Heterogeneity of SOS response expression in clinical isolates of Escherichia coli influences adaptation to antimicrobial stress.

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.

Synergistic Effect of SOS Response and GATC Methylome Suppression on Antibiotic Stress Survival in Escherichia coli.

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 Δdam ΔrecA double mutant showed no growth or delayed growth compared to the control strain. In bactericidal terms, spot tests showed that the Δdam ΔrecA double mutant was more sensitive than the ΔrecA single mutant (about 10- to 102-fold) and the wild type (about 103- to 104-fold) in both susceptible and resistant genetic backgrounds. Differences between the wild type and the Δdam ΔrecA 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.

RecA inactivation as a strategy to reverse the heteroresistance phenomenon in clinical isolates of Escherichia coli.

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 ß-lactams. The molecular basis of heteroresistance was associated with an increase in copy number of plasmid-borne resistance genes (blaTEM-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.

Burkholderia vietnamiensis causing infections in noncystic fibrosis patients in a tertiary care hospital in Mexico.

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.

Comparative analysis unravels genetic recombination events of Vibrio parahaemolyticus recA gene.

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.

Common Features and Intra-Species Variation of Cutibacterium modestum Strains, and Emended Description of the Species.

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.

Detection of Acinetobacter baumannii in fresh produce using modified magnetic nanoparticles and PCR.

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.

The molecular and phenotypic characterization of fructophilic lactic acid bacteria isolated from the guts of Apis mellifera L. derived from a Polish apiary.

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.

Clinical characteristics of bacteraemia caused by Burkholderia cepacia complex species and antimicrobial susceptibility of the isolates in a medical centre in Taiwan.

This study investigated the clinical characteristics and outcomes of bacteraemia due to Burkholderia cepacia complex (BCC) species among 54 patients without cystic fibrosis from January 2013 to February 2015. BCC isolates were identified to the species level by the Bruker Biotyper MALDI-TOF MS system and by sequencing analysis of the 16S rRNA and recA genes. Antimicrobial susceptibilities of the isolates were determined by the agar dilution method. Sequencing of the recA gene in the 54 blood isolates revealed 37 (68.5%) isolates of B. cenocepacia, 9 (16.7%) of B. cepacia, 4 (7.4%) of B. multivorans and one isolate each of B. arboris, B. pseudomultivorans, B. seminalis, and B. vietnamiensis. The overall performance of the Bruker Biotyper MALDI-TOF MS system for correctly identifying the 54 BCC isolates to the species level was 79.6%, which was better than that (16.7%) by 16S RNA sequencing analysis. Bacteraemic pneumonia (n = 23, 42.6%) and catheter-related bacteraemia (n = 21, 38.9%) were the most common types of infection. Higher rates of ceftazidime and meropenem resistance were found in B. cepacia isolates (33.3% and 22.2%, respectively) than in isolates of B. cenocepacia (21.6% and 10.8%, respectively) and other species (12.5% and 12.5%, respectively). Overall, the 30-day mortality rate was 38.9% (21/54). Bacteraemia caused by BCC species other than B. cenocepacia and B. cepacia (adjusted odds ratio [aOR] 20.005, P = 0.024) and high SOFA score (aOR 1.412, P = 0.003) were predictive of higher 30-day mortality. Different BCC species are associated with different outcomes of bacteraemia and exhibit different susceptibility patterns.

E. coli recA gene improves gene targeted homologous recombination in Mycoplasma hyorhinis.

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.

Natural Lactic Acid Bacteria Population and Silage Fermentation of Whole-crop Wheat.

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.

Performance of MALDI-ToF MS for species identification of Burkholderia cepacia complex clinical isolates.

We evaluated the performance of matrix-assisted laser desorption ionization-time of flight (MALDI-ToF) for identification of Bcc species compared with that of recA sequencing. MALDI-ToF was able of identifying 100% of Bcc isolates at the genus level, but 23.1% of Bcc isolates tested were not correctly identified at the species level. The misidentification occurred most frequently with Burkholderia contaminans (100%) and B. cepacia (33.3%).

Evaluación de los sistemas comerciales automatizados VITEK 2 y API 20NE para la identificación de organismos del complejo Burkholderia cepacia aislados de muestras clínicas / Evaluation of commercial systems VITEK 2 and API 20NE for identification of Burkholderia cepacia complex bacteria from clinical samples

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.

Molecular identification of Burkholderia cepacia complex and species distribution among cystic fibrosis patients seen at the reference center in southern Brazil / Identificação molecular do complexo Burkholderia cepacia e distribuição por espécie entre pacientes com fibrose cística em um centro de referência no sul do Brasil

Background: Burkholderia complex (Bcc) infections in cystic fibrosis (CF) patients are associated with decline in lung function and reduced survival. The potential transmissibility of Bcc among CF patients has been reported, indicating that strict segregation of CF patients with Bcc is crucial. Aims: To standardize the PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) assay in order to identify Bcc species and to establish the prevalence of Bcc species and their susceptibility profile among CF patients seen at the Hospital de Clínicas de Porto Alegre (HCPA). Methods: The classification of the clinical isolates recovered from respiratory tract specimens of CF patients as Bcc was achieved using the API-20NE® phenotypic commercial system. The identification of the Bcc species was performed using PCR-RFLP. The antimicrobial disc diffusion susceptibility testing was performed according to the CLSI (2006). Results: API-20NE® was able to identify Bcc isolates (244 specimens), such as B. cepacia, indicating that it was not able to distinguish among the Bcc species. The PCR-RFLP molecular method discriminated the eight reference Bcc species, thus validating the method for clinical isolates. Bcc prevalence determined by PCR-RFLP was 10.6% (26/244). The molecular analysis identified B. cenocepacia in 53.8% (14/26) of infected patients, B. multivorans in 15.4% (4/26), and B. vietnamiensis and B. ambifaria in 7.7% (2/26). The antibiotic resistance profile was variable among Bcc species. Conclusions: The PCR-RFLP method was validated for the identification of Bcc species. B. cenocepacia proved to be the most prevalent species among the CF patients seen at the HCPA.

Introdução: Infecções por bactérias do complexo Burkholderia cepacia (CBC) em pacientes com fibrose cística (FC) estão associadas a declínio da função pulmonar e diminuição da sobrevida. O potencial de transmissibilidade de CBC entre pacientes com FC é uma realidade, tornando-se importante a estrita segregação dos pacientes infectados.Objetivos: Padronizar a técnica de PCR-RFLP (reação em cadeia da polimerase seguida de clivagem com enzimas derestrição) para diferenciação das espécies de CBC e estabelecer a prevalência dessas espécies e seus perfis de sensibilidade em pacientes com FC atendidos no Hospital de Clínicas de Porto Alegre (HCPA). Métodos: A identificação dos isolados clínicos do trato respiratório de pacientes com FC como CBC foi feita pelo sistema deidentificação fenotípica comercial API-20NE®. A diferenciação das espécies de CBC foi realizada por PCR-RFLP, e o teste de suscetibilidade aos antimicrobianos por disco-difusão foi realizado de acordo com o CLSI (2006).Resultados: O sistema API-20NE® identificou todos os isolados do CBC (244 amostras) como B. cepacia, indicando claramente que não distingue as espécies do complexo. O método molecular de PCR-RFLP discriminou as oito espécies de referência de CBC, validando o método para isolados clínicos. A prevalência de CBC por PCR-RFLP foi de 10,6% (26/244).A análise molecular apontou B. cenocepacia colonizando em 53,8% (14/26) dos pacientes infectados, B. multivorans em 15,4% (4/26) e B. vietnamiensis e B. ambifaria em 7,7% (2/26). O perfil de resistência entre as espécies de CBC para os antibióticos testados foi variado. Conclusão: Foi validada a aplicação do método molecular PCR-RFLP para identificar espécies de CBC, e B. cenocepaciafoi a espécie mais prevalente entre os pacientes fibrocísticos atendidos no HCPA.