Antibiotic Susceptibility Test Results Research Articles

Analytical Performance Validation of Next-Generation Sequencing Based Clinical Microbiology Assays Using a K-mer Analysis Workflow.

Next-generation sequencing (NGS) enables clinical microbiology assays such as molecular typing of bacterial isolates which is now routinely applied for infection control and epidemiology. Additionally, feasibility for NGS-based identification of antimicrobial resistance (AMR) markers as well as genetic prediction of antibiotic susceptibility testing results has been demonstrated. Various bioinformatics approaches enabling NGS-based clinical microbiology assays exist, but standardized, computationally efficient and scalable sample-to-results workflows including validated quality control parameters are still lacking. Bioinformatics analysis workflows based on k-mers have been shown to allow for fast and efficient analysis of large genomics data sets as obtained from microbial sequencing applications. We here demonstrate applicability of k-mer based clinical microbiology assays for whole-genome sequencing (WGS) including variant calling, taxonomic identification, bacterial typing as well as AMR marker detection. The wet-lab and dry-lab workflows were developed and validated in line with Clinical Laboratory Improvement Act (CLIA) guidelines for laboratory-developed tests (LDTs) on multi-drug resistant ESKAPE pathogens. The developed k-mer based workflow demonstrated ≥99.39% repeatability, ≥99.09% reproducibility and ≥99.76% accuracy for variant calling and applied assays as determined by intra-day and inter-day triplicate measurements. The limit of detection (LOD) across assays was found to be at 20× sequencing depth and 15× for AMR marker detection. Thorough benchmarking of the k-mer based workflow revealed analytical performance criteria are comparable to state-of-the-art alignment based workflows across clinical microbiology assays. Diagnostic sensitivity and specificity for multilocus sequence typing (MLST) and phylogenetic analysis were 100% for both approaches. For AMR marker detection, sensitivity and specificity were 95.29 and 99.78% for the k-mer based workflow as compared to 95.17 and 99.77% for the alignment-based approach. Summarizing, results illustrate that k-mer based analysis workflows enable a broad range of clinical microbiology assays, potentially not only for WGS-based typing and AMR gene detection but also genetic prediction of antibiotic susceptibility testing results.

Susceptibility Pattern of Neisseria gonorrhoeae towards Cefixime and Ceftriaxone using Kirby-Bauer Method in Dr. Saiful Anwar General Hospital Malang

Background: Gonorrhea remains the second most common sexually transmitted infection (STI) in the world with an increasing number of cases. Oral cefixime and IM ceftriaxone are still the mainstay therapy for gonorrhea in Indonesia. However, previous studies suggested possible resistance to ceftriaxone and cefixime, which are the first-line treatment of gonorrhea. To date, there are no data available regarding the susceptibility of these antibiotics for the treatment of gonorrhea in Dr. Saiful Anwar General Hospital (RSSA) Malang. Purpose: Determine susceptibility pattern of Neisseria gonorrhoeaetowards cefixime and ceftriaxone in RSSA Malang. Methods: The samples were patients of the Outpatient Clinic of Dermatology and Venereology Department with a symptom of discharge which contained Gram-negative diplococcus after Gram staining and had a positive culture of Neisseria gonorrhoeae. Susceptibility testing for cefixime and ceftriaxone antibiotics were performed using the Kirby-Bauer method. The data are presented in percentages. Result: Antibiotic susceptibility test results showed that 80.77% of Neisseria gonorrhoeaeisolates were still susceptible to cefixime, and 80.77% of isolates were still susceptible to ceftriaxone. Conclusion: Cefixime and ceftriaxone are still effective as gonorrhea therapy in RSSA Malang.

Heteroresistant Bacteria Detected by an Extended Raman-Based Antibiotic Susceptibility Test.

Worldwide, multiresistant bacterial strains are emerging at unprecedented rates. This development seriously threatens the ability of humanity to treat even common infections, resulting in disability and death. Furthermore, this development endangers all medical achievements including cancer therapy or organ transplantations. Therefore, the World Health Organization has endorsed antimicrobial resistance as a great threat to humanity. To still allow effective treatment of patients, rapid, automated, and reliable antibiotic susceptibility testing (AST) of bacterial pathogens is essential. Thereby, speed and sensitivity of the AST results are crucial for improving patient care. Here, Raman spectroscopy as a nondestructive technique providing chemical-specific information is employed to monitor the deuterium uptake of metabolically active bacteria during antibiotic treatment, enabling fast and reliable AST. For this purpose, a bulk sample-preparation method was developed, allowing a high-throughput analysis of a significant number of cells. A protocol was developed for Gram-positive (Enterococcus faecalis) and Gram-negative (Escherichia coli) reference strains and was tested on 51 clinical isolates with well-characterized resistance phenotypes against ampicillin, ciprofloxacin, meropenem, and vancomycin. Borderline resistant and heteroresistant phenotypes were observed and further investigated. This is of critical importance as the sensitive detection of low-frequency heteroresistance in bacterial populations is a huge challenge. Such isolates seem susceptible but are resistant to treatment in vivo. Automatable analysis detects strong phenotypes within 3 h. On the basis of experimental and modeled data, heteroresistance is estimated to be detectable down to frequencies of 10-6 and investigated on clinical isolates as a proof-of-concept study, but requiring longer incubation time.

Surfactant-enhanced DNA accessibility to nuclease accelerates phenotypic β-lactam antibiotic susceptibility testing of Neisseria gonorrhoeae.

Rapid antibiotic susceptibility testing (AST) for Neisseria gonorrhoeae (Ng) is critically needed to counter widespread antibiotic resistance. Detection of nucleic acids in genotypic AST can be rapid, but it has not been successful for β-lactams (the largest antibiotic class used to treat Ng). Rapid phenotypic AST for Ng is challenged by the pathogen’s slow doubling time and the lack of methods to quickly quantify the pathogen’s response to β-lactams. Here, we asked two questions: (1) Is it possible to use nucleic acid quantification to measure the β-lactam susceptibility phenotype of Ng very rapidly, using antibiotic-exposure times much shorter than the 1- to 2-h doubling time of Ng? (2) Would such short-term antibiotic exposures predict the antibiotic resistance profile of Ng measured by plate growth assays over multiple days? To answer these questions, we devised an innovative approach for performing a rapid phenotypic AST that measures DNA accessibility to exogenous nucleases after exposure to β-lactams (termed nuclease-accessibility AST [nuc-aAST]). We showed that DNA in antibiotic-susceptible cells has increased accessibility upon exposure to β-lactams and that a judiciously chosen surfactant permeabilized the outer membrane and enhanced this effect. We tested penicillin, cefixime, and ceftriaxone and found good agreement between the results of the nuc-aAST after 15–30 min of antibiotic exposure and the results of the gold-standard culture-based AST measured over days. These results provide a new pathway toward developing a critically needed phenotypic AST for Ng and additional global-health threats.

Clinical impact of rapid susceptibility testing on Mueller-Hinton Rapid-SIR directly from urine specimens

Urinary tract infection diagnosis and management generally involves a 48-h microbiological delay to obtain the antibiotic susceptibility test (AST) results. In the context of multidrug resistance, reducing the time to obtain AST results is an essential factor, allowing for more timely appropriate treatment. We conducted a single-centre prospective study on urinary samples meeting two criteria: significant leukocyturia > 50/mm3 and exclusive presence of Gram-negative bacilli on direct examination. AST were performed by direct inoculation on Mueller-Hinton Rapid-SIR (MHR-SIR) agar. We evaluated the time to antibiotic adaptation by the antimicrobial stewardship team according to rapid AST results. Patients were subsequently excluded from the study if asymptomatic bacteria were confirmed, or in the absence of clinical data. Seventy patients were included. Mean age of patients was 68.8years (± 21.3). Empirical antibiotic treatment were mainly based on third generation cephalosporins (n = 33), fluoroquinolones (n = 15), beta-lactamin/beta-lactamase inhibitors (n = 7), fosfomycin and nitrofurantoin (n = 5, each). The average time to obtain results was 7.2h (± 1.6h). Adaptation of therapy following MHR-SIR was performed for 29 patients (41%) with early switch to oral antibiotics, de-escalation or escalation in respectively 72.3%, 30%, and 11% of cases. Time saving of MHR-SIR compared with the standard technique was 42.6 (± 16.7) h. This study showed that rapid antibiotic susceptibility test results, using MHR-SIR method directly from urine, can be obtained 40h earlier than conventional AST. The study also demonstrated significant clinical impact on the selection and reduction of the antibiotic therapy spectrum.

Antibiotic resistance pattern in patients with urinary tract infection: an observational study

Background: Urinary tract infection (UTI) is one of the most common diseases caused by bacteria in communities and hospital settings. With the irrational prescription of antibiotics and their misuse leads to constant increase in resistance. This study aims to evaluate the spectrum and antibiotic resistance pattern of uropathogens and to provide a basis for appropriate antimicrobial therapy in patients with UTI.Methods: A retrospective observational study was carried out for a period of 6 months from April 2018 to September 2018 at general hospital Kakinada. Data of 282 positive urine culture reports and their antibiotic susceptibility test results were collected from the records of the Microbiology department and were analyzed and depicted in percentages. Sample processing, identification of organisms, and pattern of antimicrobial sensitivity were carried out as per the Clinical and Laboratory Standards Institute guidelines.Results: Out of 282 positive reports, Escherichia coli was the most common isolated uropathogen with a total of 148 (52.4%) followed by Klebsiella 72 (26%), 20 (7.9%) each of Citrobacter and pseudomonas aeruginosa. E. coli were resistant to nalidixic acid (59.5%), cotrimoxazole (58.1%), ciprofloxacin (54%) amoxyclav (52.7%), ampicilin (45.9%), cefatoxime (37.8%), cefaperazone and salbactum (25.3%). Klebsiella were resistant to amoxyclav (77.7%), cotrimazole (50%), nalidixic acid (41.6%), ampicilin (44.4%), cefatoxime (27.7%), ciprofloxacin (25%). E. coli and Klebsiella were highly susceptible to nitrofurantoin, cefaperazone and salbactum, piperacillin and tazobactum, amikacin, imipenem, gentamicin.Conclusions: The majority of the isolated bacteria were resistant to many antibiotics commonly used in clinical practices. So prior culture reports and institutional antibiograms are necessary for prescribing antibiotics rationally.

Comparative proteomic analysis of sensitive and multi-drug resistant Aeromonas hydrophila isolated from diseased fish

Bacterial hemorrhagic septicemia caused by multi-drug resistant (MDR) Aeromonas hydrophila has exponentially increased in the past decade, and reached an alarming rate making it a major concern in the aquaculture industry in China. The aim of this study was to investigate the difference in the regulation of proteins expression in multi-drug resistance and susceptible A. hydrophila strains isolated from diseased fish using two-dimensional electrophoresis (2-DE) combined with mass spectrometry. 28 isolates of A. hydrophila were successfully identified by biochemical tests. Antibiotic susceptibility test results showed that all the isolates have different drug resistant patterns. A total of 61 and 17 differently expressed proteins were identified in MDR and susceptible A. hydrophila, respectively, evidencing that biological processes related to carbon metabolism, biosynthesis of secondary metabolites, microbial metabolism in diverse environments, cationic antimicrobial peptide (CAMP) resistance and propanoate metabolism were down-regulated in MDR strain, while proteins involved in biosynthesis of antibiotics, glycolysis/gluconeogenesis were highly expressed in the sensitive strain. The analysis of differentially expressed proteins from multi-drug resistance and susceptible strains suggests that a number of proteins are involved in several metabolic metabolism pathways plays an important role in A. hydrophila drug resistance. Our findings provide new insights about mechanisms involved in drug resistance and propose possible novel targets for developing alternative antibacterial drugs.

2267. Epidemiology of Candida auris Candidemia in a Teaching Hospital in North of Oman: One-Year Survival

BackgroundRecent emergence of Candida auris as a multidrug resistant fungal pathogen, is a serious concerns for public health. However, there is a paucity of reported cases from Oman. Literature search resulted in finding only 7 cases from Oman, reporting C. auris infections in the articles first published in 2017. However, the rate of isolatin is increasing.MethodsIn this study, we included the results of all positive blood cultures of C. auris in Suhar teaching hospital from May 2018 (date of first detection) till end of April 2019. Further confirmation of the species was performed by MALDI-TOF and antibiotic susceptibility test (AST) by Vitek 2 in central public health laboratory (CPHL) of Oman.ResultsWe detected 13 patients (9 females, 4 males). The mean age was 58.61% years (28–76 years). All candidemic patients had serious underlying conditions, including prolonged hospital stay or extensive and prolonged antimicrobial exposure or medical comorbidities (8 of 13). The time from hospital admission to onset of C. auris candidemia was 8–49 days, with a median of approximately 27 days. The most common isolated co- pathogen from blood culture was K. pneumonia (without regard to Coagulase-negative staphylococci). As average, every patient received 4.8 kind of different antibiotics in mean 88 doses before candidemia developed and piperacillin–tazobactam was the most common used antibiotics. AST was done just for 5 patients and revealed high-level resistance to fluconazole and Amphotricin B while, Echinocandins (anidulafungin, caspofungin) were fully sensitive and voricunazole had intermediate sensitivity. Mean duration of anti-fungal treatment was 12.5 days (5 – 26 days). 8 patients treated by Echinocandins (4/8 died), 4 by Fluconazole (3/4 died) and one without treatment discharged. 30-day all-cause mortality was 61.5%.ConclusionIn Oman, C. auris has been reported from many hospitals. Resistance to several antifungal agents and persistence in the hospital environment make this organism a potential menace for the treating physician and the infection control personnel. In our hospital, every candidemic patient should be treated with Echinocandins and assumed to be resistant to Fluconazole until proven otherwise according to results of AST.Disclosures All authors: No reported disclosures.

2182. Harnessing Direct-from-Blood MALDI-TOF (DBM) and Local Blood Stream Infection (BSI) Antibiogram Data to Direct Optimal Therapy for Gram-Negative Rod (GNR) Bacteremia

BackgroundGNR bacteremia is common in the hospital environment. Drug-resistance challenges thoughtful use of, and timely de-escalation to, regimens that limit carbapenem use. DBM accurately provides rapid identification of bacteremia pathogens, but lacks antibiotic susceptibility testing (AST) results to aid clinicians in refining therapy. We investigated whether integrating DBM identification and local BSI antibiogram data could be used to identify carbapenem-sparing regimens with predicted efficacy of ≥ 95% prior to traditional AST results.MethodsWe identified GNR blood culture results in adults from January 1, 2018 to December 31, 2018. The first isolate per bacteremia episode per patient was analyzed. AST was performed by MicroScan. We categorized isolates into 1 of 3 groups based on the most common resistance mechanisms in our population (i.e. CTX-M ESBL, AmpC): 1] Escherichia spp. and Klebsiella spp. (EK), 2] S. marscecens, P. mirabilis, E. (Klebsiella) aerogenes, C. freundii, M. morganii, E. cloacae complex, and C. non-freundii (SPEC-MEC), and 3] P. aeruginosa (PA). We compiled susceptibility rates to common antibiotics for GNR sepsis and compared results using Chi-square.Results381 GNR isolates were recovered in blood cultures from adult patients in 2018: EK 264 (69%), SPEC-MEC 66 (17%), PA 36 (9%), other 15 (4%). Susceptibility results in common GNR sepsis agents are shown in Figure 1 (*P < 0.05 between groups). Within each organism group, susceptibility was highest in the following scenarios: 1] EK: PTZ and MEM (P < 0.05 vs. FEP, TOB, CIP, and ATM), 2] SPEC-MEC: FEP, TOB, and MEM (P < 0.05 vs. PTZ and ATM), and 3] PA: comparably similar (> 90%) for PTZ, FEP, TOB, and MEM.ConclusionIntegration of DBM and antibiogram data identified species-specific carbapenem-sparing regimens that would predict ≥ 95% of patients with GNR bacteremia are on effective therapy prior to traditional AST results. In appropriate patients, this could lead to a significant reduction in carbapenem duration and avoid broad-scale carbapenem use while also optimizing time to effective therapy. This is a readily adaptable process for other centers to improve care of patients with GNR bacteremia. Our data suggest using β-lactam alternatives for GNR bacteremia, especially EK, should be done with caution. DisclosuresAlexander Lepak, MD, Paratek Pharmaceuticals: Research Grant; Tetraphase Pharmaceuticals: Research Grant.

Prolonged fever is not a reason to change antibiotics among patients with uncomplicated community-acquired acute pyelonephritis.

The study aimed to determine the pattern of fever resolution among febrile patients undergoing treatment for acute pyelonephritis (APN) and prove that switching therapy based solely on persistent fever beyond 72 hours of antibiotics treatment may be unwarranted.For the purpose of this study, non-responders were defined as those patients who had a persistent fever over 72 hours after the initiation of antibiotic therapy. Responders were defined as those patients who became afebrile in less than 72 hours after the initiation of antibiotic therapy. Clinical cure was defined as the complete resolution of all symptoms during antibiotic therapy without recurrence during the follow-up period.A total of 843 female patients with uncomplicated community-acquired APN met all inclusion criteria. The non-responder group comprised of 248 patients (29%), and the remaining patients constituted the responder group. The median initial C-reactive protein level was higher (15.6 mg/dl vs 12.6 md/dl, P < .001) and bacteremia was more frequent (31% vs 40%, P = .001) in the non-responder group. Escherichia coli (E. coli) was the most common pathogen in both groups; there was no significant difference between the groups in the etiology of APN. Antimicrobial resistance and extended spectrum β-lactamase producing strains had an increasing trend in the non-responder group but there was no significant difference between the groups.This study shows that it is difficult to identify patients at risk of uncomplicated community-acquired APN by antibiotic-resistant pathogens based exclusively on persistent fever. Patients with a prolonged fever for more than 72 hours show similar antibiotic susceptibility patterns and are not associated with adverse treatment outcomes. Therefore, switching of current antibiotics to broad-spectrum antibiotics should be reserved in this patient population until antibiotic susceptibility test results are available.

Epidemiological and Molecular Investigations on Salmonella Responsible for Gastrointestinal Infections in the Southwest of Shanghai From 1998 to 2017.

PurposeTo investigate the characteristics of gastrointestinal infections in Southwest Shanghai.MethodsClinical and epidemiological characteristics of patients with Salmonella infections between 1998 and 2017 admitted to the Jinshan Hospital in the Southwest of Shanghai were retrospectively analyzed. A total of 565 isolated Salmonella strains were classified by serotyping and pulsed field gel electrophoresis (PFGE).ResultsFrom 1998 to 2006, diarrhea was mainly caused by Vibrio parahaemolyticus followed by Shigella and Salmonella. From 2007 to 2010, Vibrio parahaemolyticus infection was the major cause of diarrhea followed by Salmonella and Shigella. From 2011 to 2017, Salmonella infections became the main cause of diarrhea after Vibrio parahaemolyticus. Salmonella infections increased from 2006 on and peaked between May and October, accounting for 82.48% of yearly infections. Patients with Salmonella infections (90.5%) had a history of eating unclean food, abdominal pain (58.05%), diarrhea ≥5 times a day (50.44%), moderate fever (24.96%) and increased fecal leukocytes (41.42%). From 1998 to 2017, infected specimens from clinical cases were dominated by Salmonella enterica serovar Typhimurium (S. Typhimurium) (21.59%) followed by Salmonella enterica serovar Enteritis (S. Enteritidis) (16.81%), Salmonella enterica serotype London (6.55%) and Salmonella group B (13.10%). Other species included Salmonella enterica serovar Thompson, Salmonella enterica serovar Saintpaul, Salmonella group D, Salmonella group C, Salmonella enterica serovar Choleraesuis and Salmonella enterica serovar Aberdeen. The PFGE classification of Salmonella serovars in 2008–2017 demonstrated that S. Enteritidis had 9 PFGE banding patterns and S. Typhimurium 16 with varying degrees of similarity among S. Enteritidis and S. Typhimurium. The results of antibiotic susceptibility tests for the 330 Salmonella strains revealed that fosfomycin had the highest sensitivity rate (97.5%) followed by levofloxacin and ceftriaxone (81%), and ampicillin/sulbactam (78.2%). The resistance to piperacillin and ciprofloxacin was 60.9 and 50.61%, respectively.ConclusionThe features of onset, epidemiological characteristics and molecular subtyping of Salmonella were conducive to clinical diagnosis, rational use of antibiotics and improved therapeutic efficacy.

Point-Counterpoint: Differences between the European Committee on Antimicrobial Susceptibility Testing and Clinical and Laboratory Standards Institute Recommendations for Reporting Antimicrobial Susceptibility Results

INTRODUCTIONAntibiotic susceptibility test results are among the most important results issued by clinical microbiology laboratories because they routinely guide critical treatment decisions. Interpretations of MIC or disk diffusion test results, such as "susceptible" or "resistant," are easily understood. Clinical laboratories also need to determine whether and how their reports will reflect more complex situations. Such situations include, first, whether there is need to administer higher or more frequent doses of antibiotic than usual for clinical efficacy; second, whether an antimicrobial is likely to be effective at a body site where it concentrates; and third, whether there is some uncertainty in the test results due to technical variability that cannot be eliminated. Two leading organizations that set standards for antimicrobial susceptibility testing, the European Committee on Antimicrobial Susceptibility Testing (EUCAST) and the Clinical and Laboratory Standards Institute (CLSI), have taken different strategies to deal with these challenges. In this Point-Counterpoint, Gunnar Kahlmeter and Christian Giske discuss how EUCAST is addressing these issues, and Thomas Kirn and Susan Sharp discuss the CLSI approach.

Clinical impact of rapid susceptibility testing on MHR-SIR directly from blood cultures

In a previous study, we demonstrated that rapid antibiotic susceptibility tests (ASTs) can be performed directly on blood culture samples tested on Mueller-Hinton Rapid agar (MHR-SIR) with a time delay of 6-8 h. Using this rapid disc diffusion method, we analysed the clinical impact associated with rapid reporting of results in our hospital setting. All patients with bloodstream infections (BSIs) related to Enterobacteriaceae or Staphylococcus aureus were prospectively included in the study. The rapid ASTs were performed by incubation of positive blood cultures on MHR-SIR for 6-8 h by direct inoculation according to BSAC recommendations. One hundred and sixty-seven patients with BSIs were included as MHR-guided adaptation therapy cases. Eighty percent had Enterobacteriaceae-related BSIs, of which 12 (9%) were ESBL producers and 20% were S. aureus-related BSIs. A urinary or intra-abdominal infection was observed in 44.3% and 19.8%, respectively, of Enterobacteriaceae-related infections. The most frequent sources of infections for S. aureus BSIs were cutaneous and endovascular, in 43% and 23% of cases, respectively. Forty-four percent of the patients benefited from therapeutic modification according to the results of the MHR-SIR AST. Thus, empirical antibiotic therapy was modified by using antibiotic therapy that had too wide a spectrum or was unsuitable in 26% and 18% of cases, respectively. Compared with the 24 h required for the reference method, the median length of time to provision of susceptibility test results by MHR-SIR was 7 h. This study showed a significant time saving (17 h) on the appropriateness of antibiotic prescription and demonstrated a significant impact regarding the choice and reduction of the spectrum of antibiotic therapy.

The Isolation of Lactococcus garvieae from Eyes of Diseased Rainbow Trout (Oncorhynchus mykiss) with Exopthalmia

In this study, fish were sampled from 9 different fish farms in the dam lakes in the Eastern Black Sea Region between January 2016 and September 2018. It was isolated twenty-two bacteria isolates from eyes of diseased rainbow trout (Oncorhynchus mykiss). All isolates identification was carried out by using conventional biochemical tests, api 20 strep tests and PCR tests. ATCC 43921 reference strain was used as a positive control. According to results of the api 20 strep test, it was determined that 20 isolates from 22 bacterial isolates were Lactococcus garvieae. The PZR test was confirmed by using the pLG-1 and pLG-2 reference genes (16S rRNA) specific for the L. garvieae species. As a result of the study, it was confirmed that there were 20 L. garvieae from 22 strains isolated from rainbow trout in the dam lakes fish farms. The antibiotic susceptibility test results showed that L. garvieae isolates were resistant %100 to streptomycin, sulfamethoxazole and sulfamethoxazole+trimethoprim, 94.2% to ampicillin, 72.5% to oxytetracycline, 55.07% to erythromycin and 43.5% to oxolinic acid, 26.09% to enrofloxacin, % 20.3 doxycycline, 8,7% to amoxicillin and florphenicol. The most effective antibiotic was found to be florfenicol, amoxicillin, doxisicline and enrofloxacin.

Next-generation sequencing of plasmid carrying blaOXA-48 in Klebsiella pneumoniae from Turkey.

A carbapenem-resistant Klebsiella pneumoniae strain was isolated in Turkey in 2012 and blaNDM-1 and blaOXA-48 genes were observed in this strain. The aim of this study was to investigate transferability of plasmid bearing blaOXA-48 in K. pneumoniae and to use whole-genome sequencing in order to understand the genetic context of plasmid. K. pneumoniae strain was used as donor in conjugation experiments. Antibiotic susceptibility profile of selected transconjugant was determined. Plasmid was isolated from transconjugant colony and was named as pKPT. Complete sequencing of the pKPT was conducted using a next-generation sequencing. Annotation of the contigs was performed using the Geneious R9, followed by finding open reading frames (ORFs) with selected web-based tools. BLAST analysis was performed at the NCBI BLAST server to determine genes showing more than 90% similarity with these ORFs. Results of antibiotic susceptibility test showed that transconjugant colony was resistant to ampicillin/sulbactam, piperacillin, and piperacillin/tazobactam. The pKPT plasmid had a length of 45,217bp and an average G + C content of 49%. Blast analysis revealed that pKPT was included in the IncL/M incompatibility group. The pKPT was found to contain blaOXA-48 within Tn1999.2 transposon without any other antibiotic resistance gene.

Determination of the Antibiotic Resistance Rates of Serratia marcescens Isolates Obtained from Various Clinical Specimens

Aim : Serratia marcescens clinical isolates are increasingly resistant to antibiotics. Therefore, the treatment of infections caused by S. marcescens becomes difficult. The aim of this study was to examine the antimicrobial resistance profiles of S. marcescens bacteria isolated from various clinical specimens according to body regions and clinics and to evaluate for enzyme production rates associated with antibiotic resistance of these isolates. Materials and Methods : Blood culture samples were incubated in a fully automated BACTEC‑FX system. Identification and antibiogram processing was carried out by fully automated VITEK 2 identification and antibiogram system. The obtained results were retrospectively screened. Results : S. marcescens was identified in a total of 158 clinical specimens. The departments where S. marcescens was most commonly identified were the Anesthesia Intensive Care Unit (25.9%), followed by Chest Diseases (19.6%). Serratia isolates were most commonly determined in blood culture (35.4%) and sputum culture (24.6%). Resistance rates to ceftriaxone and ceftazidime were 22.7% and 19.6%, respectively. However, the rate of resistance detected to cefotaxime and gentamicin (0.6%) was very low. Conclusion : Clinical isolates of Serratia exhibited highest resistance to ceftriaxone, ceftazidime, and piperacillin/tazobactam. However, it was found that the tested Serratia strains did not exhibit high resistance to other antibiotics. Our results suggest that cefotaxime and gentamicin are the most suitable antibiotics for treatment. The extended‑spectrum β‑lactamase and inducible β‑lactamase ratios were found to be decreased by 6%–7%. Although different results may be obtained from different hospitals and regions, it should not be forgotten that Serratia strains may be resistant to many antibiotics and that the results of antibiotic susceptibility testing may help plan antibiotic treatment. Keywords : Antibiotic resistance, antibiotic susceptibility, Serratia marcescens

Retrospective whole-genome sequencing analysis distinguished PFGE and drug-resistance-matched retail meat and clinical Salmonella isolates

Non-typhoidal Salmonella is a leading cause of outbreak and sporadic-associated foodborne illnesses in the United States. These infections have been associated with a range of foods, including retail meats. Traditionally, pulsed-field gel electrophoresis (PFGE) and antibiotic susceptibility testing (AST) have been used to facilitate public health investigations of Salmonella infections. However, whole-genome sequencing (WGS) has emerged as an alternative tool that can be routinely implemented. To assess its potential in enhancing integrated surveillance in Pennsylvania, USA, WGS was used to directly compare the genetic characteristics of 7 retail meat and 43 clinical historic Salmonella isolates, subdivided into 3 subsets based on PFGE and AST results, to retrospectively resolve their genetic relatedness and identify antimicrobial resistance (AMR) determinants. Single nucleotide polymorphism (SNP) analyses revealed that the retail meat isolates within S. Heidelberg, S. Typhimurium var. O5- subset 1 and S. Typhimurium var. O5- subset 2 were separated from each primary PFGE pattern-matched clinical isolate by 6-12, 41-96 and 21-81 SNPs, respectively. Fifteen resistance genes were identified across all isolates, including fosA7, a gene only recently found in a limited number of Salmonella and a ≥95 % phenotype to genotype correlation was observed for all tested antimicrobials. Moreover, AMR was primarily plasmid-mediated in S. Heidelberg and S. Typhimurium var. O5- subset 2, whereas AMR was chromosomally carried in S. Typhimurium var. O5- subset 1. Similar plasmids were identified in both the retail meat and clinical isolates. Collectively, these data highlight the utility of WGS in retrospective analyses and enhancing integrated surveillance for Salmonella from multiple sources.

Investigation of mecA gene, virulence traits and antibiotic resistance profiles in methicillin‐resistant Staphylococcus aureus isolates from dairy products

AbstractThis study was performed to determine the prevalence of methicillin‐resistant Staphylococcus aureus (MRSA) in raw milk and dairy products and the presence of virulence traits in the isolates obtained and to assess their resistance to several antibiotics. MRSA was detected by CHROMagar MRSA II in 148 (22.8%) of 650 samples. Ninety‐eight isolates were confirmed as MRSA, 86 of these isolates were phenotype‐positive, 9 were phenotype/mecA‐positive, and 3 were phenotype‐negative but mecA‐positive. However, the virulence genes were not found in any MRSA strains. According to the results of antibiotic susceptibility tests, 100, 44.9, and 29.6% of strains were resistant to penicillin, tetracycline, and clindamycin, respectively. A low prevalence (1.02%) of resistance was observed against vancomycin and chloramphenicol antibiotics. Furthermore, multidrug resistance was seen among MRSA strains isolated from cheese, butter and buttercream samples. In contrast with other studies, we identified a high‐level vancomycin‐resistant MRSA strain in one buttercream sample and the intermediate‐level vancomycin resistance in six different multiple‐resistant MRSA strains from food of dairy origin.Practical applicationsRecently, MRSA has been isolated from foods of animal origin and become one of the rising public health concerns worldwide. This study focused on the prevalence of MRSA in raw milk and dairy products, the presence of genes encoding virulence factors that allow it to cause the disease and their antibiotic susceptibility patterns. Consequently, we revealed the emergence of resistance to vancomycin and multiple antibiotics among MRSA strains. The isolation of high‐level vancomycin‐resistant MRSA strain is an important finding because vancomycin continues to be the first‐line treatment agent for MRSA infections.

Effective implementation of the Accelerate Pheno™ system for positive blood cultures.

Using conventional methods, organism identification (ID) and antibiotic susceptibility testing (AST) results are available ∼1.5–3 days after positive blood culture. New technologies can reduce this time to 8–12 h, allowing therapy to be optimized substantially sooner. To make full use of fast ID and AST results requires overcoming various hurdles to effective implementation, including restructuring laboratory workflows to optimize timeliness of results and modifying clinical pathways to respond more quickly when results are available. Efficient laboratory procedures and clinical interventions coupled with fast and accurate identification and AST results have the potential to substantially reduce overall costs and provide more-sophisticated and effective patient management.

Emergence of bla TEM resistance gene in ESBL-producing Escherichia coli clinical isolates from Health facilities in Makurdi, Benue State Nigeria

The emergence of extended-spectrum-β-lactamases (ESBLs)-producing Escherichia coli represents a serious clinical concern in healthcare. β-lactamases produced by these strains of E. coli render ineffective cephalosporins and other β-lactam antibiotics used to treat infections caused by Gram–negative bacteria. We determined the presence of ESBL in 400 clinical isolates of E. coli isolated from various clinical specimens (urine, stool, blood, sputum, throat and wound swabs) from 216 female and 184 male patients with mean age of 28.1 ± 16.8 years (age range: 2 – 71 years), who were attending 6 selected health facilities in Makurdi, Benue State Nigeria. Antibiotic susceptibility test was carried out on the isolates using Kirby Bauer diffusion method. Presence of ESBLs was determined by the double disc synergy test (DDST). Specific primers were employed to characterize the ESBL gene using PCR. The isolates showed high level of resistance to all the antibiotics tested except mipenem. Highest resistance was to penicillin 392(98.0%) followed by ceftriaxone 385(96.3%). Out of the 400 isolates, 64 (16.0%) tested positive for ESBLs by DDST method, while PCR technique confirmed 47(11.8%) to harbour bla TEM genes. Isolates from blood specimens harboured highest percentage of ESBL genes 5(26.3%) and also plasmid-mediated bla TEM genes 5(26.3%), followed by wound swabs 9(17.3). The least percentage of plasmid-mediated bla TEM genes was carried by isolates from sputum specimens 1(8.3). Age group 45 to 58 years harboured the highest percentage of bla TEM genes 15(14.6%), while female patients, 27(12.5%) carried more bla TEM resistance genes than the male patients 20(10.9%). A prevalence of 11.8% (n=47) of bla TEM resistance gene has been reported for the present study. In view of multidrug resistant ESBL-producing Escherichia coli bacteria circulating in the study location, prescription of antibiotics, especially cephalosporins should be based on laboratory results of antibiotic susceptibility tests that are carried out along with ESBL detection. Infection prevention and control strategies should be stepped up in the health facilities under study.