Novel nonsense mutation in the katA gene of a catalase-negative Staphylococcus aureus strain (original) (raw)
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International journal of medical microbiology : IJMM, 2017
We have screened 2568 healthy individuals (mostly children) for Staphylococcus aureus and Streptococcus pneumoniae nasal carriage between 2010 and 2012. Out of the isolated 751 S. aureus strains, we found one methicillin-sensitive catalase-negative S. aureus (CNSA). Our CNSA isolate possessed a novel nonsense point mutation in the katA gene leading to early truncation of the protein product. The strain was resistant to penicillin and erythromycin, but sensitive to all other tested antibiotics and carried the enterotoxin A gene. It belonged to sequence type 5 (ST5), which is a successful, worldwide spread, usually MRSA clone. Catalase has been described as a virulence factor strictly required for nasal colonisation, and this is the first case contradicting this theory, as all previous CNSA isolates derived from infections. This is the first report of a CNSA from a symptomless carrier as well as the first occurrence in Hungary.
The catalase gene differentiates between some strains of Staphylococcus aureus ssp. anaerobius
Folia Microbiologica, 2010
Staphylococcus aureus ssp anaerobius strain S10 was isolated from an outbreak of sheep abscess disease. Sequence of the catalase gene of this strain showed 99 % identity to the catalase gene (katB) sequence of the reference strain (S. aureus ssp. anaerobius strain MVF213) with mismatching of three base pairs. An important substitution located 1036 nucleotides upstream of the initiation codon from “C” in katB to “T” in the catalase gene of strain S10 originated a stop codon. The deduced protein (345 amino acids) is 105 amino acids shorter than that of katB. Partial sequence of the catalase gene of other 8 local isolates in addition to another reference strain (DSM 20714/ATCC 35844) revealed the same mutations in all local (African) strains, whereas the sequence of the reference (European) strain was typical to that of katB. Sequence of the catalase gene of S. aureus ssp. anaerobius strain S10 was deposited in GenBank under accession no. EU281993.
Role of Staphylococcus aureus catalase in niche competition against Streptococcus pneumoniae
2008
Nasal colonization by Staphylococcus aureus is a major predisposing factor for subsequent infection. Recent reports of increased S. aureus colonization among children receiving pneumococcal vaccine implicate Streptococcus pneumoniae as an important competitor for the same niche. Since S. pneumoniae uses H 2 O 2 to kill competing bacteria, we hypothesized that oxidant defense could play a significant role in promoting S. aureus colonization of the nasal mucosa. Using targeted mutagenesis, we showed that S. aureus expression of catalase contributes significantly to the survival of this pathogen in the presence of S. pneumoniae both in vitro and in a murine model of nasal cocolonization. Staphylococcus aureus causes a wide range of infections ranging from minor skin infections to life-threatening invasive diseases. The emergence of methicillin-resistant strains with high virulence potential in both hospital and community settings is contributing to a current public health crisis (9, 12, 13). A major risk factor for S. aureus infection is antecedent colonization of the nasal mucosa (19). Successful colonization depends not only on the ability of S. aureus to survive host factors (4, 6) but also on coexistence with other bacteria (16, 21). The latter concept has been underscored by two recent reports that implicate Streptococcus pneumoniae as a primary competitor for niche colonization (3, 15). Specifically, one surveillance study performed in an area where pneumococcal vaccination was not practiced showed that the S. pneumoniae carriage rate in children was negatively associated with S. aureus nasal carriage (15). The other study showed that children with recurrent otitis media vaccinated with the 7-valent pneumococcal vaccine had an increased incidence of S. aureus-related acute otitis media and S. aureus colonization after vaccination (3), suggesting that there is a natural competition for colonization between S. aureus and S. pneumoniae. S. pneumoniae produces H 2 O 2 as an antimicrobial factor to reduce competition by other upper respiratory pathogens, such as Haemophilus influenzae, Neisseria meningitides, Moraxella catarrhalis, and S. aureus (14, 16). Since S. aureus is a natural colonizer of the human nares, we hypothesized that its success derives in part from a relative resistance to H 2 O 2 killing by other microflora. Here we tested this hypothesis by generating a catalase knockout mutant strain of S. aureus and examining the role of enzymatic H 2 O 2 inactivation in niche competition with S. pneumoniae. MATERIALS AND METHODS Bacterial strains, media, and mice. S. aureus strains were cultured at 37°C in Todd-Hewitt broth (THB) or on Todd-Hewitt agar (THA) (Difco). S. pneumoniae TIGR4 was cultured in THB with 0.5% yeast extract (THY) at 37°C in a 5% CO 2 incubator. Eight-to 10-week old female CD1 mice were purchased from Charles River Laboratories, Wilmington, MA. When included, antibiotics were added at the following concentrations: 100 g ampicillin/ml, 50 g erythromycin/ml, and 100 g spectinomycin/ml. Generation of catalase-deficient S. aureus ⌬KatA mutant. In-frame allelic replacement of the S. aureus katA gene with a spectinomycin adenyltransferase (spec) cassette was performed using PCR-based methods as described previously (11), with minor modifications. Primers were designed based on the previously published S. aureus katA sequence cross-referenced to the genome of S. aureus strain N315 (10). PCR was used to amplify 500 bp upstream of katA with primers katAupF (5Ј-ATGGTCGACTATGACATCAACACTTGTAAC-3Ј) and katAupR (5Ј-TCA AATATATCCTCCTCATCCCTCCACAATTTATAATAAT-3Ј) along with 500 bp of sequence immediately downstream of katA with primers katAdownF (5Ј-AA TAACAGATTAAAAAAATTATAAATTTGATATGTAGTTTCTATA-3Ј) and katAdownR (5Ј-ATCGGATCCTACCCAGAATTACTTCGTACT-3Ј). The katAupR and katAdownF primers were constructed with 25-bp 5Ј extensions corresponding to the 5Ј and 3Ј ends of the spec gene, respectively. The upstream and downstream PCR products were then combined with a 650-bp amplicon of the complete spec gene for use as templates in a second round of PCR using primers katAupF and katAdownR. The resultant PCR amplicon, containing an in-frame substitution of katA with spec, was subcloned into temperature-sensitive vector pMAD (1) to create the knockout plasmid. This vector was transformed initially into permissive S. aureus strain RN4220 and then into S. aureus strain Newman by electroporation. Transformants were grown at 30°C and shifted to the nonpermissive temperature for plasmid replication (40°C), and differential antibiotic selection and blue-white color selection with 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) were used to identify candidate mutants. Allelic replacement of the katA allele was confirmed unambiguously by PCRs that documented targeted insertion of spec and the absence of katA in chromosomal DNA isolated from the final mutant, which was designated the ⌬KatA mutant. Complementation studies. Primers katAF_KpnI (5Ј-ATAGGTACCTCCCAT GGTAAAGCCAAGAG-3Ј) and katAR_BamHI (5Ј-ATAGGATCCTTTACGC GCACGTTAAACAC-3Ј) were used to amplify the katA gene from the chromosome of wild-type (WT) S. aureus strain Newman. The fragment was directionally cloned into the shuttle expression vector pDCerm (8), and the recombinant plasmid (pKatA) was used to transform the S. aureus ⌬KatA mutant by electroporation. For the complementation studies, the isogenic WT and ⌬KatA S. aureus strains were transformed with the control pDCerm plasmid. Strains containing the pDCerm or pKatA plasmid were maintained in THB or on THA containing erythromycin. H 2 O 2 susceptibility assay. H 2 O 2 susceptibility assays were performed using overnight S. aureus cultures grown at 37°C with shaking. Bacteria were harvested by centrifugation, suspended in phosphate-buffered saline (PBS) at a concentra
An outbreak of catalase-negative methicillin-resistant Staphylococcus aureus
The Journal of hospital infection, 2007
The wide dissemination of a major epidemic methicillin-resistant Staphylococcus aureus (MRSA) clone in Brazilian hospitals (Brazilian clone) limits the value of molecular typing techniques such as pulsed-field gel electrophoresis (PFGE) for outbreak investigation. We report the first outbreak of a catalase-negative strain of MRSA, which was initially detected by the unusual result of this phenotypical test. The outbreak occurred in the Hospital Sanatorinhos de Carapicuíba, a 237-bed secondary hospital located in São Paulo, Brazil. From May to August 2002, a total of 11 MRSA isolates were recovered from four patients in the intensive care unit. All the isolates were catalase negative and susceptible only to vancomycin and linezolid. Three of the four patients eventually died. Molecular typing demonstrated an indistinguishable PFGE pattern among the 11 isolates, with similarities to the Brazilian clone and the hospital's usual MRSA strain. This report emphasizes the importance of ...
Non-spa-Typeable Clinical Staphylococcus aureus Strains Are Naturally Occurring Protein A Mutants
Journal of Clinical Microbiology, 2009
Staphylococcus aureus is a major human pathogen responsible for increasing the prevalence of community- and hospital-acquired infections. Protein A (SpA) is a key virulence factor of S. aureus and is highly conserved. Sequencing of the variable-number tandem-repeat region of SpA ( spa typing) provides a rapid and reliable method for epidemiological studies. Rarely, non- spa -typeable S. aureus strains are encountered. The reason for this is not known. In this study, we characterized eight non- spa -typeable bacteremia isolates. Sequencing of the entire spa locus was successful for five strains and revealed various mutations of spa , all of which included a deletion of immunoglobulin G binding domain C, in which the upper primer for spa typing is located, while two strains were truly spa negative. This is the first report demonstrating that nontypeability of S. aureus by spa sequencing is due either to mutation or to a true deficiency of spa .
FEMS Microbiology Letters, 2002
The catalase gene katA of Staphylococcus xylosus was cloned. It encodes a protein of 494 amino acids with a molecular mass of 56.9 kDa, closely related to monofunctional catalases. A katA mutant still showed a relatively high catalase activity demonstrating that S. xylosus possesses more than one enzyme. By Southern blot analysis using a katA probe, a second genetic locus distinct from katA was detected that probably contained the additional catalase gene. To analyse katA expression, a transcriptional fusion of the katA promoter region to a promoterless L-galactosidase gene was integrated into the genome of S. xylosus. katA expression is induced upon entry into stationary phase, by oxygen and hydrogen peroxide. Iron and manganese depletion induced katA transcription. Comparing the resistance of S. xylosus wild-type and the katA mutant strain to hydrogen peroxide clearly showed that KatA is essential for S. xylosus to cope with hydrogen peroxide stress. Therefore, S. xylosus has at least two differentially expressed catalases.
Journal of Clinical Microbiology, 2006
One hundred seventy Staphylococcus aureus isolates, collected in 1996 to 2004, were reidentified by phenotypic and genotypic methods. One hundred ten of these (65%) were confirmed, as previously denoted, to be clumping factor (CF)-or free coagulase-deficient S. aureus, based on their phenotype. Based on the CF or coagulase production, three groups of phenotypically deficient S. aureus isolates were distinguished. Group 1 encompassed CF-positive and coagulase-deficient isolates, group 2 consisted of CF-deficient and coagulasepositive isolates, and group 3 included isolates that were CF positive, had delayed coagulase activity, and were deficient in other species-specific features. All investigated strains harbored the clfA, clfB, coa, spa, and nuc genes, but the presence of their products was not detected by the phenotypic methods. Glycopeptide susceptibility testing showed that 26 isolates (23.6%) were hetero-glycopeptide-intermediate S. aureus (hGISA) or hetero-teicoplanin-intermediate S. aureus (hTISA), based on the population analysis profile. The relatedness of the isolates was evaluated by multiple-locus variable number of tandem repeats analysis, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing. The phenotypically deficient S. aureus isolates were classified into PFGE types B (ST239-III) and D (ST246-IA) and were related to the common clones, Hungarian and Iberian, respectively, which have been widely disseminated in Poland and globally. The simultaneous occurrence of hGISA/hTISA and the CF-deficient phenotypes was found for 62.1% of isolates belonging to group 2. The majority of these isolates were assigned to the Iberian clone (PFGE type D; ST247-IA). An association between the defect in coagulase and that in thermonuclease production was observed, which concerned 59.2% of isolates of group 1. The majority of these isolates belonged to the Hungarian clone (PFGE type B; ST239-III).
Journal of Dairy Science, 2020
The most clinically relevant staphylococci in veterinary medicine are those that are coagulase-positive, namely Staphylococcus aureus. During microbiological udder health monitoring (2009-2018), a new S. aureus strain (coagulase-positive and maltose-negative) was discovered as an emerging udder pathogen during routine examinations of South African dairy herds. This study challenged the conventional microbiological diagnosis of staphylococci by comparing its results to those of the MALDI-TOF mass spectrometry and 16S rRNA sequencing. Both of these tests confirmed that the maltose-negative staphylococcus (MNS), identified as Staphylococcus pseudintermedius by conventional microbiology, was S. aureus ST2992. Multi locus sequence typing was performed on 3 of the MNS isolates and indicated that these isolates were of single origin. These strains tested positive for both MALA and MALR genes (control: S. aureus ATCC 25923). Although the α-glucosidase gene was present, it was not expressed phenotypically. The latter may be attributed to the abnormal stop codon identified in the MALA gene sequence of S. aureus ST2992 (GenBank accession number, MN531305). The newly identified MNS has a field behavior different to that of maltose-positive S. aureus, and more similar to the low virulence of non-aureus staphylococci.