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1.
Antibiotics (Basel) ; 11(11)2022 Nov 11.
Article in English | MEDLINE | ID: mdl-36421251

ABSTRACT

Small colony variants (SCVs) are clinically significant and linked to persistent infections. In this study, synchrotron-radiation-based Fourier transform infrared (SR-FTIR) is used to investigate the microspectroscopic differences between the SCVs of Staphylococcus aureus (S. aureus) and diabetic foot Staphylococcus epidermidis (S. epidermidis) in two main IR spectral regions: (3050-2800 cm-1), corresponding to the distribution of lipids, and (1855-1500 cm-1), corresponding to the distribution of protein amide I and amide II and carbonyl vibrations. SR-FTIR successfully discriminated between the two staphylococcal species and between the SCV and the non-SCV strains within the two IR spectral regions. Combined S. aureus SCVs (SCVhMu) showed a higher protein content relative to the non-SCV wild type. Complemented S. aureus SCV showed distinguishable differences from the SCVhMu and the wild type, including a higher content of unsaturated fatty acids. An increase in the CH2/CH3 ratio was detected in S. epidermidis SCV samples compared to the standard control. Protein secondary structure in standard S. epidermidis and SCVs consisted mainly of an α-helix; however, a new shoulder at 1635 cm-1, assigned to ß-sheets, was evident in the SCV. In conclusion, SR-FTIR is a powerful method that can discriminate between staphylococci species and to differentiate between SCVs and their corresponding natural strains.

2.
Microorganisms ; 10(2)2022 Feb 15.
Article in English | MEDLINE | ID: mdl-35208898

ABSTRACT

Drug alternatives to combat methicillin-resistant Staphylococcus aureus (MRSA) in human and animal healthcare are urgently needed. Recently, the recombinant bacteriophage endolysins, PRF-119 and its successor substance HY-133, have proven to be highly active against various S. aureus clonal lineages and to exhibit a very rapid bactericidal effect when standard methods for susceptibility testing are applied. Along with subsequent growth curve experiments, a re-growth phenomenon was observed in vitro necessitating its clarification for the assessment of the agent's stability and activity as well as for methodological aspects of endolysin testing in general. Distinct in vitro parameters were comparatively examined applying also scanning electron microscopy, fluorescence assays and SDS-PAGE analysis. The shape and material of the culture vessels as well as the shaking conditions were identified as factors influencing the in vitro stability and activity of HY-133. The highest function maintenance was observed in plain centrifuge tubes. Based on this, the conditions and parameters of assays for testing the antimicrobial activities of phage endolysins were determined and adjusted. In particular, shear forces should be kept to a minimum. Our results form the basis for both future test standardization and re-growth-independent experiments as prerequisites for exact determination of the antimicrobial activities of engineered endolysins.

3.
Front Microbiol ; 10: 2044, 2019.
Article in English | MEDLINE | ID: mdl-31551979

ABSTRACT

Staphylococcus aureus small-colony variants (SCVs) are associated with chronic, persistent, and relapsing courses of infection and are characterized by slow growth combined with other phenotypic and molecular traits. Although certain mechanisms have been described, the genetic basis of clinical SCVs remains often unknown. Hence, we adapted an episomal tool for rapid identification and investigation of putative SCV phenotype-associated genes via antisense gene silencing based on previously described Tnl0-encoded tet-regulatory elements. Targeting the SCV phenotype-inducing enoyl-acyl-carrier-protein reductase gene (fabI), plasmid pSN1-AS'fabI' was generated leading to antisense silencing, which was proven by pronounced growth retardation in liquid cultures, phenotype switch on solid medium, and 200-fold increase of antisense 'fabI' expression. A crucial role of TetR repression in effective regulation of the system was demonstrated. Based on the use of anhydrotetracycline as effector, an easy-to-handle one-plasmid setup was set that may be applicable to different S. aureus backgrounds and cell culture studies. However, selection of the appropriate antisense fragment of the target gene remains a critical factor for effectiveness of silencing. This inducible gene expression system may help to identify SCV phenotype-inducing genes, which is prerequisite for the development of new antistaphylococcal agents and future alternative strategies to improve treatment of therapy-refractory SCV-related infections by iatrogenically induced phenotypic switch. Moreover, it can be used as controllable phenotype switcher to examine important aspects of SCV biology in cell culture as well as in vivo.

4.
Int J Mol Sci ; 20(3)2019 Feb 07.
Article in English | MEDLINE | ID: mdl-30736446

ABSTRACT

Nasal carriage of methicillin-susceptible (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA) represents both a source and a risk factor for subsequent infections. However, existing MRSA decolonization strategies and antibiotic treatment options are hampered by the duration of administration and particularly by the emergence of resistance. Moreover, beyond classical resistance mechanisms, functional resistance as the formation of the small-colony variant (SCV) phenotype may also impair the course and treatment of S. aureus infections. For the recombinant bacteriophage endolysin HY-133, rapid bactericidal and highly selective in vitro activities against MSSA and MRSA has been shown. In order to assess the in vitro efficacy of HY-133 against the SCV phenotype, minimal inhibitory (MIC) and minimal bactericidal concentrations (MBC) were evaluated on clinical SCVs, their isogenic wild types, as well as on genetically derived and gentamicin-selected SCVs. For all strains and growth phases, HY-133 MIC and MBC ranged between 0.12 and 1 mg/L. Time-kill studies revealed a fast-acting bactericidal activity of HY-133 resulting in a ≥3 - log10 decrease in CFU/mL within 1 h compared to oxacillin, which required 4⁻24 h. Since the mode of action of HY-133 was independent of growth phase, resistance pattern, and phenotype, it is a promising candidate for future S. aureus decolonization strategies comprising rapid activity against phenotypic variants exhibiting functional resistance.


Subject(s)
Bacteriophages/physiology , Endopeptidases/genetics , Staphylococcus aureus/virology , Viral Proteins/genetics , Bacteriolysis , Endopeptidases/metabolism , Humans , Microbial Sensitivity Tests , Microbial Viability , Molecular Typing , Staphylococcal Infections/microbiology , Staphylococcus aureus/classification , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Viral Proteins/metabolism
5.
Diagn Microbiol Infect Dis ; 93(4): 362-368, 2019 Apr.
Article in English | MEDLINE | ID: mdl-30554844

ABSTRACT

Methicillin-resistant Staphylococcus aureus (MRSA) decolonization is expensive and time consuming, and new agents are necessary due to increasing resistance rates. The administration of bacteriophages or particularly their endolysins may offer an alternative treatment strategy and could provide a solution to overcome the selection pressure due to classical antibiotics. Here, the bactericidal activity was characterized for the recombinant chimeric bacteriophage endolysin HY-133 in comparison to other antimicrobials. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were determined for 2 reference strains, 24 clinical MRSA and methicillin-susceptible S. aureus (MSSA) isolates, as well as 6 isolates with high-level mupirocin resistance. Additionally, HY-133 activity against bacteria in stationary or exponential growth phase was compared in 12 isolates. Time-kill curves were performed with 2 representative isolates to investigate the pharmacodynamics until 48-h incubation time. All experiments were performed in comparison to daptomycin and mupirocin. The MIC50/90 and MBC50/90 values were in the range 0.12-0.5 mg/L for all 3 growth conditions comparable to daptomycin with 0.5/0.5 mg/L, respectively. The MBC was almost always equal the MIC and without considerable differences between MSSA and MRSA. Time-kill curves revealed a rapid bactericidal effect of HY-133 within the first 2 h, similar to daptomycin. Even with low concentrations, the recombinant endolysin HY-133 was highly active against all tested MSSA and MRSA isolates including mupirocin-resistant isolates. The application of this alternative agent may offer a future strategy for MRSA/MSSA decolonization and, potentially, for treatment purposes.


Subject(s)
Anti-Bacterial Agents/pharmacology , Endopeptidases/pharmacology , Microbial Viability/drug effects , Staphylococcus Phages/enzymology , Staphylococcus aureus/drug effects , Daptomycin/pharmacology , Endopeptidases/genetics , Microbial Sensitivity Tests , Mupirocin/pharmacology , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology
6.
Front Microbiol ; 9: 1863, 2018.
Article in English | MEDLINE | ID: mdl-30154773

ABSTRACT

Staphylococcal small-colony variants (SCVs) are invasive and persistent due to their ability to thrive intracellularly and to evade the host immune response. Thus, the course of infections due to this phenotype is often chronic, relapsing, and therapy-refractory. In order to improve treatment of patients suffering from SCV-associated infections, it is of major interest to understand triggers for the development of this phenotype, in particular for strains naturally occurring in clinical settings. Within this study, we comprehensively characterized two different Staphylococcus aureus triplets each consisting of isogenic strains comprising (i) clinically derived SCV phenotypes with auxotrophy for unsaturated fatty acids, (ii) the corresponding wild-types (WTs), and (iii) spontaneous in vitro revertants displaying the normal phenotype (REVs). Comparison of whole genomes revealed that clinical SCV isolates were closely related to their corresponding WTs and REVs showing only seven to eight alterations per genome triplet. However, both SCVs carried a mutation within the energy-coupling factor (ECF) transporter-encoding ecf module (EcfAA'T) resulting in truncated genes. In both cases, these mutations were shown to be naturally restored in the respective REVs. Since ECF transporters are supposed to be essential for optimal bacterial growth, their dysfunction might constitute another mechanism for the formation of naturally occurring SCVs. Another three triplets analyzed revealed neither mutations in the EcfAA'T nor in other FASII-related genes underlining the high diversity of mechanisms leading to the fatty acid-dependent phenotype. This is the first report on the ECF transporter as genetic basis of fatty acid-auxotrophic staphylococcal SCVs.

7.
Article in English | MEDLINE | ID: mdl-29378720

ABSTRACT

The Staphylococcus aureus small-colony variant (SCV) phenotype has been associated with relapsing and antibiotic-refractory infections. However, little is known about the activities of antibiotics on clinical SCVs. Here, we demonstrated that SCVs without detectable auxotrophies were at least as susceptible to most ß-lactam and non-ß-lactam antibiotics in vitro as their corresponding clonally identical strains with a normal phenotype. After prolonged incubation, a regrowth phenomenon has been observed in gradient diffusion inhibition zones irrespective of the strains' phenotype.


Subject(s)
Staphylococcus aureus/drug effects , beta-Lactams/pharmacology , Anti-Bacterial Agents/pharmacology , Daptomycin/pharmacology , Lincosamides/pharmacology , Linezolid/pharmacology , Microbial Sensitivity Tests , Trimethoprim/pharmacology
8.
Emerg Infect Dis ; 24(2): 242-248, 2018 02.
Article in English | MEDLINE | ID: mdl-29350135

ABSTRACT

During cefoxitin-based nasal screening, phenotypically categorized methicillin-resistant Staphylococcus aureus (MRSA) was isolated and tested negative for the presence of the mecA and mecC genes as well as for the SCCmec-orfX junction region. The isolate was found to carry a mecB gene previously described for Macrococcus caseolyticus but not for staphylococcal species. The gene is flanked by ß-lactam regulatory genes similar to mecR, mecI, and blaZ and is part of an 84.6-kb multidrug-resistance plasmid that harbors genes encoding additional resistances to aminoglycosides (aacA-aphD, aphA, and aadK) as well as macrolides (ermB) and tetracyclines (tetS). This further plasmidborne ß-lactam resistance mechanism harbors the putative risk of acceleration or reacceleration of MRSA spread, resulting in broad ineffectiveness of ß-lactams as a main therapeutic application against staphylococcal infections.


Subject(s)
Bacterial Proteins/genetics , Methicillin Resistance/genetics , Methicillin-Resistant Staphylococcus aureus/genetics , Plasmids/genetics , Aged , Gene Transfer, Horizontal , Humans , Male
9.
Antimicrob Agents Chemother ; 58(7): 3791-8, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24752255

ABSTRACT

In staphylococci, methicillin resistance is mediated by mecA-encoded penicillin-binding protein 2a (PBP2a), which has a low affinity for beta-lactams. Recently, a novel PBP2a homolog was described as being encoded by mecC, which shares only 70% similarity to mecA. To prove that mecC is the genetic determinant that confers methicillin resistance in Staphylococcus aureus, a mecC knockout strain was generated. The S. aureus ΔmecC strain showed considerably reduced oxacillin and cefoxitin MICs (0.25 and 4 µg/ml, respectively) compared to those of the corresponding wild-type methicillin-resistant S. aureus (MRSA) strain (8 and 16 µg/ml, respectively). Complementing the mutant in trans with wild-type mecC restored the resistance to oxacillin and cefoxitin. By expressing mecC and mecA in different S. aureus clonal lineages, we found that mecC mediates resistance irrespective of the genetic strain background, yielding oxacillin and cefoxitin MIC values comparable to those with mecA. In addition, we showed that mecC expression is inducible by oxacillin, which supports the assumption that a functional beta-lactam-dependent regulatory system is active in MRSA strains possessing staphylococcal cassette chromosome mec (SCCmec) type XI. In summary, we showed that mecC is inducible by oxacillin and mediates beta-lactam resistance in SCCmec type XI-carrying strains as well as in different S. aureus genetic backgrounds. Furthermore, our results could explain the comparatively low MICs for clinical mecC-harboring S. aureus isolates.


Subject(s)
Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Drug Resistance, Bacterial/genetics , Penicillin-Binding Proteins/genetics , Penicillin-Binding Proteins/metabolism , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , beta-Lactams/pharmacology , Gene Knockout Techniques , Genes, Reporter/genetics , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Methicillin-Resistant Staphylococcus aureus/drug effects , Microbial Sensitivity Tests , Plasmids/genetics , Promoter Regions, Genetic/genetics , RNA, Bacterial/genetics , Species Specificity
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