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Genomic changes in Mycoplasma pneumoniae caused by adaptation to environmental or ecologic pressures are poorly understood. We collected M. pneumoniae from children who had confirmed pneumonia in Taiwan during 2017–2020. We used whole-genome sequencing to compare these isolates with a worldwide collection of current and historical clinical strains for characterizing population structures. A phylogenetic tree for 284 strains showed that all sequenced strains consisted of 5 clades: T1–1 (sequence type [ST]1), T1–2 (mainly ST3), T1–3 (ST17), T2–1 (mainly ST2), and T2–2 (mainly ST14). We identified a putative recombination block containing 6 genes (MPN366‒371). Macrolide resistance involving 23S rRNA mutations was detected for each clade. Clonal expansion of macrolide resistance occurred mostly within subtype 1 strains, of which clade T1–2 showed the highest recombination rate and genome diversity. Functional characterization of recombined regions provided clarification of the biologic role of these recombination events in the evolution of M. pneumoniae.

Acinetobacter baumannii emerged as one of the most important pathogens that causes nosocomial infections due to its increased multidrug resistance. Identifying capsular epidemiology in A. baumannii can aid in the development of effective treatments and preventive measures against this emerging pathogen. Here we established a wzc-based method, and combined it with wzy-PCR to determine capsular types of A. baumannii causing nosocomial bacteraemia collected at two medical centres in Taiwan from 2015 to 2017. Among the 237 patients with A. baumannii bacteraemia, 98 (41.4%) isolates were resistant to carbapenems. Four prevalent capsular types (KL2, KL10, KL22, and KL52) accounted for 84.7% of carbapenem-resistant A. baumannii (CRAB) and 12.2% of non-CRAB. The rate of pneumonia, intensive care unit admission, APACHE II score, and Pitt bacteraemia score were higher in patients with KL2/10/22/52 infection than in those with non-KL2/10/22/52 infection. Patients with KL2/10/22/52 infection and patients with CRAB infection have a higher cumulative incidence of attributable and all-cause in-hospital 30-day mortality. On multivariate analysis, appropriate empirical antimicrobial therapy within 24 h was associated with a lower risk of 30-day attributable mortality in the KL2/10/22/52 isolates (odds ratio = 0.19, 95% CI: 0.06–0.66, p = 0.008) but not in non-KL2/10/22/52 isolates. Early recognition of carbapenem resistance-associated capsular types may help clinicians to promptly implement appropriate antimicrobial therapy for improving the outcomes in patients with CRAB bacteraemia.

Dissemination of multidrug-resistant, particularly tigecycline-resistant, Acinetobacter baumannii is of critical importance, as tigecycline is considered a last-line antibiotic. Acquisition of tet(X), a tigecycline-inactivating enzyme mostly found in strains of animal origin, imparts tigecycline resistance to A. baumannii. Herein, we investigated the presence of tet(X) variants among 228 tigecycline-non-susceptible A. baumannii isolates from patients at a Taiwanese hospital via polymerase chain reaction using a newly designed universal primer pair. Seven strains (3%) carrying tet(X)-like genes were subjected to whole genome sequencing, revealing high DNA identity. Phylogenetic analysis based on the PFGE profile clustered the seven strains in a clade, which were thus considered outbreak strains. These strains, which were found to co-harbor the chromosome-encoded tet(X6) and the plasmid-encoded blaOXA-72 genes, showed a distinct genotype with an uncommon sequence type (Oxford ST793/Pasteur ST723) and a new capsular type (KL129). In conclusion, we identified an outbreak clone co-carrying tet(X6) and blaOXA-72 among a group of clinical A. baumannii isolates in Taiwan. To the best of our knowledge, this is the first description of tet(X6) in humans and the first report of a tet(X)-like gene in Taiwan. These findings identify the risk for the spread of tet(X6)-carrying tigecycline- and carbapenem-resistant A. baumannii in human healthcare settings.

Acinetobacter baumannii is the main cause of nosocomial infections, and the treatment of such infections has become more difficult due to the emergence of carbapenem resistance. This study focused on major carbapenemase genes and explored the association between carbapenemase genes, sequence types (ST types), and capsular types (K types). A total of 98 carbapenem-resistant A. baumannii (CRAB) strains were collected from two hospitals, the Chang Gung Memorial Hospital-Lin Kou branch (LCGMH) in northern Taiwan and the CGMH-Kaohsiung branch (KCGMH) in southern Taiwan, from 2015–2017. Major carbapenemase genes of class A, B, and D β-lactamases were detected by polymerase chain reaction. All strains except 1 were positive for blaOXA-51-like, 76 strains (77.6%) carried blaOXA-23-like, and 25 strains (25.5%) carried blaOXA-24-like. The regional distribution showed that blaOXA-23-like was more predominant than blaOXA-24-like in both hospitals (85.3% and 60% in LCGMH and KCGMH, respectively); however, blaOXA-24-like displayed a much higher percentage in KCGMH (46.7%) than in LCGMH (16.2%). Oxford multilocus sequence typing and global optimal eBURST analysis were conducted for 59 strains. We revealed the association between blaOXA gene patterns, ST types, and K types and demonstrated that four major K types, KL2, KL10, KL22, and KL52, which were associated with specific ST types, were mainly clustered into clonal complexes CC208 and CC549 (a unique clonal complex found in Taiwan). These findings provide important information for monitoring the epidemiology and dissemination of this pathogen.

Colistin is one of the antibiotics of last resort for the treatment of carbapenem-resistant Klebsiella pneumoniae infection. This study showed that capsular type K64 (50%) and ST11 (53.9%) are the prevalent capsular and sequence types in the colistin-resistant strains in Taiwan. The interruption of transcripts (38.5%) and amino acid mutation (15.4%) in mgrB are the major mechanisms contributing to colistin resistance. In addition, novel single amino acid changes in MgrB (Stop48Tyr) and PhoQ (Leu26Pro) were observed to contribute to colistin resistance.

Klebsiella pneumoniae is an important human pathogen associated with a variety of diseases, and the prevalence of multidrug-resistant K. pneumoniae (MDRKP) is rapidly increasing. Here we determined the capsular types of 85 carbapenem-resistant K. pneumoniae (CRKP) strains by wzc sequencing and investigated the presence of carbapenemases and integrons among CRKP strains. Ten CRKP strains (12%) were positive for carbapenemase (imipenemase, 6/85 strains; K. pneumoniae carbapenemase, 3/85 strains; Verona integron-encoded metallo-β-lactamase, 1/85 strains). Capsular type K64 accounted for 32 CRKP strains (38%), followed by K62 (13%), K24 (8%), KN2 (7%), and K28 (6%). Sequence types (STs) were determined by multilocus sequence typing (MLST), and the results indicated that ST11, which accounted for 47% of these CRKP strains (40/85 strains), was the major ST. We further isolated a K64-specific capsule depolymerase (K64dep), which could enhance serum and neutrophil killing in vitro and increase survival rates for K64 K. pneumoniae -inoculated mice. The toxicity study demonstrated that mice treated with K64dep showed normal biochemical parameters and no significant histopathological changes of liver, kidney, and spleen, indicating that enzyme treatment did not cause toxicity in mice. Therefore, the findings of capsular type clustering among CRKP strains and effective treatment with capsule depolymerase for MDRKP infections are important for capsule-based vaccine development and therapy.

Statins, 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors, have been found to provide protective effects against several bacterial infectious diseases. Although the use of statins has been shown to enhance antimicrobial treated Helicobacter pylori eradication and reduce H. pylori-mediated inflammation, the mechanisms underlying these effects remain unclear. In this study, in vitro and ex vivo macrophage models were established to investigate the molecular pathways involved in statin-mediated inhibition of H. pylori-induced inflammation. Our study showed that statin treatment resulted in a dose-dependent decrease in intracellular H. pylori burden in both RAW264.7 macrophage cells and murine peritoneal exudate macrophages (PEMs). Furthermore, statin yielded enhanced early endosome maturation and subsequent activation of the autophagy pathway, which promotes lysosomal fusion resulting in degradation of sequestered bacteria, and in turn attenuates interleukin (IL)-1β production. These results indicate that statin not only reduces cellular cholesterol but also decreases the H. pylori burden in macrophages by promoting autophagy, consequently alleviating H. pylori-induced inflammation.

BACKGROUND: Klebsiella pneumoniae causing community-acquired pyogenic liver abscess complicated with metastatic meningitis and endophthalmitis has emerged recently, most frequently associated with the K1 capsular type. METHODS: A bacteriophage (NTUH-K2044-K1-1) that infects K. pneumoniae NTUH-K2044 (capsular type K1) was isolated and characterized. RESULTS: The phage infected all K1 strains, and none of the strains with other capsular types. Capsule deletion mutants were not lysed by this phage, suggesting that the capsule was essential for phage infection. Complete genome sequencing revealed the phage was a novel phiKMV-like virus. The gene-encoding capsule depolymerase was identified. The recombinant enzyme demonstrated specific lysis of the K1 capsule. Treatment with the phage or the recombinant enzyme provided significantly increased survival in mice infected with NTUH-K2044 strain, including one treated after the detection of a neck abscess by imaging. No obvious disease was observed after administration of this phage in mice. Phage was retained at detectable levels in liver, spleen, brain, and blood 24 hours after administration in mice. CONCLUSIONS: These results demonstrate this phage and its capsule depolymerase exhibit specificity for capsular type K1 and can be used for the diagnosis and treatment of K1 K. pneumoniae infections.

Background. Peptidoglycan-associated lipoprotein (Pal), murein lipoprotein (LppA), and outer membrane protein A (OmpA) are dominant outer membrane proteins (OMPs) that are released by gram-negative bacteria during sepsis. OMPs are implicated in the maintenance of cell envelope integrity. Here, we characterize the roles of these OMPs in pathogenesis during bacteremia caused by Klebsiella pneumoniae. Methods. pal-, lppA-, and ompA-deficient K. pneumoniae strains were constructed using an unmarked deletion method. Serum sensitivity, antiphagocytosis activity, outer membrane permeability, and sensitivity to anionic detergents and antimicrobial polypeptides were determined for these OMP gene deletion mutants. The ability of these OMP gene deletion mutants to induce immune responses was compared with that of the wild-type strain in a bacteremic mouse model. Results. Klebsiella pneumoniae strains deleted for pal or lppA exhibited reduced protection from serum killing and phagocytosis; perturbation to the outer membrane permeability barrier and hypersensitivity to bile salts and sodium dodecyl sulfate. The strain mutated for lppA had reduced ability to activate Toll-like receptor 4. Immunization of mice with the pal or lppA mutant provided protection against infection by the wild-type strain. Conclusions. Our findings indicate that K. pneumoniae Pal and LppA proteins are important in the maintenance of cell integrity, contribute to virulence, and could be used as attenuated vaccines.

Phagocytosis resistance is an important virulence factor in Klebsiella pneumoniae . Dictyostelium has been used to study the interaction between phagocytes and bacteria because of its similarity to mammalian macrophages. In this study, we used a Dictyostelium model to investigate genes for resistance to phagocytosis in NTUH-K2044, a strain of K. pneumoniae causing pyogenic liver abscess that is highly resistant to phagocytosis. A total of 2,500 transposon mutants were screened by plaque assay, and 29 of them permitted phagocytosis by Dictyostelium . In the 29 mutants, six loci were identified; three were capsular synthesis genes. Of the other three, one was related to carnitine metabolism, one encoded a subunit of protease ( clpX ), and one encoded a lipopolysaccharide O-antigen transporter ( wzm ). Deletion and complementation of these genes showed that only Δ clpX and Δ wzm mutants became susceptible to Dictyostelium phagocytosis, and their complementation restored the phagocytosis resistance phenotype. These two mutants were also susceptible to phagocytosis by human neutrophils and revealed attenuated virulence in a mouse model, implying that they play important roles in the pathogenesis of K. pneumoniae . Furthermore, we demonstrated that clpP , which exists in an operon with clpX , was also involved in resistance to phagocytosis. The transcriptional profile of Δ clpX was examined by microarray analysis and revealed a 3-fold lower level of expression of capsular synthesis genes. Therefore, we have identified genes involved in resistance to phagocytosis in K. pneumoniae using Dictyostelium , and this model is useful to explore genes associated with resistance to phagocytosis in heavily encapsulated bacteria.

A CMY-2-producing capsular type K2 Klebsiella pneumoniae strain (TVGHKP93) with multidrug resistance was isolated from a recurrent liver abscess in a patient who also carried a CMY-2-producing Escherichia coli strain (TVGHEC01) in the stool. TVGHKP93 retained its high virulence compared with that of the isogenic strain (TVGHKP60) with wild-type resistance from the first liver abscess. Our conjugation experiment showed the successful transfer of the bla CMY-2 -carrying plasmid from TVGHEC01 into TVGHKP60. The transconjugant showed both high virulence and the multidrug-resistant phenotype, as did TVGHKP93.

Klebsiella pneumoniae is an important pathogen that causes hospital-acquired septicemia and is associated with the recent emergence of community-acquired pyogenic liver abscess (PLA). Clinical typing suggests that K. pneumoniae infections originate from the gastrointestinal reservoir. However, the underlying mechanism remains unknown. Here, we have sought to determine how K. pneumoniae penetrates the intestinal barrier. We identified that bacteremia and PLA clinical isolates adhered to and invaded intestinal epithelial cells. Internalization of K. pneumoniae in three different human colonic cell lines was visualized by confocal microscopy and three-dimensional (3D) imaging. Using a Transwell system, we demonstrated that these K. pneumoniae isolates translocated across a polarized Caco-2 monolayer. No disruptions of transepithelial electrical resistance and altered distribution of tight junction protein ZO-1 or occludin were observed. Therefore, K. pneumoniae appeared to penetrate the intestinal epithelium via a transcellular pathway. Using specific inhibitors, we characterized the host signaling pathways involved. Inhibition by cytochalasin D and nocodazole suggested that actin and microtubule cytoskeleton were both important for K. pneumoniae invasion. A Rho inhibitor, ML141, LY294002, and an Akt1/2 inhibitor diminished K. pneumoniae invasion in a dose-dependent manner, indicating that Rho family GTPases and phosphatidylinositol 3-kinase (PI3K)/Akt signaling were required. By a mouse model of gastrointestinal colonization, in vivo invasion of K. pneumoniae into colonic epithelial cells was demonstrated. Our results present evidence to describe a possible mechanism of gastrointestinal translocation for K. pneumoniae . Cell invasion by manipulating host machinery provides a pathway for gut-colonized K. pneumoniae cells to penetrate the intestinal barrier and access extraintestinal locations to cause disease.

Background Klebsiella pneumoniae is one of the major pathogens causing hospital-acquired multidrug-resistant infections. The capsular polysaccharide (CPS) is an important virulence factor of K. pneumoniae. With 78 capsular types discovered thus far, an association between capsular type and the pathogenicity of K. pneumoniae has been observed. Methodology/Principal Findings To investigate an initially non-typeable K. pneumoniae UTI isolate NTUH-K1790N, the cps gene region was sequenced. By NTUH-K1790N cps-PCR genotyping, serotyping and determination using a newly isolated capsular type-specific bacteriophage, we found that NTUH-K1790N and three other isolates Ca0507, Ca0421 and C1975 possessed a new capsular type, which we named KN2. Analysis of a KN2 CPS− mutant confirmed the role of capsule as the target recognized by the antiserum and the phage. A newly described lytic phage specific for KN2 K. pneumoniae, named 0507-KN2-1, was isolated and characterized using transmission electron microscopy. Whole-genome sequencing of 0507-KN2-1 revealed a 159 991 bp double-stranded DNA genome with a G+C content of 46.7% and at least 154 open reading frames. Based on its morphological and genomic characteristics, 0507-KN2-1 was classified as a member of the Myoviridae phage family. Further analysis of this phage revealed a 3738-bp gene encoding a putative polysaccharide depolymerase. A recombinant form of this protein was produced and assayed to confirm its enzymatic activity and specificity to KN2 capsular polysaccharides. KN2 K. pneumoniae strains exhibited greater sensitivity to this depolymerase than these did to the cognate phage, as determined by spot analysis. Conclusions/Significance Here we report that a group of clinical strains possess a novel Klebsiella capsular type. We identified a KN2-specific phage and its polysaccharide depolymerase, which could be used for efficient capsular typing. The lytic phage and depolymerase also have potential as alternative therapeutic agents to antibiotics for treating K. pneumoniae infections, especially against antibiotic-resistant strains.

Klebsiella pneumoniae is the common cause of a global emerging infectious disease, community-acquired pyogenic liver abscess (PLA). Capsular polysaccharide (CPS) and lipopolysaccharide (LPS) are critical for this microorganism's ability to spread through the blood and to cause sepsis. While CPS type K1 is an important virulence factor in K. pneumoniae causing PLA, the role of LPS in PLA is not clear. Here, we characterize the role of LPS O antigen in the pathogenesis of K. pneumoniae causing PLA. NTUH-K2044 is a LPS O1 clinical strain; the presence of the O antigen was shown via the presence of 1,3-galactan in the LPS, and of sequences that align with the wb gene cluster, known to produce O-antigen. Serologic analysis of K. pneumoniae clinical isolates demonstrated that the O1 serotype was more prevalent in PLA strains than that in non-tissue-invasive strains (38/42 vs. 9/32, P