Unveiling a Listeria monocytogenes Outbreak in a Rabbit Farm: Clinical Manifestation, Antimicrobial Resistance, Genomic Insights and Environmental Investigation.

Listeria monocytogenes poses a threat to both human and animal health. This work describes an L. monocytogenes outbreak in a Portuguese rabbit farm, detailing the isolates' clinical manifestations, necropsy findings, and phenotypic and genomic profiles. Clinical signs, exclusively observed in does, included lethargy and reproductive signs. Post-mortem examination of does revealed splenomegaly, hepatomegaly with a reticular pattern, pulmonary congestion, and haemorrhagic lesions in the uterus, with thickening of the uterine wall and purulent greyish exudates. Positive L. monocytogenes samples were identified in fattening and maternity units across different samples, encompassing does and environmental samples. Core-genome Multi Locus Sequence Typing (cgMLST) analysis confirmed the outbreak, with the 16 sequenced isolates (lineage II, CC31, and ST325) clustering within a ≤2 allelic difference (AD) threshold. Antimicrobial susceptibility testing for five antibiotics revealed that 15 out of 19 outbreak isolates were resistant to sulfamethoxazole-trimethoprim (SXT). Concordantly, all SXT-resistant sequenced isolates were found to exclusively harbour a plasmid containing a trimethoprim-resistance gene (dfrD), along with loci linked to resistance to lincosamides (lnuG), macrolides (mphB), and polyether ionophores (NarAB operon). All sequenced outbreak isolates carried the antibiotic resistance-related genes tetM, fosX, lin, norB, lmrB, sul, and mprF. The outbreak cluster comprises isolates from does and the environment, which underscores the ubiquitous presence of L. monocytogenes and emphasizes the importance of biosecurity measures. Despite limited data on listeriosis in rabbit farming, this outbreak reveals its significant impact on animal welfare and production.

Antibacterial Potential of Symmetrical Twin-Drug 3,6-Diaminoxanthones.

Global health faces a significant issue with the rise of infectious diseases caused by bacteria, fungi, viruses, and parasites. The increasing number of multi-drug resistant microbial pathogens severely threatens public health worldwide. Antibiotic-resistant pathogenic bacteria, in particular, present a significant challenge. Therefore, there is an urgent need to identify new potential antimicrobial targets and discover new chemical entities that can potentially reverse bacterial resistance. The main goal of this research work was to create and develop a library of 3,6-disubstituted xanthones based on twin drugs and molecular extension approaches to inhibit the activity of efflux pumps. The process involved synthesizing 3,6-diaminoxanthones through the reaction of 9-oxo-9H-xanthene-3,6-diyl bis(trifluoromethanesulfonate) with various primary and secondary amines. The resulting 3,6-disubstituted xanthone derivatives were then tested for their in vitro antimicrobial properties against a range of pathogenic strains and their efficacy in inhibiting the activity of efflux pumps, biofilm formation, and quorum-sensing. Several compounds have exhibited effective antibacterial properties against the Gram-positive bacterial species tested. Xanthone 16, in particular, has demonstrated exceptional efficacy with a remarkable MIC of 11 µM (4 µg/mL) against reference strains Staphylococcus aureus ATCC 25923 and Enterococcus faecalis ATCC 29212, and 25 µM (9 µg/mL) against methicillin-resistant S. aureus 272123. Furthermore, some derivatives have shown potential as antibiofilm agents in a crystal violet assay. The ethidium bromide accumulation assay pinpointed certain compounds inhibiting bacterial efflux pumps. The cytotoxic effect of the most promising compounds was examined in mouse fibroblast cell line NIH/3T3, and two monoamine substituted xanthone derivatives with a hydroxyl substituent did not exhibit any cytotoxicity. Overall, the nature of the substituent was critical in determining the antimicrobial spectra of aminated xanthones.

An Exploratory Study on Spoilage Bacteria and Listeria monocytogenes in Fresh Salmon: Extending Shelf-Life Using Vacuum and Seasonings as Natural Preservatives.

A growing population increases the demand for food, but short shelf-lives and microbial hazards reduce supply and increase food waste. Fresh fish is highly perishable and may be consumed raw, such as salmon in sushi. This work aims to identify strategies to improve the shelf-life and safety of fresh salmon, using available methods (i.e., vacuum) and exploring the use of natural preservatives (i.e., seasonings). Vacuum packaging and good hygiene practices (which reduce initial flora) extended shelf-life up to 20 days. Carnobacterium maltaromaticum was dominant in vacuum packaging conditions and showed potential for inhibiting Listeria monocytogenes. For natural preservatives, L. monocytogenes required higher inhibitory concentrations in vitro when compared to the 10 spoilage bacteria isolated from fresh salmon fillets, presenting a minimum inhibitory concentration (MIC) of 0.13% for oregano essential oil (OEO), 10% for lemon juice, 50 mg mL-1 for garlic powder, and >10% for NaCl. A good bacteriostatic and bactericidal effect was observed for a mixture containing 5% NaCl, 0.002% OEO, 2.5% lemon juice, and 0.08 mg mL-1 garlic powder. Finally, using the salmon medium showed an adequate correlation with the commercial culture medium.

Effect of Indole-Containing Pyrazino[2,1-b]quinazoline-3,6-diones in the Virulence of Resistant Bacteria.

Drug resistance is rising to alarming levels, constituting one of the major threats to global health. The overexpression of efflux pumps and the formation of biofilms constitute two of the most common resistance mechanisms, favoring the virulence of bacteria. Therefore, the research and development of effective antimicrobial agents that can also counteract resistance mechanisms are extremely important. Pyrazino[2,1-b]quinazoline-3,6-diones, from marine and terrestrial organisms and simpler synthetic analogues, were recently disclosed by us as having relevant antimicrobial properties. In this study, using a multi-step approach, it was possible to synthesize new pyrazino[2,1-b]quinazoline-3,6-diones focusing on compounds with fluorine substituents since, to the best of our knowledge, the synthesis of fluorinated fumiquinazoline derivatives had not been attempted before. The new synthesized derivatives were screened for antibacterial activity and, along with previously synthetized pyrazino[2,1-b]quinazoline-3,6-diones, were characterized for their antibiofilm and efflux-pump-inhibiting effects against representative bacterial species and relevant resistant clinical strains. Several compounds showed relevant antibacterial activity against the tested Gram-positive bacterial species with MIC values in the range of 12.5-77 µM. Furthermore, some derivatives showed promising results as antibiofilm agents in a crystal violet assay. The results of the ethidium bromide accumulation assay suggested that some compounds could potentially inhibit bacterial efflux pumps.

Occurrence of Multidrug-Resistant Bacteria Resulting from the Selective Pressure of Antibiotics: A Comprehensive Analysis of ESBL K. pneumoniae and MRSP Isolated in a Dog with Rhinorrhea.

Because of public health concerns, much greater scrutiny is now placed on antibiotic use in pets, especially for antimicrobial agents that have human analogs. Therefore, this study aimed to characterize the phenotypic and genotypic profiles of multidrug-resistant bacteria isolated from nasal swabs samples taken from a one-year-old male Serra da Estrela dog with rhinorrhea that was treated with amikacin. An extended-spectrum ß-lactamases (ESBL) Klebsiella pneumoniae was isolated in the first sample taken from the left nasal cavity of the dog. Seven days later, methicillin-resistant (MRSP) Staphylococcus pseudintermedius was also isolated. Nevertheless, no alterations to the therapeutic protocol were performed. Once the inhibitory action of the antibiotic disappeared, the competitive advantage of the amikacin-resistant MRSP was lost, and only commensal flora was observed on both nasal cavities. The genotypic profile of extended-spectrum ß-lactamase (ESBL)-producing Klebsiella pneumoniae revealed the same characteristics and close relation to other strains, mainly from Estonia, Slovakia and Romania. Regarding MRSP isolates, although resistance to aminoglycosides was present in the first MRSP, the second isolate carried aac(6')-aph(2″), which enhanced its resistance to amikacin. However, the veterinary action was focused on the treatment of the primary agent (ESBL K. pneumoniae), and the antibiotic applied was according to its phenotypic profile, which may have led to the resolution of the infectious process. Therefore, this study highlights the importance of targeted therapy, proper clinical practice and laboratory-hospital communication to safeguard animal, human and environmental health.

Gulls in Porto Coastline as Reservoirs for Salmonella spp.: Findings from 2008 and 2023.

Gulls act as intermediaries in the exchange of microorganisms between the environment and human settlements, including Salmonella spp. This study assessed the antimicrobial resistance and molecular profiles of Salmonella spp. isolates obtained from fecal samples of gulls in the city of Porto, Portugal, in 2008 and 2023 and from water samples in 2023. Antimicrobial susceptibility profiling revealed an improvement in the prevalence (71% to 17%) and antimicrobial resistance between the two collection dates. Two isolate collections from both 2008 and 2023 underwent serotyping and whole-genome sequencing, revealing genotypic changes, including an increased frequency in the monophasic variant of S. Typhimurium. qacE was identified in 2008 and 2023 in both water and fecal samples, with most isolates exhibiting an MDR profile. The most frequently observed plasmid types were IncF in 2008 (23%), while IncQ1 predominated in 2023 (43%). Findings suggest that Salmonella spp. circulate between humans, animals, and the environment. However, the genetic heterogeneity among the isolates from the gulls' feces and the surface water may indicate a complex ecological and evolutionary dynamic shaped by changing conditions. The observed improvements are likely due to measures to reduce biological contamination and antimicrobial resistance. Nevertheless, additional strategies must be implemented to reduce the public health risk modeled by the dissemination of pathogens by gulls.

New Chalcone-Triazole Hybrids with Promising Antimicrobial Activity in Multidrug Resistance Strains.

Resistance to antibiotics is an emerging problem worldwide, which leads to an increase in morbidity and mortality rates. Several mechanisms are attributed to bacterial resistance, overexpression of efflux pumps being one of the most prominent. As an attempt to develop new effective antimicrobial drugs, which could be able to act against resistant bacterial strains and considering the antimicrobial potential of flavonoids and triazolyl flavonoid derivatives, in particular chalcones, a small library of chalcone derivatives was synthesized and evaluated for its potential to act as antimicrobials and/or adjuvants in combination with antibiotics towards resistant bacteria. Although only compound 7 was able to act as antibacterial, compounds 1, 2, 4, 5, 7, and 9 revealed to be able to potentiate the activity of antibiotics in resistant bacteria. Moreover, five compounds (3, 5-8) demonstrated to be effective inhibitors of efflux pumps in Salmonella enterica serovar Typhimurium SL1344, and four compounds (1, 3, 7, and 10) showed higher ability than reserpine to inhibit biofilm formation of resistant Staphylococcus aureus 272123. Together, our results showed the potential of these compounds regarding reversion of bacterial resistance.

Derivatives of Trimethoxybenzoic Acid and Gallic Acid as Potential Efflux Pump Inhibitors: In Silico and In Vitro Studies.

The overexpression of efflux pumps is one of the strategies used by bacteria to resist antibiotics and could be targeted to circumvent the antibiotic crisis. In this work, a series of trimethoxybenzoic acid derivatives previously described as antifouling compounds was explored for potential antimicrobial activity and efflux pump (EP) inhibition. First, docking studies on the acridine resistance proteins A and B coupled to the outer membrane channel TolC (AcrAB-TolC) efflux system and a homology model of the quinolone resistance protein NorA EP were performed on 11 potential bioactive trimethoxybenzoic acid and gallic acid derivatives. The synthesis of one new trimethoxybenzoic acid derivative (derivative 13) was accomplished. To investigate the potential of this series of 11 derivatives as antimicrobial agents, and in reverting drug resistance, the minimum inhibitory concentration was determined on several strains (bacteria and fungi), and synergy with antibiotics and EP inhibition were investigated. Derivative 10 showed antibacterial activity against the studied strains, derivatives 5 and 6 showed the ability to inhibit EPs in the acrA gene inactivated mutant Salmonella enterica serovar Typhimurium SL1344, and 6 also inhibited EPs in Staphylococcus aureus 272123. Structure-activity relationships highlighted trimethoxybenzoic acid as important for EP inhibitory activity. Although further studies are necessary, these results show the potential of simple trimethoxybenzoic acid derivatives as a source of feasible EP inhibitors.

High diversity of pathogenic Escherichia coli clones carrying mcr-1 among gulls underlines the need for strategies at the environment-livestock-human interface.

The expansion of mcr-carrying bacteria is a well-recognized public health problem. Measures to contain mcr spread have mainly been focused on the food-animal production sector. Nevertheless, the spread of MCR producers at the environmental interface particularly driven by the increasing population of gulls in coastal cities has been less explored. Occurrence of mcr-carrying Escherichia coli in gull's colonies faeces on a Portuguese beach was screened over 7 months. Cultural, molecular and genomic approaches were used to characterize their diversity, mcr plasmids and adaptive features. Multidrug-resistant mcr-1-carrying E. coli were detected for 3 consecutive months. Over time, multiple strains were recovered, including zoonotic-related pathogenic E. coli clones (e.g. B2-ST131-H22, A-ST10 and B1-ST162). Diverse mcr-1 genetic environments were mainly associated with ST2/ST4-HI2 (ST10, ST131, ST162, ST354 and ST4204) but also IncI2 (ST12990) plasmids or in the chromosome (ST656). Whole-genome sequencing revealed enrichment of these strains on antibiotic resistance, virulence and metal tolerance genes. Our results underscore gulls as important spreaders of high-priority bacteria and genes that may affect the environment, food-animals and/or humans, potentially undermining One-Health strategies to reduce colistin resistance.

Enantioselectivity of Chiral Derivatives of Xanthones in Virulence Effects of Resistant Bacteria.

Antimicrobial peptides are one of the lines of defense produced by several hosts in response to bacterial infections. Inspired by them and recent discoveries of xanthones as bacterial efflux pump inhibitors, chiral amides with a xanthone scaffold were planned to be potential antimicrobial adjuvants. The chiral derivatives of xanthones were obtained by peptide coupling reactions between suitable xanthones with enantiomerically pure building blocks, yielding derivatives with high enantiomeric purity. Among 18 compounds investigated for their antimicrobial activity against reference strains of bacteria and fungi, antibacterial activity for the tested strains was not found. Selected compounds were also evaluated for their potential to inhibit bacterial efflux pumps. Compound (R,R)-8 inhibited efflux pumps in the Gram-positive model tested and three compounds, (S,S)-8, (R)-17 and (R,S)-18, displayed the same activity in the Gram-negative strain used. Studies were performed on the inhibition of biofilm formation and quorum-sensing, to which the enantiomeric pair 8 displayed activity for the latter. To gain a better understanding of how the active compounds bind to the efflux pumps, docking studies were performed. Hit compounds were proposed for each activity, and it was shown that enantioselectivity was noticeable and must be considered, as enantiomers displayed differences in activity.

Hepatitis E virus genotype 3 in echinoderms: First report of sea urchin (Paracentrotus lividus) contamination.

Hepatitis E virus (HEV) deriving from manure application runoffs and faecal waste spill over of swine and human origin bypass wastewater treatment plants and contaminate coastal waters. Shellfish bioaccumulate enteric viruses such as HEV from fecally contaminated coastal waters and under current European Regulations, shellfish sanitary status surveillance is mandatory but only by means of bacterial faecal indicators. The sea urchins are under the same regulations and their vulnerability to fecal contamination has been pointed out. Since they are consumed raw and with no steps to control/reduce hazards, sea urchin contamination with enteric viruses can represent a food safety risk. Hence, the aim of the present study was to screen sea urchin gonads destined for human consumption for the presence of HEV. HEV was detected and quantified in gonads of sea urchins collected in north Portugal by a reverse transcription-quantitative PCR (RT-qPCR) assay targeting the ORF3 region, followed by genotyping by a nested RT-PCR targeting the ORF2 region. Sequencing and phylogenetic analysis clustered the HEV sequence within genotype 3, subgenotype e. This the first study reporting HEV contamination of sea urchins. We hypothesize that like shellfish, sea urchins can also be a food vehicle for HEV transmission to humans.

New marine-derived indolymethyl pyrazinoquinazoline alkaloids with promising antimicrobial profiles.

Due to the emergence of multidrug-resistant pathogenic microorganisms, the search for novel antimicrobials is urgent. Inspired by marine alkaloids, a series of indolomethyl pyrazino [1,2-b]quinazoline-3,6-diones was prepared using a one-pot microwave-assisted multicomponent polycondensation of amino acids. The compounds were evaluated for their antimicrobial activity against a panel of nine bacterial strains and five fungal strains. Compounds 26 and 27 were the most effective against Staphylococcus aureus ATCC 29213 reference strain with MIC values of 4 µg mL-1, and a methicillin-resistant Staphylococcus aureus (MRSA) isolate with MIC values of 8 µg mL-1. It was possible to infer that enantiomer (-)-26 was responsible for the antibacterial activity (MIC 4 µg mL-1) while (+)-26 had no activity. Furthermore, compound (-)-26 was able to impair S. aureus biofilm production and no significant cytotoxicity towards differentiated and non-differentiated SH-SY5Y cells was observed. Compounds 26, 28, and 29 showed a weak antifungal activity against Trichophyton rubrum clinical isolate with MIC 128 µg mL-1 and presented a synergistic effect with fluconazole.

River water analysis using a multiparametric approach: Portuguese river as a case study.

The Ave River in northern Portugal has a history of riverbanks and water quality degradation. The river water quality was assessed by physicochemical, biological (macroinvertebrates) and microbiological (Enterococcus spp. and Escherichia coli) parameters in six locations (A-F, point A being the nearest to the source) throughout its course during a year. Epilithic biofilms were studied through polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE). Antimicrobial susceptibility testing helped with selecting isolates (n = 149 E. coli and n = 86 enterococci) for further genetic characterization. Pursuant to physicochemical and macroinvertebrates-based parameters, the river water was of reasonable quality according to European legislation (Directive 2000/60/EC). However, the microbiological analysis showed increased fecal contamination downstream from point C. At point D, four carbapenem-resistant E. coli isolates were recovered. Paradoxically, point D was classified as a point of 'Good Water Quality' according to macroinvertebrates results. Point F presented the highest contamination level and incidence of multidrug-resistant (MDR) isolates in the water column (13 MDR enterococci out of 39 and 33 MDR E. coli out of 97). Epilithic biofilms showed higher diversity in pristine points (A and B). Thus, biological and microbiological parameters used to assess the water quality led to divergent results; an outcome that reinforces the need for a holistic evaluation.

Expert opinion on livestock antimicrobial usage indications and patterns in Denmark, Portugal and Switzerland.

We aimed at describing antimicrobial usage patterns throughout livestock production cycles, and comparing them across three countries from Northern, Central and Southern Europe. Given the difficulties to collect such detailed usage data, an expert opinion was deemed the most appropriate study design. This study provides new insights into the time periods and indications for which specific antimicrobial substances are used in different livestock sectors. Veterinary experts (n=67) from different livestock sectors (broilers, pigs, dairy cattle and veal/fattening calves) and countries (Denmark, Portugal and Switzerland) replied to a questionnaire focusing on the time periods in the production cycle when antimicrobial substances were administered, and the respective indications for treatment. Our results showed that for several antimicrobials, between-country and within-country variations exist regarding the temporal distributions of treatments and indications for use. These differences were also true for several critically important antimicrobials, which is a matter of concern. Furthermore, differences between countries were also evident regarding the antimicrobial substances licensed. Based on our results, it is recommended to establish and promote treatment guidelines, invest in the prevention of diseases during critical moments of the production cycle and target undifferentiated use of antimicrobials. Moreover, discrepancies between countries should be further investigated to better understand the factors underlying the identified patterns and to distinguish prudent from non-prudent use. The results can inform decision-making with the aim to foster antimicrobial prudent use in the veterinary setting and, therefore, protect public health from the threat of antimicrobial resistance.

Chemical Composition, Antibacterial, Antibiofilm and Synergistic Properties of Essential Oils from Eucalyptus globulus Labill. and Seven Mediterranean Aromatic Plants.

Essential oils (EOs) from Eucalyptus globulus Labill. ssp. globulus and from Mediterranean autochthonous aromatic plants - Thymus mastichina L., Mentha pulegium L., Rosmarinus officinalis L., Calamintha nepeta (L.) Savi ssp. nepeta, Cistus ladanifer L., Foeniculum vulgare L., Dittrichia viscosa (L.) Greuter ssp. viscosa - were extracted by hydrodistillation and characterized by GC-FID and NMR spectroscopy. EOs were evaluated for antimicrobial properties against several bacterial strains, using diverse methods, namely, the agar disc-diffusion method, the microdilution method, the crystal violet assay and the Live/Dead staining for assessment of biofilm formation. Potential synergy was assessed by a checkerboard method. EOs of R. officinalis and C. ladanifer showed a predominance in monoterpene hydrocarbons (> 60%); EOs of C. nepeta, M. pulegium, T. mastichina, E. globulus and F. vulgare were rich in oxygenated monoterpenes (62 - 96%) whereas EO of D. viscosa was mainly composed of oxygenated sesquiterpenes (54%). All EOs showed antimicrobial activity; M. pulegium and E. globulus generally had the strongest antimicrobial activity. EO of C. nepeta was the most promising in hampering the biofilm formation. The combinations D. viscosa/C. nepeta and E. globulus/T. mastichina were synergistic against Staphylococcus aureus. These results support the notion that EOs from the aromatic plants herein reported should be further explored as potential pharmaceuticals and/or food preservatives.

Neofiscalin A and fiscalin C are potential novel indole alkaloid alternatives for the treatment of multidrug-resistant Gram-positive bacterial infections.

Ten indole alkaloids were obtained from the marine sponge-associated fungus Neosartorya siamensis KUFA 0017. We studied the antimicrobial properties of these and of three other compounds previously isolated from the soil fungus N. siamensis KUFC 6349. Only neofiscalin A showed antimicrobial activity against Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE); with a minimum inhibitory concentration (MIC) of 8 µg mL(-1) against both strains. Another compound, fiscalin C, presented synergistic activity against MRSA when combined with oxacillin, although alone showed no antibacterial effect. Moreover, neofiscalin A, when present at sub-MICs, hampered the ability of both MRSA and VRE strains to form a biofilm. Additionally, the biofilm inhibitory concentration values of neofiscalin A against the MRSA and VRE isolates were 96 and 80 µg mL(-1), respectively. At a concentration of 200 µg mL(-1), neofiscalin A was able to reduce the metabolic activity of the biofilms by ∼50%. One important fact is that our results also showed that neofiscalin A had no cytotoxicity against a human brain capillary endothelial cell line.

Synergistic Effects Between Thioxanthones and Oxacillin Against Methicillin-Resistant Staphylococcus aureus.

The extensive use of antimicrobials is leaving medicine with few effective therapeutic options to treat many infections due to the fact that many organisms developed resistance to commonly used drugs. It is therefore pertinent to search not only for new antimicrobials but also for compounds able to restore or potentiate the activity of existing antibiotics. We have screened a library consisting of 40 (thio)xanthone derivatives for antibacterial activity and possible synergistic effects when used in combination with antibiotics. Nine out of the 40 compounds exhibited antibacterial activity against Gram-positive bacteria. Two xanthone derivatives, 1-formyl-4-hydroxy-3-methoxy (7), 2-formyl-3-hydroxy-4-methoxyxanthone (8) and the thioxanthone derivative 1-((2-(diethylamino)ethyl)amino)-4-propoxythioxanthone (10) and its hydrochloride form 13, showed activity against a methicillin-resistant Staphylococcus aureus (MRSA) isolate with minimum inhibitory concentration (MIC) values lower than 256 µg/ml. Thioxanthone 10 demonstrated antibacterial activity and also synergy when combined with ampicillin and oxacillin against MRSA. Additionally, thioxanthone 1-(piperidin-1-yl)-4-propoxythioxanthone (9), despite not having antibacterial activity presented remarkable synergy with oxacillin against MRSA; the MIC of tioxanthone 9 and oxacillin when both were in combination were 128 and 8 µg/ml, respectively. Thioxanthones 9 and 10 were also found to be synergistic when both were combined. Subsequently, docking simulations between thioxanthones 9 and 10 and the allosteric domain of penicillin-binding protein 2A (PBP2A) were undertaken in AutoDock Vina. Both compounds had the ability to bind with an allosteric domain of PBP2A, which may explain their synergy with oxacillin. These two thioxanthone derivatives with different profiles may be promising tools for restoring the activity of oxacillin against MRSA.

Microbial interaction between a CTXM-15 -producing Escherichia coli and a susceptible Pseudomonas aeruginosa isolated from bronchoalveolar lavage: influence of cefotaxime in the dual-species biofilm formation.

Two isolates, Escherichia coli ella00 and Pseudomonas aeruginosa ella01, obtained from bronchoalveolar lavage, were found to be closely associated in clusters in agar medium. Escherichia coli ella00 was multidrug resistant and CTXM-15 extended-spectrum ß-lactamase producer, while P. aeruginosa ella01 was susceptible to all antimicrobials tested. These observations impelled for further studies aimed to understand their microbial interaction. The P. aeruginosa ella01 biofilm-forming capacity was reduced and not affected when it was co-cultured with E. coli ella00 and E. coli ATCC 25922 respectively. Interestingly, the co-culture of ella isolates in the presence of high concentrations, such as 160 µg ml(-1) , of cefotaxime allowed the formation of more biofilm than in the absence of the antibiotic. As revealed by fluorescence in situ hybridization, in co-culture, P. aeruginosa ella01 survived and subsequently flourished when exposed to this third-generation cephalosporin at a concentration 10 × higher than its minimum inhibitory concentration (MIC), and this was mostly due to ß-lactamases production by E. coli ella00. In fact, it was demonstrated by high-performance liquid chromatography that cefotaxime was absent for the culture medium 4 h after application. In conclusion, we demonstrate that bacterial species can interact differently depending on the surrounding conditions (favourable or stressing), and that those interactions can switch from unprofitable to beneficial.

High prevalence of multidrug-resistant Escherichia coli and Enterococcus spp. in river water, upstream and downstream of a wastewater treatment plant.

In this study, microbial quality and antimicrobial resistance of faecal bacteria from a Portuguese river were assessed. River water samples collected upstream and downstream of a wastewater treatment plant, throughout a 3-month period, were used for the enumeration of Escherichia coli and Enterococcus spp. The highest numbers found for E. coli and enterococci were 1.1 × 104 and 1.2 × 104 colony forming units (CFU)/100 ml, respectively. In total, 144 isolates of E. coli and 144 of enterococci were recovered and tested for antimicrobial susceptibility; 104 E. coli and 78 Enterococcus spp. showed resistance to one or more antimicrobial drugs. Overall, 70 and 32 different resistance patterns were found for E. coli and enterococci, respectively. One E. coli showed resistance to imipenem and 29 isolates were extended spectrum ß-lactamase-producers. Multidrug-resistant E. coli belonged mostly to groups A, B1 and group D. Enterococcus spp. were mostly resistant to rifampicin, tetracycline, azithromycin and erythromycin; six isolates showed resistance to vancomycin, presenting the VanA phenotype. The high levels of E. coli and enterococci and the remarkable variety of antimicrobial resistance profiles, reinforces the theory that these river waters can be a pool of antimicrobial resistance determinants, which can be easily spread among different bacteria and reach other environments and hosts.

Genetic detection of extended-spectrum beta-lactamase-containing Escherichia coli isolates from birds of prey from Serra da Estrela Natural Reserve in Portugal.

Extended-spectrum beta-lactamase-containing Escherichia coli isolates were detected in 32 of 119 fecal samples (26.9%) from birds of prey at Serra da Estrela, and these isolates contained the following beta-lactamases: CTX-M-1 (n = 13), CTX-M-1 plus TEM-1 (n = 14), CTX-M-1 plus TEM-20 (n = 1), SHV-5 (n = 1), SHV-5 plus TEM-1 (n = 2), and TEM-20 (n = 1).