IgA class-switched CD27-CD21+ B cells in IgA nephropathy.

BACKGROUND: IgA nephropathy (IgAN) is characterised by the production of galactose-deficient IgA1 (GdIgA1) antibodies. As the source of pathogenic antibodies, B cells are central to IgAN pathogenesis, but the B cell activation pathways as well as the potential B cell source of dysregulated IgA-secretion remain unknown. METHODS: We carried out flow cytometry analysis of peripheral blood B cells in patients with IgA nephropathy and control subjects with a focus on IgA-expressing B cells to uncover the pathways of B cell activation in IgAN and how these could give rise to pathogenic GdIgA1 antibodies. RESULTS: In addition to global changes in the B cell landscape - expansion of naive and reduction in memory B cells - IgAN patients present with an increased frequency of IgA-expressing B cells that lack the classical memory marker CD27, but are CD21pos. IgAN patients further have an expanded population of IgApos antibody-secreting cells, which correlate with serum IgA levels. Both IgApos plasmabalsts and CD27neg B cells co-express GdIgA1. Implicating dysregulation at mucosal surfaces as the driver of such B cell differentiation, we found a correlation between lipopolysaccharide (LPS) in the serum and IgAposCD27neg B cell frequency. CONCLUSION: We propose that dysregulated immunity in the mucosa may drive de novo B cell activation within germinal centres, giving rise to IgAposCD27neg B cells and subsequently IgA-producing plasmablasts. These data integrate B cells into the paradigm of IgAN pathogenesis and allow to further investigate this pathway to uncover biomarkers and develop therapeutic interventions.

eGFRcystatinC/eGFRcreatinine ratio < 0.6 in patients with SARS-CoV-2 pneumonia: a prospective cohort study.

BACKGROUND: Shrunken Pore Syndrome (SPS), defined as a reduced ratio between two estimated filtration rates (based on cystatin C and creatinine) is an increasingly recognized risk factor for long-term mortality. Although some patients with other conditions might be erroneously identified as SPS. Our aim was to bring the focus on possible pathophysiologic mechanisms influencing the ratio in the setting of SARS-CoV-2 pneumonia and acute kidney injury. METHODS: A single-centered prospective cohort study was conducted to investigate biomarkers in symptomatic COVID-19 pneumonia patients admitted to a hospital in Latvia. Nineteen biomarkers were measured in blood and three in urine samples. Associations were sought between these biomarkers, chronic diseases and the estimated GFRcystatinC/eGFRcreatinine ratio < 0.6, mortality rates, and acute kidney injury development. Data analysis was performed using SPSS Statistics, with significance set at p < 0.05. RESULTS: We included 59 patients (average age 65.5 years, 45.8% female) admitted with COVID-19. Acute kidney injury occurred in 27.1%, and 25.4% died. Ratio < 0.6 was seen in 38.6%, associated with female sex, diabetes, hypothyroidism, and higher age. Ratio < 0.6 group had mortality notably higher - 40.9% vs. 16.2% and more cases of acute kidney injury (40.9% vs. 18.9%). Cystatin C showed strong associations with the ratio < 0.6 compared to creatinine. Urea levels and urea/creatinine ratio were higher in the ratio < 0.6 group. After excluding acute kidney injury patients, ratio < 0.6 remained associated with higher cystatin C and urea levels. Other biomarkers linked to a kidney injury as NGAL, and proteinuria did not differ. CONCLUSION: We prove that reduced ratio is common in hospitalized patients with SARS-CoV-2 pneumonia and is associated with increased mortality during hospitalization. Factors that influence this ratio are complex and, in addition to the possible shrinkage of pores, other conditions such as thickening of glomerular basal membrane, comorbidities, prerenal kidney failure and others may play an important role and should be addressed when diagnosing SPS. We highlight the need for additional diagnostic criteria for SPS and larger studies to better understand its implications in acute COVID-19 settings.

Analysis of Microorganism Colonization, Biofilm Production, and Antibacterial Susceptibility in Recurrent Tonsillitis and Peritonsillar Abscess Patients.

Background: Despite the widespread use of antibiotics to treat infected tonsils, episodes of tonsillitis tend to recur and turn into recurrent tonsillitis (RT) or are complicated by peritonsillar abscesses (PTAs). The treatment of RT and PTAs remains surgical, and tonsillectomies are still relevant. Materials and methods: In a prospective, controlled study, we analyzed the bacteria of the tonsillar crypts of 99 patients with RT and 29 patients with a PTA. We performed the biofilm formation and antibacterial susceptibility testing of strains isolated from study patients. We compared the results obtained between patient groups with the aim to identify any differences that may contribute to ongoing symptoms of RT or that may play a role in developing PTAs. Results: The greatest diversity of microorganisms was found in patients with RT. Gram-positive bacteria were predominant in both groups. Candida species were predominant in patients with a PTA (48.3% of cases). Irrespective of patient group, the most commonly isolated pathogenic bacterium was S. aureus (in 33.3% of RT cases and in 24.14% of PTA cases). The most prevalent Gram-negative bacterium was K. pneumoniae (in 10.1% of RT cases and in 13.4% of PTA cases). At least one biofilm-producing strain was found in 37.4% of RT cases and in 27.6% of PTA cases. Moderate or strong biofilm producers were detected in 16 out of 37 cases of RT and in 2 out of 8 PTA cases. There was a statistically significant association found between the presence of Gram-positive bacteria and a biofilm-formation phenotype in the RT group and PTA group (Pearson χ2 test, p < 0.001). S. aureus and K. pneumoniae strains were sensitive to commonly used antibiotics. One S. aureus isolate was identified as MRSA. Conclusions: S. aureus is the most common pathogen isolated from patients with RT, and Candida spp. are the most common pathogens isolated from patients with a PTA. S. aureus isolates are susceptible to most antibiotics. Patients with RT more commonly have biofilm-producing strains, but patients with a PTA more commonly have biofilm non-producer strains. K. pneumoniae does not play a major role in biofilm production.

In Vitro Assessment of Biofilm Production, Antibacterial Resistance of Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter spp. Obtained from Tonsillar Crypts of Healthy Adults.

BACKGROUND AND OBJECTIVE: Tonsillar crypts can be considered a reservoir for a variety of bacterial species. Some bacterial species can be considered part of the normal oropharyngeal microbiota. The roles of other pathogens, for example, the so-called non-oral and respiratory pathogens Staphylococcus aureus, Klebsiella, Pseudomonas, and Acinetobacter spp., which have strong virulence factors, biofilm production capacity, and the ability to initiate infectious diseases, are unclear. The purpose of this study was to detect the presence of S. aureus, K. pneumoniae, P. aeruginosa, and Acinetobacter spp. within the tonsillar crypts of healthy individuals, and to analyze the pathogens' biofilm production and antibacterial resistances. RESULTS: Only common oropharyngeal microbiota were cultivated from 37 participant samples (40.7%). The most commonly isolated pathogenic bacterium was S. aureus, which was isolated in 41 (45%) participant samples. K. pneumoniae was isolated in seven (7.7%) samples, Acinetobacter spp. were isolated in five (5.5%) samples, and P. aeruginosa was isolated in two (2.2%) samples. Biofilm producers predominated among the pathogenic bacteria; 51 strains were biofilm producers, and among them, 31 strains were moderate or strong biofilm producers. The tested S. aureus, K. pneumoniae, P. aeruginosa, and Acinetobacter spp. strains were sensitive to commonly used antibiotics (amoxicillin-clavulanic acid, clindamycin, or ciprofloxacin). One of the isolated S. aureus strains was MRSA. CONCLUSIONS: Biofilm is a commonly observed feature that seems to be a naturally existing form of pathogenic bacteria colonizing human tissue. S. aureus, K. pneumoniae, P. aeruginosa, and Acinetobacter spp. occasionally occur in the tonsillar crypts of healthy individuals, and, therefore, it is most likely that S. aureus, K. pneumoniae, P. aeruginosa, and Acinetobacter spp. in opportunistic tonsillar infections originate from the tonsillar crypt microbiota.

Effect of Femtosecond Laser-Irradiated Titanium Plates on Enhanced Antibacterial Activity and Preservation of Bacteriophage Stability.

Titanium (Ti) is widely recognized for its exceptional properties and compatibility with medical applications. In our study, we successfully formed laser-induced periodic surface structures (LIPSS) on Ti plates with a periodicity of 520-740 nm and a height range of 150-250 nm. To investigate the morphology and chemical composition of these surfaces, we employed various techniques, including field emission scanning electron microscopy, energy dispersive X-ray spectroscopy, atomic force microscopy, X-ray photoelectron spectroscopy, and Raman spectroscopy. Additionally, we utilized a drop-shape analyzer to determine the wetting properties of the surfaces. To evaluate the antibacterial activity, we followed the ISO 22196:2011 standard, utilizing reference bacterial cultures of Gram-positive Staphylococcus aureus (ATCC 25923) and Gram-negative Escherichia coli (ATCC 25922). The results revealed enhanced antibacterial properties against Staphylococcus aureus by more than 99% and Escherichia coli by more than 80% in comparison with non-irradiated Ti. Furthermore, we conducted experiments using the Escherichia coli bacteriophage T4 (ATCC 11303-B4) and the bacterial host Escherichia coli (ATCC 11303) to investigate the impact of Ti plates on the stability of the bacteriophage. Overall, our findings highlight the potential of LIPSS on Ti plates for achieving enhanced antibacterial activity against common bacterial strains while maintaining the stability of bacteriophages.

Successful Bacteriophage-Antibiotic Combination Therapy against Multidrug-Resistant Pseudomonas aeruginosa Left Ventricular Assist Device Driveline Infection.

There is considerable interest in the use of bacteriophages (phages) to treat Pseudomonas aeruginosa infections associated with left ventricular assist devices (LVADs). These infections are often challenging to manage due to high rates of multidrug resistance and biofilm formation, which could potentially be overcome with the use of phages. We report a case of a 54-year-old man with relapsing multidrug-resistant P. aeruginosa LVAD driveline infection, who was treated with a combination of two lytic antipseudomonal phages administered intravenously and locally. Treatment was combined with LVAD driveline repositioning and systemic antibiotic administration, resulting in a successful outcome with clinical cure and eradication of the targeted bacteria. However, laboratory in vitro models showed that phages alone could not eradicate biofilms but could prevent biofilm formation. Phage-resistant bacterial strains evolved in biofilm models and showed decreased susceptibility to the phages used. Further studies are needed to understand the complexity of phage resistance and the interaction of phages and antibiotics. Our results indicate that the combination of phages, antibiotics, and surgical intervention can have great potential in treating LVAD-associated infections. More than 21 months post-treatment, our patient remains cured of the infection.

Effect of the Biopolymer Carrier on Staphylococcus aureus Bacteriophage Lytic Activity.

The use of implant materials is always associated with the risk of infection. Moreover, the effectiveness of antibiotics is reduced due to antibiotic-resistant pathogens. Thus, selecting the appropriate alternative antimicrobials for local delivery systems is correlated with successful infection management. We evaluated immobilization of the S. aureus specific bacteriophages in clinically recognized biopolymers, i.e., chitosan and alginate, to control the release profile of the antimicrobials. The high-titre S. aureus specific bacteriophages were prepared from commercial bacteriophage cocktails. The polymer mixtures with the propagated bacteriophages were then prepared. The stability of the S. aureus bacteriophages in the biopolymer solutions was assessed. In the case of chitosan, no plaques indicating the presence of the lytic bacteriophages were observed. The titre reduction of the S. aureus bacteriophages in the Na-alginate was below 1 log unit. Furthermore, the bacteriophages retained their lytic activity in the alginate after crosslinking with Ca2+ ions. The release of the lytic S. aureus bacteriophages from the Ca-alginate matrices in the TRIS-HCl buffer solution (pH 7.4 ± 0.2) was determined. After 72 h-0.292 ± 0.021% of bacteriophages from the Ca-alginate matrices were released. Thus, sustained release of the lytic S. aureus bacteriophages can be ensured.

Anti-Biofilm Effect of Bacteriophages and Antibiotics against Uropathogenic Escherichia coli.

Escherichia coli is a common cause of biofilm-associated urinary tract infections. Bacteria inside the biofilm are more resistant to antibiotics. Six E. coli strains isolated from patients with urinary tract infections were screened for biofilm-forming capability and antimicrobial susceptibility. Two of the most significant biofilm-producing strains were selected for minimal inhibitory concentration and minimal biofilm eradication concentration in vitro testing using amoxicillin-clavulanic acid, ciprofloxacin, and three commercial bacteriophage cocktails (Pyobacteriophag, Ses, and Intesti). In case of a low phage effect, an adaptation procedure was performed. Although the biofilms formed by strain 021UR were resistant to amoxicillin-clavulanic acid and ciprofloxacin, the three phage cocktails were able to reduce biofilm formation. In contrast, phages did not affect the 01206UR strain against planktonic and biofilm-forming cells. After Pyobacteriophag adaptation, the effect improved, and, regardless of the concentration, the adapted phage cocktail could destroy both planktonic cells and the biofilm of strain 01206UR. Bacteriophages capable of killing bacteria in biofilms can be used as an alternative to antibiotics. However, each case should be considered individually due to the lack of clinical trials for phage therapy. Antimicrobial and phage susceptibility should be determined in biofilm models before treatment to achieve the desired anti-biofilm effect.

Evaluation of Staphylococcus aureus Colonization in Adult Patients Undergoing Tonsillectomy for Recurrent Tonsillitis.

BACKGROUND AND OBJECTIVES: Staphylococcus aureus (S. aureus) is often recovered from the pharynx. However, the role of this pathogen in the etiology of tonsillar inflammation is still unclear and complicated due to frequent carriage of S. aureus. The aim of the study was to evaluate the frequency and the clinical importance of S. aureus colonization and biofilm production ability in patients with recurrent tonsillitis (RT) using patient samples from tonsillar crypts during tonsillectomy, and from the throat, nasal cavity, and armpits after tonsillectomy. MATERIALS AND METHODS: A case series study was carried out at a tertiary referral center among 16 patients diagnosed with RT who were undergoing tonsillectomy. Samples from tonsillar crypts were obtained during tonsillectomy, and samples from the throat, nasal cavity, and armpit were obtained a year after surgery. An evaluation of S. aureus incidence, biofilm formation, and antibacterial susceptibility was performed. RESULTS: During tonsillectomy, 16 strains of S. aureus were isolated from 16 patients, while 15/16 S. aureus strains were biofilm producers. A year after tonsillectomy, 8 S. aureus strains were isolated from 6 out of 16 patients, while 6/8 S. aureus strains were biofilm producers. After tonsillectomy, 3 patients showed S. aureus in throat culture. CONCLUSIONS: In 10/16 cases S. aureus was the causative agent of RT, in 4/16 cases patients had a predisposition to colonization of S. aureus, and in 2/16 cases S. aureus was a part of the patients` oral microbiome. Tonsillectomy results in less frequent isolation of S. aureus strains.

Use of Phage Cocktail BFC 1.10 in Combination With Ceftazidime-Avibactam in the Treatment of Multidrug-Resistant Pseudomonas aeruginosa Femur Osteomyelitis-A Case Report.

High-energy trauma with severe bone fractures can be complicated by infection, leading to the development of osteomyelitis. Pseudomonas aeruginosa is an important causative agent of such infections because of its high virulence profile and ability to develop resistance against a wide range of antimicrobials quickly. P. aeruginosa biofilms cause treatment failure and relapsing infections. Bacteriophages are viruses that can be used to treat biofilm-associated infections. Moreover, the combination of phages with certain antimicrobials have demonstrated synergistic and additive effects. We present a case of a 21-year-old patient with relapsing multidrug-resistant (MDR) P. aeruginosa femur osteomyelitis that developed after a road accident, with a proximal right femoral Grade III B open fracture and severe soft tissue damage. Despite extensive antimicrobial treatment and multiple surgical interventions with wound debridement, the infection persisted, with subsequent development of femoral osteomyelitis with a fistula. Patient care management included femoral head excision with wound debridement, intravenous (IV) ceftazidime-avibactam, and the local application of the lytic Pseudomonas bacteriophage cocktail BFC 1.10. Nine months after the intervention, the patient did not show any clinical, radiological, or laboratory signs of inflammation; therefore, hip replacement was performed. Nevertheless, recurrent P. aeruginosa infection evolved at the distal side of the femur and was successfully treated with conventional antimicrobials. In this case, wound debridement combined with antibiotics and bacteriophages resulted in bacterial eradication of proximal femoral segment, avoiding leg amputation, but failed to treat osteomyelitis in distal bone segment. An in vitro assessment of the isolated MDR P. aeruginosa strain for biofilm formation and phage susceptibility was performed. Additionally, the antimicrobial effects of ceftazidime-avibactam and BFC 1.10 were determined on planktonic cell growth and bacterial biofilm prevention was evaluated. The isolated bacterial strains were susceptible to the bacteriophage cocktail. Strong biofilm formation was detected 6 h after inoculation. Ceftazidime-avibactam combined with BFC 1.10 was most effective in preventing planktonic cell growth and biofilm formation. In both cases, the required concentration of ceftazidime-avibactam decreased two-fold. This study demonstrates the possible use of bacteriophages and antibiotics in difficult-to-treat bone and soft tissue infections, where the additive effects of phages and antibiotics were observed.

Leucine-Rich Alpha-2-Glycoprotein (LRG-1) as a Potential Kidney Injury Marker in Kidney Transplant Recipients.

BACKGROUND Kidney transplantation is the treatment of choice for most patients with end-stage renal disease. To improve patient and transplant survival, non-invasive diagnostic methods for different pathologies are important. Leucine-rich alpha-2-glycoprotein (LRG-1) is an innovative biomarker that is elevated in cases of angiogenesis, inflammation, and kidney injury. However, there are limited data about the diagnostic role of LRG-1 in kidney transplant recipients. The aim of this study was to evaluate the association between serum LRG-1, urine LRG-1, and kidney transplant function and injury. MATERIAL AND METHODS We enrolled 35 kidney transplant recipients in the study. LRG-1 in the serum and urine was detected using ELISA. We evaluated the correlation of serum and urine LRG-1 with traditional serum and urine kidney injury markers. RESULTS A higher level of serum LRG-1 correlates with a higher level of urine LRG-1. Serum LRG-1 has a positive correlation with transplant age, serum urea, serum creatinine, serum cystatin C, proteinuria, and fractional excretion of sodium (FENa) and a negative correlation with hemoglobin and estimated glomerular filtration rate (eGFR). Urine LRG-1 has a positive correlation with serum cystatin C, proteinuria, and urine neutrophil gelatinase-associated lipocalin (NGAL). CONCLUSIONS Higher levels of serum and urine LRG-1 are associated with kidney transplant injury and functional deterioration. Thus, LRG-1 might be also as a biomarker for tubular dysfunction in patients after kidney transplantation.

S. aureus Colonization, Biofilm Production, and Phage Susceptibility in Peritoneal Dialysis Patients.

Peritonitis caused by Staphylococcusaureus is of major importance in peritoneal dialysis (PD) patients due to its great virulence profile and biofilm formation ability. Bacteriophages are a potential tool to treat peritonitis resulting from biofilm-associated infections. We screened S. aureus colonization in 71 PD patients from the nasal cavity, groin, and PD exit-site regions and analyzed clinical outcomes in these patients. We performed biofilm-formation testing of different strains and compared the isolates of one patient to detect phenotypic differences in S. aureus. Phage cocktails were used to detect S. aureus in vitro susceptibility. An adaptation procedure was performed in cases of bacterial resistance. Around 30% of PD patients (n = 21) were found to be S. aureus carriers; from these, a total of 34 S. aureus strains were isolated, of which 61.8% (n = 21) produced a strong biofilm. Phenotypic differences in strain biofilm production were detected in eight patients out of ten. All strains were sensitive to commonly used antibiotics. Broadly positive phage lytic activity (100%) was observed in six cocktails out of seven, and bacterial resistance towards phages was overcome using adaptation. Overall phages showed a promising in vitro effect in biofilm-forming S. aureus strains.