Genetic diversity of KPC-2-producing Klebsiella pneumoniae complex from aquatic ecosystems.

During the COVID-19 pandemic, the occurrence of carbapenem-resistant Klebsiella pneumoniae increased in human clinical settings worldwide. Impacted by this increase, international high-risk clones harboring carbapenemase-encoding genes have been circulating in different sources, including the environment. The blaKPC gene is the most commonly disseminated carbapenemase-encoding gene worldwide, whose transmission is carried out by different mobile genetic elements. In this study, blaKPC-2-positive Klebsiella pneumoniae complex strains were isolated from different anthropogenically affected aquatic ecosystems and characterized using phenotypic, molecular, and genomic methods. K. pneumoniae complex strains exhibited multidrug-resistant and extensively drug-resistant profiles, spotlighting the resistance to carbapenems, ceftazidime-avibactam, colistin, and tigecycline, which are recognized as last-line antimicrobial treatment options. Molecular analysis showed the presence of several antimicrobial resistance, virulence, and metal tolerance genes. In-depth analysis showed that the blaKPC-2 gene was associated with three different Tn4401 isoforms (i.e., Tn4401a, Tn4401b, and Tn4401i) and NTEKPC elements. Different plasmid replicons were detected and a conjugative IncN-pST15 plasmid harboring the blaKPC-2 gene associated with Tn4401i was highlighted. K. pneumoniae complex strains belonging to international high-risk (e.g., ST11 and ST340) and unusual clones (e.g., ST323, ST526, and ST4216) previously linked to clinical settings. In this context, some clones were reported for the first time in the environmental sector. Therefore, these findings evidence the occurrence of carbapenemase-producing K. pneumoniae complex strains in aquatic ecosystems and contribute to the monitoring of carbapenem resistance worldwide.

Detection of blaKPC gene among carbapenemase producing Klebsiella pneumoniae isolated from different clinical specimens at tertiary care hospital of Nepal.

BACKGROUND: Klebsiella pneumoniae infections have become a major cause of hospital acquired infection worldwide with the increased rate of acquisition of resistance to antibiotics. Carbapenem resistance mainly among Gram negative is an ongoing problem which causes serious outbreaks dramatically limiting treatment options. This prospective cross-sectional study was designed to detect blaKPC gene from carbapenem resistant K. pneumoniae. MATERIALS AND METHODS: A totally of 1118 different clinical specimens were screened and confirmed for KPC producing K. pneumoniae phenotypically using Meropenem (10 µg) disc. The blaKPC gene was amplified from the isolates of K. pneumoniae to detect the presence of this gene. RESULT: Of the total samples processed, 18.6% (n = 36) were K. pneumoniae and among 36 K. pneumoniae, 61.1% (n = 22/36) were meropenem resistant. This study demonstrated the higher level of MDR 91.7% (n = 33) and KPC production 47.2% (n = 17) among K. pneumoniae isolates. The blaKPC gene was detected in 8.3% (n = 3) of meropenem resistant isolates. CONCLUSION: Since the study demonstrates the higher level of MDR and KPC producing K. pneumoniae isolates that has challenged the use of antimicrobial agents, continuous microbiology, and molecular surveillance to assist early detection and minimize the further dissemination of blaKPC should be initiated. We anticipate that the findings of this study will be useful in understanding the prevalence of KPC-producing K. pneumoniae in Nepal.

Exploring the resistome, virulome, and mobilome of multidrug-resistant Klebsiella pneumoniae isolates: deciphering the molecular basis of carbapenem resistance.

BACKGROUND: Klebsiella pneumoniae, a notorious pathogen for causing nosocomial infections has become a major cause of neonatal septicemia, leading to high morbidity and mortality worldwide. This opportunistic bacterium has become highly resistant to antibiotics due to the widespread acquisition of genes encoding a variety of enzymes such as extended-spectrum beta-lactamases (ESBLs) and carbapenemases. We collected Klebsiella pneumoniae isolates from a local tertiary care hospital from February 2019-February 2021. To gain molecular insight into the resistome, virulome, and genetic environment of significant genes of multidrug-resistant K. pneumoniae isolates, we performed the short-read whole-genome sequencing of 10 K. pneumoniae isolates recovered from adult patients, neonates, and hospital tap water samples. RESULTS: The draft genomes of the isolates varied in size, ranging from 5.48 to 5.96 Mbp suggesting the genome plasticity of this pathogen. Various genes conferring resistance to different classes of antibiotics e.g., aminoglycosides, quinolones, sulfonamides, tetracycline, and trimethoprim were identified in all sequenced isolates. The highest resistance was observed towards carbapenems, which has been putatively linked to the presence of both class B and class D carbapenemases, blaNDM, and blaOXA, respectively. Moreover, the biocide resistance gene qacEdelta1 was found in 6/10 of the sequenced strains. The sequenced isolates exhibited a broad range of sequence types and capsular types. The significant antibiotic resistance genes (ARGs) were bracketed by a variety of mobile genetic elements (MGEs). Various spontaneous mutations in genes other than the acquired antibiotic-resistance genes were observed, which play an indirect role in making these bugs resistant to antibiotics. Loss or deficiency of outer membrane porins, combined with ESBL production, played a significant role in carbapenem resistance in our sequenced isolates. Phylogenetic analysis revealed that the study isolates exhibited evolutionary relationships with strains from China, India, and the USA suggesting a shared evolutionary history and potential dissemination of similar genes amongst the isolates of different origins. CONCLUSIONS: This study provides valuable insight into the presence of multiple mechanisms of carbapenem resistance in K. pneumoniae strains including the acquisition of multiple antibiotic-resistance genes through mobile genetic elements. Identification of rich mobilome yielded insightful information regarding the crucial role of insertion sequences, transposons, and integrons in shaping the genome of bacteria for the transmission of various resistance-associated genes. Multi-drug resistant isolates that had the fewest resistance genes exhibited a significant number of mutations. K. pneumoniae isolate from water source displayed comparable antibiotic resistance determinants to clinical isolates and the highest number of virulence-associated genes suggesting the possible interplay of ARGs amongst bacteria from different sources.

A challenging case of carbapenem resistant Klebsiella pneumoniae-related pyogenic liver abscess with capsular polysaccharide hyperproduction: a case report.

BACKGROUND: Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections are a major public health problem, necessitating the administration of polymyxin E (colistin) as a last-line antibiotic. Meanwhile, the mortality rate associated with colistin-resistant K. pneumoniae infections is seriously increasing. On the other hand, importance of administration of carbapenems in promoting colistin resistance in K. pneumoniae is unknown. CASE PRESENTATION: We report a case of K. pneumoniae-related pyogenic liver abscess in which susceptible K. pneumoniae transformed into carbapenem- and colistin-resistant K. pneumoniae during treatment with imipenem. The case of pyogenic liver abscess was a 50-year-old man with diabetes and liver transplant who was admitted to Abu Ali Sina Hospital in Shiraz. The K. pneumoniae isolate responsible for community-acquired pyogenic liver abscess was isolated and identified. The K. pneumoniae isolate was sensitive to all tested antibiotics except ampicillin in the antimicrobial susceptibility test and was identified as a non-K1/K2 classical K. pneumoniae (cKp) strain. Multilocus sequence typing (MLST) identified the isolate as sequence type 54 (ST54). Based on the patient's request, he was discharged to continue treatment at another center. After two months, he was readmitted due to fever and progressive constitutional symptoms. During treatment with imipenem, the strain acquired blaOXA-48 and showed resistance to carbapenems and was identified as a multidrug resistant (MDR) strain. The minimum inhibitory concentration (MIC) test for colistin was performed by broth microdilution method and the strain was sensitive to colistin (MIC < 2 µg/mL). Meanwhile, on blood agar, the colonies had a sticky consistency and adhered to the culture medium (sticky mucoviscous colonies). Quantitative real-time PCR and biofilm formation assay revealed that the CRKP strain increased capsule wzi gene expression and produced slime in response to imipenem. Finally, K. pneumoniae-related pyogenic liver abscess with resistance to a wide range of antibiotics, including the last-line antibiotics colistin and tigecycline, led to sepsis and death. CONCLUSIONS: Based on this information, can we have a theoretical hypothesis that imipenem is a promoter of resistance to carbapenems and colistin in K. pneumoniae? This needs more attention.

Phenotypic and molecular characterization of ß-lactamase-producing Klebsiella species among children discharged from hospital in Western Kenya.

BACKGROUND: The emergence and spread of ß-lactamase-producing Klebsiella spp. has been associated with a substantial healthcare burden resulting in therapeutic failures. We sought to describe the proportion of phenotypic resistance to commonly used antibiotics, characterize ß-lactamase genes among isolates with antimicrobial resistance (AMR), and assess the correlates of phenotypic AMR in Klebsiella spp. isolated from stool or rectal swab samples collected from children being discharged from hospital. METHODS: We conducted a cross-sectional study involving 245 children aged 1-59 months who were being discharged from hospitals in western Kenya between June 2016 and November 2019. Whole stool or rectal swab samples were collected and Klebsiella spp. isolated by standard microbiological culture. ß-lactamase genes were detected by PCR whilst phenotypic antimicrobial susceptibility was determined using the disc diffusion technique following standard microbiology protocols. Descriptive analyses were used to characterize phenotypic AMR and carriage of ß-lactamase-producing genes. The modified Poisson regression models were used to assess correlates of phenotypic beta-lactam resistance. RESULTS: The prevalence of ß-lactamase carriage among Klebsiella spp. isolates at hospital discharge was 62.9% (154/245). Antibiotic use during hospitalization (adjusted prevalence ratio [aPR] = 4.51; 95%CI: 1.79-11.4, p < 0.001), longer duration of hospitalization (aPR = 1.42; 95%CI: 1.14-1.77, p < 0.002), and access to treated water (aPR = 1.38; 95%CI: 1.12-1.71, p < 0.003), were significant predictors of phenotypically determined ß-lactamase. All the 154 ß-lactamase-producing Klebsiella spp. isolates had at least one genetic marker of ß-lactam/third-generation cephalosporin resistance. The most prevalent genes were blaCTX-M 142/154 (92.2%,) and blaSHV 142/154 (92.2%,) followed by blaTEM 88/154 (57.1%,) and blaOXA 48/154 (31.2%,) respectively. CONCLUSION: Carriage of ß-lactamase producing Klebsiella spp. in stool is common among children discharged from hospital in western Kenya and is associated with longer duration of hospitalization, antibiotic use, and access to treated water. The findings emphasize the need for continued monitoring of antimicrobial susceptibility patterns to inform the development and implementation of appropriate treatment guidelines. In addition, we recommend measures beyond antimicrobial stewardship and infection control within hospitals, improved sanitation, and access to safe drinking water to mitigate the spread of ß-lactamase-producing Klebsiella pathogens in these and similar settings.

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Objective: To investigate the clinical characteristics and molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolated from patients with bloodstream infections in a large tertiary-care general hospital in Southwest China. Methods: A total of 131 strains of non-repeating CRKP were collected from the blood cultures of patients who had bloodstream infections in 2015-2019. The strains were identified by VITEK-2, a fully automated microbial analyzer, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The minimum inhibitory concentration (MIC) was determined by microbroth dilution method. The common carbapenemase resistant genes and virulence factors were identified by PCR. Homology analysis was performed by multilocus sequencing typing. Whole genome sequencing was performed to analyze the genomic characteristics of CRKP without carbapenemase. Results: The 131 strains of CRKP showed resistance to common antibiotics, except for polymyxin B (1.6% resistance rate) and tigacycline (8.0% resistance rate). A total of 105 (80.2%) CRKP strains carried the Klebsiella pneumoniae carbapenemase (KPC) resistance gene, 15 (11.4%) strains carried the New Delhi Metallo-ß-lactamase (NDM) gene, and 4 (3.1%) isolates carried both KPC and NDM genes. Sequence typing (ST) 11 (74.0%) was the dominant sequence type. High detection rates for mrkD (96.2%), fimH (98.5%), entB (100%), and other virulence genes were reported. One hypervirulent CRKP strain was detected. The seven strains of CRKP that did not produce carbapenemase were shown to carry ESBL or AmpC genes and had anomalies in membrane porins OMPK35 and OMPK36, according to whole genome sequencing. Conclusion: In a large-scale tertiary-care general hospital, CRKP mainly carries the KPC gene, has a high drug resistance rate to a variety of antibiotics, and possesses multiple virulence genes. Attention should be paid to CRKP strains with high virulence.

Emergence of OXA-48-producing Klebsiella pneumoniae in Lithuania, 2023: a multi-cluster, multi-hospital outbreak.

In 2023, an increase of OXA-48-producing Klebsiella pneumoniae was noticed by the Lithuanian National Public Health Surveillance Laboratory. Whole genome sequencing (WGS) of 106 OXA-48-producing K. pneumoniae isolates revealed three distinct clusters of carbapenemase-producing K. pneumoniae high-risk clones, including sequence type (ST) 45 (n = 35 isolates), ST392 (n = 32) and ST395 (n = 28), involving six, six and nine hospitals in different regions, respectively. These results enabled targeted investigation and control, and underscore the value of national WGS-based surveillance for antimicrobial 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.

Occurrence, antibiotic resistance profiles and associated risk factors of Klebsiella pneumoniae in poultry farms in selected districts of Somalia Reginal State, Ethiopia.

BACKGROUND: Klebsiella pneumoniae is an opportunistic infection that causes production losses and death in the chicken industry. A cross-sectional study was conducted on exotic chicken breeds reared at the Jigjiga poultry farm from November 2022 to May 2023 to estimate the occurrence, associated risk factors, and antimicrobial susceptibility profiles of Klebsiella pneumoniae. The chickens were selected using systematic random sampling techniques. A total of 384 cloacal swabs were collected aseptically and transported to the laboratory for analysis. For statistical analysis, STATA® version 14.0 statistical software was used. RESULTS: From 384 examined faecal samples, 258 (67.2%) prevalences of Klebsiella pneumoniae were found. Furthermore, the association of the study's risk factors with the prevalence of Klebsiella pneumoniae was explored, and no statistically significant association was identified between sex and age. Nonetheless, relative prevalence at the age level was higher in chickens aged 12 months (67.6%) and Sasso breeds (90.0%). Similarly, male chickens and those raised for meat and egg production had a high prevalence rate of 72.5 and 75.8%, respectively. A total of 30 isolated Klebsiella pneumoniae colonies were tested in vitro for antibiotic sensitivity for six drugs, and it was shown that Klebsiella pneumoniae is moderately sensitive to Penicillin G (43.3%) while having higher resistance to Oxytetracycline (80.0%). CONCLUSIONS: The current findings revealed that the research area had the highest prevalence of Klebsiella pneumoniae, and the isolates were resistant to commonly used drugs in the study area. Thus, a long-term intervention plan, thorough research to determine a nationwide status, as well as further multi-drug resistance patterns and molecular characterization, were urged.

Intestinal carbapenem-resistant Klebsiella pneumoniae undergoes complex transcriptional reprogramming following immune activation.

Carbapenem-resistant Klebsiella pneumoniae (CR-Kp) is a significant threat to public health worldwide. The primary reservoir for CR-Kp is the intestinal tract. There, the bacterium is usually present at low density but can bloom following antibiotic treatment, mostly in hospital settings. The impact of disturbances in the intestinal environment on the fitness, survival, expansion, and drug susceptibility of this pathogen is not well-understood, yet it may be relevant to devise strategies to tackle CR-Kp colonization and infection. Here, we adopted an in vivo model to examine the transcriptional adaptation of a CR-Kp clinical isolate to immune activation in the intestine. We report that as early as 6 hours following host treatment with anti-CD3 antibody, CR-Kp underwent rapid transcriptional changes including downregulation of genes involved in sugar utilization and amino acid biosynthesis and upregulation of genes involved in amino acid uptake and catabolism, antibiotic resistance, and stress response. In agreement with these findings, treatment increased the concentration of oxidative species and amino acids in the mouse intestine. Genes encoding for proteins containing the domain of unknown function (DUF) 1471 were strongly upregulated, however their deletion did not impair CR-Kp fitness in vivo upon immune activation. Transcription factor enrichment analysis identified the global regulator cAMP-Receptor Protein, CRP, as a potential orchestrator of the observed transcriptional signature. In keeping with the recognized role of CRP in regulating utilization of alternative carbon sources, crp deletion in CR-Kp resulted in strongly impaired gut colonization, although this effect was not amplified by immune activation. Thus, following intestinal colonization, which occurs in a CRP-dependent manner, CR-Kp can rapidly respond to immune cues by implementing a well-defined and complex transcriptional program whose direct relevance toward bacterial fitness warrants further investigation. Additional analyses utilizing this model may identify key factors to tackle CR-Kp colonization of the intestine.

Klebsiella pneumoniae sequence type 147: a high-risk clone increasingly associated with plasmids carrying both resistance and virulence elements.

Introduction. The first hybrid resistance/virulence plasmid, combining elements from virulence plasmids described in hypervirulent types of Klebsiella pneumoniae with those from conjugative resistance plasmids, was described in an isolate of sequence type (ST) 147 from 2016. Subsequently, this type has been increasingly associated with these plasmids.Hypothesis or gap statement. The extent of carriage of hybrid virulence/resistance plasmids in nosocomial isolates of K. pneumoniae requires further investigation.Aim. To describe the occurrence of virulence/resistance plasmids among isolates of K. pneumoniae received by the UK reference laboratory, particularly among representatives of ST147, and to compare their sequences.Methodology. Isolates received by the laboratory during 2022 and the first half of 2023 (n=1278) were screened for virulence plasmids by PCR detection of rmpA/rmpA2 and typed by variable-number tandem repeat analysis. Twenty-nine representatives of ST147 (including a single-locus variant) from seven hospital laboratories were subjected to long-read nanopore sequencing using high-accuracy q20 chemistry to provide complete assemblies.Results. rmpA/rmpA2 were detected in 110 isolates, of which 59 belonged to hypervirulent K1-ST23, K2-ST86 and K2-ST65/375. Of the remainder, representatives of ST147 formed the largest group, with 22 rmpA/rmpA2-positive representatives (out of 47 isolates). Representatives were from 19 hospital laboratories, with rmpA/rmpA2-positive isolates from 10. Nanopore sequencing of 29 representatives of ST147 divided them into those with no virulence plasmid (n=12), those with non-New Delhi metallo-ß-lactamase (NDM) virulence plasmids (n=6) and those carrying bla NDM-5 (n=9) or bla NDM-1 (n=2) virulence plasmids. These plasmids were of IncFIB(pNDM-Mar)/IncHI1B(pNDM-MAR) replicon types. Most of the non-NDM virulence plasmids were highly similar to the originally described KpvST147L_NDM plasmid. Those carrying bla NDM-5 were highly similar to one another and to previously described plasmids in ST383 and carried an extensive array of resistance genes. Comparison of the fully assembled chromosomes indicated multiple introductions of ST147 in UK hospitals.Conclusion. This study highlights the high proportion of representatives of ST147 that carry IncFIB(pNDM-Mar)/IncHI1B(pNDM-MAR) hybrid resistance virulence plasmids. It is important to be aware of the high probability that representatives of this type carry these plasmids combining resistance and virulence determinants and of the consequent increased risk to patients.

Klebsiella pneumoniae exhibiting a phenotypic hyper-splitting phenomenon including the formation of small colony variants.

In this study, we characterized a Klebsiella pneumoniae strain in a patient with shrapnel hip injury, which resulted in multiple phenotypic changes, including the formation of a small colony variant (SCV) phenotype. Although already described since the 1960s, there is little knowledge about SCV phenotypes in Enterobacteriaceae. The formation of SCVs has been recognized as a bacterial strategy to evade host immune responses and compromise the efficacy of antimicrobial therapies, leading to persistent and recurrent courses of infections. In this case, 14 isolates with different resisto- and morpho-types were distinguished from the patient's urine and tissue samples. Whole genome sequencing revealed that all isolates were clonally identical belonging to the K. pneumoniae high-risk sequence type 147. Subculturing the SCV colonies consistently resulted in the reappearance of the initial SCV phenotype and three stable normal-sized phenotypes with distinct morphological characteristics. Additionally, an increase in resistance was observed over time in isolates that shared the same colony appearance. Our findings highlight the complexity of bacterial behavior by revealing a case of phenotypic "hyper-splitting" in a K. pneumoniae SCV and its potential clinical significance.

Analysis of virulence profiles in clinical isolates of Klebsiella pneumoniae from renal abscesses: clinical significance of hypervirulent isolates.

Introduction: Klebsiella pneumoniae can cause a wide range of infections. Hypervirulent K. pneumoniae (hvKp), particularly associated with the K1 and K2 capsular types, is an increasingly significant microorganism with the potential to cause invasive infections, including renal abscesses. Despite the rising prevalence of hvKp infections, information on renal abscesses caused by K. pneumoniae is limited, and the clinical significance of hvKp associated with specific virulence genes remains elusive. Methods: This study performed at a 1200-bed tertiary hospital sought to identify the clinical and microbiological characteristics of renal abscesses caused by K. pneumoniae, focusing on various virulence genes, including capsular serotypes and multilocus sequence typing (MLST). Results: Over an 8-year period, 64 patients with suspected renal abscesses were reviewed. Ten patients diagnosed with K. pneumoniae-related renal abscesses were ultimately enrolled in the study. Among the isolates from the 10 patients, capsular serotype K2 was predominant (40.0%), followed by K1 (30.0%). The most common sequence type by MLST was 23 (40.0%). In particular, six patients (60.0%) harbored specific genes indicative of hvKp: iucA, peg-344, rmpA, and rmpA2. Conclusions: Our findings highlight the importance of hvKp as a pathogen in renal abscesses. Although the nature of hvKp is relatively unknown, it is widely recognized as a highly virulent pathogen that can infect relatively healthy individuals of various ages and simultaneously cause infections at multiple anatomical sites. Therefore, when treating patients with K. pneumoniae-related renal abscesses, caution is necessary when considering the characteristics of hvKp, such as potential bacteremia, multi-organ abscess formation, and metastatic spread.

Respiratory carriage of hypervirulent Klebsiella pneumoniae by indigenous populations of Malaysia.

Klebsiella pneumoniae is a Gram-negative Enterobacteriaceae that is classified by the World Health Organisation (WHO) as a Priority One ESKAPE pathogen. South and Southeast Asian countries are regions where both healthcare associated infections (HAI) and community acquired infections (CAI) due to extended-spectrum ß-lactamase (ESBL)-producing and carbapenem-resistant K. pneumoniae (CRKp) are of concern. As K. pneumoniae can also exist as a harmless commensal, the spread of resistance genotypes requires epidemiological vigilance. However there has been no significant study of carriage isolates from healthy individuals, particularly in Southeast Asia, and specially Malaysia. Here we describe the genomic analysis of respiratory isolates of K. pneumoniae obtained from Orang Ulu and Orang Asli communities in Malaysian Borneo and Peninsular Malaysia respectively. The majority of isolates were K. pneumoniae species complex (KpSC) 1 K. pneumoniae (n = 53, 89.8%). Four Klebsiella variicola subsp. variicola (KpSC3) and two Klebsiella quasipneumoniae subsp. similipneumoniae (KpSC4) were also found. It was discovered that 30.2% (n = 16) of the KpSC1 isolates were ST23, 11.3% (n = 6) were of ST65, 7.5% (n = 4) were ST13, and 13.2% (n = 7) were ST86. Only eight of the KpSC1 isolates encoded ESBL, but importantly not carbapenemase. Thirteen of the KpSC1 isolates carried yersiniabactin, colibactin and aerobactin, all of which harboured the rmpADC locus and are therefore characterised as hypervirulent. Co-carriage of multiple strains was minimal. In conclusion, most isolates were KpSC1, ST23, one of the most common sequence types and previously found in cases of K. pneumoniae infection. A proportion were hypervirulent (hvKp) however antibiotic resistance was low.

Study on the inhibition activity and mechanism of Tanreqing against Klebsiella pneumoniae biofilm formation in vitro and in vivo.

Objective: To evaluate the antibacterial effect of Tanreqing (TRQ) against K. pneumoniae and its inhibition activity on bacterial biofilm formation in vitro and in vivo, and to explore the mechanism of the inhibitory effects of TRQ on K. pneumoniae biofilm formation. Methods: An in vitro biofilm model of K. pneumoniae was established, and the impact of TRQ on biofilm formation was evaluated using crystal violet staining and scanning electron microscopy (SEM). Furthermore, the clearance effect of TRQ against K. pneumoniae in the biofilm was assessed using the viable plate counting method; q-RT PCR was used to evaluate the inhibitory effect of different concentrations of TRQ on the expression of biofilm-related genes in Klebsiella pneumoniae; The activity of quorum sensing signal molecule AI-2 was detected by Vibrio harveyi bioluminescence assay; Meanwhile, a guinea pig lung infection model of Klebsiella pneumoniae was constructed, and after treated with drugs, pathological analysis of lung tissue and determination of bacterial load in lung tissue were performed. The treatment groups included TRQ group, imipenem(IPM) group, TRQ+IPM group, and sterile saline group as the control. Results: The formation of K. pneumoniae biofilm was significantly inhibited by TRQ in vitro experiments. Furthermore, when combined with IPM, the clearance of K. pneumoniae in the biofilm was notably increased compared to the TRQ group and IPM group alone. q-RT PCR analysis revealed that TRQ down-regulated the expression of genes related to biofilm formation in K. pneumoniae, specifically luxS, wbbm, wzm, and lsrK, and also inhibited the activity of AI-2 molecules in the bacterium. In vivo experiments demonstrated that TRQ effectively treated guinea pig lung infections, resulting in reduced lung inflammation. Additionally, when combined with IPM, there was a significant reduction in the bacterial load in lung tissue. Conclusion: TRQ as a potential therapeutic agent plays a great role in the treatment of K. pneumoniae infections, particularly in combination with conventional antibiotics. And TRQ can enhanced the clearance effect on the bacterium by inhibiting the K. pneumoniae biofilm formation, which provided experimental evidence in support of clinical treatment of TRQ against K. pneumoniae infections.

Co-immunization with DNA vaccines encoding yidR and IL-17 augments host immune response against Klebsiella pneumoniae infection in mouse model.

Klebsiella pneumoniae is an important gram-negative bacterium that causes severe respiratory and healthcare-associated infections. Although antibiotic therapy is applied to treat severe infections caused by K. pneumoniae, drug-resistant isolates pose a huge challenge to clinical practices owing to adverse reactions and the mismanagement of antibiotics. Several studies have attempted to develop vaccines against K. pneumoniae, but there are no licensed vaccines available for the control of K. pneumoniae infection. In the current study, we constructed a novel DNA vaccine, pVAX1-YidR, which encodes a highly conserved virulence factor YidR and a recombinant expression plasmid pVAX1-IL-17 encoding Interleukin-17 (IL-17) as a molecular adjuvant. Adaptive immune responses were assessed in immunized mice to compare the immunogenicity of the different vaccine schemes. The results showed that the targeted antigen gene was expressed in HEK293T cells using an immunofluorescence assay. Mice immunized with pVAX1-YidR elicited a high level of antibodies, induced strong cellular immune responses, and protected mice from K. pneumoniae challenge. Notably, co-immunization with pVAX1-YidR and pVAX1-IL-17 significantly augmented host adaptive immune responses and provided better protection against K. pneumoniae infections in vaccinated mice. Our study demonstrates that combined DNA vaccines and molecular adjuvants is a promising strategy to develop efficacious antibacterial vaccines against K. pneumoniae infections.

In vivo single-cell high-dimensional mass cytometry analysis to track the interactions between Klebsiella pneumoniae and myeloid cells.

In vivo single-cell approaches have transformed our understanding of the immune populations in tissues. Mass cytometry (CyTOF), that combines the resolution of mass spectrometry with the ability to conduct multiplexed measurements of cell molecules at the single cell resolution, has enabled to resolve the diversity of immune cell subsets, and their heterogeneous functionality. Here we assess the feasibility of taking CyTOF one step further to immuno profile cells while tracking their interactions with bacteria, a method we term Bac-CyTOF. We focus on the pathogen Klebsiella pneumoniae interrogating the pneumonia mouse model. Using Bac-CyTOF, we unveil the atlas of immune cells of mice infected with a K. pneumoniae hypervirulent strain. The atlas is characterized by a decrease in the populations of alveolar and monocyte-derived macrophages. Conversely, neutrophils, and inflammatory monocytes are characterized by an increase in the subpopulations expressing markers of less active cells such as the immune checkpoint PD-L1. These are the cells infected. We show that the type VI secretion system (T6SS) contributes to shape the lung immune landscape. The T6SS governs the interaction with monocytes/macrophages by shifting Klebsiella from alveolar macrophages to interstitial macrophages and limiting the infection of inflammatory monocytes. The lack of T6SS results in an increase of cells expressing markers of active cells, and a decrease in the subpopulations expressing PD-L1. By probing Klebsiella, and Acinetobacter baumannii strains with limited ability to survive in vivo, we uncover that a heightened recruitment of neutrophils, and relative high levels of alveolar macrophages and eosinophils and the recruitment of a characteristic subpopulation of neutrophils are features of mice clearing infections. We leverage Bac-CyTOF-generated knowledge platform to investigate the role of the DNA sensor STING in Klebsiella infections. sting-/- infected mice present features consistent with clearing the infection including the reduced levels of PD-L1. STING absence facilitates Klebsiella clearance.

Adhesion of Klebsiella oxytoca to bladder or lung epithelial cells is promoted by the presence of other opportunistic pathogens.

The intestinal and respiratory tracts of healthy individuals serve as habitats for a diverse array of microorganisms, among which Klebsiella oxytoca holds significance as a causative agent in numerous community- and hospital-acquired infections, often manifesting in polymicrobial contexts. In specific circumstances, K. oxytoca, alongside other constituents of the gut microbiota, undergoes translocation to distinct physiological niches. In these new environments, it engages in close interactions with other microbial community members. As this interaction may progress to co-infection where the virulence of involved pathogens may be promoted and enhance disease severity, we investigated how K. oxytoca affects the adhesion of commonly co-isolated bacteria and vice versa during co-incubation of different biotic and abiotic surfaces. Co-incubation was beneficial for the adhesion of at least one of the two co-cultured strains. K. oxytoca enhanced the adhesion of other enterobacteria strains to polystyrene and adhered more efficiently to bladder or lung epithelial cell lines in the presence of most enterobacteria strains and S. aureus. This effect was accompanied by bacterial coaggregation mediated by carbohydrate-protein interactions occurring between bacteria. These interactions occur only in sessile, but not planktonic populations, and depend on the features of the surface. The data are of particular importance for the risk assessment of the urinary and respiratory tract infections caused by K. oxytoca, including those device-associated. In this paper, we present the first report on K. oxytoca ability to acquire increased adhesive capacities on epithelial cells through interactions with common causal agents of urinary and respiratory tract infections.

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.

Surveillance and Resistance of Community-Onset Extended-Spectrum ß-Lactamase-Producing Escherichia coli and Klebsiella pneumonia in Oral and Maxillofacial Surgery Site Infections.

Background: The prevalence of community-onset infections of extended spectrum ß-lactamase (ESBL)-producing strains has increased globally, yet surveillance and resistance in patients with oral and maxillofacial surgery site infections is less investigated. Patients and Methods: A retrospective cohort study was performed to investigate risk factors and resistance of ESBL-producing Escherichia coli (ESBL-EC) and ESBL-producing Klebsiella pneumonia (ESBL-KP) among community-onset patients with oral and maxillofacial surgery during January 2010 to December 2016. Demographic features, predisposing factors, clinical outcomes, and antibiotic agent costs were analyzed. Antimicrobial susceptibility testing of nine antimicrobial agents against ESBL-KP and ESBL-EC were measured. Results: Among 2,183 cultures from infection sites in patients with oral and maxillofacial surgery site (45 cases [2.06%]) were confirmed with community-onset ESBL-KP (24; 1.10%) or ESBL-EC (21; 0.96%) infection. Multivariable analysis showed the independent risk factors for ESBL-producing bacterial infection were prior history of hospitalization (adjusted odds ratio [aOR], 10.984; 95% confidence interval [CI], 5.965-59.879; p = 0.025) and malignant condition (aOR, 3.373; 95% CI 2.947-7.634; p = 0.024). Based on antimicrobial susceptibility testing, 57.8% ESBL-KP and ESBL-EC were found receiving inappropriate antimicrobial therapy, and antibiotic agent costs were higher than non-ESBL-producing bacterial infections ($493.8 ± $367.3 vs. $304.1 ± $334.7; p = 0.031). Conclusions: Infections caused by ESBL-KP and ESBL-EC among patients in sites with oral and maxillofacial surgery are associated with prior history of hospitalization and malignant conditions. Prompt detection and appropriate antibiotic administration for community-onset infections of ESBLs are necessary for such populations.