Genotypic Identification Methods Research Articles

Comparison of Phenotypic and Genotypic Identification Methods of Pasteurella multocida Serotypes Isolated from Pigs

Background: Pasteurella multocida serotypes A and D are commonly associated with pneumonia and pleuritis in pigs. Different phenotypic techniques, such as hyaluronidase and acriflavine tests, and genotyping techniques, such as PCR, are used to distinguish between these serotypes. The objective of this study was to compare the capsular identification methods of type A and type D P. multocida isolated from pigs using both phenotypic (hyaluronidase and acriflavine tests) and genotypic (multiplex PCR) techniques.Materials, Methods & Results: A total of 44 lyophilized P. multocida isolates, obtained between 1981 and 1997 from pig farms at Rio Grande do Sul State, Brazil, were analyzed. The isolates were reactivated in Brain Heart Infusion (BHI) broth and cultured in BHI broth and blood agar supplemented with 5% sheep blood. Colony identity was further confirmed by evaluating colony morphology in blood agar and confirming the absence of growth on MacConkey agar. Bacteria in Tryptone Soy Agar (TSA) were used for the Triple Sugar Iron (TSI), Sulfide-Indole-Motility (SIM), and nitrate, glucose, lactose, sucrose and mannitol fermentation tests. For hyaluronidase test, P. multocida colonies were streaked transversally across the entire plate, approximately 3-5mm apart, in order to observe their lines of growth. Following this, a hyaluronidase producing strain of Staphylococcus aureus was heavily streaked at right angles to the P. multocida lines and the plates were incubated at 37°C for 24 h. Type A isolates were then identified as those with smaller colonies in the region adjacent to the Staphylococcus aureus streak (negative satellitism). For acriflavine test, the isolates were inoculated into tubes containing 2 mL of BHI, incubated at 37°C for 18-24 h, centrifuged (500 g for 15 min) and 1.5 mL of the supernatant was discarded. A 1:1000 solution of acriflavine neutral (0.5 mL) was then added to the residual broth containing bacteria and kept at room temperature. Solutions of acriflavine were freshly prepared each week and stored protected from light. Type D strains were identified by the appearance of a heavy flocculent precipitate within 5 min. DNA extraction by heat shock was performed prior to multiplex PCR for the detection of capsular genes hyaD-hyaC (capsular typing A) and dcbF (capsular typing D). Test of symmetry and a weighted kappa coefficient were used to evaluate correlations and to assess agreement of the results between the identification methods, respectively. Phenotypic tests showed that two isolates were type D (4.55%), 40 were type A (90.9%) and two (4.55%) were untypable isolates (4.55%) while PCR showed that 38 isolates were type A (86.36%) and six were type D (13.64%). The correlation analysis between the phenotypic and genotypic tests showed that 90.9% of the strains were identified as belonging to the same serotype by both tests and the weighted kappa coefficient (K = 0.633) indicates a substantial agreement between the two tests.Discussion: There was a disagreement between the phenotypic and genotypic results in four of the isolates (9.09%). The phenotypically untypable isolates were classified as type D by multiplex PCR. Nonetheless, we conclude that PCR testing is a more reliable method to differentiate between P. multocida serotypes A and D.

Efficacy of Phenotypic, PCR and MALDI-ToF Identification Methods for Campylobacter spp.

This study compared phenotypic and genotypic identification methods of Campylobacter spp. against the polymerase chain reaction (PCR) in terms of sensitivity, specificity, positive-predictive value and negative-predictive value. Thermophilic Campylobacter isolates were identified using conventional biochemical tests, specifically hippurate hydrolysis, matrix assisted laser desorption ionization- time of flight (MALDI-ToF) mass spectrometry and PCR with primers unique to C. jejuni and C. coli. MALDI-ToF was shown to be superior to biochemical tests for identification of C. coli but equivalent to biochemical tests for C. jejuni .

A case of abscess after BCG vaccine in an immunocompetent child without other clinical signs

Introduction: Bacille Calmette–Guérin (BCG), an attenuated strain of Mycobacterium bovis, is a rare cause of infection, with few published cases in immunocompetent individuals. Case presentation: We present the case of a cutaneous abscess in an immunocompetent infant returning from Morocco, where he received a BCG vaccination. The abscess developed at the site of inoculation in the forearm (a non-recommended site) in the absence of lymphadenopathy or systemic signs. The lesion did not recur after aspiration of the abscess and further treatment was not required. Conclusion: Infections caused by M. bovis BCG may be difficult to diagnose without systemic signs or lymphadenopathy but should be suspected in children returning from regions where BCG vaccination is widely applied. The present report suggests that abscess formation after BCG vaccination is a continuing problem, particularly in tuberculosis-endemic areas and when recommendations concerning dosage or injection techniques are not followed. Moreover, we highlight here the importance of combining phenotypic and genotypic methods for quick identification of Mycobacterium bovis BCG in abscess drainage fluids.

Genotyping methods for monitoring the epidemic evolution of A. baumannii strains.

Acinetobacter baumannii is clustered with other phenotypically similar species into what has commonly become known as the ACB complex: A. calcoaceticus, A. pittii and, A. nosocomialis. The ecology and pathology of most of these species are not well understood, mainly because current specific phenotypic techniques have, to date, been insufficient. This has inhibited both the precise identification of, as well as the ability to discriminate between, these clinically important and closely related Acinetobacter strains.However, new genotypic methods have greatly enhanced our capacity to identify the ACB complex. This has resulted in the implementation of more rational infection control programs. Several genotypic identification methods are explored in this study, including non-polymerase chain reaction (PCR)-based and PCR-based methods. These methods include ribotyping, pulsed-field gel electrophoresis, 16S rRNA identification, multilocus sequence typing, single locus sequence typing, restriction fragment length polymorphism analysis, restriction analysis of 16S-23S rRNA intergenic spacer sequences, rapid amplification of polymorphic DNA, and repetitive extragenic palindromic PCR; however, there is no current single ideal genotyping method. Each one has its own advantages and disadvantages. With this in mind we reviewed current and new genotyping methods used to characterize the Acinetobacter species.

Isolation of Bordetella species from unusual infection sites

Introduction: Bordetella hinzii has been isolated mainly from respiratory specimens and from blood of immunocompromised patients, and Bordetella trematum from ear infections or leg, arm and ankle wounds and from diabetic foot ulcers. Bordetella holmesii is instead associated with bactaeremia in young adults, mostly with underlying conditions. Only three septic arthritis cases due to this species have been described in the literature. Case presentation: Herein we describe four cases of infections due to Bordetella species that have been recovered from unusual infection sites: two cases of B. hinzii infections, one recovered from the urine of a patient with chronic prostatitis and the other from a liver cyst in an immunocompetent patient; one B. trematum case from a bone biopsy of a patient with chronic osteomyelitis of the hip; and one B. holmesii case isolated from the joint fluid of an immunocompetent patient with diagnosed septic arthritis. The organisms were identified using standard biochemical tests, by API 20 NE version 6.0, by automated system VITEK 2, by mass spectrometry using the Bruker Daltonics MicroFlex LT spectrometer with MALDI Biotyper 3.1, and by PCR amplification of 16S rRNA. Antibiotic susceptibility testing was performed using the VITEK 2 system, except for B. holmesii, for which the epsilometric method (Etest technique; bioMérieux) was used. Conclusion: We highlight the importance of isolating Bordetella species from severe infections and unusual sites, and also of combining both phenotypic and genotypic methods for definitive identification.

Phenotypic and genotypic identification of lactic acid bacteria isolated from traditional pickles of the Çubuk region in Turkey.

A total of 152 lactic acid bacteria (LAB) were isolated from pickles produced in the Ankara-Çubuk region. These isolates were clustered into eight groups on the basis of their phenotypic characteristics including cell morphology, CO2 production from glucose, growth at 10 and 45 °C, growth in 6.5 % NaCl, and growth at pH 9.6. API 50 CH carbohydrate fermentation test, 16S ribosomal RNA (rRNA) sequence analysis, and sodium dodecyl sulfate-acrylamide gel electrophoresis (SDS-PAGE) whole-cell protein profile analysis were also performed for precise identification of the isolates at the species level. Molecular identification revealed that the most prevalent LAB species involved in pickle fermentation were Pediococcus ethanolidurans (46 isolates, 30.3 %), Lactobacillus brevis (37 isolates, 24.3 %), Lactobacillus plantarum (37 isolates, 24.3 %), and Lactobacillus buchneri (15 isolates, 9.9 %). Other LAB were found in minor frequencies such as Pediococcus parvulus (8 isolates, 5.3 %), Lactobacillus namurensis (6 isolates, 3.9 %), Lactobacillus diolivorans (1 isolate, 0.7 %), Lactobacillus parabrevis (1 isolate, 0.7 %), and Enterococcus casseliflavus (1 isolate, 0.7 %). When results of phenotypic and genotypic identification methods were compared, differences in the species distribution of LAB associated with pickles were defined between the API and the 16S rRNA sequencing. The API 50 CHL test coincided with the 16S rRNA results in 71 out of the 152 tested isolates, indicating that API gave unreliable identification results. A clear correlation could not be found between the results of whole-cell SDS profiles and 16S rRNA sequencing. Therefore, molecular characterization by 16S rRNA sequencing was considered to be the most reliable method for identifying isolates. The results presented in this work provide insight in to the LAB population associated with traditional Çubuk pickles and constitute a LAB strain resource for further studies involving the development of starter cultures.

Listeria booriae sp. nov. and Listeria newyorkensis sp. nov., from food processing environments in the USA.

Sampling of seafood and dairy processing facilities in the north-eastern USA produced 18 isolates of Listeria spp. that could not be identified at the species-level using traditional phenotypic and genotypic identification methods. Results of phenotypic and genotypic analyses suggested that the isolates represent two novel species with an average nucleotide blast identity of less than 92% with previously described species of the genus Listeria. Phylogenetic analyses based on whole genome sequences, 16S rRNA gene and sigB gene sequences confirmed that the isolates represented by type strain FSL M6-0635(T) and FSL A5-0209 cluster phylogenetically with Listeria cornellensis. Phylogenetic analyses also showed that the isolates represented by type strain FSL A5-0281(T) cluster phylogenetically with Listeria riparia. The name Listeria booriae sp. nov. is proposed for the species represented by type strain FSL A5-0281(T) ( =DSM 28860(T) =LMG 28311(T)), and the name Listeria newyorkensis sp. nov. is proposed for the species represented by type strain FSL M6-0635(T) ( =DSM 28861(T) =LMG 28310(T)). Phenotypic and genotypic analyses suggest that neither species is pathogenic.

CORRELATION BETWEEN PHENOTYPIC AND GENOTYPIC IDENTIFICATION OF TRICHOPHYTON VIOLACEUM

Background: Trichophyton violaceum is an anthropophilic dermatophyte. It is the most common etiologic agent causing dermatophytosis especially tinea capitis in Egypt. As routine phenotypic identification of dermatophytes is either slow or lack specificity, improved identification methods are required. The application of PCR has made rapid and precise identification of T. violaceum possible. Aim: This study was designed to correlate between phenotypic and genotypic identification of T. violaceum. Subjects: Two hundred patients with tinea capitis (122 males and 78 females) with a mean age of 8.5±3.1 were the subjects of this study. Methods: Phenotypic identification of T. violaceum determined by observation of its macroscopic and microscopic characteristics and culture on differential media was correlated with ITS-based PCR. Results: Genotypic identification results of T. violaceum in comparison with phenotypic identification results were identical. Conclusion: Although there is no difference between the results of phenotypic and genotypic identification methods of T. violaceum isolates, genotypic method is recommended as it is rapid and accurate. Keywords: Trichophyton violaceum, identification, phenotypic, dermatophytes, PCR and genotypic.

Characterization of Klebsiella pneumoniae Strains Isolated from Urinary Tract Infection: Detection of ESBL Characteristics, Antibiotic Susceptibility and RAPD Genotyping

In this study, a hundred Klebsiella pneumoniae strains isolated from urinary tract infections were evaluated in terms of genotyping, susceptibility to certain antibiotics and detection of extended spectrum of beta lactamase (ESBL) production. The random amplified polymorphic DNA (RAPD-PCR) method was used to identify the genetic differentiation of K. pneumoniae isolates. A total of 26 different DNA bands ranging between 334 bp and 28033 bp were detected among the strains. It was found that 100 K. pneumoniae strains revealed 11 different RAPD profiles. Antibiotic susceptibility tests were conducted using a disc diffusion method against 16 antibiotics. Fifty-five different resistance profiles were determined among the strains. ESBL-productions of the strains were determined by the double disc synergy test (DDST) and ESBL E-test methods. ESBL production rates among the strains were found to be 55% by E-test method and 45% by DDST method. While ESBL-producing K. pneumoniae strains showed the greatest resistance to penicillin G (100%), followed by piperacillin (92.7%) and erythromycin (85.4%),the resistance rates of non ESBLproducing strains to those antibiotics were determined as 97.8%, 88.8% and 88.8%, respectively. Both groups of strains showed the highest sensitivity to meropenem. Based on the results obtained from the study, it was concluded that the detection of ESBL-producing strains by the E-test method was more sensitive than by the DDST method. Phenotypic and genotypic identification methods should be used together to detect ESBL presence. The RAPD-PCR method alone will not be adequate in the genotyping of the strains and alternative DNA-based methods should be used.

Rapid and accurate identification of genomic species from the Acinetobacter baumannii (Ab) group by MALDI-TOF MS

The closely related members of the Acinetobacter baumannii (Ab) group (A. baumannii, A. pittii and A. nosocomialis) are difficult to identify with phenotypic tests in diagnostic laboratories. Genotypic identification methods require special skills and most do not provide rapid results. The aim of this study was to investigate the ability of MALDI-TOF MS to identify members of the Ab group. Sixty epidemiologically unrelated Acinetobacter spp. isolates were investigated by MALDI-TOF MS: 18 A. baumannii, 17 A. pittii, 18 A. nosocomialis and seven additional isolates representing other Acinetobacter spp. All strains were verified by ARDRA, rRNA intergenic spacer (ITS), recA sequencing and b/aOXA-51. MALDI-TOF MS correctly identified all the genomic strains but erroneously identified A. nosocomialis as A. baumannii because there was no reference strain within the Bruker database. Peak analysis of individual spectra from representative strains of each member of A. baumannii, A. pittii and A. nosocomialis suggested enough differences between their protein signatures to allow accurate identification using MALDI-TOF MS. Inclusion of specific signature profiles for A. nosocomialis within the Bruker database allowed the correct identification of this genomic species. MALDI-TOF MS spectra can be used as a fast, simple and reliable method to identify members of the Ab group. The rapid and accurate identification of clinically significant Acinetobacter strains will improve insight into their epidemiology and allow for targeted therapeutic and infection control measures against clinically important strains.

Optimization of Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry Analysis for Bacterial Identification

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is a relatively new addition to the clinical microbiology laboratory. The performance of the MALDI Biotyper system (Bruker Daltonics) was compared to those of phenotypic and genotypic identification methods for 690 routine and referred clinical isolates representing 102 genera and 225 unique species. We systematically compared direct-smear and extraction methods on a taxonomically diverse collection of isolates. The optimal score thresholds for bacterial identification were determined, and an approach to address multiple divergent results above these thresholds was evaluated. Analysis of identification scores revealed optimal species- and genus-level identification thresholds of 1.9 and 1.7, with 91.9% and 97.0% of isolates correctly identified to species and genus levels, respectively. Not surprisingly, routinely encountered isolates showed higher concordance than did uncommon isolates. The extraction method yielded higher scores than the direct-smear method for 78.3% of isolates. Incorrect species were reported in the top 10 results for 19.4% of isolates, and although there was no obvious cutoff to eliminate all of these ambiguities, a 10% score differential between the top match and additional species may be useful to limit the need for additional testing to reach single-species-level identifications.

Comparative Study Using Phenotypic, Genotypic, and Proteomics Methods for Identification of Coagulase-Negative Staphylococci

Five methods were compared to determine the most accurate method for identification of coagulase-negative staphylococci (CoNS) (n = 142 strains). Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) showed the best results for rapid and accurate CoNS differentiation (99.3% of strains correctly identified). An alternative to this approach could be Vitek2 combined with partial tuf gene sequencing (100% of strains correctly identified when both methods are performed simultaneously).

Evaluation of phenotypic with genotypic methods for species identification and detection of methicillin resistant in Staphylococcus aureus.

Background:Phenotypic methods for the detection of methicillin resistance are inadequate, due to presence of hetero-resistant population and dependence of environmental factors that may affect the phenotypic expression of resistance.Aims:Present study was conducted, to evaluate the efficacy of phenotypic methods for the identification of species and mec-A mediated resistance in S. aureus with polymerase chain reaction (PCR), and to assess the prevalence of the Panton-Valentine leukocidin (pvl) toxin in methicillin resistant S. aureus (MRSA) and overall S.aureus population.Materials and Methods:A total of 200 clinical isolates of Staphylococci were subjected to phenotypic and genotypic methods for the species identification and detection of MRSA.Results:The specificity and sensitivity of conventional methods in the detection of S.aureus, was found to be 100 and 97.59% respectively. However, the performance of phenotypic methods in the detection of MRSA were: Oxacillin disc diffusion (DD)-sensitivity 70.58%, specificity 75.75%; cefoxitin DD-sensitivity 86.27%, specificity 83.33%; and oxacillin agar dilution-sensitivity 92.15%, specificity 90.90%. PVL gene was detected in all mec-A positive isolates irrespective of their types.Conclusion:Phenotypic methods still preferred for the species identification, but for the reliable detection of MRSA an algorithm should include a combination of tests and apply a genotypic method for confirmation of resistance isolates showing discrepant results. Considering the high prevalence of PVL-MRSA, we recommend PCR as assay, as it has an advantage of simultaneous detection of mec-A and pvl genes by multiplex PCR.

Comparison of phenotypic methods and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry for the identification of aero-tolerant Actinomyces spp. isolated from soft-tissue infections

Aero-tolerant Actinomyces spp. are an under-recognised cause of cutaneous infections, in part because identification using conventional phenotypic methods is difficult and may be inaccurate. Matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) is a promising new technique for bacterial identification, but with limited data on the identification of aero-tolerant Actinomyces spp. This study evaluated the accuracy of a phenotypic biochemical kit, MALDI-TOF MS and genotypic identification methods for the identification of this problematic group of organisms. Thirty aero-tolerant Actinomyces spp. were isolated from soft-tissue infections over a 2-year period. Species identification was performed by 16 s rRNA sequencing and genotypic results were compared with results obtained by API Coryne and MALDI-TOF MS. There was poor agreement between API Coryne and genotypic identification, with only 33% of isolates correctly identified to the species level. MALDI-TOF MS correctly identified 97% of isolates to the species level, with 33% of identifications achieved with high confidence scores. MALDI-TOF MS is a promising new tool for the identification of aero-tolerant Actinomyces spp., but improvement of the database is required in order to increase the confidence level of identification.

Multilocus sequence typing reveals a novel subspeciation of Lactobacillus delbrueckii

Currently, the species Lactobacillus delbrueckii is divided into four subspecies, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. indicus and L. delbrueckii subsp. lactis. These classifications were based mainly on phenotypic identification methods and few studies have used genotypic identification methods. As a result, these subspecies have not yet been reliably delineated. In this study, the four subspecies of L. delbrueckii were discriminated by phenotype and by genotypic identification [amplified-fragment length polymorphism (AFLP) and multilocus sequence typing (MLST)] methods. The MLST method developed here was based on the analysis of seven housekeeping genes (fusA, gyrB, hsp60, ileS, pyrG, recA and recG). The MLST method had good discriminatory ability: the 41 strains of L. delbrueckii examined were divided into 34 sequence types, with 29 sequence types represented by only a single strain. The sequence types were divided into eight groups. These groups could be discriminated as representing different subspecies. The results of the AFLP and MLST analyses were consistent. The type strain of L. delbrueckii subsp. delbrueckii, YIT 0080(T), was clearly discriminated from the other strains currently classified as members of this subspecies, which were located close to strains of L. delbrueckii subsp. lactis. The MLST scheme developed in this study should be a useful tool for the identification of strains of L. delbrueckii to the subspecies level.

Streptococcus spp. and related bacteria: Their identification and their pathogenic potential for chronic mastitis – A molecular approach

Streptococcus spp. and related bacteria form a large group of organisms which are associated with bovine intramammary Infections (IMI). Some of them are the well-known mastitis pathogens Streptococcus uberis and Streptococcus agalactiae. In addition, there are a considerable number of these gram-positive, catalase-negative cocci (PNC) with unclear mastitic pathogenicity such as Aerococcus viridans which make the conventional diagnostics of PNC difficult. One diagnostic, API 20 Strep (API, Biomérieux) is recommended which, as a phenotypic assay, involves a series of miniaturized biochemical tests. Recently, preference is given to genotypic identification methods. In particular, sequencing of the 16S rRNA gene allows highly reproducible and accurate identification of bacteria and permits discovery of novel, clinically relevant bacteria. As a consequence, the aim of the present study was to compare identification of IMI-associated PNC by the API method as well as by sequencing of their 16S rRNA gene (16S). Furthermore, the correlation of these bacteria to bovine chronic mastitis and their phylogeny was investigated. 102 PNC isolated from single quarter milk samples were identified by API and 16S sequencing. Considering Streptococcus uberis, Streptococcus dysgalactiae subsp. dysgalactiae and Streptococcus agalactiae, both methods generated fully concordant results. In contrast, a very high disconcordance was observed for most of the other PNC, in particular Enterococcus spp., Aerococcus viridans and the viridans streptococci were shown as apathogenic. Lactococcus garvieae was found to be an opportunistic pathogen causing IMI during late lactation. In addition, PNC isolated from milk were frequently observed together with other bacteria, in particular with Staphylococcus spp. In these cases, the levels of somatic cell counts (SCC) were determined by the specific PNC present in the sample. Considering PNC phylogeny based on 16S sequencing, 3 major clusters were observed. They included all the common mastitis pathogens (cluster I), the Lactococcus spp., Enterococcus spp. and Aerococcus spp. (cluster II) and all the viridans streptococci (cluster III).

Prevalence and Biomolecular Characterization of Campylobacter spp. Isolated from Retail Meat

We estimated the prevalence of Campylobacter spp. in retail meat (n = 352 samples; 104 chicken, 106 pork, and 142 beef) collected in Campobasso, Italy, comparing two microbiological methods. All the isolates were characterized by biomolecular techniques for epidemiological purposes. Campylobacter isolation was performed by selective culture and membrane filtration methods. Phenotypic and genotypic methods for genus and species identification were evaluated together with antimicrobial resistance and plasmid profiling. Sixty-nine (86.2%) samples were positive by selective culture, 49 (61.2%) by membrane filtration, and 38 (47.5%) by both methods. Only 74 of 80 strains were confirmed as Campylobacter spp. by PCR, and two Campylobacter coli were identified as Campylobacter jejuni. Chicken meat was more frequently contaminated than other meats. Selective culture was more sensitive than membrane filtration (85 versus 66%), and specificity of the methods was 98 and 100%, respectively. Among Campylobacter isolates from chicken meat, 86.5% were multidrug resistant. Resistance to ciprofloxacin (51.3%) and enrofloxacin (52.7%) was lower than to nalidixic acid (71.6%). C. coli strains showed the highest cross-resistance for quinolones (82.6%) and fluoroquinolones (60.9%) as well as a high resistance to tetracycline. Plasmids were isolated from six C. coli and two C. jejuni isolates, but no association was detected between antimicrobial resistance and plasmid DNA carriage. Selective culture is considered as the optimal method for Campylobacter isolation, although it was unable to detect all contaminated samples. Membrane filtration provided more specific results but with low sensitivity. A combination of both techniques may offer better results.

Identification of Staphylococcus spp. by PCR-Restriction Fragment Length Polymorphism Analysis of dnaJ Gene

A PCR-restriction fragment length polymorphism (RFLP) analysis method that analyzes a part of the dnaJ gene was designed for the rapid and accurate identification of Staphylococcus spp. XapI or Bsp143I digestion of the PCR-generated products rendered distinctive RFLP patterns that allowed 41 reference species and subspecies to be identified with a high degree of specificity. The novel method was validated by the identification of 23 clinical staphylococcal strains, and the results were compared with those obtained by other genotypic identification methods. A 100% concordance of the results was shown. Therefore, PCR-RFLP analysis of the dnaJ gene is proposed as a reliable and reproducible method for the identification of Staphylococcus spp.

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.