First detection of triazole-resistant aspergillus fumigatus harbouring the TR34/L98H Cyp51A mutation in Burkina Faso.

BACKGROUND: Triazole-resistant Aspergillus fumigatus (TRAF) isolates are a growing public health problem with worldwide distribution. Epidemiological data on TRAF is limited in Africa, particularly in West Africa. OBJECTIVES: This study aimed to screen for the environmental presence of TRAF isolates in the indoor air of two hospitals in Burkina Faso. MATERIALS AND METHODS: Air samples were collected in wards housing patients at risk for invasive aspergillosis, namely infectious diseases ward, internal medicine ward, nephrology ward, pulmonology ward, medical emergency ward and paediatric ward. Sabouraud Dextrose Agar supplemented with triazoles was used to screen the suspected TRAF isolates and EUCAST method to confirm the resistance of suspected isolates. Sequencing of cyp51A gene was used to identify the resistance mechanism of confirmed TRAF isolates. RESULTS: Of the 198 samples collected and analysed, 67 showed growth of A. fumigatus isolates. The prevalence of TRAF isolates was 3.23% (4/124). One TRAF isolate exhibited a pan-triazole resistance. Sequencing of cyp51A gene identified the TR34/L98H mutation for this pan-triazole resistant isolate. This study showed for the first time the circulation of the pan-azole resistant isolate harbouring the TR34/L98H mutation in Burkina Faso. CONCLUSIONS: These findings emphasise the need to map these TRAF isolates in all parts of Burkina Faso and to establish local and national continuous surveillance of environmental and clinical TRAF isolates in this country.

Quantitative PCR Effectively Quantifies Triazole-Susceptible and Triazole-Resistant Aspergillus fumigatus in Mixed Infections.

Increasing resistance to triazole antifungals in Aspergillus fumigatus is worrisome because of the associated high mortality of triazole-resistant A. fumigatus (TRAF) infections. While most studies have focused on single triazole-susceptible (WT) or TRAF infections, reports of TRAF cases developing mixed WT and TRAF infections have been described in several studies. However, the prevalence of mixed infections and their responses to current recommended therapies are unknown and could be inappropriate, leading to poor clinical outcomes. To address the urgent need for tools to diagnose, monitor disease development and therapy efficacies in mixed infection settings where quantification of WT versus TRAF is key, this study developed a novel qPCR assay to differentiate WT and TRAF harboring the cyp51A-TR34/L98H mutation. The proposed assay successfully quantified A. fumigatus and discriminated TRAF-TR34 in vitro and in vivo, which was achieved by increasing the yield of extracted DNA through improved homogenization and specific primers targeting the WT-sequence or TR34-insertion and a TaqMan-probe directed to A. fumigatus. The here-developed qPCR assay overcomes sensitivity issues of methodologies such as CFU counts, providing specific, reproducible, and reliable quantitative information to study and follow up the (interplay and individual) effects of mixed A. fumigatus infections on disease development and treatment responses.

Stable prevalence of triazole-resistance in Aspergillus fumigatus complex clinical isolates in a Belgian tertiary care center from 2016 to 2020.

BACKGROUND: Prevalence reports of triazole-resistance in Aspergillus fumigatus differ between countries and centers and may likewise vary over time. Continuous local surveillance programs to establish the evolving epidemiology of triazole-resistance in A. fumigatus are crucial to guide therapeutic recommendations. Here, we determined the prevalence of triazole-resistance in A. fumigatus complex culture-positive patients at the tertiary care center University Hospitals Leuven in Belgium in clinical isolates from 2016 to 2020. METHODS: All A. fumigatus complex isolates cultured from UZ Leuven patients between 2016 and 2020 were screened for triazole-resistance. Confirmation of resistance to voriconazole, posaconazole and itraconazole was performed with the European Committee for Antimicrobial Susceptibility Testing (EUCAST) broth microdilution method. Mutations in the cyp51A gene in triazole-resistant isolates were determined by sequencing. Patients were classified as susceptible or resistant cases based on their isolate's susceptibility phenotype. RESULTS: We screened 2494 A. fumigatus complex isolates from 1600 patients (320 ± 38 [SD] patients per year). The prevalence of triazole-resistance in patients was 8.3% (28/337), 6.7% (26/386), 7.0% (21/301), 7.1% (21/294) and 7.4% (21/282) in 2016, 2017, 2018, 2019 and 2020 respectively, with an overall triazole-resistance prevalence of 7.1% (85/1192; 95% CI 6.6-7.7%). The TR34/L98H mutation was the most prevalent (83.0%, 78/94) with most isolates displaying resistance to all triazole antifungals tested (94.8%, 74/78). CONCLUSION: The prevalence of triazole-resistance in A. fumigatus has remained stable from 2016 to 2020 in our center ranging between 6.7 and 8.3%, with an overall five-year prevalence of 7.1%. The environmentally associated cyp51A gene mutations were most prevalent amongst triazole-resistant isolates and conferred resistance to all antifungals tested in 73% of the isolates.

Triazole-Resistance in Environmental Aspergillus fumigatus in Latin American and African Countries.

Triazole-resistance has been reported increasingly in Aspergillus fumigatus. An international expert team proposed to avoid triazole monotherapy for the initial treatment of invasive aspergillosis in regions with >10% environmental-resistance, but this prevalence is largely unknown for most American and African countries. Here, we screened 584 environmental samples (soil) from urban and rural locations in Mexico, Paraguay, and Peru in Latin America and Benin and Nigeria in Africa for triazole-resistant A. fumigatus. Samples were screened using triazole-containing agars and confirmed as triazole-resistant by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) broth dilution reference method. Isolates were further characterized by cyp51A sequencing and short-tandem repeat typing. Fungicide presence in samples was likewise determined. Among A. fumigatus positive samples, triazole-resistance was detected in 6.9% (7/102) of samples in Mexico, 8.3% (3/36) in Paraguay, 9.8% (6/61) in Peru, 2.2% (1/46) in Nigeria, and none in Benin. Cyp51A gene mutations were present in most of the triazole-resistant isolates (88%; 15/17). The environmentally-associated mutations TR34/L98H and TR46/Y121F/T289A were prevalent in Mexico and Peru, and isolates harboring these mutations were closely related. For the first time, triazole-resistant A. fumigatus was found in environmental samples in Mexico, Paraguay, Peru, and Nigeria with a prevalence of 7-10% in the Latin American countries. Our findings emphasize the need to establish triazole-resistance surveillance programs in these countries.

Hmg1 Gene Mutation Prevalence in Triazole-Resistant Aspergillus fumigatus Clinical Isolates.

Recently, mutations in the 3-hydroxy-3-methylglutaryl-coenzyme-A-reductase-encoding gene (hmg1), a gene involved in ergosterol production, were associated with triazole-resistance in Aspergillus fumigatus. In this study, we determined the prevalence and characteristics of hmg1 mutations in a collection of clinical triazole-resistant A. fumigatus isolates collected during 2001-2019 from two international mycology reference centers: the Belgian National Reference Center for Mycosis and the Center of Expertise in Mycology Radboudumc/CWZ. Clinical isolates with and without cyp51A gene mutations and randomly selected wild-type (WT) controls were included. Isolates were characterized by in vitro susceptibility testing, cyp51A and hmg1 sequencing, and short tandem repeat typing. Available clinical records were analyzed for previous triazole exposure. In 23 isolates (24%) of the 95 triazole-resistant A. fumigatus isolates, hmg1 gene mutations were observed; including 5/23 (22%) isolates without cyp51A gene mutations and 18/72 (25%) with cyp51A mutations. Four previously described hmg1 gene mutations (E105K, G307R/D, G466V, and S541G) and two novel mutations (W273S and L304P) were found; 4/23 (17%) in the sterol-sensing-domain region. No triazole-antifungal exposure was reported in 75% (9/12) of patients harboring an isolate with hmg1 gene mutations. Three of 39 WT isolates (8%) contained a hmg1 gene mutation; E105K (2-isolates) and S541G. Hmg1 gene mutations were predominantly found in A. fumigatus with cyp51A mutations with voriconazole MICs ≥ 8 mg/L.

The Possible Role of Staphylococcus epidermidis LPxTG Surface Protein SesC in Biofilm Formation.

Staphylococcus epidermidis is the most common cause of device-associated infections. It has been shown that active and passive immunization in an animal model against protein SesC significantly reduces S. epidermidis biofilm-associated infections. In order to elucidate its role, knock-out of sesC or isolation of S. epidermidis sesC-negative mutants were attempted, however, without success. As an alternative strategy, sesC was introduced into Staphylococcus aureus 8325-4 and its isogenic icaADBC and srtA mutants, into the clinical methicillin-sensitive S. aureus isolate MSSA4 and the MRSA S. aureus isolate BH1CC, which all lack sesC. Transformation of these strains with sesC i) changed the biofilm phenotype of strains 8325-4 and MSSA4 from PIA-dependent to proteinaceous even though PIA synthesis was not affected, ii) converted the non-biofilm-forming strain 8325-4 ica::tet to a proteinaceous biofilm-forming strain, iii) impaired PIA-dependent biofilm formation by 8325-4 srtA::tet, iv) had no impact on protein-mediated biofilm formation of BH1CC and v) increased in vivo catheter and organ colonization by strain 8325-4. Furthermore, treatment with anti-SesC antibodies significantly reduced in vitro biofilm formation and in vivo colonization by these transformants expressing sesC. These findings strongly suggest that SesC is involved in S. epidermidis attachment to and subsequent biofilm formation on a substrate.

Inhibition of Staphylococcus epidermidis biofilm formation by rabbit polyclonal antibodies against the SesC protein.

Several well-studied proteins with defined roles in Staphylococcus epidermidis biofilm formation are LPXTG motif-containing proteins. Here, we investigate the possible use of the LPXTG motif-containing protein SesC (S. epidermidis surface protein C; accession no. NP_765787) as a target for antibodies to prevent biofilm formation. In vitro and in a in vivo rat model of catheter infection, gene and protein expression analysis showed that SesC is expressed more strongly in biofilm-associated cells than in planktonic cells and is expressed particularly during the late phase of in vivo biofilm formation. Polyclonal rabbit antibodies raised against SesC reduced the fibrinogen-binding ability of S. epidermidis RP62A and Staphylococcus aureus RN4220 transformants expressing SesC, inhibited in vitro biofilm formation by S. epidermidis strains 10b and 1457, and significantly reduced the numbers of bacteria in a 1-day-old in vivo biofilm (P < 0.001, one-way analysis of variance). Our findings revealed that SesC is a promising target for prevention and treatment of S. epidermidis biofilms because it affects both the primary attachment and biofilm accumulation phases. The precise role of SesC in biofilm formation remains to be identified.

The role of sigmaB in persistence of Staphylococcus epidermidis foreign body infection.

Staphylococcal biofilm formation depends on the transcription factor sigma(B). We further investigated the role of sigma(B) in biofilm formation and persistence in vitro and in vivo in a subcutaneous rat model. As expected, expression of all sigma(B) operon genes was transiently higher in the first 6 h of biofilm formation compared to planktonic bacteria, concurrent with a temporary upregulation of icaA and aap expression. However, we also observed a second upregulation of sigB expression in biofilm more than 2 days old without upregulation of icaA or aap. Biofilm formation by Staphylococcus epidermidis strains 8400 and 1457 was compared to that of isogenic mutants with inactivation of rsbU, of rsbUVW and of the entire sigma(B) operon. Both wild-type strains and the constitutively sigB-expressing rsbUVW mutant showed a strong biofilm-positive phenotype. The rsbUVW mutant biofilm was, however, thinner and more evenly spread than the wild-type biofilm. Inactivation of SigB in the rsbUVWsigB mutant or mutation of the positive regulator RsbU reduced both the number of sessile bacteria and polysaccharide intercellular adhesin (PIA) synthesis. These differences between the wild-types and their respective mutants appeared after 6 h in in vitro biofilms but only after 4 days in in vivo biofilms. Our results provide additional evidence for a role for sigma(B) in biofilm formation. They also suggest a role for sigma(B) in biofilm maturation and stability that is independent of PIA or accumulation-associated protein (Aap) and point to significant differences in the temporal development between in vitro and in vivo biofilms.

Effect of iron on the expression of sirR and sitABC in biofilm-associated Staphylococcus epidermidis.

BACKGROUND: Different gene expression patterns correlate with the altered phenotype in biofilm-associated bacteria. Iron and iron-linked genes are thought to play a key-role in biofilm formation. The expression of Fe-linked genes (sirR, sitABC operon) in Staphylococcus epidermidis, was compared in planktonic versus sessile bacteria in vitro and in vivo in a subcutaneous foreign body rat model. RESULTS: In vitro in a Fe-limited environment, the planktonic form of S. epidermidis produces siderophores and grows slower than in Fe-rich environment. The expression of sirR in planktonic bacteria, in vitro, was not different in medium without Fe or with 1 microM FeCl3. High Fe concentrations (25 microM FeCl3) increased expression of sirR transiently during the early phase of incubation. Expression of sitC in vitro, in planktonic bacteria, was inversely correlated with sirR expression in medium with 25 microM FeCl3: sitC expression decreased for the first 3 hours followed by an up regulation.In sessile bacteria in vitro, sirR expression was high and independent of the Fe concentration. The expression of sitC was not inversely correlated to sirR expression. In vivo, expression levels of sirR and of sitABC were high during the initial phase after implantation and, after a transient decrease, remained stable over a period of two weeks. CONCLUSION: Our data suggest that the expression of sirR and the regulatory effect of sirR on the sitABC operon are different in planktonic and sessile bacteria.

The effect of systemic antibiotics on the microbiological diagnosis of experimental foreign body infections caused by Staphylococcus epidermidis.

Qualitative broth culture and quantitative culture on agar were compared with quantitative polymerase chain reaction (PCR) for the diagnosis of foreign body infections (FBI) in a rat model with and without exposure to systemic antibiotics (teicoplanin and rifampin). The 3 methods had a similar and high yield without antibiotics. Antibiotics decreased the number of CFU/foreign body and increased the number of culture-negative foreign bodies and the variability of the results in quantitative culture. The yield of broth culture remained high under antibiotics although prolonged incubation (2-5 days) was required. The yield of the PCR was equivalent or even superior (for teicoplanin) to the yield of broth culture. Quantitative PCR had a higher yield and lower variability than quantitative culture and was not affected by antibiotics. The simultaneous isolation of RNA from all samples indicated viability of the bacteria. Quantitative PCR seems a promising method for the diagnosis of FBI.

Expression of biofilm-associated genes in Staphylococcus epidermidis during in vitro and in vivo foreign body infections.

The expression of the genes icaA, icaC, aap, and atlE--with a putative role in the pathogenesis of Staphylococcus epidermidis foreign body infections--and of mecA and 3 housekeeping genes (gmk, tpi, and hsp60) was examined in vitro and in vivo. In vitro expression levels of ica, atlE, and gmk were higher in sessile than in planktonic bacteria. Exposure to foreign bodies in vitro and in vivo induced a sharp increase in ica expression that was followed by a progressive decrease. The in vivo expression of aap and mecA was high during early but low during late foreign body infections. The in vivo expression of atlE and gmk remained relatively high and stable. In conclusion, biofilm genes encoding for structural elements are mainly expressed during early foreign body infections. The ica genes are associated with initial colonization but not with persistence. The constant expression of atlE and gmk may indicate a role during the entire course of foreign body infections.