Bovine Coagulase-negative Staphylococci Research Articles

Antibacterial Activity of Aqueous and Ethanol Extracts of Caesalpinia Sappan L. against Coagulase-Negative Staphylococci Isolated from Subclinical Bovine and Caprine Mastitis

This study aimed to investigate the effect of Caesalpinia sappan L. hearth wood extract against Coagulase-Negative Staphylococci (CNS) causing subclinical mastitis in dairy cows and goats. Seventeen and thirty CNS isolates were isolated from bovine and caprine mastitis samples, respectively. The phytochemicals tests performed on aqueous and ethanol extracts of C. sappan heartwood presented total phenolic (150 and 187 mg GAE/g dw), flavonoids (28 and 21 mg RE/g dw), tannins, alkaloid, saponin, and steroid. The extracts were used to determine the antibacterial effect on CNS using disc diffusion, Minimum Inhibitory Concentration (MIC), and Minimum Bactericidal Concentration (MBC) methods. The aqueous extract and ethanol extract of C. sappan contained effective compounds, including phenol, flavonoid, tannin, alkaloid, saponin, and steroid. Ethanol extract of C. sappan had a greater content of phenol, while aqueous extract of C. sappan had a greater content of flavonoid. Ethanol extracts of C. sappan showed the strongest inhibition on the CNS isolated from bovine (14.3-21.0 mm) and caprine (24.3-30.0 mm). The antimicrobial activity of ethanol C. sappan extract showed significantly higher than aqueous extract against bovine CNS (8.0 to 14.7 mm) and caprine CNS (22.3 to 29.0 mm). The C. sappan ethanol extract had the best MIC and MBC of bovine CNS (6.24 mg/mL) and caprine CNS of (1.56 mg/mL). The ethanol extracts of C. sappan showed strong antimicrobial effects against CNS isolated from both bovine and caprine mastitis.

Short communication: Search for superantigen genes in coagulase-negative staphylococci isolated from bovine milk in Canada

The objective of the present study was to look for genes encoding for superantigens in bovine coagulase-negative staphylococci (CNS) isolated from milk in Canada. We screened by PCR 71 bovine CNS isolates, obtained from the Mastitis Pathogen Culture Collection managed by the Canadian Bovine Mastitis and Milk Quality Research Network (St-Hyacinthe, QC, Canada), for the presence of 13 superantigen genes. Our results indicate that these CNS isolates did not have any of the 13 superantigen genes screened for in the present study. Thus, prevalence of those genes in CNS from milk is expected to be quite low in Canada.

Technical note: A pilot study using a mouse mastitis model to study differences between bovine associated coagulase-negative staphylococci

Coagulase-negative staphylococci (CNS) are a group of bacteria classified as either minor mastitis pathogens or commensal microbiota. Recent research suggests species- and even strain-related epidemiological and genetic differences within the large CNS group. The current pilot study investigated in 2 experiments whether a mouse mastitis model validated for bovine Staphylococcus aureus can be used to explore further differences between CNS species and strains. In a first dose titration experiment, a low inoculum dose of S. aureus Newbould 305 (positive control) was compared with increasing inoculum doses of a Staphylococcus chromogenes strain originating from a chronic bovine intramammary infection to a sham-inoculated mammary glands (negative control). In contrast to the high bacterial growth following inoculation with S. aureus, S. chromogenes was retrieved in very low levels at 24 h postinduction (p.i.). In a second experiment, the inflammation inflicted by 3 CNS strains was studied in mice. The host immune response induced by the S. chromogenes intramammary strain was compared with the one induced by a Staphylococcus fleurettii strain originating from cow bedding sawdust and by a S. chromogenes strain originating from a teat apex of a heifer. As expected, at 28 and 48 h p.i., low bacterial growth and local neutrophil influx in the mammary gland were induced by all CNS strains. As hypothesized, bacterial growth p.i. was the lowest for S. fleurettii compared with that induced by the 2 S. chromogenes strains, and the overall immune response established by the 3 CNS strains was less pronounced compared with the one induced by S. aureus. Proinflammatory cytokine profiling revealed that S. aureus locally induced IL-6 and IL-1β but not TNF-α, whereas, overall, CNS-inoculated glands lacked a strong cytokine host response but also induced IL-1β locally. Compared with both other CNS strains, S. chromogenes from the teat apex inflicted a more variable IL-1β response characterized by a more intense local reaction in several mice. This pilot study suggests that an intraductal mouse model can mimic bovine CNS mastitis and has potential as a complementary in vivo tool for future CNS mastitis research. Furthermore, it indicates that epidemiologically different bovine CNS species or strains induce a differential host innate immune response in the murine mammary gland.

High-resolution melt analysis for species identification of coagulase-negative staphylococci derived from bovine milk

Coagulase-negative staphylococci (CNS) are the most frequently isolated pathogens isolated from bovine milk. In this study, we report a rapid assay for species identification of CNS using high-resolution melt analysis (HRMA) of 16S rDNA sequences. Real-time polymerase chain reaction amplification of 16S rRNA gene fragment, spanning the variable region V1 and V2, was performed with a resulting amplicon of 215 bp. A library of distinct melt curves of reference strains of 13 common CNS species was created using HRMA. Sequencing of 16S rRNA and rpoB genes, and, when needed, tuf gene, of 100 CNS isolates obtained from Canadian Bovine Mastitis Research Network was done to determine their species identity, allowing for subsequent evaluation of the performance of HRMA for field isolates of bovine CNS. A combination of HRMA and sequencing revealed that Staphylococcus chromogenes, S. xylosus, S. simulans, and S. sciuri had multiple genotypes, complicating their resolution by HRMA. As the 3 genotypes of S. chromogenes had distinct melt curves, the 3 distinct genotypes were employed as reference strains in a blinded trial of 156 CNS isolates to identify S. chromogenes. HRMA correctly identified all S. chromogenes isolates which were later confirmed by sequencing. Staphylococcus chromogenes (68%) was most frequently found among the CNS isolates, followed by S. haemolyticus (10%) and S. xylosus (6%). The present study revealed that HRMA of 16S rRNA gene (V1–V2) could be used as a rapid, efficient, low-cost, and minimally cumbersome technique for S. chromogenes identification, the most common CNS derived from bovine milk.

Validation of amplified fragment length polymorphism genotyping for species identification of bovine associated coagulase-negative staphylococci

In many countries, coagulase-negative staphylococci (CNS) are currently the most common cause of intramammary infection in lactating cows. In order to elucidate the importance of various CNS species in udder health and milk quality, further research conducted on the species level is required. Phenotypic identification of CNS species appears to be unreliable and more accurate and reproducible genotypic methods are needed. In the current study, use of amplified fragment length polymorphism (AFLP) genotyping was validated for species identification of bovine associated CNS. An initial reference library was generated with AFLP fingerprints of 52 different CNS type and reference strains. Next, 247 bovine CNS field isolates with known species identity were analyzed. These field isolates had been previously identified by gene sequencing and were randomly divided into two subsets, i.e. a training set and a validation set. The training set was identified against the initial reference library containing only type and reference strains, which resulted in a typeability of 80.5%. Accuracy of the AFLP identifications, being the correspondence with gene sequencing results, was 95.0%. Fingerprints of the training set were then added to the initial library and identification of the validation set was done by means of this extended library. By adding bovine CNS to the library, performance of the AFLP identification method improved considerably. Final typeability and accuracy were 98.4% and 99.2%, respectively. Numerical analysis of AFLP fingerprints proves to be an accurate genotypic method for identification of CNS from bovine origin. The constructed AFLP library provides a useful identification tool for field studies on the subject of CNS.

Technical note: Use of transfer RNA-intergenic spacer PCR combined with capillary electrophoresis to identify coagulase-negative Staphylococcus species originating from bovine milk and teat apices

Coagulase-negative staphylococci (CNS) are the most frequently isolated bacteria in milk samples from cows with and without mastitis. Elucidating their relevance in bovine udder health is hampered because identification at the species level, if done at all, used to be performed based on phenotypic features. To provide a rapid, cheap, and easy-to-use genotypic technique that can be used to identify CNS species from milk and teat apices from cows, the performance of transfer RNA-intergenic spacer PCR (tDNA-PCR) in combination with capillary electrophoresis was evaluated. After updating the tDNA library with CNS reference strains, 288 field isolates were identified with tDNA-PCR and gene sequencing, and the latter was used as the reference method. The field isolates were divided in 2 groups of 144. Isolates of the first group were identified with tDNA-PCR with a typeability of 81.9% and an accuracy of 94.1%. Peak patterns of these isolates were then added to the tDNA library with species identity as determined by DNA sequencing. The second group was identified with the updated tDNA library, resulting in 91.0% typeability and 99.2% accuracy. This study showed that the updated tDNA-PCR in combination with capillary electrophoresis was almost as accurate as gene sequencing but faster and cheaper (only $3 per isolate), and is a useful tool in observational studies concerning the epidemiology of bovine CNS species.

Species identification of coagulase-negative staphylococci: Genotyping is superior to phenotyping

Coagulase-negative staphylococci (CNS) are isolated commonly from bovine milk and skin. Their impact on udder health and milk quality is debated. It has been suggested that sources and consequences of infection may differ between CNS species. Species-specific knowledge of the impact and epidemiology of CNS intramammary infections is necessary to evaluate whether species-specific infection control measures are feasible and economically justified. Accurate measurement of impact, sources, and transmission mechanisms requires accurate species level identification of CNS. Several phenotypic and genotypic methods for identification of CNS species are available. Many methods were developed for use in human medicine, and their ability to identify bovine CNS isolates varies. Typeability and accuracy of typing methods are affected by the distribution of CNS species and strains in different host species, and by the ability of test systems to incorporate information on new CNS species into their experimental design and reference database. Generally, typeability and accuracy of bovine CNS identification are higher for genotypic methods than for phenotypic methods. As reviewed in this paper, DNA sequence-based species identification of CNS is currently the most accurate species identification method available because it has the largest reference database, and because a universally meaningful quantitative measure of homology with known species is determined. Once sources, transmission mechanisms, and impact of different CNS species on cow health, productivity and milk quality have been identified through use of epidemiological data and accurate species identification methods, appropriate methods for routine use in research and diagnostic laboratories can be proposed.

Comparison of a commercialized phenotyping system, antimicrobial susceptibility testing, and tuf gene sequence-based genotyping for species-level identification of coagulase-negative staphylococci isolated from cases of bovine mastitis

In order to evaluate the usefulness of some phenotypic and genotypic methods for species identification of coagulase-negative staphylococci (CNS), isolates were obtained from bovine cases of clinical and sub-clinical mastitis from different geographical areas in Sweden. By using the Staph-Zym™ test, antimicrobial susceptibility testing, and sequencing of part of the CNS tuf gene and, when needed, part of the 16S rRNA gene we characterized 82 clinical isolates and 24 reference strains of 18 different species of staphylococci. The genotypic methods identified nine different species of CNS among the 82 milk isolates. A comparison with results obtained by tuf gene sequencing showed that Staph-Zym™ correctly identified CNS reference strains to species level more often than bovine milk CNS isolates (83% and 61%, respectively). In addition, tests supplementary to the Staph-Zym™ were frequently needed in both groups of isolates (50% of reference strains and 33% of milk isolates) to obtain an identification of the strain. It is notable that Staph-Zym™ judged two isolates as CNS, although they belonged to other species, could not give a species name in 11% of the bovine CNS isolates, and gave 28% of the isolates an incorrect species name. The present study indicates that the studied phenotypic methods are unreliable for identification of CNS from bovine intra-mammary infections.

Coagulase-negative staphylococci isolated from bovine extramammary sites and intramammary infections in a single dairy herd

Isolates of various species of coagulase-negative staphylococci (CNS) from extramammary swab samples were compared with isolates of bovine mastitis CNS species. Swab samples were taken from perineum skin and udder skin, teat apices and teat canals of lactating dairy cows of the research dairy herd of the University of Helsinki in 1999 and 2002. In addition, hands of herd staff and liners of teat cups were sampled for CNS. CNS isolates from milk samples of subclinical or clinical mastitis in the same herd were collected during 1998-2002. Species identification was performed using phenotyping (API Staph ID 32 test) and by constructing a 16 and 23S rRNA RFLP library (ribotyping). Based on phenotype, 84% of mastitis isolates and 57% of extramammary isolates were identified at species level with >90% probability. Ribotype patterns formed 24 clusters, and 15 of them included a CNS type strain. If the ribotype clusters contained isolates of both extramammary and mastitis origin, they were further typed using pulsed-field gel electrophoresis (PFGE). The predominant CNS species in mastitis, based both on phenotyping and genotyping, were Staph. chromogenes and Staph. simulans. Phenotyping failed to identify half of the extramammary isolates. Based on phenotyping, Staph. equorum and Staph. sciuri, and based on ribotyping, Staph. succinus and Staph. xylosus, were the predominant CNS species in extramammary samples. The most common species in milk samples, Staph. chromogenes, was also isolated from several extramammary samples, and five out of ten pulsotypes were shared between mastitis and extramammary isolates, indicating that strains from udder skin are highly similar. The second commonest mastitis species, Staph. simulans, was isolated only from three extramammary samples, indicating that Staph. simulans may be more specifically associated with mastitis. Consequently, the origin of CNS mastitis may vary depending on the causing CNS species.

Identification and characterization of nine novel types of small staphylococcal plasmids carrying the lincosamide nucleotidyltransferase gene lnu(A)

To date, very little is known about lincosamide resistance plasmids in staphylococci with only a single lnu(A)-carrying staphylococcal plasmid having been sequenced completely. The aim of this study was to characterize small lnu(A)-carrying plasmids isolated from bovine coagulase-negative staphylococci (CoNS). Nine CoNS isolates with MICs of the lincosamide pirlimycin of 1-4 mg/L were tested for the presence of the lnu(A) gene. Its location was determined by Southern-blot hybridization. The lnu(A)-carrying plasmids were transformed into Staphylococcus aureus RN4220 and compared by restriction mapping and subsequent sequencing. Selected plasmids were investigated for their copy number and their lnu(A) gene expression via RT real-time PCR. The lnu(A) gene was detected on plasmids in all isolates. Sequence analysis revealed that these plasmids carried a rep gene, coding for the replication initiator protein, and the resistance gene lnu(A), coding for a lincosamide nucleotidyltransferase. While the Lnu(A) proteins were closely related (91.3-100% amino acid identity), the Rep proteins differed distinctly (27.4-100% amino acid identity), but showed similarity (81.4-98.5%) to Rep proteins of other small staphylococcal resistance plasmids. Sequence features of rolling-circle plasmids, such as the single-strand (ssoA) and double-strand (dso) origins of replication, were identified. For two plasmid types detected, the lincosamide resistance level varied with regard to the amounts of lnu(A) transcripts detected. Structurally different lnu(A)-carrying plasmids were detected in various CoNS species. The detection of the same lnu(A) gene in different plasmid backbones suggested the exchange of the gene via interplasmid recombinational events.

Fusidic acid resistance, mediated by fusB, in bovine coagulase-negative staphylococci

The aim of this study was to determine the occurrence of fusidic acid resistance, mediated by the fusB gene, among coagulase-negative staphylococci (CoNS) isolated from bovine mastitis. A total of 113 CoNS isolates were screened for susceptibility to fusidic acid by using a disc diffusion method. The fusB gene was detected by using PCR and subsequent DNA sequencing. The localization of fusB was determined by hybridization. The fusB gene was detected in 3 of 11 fusidic acid-resistant bovine CoNS isolates. The organization of the fusB downstream region on a 40 kb plasmid in a Staphylococcus haemolyticus isolate (288/96) was highly similar to the previously reported organization of fusB on plasmid pUB101 from a Staphylococcus aureus isolate of human origin. The fusB gene was chromosomally located in the remaining two isolates. Fusidic acid resistance mediated by fusB is not the dominant resistance mechanism in fusidic acid-resistant CoNS studied in this work. The similarity between the organization of the fusB downstream region in S. haemolyticus (isolate 288/96) and on plasmid pUB101 from an S. aureus isolate of human origin indicates a common ancestral origin of these genes.

Bovine Coagulase-Negative Staphylococci: Biochemistry and Polymerase Chain Reaction

Hum mel, R., G. L e h man n : Bovine coagulase-negative Staphylococci: Biochemistry and Poly­ merase Chain Reaction. Acta vet. Brno, 63,1994: 133-139. Staphylococcus (S.) simulans, S. chromogenes, and S. epidermidis were the species of coa­ gulase-negative staphylococci (CNS) most frequently involved in subclinical mastitits of dai­ ry cattle. The same biochemical features characterized the successive isolates of a particular chronically infected udder quarter. Occurrence rate of the various species differed between the herds examined. In cases of clinical mastitis of cows S. xylosus, S. simulans, S. haemolyticus, and S. chromogenes predominated. A comparison between strains of the par­ ticular CNS species from infections in man and cattle did not show host attributable bioche­ mical features or profiles obtained by polymerase chain reaction using arbitrary primers (AP-PCR). Bovine mastitis, Coagulase-negative staphylococci, biochemical features, polymerase chain reaction