Hypervirulent Variants Research Articles

Screening of Klebsiella pneumoniae isolates reveals the spread of strong biofilm formers and class 1 integrons.

Klebsiella pneumoniae is a Gram-negative bacterium that can colonize, penetrate, and cause infections at several human anatomical locations. The emergence of hypervirulent K. pneumoniae and its ability to evade the immune system and develop antibiotic resistance has made it a key concern in the healthcare industry. The hypervirulent variants are increasingly involved in community-acquired infections. Therefore, it is pertinent to understand the biofilm formation potential among the clinical isolates. We acquired 225 isolates of K. pneumoniae from the Department of Microbiology, Symbiosis University Hospital and Research Centre (SUHRC), Pune, India over 1 year from March 2022- March 2023, and evaluated antimicrobial susceptibility, hypermucoviscous phenotype, virulence, and antimicrobial-resistant gene distribution in K. pneumoniae isolates and established a correlation between antimicrobial resistance and integrons. Most isolates were strong biofilm formers (76%). The isolates harbored one or more carbapenemase/ beta-lactamase encoding gene combinations. Hypermucoviscous (HMKP) isolates had considerably greater positive rates for iutA, magA, K2 serotype, rmpA, and rmpA2 than non-HMKP isolates. Isolates carrying integrons (43%) showed significantly more antibiotic resistance. The study reveals spread of strong biofilm formers with extensive virulence and antimicrobial-resistant genes, and integrons responsible for multi-drug resistance among the clinical isolates of K. pneumoniae in Pune, India, posing a threat to the public health and necessitating close surveillance, accurate diagnosis, control, and therapeutic management of infections.

An increased prevalence of carbapenem-resistant hypervirulent Klebsiella pneumoniae associated with the COVID-19 pandemic

BackgroundKlebsiella pneumoniae (Kp) is a common community-acquired and nosocomial pathogen. Carbapenem-resistant and hypervirulent (CR-hvKp) variants can emerge rapidly within healthcare facilities and impacted by other infectious agents such as COVID-19 virus. MethodsTo understand the impact of COVID-19 virus on the prevalence of CR-hvKp, we accessed Kp genomes with corresponding metadata from GenBank. Sequence types (STs), antimicrobial resistance genes, and virulence genes, and those scores and CR-hvKp were identified. We analyzed population diversity and phylogenetic characteristics of five most common STs, measured the prevalence of CR-hvKp, identified CR-hvKp subtypes, and determined associations between carbapenem resistance gene subtypes with STs and plasmid types. These variables were compared pre- and during the COVID-19 pandemic. FindingsThe proportion of CR-hvKp isolates increased within multiple STs in different continents during the COVID-19 pandemic and persistent CR-hvKp subtypes were found in common STs. blaKPC was dominant in CG258, blaKPC-2 was detected in 97 % of the ST11 CR-hvKp, blaNDM subtypes were prominent in ST147 (87.4 %) and ST307 (70.8 %); blaOXA-48 and its subtypes were prevalent in ST15 (80.5 %). The possession of carbapenemase genes was different among subclades from different origins in different periods of time within each ST. IncFIB/IncHI1B hybrid plasmids contained virulence genes and carbapenemase genes and were predominant in ST147 (67.37 %) and ST307 (56.25 %). InterpretationThe prevalence of CR-hvKp increased during the COVID-19 pandemic, which was evident by an increase in local endemic clones. This process was facilitated by the convergence of plasmids containing carbapenemase genes and virulence genes. These findings have implications for the appropriate use of antimicrobials and infection prevention and control during outbreaks of respiratory viruses and pandemic management.

RES-Xre toxin-antitoxin locus knaAT maintains the stability of the virulence plasmid in Klebsiella pneumoniae

ABSTRACT Hypervirulent Klebsiella pneumoniae isolates have been increasingly reported worldwide, especially hypervirulent drug-resistant variants owing to the acquisition of a mobilizable virulence plasmid by a carbapenem-resistant strain. This pLVPK-like mobilizable plasmid encodes various virulence factors; however, information about its genetic stability is lacking. This study aimed to investigate the type II toxin-antitoxin (TA) modules that facilitate the virulence plasmid to remain stable in K. pneumoniae. More than 3,000 TA loci in 2,000 K. pneumoniae plasmids were examined for their relationship with plasmid cargo genes. TA loci from the RES-Xre family were highly correlated with virulence plasmids of hypervirulent K. pneumoniae. Overexpression of the RES toxin KnaT, encoded by the virulence plasmid- carrying RES-Xre locus knaAT, halts the cell growth of K. pneumoniae and E. coli, whereas co-expression of the cognate Xre antitoxin KnaA neutralizes the toxicity of KnaT. knaA and knaT were co-transcribed, representing the characteristics of a type II TA module. The knaAT deletion mutation gradually lost its virulence in K. pneumoniae, whereas the stability of the plasmid in E. coli was enhanced by adding knaAT, which revealed that the knaAT operon maintained the genetic stability of the large virulence plasmid in K. pneumoniae. String tests and mouse lethality assays subsequently confirmed that a loss of the virulence plasmid resulted in reduced pathogenicity of K. pneumoniae. These findings provide important insights into the role of the RES-Xre TA pair in stabilizing virulence plasmids and disseminating virulence genes in K. pneumoniae.

Global genomic profiling of Klebsiella pneumoniae: A spatio-temporal population structure analysis

Klebsiella pneumoniae is an important clinical bacterial pathogen that has hypervirulent and multidrug-resistant variants. Uniform Manifold Approximation and Projection (UMAP) was used to cluster genomes of 16 797 K. pneumoniae strains collected, based on core genome distance, in over 100 countries during the period 1937 to 2021. A total of 60 high-density genetic clusters of strains representing the major epidemic strains were identified among these strains. Using UMAP bedding, the relationship between genetic cluster, capsular polysaccharide (KL) types and sequence type (ST) of the strains was clearly demonstrated, with some important STs, such as ST11 and ST258, found to contain multiple clusters. Strains within the same cluster often exhibited significant diverse features, such as originating from different areas and being isolated in different years, as well as carriage of different resistance and virulence genes. These data enable the routes of evolution of the globally prevalent K. pneumoniae strains to be traced. Alarmingly, carbapenem-resistant K. pneumoniae strains accounted for 51.7% of the test strains and worldwide transmission was observed. Carbapenem-resistant and hypervirulent K. pneumoniae strains are mainly reported in China; however, these strains are increasingly reported in other parts of the world. Also identified in this study were several key genetic loci that facilitate development of a new K. pneumoniae typing method to differentiate between high- and low-risk strains. In particular, the acrR, ompK35 and hha genes were predicted to play a key role in expression of the resistance and virulence phenotypes.

CpxR promotes the carbapenem antibiotic resistance of Klebsiella pneumoniae by directly regulating the expression and the dissemination of bla KPC on the IncFII conjugative plasmid

ABSTRACT Klebsiella pneumoniae is an important human pathogen known for its resistance to carbapenem antibiotics, especially the increasing carbapenem-resistant hypervirulent variants. The carbapenem resistance is mainly caused by the carbapenemase gene bla KPC which was commonly found on the IncFII transferable plasmids in K. pneumoniae ST11 isolates in regions of China. However, the mechanisms of the plasmid-carrying bla KPC regulation by the host strain are not clear. To investigate the chromosome-encoded two-component system (TCS) that regulates the carbapenem resistance of K. pneumoniae caused by bla KPC, twenty-four TCSs of a carbapenem-resistant classical K. pneumoniae ST11 clinical isolate were knocked out. The deletion mutation of the TCS regulator cpxR exhibited increased sensitivity to carbapenem, which could be restored by complementation with cpxR in trans. Electrophoretic mobility shift, isothermal titration calorimetry and DNase I footprinting results revealed that CpxR directly bound to the promoter DNA of bla KPC and the binding was abolished by disrupting the DNA-binding domain in CpxR. The subsequent in vivo assays using the lacZ reporter system and qPCR showed that CpxR upregulates the transcription of bla KPC. Notably, CpxR was also found to activate the transfer of the bla KPC-carrying IncFII plasmid between the hypervirulent K. pneumoniae and E. coli isolates, in which CpxR promoted the transcription of the tra operon via binding to its promoter region. These results provide an important insight into the regulation of the host factor CpxR in the plasmid-carrying carbapenemase gene in the classical and hypervirulent K. pneumoniae.

The genotypic and phenotypic characteristics contributing to high virulence and antibiotics resistance in Escherichia coli O25-B2-ST131 in comparison to non- O25-B2-ST131

BackgroundEscherichia coli serogroup O25b-sequence type 131 (E. coli O25-B2-ST131) is considered as multidrug-resistant and hypervirulent organism. There is lack of data about involvement of this pathogen in the children’s infection. In this study, the prevalence, and clonality, virulence capacity, and antibiotic resistance phenotype and genotype of E. coli O25-B2-ST131 compared with non-O25-B2-ST131 isolates were investigated in children with urinary tract infection in Tehran, Iran.MethodsThe E. coli isolates from urine samples were identified using conventional microbiological methods. Characterization of E. coli O25-B2-ST131 clone, antibiotic susceptibility, biofilm formation, ESBLs phenotype and genotype, serum resistance, hemolysis, hydrophobicity, and formation of curli fimbriae were done using conventional microbiological and molecular methods. Clonality of the isolates was done by rep-PCR typing.ResultsAmong 120 E. coli isolates, the highest and lowest antibiotic resistance was detected against ampicillin (92, 76.6%) and imipenem 5, (4.1%), respectively. Sixty-eight (56.6%) isolates were ESBL-producing and 58 (48.3%) isolates were considered as multi-drug resistance (MDR). The prevalence of ESBL-producing and MDR isolates in O25-B2-ST131 strains was higher compared with the non-O25-B2-ST131 strains (p value < 0.05). O25-B2-ST131 strains showed significant correlation with serum resistance and biofilm formation. Amongst the resistance and virulence genes, the prevalence of iucD, kpsMTII, cnf1, vat, blaCTX-M-15, and blaSHV were significantly higher among O25-B2-ST131 isolates in comparison with non-O25-B2-ST131 isolates (p value < 0.05). Considering a ≥ 80% homology cut-off, fifteen different clusters of the isolates were shown with the same rep-PCR pattern.ConclusionsOur results confirmed the involvement of MDR-ESBLs producing E. coli strain O25-B2-ST131 in the occurrence of UTIs among children. Source tracking and control measures seem to be necessary for containment of the spread of hypervirulent and resistance variants in children.

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.

Novel chytrid pathogen variants and the global amphibian pet trade.

Global wildlife trade spreads emerging infectious diseases that threaten biodiversity. The amphibian chytrid pathogen Batrachochytrium dendrobatidis (Bd) has caused population declines and species extinctions worldwide except in Asia. Fire-bellied toads (Bombina orientalis), exported in large numbers from Asia, are tolerant of Bd and carry hypervirulent ancestral chytrid BdAsia-1 variants. We assayed the virulence of a new isolate of BdAsia-1 on the model Australasian frog host Litoria caerulea. Infected individuals (n = 15) all showed rapid disease progression culminating in death, whereas sham-inoculated individuals (n = 10) presented no clinical signs of disease and all survived (log rank test, χ2 = 15.6, df = 1, p < 0.0001). The virulence of the new isolate of BdAsia-1 is comparable tothe one we assayed previously (χ2 = 0.0, df = 1, p = 0.91). Internationally traded wildlife, even when they appear healthy, can carry hypervirulent variants of pathogens. Once new pathogen variants escape into the environment, native species that have had no opportunity to evolve resistance to them may perish. Our study suggests that hypervirulent pathogens are being spread by the international pet trade. Notifiable wildlife diseases attributable to locally endemic pathogens often fail to generate conservation concern so are rarely subject to border surveillance or import controls. Because of the danger novel variants pose, national border control agencies need to implement disease screening and quarantine protocols to ensure the safety of their endemic fauna.

Hypervirulent Klebsiella pneumoniae Infections in Pediatric Populations in Beijing (2017-2019): Clinical Characteristics, Molecular Epidemiology and Antimicrobial Susceptibility.

Hypervirulent variants of Klebsiella pnuemoniae (hvKp) are emerging globally causing life-threatening infectious diseases; however, comprehensive studies on pediatric hvKp strains and related infections are still lacking. Clinical data were collected from medical records. Genotype (multilocus sequence typing), capsular serotype, virulence gene profile and carbapenemase of the isolates were determined by PCR and DNA sequencing. Broth microdilution method was adopted to test the antimicrobial susceptibility. Hypermucoviscosity phenotype and the virulence of the strains were evaluated by string test and Galleria mellonella larvae killing assay. Among 319 K. pneumoniae strains, 26 (8.2%) hvKp were identified, the detection rates in 2017, 2018 and 2019 were 1.8%, 5.2% and 11.3%. The majority of hvKp infections were found in school-age children and adolescents (57.7%). Pneumonia was the most common diagnosis (38.5%). Single fatal case was hvKp caused liver abscess complicated with bacteremia. hvKp were dominated by ST23 (30.8%) and ST11 (30.8%). Eight carbapenem-resistant hvKp (CR-hvKp) were found, which all belonged to ST11. Virulence gene profile revealed that ST11 hvKp might carry incomplete pLVPK-like plasmids, but they exhibited comparable in vivo virulence to the other hvKp. The infections caused by hvKp are not frequent among pediatric populations, but the detection rate of hvKp in pediatric populations is increasing rapidly in recent years. The emerging and dissemination of ST11 CR-hvKp should be monitored continuously.

Small Klebsiella pneumoniae Plasmids: Neglected Contributors to Antibiotic Resistance.

Klebsiella pneumoniae is the causative agent of community- and, more commonly, hospital-acquired infections. Infections caused by this bacterium have recently become more dangerous due to the acquisition of multiresistance to antibiotics and the rise of hypervirulent variants. Plasmids usually carry genes coding for resistance to antibiotics or virulence factors, and the recent sequence of complete K. pneumoniae genomes showed that most strains harbor many of them. Unlike large plasmids, small, usually high copy number plasmids, did not attract much attention. However, these plasmids may include genes coding for specialized functions, such as antibiotic resistance, that can be expressed at high levels due to gene dosage effect. These genes may be part of mobile elements that not only facilitate their dissemination but also participate in plasmid evolution. Furthermore, high copy number plasmids may also play a role in evolution by allowing coexistence of mutated and non-mutated versions of a gene, which helps to circumvent the constraints imposed by trade-offs after certain genes mutate. Most K. pneumoniae plasmids 25-kb or smaller replicate by the ColE1-type mechanism and many of them are mobilizable. The transposon Tn1331 and derivatives were found in a high percentage of these plasmids. Another transposon that was found in representatives of this group is the blaKPC-containing Tn4401. Common resistance determinants found in these plasmids were aac(6′)-Ib and genes coding for β-lactamases including carbapenemases.

MoS2-Modified Curcumin Nanostructures: The Novel Theranostic Hybrid Having Potent Antibacterial and Antibiofilm Activities against Multidrug-Resistant Hypervirulent Klebsiella pneumoniae.

The recent emergence of hypervirulent clinical variants of Klebsiella pneumoniae (hvKP) causing community-acquired, invasive, metastatic, life-threatening infections of lungs, pleura, prostate, bones, joints, kidneys, spleen, muscles, soft-tissues, skin, eyes, central nervous system (CNS) including extrahepatic abscesses, and primary bacteremia even in healthy individuals has posed stern challenges before the existing treatment modalities. There is therefore an urgent need to look for specific and effective therapeutic alternatives against the said bacterial infection or recurrence. A new type of MoS2-modified curcumin nanostructure has been developed and evaluated as a potential alternative for the treatment of multidrug-resistant isolates. The curcumin quantum particles have been fabricated with MoS2 via a seed-mediated hydrothermal method, and the resulting MoS2-modified curcumin nanostructures (MQCs) have been subsequently tested for their antibacterial and antibiofilm properties against hypervirulent multidrug-resistant Klebsiella pneumoniae isolates. In the present study, we found MQCs inhibiting the bacterial growth at a minimal concentration of 0.0156 μg/mL, while complete inhibition of bacterial growth was evinced at concentration 0.125 μg/mL. Besides, we also investigated their biocompatibility both in vitro and in vivo. MQCs were found to be nontoxic to the SiHa cells at a dose as high as 1024 μg/mL on the basis of the tested adhesion, spreading of the cells, and also on the various serological, biochemical, and histological investigations of the vital organs and blood of the Charles Foster Rat. These results suggest that MQCs have potent antimicrobial activities against hvKP and other drug resistant isolates and therefore may be used as broad spectrum antibacterial and antibiofilm agents.

Pathogenesis of Hypervirulent Group A Streptococcus.

Group A Streptococcus (GAS) causes common pharyngitis and skin infections and occasional severe invasive infections. This review describes the recent progress on the pathogenesis of hypervirulent GAS. CovRS mutations are frequent among invasive GAS isolates and lead to hypervirulence. GAS CovRS mutants can be selected in vivo by neutrophils. The role of protease SpeB in source-sink dynamics of wild-type GAS and hypervirulent variants is discussed. Streptolysin S and PAF acetylhydrolase Sse critically and synergistically contribute to the inhibition of neutrophil recruitment by GAS CovS mutants. CovS mutations in emm3 GAS lead to the vascular invasion and enhance systemic GAS dissemination.

Co-location of the blaKPC-2, blaCTX-M-65, rmtB and virulence relevant factors in an IncFII plasmid from a hypermucoviscous Klebsiella pneumoniae isolate

Hypervirulent variants of klebsiella pneumoniae (hvKP), which cause serious infections not only healthy individuals, but also the immunocompromised patients, have been increasingly reported recently. One conjugation of a hypermucoviscous strian SWU01 co-carried the resistance gene blaKPC-2 and virulence gene iroN by the PCR detection from three carbapenem-resistance hvKP. To know the genetic context of this plasmid. The whole genome of this strain was sequenced. We got a 162,552-bp plasmid (pSWU01) which co-carried the resistance gene blaKPC-2 and virulence gene iroN. It is composed of a typical IncFII-type backbone, five resistance genes including blaCTX-M-65, blaKPC-2, blaSHV-12, blaTEM-1 and rmtB, and several virulence relevant factors including iroN, traT and toxin-antitoxin systems. The plasmid pSWU01 co-carrying the multidrug resistance determinants and virulence relevant factors from the hypermucoviscous K. pneumoniae represents a novel therapeutic challenge.

The Fructose-Specific Phosphotransferase System of Klebsiella pneumoniae Is Regulated by Global Regulator CRP and Linked to Virulence and Growth.

Klebsiella pneumoniae is an opportunistic pathogen, and its hypervirulent variants cause serious invasive community-acquired infections. A genomic view of K. pneumoniae NTUH-2044 for the carbohydrate phosphotransferase system (PTS) found a putative fructose PTS, namely, the Frw PTS gene cluster. The deletion mutant and the complemented mutant of frwC (KP1_1992), which encodes the putative fructose-specific enzyme IIC, were constructed, and the phenotypes were characterized. This transmembrane PTS protein is responsible for fructose utilization. frwC deletion can enhance biofilm formation and capsular polysaccharide (CPS) biosynthesis but decreases the growth rate and lethality in mice. frwC expression was repressed in the cyclic AMP receptor protein (CRP) mutant. Electrophoretic mobility shift assay showed that CRP can directly bind to the promoter of frwC These results indicated that frwC expression is controlled by CRP directly and that such regulation contributes to bacterial growth, CPS synthesis, and the virulence of the Δcrp strain. The findings help elucidate fructose metabolism and the CRP regulatory mechanism in K. pneumoniae.

Grafting to manage infections of top stunting and necrogenic strains of cucumber mosaic virus in tomato

AbstractCucumber mosaic virus (CMV) lists among the most important etiological agents of tomato diseases. Some isolates of CMV function as helper virus for replication, encapsidation and transmission of satellite RNAs (satRNA), which may exacerbate symptoms induced by CMV in certain hosts. Outbreaks of CMV strains supporting hypervirulent variants of satRNAs are recurrent in tomato with devastating effects on crop production and efficient control measures are still unavailable. In this study, we examined the dynamics of infection of the CMV strains tomato top stunting (TTS) and 77 supporting replication of satRNA variants that codetermine top stunting (TTS‐satRNA) and necrotic (77‐satRNA) phenotypes in two tomato cultivars denoted Solanum lycopersicum Manduria (Sl‐Ma) and S. lycopersicum UC82 (Sl‐UC). Sl‐Ma but not Sl‐UC recovered from disease symptoms induced by CMV‐TTS while both the cultivars succumbed to the infection of CMV‐77 and its necrogenic satRNA. Ability to recover of the Sl‐Ma plants was transmitted by grafting to the susceptible genotype Sl‐UC. More interestingly, recovery was observed also against the challenge inoculation of CMV plus 77‐satRNA in plants grafted on Sl‐Ma and in self‐grafted plants of both the Sl‐Ma and Sl‐UC cultivars. Analysis of small RNAs and genes of the defence plant response based on RNA interference (RNAi) suggested that RNAi is involved in the recovery of Sl‐Ma against CMV with hypervirulent satRNAs and in scions grafted on this rootstock. The response of Sl‐Ma to the inoculation of CMV‐77 plus 77‐satRNA was compared with that of the transgenic tomato line S. lycopersicum transgenic line UCTC5.9.2 that expresses constitutively the benign variant of the satRNA denoted Tfn‐satRNA. Comparative analysis suggested that the response may operate via similar mechanisms, which involve RNAi, the graft and the presence of the satRNA.

Clinical and Genomic Analysis of Liver Abscess-Causing Klebsiella pneumoniae Identifies New Liver Abscess-Associated Virulence Genes.

Hypervirulent variants of Klebsiella pneumoniae (hvKp) that cause invasive community-acquired pyogenic liver abscess (PLA) have emerged globally. Little is known about the virulence determinants associated with hvKp, except for the virulence genes rmpA/A2 and siderophores (iroBCD/iucABCD) carried by the pK2044-like large virulence plasmid. Here, we collected most recent clinical isolates of hvKp from PLA samples in China, and performed clinical, molecular, and genomic sequencing analyses. We found that 90.9% (40/44) of the pathogens causing PLA were K. pneumoniae. Among the 40 LA-Kp, K1 (62.5%), and K2 (17.5%) were the dominant serotypes, and ST23 (47.5%) was the major sequence type. S1-PFGE analyses demonstrated that although 77.5% (31/40) of the LA-Kp isolates harbored a single large virulence plasmid varied in size, 5 (12.5%) isolates had no plasmid and 4 (10%) had two or three plasmids. Whole genome sequencing and comparative analysis of 3 LA-Kp and 3 non-LA-Kp identified 133 genes present only in LA-Kp. Further, large scale screening of the 133 genes in 45 LA-Kp and 103 non-LA-Kp genome sequences from public databases identified 30 genes that were highly associated with LA-Kp, including iroBCD, iucABCD and rmpA/A2 and 21 new genes. Then, these 21 new genes were analyzed in 40 LA-Kp and 86 non-LA-Kp clinical isolates collected in this study by PCR, showing that new genes were present 80–100% among LA-Kp isolates while 2–11% in K. pneumoniae isolates from sputum and urine. Several of the 21 genes have been proposed as virulence factors in other bacteria, such as the gene encoding SAM-dependent methyltransferase and pagO which protects bacteria from phagocytosis. Taken together, these genes are likely new virulence factors contributing to the hypervirulence phenotype of hvKp, and may deepen our understanding of virulence mechanism of hvKp.

Bacterial phospholipases C as vaccine candidate antigens against cystic fibrosis respiratory pathogens: The Mycobacterium abscessus model

BackgroundVaccine strategies represent one of the fighting answers against multiresistant bacteria in a number of clinical settings like cystic fibrosis (CF). Mycobacterium abscessus, an emerging CF pathogen, raises difficult therapeutic problems due to its intrinsic antibiotic multiresistance. MethodsBy reverse vaccinology, we identified M. abscessus phospholipase C (MA-PLC) as a potential vaccine target. We deciphered here the protective response generated by vaccination with plasmid DNA encoding the MA-PLC formulated with a tetra functional block copolymer 704, in CF (ΔF508) mice. Protection was tested against aerosolized smooth and rough (hypervirulent) variants of M. abscessus. ResultsMA-PLC DNA vaccination (days 0, 21, 42) elicited a strong antibody response. A significant protective effect was obtained against aerosolized M. abscessus (S variant) in ΔF508 mice, but not in wild-type FVB littermates; similar results were observed when: (i) challenging mice with the “hypervirulent” R variant, and; (ii) immunizing mice with purified MA-PLC protein. High IgG titers against MA-PLC protein were measured in CF patients with M. abscessus infection; interestingly, significant titers were also detected in CF patients positive for Pseudomonas aeruginosa versus P. aeruginosa-negative controls. ConclusionsMA-PLC DNA- and PLC protein-vaccinated mice cleared more rapidly M. abscessus than β-galactosidase DNA- or PBS- vaccinated mice in the context of CF. PLCs could constitute interesting vaccine targets against common PLC-producing CF pathogens like P. aeruginosa.

Morphotype transition and sexual reproduction are genetically associated in a ubiquitous environmental pathogen.

Sexual reproduction in an environmental pathogen helps maximize its lineage fitness to changing environment and the host. For the fungal pathogen Cryptococcus neoformans, sexual reproduction is proposed to have yielded hyper virulent and drug resistant variants. The life cycle of this pathogen commences with mating, followed by the yeast-hypha transition and hyphal growth, and it concludes with fruiting body differentiation and sporulation. How these sequential differentiation events are orchestrated to ensure developmental continuality is enigmatic. Here we revealed the genetic network of the yeast-to-hypha transition in Cryptococcus by analyzing transcriptomes of populations with a homogeneous morphotype generated by an engineered strain. Among this network, we found that a Pumilio-family protein Pum1 and the matricellular signal Cfl1 represent two major parallel circuits directing the yeast-hypha transition. Interestingly, only Pum1 coordinates the sequential morphogenesis events during a-α bisexual and α unisexual reproduction. Pum1 initiates the yeast-to-hypha transition, partially through a novel filament-specific secretory protein Fas1; Pum1 is also required to sustain hyphal growth after the morphological switch. Furthermore, Pum1 directs subsequent differentiation of aerial hyphae into fruiting bodies in both laboratory and clinical isolates. Pum1 exerts its control on sexual reproduction partly through regulating the temporal expression of Dmc1, the meiosis-specific recombinase. Therefore, Pum1 serves a pivotal role in bridging post-mating morphological differentiation events with sexual reproduction in Cryptococcus. Our findings in Cryptococcus illustrate how an environmental pathogen can ensure the completion of its life cycle to safeguard its long-term lineage success.

Increasing Occurrence of Antimicrobial-Resistant Hypervirulent (Hypermucoviscous) Klebsiella pneumoniae Isolates in China

New hypervirulent variants of Klebsiella pneumoniae (hvKP) are emerging globally, most of which exhibit antimicrobial susceptibility. A retrospective study was conducted in 88 patients with cultures positive for K. pneumoniae hospitalized in the Beijing You'an Hospital from April 2010 to June 2012. The clinical and molecular data of the hvKP isolates (defined as string test positive) were compared with those of the classic K. pneumoniae (cKP) isolates. Overall, 33.0% (29/88) of K. pneumoniae isolates were hvKP. Univariate analysis revealed the following risk factors for hvKP: virulence gene rmpA (odds ratio [OR], 16.92 [95% confidence interval {CI}, 4.842-59.145]), capsule antigens K1 (OR, 3.355 [95% CI, 1.153-9.768]) and K2 (OR, 9.280 [95% CI, 0.987-87.250]), alcoholic hepatitis (OR, 7.435 [95% CI, 1.397-39.572]), liver abscess (OR, 9.068 [95% CI, 1.747-47.061]), metastatic infection (OR, 2.752 [95% CI, 1.100-6.886]), community-acquired infection (OR, 10.432 [95% CI, 3.623-30.033]), sputum isolation (OR, 0.312 [95% CI, .095-1.021]), and HIV infection (<0.001 [not applicable]). Multivariate analysis implicated rmpA (OR, 17.398 [95% CI, 4.224-71.668]) and community-acquired infection (OR, 6.844 [95% CI, 1.905-24.585]) as independent risk factors. The proportion of hvKP isolates increased from April to December 2010, January to September 2011, and October 2011 to June 2012 (to 25.5%, 26.7%, and 54.5%, respectively). Resistance to 14 of 19 tested antimicrobials was found to be significantly greater in cKP compared to hvKP. Importantly, resistance to all the tested antimicrobials, except carbapenems and amikacin, was observed in a proportion of hvKP strains, 17% (5/29) of which expressed extended-spectrum β-lactamase. Furthermore, antimicrobial resistance in hvKP strains increased over time. HvKP strains are being isolated from patients in China with increasing frequency and constitute an increasing proportion of K. pneumoniae strains, indicating an increasing propensity for the acquisition of antimicrobial resistance.

Controlling Clostridium difficile

Clostridium difficile infection causes colitis and is closely associated with the use of antimicrobials, probably due to disruption of the normal bowel flora. Avoiding inappropriate antimicrobial use is the most important way to prevent this potentially life-threatening infection. Emergent hypervirulent variants of C. difficile are associated with increased transmission, morbidity and mortality and have caused epidemics in North America and Europe. Cases have also been reported in Australia so intensified surveillance and antimicrobial stewardship is more important than ever. While any antimicrobial can precipitate C. difficile, fluoroquinolones, clindamycin and cephalosporins are particularly implicated. Other drugs, such as proton pump inhibitors, have also been associated with an increased incidence of C. difficile. Severe disease is managed in hospital. For non-severe disease, treatment generally includes supportive measures and treatment with oral metronidazole. Relapse is common.