Streptococcus agalactiae isolated from clinical mastitis cases on large dairy farms in north China: phenotype, genotype of antimicrobial resistance and virulence genes

Streptococcus agalactiae (Strep. agalactiae) is bovine mastitis pathogen and has thus became a matter of concern to dairy farms worldwide in terms of economic loss. The aims of this study were to (a) determine virulence genes, and (b) characterize the antimicrobial resistance (AMR) profiles and AMR genes and (c) figure out the relationship between AMR phenotypes and genotypes of Strep. agalactiae isolated from dairy cows in north China. A total of 20 virulence genes and 23 AMR genes of 140 isolates collected from 12 farms in six provinces were studied. The antimicrobial susceptibility of 10 veterinary commonly used antimicrobials were tested using the broth microdilution method. Results showed that all the isolates harbored the virulence genes lacIV, gapC, and dltA. The isolates that harbored the genes lacIII, fbsA, hylB, and cfb exhibited the high prevalence (99.29%), followed by isolates that harbored lacI (98.57%), bibA (97.86%), cylE (97.14%), lacII (92.14%), cspA (52.14%), pavA (25%), bca (2.14%), and scpB (0.71%). The fbsB, lmb, spbI, bac, and rib genes were not detected. The virulence patterns of B (fbsA_cfb_cylE_ hylB_bibA_cspA_ gapC_dltA_lacIII/IV) and C (fbsA_cfb_ bibA _ gapC_ dltA_lacIV) were dominant, accounting for 97.86% of the isolates. The following AMR genes were prevalent: pbp1A (97.14%), tet(M) (95.00%), lnu (A) (80.71%), erm (B) (75.00%), tet(O) (72.14%), blaZ (49.29%), tet(S) (29.29%), blaTEM (25.71%), erm (A) (17.14%), erm (C) (13.57%), tet (L) (10.71%), linB (2.86%), and erm (TR) (2.86%). The pbp2b, mecA1, mecC, lnu (D), erm (F/G/Q), and mef (A) genes were not detected. Eighty percent of the isolates harbored AMR genes and were highly resistant to tetracycline, followed by macrolides (10.71%), lincosamides (9.29%) and β-lactams (4.29%). In conclusion, isolates only exhibited well correlation between tetracyclines resistance phenotype and genotype, and almost all isolates harbored intact combination of virulence genes.


Introduction
Bovine mastitis is one of the costliest diseases in the dairy industry due to the discarding of milk, costs of treatments, and even the culling of cows (Shaheen et al., 2016;Krömker and Leimbach, 2017;Gussmann et al., 2019).Streptococcus agalactiae (Strep.agalactiae), as one of the major mastitis pathogens, causing 11%-60% of mastitis cases in Brazil (Reyes et al., 2017).The implementation of the five-point mastitis control program has reduced the prevalence of Strep.agalactiae mastitis to less than 10% in dairy herds in Europe and North America (Jørgensen et al., 2016).However, the herd prevalence of Strep.agalactiae is still high in developing countries, such as Colombia (34.4%),Brazil (60%), and China (92%) (Ramıŕez et al., 2014;Bi et al., 2016;Carvalho-Castro et al., 2017).Meanwhile, the re-emergence of Strep.agalactiae mastitis in Denmark and Norway has been reported (Katholm et al., 2012;Jørgensen et al., 2016).Moreover, the harm caused by Streptococcus agalactiae to China's dairy farming industry is still very serious (Yang et al., 2016).
Strep.agalactiae is considered one of contagious pathogens that cause bovine mastitis, which can spread among cows (Thompson-Crispi et al., 2014).Once Strep.agalactiae colonize the bovine mammary gland, it obtains nutrient sources from milk for its proliferation and causes long-term and harmful effects.Therefore, the ability of metabolism and capability for adhesion, invasion, and immune evasion of Strep.agalactiae might play crucial roles in the bovine mastitis (Keefe, 2012).Strep.agalactiae harbors a great range of virulence genes encoding virulence factors, such as fbsA/B and lmb, which are involved in adhesion, cylE and hylB, which are involved in invasion, cspA, which is involved in immune evasion, and LacI/II/III/IV, which play a role in metabolism.
Antimicrobial treatment is major option for treating Strep.agalactiae inducing mastitis (Keefe, 2012).However, the excessive use of antimicrobials increased the risks of antimicrobial resistance (AMR), which is a public health concern worldwide (Flynn and Guarner, 2023).Monitoring the resistance of Strep.agalactiae associated with bovine mastitis is important to the control of AMR of the bacterium.
Investigations on virulence genes, and the phenotype and genotype of AMR can contribute to treatment decision and optimization of Strep.agalactiae control programs (Kaczorek et al., 2017).This study aims 1) to determine the antimicrobial resistance and virulence gene profiles of Strep.agalactiae, 2) to detect the AMR profiles of Strep.agalactiae under in vitro conditions, and 3) to determine the correlation between phenotypic and genotypic resistance patterns of Strep.agalactiae isolated in China.

Sample collection and identification of pathogens
Milk samples were collected from cows with clinical mastitis from large dairy farms (>500 cows) in China from 2017 to 2019 (Supplementary Table S1).Milk sampling details were provided by Gao et al. (2017).In brief, udders were disinfected before sample collection, the first three streams of milk were discarded, and 1-2 mL of quarter milk samples were aseptically collected using 50 mL sterile centrifuge tubes.The samples were packed in ice boxes and delivered to the laboratory to be processed within 10 h.
The rest of the virulence genes (spb1, dltA, bibA, gapC, and lacI/ II/III/IV) were detected using normal PCR assay.The final volume of the PCR mixture was 25 µL, and the mixture contained the template composed of 1 mL (final amount of 20 ng) of bacterial genome, 12.5 mL of premixed 2×PCR master mix (Sangon, Shanghai, China), 1 mL of primers (final concentration of 5 mmol/L), and 10.5 mL of ddH2O.The amplification program was as follows: 95°C for 5 min; 35 cycles of 95°C for 60 s; annealing temperature for 60 s; and 72°C for 10 min (Supplementary Table S2).Strep.agalactiae ATCC 13813 was used as positive control and PCR mixture without bacterial genome was used as negative control.Agarose gel electrophoresis (AGE) and UV transillumination was conducted to analyze the PCR products (Supplementary Figure S1).The virulence genes were divided into four groups: adhesion(fbsA/B, lmb, pavA), invasion(cfb, cylE, hylB, spbI), immune evasion(bac, bca, bibA, cspA, rib, scpB), and metabolism(gapC, dltA, LacI/II/III/IV).

Antimicrobial resistance gene identification
AMR genes associated with resistance to four kinds of antimicrobials: b_lactams (blaTEM, blaZ, pbp2b, mecA1, and mecC), lincosamides (lnuA, lnuD, and linB), tetracyclines (tetO/M/L/S), and macrolides (ermA/B/C/F/G/Q/TR, mefA) were detected using normal PCR assay.The final volume of the PCR mixture was 25 µL containing a template composed of 1 mL (final amount of 20 ng) of bacterial genome, 12.5 mL of premixed 2×PCR master mix (Sangon, Shanghai, China), 1 mL of primers (final concentration of 5 mmol/L), and 10.5 mL of ddH 2 O.The amplification program was as follows: 95°C for 5 min; 35 cycles of 95°C for 60 s; annealing temperature for 60 s; and 72°C for 10 min (Supplementary Table S3).Strep.agalactiae ATCC 13813 was used as positive control and PCR mixture without bacterial genome was used as negative control.Agarose gel electrophoresis (AGE) and UV transillumination was conducted to analyze the PCR products (Supplementary Figure S2).

Antimicrobial resistance testing
Antimicrobial resistance testing of all the isolates were conducted using the broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI, 2020).Strep.pneumonia ATCC 49619 and Strep.agalactiae ATCC 13813 were used as quality control strains.Antimicrobials commonly used in practice for mastitis treatment and in medicines for humans (penicillin, cefalexin, ceftiofur, cefquinome, oxacillin, clindamycin, tetracycline, enrofloxacin, amoxicillin/clavulanate, and erythromycin) were selected for antimicrobial resistance testing.

Statistical analysis
The online statistical tool VassarStats (http://www.vassarstats.net/)was used in calculating the proportion of genes and its 95% confidence interval (95% CI).Correlation calculation was performed using SPSS 26.0 (IBM Corp, Armonk, NY).The cluster of AMR genes and virulence genes were obtained using R (version 4.0.5) and the package "pheatmap" (the clustering method of "complete" and "ward.D" were used).
AMR genes can be divided into subgroups A and B. Subgroup A harbored more blaZ and tetL genes than subgroup B, and subgroup B harbored more blaTEM and ermAgenes than subgroup A (Figure 2).
The virulence genes fbsA/B encode fibrinogen-binding proteins, allowing Strep.agalactiae to bind to bMECs and extracellular proteins (Gutekunst and Eikmanns, 2004;Tenenbaum et al., 2005;Pietrocola et al., 2006;Buscetta et al., 2014).In a previous study, lmb was found to be associated with the adherence of Strep.agalactiae, but it was rarely harbored by bovine mastitis isolates (Duarte et al., 2005;Wu et al., 2016).Our results showed that the major virulence gene in charge of adhesion was fbsA, accounting for 99.29% (139/140).The low frequency or absence of pavA, fbsB, and lmb indicated that these genes are not essential to the pathogenesis of bovine mastitis.cfb encodes the CAMP factor involved in hemolytic activation (Lasagno et al., 2011).The cspA gene encoding serine protease and hemolysin encoded by cylE play crucial roles in the virulence of Strep.agalactiae (Chou et al., 2019).Hyaluronidase encoded by hylB promotes Strep.agalactiae invasion in host cells and promotes its host tissue-spreading ability (Oviedo et al., 2013;Coleman et al., 2023).Our study was consistent with the studies of Whist and Osterås (2007) and Pang et al. (2017), who indicated that cfb, cylE, and hylB were the main virulence genes of Strep.agalactiae (Whist and Osterås, 2007;Keefe, 2012).The high frequencies of virulence genes associated with invasion indicated that these genes were essential to induce clinical bovine mastitis for Strep.agalactiae (Keefe, 2012).
Immune evasion enables Strep.agalactiae to escape from host immunity killing.The a/b-C protein, as a surface protein, facilitates the invasion of Strep.agalactiae in cells and resistance to the clearance of phagocyte; the protein is encoded by bac and bca (Oviedo et al., 2013;Pulido-Colina et al., 2021).bac and bca usually appear together (Delannoy et al., 2013).The detection rate of bac is low in bovine isolates (Duarte et al., 2005).Our results showed the low detection rate of bac and bca, indicating they were not essential to bovine mastitis pathogenicity.C5a peptidase cleaving human C5a and BibA known as the C4-binding protein are encoded by scpB and bibA, respectively.Both proteins hamper the complement system, thereby reducing immune killing (Manne et al., 2020;Cullen et al., 2024).Our result indicated that bibA is the main virulence gene involved in the immune evasion of Strep.agalactiae.However, Duarte et al. (2005) revealed that 66% of Strep.agalactiae isolates from bovine harbor scpB (Duarte et al., 2005).Rib encoded by rib confers the ability of immune evasion and has been found in most isolates that caused invasive infections (Pulido-Colina et al., 2021).Consistent with our study, previous research indicated that only a small part of Strep.agalactiae isolated from bovine (20% and 26%) harbors the rib gene (B.Jain et al., 2012).Rohmer et al. (2011) assumed that bacteria evolved to access specific nutrients that hosts provided and develop pathogenicity (Rohmer et al., 2011).Lac encodes lactose operon, and dItA encodes D-alanylation of lipoteichoic acid, which is involved in the completion of the cell wall of Gram-positive bacteria.The genes were conserved in all Strep.agalactiae isolates.Glyceraldehyde-3- phosphate dehydrogenase encoded by gapC is involved in carbohydrate metabolism.Our results indicated the ability of Strep.agalactiae to use milk as a nutrient resource due to these metabolism genes (Keefe, 2012).Overall, the results of our research indicated that the integrity of the parts of the virulence genes (adhesion, invasion, immune evasion, and metabolism) mediates the pathogenesis of Strep.agalactiae.
The percentage of the isolates resistant to tetracycline was 80%, in line with the results of Gao et al. (2012) and Tomazi et al. (2018), who reported that the percentages of resistance were 72.5% in China and 68.6% in Brazil, respectively (Gao et al., 2012;Tomazi et al., 2018).The low efficacy of tetracycline in treating mastitis has been reported worldwide, and one of the reasons is its excessive use in treatment and growth promotion (Kaczorek et al., 2017).This antimicrobial should be used prudently in the treatment of mastitis.
In addition to the AMR profiles of Strep.agalactiae under in vitro conditions, genotypic AMR detection was performed for the selected AMR genes encoding different resistance mechanisms.
The lincosamides resistant genes we detected are the lnu A/D and linB genes, nucleotidyl transferases are encoded by lnu genes, resulting in enzymatic inactivation of lincosamides.The lnu gene was first identified in Enterococcus faecium and then observed in Strep.agalactiae (Arana et al., 2014;Kaczorek et al., 2017).Our results indicated that the detection rate of lnu(A) was 80.71%, which may raise concerns about the spreading of AMR genes among bacteria.
We detected four genes responsible for resistance to tetracyclines: tet(M), tet(O), tet(S).and tet(L), which encodes resistance through ribosomal protection and efflux pump (Poyart et al., 2003;Dogan et al., 2005;Gao et al., 2012).In our research, the tet(M) and tet(O) genes were predominant, which is consistent with previous study (Gao et al., 2012;Rato et al., 2013).The high detection rate of these genes can be attributed to horizontal gene transfer in the same genus of bacteria (Gao et al., 2012;Ruegg et al., 2015).
Eight genes encoding macrolide resistance were detected.erm (B) was predominant, consistent with previous reports (Loch et al., 2005;Gao et al., 2012;Rato et al., 2013).erm(B) can encode methylase, reducing the number of macrolides binding to Strep.agalactiae (Denamiel et al., 2005).erm(B) can transfer among bacteria in the same genus (Loch et al., 2005), and this feature explains the high detection rate of the gene.mef(A), harbored by only 2.86% of isolates, was examined as well.The results were consistent with those of previous research (Rato et al., 2013).
There were two isolates that exhibited resistance to lincosamides but did not harbor the examined AMR genes, the possible reason for which is that we failed to detect other resistance genes that encoding lincosamides resistance.Nevertheless, some isolates exhibited AMR genes carrying but negative in AMR phenotype.The reasons were as follows: (1) AMR genes may not transcribe nor translate because the corresponding antimicrobials were not used in bovine mastitis treatment, so they were far from a promoter or associated with a weak promoter; (2) mutations or lack of promoters induce the silencing of the AMR genes of the isolates.Advanced research is essential to discover the mechanisms of the insufficient correlation between the genotype and phenotype of AMR (Gao et al., 2012).
For the reason of insufficient controls (positive controls for each AMR or virulence genes) were used in our research, the genes that did not be detected may due to the following situations: (1) the isolates did not harbor corresponding genes, (2) the primers failed to combine to template on account of gene mutation.

Conclusion
Some isolates resistant to lincosamides did not necessarily carry any tested gene.Conversely, a large part of b-lactams, lincosamides, and macrolides sensitive isolates contained corresponding AMR genes, which may not be expressed in these isolates.Furthermore, based on almost all isolates harbored virulence genes encoded the ability of adhesion, invasion, immune evasion and metabolism, we inferred that intact combination of virulence genes is essential to the pathogenesis of Strep.agalactiae inducing bovine mastitis.

FIGURE 1
FIGURE 1Pattern of virulence genes of 140 Strep.agalactiae.white square means absence of virulence genes, red square means presence of virulence genes.

FIGURE 2
FIGURE 2Pattern of antimicrobials resistant genes of 140 Strep.agalactiae.Note: white square means absence of antimicrobials resistant genes, red square means presence of antimicrobials resistant genes.
All experiments followed the China Ministry of Science and Technology.Regulations of Experimental Animals (2008) issued by China Ministry of Science and Technology.All animal procedures were approved by the Institutional Animal Care and Use Committee of Yunnan Agricultural University (Approval No: 202403058).

TABLE 1
Prevalence of virulence genes of 140 Strep.agalactiae.

TABLE 2
Prevalence of antimicrobials resistant genes of 140 Strep.agalactiae.