Comparative study of bacterial strains and antibiotic susceptibility tests between chronic tonsillitis patients with IgA nephropathy and without nephritis

Abstract

Aim: The purpose of the present study was to detect bacterial strains and antibiotic susceptibility in chronic tonsillitis patients with IgA nephropathy (IgAN) and without nephritis, in order to provide evidence for clinical therapy and pathogenesis of IgAN. Methods: A total of 53 patients with IgAN (group A) and 53 chronic tonsillitis patients without nephritis (group B) underwent tonsillectomy. The tonsil tissues of patients were collected under sterile condition. The bacteria in the tonsil crypt of patients in both groups were isolated and identified for antibiotic susceptibility test by the manual routine of the laboratory and also with the autoScan/Microscan system. Results: There were bacteria in each specimen in both groups. The bacteria detection rate was 100%, but there was no significant difference between two groups (p > 0.05). The 522 strains of bacteria in group A and 494 strains of bacteria in group B were isolated. Streptococcus. Neisseria, Hemophilus parainfluenzae. Staphylococcus. Bacillus proteus and Streptococcus pneumoniae were detected in both groups, but there was no significant difference in the types of bacteria between the two groups (all p > 0.05). Alpha streptococcus was the most common in both groups. The antibiotic susceptibility test showed that there was no significant difference in the susceptibility to penicillin, chloramphenicol, macrolides, cephalosporin, gentamicin, amikacin sulphate, norfloxacin, ciprofloxacin, rifampicin and vancomycin between two groups (all p > 0.05). Conclusions: Alpha streptococcus in both two groups can be detected and is the most common. There was no significant difference in bacterial strains and antibiotic susceptibility between two groups.

• Antibiotic susceptibility
• bacterial strains
• IgAN
• tonsillitis

Introduction

IgA nephropathy (IgAN) is characterized by the accumulation of IgA deposits, predominantly in the glomerular mesangium,1 and represents the most common form of glomerulonephritis. Previous research indicates that the tonsils are closely related to IgAN.2 The frequent occurrence of tonsillitis or upper respiratory tract infections exacerbates hematuria or urinary findings were deteriorated in IgAN. Urinary findings were deteriorated after tonsil stimulation in patients with IgAN. Tonsillectomy can improve the urinary findings; keeps stable renal function, improve mesangial proliferation, decrease IgA deposits and have a favorable effect on long-term renal survival in some IgAN patients. Polymeric IgA deposited in glomerular mesangium were at least in part of tonsillar origin. Serum IgA concentrations are significantly decreased after tonsillectomy in patients with IgAN.3–5 It has been reported that IgA-positive plasma cells are increased in the palatine tonsils of patients with IgAN6,7 and IgA produced by tonsillar B lymphocytes binds to the glomerular mesangium in IgAN.8 Some patients with recurrent chronic tonsillitis do not suffer from renal disease. However, other patients do suffer from IgAN along with recurrent chronic tonsillitis. We therefore hypothesize that the pathogenesis of IgAN may be relevant to the type of bacteria in tonsils. The purpose of the present study was to detect bacterial strains and antibiotic susceptibility in chronic tonsillitis patients with IgAN and without nephritis, in order to provide evidence for clinical therapy and pathogenesis of IgAN.

Materials and methods

Study population

Fifty-three IgAN patients (group A; 18 males and 35 females, mean age 27.8 ± 4.3 years, range 16–49 years) and 53 controls without any renal diseases (group B; 25 males and 28 females, mean age 25.9 ± 6.5 years, range 11–45 years), who were enrolled from the Second Xiangya Hospital of Central South University from September 2005 to May 2011, participated in this study. All IgAN patients were identified through immuno histopathological examination of the subjects’ renal biopsy specimens. The histological diagnoses were confirmed by the granular deposition of predominant IgA mainly in the glomerular mesangium by immunofluorescence detection. We excluded patients with systemic lupus erythematosus, Henoch–Schönlein purpura and hepatic diseases, by means of clinical history, physical examination and laboratory test results. All control patients with chronic tonsillitis did not have hematuria and proteinuria. All patients had recurrent tonsillitis history. All patients, however, had normal renal function. All patients did not undergo treatment with steroid, immunosuppressive agents, angiotensin-converting enzyme (ACE) inhibitor, or AT1 receptor blockers.

Tonsillectomy

Patients were not allowed to use antibiotics 14 days before tonsillectomy. Tonsillectomy was performed under general anesthesia in the Department of Otorhinolaryngology, in the Second Xiangya Hospital of Central South University. After acquiring informed consent, we collected tonsillar tissues under sterile conditions for the following analyses. The study protocol was carried out in accordance with “Ethical Principles for Medical Research Involving Human Subjects” (World Medical Association Declaration of Helsinki, 2004) and approved by the ethics review committee of the Second Xiangya Hospital of Central South University and the Hunan Government Medical Research Council.

Isolation, evaluation of tonsillar bacterial strains, and the antibiotic susceptibility tests of bacteria

The identification and antibiotic susceptibility testing of bacteria, isolated from the tonsillar crypts of all study subjects, were done by the manual routine of the laboratory and also with the autoScan/Microscan system9 in the Bacterial Central Laboratory of the Second Xiangya Hospital, Central South University. These samples were cultured and the isolated bacteria were identified. Antibiotic susceptibility tests were performed on every bacterium. Collected samples of tonsillar crypts (cut surface in deep), bilateral tonsils with a microliter platinum loop and spreading it on each of the three culture media used (blood-Agar, chocolate-Agar supplemented with polivitex and MacConkey-Agar) under sterile conditions. We use one plate for each patient, with a tonsillar crypts sample on each half-plate. The media were incubated for 48 h; to the chocolate-Agar 10% of CO₂ was added. The identification of the isolated bacteria was made following the procedures described in the Manual of Clinical Microbiology.10 The antibiotic susceptibility tests were carried out in accordance with the National Committee for Clinical Laboratory Standard (NCCLS), using three antibiotics profiles adapted to the current general bacteria resistance, as follows: for rapid growing and non-exigent bacteria, combined Dade-Behring panels were used, which included the identification tests, (for Staphylococci and Enterococci, panel PC23; for Gram-negative rods, panel PC38, substituted by PUC37 in July, for including the cefuroxime test). These panels were automatically read and recorded in the AutoScan system.9 For the more exigent bacteria sensibility tests, the Kirby–Bauer disc-diffusion technique was used; the results of these tests were recorded in the AutoScan database. Statistical calculations were carried out with the Epidat program, 3.1 version.

Statistical analysis

The results are expressed as the mean ± SD. Statistical significance of differences between the two groups was determined by means of the χ² tests. Statistic significance was considered to be a p value <0.05.

Results

Bacterial detection rate

There were bacteria in each specimen in both groups. The bacterial detection rate was 100%, but there was no significant difference between two groups (p > 0.05).

Bacterial strains

The 522 strains of bacteria in IgAN cases and 494 strains of bacteria in controls were isolated. There was no significant difference between two groups (p > 0.05). Streptococcus. Neisseria. Hemophilus parainfluenzae. Staphylococcus. Bacillus proteus and Streptococcus pneumoniae were detected in both groups, but there was no significant difference in the types of bacteria between the two groups (all p > 0.05; Table 1). Alpha streptococcus was detected in both groups and was the most common.

Table 1. Bacterial strains in tonsillar crypts.

	Alpha hemolytic streptococcus	Neisseria	Haemophilus parainfluenzae	Proteus	Beta hemolysis streptococcus	Gamma-hemolysis streptococcus	Pneumococcal	Staphylococcus
IgAN cases	215*	136*	44*	39*	38*	21*	17*	12*
Controls	178	154	36	31	30	24	25	16

Notes: There was no significant difference in IgAN patients compared with controls.

*p > 0.05.

Antibiotic susceptibility testing for bacteria

Results of antibiotic susceptibility test for bacteria are given in Table 2. The antibiotic susceptibility test showed that there was no significant difference in the susceptibility to penicillin, chloramphenicol, macrolides, cephalosporin, gentamicin, amikacin sulphate, norfloxacin, ciprofloxacin, rifampicin, and vancomycin between two groups (IgAN cases and controls; all p > 0.05).

Table 2. Bacterial sensitive strains to commonly used drugs.

	Penicillin	Chloramphenicol	Macrolides	I,II cephalosporin	III,IV cephalosporin	Gentamycin	Amikacin	Norfloxacin	Ciprofloxacin	Rifampicin	Vancomycin
IgAN cases	292*	404*	298*	413*	387*	254*	476*	245*	465*	383*	375*
Controls	305	371	257	410	382	213	438	223	432	342	381

Notes: There was no significant difference in IgAN patients compared with controls.

*p > 0.05.

Discussion

Kocaturk et al.11 separated and identified bacteria in tonsils from 23 cases of chronic patients with tonsillitis and found that staphylococcus aureus was the most common bacterial strain isolated from the tonsillar crypts; Ling12 separated and identified bacteria in the tonsils of 42 cases of surgical tonsillectomy, also found that staphylococcus aureus was the most common bacterial strain isolated from tonsillar crypts; However, Liang13 separated and identified bacteria in tonsils of 42 cases of surgical tonsillectomy, found that streptococcus pneumoniae was the most common bacterial strain isolated from the tonsillar crypts; Wei14 separated and identified bacteria in the tonsils of 44 cases of children with surgical tonsillectomy, showing that alpha streptococcus was the most common bacterial strain isolated from tonsillar crypts, and were resistant to commonly used antibiotics. Rekola et al.15 reported that 33% of the IgAN patients had different groups of beta-hemolytic streptococci isolated from their throats and beta-hemolytic streptococcus was the most common bacterium in tonsils or throat. The type of the bacteria separated may be related to region or bacterial resistance. Compared to our previous experiment,16 it is also concluded that the results were the same with increased sample number. The bacteria spectrum in the two groups was not statistically different. In this study, we found that alpha streptococcus in two groups was detected and it was the most common bacterial strain isolated from the tonsillar crypts. Alpha streptococcus is a conditional pathogen that may be related to drug resistance. When antimicrobial agents are blindly used to treat chronic tonsillitis, the growth of strains susceptible to antimicrobial agents is restrained, and resistant strains are free to survive and breed bountifully in the environment for prolonged periods of time. It was said that chronic tonsillitis is endogenous infection. That is to say, pathogenic bacteria are from the normal oropharyngeal flora. As tonsillitis is induced by mixed infection, antimicrobial agents should be blindly used to treat chronic tonsillitis. Bacteria were sensitive to vancomycin, amikacin ciprofloxacin, cephalosporin, Chloromycetin, but widespread resisted to penicillin, macrolides, gentamicin, and norfloxacin, so we should choose effective antibiotics. Based on the above results, bacteria cultures should be taken from the tonsil surface with the use of throat swabs and antibiotic sensitivity tests performed to guide clinical treatment. Kunnimoto17 explored the role of virus infection in the pathogenesis of IgAN by the cell culture, PCR, and immuno-fluorescent techniques, but did not find that virus played a role in the pathogenesis of IgAN. To date, there are no comparative studies of bacterial strains and antibiotic susceptibility testing in chronic tonsillitis patients with IgAN and without nephritis. Suzuki et al.18 reported that tonsillar lymphocytes from patients with IgAN revealed a significantly higher stimulation index to H. parainfluenzae antigens (thymidine incorporation in tonsillar lymphocytes with H. parainfluenzae/thymidine incorporation in unstimulated tonsillar lymphocytes) than controls. Our experiments16 have shown that CD4 + CD25+ cells were significantly decreased and dimeric IgA-producing cells were dramatically increased in tonsils in IgAN patients compared with the controls before and after stimulation with HS-IgAN(α-hemolytic streptococcus (HS) isolated from the tonsillar crypts of IgAN patients) or HS-controls (α-hemolytic streptococcus (HS) isolated from the tonsillar crypts of controls). These indicate that immunologic abnormality of the tonsils may be related to the pathogenesis of IgAN.

We conclude that, there is no significant difference in bacterial strains and antibiotic susceptibility between the two groups. Some patients with recurrent chronic tonsillitis do not suffer from renal disease, implying that it is possible to find a balance between immunity and tolerance. However, other patients do suffer from IgAN along with recurrent chronic tonsillitis. This indicates that immunologic abnormality of the tonsils may be related to the pathogenesis of IgAN. Our results are not in line with our hypothesis. Additional studies are necessary to demonstrate other clinical parameters, other microbial and to isolate T cell or B cell clones reactive to microbial.

Declaration of interest

This study was supported in part by the foundation of Beijing Shijitan Hospital, Capital Medical University (No. 2011-C06), Hunan Provincial Natural Sciential Foundation (No. 09jj6056), and the Foundation for the Excellent Doctoral Education of Central South University (No. 2340) China.