Allium sativum and Group B Streptococcus: antimicrobial and synergistic activity

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Allium sativum and Group B Streptococcus: antimicrobial and synergistic activity

https://doi.org/10.21203/rs.3.rs-1149854/v3

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Maternal colonization by Streptococcus agalactiae or Group B Streptococcus(GBS) during pregnancy increases the risk of neonatal infection due to vertical transmission from mother to fetus before or during labor. The study aimed to evaluate the antimicrobial activity of the SP80 fraction (obtained from Allium sativum) and its synergism associated with the antibiotic against strains of GBS. The broth microdilution was used to determine the antimicrobial activity of the SP80 and to establish the MIC of SP80 (2.40 mg/mL). By using, the disk diffusion method, fifty-five clinical isolates and 1 ATCC of GBS were tested against the association of the SP80 fraction with antibiotic PEN and AMP, respectively, for synergistic assessment. The results showed that the association of SP80 + PEN did not alter the response of PEN alone. In contrast, the association of SP80 + AMP caused the mean inhibition halos to increase with a p<0.001. The SP80 fraction has antimicrobial activity against GBS, and the association with the antibiotic AMP showed a synergistic effect, which did not occur when in association with PEN.

General Microbiology

Bacteriology

Allium sativum

GBS

Streptococcus agalactiae

Neonatal Infections

Antibiotics

Synergy

Streptococcus agalactiae or Group B Streptococcus (GBS) are bacteria that inhabit the human gastrointestinal and vaginal tracts and can develop into invasive infections in the newborn during the first weeks of life (Nascimento et al. 2019).

GBS disease in newborns can be classified according to the time of onset of infection. Early-Onset Disease (EOD) occurs 12 to 48 hours after birth or up to the first 7 days and is generally due to mother-to-child transmission during childbirth, and it can cause severe problems, such as meningitis, pneumonia, and sepsis. Late-Onset Disease (LOD) occurs between a week to a few months after birth and it can cause meningitis and the contamination source may be in the infant’s home environment (Furfaro et al. 2018).

In 1996, prophylactic recommendations such as the use of antibiotics during labor were implemented to prevent GBS infections in newborns by the American College of Obstetricians and Gynecologists (ACOG)(American College of Obstetricians and Gynecologists 1996) and Centers for Disease Control and Prevention (CDC)(Centers for Disease Control and Prevention 1996) and then in 1997 by the American Academy of Pediatrics (AAP) (American Academy of Pediatrics 1997). According to the Consensus Reviews, the guidelines were revised in 2002 and 2010, and universal recommendations were issued for the collection of vaginal secretions from all pregnant women between 35 to 37 weeks of gestation (Centers for Disease Control and Prevention 2010). In 2018, the responsibility and management for updating the guidelines were transferred from the CDC to ACOG and AAP (Zhu and Lin 2021).

Currently, as part of routine prenatal care, all pregnancy, between 36 to 38 weeks of gestation, are screened for GBS. A culture from the vagina and rectum is done to optimize the identification of those who should receive appropriate intrapartum antibiotic prophylaxis (IAP) (American College of Obstetricians and Gynecologists 2020; Puopolo et al. 2019).

The use of antibiotics during pregnancy was not seen to be beneficial, but at the beginning of labor, it is (Word Health Organization 2015). The use of 5.000.000 IU (intravenous) penicillin G (PEN) is recommended, maintaining 2.500.000 IU every 4 hours, until the moment of delivery; or starting with 2 g (intravenous) ampicillin (AMP) first and then administering an additional 1 g every 4 hours, until the moment of childbirth (Word Health Organization 2015). In the case of allergic pregnant women, it is recommended 500 mg (intravenous) erythromycin every 6 hours, or 900 mg clindamycin every 8 hours; if the pregnant woman is GBS resistant, 1 g vancomycin is administered intravenously every 12 hours However, this prophylaxis does not prevent LOD (Martins et al. 2017; Morguette et al. 2018).

Although prophylactic measures are already well established by conventional medicine, the use of medicinal plants in disease prevention is quite common in developing countries like Brazil (Sistema Único de Saúde 2009). When researching the use of garlic for the treatment of GBS infection in traditional medicine, we were able to find some published articles describing positive results (Cohain 2009; Cohain 2010).

Garlic (Allium sativum L.), part of the family Liliaceae, is one of the most researched medicinal plants in the world and has been used for centuries in cooking and traditional medicine (Majewski 2014). In Brazil, this plant belongs to a list comprising 71 plants of interest to the SUS (acronym for Sistema Único de Saúde, i.e. Brazil’s Universal Health System) issued by the Ministry of Health’s National Program of Medicinal Plants and Herbal (Sistema Único de Saúde 2009).

This plant species has been used as a remedy for several diseases, such as antioxidant (Capasso 2013), antitumoral (Nicastro et al. 2015), anti-inflammatory (Lee et al. 2012), immunomodulatory (Percival 2016), antiviral (Mehrbod et al. 2009), antimicrobial (Lanzotti et al. 2014; Serrano et al. 2020), and cardiovascular protective (Castro et al. 2010) actions, as well as promoting beneficial effects in diabetic (Shang et al. 2019) and obese patients (Lee et al. 2016).

Corroborating the information on the antimicrobial actions of Allium sativum, the study carried out by Torres et al. (2021) demonstrated the pharmacological efficacy of the SP80 fraction obtained from the plant's bulbs on GBS.

The fact that the antimicrobial action of Allium sativum has been identified opens a possibility of expanding the spectrum of therapeutic options in the prevention of GBS infections, as well as an option for the association with antibiotics already established in the treatment and prevention against GBS, allowing the increase of the pharmacological efficacy or even the reduction of adverse effects caused by high doses of conventional antibiotics. In this sense, this study aimed to assess the antimicrobial activity of the SP80 fraction obtained from A. sativum L. and its association with antibiotics PEN and AMP against S. agalactiae strains.

2.1 Plant material

Garlic bulbs from A. sativum L. (Liliaceae), a purple variety, were purchased in a local market in Campos Altos, State of Minas Gerais, Brazil; Fazenda Tri "S", altitude of 1.132 m, in November 2017. For confirmation of the plant's botanical identity, samples of the bulbs were cultivated and the whole plant was subsequently provided to experts in Botany. A voucher specimen, (n° 502077), was deposited at the Herbarium Maria Eneyda P. Kaufmann Fidalgo, São Paulo/Brazil where species identity was confirmed by Dr. Domingos Sávio Rodrigues. All study/experimental protocols involving plant materials were conducted by institutional, national, and international guidelines and legislation.

2.2 Raw Garlic Extract (RGE) and SP80 fraction (Sep-Pak80%)

The SP80 fraction, obtained by purifying the RGE, on a Sep-Pak^® C18 cartridge column, was carried out as previously described by Torres et al. (2021).

2.3 Bioassays

2.3.1 Minimum Inhibitory Concentration (MIC)

The SP80 fraction, reconstituted in ultrapure water, was subjected to a liquid growth inhibition test against the S. agalactiae (ATCC^®12386^TM) strain. The assay was performed in 96-well microplates. For that, 20 µL of SP80 fraction was applied to each well at a serial dilution of two-fold microtiter broth dilution and added to 80 µL of culture medium TSB containing the microorganism in the logarithmic growth phase for a final concentration of 10³ cells/mL. After 24 hours of incubation at 35 ± 2 ºC under constant agitation, the absorbance at 595 nm was measured on a Victor 3 - 1420 (Perkin Elmer®) instrument. The MIC was considered the lowest concentration that completely inhibits the growth of the microorganism. The tests were performed in triplicate and the average of 3 readings was considered (Clinical and Laboratory Standards Institute 2012).

2.3.2 Disk Diffusion Assay

The SP80 fraction was carried out by disc diffusion test and the concentration used was established from the MIC assay results for S. agalactiae (ATCC^® 12386^TM). For antimicrobial tests were used S. agalactiae strain (ATCC^® 12386^TM) and fifty-five non-duplicate clinical isolates of S. agalactiae from positive vaginal and rectal cultures, kindly supplied by Salomão and Zoppi available in its library. These clinical isolates were confirmed by using the CAMP (Christie, Atkins, Munch-Petersen) test (Rosa-Fraile and Spellerberg 2017).

For this purpose, ready-made Petri dishes were used with the Mueller-Hinton agar (MHA) with 5% sheep blood culture medium (150 mm), supplied by Probac, and S. agalactiae strains (previously prepared in saline), using alginate swabs, at a concentration of 1.5 x 10⁸ CFU/mL, which corresponds to 0.5 in the MacFarland nephelometric scale. The strains of S. agalactiae were subjected to four treatments by inserting sterile absorbent filter paper discs immersed in the following solutions: (1) PEN (10 IU); (2) AMP (10 mcg); (3) PEN (10 IU) with 15 μL SP80 fraction; and (4) AMP (10 mcg) with 15 μL SP80 fraction. The plates, prepared as duplicates, were incubated at 35 ± 2 °C for 24 hours. The diameter of the clear zone around the disc was measured and expressed in millimeters (Clinical and Laboratory Standards Institute 2015; McFarland 1907; Torres et al. 2016).

2.4 Statistical Analysis

The statistical analysis was performed by specialized professionals from the Statistics Department of the Faculty of Medical Sciences of Santa Casa de Sao Paulo.

During planning, the sample size was (n) calculated based on the Analysis of Variance (ANOVA). A significance level of 5% (α) and test power of 80% (1- β) was adopted with a standard deviation of 5 units and a difference of 2 units, therefore finding n = 56 samples.

The results obtained were subjected to statistical analysis by using the IBM SPSS software, version 13.0, and were considered significant (p < 0.05). The non-parametric test used was Wilcoxon; whereas for paired data, T-test.

The MIC of the SP80 fraction was determined, as triplicates, by employing the microdilution method. The mean and standard deviation of the results obtained was 2.40 ± 0.42 mg/mL. This concentration was used in the disk diffusion tests.

A total of 56 S. agalactiae strains, one standard strain (ATCC^® 12386^TM), and 55 clinical isolates were tested against PEN, AMP, and SP80 fraction in association with the respective antibiotics using the disk diffusion test (Figure 1a). The results are shown as mean ± standard deviation as can be seen in the graphical (Figure 1b).

The mean results obtained were compared between the antibiotics (PEN and AMP) both isolated and associated with SP80 fraction, respectively. The statistical analysis showed that the mean for AMP, when associated with SP80 fraction, increased as compared to AMP alone (p < 0.001), a significant increase. Otherwise, the mean for PEN, when associated with the SP80 fraction did not modify as shown in Table 1.

The inhibition halos (mm) were compared between the antibiotic combinations (SP80 + AMP and SP80 + PEN), respectively, and the statistical analysis showed that the results were significant (p < 0.001). The association of SP80 + AMP showed a greater mean inhibition zone (mm) when compared to the mean value obtained for the association of SP80 + PEN for S. agalactiae (Table 2).

According to the CLSI (2015), when the inhibition halo formed by PEN and AMP is ≥ 24 mm, the strain is sensitive to the antibiotic involved. We compared the inhibition halos obtained with isolated antibiotics, and the result showed us that the strains tested are more resistant to PEN as compared to AMP (Table 3).

Two strains of S. agalactiae that were resistant to the antibiotic AMP alone when tested against the association of SP80 + AMP became sensitive, as shown in Figure 2.

Garlic is one of the most researched medicinal plants in the world and has been used in traditional medicine for many centuries for the treatment of numerous diseases (Batiha et al. 2020; Schoening et al. 2005). According to Majewski(2014), garlic’s properties result from the combination of several biologically active substances with antimicrobial properties responsible for its healing effect that aroused our interest in studying its antimicrobial activities. To that end, we evaluated their susceptibility and drug interaction when associated with the antibiotic PEN and AMP, both of which belong to the class of β-lactams, against clinical isolates of S. agalactiae.

PEN and AMP were used as they are antibiotics of the first choice used in the IAP of pregnant women (Melguizo-Rodrigues 2022). In addition, no studies were found that evaluated the interaction of these antibiotics with garlic or the prospect of finding ways to enhance the action of these antibiotics, which could aid in the prophylaxis and/or treatment of this infection. It is worth noting that our study is the first one of its kind to be published in the literature to date.

The complete phytochemical analyses of the SP80 fraction, obtained from raw garlic extract (RGE) were carried out by Torres et al. (2021) to isolate and identify the bioactive compounds presented in the garlic using reverse-phase ultrafast liquid chromatography with UV spectroscopy (RP-UFLC-UV), mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR). Some of the compounds identified in their study were y-glutamyl-S-allyl-cysteine, y-glutamyl-phenylalanine, and (E) and (Z) -ajoenes and the yields of these compounds were 8.48%, 2.77%, and 0.31%, respectively. Therefore, the current study aimed to evaluate the synergistic activity of the association of the SP80 fraction with antibiotics PEN and AMP.

The SP80 fraction was used instead of the isolated fractions as mentioned above because according to Majewski(2014), garlic’s properties result from the combination of several biologically active substances with antimicrobial properties, and because the yield of each isolated compound was very small, the ideal would be to use synthetic compounds purchased commercially, but this will be the objective of future works.

The methods used for assessing antimicrobial activity are disk diffusion and broth microdilution (Cutler et al. 2009; Gao et al. 2019; Phan et al. 2019; Pérez-Köhler et al. 2015; Rehman and Mairaj 2013). The disk diffusion method was used because it is the most used method in clinical analysis laboratories and hospitals, in Brazil, to verify antimicrobial susceptibility.

The MIC of the SP80 fraction, obtained by purifying the RGE, was determined by the broth microdilution, in triplicate. The MIC used in our tests was the lowest value that completely inhibited microbial growth and exhibited a bacteriostatic action (Schoening et al. 2005). As our work is pioneering, different extraction methods could lead to distinct biological responses. For this reason, in our study, the MIC established for the SP80 fraction was 2.40 mg/mL. This concentration was used in the disk diffusion tests.

When determining the MIC value by using the microdilution method, it was demonstrated that this garlic fraction has antimicrobial activity against S. agalactiae. This is an important finding that corroborates all other studies described in the literature regarding this plant’s antimicrobial activity (Arzanlou 2016; Cutler et al. 2009; Fratianni et al. 2016; Marchese et al. 2016; Hayes et al. 2019).

The association of the SP80 fraction with AMP was more efficient than the association of the SP80 fraction with PEN when compared to antibiotics alone, respectively. These results suggest that SP80 + AMP can act synergistically to inhibit the growth of GBS.

Work conducted by Bekka-Hadji et al. (2022) showed the synergism effect of Artemisia herba alba essential oil in association with different antibiotics, the inhibition zone diameter increased from 9.3 mm for the oil alone to 10.3 mm for the oil + fluoroquinolones combination, to 10.5 mm for the oil + penicillin combination and to 10.8 mm for the oil + carbapenems combination that corroborating with our finding, the mean of inhibition zone diameter of 56 clinical isolated of S. agalactiae strains increased from 28.46 mm for the AMP alone to 29.04 mm for the AMP + SP80 fraction combination (p<0.001).

According to the CLSI (2015), inhibition halos ≥ 24 mm formed by PEN and AMP demonstrate that the strains tested against these antibiotics, respectively, are sensitive. We compared the antimicrobial effect of PEN and AMP, isolated, respectively, against S. agalactiae strains, and of the 56 strains tested, 25 strains were resistant to PEN and another 2 were resistant to AMP, thus demonstrating that the strains tested are more resistant to PEN than they are to AMP.

There is little information regarding the mechanism behind reduced PEN susceptibility in GBS. According to Hayes et al. (2020), β-lactams exert their antibacterial effect by blocking the action of enzymes involved in cell wall synthesis. PEN inhibits bacterial peptidoglycan formation through the binding of the β-lactam ring to enzymes, known as penicillin-binding proteins (PBPs), at the DD-transpeptidase. Therefore, Chu et al. (2007) believe that modification of penicillin-blinding proteins is a possible explanation for the reduced PEN susceptibility seen with their GBS isolates.

According to Van der Linden et al. (2020), the acquisition of point mutations in PBPs genes, leading to the production of proteins with low affinity for β-lactam binding, could explain why some S. agalactiae strains can be non-susceptible to penicillin. Nevertheless, further molecular work is needed for confirmation.

Another finding was that the two strains of S. agalactiae that were resistant to the antibiotic AMP alone when tested against the association of SP80 + AMP became sensitive, the inhibition zone diameter increased from 23 mm for AMP alone to 24 mm and 25 mm for AMP + SP80 combination respectively, but it is necessary to confirm this finding by testing a larger number of strains.

Hayes et al.(2019) carried out checkerboard and time-kill assays in vitro for determining the synergistic activity of erythromycin and nisin against clinical isolates of Group B Streptococcus against invasive and colonizing GBS strains. Their results suggest that erythromycin and nisin can act synergistically to inhibit the growth of GBS. This study differs from ours in the antibiotics used and the methodology.

Garlic has a variety of bioactive compounds, including organosulfur compounds, saponins, phenolic compounds, and polysaccharides. The main ones are organosulfur compounds, especially allicin (Putnik et al. 2019; Shang et al. 2019). According to Choo et al. (2020), garlic’s antibiotic activity is mainly attributable to allicin, which acts on the destruction and inhibition of Gram-positive and negative bacteria.

In the work conducted by Torres et al. (2021), the authors showed that other compounds present in garlic, in addition to allicin, have antimicrobial activity and can be used against strains of S. agalactiae, it is enough to know how they act to inhibit these bacteria.

From this research, the SP80 fraction from A. sativum L. has in vitro antimicrobial activity against the bacteria S. agalactiae, with a MIC of 2.40 mg/mL; its association with the antibiotic AMP showed a synergistic effect, which did not occur when in association with PEN.

Based on our results, we believe that further studies should be carried out to pave the way for the development of translational research, given that this drug interaction in humans can prove beneficial in the therapy for S. agalactiae infections.

The current study reveals, in vitro, a synergy between SP80 fraction (a combination of several biologically active substances with antimicrobial properties) and AMP against clinical GBS strains and indicates the potential of antimicrobial combination therapy for use in treating clinical GBS infections.

1. American Academy of Pediatrics (1997) Revised guidelines for prevention of early-onset group B streptococcal (GBS) infection. American Academy of Pediatrics Committee on Infectious Diseases and Committee on Fetus and Newborn. Pediatrics 99:489–496.

2. American College of Obstetricians and Gynecologists. ACOG committee opinion (1996) Prevention of early-onset group B streptococcal disease in newborns. Number 173, June 1996. Committee on Obstetric Practice. American College of Obstetrics and Gynecologists. Int J Gynaecol Obstet 54:197–205.

3. American College of Obstetricians and Gynecologists. ACOG committee opinion opinion No. 797 (2020) Prevention of group B streptococcal early-onset disease in newborns. American College of Obstetricians and Gynecologistis. Obstet Gynecol 135:e51–72.

4. Arzanlou M (2016) Inhibition of streptococcal pyrogenic exotoxin B using allicin from garlic. Microb Pathog 93:166-171. https://doi.org/10.1016/j.micpath.2016.02.010

5. Batiha GES, Beshbishy AM, Wasef LG et al (2020) Chemical constituents and pharmacological activities of garlic (Allium sativum L.): a review. Nutrients 12:872. https://doi.org/10.3390/nu12030872

6. Bekka-Hadji F, Bombarda I, Djoudi F, Bakour S, Touati A (2022) Chemical composition and Synergistic potential of Mentha pulegium L. and Artemisia herba alba Asso. Essential oils and antibiotic against multi-drug resistant bacteria. Molecules 27:1095. https://doi.org/10.3390/molecules27031095

7. Brazil. Ministério da Saúde. Sistema Único de Saúde (2009) MS Elabora Relação de Plantas Medicinais de Interesse ao SUS http://bvsms.saude.gov.br/bvs/sus/pdf/marco/ms_relacao_plantas_medicinais_sus_0603.pdf.

8. Capasso A (2013) Antioxidant action and therapeutic efficacy of Allium sativum L. Molecules 18:690–700. https://doi.org/10.3390/molecules18010690

9. Castro C, Lorenzo AG, González A, Cruzado M (2010) Garlic components inhibit angiotensin II-induced cell-cycle progression and migration: involvement of cell-cycle inhibitor p27 (Kip1) and mitogen-activated protein kinase. Mol Nutr Food Res 54:781–787. https://doi.org/10.1002/mnfr.200900108

10. Centers for Disease Control and Prevention (CDC) (1996) Prevention of perinatal group B streptococcal disease: a public health perspective. MMWR Recommp Rep 45:1–24.

11. Centers for Disease Control and Prevention (CDC) (2010) Prevention of perinatal group B streptococcal disease: Revised guidelines from CDC. MMWR Recommp Rep 59:1–32.

12. Choo S, Chin VK, Wong EH et al (2020) Review: antimicrobial properties of allicin used alone or in combination with other medications. Folia Microbiol (Praha) 65:451–465. https://doi.org/10.1007/s12223-020-00786-5

13. Chu YW, Tse C, Tsang GK, So DK, Fung JT, Lo JY (2007) Invasive group B Streptococcus isolates showing reduced susceptibility to penicillin in Hong Kong. J Antimicrob Chemother 60:1407–1409. https://doi.org/10.1093/jac/dkm390

14. Clinical and Laboratory Standards Institute (CLSI) (2012) Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved Standard - Ninth Edition. CLSI document M07-A9. Wayne, PA: Clinical and Laboratory Standards Institute.

15. Clinical and Laboratory Standards Institute (CLSI) (2015) Performance standards for antimicrobial susceptibility testing; Twenty-Fifth Informational Supplement. CLSI document M100-S25. Wayne, PA: Clinical and Laboratory Standards Institute.

16. Cohain JS (2009) Long-term symptomatic group B Streptococcal vulvovaginitis: eight cases resolved with freshly cut garlic. Eur J Obstet Gynecol Reprod Biol 146:110–111. https://doi.org/10.1016/j.ejogrb.2009.05.028

17. Cohain JS (2010) Case series: symptomatic group B Streptococcus vaginitis treated with fresh garlic. Integr Med 9:40–43.

18. Cutler RR, Odent M, Hajj-Ahmad H et al (2009) In vitro activity of an aqueous allicin extract and a novel allicin topical gel formulation against Lancefield group B streptococci. J Antimicrob Chemother 63:151–154. https://doi.org/10.1093/jac/dkn457

19. Fratianni F, Riccardi R, Spigno P et al (2016) Biochemical characterization and antimicrobial and antifungal activity of two endemic varieties of garlic (Allium sativum L.) of the Campania region, southern Italy. J Med Food 19:686–691. https://doi.org/10.1089/jmf.2016.0027

20. Furfaro LL, Chang BJ, Payne MS (2018) Perinatal Streptococcus agalactiae epidemiology and surveillance targets. Clin Microbiol Rev 31:e00049–18. https://doi.org/10.1128/CMR.00049-18

21. Gao X, Chen Y, Chen Z et al (2019) Identification and antimicrobial activity evaluation of three peptides from laba garlic and the related mechanism. Food Funct 10:4486-4496.

22. Hayes K, Cotter L, O'Halloran F (2019) In vitro synergistic activity of erythromycin and nisin against clinical Group B Streptococcus isolates. J Appl Microbiol 127:1381–1390. https://doi.org/10.1111/jam.14400

23. Hayes K, O'Halloran F, Cotter L (2020) A review of antibiotic resistance in Group B Streptococcus: the story so far. Crit Rev Microbiol 46:253-269. https://doi.org/10.1080/1040841X.2020.1758626

24. Lanzotti V, Scala F, Bonanomi G (2014) Compounds from Allium species with cytotoxic and antimicrobial activity. Phytochem Rev 13:769–791.

25. Lee DY, Li H, Lim HJ, Lee HJ, Jeon R, Ryu JH (2012) Anti-inflammatory activity of sulfur-containing compounds from garlic. J Med Food 15:992–999. https://doi.org/10.1089/jmf.2012.2275

26. Lee HS, Lim WC, Lee SJ, Lee SH, Lee JH, Cho HY (2016) Antiobesity effect of garlic extract fermented by Lactobacillus plantarum BL2 in diet-induced obese mice. J Med Food 19:823–829. https://doi.org/10.1089/jmf.2016.3674

27. Majewski M (2014) Allium sativum: facts and myths regarding human health. Rocz Panstw Zakl Hig 65:1–8.

28. Marchese A, Barbieri R, Sanches-Silva A, Daglia M, Nabavi SF, Jafari NJ, Izadi M, Ajam M, Nabavi SM (2016) Antifungal and antibacterial activities of allicin: a review. Trends Food Sci Technol 52:49–56. https://doi.org/10.1016/j.tifs.2016.03.010

29. Martins ER, Pedroso-Roussado C, Melo-Cristino J, Ramirez M, Portuguese group for the study of Streptococcal infections (2017) Streptococcus agalactiae causing neonatal infections in Portugal (2005-2015): diversification and emergence of a CC17/PI-2b multidrug resistant sublineage. Front Microbiol 8:499. https://doi.org/10.3389/fmicb.2017.00499

30. McFarland J (1907) Nephelometer: An instrument for estimating the number of bacteria in suspensions used for calculating the opsonic index and for vaccines. J Am Med Assoc 14:1176–1178. https://doi.org/10.1001/jama.1907.25320140022001f

31. Mehrbod P, Amini E, Kheir MT, Pasteur Institute of IRAN (Influenza Unit) (2009) Antiviral activity of garlic extract on influenza virus. Iran J Virol 3: 19–23.

32. Melguizo-Rodrigues L, Garcia-Recio E, Ruiz C, de Luna-Bertos E, Illescas-Montes R, Costela-Ruiz VJ (2022) Biological properties and therapeutic applications of garlic and its components. Food Funct. 13:2415–2426. https://doi.org/10.1039/d1fo03180e

33. Morguette AEB, Biasi-Garben RP, Otaguiri ES et al (2018) Prevalence and antimicrobial susceptibility profile of Streptococcus agalactiae in pregnant women seen at the University Hospital of Londrina, Paraná, Brazil. Semina Cienc Biol Saude 39:77–84. https://doi.org/10.5433/1679-0367.2018v39n1p

34. do Nascimento CS, dos Santos NFB, Ferreira RCC, Taddei CR (2019) Streptococcus agalactiae in pregnant women in Brazil: Prevalence, serotypes, and antibiotic resistance. Braz J Microbiol 50:943–952. https://doi.org/10.1007/s42770-019-00129-8

35. Nicastro HL, Ross SA, Milner JA (2015) Garlic and onions: their cancer prevention properties. Cancer Prev Res (Phila) 8:181–189. https://doi.org/10.1158/1940-6207.CAPR-14-0172

36. Percival SS (2016) Aged garlic extract modifies human immunity. J Nutr 146:433S–436S. https://doi.org/10.3945/jn.115.210427

37. Pérez-Köhler B, García-Moreno F, Bayon Y, Pascual G, Bellón JM (2015) Inhibition of Staphylococcus aureus adhesion to the surface of a reticular heavyweight polypropylene mesh soaked in a combination of chlorhexidine and allicin: an in vitro study. PLoS One 10:e0126711. https://doi.org/10.1371/journal.pone.0126711

38. Phan ADT, Netzel G, Chhim P, Netzel ME, Sultanbawa Y (2019) Phytochemical characteristics and antimicrobial activity of Australian grown garlic (Allium sativum L.) cultivars. Foods 8:358. https://doi.org/10.3390/foods8090358

39. Puopolo KM, Lynfield R, Cummings JJ, AAP COMMITTEE ON FETUS AND NEWBORN, AAP COMMITTEE ON INFECTIOUS DISEASES (2019) Management of Infants at Risk for Group B Streptococcal Disease. Pediatrics 144:e20191881. https://doi.org/10.1542/peds.2019-1881

40. Putnik P, Gabrić D, Roohinejad S, Barba FJ, Granato D, Mallikarjunan K, Lorenzo JM, Bursać Kovačević D (2019) An overview of organosulfur compounds from Allium spp.: From processing and preservation to evaluation of their bioavailability, antimicrobial, and anti-inflammatory properties. Food Chem 276:680–691. https://doi.org/10.1016/j.foodchem.2018.10.068

41. Rosa-Fraile M, Spellerberg B (2017) Reliable detection of group B streptococcus in the clinical laboratory J Clin Microbiol 55(9):2590–2598. https://doi.org/10.1128/JCM.00582-17

42. Rehman F, Mairaj S (2013) Antimicrobial studies of allicin and ajoene. Int J Pharm Bio Sci 4:1095–1105.

43. Schoening TE, Wagner J, Arvand M (2005) Prevalence of erythromycin and clindamycin resistance among Streptococcus agalactiae isolates in Germany. Clin Microbiol Infect 11:579–582. https://doi.org/10.1111/j.1469-0691.2005.01180.x

44. Serrano HDA, Mariezcurrena-Berasain MA, Castillo ADCG et al (2020) Antimicrobial resistance of three common molecularly identified pathogenic bacteria to Allium aqueous extracts. Microb Pathog 142:104028. https://doi.org/10.1016/j.micpath.2020.104028

45. Torres KAM, Lima SMRR, Torres LMB, Gamberini MT, Silva Junior PID. Garlic: an alternative treatment for group B Streptococcus. Microbiol Spectr 9:e0017021. https://doi.org/10.1128/Spectrum.00170-21

46. Torres KAM, Lima SMRR, Ueda SMY (2016) Activity of the aqueous extract of Schinus terebinthifolius Raddi on strains of the Candida genus. Rev Bras Ginecol Obstet 38:593–599. https://doi.org/10.1055/s-0036-1597694

47. Shang A, Cao SY, Xu XY, Gan RY, Tang GY, Corke H, Mavumengwana V, Li HB (2019) Bioactive compounds and biological functions of garlic (Allium sativum L.). Foods 8:246. https://doi.org/10.3390/foods8070246

48. Shang A, Cao S-Y, Xu X-Y et al (2019) Bioactive compounds and biological functions of garlic (Allium sativum L.). Foods 8(7):246. https://doi.org/10.3390/foods8070246

49. Van der Linden M, Mamede R, Levina N, Helwig P, Vila-Cerqueira P, Carriço JA, Melo-Cristino J, Ramirez M, Martins ER (2020) Heterogeneity of penicillin-non-susceptible group B streptococci isolated from a single patient in Germany. J Antimicrob Chemother 75:296-299. https://doi.org/10.1093/jac/dkz465

50. Word Health Organization (WHO) (2015) Recommendation on intrapartum antibiotic administration to women with group B Streptococcus (GBS) colonization for prevention of early neonatal GBS infection. The WHO Reproductive Health Library.

51. Zhu Y, Lin X. (2021) Updates in prevention policies of early-onset group B streptococcal infection in newborns. Pediatr Neonatol 62:465–475. https://doi.org/10.1016/j.pedneo.2021.05.007

Competing Interests: The authors declare no competing interests.

Funding: This research was funded by the Research Support Foundation of the State of São Paulo (FAPESP/CeTICS) (Grant No. 2013/07467-1), by the Brazilian National Council for Scientific and Technological Development (CNPq) (Grant No. 472744/2012-7), by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Finance Code 001 and by Fundo de Amparo à Pesquisa - FCMSCSP (FAP 2017/2019).

Author Contributions: Lima SMRR: guided the work and final approval of the version to be submitted; Gamberini MT: statistical analysis; Silva-Jr PI: conception and design of the study; Rodrigues S: botanical identification of Allium sativum L.; Torres KAM: development of all experiments and writing of the manuscript.

Data Availability: All data generated or analyzed during this study are included in this published article.

Ethics declarations: The article contains no data concerning studies involving human subjects or the inclusion of identifiable human data or clinical trials; thus, no ethical approval was required.

Acknowledgments

We would like to thank the technical staff of the Faculty of Medical Sciences of Santa Casa of São Paulo (São Paulo/SP – Brazil) and the technical staff of the Laboratory for Applied Toxinology – Butantan Institute (São Paulo/SP – Brazil). We would also like to thank Salomão and Zoppi Clinical Laboratory (São Paulo - Brazil) for providing the strain S. agalactiae and also Dr. Domingos Sávio Rodrigues, for botanical identification.

Table 1: Comparison of antibiotics, both isolated and in association with the SP80 fraction against S. agalactiae (median: minimum – maximum).

AMP	28.46 (23.00 - 40.00)	*p < 0.001
SP80 + AMP	29.04 (24.00 - 40.00)	*p < 0.001
PEN	25.88 (18.00 - 40.00)	*p = 0.07
SP80 + PEN	25.53 (16.00 - 39.50)	*p = 0.07

*T-test. Significant (p < 0.05).

Table 2: Comparing antibiotics, both isolated and in association with the SP80 fraction, against S. agalactiae (mean: minimum - maximum).

SP80 + AMP	29.04 (24.00 - 40.00)	*p < 0.001
SP80 + PEN	25.53 (16.00 - 39.50)	*p < 0.001

*Wilcoxon test. Significant (p < 0.05).

Table 3: Comparison of the inhibition halo (mm) of the antibiotics PEN and AMP (isolated) against S. agalactiae.

Inhibition halo

≥ 24 mm

Inhibition halo

˂ 24 mm

PEN

31 strains

25 strains *p < 0.05

AMP

54 strains

2 strains *p < 0.1

Inhibition halo ≥ 24 mm: sensitive;

Inhibition halo ˂ 24 mm: resistant;

*Wilcoxon test. Significant (p < 0.05).

No competing interests reported.

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Allium sativum and Group B Streptococcus: antimicrobial and synergistic activity

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