HP.

Phenotypic and Molecular Characterization of Vancomycin and Linezolid Resistance in Clinical Isolates of Staphylococcus aureus

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of healthcare-associated infections and poses a significant global challenge due to its high pathogenicity and rapid spread among patients. Vancomycin and linezolid are key antibiotics for treating staphylococcal MRSA infections. Objectives: This study aimed to investigate resistance to vancomycin and linezolid in clinical isolates of S. aureus. Methods: In this study, 270 S. aureus isolates were collected from patients admitted to hospitals in Tehran and Qazvin. Strain identification was performed using biochemical methods and femA gene amplification. Resistance to methicillin and vancomycin was determined using disk diffusion, minimum inhibitory concentration (MIC) determination, and E-test methods. Methicillin resistance was also assessed by detecting the presence of the mecA and mecC genes. Resistance to vancomycin and linezolid was evaluated by examining the presence of vanA, B, C, and cfr genes using PCR and sequencing. Results: Out of 270 S. aureus isolates, 152 (56.3%) were identified as MRSA, with 144 (94.7%) of these MRSA isolates containing the mecA gene. Two (0.7%) VRSA isolates were observed, and the vanA gene was detected in both. Additionally, four VISA (1.4%) isolates were identified, but the vanA gene was not detected in any of the VISA strains. None of the isolates contained the mecC, vanB, or vanC genes. Among the 10 (3.7%) linezolid-resistant isolates, none contained the cfr gene. Conclusions: The results of this study demonstrated a high prevalence of MRSA isolates, which can lead to treatment failure with beta-lactam antibiotics. The emergence and spread of VRSA and VISA isolates pose a serious concern for the healthcare system, particularly in the treatment of MRSA infections. Therefore, the findings emphasize the need for appropriate monitoring and control measures to prevent the emergence and transmission of MRSA and VRSA strains. Vancomycin and linezolid remain suitable drugs for treating these infections.

Determination of antimicrobial susceptibility and virulence-related genes of Trueperella pyogenes strains isolated from various clinical specimens in animals

In this study, a total of 32 Trueperella pyogenes strains isolated from different disease specimens in cattle, sheep and goats were examined. Antimicrobial susceptibility of the isolates to 10 antimicrobials were determined using the E-test method and MIC values of the antimicrobials were investigated. The genes that play a role in the antimicrobial resistance or virulence of T. pyogenes were determined by PCR using gene specific primers. In the study, all the isolates were susceptible to penicillin and cephalosporin. The highest resistance rate in the isolates was determined against streptomycin (56.25%) and tetracycline (53.12%) and MIC90 values for these antimicrobials were found to be >256 μg/ml and 12 μg/ml, respectively. The ermX gene was found to be positive in 8 (80%) of 10 isolates that were resistant to macrolide group antimicrobials. Among 20 aminoglycoside resistant isolates, aadA1, aadA9, strA-strB, and aac(6’)-aph(2’’) genes were determined in 5 (25%), 14 (70%), 7 (35%) and 1 (5%) of the isolates, respectively. When the presence of virulence-related genes in the isolates was examined, nanP (93.75%), fimA (93.75%) and plo (90.62%) genes were detected in the majority of the isolates. While the cbpA gene was negative in all isolates, the fimG gene was found in a limited number of the isolates (15.62%). It was concluded that streptomycin and tetracycline resistance should be considered in T. pyogenes isolates. Also, nanP, fimA and plo genes may have an important role in the pathogenesis of the infections.

A Road Less Traveled: E-test Method for Antifungal Susceptibility Testing in Trichophyton mentagrophyte Isolates Among Patients Presenting With Dermatophytosis at a Tertiary Healthcare Center in North India.

Introduction Dermatophytosis is a common infection of the skin, hair, and nails caused by dermatophytes, a group of filamentous fungi capable of digesting and obtaining nutrients from keratin. Dermatophytes comprise three important genera: Epidermophyton, Microsporum,and Trichophyton. This study aimed to analyze the antifungal susceptibility patterns of Trichophyton mentagrophytes isolates using the epsilometer test (E-test) method. Material and methods This prospective observational study was conducted on clinically suspected cases of dermatophytosis. All samples, including skin scrapings, hair, and nails, were subjected to potassium hydroxide (KOH) examination followed by fungal culture. The Trichophyton mentagrophytes isolates were then subjected to antifungal susceptibility testing using the E-test method for the two most prescribed antifungals: itraconazole and fluconazole. Results In this study, one-third of the patients who tested positive for dermatophytosis belonged to the same family, with spouses being the most commonly affected. Tinea corporis was the most common clinical presentation, with Trichophyton mentagrophytes identified as the most common etiological agent. Itraconazole was more effective than fluconazole. Conclusion The current study demonstrated that antifungal susceptibility testing of dermatophytes using the E-test is easier and can be applied in routine laboratories as a screening method, serving as an alternative to broth microdilution.

A comparative in vitro sensitivity study of “cefepime-tazobactam” and other antibiotics against Gram-negative isolates from intensive care unit

Objectives: The major health problem today includes the emergence of multidrug-resistant (MDR) bacteria, especially extended-spectrum β lactamase, carbapenemases, and Amp C-producing Gram-negative bacilli (GNB). Our study is aimed to recognize the in vitro susceptibility pattern of cefepime/tazobactam compared to other antibiotics used against GNB in an intensive care unit (ICU) setting. Materials and Methods: We conducted a prospective observational research comprising all GNB isolated from clinical samples of patients admitted to the ICU throughout the study period from January 2021 to December 2021. All of the isolates were analyzed using “ Matrix Assisted Laser Desorption/ionization - time of flight - Mass spectrometry assay (MALDI-TOF-MS)” for identification and the Kirby-Bauer disc diffusion technique to test for susceptibility. Cefepime-tazobactam was tested by E-test (Hi-Media, Mumbai) method. The minimum inhibitory concentrations (MIC) of cefepime (as in Clinical Laboratory Standards Institute, 2021) has been utilized to elucidate the sensitivity of cefepime-tazobactam, as no criteria for cefepime-tazobactam is available. Statistical Analysis: All statistical analysis was performed using the software program IBM Statistical Package for Social Sciences (SPSS) Statistics version 20.0, with P < 0.05 considered statistically significant. Results: We included a total of 480 GNB isolated from blood, pus, body fluids, endotracheal aspirates (ETA), and sputum samples. The most common microorganism tested for susceptibility to cefepime-tazobactam was Klebsiella pneumoniae (182/480, 37.92%) followed by Acinetobacter baumannii (135/480, 28.12%), and Pseudomonas aeruginosa (94/480, 19.58%). K. pneumoniae and Enterobacter aerogenes were most resistant to all the antibiotics tested against them. K. pneumoniae was most resistant to meropenem (41/182, 22.53%), followed by imipenem (42/182, 23.08%) and cefoperazone-sulbactam (49/182, 26.92%) and was predominantly found susceptible to cefepime-tazobactam (122/182, 67.04%). Conclusions: Cefoperazone-tazobactam is a new “β-lactam/β-lactamase combination” found effective in the in vitro analysis of drug-resistant isolates of GNB.

Molecular Characterization of Resistance and Virulence Factors of Trueperella pyogenes Isolated from Clinical Bovine Mastitis Cases in China.

The present study was designed to investigate the resistance determinants and virulence factors of 45 Trueperella pyogenes isolates from clinical bovine mastitis in Hexi Corridor of Gansu, China. Minimum inhibitory concentrations (MICs) was tested by E-test method. Gene of antimicrobial resistance, virulence integrase and integron gene cassettes were determined by PCR and DNA sequencing. The T. pyogenes isolates exhibited high resistance to streptomycin (88.9%) and tetracycline (64.4%), followed by erythromycin (15.6%) and gentamicin (13.3%). Resistance to streptomycin was most commonly encoded by aadA9 (88.9%); and to tetracycline, by tetW (64.4%). Importantly, all streptomycin-resistant isolates carried aadA9 alone or in combination with aadA1, aadA11 and strA-strB. Similarly, all tetracycline-resistant isolates harbored tetW alone or in combination with tetA33. Meanwhile, ermX was detected in 13.3% isolates, only one erythromycin-resistant isolate was not identified for this gene. Moreover, all T. pyogenes isolates carried class 1 integrons, and 17.8% of them contained gene cassettes, including arrays aadA1-aadB (4.4%), aad A24-dfrA1-ORF1 (2.2%) and aadA1 (2.2%). Furthermore, all tested isolates harbored virulent genes plo and fimA, followed by fimC (88.9%), fimE (86.6%) nanP (75.6%), nanH (40.0%), cbpA (35.6%) and fimG (6.7%). To our knowledge, this is the first report of integron gene cassettes of T. pyogenes isolates from bovine mastitis cases in China. These findings are useful for developing the prevention and the virulence factors of T. pyogenes could be promising candidates for vaccine antigens for bovine mastitis caused by T. pyogenes in China.

Emergence of aztreonam/avibactam and tigecycline-resistant Pseudomonas putida group Co-producing blaIMP-1, blaAFM-4 and blaOXA-1041 with a novel sequence type ST268 in Southwestern China

ObjectivesThe emergence of carbapenem-resistant Pseudomonas putida (CRPP) has raised public awareness. This study investigated two strains from the Pseudomonas putida group that were resistant to carbapenem, tigecycline, and aztreonam-avibactam (ATM−AVI), with a focus on their microbial and genomic characteristics. MethodsWe assessed the antibiotic resistance profile using broth dilution, disk diffusion, and E-test methods. Efflux pump phenotype testing and real-time quantitative PCR were employed to evaluate efflux pump activity in tigecycline resistance, while polymerase chain reaction was utilized to detect common carbapenem genes. Additionally, whole-genome sequencing was performed to analyze genomic characteristics. The transferability of blaIMP-1 and blaAFM-4 was assessed through a conjugation experiment. Furthermore, growth kinetics and biofilm formation were examined using growth curves and crystal violet staining. ResultsBoth strains demonstrated resistance to carbapenem, tigecycline, and ATM-AVI. Notably, NMP can restore sensitivity to tigecycline. Subsequent analysis revealed that they co-produced blaIMP-1, blaAFM-4, tmexCD-toprJ, and blaOXA-1041, belonging to a novel sequence type ST268. Although they were closely related on the phylogenetic tree, they exhibited different levels of virulence. Genetic environment analysis indicated variations compared to prior studies, particularly regarding the blaIMP-1 and blaAFM-4 genes, which showed limited horizontal transferability. Moreover, it was observed that temperature exerted a specific influence on their biological factors. ConclusionWe initially identified two P. putida ST268 strains co-producing blaIMP-1, blaAFM-4, blaOXA-1041, and tmexCD-toprJ. The resistance to tigecycline and ATM-AVI can be attributed to the presence of multiple drug resistance determinants. These findings underscore the significance of P. putida as a reservoir for novel antibiotic resistance genes. Therefore, it is imperative to develop alternative antibiotic therapies and establish effective monitoring of bacterial resistance.

Silencing of Resistance Mechanism in Vancomycin-Resistance Enterococci Using Antisense RNA of vanA Gene

The World Health Organization has included the problem of antibiotic resistance among the top 10 important health problems in the world. Treatment of infectious diseases has become more difficult due to the spread of antibiotic resistance between bacteria via transposable elements. Vancomycin-resistant enterococci (VRE) are of critical medical and public health importance due to their association with serious nosocomial infections and high risk of death. One of the most important features of VREs is that they have multiple antibiotic resistance and treatment options are reduced. Therefore, new treatment methods are needed. The vanA gene constitutes the building block of the vancomycin resistance mechanism and causes high resistance to vancomycin. In this study, it was aimed to investigate the neutralization of the vancomycin resistance mechanism by creating vanA antisense RNA (asRNA). The vanA positive VRE50 strain in our culture collection which was isolated from the clinical sample, was used to amplify the vanA gene by polymerase chain reaction (PCR). The amplified vanA amplicon was inserted inversely into the pUC19 plasmid by means of the enzyme cutting sites in the primers used. The resulting plasmid was combined with the pAT392 plasmid which can replicate in gram-positive bacteria and a fusion plasmid was created. The fusion plasmid whose orientation was confirmed, was transferred to the wild strain VRE50 by electroporation method. Minimum inhibitory concentration (MIC) values of transformed VRE (tVRE50) and wild type VRE50 strains used as control were determined by the E-Test method. The vancomycin MIC value of the wild type VRE50 strain was determined as 1024 µg/mL and that of the tVRE50 strain was 32 µg/mL and it was determined that the vancomycin resistance of the tVRE50 strain decreased with asRNA (antisense RNA). Antisense RNA technology is an important method for neutralizing the expression of genes. This study showed that neutralization of the vancomycin resistance gene may provide a lower MIC value in a vancomycin-resistant enterococcus strain and lead to increased susceptibility. This new approach provides a new method for VRE treatment by neutralizing the vancomycin resistance mechanism. The result obtained in this study needs to be supported by in vivo tests.

Combinatorial effects of resazomycins and azithromycin against Neisseria gonorrhoeae

Gonorrhea is a sexually transmitted disease caused by the bacterium Neisseria gonorrhoeae. The WHO estimates 87 million new gonococcal infections occur each year making it the second most common sexually transmitted bacterial infection. When left untreated, gonorrhea can cause pelvic inflammatory disease and infertility in women. Treatment is further complicated by the fact that N. gonorrhoeae has acquired resistance to all antibiotics approved for treatment including the gold standard ceftriaxone. One way to address the growing problem of antibiotic resistance is to administer drugs in combination in the hope the two are more effective together than alone. Resazomycins, a novel family of resazurin (Rz) based antibiotics, exhibit antimicrobial activity against N. gonorrhoeae and Francisella tularensis. Here, we used E-test assays to determine if there was a synergistic bactericidal effect between resazomycins and azithromycin, which is commonly used to treat gonorrhea. Multiple clinical isolates of N. gonorrhoeae were more sensitive to azithromycin in the presence of Rz. However, additional testing in a checkerboard assay revealed no synergistic effect between azithromycin and Rz or another resazomycin, resorufin pentyl ether (RPE). Upon further investigation, we determined the conflicting results between the E-test and checkerboard method were due to differences in bacterial load. When the E-test assays were repeated using a lower inoculum of N. gonorrhoeae, increased sensitivity to azithromycin was not observed in the presence of Rz. In the future, we plan to test resazomycins in combination with other antibiotics against N. gonorrhoeae, particularly extensively drug-resistant strains. (This research was made possible by NIH Grant P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence and the NASA West Virginia Space Grant Consortium, Grant # 80NSSC20M0055)

Evidence and antibiotic resistance profiles of clinical Acinetobacter calcoaceticus-Acinetobacter baumannii (ACB) and non-ACB complex members in companion animals: A 2020–2022 retrospective study

To evaluate the frequency of Acinetobacter spp., belonging to both Acinetobacter calcoaceticus-baumannii (ACB) and non-ACB complex, and their antibiotic resistance profiles in veterinary medicine, a three-year (2020–2022) retrospective study was carried out on sick companion animals. Epidemiological data from different clinical canine, feline, and equine samples, were acquired. For each strain, MALDI-TOF MS identification and susceptibility to a panel of 11 antibiotics, by Kirby-Bauer and E-test methods, were performed. Out of 628 bacteriological examinations, 2.5% resulted positive for strains belonging to Acinetobacter genus. Frequencies of 2.3%, 1.9%, and 3% were obtained from both in-visiting and hospitalized dogs, cats, and horses, respectively. Members of ACB-complex accounted for 50% of isolates. Since all strains resulted susceptible to aminoglycosides and polymyxins, no pandrug-resistant (PDR) species were recorded. While 12.5% A. baumannii resulted extensively-drug resistant (XDR), a higher percentage of multidrug-resistant strains was recorded among non-ACB strains (35.5%) than ACB strains (25%). Susceptibility was observed in the same percentage in both groups (62.5%). All ACB strains confirmed their intrinsic resistances. Non-ACB species showed lower resistances against antipseudomonal penicillins plus beta-lactamase inhibitors (P=0.1306), III generation cephalosporins (P=0.0547), and tetracyclines (P=0.0209) than ACB species. Carbapenem-resistance was observed for XDR A. baumannii (12.5%) and, in particular for MDR non-ACB complex members (25%). To our knowledge, A. lactucae represents the first description in two sick dogs in Italy. Furthermore, our results emphasize the role of non-ACB-complex species as important zoonotic pathogens, which could be reservoirs of clinically relevant resistance profiles.

Pannonibacter anstelovis sp. nov. Isolated from Two Cases of Bloodstream Infections in Paediatric Patients.

This study describes two cases of bacteraemia sustained by a new putative Pannonibacter species isolated at the U.O.C. of Microbiology and Virology of the Policlinico of Bari (Bari, Italy) from the blood cultures of two patients admitted to the Paediatric Oncohaematology Unit. Pannonibacter spp. is an environmental Gram-negative bacterium not commonly associated with nosocomial infections. Species identification was performed using Sanger sequencing of the 16S rRNA gene and Whole-Genome Sequencing (WGS) for both strains. Genomic analyses for the two isolates, BLAST similarity search, and phylogeny for the 16S rDNA sequences lead to an assignment to the species Pannonibacter phragmitetus. However, by performing ANIb, ANIm, tetranucleotide correlation, and DNA-DNA digital hybridization, analyses of the two draft genomes showed that they were very different from those of the species P. phragmitetus. MALDI-TOF analysis, assessment of antimicrobial susceptibility by E-test method, and Analytical Profile Index (API) tests were also performed. This result highlights how environmental bacterial species can easily adapt to the human host and, especially in nosocomial environments, also gain pathogenic potential through antimicrobial resistance.

Role of MIC levels and 23S rRNA mutation sites to clarithromycin in 14-day clarithromycin bismuth quadruple therapy for Helicobacter pylori eradication: A prospective trial in Beijing

BackgroundRising clarithromycin resistance undermines Helicobacter pylori (H. pylori) treatment efficacy. We aimed to determine clarithromycin's minimum inhibitory concentration (MIC) levels and identify specific mutation sites in the 23S ribosomal subunit (23S rRNA) that predict treatment outcomes in a 14-day regimen of clarithromycin bismuth quadruple therapy (amoxicillin 1g, clarithromycin 500 mg, rabeprazole 10 mg, and colloidal bismuth pectin 200 mg). Materials and methodsWe included adult H. pylori patients who hadn't previously undergone clarithromycin-based treatment, either as initial or rescue therapy. Exclusions were made for penicillin allergy, recent use of related medications, severe illnesses, or inability to cooperate. Patients underwent a 14-day clarithromycin bismuth quadruple therapy. Gastric mucosa specimens were obtained during endoscopy before eradication. MIC against amoxicillin and clarithromycin was determined using the E-test method. The receiver operating characteristic (ROC) curve helped to find the optimal clarithromycin resistance MIC breakpoint. Genetic sequences of H. pylori 23S rRNA were identified through Sanger Sequencing. (ChiCTR2200061476) ResultsOut of 196 patients recruited, 92 met the inclusion criteria for the per-protocol (PP) population. The overall intention-to-treat (ITT) eradication rate was 80.00 % (84/105), while the modified intention-to-treat (MITT) and PP eradication rates were 90.32 % (84/93) and 91.30 % (84/92) respectively. No amoxicillin resistance was observed, but clarithromycin resistance rates were 36.19 % (38/105), 35.48 % (33/93), and 34.78 % (33/92) in the ITT, MITT, and PP populations respectively. Compared with the traditional clarithromycin resistance breakpoint of 0.25 μg/mL, a MIC threshold of 12 μg/mL predicted better eradication. Among 173 mutations on 152 sites in the 23S rRNA gene, only the 2143A > G mutation could predict eradication outcomes (p < 0.000). ConclusionsInterpretation of elevated MIC values is crucial in susceptibility testing, rather than a binary "susceptible" or "resistant" classification. The 2143A > G mutation has limited specificity in predicting eradication outcomes, necessitating further investigation into additional mutation sites associated with clarithromycin resistance.