Ceftriaxone-resistant Salmonella enterica serotype Typhi in a pregnant traveller returning from Karachi, Pakistan to Denmark, 2019.

We describe a ceftriaxone-resistant Salmonella Typhi bacteraemia in a pregnant woman returning from a family visit in Pakistan. Whole genome sequencing confirmed similarity to a Pakistani outbreak clone. Pregnancy and unawareness of this outbreak delayed appropriate antibiotic therapy. Concurrently, we detected faecal carriage of a carbapenemase-producing Escherichia coli. Awareness of the ongoing outbreak should affect empiric treatment of typhoid fever and hygiene precautions in travellers returning from Pakistan. Meropenem may be warranted in severe cases.

Clonal diversity of Cutibacterium acnes (formerly Propionibacterium acnes) in prosthetic joint infections.

Prosthetic joint infections (PJIs) are rare but feared complications following joint replacement surgery. Cutibacterium acnes is a skin commensal that is best known for its role in acne vulgaris but can also cause invasive infections such as PJIs. Some phylotypes might be associated with specific diseases, and recently, a plasmid was detected that might harbour important virulence genes. In this study, we characterized C. acnes isolates from 63 patients with PJIs (n = 140 isolates) and from the skin of 56 healthy individuals (n = 56 isolates), using molecular methods to determine the phylotype and investigate the presence of the plasmid. Single-locus sequence typing and a polymerase chain reaction designed to detect the plasmid were performed on all 196 isolates. No statistically significant differences in sequence types were seen between the two study groups indicating that the C. acnes that causes PJIs originates from the patients own normal skin microbiota. Of the 27 patients with multiple tissue samples, 19 displayed the same sequence types among all their samples. Single-locus sequence typing identified different genotypes among consecutive C. acnes isolates from four patients with recurrent infections. The plasmid was found among 17 isolates distributed in both groups, indicating that it might not be a marker for virulence regarding PJIs. Patients presenting multiple sequence types in tissue samples may represent contamination or a true polyclonal infection due to C. acnes.

Cooperative Toehold: A Mechanism To Activate DNA Strand Displacement and Construct Biosensors.

Toehold-mediated DNA strand displacement has proven powerful in the construction of various DNA circuits, DNA machines, and biosensors. So far, many new toehold activation mechanisms have been developed to achieve programmed DNA strand displacement behaviors. However, almost all those toeholds are inflexible via either a covalently attached manner or a complementary hybridization strategy, which limit the versatility of DNA devices. To solve this problem, we developed a new toehold, named "cooperative toehold", to activate DNA strand displacement. On the basis of a base stacking mechanism, the cooperative toehold is comprised of two moieties with completely independent DNA sequences between each other. The cooperative toehold enabled one to continuously tune the rate of DNA strand displacement, as well as more sophisticated strand displacement reactions. The cooperative toehold has also been employed as a universal signal translator for biosensors to qualitatively determine RNA and ATP. Moreover, as a novel specific PCR monitoring system, cooperative toehold-mediated DNA strand displacement can detect the pUC18 plasmid in genomic DNA samples with an aM detection limit.

Characteristics of ARG-carrying plasmidome in the cultivable microbial community from wastewater treatment system under high oxytetracycline concentration.

Studies on antibiotic production wastewater have shown that even a single antibiotic can select for multidrug resistant bacteria in aquatic environments. It is speculated that plasmids are an important mechanism of multidrug resistance (MDR) under high concentrations of antibiotics. Herein, two metagenomic libraries were constructed with plasmid DNA extracted from cultivable microbial communities in a biological wastewater treatment reactor supplemented with 0 (CONTROL) or 25 mg/L of oxytetracycline (OTC-25). The OTC-25 plasmidome reads were assigned to 72 antibiotic resistance genes (ARGs) conferring resistance to 13 types of antibiotics. Dominant ARGs, encoding resistance to tetracycline, aminoglycoside, sulfonamide, and multidrug resistance genes, were enriched in the plasmidome under 25 mg/L of oxytetracycline. Furthermore, 17 contiguous multiple-ARG carrying contigs (carrying ≥ 2 ARGs) were discovered in the OTC-25 plasmidome, whereas only nine were found in the CONTROL. Mapping of the OTC-25 plasmidome reads to completely sequenced plasmids revealed that the conjugative IncU resistance plasmid pFBAOT6 of Aeromonas caviae, carrying multidrug resistance transporter (pecM), tetracycline resistance genes (tetA, tetR), and transposase genes, might be a potential prevalent resistant plasmid in the OTC-25 plasmidome. Additionally, two novel resistant plasmids (containing contig C301682 carrying multidrug resistant operon mexCD-oprJ and contig C301632 carrying the tet36 and transposases genes) might also be potential prevalent resistant plasmids in the OTC-25 plasmidome. This study will be helpful to better understand the role of plasmids in the development of MDR in water environments under high antibiotic concentrations.

In vivo selection of a complex mutant TEM (CMT) from an inhibitor-resistant TEM (IRT) during ceftazidime therapy.

OBJECTIVES: A relapse from Escherichia coli bloodstream infection was observed in a patient with acute leukaemia treated with ceftazidime for 7 days for febrile neutropenia. Whereas the original E. coli isolate was resistant to ß-lactam/ß-lactamase inhibitor combinations (EC1), the relapse E. coli isolate showed a similar phenotype but with resistance extended to ceftazidime (EC2). We investigated the molecular mechanisms of ß-lactam resistance and sought if EC2 could have been selected in vivo from EC1. METHODS: EC1 and EC2 isolates were compared for antibiotic MICs, plasmid content, genotyping, ß-lactamase genes and their environment. Both isolates were conjugated with E. coli JW4111ΔampC and MICs determined for transconjugants. In addition, ceftazidime-resistant mutants were selected in vitro from EC1. RESULTS: EC1 and EC2 showed identical patterns for genotyping and resistance plasmids. PCR sequencing of blaTEM in EC1 showed the mutations M69L and N276D corresponding to TEM-35, also called inhibitor-resistant TEM (IRT)-4. In EC2, the TEM allele showed an additional mutation, R164S, known to confer resistance to ceftazidime. The combination of these three mutations was previously reported in TEM-158, described as the complex mutant TEM (CMT)-9, associated with resistance to ß-lactamase inhibitors and third-generation cephalosporins. In vitro selection of ceftazidime-resistant mutants from EC1 yielded six different CMT alleles, including TEM-158 containing the R164S mutation. CONCLUSIONS: This first known report of in vivo selection of CMT from IRT, reproduced in vitro, shows how the evolution of ß-lactamase enzymes is easily driven by antibiotic pressure, even during a short antibiotic therapy.

In vivo selection of aac(6')-Ib-cr and mutations in the gyrA gene in a clinical qnrS1-positive Salmonella enterica serovar Typhimurium DT104B strain recovered after fluoroquinolone treatment.

OBJECTIVES: To characterize the mechanisms implicated in the in vivo selection of quinolone and aminoglycoside resistance in a faecal Salmonella enterica serovar Typhimurium DT104B strain recovered after ciprofloxacin treatment of a hospitalized elderly patient with acute gastroenteritis. METHODS: Two Salmonella Typhimurium isolates were obtained before (Se6) and after (Se20) treatment and they were typed by PFGE and multilocus sequence typing (MLST). Antimicrobial susceptibility was determined by disc diffusion and agar dilution methods. Class 1, 2 and 3 integrons and resistance mechanisms were studied by PCR and sequencing. Plasmids were typed. RESULTS: Both Salmonella Typhimurium strains were resistant to tetracycline, streptomycin and sulphonamides, while Se20 was also resistant to nalidixic acid, ciprofloxacin, norfloxacin, levofloxacin, ofloxacin, amikacin, tobramycin, kanamycin and trimethoprim. PFGE and MLST showed a clonal relationship between the strains, which belonged to the sequence type ST36. Both strains contained the repC-sul2-strA-strB structure and tet(A) and qnrS1 genes, and strain Se20 also contained the aac(6')-Ib-cr gene, the Ser83-->Tyr substitution in GyrA and one class 1 integron with the dfrA17 + aadA5 gene cassette arrangement lacking qacEDelta1 + sul1. Two different transconjugants from Salmonella Se20 (TCSe20B and TCSe20L) harboured qnrS1 and sul2 genes and the class 1 integron. The TCSe20B strain also acquired the aac(6')-Ib-cr gene located on a non-typeable plasmid. qnrS1 was identified on a ColE-type plasmid and the class 1 integron on an IncI1-type plasmid. CONCLUSIONS: This is the first report of in vivo selection of the aac(6')-Ib-cr gene and the Ser83-->Tyr change in GyrA in a qnrS1-positive Salmonella Typhimurium strain after ciprofloxacin treatment; the in vitro transfer of both plasmid-mediated quinolone resistance genes was also demonstrated.

Biophysical modeling of fragment length distributions of DNA plasmids after X and heavy-ion irradiation analyzed by atomic force microscopy.

The investigation of fragment length distributions of plasmid DNA gives insight into the influence of localized energy distribution on the induction of DNA damage, particularly the clustering of double-strand breaks. We present an approach that determines the fragment length distributions of plasmid DNA after heavy-ion irradiation by using the Local Effect Model. We find a good agreement of our simulations with experimental fragment distributions derived from atomic force microscopy (AFM) studies by including experimental constraints typical for AFM. Our calculations reveal that by comparing the fragmentation in terms of fluence, we can uniquely distinguish the effect of different radiation qualities. For very high-LET irradiation using nickel or uranium ions, no difference between their fragment distributions can be expected for the same dose level. However, for carbon ions with an intermediate LET, the fragmentation pattern differs from the distribution for very high-LET particles. The results of the model calculations can be used to determine the optimal experimental parameters for a demonstration of the influence of track structure on primary radiation damage. Additionally, we compare the results of our model for two different plasmid geometries.

Biological effects of stannous chloride, a substance that can produce stimulation or depression of the central nervous system.

It was demonstrated that tin, as stannous chloride (SnCl(2)), can facilitate the neuromuscular transmission by accelerating the transmitter release from the nerve terminals in the mouse. When this salt is injected into laboratory animals, it can produce stimulation or depression of the central nervous system. Because calcium (Ca(2+)) influx into the cytoplasm is indispensable to release the transmitter, it would be possible that SnCl(2) increases the Ca(2+) influx at the nerve terminals but not by blocking the K(+) channels. SnCl(2) is known to inhibit the immune response in rodents and to induce tumor generation in thyroid gland. There is no general agreement regarding its genotoxicity and it was discussed that the effects of this salt might depend on the physicochemical conditions and the route of its administration. SnCl(2) has been used in many sectors of human interest, such as food industry and nuclear medicine. This salt is directly administered to human beings endovenously, when it is used as a reducing agent to prepare 99mTc-radiopharmaceuticals which are also used for cerebral studies. SnCl(2) is capable to promote the generation of reactive oxygen species (ROS) that are responsible for the oxidative stress. Oxidative stress has been related with aging and other neurological diseases. So, it is relevant to evaluate other biological effects of SnCl(2). We decided to study these effects using Escherichia coli mutant strains, deficient in DNA repair genes, and supercoiled plasmid DNA. We evaluated the influence of medicinal plants, metal chelating agents, and ROS scavengers against the SnCl(2) deleterious effects. Our results show that SnCl(2) produced lesions in vitro as well as in vivo. This inactivation may be due to the production of ROS. We observed that the genotoxic effect of SnCl(2) was partly inhibited or disappeared, when the treatments were done in the presence of medicinal plants, metal chelating agents, and ROS scavengers. In conclusion, these findings suggest that the SnCl(2) biological effects may be associated with the generation of ROS. Moreover, we can speculate that ROS could be associated with the detrimental effects in the brain due to exogenous or endogenous metals.

[Method for studying horizontal transfer of plasmids in Erwinia carotovora]. / Metod issledovaniia gorizontal'nogo perenosa plazmid u Erwinia carotovora.

A simple method to determine frequency characteristics of the horizontal transfer of antibiotic resistance by transconjugative plasmids of various incompatibility groups into Erwinia carotovora strains has been developed. The proposed method for selection of E. carotovora transconjugates provides for the use of selective medium with pectin and corresponding antibiotic, which permits carrying out reliable counterselection of the donor (Escherichia, Salmonella). The proposed method helped to study horizontal transfer of plasmids RP4 (Inc P), R 391 (Inc J) and pKM101 (Inc N) to some E. carotovora strains and to demonstrate their inheritance as extrachromosomal DNA in Erwinia cells.

[The antioxidative effect of procyanidins from pine bark in vitro].

OBJECTIVE: To assess the antioxidative effect of procyanidins from pine bark on free radical damage. METHODS: Hemolysis, Malonaldehyde(MDA) level of mice liver homogenates, the conformation changes of irradiated plasmid PUC18 were used as indexes. RESULTS: Procyanidins could reduce the hemolysis degree of human RBC induced by H2O2 significantly(P < 0.01), reduce the MDA level of mice liver homogenate initiated by VitC/Fe2+ remarkably(P < 0.01), and reduce the degree of single-strain break of plamid PUC18 induced by 60Co gamma significantly(P < 0.01). CONCLUSION: Procyanidins had good antioxidative function. It can prevent RBC membrane, plasmid DNA suffering from oxygen free radical damage in vitro.

Numerical analysis of Astragalus cicer microsymbionts.

Thirty-seven rhizobium strains, isolated from root nodules of Astragalus cicer (L.) (cicer milkvetch) deriving from different geographic regions, were compared with the representative strains of the known rhizobial species and genera by numerical analysis of phenotypic characteristics. Our results indicated that Astragalus cicer rhizobia were related to the bacteria of Mesorhizobium species and formed two major phena. One phenon, localized on Mesorhizobium loti branch, contained strains from Poland. Another cluster, placed in the vicinity of M. tianshanense, M. mediterraneum, M. ciceri, and M. huakuii, comprised cicer milkvetch nodule isolates from Canada, Ukraine, and one strain from Poland. The relationship of Astragalus cicer microsymbionts to bacteria of the Mesorhizobium species was also supported by phage typing.

Plasmid patterns and antimicrobial susceptibilities of Neisseria gonorrhoeae in Bandung, Indonesia.

Antimicrobial susceptibilities of Neisseria gonorrhoeae isolates from female sex workers and from men with urethritis in Bandung, Indonesia, were determined by an agar dilution technique. Typing of the Tet M plasmid in tetracycline-resistant isolates (TRNG) was performed using a polymerase chain reaction (PCR) technique and plasmid profiles of penicillinase-producing isolates (PPNG) were determined. All PPNG possessed the 4.4 MDa beta-lactamase plasmid and all TRNG showed a PCR fragment characteristic of the 'Dutch' type Tet M plasmid. Of the 50 gonococci isolates tested, all were resistant to tetracycline; 47 were TRNG, 26 were PPNG, and 6 were resistant to thiamphenicol. Chromosomal resistance to penicillin was not detected. All isolates were susceptible to ceftriaxone, ciprofloxacin, norfloxacin, ofloxacin, kanamycin, spectinomycin, and trimethoprim/sulfamethoxazole. Spectinomycin and fluoroquinolones are useful primary drugs for treatment of gonococcal infection in Bandung. Continued surveillance of antimicrobial resistance should be part of gonorrhoea control in Indonesia.

Ceftazidime-resistant Klebsiella pneumoniae and Escherichia coli bloodstream infection: a case-control and molecular epidemiologic investigation.

In a molecular, microbiologic, and case-control study to describe the epidemiology of ceftazidime-resistant Klebsiella pneumoniae and Escherichia coli bloodstream infection, 32 unique isolates were recovered over 31 months from the blood of patients hospitalized in a 900-bed hospital in Chicago. Multivariate analysis revealed cases occurred more frequently in debilitated nursing home patients with central venous catheters than in younger, healthier patients. Mortality rates were similar for cases and controls. Case-patients were less likely to die if they received appropriate antibiotic treatment within 3 days of bacteremia onset (P = .02). Pulsed-field gel electrophoresis analysis indicated a polyclonal outbreak, with strain-specific temporal and geographic clustering. Isoelectric focusing results suggested that a predominant enzyme, TEM-10, was responsible for the ceftazidime resistance. The resistance gene was usually carried on a large conjugative plasmid. The polyclonality of the resistant strains suggests that ceftazidime resistance due to TEM-10 is now endemic in Chicago.

Factors affecting the electroporation of Bacillus subtilis.

Bacillus subtilis 168 trp- was found to be transformable with the tetracycline resistance plasmid pAB124 by electroporation of whole cells, inconsistently and at very low frequencies. Supplementation of the growth medium with glycine, or particularly DL-threonine, produced cells that could be electrotransformed much more efficiently at frequencies up to 2.5 x 10(3) transformants per microgram plasmid DNA. Transformation was optimal with cells grown in medium containing a racemic mixture of the D- and L-isomers of threonine, and no transformants were obtained when pure forms of the D- and L-threonine isomers were used. The cell walls of B. subtilis grown in the presence or absence of D-, L- and DL-threonine had a similar amino acid composition which did not include threonine. A more complex biochemical explanation of the enhancement of electroporation by growth in DL-threonine is likely, and this is discussed. Lysozyme treatments to weaken the cell wall and possibly mimic the effect of DL-threonine did not yield any transformants. The effects of buffer composition and culture incubation time were also determined and the electroporation protocol optimized accordingly. The response of a range of other B. subtilis strains to electroporation by the method produced was found to be variable. In all cases, transformation was verified by recovery of the plasmid DNA from putative transformants.