Prevalence of Carbapenem-Resistant Hypervirulent Klebsiella pneumoniae and Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae in China Determined via Mouse Lethality Tests.

Objective: To investigate the epidemiology of carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-HvKP) and hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP). Methods: Totally 436 K. pneumoniae strains were collected from 7 hospitals in mainland China between 2017.01 and 2018.02. Sequence types, serotypes, antimicrobial-resistance and virulence genes were analyzed. Additionally, string test, capsule stain, Periodic Acid Schiff stain, fitness analysis, quantitative real-time PCR and mouse lethality test were also performed. Molecular combinations were used to screen putative blaKPC(+)-HvKP and Hv-blaKPC(+)-KP, followed by the confirmation of mouse lethality test. Results: Diverse detection rates were found for the virulence genes, ranging from c-rmpA (0.0%) to entB (100.0%). According to the molecular criteria, 127, 186, 9 and 26 strains were putatively denoted as HvKP, blaKPC(+)-KP, blaKPC(+)-HvKP and Hv-blaKPC(+)-KP. Mouse lethality test confirmed 2 blaKPC(+)-HvKP strains (JS184 and TZ20) and no Hv-blaKPC(+)-KP. JS184 showed K2 serotype, thin capsule, positive exopolysaccharid and string test. TZ20 presented K20 serotype, thin capsule, negative exopolysaccharide and string test. Compared with the positive control NTUH-K2044, equal galF expression and growth curves were confirmed for JS184 and TZ20. Conclusions: Molecular determination of CR-HvKP and Hv-CRKP brings remarkable bias compared with mouse lethality test. The exact prevalence of CR-HvKP is less than 1.0%, which of Hv-CRKP is much lower.

Acquisition of the Conjugative Virulence Plasmid From a CG23 Hypervirulent Klebsiella pneumoniae Strain Enhances Bacterial Virulence.

The emergence of hypervirulent and carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has become a hot topic and confounding problem for clinicians and researchers alike. Conjugative virulence plasmids have the potential to cause more threatening dissemination of hv-CRKP strains. We previously identified K2606, a CG23 clinical hypervirulent strain of Klebsiella pneumoniae harboring a conjugative virulence plasmid designated pK2606. In this study we examined hypervirulence levels using assays of biofilm formation, serum resistance, and wax larvae and mouse in vivo infection models. Moreover, to define the transfer ability of pK2606 and whether this confers hypervirulence to other strains we performed plasmid transconjugation experiments between K2606 and the ST11 CRKP strain HS11286 along with E. coli J53. We found that although biofilm formation and serum resistance were not significantly increased, the transconjugants acquired the ability of produce high level of siderophores and also caused high mortality of wax larvae and mice. Furthermore, we identified pK2606-like conjugative virulence plasmids in GenBank, providing evidence that such plasmids may have begun to spread throughout China. These findings provide an evidence base for the possible mechanisms of the emergence of hv-CRKP strains and highlight the potential of pK2606-like conjugative virulence plasmids to spread worldwide.

Co-occurrence of a novel VIM-1 and FosA3-encoding multidrug-resistant plasmid and a KPC-2-encoding pKP048-like plasmid in a clinical isolate of Klebsiella pneumoniae sequence type 11.

The spread of carbapenem-resistant Enterobacteriaceae (CRE) worldwide remains a major threat to public health. Notably, carbapenemase-encoding genes are usually located in plasmids harboring other resistance determinants, and isolates having multiple plasmids are often highly resistant to carbapenems. In this study, we characterized the genetic context of coproduction of KPC-2, VIM-1, and FosA3 via two plasmids in the multidrug-resistant Klebsiella pneumoniae sequence type 11 (ST11) isolate JS187, recovered during an outbreak of KPC-2-producing K. pneumoniae in a Chinese teaching hospital in 2008. Plasmid p187-1, coharboring blaVIM-1 and fosA3, consisted of a pKOX-R1-like backbone and two multidrug resistant (MDR) regions separated by pKHS1-like backbone sequences involving plasmid replication and stability. The MDR region 1 was a chimera composed of the blaVIM-1-bearing In916-like integron and Tn1721-like transposon, and was disrupted by sequential insertion of an IS26-based transposition unit carrying blaCTX-M-3 and a ΔTn3-like transposon bearing blaTEM-1. MDR region 2 was an IS26-array structure with fosA3 and blaSHV-12. Plasmid p187-2 harboring blaKPC-2 was closely related to pKP048. blaKPC-2 in p187-2 was carried by a Tn1721 variant, which differed from the prototype Tn1721-blaKPC-2 transposon observed in pKP048 by disruption of an IS26 at Tn3 and deletion of a 31-kb MDR fragment. Co-existence of the novel VIM-1- and FosA3-encoding MDR plasmid p187-1 and the KPC-2-encoding pKP048-like plasmid p187-2 made K. pneumoniae JS187 highly resistant to carbapenems. Moreover, p187-1 still carried genes conferring resistance to cephalosporins, fosfomycin, chloramphenicol, and quaternary ammonium, posing substantial challenges for the treatment of Enterobacteriaceae infections. Thus, monitoring the prevalence and evolution of these plasmids and/or strains is critical.

High-risk KPC-producing Klebsiella pneumoniae lack type I R-M systems.

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) have disseminated worldwide and are a major threat to public health. The multidrug-resistant (MDR)-phenotype of KPC-KP are commonly associated with the presence of high molecular weight blaKPC plasmids. Restriction-modification (R-M) systems provide bacteria with innate defense against plasmids or other infectious gene elements. As blaKPC plasmids are favored by such MDR K. pneumoniae, it was of interest to examine the co-distribution of R-M and acquired blaKPC plasmids in KPC-KP. A total of 459 clinical K. pneumoniae isolates in China and 217 global whole-genome sequences in GenBank were collected to determine the prevalence of type I R-M systems. The type I R-M systems were scarce in the KPC-positive group and high-risk Klebsiella pneumoniae clonal group 258 (CG258). The polymorphisms of type I R-M observed in K. pneumoniae revealed the ubiquity of their recognition sequences in DNA; therefore, the type I R-M systems could attack most invading DNA elements, such as blaKPC genes. Overall, this work indicated the type I R-M systems may impact the acquisition of blaKPC genes in K. pneumoniae.

The type I-E CRISPR-Cas system influences the acquisition of blaKPC-IncF plasmid in Klebsiella pneumonia.

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) have disseminated worldwide and emerged as major threats to public health. Of epidemiological significance, the international pandemic of KPC-KP is primarily associated with CG258 isolates and blaKPC-IncF plasmids. CRISPR-Cas system is an adaptive immune system that can hinder gene expansion driven by horizontal gene transfer. Because of blaKPC-IncF plasmids are favored by CG258 K. pneumoniae, it was of interest to examine the co-distribution of CRISPR and blaKPC-IncF plasmids in such isolates. We collected 459 clinical K. pneumoniae isolates in China and collected 203 global whole-genome sequences in GenBank to determine the prevalence of CRISPR-Cas systems. We observed that CRISPR-Cas system was significantly scarce in the CG258 lineage and blaKPC-positive isolates. Furthermore, the results of conjugation and plasmid stability assay fully demonstrated the CRIPSR-Cas system in K. pneumoniae could effectively hindered blaKPC-IncF plasmids invasion and existence. Notably, most blaKPC-IncF plasmids were also proved to be good targets of CRISPR owing to carry matched and functional protospacers and PAMs. Overall, our work suggests that type I-E CRISPR-Cas systems could impact the spread of blaKPC in K. pneumoniae populations, and the scarcity of CRISPR-Cas system was one of potential factors leading to the propagation of blaKPC-IncF plasmids in CG258 K. pneumoniae.

The dynamic change of neutrophil to lymphocyte ratio is predictive of pathological complete response after neoadjuvant chemotherapy in breast cancer patients.

BACKGROUND: The pre-treatment neutrophil-lymphocyte ratio (NLR) has been reported to be a predictive factor for pathologic complete response (pCR) after neoadjuvant chemotherapy (NACT) in breast cancer patients. However, whether the dynamic change of post-treatment neutrophil to lymphocyte ratio (delta-NLR) can better predict the same outcome remains unclear. MATERIALS AND METHODS: We retrospectively analyzed 242 consecutive patients affected by breast cancer and candidates of NACT. The complete blood cell counts before and after NACT were evaluated to calculate NLR. The relationships between delta-NLR and pCR, along with other clinical-pathological characteristics were analyzed. Univariate and multivariate analyses were performed using a logistic regression model. RESULTS: Of the 242 patients, 65 (26.9%) achieved a pCR. Pre-treatment NLR and post-treatment NLR were not significantly associated with pCR if analyzed separately in multivariate analyses. However, when combining together, patients with delta-NLR < 0 profile achieved a significantly higher rate of pCR compared to those with delta-NLR ≥ 0 (OR 2.84, 95% CI 1.35-5.96, p = 0.006). Additionally, the predictive value of delta-NLR was independent from common prognostic factors such as Ki-67, and molecular subtypes. CONCLUSIONS: Delta-NLR, rather than pre-treatment or post-treatment NLR is associated with pCR rate, suggesting that the dynamic change of NLR may be an important factor predicting the response to NACT in breast cancer patients.

Absence of the type I-E CRISPR-Cas system in Klebsiella pneumoniae clonal complex 258 is associated with dissemination of IncF epidemic resistance plasmids in this clonal complex.

BACKGROUND: The pandemics caused by MDR Klebsiella pneumoniae are mostly due to the global dissemination of high-risk clonal complex 258 (CC258) and related IncF epidemic plasmids. However, the factors leading to the epidemiological advantages of CC258-IncF linkage remain obscure. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas) systems, providing adaptive immunity against invading DNA, play an important role in the interactions between plasmids and hosts. OBJECTIVES: To investigate the relationship between CRISPR-Cas systems and the high-risk linkage CC258-IncF. METHODS: CRISPR-Cas loci were detected among 381 collected K. pneumoniae clinical isolates and 207 K. pneumoniae complete genomes available in GenBank. MLST was used to determine the genetic relatedness of these isolates. Nucleotide BLAST was used to search for protospacers on K. pneumoniae plasmids. RESULTS: We observed an epidemic correlation between CRISPR-Cas loci, CC258 and IncF plasmids. Interestingly, most type I-E CRISPR-Cas systems identified carried spacers matching the backbone regions of IncF plasmids. CONCLUSIONS: Our results suggest that the absence of type I-E CRISPR-Cas systems in K. pneumoniae CC258 is strongly associated with the dissemination of IncF epidemic plasmids, contributing to the global success of the international high-risk linkage CC258-IncF. Our findings provide new information regarding the dissemination and evolution of the high-risk linkage of K. pneumoniae CC258-IncF and pave the way for new strategies to address the problem of antibiotic resistance.

The golden ratio and Loshu-Fibonacci Diagram: novel research view on relationship of Chinese medicine and modern biology.

Associating geometric arrangements of 9 Loshu numbers modulo 5, investigating property of golden rectangles and characteristics of Fibonacci sequence modulo 10 as well as the two subsequences of its modular sequence by modulo 5, the Loshu-Fibonacci Diagram is created based on strict logical deduction in this paper, which can disclose inherent relationship among Taiji sign, Loshu and Fibonacci sequence modulo 10 perfectly and unite such key ideas of holism, symmetry, holographic thought and yin-yang balance pursuit from Chinese medicine as a whole. Based on further analysis and reasoning, the authors discover that taking the golden ratio and Loshu-Fibonacci Diagram as a link, there is profound and universal association existing between researches of Chinese medicine and modern biology.