Biofilm expression and antimicrobial resistance patterns of Streptococcus uberis isolated from milk samples of dairy cows in South Africa.

The research described in this Research Communication addresses the hypothesis that intramammary infections with Streptococcus uberis (S. uberis) are associated with biofilm formation, which limits antibiotic efficacy. This retrospective study investigated biofilm expression and antimicrobial resistance (AMR) patterns of 172 S. uberis infections. Isolates were recovered from milk samples of subclinical, clinical, and intramammary infection cases on 30 commercial dairy herds. We determined the presence and intensity of biofilm expression of S. uberis isolates in vitro in three somatic cell count categories to recognise their AMR patterns. An automated minimum inhibitory concentration system with a commercially available panel of 23 antimicrobial agents evaluated AMR, while biofilm determination was conducted using a microplate method. The study established that all the S. uberis isolates assessed expressed biofilm with the following varying degrees of intensities: 30 (17.8%) strong, 59 (34.9%) medium and 80 (47.3%) weak biofilms. The newly registered UBAC mastitis vaccine containing biofilm adhesion components may, therefore, be a viable option for proactive mastitis management under field conditions. No differences were identified between biofilm intensity and the three somatic cell count groups. Most S. uberis isolates indicated a high-level sensitivity to the antimicrobial agents tested. Resistances were present in 8.7, 8.1 and 7.0% cases to rifampin, minocycline and tetracycline, respectively. Multidrug resistance was observed in 6.4%, emphasising AMR to antibiotics used in human medicine only. The low overall resistance suggests that farmers adhere to the prudent use of antimicrobials in the dairy industry.

Characterisation of Staphylococci Isolated from Milk Samples of a Water Buffalo Herd.

Water buffalo produce a tenth of milk for global human consumption. Non-aureus staphylococci (NAS) are among the most commonly isolated bacteria from mastitis in water buffalo and dairy cows. These results described the initial characterisation of 17 NAS-15 Staphylococcus simulans and two Staphylococcus chromogenes from a water buffalo herd (n = 44) in South Africa. The isolates were identified by classical microbiology, MALDI-TOF, and 16S rRNA, and the disc diffusion method determined the antibiotic susceptibility. A multi-locus sequence typing scheme (MLST) was developed to determine S. simulans sequence types (ST), by defining and comparing seven housekeeping gene fragment sequences. Sequence typing confirmed all 15 S. simulans isolates from water buffalo which belonged to a single ST, genetically distant from the six bovine STs isolated from adjacent farms, which also varied, indicating no current bacterial transfer between species. The antibiotic resistance patterns of S. simulans varied between beta-lactams. The mean milk somatic cell count (SCC) for the water buffalo milk samples was 166,500 cells/mL milk. This information offers insights into the epidemiology and comparison among isolates from various origins, which leads to effective proactive mastitis strategies resulting in safe, high-quality dairy products from water buffalo and dairy cows for human consumption.

Species identification and cow risks of non-aureus staphylococci from South African dairy herds.

Detailed information on specific species of non-aureus staphylococci (NAS) has become a necessity for effective udder health control programs in South Africa. The main objective of this preliminary study was to identify the different NAS species and strains present in dairy herds in South Africa using a cost-effective method. A further objective was to investigate the effects of cow risk factors and farming systems on the NAS isolates identified. A total of 214 NAS, isolated from milk collected from 17 South African dairy herds, were identified using three diagnostic tests (API Staph test, MALDI-TOF and 16s rRNA). There was a good observed agreement between the MALDI-TOF and 16S rRNA sequencing (92.2%) and a poor observed agreement between the MALDI-TOF and API Staph (25.7%). The genetic relatedness within species was investigated in 128 of these isolates using random polymorphic amplified deoxyribonucleic acid (DNA) (RAPD), verified by multilocus sequence typing (MLST), and phylogenetic analysis and cow risk factors were investigated on species level. The main NAS species isolated were Staphylococcus chromogenes (75.2%), Staphylococcus epidermidis (9.4%) and Staphylococcus haemolyticus (8.9%). The RAPD test identified 34 Staphylococcus chromogenes, 13 Staphylococcus epidermidis and nine Staphylococcus haemolyticus strains, indicating genetic diversity amongst strains and herds. The presence of NAS intramammary infections was found to be significantly related to the farming systems, composite cow milk somatic cell count (SCC), parity and days in milk (DIM). Significantly more NAS were isolated from primiparous and from older cows. This knowledge could assist with the management of NAS on dairy farms.

The spread and antimicrobial resistance of Staphylococcus aureus in South African dairy herds - A review.

Staphylococcus aureus is internationally recognised as a principal agent of mastitis and the foremost reason for economic loss in the dairy industry. The limited data available on organism-specific antibiotic resistance surveillance in dairy cattle have stimulated the need for such a review article. The objective of this study was to review relevant literature on antimicrobial resistance of mastitis-causing staphylococci isolated from dairy cows in South Africa compared to other countries. Factors relating to the incidence of mastitis and treatment strategies in terms of the One Health concept and food security were included. The Web of Science (all databases) and relevant websites were used, and articles not written in English were excluded. The incidence of mastitis varied between South Africa and other countries. Antimicrobial resistance patterns caused by S. aureus also varied in regions within Southern Africa and those of other countries although some similarities were shown. Antimicrobial resistance differed between S. aureus bacteria that were maltose positive and negative (an emerging pathogen). The results highlighted the importance of the availability of organism-specific surveillance data of the incidence of mastitis and antibiotic resistance for specific countries and within similar climatic conditions. Accurate knowledge about whether a specific pathogen is resistant to an antibiotic within a certain climate, country, area or farm should reduce the incidence of unnecessary or incorrect treatment with antibiotics. This should enable dairy farmers to deal with these organisms in a more effective manner. Therefore such research should be ongoing.

Surveillance of Antibiotic Resistance of Maltose-Negative Staphylococcus aureus in South African Dairy Herds.

Antibiotic resistance has been reported since the 1940s in both human and veterinary medicine. Many years of monitoring milk samples in South Africa led to identification of a novel maltose-negative Staphylococcus aureus (S. aureus) strain, which appears to be an emerging pathogen. In this study, the susceptibility of this strain to antibiotics was evaluated over time, during diverse seasons in various provinces and according to somatic cell count (SCC) categories. A data set of 271 maltose-negative S. aureus isolates, from milk samples of 117 dairy herds, was examined using the disk diffusion method, between 2010 and 2017. This study also compared the susceptibility testing of 57 maltose-negative and 57 maltose-positive S. aureus isolated from 38 farms, from three provinces using minimum inhibitory concentration (MIC). The MIC results for the maltose-negative S. aureus isolates showed highest resistance to ampicillin (100%) and penicillin (47.4) and lowest resistance (1.8%) to azithromycin, ciprofloxacin and erythromycin. The maltose-negative S. aureus isolates showed overall significantly increased antibiotic resistance compared to the maltose-positive strains, as well as multidrug resistance. Producers and veterinarians should consider probability of cure of such organisms (seemingly non-chronic) when adapting management and treatment, preventing unnecessary culling.

Climatic and regional antibiotic resistance patterns of Staphylococcus aureus in South African dairy herds.

South Africa is a large country of approximately 1.22 million km2, made up of nine provinces with three climatic zones. Farming in the country is mostly defined by regional differences. Of the different organisms isolated from milk samples of dairy cows, Staphylococcus aureus poses a challenge to maintain udder health and wholesome dairy products for human consumption. Antibiotic resistant bacteria are therefore a potential health hazard. The objective of this study was to investigate the seasonal and regional relationships of antibiotic resistance of S. aureus, of which little is known. This study was undertaken to evaluate a data set of 3410 S. aureus isolates, taken from milk samples with a somatic cell count of > 400 000 cells/mL from commercial dairy herds. These isolates were tested for antimicrobial susceptibility using the Kirby Bauer method for ampicillin, cloxacillin, penicillin G, clindamycin, oxy-tetracycline, cephalexin, cefuroxime and tylosin. The samples were from 830 dairy herds, out of the estimated 2000 commercial dairy herds in South Africa. All the antibiotics tested, except for cephalosporins, showed a predicted prevalence of resistance of above 50% in most provinces, which is a concern. The lowest prevalence of resistance to the majority of the categories of antibiotics tested was present in KwaZulu-Natal during spring. The cephalosporins had the lowest levels of prevalence of bacterial resistance in Gauteng during winter. Resistance patterns of S. aureus to the eight antibiotics varied in the different seasons and provinces, possibly because of different weather conditions, and the action and spectrum of antibiotics.

Proactive udder health management in South Africa and monitoring of antibiotic resistance of Staphylococcus aureus; in dairy herds from 2001 to 2010.

Antibiotic resistance of strains of Staphylococcus aureus isolated from bovine milk is of concern internationally. The objective of this study was to investigate trends of resistance of S. aureus to antibiotics administered to dairy cows in 19 South African and one Zambian dairy herds (participating in the South African proactive udder health management programme) and to identify possible contributing factors. The resistance of S. aureus strains to eight commonly used antibiotics in South Africa from 2001 to 2010 was evaluated. Staphylococcus aureus isolates (n = 2532) were selected from cows with subclinical mastitis in 20 herds routinely sampled as part of the proactive udder health management programme. The isolates were selected from milk samples that had somatic cell counts more than 400 000 cells/mL and were tested for antibiotic resistance using a standard Kirby-Bauer test with published clinical breakpoints. The prevalence of antibiotic resistance was evaluated as a percentage of S. aureus isolates susceptible out of the total numbers for each antibiotic selected per year. Staphylococcus aureus showed a significant increase in percentage of susceptible isolates over time for all antibiotics tested except for ampicillin. The overall prevalence of mastitis did not change during the study period. However, the prevalence of mastitis caused by S. aureus (mostly subclinical cases) in the selected herds decreased numerically but not significantly. Reduction in the incidence of antibiotic resistance shown by S. aureus was presumed to be a result of the application of the proactive udder health management programme. The fact that the overall prevalence of mastitis was kept stable was possibly because of the influence of the management programme in conjunction with the return of infections caused by non-resistant strains.

Epidemiological and partial budget analysis for treatment of subclinical Staphylococcus aureus intramammary infections considering microbiological and cytological scenarios.

An innovative method was investigated to aid in the elimination of Staphylococcus aureus (S. aureus) intramammary infections (IMI) from dairy herds. A stochastic model explore the economic benefit of three-day or eight-day treatment of subclinical IMI in all S. aureus infected cows or in only those with a somatic cell count (SCC) exceeding 200,000 cells/ml. An epidemiological model was developed to run parallel to the economic model that would predict the S. aureus IMI likely to persist, develop new infections and clinical mastitis. In the economic model a first algorithm was used to consider the low prevalence (LP) scenario and made use of S. aureus prevalence information provided by retrospective analysis of microbiological and cytological results in South Africa (2008-2012). The data used considered Staphylococcus aureus prevalence from [1.495; 1.595]95% to [6.72; 6.95]95% for SCC≤200,000 and SCC>200,000 cells/ml respectively. A second algorithm considered the high prevalence (HP) scenario to evaluate a simulated situation with a 5[U1] [R12] to 25% prevalence. Scenarios of low or high transmission ratio (TR) were included in the model according to the hygiene management on the farm. Probabilities and costs were calculated over 255days. The economic models predicted average cost indices for low S. aureus IMI and low TR to vary from -3179 ZAR (South African Rands) when subclinical cases with SCC higher than 200,000 cell/ml were treated for eight days, to -3663 ZAR when all subclinical S. aureus IMI were treated for three days. With a HP and high TR of S. aureus the average cost indices changed from -18,042 ZAR when none to -5433 ZAR per 255days when all S. aureus IMI were treated for eight days. The epidemiological model in this study predicted substantial benefit of treatment mainly in high TR scenarios. New IMI decreased up to77% in the three-day and up to 91% in the eight-day treatment scenarios. In the HP scenarios, persistent IMI were reduced by 94%. The number of clinical cases predicted with no treatment for subclinical infections was higher than the total number of clinical and subclinical cases in scenarios where cows were treated three or eight days. Initial prudent treatment of subclinical IMI resulted in less overall treatments and less new, persistent and clinical cases. Combined results of economic and epidemiological models indicated that the option that cost the least did not always have the best epidemiological outcome. Models may assist in optimising and balancing decisions relating to financial and IMI.

Validity of somatic cell count as indicator of pathogen-specific intramammary infections.

The objective of this study was to determine whether somatic cell count (SCC) was an effective test, with a sensitivity exceeding 85%, to determine species-specific bacterial infections. In addition, the relation between the SCC and various udder pathogen groups was investigated. SCC thresholds of greater than 200 000 cells/mL were used in quarter and greater than 150 000 cells/mL in composite milk samples. A retrospective study was conducted on a data set for 89 635 quarter and 345 467 composite cow milk samples. Eleven SCC threshold values were used to evaluate the diagnostic efficacy for the following bacteria: Gram-positive major pathogens: Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis; Gram-negative major pathogens: Escherichia coli, Klebsiella pneumonia and Serratia spp.; minor pathogens: coagulase-negative staphylococci, Micrococcus spp., Staphylococcus pseudintermedius, Streptococcus pyogenes, Enterococcus faecalis, Enterococcus canis, Trueperella pyogenes and other Enterobacteriaceae. Sensitivity and specificity were calculated taking the effect of clustering into account with quarter milk samples. Most samples yielding major Gram-positive pathogens (88.9% in quarter and 79.9% in composite samples) and minor pathogens (61.4% in quarter and 51.7% in composite samples) had SCC greater than 200 000 cells/mL. Sensitivity of the SCC test to detect major pathogens at an SCC threshold of greater than 200 000 cells/mL in quarter samples and greater than 150 000 cells/mL in composite milk samples was 88.2% and 84.2%, respectively, but specificity was low (57.7% and 52.8%, respectively).

Somatic cell count thresholds in composite and quarter milk samples as indicator of bovine intramammary infection status.

The objective of the study was to establish an operational somatic cell count (SCC) threshold to predict the presence of intramammary infection (IMI) in composite milk samples and compare findings with those in quarter milk samples. South African dairy producers now preferred composite milk samples for herd udder health analysis because of increasing cow numbers, convenience of sampling and lower cost. A retrospective study was conducted on 345 461 composite and 89 638 quarter milk samples from South African herds. Variance estimates for the proportion of quarter samples testing positive were adjusted to account for the lack of their independence within individual cows. The IMI at SCC thresholds of 150 000 cells/mL and 200 000 cells/mL differed only by 3.26% in composite milk samples. Youden's index indicated the optimum SCC thresholds for composite and quarter milk samples as 150 000 cells/mL and 200 000 cells/mL, respectively. At 150 000 cells/mL, sensitivity (95% confidence intervals [CI]) in composite milk samples was 65.3% (64.0%, 66.6%) and specificity was 66.8% (65.7%, 67.9%); and in quarter milk samples, sensitivity at 200 000 cells/ mL was 70.8% (69.5%, 72.0%) and specificity was 63.6% (62.4%, 64.8%). The likelihood of infection for udders and quarters, respectively, was 1.034 and 1.327 at an SCC threshold of 150 000 cells/mL and 0.864 cells/mL and 1.177 cells/mL at 200 000 cells/mL. The area under the curve of the receiver operating characteristics graph was 0.7084 and 0.7277 for composite and quarter samples, respectively, indicating that the SCC test could be considered as a good indicator of IMI in both sample types.

A pathogen-specific approach towards udder health management in dairy herds: Using culture and somatic cell counts from routine herd investigations.

A dedicated udder health diagnostic programme was developed and used over a 15-year period in South Africa to analyse milk samples based on microbiological and cytological patterns within various groups and for individual cows and udder quarters in dairy herds. These pathogen-specific analyses are utilised for pro-active improvement and management of udder health in South African commercial dairy herds. The programme acts as a monitoring tool and identifies management areas at risk and individual cows with udder disease and uses both quarter and composite milk samples. Intra-mammary infection (IMI) is a dynamic situation and depending on the time a milk sample is taken, false-negative results may be obtained. A new IMI and an infection that is curing may both have low somatic cell counts (SCCs), masking the true bacterial status. SCC in individual infected udder quarters may differ greatly depending on the causative bacterial species, its pathogenicity, the host immune status and the environmental factors involved. A pathogen-specific udder health approach was followed with repeated herd tests to take account of these udder health dynamics. The results of the herd IMI investigation are applied in practice to assist veterinarians, udder health consultants and managers to make informed and specific detailed decisions at both a herd and on an individual cow basis regarding udder health.

A cost-benefit model comparing the California Milk Cell Test and Milk Electrical Resistance Test.

The indirect effects of mastitis treatment are often overlooked in cost-benefit analyses, but it may be beneficial for the dairy industry to consider them. The cost of mastitis treatment may increase when the duration of intra-mammary infections are prolonged due to misdiagnosis of host-adapted mastitis. Laboratory diagnosis of mastitis can be costly and time consuming, therefore cow-side tests such as the California Milk Cell Test (CMCT) and Milk Electrical Resistance (MER) need to be utilised to their full potential. The aim of this study was to determine the relative benefit of using these two tests separately and in parallel. This was done using a partial-budget analysis and a cost-benefit model to estimate the benefits and costs of each respective test and the parallel combination thereof. Quarter milk samples (n= 1860) were taken from eight different dairy herds in South Africa. Milk samples were evaluated by means of the CMCT, hand-held MER meter and cyto-microbiological laboratory analysis. After determining the most appropriate cut-off points for the two cow-side tests, the sensitivity and specificity of the CMCT (Se= 1.00, Sp= 0.66), MER (Se= 0.92, Sp= 0.62) and the tests done in parallel (Se= 1.00, Sp= 0.87) were calculated. The input data that were used for partial-budget analysis and in the cost-benefit model were based on South African figures at the time of the study, and on literature. The total estimated financial benefit of correct diagnosis of host-adapted mastitis per cow for the CMCT, MER and the tests done in parallel was R898.73, R518.70 and R1064.67 respectively. This involved taking the expected benefit of a correct test result per cow, the expected cost of an error per cow and the cost of the test into account. The CMCT was shown to be 11%more beneficial than the MER test, whilst using the tests in parallel was shown to be the most beneficial method for evaluating the mastitis-control programme. Therefore, it is recommended that the combined tests should be used strategically in practice to monitor udder health and promote a pro-active udder health approach when dealing with host-adapted pathogens.

Host adapted intramammary infections in pregnant heifers which were co-housed and reared on fresh milk as calves.

BACKGROUND: Heifers can calve down with intramammary infections (IMI) and udder damage. This will have a negative impact on their longevity, future milk yield and financial return. Co-housed pre-weaned calves that are fed fresh milk have the opportunity to suckle each other's teats and may infect udders of fellow heifer calves with pathogens present in milk. The prevalence of IMI in pregnant heifers in South Africa (SA) which were co-housed and reared on fresh milk as calves, is not known. Quarter secretion samples from both pregnant heifers (n = 2065) and dry cows (n = 5365) were collected for microbiological analysis from eight SA dairy herds. All heifers tested in this study were co-housed pre-weaning and were fed fresh milk as calves. RESULTS: The prevalence of coagulase negative staphylococci, Staphylococcus aureus, Streptococcus agalactiae, environmental streptococci, coliforms and samples with no bacterial growth in heifers was 26%, 0.9%, 0.08%, 1.4%, 0.4% and 66%, respectively. The overall prevalence ratio between heifers and cows for Staphylococcus aureus IMI was 0.76 (95% CI: 0.59, 0.98). Four of the individual herds had prevalence ratios of less than one (p < 0.05), one herd had a prevalence ratio of 3.15 (95% CI: 1.52, 6.32), and the remaining 3 herds had a prevalence ratio not significantly different from 1.0. Marginally significant differences were found between Staphylococcus aureus IMI in pregnant heifers compared to cows in their second and later lactations (p = 0.06, p = 0.05, respectively) but no significant differences between heifers and cows in their first lactation. CONCLUSIONS: The presence of Streptococcus agalactiae IMI in heifers came as a surprise, especially as herd infection rates were low. The high prevalence ratio of Staphylococcus aureus between heifers and cows in one herd warrants further investigation due to the potential danger of udder damage in a young cow at the start of her productive life. The IMI in heifers with host adapted pathogens can also act as a source of new IMI for lactating dairy cows.

Comparing effects of freezing at -196 °C and -20 °C on the viability of mastitis pathogens.

The aim of this study was to compare the effects of cryopreservation at approximately -196 °C in liquid nitrogen (N) and freezing at approximately -20 °C in a freezer, on the viability and survival of eight different mastitogenic bacteria inoculated in milk. Bacteria were frozen at approximately -20 °C in a freezer and cryopreserved at approximately -196 °C in liquid nitrogen. An effective preservation method was needed for follow-up samples from cows identified in the South African National Milk Recording Scheme (NMRS) with somatic cell counts above 250 000 cells/mL milk. The organisation responsible for sample collection of the NMRS milk samples also provides producers with liquid nitrogen for their semen flasks at the collection sites. This existing mode of storage and transport could therefore be utilised.Ten samples of each organism were thawed and cultured bi-weekly until week 18 for both temperature treatments. An additional sampling was performed at week 30 for samples frozen at approximately -20 °C. Freezing and cryopreservation did not impair subsequent isolation of Streptococcus dysgalactiae, Streptococcus uberis, Enterococcus faecalis, Staphylococcus aureus (STH) (phage type lytic group III) or Sta. aureus (STA) (phage typed, other than lytic group III). Survival was indicated by the isolation of bacteria from samples, and viability by the strength of growth of the bacteria isolated. The survival of Streptococcus agalactiae decreased after week 12 and Escherichia coli after week 16 of freezing, but both organisms survived under cryogenic preservation until week 18. Coagulase-negative staphylococci survived until week 18 for both freezing and cryogenic preservation.Both storage methods could thus contribute to the improvement of a pro-active approach towards udder health management in South African dairy herds.