Nosocomial transmission and rearrangement of large resistance-virulence hybrid plasmids between two bacteremic ST11 carbapenem-resistant hypervirulent Klebsiella pneumoniae strains with low fitness cost.

OBJECTIVES: To characterize nosocomial transmission and rearrangement of the resistance-virulence plasmid between two ST11-K64 carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) strains (JX-CR-hvKP-10 and JX-CR-hvKP-9) with low fitness. METHODS: Phenotypic tests were used to assess the virulence of JX-CR-hvKP-10 and JX-CR-hvKP-9. Whole-genome sequencing was used to analyze JX-CR-hvKP-10 and JX-CR-hvKP-9 chromosomes and plasmids. Fitness and conjugation experiments were also conducted using these two CR-hvKP isolates. RESULTS: Phenotypic tests indicated that both JX-CR-hvKP-10 and JX-CR-hvKP-9 were multidrug-resistant and hypervirulent K. pneumoniae. Whole-genome sequencing and clinical information demonstrated that the super large resistance-virulence fusion plasmid pJX10-1 formed precisely by the fusion of pJX9-1 and pJX9-2 via the nosocomial transmission. Interestingly pJX9-1 itself was also a classic resistance-virulence fusion plasmid by way of the blaKPC-carrying resistance plasmid and pLVPK-like virulence plasmid. Compared with classic K. pneumoniae ATCC700603, fitness analysis revealed no significant difference in growth was observed between JX-CR-hvKP-10 and JX-CR-hvKP-9. CONCLUSION: Nosocomial transmission and rearrangement of a blaKPC-harboring plasmid and a pLVPK-like virulence plasmid with a low fitness cost in ST11 K. pneumoniae enhances drug resistance and virulence simultaneously. Thus, active surveillance of this hybrid plasmid is needed to prevent these efficient resistance-virulence plasmids from disseminating in hospital settings.

mNGS helped diagnose scrub typhus presenting as a urinary tract infection with high D-dimer levels: a case report.

BACKGROUND: Scrub typhus is caused by O. tsutsugamushi and spreads through mite larvae biting the skin. Classic symptoms of the disease are eschar and lymphadenopathy. Previous reports have revealed clinical manifestations of scrub typhus, including gastrointestinal symptoms, meningoencephalitis, ocular flutter, pneumonitis, acute respiratory distress syndrome, and acute kidney injury. However, cases of scrub typhus presenting as a urinary tract infection (UTI) with high D-dimer levels could be easily misdiagnosed when clinical attention is insufficient, resulting in difficulty in making a timely diagnosis of the infection. Metagenomics next-generation sequencing (mNGS) is a revolutionary and highly sensitive method that may help in diagnosing atypical cases, even when trace amounts of pathogens are present. CASE PRESENTATION: A 52-year-old female presented with a 10-day history of fever, chills, headache and myalgia. She was initially diagnosed with influenza at a local clinic. Various antibacterials were used on the 2nd-12th day onwards; however, her symptoms persisted and were followed by increased urination duration, frequency, urgency and dysuria for 2 days. Orientia tsutsugamushi was confirmed as the pathogen responsible for the infection through mNGS analysis of her blood samples from Day 13 onwards. The patient's temperature changed remarkably 24 h after the initiation of doxycycline. Over the next 48 h (i.e., Day 15 onwards), the patient showed clinical improvement. She recovered and was discharged from the hospital. CONCLUSIONS: Scrub typhus can present atypical clinical symptoms, such as UTIs, in a febrile patient. mNGS may be a useful method for identifying O. tsutsugamushi infection in patients with atypical clinical manifestations.

Phenotypical profile and global transcriptomic profile of Hypervirulent Klebsiella pneumoniae due to carbapenemase-encoding plasmid acquisition.

BACKGROUND: Plasmids play an vital role in driving the rapid global spread of antimicrobial resistance and adaptation to changing ambient conditions. It has been suggested that the presence of plasmids can pose tremendous impacts on the host physiology. However, little is known regarding the contributions of carbapenemase-encoding plasmid carriage on the physiology and pathogenicity of hypervirulent K. pneumoniae (hvKP). RESULTS: Here we performed a transcriptomic analysis of hvKP with or without carbapenemase-encoding plasmid p24835-NDM5. The results had shown 683 genes with differential expression (false discovery rate, ≤0.001; > 2-fold change), of which 107 were up-regulated and 576 were down-regulated. Gene groups with functions relating to carbohydrate metabolism and multidrug efflux system were increased in genes with increased expression, and those relating to capsule biosynthesis and virulence factors were increased in the genes with decreased expression. In agreement with these changes, survival rate of TfpNDM-hvKP in the presence of normal human serum decreased, and competitive index (CI values) indicated significant fitness defects in the plasmid-carrying hvKP strain when co-cultured with its plasmid-free isogenic ancestor and the ATCC control. Moreover, the p24835-NDM5-containing hvKP strain retained its high neutrophil-mediated phagocytosis and murine lethality. CONCLUSION: These data indicate that hvKP responds to carbapenemase-encoding plasmid by altering the expression of genes involved in carbohydrate metabolism, antibiotic resistance, capsule biosynthesis and virulence expression. Apart from antibiotic resistance selective advantages, carbapenemase-encoding plasmid carriage may also lead to virulence change or adaption to specific habitats in hvKP strain.

Whole genome assembly and functional portrait of hypervirulent extensively drug-resistant NDM-1 and KPC-2 co-producing Klebsiella pneumoniae of capsular serotype K2 and ST86.

OBJECTIVES: To characterize an emergent carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) strain, NUHL30457, which co-produces NDM-1 and KPC-2 carbapenemases. METHODS: We performed WGS analysis on a clinical carbapenemase-producing hypervirulent K. pneumoniae (CP-hvKP) strain NUHL30457. Sequence data were analysed using comparative genomics and phylogenetics. WGS was used to perform MLST, capsular genotyping and identification of virulence and antimicrobial resistance genes. The virulence of NUHL30457 was analysed by serum killing assay, neutrophil phagocytosis and mouse lethality assay. RESULTS: The NUHL30457 strain was carbapenem resistant and belonged to ST86 and serotype K2. A significant increase in resistance to serum killing and antiphagocytosis was found in the NUHL30457 strain compared with the reference strain. The murine lethality assay showed an LD50 of 2.5 × 102 cfu for the NUHL30457 strain, indicating hypervirulence. WGS revealed that NUHL30457 has a single 5.3 Mb chromosome (57.53% G + C content) and four plasmids in the range 49.2-215.7 kb. The incompatibility group (Inc)N plasmid p30457-4 carried the blaNDM-1 and qnrS1 genes. The IncFII(K) plasmid p30457-3 also carried an array of resistance elements, including blaCTX-M-65, blaTEM-1 and blaKPC-2. The IncHI1/IncFIB plasmid p30457-1, which carried virulence genes, was identical to a pLVPK plasmid reported previously. CONCLUSIONS: To the best of our knowledge, this is the first report to isolate an ST86 hvKP strain that co-produces NDM-1 and KPC-2 carbapenemase. Further investigation is required to reinforce our understanding of the epidemiology and virulence mechanisms of this clinically significant CP-hvKP.

Isolation of Hv-CRKP with co-production of three carbapenemases (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1) and a virulence plasmid: a study from a Chinese tertiary hospital.

Background: The worldwide dissemination of K. pneumoniae isolates is a significant public health concern, as these organisms possess a unique capacity to acquire genetic elements encoding both resistance and hypervirulence. This study aims to investigate the epidemiological, resistance, and virulence characteristics of K. pneumoniae isolates that carry both virulence plasmids and blaOXA-48-like genes in a tertiary hospital in China. Methods: A total of 217 clinical isolates of carbapenem-resistant K. pneumoniae (CRKP) were collected between April 2020 and March 2022. The antimicrobial susceptibility test was conducted to evaluate the drug resistance profile. All isolates were screened for the presence of genes encoding carbapenemases (blaKPC, blaNDM, blaIMP, blaVIM, and blaOXA-48-like), ESBLs genes (blaCTX-M, blaSHV, blaTEM), and virulence plasmid pLVPK-borne genes (rmpA, rmpA2, iucA, iroB, and peg344) using polymerase chain reaction (PCR) amplification. Clonal lineages were assigned using multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). The plasmid incompatibility groups were identified using PCR-based replicon typing (PBRT). The transferability of carbapenemase-encoding plasmids and pLVPK-like virulence plasmids was assessed via conjugation. The plasmid location of rmpA2 was determined using S1-Pulsed Field Gel Electrophoresis (S1-PFGE) and southern blotting hybridization. The virulence potential of the isolates was assessed using the string test, capsular serotyping, serum killing assay and a Galleria mellonella larval infection model. Results: Of the 217 CRKP clinical isolates collected, 23% were identified as carrying blaOXA-48-like genes. All blaOXA-48-like isolates exhibited resistance to commonly used clinical antimicrobial agents, except for ceftazidime/avibactam, colistin, tigecycline, trimethoprim-sulfamethOXAzole, polymyxin B, and nitrofurantoin. The main common OXA-48-like carbapenemase enzymes were found to be blaOXA-181 and blaOXA-232. MLST and PFGE fingerprinting analysis revealed clonal transmission and plasmid transmission. OXA-48-like producing CRKP isolates mainly clustered in K64 ST11 and K47 ST15. Results of the string Test, serum killing assay (in vitro) and Galleria mellonella infection model (in vivo) indicated hypervirulence. PBRT showed that the blaOXA-181 and blaOXA-232 producing hypervirulent carbapenem-resistant Klebsiella pneumoniae (Hv-CRKP) were mainly carried on ColE-type, IncF, and IncX3. Eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1). Moreover, Southern blotting hybridization revealed that all eight isolates had a pLVPK-like virulent plasmid (138.9-216.9 kb) with an uneven number and size of plasmid. Conclusion: In our investigation, we have observed the emergence of hv-CRKP carrying blaOXA-48-like genes, which identified two genetic relationships: clonal transmission and plasmid transmission. PBRT analysis showed that these genes were mainly carried on ColE-type, IncF, and IncX3 plasmids. These isolates have been shown to be hypervirulent in vitro and in vivo. Additionally, eight clinical isolates of hv-CRKP were identified as carrying three carbapenem-resistant genes (blaKPC, blaOXA-181 or OXA-232, and blaNDM-1) and carrying a pLVPK-like virulent plasmid. Hence, our findings highlight the need for further investigation and active surveillance of hypervirulent OXA-48-like producing Hv-CRKP isolates to control their transmission.

Molecular characterization of the bla(KPC-2) gene in clinical isolates of carbapenem-resistant Klebsiella pneumoniae from the pediatric wards of a Chinese hospital.

The present study was conducted to confirm the presence of Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae associated with a nosocomial outbreak in a Chinese pediatric hospital. From July 2009 to January 2011, 124 nonduplicated K. pneumoniae isolates were collected from specimens from patients of pediatric units in the hospital. Twelve of the 124 isolates possessed the bla(KPC-2) gene and showed 7 different pulsed-field gel electrophoresis (PFGE) patterns. Meanwhile, 16S rRNA methylase, acc(6')-Ib-cr, and several types of ß-lactamases were also produced by the majority of the KPC-producing isolates. Class 1 integron-encoded intI1 integrase gene was subsequently found in all strains, and amplification, sequencing, and comparison of DNA between 5' conserved segment and 3' conserved segment region showed the presence of several known antibiotic resistance gene cassettes of various sizes. The conjugation and plasmid-curing experiments indicated some KPC-2-encoding genes were transmissible. In addition, conjugal cotransfer of multidrug-resistant phenotypes with KPC-positive phenotypes was observed in KPC-producing strains. Restriction endonuclease analysis and DNA hybridization with a KPC-specific probe showed that the bla(KPC-2) gene was carried by plasmid DNA from K. pneumoniae of PFGE pattern B. The overall results indicate that the emergence and outbreak of KPC-producing K. pneumoniae in our pediatric wards occurred in conjunction with plasmids coharboring 16S rRNA methylase and extended-spectrum ß-lactamases.

Genetic characterization and passage instability of a novel hybrid virulence plasmid in a ST23 hypervirulent Klebsiella pneumoniae.

Hypervirulent variants of Klebsiella pnuemoniae (hvKP), which causes life-threatening infections, is a global priority pathogen and frequently harbours virulence plasmids. The virulence plasmids have emerged as the predominant vehicles carrying the major pathogenic determinants of hypermucoviscosity and hypervirulence phenotypes. In the present study, we characterized a novel virulence plasmid in AP8555, an ST23 hvKP strain, which induced a metastatic infection and fatal septic shock in a critically ill patient. The serum killing assay, the quantitative biofilm formation assay, the G.mellonella infection model, and the mouse lethality assay demonstrated that AP8555 was almost as virulent as the hvKP strain NUTH-K2044. The plasmid pAP855 could be conjugated to Klebsiella quasipneumoniae ATCC700603 and E. coli J53 at a frequency of 7.2× 10-5 and 8.7× 10-7, respectively. Whole-genome sequencing and bioinformatics analysis confirmed that the plasmid was novel, clustered to the incompatibility type of IncHI1B/IncFIB/IncFII and presented high similarity to the pK2044 plasmid. In contrast, a 130-kb large-fragment insertion was observed on the plasmid, which introduced a genetic hybrid zone with multiple conjugation-related genes of type IV secretion systems (T4SS) and CcdAB toxin-antitoxin systems (TAS) to the plasmid. In the transconjugants, the presence of pAP855 had a negative impact on bacterial fitness, but enhancing the virulence-associated phenotypes. In vitro evolution experiments showed that pAP855 in the transconjugants could not be stably inherited after 10 days of passage. Our study not only reports a novel hybrid plasmid but also highlights the putative pathway of conjugative virulence plasmid formation and evolution by means of genetic rearrangement through sequence insertion. These findings indicate that structural versatility could contribute to the dissemination of cointegrate virulence plasmid, although the plasmid incurred a fitness cost. Therefore, continuous monitoring the acquisition of conjugative virulence plasmids may have critical value for plasmid research and increase awareness of hvKP.

Microbiological and Clinical Characteristics of Klebsiella pneumoniae Isolates of K57 Capsular Serotype in China.

Background: K57 Klebsiella pneumoniae (K57-KP) is associated with hypervirulence, but the basis and systematic data of K57-KP are limited. Materials and Methods: A retrospective study was conducted in 156 patients between January 2013 and January 2016. The clinical and molecular data, including antimicrobial susceptibility testing, multilocus sequence typing, antimicrobial resistance genes, and virulence determinants were assessed. Results: Among the 39 K57-KP isolates, 14 isolates (35.9%) were associated with various types of invasive infections. Diabetes, drainage, use of carbapenems and quinolone antibiotics were dependent risk factors for K57-KP infections. Sequence type (ST)412 was the most prevalent among K57-KP isolates. K57-KP isolates were more resistant to clinically often used antimicrobial agents than hvKP (K1/K2) strains, and 12.8% (5/39) of the strains were resistant to carbapenems, which all harbored blaKPC-2. The prevalence of hypermucoviscosity phenotype, aerobactin, rmpA, rmpA2, and ybts revealed 66.7%, 100%, 89.7%, 89.7%, and 30.8%, whereas wcaG, allS, magA and kfu revealed 0%, 0%, 0%, and 5.1%, which were significantly lower than that of hvKP (K1/K2). The serum sensitivity, neutrophil phagocytic rate, and biofilm formation capacity of K57-KP strains were higher than that of K1/K2. Conclusion: There were no significant differences in the prevalence of hypermucoviscosity phenotype, carriage of rmpA and aerobactin genes between K57 and K1/K2 isolates, but the composition and production of capsule polysaccharide of K57-KP may be different from that of K1/K2 strains. K57-KP isolates exhibited distinctive virulence-associated traits, most of which belonged to ST412. Physicians should enhance the management of K57-KP infections because of the emergence of more and more carbapenem-resistant K57-KP isolates.

Prevalence of the NTEKPC-I on IncF Plasmids Among Hypervirulent Klebsiella pneumoniae Isolates in Jiangxi Province, South China.

Infection caused by carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) has become a tricky health care threat in China and KPC-2 enzyme is a main factor mediating resistance to carbapenems of K. pneumoniae. Here, we report the characterization of the genetic environment of the blaKPC-2 gene in CR-hvKP clinical isolates from South China. Forty-five non-duplicated CR-hvKP isolates collected in Jiangxi Province from 2018 to 2019 were analyzed. Each of them were multidrug-resistant due to the presence not only of blaKPC-2 gene but also of other resistance determinants, including Metallo-ß-lactamases (NDM-1), extended-spectrum ß-lactamases (TEM-1, CTX-M-14, SHV-1), and plasmid-mediated quinolone resistance determinants (qnrS, aac(6')-Ib-cr). After plasmid analyses of PCR-based replicon typing (PBRT), mapping PCR, amplicon sequencing, and whole-genome sequencing (WGS) were used to analyze the genetic environment of the blaKPC-2 gene. PCR analysis of pLVPK-like plasmids, Southern Blot, and mouse lethality assay were used to characterize the virulence phenotype of K. pneumoniae. Multilocus sequence typing (MLST) analysis showed ST11 CR-hvKP was the predominant clone. In conclusion, this is the first analysis of diverse genetic structures blaKPC-2 gene in CR-hvKP isolates from south China. Both the NTEKPC-I on the IncF plasmids and pLVPK-like virulence plasmids make contributions to the formation of CR-hvKP especially ST11 which need more attention.

Emergence of Hypervirulent Ceftazidime/Avibactam-Resistant Klebsiella pneumoniae Isolates in a Chinese Tertiary Hospital.

INTRODUCTION: Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) is increasingly reported worldwide, but ceftazidime/avibactam (CAZ/AVI)-resistant hvKP isolates have rarely been observed. We attempted to characterize them in clinical CRKP isolates collected from a university hospital in China from March 2016 to March 2018. METHODS: All isolates were analyzed by antimicrobial susceptibility testing, molecular detection of antibiotic resistance determinants, multilocus sequence typing (MLST), SDS-PAGE, and pulsed-field gel electrophoresis (PFGE). The pLVPK-related genetic loci (rmpA2, terW, iutA, and silS) were screened in all CAZ/AVI-resistant CRKP isolates for the presence of virulence plasmids by PCR. Capsule typing, serum killing assay, Galleria mellonella lethality experiments, and mouse lethality assay were conducted to identify CAZ/AVI-resistant hvKP among isolates that carried all four virulence genes. RESULTS: A total of 232 CRKP isolates were collected. Overall, CAZ/AVI-resistance was found in 8.2% (19/232) CRKP isolates isolated from patients with no history of previous CAZ/AVI-based treatment. Among these, 63.2% (12/19) were metallo-ß-lactamase-producing K. pneumoniae (MBL-KP), 52.6% (10/19) were Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP), and 26.3% (5/19) produced both MBL and KPC. The presence of carbapenemase promoted a very high increase in CAZ/AVI minimum inhibitory concentration only when ompk35 and ompk36 were absent. Alarmingly, nine isolates had all four virulence genes for the presence of virulence plasmids. All nine isolates were considered to be CAZ/AVI-resistant hvKP according to the G. mellonella infection model and mouse lethality assay, with ST23 being the most common type (55.6%, 5/9). CONCLUSION: The newly emerged hypervirulent CAZ/AVI-resistant KP strain might cause a serious threat to public health, suggesting an urgent need for enhanced clinical awareness and epidemiologic surveillance.

Prevalence of Carbapenem-Resistant Klebsiella pneumoniae Co-Harboring blaKPC-Carrying Plasmid and pLVPK-Like Virulence Plasmid in Bloodstream Infections.

This study aimed to characterize carbapenem-resistant Klebsiella pneumoniae (CR-KP) co-harboring blaKPC-2-carrying plasmid and pLVPK-like virulence plasmid. Between December 2017 and April 2018, 24 CR-KP isolates were recovered from 24 patients with bacteremia. The mortality was 66.7%. Pulsed-field gel electrophoresis and multilocus sequence typing results indicated four clusters, of which cluster A (n = 21, 87.5%) belonged to ST11 and the three remaining isolates (ST412, ST65, ST23) had different pulsotypes (cluster B, C, D). The blaKPC-2-carrying plasmids all belonged to IncFIIK type, and the size ranged from 100 to 390 kb. Nineteen strains (79.2%) had a 219-kb virulence plasmid possessed high similarity to pLVPK from CG43 with serotype K2. Two strains had a 224-kb virulence plasmid resembled plasmid pK2044 from K. pneumoniae NTUH-K2044(ST23). Moreover, three strains carried three different hybrid resistance- and virulence-encoding plasmids. Conjugation assays showed that both blaKPC-2 and rmpA2 genes could be successfully transferred to E. coli J53 in 62.5% of the strains at frequencies of 4.5 × 10-6 to 2.4 × 10-4, of which three co-transferred blaKPC-2 along with rmpA2 in large plasmids. Infection assays in the Galleria mellonella model demonstrated the virulence level of these isolates was found to be consistently higher than that of classic Klebsiella pneumoniae. In conclusion, CR-KP co-harboring blaKPC-2-carrying plasmid and pLVPK-like virulence plasmid were characterized by multi-drug resistance, enhanced virulence, and transferability, and should, therefore, be regarded as a real superbug that could pose a serious threat to public health. Hence, heightened efforts are urgently needed to avoid its co-transmission of the virulent plasmid (gene) and resistant plasmid (gene) in clinical isolates.

[Construction and efficacy study of a novel 10-23 deoxyribozyme expression vector].

OBJECTIVE: To construct a novel 10-23 deoxyribozyme (10-23DZ) expression vector, identify the intracellular production of specific 10-23DZ and its inhibitory effect upon the expression of TACO gene in macrophage. METHODS: An oligonucleotide containing the primer binding sequence of mouse Moloney leukemia viral reverse transcriptase (MLV-RT), a multiple cloning site (MCS) and a stem-loop structure for termination of reverse transcription was designed and inserted into one MCS of plasmid pBUDCE4.1, downstream of the cytomegalovirus promoter (P(CMV)). The gene fragment encoding MLV-RT was inserted into another MCS of pBUDCE4.1, downstream of elongation factor 1alpha promoter (P(EF-1alpha)). The resulting plasmid was named pSDE01. Then pSDE01 was transfected into RAW264.7 cell and the expression of MLV-RT and the reverse transcriptase activity of expression product was identified by RT-PCR. To identify the cellular expression of 10-23DZ by pSDE01, a 10-23DZ targeting the TACO mRNA of macrophage was designed according to the predicted secondary structure of TACO mRNA. The expression sequence of designed 10-23DZ, DZ1, was synthesized and inserted into the MCS of ODN-PSL in pSDE01. The resulting plasmid, pSDE01-DZ1, was transfected into RAW264.7 cell and the expression of DZ1 was identified by PCR and dot-blot respectively. At 48 h after transfection of pSDE01 into RAW264.7 cells, total RNA and proteins were extracted and the TACO mRNA and protein expression level was assayed by RT-PCR and Western blotting respectively. RESULTS: Restrictive analysis and sequencing data showed that the 10-23DZ expression vector, pSDE01, was successfully constructed and could express MLV-RT with reverse transcriptase activity in cell. When transfected into RAW264.7 cells, pSDE01-DZ1 expressed DZ1 effectively and inhibited the expression of TACO gene. And TACO mRNA decreased by 77.7% (0.193 +/- 0.008 vs 0.864 +/- 0.005, P < 0.05) and TACO protein decreased by 73.3% (0.114 +/- 0.051 vs 0.427 +/- 0.043, P < 0.05). CONCLUSIONS: A novel expression vector capable of generating specific 10-23DZ in cells has been successfully constructed. And the intracellular product 10-23DZ may inhibit the expression of any target gene of interest.

Evaluation of ompK36 allele groups on clinical characteristics and virulence features of Klebsiella pneumoniae from bacteremia.

BACKGROUND/PURPOSE: This study investigated the implications of ompK36 allele groups on clinical and microbiological features of patients with Klebsiella pneumoniae bacteremia. METHODS: A total of 80 K. pneumoniae bloodstream isolates were collected and then divided into four ompK36 allele groups. Clinical characteristics, bacterial antibiotic resistance and virulence determinants were analyzed, including resistance and virulence genes, hypermucoviscosity phenotype, K capsule serotypes, biofilm formation, serum killing, neutrophil phagocytosis, and mouse lethality studies. RESULTS: 78 isolates were classified into four ompK36 variants, designated groups A (34), B (6), C (26), and D (12), respectively; 2 isolate was untypeable. OmpK36 group C isolates carried higher frequencies of K1/K2 capsule serotypes, hypermucoviscosity phenotype, rmpA gene, allS gene, iroB gene, aerobactin gene, or rmpA2 gene than non-C group isolates. OmpK36 group C isolates were significantly more virulent, as higher serum resistance, higher anti-phagocytosis and higher mouse lethality, than OmpK36 non-C group isolates, except for similar biofilm formation capability. The K20 isolates probably has low expression rates of rmpA and rmpA2 for hypermucoviscosity phenotype. The biofilm formation was significantly associated with ESBL production. OmpK36 group C isolates were more frequently detected in patients with community-acquired bloodstream infection. However, significant underlying diseases and prior use of carbapenem were highly prevalent in patients with OmpK36 non-C group isolates infection. ESBL production was apparently higher in non-C group but did not reach statistical significance. CONCLUSION: Our results suggest that the OmpK36 group C K.pneumoniae is more associated with community-acquired infection with a lower frequency of underlying illness, but with significantly more virulence in bloodstream infection. This would give a remind that clinicians should be aware of such clinical impacts of the ompK36 allele group.

High Prevalence of Plasmid-Mediated Quinolone Resistance Determinants Among Serotype K1 Hypervirulent Klebsiella pneumoniae Isolates in China.

Thirty-five serotype K1 hypervirulent Klebsiella pneumoniae (K1-hvKP) isolates collected from a Chinese hospital during the whole year of 2017 were evaluated to characterize the prevalence of the plasmid-mediated quinolone resistance (PMQR) genes. In total, 18 (51.4%) isolates were detected to carry PMQR genes, and the most frequently detected gene was qnrS1 (37.5%), followed by aac(6')-Ib-cr (15%) and qnrB4 (2.5%). Remarkably, some qnr-carrying strains had only a single or more quinolone resistance-determining region mutations in GyrA or ParC, and most exhibited high-level ciprofloxacin resistance. However, only 11.4% (4/35) isolates were resistant to quinolones. Furthermore, 34.3% (12/35) carried extended-spectrum ß-lactamase (ESBL) genes, but only 14.3% (5/35) exhibited ESBL phenotype. The most prevalent virulence genes were mrkD (100%, 21/21), followed by magA (97.1%, 34/35), wabG (97.1%, 34/35), rmpA (97.1%, 34/35), aerobactin (94.3%, 33/35), kfuB (94.3%, 33/35), ycf (91.4%, 32/35), iutA (91.4%, 32/35), rmpA2 (62.9%, 22/35), wcaG (62.9%, 22/35), ybtS (51.4%, 18/35), allS (45.7%, 16/35), and iroN (22.9%, 8/35). Multilocus sequence typing (MLST) analysis assigned the 35 K1-hvKP isolates into 5 sequence types (STs), with ST23 encompassing 77.1% of the strains. Pulsed field gel electrophoresis (PFGE) typing showed that strains closely related by MLST clustered in major PFGE clusters, of which cluster A accounts for 16 ST23 isolates and cluster B includes 11 ST23 isolates. The analysis of the transconjugants showed decreased susceptibility to quinolones and revealed a cotransfer of blaCTX-M-15 with the qnrS1 alleles. Cumulatively, our findings showed that the PMQR-producing K1-hvKP strain is covertly spreading even when K1-hvKP is rarely resistant to fluoroquinolones (FQs) according to the Clinical and Laboratory Standards Institute criteria. It is therefore critical to continuously monitor the PMQR-producing K1-hvKP epidemiology and minimize potential risks from FQ-resistant K1-hvKP.

Isocitrate lyase from Mycobacterium tuberculosis promotes survival of Mycobacterium smegmatis within macrophage by suppressing cell apoptosis.

BACKGROUND: Isocitrate lyase (ICL) was previously demonstrated to play a pivotal role in the intracellular metabolism of Mycobacterium tuberculosis (MTB). Presently several lines of evidence suggest that ICL from MTB (MTB-ICL) may play some roles in the interaction between MTB and host macrophage. However, there has been no research on the interaction between MTB-ICL and host macrophage. METHODS: MTB-icl and M. smegmatis (MS)-icl genes were amplified by polymerase chain reaction (PCR) and cloned into the E. coli-mycobacterium shuttle plasmid pUV15 to obtain recombinant shuttle plasmids pMTB-icl and pMS-icl. Following transformation into MS by electroporation, the expression of pMTB-icl and pMS-icl was verified by reverse transcriptase (RT)-PCR. The expression of recombinant plasmids derived from pUV15 when rMS was phagocytized by macrophage was also verified via fluorescence microscope. Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were used to infect RAW264.7 cells and the survival of intracellular MS was monitored by bacterial culture at 0, 24 and 48 hours after infection. The culture supernatants from macrophage infected by Ms 1 - 2c, rMS-pUV15, rMS-pMS-icl and rMS-pMTB-icl were collected and the interferon (IFN)-gamma and nitric oxide (NO) concentrations were measured by ELISA or by Griess assay, respectively. The apoptosis of macrophage was assayed by the in situ TUNEL technique. RESULTS: RT-PCR showed that both pMTB-icl and pMS-icl could be expressed in MS. Fluorescence microscopic observation showed that recombinant plasmids derived from pUV15 (pUV15-IG) could also be expressed in MS when MS were phagocytized by macrophage. Bacterial culture data demonstrated that rMS-pMTB-icl exhibited significantly increased intracellular survival in the murine macrophage cell line RAW264.7 compared with Ms 1 - 2c, rMS-pUV15 and rMS-pMS-icl. This increased intracellular survival was not accompanied by the upregulation of IFN-gamma and NO in host macrophage. But a lower apoptosis rate of macrophages infected with rMS-pMTB-icl was observed when compared with macrophages infected with other strains of MS. CONCLUSIONS: MTB-ICL could promote the intracellular survival of MS. Suppressing the apoptosis of host macrophage may be one of the important mechanisms involved in this increased intracellular survival.

[Effects of isocitrate lyase from Mycobacterium tuberculosis on the survival of Mycobacterium smegmatis in macrophage and mechanism thereof].

OBJECTIVE: To investigate the effects of isocitrate lyase (ICL) from Mycobacterium tuberculosis (MTB-icl) on the survival of Mycobacterium smegmatis (MS) in macrophage and illuminate the possible mechanisms. METHODS: MTB-icl gene was amplified by PCR and cloned into Ecoli-Mycobacterium shuttle plasmid pUV15 to obtain recombinant shuttle plasmid pUV15-icl expressing ICL-GFP. The recombinant shuttle plasmid pUV15-icl and blank plasmid pUV15 were induced into MS of the line 1-2c so as to obtain rMS-pUV15-icl and rMS-pUV15. Shuttle plasmid rMS-pUV15-IG expressing ICL-green fluorescent protein (GFP) was constructed. rMS-pUV15-IG and MS 1-2c were used to infect the murine macrophages of the line RAW264.7, fluorescence microscopy was used to observe the expression of ICL-GFP. The expression of ICL in the MS swallowed by the macrophages was verified by RT-PCR and Western blotting. Another macrophages RAW264.7 were cultured and infected with rMS-pUV15-icl and rMS-pUV15 respectively. 0, 24, and 48 hours later macrophages were collected and the number of MS colonies was calculated. The interferon (IFN)-gamma and nitrogen oxide (NO) concentrations in the culture supernatants of macrophages infected by rMS-pUV15-icl and rMS-pUV15 were measured by ELISA and Griess assay respectively. The apoptotic rate of the macrophages was assayed by in situ TUNEL technique. RESULTS: Western blotting showed that the MTB ICL protein expression of the rMS-pUV15-icl was significantly higher than that of rMS-pUVI5. Fluorescence microscopy showed green fluorescence in the RAW264.7 cells infected with rMS-pUV15-IG, but not ion the RAW264.7 cells infected with MS 1-2c. 0 h after the infection of the macrophages there was not significant difference in the MS amount in the macrophages between the rMS-pUV15-isl and rMS-pUV15 groups, and 24 h and 48 h later the MS amounts of the rMS-pUV15-icl group were (32.78 +/- 2.90) x 10(3) and (23.33 + 2.34) x 10(3) respectively, both significantly higher than those of the rMS-pUV15 group [(14.67 +/- 2.45) x 10(3) and (2.28 +/- 0.25) x 10(3) respectively, both P < 0.01]. There were not significant differences in the concentrations of IFN-gamma and NO in the culture supernatants between the 2 groups (both P >0.05). 48 h after the infection the apoptotic rate of the rMS-pUV15-icl group was significantly lower than that of the rMS-pUV15 group (P <0.01). Conclusion MTB-ICL promotes the intracellular survival of MS. Suppressing the apoptosis of the host macrophage may be one of the important mechanisms involved in this increased intracellular survival.

[Diagnostic value of anti-alpha-fodrin antibody testing for Sjögren's syndrome: a meta-analysis].

OBJECTIVE: To evaluate the diagnostic accuracy of anti-alpha-fodrin antibody for Sjögren's syndrome (SS). METHODS: Qualified literatures on evaluation of anti-alpha-fodrin antibody in diagnosis of SS in English and Chinese published between January 1997 and December 2007 were retrieved from the Cochrane Library, Medline, Embase, and China National Knowledge Infrastructure (CNKI) databases, etc. Two reviewers independently assessed the methodological quality of each study with the tool QUADAS (quality assessment of diagnostic accuracy studies). Statistical analysis was performed by employing MATLAB, Review Manager 4.2 and Meta-Disc1.4. A meta-analysis of the reported sensitivity and specificity of each study and Summary Receiver Operating Characteristic (SROC) curve was performed. RESULTS: Eighteen literatures were included at last. After testing the heterogeneity of the included articles, proper effect model was selected to calculate the pooled weighted sensitivity and specificity with 95% confidence interval: for anti-alpha-fodrin antibody IgG, the sensitivity was 0.40 [95%CI (0.37-0.43)] and the specificity was 0.82 [95%CI (0.79-0.84)], the area under the curve (AUC) of SROC was 0.8029 (SE=0.0580). Eight studies tested anti-alpha-fodrin antibody IgA, the pooled weighted sensitivity and specificity with 95% confidence interval were 0.34 [95%CI (0.30-0.38)] and 0.83 [95%CI (0.79-0.86)] respectively, the AUC of SROC was 0.6374 (SE=0.1841), the synthesis data all showed heterogeneity. The subgroups were analyzed to identify the sources of heterogeneity according to age, race, assay method, agent source, diagnostic criteria, and country. There was homogeneity among the 4 studies from China, and the 6 studies from Japan, the AUC of SROC were 0.7343 (SE=0.0448) and 0.9273 (SE=0.0394), respectively. CONCLUSION: Diagnostic criteria, agent source, assay method, age, race, and country are the important sources of heterogeneity. Anti-alpha-fodrin antibodies IgG and IgA have relatively low pooled sensitivity and relatively high pooled specificity. Negative anti-alpha-fodrin antibody has not important value in excluding SS, but positive anti-alpha-fodrin antibody may be a useful parameter in clinical diagnosis of SS.

Draft Genome Sequence of an NDM-1- and KPC-2-Coproducing Hypervirulent Carbapenem-Resistant Klebsiella pneumoniae Strain Isolated from Burn Wound Infections.

We report here the draft genome sequence of an NDM-1- and KPC-2-coproducing hypervirulent carbapenem-resistant Klebsiella pneumoniae strain, isolated from a 58-year-old male in the People's Republic of China with a burn injury.

Molecular Epidemiology and Virulence Features of Staphylococcus aureus Bloodstream Isolates in a Regional Burn Center in China, 2012-2016.

Staphylococcus aureus is known to be a predominant pathogen causing bloodstream infection (BSI) from burn units. Our study aimed to perform the clinical epidemiological analysis and virulence features of S. aureus strains isolated from the burn patients with BSI from a burn center in southeastern China during 2012-2016. A collection of 112 S. aureus isolates causing BSI from burn center of a tertiary care hospital in China was carried out. Antimicrobial susceptibility testing was conducted in accordance with the Clinical and Laboratory Standards Institute (CLSI) guidelines. Toxin gene profiles, multilocus sequence typing, staphylococcal protein A (spa) typing, accessory gene regulator (agr) locus typing, staphylococcal cassette chromosome mec (SCCmec) typing, and dendrographic analysis were used to characterize and analyze these isolates. Of 112 S. aureus isolates, 52 (46.4%) were methicillin-resistant S. aureus (MRSA) and 60 (53.6%) were methicillin-susceptible S. aureus (MSSA). ST239-SCCmec III-t030-agr I was the major prevalent clone (26 from MRSA and 6 from MSSA), which was followed by ST239-SCCmec III-t037-agr I (12, 10.0%) and ST5-SCCmec II-t002-agr I (11, 9.2%). The genotyping results showed high genetic diversity in molecular characterization and toxin gene profiles of the strains. Carriage of tsst-1 was mainly associated with ST239-SCCmec III-t030-agr I and ST30-SCCmec IV-t062-agr III, whereas lukS/F-PV was distributed in different clones. In conclusion, ST239-SCCmec III-t030-agr I is the commonest clone causing BSI among burn patients in eastern regions of China. In contrast to MRSA, polyclonality was statistically significantly higher among MSSA isolated from burn patients with BSI.