Therapeutic efficacy of a K5-specific phage and depolymerase against Klebsiella pneumoniae in a mouse model of infection.

Klebsiella pneumoniae has become one of the most intractable gram-negative pathogens infecting humans and animals due to its severe antibiotic resistance. Bacteriophages and protein products derived from them are receiving increasing amounts of attention as potential alternatives to antibiotics. In this study, we isolated and investigated the characteristics of a new lytic phage, P1011, which lyses K5 K. pneumoniae specifically among 26 serotypes. The K5-specific capsular polysaccharide-degrading depolymerase dep1011 was identified and expressed. By establishing murine infection models using bovine strain B16 (capable of supporting phage proliferation) and human strain KP181 (incapable of sustaining phage expansion), we explored the safety and efficacy of phage and dep1011 treatments against K5 K. pneumoniae. Phage P1011 resulted in a 60% survival rate of the mice challenged with K. pneumoniae supporting phage multiplication, concurrently lowering the bacterial burden in their blood, liver, and lungs. Unexpectedly, even when confronted with bacteria impervious to phage multiplication, phage therapy markedly decreased the number of viable organisms. The protective efficacy of the depolymerase was significantly better than that of the phage. The depolymerase achieved 100% survival in both treatment groups regardless of phage propagation compatibility. These findings indicated that P1011 and dep1011 might be used as potential antibacterial agents to control K5 K. pneumoniae infection.

sRNA expression profile of KPC-2-producing carbapenem-resistant Klebsiella pneumoniae: Functional role of sRNA51.

The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has significant challenges to human health and clinical treatment, with KPC-2-producing CRKP being the predominant epidemic strain. Therefore, there is an urgent need to identify new therapeutic targets and strategies. Non-coding small RNA (sRNA) is a post-transcriptional regulator of genes involved in important biological processes in bacteria and represents an emerging therapeutic strategy for antibiotic-resistant bacteria. In this study, we analyzed the transcription profile of KPC-2-producing CRKP using RNA-seq. Of the 4693 known genes detected, the expression of 307 genes was significantly different from that of carbapenem-sensitive Klebsiella pneumoniae (CSKP), including 133 up-regulated and 174 down-regulated genes. Both the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) analysis showed that these differentially expressed genes (DEGs) were mainly related to metabolism. In addition, we identified the sRNA expression profile of KPC-2-producing CRKP for the first time and detected 115 sRNAs, including 112 newly discovered sRNAs. Compared to CSKP, 43 sRNAs were differentially expressed in KPC-2-producing CRKP, including 39 up-regulated and 4 down-regulated sRNAs. We chose sRNA51, the most significantly differentially expressed sRNA in KPC-2-producing CRKP, as our research subject. By constructing sRNA51-overexpressing KPC-2-producing CRKP strains, we found that sRNA51 overexpression down-regulated the expression of acrA and alleviated resistance to meropenem and ertapenem in KPC-2-producing CRKP, while overexpression of acrA in sRNA51-overexpressing strains restored the reduction of resistance. Therefore, we speculated that sRNA51 could affect the resistance of KPC-2-producing CRKP by inhibiting acrA expression and affecting the formation of efflux pumps. This provides a new approach for developing antibiotic adjuvants to restore the sensitivity of CRKP.

Modelling lung infection with Klebsiella pneumoniae after murine traumatic brain injury.

Pneumonia is a common comorbidity in patients with severe traumatic brain injury (TBI), and is associated with increased morbidity and mortality. In this study, we established a model of intratracheal Klebsiella pneumoniae administration in young adult male and female mice, at 4 days following an experimental TBI, to investigate how K. pneumoniae infection influences acute post-TBI outcomes. A dose-response curve determined the optimal dose of K. pneumoniae for inoculation (1 x 10^6 colony forming units), and administration at 4 days post-TBI resulted in transient body weight loss and sickness behaviors (hypoactivity and acute dyspnea). K. pneumoniae infection led to an increase in pro-inflammatory cytokines in serum and bronchoalveolar lavage fluid at 24 h post-infection, in both TBI and sham (uninjured) mice. By 7 days, when myeloperoxidase + neutrophil numbers had returned to baseline in all groups, lung histopathology was observed with an increase in airspace size in TBI + K. pneumoniae mice compared to TBI + vehicle mice. In the brain, increased neuroinflammatory gene expression was observed acutely in response to TBI, with an exacerbated increase in Ccl2 and Hmox1 in TBI + K. pneumoniae mice compared to either TBI or K. pneumoniae alone. However, the presence of neuroinflammatory immune cells in the injured brain, and the extent of damage to cortical and hippocampal brain tissue, was comparable between K. pneumoniae and vehicle-treated mice by 7 days. Examination of the fecal microbiome across a time course did not reveal any pronounced effects of either injury or K. pneumoniae on bacterial diversity or abundance. Together, these findings demonstrate that K. pneumoniae lung infection after TBI induces an acute and transient inflammatory response, primarily localized to the lungs with some systemic effects. However, this infection had minimal impact on secondary injury processes in the brain following TBI. Future studies are needed to evaluate the potential longer-term consequences of this dual-hit insult.

Performance comparison of BD Phoenix CPO detect panel with Cepheid Xpert Carba-R assay for the detection of carbapenemase-producing Klebsiella pneumoniae isolates.

BACKGROUND: We aimed to compare the performance of carbapenemase classification in carbapenem-resistant Klebsiella pneumoniae (CRKP) obtained using the BD Phoenix CPO Detect panel (CPO panel) and Cepheid Xpert Carba-R assays. We analyzed 55 CRKP strains from clinical specimens collected between November 2020 and November 2022. The CPO panel was used to detect both antibiotic susceptibility and phenotypic carbapenemase classes, while Xpert Carba-R was employed to identify KPC, NDM, VIM, OXA-48, and IMP genes. Due to the limited availability of molecular kits, we arbitrarily selected 55 isolates, identified as carbapenemase-producing according to the CPO panel and with meropenem minimum inhibitory concentration values > 8 mg/L. RESULTS: According to the Xpert Carba-R assay, 16 of the 55 isolates (29.1%) were categorised as Ambler Class A (11 of which matched CPO panel Class A identification); three isolates (5.5%) were identified as Class B and 27 isolates (49.1%) as Class D (in both cases consistent with CPO panel B and D classifications). A further eight isolates (14.5%) exhibited multiple carbapenemase enzymes and were designated as dual-carbapenemase producers, while one isolate (1.8%) was identified as a non-carbapenemase-producer. The CPO panel demonstrated positive and negative percent agreements of 100% and 85.7% for Ambler Class A, 100% and 100% for Class B, and 96.4% and 100% for Class D carbapenemase detection, respectively. CONCLUSION: While the CPO panel's phenotypic performance was satisfactory in detecting Class B and D carbapenemases, additional confirmatory testing may be necessary for Class A carbapenemases as part of routine laboratory procedures.

Lipid A in outer membrane vesicles shields bacteria from polymyxins.

The continuous emergence of multidrug-resistant bacterial pathogens poses a major global healthcare challenge, with Klebsiella pneumoniae being a prominent threat. We conducted a comprehensive study on K. pneumoniae's antibiotic resistance mechanisms, focusing on outer membrane vesicles (OMVs) and polymyxin, a last-resort antibiotic. Our research demonstrates that OMVs protect bacteria from polymyxins. OMVs derived from Polymyxin B (PB)-stressed K. pneumoniae exhibited heightened protective efficacy due to increased vesiculation, compared to OMVs from unstressed Klebsiella. OMVs also shield bacteria from different bacterial families. This was validated ex vivo and in vivo using precision cut lung slices (PCLS) and Galleria mellonella. In all models, OMVs protected K. pneumoniae from PB and reduced the associated stress response on protein level. We observed significant changes in the lipid composition of OMVs upon PB treatment, affecting their binding capacity to PB. The altered binding capacity of single OMVs from PB stressed K. pneumoniae could be linked to a reduction in the lipid A amount of their released vesicles. Although the amount of lipid A per vesicle is reduced, the overall increase in the number of vesicles results in an increased protection because the sum of lipid A and therefore PB binding sites have increased. This unravels the mechanism of the altered PB protective efficacy of OMVs from PB stressed K. pneumoniae compared to control OMVs. The lipid A-dependent protective effect against PB was confirmed in vitro using artificial vesicles. Moreover, artificial vesicles successfully protected Klebsiella from PB ex vivo and in vivo. The findings indicate that OMVs act as protective shields for bacteria by binding to polymyxins, effectively serving as decoys and preventing antibiotic interaction with the cell surface. Our findings provide valuable insights into the mechanisms underlying antibiotic cross-protection and offer potential avenues for the development of novel therapeutic interventions to address the escalating threat of multidrug-resistant bacterial infections.

Three concurrent mechanisms generate gene copy number variation and transient antibiotic heteroresistance.

Heteroresistance is a medically relevant phenotype where small antibiotic-resistant subpopulations coexist within predominantly susceptible bacterial populations. Heteroresistance reduces treatment efficacy across diverse bacterial species and antibiotic classes, yet its genetic and physiological mechanisms remain poorly understood. Here, we investigated a multi-resistant Klebsiella pneumoniae isolate and identified three primary drivers of gene dosage-dependent heteroresistance for several antibiotic classes: tandem amplification, increased plasmid copy number, and transposition of resistance genes onto cryptic plasmids. All three mechanisms imposed fitness costs and were genetically unstable, leading to fast reversion to susceptibility in the absence of antibiotics. We used a mouse gut colonization model to show that heteroresistance due to elevated resistance-gene dosage can result in antibiotic treatment failures. Importantly, we observed that the three mechanisms are prevalent among Escherichia coli bloodstream isolates. Our findings underscore the necessity for treatment strategies that address the complex interplay between plasmids, resistance cassettes, and transposons in bacterial populations.

Genetic, virulence, and antimicrobial resistance characteristics associated with distinct morphotypes in ST11 carbapenem-resistant Klebsiella pneumoniae.

ST11 is the most common lineage among carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in Asia. Diverse morphotypes resulting from genetic mutations are associated with significant differences in microbial characteristics among K. pneumoniae isolates. Here, we investigated the genetic determinants and critical characteristics associated with distinct morphotypes of ST11 CRKP. An ST11-KL47 CRKP isolate carrying a pLVPK-like virulence plasmid was isolated from a patient with a bloodstream infection; the isolate had the "mcsw" morphotype. Two distinct morphotypes ("ntrd" and "msdw") were derived from this strain during in vitro passage. Whole genome sequencing was used to identify mutations that cause the distinct morphotypes of ST11 CRKP. Transmission electron microscopy, antimicrobial susceptibility tests, growth assays, biofilm formation, virulence assays, membrane permeability assays, and RNA-seq analysis were used to investigate the specific characteristics associated with different morphotypes of ST11 CRKP. Compared with the parental mcsw morphotype, the ntrd morphotype resulted from mutation of genes involved in capsular polysaccharide biosynthesis (wza, wzc, and wbaP), a result validated by gene knockout experiments. This morphotype showed capsule deficiency and lower virulence potential, but higher biofilm production. By contrast, the msdw morphotype displayed competition deficiency and increased susceptibility to chlorhexidine and polymyxin B. Further analyses indicated that these characteristics were caused by interruption of the sigma factor gene rpoN by insertion mutations and deletion of the rpoN gene, which attenuated membrane integrity presumably by downregulating the phage shock protein operon. These data expand current understanding of genetic, virulence, and antimicrobial resistance characteristics associated with distinct morphotypes in ST11 CRKP.

Genomic insights unveil the plasmid transfer mechanism and epidemiology of hypervirulent Klebsiella pneumoniae in Vietnam.

Hypervirulent Klebsiella pneumoniae (hvKp) is a significant cause of severe invasive infections in Vietnam, yet data on its epidemiology, population structure and dynamics are scarce. We screened hvKp isolates from patients with bloodstream infections (BSIs) at a tertiary infectious diseases hospital in Vietnam and healthy individuals, followed by whole genome sequencing and plasmid analysis. Among 700 BSI-causing Kp strains, 100 (14.3%) were hvKp. Thirteen hvKp isolates were identified from 350 rectal swabs of healthy adults; none from 500 rectal swabs of healthy children. The hvKp isolates were genetically diverse, encompassing 17 sequence types (STs), predominantly ST23, ST86 and ST65. Among the 113 hvKp isolates, 14 (12.6%) carried at least one antimicrobial resistance (AMR) gene, largely mediated by IncFII, IncR, and IncA/C plasmids. Notably, the acquisition of AMR conjugative plasmids facilitated horizontal transfer of the non-conjugative virulence plasmid between K. pneumoniae strains. Phylogenetic analysis demonstrated hvKp isolates from BSIs and human carriage clustered together, suggesting a significant role of intestinal carriage in hvKp transmission. Enhanced surveillance is crucial to understand the factors driving intestinal carriage and hvKp transmission dynamics for informing preventive measures. Furthermore, we advocate the clinical use of our molecular assay for diagnosing hvKp infections to guide effective management.

Infection with the multidrug-resistant Klebsiella pneumoniae New Delhi metallo-B-lactamase strain in patients with COVID-19: Nec Hercules contra plures?

Background: During the coronavirus disease 2019 (COVID-19) pandemic, in patients treated for SARS-CoV-2 infection, infections with the Klebsiella pneumoniae bacteria producing New Delhi metallo-B-lactamase (NDM) carbapenemase in the USA, Brazil, Mexico, and Italy were observed, especially in intensive care units (ICUs). This study aimed to assess the impact of Klebsiella pneumoniae NDM infection and other bacterial infections on mortality in patients treated in ICUs due to COVID-19. Methods: The 160 patients who qualified for the study were hospitalized in ICUs due to COVID-19. Three groups were distinguished: patients with COVID-19 infection only (N = 72), patients with COVID-19 infection and infection caused by Klebsiella pneumoniae NDM (N = 30), and patients with COVID-19 infection and infection of bacterial etiology other than Klebsiella pneumoniae NDM (N = 58). Mortality in the groups and chosen demographic data; biochemical parameters analyzed on days 1, 3, 5, and 7; comorbidities; and ICU scores were analyzed. Results: Bacterial infection, including with Klebsiella pneumoniae NDM type, did not elevate mortality rates. In the group of patients who survived the acute phase of COVID-19 the prolonged survival time was demonstrated: the median overall survival time was 13 days in the NDM bacterial infection group, 14 days in the other bacterial infection group, and 7 days in the COVID-19 only group. Comparing the COVID-19 with NDM infection and COVID-19 only groups, the adjusted model estimated a statistically significant hazard ratio of 0.28 (p = 0.002). Multivariate analysis revealed that age, APACHE II score, and CRP were predictors of mortality in all the patient groups. Conclusion: In patients treated for SARS-CoV-2 infection acquiring a bacterial infection due to prolonged hospitalization associated with the treatment of COVID-19 did not elevate mortality rates. The data suggests that in severe COVID-19 patients who survived beyond the first week of hospitalization, bacterial infections, particularly Klebsiella pneumoniae NDM, do not significantly impact mortality. Multivariate analysis revealed that age, APACHE II score, and CRP were predictors of mortality in all the patient groups.

A step closer to understanding how a diet high in simple carbohydrates may cause dysbiosis.

The gut microbiota is an integral part of the human metaorganism that is required to shape physiologic host immune responses including host defense against pathogens. Disease-associated gut dysbiosis has been characterized by blooms of pathobionts, which are bacterial species that can drive disease under certain conditions. Pathobionts like Enterobacteriaceae often bloom during flares of inflammatory bowel disease (IBD) and are causally linked with IBD in murine models. In this issue of the JCI, Hecht and colleagues investigated how simple carbohydrates are causally linked to the bloom of the gut pathobiont Klebsiella pneumoniae, which belong to the Enterobacteriaceae family. Notably, the presence of fiber reduced the dissemination of K. pneumoniae into the blood and liver in a colitis model. Their findings provide a diet-related mechanism for gut dysbiosis, which has implications in the management of IBD and other conditions in which gut dysbiosis is an underlying factor.

A novel chimeric vaccine containing multiple epitopes for simulating robust immune activation against Klebsiella pneumoniae.

BACKGROUND: Due to antibiotic resistance, the Klebsiella genus is linked to morbidity and death, necessitating the development of a universally protective vaccine against Klebsiella pathogens. METHODS: Core sequence analysis prioritized non-redundant host molecules and expected lipid bilayer peptides from fully sequenced Klebsiella genomes. These proteins were refined to identify epitopes, examining their immunogenicity, toxicity, solubility, and interaction with MHC alleles. Epitopes were linked to CPG ODN C274 via EAAAK, HEYGAEALERAG, and GGGS linkers to enhance immunological responses. The vaccine's tertiary structure was modelled and docked with MHC-I and MHC-II. RESULTS: Fifty-five proteins were recognized in the Vaxign collection as having remarkable features. Twenty-three proteins with potential pathogenicity were then identified. Eight options for vaccines emerged after the immunogenicity of proteins was examined. The best antigens were three proteins: MrkD, Iron-regulated lipid membrane polypeptides, and RmpA. These compounds were selected for their sensitivity. The structural protein sequences of K. pneumoniae were utilized to identify seven CTL epitopes, seven HTL epitopes, and seven LBL epitopes, respectively. The produced immunization displayed a stable contact with the receptors, based on molecular dynamic simulations lasting 250 nanoseconds. Intermolecular binding free energies also indicated the dominance of the van der Waals and electrostatic energies. CONCLUSION: In summary, the results of this study might help scientists develop a novel vaccine to prevent K. pneumoniae infections.

Differences in clinical outcomes of bloodstream infections caused by Klebsiella aerogenes, Klebsiella pneumoniae and Enterobacter cloacae: a multicentre cohort study.

BACKGROUND: Klebsiella aerogenes has been reclassified from Enterobacter to Klebsiella genus due to its phenotypic and genotypic similarities with Klebsiella pneumoniae. It is unclear if clinical outcomes are also more similar. This study aims to assess clinical outcomes of bloodstreams infections (BSI) caused by K. aerogenes, K. pneumoniae and Enterobacter cloacae, through secondary data analysis, nested in PRO-BAC cohort study. METHODS: Hospitalized patients between October 2016 and March 2017 with monomicrobial BSI due to K. aerogenes, K. pneumoniae or E. cloacae were included. Primary outcome was a composite clinical outcome including all-cause mortality or recurrence until 30 days follow-up. Secondary outcomes were fever ≥ 72 h, persistent bacteraemia, and secondary device infection. Multilevel mixed-effect Poisson regression was used to estimate the association between microorganisms and outcome. RESULTS: Overall, 29 K. aerogenes, 77 E. cloacae and 337 K. pneumoniae BSI episodes were included. Mortality or recurrence was less frequent in K. aerogenes (6.9%) than in E. cloacae (20.8%) or K. pneumoniae (19.0%), but statistical difference was not observed (rate ratio (RR) 0.35, 95% CI 0.08 to 1.55; RR 0.42, 95% CI 0.10 to 1.71, respectively). Fever ≥ 72 h and device infection were more common in K. aerogenes group. In the multivariate analysis, adjusted for confounders (age, sex, BSI source, hospital ward, Charlson score and active antibiotic therapy), the estimates and direction of effect were similar to crude results. CONCLUSIONS: Results suggest that BSI caused by K. aerogenes may have a better prognosis than E. cloacae or K. pneumoniae BSI.

Longitudinal analysis within one hospital in sub-Saharan Africa over 20 years reveals repeated replacements of dominant clones of Klebsiella pneumoniae and stresses the importance to include temporal patterns for vaccine design considerations.

BACKGROUND: Infections caused by multidrug-resistant gram-negative bacteria present a severe threat to global public health. The WHO defines drug-resistant Klebsiella pneumoniae as a priority pathogen for which alternative treatments are needed given the limited treatment options and the rapid acquisition of novel resistance mechanisms by this species. Longitudinal descriptions of genomic epidemiology of Klebsiella pneumoniae can inform management strategies but data from sub-Saharan Africa are lacking. METHODS: We present a longitudinal analysis of all invasive K. pneumoniae isolates from a single hospital in Blantyre, Malawi, southern Africa, from 1998 to 2020, combining clinical data with genome sequence analysis of the isolates. RESULTS: We show that after a dramatic increase in the number of infections from 2016 K. pneumoniae becomes hyperendemic, driven by an increase in neonatal infections. Genomic data show repeated waves of clonal expansion of different, often ward-restricted, lineages, suggestive of hospital-associated transmission. We describe temporal trends in resistance and surface antigens, of relevance for vaccine development. CONCLUSIONS: Our data highlight a clear need for new interventions to prevent rather than treat K. pneumoniae infections in our setting. Whilst one option may be a vaccine, the majority of cases could be avoided by an increased focus on and investment in infection prevention and control measures, which would reduce all healthcare-associated infections and not just one.

Ethanolamine metabolism through two genetically distinct loci enables Klebsiella pneumoniae to bypass nutritional competition in the gut.

Successful microbial colonization of the gastrointestinal (GI) tract hinges on an organism's ability to overcome the intense competition for nutrients in the gut between the host and the resident gut microbiome. Enteric pathogens can exploit ethanolamine (EA) in the gut to bypass nutrient competition. However, Klebsiella pneumoniae (K. pneumoniae) is an asymptomatic gut colonizer and, unlike well-studied enteric pathogens, harbors two genetically distinct ethanolamine utilization (eut) loci. Our investigation uncovered unique roles for each eut locus depending on EA utilization as a carbon or nitrogen source. Murine gut colonization studies demonstrated the necessity of both eut loci in the presence of intact gut microbiota for robust GI colonization by K. pneumoniae. Additionally, while some Escherichia coli gut isolates could metabolize EA, other commensals were incapable, suggesting that EA metabolism likely provides K. pneumoniae a selective advantage in gut colonization. Molecular and bioinformatic analyses unveiled the conservation of two eut loci among K. pneumoniae and a subset of the related taxa in the K. pneumoniae species complex, with the NtrC-RpoN regulatory cascade playing a pivotal role in regulation. These findings identify EA metabolism as a critical driver of K. pneumoniae niche establishment in the gut and propose microbial metabolism as a potential therapeutic avenue to combat K. pneumoniae infections.

Antimicrobial resistance and AmpC production in ESBL-producing Klebsiella pneumoniae and Klebsiella quasipneumoniae: A retrospective study in Japanese clinical isolates.

INTRODUCTION: The study of Klebsiella quasipneumoniae, Klebsiella variicola, and AmpC production in extended-spectrum ß-lactamase (ESBL)-producing Klebsiella in Japan is limited, and existing data are insufficient. This study aims to characterize Klebsiella species, determine AmpC production rates, and analyze antimicrobial resistance patterns in ESBL-producing Klebsiella isolates in Japan. METHODS: A total of 139 clinical isolates of ESBL-producing Klebsiella were collected in Japan, along with their corresponding antimicrobial susceptibility profiles. The isolates were identified using a web-based tool. ESBL genes within the isolates were identified using multiplex PCR. Screening for AmpC-producing isolates was performed using cefoxitin disks, followed by multiplex PCR to detect the presence of AmpC genes. Antimicrobial resistance patterns were analyzed across the predominant ESBL genotypes. RESULTS: The web-based tool identified 135 isolates (97.1%) as Klebsiella pneumoniae and 4 (2.9%) as K. quasipneumoniae subsp. similipneumoniae, with no instances of K. variicola detected. Among K. pneumoniae, the CTX-M-1 group emerged as the predominant genotype (83/135, 61.5%), followed by K. quasipneumoniae subsp. similipneumoniae (3/4, 75.0%). The CTX-M-9 group was the second most prevalent genotype in K. pneumoniae (45/135, 33.3%). The high resistance rates were observed for quinolones (ranging from 46.7% to 63.0%) and trimethoprim/sulfamethoxazole (78.5%). The CTX-M-1 group exhibited higher resistance to ciprofloxacin (66/83, 79.5%) compared to the CTX-M-9 group (18/45, 40.0%), a trend also observed for levofloxacin and trimethoprim/sulfamethoxazole. Among the 16 isolates that tested positive during AmpC screening, only one K. pneumoniae isolates (0.7%) were confirmed to carry the AmpC gene. CONCLUSION: Klebsiella pneumoniae with the CTX-M-1 group is the most common ESBL-producing Klebsiella in Japan and showed a low proportion of AmpC production. These isolates are resistant to quinolones and trimethoprim/sulfamethoxazole, highlighting the challenge of managing this pathogen. The findings underscore the importance of broader research and continuous monitoring to address the resistance patterns of ESBL-producing Klebsiella.

Loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing K1-ST23 hypervirulent Klebsiella pneumoniae.

OBJECTIVES: This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent Klebsiella pneumoniae (hvKp). METHODS: Here we longitudinally recovered 3 non-carbapenemase-producing K1-ST23 hvKp strains at a one-month interval (KP29105, KP29499 and KP30086) from an elderly male. Antimicrobial susceptibility testing, whole genome sequencing, transcriptomic sequencing, gene cloning, plasmid conjugation, quantitative real-time PCR (qRT-PCR), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were conducted. RESULTS: Among the 3 hvKp strains, KP29105 was resistant to the third- and fourth-generation cephalosporins, KP29499 acquired resistance to both ceftazidime-avibactam and carbapenems, while KP30086 restored its susceptibility to ceftazidime-avibactam, imipenem and meropenem but retained low-level resistance to ertapenem. KP29105 and KP29499 carried plasmid-encoded genes blaCTX-M-15 and blaCTX-M-71, respectively, but KP30086 lost both. Cloning of gene blaCTX-M-71 and conjugation experiment of blaCTX-M-71-carrying plasmid showed that the transformant and transconjugant were susceptible to ceftazidime-avibactam but had a more than 8-fold increase in MICs. Supplementation with an outer membrane permeabilizer could reduce the MIC of ceftazidime-avibactam by 32 folds, indicating that porins play a key role in ceftazidime-avibactam resistance. The OmpK35 of the 3 isolates was not expressed, and the OmpK36 of KP29499 and KP30086 had a novel amino acid substitution (L359R). SDS-PAGE and qRT-PCR showed that the expression of porin OmpK36 of KP29499 and KP30086 was significantly down-regulated compared with KP29105. CONCLUSIONS: In summary, we reported the rare ceftazidime-avibactam resistance in a non-carbapenemase-producing hvKp strain. Resistance plasmid carrying blaCTX-M-71 and mutated OmpK36 had a synergetic effect on the resistance.

Genome analyses of colistin-resistant high-risk blaNDM-5 producing Klebsiella pneumoniae ST147 and Pseudomonas aeruginosa ST235 and ST357 in clinical settings.

BACKGROUND: Colistin is a last-resort antibiotic used in extreme cases of multi-drug resistant (MDR) Gram-negative bacterial infections. Colistin resistance has increased in recent years and often goes undetected due to the inefficiency of predominantly used standard antibiotic susceptibility tests (AST). To address this challenge, we aimed to detect the prevalence of colistin resistance strains through both Vitek®2 and broth micro-dilution. We investigated 1748 blood, tracheal aspirate, and pleural fluid samples from the Intensive Care Unit (ICU), Neonatal Intensive Care Unit (NICU), and Tuberculosis and Respiratory Disease centre (TBRD) in an India hospital. Whole-genome sequencing (WGS) of extremely drug-resitant (XDR) and pan-drug resistant (PDR) strains revealed the resistance mechanisms through the Resistance Gene Identifier (RGI.v6.0.0) and Snippy.v4.6.0. Abricate.v1.0.1, PlasmidFinder.v2.1, MobileElementFinder.v1.0.3 etc. detected virulence factors, and mobile genetic elements associated to uncover the pathogenecity and the role of horizontal gene transfer (HGT). RESULTS: This study reveals compelling insights into colistin resistance among global high-risk clinical isolates: Klebsiella pneumoniae ST147 (16/20), Pseudomonas aeruginosa ST235 (3/20), and ST357 (1/20). Vitek®2 found 6 colistin-resistant strains (minimum inhibitory concentrations, MIC = 4 µg/mL), while broth microdilution identified 48 (MIC = 32-128 µg/mL), adhering to CLSI guidelines. Despite the absence of mobile colistin resistance (mcr) genes, mechanisms underlying colistin resistance included mgrB deletion, phosphoethanolamine transferases arnT, eptB, ompA, and mutations in pmrB (T246A, R256G) and eptA (V50L, A135P, I138V, C27F) in K. pneumoniae. P. aeruginosa harbored phosphoethanolamine transferases basS/pmrb, basR, arnA, cprR, cprS, alongside pmrB (G362S), and parS (H398R) mutations. Both strains carried diverse clinically relevant antimicrobial resistance genes (ARGs), including plasmid-mediated blaNDM-5 (K. pneumoniae ST147) and chromosomally mediated blaNDM-1 (P. aeruginosa ST357). CONCLUSION: The global surge in MDR, XDR and PDR bacteria necessitates last-resort antibiotics such as colistin. However, escalating resistance, particularly to colistin, presents a critical challenge. Inefficient colistin resistance detection methods, including Vitek2, alongside limited surveillance resources, accentuate the need for improved strategies. Whole-genome sequencing revealed alarming colistin resistance among K. pneumoniae and P. aeruginosa in an Indian hospital. The identification of XDR and PDR strains underscores urgency for enhanced surveillance and infection control. SNP analysis elucidated resistance mechanisms, highlighting the complexity of combatting resistance.

Thrombocytopenia in Klebsiella pneumoniae liver abscess: a retrospective study on its correlation with disease severity and potential causes.

Objective: Thrombocytopenia is commonly associated with infectious diseases and serves as an indicator of disease severity. However, reports on its manifestation in conjunction with Klebsiella pneumoniae liver abscess (KPLA) are scarce. The present study sought to elucidate the correlation between thrombocytopenia and KPLA severity and delve into the etiological factors contributing to the incidence of thrombocytopenia. Materials and methods: A retrospective analysis of the clinical data from patients with KPLA admitted between June 2012 and June 2023 was performed. Baseline characteristics, biochemical assessments, therapeutic interventions, complications, and clinical outcomes were compared between patients with and without thrombocytopenia. To investigate the potential etiologies underlying thrombocytopenia, the association between platelet count reduction and thrombophlebitis was examined, with a particular focus on platelet consumption. Furthermore, bone marrow aspiration results were evaluated to assess platelet production anomalies. Results: A total of 361 KPLA patients were included in the study, among whom 60 (17%) had concurrent thrombocytopenia. Those in the thrombocytopenia group exhibited significantly higher rates of thrombophlebitis (p = 0.042), extrahepatic metastatic infection (p = 0.01), septic shock (p = 0.024), admissions to the intensive care unit (p = 0.002), and in-hospital mortality (p = 0.045). Multivariate analysis revealed that thrombocytopenia (odds ratio, 2.125; 95% confidence interval, 1.114-4.056; p = 0.022) was independently associated with thrombophlebitis. Among the thrombocytopenic patients, eight underwent bone marrow aspiration, and six (75%) had impaired medullar platelet production. After treatment, 88.6% of thrombocytopenic patients (n = 47) demonstrated recovery in their platelet counts with a median recovery time of five days (interquartile range, 3-6 days). Conclusions: Thrombocytopenia in patients with KPLA is indicative of increased disease severity. The underlying etiologies for thrombocytopenia may include impaired platelet production within the bone marrow and augmented peripheral platelet consumption as evidenced by the presence of thrombophlebitis.

Dopamine promotes Klebsiella quasivariicola proliferation and inflammatory response in the presence of macrophages.

Background: Dopamine, a frequently used therapeutic agent for critically ill patients, has been shown to be implicated in clinical infections recently, however, the precise mechanisms underlying this association remain elusive. Klebsiella quasivariicola, a novel strain belonging to the Klebsiella species, exhibits potential pathogenic attributes. The impact of dopamine on K. quasivariicola infection has aroused our interest. Objective: Considering the contribution of host immune factors during infection, this study aimed to investigate the intricate interactions between K. quasivariicola, dopamine, and macrophages were explored. Methods: RAW264.7 cells and C57/BL6 mice were infected with K. quasivariicola, and the bacterial growth within macrophage, the production of inflammatory cytokines and the pathological changes in mice lungs were detected, in the absence or presence of dopamine. Results: Dopamine inhibited the growth of K. quasivariicola in the medium, but promoted bacterial growth when co-cultured with macrophages. The expression of proinflammatory cytokines increased in RAW 264.7 cells infected with K. quasivariicola, and a significant rise was observed upon the addition of dopamine. The infection of K. quasivariicola in mice induced an inflammatory response and lung injury, which were exacerbated by the administration of dopamine. Conclusions: Our findings suggest that dopamine may be one of the potential risk factors associated with K. quasivariicola infection. This empirical insight provides solid references for clinical precision medicine. Furthermore, an in vitro model of microbes-drugs-host immune cells for inhibitor screening was proposed to more accurately replicate the complex in vivo environment. This fundamental work had contributed to the present understanding of the crosstalk between pathogen, dopamine and host immune cells.