Paula Gómez

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.

Identification of novel vga(A)-carrying plasmids and a Tn5406-like transposon in meticillin-resistant Staphylococcus aureus and Staphylococcus epidermidis of human and animal origin.

Nine staphylococcal strains of human and animal origin with a lincomycin-resistant/erythromycin-susceptible phenotype and carrying vga genes were characterised to determine the genetic elements involved in the dissemination of these uncommon resistance genes. These strains were typed by multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) and/or spa typing. Antimicrobial susceptibility was studied by disk diffusion and agar dilution methods. Presence of the genes lnu(A), lnu(B), vga(A), vga(A)v, vga(B), vga(C), vga(E), lsa(B) and cfr was studied by PCR. Transformation experiments were carried out in all strains, and the plasmid or chromosomal gene location was determined by Southern blot analysis. Genetic environments of the vga genes were analysed by PCR mapping or inverse PCR and sequencing. Five meticillin-resistant Staphylococcus aureus (MRSA) ST398 strains and three Staphylococcus epidermidis strains harboured the gene vga(A), and one MRSA-ST8 strain contained the gene vga(A)v. One MRSA-ST398 strain, which also contained the gene lnu(A), showed the highest minimum inhibitory concentration (MIC) to lincomycin. The vga(A)v-positive strain presented lower MIC values than the vga(A)-positive strains. Presence of the pVGA plasmid was confirmed in two MRSA-ST398 strains. Four novel vga(A)-carrying plasmids were detected: pUR2355 (in two MRSA and one meticillin-susceptible S. epidermidis); pUR4128 (one MRSA); pUR3036 [one meticillin-resistant S. epidermidis (MRSE)]; and pUR3937 (one MRSE). The plasmid pUR4128 was very similar to pUR2355. Plasmids pUR3036 and pUR3937 were related and were very similar to plasmid pSE-12228-06. The gene vga(A)v was located in a transposon analogous to Tn5406. Therefore, four novel vga(A)-carrying plasmids and a variant of Tn5406 were identified in this study.

Methicillin-resistant Staphylococcus aureus of lineage ST398 as cause of mastitis in cows

The objective of this study was to analyse the prevalence and molecular characteristics of methicillin‐resistant Staphylococcus aureus (MRSA) in milk of cows with mastitis. The California mastitis test (CMT) was used to detect the presence of mastitis in all 100 cows of a farm in Brazil. The CMT was positive in milk of 115 mammary quarters from 36 cows (36%). MRSA isolates were recovered from 4 of these 36 cows with mastitis (11%), and they were further characterized (one MRSA/sample). The four MRSA isolates were typed as t011‐ST398‐agr1‐SCCmecV and presented two different pulsed‐field‐gel‐electrophoresis‐ApaI patterns. These four MRSA isolates showed resistance to tetracycline, streptomycin and ciprofloxacin, carried the mecA, blaZ, tet(K), and tet(M) resistance genes, and presented the S84L and S80F amino acid substitutions in GyrA and GrlA proteins, respectively. Two ST398 isolates exhibited resistance to gentamicin and tobramycin [with aac(6)‐aph(2”) and ant(4)‐Ia genes] and one isolate resistance to clindamycin [with lnu(B) and lsa(E) genes]; this latter isolate also carried the spectinomycin/streptomycin resistance genes spw and aadE. MRSA of lineage ST398 is worldwide spread, normally multidrug resistant and may be responsible for bovine mastitis. To our knowledge, this is the first detection of MRSA‐ST398 in Brazil.

Detection of MRSA ST3061-t843-mecC and ST398-t011-mecA in white stork nestlings exposed to human residues.

OBJECTIVES The objective of this study was to analyse the prevalence of tracheal carriage of Staphylococcus aureus/MRSA in storks and to study the resistance and virulence genes in the obtained isolates. METHODS Tracheal samples from 92 stork nestlings of two landfill-associated and two natural-habitat colonies were inoculated in specific media for S. aureus and MRSA recovery. Antimicrobial susceptibility was tested, and the presence of resistance, virulence and immune evasion cluster (IEC) genes was analysed by PCR. S. aureus isolates were characterized by spa and agr typing. Staphylococcal cassette chromosome (SCC) mec type was determined for mecC-positive isolates, and MLST was performed for 17 selected S. aureus isolates. RESULTS S. aureus isolates were identified in 32/92 samples (34.8%), and 38 isolates were recovered. The prevalence of S. aureus was higher in nestlings from landfills (24/43, 55.8%) than in those from natural habitats (8/49, 16.3%). Three birds from landfill-associated colonies carried MRSA, two with mecA-positive strains [clonal complex (CC) 5-spa-t002 and CC398-spa-t011] and one with a mecC-positive strain [sequence type (ST) 3061-CC130-spa-t843-agr-III-SCCmecXI). None of the MRSA isolates presented IEC genes. Thirty-five MSSA isolates, which showed 18 different spa types (ascribed to CC5, CC7, CC22, CC30, CC45, CC59, CC133 and CC398), were obtained. The agr types detected were I (63%), II (29%) and III (8%). Resistance and virulence genes identified in MSSA were blaZ (n = 25), erm(T) (n = 9), erm(A) (n = 1), tet(M) (n = 2), fexA (n = 3), str (n = 2), tst (n = 2), eta (n = 1) and cna (n = 15). The IEC types B, C, D and G were found in MSSA isolates, and two new STs were identified (ST3060 and ST3061). CONCLUSIONS White storks are frequently tracheal carriers of S. aureus, including ST398 isolates. MRSA isolates of lineages CC398-mecA and CC130-mecC were detected in storks from landfill-associated colonies exposed to human residues.

High prevalence of methicillin-resistant Staphylococcus aureus (MRSA) carrying the mecC gene in a semi-extensive red deer (Cervus elaphus hispanicus) farm in Southern Spain

The objective was to determine the prevalence of Staphylococcus aureus nasal carriage in red deer of a semi-extensive farm and in humans in contact with the estate animals, and to characterize obtained isolates. Nasal swabs of 65 deer and 15 humans were seeded on mannitol-salt-agar and oxacillin-resistance-screening-agar-base. Isolates were identified by microbiological and molecular methods. Antimicrobial susceptibility profile was determined for 16 antibiotics by disk-diffusion and the presence of eight antibiotic resistance genes, seven virulence genes and genes of immune-evasion-cluster (IEC) was analyzed by PCR. S. aureus was typed by PFGE-SmaI, spa, agr, SCCmec and MLST. Isolates were detected in 16 deer (24.6%). Eleven S. aureus isolates were methicillin-resistant (MRSA), and five were methicillin-susceptible (MSSA). All MRSA harbored mecC gene and were agr-III/SCCmecXI/ST1945 (four spa-t843 and seven spa-t1535). All mecC-MRSA carried blaZ-SCCmecXI and etd2, were IEC-type-E, and belonged to the same PFGE pattern. The five MSSA were typed as spa-t2420/agr-I/ST133. Regarding humans, S. aureus was recovered from six samples (40%). The isolates were MSSA and were typed as spa-t002/agr-II, spa-t012/agr-III or spa-t822/agr-III and showed different IEC types (A, B, D and F). blaZ and erm(A) genes were detected, as well as cna and tst genes. As conclusion, red deer analyzed in this study are frequent carriers of mecC-MRSA CC130 (16.9%), they are characterized by few resistance and virulence determinants, and by the presence of IEC type-E. Deer could be a source of mecC-MRSA which could potentially be transmitted to other animals, or even to humans.

First detection of methicillin-resistant Staphylococcus aureus ST398 and Staphylococcus pseudintermedius ST68 from hospitalized equines in Spain.

Eight coagulase‐positive staphylococci from equines with different pathologies obtained between 2005 and 2011 were investigated. Isolates were characterized by different molecular techniques (spa‐, agr‐, MLST), and clonal relatedness of strains was investigated by ApaI and SmaI PFGE. Anti‐microbial resistance and virulence profiles were determined. Six isolates were identified as Staphylococcus aureus, and two as Staphylococcus pseudintermedius. Of these, four isolates were methicillin‐resistant S. aureus (MRSA) ST398 and one S. pseudintermedius was mecA positive and typed as ST68. One MRSA ST398 strain was isolated in 2005 and might be one of the earliest MRSA ST398 descriptions in Spain. All 5 mecA‐positive strains were multidrug resistant and were isolated from hospitalized equines. Three MRSA ST398 strains carried the recently described transposon Tn559 within the chromosomal radC gene. The mecA‐positive S. pseudintermedius ST68 strain was also multidrug resistant and harboured the erm(B)‐Tn5405‐like element. This ST68 strain presented a clear susceptible phenotype to oxacillin and cefoxitin regardless of the presence of an integral and conserved mecA gene and mecA promoter, which enhances the need for testing the presence of this gene in routine analysis to avoid treatment failures. These data reflect the extended anti‐microbial resistance gene acquisition capacities of both bacterial species and evidence their pathogenic properties. The first detection of MRSA ST398 and S. pseudintermedius ST68 in horses in Spain is reported.

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.

Characterisation of nasal Staphylococcus delphini and Staphylococcus pseudintermedius isolates from healthy donkeys in Tunisia

REASONS FOR PERFORMING STUDY Staphylococcus intermedius group (SIG) bacteria can colonise the nares of some animals but are also emerging pathogens in humans and animals. OBJECTIVES To analyse SIG nasal carriage in healthy donkeys destined for food consumption in Tunisia and to characterise recovered isolates. METHODS Nasal swabs from 100 healthy donkeys were tested for SIG recovery, and isolates were identified by biochemical and molecular methods. Antimicrobial susceptibility of isolates was tested and detection of antimicrobial resistance and virulence genes was performed. Isolates were typed at the clonal level by multilocus sequence typing and SmaI pulsed-field gel electrophoresis. RESULTS Staphylococcus delphini and Staphylococcus pseudintermedius (included in SIG) were obtained in 19% and 2% of the tested samples, respectively, and one isolate per sample was characterised. All isolates were meticillin susceptible and mecA negative. Most S. delphini and S. pseudintermedius isolates showed susceptibility to all antimicrobials tested, with the exception of 2 isolates resistant to tetracycline (tet(M) gene) or fusidic acid. The following toxin genes were identified (percentage of isolates): lukS-I (100%), lukF-I (9.5%), siet (100%), se-int (90%), seccanine (19%) and expA (9.5%). Thirteen different pulsed-field gel electrophoresis profiles were identified among the 21 SIG isolates. Additionally, the following 9 different sequence types (STs) were detected by multilocus sequence typing, 6 of them new: ST219 (6 isolates), ST12 (5 isolates), ST220 (3 isolates), ST13, ST50, ST193, ST196, ST218 and ST221 (one isolate each). CONCLUSIONS Staphylococcus delphini and S. pseudintermedius are common nasal colonisers of donkeys, generally susceptible to the antimicrobials tested; nevertheless, these SIG isolates contain virulence genes, including the recently described exfoliative gene (expA) and several enterotoxin genes, with potential implications for public health. This is the first description of S. delphini in Tunisia. The Summary is available in Chinese - see Supporting information.

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.

Staphylococcus aureus isolated from wastewater treatment plants in Tunisia: occurrence of human and animal associated lineages

The objective was to characterize Staphylococcus aureus isolated from two wastewater treatment plants (WWTPs) located in Tunis City (Tunisia), during the period 2014-2015. Genetic lineages, antibiotic resistance mechanisms and virulence factors were determined for the recovered isolates. S. aureus isolates were recovered from 12 of the 62 wastewater samples tested (19.35%), and one isolate/sample was characterized, all of them being methicillin-susceptible (MSSA). Six spa types (t587, t674, t224, t127, t701 and t1534) were found among the 12 isolates, and the spa-t587, associated with the new sequence type ST3245, was the most predominant one (7 isolates). The remaining isolates were assigned to five clonal complexes (CC5, CC97, CC1, CC6 and CC522) according to the sequence-type determined and/or the spa-type detected. S. aureus isolates were ascribed to agrI (n = 3), agrII (n = 7) and agrIII (n = 1); however, one isolate was non-typeable. S. aureus showed resistance to (number of isolates): penicillin (12), erythromycin (7), tetracycline (one) and clindamycin (one). Among the virulence factors investigated, only one isolate harboured the tst gene, encoding the TSST-1 (toxic shock syndrome toxin 1). Despite the low number of studied isolates, the present study reports the occurrence of both human- and animal-associated S. aureus clonal complexes in WWTPs in Tunisia.