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.

Emergence of KPC-113 and KPC-114 variants in ceftazidime-avibactam-resistant Klebsiella pneumoniae belonging to high-risk clones ST11 and ST16 in South America.

Two novel variants of Klebsiella pneumoniae carbapenemase (KPC) associated with resistance to ceftazidime-avibactam (CZA) and designated as KPC-113 and KPC-114 by NCBI were identified in 2020, in clinical isolates of Klebsiella pneumoniae in Brazil. While K. pneumoniae of ST16 harbored the blaKPC-113 variant on an IncFII-IncFIB plasmid, K. pneumoniae of ST11 carried the blaKPC-114 variant on an IncN plasmid. Both isolates displayed resistance to broad-spectrum cephalosporins, ß-lactam inhibitors, and ertapenem and doripenem, whereas K. pneumoniae producing KPC-114 showed susceptibility to imipenem and meropenem. Whole-genome sequencing and in silico analysis revealed that KPC-113 presented a Gly insertion between Ambler positions 264 and 265 (R264_A265insG), whereas KPC-114 displayed two amino acid insertions (Ser-Ser) between Ambler positions 181 and 182 (S181_P182insSS) in KPC-2, responsible for CZA resistance profiles. Our results confirm the emergence of novel KPC variants associated with resistance to CZA in international clones of K. pneumoniae circulating in South America. IMPORTANCE KPC-2 carbapenemases are endemic in Latin America. In this regard, in 2018, ceftazidime-avibactam (CZA) was authorized for clinical use in Brazil due to its significant activity against KPC-2 producers. In recent years, reports of resistance to CZA have increased in this country, limiting its clinical application. In this study, we report the emergence of two novel KPC-2 variants, named KPC-113 and KPC-114, associated with CZA resistance in Klebsiella pneumoniae strains belonging to high-risk clones ST11 and ST16. Our finding suggests that novel mutations in KPC-2 are increasing in South America, which is a critical issue deserving active surveillance.

ACT-107, a novel variant of AmpC ß-lactamase from Enterobacter huaxiensis isolated from Neotropical leaf frog (Phyllomedusa distincta) inhabiting the Brazilian Atlantic Forest.

OBJECTIVES: The aim of this study was to characterise a broad-spectrum cephalosporin-resistant AmpC-positive Enterobacter huaxiensis colonising the skin of a Neotropical frog (Phyllomedusa distincta) inhabiting the Brazilian Atlantic Forest. METHODS: During a genomic surveillance study of antimicrobial resistance, we screened skin samples from P. distincta. Gram-negative bacteria growing on MacConkey agar plates supplemented with 2 µg/mL ceftriaxone were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. A cephalosporin-resistant E. huaxiensis was sequenced using the Illumina NextSeq platform. Genomic data were analysed using bioinformatics tools, whereas AmpC ß-lactamase was characterised in depth by comparative analysis of amino acids, in silico modelling, and analysis of susceptibility to ß-lactam antibiotics and combinations of ß-lactamase inhibitors. RESULTS: Whole-genome sequencing analysis revealed a novel variant of AmpC ß-lactamase belonging to the ACT family, designated ACT-107 by NCBI. This variant contains 12 novel amino acid mutations within the ACT family, 5 in the signal peptide sequence (Ile2, Met14, Tyr16, Gly18 and Thr20), and 7 in the mature protein (Gln22, His43, Cys60, Thr157, Glu225, Ala252 and Asn310). In silico modelling showed that substitutions occurring in the mature chain are localised in the solvent-accessible surface of the protein, where they are not expected to affect the ß-lactamase activity, as observed in the resistance profile. Strikingly, 'not designated' ACT variants from E. huaxiensis were clustered (> 96% identity) with ACT-107. CONCLUSION: Since E. huaxiensis has been isolated from human infection, ACT-107 requires surveillance and the attention of clinicians.

Detecting KPC-2 and NDM-1 Coexpression in Klebsiella pneumoniae Complex from Human and Animal Hosts in South America.

Reports of Gram-negative bacteria harboring multiple carbapenemase genes have increased in South America, leading to an urgent need for appropriate microbiological diagnosis. We evaluated phenotypic methods for detecting Klebsiella pneumoniae carbapenemase 2 (KPC-2) and New Delhi metallo-ß-lactamase-1 (NDM-1) coexpression in members of the K. pneumoniae complex (i.e., K. pneumoniae, K. quasipneumoniae, and K. variicola) isolated from human and animal hosts, based on inhibition of ceftazidime-avibactam (CZA) and aztreonam (ATM) by dipicolinic acid (DPA), EDTA, or avibactam (AVI). While the presence of blaKPC-2 and blaNDM-1 genes was confirmed by whole-genome sequencing, PCR, and/or GeneXpert, coexpression was successfully detected based on the following: (i) a ≥5-mm increase in the zone diameter of ATM (30 µg) disks plus AVI (4 or 20 µg) and ≥4-mm and ≥10-mm increases in the zone diameters for "CZA 50" (30 µg ceftazidime [CAZ] and 20 µg AVI) and "CZA 14" (10 µg CAZ and 4 µg AVI) disks, respectively, when we added DPA (1 mg/disk) or EDTA (5 mM) in a combined disk test (CDT); (ii) a positive ghost zone (synergism) between ATM (30 µg) and CZA 50 disks and between CZA 50 and DPA (1 mg) disks, using the double-disk synergy test (DDST) at a disk-disk distance of 2.5 cm; (iii) ≥3-fold MIC reductions of ATM and CZA in the presence of AVI (4 µg/mL), DPA (500 µg/mL), or EDTA (320 µg/mL); and (iv) immunochromatography. Although our results demonstrated that inhibition by AVI, DPA, and EDTA may provide simple and inexpensive methods for the presumptive detection of coexpression of KPC-2 and NDM-1 in members of the K. pneumoniae complex, additional studies are necessary to confirm the accuracy of these methodologies by testing other Gram-negative bacterial species and other KPC and NDM variants coexpressed by WHO critical priority pathogens detected worldwide. IMPORTANCE Alerts regarding the emergence and increase of combinations of carbapenemases in Enterobacterales in Latin America and the Caribbean have recently been issued by PAHO and WHO, emphasizing the importance of appropriate microbiological diagnosis and the effective and articulated implementation of infection prevention and control programs. In this study, we evaluated methods based on inhibition of ceftazidime (CAZ), ceftazidime-avibactam (CZA), and aztreonam (ATM) by dipicolinic acid (DPA), EDTA, and avibactam (AVI) inhibitors for the identification of KPC-2- and NDM-1-coexpression in members of the K. pneumoniae complex recovered from human and animal hosts. Our results demonstrate that inhibition by AVI, DPA, and EDTA may provide simple and inexpensive methods for the presumptive detection of coexpression of KPC-2 and NDM-1 in members of the K. pneumoniae complex.

Phylogeographical Landscape of Citrobacter portucalensis Carrying Clinically Relevant Resistomes.

During a surveillance study conducted to assess the occurrence and genomic landscape of critical priority pathogens circulating at the human-animal-environment interface in Brazil, as part of the Grand Challenges Explorations-New Approaches to Characterize the Global Burden of Antimicrobial Resistance program, two multidrug-resistant (MDR) Citrobacter portucalensis carrying blaCTX-M-15 extended-spectrum ß-lactamase (ESBL) genes, isolated from green sea turtles, were characterized. Genomic and phylogeographical analysis of C. portucalensis genomes available in public databases revealed the intercontinental dissemination of clades carrying different arrays of clinically relevant genes conferring resistance to carbapenems, broad-spectrum cephalosporins, cephamycins, aminoglycosides and fluoroquinolones, disinfectants, and heavy metals. Our observations suggest that C. portucalensis could be emerging as critical priority bacteria of both public and One Health importance worldwide. IMPORTANCE The global spread of antibiotic-resistant priority pathogens beyond the hospital setting is a critical issue within a One Health context that integrates the human-animal-environment interfaces. On the other hand, next-generation sequencing technologies along with user-friendly and high-quality bioinformatics tools have improved the identification of bacterial species, and bacterial resistance surveillance. The novel Citrobacter portucalensis species was proposed in 2017 after taxonomic reclassification and definition of the strain A60T isolated in 2008. Here, we presented genomic data showing the occurrence of multidrug-resistant C. portucalensis isolates carrying blaCTX-M-15 ESBL genes in South America. Additionally, we observed the intercontinental dissemination of clades harboring a broad resistome to clinically relevant antibiotics. Therefore, these findings highlight that C. portucalensis is a global MDR bacteria that carries intrinsic blaCMY- and qnrB-type genes and has become a critical priority pathogen due to the acquisition of clinically relevant resistance determinants, such as ESBL and carbapenemase-encoding genes.

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.

Phylogenetic MLSA and phenotypic analysis identification of three probable novel Pseudomonas species isolated on King George Island, South Shetland, Antarctica

ABSTRACT Antarctica harbors a great diversity of microorganisms, including bacteria, archaea, microalgae and yeasts. The Pseudomonas genus is one of the most diverse and successful bacterial groups described to date, but only eight species isolated from Antarctica have been characterized. Here, we present three potentially novel species isolated on King George Island. The most abundant isolates from four different environments, were genotypically and phenotypically characterized. Multilocus sequence analysis and 16S rRNA gene analysis of a sequence concatenate for six genes (16S, aroE, glnS, gyrB, ileS and rpoD), determined one of the isolates to be a new Pseudomonas mandelii strain, while the other three are good candidates for new Pseudomonas species. Additionally, genotype analyses showed the three candidates to be part of a new subgroup within the Pseudomonas fluorescens complex, together with the Antarctic species Pseudomonas antarctica and Pseudomonas extremaustralis. We propose terming this new subgroup P. antarctica. Likewise, phenotypic analyses using API 20 NE and BIOLOG® corroborated the genotyping results, confirming that all presented isolates form part of the P. fluorescens complex. Pseudomonas genus research on the Antarctic continent is in its infancy. To understand these microorganisms' role in this extreme environment, the characterization and description of new species is vital.

Extended antibiotic treatment in salmon farms select multiresistant gut bacteria with a high prevalence of antibiotic resistance genes.

The high use of antibiotics for the treatment of bacterial diseases is one of the main problems in the mass production of animal protein. Salmon farming in Chile is a clear example of the above statement, where more than 5,500 tonnes of antibiotics have been used over the last 10 years. This has caused a great impact both at the production level and on the environment; however, there are still few works in relation to it. In order to demonstrate the impact of the high use of antibiotics on fish gut microbiota, we have selected four salmon farms presenting a similar amount of fish of the Atlantic salmon species (Salmo salar), ranging from 4,500 to 6,000 tonnes. All of these farms used treatments with high doses of antibiotics. Thus, 15 healthy fish were selected and euthanised in order to isolate the bacteria resistant to the antibiotics oxytetracycline and florfenicol from the gut microbiota. In total, 47 bacterial isolates resistant to florfenicol and 44 resistant to oxytetracycline were isolated, among which isolates with Minimum Inhibitory Concentrations (MIC) exceeding 2048 µg/mL for florfenicol and 1024 µg/mL for oxytetracycline were found. In addition, another six different antibiotics were tested in order to demonstrate the multiresistance phenomenon. In this regard, six isolates of 91 showed elevated resistance values for the eight tested antibiotics, including florfenicol and oxytetracycline, were found. These bacteria were called "super-resistant" bacteria. This phenotypic resistance was verified at a genotypic level since most isolates showed antibiotic resistance genes (ARGs) to florfenicol and oxytetracycline. Specifically, 77% of antibiotic resistant bacteria showed at least one gene resistant to florfenicol and 89% showed at least one gene resistant to oxytetracycline. In the present study, it was demonstrated that the high use of the antibiotics florfenicol and oxytetracycline has, as a consequence, the selection of multiresistant bacteria in the gut microbiota of farmed fish of the Salmo salar species at the seawater stage. Also, the phenotypic resistance of these bacteria can be correlated with the presence of antibiotic resistance genes.

Phylogenetic MLSA and phenotypic analysis identification of three probable novel Pseudomonas species isolated on King George Island, South Shetland, Antarctica.

Antarctica harbors a great diversity of microorganisms, including bacteria, archaea, microalgae and yeasts. The Pseudomonas genus is one of the most diverse and successful bacterial groups described to date, but only eight species isolated from Antarctica have been characterized. Here, we present three potentially novel species isolated on King George Island. The most abundant isolates from four different environments, were genotypically and phenotypically characterized. Multilocus sequence analysis and 16S rRNA gene analysis of a sequence concatenate for six genes (16S, aroE, glnS, gyrB, ileS and rpoD), determined one of the isolates to be a new Pseudomonas mandelii strain, while the other three are good candidates for new Pseudomonas species. Additionally, genotype analyses showed the three candidates to be part of a new subgroup within the Pseudomonas fluorescens complex, together with the Antarctic species Pseudomonas antarctica and Pseudomonas extremaustralis. We propose terming this new subgroup P. antarctica. Likewise, phenotypic analyses using API 20 NE and BIOLOG® corroborated the genotyping results, confirming that all presented isolates form part of the P. fluorescens complex. Pseudomonas genus research on the Antarctic continent is in its infancy. To understand these microorganisms' role in this extreme environment, the characterization and description of new species is vital.

Alginate overproduction and biofilm formation by psychrotolerant Pseudomonas mandelii depend on temperature in Antarctic marine sediments

Background: In recent years, Antarctica has become a key source of biotechnological resources. Native microorganisms have developed a wide range of survival strategies to adapt to the harsh Antarctic environment, including the formation of biofilms. Alginate is the principal component of the exopolysaccharide matrix in biofilms produced by Pseudomonas, and this component is highly demanded for the production of a wide variety of commercial products. There is a constant search for efficient alginate-producing organisms. Results: In this study, a novel strain of Pseudomonas mandelii isolated from Antarctica was characterized and found to overproduce alginate compared with other good alginate producers such as Pseudomonas aeruginosa and Pseudomonas fluorescens. Alginate production and expression levels of the alginate operon were highest at 4°C. It is probable that this alginate-overproducing phenotype was the result of downregulated MucA, an anti-sigma factor of AlgU. Conclusion: Because biofilm formation is an efficient bacterial strategy to overcome stressful conditions, alginate overproduction might represent the best solution for the successful adaptation of P. mandelii to the extreme temperatures of the Antarctic. Through additional research, it is possible that this novel P. mandelii strain could become an additional source for biotechnological alginate production.