Comparison of identification and antimicrobial resistance pattern of Staphylococcus aureus isolated from Amassoma, Bayelsa state, Nigeria.

Background: Staphylococcus aureus is often responsible for fatal infections and recent upsurge of resistant strains has resulted in therapeutic failure. The identification of this microorganism is a major challenge to medical microbiologists in developing countries.Methods: One hundred and eighty five isolates which had been previously isolated from the nares of 185 healthy college students’ volunteers in Amassoma, Bayelsa State, South Nigeria were identified by MALDI TOF mass spectrometry, and PCR amplification of the spa gene. The identified isolates were compared with presumptive identities obtained by growth on MSA, tube coagulation and slide agglutination tests. Antimicrobial susceptibility testing of S. aureus isolates was performed by Kirby Bauer technique while MRSA was screened for by growth on chromIDTM MRSA plate and confirmed by PCR-amplification of mecA/mecC genes.Results: From the 185 staphylococci that grew with yellow colonies on MSA, 24 were positive in the slide coagulase test, while 17 were positive in the tube coagulase test; MALDI TOF mass spectrometry and PCR amplification of the spa gene showed excellent concordance with the tube test, as all tube coagulase-positive strains were identified as S. aureus, while tube coagulase-test negative isolates in all cases were designated as other staphylococcal species by MALDI-TOF mass spectrometry and were spa PCR test negative. All S. aureus isolates were susceptible to clindamycin, vancomycin, fusidic acid, rifampicin and linezolid, while observed resistance to penicillin and trimethoprim were high. Only one MRSA strain was detectedConclusion: The study confirms that the tube coagulase test is an accurate diagnostic method for identification of S. aureus, while growths on MSA and slide agglutination tests are inaccurate. We found a low prevalence of MRSA and a high rate of trimethroprim-resistance in the studied population.Keywords: Antibiotics, coagulase, identification, S. aureus

Achromobacter Species Isolated from Cystic Fibrosis Patients Reveal Distinctly Different Biofilm Morphotypes

Achromobacter species have attracted attention as emerging pathogens in cystic fibrosis. The clinical significance of Achromobacter infection is not yet fully elucidated; however, their intrinsic resistance to antimicrobials and ability to form biofilms renders them capable of establishing long-term chronic infections. Still, many aspects of Achromobacter biofilm formation remain uncharacterized. In this study, we characterized biofilm formation in clinical isolates of Achromobacter and investigated the effect of challenging the biofilm with antimicrobials and/or enzymes targeting the extracellular matrix. In vitro biofilm growth and subsequent visualization by confocal microscopy revealed distinctly different biofilm morphotypes: a surface-attached biofilm morphotype of small aggregates and an unattached biofilm morphotype of large suspended aggregates. Aggregates consistent with our in vitro findings were visualized in sputum samples from cystic fibrosis patients using an Achromobacter specific peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) probe, confirming the presence of Achromobacter biofilms in the CF lung. High antibiotic tolerance was associated with the biofilm phenotype, and biocidal antibiotic concentrations were up to 1000 fold higher than for planktonic cultures. Treatment with DNase or subtilisin partially dispersed the biofilm and reduced the tolerance to specific antimicrobials, paving the way for further research into using dispersal mechanisms to improve treatment strategies

Danish Whole-Genome-Sequenced Candida albicans and Candida glabrata Samples Fit into Globally Prevalent Clades

Candida albicans and Candida glabrata are opportunistic fungal pathogens with increasing incidence worldwide and higher-than-expected prevalence in Denmark. We whole-genome sequenced yeast isolates collected from Danish Clinical Microbiology Laboratories to obtain an overview of the Candida population in the country. The majority of the 30 C. albicans isolates were found to belong to three globally prevalent clades, and, with one exception, the remaining isolates were also predicted to cluster with samples from other geographical locations. Similarly, most of the eight C. glabrata isolates were predicted to be prevalent subtypes. Antifungal susceptibility testing proved all C. albicans isolates to be susceptible to both azoles and echinocandins. Two C. glabrata isolates presented azole-resistant phenotypes, yet all were susceptible to echinocandins. There is no indication of causality between population structure and resistance phenotypes for either species

Incidence of HACEK bacteraemia in Denmark:A 6-year population-based study

Objectives: Bacteria with common microbiological and clinical characteristics are often recognized as a particular group. The acronym HACEK stands for five fastidious genera associated with infective endocarditis (Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, and Kingella). Data on the epidemiology of HACEK are sparse. This article reports a 6-year nationwide study of HACEK bacteraemia in Denmark. Methods: Cases of HACEK bacteraemia occurring during the years 2010–2015 were retrieved from the national Danish microbiology database, covering an average surveillance population of 5.6 million per year. Results: A total of 147 cases of HACEK bacteraemia were identified, corresponding to an annual incidence of 0.44 per 100 000 population. The annual incidence for males was 0.56 per 100 000 and for females was 0.31 per 100 000. The median age was 56 years (range 0–97 years), with variation among the genera. One hundred and forty-three isolates were identified to the species level and six to the genus level: Haemophilus spp, n = 55; Aggregatibacter spp, n = 37; Cardiobacterium spp, n = 9; Eikenella corrodens n = 21; and Kingella spp, n = 27. Conclusions: This is the first study on the incidence of HACEK bacteraemia in a large surveillance population and may inspire further studies on the HACEK group. Haemophilus spp other than Haemophilus influenzae accounted for most cases of HACEK bacteraemia in Denmark, with Aggregatibacter spp in second place. Keywords: Epidemiology, Incidence, Age, Sex, Haemophilus, Aggregatibacte

Multilocus sequence phylogenetic study of the genus Haemophilus with description of Haemophilus pittmaniae sp. nov. Int J Syst Evol Microbiol

The phylogeny of human isolates of Haemophilus species was estimated based on partial sequences of four separate housekeeping genes. The clustering of each set of sequences was in accordance with speciation of the strains with few exceptions: of 108 gene fragments examined, only three appeared to have been subject to recombination events across the species barrier. Housekeeping gene similarity supported previous DNA-DNA hybridization data for the genus rather than the phylogeny inferred from 16S rRNA gene sequence comparison. The similarity of sequences of Haemophilus parainfluenzae with those of Haemophilus influenzae suggested preservation of the former species in the genus Haemophilus. Three strains representing a novel taxon were unique with respect to the four investigated gene loci. 16S rRNA gene sequence analysis suggested that this taxon belonged to the Parainfluenzae cluster. DNA-DNA hybridization data supported this generic placement. Nine strains of the novel taxon were available for analysis. They were distinct from representatives of all current species of the genus Haemophilus by conventional phenotypic characterization. Genotypic and phenotypic data show that the strains merit recognition as a novel species of Haemophilus. The name Haemophilus pittmaniae sp. nov. is proposed, with HK 85 T (=CCUG 48703 T =NCTC 13334 T ) as the type strain

Differences in Gene Expression Profiles between Early and Late Isolates in Monospecies Achromobacter Biofilm

Bacteria of genus Achromobacter are emerging pathogens in cystic fibrosis (CF) capable of biofilm formation and development of antimicrobial resistance. Evolutionary adaptions in the transition from primary to chronic infection were assessed by transcriptomic analysis of successive isolates of Achromobacter xylosoxidans from a single CF patient. Several efflux pump systems targeting antimicrobial agents were upregulated during the course of the disease, whereas all genes related to motility were downregulated. Genes annotated to subsystems of sulfur metabolism, protein metabolism and potassium metabolism exhibited the strongest upregulation. K+ channel genes were hyperexpressed, and a putative sulfite oxidase was more than 1500 times upregulated. The transcriptome patterns indicated a pivotal role of sulfur metabolism and electrical signalling in Achromobacter biofilms during late stage CF lung disease

Induction of Broad β-lactam Resistance in Achromobacter ruhlandii by Exposure to Ticarcillin Is Primarily Linked to Substitutions in Murein Peptide Ligase Mpl

Achromobacter species are emerging pathogens in cystic fibrosis with inherent resistance to several classes of antimicrobial agents. We exposed strains with wild-type antimicrobial susceptibility to ticarcillin and generated mutants with broad β-lactam resistance. Within the detection limit of the assay, the capability to develop mutational resistance was strain-specific and reproducible. Mutational resistance was observed for all three tested strains of Achromobacter ruhlandii, for one of seven strains of Achromobacter xylosoxidans, and for none of five strains of Achromobacter insuavis. All mutants were resistant to piperacillin-tazobactam, while minimal inhibitory concentration of several other β-lactams increased 4–32-fold. Whole genome sequencing identified 1–4 non-synonymous mutations in known genes per mutant. All mutants encoded amino acid substitutions in cell wall recycling proteins, primarily Mpl, and the observed resistance is probably caused by hyperproduction of OXA-114-like β-lactamases. Related, but not identical substitutions were detected in clinical strains expressing acquired antimicrobial resistance

Genetic Adaptation of Achromobacter sp. during Persistence in the Lungs of Cystic Fibrosis Patients.

Achromobacter species are increasingly isolated from the respiratory tract of cystic fibrosis patients and often a chronic infection is established. How Achromobacter sp. adapts to the human host remains uncharacterised. By comparing longitudinally collected isolates of Achromobacter sp. isolated from five CF patients, we have investigated the within-host evolution of clonal lineages. The majority of identified mutations were isolate-specific suggesting co-evolution of several subpopulations from the original infecting isolate. The largest proportion of mutated genes were involved in the general metabolism of the bacterium, but genes involved in virulence and antimicrobial resistance were also affected. A number of virulence genes required for initiation of acute infection were selected against, e.g. genes of the type I and type III secretion systems and genes related to pilus and flagellum formation or function. Six antimicrobial resistance genes or their regulatory genes were mutated, including large deletions affecting the repressor genes of an RND-family efflux pump and a beta-lactamase. Convergent evolution was observed for five genes that were all implicated in bacterial virulence. Characterisation of genes involved in adaptation of Achromobacter to the human host is required for understanding the pathogen-host interaction and facilitate design of future therapeutic interventions

Post-antibiotic Effect of Various Antibiotics against <i>Achromobacter xylosoxidans</i> Strains Isolated from Patients with Cystic Fibrosis

Objectives: Achromobacter xylosoxidans is an emerging pathogen in cystic fibrosis (CF) disease, although relatively limited is known about its clinical impact and optimal management. Pharmacodynamics such as post-antibiotic effect (PAE) are increasingly being applied to the design of antibiotic dosing regimens. In the present study, meropenem, doripenem, colistin, levofloxacin, tigecycline, tobramycin, and chloramphenicol were tested for their PAE. Materials and Methods: The in vitro activities of meropenem, doripenem, colistin, levofloxacin, tigecycline, tobramycin, and chloramphenicol were determined by the microbroth dilution technique. To determine the PAEs, A. xylosoxidans strains in the logarithmic phase of growth were exposed for 1 h to antibiotics. Recovery periods of test cultures were evaluated using viable counting after centrifugation. Results: The mean values of in vitro PAEs at 1xMIC and 4xMIC concentrations were 3.21 ± 0.66 and 3.58 ± 0.68 hours for colistin, 1.54 ± 0.46 and 2.27 ± 0.41 hours for levofloxacin, 2.34 ± 0.48 and 3.47 ± 0.64 hours for tobramycin, 1.72 ± 0.47 and 2.24 ± 0.41 hours for tigecycline 1.91 ± 0.14 and 2.13 ± 0.13 hours for chloramphenicol, respectively. The carbapenem antibiotics exhibited weak PAEs, as expected. Conclusions: This study’s findings could have important implications for the timing of doses during antimicrobial therapy with tested antibiotics