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 β-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 β-lactam resistance mechanism harbors the putative risk of acceleration or reacceleration of MRSA spread, resulting in broad ineffectiveness of β-lactams as a main therapeutic application against staphylococcal infections.
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 ⌬mecC strain showed considerably reduced oxacillin and cefoxitin MICs (0.25 and 4 g/ml, respectively) compared to those of the corresponding wild-type methicillin-resistant S. aureus (MRSA) strain (8 and 16 g/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.
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.
Aims:The aim of this study was to isolate and characterize staphylococcal isolates from diabetic foot ulcers (DFU) in Jordanian patients. Methods and Results: Selected aerobic pathogens recovered from DFU specimens and patients' nares with a focus on staphylococci were investigated. Antimicrobial susceptibilities and the prevalence of methicillin-resistant staphylococci (MRS) were determined. SCCmec types and toxigenic characteristics were analysed and spa typing was performed for methicillinresistant Staphylococcus aureus (MRSA) isolates. The relationship between toxigenic characteristics of MRSA and the Wagner ulcer grading system was statistically analysed. A total number of 87 DFU patients were recruited for the study. The DFU cultures were polymicrobial. Members of the genus Staphylococcus were the most common among DFU-associated isolates found in 48Á3% (n = 42) of all patients enrolled. Coagulase-negative staphylococci (CoNS) comprised 63Á3% of staphylococci isolated from DFUs predominated by Staphylococcus epidermidis in both DFU (7Á6%) and nares (39Á2%). Staphylococcus aureus was isolated from DFUs and nares in 14Á2 and 9Á8%, respectively, while 93 and 70% of these isolates were MRSA. Most of MRSA carried SCCmec type IV (76Á2%) while SCCmec elements were non-typeable in most methicillin resistant coagulase negative staphylococci (MR-CoNS) (61Á9%). The most frequent MRSA spa type was t386 (23Á8%). Most MRSA and MR-CoNS exhibited resistance towards aminoglycosides, fluoroquinolones and macrolides and susceptibility towards vancomycin, mupirocin and linezolid. No association was found between the possession of pvl, tst, sea and hlg toxins and Wagner ulcer grading system (P value >0Á05). Conclusions: This analysis of Jordanian DFU culture demonstrated its polymicrobial nature with predominance of Staphylococcus sp. Significance and Impact of the Study: This study is the first of its type to assess the microbiology of DFU among Jordanian patients. The results will help in the appropriate application of antimicrobial chemotherapy in the management of DFU.
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