Genomic characterisation of the population structure and antibiotic resistance of Salmonella enterica serovar Infantis in Chile, 2009-2022.

Background: Multidrug-resistant (MDR) Salmonella Infantis has disseminated worldwide, mainly linked to the consumption of poultry products. Evidence shows dissemination of this pathogen in Chile; however, studies are primarily limited to phenotypic data or involve few isolates. As human cases of Salmonella Infantis infections have substantially increased in recent years, this study aimed to characterise the genomic epidemiology and antimicrobial-resistance profiles of isolates obtained from different sources, aiming to inform effective surveillance and control measures. Methods: We sequenced 396 Salmonella Infantis genomes and analysed them with all publicly available genomes of this pathogen from Chile (440 genomes in total), representing isolates from environmental, food, animal, and human sources obtained from 2009 to 2022. Based on bioinformatic and phenotypic methods, we assessed the population structure, dissemination among different niches, and antimicrobial resistance (AMR) profiles of Salmonella Infantis in the country. Findings: The genomic and phylogenetic analyses showed that Salmonella Infantis from Chile comprised several clusters of highly related isolates dominated by sequence type 32. The HC20_343 cluster grouped an important proportion of all isolates. This was the only cluster associated with pESI-like megaplasmids, and up to 12 acquired AMR genes/mutations predicted to result in an MDR phenotype. Accordingly, antimicrobial-susceptibility testing revealed a strong concordance between the AMR genetic determinants and their matching phenotypic expression, indicating that a significant proportion of HC20_343 isolates produce extended-spectrum ß-lactamases and have intermediate fluoroquinolone resistance. HC20_343 Salmonella Infantis were spread among environmental, animal, food, and human niches, showing a close relationship between isolates from different years and sources, and a low intra-source genomic diversity. Interpretation: Our findings show a widespread dissemination of MDR Salmonella Infantis from the HC20_343 cluster in Chile. The high proportion of isolates with resistance to first-line antibiotics and the evidence of active transmission between the environment, animals, food, and humans highlight the urgency of improved surveillance and control measures in the country. As HC20_343 isolates predominate in the Americas, our results suggest a high prevalence of ESBL-producing Salmonella Infantis with intermediate fluoroquinolone resistance in the continent. Funding: Partially supported by the Food and Drug Administration (FDA) of the U.S. Department of Health and Human Services as part of an award, FDU001818, with 30% percent funded by FDA/HHS; and by Agencia de Investigación y Desarrollo de Chile (ANID) through FONDECYT de Postdoctorado Folio 3230796 and Folio 3210317, FONDECYT Regular Folio 1231082, and ANID-Millennium Science Initiative Program-ICN2021_044.

A whole-cell hypersensitive biosensor for beta-lactams based on the AmpR-AmpC regulatory circuit from the Antarctic Pseudomonas sp. IB20.

Detecting antibiotic residues is vital to minimize their impact. Yet, existing methods are complex and costly. Biosensors offer an alternative. While many biosensors detect various antibiotics, specific ones for beta-lactams are lacking. To address this gap, a biosensor based on the AmpC beta-lactamase regulation system (ampR-ampC) from Pseudomonas sp. IB20, an Antarctic isolate, was developed in this study. The AmpR-AmpC system is well-conserved in the genus Pseudomonas and has been extensively studied for its involvement in peptidoglycan recycling and beta-lactam resistance. To create the biosensor, the ampC coding sequence was replaced with the mCherry fluorescent protein as a reporter, resulting in a transcriptional fusion. This construct was then inserted into Escherichia coli SN0301, a beta-lactam hypersensitive strain, generating a whole-cell biosensor. The biosensor demonstrated dose-dependent detection of penicillins, cephalosporins and carbapenems. However, the most interesting aspect of this work is the high sensitivity presented by the biosensor in the detection of carbapenems, as it was able to detect 8 pg/mL of meropenem and 40 pg/mL of imipenem and reach levels of 1-10 ng/mL for penicillins and cephalosporins. This makes the biosensor a powerful tool for the detection of beta-lactam antibiotics, specifically carbapenems, in different matrices.

Ongoing diversification of the global fish pathogen Piscirickettsia salmonis through genetic isolation and transposition bursts.

The management of bacterial pathogens remains a key challenge of aquaculture. The marine gammaproteobacterium Piscirickettsia salmonis is the etiological agent of piscirickettsiosis and causes multi-systemic infections in different salmon species, resulting in considerable mortality and substantial commercial losses. Here, we elucidate its global diversity, evolution, and selection during human interventions. Our comprehensive analysis of 73 closed, high quality genome sequences covered strains from major outbreaks and was supplemented by an analysis of all P. salmonis 16S rRNA gene sequences and metagenomic reads available in public databases. Genome comparison showed that Piscirickettsia comprises at least three distinct, genetically isolated species of which two showed evidence for continuing speciation. However, at least twice the number of species exist in marine fish or seawater. A hallmark of Piscirickettsia diversification is the unprecedented amount and diversity of transposases which are particularly active in subgroups undergoing rapid speciation and are key to the acquisition of novel genes and to pseudogenization. Several group-specific genes are involved in surface antigen synthesis and may explain the differences in virulence between strains. However, the frequent failure of antibiotic treatment of piscirickettsiosis outbreaks cannot be explained by horizontal acquisition of resistance genes which so far occurred only very rarely. Besides revealing a dynamic diversification of an important pathogen, our study also provides the data for improving its surveillance, predicting the emergence of novel lineages, and adapting aquaculture management, and thereby contributes towards the sustainability of salmon farming.

Heavy Metal Pollution From a Major Earthquake and Tsunami in Chile Is Associated With Geographic Divergence of Clinical Isolates of Methicillin-Resistant Staphylococcus aureus in Latin America.

Methicillin-resistant Staphylococcus aureus (MRSA) is a priority pathogen listed by the World Health Organization. The global spread of MRSA is characterized by successive waves of epidemic clones that predominate in specific geographical regions. The acquisition of genes encoding resistance to heavy-metals is thought to be a key feature in the divergence and geographical spread of MRSA. Increasing evidence suggests that extreme natural events, such as earthquakes and tsunamis, could release heavy-metals into the environment. However, the impact of environmental exposition to heavy-metals on the divergence and spread of MRSA clones has been insufficiently explored. We assess the association between a major earthquake and tsunami in an industrialized port in southern Chile and MRSA clone divergence in Latin America. We performed a phylogenomic reconstruction of 113 MRSA clinical isolates from seven Latin American healthcare centers, including 25 isolates collected in a geographic area affected by an earthquake and tsunami that led to high levels of heavy-metal environmental contamination. We found a divergence event strongly associated with the presence of a plasmid harboring heavy-metal resistance genes in the isolates obtained in the area where the earthquake and tsunami occurred. Moreover, clinical isolates carrying this plasmid showed increased tolerance to mercury, arsenic, and cadmium. We also observed a physiological burden in the plasmid-carrying isolates in absence of heavy-metals. Our results are the first evidence that suggests that heavy-metal contamination, in the aftermath of an environmental disaster, appears to be a key evolutionary event for the spread and dissemination of MRSA in Latin America.

Mobile genetic elements drive the multidrug resistance and spread of Salmonella serotypes along a poultry meat production line.

The presence of mobile genetic elements in Salmonella isolated from a chicken farm constitutes a potential risk for the appearance of emerging bacteria present in the food industry. These elements contribute to increased pathogenicity and antimicrobial resistance through genes that are related to the formation of biofilms and resistance genes contained in plasmids, integrons, and transposons. One hundred and thirty-three Salmonella isolates from different stages of the production line, such as feed manufacturing, hatchery, broiler farm, poultry farm, and slaughterhouse, were identified, serotyped and sequenced. The most predominant serotype was Salmonella Infantis. Phylogenetic analyses demonstrated that the diversity and spread of strains in the pipeline are serotype-independent, and that isolates belonging to the same serotype are very closely related genetically. On the other hand, Salmonella Infantis isolates carried the pESI IncFIB plasmid harboring a wide variety of resistance genes, all linked to mobile genetic elements, and among carriers of these plasmids, the antibiograms showed differences in resistance profiles and this linked to a variety in plasmid structure, similarly observed in the diversity of Salmonella Heidelberg isolates carrying the IncI1-Iα plasmid. Mobile genetic elements encoding resistance and virulence genes also contributed to the differences in gene content. Antibiotic resistance genotypes were matched closely by the resistance phenotypes, with high frequency of tetracycline, aminoglycosides, and cephalosporins resistance. In conclusion, the contamination in the poultry industry is described throughout the entire production line, with mobile genetic elements leading to multi-drug resistant bacteria, thus promoting survival when challenged with various antimicrobial compounds.

Detección y cuantificación de SARS-CoV-2 en plantas de tratamiento de aguas residuales de diferentes ciudades de Chile: hacia la implementación de una vigilancia centinela permanente

Introducción: La cuantificación de SARS-CoV-2 en aguas residuales es una herramienta que permite determinar la tendencia de la circulación viral en un área geográfica determinada. Objetivo: Cuantificar el virus SARS-CoV-2 en 15 plantas de tratamiento de aguas residuales en diferentes ciudades de Chile para establecer una comparación con las variables de: i) casos activos por cada 100.000 habs.; ii) positividad diaria (casos nuevos); y iii) fases del plan de confinamiento. Metodología: SARS-CoV-2 se concentró a partir de muestras de aguas residuales. Para obtener el número de genomas del virus por litro se realizó una cuantificación absoluta utilizando qRT-PCR. Resultados: Entre enero y junio de 2021 se procesaron 253 muestras, siendo todas positivas para la presencia del virus. Asimismo, se logró determinar que la tasa de casos activos por cada 100.000 habs. es la variable que mejor se ajusta a las tendencias obtenidas con la cuantificación de la carga viral en las aguas residuales. Conclusiones: La cuantificación de SARS-CoV-2 en las aguas residuales de manera permanente es una herramienta eficiente para determinar la tendencia del virus en un área geográfica determinada y, en conjunto con una vigilancia genómica, puede constituirse en una vigilancia centinela ideal generando alertas sobre futuros brotes.

Background: The quantification of SARS-CoV-2 in wastewater is a tool that allows determining the trend of viral circulation in a particular geographical area. Aim: To quantify the SARS-CoV-2 virus in 15 wastewater treatment plants in different Chilean cities to establish a comparison with the variables of: i) Active cases per 100,000 inhabitants; ii) daily positivity (novel cases); and iii) phases of the lockdown strategy. Methods: SARS-CoV-2 was concentrated from wastewater samples. To obtain the number of virus genomes per liter, absolute quantification was performed using qRT-PCR. Results: Between January and June 2021, 253 samples were processed, all of which were positive for the presence of the virus. Likewise, it will be determined that the rate of active cases per 100,000 inhabitants is the variable that best fits the trends obtained with the quantification of the viral load in wastewater. Conclusions: The quantification of SARS-CoV-2 in wastewater as a continuous strategy is an efficient tool to determine the trend of the viral circulation in a delimited geographical area and, combined with genomic surveillance, it can constitute an ideal sentinel surveillance alert on future outbreaks.

Role of the multi-drug efflux systems on the baseline susceptibility to ceftazidime/avibactam and ceftolozane/tazobactam in clinical isolates of non-carbapenemase-producing carbapenem-resistant Pseudomonas aeruginosa.

Carbapenem-resistant Pseudomonas aeruginosa (CRPA) is one of the pathogens that urgently needs new drugs and new alternatives for its control. The primary strategy to combat this bacterium is combining treatments of beta-lactam with a beta-lactamase inhibitor. The most used combinations against P. aeruginosa are ceftazidime/avibactam (CZA) and ceftolozane/tazobactam (C/T). Although mechanisms leading to CZA and C/T resistance have already been described, among which are the resistance-nodulation-division (RND) efflux pumps, the role that these extrusion systems may play in CZA, and C/T baseline susceptibility of clinical isolates remains unknown. For this purpose, 161 isolates of non-carbapenemase-producing (Non-CP) CRPA were selected, and susceptibility tests to CZA and C/T were performed in the presence and absence of the RND efflux pumps inhibitor, Phenylalanine-arginine ß-naphthylamide (PAßN). In the absence of PAßN, C/T showed markedly higher activity against Non-CP-CRPA isolates than observed for CZA. These results were even more evident in isolates classified as extremely-drug resistant (XDR) or with difficult-to-treat resistance (DTR), where CZA decreased its activity up to 55.2% and 20.0%, respectively, whereas C/T did it up to 82.8% (XDR), and 73.3% (DTR). The presence of PAßN showed an increase in both CZA (37.6%) and C/T (44.6%) activity, and 25.5% of Non-CP-CRPA isolates increased their susceptibility to these two combined antibiotics. However, statistical analysis showed that only the C/T susceptibility of Non-CP-CRPA isolates was significantly increased. Although the contribution of RND activity to CZA and C/T baseline susceptibility was generally low (two-fold decrease of minimal inhibitory concentrations [MIC]), a more evident contribution was observed in a non-minor proportion of the Non-CP-CRPA isolates affected by PAßN [CZA: 25.4% (15/59); C/T: 30% (21/70)]. These isolates presented significantly higher MIC values for C/T. Therefore, we conclude that RND efflux pumps are participating in the phenomenon of baseline susceptibility to CZA and, even more, to C/T. However, the genomic diversity of clinical isolates is so great that deeper analyzes are necessary to determine which elements are directly involved in this phenomenon.

Isolation of an Extensively Drug-Resistant Pseudomonas aeruginosa exoS+/O4 Strain Belonging to the "High-Risk" Clone ST654 and Coproducer of NDM-1 and the Novel VIM-80.

The aim of this study was to investigate the genomic features of an extensively drug-resistant (XDR) Pseudomonas aeruginosa isolate (P-469) emerging in Chile. Antibiotic susceptibility was determined by disk diffusion and "colistin agar" test. Whole-genome sequencing (WGS) was performed by the Illumina NextSeq 2000 platform, and epidemiologically and clinically relevant data (i.e., sequence-type, serotype, mobile genetic elements, virulome, resistome, plasmidome, prophages, and CRISPR-Cas systems) were retrieved using multiple bioinformatic tools. The P-469 strain displayed an XDR profile, remaining susceptible to colistin. Genomic analysis revealed that this isolate belonged to the "high-risk" clone ST654 (CC654), serotype O4, and genotype exoS+. Strikingly, two CRISPR-Cas systems, five intact prophages sequences, and a broad resistome that included blaNDM-1 and the novel blaVIM-80 carbapenemase genes were predicted. Our results revealed the genomic characteristics of P. aeruginosa belonging to the high-risk clone ST654/O4 coproducing NDM-1 and VIM-80 in Chile, supporting that genomic surveillance is necessary to track the emergence and spread of epidemiologically successful WHO's critical priority pathogens in order to prevent their rapid dissemination.

Acquisition of resistance to ceftazidime-avibactam during infection treatment in Pseudomonas aeruginosa through D179Y mutation in one of two blaKPC-2 gene copies without losing carbapenem resistance.

Ceftazidime/Avibactam (CAZ/AVI) is frequently used to treat KPC-producing Pseudomonas aeruginosa (KPC-PA) and Enterobacterales. CAZ/AVI resistance is driven by several mechanisms. In P. aeruginosa this mainly occurs through alteration of AmpC, porins, and/or efflux pump overexpression, whereas in Enterobacterales it frequently occurs through D179Y substitution in the active site of KPC enzyme. This aminoacid change abolishes AVI binding to the KPC active site, hence inhibition is impaired. However, this substitution also decreases KPC-mediated resistance to carbapenems ("see-saw" effect). The goal of this work was to characterize the in vivo acquisition of CAZ/AVI resistance through D179Y substitution in a KPC-PA isolated from a hospitalized patient after CAZ/AVI treatment. Two KPC-PA isolates were obtained. The first isolate, PA-1, was obtained before CAZ/AVI treatment and was susceptible to CAZ/AVI. The second isolate, PA-2, was obtained after CAZ/AVI treatment and exhibited high-level CAZ/AVI resistance. Characterization of isolates PA-1 and PA-2 was performed through short and long-read whole genome sequencing analysis. The hybrid assembly showed that PA-1 and PA-2A had a single plasmid of 54,030 bp, named pPA-1 and pPA-2 respectively. Each plasmid harbored two copies of the bla KPC-containing Tn4401b transposon. However, while pPA-1 carried two copies of bla KPC-2, pPA-2 had one copy of bla KPC-2 and one copy of bla KPC-33, the allele with the D179Y substitution. Interestingly, isolate PA-2 did not exhibit the "see-saw" effect. The bla KPC-33 allele was detected only through hybrid assembly using a long-read-first approach. The present work describes a KPC-PA isolate harboring a plasmid-borne CAZ/AVI resistance mechanism based on two copies of bla KPC-2-Tn4401b and D179Y mutation in one of them, that is not associated with loss of resistance to carbapenems. These findings highlight the usefulness of a fine-tuned combined analysis of short and long-read data to detect similar emerging resistance mechanisms.

Humans and Hoofed Livestock Are the Main Sources of Fecal Contamination of Rivers Used for Crop Irrigation: A Microbial Source Tracking Approach.

Freshwater bodies receive waste, feces, and fecal microorganisms from agricultural, urban, and natural activities. In this study, the probable sources of fecal contamination were determined. Also, antibiotic resistant bacteria (ARB) were detected in the two main rivers of central Chile. Surface water samples were collected from 12 sampling sites in the Maipo (n = 8) and Maule Rivers (n = 4) every 3 months, from August 2017 until April 2019. To determine the fecal contamination level, fecal coliforms were quantified using the most probable number (MPN) method and the source of fecal contamination was determined by Microbial Source Tracking (MST) using the Cryptosporidium and Giardia genotyping method. Separately, to determine if antimicrobial resistance bacteria (AMB) were present in the rivers, Escherichia coli and environmental bacteria were isolated, and the antibiotic susceptibility profile was determined. Fecal coliform levels in the Maule and Maipo Rivers ranged between 1 and 130 MPN/100-ml, and 2 and 30,000 MPN/100-ml, respectively. Based on the MST results using Cryptosporidium and Giardia host-specific species, human, cattle, birds, and/or dogs hosts were the probable sources of fecal contamination in both rivers, with human and cattle host-specific species being more frequently detected. Conditional tree analysis indicated that coliform levels were significantly associated with the river system (Maipo versus Maule), land use, and season. Fecal coliform levels were significantly (p < 0.006) higher at urban and agricultural sites than at sites immediately downstream of treatment centers, livestock areas, or natural areas. Three out of eight (37.5%) E. coli isolates presented a multidrug-resistance (MDR) phenotype. Similarly, 6.6% (117/1768) and 5.1% (44/863) of environmental isolates, in Maipo and Maule River showed and MDR phenotype. Efforts to reduce fecal discharge into these rivers should thus focus on agriculture and urban land uses as these areas were contributing the most and more frequently to fecal contamination into the rivers, while human and cattle fecal discharges were identified as the most likely source of this fecal contamination by the MST approach. This information can be used to design better mitigation strategies, thereby reducing the burden of waterborne diseases and AMR in Central Chile.

Qualitative Risk Assessment for Antimicrobial Resistance among Humans from Salmon Fillet Consumption Due to the High Use of Antibiotics against Bacterial Infections in Farmed Salmon.

Background: Worldwide, aquaculture is considered as a hotspot environment for antimicrobial resistance (AMR) due to the intense use of antibiotics in its productive systems. Chile is the second largest producer of farmed salmon worldwide, and tons of antibiotics are used to control bacterial diseases, such as Salmon Rickettsial Syndrome (SRS) and Bacterial Kidney Disease (BKD). However, studies determining the risk of consuming salmon fillets that have been treated with antibiotics during the salmon production are limited. Consulting leading experts in the field could provide a knowledge base to identify and address this question and research gaps. Methods: Multisectoral risk perception of AMR through salmon fillet consumption was evaluated by eliciting expert data obtained through discussions during a workshop and from questionnaires given to experts from academia (n = 15, 63%), the public sector (n = 5, 21%), and the salmon industry (n = 4, 17%). Results: The qualitative risk analysis suggested an overall 'low' probability of AMR acquisition by consumption of salmon fillet that had been treated during the production cycle. The risk perception varied slightly between production stages in freshwater and seawater. In consensus with all sectors, this overall 'low', but existing, risk was probably associated with bacterial infections and the use of antibiotics. Conclusions: As it is essential to reduce the use of antibiotics in the Chilean salmon industry, this intersectoral approach and consensual results could favor effective implementation of targeted initiatives for the control and prevention of major bacterial diseases.

Genetic characterization of clinically relevant class 1 integrons carried by multidrug resistant bacteria (MDRB) isolated from the gut microbiota of highly antibiotic treated Salmo salar.

OBJECTIVES: The main objective of this study was the genetic characterization of clinically relevant class 1 integrons carried by multidrug resistant bacteria isolated from the intestinal microbiota of aquaculture salmon treated with high concentrations of antibiotics. METHODS: In 82 multidrug resistant bacterial isolates, the prevalence of both the conserved elements of the integrons, qacEΔ1 and sul1 genes, and the variable region (VR) was determined. Further, whole genome sequencing and complete genetic analysis was performed in VR-positive isolates. RESULTS: Despite the fact that 100% of the bacterial isolates presented the intI1 gene, only 12.3% carried the qacEΔ1 and sul1 genes and only two (2.4%) presented a VR with gene cassettes. In the Pseudomonas baetica 25P2F9 isolate, a VR carrying aac(6')31, qacH, and blaOXA-2 gene cassettes was described, whereas the VR of Aeromonas salmonicida 30PB8 isolate showed a dfrA14 gene cassette. The array of gene cassettes found in the Pseudomonas isolate appears with high frequency in clinically relevant pathogens such as Pseudomonas aeruginosa or Escherichia coli. Additionally, it was possible to determine that these integrons are contained in plasmids and coul be easily transferred. Resistome analysis demonstrated that both isolates carried a great diversity of antibiotic resistance genes, including many ß-lactamases. Even in the Aeromonas isolate a new oxacillin-hydrolyzing beta-lactamase gene was described (blaOXA-956). CONCLUSION: The presence of multidrug resistant bacteria and clinically relevant genetic elements in the salmon intestinal microbiota make the aquaculture a hotspot in the phenomenon of antibiotic resistance; therefore, the control of antibiotics used in this activity is a key point to avoid its escalation.

Real-World Performance of Susceptibility Testing for Ceftolozane/Tazobactam against Non-Carbapenemase-Producing Carbapenem-Resistant Pseudomonas aeruginosa.

Ceftolozane/tazbactam (C/T) is a potent anti-pseudomonal agent that has clinical utility against infections caused by non-carbapenemase, producing-carbapenem-resistant Pseudomonas aeruginosa (non-CP-CR-PA). Accurate, precise, and reliable antimicrobial susceptibility testing (AST) is crucial to guide clinical decisions. However, studies assessing the performance of different AST methods against non-CP-CR-PA (the main clinical niche for C/T), are lacking. Here, we evaluated performance of gradient strips (Etest and MIC test strip [MTS], and disk diffusion [DD]) using CLSI breakpoints. Additionally, we assessed the performance of DD using EUCAST breakpoints. For all susceptibility tests, we used a collection of 97 non-CP-CR-PA clinical isolates recovered from 11 Chilean hospitals. Both gradient strips and DD had acceptable performance when using CLSI breakpoints, yielding a categorical agreement (CA) of >90% and 92%, respectively. In contrast, DD using EUCAST breakpoints performed suboptimally (CA 81%). MTS yielded a higher essential agreement (EA, >90%) than Etest (84%). Importantly, the performance of all methods varied significantly when the isolates were stratified by their degree of susceptibility to other anti-pseudomonal ß-lactams. All methods had 100% CA when testing isolates that were pan-susceptible to all ß-lactams (Pan-ß-S). However, the CA markedly decreased when testing isolates resistant to all ß-lactams (Pan-ß-R). Indeed, the CA was 81% for Etest (six errors), 78% for MTS (seven errors), and 78% and 56% for DD when using CLSI (seven errors) or EUCAST breakpoints (14 errors), respectively. Our results suggest that all manual AST methods have strikingly decreased performance in the context of Pan-ß-R P. aeruginosa with potentially major clinical implications.

Isolation of Ciprofloxacin and Ceftazidime-Resistant Enterobacterales From Vegetables and River Water Is Strongly Associated With the Season and the Sample Type.

The dissemination of antibiotic-resistant bacteria (ARB) from water used for crop irrigation to vegetables is poorly studied. During a year, five farmer markets in a city in Central Chile were visited, and 478 vegetable samples (parsleys, corianders, celeries, lettuces, chards, and beets) were collected. Simultaneously, 32 water samples were collected from two rivers which are used to irrigate the vegetables produced in the area. Resistant Enterobacterales were isolated and identified. Colistin resistance gene mcr-1 and extended spectrum ß-lactamases (ESBL) were molecularly detected. The association of environmental factors was evaluated, with the outcomes being the presence of Enterobacterales resistant to four antibiotic families and the presence of multidrug resistance (MDR) phenotypes. Parsley, coriander, and celery showed the highest prevalence of resistant Enterobacterales (41.9% for ciprofloxacin and 18.5% for ceftazidime). A total of 155 isolates were obtained, including Escherichia coli (n=109), Citrobacter sp. (n=20), Enterobacter cloacae complex (n=8), Klebsiella pneumoniae (n=8), and Klebsiella aerogenes (n=1). Resistance to ampicillin (63.2%) and ciprofloxacin (74.2%) was most frequently found; 34.5% of the isolates showed resistance to third-generation cephalosporins, and the MDR phenotype represented 51.6% of the isolates. In two E. coli isolates (1.29%), the gene mcr-1 was found and ESBL genes were found in 23/62 isolates (37%), with bla CTX-M being the most frequently found in 20 isolates (32%). Resistant Enterobacterales isolated during the rainy season were less likely to be MDR as compared to the dry season. Understanding environmental associations represent the first step toward an improved understanding of the public health impact of ARB in vegetables and water.

The impaired quorum sensing response of Pseudomonas aeruginosa MexAB-OprM efflux pump overexpressing mutants is not due to non-physiological efflux of 3-oxo-C12-HSL.

Multidrug (MDR) efflux pumps are ancient and conserved molecular machineries with relevant roles in different aspects of the bacterial physiology, besides antibiotic resistance. In the case of the environmental opportunistic pathogen Pseudomonas aeruginosa, it has been shown that overexpression of different efflux pumps is linked to the impairment of the quorum sensing (QS) response. Nevertheless, the causes of such impairment are different for each analysed efflux pump. Herein, we performed an in-depth analysis of the QS-mediated response of a P. aeruginosa antibiotic resistant mutant that overexpresses MexAB-OprM. Although previous work claimed that this efflux pump extrudes the QS signal 3-oxo-C12-HSL, we show otherwise. Our results evidence that the observed attenuation in the QS response when overexpressing this pump is related to an impaired production of alkyl quinolone QS signals, likely prompted by the reduced availability of one of their precursors, the octanoate. Together with previous studies, this indicates that, although the consequences of overexpressing efflux pumps are similar (impaired QS response), the underlying mechanisms are different. This 'apparent redundancy' of MDR efflux systems can be understood as a P. aeruginosa strategy to keep the robustness of the QS regulatory network and modulate its output in response to different signals.

Pseudomonas fildesensis sp. nov., a psychrotolerant bacterium isolated from Antarctic soil of King George Island, South Shetland Islands.

The strain KG01T was isolated from a soil sample from King George Island, Antarctica. Cells of KG01T are rod-shaped and motile by means of multiple polar flagella. The absence of arginine dihydrolase activity could be a key feature to readily distinguish KG01T from its closest phylogenetic relative species. The main fatty acids of the strain include summed feature 3 (C16 : 1 ω7c and/or C15 : 0 iso 2-OH), C16 : 0 and C18 : 1 ω7c. Phylogenetic analysis based on the 16S rRNA gene sequence and on a multilocus sequence analysis (MLSA) using housekeeping genes (16S rRNA, rpoB, rpoD, gyrB) were carried out. These analyses allowed us to include the strain within the Pseudomonas fluorescens group, presenting the highest similarity of multilocus sequence with Pseudomonas veronii LMG 17761T (96.67 %). The genome of KG01T was sequenced and in silico compared with genomes of the most closely related species of the P. fluorescens group. The average nucleotide identity (ANIb) and average amino acid identity (AAI) values of the species phylogenetically closest to KG01T were less than 95-96 %, threshold currently accepted to define strain as belonging to a bacterial species, the highest scores being those to Pseudomonas veronii LMG 17761T (87.98 %) and Pseudomonas marginalis ICMP 3553T (91.90 %). Therefore, the phenotypic and genotypic analyses results, allow us to propose that KG01T represents a member of a novel species of the genus Pseudomonas, for which the name Pseudomonas fildesensis is proposed, and KG01T (=CECT 9084T;=DSM 102036T) is established as the type strain .

Analysis of the Pseudomonas aeruginosa Aminoglycoside Differential Resistomes Allows Defining Genes Simultaneously Involved in Intrinsic Antibiotic Resistance and Virulence.

High-throughput screening of transposon insertion libraries is a useful strategy for unveiling bacterial genes whose inactivation results in an altered susceptibility to antibiotics. A potential drawback of these studies is they are usually based on just one model antibiotic for each structural family, under the assumption that the results can be extrapolated to all members of said family. To determine if this simplification is appropriate, we have analyzed the susceptibility of mutants of Pseudomonas aeruginosa to four aminoglycosides. Our results indicate that each mutation produces different effects on susceptibility to the tested aminoglycosides, with only two mutants showing similar changes in the susceptibility to all studied aminoglycosides. This indicates that the role of a particular gene in the resistome of a given antibiotic should not be generalized to other members of the same structural family. Five aminoglycoside-hypersusceptible mutants inactivating glnD, hflK, PA2798, PA3016, and hpf were chosen for further analysis in order to elucidate if lower aminoglycoside susceptibility correlates with cross-hypersusceptibility to other antibiotics and with impaired virulence. Our results indicate that glnD inactivation leads to increased cross-susceptibility to different antibiotics. The mutant in this gene is strongly impaired in virulence traits such as pyocyanin production, biofilm formation, elastase activity, and swarming motility and the ability to kill Caenorhabditis elegans Thus, GlnD might be an interesting target for developing antibiotic coadjuvants with antiresistance and antivirulence properties against P. aeruginosa.