Influences of Long-Term Antibiotic Administration on Peyer's Patch Lymphocytes and Mucosal Immunoglobulin A Levels in a Mouse Model 
From the * Department of Surgery I, National
Defense Medical College, Tokorozawa, Japan; Correspondence: Kazuhiko Fukatsu, MD, Division of Basic Traumatology, National Defense Medical College Research Institute, 3-2 Namiki, Tokorozawa, Saitama, Japan 359-8513. Electronic mail may be sent to fukatsu{at}ndmc.ac.jp.
Background: Long-term antibiotic administration is sometimes
necessary to control bacterial infections during the perioperative period.
However, antibiotic administration may alter gut bacterial flora, possibly
impairing gut mucosal immunity. We hypothesized that 1 week of subcutaneous
(SC) antibiotic injections would affect Peyer's patch (PP) lymphocyte numbers
and phenotypes, as well as mucosal immunoglobulin A (IgA) levels.
Methods: Sixty-one male Institute of Cancer Research mice were
randomized to CMZ (cefmetazole 100 mg/kg, administered SC twice a day), IPM
(imipenem/cilastatin 50 mg/kg x 2), and control (saline 0.1 mL x
2) groups. After 7 days of treatment, the mice were killed and their small
intestines removed. Bacterial numbers in the small intestine were determined
using sheep blood agar plates under aerobic conditions (n = 21). PP
lymphocytes were isolated to determine cell numbers and phenotypes (CD4, CD8,
Surgical site infections and nosocomial infections are among the complications that can occur during the postoperative period. Infection itself may be the reason for undergoing surgery in some patients. Without modern methods to prevent and treat surgical infections, surgery would not exist as it is currently practiced.1 In particular, antibiotics have contributed greatly to the prophylaxis and treatment of surgical infections. Although present guidelines recommend short courses of antibiotics,2,3 long-term antibiotic administration is necessary in some cases to control bacterial infections during the perioperative period. In such cases, superinfection, adverse drug interactions, and the development of microbial resistance have been recognized as possible deleterious consequences of antibiotic use.1 In addition to these adverse effects, we hypothesized that long-term antibiotic coverage would impair gut mucosal immunity. Although the gut was long considered to be a quiescent organ under severe surgical insults, recent experimental and clinical investigations have demonstrated the gut to be a central organ in the host response.4 Particularly, maintenance of gut-associated lymphoid tissue (GALT) mass and function is important for preserving intestinal and extraintestinal mucosal immunity.5,6 Because gut commensal bacteria function to maintain the normal state of the gut mucosal immune system, it is possible that altering the microflora via systemic antibiotic administration would affect gut immunity.7 Therefore, in the present study, we examined the influences of 1 week of subcutaneous (SC) antibiotic injections on Peyer's patch (PP) lymphocyte numbers and phenotypes, as well as mucosal immunoglobulin A (IgA) levels. We selected 2 types of antibiotics, covering aerobic and anaerobic bacteria for this study. One, cefmetazole (CMZ), is a second-generation cephem. The other, imipenem/cilastatin (IPM), is a carbapenem antibiotic. Both have been widely used in surgical patients in Japan.
Animals The studies reported herein conform to guidelines for the care and use of laboratory animals established by the Animal Use and Care Committee of the National Defense Medical College. Male Institute of Cancer Research (ICR; Nippon SLC, Hamamatsu, Japan) mice were housed under controlled temperature and humidity conditions with a 12-hour:12-hour light:dark cycle. Mice were fed commercial mouse chow (CE7; Clea Japan, Tokyo, Japan; Table I) with water ad libitum for 1 week before protocol entry.
Experimental Protocol
In 40 mice, bronchoalveolar washings were obtained by lavage with 1 mL of
phosphate-buffered saline solution. The entire small intestine was harvested.
The intestinal washings were obtained by lavage with 20 mL of chilled Hanks'
balanced salt solution. Bronchoalveolar and intestinal washings were stored in
a –80°C freezer for IgA analysis. Then, PP lymphocytes were isolated
from the intestine, and their phenotypes (CD4, CD8, In another set of mice (n = 21), small intestinal washings were obtained with 20 mL of chilled saline solution. The washings were serially diluted in sterile saline solution and plated onto sheep blood agar. After inoculation for 18–24 hours at 37°C under aerobic conditions, bacterial colonies were counted.
Cell Isolation
Flow Cytometry
IgA Quantification
Statistical Analysis
Body Weight Change There were no significant differences in preexperimental body weight among the 3 groups. Body weight changes (means ± SE g) after 7 days of treatment were 1.6 ± 0.7, 1.5 ± 0.8, and 2.2 ± 0.8 in the CMZ, IPM, and control groups, respectively, with no significant differences among the 3 groups.
Number of PP and PP Lymphocytes There were no significant differences in numbers of PPs among the 3 groups. Both the CMZ and the IPM groups showed significantly lower PP cell numbers than the control group (Figure 1).
PP Cell Phenotype
Secretory IgA Levels
Bacterial Number Bacterial numbers were significantly lower in the CMZ and IPM groups than in the controls, with no significant difference between the 2 antibiotic-treated groups (Figure 3).
Indigenous microorganisms colonize the mucosal surface of the oral cavity, the upper respiratory tract, much of the gastrointestinal tract, and the urogenital tract. These organisms interact with the host's immunologic system in complex ways. Many of the details of these interactions remain unclear.8 Normal bacterial flora have the capacity to limit the growth of certain transient microbial pathogens entering their habitat, via nutrition competition, the toxicity of metabolic end products, factors such as bacteriocins, and competition for mucosal adhesion sites.9 Moreover, intestinal microbes drive GALT development and play an important role in its maintenance.7 Thus, indigenous microbial flora may function as an accessory organ in the body.10
Therefore, it is possible that long-term systemic antibiotic treatment disturbs gut microflora, resulting in impaired gut mucosal immunity. Several reports have described the influences of antibiotic therapy on gut immunity. Amoxicillin/clavulanate potassium treatments reportedly do not affect secretory IgA, lactoferrin, lysozyme, or TGF-β1 levels in human feces.11 On the other hand, De Simone et al12 demonstrated oral ofloxacin treatment to exert an immunotoxic effect on PP cells in a murine model. Thus, whether antibiotic therapy induces adverse effects on gut immunity remains controversial. In the present study, we examined the influences of systemic administration of antibiotics (CMZ and IPM) on gut immunity in a murine model. CMZ is one of the most commonly administered antibiotics in Japan for both prophylaxis and treatment of surgical infections. IPM has increasingly been used to treat severe infectious complications in surgical patients in Japan. Although we did not find significant differences in secretory IgA levels, which is consistent with the former report,13 both CMZ and IPM reduced PP lymphocyte numbers without significantly changing their phenotypes. The PP acts as an antigen detector and processor, sensitizing naïve T and B cells against intraluminal antigens.5,6 Therefore, the decrease in PP cell number in response to antibiotic therapy may reflect impairment of intestinal and extraintestinal mucosal immunity.
Clinically, this impairment may trigger bacterial translocation, systemic inflammation, pneumonia, and other disorders in surgical patients. In addition, it is possible that GALT change after antibiotic therapy is an important mechanism underlying susceptibility to infections in elderly people. Reportedly, the composition and metabolic activities of the microbiota change with age, and the bacterial flora in the feces of antibiotic-treated elderly people are further modulated.14 With regard to the impact of longer-term administration of the antibiotics, we do not have any data. Generally, the longer antibiotics are given, the more possibility of resistant species appearance exists. Therefore, longer therapy might have more deleterious effects on GALT mass and function than 7-day treatment. Enteral nutrition has been suggested as a method of preventing GALT atrophy and dysfunction.6,7 However, judging from the lack of body weight changes, the mice apparently consumed similar amounts of chow regardless of antibiotic therapy. This observation suggests that oral food intake did not restore lost GALT cell mass in this setting. Moreover, severely injured and critically ill patients are sometimes unable to take oral or enteral nutrition, such that parenteral nutrition (PN) is unavoidable. In such patients, prolonged antibiotic treatment might have more deleterious effects on GALT. Hence, preservation of normal gut microflora, through therapeutic interventions, may be needed during long-term antibiotic therapy. Such interventions may include the administration of probiotics and prebiotics. We examined bacterial numbers in small intestinal washings under aerobic conditions in this study. As expected, the antibiotic therapy significantly decreased bacterial number compared with the controls, reflecting changes in the gut microflora. Because altered gut flora have been demonstrated to affect lymphocyte proliferation,15 antibiotic-related changes in the flora may be a major mechanism responsible for GALT changes in the present setting. However, we believe that more intensive examination of gut microflora is needed to further assess the relationship between antibiotic treatment and gut immunity because anaerobic microflora are an important component of the gut barrier.13 In addition, more detailed examination of GALT changes during antibiotic administration is warranted. We examined only PPs, inductive sites of mucosal immunity, in the current study. GALT effector sites, including intraepithelial spaces and the lamina propria, as well as extraintestinal mucosal sites, need to be examined in future studies. As mentioned before, pro- and prebiotics have been expected to manipulate gut microbiota under stressful conditions.16 Administration of pro- and prebiotics with systemic antibiotic therapy may normalize gut immunity, and effects of this option should be examined. In terms of clinical standpoint, it would be interesting to examine GALT of antibiotic-treated surgical or elderly patients for comparison with that of healthy people. In conclusion, long-term antibiotic treatment reduces PP cell numbers while decreasing bacterial numbers in the small intestine. These changes may worsen susceptibility to severe infections in surgical or elderly patients. It may be important to recognize changes in gut mucosal immunity during long-term antibiotic administration.
Presented in the Premier Paper Session at the American Society for Parenteral and Enteral Nutrition Clinical Nutrition Week, February 12–15, 2006. Received for publication February 27, 2006. Accepted for publication April 27, 2006.
DiscussantNew Jersey University of Medicine & Dentistry Yaguchi and colleagues used a mouse model to study the effect of long-term (7 days) antibiotic treatment on gut flora. Although antibiotics are commonly used to control bacterial infections postoperatively, they may also place the patient at greater risk of developing microbial resistance, adverse drug interactions and possibly impair gut mucosal immunity. These issues raise the question of the effect of use of long-term antibiotics on Gut Associated Lymphoid Tissue or GALT mass and function. The authors looked at this somewhat controversial issue by examining Peyer's patch (PP) cells since a decline in PP cells due to antibiotics may reflect impaired mucosal immunity. The authors point out several important next directions in related research. Given that the current study only looked at PP, which are the inductive site of mucosal immunity, future research should look at GALT effecter sites such as intraepithelial spaces and the lamina propia as well as extra intestinal mucosal sites.
The researchers used a mouse model to determine the effect of 1 week of antibiotic injections 2 times per day (considered long-term in mice) on gut flora, in particular PP lymphocytes numbers, phenotypes and mucosal IgA. There were 3 groups of mice, a control group and 2 experimental groups. One experimental group received cefmetazole and the other imipenem/cilastin, 2 common antibiotics in Japan. Both were administered 2 times per day subcutaneously. During the week before and the week of treatment, mice received free access to chow and water. Although the results demonstrated that the control group weighed slightly more at the end of the 7 days than the 2 experimental groups, the differences were not statistically significant. There were, however, significant differences in PP lymphocyte numbers and bacterial levels in the 2 experimental groups. The mice in the 2 groups receiving antibiotics had significantly lower PP lymphocyte numbers than the control group, as well as significantly lower bacterial numbers. But, there were no significant differences in PP lymphocyte phenotype or intestinal IgA levels. Clinically, these conclusions provide insight into the affects of long-term antibiotics on gut immunity. As Dr Yaguchi points out, the decrease in PP cell numbers in the 2 experimental groups may reflect impaired intestinal immunity which could trigger bacterial translocation.
What are the natural next steps in this area of research given the findings? How can the clinician translate these results into practice? What would be the impact of longer term administration of the antibiotics?
Author's ResponseThank you for your kind comments and helpful questions. In the present study, we just examined PP lymphocyte numbers and phenotypes, mucosal IgA levels and intestinal aerobic bacterial numbers. Because PP act as GALT inductive sites, while intraepithelial and the lamina propria lymphocytes act as GALT effector sites, we need to clarify the influences of antibiotic therapy on GALT effector sites. We also feel that more intensive examination of gut microflora is needed to further assess the relationship between antibiotic treatment and gut immunity. Furthermore, because administration of pro- and prebiotics with systemic antibiotic therapy may normalize gut microflora, its effects on gut immunity should be examined. It would also be interesting to examine GALT of antibiotic-treated surgical and/or elderly patients for comparison with that of healthy people.With regard to the clinical relevance, GALT impairment due to long-term antibiotic treatment may trigger bacterial translocation, systemic inflammation, pneumonia, and other disorders in surgical patients. Moreover, it is possible that GALT change following antibiotic therapy is an important mechanism underlying susceptibility to severe infections in elderly people, because their gut flora were reported to be greatly affected by antibiotics. Thus, clinicians need to recognize changes in gut mucosal immunity during long-term antibiotic administration. As for the impact of longer administration of the antibiotics, we do not have any data and can only speculate. Because longer antibiotics may increase the possibility of resistant species appearance, longer therapy might have more deleterious effects on GALT mass and function than 7 day-treatment.
Journal of Parenteral and Enteral Nutrition, Vol. 30, No. 5,
395-399 (2006) This article has been cited by other articles:
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βTCR, 
TCR, B220; n = 40). IgA levels in the small
intestinal and bronchoalveolar washings were also measured with ELISA.
Results: Antibiotic administration decreased both bacterial number
and the PP cell yield compared with the control group. There were no
significant differences in either phenotype percentages or IgA levels at any
mucosal sites among the 3 groups. Conclusions: Long-term antibiotic
treatment reduces PP cell numbers while decreasing bacterial numbers in the
small intestine. It may be important to recognize changes in gut mucosal
immunity during long-term antibiotic administration. 
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MATERIALS AND METHODS
RESULTS
