Multidrug-resistant Gram-negative Infections Research Articles

Quaternary Ammonium Salt Derivatives of Hyperbranched Polylysine with Enhanced Antibacterial Activity against Multidrug-Resistant Gram-Negative Bacteria.

Multidrug-resistant (MDR) Gram-negative bacteria infections have gradually become a more serious health problem recently, and antibacterial drugs are urgently needed to tackle MDR Gram-negative bacteria. Herein, we synthesized a series of quaternary ammonium salt derivatives of hyperbranched polylysine (HPL-Cm-n) with different alkyl chain lengths (m = 4, 8, 12, 16) and grafting ratios (n = 5.8-21.0) of alkyl quaternary ammonium salts (Cm). HPL-Cm-ns exhibited excellent antibacterial activities against drug-sensitive E. coli and P. aeruginosa, and specifically, HPL-C12-ns were also highly active against MDR E. coli and P. aeruginosa. The cytotoxicity and hemolytic activity of HPL-Cm-ns increased with the increase in the alkyl chain length and the grafting ratio of Cm. The killing study proved that HPL-C12-9.5 had fast killing kinetics and was bactericidal toward both drug-sensitive and MDR E. coli. The mechanistic studies showed that, similar to hyperbranched polylysine (HPL), HPL-C12-9.5 killed bacteria by disrupting the cell membranes and causing leakage of the cytoplasmic contents. HPL-C12-ns might have potential as an antibacterial agent to combat MDR Gram-negative bacteria.

The Challenge of Treating Infections Caused by Metallo-β-Lactamase-Producing Gram-Negative Bacteria: A Narrative Review.

Gram-negative multidrug-resistant (MDR) bacteria, including Enterobacterales, Acinetobacter baumannii, and Pseudomonas aeruginosa, pose a significant challenge in clinical practice. Infections caused by metallo-β-lactamase (MBL)-producing Gram-negative organisms, in particular, require careful consideration due to their complexity and varied prevalence, given that the microbiological diagnosis of these pathogens is intricate and compounded by challenges in assessing the efficacy of anti-MBL antimicrobials. We discuss both established and new approaches in the treatment of MBL-producing Gram-negative infections, focusing on 3 strategies: colistin; the recently approved combination of aztreonam with avibactam (or with ceftazidime/avibactam); and cefiderocol. Despite its significant activity against various Gram-negative pathogens, the efficacy of colistin is limited by resistance mechanisms, while nephrotoxicity and acute renal injury call for careful dosing and monitoring in clinical practice. Aztreonam combined with avibactam (or with avibactam/ceftazidime if aztreonam plus avibactam is not available) exhibits potent activity against MBL-producing Gram-negative pathogens. Cefiderocol in monotherapy is effective against a wide range of multidrug-resistant organisms, including MBL producers, and favorable clinical outcomes have been observed in various clinical trials and case series. After examining scientific evidence in the management of infections caused by MBL-producing Gram-negative bacteria, we have developed a comprehensive clinical algorithm to guide therapeutic decision making. We recommend reserving colistin as a last-resort option for MDR Gram-negative infections. Cefiderocol and aztreonam/avibactam represent favorable options against MBL-producing pathogens. In the case of P. aeruginosa with MBL-producing enzymes and with difficult-to-treat resistance, cefiderocol is the preferred option. Further research is needed to optimize treatment strategies and minimize resistance.

Detection of PhoP-mediated colistin resistance in Gram-negative bacteria without mcr genes in human population in the Ho Municipality, Ghana

IntroductionAntimicrobial resistance (AMR) has become a global public health threat, with colistin emerging as a last-resort treatment option for multidrug-resistant Gram-negative infections. However, the emergence of colistin resistance, mediated by mechanisms like mutations in the PhoP gene, raises concerns about the future utility of this antibiotic. This study aimed to determine the prevalence of PhoP-mediated colistin resistance in Gram-negative bacteria isolated from the stool of residents in the Ho Municipality, Ghana. MethodsIn this cross-sectional study, 110 stool samples were collected from June 2021 to December 2022. Gram-negative bacteria were isolated, and colistin susceptibility was determined by broth microdilution. Genomic DNA from resistant isolates was extracted and sequenced using the Nanopore platform to detect the presence of the PhoP gene. ResultsOf the 107 Gram-negative isolates, 57 % were resistant to colistin. The PhoP gene was detected in 61.4 % of the colistin-resistant isolates, with the highest prevalence observed in Proteus mirabilis, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. ConclusionThe study reveals a high prevalence of PhoP-mediated colistin resistance among Gram-negative bacteria colonizing residents in the Ho Municipality, highlighting the role of the gut microbiota as a reservoir for antibiotic resistance. Continued surveillance and a collaborative One Health approach are crucial to address this growing threat.

Effectiveness and Nephrotoxicity of Polymyxin B among Intensive Care Unit Patients: A Clinical Evaluation

Polymyxin B is a reserve antibiotic, but there has been an upsurge in its use due to a rise in multidrug-resistant gram-negative bacteria. However, nephrotoxicity and resistance concerns persist, with global resistance rates reaching 29%. Effectiveness of Polymyxin B (clinical and microbiological response) and the frequency of nephrotoxicity is not well documented in resource limited settings. Research is essential to guide optimization of Polymyxin B therapy and inform the adoption of measures for early detection of kidney injury to prevent damage. This study aimed to assess the effectiveness of Polymyxin B by evaluating clinical and microbiological responses and determining nephrotoxicity incidence using KDIGO criteria in ICU patients at Moi Teaching and Referral Hospital (MTRH). A prospective observational cohort study was conducted at MTRH ICUs between December 2021 and November 2022, on patients treated with Polymyxin B. Data on demographics, comorbidities, Polymyxin B dosage regimens, clinical responses, and microbiological results were collected. Descriptive statistics summarized patient characteristics, while associations between dosage regimens and outcomes were evaluated using Fisher's exact test and multivariate regression, with a p<0.05 considered statistically significant. Forty-four patients with a mean age of 48 years were included; 66% were male, and cerebrovascular disease was the most common comorbidity. All patients had multidrug-resistant gram-negative infections qualifying for Polymyxin B therapy. Most (89%) received monotherapy, with 86% achieving a good clinical response, 7% experiencing treatment failure, and 7% dying. Doses of 20,000–25,000 IU/Kg/day were associated with microbiological eradication and good clinical response (p<0.001), while 15,000 IU/Kg/day was associated with treatment failure. Acute kidney injury occurred in 48% of patients, with 68% developing hypomagnesemia. Polymyxin B at doses of between 20,000-25,000IU/Kg/day should be considered as a starting dose due to the association with good clinical response, with alternate-day monitoring of serum creatinine levels for early detection of nephrotoxicity.

Eco-friendly drugs induce cellular changes in colistin-resistant bacteria

Abstract Background and objective As a last option, multidrug-resistant Gram-negative infections (caused by Enterobacteriaceae) are treated with the antibiotic colistin, also known as polymyxin E. Colistin-resistant superbugs predispose people to untreatable infections, possibly leading to a high mortality rate. This project aims to study the effect of Acacia nilotica aqueous extract and zinc oxide nanoparticles (ZnO-NPs) on colistin-resistant Klebsiella pneumonia (CRKP). Materials and methods ZnO-NPs were synthesized using the green method and characterized by UV-vis, Fourier transform infrared spectroscopy, and X-ray diffraction. The zone of inhibition (ZI) was measured using the agar-well diffusion method, and the minimum inhibitory concentration (MIC) and minimum bactericidal concentration were estimated to determine the antimicrobial activity of the tested compound. Scanning electron microscopy (SEM) and transmission electronic microscopy (TEM) were used to investigate the alterations in bacterial cells that were treated with the tested drugs. Results The synthesized ZnO-NPs presented good chemical and physical properties, and the plant extract and ZnO-NPs displayed a large ZI. ZnO-NPs had the lowest MIC (0.2 mg·mL−1). SEM and TEM observations revealed various morphological modifications in CRKP cells, including cell shrinkage, cell damage, cytoplasm loss, cell wall thinning, and cell death. Conclusion A. nilotica aqueous extract and ZnO-NPs could be used as alternative natural products to produce antibacterial drugs and to prevent CRKP infection.

Extended structure-activity relationship studies of the [1,2,5]oxadiazolo[3,4-b]pyrazine-containing colistin adjuvants

Antimicrobial resistance (AMR) is a formidable global health challenge. Multidrug-resistant (MDR) Gram-negative bacterial infections are of primary concern due to diminishing treatment options and high morbidity and mortality. Colistin, a polymyxin family antibiotic, is a last-resort treatment for MDR Gram-negative infections, but its wider use has resulted in escalating resistance. In 2022, using a screening approach, we discovered that a [1,2,5]oxadiazolo[3,4-b]pyrazine (ODP)-containing compound selectively re-sensitized various MDR Gram-negative bacteria to colistin. Initial structure–activity relationship (SAR) studies confirmed that bisanilino ODP compounds are colistin adjuvants with low mammalian toxicity. Herein, we report our extended SAR studies on a wide range of ODP analogs bearing alkyl- or arylalkylamines. Specifically, we discovered two new compounds, 5q and 8g, with potent colistin-potentiating activity and low mammalian toxicity in a wide range of clinically relevant pathogens.

Efficacy and safety of a preventive strategy against tuberculosis in liver transplantation recipients including the treatment of latent infection with moxifloxacin.

Preventive management of tuberculosis in liver transplantation (LT) is challenging due to difficulties in detecting and treating latent tuberculosis infection (LTBI). The aim of this study was to analyze the safety and efficacy of a screening strategy for LTBI with the inclusion of moxifloxacin as treatment. We performed a retrospective single-center study of all LTs performed between 2016 and 2019 with a minimum 4-year follow-up and a standardized protocol for the evaluation of LTBI. Pretransplant LTBI screening was performed in 191/218 (87.6%) patients, and LTBI was diagnosed in 27.2% of them. Treatment for LTBI was administered to 71.2% of the patients and included moxifloxacin in 75.6% of the cases. After a median follow-up of 1628 days, no cases of active tuberculosis occurred among moxifloxacin-treated patients. The incidence of Clostridioides difficile (0.46vs. 0.38 episodes/1000 transplant-days; p = .8) and multidrug-resistant gram-negative bacilli infection (0vs. 0.7 episodes per 1000 transplant-days; p = .08) were not significantly higher in comparison to patients who did not receive moxifloxacin. A preventive strategy based on systematic LTBI screening and moxifloxacin treatment before LT in positive cases appears safe and effective in preventing the development of tuberculosis in LT recipients. However, our findings are limited by a small sample size; thus, larger studies are required to validate our observations.

Rapid Emergence of Resistance to Broad-Spectrum Direct Antimicrobial Activity of Avibactam.

Avibactam (AVI) is a diazabicyclooctane (DBO) β-lactamase inhibitor used clinically in combination with ceftazidime. At concentrations higher than those typically achieved in vivo , it also has broad-spectrum direct antibacterial activity against Enterobacterales strains, including metallo-β-lactamase-producing isolates, mediated by inhibition of penicillin-binding protein 2 (PBP2). This activity is mechanistically similar to that of more potent novel DBOs (zidebactam, nacubactam) in late clinical development. We found that resistance to AVI emerged readily, with a mutation frequency of 2×10 -6 to 8×10 -5 . Whole genome sequencing of resistant isolates revealed a heterogeneous mutational target that permitted bacterial survival and replication despite PBP2 inhibition, in line with prior studies of PBP2-targeting drugs. While such mutations are believed to act by upregulating the bacterial stringent response, we found a similarly high mutation frequency in bacteria deficient in components of the stringent response, although we observed a different set of mutations in these strains. Although avibactam-resistant strains had increased lag time, suggesting a fitness cost that might render them less problematic in clinical infections, there was no statistically significant difference in growth rates between susceptible and resistant strains. The finding of rapid emergence of resistance to avibactam as the result of a large mutational target has important implications for novel DBOs with potent direct antibacterial activity, which are being developed with the goal of expanding cell wall-active treatment options for multidrug-resistant gram-negative infections but may be vulnerable to treatment-emergent resistance.

Pharmacokinetics and pharmacodynamics of bacteriophage therapy: a review with a focus on multidrug-resistant Gram-negative bacterial infections.

SUMMARYDespite the early recognition of their therapeutic potential and the current escalation of multidrug-resistant (MDR) pathogens, the adoption of bacteriophages into mainstream clinical practice is hindered by unfamiliarity with their basic pharmacokinetic (PK) and pharmacodynamic (PD) properties, among others. Given the self-replicative nature of bacteriophages in the presence of host bacteria, the adsorption rate, and the clearance by the host's immunity, their PK/PD characteristics cannot be estimated by conventional approaches, and thus, the introduction of new considerations is required. Furthermore, the multitude of different bacteriophage types, preparations, and treatment schedules impedes drawing general conclusions on their in vivo PK/PD features. Additionally, the drawback of acquired bacteriophage resistance of MDR pathogens with clinical and environmental implications should be taken into consideration. Here, we provide an overview of the current state of the field of PK and PD of bacteriophage therapy with a focus on its application against MDR Gram-negative infections, highlighting the potential knowledge gaps and the challenges in translation from the bench to the bedside. After reviewing the in vitro PKs and PDs of bacteriophages against the four major MDR Gram-negative pathogens, Klebsiella pneumoniae, Acinetobacter baumannii complex, Pseudomonas aeruginosa, and Escherichia coli, specific data on in vivo PKs (tissue distribution, route of administration, and basic PK parameters in animals and humans) and PDs (survival and reduction of bacterial burden in relation to the route of administration, timing of therapy, dosing regimens, and resistance) are summarized. Currently available data merit close scrutiny, and optimization of bacteriophage therapy in the context of a better understanding of the underlying PK/PD principles is urgent to improve its therapeutic effect and to minimize the occurrence of bacteriophage resistance.

Bacterial infections and outcomes of inpatients with COVID-19 in the intensive care unit during the delta-dominant phase: the worst wave of pandemic in Iran.

Epidemiological data regarding the prevalence of bacterial multidrug-resistant (MDR) Gram-negative infections in patients with COVID-19 in Iran are still ambiguous. Thus, in this study we have investigated the epidemiology, risk factors for death, and clinical outcomes of bacterial infections among patients with COVID-19 in the intensive care unit (ICU). This retrospective cohort study included patients with COVID-19 hospitalized in the ICU of a university hospital in Iran between June 2021 and December 2021. We evaluated the epidemiological, clinical, and microbiological features, outcomes and risk factors associated with death among all COVID-19 patients. Data and outcomes of these patients with or without bacterial infections were compared. Kaplan-Meier plot was used for survival analyses. In total, 505 COVID-19 patients were included. The mean age of the patients was 52.7 ± 17.6 years and 289 (57.2%) were female. The prevalence of bacterial infections among hospitalized patients was 14.9%, most of them being hospital-acquired superinfections (13.3%). MDR Klebsiella pneumoniae and Staphylococcus aureus were the most common pathogens causing respiratory infections. Urinary tract infections were most frequently caused by MDR Escherichia coli and K. pneumoniae. The overall in-hospital mortality rate of COVID-19 patients was 46.9% (237/505), while 78.7% (59/75) of patients with bacterial infections died. Infection was significantly associated with death (OR 6.01, 95% CI = 3.03-11.92, p-value <0.0001) and a longer hospital stay (p < 0.0001). Multivariate logistic regression analysis showed that Age (OR = 1.04, 95% CI = 1.03-1.06, p-value <0.0001), Sex male (OR = 1.70, 95% CI = 1.08-2.70, p-value <0.0001), Spo2 (OR = 1.99, 95% CI = 1.18-3.38, p-value = 0.010) and Ferritin (OR = 2.33, 95% CI = 1.37-3.97, p-value = 0.002) were independent risk factors associated with in-hospital mortality. Furthermore, 95.3% (221/232) of patients who were intubated died. Our findings demonstrate that bacterial infection due to MDR Gram-negative bacteria associated with COVID-19 has an expressive impact on increasing the case mortality rate, reinforcing the importance of the need for surveillance and strict infection control rules to limit the expansion of almost untreatable microorganisms.

Real-World Evidence on Use of Ceftazidime-Avibactam in the Management of Gram-Negative Infections: A Retrospective Analysis.

There is limited real-world data from India examining the treatment characteristics, safety, and efficacy of ceftazidime-avibactam against Gram-negative organisms especially multidrug-resistant pathogens including carbapenem-resistantEnterobacteralesand carbapenem-resistantPseudomonas. In this retrospective study, the real-world treatment patterns, effectiveness, and safety of ceftazidime-avibactam in treating Gram-negative infections were assessed. Data was extracted from electronic health records of adult patients admitted to the hospital with documented Gram-negative infection who had received treatment for at least 48 hours with ceftazidime-avibactam as a part of routine clinical management. Among the 189 patients, on Day 3, clinical symptom improvement was recorded in 79.6% of patients who received ceftazidime-avibactam within 72 hours of hospital admission. Clinical success was achieved in 79.5% and 76.3% of assessed patients on Day 7 and Day 14/end-of-treatment (EOT), respectively. Microbiological success was reported in 76% of patients on Day 7 and in 60.3% of patients on Day 14 or EOT. The mean treatment duration of ceftazidime-avibactam therapy was 6.92 (± 4.1) days. No new safety concerns were identified. In conclusion, this study provides real-world evidence on treatment patterns and clinical outcomes associated with ceftazidime-avibactam in India, complementing the previously reported literature. The results suggest ceftazidime-avibactam is an effective and tolerable option for the management of multidrug-resistant (MDR) Gram-negative infections in critically ill patients.

AUDIT OF INFECTION CONTROL EFFECTIVENESS AND ANTIMICROBIAL DRUGS ADMINISTRATION IN UKRAINE: A PROSPECTIVE FOLLOW-UP

Indicators of antibiotic resistance in the world and in Ukraine are steadily increasing. As of 2021, there is a national standard in Ukraine that recognizes the deescalation of antibiotic therapy, but the war in Ukraine has significantly disrupted the supply of essential services. War-related infections and antimicrobial resistance are seriously affecting the health of people in Ukraine and beyond, forming a great reservoir of multidrug-resistant gram-negative infections Aims: monitor the effectiveness of infection control and antibiotic administration in the Public Health Department of Ukraine. Methods: Based on the Department of Surgery, Anesthesiology and Intensive Care of the PDO National Medical University named after O.O. Bogomolets, a prospective observational study was carried out - an anonymous audit of doctors, anesthesiologists and doctors of other specialties who work at the Public Health Service of Ukraine from the beginning of 2024. The investigation was carried out on the online platform Google-form. After the completion of the tracking period, all completed data was transferred to an Excel table and analyzed using additional descriptive statistics for parametric data. Results: The results of 214 participants from different regions of Ukraine were collected. Half of the respondents reported that the department of infection control functions effectively - 105 (49.2 %), formally important - 85 (39.9 %), and does not function in 3 (1.5 %) Cefazolin as the first choice AB (57.7 %), using of Ceftriaxone (18.6 %) or Cefuroxime (7.9 %) as the 1st line of AB prophylaxis. About not using the protocol tell for us the choice of AB 2-line AB prevention and treatment of Gram+ and Gram-infection. The most common resistant pathogens in the Ukrainian health care sector were: Klebsiella pneumonia 140 (65.4 %) and Pseudomonas aeruginosa 99 (46.3 %) species. Most respondents also reported the expansion of three courses of AB therapy: 7-10 days in 97 (45.5 %) and 10 days and more – 85 (39.9 %) types. Conclusion: most health protection regulations in Ukraine today include the creation of infection control and approval of local documents (new clinical protocols, patient routes) from infection control and administration Instruction of AB. However, wider problems are avoided without the continuation of specific protocols, the use of AB group reserve for AB prophylaxis, the choice of non-optimal empirical AB therapy, the wider acceptance of combination therapy without indications, trivial courses therapy and type of de-escalation.

Ecthyma Gangrenosum Secondary to Multidrug-Resistant Gram-Negative Rod Infections in Leukemia Patients With Prolonged Neutropenia

Ecthyma Gangrenosum Secondary to Multidrug-Resistant Gram-Negative Rod Infections in Leukemia Patients With Prolonged Neutropenia

Global Variation in Escherichia coli mcr-1 Genes and Plasmids from Animal and Human Genomes Following Colistin Usage Restrictions in Livestock.

Antimicrobial resistance (AMR) is a significant global health threat, with multidrug-resistant (MDR) bacterial clones becoming a major concern. Polymyxins, especially colistin, have reemerged as last-resort treatments for MDR Gram-negative infections. However, colistin use in livestock has spread mobile colistin resistance (mcr) genes, notably mcr-1, impacting human health. In consequence, its livestock use was banned in 2017, originating a natural experiment to study bacterial adaptation. The aim of this work was to analyse the changes in the mcr-1 genetic background after colistin restriction across the world. This study analyses 3163 Escherichia coli genomes with the mcr-1 gene from human and livestock hosts, mainly from Asia (n = 2621) and Europe (n = 359). Genetic characterisation identifies IncI2 (40.4%), IncX4 (26.7%), and multidrug-resistant IncHI2 (18.8%) as the most common plasmids carrying mcr-1. There were differences in plasmids between continents, with IncX4 (56.6%) being the most common in Europe, while IncI2 (44.8%) was predominant in Asia. Promoter variants related to reduced fitness costs and ISApl1 showed a distinct pattern of association that appears to be associated with adaptation to colistin restriction, which differed between continents. Thus, after the colistin ban, Europe saw a shift to specialised mcr-1 plasmids as IncX4, while ISApl1 decreased in Asia due to changes in the prevalence of the distinct promoter variants. These analyses illustrate the evolution of mcr-1 adaptation following colistin use restrictions and the need for region-specific strategies against AMR following colistin restrictions.

Drug utilization evaluation of ceftazidime-avibactam in a tertiary care hospital

Background: The World Health Organization considers antimicrobial resistance a major global threat, and ceftazidime-avibactam (CAZ-AVI) is a treatment for multidrug-resistant Gram-negative infections. The FDA approved its use in 2015 for infections with limited treatment options. Methods: A retrospective study was conducted in a hospital in India to analyse the use of CAZ-AVI formulation in patients. Data was collected from electronic records and included information on patient demographics, clinical and microbiological profiles, antibiotic treatment, and patient outcomes for those in the ICU. Results: A total of 110 patients were included in the study. On assessing the results of this study, it was found that, the median age of population who received CAZ-AVI was 63 years and hypertension, diabetes mellitus were the common comorbidities present in the patients. Out of 110 subjects, 66 (60%) patients were started with a high-end antibiotic as an empirical therapy. While analysing the resistance pattern, it was found that 22 (20%) were resistant and 84 (76%) of the study subjects were sensitive to ceftazidime-avibactam. There was higher clinical success for BSI, CUTI, VAP amongst the indications of CAZ-AVI and combination of CAZ-AVI with aztreonam has given the most successful treatment outcome. The combination of CAZ-AVI with one or two high end antibiotics was observed as a common clinical practice. Conclusions: The analysis of results showed encouraging clinical cure rates. Our study results suggest that ceftazidime-avibactam could be an effective standard therapy for managing MDR Gram -negative organisms.

CTC-177, a novel drug-Fc conjugate, shows promise as an immunoprophylactic agent against multidrug-resistant Gram-negative bacterial infections.

The widespread emergence of antibiotic resistance including MDR in Gram-negative bacterial pathogens poses a critical challenge to the current antimicrobial armamentarium. To create a novel drug-Fc conjugate (DFC) that can be delivered at sustained and prolonged levels while simultaneously activating the host immune response to combat MDR Gram-negative infections. The Cloudbreak™ platform was used to develop DFCs consisting of a targeting moiety (TM) (a polymyxin-derived dimer) attached via a non-cleavable linker to an effector moiety (EM) (the Fc domain of human IgG1). In vitro activities of the DFCs were assessed by MIC testing. Neutropenic mouse models of thigh infection, septicaemia and pneumonia were used to evaluate in vivo efficacy. Pharmacokinetics were evaluated in mice and cynomolgus monkeys. A single prophylactic dose of our lead DFC, CTC-177, resulted in significantly decreased bacterial burdens and reduced inflammation comparable to daily treatment with colistin in septicaemia and pneumonia mouse models. Furthermore, CTC-177 prophylaxis was able to restore colistin efficacy in colistin-resistant septicaemia, reducing bacterial burdens beyond the limit of detection. Finally, CTC-177 displayed a long terminal half-life of over 24 and 65 h in mice and cynomolgus monkeys, respectively. These data support the continued development of Cloudbreak™ DFCs as broad-spectrum prophylactic agents against Gram-negative infections.

Analysis of mcr family of colistin resistance genes in Gram-negative isolates from a tertiary care hospital in India.

Colistin serves as the drug of last resort for combating numerous multidrug-resistant (MDR) Gram-negative infections. Its efficacy is hampered by the prevalent issue of colistin resistance, which severely limits treatment options for critically ill patients. Identifying resistance genes is crucial for controlling resistance spread, with horizontal gene transfer being the primary mechanism among bacteria. This study aimed to assess the prevalence of plasmid-mediated mcr genes associated with colistin resistance in Gram-negative bacteria, utilizing both genotypic and phenotypic tests. The clinical isolates (n=913) were obtained from a tertiary care center in Chennai, India. Colistin resistance was seen among Gram-negative isolates. These strains underwent screening for mcr-1, mcr-3, mcr-4, and mcr-5 genes via conventional PCR. Additionally, mcr-positive isolates were confirmed through Sanger sequencing and phenotypic testing. The bacterial isolates predominantly comprised Klebsiella pneumoniae (62.43%), Escherichia coli (19.71%), Pseudomonas aeruginosa (10.73%), and Acinetobacter baumannii (4.81%), along with other species. All isolates exhibited multidrug resistance to three or more antibiotic classes. Colistin resistance, determined via broth microdilution (BMD) using CLSI guidelines, was observed in 13.08% of the isolates studied. Notably, mcr-5 was detected in K. pneumoniae in PCR, despite its absence in Sanger sequencing and phenotypic tests (including the combined-disk test, colistin MIC in the presence of EDTA, and Zeta potential assays). This finding underscores the importance of employing multiple diagnostic approaches to accurately identify colistin resistance mechanisms.

Risk Factors for Multi-Drug Resistant Gram-negative Infections across a Pediatric Hospital System

Background: Infections due to antibiotic resistant bacteria are increasing worldwide and while, the epidemiology of these pathogens is well described in adults, pediatric specific data are lacking. We sought to gain an understanding of the risk factors for multi-drug resistant Gram-negative (MDRGN) infections in our pediatric population. Methods: We performed a retrospective review of pediatric patients seen at a pediatric hospital system in 2022 who had a culture-positive MDRGN, which was defined as a gram-negative bacteria resistant or intermediate to at least 1 antibiotic in ≥ 3 antibiotic groups. Repeat positive cultures for the same MDRGN were considered a single infection episode if occurring within a 14-day period. Demographic, clinical, and microbiologic data was obtained from the electronic medical record. Fisher’s exact was used for analysis. Results: One hundred and seventy-nine children had 237 infection episodes during the study period. Eighty-one patients (45%) were male and the median age was 5.3 years. The most prevalent MDRGNs included: Escherichia coli (154, 65%), Klebsiella spp (52, 22%), and Enterobacter spp (16, 7%). Escherichia coli was significantly more likely than other pathogens to be isolated from the urine (P = 0.008). Compared to multi-drug resistant E. coli, patients with a non-E. coli MDRGN were significantly more likely to have an underlying medical condition, recent hospitalization and antibiotic use (P≤0.001 for each, Table 1). A carbapenem was administered in 32% (75/237) of infection episodes. There were only 6 carbapenem resistant organisms. Conclusions: In our study, E. coli was the most frequent MDRGN. Most patients with a non-E. coli MDRGN infection episode had an underlying medical condition, recent hospitalization and antibiotic use. Carbapenem resistance was infrequent, though surveillance studies are needed to identify changing antibiotic resistance patterns and to direct prevention measures.

Ceftazidime-Avibactam Use in a Case Series of Difficult-to-Treat or Recurrent Infections in Pediatric Patients with Complex Chronic Conditions: Effectiveness and Absence of Resistance Development.

The prevalence of multidrug-resistant Gram-negative infections, particularly carbapenem-resistant strains, has become a significant global health concern. Ceftazidime-avibactam (CZA) has emerged as a promising treatment option. However, data on its efficacy and safety in children are scarce, necessitating further investigation. We conducted a descriptive case series at a tertiary hospital in Spain from February 2019 to January 2022. Pediatric patients (<16 years) treated with CZA for confirmed or suspected multidrug-resistant Gram-negative infections were included. The clinical and microbiological characteristics, treatment approaches, and outcomes were examined. Eighteen children received CZA treatment. All had complex chronic conditions, with the most frequent underlying main diseases being liver transplantation (n = 8) and biliary atresia (n = 4). The predominant type of infection for which they received CZA was intra-abdominal infection caused or suspected to be caused by OXA-48-producing Klebsiella pneumoniae. CZA was generally well tolerated. Within the first month of starting CZA therapy, two patients died, with one case directly linked to the infection's fatal outcome. Some patients needed repeated courses of therapy due to recurrent infections, yet no resistance development was noted. In summary, the use of CZA showed effectiveness and safety, while the lack of resistance development highlights CZA's potential as a primary treatment option against OXA-48-producing infections.

Antimicrobial Susceptibility Testing for Colistin: Extended Application of Novel Quantitative and Morphologic Assay Using Scanning Electron Microscopy.

Colistin (Polymyxin E) has reemerged in the treatment of MDR Gram-negative infections. Traditional Colistin AST methods have long turnaround times and are cumbersome for routine use. We present a SEM-AST technique enabling rapid detection of Colistin resistance through direct observation of morphological and quantitative changes in bacteria exposed to Colistin. Forty-four Gram-negative reference organisms were chosen based on their Colistin susceptibility profiles. Bacterial suspensions of ∼107 CFU/mL were exposed to Colistin at EUCAST-ECOFF, with controls not exposed, incubated at 37°C, and then sampled at 0, 15, 30, 60, and 120 minutes. Phosphotungstic Acid (PTA) staining was applied, followed by SEM imaging using Hitachi TM4000PlusII-Tabletop-SEM at ×2000, ×5000 and ×7000 magnifications. Bacterial viability analysis was performed for all conditions by quantifying viable and dead organisms based on PTA-staining and morphologic changes. We identified a significant drop in the percentage of viable organisms starting 30 minutes after exposure in susceptible strains, as compared to nonsignificant changes in resistant strains across all tested organisms. The killing effect of Colistin was best observed after 120 minutes of incubation with the antibiotic, with significant changes in morphologic features, including bacterial inflation, fusion, and lysis, observed as early as 30 minutes. Our observation matched the results of the gold standard-based broth microdilution method. We provide an extended application of the proof of concept for the utilization of the SEM-AST assay for Colistin for a number of clinically relevant bacterial species, providing a rapid and reliable susceptibility profile for a critical antibiotic.