Daniel Benito

Staphylococcus aureus nasal carriage, virulence traits, antibiotic resistance mechanisms, and genetic lineages in healthy humans in Spain, with detection of CC398 and CC97 strains.

S. aureus nasal carriage was investigated in 278 healthy humans, determining the antibiotic resistance mechanisms, virulence traits, and genetic lineages of recovered isolates. Nasal samples were cultured in specific media for S. aureus and methicillin-resistant S. aureus (MRSA) recovery. S. aureus was detected in 53 of 278 nasal samples (19.1%): MRSA was found in one sample (0.4%) and methicillin-susceptible S. aureus (MSSA) in the remaining 52 samples. The MRSA isolate was typed as ST1649-t701-agrI-SCCmec-IVc and only exhibited resistance to beta-lactams. A high diversity of spa types (n=37) was identified among the 52 MSSA, identifying 5 new spa-types. Multi-locus sequence typing (MLST) typing was performed in 30 selected MSSA, detecting 16 different sequence types, 2 of them being new. MSSA strains presented agr types I (30.2%), II (30.2%), III (34%), and IV (5.6%). Eleven strains showed erythromycin resistance and harbored different combinations of erm(A), erm(B), erm(C), erm(T), and msr(A) genes. Two strains exhibited ciprofloxacin resistance, and one of them presented amino acid changes in GyrA and GrlA proteins. The presence of 28 genes encoding staphylococcal toxins was investigated by PCR in all 53 S. aureus isolates. The toxic shock syndrome toxin 1 (TSST-1) gene was detected in 15 MSSA isolates (11 of them typed within the clonal complex CC30) and the gene of exfoliative toxin A in 2 strains. Different combinations of enterotoxin genes were identified among S. aureus strains. None of the S. aureus isolates harbored the Panton-Valentine leukocidin gene. Two MSSA presented the sequence-type ST398 [harboring erm(T) gene], and 2 additional isolates were typed as ST97. Interestingly, MSSA CC398 and CC97 isolates were detected. These clonal complexes are associated with food-producing animals.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) carrying the mecC gene in wild small mammals in Spain

OBJECTIVES To determine the rate of Staphylococcus aureus faecal carriage in 101 wild small mammals in Spain and to characterize the isolates obtained. METHODS Faecal samples were seeded on mannitol salt agar and ORSAB plates. The presence of the resistance genes mecA, mecC and blaZ and the new blaZ allotype associated with staphylococcal cassette chromosome mec (SCCmec) XI (blaZ-SCCmecXI) was studied by PCR. S. aureus isolates were characterized by spa typing, agr typing and multilocus sequence typing. The presence of immune evasion cluster (IEC) genes and virulence genes was analysed by PCR. RESULTS S. aureus was detected in 13/101 studied faecal samples and one isolate per positive sample was further studied. Two S. aureus isolates were methicillin-resistant S. aureus (MRSA) (recovered from wood mice, Apodemus sylvaticus) and 11 were methicillin-susceptible S. aureus (MSSA). Both MRSA isolates harboured the mecC gene and the novel blaZ-SCCmecXI, were typed as spa-t1535/agrIII/ST1945(CC130)/SCCmecXI (where ST stands for sequence type and CC stands for clonal complex), carried the exfoliative toxin etd2 gene and were IEC type E. Eight different spa types were identified among the 11 MSSA isolates (five new) and six different sequence types were identified (two new). All MSSA strains were susceptible to the antibiotics tested except one blaZ-positive penicillin-resistant isolate (spa-t120/agrII/ST15). MSSA isolates were ascribed to the CCs (number of strains) CC5 (1), CC1956 (4) and singleton (6). Nine of 11 MSSA isolates carried the cna virulence gene. Only one MSSA isolate carried IEC genes (type C). CONCLUSIONS This is the first report of MRSA carrying mecC in faecal samples of wild small mammals in Spain. These resistant isolates carried genes of the IEC system, unusual in S. aureus from animals. Wild small mammals could be a reservoir of the mecC gene with important implications for public health.

Molecular characterization and clonal diversity of methicillin-susceptible Staphylococcus aureus in milk of cows with mastitis in Brazil

Mastitis is an important disease for the dairy industry worldwide, causing economic losses and reducing milk quality and production. Staphylococcus aureus is a worldwide agent of this intramammary infection, which also causes foodborne diseases. The objective of this study was to determine the frequency of methicillin-susceptible Staphylococcus aureus (MSSA) isolates in milk of mastitis cows in Brazil and to analyze the genetic lineages and the content of antimicrobial resistance genes and virulence factors among these isolates. Fifty-six MSSA isolates were recovered from 1,484 milk samples (positive for the California mastitis test) of 518 cows from 11 different farms in Brazil (representing 51% of total Staph. aureus obtained), and they were further characterized. Methicillin-susceptible Staphylococcus aureus were isolated from 3.7% of California mastitis test-positive tested milk samples and from 6.2% of tested mastitic cows. Methicillin-susceptible Staphylococcus aureus isolates were characterized by spa typing, agr typing, and multilocus sequence typing, and resistance and virulence traits were investigated by PCR. Seven spa types were identified among MSSA (% of isolates): t127 (44.6), t605 (37.5), t002, t1784, t2066 (1.8), and 2 new ones: t10856 (10.7) and t10852 (1.8). Five distinct sequence types (ST) were detected (% of isolates): ST1 (46.4), ST126 (37.5), ST133 (10.7), ST5 (3.6), and a novel ST registered as ST2493 (1.8). Resistances were detected for streptomycin, chloramphenicol, and tetracycline. One strain contained the chloramphenicol resistance gene (fexA; included within transposon Tn558) and 3 strains contained the tetracycline resistance gene [tet(K)]. Methicillin-susceptible Staphylococcus aureus strains were susceptible to most of the antibiotics studied and lacked the virulence genes of Panton-Valentine leukocidin (lukF/S-PV), toxic shock syndrome toxin 1 (tst), exfoliative toxin A (eta), and exfoliative toxin B (etb), as well as the genes of the immune evasion cluster. Methicillin-susceptible Staphylococcus aureus isolates were detected in a relatively low proportion of cows with mastitis (6.2%) and recovered isolates presented high diversity of genetic lineages, with CC1 and CC126 the predominant clonal complexes, and CC133 also being detected. Larger epidemiological studies with molecular characterization of isolates are required to deepen the knowledge on the circulating genetic lineages among the cow population with mastitis.

Characterization of tetracycline and methicillin resistant Staphylococcus aureus strains in a Spanish hospital: is livestock-contact a risk factor in infections caused by MRSA CC398?

UNLABELLED Tetracycline-resistance (Tet(R)) has been postulated as a marker of the livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) lineage CC398. OBJECTIVES OF THE STUDY to determine the spa-types and assigned MLST clonal complexes (CCs) among all 98 MRSA-Tet(R) strains recovered during 2011-2012 (from different patients) in a Spanish Hospital, analyzing the possible correlation with livestock-contact of the patients. All 98 strains were assigned to 9 CCs: CC398 (60.2%), CC1 (19.4%), CC5 (12.2%), and other CCs (8.2%). The 98 patients were classified into three groups: (A) contact with livestock-animals (n=25); (B) no-contact with livestock-animals (n=42); (C) no information about animal contact (n=31). A significant higher percentage of CC398 strains was obtained in group A (76%) than in group B (50%) (p<0.05), being the percentage in group C of 61.3%. Most of MRSA-Tet(R)-CC398 strains presented a multi-resistance phenotype, including erythromycin, clindamycin, and ciprofloxacin, and the most prevalent detected genes were tet(M) and erm(C). Three strains presented the phenotype macrolide-susceptibility/lincosamide-resistance and contained the vga(A) gene. MRSA-CC1 strains showed higher percentages of erythromycin/clindamycin resistance (95%/89%) than MRSA-CC398 strains (58%/63%), and this resistance was usually mediated by erm(C) gene. Most of MRSA-CC5 strains showed resistance to ciprofloxacin, tobramycin/kanamycin and erythromycin. None of the strains presented the genes lukF/lukS-PV, tsst-1, eta, etb or etd. All MRSA-CC398 strains lacked the genes of the immune-evasion-cluster, but MRSA-CC1 strains carried these genes (type E). In conclusion, although MRSA CC398 is detected in a significant higher proportion in patients with livestock-contact; its detection in people without this type of contact also indicates its capacity for human-to-human transmission.

Characterization of Staphylococcus aureus strains isolated from faeces of healthy neonates and potential mother-to-infant microbial transmission through breastfeeding

Twenty-one women and their respective singleton infants participated in this study, contributing with samples of breast milk and faeces (at days 7, 14 and 35 after birth), respectively, used for Staphylococcus aureus recovery. The aim was to track the carriage of S. aureus in milk and infant faeces of mother-infant pairs, and to determine the genetic lineages of the isolates, their potential clonal relationships and their content in antimicrobial resistance, virulence and immune evasion cluster genes. The molecular characterization was performed by PCR and sequencing. Clonal relationship among mother-infant isolates was conducted by spa typing, multilocus sequence typing (MLST), and pulsed-field gel electrophoresis (PFGE). Staphylococcus aureus was isolated from milk samples of 6 of 21 mothers (16 isolates) and from faecal samples of 12 of 21 infants (25 isolates). From these 41 S. aureus recovered, 18 were methicillin-resistant (MRSA) and 23 methicillin-susceptible (MSSA). Twelve diferentes spa types and eight sequence types were detected among S. aureus. Predominant clonal complexes were CC5 (43.9%) and CC30 (36.6%). MRSA strains presented a multidrug-resistance profile, 65.2% of MSSA strains harboured tsst-1 toxin gene and 26.8% of total strains carried the cna gene. A potential mother-to-infant S. aureus transmission was demonstrated in four cases by spa typing, MLST and PFGE (transmission of t322/ST5/CC5-PFGE-A, t136/ST34/CC30-PFGE-B and t021/ST1869/CC30-PFGE-C strains). Breastfeeding seems to contribute to early S. aureus intestinal colonization in neonates what might affect the immune system development.

Animal and human Staphylococcus aureus associated clonal lineages and high rate of Staphylococcus pseudintermedius novel lineages in Spanish kennel dogs: predominance of S. aureus ST398.

Methicillin-susceptible Staphylococcus aureus (MSSA) and Staphylococcus pseudintermedius (MSSP) are gaining interest to track the evolution of emerging methicillin-resistant strains in animals and humans. We focused on the characterization of the methicillin-susceptible coagulase-positive staphylococci (MSCoPS) recovered from nasal samples of 98 healthy kennel-dogs. Isolates were typed by spa, agr, MLST and SmaI/ApaI-PFGE. Antimicrobial resistance and virulence profiles were investigated. Presence of the human-associated Immune-Evasion-Cluster (IEC) genes was analyzed in MSSA. Twenty-four MSSA, 16 MSSP and one MS Staphylococcus schleiferi subspecies coagulans were obtained. Thirteen spa-types and 12 sequence-types (STs) were detected among MSSA, with ST398 predominance (7/24, 29.2%). MSSA isolates were enclosed within 6 clonal complexes (no. of isolates): CC5 (8), CC398 (7), CC88 (4), CC45 (2), CC133 (1), and CC22 (1), and one singleton. High clonal diversity was observed among MSSP, and 14 STs (10 of them new) were detected. Twelve (50%) MSSA and 12 (75%) MSSP isolates showed resistance to at least one of the tested antimicrobials, with low MSSA penicillin resistance (5 isolates) and high MSSP tetracycline resistance (9 isolates). MSSA isolates ST398, ST133, ST1 and ST2329[new] were susceptible to all antimicrobials and were the only ones lacking the scn, chp and/or sak IEC genes. High diversity of enterotoxin genes was detected among non-ST398/ST133 MSSA isolates. MSSP showed a more homogeneous virulence genes profile. Our results give evidence that dogs can be S. aureus carriers of not only typical human associated lineages but also lineages commonly detected among other animal species. Continue surveillance on CoPS in dogs is required to unveil their role in the dissemination of clones adapted to other animal species.

Correlation Between Serum 25-Hydroxyvitamin D and Virulence Genes of Staphylococcus aureus Isolates Colonizing Children with Atopic Dermatitis

The skin of children with atopic dermatitis (AD) is colonized with Staphylococcus aureus more frequently than that of their peers. We investigated the prevalence of skin and nares colonization by S. aureus in children with AD, the virulence genes of the isolates, and their association with allergy, AD severity, and serum vitamin D (25(OH)D). This was an observational, cross‐sectional study in a sample of children diagnosed with AD in two settings in Spain. The samples were collected in 2012. Swabs from affected skin and nares were taken for microbiologic culture. The prevalence of S. aureus and presence of 17 staphylococcal virulence genes were studied using polymerase chain reaction. A total of 114 patients with a mean age of 5.7 ± 4.1 (range 3 mos to 14 yrs) were included in the study. Swabs were taken from the skin of 113 individuals with AD and from the nares of 85; 28.3% had S. aureus on the skin, which was significantly associated with positive allergen‐specific immunoglobulin E antibodies and higher Scoring Atopic Dermatitis (SCORAD) scores in the multivariate analysis. The presence of virulence factors tsst‐1, eta, cna, aur, and sec in cutaneous S. aureus isolates was associated with lower serum levels of 25(OH)D. S. aureus on nasal swabs correlated with its presence on the skin and was associated with lower 25(OH)D levels. In conclusion, S. aureus colonization is associated with allergy and severity in AD, whereas certain virulence genes are associated with lower serum 25(OH)D levels.

Molecular characterization of Staphylococcus aureus isolated from humans related to a livestock farm in Spain, with detection of MRSA-CC130 carrying mecC gene: A zoonotic case?

OBJECTIVES To conduct a study of Staphylococcus aureus carriage in members of a livestock-farmers family with different degrees of animal contact, and to characterize the recovered isolates. METHODS Nasal samples from 11 members of the family were taken in three sampling periods (every six months) (n=31), and 9 skin samples from superficial lesions were also obtained in 5 of them. Samples were analyzed for S. aureus susceptible (MSSA) and resistant to methicillin (MRSA). S. aureus isolates were tested for antibiotic-resistance phenotype and genotype and for the detection of virulence and IEC-system genes. Molecular typing of isolates was also performed (spa- and multilocus-sequence typing). RESULTS Eighteen S. aureus isolates were recovered (1 MRSA and 17 MSSA) in the 40 samples analyzed. S. aureus was detected in nasal and skin samples of 7/11 and 4/5 of tested humans, respectively. The MRSA strain was detected in the skin lesion of a farmer with high animal contact, and carried the mecC gene, and was typed as ST130-CC130-t843. The 17 MSSA isolates were ascribed to 9 different spa-types and sequence types included in the clonal complexes CC22, CC30, CC45, CC121, and in the livestock-associated lineages CC9 and CC133. Six strains harbored eta or tsst-1 genes. Three of 18 strains lacked the immune-evasion-cluster (IEC) genes (MRSA-ST130, MSSA-ST1333, and MSSA-ST133), and the remaining isolates were ascribed to IEC type-A or -B. CONCLUSIONS Animal-associated S. aureus lineages were detected in samples of the farmers family, highlighting the detection of MSSA-CC133 and mecC-MRSA-ST130.

Genetic lineages and antimicrobial resistance genotypes in Staphylococcus aureus from children with atopic dermatitis: detection of clonal complexes CC1, CC97 and CC398.

The objective was to analyse the genetic lineages of Staphylococcus aureus recovered from nasal and skin samples of atopic dermatitis (AD) paediatric patients, and to characterize the antimicrobial resistance phenotype–genotype and the immune-evasion-cluster (IEC) type of isolates. Forty S. aureus isolates from 35 patients (skin: 26; nasal samples: 14) were characterized. Isolates were submitted to spa-, agr- and multilocus sequence typing. All S. aureus strains analyzed were methicillin-susceptible (MSSA). High genetic diversity was detected among the 40 MSSA isolates (especially among skin isolates), with detection of 27 different spa-types, 20 sequence-types and 16 clonal complexes (CCs). Lineages CC30 and CC5 were predominant among nasal isolates (71% vs 23% skin). Thirteen different CCs were detected among skin isolates, with detection of clades CC1, CC9 and CC398. Antimicrobial resistance rates detected were higher in skin than in nasal isolates, especially for macrolides, aminoglycosides, lincosamides and mupirocin. MSSA strains were characterized into five IEC-types, being A, B and F the predominant ones. MSSA strains of lineages CC45 and CC5 were detected in almost all cases in AD patients with severe Scoring Atopic Dermatitis (SCORAD) and lineages CC8, and CC30 in those with mild or moderate one. As conclusion, high-clonal-diversity was detected among MSSA from AD patients, especially in skin-isolates. Colonization with S. aureus of some CCs seems more associated with AD severity than other lineages.