Draft genome sequence analysis of multidrug-resistant Salmonella enterica subsp. enterica serovar Mbandaka harboring colistin resistance gene mcr-9.1 isolated from foals in Kentucky, USA.

Horses play a significant role in the direct or indirect transmission of Salmonella to humans. Here, we report the draft genomes of multidrug-resistant Salmonella enterica subsp. enterica serovar Mbandaka YAH-F68 isolated from foals in Kentucky, USA belonging to sequence type 413 and harboring the mobile colistin resistance gene mcr-9.1.

An outbreak of blaKPC-4- and blaVIM-1-producing Klebsiella pneumoniae and Klebsiella variicola at a single hospital in South Korea.

BACKGROUND: The dissemination of Klebsiella spp. producing multiple carbapenemases has been increasingly recognized. Between July 2019 and August 2021, ten patients were found to carry Klebsiella spp. co-harboring blaKPC-4 and blaVIM-1 across multiple wards at a Korean hospital, and one isolate was recovered from a hand-washing sink, more than a year after the outbreak. This study aimed to investigate the outbreak and conduct a genomic study of these isolates. METHODS: Whole-genome sequencing, including long-read sequencing, was performed to analyze plasmid structures and mobile genetic elements (MGEs). Bioinformatics analyses were performed to trace clonal transmission chains and horizontal gene transfer. RESULTS: The findings suggested that the inter-ward spread of Klebsiella spp. seemed to be facilitated by healthcare worker contact or patient movement. Of the nine isolates collected (eight clinical and one environmental), seven (including the environmental isolate) were identified as K. pneumoniae (ST3680) and two were K. variicola (single-locus variant of ST5252). These isolates showed high genetic relatedness within their species and harbored the IncHI5B plasmid carrying both blaKPC-4 and blaVIM-1 (pKPCVIM.1). On this plasmid, blaVIM-1 was located in the Class 1 integron associated with IS1326::IS1353 (In2), and Tn4401b carrying blaKPC-4 was inserted into IS1326::IS1353, creating a novel MGE construct (In2_blaVIM-1-Tn4401b_blaKPC-4). CONCLUSION: The hospital-wide spread of blaKPC-4 and blaVIM-1 was facilitated by clonal spread and horizontal plasmid transfer. The persistence of this strain in the hospital sink suggests a potential reservoir of the strain. Understanding the transmission mechanisms of persistent pathogens is important for improving infection control strategies in hospitals.

Mechanism of synergistic action of colistin with resveratrol and baicalin against mcr-1-positive Escherichia coli.

The rising incidence of colistin (COL) resistance poses a significant challenge, undermining the therapeutic efficacy of COL against life-threatening bacterial infections. Therefore, the urgent identification and development of new therapeutics are imperative. It has been proven that combinations of antibiotics and promising non-antibiotic agents could be a potential strategy to combat infections caused by MDR pathogens. Due to various antimicrobial properties, medicinal plants have attracted significant attention, which could be promising adjuvant. In this study, we investigated the synergistic effects of combining COL with resveratrol (RST) and baicalin (BAI) against mcr-1-positive Escherichia coli through antibiotic susceptibility testing, checkerboard method and time-killing assays. The mechanisms of combination treatment were analyzed using SEM, fluorometric assays and transcriptome analysis. The molecular docking assay was conducted to elucidate potential interactions between RST, BAI and the MCR-1 protein. Finally, we assessed the in vivo efficacy of combination against mcr-1-positive Escherichia coli. The results demonstrated that the combination of RST, BAI and COL showed significant synergistic activity both in vitro and in vivo. Further mechanistic study revealed that the combination could increase the membrane-damaging ability of COL, disrupt the homeostasis of proton motive force (PMF), inhibit the activity of efflux pumps and impair ATP supply. The molecular docking revealed that RST and BAI could bind to MCR-1 stably, indicating the combination of RST and BAI may be an effective MCR-1 inhibitor. Our findings demonstrated that the combination of RST and BAI might be potential COL adjuvant, providing an alternative approach to address mcr-1-positive Escherichia coli infections.

Decoding the origins, spread, and global risks of mcr-9 gene.

BACKGROUND: The global spread of the plasmid-mediated mcr (mobilized colistin resistance) gene family presents a significant threat to the efficacy of colistin, a last-line defense against numerous Gram-negative pathogens. The mcr-9 is the second most prevalent variant after mcr-1. METHODS: A dataset of 698 mcr-9-positive isolates from 44 countries is compiled. The historical trajectory of the mcr-9 gene is reconstructed using Bayesian analysis. The effective reproduction number is used innovatively to study the transmission dynamics of this mobile-drug-resistant gene. FINDINGS: Our investigation traces the origins of mcr-9 back to the 1960s, revealing a subsequent expansion from Western Europe to the America and East Asia in the late 20th century. Currently, its transmissibility remains high in Western Europe. Intriguingly, mcr-9 likely emerged from human-associated Salmonella and exhibits a unique propensity for transmission within the Enterobacter. Our research provides a new perspective that this host preference may be driven by codon usage biases in plasmids. Specifically, mcr-9-carrying plasmids prefer the nucleotide C over T compared to mcr-1-carrying plasmids among synonymous codons. The same bias is seen in Enterobacter compared to Escherichia (respectively as their most dominant genus). Furthermore, we uncovered fascinating patterns of coexistence between different mcr-9 subtypes and other resistance genes. Characterized by its low colistin resistance, mcr-9 has used this seemingly benign feature to silently circumnavigate the globe, evading conventional detection methods. However, colistin-resistant Enterobacter strains with high mcr-9 expression have emerged clinically, implying a strong risk of mcr-9 evolving into a global "true-resistance-gene". INTERPRETATION: This study explores the mcr-9 gene, emphasizing its origin, adaptability, and dissemination potential. Given the high mcr-9 expression colistin-resistant strains was observed in clinically the prevalence of mcr-9 poses a significant challenge to drug resistance prevention and control within the One Health framework. FUNDING: This work was partially supported by the National Natural Science Foundation of China (Grant No. 32141001 and 81991533).

High-throughput screening identification of apigenin that reverses the colistin resistance of mcr-1-positive pathogens.

The plasmid-mediated gene mcr-1 that makes bacteria resistant to the antibiotic colistin is spreading quickly, which means that colistin is no longer working well to treat Gram-negative bacterial infections. Herein, we utilized a computer-aided high-throughput screening drugs method to identify the natural product apigenin, a potential mcr-protein inhibitor, which effectively enhanced the antimicrobial activity of colistin. Several assays, including a checkerboard minimum inhibitory concentration assay, a time-kill assay, the combined disk test, molecular simulation dynamics, and animal infection models assay, were conducted to verify whether apigenin enhanced the ability of colistin to fight Gram-negative bacterial infections. The results showed that apigenin improved the antimicrobial activity of colistin against multidrug-resistant Enterobacteriaceae infection. Moreover, apigenin not only did not increase the toxic effect of colistin but also had the ability to effectively inhibit the frequency of bacterial resistance mutations to colistin. Studies clearly elucidated that apigenin could interfere with the thermal stability of the protein by binding to the mcr-1 protein. Additionally, the combination of apigenin and colistin could exert multiple effects, including disrupting bacterial membranes, the generation of bacterial nitric oxide and reactive oxygen species, as well as inhibiting bacterial adenosine triphosphate production. Furthermore, the addition of apigenin was able to significantly inhibit colistin-stimulated high expression levels of the bacterial mcr-1 gene. Finally, apigenin exhibited a characteristic anti-inflammatory effect while enhancing the antimicrobial activity of colistin against mcr-1-positive Escherichia coli (E. coli) infected animals. In conclusion, as a potential lead compound, apigenin is promising in combination with colistin in the future treatment of mcr-1-positive E. coli infections.IMPORTANCEThis study found that apigenin was able to inhibit the activity of the mcr-1 protein using a high-throughput virtual screening method. Apigenin effectively enhanced the antimicrobial activity of colistin against multidrug-resistant Enterobacteriaceae, including mcr-1-positive strains, in vitro and in vivo. This study will provide new options and strategies for the future treatment of multidrug-resistant pathogen infections.

Colistin resistance among the Gram-negative nosocomial pathogens in India: a systematic review and meta-analysis.

The rapid rise of nosocomial infections and the growing ineffectiveness of frontline antibiotics against Gram-negative bacteria (GNB) have put the healthcare sector under unprecedented stress. In this scenario, colistin, an antibiotic of the polymyxin class, has become the last resort treatment option. However, the unrestricted use of colistin in the preceding decades has led to the emergence of colistin-resistant (ColR) bacterial strains. Unfortunately, comprehensive data on the prevalence of ColR nosocomial pathogens in India are scarce. This study was conducted to address this information gap. A systematic review and meta-analysis were conducted to determine the prevalence of ColR among the nosocomial GNB species in India and their geographical distribution. A systematic search of the online databases was performed and eligible studies meeting the inclusion criteria were used for qualitative synthesis. The combined event rate and 95% confidence interval were estimated using a forest plot with a random-effect model. Cochrane Q statistics and I2 statistics were used to detect possible heterogeneity. From a total of 1865 retrieved records from 4 databases, 33 studies were included in the study. Among the most common nosocomial pathogens, Klebsiella pneumoniae showed a rate of ColR at 16.1% (95% CI: 10.1 to 24.6), followed by Pseudomonas aeruginosa (13.3%) (95% CI: 9.1 to 19.2), Acinetobacter baumannii (10%) (95% CI: 7.5 to 13.2), and Escherichia coli (7.8%) (95% CI: 5.3 to 11.2). Interestingly, our analysis revealed that Enterobacter cloacae have the highest rate of ColR at 27.9% (95% CI: 12.7 to 50.9). The results indicate that the prevalence of ColR nosocomial pathogens vary among regions and over time; however, continuous monitoring, and sustained efforts are crucial to ensure the effectiveness of colistin antibiotic.

Comparative Evaluation of Colistin-Susceptibility Testing in Carbapenem-Resistant Klebsiella pneumoniae Using VITEK, Colistin Broth Disc Elution, and Colistin Broth Microdilution.

PURPOSE: The study aimed to compare the results of colistin-susceptibility testing performed using the automated VITEK system, colistin broth microdilution (BMD), and colistin broth disk elution (CBDE) methods. MATERIALS AND METHODS: This exploratory study was conducted in a tertiary care center in South India. Carbapenem-resistant Klebsiella pneumoniae (n = 49) isolates collected from a clinical microbiology laboratory over six months (March-September 2023) were used for the study. RESULTS: Among the 49 carbapenem-resistant Klebsiella pneumoniae isolates, 42 were found to be susceptible to carbapenem by all three methods. Seven isolates were found to be resistant to colistin using BMD and CBDE methods. Two isolates were incorrectly detected as colistin-susceptible, and one isolate was wrongly categorized as colistin-resistant using the automated VITEK system. CONCLUSION:  CBDE is a reliable and cost-effective method that can be adopted in the routine microbiology laboratory for colistin-susceptibility testing, as it does not require any specialized equipment or techniques and is 100% consistent with the gold standard BMD method. Although the automated VITEK system is used in most routine microbiological laboratories for antibiotic-susceptibility testing, it cannot be reliably used for colistin-susceptibility testing due to its high error rates (very major error rate of 28.5%; major error rate of 2.4%).

Limited impact of colistin resistance on mortality of intensive care patients with carbapenem-resistant bacteraemia.

BACKGROUND: Increasing incidence of carbapenem-resistant Gram-negative bacteraemia (CR-GNB) has triggered increased use of polymyxins, likely fuelling the emergence and spread of colistin resistance. AIM: To estimate the excess clinical burden of colistin resistance in intensive care patients with CR-GNB. METHODS: A cohort of patients with CR-GNB during their stay in the intensive care unit (ICU) of a university hospital in Greece over a 4-year period (2020-2023) was constructed. Competing risks survival analysis was performed to estimate the burden associated with colistin resistance. FINDINGS: Of the 177 ICU patients with CR-GNB, 134 (76%) had colistin-resistant isolates, predominantly Acinetobacter baumannii (79%), identified by broth microdilution. Patients with colistin-resistant infection were similar to those with colistin-susceptible infection with respect to age, sex, APACHE II score, Charlson comorbidity index score, Pitt bacteraemia score, prior surgery and the occurrence of polymicrobial cultures. However, patients in the colistin-resistant group had lower risk of mortality compared with those in the colistin-susceptible group (31% vs 44%, P = 0.004 at 14 days, respectively; 46% vs 56% at 28 days, respectively; P = 0.173). Multi-variable regression analysis confirmed that colistin-resistant CR-GNB was associated with significantly lower risk of inpatient death compared with colistin-susceptible CR-GNB within 14 days [cause-specific hazard ratio (csHR) 0.53, 95% CI 0.28-1.01) and 28 days (csHR 0.55, 95% CI 0.31-0.95) of infection onset. CONCLUSION: Limited impact of colistin resistance on mortality was demonstrated in a large contemporary cohort of ICU patients with CR-GNB, possibly reflecting the recent shift away from colistin-based treatment regimens.

First report of detection of mcr-1 and virulence genes in avian pathogenic Escherichia coli in the center of Algeria.

Background: Antimicrobial resistance in avian pathogenic Escherichia coli (APEC) represents a major concern in the avian industry worldwide and limited studies have investigated Colistin resistance among APEC in Algeria. Aims: Investigate antibiotic resistance, in particular, Colistin, and mediated-Colistin resistance (mcr) genes, as well as the virulence genes in APEC. Methods: One hundred E. coli were isolated from poultry suspected of colibacillosis. Antimicrobial susceptibility testing was done on 14 antibiotics by the disk diffusion method. Colistin minimum inhibitory concentration (MIC) was assessed by the broth microdilution method. Using multiplex PCR, mcr genes (mcr-1 to 5) and 7 virulence-related genes were investigated in Colistin-resistant isolates. Results: Results showed high resistance to Tetracycline (99%), Nalidixic acid (92%), Doxycycline (90%), Ampicillin (89%), Ofloxacin (74%), Sulfamethoxazole-Trimethoprim (72%), and Amoxicillin-Clavulanic acid (57%); in addition, 92% of isolates were multidrug resistant. The rate of resistance to Colistin was 27% (27/100) of which 96.3% (26/27) of isolates carried the mcr-1 gene. Twenty-five of the Colistin-resistant isolates (92.59%) had at least three virulence genes. The most frequently isolated virulence genes were: fim H (96.3%) followed by hlyF, iroN, and iss (77.7%, each), iutA and ompT were found in 59.25% and 55.5% of isolates, respectively. The most prevalent combination of virulence factors was hlyF-iss-iroN-iutA-ompT-fimH. Conclusion: This is the first report which highlighted Colistin resistance with the detection of mcr-1 in APEC isolates in the area of study. Colistin resistance and carriage of mcr-1 in virulent and multidrug-resistant isolates of E. coli are alarming and a surveillance program to limit the spread of these pathogens is mandatory.

Repurposing cetylpyridinium chloride and domiphen bromide as phosphoethanolamine transferase inhibitor to combat colistin-resistant Enterobacterales.

The emergence of plasmid-encoded colistin resistance mechanisms, MCR-1, a phosphoethanolamine transferase, rendered colistin ineffective as last resort antibiotic against severe infections caused by clinical Gram-negative bacterial pathogens. Through screening FDA-approved drug library, we identified two structurally similar compounds, namely cetylpyridinium chloride (CET) and domiphen bromide (DOM), which potentiated colistin activity in both colistin-resistant and susceptible Enterobacterales. These compounds were found to insert their long carbon chain to a hydrophobic pocket of bacterial phosphoethanolamine transferases including MCR-1, competitively blocking the binding of lipid A tail for substrate recognition and modification, resulting in the increase of bacterial sensitivity to colistin. In addition, these compounds were also found to dissipate bacterial membrane potential leading to the increase of bacterial sensitivity to colistin. Importantly, combinational use of DOM with colistin exhibited remarkable protection of test animals against infections by colistin-resistant bacteria in both mouse thigh infection and sepsis models. For mice infected by colistin-susceptible bacteria, the combinational use of DOM and colistin enable us to use lower dose of colistin to for efficient treatment. These properties render DOM excellent adjuvant candidates that help transform colistin into a highly potent antimicrobial agent for treatment of colistin-resistant Gram-negative bacterial infections and allowed us to use of a much lower dosage of colistin to reduce its toxicity against colistin-susceptible bacterial infection such as carbapenem-resistant Enterobacterales.

A review of colistin-resistant Escherichia coli isolates in the Middle East: mechanisms, epidemiology, and dissemination from different sources in humans, animals, foodand soil.

Escherichia coli is a normal gut inhabitantthat can cause various diseases ,such as intestinal, urinary tract, bladder infections and systemic infections in humans and animals. The alarming increase in profiles for extended-spectrum ß-lactamase-  and carbapenemase-producing Escherichia coli isolates is a serious problem throughout the world. Colistin is known as a lastresort agent for the treatment of Gram-negative bacterial infections. Inappropriate use of colistin and other classes of antibiotics combined with inadequate infection control, especially in developing countries, can lead to serious public health complications. The global increase in colistin resistance has been reported in many parts of the world, ,including the Middle East. Colistin is used to treat infections caused by extensively drug-resistant Gram-negative bacteria. There are few reliable epidemiologic data on colistin-resistant E. coli isolates, and information on colistin-resistant E. coli from Asia, the largest, most populous, and most diverse continent in the world, is generally limited compared with Europe and the United States.  The data in this review article were compiled from related articles associated with isolated colistin-resistant Escherichia coli (E. coli) isolates from humans, animals, and food-producing animals. In the Middle East, colistin-resistant E. coli isolates were reported from Turkey, Egypt, Saudi Arabia, Algeria, Iran, Iraq, Bahrain, Qatar, Oman, Kuwait, Israel, and Lebanon between 2010 and 2023. While colistin resistance is most commonly observed in E. coli isolates, data have shown that mcr genes are the most common genes associated with colistin resistance in E. coli isolatescompared to mutations in pmrAsB, phoQ, and mgrB genes.

Characterization of a colistin resistant, hypervirulent hospital isolate of Acinetobacter courvalinii from Canada.

Non-baumannii Acinetobacter spp. are becoming more prevalent in clinical settings including those that present resistance to last-resort antibiotics such as colistin. AB222-IK40 is an Acinetobacter courvalinii strain isolated from the Ottawa Hospital Research Institute located in Ottawa, Canada. To our knowledge, it is the first report of clinical A. courvalinii in Canada. Based on the susceptibility profile, AB222-IK40 is resistant to colistin and non-susceptible to ertapenem. Whole-genome sequencing allowed for genomic investigation into colistin resistance mechanisms. No previously identified mechanism(s) were observed, but a mobile colistin resistance (mcr)-like gene and a UDP-glucose dehydrogenase gene were identified. Based on phylogenomic analyses, the mcr-like gene is an intrinsic phosphoethanolamine transferase. This gene family is implicated in one of the many mechanisms responsible for colistin resistance in Acinetobacter baumannii as well as Acinetobacter modestus. UDP-glucose dehydrogenase is involved in colistin resistance in Enterobacterales and has been shown to be involved in capsule formation in A. baumannii. Global lipidomics revealed greater abundance of phosphatidyl-myo-inositol and lyso-phosphatidyl ethanolamine moieties in the membrane of A. courvalinii than in A. baumannii. Lipidomic profiles showed differences that were probably responsible for the colistin resistance phenotype in AB222-IK40. This isolate was also hypervirulent based on survival assays in Galleria mellonella. As this is the first report of A. courvalinii from a hospital in Canada, this species may be an emerging clinical pathogen, and therefore, it is important to understand this mechanism of its colistin resistance and hypervirulence.

Cinnamaldehyde and baicalin reverse colistin resistance in Enterobacterales and Acinetobacter baumannii strains.

PURPOSE: Colistin is used as a last resort antibiotic against infections caused by multidrug-resistant gram-negative bacteria, especially carbapenem-resistant bacteria. However, colistin-resistance in clinical isolates is becoming more prevalent. Cinnamaldehyde and baicalin, which are the major active constituents of Cinnamomum and Scutellaria, have been reported to exhibit antibacterial properties. The aim of this study was to evaluate the capacity of cinnamaldehyde and baicalin to enhance the antibiotic activity of colistin in Enterobacterales and Acinetobacter baumannii strains. METHODS: The MICs of colistin were determined with and without fixed concentrations of cinnamaldehyde and baicalin by the broth microdilution method. The FIC indices were also calculated. In addition, time-kill assays were performed with colistin alone and in combination with cinnamaldehyde and baicalin to determine the bactericidal action of the combinations. Similarly, the effects of L-arginine, L-glutamic acid and sucrose on the MICs of colistin combined with cinnamaldehyde and baicalin were studied to evaluate the possible effects of these compounds on the charge of the bacterial cell- wall. RESULTS: At nontoxic concentrations, cinnamaldehyde and baicalin partially or fully reversed resistance to colistin in Enterobacterales and A. baumannii. The combinations of the two compounds with colistin had bactericidal or synergistic effects on the most resistant strains. The ability of these agents to reverse colistin resistance could be associated with bacterial cell wall damage and increased permeability. CONCLUSION: Cinnamaldehyde and baicalin are good adjuvants for the antibiotic colistin against Enterobacterales- and A. baumannii-resistant strains.

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.

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.

Colistin resistance in ESBL- and Carbapenemase-producing Escherichia coli and Klebsiella pneumoniae clinical isolates in Cambodia.

OBJECTIVES: Despite the critical importance of colistin as a last-resort antibiotic, limited studies have investigated colistin resistance in human infections in Cambodia. This study aimed to investigate the colistin resistance and its molecular determinants among Extended-spectrum beta-lactamase (ESBL)- and carbapenemase-producing (CP) Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) isolated in Cambodia between 2016 and 2020. METHODS: E. coli (n = 223) and K. pneumoniae (n = 39) were tested for colistin minimum inhibitory concentration (MIC) by broth microdilution. Resistant isolates were subjected to polymerase chain reaction (PCR) for detection of mobile colistin resistance genes (mcr) and chromosomal mutations in the two-component system (TCS). RESULTS: Eighteen isolates (10 K. pneumoniae and 8 E. coli) revealed colistin resistance with a rate of 5.9% in E. coli and 34.8% in K. pneumoniae among ESBL isolates, and 1% in E. coli and 12.5% in K. pneumoniae among CP isolates. The resistance was associated with mcr variants (13/18 isolates, mcr-1, mcr-3, and mcr-8.2) and TCS mutations within E. coli and K. pneumoniae, with the first detection of mcr-8.2 in Cambodia, the discovery of new mutations potentially associated to colistin resistance in the TCS of E. coli (PhoP I47V, PhoQ N352K, PmrB G19R, and PmrD G85R) and the co-occurrence of mcr genes and colistin resistance conferring TCS mutations in 11 of 18 isolates. CONCLUSIONS: The findings highlight the presence of colistin resistance in ESBL- and CP- Enterobacteriaceae involved in human infections in Cambodia as well as chromosomal mutations in TCS and the emergence of mcr-8.2 in E. coli and K. pneumoniae. It underscores the need for continuous surveillance, antimicrobial stewardship, and control measures to mitigate the spread of colistin resistance.

Emergence of mcr-4.3 genes in a novel Shewanella specie isolated from the Arctic environment.

Mobile colistin resistance (mcr) genes are pivotal contributors to last-line of antimicrobial resistance in human infections. Shewanella, historically recognized as a natural environmental bacterium with metal reduction capabilities, recently has been observed in clinical settings. However, limited knowledge has been explored on genetic differences between strains from non-clinical and clinical strains. In this study, we conducted the whole genome sequencing on six Arctic strains, illustrated the phylogenetic relationships on published 393 Shewanella strains that categorized the genus into four lineages (L1 to L4). Over 86.4% of clinical strain group (CG) strains belonged to L1 and L4, carrying mcr-4 genes and a complete metal-reduction pathways gene cluster. Remarkably, a novel Arctic Shewanella strain in L3, exhibits similar genetic characteristics with CG strains that carried both mcr-4 genes and a complete metal reduction pathway gene cluster. It raised concerns about the transmission ability from environment to clinic setting causing in the potential infections, and emphasized the need for monitoring the emerging strains with human infections.

Genomic Characteristics of an Extensive-Drug-Resistant Clinical Escherichia coli O99 H30 ST38 Recovered from Wound.

Background: Antibiotic-resistant Escherichia coli is one of the major opportunistic pathogens that cause hospital-acquired infections worldwide. These infections include catheter-associated urinary tract infections (UTIs), ventilator-associated pneumonia, surgical wound infections, and bacteraemia. Objectives: To understand the mechanisms of resistance and prevent its spread, we studied E. coli C91 (ST38), a clinical outbreak strain that was extensively drug-resistant. The strain was isolated from an intensive care unit (ICU) in one of Kuwait's largest hospitals from a patient with UTI. Methods: This study used whole-genome sequencing (Illumina, MiSeq) to identify the strain's multi-locus sequence type, resistance genes (ResFinder), and virulence factors. This study also measured the minimum inhibitory concentrations (MIC) of a panel of antibiotics against this isolate. Results: The analysis showed that E. coli C-91 was identified as O99 H30 ST38 and was resistant to all antibiotics tested, including colistin (MIC > 32 mg/L). It also showed intermediate resistance to imipenem and meropenem (MIC = 8 mg/L). Genome analysis revealed various acquired resistance genes, including mcr-1, bla CTX-M-14, bla CTX-M-15, and bla OXA1. However, we did not detect bla NDM or bla VIM. There were also several point mutations resulting in amino acid changes in chromosomal genes: gyrA, parC, pmrB, and ampC promoter. Additionally, we detected several multidrug efflux pumps, including the multidrug efflux pump mdf(A). Eleven prophage regions were identified, and PHAGE_Entero_SfI_NC was detected to contain ISEc46 and ethidium multidrug resistance protein E (emrE), a small multidrug resistance (SMR) protein family. Finally, there was an abundance of virulence factors in this isolate, including fimbriae, biofilm, and capsule formation genes. Conclusions: This isolate has a diverse portfolio of antimicrobial resistance and virulence genes and belongs to ST38 O99 H30, posing a serious challenge to treating infected patients in clinical settings.

Genetic and structural basis of colistin resistance in Klebsiella pneumoniae: Unravelling the molecular mechanisms.

OBJECTIVE: Antimicrobial resistance (AMR), together with multidrug resistance (MDR), mainly among Gram-negative bacteria, has been on the rise. Colistin (polymyxin E) remains one of the primary available last resorts to treat infections caused by MDR bacteria during the rapid emergence of global resistance. As the exact mechanism of bacterial resistance to colistin remains undetermined, this study warranted elucidation of the underlying mechanisms of colistin resistance and heteroresistance among carbapenem-resistant Klebsiella pneumoniae isolates. METHODS: Molecular analysis was carried out on the resistant isolates using a genome-wide characterisation approach, as well as MALDI-TOF mass spectrometry, to identify lipid A. RESULTS: Among the 32 carbapenem-resistant K. pneumoniae isolates, several isolates showed resistance and intermediate resistance to colistin. The seven isolates with intermediate resistance exhibited the "skip-well" phenomenon, attributed to the presence of resistant subpopulations. The three isolates with full resistance to colistin showed ions using MALDI-TOF mass spectrometry at m/z of 1840 and 1824 representing bisphosphorylated and hexa-acylated lipid A, respectively, with or without hydroxylation at position C'-2 of the fatty acyl chain. Studying the genetic environment of mgrB locus revealed the presence of two insertion sequences that disrupted the mgrB locus in the three colistin-resistant isolates: IS1R and IS903B. CONCLUSIONS: Our findings show that colistin resistance/heteroresistance was inducible with mutations in chromosomal regulatory networks controlling the lipid A moiety and insertion sequences disrupting the mgrB gene, leading to elevated minimum inhibitory concentration values and treatment failure. Different treatment strategies should be employed to avoid colistin heteroresistance-linked treatment failures, mainly through combination therapy using colistin with carbapenems, aminoglycosides, or tigecycline.