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Amoxicillin (aminopenicillin) is the drug of first choice for the treatment of respiratory tract infections.

This beta-lactam antibiotic with bactericidal action is widely prescribed in clinical practice, particularly when a patient's airways are compromised [32].

Knowledge on the influence of infectious processes and antimicrobial agents on the gastrointestinal ecosystem is highly important clinically, since this microbiota has functions that affect host health.

We investigated the influence of respiratory tract infections and of amoxicillin therapy on the normal intestinal microbiota of patients.

A prospective study was carried out on 42 individuals distributed into two Groups: Group 1 (G1) : 22 patients with bacterial infections of the respiratory tract (sinusitis, pneumonia), of both sexes, 19 to 50 years old, seen at the emergency department of the Hospital das Clinicas of the School of Medicine of the Universidade Estadual Paulista (UNESP) in Botucatu, SP, Brazil, from July to December 2002. Control Group (CG) : 20 blood donors of both sexes, 18 to 50 years old, screened at the blood center of the same institution over the same period. Criteria for inclusion : patients of both sexes, aged 18 years and over, with epidemiological, clinical, and imaging diagnosis of acute bacterial infectious diseases of the respiratory tract. Criteria for exclusion : pregnancy; lactation; women with hormonal disorders; individuals with other underlying diseases; use of medication, particularly antimicrobials, within the past 30 days. The diagnoses of infection with bacterial microorganisms were based on clinical and epidemiological data and on nonspecific supplementary exams (complete blood counts, chest and/or sinus radiographs).

Blood counts revealing leukocytosis and radiographic tests showing sinus opacity (sinusitis cases) and/or revealing lung condensation (pneumonia cases) were considered indicative of bacterial infection. The nutritional assessment took into account dietary aspects and anthropometric measurements (weight and height).

Three-day intake records were used for food intake assessment. The calculations of total calories, carbohydrates, proteins, lipids, and other specific nutrients ingested were performed with the program Virtual Nutri [33].

In each group, the nutritional classification was based on body mass index (BMI) [34].

The intestinal bacterial microbiota was assessed with the method proposed by Sutter et al.

Stool samples were collected from both groups in sterile containers with Transbac transport medium (Probac). The interval between sample collection and laboratory handling did not exceed 1 h [36,37]. Three stool samples were collected from patients in Group 1: before treatment (time point T 1 ), at the end of treatment (time point T 2 ), and 30 days after treatment (time point T 3 ).

Only one sample was collected from each individual in the control group (time point T 0 ).

A 1-g aliquot was taken from each sample and transferred into a screw-capped test tube containing 9 mL of Stuart transport medium (Oxoid). After homogenization, successive dilutions up to 10 -8 were prepared using the same eluent.

Kanamycin-vancomycin blood agar, Bifidobacterium medium, and Lactobacillus selective medium (Probac) were the selective culture media used

for

Once inoculated and identified, the plates were placed into GasPak anaerobic jars (Permution) and incubated at 37?C for 48 h in a low-oxygen and high-carbon dioxide atmosphere generated by an Anaerobac system (Probac).

After that, the plates were evaluated for bacterial growth and colony aspect. Colony-forming units (CFUs) were counted for each plate, and the mean values for each type of microorganism were calculated.

Microorganism concentration was expressed as log 10 CFU/g of feces. Observations related to colony morphology, Gram staining, and catalase testing were recorded for each plate. Mean values and standard deviations of log-transformed data and mean values at the original scale were calculated for Bacteroides, Bifidobacterium , and Lactobacillus counts [38].

Time points T 1 , T 2 , and T 3 (Group 1) were compared by using Friedman's nonparametric test with calculations of c 2 and p statistics.

Comparisons of both groups at each time point were performed with the t -test for two independent samples (using log-transformed counts) with calculations of t and p statistics and/or Mann-Whitney's nonparametric method.

The comparisons were considered significant

whenever

The study was approved by the Research Ethics Committee of Hospital das Clinicas of the School of Medicine of UNESP in Botucatu.

The distribution of subjects by sex was homogeneous in both groups (50% males and 50% females). there was predominance of the 41 to 50 year (45%) and 21 to 30 year (30%) age ranges, whereas in Group 1 the 21 to 30 year (40%) and 41 to 50 year (30%) ranges predominated.

In both groups, eutrophic individuals (BMI = 18.5 to 24.9) were the most frequent.

The food consumption records did not reveal significant differences between the groups in terms of macronutrients, fibers, or micronutrients. When each experimental time point (T 1 , T 2 , T 3 ) in Group 1 was compared with T 0 of the controls, a significant (p Bacteroides spp. /g of feces at T 1 , demonstrating that the infectious process affecting the respiratory tract of patients influenced their intestinal microorganism populations (Table 1).

When each experimental time point (T 1 , T 2 , T 3 ) in Group 1 was compared with T 0 of the controls, a significant (p Bifidobacterium spp.

at T 2 , demonstrating that treatment with amoxicillin influenced the intestinal population of these microorganisms (Table 2).

in Group 1, the number of CFU/g of feces at T 2 was smaller than that at T 1 , which was smaller than that at T 3 (p 1 , T 2 , T 3 ) of Group 1 was compared with T 0 of the controls, significantly fewer Lactobacillus spp.

CFU/g of feces were found for time points T 1 (p = 0.05) and T 2 (p. Amoxicillin, however, did not prevent a quantitative recovery of Bacteroides spp. Thirty days after the end of treatment (T 3 ), the concentrations of Bifidobacterium and Lactobacillus had recovered their normal values (Figure 1).

The gastrointestinal ecosystem is an ample field for research and has long been the focus of interest of investigators. Although several studies have investigated the gastrointestinal microbiota [39-46], little information is available on the effect of some diseases and on the use of antimicrobials.

To help fill this gap, we investigated the influence of bacterial infections of the respiratory tract and of amoxicillin therapy on intestinal populations of the bacterial genera Bifidobacterium , Lactobacillus , and Bacteroides . Several factors can influence the intestinal microbiota, including host age.

Such changes, however, are more pronounced at birth, when the flora is established and the gastrointestinal tract is colonized with microorganisms ingested with food and acquired through contact with the environment [17,47,48].

Aging also promotes changes to the intestinal microbiota, significantly reducing the quantity of Bifidobacterium spp. and increasing the numbers of Lactobacillus spp., Clostridium perfringens, Escherichia coli ,

and

[17] Several authors have pointed out, however, that in the human adult phase the intestinal flora remains relatively stable, both qualitatively and quantitatively [3,9,12,17,47,49,50]. We found that age did not affect the qualitative or quantitative composition of the intestinal microbiota. Another factor that has been suggested to influence this microbiota is diet [2,6,39,46,51]. Based on food consumption records and nutritional diagnosis, we did not find that diet had an influence on the intestinal microbiotic composition.

However, all the individuals that were investigated lived in the same geographic location and had very similar diets. Over the past years, a few studies have revealed the influence of certain inflammatory processes on the gastrointestinal microbiota. The relationship between rheumatoid arthritis and intestinal microbiota has been a focus of great interest for researchers, who have found patients with rheumatoid arthritis to harbor fecal floras significantly different from those of normal individuals [25,52,53].

A number of studies are currently being conducted on intestinal microbiota and allergic diseases, and reductions in microorganisms of the genera Lactobacillus, Bifidobacterium , and Bacteroides have been detected [54,55].

Alterations in the intestinal microbiota have also been observed in diarrhea episodes [48].

We also detected changes in the intestinal microbiota accompanying

bacterial

The extent of these changes depends not only on the spectrum of action of the antimicrobial agent administered, but also on its degree of absorption, administration route, and possible enzymatic inactivation and/or ability to attach to water and to intestinal material [7]. During the past decade, several researchers have investigated the effects of various antimicrobial agents.

Amoxicillin has been found to cause important alterations to the intestinal microbiota, affecting several groups of microorganisms both in patients and normal individuals. Changes include an increase in the number of enterobacteria and Bacteroides spp. [58], the emergence of resistant strains of enterobacteria [31,58,59], overgrowth of C. [60], a reduction in the anaerobic microflora [61], and a reduction in the number of bifidobacteria, lactobacilli, and clostridia [62].

In our study, the administration of amoxicillin led to a significant decrease in the CFU of Bifidobacterium spp. /g of feces, though it did not prevent the recovery of Bacteroides spp.

Thirty days after the end of treatment, the concentrations of all three microorganisms had returned to their normal values. Identifying gastrointestinal microbiota imbalances caused by infectious processes and by the use of antimicrobials is thus quite important, as this microbiota has a decisive role in health maintenance.

Any quantitative change in this group of microorganisms may have serious effects on the ecological balance of the intestinal microbiota, with detrimental consequences for the host. The intestinal microbiota plays a central role in maintaining the host's health, and it can be adversely affected by bacterial infectious processes occurring in the respiratory tract, as revealed by the significant decrease in the CFU of Bacteroides spp. The amoxicillin antibacterial use of amoxicillin also affected the intestinal microbiota, significantly decreasing the CFU of Bifidobacterium spp.

We expect that these results will encourage additional studies on the infectious processes in the Brazilian population, since the investigations available for comparison have been conducted in countries where socioeconomic and cultural conditions

differ

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Armando da Cunha, 471, Campo Grande MS 79051-040, Brazil E-mail: monreal@brturbo.com. Received on 25 February 2005; revised 12

June

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