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The antimicrobial activity of zinc against group B Streptococcus is strain-dependent across diverse sequence types, capsular serotypes, and invasive versus colonizing isolates

The antimicrobial activity of zinc against group B Streptococcus is strain-dependent across... Background: Streptococcus agalactiae or Group B Streptococcus (GBS) is an encapsulated gram-positive bacterial pathobiont that commonly colonizes the lower gastrointestinal tract and reproductive tract of human hosts. This bac- terium can infect the gravid reproductive tract and cause invasive infections of pregnant patients and neonates. Upon colonizing the reproductive tract, the bacterial cell is presented with numerous nutritional challenges imposed by the host. One strategy employed by the host innate immune system is intoxication of bacterial invaders with certain transition metals such as zinc. Methodology: Previous work has demonstrated that GBS must employ elegant strategies to circumnavigate zinc stress in order to survive in the vertebrate host. We assessed 30 strains of GBS from diverse isolation sources, capsular serotypes, and sequence types for susceptibility or resistance to zinc intoxication. Results: Invasive strains, such as those isolated from early onset disease manifestations of GBS infection were signifi- cantly less susceptible to zinc toxicity than colonizing strains isolated from rectovaginal swabs of pregnant patients. Additionally, capsular type III (cpsIII) strains and the ST-17 and ST-19 strains exhibited the greatest resilience to zinc stress, whereas ST-1 and ST-12 strains as well as those possessing capsular type Ib (cpsIb) were more sensitive to zinc intoxication. Thus, this study demonstrates that the transition metal zinc possesses antimicrobial properties against a wide range of GBS strains, with isolation source, capsular serotype, and sequence type contributing to susceptibility or resistance to zinc stress. Keywords: Antimicrobial, Metal, Zinc, Streptococcus agalactiae, Group B Streptococcus Introduction Group B Streptococcus (GBS), or Streptococcus agalac- tiae, infections are one of the top five leading causes of neonatal mortality. GBS infection induces chorioamnio- *Correspondence: jennifer.a.gaddy@vumc.org nitis, preterm prelabor rupture of the gestational mem- Department of Medicine, Division of Infectious Diseases, Vanderbilt University Medical Center, A2200 Medical Center North, 1161 21st branes (PPROM), preterm birth, and both maternal Avenue South, Nashville, TN 37232, U.S.A. and neonatal sepsis [1]. GBS disease in neonates often Full list of author information is available at the end of the article © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Francis et al. BMC Microbiology (2022) 22:23 Page 2 of 14 manifests as early-onset or late-onset sepsis, pneumonia, dissemination in a mouse model of invasive disease in the or meningitis and can lead to death [2]. blood, heart, liver, and bladder. Together, these results GBS is an encapsulated gram-positive bacterium indicate that zinc is an important innate immune anti- that colonize the urogenital and/or lower gastrointesti- microbial strategy employed against GBS, and that GBS nal tract of healthy women and colonization rates vary resistance to zinc toxicity is critical for full virulence. between 15 and 40% depending on geographical region While zinc has been implicated in modulating the [3–5]. During infection of the upper female reproductive immune response to infection [15], the role zinc plays in tract, GBS ascends the vagina to the cervix and then to the context of perinatal-related GBS infections has not the uterus where the bacteria can cross the gestational yet been elucidated. In this study, we advanced previous membrane barrier. GBS infection of these extraplacen- findings by analyzing the antimicrobial effects of zinc tal, gestational membranes (i.e., chorioamnionitis) can against a panel of clinical GBS strains that vary by cap- provoke inflammation that triggers labor and/or causes sular serotype, sequence type (ST), isolation source, and PPROM [1]. Infection of the fetus by GBS can also lead clinical presentation. We observed strain-specific vari - to stillbirth or neonatal sepsis. GBS can cause early-onset ation in susceptibility to zinc intoxication with specific neonatal disease (EOD) (within the first week of life), of differences between invasive and colonizing strains and which 90% occurs 0-3 days after birth, or late-onset dis- across different capsular serotypes and STs. ease (LOD), which occurs one week to three months after birth [2]. Methods Current recommendations for prevention of EOD focus Bacterial strains and culture conditions on maternal GBS screening at 35-37 week’s gestation A bank of 27 GBS clinical strains were provided by Dr. H. and the use of intrapartum antibiotic prophylaxis (IAP). Dele Davies following recovery from neonates with inva- Beta-lactam antibiotics such as penicillin and ampicil- sive disease [16] and colonized mothers sampled before lin, are used in GBS positive expecting mothers [6]. Cur- and after childbirth [17] for use in this study (Table  1). rently, IAP has not been as effective for the treatment and These strains were all isolated from separate patients and prevention of LOD, nor has it reduced the incidence of were previously characterized using multilocus sequence PPROM, preterm birth, or stillbirth [6, 7]. In addition, typing and cps typing [18, 19]. Three common laboratory the rise in antibiotic resistant GBS strains is also of grave reference strains (A909, NEM316, and COH1; American concern [8]. Thus, developing new interventions to con - Type Culture Collection) were also evaluated. Bacterial trol antibacterial resistance is of great importance. Subse- strains were cultured on tryptic soy agar (TSA) plates quently, these innovations will decrease the incidence of supplemented with 5% sheep blood at 37 °C overnight maternal-fetal GBS infections and complications. followed by inoculation into brain-heart infusion broth Zinc, copper, and iron are transition metals that are (BHI) and incubation in aerobic conditions (ambient air, essential micronutrients for all cells. In both eukaryotes shaking at 200 rpm) at 37 °C. After 24 h, bacterial density and prokaryotes, transition metals serve as co-factors was measured spectrophotometrically to determine the for enzymes that perform critical cellular processes [9]. optical density at 600 nm (OD ). These bacterial cul - These metal ions, though required, can become toxic to tures were used for growth and viability assays. bacteria at high concentrations [10]. The host exploits this specifically within innate immune cells, such as mac - Evaluation of bacterial growth rophages and neutrophils, which load the phagosome GBS growth was determined by a spectrophotometric 2+ with divalent zinc cations (Zn ) to intoxicate phagocy- reading of optical density (OD) at OD as previously tosed bacteria [11, 12]. To circumnavigate this, bacteria described [20]. Briefly, GBS cultures were grown over - must regulate metal import and export machinery to night and diluted at 1:10 in fresh BHI medium; 100 μL of maintain normal zinc levels for growth and survival [13]. 1:10 diluted cultures were added to each well in a 96-well Recent work demonstrates that GBS employs a rep- plate. Increasing concentrations of divalent zinc ions 2+ ertoire of factors to facilitate metal homeostasis and (Zn ) in the form of zinc chloride (Z nCl ) (0, 125, 250, promote bacterial survival in the vertebrate host. Spe- 500, 750, 1000, 2500, 5000 μM) were added to the culture cifically, GBS require efflux determinants to overcome media. These concentrations were chosen because they metal stress and promote survival. Sullivan and col- represent a range of physiologically relevant concentra- leagues reported that GBS elaborates the CzcD efflux tions often encountered in the host-pathogen environ- system, activated by the SczA response regulator to ment in vivo [11]. The plates were incubated statically in manage intracellular zinc levels [14]. Furthermore, they 5% CO at 37 °C overnight. The following day, bacterial demonstrated that the CzcD and SczA systems are criti- density was estimated via OD . Three fresh biologi - cal for zinc resistance, survival within macrophages, and cal replicates were assessed with 1-3 technical replicates F rancis et al. BMC Microbiology (2022) 22:23 Page 3 of 14 within each biological replicate, and the OD values Results were normalized to a blank control of sterile, uninocu- High concentrations of zinc suppress bacterial growth lated bacteriological medium (BHI). in many clinical GBS isolates Previous reports indicate that zinc has antimicrobial Statistical analyses activities against GBS [14]. To enhance the generalizabil- Statistical analyses were performed using Mann-Whitney ity of these findings, we sought to test a larger number U for MIC studies, and either Student’s t-test or one-way of GBS strains, thereby capturing more isolates (both ANOVA with either Tukey’s or Dunnett’s post hoc cor- colonizing and invasive) across diverse capsular sero- rection for multiple comparisons for bacterial growth types and genetic STs. We also investigated the effects assays. All reported P values were adjusted to account for of increasing zinc concentration exposure. Out of the multiple comparisons. P values of ≤0.05 were considered 30 GBS strains screened, 4 strains (GB0083, GB0561, significant. All data analyzed in this work were derived GB0651, and A909) exhibited significant inhibition of from at least three biological replicates. Statistical analy- bacterial growth when treated with 125 μM zinc (Table 1; ses were performed using GraphPad Prism 9 software P < 0.05, Student’s t test, compared to medium alone (GraphPad Prism Software Inc., La Jolla, California). control cultures). All of these strains were classified as Table 1 Strain identifier, strain type, Isolation source, capsular type, and sequence type (ST) of clinical strains of Streptococcus agalactiae used in this study and the minimum inhibitory concentration (MIC) of zinc chloride required to suppress growth (as determined by OD ) by comparison with growth in medium alone lacking zinc supplementation (P < 0.05, Student’s t test) Strain Identifier Strain Type Sequence Type Capsular Serotype Isolation Growth Source MIC GB0002 Colonizing ST-23 cpsIa Vaginal/rectal colonization 2500 μM GB0012 Colonizing ST-1 cpsV Vaginal/rectal colonization 500 μM GB0037 Invasive ST-1 cpsV EOD/sepsis 1000 μM GB0064 Invasive ST-17 cpsIII EOD/sepsis 2500 μM GB0066 Invasive ST-19 cpsIII EOD/sepsis > 5000 μM GB0069 Invasive ST-17 cpsIII EOD/sepsis 2500 μM GB0079 Invasive ST-19 cpsIII EOD/sepsis > 5000 μM GB0083 Colonizing ST-1 cpsVI Vaginal/rectal colonization 125 μM GB0112 Colonizing ST-12 cpsIII Vaginal/rectal colonization 500 μM GB0115 Colonizing ST-17 cpsIII Vaginal/rectal colonization 2500 μM GB0241 Colonizing ST-23 cpsV Vaginal/rectal colonization 1000 μM GB0285 Colonizing ST-12 cpsII Vaginal/rectal colonization 2500 μM GB0291 Colonizing ST-12 cpsII Vaginal/rectal colonization 2500 μM GB0374 Invasive ST-12 cpsIb EOD/sepsis 2500 μM GB0377 Invasive ST-19 cpsIII EOD/sepsis > 5000 μM GB0390 Invasive ST-23 cpsIa EOD/sepsis > 5000 μM GB0397 Invasive ST-23 cpsIII EOD/sepsis 1000 μM GB0411 Invasive ST-17 cpsIII EOD/sepsis 750 μM GB0418 Invasive ST-17 cpsIII EOD/sepsis 2500 μM GB0438 Invasive ST-12 cpsIb LOD/sepsis 1000 μM GB0561 Colonizing ST-19 cpsV Vaginal/rectal colonization 125 μM GB0571 Colonizing ST-19 cpsIII Vaginal/rectal colonization 250 μM GB0590 Colonizing ST-19 cpsIII Vaginal/rectal colonization > 5000 μM GB0651 Colonizing ST-19 cpsIb Vaginal/rectal colonization 125 μM GB0653 Colonizing ST-12 cpsII Vaginal/rectal colonization 1000 μM GB0654 Colonizing ST-17 cpsIII Vaginal/rectal colonization 2500 μM GB0663 Colonizing ST-19 cpsIII Vaginal/rectal colonization > 5000 μM NEM316 Invasive ST-23 cpsIII EOD/sepsis 500 μM COH1 Invasive ST-17 cpsIII Blood 750 μM A909 Invasive ST-7 cpsIa Blood/sepsis 125 μM Francis et al. BMC Microbiology (2022) 22:23 Page 4 of 14 Fig. 1 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 (OD ). At 0, 250, 500, 750, 1000, 2500, and 5000 μM concentrations of zinc, colonizing strains of GBS (circles) showed no significant differences in OD values across strain type 600 F rancis et al. BMC Microbiology (2022) 22:23 Page 5 of 14 colonizing strains except the single laboratory strain GB0411 (invasive) and COH1 (laboratory) exhibited sig- (A909). At 250 μM zinc, an additional colonizing strain nificant inhibition of bacterial growth compared to the (GB0571) exhibited significant inhibition of bacterial medium only control (Table  1; P < 0.05, Student’s t test). growth compared to cultures grown in medium alone The growth of 5 additional strains (GB0037, GB0397, (Table 1; P < 0.05, Student’s t test). Growth of three addi- GB0438, GB0241, GB0653), including three invasive and tional strains, two colonizing strains and one laboratory two colonizing isolates, respectively, was inhibited when strain (GB0012, GB0112, and NEM316, respectively), was treated with 1000 μM zinc (Table  1; P < 0.05, Student’s t inhibited when treated with a concentration of 500 μM test, compared to medium alone control cultures). The zinc (Table  1; P < 0.05, Student’s t test, compared to growth of 9 additional strains (GB002, GB0115, GB0285, medium alone control cultures). At 750 μM zinc, strains GB0291, GB0654, GB0064, GB0069, GB0374, GB0418), Fig. 2 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 (OD ). 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 OD values in each zinc concentration compared to OD values for each strain in medium alone). Statistical 600 600 significance was determined by paired Student’s t test (n = 3 biological replicates) Francis et al. BMC Microbiology (2022) 22:23 Page 6 of 14 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 col- onizing 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 con- trol cultures). GBS colonizing and invasive strain types differ in susceptibility to zinc intoxication Because zinc has been shown to be a crucial antimicro- bial strategy deployed by the innate immune system [10– Fig. 3 Minimum Inhibitory Concentration (MIC) of zinc chloride 12], we hypothesized that there could be differences in required to inhibit GBS growth across colonizing or invasive strains. susceptibility to zinc intoxication between colonizing and Analysis of susceptibility to zinc-associated growth inhibition in invasive GBS strains. To test this, we stratified the GBS invasive vs. clinical isolates of Group B Streptococcus (GBS). GBS strains strains by clinical phenotype. Strains were classified as isolated from colonized patients, or patients experiencing invasive disease were grown in increasing concentrations of zinc chloride and “colonizing” if they were recovered from asymptomatic mean MIC was calculated. Significant differences were determined women sampled before or after childbirth, whereas “inva- by Mann-Whitney U test (P < 0.05). Dotted line indicates upper limit sive” strains were isolated from babies with GBS disease. of detection Strains were exposed to increasing concentrations of zinc chloride and growth was measured after 24 h of static incubation in 5% C O at 37 °C (Fig. 1). In medium alone, the mean OD measurement was calculated as 0.34 for growth compared to colonizing strains (P = 0.0378, colonizing strains and 0.33 for invasive strains, results paired Student’s t test; P = 0.1160, Mann-Whitney U that were statistically indistinguishable (P = 0.1627, test). Exposure to 1000 μM zinc resulted in a 25% mean Mann-Whitney U test). Similarly, no significant differ - decrease in colonizing strain growth, compared to only a ence was noted in raw OD values between colonizing 9% in invasive strains (P = 0.0486, paired Student’s t test; and invasive strains at 125, 250, 500, 750, 1000, 2500, or P = 0.2328, Mann-Whitney U test). Exposure to 2500 μM 5000 μM zinc exposure. However, calculation of percent zinc resulted in a 47% mean decrease in colonizing strain growth of each strain (comparing growth of a specific growth, compared to a 24% percent decrease in invasive strain at each zinc concentration compared to growth strains (P = 0.00781, paired Student’s t test; P = 0.0555, of that strain in medium alone) revealed that invasive Mann-Whitney U test). At a concentration of 5000 μM strains exhibited a significantly enhanced mean growth zinc, no significant difference in growth was observed when treated with 250, 500, 1000, and 2500 μM zinc (12, between colonizing and invasive strains, largely because 12, 14, 19, and 36%, respectively) compared to colonizing the growth of most strains was significantly inhibited at strains (P < 0.05, Mann-Whitney U test, Fig. 2). Exposure this concentration. Comparison of minimal inhibitory to 250 μM zinc resulted in invasive strains having 13% concentrations (MIC) of zinc to repress growth for colo- higher percent growth compared to colonizing strains, a nizing versus invasive strains, revealed colonizing strains result that was statistically significant (P = 0.0143, paired have a mean MIC of 1875 μM zinc, whereas invasive Student’s t test; P = 0.0329, Mann-Whitney U test). Expo- strains have a mean MIC of 3145 μM zinc (Fig.  3), a 68% sure to 500 μM zinc resulted in a 14% higher percent (See figure on next page.) Fig. 4 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 (OD ). 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) F rancis et al. BMC Microbiology (2022) 22:23 Page 7 of 14 Fig. 4 (See legend on previous page.) Francis et al. BMC Microbiology (2022) 22:23 Page 8 of 14 contribute to alterations in susceptibility to zinc toxicity. increase which was statistically significant (P = 0.0402, To test this, we stratified strains based on capsular sero - Mann-Whitney U test). type and analyzed growth in cultures exposed to increas- ing concentration of zinc (Fig.  5). At concentrations Susceptibility to zinc toxicity differs across GBS strains as low as 125 μM of zinc, capsular serotype III (cpsIII) from varying isolation sources strains emerged as having enhanced growth compared Because differences were observed between invasive to the cpsIb and cpsII strains. Notably, the cpsIII strains and colonizing strains, we hypothesized that the source also exhibited enhanced growth at 250, 500, 750, 1000, of bacterial strain isolation could contribute to zinc and 2500 μM zinc compared to the cpsIb strains (P < 0.05, intoxication susceptibility due to GBS adaptation to the one-way ANOVA). At 2500 μM zinc, cpsIII strains exhib- ecology of the specific host niche. To test this, we strat - ited the highest mean growth (mean OD = 0.267), fol- ified strains into categories based on source of strain lowed by strains with cpsIa (mean OD = 0.246), cpsV isolation (Fig.  4) such as blood from neonatal early (mean OD = 0.200), cpsII (mean OD = 0.126), and onset disease (EOD), late onset disease (LOD), rectal 600 600 cpsIb (mean OD = 0.107). The cpsVI strains were most or vaginal swabs (Vaginal/Rectal), or adult blood from sensitive to zinc toxicity at a concentration of 2500 μM septic patients (Blood/Sepsis). Results indicate that at (mean OD = 0.100) compared to other capsular sero- concentrations of 250, 500, 750, 1000, and 2500 μM types. However, at concentrations of 125, 250, 500, 750, zinc, isolates from EOD have significantly enhanced and 1000 μM zinc, cpsIb isolates consistently exhibited growth (12, 13, 13, 27, and 39%, respectively) compared the lowest growth, or greatest level of inhibition, among to vaginal/rectal isolates (P < 0.05, one-way ANOVA). all capsular serotypes tested, underscoring their suscepti- At concentration of 250 μM zinc, LOD isolates were bility to zinc intoxication (P < 0.05, one-way ANOVA). At significantly more susceptible to zinc toxicity than a concentration of 5000 μM zinc, no statistically signifi - those isolated from blood/sepsis (P < 0.05, one-way cant differences were observed between capsular types, a ANOVA). At 500 μM zinc, blood/sepsis isolates were result that is likely due to a threshold effect of all strains significantly more tolerant of zinc stress than their vag - experiencing significant growth inhibition. inal/rectal or LOD isolate counterparts (P < 0.05, one- Because our results have shown that colonizing strains way ANOVA). At 750 μM zinc, blood/sepsis and EOD may be more susceptible to zinc toxicity, and the cpsIII isolates were significantly less susceptible to zinc stress strains exhibited low susceptibility to zinc toxicity, we than rectovaginal isolates. At 1000 μM zinc, EOD and stratified the cpsIII strains into invasive versus colonizing blood/sepsis isolates were significantly less susceptible strains to ascertain if there were any differences in these than rectovaginal isolates. However, GBS isolated from two cohorts (Fig. 6). Mann-Whitney U analyses revealed LOD were significantly more susceptible than strains no statistically significant difference between invasive from the other three isolation sites. and colonizing cpsIII strains (P = 0.4401). GBS capsular serotype confers varying susceptibility GBS susceptibility to zinc toxicity varies across sequence to zinc intoxication types Capsular serotypes have been implicated as an impor- Because different GBS sequence types (STs) are associ- tant virulence factor that aids in evasion of the innate ated with maternal colonization and neonatal disease immune response [21]. Additionally, capsular serotype [24], it is possible that strains of different STs have III strains are associated with higher rates of invasive variable mechanisms to facilitate metal homeostasis. neonatal disease [22] and account for the majority of To test this hypothesis, we stratified GBS strains by late-onset meningitis cases in neonates [23]. Because ST and analyzed the growth under varying concentra- invasive strains and isolates from EOD were less suscep- tions of zinc (Fig.  7). At 2500 μM zinc, ST-17, ST-19, tible to zinc toxicity than rectovaginal colonizing strains, and ST-23 remained the least susceptible to zinc we hypothesized that capsular serotype variation could (See figure on next page.) Fig. 5 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 (OD ). 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) F rancis et al. BMC Microbiology (2022) 22:23 Page 9 of 14 Fig. 5 (See legend on previous page.) Francis et al. BMC Microbiology (2022) 22:23 Page 10 of 14 Streptococcus spp. [23]. Bacteria that have the capability to survive and replicate inside the phagosome of mac- rophages employ and regulate metal transport machinery to maintain metal homeostasis. For example, the notori- ous intracellular pathogen, Mycobacterium tuberculosis, has been shown to upregulate P-type ATPases which act in heavy metal efflux to counteract the toxic, high extra - cellular zinc levels of the phagosome [26]. In fact, metal intoxication survival is a critical virulence trait in S. pneu- moniae and S. pyogenes [27–30] as well as GBS [14]. In this study, we sought to determine the differences in sus - ceptibility or resistance to zinc toxicity in 30 strains of GBS, spanning diverse capsular serotypes, STs, isolation source, and disease manifestation. Our work demonstrates that GBS colonizing and Fig. 6 Minimum Inhibitory Concentration (MIC) of zinc chloride invasive strain types differ in susceptibility to zinc required to inhibit GBS growth across colonizing or invasive strains intoxication. Specifically, invasive strains exhibit of capsular serotype III (cpsIII) isolates. Analysis of susceptibility to diminished susceptibility to zinc toxicity compared zinc-associated growth inhibition in invasive vs. clinical isolates of Group B Streptococcus (GBS). GBS strains isolated from colonized to colonizing strains, indicating invasive strains may patients, or patients experiencing invasive disease were grown have acquired adaptations to survive metal intoxication in increasing concentrations of zinc chloride and mean MIC was strategies imposed within the vertebrate host during calculated. Mann-Whitney U test revealed no statistically significant invasive infections. GBS strains from varying isolation differences between colonizing and invasive strains of the cpsIII sources also exhibit varying susceptibility to zinc intox- cohort (P = 0.4401). Dotted line indicates upper limit of detection ication. In particular, strains isolated from early onset disease manifestations have significantly enhanced tolerance of zinc intoxication compared to rectovagi- intoxication compared to ST-1, ST-7, ST-12 (P < 0.05, nal colonizing isolates. Again, similar to the results one-way ANOVA). At 5000 μM zinc, ST-19 remained observed with colonizing versus invasive isolates, this the least susceptible to zinc intoxication compared to could reflect strain-specific adaptation to the ecological all other strains tested (P < 0.05, one-way AN OVA). niche of the host. The human vaginal environment is Interestingly, the ST-19 isolates that exhibited the rich in S100A-family proteins, especially during infec- highest resistance to zinc intoxication were all classi- tious processes, and these proteins bind and seques- fied as cpsIII strains. ter nutrient metals, such as zinc [31–34]. It is possible that the GBS-colonized vaginal mucosa represents an Discussion environment with low zinc availability, thus there is The battle for essential nutrient metals between the verte - no selective pressure for colonizing strains to develop brate host and invading pathogen has been closely linked strategies to circumnavigate high zinc concentrations. to virulence [25]. Starvation of transition metals is detri- Conversely, numerous studies have shown that circu- mental to bacteria, however, high concentrations of met- lating innate immune cells such as macrophages and als like zinc also have antimicrobial effects [10, 12]. Zinc neutrophils use zinc intoxication as a strategy to inhibit plays an important antimicrobial role in innate immune invading microbes [11, 35–37]. It is possible that inva- defense against several pathogens, including a variety of sive strains have undergone selection for survival in (See figure on next page.) Fig. 7 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 (OD ). 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) F rancis et al. BMC Microbiology (2022) 22:23 Page 11 of 14 Fig. 7 (See legend on previous page.) Francis et al. BMC Microbiology (2022) 22:23 Page 12 of 14 high zinc environments, yielding strains with greater circumstances. Thus, some “colonizing” strains might resistance to the toxicity of this transition metal. be misclassified because they have unrecognized Our results indicate that GBS capsular serotype also invasive potential. Such a misclassification could bias confers varying susceptibility to zinc intoxication. Spe- towards a null hypothesis (contributing to a type II cifically, cpsIII isolates were less susceptible to zinc error). Additional genomics studies are also war- intoxication, whereas cpsIb isolates were much more ranted to identify genetic traits linked to zinc resist- susceptible to zinc toxicity. This is interesting because ance, particularly in the more virulent lineages. An cpsIII is the predominant capsular serotype responsible additional limitation includes the medium sample size for invasive neonatal infections. Additionally, there is which should be expanded in future studies to include growing evidence that bacterial exopolysaccharides have more representation in other capsular serotypes and strong binding properties for metal sorption [38, 39]. sequence types to draw broader conclusions across a Additionally, production of specific exopolysaccharides larger number of GBS strains. can promote cellular survival of metal stress [40–42]. u Th s, it remains possible that the variations in capsular Conclusions polysaccharide production responsible for alterations In conclusion, we report strain variations within a cohort in capsular serotype of the surveyed GBS strains could of GBS strains with respect to susceptibility to zinc intox- contribute to alternate binding of excess metals, thereby ication across STs, capsular serotypes, isolation source, altering GBS susceptibility to intoxication with metals, and invasive versus colonizing strains. Invasive isolates such as zinc. demonstrated greater resistance to zinc toxicity com- Our study also revealed that different sequence types pared to colonizing strains. Additionally, ST-1 and ST-12 of GBS had varying susceptibilities to zinc intoxication. were highly susceptible to zinc stress, while ST-17, ST-19, Specifically, ST-1 and ST-12 were highly susceptible to and ST-23 were much more resistant to zinc intoxication. zinc stress, while ST-17, ST-19, and ST-23 were much cpsIII isolates were less susceptible to zinc intoxication, more resistant to zinc intoxication. A cross-continental whereas cpsIb isolates were much more susceptible to study revealed that GBS ST-1 and ST-19 are associated zinc toxicity. Our study is a pilot study that is hamstrung with asymptomatic colonization, while ST-17 is predomi- by the relatively small number of strains. Future stud- nantly associated with invasive neonatal disease [24]. ies will require an expansion to include genetic studies ST-23 was associated with both rectovaginal carriage and a larger number of strains and diverse capsular and and invasive GBS disease [24]. By contrast, STs 1, 17, 19 sequence types, as well as GBS strains from non-perina- and 23, were all found to colonize pregnant women at tal sources and distinct geographic locations. higher rates in different patient populations [18]. Addi - tionally, ST-17 (specifically capsular serotype III strains Abbreviations in this clade) was linked to EOD and strongly associ- GBS: Group B Streptococcus; PPROM: preterm prelabor rupture of the ated with LOD and meningitis [19, 43, 44]. This finding gestational membranes; EOD: Early onset disease; LOD: Late onset disease; 2+ IAP: Intrapartum antibiotic prophylaxis; Zn : Zinc; ZnCl : Zinc chloride; ST: further supports a model in which invasive strains are Sequence type; TSA: Tryptic soy agar; OD : Optical density at 600 nm; MIC: likely undergoing positive selection for zinc resistance Minimum inhibitory concentration; cps: Capsular polysaccharide; rpm: Rota- as a critical virulence factor to overcome innate immune tions per minute; nm: Nanometer; μM: Micromolar. defenses which employ zinc intoxication as an antimicro- Acknowledgments bial strategy [45]. Interestingly, in our study, ST-19 strains Not applicable. (largely colonizing strains) that were most resistant to Authors’ contributions zinc intoxication were all cpsIII strains, underscoring the SDM curated and validated the clinical strains for this study. JDF, MAG, JL, and relationship between capsular polysaccharide production JAG conceptualized and performed the wet-bench experiments. JDF, MAG, JL, and zinc resistance in GBS. SAM, GK, DMA, SDM, and JAG analyzed results and interpreted data. JDF, MAG, JL, SAM, GK, DMA, SDM, and JAG wrote and edited the manuscript for critical content. All authors have read and approved the manuscript and have given Limitations of the study their consent to publish this work. There are several limitations of our study including Funding the clinical definitions of “colonizing” versus “invasive” This work was supported by the National Institutes of Health grant R01 strains which can be imperfect. Isolating strains from HD090061 (to J.A.G.), R35GM133602 (to S.D.T.), T32 HL007411-36S1 (support- invasive neonatal infections proves that such strains ing J.L.), and 2T32AI112541-06 (supporting J.D.F.). Additional support was provided by the Department of Veterans Affairs Office of Research BX005352 are capable of causing perinatal infection. However, (to J.A.G.). Core Services were performed through both Vanderbilt University simply because a “colonizing” strain was isolated from Medical Center’s Digestive Disease Research Center supported by NIH grant a rectal or vaginal swab does not mean it would be P30DK058404 Core Scholarship and Vanderbilt Institute for Clinical and Translational Research program supported by the National Center for Research incapable of causing invasive disease under different F rancis et al. BMC Microbiology (2022) 22:23 Page 13 of 14 Resources, Grant UL1 RR024975-01, and the National Center for Advancing streptococcal disease in the United States, 2006 to 2015: multistate labo- Translational Sciences, Grant 2 UL1 TR000445-06, which supports access to ratory and population-based surveillance. JAMA Pediatr. 2019;173:224– core facilities and biostatistics support. The content is solely the responsibility 33. https:// doi. org/ 10. 1001/ jamap ediat rics. 2018. 4826. of the authors and does not necessarily represent the official views of the NIH 8. Shipitsyna E, Shalepo K, Zatsiorskaya S, Krysanova A, Razinkova M, or any of the other supporters. Grigoriev A, et al. Significant shifts in the distribution of vaccine capsular polysaccharide types and rates of antimicrobial resistance of perinatal Availability of data and materials group B streptococci within the last decade in St. Petersburg, Russia. Eur The datasets used and/or analyzed during the current study available from the J Clin Microbiol Infect Dis. 2020;39:1487–93. https:// doi. org/ 10. 1007/ corresponding authors upon reasonable request.s10096- 020- 03864-1. 9. Andreini C, Bertini I, Cavallaro G, Holliday GL, Thornton JM. Metal ions in biological catalysis: from enzyme databases to general princi- Declarations ples. J Biol Inorg Chem. 2008;13:1205–18. https:// doi. org/ 10. 1007/ s00775- 008- 0404-5. Ethics approval and consent to participate 10. Djoko KY, Ong CY, Walker MJ, McEwan AG. The role of copper and zinc The secondary use of de-identified or coded samples is not considered toxicity in innate immune defense against bacterial pathogens. J Biol research involving human subjects under 45 CFR 46. Biospecimens (bacterial Chem. 2015;290:18954–61. https:// doi. org/ 10. 1074/ jbc. R115. 647099. strains) used in this study were deidentified and need for consent was waived 11. Wagner D, Maser J, Lai B, Cai Z, Barry CE 3rd, Bentrup HZ, et al. Elemental by the IRB in accordance with federal regulation (45 CFR 46, Department of analysis of Mycobacterium avium-, mycobacterium tuberculosis-, and Health and Human Services, Authority: 5 U.S.C. 301; 42 U.S.C. 289(a); 42 U.S.C. mycobacterium smegmatis-containing phagosomes indicates pathogen- 300v-1(b)). induced microenvironments within the host cell’s endosomal system. J Immunol. 2005;174:1491–500. https:// doi. org/ 10. 4049/ jimmu nol. 174.3. Consent for publication Not applicable. 12. Stafford SL, Bokil NJ, Achard MES, Kapetanovic R, Schembri MA, McEwan AGS, et al. Metal ions in macrophage antimicrobial pathways: emerging Competing interests roles for zinc and copper. Biosci Rep. 2013;33. https:// doi. org/ 10. 1042/ The authors declare no conflicts of interest. The authors declare no competing BSR20 130014. interests. 13. Hood MI, Skaar EP. Nutritional immunity: transition metals at the patho- gen–host interface. Nat Rev Microbiol. 2012;10:525–37. https:// doi. org/ Author details 10. 1038/ nrmic ro2836. Department of Pathology, Microbiology and Immunology, Vanderbilt 14. Sullivan MJ, Goh KGK, Ulett GC. Cellular Management of Zinc in Group University Medical Center, Nashville, TN 37212, USA. South African Medical B Streptococcus Supports Bacterial Resistance against Metal Intoxication Research Council Vaccines and Infectious Diseases Analytics Research Unit, and Promotes Disseminated Infection. mSphere. 2021;6. https:// doi. org/ Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, 10. 1128/ mSphe re. 00105- 21. South Africa. Department of Clinical Microbiology, Christian Medical Col- 15. Bonaventura P, Benedetti G, Albarède F, Miossec P. Zinc and its role in lege, Vellore, India. Department of Medicine, Division of Infectious Diseases, immunity and inflammation. Autoimmun Rev. 2015;14:277–85. https:// Vanderbilt University Medical Center, A2200 Medical Center North, 1161 doi. org/ 10. 1016/j. autrev. 2014. 11. 008. 21st Avenue South, Nashville, TN 37232, U.S.A.. Department of Obstetrics 16. Davies HD, Adair C, McGreer A, Ma D, Robertson S, Mucenski M, et al. and Gynecology, Vanderbilt University Medical Center Nashville, Nashville, TN Antibodies to capsular polysaccharides of group B Streptococcus in preg- 37232, USA. Department of Microbiology and Molecular Genetics, Michigan nant Canadian women: relationship to colonization status and infection State University, East Lansing, MI 48824, USA. Department of Veterans Affairs, in the neonate. J Infect Dis. 2001;184:285–91. https:// doi. org/ 10. 1086/ Tennessee Valley Healthcare Systems, Nashville, TN 37212, USA. 17. Spaetgens R, DeBella K, Ma D, Robertson S, Mucenski M, Davies HD. Peri- Received: 28 August 2021 Accepted: 13 December 2021 natal antibiotic usage and changes in colonization and resistance rates of group B streptococcus and other pathogens. 2002;100:525–33. https:// doi. org/ 10. 1016/ s0029- 7844(02) 02068-9. 18. Manning SD, Springman AC, Lehotzky E, Lewis MA, Whittam TS, Davies HD. Multilocus sequence types associated with neonatal group B strepto- References coccal sepsis and meningitis in Canada. J Clin Microbiol. 2009;47:1143–8. 1. Patras KA, Nizet V. Group B streptococcal maternal colonization and https:// doi. org/ 10. 1128/ JCM. 01424- 08. neonatal disease: molecular mechanisms and preventative approaches. 19. Manning SD, Lewis MA, Springman AC, Lehotzky E, Whittam TS, Davies Front Pediatr. 2018;6:27. https:// doi. org/ 10. 3389/ fped. 2018. 00027. HD. Genotypic diversity and serotype distribution of group B strepto- 2. Raabe VN, Shane AL. Group B Streptococcus (Streptococcus agalac- coccus isolated from women before and after delivery. Clin Infect Dis. tiae). Microbiol Spectr. 2019;7. https:// doi. org/ 10. 1128/ micro biols pec 2008;46:1829–37. https:// doi. org/ 10. 1086/ 588296. GPP3-0007-2018. 20. Lu J, Francis JD, Guevara MA, Moore RE, Chambers SA, Doster RS, 3. Campbell JR, Hillier SL, Krohn MA, Ferrieri P, Zaleznik DF, Baker CJ. Group et al. Antibacterial and anti-biofilm activity of the human breast Milk B streptococcal colonization and serotype-specific immunity in pregnant glycoprotein Lactoferrin against group B Streptococcus. Chembiochem. women at delivery. Obstet Gynecol. 2000;96:498–503. https:// doi. org/ 10. 2021;22(12):2124–33. https:// doi. org/ 10. 1002/ cbic. 20210 0016. 1016/ s0029- 7844(00) 00977-7. 21. Rajagopal L. Understanding the regulation of group B streptococcal viru- 4. Stoll BJ, Schuchat A. Maternal carriage of group B streptococci in develop- lence factors. Future Microbiol. 2009;4:201–21. https:// doi. org/ 10. 2217/ ing countries. Pediatr Infect Dis J. 1998;17:499–503. https:// doi. org/ 10. 17460 913.4. 2. 201. 1097/ 00006 454- 19980 6000- 00013. 22. Alhhazmi A, Pandey A, Tyrrell GJ. Identification of group B Streptococcus 5. Armistead B, Oler E, Adams Waldorf K, Rajagopal L. The double life of capsule type by use of a dual phenotypic/genotypic assay. J Clin Micro- group B Streptococcus: asymptomatic colonizer and potent pathogen. J biol. 2017;55:2637–50. https:// doi. org/ 10. 1128/ JCM. 00300- 17. Mol Biol. 2019;431:2914–31. https:// doi. org/ 10. 1016/j. jmb. 2019. 01. 035. 23. Bellais S, Six A, Fouet A, Longo M, Dmytruk N, Glaser P, et al. Capsu- 6. Schrag SJ, Verani JR. Intrapartum antibiotic prophylaxis for the prevention lar switching in group B Streptococcus CC17 hypervirulent clone: a of perinatal group B streptococcal disease: experience in the United future challenge for polysaccharide vaccine development. J Infect Dis. States and implications for a potential group B streptococcal vaccine. 2012;206:1745–52. https:// doi. org/ 10. 1093/ infdis/ jis605. Vaccine. 2013;31(Suppl 4):D20–6. https:// doi. org/ 10. 1016/j. vacci ne. 2012. 24. Jones N, Bohnsack JF, Takahashi S, Oliver KA, Chan M-S, Kunst F, et al. 11. 056. Multilocus sequence typing system for group B streptococcus. J Clin 7. Nanduri SA, Petit S, Smelser C, Apostol M, Alden NB, Harrison LH, Microbiol. 2003;41:2530–6. https:// doi. org/ 10. 1128/ JCM. 41.6. 2530- 2536. et al. Epidemiology of invasive early-onset and late-onset group B 2003. Francis et al. BMC Microbiology (2022) 22:23 Page 14 of 14 25. Palmer LD, Skaar EP. Transition metals and virulence in Bacteria. Streptococcus) causing invasive neonatal disease. Proc Natl Acad Sci U S A. Annu Rev Genet. 2016;50:67–91. https:// doi. org/ 10. 1146/ annur 1989;86:4731–5. https:// doi. org/ 10. 1073/ pnas. 86. 12. 4731. ev- genet- 120215- 035146. 44. Lin F-YC, Whiting A, Adderson E, Takahashi S, Dunn DM, Weiss R, et al. 26. Botella H, Peyron P, Levillain F, Poincloux R, Poquet Y, Brandli I, et al. Phylogenetic lineages of invasive and colonizing strains of serotype III Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in group B streptococci from neonates: a multicenter prospective study. J human macrophages. Cell Host Microbe. 2011;10:248–59. https:// doi. org/ Clin Microbiol. 2006;44:1257–61. https:// doi. org/ 10. 1128/ JCM. 44.4. 1257- 10. 1016/j. chom. 2011. 08. 006.1261. 2006. 27. Shafeeq S, Kuipers OP, Kloosterman TG. The role of zinc in the inter- 45. Ong CY, Gillen CM, Barnett TC, Walker MJ, McEwan AG. An antimicrobial play between pathogenic streptococci and their hosts. Mol Microbiol. role for zinc in innate immune defense against group a streptococcus. J 2013;88:1047–57. https:// doi. org/ 10. 1111/ mmi. 12256. Infect Dis. 2014;209:1500–8. https:// doi. org/ 10. 1093/ infdis/ jiu053. 28. Turner AG, Ong C-LY, Walker MJ, Djoko KY, McEwan AG. Transition metal homeostasis in Streptococcus pyogenes and Streptococcus pneumoniae. Publisher’s Note Adv Microb Physiol. 2017;70:123–91. https:// doi. org/ 10. 1016/ bs. ampbs. Springer Nature remains neutral with regard to jurisdictional claims in pub- 2017. 01. 002. lished maps and institutional affiliations. 29. Danilova TA, Danilina GA, Adzhieva AA, Vostrova EI, Zhukhovitskii VG, Cheknev SB. Inhibitory effect of copper and zinc ions on the growth of Streptococcus pyogenes and Escherichia coli biofilms. Bull Exp Biol Med. 2020;169:648–52. https:// doi. org/ 10. 1007/ s10517- 020- 04946-y. 30. Makthal N, Kumaraswami M. Zinc’ing it out: zinc homeostasis mecha- nisms and their impact on the pathogenesis of human pathogen group a Streptococcus. Metallomics. 2017;9:1693–702. https:// doi. org/ 10. 1039/ c7mt0 0240h. 31. Leizer J, Nasioudis D, Forney LJ, Schneider GM, Gliniewicz K, Boester A, et al. Properties of epithelial cells and vaginal secretions in pregnant women when lactobacillus crispatus or lactobacillus iners dominate the vaginal microbiome. Reprod Sci. 2018;25:854–60. https:// doi. org/ 10. 1177/ 19337 19117 698583. 32. Yano J, Lilly E, Barousse M, Fidel PL. Epithelial cell-derived S100 calcium- binding proteins as key mediators in the hallmark acute neutrophil response during Candida vaginitis. Infect Immun. 2010;78:5126–37. https:// doi. org/ 10. 1128/ IAI. 00388- 10. 33. Haley KP, Delgado AG, Piazuelo MB, Mortensen BL, Correa P, Damo SM, et al. The human antimicrobial protein calgranulin C participates in control of helicobacter pylori growth and regulation of virulence. Infect Immun. 2015;83(7):2944–56. https:// doi. org/ 10. 1128/ IAI. 00544- 15. 34. Damo SM, Kehl-Fie TE, Sugitani N, Holt ME, Rathi S, Murphy WJ, et al. Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens. Proc Natl Acad Sci U S A. 2013;110:3841–6. https:// doi. org/ 10. 1073/ pnas. 12203 35. Neyrolles O, Wolschendorf F, Mitra A, Niederweis M. Mycobacteria, metals, and the macrophage. Immunol Rev. 2015;264:249–63. https:// doi. org/ 10. 1111/ imr. 12265. 36. Neyrolles O, Mintz E, Catty P. Zinc and copper toxicity in host defense against pathogens: mycobacterium tuberculosis as a model example of an emerging paradigm. Front Cell Infect Microbiol. 2013;3:89. https:// doi. org/ 10. 3389/ fcimb. 2013. 00089. 37. von Pein JB, Stocks CJ, Schembri MA, Kapetanovic R, Sweet MJ. An alloy of zinc and innate immunity: Galvanising host defence against infection. Cell Microbiol. 2021;23:e13268. https:// doi. org/ 10. 1111/ cmi. 13268. 38. Maalej H, Hmidet N, Boisset C, Buon L, Heyraud A, Nasri M. Optimization of exopolysaccharide production from pseudomonas stutzeri AS22 and examination of its metal-binding abilities. J Appl Microbiol. 2015;118:356– 67. https:// doi. org/ 10. 1111/ jam. 12688. 39. De Philippis R, Colica G, Micheletti E. Exopolysaccharide-producing cyanobacteria in heavy metal removal from water: molecular basis and practical applicability of the biosorption process. Appl Microbiol Biotech- Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : nol. 2011;92:697–708. https:// doi. org/ 10. 1007/ s00253- 011- 3601-z. 40. Mukherjee P, Mitra A, Roy M. Halomonas Rhizobacteria of Avicennia fast, convenient online submission marina of Indian Sundarbans promote Rice growth under saline and thorough peer review by experienced researchers in your field heavy metal stresses through exopolysaccharide production. Front Microbiol. 2019;10:1207. https:// doi. org/ 10. 3389/ fmicb. 2019. 01207. rapid publication on acceptance 41. Polak-Berecka M, Szwajgier D, Waśko A. Biosorption of Al(+3) and cd(+2) support for research data, including large and complex data types by an exopolysaccharide from lactobacillus rhamnosus. J Food Sci. • gold Open Access which fosters wider collaboration and increased citations 2014;79:T2404–8. https:// doi. org/ 10. 1111/ 1750- 3841. 12674. 42. Mitra A, Chatterjee S, Kataki S, Rastogi RP, Gupta DK. Bacterial tolerance maximum visibility for your research: over 100M website views per year strategies against lead toxicity and their relevance in bioremediation application. Environ Sci Pollut Res Int 2021;28:14271–14284. oi: https:// At BMC, research is always in progress. doi. org/ 10. 1007/ s11356- 021- 12583-9. Learn more biomedcentral.com/submissions 43. Musser JM, Mattingly SJ, Quentin R, Goudeau A, Selander RK. Identifica- tion of a high-virulence clone of type III Streptococcus agalactiae (group B http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png BMC Microbiology Springer Journals

The antimicrobial activity of zinc against group B Streptococcus is strain-dependent across diverse sequence types, capsular serotypes, and invasive versus colonizing isolates

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Abstract

Background: Streptococcus agalactiae or Group B Streptococcus (GBS) is an encapsulated gram-positive bacterial pathobiont that commonly colonizes the lower gastrointestinal tract and reproductive tract of human hosts. This bac- terium can infect the gravid reproductive tract and cause invasive infections of pregnant patients and neonates. Upon colonizing the reproductive tract, the bacterial cell is presented with numerous nutritional challenges imposed by the host. One strategy employed by the host innate immune system is intoxication of bacterial invaders with certain transition metals such as zinc. Methodology: Previous work has demonstrated that GBS must employ elegant strategies to circumnavigate zinc stress in order to survive in the vertebrate host. We assessed 30 strains of GBS from diverse isolation sources, capsular serotypes, and sequence types for susceptibility or resistance to zinc intoxication. Results: Invasive strains, such as those isolated from early onset disease manifestations of GBS infection were signifi- cantly less susceptible to zinc toxicity than colonizing strains isolated from rectovaginal swabs of pregnant patients. Additionally, capsular type III (cpsIII) strains and the ST-17 and ST-19 strains exhibited the greatest resilience to zinc stress, whereas ST-1 and ST-12 strains as well as those possessing capsular type Ib (cpsIb) were more sensitive to zinc intoxication. Thus, this study demonstrates that the transition metal zinc possesses antimicrobial properties against a wide range of GBS strains, with isolation source, capsular serotype, and sequence type contributing to susceptibility or resistance to zinc stress. Keywords: Antimicrobial, Metal, Zinc, Streptococcus agalactiae, Group B Streptococcus Introduction Group B Streptococcus (GBS), or Streptococcus agalac- tiae, infections are one of the top five leading causes of neonatal mortality. GBS infection induces chorioamnio- *Correspondence: jennifer.a.gaddy@vumc.org nitis, preterm prelabor rupture of the gestational mem- Department of Medicine, Division of Infectious Diseases, Vanderbilt University Medical Center, A2200 Medical Center North, 1161 21st branes (PPROM), preterm birth, and both maternal Avenue South, Nashville, TN 37232, U.S.A. and neonatal sepsis [1]. GBS disease in neonates often Full list of author information is available at the end of the article © The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Francis et al. BMC Microbiology (2022) 22:23 Page 2 of 14 manifests as early-onset or late-onset sepsis, pneumonia, dissemination in a mouse model of invasive disease in the or meningitis and can lead to death [2]. blood, heart, liver, and bladder. Together, these results GBS is an encapsulated gram-positive bacterium indicate that zinc is an important innate immune anti- that colonize the urogenital and/or lower gastrointesti- microbial strategy employed against GBS, and that GBS nal tract of healthy women and colonization rates vary resistance to zinc toxicity is critical for full virulence. between 15 and 40% depending on geographical region While zinc has been implicated in modulating the [3–5]. During infection of the upper female reproductive immune response to infection [15], the role zinc plays in tract, GBS ascends the vagina to the cervix and then to the context of perinatal-related GBS infections has not the uterus where the bacteria can cross the gestational yet been elucidated. In this study, we advanced previous membrane barrier. GBS infection of these extraplacen- findings by analyzing the antimicrobial effects of zinc tal, gestational membranes (i.e., chorioamnionitis) can against a panel of clinical GBS strains that vary by cap- provoke inflammation that triggers labor and/or causes sular serotype, sequence type (ST), isolation source, and PPROM [1]. Infection of the fetus by GBS can also lead clinical presentation. We observed strain-specific vari - to stillbirth or neonatal sepsis. GBS can cause early-onset ation in susceptibility to zinc intoxication with specific neonatal disease (EOD) (within the first week of life), of differences between invasive and colonizing strains and which 90% occurs 0-3 days after birth, or late-onset dis- across different capsular serotypes and STs. ease (LOD), which occurs one week to three months after birth [2]. Methods Current recommendations for prevention of EOD focus Bacterial strains and culture conditions on maternal GBS screening at 35-37 week’s gestation A bank of 27 GBS clinical strains were provided by Dr. H. and the use of intrapartum antibiotic prophylaxis (IAP). Dele Davies following recovery from neonates with inva- Beta-lactam antibiotics such as penicillin and ampicil- sive disease [16] and colonized mothers sampled before lin, are used in GBS positive expecting mothers [6]. Cur- and after childbirth [17] for use in this study (Table  1). rently, IAP has not been as effective for the treatment and These strains were all isolated from separate patients and prevention of LOD, nor has it reduced the incidence of were previously characterized using multilocus sequence PPROM, preterm birth, or stillbirth [6, 7]. In addition, typing and cps typing [18, 19]. Three common laboratory the rise in antibiotic resistant GBS strains is also of grave reference strains (A909, NEM316, and COH1; American concern [8]. Thus, developing new interventions to con - Type Culture Collection) were also evaluated. Bacterial trol antibacterial resistance is of great importance. Subse- strains were cultured on tryptic soy agar (TSA) plates quently, these innovations will decrease the incidence of supplemented with 5% sheep blood at 37 °C overnight maternal-fetal GBS infections and complications. followed by inoculation into brain-heart infusion broth Zinc, copper, and iron are transition metals that are (BHI) and incubation in aerobic conditions (ambient air, essential micronutrients for all cells. In both eukaryotes shaking at 200 rpm) at 37 °C. After 24 h, bacterial density and prokaryotes, transition metals serve as co-factors was measured spectrophotometrically to determine the for enzymes that perform critical cellular processes [9]. optical density at 600 nm (OD ). These bacterial cul - These metal ions, though required, can become toxic to tures were used for growth and viability assays. bacteria at high concentrations [10]. The host exploits this specifically within innate immune cells, such as mac - Evaluation of bacterial growth rophages and neutrophils, which load the phagosome GBS growth was determined by a spectrophotometric 2+ with divalent zinc cations (Zn ) to intoxicate phagocy- reading of optical density (OD) at OD as previously tosed bacteria [11, 12]. To circumnavigate this, bacteria described [20]. Briefly, GBS cultures were grown over - must regulate metal import and export machinery to night and diluted at 1:10 in fresh BHI medium; 100 μL of maintain normal zinc levels for growth and survival [13]. 1:10 diluted cultures were added to each well in a 96-well Recent work demonstrates that GBS employs a rep- plate. Increasing concentrations of divalent zinc ions 2+ ertoire of factors to facilitate metal homeostasis and (Zn ) in the form of zinc chloride (Z nCl ) (0, 125, 250, promote bacterial survival in the vertebrate host. Spe- 500, 750, 1000, 2500, 5000 μM) were added to the culture cifically, GBS require efflux determinants to overcome media. These concentrations were chosen because they metal stress and promote survival. Sullivan and col- represent a range of physiologically relevant concentra- leagues reported that GBS elaborates the CzcD efflux tions often encountered in the host-pathogen environ- system, activated by the SczA response regulator to ment in vivo [11]. The plates were incubated statically in manage intracellular zinc levels [14]. Furthermore, they 5% CO at 37 °C overnight. The following day, bacterial demonstrated that the CzcD and SczA systems are criti- density was estimated via OD . Three fresh biologi - cal for zinc resistance, survival within macrophages, and cal replicates were assessed with 1-3 technical replicates F rancis et al. BMC Microbiology (2022) 22:23 Page 3 of 14 within each biological replicate, and the OD values Results were normalized to a blank control of sterile, uninocu- High concentrations of zinc suppress bacterial growth lated bacteriological medium (BHI). in many clinical GBS isolates Previous reports indicate that zinc has antimicrobial Statistical analyses activities against GBS [14]. To enhance the generalizabil- Statistical analyses were performed using Mann-Whitney ity of these findings, we sought to test a larger number U for MIC studies, and either Student’s t-test or one-way of GBS strains, thereby capturing more isolates (both ANOVA with either Tukey’s or Dunnett’s post hoc cor- colonizing and invasive) across diverse capsular sero- rection for multiple comparisons for bacterial growth types and genetic STs. We also investigated the effects assays. All reported P values were adjusted to account for of increasing zinc concentration exposure. Out of the multiple comparisons. P values of ≤0.05 were considered 30 GBS strains screened, 4 strains (GB0083, GB0561, significant. All data analyzed in this work were derived GB0651, and A909) exhibited significant inhibition of from at least three biological replicates. Statistical analy- bacterial growth when treated with 125 μM zinc (Table 1; ses were performed using GraphPad Prism 9 software P < 0.05, Student’s t test, compared to medium alone (GraphPad Prism Software Inc., La Jolla, California). control cultures). All of these strains were classified as Table 1 Strain identifier, strain type, Isolation source, capsular type, and sequence type (ST) of clinical strains of Streptococcus agalactiae used in this study and the minimum inhibitory concentration (MIC) of zinc chloride required to suppress growth (as determined by OD ) by comparison with growth in medium alone lacking zinc supplementation (P < 0.05, Student’s t test) Strain Identifier Strain Type Sequence Type Capsular Serotype Isolation Growth Source MIC GB0002 Colonizing ST-23 cpsIa Vaginal/rectal colonization 2500 μM GB0012 Colonizing ST-1 cpsV Vaginal/rectal colonization 500 μM GB0037 Invasive ST-1 cpsV EOD/sepsis 1000 μM GB0064 Invasive ST-17 cpsIII EOD/sepsis 2500 μM GB0066 Invasive ST-19 cpsIII EOD/sepsis > 5000 μM GB0069 Invasive ST-17 cpsIII EOD/sepsis 2500 μM GB0079 Invasive ST-19 cpsIII EOD/sepsis > 5000 μM GB0083 Colonizing ST-1 cpsVI Vaginal/rectal colonization 125 μM GB0112 Colonizing ST-12 cpsIII Vaginal/rectal colonization 500 μM GB0115 Colonizing ST-17 cpsIII Vaginal/rectal colonization 2500 μM GB0241 Colonizing ST-23 cpsV Vaginal/rectal colonization 1000 μM GB0285 Colonizing ST-12 cpsII Vaginal/rectal colonization 2500 μM GB0291 Colonizing ST-12 cpsII Vaginal/rectal colonization 2500 μM GB0374 Invasive ST-12 cpsIb EOD/sepsis 2500 μM GB0377 Invasive ST-19 cpsIII EOD/sepsis > 5000 μM GB0390 Invasive ST-23 cpsIa EOD/sepsis > 5000 μM GB0397 Invasive ST-23 cpsIII EOD/sepsis 1000 μM GB0411 Invasive ST-17 cpsIII EOD/sepsis 750 μM GB0418 Invasive ST-17 cpsIII EOD/sepsis 2500 μM GB0438 Invasive ST-12 cpsIb LOD/sepsis 1000 μM GB0561 Colonizing ST-19 cpsV Vaginal/rectal colonization 125 μM GB0571 Colonizing ST-19 cpsIII Vaginal/rectal colonization 250 μM GB0590 Colonizing ST-19 cpsIII Vaginal/rectal colonization > 5000 μM GB0651 Colonizing ST-19 cpsIb Vaginal/rectal colonization 125 μM GB0653 Colonizing ST-12 cpsII Vaginal/rectal colonization 1000 μM GB0654 Colonizing ST-17 cpsIII Vaginal/rectal colonization 2500 μM GB0663 Colonizing ST-19 cpsIII Vaginal/rectal colonization > 5000 μM NEM316 Invasive ST-23 cpsIII EOD/sepsis 500 μM COH1 Invasive ST-17 cpsIII Blood 750 μM A909 Invasive ST-7 cpsIa Blood/sepsis 125 μM Francis et al. BMC Microbiology (2022) 22:23 Page 4 of 14 Fig. 1 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 (OD ). At 0, 250, 500, 750, 1000, 2500, and 5000 μM concentrations of zinc, colonizing strains of GBS (circles) showed no significant differences in OD values across strain type 600 F rancis et al. BMC Microbiology (2022) 22:23 Page 5 of 14 colonizing strains except the single laboratory strain GB0411 (invasive) and COH1 (laboratory) exhibited sig- (A909). At 250 μM zinc, an additional colonizing strain nificant inhibition of bacterial growth compared to the (GB0571) exhibited significant inhibition of bacterial medium only control (Table  1; P < 0.05, Student’s t test). growth compared to cultures grown in medium alone The growth of 5 additional strains (GB0037, GB0397, (Table 1; P < 0.05, Student’s t test). Growth of three addi- GB0438, GB0241, GB0653), including three invasive and tional strains, two colonizing strains and one laboratory two colonizing isolates, respectively, was inhibited when strain (GB0012, GB0112, and NEM316, respectively), was treated with 1000 μM zinc (Table  1; P < 0.05, Student’s t inhibited when treated with a concentration of 500 μM test, compared to medium alone control cultures). The zinc (Table  1; P < 0.05, Student’s t test, compared to growth of 9 additional strains (GB002, GB0115, GB0285, medium alone control cultures). At 750 μM zinc, strains GB0291, GB0654, GB0064, GB0069, GB0374, GB0418), Fig. 2 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 (OD ). 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 OD values in each zinc concentration compared to OD values for each strain in medium alone). Statistical 600 600 significance was determined by paired Student’s t test (n = 3 biological replicates) Francis et al. BMC Microbiology (2022) 22:23 Page 6 of 14 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 col- onizing 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 con- trol cultures). GBS colonizing and invasive strain types differ in susceptibility to zinc intoxication Because zinc has been shown to be a crucial antimicro- bial strategy deployed by the innate immune system [10– Fig. 3 Minimum Inhibitory Concentration (MIC) of zinc chloride 12], we hypothesized that there could be differences in required to inhibit GBS growth across colonizing or invasive strains. susceptibility to zinc intoxication between colonizing and Analysis of susceptibility to zinc-associated growth inhibition in invasive GBS strains. To test this, we stratified the GBS invasive vs. clinical isolates of Group B Streptococcus (GBS). GBS strains strains by clinical phenotype. Strains were classified as isolated from colonized patients, or patients experiencing invasive disease were grown in increasing concentrations of zinc chloride and “colonizing” if they were recovered from asymptomatic mean MIC was calculated. Significant differences were determined women sampled before or after childbirth, whereas “inva- by Mann-Whitney U test (P < 0.05). Dotted line indicates upper limit sive” strains were isolated from babies with GBS disease. of detection Strains were exposed to increasing concentrations of zinc chloride and growth was measured after 24 h of static incubation in 5% C O at 37 °C (Fig. 1). In medium alone, the mean OD measurement was calculated as 0.34 for growth compared to colonizing strains (P = 0.0378, colonizing strains and 0.33 for invasive strains, results paired Student’s t test; P = 0.1160, Mann-Whitney U that were statistically indistinguishable (P = 0.1627, test). Exposure to 1000 μM zinc resulted in a 25% mean Mann-Whitney U test). Similarly, no significant differ - decrease in colonizing strain growth, compared to only a ence was noted in raw OD values between colonizing 9% in invasive strains (P = 0.0486, paired Student’s t test; and invasive strains at 125, 250, 500, 750, 1000, 2500, or P = 0.2328, Mann-Whitney U test). Exposure to 2500 μM 5000 μM zinc exposure. However, calculation of percent zinc resulted in a 47% mean decrease in colonizing strain growth of each strain (comparing growth of a specific growth, compared to a 24% percent decrease in invasive strain at each zinc concentration compared to growth strains (P = 0.00781, paired Student’s t test; P = 0.0555, of that strain in medium alone) revealed that invasive Mann-Whitney U test). At a concentration of 5000 μM strains exhibited a significantly enhanced mean growth zinc, no significant difference in growth was observed when treated with 250, 500, 1000, and 2500 μM zinc (12, between colonizing and invasive strains, largely because 12, 14, 19, and 36%, respectively) compared to colonizing the growth of most strains was significantly inhibited at strains (P < 0.05, Mann-Whitney U test, Fig. 2). Exposure this concentration. Comparison of minimal inhibitory to 250 μM zinc resulted in invasive strains having 13% concentrations (MIC) of zinc to repress growth for colo- higher percent growth compared to colonizing strains, a nizing versus invasive strains, revealed colonizing strains result that was statistically significant (P = 0.0143, paired have a mean MIC of 1875 μM zinc, whereas invasive Student’s t test; P = 0.0329, Mann-Whitney U test). Expo- strains have a mean MIC of 3145 μM zinc (Fig.  3), a 68% sure to 500 μM zinc resulted in a 14% higher percent (See figure on next page.) Fig. 4 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 (OD ). 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) F rancis et al. BMC Microbiology (2022) 22:23 Page 7 of 14 Fig. 4 (See legend on previous page.) Francis et al. BMC Microbiology (2022) 22:23 Page 8 of 14 contribute to alterations in susceptibility to zinc toxicity. increase which was statistically significant (P = 0.0402, To test this, we stratified strains based on capsular sero - Mann-Whitney U test). type and analyzed growth in cultures exposed to increas- ing concentration of zinc (Fig.  5). At concentrations Susceptibility to zinc toxicity differs across GBS strains as low as 125 μM of zinc, capsular serotype III (cpsIII) from varying isolation sources strains emerged as having enhanced growth compared Because differences were observed between invasive to the cpsIb and cpsII strains. Notably, the cpsIII strains and colonizing strains, we hypothesized that the source also exhibited enhanced growth at 250, 500, 750, 1000, of bacterial strain isolation could contribute to zinc and 2500 μM zinc compared to the cpsIb strains (P < 0.05, intoxication susceptibility due to GBS adaptation to the one-way ANOVA). At 2500 μM zinc, cpsIII strains exhib- ecology of the specific host niche. To test this, we strat - ited the highest mean growth (mean OD = 0.267), fol- ified strains into categories based on source of strain lowed by strains with cpsIa (mean OD = 0.246), cpsV isolation (Fig.  4) such as blood from neonatal early (mean OD = 0.200), cpsII (mean OD = 0.126), and onset disease (EOD), late onset disease (LOD), rectal 600 600 cpsIb (mean OD = 0.107). The cpsVI strains were most or vaginal swabs (Vaginal/Rectal), or adult blood from sensitive to zinc toxicity at a concentration of 2500 μM septic patients (Blood/Sepsis). Results indicate that at (mean OD = 0.100) compared to other capsular sero- concentrations of 250, 500, 750, 1000, and 2500 μM types. However, at concentrations of 125, 250, 500, 750, zinc, isolates from EOD have significantly enhanced and 1000 μM zinc, cpsIb isolates consistently exhibited growth (12, 13, 13, 27, and 39%, respectively) compared the lowest growth, or greatest level of inhibition, among to vaginal/rectal isolates (P < 0.05, one-way ANOVA). all capsular serotypes tested, underscoring their suscepti- At concentration of 250 μM zinc, LOD isolates were bility to zinc intoxication (P < 0.05, one-way ANOVA). At significantly more susceptible to zinc toxicity than a concentration of 5000 μM zinc, no statistically signifi - those isolated from blood/sepsis (P < 0.05, one-way cant differences were observed between capsular types, a ANOVA). At 500 μM zinc, blood/sepsis isolates were result that is likely due to a threshold effect of all strains significantly more tolerant of zinc stress than their vag - experiencing significant growth inhibition. inal/rectal or LOD isolate counterparts (P < 0.05, one- Because our results have shown that colonizing strains way ANOVA). At 750 μM zinc, blood/sepsis and EOD may be more susceptible to zinc toxicity, and the cpsIII isolates were significantly less susceptible to zinc stress strains exhibited low susceptibility to zinc toxicity, we than rectovaginal isolates. At 1000 μM zinc, EOD and stratified the cpsIII strains into invasive versus colonizing blood/sepsis isolates were significantly less susceptible strains to ascertain if there were any differences in these than rectovaginal isolates. However, GBS isolated from two cohorts (Fig. 6). Mann-Whitney U analyses revealed LOD were significantly more susceptible than strains no statistically significant difference between invasive from the other three isolation sites. and colonizing cpsIII strains (P = 0.4401). GBS capsular serotype confers varying susceptibility GBS susceptibility to zinc toxicity varies across sequence to zinc intoxication types Capsular serotypes have been implicated as an impor- Because different GBS sequence types (STs) are associ- tant virulence factor that aids in evasion of the innate ated with maternal colonization and neonatal disease immune response [21]. Additionally, capsular serotype [24], it is possible that strains of different STs have III strains are associated with higher rates of invasive variable mechanisms to facilitate metal homeostasis. neonatal disease [22] and account for the majority of To test this hypothesis, we stratified GBS strains by late-onset meningitis cases in neonates [23]. Because ST and analyzed the growth under varying concentra- invasive strains and isolates from EOD were less suscep- tions of zinc (Fig.  7). At 2500 μM zinc, ST-17, ST-19, tible to zinc toxicity than rectovaginal colonizing strains, and ST-23 remained the least susceptible to zinc we hypothesized that capsular serotype variation could (See figure on next page.) Fig. 5 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 (OD ). 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) F rancis et al. BMC Microbiology (2022) 22:23 Page 9 of 14 Fig. 5 (See legend on previous page.) Francis et al. BMC Microbiology (2022) 22:23 Page 10 of 14 Streptococcus spp. [23]. Bacteria that have the capability to survive and replicate inside the phagosome of mac- rophages employ and regulate metal transport machinery to maintain metal homeostasis. For example, the notori- ous intracellular pathogen, Mycobacterium tuberculosis, has been shown to upregulate P-type ATPases which act in heavy metal efflux to counteract the toxic, high extra - cellular zinc levels of the phagosome [26]. In fact, metal intoxication survival is a critical virulence trait in S. pneu- moniae and S. pyogenes [27–30] as well as GBS [14]. In this study, we sought to determine the differences in sus - ceptibility or resistance to zinc toxicity in 30 strains of GBS, spanning diverse capsular serotypes, STs, isolation source, and disease manifestation. Our work demonstrates that GBS colonizing and Fig. 6 Minimum Inhibitory Concentration (MIC) of zinc chloride invasive strain types differ in susceptibility to zinc required to inhibit GBS growth across colonizing or invasive strains intoxication. Specifically, invasive strains exhibit of capsular serotype III (cpsIII) isolates. Analysis of susceptibility to diminished susceptibility to zinc toxicity compared zinc-associated growth inhibition in invasive vs. clinical isolates of Group B Streptococcus (GBS). GBS strains isolated from colonized to colonizing strains, indicating invasive strains may patients, or patients experiencing invasive disease were grown have acquired adaptations to survive metal intoxication in increasing concentrations of zinc chloride and mean MIC was strategies imposed within the vertebrate host during calculated. Mann-Whitney U test revealed no statistically significant invasive infections. GBS strains from varying isolation differences between colonizing and invasive strains of the cpsIII sources also exhibit varying susceptibility to zinc intox- cohort (P = 0.4401). Dotted line indicates upper limit of detection ication. In particular, strains isolated from early onset disease manifestations have significantly enhanced tolerance of zinc intoxication compared to rectovagi- intoxication compared to ST-1, ST-7, ST-12 (P < 0.05, nal colonizing isolates. Again, similar to the results one-way ANOVA). At 5000 μM zinc, ST-19 remained observed with colonizing versus invasive isolates, this the least susceptible to zinc intoxication compared to could reflect strain-specific adaptation to the ecological all other strains tested (P < 0.05, one-way AN OVA). niche of the host. The human vaginal environment is Interestingly, the ST-19 isolates that exhibited the rich in S100A-family proteins, especially during infec- highest resistance to zinc intoxication were all classi- tious processes, and these proteins bind and seques- fied as cpsIII strains. ter nutrient metals, such as zinc [31–34]. It is possible that the GBS-colonized vaginal mucosa represents an Discussion environment with low zinc availability, thus there is The battle for essential nutrient metals between the verte - no selective pressure for colonizing strains to develop brate host and invading pathogen has been closely linked strategies to circumnavigate high zinc concentrations. to virulence [25]. Starvation of transition metals is detri- Conversely, numerous studies have shown that circu- mental to bacteria, however, high concentrations of met- lating innate immune cells such as macrophages and als like zinc also have antimicrobial effects [10, 12]. Zinc neutrophils use zinc intoxication as a strategy to inhibit plays an important antimicrobial role in innate immune invading microbes [11, 35–37]. It is possible that inva- defense against several pathogens, including a variety of sive strains have undergone selection for survival in (See figure on next page.) Fig. 7 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 (OD ). 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) F rancis et al. BMC Microbiology (2022) 22:23 Page 11 of 14 Fig. 7 (See legend on previous page.) Francis et al. BMC Microbiology (2022) 22:23 Page 12 of 14 high zinc environments, yielding strains with greater circumstances. Thus, some “colonizing” strains might resistance to the toxicity of this transition metal. be misclassified because they have unrecognized Our results indicate that GBS capsular serotype also invasive potential. Such a misclassification could bias confers varying susceptibility to zinc intoxication. Spe- towards a null hypothesis (contributing to a type II cifically, cpsIII isolates were less susceptible to zinc error). Additional genomics studies are also war- intoxication, whereas cpsIb isolates were much more ranted to identify genetic traits linked to zinc resist- susceptible to zinc toxicity. This is interesting because ance, particularly in the more virulent lineages. An cpsIII is the predominant capsular serotype responsible additional limitation includes the medium sample size for invasive neonatal infections. Additionally, there is which should be expanded in future studies to include growing evidence that bacterial exopolysaccharides have more representation in other capsular serotypes and strong binding properties for metal sorption [38, 39]. sequence types to draw broader conclusions across a Additionally, production of specific exopolysaccharides larger number of GBS strains. can promote cellular survival of metal stress [40–42]. u Th s, it remains possible that the variations in capsular Conclusions polysaccharide production responsible for alterations In conclusion, we report strain variations within a cohort in capsular serotype of the surveyed GBS strains could of GBS strains with respect to susceptibility to zinc intox- contribute to alternate binding of excess metals, thereby ication across STs, capsular serotypes, isolation source, altering GBS susceptibility to intoxication with metals, and invasive versus colonizing strains. Invasive isolates such as zinc. demonstrated greater resistance to zinc toxicity com- Our study also revealed that different sequence types pared to colonizing strains. Additionally, ST-1 and ST-12 of GBS had varying susceptibilities to zinc intoxication. were highly susceptible to zinc stress, while ST-17, ST-19, Specifically, ST-1 and ST-12 were highly susceptible to and ST-23 were much more resistant to zinc intoxication. zinc stress, while ST-17, ST-19, and ST-23 were much cpsIII isolates were less susceptible to zinc intoxication, more resistant to zinc intoxication. A cross-continental whereas cpsIb isolates were much more susceptible to study revealed that GBS ST-1 and ST-19 are associated zinc toxicity. Our study is a pilot study that is hamstrung with asymptomatic colonization, while ST-17 is predomi- by the relatively small number of strains. Future stud- nantly associated with invasive neonatal disease [24]. ies will require an expansion to include genetic studies ST-23 was associated with both rectovaginal carriage and a larger number of strains and diverse capsular and and invasive GBS disease [24]. By contrast, STs 1, 17, 19 sequence types, as well as GBS strains from non-perina- and 23, were all found to colonize pregnant women at tal sources and distinct geographic locations. higher rates in different patient populations [18]. Addi - tionally, ST-17 (specifically capsular serotype III strains Abbreviations in this clade) was linked to EOD and strongly associ- GBS: Group B Streptococcus; PPROM: preterm prelabor rupture of the ated with LOD and meningitis [19, 43, 44]. This finding gestational membranes; EOD: Early onset disease; LOD: Late onset disease; 2+ IAP: Intrapartum antibiotic prophylaxis; Zn : Zinc; ZnCl : Zinc chloride; ST: further supports a model in which invasive strains are Sequence type; TSA: Tryptic soy agar; OD : Optical density at 600 nm; MIC: likely undergoing positive selection for zinc resistance Minimum inhibitory concentration; cps: Capsular polysaccharide; rpm: Rota- as a critical virulence factor to overcome innate immune tions per minute; nm: Nanometer; μM: Micromolar. defenses which employ zinc intoxication as an antimicro- Acknowledgments bial strategy [45]. Interestingly, in our study, ST-19 strains Not applicable. (largely colonizing strains) that were most resistant to Authors’ contributions zinc intoxication were all cpsIII strains, underscoring the SDM curated and validated the clinical strains for this study. JDF, MAG, JL, and relationship between capsular polysaccharide production JAG conceptualized and performed the wet-bench experiments. JDF, MAG, JL, and zinc resistance in GBS. SAM, GK, DMA, SDM, and JAG analyzed results and interpreted data. JDF, MAG, JL, SAM, GK, DMA, SDM, and JAG wrote and edited the manuscript for critical content. All authors have read and approved the manuscript and have given Limitations of the study their consent to publish this work. There are several limitations of our study including Funding the clinical definitions of “colonizing” versus “invasive” This work was supported by the National Institutes of Health grant R01 strains which can be imperfect. Isolating strains from HD090061 (to J.A.G.), R35GM133602 (to S.D.T.), T32 HL007411-36S1 (support- invasive neonatal infections proves that such strains ing J.L.), and 2T32AI112541-06 (supporting J.D.F.). Additional support was provided by the Department of Veterans Affairs Office of Research BX005352 are capable of causing perinatal infection. However, (to J.A.G.). Core Services were performed through both Vanderbilt University simply because a “colonizing” strain was isolated from Medical Center’s Digestive Disease Research Center supported by NIH grant a rectal or vaginal swab does not mean it would be P30DK058404 Core Scholarship and Vanderbilt Institute for Clinical and Translational Research program supported by the National Center for Research incapable of causing invasive disease under different F rancis et al. BMC Microbiology (2022) 22:23 Page 13 of 14 Resources, Grant UL1 RR024975-01, and the National Center for Advancing streptococcal disease in the United States, 2006 to 2015: multistate labo- Translational Sciences, Grant 2 UL1 TR000445-06, which supports access to ratory and population-based surveillance. JAMA Pediatr. 2019;173:224– core facilities and biostatistics support. The content is solely the responsibility 33. https:// doi. org/ 10. 1001/ jamap ediat rics. 2018. 4826. of the authors and does not necessarily represent the official views of the NIH 8. Shipitsyna E, Shalepo K, Zatsiorskaya S, Krysanova A, Razinkova M, or any of the other supporters. Grigoriev A, et al. Significant shifts in the distribution of vaccine capsular polysaccharide types and rates of antimicrobial resistance of perinatal Availability of data and materials group B streptococci within the last decade in St. Petersburg, Russia. Eur The datasets used and/or analyzed during the current study available from the J Clin Microbiol Infect Dis. 2020;39:1487–93. https:// doi. org/ 10. 1007/ corresponding authors upon reasonable request.s10096- 020- 03864-1. 9. Andreini C, Bertini I, Cavallaro G, Holliday GL, Thornton JM. Metal ions in biological catalysis: from enzyme databases to general princi- Declarations ples. J Biol Inorg Chem. 2008;13:1205–18. https:// doi. org/ 10. 1007/ s00775- 008- 0404-5. Ethics approval and consent to participate 10. Djoko KY, Ong CY, Walker MJ, McEwan AG. The role of copper and zinc The secondary use of de-identified or coded samples is not considered toxicity in innate immune defense against bacterial pathogens. J Biol research involving human subjects under 45 CFR 46. Biospecimens (bacterial Chem. 2015;290:18954–61. https:// doi. org/ 10. 1074/ jbc. R115. 647099. strains) used in this study were deidentified and need for consent was waived 11. Wagner D, Maser J, Lai B, Cai Z, Barry CE 3rd, Bentrup HZ, et al. Elemental by the IRB in accordance with federal regulation (45 CFR 46, Department of analysis of Mycobacterium avium-, mycobacterium tuberculosis-, and Health and Human Services, Authority: 5 U.S.C. 301; 42 U.S.C. 289(a); 42 U.S.C. mycobacterium smegmatis-containing phagosomes indicates pathogen- 300v-1(b)). induced microenvironments within the host cell’s endosomal system. J Immunol. 2005;174:1491–500. https:// doi. org/ 10. 4049/ jimmu nol. 174.3. Consent for publication Not applicable. 12. Stafford SL, Bokil NJ, Achard MES, Kapetanovic R, Schembri MA, McEwan AGS, et al. Metal ions in macrophage antimicrobial pathways: emerging Competing interests roles for zinc and copper. Biosci Rep. 2013;33. https:// doi. org/ 10. 1042/ The authors declare no conflicts of interest. The authors declare no competing BSR20 130014. interests. 13. Hood MI, Skaar EP. Nutritional immunity: transition metals at the patho- gen–host interface. Nat Rev Microbiol. 2012;10:525–37. https:// doi. org/ Author details 10. 1038/ nrmic ro2836. Department of Pathology, Microbiology and Immunology, Vanderbilt 14. Sullivan MJ, Goh KGK, Ulett GC. Cellular Management of Zinc in Group University Medical Center, Nashville, TN 37212, USA. South African Medical B Streptococcus Supports Bacterial Resistance against Metal Intoxication Research Council Vaccines and Infectious Diseases Analytics Research Unit, and Promotes Disseminated Infection. mSphere. 2021;6. https:// doi. org/ Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, 10. 1128/ mSphe re. 00105- 21. South Africa. Department of Clinical Microbiology, Christian Medical Col- 15. Bonaventura P, Benedetti G, Albarède F, Miossec P. Zinc and its role in lege, Vellore, India. Department of Medicine, Division of Infectious Diseases, immunity and inflammation. Autoimmun Rev. 2015;14:277–85. https:// Vanderbilt University Medical Center, A2200 Medical Center North, 1161 doi. org/ 10. 1016/j. autrev. 2014. 11. 008. 21st Avenue South, Nashville, TN 37232, U.S.A.. Department of Obstetrics 16. Davies HD, Adair C, McGreer A, Ma D, Robertson S, Mucenski M, et al. and Gynecology, Vanderbilt University Medical Center Nashville, Nashville, TN Antibodies to capsular polysaccharides of group B Streptococcus in preg- 37232, USA. Department of Microbiology and Molecular Genetics, Michigan nant Canadian women: relationship to colonization status and infection State University, East Lansing, MI 48824, USA. Department of Veterans Affairs, in the neonate. J Infect Dis. 2001;184:285–91. https:// doi. org/ 10. 1086/ Tennessee Valley Healthcare Systems, Nashville, TN 37212, USA. 17. Spaetgens R, DeBella K, Ma D, Robertson S, Mucenski M, Davies HD. Peri- Received: 28 August 2021 Accepted: 13 December 2021 natal antibiotic usage and changes in colonization and resistance rates of group B streptococcus and other pathogens. 2002;100:525–33. https:// doi. org/ 10. 1016/ s0029- 7844(02) 02068-9. 18. Manning SD, Springman AC, Lehotzky E, Lewis MA, Whittam TS, Davies HD. Multilocus sequence types associated with neonatal group B strepto- References coccal sepsis and meningitis in Canada. J Clin Microbiol. 2009;47:1143–8. 1. Patras KA, Nizet V. Group B streptococcal maternal colonization and https:// doi. org/ 10. 1128/ JCM. 01424- 08. neonatal disease: molecular mechanisms and preventative approaches. 19. Manning SD, Lewis MA, Springman AC, Lehotzky E, Whittam TS, Davies Front Pediatr. 2018;6:27. https:// doi. org/ 10. 3389/ fped. 2018. 00027. HD. Genotypic diversity and serotype distribution of group B strepto- 2. Raabe VN, Shane AL. Group B Streptococcus (Streptococcus agalac- coccus isolated from women before and after delivery. Clin Infect Dis. tiae). Microbiol Spectr. 2019;7. https:// doi. org/ 10. 1128/ micro biols pec 2008;46:1829–37. https:// doi. org/ 10. 1086/ 588296. GPP3-0007-2018. 20. Lu J, Francis JD, Guevara MA, Moore RE, Chambers SA, Doster RS, 3. Campbell JR, Hillier SL, Krohn MA, Ferrieri P, Zaleznik DF, Baker CJ. Group et al. Antibacterial and anti-biofilm activity of the human breast Milk B streptococcal colonization and serotype-specific immunity in pregnant glycoprotein Lactoferrin against group B Streptococcus. Chembiochem. women at delivery. Obstet Gynecol. 2000;96:498–503. https:// doi. org/ 10. 2021;22(12):2124–33. https:// doi. org/ 10. 1002/ cbic. 20210 0016. 1016/ s0029- 7844(00) 00977-7. 21. Rajagopal L. Understanding the regulation of group B streptococcal viru- 4. Stoll BJ, Schuchat A. Maternal carriage of group B streptococci in develop- lence factors. Future Microbiol. 2009;4:201–21. https:// doi. org/ 10. 2217/ ing countries. Pediatr Infect Dis J. 1998;17:499–503. https:// doi. org/ 10. 17460 913.4. 2. 201. 1097/ 00006 454- 19980 6000- 00013. 22. Alhhazmi A, Pandey A, Tyrrell GJ. Identification of group B Streptococcus 5. Armistead B, Oler E, Adams Waldorf K, Rajagopal L. The double life of capsule type by use of a dual phenotypic/genotypic assay. J Clin Micro- group B Streptococcus: asymptomatic colonizer and potent pathogen. J biol. 2017;55:2637–50. https:// doi. org/ 10. 1128/ JCM. 00300- 17. Mol Biol. 2019;431:2914–31. https:// doi. org/ 10. 1016/j. jmb. 2019. 01. 035. 23. Bellais S, Six A, Fouet A, Longo M, Dmytruk N, Glaser P, et al. Capsu- 6. Schrag SJ, Verani JR. Intrapartum antibiotic prophylaxis for the prevention lar switching in group B Streptococcus CC17 hypervirulent clone: a of perinatal group B streptococcal disease: experience in the United future challenge for polysaccharide vaccine development. J Infect Dis. States and implications for a potential group B streptococcal vaccine. 2012;206:1745–52. https:// doi. org/ 10. 1093/ infdis/ jis605. Vaccine. 2013;31(Suppl 4):D20–6. https:// doi. org/ 10. 1016/j. vacci ne. 2012. 24. Jones N, Bohnsack JF, Takahashi S, Oliver KA, Chan M-S, Kunst F, et al. 11. 056. Multilocus sequence typing system for group B streptococcus. J Clin 7. Nanduri SA, Petit S, Smelser C, Apostol M, Alden NB, Harrison LH, Microbiol. 2003;41:2530–6. https:// doi. org/ 10. 1128/ JCM. 41.6. 2530- 2536. et al. Epidemiology of invasive early-onset and late-onset group B 2003. Francis et al. BMC Microbiology (2022) 22:23 Page 14 of 14 25. Palmer LD, Skaar EP. Transition metals and virulence in Bacteria. Streptococcus) causing invasive neonatal disease. Proc Natl Acad Sci U S A. Annu Rev Genet. 2016;50:67–91. https:// doi. org/ 10. 1146/ annur 1989;86:4731–5. https:// doi. org/ 10. 1073/ pnas. 86. 12. 4731. ev- genet- 120215- 035146. 44. Lin F-YC, Whiting A, Adderson E, Takahashi S, Dunn DM, Weiss R, et al. 26. Botella H, Peyron P, Levillain F, Poincloux R, Poquet Y, Brandli I, et al. Phylogenetic lineages of invasive and colonizing strains of serotype III Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in group B streptococci from neonates: a multicenter prospective study. J human macrophages. Cell Host Microbe. 2011;10:248–59. https:// doi. org/ Clin Microbiol. 2006;44:1257–61. https:// doi. org/ 10. 1128/ JCM. 44.4. 1257- 10. 1016/j. chom. 2011. 08. 006.1261. 2006. 27. Shafeeq S, Kuipers OP, Kloosterman TG. The role of zinc in the inter- 45. Ong CY, Gillen CM, Barnett TC, Walker MJ, McEwan AG. An antimicrobial play between pathogenic streptococci and their hosts. Mol Microbiol. role for zinc in innate immune defense against group a streptococcus. J 2013;88:1047–57. https:// doi. org/ 10. 1111/ mmi. 12256. Infect Dis. 2014;209:1500–8. https:// doi. org/ 10. 1093/ infdis/ jiu053. 28. Turner AG, Ong C-LY, Walker MJ, Djoko KY, McEwan AG. Transition metal homeostasis in Streptococcus pyogenes and Streptococcus pneumoniae. Publisher’s Note Adv Microb Physiol. 2017;70:123–91. https:// doi. org/ 10. 1016/ bs. ampbs. Springer Nature remains neutral with regard to jurisdictional claims in pub- 2017. 01. 002. lished maps and institutional affiliations. 29. Danilova TA, Danilina GA, Adzhieva AA, Vostrova EI, Zhukhovitskii VG, Cheknev SB. Inhibitory effect of copper and zinc ions on the growth of Streptococcus pyogenes and Escherichia coli biofilms. Bull Exp Biol Med. 2020;169:648–52. https:// doi. org/ 10. 1007/ s10517- 020- 04946-y. 30. Makthal N, Kumaraswami M. Zinc’ing it out: zinc homeostasis mecha- nisms and their impact on the pathogenesis of human pathogen group a Streptococcus. Metallomics. 2017;9:1693–702. https:// doi. org/ 10. 1039/ c7mt0 0240h. 31. Leizer J, Nasioudis D, Forney LJ, Schneider GM, Gliniewicz K, Boester A, et al. Properties of epithelial cells and vaginal secretions in pregnant women when lactobacillus crispatus or lactobacillus iners dominate the vaginal microbiome. Reprod Sci. 2018;25:854–60. https:// doi. org/ 10. 1177/ 19337 19117 698583. 32. Yano J, Lilly E, Barousse M, Fidel PL. Epithelial cell-derived S100 calcium- binding proteins as key mediators in the hallmark acute neutrophil response during Candida vaginitis. Infect Immun. 2010;78:5126–37. https:// doi. org/ 10. 1128/ IAI. 00388- 10. 33. Haley KP, Delgado AG, Piazuelo MB, Mortensen BL, Correa P, Damo SM, et al. The human antimicrobial protein calgranulin C participates in control of helicobacter pylori growth and regulation of virulence. Infect Immun. 2015;83(7):2944–56. https:// doi. org/ 10. 1128/ IAI. 00544- 15. 34. Damo SM, Kehl-Fie TE, Sugitani N, Holt ME, Rathi S, Murphy WJ, et al. Molecular basis for manganese sequestration by calprotectin and roles in the innate immune response to invading bacterial pathogens. Proc Natl Acad Sci U S A. 2013;110:3841–6. https:// doi. org/ 10. 1073/ pnas. 12203 35. Neyrolles O, Wolschendorf F, Mitra A, Niederweis M. Mycobacteria, metals, and the macrophage. Immunol Rev. 2015;264:249–63. https:// doi. org/ 10. 1111/ imr. 12265. 36. Neyrolles O, Mintz E, Catty P. Zinc and copper toxicity in host defense against pathogens: mycobacterium tuberculosis as a model example of an emerging paradigm. Front Cell Infect Microbiol. 2013;3:89. https:// doi. org/ 10. 3389/ fcimb. 2013. 00089. 37. von Pein JB, Stocks CJ, Schembri MA, Kapetanovic R, Sweet MJ. An alloy of zinc and innate immunity: Galvanising host defence against infection. Cell Microbiol. 2021;23:e13268. https:// doi. org/ 10. 1111/ cmi. 13268. 38. Maalej H, Hmidet N, Boisset C, Buon L, Heyraud A, Nasri M. Optimization of exopolysaccharide production from pseudomonas stutzeri AS22 and examination of its metal-binding abilities. J Appl Microbiol. 2015;118:356– 67. https:// doi. org/ 10. 1111/ jam. 12688. 39. De Philippis R, Colica G, Micheletti E. Exopolysaccharide-producing cyanobacteria in heavy metal removal from water: molecular basis and practical applicability of the biosorption process. Appl Microbiol Biotech- Re Read ady y to to submit y submit your our re researc search h ? Choose BMC and benefit fr ? Choose BMC and benefit from om: : nol. 2011;92:697–708. https:// doi. org/ 10. 1007/ s00253- 011- 3601-z. 40. Mukherjee P, Mitra A, Roy M. Halomonas Rhizobacteria of Avicennia fast, convenient online submission marina of Indian Sundarbans promote Rice growth under saline and thorough peer review by experienced researchers in your field heavy metal stresses through exopolysaccharide production. Front Microbiol. 2019;10:1207. https:// doi. org/ 10. 3389/ fmicb. 2019. 01207. rapid publication on acceptance 41. Polak-Berecka M, Szwajgier D, Waśko A. Biosorption of Al(+3) and cd(+2) support for research data, including large and complex data types by an exopolysaccharide from lactobacillus rhamnosus. J Food Sci. • gold Open Access which fosters wider collaboration and increased citations 2014;79:T2404–8. https:// doi. org/ 10. 1111/ 1750- 3841. 12674. 42. Mitra A, Chatterjee S, Kataki S, Rastogi RP, Gupta DK. Bacterial tolerance maximum visibility for your research: over 100M website views per year strategies against lead toxicity and their relevance in bioremediation application. Environ Sci Pollut Res Int 2021;28:14271–14284. oi: https:// At BMC, research is always in progress. doi. org/ 10. 1007/ s11356- 021- 12583-9. Learn more biomedcentral.com/submissions 43. Musser JM, Mattingly SJ, Quentin R, Goudeau A, Selander RK. Identifica- tion of a high-virulence clone of type III Streptococcus agalactiae (group B

Journal

BMC Microbiology – Springer Journals

Published: Jan 13, 2022

Keywords: Antimicrobial; Metal; Zinc; Streptococcus agalactiae; Group B Streptococcus

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