Hypervirulent Isolates Research Articles

Invasive Group A Streptococcus Hypervirulent M1UK Clone, Canada, 2018-2023.

To determine invasive group A Streptococcus trends in Canada, we characterized emm1 isolates collected during 2018-2023. The percentage of hypervirulent M1UK lineage isolates increased significantly, from 22.1% in 2018 to 60.2% in 2023. Genomic analysis identified geographically and temporally associated clusters and genes associated with virulent bacteriophage acquisition.

The Streptococcus agalactiae LytSR two-component regulatory system promotes vaginal colonization and virulence in vivo.

Streptococcus agalactiae (or group B Streptococcus, GBS) is a leading cause of neonatal sepsis and meningitis globally. To sense and respond to variations in its environment, GBS possesses multiple two-component regulatory systems (TCSs), such as LytSR. Here, we aimed to investigate the role of LytSR in GBS pathogenicity. We generated an isogenic lytS knockout mutant in a clinical GBS isolate and used a combination of phenotypic in vitro assays and in vivo murine models to investigate the contribution of lytS to the colonization and invasive properties of GBS. Deletion of the lytS gene in the GBS chromosome resulted in significantly higher survival rates in mice during sepsis, accompanied by reduced bacterial loads in blood, lung, spleen, kidney, and brain tissues compared to infection with the wild-type strain. In a mouse model of GBS vaginal colonization, we also observed that the lytS knockout mutant was cleared more readily from the vaginal tract compared to its wild-type counterpart. Interestingly, lower levels of proinflammatory cytokines were found in the serum of mice infected with the lytS mutant. Our results demonstrate that the LytSR TCS plays a key role in GBS tissue invasion and pathogenesis, and persistence of mucosal colonization.IMPORTANCEStreptococcus agalactiae (group B Streptococcus, or GBS) is a common commensal of the female urogenital tract and one of WHO's priority pathogens. The bacterium has evolved mechanisms to adapt and survive in its host, many of which are regulated via two-component signal transduction systems (TCSs); however, the exact contributions of TCSs toward GBS pathogenicity remain largely obscure. We have constructed a TCS lytS-deficient mutant in a CC-17 hypervirulent GBS clinical isolate. Using murine models, we showed that LytSR regulatory system is essential for vaginal colonization via promoting biofilm production. We also observed that lytS deficiency led to significantly attenuated virulence properties and lower levels of proinflammatory cytokines in blood. Our findings are of significant importance in that they unveil a previously unreported role for LytSR in GBS and pave the way toward a better understanding of its ability to transition from an innocuous commensal to a deadly pathogen.

Emergence of extensively drug-resistant hypervirulent Acinetobacter baumannii isolated from patients with bacteremia: bacterial phenotype and virulence analysis

ObjectivesIndividuals infected with extensively drug-resistant (XDR) Acinetobacter baumannii are difficult to cure and have a high mortality rate. In this study, we compared the genomic and phenotypic differences between XDR and non-multidrug-resistant (MDR) A. baumannii and further characterized hypervirulent XDR A. baumannii. MethodsA total of 1,403 Acinetobacter isolates were collected from patients with bacteremia between 1997 and 2015. Antimicrobial susceptibility test was performed to categorize isolates into non-MDR, MDR, and XDR groups. The presence of selected virulence-associated genes was determined by PCR. Bacterial phenotypes, including iron acquisition, biofilm formation, capsule production, and virulence to larvae and mice, were determined. ResultsMultilocus sequence types revealed the high prevalence of ST2 (81.6%) and ST129 (18.4%) among 49 XDR isolates and sequence types of 18 non-MDR isolates were more diverse. Virulence-associated phenotypic assays showed that XDR isolates had higher iron acquisition ability and capsule production and higher virulence to Galleria mellonella larvae. However, their biofilm formation ability was lower compared to that of non-MDR isolates. XDR isolates contained more virulence genes, tonB, hemO, abaI, and ptk, while non-MDR isolates contained more pld and ompA. Twenty-one XDR isolates caused <20% larvae survival rate after 7 days post-infection were defined as hypervirulent XDR isolates. Among them, isolates 1677 (ST129) and 929-1 (ST2) also caused the death of all infected mice within two days. ConclusionSome subpopulations of highly drug-resistant ST2 also exhibit high virulence. Therefore, it is of utmost importance to continue monitoring the spread of hypervirulent XDR A. baumannii isolates.

The Comparative Characterization of a Hypervirulent Acinetobacter baumannii Bacteremia Clinical Isolate Reveals a Novel Mechanism of Pathogenesis.

Acinetobacter baumannii is an opportunistic Gram-negative pathogen with exquisite survival capabilities under various environmental conditions and displays widespread resistance to common antibiotics. A. baumannii is a leading cause of nosocomial infections that result in high morbidity and mortality rates. Accordingly, when multidrug resistance rates surpass threshold levels, the percentage of A. baumannii clinical isolates surges. Research into A. baumannii has increased in the past decade, and multiple mechanisms of pathogenesis have been identified, including mechanisms underlying biofilm development, quorum sensing, exotoxin production, secretion system utilization, and more. To date, the two gold-standard strains used to investigate different aspects of A. baumannii pathogenesis include ATCC 17978 and ATCC 19606. Here, we report a comparative characterization study of three additional A. baumannii clinical isolates obtained from different infection types and derived from different anatomical regions of infected patients. The comparison of three clinical isolates in addition to the ATCC strains revealed that the hypervirulent bacteremia clinical isolate, known as HUMC1, employs a completely different mechanism of pathogenesis when compared to all its counterparts. In stark contrast to the other genetic variants, the hypervirulent HUMC1 isolate does not form biofilms, is antibiotic-susceptible, and has the capacity to reach higher levels of quorum compared to the other clinically relevant strains. Our data also reveal that HUMC1 does not shed endotoxin into the extracellular milieu, rather secretes the evolutionarily conserved, host-mimicking, Zonula occludens toxin (Zot). Taken together, our hypothesis that HUMC1 cells have the ability to reach higher levels of quorum and lack biofilm production and endotoxin shedding, accompanied by the substantial elaboration of Zot, suggests a novel mechanism of pathogenesis that appears to afford the hypervirulent pathogen with stealth-like capabilities when disseminating through the circulatory system in a state of bacteremia.

Deciphering the relative importance of genetic elements in hypervirulent Klebsiella pneumoniae to guide countermeasure development

Quantitating the contribution of phenotype-responsible elements in hypervirulent Klebsiella pneumoniae is needed. Isogenic mutants of four hypervirulent clinical isolates that produced K1 (ST23), K2 (ST86), K20 (ST1544), or K54 (ST29) capsules (mean 2.2 log10 LD50 (range 1.5-2.9)) were created to measure the effects on LD50 in a murine model of the hypervirulence-associated plasmid (pVir), iucA, prmpA, prmpA2 (truncated), irp2, and clbBC. Curing pVir had the greatest increase in survival (mean LD50 to 7.6 (range 7.0-9.0, p≤0.0001), a dosage comparable to classical K.pneumoniae. Results also showed increased mean LD50s for ΔprmpA (5.9, p≤0.0001), ΔiucA (3.6, p≤0.0001), Δirp2 (3.4), ΔrmpAΔiucA (6.3, p≤0.0001), and ΔpVirΔirp2 (8.7, p≤0.0001). Notably ΔpVir had an additional mean LD50 increase of 1.3 compared to the pVir-encoded ΔprmpAΔiucA (p≤0.01), suggesting presence of additional pVir-virulence genes. Truncated pRmpA2 did not contribute to virulence. Odd ratios in the absence of pVir/yersiniabactin, pVir, pRmpA/aerobactin, pRmpA, aerobactin, yersiniabactin, and colibactin demonstrated a 250-fold, 67-fold, 20-fold, 16.7-fold, 9.6-fold, and 1.7-fold decrease in lethality respectively. These data can guide countermeasure development. This work was supported by NIH R21 AI123558-01 and 1R21AI141826-01A1 (Dr. Russo) and the Department of Veterans Affairs VA Merit Review (I01 BX004677-01) (Dr. Russo). This study was also partially funded by the U.S. Defense Health Program (DHP) Operations and Maintenance.

RNA interactome of hypervirulent Klebsiella pneumoniae reveals a small RNA inhibitor of capsular mucoviscosity and virulence

Hypervirulent Klebsiella pneumoniae (HvKP) is an emerging bacterial pathogen causing invasive infection in immune-competent humans. The hypervirulence is strongly linked to the overproduction of hypermucoviscous capsule, but the underlying regulatory mechanisms of hypermucoviscosity (HMV) have been elusive, especially at the post-transcriptional level mediated by small noncoding RNAs (sRNAs). Using a recently developed RNA interactome profiling approach iRIL-seq, we interrogate the Hfq-associated sRNA regulatory network and establish an intracellular RNA-RNA interactome in HvKP. Our data reveal numerous interactions between sRNAs and HMV-related mRNAs, and identify a plethora of sRNAs that repress or promote HMV. One of the strongest HMV repressors is ArcZ, which is activated by the catabolite regulator CRP and targets many HMV-related genes including mlaA and fbp. We discover that MlaA and its function in phospholipid transport is crucial for capsule retention and HMV, inactivation of which abolishes Klebsiella virulence in mice. ArcZ overexpression drastically reduces bacterial burden in mice and reduces HMV in multiple hypervirulent and carbapenem-resistant clinical isolates, indicating ArcZ is a potent RNA inhibitor of bacterial pneumonia with therapeutic potential. Our work unravels a novel CRP-ArcZ-MlaA regulatory circuit of HMV and provides mechanistic insights into the posttranscriptional virulence control in a superbug of global concern.

Characterization of a colistin resistant, hypervirulent hospital isolate of Acinetobacter courvalinii from Canada.

Non-baumannii Acinetobacter spp. are becoming more prevalent in clinical settings including those that present resistance to last-resort antibiotics such as colistin. AB222-IK40 is an Acinetobacter courvalinii strain isolated from the Ottawa Hospital Research Institute located in Ottawa, Canada. To our knowledge, it is the first report of clinical A. courvalinii in Canada. Based on the susceptibility profile, AB222-IK40 is resistant to colistin and non-susceptible to ertapenem. Whole-genome sequencing allowed for genomic investigation into colistin resistance mechanisms. No previously identified mechanism(s) were observed, but a mobile colistin resistance (mcr)-like gene and a UDP-glucose dehydrogenase gene were identified. Based on phylogenomic analyses, the mcr-like gene is an intrinsic phosphoethanolamine transferase. This gene family is implicated in one of the many mechanisms responsible for colistin resistance in Acinetobacter baumannii as well as Acinetobacter modestus. UDP-glucose dehydrogenase is involved in colistin resistance in Enterobacterales and has been shown to be involved in capsule formation in A. baumannii. Global lipidomics revealed greater abundance of phosphatidyl-myo-inositol and lyso-phosphatidyl ethanolamine moieties in the membrane of A. courvalinii than in A. baumannii. Lipidomic profiles showed differences that were probably responsible for the colistin resistance phenotype in AB222-IK40. This isolate was also hypervirulent based on survival assays in Galleria mellonella. As this is the first report of A. courvalinii from a hospital in Canada, this species may be an emerging clinical pathogen, and therefore, it is important to understand this mechanism of its colistin resistance and hypervirulence.

Microbiological and molecular genetic characteristics of Klebsiella pneumoniae isolates, extracted under conditions of cardiac surgery hospital

Klebsiella pneumoniae is one of the main causative agents of healthcare-associated infections (HAI) worldwide. The study was aimed at molecular characterization of beta-lactam antibiotic resistance in K. pneumoniae isolated in the cardiac surgery hospital, identify hypervirulent isolates and assess isolate genetic relatedness. K. pneumoniae isolated from the clinical material of patients (n = 50) at cardiac surgery departments as well as anesthesiology and reanimation department treated at the cardiovascular surgery hospital in Perm from July 2021 to December 2022 were analyzed. Bacteriological studies were performed on an automatic analyzer WalkAway-96Plus (Beckman Coulter, USA). The sensitivity to thirteen antimicrobials, production of extended-spectrum beta-lactamases (ESBL) and multidrug resistance (MDR) phenotype were assessed. The string test was used to screen hypermucoviscous K. pneumoniae. Isolate relatedness (ERIC-PCR), presence of beta-lactamase genes (blaTEM, blaCTX-M, blaSHV, blaOXA, blaKPC, blaVIM-2, blaIMP-1, blaNDM-1), as well as genes associated with hypervirulence (prmpA, iucA, peg-344, wzyK2, magA) were analyzed by PCR. Amplifications were performed in a DNA Engine Dyad Thermal Cycler (Bio-Rad, USA). Gel documentation system Gel-Doc XR (Bio-Rad, USA) was used for band visualization and data documentation. According to the study results, it was found that carbapenem-resistant isolates accounted for almost 76% of all isolates, the ESBL-phenotype had 90%, and MDR-phenotype — 88% of studied isolates. According to the string test results, 8% of K. pneumoniae isolates had a hypermucoviscous phenotype. As for genes associated with hypervirulence, it was shown that the iucA gene was the most common, amplified in 54% of isolates, prmpA was detected in 38% of isolates, 48% of isolates had wzyK2 or magA genes encoding capsular type K1/K2. Almost a third of K. pneumoniae samples contained a combination of the rmpA and iucA genes, and they were isolated more often in the group of patients with active infection. Only carbapenem-sensitive microorganisms had hypermucoviscous phenotype, K1 capsular type, and a combination of prmpA, iucA, peg-344 genes, whereas the K2 capsular type was most common among carbapenem-resistant K. pneumoniae. Based on molecular genetic typing, it was found that 24% of K. pneumoniae isolates were assigned to four genome groups of identical cultures, 76% of the isolates had individual profiles. Closely related isolates were found among patients from different departments that indicates a potential pathogen circulation in the closed circuit “surgery department–department of anesthesiology” and “reanimation–surgery department”.

Analysis of virulence profiles in clinical isolates of Klebsiella pneumoniae from renal abscesses: clinical significance of hypervirulent isolates.

Klebsiella pneumoniae can cause a wide range of infections. Hypervirulent K. pneumoniae (hvKp), particularly associated with the K1 and K2 capsular types, is an increasingly significant microorganism with the potential to cause invasive infections, including renal abscesses. Despite the rising prevalence of hvKp infections, information on renal abscesses caused by K. pneumoniae is limited, and the clinical significance of hvKp associated with specific virulence genes remains elusive. This study performed at a 1200-bed tertiary hospital sought to identify the clinical and microbiological characteristics of renal abscesses caused by K. pneumoniae, focusing on various virulence genes, including capsular serotypes and multilocus sequence typing (MLST). Over an 8-year period, 64 patients with suspected renal abscesses were reviewed. Ten patients diagnosed with K. pneumoniae-related renal abscesses were ultimately enrolled in the study. Among the isolates from the 10 patients, capsular serotype K2 was predominant (40.0%), followed by K1 (30.0%). The most common sequence type by MLST was 23 (40.0%). In particular, six patients (60.0%) harbored specific genes indicative of hvKp: iucA, peg-344, rmpA, and rmpA2. Our findings highlight the importance of hvKp as a pathogen in renal abscesses. Although the nature of hvKp is relatively unknown, it is widely recognized as a highly virulent pathogen that can infect relatively healthy individuals of various ages and simultaneously cause infections at multiple anatomical sites. Therefore, when treating patients with K. pneumoniae-related renal abscesses, caution is necessary when considering the characteristics of hvKp, such as potential bacteremia, multi-organ abscess formation, and metastatic spread.

Kinetic and proteomic studies in milk show distinct patterns among major Listeria monocytogenes clones

Listeria monocytogenes, a contaminant of raw milk, includes hypervirulent clonal complexes (CC) like CC1, CC4, and CC6, highly overrepresented in dairy products when compared to other food types. Whether their higher prevalence in dairy products is the consequence of a growth advantage in this food remains unknown. We examined growth kinetics of five L. monocytogenes isolates (CC1, CC4, CC6, CC9, and CC121) at 37 and 4 °C in ultra-high temperature (UHT) milk and raw milk. At 4 °C, hypovirulent CC9 and CC121 isolates exhibit better growth parameters in UHT milk compared to the hypervirulent CC1, CC4, and CC6 isolates. CC9 isolate in raw milk at 4 °C exhibited the fastest growth and the highest final concentrations. In contrast, hypervirulent isolates (CC1, CC4, and CC6) displayed better growth rates in UHT milk at 37 °C, the mammalian host temperature. Proteomic analysis of representative hyper- (CC1) and hypovirulent (CC9) isolates showed that they respond to milk cues differently with CC-specific traits. Proteins related to metabolism (such as LysA or different phosphotransferase systems), and stress response were upregulated in both isolates during growth in UHT milk. Our results show that there is a Listeria CC-specific and a Listeria CC-common response to the milk environment. These findings shed light on the overrepresentation of hypervirulent L. monocytogenes isolates in dairy products, suggesting that CC1 and CC4 overrepresentation in dairy products made of raw milk may arise from contamination during or after milking at the farm and discard an advantage of hypervirulent isolates in milk products when stored at refrigeration temperatures.

One-year surveillance for hypervirulent Klebsiella pneumoniae detected carbapenem-resistant superbugs.

Hypervirulent Klebsiella pneumoniae (hvKp) can cause infections in clinically healthy people, such as young and immunocompetent patients. Genes involved in the capsule synthesis or those encoding the siderophores have been adopted as predictors of hvKp. Certain sequence types, such as ST23 and ST86, have been associated with hvKp strains, too. The aim of this study was to investigate the presence of hvKp among 354 K. pneumoniae strains isolated from clinical samples of patients admitted to an Italian 900-bed hospital between 21 May 2021 and April 2022. All the isolates were screened by PCR for the amplification of virulence loci. Whole genome sequencing was performed in strains tested positive for at least one target gene. Thirteen out of 354 (3.7%) were hvKp. Five were wild type and belonged to the hypervirulent clones ST23, ST86, ST5, and ST375 and to the new clone ST6310. Six strains carried the blaKPC gene: three belonged to ST101, two to ST512, and one to ST395. Two isolates were ST147 and carried the blaNDM gene. Although hvKp isolation is not frequent, their presence should be systematically investigated to avoid the spreading of both virulent strains and strains with combined increase in virulence and resistance to antibiotics. PCR-based protocols are essential for surveillance of these strains, which do not always show a recognizable phenotype. Moreover, hvKp strains were isolated also from patients without history of recent foreign travels, indicating an increased spreading of these strains as well as an underestimated of their circulation so far.IMPORTANCEKlebsiella pneumoniae is a healthcare-associated pathogen frequently resistant to antibiotics. Hypervirulent strains of pneumoniae (hvKp) can spread from the primary site of infection to multiple sites causing life-threatening infections also in young otherwise healthy individuals. This study described the isolation of 13 isolates of K. pneumoniae with increased virulence in a large tertiary hospital over a 1-year period. Among them, eight strains were multidrug resistant and hypervirulent. Although these hypervirulent strains are still rare in Italy, their presence is particularly concerning since they can cause difficult-to-treat life-threatening infections. Moreover, not all the hypervirulent isolates were positive by the string test, so hvKp isolates were not always phenotypically detectable. Molecular biology techniques such as PCR amplification and next generation sequencing are therefore necessary for the detection of hvKp isolates, and surveillance programs exploiting molecular techniques are highly desirable.

Involvement of Tn3 transposon in formation and transmission of hypervirulent and carbapenem-resistant Klebsiella pneumoniae

ABSTRACT Hypervirulent and carbapenem-resistant Klebsiella pneumoniae poses a severe threat to public health for its high pathogenicity, transmissibility, and drug resistance. This study aims to explore the evolutionary path and the role of Tn3 transposon in the virulence and carbapenem resistance transmission of K. pneumoniae . Bioinformatics analysis and some experimental tests such as plasmid conjugation experiments, antimicrobial susceptibility testing, and virulence-associated tests were performed to explore the virulence and drug resistance transmission. The complete genome sequencing, S1 nuclease pulsed-field gel electrophoresis, and bioinformatics analysis were used to investigate their transmission mechanism mediated by Tn3 transposons. Three hypervirulent and carbapenem-resistant K. pneumoniae isolates, which were obtained from different patients at different time points in the same ward, harbored a virulence plasmid and a carbapenem resistance plasmid. They were confirmed to have first evolved from hypervirulent isolates and then acquired a bla KPC -positive plasmid (CR-hvKp evolutionary pattern). The non-conjugative virulence plasmid pVir could be transferred to other bacterial strains via mobilization by the conjugative IncN/U-type plasmid pKPC, as well as by fusing with the conjugative pKPC plasmid (mediated by Tn3-based homologous recombination) to be self-transmissible, thus transferring drug resistance and virulence. The cointegration, pVir/KPC fusion plasmid, was further resolved into pVir and pKPC between the duplicated copies of the Tn3 transposon resolution site mediated by site-specific recombination. Therefore, Tn3 transposons can mediate hypervirulent and carbapenem-resistant K. pneumoniae strains transferring drug resistance and virulence to other bacteria. We must be vigilant to emerging transposon-mediated hypervirulent and carbapenem-resistant pathogens. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae (CRKP) is resistant to most common antibiotics, becoming the most important and prevalent nosocomial opportunity pathogen. Besides, K. pneumoniae can also cause severe community-acquired infections, such as primary liver abscess and endophthalmitis. These pathogens are commonly referred to as hvKp. CRKP and hvKp have evolved separately, each occupying its own clonal lineage and exhibiting a variety of properties. Our study provides important insights into the evolutionary events related to the arousal of virulence and drug resistance in K. pneumoniae through plasmid transmission, mediated by Tn3 transposon. Our study also provides evidence that multiple mechanisms contribute to the successful transfer of non-conjugative virulence plasmid, and the involvement of transposons enhances the efficiency. A good knowledge of its transmission mechanisms is fundamental to finding effective strategies to combat these threatening pathogens. Transposons are widely present in bacteria, spreading resistance and virulence genes between the environment and humans. Therefore, emerging transposon-mediated hypervirulent and carbapenem-resistant pathogens should be highly valued.

Molecular Detection of Key Virulence-associated Genes and Phenotypic Analysis of Virulence Traits of Klebsiella pneumoniae Clinical Isolates from Kenya

Klebsiella pneumoniae is an opportunistic pathogen and a major cause of nosocomial infections. Phenotypic analysis of virulence and molecular typing of virulence-associated genes are powerful approaches to understanding Klebsiella pneumoniae infection biology. This study subjected 102 clinical Klebsiella pneumoniae isolates to virulence gene screening and phenotypic analysis of serum resistance, biofilm formation, and hypermucoviscosity. The virulence genes mrkD, ybtS, wcaJ, entD, and rmpA had a prevalence of 95.1%, 30.4%, 27.5%, 22.5%, and 0.98%, respectively. 54.9%, 36.3%, and 8.8% were serum resistant, intermediate, and susceptible, respectively. There was no significant correlation between the presence of mrkD, ybtS, wcaJ, entD, and rmpA genes and serum non-susceptibility. 53.9%, 22.5%, 6.9%, and 16.7% were strong, moderate, weak, and non-biofilm formers, respectively. The biofilm-forming phenotype was significantly correlated with mrkD (P= 0.000098) and ybtS (P=0.032) gene presence. In addition, 11.8 % of the isolates had the hypermucoviscous phenotype indicating hypervirulence. All of these hypervirulent isolates were positive for the mrkD gene and were significantly associated with the presence of the wcaJ gene (P = 0.000085). These results indicate a positive association between virulence genes with biofilm formation and hypervirulence. In conclusion, Klebsiella pneumoniae isolates circulating in Kenya are predominantly serum non-susceptible and biofilm formers. mrkD, ybtS, and wcaJ genes were identified as key genes influencing biofilm formation and hypervirulence and would be good targets for vaccine development to reduce the severity of Klebsiella pneumoniae infections in Kenya.

Clinical and Genomic Characterization of a Cohort of Patients With Klebsiella pneumoniae Bloodstream Infection.

The clinical and microbial factors associated with Klebsiella pneumoniae bloodstream infections (BSIs) are not well characterized. Prior studies have focused on highly resistant or hypervirulent isolates, limiting our understanding of K. pneumoniae strains that commonly cause BSI. We performed a record review and whole-genome sequencing to investigate the clinical characteristics, bacterial diversity, determinants of antimicrobial resistance, and risk factors for in-hospital death in a cohort of patients with K. pneumoniae BSI. We identified 562 patients at Massachusetts General Hospital with K. pneumoniae BSIs between 2016 and 2022. We collected data on comorbid conditions, infection source, clinical outcomes, and antibiotic resistance and performed whole-genome sequencing on 108 sequential BSI isolates from 2021 to 2022. Intra-abdominal infection was the most common source of infection accounting for 34% of all BSIs. A respiratory tract source accounted for 6% of BSIs but was associated with a higher in-hospital mortality rate (adjusted odds ratio, 5.4 [95% confidence interval, 2.2-12.8]; P < .001 for comparison with other sources). Resistance to the first antibiotic prescribed was also associated with a higher risk of death (adjusted odds ratio, 5.2 [95% confidence interval, 2.2-12.4]; P < .001). BSI isolates were genetically diverse, and no clusters of epidemiologically and genetically linked cases were observed. Virulence factors associated with invasiveness were observed at a low prevalence, although an unexpected association between O-antigen type and the source of infection was found. These observations demonstrate the versatility of K. pneumoniae as an opportunistic pathogen and highlight the need for new approaches for surveillance and the rapid identification of patients with invasive antimicrobial-resistant K. pneumoniae infection.

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.

Genomic Epidemiology of Carbapenem-Resistant Klebsiella in Qatar: Emergence and Dissemination of Hypervirulent Klebsiella pneumoniae Sequence Type 383 Strains.

The emergence of carbapenem-resistant, hypervirulent Klebsiella pneumoniae is a new threat to health care. We studied the molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae isolates in Qatar using whole-genome sequence data. We also characterized the prevalence and genetic basis of hypervirulent phenotypes and established the virulence potential using a Galleria mellonella model. Of 100 Klebsiella isolates studied, NDM and OXA-48 were the most common carbapenemases. Core genome single-nucleotide polymorphism (SNP) analysis indicated the presence of diverse sequence types and clonal lineages; isolates belonging to Klebsiella quasipneumoniae subsp. quasipneumoniae sequence type 196 (ST196) and ST1416 may be disseminated among several health care centers. Ten K. pneumoniae isolates carried rmpA and/or truncated rmpA2, and 2 isolates belonged to KL2, indicating low prevalence of classical hypervirulent isolates. Isolates carrying both carbapenem resistance and hypervirulence genes were confined mainly to ST231 and ST383 isolates. One ST383 isolate was further investigated by MinION sequencing, and the assembled genome indicated that blaNDM was located on an IncHI1B-type plasmid (pFQ61_ST383_NDM-5) which coharbored several virulence factors, including the regulator of the mucoid phenotype (rmpA), the regulator of mucoid phenotype 2 (rmpA2), and aerobactin (iucABCD and iutA), likely resulting from recombination events. Comparative genomics indicated that this hybrid plasmid may be present in two additional Qatari ST383 isolates. Carbapenem-resistant, hypervirulent K. pneumoniae ST383 isolates pose an emerging threat to global health due to their simultaneous hypervirulence and multidrug resistance.

Hypermucoviscosity Regulator RmpD Interacts with Wzc and Controls Capsular Polysaccharide Chain Length.

Klebsiella pneumoniae is a leading cause of nosocomial infections, including pneumonia, bacteremia, and urinary tract infections. Treatment options are increasingly restricted by the high prevalence of resistance to frontline antibiotics, including carbapenems, and the recently identified plasmid-conferred colistin resistance. The classical pathotype (cKp) is responsible for most of the nosocomial infections observed globally, and these isolates are often multidrug resistant. The hypervirulent pathotype (hvKp) is a primary pathogen capable of causing community-acquired infections in immunocompetent hosts. The hypermucoviscosity (HMV) phenotype is strongly associated with the increased virulence of hvKp isolates. Recent studies demonstrated that HMV requires capsule (CPS) synthesis and the small protein RmpD but is not dependent on the increased amount of capsule associated with hvKp. Here, we identified the structure of the capsular and extracellular polysaccharide isolated from hvKp strain KPPR1S (serotype K2) with and without RmpD. We found that the polymer repeat unit structure is the same in both strains and that it is identical to the K2 capsule. However, the chain length of CPS produced by strains expressing rmpD demonstrates more uniform length. This property was reconstituted in CPS from Escherichia coli isolates that possess the same CPS biosynthesis pathway as K. pneumoniae but naturally lack rmpD. Furthermore, we demonstrate that RmpD binds Wzc, a conserved capsule biosynthesis protein required for CPS polymerization and export. Based on these observations, we present a model for how the interaction of RmpD with Wzc could impact CPS chain length and HMV. IMPORTANCE Infections caused by Klebsiella pneumoniae continue to be a global public health threat; the treatment of these infections is complicated by the high frequency of multidrug resistance. K. pneumoniae produces a polysaccharide capsule required for virulence. Hypervirulent isolates also have a hypermucoviscous (HMV) phenotype that increases virulence, and we recently demonstrated that a horizontally acquired gene, rmpD, is required for HMV and hypervirulence but that the identity of the polymeric product(s) in HMV isolates is uncertain. Here, we demonstrate that RmpD regulates capsule chain length and interacts with Wzc, a part of the capsule polymerization and export machinery shared by many pathogens. We further show that RmpD confers HMV and regulates capsule chain length in a heterologous host (E. coli). As Wzc is a conserved protein found in many pathogens, it is possible that RmpD-mediated HMV and increased virulence may not be restricted to K. pneumoniae.

A panel of diverse Klebsiella pneumoniae clinical isolates for research and development.

Klebsiella pneumoniae are a leading cause of healthcare-associated infections worldwide. In particular, strains expressing extended-spectrum β-lactamases (ESBLs) and carbapenemases pose serious treatment challenges, leading the World Health Organization (WHO) to designate ESBL and carbapenem-resistant Enterobacteriaceae as 'critical' threats to human health. Research efforts to combat these pathogens can be supported by accessibility to diverse and clinically relevant isolates for testing novel therapeutics. Here, we describe a panel of 100 diverse K. pneumoniae isolates that are publicly available to assist the research community in this endeavour. Whole-genome sequencing (WGS) was performed on 3878 K. pneumoniae clinical isolates housed at the Multidrug-Resistant Organism Repository and Surveillance Network. The isolates were cultured from 63 facilities in 19 countries between 2001 and 2020. Core-genome multilocus sequence typing and high-resolution single-nucleotide polymorphism-based phylogenetic analyses captured the genetic diversity of the collection and were used to select the final panel of 100 isolates. In addition to known multidrug-resistant (MDR) pandemic lineages, the final panel includes hypervirulent lineages and isolates with specific and diverse resistance genes and virulence biomarkers. A broad range of antibiotic susceptibilities, ranging from pan-sensitive to extensively drug-resistant isolates, are described. The panel collection, and all associated metadata and genome sequences, are available at no additional cost and will be an important resource for the research community and for the design and development of novel antimicrobial agents and diagnostics against this important pathogen.

Investigation of Virulence Genes and Carbapenem Resistance Genes in Hypervirulent and Classical Isolates of Klebsiella pneumoniae Isolated from Various Clinical Specimens

Klebsiella pneumoniae is a global pathogen that can cause hospital-acquired and community-acquired infections and is known for its resistance to antibiotics. The pathotype, which is defined as hypervirulent K.pneumoniae (hvKp) is more lethal than classical K.pneumoniae (cKp) isolates and causes many community-acquired infections such as liver abscess, endophthalmitis, pneumonia in healthy young adults. There are no clear clinical or microbiological criteria to define hvKp. String test showing hypermucoviscosity and the iucA gene encoding aerobactin, a siderophore, were used to demonstrate hypervirulence. In this study, it was aimed to investigate the presence of various virulence genes and carbapenem resistance genes in the isolates of K.pneumoniae isolated from various clinical samples in our laboratory and classified as classical and hypervirulent by string test and also to detect the presence of various virulence and carbapenem resistance genes in hvKp isolates. Presence of four virulence genes (fimH-1, rmpA, magA, iucA), K1-K2 serotypes in all isolates and five carbapenem resistance genes (blaOXA-48, blaKPC, blaIMP, blaVIM, blaNDM-1) in carbapenem resistant isolates were investigated with polymerase chain reaction (PCR) method. Forty-five percent of the isolates were defined as hvKp and 55% as cKp. The fimH-1 gene was found to be positive in 94% of the isolates, the iucA gene in 37%, the magA gene (K1) in 34%, the rmpA gene in 5%, and the K2 serotype in 3% of the isolates. iucA gene was positive in 68.9% of hvKp isolates and 10.9% of cKp isolates, and the presence of iucA gene in hvKp isolates was statistically significant compared to cKp isolates (p<0.05). magA gene and K1 serotype were detected in 28.9% of hvKp isolates and 38.2% of cKp isolates. Although the magA gene ratio was high in cKp isolates, this difference was not statistically significant (p> 0.05). fimH-1 gene was found positive in 93.3% of hvKp isolates and 94.5% of cKp isolates. The rmpA gene was positive in 8.9% of hvKp isolates and 1.8% of cKp isolates. The K2 serotype was positive in 4.4% of hvKp isolates and 1.8% of cKp isolates. Although there was no statistical difference in antibiotic susceptibility between hvKp and cKp isolates; ceftriaxone, cefuroxime, ceftazidime, amikacin, cefoxitin, ertapenem, cefuroxime axetil were found to be more sensitive in hvKp isolates. Ciprofloxacin and trimethoprim sulfamethoxazole were found to be more sensitive in hvKp isolates than cKp isolates, and the difference was statistically significant (p<0.05). Although gentamicin, amoxicillin, piperacillintazobactam were not statistically significant in the cKp group, they were more sensitive than the hvKp group (p> 0.05). Carbapenem resistance were found to be 65.7% in cKp and 34.3% in hvKp isolates. Although not statistically significant, hvKp isolates were found to be more sensitive to carbapenems. The most common gene among 35 carbapenem resistant isolates was blaOXA-48 detected in 29 isolates. While the blaKPC gene was detected in five isolates, blaIMP, blaVIM and blaNDM-1 were not detected in any isolates. Sixty nine percent of blaOXA-48 positive samples were found to be in cKp isolates and 31% in hvKp isolates. It was determined that all of the blaKPC positive isolates were hvKp isolates. It was concluded that the string test and virulence factors alone would not be sufficient to show hypervirulence, and that more than one virulence factor combination should be shown in the presence of clinical features of hypervirulent infections to show hypervirulence.

Clostridioides difficile, a New "Superbug".

Clostridioides difficile is a Gram-positive, spore-forming, anaerobic bacterium. The clinical features of C. difficile infections (CDIs) can vary, ranging from the asymptomatic carriage and mild self-limiting diarrhoea to severe and sometimes fatal pseudomembranous colitis. C. difficile infections (CDIs) are associated with disruption of the gut microbiota caused by antimicrobial agents. The infections are predominantly hospital-acquired, but in the last decades, the CDI patterns have changed. Their prevalence increased, and the proportion of community-acquired CDIs has also increased. This can be associated with the appearance of hypervirulent epidemic isolates of ribotype 027. The COVID-19 pandemic and the associated antibiotic overuse could additionally change the patterns of infections. Treatment of CDIs is a challenge, with only three appropriate antibiotics for use. The wide distribution of C. difficile spores in hospital environments, chronic persistence in some individuals, especially children, and the recent detection of C. difficile in domestic pets can furthermore worsen the situation. "Superbugs" are microorganisms that are both highly virulent and resistant to antibiotics. The aim of this review article is to characterise C. difficile as a new member of the "superbug" family. Due to its worldwide spread, the lack of many treatment options and the high rates of both recurrence and mortality, C. difficile has emerged as a major concern for the healthcare system.