Description of Corynebacterium hiratae sp. nov. isolated from a human tissue bone a novel member of Corynebacterium Genus.

BACKGROUND: Corynebacterium spp. are widely disseminated in the environment, and they are part of the skin and mucosal microbiota of animals and humans. Reports of human infections by Corynebacterium spp. have increased considerably in recent years and the appearance of multidrug resistant isolates around the world has drawn attention. OBJECTIVES: To describe a new species of Corynebacterium from human tissue bone is described after being misidentified using available methods. METHODS: For taxonomic analyses, phylogenetic analysis of 16S rRNA and rpoB genes, in silico DNA-DNA hybridization, average nucleotide and amino acid identity, multilocus sequence analysis, and phylogenetic analysis based on the complete genome were used. FINDINGS: Genomic taxonomic analyzes revealed values of in silico DNA-DNA hybridization, average nucleotide and amino acids identity below the values necessary for species characterization between the analyzed isolates and the closest phylogenetic relative Corynebacterium aurimucosum DSM 44532T. MAIN CONCLUSIONS: Genomic taxonomic analyzes indicate that the isolates analyzed comprise a new species of the Corynebacterium genus, which we propose to name Corynebacterium hiratae sp. nov. with isolate 332T (= CBAS 826T = CCBH 35,014T) as the type strain.

Comparison of two MALDI-TOF MS systems for the identification of clinically relevant anaerobic bacteria in Argentina.

The aim of this study was to compare the performance of two MALDI-TOF MS systems in the identification of clinically relevant strict anaerobic bacteria. The 16S rRNA gene sequencing was the gold standard method when discrepancies or inconsistencies were observed between platforms. A total of 333 isolates were recovered from clinical samples of different centers in Buenos Aires City between 2016 and 2021. The isolates were identified in duplicate using two MALDI-TOF MS systems, BD Bruker Biotyper (Bruker Daltonics, Bremen, Germany) and Vitek MS (bioMèrieux, Marcy-l'Etoile, France). Using the Vitek MS system, the identification of anaerobic isolates yielded the following percentages: 65.5% (n: 218) at the species or species-complex level, 71.2% (n: 237) at the genus level, 29.4% (n: 98) with no identification and 5.1% (n: 17) with misidentification. Using the Bruker Biotyper system, the identification rates were as follows: 85.3% (n: 284) at the species or species-complex level, 89.7% (n: 299) at the genus level, 14.1% (n: 47) with no identification and 0.6% (n: 2) with misidentification. Differences in the performance of both methods were statistically significant (p-values <0.0001). In conclusion, MALDI-TOF MS systems speed up microbial identification and are particularly effective for slow-growing microorganisms, such as anaerobic bacteria, which are difficult to identify by traditional methods. In this study, the Bruker system showed greater accuracy than the Vitek system. In order to be truly effective, it is essential to update the databases of both systems by increasing the number of each main spectrum profile within the platforms.

Actinomyces europaeus (Gleimia europaea) asociado con absceso cerebral: comunicación de tres casos / Actinomyces europaeus (Gleimia europaea) associated with brain abscess: A reportof three cases

Resumen El absceso cerebral es una infección focal caracterizada por acumulación de pus enel parénquima cerebral; su diagnóstico es de urgencia debido a la alta mortalidad que acarrea.Presentamos tres casos de pacientes con abscesos cerebrales con foco otogénico de origen poli-microbiano, que presentaron en común el aislamiento de Actinomyces europaeus, agente nodescrito hasta el momento en esta localización. A. europaeus fue identificado por la metodo-logía convencional, por espectrometría de masas por desorción/ionización asistida por matriz(MALDI-TOF MS) y por secuenciación del gen ARNr 16S. La sensibilidad antibiótica se evaluó porel método epsilométrico. Todos los aislados presentaron sensibilidad a penicilina, vancomicinay linezolid, mientras que la sensibilidad a clindamicina y eritromicina fue variable. La iden-tificación por MALDI-TOF MS permitió arribar a nivel de especie de forma rápida y confiabley dar una respuesta oportuna y efectiva, evitando el retraso en el tratamiento, lo que sueleincrementar la morbimortalidad del cuadro clínico.

Interspecies interactions in mixed-species biofilms formed by Enterococcus faecalis and gram-negative bacteria isolated from polymicrobial diabetic foot ulcers.

Diabetic foot ulcers (DFU) are exacerbated by bacterial colonisation. Here, a high prevalence of Enterococcus faecalis was observed in DFU patients from an Argentinean hospital. E. faecalis was frequently co-isolated with Escherichia coli, Morganella morganii, and Pseudomonas aeruginosa. The effect of interspecies interactions on bacterial growth was investigated in mixed-species macrocolony biofilms developed in Lubbock-Glc-agar. Similar cell counts were found for E. faecalis and M. morganii growing in mixed and single-species biofilms. An E. faecalis strain showed 1 Log higher cell counts in mixed biofilms with E. coli. Remarkably, E. faecalis strains showed 2 to 4 Log higher cell counts in mixed biofilms with P. aeruginosa. This effect was not observed in planktonic growth or biofilms developed in tryptic soy agar. The present findings reveal bacterial interactions that benefit E. faecalis in mixed-species biofilms, mainly with P. aeruginosa, in a medium that partially mimics the nutrients found in DFU.

Corynebacterium guaraldiae sp. nov.: a new species of Corynebacterium from human infections.

Non-diphtheria Corynebacterium species (NDC) belonging to the human skin and mucosa microbiota are frequently neglected as contaminants. However, reports of human infections by Corynebacterium spp. have increased considerably in recent years. In this study, a group of six NDC isolates of urine (n = 5) and sebaceous cyst (n = 1) from two South American countries were identified at genus level or misidentified based on API® Coryne and genetic/molecular analyses. The 16S rRNA (99.09-99.56%) and rpoB (96.18-97.14%) gene sequence similarities of the isolates were higher when compared with Corynebacterium aurimucosum DSM 44532 T. Multilocus sequence analysis (MLSA) indicated that these six NDC isolates compose a distinctive phylogenetic clade. Genome-based taxonomic analysis with the whole-genome sequences was able to separate these six isolates from other known Corynebacterium type strains. Average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridization (dDDH) values between closely related type strains and the six isolates were considerably lower than the currently recommended threshold values for species circumscription. Phylogenetic and genomic taxonomy analyses indicated these microorganisms as a novel Corynebacterium species, for which we formally propose the name Corynebacterium guaraldiae sp. nov. with isolate 13T (= CBAS 827T = CCBH 35012T) as type strain.

[Actinomyces europaeus (Gleimia europaea) associated with brain abscess: A report of three cases]. / Actinomyces europaeus (Gleimia europaea) asociado con absceso cerebral: comunicación de tres casos.

A brain abscess is a focal infection characterized by a collection of pus in the brain parenchyma. It is a life-threatening condition that should be diagnosed and treated as soon as possible. We report here three cases of patients with otogenic brain abscesses of polymicrobial origin that had in common the isolation of Actinomyces europaeus, which has not been previously described in this location. A. europaeus was identified by the conventional methodology, matrix-associated laser deionization-time of flight mass spectrometry (MALDI-TOF MS) and 16S rRNA gene sequencing. Antibiotic susceptibility was evaluated by the epsilometric method, and all isolates showed sensitivity to penicillin, vancomycin and linezolid, whereas susceptibility to clindamycin and erythromycin was variable. MALDI-TOF MS identification allowed a quick and reliable species level identification in order to provide a rapid and effective response to avoid treatment delay that could lead to increased morbidity and even mortality.

[Vibrio vulnificus infection of the skin and soft parts. Communication of a case diagnosed in Argentina]. / Infección de piel y partes blandas por Vibrio vulnificus. Comunicación de un caso diagnosticado en Argentina.

Vibrio vulnificus is one of the most virulent Vibrio species known. It is a bacterium with universal distribution. The first case registered in Uruguay occurred in 2001 and, since then, several infections have occurred per year. Recently, in this country, V. vulnificus was responsible for a fatal soft tissue infection. Although no cases of human infection with this species have been reported in Argentina, researchers have recently identified V. vulnificus in samples associated with microplankton in the Rio Negro estuary. We present the case of a severe skin and soft tissue infection by V. vulnificus from an open wound in a patient in contact with a marine aquatic environment on the coast of the River Plate, in Uruguay. Isolation of vibrios from wound specimens can cause rapidly progressing tissue damage, particularly V. vulnificus which has a high mortality rate without early and appropriate intervention. In our case, the rapid identification of the microorganism allowed us to support the empirical treatment used, which a good clinical evolution.

Vibrio vulnificus es una de las especies de Vibrio más virulentas que se conocen. Es una bacteria de distribución universal. El primer caso registrado en Uruguay se produjo en 2001, y desde entonces ocurren varias infecciones por año. Recientemente, en ese país, V. vulnificus fue responsable de una infección de partes blandas de curso letal. Aunque no han sido comunicados casos de infección humana por esta especie en Argentina, se ha identificado recientemente Vibrio vulnificus en muestras asociadas con microplancton en el estuario del Río Negro. Presentamos el caso de una infección grave de piel y partes blandas por V. vulnificus a partir de una herida abierta en un paciente en contacto con medio acuático marino en la costa de Uruguay del Río de la Plata. El aislamiento de vibrios en muestras de heridas puede causar un daño en los tejidos con rápida progresión, en particular V. vulnificus, que tiene una alta mortalidad sin la precoz y apropiada intervención. En nuestro caso, la rápida identificación del microorganismo permitió avalar el tratamiento empírico utilizado, con una buena evolución clínica.

Empiema necessitatis por Campylobacter rectus. Identificación rápida por MALDI-TOF MS / Empyema necessitatis caused by Campylobacter rectus. Rapid identification by MALDI-TOF MS

Resumen El empiema necessitatis (EN) constituye una muy rara complicación de un empiema pleural en el cual la infección se extiende a los tejidos blandos adyacentes. La etiología por anaerobios es muy infrecuente y se da en el curso de infecciones crónicas. Se presenta el primer caso de empiema necessitatis por Campylobacter rectus. La identificación de este agente se efectuó por espectrometría de masas (MALDI-TOF MS) y su sensibilidad antimicrobiana se determinó por el método epsilométrico.

Abstract Empyema necessitatis (EN) is a very rare complication of a pleural empyema, in which the infection extends to adjacent soft tissues. Anaerobic bacteria are very rare etiologic agents of EN, which occurs in the course of chronic infections. We present the first case of empyema necessitatis caused by Campylobacter rectus. Bacterial identification was carried out by mass spectrometry (MALDI-TOF MS) and antimicrobial susceptibility was determined by the epsilometer method.

Infección de piel y partes blandas por Vibrio vulnificus. Comunicación de un caso diagnosticado en Argentina / Vibrio vulnificus infection of the skin and soft parts. Communication of a case diagnosed in Argentina

Resumen Vibrio vulnificus es una de las especies de Vibrio más virulentas que se conocen. Es una bacteria de distribución universal. El primer caso registrado en Uruguay se produjo en 2001, y desde entonces ocurren varias infecciones por año. Recientemente, en ese país, V. vulnificus fue responsable de una infección de partes blandas de curso letal. Aunque no han sido comunicados casos de infección humana por esta especie en Argentina, se ha identificado recientemente Vibrio vulnificus en muestras asociadas con microplancton en el estuario del Río Negro. Presentamos el caso de una infección grave de piel y partes blandas por V. vulnificus a partir de una herida abierta en un paciente en contacto con medio acuático marino en la costa de Uruguay del Río de la Plata. El aislamiento de vibrios en muestras de heridas puede causar un daño en los tejidos con rápida progresión, en particular V. vulnificus, que tiene una alta mortalidad sin la precoz y apropiada intervención. En nuestro caso, la rápida identificación del microorganismo permitió avalar el tratamiento empírico utilizado, con una buena evolución clínica.

Abstract Vibrio vulnificus is one of the most virulent Vibrio species known. It is a bacterium with universal distribution. The first case registered in Uruguay occurred in 2001 and, since then, several infections have occurred per year. Recently, in this country, V. vulnificus was responsible for a fatal soft tissue infection. Although no cases of human infection with this species have been reported in Argentina, researchers have recently identified V. vulnificus in samples associated with microplankton in the Rio Negro estuary. We present the case of a severe skin and soft tissue infection by V. vulnificus from an open wound in a patient in contact with a marine aquatic environment on the coast of the River Plate, in Uruguay. Isolation of vibrios from wound specimens can cause rapidly progressing tissue damage, particularly V. vulnificus which has a high mortality rate without early and appropriate intervention. In our case, the rapid identification of the microorganism allowed us to support the empirical treatment used, which a good clinical evolution.

Dolosigranulum pigrum en absceso corneal / Dolosigranulum pigrum in corneal abscess

Resumen Dolosigranulum pigrum es un coco gram positivo, anaerobio facultativo, que forma parte de la microbiota oral y del tracto respiratorio superior. Aunque los reportes de infecciones por este microorganismo son escasos, se lo ha asociado a un amplio espectro de enfermedades infecciosas. Se describe el caso de un hombre adulto con un absceso corneal del que se aisló D. pigrum. El microorganismo fue identificado por espectrometría de masas (MALDI-TOF MS) y secuenciación del gen 16S ARNr. A su vez, se logró la identificación presuntiva mediante pruebas fenotípicas claves, como la disposición en racimos en la coloración de Gram, la prueba negativa de la catalasa, la producción de pirrolidonil arilamidasa y leucina aminopeptidasa, el crecimiento en NaCl al 6,5% y la hidrólisis de esculina. Los datos de la literatura y el presente caso respaldan la asociación del microorganismo con infecciones oculares, a menudo de curso destructivo, principalmente en pacientes de edad avanzada.

Abstract Dolosigranulum pigrum is a gram-positive, facultatively anaerobic coccus, which is part of the oral and upper respiratory tract microbiota. Although reports of infections by this microorganism are scarce, it has been associated with a wide spectrum of infectious diseases. The case of an elderly man with a lower corneal abscess, in which Dolosigranulum pigrum was isolated, is described. The microorganism was identified by mass spectrometry (MALDI-TOF MS) and by the sequencingof the 16S rRNAgene. Furthermore, the presumptive identification of the causative agent was achieved by using key phenotypic tests such as the cluster arrangement in Gram stain, the negative catalase test, the production of pyrrolidonyl arylamidase and leucine aminopeptidase activity, the growth in 6.5% NaCl and esculin hydrolysis. The data from the literature (and the present case) support the association of the microorganism with ocular infections, which often take a destructive course, mainly in elderly patients.

[Empyema necessitatis caused by Campylobacterrectus. Rapid identification by MALDI-TOF MS]. / Empiema necessitatis por Campylobacter rectus. Identificación rápida por MALDI-TOF MS.

Empyema necessitatis (EN) is a very rare complication of a pleural empyema, in which the infection extends to adjacent soft tissues. Anaerobic bacteria are very rare etiologic agents of EN, which occurs in the course of chronic infections. We present the first case of empyema necessitatis caused by Campylobacter rectus. Bacterial identification was carried out by mass spectrometry (MALDI-TOF MS) and antimicrobial susceptibility was determined by the epsilometer method.

The dilemma of identifying Peptoniphilus species by using two MALDI-TOF MS systems.

Two commercial MALDI-TOF MS systems were used to identify 18 isolates, belonging to the Peptoniphilus genus; also the 16S rRNA sequencing identity was compared against the MALDI-TOF MS system results. Bruker Biotyper system provided higher accuracy than Vitek MS system, however, adding spectra could allow a more reliable species level identification.

[Dolosigranulum pigrum in corneal abscess]. / Dolosigranulum pigrum en absceso corneal.

Dolosigranulum pigrum is a gram-positive, facultatively anaerobic coccus, which is part of the oral and upper respiratory tract microbiota. Although reports of infections by this microorganism are scarce, it has been associated with a wide spectrum of infectious diseases. The case of an elderly man with a lower corneal abscess, in which Dolosigranulum pigrum was isolated, is described. The microorganism was identified by mass spectrometry (MALDI-TOF MS) and by the sequencing of the 16S rRNA gene. Furthermore, the presumptive identification of the causative agent was achieved by using key phenotypic tests such as the cluster arrangement in Gram stain, the negative catalase test, the production of pyrrolidonyl arylamidase and leucine aminopeptidase activity, the growth in 6.5% NaCl and esculin hydrolysis. The data from the literature (and the present case) support the association of the microorganism with ocular infections, which often take a destructive course, mainly in elderly patients.

Infections due to Vagococcus spp. Microbiological and clinical aspects and literature review.

INTRODUCTION: An increase in recent years in the isolation of Vagococcus spp. is suggestive of emerging infection by this pathogen in our hospital. METHODS: Prospective, descriptive study. PERIOD: July 2014-January 2019. Phenotypic identification of 15 isolates of Vagococcus spp. was performed by conventional biochemical tests, automated methodology and mass spectrometry (MALDI-TOF MS). Molecular identification was achieved by sequencing the 16S rRNA gene. The Vitek™ 2C automated system was used to test antibiotic susceptibility. RESULTS: The molecular method identified 11 Vagococcus fluvialis, one Vagococcus lutrae and three Vagococcus spp. MALDI-TOF MS facilitated the rapid recognition of the genus. The most active antibiotics were ampicillin, trimethoprim/sulfamethoxazole, vancomycin, teicoplanin and linezolid. Most of the cases of isolation were associated with skin and soft tissue or osteoarticular infections in patients with diabetes. CONCLUSION: This article is the most extensive review of cases of Vagococcus spp. infection reported in the literature and highlights the microbiological and clinical aspects of this pathogen.

Infecciones por Vagococcus spp. Aspectos microbiológicos y clínicos y revisión de la literatura / Infections due to Vagococcus spp. Microbiological and clinical aspects and literature review

Introducción: En los últimos años un aumento en la frecuencia de aislamiento de Vagococcus spp. estaría indicando la urgencia de la infección por este patógeno en nuestra institución. Métodos: Estudio prospectivo y descriptivo que abarca el periodo de julio de 2014 a enero de 2019. La identificación fenotípica de 15 aislados de Vagococcus spp. se realizó por pruebas bioquímicas convencionales, por metodología automatizada y por espectrometría de masas (MALDI-TOF MS). La identificación molecular por secuenciación del gen ARNr 16S. La sensibilidad antibiótica fue realizada utilizando el sistema automatizado Vitek® 2C. Resultados: Se identificaron 11 Vagococcus fluvialis, un Vagococcus lutrae y tres Vagococcus spp. por metodología molecular. MALDI-TOF MS permitió el rápido reconocimiento de este género. Los antibióticos más activos fueron ampicilina, trimetoprima/sulfametoxazol, vancomicina, teicoplanina y linezolid. La mayoría de los aislamientos se asociaron con infecciones en la piel y partes blandas u osteoarticulares en pacientes diabéticos. Conclusión: Esta comunicación representa la mayor revisión de casos de infecciones por Vagococcus spp. reportados en la literatura, en la que se destacan los aspectos microbiológicos y clínicos de este patógeno.(AU)

Introduction: An increase in recent years in the isolation of Vagococcus spp. is suggestive of emerging infection by this pathogen in our hospital. Methods: Prospective, descriptive study. Period: July 2014-January 2019. Phenotypic identification of 15 isolates of Vagococcus spp. was performed by conventional biochemical tests, automated methodology and mass spectrometry (MALDI-TOF MS). Molecular identification was achieved by sequencing the 16S rRNA gene. The Vitek™ 2C automated system was used to test antibiotic susceptibility. Results: The molecular method identified 11 Vagococcus fluvialis, one Vagococcus lutrae and three Vagococcus spp. MALDI-TOF MS facilitated the rapid recognition of the genus. The most active antibiotics were ampicillin, trimethoprim/sulfamethoxazole, vancomycin, teicoplanin and linezolid. Most of the cases of isolation were associated with skin and soft tissue or osteoarticular infections in patients with diabetes. Conclusion: This article is the most extensive review of cases of Vagococcus spp. infection reported in the literature and highlights the microbiological and clinical aspects of this pathogen.(AU)

Corynebacterium phoceense - a rare Corynebacterium species isolated from a urine sample.

Corynebacterium spp. are Gram-positive rods that are recognized to cause opportunistic diseases under certain predisposing clinical conditions. Some species have been described in urinary tract infections. In this report we document a new episode of urinary tract infection caused by Corynebacterium phoceense and describe the whole-genome sequencing, phenotypic characteristics and mass spectra obtained by matrix-assisted desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Based on genome identification and DNA-to-DNA hybridization, we can assume that our strain is the second isolate of C. phoceense to be described in a urine sample. No other infectious diseases have been reported to be associated with this species.

Role of Tonsillar Chronic Inflammation and Commensal Bacteria in the Pathogenesis of Pediatric OSA.

Immune responses at the boundary between the host and the world beyond are complex and mucosal tissue homeostasis relies on them. Obstructive sleep apnea (OSA) is a syndrome suffered by children with hypertrophied tonsils. We have previously demonstrated that these tonsils present a defective regulatory B cell (Breg) compartment. Here, we extend those findings by uncovering the crucial role of resident pro-inflammatory B and T cells in sustaining tonsillar hypertrophy and hyperplasia by producing TNFα and IL17, respectively, in ex vivo cultures. Additionally, we detected prominent levels of expression of CD1d by tonsillar stratified as well as reticular epithelium, which have not previously been reported. Furthermore, we evidenced the hypertrophy of germinal centers (GC) and the general hyperplasia of B lymphocytes within the tissue and the lumen of the crypts. Of note, such B cells resulted mainly (IgG/IgM)+ cells, with some IgA+ cells located marginally in the follicles. Finally, by combining bacterial culture from the tonsillar core and subsequent identification of the respective isolates, we determined the most prevalent species within the cohort of OSA patients. Although the isolated species are considered normal oropharyngeal commensals in children, we confirmed their capacity to breach the epithelial barrier. Our work sheds light on the pathological mechanism underlying OSA, highlighting the relevance taken by the host immune system when defining infection versus colonization, and opening alternatives of treatment.

[Corynebacterium kroppenstedtii breast infections: Report of four cases]. / Infecciones mamarias por Corynebacterium kroppenstedtii: comunicación de 4 casos.

Corynebacterium kroppenstedtii is an immobile, non-sporulated, glucose-fermenting and lipophilic gram-positive rod of the skin microbiota. In recent years, numerous isolates of this species have been reported mainly in breast infections, such as abscesses and granulomatous mastitis. We present here four cases of C. kroppenstedtii infections isolated from breast aspiration samples in women. C. kroppenstedtii was identified by conventional methodology and mass spectrometry (MALDI-TOF MS). Using the epsilometric method, these isolates showed susceptibility to penicillin, ceftriaxone, minocycline, ciprofloxacin, and vancomycin, and variable susceptibility to clindamycin and trimethoprim sulfamethoxazole. Due to the association of C. kroppenstedtii with mammary infections, the identification at the species level of those corynebacteria isolated from this location is highly advisable in order to reach the final diagnosis and to test the antimicrobial susceptibility in order to apply the appropriate antibiotic treatment.

[Analysis of the diversity of Actinomyces/Actinotignum clinical isolates in a university hospital]. / Análisis de la diversidad de aislados clínicos de Actinomyces/Actinotignum en un hospital universitario.

Actinomyces and related genera are grampositive bacilli, opportunistic pathogens, which have been mainly involved in endogenous infections. However, due to the complexity in identifying them for most clinical laboratories, there is scant knowledge about their real clinical significance. In this work, 166 isolates of 13 different species of Actinomyces/Actinotignum species recovered from clinical samples of patients treated in a university hospital were studied. The identification was performed by MALDI-TOF MS and molecular identification. MALDI-TOF MS identified 91.57% of the isolates (152/166) at the species level using a score ≥ 1.7 and 3.61% (6/166) of the isolates were identified only at the gender level with a score ≥ 1.5. MALDI-TOF MS did not yield reliable identification results for 4.82% (8/166) of the isolates. Actinomyces/Actinotignum species were isolated from: soft tissue (n: 47), urine samples (n: 35), head / neck abscesses (n: 19), genital abscesses (n: 11), blood samples (n: 10), breast abscesses (n: 8), osteoarticular samples (n: 6), abdominal/ascitic fluids (n: 3), abdominal abscesses (n: 5), sputum/BAL (n: 4), brain abscesses (n: 3), and others (n: 15). The results obtained from the statistical analysis showed a high differential frequency (> 2) for the location/species association: urine/A. schaalii/sanguinis; brain abscesses/A. europaeus; osteoarticular samples/A. urogenitalis; abdominal abscesses/ A. turicensis; respiratory samples/A. naeslundii/viscosus. This information provides a greater understanding of the clinical and epidemiological relevance of these species. The pathogenic role of Actinomyces spp. will be increasingly revealed as these microorganisms could be recognized thanks to prolonged culture and the advances in identification technology facilitated by MALDI-TOF MS.

Differentiation of non-aureus staphylococci species isolated from bovine mastitis by PCR-RFLP of groEL and gap genes in comparison to MALDI-TOF mass spectrometry.

Intramammary infections (IMI) cause serious economic losses for farmers and the dairy industry. Cases of subclinical mastitis are commonly the result of infection by minor pathogens such as non-aureus staphylococci (NAS), so their correct identification is important for appropriate therapeutic intervention and management. The aim of this study was to assess the reliability of PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) of the groEL and gap genes to discriminate between bovine-associated NAS species, using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) as the reference method. MALDI-TOF MS was able to correctly identify 112 NAS isolates from bovine IMI at species level out of a total of 115 (97.4%). These results were considered definitive and thus compared with those from the PCR-RFLP analyses. Only 50% (56/112) of the samples classified through groEL PCR-RFLP matched the molecular identity determined by MALDI-TOF MS, whereas coincidence rose to 96.4% (108/112) when comparing results from gap PCR-RFLP and the spectral analysis. This study demonstrates that gap PCR-RFLP is a useful and reliable tool for the identification of NAS species isolated from bovine mastitis.