The prevalence and mechanisms of heteroresistance to ceftazidime/avibactam in KPC-producing Klebsiella pneumoniae.

OBJECTIVES: To investigate the prevalence and mechanisms of ceftazidime/avibactam heteroresistance in KPC-producing Klebsiella pneumoniae (KPC-KP) isolates, as well as the role of heteroresistance in the transition of ceftazidime/avibactam susceptibility to resistance. METHODS: Clinical KPC-KP isolates were obtained from a tertiary hospital in China from 2016 to 2017 and 2019 to 2020. Antimicrobial susceptibility was determined by the broth microdilution method. Population analysis profiles were used to assess ceftazidime/avibactam heteroresistance. WGS and molecular cloning were conducted to reveal heteroresistance mechanisms and molecular characteristics. RESULTS: The findings indicated that the transition of ceftazidime/avibactam susceptibility to resistance during the treatment of KPC-KP infection is primarily attributed to the heteroresistance exhibited by KPC-KP isolates towards ceftazidime/avibactam. Among 355 ceftazidime/avibactam-susceptible KPC-KP isolates (indicating a resistance rate of 0%), 41 (11.55%) exhibited ceftazidime/avibactam heteroresistance, with the primary mechanism being the presence of KPC mutant subpopulations. These KPC variants, arising from point mutations, deletions and insertions, significantly increased ceftazidime/avibactam resistance while alongside enhanced carbapenem susceptibility. Notably, 11 new KPC variants were identified. Furthermore, four heteroresistant isolates were caused by mixed infection involving subpopulations carrying NDM-1 or NDM-5. Phylogenetic analysis indicated that the clonal spread of ST11-KL64 KPC-KP may be correlated with the prevalence of heteroresistance. CONCLUSIONS: Ceftazidime/avibactam heteroresistance, primarily driven by pre-existing KPC variants, underscores the importance of considering heteroresistance in ceftazidime/avibactam therapeutics. Awareness of these dynamics is crucial for the effective and sustainable clinical application of ceftazidime/avibactam.

Carbapenemase-loaded outer membrane vesicles protect Pseudomonas aeruginosa by degrading imipenem and promoting mutation of antimicrobial resistance gene.

AIMS: To investigate the effect of Klebsiella pneumoniae carbapenemase (KPC)-loaded outer membrane vesicles (OMVs) in protecting Pseudomonas aeruginosa against imipenem treatment and its mechanism. METHODS: The OMVs of carbapenem-resistant Klebsiella pneumonia (CRKP) were isolated and purified from the supernatant of bacterial culture by using ultracentrifugation and Optiprep density gradient ultracentrifugation. The transmission electron microscope, bicinchoninic acid, PCR and carbapenemase colloidal gold assays were applied to characterize the OMVs. Bacterial growth and larvae infection experiments were performed to explore the protective function of KPC-loaded OMVs for P. aeruginosa under imipenem treatment. Ultra-performance liquid chromatography, antimicrobial susceptibility testing, whole-genome sequencing and bioinformatics analysis were used to investigate the mechanism of P. aeruginosa resistance phenotype mediated by OMVs. RESULTS: CRKP secreted OMVs loaded with KPC, which protect P. aeruginosa from imipenem through hydrolysis of antibiotics in a dose- and time-dependent manner. Furthermore, carbapenem-resistant subpopulations were developed in P. aeruginosa by low concentrations of OMVs that were confirmed to inadequately hydrolyze imipenem. Interestingly, none of the carbapenem-resistant subpopulations obtained the exogenous antibiotic resistance genes, but all of them possessed OprD mutations, which was consistent with the mechanism of P. aeruginosa induced by sub-minimal inhibitory concentrations of imipenem. CONCLUSIONS: OMVs containing KPC provide a novel route for P. aeruginosa to acquire an antibiotic-resistant phenotype in vivo.

Ceftazidime-Decorated Gold Nanoparticles: a Promising Strategy against Clinical Ceftazidime-Avibactam-Resistant Enterobacteriaceae with Different Resistance Mechanisms.

Nanoparticle-based antibiotic delivery systems are essential in combating antibiotic-resistant bacterial infections arising from acquired resistance and/or biofilm formation. Here, we report that the ceftazidime-decorated gold nanoparticles (CAZ_Au NPs) can effectively kill clinical ceftazidime-avibactam-resistant Enterobacteriaceae with various resistance mechanisms. Further study of underlying antibacterial mechanisms suggests that CAZ_Au NPs can damage the bacterial cell membrane and increase the level of intracellular reactive oxygen species. Moreover, CAZ_Au NPs show great potential in inhibiting biofilm formation and eradicating mature biofilms via crystal violet and scanning electron microscope assays. In addition, CAZ_Au NPs demonstrate excellent performance in improving the survival rate in the mouse model of abdominal infection. In addition, CAZ_Au NPs show no significant toxicity at bactericidal concentrations in the cell viability assay. Thus, this strategy provides a simple way to drastically improve the potency of ceftazidime as an antibiotic and its use in further biomedical applications.

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.

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.

Helicobacter macacae MazF interplays with Escherichia coli homologs and enhances antibiotic tolerance.

BACKGROUND: Toxin-antitoxin systems are highly variable, even among strains of the same bacterial species. The MazEF toxin-antitoxin system is found in many bacteria and plays important roles in various biological processes such as antibiotic tolerance and phage defense. However, no interplay of MazEF systems between different species was reported. MATERIALS AND METHODS: MazEF toxin-antitoxin system of Helicobacter macacae was examined in three Escherichia coli strains with and without endogenous MazEF knockout. In vivo toxicity, antibiotic tolerance, and live/dead staining followed by flowcytometry analysis were performed to evaluate the functionality and interplay of the toxin-antitoxin system between the two species. RESULTS: Controlled ectopic expression of MazF of H. macacae (MazFhm) in E. coli did not affect its growth. However, in endogenous MazEF knockout E. coli strains, MazFhm expression caused a sharp growth arrest. The toxicity of MazFhm could be neutralized by both the antitoxin of MazE homolog of H.macacae and the antitoxin of MazE of E. coli, indicating interplay of MazEF toxin-antitoxin systems between the two species. Induced expression of MazFhm enhanced tolerance to a lethal dose of levofloxacin, suggesting enhanced persister formation, which was further confirmed by live/dead cell staining. CONCLUSIONS: The MazEF toxin-antitoxin system of H. macace enhances persister formation and thus antibiotic tolerance in E. coli. Our findings reveal an interplay between the MazEF systems of H. macacae and E. coli, emphasizing the need to consider this interaction while evaluating the toxicity and functionality of MazF homologs from different species in future studies.

A potential strategy against clinical carbapenem-resistant Enterobacteriaceae: antimicrobial activity study of sweetener-decorated gold nanoparticles in vitro and in vivo.

BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) present substantial challenges to clinical intervention, necessitating the formulation of novel antimicrobial strategies to counteract them. Nanomaterials offer a distinctive avenue for eradicating bacteria by employing mechanisms divergent from traditional antibiotic resistance pathways and exhibiting reduced susceptibility to drug resistance development. Non-caloric artificial sweeteners, commonly utilized in the food sector, such as saccharin, sucralose, acesulfame, and aspartame, possess structures amenable to nanomaterial formation. In this investigation, we synthesized gold nanoparticles decorated with non-caloric artificial sweeteners and evaluated their antimicrobial efficacy against clinical CRE strains. RESULTS: Among these, gold nanoparticles decorated with aspartame (ASP_Au NPs) exhibited the most potent antimicrobial effect, displaying minimum inhibitory concentrations ranging from 4 to 16 µg/mL. As a result, ASP_Au NPs were chosen for further experimentation. Elucidation of the antimicrobial mechanism unveiled that ASP_Au NPs substantially elevated bacterial reactive oxygen species (ROS) levels, which dissipated upon ROS scavenger treatment, indicating ROS accumulation within bacteria as the fundamental antimicrobial modality. Furthermore, findings from membrane permeability assessments suggested that ASP_Au NPs may represent a secondary antimicrobial modality via enhancing inner membrane permeability. In addition, experiments involving crystal violet and confocal live/dead staining demonstrated effective suppression of bacterial biofilm formation by ASP_Au NPs. Moreover, ASP_Au NPs demonstrated notable efficacy in the treatment of Galleria mellonella bacterial infection and acute abdominal infection in mice, concurrently mitigating the organism's inflammatory response. Crucially, evaluation of in vivo safety and biocompatibility established that ASP_Au NPs exhibited negligible toxicity at bactericidal concentrations. CONCLUSIONS: Our results demonstrated that ASP_Au NPs exhibit promise as innovative antimicrobial agents against clinical CRE.

The Properties of Linezolid, Rifampicin, and Vancomycin, as Well as the Mechanism of Action of Pentamidine, Determine Their Synergy against Gram-Negative Bacteria.

Combining pentamidine with Gram-positive-targeting antibiotics has been proven to be a promising strategy for treating infections from Gram-negative bacteria (GNB). However, which antibiotics pentamidine can and cannot synergize with and the reasons for the differences are unclear. This study aimed to identify the possible mechanisms for the differences in the synergy of pentamidine with rifampicin, linezolid, tetracycline, erythromycin, and vancomycin against GNB. Checkerboard assays were used to detect the synergy of pentamidine and the different antibiotics. To determine the mechanism of pentamidine, fluorescent labeling assays were used to measure membrane permeability, membrane potential, efflux pump activity, and reactive oxygen species (ROS); the LPS neutralization assay was used to evaluate the target site; and quantitative PCR was used to measure changes in efflux pump gene expression. Our results revealed that pentamidine strongly synergized with rifampicin, linezolid, and tetracycline and moderately synergized with erythromycin, but did not synergize with vancomycin against E. coli, K. pneumoniae, E. cloacae, and A. baumannii. Pentamidine increased the outer membrane permeability but did not demolish the outer and inner membranes, which exclusively permits the passage of hydrophobic, small-molecule antibiotics while hindering the entry of hydrophilic, large-molecule vancomycin. It dissipated the membrane proton motive force and inactivated the efflux pump, allowing the intracellular accumulation of antimicrobials that function as substrates of the efflux pump, such as linezolid. These processes resulted in metabolic perturbation and ROS production which ultimately was able to destroy the bacteria. These mechanisms of action of pentamidine on GNB indicate that it is prone to potentiating hydrophobic, small-molecule antibiotics, such as rifampicin, linezolid, and tetracycline, but not hydrophilic, large-molecule antibiotics like vancomycin against GNB. Collectively, our results highlight the importance of the physicochemical properties of antibiotics and the specific mechanisms of action of pentamidine for the synergy of pentamidine-antibiotic combinations. Pentamidine engages in various pathways in its interactions with GNB, but these mechanisms determine its specific synergistic effects with certain antibiotics against GNB. Pentamidine is a promising adjuvant, and we can optimize drug compatibility by considering its functional mechanisms.

Activation of unfolded protein response overcomes Ibrutinib resistance in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most widespread type of non-Hodgkin lymphoma (NHL). As the most aggressive form of the DLBCL, the activated B-cell-like (ABC) subtype is often resistant to standard chemotherapies. Bruton's tyrosine kinase (BTK) inhibitor ibrutinib provides a potential therapeutic approach for the DLBCL but fails to improve the outcome in the phase III trial. In the current study, we investigated the molecular mechanisms underlying ibrutinib resistance and explored new combination therapy with ibrutinib. We generated an ibrutinib-resistant ABC-DLBCL cell line (OCI-ly10-IR) through continuous exposure to ibrutinib. Transcriptome analysis of the parental and ibrutinib-resistant cell lines revealed that the ibrutinib-resistant cells had significantly lower expression of the unfolded protein response (UPR) marker genes. Overexpression of one UPR branch-XBP1s greatly potentiated ibrutinib-induced apoptosis in both sensitive and resistant cells. The UPR inhibitor tauroursodeoxycholic acid (TUDCA) partially reduced the apoptotic rate induced by the ibrutinib in sensitive cells. The UPR activator 2-deoxy-D-glucose (2-DG) in combination with the ibrutinib triggered even greater cell growth inhibition, apoptosis, and stronger calcium (Ca2+) flux inhibition than either of the agents alone. A combination treatment of ibrutinib (15 mg·kg-1·d-1, po.) and 2-DG (500 mg/kg, po, b.i.d.) synergistically retarded tumor growth in NOD/SCID mice bearing OCI-ly10-IR xenograft. In addition, ibrutinib induced the UPR in the sensitive cell lines but not in the resistant cell lines of the DLBCL. There was also a combined synergistic effect in the primary resistant DLBCL cell lines. Overall, our results suggest that targeting the UPR could be a potential combination strategy to overcome ibrutinib resistance in the DLBCL.

Haemophilus seminalis sp. nov., isolated from human semen.

Two Haemophilus-like isolates with similar biochemical characteristics, designated strains SZY H1T and SZY H2, were isolated from human semen specimens. Cells were Gram-negative, non-motile, non-acid-fast, pleomorphic rods or coccobacilli. The major fatty acids (>10 %) were C16 : 0, C14 : 0, iso-C16 : 0 and/or C14 : 0 3-OH and C16 : 1 ω6c and/or C16 : 1 ω7c. The polar lipids were determined to be phosphatidylethanolamine, phosphatidylglycerol, an unidentified phospholipid, an unidentified aminophospholipid, two unidentified polar lipids and four unidentified aminolipids. The major polyamine was found to be cadaverine. The near-full-length (1462 nt) 16S rRNA gene sequences analysis showed the two isolates were nearly identical (>99.8 %), and closely matched Haemophilus haemolyticus ATCC 33390T with 98.9-99.1 % sequence similarities. Phylogenetic analysis based on 16S rRNA gene sequences and concatenation of 30 protein markers also revealed that the isolates clustered together with H. haemolyticus ATCC 33390T, and formed a distinct lineage well separated from the other members of the genus Haemophilus. Further, the average nucleotide identity values between the two isolates and their related species were below the established cut-off values for species delineation (95 %). Based on these findings, the two isolates are considered to represent a new species of the genus Haemophilus, for which name Haemophilus seminalis sp. nov. is proposed. The type strain is SZY H1T (=NBRC 113782T=CGMCC 1.17137T).

Antiparasitic nitazoxanide potentiates colistin against colistin-resistant Acinetobacter baumannii and Escherichia coli in vitro and in vivo.

IMPORTANCE: Colistin is used as a last resort in many infections caused by multidrug-resistant Gram-negative bacteria; however, colistin-resistant (COL-R) is on the rise. Hence, it is critical to develop new antimicrobial strategies to overcome COL-R. We found that nitazoxanide (NTZ) combined with colistin showed notable synergetic antibacterial activity. These findings suggest that the NTZ/colistin combination may provide an effective alternative route to combat COL-R A. baumannii and COL-R Escherichia coli infections.

ISAba1 mediated intrinsic chromosomal oxacillinase amplification confers carbapenem resistance in Acinetobacter baumannii.

Tandem amplification of carbapenemase genes increases gene copy number and enhances carbapenem resistance. These amplifications are often heterogeneous, transient, and located on plasmids, which also contribute to heteroresistance. Amplification of encoding genes is especially important for enzymes with low hydrolysis activity, which are often overlooked. Here, we reported an intrinsic oxacillinase oxaAb amplification flanked by ISAba1. The amplification is in the chromosome and contains up to twenty-five repeats. We provided genomic, transcriptomic, and proteomic evidence that the amplification resulted in oxacillinase overproduction. Notably, no point mutations of oxaAb were found during the amplification process. Strains of A. baumannii with intrinsic amplified or external transformed ISAba1-oxaAb exhibited higher meropenem hydrolysis activity. Furthermore, the number of repeats in the amplification decreased gradually over a period of 21 days cultured with carbapenem withdrawal. However, upon re-exposure to meropenem, the ISAba1 flanked oxaAb responded rapidly, with repeat numbers reaching or exceeding pre-carbapenem withdrawal levels within 24 hours. Taken together, these findings suggest that ISAba1-mediated gene amplification and overproduction of intrinsic low-activity oxacillinase oxaAb resulted in carbapenem resistance.

Bithionol Restores Sensitivity of Multidrug-Resistant Gram-Negative Bacteria to Colistin with Antimicrobial and Anti-biofilm Effects.

Being among the few last-resort antibiotics, colistin (COL) has been used to treat severe infectious diseases, such as those caused by multidrug-resistant Gram-negative bacteria (MDR GNB). However, the appearance of colistin-resistant (COL-R) GNB has been frequently reported. Therefore, novel antimicrobial strategies need to be urgently sought to address this resistance challenge. In the present study, antimicrobial drug screening conducted revealed that bithionol (BT), approved by the Food and Drug Administration and used as an anthelminthic drug for paragonimiasis, exhibited a synergistic antibacterial effect with COL. Clinically isolated COL-R GNB were used as candidates to evaluate the synergistic antibacterial activity. The results revealed that BT could significantly reverse the sensitivity of COL-R GNB to COL. Furthermore, the combined application of BT and COL can reduce bacterial biofilm formation and have a scavenging effect on the mature biofilm in vitro. The damage caused to the bacterial cell membrane integrity by the BT/COL combination was observed under a fluorescence microscope. The fluorescence intensity of reactive oxygen species also increased in the experimental group. The BT/COL combination also exhibited a synergistic antibacterial effect in vivo. Importantly, BT was confirmed to be safe at the highest concentrations that exerted synergistic effects on all tested strains. In conclusion, our findings demonstrated that BT exerted synergistic antimicrobial and anti-biofilm effects when combined with COL against MDR organisms, especially COL-R GNB, in vitro and in vivo. The findings thus provide a reference for the clinical response to the serious challenge of MDR GNB and the exploitation of the potential antibacterial activities of existing clinical non-antibacterial drugs.

When Combined with Pentamidine, Originally Ineffective Linezolid Becomes Active in Carbapenem-Resistant Enterobacteriaceae.

The increasing prevalence of carbapenem-resistant Enterobacteriaceae (CRE) and their biofilm-relevant infections pose a threat to public health. The drug combination strategy provides a new treatment option for CRE infections. This study explored the synergistic antibacterial, antibiofilm activities as well as the in vivo efficacy against CRE of pentamidine combined with linezolid. This study further revealed the possible mechanisms underlying the synergy of the combination. The checkerboard and time-kill assays showed that pentamidine combined with linezolid had significant synergistic antibacterial effects against CRE strains (9/10). Toxicity assays on mammal cells (mouse RAW264.7 and red blood cells) and on Galleria mellonella confirmed that the concentrations of pentamidine and/or linezolid that were used were relatively safe. Antibiofilm activity detection via crystal violet staining, viable bacteria counts, and scanning electron microscopy demonstrated that the combination enhanced the inhibition of biofilm formation and the elimination of established biofilms. The G. mellonella infection model and mouse thigh infection model demonstrated the potential in vivo efficacy of the combination. In particular, a series of mechanistic experiments elucidated the possible mechanisms for the synergy in which pentamidine disrupts the outer membranes, dissipates the membrane potentials, and devitalizes the efflux pumps of CRE, thereby facilitating the intracellular accumulation of linezolid and reactive oxygen species (ROS), which ultimately kills the bacteria. Taken together, when combined with pentamidine, which acts as an outer membrane permeabilizer and as an efflux pump inhibitor, originally ineffective linezolid becomes active in CRE and exhibits excellent synergistic antibacterial and antibiofilm effects as well as a potential therapeutic effect in vivo on CRE-relevant infections. IMPORTANCE The multidrug resistance and biofilm formation of Gram-negative bacteria (GNB) may lead to incurable "superbug" infections. Drug combinations, with the potential to augment the original treatment ranges of drugs, are alternative treatment strategies against GNB. In this study, the pentamidine-linezolid combination showed notable antibacterial and antibiofilm activity both in vitro and in vivo against the problem carbapenem-resistant Enterobacteriaceae (CRE). Pentamidine is often used as an antiprotozoal and antifungal agent, and linezolid is a defensive Gram-positive bacteria (GPB) antimicrobial. Their combination expands the treatment range to GNB. Hence, the pentamidine-linezolid pair may be an effective treatment for complex infections that are mixed by GPB, GNB, and even fungi. In terms of mechanism, pentamidine inhibited the outer membranes, membrane potentials, and efflux pumps of CRE. This might be a universal mechanism by which pentamidine, as an adjuvant, potentiates other drugs, similar to linezolid, thereby having synergistic antibacterial effects on CRE.

Thymol-Decorated Gold Nanoparticles for Curing Clinical Infections Caused by Bacteria Resistant to Last-Resort Antibiotics.

Multidrug-resistant bacteria pose a tremendous challenge to public health worldwide. Many bacteria resistant to last-resort antibiotics due to antibiotic misuse have been recently reported, which may give rise to serious infections without effective treatment. Therefore, it is imperative to develop novel antimicrobial strategies. Natural phenols are known to increase bacterial membrane permeability and are potential candidates for the development of new antimicrobial agents. In this study, gold nanoparticles (Au NPs) carrying natural phenols were synthesized to combat bacteria resistant to last-resort antibiotics. Transmission electron microscopy, dynamic light scattering, zeta potential, and UV-visible spectra were used to characterize the synthesized Au NPs, which showed good monodispersity and uniform particle size. Evaluation of antibacterial activity using the broth microdilution method revealed that thymol-decorated gold nanoparticles (Thymol_Au NPs) had a broad antibacterial spectrum and higher bactericidal effects than last-resort antibiotics against last-resort-antibiotic-resistant bacteria. Considering the underlying antibacterial mechanism, the results showed that Thymol_Au NPs destroyed bacterial cell membranes. Further, Thymol_Au NPs were effective in treating mouse abdominal infections and exhibited acceptable biocompatibility without any significant toxicity in cell viability and histopathological assays, respectively, at most bactericidal concentrations. However, attention should be paid to changes in white blood cells, reticulocyte percentages, and superoxide dismutase activity during Thymol_Au NP treatment. In conclusion, Thymol_Au NPs have the potential for treating clinical infections caused by bacteria resistant to last-resort antibiotics. IMPORTANCE Excessive use of antibiotics can lead to bacterial resistance and the development of multidrug-resistant bacteria. Antibiotic misuse can also promote resistance against last-resort antibiotics. It is thus crucial to develop alternatives to antibiotics to retard the development of multidrug resistance. In recent years, the use of several nanodosage forms of antibacterial drugs has been investigated. These agents kill bacteria through a variety of mechanisms and avoid the problem of resistance. Among them, Au NPs, which are safer to use for medical applications than other metal nanoparticles, have attracted interest as potential antibacterial agents. To combat bacterial resistance to last-resort antibiotics and mitigate the problem of antimicrobial resistance, it is important and meaningful to develop antimicrobial agents based on Au NPs.

Long-Read- and Short-Read-Based Whole-Genome Sequencing Reveals the Antibiotic Resistance Pattern of Helicobacter pylori.

The rates of antibiotic resistance of Helicobacter pylori are increasing, and the patterns of resistance are region and population specific. Here, we elucidated the antibiotic resistance pattern of H. pylori in a single center in China and compared short-read- and long-read-based whole-genome sequencing for identifying the genotypes. Resistance rates of 38.5%, 61.5%, 27.9%, and 13.5% against clarithromycin, metronidazole, levofloxacin, and amoxicillin were determined, respectively, while no strain was resistant to tetracycline or furazolidone. Single nucleotide variations (SNVs) in the 23S rRNA and GyrA/B genes revealed by Illumina short-read sequencing showed good diagnostic abilities for clarithromycin and levofloxacin resistance, respectively. Nanopore long-read sequencing also showed a good efficiency in elucidating SNVs in the 23S rRNA gene and, thus, a good ability to detect clarithromycin resistance. The two technologies displayed good consistency in discovering SNVs and shared 76% of SNVs detected in the rRNA gene. Taking Sanger sequencing as the gold standard, Illumina short-read sequencing showed a slightly higher accuracy for discovering SNVs than Nanopore sequencing. There are two copies of the rRNA gene in the genome of H. pylori, and we found that the two copies were not the same in at least 26% of the strains tested, indicating their heterozygous status. Especially, three strains harboring a 2143G/A heterozygous status in the 23S rRNA gene, which is the most important site for clarithromycin resistance, were found. In conclusion, our results provide evidence for an empirical first-line treatment for H. pylori eradication in clinical settings. Moreover, we show that Nanopore sequencing is a potential tool for predicting clarithromycin resistance. IMPORTANCE Helicobacter pylori resistance has been increasing in recent years. The resistance profile, which is important for empirical treatment, is region and population specific. We found high rates of resistance to metronidazole, clarithromycin, and levofloxacin in H. pylori in our center, while no resistance to tetracycline or furazolidone was found. These results provide a reference for local physicians prescribing antibiotics for H. pylori eradication. Nanopore sequencing recently appeared to be a promising technology for elucidating whole-genome sequences, which generates long sequencing reads and is time-efficient and portable. However, a relatively higher error rate of sequencing reads was also found. In this study, we compared Nanopore sequencing and Illumina sequencing for revealing single nucleotide variations in the 23S rRNA gene, which determines clarithromycin resistance, and we found that although there were a few false discoveries, Nanopore sequencing showed good consistency with Illumina sequencing, indicating that it is a potential tool for predicting clarithromycin resistance.

Clinical and Molecular Characteristics and Antibacterial Strategies of Klebsiella pneumoniae in Pyogenic Infection.

Treatment of Klebsiella pneumoniae causing pyogenic infections is challenging. The clinical and molecular characteristics of Klebsiella pneumoniae causing pyogenic infections are poorly understood, and antibacterial treatment strategies are limited. We analyzed the clinical and molecular characteristics of K. pneumoniae from patients with pyogenic infections and used time-kill assays to reveal the bactericidal kinetics of antimicrobial agents against hypervirulent K. pneumoniae (hvKp). A total of 54 K. pneumoniae isolates were included, comprising 33 hvKp and 21 classic K. pneumoniae (cKp) isolates, and the hvKp and cKp isolates were identified using five genes (iroB, iucA, rmpA, rmpA2, and peg-344) that have been applied as hvKp strain markers. The median age of all cases was 54 years (25th and 75th percentiles, 50.5 to 70), 62.96% of individuals had diabetes, and 22.22% of isolates were sourced from individuals without underlying disease. The ratios of white blood cells/procalcitonin and C-reactive protein/procalcitonin were potential clinical markers for the identification of suppurative infection caused by hvKp and cKp. The 54 K. pneumoniae isolates were classified into 8 sequence type 11 (ST11) and 46 non-ST11 strains. ST11 strains carrying multiple drug resistance genes have a multidrug resistance phenotype, while non-ST11 strains carrying only intrinsic resistance genes are generally susceptible to antibiotics. Bactericidal kinetics revealed that hvKp isolates were not easily killed by antimicrobials at susceptible breakpoint concentrations compared with cKp. Given the varied clinical and molecular features and the catastrophic pathogenicity of K. pneumoniae, it is critical to determine the characteristics of such isolates for optimal management and effective treatment of K. pneumoniae causing pyogenic infections. IMPORTANCE Klebsiella pneumoniae may cause pyogenic infections, which are potentially life-threatening and bring great challenges for clinical management. However, the clinical and molecular characteristics of K. pneumoniae are poorly understood, and effective antibacterial treatment strategies are limited. We analyzed the clinical and molecular features of 54 isolates from patients with various pyogenic infections. We found that most patients with pyogenic infections had underlying diseases, such as diabetes. The ratio of white blood cells to procalcitonin and the ratio of C-reactive protein to procalcitonin were potential clinical markers for differentiating hypervirulent K. pneumoniae strains from classical K. pneumoniae strains that cause pyogenic infections. K. pneumoniae isolates of ST11 were generally more resistant to antibiotics than non-ST11 isolates. Most importantly, hypervirulent K. pneumoniae strains were more tolerant to antibiotics than classic K. pneumoniae isolates.

Genomic Characterization of a Carbapenemase-Producing, Extensively Drug-Resistant Klebsiella michiganensis Strain from a Renal Abscess Patient.

We describe an extensively drug-resistant Klebsiella michiganensis strain, Kmfe267, which was originally isolated from a renal abscess patient. The strain carries the blaNDM-5 gene, which encodes a New Delhi metallo-ß-lactamase. The complete genome of the strain contains a 5.9-Mb chromosome and 5 plasmids.

Clinical Characteristics of Immune Response in Asymptomatic Carriers and Symptomatic Patients With COVID-19.

The pandemic of coronavirus disease 2019 (COVID-19) has emerged as a major public health challenge worldwide. A comprehensive understanding of clinical characteristics and immune responses in asymptomatic carriers and symptomatic patients with COVID-19 is of great significance to the countermeasures of patients with COVID-19. Herein, we described the clinical information and laboratory findings of 43 individuals from Hunan Province, China, including 13 asymptomatic carriers and 10 symptomatic patients with COVID-19, as well as 20 healthy controls in the period from 25 January to 18 May 2020. The serum samples of these individuals were analyzed to measure the cytokine responses, receptor-binding domain (RBD), and nucleocapsid (N) protein-specific antibody titers, as well as SARS-CoV-2 neutralizing antibodies (nAbs). For cytokines, significantly higher Th1 cytokines including IL-2, IL-8, IL-12p70, IFN-γ, and TNF-α, as well as Th2 cytokines including IL-10 and IL-13 were observed in symptomatic patients compared with asymptomatic carriers. Compared with symptomatic patients, higher N-specific IgG4/IgG1 ratio and RBD-specific/N-specific IgG1 ratio were observed in asymptomatic carriers. Comparable nAbs were detected in both asymptomatic carriers and symptomatic patients with COVID-19. In the symptomatic group, nAbs in patients with underlying diseases were weaker than those of patients without underlying diseases. Our retrospective study will enrich and verify the clinical characteristics and serology diversities in asymptomatic carriers and symptomatic patients with COVID-19.

Clinical and Microbiological Characterization of Bloodstream Infections Caused by Mycoplasma hominis: An Overlooked Pathogen.

INTRODUCTION: Bloodstream infection (BSI) is associated with high mortality rates. Mycoplasma hominis, which rarely causes extragenital infections, has been shown to induce BSI and presents a clinical diagnostic and therapeutic challenge. METHODS: In this study, we investigated the clinical characteristics, antibiotic resistance, and multilocus sequence typing (MLST) of eight BSI cases caused by M. hominis in South China from January 2018 to October 2021. RESULTS: Underlying immunosuppression and genitourinary tract surgery are important risk factors for M. hominis BSI. The most prevalent clinical symptoms and signs were fever. Additional findings included elevated neutrophil count and C-reactive protein level. Furthermore, in this study, all the patients had erythrocytopenia. M. hominis harbored the highest rate of resistance to levofloxacin (75.0%), followed by sparfloxacin (50.0%), and gatifloxacin (37.5%). gyrA S153L was the most frequent mutation in levofloxacin-resistant strains, followed by parC S91I. parC K144R may be related to resistance to gatifloxacin and sparfloxacin. Eight strains showed sensitivity to all the other antibiotics analyzed (doxycycline, minocycline, josamycin, and clindamycin). MLST was performed in seven isolates, and seven new sequence types were described. We compared our isolates with all M. hominis strains from the PubMLST database, and one major clonal complex and eight singletons were identified. CONCLUSIONS: Our study clarified and expanded the clinical features and antibiotic resistance of M. hominis BSI. These findings are useful for the clinical diagnosis and control of M. hominis BSI.