Investigating the Unfavored Factors That Interfere MALDI-TOF Based AI in Predicting Antibiotic Resistance

Combining Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) spectra data and artificial intelligence (AI) has been introduced for rapid prediction on antibiotic susceptibility test (AST) of S. aureus. Based on the AI predictive probability, the cases with probabilities between low and high cut-offs are defined as “grey zone”. We aimed to investigate the underlying reasons of unconfident (grey zone) or wrong predictive AST. A total 479 S. aureus isolates were collected, analyzed by MALDI-TOF, and AST prediction, standard AST were obtained in a tertiary medical center. The predictions were categorized into the correct prediction group, wrong prediction group, and grey zone group. We analyzed the association between the predictive results and the demographic data, spectral data, and strain types. For MRSA, larger cefoxitin zone size was found in the wrong prediction group. MLST of the MRSA isolates in the grey zone group revealed that uncommon strain types composed 80%. Amid MSSA isolates in the grey zone group, the majority (60%) was composed of over 10 different strain types. In predicting AST based on MALDI-TOF AI, uncommon strains and high diversity would contribute to suboptimal predictive performance.

Bacteriological Identification among Patients Attending at Jimma University Veterinary Clinic

Findings of conducted study showed that the predominant bacteria in Jimma University Veterinary Clinic. Among predominant bacteria identified includes; Escherichia coli 46 (34.3%), Klebsiella 23(17.2%), followed by Staphylococcus aureus 40(29.9%) and Streptococcus spp 25(18.7%). Among 4 species of bacteria isolated, 2 were gram negative and 2 of them was gram positive bacteria. Among the antibiotic susceptibility test, Staphylococcus aureus showed the total resistant on penicillin while tetracycline showed to have the highest sensitivity. Escherichia coli showed the total resistant on gentamicin (100%). Veterinary Clinic is at risk of acquiring bacterial infections due to the presence of mentioned bacteria during this study. The big issue is that these infections resist to antibiotics used in treatment according to results of the study. Health care workers in Veterinary Clinic should improve hygiene to control clinic acquired infections and much attention should be done during diagnosis to improve treatment of patients.

A New Colorimetric Method for Rapid Detection of Antibiotic Resistance in Escherichia coli Isolates

Background: The quick diagnosis and early initiation of antibiotic therapy in bacteria-induced infections is of paramount importance. Accordingly, the rapid identification of the causative agent, the short-term results of antibiotic sensitivity, the selection and use of right antibiotics for treatment further highlights the significance of this issue. Objectives: This study aimed to develop a new susceptibility testing method to provide rapid results in Escherichia coli clinical isolates and report the antibiotic susceptibility test results to clinicians in a short period. Methods: In the study, one hundred and ten E. coli clinical isolates were tested. In this regard, antibiotics recommended by the "Clinical and Laboratory Standards Institute (CLSI)" for testing the sensitivity of E. coli isolates, including amoxicillin-clavulanate, cefixime, ceftriaxone, ertapenem, ciprofloxacin, gentamicin, trimethoprim-sulfamethoxazole, and nitrofurantoin were tested. For quality control, E. coli ATCC25922, E. coli ATCC35218, Staphylococcus aureus ATCC29213, and E. coli 13846NTCC strains were used. The broth microdilution method recommended by CLSI was used as the reference method. Minimum inhibitory concentration values were determined, and antimicrobial susceptibilities were then determined according to the “European Committee on Antimicrobial Susceptibility Testing (EUCAST)” criteria. In the next phase, the results of the resazurin microplate method (RMM) were compared. Results: The comparison of the RMM developed in the present study with the reference method revealed that the calculated essential agreement ratios for eight antibiotics varied from 82.72 to 100%, and the categorical agreement values ranged from 95.45 to100%. Conclusions: According to the findings, the RMM results were highly in agreement with the results of the reference method. RMM allows the detection of antibiotic susceptibility quickly (e.g., within 5 hours) as such it is preferred, especially for laboratories with limited facilities. However, further multi-center studies are recommended to use this method in routine laboratories.

Detection of Extended Spectrum β-Lactamases (ESBL) Producing Bacteria in Sepsis Suspected Neonates

Introduction: Neonatal sepsis is a clinical syndrome that is caused when the bloodstream of an infant is invaded by bacteria in the first month after birth. Objective: The objective of the study was to identify bacteria involved in the infection and to determine “extended-spectrum beta-lactamase” (ESBL) producing bacteria from blood samples of sepsis suspected neonates in the Neonatal Intensive Care Unit and Special Care Baby Unit. Methods: This cross-sectional study was conducted from January to July 2019 at Microbiology laboratory of Paropakar Maternity and Women’s Hospital. A total of 380 venous blood specimens were included in the study. The blood culture was performed and organisms were identified with standard microbiological methods. The Antibiotic susceptibility test was performed using the modified Kirby Bauer disk diffusion method. Screening of the organisms was done using cefotaxime and ceftazidime antibiotic disc and confirmation of ESBL was done by combined disk test. The data were considered statistically significant if the p-value was < 0.05. Results: Out of a total of 380 blood specimens, the prevalence of neonatal sepsis was found to be 21.05% among which 57.5% were EOS type and 42.5% were LOS type. In EOS, E. coli (72.73%) was the predominant isolate while CoNS (100%) was the predominant isolate in LOS. Of the total 80 isolates, 65% isolates were found multidrug-resistant (MDR) whereas 58.75% of isolates were found to be ESBL producers. Conclusions: This study concludes that routine bacterial surveillance and study of their resistance patterns is an essential component of the neonatal care unit. Keywords: Extended-spectrum β-Lactamases; neonates; neonate intensive care unit; special care baby unit; sepsis.

Toxin gene profiles and antimicrobial resistance of Clostridioides difficile infection: a single tertiary care center study in Iran

Background and Objectives: Due to the reduced susceptibility of clinical Clostridioides difficile strains in hospitals to var- ious antimicrobial agents, the importance of antimicrobial susceptibility testing (ASTs) has increased. This study aimed to investigate the toxin gene profiles and the antimicrobial resistance of C. difficile isolated from hospitalized patients suspected of having Clostridioides difficile infection (CDI) in Tehran, Iran. Materials and Methods: The stool samples were obtained from a hospitalized patients. The samples were shocked by al- cohol and the patients cultured on cycloserine-cefoxitin-fructose agar in anaerobic Conditions. Toxin assay was performed for detection of toxinogenic isolates. An antibiotic susceptibility test was done. Furthermore, their genome was extracted for PCR to confirm C. difficile and detect toxin gene profile. Results: Toxigenic C. difficile were identified in 21 of the 185 stool samples (11.3%). PCR detected seven toxin gene profiles; the highest prevalence was related to tcdA+B+, cdtA+B- toxin gene profile (57.1%). There were 14.3% and 28.6% resistant rates of the isolates towards vancomycin and metronidazole with the toxin gene profiles; tcdA+B+, cdtA±B+; and tc- dA+B-, cdtA-B+. All resistant isolates to moxifloxacin, clindamycin, and tetracycline were belonged to the toxin gene profiles; tcdA+B+, cdtA+B+; tcdA+B+, cdtA+B-, and tcdA-B+, cdtA+B-. Conclusion: Relative high resistance was detected towards metronidazole and vancomycin, although, still have acceptable activity for CDI treatment. However, a proper plan for the use of antibiotics and more regular screening of C. difficile anti- biotic resistance seems necessary.

Detection of invA virulence gene of multidrug-resistant Salmonella species isolated from the cloacal swab of broiler chickens in Blitar district, East Java, Indonesia

Background and Aim: The increasing number of multidrug-resistant (MDR) Salmonella species on poultry farms in Indonesia has caused concern regarding human health. This study was conducted to determine the presence of the virulence gene invA in MDR Salmonella species isolated from the cloacal swab of broiler chickens in Blitar district, East Java Province, Indonesia. Materials and Methods: Cloacal swab samples were collected by purposive sampling from 15 farms in four districts. Isolation and identification of bacteria were performed using standard microbiological techniques. Confirmation of MDR isolates was done using five different classes of antibiotics, including the beta-lactam, aminoglycoside, fluoroquinolone, phenicol, and monobactam groups. An antibiotic susceptibility test was conducted using the Kirby–Bauer disk diffusion method, and a polymerase chain reaction method was used to screen for the presence of invA. Results: It was observed that 32.26% (50/155) of the samples were positive for Salmonella species. Of these 50 Salmonella isolates, 7 (14%) were identified as MDR strains. An important finding was the detection of invA in all the seven MDR Salmonella strains (100%) isolated from the cloacal swab of broiler chickens in Blitar district, East Java Province. Conclusion: Veterinarians have an extremely important role in monitoring the use of antibiotics in farm animals to mitigate the rapid spread of MDR organisms in our environment, which can otherwise cause serious economic losses and also public health issues.

Comparison of Colistin MIC by Microbroth Dilution, E-Test and Vitek-2, in Isolates of Acinetobacter spp. Isolated from Bloodstream Infections

Background: Acinetobacter species are leading cause of nosocomial infections, causing significant morbidity and mortality globally including India. Being persistent in the hospital environment and rapidly developing resistance to a wide variety of antibiotics are the most important features of this pathogen. The present study aimed to compare Colistin MIC of Acinetobacter species isolated from the blood samples by E test and Vitek 2 to the standard broth micro dilution test. Methodology: Two antibiotic susceptibility test methods, The Vitek-2 and the E test, against the reference broth micro dilution method in terms of the various parameters such as Reproducibility, reliability, cost and time effectiveness. Data obtained from the current study regarding antimicrobial resistance of Acinetobacter species recovered from clinical specimens referred to microbiology laboratory of SKIMS and was analyzed by using SPSS20.0. Results: Out of 100 isolates of Acinetobacter species analyzed from blood specimens the distribution of Acinetobacter species according to different clinical diagnosis of patients 89% were A. baumannii and 11% were A. lwoffii. Seventy three percent of them were from males and 27% of them were from females with a mean age of 39.6 (SD±27.46). Regarding the specimen and isolate sources, the majority were from ICU (54%), Surgical ward (26%), Medical ward (16%) and 4% from Outpatient department of SKIMS. Significant descending trends of antimicrobial resistance was shown for Amoxicillin/Clavulanic acid, Cefoperazone/ Sulbactam combination, Cotrimoxazole (100%), Levofloxacin (92%) Piperacillin/Tazobactam, Ciprofloxacin (90%), Cephalosporins (>80%), Imipenem and Meropenem (76%), Amikacin (68%), Gentamycin (67%), Tigecycline (11%) and 0% for Colistin respectively. Conclusion: from the study it could be concluded that the best reference method for testing susceptibility to the Polymyxins still remains to be defined. However, in routine clinical practice in most regions worldwide, where a reference method can hardly be implemented, the interpretation of Colistin susceptibility should preferably be based on results of automated systems such as Vitek-2 or the E test. The micro broth Dilution method remains the most reliable and reproducible, however most tedious and time-consuming method. Colistin remains a very effective, least resisted drug for MDR Acinetobacter species as compared by all the three methods. Keywords: Acinetobacter species; Antimicrobial resistance; Colistin; E test and Vitek 2.

Probiotic Bifidobacterium Isolated from Chicken Intestine with PVP Showed Synergistic Effects on Reduction of AFB1

Food contamination with aflatoxin is one of the most important concerns of health professionals. One of the best ways to reduce aflatoxin content in food is to use probiotics. Therefore, this study was performed to isolate Bifidobacterium from the chick's intestine and its probiotic activities and also its application with Polyvinylpyrrolidone (PVP) to reduce aflatoxin B1 (AFB1) toxin in the medium were investigated. Samples were isolated from the chicken intestine. After preparing the samples, Bifidobacterium was isolated and identified using biochemical and molecular methods. To measure probiotic activities, pH, bile, and salt tolerance tests were used. Then, the antimicrobial activity of isolate against gastrointestinal pathogens and the antibiotic susceptibility test were done. Then, the effect of selected isolate and PVP on reducing AFB1 in the medium was studied using ELISA and HPLC. Biochemical and molecular evaluations indicated isolation of B. bifidum strain from chicken intestine. The selected strain showed antimicrobial activities on S. enterica, E. coli, and P. vulgaricus and was found to be resistant against Amikacin, Ampicillin, Erythromycin, and Ceftazidine antibiotics. The selected strain showed the ability to reduce the concentration of AFB1 in the medium (50% reduction) and when used in combination with PVP showed the synergistic effects in reducing the concentration of AFB1 from the medium (up to 90%). In conclusion, it was found that selected B. bifidum strain together with PVP could have synergistic effects in reducing AFB1 toxin in medium up to 90%.

Whole-Genome Analysis of Multidrug-Resistant Salmonella Enteritidis Strains Isolated from Poultry Sources in Korea

The Salmonella Enterica subsp. Enterica serovar Enteritidis is one of main serovars isolated from human patients with food poisoning and poultry without clinical signs. Consumption of poultry products contaminated with Salmonella Enteritidis is a common source of human salmonellosis; 82 Salmonella spp. were isolated from 291 samples of retail chicken meat, 201 one-day-old chicks, 30 internal organs of chickens, 156 chicken eggs, 100 duck eggs, 38 straw bedding samples, 18 samples of retail duck meat, and 19 swab samples from slaughterhouses in 2019 and 2020. An antibiotic susceptibility test was performed for all isolates, revealing 33 multidrug-resistant (MDR) strains. The whole genome of 33 MDR strains isolated in 2019 and 2020 and 10 strains isolated in 2011, 2012, and 2017 was sequenced using the MinION sequencing protocol. Within these 43 samples, 5 serovars were identified: S. Enteritidis, S. Agona, S. Virchow, S. Albany, and S. Bareilly. The most common serovar was S. Enteritidis (26/43), which showed the highest resistance to ampicillin (100%), followed by nalidixic acid (90%) and colistin (83%). Core genome multilocus sequence typing analysis showed that the S. Enteritidis strains isolated from different sources and in different years were clustered together. In addition, the S. Enteritidis strains isolated since 2011 consistently harbored the same antibiotic resistance patterns.