Ambreen LEGHARI, Shakeel Ahmed LAKHO, Faiz Muhammad KHAND, Khaliq ur Rehman BHUTTO, Sameen Qayoom LONE, Muhammad Tahir ALEEM, Iqra BANO, Muhammad Ali CHANDIO, Jan Muhammad SHAH, LIN Hui-xing, FAN Hong-jie,

1 MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, P.R.China

2 Shaheed Benazir Bhutto University of Veterinary and Animal Sciences, Sakrand 67210, Pakistan

3 Central Veterinary Diagnostic Laboratory, Tando Jam 70050, Pakistan

4 Jiangsu Co-innovation Center for the Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, Yangzhou 225009, P.R.China

Abstract Streptococcus agalactiae is one of the most common pathogens that cause bovine mastitis worldwide.Identifying pathogen prevalence and virulence factors is critical for developing prevention and control approaches.Herein, 1 161 milk samples from various dairy farms in China (n=558) and Pakistan (n=603) were collected between 2019–2021 and were subjected to S.agalactiae isolation.Prevalence, serotyping, virulence genes, and antibiotic-resistant genes of S.agalactiae were evaluated by PCR assay.All isolates were characterized for haemolysis, biofilm production, cytotoxicity, adhesion, and invasion on bovine mammary epithelial cells.The prevalence of S.agalactiae-induced mastitis in cattle was found to be considerably higher in Pakistan than in China.Jiangsu and Sindh provinces had the highest area-wise prevalence in China and Pakistan, respectively.Serotypes Ia and II were prevalent in both countries, whereas serotype III was found only in Pakistan.Moreover, all isolates tested positive for PI-2b gene but negative for PI-1 and PI-2a genes.All isolates harboured cfb, cylE, hylB, and fbsB virulent genes, whereas many of them lacked bibA, rib and bca.However, the absence of bac and scp genes in Chinese isolates and cspA in Pakistani isolates was noted, while spb1 and lmb were not detected in isolates of both countries.Pakistani isolates, particularly serotype Iapositive, had a considerably higher ability to produce biofilm, haemolysis, cytotoxicity, adhesion, and invasion than Chinese isolates.Most of the isolates were phenotypically resistant to tetracycline, erythromycin, and clindamycin and genotypic resistance was confirmed by the presence of ermA, ermB, tetM and tetO genes.Our study highlights theantimicrobial resistance profile and virulence-related factors contributing to the epidemiological spread of mastitis-causing S.agalactiae in China and Pakistan.The findings may facilitate future studies designed to develop improved treatment and control strategies against this pathogen.

Keywords: Streptococcus agalactiae, mastitis, epidemiology, virulence characterization, antibiotic resistance

1.Introduction

Bovine mastitis is the most prevalent and economically important disease of dairy animals worldwide (De Vliegheret al.2012).This disease is characterized by infection of the mammary glands resulting in physical, chemical, pathological, and bacteriological changes in glandular tissues and milk composition (Zhao and Lacasse 2008).It causes considerable economic loss by negatively impacting animal health and welfare, as well as milk quality and quantity (Romeroet al.2018).Epidemiologically, mastitis is considered a multifactorial disease that involves a complex interaction among three major factors: the infectious agent, the host, and the environment (De Vliegheret al.2012).Bacterial pathogens contribute 70% to the aetiology of this disease.Non-infectious causes, such as physical trauma and mechanical injuries to the mammary glands, accounts for 30% of the cases (Sharunet al.2021).Mammary gland infection occurs when bacteria get a chance to invade the teat canal and reach upper milk-tract.The most commonly isolated pathogens from mastitis-affected cattle areStreptococci,Staphylococci,Escherichiacoli, andKlebsiellaspecies (Aliet al.2017; Gaoet al.2017).The prevalence of mastitis has been documented in several Asian countries including China (Liet al.2009; Heet al.2020), Pakistan (Aliet al.2021), India (Bangaret al.2015), Egypt (El-Behiryet al.2015), and Bangladesh (Sarkeret al.2013).In Pakistan, average prevalence of this disease in different parts of the country ranges from 20 to 60%, whereas among different regions in China, the average prevalence ranges from 40 to 65% (Zhanget al.2005; Liet al.2009).Therefore, understanding the prevalence of mastitis in China and Pakistan and identifying the virulence factors of the pathogen is important for developing infectioncontrol strategies and implementing suitable therapeutic measures (Cheng and Han 2020).

Among various bacteriological aetiologies of mastitis,Streptococcus agalactiaeis the major pathogen that induces intramammary infection (Pereiraet al.2010; Kabelitzet al.2021).Streptococcus agalactiaeis a Grampositive, facultatively aerobic and encapsulated coccus that is also known as Group BStreptococcus(GBS) according to the Lancefield classification.Furthermore,S.agalactiaeis believed to cause infections in a range of animals including humans (Pereiraet al.2010; Kabelitzet al.2021).It is the leading cause of invasive bacterial diseases in human new-borns, such as meningitis, neonatal sepsis, and pneumonia (Sridharet al.2015).GBS colonization, persistence, translocation, and invasion of host barriers are largely dependent on their multiple virulence factors and their ability to adhere to host cells.During the onset of infection, the adhesion of bacterium to the epithelial cells has a significant role in its pathogenesis (Lindahlet al.2005).Moreover, the biofilmforming property ofS.agalactiaeis considered a major virulence factor influencing the survival and persistence in both the environment and the host (Rosini and Margarit 2015).A variety of regulatory proteins and surfacelocalized proteins fromS.agalactiaehave been identified as virulence factors and have been found to play critical role in adhesion, tissue damage and immune evasion (Herbertet al.2004; Burchamet al.2019).These genes includefbs(fibrinogen-binding protein),bca(α-subunit of C protein),scpB(C5a peptidase),bac(β-subunit of C protein),lmb(laminin-binding surface protein),rib(resistant to protease immunity), surface protein (spb1), andcsp(cell surface protease).In addition,S.agalactiaealso produces many toxins, including CAMP factor (cfb), hyaluronidase (hylB) and haemolysins which help the pathogen to enter host cells and survive and spread within them (Rajagopal 2009).InS.agalactiae, serotypes are classified based on the presence of a polysaccharide capsule, a major virulence factor ofS.agalactiae, which helps the bacterium to evade host’s defence mechanisms by interfering with phagocytic cells.Capsular serotyping is the most commonly used method in epidemiological studies ofS.agalactiae.There are 10 knownS.agalactiaeserotypes based on distinct capsule compositions (Ia, Ib, and II to IX) and their distribution is directly linked to their geographical locations (Yaoet al.2013; Reyeset al.2017).Serotyping ofS.agalactiaeisolates is important for understanding local epidemiology and for monitoring for serotype replacement or capsular switching.The distribution of serotypes varies by country, indicates that one size may not fit all in understanding and preventingS.agalactiaeinfections (Reyeset al.2017).Therefore, it is necessary to understand the serotyping ofS.agalactiaeand the molecular characterization of virulent strains to design improved mastitis-control strategies in both countries, i.e., China and Pakistan.

Antibiotic treatment is one of the most commonly used mastitis-controlling practice; however, majority of these drugs are currently reported to be ineffective due to the emergence of antibiotic-resistant bacterial pathogens (Oliver and Murinda 2012).The widespread use of antibiotics in livestock and humans causes bacteria to mutate, allowing them to survive and propagate as antibiotic-resistant bacteria.Antibiotic resistance has reached dangerously high levels around the world, which led to a decline in the treatment options available to diseased animals and consequently resulted in increased morbidity, mortality, and treatment costs.Thus, identifying the pattern of antibiotic resistance in the mastitis-causing pathogen is critical for developing alternate prevention and control strategies.

This study aimed at investigating molecular epidemiology ofS.agalactiaeisolates from various dairy farms located in China and Pakistan.We also elucidated the relationships betweenS.agalactiaeisolates from both countries by characterizing capsular serotyping, pilus typing and various virulent factors such as growth ability of isolates in bovine milk, haemolytic ability, biofilmformation ability, cytotoxicity, adhesion, and invasion on bovine mammary epithelial cells (BMECs).In addition, the antibiogram profile of the isolates was also investigated.

2.Materials and methods

2.1.Collection of milk samples

Between 2019 and 2021, 558 milk samples were collected from cows with clinical and subclinical mastitis from 25 dairy farms located in different provinces of China namely, Jiangsu (n=135), Shanghai (n=108), Anhui (n=70), Shandong (n=90), Zhejiang (n=85), and Jiangxi (n=70).Similarly, 603 milk samples were collected from infected cattle of 26 dairy farms located in three Pakistani provinces, namely Sindh (n=304), Punjab (n=163), and Baluchistan (n=136).All the Chinese milk samples were transported in aseptic conditions to the Laboratory of Veterinary Microbiology and Immunology, Nanjing Agricultural University, China, whereas Pakistani samples were transported to the Central Veterinary and Diagnostic Laboratory, Tando Jam, Sindh, Pakistan within 24–48 h on ice packs.Before collecting milk samples, all teats were washed with 70% ethanol, foremilk was discarded, and 5 mL of milk from each teat was collected, labelled, and stored at 4°C until further experimentation.

2.2.Isolation of S.agalactiae from milk samples

Milk samples were mixed and streaked on Todd Hewitt agar (THA) plates using a sterilized wire loop.Plates were incubated at 37°C for 24 h.Suspected colonies were further streaked on 5% sheep blood agar to check the haemolytic property.Bacteria which showed betahaemolysis were harvested from the blood agar media and were characterized through Gram’s staining.Grampositive isolates were subsequently inoculated into Todd Hewitt Broth (THB; Oxoid Ltd., Basingstoke, Hampshire, UK) and incubated at 37°C for 24 h.Isolates were saved at –80°C in THB and glycerol solution at a ratio of 80:20 (v/v) for subsequent assays.

2.3.Molecular identification, serotyping, virulence genes and antibiotic resistance profiling of S.agalactiae by PCR assay

Pure bacterial isolates were sub-cultured into THB and incubated in a shaker incubator at 37°C for 24 h.Afterwards, the cultures were centrifuged at 4 000 r min–1for 25 min to collect bacterial pellets.The genomic DNA from each isolate was then extracted using a QIAmp DNA Kit (Qiagen, Duesseldorf, Germany) according to the kit protocols.The concentration of purified DNA was quantified using a NanoDrop 2000c (Thermo Fischer Scientific Inc., USA) and then stored at –20°C for subsequent experiments.The molecular identification ofS.agalactiaewas performed by PCR amplification of species-specific parts of the gene encoding, the 16S rRNA gene with oligonucleotide primers (Appendix A) as described previously (Hassanet al.2000; Meiri-Bendeket al.2002).The isolates were further characterized by serogrouping by the determination of specific capsular polysaccharides (Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX) (Appendix A).In addition, virulence genes (cfb,cylE,fbsB,hylB,cspA,bibA,lmb,scpB,bca,spb1, andbac) (Appendix B), pilus genes (PI-1, PI-2a, and PI-2b) and antibiotic resistance genes (ermA,ermB,mefA,tetK,tetM, andtetO) (Appendix C) were also investigated from the isolates by PCR assay using gene-specific primers as described elsewhere (Carvalho-Castroet al.2017; Kannikaet al.2017; Panget al.2017).In brief, a PCR mixture of 10 μL containing 5 μL PCR GoTaqGreen Mix (Promega, Leiden, Netherlands), 1 μL of the DNA template and 0.5 μmol L–1of each of the forward primer and the reverse primer was amplified using a thermal cycler (Applied Biosystems, USA).The PCR cycling parameters were as follows: initial denaturation at 96°C for 1 min, followed by denaturation, annealing, and elongation temperatures and times of 96°C for 10 s,55 to 72°C (according to the melting temperature of the corresponding primer) for 30 s, and 74°C for 1 to 30 s (according to the amplicon length of the target gene), respectively, for 35 cycles.

2.4.Assessment of the growth ability of S.agalactiae isolates

The drop-plate method was used to assess the ability of bacterial isolates to grow in bovine milk as described previously (Panget al.2017).Briefly, each isolate was cultured in THB media at 37°C in a shaker incubator until an absorbance of 0.5 at an OD600was achieved.The culture was then diluted in 2 mL of sterile bovine milk or PBS (control) and subsequently incubated in a shaker incubator adjusted to 80 r min–1at 37°C for a maximum of 12 h.Bacterial viability was assessed by preparing serial dilution of milk with PBS and plated onto THA plates, and viable colony-forming units per mL of culture (CFU mL–1) were quantified.Experiments were conducted in triplicates.

The ability of all isolates to grow in THB was evaluated in the same way as described elsewhere (Panget al.2015).In brief, bacterial culture was grown in THB until an absorbance of 0.5 at OD600was obtained and subsequently a dilution of 1:100 was made in 1 mL THB.Next, 200 μL of culture volume or THB alone (control) was inoculated to the wells of a 96-well polystyrene tissue culture plate and incubated at 37°C for 6 or 12 h.Bacterial culture was evaluated by spectrophotometric reading (OD600) using a microplate spectrophotometer (Bio-Rad Laboratories, USA).The data are presented as the OD obtained at 600 nm wavelength, normalized against the OD600of the control group.Three independent experiments were performed.

2.5.Measurement of the biofilm production capability of S.agalactiae

Bacterial attachment and surface growth on polystyrene microtiter plates were investigated as stated earlier (Panget al.2017) with slight modification.Briefly, bacteria were cultivated in THB until an absorbance of 0.5 at OD600was achieved followed by dilution to a concentration of 1:1 000 in THB.A total of 200 μL volume of bacterial culture or THB alone (control) was dispensed into a 96-wells plate (Corning, Inc., NY) followed by incubation at 37°C for 24 h.Following 24 h of incubation at 37°C, the OD600of each culture was measured to ensure that all cells had reached a stationary phase with a similar OD600the suspensions were aspirated, and planktonic cells were eliminated by washing the plates three times with PBS.The surface adherent biofilms were then fixed with methanol for 20 min and the plates were then rinsed three times with PBS.Subsequently, biofilms were stained with 100 μL of 0.1% crystal violet for 30 min and the wells were washed twice with PBS and then air-dried.The stained biomass was resuspended for quantification in ethanol/acetone (80:20) and each sample was measured at OD595on a microplate reader (Bio-Rad, USA) for the biofilm quantitation.The assays were performed in triplicate.

2.6.Haemolytic activity measurement

To evaluate the haemolytic phenotype ofS.agalactiaeisolates, they were cultured on Columbia blood agar base (BD, Difco) containing 5% sheep blood for 24 h.Next, bacterial culture filtrates (grown for 18 h in THB medium at 37°C with shaking at 180 r min–1) were used to check the haemolytic activity of all isolates as described elsewhere (Bannoet al.2017).Briefly, each well, containing 200 μL of culture filtrates in 96-well plates, was seeded with cells at 1×109CFU mL–1density, with the subsequent addition of 100 μL (2%) sheep erythrocytes and then incubated at 37°C for 1 h.The reaction was then stopped by incubating the plate at 4°C for 12 h.The samples were then centrifuged for 5 min at 10 000×g, and 100 μL of the supernatants were transferred into a 96-well flat-bottom plate.Aliquots of 100 μL of normal saline as negative control and 1% (v/v) Triton-X 100 as positive control were added to an equivalent volume of sheep erythrocytes (2%).A 100-μL aliquot of the supernatant was collected from each well and the haemoglobin release was recorded at OD540.The percentage of haemolysis in each sample was found as follows:

where Asampleis the absorbance measured for each experimental condition and Anegativeand Apositiveare the absorbances of negative and positive controls respectively (Papazafiriet al.2005).

2.7.Cytotoxicity evaluation

The cytotoxicity ofS.agalactiaeisolates on bovine mammary epithelial cells (BMECs) was quantified using a Lactate Dehydrogenase (LDH) Assay Kit (Beyotime Biotechnology, Shanghai, China).Briefly, BMECs were incubated in 96-well plates with bacterial suspension (100 μL) at a multiplicity of infection (MOI) of 1, 10 and 100, separately.Non-infected cells and bacteria, without a BMEC monolayer, were used as negative controls, whereas cells lysed with 100 μL of lysis solution were used as a positive control.After 3 h of incubation, the lactate dehydrogenase released from the cells was measured at OD490.Finally, the percentage of cytotoxicity was determined using the formula presented in the kit protocol.

2.8.Adhesion and invasion assays

The assay of adhesion and invasion was performed as described previously (Rubenset al.1992; Panget al.2017).Briefly,S.agalactiaeadhesion was determined by growing bacteria in THB to an absorbance of 0.3 to 0.5 at OD650and incubating them on a monolayer of BMEC in a 24-well plate at 1×107CFU/well for 2 h to achieve 1:1 infection.Cells were washed five times with PBS before being detached with 0.8 mL of 0.025% Triton-X 100.The diluted aliquots were plated on THB with 5% sheep blood and incubated for 24 h at 37°C.The CFU on the plates were counted and each test was repeated four times.Calculation of attachedS.agalactiaewas calculated as total (attached and invaded) CFU minus invaded CFU.

Similarly, for invasion assay,S.agalactiaewas grown in THB to an OD650of 0.3 to 0.5 and incubated on the monolayer of BMEC in a 24-well plate at 1×107CFU/well to achieve 1:1 infection for 2 h.The cells were then washed with PBS before 1 mL DMEM medium containing 10% fetal bovine serum, 100 μg gentamycin and 50 μg penicillin G per mL were added to each well of the plate and incubated in a CO2incubator for 2 h.After washing the cells five times, 0.8 mL of 0.025% Triton-X 100 was added to detach them.The diluted aliquots were plated on THB with 5% sheep blood and incubated for 24 h at 37°C.The CFU on the plates were counted and each test was repeated four times.

2.9.Evaluation of antibiotic resistance profile of S.agalactiae isolates by disc diffusion method

Among all confirmedS.agalactiaeisolates from China and Pakistan, we randomly selected 200 isolates (n=100 from China;n=100 from Pakistan) for the evaluation of antibiotic resistance patterns.Antibiotic resistance was tested by the disc diffusion method using Mueller-Hinton agar (Merck, Germany) and commercially available discs (Oxoid, United Kingdom) according to the guidelines of Clinical and Laboratory Standards Institute (CLSI).A panel of 14 antibiotics was tested: penicillin (10 IU), oxacillin (1 μg), amoxillin (25 μg), lincomycin (15 μg), cephalothin (30 μg), ceftriaxone (25 μg), gentamycin (10 μg), kanamycin (10 μg), clindamycin (10 μg), bacitracin (30 μg), erythromycin (15 μg), tetracycline (30 μg), rifampicin (30 μg), and ampicillin (10 μg).IsolatedS.agalactiaebacteria were inoculated onto a Muller-Hinton agar plate, supplemented with 5% sheep blood.Antibiotic discs were placed on the agar surface and plates were incubated for 18 h at 37°C.Control was grown on Mueller-Hinton agar without antibiotic disc.After incubation, the diameters of the inhibition zone on the Petri dishes were measured using a clean ruler and then theS.agalactiaeisolates were scored as susceptible, intermediate, or resistant according to the criteria of CLSI.

2.10.Statistical analysis

The virulence characteristics in different groups were analysed by two-way ANOVA (analysis of variance), followed by Fisher’s LSD test for multiple comparison using GraphPad Prism Software, version 6 (La Jolla, CA, USA).The error bars in the figures show the standard errors of multiple replicates.Significant values obtained are presented as:＊,P<0.05;＊＊,P<0.01; “ns”, nonsignificance,P>0.05.

3.Results

3.1.Prevalence of S.agalactiae in different dairy herds in China and Pakistan

The overall prevalence ofS.agalactiaeisolated from milk samples of cows with clinical and subclinical mastitis, belonging to various areas of China and Pakistan, was 18.64%, and 27.53% respectively.The area-wise highest prevalence ofS.agalactiaein China was observed in Jiangsu Province (29.63%) followed by Anhui (21.43%), Jiangxi (18.57%), Zhejiang (17.65%), Shandong (13.33%) and Shanghai (8.33%) provinces.A total of 166 isolates (27.53%) from 603 cattle, belonging to different dairy farms across three provinces of Pakistan (Table 1), were identified asS.agalactiae.Among 166 cattle, Sindh (30.92%) and Punjab (26.38%) were the provinces of most prevalence ofS.agalactiaefollowed by Baluchistan (21.32%).This data indicated that the overall prevalence of mastitis caused byS.agalactiaein the cattle populations of selected areas was considerably higher in Pakistan than that in China (Table 1).

3.2.Characterization of capsular serotypes and distribution of pilus-islands of S.agalactiae isolated from different dairy farms in China and Pakistan

Of the 266 collectedS.agalactiaeisolates from selected areas of China and Pakistan, the most predominant serotype was serotype Ia (53.85% in China and 44.58% in Pakistan) followed by serotype II (30.77% in China and 25.30% in Pakistan), serotype III (0% in China and 9.04% in Pakistan) and non-typeable serotype (15.38% in China and 17.47% in Pakistan) (Table 2).Serotype III was isolated only from Pakistani isolates.Moreover, serotype Ia and serotype II were predominant in Chinese isolates.The serotypes IV, V, VI, VII, VIII, and IX were not found in any of the isolates from both countries.All of the isolates were further investigated for pilus typing (P, I-1, PI-2a and PI-2b).Each isolate from either country tested positive for the PI-2b gene but negative for the PI-1 and PI-2a genes (Table 2; Appendix D).According to these findings, the most common serotypes and pilus gene in both countries were serotypes Ia and II and P1-2b.

Table 1 Prevalence of Group B Streptococcus (GBS) isolated from dairy farms in China and Pakistan

Table 2 Polysaccharide capsular serotypes and virulent genes distribution in Streptococcus agalactiae isolates from China and Pakistan

3.3.Distribution of virulence factors in bovine S.agalactiae isolated in China and Pakistan

To investigate whetherS.agalactiaeisolates varied in virulence characters, a panel of virulence-related genes was studied.The virulence gene profile of allS.agalactiaestrains, isolated from different dairy farms in China and Pakistan, is summarized in Table 2.It shows thatcfb,cylE,HylB,fbsB,bibA,rib, andbcawere widely distributed among differentS.agalactiaeisolates of both countries.Streptococcusagalactiaeisolated from China were positive for the virulence genes tested (cfb(100%),cylE(100%),hylB(100%),fbsB(100%),bibA(46.15%),rib(19.23%),bca(23.08%), andcspA(18.27%)) exceptbacandscpwhile the Pakistani isolates also carried examined genes (cfb(100%),cylE(100%),HylB(100%),fbsB(100%),bibA(31.33%),rib(21.59%),bca(24.10%),scp(13.25%), andbac(7.83%)) exceptcspA.However,spb1andlmbwere not detected in any isolate from either country (Table 2; Appendix E).These observations point out that genescfb,cylE,HylB, andfbsBwere present in allS.agalactiaebacteria isolated from China and Pakistan.

3.4.Growth ability of S.agalactiae isolates from China and Pakistan

The 270S.agalactiaeisolates were divided into two groups based on their geographical origin: China and Pakistan and serotype, serotype Ia and serotype II.The growth ability of allS.agalactiaeisolates in milk and THB media was determined separately and compared among different groups.No significant difference in bacterial growth among Chinese and Pakistani groups was observed when bacteria were cultured in milk and THB separately (Fig.1-A).Furthermore, the average bacterial growth of both groups was higher after culturing for 12 h.Bacterial concentrations of serotype Ia and serotype II groups were found non-significant in both milk and THB.Additionally, it was observed that when bacteria were cultured in milk for 12 h, the bacterial concentration of serotype Ia in the Chinese group was higher than that of the Pakistani group, but the difference was statistically insignificant (Fig.1-A and B).

Fig.1 Growth ability, biofilm formation ability and haemolytic activity of Streptococcus agalactiae isolated from different dairy farms in China and Pakistan.A, the growth of S.agalactiae strains in Todd Hewitt Broth was assessed by detecting the absorbance of bacterial cultures at OD600.B, the growth of S.agalactiae strains in milk was determined using the drop plate method.C, biofilm formation ability of S.agalactiae evaluated by crystal violet staining using 96-well plates.D, haemolytic activity of S.agalactiae strains.Data are mean±SE.＊, P< 0.05; “ns”, non-significant, P>0.05.

3.5.Biofilm formation capability of S.agalactiae isolates from China and Pakistan

Crystal-violet staining was used to assess each isolate’s ability to form a biofilm.The results revealed that all isolates ofS.agalactiae, from both China and Pakistan, could form biofilm.Pakistani serotype Ia showed the strongest ability to form biofilm, which was statistically significant (P<0.05) compared to Chinese serotype Ia.The biofilm formed by the Pakistani isolates with serotype II was weaker and statistically insignificant than that formed by the Chinese serotype II isolates.However, the statistical difference between serotypes Ia and II was insignificant in both countries (Fig.1-C).

3.6.Haemolytic activity of S.agalactiae bacteria isolated from dairy farms in China and Pakistan

The haemolytic ability ofS.agalactiaeisolates was evaluated after being cultivated on blood agar plates.All of the isolates from different areas of China and Pakistan exhibited complete haemolytic rings (Fig.1-D).Furthermore, a sheep erythrocytes lysis assay was used to compare the haemolytic activity of Chinese and Pakistani isolates.Results demonstrated that the haemoglobin released from sheep erythrocytes by Pakistani and ChineseS.agalactiaeisolates was not greater than 70% of the value of the positive control.Pakistani serotype Ia exhibited significantly higher haemolysis (P<0.05) than Chinese serotypes Ia and II.In contrast, statistically insignificant differences were found between serotypes Ia and II of either country.Overall, the Pakistani groups had higher percentage haemolysis compared to Chinese groups.

3.7.Cytotoxicity analysis of S.agalactiae isolates from China and Pakistan using BMECs

The cytotoxic effect ofS.agalactiaeisolates was evaluated based on the measurement of LDH released from the cytosol of BMECs in the presence of various concentrations (MOI of 10, 50 or 100) ofS.agalactiae.Results indicated that all isolates from Pakistan and China were cytotoxic to BMECs in a dose-dependent manner, with the cytotoxic effect becoming stronger as the MOI increased.When the MOI was 10, the cytotoxic effect of all isolates was found to a lesser extent, whereas at the MOI of 50, the cytotoxic effect of all isolates was slightly higher but statistically insignificant.At an MOI of 100, the cytotoxicity of Pakistani serotype Ia was significantly higher than that of the Chinese serotype Ia (P<0.05).However, insignificant difference in cytotoxic effect between serotypes Ia and II of either country was noted (Fig.2).

Fig.2 In vitro cytotoxicity of Streptococcus agalactiae isolates from China and Pakistan determined by measuring the lactate dehydrogenase (LDH) activity from bovine mammary epithelial cells (BMECs).The cytotoxicity of Pakistani serotypes Ia and II was compared to Chinese serotypes Ia and II at different multiplicities of infection (MOIs) of 10, 50, and 100.The P-values for each comparison were calculated as follows: Pakistani serotype Ia vs.Chinese serotype Ia (MOI 10: P=0.939; MOI 50: P=0.597; MOI 100: P=0.032); Pakistani serotype Ia vs.Chinese serotype II (MOI 10: P=0.523; MOI 50: P=0.523; MOI 100: P=0.142); Pakistani serotype II vs.Chinese serotype Ia (MOI 10: P=0.991; MOI 50: P>0.999; MOI 100: P=0.357), and Pakistani serotype II vs.Chinese serotype II (MOI 10: P=0.679; MOI 50: P=0.998; MOI 100: P=0.893).Data are pooled from three independent experiments each containing at least quadruplicate samples.Data are mean±SE.＊, P<0.05;“ns”, non-significant, P>0.05.

3.8.Ability of S.agalactiae to adhere to and invade BMECs

We further examined whether different serotypes ofS.agalactiaebacteria isolated from selected areas of Pakistan and China might adhere to and invade BMECs with distinct efficiencies, since these processes are critical early steps in the pathogenesis of illnesses caused by these bacteria (Fig.3-A).BMECs were challenged withS.agalactiaeisolates and bacterial adherence and invasion were quantified.Results showed that all examinedS.agalactiaebacteria adhered to and invaded BMECs cell monolayers.The adhesion and invasion ability of serotypes Ia and II of either country was insignificant (Fig.3-B).However, Pakistani serotype Ia adhered to and invaded BMECs at significantly higher levels compared to Chinese serotypes Ia and II (P<0.05).

Fig.3 Adhesion and invasion of Streptococcus agalactiae isolates from China and Pakistan.A, adherence of S.agalactiae to bovine mammary epithelial cells (BMECs).B, invasion of S.agalactiae to BMECs evaluated using gentamycin protection assays.Data are pooled from three independent experiments each containing at least quadruplicate samples.Data are mean±SE.＊, P< 0.05; ＊＊, P< 0.01; “ns”, non-significant.

3.9.Phenotypic and genotypic antimicrobial susceptibility of S.agalactiae isolates

Table 3 shows the resistant and susceptible pattern ofS.agalactiaeisolates against 13 antibiotics.The highest percentages of phenotypic resistance inS.agalactiaeisolated from China and Pakistan were recorded against tetracycline (94% in China and 95% in Pakistan), erythromycin (95% in China and 91% in Pakistan), and clindamycin (70% in China and 72% in Pakistan), followed by rifampicin (21% in China and 27% in Pakistan), bacitracin (17% in China and 27% in Pakistan) and kanamycin (12% in China and 20% in Pakistan).However, the isolates from both countries showed high susceptibility to penicillin (95% in China and 70% in Pakistan), oxacillin (94% in China and 71% in Pakistan), gentamycin (93% in China and 80% in Pakistan), ampicillin (91% in China and 75% in Pakistan), lincomycin (91% in China and 81% in Pakistan), cephalothin (90% in China and 70% in Pakistan), ceftriaxone (86% in China and 60% in Pakistan), kanamycin (81% in China and 65% in Pakistan), and amoxicillin (80% in China and 71% in Pakistan).The differences in the prevalence of apparent phenotypic tetracycline, erythromycin and clindamycin resistance between Chinese and Pakistani isolates was only 1 to 4 percentage points.

The genotypic analysis of isolates that showed phenotypical resistance to antibiotics was done using PCR assay.Concerning erythromycin resistance, the most common mechanism of resistance, observed in isolates, was the methylation of target encoded byermA(84% in China and 73% in Pakistan) andermB(94% in China and 91% in Pakistan) genes.The tetracycline resistance genes revealed that the efflux pump mediated by ribosomal protection genestetMandtetOwas recovered in the majority of the tetracycline-resistant isolates (tetM: China 53%, Pakistan 68%;tetO: China90%, Pakistan 91%), whereastetKgene was detected only in Chinese isolates (12%) (Table 4).Moreover, none of the tested strains was positive for the efflux pump mediated bymefAgenes, which are related to macrolides resistance.The PCR products of isolates with resistant genes were distinguished by agarose gel electrophoresis as shown in Appendix F.

Table 3 Antibiotic susceptibility profile of bovine Streptococcus agalactiae isolated in China (n=100) and Pakistan (n=100)

Table 4 Prevalence of antibiotic resistant genes in Streptococcus agalactiae isolated from dairy herds in China and Pakistan

4.Discussion

Bovine mastitis is a major veterinary and economic problem in many parts of the world, including China and Pakistan (Liet al.2009; Aliet al.2011; Heet al.2020; Aliet al.2021).In this study, we investigated the prevalence and molecular characterization ofS.agalactiaeisolated from milk samples of different areas in China and Pakistan.Moreover, the capsular-typing and virulence characteristics, such as the ability of bacteria to grow in milk, ability to form a biofilm, haemolytic activity, cytotoxicity, adhesion to and invasion on BMECs were investigated and compared between both countries to identify the most virulent serotypes capable of host immune escape, colonization in mammary gland tissues and causing mastitis.

Of the 558 and 603 milk samples isolated from China and Pakistan respectively, only 104 from China and 166 from Pakistan were genotypically confirmed to beS.agalactiae.This result was not entirely unexpected because the PCR method used here is less prone to error than the previously used phenotypic identification method.Previous studies also reported that the prevalence of mastitis varied with geographical regions as well as with differences in management practices adopted in dairy farms (Aliet al.2014).The prevalence of bovine mastitis in selected areas of cattle population was in partial agreement with several global studies conducted in India (Bangaret al.2015), China (Liet al.2009), North America (Middletonet al.2005), Europe (Pieperset al.2010), Ethiopia (Birhanuet al.2017), and Poland (Sztachanskaet al.2016).Gram-positive pathogens such asStreptococcusandStaphylococcusspecies have been identified as the primary causative agentsof bovine mastitis.Streptococcusagalactiaehas been reported to be sporadic in several countries, including Belgium, Denmark, Norway, and the United Kingdom (Andersenet al.2003; Oster?set al.2006; Bradleyet al.2007; Pieperset al.2007).Our findings are consistent with previous studies, which have found high prevalence ofS.agalactiae-induced mastitis in several parts of the world, including parts of Europe (Germany: 29% of herds positive (Tenhagenet al.2006)) and the Americas (Brazil: 60% of herds positive (Duarteet al.2004)); Uruguay: 11% of herds positive (Gianneechini Ret al.2002); New York, USA: 10% of cows positive (Wilsonet al.1997).According to the current investigation, bovine mastitis is prevalent in dairy farms belonging to various regions in China and Pakistan andS.agalactiaecould be the dominant cause of this disease, suggesting that mastitis controls will need to be adopted to improve animal welfare and farm profitability.

We further examined molecular serotyping of the isolatedS.agalactiaestrains.Of all the tested serotypes (Ia, Ib, II, III, IV, V, VI, VII, VIII, and IX), serotypes Ia and II were found to be the most prevalent in both China and Pakistan, whereas serotype III was only found in Pakistani isolates.An earlier study identified capsular types similar to those found in this study, including serotype III inS.agalactiaebacteria isolated from Brazilian dairy farms (Duarteet al.2004).However, our findings differed from those of another study, which found a higher frequency of serotypes V and NT strains inS.agalactiaeisolates (Pintoet al.2013).The dominance of serotype Ia in our study is quite intriguing since this serotype is predominant in humans.However, the identification of serotype Ia in cattle has been demonstrated in eastern China, Germany, and the United States (Merlet al.2003; Doganet al.2005; Shomeet al.2012; Yanget al.2013).These findings appear to confirm the hypothesis that the presence of specific serotypes may be due to strain origins in different geographical locations, highlighting the significance of serotyping studies in choosing suitable preventative measures that are effective across a broad geographic area.

A variety of virulence genes were analysed to further understand the virulence determinants of GBS isolates.Many previous studies have examined the molecular epidemiology of putative virulence factors in GBS.Of the nine genes examined forS.agalactiae, four (cfb,cylE,HylB, andfbsB) were determined to be present in allS.agalactiaebacteria isolated from China and Pakistan, suggesting that they are conserved inS.agalactiaestrains.These results were unsurprising because their presence had been previously confirmed in bovine strains in countries such as Brazil, India and Egypt and the frequency of occurrence of these genes is local in character (Shomeet al.2012; Bangaret al.2015; El-Behiryet al.2015; Carvalho-Castroet al.2017).The situation differs with respect to the genespb1andlmb, whose presence was not detected in any of the analysed isolates.Our study confirms prior research that primarily identified thebcaandribgenes in serotypes Ia and II.Although we detected differences in the frequency of some genes, the encoded proteins may be differentially expressed (Rosinski-Chupinet al.2013) and thus, differences in pathogenicity could be attributable to differences in gene expression.The absence ofbacandscpin Chinese isolates andcspA in Pakistani isolates was noted.It has been reported that thebacgene was always found with thebcagene, but thebcagene might be present while thebacgene was missing (Delannoyet al.2013).Moreover, the variability in the presence or absence of thecspAgene in this study is consistent with an earlier study in which GBS serotypes isolated from the tilapia farms in Thailand showed variation in the existence of thecspAgene (Kannikaet al.2017).Consistent with the earlier investigations (Yanget al.2013; Springmanet al.2014), our observations revealed that allS.agalactiaeisolates harboured the gene PI-2b.Earlier research has demonstrated that vaccines containing the PI-2b protein components could be effective in preventing GBS infections (Margaritet al.2009); thus, the conserved PI-2b could be considered a potential vaccine candidate to develop subunit vaccines against bovine mastitis.

Streptococcusagalactiaemust propagate competently in milk to survive, colonize the bovine mammary glands and produce virulence factors (Panget al.2017).This study observed that all bovine isolates reproduced efficiently in milk as well as in THB media.Nevertheless, the GBS bacteria isolated from various regions demonstrated less variation in milk.These findings indicate that bacterial ability to proliferate in cattle milk may contribute to better adaptation of these isolates to bovine herds.

The ability of bacteria to form biofilms is a crucial phenomenon that allows them to survive for long periods in both natural ecosystems and animal hosts (Rosini and Margarit 2015).Bacteria growing in biofilms have shown 10–1 000 times more antibiotic resistance than planktonic cells (Costertonet al.1999; Royet al.2018).Our finding revealed that all bovine GBS isolated from different areas in China and Pakistan could form biofilm.A subset of serotype Ia isolates belonging to Pakistan was shown to form stronger biofilms than the rest of tested isolates.The differences in the biofilm formation ability among different groups can be explained by facts that isolated strains belong to different geographical origins and that husbandry conditions and herd management differ among dairy farms.Furthermore, a positive correlation was found between biofilm formation and adherence to BMECs.The adhesion ability of GBS bacteria to BMECs is critical for bacterial colonization in the mammary glands despite the flow of milk, which results in the excretion of planktonic bacterial cells (Leigh 1999).Earlier research has shown that pili are crucial for bacterial cell adhesion to host epithelial cells which is the first step in the formation of a biofilm (Costertonet al.1999; Konto-Ghiorghiet al.2009) and that adherence can be prevented by PI-2b proteinspecific antibodies (Sharmaet al.2013).Surprisingly, the current study found that all bovine isolates containing PI-2b had strong biofilm formation and adhesion abilities.

The haemolytic activity and cytotoxicity ofS.agalactiaeisolates were also investigated in this study.It was demonstrated that all isolates showed cytotoxicity towards BMECs, however, serotype Ia-positive Pakistani isolates exhibited higher cytotoxic activity than other groups.Similar to cytotoxicity, Pakistani isolates showed significantly higher haemolysis towards sheep erythrocytes compared to Chinese isolates.These findings suggest that haemolytic activity and cytotoxicity may be required for the infection ofS.agalactiaeisolates.Conversely, it has been shown thatS.agalactiaewith high haemolytic activity, were not cytotoxic to host cells (Leclercqet al.2016).Nevertheless, it is likely that differences underlying host–pathogen interaction at the cell–cell interface or unique virulence traits expressed by the pathogen, may contribute to the haemolytic activity as well as cytotoxicity ofS.agalactiaetowards host cells.

Studies on antimicrobial resistance are important for the rational use of antibiotics and for limiting the development of drug-resistant pathogens (Oliver and Murinda 2012).In this study, antimicrobial resistance profiles ofS.agalactiaeisolates revealed high resistance to tetracycline, erythromycin, and clindamycin drugs.These findings are comparable to those found in previous studies onStreptococciisolated from bovine mastitis (Namet al.2009; Jainet al.2012).However, the tetracycline data from this study is quite high when compared to a report from Uruguay, which showed 0% resistance inS.agalactiae(Gianneechini R Eet al.2002).It is believed that the high prevalence of tetracycline, erythromycin, and clindamycin resistance could be associated with extensive usage of these antibiotics in the veterinary field without control or supervision either in treatment or even prophylaxis (Chohanet al.2006).β-Lactams are known as the first-line antimicrobial agents when treatingStreptococcaludder infections (Erskineet al.2002).Previously, several studies have demonstrated that β-lactams were found to be uniformly effective againstStreptococcusisolates (Erskineet al.2002; Gianneechini R Eet al.2002; Rossittoet al.2002).Similarly, in the current study, more than 70% of isolates showed susceptibilities to penicillin, ampicillin and oxacillin.In GBS, tetracycline resistance is primarily due to ribosomal protection and encoded bytetMandtetO(Jaramillo-Jaramilloet al.2018).In our study,tetOwas the most common resistance gene in bovine isolates from China and Pakistan.These results are consistent with those from the USA, where bovine GBS isolates predominantly carriedtetOgene (Doganet al.2005).However, the frequent presence of the genetetMamong the analysed strains is surprising because a previous report stated that this gene had been associated with strains ofS.agalactiaeisolated from humans (Doganet al.2005).In contrast, studies conducted in China and the United States have confirmed frequent presence of this gene among bovine isolates, which can be explained by horizontal gene transfer within the same genus (Gaoet al.2012; Ruegget al.2015).Two genes of theermclass,ermAandermB, were selected for analysis.These genes encode methylase, which reduces the binding of antibiotics to the target site, both in the macrolides group and lincosamides (Lochet al.2005).In our study, the geneermBwas dominant and our results are similar to those obtained by other authors (Lochet al.2005; Gaoet al.2012).According to Lochet al.(2005), this result can be explained by the location of this gene on transposons, which are transferred to different bacteria, includingStreptococcus,viahorizontal gene transfer.Additionally, we examined the presence of the genemefA, encoding the efflux pump responsible for resistance to 14- and 15-membered macrolides.The presence of this gene has previously been confirmed in human isolates ofS.pneumoniae(Szczypaet al.2013).Nevertheless, this gene was not detected among the strains we analysed, and this result is consistent with previous reports (Lochet al.2005; Schmitt-van de Leemput and Zadoks 2007; Ratoet al.2013).Moreover, the presence of antibiotic resistance genes in the analysed isolates demand further research to evaluate the occurrence of point mutations in these genes.

5.Conclusion

Our study elucidated the epidemiology, virulence factors and antibiotic resistance profile ofS.agalactiaeisolated from cattle in China and Pakistan.Our findings showed that the prevalence ofS.agalactiaewas considerably higher in Pakistan than in China.Pakistani isolates, particularly serotype Ia positive, were found distinct from Chinese isolates in the expression of several virulence-related characteristics such as growth ability in milk, biofilm-forming ability, adhesion and invasion abilities, and cytotoxicity to BMECs.Phenotypically, the isolatedS.agalactiaeappeared to be highly resistant to tetracycline, erythromycin, and clindamycin antibiotics with susceptibility to penicillin, oxacillin, gentamycin, ampicillin, amoxicillin, lincomycin, cephalothin, and ceftriaxone.TheermA,ermB,tetMandtetOantibiotic-resistant genes were identified in the majority of isolates from dairy cows’ milk in China and Pakistan, indicating a theoretical potential for horizontal transfer of these resistance genes on dairy farms.This study contributes to a better understanding of the virulent characteristics as well as the antibioticresistant profile of bovineS.agalactiaein both countries, which will be useful in future studies aimed at developing control and treatment approaches againstS.agalactiae.However, the role of the host immune response against these virulence factors warrants further research.

Acknowledgements

This study was supported by the National Key Research and Development Program of China (2021YFD1800400), the National Natural Science Foundation of China (31872480), the Jiangsu Agriculture Science and Technology Innovation Fund of China (CX(19)2020), and the Priority Academic Program Development of Jiangsu Higher Education Institutions, China (PAPD).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Ethical approval

The Ethical Committee of Nanjing Agricultural University, China and Central Veterinary and Diagnostic Laboratory, Tando Jam, Pakistan gave the approval for the project conducted from 2019 to 2021.Animal care and experimental techniques strictly adhered to the rules set by the Chinese and Pakistani Councils for animal experimentation.The animals were not harmed and were properly cared during the collection of milk samples.

Appendicesassociated with this paper are available on https://doi.org/10.1016/j.jia.2022.10.004