Synergy Test Research Articles

Abstract 3942: 3D models reveal novel drug synergies and mevalonate pathway activation in Ewing sarcoma

Abstract Ewing sarcoma (ES) is a rare but aggressive cancer, with survival rates of only about 30% in metastatic cases. Its rarity and severity highlight the need for focused research, yet progress is limited by scarce patient samples, few animal models, and minimal preclinical testing platforms. Conventional 2D cell cultures often fail to replicate primary tumor gene expression and drug sensitivity, while patient-derived xenografts (PDXs), though more accurate, require extensive resources and tend to lose tumor-specific traits when cultured in 2D. We established and standardized a spheroid culture system for ES and developed a semi-automated drug-screening platform for tumor spheroids. This platform enables drug testing in a 3D environment, offering more relevant insights than 2D models and helping to define drug concentration ranges for testing on tumoroids and patient samples. Bulk RNA sequencing revealed significant transcriptional differences between 3D cell line models (including spheroids and encapsulated 3D constructs) and traditional 2D cultures. The 3D constructs were created using extrusion bioprinting, which ensures uniform cell distribution and high density. Notably, the mevalonate pathway was activated in all 3D models used. ES is among the tumors with the highest expression of mevalonate pathway genes, making its activation in 3D models a promising replication of in vivo tumor characteristics. This activation underscores the pathway’s importance in ES metabolism and its potential as a therapeutic target. Building on these findings, we validated the encapsulated 3D models using PDX-derived cells, confirming consistent mevalonate pathway upregulation across these constructs. These models further demonstrated the spheroid platform’s capacity to accurately predict effective drug concentration ranges, which proved optimal for testing PDX-derived cells and may extend to other primary materials. Next, we conducted synergy tests using concentration matrices in both spheroid and 2D cultures, systematically exploring different drug concentration combinations. Based on transcriptional data and earlier studies, we opted to test interactions between mevalonate pathway inhibitors (statins), and apoptosis inhibitors, specifically BCL2 family inhibitors. Remarkably, we observed a strong synergy between BCL-xL inhibitors and statins, but only within the physiologically relevant 3D environments. These findings underscore the value of 3D models as potent tools for uncovering therapeutic combinations missed by conventional approaches. By more accurately reflecting key aspects of tumor behavior, these models enhance the reliability of drug response predictions and enable targeted treatment strategies. Advanced 3D models could accelerate the translation of preclinical discoveries into clinical treatments for Ewing sarcoma, offering new possibilities for tackling this aggressive cancer. Citation Format: Branka Radic Sarikas, Marica Markovic, Mathias Ilg, Caterina Sturtzel, Martha Zylka, Didier Surdez, Martin Metzelder, Martin Distel, Aleksandr Ovsianikov, Florian Halbritter, Heinrich Kovar. 3D models reveal novel drug synergies and mevalonate pathway activation in Ewing sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3942.

Diagnostic algorithm for the detection of carbapenemases and extended-spectrum β-lactamases in carbapenem-resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa can acquire carbapenem resistance through various mechanisms, including genomic mutations leading to the overexpression of efflux pumps, intrinsic AmpC-β-lactamase, and/or reduced permeability, and/or through the acquisition of plasmid-mediated carbapenemases and/or extended-spectrum-β-lactamases (ESBLs). Unfortunately, carbapenemase/ESBL-producing-carbapenem-resistant-P. aeruginosa (CP/ESBL-CRPA) cannot be differentiated from non-CP/ESBL-CRPA based solely on susceptibility testing results of conventional β-lactam (BL)-antibiotics. Knowing that these two groups display different activity profiles toward novel BL/β-lactamase-inhibitor (BLI) combinations, we developed and verified a cost-effective and easy-to-use diagnostic algorithm for screening and differentiation of carbapenemase and ESBL production in CRPA. We determined disc diffusion inhibition zones and gradient strip minimal inhibitory concentration values of 136 whole-genome-sequenced CRPA (70 metallo-β-lactamase-[MBL-], 1 GES-5-, 1 KPC-2-, 12 ESBL-, and 53 AmpC-hyper-producing isolates). We used the following BL-BLI combinations: ceftolozane-tazobactam (C-T), ceftazidime-avibactam, imipenem-relebactam (I-R), meropenem-vaborbactam, cefepime-enmetazobactam (C-E), and aztreonam-avibactam. We also included a lateral flow immunoassay (Carba-5, NG-Biotech) for confirmation of MBL production and double disc synergy testing (DDST) to improve ESBL detection. C-T was the most effective screening antibiotic for distinguishing MBL and ESBL producers from AmpC-hyperproducing CRPA, achieving a sensitivity of 100% for both MBL and ESBL producers. I-R reliably confirmed MBL production in C-T positive screened CRPAs, with a sensitivity of 92.8% and specificity of 100%. Incorporating Carba-5 into the phenotypic algorithm improved sensitivity for confirming MBL production to 100%. For the remaining C-T positive but I-R negative isolates, C-E showed 75% sensitivity and 78.6% specificity in detecting ESBL production. The DDST further confirmed ESBL production in six out of nine ESBL producers (66.6%). In conclusion, we established a simple and cost-effective diagnostic algorithm, enabling screening and confirmation of carbapenemase and ESBL production in CRPA.IMPORTANCECarbapenem-resistant Pseudomonas aeruginosa (CRPA) is a major global health threat, and rapid identification of its resistance mechanisms is crucial for effective treatment and infection control. Differentiating between carbapenemase-producing (CP), extended-spectrum β-lactamase-producing (ESBL), and AmpC-hyperproducing CRPA is challenging, as conventional susceptibility testing cannot reliably distinguish these resistance mechanisms. Our study presents a simple, cost-effective, and easy-to-implement phenotypic diagnostic algorithm that enables accurate screening and confirmation of CP and ESBL production in CRPA. This method is particularly valuable for laboratories lacking access to molecular diagnostics, as it provides a practical alternative for routine testing. By facilitating the early detection of resistant P. aeruginosa strains, this approach has the potential to improve patient outcomes, optimize antimicrobial therapy, and enhance global surveillance efforts against multidrug-resistant pathogens.

In Vitro Assessment on the Synergistic Antibacterial Effect of Ginger (Zingiber officinale Roscoe) Extract with Clindamycin Antibiotic against Staphylococcus aureus

Objectives: Examine if there is a synergistic antibacterial effect of Ginger (Zingiber officinale Roscoe) extract with clindamycin antibiotic against S. aureus.  Methods: The ginger extract was prepared by air-drying 50 grams of ginger and macerating it in 500 mL of 95% ethanol for 24 hours, followed by filtration and evaporation to obtain a condensed extract. Staphylococcus aureus colonies were inoculated on nutrient agar and incubated at 37°C for 18 to 24 hours. A 1 mg/mL clindamycin suspension was prepared from 300 mg capsules. MHA plates were inoculated with S. aureus, and filter paper discs labeled Clindamycin (C), Ginger (G), and combinations (GC50, GC75, GC100) were placed on the agar. The plates were incubated at 37°C for 18 to 24 hours, and the zones of inhibition were measured using the Kirby-Bauer Test to assess the synergy through mean ZOI and cooperative effect synergy testing.  Results: The results of the study indicate that the combination of ginger extract and Clindamycin generally showed antagonistic interactions. However, GC100 (100% ginger extract and Clindamycin) had a mean zone of inhibition (ZOI) closest to Clindamycin alone, suggesting potential antibacterial benefits at higher concentrations of ginger extract. Further analysis using cooperative effect synergy models confirmed the lack of synergistic interaction. Pairwise comparisons revealed that GC50 (50% ginger extract and Clindamycin) had a significant difference in effectiveness compared to Clindamycin alone (p-value: 0.045), while GC75 (75% ginger extract and Clindamycin) and GC100 did not exhibit significant differences.  Conclusion: These findings underscore the complexity of combining natural extracts with antibiotics. Although the combination did not enhance antibacterial efficacy, the potential of high-concentration ginger extract to act as a complementary treatment warrants further investigation to develop effective strategies against antibiotic-resistant S. aureus.

Public health threat of antimicrobial resistance and virulence genes in Escherichia coli from human-chicken transmission in Egypt

Escherichia coli (E. coli) infections cause significant losses in the poultry industry and pose zoonotic risks due to rising antimicrobial resistance (AMR) and virulence factors. This study investigates E. coli prevalence, AMR, and virulence genes (papC, vgrG1, iss) in Egyptian chickens and farm workers. A total of 35 dead chickens from 14 flocks and 17 farm workers urine samples were examined bacteriologically to investigate E. coli presence followed by biochemical identification. Antimicrobial susceptibility testing was performed on 14 antibiotics using the disk diffusion method on Mueller–Hinton agar, following ‘Clinical and Laboratory Standards Institute (CLSI) (2020) guidelines with Extended-spectrum β-lactamase (ESBL) activity evaluated via the Double Disc Synergy Test (DDST) with ceftazidime, cefotaxime, and their clavulanate combinations following CLSI protocols. virulence genes were detected through polymerase chain reaction (PCR) and phylogenetic analysis of the vgrG1 gene evaluated genetic relatedness between the chicken and human isolates. The study analysed 52 samples, identifying E. coli in 18 chicken organs (51.4%) and 11 human urine samples (64.7%), with no significant difference. various antimicrobic sensitivity profiles were identified phenotypically among all isolates in which 29 isolates, 58.6% were ESBL-producing, and 96.5% exhibited multidrug resistance (MDR), with chicken isolates showing higher resistance overall. virulence genes were detected in similar proportions across the isolates highlighting significant public health risks due to resistant and virulent E. coli. This study emphasized the public health risks of multidrug-resistant E. coli with virulence genes, highlighting potential zoonotic transmission and antibiotic use and food safety.

Design, synthesis and optimization of TarO inhibitors as multifunctional antibiotics against Methicillin-resistant Staphylococcus aureus

UDP-N-acetylglucosamine-undecaprenyl-phosphate N-acetylglucosaminephosphotransferase (TarO) has been found to simultaneously contribute to β-lactam resistance and virulence of Methicillin-resistant Staphylococcus aureus (MRSA). However, optimization of hit compounds targeting TarO has been hindered due to their high lipophilicity and the poor correlation between the enzyme activity inhibition and β-lactam sensitization. In this study, 31 analogues of Tarocin A were designed, synthesized and evaluated by a luminescence-based reporter preliminary screening. In the subsequent β-lactams synergy test, a good correlation was observed between the results obtained from these two methods. Finally, analog 18a with more potential against TarO and an improved hydrophilicity (clogP = 3.2) was obtained. Compared with Tarocin A, 18a shows stronger β-lactam sensitizing and anti-biofilm activities in vitro, as well as potent anti-virulence and synergistic potency with imipenem in vivo. These results suggest that TarO is a promising target for combating MRSA, and 18a can serve as a lead molecule.

Genetic diversity and antibiogram of ESBL-producing Escherichia coli isolated from apparently healthy birds sold at two selected live bird markets in Nigeria.

Live bird markets (LBMs) play a crucial role in the poultry value chain. However, there is a significant threat of antibiotic resistance development via this chain. This study aimed to characterise ESBL-producing Escherichia coli (E. coli) from cloacal samples of apparently healthy ducks and pigeons, determine their antibiotic resistance profile and carriage of ESBL genes. Sasa and Molete LBMs were selected for this study. Three hundred and forty cloacal swabs (170 each from ducks and pigeons) were sampled and isolation of E. coli was done using the streak plate method. Resistance to a panel of 10 antibiotics was determined using the disc diffusion method and phenotypic ESBL production was carried out using the double disc synergy test (DDST). Detection of ESBL genes in the isolates was done using PCR amplification method. Out of 340 samples, 22.9% (n = 78) tested positive for E. coli. Among these, 38.5% (n = 30) were positive for ESBL production. The thirty ESBL-producing isolates showed varying level of resistance to the tested antibiotics, with the highest level of resistance observed to imipenem in both ducks and pigeons. Twenty of the ESBL producers showed multidrug-resistant phenotypes. blaCTX-M which was detected in 19 isolates (63.3%) was the most predominant ESBL gene among the isolates, while 15/30 (50.0%) carried blaSHV and 6/30 (20.0%) carried blaTEM. Thirteen (43.3%) and two (6.6%) isolates co-harboured two and all the three target ESBL genes, respectively. This study has shown that LBMs in Ibadan are a repository of multidrug-resistant and ESBL-producing E. coli, hence urgent measures need to be taken to monitor and control the use of antibiotics in LBMs to mitigate this risk.

The Study of Escherichia coli as Antimicrobial-Resistant Sentinel Microorganism Isolated in the Farms of Three Districts of Ankara by MALDI-TOF MS and Genomic Analysis.

One Health is a unified approach that aims to sustain and maintain the health of humans, animals and the ecosystem. The environment plays an important role in the spread of resistance genes, as it is an unlimited source of antimicrobial resistance genes. Escherichia coli can acquire and spread resistance genes from its environment. Extended-spectrum beta-lactamase (ESBL)-producing E. coli is a global concern because it can hydrolyse many beta-lactam antibiotics. In this study, the aim was to isolate E. coli from faeces and soil samples collected from cattle, sheep and poultry in three districts (Golbası, Haymana and Cubuk) where livestock (cattle, sheep and poultry) farming activities are intensively carried out. In addition, the antibiotic susceptibility of the isolated E. coli was to be determined using phenotypic and genotypic methods and the presence of ESBLs was to be determined using a double-disc synergy test. All 120 E. coli isolates were confirmed by the MALDI-TOF MS method. The resistance rates of all isolates were as follows: ampicillin, 12.5%; trimethoprim/sulfamethoxazole, 6.6%; cefazolin, 0.83%; ciprofloxacin, 2.5%; ceftazidime, 0.83%; cefotaxime, 1.6% and ceftriaxone, 1.6%. Cefazolin (99.1%) and trimethoprim/sulfamethoxazole (0.83%) were determined to have intermediate susceptibility. Only one E. coli strain was found to be ESBL positive via phenotypic methods, and whole-genome analysis was performed on this strain. As a result of whole-genome analysis, ESBL-related CTX-M-14 and TEM-1 genes were found in the plasmids. This is the first study on the determination of antibiotic susceptibility and the presence of ESBL in E. coli isolated from the soil and faeces samples of farms in these regions. More studies are needed to determine and understand antibiotic resistance and ESBL positivity in environmental samples. Therefore, the One Health approach should be emphasised.

Helicid: A novel Anti-Staphylococcus aureus adjuvant.

Helicid: A novel Anti-Staphylococcus aureus adjuvant.

Investigating the broad clonal diversity of β-lactamases producing Enterobacteriaceae strains recovered from bloodstream infection in five hospitals

Background: Detecting β-lactamases-producing Enterobacteriaceae is underestimated due to difficulties in clinical laboratory procedures. Objectives: Identify β-lactamases in Enterobacteriaceae recovered from bloodstream infection patients in five different hospitals. Methods: Phenotypic and genetic methods were used. PCR were used to detect blaTEM, blaSHV, blaCTX-M, blaGES, blaAmpC, blaCMY, blaFOX, blaDHA, blaKPC and blaNDM genes and ERIC-PCR investigated the genetic diversity. Findings: K. pneumoniae, Enterobacter cloacae complex, Enterobacter aerogenes, and Escherichia coli were recovered. ESBL production was detected in 56.8% (Double-Disk Synergy Test) and 55.6% (Etest) of all strains. Modified Hodge Test was positive for K. pneumoniae (39.2%) and Enterobacter spp. (11.1%). ESBL genes were observed in 79.5% of all strains, blaTEM in 61.3% and blaSHV in 42%. The simultaneous presences of ESBL-encoding genes were observed (37.5%). The gene blaKPC was detected in 45% of K. pneumoniae and 18.5% of Enterobacter strains. Main conclusions: Phenotypic and molecular tests detected several types of β-lactamases among the bacterial strains recovered. The obtained results also suggest that several clonal populations circulate among hospitals, and some strains were genetically identical, showing that cross infection practices must be used for all patients, in all healthcare services.

Detection of Extended Spectrum Beta-lactamase Genes in Klebsiella pneumoniae Isolates from Urine and Sputum Samples in Nasarawa State, Nigeria: A Phenotypic and Genotypic Study

The rise of extended spectrum beta-lactamase (ESBL) producing Klebsiella pneumoniae presents a considerable challenge to healthcare systems globally, including those in Nasarawa State, Nigeria. This study aimed at determine the phenotypic and genotypic detection of extended spectrum beta-lactamase genes in K. pneumoniae isolates from urine and sputum specimen in Nasarawa state. Out of the twenty eight (28) Klebsiella pneumoniae isolates subjected for ESBL production using Double Dics Synergy Test and were confirmed by combine Disc Method, 12 Klebsiella pneumoniae isolates were ESBL producers. Genomic DNA from ESBL producing K. pneumoniae isolates were extracted and amplified using the Polymerase Chain Reaction (PCR) with universal primer for 16srRNA while specific primers of blaCTX-M, blaTEM, and blaSHV were used. Molecular analysis identified blaCTX-M and blaSHV as the predominant ESBL gene, detected in all 12(100%) ESBL producing isolates followed by blaTEM 9(75.0%). This study underscores the urgent need for enhanced surveillance and infection control measures to curb the spread of ESBL-producing Klebsiella pneumoniae in Nasarawa State. Furthermore, efforts to optimize antibiotic stewardship programs and promote rational antimicrobial use are essential to preserve the efficacy of existing antibiotics.

Ceftaroline + Rifampin Versus Vancomycin + Rifampin in the Treatment of Methicillin-Resistant Staphylococcus aureus Meningitis in an Experimental Rabbit Model.

Background/Aim: To compare the effectiveness ceftaroline-rifampicin (CR) and vancomycin-rifampicin (VR), against methicillin-resistant Staphylococcus aureus (MRSA) in a rabbit meningitis model, to compare the effects on brain tissues in terms of inflammation and apoptosis and to test the antibiotics via in vitro time-kill and synergy tests. Method: Meningitis was induced using MRSA strain ATCC 43300. After 28 hours, the rabbits were split into three groups: control, VR, and CR. A CSF culture was taken at the start (T0) and end of treatment (EOT)-the 24th hour of treatment. At EOT, the animals' brain tissues were examined for inflammation and apoptosis. The study strain was tested for a 24-hour time kill assay. Results: At the EOT, statistically significant differences were observed between the treatment groups in terms of reducing the cerebrospinal fluid (CSF) bacterial count, achieving partial or complete treatment response, and exhibiting lower levels of neuronal apoptosis compared with the control group. However, there was no significant difference in all three parameters and in survival between the two treatment groups. The CR group exhibited a noticeable decrease in inflammation than the control group, but no significant difference was found between the control group versus VR and VR versus CR group. Rifampicin did not improve antibacterial efficacy in the in vitro time-kill assay. Conclusion: The CR arm showed better complete response and inflammation, but both treatments were similar in other parameters. CR combination was found as effective as VR combination for treating MRSA meningitis.

Pharmacodynamic target attainment of the synergism of ceftazidime-avibactam in combination with amikacin against OXA-producing extensively drug-resistant or pan drug-resistant (XDR/PDR) Pseudomonas aeruginosa.

To investigate the pharmacodynamic target attainment of ceftazidime-avibactam (CZA) in combination with amikacin against OXA-producing extensively drug-resistant/ pan-drug-resistant Pseudomonas aeruginosa (XDR/PDR-PA). The minimum inhibitory concentrations (MICs) of CZA and amikacin against OXA-producing XDR/PDR-PA were determined by the checkerboard method, and the combined inhibitory index (FICI) was calculated to evaluate whether the combination of the two antimicrobials has a synergistic effect on OXA-producing XDR/PDR-PA in vitro. The pharmacokinetic (PK) and pharmacodynamic (PD) parameters of CZA and amikacin were combined by Monte Carlo simulation (MCS) to evaluate the cumulative fraction of response (CFR) of the two antimicrobials for the treatment of OXA-producing XDR/PDR-PA infection. The results of synergy tests of CZA in combination with amikacin suggested that 77.3% of XDR/PDR-PA showed synergistic effects. When the PK/PD target was greater than 50, CFR was 97.84% for CZA 2.5g q8h when CZA in combination with amikacin. CZA in combination with AMK has a synergistic effect in vitro and could be a potential option for treating OXA-producing XDR/PDR-PA infections.

Genetic study of Salmonella spp. Producting Betalatimase (ESBLs)

Ten isolates were collected from different clinical sources from laboratory in medicine century . These isolates were belonging to the genus Salmonella depending on morphological and biochemical tests . The antibiotic scussptibility tests against 10 antibiotics were examined , and it was found that the 60% isolates have multiple resistant to antibiotic ,(70%) of isolates were resistant to ampicillin,(50%) were resistant to augmentin ,(40%) were resistant to ceftriaxone ,(20%) were resistant to cefotaxime and (10%) were resistant to ciprofloxacin and tetracycline while all isolates showed sensitivity to piperacillin, imipenem, amikacin and erythromycin .The ability of Salmonela isolates to produce ?-lactamase enzymes were tested using iodometric method , and the results showed that all isolates produced this enzyme.The ability of these isolates to produce Extended-Spectrum ?-lactamase (ESBLs)were also determined by double disc synergy test , only five isolates produced these enzyme. Agarose gel electrophoresis showed that Salmonella isolates ?-lactamase producer have two small plasmid bands . Transformation experiments revealed that these plasmids were capable to transform E. coli MM294, an observation which indicates the ability of these plasmids to show their expression in more than one host.

Evaluation of two gradient diffusion tests to determine susceptibility to aztreonam and ceftazidime-avibactam in combination.

The combination of aztreonam and ceftazidime-avibactam (ATM-CZA) is a last resort regimen against recalcitrant infections caused by metallo-β-lactamase (MBL)-producing organisms. Susceptibility testing is warranted due to emerging resistance to the combination, but there are no widely implemented methods for use in clinical laboratories. Here, we used a cohort of 100 Enterobacterales, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia strains, including 68 MBL producers, to evaluate the performance of two ETEST strip-based synergy testing methods: the side-by-side (SS) method with an ATM ETEST placed next to a CZA ETEST (10 mm apart) and the strip cross (SX) method with a CZA ETEST placed perpendicularly on top of the ATM ETEST (at the 8 µg/mL mark). By reference broth microdilution (BMD), 89.1% (41/46) of the Enterobacterales, 15% (3/20) of the P. aeruginosa, and 97.1% (33/34) of the S. maltophilia isolates tested susceptible to the ATM-CZA combination. The SS method yielded 72% categorical agreement with BMD and 28 major errors (ME, 36.4%). Initial testing with the SX method yielded three ME , of which one was resolved upon repeat testing, yielding a final categorical agreement of 98% with BMD with two ME (2.6%). The SX method also yielded 100% reproducibility across three brands of Mueller Hinton agar (BD, Hardy, Remel). Our study demonstrates that the SX method is accurate, precise, and feasible for clinical laboratories to perform ATM-CZA susceptibility testing to guide use of the combination for treatment of multidrug-resistant gram-negative pathogens.

PREVALENCE, CHARACTERIZATION, AND EMERGENCE OF EXTENDED-SPECTRUM Β-LACTAMASE PRODUCING- AND CARBAPENEM-RESISTANT GRAM-NEGATIVE BACTERIA ISOLATED FROM HOUSEFLIES (Musca domestica) IN TUNISIA

Houseflies (Musca domestica) live in close contact with humans. They are carriers of human pathogenic bacteria in the digestive tract and on their body. This study aimed to assess the prevalence of antibiotic-resistant Gram-negative bacteria in flies.Sixty-one isolates were collected from 100 houseflies at three different locations: a laying hen farm, a market, and three houses, comprising 23 Escherichia coli, 31 Klebsiella pneumoniae and 7 Pseudomonas aeruginosa. Antimicrobial sensitivity was determined by the disk diffusion method, and the ESBL-producing isolates were screened by the double-disc synergy test. β-lactamase genes, associated resistance genes, and integrons were studied by PCR.The ESBL-producing isolates comprised14.8% (9/61) of the isolates, seven K. pneumoniae isolates, and two E. coli isolates. The highest rate of ESBL-producing strains was observed in houses (7/22; 31.8%), followed by the market (2/43; 4.7%). Multi-drug-resistant bacteria were detected in 19/61 (31.2%) insects. Third-generation cephalosporin-resistant isolates (n= 30) were used to identify the resistance genes. The following resistance genes were identified in the isolates; blaCTX-M-G-1 (76.7%, 23/30), blaSHV-1 (43.3%, 13/30), blaTEM-1 (36.7%, 11/30), blaIMP (16.7%, 5/30), blaOXA-48 (10%, 3/30) and blaNDM (3.3%, 1/30). The quinolone resistance genes qnrs, aac(6′)-Ib-cr, qnrB and qnrA were found in 11, 11, 7 and 5 isolates, respectively. Integron 1 (intI1) was detected in 15 (50%) isolates, qacEΔ1+sul1 was identified in ten intI1-positive isolates. Class 2 integron was detected in three isolates. Houseflies collected from houses and markets may be implicated in the spread of multi-drug resistant bacteria which constitute a considerable threat to human public health. The ESBLs in flies reflect the contamination status of the environment and can be used as indicators of contamination.

Tossing the coin of extended-spectrum β-lactamase: prevalence of extended-spectrum β-lactamase-producing Klebsiella pneumoniae isolated from patients with sepsis.

Background. Klebsiella pneumoniae is part of the ESKAPE (Enterococcus faecium, Staphylococcus aureus, K. pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) group of multidrug-resistant (MDR) pathogens. K. pneumoniae is the leading cause of antimicrobial resistance-associated mortality and the second leading cause of nosocomial bloodstream infections (BSIs), globally and in sub-Saharan Africa. Therefore, it was aimed to determine the antibiotic resistance patterns of K. pneumoniae isolated from blood cultures of patients with features of sepsis at Mulago National Referral Hospital, Uganda. Methods. The cross-sectional study on patients with features of sepsis utilized K. pneumoniae (n=30) isolated from positive blood culture specimens. The antibiotic resistance profile was determined by the Clinical and Laboratory Standards Institute's Kirby-Bauer disc diffusion method, which was used to classify the isolates as susceptible, intermediate and resistant. K. pneumoniae isolates that were resistant to third-generation cephalosporins were subjected to extended-spectrum β-lactamase (ESBL) screening and confirmation using the double-disc synergy test using cefotaxime, ceftazidime, ceftriaxone, cefotaxime-clavulanic acid and ceftazidime-clavulanic acid. The results were analysed for frequencies. Results. K. pneumoniae isolates showed emerging resistance to imipenem at 13% (4 out of 30) followed by amikacin at 17% (5 out of 30). There was intermediate resistance to gentamycin at 60% (18 out of 30). However, K. pneumoniae showed the highest resistance to piperacillin at 100% (30 out of 30) followed by sulphamethoxazole-trimethoprim and cefepime, both showing a percentage of 97% (29 out of 30). Up to 16 out of 30 (53.3%) of K. pneumoniae were positive for ESBL production, whilst 14 out of 30 (46.7%) were negative. Conclusion. There was a high prevalence of antibiotic-resistant ESBL-producing K. pneumoniae isolates from BSI of patients with features of sepsis in Uganda's Mulago National Referral Hospital.

Yearly incidence of acute childhood gastroenteritis in Nigeria: Implicated pathogens predominantly harbor blaCTXM and blaTEM genes.

Routine use of antibiotics for infectious diarrhea in children is associated with the risk of increasing antibiotic resistance in developing countries. This work aimed to study the predominant extended spectrum beta-lactamase (ESBL) genes among bacteria pathogens implicated in acute childhood gastroenteritis in a tertiary hospital in Nigeria. The stool samples of children diagnosed with acute gastroenteritis were collected. Isolation and identification of bacterial pathogens from the stool samples using standard microbiological and molecular sequencing methods. Pure cultures of the probable bacteria pathogens were subjected to antibiotics susceptibility profiling using the Kirby-Bauer Disk Diffusion Method and also screened for ESBL and AmpC using the Modified Double Disc Synergy Test. Primers for 5 different ESBL genes associated with beta-lactam antibiotic resistance were amplified and sequenced. Out of the 62 isolates, the highest number of organisms identified within the isolates were Bacillus sp at 38.7% (24) followed by Alcaligenes sp at 37% (23). Resistance to cefepime and ceftazidime were recorded at 50.8% (30) each. Ceftriaxone and cefotaxime were resisted in 47.4% (28) of the isolates. Out of 34 isolates resistant to all the cephalosporins used, 41.2% (14) were ESBL-producing, of which blaCTXM-1 and blaCTXM-2 were detected in 85.7%, while blaTEM was seen in 64.3%. blaCTXM and blaTEM may be the predominant ESBL genes haboured in the bacteria pathogens implicated in the yearly incidence of acute childhood gastroenteritis in Nigeria. This may be due to the widespread use of antibiotics in treating this disease.

Faecal Carriage of Multidrug Resistant Enterobacterales and Associated Factors among Neonates Admitted at Tertiary Hospital in Dar es Salaam, Tanzania

Background: Hospitalised neonates are at increased risk of carrying extended-spectrum β-lactamase-producing Enterobacterales (ESBL-PE) and carbapenemase-producing Enterobacterales (CPE), possibly leading to invasive infections. This study determined the faecal carriage of ESBL-PE, CPE, and associated factors among neonates at Muhimbili National Hospital (MNH). Methods: A hospital-based cross-sectional study was conducted among neonates aged ≤ 28 days admitted at MNH. The participants’ data and rectal swab samples were collected. Samples were processed to detect ESBL-PE and CPE. Results were confirmed using the double-disc diffusion synergy test and modified carbapenem inactivation method, respectively. An antimicrobial susceptibility test was performed using the Kirby Bauer disk diffusion method. Results: Three hundred forty neonates with a median age of 3 days (IQR: 2-9) were enrolled. The carriage rate of ESBL-E and CPE was 39.4%(134/340) and 1.8%(6/340), respectively. Klebsiella pneumoniae (66.9%) and Escherichia coli (66.7%) were the common isolates for ESBL-PE and CPE, respectively. The factors independently associated with ESBL-PE carriage were antibiotic use (aOR 2.73, 95% CI: 1.38-5.39, p=.04), age increase (aOR 1.09, 95% CI: 1.02-1.15, p=.006), prolonged hospitalisation (aOR 2.92, 95% CI: 1.17-7.29, p=.02), and neonate-sucking their fingers (aOR 2.98, 95% CI: 1.04-8.58, p=.04). The study observed a trend of CPE carriage toward neonates with prolonged hospitalisation (p=.05). ESBL-PE low resistance was observed to meropenem (0.9%), amikacin (2.7%-6.7%), and gentamicin (19.4% to 100 %). Conclusions: The study revealed a relatively high carriage rate of multidrug resistant Enterobacterales among neonates admitted to a tertiary hospital. These findings underscore the importance of continuous surveillance of ESBL-PE and CPE to prevent infections and limit their potential transmission within hospital settings and the community.

Comparison of Phenotypic and Genotypic Detection of Drug Resistance in Acinetobacter baumannii in a Tertiary Care Hospital.

Background Acinetobacter baumannii (A. baumannii) is a commoncauseof nosocomial infection. Multidrug-resistant A. baumannii is a life-threatening and therapeutic challenge, especially in critically ill and vulnerable patients. Drug resistance in A. baumannii is conferred by various underlying mechanisms. This prospective cross-sectional study aims to study the comparison of the phenotypic MBL-E test and molecular tests conferring drug resistance to A. baumannii. Materials and methods Different clinical samples were collected in a time period of two years. Isolated A. baumannii strains were studied for the drug-resistance profile by the Kirby disc method. These drug-resistant isolates were further subjected to metallo-beta-lactamase (MBL) production by molecular detection of OXA-48, NDM, and VIM genesand phenotypic methods by the double-disk synergy test, modified Hodge test, and MBL-E test. Results A total of 104 A. baumannii isolates were obtained from 3965 samples. Ninety-three (89.4%) of these 104 isolates were found to be drug-resistant which were further analyzed by phenotypic methods for MBL production which showed a detection range of 36.54%-89.42% as compared to a molecular method where detection was observed as 56 (60%). Conclusion Molecular detection of drug-resistance conferring genes can be atime-effective method as compared to phenotypic detection. However, genetic methods have their own limitation and additional research empaneling in a single test is required.

P-1119. The role of Carbapenemase gene detection and synergy testing in invasive infections caused by Carbapenem resistant Enterobacterales in the pediatric population at a quaternary hospital in South India

Abstract Background The incidence and prevalence of carbapenem-resistant Enterobacteriaceae (CRE) infections in children is increasing globally. However, there are limited studies that address clinical, microbiological profiles, molecular and synergy testing of these CRE infections, in this population. Our study was aimed at studying the clinical profile, microbiological and molecular diagnoses, and synergy testing of CRE bacteremia in children admitted to the intensive care unit (ICU). Methods Medical records of children admitted to the ICU with CRE bacteraemia over a year were reviewed. Their clinical background, microbiological diagnoses, molecular profile, antibiotic resistance patterns, synergy testing, and clinical outcomes were analyzed. Carbapenemase genes were detected using Gene X pert (Cepheid) and synergy testing was carried out on those isolates with New Delhi Metalloproteinase (NDM). NDM-1 or NDM + Oxacillinase (OXA) OXA-48 genes. The results were expressed as fractional inhibitory concentration index (FICI) and the same was correlated with treatment using Ceftazidime- Avibactam+/- Aztreonam. Results 98 children with blood stream infections (40 in neonatal ICU and 58 pediatric ICU) were identified. K. pneumoniae accounted for a majority of BSI’s (n-34 in neonatal ICU and n- 47 in pediatric ICU) with most of them expressing the NDM gene (50%). The overall Colistin resistance across both ICU’s was (MIC > 2 ug ml) was 0.03%. Synergy to Ceftazidime-Avibactam- Aztreonam could be demonstrated in a majority of CREs which were NDM / NDM+OXA-48 producers (82.3% K. pneumoniae in neonatal ICU and 90% in pediatric ICU). Eight neonates (20%) and seventeen children (29%) succumbed due to other comorbidities, all of whom had NDM Klebsiella pneumoniae bacteraemia (n-17, 100%). Conclusion NDM producing Klebsiella pneumoniae remains the commonest identified BSI in neonatal and paediatric patients in the intensive care and was isolated in all children with adverse outcomes. Infection control, antimicrobial stewardship protocols and Carbapenemase gene detection with targeted synergy and directed therapy is recommended for favorable clinical outcomes of BSI’s in the critically ill children. Disclosures All Authors: No reported disclosures