Culture-based bacterial evaluation of the appendix lumen and antibiotic susceptibility of acute appendicitis in Japan: A single-center retrospective analysis

The question of whether to perform an appendectomy or conservative treatment for acute appendicitis can differ depending on the facility or surgeon, but antibiotic treatment is administered regardless of whether an appendectomy or conservative treatment is selected. We investigated the contemporary bacteriology for acute appendicitis and evaluated the antibiotic sensitivity of the bacteria that are currently associated with appendicitis. We retrospectively analyzed the bacterial culture results and antibiotic susceptibility of 141 patients who underwent appendicitis surgery, including the identification and antimicrobial susceptibility of the cultured bacteria within the excised appendices. Bacterial cultures were positive in 131 cases (92.9%). The most commonly isolated bacteria were Escherichia coli (90 isolates, 66.7%), followed by Enterococcus species (n = 19, 14.5%), Pseudomonas aeruginosa (n = 18, 13.7%), Streptococcus species (n = 15, 11.5%), and Klebsiella species (n = 8, 6.1%). Eight strains (8.8%) of E coli were extended-spectrum β-lactamase producers, and ten strains (11.1%) were fluoroquinolone-resistant. Tazobactam/piperacillin and meropenem inhibited the growth of 100% of the major identified bacteria. The patients with appendicoliths had a significantly higher bacterial culture rate. Enterococcus species were frequently isolated from the patients with complicated appendicitis. For the antibiotic treatment of appendicitis, it is essential to understand the patient’s microbiological profile and antibiotic susceptibilities. Research from Asian countries such as Japan can enhance our knowledge of regional antibiotic resistance patterns and inform effective treatment strategies.


Introduction
Acute appendicitis is a common disease that often requires emergency surgery.According to a systematic review with a meta-analysis, surgical resection continues to be the preferred treatment, although antibiotic therapy alone may be considered for specific patients. [1]Antibiotics-only treatment has usually involved broad-spectrum antibiotics, including ertapenem and combinations of multiple antibiotics.Although this approach can effectively target the primary microorganisms that are responsible for acute appendicitis, there are concerns about the potential development of multidrug resistance; given the global tendency to overuse antibiotics, this is a worrisome issue.
The selection of appropriate antibiotics for acute appendicitis is often empirical, as obtaining a culture specimen is challenging unless the peritoneal cavity is accessed (except in cases of bacteremia).Common bacteria in appendicitis encompass Escherichia coli, Bacteroides, Streptococci, and Pseudomonas. [2]n Japan, there are no specific guidelines for the use of antibiotics in the treatment of acute appendicitis.Notably, widely accepted global guidelines such as those from the Surgical Infection Society and the Infectious Disease Society of America (SIS-IDSA) [3] and the World Society of Emergency Surgery (WSES) [4] cannot be directly applied in Japan and other Asian countries due to the high prevalence of extended-spectrum β-lactamase (ESBL)-producing and fluoroquinolone-resistant E coli in this region.The cautious selection of empirical antibiotics is therefore essential.
We conducted the present study to delve into the microbiology of patients diagnosed with acute appendicitis who underwent treatment with surgery combined with antibiotic therapy.The primary aims of the study were to identify the bacteria commonly found in the appendix lumen during acute appendicitis and to evaluate the susceptibility of these bacteria to various antibiotics.

Materials and methods
We retrospectively reviewed the medical records of the patients who underwent appendectomy and were histopathologically diagnosed as having acute appendicitis at Showa Inan General Hospital (Komagane, Japan) between April 2014 and September 2022.Patients who underwent interval appendectomy and those without intraoperative cultures of the lumen of the excised appendix were excluded from the study.At our hospital, the diagnosis of appendicitis is comprehensively determined based on clinical findings, blood sampling data, and imaging (computed tomography or ultrasonography).All resected appendices in the present series were evaluated histopathologically.Laparoscopic appendectomy is typically performed for patients who are eligible for surgery at our hospital, whereas open laparotomy is performed in cases with extensive abscesses.We performed the patients' surgeries by the single-incision plus one-port laparoscopic method, with an additional port added depending on intraoperative findings.Aspiration or swabbing of the lumen of the excised appendix was performed in each patient.The swabbing was performed using Seed-Swab γ-3 (Eiken Chemical Co., Ltd., Tokyo, Japan), a flocked swab with a modified liquid Amies microbiology transport medium.These specimens were sent for culture and sensitivity testing to identify bacterial growth and the assessment of antibiotic sensitivity.
Each culture was subjected to antimicrobial susceptibility testing in our hospital's clinical laboratory.The bacteria were cultured on blood agar, chocolate agar, Brucella HK agar, and bromothymol blue lactate agar.The autoed MicroScan WalkAway 40plus (Beckman Coulter, Brea, CA) was used to identify pathogens and perform the antibiotic susceptibility tests.Based on the susceptibility reports, antibiotics considered to be effective were then identified.The antimicrobial susceptibility tests were performed according to the criteria of the Clinical and Laboratory Standards Institute.
The timing of the surgery depended on the decision of the physician in charge at the patient's first visit or the surgeon in charge of the surgery.Emergency surgery was performed in cases in which antibiotic treatment was administered but the patient's symptoms worsened after his or her admission.
The following parameters were evaluated: the patients' preoperative characteristics, including age, sex, antibiotic administration status, and time from diagnosis to surgery; the surgical procedure, including open or laparoscopic appendectomy; the histological or clinical classification of appendicitis, including phlegmonous and gangrenous; and bacterial cultures with antibiotic susceptibility, including specimens of bacterial cultures obtained by aspirating or swabbing the lumen of the excised appendix specimen.
Statistical analyses were performed with JMP ver.15.2.0 (SAS Institute, Cary, NC).Fisher exact test was used as appropriate for the statistical analysis.Statistical significance was considered as a P value < .05.The study was approved by the institutional review board of Showa Inan General Hospital (No. 2021-06).The requirement for patients' written informed consent was waived because this was a retrospective study.This study was conducted in accord with the STROBE guidelines and with the latest version of the Declaration of Helsinki.All authors had access to the study data and reviewed and approved the final manuscript.

Results
During the study period, a total of 320 patients were admitted to our hospital with acute appendicitis.Among them, 209 patients underwent appendectomy, and appendiceal lumen culture testing was conducted in 141 patients (Table 1).The ages of the patients (82 [58%] men, 59 [42%] women) ranged from 3 to 94 years (mean 41 years).The result of a bacterial culture was positive in 131 cases (93%).In all patients, intravenous antibiotics were administered from the day of admission.The initial antibiotic was cephem.Of the cases in which cephem was selected, ceftriaxone was used in 5 patients, and cefmetazole (CMZ) was used in the other 136 patients.Twenty-four patients who underwent surgery within 4 hours of their visit were not given preoperative antibiotics.The remaining 117 patients received preoperative antibiotic administration.
The preoperative in-hospital stay ranged from 0 to 8 days (mean 0 day), and 122 patients underwent the appendicitis surgery within 24 hours of admission.Antibiotic treatment for ≥24 hours before surgery was administered to 19 patients.During the in-hospital waiting period, 3 patients were deemed to have worsened or were deemed unable to wait based on their physical assessments, necessitating emergency surgery.A total of 140 patients (99%) underwent laparoscopic appendectomy, and the remaining patient (1%) underwent an open appendectomy.Based on their intraoperative and pathological findings, 45 patients were diagnosed with complicated appendicitis.
The bacterial culture was positive in 131 patients (92.9%).The bacterial culture rate decreased significantly in the group of patients who underwent antibiotic treatment for >24 hours before their surgery.Preoperative antibiotic administration, especially for durations exceeding 24 hours, appears to correlate with a decreased positivity rate in bacterial cultures.The group of patients with appendicoliths had a significantly higher bacterial culture rate.Neither complicated appendicitis nor SSI was associated with a positive culture (Table 4).
When preoperative antibiotic treatment was administered for >24 hours, the detection rate of E coli (non-ESBL) decreased to 26%.E coli (ESBL) decreased, but the difference was not significant.However, Enterococcus species, P aeruginosa, Streptococcus species, and K pneumoniae did not show a decrease even with preoperative antibiotic treatment for >24 hours (Table 5).Enterococcus species were detected significantly more frequently in patients with complicated appendicitis (22.9%) compared to those with simple appendicitis (8.6%).There were no significant differences in the detection of other bacteria between the simple appendicitis and complicated appendicitis cases (Table 6).

Discussion
We retrospectively analyzed the microbiological profiles and antibiotic susceptibilities of appendicitis in a series of 141 patients.E coli was the most common pathogen identified (68.7% of all isolates), similar to previous appendicitis findings. [2,5,6][9] Enterococcus species and Streptococcus species were the most frequently isolated Gram-positive organisms. [5]][9] The reported isolation rate of K pneumonia is 4.8% to 13%. [5,7,8]lthough E coli showed high susceptibility (96%) to commonly used cephalosporins, quinolones showed 83.4% efficacy.Enterococcus species, which are resistant to cephalosporins, require penicillin-based antibiotics.In the present study, Enterococcus species was frequently observed in the cases of complicated appendicitis, and penicillin-based antibiotics should be considered for complicated appendicitis.P aeruginosa, which is not resolved by CMZ, flomoxef, or cefazolin, necessitates broad-spectrum antibiotics such as TAZ/PIPC and MEPN for effective treatment. [6]e also observed that the rate of bacteria culture positivity was higher in the acute appendicitis patients with appendicoliths.Hattori et al reported that the numbers of aerobes and anaerobes were increased in patients with appendicoliths, [6,9] and we thus speculate that the increased number of bacterial species may have been induced by the appendiceal lumen obstruction.In large random controlled trials, the presence of an appendicolith has been shown to be associated with a more complicated course of the disease. [10]n the management of patients with acute appendicitis, the selection of suitable antibiotics plays a significant role, particularly in patients experiencing infectious complications postsurgery and those undergoing nonsurgical treatment.The proper antibiotic choice should encompass agents that demonstrate effectiveness against both facultative and aerobic Gramnegative organisms as well as anaerobic organisms. [11]The commonly employed third-generation cephalosporin for empirical antibiotic treatment has proven efficacy against a wide range of microorganisms, including E coli.Our present findings suggest that cephalosporins are generally suitable, with the exception of efficacy against P aeruginosa.In cases involving P aeruginosa, treatment options such as TAZ/PIPC and carbapenem may be considered.
Globally accepted guidelines such as the 2017 SIS-IDSA guidelines, [3] which are based on the results of clinical trials of antibiotics for intra-abdominal infections including appendicitis, recommend using cefotaxime or ceftriaxone plus metronidazole or ertapenem as the preferred agents for the initial empiric therapy of lower-risk patients.Guidelines and recent reports do not recommend broad-spectrum regimens with activity against P aeruginosa and fluoroquinolone-resistant and ESBL-producing bacteria for patients with mild-tomoderate community-acquired infections unless antimicrobial resistance risk factors exist, such as recent antimicrobial exposure, a past infection with a resistant strain, or a high prevalence of resistance in the patient's community or in recent areas of travel. [3,12]However, the 2020 WSES Jerusalem guidelines [4] recommend a single preoperative dose of broad-spectrum antibiotics in patients with acute appendicitis undergoing an appendectomy.
ESBL prevalence in the West Pacific and Southeast Asia (46% and 22%, respectively) exceeds that in Europe and the Americas (4% and 2%, respectively). [13]Furthermore, the incidence of fluoroquinolone-resistant E coli in Asia is notably higher, ranging from 5.7% to 32.8%, compared to Europe (1.4-23.3%)and North America (2.8-8.9%). [14]In Japan, ESBL-producing E coli and fluoroquinolone-resistant E coli constitute 9.5% to 15% and 15.6% to 30% of all E coli isolates, respectively. [7,15]In this study, the frequency of ESBL-producing E coli was 8.8%, and the frequency of fluoroquinolone-resistant E coli was 11.1%.While these rates fell below those reported in previous studies, they were not insignificant.Yukumi et al [7] reported third-generation and fourth-generation CEPs or fluoroquinolone-based regimens seem to not be appropriate as a first choice in Japan or other Asian countries.The prevalence of ESBL and fluoroquinolone-resistant E coli in Asia challenges the applicability of these guidelines, thereby emphasizing the importance of region-specific considerations.Our study has certain limitations to consider.It was a retrospective analysis involving a relatively small cohort (n = 141) and was conducted exclusively at a surgical department within a local community hospital in Japan.Consequently, the findings are specific to our regional context and may undergo alterations over time.A comprehensive assessment of different antibiotics should be conducted in a prospective trial.

Conclusion
Because antibiotic administration has emerged as a reasonable first-line treatment for patients with uncomplicated appendicitis, an understanding of the role of patient' microbiological profiles and antibiotic susceptibilities in acute appendicitis is of substantial importance.Studies from Asian countries such as Japan can deepen our understanding of regional antibiotic resistance patterns and guide effective therapeutic strategies.

Table 1
The patients' characteristics.

Table 2
Bacterial isolates from 131 patients.

Table 3
Antibiotic susceptibility of major identified bacteria.

Table 4
Associations between patient characteristics and culture-positive rates.

Table 5
Detection rate of bacteria in the patients with or without antibiotic treatment for ≥24 hours before surgery.

Table 6
Microorganisms cultured from the simple appendicitis and complicated appendicitis patients.