Genomic and phenotypic profiling of Staphylococcus aureus isolates from bovine mastitis for antibiotic resistance and intestinal infectivity

Prevalence of antibiotic resistance in S. aureus isolates

Out of the 43 isolates, 6 isolates showed either single antibiotic resistance (5/6) or multi-antibiotic resistance (1/6) (Fig. 1a and Table S3a). The isolates Sa3 and Sa9 showed resistance to tetracycline, whereas isolates Sa3489, Sa3493, and Sa3603 were resistant to lincomycin. Isolate Sa1158c showed resistance to multiple classes of antibiotics including ampicillin, gentamycin, kanamycin, penicillin, tetracycline, ticarcillin, and ceftiofur. Intermediate responses to lincomycin and spectinomycin were observed from 39.5% and 46.5% of the isolates, respectively (Fig. 1b). Eight different sequence types (ST) covering the 43 isolates were identified where 34 of the isolates either belonged to ST151 or ST352 (Table S1). The two tetracycline-resistant and three lincomycin-resistant isolates were from ST151 and ST352 respectively, whereas the multi-drug-resistant isolate belonged to ST8. Clinically important ABR genes were identified from the whole genome data. For instance, tetracycline-resistant genes (tet(K); 1/43, tet(M); 3/43 and tet(38); 43/43), lincomycin-resistant genes (lnu(A); 3/43), aminoglycoside-resistant genes (aac(6'); 1/43, aph(3'); 43/43, aac3; 43/43), ß-lactam and cephalosporin-resistant genes (blaI, blaR, blaZ; 1/43, mecA; 1/43), and multi-drug resistant regulators (arlR, arlS, mgrA; 43/43) were evident.

Fig. 1figure 1

Response of 43 S. aureus isolates against antibiotics. a List of antibiotic-resistant isolates. b Bacterial responses toward 24 antibiotics. The S. aureus isolates were subjected to Kirby-Bauer disc diffusion susceptibility tests. The scores based on CLSI guidelines for susceptibility or resistance to an antibiotic were generated for each isolate. Abbreviations used—AK: Amikacin; AMP: Ampicillin; APR: Apramycin; B: Bacitracin; CZ: Cefazolin; CTX: Cefotaxime; CF: Ceftiofur; C: Chloramphenicol; CIP: Ciprofloxacin; E: Erythromycin; CN: Gentamycin; K: Kanamycin; MY: Lincomycin; N: Neomycin; OFX: Ofloxacin; P: Penicillin; SH: Spectinomycin; S: Streptomycin; TET: Tetracycline; TIC: Ticarcillin; TIL: Tilmicosin; TOB: Tobramycin; SXT: Trimethoprim/Sulfamethoxazole; VA: Vancomycin. The experiment was performed in triplicates and repeated thrice to ensure reproducibility

EtBr efflux assay and Nitrocefin assay were performed to assess efflux pump activity and ß-lactamase enzyme activity, respectively in all the isolates (Table S3b). The tetracycline-resistant Sa3 and Sa9, lincomycin-resistant Sa3489, and the multi-drug-resistant Sa1158c showed active efflux pump activity. For instance, isolates Sa3, Sa9, Sa3489, and Sa1158c extruded 50% of the EtBr in 295.2 sec, 1067 sec, 3443 sec, and 271.9 sec, respectively (Fig. 2a, see the tabular data). The ß-lactamase enzyme activity was observed only in the multi-drug resistant isolate (50.36 U/mL) (Fig. 2b, see the tabular data). Genome analysis indicated the presence of genes associated with MFS efflux pumps (norA; 43/43, norB; 43/43, lmrS; 43/43, tet(38); 43/43, tet(K); 1/43, tet(M); 3/43), MATE efflux pumps (mepR, mepA, mepB; 43/43), and ß-lactamase enzyme activity (blaI, blaR, blaZ; 1/43). The list of genes associated with antibiotic resistance is provided in Tables 1 and S4.

Fig. 2figure 2

Antibiotic-resistance mechanisms and virulence factors in S. aureus isolates. a Assessment of efflux pump activity in five antibiotic-resistant and five susceptible isolates. EtBr efflux assay was performed using 3 µg/mL of EtBr and 30 µg/mL of CPZ. The fluorescent intensity (530 nm/590 nm) was monitored for 60 min after reenergizing the bacterial cells to trigger EtBr efflux with glucose (0.4% v/v). b Assessment of ß-lactamase enzyme activity in the isolates. The isolates were subjected to a Nitrocefin assay where the absorbance of the cell-free extract mixed with Nitrocefin was detected at 490 nm for 15 min. c Distribution of hemolysin manifestation by the 43 isolates. d Alpha hemolysin manifestation by Sa30. Each isolate was cultured in TSA plates with 5% sheep blood for 24 h. The hemolysis was detected visually by the translucency around the bacterial colony. e Distribution of biofilm-forming ability by the 43 isolates. The biofilm formation was assessed using a crystal violet assay. All isolates were classified into weak, moderate, and strong biofilm-formers based on their biofilm-forming ability. f Fluorescence microscopic image of extracellular polymeric substances (EPS) and Sa30 cells. A green (505 nm) filter was used to acquire the GFP-labeled Sa30 biofilms using a high-content screening microscope, Cell Discoverer 7. Alphabets (in tabular data) indicate a significant difference (p < 0.05). ‘N/A’ stands for not applicable. More data on the resistance mechanisms and virulence factors are provided in Tables S3 and S5. The experiment was performed in quadruplicates and repeated thrice to ensure reproducibility

Table 1 List of the genes associated with antibiotic resistance and adherence in the six antibiotic-resistant isolates (Sa3, Sa9, Sa1158c, Sa3489, Sa3493, and Sa3603), and five antibiotic-susceptible isolates (Sa11, Sa14, Sa30, Sa3014, and Sa3154)Virulence profile of S. aureus isolates

The 43 isolates were cultured on blood agar plates and checked for hemolysis. All the isolates either produced alpha-hemolysin (34/43) (encoded by hla) or beta-hemolysin (9/43) (encoded by hlb) (Fig. 2c,d). Beta-hemolysis was observed only in the members of ST151 and ST8. Crystal violet assay confirmed biofilm-forming ability in all isolates. Specifically, 23.26% of the isolates showed strong biofilm formation, 55.81% of them were moderate biofilm formers, whereas the rest formed weak biofilms (Fig. 2e,f and Table S5). None of the isolates except Sa16, Sa23, and Sa27 from ST352 and ST151 were strong biofilm formers, whereas, isolates from ST8 and ST2270 formed strong biofilms. Common virulence genes included: two-component leukotoxins, including gamma-hemolysin (Hlg, encoded by hlgA, hlgB, and hlgC), and leukotoxin D (LukD, encoded by lukD). None of the isolates had pyrogenic toxin superantigen (PTSAg) genes except for Sa3154 which contained enterotoxin C, enterotoxin L, and toxic shock syndrome toxin-associated sec, sell, and tsst-1 genes, respectively. Adhesins that are involved in biofilm formation were also identified in all isolates. For instance, fibronectin-binding proteins, fnbA, and fnbB were observed in 60.46% of the isolates. Clumping factor A (clfA), a cell-wall anchored protein was identified in 58.13% of the isolates, whereas, clfB, a fibrinogen-binding adhesin was found in 95.56% of the isolates. Accessory gene regulator (agr) and staphylococcal accessory regulator (sarA) system associated with quorum sensing [11] was identified among the isolates as well. Genes associated with intercellular adhesion such as icaA, icaB, icaC, icaD, and icaR were evident in all 43 isolates. The presence of the staphylococcal protein A (spa) gene, the product of which plays an important role in colonization and immune invasion [12] was found in 17 isolates including 4 ABR isolates. Ssp serine protease (encoded by sspA) that contributes to in vivo growth and survivability [13] was identified in all the isolates. The second immunoglobulin-binding protein (Sbi) which is a multifunctional immune invasion factor [14] was observed in 34 isolates. Moreover, all the isolates had bovine immune invasion factors such as serotype 8 capsular polysaccharide (Cap), and adenosine synthase A (AdsA). The list of genes associated with adherence, toxin/enzyme production, and immune invasion is provided in Tables 12, and S6.

Table 2 List of the genes associated with toxin and enzyme production, and immune invasionInternalization of the S. aureus isolates in human intestinal epithelial cells

Five antibiotic-resistant isolates (Sa3, Sa9, Sa1158c, Sa3489, and Sa3603), and five antibiotic-susceptible isolates (Sa11, Sa14, Sa30, Sa3014, and Sa3154) were tested for internalization in Caco-2 cells. All the isolates showed significantly (p < 0.05) higher internalization in Caco-2 cells in comparison to the reference strain, Sa ATCC 25923 (Fig. 3a). For instance, >8 log10 cfu/well of Sa14, Sa1158c, Sa3014, and Sa3603, and >6 log10 cfu/well of Sa3, Sa9, Sa11, Sa30, Sa3154, and Sa3489 were recovered from the Caco-2 cells, which was ~5.22 log10 higher (p < 0.05) than the reference strain. High-content microscopy confirmed the internalization of Sa30 and the death of Caco-2 cells (Fig. 3b(i-viii)). Cysteine proteases, staphopain B (SspB), and staphopain C (SspC) which are often associated with biofilm formation and intracellular colonization of S. aureus [15, 16] were identified in all isolates (Tables 1 and S6).

Fig. 3figure 3

Internalization of S. aureus isolates and response to antibiotic treatment in Caco-2 cells. a Intracellular survivability of S. aureus isolates in Caco-2 cells. Approximately 2 × 104 Caco-2 cells/well of a 96-well plate were exposed to the isolates maintained at 0.5 Macfarland standard (1.5 X 108 cells/ mL). The cells were washed with PBS and subjected to gentamicin (10 µg/mL) to remove extracellular bacteria. The cells were washed further after 4 h of incubation and lysed using 0.5% (v/v) of Triton-X. Colony-forming units were determined using the drop culture method. b Epifluorescence microscopic images of non-infected control and Sa30-infected Caco-2 cells. After 4 h incubation of the Caco-2 cells exposed to bacteria, 30 µL of Hoechst-PI cocktail was added and incubated for 30 min in dark. These wells were imaged using an epifluorescence microscope with blue, green, and red filters. i-ii) Hoechst 33342 staining of non-infected and Sa30-infected Caco-2 cells. iii-iv) GFP-labeled Sa30 internalization in Caco-2 cells. Non-infected cells were considered as a control. v-vi) PI staining of infected and non-infected cells. vii-viii) Overlap of Hoechst and PI stained Sa30-infected and non-infected Caco-2 cells. The images confirmed Sa30 internalization and infection of the Caco-2 cells leading to cell death. c Antibiotic efficiency against intracellular Sa1158c, Sa30, and Sa3. The infected Caco-2 cells were exposed to ampicillin (AMP) (10 µg/mL), kanamycin (K) (30 µg/mL), streptomycin (S) (10 µg/mL), chloramphenicol (C) (30 µg/mL), tetracycline (TET) (30 µg/mL), and ceftiofur (CF) (30 µg/mL). The plates were incubated for 24 h, followed by washing, lysing of the cells, and drop-culture method to enumerate the cfu/well. Average values plotted in the graph with different alphabets indicate a significant difference (p < 0.05). ‘IF-control’ stands for infected Caco-2 cells without antibiotic treatment. The experiment was performed in quadruplicates and repeated thrice to ensure reproducibility

The Caco-2 cells with internalized S. aureus were exposed to antibiotics to check for their colonization-remediation efficiency. The aminoglycosides and ß-lactam antibiotics failed to show effectiveness against any of the intracellular S. aureus whereas, chloramphenicol, tetracycline, and ceftiofur were comparatively more effective (p < 0.05) against all the antibiotic-susceptible and lincosamide resistant isolates. However, none of these antibiotics could reduce the intracellular bacterial load by more than 2.5 log10. For instance, chloramphenicol, tetracycline, and ceftiofur showed a 2.3-2.5 log10 reduction (p < 0.05) of Sa30 colonization (Fig. 3c). Moreover, no antibiotic demonstrated a significant reduction in the aminoglycoside/ß-lactam/tetracycline/cephalosporin-resistant Sa1158c colonization except chloramphenicol which showed a 2.42 log10 reduction (p < 0.05) (Fig. 3c). Similarly, tetracycline failed to show any efficiency against the tetracycline-resistant Sa3 and Sa9, while chloramphenicol and ceftiofur significantly reduced their colonization by ~2.35 log10 (Figs. 3c and S1a).

Pathogenicity of the S. aureus isolates in Caenorhabditis elegans model of intestinal infection

Infection of C. elegans with the selected isolates had a significant effect on the lifespan of worms when compared with non-infected or infected with reference strain (Sa ATCC 25923). For instance, 100% death in the infected worms was observed by the 15th day post-infection, whereas ~77.5%, and ~60% of the non-infected and Sa ATCC 25923 infected worms respectively, were alive on the 15th day (Fig. 4a). High-content microscopy confirmed Sa30 accumulation in the pharynx, intestinal lumen, rectum, and anus of the worms within 24 h post-infection (Fig. 5a). After 48 h of infection, Sa30 accumulation was observed throughout the digestive tract of C. elegans leading to intestinal epithelium destruction, and complete degradation of internal organs (Figs. 5b-d and S2a-i). The multiplication of Sa30 in C. elegans was evident with increased fluorescence when the microscopic images of 24 h and 48 h post-infection periods were compared.

Fig. 4figure 4

Life-span of S. aureus infected C. elegans and assessment of antibiotic efficiency. a Life-span assay of S. aureus infected and non-infected C. elegans. The worms were grown to the L4 stage and exposed to 1.5 × 108 cells of S. aureus isolates for 48 h. The worms were washed, transferred to NGM plates with E. coli OP50 lawns, and monitored for survivability for 20 days. The mantel-Cox test was used to find the statistical significance (p < 0.05). b-d Antibiotic efficiency against b Sa1158c, c Sa3, and d Sa30 infection in C. elegans. The infected worms were subjected to ampicillin (AMP) (10 µg/mL), kanamycin (K) (30 µg/mL), streptomycin (S) (10 µg/mL), chloramphenicol (C) (30 µg/mL), tetracycline (TET) (30 µg/mL), and ceftiofur (CF) (30 µg/mL) for 24 h. The worms were washed and transferred to NGM plates with E. coli OP50 lawns. The survivability of the worms was monitored for 20 days. Average values plotted in the graph with different alphabets indicate a significant difference (p < 0.05). ‘NIF-Worms’ and ‘IF-Worm UT’ stands for non-infected worms and infected untreated worms, respectively. The experiment was performed in quadruplicates and repeated thrice to ensure reproducibility

Fig. 5figure 5

Microscopic images of non-infected, and antibiotic-treated/untreated Sa30-infected C. elegans. Epifluorescence images of a Sa30 infected (24 h post-infection), b non-infected, c Sa30 infected (48 h post-infection), and d dead infected worms. Sa30 accumulation was observed in the pharynx, intestinal lumen, rectum, and anus of the worms 24 h post-infection. The increased fluorescence 48 h post-infection throughout the digestive tract of the worms suggested Sa30 multiplication leading to intestinal epithelium destruction. The antibiotics e ampicillin, f kanamycin, g streptomycin, h chloramphenicol, i tetracycline, and j ceftiofur were exposed to Sa30 infected worms for 24 h. The worms were washed and resuspended in S-basal media in a 96-well plate. Green (505 nm) and red (583 nm) filter combinations were used to acquire the green autofluorescence of C. elegans and RFP-labeled Sa30. The epifluorescence images were captured using the Cell Discoverer 7

The 24 h treatments with ampicillin, streptomycin, kanamycin, and chloramphenicol failed to improve (p > 0.05) the lifespan of C. elegans infected by any of the 10 selected isolates (Figs. 4b-d and S1b-h). For instance, 20-31% of the worms infected with Sa3 and Sa1158c were alive (p > 0.05) irrespective of treatment by these antibiotics on the 10th day (Fig. 4b,c). Tetracycline failed to improve (p > 0.05) the survivability of Sa3 and Sa1158c infected worms on the 10th day either, while ceftiofur treatment was comparatively effective (p < 0.05) against both these isolates. Only 25-29% of the Sa30 infected worms were alive after ampicillin, streptomycin, kanamycin, and chloramphenicol treatment on the 10th day which was insignificant (p > 0.05) when compared to untreated control (Fig. 4d). The low antibiotic efficiency was verified through higher fluorescence showing Sa30 accumulation in the pharynx, intestinal lumen, and anus of the worms (Fig. 5e-h). In comparison, 45% of the Sa30 infected worms were alive after tetracycline treatment, which was supported by low fluorescence in the pharynx, rectum, and anus regions suggesting reduced Sa30 accumulation (Fig. 5i). A significant (p < 0.05) improvement in the survivability of Ceftiofur-treated Sa30 (63% until the 10th day) infected worms was observed which was confirmed by a negligible accumulation of the isolate in the pharynx, and rectum regions (Fig. 5j).

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