Standard Antibiotic Susceptibility Testing Research Articles

Label-free single-cell antimicrobial susceptibility testing in droplets with concentration gradient generation.

Bacterial communities exhibit significant heterogeneity, resulting in the emergence of specialized phenotypes that can withstand antibiotic exposure. Unfortunately, the existence of subpopulations resistant to antibiotics often goes unnoticed during treatment initiation. Thus, it is crucial to consider the concept of single-cell antibiotic susceptibility testing (AST) to tackle bacterial infections. Nevertheless, its practical application in clinical settings is hindered by its inability to conduct AST efficiently across a wide range of antibiotics and concentrations. This study introduces a droplet-based microfluidic platform designed for rapid single-cell AST by creating an antibiotic concentration gradient. The advantage of a microfluidic platform is achieved by executing bacteria and antibiotic mixing, cell encapsulation, incubation, and enumeration of bacteria in a seamless workflow, facilitating susceptibility testing of each antibiotic. Firstly, we demonstrate the rapid determination of minimum inhibitory concentration (MIC) of several antibiotics with Gram-negative E. coli and Gram-positive S. aureus, which enables us to bypass the time-consuming bacteria cultivation, speeding up the AST in 3 h from 1 to 2 days of conventional methods. Additionally, we assess 10 clinical isolates including methicillin-resistant Staphylococcus aureus (MRSA) and multidrug-resistant Staphylococcus aureus (MDRSA) against clinically important antibiotics for analyzing the MIC, compared to the gold standard AST method from the United States Clinical and Laboratory Standards Institute (CLSI), which becomes available only after 48 h. Furthermore, by monitoring single cells within individual droplets, we have found a spectrum of resistance levels among genetically identical cells, revealing phenotypic heterogeneity within isogenic populations. This discovery not only advances clinical diagnostics and treatment strategies but also significantly contributes to the field of antibiotic stewardship, underlining the importance of our approach in addressing bacterial resistance.

Platinum nanoparticles-based electrochemical H2O2 sensor for rapid antibiotic susceptibility testing

With the increase of antimicrobial resistance, rapid antibiotic susceptibility testing (AST) to guide precise antibiotic administration has become increasingly important. However, current gold standard AST approaches tend to take up to 24–48 h. In this work, based on the nature of catalase-positive bacteria decomposing H2O2, we developed a rapid, portable, straightforward, and cost-effective phenotypic AST approach by detecting residual H2O2 using a Pt nanoparticles-based electrochemical sensor. The pulse current of the sensor exhibited a linear increase with rising H2O2 concentration, demonstrating a high sensitivity of ∼382.2 μA cm−2 mM−1. This approach showed superb diagnostic performance, with an area under the curve of 1 for 24 clinical samples of Escherichia coli and Staphylococcus aureus, with a total detection time of 60 and 45 min, respectively. Furthermore, the performance of the sensor showed no degradation even after 100 detections, promising a substantial reduction in AST costs. Overall, the proposed approach exhibited immense potential for diagnosing bacterial antibiotic resistance.

Evaluation of Direct Antimicrobial Susceptibility Testing of Gram-Negative Bacilli and Staphylococcus aureus from Positive Pediatric Blood Culture Bottles Using BD Phoenix M50.

Bloodstream infections (BSIs) are life-threatening infections for which a timely initiation of appropriate antimicrobial therapy is critical. Antibiotic susceptibility testing (AST) directly performed on positive blood culture broths can help initiate targeted antibiotic therapy sooner than the standard AST performed on colonies isolated on solid media after overnight incubation. Faster antimicrobial susceptibility testing (AST) results can improve clinical outcomes, and reduce broad-spectrum antimicrobial consumption and healthcare-associated costs in sepsis. In this study, we evaluated the accuracy of a direct AST inoculation method on the BD Phoenix M50 system using serum separator tubes to harvest bacteria from positive pediatric blood culture bottles. Direct AST was performed on 132 monomicrobial pediatric blood culture bottles that were positive for Enterobacterales (65; 49.2%), Staphylococcus aureus (46; 34.8%), and non-fermenting Gram-negative bacilli (21; 16%). Overall, the categorical and essential agreements between the direct method and standard method were 99.6% and 99.8%, respectively. Very major, major, and minor error rates were 0.1%, 0.09%, and 0.20% respectively. Direct AST performed on pediatric blood culture bottles using BD Phoenix M50 can quickly provide accurate susceptibility information to guide antimicrobial therapy in patients with BSI.

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.

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.

High prevalence of heteroresistance in Staphylococcus aureus is caused by a multitude of mutations in core genes.

Heteroresistance (HR) is an enigmatic phenotype where, in a main population of susceptible cells, small subpopulations of resistant cells exist. This is a cause for concern, as this small subpopulation is difficult to detect by standard antibiotic susceptibility tests, and upon antibiotic exposure the resistant subpopulation may increase in frequency and potentially lead to treatment complications or failure. Here, we determined the prevalence and mechanisms of HR for 40 clinical Staphylococcus aureus isolates, against 6 clinically important antibiotics: daptomycin, gentamicin, linezolid, oxacillin, teicoplanin, and vancomycin. High frequencies of HR were observed for gentamicin (69.2%), oxacillin (27%), daptomycin (25.6%), and teicoplanin (15.4%) while none of the isolates showed HR toward linezolid or vancomycin. Point mutations in various chromosomal core genes, including those involved in membrane and peptidoglycan/teichoic acid biosynthesis and transport, tRNA charging, menaquinone and chorismite biosynthesis and cyclic-di-AMP biosynthesis, were the mechanisms responsible for generating the resistant subpopulations. This finding is in contrast to gram-negative bacteria, where increased copy number of bona fide resistance genes via tandem gene amplification is the most prevalent mechanism. This difference can be explained by the observation that S. aureus has a low content of resistance genes and absence of the repeat sequences that allow tandem gene amplification of these genes as compared to gram-negative species.

THE PERSPECTIVE OF N-CHLOROTAURINE INFLUENCE ON MULTI-DRUF RESISTANT MICROORGANISMS ASSOCIATED WITH DENTAL IMPLANTATION FAILURE

Introduction: Microorganisms colonizing the surface of dental implants can form biofilms that protect their microcolonies from mechanical impact, antimicrobial compounds, and immune factors. Prophylactic use of antibiotics during dental implantation can exert selective pressure on the development of antimicrobial resistance. In this context, N-chlorotaurine (NCT) is a promising compound to combat resistant agents.
 The aim is to investigate the efficacy of NCT against pathogens associated with dental implant complications.
 Materials and methods. A buffered 1% NCT solution adjusted to pH 7.1 was used for the study. Multidrug-resistant cultures (MDR) were identified. Determination of NCT activity was carried out by the agar and suspension method.
 The results. After standard antibiotic susceptibility testing, cultures classified as MDR were selected. 12 cultures were included in the MDR group, including seven S. aureus, three K. pneumoniae and three A. baumannii. MDR cultures of S. aureus had MRSA profile. Production of carbapenemases was confirmed in all MDR K. pneumoniae isolates, 2 of them showed production of metallo-β-lactamases. MDR isolates A. baumannii were also producers of metallo-β-lactamases. All 12 investigated MDR cultures were sensitive to NCT, the diameter of the zones of growth retardation was at least 15 mm. No significant difference was found between the effectiveness of 1% NCT solution against microorganisms with different mechanisms of drug resistance (t-test, p>0.05). When conducting studies in a liquid environment, a 1% NCT solution showed similar results, all MDR cultures were sensitive and showed a decrease in the microbial load (CFU/ml) by at least 2 log10 steps already 15 minutes after the start of the study. Notably, there was no significant difference in the efficacy of NCT against conditionally susceptible and MDR agents.
 Conclusions. Reducing the threat and burden of MDR pathogens is one of the goals announced by WHO and the Centres for Disease Control. Our study clearly confirms that various antibiotic resistance mechanisms do not affect the bactericidal activity of NCT, making it promising for the treatment of resistant infections.

Antimicrobial Susceptibility Pattern of Bacterial Isolates in Patients with Lower Respiratory Tract Infections

Lower respiratory tract infection (LRI) is one of the global public health problems accounting for millions of deathsannually. The present study aim is to determine bacterial pathogens' bacteriological profile and antibiotic susceptibility patternaffecting the patients with LRI attending a tertiary care hospital. Two hundred and eight sputum samples were collected frompatients with signs and symptoms of LRI. The samples were processed in Microbiology laboratory for bacterial pathogens.Bacterial isolates were identified using standard microbiological techniques and antibiotic susceptibility testing was performedusing the Kirby-Bauer disc diffusion method. Klebsiella pneumoniae (39.6%), followed by Pseudomonas (32%), were the mostpredominant bacteria isolated in the present study. The other bacteria isolated were Escherichia coli (16.9%), Staphylococcusaureus (5.6%), Streptococcus species (1.8%), and Acinetobacter (3.7%). Gram-negative bacteria showed high drug resistance toampicillin and cephalosporins and maximum susceptibility to piperacillin-tazobactam, amikacin, and gentamicin. Most of theisolates showed resistance to the Beta-lactam group of antibiotics. The isolated bacterial pathogens' resistance profile indicatesthe need for the appropriate diagnosis and antibiotic susceptibility testing before managing the cases.

A novel inductively coupled capacitor wireless sensor system for rapid antibiotic susceptibility testing

BackgroundThe increasing prevalence and severity of antimicrobial resistance (AMR) present a major challenge to our healthcare system. Rapid detection of AMR is essential for lifesaving under emergent conditions such as sepsis. The current gold standard phenotypic antibiotic susceptibility testing (AST) takes more than a day to obtain results. Genotypic ASTs are faster (hours) in detecting the presence of resistance genes but require specific probes/knowledge of each AMR gene and do not provide specific information at the phenotype level. To address this unmet challenge, we developed a new rapid phenotypic AST.ResultWe designed a new electrochemical biosensor based on the concept of magnetically coupled LC sensors. The engineered LC sensors can be placed in 96-well plates and communicate the reading remotely with a receiver coil for signal analysis. The sensors were validated by monitoring the growth of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa in the presence and absence of different antibiotics. Drug-resistant strains were used as controls. Bacterial growth was detected within 30 min after inoculation, allowing rapid determination of antibiotic susceptibility at the phenotype level. The sensor also functions in the presence of host proteins when tested with 2% FBS in growth media.ConclusionsWith the compatibility with 96-well plates, this label-free rapid 30-min AST has the potential for low-cost applications with simple integration into the existing workflow in clinical settings.

Identifying macroplastic pathobiomes and antibiotic resistance in a subtropical fish farm

Macroplastics are ubiquitous in aquaculture ecosystems. However, to date the potential role of plastics as a support for bacterial biofilm that can include potential human pathogenic bacteria (PHPB) and antibiotic-resistant bacteria (ARB) has been largely overlooked. In this study, we used a combination of metabarcoding and standard antibiotic susceptibility testing to study the pathobiome and resistome of macroplastics, fish guts and the environment in a marine aquaculture farm in Mauritius. Aquaculture macroplastics were found to be higher in PHPB, dominated by the Vibrionaceae family (0.34 % of the total community), compared with environmental samples. Moreover, isolates from aquaculture plastics showed higher significant multiple antibiotic resistance (MAR) compared to non-plastic samples of seawater, sediment and fish guts. These results suggest that plastics act as a reservoir and fomite of PHPB and ARB in aquaculture, potentially threatening the health of farmed fish and human consumers.

P10 Impact of miniaturization and early readout on antimicrobial susceptibility tests

Abstract Background New technologies offer the promise of faster, more accessible antibiotic susceptibility testing. UK policy includes the development and introduction of innovative diagnostics to tackle antimicrobial resistance, and whilst many new technologies are emerging, we still have limited evidence of their performance. Many of these emerging technologies are changing two core aspects of antimicrobial susceptibility tests (AST). Firstly, using microfluidic platforms to miniaturize standard antibiotic susceptibility testing and secondly measuring susceptibility at earlier timepoints, here, we report a comprehensive assessment of 1-mL volume broth microdilution with an early endpoint readout, proving that miniaturization and earlier timepoints of conventional antibiotic susceptibility testing is feasible and accurate. Methods The test measures bacterial growth in 1-mL capillaries in the presence of the metabolic dye resazurin. All other aspects of the test remain standard. Images of the capillaries are taken every 5 minutes over 18 h to determine accuracy at different timepoints during incubation at 37°C. Over a total of 3 sites in the UK, Spain and Thailand, we have tested 367 isolates from clinical samples for 8–17 antibiotics. Results Preliminary data shows that for 99 Gram-negative isolates tested in Thailand, after 6-h incubation, accuracy for antibiotics is as follows: cefotaxime 96%, ceftazidime 92%, meropenem 93%, amikacin 94%, amoxicillin–clavulanic acid 79%, cefuroxime 97%, cefoxitin 80%, ciprofloxacin 84%, gentamicin 93% and trimethoprim 97%. Further testing is ongoing, and isolates from urine samples are currently being collected from Basingstoke and North Hampshire hospital to expand the type of organism tested. Conclusions Unlike many microfluidic systems, the dip and test microcapillary–based broth microdilution test is manufactured in bulk, allowing rapid and testing of large sample sets with minimal investment. An understanding of the effect of miniaturization is vital to developers of microfluidic phenotypic antibiotic susceptibility tests and can provide confidence in emerging systems.

Prevalence, Antibiotic Resistance and Associated Factors of Neisseria gonorrhoeae Among Patients Attending Non-Profitable Private Clinics in Mekelle, Tigrai, Ethiopia.

Globally, Neisseria gonorrhoeae is the second most common cause of bacterial sexually transmitted diseases. The prominent predicament of this bacterium is its complications, non-susceptibility for many drugs, and aggravated transmission of other sexually transmitted infections. There is limited information about the prevalence, antibiotic resistance, and risk factors of N. gonorrhoeae in Tigrai, Ethiopia. Therefore, we aimed to determine the prevalence, antibiotic resistance, and risk factors of N. gonorrhoeae among patients attending non-profitable private clinics in Mekelle, Tigrai, Ethiopia. A cross-sectional study among 229 patients was conducted from February to June 2018. The socio-demographic data and associated factors were gathered using structured questionnaire, and swabs were taken from urethra and cervix of males and females, respectively. Specimens were inoculated on standard bacteriological culture media and antibiotic susceptibility testing was performed using Kirby-Bauer disc diffusion technique following the Clinical and Laboratory Standard Institute. Data were analyzed using Statistical Package for Social Sciences Version 21. The level of significance at p-value <0.05 was considered statistically significant. The overall prevalence of N. gonorrhoeae was 23 (10.04%). High prevalence rates of N. gonorrhoeae were observed in females, urban residents and married ones. N. gonorrhoeae had shown statistically significant association with HIV positive, previous history of STIs, shisha users, Khat (Catha edulis) users, condom non-users and having more than two sexual partners. All isolates showed resistance to penicillin followed by tetracycline 16 (69.6%) and ciprofloxacin 8 (34.8%). Four isolates (7.4%) exhibited resistance to azithromycin with no resistance to ceftriaxone. Twelve (52.2%) isolates showed multidrug resistance (MDR). The prevalence of N. gonorrhoeae and drug resistance, including multidrug resistance, was high in the study. Multiple factors were associated with the acquisition of N. gonorrhoeae. Therefore, behavioral change and communication should be strengthened.

Antimicrobial susceptibility pattern of an emerging multidrug resistant &lt;em&gt;Acinetobacter baumannii &lt;/em&gt;from intensive care units of a tertiary care hospital

Introduction: Acinetobacter baumannii is one of the most challenging gram negative, lactose non-lactose fermenting bacilli with ability to cause nosocomial infections and develop resistance to many classes of antibiotics. Multidrug-resistant (MDR) A. baumannii is a well known agent causing life-threatening infections with limited therapeutic options. In recent years, the numbers of reported carbapenem-resistant Acinetobacter spp. are increasing, which has further narrowed the treatment options and emerging as extensively drug resistant (XDR) strain. There are only a few effective drugs such as colistin, polymyxin B, and tigecycline. But given the side effects and cost effectiveness, treatment of resistant nosocomial infections by A. baumanii is the biggest challenge for clinicians and policy makers all over the world.Aim:1. To know the antibiotic susceptibility pattern of Acinetobacter baumnii2. To know the prevalence of multidrug resistant Acinetobacter baumanii in critical areas of our tertiary care centre.Methodology: Clinical samples from both critical and non critical areas were processed routinely. Only non lactose fermenting colonies were included for the study and Acinetobacter baumanii isolates were identified using standard culture method and antibiotic susceptibility testing done by disk diffusion method.Results: Of 308 NLFs, 160 were Acinetobacter baumanii. 106 (66.25%) samples were from ICU and 54 (33.75%) from non critical areas. Commonest sample was endotracheal secretion 72(45%) followed by blood 38(23.75) and others. 70% of our isolates were resistant to Aminoglycosides. Among Fluoroquinolones, maximum resistance was shown for Ciprofloxacin. 59% of the isolates were resistant to Piperacillin-tazobactum and their resistance to Cephalosporins ranged from 60-80%. 42 (39.62% of our isolates were Multidrug resistant (MDR) and 19 (17.92%) were extensively drug resistant (XDR).Conclusion: Multidrug resistant and extensively drug resistant strains of Acinetobacter baumanii are on the rise and slowly getting established in our healthcare set up especially the critical areas. Treatment options are very few and limited. Thus leaving us with call to take stringent measures to stop the inadvertent use of antibiotics and to review our infection control policies.

Antibiotic-resistant pathogenic bacterial isolates from patients attending the outpatient department of university of Cape Coast hospital, Ghana: A retrospective study between 2013-2015.

Uropathogenic Escherichia coli (E. coli) is an important urinary tract infection (UTI) that has been associated with both complicated and uncomplicated disease conditions. The global emergence of multiple drug-resistant (MDR) and extended-spectrum β-lactamase (ESBL) is of public health concern as the resistance limits the current treatment options. The objective of this study was to analyze the antibiotic-resistant patterns among the uropathogenic E. coli isolates at the University of Cape Coast (UCC) hospital between 2013 and 2015 as baseline data to understand the current antibiotic resistance situation within UCC and its environs. A retrospective cross-sectional study of bacteria isolates at UCC hospital from January 2013 to December 2015 were analyzed. A standard biochemical and antibiotic susceptibility tests were performed using Kirby-Bauer NCCLs modified disc diffusion technique. The network of interaction between pathogenic isolates and antibiotic resistance was performed using Cytoscape software. Statistical significance was tested using ANOVA and one-sample Wilcoxon test. The overall E. coli prevalence was 15.76% (32/203); females had the highest infection of 17.33% (26/150) compared to male subjects who had 11.32% (6/53) out of all the pathogenic infections. The E. coli prevalence among the age categories were 2/21 (9.52%), 27/154 (17.53%) and 4/21 (19.05%) among ≤20 years, 21-40 years and 41-60 years respectively. The isolated resistant pathogens exhibited different antibiotic resistance patterns. An interaction network of nodes connecting to other nodes indicating positive correlations between the pathogens and antibiotic resistance was established. Escherichia coli, Citrobacter spp, Klebsiella spp among other isolated pathogens formed higher centrality in the network of interaction with antibiotic resistance. The individual E. coli isolates showed a significant difference in the mean ± SD (95% CI) pattern of antibiotic resistance, 2.409±1.205 (1.828-2.990), χ2 = 36.68, p<0.0001. In conclusion, the study reports the interaction of E. coli isolates at UCC hospital and its antibiotic-resistant status between 2013 and 2015. This data forms the baseline information for assessing the current antibiotic status in UCC and its environs.

Investigating Unfavorable Factors That Impede MALDI-TOF-Based AI in Predicting Antibiotic Resistance.

The combination of 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 testing (AST) of Staphylococcus aureus. Based on the AI predictive probability, cases with probabilities between the low and high cut-offs are defined as being in the “grey zone”. We aimed to investigate the underlying reasons of unconfident (grey zone) or wrong predictive AST. In total, 479 S. aureus isolates were collected and analyzed by MALDI-TOF, and AST prediction and standard AST were obtained in a tertiary medical center. The predictions were categorized as 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 methicillin-resistant S. aureus (MRSA), a larger cefoxitin zone size was found in the wrong-prediction group. Multilocus sequence typing of the MRSA isolates in the grey-zone group revealed that uncommon strain types comprised 80%. Of the methicillin-susceptible S. aureus (MSSA) isolates in the grey-zone group, the majority (60%) comprised over 10 different strain types. In predicting AST based on MALDI-TOF AI, uncommon strains and high diversity contribute to suboptimal predictive performance.

New update on molecular diversity of clinical Staphylococcus aureus isolates in Iran: antimicrobial resistance, adhesion and virulence factors, biofilm formation and SCCmec typing.

Staphylococcus aureus is often considered as a potential pathogen and resistant to a wide range of antibiotics. The pathogenicity of this bacterium is due to the presence of multiple virulence factors and the ability to form biofilm. SCCmec types I, II and III are mainly attributed to HA-MRSA, while SCCmec types IV and V have usually been reported in CA-MRSA infections. In this study, we performed a cross-sectional study to determine the antimicrobial resistance, adhesion and virulence factors, biofilm formation and SCCmec typing of clinical S. aureus isolates in Iran. S. aureus isolates were identified using microbiological standard methods and antibiotic susceptibility tests were performed as described by the Clinical and Laboratory Standards Institute (CLSI) guidelines. Inducible resistance phenotype and biofilm formation were determined using D-test and tissue culture plate methods, respectively. Multiplex-PCRs were performed to detect adhesion and virulence factors, antibiotic resistance genes, biofilm formation and SCCmec typing by specific primers. Among 143 clinical samples, 67.8% were identified as MRSA. All isolates were susceptible to vancomycin. The prevalence of cMLSB, iMLSB and MS phenotypes were 61.1%, 22.2% and 14.8%, respectively. The TCP method revealed that 71.3% of isolates were able to form biofilm. The predominant virulence and inducible resistance genes in both MRSA and MSSA isolates were related to sea and ermC respectively. SCCmec type III was the predominant type. Data show the high prevalence rates of virulence elements among S. aureus isolates, especially MRSA strains. This result might be attributed to antibiotic pressure, facilitating clonal selection.

A Study on bacteriological profile and the antibiotic susceptibility pattern in cases of chronic suppurative otitis media in Haroti region

Introduction: Chronic suppurative otitis media (CSOM) is one of the common hearing problems which can cause many complications if not treated properly. Hence, the study aims to identify the bacterial isolates causing CSOM and to study their antimicrobial susceptibility pattern. Materials and Methods: The study was carried out in a tertiary care hospital in Kota from May 2019 to January 2020. Three hundred and seventy patients of CSOM with discharge attending the ENT outpatient department were included in the study. Swabs were taken and sent immediately to the microbiology laboratory and processed. Bacterial isolates were identified using standard methods and antibiotic susceptibility testing was done. Results: Pseudomonas was the most predominant bacteria isolated followed by Staphylococcus aureus, Coagulase-negative Staphylococcus, Enterobacter spp., Proteus spp., Streptococcus pneumonia, Klebsiella spp., Enterococcus and Acinetobacter spp. Conclusions: The present study helped in knowing the bacteriological profile of the cases of CSOM. Empirical treatment may not be successful in all cases because of the emergence of resistant organisms. Hence, it is advisable to do an antibiogram before starting the treatment.

Miniaturised broth microdilution for simplified antibiotic susceptibility testing of Gram negative clinical isolates using microcapillary devices.

Antibiotic resistance is a major global challenge. Although microfluidic antibiotic susceptibility tests (AST) offer great potential for rapid and portable testing to inform correct antibiotic selection, the impact of miniaturisation on broth microdilution (BMD) is not fully understood. We developed a 10-plex microcapillary based broth microdilution using resazurin as a colorimetric indicator for bacterial growth. Each capillary had a 1 microlitre capillary volume, 100 times smaller than microplate broth microdilution. The microcapillary BMD was compared to an in-house standard microplate AST and commercial Vitek 2 system. When tested with 25 uropathogenic isolates (20 Escherichia coli and 5 Klebsiella pneumoniae) and 2 reference E. coli, these devices gave 96.1% (441/459 isolate/antibiotic combinations) categorical agreement, across 17 therapeutically beneficial antibiotics, compared to in-house microplate BMD with resazurin. A further 99 (50 E. coli and 49 K. pneumoniae) clinical isolates were tested against 10 antibiotics and showed 92.3% categorical agreement (914/990 isolate/antibiotic combinations) compared to the Vitek 2 measurements. These microcapillary tests showed excellent analytical agreement with existing AST methods. Furthermore, the small size and simple colour change can be recorded using a smartphone camera or it is feasible to follow growth kinetics using very simple, low-cost readers. The test strips used here are produced in large batches, allowing hundreds of multiplex tests to be made and tested rapidly. Demonstrating performance of miniaturised broth microdilution with clinical isolates paves the way for wider use of microfluidic AST.

English

BACKGROUND Urinary tract infection (UTI) is a very important reason for increased hospital stay, increased morbidity and mortality in the hospital setting. Annually, about 150 million people are diagnosed with UTI worldwide.1The fluoroquinolones have assumed an important role in the therapy of UTI. But the prevalence of urinary tract infections caused by fluoroquinolone-resistant bacteria has been increasing over the years. The purpose of this study was to evaluate fluoroquinolone resistance pattern among the bacterial pathogens causing urinary tract infections. METHODS This is a cross-sectional descriptive study. A total of 200 urine samples were collected from clinically suspected UTI patients, attending out-patient department (OPDs) and were admitted to various wards of our hospital. The samples were inoculated using the standard loop, incubated aerobically, isolates were identified by standard biochemical reactions and antibiotic susceptibility testing was performed. RESULTS Out of 200 samples, the most common organism isolated was Escherichia coli (57 %), followed by Klebsiella pneumoniae subsp pneumonia (27 %). 93.9 % of Escherichia coli and 100 % of Klebsiella pneumoniae subsp pneumoniae isolates were resistant to ampicillin. The present study also documented high level of fluoroquinolone resistance among almost all common uropathogens. Among Escherichia coli and Klebsiella pneumoniae subsp pneumoniae; ciprofloxacin resistance was 77.2 % and 85.2 % respectively. Quinolone resistance was found to be more common in hospitalised patients and catheterized patients. According to our study, although levofloxacin is far effective than ciprofloxacin for uropathogens, the resistance to both are increasing. CONCLUSIONS The results presented in this study indicate that it is time to reconsider the frequent use of fluoroquinolones. Use of fluoroquinolones to treat patients before getting the sensitivity pattern of the isolate should be stopped. It is mandatory to formulate antimicrobial policy according to the local resistance pattern. Resistance pattern must be assessed in hospital and laboratory-based surveillance studies. KEY WORDS Urinary Tract Infections, Escherichia coli, Antibiotic Sensitivity, Fluoroquinolones

Bicarbonate modulates delafloxacin activity against MDR Staphylococcus aureus and Pseudomonas aeruginosa.

To investigate the utility of recently approved delafloxacin and other fluoroquinolones against leading MDR bacterial pathogens under physiologically relevant conditions. MIC and MBC assays were conducted for MDR strains of Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii and Klebsiella pneumoniae in the standard antibiotic susceptibility testing medium CAMHB, amended Roswell-Park Memorial Institute tissue culture medium (RPMI+) or 20% fresh human whole blood. In vivo correlation of in vitro findings was performed in a murine P. aeruginosa pneumonia model. Mechanistic bases for the findings were explored by altering media conditions and with established fluoroquinolone accumulation assays. Fluoroquinolone MICs were increased in RPMI+ compared with CAMHB for all four MDR pathogens. Specifically, delafloxacin MICs were increased 32-fold versus MDR S. aureus and 8-fold versus MDR P. aeruginosa. MBC assays in 20% human whole blood and a murine MDR P. aeruginosa pneumonia model both confirmed that delafloxacin activity was reduced under physiological conditions. Bicarbonate (HCO3-), a key component of host physiology found in RPMI+ but absent from CAMHB, dictated delafloxacin susceptibility in CAMHB and RPMI+ by impairing its intracellular accumulation. Standard in vitro antibiotic susceptibility testing conditions overpredicted the effectiveness of delafloxacin against MDR pathogens by failing to capture the role of the biological buffer HCO3- to impair delafloxacin accumulation. This work showcases limitations of our current antibiotic susceptibility testing paradigm and highlights the importance of understanding host microenvironmental conditions that impact true clinical efficacy.