Standard Susceptibility Testing Research Articles

Insecticide Resistance in Aedes aegypti from the National Capital Region of the Philippines

Human arboviral diseases such as dengue, chikungunya, and Zika can be transmitted by the mosquito Aedes aegypti. The insecticide-based vector control strategy is critical in reducing transmission of these Aedes-borne diseases but is threatened mainly by the emergence of insecticide resistance. Adult Ae. aegypti from the National Capital Region (NCR), Philippines, were subjected to bioassays to determine their susceptibility to diagnostic doses of pyrethroid, organochlorine, and organophosphate insecticides following the standard World Health Organization insecticide susceptibility test. This study reports the detection of insecticide resistance to pyrethroids and organochlorine in Ae. aegypti from the Philippines for the first time. Most of the Ae. aegypti populations from NCR exhibited phenotypic resistance to permethrin, etofenprox, and DDT. Varying resistance levels to deltamethrin, cyfluthrin, and lambda-cyhalothrin were observed in the different mosquito populations, while all populations tested to malathion were susceptible to this organophosphate. This finding should alert public health authorities to consider modifying the existing vector management package for greater control efficacy. Best practices proven to prevent or delay the development of insecticide resistance, such as insecticide rotation, should also be implemented, while alternative chemicals with a different mode of action should be explored to ensure the continuing efficacy of program interventions.

Verification of the Mycoplasma IST3 for urogenital mycoplasma culture in comparison to the Mycoplasma IST2

Background: Unlike the Mycoplasma IST2 kit (bioMérieux), the Mycoplasma IST3 kit has been updated to comply with the standardized antimicrobial susceptibility test (AST) method for Ureaplasma spp. (Up) and Mycoplasma hominis (Mh). We aimed to verify the use of the Mycoplasma IST3 kit for genital mycoplasma cultures. Methods: From September 2023 to January 2024, the R1 medium remaining after inoculation with IST2 was refrigerated until the next day. For IST2-positive samples, 300 μL of residual R1 medium was inoculated into the IST3. Species identification, enumeration, and AST results obtained using IST3 were compared with those obtained using IST2. Results: A total of 48 IST2-positive samples were inoculated into IST3, including 35, 1, and 12 Up-only, Mh-only, and both Up- and Mh-positive samples, respectively. Among Up-only samples, 2.8%, 91.4%, and 100.0% were susceptible to ciprofloxacin, tetracycline, and erythromycin, respectively. With IST3, 45 (93.8%) samples grew genital mycoplasmas; 42 (89.4%) of the 47 Up-positive samples and 6 (46.2%) of the 13 Mh-positive samples showed growth of the same organisms. All seven samples that failed to grow Mh were from mixed cultures, of which four Mh concentrations of < 104/mL. Up was susceptible to levofloxacin, tetracycline, and erythromycin at the rates of 64.3 %, 88.1 %, and 95.2 %, respectively. Conclusion: IST3 showed good performance in detecting genital Mycoplasma except for its tendency to not detect Mh of low concentrations in mixed cultures. IST3 is preferable to IST2 because it can accurately screen for erythromycin resistance in Up and reduce falseresistances for fluoroquinolone.

Activity of antibiotics against Burkholderia cepacia complex in artificial sputum medium.

Burkholderia cepacia complex (Bcc) is a collection of intrinsically drug-resistant Gram-negative bacteria that cause life-threatening disease in people with cystic fibrosis (CF). Standard antimicrobial susceptibility testing methods have poor predictive value for clinical outcomes in Bcc infections, probably due in part to differences between in vitro testing conditions and the environment in which Bcc grow in the lungs of people with CF. To compare the activity of commonly used antibiotics under standard in vitro testing conditions with activity in conditions mimicking those found in vivo. Two Bcc strains were grown alone and with six different antibiotics (minocycline, ceftazidime, meropenem, tobramycin, levofloxacin, trimethoprim-sulfamethoxazole) in two different media: standard cation-adjusted Mueller-Hinton broth and an artificial sputum medium designed to simulate the environment in the lungs of people with CF through addition of components including mucin, free DNA and amino acids. Two different starting conditions were used for time-kill assays: a standard ∼5 × 106 cfu/mL inoculum, and a high-density inoculum in which bacteria were grown for 72 hours before addition of antibiotics. Growth detection was performed by colony enumeration and by detection of resazurin reduction. There were major discrepancies between standard susceptibility results and activity in our models. Some antibiotics, including ceftazidime, showed minimal activity in all time-kill assays despite low minimal inhibitory concentrations, while others, notably tobramycin, were more active in high-density growth conditions than in standard time-kill assays. This work underscores the urgent need to develop more clinically relevant susceptibility testing approaches for Bcc.

Vulvovaginal candidiasis, an increasing burden to women in the tropical regions attending Bharatpur Hospital, Chitwan

Vulvovaginal candidiasis is a yeast infection commonly caused by the overgrowth of Candida species in and around the vulva and vagina. Abnormal vaginal discharge, itching and irritation, swelling and redness of the vaginal area, pain during sexual intercourse, and dyspareunia are important clinical findings of the infection. Currently, the infection is one of the growing burdens to married women. Moreover, the infection with antifungal-resistant Candida species adds challenges to managing the disease. Hence, this study was conducted to identify the different Candida species causing vulvovaginal candidiasis and to determine its susceptibility pattern against different antifungal drugs. A hospital-based cross-sectional and quantitative study was conducted for the period of six months from September 2022 to March 2023 among symptomatic married women in the Gynecology and Obstetrics Department of Bharatpur Hospital, Chitwan. A total of 300 symptomatic cases were enrolled in the study. Candida species were isolated from vaginal swabs following standard microbiological procedures and antifungal susceptibility testing was performed with different antifungal agents. The total prevalence of vulvovaginal candidiasis was found to be 37.3 % (112/300). Among different isolates, Candida albicans was found to be the most predominant (52.6 %), followed by Candida glabrata (29.3 %) among non-albicans. Women from the age group 25–35 years were found to be more infected (47.3 %) and the relationship between contraceptive use and vulvovaginal candidiasis was found to be statistically significant (p < 0.05). Candida species showed higher susceptibility toward Amphotericin-B (68.1 %), followed by fluconazole (Diflucan), and Clotrimazole (50.9 %). Whereas the least susceptibility was observed to Voriconazole (27.6 %) and Itraconazole (35.30 %). Candida albicans was comparatively more susceptible to different antifungal drugs than non-albicans species. Candida parapsilosis was only susceptible to Amphotericin-B and the increasing incidence of vaginal candidiasis due to non-albicans Candida indicates the need for routine speciation of Candida.

Prevalence and antimicrobial susceptibility profile of Salmonella isolated from vegetable farms fertilized with animal manure in Addis Ababa Ethiopia

The resistance of foodborne pathogens to antimicrobial agents is a potential danger to human health. Hence, establishing the status of good agricultural practices (GAPs) and the antimicrobial susceptibility of major foodborne pathogens has a significant programmatic implication in planning interventions. The objective of this study was to assess the gap in attaining GAP and estimate the prevalence and antimicrobial susceptibility profile of Salmonella in vegetable farms fertilized with animal manure in Addis Ababa, Ethiopia. A total of 81 vegetable farms from four sub-cities in Addis Ababa were visited, and 1119 samples were collected: soil (n = 271), manure (n = 375), vegetables (n = 398), and dairy cattle feces (n = 75). Additional data were collected using a structured questionnaire. Isolation of Salmonella was done using standard microbiology techniques and antimicrobial susceptibility testing was conducted using disk diffusion assays. Carriage for antimicrobial resistance genes was tested using polymerase chain reaction (PCR). Among the 81 vegetable farms visited, 24.7% used animal manure without any treatment, 27.2% used properly stored animal manure and 80.2% were easily accessible to animals. The prevalence of Salmonella was 2.3% at the sample level, 17.3% at the vegetable farm level, and 2.5% in vegetables. The highest rate of resistance was recorded for streptomycin, 80.7% (21 of 26), followed by kanamycin, 65.4% (17 of 26), and gentamicin, 61.5% (16 of 26). Multidrug resistance was detected in 61.5% of the Salmonella isolates. Vegetable farms have a gap in attaining GAPs, which could contribute to increased contamination and the transfer of antimicrobial resistance to the vegetables. The application of GAPs, including proper preparation of compost and the appropriate use of antimicrobials in veterinary practices, are recommended to reduce the emergence and spread of antimicrobial resistance.

Antifungal heteroresistance causes prophylaxis failure and facilitates breakthrough Candida parapsilosis infections.

Breakthrough fungal infections in patients on antimicrobial prophylaxis during allogeneic hematopoietic cell transplantation (allo-HCT) represent a major and often unexplained cause of morbidity and mortality. Candida parapsilosis is a common cause of invasive candidiasis and has been classified as a high-priority fungal pathogen by the World Health Organization. In high-risk allo-HCT recipients on micafungin prophylaxis, we show that heteroresistance (the presence of a phenotypically unstable, low-frequency subpopulation of resistant cells (~1 in 10,000)) underlies breakthrough bloodstream infections by C. parapsilosis. By analyzing 219 clinical isolates from North America, Europe and Asia, we demonstrate widespread micafungin heteroresistance in C. parapsilosis. Standard antimicrobial susceptibility tests, such as broth microdilution or gradient diffusion assays, which guide drug selection for invasive infections, fail to detect micafungin heteroresistance in C. parapsilosis. To facilitate rapid detection of micafungin heteroresistance in C. parapsilosis, we constructed a predictive machine learning framework that classifies isolates as heteroresistant or susceptible using a maximum of ten genomic features. These results connect heteroresistance to unexplained antifungal prophylaxis failure in allo-HCT recipients and demonstrate a proof-of-principle diagnostic approach with the potential to guide clinical decisions and improve patient care.

Sensitivity and specificity of Nanopore sequencing for detecting carbapenem and 3rd-generation cephalosporin-resistant Enterobacteriaceae in urine samples: Real-time simulation with public antimicrobial resistance gene database

ObjectivesTo evaluate the accuracy of beta-lactamase gene detection directly from urine samples by Nanopore sequencing. MethodsDNA was extracted from bacterial pellets in spun urine. The purified DNA was then sequenced in native form by a Nanopore sequencer (MinION) to identify the organisms and beta-lactamase genes. Results were compared to routine urine cultures and standard antimicrobial susceptibility tests (AST). ResultsWe processed 60 urine samples of which routine cultures grew Enterobacteriaceae, including 28 carbapenem-resistant (CRE), 17 extended-spectrum beta-lactamase (ESBL) or AmpC producing, and 15 non-ESBL/AmpC phenotypes. We excluded 7 samples with extremely low DNA amounts (<1 ng/μl) for a final case of 53 in total. The sensitivity of antimicrobial resistance gene detection within 6 h, the optimal duration from real-time simulation, of Nanopore sequencing for the diagnosis of carbapenem-resistant and ceftriaxone-resistant phenotypes was 73.9 % (95%CI 56.0–91.9 %) and 81.1 % (95%CI 68.5–93.7 %), while the specificity was 96.7 % (95%CI 90.2–100.0 %) and 56.3 % (95%CI 31.9–80.6 %), respectively. The median times for MinION to generate DNA reads containing carbapenemase and ESBL/AmpC genes were 93 min (IQR 17–245.5) and 99 min (IQR 31.25–269.75) after sequencing commencement, respectively. ConclusionsNanopore sequencing can identify bacterial genotypic resistance in urine and may enable clinicians to adjust antimicrobial therapy earlier than routine AST.

Antibiotic Resistance Profiles of Extended-Spectrum β-Lactamase (ESBL)- and Metallo-β-Lactamase (MBL)-Producing Klebsiella pneumoniae Isolates From Diabetic Foot Ulcers: Implications for Treatment Strategies.

Background Diabetic foot ulcers (DFUs) are prevalent complications of diabetes mellitus, often leading to severe infections and adverse clinical outcomes. Klebsiella pneumoniae, a gram-negative bacterium, has emerged as a significant causative agent in DFU infections, raising concerns due to its increasing antibiotic resistance, particularly in extended-spectrum β-lactamase (ESBL) and metallo-β-lactamase (MBL) production. Aim This study aimed to comprehensively assess the prevalence, antibiotic resistance profiles, and clinical correlates of ESBL- and MBL-producing K. pneumoniae isolates specifically derived from DFUs. Methods A cross-sectional observational study was conducted at Krishna Vishwa Vidyapeeth from January 2023 to June 2023, involving 126 patients diagnosed with DFUs. Clinical and demographic data were collected, and wound swabs underwent microbiological analysis. Phenotypic detection methods were employed to identify ESBL and MBL production, followed by standardized antibiotic susceptibility testing. Results Among the 126 isolates tested, 36 (28.6%) were identified as ESBL-producing and 21 (16.7%) as MBL-producing strains. ESBL-producing isolates exhibited high resistance rates to antibiotics such as ampicillin (92.3%), amoxicillin-acid (84.6%), and cephalosporins, including ceftriaxone (76.9%), and cefepime (73.8%). MBL-producing isolates demonstrated even broader resistance profiles, including resistance to fluoroquinolones (ciprofloxacin, 60.0%; levofloxacin, 57.1%), aminoglycosides (gentamicin, 42.9%), and carbapenems (meropenem, 38.1%; imipenem, 35.7%). Conclusion This study identifies a significant prevalence of ESBL- and MBL-producing K. pneumoniae in DFUs, showcasing high antibiotic resistance rates. Comorbidities correlate significantly with the presence of resistant isolates, necessitating treatment strategies for effective management.

The Oral Cavity-Another Reservoir of Antimicrobial-Resistant Staphylococcus aureus?

Staphylococcus aureus is one of the most common potentially pathogenic bacteria that may asymptomatically colonize many sites of healthy carriers. Non-nasal carriage, especially in the oral cavity, and its role in transmitting antimicrobial-resistant S. aureus strains in the healthcare community, is poorly understood. This study aimed to assess the prevalence and antimicrobial susceptibility of S. aureus in both oral and nasal cavities among preclinical dentistry students. A total of 264 oral and nasal swabs were taken from 132 participants, and all specimens were cultured using standard diagnostic procedures and antimicrobial susceptibility testing (EUCAST). The prevalence of S. aureus exclusively in the nasal (11.4%) or oral (9.1%) cavity was comparable, while concurrent oral and nasal carriage was present in 27.3% of participants. Although antibiotic resistance rates observed in both oral and nasal isolates were similar (ranging from 2.7% to 95.5%), 16.7% of carriers exhibited distinct antibiotic resistance profiles between oral and nasal isolates. Three (2.7%) methicillin-resistant S. aureus (MRSA) were isolated from the mouth and nose but multidrug resistance (27.3%) was more frequent in the oral than in the nasal isolates: 34% and 21.1%, respectively. This study demonstrated that preclinical dentistry students have a similar rate of oral S. aureus carriage as the nasal carriage rate, and that the oral cavity can be colonized by antimicrobial-resistant strains that do not originate from the nose. Consequently, the oral cavity seems to be an unjustly overlooked body site in screening for S. aureus carriage.

Efficacy of sodium hypochlorite in overcoming antimicrobial resistance and eradicating biofilms in clinical pathogens from pressure ulcers.

Sodium hypochlorite (NaOCl) is widely recognized for its broad-spectrum antimicrobial efficacy in skin wound care. This study investigates the effectiveness of NaOCl against a range of bacterial and fungal isolates from pressure ulcer (PU) patients. We analyzed 20 bacterial isolates from PU patients, comprising carbapenem-resistant Klebsiella pneumoniae (CRKP), multidrug-resistant Acinetobacter baumannii (MDRAB), methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible Staphylococcus aureus (MSSA), along with 5 Candida albicans isolates. Antibiotic resistance profiles were determined using standard susceptibility testing. Whole-genome sequencing (WGS) was employed to identify antimicrobial resistance genes (ARGs) and disinfectant resistance genes (DRGs). Genetic determinants of biofilm formation were also assessed. The antimicrobial activity of NaOCl was evaluated by determining the minimum inhibitory concentration (MIC) and the minimal biofilm eradication concentration (MBEC) for both planktonic and biofilm-associated cells. CRKP and MDRAB showed resistance to fluoroquinolones and carbapenems, while MRSA exhibited resistance to β-lactams and levofloxacin. MSSA displayed a comparatively lower resistance profile. WGS identified significant numbers of ARGs in CRKP and MDRAB, with fewer DRGs compared to MRSA and MSSA. All isolates possessed genes associated with fimbriae production and adhesion, correlating with pronounced biofilm biomass production. NaOCl demonstrated substantial antimicrobial activity against both planktonic cells and biofilms. The MIC90 for planktonic bacterial cells was 0.125 mg/mL, and the MBEC90 ranged from 0.225 to 0.5 mg/mL. For planktonic C. albicans, the MIC90 was 0.150 mg/mL, and the MBEC90 was 0.250 mg/mL. These results highlight the challenge in treating biofilm-associated infections and underscore the potential of NaOCl as a robust antimicrobial agent against difficult-to-treat biofilm infections at concentrations lower than those typically found in commercial disinfectants.

Clinical Pilot of Bacterial Transcriptional Profiling as a Combined Genotypic and Phenotypic Antimicrobial Susceptibility Test.

Antimicrobial resistance is a growing health threat, but standard methods for determining antibiotic susceptibility are slow and can delay optimal treatment, which is especially consequential in severe infections such as bacteremia. Novel approaches for rapid susceptibility profiling have emerged that characterize either bacterial response to antibiotics (phenotype) or detect specific resistance genes (genotype). GoPhAST-R is a novel assay, performed directly on positive blood cultures, that integrates rapid transcriptional response profiling with detection of key resistance gene transcripts, thereby providing simultaneous data on both phenotype and genotype. Here, we performed the first clinical pilot of GoPhAST-R on 42 positive blood cultures: 26 growing Escherichia coli, 15 growing Klebsiella pneumoniae, and 1 with both. An aliquot of each positive blood culture was exposed to 9 different antibiotics, lysed, then underwent rapid transcriptional profiling on the NanoString® platform; results were analyzed using an in-house susceptibility classification algorithm. GoPhAST-R achieved 95% overall agreement with standard antimicrobial susceptibility testing methods, with the highest agreement for beta-lactams (98%) and the lowest for fluoroquinolones (88%). Epidemic resistance genes including the extended spectrum beta-lactamase bla CTX-M-15 and the carbapenemase bla KPC were also detected within the population. This study demonstrates the clinical feasibility of using transcriptional response profiling for rapid resistance determination, although further validation with larger and more diverse bacterial populations will be essential in future work. GoPhAST-R represents a promising new approach for rapid and comprehensive antibiotic susceptibility testing in clinical settings.

Antibiotics Sensitivity Reaction of salmonella Species Isolated from Ready-to-Eat Porridge Beans Sold in Federal Polytechnic of Oil and Gas Bonny of Rivers State

Salmonella specie is one of the four key global causes of diarrhoeal diseases. In this research we investigated the frequency occurrence of salmonella species isolated from ready-to-eat porridge beans sold from vendors in the proximity of federal polytechnic of oil and gas bonny island. A total of 40 samples were purchased from four different food vendors in four different spots [vendor 1(The first spot), vendor 11(second spot), vendor 111(third spot) and vendor 1V (fourth spot)] between February and May 2024.All samples were processed and analysed using standard culturebased, biochemical methods and antibiotic susceptibility test assays to confirm salmonella isolates. The highest total occurrence of heterotrophic bacteria population densities were: vendor1 4.0.0×105CFU/g; vendor11 4.4×107CFU/g, vendor111 4.5×102CFU/g and vendor 1V 4.2×109CFU/g. The highest total occurrence of salmonella population density were, for vendor1 3.9×102, Vendor11 2.7×104 , Vendor111 4.1×105,Vendor1V 4.4×103 respectively. Antibiotics susceptibility test was performed for the isolates which exhibited that all of them were susceptible to Ciprofloxacin (CPR)-5 g, Nitrofurantoin (NIT)-30 g, Oflotaxin (OFL)-5 g, were susceptible to Salmonella species. While Gentamicin (GEN)-10 g, and Cefuroxime (CXM)-5 g, were intermediate and Finally, Augmentin (AUG)-30 g, Cefuroxime (CAZ)-30 g, and Cefuroxime (CRX)-30 g were resistant to Salmonella species. Therefore, it can be stated that ready-to-eat porridge beans sold in the proximity of FPOG environment are possible route of transmission for Salmonella species. However, due to lack of intense antibiotic resistance among these bacteria, most of them can be treated with the antibiotics available in the market. Nonetheless, strict monitoring and regular surveillance is necessary.

Genome analyses of colistin-resistant high-risk blaNDM-5 producing Klebsiella pneumoniae ST147 and Pseudomonas aeruginosa ST235 and ST357 in clinical settings

BackgroundColistin is a last-resort antibiotic used in extreme cases of multi-drug resistant (MDR) Gram-negative bacterial infections. Colistin resistance has increased in recent years and often goes undetected due to the inefficiency of predominantly used standard antibiotic susceptibility tests (AST). To address this challenge, we aimed to detect the prevalence of colistin resistance strains through both Vitek®2 and broth micro-dilution. We investigated 1748 blood, tracheal aspirate, and pleural fluid samples from the Intensive Care Unit (ICU), Neonatal Intensive Care Unit (NICU), and Tuberculosis and Respiratory Disease centre (TBRD) in an India hospital. Whole-genome sequencing (WGS) of extremely drug-resitant (XDR) and pan-drug resistant (PDR) strains revealed the resistance mechanisms through the Resistance Gene Identifier (RGI.v6.0.0) and Snippy.v4.6.0. Abricate.v1.0.1, PlasmidFinder.v2.1, MobileElementFinder.v1.0.3 etc. detected virulence factors, and mobile genetic elements associated to uncover the pathogenecity and the role of horizontal gene transfer (HGT).ResultsThis study reveals compelling insights into colistin resistance among global high-risk clinical isolates: Klebsiella pneumoniae ST147 (16/20), Pseudomonas aeruginosa ST235 (3/20), and ST357 (1/20). Vitek®2 found 6 colistin-resistant strains (minimum inhibitory concentrations, MIC = 4 μg/mL), while broth microdilution identified 48 (MIC = 32–128 μg/mL), adhering to CLSI guidelines. Despite the absence of mobile colistin resistance (mcr) genes, mechanisms underlying colistin resistance included mgrB deletion, phosphoethanolamine transferases arnT, eptB, ompA, and mutations in pmrB (T246A, R256G) and eptA (V50L, A135P, I138V, C27F) in K. pneumoniae. P. aeruginosa harbored phosphoethanolamine transferases basS/pmrb, basR, arnA, cprR, cprS, alongside pmrB (G362S), and parS (H398R) mutations. Both strains carried diverse clinically relevant antimicrobial resistance genes (ARGs), including plasmid-mediated blaNDM-5 (K. pneumoniae ST147) and chromosomally mediated blaNDM-1 (P. aeruginosa ST357).ConclusionThe global surge in MDR, XDR and PDR bacteria necessitates last-resort antibiotics such as colistin. However, escalating resistance, particularly to colistin, presents a critical challenge. Inefficient colistin resistance detection methods, including Vitek2, alongside limited surveillance resources, accentuate the need for improved strategies. Whole-genome sequencing revealed alarming colistin resistance among K. pneumoniae and P. aeruginosa in an Indian hospital. The identification of XDR and PDR strains underscores urgency for enhanced surveillance and infection control. SNP analysis elucidated resistance mechanisms, highlighting the complexity of combatting resistance.

Practice of standardization of CLSI M45 A3 antimicrobial susceptibility testing of Infrequently Isolated or Fastidious Bacteria strains isolated from blood specimens in Guangdong Province 2017-2021.

The concentration of antimicrobial agents in environments like water and food has increased rapidly, which led to a rapid increase in antimicrobial resistance levels in the environment. Monitoring of bacterial resistance levels is considered as a necessary means to control the bacterial resistance. Reference standards are critical for antimicrobial susceptibility testing. CLSI M45 A3 standard defines pathogenic microorganisms that cause infections less frequently than those covered by CLSI M02, M07, and M100 as Infrequently Isolated or Fastidious Bacteria and specifies antimicrobial susceptibility testing methods. Our study investigated the epidemiology and antimicrobial susceptibility testing data of Infrequently Isolated or Fastidious Bacteria strains isolated from blood specimens in 70 hospitals in Guangdong Province between 2017 and 2021. We defined testing methods other than those specified in CLSI M45 A3 as "Non-Standardized." The proportion of standardized antimicrobial susceptibility testing for penicillin increased significantly (Corynebacterium spp. 17.4% vs. 50.0% p < 0.05; Micrococcus spp. 50.0% vs. 77.8% p < 0.05; Abiotrophia spp. and Granulicatella spp. 21.4% vs. 90.9% p < 0.001), while for cefotaxime (Corynebacterium spp. 0.0% vs. 45.2% p < 0.05; Abiotrophia spp. and Granulicatella spp. 0.0% vs. 14.3% p = 0.515) and vancomycin increased finitely. Non-standardized methods were used for all other antimicrobials. Due to limitations in the economic and medical environment, some clinical laboratories are unable to fully comply with CLSI M45 A3 standard. We recommend that CLSI should add breakpoints for disk diffusion method to improve the standardization of antimicrobial susceptibility testing.

Antibiotic susceptibility profile of Pseudomonas species isolated from clinical specimens to access, watch and reserve drugs across various hospital settings at a tertiary care hospital of central India

Background and Objectives: Over the last decade, hospital-acquired infections, particularly in the critical care setting, have become more common, with Gram-negative bacterial infections having the highest prevalence. This study aims to determine the prevalence and antibiotic susceptibility pattern of Pseudomonas species to WHO’s, aware class of antibiotics, which are commonly prescribed across various ICU’s, medical and surgical wards of our tertiary care teaching hospital. Materials and Methods: This prospective study conducted from January 2021 to June 2022 at a tertiary care centre of cen- tral India identified Pseudomonas species from clinical samples using standard procedures and antimicrobial susceptibility testing performed as per Clinical Laboratory Standards Institute (CLSI) guidelines (M100; 32th Edition). Results: A total of 1490 non duplicate Pseudomonas species isolates were grown from 21,019 culture positive clinical sam- ples, of which 1247 were Pseudomonas aeruginosa. Out of these 1247 Pseudomonas aeruginosa 384 were MDR (30.7%). Pseudomonas aeruginosa were most commonly isolated from the pus samples (85%). ICU isolates were significantly more resistant to antibiotics than those from other units. P. aeruginosa strains from ICUs showed the highest rates of resistance to ceftazidime (93.9%). Reserve drug colistin showed good susceptibility (98.2%). All the 18 colistin resistant strains were found to be negative for plasmid mediated mcr-1,2,3 genes. Conclusion: The study shall help to generate and disseminate the data so that proper antibiotic policy can be made for judi- cious use of Access, Watch and Reserve antibiotics and antibiotic de-escalation plan can be put forth.

Prevalence, Risk Factors and Antimicrobial Resistance Patterns of Candida albicans and Co-Infectious Agents among Female Sex Workers in Lagos, Nigeria

Background: An upsurge in the number of female sex workers (FSW) recently occurred in Lagos due to the harsh economic situation. This study is aimed at providing essential data on the carriage and antimicrobial susceptibility of Candida albicans and co-infections with other urogenital pathogens isolated from sex workers in Lagos. Methods: Two hundred female sex workers (FSWs) participated in this study conducted in Lagos, Nigeria, between September 2019 and February 2020. Urine and high vaginal swab (HVS) samples were collected from each participant, resulting in a total of 400 samples. Sociodemographic data were obtained through structured questionnaires. All samples were analyzed microbiologically, and the isolated microorganisms were subjected to standardized antibiotic and antifungal susceptibility testing. Results: Candida albicans were detected in 95 (47.5%) and 86 (43%) urine and HVS samples of FSWs, with no significant difference between the two samples. Three bacteria genera and a protozoan co-infection with C. albicans: Klebsiella-Candida, Staphylococcus-Candida, Candida-Proteus, and Trichomonas vaginalis-Candida, respectively. Risk factors like age, sexual, and hygiene behaviour predispose to vulvovaginal candidiasis. Amphotericin-B was the most effective antifungal drug against C. albicans, followed by Caspofungin and Fluconazole. Metronidazole was effective against all the T. vaginalis recovered, while Ciprofloxacin was the most effective against all bacterial strains. Conclusion: The study found a high prevalence of Candida albicans in urine and HVS samples of FSWs. Over 80% of C. albicans were susceptible to Amphotericin-B, Caspofungin, and Fluconazole, while Ciprofloxacin and Metronidazole were effective for co-infected bacterial and protozoan agents, respectively. FSWs pose a high risk for sexually transmitted infections and bacterial vaginosis transmission.

Fosfomycin susceptibility testing and resistance mechanisms in Enterobacterales in South Africa.

Fosfomycin treatment of urinary tract infections is increasingly attractive due to escalating antibiotic resistance rates among urinary pathogens. Standard antibiotic susceptibility testing methods perform poorly for fosfomycin as there is poor correlation between susceptibility results and clinical outcomes in urinary pathogens other than Escherichia coli. We evaluated the performance of fosfomycin susceptibility testing in E. coli and Klebsiella pneumoniae to determine whether fosfomycin susceptibility is associated with molecular resistance mechanisms. Forty-six each of E. coli and K. pneumoniae clinical isolates were obtained from a tertiary hospital in South Africa, from 01 June 2017 to 31 January 2018. Agar dilution, disk diffusion, and gradient diffusion were performed and interpreted using the Clinical Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing guidelines. Molecular resistance mechanisms were identified by whole genome sequence analysis. Disk diffusion and gradient diffusion were accurate alternatives for fosfomycin susceptibility testing in E. coli (98% categorical agreement), but not in K. pneumoniae (47% categorical agreement). All E. coli isolates contained at least one resistance mechanism, but only one isolate with a fosA gene was resistant. In K. pneumoniae, 63% (29/46) and 70% (32/46) of isolates were susceptible to fosfomycin, using Clinical Laboratory Standards Institute and European Committee on Antimicrobial Susceptibility Testing breakpoints, respectively, despite all isolates containing a fosA gene and a uhpT mutation. A better understanding of fosfomycin susceptibility and improved antibiotic susceptibility testing tools could improve diagnostic capability and clinical guidelines for fosfomycin treatment of urinary tract infections. This study highlights the importance of adhering to interpretive guidelines when performing antimicrobial susceptibility testing and the need for simplified, accurate and standardised susceptibility testing methodology and interpretation for fosfomycin in Enterobacterales organisms.

Performance of ePlex® blood culture identification panels in clinical isolates and characterization of antimicrobial stewardship opportunities

We assessed the performance of GenMark's ePlex® Blood Culture Identification (BCID) Panels for overall agreement of organism identification and resistance mechanism detection with standard microbiologic methods. This study included patients with a positive blood culture from May 2020 to January 2021. The primary outcomes were to assess concordance of ePlex® organism identification with standard identification methods and concordance of ePlex® genotypic resistance mechanism detection with standard phenotypic susceptibility testing. Secondary outcomes included panel specific performance and characterization of antimicrobial stewardship opportunities. The overall identification concordance rate in 1276 positive blood cultures was 98.1%. The overall concordance for the presence of resistance markers was 98.2% and concordance for the absence of resistance markers was 100%. A majority of ePlex® results (69.5%) represented opportunities for potential antimicrobial stewardship intervention. High concordance rates between the ePlex® BCID panels and standard identification and susceptibility methods enable utilization of results to guide rapid antimicrobial optimization.

Recommendation of a standardized broth microdilution method for antimicrobial susceptibility testing of Avibacterium paragallinarum and resistance monitoring.

Avibacterium paragallinarum is an important pathogen in veterinary medicine that causes infectious coryza in chickens. Since antibiotics are often used for treatment and resistance of the pathogen is known, targeted therapy should be given after resistance testing of the pathogen. Unfortunately, there is currently no accepted method in standards that allows susceptibility testing of this fastidious pathogen. Therefore, we have worked out a method that allows harmonized susceptibility testing of the pathogen. The method meets the requirements of the CLSI and could be used by diagnostic laboratories.

Relative quantification of the recA gene for antimicrobial susceptibility testing in response to ciprofloxacin for pathogens of concern

Antimicrobial resistance (AR) is one of the greatest threats to global health and is associated with higher treatment costs, longer hospital stays, and increased mortality. Current gold standard antimicrobial susceptibility tests (AST) rely on organism growth rates that result in prolonged time-to-answer for slow growing organisms. Changes in the cellular transcriptome can be rapid in the presence of stressors such as antibiotic pressure, providing the opportunity to develop AST towards transcriptomic signatures. Here, we show that relative quantification of the recA gene is an indicator of pathogen susceptibly when select species are challenged with relevant concentrations of ciprofloxacin. We demonstrate that ciprofloxacin susceptible strains of Y. pestis and B. anthracis have significant increases in relative recA gene expression after 15 min of exposure while resistant strains show no significant differences. Building upon this data, we designed and optimized seven duplex RT-qPCR assays targeting the recA and 16S rRNA gene, response and housekeeping genes, respectively, for multiple biothreat and ESKAPE pathogens. Final evaluation of all seven duplex assays tested against 124 ciprofloxacin susceptible and resistant strains, including Tier 1 pathogens, demonstrated an overall categorical agreement compared to microbroth dilution of 97% using a defined cutoff. Testing pathogen strains commonly associated with urinary tract infections in contrived mock sample sets demonstrated an overall categorical agreement of 96%. These data indicate relative quantification of a single highly conserved gene accurately determines susceptibility for multiple bacterial species in response to ciprofloxacin.