Identification, characterization and antibiotic susceptibility testing for Bacillus species

Background and Objectives: With the increase in immunosuppressed patients and antimicrobial misuse, Bacillus species have risen as opportunistic pathogens in hospitalized patients. The present study aimed at comparing chromogenic media, automated identification cards versus MALDI-TOF as the gold standard method for identification of different Bacillus species and determining minimal inhibitory concentration (MIC) of different antibiotics by broth microdilution method (BMD) to suggest recommendations for Bacillus treatment. Materials and Methods: The study included 30 Bacillus species isolates recovered from normally sterile sites of the human body and were subjected to identification by MALDI-TOF, Vitek-2c, and HiCrome Bacillus Agar. BMD test was performed to determine the MIC of vancomycin, gentamicin, and ciprofloxacin. Results: Our study showed B. cereus was the most commonly isolated species (76.66%) followed by B. subtilis (23.33%). Regarding the different methods of identification, the highest agreement with MALDI-TOF was exhibited by HiCrome agar without polymyxin B (93.3%) followed by Vitek-2C and HiCrome agar with polymyxin with an agreement of 83.3%. Concerning the antibiogram, the tested isolates showed a susceptibility of 93.3%, 86.6%, and 83.3% towards vancomycin, gentamicin, and ciprofloxacin respectively. Conclusion: In conclusion, we spotlight that Bacillus species should no longer be considered contaminant bacteria in cultures, particularly in immunosuppressed patients. HiCrome Bacillus Agar without polymyxin B displayed the highest agreement with MALDI-TOF. Hence, it represents a good option for identification for routine laboratories where expensive instru-ments are unavailable. The high susceptibility towards the tested antibiotics can suggest the possibility of empirical use of vancomycin, gentamicin, and ciprofloxacin.


INTRODUCTION
Bacillus species are Gram-positive, spore-forming, rod-shaped, aerobic or facultative anaerobic bacteria (1). Predisposing factors responsible for hu-man infections with Bacillus spp. other than Bacillus anthracis include chronic alcoholism, presence of intravascular devices, intravenous drug abuse, and trauma (2).
Although Bacillus species are uncommonly rehttp://ijm.tums.ac.ir IRAN. J. MICROBIOL. Volume 14 Number 4 (August 2022) 484-494 2 ported as major human pathogens, they have been frequently reported among debilitated patients including hospitalized patients. The major drawback of the inadequate reporting of Bacillus spp. infections by clinical microbiology laboratories are the fact that most of these bacteria are saprophytic and their isolation in human clinical specimens is neglected as laboratory contaminants (3). The clinical range of infections caused by Bacillus spp. include bacteraemia, endocarditis, wound infections, eye infection, respiratory tract infections, urinary tract infections and gastrointestinal tract, food poisoning, and meningitis (3,4). Laboratory identification of Bacillus spp. includes certain biochemical reactions and physiological properties such as anaerobically growth, motility, catalase production, citrate utilization, nitrate reduction, and glucose fermentation (3).
To overcome the limitations of conventional plating methods, selective chromogenic media are now widely used for the identification and differentiation of B. cereus from other members of the group. These media contain synthetic fluorogenic and chromogenic substrates that are cleaved by specific enzymatic activities (5).
Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), which can be used to analyze the protein composition of a bacterial cell, has arisen as a new technology for species identification. MALDI-TOF MS is suitable for high-throughput and rapid diagnostics at low costs and can be considered an alternative for conventional biochemical and molecular identification systems in a conventional microbiological laboratory (6).
The present study aimed at comparing chromogenic media, automated identification cards versus MALDI-TOF as the gold standard method for identification of different Bacillus species and determining minimal inhibitory concentration of different antibiotics by broth microdilution method (BMD) to suggest recommendations for Bacillus treatment. Study population and inclusion criteria. Thirty (30) non-duplicate pure Bacillus species isolates were recovered from sterile sites of the human body e.g. blood cultures, body fluids, and infected deep site surgical wounds. We collected isolates suggested to be pathogenic from ICU patients with a history of immunosuppression such as the use of immunosuppressive drugs and those with debilitating diseases. Also, we selected patients to be enrolled in our study based on their clinical data such as symptoms suggesting infections like bacteremia and deep-seated wound infections. Isolates were preliminarily identified as Bacillus spp. by Gram stain and biochemical tests that included positive Oxidase test, positive Catalase test, and positive Motility test in accordance with the identification procedures followed at the microbiology laboratory (3).

MATERIALS AND METHODS
All the 30 isolates of Bacillus spp. were identified by Matrix-Assisted Laser Desorption Ionisation -Time of Flight (MALDI-TOF MS) (BioMérieux SA, Marcy l'Etoile, France).
Isolates were subcultured on 5% sheep blood agar and incubated for 24 h at 35°C in 5 to 10% CO . Then, a thin film of fresh colony growth was picked by VITEK PICKME TM pen and NIBS and smeared directly onto a well of the VITEK MS-DS TM 48-welled target slides plate, then 1 µL of ready-to-use ɑ-cyano-4-hydroxycinnamic acid (VITEK MS-CHCA TM matrix) was added directly to the organism on the target slide and allowed to dry for 1-2 minutes to absorb energy from the VITEK MS laser and transfered it to the microorganisms to enable ionization (Fig. 1). As recommended by the manufacturer's instructions, the Escherichia coli ATCC 8739 strain, used as a calibrator and internal ID control, was inoculated on the calibration spots of each acquisition group (a small spot in the middle of each acquisition group) (7).
2. Vitek@2C BCL ID card for Bacillus species (bioMérieux SA, Marcy l'Etoile, France): The bacterial suspensions were prepared from fresh colonies in 5 ml of sterile saline to be adjusted using the VITEK2 DensiChek (bioMérieux SA, Marcy l'Etoile, France) to a McFarland standard 2.0with accepted range of 1. 8  3. Chromogenic media for Bacillus spp. (HI-Crome TM Bacillus Agar, India). The media was prepared and dispensed in screw-capped flasks. Briefly, flasks were boiled and sterilized by autoclaving for 15 minutes. After boiling, the medium was homogenized under continuous stirring and cooled to 50°C. In the case of preparing media supplemented with polymyxin, aseptically rehydrated content of 1 vial of polymyxin B was added to one liter. Then, the fresh 24 hours colonies were sub-cultured on two culture plates, one chromogenic media supplemented with polymyxin B (10 gm per liter) and another without polymyxin B (Fig. 3).
As recommended by the manufacturer's instructions, polymyxin B was not added to improve the recovery of Bacillus species beyond the B. cereus group (9). Also, the Enterococcus faecalis ATCC 29212 strain can be used for quality control (Fig. 4).
Fifty microliters (50 μl) of Mueller Hinton broth (MHB) were added to the wells in 96-well plates, and then each antibiotic was added in serial dilution in a row, with starting drug concentration of 64 µg/ml for vancomycin, gentamicin and 32 µg/ml for cipro-floxacin. Then were serially diluted, so that the starting concentration and final dilution covered fourfold above the resistant breakpoint and fourfold below the sensitive one, finally, five microliters (5 μl) of bacterial suspension adjusted to a 0.5 McFarland turbidity standard was inoculated into each antibiotic containing well. Staphylococcus aureus ATCC® 29213 was used as a quality control strain as recommended by CLSI M45 (2016) (10).

Data management and atatistical analysis.
The collected data was revised, coded, tabulated and introduced to a PC using the Statistical Package for Social Science (IBM Corp. Released 2019. IBM SPSS Statistics for Windows, Version 25.0 Armonk, NY: IBM Corp). Data were expressed as both numbers and percentages for categorized data.
The following tests were done: 1.
Chi-square test to study the association between each 2 variables or comparison between 2 independent groups as regards the categorized data. 2.
Cohen's Kappa statistics was used to measure the agreement between every two techniques. The agreement was interpreted as follows: Kappa's 0.80-1.00 is excellent, 0.61 to< 0.80 is good, 0.40-0.60 is moderate, Fair if 0.21-<0.40, and poor if below 0.20.
The probability of error at 0.05 was considered significant, while at 0.01 and 0.001 are highly significant.
3. Diagnostic validity test was assessed by calculating the following: a. The diagnostic sensitivity: It is the percentage of diseased cases truly diagnosed (TP) among total diseased cases (TP+FN)  non-diseased truly excluded by the test (TN) among total non-diseased cases (TN+FP). c. The predictive value for a positive test: It is the percentage of cases truly diagnosed among total positive cases.
d. The predictive value for a negative test: It is the percentage of cases truly negative among total negative cases.

RESULTS
The present study was conducted on 30 Bacillus species isolates that were recovered from different clinical specimens submitted to the Central Microbiology Laboratory, Ain Shams University Hospitals for culture and antimicrobial susceptibility testing. Isolates were identified biochemically following the microbiology laboratory procedures and were tested for further identification including MALDI-TOF as the reference method, VITEK2-C and chromogenic agar media and also tested for their susceptibility to different antimicrobial agents by the MIC method. As regards types of samples in our study, 80% (24/30) were isolated from blood culture and 20% (6/30) were isolated from a deep wound, also 71.4% (5/7) of B. subtilis were isolated from wound culture and 28.6% (2/7) of B. subtilis were isolated from blood culture, while 91.4% (21/23) of B. cereus were isolated from blood culture and 8.6% (2/23) of B. cereus were isolated from wound culture. The types of samples enrolled in our study are summarized in Fig. 4.  As for the diagnostic performance for each identification method performed in the current study, we found that the VITEK 2c system and chromogenic media supplemented with polymyxin B for B. cereus had specificity, sensitivity, and negative predictive value, and a positive predictive value of 100% each. While for B. subtilis, the method had specificity, sensitivity, negative predictive value and positive predictive value of 100%, 28.6%, 82.1%, and 100% respectively. While the chromogenic media without polymyxin B for B. cereus had specificity, sensitivity, negative predictive value and positive predictive value of 100% for each. B. subtilis had specificity, and a positive predictive value of 100% with better sensitivity and negative predictive values of 71.4% and 92% respectively.
Regarding the agreement in the identification results of the Bacillus species among Vitek MS and Vitek2c system methods, data in Table 3 showed a statistically fair/good agreement (Kappa = 0.58). Both methods showed 100% agreement in the identification of B. cereus and 83.3% agreement in B. subtilis isolates.
Also, a statistically fairly good agreement was observed between Vitek MS and Hicrome Agar with supplement method regarding the identification results of the Bacillus spp. (Kappa = 0.58) is shown in Table 4. Both methods showed 100% agreement in the identification of B. cereus and 83.3% agreement in B. subtilis isolates.  Concerning the MIC results of the three tested antibiotics, the MIC of the two resistant isolates for vancomycin were 16 and 64 ug/ml. Whilst the MIC of the four resistant isolates for gentamicin ranged from 32 to 64 ug/ml. As for ciprofloxacin, the MIC of the five resistant isolates ranged from 8 to 32 ug/ml. Table 6 summarizes the results of the MIC of the thirty studied isolates.

DISCUSSION
Although Bacillus spp. have not been recognized a major human pathogen, but with recent advances in medical technology and increased number of immunosuppressed patients, they have been increasingly recognized as opportunistic pathogens in hospital-   (3,11). The identification of species in the genus Bacillus by classical methods is often difficult, despite still prevailing in many microbiology laboratories, due to similarities among closely related species that share a pattern of morphological, biochemical, and genetic characteristics. The use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) as a diagnostic technique for Bacillus spp. identification has been applied for addressing the challenges associated with the identification of these organisms (12).
We conducted the present study in Ain Shams University Hospitals in collaboration with 57357 hospitals during the period between December 2018 and December 2019. A total of 30 Bacillus isolates were recovered from different clinical specimens (blood and deep wound swab) from the clinically diagnosed ICU bacteremic patients and those with infected deep-seated wounds respectively. Isolates were preliminarily identified phenotypically by Gram stain, oxidase, catalase, and motility test. They were further subjected to subspecies identification by MAL-DI-TOF MS, Vitek@2C ID cards, and chromogenic agar media. We performed minimal inhibitory concentration measurement of the following antibiotics (vancomycin, gentamicin, ciprofloxacin) by BMD to suggest recommendations for Bacillus treatment.
Regarding the results of MALDI-TOF, the present study showed that 23/30 (76.66%) were Bacillus cereus and 7/30 (23.33%) were Bacillus subtilis. Eighty percent of the isolates were recovered from blood culture while twenty percent were recovered from deep wounds. In a similar fashion, Fujita and Nishiura (2018) from Japan who did their research during subtilis at a high rate as well (14).
We noticed that B. cereus displayed several group-specific peaks at 3417, 6699 Da. The intensity of which varied depending on the degree of sporulation. Comparable findings were reported by Lasch and colleagues (2009) in Germany who worked on veterinarian strains and they noted that the B. cereus group members B. anthracis, B. cereus, and B. thuringiensis displayed several group-specific peaks at 3,683, 4,334, 5,171, 5,886, and 7,368 Da respectively (15).
Using HiCrome Bacillus Agar supplemented with polymyxin B, 83.3 % of total isolates were well identified with 76.66% (23/30) Bacillus cereus and 6.6% (2/30) Bacillus subtilis. It is worthy to note that the presence of polymyxin B supplement resulted in inhibition of 16.6 % (5/30) of total isolates showing no growth. While in absence of the polymyxin B supplement, the performance of HiCrome Bacillus Agar improved and 93.3 % of total isolates were successfully identified with 76.66% (23/30) were Bacillus cereus and 16.6% (5/30) were Bacillus subtilis with lowering the inhibition rate to 6.6% (2/30). In Argentina, a study done by Alippi and Abrahamovich (2019) on eighty-three isolates of B. cereus of honey coincided with our findings and reported that HiCrome media correctly identified all their isolates (9). Among the tested methods, HiCrome Bacillus Agar without polymyxin B displayed the highest agreement with our reference method. Hence, it represents a good option for Bacillus identification in routine laboratories where expensive and sophisticated instruments are unavailable.
As for the results of antimicrobial testing using the BMD Method, out of the 30 studied isolates, 28 (93.3%), 26 (86.6%), and 25 (83.3%) were susceptible to vancomycin, gentamicin and ciprofloxacin respectively. Similarly, Celandroni and his team in their study stated that all Bacillus species were susceptible to vancomycin and ciprofloxacin by MIC using E-test (14). Based on our reported results, this high susceptibility towards the examined antibiotics can propose their potential effect as empirical therapy. However, we must consider performing antimicrobial susceptibility to avoid the phenomenon of antimicrobial resistance.
On searching several databases, we found that similar studies were lacking and most of the available studies were not concerned about the medical aspect. Instead, almost all the researchers' interests were directed toward environmental, pharmaceutical and food studies. Hence, we faced difficulties to find counterparts with whom we could compare our results. Very few studies regarding the antimicrobial susceptibility of clinical Bacillus species were available. Several case reports had recorded successful clinical outcomes using various antibiotic combinations. A case report by Aygun et al. (2016) in Istanbul University, Turkey stated that Bacillus cereus was isolated in blood cultures obtained from a 16-monthold male patient and vancomycin, meropenem, and amikacin sulfate were started empirically and continued for 14 days till recovery (16).
Likewise, Hillard and colleagues (2003) from the USA stated that a case of bacteremia caused by B. cereus in a 19-day-old preterm neonate was successfully treated with vancomycin, aminoglycosides, meropenem, and clindamycin (17).
In a retrospective study between April 2003 and March 2012, Ikeda and his team (2015) from Japan investigated the antimicrobial susceptibility of B. cereus in bacteremic patients. Their 29 isolates expressed no resistance towards vancomycin, gentamicin, and imipenem. However, 65.5% and 10.3% were resistant to clindamycin and levofloxacin respectively (19).
In our study, we highlighted the value of Bacillus spp. as an opportunistic pathogen affecting those with immunocompromised status. To our knowledge, we are one among the few studies to compare all these methodologies for the identification of Bacillus species. Moreover, medical studies regarding Bacillus spp. in humans and scarce and most of the available studies are concerned about those in the food industry as well as of the environmental source. Thus, we felt the urge to withdraw the attention to such a neglected organism that is not receiving enough attentiveness.
It is worthy to note that although the majority of our isolates were susceptible to almost all the tested antibiotics. However, this low rate of resistance can raise the awareness of the possibility of development of higher rate resistance especially in the era of multidrug resistance. Thus, further studies are recommended to follow up the resistance pattern of the Bacillus species. Molecular studies, as well, are highly encouraged for better understanding of the genetic basis of resistance.

CONCLUSION
Bacillus spp. should no longer be considered as contaminating bacteria in cultures especially in patients with a history of immunosuppression such as the use of immunosuppressive drugs and ICU patients with a debilitating disease. Hence testing of isolates from normally sterile sources (eg, deep tissue, CSF, multiple positive blood cultures) may be warranted.
B. cereus was the most frequently isolated Bacillus spp. (76.66%) followed by B. subtilis (23.33%). The majority of B. cereus isolates were from blood cultures from immunocompromised patients mostly on steroid therapy, while B. subtilis was isolated from surgical site infection.
As regards the different methods of identification in the present study, HiCrome agar without polymyxin B exhibited the highest agreement (93.3%) which was comparable with our gold standard method ''MALDI-TOF'' followed by Vitek-2C and Hi-Crome agar with polymyxin with an agreement of 83.3% each. We found that the addition of HiCrome polymyxin B selective supplement -according to the manufacturer's instruction -has no additional value in the identification of Bacillus species, especially with inhibition of growth of B. subtilis. Also, using the chromogenic media can aid in correctly identifying a large number of isolates using the same plate instead of using a large battery of biochemical reactions which can be tiresome. Thus, we recommend using HiCrome agar without polymyxin as a practical, easy and affordable method for the identification of Bacillus even in those laboratories with limited resources. However, further studies using different types of Bacillus isolates should be conducted to test the capability of HiCrome media for the identification of other types.
Concerning the antibiogram of the studied isolates using BMD, the tested isolates showed a susceptibility of 93.3%, 86.6%, and 83.3% towards vancomycin, gentamicin, and ciprofloxacin respectively. Further clinical studies on a wider scale with a large number of patients are highly recommended to verify the effectiveness of the suggested empirical therapy reported in the current study.
It is worthy to note that although the majority of our isolates were susceptible to almost all the tested antibiotics. However, this low rate of resistance can raise the awareness of the possibility of developing a higher rate of resistance, especially in the current era of multidrug resistance. Thus, further studies are recommended to follow up on the resistance pattern of the Bacillus species. And molecular studies, as well, are highly encouraged for a better understanding of the genetic basis of resistance.