Frecuencia de E. coli y Klebsiella spp productoras de betalactamasas en cultivos procesados en un laboratorio clínico / Frequency of E. coli and betalactamase-producing Klebsiella spp in cultures processed in a clinical laboratory / Frequência de E. coli e Klebsiella spp produtoras de betalactamase em culturas processadas em laboratório clínico

Introducción: la resistencia antibiótica en bacterias patógenas como Escherichia coli y Klebsiella spp. productoras de betalactamasas, han surgido como un problema global de salud pública. Su presencia, se asocia con infecciones intrahospitalarias y comunitarias, aumentando la morbilidad y la mortalidad de los pacientes. Objetivo: determinar la frecuencia de E.coli y Klebsiella spp productoras de betalactamasas en cultivos procesados en un laboratorio clínico. Métodos: se realizó un estudio descriptivo de diseño documental. La muestra estuvo constituida por un total de 1465 resultados de cultivos positivos para Escherichia coli o Klebsiella spp. en el periodo 2022. Para la recolección de la información, se tuvo acceso a la base de datos anonimizada del laboratorio en una hoja de Excel para su posterior análisis. Los datos fueron tabulados en SPSS versión 25. Resultados: el análisis de bacterias productoras de BLEE mostró una positividad del 22,3% en E. coli y 46,1% en Klebsiella spp. E. coli presentó mayor frecuencia de negativos (77,7%) en comparación con Klebsiella spp. La presencia de E. coli fue más común en muestras de orina (90,6%) y en otras muestras como esputo y heridas cutáneas (21,3%). Se evaluaron 8 antibióticos, y se destacó la alta sensibilidad para amikacina (AK) (99,6% y 98,0%) y elevada resistencia ampicilina (AM) (91,5% y 100%) en ambas especies. Ciprofloxacino (CIP) y Trimetropin/Sulfametoxazol (STX) mostraron relativa frecuencia mayor de resistencia. Conclusión: los resultados muestran una alta frecuencia de bacterias productoras de BLEE en E. coli y Klebsiella spp., con una mayor prevalencia en Klebsiella spp. Además, la resistencia a AM, CIP y STX destaca la importancia de una gestión adecuada de la resistencia antimicrobiana.

Introduction: antibiotic resistance in pathogenic bacteria such as Escherichia coli and Klebsiella spp. producing beta-lactamases has emerged as a global public health problem. Their presence has been associated with both hospital-acquired and community-acquired infections, leading to increased morbidity and mortality in patients. Objective: to determine the frequency of betalactamase-producing E. coli and Klebsiella spp. in cultures processed in a clinical laboratory. Methods: a descriptive documentary design study was conducted. The sample consisted of a total of 1465 positive culture results for Escherichia coli or Klebsiella spp. in the year 2022. Data collection involved accessing the laboratory's anonymized database in an Excel sheet for subsequent analysis. The data were tabulated in SPSS version 25. Results: the analysis of ESBL-producing bacteria showed a positivity of 22.3% in E. coli and 46.1% in Klebsiella spp. E. coli showed a higher frequency of negatives (77.7%) compared to Klebsiella spp. The presence of E. coli was more common in urine samples (90.6%) and in other samples such as sputum and skin wounds (21.3%). Eight antibiotics were evaluated, with high sensitivity noted for amikacin (AK) (99.6% and 98.0%) and high resistance for ampicillin (AM) (91.5% and 100%) in both species. Ciprofloxacin (CIP) and Trimethoprim/Sulfamethoxazole (STX) showed a relatively higher frequency of resistance. Conclusion: the results show a high frequency of ESBL-producing bacteria in E. coli and Klebsiella spp., with a higher prevalence in Klebsiella spp. Furthermore, the resistance to AM, CIP, and STX highlights the importance of proper management of antimicrobial resistance.

Introdução: a resistência antibiótica em bactérias patogênicas como Escherichia coli e Klebsiella spp., produtoras de beta-lactamases, emergiu como um problema de saúde pública global. Sua presença tem sido associada a infecções hospitalares e comunitárias, aumentando a morbidade e a mortalidade dos pacientes. Objetivo: determinar a frequência de E. coli e Klebsiella spp. produtoras de betalactamase em culturas processadas em laboratório clínico. Métodos: foi realizado um estudo descritivo de design documental. A amostra consistiu em um total de 1465 resultados de cultura positiva para Escherichia coli ou Klebsiella spp. no ano de 2022. A coleta de dados envolveu o acesso ao banco de dados anonimizado do laboratório em uma planilha do Excel para análise subsequente. Os dados foram tabulados na versão 25 do SPSS. Resultados: a análise de bactérias produtoras de BLEE mostrou uma positividade de 22,3% em E. coli e 46,1% em Klebsiella spp. E. coli apresentou uma frequência maior de resultados negativos (77,7%) em comparação com Klebsiella spp. A presença de E. coli foi mais comum em amostras de urina (90,6%) e em outras amostras, como escarro e feridas na pele (21,3%). Foram avaliados oito antibióticos, com alta sensibilidade observada para amicacina (AK) (99,6% e 98,0%) e alta resistência para ampicilina (AM) (91,5% e 100%) em ambas as espécies. Ciprofloxacina (CIP) e Trimetoprima/Sulfametoxazol (STX) mostraram uma frequência relativamente maior de resistência. Conclusão: os resultados mostram uma alta frequência de bactérias produtoras de BLEE em E. coli e Klebsiella spp., com uma maior prevalência em Klebsiella spp. Além disso, a resistência a AM, CIP e STX destaca a importância da adequada gestão da resistência antimicrobiana.

Caracterización molecular de Escherichia coli diarreogénica proveniente de población pediátrica ambulatoria con diarrea, atendida en dos hospitales de Buenos Aires, Argentina / Molecular characterization of diarrheagenic Escherichia coli from an outpatient pediatric population with diarrhea attended in two hospitals from Buenos Aires, Argentina

Resumen Escherichia coli diarreogénica abarca un grupo heterogéneo de patotipos o variantes patogénicas que comparten características fenotípicas con marcadas diferencias en genes de virulencia, sitios de colonización, patogenia, presentación clínica y epidemiología de la infección. Los patotipos más estudiados son E.coli productora de toxina Shiga (STEC), E.coli enterotoxigénica (ETEC), E.coli enteropatogénica (EPEC), E.coli enteroagregativa (EAEC) y E.coli enteroinvasiva (EIEC). El objetivo del estudio fue caracterizar los aislamientos de E.coli diarreogénica provenientes de población pediátrica ambulatoria con diarrea, atendida en dos hospitales públicos de Buenos Aires, Argentina. Los patotipos de E.coli diarreogénica se investigaron mediante la amplificación de fragmentos de genes de virulencia característicos: intimina (eae), toxina termolábil (lt), toxinas termoestables (stp, sth), antígeno plasmídico de invasiónH (ipaH), activador transcripcional R (aggR) y toxinas Shiga (stx1, stx2). La subtipificación molecular de aislamientos se realizó mediante PFGE (XbaI). E.coli diarreogénica fue detectada en el 14% (84/601) de los casos. El patotipo EAEC fue prevalente, mientras que ETEC, STEC, EPEC y EIEC fueron hallados en menor proporción. Los aislamientos de EAEC presentaron un alto grado de diversidad genética. Todos los patotipos fueron hallados en niños menores de 5años, mientras que solamente EAEC, EIEC y ETEC fueron detectados en población de mayor edad. Futuros estudios que incluyan la caracterización de aislamientos a partir de un mayor número de genes y población de otras áreas geográficas serán necesarios para determinar la relevancia de E.coli diarreogénica en Argentina.

Abstract Diarrheagenic Escherichia coli comprises a heterogeneous group of pathotypes or pathogenic variants that share phenotypic characteristics with marked differences in virulence genes, colonization sites, pathogenesis, clinical presentation, and epidemiology of infection. The most studied pathotypes are Shiga toxin-producing E.coli (STEC), enterotoxigenic E.coli (ETEC), enteropathogenic E.coli (EPEC), enteroaggregative E.coli (EAEC), and enteroinvasive E.coli (EIEC). The objective of the study was to characterize the isolates of diarrheagenic E.coli from an outpatient pediatric population with diarrhea attended in two public hospitals from Buenos Aires, Argentina. Diarrheagenic E.coli pathotypes were investigated by amplifying characteristic virulence gene fragments: intimin (eae), heat-labile toxin (lt), heat-stable toxins (stp, sth), invasion plasmid antigen H (ipaH), transcriptional activator R (aggR) and Shiga toxins (stx1, stx2). Molecular subtyping of isolates was performed using PFGE (XbaI). Diarrheagenic E.coli was detected in 14% (84/601) of cases. The EAEC pathotype was prevalent, while ETEC, STEC, EPEC and EIEC were found in a lower proportion. EAEC isolates exhibited a high degree of genetic diversity. All pathotypes were found in children under 5years of age, while only EAEC, EIEC and ETEC were detected in the older population. Future studies that include the characterization of isolates from a greater number of genes and populations from other geographical areas will be necessary to determine the relevance of diarrheagenic E.coli in Argentina.

Detection of plasma anti-lipopolysaccharide (LPS)antibodies against enterohemorrhagic Escherichia coli(EHEC) in asymptomatic kindergarten teachers fromBuenos Aires province

Resumen En Argentina, el síndrome urémico hemolítico causado por Escherichia coli enterohemorrágica (EHEC) tiene la más alta incidencia del mundo. Las infecciones por EHEC tienen un comportamiento endemoepidémico y causan del 20 al 30% de los síndromes de diarrea sanguinolenta en niños menores de 5 años. En el período 2016-2020, se notificaron 272 nuevos casos por año al Sistema de Vigilancia de Salud Nacional. Múltiples factores son responsables de la alta incidencia de SUH en Argentina, incluyendo la transmisión persona-persona. Con el objetivo de detectar posibles portadores asintomáticos de EHEC, realizamos un estudio preliminar de la frecuencia de anticuerpos antilipopolisacáridos contra los serotipos de EHEC más prevalentes en Argentina. El estudio se realizó con muestras de plasma obtenidas de 61 maestras y maestros de jardines de infantes de 26 instituciones del distrito de José C. Paz, localizado en el área suburbana de la provincia de Buenos Aires, Argentina. El 51% de las muestras presentaron anticuerpos contra los serotipos de lipopolisacáridos O157, O145, O121 y O103; el 6,4% de las muestras positivas tuvieron el isotipo IgM (n=2), el 61,3% el isotipo IgG (n=19) y el 32,3% los isotipos IgM e IgG (n=10). Dado que los anticuerpos antilipopolisacáridos presentan usualmente una duración corta, la detección de IgM específica podría indicar una infección reciente. Además, el alto porcentaje de muestras positivas hallado podría indicar una exposición frecuente a las cepas de EHEC en la cohorte estudiada. Asimismo, la gran población de adultos portadores asintomáticos de estas cepas patógenas podría contribuir al comportamiento endémico, a través de la transmisión persona-persona. El perfeccionamiento de programas educacionales continuos en jardines de infantes podría constituir una medida importante para reducir los casos de síndrome urémico hemolítico, no solo en Argentina, sino también en el mundo.

Abstract In Argentina, hemolytic uremic syndrome (HUS) caused by EHEC has the highest incidence in the world. EHEC infection has an endemo-epidemic behavior, causing 20-30% of acute bloody diarrhea syndrome in children under 5 years old. In the period 2016-2020, 272 new cases per year were notified to the National Health Surveillance System. Multiple factors are responsible for HUS incidence in Argentina including person-to-person transmission. In order to detect possible EHEC carriers, we carried out a preliminary study of the frequency of kindergarten teachers with anti-LPS antibodies against the most prevalent EHEC serotypes in Argentina. We analyzed 61 kindergarten teachers from 26 institutions from José C. Paz district, located in the suburban area of Buenos Aires province, Argentina. Fifty-one percent of the plasma samples had antibodies against O157, O145, O121 and O103 LPS: 6.4% of the positive samples had IgM isotype (n=2), 61.3% IgG isotype (n=19) and 32.3% IgM and IgG (n=10). Given that antibodies against LPS antigens are usually short-lived specific IgM detection may indicate a recent infection. In addition, the high percentage of positive samples may indicate a frequent exposure to EHEC strains in the cohort studied, as well as the existence of a large non-symptomatic population of adults carrying pathogenic strains that could contribute to the endemic behavior through person-to-person transmission. The improvement of continuous educational programs in kindergarten institutions could be a mandatory measure to reduce HUS cases not only in Argentina but also globally.

Application of multi-disciplinary team mode in prevention and control of multidrug resistant organism infection in lung transplant recipients / 器官移植

<b>Objective</b> To evaluate the effectiveness of multi-disciplinary team (MDT) mode in the prevention and control of multidrug resistant organism (MDRO) infection in lung transplant recipients. <b>Methods</b> Lung transplant recipients admitted to the hospital from 2019 to 2022 were enrolled. MDT expert group was established in January, 2020. A series of prevention and control measures were conducted. The implementation rate of MDRO prevention and control measures and the detection rate of MDRO on the environmental surface from 2020 to 2022, and the detection rate of MDRO in lung transplant recipients from 2019 to 2022 were analyzed. <b>Results</b> The overall implementation rate of MDRO prevention and control measures for medical staff was increased from 64.9% in 2020 to 91.6% in 2022, showing an increasing trend year by year (<i>P</i><0.05). The detection rate of MDRO on the environmental surface was decreased from 28% in 2020 to 9% in 2022, showing a downward trend year by year (<i>P</i><0.05). The detection rate of MDRO in lung transplant recipients was decreased from 66.7% in 2019 to 44.3% in 2022, showing a decreasing trend year by year (<i>P</i><0.001). <b>Conclusions</b> MDT mode management may enhance the implementation of MDRO prevention and control measures for medical staff, effectively reduce the infection rate of MDRO in lung transplant recipients and the detection rate of MDRO on the environmental surface, which is worthy of widespread application.

Isolation of Enterobacteriaceae strains carrying mcr-1 resistance gene from Shanghai wastewater treatment plants and quantification of their copy number / 上海预防医学

ObjectiveTo provide technical support for the molecular surveillance of pathogenic bacteria strains carrying mobile colistin resistance-1 （mcr⁃1） gene isolate from inlet of wastewater treatment plants （WWTP）. MethodsThe Enterobacteriaceae strains carrying mcr⁃1 resistance gene isolate from inlet of WWTP during April 1 to June 30， 2023 in Shanghai were cultured on blood-rich and SS culture medium and were identified using a mass spectrometry analyzer. The mcr⁃1 gene and copy number were detected by real-time fluorescence quantitative PCR. Drug susceptibility test was performed by microbroth dilution method. The copy numbers of Escherichia coli carrying mcr⁃1 gene isolated from wastewater and human fecel were statistically analyzed by SPSS 25.0. ResultsA total of 14 strains carrying the mcr⁃1 gene were isolated from 49 WWTP samples， and the positive isolation rate was 28.6%， including 12 non-diarrheal E. coli strains and 2 Klebsiella pneumoniae strains. The drug susceptibility results showed that all 14 strains were multi-drug resistant bacteria. They were all sensitive to imipenem and tigecycline， but were ampicillin- and cefazolin-resistant. There was no significant difference in the copy number between human-sourced diarrheal E. coli and wastewater-sourced non-diarrheal E. coli （t=0.647， P>0.05）. ConclusionThe isolation and identification of strains carrying the mcr⁃1 gene from inlet of WWTP samples were firstly established in Shanghai. The multi-drug resistance among the isolated strains is severe. To effectively prevent and control the spread of colistin-resistant bacteria， more attention should be paid to the surveillance of mcr⁃1 gene.

Analysis of virulence and drug resistance characteristics of diarrheagenic Escherichia coli isolated from patients with infectious diarrhea in a hospital in Beijing / 临床检验杂志

Objective To investigate the virulence and drug resistance characteristics of diarrheagenic Escherichia coli isolated from patients with infectious diarrhea in our hospital.Methods The preliminary identification of microbes was carried out by the VITEK-MS microbial mass spectrometry detection system and virulence genes were detected by the multiplex real-time PCR.Five types of diarrhea-genic Escherichia coli(DEC)clinically isolated from patients with infectious diarrhea in our hospital were identified.The drug resist-ance characteristics of DEC strains were detected by the microbroth dilution and E-test.The drug-resistant molecular characteristics were analyzed by the next-generation sequencing and bioinformatics.The Fisher exact probability method was used for statistical analy-sis.Results The detection rate of DEC in our hospital was 11.9%,with enteroaggregative E.coli(EAEC)accounting for 37.5%,a-typical enteropathogenic Escherichia coli(EPEC)accounting for 34.38%,enterotoxigenic E.coli(ETEC)accounting for 25.0%,and enteroinvasive E.coli(EIEC)accounting for 3.12%.None of enterohemorrhagic E.coli(EHEC)strain was detected.The resistance rates of 32 DEC strains to ampicillin,tetracycline,and trimethoprim/sulfamethoxazole were 53.12%,43.75%,and 37.5%,respec-tively.ESBLs(+)strains accounted for 18.75%,and the detection rate of multidrug-resistant strains was 83.83%,significantly higher than that of ESBLs(-)strains(P=0.042).A total of 25 ST genotypes were obtained from 32 DEC strains.The dominant genotypes were ST10(4 strains,12.5%),followed by ST28(2 strains,6.25%),ST31(2 strains,6.25%),ST3153(2 strains,6.25%),and the other 21 genotypes(1 strain,3.13%).One carbapenem resistant strain carrying the blaNDM-1 gene was detected in EAEC.Conclu-sion Four virulence genes such as aggR,pic,astA,and eae,are more common in the DEC of patients with infectious diarrhea in our hospital,with EAEC and EPEC as the main subtypes.The genotypes are highly polymorphic,and multidrug-resistant strains have been detected.

Antibacterial performance of cerium oxide nanoenzyme against Escherichia coli / 中国组织工程研究

BACKGROUND:The increase in multi-drug resistant bacterial infections has become a major problem in modern healthcare due to the development of bacterial resistance to antibiotics and the development of new antibacterial alternative drug materials is of great importance. OBJECTIVE:To synthesize and perform a series of characterization of a CeO2 nanoenzyme to investigate its biocompatibility and antibacterial properties against Escherichia coli. METHODS:CeO2 nanoenzymes were synthesized using a hydrothermal method.The morphology,product composition,and chemical composition were analyzed using characterization methods such as X-ray diffraction,X-ray photoelectron spectroscopy,Fourier infrared analysis,Raman spectroscopy,scanning electron microscopy,and transmission electron microscopy.The peroxide-mimetic enzyme activity of CeO2 nanoenzymes was characterized using TMB color development assay.The toxic effect of CeO2 nanoenzymes at different concentrations(10,25,and 50 μg/mL)on mouse fibroblast L929 cells was evaluated using the CCK-8 assay.The antibacterial properties of CeO2 nanoenzymes against Escherichia coli under different conditions were evaluated using the plate coating method.Changes in intra-bacterial reactive oxygen species after treatment with different conditions were detected using a reactive oxygen species detection kit. RESULTS AND CONCLUSION:(1)The morphology of the synthesized CeO2 nanoparticles was rod-shaped,with Ce3+ accounting for 29.87%of the total Ce3+/Ce4+ and an average grain size of 7.4 nm.In a slightly acidic environment containing TMB and pH=5.5,CeO2 nanoenzymes mixed with H2O2 showed excellent peroxidase activity,but did not show peroxidase simulated activity at pH=7.4.(2)There was no statistically significant difference in the toxic effects of CeO2 nanoparticles at various mass concentrations on mouse fibroblast L929 cells.(3)In a slightly acidic environment at pH 5.5,Escherichia coli was inhibited to a certain extent in the presence of CeO2 nanoenzyme alone at a concentration of 10 μg/mL,with a decrease in CFU results of about 0.5 log(P＜0.01);in a slightly acidic environment containing 50 μmol/L H2O2,CeO2 nanoenzyme showed excellent antibacterial effects against Escherichia coli,with a decrease in Escherichia coli CFU results of by about 1.5 log(P＜0.001).After CeO2 nanoenzymes interacted with Escherichia coli,the level of reactive oxygen species in Escherichia coli increased(P＜0.05);after CeO2 nanoenzymes interacted with Escherichia coli together with H2O2,the level of reactive oxygen species in Escherichia coli increased significantly(P＜0.001).(4)The results show that the CeO2 nanoenzymes have good biocompatibility,are inherently antibacterial,and can exhibit peroxidase activity in a slightly acidic environment containing low concentrations of H2O2,and generate reactive oxygen species to kill bacteria,thus showing excellent antibacterial effects.

Influence of Escherichia coli in the intestine on short-term prognosis in patients with acute ischemic stroke:a primary study / 中华老年心脑血管病杂志

Objective To detect the content of intestinal Escherichia coli(E.coli)in patients with acute ischemic stroke(AIS)and to analyze the relationship between the bacteria and short-term prognosis of patients with cerebral stroke.Methods A total of 75 elderly AIS patients admitted to our department from March to December 2022 were enrolled,and divided into good prognosis group(47 cases)and poor prognosis group(28 cases)according to the results of modified Rankin scale 3 months after discharge.Multivariate logistic regression analysis was used to analyze the factors affecting the prognosis of the patients,and ROC curve analysis was employed to analyze the predictive value of the intestinal bacterial content for the short-term prognosis of stroke pa-tients.Results The NIHSS score at admission and E.coli content were significantly higher in the poor prognosis group than the good prognosis group(P＜0.01).Multivariate logistic regression analysis showed that NIHSS score at admission(OR=1.302,95％CI:1.077-1.573,P=0.006)and E.coli content(OR=2.803,95％CI:1.454-5.404,P=0.002)were independent risk factors for short-term poor prognosis in the AIS patients.ROC curve analysis indicated that the AUC value was 0.758 for E.coli content,0.718 for NIHSS score,and 0.818 when the 2 indicators combined together.Conclusion Intestinal E.coli content and NIHSS score may be related to the poor prog-nosis of elderly AIS patients.The higher the content of E.coli is,the worse the recovery of neuro-logical function,which affects the short-term prognosis of the patients.

Role of uropathogenic Escherichia coli virulence factor TcpC in immune evasion and its pathogenic mechanism / 中华微生物学和免疫学杂志

Objective:To investigate the role of TcpC, a virulence factor of uropathogenic Escherichia coli (UPEC), in immune evasion, and analyze its related pathogenic mechanism. Methods:C57BL/6 mice were injected with 10 9 colony-forming unit of wild-type (CFT073 wt) or tcpc gene-knockout (CFT073 Δ tcpc) UPEC CFT073 strains from urethra into bladder to construct a mouse model of pyelonephritis. These mice were sacrificed 5 d after infection and their kidneys were taken to observe the gross pathological changes. Hematoxylin-eosin staining was used to observe histopathological changes in kidney tissues and immunohistochemistry was performed to locate TcpC in kidney tissues. The bacterial loads in urine samples of UPEC infected-mice were counted by ten-fold dilution method, and the presence of tcpc gene in the genomic DNA of bacteria from CFT073-infected mouse kidney or urine samples was measured by PCR. The expression of TcpC at mRNA level was detected by qRT-PCR after infecting dendritic cells with CFT073 wt strains. The influences of UPEC infection on the activation of NF-κB signaling pathway and the secretion of proinflammatory factors by dendritic cells were analyzed by Western blot and ELISA, respectively. The viability of UPEC strains in dendritic cells were observed by laser confocal microscope. Results:Compared with the CFT073 Δ tcpc group, the mice in the CFT073 wt group had obvious abscess in the kidneys as well as massive neutrophil infiltration and abundant TcpC in kidney tissues. The bacterial loads in the urine of CFT073 wt-infected mice were significantly higher than those in the urine of CFT073 Δ tcpc mice. PCR results showed that tcpc gene was successfully amplified from mouse kidney and urine samples. Increased expression of TcpC at both mRNA and protein levels was detected in CFT073 wt-infected dendritic cells. CFT073 wt infection inhibited the phosphorylation of NF-κB p50 and the production of proinflammatory factors in dendritic cells. TcpC promoted the survival of CFT073 wt in dendritic cells. Conclusions:TcpC expression increases significantly during CFT073 wt infection or in mice with CFT073 wt-induced pyelonephritis. It promotes the survival of CFT073 wt in dendritic cells by inhibiting the activation of NF-κB signaling pathway and reducing the secretion of pro-inflammatory cytokines. TcpC is involved in the pathogenesis of UPEC and immune evasion.

Construction of luminescent bacteriophage using CRISPR technology and its application in Escherichia coli indentification / 中华检验医学杂志

Objective:To construct a recombinant bioluminescent bacteriophage (HT7) targeting Escherichia coli, and evaluate its ability to identify Escherichia coli. Methods:Initially, pCRISPR-sg (1-10) and PFN-1000 plasmid strains were constructed by genetic engineering, and the most efficient small guild RNA (sgRNA) were screened by bilayer plate. By the gene editing technique, which comprised homologous recombination and clustered regularly interspaced short palin dromic repeats (CRISPR)-Cas system, the Nanoluc luciferase gene was integrated into the downstream non-coding region of 10A gene of T7 phage, to constructe the bioluminescent phage HT7 successfully. The difference of biological characteristics between HT7 phage and T7 phage was evaluated by plaque assay and liquid amplification assay. In addition, 51 strains of Escherichia coli, 20 strains of Klebsiella pneumoniae, 14 strains of Staphylococcus aureus, 6 strains of Enterococcus faecium, 5 strains of Enterococcus faecalis, 3 strains of Acinetobacter baumannii and 1 strain of Pseudomonas aeruginosa were collected and isolated to evaluate the limit of detection and specificity of HT7 phage. Results:Among the 10 CRISPR-targeted cleavage systems constructed, sgRNA8 exhibited the highest cleavage efficiency, with a cleavage rate of 0.18. After three rounds of recombination screening using the pCas9/pCRISPR/PFN-1000 triple-plasmid system, PCR validation yielded recombinant phage bands at 2 798 bp, indicating the successful construction of the HT7 phage. The recombinant phage showed significant differences in biological characteristics in terms of lysis efficiency ( P<0.001), one-step growth curve ( P=0.001), and infection multiplicity ( P=0.031). Both lysis burst time and log growth node were extended by 10 min, with the optimal infection multiplicity being 0.1. Clinical sample testing identified lysis of 6 strains of Escherichia coli within 4.5 h, while other strains remained unaffected, with detection of pathogenic bacteria below 10 CFU/ml. Conclusions:The developed pCas9/pCRISPR/PFN-1000 triple-plasmid editing system efficiently edits the bacteriophage genome. The constructed HT7 fluorescent bacteriophage enables the detection of Escherichia coli below 10 CFU/ml within 4.5 hours, demonstrating low detection limits and high detection specificity.

Drug resistance and homology of diarrheagenic Escherichia coli in sentinel hospital of Fengxian District of Shanghai， 2019‒2022 / 上海预防医学

ObjectiveWe conducted a drug resistance and homology analysis of diarrheagenic Escherichia coli （DEC） in Fengxian District of Shanghai in order to provide a basis for clinical rational drug use， risk monitoring and early warning. MethodsDEC were isolated from diarrheal patients in Fengxian District， Shanghai from 2019 to 2022. The minimum inhibitory concentrations （MIC） of 21 drugs to the DEC were determined. Genotyping and homology analysis were conducted with pulsed-field gel electrophoresis （PFGE）. ResultsThe DEC detection rate of diarrhea cases was 18.99% （131/690）， including enteroaggregative E.coli （EAEC） 64.89% （85/131）， enterotoxigenic E.coli （ETEC） 22.14% （29/131）， enteropathogenic E.coli （EPEC） 12.21% （16/131）， and enterohemorrhagic E.coli （EHEC） 0.76%（1/131）. The DEC detection showed obvious seasonal characteristics with a high incidence in summer. The DEC multidrug resistance rate was 66.41% with a total of 65 drug resistance profiles. The five antimicrobial drugs with the highest resistance rate were ampicillin （60.31%）， nalidixic acid （51.91%）， cefazolin （50.38%）， tetracycline （44.27%）， and cotrimoxazole （35.11%）. The rate of DEC resistance to levofloxacin was significantly increased from 2019 to 2022. Cluster analysis showed that the similarity of 85 EAEC cluster was 58.4%‒100.0%， and 69 band patterns were obtained. The similarity of 29 ETEC cluster was 58.5%‒100.0%， and 13 band patterns were obtained， including 2 dominant band types. The similarity of 16 EAEC clusters was 53.9%‒100.0%， and 15 band patterns were obtained. Five groups of homologous strains were found， consistent with the resistance phenotypes. ConclusionAmong the diarrhea cases， the DEC epidemic intensity is high， the drug resistance situation is severe， and the risk of outbreak infection is high in Fengxian District， Shanghai. Therefore， health monitoring and prevention need to be strengthened.

The killing effects of gentamicin on uropathogenic Escherichia coli and its cytotoxicities in in vitro experiments of urinary tract infection / 西安交通大学学报(医学版)

【Objective】 To compare the killing effects of different concentrations of gentamicin (0, 10, 20, 50, 100, and 200 μg/mL) on uropathogenic Escherichia coli (UPEC) and its cytotoxicities on urinary urothelial cells and inflammatory cells such as macrophages in vitro. 【Methods】 The killing effects of different concentrations of gentamicin on different amounts (108, 107, and 106) of UPEC strain J96 were compared. The cytotoxicities of different concentrations of gentamicin on primary cultured male C57BL/6 mouse renal tubular epithelial cells, mouse macrophages and human bladder epithelial cell line J82 at different time points (2 h and 24 h) were detected by CCK-8 assay. According to the experiments above, we chose appropriate gentamicin concentrations and incubation time in in vitro cell culture experiments to verify J96 adhesion and invasion to mouse renal tubular epithelial cells or phagocytosis and clearance of J96 by mouse macrophages. 【Results】 The killing effect of gentamicin (≥10 μg/mL) on J96 was stronger than that of 1% P/S (P<0.000 1). High concentrations of gentamicin (≥100 μg/mL) could kill up to 108 J96 within 30 min. 50 μg/mL gentamincin treatment for 2 h was cytotoxic for human bladder epithelial cell line J82 (P<0.05). 【Conclusion】 The appropriate concentration and duration of gentamicin treatment for different cells in vitro were determined. Urothelial cells, especially human bladder epithelial cell line J82, were more sensitive to gentamicin.

Molecular characteristics of enteropathogenic Escherichia coli isolates from livestock at a monitoring point in Jiangsu Province / 中国人兽共患病学报

This study was aimed at analyzing the molecular characteristics of enteropathogenic Escherichia coli(E.coli)strains isolated from domestic animals at a surveillance site in Jiangsu province and evaluating their potential pathogenicity,to provide evidence supporting the surveillance,prevention,and control of infectious diarrhea.Thirty-seven EPEC strains isolated from domestic animals at this surveillance site were characterized by whole genome sequencing.All EPEC strains isolated from local livestock were aEPEC,which has a variety of serotypes and carries a variety of virulence genes associated with diarrhea.Nine ST types with regional epidemic characteristics were identified.Five eae gene subtypes were found,among which β1 was dominant and was also the most common strain in patients with diarrhea.According to analysis of the characteristics of 37 EPEC strains,all EPEC strains from local livestock were aEPEC,thus posing a potential threat to public health.Monitoring of livestock feces and the breeding environment must be strengthened in the surveillance of infectious diarrhea.

Changes in the isolation and drug resistance rate of Escherichia coli in a hospital over the past 10 years / 中国医科大学学报

Objective To summarize the isolation and drug resistance rate of Escherichia coliin The First Hospital of China Medical University over the past 10 years,in order to provide evidence for the efficacies of clinical anti-infection treatments.Methods The data was collected from Escherichia coli isolated from patients treated at The First Hospital of China Medical University between 2013 and 2022.VITEK 2 and VITEK MS were used for bacterial identification,VITEK2 and KB method were used for drug sensi-tivity testing,and WHONET 5.6 software was used for analysis.Results From 2013 to 2022,6 845 strains were isolated,including 80.5%from inpatients and 19.5%from emergency and outpatients.The specimens were most commonly found in the urine(57.8%),blood(15.0%),secretions(9.2%),and drainage fluid(8.1%).The isolation rate of extended-spectrumβ-lactamase(ESBL)producing Escherichia coli was 57.2%(54.3%to 61.5%).The drug resistance rate of Escherichia coli to carbapenems was low,at only 1.2%(0.2%to 2.6%).Conclusion Escherichia coli remains an important pathogen in clinical infections,with varying degrees of resist-ance to multiple antibiotics,and the resistance rate is increasing.Clinical physicians should pay sufficient attention to this issue.

Clinical characteristics and risk factors of 209 cases of urinary tract infection after kidney transplantation / 器官移植

Objective To investigate clinical characteristics and risk factors of different stages of urinary tract infection after kidney transplantation. Methods Clinical data of 209 kidney transplant recipients were retrospectively analyzed. According to time points of postoperative follow-up, all recipients were divided into 3 stages: within 1 month post-kidney transplantation, 1-6 months post-kidney transplantation, and 7-12 months post-kidney transplantation. The incidence of urinary tract infection, urine culture results of recipients with urinary tract infection and drug resistance characteristics of common pathogens during different stages after kidney transplantation were analyzed. The strains of patients with recurrent urinary tract infection were identified. The risk factors of urinary tract infection and the effect of urinary tract infection on renal allograft function were analyzed. Results The urinary tract infection rate was 90.0% in the first stage, 49.3% in the second stage and 22.5% in the third stage. The urinary tract infection rates of male recipients undergoing living-related organ donation in the second and third stages were lower than those of female recipients (both P<0.05). Urine culture test yielded positive results in 60 cases, and 84 strains of pathogenic bacteria were detected, mainly Gram-negative bacteria, among which Klebsiella pneumoniae accounted for the highest proportion. Sixty-six recipients had recurrent urinary tract infection, and the detected pathogens included Klebsiella pneumoniae, Escherichia coli and Candida glabrata, etc. Univariate analysis showed that postoperative use of antithymocyte globulin was the risk factor for urinary tract infection in the first stage. Preoperative urinary tract infection and donor type were the risk factors for urinary tract infection in the second stage. Gender and age of the recipients were the risk factors for urinary tract infection in the third stage. Multivariate analysis revealed that postoperative use of antithymocyte globulin was the risk factor for urinary tract infection in the first stage. Gender and age of the recipients were the risk factors for urinary tract infection in the third stage (all P<0.05). In the third stage, 65 cases were cured and 38 cases were not cured. In the treated recipients, the serum creatinine level and white blood cell count were decreased after corresponding treatment than those before treatment (both P<0.05). Conclusions Gram-negative bacteria are the main pathogens of urinary tract infection in kidney transplant recipients, and drug resistance is relatively high. Postoperative use of antithymocyte globulin, female and old age are the risk factors for urinary tract infection in kidney transplant recipients.

Mechanisms of Transcription-translation Coupling in Escherichia coli / 生物化学与生物物理进展

In prokaryotes like Escherichia coli (E. coli), transcription tends to be coupled with translation, which is usually manifested in the mutual regulation of transcription and translation such as transcription polarity, transcription attenuation and synchronization of transcription and translation rates. Indirect coupling and physical coupling are two different models of the coupling. Indirect coupling maintained by the alarmone (p)ppGpp may require the assistance of DksA and TufA proteins. Physical coupling could be divided into those mediated by NusG or RfaH factors and those induced via “collision” under non-factor condition. Changes in transcription or translation in response to pressure will lead to mutual transitions among several coupling modes. Coupling is necessary for normal gene expression, and its release will contribute to adverse events such as transcription termination, R-loop formation, conflict between replication and transcription and mRNA cleavage. The related technologies of structural biology have clearly demonstrated the structural details and characteristics of partial coupled expressomes. These technologies, combined with methods like multiomics analysis, will provide deeper insights into the coupling. Significantly, the study of the coupling may bring new ideas for development of the targeted antibiotics.

Development and verification of an ELISA method for detection of process-specific residual host protein in biological preparation / 中国生物制品学杂志

@#Objective To develop and verify a double-antibody sandwich ELISA method for the detection of process-specific E.coli residual protein in recombinant biological preparations.Methods Taking the production and purification process of glucagon-like peptide(GLP)expressed by E.coli as the specific process model,the same process was used to intercept the residual protein of empty E.coli(normal E.coli that does not express recombinant protein). One female New Zealand white rabbit and six female Kunming mice were immunized with the residual protein as the immunogen. Using the IgG antibody purified from rabbit immune serum as the coating antibody,mouse immune serum as the second sandwich antibody,and antimouse IgG-HRP as the enzyme-labeled secondary antibody,a double antibody sandwich ELISA method for process-specific residual protein of E.coli was established. The specificity,accuracy and precision of the method were verified,and the limit of detection(LOD)was determined. Simultaneously,the developed method and the commercial E.coli host protein residue detection kit were used to quantitatively determine the residual protein of purified GLP preparation.Results After a series of gradient dilution of process-specific residual protein with known concentration,the sensitivity of this ELISA method reached 338 pg/mL. No cross reaction occurred in the detection of CHO and yeast cell lysis protein by this method,the recoveries of samples with low,medium and high concentrations were all in the range of 80% — 120%,and the intra-assay and inter-assay CVs of the empty E.coli interception standard with low,medium and high concentrations were all less than15%. For the residual protein in GLP preparation,about 62% of the residual proteins were not detected by the commercial non-process-specific ELISA kit compared with the total amount of residual proteins detected by the developed method,and these residual proteins should be the process-specific residual proteins.Conclusion The double antibody sandwich ELISA method developed in this study has high sensitivity,strong specificity,good accuracy and precision for the detection of process-specific E.coli residual protein,which can meet the detection requirements that the residual protein is less than0. 01% — 0. 1% in biological preparations.

In vitro susceptibility to fosfomycin in clinical and environmental extended-spectrum beta-lactamase producing and/or ciprofloxacin-non-susceptible Escherichia coli isolates

ABSTRACT Extended-spectrum beta-lactamase producing and ciprofloxacin-non-susceptible Escherichia coli are clinical and environmental issues. We evaluated the susceptibility profile of fosfomycin in non-susceptible E. coli isolated from urine and the environment. We measured the activity of fosfomycin against 319 and 36 E. coli strains from urine and environmental isolates, respectively, collected from rivers. Fosfomycin resistance profiles were investigated using the minimal inhibitory concentration (MIC), according to the Clinical and Laboratory Standards Institute (CLSI) and the European Committee for Antimicrobial Susceptibility Testing (EUCAST) guidelines. Antibiotic susceptibility testing revealed that 5% and 6.6% of urine samples were non-susceptible to fosfomycin according to CLSI and EUCAST guidelines, respectively. The fosfomycin MIC50/90 was 0.5/4 mg/L. Of the 36 E. coli isolates from river water, 11.1% and 13,8% were non-susceptible to fosfomycin according to CLSI and EUCAST, respectively (range ≤0.25 ≥512 mg/L). All the isolates with MIC ≥512 mg/L for fosfomycin showed the fosA3 gene. Fosfomycin resistance was more frequent in the environment than in clinical samples.

Hospitalizaciones por diarrea con sangre en un centro pediátrico de referencia en Uruguay / Hospitalizations due to bloody diarrhea in a pediatric reference center in Uruguay / Internações por diarreia sanguinolenta em um centro de referência pediátrica no Uruguai

Introducción: La diarrea con sangre es un motivo frecuente de admisión hospitalaria en niños, con gastroenteritis aguda; en la mayoría de los casos se tratan de infecciones leves y autolimitadas, pero pueden producirse complicaciones graves. Objetivos: Describir la etiología y características clínico- evolutivas de los niños menores de 15 años hospitalizados por diarrea con sangre en el Hospital Pediátrico, Centro Hospitalario Pereira Rossell entre los años 2012- 2023. Materiales y métodos: Estudio retrospectivo mediante revisión de historias y registros de laboratorio. Variables: demográficas, estado nutricional, hidratación, motivos de hospitalización, ingreso unidades de cuidados intensivos (UCI), enteropatógenos, tratamientos, evolución. Resultados: Se incluyeron 229 niños, mediana de edad de 8 meses; sexo masculino 61%; eutróficos 88%, bien hidratados 55%, con comorbilidades 11%, prematurez 6,5%. El motivo de hospitalización fue diarrea con sangre/disentería sin otro síntoma 45%. Se solicitó coprovirológico/coprocultivo en 98% y detección por técnicas de ácidos nucleicos en materia fecal 5,2%. Se identificó al menos un agente patógeno en 34,3%: Shigella sp. 38%; Salmonella sp. 19,5%; coinfecciones en 12%. Se indicaron antibióticos a 86%; ceftriaxona 62%, azitromicina 35%. Ingresaron a UCI 6,5% (15), presentaron complicaciones 10/14, fallo renal agudo 5 y alteraciones del medio interno 3. La mayoría presentó buena evolución. Conclusiones: La diarrea con sangre/disentería continúa siendo una causa importante de hospitalización afectando en su mayoría a niños sanos menores de 5 años. Los patógenos detectados con mayor frecuencia fueron bacterias principalmente Shigella sp., Salmonella sp. y E coli diarreogénicas. Se reportó alta prescripción de antibióticos, cumpliendo en la mayoría de los casos con las recomendaciones.

Introduction: Bloody diarrhea is a common reason for hospital admission in children with acute gastroenteritis; In most cases these are mild and self-limiting infections, but serious complications can occur. Goals: To describe the etiology and clinical-evolutionary characteristics of children under 15 years of age hospitalized for bloody diarrhea at the Pediatric Hospital, Centro Hospitalario Pereira Rossell between the years 2012-2023. Materials and methods: Retrospective study through review of histories and laboratory records. Variables: demographics, nutritional status, hydration, reason for hospitalization, intensive care unit (ICU) admission, enteropathogens, treatments, evolution. Results: 229 children were included, median age 8 months; male sex 61%; eutrophic 88%, well hydrated 55%, with comorbidities 11%, prematurity 6.5%. The reason for hospitalization was bloody diarrhea/dysentery without other symptoms 45%. Coprovirological/coproculture was requested in 98% and detection by nucleic acid techniques in fecal matter was requested in 5,2%. At least one pathogenic agent was identified in 34,3%: Shigella sp. 38%; Salmonella sp 19,5%; coinfections in 12%. Antibiotics were indicated for 86%; ceftriaxone 62%, azithromycin 35%. Were admitted to the ICU 6,5% (15), 10/14 had complications, 5 had acute kidney failure and 3 had alterations in the internal environment. The majority had a good evolution. Conclusions: Bloody diarrhea/dysentery continues to be an important cause of hospitalization, affecting mostly healthy children under 5 years of age. The most frequently detected pathogens were bacteria, mainly Shigella sp., Salmonella sp. and diarrheagenic E coli. High prescription of antibiotics was reported, complying in most cases with the recommendations.

Introdução: A diarreia com sangue é um motivo comum de internação hospitalar em crianças com gastroenterite aguda; Na maioria dos casos, estas são infecções leves e autolimitadas, mas podem ocorrer complicações graves. Metas: Descrever a etiologia e as características clínico-evolutivas de crianças menores de 15 anos internadas por diarreia sanguinolenta no Hospital Pediátrico Centro Hospitalario Pereira Rossell entre os anos de 2012-2023. Materiais e métodos: Estudo retrospectivo por meio de revisão de histórias e registros laboratoriais. Variáveis: dados demográficos, estado nutricional, hidratação, motivo da internação, internação em unidade de terapia intensiva (UTI), enteropatógenos, tratamentos, evolução. Resultados: foram incluídas 229 crianças, mediana de idade 8 meses; sexo masculino 61%; eutrófico 88%, bem hidratado 55%, com comorbidades 11%, prematuridade 6,5%. O motivo da internação foi diarreia sanguinolenta/disenteria sem outros sintomas 45%. O estudo coprovirologico/coprocultivo foi solicitado em 98% e a detecção por técnicas de ácidos nucleicos em matéria fecal foi solicitada em 5,2%. Pelo menos um agente patogênico foi identificado em 34,3%: Shigella sp. 38%; Salmonella sp. 19,5%; coinfecções em 12%. Os antibióticos foram indicados para 86%; ceftriaxona 62%, azitromicina 35%. Foram internados em UTI 6,5% (15), 10/14 apresentaram complicações, 5 tiveram insuficiência renal aguda e 3 apresentaram alterações no meio interno, a maioria teve boa evolução. Conclusões: A diarreia/disenteria com sangue continua a ser uma causa importante de hospitalização, afetando sobretudo crianças saudáveis ​​com menos de 5 anos de idade. Os patógenos mais frequentemente detectados foram bactérias, principalmente Shigella sp., Salmonella sp. e E. coli diarreiogênica. Foi relatada elevada prescrição de antibióticos, cumprindo na maioria dos casos as recomendações.

Nueve años de tendencia en la resistencia a ciprofloxacina por Escherichia coli: estudio transversal en un hospital de Colombia / Nine-year trend in Escherichia coli resistance to ciprofloxacin: cross-sectional study in a hospital in Colombia / Tendência de nove anos na resistência da Escherichia coli à ciprofloxacina: um estudo transversal em um hospital na Colômbia

Resumen: La ciprofloxacina es un antibiótico de importancia crítica para la salud humana. El aumento de la resistencia de Escherichia coli a ciprofloxacina es un problema de salud pública global por su importancia en el tratamiento de infecciones urinarias complicadas y otras infecciones graves; sin embargo, su prescripción es alta en el caribe colombiano. El objetivo fue determinar la tendencia de resistencia de E. coli a ciprofloxacina en un hospital colombiano de alta complejidad. A partir de reportes de antibiogramas, los aislados fueron categorizados según los criterios del Instituto de Normas Clínicas y de Laboratorio de los Estados Unidos para cada año estudiado; se calcularon proporciones y se exploraron diferencias en la sensibilidad con pruebas χ2. Se utilizó la prueba de Cochran-Armitage para evaluar la tendencia de la resistencia. Valores de p ≤ 0,05 se consideraron significativos. Se analizaron 6.848 aislados, encontrándose una resistencia de 49,31%. Según el origen, la resistencia más alta fue en muestras comunitarias (51,96% - IC95%: 50,51; 53,41), y por tipo de muestra, en piel y tejidos (61,76% - IC95%: 56,96; 66,35) y orina (48,97% - IC95%: 47,71; 50,23). Se halló una tendencia al aumento en la resistencia por año (p < 0,0001), en muestras comunitarias (p = 0,0002) y en orina (p < 0,0001). La resistencia a ciprofloxacina es alta y tiende al aumento en comunidad y en orina, superando el límite establecido para su uso a nivel ambulatorio, lo que es preocupante por la alta prescripción de fluoroquinolonas en la localidad.

Abstract: Ciprofloxacin is a critically important antibiotic for human health. The increase of Escherichia coli resistance to ciprofloxacin is a global public health problem due to its importance in the treatment of complicated urinary tract infections and other serious infections; however, its prescription is high in the Colombian Caribbean. The objective was to determine the resistance trend of E. coli to ciprofloxacin in a Colombian hospital of high complexity. From antibiogram reports, isolates were categorized according to Clinical and Laboratory Standards Institute criteria for each year studied; proportions were calculated and differences in sensitivity were explored using the χ2 test. The Cochran-Armitage test was used to evaluate the resistance trend. Significance was considered when p-value ≤ 0.05. In total, 6,848 isolates were analyzed, and 49.31% resistance was found. According to origin, the highest resistance was in community samples (51.96% - 95%CI: 50.51; 53.41), and by type of sample, in skin and tissues (61.76% - 95%CI: 56.96; 66.35) and urine (48.97% - 95%CI: 47.71; 50.23). Increasing trends were observed for resistance per year (p < 0.0001), community samples (p = 0.0002) and urine (p < 0.0001). Resistance to ciprofloxacin is high and tends to increase in the community and in urine, exceeding the limit established for its use at the ambulatory level, which is of concern due to the high prescription of fluoroquinolones in the locality.

Resumo: A ciprofloxacina é um antibiótico extremamente importante para a saúde humana. O aumento da resistência da Escherichia coli à ciprofloxacina é um problema de saúde pública global devido à sua importância no tratamento de infecções complicadas do trato urinário e outras infecções graves; no entanto, sua prescrição é alta no caribe colombiano. O objetivo foi determinar a tendência de resistência da E. coli à ciprofloxacina em um hospital colombiano de alta complexidade. A partir de relatórios de antibiogramas, os isolados foram categorizados de acordo com os critérios do Instituto de Padrões Clínicos e Laboratoriais dos Estados Unidos para cada ano estudado; as proporções foram calculadas e as diferenças de sensibilidade foram exploradas com os testes χ2. O teste de Cochran-Armitage foi usado para avaliar a tendência de resistência. Os valores de p ≤ 0,05 foram considerados significativos. Um total de 6.848 isolados foi testado e foi encontrada uma taxa de resistência de 49,31%. Por origem, a resistência foi mais alta em amostras comunitárias (51,96% - IC95%: 50,51; 53,41) e, por tipo de amostra, em pele e tecidos (61,76% - IC95%: 56,96; 66,35) e urina (48,97% - IC95%: 47,71; 50,23). Foi encontrada uma tendência de aumento na resistência por ano (p < 0,0001), em amostras da comunidade (p = 0,0002) e na urina (p < 0,0001). A resistência à ciprofloxacina é alta e tende a aumentar na comunidade e na urina, excedendo o limite estabelecido para uso ambulatorial, o que é preocupante, dada a alta prescrição de fluoroquinolonas na localidade.