OmpK35 and OmpK36 are the major outer membrane porins of Klebsiella pneumoniae. In this study, a virulent clinical isolate was selected to study the role of these two porins in antimicrobial resistance and virulence. The single deletion of ompK36 (⌬ompK36) resulted in MIC shifts of cefazolin, cephalothin, and cefoxitin from susceptible to resistant, while the single deletion of ompK35 (⌬ompK35) had no significant effect. A double deletion of ompK35 and ompK36 (⌬ompK35/36) further increased these MICs to high-level resistance and led to 8-and 16-fold increases in the MICs of meropenem and cefepime, respectively. In contrast to the routine testing medium, which is of high osmolarity, susceptibility tests using low-osmolarity medium showed that the ⌬ompK35 mutation resulted in a significant (>4-fold) increase in the MICs of cefazolin and ceftazidime, whereas a ⌬ompK36 deletion conferred a significantly (4-fold) lower increase in the MIC of cefazolin. In the virulence assays, a significant (P < 0.05) defect in the growth rate was found only in the ⌬ompK35/36 mutant, indicating the effect on metabolic fitness. A significant (P < 0.05) increase in susceptibility to neutrophil phagocytosis was observed in both ⌬ompK36 and ⌬ompK35/36 mutants. In a mouse peritonitis model, the ⌬ompK35 mutant showed no change in virulence, and the ⌬ompK36 mutant exhibited significantly (P < 0.01) lower virulence, whereas the ⌬ompK35/36 mutant presented the highest 50% lethal dose of these strains. In conclusion, porin deficiency in K. pneumoniae could increase antimicrobial resistance but decrease virulence at the same time.

In Klebsiella pneumoniae liver abscess (KP-LA), K. pneumoniae K2 is the most frequently isolated serotype after K1, but this serotype has been much less studied. In the present study, the molecular types sequences type (MLST) of serotype K2 isolates from three different regions in Asia were identified and the virulence of these isolates was investigated. Eight different MLSTs were found among 26 isolates (ST 65, 66, 86, 373, 374, 375, 380, and 434). There were two major MLST groups, ST-65-like (42%) and ST86-like (46%). No isolates contained allS while all isolates contained rmpA. The prevalence of aerobactin gene and kfu were 25/26 (96%) and 3/26 (11.5%) respectively. Although liver abscess isolates were generally more resistant (11/15 isolates) to serum killing, there was no specific distribution of serum killing resistant or susceptible ST types between stool carriage and liver abscess isolates. Neutrophil phagocytosis showed that the liver abscess and carriage isolates varied in their susceptibility to phagocytosis. Strains with resistance to both neutrophil phagocytosis and serum killing were generally hypervirulent with lethality at LD50 < 103 colony forming units by intraperitoneal injection. In conclusion, Anti-phagocytosis and resistance to serum killing are two parameters that most predict hyperviurlence in serotype K2 isolates. Unlike serotype K1 KP-LA that mainly belong to ST-23, ST-65-like and −86-like are the two major MLST types among serotype K2 isolates from Singapore, Hong Kong and Taiwan.

Tigecycline is regarded as a last-resort treatment for carbapenem-resistant Klebsiella pneumoniae (CRKP) infections, but increasing numbers of tigecycline-resistant K. pneumoniae isolates have been reported. The tigecycline resistance mechanisms in CRKP are undetermined. This study aimed to elucidate the mechanisms underlying tigecycline resistance in 16 tigecycline-and carbapenem-resistant K. pneumoniae (TCRKP) isolates. Mutations in tigecycline resistance determinant genes [ramR, acrR, oqxR, tet(A), tet(L), tet(X), tet(M), rpsJ]were assessed by PCR amplicon sequencing, and mutations in ramR and tet(A) exhibited high prevalences individually (81%) and in combination (63%). Eight functional ramR mutation profiles reducing tigecycline sensitivity were verified by plasmid complementation of wild-type and mutant ramR. Using a site-specific mutant, the most frequent RamR mutation, A19V (60%), had no significant effect on tigecycline susceptibility or the upregulation of ramA and acrA. Two tet(A) variants with double frameshift mutations, type 1 and type 2, were identified; type 2 tet(A) is novel. A parent strain transformed with a plasmid carrying type 1 or type 2 tet(A) increased the tigecycline MIC by 8-fold or 4-fold, respectively. Synergistic effects were observed in strains harboring no ramR gene and a mutated tet(A), with an 8-fold increase in the tigecycline MIC compared with that in strains harboring only mutated tet(A) being seen. Overall, mutations in the ramR and tet(A) efflux genes constituted the major tigecycline resistance mechanisms among the studied TCRKP isolates. The identification of strains exhibiting the combination of a ramR deficiency and widespread mutated tet(A) is concerning due to the possible dissemination of increased tigecycline resistance in K. pneumoniae.

Loss of OmpK36 in K. pneumoniae resulted in increased antimicrobial resistance, increased susceptibility to neutrophil phagocytosis, increased resistance to serum killing and reduced virulence.

Resistance to carbapenems has been documented by the production of carbapenemase or the loss of porins combined with extended-spectrum β-lactamases or AmpC β-lactamases. However, no complete comparisons have been made regarding the contributions of each resistance mechanism towards carbapenem resistance. In this study, we genetically engineered mutants of Klebsiella pneumoniae with individual and combined resistance mechanisms, and then compared each resistance mechanism in response to ertapenem, imipenem, meropenem, doripenem and other antibiotics. Among the four studied carbapenems, ertapenem was the least active against the loss of porins, cephalosporinases and carbapenemases. In addition to the production of KPC-2 or NDM-1 alone, resistance to all four carbapenems could also be conferred by the loss of two major porins, OmpK35 and OmpK36, combined with CTX-M-15 or DHA-1 with its regulator AmpR. Because the loss of OmpK35/36 alone or the loss of a single porin combined with bla CTX-M-15 or bla DHA-1-ampR expression was only sufficient for ertapenem resistance, our results suggest that carbapenems other than ertapenem should still be effective against these strains and laboratory testing for non-susceptibility to other carbapenems should improve the accurate identification of these isolates.

Serotype K1 Klebsiella pneumoniae is a major cause of liver abscesses and endophthalmitis. This study was designed to identify the role of neutrophils in the development of distant metastatic complications that were caused by serotype K1 K. pneumoniae. An in vitro cellular model was used to assess serum resistance and neutrophil-mediated killing. BALB/c mice were injected with neutrophils containing phagocytosed K. pneumoniae. Serotype K1 K. pneumoniae was significantly more resistant to serum killing, neutrophil-mediated phagocytosis and intra-cellular killing than non-K1 isolates (p<0.01). Electron microscopic examination had similar findings as in the bioassay findings. Intraperitoneal injection of neutrophils containing phagocytosed serotype K1 K. pneumoniae led to abscess formation in multiple sites including the subcutaneous tissue, lung, and liver, whereas no abscess formation was observed in mice injected with non-K1 isolates. The resistance of serotype K1 K. pneumoniae to complement- and neutrophil-mediated intracellular killing results in the dissemination of K. pneumoniae via the bloodstream. Escape from neutrophil intracellular killing may contribute to the dissemination and establishment of distant metastases. Thus, neutrophils play a role as a vehicle for helping K. pneumoniae and contributing to the establishment of liver abscess and distant metastatic complications.

eIn this study, a novel colloidal gold-based immunochromatographic strip (ICS) containing anti-Klebsiella pneumoniae capsular polysaccharide polyclonal antibodies was developed to specifically detect K. pneumoniae serotypes K1 and K2. Capsular polysaccharide K1 and K2 antigens were first used to produce polyclonal anti-K1 and anti-K2 antibodies. Reference strains with different serotypes, nontypeable K. pneumoniae strains, and other bacterial species were then used to assess the sensitivity and specificity of these test strips. The detection limit was found to be 10 5 CFU, and the ICSs were stable for 6 months when stored at room temperature. No false-positive or false-negative results were observed, and equivalent results were obtained compared to those of more conventional test methods, such as PCR or serum agglutination. In conclusion, the ICS developed here requires no technical expertise and allows for the specific, rapid, and simultaneous detection of K. pneumoniae serotypes K1 and K2.K lebsiella pneumoniae is a Gram-negative, rod-shaped bacterium that is found in the normal flora of the mouth, skin, and intestines. Recently, a new type of invasive infection caused by K. pneumoniae has emerged as a global disease (1). There are 77 different serotypes of the capsular polysaccharides (CPSs) of K. pneumoniae (2), with K2 and K21 being the most prevalent in western countries (3). Recently, by genotyping capsular polysaccharide genes, 79 capsular types were distinguished (4, 5). In contrast, the K1 and K2 serotypes are most often associated with bacteremia, liver abscess, and community-acquired pneumonia in Asia (3). However, reports of this new invasive syndrome have recently been on the rise in western countries (6, 7). In addition to causing human disease, K. pneumoniae serotypes K1 and K2 have also been detected in contaminated animal milk (8). Capsular serotypes have typically been identified by using either a capsular swelling test or countercurrent immunoelectrophoresis (3, 9). However, these conventional methods are time-consuming, require extensive materials and manpower, and often produce falsepositive or false-negative results that are caused by cross-reactions. Another option is PCR, but this analysis requires a long time for preparation, expensive equipment, and skilled technicians, and previous studies have recommended that rapid detection is ideal for early clinical diagnosis and treatment (1).To address these concerns, this study involved the development of a colloidal gold-based immunochromatographic strip (ICS) that contained anti-K. pneumoniae capsular polysaccharide polyclonal antibodies (pAbs) and was able to detect K. pneumoniae serotypes K1 and K2. This testing kit provides a sensitive, rapid, and simultaneous method to detect K. pneumoniae serotypes K1 and K2. MATERIALS AND METHODSBacterial strains. The various serotypes of K. pneumoniae that were used here were obtained from our previous studies in Taiwan and at the Serum Institute in Denmark. The panel of bacterial strains contained al...

Aims:  Understanding the metabolism of lactose and galactose and their regulation in Lactobacillus rhamnosus. Methods and Results:  A gene cluster containing nine open reading frames (ORFs) involved in the metabolism of lactose and galactose in Lact. rhamnosus TCELL‐1 was sequenced and characterized. The order of the ORFs was lacTEGF and galKETRM. Northern blotting experiments revealed that the gene cluster could be transcribed as one lacTEGF–galKETRM mRNA though three major transcripts (lacTEGF, galKETRM and galETRM) were detected for the gene cluster. The transcription of the lac or gal operon was independently induced in the presence of lactose or galactose. Northern blotting and primer extension experiments found the presence of four putative promoters upstream from the ORFs lacT (lacTp), galK (galKp1 and galKp2) and galE (galEp). The measurements of enzymatic activities of GalK, GalE and GalT suggested that the expression of the gal operon was subjected to a galactose activation and glucose repression mechanism. Conclusions:  In Lact. rhamnosus TCELL‐1, the galactose moiety of lactose could be metabolized by two alternative pathways (the Leloir and the tagatose 6‐phosphate pathways) whereas galactose metabolism could be mediated by the Leloir pathway. Significance and Impact of the Study:  This work provides important information about sugar metabolism in Lact. rhamnosus.