Journal of medical microbiology最新文献

Introduction. Klebsiella spp. are important bacteria that colonize the human intestine, especially in preterm infants; they can induce local and systemic disease under specific circumstances, including inflammatory bowel disease, necrotizing enterocolitis and colorectal cancer.Hypothesis. Klebsiella spp. colonized in the intestine of the neonates in the neonatal intensive care unit (NICU) may be associated with disease and antibiotic resistance, which will be hazardous to the children.Aim. Our aim was to know about the prevalence, antimicrobial resistance and genome characteristics of Klebsiella spp. in neonate carriers.Methodology. Genome sequencing and analysis, and antimicrobial susceptibility testing were mainly performed in this study.Results. The isolation rates of Klebsiella spp. strains were 3.7% (16/436) in 2014 and 4.3% (18/420) in 2021. Cases with intestinal-colonized Klebsiella spp. were mainly infants with low birth weights or those with pneumonia or hyperbilirubinemia. According to the core-pan genomic analysis, 34 stains showed gene polymorphism and a sequence type (ST) of an emerging high-risk clone (ST11). Eight strains (23.5%) were found to be resistant to 2 or more antibiotics, and 46 genes/gene families along with nine plasmids were identified that conferred resistance to antibiotics. In particular, the two strains were multidrug-resistant. Strain A1256 that is related to Klebsiella quasipneumoniae subsp. similipneumoniae was uncommon, carrying two plasmids similar to IncFII and IncX3 that included five antibiotic resistance genes.Conclusion. The prevention and control of neonatal Klebsiella spp. colonization in the NICU should be strengthened by paying increased attention to preventing antimicrobial resistance in neonates.

Introduction. Sporotrichosis is a subcutaneous infection caused by dimorphic Sporothrix species embedded in the clinical clade. Fungi have virulence factors, such as biofilm and melanin production, which contribute to their survival and are related to the increase in the number of cases of therapeutic failure, making it necessary to search for new options.Gap statement. Proton pump inhibitors (PPIs) have already been shown to inhibit the growth and melanogenesis of other fungi.Aim. Therefore, this study aimed to evaluate the effect of the PPIs omeprazole (OMP), rabeprazole (RBP), esomeprazole, pantoprazole and lansoprazole on the susceptibility and melanogenesis of Sporothrix species, and their interactions with itraconazole, terbinafine and amphotericin B.Methodology. The antifungal activity of PPIs was evaluated using the microdilution method, and the combination of PPIs with itraconazole, terbinafine and amphotericin B was assessed using the checkerboard method. The assessment of melanogenesis inhibition was assessed using grey scale.Results. The OMP and RBP showed significant MIC results ranging from 32 to 256 µg ml^-1 and 32 to 128 µg ml^-1, respectively. Biofilms were sensitive, with a significant reduction (P<0.05) in metabolic activity of 52% for OMP and 50% for RBP at a concentration of 512 µg ml^-1 and of biomass by 53% for OMP and 51% for RBP at concentrations of 512 µg ml^-1. As for the inhibition of melanogenesis, only OMP showed inhibition, with a 54% reduction.Conclusion. It concludes that the PPIs OMP and RBP have antifungal activity in vitro against planktonic cells and biofilms of Sporothrix species and that, in addition, OMP can inhibit the melanization process in Sporothrix species.

Introduction. Commensal Neisseria spp. are highly prevalent in the oropharynx as part of the healthy microbiome. N. meningitidis can colonise the oropharynx too from where it can cause invasive meningococcal disease. To identify N. meningitidis, clinical microbiology laboratories often rely on Matrix Assisted Laser Desorption/Ionisation Time of Flight Mass Spectrometry (MALDI-TOF MS).Hypothesis/Gap statement. N. meningitidis may be misidentified by MALDI-TOF MS.Aim. To conduct genomic surveillance of oropharyngeal Neisseria spp. in order to: (i) verify MALDI-TOF MS species identification, and (ii) characterize commensal Neisseria spp. genomes.Methodology. We analysed whole genome sequence (WGS) data from 119 Neisseria spp. isolates from a surveillance programme for oropharyngeal Neisseria spp. in Belgium. Different species identification methods were compared: (i) MALDI-TOF MS, (ii) Ribosomal Multilocus Sequence Typing (rMLST) and (iii) rplF gene species identification. WGS data were used to further characterize Neisseria species found with supplementary analyses of Neisseria cinerea genomes.Results. Based on genomic species identification, isolates from the oropharyngeal Neisseria surveilence study were composed of the following species: N. meningitidis (n=23), N. subflava (n=61), N. mucosa (n=15), N. oralis (n=8), N. cinerea (n=5), N. elongata (n=3), N. lactamica (n=2), N. bacilliformis (n=1) and N. polysaccharea (n=1). Of these 119 isolates, four isolates identified as N. meningitidis (n=3) and N. subflava (n=1) by MALDI-TOF MS, were determined to be N. polysaccharea (n=1), N. cinerea (n=2) and N. mucosa (n=1) by rMLST. Phylogenetic analyses revealed that N. cinerea isolates from the general population (n=3, cluster one) were distinct from those obtained from men who have sex with men (MSM, n=2, cluster two). The latter contained genomes misidentified as N. meningitidis using MALDI-TOF MS. These two N. cinerea clusters persisted after the inclusion of published N. cinerea WGS (n=42). Both N. cinerea clusters were further defined through pangenome and Average Nucleotide Identity (ANI) analyses.Conclusion. This study provides insights into the importance of genomic genus-wide Neisseria surveillance studies to improve the characterization and identification of the Neisseria genus.

Introduction. Tuberculosis (TB), an infectious disease caused by Mycobacterium tuberculosis (M. tb), remains a significant global public health concern. It is crucial to develop more effective vaccines for TB in order to achieve global control of the disease. Extracellular vesicles (EVs) are spherical membrane-bound structures released by pathogens and host cells. During the course of an infection, both pathogen- and host-derived EVs are produced and play important roles in determining the course of the infection. EVs offer intriguing tools as potential vaccines due to their ability to deliver multiple pathogen or host antigens.Hypothesis /Gap Statement. We hypothesized that EVs derived from M. tb and EVs from M. tb-infected macrophages may serve as potential vaccine candidates against M. tb infection.Aim. This study aims to compare the immunogenicity and immune protection between M. tb EVs and M. tb-infected macrophage-derived EVs.Methodology. In this study, EVs were extracted from culture supernatants of M. tb and M. tb-infected macrophages, respectively. Mass spectrometry was employed to explore the antigen composition of H37Rv-Mφ-EVs and H37Rv-EVs. Cytokine profiling and antibody response assays were used to analyse the immunogenicity offered by EVs. Additionally, we used histological examination to evaluate and protective efficacy of the EVs.Results. Our results demonstrated that mice immunized by EVs released from M. tb-infected macrophages induced stronger inflammatory cytokine response than M. tb. Moreover, EVs from M. tb-infected macrophages reinforced T-cell activation and antibody response compared to M. tb EVs. Proteomic analysis revealed that EVs from M. tb-infected macrophages containing immunodominant cargos have stronger binding ability with major histocompatibility complex molecules, which may contribute to the protection from M. tb infection. Indeed, immunization of EVs released from M. tb-infected macrophages significantly reduced the bacterial load and better protection against M. tb infection than EVs from M. tb. Importantly, the selected antigens (Ag85B, ESAT-6 and the Rv0580c) from EVs of M. tb-infected macrophages exhibited effective immunogenicity.Conclusion. Our results suggested that EVs derived from M. tb-infected macrophages might serve as a proper antigenic library for vaccine candidates against M. tb challenge.

Introduction. Antimicrobial resistance (AMR) is recognized as an important global health risk, associated with increased mortality, morbidity and healthcare costs. Antimicrobial stewardship (AMS) involves a coherent set of processes that promote the rational use of antimicrobials.Gap statement. An AMS programme should be adapted and developed according to the available resources of a facility. This requires an analysis of the core AMS elements that are already in place and the resources available.Aim. This study aimed to assess the readiness of a tertiary healthcare facility and staff towards implementing an antimicrobial stewardship programme (ASP).Methodology. This study focused on two aspects during an AMS pre-implementation phase. A situational or strengths, weaknesses, opportunities, and threats analysis was conducted based on (1) a questionnaire on attitudes and perceptions of pharmacists, clinicians and nurses towards AMR and AMS and (2) a situational analysis on the readiness of the facility.Results. The questionnaire, which was available for completion between September 2021 and December 2021, was sent to a total of 3100 healthcare professionals (HCPs). Thirty-two (1.0 %) HCPs comprising 2 pharmacists, 16 clinicians and 14 nurses completed the questionnaire. Of the total participants, 31 (96.9 %) viewed AMR as a problem in South African hospitals and 29 (90.6 %) perceived AMR as a problem at their facility. The majority (n = 29, 90.6 %) of the participants were familiar with the term AMS, and 26 (81.3 %) participants agreed to willingly participate in any initiatives involving antimicrobial use at the facility. The situational analysis depicted existing strengths in terms of AMS structures such as the formation of an AMS committee and information and technology systems at the HCP's disposal. Weaknesses included the limited number of AMS activities being carried out and poor participation from HCPs within the AMS team.Conclusion. A pre-implementation phase in the building of an ASP can greatly assist in finding gaps for improvement, which can then be addressed in the implementation phase. Furthermore, the pre-implementation phase provides a baseline to measure improvements once the implementation phase has been instituted.

Introduction. Aspergillus flavus and Fusarium keratoplasticum are common causative pathogens of fungal keratitis (FK), a severe corneal disease associated with significant morbidity and vision loss. Escalating incidence of antifungal resistance to available antifungal drugs poses a major challenge to FK treatment. Cold atmospheric plasma (CAP) is a pioneering nonpharmacologic antimicrobial intervention that has demonstrated potential as a broad-spectrum antifungal treatment.Gap statement. Previous research highlights biofilm-associated resistance as a critical barrier to effective FK treatment. Although CAP has shown promise against various fungal infections, its efficacy against biofilm and conidial forms of FK pathogens remains inadequately explored.Aim. This study aims to investigate the antifungal efficacy of CAP against clinical fungal keratitis isolates of A. flavus and F. keratoplasticum in vitro.Methodology. Power parameters (22-27 kV_pp, 300-400 Hz and 20-80 mA) of a dielectric barrier discharge CAP device were optimized for inactivation of A. flavus biofilms. Optimal applied voltage and total current were applied to F. keratoplasticum biofilms and conidial suspensions of A. flavus and F. keratoplasticum. The antifungal effect of CAP treatment was investigated by evaluating fungal viability through means of metabolic activity, c.f.u. enumeration (c.f.u. ml^-1) and biofilm formation.Results. For both fungal species, CAP exhibited strong time-dependent inactivation, achieving greater than 80 % reduction in metabolic activity and c.f.u. ml^-1 within 300 s or less, and complete inhibition after 600 s of treatment.Conclusion. Our findings indicate that CAP is a promising broad-spectrum antifungal intervention. CAP treatment effectively reduces fungal viability in both biofilm and conidial suspension cultures of A. flavus and F. keratoplasticum, suggesting its potential as an alternative treatment strategy for fungal keratitis.

Introduction. Persister cells are transiently non-growing antibiotic-tolerant bacteria that cause infection relapse, and there is no effective antibiotic therapy to tackle these infections.Gap statement. High-throughput assays in drug discovery are biased towards detecting drugs that inhibit bacterial growth rather than killing non-growing bacteria. A new and simple assay to discover such drugs is needed.Aim. This study aims to develop a simple and high-throughput assay to identify compounds with antimicrobial activity against persister cells and use it to identify molecular motifs with such activity.Methodology. We quantified Staphylococcus aureus persister cells by enumeration of colony forming units after 24 h ciprofloxacin treatment. We first quantified how the cell concentration, antibiotic concentration, growth phase and presence/absence of nutrients during antibiotic exposure affected the fraction of persister cells in a population. After optimizing these parameters, we screened the antimicrobial activity of compound fragments to identify molecular structures that have activity against persister cells.Results. Exponential- and stationary-phase cultures transferred to nutrient-rich media displayed a bi-phasic time-kill curve and contained 0.001-0.07% persister cells. A short rifampicin treatment resulted in 100% persister cells for 7 h, after which cells resumed activity and became susceptible. Stationary-phase cultures displayed a low but constant death rate but ultimately resulted in similarly low survival rates as the exponential-phase cultures after 24 h ciprofloxacin treatment. The persister phenotype was only maintained in most of the population for 24 h if cells were transferred to a carbon-free minimal medium before exposure to ciprofloxacin. Keeping cells starved enabled the generation of high concentrations of S. aureus cells that tolerate 50× MIC ciprofloxacin, and we used this protocol for rapid screening for biocidal antibiotics. We identified seven compounds from four structural clusters with activity against antibiotic-tolerant S. aureus. Two compounds were moderately cytotoxic, and the rest were highly cytotoxic.Conclusion. Transferring a stationary-phase culture to a carbon-free minimal medium for antimicrobial testing is a simple strategy for high-throughput screening for new antibiotics that kill persister cells. We identified molecule fragments with such activity, but further screening is needed to identify motifs with lower general cytotoxicity.

Introduction. Maternal screening tests and prophylactic antibiotics are important to prevent neonatal and infant group B streptococcal (GBS) infections.Hypothesis/Gap Statement. The performance of enrichment broth media for GBS screening that are available in Japan is unclear. Whole-genome data of GBS isolates from pregnant women in Japan is lacking.Aim. The aim of this study was to compare the protocol performance of six enrichment broths and two subculture agar plates, which were all available in Japan, for GBS detection. In addition, we showed whole-genome data of GBS isolates from pregnant women in Japan.Methodology. We collected 133 vaginal-rectal swabs from pregnant women visiting clinics and hospitals in Nagasaki Prefecture, Japan, and compared the protocol performance of 6 enrichment broths and 2 subculture agar plates. All GBS isolates collected in this study were subjected to whole-genome sequencing analysis.Results. We obtained 133 vaginal-rectal swabs from pregnant women at 35-37 weeks of gestation from 8 private clinics and 2 local municipal hospitals within Nagasaki Prefecture, Japan. The detection rate of the protocol involving the six enrichment broths and subsequent subcultures varied between 95.5 and 100 %, depending on the specific choice of enrichment broth. The GBS carriage rate among pregnant women in this region was 18.8 %. All 25 isolates derived from the swabs were susceptible to penicillin, whereas 48 and 36 % of the isolates demonstrated resistance to erythromycin and clindamycin, respectively. The distribution of serotypes was highly diverse, encompassing seven distinct serotypes among the isolates, with the predominant serotype being serotype V (n = 8). Serotype V isolates displayed a tendency towards increased resistance to erythromycin and clindamycin, with all resistant isolates containing the ermB gene.Conclusion. There was no difference in performance among the culture protocols evaluated in this study. GBS strains isolated from pregnant women appeared to have greater genomic diversity than GBS strains detected in neonates/infants with invasive GBS infections. To confirm this result, further studies with larger sample sizes are needed.

Introduction. Pre-existing fluoroquinolones (FQs) resistance is a major threat in treating multidrug-resistant (MDR) tuberculosis. Sitafloxacin (Sfx) is a new broad-spectrum FQ.Hypothesis. Sfx is more active against drug-resistant Mycobacterium tuberculosis (Mtb) isolates.Aim. To determine whether there is cross-resistance between Sfx and ofloxacin (Ofx), levofloxacin (Lfx) and moxifloxacin (Mfx) in MDR Mtb.Methods. A total of 106 clinical Mtb isolates, including 23 pan-susceptible and 83 MDR strains, were analysed for Sfx, Lfx and Mfx resistance using MIC assay. The isolates were also subjected to whole-genome sequencing to analyse drug-resistant genes.Results. Sfx exhibited the most robust inhibition activity against Mtb clinical isolates, with a MIC₅₀ of 0.0313 µg ml^-1 and MIC₉₀ of 0.125 µg ml^-1, which was lower than that of Mfx (MIC₅₀ = 0.0625 µg ml^-1, MIC₉₀ = 1 µg ml^-1) and Lfx (MIC₅₀ = 0.125 µg ml^-1, MIC₉₀ = 2 µg ml^-1). We determined the tentative epidemiological cut-off values as 0.5 µg ml^-1 for Sfx. Also, 8.43% (7/83), 43.37% (36/83), 42.17% (35/83) and 51.81% (43/83) MDR strains were resistant to Sfx, Mfx, Lfx and Ofx, respectively. Cross-resistance between Ofx, Lfx and Mfx was 80.43% (37/46). Only 15.22% (7/46) of the pre-existing FQs resistance isolates were resistant to Sfx. Among the 30 isolates with mutations in gyrA or gyrB, 5 (16.67%) were Sfx resistant. The combination of Sfx and rifampicin could exert partial synergistic effects, and no antagonism between Sfx and six clinically important anti-Mtb antibiotics was evident.Conclusion. Sfx exhibited superior activity against MDR isolates comparing to Lfx and Mfx, and could potentially overcome the majority pre-existing FQs resistance in Mtb strains.

Background. Pseudomonas aeruginosa is an invasive organism that frequently causes severe tissue damage in diabetic foot ulcers.Gap statement. The characterisation of P. aeruginosa strains isolated from diabetic foot infections has not been carried out in Tunisia.Purpose. The aim was to determine the prevalence of P. aeruginosa isolated from patients with diabetic foot infections (DFIs) in Tunisia and to characterize their resistance, virulence and molecular typing.Methods. Patients with DFIs admitted to the diabetes department of the International Hospital Centre of Tunisia, from September 2019 to April 2021, were included in this prospective study. P. aeruginosa were obtained from the wound swabs, aspiration and soft tissue biopsies during routine clinical care and were confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Antimicrobial susceptibility testing, serotyping, integron and OprD characterization, virulence, biofilm production, pigment quantification, elastase activity and molecular typing were analysed in all recovered P. aeruginosa isolates by phenotypic tests, specific PCRs, sequencing, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing.Results. Sixteen P. aeruginosa isolates (16.3 %) were recovered from 98 samples of 78 diabetic patients and were classified into 6 serotypes (O:11 the most frequent), 11 different PFGE patterns and 10 sequence types (three of them new ones). The high-risk clone ST235 was found in two isolates. The highest resistance percentages were observed to netilmicin (69 %) and cefepime (43.8 %). Four multidrug-resistant (MDR) isolates (25 %) were detected, three of them being carbapenem-resistant. The ST235-MDR strain harboured the In51 class 1 integron (intI1 +aadA6+orfD+qacED1-sul1). According to the detection of 14 genes involved in virulence or quorum sensing, 5 virulotypes were observed, including 5 exoU-positive, 9 exoS-positive and 2 exoU/exoS-positive strains. The lasR gene was truncated by ISPpu21 insertion sequence in one isolate, and a deletion of 64 bp in the rhlR gene was detected in the ST235-MDR strain. Low biofilm, pyoverdine and elastase production were detected in all P. aeruginosa; however, the lasR-truncated strain showed a chronic infection phenotype characterized by loss of serotype-specific antigenicity, high production of phenazines and high biofilm formation.Conclusions. Our study demonstrated for the first time the prevalence and the molecular characterization of P. aeruginosa strains from DFIs in Tunisia, showing a high genetic diversity, moderate antimicrobial resistance, but a high number of virulence-related traits, highlighting their pathological importance.