Letters in Applied Microbiology ISSN 0266-8254 ORIGINAL ARTICLE Bacteriocin-like inhibitory activities of seven Lactobacillus delbrueckii subsp. bulgaricus strains against antibiotic susceptible and resistant Helicobacter pylori strains L. Boyanova, G. Gergova, R. Markovska, D. Yordanov and I. Mitov Department of Medical Microbiology, Medical University of Sofia, Sofia, Bulgaria Significance and Impact of the Study: In this study, anti-Helicobacter pylori activity of seven Lactobacil- lus delbrueckii subsp. bulgaricus (GLB) strains was evaluated by four cell-free supernatant (CFS) types. The GLB strains produced heat-stable bacteriocin-like inhibitory substances (BLISs) with a strong anti-H. pylori activity and some neutralized, catalase- and heat-treated CFSs inhibited >83% of the test strains. Bacteriocin-like inhibitory substance production of GLB strains can render them valuable probiotics in the control of H. pylori infection. Keywords Abstract antibiotics, bacteriocins, Helicobacter, Lactobacillus, probiotics. The aim of the study was to detect anti-Helicobacter pylori activity of seven Lactobacillus delbrueckii subsp. bulgaricus (GLB) strains by four cell-free Correspondence supernatant (CFS) types. Activity of non-neutralized and non-heat-treated Lyudmila Boyanova, Department of Medical (CFSs1), non-neutralized and heat-treated (CFSs2), pH neutralized, catalase- Microbiology, Medical University of Sofia, treated and non-heat-treated (CFSs3), or neutralized, catalase- and heat-treated Zdrave Street 2, 1431 Sofia, Bulgaria. (CFSs4) CFSs against 18 H. pylori strains (11 of which with antibiotic E-mail: [email protected] resistance) was evaluated. All GLB strains produced bacteriocin-like inhibitory 2017/1069: received 3 June 2017, revised 27 substances (BLISs), the neutralized CFSs of two GLB strains inhibited >81% of August 2017 and accepted 25 September test strains and those of four GLB strains were active against >71% of 2017 antibiotic resistant strains. Two H. pylori strains were BLIS resistant. The heating did not reduce the CFS activity. Briefly, all GLB strains evaluated doi:10.1111/lam.12807 produced heat-stable BLISs, although GLB and H. pylori strain susceptibility patterns exhibited differences. Bacteriocin-like inhibitory substance activity can be an advantage for the probiotic choice for H. pylori infection control. adhesion; secretion of lactic acid, bacteriocins, autolysins Introduction and suppression of H. pylori-associated IL-8 production Helicobacter pylori is linked to severe gastroduodenal dis- (Ruggiero 2014). An important quality of some lacto- eases, however, increasing antibiotic resistance of the bac- bacilli is their ability to produce bacteriocins or bacteri- teria is the main cause of eradication failure of the ocin-like inhibitory substances (BLISs). The antibacterial infection (Boyanova et al. 2016a). effect of these ribosomally synthesized and extracellularly Lactic acid bacteria (LAB) are widely used as starter released antimicrobial peptides is due to membrane per- cultures in many fermented foods and as natural biop- meabilization of the bacterial cells, resulting in cytoplas- reservatives (Montalban-Lopez et al. 2011; Kaskoniene_ mic content release, membrane potential depolarization et al. 2017). The LAB also possess many health-promoting and, finally, to cell death (Drider et al. 2006; Simova activities. For instance, Lactobacillus delbrueckii subsp. et al. 2009). Bacteriocin-like inhibitory substances can bulgaricus exhibits antibacterial, immunostimulatory and inhibit pathogenic bacteria such as Listeria monocytogenes antimutagenic properties (van de Guchte et al. 2006; Boy- (Scatassa et al. 2017). anova et al. 2009). Lactobacillus delbrueckii subsp. bulgaricus strains can Anti-H. pylori activity of lactobacilli may involve: direct suppress the growth of clinically important bacteria such inhibition; decrease in H. pylori urease activity and as Clostridium difficile, Escherichia coli, Streptococcus Letters in Applied Microbiology 65, 469--474 © 2017 The Society for Applied Microbiology 469 Seven Lactobacillus L. Boyanova et al. mutans and Pseudomonas aeruginosa through a competi- zone diameters of >13 mm) was found with both CFSs3 tive adhesion inhibition, organic acid production, bacteri- and CFSs4 of two (GLB44 and GLB47) Lactobacillus ocins production and immunomodulation (van de strains. Guchte et al. 2006; Boyanova et al. 2009). Lactobacillus The CFSs retained their activity against H. pylori strains delbrueckii subsp. bulgaricus clinical benefits in H. pylori after treatment at 100°C for 10 min. The differences in infection treatment have been reported. In Thailand, add- the activities of the non-heat-treated CFSs1 and the heat- ing L. delbrueckii subsp. bulgaricus and Streptococcus ther- treated CFS2s as well as between the non-heat-treated mophilus either before or before and after 1-week tailored CFSs3 and the heat-treated CFSs4 were not statistically triple therapy significantly improved eradication rates significant (P > 0Á05). (Tongtawee et al. 2015). No H. pylori strain was resistant to the CFSs of all GLB However, there are scanty reports on L. delbrueckii strains evaluated. The growth of >71% (71Á4–87Á5%) of subsp. bulgaricus effects on H. pylori and only single stud- the antibiotic resistant H. pylori strains was suppressed by ies on the BLIS producing strains in the subspecies both neutralized and catalase-treated CFSs (CFSs3 and (Miteva et al. 1998; Simova et al. 2009). CFSs4) of four GLB strains. Only two H. pylori strains, The aim of this study was to evaluate anti-H. pylori both of which were antibiotic resistant, exhibited no sus- activity of cell-free supernatants (CFS) from seven L. del- ceptibility to all the neutralized, catalase- and heat-treated brueckii subsp. bulgaricus (GLB) strains of four types CFSs (CFSs4). One of the strains was metronidazole resis- against numerous clinical H. pylori strains and to look for tant and was susceptible to five CFSs2 but to none of the CFS susceptibility variations. CFS4 from the seven GLB strains. The other strain (triple resistant to amoxicillin, metronidazole and ciprofloxacin) was susceptible to only 1 CFS2 (of GLB14) and to none Results and discussion of the seven CFSs4 tested. Of the 18 H. pylori strains evaluated, 1 (5Á6%) was amox- Most LAB bacteriocins are small cationic and heat- icillin resistant, 5 (27Á8%) strains were metronidazole stable peptides with higher antibacterial activity at lower resistant, 4 (22Á2%) strains were quinolone resistant and (<5) pH compared to higher pH values (Zacharof and no strain was tetracycline resistant. High (in six strains, Lovitt 2012). Class I and II LAB bacteriocins are heat- 33Á3% of the strains) clarithromycin resistance rate was stable (Zacharof and Lovitt 2012). Class IIa bacteriocins found. In our prior study on 84 phenotypically resistant/ are the major antibacterial peptide group of the LAB intermediately susceptible to clarithromycin strains by (Drider et al. 2006). allele-specific PCR and 30-mismatched PCR, the resistance In this study, the anti-H. pylori activity of the non-neu- was confirmed in all strains and the prevalence of tralized and non-heat-treated CFSs (CFSs1) was higher A2143G, A2142G, A2143G+A2142G and A2142C muta- (against 95Á6%, 65/68 of the CFS-H. pylori combinations) tions was 64Á3, 23Á8, 10Á7 and 1Á2% respectively (Boy- than that (against 75Á9%, 63/83, P = 0Á001) of the neu- anova et al. 2016b). 7 (38Á9%) H. pylori strains had single tralized and non-heat-treated CFSs (CFSs3). The activity antibiotic resistance, 3 (16Á7%) strains had double resis- of the non-neutralized heat-treated CFSs (CFSs2) was tance and 1 (5Á6%) strain exhibited triple resistance. slightly higher (against 88Á2%, 60/68 of the CFS-H. pylori In our previous study, performed with whole L. del- combinations) than that (against 76Á8%, 63/82, P = 0Á07) brueckii subsp. bulgaricus cells and six different L. del- of the neutralized and heat-treated CFSs (CFSs4). The brueckii subsp. bulgaricus strains, three strains suppressed results imply effects of organic acids or other bioactive the growth of >86% of H. pylori strains at low pH values substances of the lactobacilli as well as a higher BLIS and two strains suppressed the growth of >53% of the activity at lower pH values (Zacharof and Lovitt 2012). test strains at neutralized pH values (Boyanova et al. Notably, the CFSs of all seven GLB stains exhibited 2009). anti-H. pylori activity as well as thermostability at 100°C In the present work, the CFSs at their natural pH for 10 min, suggesting the presence of heat-stable BLISs. (CFSs1 and CFSs2) inhibited the growth of H. pylori Simova et al. (2009) found a bacteriocin (bulgaricin strains tested in 95Á6% (65/68) and 88Á2% (60/68, BB18) with anti-H. pylori activity. The bacteriocin was P = 0Á21) respectively, of the CFS-H. pylori combinations heat-resistant (at 121°C for 15 min), stable at pH from 2 (Table 1). Mean inhibitory zone diameters were similar to 10 and protease sensitive. and between 13Á4 and 16Á4 mm. The neutralized and In this study, the activity of the neutralized and cata- catalase-treated CFSs3 and CFSs4 inhibited 75Á9% (63/83) lase-treated CFSs (CFSs3 and CFSs4) of all GLB strains and 76Á8% (63/82, P = 0Á89) respectively, of the CFS-H. was similar with regard to the mean H. pylori inhibitory pylori combinations. The highest activity (growth inhibi- diameters (11Á8–15Á0 mm). It is important, however, that tion of >81% of the test strains with mean inhibitory the CFSs3