Genome analysis of tigecycline-resistant Acinetobacter baumannii reveals nosocomial lineage shifts and novel resistance mechanisms.

OBJECTIVES: To investigate the characteristics and clonal dynamics of tigecycline-resistant Acinetobacter baumannii (TRAB) isolates from a Chinese hospital from 2016 to 2021. METHODS: A total of 64 TRAB isolates were screened and WGS was performed. Phylogenetic analysis and non-polymorphic mutation analysis were used to analyse their clonal dynamics and tigecycline resistance-related mutations. RT-PCR was used to analyse the expression of the resistance-nodulation cell-division (RND) efflux pump genes adeB and adeJ. Gene cloning was used to explore the effect of tet(39) variants on tigecycline resistance. RESULTS: Most TRAB isolates were found to be MDR, with 95% (61/64) of the isolates showing resistance to carbapenems. These TRAB isolates were classified into three primary genetic clusters based on core-genome SNPs. The KL2 cluster persisted throughout the study period, whereas the KL7 cluster emerged in 2019 and became the dominant clone. The KL7 cluster carried more antimicrobial resistance genes than the other two clusters. The predominant tigecycline resistance mechanism of the KL2 cluster and KL7 cluster was IS insertion in adeN (82.1%, 23/28) and genetic alterations in adeS (76.2%, 16/21), respectively. Eleven novel AdeS mutations were identified associated with elevated AdeB expression and tigecycline resistance. Moreover, we characterized a plasmid-borne tet(39) variant with an Ala-36-Thr substitution that synergizes with the RND efflux pump to confer high-level tigecycline resistance. CONCLUSIONS: This work provides important insights into the diverse mechanisms associated with tigecycline resistance in A. baumannii, highlighting a pressing need for further monitoring of ST2-KL7 A. baumannii in clinical settings.

Evaluation of resazurin microplate method for rapid detection of vancomycin and linezolid resistance in Enterococcus faecalis and Enterococcus faecium clinical isolates.

BACKGROUND: Vancomycin and linezolid resistance among enterococci is an increasing problem due to a lack of alternative antibiotics. Early identification of vancomycin-resistant and linezolid-resistant strains can help prevent the spread of resistance to these antibiotics. Hence, early, rapid and accurate detection of vancomycin and linezolid resistance is critical. OBJECTIVES: The resazurin microplate method (RMM) was developed for detecting vancomycin and linezolid susceptibility among Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) clinical isolates, and its performance was further evaluated. METHODS: A total of 209 non-duplicate clinical isolates and three strains from the faeces of domestic animals, including 142 E. faecalis (71 linezolid non-susceptible and 71 linezolid susceptible) and 70 E. faecium (23 vancomycin non-susceptible, 23 vancomycin susceptible, 12 linezolid non-susceptible and 12 linezolid susceptible), were tested using RMM. RESULTS: The susceptibility of E. faecium to vancomycin was detected within 5 h, with high susceptibility (23/23) and specificity (23/23). The susceptibility of E. faecalis and E. faecium to linezolid was detected within 4 h, with specificities of 98.59% and 100% and susceptibilities of 94.37% and 58.33% for E. faecalis and E. faecium, respectively. CONCLUSIONS: RMM had a good positive predictive value for the detection of vancomycin-non-susceptible E. faecium and linezolid-non-susceptible E. faecalis. It thus has the potential to become an alternative method for the rapid screening of these resistant pathogens in clinical practice.

Emergence of tet(X2) in Acinetobacter pittii confers clinical resistance to tigecycline.

OBJECTIVES: To characterize a novel transposon Tn7533 carrying the tet(X2) gene in a tigecycline-resistant Acinetobacter pittii BM4623 of clinical origin. METHODS: Gene knockout and in vitro cloning were used to verify the function of tet(X2). WGS and comparative genomic analysis were used to explore the genetic characteristics and molecular evolution of tet(X2). Inverse PCR and electroporation experiments were used to evaluate the excision and integration capabilities of Tn7533. RESULTS: A. pittii BM4623 belonged to a novel ST, ST2232 (Pasteur scheme). Knockout of tet(X2) in BM4623 restored its susceptibility to tigecycline. Cloning of the tet(X2) gene into Escherichia coli DH5α and Acinetobacter baumannii ATCC 17978 resulted in 16-fold or more increases in MICs of tigecycline. Sequence analysis showed that the region upstream of tet(X2) exhibited a high degree of diversity, while there was a 145 bp conserved region downstream of tet(X2). tet(X2) in BM4623 was located on a novel composite transposon Tn7533, which also contains multiple resistance genes including blaOXA-58. Tn7533 could be excised from the chromosome to form a circular intermediate and transferred into A. baumannii ATCC 17978 by electroporation. CONCLUSIONS: Our study demonstrates that tet(X2) is a determinant conferring clinical resistance to tigecycline in Acinetobacter species. The emergence of Tn7533 may lead to the potential dissemination of tigecycline and carbapenem resistance in Acinetobacter, which requires continuous monitoring.

Acetylcysteine increases sensitivity of ceftazidime-avibactam-resistant enterobacterales with different enzymatic resistance to ceftazidime-avibactam in vitro and in vivo.

BACKGROUND: Ceftazidime-avibactam (CZA) improves treatment outcomes for infections caused by carbapenem-resistant organisms, but has led to serious bacterial resistance. Acetylcysteine (NAC) is an approved medication that protects the respiratory tract through antioxidant and anti-inflammatory effects. RESULTS: This study found that NAC combined with CZA effectively inhibits the growth of CZA-resistant clinical Enterobacterales strains. The CZA/NAC combination inhibits biofilm formation in vitro and decreases bacterial burden in a mouse thigh infection model. The combination is biocompatible and primarily increases cell membrane permeability to cause bacterial death. CONCLUSIONS: These findings prove that the CZA/NAC combination has potential as a treatment for CZA-resistant Enterobacterales infections.

PAM-1: an antimicrobial peptide with promise against ceftazidime-avibactam resistant Escherichia coli infection.

Introduction: Antibiotic misuse and overuse have led to the emergence of carbapenem-resistant bacteria. The global spread of resistance to the novel antibiotic combination ceftazidime-avibactam (CZA) is becoming a severe problem. Antimicrobial peptide PAM-1 offers a novel approach for treating infections caused by antibiotic-resistant bacteria. This study explores its antibacterial and anti-biofilm activities and mechanisms against CZA-resistant Escherichia. Coli (E. coli), evaluating its stability and biosafety as well. Methods: The broth microdilution method, growth curve analysis, crystal violet staining, scanning electron microscopy, and propidium iodide staining/N-phenyl-1-naphthylamine uptake experiments were performed to explore the antibacterial action and potential mechanism of PAM-1 against CZA-resistant E. coli. The biosafety in diverse environments of PAM-1 was evaluated by red blood cell hemolysis, and cytotoxicity tests. Its stability was further assessed under different temperatures, serum concentrations, and ionic conditions using the broth microdilution method to determine its minimum inhibitory concentration (MIC). Galleria mellonella infection model and RT-qPCR were used to investigate the in vivo antibacterial and anti-inflammatory effects. Results and discussion: In vitro antibacterial experiments demonstrated that the MICs of PAM-1 ranged from 2 to 8 µg/mL, with its effectiveness sustained for a duration of 24 h. PAM-1 exhibited significant antibiofilm activities against CZA-resistant E. coli (p < 0.05). Furthermore, Membrane permeability test revealed that PAM-1 may exert its antibacterial effect by disrupting membrane integrity by forming transmembrane pores (p < 0.05). Red blood cell hemolysis and cytotoxicity tests revealed that PAM-1 exerts no adverse effects at experimental concentrations (p < 0.05). Moreover, stability tests revealed its effectiveness in serum and at room temperature. The Galleria mellonella infection model revealed that PAM-1 can significantly improve the survival rate of Galleria mellonella (>50%)for in vivo treatment. Lastly, RT-qPCR revealed that PAM-1 downregulates the expression of inflammatory cytokines (p < 0.05). Overall, our study findings highlight the potential of PAM-1 as a therapeutic agent for CZA-resistant E. coli infections, offering new avenues for research and alternative antimicrobial therapy strategies.

Azomycin Orchestrate Colistin-Resistant Enterobacter cloacae Complex's Colistin Resistance Reversal In Vitro and In Vivo.

The Enterobacter cloacae complex (ECC) is a group of nosocomial pathogens that pose a challenge in clinical treatment due to its intrinsic resistance and the ability to rapidly acquire resistance. Colistin was reconsidered as a last-resort antibiotic for combating multidrug-resistant ECC. However, the persistent emergence of colistin-resistant (COL-R) pathogens impedes its clinical efficacy, and novel treatment options are urgently needed. We propose that azomycin, in combination with colistin, restores the susceptibility of COL-R ECC to colistin in vivo and in vitro. Results from the checkerboard susceptibility, time-killing, and live/dead bacterial cell viability tests showed strong synergistic antibacterial activity in vitro. Animal infection models suggested that azomycin-colistin enhanced the survival rate of infected Galleria mellonella and reduced the bacterial load in the thighs of infected mice, highlighting its superior in vivo synergistic antibacterial activity. Crystal violet staining and scanning electron microscopy unveiled the in vitro synergistic antibiofilm effects of azomycin-colistin. The safety of azomycin and azomycin-colistin at experimental concentrations was confirmed through cytotoxicity tests and an erythrocyte hemolysis test. Azomycin-colistin stimulated the production of reactive oxygen species in COL-R ECC and inhibited the PhoPQ two-component system to combat bacterial growth. Thus, azomycin is feasible as a colistin adjuvant against COL-R ECC infection.

Restoring Colistin Sensitivity and Combating Biofilm Formation: Synergistic Effects of Colistin and Usnic Acid against Colistin-Resistant Enterobacteriaceae.

Colistin (COL), the last line of defense in clinical medicine, is an important therapeutic option against multidrug-resistant Gram-negative bacteria. In this context, the emergence of colistin-resistant (COL-R) bacteria mediated by broad-spectrum efflux pumps, mobile genetic elements, and biofilm formation poses a significant public health concern. In response to this challenge, a novel approach of combining COL with usnic acid (UA) has been proposed in this study. UA is a secondary metabolite derived from lichens and is well-known for its anti-inflammatory properties. This study aimed to investigate the synergistic effects of UA and COL against COL-R Enterobacteriaceae both in vitro and in vivo. The exceptional synergistic antibacterial activity exhibited by the combination of COL and UA was demonstrated by performing a comprehensive set of assays, including the checkerboard assay, time-dependent killing assay, and Live/Dead bacterial cell viability assay. Furthermore, crystal violet staining and scanning electron microscopy assays revealed the inhibitory effect of this combination on the biofilm formation. Mechanistically, the combination of UA and COL exacerbated cell membrane rupture, induced DNA damage, and generated a significant amount of reactive oxygen species, which ultimately resulted in bacterial cell death. In addition, erythrocyte hemolysis and cell viability tests confirmed the biocompatibility of the combination. The evaluation of the COL/UA combination in vivo using Galleria mellonella larvae and a mouse infection model showed a significant improvement in the survival rate of the infected larvae as well as a reduction in the bacterial load in the mouse thigh muscle. These findings, for the first time, provide strong evidence for the potential application of COL/UA as an effective alternative therapeutic option to combat infections caused by COL-R Enterobacteriaceae strains.

A novel transposon Tn7540 carrying blaNDM-9 and fosA3 in chromosome of a pathogenic multidrug-resistant Salmonella enterica serovar Indiana isolated from human faeces.

OBJECTIVES: Emergence of multidrug-resistant (MDR) Salmonella enterica serovar Indiana has raised global concern. Mobile genetic elements (MGEs) play vital roles in accelerating the dissemination of resistance genes in bacteria communities. The study aims to improve our understanding of the underlying resistance mechanisms and characterize the MGEs in a MDR S. Indiana isolate. METHODS: Here, we report the characteristics of a MDR pathogenic S. Indiana isolate. The antimicrobial susceptibility pattern of S. Indiana QT6365 was determined. The genomic structure of the chromosome and the plasmid, serotype, and multi-locus sequence type were analysed by whole genome sequencing. The circular form derived from IS26-flanked transposon was confirmed by reverse polymerase chain reaction and sequencing. RESULTS: S. Indiana QT6365 exhibited resistance to all tested antimicrobials except for aztreonam, amikacin, polymyxin, and tigecycline, was defined as MDR, and belonged to ST17. S. Indiana QT6365 was closely related with food resource S. Indiana C629 with similar resistance gene profiles. Multiple resistance genes are mainly carried by a novel transposon Tn7540 located on the chromosome and an IncHI2/HI2A/N plasmid. Sequence analysis and the formed circular intermediate suggested Tn7540 might be generated through homologous recombination by IS26-bounded translocatable units (IS26-fosA-IS26-intI1-dfrA12-aadA2-sul1-ISCR1-blaNDM-9-IS26). CONCLUSIONS: To the best of our knowledge, this is the first report of the novel chromosomal transposon possessing blaNDM-9 and fosA3 in S. Indiana isolated from human specimen, which might facilitate the dissemination of resistance genes and should arouse serious awareness.

Octominin: An antimicrobial peptide with antibacterial and anti-inflammatory activity against carbapenem-resistant Escherichia coli both in vitro and in vivo.

OBJECTIVES: The emergence of carbapenem-resistant Escherichia coli (CREC) is a global concern as its prevalence restricts treatment options and poses a considerable threat to public health. In this study, in vitro and in vivo activity of the antimicrobial peptide Octominin against CREC was investigated to reveal possible mechanisms of action. Furthermore, its safety and factors influencing its antibacterial effect were assessed. Additionally, the anti-inflammatory effects of Octominin were examined. METHODS: The antimicrobial activity of Octominin against 11 strains of CREC was determined using the broth microdilution method, growth curve, and time-kill assay. Its possible mechanism of action was unraveled using the propidium iodide and N-phenyl-1-naphthylamine fluorochrome and lipopolysaccharide-binding assays. To understand the safety and stability of Octominin, its cytotoxicity, hemolysis, and antibacterial activity under various conditions (i.e, temperature, ions) were estimated. Additionally, a Galleria mellonella infection model was utilized to evaluate the efficacy of Octominin in vivo, and qRT-PCR was performed to assess its effect on the expression of proinflammatory cytokines. RESULTS: Octominin displayed a significant antibacterial effect, with MICs of 4-8 µg/mL and MBCs of 8-16 µg/mL. Octominin exerted its antibacterial effect by disrupting bacterial membranes. Cytotoxicity and hemolysis tests demonstrated the potential application of Octominin in vivo. The G. mellonella infection model asserted the in vivo efficacy of Octominin. Furthermore, Octominin inhibited the expression of proinflammatory cytokines. Although the temperature had little effect on its the activity, serum and ions reduced activity. CONCLUSION: Octominin is a promising alternative agent with remarkable antibacterial and anti-inflammatory effects for treating infections caused by CREC.

Phage resistance formation and fitness costs of hypervirulent Klebsiella pneumoniae mediated by K2 capsule-specific phage and the corresponding mechanisms.

Introduction: Phage is promising for the treatment of hypervirulent Klebsiella pneumoniae (hvKP) infections. Although phage resistance seems inevitable, we found that there still was optimization space in phage therapy for hvKP infection. Methods: The clinical isolate K. pneumoniae FK1979 was used to recover the lysis phage ΦFK1979 from hospital sewage. Phage-resistant bacteria were obtained on LB agar and used to isolate phages from sewage. The plaque assay, transmission electron microscopy (TEM), multiplicity of infection test, one-step growth curve assay, and genome analysis were performed to characterize the phages. Colony morphology, precipitation test and scanning electron microscope were used to characterize the bacteria. The absorption test, spot test and efficiency of plating (EOP) assay were used to identify the sensitivity of bacteria to phages. Whole genome sequencing (WGS) was used to identify gene mutations of phage-resistant bacteria. The gene expression levels were detected by RT-qPCR. Genes knockout and complementation of the mutant genes were performed. The change of capsules was detected by capsule quantification and TEM. The growth kinetics, serum resistance, biofilm formation, adhesion and invasion to A549 and RAW 264.7 cells, as well as G. mellonella and mice infection models, were used to evaluate the fitness and virulence of bacteria. Results and discussion: Here, we demonstrated that K2 capsule type sequence type 86 hvKP FK1979, one of the main pandemic lineages of hvKP with thick capsule, rapidly developed resistance to a K2-specific lysis phage ΦFK1979 which was well-studied in this work to possess polysaccharide depolymerase. The phage-resistant mutants showed a marked decrease in capsule expression. WGS revealed single nucleotide polymorphism (SNP) in genes encoding RfaH, galU, sugar glycosyltransferase, and polysaccharide deacetylase family protein in the mutants. RfaH and galU were further identified as being required for capsule production and phage sensitivity. Expressions of genes involved in the biosynthesis or regulation of capsule and/or lipopolysaccharide significantly decreased in the mutants. Despite the rapid and frequent development of phage resistance being a disadvantage, the attenuation of virulence and fitness in vitro and in vivo indicated that phage-resistant mutants of hvKP were more susceptible to the immunity system. Interestingly, the newly isolated phages targeting mutants changed significantly in their plaque and virus particle morphology. Their genomes were much larger than and significantly different from that of ΦFK1979. They possessed much more functional proteins and strikingly broader host spectrums than ΦFK1979. Our study suggests that K2-specific phage has the potential to function as an antivirulence agent, or a part of phage cocktails combined with phages targeting phage-resistant bacteria, against hvKP-relevant infections.