Whole-plasmid analysis of NDM-1-producing Acinetobacter seifertii isolate and its fitness in several Acinetobacter species.

OBJECTIVES: The aim of this study is to characterize an NDM-1-producing Acinetobacter seifertii isolates from a patient in South Korea. METHODS: Antibiotic susceptibility testing and genotyping using multigene sequencing were performed and whole plasmid sequences were determined. RESULTS: The genotype of A. seifertii was ST1899, and was resistant to ceftazidime, trimethoprim-sulfamethoxazole, and piperacillin-tazobactam, in addition to carbapenem. blaNDM-1 was surrounded by ISAba125 insertion sequence within the structure of Tn125 in the 47 kb-sized plasmid. The plasmid exhibited a structure similar to that of other plasmids of diverse Acinetobacter species found worldwide. Transconjugation and growth curve indicated that the plasmid was adapted to A. seifertii rather than other closely related Acinetobacter species. CONCLUSION: Acquisition of the carbapenem resistance by horizontal transfer of the blaNDM-1-carrying plasmid from another Acinetobacter species with no growth defect.

Delivery of endolysin across outer membrane of Gram-negative bacteria using translocation domain of botulinum neurotoxin.

The emergence of multidrug resistant pathogens has outpaced the development of new antibiotics, leading to renewed interest in endolysins. Endolysins have been investigated as novel biocontrol agents for Gram-positive bacteria. However, their efficacy against Gram-negative species is limited by the barrier presented by their outer membrane, which prevents endolysin access to the peptidoglycan substrate. Here, we used the translocation domain of botulinum neurotoxin (BoNT) to deliver endolysin across the outer membrane of Gram-negative bacteria. The translocation domain selectively interacts with and penetrates membranes composed of anionic lipids, which have been used in nature to deliver various proteins into animal cells. In addition to the BoNT translocation domain, we have fused bacteriophage-derived receptor binding protein to endolysins. This allows the attached protein to efficiently bind to a broad spectrum of Gram-negative bacteria. By attaching these target-binding and translocation machinery to endolysins, we aimed to develop an engineered endolysin with broad-spectrum targeting and enhanced antibacterial activity against Gram-negative species. To validate our strategy, we designed engineered endolysins using two well-known endolysins, T5 and LysPA26, and tested them against 23 strains from six species of Gram-negative bacteria, confirming that our machinery can act broadly. In particular, we observed a 2.32 log reduction in 30 minutes with only 0.5 µM against an A. baumannii isolate. We also used the SpyTag/SpyCatcher system to easily attach target-binding proteins, thereby improving its target binding ability. Overall, our newly developed endolysin engineering strategy may be a promising approach to control multidrug-resistant Gram-negative bacterial strains.

Polymyxin B nonapeptide potentiates the eradication of Gram-negative bacterial persisters.

Antibiotic-resistant Gram-negative bacteria remain a globally leading cause of bacterial infection-associated mortality, and it is imperative to identify novel therapeutic strategies. Recently, the advantage of using antibacterials selective against Gram-negative bacteria has been demonstrated with polymyxins that specifically target the lipopolysaccharides of Gram-negative bacteria. However, the severe cytotoxicity of polymyxins limits their clinical use. Here, we demonstrate that polymyxin B nonapeptide (PMBN), a polymyxin B derivative without the terminal amino acyl residue, can significantly enhance the effectiveness of commonly used antibiotics against only Gram-negative bacteria and their persister cells. We show that although PMBN itself does not exhibit antibacterial activity or cytotoxicity well above the 100-fold minimum inhibitory concentration of polymyxin B, PMBN can increase the potency of co-treated antibiotics. We also demonstrate that using PMBN in combination with other antibiotics significantly reduces the frequency of resistant mutant formation. Together, this work provides evidence of the utilities of PMBN as a novel potentiator for antibiotics against Gram-negative bacteria and insights for the eradication of bacterial persister cells during antibiotic treatment. IMPORTANCE: The significance of our study lies in addressing the problem of antibiotic-resistant Gram-negative bacteria, which continue to be a global cause of mortality associated with bacterial infections. Therefore, identifying innovative therapeutic approaches is an urgent need. Recent research has highlighted the potential of selective antibacterials like polymyxins, which specifically target the lipopolysaccharides of Gram-negative bacteria. However, the clinical use of polymyxins is limited by their severe cytotoxicity. This study unveils the effectiveness of polymyxin B nonapeptide (PMBN) in significantly enhancing the eradication of persister cells in Gram-negative bacteria. Although PMBN itself does not exhibit antibacterial activity or cytotoxicity, it remarkably reduces persister cells during the treatment of antibiotics. Moreover, combining PMBN with other antibiotics reduces the emergence of resistant mutants. Our research emphasizes the utility of PMBN as a novel potentiator to decrease persister cells during antibiotic treatments for Gram-negative bacteria.

Colistin heteroresistance in Citrobacter freundii clinical isolates from Republic of Korea.

We investigated colistin heteroresistance in Citrobacter freundii isolates from Korean hospitals. Using population analysis profiling (PAP), we detected colistin heteroresistance in 31.3% of isolates. Among these, ST217 was the most prevalent clone (58.5%), particularly within colistin-heteroresistant isolates (80.0%). Interestingly, the second most common clone, ST248, was not found in heteroresistant isolates. We identified amino acid changes in PhoQ, PmrA, and PmrB, along with mRNA overexpression in pmrB and arnD. Colistin monotherapy showed no efficacy, but a combination of colistin and ciprofloxacin successfully eradicated all five isolates, even at 0.5 × minimum inhibitory concentrations. This study underscores the high prevalence of colistin heteroresistance in C. freundii isolates, limiting the effectiveness of colistin monotherapy. Combining colistin with ciprofloxacin may offer a viable treatment option for C. freundii infections.

Conversion to colistin susceptibility by tigecycline exposure in colistin-resistant Klebsiella pneumoniae and its implications to combination therapy.

OBJECTIVES: This study investigated the effect of tigecycline exposure on susceptibility of colistin-resistant Klebsiella pneumoniae isolates to colistin and explored the possibility of antibiotic combination at low concentrations to treat colistin-resistant K. pneumoniae isolates. METHODS: Twelve tigecycline-resistant (TIR) mutants were induced in vitro from wild-type, colistin-resistant, and tigecycline-susceptible K. pneumoniae isolates. Antibiotic susceptibility was determined using the broth microdilution method. The deduced amino acid alterations were identified for genes associated with colistin resistance, lipid A biosynthesis, and tigecycline resistance. Expression levels of genes were compared between wild-type stains and TIR mutants using quantitative real-time polymerase chain reaction (PCR). Lipid A modification was explored using MALDI-TOF mass spectrometry. Time-killing assay was performed to assess the efficiency of combination therapy using low concentrations of colistin and tigecycline. RESULTS: All TIR mutants except one were converted to be susceptible to colistin. These TIR mutants had mutations in the ramR gene and increased expression levels of ramA. Three genes associated with lipid A biosynthesis, lpxC, lpxL, and lpxO, were also overexpressed in TIR mutants, although no mutation was observed. Additional polysaccharides found in colistin-resistant, wild-type strains were modified in TIR mutants. Colistin-resistant K. pneumoniae strains were eliminated in vitro by combining tigecycline and colistin at 2 mg/L. In this study, we found that tigecycline exposure resulted in reduced resistance of colistin-resistant K. pneumoniae to colistin. Such an effect was mediated by regulation of lipid A modification involving ramA and lpx genes. CONCLUSION: Because of such reduced resistance, a combination of colistin and tigecycline in low concentrations could effectively eradicate colistin-resistant K. pneumoniae strains.

Roles of crrAB two-component regulatory system in Klebsiella pneumoniae: growth yield, survival in initial colistin treatment stage, and virulence.

OBJECTIVES: Alternation of the colistin resistance-regulating two-component regulatory system (crrAB) is a colistin-resistance mechanism in Klebsiella pneumoniae (K. pneumoniae), but its role in bacteria is not fully understood. METHODS: Twelve colistin-susceptible K. pneumoniae clinical isolates were included in this study: six crrAB-positive and six crrAB-negative. We deleted the crrAB genes from two crrAB-positive isolates and complemented them. We measured the growth yields by determining growth curves in lysogeny broth and minimal media with or without Fe2+. In vitro selection rates for colistin resistance were determined by exposure to colistin, and survival rates against high concentrations of colistin (20 mg/L) at the early stage of growth (20 min) were investigated. Virulence was determined using a serum bactericidal assay and Galleria mellonella larval infection. RESULTS: The presence of crrAB was not associated with colistin resistance and did not increase the in vitro selection rate of colistin resistance after exposure. The growth yield of crrAB-positive isolates was higher in lysogeny broth media and increased when Fe2+ was added to minimal media. The crrAB-positive isolates showed higher survival rates in the early stages of exposure to high colistin concentrations. Decreased serum resistance was identified in the crrAB-deleted mutants. More G. mellonella larvae survived when infected by crrAB-deleted mutants, and higher survival rates of bacteria were identified within the larvae infected with wild-type than crrAB-deletant isolates. CONCLUSION: Through rapid response to external signals, crrAB would provide advantages for K. pneumoniae survival by increasing the final growth yield and initial survival against colistin treatment. This may partly contribute to the bacterial virulence.

Extensively drug-resistant Enterobacter ludwigii co-harbouring MCR-9 and a multicopy of blaIMP-1 in South Korea.

In this study, we describe an Enterobacter ludwigii clinical isolate that is resistant to both carbapenems and colistin in South Korea. Antimicrobial susceptibility testing revealed that E. ludwigii CRE2104-31 was non-susceptible to all tested antibiotics except fosfomycin. Whole genome sequencing identified a 323-kbp IncHI2 plasmid, pCRE2104-31a, that was co-harbouring mobile colistin resistance (mcr)-9.1 and blaIMP-1. In comparison with other full plasmids, pCRE2104-31a exhibited the closest similarity to a plasmid from the Klebsiella pneumoniae strain CNR48 from France, with 19.9% query coverage and 99% identity. Notably, we observed five tandem repeats of blaIMP-1 and aac(6')-Il genes, accompanied by multiple attCs within a class I integron on the Tn402-like transposon. The unit of blaIMP-1-attC-aac(6')-Il-attC might have accumulated due to multiple convergent events. In addition to mcr-9.1 and blaIMP-1, various other antibiotic resistance-associated genes were identified in the plasmid, as follows: blaTEM-1B, aph(3')-I, aph(3')-Ia, aac(6')-Il, aac(6')-IIc, aac(6')-IIa, aph(6)-Id, aph(3'')-Ib, aadA2b, aac(6')-Ib3, sul, dfrA19, qnrB2, aac(6')-Ib-cr, ere(A), and qacE. A conjugation assay showed that the mcr-9.1/blaIMP-1-co-bearing plasmid was self-transmissible to E. coli J53. However, colistin and carbapenem resistance could not be transferred to E. coli due to high incompatibility. The convergence of mcr and carbapenemase genes is thought to be host-dependent among Enterobacteriaceae. The emergence of extensively drug-resistant E. ludwigii co-harbouring MCR-9.1 and a multicopy of blaIMP-1 would pose a significant threat within the compatible Enterobacteriaceae.

Antibiotic Efficacy in Escherichia coli and Klebsiella pneumoniae Under Nutrient Limitation and Effectiveness of Colistin-Based Antibiotic Combinations to Eradicate Persister Cells.

Persister cells are responsible for recurrent or chronic infections resulting in antibiotic treatment failure. We aimed to investigate antibiotic efficacy in Escherichia coli and Klebsiella pneumoniae strains with limited metabolic activity. Bacterial cells cultured in nutrient-limited media showed characteristic persister phenotypes, including low intracellular ATP concentration, maintenance of antibiotic susceptibility, and an increase of (p)ppGpp levels. Amikacin showed no bactericidal activity under nutrient limitation conditions; however, metabolism-dependent ciprofloxacin exhibited metabolism-independent activity. The activity of colistin was metabolism-dependent, but it was retained under limited nutrient conditions. Nutrient limitation and antibiotic stress were related to the SOS response through recA expression in all four strains of E. coli and K. pneumoniae. However, the mRNA expression patterns of relA and spoT (associated with (p)ppGpp synthesis) and hpf and rpoS (downstream target genes of (p)ppGpp signaling) varied according to bacterial species, strain, and antibiotics, indicating diverse responses to nutrient stress in various persister cells. We also investigated the efficacy of antibiotic combinations to eradicate persister cells. As a result, colistin-based combinations were effective in the eradication of both E. coli and K. pneumoniae persister cells. In this study, persister cells were shown to be induced by metabolic stress, reducing antibiotic efficacy. We identified that combinations of colistin with amikacin or ciprofloxacin were effective to eliminate E. coli and K. pneumoniae persister cells.

Multiple heteroresistance to tigecycline and colistin in Acinetobacter baumannii isolates and its implications for combined antibiotic treatment.

BACKGROUND: We investigated the presence of heteroresistance against both tigecycline and colistin in Acinetobacter baumannii and then evaluated the effectiveness of combined antibiotic treatment given the existence of discrete tigecycline- and colistin-resistant subpopulations. METHODS: We performed population analysis profiling (PAP) to evaluate the degree of composite heteroresistance in A. baumannii isolates, with the extent of this resistance quantified using subsequent antibiotic susceptibility testing. We then evaluated the amino acid sequence of PmrBAC and the relative mRNA expression levels of pmrB. Finally, we investigated the combined antibiotic efficacy of tigecycline and colistin in multiple-heteroresistant isolates using dual PAP and in vitro time-killing assays. RESULTS: All tigecycline-heteroresistant A. baumannii isolates, with the exception of one colistin-resistant isolate, were also heteroresistant to colistin. Evaluations of the colistin-resistant subpopulations revealed amino acid alterations in PmrA and PmrB and increased expression of pmrB. All tigecycline-resistant subpopulations were susceptible to colistin, and all colistin-resistant subpopulations were susceptible to tigecycline. Dual PAP analysis using tigecycline and colistin showed no heteroresistance, and in vitro time-killing assays revealed that a combination of these two antibiotics effectively eliminated the bacterial cells. CONCLUSION: Our results suggest that multiple heteroresistance to tigecycline and colistin is highly prevalent among A. baumannii clinical isolates and that these resistant subpopulations exist independently in single multiple heteroresistant isolates. Therefore, our findings may explain the success of combined antibiotic therapies in these infections.

Lipid A modification-induced colistin-resistant Klebsiella variicola from healthy adults.

Background. Klebsiella variicola was once recognised as a benign plant-endosymbiont but recent case reports suggest that it is a newly emerging Gram-negative pathogen related to opportunistic infection of multiple sites in humans.Methods. Antimicrobial susceptibility testing was performed using broth microdilution method. To identify colistin resistance mechanisms, phoPQ, pmrAB, and mgrB were sequenced and their mRNA expression was analysed using quantitative real-time PCR. In addition, we tried to detect crrAB and mcr. The lipid A moieties of colistin-susceptible and -resistant isolates were analysed using MALDI-TOF.Results. Among the two K. variicola isolates, one is colistin-resistant, and another is colistin-susceptible. The colistin-resistant K. variicola isolate showed no mutations in phoPQ, pmrAB, and mgrB, and crrAB and mcr were not identified. However, its phoQ and pbgP expression was significantly higher and amino-arabinosylated lipid A with hexa-acylated species in lipopolysaccharide was identified.Conclusions. We found that colistin resistance in K. variicola was mediated by the modification of lipid A. Although the isolate was obtained from faecal samples of healthy adults, colistin-resistant K. variicola challenges public health as an opportunistic pathogen.

Treatment of Colistin Dependence-Developing Acinetobacter baumannii with Antibiotic Combinations at Subinhibitory Concentrations.

Recent studies have revealed that colistin dependence frequently develops in colistin-susceptible Acinetobacter baumannii isolates. Despite resistance in parental strains, colistin-dependent mutants showed increased susceptibility to several antibiotics, which suggests the possibility of developing strategies to eliminate multidrug-resistant (MDR) A. baumannii. We investigated in vitro and in vivo efficacy of combinations of colistin and other antibiotics using MDR A. baumannii strains H08-391, H06-855, and H09-94, which are colistin-susceptible but develops colistin dependence upon exposure to colistin. An in vitro time-killing assay, a checkerboard assay, and an antibiotic treatment assay using Galleria mellonella larvae were performed. Although a single treatment of colistin at a high concentration did not prevent colistin dependence, combinations of colistin with other antibiotics at subinhibitory concentrations, especially amikacin, eradicated the strains by inhibiting the development of colistin dependence, in the in vitro time-killing assay. Only 40% of G. mellonella larvae infected by A. baumannii survived with colistin treatment alone; however, all or most of them survived following treatment with the combination of colistin and other antibiotics (amikacin, ceftriaxone, and tetracycline). Our results suggest the possibility of the combination of colistin and amikacin or other antibiotics as one of therapeutic options against A. baumannii infections by eliminating colistin-dependent mutants.

Species identification, antibiotic resistance, and virulence in Enterobacter cloacae complex clinical isolates from South Korea.

This study aimed to identify the species of Enterobacter cloacae complex (ECC) isolates and compare the genotype, antibiotic resistance, and virulence among them. A total of 183 ECC isolates were collected from patients in eight hospitals in South Korea. Based on partial sequences of hsp60 and phylogenetic analysis, all ECC isolates were identified as nine species and six subspecies. Enterobacter hormaechei was the predominant species (47.0%), followed by Enterobacter kobei, Enterobacter asburiae, Enterobacter ludiwigii, and Enterobacter roggenkampii. Multilocus sequence typing analysis revealed that dissemination was not limited to a few clones, but E. hormaechei subsp. xiangfangensis, E. hormaechei subsp. steigerwaltii, and E. ludwigii formed large clonal complexes. Antibiotic resistance rates were different between the ECC species. In particular, E. asburiae, E. kobei, E. roggenkampii, and E. cloacae isolates were highly resistant to colistin, whereas most E. hormaechei and E. ludwigii isolates were susceptible to colistin. Virulence was evaluated through serum bactericidal assay and the Galleria mellonella larvae infection model. Consistency in the results between the serum resistance and the G. mellonella larvae infection assay was observed. Serum bactericidal assay showed that E. hormaechei, E. kobei, and E. ludwigii were significantly more virulent than E. asburiae and E. roggenkampii. In this study, we identified the predominant ECC species in South Korea and observed the differences in antibiotic resistance and virulence between the species. Our findings suggest that correct species identification, as well as continuous monitoring is crucial in clinical settings.

Reduced virulence in tigecycline-resistant Klebsiella pneumoniae caused by overexpression of ompR and down-regulation of ompK35.

BACKGROUND: The development of tigecycline resistance in hypervirulent Klebsiella pneumoniae strains has resulted in decreased virulence that is associated with reduced production of capsular polysaccharides (CPS). In this study, we investigated the mechanisms that link tigecycline susceptibility to decreased virulence. METHODS: We compared transcriptomes from tigecycline-susceptible wild-type strains and tigecycline-resistant mutants using mRNA sequencing. ompR-overexpressed and ompR-deleted mutants were constructed from wild-type strains and tigecycline-resistant mutants, respectively. Antibiotic susceptibility tests were performed, and string tests and precipitation assays were conducted to identify phenotypic changes related to tigecycline susceptibility and ompR expression. Bacterial virulence was assessed by serum resistance and Galleria mellonella infection assays. RESULTS: Transcriptomic analyses demonstrated a significant decrease in the expression of ompK35 in the tigecycline-resistant mutants. We observed that tigecycline-resistant mutants overexpressed ompR, and that the expression of ompK35 was regulated negatively by ompR. While tigecycline-resistant mutants and ompR-overexpressed mutants exhibited reduced hypermucoviscosity and virulence, deletion of ompR from tigecycline-resistant mutants restored their hypermucoviscosity and virulence. CONCLUSIONS: In hypervirulent K. pneumoniae strains, ompR expression, which is regulated by exposure to tigecycline, may affect the production of CPS, leading to bacterial virulence.

Exploitation of a novel adjuvant for polymyxin B against multidrug-resistant Acinetobacter baumannii.

BACKGROUND: Although polymyxin has been used as a last-resort antibiotic against resistant bacteria, its use is restricted due to nephrotoxicity and neurotoxicity. While the present antibiotic resistance issue compels clinicians to reconsider polymyxin use in severe illness cases, polymyxin-resistant microorganisms exert an effect. OBJECTIVES: To address the issue of antibiotic resistance, the cycle of developing new antibiotics to counteract emerging resistance must be discontinued. Here we tried to develop novel therapies that do not rely on direct antimicrobial activity and thus do not promote antibiotic resistance. METHODS: By a high-throughout screening system based on bacterial respiration, chemical compounds accelerating the antimicrobial effects of polymyxin B were screened. In vitro and in vivo tests were performed to validate adjuvanticity. In addition, membrane depolarization and total transcriptome analysis were used to determine molecular mechanisms. RESULTS: PA108, a newly discovered chemical compound, was used to eradicate polymyxin-resistant A. baumannii and three other species in the presence of polymyxin B at concentrations less than the MIC. Since this molecule lacks self-bactericidal action, we hypothesized that PA108 acts as an antibiotic adjuvant, enhancing the antimicrobial activity of polymyxin B against resistant bacteria. At working concentrations, no toxicity was observed in cell lines or mice, although co-treatment with PA108 and polymyxin B increased survival of infected mouse and decreased bacterial loads in organs. CONCLUSIONS: Boosting antibiotic efficiency through the use of antibiotic adjuvants holds significant promise for tackling the rise in bacterial antibiotic resistance.

Development mechanism of resistant population post-exposure to tigecycline in tigecycline-heteroresistant Acinetobacter baumannii strain.

Tigecycline heteroresistance is highly prevalent in Acinetobacter baumannii clinical isolates, reducing the efficacy of tigecycline treatment. This study investigated the population dynamics of A. baumannii with tigecycline heteroresistance to determine the origin of resistance that occurs over time after antibiotic exposure. Tigecycline heteroresistance was imitated by mixing tigecycline-susceptible and -resistant A. baumannii isolates in a 1:10-6 ratio, and confirmed using population analysis profiling. Growth curves and an in-vitro competition assay found no difference in bacterial fitness between tigecycline-resistant and -susceptible populations. The green fluorescent protein (GFP) expression system and flow cytometry were used to monitor the population dynamics of the heteroresistant population, while differentiating the resistant population from the susceptible population. The mimicked tigecycline heteroresistance was confirmed to be reproducible and stable without tigecycline. The GFP-expressing population (i.e. the resistant population) nearly went undetected because it only represented approximately 10-6 of the entire population. However, when the mimicked tigecycline-heteroresistant strain was treated with tigecycline, most subpopulations expressing GFP were detected. The surviving A. baumannii population, upon exposure to tigecycline, exhibited a high minimum inhibitory concentration for tigecycline, equivalent to that of tigecycline-resistant isolates that were used to mimic heteroresistance. These results indicate that the development of resistance in tigecycline-heteroresistant A. baumannii strains, resulting in decreased antibiotic efficacy, may depend on the selection of a pre-existing resistant subpopulation.

ColE-type plasmid bearing blaOXA-232 increases persister cell formation.

OBJECTIVES: Bacterial persister cells are a sub-population of cells that are tolerant to high concentrations of antibiotics. In this study, we investigated the effect of plasmids bearing carbapenemase genes on persister cell formation. METHODS: Three plasmids, IncX3-type plasmid with blaNDM-1, IncN-type plasmid with blaKPC-2, and ColE-type plasmid with blaOXA-232, were transformed into Escherichia coli MG1655. For the ColE-type plasmid (pM5_OXA232), gene-deletion plasmids were constructed and transformed into the MG1655. Persister assays were performed against ciprofloxacin and amikacin, and expression levels of relA and spoT were measured for the wild-type E. coli and all transformants. RESULTS: Unlike the other two plasmids, transformation of ColE-type plasmid (pM5_OXA232) caused a significant increase in the formation of persister cells. Compared with transformants that harboured intact pM5_OXA232, transformants that harboured plasmids with deletions of gene(s), vbhA, hypothetical gene, or a mobile gene cassette showed decreased persister cell formation. Expression levels of relA and spoT exhibited patterns similar to those of persister cell formation rates, particularly against ciprofloxacin. CONCLUSION: In this study, we showed that a small ColE-type plasmid bearing blaOXA-232 has an effect on persister cell formation, possibly contributing to the dissemination of low-level carbapanemase.

Improvement of transformation efficiency in hypermucoviscous Klebsiella pneumoniae using citric acid.

Transformation efficiency is very low in hypermucoviscous Kllebsiella pneumoniae owing to high amount of capsular polysaccharide. Here, we propose a method to improve the transformation in hypermucoviscous K. pneumoniae using citric acid. An increased number of recovered transformants was observed in the strains cultured in the presence of citric acid.