Genomic Study on Blood Culture Isolates From Patients With Staphylococcus Infection-associated Glomerulonephritis

Abstract

Introduction: Staphylococcus infection-associated glomerulonephritis (SAGN), is an autoimmune sequela of infection affecting a subset of infected patients without specific predictive factors, frequently presenting with acute nephritic syndrome and propensity for chronic kidney disease. We performed a comparative genotypic and phenotypic analysis of S. aureus isolates from patients that did and those that did not develop SAGN.

Methods: We had 22 culture-proven cases of SAGN from Ohio State University Wexner Medical Center (OSUWMC) from 2004 to 2016, 9 of 22 being blood cultures, with archived isolates. These, along with blood culture isolates from 12 patients with no clinical evidence of SAGN (between ages 40 to 80 years) over the same period were used for genotyping. For host demographic comparison, we used all available SAGN cases (n = 85, including those with positive cultures other than blood; and patients with kidney biopsies received from referring hospitals) and all OSUWMC patients with positive Staphylococcus cultures without glomerulonephritis (GN) (n = 23,496).

Results: Multiple sequence types (STs) suggesting strain diversity was seen in the GN isolates with mainly clonal complexes (CC) 5 and 59. Mutations in the agr operon were identified in significantly higher number of the GN isolates (83%) than non-GN isolates (16%). Significant differences in β-hemolysis and biofilm formation was also observed between the groups.

Conclusion: The functionality of these agr mutants remains to be seen, but the presently known effects of reduced agr function, namely increased surface adhesins, biofilm formation, and persistent bacteremia could be important microbial factors predisposing to SAGN and testing for them early during infection could help to predict its development.

Keywords: MRSA; SAGN; bacterial isolates; biofilm assay; hemolysis; whole genome sequencing.

Graphical abstract

Figure 1

Flowchart showing total number of patients with and without GN and those available for genotyping studies. GN, glomerulonephritis; SAGN, Staphylococcus infection-associated glomerulonephritis.

Figure 2

(a) Age distribution by GN status. Overall distribution is similar for patients with and without glomerulonephritis, showing a higher prevalence of Staphylococcal infections in older adults. (b) Sex distribution by GN status. GN, glomerulonephritis.

Figure 3

Comparison of laboratory parameters between GN (n = 22) and non-GN (n = 96) groups. (a) The median serum creatinine values were higher in the patients with GN as compared with patients without GN at both time points. There was an increase in median serum creatinine level by 0.3 mg/dl in the GN group, indicating a worsening renal function in these patients, but these were not statistically significant. (b) Total white blood cell counts, (c) differential lymphocyte, and (d) differential neutrophil counts did not show significant differences between the 2 groups at either of the 2 time points. GN, glomerulonephritis.

Figure 4

(a) Antimicrobial resistance genes were identified by K-mer search or BLAT from sources, including PATRIC, CARD, and NDARO. Red indicates the gene was detectable in the sample, blue indicates no gene was detected. (b) Virulence factors were identified by BLAT from sources VFDB or Victors. Genes present in all samples were filtered out since they were not informative. Red indicates the gene was detectable in the sample, blue indicates no gene was detected. VFDB, virulence factor database.

Figure 5

Reduced hemolytic activity in isolates from patients with GN. (a) Staphyloccus aureus isolates were spotted on 5% sheep blood agar to measure a zone of clearing due to lysis of erythrocytes. The cleared zone was measured along with the diameter of the spotted S. aureus colony to calculate hemolysis activity. Results obtained are from 3 independent experiments performed in triplicates. The data are represented as box and whiskers plot indicating the minimum and maximum values of hemolysis activity. (One-way ANOVA analysis, ∗∗∗∗P < 0.0001). (b) Grouping averages of hemolytic activity of isolates from GN+ and GN- patients resulted in decreased hemolytic activity in isolates from GN+ patients compared with GN−. Each data point represents the average of results obtained from 3 independent experiment perfomed in triplicates. Data are presented as mean ± SD. (∗P < 0.05. Unpaired t test with Welch’s correction). ANOVA, analysis of variance; GN, glomerulonephritis.

Figure 6

Comparative analyses on hemolysis, biofilm formation, and PNAG production between GN+ and GN− isolates within CC5 clonal complex. Data obtained from in vitro (a) hemolysis, (c) biofilm, and (e) PNAG production assays were grouped as isolates from GN- (gray) and GN+ (red) within CC5. The averages for each group were taken for comparison. On average, isolates from GN+ group within CC5 exhibited (b) decreased hemolytic activity and (d) increased biofilm production, while there was no significant difference in (f) PNAG production. Results represent an average of 3 independent experiments performed in triplicates. Data are presented as mean ± SD. (∗∗P < 0.01, P ∗∗∗<0.001. Unpaired t test with Welch’s correction). CC, clonal complex; GN, glomerulonephritis; ns, not significant; PNAG, Poly-N-acetyl-β-(1-6)-glucosamine; SAGN, Staphylococcus infection-associated glomerulonephritis.

Figure 7

Comparative analyses on hemolysis, biofilm formation, and PNAG production between GN+ and GN− isolates within CC59 clonal complex. Data obtained from in vitro (a) hemolysis, (c) biofilm, and (e) PNAG production assays were grouped as isolates from GN- (gray) and GN+ (red) within CC5. The averages for each group were taken for comparison. On average, isolates from GN+ group within CC5 exhibited (b) decreased hemolytic activity, while there was no significant difference in (d) biofilm and (f) PNAG production. Results represent an average of 3 independent experiments performed in triplicates. Data are presented as mean ± SD. (∗∗P < 0.01. Unpaired t test with Welch’s correction). CC, clonal complex; GN, glomerulonephritis; ns, not significant; PNAG, Poly-N-acetyl-β-(1-6)-glucosamine; SAGN, Staphylococcus infection-associated glomerulonephritis.