Fimbrial Gene Research Articles

Salmonella enterica subsp. diarizonae Harboring ST233, ST1263, and ST1845 in Children.

ObjectiveThis study aims to analyze the molecular epidemiology, resistance, and pathogenicity of Salmonella enterica subsp. diarizonae isolated from children.MethodsWhole genome sequencing was carried out, and molecular serotypes, sequence types, resistance genes, and virulence genes of S. enterica subsp. diarizonae isolates were analyzed. Antimicrobial susceptibility test was determined by commercialized microdilution method.ResultsA total of three isolates of S. enterica subsp. diarizonae were isolated during 2015 to 2020. The molecular serotypes of the three strains were 61:c:z35, 61:l,v:1,5,7:[z57], and 65:k:z, respectively, and the sequence types were ST1845, ST233, and ST1263. All the three isolates were susceptible to ceftriaxone, ceftazidime, cefepime, amoxycillin/clavulanic acid, piperacillin/tazobactam, ertapenem, imipenem, levofloxacin, and trimethoprim/sulfamethoxazole. No other resistant gene was detected except aac(6’)-Iaa. There were no resistant plasmids detected in all the three isolates. A total of 76 genes were present in all isolates, containing 49 genes of Type III Secretion System (T3SS) mediated by SPI-1and SPI-2, 13 genes of adherence (type 1 fimbriae, Agf, and MisL-related genes), 11 genes of iron uptake (Yersiniabactin), two genes of magnesium uptake, and one gene of typhoid toxin(cdtB).ConclusionThe serotypes and sequence types of S. enterica subsp. diarizonae isolates were rarely reported in children; all the S. enterica subsp. diarizonae isolates were susceptible to detected antibiotics; T3SS, adherence, iron uptake, magnesium uptake, and typhoid toxin were responsible for pathogenicity of the S. enterica subsp. diarizonae isolates in children.

Molecular epidemiology of Bordetella pertussis and analysis of vaccine antigen genes from clinical isolates from Shenzhen, China

BackgroundAlthough pertussis cases globally have been controlled through the Expanded Programme on Immunization (EPI), the incidence of pertussis has increased significantly in recent years, with a “resurgence” of pertussis occurring in developed countries with high immunization coverage. Attracted by its fast-developing economy, the population of Shenzhen has reached 14 million and has become one of the top five largest cities by population size in China. The incidence of pertussis here was about 2.02/100,000, far exceeding that of the whole province and the whole country (both < 1/100,000). There are increasing numbers of reports demonstrating variation in Bordetella pertussis antigens and genes, which may be associated with the increased incidence. Fifty strains of Bordetella pertussis isolated from 387 suspected cases were collected in Shenzhen in 2018 for genotypic and molecular epidemiological analysis.MethodsThere were 387 suspected cases of pertussis enrolled at surveillance sites in Shenzhen from June to August 2018. Nasopharyngeal swabs from suspected pertussis cases were collected for bacterial culture and the identity of putative Bordetella pertussis isolates was confirmed by real-time PCR. The immunization history of each patient was taken. The acellular pertussis vaccine (APV) antigen genes for pertussis toxin (ptxA, ptxC), pertactin (prn) and fimbriae (fim2 and fim3) together with the pertussis toxin promoter region (ptxP) were analyzed by second-generation sequencing. Genetic and phylogenetic analysis was performed using sequences publicly available from GenBank, National Institutes of Health, Bethesda, MD, USA (https://www.ncbi.nlm.nih.gov/genbank/). The antimicrobial susceptibility was test by Kirby-Bauer disk diffusion.ResultsFifty strains of Bordetella pertussis were successfully isolated from nasopharyngeal swabs of 387 suspected cases, with a positivity rate of 16.79%, including 28 males and 22 females, accounting for 56.0% and 44.0% respectively. Thirty-eight of the 50 (76%) patients were found to be positive for B. pertussis by culture. Among the positive cases with a history of vaccination, 30 of 42 (71.4%) cases had an incomplete pertussis vaccination history according to the national recommendation. Three phylogenetic groups (PG1-PG3) were identified each containing a predominant genotype. The two vaccines strains, CS and Tohama I, were distantly related to these three groups. Thirty-one out of fifty (62%) isolates belonged to genotype PG1, with the allelic profile prn2/ptxC2/ptxP3/ptxA1/fim3-1/fim2-1. Eighteen out of fifty (36%) isolates contained the A2047G mutation and were highly resistant to erythromycin, and all belonged to genotype PG3 (prn1/ptxA1/ptxP1/ptxC1/fim3-1/fim2-1), which is closely related to the recent epidemic strains found in northern China.ConclusionsThe positive rate of cases under one-year-old was significantly higher than that of other age groups and should be monitored. The dominant antigen genotypes of 50 Shenzhen isolates are closely related to the epidemic strains in the United States, Australia and many countries in Europe. Despite high rates of immunization with APV, epidemics of pertussis have recently occurred in these countries. Therefore, genomic analysis of circulating isolates of B. pertussis should be continued, for it will benefit the control of whooping cough and development of improved vaccines and therapeutic strategies.

Escherichia coli Causing Recurrent Urinary Tract Infections: Comparison to Non-Recurrent Isolates and Genomic Adaptation in Recurrent Infections.

Recurrent urinary tract infection (rUTI) remains a major problem for many women and therefore the pursuit for genomic and phenotypic traits which could define rUTI has been ongoing. The present study applied a genomic approach to investigate recurrent urinary tract infections by comparative analyses of recurrent and non-recurrent Escherichia coli isolates from general practice. From whole-genome sequencing data, phylogenetic clustering and genomic traits were studied on a collection of isolates which caused recurrent infection compared to non-recurrent isolates. In addition, genomic variation between the 1st and following infection was studied on a subset of the isolates. Evidence of limited adaptation between the recurrent infections based on single nucleotide polymorphism analyses with a range of 0–13 non-synonymous single nucleotide polymorphisms (SNPs) between the paired isolates. This included an overrepresentation of SNPs in metabolism genes. We identified several genes which were more common in rUTI isolates, including nine fimbrial genes, however, not significantly after false-discovery rate. Finally, the results show that recurrent isolates of the present dataset are not distinctive by variation in the core genome, and thus, did not cluster distinct from non-rUTI isolates in a SNP phylogeny.

Diagnostic Value of the Fimbriae Distribution Pattern in Localization of Urinary Tract Infection.

Urinary tract infections (UTIs) are one of the most common infectious diseases. UTIs are mainly caused by uropathogenic Escherichia coli (UPEC), and are either upper or lower according to the infection site. Fimbriae are necessary for UPEC to adhere to the host uroepithelium, and are abundant and diverse in UPEC strains. Although great progress has been made in determining the roles of different types of fimbriae in UPEC colonization, the contributions of multiple fimbriae to site-specific attachment also need to be considered. Therefore, the distribution patterns of 22 fimbrial genes in 90 UPEC strains from patients diagnosed with upper or lower UTIs were analyzed using PCR. The distribution patterns correlated with the infection sites, an XGBoost model with a mean accuracy of 83.33% and a mean area under the curve (AUC) of the receiver operating characteristic (ROC) of 0.92 demonstrated that fimbrial gene distribution patterns could predict the localization of upper and lower UTIs.

Frequency of k99, stx1, and stx2 Virulence Factors in Escherichia coli isolated from Diarrheic and Clinically Healthy Suckling Calves in Sistan and Baluchistan Province, Iran.

It is necessary to understand the frequency of virulence factor-encoding genes in the assessment of the carriage proportion. Moreover, it is required in the characterization of major unique antigens that are useful in the development of effective immunological-based preventive measures. The current study aimed to evaluate the frequency of three encoding-virulence genes associated with Enterotoxigenic (ET) and Shigatoxigenic Escherichia coli (E. coli/EC) pathotypes (k99, stx1, and stx2) in North of Sistan and Baluchistan Province, Iran. The frequency of k99, stx1, and stx2 was determined via polymerase chain reaction among E. coli isolates collected from the feces of the clinically healthy suckling (n=50) and diarrheic calves (n=50). The k99 gene was absent in all isolates, and the frequencies of the E. coli containing stx1 and stx2 or both stx1 and stx2 were estimated at 8%, 14%, and 4%, respectively, in the clinically healthy suckling calves (P&amp;gt;0.05), compared to 24%, 16%, and 6% in diarrheic animals (p &amp;lt;0.05). Among the three studied genes, there was a statistically significant difference between clinically healthy suckling and diarrheic calves in terms of the frequency of E. coli isolatescontaining stx1. On the other hand, the results of this study indicated that k99 was not a major fimbrial antigen-encoding gene in the ETECpopulation in the region. It is assumed that in any health measure intended to control the pathogen, other genes involved with encoding fimbriae should also be considered. The noticeable high frequency of E. coli isolates bearing stx1 and/or stx2 virulence elementsboth in clinically healthy and diarrheic suckling calves in this study isa concern for public health. Accordingly, it is recommended that further epidemiological studies be conducted on the role of the stx1 gene in the diarrhea of suckling calves in Sistan and Baluchistan Province, Iran.

An Investigation of the Male Genitourinary Abscess Originated from Urinary Tract in a Tertiary Hospital, Shanghai, China, from 2004 to 2019.

BackgroundMale genitourinary abscess is one of the serious complications of urinary tract infections (UTIs). There were few researches on the clinical and pathogenic characteristics of male genitourinary abscess.Patients and MethodsA retrospective observational study was conducted between January 2004 and April 2019. Male patients with genitourinary abscess originated from urinary tract, including sites of scrotum, testis, epididymis, spermatic cord, and prostate, were enrolled. Clinical and microbial records were collected and analyzed, and antimicrobial susceptibility testings were performed according to CLSI standard. Whole-genome sequencing was applied to detect the β-lactamase genes and virulence genes, as well as to determine the multilocus-sequence typing (MLST) of the collected Klebsiella pneumoniae (K. pneumoniae) isolates.ResultsA total of 22 male patients were included. The main clinical symptoms were fever (86.4%), scrotal swelling (68.2%), local skin warmth (59.1%) and ulceration (45.5%). Urinary irritation was often presented in prostate involved abscess. Ultrasound features had a 94.7% positive rate. Surgical treatment, including abscess drainage, was helpful to the prognosis. No matter where the specimens obtained from, including blood, urine or pus, multidrug-resistant K. pneumoniae was the dominant (11 cases, 50.0%) microorganism in positive cultures. Nine of eleven K. pneumoniae isolates had been preserved and recovered. As for MLST typing, all the nine available isolates of K. pneumoniae belonged to the ST11 type and characterized with blaKPC-2 carbapenemase gene. Virulence genes rmpA2, ybtS, kfuC, wzi, aerobactin genes (iucABCD and iutA) and type 3 fimbriae genes (mrkAD) were identified in all the K. pneumoniae isolates.ConclusionIt seemed that more patients under 35 years old were vulnerable to genitourinary abscess. There was an increasing trend that multidrug-resistant K. pneumoniae isolates with multiple virulence genes were involved in male genitourinary abscess. Prompt and proper antibiotic use, combined with adequate drainage of the abscess, was important to prognosis.

Role of long polar fimbriae type 1 and 2 in pathogenesis of mammary pathogenic Escherichia coli

Escherichia coli is a leading cause of bovine mastitis worldwide. The bacteria can rapidly grow in milk and elicit a strong lipopolysaccharide (LPS)/toll-like receptor-4 (TLR4)-dependent inflammatory response. Recently, the long polar fimbriae (LPF) were identified as a promising virulence factor candidate widely distributed in mammary pathogenic E. coli (MPEC) strains. Mammary pathogenic E. coli possess 2 lpf loci encoding LPF1 and LPF2, respectively. By deleting the major fimbrial subunit gene, lpfA, we found that both LPF1 and LPF2 contribute to MPEC adhesion, invasion, and biofilm formation in vitro. The lpf1A and lpf2A mutants showed reduced cytotoxicity in our in vitro cell infection model. Furthermore, we observed that LPF2 induced a mild TLR4-independent proinflammatory response. The median lethal dose (LD50) of both ∆lpf2A and ∆lpf1A∆lpf2A mutants to BALB/c mice increased by 0.38 and 0.15 logs, respectively, whereas that of wild-type strain MPJS13 was 8.69 logs. In contrast, LPF1 deficiency significantly enhanced the LPS/TLR4-mediated inflammatory response in mammary epithelial cells, and the LD50 of the mutant decreased to 8.18 logs. In conclusion, our data suggested that LPF are important in MPEC colonization of mammary cells and may provide a benefit to bacterial intracellular survival that induces persistent bovine mastitis.

Exacerbation of AMD Phenotype in Lasered CNV Murine Model by Dysbiotic Oral Pathogens.

Emerging evidence underscores an association between age-related macular degeneration (AMD) and periodontal disease (PD), yet the biological basis of this linkage and the specific role of oral dysbiosis caused by PD in AMD pathophysiology remains unclear. Furthermore, a simple reproducible model that emulates characteristics of both AMD and PD has been lacking. Hence, we established a novel AMD+PD murine model to decipher the potential role of oral infection (ligature-enhanced) with the keystone periodontal pathogen Porphyromonas gingivalis, in the progression of neovasculogenesis in a laser-induced choroidal-neovascularization (Li-CNV) mouse retina. By a combination of fundus photography, optical coherence tomography, and fluorescein angiography, we documented inflammatory drusen-like lesions, reduced retinal thickness, and increased vascular leakage in AMD+PD mice retinae. H&E further confirmed a significant reduction of retinal thickness and subretinal drusen-like deposits. Immunofluorescence microscopy revealed significant induction of choroidal/retinal vasculogenesis in AMD+PD mice. qPCR identified increased expression of oxidative-stress, angiogenesis, pro-inflammatory mediators, whereas antioxidants and anti-inflammatory genes in AMD+PD mice retinae were notably decreased. Through qPCR, we detected Pg and its fimbrial 16s-RrNA gene expression in the AMD+PD mice retinae. To sum-up, this is the first in vivo study signifying a role of periodontal infection in augmentation of AMD phenotype, with the aid of a pioneering AMD+PD murine model established in our laboratory.

Molecular characterization of KPC-2-producing Klebsiella pneumoniae ST258 isolated from bovine mastitis.

Bovine mastitis, an inflammation of the mammary gland of dairy cattle, is the most prevalent disease causing economically important losses, reduced milk production, early culling, veterinary expenses, and higher death rates. Bovine mastitis infections are the main cause for the use of antibiotics; however, the emergence of multidrug-resistant bacteria and the poor or nil response to antibiotics has become a critical global health problem. The goal of this study was the characterization of bacterial infections associated with clinical bovine mastitis. All the isolates were multidrug-resistant and were negative for the production of extended spectrum β-lactamases. However, all isolates were identified as carbapenemase-producing organisms by the Carba NP test. The carbapenemase identified was the product of the KPC-2 gene. The isolates were identified as Klebsiella pneumoniae and contained virulence genes for fimbriae, lipopolysaccharides, nitrogen starvation genes, and siderophores. Sixty-nine percent of the KPC-2-producing isolates had the same plasmid profile, although the genetic mobilization of resistance by bacterial conjugation was unsuccessful. The carbapenemase corresponded to the plasmid-borne KPC-2 gene identified by Southern blot hybridization. The assay showed a positive signal in the 90 kb (69% of the isolates), 165 kb (31% of the isolates), and 130 kb (6% of the isolates) plasmids. The IncFIIy and IncFIIk replicons were detected among these K. pneumoniae isolates. The PFGE and MLST analysis showed that all of the isolates are comprised by two clones (A and B) belonging to Sequence Type 258. This is the first report of K. pneumoniae producing carbapenemase KPC-2 isolated from bovine mastitis.

Targeting effect of berberine on type I fimbriae of Salmonella Typhimurium and its effective inhibition of biofilm.

As a primary cause of food contamination and human diseases, Salmonella Typhimurium can easily form a biofilm that is difficult to remove from food surfaces, and often causes significant invisible threats to food safety. Although berberine has been widely used as an anti-infective drug in traditional medicine, some basic principles underlying its mechanism, especially the interaction between berberine and type I fimbriae genes, has not been verified yet. In this study, two strains of major fimbrial gene mutants (ΔfimA and ΔfimH) were constructed to demonstrate the possible action of berberine on type I fimbriae genes. The broth microdilution method was used to determine the antibacterial activity of berberine against selected strains (WT, ΔfimA, and ΔfimH). Cell agglutination experiments revealed that the number of S. Typhimurium type I fimbriae reduced after berberine treatment, which was consistent with transmission electron microscopy results. Quantitative real-time PCR experiments also confirmed that berberine reduced fimA gene expression, indicating a certain interaction between berberine and fimA gene. Furthermore, confocal laser scanning microscopy imaging of biofilm clearly revealed that berberine prevents biofilm formation by reducing the number of type I fimbriae. Overall, it is well speculated for us that berberine could be an excellent combating-biofilm drug in clinical microbiology and food preservation. KEY POINTS: • Reduce the number of fimbriae. • Berberine targeting fimA. • Effective biofilm inhibitor.

Functional Insights From KpfR, a New Transcriptional Regulator of Fimbrial Expression That Is Crucial for Klebsiella pneumoniae Pathogenicity

Although originally known as an opportunistic pathogen, Klebsiella pneumoniae has been considered a worldwide health threat nowadays due to the emergence of hypervirulent and antibiotic-resistant strains capable of causing severe infections not only on immunocompromised patients but also on healthy individuals. Fimbriae is an essential virulence factor for K. pneumoniae, especially in urinary tract infections (UTIs), because it allows the pathogen to adhere and invade urothelial cells and to form biofilms on biotic and abiotic surfaces. The importance of fimbriae for K. pneumoniae pathogenicity is highlighted by the large number of fimbrial gene clusters on the bacterium genome, which requires a coordinated and finely adjusted system to control the synthesis of these structures. In this work, we describe KpfR as a new transcriptional repressor of fimbrial expression in K. pneumoniae and discuss its role in the bacterium pathogenicity. K. pneumoniae with disrupted kpfR gene exhibited a hyperfimbriated phenotype with enhanced biofilm formation and greater adhesion to and replication within epithelial host cells. Nonetheless, the mutant strain was attenuated for colonization of the bladder in a murine model of urinary tract infection. These results indicate that KpfR is an important transcriptional repressor that, by negatively controlling the expression of fimbriae, prevents K. pneumoniae from having a hyperfimbriated phenotype and from being recognized and eliminated by the host immune system.

Plasmids shape the diverse accessory resistomes of Escherichia coli ST131.

The human gut microbiome includes beneficial, commensal and pathogenic bacteria that possess antimicrobial resistance (AMR) genes and exchange these predominantly through conjugative plasmids. Escherichia coli is a significant component of the gastrointestinal microbiome and is typically non-pathogenic in this niche. In contrast, extra-intestinal pathogenic E. coli (ExPEC) including ST131 may occupy other environments like the urinary tract or bloodstream where they express genes enabling AMR and host cell adhesion like type 1 fimbriae. The extent to which commensal E. coli and uropathogenic ExPEC ST131 share AMR genes remains understudied at a genomic level, and we examined this here using a preterm infant resistome. We found that individual ST131 had small differences in AMR gene content relative to a larger shared resistome. Comparisons with a range of plasmids common in ST131 showed that AMR gene composition was driven by conjugation, recombination and mobile genetic elements. Plasmid pEK499 had extended regions in most ST131 Clade C isolates, and it had evidence of a co-evolutionary signal based on protein-level interactions with chromosomal gene products, as did pEK204 that had a type IV fimbrial pil operon. ST131 possessed extensive diversity of selective type 1, type IV, P and F17-like fimbriae genes that was highest in subclade C2. The structure and composition of AMR genes, plasmids and fimbriae vary widely in ST131 Clade C and this may mediate pathogenicity and infection outcomes.

A Cross-Sectional Cohort Study of Extended-Spectrum-Beta-Lactamase-Producing Enterobacterales in Patients with Traveler's Diarrhea.

Patients with traveler's diarrhea (TD) can acquire extended-spectrum-beta-lactamase (ESBL)-producing members of the Enterobacterales (EPE) during travel to areas of endemicity. The aim of the present study was to investigate the prevalence and characteristics of EPE carriage in travelers from southern Sweden who were sampled for bacterial diagnostics of TD compared to those of EPE carriage 10 years ago. Clinical samples sent for culture of common causes of bacterial enterocolitis, if the referral stated foreign travel, were included in the study. Antimicrobial susceptibility testing was done according to the EUCAST disk diffusion test method. EPE strains were subjected to whole-genome sequencing (WGS). Eighty-four of 303 patients carried a total of 92 ESBL-producing members of the Enterobacterales The overall prevalence of EPE in tested samples was thus 28%, compared to 24% 10 years earlier (P = 0.33). Among 86 strains available for WGS, 47 different sequence types (STs) were identified, and there were only 5 ST131 strains. Of the 79 Escherichia coli isolates, 76% carried at least one fim (type 1 fimbria) gene, 29% carried at least one pap (p-fimbriae) gene, and 43% were extraintestinal pathogenic E. coli (ExPEC) or uropathogenic E. coli (UPEC). Over half of the E. coli strains (57%) were intestinal pathogenic E. coli, most commonly enteroaggregative E. coli (EAEC) (33%), and enteroinvasive E. coli EIEC (22%). A relatively high proportion of patients with traveler's diarrhea carry EPE, but there was no significant increase compared to 10 years ago. Most E. coli strains were intestinal pathogenic strains. A comparatively high proportion of the strains were ExPEC/UPEC, many expressing the virulence genes pap and/or fim (This project was assigned ClinicalTrials.gov number NCT03866291.).

Analysis of the Correlation Between Antibiotic Resistance Patterns and Virulence Determinants in Pathogenic Klebsiella pneumoniae Isolates from Egypt.

Klebsiella pneumoniae is responsible for a plethora of infections involving multiple body systems. This study investigated K. pneumoniae clinical isolates for virulence-associated characters and antibiotic resistance. First, antibiotic sensitivity was determined for 40 K. pneumoniae clinical isolates. Some virulence and resistance-associated factors were studied phenotypically and genotypically. Multiple resistance profiles were observed (multidrug resistant [MDR; 42.5%], extensive drug resistant [XDR; 35%], and pandrug resistant [PDR; 5%]). Moreover, CTX-M-1, TEM, qnrS, and qnrA genes were detected in 70%, 30%, 60%, and 30% of selected isolates, respectively, and 40% of tested isolates were extended-spectrum β-lactamases (ESBLs) producers. Interestingly, all ESBLs producers harbored class 1 integrase gene (IntI1), while 60% of ESBLs producers harbored both CTX-M-1 and TEM. All tested isolates were capsulated while 87.5% were biofilm producers. Fimbriae were detected in 90% of tested isolates (all were biofilm producers and type 3 fimbriae adhesion gene [mrkD] positive). Sequence analysis of OXA-48, qnrS, and IntI1 revealed 100% identity with published sequences, while sequencing of qnrA, OmpK-35, and iron regulatory protein gene (irp2) showed minor variations in the form of one or few single-nucleotide polymorphism. Altogether, the current study revealed that all MDR, XDR, and PDR K. pneumoniae isolates were multivirulent and all harbored 3-5 virulence genes and 2-9 antimicrobial resistance genes and exhibited 8 and 10 different virulence and antimicrobial resistance profiles, respectively. In this study, we also report a positive correlation between some virulence genes and antimicrobial resistance genes among K. pneumoniae tested isolates.

Use of Fimbrial Rod for F18ab Fimbriae+ STEC Colonization to Host Cells.

Type 1 fimbriae are important virulence determinants of some Gram-negative pathogens, which promote bacterial colonization. The fimbrial rod is primarily composed of multiple copies of the major fimbrial subunit FimA. FimH adhesin, however, is present as a fibrillar tip structure that drive bacteria binding to host cellular mannose containing receptor. Here, we provide protocols to evaluate and compare the function of type 1 fimbrial subunits in F18ab fimbriae+ Shiga toxin-producing Escherichia coli (STEC). We found that both FimA and FimH are required for bacterial adhesion, invasion, and biofilm formation. Deleting fimA gene showed much more reduction in bacterial adhesion and invasion to porcine intestinal columnar epithelial cells IPEC-J2, than that of fimH mutant. Biofilm formation was significantly reduced in both mutants with an equal level. In addition, qPCR demonstrated that either fimA or fimH deletion down-regulated the bacterial flagella and F18 fimbriae genes expression, while up-regulated adhesin was involved in diffuse adherence-I (AIDA-I) gene expression, suggesting the co-regulation of cell surface-localized adhesins in F18ab fimbriae+ STEC.

Emergence of NDM-5-Producing Carbapenem-Resistant Klebsiella pneumoniae and SIM-Producing Hypervirulent Klebsiella pneumoniae Isolated from Aseptic Body Fluid in a Large Tertiary Hospital, 2017-2018: Genetic Traits of blaNDM-Like and blaSIM-Like Genes as Determined by NGS.

PurposeTo characterize the clinical, resistance, and virulence features of carbapenem-resistant Klebsiella pneumonaie (CRKP) and hypervirulent Klebsiella pneumoniae (hvKP) and also provide an effective selection of drug in CRKP and hvKP treatment.Materials and MethodsTwelve strains were collected and investigated these isolates for their antimicrobial susceptibility and molecular features. Resistance mechanisms, virulence-associated genes, multilocus sequence typing (MLST), and serotypes were detected by PCR and sequencing. Next general sequencing (NGS) was carried out to determine the features of carbapenem resistance and virulence. The synergistic activity of tigecycline–imipenem (TGC+IPM), tigecycline–meropenem (TGC+MEM), and tigecycline–aztreonam (TGC+ATM) combinations were performed by microdilution checkerboard method.ResultsEleven CRKP and one hvKP strains were collected. All strains showed highly sensitive rates to tigecycline (TGC) and amikacin (AMK). NDM (33.3%, 4/12) was the main resistance mechanism and MLST assigned 3 of them to ST11. CTX-M-producing (n = 1) and KPC-2-producing (n = 1) isolates belonged to ST147 and ST11, respectively. The MICs of ATM and quinolones in NDM-1 CRKP and NDM-5 CRKP strains were different. The serotype of the majority strains was KL22KL137 (58.3%, 7/12), hvKP stain belonged to K64. CRKP strains harbored plasmid-mediated quinolone resistance genes (oqxA, oqxB, qnrS, qnrB), β-lactams (blaCTX-M-3), aminoglycosides, type I and type III fimbriae genes, siderophore genes, and transporter and pumps. SIM-producing ST1764 K64 showed typical features of hvKP, showing hypermucoviscosity phenotype. The virulence genes, including rmpA2, alls and aerobactin genes, linked to hvKP, were found in ST1764 hvKP. hvKP was sensitive to quinolone; also, oqxA gene was detected. All TGC combinations showed highly synergistic effects and TGC+IPM was more effective treatment.ConclusionWe first identified the NDM-5-producing ST690 CRKP and SIM-producing ST1764 hvKP strains in Shanxi province. Tigecycline-carbapenem combinations were available treatments for CRKP.

Genetic adhesion profiles and adhesive variability of uropathogenic &lt;i&gt;Escherichia coli&lt;/i&gt; strains

Our study was aimed at investigating prevalence of adhesion genetic determinants among uropathogenic E. coli strains and assessing their correlation with level of specific and non-specific adhesion. E. coli bacterial cultures (n = 33) isolated from patients with urinary tract infection were examined. A phylogenetic group of strains was detected by using Clermont quadriplex-PCR method. Detection of fimbrial and afimbrial adhesin genes was carried out with end-point PCR. Level of erythrocyte-specific, non-specific hydrophobic and hydrophilic adhesion as well as biofilm formation were estimated by using standard methods. Adhesin genes were detected with the following frequencies: fimH — 75.76%, flu — 66.67%, iha — 39.40%, papC — 33.33%. Each of the genes sfaDE, upaG, afa/draBC and yqi was found with frequency 18.18%, whereas eaeA was not detected. Seven strains (21.21%) carried solely fimbrial adhesin genes, three strains (9.09%) — afimbrial adhesin genes, and twenty-one strains (63.64%) had genes of both adhesin types. Twenty-three individual adhesion genotypes were found among thirty-three UPEC strains. A combination of at least four genes were detected in 45.45% strains, among which 60% belonged to phylogroup B2. Odds ratio for adhesin gene prevalence in B2 group was calculated. It was shown that in B2 group yqi and sfaDE genes were detected by 14-fold more frequently (OR = 14.286) than in other phylogroups, and flu gene was observed at 10-fold higher rate (OR = 10.000). No correlation between such genes and level of adhesion to erythrocytes was found, whereas fimH+, papC+ and upaG+ strains had higher level of non-specific hydrophilic adhesion. It was shown that fimbrial adhesins accounted for bacterial adhesion and biofilm formation stronger than afimbrial ones. Thicker biofilm tended to form on latex catheter surface for strains with positive genetic profile for adhesin gene carriers.

Enzyme-assisted extraction of flavanones from citrus pomace: Obtention of natural compounds with anti-virulence and anti-adhesive effect against Salmonella enterica subsp. enterica serovar Typhimurium

Food poisoning has been recognized as a global challenge, causing illness in millions of people. Salmonella enterica subsp. enterica serovar Typhimurium is a major foodborne pathogen causing disease in humans by expressing virulence genes to invade the host epithelial cells. Thus, the search for natural compounds with anti-virulence and anti-adhesive effect could help to mitigate Salmonella infection. This study aimed to determine the effect of flavanones on S. Typhimurium virulence and adhesion to Caco-2 cells. Flavanones were obtained from citrus pomace (CP) by enzyme-assisted (CP-E) and conventional hydroalcoholic (CP–H) extraction methods, which produced extracts rich in aglycones and glycosylated flavanones, respectively. CP-E and aglycone standards (naringenin and hesperetin) decreased bacterial growth (25–90%), motility (50–90%), and adhesion to Caco-2 cells (25–50%). CP-H and glycosylated flavanones (naringin and hesperidin) affected only bacterial adhesion (decreased 20–75%). Agglutination assays demonstrated the binding of extracts and pure flavanones to S. Typhimurium adhesion structures. Fimbrial gene expression (fimA and fimZ) was downregulated in the presence of CP-H, CP-E, and aglycones. Both CP extracts decreased the expression of genes associated with cell invasion (hilA, ssrB and sopD). These results suggest that CP extracts and flavanones inhibit S. Typhimurium adhesion by interacting with fimbriae and flagella structures and downregulating fimbrial and virulence genes. CP-E and aglycone flavanones, rarely found in nature, exhibited superior inhibitory effects on bacterial viability, motility, adhesion and virulence gene expression than glycosylated flavanones. The outcomes of this research advance the understanding of how aglycone and glycosylated flavanones impact S. Typhimurium virulence activity and adhesion, contributing to their application as functional food ingredients for the prevention of adhesion, invasion, and the development of this pathogen in the gut.

Exposure to Moderate Glycosuria Induces Virulence of Group B Streptococcus.

To explore whether glycosuria induces virulence of uropathogens, in turn facilitating urinary tract infection (UTI), we exposed group B Streptococcus (GBS) strain 10/84 to human urine plain or with 300 mg/dL glucose (mimicking moderate glycosuria). Exposure to moderate glycosuria significantly augmented bacterial growth, kidney bacterial burden in a mouse model of ascending UTI, and virulence characteristics and expression of corresponding genes. Exposure to glycosuria increased GBS adherence to human bladder epithelial cell line and expression of corresponding PI2a fimbrial gene, antimicrobial peptide LL-37 resistance and bacterial surface charge modulating dltA, and GBS hemolytic ability and expression of genes encoding pore-forming toxins.

The sialidase inhibitor, DANA, reduces Porphyromonas gingivalis pathogenicity and exerts anti-inflammatory effects: An in vitro and in vivo experiment.

Sialidase has an important role in the pathogenesis of periodontitis and Porphyromonas gingivalis is a sialidase-producing organism implicated in periodontitis development. The aim of this study was to evaluate the anti-virulence and anti-inflammatory properties of the sialidase inhibitor, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA), in vitro and in vivo. The effects of DANA on P. gingivalis sialidase and cell viability were determined, and the effects of DANA on P. gingivalis virulence were evaluated by assessment of growth curves, cell morphology, biofilm formation, fimbriae gene expression, and gingipains and lipopolysaccharide (LPS) activity. Anti-inflammatory effects of DANA on LPS-induced macrophages were assessed by measurement of tumor necrosis factor-alpha (TNF-α), interleukin (IL-1β), inducible nitric oxide synthase (iNOS) secretions. The effect of DANA on P. gingivalis-induced periodontitis in rats was analyzed by radiography, stereoscopic microscopy, histopathology, and immunohistochemistry. Sialidase inhibition rate of 1mM DANA was 72.01%. Compared with untreated controls, treatment with DANA inhibited P. gingivalis growth and biofilm formation, and significantly decreased expression of the fimA, fimR, and fimS genes, as well as gingipains activity. DANA did not influence macrophage viability, but significantly inhibited TNF-α, IL-1β, and iNOS production in LPS-stimulated macrophages. In the periodontitis rat model, DANA prevented alveolar bone absorption and inhibited TNF-α and IL-1β production. DANA can reduce the growth, the biofilm formation and the virulence of P. gingivalis and exhibits anti-inflammatory effects, as well as effects against rat periodontitis, suggesting that DANA should be considered for development as a new adjunctive treatment for periodontitis.