Identification of bovine-associated coagulase-negative staphylococci by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a direct transfer protocol

Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry accurately identifies both selected bacteria and bacteria in select clinical situations. It has not been evaluated for routine use in the clinic. We prospectively analyzed routine MALDI-TOF mass spectrometry identification in parallel with conventional phenotypic identification of bacteria regardless of phylum or source of isolation. Discrepancies were resolved by 16S ribosomal RNA and rpoB gene sequence-based molecular identification. Colonies (4 spots per isolate directly deposited on the MALDI-TOF plate) were analyzed using an Autoflex II Bruker Daltonik mass spectrometer. Peptidic spectra were compared with the Bruker BioTyper database, version 2.0, and the identification score was noted. Delays and costs of identification were measured. Of 1660 bacterial isolates analyzed, 95.4% were correctly identified by MALDI-TOF mass spectrometry; 84.1% were identified at the species level, and 11.3% were identified at the genus level. In most cases, absence of identification (2.8% of isolates) and erroneous identification (1.7% of isolates) were due to improper database entries. Accurate MALDI-TOF mass spectrometry identification was significantly correlated with having 10 reference spectra in the database (P=.01). The mean time required for MALDI-TOF mass spectrometry identification of 1 isolate was 6 minutes for an estimated 22%-32% cost of current methods of identification. MALDI-TOF mass spectrometry is a cost-effective, accurate method for routine identification of bacterial isolates in or =10 reference spectra per bacterial species and a 1.9 identification score (Brucker system). It may replace Gram staining and biochemical identification in the near future.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been found to be an accurate, rapid, and inexpensive method for the identification of bacteria and yeasts. Previous evaluations have compared the accuracy, time to identification, and costs of the MALDI-TOF MS method against standard identification systems or commercial panels. In this prospective study, we compared a protocol incorporating MALDI-TOF MS (MALDI protocol) with the current standard identification protocols (standard protocol) to determine the performance in actual practice using a specimen-based, bench-by-bench approach. The potential impact on time to identification (TTI) and costs had MALDI-TOF MS been the first-line identification method was quantitated. The MALDI protocol includes supplementary tests, notably for Streptococcus pneumoniae and Shigella, and indications for repeat MALDI-TOF MS attempts, often not measured in previous studies. A total of 952 isolates (824 bacterial isolates and 128 yeast isolates) recovered from 2,214 specimens were assessed using the MALDI protocol. Compared with standard protocols, the MALDI protocol provided identifications 1.45 days earlier on average (P < 0.001). In our laboratory, we anticipate that the incorporation of the MALDI protocol can reduce reagent and labor costs of identification by $102,424 or 56.9% within 12 months. The model included the fixed annual costs of the MALDI-TOF MS, such as the cost of protein standards and instrument maintenance, and the annual prevalence of organisms encountered in our laboratory. This comprehensive cost analysis model can be generalized to other moderate- to high-volume laboratories.

A longitudinal study in 3 dairy herds was conducted to profile the distribution of coagulase-negative Staphylococcus (CNS) species causing bovine intramammary infection (IMI) using molecular identification and to gain more insight in the pathogenic potential of CNS as a group and of the most prevalent species causing IMI. Monthly milk samples from 25 cows in each herd as well as samples from clinical mastitis were collected over a 13-mo period. Coagulase-negative staphylococci were identified to the species level using transfer-RNA intergenic spacer PCR. The distribution of CNS causing IMI was highly herd-dependent, but overall, Staphylococcus chromogenes, Staphylococcus xylosus, Staphylococcus cohnii, and Staphylococcus simulans were the most prevalent. No CNS species were found to cause clinical mastitis. The effect of the most prevalent species on the quarter milk somatic cell count (SCC) was analyzed using a linear mixed model, showing that Staph. chromogenes, Staph. simulans, and Staph. xylosus induced an increase in the SCC that is comparable with that of Staphylococcus aureus. Almost all CNS species were able to cause persistent IMI, with Staph. chromogenes causing the most persistent infections. In conclusion, accurate species identification cannot be ignored when studying the effect of CNS on udder health, as the effect on SCC differs between species and species distribution is herd-specific. Staphylococcus chromogenes, Staph. simulans, and Staph. xylosus seem to be the more important species and deserve special attention in further studies. Reasons for herd dependency and possible cow- and quarter-level risk factors should be examined in detail for the different species, eventually leading to cost-benefit analyses for management changes and, if needed, treatment recommendations. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.