Hypervirulent Strains Research Articles

Transcriptional alteration of NF-κB-associated long noncoding RNAs in the stomach of Helicobacter pylori-infected and non-infected patients.

Helicobacter pylori could colonize the gastric mucosa and cause gastritis, ulcers and cancer. Numerous virulence factors have been identified in this bacterium that play important roles in promoting gastric disorders. Although the interaction of long noncoding RNAs (lncRNAs) with transcription, processing, and translation of genes associated with different diseases are described, their interaction with the inflammatory genes and H. pylori infection in the gastric tissue is not well known. This study compared changes in common NF-κB-regulatory lncRNAs in the gastric tissue of H. pylori-infected and non-infected patients with gastritis. Moreover, a link between the virulence entity of the strains and the transcriptional changes was analyzed. Two groups of infected and non-infected patients with chronic gastritis were included in the study. Genotyping of the H. pylori strains was done by PCR and relative changes in the expression of NF-κB and regulatory lncRNAs, lincRNA-p21, MALAT1, NKILA, were measured by relative quantitative real time-PCR. Transcriptional levels of MALAT1, lincRNA-p21, and NKILA genes decreased in the infected patients compared with the non-infected patients, which was significantly linked with increased NF-κB gene expression. Our results showed that a hypervirulent strain of H. pylori with oipA"on"/HP-NAP+/iceA1+/iceA2+/vacA s1m1/s1m2+/cagA+ genotype can promote a higher level of NF-κB transcription in the inflamed tissue. H. pylori infection could promote down-regulation of lincRNA-p21, MALAT1 and NKILA in the infected gastric tissue in correlation with NF-κB upregulation. More detailed studies are needed to show a link between the virulence genes and their impact on the regulation of lncRNAs in the stomach.

2145. Antagonistic Activity of Lacticseibacillus rhamnosus R3 Against Carbapenem-Resistant Acinetobacter baumannii Strain: A Promising Therapeutic Alternative

Abstract Background Acinetobacter baumannii (AB) is a recognized nosocomial pathogen of critical importance Most circulating AB strains are carbapenem-resistant (CRAB), which severely complicates therapeutics with available antibiotics. Probiotic lactic acid bacteria constitute a promising therapeutic alternative. Previously, we determined that Lacticaseibacillus rhamnosus R3 (LR-R3) exerts a strong inhibitory capacity on the model strain A118 (susceptible to antibiotics). In this analysis, the antimicrobial activity of LR-R3 against type CRAB hypervirulent strain AB5075 was evaluated. Figure 1 A) Transwell migration assay. AB5075 viability after 24h exposure to LR-R3 CFCM. B) CFCM activity obtained from 96h culture of LR-R3 on CRAB strains. Methods The inhibitory activity of cell pellet and its cell-free supernatants (CFCM) were determined, respectively, by the soft-agar overlay method, CFCM activity, and transwell migration assay. Also, killing assays and scanning microscopy imaging were performed under co-culture conditions. Lastly, AB5075 transcriptional response when exposed to LR-R3 was assessed by RT-qPCR. Results The lactic acid bacteria LR-R3 demonstrated a strong inhibitory capacity on AB5075 strain (inhibition halo diameter, DHI >20 mm), with loss of cell viability at 4h of co-culture conditions. The CFCM of LR-R3 also showed similar antimicrobial activity on AB5075 (Fig 1A) and against others CRAB strains (Fig 1B), suggesting that the inhibitory effect observed is due to the presence of a compound or metabolite produced and secreted by the lactobacillus. Microscopy images showed increase formation of outer membrane vesicles (OMVs) and the presence of nanotubes. In addition, significant changes in the expression levels of the studied genes were observed in AB5075 in co-culture with LR-R3: increase in the expression of genes involved in biofilm (csuAB, csuB, ompA); and decrease in the expression of genes involved in the synthesis and utilization of iron (bauA, exbD, bfnA, basE, pirA), and genes related to fatty acid and lipid metabolism (paaA, paaB). Conclusion The results obtained allowed the identification of Lcb. rhamnosus R3 as a probiotic strain with antagonistic activity on the nosocomial pathogen A. baumannii. In addition, it was shown that LR-R3 alters iron and fatty acid metabolism, by virtue of this interaction. This work constitutes the basis for future studies necessary to elucidate the mechanism involved. Disclosures Robert A. bonomo, MD, Entasis, Merck, VenatoRx, Wockhardt: Grant/Research Support

Current insight into klebsiella infection in children

Klebsiella pneumoniae is an important causative agent of nosocomial and community-acquired infections in children. The relevance of Klebsiella infection has increased in recent years due to the spread of multiresistant and hypervirulent strains that can cause invasive forms of infection (sepsis, meningitis, liver abscess, etc.). Strains of Kl. pneumoniae, producing extended-spectrum β-lactamase (ESBL), as well as carbapenem-resistant strains, are the cause of nosocomial outbreaks with high mortality. The choice of antibacterial drugs for the treatment of invasive forms of infection is limited and depends on the spectrum of sensitivity to antimicrobial drugs of Klebsiella clinical isolates circulating in the hospital and the age of the child. Due to the high rate of ESBL production, carbapenems are the drugs of choice in the empirical therapy of invasive forms of infection.

Genome Analysis of Klebsiella pneumoniae Reveals International High-Risk Pandemic MDR Clones Emerging in Tertiary Healthcare Settings in Uganda.

Klebsiella pneumoniae is a threat to public health due to its continued evolution. In this study, we investigated the evolution, convergence, and transmission of hypervirulent and multi-drug resistant (MDR) clones of K. pneumoniae within healthcare facilities in Uganda. There was high resistance to piperacillin (90.91%), cefuroxime (86.96%), ceftazidime (84.62%), cefotaxime (84.00%), amoxicillin/clavulanate (75%), nalidixic acid (73.68%), and nitrofurantoin (71.43%) antibiotics among K. pneumoniae isolates. The isolates were genetically diverse, consisting of 20 different sequence types (STs) and 34 K-serotype groups. Chromosomal fosA (for fosfomycin) and oqxAB efflux pump genes were detected in all isolates. Two carbapenem resistance genes, blaNDM-5 and blaOXA-181 plus extended-spectrum beta-lactamase (blaCTX-M-15) gene (68.12%), quinolone-resistant genes qnrS1 (28.99%), qnrB1 (13.04%), and qnrB6 (13.04%) and others were found. All, except three of the isolates, harbored plasmids. While the isolates carried a repertoire of virulence genes, only two isolates carried hypervirulent genes demonstrating a low prevalence (2.90%) of hypervirulent strains. Our study demonstrated genetically diverse populations of K. pneumoniae, low levels of carbapenem resistance among the isolates, and no convergence of MDR and hypervirulence. Emerging high-risk international pandemic clones (ST11, ST14, ST147, ST 86 and ST307) were detected in these healthcare settings which are difficult to treat.

Complete Genome Resource of a Hypervirulent Xanthomonas oryzae pv. oryzae Strain YNCX Isolated from Yunnan Plateau Japonica Rice.

Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight (BLB), is one of the most destructive bacterial pathogens in rice production worldwide. Although several complete genome sequences of Xoo strains have been released in public databases, they are mainly isolated from low-altitude indica rice cultivating areas. Here, a hypervirulent strain, YNCX, isolated from the high-altitude japonica rice-growing region in Yunnan Plateau, was used to extract genomic DNA for PacBio sequencing and Illumina sequencing. After assembly, a high-quality complete genome consisting of a circular chromosome and six plasmids was generated. The genome sequence of YNCX provides a valuable resource for high-altitude races and enables the identification of new virulence TALE effectors, contributing to a better understanding of rice-Xoo interactions.

Evaluating the incidence of ampC-lactamase genes, biofilm formation, and antibiotic resistance among hypervirulent and classical Klebsiella pneumoniae strains.

Both immunocompetent and healthy individuals can become life-threateningly ill when exposed to the hypervirulent (hvKp) strains of Klebsiella pneumoniae (Kp). The main objectives of this study were to evaluate the presence of ampC-lactamase genes, biofilm formation, and antibiotic resistance in clinical strains of hvKp and cKp (classical K. pneumoniae). Kp strains were collected from patients referred to Shahidzadeh Hospital in Behbahan city, Khuzestan Province, Iran. Several techniques were used to identify hvKp. The hypermucoviscosity phenotype was determined using the string test. Isolates that developed dark colonies on tellurite agar were assumed to be hvKp strains. If any of the iucA, iutA, or peg-344 genes were detected, the isolates were classified as hvKp. Phenotypic and genotypic detection of AmpC β-lactamases of hvKp strains was performed by combined disk method and PCR, respectively. In addition, crystal violet staining was used to determine the biofilm formation of these isolates. For this study, 76 non-duplicative isolates of Kp were collected. Overall, 22 (28.94%) strains had positive string test results, and 31 (40.78%) isolates were grown in tellurite-containing medium. The genes iucA and iutA or peg-344 were found in 23.68% of all Kp strains and in 50% of tellurite-resistant isolates, respectively. The most effective antibiotics against hvKp isolates were tetracycline (85.52%) and chloramphenicol (63.15%). Using the cefoxitin disc diffusion method, we observed that 56.57% (43/76) of the strains were AmpC producer. A total of 30.26% (n=23/76) of the isolates tested positive for at least one ampC gene, including blaDHA (52.63%, n=40), blaCIT (40.78%, n=31), blaACC (19.76%, n=15), blaMOX (25%, n=19), and blaFOX (43.42%, n=33). Biofilm formation analysis revealed that most hvKp isolates were weak (n=6, 40%) and moderate (n=5, 33.33%) biofilm producers. Healthcare practitioners should consider the possibility of the existence and acquisition of hvKp everywhere. The exact mechanisms of bacterial acquisition are also unknown, and it is unclear whether the occurrence of infections is related to healthcare or not. Thus, there are still many questions about hvKp that need to be investigated.

Phylogenetic analysis of the Klebsiella pneumoniae uge gene in local microbiological monitoring

The aim of the study was to evaluate the data of phylogenetically analyzed nucleotide sequences from the K. pneumoniae strain uge genes carried out in the perinatal center. Materials and methods. Fifty-six sequences of the K. pneumoniae uge gene were analyzed. The uge gene was detected by real-time PCR using DT light amplifier (Russia). Results. The rate of K. pneumoniae strains among patients of obstetric and gynecological departments in 2020–2023 averaged 1.4%. In pediatric hospitals, the isolation of K. pneumoniae strains comprised 12–14% in 2020, 2021 and 2023. In 2022, a fourfold decrease in detected K. pneumoniae strains was recorded. Phylogenetic analysis showed that the nucleotide sequences were significantly grouped into 14 clusters. The K. pneumoniae virulence factor uge gene is found in 64.3% cases. The nucleotide sequences allowed to detect heterogeneous K. pneumoniae strain population isolated from patients at the perinatal center from 2019 to 2023 analyzed by the phylogenetic method. There are clusters that combine K. pneumoniae genovariants isolated in 2019 and not replenished with new isolates, which confirms the effectiveness of ongoing anti-epidemic measures, enhanced during COVID-19 spread, excluding the transmission of an infectious agent from a source to a susceptible organism in nosocomial environment. P249Q, N279L amino acid substitutions within the uge gene were determined, which distinguish hypervirulent strains from those with a lower degree of pathogenicity. Out of five mother-child pairs, in four — nucleotide sequences of K. pneumoniae strain uge gene were genetically closer to each other than to other strains isolated from patients of the Medical Departments suggesting about a high degree of their relationship, highly likely indicating that the source of the strain for the child was the paired mother, and not the patients of the departments or the staff of the institution. In one mother-child pair, K. pneumoniae strains belonged to different clusters. The isolate obtained on 09/06/2021 from neonatal faeces (age: 7 days old) was grouped with strains isolated on 12/12/2020 from a patient in the maternity ward and in Laos in 2013 (CP035196). K. pneumoniae isolated on 06/04/2021 from the urine of a woman, was significantly grouped with strains included in cluster 13. Conclusion. An opportunity for improving local microbiological monitoring by sequencing and phylogenetic analysis of K. pneumoniae uge gene has been demonstrated. Assessment of changes in the intraspecies population pattern of HCAI pathogens is necessary for a timely and reasonable response to the deterioration of the epidemiological situation.

Transcriptomic and phenotype analysis revealed the role of rpoS in stress resistance and virulence of a novel ST3355 ESBL-producing hypervirulent Klebsiella pneumoniae isolate.

An extended-spectrum beta-lactamase (ESBL)-hypervirulent Klebsiella pneumoniae (HvKP) strain HKE9 was isolated from the blood in an outpatient. The effect of the global regulatory factor RpoS on antimicrobial resistance, pathogenicity, and environmental adaptability was elucidated. HKE9 is a novel ST3355 (K20/O2a) hypervirulent strain with a positive string test and resistant to cephems except cefotetan. It has a genome size of 5.6M, including two plasmids. CTX-M-15 was found in plasmid 2, and only ompk37 was found in the chromosome. HKE9 could produce bacterial siderophores, and genes of enterobactin, yersiniabactin, aerobactin, and salmochelin have been retrieved in the genome. As a global regulatory factor, knockout of rpoS did not change antimicrobial resistance or hemolytic phenotype while increasing the virulence to Galleria mellonella larvae and showing higher viscosity. Moreover, rpoS knockout can increase bacterial competitiveness and cell adhesion ability. Interestingly, HKE9-M-rpoS decreased resistance to acidic pH, high osmotic pressure, heat shock, and ultraviolet and became sensitive to disinfectants (H2O2, alcohol, and sodium hypochlorite). Although there were 13 Type 6 secretion system (T6SS) core genes divided into two segments with tle1 between segments in the chromosome, transcriptomic analysis showed that rpoS negatively regulated T4SS located on plasmid 2, type 1, and type 3 fimbriae and positively regulate genes responsible for acidic response, hyperosmotic pressure, heat shock, oxidative stress, alcohol and hypochlorous acid metabolism, and quorum sensing. Here, this novel ST3355 ESBL-HvKP strain HKE9 may spread via various clonal types. The important regulation effect of rpoS is the enhanced tolerance and resistance to environmental stress and disinfectants, which may be at the cost of reducing virulence and regulated by T4SS.

The Effect of Subinhibitory Concentration of Metronidazole on the Growth and Biofilm Formation on Toxigenic Clostridioides difficile Strains Belonging to Different Ribotypes.

Clostridioides difficile is a predominant nosocomial pathogen within the healthcare setting able to produce biofilms. Sub-minimum inhibitory concentrations (sub-MICs) of antibiotics trigger mechanisms affecting bacterial virulence, including increased adhesion and biofilm formation. The aim of this study was to investigate how sub-MICs of metronidazole affect the biofilm formation of C. difficile strains. We tested 14 reference and clinical C. difficile strains, including hypervirulent strains of RT027. The MICs of metronidazole for the tested strains were determined using the broth microdilution method. Biofilm formation was evaluated using confocal laser scanning microscopy. The C. difficile strains belonging to RT027 produced the highest amounts of biofilm. The results of confocal laser scanning microscopy showed that all the tested C. difficile strains developed larger biofilms with diversified architectures upon exposure to sub-MICs of metronidazole. In our study, we reveal that sub-MIC concentrations of metronidazole affect the biofilm formation of clinical and reference strains of C. difficile. Importantly, metronidazole induces biofilm formation via hypervirulent RT027 strains.

Extensive Expression of the Virulome Related to Antibiotic Genotyping in Nosocomial Strains of Klebsiella pneumoniae.

The emergence of hyper-virulent and multidrug-resistant (MDR) strains of Klebsiella pneumoniae isolated from patients with hospital- and community-acquired infections is a serious health problem that increases mortality. The molecular analysis of virulome expression related to antimicrobial-resistant genotype and infection type in K. pneumoniae strains isolated from patients with hospital- and community-acquired infections has been poorly studied. In this study, we analyzed the overall expression of the virulence genotype associated with the antimicrobial resistance genotype and pulse field gel electrophoresis (PFGE) type (PFtype) in K. pneumoniae. We studied 25 strains of K. pneumoniae isolated from patients who developed bacteremia and pneumonia during their hospital stay and 125 strains from outpatients who acquired community-acquired infections. Susceptibility to 12 antimicrobials was determined by Kirby-Bauer. The identification of K. pneumoniae and antibiotic-resistance genes was performed using polymerase chain reaction (PCR). To promote the expression of the virulence genes of K. pneumoniae, an in vitro infection model was used in human epithelial cell lines A549 and A431. Bacterial RNA was extracted with the QIAcube robotic workstation, and reverse transcription to cDNA was performed with the Reverse Transcription QuantiTect kit (Qiagen). The determination of the expression of the virulence genes was performed by real-time PCR. In addition, 57.3% (n = 86) of the strains isolated from patients with hospital- and community-acquired infections were multidrug-resistant (MDR), mainly to beta-lactam antibiotics (CB, AM, CFX, and CF), aminoglycosides (GE), quinolones (CPF and NOF), nitrofurantoin (NF), and sulfamethoxazole/trimethoprim (SXT). The most frequently expressed genes among strains isolated from hospital- and community-acquired infections were adhesion-type, ycfm (80%), mrkD (51.3%), and fimH (30.7%); iron uptake, irp2 (84%), fyuA (68.7%), entB (64.7%), and irp1 (56.7%); and protectins, rpmA (26%), which were related to antibiotic-resistance genes, blaTEM (96%), blaSHV (64%), blaCITM (52.6%), blaCTXM-1 (44.7%), tetA (74%), sul1 (57.3%), aac(3)-IV (40.7%), and aadA1 (36%). The results showed the existence of different patterns of expression of virulome related to the genotype of resistance to antimicrobials and to the PFtypes in the strains of K. pneumoniae that cause hospital- and community-acquired infections. These findings are important and may contribute to improving medical treatment strategies against infections caused by K. pneumoniae.

Bacteriophages with depolymerase activity in the control of antibiotic resistant Klebsiella pneumoniae biofilms

Klebsiella pneumoniae is associated with a variety of infections, such as pneumonia, urogenital infection, liver abscess, and bloodstream infection. It is especially dangerous for patients in medical facilities, where it can cause ventilator-associated pneumonia or intensive care unit-acquired pneumonia. The emergence of multidrug-resistant and hypervirulent strains as well as the ability to form biofilms on various medical devices complicates the treatment of such infections and makes the use of antibiotics ineffective. The application of bacteriophages is a promising alternative for combating Klebsiella pneumoniae biofilms. In the present study a cocktail of 3 bacteriophages with depolymerase activity was used to control antibiotic resistant Klebsiella pneumoniae biofilms in vitro. Biofilms were examined using optical and scanning electron microscopy. The obtained results demonstrate that the studied bacteriophage cocktail can effectively disrupt Klebsiella pneumoniae biofilms.

C. difficile intoxicates neurons and pericytes to drive neurogenic inflammation.

Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections1,2. The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization3-6, but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4)in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.

Regulation of biofilm formation in Klebsiella pneumoniae.

Klebsiella pneumoniae is an important Gram-negative opportunistic pathogen that is responsible for a variety of nosocomial and community-acquired infections. Klebsiella pneumoniae has become a major public health issue owing to the rapid global spread of extensively-drug resistant (XDR) and hypervirulent strains. Biofilm formation is an important virulence trait of K. pneumoniae. A biofilm is an aggregate of microorganisms attached to an inert or living surface by a self-produced exo-polymeric matrix that includes proteins, polysaccharides and extracellular DNA. Bacteria within the biofilm are shielded from antibiotics treatments and host immune responses, making it more difficult to eradicate K. pneumoniae-induced infection. However, the detailed mechanisms of biofilm formation in K. pneumoniae are still not clear. Here, we review the factors involved in the biofilm formation of K. pneumoniae, which might provide new clues to address this clinical challenge.

Description of extraintestinal pathogenic Escherichia coli based on phylogenetic grouping, virulence factors, and antimicrobial susceptibility.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a recently recognized and highly diverse pathotype of E. coli. Its significance as a pathogen has increased due to the emergence of hypervirulent and multidrug-resistant (MDR) strains. The aim of this study was to characterize ExPEC isolates from humans based on their phylogenetic group, virulence factor profile, and antimicrobial susceptibility. The isolates were collected from patients with extraintestinal infections caused by E. coli, including urinary tract infections, bacteremia, and surgical site infections. The E. coli phylogenetic groups were determined using multiplex PCR. Additionally, the isolates were evaluated for their biofilm-forming abilities, susceptibility to antimicrobial agents, and presence of virulence genes. In this study, the isolates were classified into four phylogenetic groups: A (48.3%), B2 (25.8%), D (19.35%), and B1 (6.45%). All isolates exhibited at least one of the ten analyzed virulence factors. However, there was no direct evidence linking a specific phylogenetic group to a particular virulence factor. Nevertheless, the presence of the fimH, fyuA, ompT, traT, and kpsMTII virulence genes was correlated with the production of strong biofilms, multidrug resistance (MDR), and the production of alpha hemolysin. This study provides a description of the phylogenetic groups in ExPEC and their potential association with virulence factor profiles and antimicrobial susceptibility.

Australian Meningococcal Surveillance Programme Annual Report, 2022.

In Australia, both probable and laboratory-confirmed cases of invasive meningococcal disease (IMD) are reported to the National Notifiable Diseases Surveillance System (NNDSS). Compared to 2021, the number of IMD notifications in 2022 increased by 81% to 127, alongside the easing of COVID-19 containment measures. Laboratory confirmation occurred in 95% of these cases, with 51% (62/121) diagnosed by bacterial culture and 49% (59/121) by nucleic acid amplification testing. The serogroup was determined for 97% of laboratory-confirmed cases (117/121): serogroup B (MenB) accounted for 83% of infections (100/121); MenW for 4% (5/121); MenY for 10% (12/121); no infections were attributed to MenC disease. Fine typing was available on 67% of the cases for which the serogroup was determined (78/117). In MenB isolates, 27 porA types were detected, the most prevalent of which were P1.7-2,4 (18%;11/62), P1.22,14 (15%; 9/62), P1.18-1,34 (10%; 6/62) and P1.7,16-26 (10%; 6/62). All five MenW infections identified as porA type P1.5,2 with different MLST sequence types (ST): 11, 574, 1287, 12351, 13135 all belonging to clonal complex 11, the hypervirulent strain reported in outbreaks in Australia and overseas. In MenY, the predominant porA type was P1.5-1,10-1 (73%; 8/11), ST 1655 and from clonal complex 23. Children less than 5 years of age and people aged 15-19 years were overrepresented with IMD notifications, accounting for 22% (27/121) and 23% (28/121) of laboratory-confirmed cases respectively. Fifteen percent of laboratory-confirmed notifications (18/121) were in persons aged 45-64 years. MenB infections were detected in all age groups but predominated in persons aged 15-19 years (93% of IMD in this age group; 26/28) and comprised 89% (24/27) of infections in children aged less than 5 years. MenW infections were markedly reduced in 2022, accounting for two IMD detections in children 1-4 years (2/16) and sporadic detections in other older age groups. MenY infections were largely detected in adults aged 45-64 years, accounting for 28% of IMD in this age group (5/18). All 62 cultured IMD isolates had antimicrobial susceptibility testing performed. Minimum inhibitory concentration (MIC) values were categorised using Clinical Laboratory Standards Institute (CLSI) interpretative criteria: 5% (3/62) were defined as penicillin resistant (MIC value ≥ 0.5 mg/L); 71% (44/62) had intermediate susceptibility to penicillin (MIC values 0.125 and 0.25 mg/L) and 24% (15/62) were susceptible to penicillin. All isolates were susceptible to ceftriaxone, ciprofloxacin and rifampicin.

Urine-mediated suppression of Klebsiella pneumoniae mucoidy is counteracted by spontaneous Wzc variants altering capsule chain length.

Klebsiella pneumoniae is a hospital-associated pathogen primarily causing urinary tract infections (UTIs), pneumonia, and septicemia. Two challenging lineages include the hypervirulent strains, causing invasive community-acquired infections, and the carbapenem-resistant classical strains, most frequently isolated from UTIs. While hypervirulent strains are often characterized by a hypermucoid phenotype, classical strains usually present with low mucoidy. Since clinical UTI isolates tend to exhibit limited mucoidy, we hypothesized that environmental conditions may drive K. pneumoniae adaptation to the urinary tract and select against mucoid isolates. We found that both hypervirulent K. pneumoniae and classical Klebsiella UTI isolates significantly suppressed mucoidy when cultured in urine without reducing capsule abundance. A genetic screen identified secondary mutations in the wzc tyrosine kinase that overcome urine-suppressed mucoidy. Over-expressing Wzc variants in trans was sufficient to boost mucoidy in both hypervirulent and classical Klebsiella UTI isolates. Wzc is a bacterial tyrosine kinase that regulates capsule polymerization and extrusion. Although some Wzc variants reduced Wzc phospho-status, urine did not alter Wzc phospho-status. Urine does, however, increase K. pneumoniae capsule chain length diversity and enhance cell-surface attachment. The identified Wzc variants counteract urine-mediated effects on capsule chain length and cell attachment. Combined, these data indicate that capsule chain length correlates with K. pneumoniae mucoidy and that this extracellular feature can be fine-tuned by spontaneous Wzc mutations, which alter host interactions. Spontaneous Wzc mutation represents a global mechanism that could fine-tune K. pneumoniae niche-specific fitness in both classical and hypervirulent isolates. IMPORTANCE Klebsiella pneumoniae is high-priority pathogen causing both hospital-associated infections, such as urinary tract infections, and community-acquired infections. Clinical isolates from community-acquired infection are often characterized by a tacky, hypermucoid phenotype, while urinary tract isolates are usually not mucoid. Historically, mucoidy was attributed to capsule overproduction; however, recent reports have demonstrated that K. pneumoniae capsule abundance and mucoidy are not always correlated. Here, we report that human urine suppresses K. pneumoniae mucoidy, diversifies capsule polysaccharide chain length, and increases cell surface association. Moreover, specific mutations in the capsule biosynthesis gene, wzc, are sufficient to overcome urine-mediated suppression of mucoidy. These Wzc variants cause constitutive production of more uniform capsular polysaccharide chains and increased release of capsule from the cell surface, even in urine. These data demonstrate that K. pneumoniae regulates capsule chain length and cell surface attachment in response host cues, which can alter bacteria-host interactions.

Klebsiella pneumoniae outer membrane vesicles induce strong IL-8 expression via NF-κB activation in normal pulmonary bronchial cells.

Outer membrane vesicles (OMVs) derived from bacteria are known to play a crucial role in the interactions between bacteria and their environment, as well as bacteria-bacteria and bacteria-host interactions.Specifically, OMVs derived from Klebsiella pneumoniae have been implicated in contributing to the pathogenesis of this bacterium.Hypervirulent Klebsiella pneumoniae (hvKp) has emerged as a global pathogen of great concern due to its heightened virulence compared to classical K. pneumoniae (cKp), and its ability to cause community-acquired infections, even in healthy individuals.The objective of this study was to investigate potential differences between hvKp-derived OMVs and cKp-derived OMVs in their interactions with microorganisms and host cells. Four strains of K. pneumoniae were used to produce OMVs: hvKp strain NTUH-K2044 (K1, ST23), hvKp clinical strain AP8555, and two cKP clinical strains C19 and C250. To examine the morphology and size of the bacterial OMVs, transmission electron microscopy (TEM) was utilized. Additionally, dynamic light scattering (DLS) was used to analyze the size characterization of the OMVs.The normal pulmonary bronchial cell line HBE was exposed to OMVs derived from hvKp and cKP. Interleukin 8 (IL-8) messenger RNA (mRNA) expression was assessed using reverse transcription-polymerase chain reaction (RT-PCR), while IL-8 secretion was analyzed using enzyme-linked immunosorbent assay (ELISA).Furthermore, the activation of nuclear factor kappa B (NF-κB) was evaluated using both Western blotting and confocal microscopy. After purification, OMVs appeared as electron-dense particles with a uniform spherical morphology when observed through TEM.DLS analysis indicated that hvKp-derived OMVs from K2044 and AP8555 measured an average size of 116.87±4.95nm and 96.23±2.16nm, respectively, while cKP-derived OMVs from C19 and C250 measured an average size of 297.67±26.3nm and 325±6.06nm, respectively. The average diameter of hvKp-derived OMVs was smaller than that of cKP-derived OMVs.A total vesicular protein amount of 47.35mg, 41.90mg, 16.44mg, and 12.65mg was generated by hvKp-K2044, hvKp-AP8555, cKP-C19, and cKP-C250, respectively, obtained from 750mL of culture supernatant. Both hvKp-derived OMVs and cKP-derived OMVs induced similar expression levels of IL-8 mRNA and protein. However, IL-8 expression was reduced when cells were exposed to BAY11-7028, an inhibitor of the NF-κB pathway.Western blotting and confocal microscopy revealed increased phosphorylation of p65 in cells exposed to OMVs. Klebsiella pneumoniae produces outer membrane vesicles (OMVs) that play a key role in microorganism-host interactions. HvKp, a hypervirulent strain of K. pneumoniae, generates more OMVs than cKP.The average size of OMVs derived from hvKp is smaller than that of cKP-derived OMVs.Despite these differences, both hvKp-derived and cKP-derived OMVs induce a similar level of expression of IL-8 mRNA and protein.OMVs secreted by K. pneumoniae stimulate the secretion of interleukin 8 by activating the nuclear factor NF-κB.

Characteristics of Clostridioides difficile isolates circulating in the Slovak hospitals

Clostridioides difficile is a frequent cause of hospital-acquired diarrhea associated with antibiotics. Hypervirulent strains of Clostridioides difficile are associated with a severe course of Clostridioides difficile infection and a higher mortality. We have confirmed 322 non-duplicate Clostridioides difficile isolates in stool samples of patients hospitalized in three Slovak hospitals providing primary health care between January and December 2021 and determined genes encoding toxins, ribotypes and susceptibility to antimicrobials. 93.7% of them were toxigenic and co-occurrence of genes tcdA, tcdB, cdtA, cdtB, which encode toxins, was detected in 71.4% isolates. Nosocomially associated ribotypes 176, 001, and 027 were confirmed in 61.1%, 10.2%, and 5.2% of isolates, respectively, and found to be the most frequent. Although ribotype 176 predominated in all three hospitals, the highest incidence was recorded in Košice (73.5%). Results of antimicrobial susceptibility testing revealed 65.8% resistance rate for rifampicin, 5.2% for vancomycin, 1.5% for metronidazole, 1.2% for teicoplanin and 0.6% for doxycycline. The correlation of frequent ribotypes and results of antimicrobial susceptibility testing points to the predominance of rifampicin resistance in isolates belonging to ribotypes 176 and 027. The above results reflect the problematic situation in our hospitals during the COVID-19 pandemic, that was related to the misuse of broad-spectrum antibiotics and impossibility to follow epidemiological measures to prevent the spread of hypervilulent isolates.

Evolutionary mechanisms of virus variability

ABSTRACT. The evolutionary changes of viruses are primarily associated with the replication processes of viruses containing deoxyribonucleic and ribonucleic acids, which differ significantly. The genomes of most viruses containing ribonucleic acid are replicated with much less accuracy compared to the genomes of viruses containing deoxyribonucleic acid. Comparing the number of mutations in an infected cell reflects an inverse relationship between genome size and the frequency of mutations carried out by these two categories of viruses. Viruses with double-stranded deoxyribonucleic acid genomes have a low mutation rate compared to single-stranded genomes. The genome of viruses is not a stable unique structure, but rather an average, variable number of different amino acid sequences. It is in the virus population that a high mutation rate is maintained, and low variability is not beneficial for the preservation of viruses in nature. Some animal species may be intermediate hosts when new epidemic viruses appear. The introduction of non-viral nucleic acid into the viral genome can also contribute to the evolutionary changes of the virus, lead to the formation of defective genomes or to the emergence of hypervirulent strains. Viral genomes encode numerous molecules that modulate a wide range of protective immune mechanisms. The variability of viruses is also facilitated by the simultaneous integration of several proviral genomes into one cell, which activates the processes of recombination and genetic shift. An important evolutionary point may be the conversion of ribonucleic acid ribose into deoxyribose of deoxyribonucleic acid, which increases the stability of nucleic acids by more than 100 times. Horizontal gene transfer between viruses that infect different hosts is a central feature of the evolution of viruses containing ribonucleic acid. Eukaryote viruses with single-stranded deoxyribonucleic acid probably evolved from bacterial plasmids after they acquired capsid protein genes from the (+) ribonucleic acid chain of viruses. In addition to megaviruses and adenoviruses, polintons are likely precursors to bidnaviruses and virophages.

Comparative genomics and virulome analysis reveal unique features associated with clinical strains of Klebsiella pneumoniae and Klebsiella quasipneumoniae from Trinidad, West Indies.

Klebsiella pneumoniae and Klebsiella quasipneumoniae are closely related human pathogens of global concern. The more recently described K. quasipneumoniae shares similar morphological characteristics with K. pneumoniae and is commonly misidentified as this species using traditional laboratory techniques. The vast mobilome in these pathogenic bacteria influences the dissemination of virulence factors in high-risk environments and it is, therefore, critical to monitor strains for developing effective clinical management strategies. Herein, this study utilized Illumina sequencing to characterize the whole genomes of nine clinical K. pneumoniae and one K. quasipneumoniae isolate obtained from patients of 3 major hospitals in Trinidad, West Indies. Reconstruction of the assembled genomes and implementation of several bioinformatic tools revealed unique features such as high pathogenicity islands associated with the isolates. The K. pneumoniae isolates were categorized as classical (n = 3), uropathogenic (n = 5), or hypervirulent (n = 1) strains. In silico multilocus sequence typing, and phylogenetic analysis showed that isolates were related to several international high-risk genotypes, including sequence types ST11, ST15, ST86, and ST307. Analysis of the virulome and mobilome of these pathogens showed unique and clinically important features including the presence of genes associated with Type 1 and Type 3 fimbriae, the aerobactin and yersiniabactin siderophore systems, the K2 and O1/2, and the O3 and O5 serotypes. These genes were either on or in close proximity to insertion sequence elements, phage sequences, and plasmids. Several secretion systems including the Type VI system and relevant effector proteins were prevalent in the local isolates. This is the first comprehensive study investigating the genomes of clinical K. pneumoniae and K. quasipneumoniae isolates from Trinidad, West Indies. The data presented illustrate the diversity of Trinidadian clinical K. pneumoniae isolates as well as significant virulence biomarkers and mobile elements associated with these isolates. Additionally, the genomes of the local isolates will add to global databases and thus can be used in future surveillance or genomic studies in this country and the wider Caribbean region.