Demonstration of IS711 transposition in Brucella ovis and Brucella pinnipedialis

<p>Abstract</p> <p>Background</p> <p>The <it>Brucella </it>genome contains an insertion sequence (IS) element called IS<it>711 </it>or IS<it>6501</it>, which is specific to the genus. The copy number of IS<it>711 </it>varies in the genome of the different <it>Brucella </it>species, ranging from 7 in <it>B. abortus</it>, <it>B. melitensis </it>and <it>B. suis </it>to more than 30 in <it>B. ovis </it>and in <it>Brucella </it>strains isolated from marine mammals. At present, there is no experimental evidence of transposition of IS<it>711</it>, but the occurrence of this element with a high copy number in some species, and the isolation of <it>Brucella </it>strains with "ectopic" copies of IS<it>711 </it>suggested that this IS could still transpose.</p> <p>Results</p> <p>In this study we obtained evidence of transposition of IS<it>711 </it>from the <it>B. ovis </it>and <it>B. pinnipedialis </it>chromosomes by using the "transposon trap" plasmid pGBG1. This plasmid expresses resistance to tetracycline only if the repressor gene that it contains is inactivated. The strains <it>B. melitensis </it>16 M, <it>B. abortus </it>RB51, <it>B. ovis </it>BOC22 (field strain) and <it>B. pinnipedialis </it>B2/94, all containing the plasmid pGBG1, were grown in culture media with tetracycline until the appearance of tetracycline resistant mutants (Tc^R). Tc^Rmutants due to IS<it>711 </it>transposition were only detected in <it>B. ovis </it>and <it>B. pinnipedialis </it>strains.</p> <p>Conclusion</p> <p>Four different copies of IS<it>711 </it>were found to transpose to the same target sequence in the plasmid pGBG1. This demonstrated that IS<it>711 </it>are active <it>in vivo</it>, specially in <it>Brucella </it>species with a high number of IS<it>711 </it>copies as <it>B. ovis </it>and <it>B. pinnipedialis</it>.</p

Alterations of OprD in carbapenem-intermediate and -susceptible strains of Pseudomonas aeruginosa isolated from patients with bacteremia in a Spanish multicenter study

Spanish Network for Research in Infectious Diseases (REIPI).-- et al.We investigated the presence of OprD mutations in 60 strains of metallo-ß-lactamase-negative Pseudomonas aeruginosa intermediately susceptible (IS [n = 12]; MIC = 8 μg/ml) or susceptible (S [n = 48]; MICs ≤ 1 to 4 μg/ml) to imipenem and/or meropenem that were isolated from patients with bacteremia in order to evaluate their impact on carbapenem susceptibility profiles. The presence of mutations in oprD was detected by sequencing analysis. OprD expression was assessed by both outer membrane protein (OMP) analysis and real-time PCR (RT-PCR). Fourteen (23%) isolates had an OprD identical to that of PAO1, and OprD modifications were detected in 46 isolates (77%). Isolates were classified as OprD “full-length types” (T1 [n = 40, including both wild-type OprD and variants showing several polymorphisms]) and OprD “deficient types” (T2 [n = 3 for OprD frameshift mutations] and T3 [n = 17 for premature stop codons in oprD]). RT-PCR showed that 5 OprD type T1 isolates presented reduced transcription of oprD (0.1- to 0.4-fold compared to PAO1), while oprD levels increased more than 2-fold over that seen with PAO1 in 4 OprD type T1 isolates. A total of 50% of the isolates belonging to OprD “deficient types” were susceptible to both carbapenems, and 40% were susceptible to meropenem and intermediately susceptible to imipenem. Only one isolate (5%) within this group was intermediately susceptible to both carbapenems, and one (5%) was susceptible to imipenem and intermediately susceptible to meropenem. We concluded that OprD inactivating mutations in clinical isolates of P. aeruginosa are not restricted only to carbapenem-resistant isolates but are also found in isolates with imipenem or meropenem MICs of only 0.06 to 4 μg/ml.This work was supported by the Ministerio de Ciencia e Innovación of Spain and Instituto de Salud Carlos III, through the Spanish Network for the Research in Infectious Diseases (REIPI C03/14 and RD06/0008), and grants PI08/0802, PS09/00033, and PI08/0276.Peer reviewe

Isolation of VIM-2-producing Pseudomonas monteilii clinical strains disseminated in a tertiary hospital in northern Spain

We describe here the occurrence of blaVIM-2 in 10 carbapenem-resistant Pseudomonas monteilii strains isolated from different clinical samples from patients at the University Hospital Marqués de Valdecilla in northern Spain. All the blaVIM-2-harboring P. monteilii isolates possessed a class 1 integron, with the cassette array [intI1_blaVIM-2_aac(6')-Ib_qacEΔ1_sul1]. Our results show the emergence of VIM-2-producing multidrug-resistant species other than Pseudomonas aeruginosa or Pseudomonas putida in a Spanish hospital. P. monteilii, although sporadically isolated, should also be considered an important metallo-β-lactamase (MBL) reservoir

Prevalence and Population Diversity of Listeria monocytogenes Isolated from Dairy Cattle Farms in the Cantabria Region of Spain

Listeria monocytogenes is an opportunistic pathogen that is widely distributed in the environment. Here we show the prevalence and transmission of L. monocytogenes in dairy farms in the Cantabria region, on the northern coast of Spain. A total of 424 samples was collected from 14 dairy farms (5 organic and 9 conventional) and 211 L. monocytogenes isolates were recovered following conventional microbiological methods. There were no statistically significant differences in antimicrobial resistance ratios between organic and conventional farms. A clonal relationship among the isolates was assessed by pulsed field gel electrophoresis (PFGE) analysis and 64 different pulsotypes were obtained. Most isolates (89%, n = 187) were classified as PCR serogroup IVb by using a multiplex PCR assay. In this case, 45 isolates of PCR serogroup IVb were whole genome-sequenced to perform a further analysis at genomic level. In silico MLST analysis showed the presence of 12 sequence types (ST), of which ST1, ST54 and ST666 were the most common. Our data indicate that the environment of cattle farms retains a high incidence of L. monocytogenes, including subtypes involved in human listeriosis reports and outbreaks. This pathogen is shed in the feces and could easily colonize dairy products, as a result of fecal contamination. Effective herd and manure management are needed in order to prevent possible outbreaks.This work was supported by Research Project grants RTA08-099, RTA2008-00080-C02, RTA2014-00045-C03-01 (INIA and FEDER) from the Spanish Ministry of Economy and Competitiveness and RTI2018-098267-R-C31 (INIA and FEDER) from the Spanish Ministry of Science and Innovation.S

Co-Existence of blaNDM-1, blaOXA-23, blaOXA-64, blaPER-7 and blaADC-57 in a Clinical Isolate of Acinetobacter baumannii from Alexandria, Egypt

The increasing rates of antimicrobial resistance among carbapenem-resistant Acinetobacter baumannii in the Middle East and North Africa are one of the major concerns for healthcare settings. We characterised the first A. baumannii isolate harbouring five β-lactamases identified in Egypt. The isolate Ale25 was obtained from an ICU patient of a hospital from Alexandria. The isolate was phenotypically and genotypically screened for carbapenemase genes. The isolate was resistant to carbapenems, aminoglycosides, fluoroquinolones and cefiderocol. Whole-Genome Sequencing identified five β-lactamase genes, blaNDM-1, blaOXA-23, blaOXA-64, blaPER-7 and blaADC-57, together with other antibiotic resistance genes, conferring resistance to sulfonamides, macrolides, tetracyclines, rifamycin and chloramphenicol. Virulome analysis showed the presence of genes involved in adhesion and biofilm production, type II and VI secretion systems, exotoxins, etc. Multi-Locus Sequence Typing analysis identified the isolate as Sequence Types 113Pas and 2246Oxf, belonging to International Clone 7. Sequencing experiments revealed the presence of four plasmids of 2.7, 22.3, 70.4 and 240.8 Kb. All the β-lactamase genes were located in the chromosome, except the blaPER-7, gene which was found within the plasmid of 240.8 Kb. This study highlights the threat of the emergence and dissemination of these types of isolates.This research was funded by the MINISTRY OF SCIENCE AND INNOVATION (MCIN/AEI/10.13039/501100011033), grant number PID2020-116495RB-I00; the DEPARTMENT OF EDUCATION OF THE BASQUE GOVERNMENT (Research Groups of the Basque University System 2021), grant number Group IT1578-22, GIC21/18; and the ARAB ACADEMY FOR SCIENCE, TECHNOLOGY AND MARITIME TRANSPORT, grant number 2072

Whole-Genome Sequence of Serratia liquefaciens HUMV-21, a Cytotoxic, Quorum-Sensing, and Biofilm-Producing Clinical Isolate

A clinical isolate of Serratia liquefaciens (strain HUMV-21) was obtained from a skin ulcer of an adult patient. We report here its complete genome assembly using PacBio single-molecule real-time (SMRT) sequencing, which resulted in a single circular chromosome with 5.3 Mb. About 5,844 protein-coding genes are predicted from this assembly

Construction of a new class of tetracycline lead structures with potent antibacterial activity through biosynthetic engineering

Antimicrobial resistance and the shortage of novel antibiotics have led to an urgent need for new antibacterial drug leads. Several existing natural product scaffolds (including chelocardins) have not been developed because their suboptimal pharmacological properties could not be addressed at the time. It is demonstrated here that reviving such compounds through the application of biosynthetic engineering can deliver novel drug candidates. Through a rational approach, the carboxamido moiety of tetracyclines (an important structural feature for their bioactivity) was introduced into the chelocardins, which are atypical tetracyclines with an unknown mode of action. A broad-spectrum antibiotic lead was generated with significantly improved activity, including against all Gram-negative pathogens of the ESKAPE panel. Since the lead structure is also amenable to further chemical modification, it is a platform for further development through medicinal chemistry and genetic engineering

Biological Markers of Pseudomonas aeruginosa Epidemic High-Risk Clones

A limited number of Pseudomonas aeruginosa genotypes (mainly ST-111, ST-175, and ST-235), known as high-risk clones, are responsible for epidemics of nosocomial infections by multidrug-resistant (MDR) or extensively drug-resistant (XDR) strains worldwide. We explored the potential biological parameters that may explain the success of these clones. A total of 20 isolates from each of 4 resistance groups (XDR, MDR, ModR [resistant to 1 or 2 classes], and MultiS [susceptible to all antipseudomonals]), recovered from a multicenter study of P. aeruginosa bloodstream infections performed in 10 Spanish hospitals, were analyzed. A further set of 20 XDR isolates belonging to epidemic high-risk clones (ST-175 [n = 6], ST-111 [n = 7], and ST-235 [n = 7]) recovered from different geographical locations was also studied. When unknown, genotypes were documented through multilocus sequence typing. The biological parameters evaluated included twitching, swimming, and swarming motility, biofilm formation, production of pyoverdine and pyocyanin, spontaneous mutant frequencies, and the in vitro competition index (CI) obtained with a flow cytometry assay. All 20 (100%) XDR, 8 (40%) MDR, and 1 (5%) ModR bloodstream isolate from the multicenter study belonged to high-risk clones. No significant differences were observed between clonally diverse ModR and MultiS isolates for any of the parameters. In contrast, MDR/XDR high-risk clones showed significantly increased biofilm formation and mutant frequencies but significantly reduced motility (twitching, swimming, and swarming), production of pyoverdine and pyocyanin, and fitness. The defined biological markers of high-risk clones, which resemble those resulting from adaptation to chronic infections, could be useful for the design of specific treatment and infection control strategies

Automatic classification of Candida species using Raman spectroscopy and machine learning

One of the problems that most affect hospitals is infections by pathogenic microorganisms. Rapid identification and adequate, timely treatment can avoid fatal consequences and the development of antibiotic resistance, so it is crucial to use fast, reliable, and not too laborious techniques to obtain quick results. Raman spectroscopy has proven to be a powerful tool for molecular analysis, meeting these requirements better than traditional techniques. In this work, we have used Raman spectroscopy combined with machine learning algorithms to explore the automatic identification of eleven species of the genus Candida, the most common cause of fungal infections worldwide. The Raman spectra were obtained from more than 220 different measurements of dried drops from pure cultures of each Candida species using a Raman Confocal Microscope with a 532 nm laser excitation source. After developing a spectral preprocessing methodology, a study of the quality and variability of the measured spectra at the isolate and species level, and the spectral features contributing to inter-class variations, showed the potential to discriminate between those pathogenic yeasts. Several machine learning and deep learning algorithms were trained using hyperparameter optimization techniques to find the best possible classifier for this spectral data, in terms of accuracy and lowest possible overfitting. We found that a one-dimensional Convolutional Neural Network (1-D CNN) could achieve above 80 % overall accuracy for the eleven classes spectral dataset, with good generalization capabilities.This work was supported by the R + D projects INNVAL19/17 (funded by Instituto de Investigación Valdecilla-IDIVAL), PID2019-107270RB-C21 (funded by MCIN/ AEI /10.13039/501100011033) and by Plan Nacional de I + D + and Instituto de Salud Carlos III (ISCIII), Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases (REIPI RD16/0016/0007), CIBERINFEC (CB21/13/00068), CIBER-BBN (BBNGC1601), cofinanced by European Development Regional Fund “A way to achieve Europe”. A. A. O.-S was financially supported by the Miguel Servet II program (ISCIII-CPII17-00011)