Rapid evolution of colistin resistance in a bioreactor model of infection of Klebsiella pneumoniae.

Colistin remains an important antibiotic for the therapeutic management of drug-resistant Klebsiella pneumoniae. Despite the numerous reports of colistin resistance in clinical strains, it remains unclear exactly when and how different mutational events arise resulting in reduced colistin susceptibility. Using a bioreactor model of infection, we modelled the emergence of colistin resistance in a susceptible isolate of K. pneumoniae. Genotypic, phenotypic and mathematical analyses of the antibiotic-challenged and un-challenged population indicates that after an initial decline, the population recovers within 24 h due to a small number of "founder cells" which have single point mutations mainly in the regulatory genes encoding crrB and pmrB that when mutated results in up to 100-fold reduction in colistin susceptibility. Our work underlines the rapid development of colistin resistance during treatment or exposure of susceptible K. pneumoniae infections having implications for the use of cationic antimicrobial peptides as a monotherapy.

Colistin-, cefepime-, and levofloxacin-resistant Salmonella enterica serovars isolated from Egyptian chicken carcasses.

OBJECTIVES: The emergence of multidrug-resistant (MDR) Salmonella strains, especially resistant ones toward critically important antimicrobial classes such as fluoroquinolones and third- and fourth-generation cephalosporins, is a growing public health concern. The current study, therefore, aimed to determine the prevalence, and existence of virulence genes (invA, stn, and spvC genes), antimicrobial resistance profiles, and the presence of ß-lactamase resistance genes (blaOXA, blaCTX-M1, blaSHV, and blaTEM) in Salmonella strains isolated from native chicken carcasses in Egypt marketed in Mansoura, Egypt, as well as spotlight the risk of isolated MDR, colistin-, cefepime-, and levofloxacin-resistant Salmonella enterica serovars to public health. METHODS: One hundred fifty freshly dressed native chicken carcasses were collected from different poultry shops in Mansoura City, Egypt between July 2022 and November 2022. Salmonella isolation was performed using standard bacteriological techniques, including pre-enrichment in buffered peptone water (BPW), selective enrichment in Rappaport Vassiliadis broth (RVS), and cultivating on the surface of xylose-lysine-desoxycholate (XLD) agar. All suspected Salmonella colonies were subjected to biochemical tests, serological identification using slide agglutination test, and Polymerase Chain Reaction (PCR) targeting the invasion A gene (invA; Salmonella marker gene). Afterward, all molecularly verified isolates were screened for the presence of virulence genes (stn and spvC). The antimicrobial susceptibility testing for isolated Salmonella strains towards the 16 antimicrobial agents tested was analyzed by Kirby-Bauer disc diffusion method, except for colistin, in which the minimum inhibition concentration (MIC) was determined by broth microdilution technique. Furthermore, 82 cefotaxime-resistant Salmonella isolates were tested using multiplex PCR targeting the ß-lactamase resistance genes, including blaOXA, blaCTX-M1, blaSHV, and blaTEM genes. RESULTS: Salmonella enterica species were molecularly confirmed via the invA Salmonella marker gene in 18% (27/150) of the freshly dressed native chicken carcasses. Twelve Salmonella serotypes were identified among 129 confirmed Salmonella isolates with the most predominant serotypes were S. Kentucky, S. Enteritidis, S. Typhimurium, and S. Molade with an incidence of 19.4% (25/129), 17.1% (22/129), 17.1% (22/129), and 10.9% (14/129), respectively. All the identified Salmonella isolates (n = 129) were positive for both invA and stn genes, while only 31.8% (41/129) of isolates were positive for the spvC gene. One hundred twenty-one (93.8%) of the 129 Salmonella-verified isolates were resistant to at least three antibiotics. Interestingly, 3.9%, 14.7%, and 75.2% of isolates were categorized into pan-drug-resistant, extensively drug-resistant, and multidrug-resistant, respectively. The average MAR index for the 129 isolates tested was 0.505. Exactly, 82.2%, 82.2%, 63.6%, 51.9%, 50.4%, 48.8%, 11.6%, and 10.1% of isolated Salmonella strains were resistant to cefepime, colistin, cefotaxime, ceftazidime/clavulanic acid, levofloxacin, ciprofloxacin, azithromycin, and meropenem, respectively. Thirty-one out (37.8%) of the 82 cefotaxime-resistant Salmonella isolates were ß-lactamase producers with the blaTEM as the most predominant ß-lactamase resistance gene, followed by blaCTX-M1 and blaOXA genes, which were detected in 21, 16, and 14 isolates respectively). CONCLUSION: The high prevalence of MDR-, colistin-, cefepime-, and levofloxacin-resistant Salmonella serovars among Salmonella isolates from native chicken is alarming as these antimicrobials are critically important in treating severe salmonellosis cases and boost the urgent need for controlling antibiotic usage in veterinary and human medicine to protect public health.

Genomic and phenotypic inconsistencies in Pseudomonas aeruginosa resistome among intensive care patients.

Objective: Pseudomonas aeruginosa, a difficult-to-manage nosocomial pathogen, poses a serious threat to clinical outcomes in intensive care (ICU) patients due to its high antimicrobial resistance (AMR). To promote effective management, it is essential to investigate the genomic and phenotypic differences in AMR expression of the isolates. Methods: A prospective observational study was conducted from July 2022 to April 2023 at Liepaja Regional Hospital in Latvia. The study included all adult patients who were admitted to the ICU and had a documented infection with P. aeruginosa, as confirmed by standard laboratory microbiological testing and short-read sequencing. Since ResFinder is the only sequencing-based database offering antibacterial susceptibility testing (AST) data for each antibiotic, we conducted a comparison of the resistance profile with the results of phenotypic testing, evaluating if ResFinder met the US Food and Drug Administration (FDA) requirements for approval as a new AMR diagnostic test. Next, to improve precision, AST data from ResFinder was compared with two other databases - AMRFinderPlus and RGI. Additionally, data was gathered from environmental samples to inform the implementation of appropriate infection control measures in real time. Results: Our cohort consisted of 33 samples from 29 ICU patients and 34 environmental samples. The presence of P. aeruginosa infection was found to be associated with unfavourable clinical outcomes. A third of the patient samples were identified as multi-drug resistant isolates. Apart from resistance against colistin, significant discrepancies were observed when phenotypic data were compared to genotypic data. For example, the aminoglycoside resistance prediction of ResFinder yielded a major errors value of 3.03% for amikacin, which was marginally above the FDA threshold. Among the three positive environmental samples, one sample exhibited multiple AMR genes similar to the patient samples in its cluster. Conclusion: Our findings underscore the importance of utilizing a combination of diagnostic methods for the identification of resistance mechanisms, clusters, and environmental reservoirs in ICUs.

Antimicrobial resistance genes harbored in invasive Acinetobacter calcoaceticus-baumannii complex isolated from Korean children during the pre-COVID-19 pandemic periods, 2015-2020.

Background: Acinetobacter baumannii (AB) has emerged as one of the most challenging pathogens worldwide, causing invasive infections in the critically ill patients due to their ability to rapidly acquire resistance to antibiotics. This study aimed to analyze antibiotic resistance genes harbored in AB and non-baumannii Acinetobacter calcoaceticus-baumannii (NB-ACB) complex causing invasive diseases in Korean children. Methods: ACB complexes isolated from sterile body fluid of children in three referral hospitals were prospectively collected. Colistin susceptibility was additionally tested via broth microdilution. Whole genome sequencing was performed and antibiotic resistance genes were analyzed. Results: During January 2015 to December 2020, a total of 67 ACB complexes were isolated from sterile body fluid of children in three referral hospitals. The median age of the patients was 0.6 (interquartile range, 0.1-7.2) years old. Among all the isolates, 73.1% (n=49) were confirmed as AB and others as NB-ACB complex by whole genome sequencing. Among the AB isolates, only 22.4% susceptible to carbapenem. In particular, all clonal complex (CC) 92 AB (n=33) showed multi-drug resistance, whereas 31.3% in non-CC92 AB (n=16) (P<0.001). NB-ACB showed 100% susceptibility to all classes of antibiotics except 3rd generation cephalosporin (72.2%). The main mechanism of carbapenem resistance in AB was the bla oxa23 gene with ISAba1 insertion sequence upstream. Presence of pmr gene and/or mutation of lpxA/C gene were not correlated with the phenotype of colistin resistance of ACB. All AB and NB-ACB isolates carried the abe and ade multidrug efflux pumps. Conclusions: In conclusion, monitoring and research for resistome in ACB complex is needed to identify and manage drug-resistant AB, particularly CC92 AB carrying the bla oxa23 gene.

Genomic analysis of carbapenem- and colistin-resistant Klebsiella pneumoniae complex harbouring mcr-8 and mcr-9 from individuals in Thailand.

The surge in mobile colistin-resistant genes (mcr) has become an increasing public health concern, especially in carbapenem-resistant Enterobacterales (CRE). Prospective surveillance was conducted to explore the genomic characteristics of clinical CRE isolates harbouring mcr in 2015-2020. In this study, we aimed to examine the genomic characteristics and phonotypes of mcr-8 and mcr-9 harbouring carbapenem-resistant K. pneumoniae complex (CRKpnC). Polymerase chain reaction test and genome analysis identified CRKpnC strain AMR20201034 as K. pneumoniae (CRKP) ST147 and strain AMR20200784 as K. quasipneumoniae (CRKQ) ST476, harbouring mcr-8 and mcr-9, respectively. CRKQ exhibited substitutions in chromosomal-mediated colistin resistance genes (pmrB, pmrC, ramA, and lpxM), while CRKP showed two substitutions in crrB, pmrB, pmrC, lpxM and lapB. Both species showed resistance to colistin, with minimal inhibitory concentrations of 8 µg/ml for mcr-8-carrying CRKP isolate and 32 µg/ml for mcr-9-carrying CRKQ isolate. In addition, CRKP harbouring mcr-8 carried blaNDM, while CRKQ harbouring mcr-9 carried blaIMP, conferring carbapenem resistance. Analysis of plasmid replicon types carrying mcr-8 and mcr-9 showed FIA-FII (96,575 bp) and FIB-HI1B (287,118 bp), respectively. In contrast with the plasmid carrying the carbapenemase genes, the CRKQ carried blaIMP-14 on an IncC plasmid, while the CRKP harboured blaNDM-1 on an FIB plasmid. This finding provides a comprehensive insight into another mcr-carrying CRE from patients in Thailand. The other antimicrobial-resistant genes in the CRKP were blaCTX-M-15, blaSHV-11, blaOXA-1, aac(6')-Ib-cr, aph(3')-VI, ARR-3, qnrS1, oqxA, oqxB, sul1, catB3, fosA, and qacE, while those detected in CRKQ were blaOKP-B-15, qnrA1, oqxA, oqxB, sul1, fosA, and qacE. This observation highlights the importance of strengthening official active surveillance efforts to detect, control, and prevent mcr-harbouring CRE and the need for rational drug use in all sectors.

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

AIM: 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. METHODS AND RESULTS: 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.

Potent synergy and sustained bactericidal activity of polymyxins combined with Gram-positive only class of antibiotics versus four Gram-negative bacteria.

BACKGROUND: Gram-negative bacteria (GNB) are becoming increasingly resistant to a wide variety of antibiotics. There are currently limited treatments for GNB, and the combination of antibiotics with complementary mechanisms has been reported to be a feasible strategy for treating GNB infection. The inability to cross the GNB outer membrane (OM) is an important reason that a broad spectrum of Gram-positive only class of antibiotics (GPOAs) is lacking. Polymyxins may help GPOAs to permeate by disrupting OM of GNB. OBJECTIVE: To identify what kind of GPOAs can be aided to broaden their anti-GNB spectrum by polymyxins, we systematically investigated the synergy of eight GPOAs in combination with colistin (COL) and polymyxin B (PMB) against GNB in vitro. METHODS: The synergistic effect of COL or PMB and GPOAs combinations against GNB reference strains and clinical isolates were determined by checkerboard tests. The killing kinetics of the combinations were assessed using time-kill assays. RESULTS: In the checkerboard tests, polymyxins-GPOAs combinations exert synergistic effects characterized by species and strain specificity. The synergistic interactions on P. aeruginosa strains are significantly lower than those on strains of A. baumannii, K. pneumoniae and E. coli. Among all the combinations, COL has shown the best synergistic effect in combination with dalbavancin (DAL) or oritavancin (ORI) versus almost all of the strains tested, with FICIs from 0.16 to 0.50 and 0.13 to < 0.28, respectively. In addition, the time-kill assays demonstrated that COL/DAL and COL/ORI had sustained bactericidal activity. CONCLUSIONS: Our results indicated that polymyxins could help GPOAs to permeate the OM of specific GNB, thus showed synergistic effects and bactericidal effects in the in vitro assays. In vivo combination studies should be further conducted to validate the results of this study.

Colistin: Lights and Shadows of an Older Antibiotic.

The emergence of antimicrobial resistance represents a serious threat to public health and for infections due to multidrug-resistant (MDR) microorganisms, representing one of the most important causes of death worldwide. The renewal of old antimicrobials, such as colistin, has been proposed as a valuable therapeutic alternative to the emergence of the MDR microorganisms. Although colistin is well known to present several adverse toxic effects, its usage in clinical practice has been reconsidered due to its broad spectrum of activity against Gram-negative (GN) bacteria and its important role of "last resort" agent against MDR-GN. Despite the revolutionary perspective of treatment with this old antimicrobial molecule, many questions remain open regarding the emergence of novel phenotypic traits of resistance and the optimal usage of the colistin in clinical practice. In last years, several forward steps have been made in the understanding of the resistance determinants, clinical usage, and pharmacological dosage of this molecule; however, different points regarding the role of colistin in clinical practice and the optimal pharmacokinetic/pharmacodynamic targets are not yet well defined. In this review, we summarize the mode of action, the emerging resistance determinants, and its optimal administration in the treatment of infections that are difficult to treat due to MDR Gram-negative bacteria.

High Prevalence of Colistin-Resistant Encoding Genes Carriage among Patients and Healthy Residents in Vietnam.

Background and Objectives: We aimed to investigate the carriage of colistin-resistant genes among both patients with a history of antibiotic exposure and apparently healthy adults with no recent healthcare contact. Materials and Methods: Stool swabs were collected from healthy people, and specimens were collected at the infection foci from the patients. Eleven primer/probe sets were used to perform the Multiplex Real-Time PCR assay with the QuantiNova Multiplex Probe PCR kit for screening the carriage of colistin-resistant genes (mcr-1 to mcr-10) and 16S rRNA gene as internal control. Results: In total, 86 patients and 96 healthy residents were included. Twenty two patients (25.9%) were positive with at least one colistin-resistance encoding gene. The mcr-1 gene was the most frequent (16.5%), followed by mcr-9, mcr-6, and mcr-4 genes, where the prevalence was 11.8%, 10.6%, and 9.4%, respectively. No patient was positive with mcr-3, mcr-7, and mcr-8 genes. Eight patients (9.4%) were positive with multiple colistin-encoding genes. Twenty-three healthy people (24.0%) were positive with at least one colistin-resistance encoding gene, and the mcr-10 gene was the most frequent (27.0%), followed by the mcr-1, mcr-8, and mcr-9 genes, where the prevalence was 24.3%, 21.6%, and 13.5%, respectively. No person was positive with the mcr-2 and mcr-5 genes. Conclusions: Our findings underscore the urgent need for enhanced surveillance, infection control measures, and stewardship interventions to mitigate the spread of colistin resistance in Vietnam.

Synergistic antibacterial activity of ceftazidime-avibactam in combination with colistin, gentamicin, amikacin, and fosfomycin against carbapenem-resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CPKP) infections seriously threaten global public health. The main objective of this study was to assess the in-vitro synergistic activity of ceftazidime-avibactam (CZA) in combination with colistin (COL), amikacin (AK), gentamicin (GEN), and fosfomycin (FOS) against CPKP isolates. The secondary goal was to determine the antibiotic susceptibility performance of BD Phoenix. OXA-48 (49.1%) was the predominant carbapenemase, followed by KPC (29.1%). We used the broth microdilution (BMD) method to determine the minimum inhibitory concentrations (MICs) of CZA, COL, AK, and GEN. Meanwhile, the MICs of FOS were determined by the agar dilution (AD) method. To examine the antibacterial activity of CZA, we conducted a checkerboard assay (CBA) with COL, AK, GEN, and FOS against CRKP isolates. We randomly selected three strains and performed synergy testing via time-kill assay (TKA). CRKP isolates were 89.1% susceptible to CZA, 16.4% to COL, 21.8% to GEN, and 29.1% to AK using BMD, 47.3% to FOS by AD. The most synergistic effects were observed in the combination of CZA-COL (78.2%) and CZA-FOS (63.6%). Given the limited therapeutic options for treating severe CRKP infections, combining CZA with COL and FOS may enhance in-vitro activity against clinical CRKP isolates.

Co-existence of two plasmids harboring transferable resistance-nodulation-division pump gene cluster, tmexCD1-toprJ1, and colistin resistance gene mcr-8 in Klebsiella pneumoniae.

BACKGROUND: The emergence of plasmid-mediated mobile colistin resistance (mcr) gene poses a great challenge to the clinical application of polymyxins. To date, mcr-1 to mcr-10 have been found in animals, humans, and the environment. Among them, mcr-8 was first identified in Klebsiella pneumoniae (K. pneumoniae) of swine origin, and then mcr-8.1 to mcr-8.5 were successively identified. Notably, K. pneumoniae is the major host of the mcr-8 gene in both animals and humans. This study aims to explore the characteristics of K. pneumoniae strains carrying the mcr-8 gene and tmexCD1-toprJ1 gene cluster and investigate the correlation between these two antibiotic resistance genes. METHODS: The isolates from the poultry farms and the surrounding villages were identified by mass spectrometer, and the strains positive for mcr-1 to mcr-10 were screened by polymerase chain reaction (PCR). The size of the plasmid and the antimicrobial resistance genes carried were confirmed by S1-nuclease pulsed-field gel electrophoresis (S1-PFGE) and Southern hybridization, and the transferability of the plasmid was verified by conjugation experiments. Antimicrobial susceptibility testing (AST) and whole genome sequencing (WGS) were used to characterize the strains. RESULTS: Two K. pneumoniae isolates (KP26 and KP29) displaying polymyxin resistance were identified as mcr-8 gene carriers. Besides that, tigecycline-resistant gene cluster tmexCD1-toprJ1 was also found on the other plasmid which conferred strain resistance to tigecycline. Through epidemiological analysis, we found that the mcr-8 gene has dispersed globally, circulating in the human, animals, and the environment. Furthermore, our analysis suggests that the coexistence of mcr-8 and tmexCD1-toprJ1 on a single plasmid might evolved through plasmid recombination. CONCLUSIONS: Although the mcr-8 and tmexCD1-toprJ1 gene clusters in the two strains of K. pneumoniae in this study were on two different plasmids, they still pose a potential threat to public health, requiring close monitoring and further study.

Integrated Analysis of Patient Networks and Plasmid Genomes to Investigate a Regional, Multispecies Outbreak of Carbapenemase-Producing Enterobacterales Carrying Both blaIMP and mcr-9 Genes.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) are challenging in healthcare, with resistance to multiple classes of antibiotics. This study describes the emergence of imipenemase (IMP)-encoding CPE among diverse Enterobacterales species between 2016 and 2019 across a London regional network. METHODS: We performed a network analysis of patient pathways, using electronic health records, to identify contacts between IMP-encoding CPE-positive patients. Genomes of IMP-encoding CPE isolates were overlaid with patient contacts to imply potential transmission events. RESULTS: Genomic analysis of 84 Enterobacterales isolates revealed diverse species (predominantly Klebsiella spp, Enterobacter spp, and Escherichia coli); 86% (72 of 84) harbored an IncHI2 plasmid carrying blaIMP and colistin resistance gene mcr-9 (68 of 72). Phylogenetic analysis of IncHI2 plasmids identified 3 lineages showing significant association with patient contacts and movements between 4 hospital sites and across medical specialties, which was missed in initial investigations. CONCLUSIONS: Combined, our patient network and plasmid analyses demonstrate an interspecies, plasmid-mediated outbreak of blaIMPCPE, which remained unidentified during standard investigations. With DNA sequencing and multimodal data incorporation, the outbreak investigation approach proposed here provides a framework for real-time identification of key factors causing pathogen spread. Plasmid-level outbreak analysis reveals that resistance spread may be wider than suspected, allowing more interventions to stop transmission within hospital networks.SummaryThis was an investigation, using integrated pathway networks and genomics methods, of the emergence of imipenemase-encoding carbapenemase-producing Enterobacterales among diverse Enterobacterales species between 2016 and 2019 in patients across a London regional hospital network, which was missed on routine investigations.

The impact of colistin-based regimens on mortality compared to other antimicrobials in patients with carbapenem-resistant Enterobacterales bacteremia in South African hospitals: a cross-sectional study.

BACKGROUND: Treatment of carbapenem-resistant Enterobacterales (CRE) infections in low-resource settings is challenging particularly due to limited treatment options. Colistin is the mainstay drug for treatment; however, nephrotoxicity and neurotoxicity make this drug less desirable. Thus, mortality may be higher among patients treated with alternative antimicrobials that are potentially less efficacious than colistin. We assessed mortality in patients with CRE bacteremia treated with colistin-based therapy compared to colistin-sparing therapy. METHODS: We conducted a cross-sectional study using secondary data from a South African national laboratory-based CRE bacteremia surveillance system from January 2015 to December 2020. Patients hospitalized at surveillance sentinel sites with CRE isolated from blood cultures were included. Multivariable logistic regression modeling, with multiple imputations to account for missing data, was conducted to determine the association between in-hospital mortality and colistin-based therapy versus colistin-sparing therapy. RESULTS: We included 1 607 case-patients with a median age of 29 years (interquartile range [IQR], 0-52 years) and 53% (857/1 607) male. Klebsiella pneumoniae caused most of the infections (82%, n=1 247), and the most common carbapenemase genes detected were blaOXA-48-like (61%, n=551), and blaNDM (37%, n=333). The overall in-hospital mortality was 31% (504/1 607). Patients treated with colistin-based combination therapy had a lower case fatality ratio (29% [152/521]) compared to those treated with colistin-sparing therapy 32% [352/1 086]) (p=0.18). In our imputed model, compared to colistin-sparing therapy, colistin-based therapy was associated with similar odds of mortality (adjusted odds ratio [aOR] 1.02; 95% confidence interval [CI] 0.78-1.33, p=0.873). CONCLUSION: In our resource-limited setting, the mortality risk in patients treated with colistin-based therapy was comparable to that of patients treated with colistin-sparing therapy. Given the challenges with colistin treatment and the increasing resistance to alternative agents, further investigations into the benefit of newer antimicrobials for managing CRE infections are needed.

Comparison of Colistin Susceptibility via Two Different Methods in Gram-Negative Extensive Drug-Resistance Isolates from ICU Patients.

OBJECTIVE: To compare the susceptibility of colistin by two methods in extensive drug-resistant (XDR) Gram-negative isolates from ICU patients. STUDY DESIGN: Cross-sectional comparative analysis. Place and Duration of the Study: Department of Microbiology, Combined Military Hospital Karachi, Pakistan, from August 2022 to February 2023. METHODOLOGY: A total of 100 clinical specimens received from the intensive care unit yielded growth of extensively drug-resistant gram-negative bacteria, which were evaluated for polymyxin E susceptibility. The agar dilution method was compared with the reference broth microdilution (BMD) method. Minimum inhibitory concentration (MIC) was noted for both methods. RESULTS: Comparison of the MIC method by agar dilution showed a 90% correlation with the reference method of broth microdilution. With MICs within the acceptable range of the clinical and laboratory standards institute (CLSI) recommendations, 89 isolates were susceptible to colistin, whereas only 11 remained resistant. Polymyxin E's MIC 50 and MIC 90 were determined to be 1 and 2 µg/ml, respectively, with 97% susceptibility. CONCLUSION: Agar dilution susceptibility method can be used for screening purposes for the susceptibility testing of polymyxin E. This method is reliable and can easily identify the heteroresistance. KEY WORDS: Extensively drug-resistant, Broth microdilution, Multidrug-resistant, Agar dilution, Minimum inhibitory concentration, Colony forming unit.

ß-lactamase expression induces collateral sensitivity in Escherichia coli.

Major antibiotic groups are losing effectiveness due to the uncontrollable spread of antimicrobial resistance (AMR) genes. Among these, ß-lactam resistance genes -encoding ß-lactamases- stand as the most common resistance mechanism in Enterobacterales due to their frequent association with mobile genetic elements. In this context, novel approaches that counter mobile AMR are urgently needed. Collateral sensitivity (CS) occurs when the acquisition of resistance to one antibiotic increases susceptibility to another antibiotic and can be exploited to eliminate AMR selectively. However, most CS networks described so far emerge as a consequence of chromosomal mutations and cannot be leveraged to tackle mobile AMR. Here, we dissect the CS response elicited by the acquisition of a prevalent antibiotic resistance plasmid to reveal that the expression of the ß-lactamase gene blaOXA-48 induces CS to colistin and azithromycin. We next show that other clinically relevant mobile ß-lactamases produce similar CS responses in multiple, phylogenetically unrelated E. coli strains. Finally, by combining experiments with surveillance data comprising thousands of antibiotic susceptibility tests, we show that ß-lactamase-induced CS is pervasive within Enterobacterales. These results highlight that the physiological side-effects of ß-lactamases can be leveraged therapeutically, paving the way for the rational design of specific therapies to block mobile AMR or at least counteract their effects.

Colistin-resistance genes in Escherichia coli isolated from patients with urinary tract infections.

BACKGROUND: The incidence of antimicrobial resistance is alarmingly high because it occurs in humans, environment, and animal sectors from a "One Health" viewpoint. The emergence of plasmid-carried mobile colistin-resistance (MCR) genes limits the efficacy of colistin, which is the last-line treatment for multidrug resistance (MDR) against gram-negative infections. OBJECTIVES: The current study aimed to investigate emergence of colistin-resistance (MCR 1-5) genes in E. coli isolated from patients with urinary tract infections (UTIs) in Jordan. METHODS: E. coli (n = 132) were collected from urine specimens. The E. coli isolated from human UTI patients were examined the resistance to colistin based on the presence of MCR (1-5). All isolates were tested against 20 antimicrobials using the standard disk diffusion method. The broth microdilution technique was used to analyze colistin resistance. In addition, the MCR (1-5) genes were detected using multiplex PCR. RESULTS: Out of the 132 isolates, 1 isolate was colistin-resistant, having a minimum inhibitory concentration of 8 µg/mL and possessing MCR-1. All the E. coli isolates showed high resistance to penicillin (100%), amoxicillin (79.55%), cephalexin (75.76%), nalidixic acid (62.88%), tetracycline (58.33%), or cefepime (53.79). CONCLUSION: To our knowledge, this is the first report on the presence of plasmid-coded MCR-1 in E. coli from a patient with UTIs in Jordan. This is a problematic finding because colistin is the last-line drug for the treatment of infections caused by MDR gram-negative bacteria. There is a crucial need to robustly utilize antibiotics to control and prevent the emergence and prevalence of colistin-resistance genes.

Modification of the Antibiotic, Colistin, with Dextrin Causes Enhanced Cytotoxicity and Triggers Apoptosis in Myeloid Leukemia.

Introduction: Acute myeloid leukemia (AML) remains difficult to treat due to its heterogeneity in molecular landscape, epigenetics and cell signaling alterations. Precision medicine is a major goal in AML therapy towards developing agents that can be used to treat patients with different 'subtypes' in combination with current chemotherapies. We have previously developed dextrin-colistin conjugates to combat the rise in multi-drug resistant bacterial infections and overcome dose-limiting nephrotoxicity. Recent evidence of colistin's anticancer activity, mediated through inhibition of intracellular lysine-specific histone demethylase 1 (LSD1/KDM1A), suggests that dextrin-colistin conjugates could be used to treat cancer cells, including AML. This study aimed to evaluate whether dextrin conjugation (which reduces in vivo toxicity and prolongs plasma half-life) could enhance colistin's cytotoxic effects in myeloid leukemia cell lines and compare the intracellular uptake and localization of the free and conjugated antibiotic. Results: Our results identified a conjugate (containing 8000 g/mol dextrin with 1 mol% succinoylation) that caused significantly increased toxicity in myeloid leukemia cells, compared to free colistin. Dextrin conjugation altered the mechanism of cell death by colistin, from necrosis to caspase 3/7-dependent apoptosis. In contrast, conjugation via a reversible ester linker, instead of an amide, had no effect on the mechanism of the colistin-induced cell death. Live cell confocal microscopy of fluorescently labelled compounds showed both free and dextrin-conjugated colistins were endocytosed and co-localized in lysosomes, and increasing the degree of modification by succinoylation of dextrin significantly reduced colistin internalization. Discussion: Whilst clinical translation of dextrin-colistin conjugates for the treatment of AML is unlikely due to the potential to promote antimicrobial resistance (AMR) and the relatively high colistin concentrations required for anticancer activity, the ability to potentiate the effectiveness of an anticancer drug by polymer conjugation, while reducing side effects and improving biodistribution of the drug, is very attractive, and this approach warrants further investigation.

In-vitro evaluation of different antimicrobial combinations with and without colistin against carbapenem-resistant Acinetobacter baumannii clinical isolates.

BACKGROUND: Carbapenem-resistant Acinetobacter baumannii (CRAB) infections are one of the most common causes of nosocomial infections and have high mortality rates due to difficulties in treatment. In this study, the in vitro synergistic interactions of the colistin (CT)-meropenem (MEM) combination and patient clinical outcomes were compared in CRAB-infected patients that receive CT-MEM antimicrobial combination therapy. In addition, in vitro synergistic interactions of MEM-ertapenem (ETP), MEM-fosfomycin (FF) and CT-FF antimicrobial combinations were investigated. Finally, the epsilometer (E) test and checkerboard test results were compared and the compatibility of these two tests was evaluated. METHODS: Twenty-one patients were included in the study. Bacterial identification was performed with MALDI-TOF, and antimicrobial susceptibility was assessed with an automated system. Synergy studies were performed using the E test and checkerboard method. RESULTS: For the checkerboard method, the synergy rates for CT-MEM, MEM-FF, MEM-ETP and CT-FF were 100%, 52.3%, 23.8% and 28.5%, respectively. In the E test synergy tests, synergistic effects were detected for two isolates each in the CT-MEM and CT-FF combinations. Microbial eradication was achieved in nine (52.9%) of the 17 patients that received CT-MEM combination therapy. The agreement between the E test and the checkerboard test was 6.5%. CONCLUSIONS: A synergistic effect was found with the checkerboard method for the CT-MEM combination in all isolates in our study, and approximately 70% of the patients benefited from treatment with this combination. In addition, more than half of the isolates showed a synergistic effect for the MEM-FF combination. Combinations of CT-MEM and MEM-FF may be options for the treatment of CRAB infections. However, a comprehensive understanding of the potential of the microorganism to develop resistant mutants under applied exposures, as well as factors that directly affect antimicrobial activity, such as pharmacokinetics/pharmacodynamics, is essential for providing treatment advice. We found a low rate of agreement between the E test method and the checkerboard test method in our study, in contrast to the literature. Comprehensive studies that compare clinical results with methods are needed to determine the ideal synergy test and interpretation method.

In Vivo and in Vitro activity of colistin-conjugated bimetallic silver-copper oxide nanoparticles against Pandrug-resistant Pseudomonas aeruginosa.

BACKGROUND: Addressing microbial resistance urgently calls for alternative treatment options. This study investigates the impact of a bimetallic formulation containing colistin, silver, and copper oxide on a pandrug-resistant, highly virulent Pseudomonas aeruginosa (P. aeruginosa) isolate from a cancer patient at the National Cancer Institute, Cairo University, Egypt. METHODS: Silver nanoparticles (Ag NPs), copper oxide nanoparticles (CuO NPs), and bimetallic silver-copper oxide nanoparticles (Ag-CuO NPs) were synthesized using gamma rays, combined with colistin (Col), and characterized by various analytical methods. The antimicrobial activity of Col-Ag NPs, Col-CuO NPs, and bimetallic Col-Ag-CuO NPs against P. aeruginosa was evaluated using the agar well diffusion method, and their minimum inhibitory concentration (MIC) was determined using broth microdilution. Virulence factors such as pyocyanin production, swarming motility, and biofilm formation were assessed before and after treatment with bimetallic Col-Ag-CuO NPs. The in vivo efficacy was evaluated using the Galleria mellonella model, and antibacterial mechanism were examined through membrane leakage assay. RESULTS: The optimal synthesis of Ag NPs occurred at a gamma ray dose of 15.0 kGy, with the highest optical density (OD) of 2.4 at 375 nm. Similarly, CuO NPs had an optimal dose of 15.0 kGy, with an OD of 1.5 at 330 nm. Bimetallic Ag-CuO NPs were most potent at 15.0 kGy, yielding an OD of 1.9 at 425 nm. The MIC of colistin was significantly reduced when combined with nanoparticles: 8 µg/mL for colistin alone, 0.046 µg/mL for Col-Ag NPs, and 0.0117 µg/mL for Col-Ag-CuO NPs. Bimetallic Col-Ag-CuO NPs reduced the MIC four-fold compared to Col-Ag NPs. Increasing the sub-inhibitory concentration of bimetallic nanoparticles from 0.29 × 10-2 to 0.58 × 10-2 µg/mL reduced P. aeruginosa swarming by 32-64% and twitching motility by 34-97%. At these concentrations, pyocyanin production decreased by 39-58%, and biofilm formation was inhibited by 33-48%. The nanoparticles were non-toxic to Galleria mellonella, showing 100% survival by day 3, similar to the saline-treated group. CONCLUSIONS: The synthesis of bimetallic Ag-CuO NPs conjugated with colistin presents a promising alternative treatment for combating the challenging P. aeruginosa pathogen in hospital settings. Further research is needed to explore and elucidate the mechanisms underlying the inhibitory effects of colistin-bimetallic Ag-CuO NPs on microbial persistence and dissemination.

Widespread fungal-bacterial competition for magnesium lowers bacterial susceptibility to polymyxin antibiotics.

Fungi and bacteria coexist in many polymicrobial communities, yet the molecular basis of their interactions remains poorly understood. Here, we show that the fungus Candida albicans sequesters essential magnesium ions from the bacterium Pseudomonas aeruginosa. To counteract fungal Mg2+ sequestration, P. aeruginosa expresses the Mg2+ transporter MgtA when Mg2+ levels are low. Thus, loss of MgtA specifically impairs P. aeruginosa in co-culture with C. albicans, but fitness can be restored by supplementing Mg2+. Using a panel of fungi and bacteria, we show that Mg2+ sequestration is a general mechanism of fungal antagonism against gram-negative bacteria. Mg2+ limitation enhances bacterial resistance to polymyxin antibiotics like colistin, which target gram-negative bacterial membranes. Indeed, experimental evolution reveals that P. aeruginosa evolves C. albicans-dependent colistin resistance via non-canonical means; antifungal treatment renders resistant bacteria colistin-sensitive. Our work suggests that fungal-bacterial competition could profoundly impact polymicrobial infection treatment with antibiotics of last resort.