A New Filter Based Cultivation Approach for Improving Aspergillus Identification using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) is widely used in clinical laboratories for routine identification of bacteria and yeasts. However, methodological difficulties are still apparent when applied to filamentous fungi. The liquid cultivation method recommended by Bruker Daltonics GmbH for identification of filamentous fungi by MALDI-TOF MS is labour intensive and time-consuming. In this study, growth of Aspergillus species on different (porous) surfaces was investigated with the aim to develop a more reliable, quicker and less laborious identification method using MALDI-TOF MS. Mycelial growth without sporulation mimicking liquid cultivation and reliable MALDI-TOF MS spectra were obtained when A. fumigatus strains were grown on and in between a polycarbonate membrane filter on Sabouraud dextrose agar. A database of in-house reference spectra was created by growing Aspergillus reference strains (mainly focusing on sections Fumigati and Flavi) under these selected conditions. A test set of 50 molecularly identified strains grown under different conditions was used to select the best growth condition for identification and to perform an initial validation of the in-house database. Based on these results, the cultivation method on top of a polycarbonate filter proved to be most successful for species identification. This method was therefore selected for the identification of two sets of clinical isolates that mainly consisted of Aspergilli (100 strains originating from Indonesia, 70 isolates from Qatar). The results showed that this cultivation method is reliable for identification of clinically relevant Aspergillus species, with 67% and 76% correct identification of strains from Indonesia and Qatar, respectively. In conclusion, cultivation of Aspergilli on top of a polycarbonate filter showed improved results compared to the liquid cultivation protocol recommended by Bruker in terms of percentage of correct identification, ease of MSP creation, time consumption, cost and labour intensity. This method can be reliably applied for identification of clinically important Aspergilli and has potential for identification of other filamentous fungi.

Antibiotic Resistance Patterns in Cervical Microbes of Gilts and Sows

Extenders for boar semen contain antibiotics, which may induce antimicrobial resistance (AMR) in inseminated females. The objective was to investigate AMR of bacteria isolated from the cervix of sows and gilts in standing heat, representing females previously exposed to antibiotics in the semen extender and non-exposed females, respectively. Cervical swabs were taken from 30 multiparous sows and 30 gilts prior to their first insemination. After culturing on agar plates, bacterial isolates were identified by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry and antimicrobial minimum inhibitory concentrations (MIC) were determined. Differences in antibiotic resistance between sows and gilts were analyzed by Chi-squared or Fisher’s exact test. Bacteria isolated were mostly Staphylococcus spp., Streptococcus spp. and Corynebacterium spp. Higher MICs were observed for isolates from sows than from gilts. Most (>80%) Corynebacterium spp. were resistant to clindamycin; small numbers (<20%) were resistant to gentamicin, penicillin, vancomycin, ciprofloxacin and rifampicin, with no differences between gilts and sows. Corynebacterium from gilts were more often resistant to tetracycline than those from sows (25% vs. 4.17%; p = 0.04). In conclusion, bacteria from the porcine cervix showed low resistance to most antibiotics except for clindamycin, but antibacterial resistance may increase with increasing parity.

Boot Swabs to Evaluate Cleaning and Disinfection Success in Poultry Barns

Due to the relevance of cleaning and disinfection in farm hygiene management, accurate evaluation of the success of such procedures remains a fundamental challenge for producers. This study aimed to use boot swab sampling to quantify the effects of such practices in poultry barns. For this purpose, the counts of both the total and fecal indicator bacteria were detected after the application of a cleaning and disinfection protocol in identical barns that were occupied by turkeys and broilers. Boot swab samples were compared to an established agar contact plating method to evaluate disinfection success. Statistical analyses showed no correlations between the bacterial counts that were obtained with either method. In contrast to the agar contact plating method, boot swab sampling permitted the assessment of the hygienic conditions of the barn floors before and after cleaning procedures. Furthermore, according to observations with the boot swab method, factors related to the species being farmed influenced the initial bacterial loads but did not affect the effectiveness of cleaning and disinfection. Species identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) also suggested that non-fecal bacteria grow on selective media. Further studies should validate the use of this sampling technique by comparing different cleaning and disinfection protocols.

Performance and Impact on Initial Antibiotic Choice of Direct Identification of Pathogens from Pediatric Blood Culture Bottles Using an In-House MALDI-TOF MS Protocol

We demonstrate the performance and early impact on the antimicrobial management of bloodstream infections of an inexpensive, in-house preparation method for direct identification of bloodstream pathogens in pediatric blood culture bottles by matrix-assisted laser desorption/ionization–time-of-flight mass spectrometry.

Rapid identification of mcr-1-positive Escherichia coli from patient urine using a novel lipid-based MALDI-TOF-MS assay

Mobilized colistin resistance (mcr) genes confer resistance to colistin, a last-resort antibiotic for multidrug-resistant Gram-negative infections. In this case report, we describe a novel lipid-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) diagnostic used to rapidly identify an mcr-1-positive Escherichia coli directly from a patient with a urinary tract infection without the need for ex vivo growth.