Antimicrobial Activity of Clinically Isolated Bacterial Species Against Staphylococcus aureus

Abstract

Bacteria often exist in polymicrobial communities where they compete for limited resources. Intrinsic to this competition is the ability of some species to inhibit or kill their competitors. This phenomenon is pervasive throughout the human body where commensal bacteria block the colonization of incoming microorganisms. In this regard, molecular epidemiological and microbiota-based studies suggest that species-specific interactions play a critical role in the prevention of nasal colonization of the opportunistic pathogen Staphylococcus aureus. Despite this, S. aureus exists as part of the microbiota of ∼25% of the population, suggesting that the interplay between S. aureus and commensals can be complex. Microbiota studies indicate that several bacterial genera are negatively correlated with S. aureus colonization. While these studies paint a broad overview of bacterial presence, they often fail to identify individual species-specific interactions; a greater insight in this area could aid the development of novel antimicrobials. As a proof of concept study designed to identify individual bacterial species that possess anti-S. aureus activity, we screened a small collection of clinical isolates from the Walter Reed National Military Medical Center for the ability to inhibit multiple S. aureus strains. We found that the majority of the isolates (82%) inhibited at least one S. aureus strain; 23% inhibited all S. aureus strains tested. In total, seven isolates mediated inhibitory activity that was independent of physical contact with S. aureus, and seven isolates mediated bactericidal activity. 16S rRNA based-sequencing revealed that the inhibitory isolates belonged to the Acinetobacter, Agromyces, Corynebacterium, Microbacteria, Mycobacterium, and Staphylococcus genera. Unexpectedly, these included seven distinct Acinetobacter baumannii isolates, all of which showed heterogeneous degrees of anti-S. aureus activity. Defined mechanistic studies on specific isolates revealed that the inhibitory activity was retained in conditioned cell free medium (CCFM) derived from the isolates. Furthermore, CCFM obtained from S. saprophyticus significantly decreased mortality of S. aureus-infected Galleria mellonella caterpillars. While future studies will seek to define the molecular mechanisms of the inhibitory activities, our current findings support the study of polymicrobial interactions as a strategy to understand bacterial competition and to identify novel therapeutics against S. aureus and other pathogens.

Keywords: MRSA; Staphylococcus aureus; bacterial interaction; clinical isolates; polymicrobial interactions.

FIGURE 1

Bacterial species isolated from patients at the Walter Reed National Military Medical Center inhibit Staphylococcus aureus growth. (A) Venn Diagram of anti-S. aureus activity: thirty-four clinical isolates were screened for anti-S. aureus activity in an in vitro bacterial interaction assays against S. aureus strains 2014.N, LAC and Mu50. 28/34 isolates tested possessed anti-S. aureus activity against at least one S. aureus strain. 6/34 possessed no activity against S. aureus. 8/34 inhibited growth of all S. aureus strains tested while the remaining 20 strains showed activity against one or two of the S. aureus strains as indicated in the Venn Diagram. (B) Representative Bacterial Interaction Assays: C. aurimucosum (C. aur-1), M. yunnanensis (M. yun), or S. saprophyticus (S. sap) were spotted onto an agar plate that had been seeded with the indicated S. aureus strains (2014.N, LAC, or Mu50). Plates were incubated at 28°C, and images were taken at 24, 72, and 120 h (24 and 120 h images are shown). Images are representative of three independent biological replicates. Scale bar = 10 mm and is the same in the corresponding 24 h and 120 images; in some cases the 120 h spots appear larger than the 24 h spots due to growth of the bacteria within the spots.

FIGURE 2

Clinical isolates mediate strain-specific anti-S. aureus activity. (A) Heat map showing the activity of the indicated clinical isolate against the indicated S. aureus strains: The Zone of Clearance (ZOC) was defined as the distance between the edge of the clinical isolate spot to the visible edge of the S. aureus ring of clearance. The ZOC was measured using ImageJ software (NCBI) and each value within a box represents the arithmetic mean (in millimeters) of three independent biological replicates measured temporally (24, 72, and 120 h). Clinical isolate species names have been abbreviated as follows: C. aurimucosum (C. aur), C. striatum (C. str), C. amycolatum (C. amy), C. jeikeium (C. jei), C. tuberculostearicum (C. tub), S. epidermidis (S. epi), S. saprophyticus (S. sap), S. hominis (S. hom), A. baumannii (A. bau), M. paraoxydans (M. par), M. yunnanensis (M. yun), and Agromyces sp. 3098BRRJ (Agro sp.). Additionally, a number preceded by a – is used to indicate an independent isolate of the indicated species. (B) Example of strain-specific activity: S. epidermidis (S. epi-1) and S. hominis (S. hom) were co-incubated with agar plates seeded with S. aureus strains 2014.N, LAC or Mu50. Images of the ZOC were taken after 24 and 120 h of incubation at 28°C and are representative of three independent biological replicates. Scale bar = 10 mm and is the same in the corresponding 24 h and 120 images; in some cases the 120 h spots appear larger than the 24 h spots due to growth of the bacteria within the spots.

FIGURE 3

Independent Acinetobacter baumannii isolates produce strain-specific anti-S. aureus activity. Individual A. baumannii (A. bau-2 and A. bau-6) isolates were spotted onto an agar plate that had been seeded with various S. aureus strains (2014.N, LAC, or Mu50). Plates were incubated at 28°C, and images were taken at 24, 72, and 120 h (24 and 120 h images are shown). Images are representative of three independent biological replicates. Scale bar = 10 mm and is the same in the corresponding 24 h and 120 images; in some cases the 120 h spots appear larger than the 24 h spots due to growth of the bacteria within the spots.

FIGURE 4

Select clinical isolates mediate contact-independent anti-S. aureus activity. (A) Strongly inhibitory clinical isolates (17/28) were defined as follows: visibly transparent ZOC of at least 2 mm with a defined edge. (B) A 0.2 μm filter was placed on top of BHIT agar plates seeded with S. aureus (S. epi-1 and S. sap were incubated with S. aureus LAC, C. amy-2 was incubated with S. aureus Mu50, and C. aur-1 was incubated with S. aureus 2014.N). A clinical isolate (as described above) was then spotted on top of the filter paper such that the clinical isolate and the S. aureus seeded agar plate were physically separated. Images of the ZOC were taken after 120 h of incubation at 28°C. Images are representative of three independent biological replicates. Scale bar = 10 mm.

FIGURE 5

Select clinical isolates mediate bacteriostatic or bactericidal activity against S. aureus. The number of S. aureus CFU seeded within the agar (T0) was compared to the number of S. aureus found after 48 h of incubation. S. aureus CFU numbers were determined from directly adjacent to the clinical isolate spot (Inside ZOC) or from outside the ZOC and the fold change from T0 was calculated. Each box represents the data from three independent biological replicates; the horizontal line is plotted at the arithmetic mean and the length of the box represents the range. A dotted line is indicated at 1, which would represent a purely bacteriostatic interaction where the numbers of S. aureus at T0 and T48 were unchanged. Values below the line indicate a decrease in S. aureus at the 48-h time point, which indicates bactericidal activity.

FIGURE 6

Anti-S. aureus activity is retained in Conditioned Cell Free Medium. S. aureus strains LAC or Mu50 were spread on the BHI agar surface and a sterile disk was placed in the center of the plate. Fifty μl of concentrated CCFM prepared from C. amy-2; (incubated with Mu50), S. sap (incubated with LAC), and S. epi-3 (incubated with LAC) that was Heat-Treated (H+) or maintained at room temperature (H–) prior to use was inoculated onto the disk and allowed to dry. Images of the ZOC were taken after 72 h of incubation. Images are representative of three independent biological replicates. Scale bar = 10 mm.

FIGURE 7

Staphylococcus aureus-infected Galleria mellonella. Galleria mellonella caterpillars were divided into the indicated groups and were monitored for survival over 120 h at 37°C after the indicated inoculations. (A,B) Untouched and PBS injected Galleria served as negative controls and the indicated doses of 2014.N, LAC and Mu50 were tested. (C) For the treatment experiments, untouched caterpillars were maintained. Additionally, PBS injected caterpillars were treated 1-h post S. aureus infection with CCFM prepared from BHIT in which no bacteria had been cultured to ensure no effect of dual injection on survival. Caterpillars that were injected with 10⁶ CFU of S. aureus strain LAC were treated 1-h post infection with BHIT CCFM (sham treatment) or S. epi-3 or S. sap derived CCFM. Kaplan-Meier survival curves were compared (excluding negative controls) using the Mantel–Cox test with Holm’s correction for multiple comparisons. In (A,B), the three S. aureus strains were compared to identify statistically significant differences in Galleria mortality; untouched and PBS dosed caterpillars were not included in the comparisons. In (A), Mu50 was significantly different than both 2014.N and LAC. In (B), significant differences between the various groups are indicated. In (C), BHIT treated caterpillars were compared to CCFM treated (S. epi-3 or S. sap) Galleria to identify difference; only S. sap was significantly different. Asterisks signifying the P value as follows: ^∗P < 0.05; ^∗∗P < 0.01; ^∗∗∗P < 0.001; ^∗∗∗∗P < 0.0001.