Marine Bacillus pumilus substances exhibit antimicrobial effect on multidrug-resistant Staphylococcus aureus.

AIMS: This study aimed to assess the antimicrobial potential of Bp1-AdE, produced by Bacillus pumilus 64-1, and to investigate its mode of action against Staphylococcus aureus and methicillin-resistant S. aureus (MRSA). METHODS AND RESULTS: Bp-1AdE, derived from sponge-associated B. pumilus, exhibited bactericidal activity at 1 550 µg ml-1 against S. aureus ATCC29213 and MRSA strains. Light and fluorescence microscopy revealed drastic cell lysis of S. aureus treated with Bp-1AdE. Scanning and transmission electron microscopy suggested that Bp-1AdE disrupts the cytoplasmic membrane. Toxicity assays showed that Bp-1AdE was non-toxic to Tenebrio molitor larvae. Liquid chromatography-mass spectrometry and Global Natural Product Social spectral libraries identified four substances within Bp-1AdE, including aliphatic alcohols [3,4-dipentylhexane-2,5-diol and 1,1'-(4,5-dibutyl-3,6-dimethylcyclohexane-1,2-diyl)bis(ethan-1-one)] and terpenoids (cholic acid and canrenone). CONCLUSIONS: Bp-1AdE demonstrated selective toxicity and bactericidal activity, highlighting its potential for controlling infections caused by multidrug-resistant S. aureus strains.

Mammaliicoccus sciuri's Pan-Immune System and the Dynamics of Horizontal Gene Transfer Among Staphylococcaceae: a One-Health CRISPR Tale.

Recently emancipated from the Staphylococcus genus due to genomic differences, Mammaliicoccus sciuri, previously classified as an occasional pathogen, emerges as a significant player in the landscape of resistance gene dissemination among Staphylococcaceae. Despite its classification, its role remained enigmatic. In this study, we delved into the genomic repertoire of M. sciuri to unravel its contribution to resistance and virulence gene transfer in the context of One Health. Through comprehensive analysis of publicly available genomes, we unveiled a diverse pan-immune system adept at defending against exogenous genetic elements, yet concurrently fostering horizontal gene transfer (HGT). Specifically, exploration of CRISPR-Cas systems, with spacer sequences as molecular signatures, elucidated a global dissemination pattern spanning environmental, animal, and human hosts. Notably, we identified the integration of CRISPR-Cas systems within SCCmecs (Staphylococcal Cassette Chromosome mec), harboring key genes associated with pathogenicity and resistance, especially the methicillin resistance gene mecA, suggesting a strategic adaptation to outcompete other mobile genetic elements. Our findings underscored M. sciuri's active engagement in HGT dynamics and evolutionary trajectories within Staphylococcaceae, emphasizing its central role in shaping microbial communities and highlighting the significance of understanding its implications in the One Health framework, an interdisciplinary approach that recognizes the interconnectedness of human, animal, and environmental health to address global health challenges.

Staphylococcus haemolyticus: An updated review on nosocomial infections, antimicrobial resistance, virulence, genetic traits, and strategies for combating this emerging opportunistic pathogen.

Staphylococcus haemolyticus, a key species of the Staphylococcus genus, holds significant importance in healthcare-associated infections, due to its notable resistance to antimicrobials, like methicillin, and proficient biofilms-forming capabilities. This coagulase-negative bacterium poses a substantial challenge in the battle against nosocomial infections. Recent research has shed light on Staph. haemolyticus genomic plasticity, unveiling genetic elements responsible for antibiotic resistance and their widespread dissemination within the genus. This review presents an updated and comprehensive overview of the clinical significance and prevalence of Staph. haemolyticus, underscores its zoonotic potential and relevance in the one health framework, explores crucial virulence factors, and examines genetics features contributing to its success in causing emergent and challenging infections. Additionally, we scrutinize ongoing studies aimed at controlling spread and alternative approaches for combating it.

Antimicrobial resistance and biotechnological potential of plastic-associated bacteria isolated from an urban estuary.

Plastics have quickly become one of the major pollutants in aquatic environments worldwide and solving the plastic pollution crisis is considered a central goal of modern society. In this study, 10 different plastic samples, including high- and low-density polyethylene and polypropylene, were collected from a deeply polluted urban estuary in Brazil. By employing different isolation and analysis approaches to investigate plastic-associated bacteria, a predominance of potentially pathogenic bacteria such as Acinetobacter, Aeromonas, and Vibrio was observed throughout all plastic samples. Bacteria typically found in the aquatic environment harboured clinically relevant genes encoding resistance to carbapenems (blaKPC ) and colistin (such as mcr-3 and mcr-4), along with genetic determinants associated with potentially active gene mobilization. Whole genome sequencing and annotation of three plastic-associated Vibrio strains further demonstrated the carriage of mobile genetic elements and antimicrobial resistance and virulence genes. On the other hand, bacteria isolated from the same samples were also able to produce esterases, lipases, and bioemulsifiers, thus highlighting that the plastisphere could also be of special interest from a biotechnological perspective.

Tenebrio molitor as a model system to study Staphylococcus spp virulence and horizontal gene transfer.

Invertebrates can provide a valuable alternative to traditional vertebrate animal models for studying bacterial and fungal infections. This study aimed to establish the larvae of the coleoptera Tenebrio molitor (mealworm) as an in vivo model for evaluating virulence and horizontal gene transfer between Staphylococcus spp. After identifying the best conditions for rearing T. molitor, larvae were infected with different Staphylococcus species, resulting in dose-dependent killing curves. All species tested killed the insects at higher doses, with S. nepalensis and S. aureus being the most and least virulent, respectively. However, only S. nepalensis was able to kill more than 50% of larvae 72 h post-infection at a low amount of 105 CFU. Staphylococcus infection also stimulated an increase in the concentration of hemocytes present in the hemolymph, which was proportional to the virulence. To investigate T. molitor's suitability as an in vivo model for plasmid transfer studies, we used S. aureus strains as donor and recipient of a plasmid containing the gentamicin resistance gene aac(6')-aph(2″). By inoculating larvae with non-lethal doses of each, we observed conjugation, and obtained transconjugant colonies with a frequency of 1.6 × 10-5 per donor cell. This study demonstrates the potential of T. molitor larvae as a reliable and cost-effective model for analyzing the virulence of Staphylococcus and, for the first time, an optimal environment for the plasmid transfer between S. aureus carrying antimicrobial resistance genes.

Immunoproteomic and immunoinformatic approaches identify secreted antigens and epitopes from Staphylococcus saprophyticus.

Urinary tract infections (UTIs) are common human infections that compromise women's health around the world, even though they can affect men and women of all ages. Bacterial species are the primary causative agents of UTIs, while Staphylococcus saprophyticus, a gram-positive bacterium, is especially important for uncomplicated infections in young women. Despite the number of antigenic proteins identified in Staphylococcus aureus and other bacteria of the genus, there is no immunoproteomic study in S. saprophyticus. In this context, since pathogenic microorganisms secrete important proteins that interact with hosts during infection, the present work aims to identify the exoantigens from S. saprophyticus ATCC 15305 by immunoproteomic and immunoinformatic approaches. We identified 32 antigens on the exoproteome of S. saprophyticus ATCC 15305 by immunoinformatic tools. By using 2D-IB immunoproteomic analysis, it was possible to identify 3 antigenic proteins: transglycosylase IsaA, enolase and the secretory antigen Q49ZL8. In addition, 5 antigenic proteins were detected by immunoprecipitation (IP) approach, where the most abundant were bifunctional autolysin and transglycosylase IsaA proteins. The transglycosylase IsaA was the only protein detected by all the tools approaches used in this study. In this work it was possible to describe a total of 36 S. saprophyticus exoantigens. Immunoinformatic analysis allowed the identification of 5 exclusive linear B cell epitopes from S. saprophyticus and 5 epitopes presenting homology with other bacteria that cause UTIs. This work describes, for the first time, the profile of exoantigens secreted by S. saprophyticus and can contribute to the identification of new diagnostic targets of UTIs, as well as to develop vaccines and immunotherapies against bacterial urinary infections.

Unusual carriage of virulence genes sasX/sesI/shsA by nosocomial Staphylococcus haemolyticus from Brazil.

Background: Staphylococcus haemolyticus is an emerging threat in the nosocomial environment but only some virulence factors are known. Materials & methods: The frequency of the sasX gene (or orthologues sesI/shsA), encoding an invasiveness-related surface-associated protein, in S. haemolyticus was detected in different hospitals in Rio de Janeiro. Results: 9.4% of strains were sasX/sesI/shsA-positive, some were in the context of the ΦSPß-like prophage and devoid of CRISPR systems, indicating potential transferability of their virulence genes. Gene sequencing evidenced that Brazilian S. haemolyticus harbored sesI, instead of the usual sasX, while S. epidermidis had sasX instead of sesI, suggesting horizontal acquisition. Conclusion: The contexts of Brazilian sasX/sesI/shsA favor transfer, which is alarming given the difficulty in treating infections caused by S. haemolyticus.

Dispersion and persistence of antimicrobial resistance genes among Staphylococcus spp. and Mammaliicoccus spp. isolated along a swine manure treatment plant.

Staphylococcus spp. and Mammaliicoccus spp. colonize the skin and mucosa of humans and other animals and are responsible for several opportunistic infections. Staphylococci antibiotic resistance may be present in the environment due to the spread of treated and untreated manure from the livestock industry due to antibiotic use to disease control or growth promoter. In this work, we analyzed the species distribution and antimicrobial susceptibility of Staphylococcus and Mammaliicoccus species along different sites of a swine manure treatment plant from Southeastern Brazil. Bacterial colonies were obtained on mannitol salt agar, selected after catalase test and Gram staining, and finally identified by mass spectrometry and sequencing of the tuf gene. According to the results, S.cohnii and S. simulans were the most prevalent species. Antibiotic resistance test revealed that several strains were resistant to multiple drugs, with high levels of chloramphenicol resistance (98%), followed by erythromycin (79%), tetracycline (73%), gentamicin (46%), ciprofloxacin (42%), cefoxitin (18%), sulfamethoxazole + trimethoprim (12%), and linezolid (4%). In addition, gene detection by PCR showed that all strains carried at least 2 resistance genes and one of them carried all 11 genes investigated. Using the GTG5-PCR approach, a high genetic similarity was observed between some strains that were isolated from different points of the treatment plant. Although some were seemingly identical, differences in their resistance phenotype and genotype suggest horizontal gene transfer. The presence of resistant bacteria and resistance genes along the treatment system highlights the potential risk of contamination by people in direct contact with these animals and the soil since the effluent is used as a biofertilizer in the surrounding environment.

Coronary atherosclerosis and periodontitis have similarities in their clinical presentation.

Background: Periodontitis (PE) and coronary heart disease (CHD) possess multiple mechanisms for a putative association. This case-control study compared the periodontal status among CHD subjects to controls without CHD, while also investigating atheroma invasion by known periodontal pathogens. Methods: 161 subjects participated in this study were divided into three CHD groups: No CHD, chronic CHD, acute CHD. Additional analysis involved grouping subjects according to number of atheromas: no atheroma, 1-4 atheromas, 5-18 atheromas. Data were collected from medical records, periodontal examinations, and questionnaires that included demographic, behavioral, and oral health variables. Angiographic catheterizations were analyzed according to the number of atheroma lesions, lesion size, lesion location, and atheroma lesion stability. Lipoprotein profile, inflammatory markers and cells were analyzed. The microbiological branch added 30 individuals who had their atheroma lesion and subgingival plaque analyzed using polymerase chain reaction probes against the 16 s region, red complex and Aggregatibacter actinomycetemcomitans' DNA. Results: Subjects with CHD had high levels of systemic inflammatory markers and low levels of high-density lipoproteins compared to subjects without CHD. Subjects without CHD and clear coronaries had a prevalence of mild CAL, while individuals with more atheroma lesions had advanced CAL and more active PE. Subjects with more advanced CAL were 4 times more likely to have CHD compared to subjects with less, which is comparable to smoking. Only 4 subjects had the screened pathogens detected in atheroma, although these subjects also have the screened pathogens in subgingival plaque. However, 80% of atheromas had bacteria. Conclusions: CHD and PE showed similarities in progression while active PE led to more atheroma lesions that also tended to be larger in size.

Interspecies transfer of plasmid-borne gentamicin resistance between Staphylococcus isolated from domestic dogs to Staphylococcus aureus.

As preconized by the One Health concept, the intimate relationship between pets and owners is a common source for the trade of microorganisms with zoonotic potential, and with them, antimicrobial resistance genes. In this work, we evaluated the presence of antimicrobial resistance genes, that are usually within mobile genetic elements, in a laboratory collection of 79 canine Staphylococcus strains, mostly Staphylococcus pseudintermedius and Staphylococcus coagulans. Resistance to tetracycline was observed in 34% of the strains, followed by resistance to erythromycin (21%) and gentamicin (19%). These phenotypes were partially correlated with the presence of the tetracycline resistance genes tet(M) and tet(K) in 64% and 44% of all strains, respectively; erythromycin resistance genes erm(A) and erm(C) in 53% and 23%; and gentamicin resistance gene aac(6')-aph(2″) in 26% of the strains. At least 45% of the strains harbored high- and/or low-molecular weight plasmids, whose transfer may be facilitated by their widespread biofilm-forming capacity, and absence of restrictive CRISPR systems. We selected eight plasmid-bearing and multidrug resistant strains, which were submitted to plasmid curing by stress with SDS. No strain lost resistance during stressing cultivation but, by conjugation experiments, the S. pseudintermedius strain 27 transferred its plasmid-borne resistance to gentamicin, conferred by the aac(6')-aph(2″) gene, to Staphylococcus aureus. The frequent empirical use of gentamicin to treat skin and ear infections in domestic dogs is likely to select resistant strains. Also, as demonstrated by our study, these strains can serve as gene reservoirs for human pathogens, such as S. aureus.

shsA: A novel orthologous of sasX/sesI virulence genes is detected in Staphylococcus haemolyticus Brazilian strains.

The surface protein SasX, has a key role in methicillin-resistant Staphylococcus aureus (MRSA) colonization and pathogenesis, and has been associated with the epidemic success of some MRSA clones. To date, only one SasX homologous protein, named SesI, has been described in Staphylococcus epidermidis. In this work, we analyze the occurrence of the sasX gene and its genetic environment in Staphylococcus haemolyticus S. haemolyticus clinical strains (n = 62) were screened for the presence of the sasX gene and its carrier, the prophage Φ SPß-like. A deep characterization was done in one strain (MD43), through which we determined the complete nucleotide sequence for the S. haemolitycus sasX-like gene. Whole genome sequencing of strain MD43 was performed, and the gene, termed here because of its unique attributes, shsA, was mapped to the Φ SPß-like prophage sequence. The shsA gene was detected in 33 out of 62 strains showing an average identity of 92 and 96% with the sasX and sesI genes and at the amino acid level, 88% identity with SasX and 92% identity with SesI. The ~124Kb Φ SPß-like prophage sequence showed a largely intact prophage compared to its counterpart in S. epidermidis strain RP62A, including the sesI insertion site. In conclusion, we identified a new sasX ortholog in S. haemolyticus (shsA). Its horizontal spread from this reservoir could represent an emergent threat in healthcare facilities since so far, no S. aureus sasX+ strains have been reported in Brazil.

Antibiotic and Heavy Metal Susceptibility of Non-Cholera Vibrio Isolated from Marine Sponges and Sea Urchins: Could They Pose a Potential Risk to Public Health?

Vibrio is an important human and animal pathogen that can carry clinically relevant antibiotic resistance genes and is present in different aquatic environments. However, there is a knowledge gap between antibiotic and heavy metal resistance and virulence potential when it is part of the microbiota from marine invertebrates. Here, we aimed to evaluate these characteristics and the occurrence of mobile genetic elements. Of 25 non-cholera Vibrio spp. from marine sponges and sea urchins collected at the coastlines of Brazil and France analyzed in this study, 16 (64%) were non-susceptible to antibiotics, and two (8%) were multidrug-resistant. Beta-lactam resistance (blaSHV) and virulence (vhh) genes were detected in sponge-associated isolates. The resistance gene for copper and silver (cusB) was detected in one sea urchin isolate. Plasmids were found in 11 (44%) of the isolates. This new information allows a better comprehension of antibiotic resistance in aquatic environments, since those invertebrates host resistant Vibrio spp. Thus, Vibrio associated with marine animals may pose a potential risk to public health due to carrying these antibiotic-resistant genes.

Characterization of biofilms and antimicrobial resistance of coagulase-negative Staphylococcus species involved with subclinical mastitis.

Biofilm formation is a central feature to guarantee staphylococcal persistence in hosts and is associated with several diseases that are difficult to treat. In this research paper, biofilm formation and antimicrobial susceptibility were investigated in staphylococcal strains belonging to several species. These strains were isolated from the milk of cows with subclinical mastitis and most of them were coagulase-negative, with the prevalence of Staphylococcus chromogenes. High genetic diversity was observed among the strains by pulsed field gel electrophoresis. Antimicrobial resistance was assessed by disk diffusion and more than 50% of the strains were resistant to ampicillin and penicillin G, with multi-resistance profiles (13.6%) also being observed. Most strains (65.9%) formed biofilms when cultivated in BHI supplemented with 1% glucose. Most strains (72.7%) carried the intercellular adhesion gene (icaA), while less than half (36.3%) carried the biofilm-associated protein gene (bap). Concentrations of up to 10xMIC of erythromycin and tetracycline were not sufficient to suppress cell viability in preformed biofilms. Our results revealed that a genetically diverse group of biofilm-forming Staphylococcus species can be involved in subclinical mastitis. Since high antimicrobial concentrations cannot eradicate biofilm cells in vitro, their use in dairy animals may be ineffective in controlling infections, while supporting selection of resistant microorganisms. These data reinforce the need for alternative therapies aiming at disrupting biofilms for effective disease control.

Antimicrobial and antibiofilm activities of marine sponge-associated bacteria against multidrug-resistant Staphylococcus spp. isolated from canine skin.

Dogs play important roles in our society, thus the concern for their health becomes imperative. Staphylococcus spp. are commensal bacterium frequently isolated from canine skin and recognized as zoonotic agents. These bacteria have been becoming increasingly resistant to antimicrobials used to treat infections and to produce biofilm, which further increases their virulence capability and resistance. In this context, sponges-associated bacteria are known as prolific sources of substances with antimicrobial activities, representing a potential to integrate the arsenal of drugs for clinical use. In this study, 121 strains of Staphylococcus isolated from healthy or infected dogs were characterized according to their resistance to antimicrobials, as well as to their biofilm production ability. From the total of strains, 82 were resistant to at least one antimicrobial and 40 were multidrug-resistant (MDR). Furthermore, 117 out of 121 were capable to produce biofilm, and within those 36 were classified as strong biofilm producers. A set of fifteen bacterial strains previously isolated from marine sponges were also evaluated for antimicrobial and antibiofilm activities. Among the marine bacteria with antimicrobial activity, eight inhibited the growth of more than 50% of the MDR Staphylococcus. In addition, the cell-free supernatant obtained from five sponge-associated bacteria cultures was able to disaggregate more than 50% of the mature biofilm staphylococcal cells. The organic extracts (256 µg/mL) from two potential strains, Pseudomonas fluorescens H40 and H41, dissociated the biofilm of a strain classified as MDR and strong biofilm producer in 88.5% and 91.3%, respectively. These marine Pseudomonas strains also exhibited a strong activity of antimicrobial and antibiofilm substances. The results suggest that the sponge-associated bacteria analyzed could be potential sources of antimicrobial and antibiofilm substances against MDR and biofilm producers Staphylococcus isolated from canine skin.

Staphylococcus nepalensis, a commensal of the oral microbiota of domestic cats, is a reservoir of transferrable antimicrobial resistance.

Staphylococcus nepalensis is a commensal bacterium from the oral microbiota of domestic cats, with a still obscure clinical importance. In this work, we analysed the ability of feline strains of S. nepalensis to transfer antimicrobial resistance genes to Staphylococcus aureus isolated from humans through plasmids. To this end, we first analysed all publicly available genomes from cat staphylococci using computational methods to build a pan-resistome. Genes that encode resistance to erythromycin, gentamicin, mupirocin and tetracycline, common to human and cat staphylococci and previously described to be located in mobile genetic elements, were chosen for the next analyses. We studied 15 strains of S. nepalensis, which were shown to be genetically different by GTG5-PCR. As observed by disc diffusion, resistance to tetracycline was widespread (80 %), followed by resistance to erythromycin (40 %), gentamicin (27 %) and mupirocin (7 %). The strains were positive for several antimicrobial resistance genes and more than half of them harboured plasmids. The loss of plasmids and resistance genes in some strains were induced by stress with SDS. Through conjugation experiments, we observed that these plasmids can be transferred to S. aureus, thus increasing its potential to resist drug therapy. Our findings show that S. nepalensis, an underestimated inhabitant of the cat microbiota, can be a reservoir of antimicrobial resistance genes for S. aureus and, like many other staphylococci, be an overlooked and silent threat to their animal hosts and humans living with them.

Shewanella harboring antimicrobial and copper resistance genes in sea urchins (Paracentrotus lividus) from the Crozon peninsula (Brittany, France).

Shewanella is a genus of aquatic non-fermenting Gram-negative bacteria with increasing numbers of reports of infections in humans and appearance of antimicrobial resistant strains. Cases of infection show a relatively strong association with seafood consumption or exposure to seawater. This study aimed to analyze Shewanella spp. isolated from the sea urchin Paracentrotus lividus collected from the Crozon peninsula (France) with the intention of obtaining insights into the role of this genus as a reservoir of antimicrobial and heavy metal resistance genes. Five among seven Shewanella isolates were resistant to antimicrobials, mainly to broad spectrum beta-lactams. Four isolates displayed multiple resistance to at least three of these antimicrobial classes: broad spectrum beta-lactams, aminoglycosides, macrolide, quinolones and/or tetracycline. Three antimicrobial resistance genes were detected in just one isolate encoding resistance to beta-lactam (blaSHV and blaTEM-1) and macrolide (ermB). In addition, the copper resistance gene cusB, was observed in this isolate which is also a plasmid carrier. Another copper resistance encoding gene, copA, was found among the isolates. These results indicate that the multidrug-resistant (MDR) Shewanella isolates and resistance genes could be potential risks to public health, due to the carrying of these MDR bacteria by sea urchins through human consumption.

Underrated Staphylococcus species and their role in antimicrobial resistance spreading.

The increasing threat of antimicrobial resistance has shed light on the interconnection between humans, animals, the environment, and their roles in the exchange and spreading of resistance genes. In this review, we present evidences that show that Staphylococcus species, usually referred to as harmless or opportunistic pathogens, represent a threat to human and animal health for acting as reservoirs of antimicrobial resistance genes. The capacity of genetic exchange between isolates of different sources and species of the Staphylococcus genus is discussed with emphasis on mobile genetic elements, the contribution of biofilm formation, and evidences obtained either experimentally or through genome analyses. We also discuss the involvement of CRISPR-Cas systems in the limitation of horizontal gene transfer and its suitability as a molecular clock to describe the history of genetic exchange between staphylococci.

Staphylococcus saprophyticus Proteomic Analyses Elucidate Differences in the Protein Repertories among Clinical Strains Related to Virulence and Persistence.

Staphylococcus saprophyticus is a Gram-positive and coagulase negative cocci that composes the skin microbiota and can act as an opportunistic agent causing urinary tract infections, being more frequent in sexually active young women. The ability of a pathogen to cause infection in the host is associated to its ability to adhere to host cells and to survive host immune defenses. In this work, we presented the comparative proteomic profile of three S. saprophyticus strains. It was possible to characterize differences in the proteome content, specially related to expression of virulence factors. We compiled this data and previous data and we detected one strain (9325) possessing higher production and secretion of proteins related to virulence. Our results show that phenotypic, genotypic, and proteomic differences reflect in the ability to survive during interaction with host cells, since the 9325 strain presented a higher survival rate after macrophage interaction. In counterpart, the 7108 strain that possesses lower content of proteins related to virulence presented higher ability to form biofilm suggesting that this strain can be better adapted to persist in the host and in the environment. Our work describes, for the first time, proteomic flexibility among S. saprophyticus strains, reflecting in virulence and persistence.

The influence of pH on Staphylococcus saprophyticus iron metabolism and the production of siderophores.

Staphylococcus saprophyticus is a gram-positive coagulase negative bacteria which shows clinical importance due to its capability of causing urinary tract infections (UTI), as well as its ability to persist in this environment. Little is known about how S. saprophyticus adapts to the pH shift that occurs during infection. Thus, in this study we aim to use a proteomic approach to analyze the metabolic adaptations which occur as a response by S. saprophyticus when exposed to acid (5.5) and alkaline (9.0) pH environments. Proteins related to iron storage are overexpressed in acid pH, whilst iron acquisition proteins are overexpressed in alkaline pH. It likely occurs because iron is soluble at acid pH and insoluble at alkaline pH. To evaluate if S. saprophyticus synthesizes siderophores, CAS assays were performed, and the results confirmed their production. The chemical characterization of siderophores demonstrates that S. saprophyticus produces carboxylates derived from citrate. Of special note is the fact that citrate synthase (CS) is down-regulated during incubation at acid pH, corroborating this result. This data was also confirmed by enzymatic assay. Our results demonstrate that iron metabolism regulation is influenced by different pH levels, and show, for the first time, the production of siderophores by S. saprophyticus. Enzymatic assays suggest that citrate from the tricarboxylic acid cycle (TCA) is used as substrate for siderophore production.

Short communication: Diversity of species and transmission of antimicrobial resistance among Staphylococcus spp. isolated from goat milk.

The increasing production of goat milk and its derivatives is affected by the occurrence of intramammary infections, which are highly associated with the presence of Staphylococcus species, including some with zoonotic potential. Staphylococci in general can exchange mobile genetic elements, a process that may be facilitated by the isolate's capacity of forming biofilms. In this study we identified, to the species level, Staphylococcus isolated from goat milk samples by MALDI-TOF and confirmed the identification by sequencing housekeeping genes (rrs and tuf). Eight species were identified, more than half being either Staphylococcus epidermidis or Staphylococcus lugdunensis. The isolates were shown by pulsed-field gel electrophoresis to be genetically diverse between the studied herds. Resistance to ampicillin and penicillin was widespread, and 2 Staph. epidermidis isolates contained the methicillin-resistance gene mecA. Most of the isolates that were resistant to at least 1 of the 13 antimicrobials tested harbored plasmids, one of which was demonstrated to be conjugative, being transferred from a Staph. epidermidis to a Staphylococcus aureus strain. Biofilm formation was observed in almost every isolate, which may contribute to their capacity of exchanging antimicrobial resistance genes in addition to acting as a physical barrier to the access of drugs. Our results showed that antimicrobial resistance among goat staphylococci may be emerging in a process facilitated by the exchange of mobile genetic elements between the bacteria and the establishment of biofilms, which calls for careful monitoring and more effective control therapies.