Annals of Clinical Microbiology, The official Journal of the Korean Society of Clinical Microbiology
Kwangjin Ahn1
, Hyunju Choi1, Taesic Lee2, Young Uh1
1Department of Laboratory Medicine, Yonsei University Wonju College of Medicine, Wonju, Korea
2Department of Family Medicine, Yonsei University Wonju College of Medicine, Wonju, Korea
Correspondence to Young Uh, E-mail: u931018@yonsei.ac.kr
Ann Clin Microbiol 2025;28(2):1. https://doi.org/10.5145/ACM.2025.28.2.1
Received on 4 March 2025, Revised on 7 April 2025, Accepted on 14 April 2025, Published on 19 May 2025.
Copyright © Korean Society of Clinical Microbiology.
This is an Open Access article which is freely available under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND) (https://creativecommons.org/licenses/by-nc-nd/4.0/).
Background: Identifying Bacillus spp. and performing antimicrobial susceptibility testing (AST) is challenging because of their diversity and limited clinical laboratory resources. We investigated the isolation frequency and antimicrobial susceptibility of Bacillus spp. over a 4-year period.
Methods: Bacillus isolates collected between 2020 and 2024 were identified using matrixassisted laser desorption ionization-time of flight mass spectrometry, and AST was performed using the Pos Breakpoint Combo Panel Type 28 (Beckman Coulter).
Results: Species among total isolates (n = 432) were B. cereus (25.1%), B. subtilis (11.8%), B. licheniformis (10.8%), B. pumilus (7.4%), B. simplex (6.9%), B. circulans (6.4%), and B. amyloliquefaciens ssp. plantarum (5.9%). Overall, 65% of all Bacillus isolates were obtained from patients aged ≥ 60 years. The isolation ratios of sterile body fluids, including blood, to non-sterile specimens, in decreasing order, were: B. licheniformis, 4.5; B. subtilis, 2.4; B. pumilus, 2.0; B. amyloliquefaciens ssp. plantarum, 2.0; B. circulans, 1.2; B. thuringiensis, 1.0; B. cereus, 0.76; B. simplex, 0.56; and B. infantis, 0.43. The overall antimicrobial resistance rates were as follows: penicillin, 57.1%; ampicillin, 52.4%; clindamycin, 31.6%; erythromycin, 9.0%; cotrimoxazole, 6.4%; tetracycline, 2.3%; ciprofloxacin, 1.9%; rifampicin, 1.1%; levofloxacin, 0.9%; vancomycin, 0.4%; gentamicin, 0.4%; and imipenem, 0.4%. Penicillin resistance was particularly high in B. thuringiensis (100%), B. cereus (93.5%), and B. licheniformis (71.4%). Clindamycin resistance was high in B. circulans, B. licheniformis, and B. pumilus at 81.8%, 71.4%, and 64.3%, respectively.
Conclusion: Accurate identification and AST of Bacillus spp. are essential when they are isolated from invasive infections, as resistance profiles and isolated species vary significantly depending on the specimens.
Bacillus species, Bacteriological techniques, Microbial sensitivity tests
Aerobic endospore-forming Bacillus species are widely distributed in nature and are commonly found in food and environmental sources [1]. The spores of these species are resilient and can spread through the air, dust, and aerosols, thereby contributing to the contamination of hospital environments. Clinical Bacillus isolates were initially considered mere contaminants and non-pathogenic. However, its pathogenic potential has been recognized, particularly in the Bacillus cereus group, which includes B. cereus, B. anthracis, and B. thuringiensis [2]. Patients with intravenous drug abuse, trauma, implanted medical devices, malignancy, neutropenia, or corticosteroid therapy are vulnerable to severe Bacillus infections [3].
Although most Bacillus spp. are considered contaminants, these organisms should be identified at the species level when they are isolated from sterile body fluids, including blood, and are predominantly isolated from adequately collected clinical specimens. The accurate identification and antimicrobial susceptibility testing (AST) of Bacillus species are difficult because of the extensive diversity of these organisms and the limited commercial identification and AST systems available in clinical laboratories [4,5]. Among Bacillus spp., the most clinically relevant is B. cereus, a gram-positive spore-forming bacterium that is widely distributed in the environment. Although primarily associated with foodborne illnesses, its pathogenicity, which is related to the production of tissue-destructive exotoxins, has been reported to cause fatal infections [6]. Various Bacillus species cause infections in extra-intestinal organs and are frequently detected in wounds [7].
With the widespread use of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) in clinical laboratories, the identification of various Bacillus spp. that were previously unidentifiable at the species level has become possible, making it difficult to determine their clinical significance. As Bacillus strains can be reported to the species level, clinical laboratories are faced with the challenge of whether to perform AST for Bacillus species. The Clinical and Laboratory Standards Institute (CLSI) recommends AST for Bacillus isolates from normally sterile sources, such as deep tissue, cerebrospinal fluid, and multiple positive blood cultures, particularly in patients with an implanted prosthetic device, immunosuppression, or a history of intravenous drug abuse [8]. The interpretive criteria for minimal inhibitory concentration (MIC) values of Bacillus species are adapted from those for Staphylococcus species as published in the CLSI document M100 [9]. The key citations used in the derivation of interpretive criteria are based on previously reported researches on AST of Bacillus spp. [9–11].
An increasing prevalence of multidrug-resistant Bacillus species isolated from the stool samples of patients with gastrointestinal infections has been reported, and multidrug resistance has been observed in B. cereus isolates identified in freshly collected food samples, including milk [1,12]. Additionally, certain species such as B. coagulans and B. subtilis have been utilized as probiotics [13,14]. These bacteria may possess intrinsic antimicrobial resistance or have the potential to horizontally transfer resistance genes [15–17]. As Bacillus spp. are increasingly associated with normal human life, there is a growing need for more clinical data on these organisms.
This study aimed to investigate the isolation frequency by age group, specimen type, and antimicrobial susceptibility of Bacillus isolates over the past four years (2020–2024).
It is a retrospective surveillance study based on the laboratory records. It was described according to the Microbiology Investigation Criteria for Reporting Objectively: a framework for the reporting and interpretation of clinical microbiology data available at: https://bmcmedicine.biomedcentral.com/articles/10.1186/s12916-019-1301-1.
From August 2020 to July 2024, Bacillus isolates identified during routine clinical practice at Wonju Severance Christian Hospital (WSCH) were collected from the laboratory information system, and all data were automatically de-identified.
Bacillus species were identified by Gram staining, colony morphology analysis, and MALDI-TOF MS using a Bruker Biotyper (Bruker Daltonics GmbH & Co.). When species-level identification could not be clearly determined, further identification was not performed using additional methods. In such cases, the isolates were classified and analyzed as “Bacillus species.” Specimens were classified as either sterile body f luids (blood, cerebrospinal fluid, peritoneal fluid, pleural fluid, and synovial fluid) or non-sterile (catheter, drain, and wound). AST was performed using the Pos Breakpoint Combo Panel Type 28 (Beckman Coulter), and the results were interpreted according to CLSI recommendations [8]. Ampicillin, penicillin, ciprofloxacin, levofloxacin, clindamycin, erythromycin, tetracycline, cotrimoxazole, gentamicin, imipenem, rifampicin, and vancomycin have been reported previously. As of February 1, 2024, the AST for Bacillus species transitioned from the MicroScan WalkAway 96 plus system (Siemens Healthcare Diagnostics Inc.) to the DxM 1096 MicroScan WalkAway system (Beckman Coulter). For the AST of gram-positive bacteria using these systems, modifying the organism designation to Bacillus species is necessary during the electronic data entry process after generating the predefined barcode required for recognition of the AST testing panel. If the organism designation is missed, AST results for ampicillin, imipenem, and levofloxacin may not be reported. Data were analyzed using IBM SPSS Statistics (version 25.0; IBM Corp.).
The Department of Laboratory Medicine at WSCH consistently participates in Proficiency Testing and External Quality Assurance programs organized by the Korean Association of External Quality Assessment Services. Additionally, the department holds accreditation through the Outstanding Laboratory Accreditation Program administered by the Laboratory Medicine Foundation and Korean Society for Laboratory Medicine.
Only one of the initially isolated Bacillus strains was included in the data analysis if the same Bacillus species was identified in multiple blood culture bottles from the same patient. If Bacillus species were identified in different specimen types from the same patient, each case was analyzed separately.
After excluding duplicates, a final set of 432 Bacillus isolates (502) were identified. Among the Bacillus isolates identified to the species level, the most common Bacillus species were B. cereus (25.1%), B. subtilis (11.8%), B. licheniformis (10.8%), B. pumilus (7.4%), B. simplex (6.9%), B. circulans (6.4%), B. amyloliquefaciens ssp. plantarum (5.9%), B. infantis (4.9%), B. thuringiensis (4.9%), and B. sonorensis (4.4%). The male-to-female isolation ratio of all Bacillus isolates was 1.17:1, with no statistically significant differences according to sex for any species (chi-square test, P = 0.492) (Table 1). The isolation frequency of Bacillus spp. by age group, in decreasing order, were 60s (23.8%), 70s (20.8%), ≥ 80s (20.4%), and 50s (14.8%), showing a higher isolation rate in older individuals. However, the Kruskal–Wallis rank sum test revealed no statistically significant differences in the age of the patients across the identified species (P = 0.222) (Table 1). The ratio of sterile to non-sterile specimens isolated from all Bacillus spp. was 0.58 (159/273). Among the Bacillus spp. with at least 10 isolates, the ratios of sterile to non-sterile specimens were as follows: B. licheniformis, 4.5; B. subtilis, 2.4; B. pumilus, 2.0; B. amyloliquefaciens ssp. plantarum, 2.0; B. circulans, 1.2; B. thuringiensis, 1.0; B. cereus, 0.76; B. simplex, 0.56; and B. infantis, 0.43 (Table 2).
Table 1. Basic characteristics of isolated Bacillus species
| Isolates | Patients’ information | Total No. (%)b | |||
|---|---|---|---|---|---|
| Sex female:male | Age (yr)a M (IQR) | ||||
| Bacillus amyloliquefaciens | 0:2 | 82 | (80–85) | 2 | (0.5/1.0) |
| Bacillus amyloliquefaciens ssp. plantarum | 8:4 | 74 | (64–78) | 12 | (2.8/5.9) |
| Bacillus asahii | 0:1 | 76 | 1 | (0.2/0.5) | |
| Bacillus cereus | 23:28 | 61 | (45–76) | 51 | (11.8/25.1) |
| Bacillus circulans | 7:6 | 61 | (53–74) | 13 | (3.0/6.4) |
| Bacillus clausii | 1:0 | 47 | 1 | (0.2/0.5) | |
| Bacillus cytotoxicus | 0:1 | 75 | 1 | (0.2/0.5) | |
| Bacillus flexus | 2:2 | 60 | (31–79) | 4 | (0.9/2.0) |
| Bacillus gibsonii | 2:0 | 68 | (51–84) | 2 | (0.5/1.0) |
| Bacillus halosaccharovorans | 0:3 | 52 | (33–62) | 3 | (0.7/1.5) |
| Bacillus idriensis | 1:1 | 65 | (55–75) | 2 | (0.5/1.0) |
| Bacillus indicus | 1:0 | 78 | 1 | (0.2/0.5) | |
| Bacillus infantis | 2:8 | 74 | (62–83) | 10 | (2.3/4.9) |
| Bacillus licheniformis | 12:10 | 74 | (67–80) | 22 | (5.1/10.8) |
| Bacillus mojavensis | 0:3 | 66 | (63–76) | 3 | (0.7/1.5) |
| Bacillus oceanisediminis | 1:0 | 82 | 1 | (0.2/0.5) | |
| Bacillus pumilus | 7:8 | 69 | (56–77) | 15 | (3.5/7.4) |
| Bacillus simplex | 7:7 | 67 | (60–74) | 14 | (3.2/6.9) |
| Bacillus sonorensis | 4:5 | 65 | (59–84) | 9 | (2.1/4.4) |
| Bacillus subtilis | 11:13 | 66 | (52–74) | 24 | (5.6/11.8) |
| Bacillus thuringiensis | 4:6 | 58 | (43–66) | 10 | (2.3/4.9) |
| Bacillus vallismortis | 0:1 | 80 | 1 | (0.2/0.5) | |
| Bacillus velezensis | 1:0 | 81 | 1 | (0.2/0.5) | |
| Bacillus species | 105:124 | 66 | (53–77) | 229 | (53.0) |
| Total | 199:233 | 67 | (53–77) | 432 | (100.0) |
aIf the number of isolation is one, only the patient’s age is presented. If the number of isolation is two, a mean of ages is indicated with the two patients as an age range. For three or more isolations, median (M) and interquartile range (IQR) are reported.
bThe number before the slash in parentheses is the frequency percentage including Bacillus species, and the number after the slash in parentheses is the frequency percentage excluding Bacillus species.
Table 2. Isolation frequency of Bacillus species according to specimen type
| Bacillus species (No. of isolates) | Blood | CSF | Peritoneal F | Pleural F | Synovial F | Catheter | Drain | Wound | Othersa |
|---|---|---|---|---|---|---|---|---|---|
| B. amyloliquefaciens (2) | 2(100.0) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B. amyloliquefaciens ssp. plantarum (12) | 8(66.7) | 0 | 0 | 0 | 0 | 0 | 3(25.0) | 1(8.3) | 0 |
| B. asahii (1) | 0 | 0 | 0 | 0 | 0 | 0 | 1(100.0) | 0 | 0 |
| B. cereus (51) | 13(25.5) | 0 | 7(13.7) | 2(3.9) | 0 | 0 | 4(7.8) | 24(47.1) | 1(2.0) |
| B. circulans (13) | 4(30.8) | 1(7.7) | 1(7.7) | 1(7.7) | 0 | 1(7.7) | 1(7.7) | 4(30.8) | 0 |
| B. clausii (1) | 1(100.0) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B. cytotoxicus (1) | 0 | 0 | 0 | 0 | 0 | 0 | 1(100) | 0 | 0 |
| B. flexus (4) | 1(25.0) | 0 | 0 | 0 | 0 | 1(25.0) | 1(25.0) | 1(25.0) | 0 |
| B. gibsonii (2) | 2(100.0) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B. halosaccharovorans (3) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 2(66.7) | 1(33.3) |
| B. idriensis (2) | 1(50.0) | 0 | 0 | 0 | 0 | 0 | 0 | 1(50.0) | 0 |
| B. indicus (1) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1(100.0) | 0 |
| B. infantis (10) | 3(30.0) | 0 | 0 | 0 | 0 | 4(40.0) | 2(20.0) | 0 | 1(10.0) |
| B. licheniformis (22) | 16(72.7) | 0 | 0 | 1(4.5) | 1(4.5) | 1(4.5) | 2(9.1) | 1(4.5) | 0 |
| B. mojavensis (3) | 2(66.7) | 0 | 0 | 0 | 0 | 0 | 1(33.3) | 0 | 0 |
| B. oceanisediminis (1) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1(100) | 0 |
| B. pumilus (15) | 7(46.7) | 0 | 0 | 2(13.3) | 1(6.7) | 0 | 0 | 5(33.3) | 0 |
| B. simplex (14) | 5(35.7) | 0 | 0 | 0 | 0 | 2(14.3) | 2(14.3) | 3(21.4) | 2(14.3) |
| B. sonorensis (9) | 2(22.2) | 0 | 0 | 1(11.1) | 0 | 1(11.1) | 3(33.3) | 2(22.2) | 0 |
| B. subtilis (24) | 15(62.5) | 0 | 0 | 2(8.3) | 0 | 0 | 4(16.7) | 3(12.5) | 0 |
| B. thuringiensis (10) | 4(40.0) | 0 | 0 | 1(10.0) | 0 | 0 | 0 | 5(50.0) | 0 |
| B. vallismortis (1) | 0 | 0 | 0 | 1(100) | 0 | 0 | 0 | 0 | 0 |
| B. velezensis (1) | 1(100.0) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Bacillus species (229) | 44(19.2) | 1(0.4) | 4(1.7) | 1(0.4) | 0 | 44(19.2) | 48(21.0) | 86(37.6) | 1(0.4) |
| Total | 131(30.3) | 2(0.5) | 12(2.8) | 12(2.8) | 2(0.5) | 54(12.5) | 73(16.9) | 140(32.4) | 6(1.4) |
Values are presented as n or n (%).
aUrine (2), bile fluid (2), bronchial washing (1), and liver abscess (1) are included.
Abbreviations: CSF, cerebrospinal fluid; F, fluid.
The overall rates of antimicrobial resistance decreased in the following order: penicillin G, 57.1%; ampicillin, 52.4%; clindamycin, 31.6%; erythromycin, 9.0%; cotrimoxazole, 6.4%; tetracycline, 2.3%; ciprofloxacin, 1.9%; rifampicin, 1.1%; levofloxacin, 0.9%; vancomycin, 0.4%; gentamicin, 0.4%; and imipenem, 0.4%. The only isolate showing vancomycin resistance was B. circulans strain isolated from the pleural fluid of a 59-year-old man with a vancomycin MIC of 32 mg/L, which was resistant to all tested antimicrobial agents except rifampicin (MIC 2 mg/L, intermediate). Penicillin resistance rates were particularly high in B. thuringiensis (100%), B. cereus (93.5%), and B. licheniformis (71.4%), with the ampicillin resistance rates similar to those of penicillin. Clindamycin resistance was high in B. circulans, B. licheniformis, and B. pumilus at rates of 81.8%, 71.4%, and 64.3%, respectively. Among the two B. cereus isolates resistant to ciprofloxacin, one exhibited intermediate resistance, and the other was resistant to levofloxacin (Table 3).
Table 3. Resistance rate of antimicrobial agents according to Bacillus species
| Bacillus speciesb | VAN (266)a | PEN (266) | TET (266) | CLI (266) | ERY (266) | SXT (266) | CIP (266) | GEN (266) | RIF (266) | IMP (231) | AMP (231) | LEV (231) |
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| B. amyloliquefaciens (2/2) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B. amyloliquefaciens ssp. plantarum (11/9) | 0 | 9.1 | 9.1 | 9.1 | 0 | 18.2 | 0 | 0 | 0 | 0 | 0 | 0 |
| B. asahii (1/1) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B. cereus (46/36) | 0 | 93.5 | 4.3 | 0 | 0 | 17.4 | 4.3 | 0 | 0 | 2.8 | 91.7 | 2.8 |
| B. circulans (11/7) | 9.1 | 27.3 | 9.1 | 81.8 | 27.3 | 18.2 | 9.1 | 9.1 | 0 | 0 | 0 | 0 |
| B. clausii (1/1) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B. cytotoxicus (1/1) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B. flexus (1/0) | 0 | 0 | 0 | 0 | 100 | 0 | 0 | 0 | 0 | – | – | – |
| B. gibsonii (2/2) | 0 | 0 | 0 | 50.0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B. halosaccharovorans (1/0) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | – | – | – |
| B. idriensis (1/1) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B. infantis (4/4) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B. licheniformis (21/19) | 0 | 71.4 | 0 | 71.4 | 28.6 | 0 | 0 | 0 | 0 | 0 | 73.7 | 0 |
| B. mojavensis (3/2) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B. oceanisediminis (1/1) | 0 | 100 | 0 | 100 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B. pumilus (14/13) | 0 | 0 | 7.1 | 64.3 | 7.1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B. simplex (7/7) | 0 | 28.6 | 0 | 0 | 14.3 | 0 | 0 | 0 | 0 | 0 | 28.6 | 0 |
| B. sonorensis (8/7) | 0 | 75.0 | 0 | 25.0 | 12.5 | 0 | 0 | 0 | 0 | 0 | 14.3 | 0 |
| B. subtilis (21/16) | 0 | 4.8 | 0 | 28.6 | 0 | 0 | 0 | 0 | 4.8 | 0 | 6.3 | 0 |
| B. thuringiensis (9/8) | 0 | 100 | 0 | 0 | 11.1 | 33.3 | 11.1 | 0 | 0 | 0 | 100 | 0 |
| B. vallismortis (1/1) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| B. velezensis (1/1) | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Bacillus species (98/92) | 0 | 72.4 | 1.0 | 40.8 | 10.2 | 2.0 | 1.0 | 0 | 2.0 | 0 | 67.4 | 1.1 |
| Total | 0.4 | 57.1 | 2.3 | 31.6 | 9.0 | 6.4 | 1.9 | 0.4 | 1.1 | 0.4 | 52.4 | 0.9 |
Values are presented as %.
aNumber in parenthesis refers to the tested number of isolates.
bThe number before the slash in parentheses is the number of 266 strains tested against nine antimicrobial agents excluding imipenem, ampicillin, and levofloxacin, and the number after the slash in parentheses is the number of strains tested against all 12 antimicrobial agents.
Abbreviations: VAN, vancomycin; PEN, penicillin G; TET, tetracycline; CLI, clindamycin; ERY, erythromycin; SXT, cotrimoxazole; CIP, ciprofloxacin; GEN, gentamicin; RIF, rifampicin; IMP, imipenem; AMP, ampicillin; LEV, levofloxacin.
In this study, the most frequently isolated Bacillus species was B. cereus (25.1%), followed by B. subtilis, B. licheniformis, B. pumilus, B. simplex, B. circulans, B. amyloliquefaciens ssp. plantarum, B. infantis, B. thuringiensis, and B. sonorensis (ranging from 4.4% to 11.8%).
B. cereus is a well-known cause of foodborne illness outbreaks because its spores can survive at cooking temperatures and cleaning procedures. Recently, B. cereus has been increasingly recognized as an opportunistic pathogen that causes localized wounds, eye, and systemic infections [18]. The pathogenicity of B. cereus, whether intestinal or non-intestinal, is closely associated with adhesion and tissue destructive/reactive secreted toxins, such as hemolysins, phospholipases, proteases, emesis-inducing toxins, and pore-forming enterotoxins [18,19]. B. thuringiensis strains are invertebrate pathogens that have been used as biopesticides, and some strains have been reported to cause bacteremia, mainly in immunocompromised individuals [20].
The Bacillus subtilis group included B. subtilis, B. licheniformis, B. pumilus, B. amyloliquefaciens, and B. sonorensis. B. subtilis is generally considered non-pathogenic and harmless to human health. It has also been used as a probiotic [21]. However, true infections, such as ocular infections, traumatic wound infections, meningitis, and bacteremia have also been reported [22]. B. licheniformis bacteremia is associated with the presence of long-term central venous catheters, especially in immunocompromised patients [23]. Although rare, B. pumilus has been reported to cause food poisoning, septic arthritis, cutaneous infections, and bacteremia [24]. Fan et al. [25] proposed that the B. amyloliquefaciens clade should be considered as a taxonomic unit above the species level, designated as “operational group B. amyloliquefaciens”. This group consists of soil-borne B. amyloliquefaciens, and plant associated B. siamensis and B. velezensis, whose members are closely related. These species undergo genomic changes as they adapt to the plant-associated lifestyles. B. sonorensis is closely related to B. licheniformis [26]. B. simplex is an environmental microorganism primarily found in soil and rarely causes infections in humans [27].
The Bacillus circulans group included B. circulans, B. firmus, and B. coagulans, B. circulans is commonly found in soil, and its non-pathogenic strains are utilized in industrial enzyme production, particularly of proteinases [28]. However, pathogenic strains of B. circulans have been implicated in multiple human infections, such as septicemia, wounds, and abscesses, particularly in immunocompromised individuals [28]. B. infantis was first isolated from a patient with neonatal sepsis and was closely related to B. firmus (98.2% sequence similarity) [29].
Identification of Bacillus species was based on Gram staining, an examination of colony morphology, and MALDI-TOF MS analysis. When MALDI-TOF MS was used to identify the Bacillus species, the most commonly isolated species in clinical laboratories were B. cereus, B. pumilus, B. subtilis, B. licheniformis, and B. simplex [30]. Although there are over 280 species in the Bacillus genus [19], the number of Bacillus species in the MALDI-TOF MS database library is much smaller, even though protein profile codes (universally unique identifiers) are continuously being added. Therefore, Bacillus spp. that can be identified by MALDI-TOF MS may vary depending on the version of the database library.
Bacillus species were detected in a range of specimen types. With respect to analysis based on the ratio of sterile to non-sterile specimens, although we obtained an average value of 0.58, there was a considerable variation among species, with ratios ranging from 0.43 to 4.5 among species for which more than 10 isolates were obtained. The reporting criteria for the identification level and AST of Bacillus species in clinical microbiology laboratories may vary depending on the type of specimen, colony count, and the clinical condition of the patient. When Bacillus species are isolated from non-sterile sites with low colony counts, descriptive identification based on Gram staining may be sufficient. In contrast, when numerous Bacillus spp. are isolated from open wounds with ground contamination, species-level identification and AST are required [31]. Cotton et al. [32] reported a significant difference in the frequency of recurrent bacteremia between 9.2% (positive in two of two bottles) and 3.3% (positive in one of two bottles) according to the degree of initial blood culture bottle positivity. Bacillus bacteremia occurs frequently in immunosuppressed patients, such as those with cancer or leukemia, and is considered an independent risk factor in patients with a central venous catheter and the use of extended-spectrum cephalosporins within one month [33].
When Bacillus species are isolated from clinical specimens, distinguishing between contamination and true infection is challenging for the following reasons: First, no clear criteria have been established based on specimen type to differentiate contamination from infection. Second, testing for the diverse toxins produced by Bacillus species in clinical laboratories is impractical. Third, the clinical characteristics of most Bacillus species remain poorly understood, with the exception of well-known pathogens, such as B. anthrax and B. cereus.
Our study revealed a marked variability in antimicrobial susceptibility patterns among Bacillus species. Therefore, when clinicians suspect a true infection due to Bacillus spp., the AST results can be of great help in treatment. Most B. cereus strains are intrinsically resistant to penicillin, cephalosporins, and β-lactamase inhibitor combinations because they produce three different β-lactamases (penicillinase, cephalosporinase, and metallolactamase) [34–36]. Therefore, β-lactam agents should be avoided in the empirical coverage of patients, especially in immunocompromised patients who have had Bacillus spp. isolated, until AST results are available. Additionally, in this study, B. licheniformis, B. pumilus, B. subtilis, and B. sonorensis, which belong to the B. subtilis group, were more resistant to clindamycin than other Bacillus species. B. circulans strains resistant to common antibiotics pose a threat as they can lead to the formation of more dangerous multidrug-resistant strains. Ligozzi et al. [37] reported that a vancomycin-resistant clinical isolate of B. circulans was associated with the acquisition of a vanA gene cluster located on the chromosome, showing a high degree of homology with that of enterococci. However, the cluster was not carried by Tn1546 and was borne by the chromosome.
This study has several limitations. First, 53.0% of the isolates could not be identified at the species level. Although molecular methods provide the most accurate identification, clinical laboratories often lack standardized AST guidelines and interpretation criteria for newly identified or rare organisms. As our laboratory prioritized rapid reporting to support patient care, the results were communicated to physicians even when full species-level identification was unavailable. Second, the AST results were not uniformly reported. The AST system requires manual organism designation to ensure proper reporting of key antibiotics, such as imipenem, ampicillin, and levofloxacin. Owing to this limitation, 13.2% of the 266 isolates tested lacked AST results for these antibiotics (Table 3), highlighting the need for improved system integration.
Bacillus species are increasingly integrated into various aspects of human life, leading to more frequent human contact. Most Bacillus species exhibit antibiotic resistance and have the potential to transfer antibiotic resistance genes to humans, suggesting that antibiotic residues may enter consumer food products and the human food chain. Therefore, continuous monitoring of the isolation frequency and AST results of Bacillus species isolated from clinical specimens is necessary.
This study focused on the analysis of previous identification records of Bacillus and did not involve human participants or the collection of individual patient information. Given the study design, the Institutional Review Board (IRB) waived the requirement for informed consent. The study was conducted in compliance with the principles of the Declaration of Helsinki and was approved by the IRB of WSCH (approval no. CR324123) on February 4, 2025.
Young Uh has been a statistical editor of the Annals of Clinical Microbiology since 2024. However, he was not involved in the review process of this article. No other potential conflicts of interest relevant to this article were reported.
None.
The datasets generated during the current study are available from the corresponding author upon request.
1. Haque MA, Wang F, Chen Y, Hossen F, Islam MA, Hossain MA, et al. Bacillus spp. contamination: a novel risk originated from animal feed to human food chains in South-Eastern Bangladesh. Front Microbiol 2022;12:783103.
2. Ehling-Schulz M, Lereclus D, Koehler TM. The Bacillus cereus group: Bacillus species with pathogenic potential. Microbiol Spectr 2019;7:10.1128/microbiolspec.gpp3-0032-2018.
3. Sliman R, Rehm S, Shlaes DM. Serious infections caused by Bacillus species. Medicine (Baltimore) 1987;66:218-23.
4. Navas M, Pincus DH, Wilkey K, Sercia L, LaSalvia M, Wilson D, et al. Identification of aerobic Gram-positive bacilli by use of Vitek MS. J Clin Microbiol 2014;52:1274-7.
5. Mills E, Sullivan E, Kovac J. Comparative analysis of Bacillus cereus group isolates’ resistance using disk diffusion and broth microdilution and the correlation between antimicrobial resistance phenotypes and genotypes. Appl Environ Microbiol 2022;88:e0230221.
6. Bottone EJ. Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev 2010;23:382-98.
7. Ihde DC and Armstrong D. Clinical spectrum of infection due to Bacillus species. Am J Med 1973;55:839-45.
8. Clinical and Laboratory Standards Institute. Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria. 3rd ed. CLSI supplement M45. Wayne, PA; CLSI: 2016.
9. Andrews JM and Wise R. Susceptibility testing of Bacillus species. J Antimicrob Chemother 2002;49:1040-2.
10. Citron DM and Appleman MD. In vitro activities of daptomycin, ciprofloxacin, and other antimicrobial agents against the cells and spores of clinical isolates of Bacillus species. J Clin Microbiol 2006;44:3814-8.
11. Coonrod JD, Leadley PJ, Eickhoff TC. Antibiotic susceptibility of Bacillus species. J Infect Dis 1971;123:102-5.
12. Adamski P, Byczkowska-Rostkowska Z, Gajewska J, Zakrzewski AJ, Kłębukowska L. Prevalence and antibiotic resistance of Bacillus sp. isolated from raw milk. Microorganisms 2023;11:1065.
13. Elshaghabee FMF, Rokana N, Gulhane RD, Sharma C, Panwar H. Bacillus as portential probiotics: status, concerns, and future perspectives. Front Microbiol 2017;8:1490.
14. Payne J, Bellmer D, Jadeja R, Muriana P. The potential of Bacillus species as probiotics in the food industry: a review. Foods 2024;13:2444.
15. Jin X, Zhang L, Cao Y, Dia Z, Ge X, Cai R, et al. Antibiotic resistance characterization, virulence factors and molecular characteristics of Bacillus species isolated from probiotic preparations in China. J Glob Antimicrob Resist 2024:S2213-7165(24)00124-3.
16. Cui Y, Wang S, Ding S, Shen J, Zhu K. Toxins and mobile antimicrobial resistance genes in Bacillus probiotics constitute a potential risk for One Health. J Hazard Mater 2020;385:121266.
17. Lin YC, Chen EHL, Chen RPY, Dunny GM, Hu WS, Lee KT. Probiotic Bacillus affects Enterococcus faecalis antibiotic resistance transfer by interfering with pheromone signaling cascades. Appl Environ Microbiol 2021;87:e0044221.
18. Bianco A, Capozzi L, Monno MR, Del Sambro L, Manzulli V, Pesole G, et al. Characterization of Bacillus cereus group isolates from human bacteremia by whole-genome sequencing. Front Microbiol 2021;11:599524.
19. Gupta RS, Patel S, Saini N, Chen S. Robust demarcation of 17 distinct Bacillus species clades, proposed as novel Bacillaceae genera, by phylogenomics and comparative genomic analyses: description of Robertmurraya kyonggiensis sp. nov. and proposal for an emended genus Bacillus limiting it only to the members of the Subtilis and Cereus clades of species. Int J Syst Evol Microbiol 2020;70:5753-98.
20. Baindara P and Aslam B. Editorial: Bacillus spp. – transmission, pathogenesis, host-pathogen interaction, prevention and treatment. Front Microbiol 2023;14:1307723.
21. Kovács ÁT. Bacillus subtilis. Trends Microbiol 2019;27:724-5.
22. Ishikawa K, Hasegawa R, Furukawa K, Kawai F, Uehara Y, Ohkusu K, et al. Recurrent Bacillus subtilis var. natto bacteremia and review of the literature on Bacillus subtilis: the first case report. Am J Case Rep 2024;25:e942553.
23. Blue SR, Singh VR, Saubolle MA. Bacillus licheniformis bacteremia: five cases associated with indwelling central venous catheters. Clin Infect Dis 1995;20:629-33.
24. Patel D, Ahmad A, Maheshwari P, Bird B, Albert A. Bacillus pumilus-borne food poisoning in an immunosuppressed host. Am J Med 2021;134:e573-4.
25. Fan B, Blom J, Klenk HP, Borriss R. Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamensis form an “Operational group B. amyloliquefaciens” within the B. subtilis species complex. Front Microbiol 2017;8:22.
26. Palmisano MM, Nakamura LK, Duncan KE, Istock CA, Cohan FM. Bacillus sonorensis sp. nov., a close relative of Bacillus licheniformis, isolated from soil in the Sonoran Desert, Arizona. Int J Syst Evol Microbiol 2001;51:1671-9.
27. Xaplanteri P, Serpanos DS, Dorva E, Beqo-Rokaj T, Papadogeorgaki E, Lekkou A. Bacillus simplex as the most probable culprit of penetrating trauma infection: a case report. Pathogens 2022;11:1203.
28. Hossain MI, Saleh NUA, Numan A, Hossain MM, Uddin MA, Hossain MS. Bombyx mori as a model for Niallia circulans pathogenicity. Drug Discov Ther 2023;17:18-25.
29. Ko KS, Oh WS, Lee MY, Lee JH, Lee H, Peck KR, et al. Bacillus infantis sp. nov. and Bacillus idriensis sp. nov., isolated from a patient with neonatal sepsis. Int J Syst Evol Microbiol 2006;56:2541-4.
30. Celandroni F, Salvetti S, Gueye SA, Mazzantini D, Lupetti A, Senesi S, et al. Identification and pathogenic potential of clinical Bacillus and Paenibacillus isolates. PLoS One 2016;11:e0152831.
31. Wong MT and Dolan MJ. Significant infections due to Bacillus species following abrasions associated with motor vehicle-related trauma. Clin Infect Dis 1992;15:855-7.
32. Cotton DJ, Gill VJ, Marshall DJ, Gress J, Thaler M, Pizzo PA. Clinical features and therapeutic interventions in 17 cases of Bacillus bacteremia in an immunosuppressed patient population. J Clin Microbiol 1987;25:672-4.
33. Ko JH, Kang CI, Lee WJ, Huh K, Yoo JR, Kim K, et al. Clinical features and risk factors for development of Bacillus bacteremia among adult patients with cancer: a case-control study. Support Care Cancer 2015;23:377-84.
34. Hussain M, Pastor FI, Lampen JO. Cloning and sequencing of the blaZ gene encoding β-lactamase III, a lipoprotein of Bacillus cereus 569/H. J Bacteriol 1987;169:579-86.
35. Weber DJ, Saviteer SM, Rutala WA, Thomann CA. In vitro susceptibility of Bacillus spp. to selected antimicrobial agents. Antimicrob Agents Chemother 1988;32:642-5.
36. Torkar KG and Bedenić B. Antimicrobial susceptibility and characterization of metallo-β-lactamases, extended-spectrum β-lactamases, and carbapenemases of Bacillus cereus isolates. Microb Pathog 2018;118:140-5.
37. Ligozzi M, Lo Cascio G, Fontana R. vanA gene cluster in a vancomycin-resistant clinical isolate of Bacillus circulans. Antimicrob Agents Chemother 1998;42:2055-9.
1. Haque MA, Wang F, Chen Y, Hossen F, Islam MA, Hossain MA, et al. Bacillus spp. contamination: a novel risk originated from animal feed to human food chains in South-Eastern Bangladesh. Front Microbiol 2022;12:783103.
2. Ehling-Schulz M, Lereclus D, Koehler TM. The Bacillus cereus group: Bacillus species with pathogenic potential. Microbiol Spectr 2019;7:10.1128/microbiolspec.gpp3-0032-2018.
3. Sliman R, Rehm S, Shlaes DM. Serious infections caused by Bacillus species. Medicine (Baltimore) 1987;66:218-23.
4. Navas M, Pincus DH, Wilkey K, Sercia L, LaSalvia M, Wilson D, et al. Identification of aerobic Gram-positive bacilli by use of Vitek MS. J Clin Microbiol 2014;52:1274-7.
5. Mills E, Sullivan E, Kovac J. Comparative analysis of Bacillus cereus group isolates’ resistance using disk diffusion and broth microdilution and the correlation between antimicrobial resistance phenotypes and genotypes. Appl Environ Microbiol 2022;88:e0230221.
6. Bottone EJ. Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev 2010;23:382-98.
7. Ihde DC and Armstrong D. Clinical spectrum of infection due to Bacillus species. Am J Med 1973;55:839-45.
8. Clinical and Laboratory Standards Institute. Methods for antimicrobial dilution and disk susceptibility testing of infrequently isolated or fastidious bacteria. 3rd ed. CLSI supplement M45. Wayne, PA; CLSI: 2016.
9. Andrews JM and Wise R. Susceptibility testing of Bacillus species. J Antimicrob Chemother 2002;49:1040-2.
10. Citron DM and Appleman MD. In vitro activities of daptomycin, ciprofloxacin, and other antimicrobial agents against the cells and spores of clinical isolates of Bacillus species. J Clin Microbiol 2006;44:3814-8.
11. Coonrod JD, Leadley PJ, Eickhoff TC. Antibiotic susceptibility of Bacillus species. J Infect Dis 1971;123:102-5.
12. Adamski P, Byczkowska-Rostkowska Z, Gajewska J, Zakrzewski AJ, Kłębukowska L. Prevalence and antibiotic resistance of Bacillus sp. isolated from raw milk. Microorganisms 2023;11:1065.
13. Elshaghabee FMF, Rokana N, Gulhane RD, Sharma C, Panwar H. Bacillus as portential probiotics: status, concerns, and future perspectives. Front Microbiol 2017;8:1490.
14. Payne J, Bellmer D, Jadeja R, Muriana P. The potential of Bacillus species as probiotics in the food industry: a review. Foods 2024;13:2444.
15. Jin X, Zhang L, Cao Y, Dia Z, Ge X, Cai R, et al. Antibiotic resistance characterization, virulence factors and molecular characteristics of Bacillus species isolated from probiotic preparations in China. J Glob Antimicrob Resist 2024:S2213-7165(24)00124-3.
16. Cui Y, Wang S, Ding S, Shen J, Zhu K. Toxins and mobile antimicrobial resistance genes in Bacillus probiotics constitute a potential risk for One Health. J Hazard Mater 2020;385:121266.
17. Lin YC, Chen EHL, Chen RPY, Dunny GM, Hu WS, Lee KT. Probiotic Bacillus affects Enterococcus faecalis antibiotic resistance transfer by interfering with pheromone signaling cascades. Appl Environ Microbiol 2021;87:e0044221.
18. Bianco A, Capozzi L, Monno MR, Del Sambro L, Manzulli V, Pesole G, et al. Characterization of Bacillus cereus group isolates from human bacteremia by whole-genome sequencing. Front Microbiol 2021;11:599524.
19. Gupta RS, Patel S, Saini N, Chen S. Robust demarcation of 17 distinct Bacillus species clades, proposed as novel Bacillaceae genera, by phylogenomics and comparative genomic analyses: description of Robertmurraya kyonggiensis sp. nov. and proposal for an emended genus Bacillus limiting it only to the members of the Subtilis and Cereus clades of species. Int J Syst Evol Microbiol 2020;70:5753-98.
20. Baindara P and Aslam B. Editorial: Bacillus spp. – transmission, pathogenesis, host-pathogen interaction, prevention and treatment. Front Microbiol 2023;14:1307723.
21. Kovács ÁT. Bacillus subtilis. Trends Microbiol 2019;27:724-5.
22. Ishikawa K, Hasegawa R, Furukawa K, Kawai F, Uehara Y, Ohkusu K, et al. Recurrent Bacillus subtilis var. natto bacteremia and review of the literature on Bacillus subtilis: the first case report. Am J Case Rep 2024;25:e942553.
23. Blue SR, Singh VR, Saubolle MA. Bacillus licheniformis bacteremia: five cases associated with indwelling central venous catheters. Clin Infect Dis 1995;20:629-33.
24. Patel D, Ahmad A, Maheshwari P, Bird B, Albert A. Bacillus pumilus-borne food poisoning in an immunosuppressed host. Am J Med 2021;134:e573-4.
25. Fan B, Blom J, Klenk HP, Borriss R. Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamensis form an “Operational group B. amyloliquefaciens” within the B. subtilis species complex. Front Microbiol 2017;8:22.
26. Palmisano MM, Nakamura LK, Duncan KE, Istock CA, Cohan FM. Bacillus sonorensis sp. nov., a close relative of Bacillus licheniformis, isolated from soil in the Sonoran Desert, Arizona. Int J Syst Evol Microbiol 2001;51:1671-9.
27. Xaplanteri P, Serpanos DS, Dorva E, Beqo-Rokaj T, Papadogeorgaki E, Lekkou A. Bacillus simplex as the most probable culprit of penetrating trauma infection: a case report. Pathogens 2022;11:1203.
28. Hossain MI, Saleh NUA, Numan A, Hossain MM, Uddin MA, Hossain MS. Bombyx mori as a model for Niallia circulans pathogenicity. Drug Discov Ther 2023;17:18-25.
29. Ko KS, Oh WS, Lee MY, Lee JH, Lee H, Peck KR, et al. Bacillus infantis sp. nov. and Bacillus idriensis sp. nov., isolated from a patient with neonatal sepsis. Int J Syst Evol Microbiol 2006;56:2541-4.
30. Celandroni F, Salvetti S, Gueye SA, Mazzantini D, Lupetti A, Senesi S, et al. Identification and pathogenic potential of clinical Bacillus and Paenibacillus isolates. PLoS One 2016;11:e0152831.
31. Wong MT and Dolan MJ. Significant infections due to Bacillus species following abrasions associated with motor vehicle-related trauma. Clin Infect Dis 1992;15:855-7.
32. Cotton DJ, Gill VJ, Marshall DJ, Gress J, Thaler M, Pizzo PA. Clinical features and therapeutic interventions in 17 cases of Bacillus bacteremia in an immunosuppressed patient population. J Clin Microbiol 1987;25:672-4.
33. Ko JH, Kang CI, Lee WJ, Huh K, Yoo JR, Kim K, et al. Clinical features and risk factors for development of Bacillus bacteremia among adult patients with cancer: a case-control study. Support Care Cancer 2015;23:377-84.
34. Hussain M, Pastor FI, Lampen JO. Cloning and sequencing of the blaZ gene encoding β-lactamase III, a lipoprotein of Bacillus cereus 569/H. J Bacteriol 1987;169:579-86.
35. Weber DJ, Saviteer SM, Rutala WA, Thomann CA. In vitro susceptibility of Bacillus spp. to selected antimicrobial agents. Antimicrob Agents Chemother 1988;32:642-5.
36. Torkar KG and Bedenić B. Antimicrobial susceptibility and characterization of metallo-β-lactamases, extended-spectrum β-lactamases, and carbapenemases of Bacillus cereus isolates. Microb Pathog 2018;118:140-5.
37. Ligozzi M, Lo Cascio G, Fontana R. vanA gene cluster in a vancomycin-resistant clinical isolate of Bacillus circulans. Antimicrob Agents Chemother 1998;42:2055-9.