Antibiotic Resistance Markers Research Articles

Genomic perspective of Salmonella enterica isolated from farm animals in Sinaloa, Mexico

Farm animals play a crucial role as a primary source of sustenance for human consumption. However continues to be one of the major food-borne pathogens from a public health standpoint. Its persistent global concern surrounding stems from its capability to induce foodborne illnesses. Unraveling the genetic characteristics of this pathogen stands as a pivotal step, shedding light on the intricate biology of and aiming to mitigate its prevalence. Within this context, our study aims to delve into the genomic traits and population structure of Mexican isolates derived from farm animals. We performed next-generation sequencing across 72 genomes. Genomic data was analyzed to determine virulence and antibiotic resistance markers. In addition, a phylogenetic tree and a spanning tree was constructed. The study revealed a diversity of 18 serovars linked to a singular ST, with prominent serovars being Oranienburg, Give, and Saintpaul. Across all isolates, an extensive array of virulence-related genes was identified. Interestingly, 95% of the isolates displayed exclusive resistance to aminoglycosides, while the remaining exhibited multidrug resistance to tetracycline and chloramphenicol. Notably, a substantial prevalence of prophages in the genomes was observed, accounting for 94% and totaling 183 sequences. The serovar Give stood out with the highest number of sequences, featuring Vibrio X29 and Escher RCS47 as the most prevalent phages. This comprehensive analysis provides valuable insights into the intricate world of , paving the way for enhanced understanding and targeted interventions.

Metagenome-assembled genomes (MAGs) suggest an acetate-driven protective role in gut microbiota disrupted by Clostridioides difficile

Clostridioides difficile may have a negative impact on gut microbiota composition in terms of diversity and abundance, thereby triggering functional changes supported by the differential presence of genes involved in significant metabolic pathways, such as short-chain fatty acids (SCFA). This work has evaluated shotgun metagenomics data regarding 48 samples from four groups classified according to diarrhea acquisition site (community- and healthcare facility-onset) and positive or negative Clostridioides difficile infection (CDI) result. The metagenomic-assembled genomes (MAGs) obtained from each sample were taxonomically assigned for preliminary comparative analysis concerning differences in composition among groups. The predicted genes involved in metabolism, transport, and signaling remained constant in microbiota members; characteristic patterns were observed in MAGs and genes involved in SCFA butyrate and acetate metabolic pathways for each study group. A decrease in genera and species, as well as relative MAG abundance with the presence of the acetate metabolism-related gene, was evident in the HCFO/- group. Increased antibiotic resistance markers (ARM) were observed in MAGs along with the genes involved in acetate metabolism. The results highlight the need to explore the role of acetate in greater depth as a potential protector of the imbalances produced by CDI, as occurs in other inflammatory intestinal diseases.

Scalable protein production by Komagataella phaffii enabled by ARS plasmids and carbon source-based selection

BackgroundMost recombinant Komagataella phaffii (Pichia pastoris) strains for protein production are generated by genomic integration of expression cassettes. The clonal variability in gene copy numbers, integration loci and consequently product titers limit the aptitude for high throughput applications in drug discovery, enzyme engineering or most comparative analyses of genetic elements such as promoters or secretion signals. Circular episomal plasmids with an autonomously replicating sequence (ARS), an alternative which would alleviate some of these limitations, are inherently unstable in K. phaffii. Permanent selection pressure, mostly enabled by antibiotic resistance or auxotrophy markers, is crucial for plasmid maintenance and hardly scalable for production. The establishment and use of extrachromosomal ARS plasmids with key genes of the glycerol metabolism (glycerol kinase 1, GUT1, and triosephosphate isomerase 1, TPI1) as selection markers was investigated to obtain a system with high transformation rates that can be directly used for scalable production processes in lab scale bioreactors.ResultsIn micro-scale deep-well plate experiments, ARS plasmids employing the Ashbya gossypii TEF1 (transcription elongation factor 1) promoter to regulate transcription of the marker gene were found to deliver high transformation efficiencies and the best performances with the reporter protein (CalB, lipase B of Candida antarctica) for both, the GUT1- and TPI1-based, marker systems. The GUT1 marker-bearing strain surpassed the reference strain with integrated expression cassette by 46% upon re-evaluation in shake flask cultures regarding CalB production, while the TPI1 system was slightly less productive compared to the control. In 5 L bioreactor methanol-free fed-batch cultivations, the episomal production system employing the GUT1 marker led to 100% increased CalB activity in the culture supernatant compared to integration construct.ConclusionsFor the first time, a scalable and methanol-independent expression system for recombinant protein production for K. phaffii using episomal expression vectors was demonstrated. Expression of the GUT1 selection marker gene of the new ARS plasmids was refined by employing the TEF1 promoter of A. gossypii. Additionally, the antibiotic-free marker toolbox for K. phaffii was expanded by the TPI1 marker system, which proved to be similarly suited for the use in episomal plasmids as well as integrative expression constructs for the purpose of recombinant protein production.

A dual-channel electrochemical biosensor enables concurrent detection of pathogens and antibiotic resistance

Diarrheagenic E. coli infections, commonly treated with β-lactam antibiotics, contribute to antibiotic resistance - a pressing public health concern. Rapid monitoring of pathogen antibiotic resistance is vital to combat antimicrobial spread. Current bacterial diagnosis methods identify pathogens or determine antibiotic resistance separately, necessitating multiple assays. There is an urgent need for tools that simultaneously identify infectious agents and their antibiotic resistance at the point of care (POC). We developed an integrated electrochemical chip-based biosensor for detecting enteropathogenic E. coli (EPEC), a major neonatal diarrheal pathogen, using an antibody against a virulence marker, termed EspB, and the β-lactam resistance marker, β-lactamase. A dual-channel microfabricated chip, bio-functionalized with a specific EspB monoclonal antibody, and nitrocefin, a β -lactamase substrate was utilized. The chip facilitated electrochemical impedance spectroscopy (EIS)-based detection of EspB antigen and EspB-expressing bacteria. For β-lactam resistance profiling, a second channel enabled differential-pulse voltammetric (DPV) measurement of hydrolyzed nitrocefin. EIS-based detection of EspB antigen was calibrated (LOD: 4.3 ng/mL ±1 and LOQ: 13.0 ng/mL ±3) as well as DPV-based detection of the antibiotic resistance marker, β-lactamase (LOD: 3.6 ng/mL ±1.65 and LOQ: 10 ng/mL ±4). The integrated EIS and DPV biosensor was employed for the simultaneous detection of EspB-expressing and β-lactamase-producing bacteria. The combined readout from both channels allowed the distinction between antibiotic-resistant and -sensitive pathogenic bacteria. The integrated electrochemical biosensor successfully achieved simultaneous, rapid detection of double positive EspB- and β-lactamase-expressing bacteria. Such distinction enabled by a portable device within a short assay time and a simplified sample preparation, may be highly valuable in mitigating the spread of AMR. This new diagnostic tool holds promise for the development of POC devices in clinical diagnosis.

Identification of antibiotic-resistance markers of Edwardsiella tarda using aptamers

Edwardsiella tarda poses significant challenges in the aquaculture sector due to its antibiotic resistance, leading to substantial economic loss in the aquaculture industry. Therefore, it is imperative to identify antibiotic-resistance markers to prevent and control E. tarda. In the present study, we used tetracycline-sensitive (S) and resistant (R) E. tarda strains and six non-target bacterial strains, to develop aptamers through the Systematic Evolution of Ligands by Exponential Enrichment technique. We then conducted comparative analysis of these aptamers based on their relative importance index values and selected six candidate sequences (M1, M13, M15, N57, N69, and N71) for affinity verification. The results showed that M1 had significantly higher affinity towards both the S and R strains compared to the non-target strains. M13 and M15 had significantly higher affinities for the S strain compared to the R strain and non-target strains, and N57, N69, and N71 had significantly higher affinities for R strains relative to the sensitive and non-target strains. Subsequent analysis indicated that N57 and N69 specifically recognized unique sites in the R strain. Therefore, our study presents a novel approach for detecting and controlling antibiotic-resistant strains of E. tarda and identification of drug-resistant markers in bacteria.

Identifying functional multi-host shuttle plasmids to advance synthetic biology applications in Mesorhizobium and Bradyrhizobium.

Ammonia availability has a crucial role in agriculture as it ensures healthy plant growth and increased crop yields. Since diazotrophs are the only organisms capable of reducing dinitrogen to ammonia, they have great ecological importance and potential to mitigate the environmental and economic costs of synthetic fertilizer use. Rhizobia are especially valuable being that they can engage in nitrogen-fixing symbiotic relationships with legumes, and they demonstrate great diversity and plasticity in genomic and phenotypic traits. However, few rhizobial species have sufficient genetic tractability for synthetic biology applications. This study established a basic genetic toolbox with antibiotic resistance markers, multi-host shuttle plasmids and a streamlined protocol for biparental conjugation with Mesorhizobium and Bradyrhizobium species. We identified two repABC origins of replication from Sinorhizobium meliloti (pSymB) and Rhizobium etli (p42d) that were stable across all three strains of interest. Furthermore, the NZP2235 genome was sequenced and phylogenetic analysis determined its reclassification to Mesorhizobium huakuii. These tools will enable the use of plasmid-based strategies for more advanced genetic engineering projects and ultimately contribute towards the development of more sustainable agriculture practices by means of novel nitrogen-fixing organelles, elite bioinoculants, or symbiotic association with nonlegumes.

Orfamide production in Pseudomonas protegens CHA0T promotes rhizospheric colonization and influences assemblage of the bacterial community of wheat roots in soil.

Orfamides (Ofa) are a family of closely related cyclic lipopeptides (cLPs) produced by different soil-inhabiting and plant beneficial strains of the Pseudomonas protegens subgroup. Orfamides are required for swarming motility, they mediate toxicity on oomycetes, algae, and insects, and they can act as ISR elicitors. Ofa biosynthesis depends on a non-ribosomal peptide synthetase gene cluster whose expression is tightly controlled by the post-transcriptional regulatory cascade Gac-Rsm. Although the biological properties and physiological role of Ofa have been well characterized in vitro, the role of Ofa in the fitness of the producing bacterium in its niche has not been studied so far. Here, we used an Ofa biosynthesis-deficient mutant (ΔofaABC) derived from the biocontrol model strain P. protegens CHA0T to explore the relevance of this cLP in soil for the survival of strain CHA0T, its contribution to the bacterial competitiveness for colonization of wheat roots, and its impact on the corresponding rhizobacterial community structure. To facilitate CFU counts in non-sterile soil and root samples we used chromosomally tagged versions of CHA0T and its ΔofaABC mutant with mini-Tn7 constructs carrying antibiotic resistance markers. We confirmed that the tagged Ofa− mutant does not show drop-collapse activity neither it can swarm in semi-solid medium. We also confirmed that Ofa can be exploited in vitro as a social good by the Ofa− mutant to resume swarming in the presence of CHA0T cells. We found that Ofa production did not confer any advantage for bacterial survival along a period of 5 weeks after soil inoculation. However, we observed that Ofa production was associated with a higher competitiveness for colonization of the wheat rhizosphere. Expression of the ofa biosynthetic operon, by means of a transcriptional Pofa-gfp reporter fusion, was detected in the rhizoplane of colonized wheat roots, suggesting that Ofa is produced in situ on the surface of colonized wheat roots. Finally, we observed that seed inoculation with the wild type Ofa producer strain CHA0T resulted in a differential assemblage of the bacterial community of the wheat rhizosphere with respect to that of seeds bacterized with the Ofa deficient mutant. Overall, we provide evidence that Ofa is important for the competitiveness of P. protegens CHA0T cells to colonize the surface of wheat roots and that Ofa production in the rhizosphere can impact the structure of the assembled bacterial microbiome.

Protective effects of different lyoprotectants on survival of clinical bacterial isolates in a hospital biobank

Nowadays the significant role of biobanks in medical, diagnostic, industrial, and environmental research is well known. Bacterial biobanks could be used as a good resource for designing new treatments, biomedical and industrial researches, and laboratory diagnostics. To have a collection of bacteria from clinical samples and maintain their long-term viability, their preservation needs appropriate protective agents, like cryoprotectants and lyoprotectants. In this study, we collected and characterized Gram-negative and Gram-positive bacteria carrying important antibiotic resistance markers from different clinical samples of hospitalized children. Sucrose (10%), skimmed milk (10%), skimmed milk plus sodium glutamate (10% + 1%), and bovine serum albumin (BSA, 10%) were used as lyoprotectants during the freeze-drying procedure. The survival rate of the lyophilized samples was calculated by dilution plating and measuring the colony forming unit (CFU) after 3 months of storage. The culture analysis results indicated that 25 of the 27 studied bacterial genera (Dilutions 10−3 to 10−6), including Shigella, Methicillin-resistant S. aureus, Acinetobacter spp., Escherichia spp., Pseudomonas spp., Klebsiella spp., Enterococcus spp., were recovered in cultured fractions from all preservation conditions, while 2 genera were only detected in a single preservation condition (2/27, 7.4%). Based on the results, sucrose (10%) and skimmed milk (10%) presented the most protective features. The survival rates varied significantly according to types of the bacteria. Collectively, our results showed a diversity in the recovery of different bacterial genera after lyophilization. While statistically no significant difference was detected among the studied protective agents, sucrose (10%) and skimmed milk (10%) exhibited more effective lyoprotective properties for both Gram-positive and Gram-negative bacteria among the clinical isolates in our study.

Plasmid-free cheater cells commonly evolve during laboratory growth.

It has been nearly a century since the isolation and use of penicillin, heralding the discovery of a wide range of different antibiotics. In addition to clinical applications, such antibiotics have been essential laboratory tools, allowing for selection and maintenance of laboratory plasmids that encode cognate resistance genes. However, antibiotic resistance mechanisms can additionally function as public goods. For example, extracellular beta-lactamases produced by resistant cells that subsequently degrade penicillin and related antibiotics allow neighboring plasmid-free susceptible bacteria to survive antibiotic treatment. How such cooperative mechanisms impact selection of plasmids during experiments in laboratory conditions is poorly understood. Here, we show in multiple bacterial species that the use of plasmid-encoded beta-lactamases leads to significant curing of plasmids in surface-grown bacteria. Furthermore, such curing was also evident for aminoglycoside phosphotransferase and tetracycline antiporter resistance mechanisms. Alternatively, antibiotic selection in liquid growth led to more robust plasmid maintenance, although plasmid loss was still observed. The net outcome of such plasmid loss is the generation of a heterogenous population of plasmid-containing and plasmid-free cells, leading to experimental confounds that are not widely appreciated.IMPORTANCEPlasmids are routinely used in microbiology as readouts of cell biology or tools to manipulate cell function. Central to these studies is the assumption that all cells in an experiment contain the plasmid. Plasmid maintenance in a host cell typically depends on a plasmid-encoded antibiotic resistance marker, which provides a selective advantage when the plasmid-containing cell is grown in the presence of antibiotic. Here, we find that growth of plasmid-containing bacteria on a surface and to a lesser extent in liquid culture in the presence of three distinct antibiotic families leads to the evolution of a significant number of plasmid-free cells, which rely on the resistance mechanisms of the plasmid-containing cells. This process generates a heterogenous population of plasmid-free and plasmid-containing bacteria, an outcome which could confound further experimentation.

An intein-based biosensor to measure protein stability in vivo.

Biosensors to measure protein stability in vivo are valuable tools for a variety of applications. Previous work has demonstrated that a tripartite design, whereby a protein of interest (POI) is inserted within a reporter, can link POI stability to reporter activity. Inteins are translated within other proteins and excised in a self-mediated protein splicing reaction. Here, we developed a novel folding biosensor where a POI is inserted within an intein, which is subsequently translated within an antibiotic resistance marker. We showed that protein splicing is required for antibiotic resistance and that housing a stable POI within the intein, compared to an unstable variant, results in a 100,000-fold difference in survival. Further, using a fluorescent protein that matures slowly as the POI, we developed a reporter with two simultaneous readouts for protein folding. Finally, we showed that co-expression of GroEL can significantly increase the activity of both reporters, further verifying that protein folding factors can act on the POI in the biosensor. As a whole, our work provides a new twist on the traditional tripartite approach to measuring protein stability in vivo.

CRISPR-like sequences association with antibiotic resistance and biofilm formation in Helicobacter pylori clinical isolates

Role of clustered regularly interspaced short palindromic repeats (CRISPR)-like sequences in antibiotic resistance and biofilm formation isn't clear. This study investigated association of CRISPR-like sequences with antibiotic resistance and biofilm formation in H. pylori isolates. Thirty-six of H. pylori isolates were studied for existence of CRISPR-like sequences using PCR method and their correlation with biofilm formation and antibiotic resistance. Microtiter-plate technique was utilized for investigating antibiotic resistance profile of isolates against amoxicillin, tetracycline, metronidazole and clarithromycin. Biofilm formation of isolates was analyzed by microtiter-plate-based-method. Out of 23 CRISPR-like positive isolates, 19 had ability of biofilm formation and 7 of 13 CRISPR-like negative isolates were able to form biofilm (Pvalue = 0.445). In CRISPR-like positive isolates, 11 (48%), 18 (78%), 18 (78%) and 23 (100%) were resistant to amoxicillin, tetracycline, metronidazole and clarithromycin, respectively. Since CRISPR-like sequences have role in antibiotic resistance, may be applied as genetic markers of antibiotic resistance. But there was no substantial correlation between biofilm formation and existence of CRISPR-like sequences. These results indicate possible importance of CRISPR-like sequences on acquisition of resistance to antibiotics in this bacterium.

ESCHERICHIA COLI phenotypic characteristics and antagonistic activity in opisthorchiasis invasion

Opisthorchis felineus invasion in human causes inflammatory and dyskinetic disorders of the gastrointestinal tract accompanied by altered phenotypic characteristics in colon microbiota. The aim of research — study an impact of the Escherichia coli isolate phenotypic characteristics on Klebsiella spp. bacteria, isolated from colonic contents of patients with diagnosed opisthorchiasis as well as E. coli antagonistic activity. Materials and methods. The phenotypic properties of 54 E. coli isolates and 8 genus Klebsiella isolates obtained from colonic contents of patients with diagnosed opisthorchiasis were assessed. Identification of isolates and analysis of proteomic profiles were performed using Maldi BioTyper 3.0 software. 204 co-cultivation datasets were analyzed investigating antagonistic activity of E. coli isolates with varying properties on Klebsiella spp. E. coli and Klebsiella spp. isolates were examined by whole genome sequencing. Results. E. coli bacteria with typical phenotypic characteristics showed significantly more prominent antagonistic activity against Klebsiella spp. A significantly higher level of antagonistic activity against K. oxytoca bacteria vs K. pneumoniae strains. The proteomic bacterial strain profiles were divided into clusters depending on the level of antagonistic activity. E. coli molecular serotyping for O- and H-antigens revealed the genes of enterotoxigenic, enteroinvasive and extraintestinal pathogens in 60.0% of cases. Strains with the highest antagonistic activity index, which are carriers of the genes typical to enterotoxigenic E. coli sequence serotypes O6:H1 and O6:H5, were identified. The genome of such strains consisted of the largest number of virulence gene complexes: adhesins, invasins, toxins, bacteriocins. Multilocus sequence typing and sequence serotyping of E. coli and K. pneumoniae strains established their heterogeneity; K. oxytoca isolates were identified as ST242 and ST176. All strains were characterized by homology of antibiotic resistance markers (oqxA, oqxB, fosA) and a variety of beta-lactam resistance gene variants. Conclusion. It was found that E. coli isolates with typical phenotypic characteristics and carriers of virulence gene complexes exhibited significantly more pronounced antagonistic activity against Klebsiella spp. isolated from colonic contents of patients with diagnosed opisthorchiasis.

Genetic transformation of the frog-killing chytrid fungus Batrachochytrium dendrobatidis

Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis, is decimating amphibian populations around the world. Bd belongs to the chytrid lineage, a group of early-diverging fungi that are widely used to study fungal evolution. Like all chytrids, Bd develops from a motile form into a sessile, growth form, a transition that involves drastic changes in its cytoskeletal architecture. Efforts to study Bd cell biology, development, and pathogenicity have been limited by the lack of genetic tools with which to test hypotheses about underlying molecular mechanisms. Here, we report the development of a transient genetic transformation system for Bd. We used electroporation to deliver exogenous DNA into Bd cells and detected transgene expression for up to three generations under both heterologous and native promoters. We also adapted the transformation protocol for selection using an antibiotic resistance marker. Finally, we used this system to express fluorescent protein fusions and, as a proof of concept, expressed a genetically encoded probe for the actin cytoskeleton. Using live-cell imaging, we visualized the distribution and dynamics of polymerized actin at each stage of the Bd life cycle, as well as during key developmental transitions. This transformation system enables direct testing of key hypotheses regarding mechanisms of Bd pathogenesis. This technology also paves the way for answering fundamental questions of chytrid cell, developmental, and evolutionary biology.

Plasmid content of carbapenem resistant Acinetobacter baumannii isolates belonging to five International Clones collected from hospitals of Alexandria, Egypt.

Multidrug resistant Acinetobacter baumannii is one of the most important nosocomial pathogens worldwide. During the last decades it has become a major threat for healthcare settings due to the high antibiotic resistance rates among these isolates. Many resistance determinants are coded by conjugative or mobilizable plasmids, facilitating their dissemination. The majority of plasmids harbored by Acinetobacter species are less than 20 Kb, however, high molecular weight elements are the most clinically relevant since they usually contain antibiotic resistance genes. The aim of this work was to describe, classify and determine the genetic content of plasmids harbored by carbapem resistant A. baumannii isolates belonging to predominant clonal lineages circulating in hospitals from Alexandria, Egypt. The isolates were subjected to S1-Pulsed Field Gel Electrophoresis experiments to identify high molecular weight plasmids. To further analyze the plasmid content and the genetic localization of the antibiotic resistance genes, isolates were sequenced by Illumina Miseq and MinION Mk1C and a hybrid assembly was performed using Unicycler v0.5.0. Plasmids were detected with MOBsuite 3.0.3 and Copla.py v.1.0 and mapped using the online software Proksee.ca. Replicase genes were further analyzed through a BLAST against the Acinetobacter Plasmid Typing database. Eleven plasmids ranging in size from 4.9 to 205.6 Kb were characterized and mapped. All isolates contained plasmids, and, in many cases, more than two elements were identified. Antimicrobial resistance genes such as bla OXA-23, bla GES-like, aph(3')-VI and qacEΔ1 were found in likely conjugative large plasmids; while virulence determinants such as septicolysin or TonB-dependent receptors were identified in plasmids of small size. Some of these resistance determinants were, in turn, located within transposons and class 1 integrons. Among the identified plasmids, the majority encoded proteins belonging to the Rep_3 family, but replicases of the RepPriCT_1 (Aci6) family were also identified. Plasmids are of high interest as antibiotic resistance control tools, since they may be used as genetic markers for antibiotic resistance and virulence, and also as targets for the development of compounds that can inhibit transfer processes.

Filamentous prophage Pf4 promotes genetic exchange in Pseudomonas aeruginosa.

Filamentous prophages are widespread among bacteria and play crucial functions in virulence, antibiotic resistance, and biofilm structures. The filamentous Pf4 particles, extruded by an important pathogen Pseudomonas aeruginosa, can protect producing cells from adverse conditions. Contrary to the conventional belief that the Pf4-encoding cells resist reinfection, we herein report that the Pf4 prophage is reciprocally and commonly exchanged within P. aeruginosa colonies, which can repair defective Pf4 within the community. By labeling the Pf4 locus with antibiotic resistance and fluorescence markers, we demonstrate that the Pf4 locus is frequently exchanged within colony biofilms, in artificial sputum media, and in infected mouse lungs. We further show that Pf4 trafficking is a rapid process and capable of rescuing Pf4-defective mutants. The Pf4 phage is highly adaptable and can package additional DNA doubling its genome size. We also report that two clinical P. aeruginosa isolates are susceptible to the Pf4-mediated exchange, and the Pf5 prophage can be exchanged between cells as well. These findings suggest that the genetic exchanging interactions by filamentous prophages may facilitate defect rescue and the sharing of prophage-dependent benefits and costs within the P. aeruginosa community.

Antibiotic resistance, plasmids, and virulence-associated markers in human strains of Campylobacter jejuni and Campylobacter coli isolated in Italy.

Campylobacteriosis, a prevalent foodborne gastrointestinal infection in Europe, is primarily caused by Campylobacter jejuni and Campylobacter coli, with rising global concerns over antimicrobial resistance in these species. This study comprehensively investigates 133 human-origin Campylobacter spp. strains (102 C. jejuni and 31 C. coli) collected in Italy from 2013 to 2021. The predominant Multilocus Sequence Typing Clonal complexes (CCs) were ST-21 CC and ST-206 CC in C. jejuni and ST-828 CC in C. coli. Ciprofloxacin and tetracycline resistance, mainly attributed to GyrA (T86I) mutation and tet(O) presence, were prevalent, while erythromycin resistance was associated with 23S rRNA gene mutation (A2075G), particularly in C. coli exhibiting multidrug-resistant pattern CipTE. Notable disparities in virulence factors among strains were observed, with C. jejuni exhibiting a higher abundance compared to C. coli. Notably, specific C. jejuni sequence types, including ST-21, ST-5018, and ST-1263, demonstrated significantly elevated counts of virulence genes. This finding underscores the significance of considering both the species and strain-level variations in virulence factor profiles, shedding light on potential differences in the pathogenicity and clinical outcomes associated with distinct C. jejuni lineages. Campylobacter spp. plasmids were classified into three groups comprising pVir-like and pTet-like plasmids families, exhibiting diversity among Campylobacter spp. The study underscores the importance of early detection through Whole Genome Sequencing to identify potential emergent virulence, resistance/virulence plasmids, and new antimicrobial resistance markers. This approach provides actionable public health data, supporting the development of robust surveillance programs in Italy.

A supernumerary synthetic chromosome in Komagataella phaffii as a repository for extraneous genetic material

BackgroundKomagataella phaffii (Pichia pastoris) is a methylotrophic commercially important non-conventional species of yeast that grows in a fermentor to exceptionally high densities on simple media and secretes recombinant proteins efficiently. Genetic engineering strategies are being explored in this organism to facilitate cost-effective biomanufacturing. Small, stable artificial chromosomes in K. phaffii could offer unique advantages by accommodating multiple integrations of extraneous genes and their promoters without accumulating perturbations of native chromosomes or exhausting the availability of selection markers.ResultsHere, we describe a linear “nano”chromosome (of 15–25 kb) that, according to whole-genome sequencing, persists in K. phaffii over many generations with a copy number per cell of one, provided non-homologous end joining is compromised (by KU70-knockout). The nanochromosome includes a copy of the centromere from K. phaffii chromosome 3, a K. phaffii-derived autonomously replicating sequence on either side of the centromere, and a pair of K. phaffii-like telomeres. It contains, within its q arm, a landing zone in which genes of interest alternate with long (approx. 1-kb) non-coding DNA chosen to facilitate homologous recombination and serve as spacers. The landing zone can be extended along the nanochromosome, in an inch-worming mode of sequential gene integrations, accompanied by recycling of just two antibiotic-resistance markers. The nanochromosome was used to express PDI, a gene encoding protein disulfide isomerase. Co-expression with PDI allowed the production, from a genomically integrated gene, of secreted murine complement factor H, a plasma protein containing 40 disulfide bonds. As further proof-of-principle, we co-expressed, from a nanochromosome, both PDI and a gene for GFP-tagged human complement factor H under the control of PAOX1 and demonstrated that the secreted protein was active as a regulator of the complement system.ConclusionsWe have added K. phaffii to the list of organisms that can produce human proteins from genes carried on a stable, linear, artificial chromosome. We envisage using nanochromosomes as repositories for numerous extraneous genes, allowing intensive engineering of K. phaffii without compromising its genome or weakening the resulting strain.

K fragment as a polymerase chain reaction-based vector for antibiotic resistance gene hunting

Antimicrobial resistance (AMR) has emerged as an escalating health issue in the global public health arena. To evaluate and predict AMR, it is of utmost importance to identify and characterize both the known and unknown genes responsible for AMR. While known genes can be readily detected, identifying unknown genes present a challenge. In this study, we developed the plasmid K fragment (pKF) by modifying the pUC19 vector, specifically by removing the multiple cloning site and introducing a Prom-RBS sequence. pKF was used for amplification of K fragment that contains a ribosomal binding site (RBS), and promoter at both ends and plasmid origin of replication. The functionality of added Prom-RBS sequence and K fragment as a cloning vector was tested by cloning chloramphenicol resistance gene amplicon and erythromycin resistance gene from genomic DNA, respectively. The cloning experiment demonstrated the usability of this newly developed cloning method with K fragment. K fragment is an innovative vector that can be easily obtained through amplification by polymerase chain reaction and lacks antibiotic resistance markers. This novel approach is convenient to use since it allows cloning of resistance genes at all orientations and this flexibility can be maneuvered by changing restriction enzymes for primers and fragments. With these distinctive features, this vector stands out to be a versatile tool for cloning both known and unknown resistance genes, and the improved method with K fragment enables the microbiological and molecular characterization of cloned genes. K fragment can be utilized for cloning of resistance genes in bacteria originated in different environments without having to perform bacterial culture. We believe that the convenience brought by this technique could lend itself efficient in the battle against the growing AMR crisis through pre-emptive identification of resistance genes.

The emergence of clonally diverse carbapenem-resistant Enterobacter cloacae complex in West Bengal, India: a dockyard of β-lactamases periling nosocomial infections.

Carbapenem-resistant Enterobacter cloacae complex (CRECC) constitutes a global public health threat challenging clinical treatment and infection control, especially in low- and middle-income countries such as India. We analyzed the antimicrobial susceptibility, major β-lactamase genes, plasmid profiles, and genetic relatedness to understand the molecular epidemiology of CRECC clinical isolates (n = 44) in West Bengal, India, during 2021-2022. The majority (> 55%) of the isolates were resistant to fluoroquinolones, aminoglycosides, and co-trimoxazole, even > 20% for tigecycline and > 35% were extensively drug-resistant. Co-β-lactamase production was categorized into twenty-seven types, importantly NDM (84%), OXA-48 (40%), TEM (61%), CTX-M (46%), OXA-1 (55%), and MIR (27%). The NDM-1 and OXA-181 were major variants with the first observations of NDM-24 and -29 variants in India. Wide-range of plasmids (2 to > 212kb) were harbored by the β-lactamase-producing isolates: small (91%), medium (27%), large (9%), and mega (71%). IncX3, ColE1, and HI2 were noted in about 30% of isolates, while IncF and R were carried by < 20% of isolates. The clonally diverse CRECC isolates were noted to cause cross-infections, especially at superficial site, bloodstream, and urinary-tract. This is the first molecular surveillance on CRECC in India. The study isolates serve as the dockyard of NDM, TEM, and CTX-M harboring a wide range of plasmids. The outcomes of the study may strengthen local and national policies for infection prevention and control practices, clarifying the genetic diversity among CRECC. Extensive genomic study may further intersect the relationships between these different plasmids, especially with their sizes, types, and antibiotic resistance markers.

Mouse genome rewriting and tailoring of three important disease loci

Genetically engineered mouse models (GEMMs) help us to understand human pathologies and develop new therapies, yet faithfully recapitulating human diseases in mice is challenging. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences, which control spatiotemporal gene expression patterns and splicing in many human diseases1,2. Including regulatory extensive genomic regions, which requires large-scale genome engineering, should enhance the quality of disease modelling. Existing methods set limits on the size and efficiency of DNA delivery, hampering the routine creation of highly informative models that we call genomically rewritten and tailored GEMMs (GREAT-GEMMs). Here we describe ‘mammalian switching antibiotic resistance markers progressively for integration’ (mSwAP-In), a method for efficient genome rewriting in mouse embryonic stem cells. We demonstrate the use of mSwAP-In for iterative genome rewriting of up to 115 kb of a tailored Trp53 locus, as well as for humanization of mice using 116 kb and 180 kb human ACE2 loci. The ACE2 model recapitulated human ACE2 expression patterns and splicing, and notably, presented milder symptoms when challenged with SARS-CoV-2 compared with the existing K18-hACE2 model, thus representing a more human-like model of infection. Finally, we demonstrated serial genome writing by humanizing mouse Tmprss2 biallelically in the ACE2 GREAT-GEMM, highlighting the versatility of mSwAP-In in genome writing.