Discovering Novel Antimicrobial Peptides in Generative Adversarial Network

Due to the growing number of clinical antibiotic resistance cases in recent years, novel antimicrobial peptides (AMPs) can become ideal for next-generation antibiotics. This study trained a deep convolutional generative adversarial network (GAN) with known AMPs to generate novel AMP candidates. The quality of the GAN-designed peptides was evaluated in silico, and eight of them named GAN-pep 1~8 were chosen to be synthesized for further experiments. Disk diffusion testing and minimum inhibitory concentration (MIC) determination were used to determine the antibacterial effects of the synthesized GAN-designed peptides. Seven out of the eight synthesized GAN-designed peptides showed antibacterial activities. Additionally, GAN-pep 3 and GAN-pep 8 had a broad spectrum of antibacterial effects. Both of them were also effective against antibiotic-resistant bacteria strains such as methicillin-resistant Staphylococcus aureus (S. aureus) and carbapenem-resistant Pseudomonas aeruginosa (P. aeruginosa). GAN-pep 3, the most promising GAN-designed peptide candidate, had low MICs against all the tested bacteria.

Surveillance of Antimicrobial Susceptibility of Anaerobe Clinical Isolates in Southeast Austria: Bacteroides fragilis Group Is on the Fast Track to Resistance

Anaerobic bacteria play an important role in human infections. Bacteroides spp. are some of the 15 most common pathogens causing nosocomial infections. We present antimicrobial susceptibility testing (AST) results of 114 Gram-positive anaerobic isolates and 110 Bacteroides-fragilis-group-isolates (BFGI). Resistance profiles were determined by MIC gradient testing. Furthermore, we performed disk diffusion testing of BFGI and compared the results of the two methods. Within Gram-positive anaerobes, the highest resistance rates were found for clindamycin and moxifloxacin (21.9% and 16.7%, respectively), and resistance for beta-lactams and metronidazole was low (<1%). For BFGI, the highest resistance rates were also detected for clindamycin and moxifloxacin (50.9% and 36.4%, respectively). Resistance rates for piperacillin/tazobactam and amoxicillin/clavulanic acid were 10% and 7.3%, respectively. Two B. fragilis isolates were classified as multi-drug-resistant (MDR), with resistance against all tested beta-lactam antibiotics. The comparative study of 109 BFGI resulted in 130 discrepancies in 763 readings (17%) with a high number of Very Major Errors (VME) and Major Errors (ME). In summary, resistance rates, with the exception of clindamycin and moxifloxacin, are still low, but we are facing increasing resistance rates for BFGI. Surveillance studies on a regular basis are still recommended.

Predictive value of direct disk diffusion testing from positive blood cultures in a children’s hospital and its utility in antimicrobial stewardship

Background Accurate and early susceptibility results could reduce overuse of broad-spectrum antibiotics for empiric treatment of bacteremia. Direct disk diffusion testing (dDD) using non-standardized inocula directly from blood cultures could facilitate earlier narrowing of antibiotics. Methods To determine the predictive value of dDD compared with standardized antimicrobial susceptibility testing (AST), we performed a retrospective cohort study of 582 blood cultures from 495 pediatric patients with bacteremia. Positive and negative predictive value (PPV: number of isolates susceptible by both dDD and AST divided by the total number of isolates susceptible by dDD; NPV: number of isolates not susceptible [either intermediate or resistant] by both dDD and AST divided by the total number of isolates not susceptible by dDD), sensitivity, specificity, and 95% confidence interval were calculated for each bacterium-antibiotic combination. We evaluated the Antibiotic Spectrum Index of prescribed antibiotics to assess change in antibiotic prescribing after availability of Gram stain, dDD, and AST results. Results dDD results were available a median of 21 hours before AST results. dDD had PPVs of ≥96% for most organism-antibiotic pairs, including 100% (CI 96-100%) for Staphylococcus aureus and oxacillin and 99% (CI 93%-100%) for Enterobacterales and ceftriaxone. NPVs of dDD were variable and frequently lower than PPV. Very major errors and major errors occurred in 31/5454 (0.6%) and 231/5454 (4.2%) organism-antibiotic combinations, respectively. Antibiotics were narrowed in 30% of cases after dDD result and a further 25% of cases after AST result. Conclusions dDD is highly predictive of susceptibility for many common organism-antibiotic combinations and provides actionable information one day earlier than standard susceptibility approaches. dDD has the potential to facilitate earlier de-escalation to narrow-spectrum antibiotic treatment.

Quinolone Susceptibility in Salmonella Isolates Based on Minimum Inhibitory Concentration Determination

Introduction Typhoid fever, caused by Salmonella typhi and paratyphi, is a generalized infection with case fatality of about 10%. The symptoms may be severe, with life threatening sequelae of infection in a proportion of cases. Antimicrobial agents are the mainstay of therapy in enteric fever so as to prevent the complications associated with severe illness and mortality in the patients. Fluoroquinolones (e.g., ciprofloxacin) are very effective against completely susceptible Salmonella bacteria. However, their efficacy is doubtful once any resistance is detected. Pefloxacin testing has ultimately helped in the accurate identification of quinolone susceptibility for a better therapeutic success rate. In the present study we have tried to evaluate the quinolone susceptibility in Salmonella isolates based on minimum inhibitory concentration (MIC) determination. Materials and Methods The method used in the study is quinolone susceptibility in Salmonella isolates based on MIC determination. Salmonella isolates show intermediate susceptibility to ciprofloxacin using disk diffusion. Both ciprofloxacin and pefloxacin MIC evaluation has been done to corroborate the results with pefloxacin disk diffusion testing. Results There was a positive correlation between the susceptibility to ciprofloxacin and pefloxacin. However, the isolates with intermediate susceptibility had variations in terms of susceptibility to pefloxacin. MIC values for pefloxacin and our findings suggested that pefloxacin susceptible on disk diffusion as per Clinical and Laboratory Standards Institute guidelines showed lower values for MIC using Pefloxacin HICOMB test and pefloxacin resistant isolates showed higher MIC values.

Implications of Oxacillin-Resistant, mecA-Negative Staphylococcus aureus Detected in NICU MRSA Surveillance Cultures

Background: Weekly surveillance to identify neonatal intensive care unit (NICU) infants with methicillin-resistant S. aureus (MRSA) nasal colonization was performed using Remel Spectra MRSA chromogenic media. An increased MRSA colonization rate from baseline was detected in 2019, prompting additional review of all positive MRSA NICU screening cultures from 2019. Methods: A subset of 23 positive cultures were interrogated in detail. Species-level identification was confirmed using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) with a Bruker Biotyper. Penicillin-binding protein 2a (PBP2a) testing was performed using the Alere culture colony test, and cefoxitin and oxacillin susceptibility were assessed via Kirby-Bauer disk-diffusion methods (for the purpose of this analysis, oxacillin zone sizes 18 mm were considered susceptible). Molecular detection of mecA and mecC genes using PCR was performed. Results: All 23 isolates in the subset group were confirmed as S. aureus based on MALDI-TOF testing. Moreover, 8 isolates (35%) were confirmed as MRSA based on cefoxitin susceptibility, positive rapid PBP2a testing, and mecA PCR results. Overall, 15 isolates (65%) tested cefoxitin-susceptible and PBP2a negative with negative mecA and mecC gene testing. Of these, 1 (7%) tested oxacillin-susceptible based on disk-diffusion testing, consistent with methicillin-susceptible S. aureus (MSSA). The remaining 14 isolates (93%) tested oxacillin resistant based on oxacillin zone size. Conclusions: Our findings indicate the detection of mecA/mecC negative S. aureus isolates demonstrating oxacillin resistance and growth on Remel Spectra MRSA chromogenic media. These results have important implications for infection prevention surveillance efforts to detect MRSA and raise questions regarding optimal antibiotic therapy in patients with isolates displaying this phenotype.Funding: NoneDisclosures: None

Impact of Amoxicillin treatment on E. coli in dairy cow faeces

Introduction: There has been a global drive for increased antimicrobial stewardship. In the UK, this drive has focused on decreased usage of antimicrobials, specifically HP-CIAs. It is well known that antimicrobials are selection drivers for antimicrobial resistance. How a given dose will impact the prevalence of resistance in a microbiome, and therefore the associated risk of a resistant infection is less understood. Questions also remain around the impact of antimicrobial therapy on resistance across a herd, if selection occurs within an animal receiving treatment, what is the potential risk of this resistance spreading throughout other animals? Methods: E. COLI ISOLATION Tryptone Bile X-Glucuronide (TBX) media was used for selective growth of non – toxigenic E. coli from dairy cow faeces. SUSCEPTIBILITY TESTING EUCAST Disk Diffusion testing guidelines were followed to determine susceptibility of faecal isolates to a panel of 8 antimicrobials from 5 different classes. ISOLATE FINGERPRINTING To determine clonality of faecal isolates ERIC-PCR was used as an efficient method to provide a genomic fingerprint. Results This work is ongoing. Current work suggests that low frequency Amoxicillin treatment has no significant selection for resistance. However, there appears to be some instances of co-selection for Streptomycin, Tetracycline and Sulphonamide resistance, and several multi drug resistance isolates have been identified. More work needs to be done to confirm the impact of low frequency Amoxicillin treatment on E. coli resistance and identify the mechanism behind suspected co-selection and multi drug resistance.

Carbapenem Resistance in Non-Fermenters: An Overview

This study was conducted with interest in increasing carbapenem resistance in non-fermenters: an important causative agent of nosocomial infection and to standardize the methods for interpretation of their resistance. The aim of this study is to perform disk diffusion testing and minimal inhibitory concentration technique for the identification of carbapenem resistance for imipenem and meropenem. The isolates found resistant to carbapenems were confirmed with the modified Hodge test. The genes responsible for carbapenem resistance were identified by both phenotypic and genotypic methods. Out of 240 non-fermenters isolated 20% showed resistance to carbapenem by disk diffusion. Only 7% showed resistance by the micro broth dilution technique of minimum inhibitory concentration. 3% were panning drug-resistant. Out of 16 carbapenem-resistant isolates, 5 were found to have KPC (Klebsiella pneumonia carbapenem) genes, 9 had MBL (Metallo beta-lactamase) genes and 2 had KPC+MBL genes and none were found to have Amp C and OXA-48 genes phenotypically. Genotypically all the KPC strains had KPC genes and out of 9 MBL strains, 6 had VIM and the remaining 3 strains were negative for both IMP and VIM gene. In conclusion, the interpretation of susceptibility for carbapenems should not be made only with disk diffusion testing. Always check for Minimal inhibitory concentration methods and determination of genes responsible for carbapenem resistance, a double-disc synergy test goes in hand with genotypic detection.

In vitro synergy testing of prodigiosin in combination with inhibitors of cell wall synthesis against Mycobacterium smegmatis

The cell wall is not a target of currently used therapeutics as Mycobacterium are considered naturally resistant to most β-lactam antibiotics. Therefore, combinations of conventional antibiotics with antibiotic activity-enhancing compounds offer a productive treatment strategy and address the widespread emergence of antibiotic-resistant strains. The first area of research was the study of a comparative analysis of disk diffusion testing and the broth dilution method for evaluating the susceptibility of M. smegmatis to antimicrobial agents. A comparative analysis of the susceptibility to antimicrobial agents alone showed that M. smegmatis was the most susceptible to ceftriaxone and kanamycin, and moderately sensitive to vancomycin and prodigiosin. Compared to the susceptibility of the antibacterial combinations, the isolate was not susceptible to antibacterial combinations with prodigiosin in disk diffusion testing. The second area of research was the study of the synergic activity of prodigiosin of S. marcescens and inhibitors of cell wall synthesis manifested by their simultaneous effect on M. smegmatis. The greatest increase in the sensitivity of test-culture of mycobacteria occurred with ampicillin, benzylpenicillin, cephazolin and ceftriaxone in combination with prodigiosin of S. marcescens. The presented combination of antibiotics and prodigiosin reduce the required concentration of the antibiotic and by amplifying the effect of compounds inhibiting cell wall synthesis, thereby giving lower FICI values. These data indicate the possibility of using prodigiosin as a promising candidate for the development of "accompaniment-preparations" for antibiotics for the additional therapy of infectious diseases caused by Mycobacterium spp. and can suspend the likelihood of developing resistance to antibiotics. The cell wall is not a target of currently used therapeutics as Mycobacterium are considered naturally resistant to most β-lactam antibiotics. Therefore, combinations of conventional antibiotics with antibiotic activity-enhancing compounds offer a productive treatment strategy and address the widespread emergence of antibiotic-resistant strains. The first area of research was the study of a comparative analysis of disk diffusion testing and the broth dilution method for evaluating the susceptibility of M. smegmatis to antimicrobial agents. A comparative analysis of the susceptibility to antimicrobial agents alone showed that M. smegmatis was the most susceptible to ceftriaxone and kanamycin, and moderately sensitive to vancomycin and prodigiosin. Compared to the susceptibility of the antibacterial combinations, the isolate was not susceptible to antibacterial combinations with prodigiosin in disk diffusion testing. The second area of research was the study of the synergic activity of prodigiosin of S. marcescens and inhibitors of cell wall synthesis manifested by their simultaneous effect on M. smegmatis. The greatest increase in the sensitivity of test-culture of mycobacteria occurred with ampicillin, benzylpenicillin, cephazolin and ceftriaxone in combination with prodigiosin of S. marcescens. The presented combination of antibiotics and prodigiosin reduce the required concentration of the antibiotic and by amplifying the effect of compounds inhibiting cell wall synthesis, thereby giving lower FICI values. These data indicate the possibility of using prodigiosin as a promising candidate for the development of "accompaniment-preparations" for antibiotics for the additional therapy of infectious diseases caused by Mycobacterium spp. and can suspend the likelihood of developing resistance to antibiotics.

Hybrid Assembly from a Pathogenic Methicillin- and Multidrug-Resistant Staphylococcus pseudintermedius Strain Isolated from a Case of Canine Otitis in Spain

Here we report the genome assembly, using a hybrid approach with Illumina and Nanopore sequencing, of a pathogenic Staphylococcus pseudintermedius strain isolated from a case of canine otitis. Genome assembly confirmed the antimicrobial resistance profile (disk diffusion testing) with specific genes and mutations.