The OprF porin as a potential target for the restoration of carbapenem susceptibility in Pseudomonas aeruginosa expressing acquired carbapenemases.

Carbapenem resistance in Pseudomonas aeruginosa is primarily due to the acquisition of carbapenemases and is often associated with a diminution of the membrane permeability. The outer membrane protein, OprD, is a well-known route, by which carbapenems, predominantly imipenem, can enter the cell, and its loss has been associated with reduced susceptibility to imipenem. In this study, we investigated the antibiotic susceptibility patterns of isogenic P. aeruginosa mutants containing various acquired ß-lactamases, including carbapenemases, in a porin-depleted background. We identified that the deletion of oprF was associated with some recovery of susceptibility to carbapenems.

Reduced susceptibility to aztreonam-avibactam conferred by acquired AmpC-type ß-lactamases in PBP3-modified Escherichia coli.

PURPOSE: Carbapenemase-producing Enterobacterales are a growing threat, and very few therapeutic options remain active against those multidrug resistant bacteria. Aztreonam is the molecule of choice against metallo-beta-lactamases (MBL) producers since it is not hydrolyzed by those enzymes, but the co-production of acquired plasmidic cephalosporinases or extended-spectrum ß-lactamases leading to aztreonam resistance may reduce the efficacy of this molecule. Hence, the development of the aztreonam-avibactam (AZA) combination provides an interesting therapeutic alternative since avibactam inhibits the activity of both cephalosporinases and extended-spectrum ß-lactamases. However, structural modifications of penicillin binding protein PBP3, the target of aztreonam, may lead to reduced susceptibility to aztreonam-avibactam. METHODS: Here the impact of various plasmid-encoded AmpC-type ß-lactamases (ACC-1, ACT-7, ACT-17, CMY-2, CMY-42, DHA-1, FOX-1, and FOX-5) on susceptibility to aztreonam-avibactam was evaluated using isogenic E. coli MG1655 strains harboring insertions in PBP3 (YRIN and YRIK). The inhibitory activity of various ß-lactamase inhibitors (clavulanic acid, tazobactam, avibactam, relebactam, and vaborbactam) were also compared against these enzymes. RESULTS: Hence, we showed that reduced susceptibility to AZA was due to the combined effect of both AmpC production and amino acid insertions in PBP3. The highest resistance level was achieved in strains possessing the insertions in PBP3 in association with the production of ACT-7, ACC-1, or CMY-42. CONCLUSION: Although none of the recombinant strains tested displayed clinical resistance to aztreonam-avibactam, our data emphasize that the occurrence of such profile might be of clinical relevance for MBL-producing strains.

No evidence for a relationship between farm or transformation process locations and antibiotic resistance patterns of Pseudomonas population associated with rainbow trout (Oncorhynchus mykiss).

AIMS: Study the relationship between antibiotic resistance patterns of Pseudomonas isolated from farmed rainbow trout fillets and farm or transformation process locations. METHODS AND RESULTS: Pseudomonas strains were isolated from rainbow trout sampled in two differently located farms and filleted in laboratory or in a processing factory. One hundred and twenty-five isolates were confirmed as belonging to Pseudomonas using CFC selective media, Gram staining, oxidase test and quantitative polymerase chain reaction methods. Fifty-one isolates from separate fish fillets were further identified using MALDI-TOF mass spectrometry, and the minimal inhibitory concentrations (MIC) of 11 antibiotics were also determined by microdilution method. Most of the isolates belonged to the Pseudomonas fluorescens group (94.1%), and no relationship was established between antibiotic resistance patterns and sampling locations (farms or filleting areas). Multiple resistance isolates with high MIC values (from 64 µg ml-1 to more than 1024 µg ml-1 ) were identified. CONCLUSIONS: Antibiotic resistance patterns found in Pseudomonas isolates were not influenced by farms or transformation process locations. Seven isolates were found highly resistant to four different antibiotic classes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study does not provide evidence of a relationship between farm or transformation process locations on antibiotic resistance patterns of Pseudomonas population.

Evaluation of matrix-assisted laser desorption ionization-time of flight mass spectrometry for identification of human oral Capnocytophaga species.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was evaluated for rapid identification of cfxA PCR positive and negative Capnocytophaga strains. Colonies were grown on blood agar, incubated anaerobically at 37 °C for 48 h, and were then evaluated by MALDI-TOF MS and 16S rRNA gene sequencing. Both methods identified all colonies to the genus level. The MALDI-TOF MS method gave the same result, at the species level, as 16S rRNA gene sequencing for 41/53 Capnocytophaga sp. strains (77.4%), but the limit of this technique was the absence of some species (C. leadbetteri, C. AHN) in the Biotyper-Bruker® database used in this study. Distinction between the cefotaxime resistant and susceptible strains was unsuccessful using the MALDI-TOF MS method. This technique had low discriminatory power to rapidly detect beta-lactamase-producing Capnocytophaga strains in clinical samples. However, the results from a score-oriented dendrogram confirmed MALDI-TOF MS is a rapid, inexpensive, and reliable method for Capnocytophaga species identification. Enrichment of the reference database used (Biotyper®) will improve future results.

Antimicrobial susceptibility profile and molecular characterization of Vibrio parahaemolyticus strains isolated from imported shrimps.

Vibrio parahaemolyticus is a threat to human health and one of the leading bacterial causes of seafood-borne infection worldwide. This pathogen is autochtonous in the marine environment and is able to acquire antimicrobial resistance (AMR) mechanisms, which is a global concern. However, the emergence of AMR V. parahaemolyticus strains in seafood is still understudied, as interpretation criteria for this species for antimicrobial susceptibility tests are limited in the literature. In this study, we investigated the susceptibility profiles to clinically important antibiotics and the associated genetic determinants of V. parahaemolyticus isolates cultured from imported shrimps. Based on the analysis of the resistance phenotypes of 304 V. parahaemolyticus isolates, we have defined experimental epidemiological cutoff values (COWT) for 14/15 antibiotics tested. We observed that 19.1% of the bacterial isolates had acquired resistance to at least one antibiotic class. The highest number of resistance was associated with tetracycline (14.5% of the strains) and trimethoprim-sulfamethoxazole (3.6%). Moreover, seven strains were multidrug-resistant (MDR, resistant to at least three antibiotic classes). The most frequently identified genes in these strains were aph(3″)-Ib/aph(6)-Id (aminoglycoside resistance), sul2 (sulfonamide), tet(59) (tetracycline), and floR (chloramphenicol). The SXT/R391 family ICE and class 1 integron-integrase genes were detected by PCR in three and one MDR V. parahaemolyticus strains, respectively. Consequently, V. parahaemolyticus in seafood can act as a reservoir of AMR, constituting a health risk for the consumer.IMPORTANCEOur study on "Antimicrobial Resistance Profiles and Genetic Determinants of Vibrio parahaemolyticus Isolates from Imported Shrimps" addresses a critical gap in understanding the emergence of antimicrobial resistance (AMR) in this seafood-associated pathogen. Vibrio parahaemolyticus is a major cause of global seafood-borne infections, and our research reveals that 19.1% of isolates from imported shrimps display resistance to at least one antibiotic class, with multidrug resistance observed in seven strains. Importantly, we establish experimental epidemiological cutoff values for antibiotic susceptibility, providing valuable criteria specific to V. parahaemolyticus. Our findings underscore the potential risk to consumers, emphasizing the need for vigilant monitoring and intervention strategies. This study significantly contributes to the comprehension of AMR dynamics in V. parahaemolyticus, offering crucial insights for global public health. The dissemination of our research through Microbiology Spectrum ensures broad accessibility and impact within the scientific community and beyond.

Comparison of Stomaching versus Rinsing for Recovering Bacterial Communities from Rainbow Trout (Oncorhynchus mykiss) Fillets.

ABSTRACT: The use of high-throughput methods allows a better characterization of food-related bacterial communities. However, such methods require large amounts of high-quality bacterial DNA, which may be a challenge when dealing with a complex matrix that has a low concentration of bacteria, such as fresh fish fillets. Therefore, the choice of method used to recover bacteria from a food matrix in a cost-effective way is critical, yet little information is available on the performance of commonly used methods. We assessed the recovery capacity of two such methods: stomaching and mechanical rinsing. The efficiency of the methods was evaluated through quantitative recovery and compatibility with end-point quantitative PCR (qPCR). Fresh rainbow trout (Oncorhynchus mykiss) fillets were inoculated with a bacterial marker, Brochothrix thermosphacta, at different concentrations (7.52 to 1.52 log CFU/g). The fillets were processed by one of the two methods, and the recovery of the marker in the suspensions was assessed by plate counting and qPCR targeting B. thermosphacta-rpoC. The same analyses were performed on six noninoculated fresh fillets. Stomaching and mechanical rinsing allowed efficient and repeatable recovery of the bacterial communities from the 42 inoculated fillets. No significant differences in recovery ratios were observed between the marker enumerated in the inoculation suspensions and in the corresponding recovery suspensions after rinsing and stomaching. However, the stomaching method allowed too many particles to pass through the filters bag, making necessary a limiting supplementary filtration step. As a consequence, only the rinsing recovery method allowed proper PCR quantification of the inoculated B. thermosphacta. The mean recovered bacterial level of the fillets was approximately 3 log CFU/g. It seems more relevant and cost-effective to recover the endogenous bacterial microbiota of a fish fillet structure using the rinsing method rather than the stomaching method.

Antibiotic Resistance Genes and Bacterial Communities of Farmed Rainbow Trout Fillets (Oncorhynchus mykiss).

The rise of antibiotic resistance is not only a challenge for human and animal health treatments, but is also posing the risk of spreading among bacterial populations in foodstuffs. Farmed fish-related foodstuffs, the food of animal origin most consumed worldwide, are suspected to be a reservoir of antibiotic resistance genes and resistant bacterial hazards. However, scant research has been devoted to the possible sources of diversity in fresh fillet bacterial ecosystems (farm environment including rivers and practices, and factory environment). In this study bacterial communities and the antibiotic resistance genes of fresh rainbow trout fillet were described using amplicon sequencing of the V3-V4 region of the 16S rRNA gene and high-throughput qPCR assay. The antibiotic residues were quantified using liquid chromatography/mass spectrometry methods. A total of 56 fillets (composed of muscle and skin tissue) from fish raised on two farms on the same river were collected and processed under either factory or laboratory sterile filleting conditions. We observed a core-bacterial community profile on the fresh rainbow trout fillets, but the processing conditions of the fillets has a great influence on their mean bacterial load (3.38 ± 1.01 log CFU/g vs 2.29 ± 0.72 log CFU/g) and on the inter-individual diversity of the bacterial community. The bacterial communities were dominated by Gamma- and Alpha-proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria. The most prevalent genera were Pseudomonas, Escherichia-Shigella, Chryseobacterium, and Carnobacterium. Of the 73 antibiotic residues searched, only oxytetracycline residues were detected in 13/56 fillets, all below the European Union maximum residue limit (6.40-40.20 µg/kg). Of the 248 antibiotic resistance genes searched, 11 were found to be present in at least 20% of the fish population (tetracycline resistance genes tetM and tetV, ß-lactam resistance genes bla DHA and bla ACC, macrolide resistance gene mphA, vancomycin resistance genes vanTG and vanWG and multidrug-resistance genes mdtE, mexF, vgaB and msrA) at relatively low abundances calculated proportionally to the 16S rRNA gene.