Journal of medical microbiology最新文献

Contemporary pharmaceutical design often incorporates functional groups to improve the pharmacokinetic/pharmacodynamic profile of a desired active chemical entity. These compounds are known as prodrugs. While prodrug enhancements may improve a medication's clinical utility, they often limit the ability for in vitro testing of the active drug. Published protocols suggest that commercially available phosphatase enzymes can provide a straightforward and cost-effective way to access the active components of phosphoramidate prodrugs. Here, we demonstrate that commercial phosphatases lack reproducibility in generating the active antibiotic ceftaroline from its prodrug ceftaroline fosamil (Teflaro^®). We propose that previously reported successes with phosphatase-mediated conversion are due to the purification of natural ceftaroline fosamil degradation products or batch-dependent isozyme contaminants present in alkaline phosphatases obtained from biological sources. Here, we demonstrate the chemical/thermodegradation to provide a robust, non-enzymatic source of the ceftaroline free base. This efficient method can be readily adapted to expand the availability of deprotected thermostable commercial pharmaceutical compounds for in vitro testing and research purposes.

Introduction. Blood cultures are an important test for the diagnosis of sepsis, and the time to an accurate result can allow for optimized antibiotic therapy. The 'choc drop' is a commonly performed, reliable and cheap rapid identification method, using a drop of positive blood culture broth incubated for a short period (2-4 h) before performing matrix-assisted laser desorption/ionization time-of-flight MS on any early growth/microbiological veil present.Hypothesis. Due to the short incubation time (~2 h), agar plates may not reach the target temperature for optimal bacterial growth, leading to reduced accuracy of identification. Pre-warmed agar plates may allow for target temperature attainment more rapidly and therefore lead to more accurate identification in short-term incubation.Aim. To investigate if pre-warmed chocolate agar used for choc drop identification resulted in increased rates of correct identification.Methodology. In this study, baseline performance of choc drop identification using room temperature chocolate agar plates (n=542) was compared with an interventional period of pre-warmed chocolate agar plates (n=426).Results. Pre-warmed plates showed an increase in correct identification rate of 16.1% [95 % confidence interval (CI) 9.8%-22.4%, P=0.0013], largely due to a 17.1% (95% CI 11.1%-23.1%, P<0.0001) decrease in Gram-positive bacteria returning 'no identification'. There were slightly more errors in the interventional (n=11, 2.8%) compared with the baseline period (n=2, 0.2%).Conclusions. Pre-warming plates appears to be a cheap, effective and easy to implement method for improving the diagnostic yield of short-term incubation for rapid blood culture identification.

Introduction. Azoles are extensively employed as clinical antifungal agents; however, their long-term and widespread application contributed to the progressive emergence of azole resistance. A significant increase in infections caused by azole-resistant Nakaseomyces glabrata highlights the need for novel therapeutic strategies.Hypothesis/Gap Statement. The efficacy of hexyl-aminolevulinate ethosome-mediated antimicrobial photodynamic therapy (HAL-ES-aPDT) against drug-resistant fungal pathogens, as well as its underlying mechanisms, remains to be elucidated. Our previous studies have demonstrated that HAL-ES-aPDT, which utilizes the photosensitizer HAL-ES, eliminates pathogens via photochemical reactions.Aim. This study aimed to evaluate the effects and mechanisms of HAL-ES-aPDT on clinical isolates of N. glabrata exhibiting varying levels of azole susceptibility, with a focus on changes in resistance and virulence.Methodology. Several clinical isolates of N. glabrata were collected, and the effects and mechanisms of HAL-ES-aPDT treatment on azole-resistant strains were investigated.Results. HAL-ES-aPDT reduced N. glabrata tolerance to environmental stress and reversed azole resistance by inhibiting drug efflux and downregulating genes encoding the target enzymes. It also attenuated in vivo virulence by downregulating the expression of the epithelial adhesin gene EPA1.Conclusion. These results confirm the efficacy of HAL-ES-aPDT against azole-resistant N. glabrata and provide novel mechanistic insights that may facilitate the development of effective therapeutic interventions for resistant fungal infections.

Introduction. Pseudomonas aeruginosa has a high propensity to develop drug resistance and is a leading cause of bacterial keratitis, particularly amongst contact lens (CL) wearers. Although CL wearers are advised to regularly disinfect their CLs with fresh multi-purpose disinfection solutions, adherence is often poor. This raises the possibility that P. aeruginosa may develop resistance to multi-purpose disinfection solutions when exposed to sub-inhibitory concentrations of disinfection solutions.Aim. We therefore investigated the survivability of a CL-associated keratitis isolate of P. aeruginosa (PA76203) in a commercially available multi-purpose disinfection solution (Opti-Free RepleniSH) when pre-treated with a sub-inhibitory concentration of solution.Methodology. Survival of PA76203 in 100% (v/v) disinfectant solution was evaluated for up to 6 h with both untreated control cultures and cultures pre-treated with 30% (v/v) solution, using bacterial colony count data. Transcriptomic and proteomic analysis of the model strain P. aeruginosa PAO1 was conducted to evaluate genes and proteins associated with growth in 30% (v/v) solution.Results. Untreated PA76203 was undetectable in the disinfection solution after 10 min incubation, whereas pretreated PA76203 was detectable at 6 h (P<0.05), indicating the acquisition of a tolerance phenotype. Transcriptomic and proteomic data from PAO1 treated with a sub-inhibitory concentration of disinfectant revealed 85 significantly differentially expressed genes and 342 differentially abundant proteins, respectively. Genes and proteins involved in LPS lipid A modifications (including arnA, which encodes the first functional enzyme in a lipid A modification operon) were shown to be upregulated in the pre-treated condition compared to the untreated control. The tolerance phenotype was not maintained in a strain of P. aeruginosa with a non-functional arnA gene.Conclusion. Exposure of P. aeruginosa to sub-inhibitory concentrations of disinfection solution enhances tolerance to previously lethal concentrations of solution and is positively associated with upregulation of genes involved in LPS lipid A modifications.

Introduction. Pseudomonas aeruginosa is a bacterial pathogen that causes acute infections in burned patients which can be very difficult to treat due to the high frequency of antibiotic-resistant strains. In 2019, whole-genome analysis led to the identification of five P. aeruginosa phylogroups including both clinical and environmental isolates. One of these phylogroups (Group 3) is highly genetically divergent and has been recently reclassified as Pseudomonas paraeruginosa based on genomic analysis, although there is no phenotypic difference with other P. aeruginosa phylogroups.Hypothesis. We propose that by using phylogroup-specific sequences, a multiplex PCR could be devised to detect Group 3 strains among clinical isolates from burned patients.Aim. To design a multiplex PCR that can be used to classify strains belonging to phylogroups 1, 2, 3 or 5. It is used to study a collection of 136 P. aeruginosa isolates obtained from 136 burned patients treated in a tertiary hospital in Mexico City during a 10-year period (2011 to 2021) to detect strains belonging to Group 3.Methodology. A multiplex PCR was devised to classify into four of the five P. aeruginosa phylogroups a collection of 136 strains isolated from burned patients. The genome sequence of the three strains belonging to Group 3 or P. paraeruginosa (P701, P1543 and P1565) was analysed bioinformatically, and their production of virulence factors was determined using reported microbiological procedures. Mutant complementation by genes carried in plasmids was performed to characterize the P1543-defective LasR protein. The MIC for 12 antibiotics of the 3 identified Group 3 strains was determined using standard techniques.Results. Using this multiplex PCR, we identified three strains belonging to the PA7 group (2%), which were sequenced and phenotypically characterized. We determined their production of virulence factors such as elastase, motility, biofilm formation, pyocyanin, rhamnolipids and their antibiotic resistance profile and showed that one of these strains (P1543) harbours a point mutation that inactivates lasR which causes elastase deficiency and lack of swarming motility and biofilm formation.Conclusion. These results show that P. aeruginosa Group 3 strains, or P. paraeruginosa isolates, have the same physiopathology as the most common P. aeruginosa phylogroup.

Introduction. Obstructive sleep apnoea syndrome (OSAS) is characterized by chronic intermittent hypoxia (CIH), which contributes to systemic complications, including metabolic and gastrointestinal disorders. Emerging evidence suggests a critical role of the gut microbiota in mediating these effects; however, the impact of CIH on the gut microbiota remains poorly understood.Gap Statement. While CIH is associated with systemic metabolic dysfunction, the specific alterations in gut microbiota composition and function induced by CIH remain understudied. Filling this knowledge gap could elucidate microbiota-mediated mechanisms of OSAS pathogenesis and identify therapeutic targets.Aim. To investigate the effects of CIH on the gut microbiota structure and functional pathways in a mouse model of OSAS.Methodology. Male C57BL/6 mice were exposed to normoxia (NM) or CIH conditions for 6 weeks. Faecal samples were collected via stress defecation before intervention (NM0 and CIH0 groups) and after 6 weeks (NM6 and CIH6 groups). Gut microbiota composition was assessed using 16S rRNA gene sequencing, and functional potential was predicted via Phylogenetic Investigation of Communities by Reconstruction of Unobserved States 2.Results. A total of 40 faecal samples (10 mice/group) were analysed. No significant differences in microbiota composition, alpha diversity or beta diversity were observed between groups before intervention. CIH significantly altered gut microbiota composition and abundance. At the genus level, Bacteroides abundance increased (rank-biserial=0.558, P=0.014) in CIH6 mice, while Bifidobacterium (Cohen's d=1.779, P=0.002), Helicobacter (rank-biserial=0.609, P=0.007) and Prevotella (rank-biserial=0.541, P=0.0173) decreased. Linear discriminant analysis effect size (LEfSe) and random forest model analyses identified these genera as key discriminators of microbiota composition. Kyoto Encyclopedia of Genes and Genomes functional prediction revealed 28 significantly altered tertiary metabolic pathways in CIH6 mice, including biotin, lipoic acid, beta-alanine and cyanoamino acid metabolism.Conclusion. CIH induces gut microbiota dysbiosis, disrupts short-chain fatty acid-producing bacteria and impacts multiple metabolic pathways. This study provides evidence linking gut microbiota alterations to OSAS pathogenesis and offers a theoretical foundation for targeting the microbiome as a potential therapeutic strategy for CIH-related disorders.

Introduction. Biofilms are a primary form of device-associated infections and typically exhibit high tolerance to antimicrobial agents. In biofilms formed by multiple microbial species, microorganisms may show even greater tolerance, complicating treatment. There is evidence that meropenem (MEPM) tolerance in Escherichia coli is increased in dual-species biofilms with Candida albicans, and effective treatments have not been established.Hypothesis/Gap Statement. If the presence of viable C. albicans increases the MEPM tolerance of E. coli in mature biofilms, then the killing of C. albicans will attenuate the MEPM tolerance of E. coli.Aim. We evaluated the effectiveness of various antifungal combination treatments against dual-species biofilms of E. coli and C. albicans in vitro and in vivo.Methodology. The reduction in the number of viable cells in dual-species mature biofilms formed by E. coli and C. albicans was evaluated after treatment with a combination of antifungal drugs (fluconazole, amphotericin B and micafungin) and MEPM. In addition, the in vivo effects of combination therapy were assessed using a silkworm biofilm infection model.Results. The combination of amphotericin B and MEPM reduced the viable cell counts of both E. coli and C. albicans within dual-species biofilms. In contrast, the combination of fluconazole and MEPM did not reduce the viable cell count of either species, whereas the combination of micafungin and MEPM reduced C. albicans only. The reduction in viable C. albicans counts by micafungin was less than that by amphotericin B, suggesting that micafungin did not affect the tolerance of E. coli. The combination of amphotericin B and MEPM also reduced the viable cell counts of both E. coli and C. albicans in the in vivo model.Conclusion. These findings suggest that the combination of amphotericin B and antibacterial agents is a potential treatment option to reduce the C. albicans-induced bacterial tolerance for catheter-related infections involving C. albicans co-infection.

Introduction. Allogeneic haematopoietic stem cell transplantation (allo-HSCT) is a treatment option for haematological malignancies. Allo-HSCT patients are more susceptible to viral infections due to immunosuppression.Gap Statement. The bone marrow transplant programme in Qatar, established within the past decade, remains in a developmental phase. A thorough evaluation of infection-related outcomes is essential to identify areas for improvement and to enhance infection control measures.Aim. This study aims to estimate the incidence, clinical impact and outcomes of viral respiratory tract infection (vRTI) in allo-HSCT recipients.Methodology. A total of 64 allo-HSCT patients were included in this study. Respiratory samples were collected from patients presenting with respiratory symptoms or during episodes of febrile neutropenia without an identified source. Nasopharyngeal swabs were the primary sampling method for upper respiratory tract infections (URTIs), while sputum, bronchoalveolar lavage or tracheal aspirates were obtained in patients with lower respiratory tract involvement or those requiring mechanical ventilation. During the high-risk COVID-19 pandemic period, pre-admission screening for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was routinely performed for all patients undergoing transplantation or chemotherapy. All samples were assessed using multiplex PCR assays. Viral agents, outcomes of vRTI occurring in the period of 1 year after allo-HSCT, clinical symptoms, infection-related complications and risk factors were reviewed.Result. A total of 64 allo-HSCT patients were reviewed; 41 of them (53%) had vRTI. SARS-CoV-2, respiratory syncytial virus (RSV), rhinovirus, influenza and parainfluenza were the most common viruses. Importantly, 19 patients (46.3%) had URTI, and 22 patients (53.7%) progressed to pneumonia. The risk of vRTI was significantly related to non-corticosteroid immune suppressants (P≤0.01) and to lymphopenia (P≤0.05). RSV (66.7%), coronaviruses (229E, NL63, OC43 and HKU1) (60%) and rhinovirus (58.3%) were the most dominant viruses associated with the development of pneumonia. Thirteen patients (20.3%) were admitted to the ICU; eight of them were associated with vRTI (61.5%). Ten deaths were reported (15.6%); vRTI was the primary cause of death in one of the deceased patients.Conclusion. Early detection and intervention strategies are crucial in mitigating the impact of these infections. The immune alteration effect of prophylaxis immune suppressants and antiviral therapy exacerbates the risk of infections among allo-HSCT patients. Novel antiviral approaches based on enhancing antiviral immune responses, vaccines and non-pharmaceutical preventive strategies are required to improve outcomes in allo-HSCT patients.

Introduction. Molecular epidemiological and phenotypic analyses of Listeria monocytogenes strains can inform our understanding of factors that influence onward transmission, virulence potential and ability to control the pathogen in foods or in clinical settings. Bile acids represent a host-specific barrier to microbial colonization of the gastrointestinal tract and are, therefore, not encountered in the external environment. We tested the hypothesis that tolerance of bile acids may be an evolutionary adaptation across L. monocytogenes clonal complexes (CCs), which varies with genotype and/or is associated with clinical isolates.Hypothesis. We hypothesized that strains of L. monocytogenes may differ in bile tolerance (a potential virulence-associated trait) and herein examine this phenomenon according to CC, strain origin and genotype.Aim. To assess 205 genome-sequenced isolates of L. monocytogenes for tolerance of porcine bile acids and bile salt hydrolase (BSH) activity.Methodology. Survival of L. monocytogenes strains was determined following exposure to porcine bile acids under conditions that mimic the small intestinal environment. BSH activity was assayed against pure taurodeoxycholic acid and glycodeoxycholic acid using an agar plate deconjugation assay. Genomes were analysed for polymorphisms in known bile tolerance loci.Results. Isolates demonstrated distinct inter-individual variances in bile tolerance under anaerobic conditions that mimic the intestinal environment. Strains isolated from cases of human disease were significantly more bile-tolerant than those isolated from natural environments or foods. There was no correlation between levels of bile tolerance and the size of bile precipitation zones on the BSH agar plate assay. No significant patterns were seen upon analysis of known or putative bile tolerance loci; however, individual strains with naturally occurring sigB operon mutations demonstrated reduced bile tolerance.Conclusion. L. monocytogenes strains isolated from clinical cases of listeriosis demonstrated elevated bile resistance consistent with a likely enhanced capacity to cause gastrointestinal infection preceding invasive disease. The data suggest the potential importance of bile tolerance in L. monocytogenes infection and highlight underlying molecular mechanisms by which strains vary in their natural levels of bile tolerance.