Patrícia Aparecida de Campos Braga

Bacterial identification: from the agar plate to the mass spectrometer

For more than a century, bacteria and fungi have been identified by isolation in culture followed by enzymatic reactions and morphological analyses. The identification of environmental microorganisms, however, remains a challenge because biochemical and staining protocols for bacteria identification are tedious, usually stepwise, can be long (days) and are prone to errors. Molecular techniques based on DNA amplification and/or sequencing provide more secure molecular identification of specific bacteria, but identification based on mass spectrometry (MS), mainly on MALDI-MS, has been shown to be an alternative accurate and fast method able to identify unknown bacteria on the genus, species and even subspecies level based profiles of proteins and peptides derived from whole bacterial cells. Breakthroughs such as non-culture-based identification of bacteria from biological fluids and MS detection of antibiotic resistance have recently been reported. This review provides an overview of the traditional bacterial and fungal identification workflow and discusses the recent introduction of MS as a powerful tool for the identification of microorganisms. Principles and applications of MS, followed by the use of high-quality databases with dedicated algorithms, are discussed for routine microbial diagnostics, mainly in human clinical settings and in veterinary medicine.

Identification of Coagulase-Negative Staphylococci from Bovine Intramammary Infection by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT Coagulase-negative staphylococci (CoNS) are among the main pathogens causing bovine intramammary infection (IMI) in many countries. However, one of the limitations related to the specific diagnosis of CoNS is the lack of an accurate, rapid, and convenient method that can differentiate the bacterial species comprising this group. The aim of this study was to evaluate the ability of matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) to accurately identify CoNS species in dairy cow IMI. In addition, the study aimed to determine the frequency of CoNS species causing bovine IMI. A total of 108 bacterial isolates were diagnosed as CoNS by microbiological cultures from two milk samples collected from 21 dairy herds; the first sample was collected at the cow level (i.e., 1,242 composite samples from all quarters), while the second sample was collected at the mammary quarter level (i.e., 1,140 mammary samples collected from 285 cows). After CoNS isolation was confirmed by microbiological culture for both samples, all CoNS isolates (n = 108) were genotypically differentiated by PCR restriction fragment length polymorphism (RFLP) analysis of a partial groEL gene sequence and subjected to the MALDI-TOF MS identification procedure. MALDI-TOF MS correctly identified 103 (95.4%) of the CoNS isolates identified by PCR-RFLP at the species level. Eleven CoNS species isolated from bovine IMI were identified by PCR-RFLP, and the most prevalent species was Staphylococcus chromogenes (n = 80; 74.1%). In conclusion, MALDI-TOF MS may be a reliable alternative method for differentiating CoNS species causing bovine IMI.

Identification of Corynebacterium spp. isolated from bovine intramammary infections by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Corynebacterium species (spp.) are among the most frequently isolated pathogens associated with subclinical mastitis in dairy cows. However, simple, fast, and reliable methods for the identification of species of the genus Corynebacterium are not currently available. This study aimed to evaluate the usefulness of matrix-assisted laser desorption ionization/mass spectrometry (MALDI-TOF MS) for identifying Corynebacterium spp. isolated from the mammary glands of dairy cows. Corynebacterium spp. were isolated from milk samples via microbiological culture (n=180) and were analyzed by MALDI-TOF MS and 16S rRNA gene sequencing. Using MALDI-TOF MS methodology, 161 Corynebacterium spp. isolates (89.4%) were correctly identified at the species level, whereas 12 isolates (6.7%) were identified at the genus level. Most isolates that were identified at the species level with 16 S rRNA gene sequencing were identified as Corynebacterium bovis (n=156; 86.7%) were also identified as C. bovis with MALDI-TOF MS. Five Corynebacterium spp. isolates (2.8%) were not correctly identified at the species level with MALDI-TOF MS and 2 isolates (1.1%) were considered unidentified because despite having MALDI-TOF MS scores >2, only the genus level was correctly identified. Therefore, MALDI-TOF MS could serve as an alternative method for species-level diagnoses of bovine intramammary infections caused by Corynebacterium spp.

Non-culture-based identification of mastitis-causing bacteria by MALDI-TOF mass spectrometry

The purpose of this study was to evaluate the detection limit of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for direct identification, without previous microbiological culture, of bovine mastitis-causing bacteria from milk samples. Milk samples (n = 15) were experimentally contaminated with Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae, and Escherichia coli to have bacterial counts ranging from 103 to 109 cfu/mL. These contaminated milk samples were subjected to a preparation protocol for bacterial ribosomal protein extraction using the MALDI Sepsityper kit (Bruker Daltonik, Bremen, Germany), which allowed MALDI-TOF MS coupled with Biotyper software (Bruker Daltonik) to identify bacterial fingerprints based on intact ribosomal proteins. The ability of MALDI-TOF MS to correctly identify bacterial strains from experimentally contaminated milk (without previous microbiological culture) depended on the bacterial count of the samples and on the species of the bacteria evaluated. Adequate identification at the bacterial species level (score ≥2.0) directly from milk samples required bacterial counts in the following ranges: ≥106 cfu/mL of Staph. aureus, ≥107 cfu/mL of E. coli, and ≥108 cfu/mL of Strep. agalactiae, Strep. dysgalactiae, and Strep. uberis. We concluded that direct identification of mastitis-causing pathogens is possible for Staph. aureus, E. coli, Strep. agalactiae, Strep. dysgalactiae, and Strep. uberis, but correct identification depended on the bacterial count in the milk samples.

Microorganisms in cryopreserved semen and culture media used in the in vitro production (IVP) of bovine embryos identified by matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS)

Commercial cattle breeders produce their own herd offspring for the dairy and beef market using artificial insemination. The procedure involves sanitary risks associated with the collection and commercialization of the germplasm, and the in vitro production and transfer of the bovine embryos must be monitored by strict health surveillance. To avoid the spreading of infectious diseases, one must rely on using controlled and monitored germplasm, media, and reagents that are guaranteed free of pathogens. In this article, we investigated the use of a new mass spectrometric approach for fast and accurate identification of bacteria and fungi in bovine semen and in culture media employed in the embryo in vitro production process. The microorganisms isolated from samples obtained in a commercial bovine embryo IVP setting were identified in a few minutes by their conserved peptide/protein profile, obtained applying matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS), matched against a commercial database. The successful microorganisms MS identification has been confirmed by DNA amplification and sequencing. Therefore, the MS technique seems to offer a powerful tool for rapid and accurate microorganism identification in semen and culture media samples.

Moxidectin residues in lamb tissues: Development and validation of analytical method by UHPLC-MS/MS

The development and validation of a throughput method for the quantitation of moxidectin residues in lamb target tissues (muscle, kidney, liver and fat) was conducted using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). To achieve higher recovery of the analyte from the matrices, a modified QuEChERS method was used for sample preparation. The chromatographic separation was achieved using a Zorbax Eclipse Plus C18 RRHD column with a mobile phase comprising 5mM ammonium formate solution +0.1% formic acid (A) and acetonitrile +0.1% formic acid (B) in a linear gradient program. Method validation was performed based on the Commission Decision 2002/657/EC and VICH GL49. To quantify the analyte, matrix-matched analytical curves were constructed with spiked blank tissues, with a limit of quantitation of 5ngg-1 and limit of detection of 1.5ngg-1 for all matrices. The linearity, decision limit, detection capability accuracy, and inter- and intra-day repeatability of the method are reported. The method was successfully applied to incurred lamb tissue samples (muscle, liver, kidney and fat) in a concentration range from 5 to 200μgkg-1, which demonstrated its suitability for monitoring moxidectin residues in lamb tissues in health surveillance programs, as well as for pharmacokinetics and residue depletion studies.

Evaluation of the leaching of florfenicol from coated medicated fish feed into water

Florfenicol is one of the most-used antimicrobial agents in global fish farming. Nevertheless, in most countries, its use is not conducted in accordance with good practices. The aim of this work was to evaluate the leaching of florfenicol from coated fish feed into the water. Analytical methods were developed and validated for the quantitation of florfenicol in medicated feed and water by UHPLC-MS/MS. Florfenicol residues in the water were quantified after 5- and 15-min exposures of the medicated feed in the water at 22 and 28 °C and at pH 4.5 and 8.0. The influence of pellet size and three coating agents (vegetable oil, carboxymethylcellulose, and low-methoxylated pectin) on the leaching of the drug was also assessed. Pellet size, coating agent, water temperature, and time of exposure significantly (p < 0.05) affected florfenicol leaching, while water pH did not interfere with the leaching. Coating with vegetable oil was the most efficient method to reduce florfenicol leaching, while coating with carboxymethylcellulose presented the highest leaching (approximately 60% after 15 min at 28 °C). Thus, the coating agent has a significant effect on the florfenicol leaching rate and, consequently, on the necessary dose of the drug to be administered. Moreover, it is worth mentioning that higher florfenicol leaching will pose a greater risk to environmental health, specifically in terms of the development of bacteria resistant to florfenicol. Additional studies are needed with other polymers and veterinary drugs used in medicated feed for fish farming.