Detection of Methicillin-Susceptible Staphylococcus aureus ST398 and ST133 Strains in Gut Microbiota of Healthy Humans in Spain

Fecal samples of 100 healthy humans were tested for Staphylococcus aureus recovery. Fifteen samples (15 %) contained S. aureus, all methicillin-susceptible (MSSA), being one isolate/sample further studied. These 15 isolates were characterized by spa and agr typing as well as multi-locus sequence typing. High diversity of spa types (n?=?11) and sequences types (n?=?8) was detected. Two S. aureus of lineages ST398 or ST133 were detected, and six isolates were ascribed to clonal complex 30 (CC30). Strains were susceptible to most of the 17 antimicrobial agents tested with exceptions: erythromycin/clindamycin (three strains, containing erm(C) and/or erm(A) + mph(C) genes) and tobramycin and mupirocin (one strain containing ant(4′)-Ia + mup(A) genes). The presence of 18 staphylococcal enterotoxin genes was studied by PCR, and isolates were negative for lukF/lukS-PV genes, although strain ST133 harbored the lukD-lukE + lukM genes. Other virulence genes detected were (number of strains): tsst-1 (6), hla (15), hlb (9), hld (15), hlg (6), hlgv (9), cna (2), aur (14), and egc-like cluster (3). Analysis of immune evasion cluster genes showed six types, highlighting their absence in two strains of lineages ST133 and ST5. A high clonal diversity of MSSA strains was identified in the intestinal microbiota of healthy humans, being CC30 the most frequent one. This is the first report of MSSA ST133 and ST398 isolates in gut microbiota of healthy humans.     

Staphylococcus aureus CC398 Clade Associated with Human-to-Human Transmission

Staphylococcus aureus clonal complex 398 (CC398) isolates colonize livestock and can spread to human contacts. Genetic analysis of isolates epidemiologically associated with human-to-human, but not livestock, transmission in multiple countries and continents identified a common clade that was negative for tet(M) and positive for bacteriophage ϕ3. Another group of human-to-human-transmitted isolates belonged to the common livestock-associated clade but had acquired a unique ϕ7 bacteriophage.     

Rapid Differentiation between Livestock-Associated and Livestock-Independent Staphylococcus aureus CC398 Clades

Staphylococcus aureus clonal complex 398 (CC398) isolates cluster into two distinct phylogenetic clades based on single-nucleotide polymorphisms (SNPs) revealing a basal human clade and a more derived livestock clade. The scn and tet(M) genes are strongly associated with the human and the livestock clade, respectively, due to loss and acquisition of mobile genetic elements. We present canonical single-nucleotide polymorphism (canSNP) assays that differentiate the two major host-associated S. aureus CC398 clades and a duplex PCR assay for detection of scn and tet(M). The canSNP assays correctly placed 88 S. aureus CC398 isolates from a reference collection into the human and livestock clades and the duplex PCR assay correctly identified scn and tet(M). The assays were successfully applied to a geographically diverse collection of 272 human S. aureus CC398 isolates. The simple assays described here generate signals comparable to a whole-genome phylogeny for major clade assignment and are easily integrated into S. aureus CC398 surveillance programs and epidemiological studies.     

Comparative Secretome Analyses of Human and Zoonotic Staphylococcus aureus Isolates CC8, CC22, and CC398

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Highlights

• •Proteogenomics and secretome comparison of human and zoonotic Staphylococcus aureus lineages.
• •869 secreted proteins identified in eight S. aureus isolates of CC8, CC22 and CC398.
• •CC398 lower secretion of surface proteins and higher secretion of hemolysins and exoenzymes.
• •Regulatory differences in the secretomes could be linked to lower SigB activity in CC398.

     

Transmission of Methicillin-Resistant Staphylococcus aureus CC398 from Livestock Veterinarians to Their Household Members

There are indications that livestock-associated MRSA CC398 has a reduced human-to-human transmissibility, limiting its impact on public health and justifying modified control measures. This study determined the transmissibility of MRSA CC398 from livestock veterinarians to their household members in the community as compared to MRSA non-CC398 strains. A one-year prospective cohort study was performed to determine the presence of MRSA CC398 in four-monthly nasal and oropharyngeal samples of livestock veterinarians (n  =  137) and their household members (n  =  389). In addition, a cross-sectional survey was performed to detect the presence of MRSA non-CC398 in hospital derived control patients (n  =  20) and their household members (n  =  41). Staphylococcus aureus isolates were genotyped by staphylococcal protein A (spa) typing and multiple-locus variable-number tandem repeat analysis (MLVA). Mean MRSA CC398 prevalence over the study period was 44% (range 41.6–46.0%) in veterinarians and 4.0% (range 2.8–4.7%) in their household members. The MRSA CC398 prevalence in household members of veterinarians was significantly lower than the MRSA non-CC398 prevalence in household members of control patients (PRR 6.0; 95% CI 2.4–15.5), indicating the reduced transmissibility of MRSA CC398. The impact of MRSA CC398 appears to be low at the moment. However, careful monitoring of the human-to-human transmissibility of MRSA CC398 remains important.     

Rise of CC398 Lineage of Staphylococcus aureus among Infective Endocarditis Isolates Revealed by Two Consecutive Population-Based Studies in France

Staphylococcus aureus isolates from two prospective studies on infective endocarditis (IE) conducted in 1999 and 2008 and isolated from non-IE bacteremia collected in 2006 were spa-typed and their virulence factors were analyzed with a microarray. Both populations were genetically diverse, with no virulence factors or genotypes significantly more associated with the IE isolates compared with the non-IE isolates. The population structure of the IE isolates did not change much between 1999 and 2008, with the exception of the appearance of CC398 methicillin-susceptible Staphylococcus aureus (MSSA) isolates responsible for 5.6% of all cases in 2008. In 1999, this lineage was responsible for no cases. The increasing prevalence of S. aureus in IE is apparently not the result of a major change in staphylococcal population structure over time, with the exception of the emerging CC398 MSSA lineage.     

Nasal Colonization of Humans with Methicillin-Resistant Staphylococcus aureus (MRSA) CC398 with and without Exposure to Pigs

Background

Studies in several European countries and in North America revealed a frequent nasal colonization of livestock with MRSA CC398 and also in humans with direct professional exposure to colonized animals. The study presented here addresses the question of further transmission to non exposed humans.

Methods

After selecting 47 farms with colonized pigs in different regions of Germany we sampled the nares of 113 humans working daily with pigs and of their 116 non exposed family members. The same was performed in 18 veterinarians attending pig farms and in 44 of their non exposed family members. For investigating transmission beyond families we samples the nares of 462 pupils attending a secondary school in a high density pig farming area. MRSA were detected by direct culture on selective agar. The isolates were typed by means of spa-sequence typing and classification of SCCmec elements. For attribution of spa sequence types to clonal lineages as defined by multi locus sequence typing we used the BURP algorithm. Antibiotic susceptibility testing was performed by microbroth dilution assay.

Results

At the farms investigated 86% of humans exposed and only 4.3% of their family members were found to carry MRSA exhibiting spa-types corresponding to clonal complex CC398. Nasal colonization was also found in 45% of veterinarians caring for pig farms and in 9% of their non exposed family members. Multivariate analysis revealed that antibiotic usage prior to sampling beard no risk with respect to colonization. From 462 pupils only 3 were found colonized, all 3 were living on pig farms.

Conclusion

These results indicate that so far the dissemination of MRSA CC398 to non exposed humans is infrequent and probably does not reach beyond familial communities.     

Genetic Organization of the mecA Region in Methicillin-Susceptible and Methicillin-Resistant Strains of Staphylococcus sciuri

A homolog of the Staphylococcus aureus methicillin resistance gene mecA was recently shown to be ubiquitous in independent isolates of the animal species Staphylococcus sciuri. The mecA gene homolog and regions flanking it were cloned and sequenced from four strains of S. sciuri: strain K1 (ATCC 29062), a representative of S. sciuri subsp. sciuri; two strains (K3 and K8) representing S. sciuri subsp. rodentius; and strain K11, a representative of S. sciuri subsp. carnaticum. Strains K1 and K11 were susceptible to methicillin, while strains K3 and K8 showed heterogeneous resistance. The mecA genes of strains K1 and K11 and one of the two copies of mecA (mecA1) present in strain K3 had virtually identical DNA sequences in the mecA gene and were similar in genetic organization in the flanking regions. In contrast, the single copy of mecA in strain K8 and the second copy of mecA (mecA2) in strain K3 had mecA DNA sequences identical to that of S. aureus mecA, and the mecA region in these two strains was also similar to that of the same region in the S. aureus strain used for comparison. Interestingly, an open reading frame defining an N-terminal truncated polypeptide, NTORF101, with a high degree of homology to a DNA segment in the hypervariable region of methicillin-resistant S. aureus (and also similar to the Escherichia coli gene ugpQ) was also identified downstream of the mecA homolog of strain K11, representing S. sciuri subsp. carnaticum. The ugpQ-like gene is not present in methicillin-susceptible strains of S. aureus. The presence of such a ugpQ-like gene together with the homolog of mecA in strain K11 supports the speculation that these genetic elements may be evolutionary relatives and/or precursors of the genetic determinant of methicillin resistance in S. aureus.     

Methicillin Resistance Transfer from Staphylocccus epidermidis to Methicillin-Susceptible Staphylococcus aureus in a Patient during Antibiotic Therapy

Background

The mecA gene, encoding methicillin resistance in staphylococci, is located on a mobile genetic element called Staphylococcal Cassette Chromosome mec (SCCmec). Horizontal, interspecies transfer of this element could be an important factor in the dissemination of methicillin-resistant S. aureus (MRSA). Previously, we reported the isolation of a closely related methicillin-susceptible Staphylococcus aureus (MSSA), MRSA and potential SCCmec donor Staphylococcus epidermidis isolate from the same patient. Based on fingerprint techniques we hypothesized that the S. epidermidis had transferred SCCmec to the MSSA to become MRSA. The aim of this study was to show that these isolates form an isogenic pair and that interspecies horizontal SCCmec transfer occurred.

Methodology/Results

Whole genome sequencing of both isolates was performed and for the MSSA gaps were closed by conventional sequencing. The SCCmec of the S. epidermidis was also sequenced by conventional methods. The results show no difference in nucleotide sequence between the two isolates except for the presence of SCCmec in the MRSA. The SCCmec of the S. epidermidis and the MRSA are identical except for a single nucleotide in the ccrB gene, which results in a valine to alanine substitution. The main difference with the closely related EMRSA-16 is the presence of SaPI2 encoding toxic shock syndrome toxin and exfoliative toxin A in the MSSA-MRSA pair. No transfer of SCCmec from the S. epidermidis to the MSSA could be demonstrated in vitro.

Conclusion

The MSSA and MRSA form an isogenic pair except for SCCmec. This strongly supports our hypothesis that the MRSA was derived from the MSSA by interspecies horizontal transfer of SCCmec from S. epidermidis O7.1.     

Detecting the Enterotoxigenicity of Staphylococcus aureus Strains

An optimal sensitivity plate method for examining large number of staphylococcal strains for production of the known enterotoxins (A-E) is presented. Small volumes of relatively concentrated enterotoxin are produced by the semi-solid agar, cellophane-over-agar, or sac culture techniques. Detection of the enterotoxin in the supernatant fluid is accomplished with the optimal sensitivity plate method. In this method small plastic petri dishes (50 mm) were used for a modified Ouchterlony of high sensitivity.     

Proteome of a methicillin-resistant Staphylococcus aureus clinical strain of sequence type ST398

Proteomics is a powerful tool to analyze the differences in gene expression of bacterial strains. Staphylococcus aureus has long been recognized as an important pathogen in human disease. In order to investigate this pathogen, the proteome of a clinical methicillin-resistant S. aureus (MRSA) strain of the sequence type ST398 was determined using 2-DE. Using 2-DE we obtained a total of 105 spots the MRSA strain. Furthermore in correlation with bioinformatic databases, they allowed accurate identification and characterization of proteins, resulting in 227 identified proteins. There were found proteins related to basic function of the cell, but also proteins related to virulence like catalase, specific of S. aureus species, and proteins related to antibiotic resistance. Proteins associated with antibiotic resistance or virulence factors are related to genomic databases. The most abundant classes identified involved glycolysis, energy production, one-carbon metabolism, and oxidation-reduction process, all of which reflect an active metabolism. These results highlight the importance of proteomics to deepen in the knowledge of protein expression of MRSA strain of the lineage ST398, microorganism with diverse and important resistance mechanisms. With this proteome map we have an essential tool for a better understanding of this pathogen and providing new data for protein databases. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Colonization and transmission of methicillin-resistant Staphylococcus aureus ST398 in nursery piglets

A transmission experiment was performed to evaluate the spread of methicillin-resistant Staphylococcus aureus (MRSA) ST398 in nursery piglets. Reproduction ratios (R(0)) in three experimental groups were found to vary between 3.92 and 52.54, indicating that after introduction, MRSA ST398 will spread easily among weaned piglets, with a tendency to become established.     

Recent Emergence of Staphylococcus aureus Clonal Complex 398 in Human Blood Cultures

Background

Recently, a clone of MRSA with clonal complex 398 (CC398) has emerged that is related to an extensive reservoir in animals, especially pigs and veal calves. It has been reported previously that methicillin-susceptible variants of CC398 circulate among humans at low frequency, and these have been isolated in a few cases of bloodstream infections (BSI). The purpose of this study was to determine the prevalence of S. aureus CC398 in blood cultures taken from patients in a geographic area with a high density of pigs.

Methodology/Principal Findings

In total, 612 consecutive episodes of S. aureus BSI diagnosed before and during the emergence of CC398 were included. Three strains (2 MSSA and 1 MRSA) that were isolated from bacteremic patients between 2010–2011 were positive in a CC398 specific PCR. There was a marked increase in prevalence of S. aureus CC398 BSI isolated between 2010–2011 compared to the combined collections that were isolated between 1996–1998 and 2002–2005 (3/157, 1.9% vs. 0/455, 0.0%; p = 0.017).

Conclusions/Significance

In conclusion, in an area with a relative high density of pigs, S. aureus CC398 was found as a cause of BSI in humans only recently. This indicates that S. aureus CC398 is able to cause invasive infections in humans and that the prevalence is rising. Careful monitoring of the evolution and epidemiology of S. aureus CC398 in animals and humans is therefore important.     

Lactate Dehydrogenase Activity in Certain Strains of Staphylococcus aureus

Lactate dehydrogenase (LDH) was studied in phage-propagating strains 29, 3A, 6, 81, and 42D of Staphylococcus aureus selected from the five groups in the International-Blair series. Cells were cultivated in Brain Heart Infusion (Difco) under nearly anaerobic conditions and were harvested near the end of the log phase. LDH activity was maximal at the end of the exponential growth period and was measured spectrophotometrically by reduction of p-nitro-blue tetrazolium, with phenazine methosulfate as a coupling agent. Crude enzyme extracts were prepared both by an acetone extraction technique and by sonic treatment. LDH activity for these enzyme preparations was determined by the colorimetric method mentioned and also by measuring the rate of nicotinamide adenine dinucleotide reduction at 340 mmu. The order of activity observed, by use of both assay methods, was 29 > 81 > 6 > 3A > 42D. LDH forms (possibly isoenzymes) for each of 15 strains, which represent the five phage-propagating groups of the International-Blair series, were separated by acrylamide gel electrophoresis. Five forms were distinguished and arbitrarily numbered on the basis of their rate of migration, no. 5 being the slowest component. No one strain had more than four, nor fewer than two, LDH forms. Form 3 appeared in 13 of the 15 strains and was followed in frequency by no. 2, 1, 4, and 5.     

Surfaceome and exoproteome of a clinical sequence type 398 methicillin resistant Staphylococcus aureus strain

For many years Staphylococcus aureus has been recognized as an important human pathogen. In this study, the surfacome and exoproteome of a clinical sample of MRSA was analyzed. The C2355 strain, previously typed as ST398 and spa-t011 and showing a phenotype of multiresistance to antibiotics, has several resistance genes. Using shotgun proteomics and bioinformatics tools, 236 proteins were identified in the surfaceome and 99 proteins in the exoproteome. Although many of these proteins are related to basic cell functions, some are related to virulence and pathogenicity like catalase and isdA, main actors in S. aureus infection, and others are related to antibiotic action or eventually resistance like penicillin binding protein, a cell-wall protein. Studying the proteomes of different subcellular compartments should improve our understanding of this pathogen, a microorganism with several mechanisms of resistance and pathogenicity, and provide valuable data for bioinformatics databases.