Proteomics-based screening and antibiotic resistance assessment of clinical and sub-clinical Brucella species: An evolution of brucellosis infection control

Brucellae are intracellular sneaky bacteria and they can elude the host’s defensive mechanisms, resulting in therapeutic failure. Therefore, the goal of this investigation was to rapid identification of Brucella species collected from animals and humans in Saudi Arabia, as well as to evaluate their resistance to antibiotics. On selective media, 364 animal samples as well as 70 human blood samples were cultured. Serological and biochemical approaches were initially used to identify a total of 25 probable cultured isolates. The proteomics of Brucella species were identified using the MALDI Biotyper (MBT) system, which was subsequently verified using real-time polymerase chain reaction (real-time PCR) and microfluidic electrophoresis assays. Both Brucella melitensis (B. melitensis) and Brucella abortus (B. abortus) were tested for antimicrobial susceptibility using Kirby Bauer method and the E-test. In total, 25 samples were positive for Brucella and included 11 B. melitensis and 14 B. abortus isolates. Twenty-two out of 25 (88%) and 24/25 (96%) of Brucella strains were recognized through the Vitek 2 Compact system. While MBT was magnificently identified 100% of the strains at the species level with a score value more than or equal to 2.00. Trimethoprim-sulfamethoxazole, rifampin, ampicillin-sulbactam, and ampicillin resistance in B. melitensis was 36.36%, 31.82%, 27.27%, and 22.70%, respectively. Rifampin, trimethoprim-sulfamethoxazole, ampicillin, and ampicillin-sulbactam resistance was found in 35.71%, 32.14%, 32.14%, and 28.57% of B. abortus isolates, correspondingly. MBT confirmed by microfluidic electrophoresis is a successful approach for identifying Brucella species at the species level. The resistance of B. melitensis and B. abortus to various antibiotics should be investigated in future studies.

Laboratory identification and clinical characteristics of Streptococcus bovis/Streptococcus equinus complex bacteremia: a retrospective, multicenter study in Hiroshima, Japan

Background Bacteremia due to the Streptococcus bovis/Streptococcus equinus complex (SBSEC) is associated with specific diseases, such as colorectal cancer and infective endocarditis. This study aimed to evaluate the clinical characteristics of SBSEC bacteremia and the accuracy of identification of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and phenotypic identification systems for SBSEC isolates. Methods We analyzed patients with SBSEC bacteremia retrospectively between 2012 and 2019 at three hospitals in Japan. We re-identified each SBSEC isolate using sequencing superoxide dismutase (sodA) analysis, MALDI-TOF MS using the MALDI Biotyper, and phenotypic identification using the VITEK2. Results During the study period, 39 patients with SBSEC bacteremia were identified. S. gallolyticus subsp. pasteurianus (SGSP, n = 29), S. gallolyticus subsp. gallolyticus (SGSG, n = 5), S. lutetiensis (SL, n = 4), and S. infantarius subsp. infantarius (n = 1) were identified using sodA sequencing analysis. Primary bacteremia (36%) was the most common cause of bacteremia, followed by infective endocarditis (26%) and biliary tract infections (23%). Colorectal cancer was associated significantly with SGSG bacteremia, while the sources of bacteremia were similar in each SBSEC subspecies. The MALDI Biotyper was significantly more accurate in identifying the SBSEC isolates at the subspecies level compared to the VITEK2 (92% vs. 67%, P = 0.010). In contrast, there were no significant differences in the rates of correct identification of the SBSEC isolates at the species level between the MALDI Biotyper and the VITEK2 (100% vs. 87%, P = 0.055). Conclusions Bacteremia with SGSG was associated with colorectal cancer, and the sources of bacteremia were similar in each SBSEC subspecies. The MALDI-TOF MS was significantly more accurate in identifying SBSEC isolates at the subspecies level than the phenotypic identification systems. The accurate identification of SBSEC isolates using the MALDI-TOF MS and phenotypic identification systems was sufficient at the species level, but it was insufficient at the subspecies level. Therefore, it may be reasonable for clinicians to perform echocardiographies and colonoscopies in all patients with SBSEC bacteremia.

Detection of Colistin Resistance in Pseudomonas aeruginosa Using the MALDIxin Test on the Routine MALDI Biotyper Sirius Mass Spectrometer

Colistin is frequently a last resort treatment for Pseudomonas aeruginosa infections caused by multidrug-resistant (MDR) and extensively drug resistant (XDR) strains, and detection of colistin resistance is essential for the management of infected patients. Therefore, we evaluated the recently developed MALDIxin test for the detection of colistin resistance in P. aeruginosa clinical strains using the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system. The test is based on the detection by mass spectrometry of modified lipid A by the addition of 4-amino-l-arabinose (l-ara4N) molecules on one or two phosphate groups, in strains resistant to colistin. Overproduction of l-Ara4N molecules is mainly due to the constitutive activation of the histidine kinase (PmrB) or the response regulator (PmrA) following an amino-acid substitution in clinical strains. The performance of the test was determined on a panel of 14 colistin-susceptible and 14 colistin-resistant P. aeruginosa clinical strains, the reference strain PAO1 and positive control mutants PmrB (V28G), PmrB (D172), PhoQ (D240–247), and ParR (M59I). In comparison with the broth microdilution (BMD) method, all the susceptible strains (n=14) and 8/14 colistin-resistant strains were detected in less than 1h, directly on whole bacteria. The remaining resistant strains (n=6) were all detected after a short pre-exposure (4h) to colistin before sample preparation. Validation of the method on a larger panel of strains will be the next step before its use in diagnostics laboratories. Our data showed that the MALDIxin test offers rapid and efficient detection of colistin resistant P. aeruginosa and is thus a valuable diagnostics tool to control the spread of these emerging resistant strains.

Rapid detection by MALDI-TOF MS of isolates from cystic fibrosis patients belonging to the epidemic clones Achromobacter xylosoxidans ST137 or Achromobacter ruhlandii DES.

Objective: Achromobacter spp. are increasingly reported among cystic fibrosis patients. Genotyping requires time consuming methods such as Multilocus-Sequence-Typing or Pulsed-Field-Gel-Electrophoresis. Therefore, data on the prevalence of the multiresistant epidemic clones, especially A. xylosoxidans ST137 (AxST137) and the Danish Epidemic Strain A. ruhlandii (DES) are lacking. We recently developed and published a database for Achromobacter species identification by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS, Bruker Daltonics). The aim of this study was to evaluate the ability of the MALDI-TOF MS to distinguish these multiresistant epidemic clones within Achromobacter species. Methods: All the spectra of A.xylosoxidans (n=1571) and A.ruhlandii (n=174) used to build the local database were analysed by ClinProTools™, MALDI Biotyper® PCA, MALDI Biotyper ® dendrogram and flexAnalysis™ softwares for biomarker peaks detection. Two-hundred-two isolates (including 48 isolates of AxST137 and 7 of DES) were tested. Results: Specific biomarker peaks were identified: absent peak at m/z 6651 for AxST137 isolates and present peak at m/z 9438 for DES isolates. All tested isolates were well typed by our local database and clustered within distinct groups (ST137 or non-ST137 and DES or non-DES) no matter the MALDI-TOF software or only by simple visual inspection of the spectra by any user. Conclusions: The use of MALDI-TOF MS allowed identifying isolates of A. xylosoxidans belonging to the AxST137 clone which spread in France and Belgium (the Belgian epidemic clone) and of A. ruhlandii belonging to the DES clone. This tool will help implementation of segregation measures to avoid inter-patient transmission of these resistant clones.

A multidrug-resistant Stenotrophomonas maltophilia clinical isolate from Kamuzu Central Hospital, Malawi

BackgroundStenotrophomonas maltophilia is a significant opportunistic pathogen that is associated with high mortality in immunocompromised individuals. In this study, we describe a multidrug-resistant (MDR) S. maltophilia clinical isolate from Kamuzu Central Hospital (KCH), Lilongwe, Malawi.MethodsA ceftriaxone and meropenem nonsusceptible isolate (Sm-MW08), recovered in December 2017 at KCH, was referred to the National Microbiology Reference Laboratory for identification. In April 2018, we identified the isolate using MALDI Biotyper mass spectrometry and determined its antimicrobial susceptibility profile using microdilution methods. Sm-MW08 was analysed by S1- PFGE, PCR, and Sanger sequencing, in order to ascertain the genotypes that were responsible for the isolate`s multidrug-resistance (MDR) phenotype.ResultsSm-MW08 was identified as S. maltophilia and exhibited resistance to a range of antibiotics, including all β-lactams, aminoglycosides (except arbekacin), chloramphenicol, minocycline, fosfomycin and fluoroquinolones, but remained susceptible to colistin and trimethoprim-sulfamethoxazole. The isolate did not harbour any plasmid but did carry chromosomally-encoded blaL1 metallo-βlactamase and blaL2 β-lactamase genes; this was consistent with the isolate’s resistance profile. No other resistance determinants were detected, suggesting that the MDR phenotype exhibited by Sm-MW08 was innate.ConclusionHerein, we have described an MDR S. maltophilia from KCH in Malawi, that was resistant to almost all locally available antibiotics. We therefore recommend the practice of effective infection prevention measures to curtail spread of this organism.

A NEW APPLICATION OF MALDI BIOTYPER FOR A POTENTIAL USE ON THERAPEUTIC DRUG MONITORING OF VORICONAZOLE

We present a proof of concept study on the use of MALDI Biotyper to detect and monitor the levels of voriconazole in human serum. A simple extraction-concentration method and a MALDI Biotyper protocol were developed and a parent ion of voriconazole (1 H+) could be detected and quantified with good reproducibility. Our results point to a new application of MALDI Biotyper for therapeutic drug monitoring.

Polymicrobial Necrotizing Fasciitis in a Dog: The Involvement of Macrococcus caseolyticus, Proteus mirabilis, and Escherichia coli

A male mixed breed dog was presented with two large wounds, extending the epidermis, dermis, and fascia: one at the dorsum of the thoracolumbar region and the other at the lumbosacral area. Lesions had extended inconspicuously to the dorsum of thorax affecting a large area, which showed regions with necrotic and crepitating foci after shaving. Based on histopathological and bacterial culture examinations, polymicrobial necrotizing fasciitis (NF) was diagnosed. Using the Bruker MALDI Biotyper identification technique, Macrococcus caseolyticus, Proteus mirabilis, and Escherichia coli were identified. Hitherto, there is no report on these bacteria linking them simultaneously to NF in a dog. In addition, the authors highlight other microbes associated with NF in humans and animals.

Antibiotic resistance features in Klebsiella pneumoniae and Escherichia coli strains isolated from hospital and community acquired urinary tract infections

A total number of 35 strains (n=23 of K. pneumoniae and n=12 of E.coli) were isolated in May 2017 from patients with UTI, hospitalized in the National Institute for Cardiovascular Diseases Prof. C.C. Iliescu and from community infections (CA) diagnosed in Central Reference Synevo-Medicover Laboratory from Bucharest. The hospital strains were identified by BD Phoenix and the CA ones by mass spectrometry using MALDI Biotyper. The antibiotic susceptibility was determined by agar disk diffusion (CLSI, 2017) and automated methods (BD Phoenix and Vitek II system). For molecular characterization, all strains were analyzed be using PCR amplification. The investigated strains revealed the presence of tetracycline resistance gene, i.e. tet(A) (67% in E. coli and 45% of K. pneumoniae strains), tet(D) (8% of E. coli and 5% of K. pneumoniae strains), carbapenemase genes (blaOXA-48 in 40% of the K. pneumoniae strains); blaTEM (25% of E. coli strains and 10% of K. pneumoniae strains).