Genetic Diversification and Resistome of Coagulase-Negative Staphylococci from Nostrils of Healthy Dogs and Dog-Owners in La Rioja, Spain.

Coagulase-negative staphylococci (CoNS) species in healthy dogs and their owners could be transferred between these hosts and carry diverse antimicrobial resistance (AMR) genes of public health concern. This study determined the frequency, diversity, and AMR genes of nasal CoNS from healthy dogs and in-contact people as well as the rate of intra-household (between healthy dogs and dog-owners) transmission of CoNS. Nasal samples were collected and processed from 34 dogs and 41 humans from 27 households, and CoNS identification was done by MALDI-TOF-MS. The AMR determinants and genetic lineages were determined by PCR/sequencing. A total of 216 CoNS isolates were initially obtained and identified, and the AMR phenotypes were determined. From these, 130 non-repetitive CoNS were selected (one isolate of each species per sample or more than one if they presented different AMR phenotypes) and further characterized. The predominant species from dog carriers were S. epidermidis (26.5%), S. hominis (8.8%), and S. cohnii (8.8%), whereas in the human carriers, the predominant ones were S. epidermidis (80.4%), S. lugdunensis (9.8%), and S. hominis (9.8%). Intra-host species diversity (>one CoNS species) was detected in 37.5% of dogs and 21.6% of dog-owners. Conversely, 50% of dogs and 70.3% of dog-owners had intra-species AMR diversity (2-4 AMR-CoNS profiles). About 20% were susceptible to all antimicrobial agents tested, 31.5% displayed a multidrug resistance phenotype, and 17.4% were mecA-positive, located in SCCmec type V (24.2%), III (18.1%), IVc (12.1%), and II (6.1%). The other mec-A positive CoNS isolates (39.5%) had non-typeable SCCmec. The highest AMR rates were found against erythromycin (32.3%/mph(C), msr(A)) and mupirocin (20.8%/mupA), but the resistance rates for other antimicrobial agents were <10% each. Remarkably, one linezolid-resistant S. epidermidis-ST35 isolate was identified and mediated by four amino acid substitutions in L3 and one in L4 ribosomal proteins. Dogs and dog-owners as carriers of S. epidermidis with similar AMR patterns and genetic lineages (ST59, ST61, ST166 and ST278) were detected in four households (14.8%). Diverse CoNS carriage and moderate level of AMR were obtained from this study. The detection of CoNS carrying diverse SCCmec elements and intra-species AMR diversity highlights the roles of dog ownership in the potential transmission of antimicrobial-resistant CoNS in either direction.

Unexpected role of pig nostrils in the clonal and plasmidic dissemination of extended-spectrum beta-lactamase-producing Escherichia coli at farm level.

The presence of methicillin-resistant or -susceptible S. aureus in pig nostrils has been known for a long time, but the occurrence of extended-spectrum beta-lactamase (ESBL)-producing E. coli has hardly been investigated. Here, we collected 25 E. coli recovered from nasal samples of 40 pigs/10 farmers of four farms. Nine ESBL-producing isolates belonging to ST48, ST117, ST847, ST5440, ST14914 and ST10 were retrieved from seven pigs. All blaESBL genes (blaCTX-M-32,blaCTX-M-14,blaCTX-M-1,blaCTX-M-65, and blaSHV-12) were horizontally transferable by conjugation through plasmids belonging to IncI1 (n=3), IncX1 (n=3) and IncHI2 (n=1) types. IncI1-plasmids displayed different genetic environments: i) IS26-blaSHV-12-deoR-IS26, ii) wbuC-blaCTX-M-32-ISKpn26 (IS5), and iii) IS930-blaCTX-M-14-IS26. The IncHI2-plasmid contained the genetic environment IS903-blaCTX-M-65-fipA with multiple resistance genes associated either to: a) Tn21-like transposon harbouring genes conferring aminoglycosides/beta-lactams/chloramphenicol/macrolides resistance located on two atypical class 1 integrons with an embedded ΔTn5393; or b) Tn1721-derived transposon displaying an atypical class 1 integron harbouring aadA2-arr3-cmlA5-blaOXA-10-aadA24-dfrA14, preceding the genetic platform IS26-blaTEM-95-tet(A)-lysR-floR-virD2-ISVsa3-IS3075-IS26-qnrS1, as well as the tellurite resistance module. Other plasmids harbouring clinically relevant genes were detected, such as a ColE-type plasmid carrying the mcr-4.5 gene. Chromosomally encoded genes (fosA7) or integrons (intI1-dfrA1-aadA1-qacE-sul1/intI1-IS15-dfrA1-aadA2) were also identified. Finally, an IncY plasmid harbouring a class 2 integron (intI2-dfrA1-sat2-aadA1-qacL-IS406-sul3) was detected but not associated with a blaESBL gene. Our results evidence that pig nostrils might favour the spread of ESBL-E. coli and mcr-mediated colistin-resistance. Therefore, enhanced monitoring should be considered, especially in a sector where close contact between animals in intensive farming increases the risk of spreading antimicrobial resistance.

Comparative genomics of Staphylococcus aureus strains from wild birds and pig farms elucidates levels of mobilomes, antibiotic pressure and host adaptation.

OBJECTIVES: This study characterized the resistome, mobilome and phylogenomic relatedness of Staphylococcus aureus strains previously obtained from healthy nestling storks (HNS), pigs (HP) and pig farmers (HPF) to analyse possible transmission pathways of S. aureus with implications for the spread of antimicrobial resistance. METHODS: The genomic contents of 52 S. aureus strains obtained from the nasal cavity of HNS, HP and HPF in Spain were sequenced using the Illumina NextSeq platform to characterize their resistome, virulome and mobile genetic elements. The relatedness of strains was assessed by core-genome single nucleotide polymorphisms (SNPs). RESULTS: The frequencies of multidrug-resistance phenotype and transposons were significantly lower in strains from HNS than in those from HP and HPF (P < 0.005). However, the presence of human immune evasion cluster genes in S. aureus strains from HNS was significantly higher than in those from HP and HPF (P < 0.005). Interestingly, the frequencies of plasmids and phages were not significantly associated with the host (P > 0.05). The phylogenetic analysis identified a cluster of all the MSSA-CC398 strains carrying φSa3 and ermT on rep13 separately from the two MRSA-CC398 strains (carrying ermT on repUS18). Highly related MRSA-CC398 strains were detected in some pigs and related farmers (<10 SNPs). CONCLUSION: This study confirms high-level antibiotic selection in S. aureus in HP and HPF in comparison to HNS. Furthermore, our findings highlight the continuous transmission of MRSA-CC398 in the pig-to-human interface and MSSA-CC398 with human adaptation markers in HNS. Molecular surveillance of S. aureus using the One Health model is required to establish appropriate control strategies.

Nasal staphylococci microbiota and resistome in healthy adults in La Rioja, northern Spain: High frequency of toxigenic S. aureus and MSSA-CC398 subclade.

This study determined the nasal staphylococci diversity and characterized their resistome, with a focus on the mobilome of methicillin-susceptible Staphylococcus aureus (MSSA)-CC398 subclade from healthy adults in La Rioja (northern Spain). Nasal staphylococci recovered from 57 healthy individuals (HI) were identified (MALDI-TOF-MS) and their antimicrobial resistance, virulence determinants and genetic lineages were studied. The relatedness of MSSA-CC398 isolates was assessed by core-genome single-nucleotide-polymorphisms (SNPs). One-hundred-forty-three non-repetitive staphylococci were obtained from most HI (98.2%), of which S. epidermidis (87.7%) and S. aureus (36.8%) were the predominant species. About 15% of the 27 S. aureus and 30.1% of the 116 coagulase-negative staphylococci (CoNS) isolates presented a multidrug resistance (MDR) phenotype. All S. aureus isolates were MSSA but 30.2% of CoNS isolates were mecA-positive and carried SCCmec types III, IV, and V. The highest non-beta-lactam resistance (frequency/genes) in S. aureus and CoNS were: erythromycin-clindamycin-inducible (25.9%/ermT, ermC) and mupirocin (30.1%/mupA), respectively. About 85% of S. aureus isolates carried relevant virulence genes. Eight clonal complexes (CCs) of MSSA were identified, of which CC398 was the predominant (33.3%). About 78% of the CC398 isolates harboured rep13-bound ermT gene, however, one carried a rep10-bound ermC gene. Only the ermT-positive MSSA-CC398 isolates were closely related (<50 SNPs) and carried the φSa3. Diverse MDR-S. epidermidis isolates were identified which included the lineages ST59 and ST210. The high rate of toxigenic S. aureus and of MSSA-CC398 subclade highlight the ability of HI to carry and transmit virulent isolates. Moreover, the high frequency of MDR-CoNS, often linked with SCCmec, needs to be monitored for their potential human health implications.

Within-Host Diversity of Coagulase-Negative Staphylococci Resistome from Healthy Pigs and Pig Farmers, with the Detection of cfr-Carrying Strains and MDR-S. borealis.

The ecology and diversity of resistome in coagulase-negative staphylococci (CoNS) from healthy pigs and pig farmers are rarely available as most studies focused on the livestock-associated methicillin-resistant S. aureus. This study aims to characterize the antimicrobial resistance (AMR) mechanisms, intra-host species diversity (more than one species in a host), and intra-species AMR diversity (same species with more than one AMR profile) in CoNS recovered from the nasal cavities of healthy pigs and pig farmers. One-hundred-and-one CoNS strains previously recovered from 40 pigs and 10 pig farmers from four Spanish pig farms were tested to determine their AMR profiles. Non-repetitive strains were selected (n = 75) and their AMR genes, SCCmec types, and genetic lineages were analyzed by PCR/sequencing. Of the non-repetitive strains, 92% showed a multidrug resistance (MDR) phenotype, and 52% were mecA-positive, which were associated with SCCmec types V (46.2%), IVb (20.5%), and IVc (5.1%). A total of 28% of the pigs and pig farmers had intra-host species diversity, while 26% had intra-species AMR diversity. High repertoires of AMR genes were detected, including unusual ones such as tetO, ermT, erm43, and cfr. Most important was the detection of cfr (in S. saprophyticus and S. epidermidis-ST16) in pigs and pig farmers; whereas MDR-S. borealis strains were identified in pig farmers. Pig-to-pig transmission of CoNS with similar AMR genes and SCCmec types was detected in 42.5% of pigs. The high level of multidrug, within-host, and intra-species resistome diversity in the nasal CoNS highlights their ability to be AMR gene reservoirs in healthy pigs and pig farmers. The detection of MDR-S. borealis and linezolid-resistant strains underscore the need for comprehensive and continuous surveillance of MDR-CoNS at the pig farm level.

Detection and evaluation of the antimicrobial activity of Micrococcin P1 isolated from commensal and environmental staphylococcal isolates against MRSA.

BACKGROUND: Bacteriocins (of different origins) have been proposed as promising alternatives to face antimicrobial resistance-associated health problems. Isolates of the Staphylococcus genus are well-known bacteriocin producers, especially coagulase-negative species. METHODS: Twenty-eight bacteriocin-producing staphylococcal isolates were selected from a previous study for in-depth characterisation. The antimicrobial activities (AA) of the producing isolates were studied by the spot-on-lawn method and their crude cell-free supernatants (CFS) and butanol extracts (BT) were evaluated by agar diffusion assays against six indicator bacteria, including multidrug-resistant and zoonotic isolates (such as Listeria monocytogenes or methicillin-resistant Staphylococcus aureus [MRSA]). RESULTS: Six bacteriocin-producing isolates showed AA in their CFS, whereas all staphylococcal BT extracts inhibited at least one of the tested indicator bacteria. Micrococcin P1 (MP1) bacteriocin was detected by mass spectrometry in four producing isolates: Staphylococcus aureus-C5802, Staphylococcus hominis-C5835, Staphylococcus sciuri-X3041, and -X3011. Growth curves performed with CFS and BT extracts of the four MP1 producers revealed a strong AA profile against MRSA and Listeria monocytogenes, even when considerably diluted. Moreover, synergism between the BT extract of MP1-producing Staphylococcus sciuri-X3041 and several antibiotics against an MRSA indicator was observed: BT-clindamycin (> 80%) and BT-oxacillin (30%) combinations. For the BT-chloramphenicol combination, synergism and near synergism values were observed in 37% of the combinations. Competition studies revealed potent inhibitory effects of the MP1-producing isolates against the MRSA indicator. CONCLUSION: These results help to identify Staphylococcus isolates or their bacteriocins as interesting candidates for potential future applications.

Genomic Analysis of Bacteriocin-Producing Staphylococci: High Prevalence of Lanthipeptides and the Micrococcin P1 Biosynthetic Gene Clusters.

Bacteriocins are antimicrobial peptides produced by bacteria. This study aimed to in silico analyze the presence of bacteriocin gene clusters (BGCs) among the genomes of 22 commensal Staphylococcus isolates from different origins (environment/human/food/pet/wild animals) previously identified as bacteriocin producers. The resistome and plasmidome were studied in all isolates. Five types of BGC were detected in 18 genomes of the 22 bacteriocin-producing staphylococci included in this study: class I (Lanthipeptides), class II, circular bacteriocins, the non-ribosomal-peptide lugdunin and the thiopeptide micrococcin P1 (MP1). A high frequency of lanthipeptides was detected in this collection: BGC variants of BSA, bacCH91, and epilancin15X were identified in two Staphylococcus aureus and one Staphylococcus warneri isolates from food and wild animals. Moreover, two potentially new lanthipeptide-like BGCs with no identity to database entries were found in Staphylococcus epidermidis and Staphylococcus simulans from food and wild animal, respectively. Interestingly, four isolates (one S. aureus and one Staphylococcus hominis, environmental origin; two Staphylococcus sciuri, food) carried the MP1 BGC with differences to those previously described. On the other hand, seven of the 22 genomes (~32%) lacked known genes related with antibiotic or disinfectant-acquired resistance mechanisms. Moreover, the potential carriage of plasmids was evaluated, and several Rep-proteins were identified (~73% of strains). In conclusion, a wide variety of BGCs has been observed among the 22 genomes, and an interesting relationship between related Staphylococcus species and the type of bacteriocin has been revealed. Therefore, bacteriocin-producing Staphylococcus and especially coagulase-negative staphylococci (CoNS) can be considered good candidates as a source of novel bacteriocins.

Detection and genetic characterization of blaESBL-carrying plasmids of cloacal Escherichia coli isolates from white stork nestlings (Ciconia ciconia) in Spain.

OBJECTIVES: This study aimed to characterize Escherichia coli isolates from cloacal samples of white stork nestlings, with a special focus on extended-spectrum ß-lactamases (ESBLs)-producing E. coli isolates and their plasmid content. METHODS: Cloacal samples of 88 animals were seeded on MacConkey-agar and chromogenic-ESBL plates to recover E. coli and ESBL-producing E. coli. Antimicrobial susceptibility was screened using the disc diffusion method, and the genotypic characterization was performed by polymerase chain reaction (PCR) and subsequent sequencing. S1 nuclease Pulsed-Field-Gel-Electrophoresis (PFGE), Southern blotting, and conjugation essays were performed on ESBL-producing E. coli, as well as whole-genome sequencing by short- and long-reads. The four blaESBL-carrying plasmids were completely sequenced. RESULTS: A total of 113 non-ESBL-producing E. coli isolates were collected on antibiotic-free MacConkey-agar, of which 27 (23.9%) showed a multidrug-resistance (MDR) phenotype, mainly associated with ß-lactam-phenicol-sulfonamide resistance (blaTEM/cmlA/floR/sul1/sul2/sul3). Moreover, four white stork nestlings carried ESBL-producing E. coli (4.5%) with the following characteristics: blaSHV-12/ST38-D, blaSHV-12/ST58-B1, blaCTX-M-1/ST162-B1, and blaCTX-M-32/ST155-B1. Whole-genome sequencing followed by Southern blot hybridizations on S1-PFGE gels in ESBL-positive isolates proved that the blaCTX-M-1 gene and one of the blaSHV-12 genes were carried by IncI1/pST3 plasmids, while the second blaSHV-12 gene and the blaCTX-M-32 gene were located on IncF plasmids. The two blaSHV-12 genes and the two blaCTX-M genes had similar but non-identical close genetic environments, as all four genes were flanked by a variety of insertion sequences. CONCLUSION: The role played by several genetic platforms in the mobility of ESBL genes allows for interchangeability on a remarkably small scale (gene-plasmid-clones), which may support the spread of ESBL genes.

Antimicrobial resistome of coagulase-negative staphylococci from nasotracheal cavities of nestlings of Ciconia ciconia in Southern Spain: Detection of mecC-SCCmec type-XI-carrying S. lentus.

The antimicrobial resistance (AMR) genes of 268 non-duplicated coagulase-negative staphylococci (CoNS) previously obtained from nasotracheal cavities of nestling storks were characterized. They included S. sciuri isolates (n = 191), and non-sciuri-CoNS isolates (NSc-CoNS, n = 77). All S. sciuri carried the intrinsic salA gene (for clindamycin-resistance) and so, clindamycin was not considered for general analysis in this species. About 71.7%/41.6% of the S. sciuri/NSc-CoNS isolates were susceptible to all antibiotics tested; moreover, 14.1%/16.9% and 3.1%/20.8% of S. sciuri/NSc-CoNS showed single antibiotic resistance and multidrug resistance (MDR) phenotype, respectively. Of the ten mecA-positive CoNS isolates, six were associated with SCCmec types-III, -IV or -V elements. Remarkably was the detection of one MDR-S. lentus isolate carrying both mecA and mecC genes, as well as the SCCmec type-XI element. MDR-CoNS was relatively higher in nestlings of parent storks foraging in landfills (21.3%) than those in natural areas (9.7%) (χ2 = 3.421, df=1, p = 0.064). AMR phenotypes (and genes detected) include penicillin (blaZ, blaARL), erythromycin-clindamycin-constitutive (ermA, ermC, ermT), clindamycin (lnuA, salA, vgaA), erythromycin (msrA, mphC), tetracycline (tetK, tetL, tetM), tobramycin (ant4'), tobramycin-gentamicin (aac6'-aph2″), sulfamethoxazole-trimethoprim (dfrA, dfrG, dfrK), chloramphenicol (fexA, fexB, catPC221), and mupirocin (mupA). Interestingly, one S. epidermidis isolate carried the ermT gene. About 29.9% of nestlings harboured more than one non-duplicated CoNS (with varied 2-5 AMR profiles). This study demonstrated that most of the CoNS isolates were susceptible to all the antibiotics tested (63.1%). However, AMR genes of public health importance were found, including the mecC-mediated methicillin resistance trait.

Comprehensive Approaches for the Search and Characterization of Staphylococcins.

Novel and sustainable approaches are required to curb the increasing threat of antimicrobial resistance (AMR). Within the last decades, antimicrobial peptides, especially bacteriocins, have received increased attention and are being explored as suitable alternatives to antibiotics. Bacteriocins are ribosomally synthesized antimicrobial peptides produced by bacteria as a self-preservation method against competitors. Bacteriocins produced by Staphylococcus, also referred to as staphylococcins, have steadily shown great antimicrobial potential and are currently being considered promising candidates to mitigate the AMR menace. Moreover, several bacteriocin-producing Staphylococcus isolates of different species, especially coagulase-negative staphylococci (CoNS), have been described and are being targeted as a good alternative. This revision aims to help researchers in the search and characterization of staphylococcins, so we provide an up-to-date list of bacteriocin produced by Staphylococcus. Moreover, a universal nucleotide and amino acid-based phylogeny system of the well-characterized staphylococcins is proposed that could be of interest in the classification and search for these promising antimicrobials. Finally, we discuss the state of art of the staphylococcin applications and an overview of the emerging concerns.

Nasal staphylococci community of healthy pigs and pig-farmers in Aragon (Spain). Predominance and within-host resistome diversity in MRSA-CC398 and MSSA-CC9 lineages.

This study investigated the diversity and carriage rate of nasal Staphylococcus spp., and within-host variability of antimicrobial resistance (AMR), virulence determinants, immune evasion cluster (IEC) types and genetic lineages of S. aureus isolates. Also, the co-carriage rate of CoNS with S. aureus in the same nasal niche of healthy pigs and pig-farmers were studied in four pig-farms (A-D) in Aragon (Spain). Nasal samples of 40 pigs (10 pigs/farm) and 10 pig-farmers (2-3/farm) were collected for staphylococci recovery and isolates (up to 9 per sample) were identified by MALDI-TOF-MS. The virulence and AMR genes and spa-types of S. aureus isolates were investigated by PCR/sequencing. Of the 243 staphylococci identified (10 different species), 142 were S. aureus and 51 distinct isolates were selected for further characterization (that corresponded to one S. aureus/sample or more than one if they showed different AMR phenotypes). The highest carriage rate in pigs was S. aureus (65%) and S. chromogenes (22.5%), whereas in the pig-farmers, S. aureus (80%) and S. epidermidis (40%). Methicillin-resistant S. aureus (MRSA) were detected in 60% of pigs and 70% of pig-farmers. Only six S. aureus isolates were methicillin-susceptible (MSSA), all from farm-C. A multidrug resistance (MDR) phenotype was detected in all MRSA and in 83.3% of the MSSA isolates. All MRSA isolates were CC398 with spa-type t011 being the predominant (92.7%), while t034, t1451 (only in pig-farmers) and t4571 (in pigs) were also found. MSSA-CC9 isolates (t191, t1430) were detected in farm-C. All S. aureus isolates were negative for luk-S/F-PV, tst, and scn genes, except one MSSA-CC45-t065-IEC-type C isolate from a pig-farmer. About 34.6% and 75.0% of the pigs and pig-farmers S. aureus carriers, respectively, harboured within-host varied spa-types or resistomes. Moreover, 40% of pigs and pig-farmers with MRSA-CC398 had no CoNS nasal co-carriage, and 23.3% had ≥2 CoNS carriage. Conversely, only 16.7% of MSSA carriers had no CoNS co-carriage, whereas 50% had ≥2 CoNS carriages. The very high MRSA level and within-host resistome diversities highlight the need for multiple samplings to account for the dynamics of AMR crisis and control of inter-host transmission of S. aureus in pig-farms using "One Health" approach.

Detection of antimicrobial producing Staphylococcus from migratory birds: Potential role in nasotracheal microbiota modulation.

A collection of 259 staphylococci of 13 different species [212 coagulase-negative (CoNS) and 47 coagulase-positive (CoPS)] recovered from nasotracheal samples of 87 healthy nestling white storks was tested by the spot-on-lawn method for antimicrobial-activity (AA) against 14 indicator bacteria. Moreover, extracts of AP isolates were obtained [cell-free-supernatants (CFS) both crude and concentrated and butanol extracts] and tested against the 14 indicator bacteria. The microbiota modulation capacity of AP isolates was tested considering: (a) intra-sample AA, against all Gram-positive bacteria recovered in the same stork nasotracheal sample; (b) inter-sample AA against a selection of representative Gram-positive bacteria of the nasotracheal microbiota of all the storks (30 isolates of 29 different species and nine genera). In addition, enzymatic susceptibility test was carried out in selected AP isolates and bacteriocin encoding genes was studied by PCR/sequencing. In this respect, nine isolates (3.5%; seven CoNS and two CoPS) showed AA against at least one indicator bacteria and were considered antimicrobial-producing (AP) isolates. The AP isolates showed AA only for Gram-positive bacteria. Three of these AP isolates (S. hominis X3764, S. sciuri X4000, and S. chromogenes X4620) revealed AA on all extract conditions; other four AP isolates only showed activity in extracts after concentration; the remaining two AP isolates did not show AA in any of extract conditions. As for the microbiota modulation evaluation, three of the nine AP-isolates revealed intra-sample AA. It is to highlight the potent inter-sample AA of the X3764 isolate inhibiting 73% of the 29 representative Gram-positive species of the nasotracheal stork microbiota population. On the other hand, enzymatic analysis carried out in the two highest AP isolates (X3764 and X4000) verified the proteinaceous nature of the antimicrobial compound and PCR analysis revealed the presence of lantibiotic-like encoding genes in the nine AP isolates. In conclusion, these results show that nasotracheal staphylococci of healthy storks, and especially CoNS, produce antimicrobial substances that could be important in the modulations of their nasal microbiota.

Nasotracheal Microbiota of Nestlings of Parent White storks with Different Foraging Habits in Spain.

Migratory storks could be vectors of transmission of bacteria of public health concern mediated by the colonization, persistence and excretion of such bacteria. This study aims to determine genera/species diversity, prevalence, and co-colonization indices of bacteria obtained from tracheal (T) and nasal (N) samples from storks in relation to exposure to point sources through foraging. One-hundred and thirty-six samples from 87 nestlings of colonies of parent white storks with different foraging habits (natural habitat and landfills) were obtained (84 T-samples and 52 N-samples) and processed. Morphologically distinct colonies (up to 12/sample) were randomly selected and identified by MALDI-TOF-MS. About 87.2% of the total 806 isolates recovered were identified: 398 from T-samples (56.6%) and 305 from N-samples (43.4%). Among identified isolates, 17 genera and 46 species of Gram-positive and Gram-negative bacteria were detected, Staphylococcus (58.0%) and Enterococcus (20.5%) being the most prevalent genera. S. sciuri was the most prevalent species from T (36.7%) and N (34.4%) cavities of total isolates, followed by E. faecalis (11.1% each from T and N), and S. aureus [T (6.5%), N (13.4%)]. Of N-samples, E. faecium was significantly associated with nestlings of parent storks foraging in landfills (p = 0.018). S. sciuri (p = 0.0034) and M. caseolyticus (p = 0.032) from T-samples were significantly higher among nestlings of parent storks foraging in natural habitats. More than 80% of bacterial species in the T and N cavities showed 1-10% co-colonization indices with one another, but few had ≥ 40% indices. S. sciuri and E. faecalis were the most frequent species identified in the stork nestlings. Moreover, they were highly colonized by other diverse and potentially pathogenic bacteria. Thus, storks could be sentinels of point sources and vehicles of bacterial transmission across the "One Health" ecosystems.

Clonal relatedness of coagulase-positive staphylococci among healthy dogs and dog-owners in Spain. Detection of multidrug-resistant-MSSA-CC398 and novel linezolid-resistant-MRSA-CC5.

Introduction: Nasal carriage of coagulase-positive staphylococci (CoPS) in healthy dogs could indicate increased risks of colonization for in-contact people or vice versa. This study determined the nasal carriage rate of CoPS among healthy dogs and in-contact people, their genotypic characteristics and phylogenetic relatedness. Methods: Nasal samples were collected from 27 households (34 dogs and 41 humans) in Spain. Staphylococci were identified by MALDI-TOF-MS, their antimicrobial resistance (AMR) genes and spa-types were tested by PCR/sequencing. The relatedness of CoPS from the same households was assessed by core genome single nucleotide polymorphisms (SNPs) analyses. Results: Staphylococcus aureus carriage was found in 34.1% of humans (including one methicillin-resistant S. aureus MRSA-CC5-t2220-SCCmec type-IV2B) and 5.9% of dogs; Staphylococcus pseudintermedius in 2.4% of humans and 32.4% of dogs; while Staphylococcus coagulans was only detected in dogs (5.4%). Remarkably, one human co-carried S. aureus/S. pseudintermedius, while a dog co-carried the three CoPS species. Household density was significantly associated with S. pseudintermedius carriage in households with > than 1 dog and >than 1 human (OR = 18.10, 95% CI: 1.24-260.93, p = 0.034). Closely related (<15 SNPs) S. aureus or S. pseudintermedius were found in humans or dogs in three households. About 56.3% S. aureus carriers (dog or human) harboured diverse within-host spa-types or AMR genotypes. Ten clonal complexes (CCs) were detected among the S. aureus, of which methicillin-susceptible S. aureus-CC398-IEC-type C (t1451 and t571) was the most frequent, but exclusive to humans. S. aureus and S. pseudintermedius isolates harboured resistance genes or mutations associated to 9 classes of antimicrobials including linezolid (G2261A & T1584A point mutations in 23S rDNA). The S. coagulans isolates were susceptible to all antimicrobials. Most of the S. pseudintermedius carried lukS/F-I, siet, and sient genes, and all S. aureus were negative for lukS/F-PV, tst-1, eta and etb genes. Discussion: Clonally related human-to-human MSSA and dog-to-human MSSP were found. The detection of the MSSA-CC398 clade highlights the need for its continuous surveillance from One Health perspective.

Staphylococcus aureus Carriage in the Nasotracheal Cavities of White Stork Nestlings (Ciconia ciconia) in Spain: Genetic Diversity, Resistomes and Virulence Factors.

The molecular ecology of Staphylococcus aureus in migratory birds (such as white storks) is necessary to understand their relevance in the "One Health" ecosystems. This study determined the nasotracheal carriage rates of S. aureus from white storks in Southern Spain and genetically characterized the within-host diversity. A collection of 67 S. aureus strains, previously obtained from 87 white stork nestlings (52 nasal and 85 tracheal samples) fed by their parents with food foraged in natural and landfill habitats, were tested for their antimicrobial resistance (AMR) phenotypes. Moreover, the AMR genotypes, immune evasion cluster (IEC), virulence genes and the detection of CC398 lineage were studied by PCR. The spa types and multilocus-sequencing-typing (MLST) were also determined by PCR and sequencing. Staphylococcus aureus carriage was found in 31% of storks (36.5%/11.9% in nasal/tracheal samples). All isolates were methicillin-susceptible (MSSA) and 8.8% of them were also susceptible to all tested antibiotics. The AMR phenotype/percentage/genes detected were as follows: penicillin/79.1%/blaZ; erythromycin-clindamycin-inducible/19.1%/ermA, ermT; tetracycline/11.9%/tetK; clindamycin/4.5%/lnuA and ciprofloxacin/4.5%. Twenty-one different spa types, including 2 new ones (t7778-ST15-CC15 and t18009-ST26-CC25), were detected and ascribed to 11 clonal complexes (CCs). MSSA-CC398 (8.2%), MSSA-CC15 (7.1%) and MSSA-ST291 (5.9%) were the most prevalent lineages in storks. Moreover, tst-positive (MSSA-CC22-t223 and MSSA-CC30-t1654), eta-positive (MSSA-CC9-t209) and etb-positive strains (MSSA-CC45-t015) were detected in four storks. The 18.5% of storks harboured distinct MSSA strains (with different lineages and/or AMR genes). Nestlings of storks foraging in landfills (10 CCs) had more diverse S. aureus strains than those of parents foraging in natural habitats (3 CCs). Low level of AMR was demonstrated among S. aureus strains. The predominance of MSSA-CC398 (an emergent clade) and toxigenic MSSA strains in stork nestlings highlight the need for continuous surveillance of S. aureus in wild birds.

Nasotracheal enterococcal carriage and resistomes: detection of optrA-, poxtA- and cfrD-carrying strains in migratory birds, livestock, pets, and in-contact humans in Spain.

This study determined the carriage rates and antimicrobial resistance (AMR) genes of enterococci from nasotracheal samples of three healthy animal species and in-contact humans. Nasal samples were collected from 27 dog-owning households (34 dogs, 41 humans) and 4 pig-farms (40 pigs, 10 pig-farmers), and they were processed for enterococci recovery (MALDI-TOF-MS identification). Also, a collection of 144 enterococci previously recovered of tracheal/nasal samples from 87 white stork nestlings were characterized. The AMR phenotypes were determined in all enterococci and AMR genes were studied by PCR/sequencing. MultiLocus-Sequence-Typing was performed for selected isolates. About 72.5% and 60% of the pigs and pig-farmers, and 29.4% and 4.9%, of healthy dogs and owners were enterococci nasal carriers, respectively. In storks, 43.5% of tracheal and 69.2% of nasal samples had enterococci carriages. Enterococci carrying multidrug-resistance phenotype was identified in 72.5%/40.0%/50.0%/23.5%/1.1% of pigs/pig-farmers/dogs/dogs' owners/storks, respectively. Of special relevance was the detection of linezolid-resistant enterococci (LRE) in (a) 33.3% of pigs (E. faecalis-carrying optrA and/or cfrD of ST59, ST330 or ST474 lineages; E. casseliflavus-carrying optrA and cfrD); (b) 10% of pig farmers (E. faecalis-ST330-carrying optrA); (c) 2.9% of dogs (E. faecalis-ST585-carrying optrA); and (d) 1.7% of storks (E. faecium-ST1736-carrying poxtA). The fexA gene was found in all optrA-positive E. faecalis and E. casseliflavus isolates, while fexB was detected in the poxtA-positive E. faecium isolate. The enterococci diversity and AMR rates from the four hosts reflect differences in antimicrobial selection pressure. The detection of LRE carrying acquired and transferable genes in all the hosts emphasizes the need to monitor LRE using a One-Health approach.

Comparative review of the nasal carriage and genetic characteristics of Staphylococcus aureus in healthy livestock: Insight into zoonotic and anthroponotic clones.

Given the central role of livestock in understanding the genomic epidemiology of S. aureus, the present study systematically reviewed and synthesized data on the nasal S. aureus carriage, resistance patterns to critical antimicrobial agents, virulence factors and genetic lineages among healthy livestock. Bibliographical databases were searched for published studies from May 2003 to May 2022 on nasal S. aureus carriage, their phenotypic and genetic characteristics among healthy pigs (A), sheep and goats (B), cattle (C), poultry (D), camels (E) and buffaloes (F). Special focus was given to the prevalence of nasal MRSA, MRSA-CC398, MRSA-CC9, mecC-MRSA, MSSA-CC398, and resistance to linezolid (LZDR), chloramphenicol (CLOR) and tetracycline (TETR) in S. aureus isolates. Of the 5492 studies identified, 146 comprised groups A(83)/B(18)/C(33)/D(4)/E(5)/F(3), and were found eligible. The overall pooled nasal prevalence of MRSA in healthy livestock was 13.8% (95% CI: 13.5-14.1) among a pooled 48,154 livestock population. Specifically, the pooled prevalence in groups A to F were: 16.0% (95% CI: 15.6-16.4), 3.7% (95% CI: 2.9-4.6), 13.6% (95% CI: 12.8-14.4), 5.8% (95% CI: 5.1-6.5), 7.1% (95% CI: 6.1-10.7), and 2.8% (95% CI: 1.5-4.8), respectively. These values varied considerably by continent. Varied pooled prevalences of CC398 lineage with respect to MRSA isolates were obtained, with the highest from pigs and cattle (>70%). Moreover, other classical animal-adapted MRSA as well as MSSA-CC398-t1928 were reported. TETR-MSSA was lowest in cattle (18.9%) and highest in pigs (80.7%). LZDR-S. aureus was reported in 8 studies (mediated by optrA and cfr), mainly in pigs (n = 4), while CLOR-S. aureus was reported in 32 studies. The virulence genes luk-S/F-PV, tst, etd, sea, see were sparsely reported, and only in non-CC398-MRSA lineages. Certain S. aureus clones and critical AMR appeared to have predominance in some livestock, as in the case of pigs that are high nasal carriers of MRSA-CC398 and -CC9, and MSSA-CC398. These findings highlight the need for adequate prevention against the transmission of zoonotic S. aureus lineages to humans.

Beyond the Wild MRSA: Genetic Features and Phylogenomic Review of mecC-Mediated Methicillin Resistance in Non-aureus Staphylococci and Mammaliicocci.

Methicillin resistance, mediated by the mecA gene in staphylococci and mammaliicocci, has caused tremendous setbacks in the use of antibiotics in human and veterinary medicine due to its high potential of presenting the multidrug resistance (MDR) phenotype. Three other mec analogs exist, of which the mecC has evolutionary been associated with methicillin-resistant Staphylococcus aureus (MRSA) in wild animals, thus loosely referred to as the wild MRSA. In this study, we present an epidemiological review and genomic analysis of non-aureus staphylococci and mammaliicocci that carry the mecC-mediated methicillin resistance trait and determine whether this trait has any relevant link with the One Health niches. All previous studies (2007 till 2023) that described the mecC gene in non-aureus staphylococci and mammaliicocci were obtained from bibliometric databases, reviewed, and systematically analyzed to obtain the antimicrobial resistance (AMR) and virulence determinants, mobilome, and other genetic contents. Moreover, core genome single-nucleotide polymorphism analysis was used to assess the relatedness of these strains. Of the 533 articles analyzed, only 16 studies (on livestock, environmental samples, milk bulk tanks, and wild animals) were eligible for inclusion, of which 17 genomes from 6 studies were used for various in silico genetic analyses. Findings from this systematic review show that all mecC-carrying non-aureus staphylococci were resistant to only beta-lactam antibiotics and associated with the classical SCCmec XI of S. aureusLGA251. Similarly, two studies on wild animals reported mecC-carrying Mammaliicoccus stepanovicii associated with SCCmec XI. Nevertheless, most of the mecC-carrying Mammaliicoccus species presented an MDR phenotype (including linezolid) and carried the SCCmec-mecC hybrid associated with mecA. The phylogenetic analysis of the 17 genomes revealed close relatedness (<20 SNPs) and potential transmission of M. sciuri and M. lentus strains in livestock farms in Algeria, Tunisia, and Brazil. Furthermore, closely related M. sciuri strains from Austria, Brazil, and Tunisia (<40 SNPs) were identified. This systematic review enhances our comprehension of the epidemiology and genetic organization of mecC within the non-aureus staphylococci and mammaliicocci. It could be hypothesized that the mecC-carrying non-aureus staphylococci are evolutionarily related to the wild MRSA-mecC. The potential implications of clonal development of a lineage of mecA/mecC carrying strains across multiple dairy farms in a vast geographical region with the dissemination of MDR phenotype is envisaged. It was observed that most mecC-carrying non-aureus staphylococci and mammaliicocci were reported in mastitis cases. Therefore, veterinarians and veterinary microbiology laboratories must remain vigilant regarding the potential existence of mecA/mecC strains originating from mastitis as a potential niche for this resistance trait.

Nasal Staphylococcus aureus and S. pseudintermedius carriage in healthy dogs and cats: a systematic review of their antibiotic resistance, virulence and genetic lineages of zoonotic relevance.

The molecular ecology of Staphylococcus aureus, Staphylococcus pseudintermedius and their methicillin-resistant strains in healthy dogs and cats could serve as good models to understand the concept of bacterial zoonosis due to animal companionship. This study aims to provide insights into pooled prevalence, genetic lineages, virulence and antimicrobial resistance (AMR) among healthy dogs and cats. Original research and brief communication articles published from 2001 to 2021 that reported the nasal detection of S. aureus and S. pseudintermedius in healthy dogs and cats in the community, homes and outside veterinary clinics were examined and analysed. Forty-nine studies were eligible and included in this systematic review. The pooled prevalence of nasal carriage of S. aureus/methicillin-resistant S. aureus (MRSA) in healthy dogs and cats were 10.9% (95% CI: 10.1-11.9)/2.8% (95% CI: 2.4-3.2) and 3.2% (95% CI: 1.9-4.8)/0.5% (95% CI: 0.0-1.1), respectively. Conversely, the pooled prevalence of S. pseudintermedius/methicillin-resistant S. pseudintermedius (MRSP) in healthy dogs and cats were 18.3% (95% CI: 17.1-19.7)/3.1% (95% CI: 2.5-3.7) and 1.3% (95% CI: 0.6-2.4)/1.2% (95% CI: 0.6-2.3), respectively. Although highly diverse genetic lineages of S. aureus were detected in healthy dogs and cats, MSSA-CC1/CC5/CC22/CC45/CC121/CC398 and MRSA-CC5/CC93/CC22/CC30 were mostly reported in dogs; and MSSA-CC5/CC8/CC15/CC48 and MRSA-CC22/CC30/CC80 in cats. Of note, MSSA-CC398 isolates (spa-types t034 and t5883) were detected in dogs. Genetic lineages often associated with MSSP/MRSP were ST20/ST71, highlighting the frequent detection of the epidemic European MRSP-ST71 clone in dogs. S. aureus isolates carrying the luk-S/F-PV, tst, eta, etb and etd genes were seldomly detected in dogs, and luk-S/F-PV was the unique virulence factor reported in isolates of cats. S. pseudintermedius isolates harbouring the luk-S/F-I, seint and expA genes were frequently found, especially in dogs. High and diverse rates of AMR were noted, especially among MRSA/MRSP isolates. There is a need for additional studies on the molecular characterization of isolates from countries with under-studied nasal staphylococci isolates.

Antimicrobial Resistance and Antimicrobial Activity of Staphylococcus lugdunensis Obtained from Two Spanish Hospitals.

Staphylococcus lugdunensis is a coagulase-negative-staphylococci (CoNS) that lately has gained special attention in public health as a human pathogen and also as a bacteriocin-producer bacteria. In this study, we characterized 56 S. lugdunensis isolates recovered from human samples in two Spanish hospitals. Antimicrobial susceptibility testing was performed and antimicrobial resistance and virulence genotypes were determined. Antimicrobial activity (AA) production was evaluated by the spot-on-lawn method against 37 indicator bacteria, including multidrug-resistant (MDR) isolates, and the presence of the lugD gene coding for lugdunin bacteriocin was analyzed by PCR. The antibiotic resistance detected was as follows (% resistance/genes detected): penicillin (44.6%/blaZ), oxacillin (1.8%/mecA on SCCmec-V), erythromycin-clindamycin inducible (7.1%/erm(C), msrA), tetracycline (5.3%/tetK), gentamicin and/or tobramycin (3.6%/ant(4')-Ia, acc(6')-aph(2″)), and fosfomycin (21.4%). A MDR phenotype was detected in 5% of isolates. Twenty-one of the S. lugdunensis isolates showed susceptibility to all 20 antibiotics tested (37.5%). The screening for AA revealed 23 antimicrobial producer (AP) isolates with relevant inhibition against coagulase-positive-staphylococci (CoPS), including both methicillin-susceptible and -resistant S. aureus. The lugD gene was detected in 84% of the 56 S. lugdunensis isolates. All of the AP S. lugdunensis isolates (n = 23) carried the lugD gene and it was also detected in 24 of the non-AP isolates, suggesting different gene expression levels. One of the AP isolates stood out due to its high antimicrobial activity against more than 70% of the indicator bacteria tested, so it will be further characterized at genomic and proteomic level.