High concentrations of zinc suppress bacterial growth in many clinical GBS isolates

Previous reports indicate that zinc has antimicrobial activities against GBS [14]. To enhance the generalizability of these findings, we sought to test a larger number of GBS strains, thereby capturing more isolates (both colonizing and invasive) across diverse capsular serotypes and genetic STs. We also investigated the effects of increasing zinc concentration exposure. Out of the 30 GBS strains screened, 4 strains (GB0083, GB0561, GB0651, and A909) exhibited significant inhibition of bacterial growth when treated with 125 μM zinc (Table 1; P < 0.05, Student’s t test, compared to medium alone control cultures). All of these strains were classified as colonizing strains except the single laboratory strain (A909). At 250 μM zinc, an additional colonizing strain (GB0571) exhibited significant inhibition of bacterial growth compared to cultures grown in medium alone (Table 1; P < 0.05, Student’s t test). Growth of three additional strains, two colonizing strains and one laboratory strain (GB0012, GB0112, and NEM316, respectively), was inhibited when treated with a concentration of 500 μM zinc (Table 1; P < 0.05, Student’s t test, compared to medium alone control cultures). At 750 μM zinc, strains GB0411 (invasive) and COH1 (laboratory) exhibited significant inhibition of bacterial growth compared to the medium only control (Table 1; P < 0.05, Student’s t test). The growth of 5 additional strains (GB0037, GB0397, GB0438, GB0241, GB0653), including three invasive and two colonizing isolates, respectively, was inhibited when treated with 1000 μM zinc (Table 1; P < 0.05, Student’s t test, compared to medium alone control cultures). The growth of 9 additional strains (GB002, GB0115, GB0285, GB0291, GB0654, GB0064, GB0069, GB0374, GB0418), including 5 colonizing and 4 invasive strains, respectively, was significantly inhibited when treated with 2500 μM zinc (Table 1; P < 0.05, Student’s t test, compared to medium alone control cultures). Finally, the growth of six strains (GB0066, GB0079, GB0377, GB0390, GB0590, and GB0663), including four invasive strains and two colonizing strains, respectively, was unaffected when treated with concentrations of zinc up to 5000 μM (Table 1; P > 0.05, Student’s t test, compared to medium alone control cultures).

GBS colonizing and invasive strain types differ in susceptibility to zinc intoxication

Because zinc has been shown to be a crucial antimicrobial strategy deployed by the innate immune system [10,11,12], we hypothesized that there could be differences in susceptibility to zinc intoxication between colonizing and invasive GBS strains. To test this, we stratified the GBS strains by clinical phenotype. Strains were classified as “colonizing” if they were recovered from asymptomatic women sampled before or after childbirth, whereas “invasive” strains were isolated from babies with GBS disease. Strains were exposed to increasing concentrations of zinc chloride and growth was measured after 24 h of static incubation in 5% CO2 at 37 °C (Fig. 1). In medium alone, the mean OD600 measurement was calculated as 0.34 for colonizing strains and 0.33 for invasive strains, results that were statistically indistinguishable (P = 0.1627, Mann-Whitney U test). Similarly, no significant difference was noted in raw OD600 values between colonizing and invasive strains at 125, 250, 500, 750, 1000, 2500, or 5000 μM zinc exposure. However, calculation of percent growth of each strain (comparing growth of a specific strain at each zinc concentration compared to growth of that strain in medium alone) revealed that invasive strains exhibited a significantly enhanced mean growth when treated with 250, 500, 1000, and 2500 μM zinc (12, 12, 14, 19, and 36%, respectively) compared to colonizing strains (P < 0.05, Mann-Whitney U test, Fig. 2). Exposure to 250 μM zinc resulted in invasive strains having 13% higher percent growth compared to colonizing strains, a result that was statistically significant (P = 0.0143, paired Student’s t test; P = 0.0329, Mann-Whitney U test). Exposure to 500 μM zinc resulted in a 14% higher percent growth compared to colonizing strains (P = 0.0378, paired Student’s t test; P = 0.1160, Mann-Whitney U test). Exposure to 1000 μM zinc resulted in a 25% mean decrease in colonizing strain growth, compared to only a 9% in invasive strains (P = 0.0486, paired Student’s t test; P = 0.2328, Mann-Whitney U test). Exposure to 2500 μM zinc resulted in a 47% mean decrease in colonizing strain growth, compared to a 24% percent decrease in invasive strains (P = 0.00781, paired Student’s t test; P = 0.0555, Mann-Whitney U test). At a concentration of 5000 μM zinc, no significant difference in growth was observed between colonizing and invasive strains, largely because the growth of most strains was significantly inhibited at this concentration. Comparison of minimal inhibitory concentrations (MIC) of zinc to repress growth for colonizing versus invasive strains, revealed colonizing strains have a mean MIC of 1875 μM zinc, whereas invasive strains have a mean MIC of 3145 μM zinc (Fig. 3), a 68% increase which was statistically significant (P = 0.0402, Mann-Whitney U test).

Fig. 1
figure1

Analysis of susceptibility to zinc-associated growth inhibition in invasive vs. clinical isolates of Group B Streptococcus (GBS). GBS strains isolated from colonized patients, or patients experiencing invasive disease were grown in medium alone (Medium Alone) or increasing concentrations of zinc chloride (125 μM, 250 μM, 500 μM, 750 μM, 1000 μM, 2500 μM, 5000 μM). Bacterial growth was measured at 24 h post-inoculation as an optical density at 600 nm absorbance (OD600). At 0, 250, 500, 750, 1000, 2500, and 5000 μM concentrations of zinc, colonizing strains of GBS (circles) showed no significant differences in OD600 values across strain type

Fig. 2
figure2

Analysis of percent growth of invasive vs. clinical isolates of Group B Streptococcus (GBS) when cultured under increasing concentrations of zinc. GBS strains isolated from colonized patients, or patients experiencing invasive disease were grown in medium alone (Medium Alone) or increasing concentrations of zinc chloride (125 μM, 250 μM, 500 μM, 750 μM, 1000 μM, 2500 μM). Bacterial growth was measured at 24 h post-inoculation as an optical density at 600 nm absorbance (OD600). At 250, 500, 1000, and 2500 μM concentrations of zinc, invasive strains of GBS (circles) showed significantly higher percent growth compared to medium alone (as calculated by mean percent growth of three biological replicates for each strain, comparing OD600 values in each zinc concentration compared to OD600 values for each strain in medium alone). Statistical significance was determined by paired Student’s t test (n = 3 biological replicates)

Fig. 3
figure3

Minimum Inhibitory Concentration (MIC) of zinc chloride required to inhibit GBS growth across colonizing or invasive strains. Analysis of susceptibility to zinc-associated growth inhibition in invasive vs. clinical isolates of Group B Streptococcus (GBS). GBS strains isolated from colonized patients, or patients experiencing invasive disease were grown in increasing concentrations of zinc chloride and mean MIC was calculated. Significant differences were determined by Mann-Whitney U test (P < 0.05). Dotted line indicates upper limit of detection

Susceptibility to zinc toxicity differs across GBS strains from varying isolation sources

Because differences were observed between invasive and colonizing strains, we hypothesized that the source of bacterial strain isolation could contribute to zinc intoxication susceptibility due to GBS adaptation to the ecology of the specific host niche. To test this, we stratified strains into categories based on source of strain isolation (Fig. 4) such as blood from neonatal early onset disease (EOD), late onset disease (LOD), rectal or vaginal swabs (Vaginal/Rectal), or adult blood from septic patients (Blood/Sepsis). Results indicate that at concentrations of 250, 500, 750, 1000, and 2500 μM zinc, isolates from EOD have significantly enhanced growth (12, 13, 13, 27, and 39%, respectively) compared to vaginal/rectal isolates (P < 0.05, one-way ANOVA). At concentration of 250 μM zinc, LOD isolates were significantly more susceptible to zinc toxicity than those isolated from blood/sepsis (P < 0.05, one-way ANOVA). At 500 μM zinc, blood/sepsis isolates were significantly more tolerant of zinc stress than their vaginal/rectal or LOD isolate counterparts (P < 0.05, one-way ANOVA). At 750 μM zinc, blood/sepsis and EOD isolates were significantly less susceptible to zinc stress than rectovaginal isolates. At 1000 μM zinc, EOD and blood/sepsis isolates were significantly less susceptible than rectovaginal isolates. However, GBS isolated from LOD were significantly more susceptible than strains from the other three isolation sites.

Fig. 4
figure4

Analysis of susceptibility to growth inhibition by zinc intoxication based on isolation source. Group B Streptococcus (GBS) strains isolated from recto-vaginal swabs (Vaginal/Rectal, circles), early onset disease in neonates (EOD, squares), late onset disease in neonates (LOD, triangles), or blood/sepsis in adults (Blood/Sepsis, inverted triangles) were grown in medium alone (Medium Alone) or increasing concentrations of zinc chloride (125 μM, 250 μM, 500 μM, 750 μM, 1000 μM, 2500 μM, 5000 μM). Bacterial growth was measured at 24 h post-inoculation as an optical density at 600 nm absorbance (OD600). At 250, 500, 750, 1000 and 2500 μM concentrations of zinc, rectovaginal strains of GBS (circles) exhibited greater growth inhibition than strains isolated from early onset disease in neonates (squares), as determined by one-way ANOVA with Tukey’s post hoc multiple correction (*P < 0.05, and **P < 0.01, n = 3 biological replicates)

GBS capsular serotype confers varying susceptibility to zinc intoxication

Capsular serotypes have been implicated as an important virulence factor that aids in evasion of the innate immune response [21]. Additionally, capsular serotype III strains are associated with higher rates of invasive neonatal disease [22] and account for the majority of late-onset meningitis cases in neonates [23]. Because invasive strains and isolates from EOD were less susceptible to zinc toxicity than rectovaginal colonizing strains, we hypothesized that capsular serotype variation could contribute to alterations in susceptibility to zinc toxicity. To test this, we stratified strains based on capsular serotype and analyzed growth in cultures exposed to increasing concentration of zinc (Fig. 5). At concentrations as low as 125 μM of zinc, capsular serotype III (cpsIII) strains emerged as having enhanced growth compared to the cpsIb and cpsII strains. Notably, the cpsIII strains also exhibited enhanced growth at 250, 500, 750, 1000, and 2500 μM zinc compared to the cpsIb strains (P < 0.05, one-way ANOVA). At 2500 μM zinc, cpsIII strains exhibited the highest mean growth (mean OD600 = 0.267), followed by strains with cpsIa (mean OD600 = 0.246), cpsV (mean OD600 = 0.200), cpsII (mean OD600 = 0.126), and cpsIb (mean OD600 = 0.107). The cpsVI strains were most sensitive to zinc toxicity at a concentration of 2500 μM (mean OD600 = 0.100) compared to other capsular serotypes. However, at concentrations of 125, 250, 500, 750, and 1000 μM zinc, cpsIb isolates consistently exhibited the lowest growth, or greatest level of inhibition, among all capsular serotypes tested, underscoring their susceptibility to zinc intoxication (P < 0.05, one-way ANOVA). At a concentration of 5000 μM zinc, no statistically significant differences were observed between capsular types, a result that is likely due to a threshold effect of all strains experiencing significant growth inhibition.

Fig. 5
figure5

Analysis of susceptibility to growth inhibition by zinc intoxication in diverse capsular serotypes of Group B Streptococcus (GBS). GBS strains isolated with a span of capsular serotypes (cpsIa, black circles; cpsIb, squares; cpsII, triangles; cpsIII, inverted triangles; cpsV, diamonds; cpsVI, open circles) were grown in medium alone or increasing concentrations of zinc chloride (125 μM, 250 μM, 500 μM, 750 μM, 1000 μM, 2500 μM, 5000 μM). Bacterial growth was measured at 24 h post-inoculation as an optical density at 600 nm absorbance (OD600). At 125, 250, 500, 750, 1000 and 2500 μM concentrations of zinc, cpsIII strains of GBS (inverted triangles) exhibited less susceptibility to zinc intoxication than other capsular serotypes, as determined by one-way ANOVA with Tukey’s post hoc multiple correction (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 3 biological replicates)

Because our results have shown that colonizing strains may be more susceptible to zinc toxicity, and the cpsIII strains exhibited low susceptibility to zinc toxicity, we stratified the cpsIII strains into invasive versus colonizing strains to ascertain if there were any differences in these two cohorts (Fig. 6). Mann-Whitney U analyses revealed no statistically significant difference between invasive and colonizing cpsIII strains (P = 0.4401).

Fig. 6
figure6

Minimum Inhibitory Concentration (MIC) of zinc chloride required to inhibit GBS growth across colonizing or invasive strains of capsular serotype III (cpsIII) isolates. Analysis of susceptibility to zinc-associated growth inhibition in invasive vs. clinical isolates of Group B Streptococcus (GBS). GBS strains isolated from colonized patients, or patients experiencing invasive disease were grown in increasing concentrations of zinc chloride and mean MIC was calculated. Mann-Whitney U test revealed no statistically significant differences between colonizing and invasive strains of the cpsIII cohort (P = 0.4401). Dotted line indicates upper limit of detection

GBS susceptibility to zinc toxicity varies across sequence types

Because different GBS sequence types (STs) are associated with maternal colonization and neonatal disease [24], it is possible that strains of different STs have variable mechanisms to facilitate metal homeostasis. To test this hypothesis, we stratified GBS strains by ST and analyzed the growth under varying concentrations of zinc (Fig. 7). At 2500 μM zinc, ST-17, ST-19, and ST-23 remained the least susceptible to zinc intoxication compared to ST-1, ST-7, ST-12 (P < 0.05, one-way ANOVA). At 5000 μM zinc, ST-19 remained the least susceptible to zinc intoxication compared to all other strains tested (P < 0.05, one-way ANOVA). Interestingly, the ST-19 isolates that exhibited the highest resistance to zinc intoxication were all classified as cpsIII strains.

Fig. 7
figure7

Analysis of susceptibility to growth inhibition by zinc intoxication in diverse sequence types of Group B Streptococcus (GBS). GBS strains of varying sequence types (ST-1, black circles; ST-7, squares; ST-12, triangles; ST-17, inverted triangles; ST-19, diamonds; ST-23, open circles) were grown in medium alone or increasing concentrations of zinc chloride (500 μM, 750 μM, 1000 μM, 2500 μM, 5000 μM). Bacterial growth was measured at 24 h post-inoculation as an optical density at 600 nm absorbance (OD600). In medium alone and at 500 and 750 μM concentrations of zinc, no differences in susceptibility were observed. However, at 1000, 2500, and 5000 μM zinc concentrations, ST-1 and ST-7 strains exhibited the highest susceptibility to zinc intoxication than other sequence types. At 2500 μM zinc concentration, ST-17 and ST-19 emerged as the least susceptible strain types. At 5000 μM zinc concentration, ST-19 remained the least susceptible strain type. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc multiple correction (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n = 3 biological replicates)

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