Detection of pathogenic gram-negative bacteria using an antimicrobial peptides-modified bipolar electrode-electrochemiluminescence platform.

Real-time, label-free detection of gram-negative bacteria with high selectivity and sensitivity is demonstrated using a bipolar electrode-electrochemiluminescence (BPE-ECL) platform. This platform utilizes anode luminescence and cathode modification of antimicrobial peptides (AMPs) to effectively capture bacteria. Magainin I, basic AMP from Xenopus skin, boasting an α-helix structure, exhibits a preferential affinity for the surface of gram-negative pathogens. The covalent attachment of the peptide's C-terminal carboxylic acid to the free amines of a previously thiolated linker ensures its secure immobilization onto the surface of the interdigitated gold-plated cathode of BPE. The AMP-modified BPE sensor, when exposed to varying concentrations of gram-negative bacteria, produces reproducible ECL intensities, allowing for the detection of peptide-bacteria interactions within the range 1 to 104 CFU mL-1. Furthermore, this AMP-modified BPE sensor demonstrates a selective capacity to detect Escherichia coli O157:H7 amidst other gram-negative strains, even at a concentration of 1-CFU mL-1. This study underscores the high selectivity of Magainin I in bacterial detection, and the AMP-modified BPE-ECL system holds significant promise for rapid detection of gram-negative bacteria in various applications. The AMP-modified BPE sensor generated reproducible ECL intensity that detected peptide-bacteria interactions in the range 1 to 104 CFU mL-1. The AMP-modified BPE sensor also selectively detected E. coli O157:H7 from other gram-negative strains at a concentration of 1-CFU mL-1. In this paper, AMP demonstrated high selectivity in bacterial detection. The AMP-modified BPE-ECL system prepared has a great potential for application in the field of rapid detection of gram-negative bacteria.

The epidemiology of gram-negative bacteremia in Lebanon: a study in four hospitals.

INTRODUCTION: Gram-negative bacteremia is a life-threatening infection with high morbidity and mortality. Its incidence is rising worldwide, and treatment has become more challenging due to emerging bacterial resistance. Little data is available on the burden and outcome of such infections in Lebanon. METHODS: We conducted this retrospective study in four Lebanese hospitals. Data on medical conditions and demographics of 2400 patients diagnosed with a bloodstream infection based on a positive blood culture were collected between January 2014 and December 2020. RESULTS: Most bacteremias were caused by Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter baumannii, with the more resistant organisms being hospital-acquired. Third-generation cephalosporin and quinolone resistance was steady throughout the study, but carbapenem resistance increased. Mortality with such infections is high, but carbapenem resistance or infection with Pseudomonas or Acinetobacter species were significant risk factors for poor outcomes. CONCLUSION: This is the first multi-center study from Lebanon on gram-negative bacteremia, resistance patterns, and factors associated with a poor outcome. More surveillance is needed to provide data to guide empirical treatment for bacteremia in Lebanon.

First report of carbapenems encoding multidrug-resistant gram-negative bacteria from a pediatric hospital in Gaza Strip, Palestine.

BACKGROUND: The worldwide prevalence of multi-drug resistance (MDR) in Gram-negative bacteria (GNB), particularly related to extended-spectrum beta-lactamases (ESBLs) and carbapenemases, poses significant global public health and clinical challenges. OBJECTIVES: To characterize ESBL-producing Gram-negative bacilli, within a pediatric hospital in Gaza using whole genome sequencing (WGS). METHODS: A total of 158 clinical isolates of Gram-negative bacilli were collected from Al-Nasser Pediatric Hospital. These isolates were tested for ESBL production using the double disk synergy test. The antibiotic susceptibility profile was determined using the Kirby Bauer method following the Clinical and Laboratory Standard Institute guidelines. Selected 15 phenotypically MDR isolates were whole-genome sequenced and characterized for their genome-based species identity and antibiotic resistance gene profile. RESULTS: Of the 158 isolates, 93 (58.9%) were positive for ESBL production. The frequency of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Acinetobacter baumannii, Proteus mirabilis, and Serratia marcescens was 50%, 22.7%, 22.7%, 1.8%, 1.2%, and 1.2% respectively. The prevalence of ESBL among urine, pus, blood, and sputum was 64%, 44%, 23%, and 63.6%, respectively. Chloramphenicol, Imipenem, and Meropenem were the most effective antibiotics against ESBL producers. In sequenced isolates,  an average of six anti-microbial resistance (AMR) genes were noted per isolate, where one of them carried up to 13 antibiotic resistance genes. Carbapenem resistance genes such as blaKPC-2(6.6%), blaPDC-36/12 (6.6%), and blaPOM-1 (6.6%) were detected. All the sequenced E. coli isolates (n = 8) showed multiple resistance genes, mainly against ß-lactamase (25.0%), aminoglycosides (37.5%), sulfonamides (37.5%), and genes conferring resistance to tetracyclines (25.0). CONCLUSION: Our results showed a high prevalence of ESBL-producing GNB isolated from a pediatric hospital in the Gaza Strip. Various antibiotic resistance genes were identified, including those encoding ESBL and carbapenems. The results highlight the significant challenge posed by MDR in GNB and emphasize the need for effective antibiotic strategies. Given the high endemicity observed in various studies from Palestine, it is important to conduct clinical and molecular epidemiology research to identify risk factors, transmission patterns, and clinical outcomes associated with GNB strains that carry ESBL and carbapenem resistance genes.

An investigation of scattered light integrating collector technology for rapid blood culture sensitivity testing.

Introduction. Sepsis rates are increasing, with Gram-negative organisms representing a large proportion of bloodstream infections. Rapid antibiotic administration, alongside diagnostic investigations, is required for the effective management of these patients.Gap statement. Current diagnostics take ~48 h for a final report; therefore, rapid diagnostics are required.Aim. This study investigated a novel antibiotic sensitivity method, the scattered light integrating collector (SLIC), combined with a rapid identification method using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) technology to determine if an accurate identification and susceptibility result can be provided within 4 h of a positive blood culture report.Methodology. A total of 47 blood cultures containing Gram-negative bacteria from 46 patients were processed using the MALDI-TOF Biotyper Sepsityper for identification directly from the blood and the SLIC instrument for susceptibility testing. All organisms were also tested using the current standard workflow used in the host laboratory. Categorical agreement (CA), major errors (MaEs) and very major errors (VMEs) were determined.Results. SLIC produced susceptibility results with a 71.9% CA, 30.6% MaE and 17.5% VME. The median difference in time to the final result was 44.14 (43 : 05-45 : 15) h earlier compared to the current method.Conclusion. We conclude that SLIC was unable to consistently provide sufficiently accurate antibiotic susceptibility results compared to the current standard method.

Risk Factors Associated with the Colonization of Multidrug-Resistant Gram-Negative Bacteria Upon Admission to the Intensive Care Unit: A Cross-sectional Study.

BACKGROUND: Multidrug-resistant Gram-negative bacteria (MDR-GNB) are prevalent in intensive care units (ICUs), leading to increased morbidity and mortality. Limited data on MDR-GNB in Indonesia prompted this study to determine the prevalence and risk factors associated with MDR-GNB colonization, enhancing screening strategies, and acquiring phenotypic and genotypic data on these bacteria. METHODS: This analytical cross-sectional observational study included participants who met the criteria and were admitted to the ICU at Dr. Cipto Mangunkusumo Hospital from January to December 2022. We used multivariate analysis on the findings from rectal swab screening, sociodemographic, clinical, and microbiological examinations. RESULTS: Out of 108 participants, 172 cultures comprised 165 Gram-negative isolates, four yeasts, and three with no growth. The prevalence of patients colonized with MDR-GNB was 51.85% (56/108), and the prevalence of MDR-GNB isolates was 39.53% (68/172), with the most common MDR-GNB being Escherichia coli (29.65%) and Klebsiella pneumoniae (19.44%). The most resistant gene found in ESBL was CTX-M (75%), and the carbapenemase producer gene was NDM (5.88%). Risk factors associated with MDR-GNB colonization were the length of stay before admission to the ICU (p = 0.003) and a history of previous antibiotic therapy (p = 0.036). CONCLUSION: In this study, two risk factors were associated with the occurrence of MDR-GNB colonization, with the prevalence of MDR-GNB colonization in patients initially admitted to the ICU still quite high. Therefore, selecting screening patients based on risk factors at the time of initial admission to the ICU is crucial for infection control programs.

Emergence of resistance to last-resort antimicrobials in bacteremia patients: A multicenter analysis of bloodstream pathogens in Korea.

This study retrospectively reviewed the microbiological and clinical characteristics of patients diagnosed with bacteremia. Results from the first positive blood cultures were consecutively collected from July 2022 to June 2023 at a public secondary hospital, a university-affiliated tertiary hospital, and a university-affiliated secondary hospital in the Seoul metropolitan area. Antibiotic spectrum coverage (ASC) scores were calculated on the day the blood culture was performed (B0) and on two days after the blood culture results were reported (R+2). A total of 3,397 isolates were collected from 3,094 patients. Among these, 949 isolates obtained from 893 patients were classified as multidrug-resistant organisms (MDRO), including 170 imipenem-resistant gram-negative bacteria, 714 methicillin-resistant staphylococci, and 65 vancomycin-resistant enterococci. Interestingly, 13 and 42 gram-positive isolates were resistant to linezolid and quinupristin/dalfopristin, respectively. Moreover, 44 and 181 gram-negative isolates were resistant to amikacin and tigecycline, respectively. The proportion of ASC scores corresponding to broad or extremely broad-spectrum coverage was not significantly different between MDRO and non-MDRO groups at B0 (p = 0.0925). However, it increased in the MDRO group at R+2 (p <0.001). This study found that resistance to last-resort antimicrobials is emerging. Therefore, developing and incorporating molecular diagnostics using a wide range of resistance targets may facilitate rapid, tailored antimicrobial treatments.

Comparison of Zybio Kit and saponin in-house method in rapid identification of bacteria from positive blood cultures by EXS2600 matrix-assisted laser desorption ionization time-of-flight mass spectrometry system.

We evaluated the performance of Zybio EXS2600 matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Zybio Inc., Chongqing, China) for the identification of bacteria from positive blood culture (BC) bottles using Blood Culture Positive Sample Pretreatment Kit (Zybio Inc., Chongqing, China) in comparison to an in-house saponin method. Following a positive signal by the BACTEC™ FX system, confirmation of identification was achieved using subcultured growing biomass used for MALDI-TOF MS analysis. A total of 94 positive BC bottles with 97 bacterial isolates were analyzed. The overall identification rates at the genus and species levels for the saponin method were 89.7% (87/97) and 74.2% (72/97), respectively. With the Zybio Kit, 88.7% (86/97) and 80.4% (78/97) of microorganisms were correctly identified to the genus and species levels, respectively. The saponin method identified 65.3% (32/49) of Gram-positive bacteria at the species level, whereas the Zybio Kit achieved a higher species-level identification rate of 79.6% (39/49) (p = 0.1153). The saponin method with additional on-plate formic acid extraction showed a significantly higher overall identification rate in comparison to the saponin method without that step for both genus (87.6% [85/97] vs. 70.1% [68/97], p = 0.0029) and species level (70.1% [68/97] vs. 46.4% [45/97], p = 0.0008). Identification rates of Gram-negative bacteria showed a higher identification rate, however, not statistically significant with additional Zybio Kit protocol step on both genus (85.4% [41/48] vs. 81.3% [39/48], p = 0.5858) and species level (77.1% [37/48] vs. 75% [36/48], p = 0.8120). Zybio Kit could offer an advantage in species-level identification, particularly for Gram-positive bacteria. The inclusion of on-plate formic acid extraction in the saponin method notably enhanced identification at both genus and species levels for Gram-positive bacteria. The extended protocol provided by the Zybio Kit could potentially offer an advantage in the identification of Gram-negative bacteria at both genus and species levels. Enhancements to the Zybio EXS2600 MALDI-TOF instrument software database are necessary.

Antibiotic Resistance Pattern in Intensive Care Unit of a Tertiary Care Hospital, Nepal.

Background Intensive care unit (ICU) is the especial department of the hospital where critically ill patients are treated with the unique type of technologies to revert back to functional by body's own mechanism. Therefore, there are lots of external intervention with chance of getting bacterial infections. Antibiotics are medicines used to prevent and treat such bacterial infections. However, due to selective broad spectrum antibiotic pressure there is great chances to develop antimicrobial resistance at any time during hospital stay in intensive care unit. Objective To find out the antibiotic resistance pattern among Gram negative bacteria in Intensive Care Unit. Method A Descriptive cross-sectional study was conducted in Department of Microbiology of Tertiary care center for 18 months On the basis of previous sample load census method was used to include 500 sample from intensive care unit during study period. Among them only Gram negative bacteria were included in the study. All the samples were processed following standard methodology. Result Out of 500 samples, growth was observed in 451 (90.2%) samples. Among all the isolates Escherichia coli (29.6%) was predominant organism. It had shown high resistance towards Ciprofloxacin (93.5%) even in urine sample Ciprofloxacin (86.9%). Conclusion Our study showed Escherichia coli as a major organism in intensive care unit. This was resistant to commonly used oral antibiotic leaving restricted option for use of higher antibiotics. Therefore, continuous surveillance of such bacterial pathogen is warranted with implementation of effective Infection Prevention and Control measures in Health Care setting with emphasis to critical care units.

Microbiology of catheter-associated bloodstream infection: differences according to catheter type.

OBJECTIVES: Catheter-associated bloodstream infections (CABSI) cause preventable morbidity. We compared the microbiological etiology of CABSI across different types of central and peripherally-inserted catheters. METHODS: We analyzed prospectively collected CABSI data in a 2100-bed hospital network in Switzerland between 2016 and 2022. We included: short-term non-tunneled central venous catheters (CVC); long-term catheters (tunneled, or peripherally-inserted central catheters); arterial catheters; dialysis catheters; and peripheral venous catheters (PVC). We used multivariable logistic regression models to describe the risk of Staphylococcus aureus and Gram-negative pathogens according to catheter type. RESULTS: Overall, 416 CABSI episodes were included, including 60 episodes of S. aureus and 92 episodes of Gram-negative CABSI. Microbiological profiles differed between catheter types. Together, PVC and dialysis catheters accounted for 43/60 (72%) of all S. aureus CABSI. After adjusting for age, sex, and hematology/oncology care, the odds of S. aureus were higher for hemodialysis catheters (odds ratio [OR] 17.3, 95% confidence interval [CI] 5.75-52.2, P <0.01) and PVC (OR 2.96, 95% CI 1.22-7.20, P = 0.02) compared to short-term non-tunneled CVC. Odds of Gram-negative organism as the cause of CABSI were higher in long-term catheters versus short-term non-tunneled CVC (OR 2.70, 95% CI 1.37-5.24). CONCLUSIONS: CABSI in catheters other than short-term non-tunneled CVC is more commonly caused by virulent organisms including S. aureus and Gram-negative bacteria. Catheter type should be considered when selecting empirical antimicrobial therapies.

Validation of short culture method for rapid bacterial identification of blood cultures via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: Development of rapid bacterial identification from blood cultures has been an area of intense study in diagnostic microbiology. Shortened turnaround time coupled with antimicrobial stewardship interventions have been shown to improve patient outcomes and decrease healthcare-associated costs. OBJECTIVES: We report the validation of a short incubation method for Gram-positive and Gram-negative bacterial identification utilizing MALDI-TOF MS without additional instrumentation, processing or cost compared with current practice. METHODS: Prospective, observational, single-centre study in a quaternary care academic hospital encompassing 376 blood cultures subjected to bacterial identification after short incubation periods of 3-4 and 6-8 h. RESULTS: There was 97.5% species-level identification agreement with tests undertaken after 3-4 h incubation with 83.6% isolates identified, and 99.7% species-level identification agreement after 6-8 h incubation with 96.7% isolates identified. CONCLUSIONS: The short incubation method provides a rapid MALDI-TOF MS bacterial identification method, reducing turnaround time by 10-18 h compared with standard practice without additional cost, processing or instrumentation.

Evaluating the potential of vancomycin-modified magnetic beads as a tool for sample preparation in diagnostic assays.

Vancomycin-functionalized micro- or nanoparticles are frequently used for isolation and enrichment of bacteria from various samples. Theoretically, only Gram-positive organisms should adhere to the functionalized surfaces as vancomycin is an antibiotic targeting a peptidoglycan precursor in the cell wall, which in Gram-negative bacteria is shielded by the outer cell membrane. In the literature, however, it is often reported that Gram-negative bacteria also bind efficiently to the vancomycin-modified particles. The goal of our study was to identify the underlying cause for these different findings. For each species several strains, including patient isolates, were investigated, and effects such as day-to-day reproducibility, particle type, and the antimicrobial effect of vancomycin-coupled beads were explored. Overall, we found that there is a strong preference for binding Gram-positive organisms, but the specific yield is heavily influenced by the strain and experimental conditions. For Staphylococcus aureus average yields of approximately 100% were obtained. Respectively, yields of 44% for Staphylococcus cohnii, 22% for Staphylococcus warneri, 17% for Enterococcus faecalis and 5% for vancomycin-sensitive Enterococcus faecium were found. Yields for Gram-negative species (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii) and vancomycin-resistant Enterococcus faecium were below 3%. Our results indicate that the interaction between vancomycin and the D-alanine-D-alanine terminus of the peptidoglycan precursor in the bacterial cell wall is the dominant force responsible for the adherence of the bacteria to the particle surface. It needs to be considered though, that other factors, such as the specific molecules presented on the bacterial surface, as well as the pH, and the ion concentrations in the surrounding medium will also play a role, as these can lead to attractive or repulsive electrostatic forces. Last but not least, when using colony forming unit-based quantification for determining the yields, the influence of cell cluster formation and different sensitivities towards the antimicrobial effect of the vancomycin beads between species and strains needs to be considered.

Examining the prevalence and antimicrobial resistance profiles of multidrug-resistant bacterial isolates in wound infections from Indonesian patients.

The emergence of multidrug-resistant (MDR) infections in wounds is a significant public health issue. The aim of this study was to investigate the prevalence and antimicrobial resistance profiles of MDR bacterial isolates in wound infections. Through a cross-sectional study, 1,035 bacterial isolates were collected from wound infection patients at Tugurejo Hospital in Semarang, Indonesia, over a three-year period (from January 2020 to December 2022). Initial identification involved Gram staining and colony morphology assessment, followed by biochemical assays and antimicrobial susceptibility testing using the VITEK®2 Compact system. Gram-negative bacteria constituted the majority of isolates (60.77%, n=629). The predominant strains included were Staphylococcus spp. (30.92%, n=320), Escherichia coli (18.45%, n=191), and Klebsiella pneumoniae (13.04%, n=135). Notably, Gram-negative bacteria exhibited a significantly higher likelihood of MDR development compared to their Gram-positive counterparts (p<0.001), with Gram-negative bacteria having a 2.05 times higher probability of acquiring MDR. These findings underscore the urgent need for comprehensive surveillance of antimicrobial resistance patterns and the implementation of tailored antimicrobial stewardship programs to address the pressing public health challenge of MDR wound infections. Further research is warranted to elucidate the complex interplay of factors contributing to MDR development in wound infections, thereby informing targeted intervention strategies and improving patient outcomes.

Retrospective evaluation of rapid genotypic ID and phenotypic AST systems on positive blood culture turnaround time and simulated potential impacts on bloodstream infection management.

BACKGROUND: Bloodstream infections are linked to heightened morbidity and mortality rates. The consequences of delayed antibiotic treatment can be detrimental. Effective management of bacteraemia hinges on rapid antimicrobial susceptibility testing. OBJECTIVES: This retrospective study examined the influence of the VITEK® REVEAL™ Rapid AST system on positive blood culture (PBC) management in a French tertiary hospital. MATERIALS AND METHODS: Between November 2021 and March 2022, 79 Gram-negative monomicrobial PBC cases underwent testing with both VITEK®REVEAL™ and VITEK®2 systems. RESULTS: The study found that VITEK®REVEAL™ yielded better results than the standard of care, significantly shortening the time to result (7.0 h compared to 9.6 h) as well as the turnaround time (15 h compared to 31.1 h) when applied for all isolates. CONCLUSIONS: This study implies that the use of VITEK®REVEAL™ enables swift adaptations of antibiotic treatment strategies. By considerably minimizing the turnaround time, healthcare professionals can promptly make necessary adjustments to therapeutic regimens. Notably, these findings underscore the potential of VITEK®REVEAL™ in expediting appropriate antibiotic interventions, even in less ideal conditions. Further studies in varied laboratory contexts are required to validate these encouraging outcomes.

In treacherous waters: detection of colistin-resistant bacteria in water and plastic litter from a recreational estuary.

Colistin resistance poses a major therapeutic challenge and resistant strains have now been reported worldwide. However, the occurrence of such bacteria in aquatic environments is considerably less understood. This study aimed to isolate and characterize colistin-resistant strains from water and plastic litter collected in an urban recreational estuary. Altogether, 64 strains with acquired colistin resistance were identified, mainly Acinetobacter spp. and Enterobacter spp. From these, 40.6% were positive for at least one mcr variant (1-9), 26.5% harbored, extended-spectrum beta-lactamases, 23.4% harbored, sulfonamide resistance genes, and 9.3% harbored, quinolone resistance genes. merA, encoding mercury resistance, was detected in 10.5% of these strains, most of which were also strong biofilm producers. The minimum inhibitory concentration toward colistin was determined for the mcr-positive strains and ranged from 2 to ≥512 µg ml-1. Our findings suggest that Gram-negative bacteria highly resistant to a last-resort antimicrobial can be found in recreational waters and plastic litter, thereby evidencing the urgency of the One Health approach to mitigate the antimicrobial resistance crisis.

Antimicrobial resistance profile and associated factors of hospital-acquired gram-negative bacterial pathogens among hospitalized patients in northeast Ethiopia.

BACKGROUND: Antimicrobial resistance is a major global public health issue. Infections caused by resistant species are associated with higher mortality rates, longer hospital stays, medication failure, and rising medical costs. The World Health Organisation has declared multidrug resistance-associated infections as an epidemic of public health concern. OBJECTIVE: This study aimed to evaluate the antimicrobial resistance profile and associated factors of hospital-acquired Gram-negative bacterial pathogens among hospitalized patients in Northeast Ethiopia. MATERIALS AND METHODS: A health facility-based cross-sectional study was conducted among hospitalized patients from March 2021 to February 2022. About 810 clinical specimens were collected, transported, and processed from admitted patients following the standard bacteriological procedures. The clinical samples were inoculated onto blood agar, MacConkey agar, and chocolate agar. Furthermore, the species identification was done using gram reactions, colony morphology, and color and biochemical tests. Antimicrobial susceptibility tests, extended-spectrum beta-lactamase, and carbapenemase production were performed as per the clinical laboratory standard institute guidelines. For analysis, the information was entered into Epi-data and exported to SPSS. A P value of < 0.05 with a 95% confidence interval was considered as a statistically significant association. RESULTS: Out of 810 clinical specimens, 285/810 (35.2%) developed bacterial infections. From the isolated bacteria, E. coli was the predominant bacteria accounting for 78/285 (27.4%) followed by K. pneumoniae, 69/285(24.42%), whereas P. vulgaris accounted for the least, 7/285 (2.5%). Overall, 132/285 (46.3%) and 99/285 (34.7%) of culture-positive patients were infected by extended-spectrum beta-lactamase and carbapenemase-producing bacteria. The overall multidrug resistance rate of the isolated bacteria was 89.4%. The highest antibiotic resistance rates were detected for doxycycline (92.9%), amoxicillin-clavulanic acid (83.9%), and ampicillin (93%). The least antibiotic resistance rate was observed for meropenem at 41.1% and amikacin at 1.7%, respectively. CONCLUSIONS AND RECOMMENDATIONS: In the study area, significant health concerns include a range of hospital-acquired bacterial infections associated with elevated rates of multidrug resistance, Extended-spectrum beta-lactamase (ESBL), and carbapenemase-producing bacterial pathogens. Consequently, it is recommended to conduct drug-susceptibility testing of isolates and molecular detection at a national level to optimize antibiotic usage for treating prevalent bacterial infections in this area.

Increasing trends of antibiotic resistance in Uganda: analysis of the national antimicrobial resistance surveillance data, 2018-2021.

BACKGROUND: Continuous monitoring of antimicrobial resistance (AMR) in Uganda involves testing bacterial isolates from clinical samples at national and regional hospitals. Although the National Microbiology Reference Laboratory (NMRL) analyzes these isolates for official AMR surveillance data, there's limited integration into public health planning. To enhance the utilization of NMRL data to better inform drug selection and public health strategies in combating antibiotic resistance, we evaluated the trends and spatial distribution of AMR to common antibiotics used in Uganda. METHODS: We analyzed data from pathogenic bacterial isolates from blood, cerebrospinal, peritoneal, and pleural fluid from AMR surveillance data for 2018-2021. We calculated the proportions of isolates that were resistant to common antimicrobial classes. We used the chi-square test for trends to evaluate changes in AMR resistance over the study period. RESULTS: Out of 537 isolates with 15 pathogenic bacteria, 478 (89%) were from blood, 34 (6.3%) were from pleural fluid, 21 (4%) were from cerebrospinal fluid, and 4 (0.7%) were from peritoneal fluid. The most common pathogen was Staphylococcus aureus (20.1%), followed by Salmonella species (18.8%). The overall change in resistance over the four years was 63-84% for sulfonamides, fluoroquinolones macrolides (46-76%), phenicols (48-71%), penicillins (42-97%), ß-lactamase inhibitors (20-92%), aminoglycosides (17-53%), cephalosporins (8.3-90%), carbapenems (5.3-26%), and glycopeptides (0-20%). There was a fluctuation in resistance of Staphylococcus aureus to methicillin (60%-45%) (using cefoxitin resistance as a surrogate for oxacillin resistance) Among gram-negative organisms, there were increases in resistance to tetracycline (29-78% p < 0.001), ciprofloxacin (17-43%, p = 0.004), ceftriaxone (8-72%, p = 0.003), imipenem (6-26%, p = 0.004), and meropenem (7-18%, p = 0.03). CONCLUSION: The study highlights a concerning increase in antibiotic resistance rates over four years, with significant increase in resistance observed across different classes of antibiotics for both gram-positive and gram-negative organisms. This increased antibiotic resistance, particularly to commonly used antibiotics like ceftriaxone and ciprofloxacin, makes adhering to the WHO's Access, Watch, and Reserve (AWaRe) category even more critical. It also emphasizes how important it is to guard against the growing threat of antibiotic resistance by appropriately using medicines, especially those that are marked for "Watch" or "Reserve."

Prevalence and antimicrobial resistance of bacterial meningitis in China from 2017 to 2021: a multicenter retrospective study.

INTRODUCTION: This study aims to investigate the changing epidemiology and antimicrobial susceptibility of bacteria isolated from cerebrospinal fluid (CSF) in the Shandong region. METHODOLOGY: We conducted a retrospective analysis of bacterial distribution and resistance patterns in CSF samples, utilizing data from the SPARSS network and analyzed with WHONET 5.6 software. RESULTS: A total of 3968 pathogenic bacterial strains were isolated, consisting of 70.6% Gram-positive bacteria, 27.2% Gram-negative bacteria, and 0.2% fungi. The six most commonly detected bacteria were coagulase-negative staphylococcus, Acinetobacter baumannii, Klebsiella pneumoniae, Streptococcus pneumoniae, Escherichia coli, and staphylococcus aureus. Analysis revealed gender and seasonal variations in the distribution of CSF pathogens, with a higher incidence observed in males and during autumn compared to other seasons. The susceptibility profiles of these bacterial species varied significantly, with many exhibiting multidrug resistances. A. baumannii showed a high resistance rate to cephalosporins and carbapenems but was sensitive to tigecycline and polymyxins. For treating multidrug-resistant A. baumannii infections, polymyxin-based combinations with tigecycline or sulbactam are recommended for adults, while tigecycline combined with meropenem is suggested for children. Enterobacteriaceae species were generally sensitive to carbapenems, such as meropenem and other carbapenems that can penetrate the blood-brain barrier can be recommended. Linezolid and vancomycin are the first choice for treating common gram-positive bacterial infections. CONCLUSIONS: The high resistance rates observed among common CSF isolates and their varied distributions across different demographics highlight the necessity for customized treatment strategies.

Augmented pathogen detection in brain abscess using metagenomic next-generation sequencing: a retrospective cohort study.

Brain abscess is a severe infection characterized by the accumulation of pus within the brain parenchyma. Accurate identification of the causative pathogens is crucial for effective treatment and improved patient outcomes. This 10-year retrospective, single-center study aimed to compare the detection performance of conventional culture methods and metagenomic next-generation sequencing (mNGS) in brain abscess. We reviewed 612 patients diagnosed with brain abscess and identified 174 cases with confirmed etiology. The median age was 52 years, with 69.5% males. Culture tests predominately identified gram-positive bacteria, particularly Streptococcus spp. Gram-negative bacteria, including Klebsiella spp., were also detected. However, mNGS revealed a more diverse pathogen spectrum, focusing on anaerobes (e.g., Fusobacterium spp., Parvimonas spp., Porphyromonas spp., Prevotella spp., and Tannerella spp.). mNGS exhibited significantly higher overall pathogen-positive rates in pus samples (85.0% vs 50.0%, P = 0.0181) and CSF samples (84.2% vs 7.9%, P < 0.0001) compared to culture. Furthermore, the detection rates for anaerobes displayed a notable disparity, with mNGS yielding significantly higher positive detections in both pus samples (50.0% vs 10%, P = 0.0058) and CSF samples (18.4% vs 0%, P = 0.0115) when compared to culture methods. The assistance of mNGS in pathogen detection, particularly anaerobes in brain abscess, was evident in our findings. mNGS demonstrated the ability to identify rare and fastidious pathogens, even in culture-negative cases. These results emphasize the clinical value of mNGS as a supplement for brain abscess, enabling more comprehensive and accurate pathogen identification.IMPORTANCEThe accurate identification of pathogens causing brain abscess is crucial for effective treatment and improved patient outcomes. In this 10-year retrospective study, the detection performance of conventional culture methods and metagenomic next-generation sequencing (mNGS) was compared. The study analyzed 612 patients with brain abscess and confirmed etiology in 174 cases. The results showed that culture tests predominantly identified gram-positive bacteria, while mNGS unveiled a broader diverse pathogen spectrum, particularly anaerobes. The mNGS method exhibited significantly higher overall rates of pathogen positivity both in pus and cerebrospinal fluid (CSF) samples, surpassing the culture methods. Notably, mNGS detected a significantly higher number of anaerobes in both pus and CSF samples compared to culture methods. These findings underscore the clinical value of mNGS as a supplement for brain abscess diagnosis, enabling more comprehensive and accurate pathogen identification, particularly for rare and fastidious pathogens that evade detection by conventional culture methods.

Evaluation of an expanded antibiotic resistance gene panel on prediction of antimicrobial susceptibility results for Gram-negative bacteria in blood cultures.

The QIAstat-Dx BCID Panels (RUO) ("QIAstat," QIAGEN, Hilden, Germany) for identification of 13 Gram-negative bacteria and 18 antimicrobial resistance (AMR) gene groups was evaluated. The study was conducted in two phases; in phase 1, analytical performance was evaluated against 154 challenge isolates against whole genome sequencing data. In this phase, sensitivity and specificity of organism identification calls were 153/154 (99.3%) and 1,748/1,749 (99.8%), respectively. For AMR genes, sensitivity was 434/435 (99.8%) and specificity was 2,334/2,337 (99.9%). One false-negative blaIMP, one false-positive blaCTX-M, and two false-positive aac-6'-lb detections were noted in this challenge set of organisms. In phase 2, 101 clinical blood culture isolates of Gram-negative rods were evaluated by the multiplexed PCR versus reference broth microdilution, for the ability of identification combined with AMR genes to predict final susceptibility results. Negative predictive values were 92.8% for ampicillin resistance (100% for Escherichia coli), 93.4% for ceftriaxone, 97.4% for ceftazidime, and 98.7% for cefepime. In constrast, negative predictive values for current standard of care (identification plus detection of blaCTX-M) ranged from 56.5% to 88.8%. This study demonstrated additive value of additional beta-lactamase genes for bacteria isolated from blood cultures. IMPORTANCE: Prediction of Gram-negative bacteria resistance through detection of resistance genes is complex. This study evaluated a novel, direct-from-blood or bacterial isolate multiplexed PCR for the detection of 17 resistance genes, and evaluated the prediction of antimicrobial susceptibility.

A comparative study of a rapid phenotypic antimicrobial susceptibility testing system directly from positive blood cultures to the disk diffusion and VITEK 2 methods.

BACKGROUND: Sepsis is a life-threatening condition that impacts 49 million people annually and causes 11 million deaths worldwide. Surviving bloodstream infections (BSIs) depends on the rapid administration of effective antimicrobial treatment, underscoring a need for rapid antimicrobial susceptibility testing (AST). AIM: To evaluate the performance of Quantamatrix's dRAST v2.5 system (Seoul, South Korea) for AST directly from positive blood cultures as compared to the Disk-Diffusion (DD) and VITEK 2 methods. METHODS: The study included 191 positive blood cultures from clinical samples and spiked blood culture bottles. Following Gram staining and species-level identification, AST was performed by VITEK 2 and standard DD methods using CLSI (2021) interpretation. RESULTS: dRAST demonstrated very good AST performance for a Gram-negative isolate, and good performance for Gram-positive isolates, meeting CLSI criteria for the acceptance of a new method. Antimicrobials that were not considered verified compared to VITEK 2 and DD were cefazolin, ceftazidime, meropenem, and trimethoprim/sulfamethoxazole for Gram-negatives and clindamycin, erythromycin, penicillin, and oxacillin for Gram-positives. dRAST ESBL detection results were strongly correlated with the ESBL phenotypes obtained with other methods. Additional resistance mechanisms were in concordance with traditional tests. CONCLUSIONS: dRAST demonstrated good AST performance, meeting CLSI criteria for most relevant antibiotics. dRAST was associated with a significant reduction in time-to-results, labor, and the subjectivity of result analyses, making it a valuable addition to efforts supporting the treatment of patients with bacteremia. AST (antimicrobial susceptibility test), blood culture, dRAST, rapid methods, sepsis, turnaround time (TAT).