Susceptibility Testing Methods Research Articles

A novel rapid bioluminescence-based antimicrobial susceptibility testing method based on adenosine triphosphate consumption.

Standard, phenotypic antimicrobial susceptibility testing (AST) methods require 16-20 h of incubation and are considered as the bottleneck in providing timely input for appropriate antimicrobial treatment. In this study, a novel adenosine triphosphate (ATP)-bioluminescence-based method which allows rapid AST within 3 h was described. Standard AST was performed for 56 Enterobacterales isolates using EUCAST disk diffusion (DD) methodology. For the bioluminescence-based rapid AST, suspensions of bacteria were prepared using Mueller-Hinton broth to obtain a turbidity of 0.5 McFarland. The suspensions were distributed into 96-well microtiter plates. ATP (20 mM) and fixed concentrations of different antibiotics were added. Following incubation at 37°C for 1 h, a luminescent reaction mixture, including the substrate luciferin and luciferase enzyme solutions, was added. The chemiluminescence was monitored using an imaging system. Light production demonstrated the presence of ATP, indicating that the isolate was susceptible to the antibiotic in the well. Absence or decrease of light intensity, compared with the growth control well, indicated the use of ATP as an indirect measure of bacterial growth, and therefore resistance to the antibiotic in the well. The novel AST method was tested using a total of 348 test wells. Concordance was achieved for 290 (83.3%) of the tests, whereas 52 (14.9%) and 6 (1.7%) tests caused minor and major errors, respectively. In this study, a bioluminescence-based rapid AST was developed based on the consumption of ATP by bacteria. Our method's uniqueness relies on determining ATP consumption by microorganisms in the presence or absence of an antibiotic. The novel AST method described in this study lays the groundwork for obtaining rapid results, which should be considered as a proof of concept. With further optimization studies, this novel method can provide higher accuracy and be introduced into clinical practice as a routine AST method.

High Fosfomycin Susceptibility in Escherichia Coli Recovered from Urine in Brazil

Urinary Tract Infections (UTIs) are widespread globally, with a notably higher incidence in women. In 2018, the European Medicines Agency (EMA) endorsed the use of fluoroquinolones for uncomplicated UTI treatment. Following this recommendation, several international agencies adopted similar guidelines. Consequently, older antimicrobial agents like fosfomycin emerged as primary treatment options for UTIs. In this context, our study aimed to evaluate the susceptibility of Escherichia coli strains from urine samples to various recommended UTI antibiotics. These strains were collected between January 2017 and July 2020 in São Paulo, Brazil. We utilized the disk-diffusion method for antimicrobial susceptibility testing, interpreting the results according to BRCAST/EUCAST guidelines. Out of the 86,957 urine cultures undertaken during this timeframe, 10,041 yielded E. coli isolates. Of these, 8,655 were tested against fosfomycin, with 99.0% (8,572 strains) found to be susceptible. Additionally, susceptibility rates for other drugs were as follows: nitrofurantoin (95.8%), amoxicillin/clavulanic acid (83.9%), ciprofloxacin (65.1%), norfloxacin (65.6%), and levofloxacin (67.7%). Notably, of the 571 ESBL-positive strains, 94.0% were susceptible to fosfomycin. It’s important to mention a slight decline in fosfomycin susceptibility observed during this period. This finding underscores the importance of continuous monitoring for fosfomycin resistance and rational usage of the drug.

Contamination of Kazakhstan cheeses originating from Escherichia coli and its resistance to antimicrobial drugs

Background and Aim: Escherichia coli, a commensal intestine bacterium of vertebrates, is widely distributed in the environment and indicates the microbiological quality of food products in relation to coliforms. In addition, virulent strains, particularly E. coli O157:H7, cause outbreaks of toxic infections caused by consuming dairy products. Because food safety studies regarding E. coli have not been conducted in Central Asia, this research aimed to study the characteristics of contamination, microbiological and genotypic properties, and resistance to antimicrobial agents of E. coli strains that contaminate various types of commercialized cheeses originating from Kazakhstan. Materials and Methods: In retail outlets, 207 samples of three types of cheese produced by 22 industrial and eight small enterprises in the central, eastern, southern, and northern regions of Kazakhstan were selected in 2020–2023. E. coli contamination was examined using standard microbiological, mass spectrometric, and molecular genetic methods. The discodiffuse European Committee on Antimicrobial Susceptibility Testing method was used to test the resistance of the identified E. coli isolates (65/207; 31.4%) to 20 antibacterial drugs. The Shiga toxin-producing E. coli (VT1 and VT2) and E. coli O157:H7 (eae) genes were investigated in all E. coli isolates using multiplex polymerase chain reaction. Results: An average of 31.4% samples of commercial Kazakhstani cheeses of various types were found to be contaminated with E. coli in almost all geographical regions of Kazakhstan, regardless of the productivity of the dairy enterprises. Soft cheeses produced by small farms (80% of samples) packaged at the retail site (100%) were the most contaminated with E. coli. The microbiological index (colony-forming unit/g) was unsatisfactory and unsuitable in 6.2% of such cheese samples. For the first time in Central Asia, the enteropathogenic strain E. coli O157:H7 was detected in 0.5% of cheese samples. E. coli isolates from cheese samples were resistant to 65% of antibacterial drugs and contained resistance genes to β-lactams, sulfonamides, and quinolones groups. At the same time, 25% of the E. coli isolates were multi-resistant to three or more antimicrobial agents. Conclusion: The high level of contamination caused by multi-antibiotic resistant E. coli strains, including pathogenic pathogens, poses a risk to public health and highlights the need for further research on the monitoring and control of coliform enteropathogens in food products. Keywords: antibiotic resistance, Escherichia coli O157:H7, Kazakhstan cheese, microbial contamination.

Anti-microbial efficacy of a scientifically developed and standardized herbal-alcohol sanitizer.

Hands are the primary mode of transmission of microbe-based infections, as they harbor normal microbiota and pathogenic microbes. SARS-CoV-2 has endangered lives worldwide, and WHO has recommended good hygiene practices, especially hand hygiene. In addition, other infectious diseases like diphtheria, measles, tuberculosis, HIV, malaria, etc. are spreading in the shadow of the COVID-19 pandemic. The anti-microbial efficiency of two in-house developed herbal-alcohol based hand sanitizers containing Azadirachta indica, Citrus limon, Zingiber officinale, and Aloe vera (HS1) and Zingiber officinale replaced with Ocimum sanctum (HS2) was evaluated. HS1, with Zingiber officinale, and HS2, with Ocimum sanctum, herbal sanitizers showcased in-vitro anti-viral activity on MDCK cells using the reference strain of influenza A virus, A/PR/8/34 (H1N1), and reduced 99.99% of microbial load within 30s of contact time, estimated by the Antimicrobial Susceptibility Testing Method. On volunteers, HS1 and HS2 were more effective than alcohol-based WHO sanitizers. Moreover, HS2 sanitizer is more effective against viruses and has better efficiency and hedonic qualities in volunteers than HS1. These sanitizers don't irritate or dry up the skin and have a longer shelf life. Overall, findings reveal that herbal-alcohol-based sanitizers are promising hand hygiene products with the capability of reducing microbial load.

Antibiotic resistance determination using Enterococcus faecium whole-genome sequences: a diagnostic accuracy study using genotypic and phenotypic data

DNA sequencing could become an alternative to invitro antibiotic susceptibility testing (AST) methods for determining antibiotic resistance by detecting genetic determinants associated with decreased antibiotic susceptibility. Here, we aimed to assess and improve the accuracy of antibiotic resistance determination from Enterococcus faecium genomes for diagnosis and surveillance purposes. In this retrospective diagnostic accuracy study, we first conducted a literature search in PubMed on Jan14,2021, to compile a catalogue of genes and mutations predictive of antibiotic resistance in E faecium. We then evaluated the diagnostic accuracy of this database to determine susceptibility to 12 different, clinically relevant antibiotics using a diverse population of 4382 E faecium isolates with available whole-genome sequences and invitro culture-based AST phenotypes. Isolates were obtained from various sources in 11 countries worldwide between 2000 and 2018. We included isolates tested with broth microdilution, Vitek 2, and disc diffusion, and antibiotics with at least 50 susceptible and 50 resistant isolates. Phenotypic resistance was derived from raw minimum inhibitory concentrations and measured inhibition diameters, and harmonised primarily using the breakpoints set by the European Committee on Antimicrobial Susceptibility Testing. A bioinformatics pipeline was developed to process raw sequencing reads, identify antibiotic resistance genetic determinants, and report genotypic resistance. We used our curated database, as well as ResFinder, AMRFinderPlus, and LRE-Finder, to assess the accuracy of genotypic predictions against phenotypic resistance. We curated a catalogue of 228 genetic markers involved in resistance to 12 antibiotics in E faecium. Very accurate genotypic predictions were obtained for ampicillin (sensitivity 99·7% [95% CI 99·5-99·9] and specificity 97·9% [95·8-99·0]), ciprofloxacin (98·0% [96·4-98·9] and 98·8% [95·9-99·7]), vancomycin (98·8% [98·3-99·2] and 98·8% [98·0-99·3]), andlinezolid resistance (after re-testing false negatives: 100·0% [90·8-100·0] and 98·3% [97·8-98·7]). High sensitivity was obtained for tetracycline (99·5% [99·1-99·7]), teicoplanin (98·9% [98·4-99·3]), and high-level resistance to aminoglycosides (97·7% [96·6-98·4] for streptomycin and 96·8% [95·8-97·5] for gentamicin), although at lower specificity (60-90%). Sensitivity was expectedly low for daptomycin (73·6% [65·1-80·6]) and tigecycline (38·3% [27·1-51·0]), for which the genetic basis of resistance is not fully characterised. Compared with other antibiotic resistance databases and bioinformatic tools, our curated database was similarly accurate at detecting resistance to ciprofloxacin and linezolid and high-level resistance to streptomycin and gentamicin, but had better sensitivity for detecting resistance to ampicillin, tigecycline, daptomycin, and quinupristin-dalfopristin, and better specificity for ampicillin, vancomycin, teicoplanin, and tetracycline resistance. In a validation dataset of 382 isolates, similar or improved diagnostic accuracies were also achieved. To our knowledge, this work represents the largest published evaluation to date of the accuracy of antibiotic susceptibility predictions from E faecium genomes. The results and resources will facilitate the adoption of whole-genome sequencing as a tool for the diagnosis and surveillance of antimicrobial resistance in E faecium. A complete characterisation of the genetic basis of resistance to last-line antibiotics, and the mechanisms mediating antibiotic resistance silencing, are needed to close the remaining sensitivity and specificity gaps in genotypic predictions. Wellcome Trust, UK Department of Health, British Society for Antimicrobial Chemotherapy, Academy of Medical Sciences and the Health Foundation, Medical Research Council Newton Fund, Vietnamese Ministry of Science and Technology, and European Society of Clinical Microbiology and Infectious Disease.

Antifungal susceptibility profile and local epidemiological cut-off values of Yarrowia (Candida) lipolytica: an emergent and rare opportunistic yeast.

Yarrowia lipolytica, also known as Candida lipolytica, is an emerging opportunistic "rare pathogenic yeast". Due to the limited data on its antifungal susceptibility, the clinical treatments become challenging. Based on the China Hospital Invasive Fungal Surveillance Network (2009-2022), we conducted a comprehensive multi-method study on clinical isolates from various central hospitals. This study is currently the largest study carried out to assess the antifungal susceptibility of Y. lipolytica. It is also the first to establish local epidemiological cut-off values (L-ECOFFs), identify its ERG11 mutations, and assess the consistency between the three prevalent commercial antifungal susceptibility testing methods and the broth microdilution method. We recommend the Sensititre YeastOne as the best option for antifungal susceptibility testing for Y. lipolytica, followed by the ATB FUNGUS 3. Nevertheless, practitioners should use the MIC test strip with discretion.

Prevalence of Multi Drug Resistance Escherichia coli Bacteriuria among Antenatal Women in a Tertiary Hospital in Enugu, Nigeria

Background: Urinary Tract Infection (UTI) due to Escherichia coli (E. coli) is a common health problem among women. Pregnant women are at increased risk due to the physiological changes associated with pregnancy. The present study was designed to determine the prevalence of E. coli UTI and its antibiotic susceptibility pattern among pregnant women at the antenatal clinic of the Enugu State University of Science and Technology Teaching Hospital, Enugu, Nigeria.
 Materials and Methods: A cross sectional study was carried out on a total of 200 pregnant women with and without symptoms of UTI. Mid-stream urine samples were collected and inoculated into Cysteine Lactose Electrolyte Deficient (CLED) and MacConkay. Colony counts > 102 cfu/ml of urine were regarded as significant bacteriuria. Pure isolates of bacterial pathogen were characterized by colony morphology, gram-stain and standard biochemical procedures. The method of agar disc diffusion susceptibility testing was used to determine the antibiotic resistance and sensitivity of the isolates.
 Results: The overall prevalence of UTI was 15.4% while 48.5% were symptomatic cases and 5.0% asymptomatic cases. E,Coli was the most frequently isolated organism with a prevalence of 53.4% (111 samples) while other pathogens accounted for 46.6% (89 samples). E. coli showed the highest resistance to the antibiotic Ampicillin 100%, 78% to Tetracycline and 70% to Rocephine but a low resistance of 16.7% against Nitrofurantoin and Ceftriazone respectively.
 Conclusion: UTI due to E. coli in pregnant women in Enugu is relatively high and shows high resistance to routine antibiotics. It is therefore, strongly recommended to undertake a routine culture as part of the antenatal care for pregnant women.

Rapid detection of drug-resistant Mycobacterium tuberculosis by Modified MODS assay suitable for resource-poor settings.

Cross contamination and biosafety are concerns with the microscopic observation drug susceptibility assay. To address these issues, we modified the MODS technique in the current study. Two hundred and seventy-five samples were processed on LJ media and drug susceptibility was performed by the Indirect agar proportion method. A modified MODS test was done in tissue culture bottles. GenoType MTBDRplus assay was performed to detect the resistance and mutational pattern associated with the resistances. Sensitivity, specificity, positive predictive value, and negative predictive value for the detection of tuberculosis by modified MODS were 97.44%, 80.00%, 97.44%, and 80.00% respectively. The perfect agreement was seen between modified MODS and the Indirect agar proportion method for drug susceptibility testing of isoniazid (kappa = 0.923) and rifampicin (kappa = 1). The contamination rate, cost and TAT for modified MODS were less as compared to the solid media. In the case of MDR-TB isolates S531L (66.66%) was the most prevalent mutation in the rpoB gene followed by S315T2 mutation (58.33%) and T8C (41.66%) in katG and inhA gene respectively. In hetero-resistant strains, C-15T mutation (37.50%) was the most common followed by A-16G (12.50%) in the inhA gene. In INH mono-resistant strains only two mutations were observed i.e., S-315T1(50%) and C-15T (50%) in the katG and inhA genes respectively. Modified MODS proved to be cost-effective and user-friendly, with minimal risk to the handler and no cross-contamination between samples were observed. Hence, it can be used in low-income countries for early detection of tuberculosis and its resistance.

Drug-Resistant Characteristics, Genetic Diversity, and Transmission Dynamics of Multidrug-Resistant Mycobacterium tuberculosis in Jiangxi, China.

In this study, we aimed to determine the transmission pattern of multidrug-resistant tuberculosis (MDR-TB) isolates circulating in Jiangxi Province with whole-genome sequencing (WGS). In addition, we also sought to describe mutational resistome of MDR-TB isolates. A total of 115 MDR-TB isolates determined by the phenotypic proportion method of drug susceptibility testing between January 2018 and December 2022 from provincial drug surveillance (DRS) in Jiangxi were included in our analysis. The demographic data and treatment history were extracted from the National TB Registry System. WGS was used to analyze the genotypic characteristics of drug resistance and transmissions. About 62.6% of MDR-TB strains were isolated from cases that received previous anti-tuberculosis treatment. According to the WGS results, 96.5% were genotypic MDR-TB, and more than half of MDR-TB isolates tested were also resistant to streptomycin (59.1%), ethambutol (56.5%), and fluroquinolones (53.0%), while resistance to cycloserine and linezolid was lowest, only in two (1.7%) and one (0.9%) isolate, respectively. Ser450Leu in rpoB (57.9%), Ser315Thr in katG (74.1%), Met306Val in embB (40.0%), Lys43Arg in rpsL (75.0%), Ala90Val in gyrA (32.8%) were predominant mutant types among the rifampin-, isoniazid-, ethambutol-, streptomycin-, fluoroquinolones-resistant isolates, respectively. Lineage 2 (East Asian genotype) occurred at the highest frequency with 97 cases (84.3%), followed by lineage 4 (Euro-American genotype) with 18 cases (15.7%). Additionally, 5 clusters consisting of 10 isolates were identified in the present study, demonstrating a clustering rate of 8.7%. MDR/Rifampicin-Resistant (RR)-TB epidemic in this region is driven by lineage 2 clade that also show higher resistance to other anti-tuberculosis drugs. Lower cluster rates compared with a relatively higher proportion of new MDR-TB cases indicate that a considerable number of MDR-TB cases remain undiagnosed.

Molecular detection of multidrug and methicillin resistance in Staphylococcus aureus isolated from wild pigeons (Columba livia) in South Africa

Staphylococcus aureus is an important human and veterinary pathogen. The present study aimed to determine the prevalence of antibiotic resistance among S. aureus isolated from samples obtained from free-flying wild pigeons and houseflies from different locations surrounding a local hospital in the Greater Durban area in KwaZulu-Natal Province, South Africa. Environmental fecal samples were obtained from wild pigeons that inhabits the grounds of a local public hospital located on the South Beach area, Durban, South Africa. Housefly samples were collected from three different locations (Kenneth Stainbank Nature Reserve, Montclair/Clairwood, and Glenwood/Berea) in the greater Durban area, all within a close proximity to the hospital. Following enrichment, identification, and antimicrobial resistance profiling, S. aureus isolates were subjected to DNA extraction using the boiling method. It was found that 57 out of 252 samples (22.62%) were positive for S. aureus. The Kirby-Bauer disk diffusion method of antibiotic susceptibility testing was performed and revealed that antibiotic resistance rates to penicillin and rifampicin were the most common, with both returning 48 (84.2%) out of the 57 S. aureus isolates being resistant to penicillin and rifampicin. Antibiotic resistance rates to clindamycin, linezolid, erythromycin, tetracycline, cefoxitin, and ciprofloxacin were 82.5%, 78.9%, 73.7%, 63.2%, 33.3%, and 15.8% respectively. Antibiotic resistance genes were detected using primer-specific PCR and it was found that the prevalence rates of tetM, aac(6′)–aph(2″), mecA, tetK, ermc, and blaZ genes were 66.7%, 40.4%, 40.4%, 38.6%, 24.6%, and 3.51% respectively. Statistical analysis revealed significant (p < 0.05) relationships between the tetM, aac(6′)–aph(2″), and ermC genes and all parameters tested. A significant correlation between the aac(6′)–aph(2″) gene and the tetM (0.506) and ermC (−0.386) genes was identified. It was found that 23 (40.3%) S. aureus isolates were mecA positive, of which 10 (52.6%) out of 19 cefoxitin-resistant isolates were mecA positive and 13 (35.1%) out of 37 cefoxitin-sensitive isolates were mecA positive. The results of the present study demonstrated the detection of methicillin and multidrug resistant S. aureus isolated from samples obtained from wild pigeons and houseflies in the surroundings of a local public hospital in the Greater Durban area in South Africa. The findings of the study may account for the emergence of multidrug-resistant staphylococcal infections. The findings highlight the significant role of wild pigeons and houseflies in the spread of drug-resistant pathogenic S. aureus including MRSA. The conclusions of the present study highlight the improtant role of wildlife and the environment as interconnected contributors of One Health.

Impact of Accelerated Antimicrobial Testing on Clinical Outcomes in Critically Ill Septicaemia Patients: A Prospective Observational Study form a Tertiary Care Centre in Andhra Pradesh, India

Introduction: Bloodstream infection is a major cause of mortality and morbidity worldwide. Timely reporting of blood culture results is of utmost importance for better patient outcomes. The recently introduced Rapid Antimicrobial Susceptibility Testing (RAST) method is poised to profoundly influence clinical outcomes. Aim: To compare the results of RAST with Standard Antimicrobial Susceptibility Testing (SAST) and evaluate the impact of RAST reporting on the clinical management of septicaemia patients. Materials and Methods: This prospective, observational study was conducted in the Department of Microbiology, Government Medical College, Kurnool, Andhra Pradesh, India from May 2021 to September 2022. All positive blood culture bottles with only a single morphotype in gram staining were further processed using the RAST method, followed by conventional identification and SAST. Categorical agreement and disagreement between the RAST and SAST results were compared, along with the difference in the time at which results were available. Results: Out of a total of 1,146 blood cultures received, 228 were flagged as positive. A total of 514 isolate and antimicrobial agent combinations were evaluated, of which 496 (96.5%) showed categorical agreement. Only 18 (3.5%) showed categorical disagreement, with the majority being Major Errors (ME) (1.56%), followed by Very Major Errors (VME) (0.97%) and minor Errors (mE) (0.97%). Conclusion: RAST results demonstrated strong concurrence with SAST results. RAST is affordable, fast, and flexible and can potentially lead to a considerably shortened time for AST results to reach the bedside of the patient. This enables rapid modifications and adjustments in antibiotic therapy, including both escalation and de-escalation.

Rapid identification of bacterial isolates using microfluidic adaptive channels and multiplexed fluorescence microscopy.

We demonstrate the rapid capture, enrichment, and identification of bacterial pathogens using Adaptive Channel Bacterial Capture (ACBC) devices. Using controlled tuning of device backpressure in polydimethylsiloxane (PDMS) devices, we enable the controlled formation of capture regions capable of trapping bacteria from low cell density samples with near 100% capture efficiency. The technical demands to prepare such devices are much lower compared to conventional methods for bacterial trapping and can be achieved with simple benchtop fabrication methods. We demonstrate the capture and identification of seven species of bacteria with bacterial concentrations lower than 1000 cells per mL, including common Gram-negative and Gram-positive pathogens such as Escherichia coli and Staphylococcus aureus. We further demonstrate that species identification of the trapped bacteria can be undertaken in the order of one-hour using multiplexed 16S rRNA-FISH with identification accuracies of 70-98% with unsupervised classification methods across 7 species of bacteria. Finally, by using the bacterial capture capabilities of the ACBC chip with an ultra-rapid antimicrobial susceptibility testing method employing fluorescence imaging and convolutional neural network (CNN) classification, we demonstrate that we can use the ACBC chip as an imaging flow cytometer that can predict the antibiotic susceptibility of E. coli cells after identification.

Vibrio species: development of EUCAST susceptibility testing methods and MIC and zone diameter distributions on which to determine clinical breakpoints.

Most human infections caused by Vibrio spp. do not warrant antimicrobial treatment but in severe cases, targeted antimicrobial treatment can be lifesaving. For Vibrio spp., standardized antimicrobial susceptibility testing (AST) guidelines with EUCAST methodology are lacking. In this study, we aimed to produce data suitable for EUCAST to establish clinical MIC breakpoints and zone diameter correlates for Vibrio spp. An intercontinental collection (N = 524) comprising five important Vibrio spp. (V. alginolyticus, V. cholerae, V. fluvialis, V. parahaemolyticus and V. vulnificus) was organized. All isolates were subjected to broth microdilution (BMD) against 11 antimicrobial agents according to ISO 20776-1 using unsupplemented Mueller-Hinton broth on freeze-dried Sensititre panels (Thermo Scientific, UK), and most isolates (n = 371) were also tested with disc diffusion according to EUCAST methodology for non-fastidious organisms. Aggregated results were used to generate MIC and zone diameter distributions and to prepare graphs of MIC-zone diameter correlation. Based on these results, the EUCAST Steering Committee determined clinical susceptible (S) and resistant (R) MIC (mg/L) breakpoints (S≤/R>) for the five Vibrio spp. for piperacillin/tazobactam (1/1), cefotaxime (0.25/0.25), ceftazidime (1/1), meropenem (0.5/0.5), ciprofloxacin (0.25/0.25), levofloxacin (0.25/0.25), azithromycin (4/4), doxycycline (0.5/0.5) and trimethoprim/sulfamethoxazole (0.25/0.25). The corresponding zone diameter breakpoints were identified. We demonstrated the validity of using standard BMD and EUCAST disc diffusion methodology for AST of five Vibrio spp., and generated suitable data to allow EUCAST to determine clinical MIC and zone diameter breakpoints for five pathogenic Vibrio spp., including both non-toxigenic and toxigenic V. cholerae.

Prevalence of piperacillin/tazobactam resistance in invasive Haemophilus influenzae in Germany.

Haemophilus influenzae (Hi) is a Gram-negative bacterium that may cause sepsis or meningitis, treatment of which mainly includes β-lactam antibiotics. Since 2019 EUCAST breakpoints for piperacillin/tazobactam have been available. Little is known about the prevalence and mechanisms of piperacillin/tazobactam resistance in Hi. To provide reliable prevalence data for piperacillin/tazobactam resistance in Hi in Germany, to evaluate different antibiotic susceptibility testing methods and to examine possible resistance mechanisms. According to EUCAST breakpoints, the MIC for piperacillin/tazobactam resistance is >0.25 mg/L. All invasive Hi in Germany from 2019 were examined by gradient agar diffusion (GAD) for piperacillin/tazobactam susceptibility. Piperacillin/tazobactam broth microdilution (BMD), piperacillin GAD on tazobactam-containing agar [piperacillin GAD on Mueller-Hinton agar with horse blood (MH-F)/tazobactam) and piperacillin/tazobactam agar dilution (AD) were used for confirmation. Phenotypic testing was complemented by ftsI sequencing. Piperacillin/tazobactam GAD resulted in 2.9% (21/726) resistant Hi. BMD did not confirm piperacillin/tazobactam resistance. Two strains were found resistant by AD, of which one was also resistant using piperacillin GAD on MH-F/tazobactam. Overall, we found two strains with a piperacillin/tazobactam MIC >0.25 mg/L in at least two different tests (0.3%). Both were β-lactamase-producing amoxicillin/clavulanate-resistant with PBP3 mutations characterized as group III-like+. Relevant PBP3 mutations occurred in six strains without phenotypic piperacillin/tazobactam resistance. These mutations suggest a reduced efficacy of β-lactam antibiotics in these isolates. Piperacillin/tazobactam resistance prevalence in invasive Hi is low in Germany. Reduced susceptibility was correlated with PBP3 mutations, in particular with group III mutations.

Identification and antifungal susceptibility testing of Candida haemulonii complex isolated from clinical samples.

Candida haemulonii complex (Candida haemulonii [I], Candida duobushaemulonii [II], and Candida haemulonii var. vulnera [III]) has become relevant in recent times, not so much because of a high incidence in human clinical sample cultures but because of its remarkable antifungal resistance. The objective of this study was to evaluate several methods for the identification of this uncommon species of Candida. Ten isolates of C. haemulonii were identified by biochemical and proteomic methods, and their antifungal susceptibility testing was performed by both commercial and reference methods. MALDI-TOF MS (Vitek MS and Vitek MS PRIME) and Vitek2 correctly identified these genera but API method did not. There was a good correlation between the commercial methods and the reference methods for the AST. In conclusion Vitek MS, Vitek MS PRIME, and Vitek2 systems, but not API32C, are reliable for identification of C. haemulonii complex. Furthermore, MALDI-TOF MS systems could identify to the subspecies level. Commercial methods for antifungal susceptibility testing are valid for the study of this species and confirm amphotericin B and to azole resistance.

Comparative Evaluation of Broth Microdilution With Disc Diffusion and VITEK 2 for Susceptibility Testing of Colistin on Multidrug-Resistant Gram-Negative Bacteria.

Background The rise of antibiotic resistance, particularly in Gram-negative bacteria, poses a significant global health threat. Colistin, a last-resort antibiotic, has witnessed renewed use. However, accurate susceptibility testing for colistin is challenging, with various methods available, leading to potential discrepancies. Ensuring reliable testing is crucial for effective patient treatment and antimicrobial stewardship. This study addresses the need to compare different colistin susceptibility testing methods, providing insights into their accuracy and relevance in clinical settings. Methods In this one-year prospective observational cross-sectional study conducted atIndira Gandhi Institute of Medical Sciences (IGIMS), Bihar, India, atertiary care hospital from July 2021 to June 2022, we aimed to evaluate the concordance between two widely used methods, VITEK 2 and Disc Diffusion, for antibiotic susceptibility testing in clinical multidrug-resistant Gram-negative bacterial isolates. These isolates, including species like Klebsiella pneumoniae, Acinetobacter baumannii, Klebsiella oxytoca, Pseudomonas aeruginosa, Citrobacter freundii, and Escherichia coli, were isolated from various clinical specimens. After rigorous species-level identification and quality control measures, antibiotic susceptibility testing was performed using both methods, and their agreement was assessed through Percentage Agreement analysis. Results In our study, we isolated and identified bacterial isolates from 105 patients, with a mean age of 47.30 years, demonstrating a wide age range. Pus samples were the most common type (25.7%), and K. pneumoniae was the most prevalent organism (45.7%). Antibiotic resistance patterns revealed significant challenges in treating infections caused by K. pneumoniae and A. baumannii, with resistance rates exceeding 70% for certain antibiotics. Among the 48 isolates of K. pneumoniae, the agreement was 93.8%, with 89.6% being sensitive and 6.3% being resistant by Disc Diffusion, while VITEK 2 indicated 0% resistance. E. coli isolates (n=21) had an agreement of 90.5%, with 90.5% sensitivity and 9.5% resistance by Disc Diffusion, and no resistance by VITEK 2. Conclusion The comparative analysis of antibiotic susceptibility testing methods reveals the superior performance of the VITEK 2 system, particularly in sensitivity and negative predictive value, emphasizing its potential as a reliable tool for guiding antibiotic therapy decisions.

Prevalence of antibiotic resistance in Clostridium difficile: A meta-analysis

In recent years, the resistance of Clostridium difficile to various common mainstream antibiotics cannot be ignored, the incidence of CDI in many areas is also increasing year by year, posing a great threat to public safety. Literature collection was carried out on 6 open databases, and high-quality literature data conforming to classification criteria were screened and extracted. The WPR and WPF of each antibacterial drug were calculated using the random effects model. Subgroup analysis was used to investigate the influence of regional and experimental methods. Results: A total of 40 studies were included. The WPR of vancomycin was 0.0372, 95%CI: [0.0148; 0.0671], I: 93.7%, WPF was 0.2195, I: 94.0%. The WPR of fidaxomicin was 0.0051, 95%CI: [0.0000; 0.0205], I: 82.7%, WPF was 0.1757, I: 93.6%. The WPR of metronidazole was 0.0318, 95%CI: [0.0056; 0.0727], I: 91.6%. Among the three antibiotics, the highest resistance of C. difficile to metronidazole were mainly distributed in the Middle East and Africa. The conclusion seems to indicate that C. difficile is most sensitive to Fidaxomicin, and the clinical cure rate is better than vancomycin. However, the clinical cure failure rate increased significantly from 2007 to 2019, while the drug resistance rate did not change significantly, suggesting that other factors besides drug resistance may affect the therapeutic effect of fidaxomicin on CDI. There was no significant difference in the results of AGAR dilution and e test, the main methods for drug susceptibility testing.

Development of an agar-based screening method for terbinafine, itraconazole, and amorolfine susceptibility testing of Trichophyton spp.

Resistance in dermatophytes is an emerging global public health issue. We, therefore, developed an agar-based method for screening Trichophyton spp. susceptibility to terbinafine (TRB), itraconazole (ITC), and amorolfine (AMF) and validated it using molecularly characterized isolates. Α total of 40 Trichophyton spp. isolates, 28 TRB wild type (WT) (13 T. rubrum, 10 T. mentagrophytes, 5 T. interdigitale) and 12 TRB non-WT (7 T. rubrum, 5 T. indotineae) with different alterations in the squalene epoxidase (SQLE) gene, were used. The optimal test conditions (inoculum and drug concentrations, incubation time, and temperature) and stability over time were evaluated. The method was then applied for 86 WT Trichophyton spp. clinical isolates (68 T. rubrum, 7 T. interdigitale, 6 T. tonsurans, 5 T. mentagrophytes) and 4 non-WT T. indotineae. Optimal growth of drug-free controls was observed using an inoculum of 20 µL 0.5 McFarland after 5-7 days of incubation at 30°C. The optimal concentrations that prevented the growth of WT isolates were 0.016 mg/L of TRB, 1 mg/L of ITC, and 0.25 mg/L of AMF, whereas 0.125 mg/L of TRB was used for the detection of Trichophyton strong SQLE mutants (MIC ≥0.25 mg/L). The agar plates were stable up to 4 months. Inter-observer and inter-experimental agreement were 100%, and the method successfully detected TRB non-WT Trichophyton spp. strains showing 100% agreement with the reference EUCAST methodology. An agar-based method was developed for screening Trichophyton spp. in order to detect TRB non-WT weak and strong mutant isolates facilitating their detection in non-expert routine diagnostic laboratories.

High-Throughput Monitoring of Pathogenic Fungal Growth Using Whole Slide Imaging for Rapid Antifungal Susceptibility Assessment

Invasive fungal infections are a major health threat with high morbidity and mortality, highlighting the urgent need for rapid diagnostic tools to detect antifungal resistance. Traditional culture-based antifungal susceptibility testing (AFST) methods often fall short due to their lengthy process. In our previous research, we developed a whole-slide imaging (WSI) technique for the high-throughput assessment of bacterial antibiotic resistance. Building on this foundation, this study expands the application of WSI by adapting it for rapid AFST through high-throughput monitoring of the growth of hundreds of individual fungi. Due to the distinct “budding” growth patterns of fungi, we developed a unique approach that utilizes specific cell number change to determine fungi replication, instead of cell area change used for bacteria in our previous study, to accurately determine the growth rates of individual fungal cells. This method not only accelerates the determination of antifungal resistance by directly observing individual fungal cell growth, but also yields accurate results. Employing Candida albicans as a representative model organism, reliable minimum inhibitory concentration (MIC) of fluconazole inhibiting 100% cells of Candida albicans (denoted as MIC100) was obtained within 3h using the developed method, while the modified broth dilution method required 72h for the similar reliable result. In addition, our approach was effectively utilized to test blood culture samples directly, eliminating the need to separate the fungi from whole blood samples spiked with Candida albicans. These features indicate the developed method holds great potential serving as a general tool in rapid antifungal susceptibility testing and MIC determination.

Intervention with impact: Reduced isolation of methicillin-resistant Staphylococcus pseudintermedius from dogs following the introduction of antimicrobial prescribing legislation in Germany.

Legislation was introduced in Germany in 2018, requiring bacterial culture and antimicrobial susceptibility testing before the prescription of fluoroquinolones and third-generation cephalosporins to dogs. We hypothesised that, following this intervention, the number of clinical samples testing positive for methicillin-resistant Staphylococcus pseudintermedius (MRSP) would reduce. Reports of S. pseudintermedius isolated from canine clinical samples by three German veterinary diagnostic microbiology laboratories during the 38 months before the introduction of the legislation and the 46 months after were compared. Bacterial identification was performed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry, and antimicrobial susceptibility testing followed recognised recommendationsbut with changes during the study period. Among a total of 120,571 S. pseudintermedius isolates, MRSP accounted for 7.1% overall. Following the legislative intervention, monthly submissions yielding S. pseudintermedius increased at all three laboratories. The MRSP percentage was lower in the period after the intervention in two of the three laboratories (p < 0.001); in the third laboratory, there was no change between periods, but a year-on-year reduction in MRSP percentages occurred after the intervention (p = 0.0004). Changing susceptibility testing methods limited the direct comparison of resistance patterns among laboratories. The reduction in MRSP in canine clinical samples following the introduction of this legislation suggests a positive impact of compulsory laboratory testing on reducing antimicrobial resistance.