Ceftazidima/avibactam frente a otros antimicrobianos para el tratamiento de bacteriemias por Enterobacterales productores de carbapenemasa KPC: datos de la vida real en un hospital universitario de Argentina / Ceftazidime/avibactam versus other antibiotics for the treatment of KPC carbapenemase-producing Enterobacterales bacteremia: real-life data from a university hospital in Argentina

INTRODUCCIÓN: Las bacteriemias por Enterobacterales productores de carbapenemasa KPC (EPC-KPC) presentan una mortalidad elevada y opciones terapéuticas limitadas. OBJETIVOS: Describir y comparar la evolución de los pacientes con bacteriemia por EPC-KPC tratados con ceftazidima/avibactam (CA) frente a otros antimicrobianos (OA). PACIENTES Y MÉTODOS: Estudio prospectivo y retrospectivo de casos y controles. Se incluyeron pacientes adultos con bacteriemia por EPC-KPC, con una proporción entre casos tratados con CA y controles tratados con OA. de 1:2. Se analizaron variables clínicas, epidemiológicas y de evolución. RESULTADOS: Se incluyeron 48 pacientes (16 CA y 32 OA). Los casos se encontraban más frecuentemente neutropénicos (50 vs.16%, p = 0,012); asimismo, presentaron medianas de score de APACHE II más altas y de score de Pitt más bajas. El 65% de la cohorte total presentó un foco clínico y Klebsiellapneumoniae fue el microorganismo más frecuentemente aislado. Los casos recibieron una mayor proporción de tratamiento antimicrobiano empírico adecuado (81 vs. 53%, p = 0,05). La antibioterapia dirigida en casos y controles fue combinada en 38 y 91%, p = 0,009. Los casos presentaron menor mortalidad al día 7 y al día 30 relacionada a infección (0 vs. 22%, p = 0,04 y 0 vs. 34%, p = 0,008). Solo los controles desarrollaron shock, ingresaron a la unidad de cuidados intensivos y presentaron bacteriemia de brecha. CONCLUSIÓN: CA mostró beneficio clínico frente a OA para el tratamiento de pacientes con bacteriemia por EPC-KPC.

BACKGROUND: KPC-producing Enterobacterales bacteremia (KPCCPE) is associated with a high mortality rate and limited therapeutic options. AIM: To describe and compare the outcome of patients with KPC-CPE bacteremia treated with ceftazidime/avibactam (CA) versus other antibiotics (OA). METHODS: Prospective and retrospective cases and control study performed in adult patients with KPC-CPE bacteremia, with a 1:2 ratio between cases treated with CA. and controls treated with OA. Clinical, epidemiological, and outcome variables were analyzed. RESULTS: Forty-eight patients (16 CA and 32 OA) were included. Cases were more frequently neutropenic (50 vs. 16%, p = 0.012), presented higher median APACHE II score and lower Pitt score. Of the total cohort, 65% had a clinical source, and Klebsiella pneumoniae was the most frequently isolated microorganism. Cases received more adequate empirical antibiotic treatment (81 vs. 53%, p = 0.05). Targeted antibiotic therapy in cases and controls was combined in 38 and 91%, p = 0.009. Cases had a lower 7-day mortality and 30-day infection-related mortality (0 vs. 22%, p = 0.04 and 0 vs. 34%, p = 0.008). Only controls developed shock, were admitted to the intensive care unit, and had breakthrough bacteremia. CONCLUSION: CA. showed clinical benefit over OA in the treatment of patients with EPC-KPC bacteremia.

Clinical distribution and drug resistance characteristics of carbapenem-resistant Klebsiella pneumoniae / 中华预防医学杂志

To explore the clinical distribution and drug resistance characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP), in order to provide reference for the prevention and treatment of CRKP infection. Retrospective analysis was performed on 510 clinical isolates of CRKP from January 2017 to December 2021, and strain identification and drug sensitivity tests were conducted by MALDI-TOF mass spectrometer and VITEK-2 Compact microbial drug sensitivity analyzer. The carbapenemase phenotype of CRKP strain was detected by carbapenemase inhibitor enhancement test. The CRKP strain was further categorized by immunochromogenic method and polymerase chain reaction (PCR) was used for gene detection. The results showed that 302 strains (59.2%) were derived from sputum, 127 strains (24.9%) from urine and 47 strains (9.2%) from blood. 231 (45.3%) were mainly distributed in intensive care, followed by 108 (21.2%) in respiratory medicine and 79 (15.5%) in neurosurgery. Drug susceptibility test result shows that the resistant rate of tigecycline increased from 1.0% in 2017 to 10.1% in 2021, the difference was statistically significant (χ2=14.444,P<0.05). The results of carbapenemase inhibitor enhancement test showed that 461 carbapenemase strains (90.4%) of 510 CRKP strains, including 450 serinase strains (88.2%), 9 metalloenzyme strains (1.8%), and 2 strains (0.4%) produced both serine and metalloenzyme. 49 strains (9.6%) did not produce enzymes. Further typing by immunochromogenic assay showed that 461 CRKP strains were KPC 450 (97.6%) and IMP 2 (0.4%). 7 NDM (1.5%); 2 strains of KPC+NDM (0.4%); PCR results were as follows: 450 strains of blaKPC (97.6%), 2 strains of blaIMP (0.4%), 7 strains of blaNDM (1.5%), and 2 strains of blaKPC+NDM (0.4%). In conclusion, CRKP strains mainly originated from sputum specimens and distributed in intensive care department, and the drug resistance characteristics were mainly KPC type in carbapenemase production. Clinical microbiology laboratory should strengthen the monitoring of CRKP strains, so as to provide reference for preventing CRKP infection and reducing the production of bacterial drug resistance.

Study on the features of Clostridioides difficile infection among diarrhea patients in Kunming from 2018 to 2020 / 中华流行病学杂志

Objective: We analyze the characteristics of Clostridioides difficile (C. difficile) infection among diarrhea patients in Kunming from 2018 to 2020 and provide evidence for follow-up surveillance and prevention. Methods: A total of 388 fecal samples of diarrhea patients from four sentinel hospitals in Yunnan Province from 2018 to 2020 were collected. Real-time quantitative PCR was used to detect the fecal toxin genes of C. difficile. The positive fecal samples isolated the bacteria, and isolates were identified by mass spectrometry. The genomic DNA of the strains was extracted for multi-locus sequence typing (MLST). The fecal toxin, strain isolation, and clinical patient characteristics, including co-infection with other pathogens, were analyzed. Results: Among the 388 fecal samples, 47 samples with positive reference genes of C. difficile were positive, with a total positive rate of 12.11%. There were 4 (8.51%) non-toxigenic and 43 (91.49%) toxigenic ones. A total of 18 strains C. difficile were isolated from 47 positive specimens, and the isolation rate of positive specimens was 38.30%. Among them, 14 strains were positive for tcdA, tcdB, tcdC, tcdR, and tcdE. All 18 strains of C. difficile were negative for binary toxins. The MLST results showed 10 sequence types (ST), including 5 strains of ST37, accounting for 27.78%; 2 strains of ST129, ST3, ST54, and ST2, respectively; and 1 strain of ST35, ST532, ST48, ST27, and ST39, respectively. Fecal toxin gene positive (tcdB+) results were statistically associated with the patient's age group and with or without fever before the visit; positive isolates were only statistically associated with the patient's age group. In addition, some C. difficile patients have co-infection with other diarrhea-related viruses. Conclusions: The infection of C. difficile in diarrhea patients in Kunming is mostly toxigenic strains, and the high diversity of strains was identified using the MLST method. Therefore, the surveillance and prevention of C. difficile should be strengthened.

Molecular epidemiology of clinical isolation of carbapenem-resistant Enterobacterales and application of carbapenemase inhibitor enhancement test / 中南大学学报(医学版)

OBJECTIVES@#The prevalence of carbapenem-resistant Enterobacterales (CRE) presents a significant challenge in clinical anti-infective treatment. This study aims to investigate drug resistance and the molecular epidemiological characteristics of CRE in our area. Additionally, we seek to evaluate practicality of utilizing carbapenemase inhibitor enhancement test in clinical laboratory.@*METHODS@#Non-repeated CREs isolated from clinical specimens at Xiangya Hospital, Central South University, were collected. Minimum inhibitory concentration (MIC) combined with Kirby-Bauer (KB) assay was used to detect the drug susceptibility of the strains, and 13 carbapenemase-producing genes were detected by PCR. The phenotype of 126 strains of carbapenemase-producing Enterobacterales identified by PCR was detected by the carbapenemase inhibitor enhancement test to understand the agreement between the method and the gold standard PCR results.@*RESULTS@#Among 704 CRE strains examined, we observed significant drug resistance in 501 strains dentified as carbapenemase-producing Enterobacterales (CPE). Klebsiella pneumoniae was the predominant CPE strain, followed by Enterobacter cloacae and Escherichia coli. A total of 9 carbapenemase types were detected, including Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), Verona integron- encoded metallo-β-lactamases (VIM), imipenemase (IMP), oxacillinase-48 (OXA-48), and rare imipenem-hydrolyzing β-lactamase (IMI), adelaide imipenemase (AIM), Bicêtre carbapenemase (BIC), and guiana extended-spectrum β-lactamase (GES). The detection rate of KPC serine carbapenemase was 61.7% (309/501). The carbapenemase inhibitor enhancement test exhibited a 100% consistency rate for the strains producing Class A serine carbapenemase and/or Class B metallo-β-lactamases.@*CONCLUSIONS@#CRE strains in Changsha, Hunan, China, are wide distribution and exhibit carbapenemase production. The main mechanism of carbapenem resistance in these bacterias is predominatly attributed to the production of KPC serine carbapenemase. The presence of GES and IMI genes carried by Enterobacterales has been detected for the first time in this region. The carbapenemase inhibitor enhancement test has been proven to be an accurate method for detecting CRE producing Class A serine carbapenemase and/or Class B metallo-β-lactamases. This method offers simpicity of operation and ease of results interpretation, making it weel-suited meeting the clinical microbiology laboratory's reguirements for the detection of serine carbapenemase and metallo-β-lactamases.

The fliL gene significantly affects the motility and sporulation abilities of Clostridioides difficile / 生物工程学报

Flagella are the main motility structure of Clostridioides difficile that affects the adhesion, colonization, and virulence of C. difficile in the human gastrointestinal tract. The FliL protein is a single transmembrane protein bound to the flagellar matrix. This study aimed to investigate the effect of the FliL encoding gene flagellar basal body-associated FliL family protein (fliL) on the phenotype of C. difficile. The fliL gene deletion mutant (ΔfliL) and its corresponding complementary strains (: : fliL) were constructed using allele-coupled exchange (ACE) and the standard molecular clone method. The differences in physiological properties such as growth profile, antibiotic sensitivity, pH resistance, motility, and spore production ability between the mutant and wild-type strains (CD630) were investigated. The ΔfliL mutant and the : : fliL complementary strain were successfully constructed. After comparing the phenotypes of strains CD630, ΔfliL, and : : fliL, the results showed that the growth rate and maximum biomass of ΔfliL mutant decreased than that of CD630. The ΔfliL mutant showed increased sensitivity to amoxicillin, ampicillin, and norfloxacin. Its sensitivity to kanamycin and tetracycline antibiotics decreased, and the antibiotic sensitivity partially returned to the level of CD630 strain in the : : fliL strain. Moreover, the motility was significantly reduced in the ΔfliL mutant. Interestingly, the motility of the : : fliL strain significantly increased even when compared to that of the CD630 strain. Furthermore, the pH tolerance of the ΔfliL mutant significantly increased or decreased at pH 5 or 9, respectively. Finally, the sporulation ability of ΔfliL mutant reduced considerably compared to the CD630 strain and recovered in the : : fliL strain. We conclude that the deletion of the fliL gene significantly reduced the swimming motility of C. difficile, suggesting that the fliL gene is essential for the motility of C. difficile. The fliL gene deletion significantly reduced spore production, cell growth rate, tolerance to different antibiotics, acidity, and alkalinity environments of C. difficile. These physiological characteristics are closely related to the survival advantage in the host intestine, which is correlated with its pathogenicity. Thus, we suggested that the function of the fliL gene is closely related to its motility, colonization, environmental tolerance, and spore production ability, which consequently affects the pathogenicity of C. difficile.

Genetic and Phenotypic Variation of Campylobacter jejuni NCTC11168 Caused by flhA Mutation during Laboratory Passage / 生物医学与环境科学（英文）

OBJECTIVE@#Campylobacter jejuni NCTC11168 is commonly used as a standard strain for flagellar biosynthesis research. In this report, two distinguished phenotypic isolates (CJ1Z, flhA mutant strain, lawn; CJ2S, flhA complemented strain, normal colony) appeared during laboratory passages for NCTC11168.@*METHODS@#Phenotypic assessments, including motility plates, transmission electron microscopy, biofilm formation assay, autoagglutination assay, and genome re-sequencing for these two isolates (CJ1Z, flhA mutant strain; CJ2S, flhA complemented strain) were carried out in this study.@*RESULTS@#Transmission electron microscopy revealed that the flagellum was lost in CJ1Z. Phenotypic assessments and genome sequencing of the two isolates were performed in this study. The capacity for biofilm formation, colony auto-agglutination, and isolate motility was reduced in the mutant CJ1Z. Comparative genomic analysis indicated a unique native nucleotide insertion in flhA (nt, 2154) that caused the I719Y and I720Y mutations and early truncation in flhA.@*CONCLUSION@#FlhA has been found to influence the expression of flagella in C. jejuni. To the best of our knowledge, this is the first study to describe the function of the C-terminal of this protein.

Efficient production of L-asparaginase in Bacillus licheniformis by optimizing expression elements and host / 生物工程学报

L-asparaginase (L-ASN) is widely applied in the treatment of malignant tumor and low-acrylamide food production, however, the low expression level hampers its application. Heterologous expression is an effective strategy to increase the expression level of target enzymes, and Bacillus is generally used as the host for efficient production of enzymes. In this study, the expression level of L-asparaginase in Bacillus was enhanced through optimization of expression element and host. Firstly, five signal peptides (SPSacC, SPAmyL, SPAprE, SPYwbN and SPWapA) were screened, among which SPSacC showed the best performance, reaching an activity of 157.61 U/mL. Subsequently, four strong promoters (P43, PykzA-P43, PUbay and PbacA) from Bacillus were screened, and tandem promoter PykzA-P43 showed the highest yield of L-asparaginase, which was 52.94% higher than that of control strain. Finally, three Bacillus expression hosts (B. licheniformis Δ0F3 and BL10, B. subtilis WB800) were investigated, and the maximum L-asparaginase activity, 438.3 U/mL, was reached by B. licheniformis BL10, which was an 81.83% increase compared with that of the control. This is also the highest level of L-asparaginase in shake flask reported to date. Taken together, this study constructed a B. licheniformis strain BL10/PykzA-P43-SPSacC-ansZ capable of efficiently producing L-asparaginase, which laid the foundation for industrial production of L-asparaginase.

Targeted innovative design of Bt Cry toxin insecticidal mimics / 生物工程学报

Bt Cry toxin is the mostly studied and widely used biological insect resistance protein, which plays a leading role in the green control of agricultural pests worldwide. However, with the wide application of its preparations and transgenic insecticidal crops, the resistance to target pests and potential ecological risks induced by the drive are increasingly prominent and attracting much attention. The researchers seek to explore new insecticidal protein materials that can simulate the insecticidal function of Bt Cry toxin. This will help to escort the sustainable and healthy production of crops, and relieve the pressure of target pests' resistance to Bt Cry toxin to a certain extent. In recent years, the author's team has proposed that Ab2β anti-idiotype antibody has the property of mimicking antigen structure and function based on the "Immune network theory" of antibody. With the help of phage display antibody library and specific antibody high-throughput screening and identification technology, Bt Cry toxin antibody was designed as the coating target antigen, and a series of Ab2β anti-idiotype antibodies (namely Bt Cry toxin insecticidal mimics) were screened from the phage antibody library. Among them, the lethality of Bt Cry toxin insecticidal mimics with the strongest activity was close to 80% of the corresponding original Bt Cry toxin, showing great promise for the targeted design of Bt Cry toxin insecticidal mimics. This paper systematically summarized the theoretical basis, technical conditions, research status, and discussed the development trend of relevant technologies and how to promote the application of existing achievements, aiming to facilitate the research and development of green insect-resistant materials.

Prokaryotic expression and biological activities of the hemolysin BL subunit of a pathogenic Bacillus cereus of cattle origin / 生物工程学报

Bacillus cereus belongs to Gram-positive bacteria, which is widely distributed in nature and shows certain pathogenicity. Different B. cereus strains carry different subsets of virulence factors, which directly determine the difference in their pathogenicity. It is therefore important to study the distribution of virulence factors and the biological activity of specific toxins for precise prevention and control of B. cereus infection. In this study, the hemolysin BL triayl was expressed, purified, and characterized. The results showed that the bovine pathogenic B. cereus hemolysin BL could be expressed and purified in the prokaryotic expression system, and the bovine pathogenic B. cereus hemolysin BL showed hemolysis, cytotoxicity, good immunogenicity and certain immune protection in mice. In this study, the recombinant expression of hemolysin BL triayl was achieved, and the biological activity of hemolysin BL of bovine pathogenic ceroid spore was investigated. This study may facilitate further investigating the pathogenic mechanism of B. cereus hemolysin BL and developing a detection method for bovine pathogenic B. cereus disease.

Visualization method of type Ⅳ pili and its application in the study of pili function / 生物工程学报

As an important protein structure on the surface of bacteria, type Ⅳ pili (TFP) is the sensing and moving organ of bacteria. It plays a variety of roles in bacterial physiology, cell adhesion, host cell invasion, DNA uptake, protein secretion, biofilm formation, cell movement and electron transmission. With the rapid development of research methods, technical equipment and pili visualization tools, increasing number of studies have revealed various functions of pili in cellular activities, which greatly facilitated the microbial single cell research. This review focuses on the pili visualization method and its application in the functional research of TFP, providing ideas for the research and application of TFP in biology, medicine and ecology.

Protein Containing the GGDEF Domain Affects Motility and Biofilm Formation in Vibrio cholerae and is Negatively Regulated by Fur and HapR / 生物医学与环境科学（英文）

OBJECTIVE@#This study aimed to investigate whether the VCA0560 gene acts as an active diguanylate cyclase (DGC) in Vibrio cholerae and how its transcription is regulated by Fur and HapR.@*METHODS@#The roles of VCA0560 was investigated by utilizing various phenotypic assays, including colony morphological characterization, crystal violet staining, Cyclic di-GMP (c-di-GMP) quantification, and swimming motility assay. The regulation of the VCA0560 gene by Fur and HapR was analyzed by luminescence assay, electrophoretic mobility shift assay, and DNase I footprinting.@*RESULTS@#VCA0560 gene mutation did not affect biofilm formation, motility, and c-di-GMP synthesis in V. cholerae, and its overexpression remarkably enhanced biofilm formation and intracellular c-di-GMP level but reduced motility capacity. The transcription of the VCA0560 gene was directly repressed by Fur and the master quorum sensing regulator HapR.@*CONCLUSION@#Overexpressed VCA0560 functions as an active DGC in V. cholerae, and its transcription is repressed by Fur and HapR.

Impact of nitrogen and phosphorus fertilizers on Cry1Ac protein contents in transgenic cotton / Impacto de fertilizantes de nitrogênio e fósforo nos conteúdos de proteína Cry1Ac em algodão transgênico

Abstract Application of different fertilizers to check the efficiency of expression of Bt (Bacillus thuringiensis) gene in one of the leading commercialized crops (cotton) against Lepidopteran species is of great concern. The expression of Cry protein level can be controlled by the improvement of nutrients levels. Therefore, the myth of response of Cry toxin to different combinations of NP fertilizers was explored in three Bt cotton cultivars. Combinations include three levels of nitrogen and three levels of phosphorus fertilizers. Immunostrips and Cry gene(s) specific primer based PCR (Polymerase Chain Reaction) analysis were used for the presence of Bt gene that unveiled the presence of Cry1Ac gene only. Further, the ELISA (enzyme-linked immunosorbent assay) kit was used to quantify the expression of Cry1Ac protein. Under various NP fertilizers rates, the level of toxin protein exhibited highly significant differences. The highest toxin level mean was found to be 2.3740 and 2.1732 µg/g under the treatment of N150P75 kg ha-1 combination while the lowest toxin level mean was found to be 0.9158 and 0.7641 µg/g at the N50P25 kg ha-1 level at 80 and 120 DAS (Days After Sowing), respectively. It was concluded from the research that the usage of NP fertilizers has a positive relation with the expression of Cry1Ac toxin in Bt cotton. We recommend using the N150P50 kg ha-1 level as the most economical and practicable fertilizer instead of the standard dose N100P50 kg ha-1 to get the desired level of Cry1Ac level for long lasting plant resistance (<1.5). The revised dose of fertilizer may help farmers to avoid the cross-resistance development in contradiction of insect pests.

Resumo A aplicação de diferentes fertilizantes para verificar a eficiência da expressão do gene Bt (Bacillus thuringiensis) em uma das principais culturas comercializadas (algodão) contra espécies de lepidópteros é uma grande preocupação. A expressão do nível de proteína Cry pode ser controlada pela melhoria dos níveis de nutrientes. Portanto, o mito da resposta da toxina Cry a diferentes combinações de fertilizantes NP foi explorado em três cultivares de algodão Bt. As combinações incluem três níveis de nitrogênio e três níveis de fertilizantes de fósforo. A análise de PCR (reação em cadeia da polimerase) específica para o gene (s) Immunostrips e Cry (s) foi usada para a presença do gene Bt que revelou a presença do gene Cry1Ac apenas. Além disso, o kit ELISA (ensaio de imunoabsorção enzimática) foi usado para quantificar a expressão da proteína Cry1Ac. Sob várias taxas de fertilizantes NP, o nível de proteína de toxina exibiu diferenças altamente significativas. A média do nível mais alto de toxina foi de 2,3740 e 2,1732 µg / g sob o tratamento da combinação N150P75 kg ha-1, enquanto a média do nível mais baixo de toxina foi de 0,9158 e 0,7641 µg / g no nível de N50P25 kg ha-1 em 80 e 120 DAS (dias após a semeadura), respectivamente. Concluiu-se com a pesquisa que o uso de fertilizantes NP tem relação positiva com a expressão da toxina Cry1Ac no algodão Bt. Recomendamos o uso do nível de N150P50 kg ha-1 como o fertilizante mais econômico e praticável em vez da dose padrão N100P50 kg ha-1 para obter o nível desejado de nível de Cry1Ac para resistência de planta de longa duração (<1,5). A dose revisada de fertilizante pode ajudar os agricultores a evitar o desenvolvimento de resistência cruzada em contradição com as pragas de insetos.

Primeros aislados de Enterobacter cloacae complex co-productores de KPC y NDM en un hospital de segundo nivel en la Cuidad de Panamá / First isolates of Enterobacter cloacae complex co-producing KPC and NDM in a second level hospital in City of Panama

Resumen La aparición de Enterobacterales co-productores de dos o más carbapenemasas han despertado las alertas sanitarias en Latinoamérica. Las enterobacterias co-productoras de carbapenemasas KPC y NDM-1 son resistentes a casi todos los antibacterianos existentes. Panamá ha reportado la presencia de carbapenemasas KPC desde 2010 y NDM desde 2011; sin embargo, Enterobacterales con doble producción de carbapenemasas es un fenómeno reciente en nuestros hospitales. Presentamos los dos primeros aislados de Enterobacter cloacae complex co-productores de KPC y NDM, en un hospital de segundo nivel de la Ciudad de Panamá. El reforzamiento de los sistemas de vigilancia epidemiológica en los hospitales permite realizar una detección oportuna de estas nuevas combinaciones de mecanismos de resistencia; para así, implementar medidas de prevención y control de brotes.

Abstract Enterobacterales co-producing carbapenemases have awakened health alerts in Latin America. Carbapenemase-producing Enterobacterales harboring KPC and NDM-1 are resistant to almost all existing antibiotics. Panama reports KPC since 2010, and NDM since 2011, however, Enterobacterales with double carbapenemase production is new to our hospitals. We present the first two isolates of Enterobacter cloacae complex co-producing KPC and NDM, in a second level hospital in Panama City. Strengthening epidemiological surveillance systems in hospitals allows to carry out timely detection of these new combinations of resistance; to implement outbreak prevention and control measures.

Resistencia a colistina en aislados de Klebsiella pneumoniae resistente a carbapenémicos en un hospital pediátrico de Corrientes / Colistin resistance in carbapenem-resistant Klebsiella pneumoniae isolates from a pediatric hospital from Corrientes, Argentina

INTRODUCCIÓN: Existe un incremento de las infecciones por Klebsiella pneumoniae resistente a carbapenémicos (KPRC) en la población pediátrica y los datos epidemiológicos son limitados. OBJETIVOS: Conocer la frecuencia de KPRC en pacientes pediátricos, determinar la actividad in vitro de colistina y detectar el gen mcr-1 en dichos aislados. MATERIALES Y MÉTODOS: Se estudiaron 220 aislados de K. pneumoniae en un hospital pediátrico durante los años 2018 y 2019. La susceptibilidad antimicrobiana se determinó por microdilución en caldo según CLSI y EUCAST. Los genes blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-48 y mcr-1 se analizaron mediante reacción de polimerasa en cadena (RPC). RESULTADOS: El 9,5% (n: 21) de los aislados fueron caracterizados como KPRC, donde se observó una resistencia a colistina de 47,6% (10/21) con valores de CIM50 de 2 μg/mL y CIM90 de > 4 μg/mL. En todos los aislados de KPRC se caracterizó el gen blaKPC y no se detectó el gen mcr-1. El perfil de resistencia observado en otros antimicrobianos fue el siguiente: gentamicina 100% (n: 21), ciprofloxacina 100% (n: 21), cotrimoxazol 100% (n: 21) y amikacina 19% (n: 4). Se observó 100% de sensibilidad a tigeciclina y ceftazidima/avibactam. CONCLUSIÓN: Este estudio demuestra un valor significativo de la resistencia a colistina en comparación a ceftazidima/avibactam y tigeciclina.

BACKGROUND: There is an increase of carbapenem-resistant Klebsiella pneumoniae (CRKP) infections in the pediatric population and epidemiological data are limited. Aim: To calculate the frequency of CRKP in pediatric patients, to determine the in vitro activity of colistin and to detect the presence of mcr-1 gene in said isolates. METHODS: 220 isolates of K. pneumoniae were studied in a pediatric hospital between January 2018 and December 2019. Antimicrobial susceptibility was determined by microdilution in broth according to guidelines of CLSI and EUCAST. The genes blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-48 and mcr-1 were detected by polymerase chain reaction (PCR). RESULTS: 9.5% (n: 21) of the isolates were characterized as CRKP, where was observed a resistance to colistin of 47.6% (10/21) with values of MIC50 of 2 μg/mL and MIC90 of ≥ 4 μg/mL. In 100% of CRKP strains the blaKPC gene was detected and the mcr-1 gene was not found. The resistance profile to other antimicrobials was as follow: gentamicin 100% (n: 21), trimethoprim/sulfamethoxazole 100% (n: 21), ciprofloxacin 100% (n: 21), amikacin 19% (n: 4). All of the isolates were sensitive to ceftazidime/avibactam and tigecycline. CONCLUSION: This study demonstrates a significant value of resistance to colistin in pediatric patients compared to other last line antimicrobial such as ceftazidime/avibactam and tigecycline.

Transcriptomic analysis of the ΔPaLoc mutant of Clostridioides difficile and verification of its toxicity / 中华预防医学杂志

Objective: Comparative analyses of wild-type Clostridioides difficile 630 (Cd630) strain and pathogenicity locus (PaLoc) knockout mutant (ΔPaLoc) by using RNA-seq technology. Analysis of differential expression of Cd630 wild-type strain and ΔPaLoc mutant strain and measurement of its cellular virulence changes. Lay the foundation for the construction of an toxin-attenuated vaccine strain against Clostridioides difficile. Methods: Analysis of Cd630 and ΔPaLoc mutant strains using high-throughput sequencing (RNA-seq). Clustering differentially expressed genes and screening differentially expressed genes by DESeq software. Further analysis of differential genes using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Finally, cytotoxicity assays of ΔPaLoc and Cd630 strains were performed in the African monkey kidney epithelial cell (Vero) and the human colonic cell (Caco-2) lines. Results: The transcriptome data showed that the ΔPaLoc mutant toxin genes tcdA and tcdB were not transcribed. Compared to the wild-type strain, CD630_36010, CD630_020910,CD630_02080 and cel genes upregulated 17.92,11.40,8.93 and 7.55 fold, respectively. Whereas the hom2 (high serine dehydrogenase), the CD630_15810 (spore-forming protein), CD630_23230 (zinc-binding dehydrogenase) and CD630_23240 (galactitol 1-phosphate 5-dehydrogenase) genes were down-regulated by 0.06, 0.075, 0.133 and 0.183 fold, respectively. The GO and KEGG enrichment analyses showed that the differentially transcribed genes in ΔPaLoc were enriched in the density-sensing system, ABC transport system, two-component system, phosphotransferase (PTS) system, and sugar metabolism pathway, as well as vancomycin resistance-related pathways. Cytotoxicity assays showed that the ΔPaLoc mutant strain lost its virulence to Vero and Caco-2 cells compared to the wild-type Cd630 strain. Conclusion: Transcriptional sequencing analysis of the Cd630 and ΔPaLoc mutant strains showed that the toxin genes were not transcribed. Those other differential genes could provide a reference for further studies on the physiological and biochemical properties of the ΔPaLoc mutant strain. Cytotoxicity assays confirmed that the ΔPaLoc mutant lost virulence to Vero and Caco-2 cells, thus laying the foundation for constructing an toxin-attenuated vaccine strain against C. difficile.

Heparin-binding hemagglutinin as a composition antigen of tuberculosis vaccine exerts protective immune effects by inducing IL-17 / 中华预防医学杂志

To explore the protective immune effect induced by mucosal delivery heparin-binding hemagglutinin (HBHA)-a candidate vaccine antigen of Mycobacterium tuberculosis. Female C57BL/6 mice were between 6 and 8 weeks of age before experimental use. Thirty mice received different immunization strategies and were randomly divided into the control group, the early secreting antigen target-6 (ESAT-6) intranasal immunization group, the HBHA intranasal immunization group, the BCG priming PBS control group, or BCG priming HBHA boost group, 6 mice in each group. In order to analyzed the immune effect, the concentrations of plasma Interleukin-17A (IL-17A) and other cytokines were measured by ELISA. Quantitative real-time PCR analyses were performed to detect the relative quantity (RQ) mRNA of IL-17A in the lung. The lung tissue sections were stained to detect the formation of the tertiary lymphoid structures. The chemokines contributed to formation of the tertiary lymphoid structures were also measured. Flow cytometry was used to detect the frequency of Th1 and Th17 cells in the system. Sixty mice in the BCG priming PBS control group and the BCG priming HBHA boost group were sacrificed at different time points after infection to count the lung bacterial burden. The concentrations of plasma IL-17A and relative quantity of lung IL-17A mRNA were highest in the BCG priming HBHA boost group [(14.76±4.73) pg/mL,RQ (12.27±6.71)], which was significantly higher than the control group [(5.57±2.95) pg/mL,RQ (1.30±0.97)] (t=4.213, P<0.001; t=5.984, P<0.001), and also significantly higher than the BCG priming PBS control group [(6.81±2.18) pg/mL,RQ (1.44±1.16)] (t=3.646 P=0.001; t=6.185 P<0.001). Compared with the BCG priming PBS control group (0.38±0.38)% the frequency of spleen Th17 cells were also significantly increased (t=-0.280 , P=0.048) in the BCG-primary HBHA boost group (1.02±0.34)%. In addition, HBHA boosting could promote better formation of the tertiary lymphoid structures in the lung, and decrease the bacterial load on the early stage after BCG challenge. Collectively, mucosal delivery of HBHA can effectively enhance the protective effect after BCG vaccination, and it is a potential candidate vaccine component.

Perifosine inhibits biofilm formation of Pseudomonas aeruginosa by interacting with PqsE protein / 中华预防医学杂志

To explore the biofilm inhibitory efficacy of perifosine against Pseudomonas aeruginosa (P. aeruginos) and its mechanisms. Twenty-fourwell plate was used to form biofilms at the bottom and crystal violet staining was used to determine the biofilm inhibitory effects of perifosine against P. aeruginosa, the wells without perifosine was set as control group. Glass tubes combined with crystal violet staining was used to detect the gas-liqud interface related bioiflm inhibitory effects of perifosine, the wells without perifosine was set as control group. Time-growth curved was used to detect the effects of perifosine on the bacteial planktonic cells growth of P. aeruginosa, the wells without perifosine was set as control group. The interaction model between perifosine and PqsE was assessed by molecular docking assay. The inhibitory effects of perifosine on the catalytic activity of PqsE was determined by detection the production of thiols, the wells without perifosine was set as control group. Binding affinity between perifosine and PqsE was detected by plasma surface resonance. The biofims at the bottom of the microplates and air-liquid interface were effectively inhibited by perifosine at the concentration of 4-8 μg/ml. There was no influence of perifosine on the cells growth of P. aeruginosa. The resuts of molecular docking assay indicates that perifosine could interacted with PqsE with the docking score of -10.67 kcal/mol. Perifosine could inhibit the catalytic activity of PqsE in a dose-dependent manner. The binding affinity between perifosine and PqsE was comfirmed by plasma surface resonance with KD of 6.65×10-5mol/L. Perifosine could inhibited the biofilm formation of P. aeruginosa by interacting with PqsE.

Advances in receptor-mediated resistance mechanisms of Lepidopteran insects to Bacillus thuringiensis toxin / 生物工程学报

Bacillus thuringiensis is widely used as an insecticide which is safe and environmentally friendly to humans and animals. One of the important insecticidal mechanisms is the binding of Bt toxins to specific toxin receptors in insect midgut and forming a toxin perforation which eventually leads to insect death. The resistance of target pests to Bt toxins is an important factor hampering the long-term effective cultivation of Bt crops and the continuous use of Bt toxins. This review summarizes the mechanism of insect resistance to Bt toxins from the perspective of important Bt toxin receptors in midgut cells of Lepidopteran insects, which may facilitate the in-depth study of Bt resistance mechanism and pest control.

Effects of deleting peptidoglycan hydrolase genes on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease / 生物工程学报

In order to explore the effect of peptidoglycan hydrolase on the viable cell counts of Bacillus amyloliquefaciens and the yield of alkaline protease, five peptidoglycan hydrolase genes (lytC, lytD, lytE, lytF and lytG) of B. amyloliquefaciens TCCC111018 were knocked out individually. The viable cell counts of the bacteria and their alkaline protease activities before and after gene deletion were determined. The viable cell counts of the knockout mutants BA ΔlytC and BA ΔlytE achieved 1.67×106 CFU/mL and 1.44×106 CFU/mL respectively after cultivation for 60 h, which were 32.5% and 14.3% higher than that of the control strain BA Δupp. Their alkaline protease activities reached 20 264 U/mL and 17 265 U/mL, respectively, which were 43.1% and 27.3% higher than that of the control strain. The results showed that deleting some of the peptidoglycan hydrolase genes effectively maintained the viable cell counts of bacteria and increased the activity of extracellular enzymes, which may provide a new idea for optimization of the microbial host for production of industrial enzymes.

Construction of EF-G knockdown strain of Mycobacterium smegmatis and drug resistance analysis / 生物工程学报

As the only translational factor that plays a critical role in two translational processes (elongation and ribosome regeneration), GTPase elongation factor G (EF-G) is a potential target for antimicrobial agents. Both Mycobacterium smegmatis and Mycobacterium tuberculosis have two EF-G homologous coding genes, MsmEFG1 (MSMEG_1400) and MsmEFG2 (MSMEG_6535), fusA1 (Rv0684) and fusA2 (Rv0120c), respectively. MsmEFG1 (MSMEG_1400) and fusA1 (Rv0684) were identified as essential genes for bacterial growth by gene mutation library and bioinformatic analysis. To investigate the biological function and characteristics of EF-G in mycobacterium, two induced EF-G knockdown strains (Msm-ΔEFG1(KD) and Msm-ΔEFG2(KD)) from Mycobacterium smegmatis were constructed by clustered regularly interspaced short palindromic repeats interference (CRISPRi) technique. EF-G2 knockdown had no effect on bacterial growth, while EF-G1 knockdown significantly retarded the growth of mycobacterium, weakened the film-forming ability, changed the colony morphology, and increased the length of mycobacterium. It was speculated that EF-G might be involved in the division of bacteria. Minimal inhibitory concentration assay showed that inhibition of EF-G1 expression enhanced the sensitivity of mycobacterium to rifampicin, isoniazid, erythromycin, fucidic acid, capreomycin and other antibacterial agents, suggesting that EF-G1 might be a potential target for screening anti-tuberculosis drugs in the future.