High sugar diets can increase susceptibility to bacterial infection in Drosophila melanogaster.

Overnutrition with dietary sugar can worsen infection outcomes in diverse organisms including insects and humans, through generally unknown mechanisms. In the present study, we show that adult Drosophila melanogaster fed high-sugar diets became more susceptible to infection by the Gram-negative bacteria Providencia rettgeri and Serratia marcescens. We found that P. rettgeri and S. marcescens proliferate more rapidly in D. melanogaster fed a high-sugar diet, resulting in increased probability of host death. D. melanogaster become hyperglycemic on the high-sugar diet, and we find evidence that the extra carbon availability may promote S. marcescens growth within the host. However, we found no evidence that increased carbon availability directly supports greater P. rettgeri growth. D. melanogaster on both diets fully induce transcription of antimicrobial peptide (AMP) genes in response to infection, but D. melanogaster provided with high-sugar diets show reduced production of AMP protein. Thus, overnutrition with dietary sugar may impair host immunity at the level of AMP translation. Our results demonstrate that dietary sugar can shape infection dynamics by impacting both host and pathogen, depending on the nutritional requirements of the pathogen and by altering the physiological capacity of the host to sustain an immune response.

Biodegradation and bioaugmentation of tetracycline by Providencia stuartii TX2: Performance, degradation pathway, genetic background, key enzymes, and application risk assessment.

The antibiotic tetracycline (TC) is an emerging pollutant frequently detected in various environments. Biodegradation is a crucial approach for eliminating TC contamination. However, only a few efficient TC-degrading bacteria have been isolated, and the molecular mechanisms of TC degradation, as well as their application potential, remain poorly understood. This study isolated a novel TC-degrading bacterium, Providencia stuartii TX2, from the intestine of black soldier fly larvae. TX2 exhibited remarkable performance, degrading 72.17 % of 400 mg/L TC within 48 h. Genomic analysis of TX2 unveiled the presence of antibiotic resistance genes and TC degradation enzymes. Transcriptomic analysis highlighted the roles of proteins related to efflux pumps, enzymatic transformation, adversity resistance, and unknown functions. Three TC degradation pathways were proposed, with TC being transformed into 27 metabolites through epimerization, hydroxylation, oxygenation, ring opening, and de-grouping, reducing TC toxicity. Additionally, TX2 significantly enhanced TC biodegradation in four TC-contaminated environmental samples and reduced antibiotic resistance genes and mobile genetic elements in chicken manure. This research provides insights into the survival and biodegradation mechanisms of Providencia stuartii TX2 and evaluates its potential for environmental bioremediation.

IMD-mediated innate immune priming increases Drosophila survival and reduces pathogen transmission.

Invertebrates lack the immune machinery underlying vertebrate-like acquired immunity. However, in many insects past infection by the same pathogen can 'prime' the immune response, resulting in improved survival upon reinfection. Here, we investigated the mechanistic basis and epidemiological consequences of innate immune priming in the fruit fly Drosophila melanogaster when infected with the gram-negative bacterial pathogen Providencia rettgeri. We find that priming in response to P. rettgeri infection is a long-lasting and sexually dimorphic response. We further explore the epidemiological consequences of immune priming and find it has the potential to curtail pathogen transmission by reducing pathogen shedding and spread. The enhanced survival of individuals previously exposed to a non-lethal bacterial inoculum coincided with a transient decrease in bacterial loads, and we provide strong evidence that the effect of priming requires the IMD-responsive antimicrobial-peptide Diptericin-B in the fat body. Further, we show that while Diptericin B is the main effector of bacterial clearance, it is not sufficient for immune priming, which requires regulation of IMD by peptidoglycan recognition proteins. This work underscores the plasticity and complexity of invertebrate responses to infection, providing novel experimental evidence for the effects of innate immune priming on population-level epidemiological outcomes.

Discovery of a novel symbiotic lineage associated with a hematophagous leech from the genus Haementeria.

Similarly to other strict blood feeders, leeches from the Haementeria genus (Hirudinida: Glossiphoniidae) have established a symbiotic association with bacteria harbored intracellularly in esophageal bacteriomes. Previous genome sequence analyses of these endosymbionts revealed co-divergence with their hosts, a strong genome reduction, and a simplified metabolism largely dedicated to the production of B vitamins, which are nutrients lacking from a blood diet. 'Candidatus Providencia siddallii' has been identified as the obligate nutritional endosymbiont of a monophyletic clade of Mexican and South American Haementeria spp. However, the Haementeria genus includes a sister clade of congeners from Central and South America, where the presence or absence of the aforementioned symbiont taxon remains unknown. In this work, we report on a novel bacterial endosymbiont found in a representative from this Haementeria clade. We found that this symbiont lineage has evolved from within the Pluralibacter genus, known mainly from clinical but also environmental strains. Similarly to Ca. Providencia siddallii, the Haementeria-associated Pluralibacter symbiont displays clear signs of genome reduction, accompanied by an A+T-biased sequence composition. Genomic analysis of its metabolic potential revealed a retention of pathways related to B vitamin biosynthesis, supporting its role as a nutritional endosymbiont. Finally, comparative genomics of both Haementeria symbiont lineages suggests that an ancient Providencia symbiont was likely replaced by the novel Pluralibacter one, thus constituting the first reported case of nutritional symbiont replacement in a leech without morphological changes in the bacteriome. IMPORTANCE: Obligate symbiotic associations with a nutritional base have likely evolved more than once in strict blood-feeding leeches. Unlike those symbioses found in hematophagous arthropods, the nature, identity, and evolutionary history of these remains poorly studied. In this work, we further explored obligate nutritional associations between Haementeria leeches and their microbial symbionts, which led to the unexpected discovery of a novel symbiosis with a member of the Pluralibacter genus. When compared to Providencia siddallii, an obligate nutritional symbiont of other Haementeria leeches, this novel bacterial symbiont shows convergent retention of the metabolic pathways involved in B vitamin biosynthesis. Moreover, the genomic characteristics of this Pluralibacter symbiont suggest a more recent association than that of Pr. siddallii and Haementeria. We conclude that the once-thought stable associations between blood-feeding Glossiphoniidae and their symbionts (i.e., one bacteriome structure, one symbiont lineage) can break down, mirroring symbiont turnover observed in various arthropod lineages.

Dissemination of extensively drug-resistant NDM-producing Providencia stuartii in Europe linked to patients transferred from Ukraine, March 2022 to March 2023.

BackgroundThe war in Ukraine led to migration of Ukrainian people. Early 2022, several European national surveillance systems detected multidrug-resistant (MDR) bacteria related to Ukrainian patients.AimTo investigate the genomic epidemiology of New Delhi metallo-ß-lactamase (NDM)-producing Providencia stuartii from Ukrainian patients among European countries.MethodsWhole-genome sequencing of 66 isolates sampled in 2022-2023 in 10 European countries enabled whole-genome multilocus sequence typing (wgMLST), identification of resistance genes, replicons, and plasmid reconstructions. Five bla NDM-1-carrying-P. stuartii isolates underwent antimicrobial susceptibility testing (AST). Transferability to Escherichia coli of a bla NDM-1-carrying plasmid from a patient strain was assessed. Epidemiological characteristics of patients with NDM-producing P. stuartii were gathered by questionnaire.ResultswgMLST of the 66 isolates revealed two genetic clusters unrelated to Ukraine and three linked to Ukrainian patients. Of these three, two comprised bla NDM-1-carrying-P. stuartii and the third bla NDM-5-carrying-P. stuartii. The bla NDM-1 clusters (PstCluster-001, n = 22 isolates; PstCluster-002, n = 8 isolates) comprised strains from seven and four countries, respectively. The bla NDM-5 cluster (PstCluster-003) included 13 isolates from six countries. PstCluster-001 and PstCluster-002 isolates carried an MDR plasmid harbouring bla NDM-1, bla OXA-10, bla CMY-16, rmtC and armA, which was transferrable in vitro and, for some Ukrainian patients, shared by other Enterobacterales. AST revealed PstCluster-001 isolates to be extensively drug-resistant (XDR), but susceptible to cefiderocol and aztreonam-avibactam. Patients with data on age (n = 41) were 19-74 years old; of 49 with information on sex, 38 were male.ConclusionXDR P. stuartii were introduced into European countries, requiring increased awareness and precautions when treating patients from conflict-affected areas.

The endosymbiont Spiroplasma poulsonii increases Drosophila melanogaster resistance to pathogens by enhancing iron sequestration and melanization.

Facultative endosymbiotic bacteria, such as Wolbachia and Spiroplasma species, are commonly found in association with insects and can dramatically alter their host physiology. Many endosymbionts are defensive and protect their hosts against parasites or pathogens. Despite the widespread nature of defensive insect symbioses and their importance for the ecology and evolution of insects, the mechanisms of symbiont-mediated host protection remain poorly characterized. Here, we utilized the fruit fly Drosophila melanogaster and its facultative endosymbiont Spiroplasma poulsonii to characterize the mechanisms underlying symbiont-mediated host protection against bacterial and fungal pathogens. Our results indicate a variable effect of S. poulsonii on infection outcome, with endosymbiont-harboring flies being more resistant to Rhyzopus oryzae, Staphylococcus aureus, and Providencia alcalifaciens but more sensitive or as sensitive as endosymbiont-free flies to the infections with Pseudomonas species. Further focusing on the protective effect, we identified Transferrin-mediated iron sequestration induced by Spiroplasma as being crucial for the defense against R. oryzae and P. alcalifaciens. In the case of S. aureus, enhanced melanization in Spiroplasma-harboring flies plays a major role in protection. Both iron sequestration and melanization induced by Spiroplasma require the host immune sensor protease Persephone, suggesting a role of proteases secreted by the symbiont in the activation of host defense reactions. Hence, our work reveals a broader defensive range of Spiroplasma than previously appreciated and adds nutritional immunity and melanization to the defensive arsenal of symbionts. IMPORTANCE: Defensive endosymbiotic bacteria conferring protection to their hosts against parasites and pathogens are widespread in insect populations. However, the mechanisms by which most symbionts confer protection are not fully understood. Here, we studied the mechanisms of protection against bacterial and fungal pathogens mediated by the Drosophila melanogaster endosymbiont Spiroplasma poulsonii. We demonstrate that besides the previously described protection against wasps and nematodes, Spiroplasma also confers increased resistance to pathogenic bacteria and fungi. We identified Spiroplasma-induced iron sequestration and melanization as key defense mechanisms. Our work broadens the known defense spectrum of Spiroplasma and reveals a previously unappreciated role of melanization and iron sequestration in endosymbiont-mediated host protection. We propose that the mechanisms we have identified here may be of broader significance and could apply to other endosymbionts, particularly to Wolbachia, and potentially explain their protective properties.

Discovery of antibacterial biogenic magnetosome nanoparticles from Providencia sp. MTBPRB-1: Screening, purification and characterization.

Bacterial species referred to as magnetotactic bacteria (MTB) biomineralize iron oxides and iron sulphides inside the cell. Bacteria can arrange themselves passively along geomagnetic field lines with the aid of these iron components known as magnetosomes. In this study, magnetosome nanoparticles, which were obtained from the taxonomically identified MTB isolate Providencia sp. PRB-1, were characterized and their antibacterial activity was evaluated. An in vitro test showed that magnetosome nanoparticles significantly inhibited the growth of Staphylococcus sp., Pseudomonas aeruginosa, and Klebsiella pneumoniae. Magnetosomes were found to contain cuboidal iron crystals with an average size of 42 nm measured by particle size analysis and scanning electron microscope analysis. The energy dispersive X-ray examination revealed that Fe and O were present in the extracted magnetosomes. The extracted magnetosome nanoparticles displayed maximum absorption at 260 nm in the UV-Vis spectrum. The distinct magnetite peak in the Fourier transform infrared (FTIR) spectroscopy spectra was observed at 574.75 cm-1. More research is needed into the intriguing prospect of biogenic magnetosome nanoparticles for antibacterial applications.

Identification of a novel Providencia species showing multi-drug-resistant in three patients with hospital-acquired infection.

Providencia species are important opportunistic pathogens for humans and are associated with several infectious diseases. In this study, we found three clinical strains belonging to a novel Providencia species, namely Providencia huashanensis, including strains CRE-3FA-0001T, CRE-138-0026, and CRE-138-0111. These strains were recovered from three patients, and all of them were associated with nosocomial infections, including incision infection, urinary tract infection, and intracranial infection. The three strains showed high-level resistance to many types of antimicrobials, including amikacin, aztreonam, ceftazidime, cefepime, ciprofloxacin, colistin, polymyxin B, imipenem, meropenem, ceftazidime-avibactam, imipenem-relebactam. Investigation of the resistance mechanism revealed that acquired resistance genes such as blaKPC, blaNDM, blaPER, blaOXA, aac, ant, and qnrD, played an important role in the multi-drug-resistant phenotype for the three strains. The phylogenetic trees were reconstructed based on the 16S rRNA gene sequences, multi-locus sequence analysis, and core single nucleotide polymorphisms. The genome sequence of the strains had a range of 83.5%-85.8% average nucleotide identity and 21%-25.5% in silico DNA-DNA hybridization scores with other Providencia type strains. The average nucleotide identity and in silico DNA-DNA hybridization values and the phylogenetic trees indicated that the strains CRE-3FA-0001T, CRE-138-0026, and CRE-138-0111 strains should be considered as a novel species of the genus Providencia, for which the name P. huashanensis sp. nov. is proposed. The type strain is CRE-3FA-0001T = China Center for Type Culture Collection AB 2023186T = Korean Collection for Type Cultures 8373T.

Novel Providencia xianensis sp. nov.: A multidrug-resistant species identified in clinical infections.

Providencia genus is known to harbor certain opportunistic pathogens capable of causing human infections. Here, we report two strains of multidrug-resistant bacteria initially identified as Providencia rettgeri by mass spectrometry, but genome analysis revealed their ANI (79.84-84.20%) and dDDH (21.1-25.6%) values to fall below the accepted species threshold for known Providencia species. We therefore propose that these isolates be recognized as a novel species, Providencia xianensis sp. nov. Alarmingly, both strains, isolated from locations far apart, exhibited resistance to last-resort antibiotics, indicating their possible wide distribution, underscoring the urgency for immediate attention and enhanced surveillance for this emerging multidrug-resistant pathogen.

Biodegradation pathway and mechanism of tri (2-chloropropyl) phosphate by Providencia rettgeri.

Tri (2-chloropropyl) phosphate (TCPP) was an emerging contaminant of global concern because of its frequent occurrence, potential toxic effects, and persistence in the environment. Microbial degradation might be an efficient and safe removal method, but limited information was available. In this study, Providencia rettgeri was isolated from contaminated sediment and showed it could use TCPP as unique phosphorus source to promote growth, and decompose 34.7% of TCPP (1 mg/L) within 5 days. The microbial inoculation and the initial concentration of TCPP could affect the biodegradation efficient. Further study results indicated that TCPP decomposition by Providencia rettgeri was mainly via phosphoester bond hydrolysis, evidenced by the production of bis (2-chloropropyl) phosphate (C6H13Cl2PO4) and mono-chloropropyl phosphate (C3H8ClPO4). Both intracellular and extracellular enzymes could degrade TCPP, but intracellular degradation was dominant in the later reaction stage, and the presence of Cu2+ ions had a promoting effect. These findings developed novel insights into the potential mechanism of TCPP microbial degradation.

Identification of Providencia spp. clinical isolates co-producing carbapenemases IMP-27, OXA-24, and OXA-58 in Mexico.

Providencia rettgeri, belonging to the genus Providencia, had gained significant interest due to its increasing prevalence as a common pathogen responsible for healthcare-associated infections in hospitals. P. rettgeri isolates producing carbapenemases have been reported to reduce the efficiency of carbapenems in clinical antimicrobial therapy. However, coexistence with other resistance determinants is rarely reported. The goal of this study was the molecular characterization of carbapenemase-producing Providencia spp. clinical isolates. Among 23 Providencia spp. resistant to imipenem, 21 were positive to blaNDM-1; one positive to blaNDM-1 and blaOXA-58 like; and one isolate co-producing blaIMP-27, blaOXA-24/40 like, and blaOXA-58 like were identified. We observed a low clonal relationship, and the incompatibility groups Col3M and ColRNAI were identified in the plasmid harboring blaNDM-1. We report for the first time a P. rettgeri strain co-producing blaIMP-27, blaOXA-24-like, and blaOXA-58 like. The analysis of these resistance mechanisms in carbapenemase co-producing clinical isolates reflects the increased resistance.

Defining a metagenomic threshold for detecting low abundances of Providencia alcalifaciens in canine faecal samples.

Introduction: Acute haemorrhagic diarrhoea syndrome (AHDS) in dogs is a condition of unknown aetiology. Providencia alcalifaciens is suspected to play a role in the disease as it was commonly found in dogs suffering from AHDS during a Norwegian outbreak in 2019. The role of this bacterium as a constituent of the canine gut microbiota is unknown, hence this study set out to investigate its occurrence in healthy dogs using metagenomics. Materials and methods: To decrease the likelihood of false detection, we established a metagenomic threshold for P. alcalifaciens by spiking culture-negative stool samples with a range of bacterial dilutions and analysing these by qPCR and shotgun metagenomics. The detection limit for P. alcalifaciens was determined and used to establish a metagenomic threshold. The threshold was validated on naturally contaminated faecal samples with known cultivation status for P. alcalifaciens. Finally, the metagenomic threshold was used to determine the occurrence of P. alcalifaciens in shotgun metagenomic datasets from canine faecal samples (n=362) collected in the HUNT One Health project. Results: The metagenomic assay and qPCR had a detection limit of 1.1x103 CFU P. alcalifaciens per faecal sample, which corresponded to a Cq value of 31.4 and 569 unique k-mer counts by shotgun metagenomics. Applying this metagenomic threshold to 362 faecal metagenomic datasets from healthy dogs, P. alcalifaciens was found in only 1.1% (95% CI [0.0, 6.8]) of the samples, and then in low relative abundances (median: 0.04%; range: 0.00 to 0.81%). The sensitivity of the qPCR and shotgun metagenomics assay was low, as only 40% of culture-positive samples were also positive by qPCR and metagenomics. Discussion: Using our detection limit, the occurrence of P. alcalifaciens in faecal samples from healthy dogs was low. Given the low sensitivity of the metagenomic assay, these results do not rule out a significantly higher occurrence of this bacterium at a lower abundance.

Genomic revisitation and reclassification of the genus Providencia.

Members of Providencia, although typically opportunistic, can cause severe infections in immunocompromised hosts. Recent advances in genome sequencing provide an opportunity for more precise study of this genus. In this study, we first identified and characterized a novel species named Providencia zhijiangensis sp. nov. It has ≤88.23% average nucleotide identity (ANI) and ≤31.8% in silico DNA-DNA hybridization (dDDH) values with all known Providencia species, which fall significantly below the species-defining thresholds. Interestingly, we found that Providencia stuartii and Providencia thailandensis actually fall under the same species, evidenced by an ANI of 98.59% and a dDDH value of 90.4%. By fusing ANI with phylogeny, we have reclassified 545 genomes within this genus into 20 species, including seven unnamed taxa (provisionally titled Taxon 1-7), which can be further subdivided into 23 lineages. Pangenomic analysis identified 1,550 genus-core genes in Providencia, with coenzymes being the predominant category at 10.56%, suggesting significant intermediate metabolism activity. Resistance analysis revealed that most lineages of the genus (82.61%, 19/23) carry a high number of antibiotic-resistance genes (ARGs) and display diverse resistance profiles. Notably, the majority of ARGs are located on plasmids, underscoring the significant role of plasmids in the resistance evolution within this genus. Three species or lineages (P. stuartii, Taxon 3, and Providencia hangzhouensis L12) that possess the highest number of carbapenem-resistance genes suggest their potential influence on clinical treatment. These findings underscore the need for continued surveillance and study of this genus, particularly due to their role in harboring antibiotic-resistance genes. IMPORTANCE: The Providencia genus, known to harbor opportunistic pathogens, has been a subject of interest due to its potential to cause severe infections, particularly in vulnerable individuals. Our research offers groundbreaking insights into this genus, unveiling a novel species, Providencia zhijiangensis sp. nov., and highlighting the need for a re-evaluation of existing classifications. Our comprehensive genomic assessment offers a detailed classification of 545 genomes into distinct species and lineages, revealing the rich biodiversity and intricate species diversity within the genus. The substantial presence of antibiotic-resistance genes in the Providencia genus underscores potential challenges for public health and clinical treatments. Our study highlights the pressing need for increased surveillance and research, enriching our understanding of antibiotic resistance in this realm.

Bioflocculation of pollutants in wastewater using flocculant derived from Providencia huaxiensis OR794369.1.

BACKGROUND: Water pollution has become a major environmental and health concern due to increasing population and industrialisation. Microbial flocculants are promising agents for treatment of contaminated water owing to their effectiveness, eco-friendliness, and high biosafety levels. In this study, culture conditions of Providencia huaxiensis OR794369.1 were optimised and its bioflocculant was extracted, characterised and used to treat wastewater. RESULTS: The maximum flocculating activity of 92% and yield of 3.5 g/L were obtained when cultivation conditions were: 3% inoculum size, starch, casein, initial pH of 6, cultivation temperature of 30 oC and 72 h of fermentation. The bioflocculant is an amorphous glycoprotein biomolecule with 37.5% carbohydrates, 27.9% protein, and 34.6% uronic acids. It is composed of hydroxyl, amino, alkanes, carboxylic acid and amines groups as its main functional structures. It was found to be safe to use as it demonstrated non-cytotoxic effects on bovine dermis and African green monkey kidney cells, illustrating median inhibitory concentration (IC50) values of 180 and > 500 µg/mL on both cell lines, respectively. It demonstrated the removal efficiencies of 90% on chemical oxygen demand (COD), 97% on biological oxygen demand (BOD) and 72% on Sulphur on coal mine wastewater. It also revealed the reduction efficacies of 98% (COD) and 92% (BOD) and 70% on Sulphur on domestic wastewater. CONCLUSION: The bioflocculant was effective in reducing pollutants and thus, illustrated potential to be used in wastewater treatment process as an alternative.

Genomic landscape of NDM-1 producing multidrug-resistant Providencia stuartii causing burn wound infections in Bangladesh.

The increasing antimicrobial resistance in Providencia stuartii (P. stuartii) worldwide, particularly concerning for immunocompromised and burn patients, has raised concern in Bangladesh, where the significance of this infectious opportunistic pathogen had been previously overlooked, prompting a need for investigation. The two strains of P. stuartii (P. stuartii SHNIBPS63 and P. stuartii SHNIBPS71) isolated from wound swab of two critically injured burn patients were found to be multidrug-resistant and P. stuartii SHNIBPS63 showed resistance to all the 22 antibiotics tested as well as revealed the co-existence of blaVEB-6 (Class A), blaNDM-1 (Class B), blaOXA-10 (Class D) beta lactamase genes. Complete resistance to carbapenems through the production of NDM-1, is indicative of an alarming situation as carbapenems are considered to be the last line antibiotic to combat this pathogen. Both isolates displayed strong biofilm-forming abilities and exhibited resistance to copper, zinc, and iron, in addition to carrying multiple genes associated with metal resistance and the formation of biofilms. The study also encompassed a pangenome analysis utilizing a dataset of eighty-six publicly available P. stuartii genomes (n = 86), revealing evidence of an open or expanding pangenome for P. stuartii. Also, an extensive genome-wide analysis of all the P. stuartii genomes revealed a concerning global prevalence of diverse antimicrobial resistance genes, with a particular alarm raised over the abundance of carbapenem resistance gene blaNDM-1. Additionally, this study highlighted the notable genetic diversity within P. stuartii, significant informations about phylogenomic relationships and ancestry, as well as potential for cross-species transmission, raising important implications for public health and microbial adaptation across different environments.

fosA11, a novel chromosomal-encoded fosfomycin resistance gene identified in Providencia rettgeri.

This study investigated resistance genes corresponding to the fosfomycin resistance phenotype in clinical isolate Providencia rettgeri W986, as well as characterizing the enzymatic activity of FosA11 and the genetic environment. Antimicrobial susceptibility testing was performed using the agar microdilution method based on the Clinical and Laboratory Standards Institute guidelines. The whole genomic sequence of Providencia rettgeri W986 was obtained using Illumina sequencing and the PacBio platform. The fosA-11 gene was amplified by PCR and cloned into the pUCP20 vector. The recombinant strain pCold1-fosA11-BL21 was expressed to extract the target protein, and absorbance photometry was applied for enzymatic parameter determination. Minimal inhibitory concentration (MIC) tests showed that W986 conferred fosfomycin resistance and was inhibited by phosphonoformate, thereby indicating the presence of a FosA protein. A novel resistance gene designated as fosA11 was identified by whole-genome sequencing and bioinformatics analysis, and it shared 54.41%-64.23% amino acid identity with known FosA proteins. Cloning fosA11 into Escherichia coli obtained a significant increase (32-fold) in the MIC with fosfomycin. Determination of the enzyme kinetics showed that FosA11 had a high catalytic effect on fosfomycin, with Km = 18 ± 4 and Kcat = 56.1 ± 3.2. We also found that fosA11 was located on the chromosome, but the difference in the GC content between the chromosome and fosA11 was dubious, and thus further investigation is required. In this study, we identified and characterized a novel fosfomycin inactivation enzyme called FosA11. The origin and prevalence of the fosA11 gene in other bacteria require further investigation.IMPORTANCEFosfomycin is an effective antimicrobial agent against Enterobacterales strains. However, the resistance rate of fosfomycin is increasing year by year. Therefore, it is necessary to study the deep molecular mechanism of bacterial resistance to fosfomycin. We identified a novel chromosomal fosfomycin glutathione S-transferase, FosA11 from Providencia rettgeri, which shares a very low identity (54.41%-64.23%) with the previously known FosA and exhibits highly efficient catalytic ability against fosfomycin. Analysis of the genetic context and origin of fosA11 displays that the gene and its surrounding environments are widely conserved in Providencia and no mobile elements are discovered, implying that FosA11 may be broadly important in the natural resistance to fosfomycin of Providencia species.

Use of MALDI-TOF for identification and surveillance of gram-negative bacteria in captive wild psittacines / Uso do MALDI-TOF para identificação e monitoramento de bactérias Gram-negativas em psitacídeos cativos

Microbiological studies of the sanitary and health status of psittacine birds that will be reintroduced is important in evaluating whether these animals act as carriers of pathogenic agents to other animals and humans. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a faster and more accurate method to identify bacteria than conventional microbiology methods. The aim of this study was to evaluate the health status of psittacines housed in captivity, by assessment of Gram-negative bacteria from fecal microbiota through MALDI- TOF MS identification. The results indicate high frequency of Gram-negative bacteria in feces (96.5%), especially from the Enterobacteriaceae family (88.7%). The most prevalent bacteria were Escherichia coli (39.0%), Proteus vulgaris (12.2%), Klebsiella spp. (12.1%) and Raoultella ornithinolytica (8.7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. and Escherichia hermannii were isolated with lower frequency. . All these agents are potentially pathogenic for parrots and can cause systemic infections in other animals and humans. These findings reinforce that MALDI- TOF MS proved to be a rapid and accurate method of identification of the microorganism and evaluation of the health status of psittacines, providing relevant data to assist decision-making regarding the sanitary protocols in wildlife centers, and possible future reintroduction of wild birds.

Estudos microbiológicos da sanidade de psitacídeos que serão reintroduzidos são importantes para avaliar se esses animais atuam como portadores de agentes patogênicos para outros animais e humanos. A espectrometria de massa por ionização/dessorção de matriz assistida por laser/tempo de vôo (MALDI-TOF MS) é um método mais rápido e preciso para identificar bactérias na comparação com métodos convencionais de microbiologia. O objetivo deste estudo foi avaliar o estado de saúde de psitacídeos cativos, identificando bactérias Gram-negativas da microbiota fecal por MALDI -TOF MS. Os resultados indicaram alta frequência de bactérias Gram-negativas nas fezes (96,5%), principalmente da família Enterobacteriaceae (88,7%). As mais prevalentes foram Escherichia coli (39,0%), Proteus vulgaris (12,2%), Klebsiella spp. (12,1%) e Raoultella ornithinolytica (8,7%). Proteus hauseri, Citrobacter spp., Morganella morgannii, Providencia rettgeri, Enterobacter spp. e Escherichia hermannii foram isolados com menor frequência. Todos esses agentes são potencialmente patogênicos para os papagaios e podem causar infecções sistêmicas em outros animais e seres humanos. Esses achados reforçam que o MALDI- TOF MS é um método rápido e preciso de identificação do microrganismo e avaliação do estado de saúde dos psitacídeos, fornecendo dados relevantes para auxiliar na tomada de decisões sobre os protocolos sanitários em centros de triagem de animais selvagens e sobre a possibilidade de reintrodução futura.

First report of the aac(6)-Ib-cr gene in Providencia stuartii isolates in Brazil

Abstract INTRODUCTION: The aac(6')-Ib-cr and bla KPC genes are spreading among Enterobacteriaceae species, including Providencia stuartii, in some countries of world. METHODS: These genes were investigated in 28 P. stuartii isolates from a public hospital in Recife, Pernambuco, Brazil, by PCR and sequencing. RESULTS: The aac(6')-Ib-cr gene was detected in 16 resistant isolates, and the bla KPC gene was seen in 14. CONCLUSIONS: The presence of these genes in P. stuartii multi- and extensively drug-resistant isolates indicates that the resistance arsenal of this species is increasing, thus limiting the therapeutic options.

Análise dos fatores de virulência e do potencial patogênico em amostras de providencia alcalifaciens com capacidade de invadir ou näo células cultivadas in vitro / Analysis of the virulence factors and the pathogenic potential of Providencia alcalifaciens strains presenting or not invasive ability to in vitro cultured cells

Providencia alcaffaciens é um enteropatógeno invasor e tem sido isolado) de casos de diarréia em crianças e adultos] em países erre desenvolvimento. Estudos anteriores realizados em nosso laboratório demonstraram que as amostras invasoras eram capazes de aderir a células HeLa. A análise desta interação à microscopia eletrônica de varredura revelou uma discreta projeção de membrana e formação de pedestal nos sítios de aderência bacteriana Entretanto não existem ainda relatos sobre os componentes bacterianos envolvidos na expressão, do fenótipo de aderência) e invasão de P. alcalifacíens Portantoa os objetivos deste estudo foram pesquisar os fatores de virulência relacionados ao fenótipo de aderência e invasão em amostras de P. alcalifacíens, bem como avaliar a presença de outros mecanismos de patogenicidade em amostras invasoras (INV+) e não invasoras (INV; isoladas de casos de diarréia, em São Paulo. Neste trabalho foram analisadas 30 amostras sendo que em I 5 amostras foi confirmada a capacidade de aderir e invadir células HeLa previamente identificada, Todas estas 15 amostras foram também capazes de aderir e invadir células Caco-2 apresentando um padrão de aderência semelhante ao tipo localizada (AL de Escherichia coli enteropatogênica (EPEC', Entretanto ai interação as células Caco-2I ocorreu erre menor freqüência do que a observada em células HeLa. Nas demais amostras, não...(au)