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Silver nanoparticles (AgNPs) are of potential interest because of their effective antibacterial and antiviral activities. Capping agents are used for exhibiting a better antibacterial activity than uncapped Ag NPs. There are very few reports that have shown the usage of AgNPs for in-vivo antibacterial therapy. Citrate-capped silver nanoparticles were synthesized chemically by citrate reduction method; the size of Cit-AgNPs was determined by an atomic force microscope (AFM) and was between 15 - 90 nm. Acinetobacter baumannii (A. baumannii) isolates were the only sensitive species to Cit-AgNPs. MICs and MBC of Cit-AgNPs were determined by using A. baumannii. The results showed an additive effect of Cit-AgNPs. Four mice groups were infected with a sub-lethal dose of A. baumannii intraperitoneally, IP. The single daily dose of Cit-AgNPs and imipenem plus Cit-AgNPs combination were administered IP. Imipenem and phosphate buffer saline (PBS) was used as positive control and negative control, respectively. Interestingly, only the PBS-treated group showed growth of A. baumannii in the liver and spleen of sacrificed mice. Histopathologically, Cit-AgNPs showed antibacterial activity and had an additive effect when combined with imipenem in vivo and in vitro. Moreover, the Cit-AgNPs showed dose-dependent activity and the organs differed in the illumination of the toxicity effect of Cit-AgNPs even after high dose administration. In conclusion, Cit-capped AgNPs had antibacterial activity against multiple drug resistant (MDR) A. baumannii but not against K. pneumoniae and E. coli. Cit-capped AgNPs increased the inhibition zone of imipenem in additive effect; the minimum inhibitory concentration and the minimum bactericidal concentration of Cit-capped AgNPs were relatively low. Cit-capped AgNPs eliminated A. baumannii infection in vivo when it was given alone or in combination with imipenem. The cytotoxicity of Cit-AgNPs was dose-dependent and the organs differed in the illumination of the inflammatory effect of Cit-AgNPs after high dose administration. It is not recommended to use Cit-capped AgNPs systemically despite their valuable additive antibacterial effect especially with a high dose and the combination with imipenem, Topical administration needs to be evaluated.

Identification of Vibrio harveyi, Vibrio ichthyoenteri, and Photobacterium damselae isolated from olive flounder Paralichthys olivaceus in Korea by multiplex PCR developed using the rpoB gene. Keywords: bacterial fish pathogen; conventional PCR; quantitative PCR; recA gene; Vibrio scophthalmi EN bacterial fish pathogen conventional PCR quantitative PCR recA gene Vibrio scophthalmi 373 378 6 01/17/22 20220201 NES 220201 Since it was first isolated from the intestine of the turbot (Cerdà-Cuéllar et al., 1997), I Vibrio scophthalmi i has been considered an opportunistic pathogen in several fish species (Gauger et al., 2006; Valdenegro-Vega et al. 2013). Bacterial fish pathogen, quantitative PCR, conventional PCR, recA gene, Vibrio scophthalmi. [Extracted from the article]

2-years-old plants of Pyrus communis showing symptoms of fire blight disease were sampled in an orchard in Tuscany (Italy) during Autumn 2020. Plants were obtained the previous spring from a commercial nursery located in a region where the disease is present since 1994. The collected material was processed in the lab in order to verify the presence of the bacterium Erwinia amylovora, the causal agent of fire blight. Pure isolates showing white mucoid colonies and levan producers on Levan medium were putatively assimilated to E. amylovora. DNA was extracted from the cultures and analysed with three molecular assays, including duplex PCR of the 29-Kb plasmid pEA29 and the ams chromosomal region, sequencing of the 16S rDNA and recA gene regions, two real-time PCR assays on symptomatic plant tissues. All tests confirmed the presence of E. amylovora. Symptomatic and surrounding plants were removed and immediately destroyed according to the regional phytosanitary protocol. This outcome poses a serious threat for fruit orchards in the area.

2-years-old plants of Pyrus communis showing symptoms of fire blight disease were sampled in an orchard in Tuscany (Italy) during Autumn 2020. Plants were obtained the previous spring from a commercial nursery located in a region where the disease is present since 1994. The collected material was processed in the lab in order to verify the presence of the bacterium Erwinia amylovora, the causal agent of fire blight. Pure isolates showing white mucoid colonies and levan producers on Levan medium were putatively assimilated to E. amylovora. DNA was extracted from the cultures and analysed with three molecular assays, including duplex PCR of the 29-Kb plasmid pEA29 and the ams chromosomal region, sequencing of the 16S rDNA and recA gene regions, two real-time PCR assays on symptomatic plant tissues. All tests confirmed the presence of E. amylovora. Symptomatic and surrounding plants were removed and immediately destroyed according to the regional phytosanitary protocol. This outcome poses a serious threat for fruit orchards in the area. [ABSTRACT FROM AUTHOR]

In recent years, new evidence has shown that the SOS response plays an important role in the response to antimicrobials, with involvement in the generation of clinical resistance. Here we evaluate the impact of heterogeneous expression of the SOS response in clinical isolates of Escherichia coli on response to the fluoroquinolone, ciprofloxacin. In silico analysis of whole genome sequencing data showed remarkable sequence conservation of the SOS response regulators, RecA and LexA. Despite the genetic homogeneity, our results revealed a marked differential heterogeneity in SOS response activation, both at population and single-cell level, among clinical isolates of E. coli in the presence of subinhibitory concentrations of ciprofloxacin. Four main stages of SOS response activation were identified and correlated with cell filamentation. Interestingly, there was a correlation between clinical isolates with higher expression of the SOS response and further progression to resistance. This heterogeneity in response to DNA damage repair (mediated by the SOS response) and induced by antimicrobial agents could be a new factor with implications for bacterial evolution and survival contributing to the generation of antimicrobial resistance. [ABSTRACT FROM AUTHOR]

Silver nanoparticles (AgNPs) are of potential interest because of their effective antibacterial and antiviral activities. Capping agents are used for exhibiting a better antibacterial activity than uncapped Ag NPs. There are very few reports that have shown the usage of AgNPs for in-vivo antibacterial therapy. Citrate-capped silver nanoparticles were synthesized chemically by citrate reduction method; the size of Cit-AgNPs was determined by an atomic force microscope (AFM) and was between 15 - 90 nm. Acinetobacter baumannii (A. baumannii) isolates were the only sensitive species to Cit- AgNPs. MICs and MBC of Cit-AgNPs were determined by using A. baumannii. The results showed an additive effect of Cit-AgNPs. Four mice groups were infected with a sub-lethal dose of A. baumannii intraperitoneally, IP. The single daily dose of Cit-AgNPs and imipenem plus Cit-AgNPs combination were administered IP. Imipenem and phosphate buffer saline (PBS) was used as positive control and negative control, respectively. Interestingly, only the PBS-treated group showed growth of A. baumannii in the liver and spleen of sacrificed mice. Histopathologically, Cit-AgNPs showed antibacterial activity and had an additive effect when combined with imipenem in vivo and in vitro. Moreover, the Cit-AgNPs showed dose-dependent activity and the organs differed in the illumination of the toxicity effect of Cit-AgNPs even after high dose administration. In conclusion, Cit-capped AgNPs had antibacterial activity against multiple drug resistant (MDR) A. baumannii but not against K. pneumoniae and E. coli. Cit-capped AgNPs increased the inhibition zone of imipenem in additive effect; the minimum inhibitory concentration and the minimum bactericidal concentration of Citcapped AgNPs were relatively low. Cit-capped AgNPs eliminated A. baumannii infection in vivo when it was given alone or in combination with imipenem. The cytotoxicity of Cit-AgNPs was dosedependent and the organs differed in the illumination of the inflammatory effect of Cit-AgNPs after high dose administration. It is not recommended to use Cit-capped AgNPs systemically despite their valuable additive antibacterial effect especially with a high dose and the combination with imipenem, Topical administration needs to be evaluated. [ABSTRACT FROM AUTHOR]

Background: Streptococcus pneumoniae identification has traditionally been based on two biochemical tests, susceptibility of pneumococci to optochin and solubility in bile-salt solution. Due to slowness and sometimes difficulty in interpretation, the bile solubility test has fallen into disuse. The main objective of this work was to assess the current effectiveness of the optochin susceptibility test in pneumococcal identification in clinical practice. Materials and Methods: Overall 126 viridans group streptococci consecutively isolated from respiratory samples were analyzed using the optochin susceptibility test by picking one colony from the culture. Sixty-two were initially considered optochin susceptible, and 64 were considered optochin resistant and analyzed with the bile solubility test. If a discrepancy between the tests was observed (i.e., whether an isolate was optochin susceptible and bile insoluble or optochin resistant and bile soluble), then the optochin susceptibility test was repeated, adjusting the inoculum to a McFarland standard of 0.5. Species were identified by sequencing the lytA and recA genes. Results: Twelve discrepancies were initially observed. The result of the repeated optochin test showed that the initial optochin test of 4 isolates had been wrongly interpreted. Of the remaining 8 discrepancies, 2 optochin-resistant bile-soluble isolates were identified by gene sequencing as S. pneumoniae, and of the 6 optochin-susceptible bile-nonsoluble isolates, 3 were identified as Streptococcus mitis and 3 as Streptococcus pseudopneumoniae. Conclusions: The optochin test correctly identified 90.5% of all recent viridans group streptococci clinical isolates which include both optochin susceptible (62/126 = 49.2%) and optochin resistant (64/126 = 50.8%) strains. Of the group of optochin susceptible viridans, 87.5% were correctly identified, and 93.5% of the optochin resistant group were correctly identified. However, this technique does not correctly differentiate between S. pneumoniae from other viridans group streptococci in the clinical setting. Additional testing is needed for that identification. [ABSTRACT FROM AUTHOR]

In interspecific hybrids a decrease in genetic exchanges between chromosomes from different species and negative interactions between functionally incompatible loci limit genetic variation among the progeny. Earlier the fraction of crossover genotypes was increased in intraspecific hybrids of the cultivated tomato Solanum lycopersicum with the expression of the recA Escherichia coli gene. In this research transgenic hybrids between S. lycopersicum and some wild tomato species were obtained in order to study the effect of recA gene expression on the frequency of crossovers in the progeny of interspecific hybrids. Interspecific hybrids showed negative interactions between the loci of the cultivated tomato and the S. cheesmaniae, S. pimpinellifolium, and S. habrochaites species. In the latter case it resulted in semilethal necrosis. The recA gene expression in interspecific hybrids with S. cheesmaniae partially compensates among the F2 progeny the deficiency of recessive genotypes at the Wv:wv locus of chromosome 2. In general, the fractions of crossover genotypes in the F2 progeny were higher in transgenic interspecific hybrids than in control non-transgenic hybrids of the same combination of crossing, but lower than in intraspecific S. lycopersicum hybrids. [ABSTRACT FROM AUTHOR]

This paper describes taxonomic position, phylogeny, and phenotypic properties of 14 lactic acid bacteria (LAB) originating from an Apis mellifera guts. Based on the 16S rDNA and recA gene sequence analyses, 12 lactic acid bacteria were assigned to Lactobacillus kunkeei and two others were classified as Fructobacillus fructosus. Biochemically, all isolated lactic acid bacteria showed typical fructophilic features and under anaerobic conditions grew well on fructose, but poorly on glucose. Fast growth of bacteria on glucose was noted in the presence of oxygen or fructose as external electron acceptors. The residents of honeybee guts were classified as heterofermentative lactic acid bacteria. From glucose, they produced almost equimolar amounts of lactic acid, acetic acid, and trace amounts of ethanol. Furthermore, they inhibited the growth of the major honeybee pathogen, Paenibacillus larvae, meaning that the LAB studied may have the health-conferring properties of probiotics. [ABSTRACT FROM AUTHOR]

The aim of this study was to evaluate probiotic properties of antimicrobial Lactobacillus plantarum VJC38 in vitro. L. plantarum VJC38 was isolated from the crop of broiler chicken and characterized using dnaK gene sequence. The inhibitory activities of L. plantarum VJC38 against bacterial and fungal pathogens were evaluated. Antifungal compounds secreted by the strain VJC38 were identified using Gas Chromatography and Mass Spectrometry (GC-MS). The strain was evaluated for its tolerance to low pH, resistance to bile salts, auto-aggregation, co-aggregation with pathogenic Escherichia coli, cell surface hydrophobicity, cholesterol lowering activity, β-galactosidase production, adhesion ability to Caco-2 cells, mucin degradation, hemolytic activity and biogenic amine production. Phylogenetic analysis of dnaK gene of bacterial strain VJC38 showed 99% sequence similarity to Lactobacillus plantarum var. plantarum. It showed effective inhibition against food spoiling and pathogenic organisms like Escherichia coli, Listeria monocytogenes, Staphylococcus aureus, Aspergillus niger, Penicillium expansum and Eurotium species. The antifungal compound phenol- 2,4-bis(1,1-dimethylethyl) (PD) was identified in the culture filtrate of L. plantarum VJC38 and reported to have inhibition against Aspergillus species. L. plantarum VJC38 exhibited tolerance to low pH, resistance to bile salts, bile salt hydrolase activity, auto-aggregation (87.5%), co-aggregation with Escherichia coli (55.7%), cholesterol lowering activity (64%), β-galactosidase production (1206 MU), adherence to Caco-2 cells (11%), negative for mucin degradation, hemolytic activity and biogenic amine production. L. plantarum VJC38 could be a good candidate for further investigation in vivo to elucidate its health benefits and to evaluate its technological properties as a bio-protective strain. [ABSTRACT FROM AUTHOR]

Mycoplasma hyorhinis is an opportunistic pathogen of pigs. Recently, it has been shown to transform cell cultures, increasing the attention of the researchers. Studies on the pathogenesis require specific genetic tool that is not yet available for the pathogen. To address this limitation, we constructed two suicide plasmids pGEMT-tetM/LR and pGEMT-recA-tetM/LR having a tetracycline resistance marker flanked by two hemolysin gene arms. The latter plasmid encodes an E. coli recA , a gene involved in DNA recombination, repair and maintenance of DNA. Using inactivation of the hemolysin gene, which results in a detectable and measurable phenotype, we found that each plasmid can disrupt the hemolysin gene of M. hyorhinis through a double cross-over homologous recombination. However, inclusion of the E. coli recA gene in the construct resulted in 9-fold increase in the frequency of hemolysin gene mutants among the screened tetracycline resistance colonies. The resultant hemolysin mutant strain lacks the ability to lyse mouse bed blood cells (RBC) when tested in vitro ( p < 0.001). The host-plasmid system described in this study, has applications for the genetic manipulation of this pathogen and potentially other mycoplasmas. [ABSTRACT FROM AUTHOR]

Las especies del complejo Burkholderia cepacia (CBC) son capaces de causar infecciones crónicas del tracto respiratorio en pacientes con fibrosis quística y en otros individuos inmunocomprometidos. La mayoría de estas especies exhiben alta resistencia a la terapia antibiótica, lo que genera la necesidad de una detección rápida y precisa para poder implementar estrategias de control adecuadas. En este trabajo se utilizó la técnica de reacción en cadena de la polimerasa (PCR) para amplificar el gen recA (PCR-recA), con el fin de identificar microorganismos pertenecientes al CBC. Con este método molecular como referencia, se evaluó la sensibilidad (S) y la especificidad (E) de dos sistemas de identificación comerciales automatizados, VITEK 2 y API 20NE (bioMérieux®), así como también el valor de las pruebas bioquímicas manuales más representativas para la identificación de estos microorganismos. El método VITEK 2 presentó una S del 71,1 % y una E del 100 %; para el método API 20NE, estos valores fueron 69,7 % y 90,2 %, respectivamente. En cuanto a las pruebas fenotípicas manuales, los resultados obtenidos fueron más heterogéneos, lo que posiblemente se deba a que estas bacterias podrían sufrir presión selectiva para sobrevivir en pacientes crónicos y perder factores fenotípicos característicos. La técnica de PCR-recA resultó de fácil implementación, por lo que cabe considerar a esta técnica de identificación como una opción viable, aun en laboratorios de diagnóstico clínico de mediana complejidad.Species belonging to the Burkholderia cepacia complex (BCC) are capable of causing chronic respiratory tract infections in patients suffering from cystic fibrosis as wel as in immunocompromised individuals. Most of these species are highly resistant to antibiotic therapy, generating the need for their rapid and accurate detection for the proper treatment and clinical management of these patients. In this wok, the polymerase chain reaction (PCR) technique based on the amplification of the recA gene (PCR-recA) was applied for an accurate identification of bacteria belonging to the BCC. Sensitivity (S) and specificity (E) of two biochemically-based commercial automated systems, API 20NE and VITEK 2 (bioMérieux®), and of the most representative biochemical manual tests for the identification of the Burkholderia cepacia complex were herein evaluated. The commercial systems VITEK 2 and API 20NE showed the following sensitivity and specificity vaues for identification to the species level, S: 71.1 %, E: 100 %, S: 69.7 %, E: 90.2 %, respectively. More complex results were observed for phenotypic manual tests, since BCC bacteria can undergo selective pressure to survive in chronic patients causing the loss of their typical phenotypic characteristics. The PCR-recA technique was easy to implement even in medium-complexity clinical diagnostic laboratories.

Burkholderia cepacia complex (Bcc) species are opportunistic pathogens widely distributed in the environment and often infect people with cystic fibrosis (CF). This study aims to determine which genomovars of the Bcc can cause infections in non-CF patients from a tertiary care hospital in Mexico and if they carry virulence factors that could increase their pathogenicity. We identified 23 clinical isolates that carry the recA gene. Twenty-two of them belongs to the genomovar V (B. vietnamiensis) and one to the genomovar II (B. multivorans). Thirteen pulsotypes were identified among 22 B. vietnamiensis isolates. All clinical isolates produced biofilm were motile and cytotoxic on murine macrophage-like RAW264.7 and in A549 human lung epithelial cells. In conclusion, B. vietnamiensis causes infections in non-CF patients in a tertiary care hospital in Mexico, rapid identification of this pathogen can help physicians to establish a better antimicrobial treatment. [ABSTRACT FROM AUTHOR]

• Clinical heteroresistance is associated with acquired resistance gene copy number. • Clinical heteroresistance is dependent on RecA activity. • RecA constitutes a promising target for combating heteroresistance. RecA inhibition could be an important strategy to combat antimicrobial resistance because of its key role in the SOS response, DNA repair and homologous recombination contributing to bacterial survival. This study evaluated the impact of RecA inactivation on heteroresistance in clinical isolates of Escherichia coli and their corresponding recA -deficient isogenic strains to multiple classes of antimicrobial agents. A high frequency (>30%) of heteroresistance was observed in this collection of clinical isolates. Deletion of the recA gene led to a marked reduction in heteroresistant subpopulations, especially against quinolones or β-lactams. The molecular basis of heteroresistance was associated with an increase in copy number of plasmid-borne resistance genes (bla TEM-1B) or tandem gene amplifications (qnrA1). Of note, in the absence of the recA gene, the increase in copy number of resistance genes was suppressed. This makes the recA gene a promising target for combating heteroresistance. [Display omitted] [ABSTRACT FROM AUTHOR]

A total of 113 lactic acid bacteria (LAB) were isolated from crop, gizzard, intestine and ceca regions of broiler chicken. ThirteenLactobacillusisolates were selected based on their ability to grow on acidic pH and inhibition of enteric bacterial pathogens. They were identified using 16S rRNA andrecAgene sequencing and evaluated for probiotic propertiesin vitro. These isolates showed effective inhibition against enteric bacterial pathogens likeEscherichia coliand fungal pathogens, such asAspergillus niger, A. flavus, Penicillium expansum, P. roqueforti, Candida albicansandEurotiumspecies. Analysis of 16S rRNA andrecAgene sequences revealed that isolates belonged toLactobacillus plantarum. They exhibited tolerance to bile salts (0.4%), gastric and intestinal conditions with bile salt hydrolase activity, suggesting their ability to survive during gastrointestinal transit and exert their probiotic action on host organism. They showed cell surface hydrophobicity (39–58%), auto-aggregation (60–80%) and co-aggregation with enteric pathogens likeE. coli(49–62%) andListeria monocytogenes(26–38%). Thus, they could potentially be bactericidal to pathogens and prevent their colonization on intestinal epithelium. They are non-pathogenic (γ-hemolytic) and negative for mucin degradation. Among these isolates,L. plantarumVJI21 and VJC1 showed more adherence to HT-29 cells than the enteric pathogenE. coliand prevented pathogen adherence to intestinal cells.L. plantarumVJI21 and VJC1, having potent probiotic properties, are considered good candidates for further studiesin vivotowards the development of probiotic feed supplement in the poultry industry. [ABSTRACT FROM PUBLISHER]

The Escherichia coli RecA protein (RecAp) has been demonstrated to induce mutagenesis in yeast cells, although there is still little information on the role of the RecAp in yeast recombination events. We evaluated spontaneous and induced general recombination in vegetative and meiotic cells of the XS2316 strain of the yeast Saccharomyces cerevisiae bearing the recA gene. We found that RecAp decreased reciprocal recombination, gene conversion and intrachromosomal recombination and promoted an increase in error-prone processes in both vegetative and meiotic cells, while its negative effect on meiotic recombination blocked ascospore formation.

Vibrio parahaemolyticus is a gram-negative bacterium capable of causing diseases in humans and aquatic animals. The global relationships among V. parahaemolyticus genomes have been studied using multilocus sequence typing (MLST). Recently, the MLST gene recA has shown difficulties in amplification and/or a larger PCR fragment for some V. parahaemolyticus genomes due to genetic recombination. We aimed to investigate these recombination events of recA gene by analyzing 500 publicly available whole genomes from the NCBI database. The genomes with untypable recA genes were separated using BIGSdb and CGEMLST 2.0 servers, followed by annotation with RAST and NCBI pipelines. Moreover, the variable nature of V. parahaemolyticus was investigated by wgMLST analysis. The hypothetical proteins in recombinant regions were analyzed with VCIMPred tool. In the results, 3 genomes were detected with recA gene recombination, in which 2 were associated with phages and 1 to an AHPND causing strain. All 3 recombinant regions had a G + C content of 39%–40% with 15–30 ORFs, including a newly incorporated recA gene. These acquired recA genes were closely related to 3 different genera namely Aliivibrio , Photobacterium , and Vibrio. The wgMLST analysis indicated genetic recombination events occur independently among V. parahaemolyticus on a global scale. The in silico analysis revealed 4 hypothetical proteins associated with virulence factors in recombinant regions. The present study confirms, recombination events of V. parahaemolyticus recA gene, are diverse and may have an impact on the evolutionary process. Moreover, understanding these genetic recombination events of the recA gene is necessary to determine their STs and, therefore assessing epidemiological relationships. • Genetic recombination event of recA gene for AHPND causing V. parahaemolyticus identified for the first time. • Evidence on possible interspecies and/or genera level recombination of V. parahaemolyticus MLST gene recA • Hypothetical proteins in recombinant regions shown to possess virulent related functions. [ABSTRACT FROM AUTHOR]

Deletion of recA gene in Francisella tularensis 15/10 genome leads to the increase in its sensitivity to ultraviolet irradiation, reduction of the homologous recombination capacity, and a slight decline of virulence for mice. Efficacy of Francisella tularensis 15/10ΔrecA reproduction within microphage-like cells, its resistance to normal rabbit serum, and protective properties on the mouse model of tularemia are the same as in original Francisella tularensis 15/10.

The suppression of the SOS response has been shown to enhance the in vitro activity of quinolones. Furthermore, Dam-dependent base methylation has an impact on susceptibility to other antimicrobials affecting DNA synthesis. Here, we investigated the interplay between these two processes, alone and in combination, in terms of antimicrobial activity. A genetic strategy was used employing single- and double-gene mutants for the SOS response ( recA gene) and the Dam methylation system ( dam gene) in isogenic models of Escherichia coli both susceptible and resistant to quinolones. Regarding the bacteriostatic activity of quinolones, a synergistic sensitization effect was observed when the Dam methylation system and the recA gene were suppressed. In terms of growth, after 24 h in the presence of quinolones, the Δ dam Δ recA double mutant showed no growth or delayed growth compared to the control strain. In bactericidal terms, spot tests showed that the Δ dam Δ recA double mutant was more sensitive than the Δ recA single mutant (about 10- to 10 2 -fold) and the wild type (about 10 3 - to 10 4 -fold) in both susceptible and resistant genetic backgrounds. Differences between the wild type and the Δ dam Δ recA double mutant were confirmed by time-kill assays. The suppression of both systems, in a strain with chromosomal mechanisms of quinolone resistance, prevents the evolution of resistance. This genetic and microbiological approach demonstrated the enhanced sensitization of E. coli to quinolones by dual targeting of the recA (SOS response) and Dam methylation system genes, even in a resistant strain model.

Winter wheat is a suitable crop to be ensiled for animal feed and China has the largest planting area of this crop in the world. During the ensiling process, lactic acid bacteria (LAB) play the most important role in the fermentation. We investigated the natural population of LAB in whole-crop wheat (WCW) and examined the quality of whole-crop wheat silage (WCWS) with and without LAB inoculants. Two Lactobacillus plantarum subsp. plantarum strains, Zhengzhou University 1 (ZZU 1) selected from corn and forage and grass 1 (FG 1) from a commercial inoculant, were used as additives. The silages inoculated with LAB strains (ZZU 1 and FG 1) were better preserved than the control, with lower pH values (3.5 and 3.6, respectively) (p<0.05) and higher contents of lactic acid (37.5 and 34.0 g/kg of fresh matter (FM), respectively) (p<0.05) than the control. Sixty LAB strains were isolated from fresh material and WCWS without any LAB inoculation. These LAB strains were divided into the following four genera and six species based on their phenotypic, biochemical and phylogenetic characteristics: Leuconostoc pseudomesenteroides, Leuconostoc citreum, Weissella cibaria, Lactococcus lactis subsp. lactis, Lactobacillus buchneri, and Lactobacillus plantarum subsp. plantarum. However, the prevalent LAB, which was predominantly heterofermentative (66.7%), consisted of Leuconostoc pseudomesenteroides, Leuconostoc citreum, Weissella cibaria, and Lactobacillus buchneri. This study revealed that most of isolated LAB strains from control WCWS were heterofermentative and could not grow well at low pH condition; the selective inoculants of Lactobacillus strains, especially ZZU 1, could improve WCWS quality significantly. [ABSTRACT FROM AUTHOR]

Live vaccine used in Russia against tularemia is based on Francisella tularensis subsp. holarctica NIIEG vaccine strain 15. This vaccine is highly effective, but fairly unstable. Therefore, the development of stable live tularemia vaccine with minimal side effects is rather pressing. F. tularensis iglC gene is required for intracellular production of the microbe, and recA gene is crucial for homologous recombination. Using methodology for allele removal, we deleted one copy of iglC gene and both copies of recA gene in F. tularensis vaccine strain. To achieve replacement of intact F. tularensis chromosome segments with modified segments, we constructed a novel pGM5 suicide vector based on pHV33 bireplicon plasmid. Modified chromosome segments contained a F. tularensis DNA fragment without 545 bp of iglC structural gene segment (in pGMΔiglC plasmid) and lacked a 1060-bp DNA fragment containing recA structural gene segment (pGMΔrecA plasmid). The constructed 15/23-lΔrecA mutant was capable to reproduce in macrophage-like J77A.l cell line with proliferation efficiency eight to ten times higher than parent vaccine strain 15. BALB/c mouse responded to immunization by 15/23-lΔrecA strain with a slight (about 2%) weight decrease compared to infection with strain 15 (about 14%). Bacteria of 15/23-lΔrecA strain were virtually incapable of maintaining germination in BALB/c murine spleen 14 days after invasion. In turn, bacteria of strain 15 were detected in murine spleen even after 21 days. Thus, F. tularensis 15/23-lΔrecA strain, which results in a lighter organismal reaction, can be successfully used as a basis for construction of stable live tularemia vaccines with minimal side effects. [ABSTRACT FROM AUTHOR]

Abstract: The aim of this study was to establish the identification ability of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) for bacteria of Burkholderia cepacia complex (Bcc) and to compare these results with those obtained by a molecular method (PCR-RFLP). A total of 57 isolates was used in the study. Isolates were collected from 31 patients attending the Regional Cystic Fibrosis Unit from January 2001 to December 2005. For phenotypic identification, both automated and manual systems were used. Using mass spectrometry, we identified all 57 isolates, previously identified by molecular method. Of these, 28 isolates were identified as B. cenocepacia, although not differentiated further into lineages. Moreover, other isolates were identified as B. cepacia (12 isolates), B. stabilis (12 isolates), and B. vietnamiensis (5 isolates). Our data indicate a good correlation between the two approaches. [Copyright &y& Elsevier]

Clin Microbiol Infect 2012; 18: 1089-1096 Abstract We sequenced the evolutionarily conserved genes 16S rRNA, atpD, tuf, and recA from Streptococcus pseudopneumoniae, Streptococcus pneumoniae, Streptococcus mitis, and Streptococcus oralis. Phylogenetic analysis revealed that recA provided good resolution between these species, including discrimination of the novel species S. pseudopneumoniae. By contrast, the more conserved 16S rRNA, tuf and atpD are not sufficiently discriminatory. Therefore, recA sequences were used to develop a real-time PCR assay with a locked nucleic acid-mediated TaqMan probe for the specific detection and identification of S. pseudopneumoniae. The PCR assay showed excellent specificity and a detection limit of <10 genome copies for the detection and identification of S. pseudopneumoniae strains, which makes it a promising tool for molecular identification and epidemiological studies. In conclusion, this article describes for the first time a PCR assay for the specific identification of S. pseudopneumoniae. [ABSTRACT FROM AUTHOR]

This work is concerned with the role of alkylhydroxybenzenes (AHBs), chemical analogs of the autoregulatory microbial d factors, on the development of the stress response of bacterial cells to UV irradiation, including SOS system induction, preservation of cell viability, and S → R phase transitions of the Escherichia coli test strain with the bioluminescence genes cloned under the control of the recA gene promoter. UV irradiation, a natural stress factor, and an increase in AHB concentrations were found to elicit uniform responses in bacteria, indicating that AHBs function as alarmones, i.e., alarm signals. It was revealed that preincubating bacteria with alkylhydroxybenzenes considerably enhanced their viability upon irradiation with lethal UV doses; this was accompanied by a relative decrease in the SOS response activity and a concomitant increase in the frequency of phase transitions. The efficiency of the protective action of AHBs increased with an increase in their hydrophobicity degree. The probable mechanism of the protective effect of AHBs is discussed, based on their capacity for the interaction with biopolymers, which results in changing their structural organization and conferring resistance to a broad spectrum of stress factors. Such a 'passive' protective mechanism reduces the susceptibility of DNA to UV irradiation, causing a decrease in the parameters related to the SOS system induction that is responsible for the 'active' protective mechanism in bacterial cells. As a result, viability retention under the lethal influence of UV irradiation is possible at minimal values of repair activity and is accompanied by an increase in the phenotypic variability of the surviving part of a bacterial population. [ABSTRACT FROM AUTHOR]

A total of 140 lactic acid bacteria (LAB) strains were isolated from corn, alfalfa, clover, sainfoin, and Indian goosegrass in the Tibetan Plateau. According to phenotypic and chemotaxonomic characteristics, 16S rDNA sequence, and recA gene PCR amplification, these LAB isolates were identified as belonging to five genera and nine species. Corn contained more LAB species than other forage crops. Leuconostoc pseudomesenteroides, Lactococcus lactis subsp. lactis, Lactobacillus brevis, and Weissella paramesenteroides were dominant members of the LAB population on alfalfa, clover, sainfoin, and Indian goosegrass, respectively. The comprehensive 16S rDNA and recA-based approach effectively described the LAB community structure of the relatively abundant LAB species distributed on different forage crops. This is the first report describing the diversity and natural populations of LAB associated with Tibetan forage crops, and most isolates grow well at or below 10°C. The results will be valuable for the future design of appropriate inoculants for silage fermentation in this very cold area. [ABSTRACT FROM AUTHOR]

Abstract: It has been shown that photocatalytic TiO2 nanoparticles (NPs) can be used as an efficient anti-microbial agent under UV light due to generation of reactive oxygen species (ROS), while Shewanella oneidensis MR-1 is a metal-reducing bacterium highly susceptible to UV radiation. Interestingly, we found that the presence of Cu-doped TiO2 NPs in the cultural medium dramatically increased the survival rates (based on colony-forming unit) of strain MR-1 by over 10,000-fold (incubation without shaking) and ~200 fold (incubation with shaking) after a 2-h exposure to UV light. Gene expression results (via qPCR measurement) indicated that the DNA repair gene recA in MR-1 was significantly induced by UV exposure (indicating cellular damage under UV stress), but the influence of NPs on recA expression was not statistically evident. Plausible explanations to NP attenuation of UV stresses are: 1. TiO2 based NPs are capable of scattering and absorbing UV light and thus create a shading effect to protect MR-1 from UV radiation; 2. more importantly, Cu-doped TiO2 NPs can co-agglomerate with MR-1 to form large flocs that improves cells'' survival against the environmental stresses. This study improves our understanding of NP ecological impacts under natural solar radiation and provides useful insights to application of photocatalytic-NPs for bacterial disinfection. [Copyright &y& Elsevier]

Abstract: Spanish dry-cured ham is an uncooked meat product highly appreciated due to its characteristics flavour. In this study, we examined the accuracy of biochemical tests and 16S rDNA sequencing in the identification of 56 staphylococcal strains isolated during industrial Spanish dry-cured ham processes. Important differences were observed comparing genotypic and phenotypic data. Staphylococcus xylosus was the prevalent species identified by biochemical methods (87.5%), however, sequencing of the 16S rDNA resulted in an unambiguous identification of Staphylococcus equorum (73.2%) and Staphylococcus vitulinus (8.9%) strains. Reliable identification of meat staphylococci, mainly among S. xylosus and S. equorum strains could be also achieved by means of recA gene sequence comparison. Two degenerate primers previously described for lactic acid bacteria were used to amplify an internal fragment of the recA gene. This fragment was amplified from twelve staphylococcal type strains representing frequent meat species. The results indicated that recA sequencing is an adequate method to discriminate among meat staphylococci. In addition, S. xylosus and S. equorum strains could be more accurately discriminated by recA sequencing than 16S rDNA or sodA sequencing. The S. equorum sequence diversity showed at the intra-species level by recA gene sequencing confirmed the high heterogeneity described among S. equorum strains. [Copyright &y& Elsevier]

Abstract: The use of an artificial consortium composed of selected hydrogen-producing species, instead of a natural anaerobic sludge, has been proposed for biohydrogen production. In order to monitor such a consortium composed of different Clostridium spp., new protocols were tested for two different assays, FISH and qPCR. New species-specific FISH probes and qPCR primer sets were developed and optimised for three strains: Clostridium butyricum, Clostridium felsineum and Clostridium pasteurianum, that were used in a consortium. Application of a fast two-step FISH protocol, with pre-treatment step at 90 °C for 5 min and a subsequent hybridisation step at higher temperature (55 °C) for 20 min resulted in a much shorter analytical time compared to the standard FISH procedure (46 °C for 2–3 h) and gave a high hybridisation performance. Moreover, to accurately quantify each microorganism by qPCR assay, two innovations were tested: the direct use of cell lysates (omitting the DNA extraction step) and the use of two alternative molecular markers, recA and gyrA. These markers are present in single copies in the genome, whereas there are multiple copies of the ribosomal operons. This resulted in the development of accurate, reliable and fast FISH and qPCR assays for routine monitoring of the dynamics of artificial hydrogen-producing microbial consortia. Moreover, both techniques can be easily adapted to new Clostridium strains. [Copyright &y& Elsevier]

Abstract: One hundred and fifty-six strains isolated from corn (Zea mays L.), forage paddy rice (Oryza sativa L.), sorghum (Sorghum bicolor L.) and alfalfa (Medicago sativa L.) silages prepared on dairy farms were screened, of which 110 isolates were considered to be lactic acid bacteria (LAB) according to their Gram-positive and catalase-negative characteristics and, mainly, the lactic acid metabolic products. These isolates were divided into eight groups (A–H) based on the following properties: morphological and biochemical characteristics, γ-aminobutyric acid production capacity, and 16S rRNA gene sequences. They were identified as Weissella cibaria (36.4%), Weissella confusa (9.1%), Leuconostoc citreum (5.3%), Leuconostoc lactis (4.9%), Leuconostoc pseudomesenteroides (8.0%), Lactococcus lactis subsp. lactis (4.5%), Lactobacillus paraplantarum (4.5%) and Lactobacillus plantarum (27.3%). W. cibaria and W. confusa were mainly present in corn silages, and L. plantarum was dominant on sorghum and forage paddy rice silages, while L. pseudomesenteroides, L. plantarum and L. paraplantarum were the dominant species in alfalfa silage. The corn, sorghum and forage paddy rice silages were well preserved with lower pH values and ammonia-N concentrations, but had higher lactic acid content, while the alfalfa silage had relatively poor quality with higher pH values and ammonia-N concentrations, and lower lactic acid content. The present study confirmed the diversity of LAB species inhabiting silages. It showed that the differing natural populations of LAB on these silages might influence fermentation quality. These results will enable future research on the relationship between LAB species and silage fermentation quality, and will enhance the screening of appropriate inoculants aimed at improving such quality. [Copyright &y& Elsevier]

Aims: To evaluate the usefulness of partial recA sequences for the identification of Aeromonas strains at the genotype level. Methods and Results: A partial recA sequence was obtained from 21 type or reference strains and 33 Aeromonas isolates, collected in the South of Switzerland from human, animal and aquatic environments. The 272 bp long recA fragments showed a mean interspecies divergence of 7·8% and allowed the classification of strains at genotype level. However, some discrepancies could be observed with other gene sequence based analyses in the classification of some strains. Conclusions: The 272 bp long recA fragment is a good molecular marker to infer taxonomy of members of the genus Aeromonas, even if the primers we chose for the amplification did not allow its direct sequencing. Significance and Impact of the Study: In the genus Aeromonas, nucleotide sequences of some protein-encoding genes have already been evaluated as molecular markers to be used in taxonomical and epidemiological researches. This study suggests the usefulness of a recA fragment as a further sequence to investigate for these purposes. [ABSTRACT FROM AUTHOR]

Abstract: Conventional phenotypic methods sometimes lead to misidentification of some heterofermentative wine lactobacilli such as Lactobacillus hilgardii, Lactobacillus buchneri, and Lactobacillus brevis. We establish the specificity of 16S rDNA sequencing in the differentiation of these species and in the rejection of the Lactobacillus vermiforme species name. Moreover, we succeeded in differentiating these heterofermentative species by means of recA gene sequence comparison. Short homologous regions were amplified by PCR with degenerate consensus primers, sequenced, and 280 bp were analysed and considered for the inference of phylogenetic trees. The phylogram obtained was coherent and clearly separated the three species. The recA gene sequence was a reliable and useful method that allowed a good discrimination among closely related species. The validity of the recA gene sequence, restriction fragment length polymorphism (RFLP) analysis of the PCR-amplified 16S–23S rDNA intergenic spacer region (ISR), and random amplified polymorphic DNA (RAPD) to study the L. hilgardii intraspecies heterogeneity was tested in five strains isolated from stuck wine fermentations at the same winery in the same vintage. The results indicated that L. hilgardii is a heterogeneous species. Since L. hilgardii is a malolactic species that can influence the final quality of the wine, the presence of oenological relevant genes, such as those involved in ethyl carbamate or biogenic amine production, was investigated. [Copyright &y& Elsevier]

Abstract: The application of real-time PCR targeting the multicopy 16S rRNA gene and the single copy recA gene was evaluated for the enumeration of bifidobacteria in 29 probiotic products claimed to contain these organisms. Both assays relied on the use of genus-specific primers and the non-specific SYBR Green I chemistry. For both applications, the calibration curve was constructed using the type strain of Bifidobacterium animalis subsp. lactis. Upon correction with a factor corresponding to the 16S rRNA gene copy number, both assays generally produced comparable enumeration results. Only in exceptional cases, differences between both gene targets were found in probiotic products containing low amounts of bifidobacteria in which case the quantification of the multicopy 16S rRNA gene turned out to be more sensitive than the recA-based assay. On the other hand, the use of the latter single copy gene in real-time PCR quantification offers the advantage that no prior knowledge of bacterial content is required when using genus-specific primers, since no correction for multiple gene copies has to be performed. Only 11 of the analysed products (38%), including one dairy based product and ten dried products, contained a minimal Bifidobacterium concentration of 106 CFU per ml or g of product. Depending on the application, both assays proved to be rapid and reproducible alternatives for culture-based detection and quantification of bifidobacteria in probiotic products. [Copyright &y& Elsevier]

Abstract: The aim of this study was to investigate the validity of recA gene as a molecular marker for the reliable discrimination and classification of dairy propionibacteria and the closely related species. Regions of the recA gene, varying in size between 613 and 677 nucleotides, were sequenced for Propionibacterium acidipropionici, P. cyclohexanicum, P. freudenreichii, P. jensenii, P. microaerophilum and P. thoenii using degenerate consensus primers constructed by aligning recA sequences of some actinobacteria. The 16S rRNA encoding genes for the type and reference strains of the species P. acidipropionici, P. jensenii and P. thoenii were also sequenced to remove ambiguous positions present in the current database reports, such to improve the classification scheme of reference. As found for other bacterial species, recA sequences permitted a better distinction among the dairy propionibacteria considered than 16S rRNA gene. However, the topology of phylogenetic trees constructed on the recA gene regions sequenced and their putative translations appeared rather different and less statistically valid than the 16S rRNA gene tree. In addition, the possibility of designing PCR-based identification and detection tests on the new recA sequences was demonstrated by assessing specific amplification protocols for P. cyclohexanicum and P. microaerophilum. [Copyright &y& Elsevier]

Abstract: Most of group II introns are found in intergenes and CDSs with unknown functions, but not in housekeeping genes. In particular, no group II intron within the housekeeping recA gene has been reported either in eukaryotic genomes or in prokaryotic genomes. In this study, we found that the recA gene of the thermophilic Geobacillus kaustophilus genome is interrupted by a group II intron (Gk. Int1), and that Gk.Int1 can splice in temperatures above 70 °C in vivo. Here, we report the first prokaryotic group II intron to be found in a housekeeping gene, the characteristics of its self-splicing in vivo and in vitro, and our conclusion that the recA gene functions through the self-splicing of Gk.Int1. It is suggested that the amelioration of Gk.Int1 intron has occurred recently, and that it is still in the process of evolution to the recipient genome. [Copyright &y& Elsevier]

Abstract: As an initial step to understand the mobile nature of class II mercury resistance transposon TnMERI1, the effect of the recA gene on translocation of mini-TnMERI1 was evaluated. A higher transposition frequency in the LE392 strain (2.4±1.2×10−5) than in the recA-deficient DH1 strain (1.2±0.8×10−6) indicated participation of the recA gene in mini-TnMERI1 transposition. Introduction of the recA gene into the DH1 strain complemented the transposition frequency at the same level as in LE392 and confirmed participation of the recA gene in transposition. However, treatment of cells by stress agents, including irradiation of up to 3000Jm−2 UV doses, did not alter the transposition frequency and suggested independence of RecA from the SOS stress response. Further analysis of transconjugants indicated participation of RecA in the resolution of the cointegrate structure of the transposon. These results suggested that RecA is a constitutive cellular factor that increases translocation of mini-TnMERI1 and may participate in dissemination of TnMERI1-like transposons. [Copyright &y& Elsevier]

The 3.5 kb nucleotide fragment, including the recA gene and its downstream recX-like gene, has been isolated from a genomic library by dot-blot hybridization with the Mycobacterium smegmatis recA gene. The recA gene, consisting of 1047 base pairs (bp), encodes a polypeptide of 348 amino acids while the recX-like gene, consisting of 450 bp, encodes a shorter polypeptide of 149 amino acids. Both the deduced amino acid sequences of recA and recX resemble those of the recA and recX genes from other bacteria. The cloned Amycolatopsis mediterranei U32 recA gene conferred partial resistance to ethyl methane sulfonate when expressed in E. coli with the lacZ promoter. [ABSTRACT FROM AUTHOR]

Using primer-extension analysis we identified two transcription start sites for the recA gene in Streptomyces rimosus. A longer, weak transcript is initiated from the distal SEP promoter that contains a Cheo box like sequence: GAAC-N4-ATTC. However, the major start site of transcription is a G at position −36 and this shorter transcript significantly increases in response to DNA damage by UV-light. The −35 box (TTGTCA) and −10 box (TAGCGT) of the strong recA promoter are only 11 bp apart and this proximal promoter is almost identical to the strong, DNA damage-inducible promoter of Mycobacterium tuberculosis recA gene. We inspected the Streptomyces coelicolor database and found this type of promoter in the upstream regions of many (potentially) UV-inducible genes as well as some other genes/ORFs. Moreover, the DNA sequence between the predicted −35 and −10 boxes is also partially conserved. The consensus sequence for this new type of promoter in Streptomyces is: TTGTCAGTGGC-N6-TAGggT. [Copyright &y& Elsevier]

Cutibacterium modestum is a new species coined in 2020 as the fifth species of genus Cutibacterium, which includes Cutibacterium acnes. The species is predicted as a minor but common member of skin microbiome and includes a group tentatively named as "Propionibacterium humerusii". The description of the species has been provided only with a single strain. To establish the characteristics of C. modestum and search for possible disease-related subtypes, we investigated the biochemical characteristics of eight live strains and performed in silico comparison of nine genomes. The common features, which included the morphology of Gram-stain positive short rods, the negativity of phenylalanine arylamidase, and several unique MALDI-TOF MS spectral peaks, were considered useful in laboratory identification. Pairwise comparisons of the genomes by in silico DNA–DNA hybridization showed similarity values of 98.1% or larger, which were far higher than the subspecies cutoff of 79–80%. The 16S rRNA gene sequences of thirteen isolates and genomes were identical. Their recA gene sequences were identical except for two strains, HM-510 (HL037PA2) and Marseille-P5998, which showed unique one-nucleotide polymorphisms. The biochemical features using API kits were slightly different among the isolates but far closer than those of the nearest other species, C. acnes and Cutibacterium namnetense. Spectra of MALDI-TOF mass spectrometry showed slight differences in the presence of m/z 10,512 (10 kD chaperonin GroS) and three other peaks, further clustering the eight isolates into three subtypes. These results indicated that these isolates did not separate to form subspecies-level clusters, but subtyping is possible by using recA gene sequences or MALDI-TOF mass spectrometry spectra. Moreover, this work has confirmed that a group "P. humerusii" is included in C. modestum. [ABSTRACT FROM AUTHOR]

The recA gene of Aquaspirillum magnetotacticum has been isolated from a genomic library and introduced into a recA mutant strain of Escherichia coli K12. The cloned gene complemented both the recombination and DNA repair deficiency of the host and its protein product promoted the proteolytic cleavage of the LexA protein. A protein whose molecular weight is similar to that of the RecA protein of E. coli was associated with the cloned sequence. [ABSTRACT FROM AUTHOR]

The DNA sequences of the recA gene from 25 strains of bacteria are known. The evolution of these recA gene sequences, and of the derived RecA protein sequences, is examined, with special reference to the effect of variations in genomic G + C content. From the aligned RecA protein sequences, phylogenetic trees have been drawn using both distance matrix and maximum parsimony methods. There is a broad concordance between these trees and those derived from other data (largely 16S ribosomal RNA sequences). There is a fair degree of certainty in the relationships among the 'Purple' or Proteobacteria, but the branching pattern between higher taxa within the eubacteria cannot be reliably resolved with these data. [ABSTRACT FROM AUTHOR]

Foodborne pathogens represent a major public health concern. In this respect, Acinetobacter baumannii is emerging as multi-drug resistant food pathogen. Screening of A. baumannii in food is a lengthy process. The aim of this study is to develop a fast and efficient protocol for detecting A. baumannii as well as other potential pathogens in fresh food. A. baumannii was collected from strawberry samples (n = 93) and were identified by DNA sequencing and polymerase chain reaction (PCR; recA). Hydrophobic magnetic nanoparticles (OA-MNP) were synthesized and tested for capturing A. baumannii and other bacteria from broth medium. Their ability to immobilize bacteria was assessed and the accuracy of PCR performed on immobilized bacteria was compared against control. A. baumannii (n = 14) was isolated form fresh produce as confirmed by sequencing and PCR. OA-MNPs were able to capture A. baumannii as well as other bacteria from broth medium. Intact DNA was extracted from immobilized bacteria and was successfully recruited for subsequent PCR. In conclusion, OA-MNP can be used for immobilizing food pathogens from broth medium. PCR can be performed using DNA extracted from immobilized bacteria for identification. The proposed analysis procedure may shorten the time required for detection of bacterial contamination in food. Image 1 • The potential of fresh strawberries were investigated as a reservoir for the antibiotic resistant A. baumanii. • The molecular and sequencing confirmation of A. baumanii were performed. • Modified magnetic nanoparticles were synthesized and recruited as a dispersed solid phase extraction material for bacteria. • The compatibility of magnetic nanoparticles with PCR was tested. [ABSTRACT FROM AUTHOR]