Four New Isocoumarins from the Mangrove Fungus Alternaria Malorum with Antimicrobial Activities.

Four new isocoumarins, alternariethers A-C (1-3) and alternariester (4) were separated from the fermentation of the fungus Alternaria malorum FL39, purified from Myoporum bontioides. Their structures were ascertained using NMR and HR-ESI-MS spectroscopy. For compound 4, the absolute configuration was solved with the help of ECD calculation and the DP4+ method. Compared with the positive control triadimefon, compound 1 showed more potent antifungal effects on Colletotrichum musae. The antifungal effects of compounds 1, 2, and 3 on Fusarium oxysporum and Fusarium graminearum, of compound 4 on F. oxysporum, were equal to those of triadimefon. Except for compound 4 which was inactive against Escherichia coli with O78 serotype, all compounds showed moderate or weak antibacterial activity against Staphylococcus aureus ATCC 6538 and E. coli with O6 or O78 serotype.

Acquisition of a stable and transferable plasmid coharbouring hypervirulence and MDR genes with low fitness cost: Accelerating the dissemination of ST11-KL64 CR-HvKP.

OBJECTIVES: This study aimed to delineate the ability of a plasmid, pS130-4, which harboured both hypervirulence and multidrug resistance genes, to disseminate within Klebsiella pneumoniae, as well as its potential formation mechanism. METHODS: We employed whole-genome sequencing to decipher the genetic architecture of pS130-4. Its capability to conjugate and transfer was assessed through a series of experiments, including plasmid stability, competitive growth, and growth curve analysis. Its expression stability was further evaluated using drug sensitivity, larval survival, and biofilm formation tests. RESULTS: pS130-4 contained four intact modules typical of self-transmissible plasmids. BLAST analysis revealed a sequence identity exceeding 90% with other plasmids from a variety of hosts, suggesting its broad prevalence. Our findings indicated the plasmid's formation resulted from IS26-mediated recombination, leading us to propose a model detailing the creation of this conjugative fusion plasmid housing both blaKPC-2 and hypervirulence genes. Our conjugation experiments established that pS130-4, when present in the clinical strain S130, was self-transmissible with an estimated efficiency between 10-5 and 10-4. Remarkably, pS130-4 showcased a 90% retention rate and did not impede the growth of host bacteria. Galleria mellonella larval infection assay demonstrated that S130 had pronounced toxicity when juxtaposed with high-virulence control strain NTUH-K2044 and low-toxicity control strain ATCC700603. Furthermore, pS130-4's virulence remained intact postconjugation. CONCLUSION: A fusion plasmid, encompassing both hypervirulence and multidrug resistance genes, was viable within K. pneumoniae ST11-KL64 and incurred minimal fitness costs. These insights underscored the criticality of rigorous monitoring to pre-empt the escalation and distribution of this formidable super-plasmid.

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.

Four new diphenyl ether derivatives from a mangrove endophytic fungus Epicoccum sorghinum.

Four new diphenyl ethers, named epicoccethers K-N (1-4), were purified from the fermentation medium of a fungus Epicoccum sorghinum derived from Myoporum bontioides, and identified through HR-ESI-MS and NMR spectral analysis. Except that compound 1 showed moderate antifungal activity against Penicillium italicum and Fusarium graminearum, the other three compounds showed stronger activity against them than triadimefon. All of them showed moderate or weak antibacterial activity towards Staphylococcus aureus and Escherichia coli with O6 and O78 serotypes except that 3 was inactive to E. coli O6.

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.

Distribution of type VI secretion system (T6SS) in clinical Klebsiella pneumoniae strains from a Chinese hospital and its potential relationship with virulence and drug resistance.

OBJECTIVES: The type VI secretion system (T6SS) in Klebsiella pneumoniae strains isolated from the bloodstream, intestinal, the pyogenic liver abscess has been reported. Here we aimed to characterize T6SS in 248 Klebsiella pneumoniae isolates with all kinds of specimens from a Chinese hospital and to investigate the potential association of T6SS with virulence and drug resistance. METHODS: T6SS genes, capsular serotyping genes, drug resistance genes, and virulence genes were identified by polymerase chain reaction (PCR). Antibiotic susceptibilities were examined by the disk diffusion method. To assess biofilm formation of these clinical Klebsiella pneumoniae isolates, 96-well microtiter plate assays were performed. MLST was used to analyze the genotypes of these Klebsiella pneumoniae isolates. RESULTS: The frequency of T6SS genes among the clinical Klebsiella pneumoniae isolates was 72.2%. The T6SS-positive isolates displayed higher resistance to piperacillin-tazobactam, ciprofloxacin, levofloxacin, meropenem than the T6SS-negative isolates (P < 0.05). The T6SS-positive isolates formed significantly more biofilm mass than the T6SS-negative isolates (mean ± standard deviation [SD], 0.3 ± 0.09 vs.0.16 ± 0.06; P < 0.01). Compared to the T6SS-negative isolates, the T6SS-positive isolates had a higher frequency of virulence genes (rmpA, fimH, entB, kfu, ybtS) and the pLVPK-like plasmid (P < 0.05). CONCLUSION: In conclusion, the prevalence of the type VI secretion system is high in clinical Klebsiella pneumoniae isolates in a Chinese teaching hospital. T6SS-positive strains show higher biofilm-forming activity with high drug resistance and exhibit higher virulence potential.

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.

Rapid Detection to Differentiate Hypervirulent Klebsiella pneumoniae (hvKp) From Classical K. pneumoniae by Identifying peg-344 With Loop-Mediated Isothermal Amplication (LAMP).

OBJECTIVES: To establish a rapid molecular diagnostics of hvKp using the peg-344 loop-mediated isothermal amplification technique (LAMP). METHODS: In all, 28 K. pneumoniae strains isolated from the blood of patients were used for the peg-344 LAMP. K. pneumoniae NTUH-K2044 and K. pneumoniae ATCC700603 were used as positive control and negative control, respectively. For comparison, all the results were detected in a polymerase chain reaction (PCR), which was considered the gold standard for the detection of the gene. Mouse lethality assay, and Serum killing assay were also used to determine the virulence phenotype of K. pneumoniae. RESULTS: We determined the specificity and sensitivity of the primers for peg-344 detection in the LAMP reactions. This LAMP assay was able to specifically differentiate hvKp from classical K. pneumoniae (cKp) at 65°C, which was 100-fold more sensitive than a PCR assay for peg-344 detection. The virulence phenotype of K. pneumoniae detected by LAMP was as precise as by Mouse lethality assay and Serum killing assay. CONCLUSION: The LAMP assay is easy to perform and rapid. Therefore, it can be routinely applied to differentiate hvKp from cKp in the clinical laboratory.

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.