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.     

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.     

Antimicrobial susceptibility of coagulase-negative staphylococci isolated from bovine milk samples

The aim of this study was to examine whether antimicrobial resistance profiles of coagulase-negative Staphylococcus (CNS) species isolated from milk of dairy cows differed between bacterial species, and to compare results obtained by phenotypic and genotypic profiling of resistance to penicillin, oxacillin and macrolide-lincosamide (ML) antibiotics. Of 170 CNS isolates, 83 (48.8%) were phenotypically susceptible to all antimicrobial agents tested in minimum inhibitory concentration (MIC) assays, 40.6% expressed resistance to a single compound or a single class of compounds, and 10.6% to multiple drug classes. Nine percent, 68%, 19%, 4% and 1% of isolates were negative for all resistance genes tested by PCR or positive for one, two, three or four resistance genes, respectively. Phenotypic resistance and detection of resistance genes other than blaZ were relatively rare in Staphylococcus chromogenes, which was the most common CNS species (36% of 170 genotypically identified isolates). In Staphylococcus epidermidis, which was the second most common CNS species (14% of isolates), phenotypic penicillin resistance was significantly more common than in other CNS species. Almost half of the S. epidermidis isolates carried multiple resistance genes and 30% carried the methicillin resistance gene mecA. Survival analysis using MIC values showed significant associations between phenotypic and genotypic resistance profiles. We conclude that CNS species from bovine milk differ significantly in phenotypic and genotypic antimicrobial resistance profiles, which has implications for treatment and management decisions.     

Macrolide and lincosamide resistance genes of environmental streptococci from bovine milk

Environmental streptococcus isolates from bovine milk were identified to the species and strain level and screened for resistance to macrolide and lincosamide antibiotics by phenotypic and genotypic methods. Isolates were tested for resistance to erythromycin and pirlimycin by broth microdilution assays. Presence of ribosomal methylase genes (ermA, ermB, ermC) and efflux pump genes (mefA/E, msrA/C) was detected by polymerase chain reaction (PCR). Resistance to pirlimycin (minimum inhibitory concentration (MIC) = 8microg/ml) was detected in 6 of 13 Enterococcus isolates that were identified as E. faecium by API20Strep typing. msrC was detected in 10 enterococcal isolates but the detection of msrC was not associated with phenotypic resistance. msrC negative isolates were reclassified as Enterococcus mundtii based on sequencing of housekeeping genes. Resistance to erythromycin and pirlimycin (MIC > 16microg/ml) was detected in 4 of 4 Streptococcus dysgalactiae and 12 of 20 Streptococcus uberis isolates and was encoded by ermB. All Streptococcus isolates tested negative for ermA, ermC, mefA/E and msrA/C. Among ermB positive streptococci, three alleles were identified based on a 527 bp gene fragment. Each allele was detected in at least two herds. The same alleles have also been detected in other bacterial species from bovine and non-bovine hosts and farm soil, suggesting a theoretical potential for horizontal transfer of macrolide resistance genes on dairy farms.     

Reclassification of German, British and Dutch isolates of so-called Pasteurella multocida obtained from pneumonic calf lungs

The taxonomic relationship of 131 strains previously identified as Pasteurella multocida obtained from calf pneumonia in West Germany, United Kingdom and Netherlands was investigated by extended phenotypic and limited genotypic characterization. Twenty-four strains were classified as P. multocida ssp. multocida, 15 strains as P. avium biovar 2 and 13 strains as P. canis biovar 2. Sixty-five and five strains were tentatively classified as ornithine negative P. multocida ssp. multocida and P. multocida ssp. septica, respectively. Genetic investigations showed that ornithine negative strains of P. multocida were related on species level. Less genomic binding was found between an ornithine negative strain of P. multocida ssp. septica and the type strains of the three subspecies of P. multocida. The taxonomic position of ornithine negative strains of P. multocida is still under investigation. The taxonomic position of the remaining nine strains is uncertain underlining the need for genotypic characterization within the genus Pasteurella to aid in defining single species by phenotypic tests.     

Characterization of Staphylococcus aureus strains isolated from bovine mastitis in Japan

Fifty-three strains of Staphylococcus aureus isolated from cows affected with mastitis from 21 prefectures in Japan were characterized by phenotypic and genotypic methods. Thirty-three (62.3%) strains showed biotype K-beta+CV:A, coagulase type VI, and sensitivity to bovine phages of group III or IV. These 33 strains could be subdivided into two groups on the basis of the production of staphylococcal enterotoxins (SEs) and on toxic shock syndrome toxin-1 (TSST-1). By pulsed-field gel electrophoresis, the 16 SEC- and TSST-1-producing strains showed similar patterns that differed by only a few fragments, suggesting that they were genetically closely related. Fifteen of 17 non SEC-producing strains which did not produce any other SEs and TSST-1 were genetically different from the SEC-producing strains and showed genetic diversity.     

Virulence markers and genetic relationships of Shiga toxin-producing Escherichia coli strains from serogroup O111 isolated from cattle

Shiga toxin-producing Escherichia coli (STEC) strains isolated from healthy cattle (O111:NM, seven strains; O111:H8, three strains) in Brazil were studied and compared to previously characterized human strains in regard to their phenotypic and genotypic characteristics to evaluate their pathogenic potential. Most bovine STEC O111 strains were isolated from dairy calves, and strains with genotypes stx1 alone and stx1/stx2 (variant stx2) occurred in different regions. Irrespective of the stx genotype, all strains were positive for eae theta, alpha variants of tir, espA and espB, and for ler, qseA, iha, astA and efa1 genes. Only one strain was negative for EHEC-hlyA and all strains were negative for iha, saa and espP genes and for EAF and bfpA, genetic markers of EPEC. Except for the presence of stx2, bovine strains showed the same profile of putative virulence genes found among the human strains. Similar biochemical behavior was identified among the strains analysed. Two bovine STEC strains produced the localized adherence (LA) phenotype in 6-h tests with Caco-2 (human enterocyte) cells. Intimate attachment (judged by the FAS test) was found in 9 out of 10 bovine strains as it was observed for the human STEC strains. RAPD-PCR analysis showed two distinct RAPD groups among the STEC O111 strains examined. Despite the relative low frequency of STEC O111 strains recovered from cattle no differences in their pathogenic potential were observed compared to some strains isolated from human diarrhea, suggesting that healthy cattle may be a potential source of infection for humans in Brazil.     

Differentiation of Pasteurella multocida isolates from cases of atrophic rhinitis in pigs from Zimbabwe by RAPD and ribotyping

Atrophic rhinitis in pigs is rarely reported in Southern Africa. To determine the relationship between Pasteurella multocida clones from clinical cases of atrophic rhinitis, twenty-one strains were characterised by selected phenotypic and genotypic methods. Biochemical analysis classified 18 strains as P. multocida subspecies multocida, whilst the remainder were grouped into separate unassigned biotypes. Capsular groups A (16/21) and D (l/21) were found among the isolates by PCR. Four ribotype patterns were obtained following HpaII ribotyping, whilst random amplification of polymorphic DNA (RAPD) revealed three main clusters. However, subclusters were also noted for each RAPD cluster. Our results indicate that RAPD offers a better discrimination of strains than ribotyping and that none of the phenotypic characters were directly related to the genotypic clusters.     

Detection of urease-negative Bordetella bronchiseptica from the field

Four urease-negative Bordetella bronchiseptica isolates originating from pigs were examined by phenotypic and molecular methods. The phenotypic properties of the isolates were in harmony with the data of the literature, except for the lack of urease activity in conventional tube test, API 20 NE and Diatabs? assays. Using genotypic methods, the urease-negative isolates did not differ from the urease-positive reference strain. They were positive in species-specific and ureC PCR, and all strains showed uniform bands in PCR-RFLP studies of flaA genes. The reason for the lack of urease activity, a characteristic considered species specific for B. bronchiseptica, needs to be studied further. The finding underlines the significance of genotyping when the phenotypic identification of B. bronchiseptica seems questionable.     

Genetic diversity of Pasteurella species isolated from European vespertilionid bats

Pasteurella are an important cause of fatal infections in free-ranging bats, but the genetic diversity of bat-derived strains is unclear. In the current study, 81 Pasteurella strains associated with pneumonia, severe organ necroses and systemic infection in free-ranging European vespertilionid bats were characterized by biochemical and molecular typing methods. Genetic relationships and subspecies status of Pasteurella multocida strains were determined by comparative 16S rDNA and rpoB gene sequence analysis. In addition, 30 representatives of the bat-derived P. multocida strains were selected based on phenotypic and genotypic tests to be compared by pulsed-field gel electrophoresis using SmaI. Most (85%) of the Pasteurella strains obtained from free-ranging bats in this study represented P. multocida ssp. septica. P. multocida ssp. multocida and Pasteurella species B were also identified in a small number of isolates. PFGE analysis correlated well with the sequencing results and revealed a high genetic diversity among bat-derived strains of P. multocida ssp. septica. Strains sharing identical or closely related SmaI fragment patterns were cultured from bats of different species, geographic origins, and years of isolation. The presence of numerous different P. multocida strains allows the assumption that Pasteurella infections in vespertilionid bats are not solely based on intra- but also on inter-species transmission. And indeed, our results present evidence of P. multocida infections in bats following cat predation.     

Species and strain specific identification of lactic acid bacteria in complex microflora

The identification of lactic acid bacteria in a complex microbiota using bacteriological culture in combination with phenotypic and genotypic identification techniques is laborious and time-consuming. New molecular methods permit a fast and culture-independent characterisation of such microbiota. Denaturing gradient gel electrophoresis (DGGE) of PCR fragments of the 16S rRNA gene has been proven to be a suitable tool. Here the use of PCR-DGGE with group specific primers is described to investigate the dynamic of sourdough microbiota from addition of the starter until the microbiota remained stable. Species were identified by applying an identification ladder obtained from reference strains or by sequence analysis of the PCR fragments. Furthermore, a method for detection of strains in complex microbiota is described. A strain specific chromosomal DNA fragment of Lactobacillus paracasei LTH 2579 was isolated applying the subtraction hybridisation. Based on the acquired target sequence a specific PCR system was established and combined with a PCR system specific for the species L. paracasei. Use of this detection system permitted to identify and quantitatively detect L. paracasei LTH 2579 in fermented sausages and upon consumption in faecal samples.     

Virulence factors and genetic relatedness of Escherichia coli strains isolated from pigs with post-weaning diarrhea

Forty-six Escherichia coli strains isolated from post-weaning diarrhea of pigs were analysed for their phenotypic and genotypic properties. The isolates were of serogroups O138, O139, and O141 and most of them possessed hemolytic activities. PCR analysis showed that 34 of the isolates harboured the genes for shiga toxin 2e and 32 strains possessed the genes for heat-stable enterotoxins I and II. Ten strains had the fedA gene of F18 fimbriae. The genetic relationships among all isolates were tested by random amplified polymorphic DNA (RAPD) and enterobacterial repetitive intergenic consensus (ERIC) PCR analyses. Using the RAPD test with two different primers, six fingerprints were distinguished whereas the ERIC analysis revealed only three DNA patterns. Some strains possessing identical phenotypic and genotypic virulence determinants exhibited distinct RAPD profiles and some isolates with different pathogenic markers showed the same RAPD and ERIC pictures. Thus, RAPD, and to a less extent ERIC techniques, revealed intra- and interserogroup genotypic variations among the E. coli strains analyzed.     

Characterization of Bordetella bronchiseptica strains using phenotypic and genotypic markers

Thirty-five strains of Bordetella bronchiseptica, recovered primarily from pigs, rabbits, dogs, cats and humans, were characterized by phenotypic and genotypic markers. Biochemical typing only showed variation in the ability to reduce nitrate to nitrite. OMP profiles from virulent strains showed variations in the region of 85-95kDa, which lead us to describe five OMP-types alpha, beta, gamma, delta and varepsilon. Genotypic markers included the presence of IS1001, and polymorphisms in the flagellin gene (flaA) and pertussis toxin (PT) promoter region. The IS1001 was detected in 16 isolates (2 from humans and 10 from pigs) but was absent in rabbit isolates. The restriction profiles of the flaA gene allowed us to differentiate the strains into types A-C. The PT types were characterized by an RFLP assay and could be typed through patterns III-V. There was no apparent association between the flaA or PT types and the origin of the isolates. Eleven groups of isolates were identified on the basis of specific combinations of the analyzed markers. The combination of phenotypic and genotypic tests used could be useful in characterizing isolates and differentiating between certain clonal types of B. bronchiseptica.     

Detection of central nervous system tissues in meat products: validation and standardization of a real-time PCR-based detection system

Several phenotypic as well as genotypic methods have been published describing the detection of central nervous system (CNS) tissues that are part of the bovine spongiform encephalopathy (BSE) risk material in food products. However, none of these methods is able to differentiate between CNS tissue of the banned ruminant species and tissues of other animal species. A quantitative and species-specific real-time RT-PCR method has been developed that enables the reliable identification of CNS tissues in meat and meat products. This method is based on a messenger (m)RNA assay that uses bovine, ovine and caprine glial fibrillary acidic protein (GFAP) encoding gene sequences as markers. The in-house validation studies evaluated the tissue specificity of up to 15 bovine tissues and the standardization of absolute as well as relative quantitative measurement. The specific amplification of spinal cord and brain tissue GFAP cDNA has been shown previously. In addition, two commercially available ELISA kits were used for the comparative analysis of artificially contaminated minced meat. Small quantities of bovine brain that had been stored over the recommended period of 14 days were examined. The real-time PCR method proved to be suitable for the detection of 0.1% CNS tissue. No false negative results were observed. The quantitative detection of GFAP mRNA using real-time RT-PCR seems a suitable tool in routine diagnostic testing that assesses the illegal use of CNS tissue in meat and meat products. The stability of the selected target region of the GFAP mRNA also allows the detection of CNS tissues after the meat has been processed.     

Genotyping of Staphylococcus aureus isolated from bovine mastitis.

The present study was designed to comparatively investigate 25 Staphylococcus aureus strains isolated from bovine subclinical mastitis. The S. aureus strains, obtained from six different farms at five locations in one region of Germany, were characterized by phenotypic and genotypic methods. The S. aureus could be identified and further characterized by their cultural, biochemical and hemolytic properties. To analyze the epidemiological relationship the isolates were subjected to DNA fingerprinting by macrorestriction analysis of their chromosomal DNA, by PCR amplification of the gene encoding the 16S-23S rRNA intergenic spacer, by PCR amplification of the gene encoding the IgG binding region and the X region of protein A and by amplifying, and subsequent, digestion of the gene encoding staphylococcal coagulase. The macrorestriction analysis revealed five DNA restriction patterns with DNA patterns I, III and IV occurring in three, four, and three different farms, respectively. In addition, clones with different DNA patterns could be found within one herd. The PCR products for the spacer DNA, the spa gene encoding the X region of protein A and the coa gene encoding coagulase corresponded mostly to the pattern observed by DNA fingerprinting. Amplification of the gene encoding the IgG binding region revealed sizes of 620 bp for 20 of the isolates and 280 bp for four isolates indicating, for the latter, a deletion of segments in this region. These findings show, that single, widely distributed clones seemed to be responsible for cases of bovine subclinical mastitis found in one region of Germany.     

Detection of intercellular adhesion genes and biofilm production in <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> isolated from bovine subclinical mastitis

Biofilm production by Staphylococcus aureus, an important virulence factor was investigated employing phenotypic and genotypic methods. A total of 102 S. aureus isolates from bovine subclinical mastitis cases were included in the study. Maximum number of biofilm producing strains were detected by Congo red agar (CRA) method (48.03%) followed by tube method (36.27%). Tissue culture plate method (TCP) without and with destaining identified 19.60 and 29.41% of S. aureus as biofilm producers, respectively. A polymerase chain reaction for detection of intercellular adhesion genes, icaA and icaD, responsible for biofilm formation was standardized. Of the 102 S. aureus isolates investigated, 36 (35.29%) strains revealed presence of both the genes. Considering polymerase chain reaction as a standard test, CRA and TCP without destaining were the most sensitive and specific, respectively. PCR technique standardized for detection of the icaA and icaD genes is reliable for identifying biofilm producing potential of S. aureus which may help in rapid detection of biofilm-producer Staphylococci. This would allow the early application of control measures.     

Molecular analyses of mycobacteria other than the M. tuberculosis complex isolated from Northern Ireland cattle

Mycobacteria other than the Mycobacterium tuberculosis complex (MOTT), isolated from Northern Ireland cattle, were identified by PCR amplification of the 16S rRNA gene, and subsequent reverse cross blot hybridisation and sequence analyses. Elucidation of the MOTT species was to facilitate specificity testing of new and existing diagnostic test reagents for bovine tuberculosis. The presence of the genes for potential diagnostic antigens: MPB70, MPB64, ESAT-6 and CFP-10 in the isolated MOTT species was investigated. Molecular analyses of cultured isolates from bovine lymph node specimens of 48 cattle identified a wide variety of mycobacterial species including Mycobacterium nonchromogenicum, Mycobacterium malmoense, Mycobacterium bohemicum, Mycobacterium paratuberculosis, Mycobacterium avium, Mycobacterium kansasii, Mycobacterium holsaticum, Mycobacterium palustre, Mycobacterium sp. IWGMT 90210, Mycobacterium sp. LIV-2129, a potentially novel mycobacterial species (EMBL/GenBank/DDBJ Accession Number AJ617495) and Rhodococcus equi. Apart from M. kansasii, the results of traditional (standard phenotypic and biochemical) and molecular identification methods did not correlate well, with traditional methods identifying fewer species. Most of the species identified were either recognised pathogenic or potential pathogenic species. The genes for ESAT-6, CFP-10 and, unusually, MPB64 were detected in M. kansasii only. The MPB70 gene was not detected in any of the species. This study supported restricted species distribution of these genes as well as identifying a different range of MOTT species that could be included in specificity testing of new diagnostic reagents for bovine tuberculosis.     

Whole genome sequencing of methicillin-resistant and methicillin-sensitive Staphylococcus aureus isolated from 4 horses in a veterinary teaching hospital and its ambulatory service

Genomic characterization was conducted on 2 methicillin-resistant Staphylococcus aureus (MRSA) strains isolated from 2 horses hospitalized during an overlapping period of time and 2 methicillin-sensitive S. aureus (MSSA) strains isolated from 2 distinct horses. Phylogenetic proximity was traced and the genotypic and phenotypic characteristics of the antimicrobial resistance of the strains were compared.Whole genome sequencing of MRSA strains for this report was similar but differed from whole genome sequencing of MSSA strains. The MRSA strains were closely related, belonging to sequence type (ST) 612, spa type t1257, and SCCmec type IVd2B. The MSSA strains were also closely related, belonging to ST1660, spa type t3043, and having no detectable staphylococcal cassette chromosome mec elements. All MSRA and MSSA strains were Panton-Valentine leukocidin negative. There were discrepancies in the genotypic analysis and the antimicrobial susceptibility testing (phenotypic analysis) of MRSA strains for rifampin, trimethoprim-sulfamethoxazole, gentamicin, amikacin, and enrofloxacin.     

Three unique groups of spirochetes isolated from digital dermatitis lesions in UK cattle

Bovine digital dermatitis (BDD) is a severe infectious cause of lameness which has spread through dairy cattle populations worldwide, causing serious welfare and agricultural problems. Spirochetes are the main organisms implicated and have previously proven difficult to isolate. This study aimed to isolate and characterise the range of spirochetes associated with BDD in the UK. Twenty-three spirochete isolates were obtained from 30 BDD lesions, which by 16S rRNA gene and flaB2 gene analysis clustered within the genus Treponema as three phylogroups; groups 1 (Treponema medium/Treponema vincentii-like), 2 (Treponema phagedenis-like) and 3 (Treponema denticola/Treponema putidum-like). The treponemes displayed large genotypic and phenotypic diversity between phylogroups and differed from named treponeme species. A previously isolated contagious ovine digital dermatitis spirochete was located within one of the three phylogroups, group 3, and could also be identified within this group on the basis of phenotype testing, suggesting BDD and contagious ovine digital dermatitis may share the same aetiological agent. A strain isolated from a bovine interdigital dermatitis lesion, could be identified as part of BDD isolate group 2, suggesting bovine interdigital dermatitis and BDD may have the same causative agent. Two common enzyme activities, C4 esterase and C8 esterase lipase, were identified in all BDD associated treponemes suggesting common metabolic pathways for sharing this novel niche or even common virulence traits. Further studies are required to determine whether the three groups of novel treponemes are representative of new treponeme taxa and to delineate how they interact with bovine tissues to cause disease.     

Comparative evaluation of Vitek gram-positive identification system and API Rapid Strep system for identification of Streptococcus species of bovine origin

The Vitek Gram-positive identification system (GPI, Vitek Systems, Inc., Hazelwood, MO) and the API Rapid Strep system (Analytab Products, Plainview, NY) were evaluated for species identification of streptococci isolated from bovine mammary glands and compared to conventional biochemical methods. A total of 144 strains including Streptococcus uberis (60), S. dysgalactiae (32), S. agalactiae (15), S. bovis (15), Enterococcus faecium (10) and Ent. faecalis (12) were evaluated. All reference strains were identified correctly by both systems. Vitek GPI card system identified 94.4% of strains, including 95% of S. uberis, 93.8% of S. dygalactiae, 93.3% of S. agalactiae and S. bovis II, 90% of Ent. faecium and 100% of Ent. faecalis. Majority of strains were identified with a 90-99% level of confidence, with an average of 8 h needed for identification. The API Rapid Strep system identified 96.5% of strains correctly, including 95% of S. 96.9% of S. dysgalactiae, 93.3% of S. agalactiae, and 100% of S. bovis II, Ent. faecium, and Ent. faecalis. Majority of strains were identified with excellent level of identification. With the exception of S. uberis, most strains were identified at 4 h of incubation.