Recurrence, Microevolution, and Spatiotemporal Dynamics of Legionella pneumophila Sequence Type 1905, Portugal, 2014-2022.

We investigated molecular evolution and spatiotemporal dynamics of atypical Legionella pneumophila serogroup 1 sequence type 1905 and determined its long-term persistence and linkage to human disease in dispersed locations, far beyond the large 2014 outbreak epicenter in Portugal. Our finding highlights the need for public health interventions to prevent further disease spread.

Alkyl deoxyglycoside-polymyxin combinations against critical priority carbapenem-resistant gram-negative bacteria.

The escalating antimicrobial resistance crisis urges the development of new antibacterial treatments with innovative mechanisms of action, particularly against the critical priority carbapenem-resistant Acinetobacter baumannii (CRAB), Pseudomonas aeruginosa (CRPA) and Enterobacteriaceae (CRE). Membrane-disrupting dodecyl deoxyglycosides have been reported for their interesting phosphatidylethanolamine-associated bactericidal activity against Gram-positive strains; however, their inability to penetrate the Gram-negative outer membrane (OM) renders them useless against the most challenging pathogens. Aiming to repurpose alkyl deoxyglycosides against Gram-negative bacteria, this study investigates the antimicrobial effects of five reference compounds with different deoxygenation patterns or anomeric configurations in combination with polymyxins as adjuvants for enhanced OM permeability. The generation of the lead 4,6-dideoxy scaffold was optimized through a simultaneous dideoxygenation step and applied to the synthesis of a novel alkyl 4,6-dideoxy C-glycoside 5, herein reported for the first time. When combined with subtherapeutic colistin concentrations, most glycosides demonstrated potent antimicrobial activity against several multidrug-resistant clinical isolates of CRAB, CRE and CRPA exhibiting distinct carbapenem resistance mechanisms, together with acceptable cytotoxicity against human HEK-293T and Caco-2 cells. The novel 4,6-dideoxy C-glycoside 5 emerged as the most promising prototype structure for further development (MIC 3.1 µg/mL when combined with colistin 0.5 µg/mL against CRPA or 0.25 µg/mL against several CRE and CRAB strains), highlighting the potential of C-glycosylation for an improved bioactive profile. This study is the first to show the potential of IM-targeting carbohydrate-based compounds for the treatment of infections caused by MDR Gram-negative pathogens of clinical importance.

Genomic and epidemiological insight of an outbreak of carbapenemase-producing Enterobacterales in a Portuguese hospital with the emergence of the new KPC-124.

BACKGROUND: Carbapenemase-producing Enterobacterales (CPE) is an increasing problem in healthcare settings. This study aimed to identify the source of a CPE outbreak that occurred in 2022, in a tertiary hospital in the North of Portugal, to identify exposed patients, and to assess the risk of becoming CPE-positive following hospital admission. METHODS: A multi-disciplinary investigation was conducted including descriptive, analytical, and molecular epidemiology, environmental screening, and assessment of infection control measures. Clinical and environmental isolates were analyzed using whole-genome sequencing and phylogenetic analysis. Additionally, a prospective observational cohort study was conducted to further investigate the risk factors associated with the emergence of new cases in cohorts of CPE-negative admitted patients. RESULTS: We observed the presence of multispecies KPC-, IMP-, and/or NDM-producing isolates. Genetically indistinguishable clinical and environmental isolates were found on the same room/ward. The ST45 KPC-3-producing Klebsiella pneumoniae clone was the responsible for the outbreak. During patients' treatment, we detected the emergence of resistance to ceftazidime-avibactam, associated with mutations in the blaKPC-3 gene (blaKPC-46, blaKPC-66 and blaKPC-124, the last variant never previously reported), suggesting a vertical evolutionary trajectory. Patients aged ≥ 75 years, hygiene/feeding-care dependent, and/or subjected to secretion aspiration were risk factors for CPE colonization after hospital admission. Additionally, cases with previous admission to the emergency department suggest that CPE dissemination may occur not only during hospitalization but also in the emergency department. CONCLUSION: Overall, the study highlights that selection pressure with antibiotics, like ceftazidime-avibactam, is a contributing factor to the emergence of new ß-lactamase variants and antibiotic resistance. It also shows that the hospital environment can be a significant source of CPE transmission, and that routine use of infection control measures and real-time molecular epidemiology investigations are essential to ensure the long-term termination of CPE outbreaks and prevent future resurgences.

Antimicrobial activity of prophage endolysins against critical Enterobacteriaceae antibiotic-resistant bacteria.

Enterobacteriaceae species are part of the 2017 World Health Organization antibiotic-resistant priority pathogens list for development of novel medicines. Multidrug-resistant Klebsiella pneumoniae is an increasing threat to public health and has become a relevant human pathogen involved in life-threatening infections. Phage therapy involves the use of phages or their lytic endolysins as bioagents for the treatment of bacterial infectious diseases. Gram-negative bacteria have an outer membrane, making difficult the access of endolysins to the peptidoglycan. Here, three endolysins from prophages infecting three distinct Enterobacterales species, Kp2948-Lys from K. pneumoniae, Ps3418-Lys from Providencia stuartii, and Kaer26608-Lys from Klebsiella aerogenes, were purified and exhibited antibacterial activity against their specific bacterium species verified by zymogram assays. These three endolysins were successfully associated to liposomes composed of dimyristoyl phosphatidyl choline (DMPC), dioleoyl phosphatidyl ethanolamine (DOPE) and cholesteryl hemisuccinate (CHEMS) at a molar ratio (4:4:2), with an encapsulation efficiency ranging from 24 to 27%. Endolysins encapsulated in liposomes resulted in higher antibacterial activity compared to the respective endolysin in the free form, suggesting that the liposome-mediated delivery system enhances fusion with outer membrane and delivery of endolysins to the target peptidoglycan. Obtained results suggest that Kp2948-Lys appears to be specific for K. pneumoniae, while Ps3418-Lys and Kaer26608-Lys appear to have a broader antibacterial spectrum. Endolysins incorporated in liposomes constitute a promising weapon, applicable in the several dimensions (human, animals and environment) of the One Health approach, against multidrug-resistant Enterobacteriaceae.

Intersectoral collaboration in a One Health approach: Lessons learned from a country-level simulation exercise.

Intersectoral collaboration is an essential component of the One Health (OH) approach, which recognises the interconnectedness of the health of humans, animals, and the environment. The OH European Joint Programme (OHEJP) developed a national foodborne outbreak table-top simulation exercise (SimEx) to practice OH capacity and interoperability across the public health, animal health, and food safety sectors, improving OH preparedness for future disease outbreaks. The Portuguese OHEJP SimEx highlighted strengths and weaknesses regarding the roles and functions of available systems, the constraints of existing legislation, the importance of harmonisation and data sharing, and the creation of common main messages adapted to each target sector. However, there is still a long way to go to ensure cooperation among the Public Health, Animal Health, and Food Safety sectors, as a OH approach relies not only on the awareness of "field experts" but also on political and organisational willingness and commitment.

Prevention, protocols, and lab capacity: lessons from a norovirus outbreak in the Algarve.

This brief report presents the findings of an epidemiological investigation into a large-scale outbreak of norovirus gastroenteritis that occurred in a hotel in Algarve, Portugal, in August 2022. A total of 244 cases were reported, primarily affecting Portuguese families, with the parents aged 40-50 years and the children aged 0-19 years. Reported symptoms included vomiting, nausea, abdominal pain, and diarrhoea. Norovirus genotype GI.3 [P3] was detected in stool samples from eight probable cases, while food samples tested negative for norovirus and common pathogenic bacteria. The investigation data collected suggest that the source of the outbreak was likely in the hotel's common areas, with subsequent person-to-person transmission in other areas. The final report emphasizes the importance of improving outbreak prevention and control measures, including the development of a foodborne outbreak investigation protocol, the establishment of an outbreak response team, and the enhancement of regional laboratory capacity.

Snapshot of resistome, virulome and mobilome in aquaculture.

Aquaculture environments can be hotspots for resistance genes through the surrounding environment. Our objective was to study the resistome, virulome and mobilome of Gram-negative bacteria isolated in seabream and bivalve molluscs, using a WGS approach. Sixty-six Gram-negative strains (Aeromonadaceae, Enterobacteriaceae, Hafniaceae, Morganellaceae, Pseudomonadaceae, Shewanellaceae, Vibrionaceae, and Yersiniaceae families) were selected for genomic characterization. The species and MLST were determined, and antibiotic/disinfectants/heavy metals resistance genes, virulence determinants, MGE, and pathogenicity to humans were investigated. Our study revealed new sequence-types (e.g. Aeromonas spp. ST879, ST880, ST881, ST882, ST883, ST887, ST888; Shewanella spp. ST40, ST57, ST58, ST60, ST61, ST62; Vibrio spp. ST206, ST205). >140 different genes were identified in the resistome of seabream and bivalve molluscs, encompassing genes associated with ß-lactams, tetracyclines, aminoglycosides, quinolones, sulfonamides, trimethoprim, phenicols, macrolides and fosfomycin resistance. Disinfectant resistance genes qacE-type, sitABCD-type and formA-type were found. Heavy metals resistance genes mdt, acr and sil stood out as the most frequent. Most resistance genes were associated with antibiotics/disinfectants/heavy metals commonly used in aquaculture settings. We also identified 25 different genes related with increased virulence, namely associated with adherence, colonization, toxins production, red blood cell lysis, iron metabolism, escape from the immune system of the host. Furthermore, 74.2 % of the strains analysed were considered pathogenic to humans. We investigated the genetic environment of several antibiotic resistance genes, including blaTEM-1B, blaFOX-18, aph(3″)-Ib, dfrA-type, aadA1, catA1-type, tet(A)/(E), qnrB19 and sul1/2. Our analysis also focused on identifying MGE in proximity to these genes (e.g. IntI1, plasmids and TnAs), which could potentially facilitate the spread of resistance among bacteria across different environments. This study provides a comprehensive examination of the diversity of resistance genes that can be transferred to both humans and the environment, with the recognition that aquaculture and the broader environment play crucial roles as intermediaries within this complex transmission network.

Bioguided Identification of Active Antimicrobial Compounds from Asphodelus bento-rainhae and Asphodelus macrocarpus Root Tubers.

Root tubers of Asphodelus bento-rainhae subsp. bento-rainhae (AbR), a vulnerable endemic species, and Asphodelus macrocarpus subsp. macrocarpus (AmR) have traditionally been used in Portugal to treat inflammatory and infectious skin disorders. The present study aims to evaluate the in vitro antimicrobial activity of crude 70% and 96% hydroethanolic extracts of both medicinal plants, specifically against multidrug-resistant skin-related pathogens, to identify the involved marker secondary metabolites and also to assess the pre-clinical toxicity of these medicinal plant extracts. Bioguided fractionation of the 70% hydroethanolic extracts of both species using solvents of increasing polarity, namely diethyl ether (DEE: AbR-1, AmR-1), ethyl acetate (AbR-2, AmR-2) and aqueous (AbR-3, AmR-3) fractions, enabled the identification of the DEE fractions as the most active against all the tested Gram-positive microorganisms (MIC: 16 to 1000 µg/mL). Furthermore, phytochemical analyses using TLC and LC-UV/DAD-ESI/MS techniques revealed the presence of anthracene derivatives as the main constituents of DEE fractions, and five known compounds, namely 7'-(chrysophanol-4-yl)-chrysophanol-10'-C-beta-D-xylopyranosyl-anthrone (p), 10,7'-bichrysophanol (q), chrysophanol (r), 10-(chrysophanol-7'-yl)-10-hydroxychrysophanol-9-anthrone (s) and asphodelin (t), were identified as the main marker compounds. All these compounds showed high antimicrobial activity, particularly against Staphylococcus epidermidis (MIC: 3.2 to 100 µg/mL). Importantly, no cytotoxicity against HepG2 and HaCaT cells (up to 125 µg/mL) for crude extracts of both species and genotoxicity (up to 5000 µg/mL, with and without metabolic activation) for AbR 96% hydroethanolic extract was detected using the MTT and Ames tests, respectively. Overall, the obtained results contribute to the concrete validation of the use of these medicinal plants as potential sources of antimicrobial agents in the treatment of skin diseases.

Rapid cross-border emergence of NDM-5-producing Escherichia coli in the European Union/European Economic Area, 2012 to June 2022.

Whole genome sequencing data of 874 Escherichia coli isolates carrying bla NDM-5 from 13 European Union/European Economic Area countries between 2012 and June 2022 showed the predominance of sequence types ST167, ST405, ST410, ST361 and ST648, and an increasing frequency of detection. Nearly a third (30.6%) of these isolates were associated with infections and more than half (58.2%) were predicted to be multidrug-resistant. Further spread of E. coli carrying bla NDM-5 would leave limited treatment options for serious E. coli infections.

Identification of Marker Compounds and In Vitro Toxicity Evaluation of Two Portuguese Asphodelus Leaf Extracts.

The leaves of Asphodelus bento-rainhae subsp. bento-rainhae, an endemic Portuguese species, and Asphodelus macrocarpus subsp. macrocarpus have been used as food, and traditionally as medicine, for treating ulcers, urinary tract, and inflammatory disorders. The present study aims to establish the phytochemical profile of the main secondary metabolites, together with the antimicrobial, antioxidant and toxicity assessments of both Asphodelus leaf 70% ethanol extracts. Phytochemical screenings were conducted by the TLC and LC-UV/DAD-ESI/MS chromatographic technique, and quantification of the leading chemical classes was performed by spectrophotometric methods. Liquid-liquid partitions of crude extracts were obtained using ethyl ether, ethyl acetate, and water. For in vitro evaluations of antimicrobial activity, the broth microdilution method, and for the antioxidant activity, the FRAP and DPPH methods were used. Genotoxicity and cytotoxicity were assessed by Ames and MTT tests, respectively. Twelve known compounds including neochlorogenic acid, chlorogenic acid, caffeic acid, isoorientin, p-coumaric acid, isovitexin, ferulic acid, luteolin, aloe-emodin, diosmetin, chrysophanol, and ß-sitosterol were identified as the main marker compounds, and terpenoids and condensed tannins were found to be the major class of secondary metabolites of both medicinal plants. The ethyl ether fractions demonstrated the highest antibacterial activity against all the Gram-positive microorganisms, (MIC value of 62 to 1000 µg/mL), with aloe-emodin as one of the main marker compounds highly active against Staphylococcus epidermidis (MIC value of 0.8 to 1.6 µg/mL). Ethyl acetate fractions exhibited the highest antioxidant activity (IC50 of 800 to 1200 µg/mL, respectively). No cytotoxicity (up to 1000 µg/mL) or genotoxicity/mutagenicity (up to 5 mg/plate, with/without metabolic activation) were detected. The obtained results contribute to the knowledge of the value and safety of the studied species as herbal medicines.

First comparative genomic characterization of the MSSA ST398 lineage detected in aquaculture and other reservoirs.

Staphylococcus aureus ST398 can cause diseases in several different animals. In this study we analyzed ten S. aureus ST398 previously collected in three different reservoirs in Portugal (humans, gilthead seabream from aquaculture and dolphin from a zoo). Strains tested against sixteen antibiotics, by disk diffusion or minimum inhibitory concentration, showed decreased susceptibility to benzylpenicillin (all strains from gilthead seabream and dolphin) and to erythromycin with an iMLSB phenotype (nine strains), and susceptibility to cefoxitin (methicillin-susceptible S. aureus, MSSA). All strains from aquaculture belonged to the same spa type, t2383, whereas strains from the dolphin and humans belonged to spa type t571. A more detailed analysis using single nucleotide polymorphisms (SNPs)-based tree and a heat map, showed that all strains from aquaculture origin were highly related with each other and the strains from dolphin and humans were more distinct, although they were very similar in ARG, VF and MGE content. Mutations F3I and A100V in glpT gene and D278E and E291D in murA gene were identified in nine fosfomycin susceptible strains. The blaZ gene was also detected in six of the seven animal strains. The study of the genetic environment of erm(T)-type (found in nine S. aureus strains) allowed the identification of MGE (rep13-type plasmids and IS431R-type), presumably involved in the mobilization of this gene. All strains showed genes encoding efflux pumps from major facilitator superfamily (e.g., arlR, lmrS-type and norA/B-type), ATP-binding cassettes (ABC; mgrA) and multidrug and toxic compound extrusion (MATE; mepA/R-type) families, all associated to decreased susceptibility to antibiotics/disinfectants. Moreover, genes related with tolerance to heavy metals (cadD), and several VF (e.g., scn, aur, hlgA/B/C and hlb) were also identified. Insertion sequences, prophages, and plasmids made up the mobilome, some of them associated with ARG, VF and genes related with tolerance to heavy metals. This study highlights that S. aureus ST398 can be a reservoir of several ARG, heavy metals resistance genes and VF, which are essential in the adaption and survival of the bacterium in the different environments and an active agent in its dissemination. It makes an important contribution to understanding the extent of the spread of antimicrobial resistance, as well as the virulome, mobilome and resistome of this dangerous lineage.

Portable Differential Detection of CTX-M ESBL Gene Variants, blaCTX-M-1 and blaCTX-M-15, from Escherichia coli Isolates and Animal Fecal Samples Using Loop-Primer Endonuclease Cleavage Loop-Mediated Isothermal Amplification.

Cefotaximase-Munich (CTX-M) extended-spectrum beta-lactamase (ESBL) enzymes produced by Enterobacteriaceae confer resistance to clinically relevant third-generation cephalosporins. CTX-M group 1 variants, CTX-M-1 and CTX-M-15, are the leading ESBL-producing Enterobacteriaceae associated with animal and human infection, respectively, and are an increasing antimicrobial resistance (AMR) global health concern. The blaCTX-M-1 and blaCTX-M-15 genes encoding these variants have an approximate nucleotide sequence similarity of 98.7%, making effective differential diagnostic monitoring difficult. Loop-primer endonuclease cleavage loop-mediated isothermal amplification (LEC-LAMP) enables rapid real-time multiplex pathogen detection with single-base specificity and portable on-site testing. We have developed an internally controlled multiplex CTX-M-1/15 LEC-LAMP assay for the differential detection of blaCTX-M-1 and blaCTX-M-15. Assay analytical specificity was established using a panel of human, animal, and environmental Escherichia coli isolates positive for blaCTX-M-1 (n = 18), blaCTX-M-15 (n = 35), and other closely related blaCTX-Ms (n = 38) from Ireland, Germany, and Portugal, with analytical sensitivity determined using probit regression analysis. Animal fecal sample testing using the CTX-M-1/15 LEC-LAMP assay in combination with a rapid DNA extraction protocol was carried out on porcine fecal samples previously confirmed to be PCR-positive for E. coli blaCTX-M. Portable instrumentation was used to further analyze each fecal sample and demonstrate the on-site testing capabilities of the LEC-LAMP assay with the rapid DNA extraction protocol. The CTX-M-1/15 LEC-LAMP assay demonstrated complete analytical specificity for the differential detection of both variants with sensitive low-level detection of 8.5 and 9.8 copies per reaction for blaCTX-M-1 and blaCTX-M-15, respectively, and E. coli blaCTX-M-1 was identified in all blaCTX-M positive porcine fecal samples tested. IMPORTANCE CTX-M ESBL-producing E. coli is an increasing AMR public health issue with the transmission between animals and humans via zoonotic pathogens now a major area of interest. Accurate and timely identification of ESBL-expressing E. coli CTX-M variants is essential for disease monitoring, targeted antibiotic treatment and infection control. This study details the first report of portable diagnostics technology for the rapid differential detection of CTX-M AMR markers blaCTX-M-1 and blaCTX-M-15, facilitating improved identification and surveillance of these closely related variants. Further application of this portable internally controlled multiplex CTX-M-1/15 LEC-LAMP assay will provide new information on the transmission and prevalence of these CTX-M ESBL alleles. Furthermore, this transferable diagnostic technology can be applied to other new and emerging relevant AMR markers of interest providing more efficient and specific portable pathogen detection for improved epidemiological surveillance.

Genomic Analysis of a mcr-9.1-Harbouring IncHI2-ST1 Plasmid from Enterobacter ludwigii Isolated in Fish Farming.

This study analyzed the resistome, virulome and mobilome of an MCR-9-producing Enterobacter sp. identified in a muscle sample of seabream (Sparus aurata), collected in a land tank from multitrophic fish farming production. Average Nucleotide Identity analysis identified INSAq77 at the species level as an Enterobacter ludwigii INSAq77 strain that was resistant to chloramphenicol, florfenicol and fosfomycin and was susceptible to all other antibiotics tested. In silico antimicrobial resistance analyses revealed genes conferring in silico resistance to ß-lactams (blaACT-88), chloramphenicol (catA4-type), fosfomycin (fosA2-type) and colistin (mcr-9.1), as well as several efflux pumps (e.g., oqxAB-type and mar operon). Further bioinformatics analysis revealed five plasmid replicon types, including the IncHI2/HI2A, which are linked to the worldwide dissemination of the mcr-9 gene in different antibiotic resistance reservoirs. The conserved nickel/copper operon rcnR-rcnA-pcoE-ISSgsp1-pcoS-IS903-mcr-9-wbuC was present, which may play a key role in copper tolerance under anaerobic growth and nickel homeostasis. These results highlight that antibiotic resistance in aquaculture are spreading through food, the environment and humans, which places this research in a One Health context. In fact, colistin is used as a last resort for the treatment of serious infections in clinical settings, thus mcr genes may represent a serious threat to human health.

Ongoing monkeypox virus outbreak, Portugal, 29 April to 23 May 2022.

Up to 27 May 2022, Portugal has detected 96 confirmed cases of monkeypox. We describe 27 confirmed cases (median age: 33 years (range: 22-51); all males), with an earliest symptom onset date of 29 April. Almost all cases (n = 25) live in the Lisbon and Tagus Valley health region. Most cases were neither part of identified transmission chains, nor linked to travel or had contact with symptomatic persons or with animals, suggesting the possible previously undetected spread of monkeypox.

Antimicrobial Resistance and Molecular Epidemiology of Staphylococcus aureus from Hunters and Hunting Dogs.

Several studies have showed that a dog-to-human transmission of Staphylococcus aureus occurs. Hunting dogs do not have as much contact with their owners as dogs that live in the same household as the owners; however, these dogs have contact with their owners during hunting activities as well as when hunting game; therefore, we aimed to isolate S. aureus from hunters and their hunting dogs to investigate a possible S. aureus transmission. Nose and mouth samples were collected from 30 hunters and their 78 hunting dogs for staphylococcal isolation. The species identification was performed using MALDI-TOF. The antimicrobial susceptibility profiles were accessed using the Kirby-Bauer method and respective antimicrobial resistance genes were investigated by PCR. Multilocus sequence typing (MLST) and spa- and agr-typing was performed in all S. aureus isolates. S. aureus were detected in 10 (30%) human samples and in 11 (15.4%) dog samples of which 11 and 5 were methicillin-resistant S. aureus (MRSA). Other staphylococci were identified, particularly, S. pseudintermedius. Most S. aureus isolates were resistant to penicillin, erythromycin, and tetracycline. Evidence of a possible transmission of S. aureus between human and dogs was detected in three hunters and their dogs. S. aureus isolates were ascribed to 10 STs and 9 spa-types. A moderate colonization of S. aureus in hunting dogs and their owners was detected in this study. A few dog-to-dog and dog-to-human possible transmissions were identified.

Staphylococcus aureus and Methicillin-Resistant Coagulase-Negative Staphylococci in Nostrils and Buccal Mucosa of Healthy Camels Used for Recreational Purposes.

Several different species of animals host staphylococci as normal microbiota. These animals can be a source of staphylococci zoonotic infections. People with routine or occupational exposure to infected/colonized animals are at risk of a potential transmission. Therefore, we aimed to investigate the presence of S. aureus and other staphylococci in camels used for recreational purposes as well as their antimicrobial resistance, virulence factors and genetic lineages. A total of 172 samples were collected from 86 healthy camels (nose and mouth) from different farms located in the Canary Islands, Spain. Antimicrobial susceptibility testing was performed against 14 antimicrobial agents. The presence of virulence genes was studied by PCR. Multilocus sequence typing, spa typing and agr typing were performed in all S. aureus isolates. From the 86 camels tested, 42 staphylococci were isolated, of which there were 11 S. aureus, 13 S. lentus, 12 S. sciuri, 3 S. xylosus, S. epidermidis, S. hominis and S. chromogenes. Staphylococci isolates were resistant to penicillin, ciprofloxacin, clindamycin and fusidic acid. All S. aureus isolates harbored the hla, hlb and hld virulence genes. S. aureus isolates were ascribed to three sequence types (STs) and three spa types. All S. aureus isolates belonged to agr type III. Camels from Gran Canaria used in recreational purposes have a moderate prevalence of S. aureus and other coagulase-negative staphylococci. Nevertheless, S. aureus isolates are susceptible to almost all antibiotics tested.

A One Health Approach Molecular Analysis of Staphylococcus aureus Reveals Distinct Lineages in Isolates from Miranda Donkeys (Equus asinus) and Their Handlers.

Donkeys (Equus asinus) are in decline in Europe. Occupational exposure to farm animals has been associated with increased staphylococci carriage. We aimed to isolate S. aureus and coagulase-negative staphylococci (CoNS) from donkeys and handlers and characterize the antimicrobial resistance profiles and genetic lineages of S. aureus strains. Oral and nasal swab samples were collected from 49 Miranda donkeys and 23 handlers from 15 different farms. Staphylococci species were identified by MALDI-TOF MS. The presence of antimicrobial resistance genes and virulence factors was investigated by PCR. Molecular typing was performed in S. aureus isolates. From the 49 donkey samples, 4 S. aureus (8.2%) and 21 CoNS (42.9%) were isolated. Ten handlers (43.5%) were carriers of S. aureus and 4 (17.4%) carried CoNS. The CoNS isolates showed resistance to several classes of antimicrobials encoded by the mecA, aph (3')-IIIa, ant (4')-Ia, tetM, tetK, lnuA, ermB, ermC, dfrA and dfrG genes. S. aureus isolates were resistant to penicillin, aminoglicosides and tetracycline harboring the blaZ, aph (3')-IIIa, tetL, tetM and tetK genes. All S. aureus isolates from donkeys belonged to ST49 and spa-type t208 while the strains isolated from the handlers were ascribed to 3 STs and 7 spa-types. However, human isolates were from different STs than the donkey isolates. Donkeys are mainly colonized by methicillin-resistant S. sciuri. S. aureus transmission between donkeys and their handlers appears not to have occurred since the isolates belonged to different genetic lineages.

Nocturnal Birds of Prey as Carriers of Staphylococcus aureus and Other Staphylococci: Diversity, Antimicrobial Resistance and Clonal Lineages.

Owls are nocturnal predators that inhabit urbanized and farmlands. They are in direct contact with other animals, both livestock and small wild rodents that they mostly feed on. Staphylococci can be both commensal and pathogenic bacteria that are widespread across the various ecological niches. We aimed to isolate staphylococci from owls and to characterize their antimicrobial resistance, virulence factors and genetic lineages. Swab samples were collected from the throat and cloaca of 114 owls admitted to two rehabilitation centers in Portugal. The identification of staphylococci species was performed by MALDI-TOF. Staphylococci antimicrobial resistance and virulence genes were investigated by means of the disk diffusion method and PCR. Staphylococcus aureus isolates were characterized by MLST, agr and spa-typing. Of the tested animals, 66 isolates were recovered, including 10 different species of staphylococci, of which 25 were coagulase-positive (CoPS) and 41 were coagulase-negative (CoNS). Twenty-three S. aureus were isolated, of which one mecC-MRSA was identified. The isolates were mainly resistant to penicillin, aminoglycosides, clindamycin and tetracycline. mecC-MRSA belonged to ST1245 and spa-type t843 and the remaining S. aureus were ascribed to 12 STs and 15 spa types. A high diversity of clonal lineages was identified among the S. aureus isolated from wild owls. Owls feed mainly on small rodents often exposed to waste and anthropogenic sources, which may explain the moderate prevalence of S. aureus in these animals.

Outbreak of KPC-producing Klebsiella pneumoniae at a Portuguese university hospital: Epidemiological characterization and containment measures.

Background: KPC-producing K pneumoniae (KPC-Kp) is a public health problem with important clinical and epidemiological implications. We describe an outbreak of KPC-Kp at vascular surgery and neurosurgery wards in a central hospital in Porto, Portugal. Methods: A case of KPC-Kp was considered to be a patient positive for KPC-Kp with strong epidemiological plausibility of having acquired this microorganism in the affected wards and/or with genetic relationship ≥92% between KPC-Kp isolates. Active surveillance cultures (ASCs) and real-time polymerase chain reaction were used for the detection of carbapenemase genes through rectal swab in a selected population. Molecular analysis was performed using pulsed-field gel electrophoresis at the National Reference Laboratory. Patient risk factors were collected from the electronic medical record system. Information regarding outbreak containment strategy was collected from the Infection Control Unit records. Results: Of the 16 cases, 11 (69%) were identified through active screening, representing 1.4% of the total 766 ASCs collected. The most frequent risk factors identified were previous admission (63%), antibiotic exposure in the past 6 months (50%), and immunodepression (44%). The length of stay until KPC-Kp detection was high (0-121 days, mean 35.6), as was the total length of stay (5-173 days, mean 56.6). Three patients (19%) were infected by KPC-Kp, 2 of whom died. One previously colonized patient died later because of KPC-Kp infection. Conclusions: Multifactorial strategy based on contact precautions (with patient and healthcare professional cohorts) and ASC, as well as Antibiotic Stewardship Program reinforcement, allowed to contain this KPC-Kp outbreak.

Distribution and Clonal Diversity of Staphylococcus aureus and Other Staphylococci in Surface Waters: Detection of ST425-t742 and ST130-t843 mecC-Positive MRSA Strains.

Natural aquatic environments represent one of the most important vehicles of bacterial dissemination. Therefore, we aimed to isolate staphylococci from surface waters and to investigate the presence of antimicrobial resistance genes and virulence factors as well as the genetic lineages of all Staphylococcus aureus isolates. Staphylococci were recovered from water samples collected from 78 surface waters, including rivers, streams, irrigation ditches, dams, lakes, and fountains. The presence of antimicrobial resistance genes and virulence factors was investigated by PCR. Multilocus sequence typing and spa-typing were performed in all S. aureus isolates. From the 78 water samples, 33 S. aureus, one S. pseudintermedius, and 51 coagulase-negative staphylococci (CoNS) were identified. Among the S. aureus isolates, four MRSA were identified, and all harbored the mecC gene. Fourteen S. aureus were susceptible to all antimicrobials tested and the remaining showed resistance to penicillin, erythromycin and/or tetracycline encoded by the blaZ, ermT, msr(A/B), tetL, and vgaA genes. Regarding the clonal lineages, one mecC-MRSA isolate belonged to spa-type t843 and sequence type (ST) 130 and the other three to t742 and ST425. The remaining S. aureus were ascribed 14 spa-types and 17 sequence types. Eleven species of CoNS were isolated: S. sciuri, S. lentus, S. xylosus, S. epidermidis, S. cohnii spp. urealyticus, S. vitulinus, S. caprae, S. carnosus spp. Carnosus, S. equorum, S. simulans, and S. succinus. Thirteen CoNS isolates had a multidrug resistance profile and carried the following genes: mecA, msr(A/B), mph(C), aph(3')-IIIa, aac(6')-Ie-aph(2'')-Ia, dfrA, fusB, catpC221, and tetK. A high diversity of staphylococci was isolated from surface waters including mecCMRSA strains and isolates presenting multidrug-resistance profiles. Studies on the prevalence of antibiotic-resistant staphylococci in surface waters are still very scarce but extremely important to estimate the contribution of the aquatic environment in the spread of these bacteria.