Conserved FimK Truncation Coincides with Increased Expression of Type 3 Fimbriae and Cultured Bladder Epithelial Cell Association in Klebsiella quasipneumoniae.

Klebsiella spp. commonly cause both uncomplicated urinary tract infection (UTI) and recurrent UTI (rUTI). Klebsiella quasipneumoniae, a relatively newly defined species of Klebsiella, has been shown to be metabolically distinct from Klebsiella pneumoniae, but its type 1 and type 3 fimbriae have not been studied. K. pneumoniae uses both type 1 and type 3 fimbriae to attach to host epithelial cells. The type 1 fimbrial operon is well conserved between Escherichia coli and K. pneumoniae apart from fimK, which is unique to Klebsiella spp. FimK contains an N-terminal DNA binding domain and a C-terminal phosphodiesterase (PDE) domain that has been hypothesized to cross-regulate type 3 fimbriae expression via modulation of cellular levels of cyclic di-GMP. Here, we find that a conserved premature stop codon in K. quasipneumoniae fimK results in truncation of the C-terminal PDE domain and that K quasipneumoniae strain KqPF9 cultured bladder epithelial cell association and invasion are dependent on type 3 but not type 1 fimbriae. Further, we show that basal expression of both type 1 and type 3 fimbrial operons as well as cultured bladder epithelial cell association is elevated in KqPF9 relative to uropathogenic K. pneumoniae TOP52. Finally, we show that complementation of KqPF9ΔfimK with the TOP52 fimK allele reduced type 3 fimbrial expression and cultured bladder epithelial cell attachment. Taken together these data suggest that the C-terminal PDE of FimK can modulate type 3 fimbrial expression in K. pneumoniae and its absence in K. quasipneumoniae may lead to a loss of type 3 fimbrial cross-regulation. IMPORTANCE K. quasipneumoniae is often indicated as the cause of opportunistic infections, including urinary tract infection, which affects >50% of women worldwide. However, the virulence factors of K. quasipneumoniae remain uninvestigated. Prior to this work, K. quasipneumoniae and K. pneumoniae had only been distinguished phenotypically by metabolic differences. This work contributes to the understanding of K. quasipneumoniae by evaluating the contribution of type 1 and type 3 fimbriae, which are critical colonization factors encoded by all Klebsiella spp., to K. quasipneumoniae bladder epithelial cell attachment in vitro. We observe clear differences in bladder epithelial cell attachment and regulation of type 3 fimbriae between uropathogenic K. pneumoniae and K. quasipneumoniae that coincide with a structural difference in the fimbrial regulatory gene fimK.

Colonization of White-Tailed Deer (Odocoileus virginianus) from Urban and Suburban Environments with Cephalosporinase- and Carbapenemase-Producing Enterobacterales.

Wildlife play a role in the acquisition, maintenance, and dissemination of antimicrobial resistance (AMR). This is especially true at the human-domestic animal-wildlife interface, like urbanized areas, where interactions occur that can promote the cross-over of AMR bacteria and genes. We conducted a 2-year fecal surveillance (n = 783) of a white-tailed deer (WTD) herd from an urban park system in Ohio to identify and characterize cephalosporin-resistant and carbapenemase-producing bacteria using selective enrichment. Using generalized linear mixed models we found that older (OR = 2.3, P < 0.001), male (OR = 1.8, P = 0.001) deer from urbanized habitats (OR = 1.4, P = 0.001) were more likely to harbor extended-spectrum cephalosporin-resistant Enterobacterales. In addition, we isolated two carbapenemase-producing Enterobacterales (CPE), a Klebsiella quasipneumoniae harboring blaKPC-2 and an Escherichia coli harboring blaNDM-5, from two deer from urban habitats. The genetic landscape of the plasmid carrying blaKPC-2 was unique, not clustering with other reported plasmids encoding KPC-2, and only sharing 78% of its sequence with its nearest match. While the plasmid carrying blaNDM-5 shared sequence similarity with other reported plasmids encoding NDM-5, the intact IS26 mobile genetic elements surrounding multiple drug resistant regions, including the blaNDM-5, has been reported infrequently. Both carbapenemase genes were successfully conjugated to a J53 recipient conferring a carbapenem-resistant phenotype. Our findings highlight that urban environments play a significant role on the transmission of AMR bacteria and genes to wildlife and suggest WTD may play a role in the dissemination of clinically and epidemiologically relevant antimicrobial resistant bacteria. IMPORTANCE The role of wildlife in the spread of antimicrobial resistance is not fully characterized. Some wildlife, including white-tailed deer (WTD), can thrive in suburban and urban environments. This may result in the exchange of antimicrobial resistant bacteria and resistance genes between humans and wildlife, and lead to their spread in the environment. We found that WTD living in an urban park system carried antimicrobial resistant bacteria that were important to human health and resistant to antibiotics used to treat serious bacterial infections. This included two deer that carried bacteria resistant to carbapenem antibiotics which are critically important for treatment of life-threatening infections. These two bacteria had the ability to transfer their AMR resistance genes to other bacteria, making them a threat to public health. Our results suggest that WTD may contribute to the spread of antimicrobial resistant bacteria in the environment.

First detection of Klebsiella quasipneumoniae producing OXA-181 carbapenemase in Malaysia.

The emergence of carbapenemase-producing Enterobacteriaceae has become a major global concern. OXA-48-like carbapenemase gene and its variants have been increasingly reported worldwide. This study reported the first OXA-181-producing Klebsiella quasipneumoniae isolate in Malaysia. This bacterium was isolated from blood specimen of a three-year-old boy with Alagille syndrome who had liver biopsy on October 2016. He had undergone liver transplant in India ten months previously. The genotypic and phenotypic characteristics of the strain were elucidated in this study. To our best knowledge, this is the first report of OXA-181-producing K. quasipneumoniae in Malaysia.

Klebsiella quasipneumoniae Provides a Window into Carbapenemase Gene Transfer, Plasmid Rearrangements, and Patient Interactions with the Hospital Environment.

Several emerging pathogens have arisen as a result of selection pressures exerted by modern health care. Klebsiella quasipneumoniae was recently defined as a new species, yet its prevalence, niche, and propensity to acquire antimicrobial resistance genes are not fully described. We have been tracking inter- and intraspecies transmission of the Klebsiella pneumoniae carbapenemase (KPC) gene, blaKPC, between bacteria isolated from a single institution. We applied a combination of Illumina and PacBio whole-genome sequencing to identify and compare K. quasipneumoniae from patients and the hospital environment over 10- and 5-year periods, respectively. There were 32 blaKPC-positive K. quasipneumoniae isolates, all of which were identified as K. pneumoniae in the clinical microbiology laboratory, from 8 patients and 11 sink drains, with evidence for seven separate blaKPC plasmid acquisitions. Analysis of a single subclade of K. quasipneumoniae subsp. quasipneumoniae (n = 23 isolates) from three patients and six rooms demonstrated seeding of a sink by a patient, subsequent persistence of the strain in the hospital environment, and then possible transmission to another patient. Longitudinal analysis of this strain demonstrated the acquisition of two unique blaKPC plasmids and then subsequent within-strain genetic rearrangement through transposition and homologous recombination. Our analysis highlights the apparent molecular propensity of K. quasipneumoniae to persist in the environment as well as acquire carbapenemase plasmids from other species and enabled an assessment of the genetic rearrangements which may facilitate horizontal transmission of carbapenemases.

Clinical characteristics in blood stream infections caused by Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae: a comparative study, Japan, 2014-2017.

BACKGROUND: Klebsiella variicola and K. quasipneumoniae are new species distinguishable from K. pneumoniae but they are often misidentified as K. pneumoniae in clinical settings. Several reports have demonstrated the possibility that the virulence factors and clinical features differ among these three phylogroups. In this study, we aimed to clarify whether there were differences in clinical and bacterial features between the three phylogroups isolated from patients with bloodstream infections (BSIs) in Japan. METHODS: Isolates from all patients with BSIs caused by K. pneumoniae admitted to two hospitals between 2014 and 2017 (n = 119) were included in the study. Bacterial species were identified via sequence analysis, and their virulence factors and serotypes were analyzed via multiplex PCR results. Clinical data were retrieved from medical records. RESULTS: Of the 119 isolates, 21 (17.7%) were identified as K. variicola and 11 (9.2%) as K. quasipneumoniae; K1 serotype was found in 16 (13.4%), and K2 serotype in 13 (10.9%). Significant differences in the prevalence of rmpA, iutA, ybtS, entB and kfu (p < 0.001), and allS genes (p < 0.05) were found between the three phylogroups. However, there were no significant differences in clinical features, including the 30-day mortality rate, between the three organisms, although K. variicola was more frequently detected in patients over 80 years old compared with other Klebsiella species (p < 0.005), and K. quasipneumoniae more frequently occurred in patients with malignancy (p < 0.05). CONCLUSIONS: Our findings demonstrated the differences in bacterial pathogenicity and clinical features among these three phylogroups. Further epidemiological studies into BSI caused by Klebsiella species are warranted.

Liver Abscess Caused by Infection with Community-Acquired Klebsiella quasipneumoniae subsp. quasipneumoniae.

We report a case of pyogenic liver abscess caused by community-acquired Klebsiella quasipneumoniae subsp. quasipneumoniae. The infecting isolate had 2 prominent features of hypervirulent K. pneumoniae strains: the capsular polysaccharide synthesis region for K1 serotype and the integrative and conjugative element ICEKp1, which encodes the virulence factors yersiniabactin, salmochelin, and RmpA.

Genomic identification of nitrogen-fixing Klebsiella variicola, K. pneumoniae and K. quasipneumoniae.

It was difficult to differentiate Klebsiella pneumoniae, K. quasipneumoniae and K. variicola by biochemical and phenotypic tests. Genomics increase the resolution and credibility of taxonomy for closely-related species. Here, we obtained the complete genome sequence of the K. variicola type strain DSM 15968(T) (=F2R9(T)). The genome of the type strain is a circular chromosome of 5,521,203 bp with 57.56% GC content. From 540 Klebsiella strains whose genomes had been publicly available as at 3 March 2015, we identified 21 strains belonging to K. variicola and 8 strains belonging to K. quasipneumoniae based on the genome average nucleotide identities (ANI). All the K. variicola strains, one K. pneumoniae strain and five K. quasipneumoniae strains contained nitrogen-fixing genes. A phylogenomic analysis showed clear species demarcations for these nitrogen-fixing bacteria. In accordance with the key biochemical characteristics of K. variicola, the idnO gene encoding 5-keto-D-gluconate 5-reductase for utilization of 5-keto-D-gluconate and the sorCDFBAME operon for catabolism of L-sorbose were present whereas the rbtRDKT operon for catabolism of adonitol was absent in the genomes of K. variicola strains. Therefore, the genomic analyses supported the ANI-based species delineation; the genome sequence of the K. variicola type strain provides the reference genome for genomic identification of K. variicola, which is a nitrogen-fixing species.

Characterisation of highly virulent and colistin-resistant ST367-KL1 Klebsiella quasipneumoniae subsp. similipneumoniae Strain.

OBJECTIVES: To elucidate the characteristics of a colistin-resistant and hypervirulent Klebsiella quasipneumoniae subsp. similipneumoniae strain (KP8) using whole genome sequencing and various phenotypic assays. METHODS: Antimicrobial susceptibility testing was performed using broth microdilution. Whole genome sequencing and comparative genomics were utilised to elucidate genomic characteristics. Phenotypic assays to evaluate virulence factors included measurements of mucosal viscosity, biofilm production, siderophore production, infection of A549 cells, serum-killing assays, and Galleria mellonella infection models. RESULTS: Whole-genome sequencing revealed that the strain (KP8) belongs to sequence type 367 (ST367) and capsular type 1 (KL1), and it harbours several virulence genes, including regulator of mucoid phenotype (rmpA/A2), salmochelin (iroBCDN) and aerobactin (iucABCDiutA). Antibiotic susceptibility tests showed that KP8 was resistant to colistin. Genome analysis showed that the colistin resistance of KP8 might be related to amino acid insertions in pmrB (L215_D217, insL) and pagP (M1_S3, insV). Importantly, KP8 demonstrated comparable mucosal viscosity, biofilm production capacity, siderophore production levels to hvKP. Serum-killing experiments, A549 cell infection models, and G. mellonella infection models further indicated that KP8 displayed high virulence, akin to the hypervirulent strain NUTH-K2044. Notably, global genome analysis of the K. quasipneumoniae subsp. similipneumoniae strains highlighted that the ST367 lineage has a higher tendency to carry virulence-associated genes compared to other sequence types. The prevalence of virulence-associated factors concentrated within Chinese ST367 isolates reinforces this observation. CONCLUSION: These findings further enhance our understanding of the resistance and pathogenicity of ST367 K. quasipneumoniae subsp. similipneumoniae strain and also providing a broader perspective on the global epidemiological landscape.

Carbapenemase-producing Klebsiella quasipneumoniae ST688 (NDM-1) and Klebsiella michiganensis ST40 (KPC-2) in food destined for hospitalized patients.

OBJECTIVES: Klebsiella spp. are leading causes of nosocomial infections. Their ability to harbour antimicrobial resistance genes makes them an important public health threat. This study aimed to report the genomic background of carbapenemase-producing Klebsiella quasipneumoniae (HV55B) and Klebsiella michiganensis (HV55D) strains isolated from fresh vegetable destined for inpatients. METHODS: Microbiological and molecular methods were used to isolate and identify the strains, which were submitted to the antimicrobial susceptibility test and pH tolerance assays. Whole genome sequencing (WGS) was performed on MiSeq and NextSeq platforms, and online available tools were applied to bioinformatic analysis of clinically relevant information. RESULTS: Both isolates were considered multidrug-resistant and tolerated pH ≥ 4 for 24 h. HV55B belonged to sequence type (ST) ST668, and presented a broad resistome and plasmids from four incompatibility groups. HV55D belonged to ST40. Both strains HV55B and HV55D were genetically close to isolates responsible for human infections around the world, which stands for the plausibility of such bacteria to cause disease in patients of the studied institution. CONCLUSIONS: Our results confirm the presence of carbapenemase-producing Klebsiella spp. in fresh foodstuffs intended for inpatients consumption. The genomes characterized here also provide clinically and genomically relevant information to forthcoming epidemiological surveillance studies.

Whole genome sequencing reveals complex resistome features of Klebsiella pneumoniae isolated from patients at major hospitals in Trinidad, West Indies.

OBJECTIVES: Antibiotic-resistant Klebsiella pneumoniae is a human pathogen of major global concern due to its ability to cause multiple severe diseases that are often difficult to treat therapeutically. This study aimed to investigate the resistome of local clinical K. pneumoniae isolates. METHODS: Herein, we used a whole genome sequencing approach and bioinformatics tools to reconstruct the resistome of 10 clinical K. pneumoniae isolates and one clinical isolate of the closely related Klebsiella quasipneumoniae obtained from patients from three major hospitals in Trinidad, West Indies. RESULTS: The results of the study revealed the presence of a complex antibiotic-resistant armoury among the local isolates with multiple resistance mechanisms involving (i) inactivation of antibiotics, (ii) efflux pumps, (iii) antibiotic target alteration, protection, and replacement against antibiotics, and (iv) altered porin protein that reduced the permeability to antibiotics. Several resistance genes such as blaCTX-M-15, blaTEM-1B, blaSHV-28, blaKPC-2, oqxA, sul1, tetD, aac(6')-Ib-cr5, aph(6)-Id, and fosA6, which are known to confer resistance to antibiotics used to treat K. pneumoniae infections. In most cases, the resistance genes were flanked by mobile elements, including insertion sequences and transposons, which facilitate the spread of these genetic features among related organisms. CONCLUSION: This is the first comprehensive study to thoroughly investigate the resistome of clinical K. pneumoniae isolates and K. quasipneumoniae from Trinidad, West Indies. These findings suggest that monitoring K. pneumoniae and its genome-wide antibiotic resistance features in clinical strains would be of critical importance for guiding antibiotic stewardship programs and improving regional disease management systems for this pathogen.

Coexistence of plasmid-mediated tmexCD2-toprJ2, blaIMP-4, and blaNDM-1 in Klebsiella quasipneumoniae.

Klebsiella quasipneumoniae is a potential pathogen that has not been studied comprehensively. The emergence of multidrug-resistant (MDR) K. quasipneumoniae, specifically strains resistant to tigecycline and carbapenem, presents a significant challenge to clinical treatment. This investigation aimed to characterize MDR K. quasipneumoniae strain FK8966, co-carrying tmexCD2-toprJ2, blaIMP-4, and blaNDM-1 by plasmids. It was observed that FK8966's MDR was primarily because of the IncHI1B-like plasmid co-carrying tmexCD2-toprJ2 and blaIMP-4, and an IncFIB(K)/IncFII(K) plasmid harboring blaNDM-1. Furthermore, the phylogenetic analysis revealed that IncHI1B-like plasmids carrying tmexCD2-toprJ2 were disseminated among different bacteria, specifically in China. Additionally, according to the comparative genomic analysis, the MDR regions indicated that the tmexCD2-toprJ2 gene cluster was inserted into the umuC gene, while blaIMP-4 was present in transposon TnAs3 linked to the class 1 integron (IntI1). It was also observed that an ΔTn3000 insertion with blaNDM-1 made a novel blaNDM-1 harboring IncFIB(K)/IncFII(K) plasmid. The antimicrobial resistance prevalence and phylogenetic analyses of K. quasipneumoniae strains indicated that FK8966 is a distinct MDR branch of K. quasipneumoniae. Furthermore, CRISPR-Cas system analysis showed that many K. quasipneumoniae CRISPR-Cas systems lacked spacers matching the two aforementioned novel resistance plasmids, suggesting that these resistance plasmids have the potential to disseminate within K. quasipneumoniae. Therefore, the spread of MDR K. quasipneumoniae and plasmids warrants further attention.IMPORTANCEThe emergence of multidrug-resistant K. quasipneumoniae poses a great threat to clinical care, and the situation is exacerbated by the dissemination of tigecycline- and carbapenem-resistant genes. Therefore, monitoring these pathogens and their resistance plasmids is urgent and crucial. This study identified tigecycline- and carbapenem-resistant K. quasipneumoniae strain, FK8966. Furthermore, it is the first study to report the coexistence of tmexCD2-toprJ2, blaIMP-4, and blaNDM-1 in K. quasipneumoniae. Moreover, the CRISPR-Cas system of many K. quasipneumoniae lacks spacers that match the plasmids carried by FK8966, which are crucial for mediating resistance against tigecycline and carbapenems, indicating their potential to disseminate within K. quasipneumoniae.

Genome sequence of the Klebsiella quasipneumoniae bacteriophage EKq1 with activity against Klebsiella pneumoniae.

We describe the genome of a lytic phage EKq1 isolated on Klebsiella quasipneumoniae, with activity against Klebsiella pneumoniae. EKq1 is an unclassified representative of the class Caudoviricetes, similar to Klebsiella phages VLCpiS8c, phiKp_7-2, and vB_KleS-HSE3. The 48,244-bp genome has a GC content of 56.43% and 63 predicted protein-coding genes.

Longitudinal monitoring reveals the emergence and spread of blaGES-5-harboring carbapenem-resistant Klebsiella quasipneumoniae in a Hong Kong hospital wastewater discharge line.

Testing hospital wastewater (HWW) is potentially an effective, long-term approach for monitoring trends in antimicrobial resistance (AMR) patterns in health care institutions. Over a year, we collected wastewater samples from the clinical and non-clinical sites of a tertiary hospital and from a downstream wastewater treatment plant (WWTP). We focused on the extent of carbapenem resistance among Enterobacteriaceae isolates given their clinical importance. Escherichia coli and Klebsiella spp. were the most frequently isolated Enterobacteriaceae species at all sampling sites. Additionally, a small number of isolates belonging to ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species), except K. pneumoniae, were detected. Of the 232 Klebsiella spp. isolates, 100 (43.1 %) were multi-drug resistant (MDR), with 46 being carbapenem-resistant. Most of these carbapenem-resistant isolates were K. quasipneumoniae (CRKQ) (n = 44). All CRKQ isolates were isolated from the wastewater of a clinical site that includes intensive care units, which also yielded significantly more multi-drug resistant isolates compared to all other sampling sites. Among the CRKQ isolates, blaGES-5 genes (n = 42) were the primary genetic determinant of carbapenem resistance. Notably, three different CRKQ isolates, collected within the same month in HWW and the influent and effluent flow of the WWTP, shared >99 % sequence similarity between their blaGES-5 genes and between their flanking regions and upstream integron-integrase region. The influent isolate was phylogenetically close to K. quasipnuemoniae isolates from wastewater collected in Japan. Its blaGES-5 gene and surrounding sequences were > 99 % identical to blaGES-24 genes found in the Japanese isolates. Our results suggest that testing samples from sites located closer to hospitals could support antibiotic stewardship programs compared to samples collected further downstream. Moreover, testing samples collected regularly from WWTPs may reflect the local and global spread of pathogens and their resistances.

Within-host acquisition of colistin-resistance of an NDM-producing Klebsiella quasipneumoniae subsp. similipneumoniae strain through the insertion sequence-903B-mediated inactivation of mgrB gene in a lung transplant child in China.

Background: Colistin, as the antibiotic of "last resort" for carbapenem-resistant Klebsiella, develop resistance during administration of this antimicrobial agent. We identified an NDM-1-producing Klebsiella quasipneumonuae subsp. similipneumoniae (KQSS) strain KQ20605 recovered from a child, which developed resistance to colistin (KQ20786) through acquiring an IS903B element between the -27th and -26th bp of mgrB promoter region after 6-day colistin usage. Objectives: The aim of this study is to explore the source of IS903B in the disruptive mgrB gene and its underlying mechanisms. Materials and methods: Antibiotics susceptibility testing was conducted via microbroth dilution method. The in vitro colistin-induced experiment of KQ20605 was performed to mimic the in vivo transition from colistin-sensitive to resistant. Whole-genome sequencing was used to molecular identification of colistin resistance mechanism. Results: The IS903B element integrated into mgrB gene of KQ20786 had a 100% nucleotide identity and coverage match with one IS903B on plasmid IncR, and only 95.1% (1005/1057) identity to those on chromosome. In vitro, upon the pressure of colistin, KQ20605 could also switch its phenotype from colistin-sensitive to resistant with IS elements (e.g., IS903B and IS26) frequently inserted into mgrB gene at "hotspots", with the insertion site of IS903B nearly identical to that of KQ20786. Furthermore, IS26 elements in this isolate were only encoded by plasmids, including IncR and conjugative plasmid IncN harboring bla NDM. Conclusion: Mobilizable IS elements on plasmids tend to be activated and integrated into mgrB gene at "hotspots" in this KQSS, thereby causing the colistin resistance emergence and further dissemination.

The evolutionary mechanism of non-carbapenemase carbapenem-resistant phenotypes in Klebsiella spp.

Antibiotic resistance is driven by selection, but the degree to which a bacterial strain's evolutionary history shapes the mechanism and strength of resistance remains an open question. Here, we reconstruct the genetic and evolutionary mechanisms of carbapenem resistance in a clinical isolate of Klebsiella quasipneumoniae. A combination of short- and long-read sequencing, machine learning, and genetic and enzymatic analyses established that this carbapenem-resistant strain carries no carbapenemase-encoding genes. Genetic reconstruction of the resistance phenotype confirmed that two distinct genetic loci are necessary in order for the strain to acquire carbapenem resistance. Experimental evolution of the carbapenem-resistant strains in growth conditions without the antibiotic revealed that both loci confer a significant cost and are readily lost by de novo mutations resulting in the rapid evolution of a carbapenem-sensitive phenotype. To explain how carbapenem resistance evolves via multiple, low-fitness single-locus intermediates, we hypothesised that one of these loci had previously conferred adaptation to another antibiotic. Fitness assays in a range of drug concentrations show how selection in the antibiotic ceftazidime can select for one gene (blaDHA-1) potentiating the evolution of carbapenem resistance by a single mutation in a second gene (ompK36). These results show how a patient's treatment history might shape the evolution of antibiotic resistance and could explain the genetic basis of carbapenem-resistance found in many enteric-pathogens.

Accurate Identification of Klebsiella variicola by MALDI-TOF Mass Spectrometry in Clinical Microbiology Laboratories.

Klebsiella variicola is a pathogen that is increasingly recognized as being associated with human infections, but the methods available to clinical microbiology laboratories for accurate identification are limited. In this study, we assessed the accuracy of identification of K. variicola by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using genetic identification with multiplex PCR as the reference method. Antimicrobial susceptibilities and virulence of K. variicola strains were also investigated. Fifty-five Klebsiella pneumoniae, 26 K. variicola, and 2 Klebsiella quasipneumoniae clinical strains were used for evaluation. Both MALDI Biotyper with library version 9 and Klebsiella MALDI TypeR, a web-based species identification tool using MALDI-TOF data, accurately identified all K. variicola strains. In addition, two strains of K. quasipneumoniae were accurately identified with Klebsiella MALDI TypeR. Whole-genome sequencing confirmed the accurate identification to the subspecies level by Klebsiella MALDI TypeR for four strains (two strains each of K. variicola subsp. variicola and K. quasipneumoniae subsp. similipneumoniae). While 13 strains, 3 strains, and 1 strain of K. pneumoniae showed nonsusceptibility to ampicillin-sulbactam, ceftriaxone, and meropenem, respectively, all strains of K. variicola were susceptible to all tested antimicrobial agents. Although two K. variicola strains were positive for the string test, no K. variicola strains harbored any of the genes associated with hypervirulence of K. pneumoniae. Accurate identification of the K. pneumoniae complex, including K. variicola, by MALDI-TOF in clinical microbiology laboratories is expected to clarify the clinical characteristics of each species in the future. IMPORTANCE Recent widespread use of bacterial whole-genome sequencing analysis has resulted in the proposal of novel bacterial species and reclassification of taxonomy. Accurate methods for identification of bacterial species in clinical microbiology laboratories are essential to accumulate information on the clinical characteristics of each bacterial species. Klebsiella variicola is a member of the Klebsiella pneumoniae complex, and its association with human infections has been increasingly recognized, but accurate identification methods approved for use in clinical microbiology laboratories have been limited thus far. The findings of the present study suggest that K. variicola can be accurately identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry using updated library or web-based identification tools. Accurate identification will promote exploration of clinical characteristics of K. variicola.

Reduction of gastrointestinal tract colonization by Klebsiella quasipneumoniae using antimicrobial protein KvarIa.

BACKGROUND: Klebsiella quasipneumoniae is an opportunistic pathogen causing antibiotic-resistant infections of the gastrointestinal tract in many clinical cases. Orally delivered bioactive Klebsiella-specific antimicrobial proteins, klebicins, could be a promising method to eradicate Klebsiella species infecting the gut. METHODS: Mouse infection model was established based on infection of antibiotic-treated BALB/C mice with K. quasipneumoniae strain DSM28212. Four study groups were used (3 animals/group) to test the antimicrobial efficacy of orally delivered klebicin KvarIa: vehicle-only group (control, phosphate-buffered saline), and other three groups with bacteria, antibiotic therapy and 100 µg of uncoated Kvarla, 100 µg coated KvarIa, 1000 µg coated-KvarIa. Because of the general sensitivity of bacteriocins to gastroduodenal proteases, Kvarla doses were coated with Eudragit®, a GMP-certified formulation agent that releases the protein at certain pH. The coating treatment was selected based on measurements of mouse GI tract pH. The quantity of Klebsiella haemolysin gene (khe) in faecal samples of the study animals was used to quantify the presence of Klebsiella. RESULTS: GI colonization of K. quasipneumoniae was achieved only in the antibiotic-treated mice groups. Significant changes in khe marker quantification were found after the use of Eudragit® S100 formulated klebicin KvarIa, at both doses, with a significant reduction of K. quasipneumoniae colonization compared to the vehicle-only control group. CONCLUSIONS: Mouse GI tract colonization with K. quasipneumoniae can be achieved if natural gut microbiota is suppressed by prior antibiotic treatment. The study demonstrates that GI infection caused by K. quasipneumoniae can be significantly reduced using Eudragit®-protected klebicin KvarIa.

Wastewater-based surveillance in Italy leading to the first detection of mcr-10-positive Klebsiella quasipneumoniae.

Wastewater-based surveillance enabled the first detection of the mobile colistin resistance gene mcr-10 in Italy. This plasmid-borne resistance gene was found in strains of Klebsiella quasipneumoniae isolated from samples of human raw sewage collected over several months. Although the isolates were phenotypically susceptible to colistin, the emergence of mcr-10 is concerning due to the highly variable expression of the gene and the potential for horizontal transfer to other species. In addition, the strains also carried an extended-spectrum ß-lactamase gene and were phenotypically resistant to several beta-lactams. This study highlights the value of wastewater-based surveillance as an effective tool to monitor the emergence of antimicrobial resistance in strains circulating in the community and the environment.

Case Report of bla NDM-7-Harboring IncX3 Plasmid in ST196 Klebsiella quasipneumoniae in China.

Klebsiella quasipneumoniae isolate SBH035 was recovered from a patient in Jiangsu Province, China. The isolate showed resistance to ampicillin, cefazolin, cefotaxime, meropenem, ceftazidime-avibactam, and fosfomycin. The carbapenemase-encoding gene bla NDM-7 was identified, and whole genome sequencing analysis indicated that bla NDM-7 was located in an IncX3 plasmid with a conserved structure of IS26-ΔcutA-tat-trpF-ble MBL -bla NDM-7-ISAba125-IS3000-ΔTn2. To date, this is the first identification of a bla NDM-7-harboring IncX3 plasmid in ST196 K. quasipneumoniae from a patient in China. Greater attention to controlling the dissemination of IncX3 plasmids is needed owing to potential horizontal transfer via mobile genetic elements.

Characterisation of clinical carbapenem-resistant K1 Klebsiella quasipneumoniae subsp. similipneumoniae strains harbouring a virulence plasmid.

The continuous emergence of carbapenem-resistant and hypervirulent Klebsiella pneumoniae (CR-hvKP) poses a great challenge to human health owing to the associated extremely high morbidity and mortality. Klebsiella quasipneumoniae is a newly described bacterial species that is often misidentified as K. pneumoniae. Clinical K. quasipneumoniae strains have been reported worldwide, among which multidrug-resistant lineages have become a severe health problem, while less has been understood on this important pathogen. In this study, we characterised three clinical carbapenem-resistant K. quasipneumoniae subsp. similipneumoniae isolates belonging to sequence type 367 (ST367) and capsular type K1 and containing several virulence genes, including salmochelin (iroBCDN), aerobactin (iucABCDiutA) and regulator of mucoid phenotype (rmpA/A2), as well as some resistance genes, including blaKPC-2, blaTEM-1, blaOKP-B-9 and oqxAB. These carbapenem-resistant K. quasipneumoniae subsp. similipneumoniae strains containing virulence genes exhibited a higher level of virulence and serum resistance than a classical K. pneumoniae strain, while their virulence levels were slightly lower compared with typical ST11 CR-hvKP and ST23 K1 hvKP strains. This study reports for the first time the genetic and virulence characterisation of clinical K. quasipneumoniae subsp. similipneumoniae strains that simultaneously contained blaKPC-2 and virulence genes, contributing to a better understanding of their resistance and pathogenicity as well as for epidemic surveillance worldwide.