To investigate the effect and the mechanism of ppk1 gene deletion on the drug susceptibility of uropathogenic Escherichia coli producing extended-spectrum beta-lactamases (ESBLs-UPEC). The study was an experimental study. From March to April 2021, a strain of ESBLs-UPEC (genotype was TEM combined with CTX-M-14) named as UE210113, was isolated from urine sample of the patient with urinary tract infection in the Laboratory Department of Guangzhou Eighth People's Hospital, meanwhile its ppk1 gene knock-out strain Δpk1 and complemented strain Δpk1-C were constructed by suicide plasmid homologous recombination technique, which was used to study the effect of ppk1 gene on ESBLs-UPEC drug sensitivity and its mechanism. The drug susceptibility of UE210113, Δpk1, and Δpk1-C were measured by Vitek2 Compact System and broth microdilution method. The quantitative expression of ESBLs, outer membrane protein and multidrug efflux systems encoding genes of UE210113, Δpk1 and Δpk1-C were performed by using qRT-PCR analysis. By using two independent sample Mann-Whitney U test, the drug susceptibility results showed that, compared with UE210113 strain, the sensitivities of Δpk1 to ceftazidime, cefepime, tobramycin, minocycline and cotrimoxazole were enhanced (Z=-2.121,P<0.05;Z=-2.236,P<0.05;Z=-2.236,P<0.05;Z=-2.121,P<0.05), and the drug susceptibility of Δpk1-C restored to the same as which of UE210113 (Z=0,P>0.05). The expression levels of ESBLs-enconding genes bla_TEM and bla_CTX-M-14 in Δpk1 were significantly down-regulated compared with UE210113, but the expression was not restored in Δpk1-C. The expression of outer membrane protein gene omp F in Δpk1 was significantly up-regulated, while the expression of omp A and omp C were down-regulated. The results showed that the expression of multidrug efflux systems encoding genes tol C, mdt A and mdtG were down-regulated in Δpk1 compared with UE210113. The expression of all of the outer membrane protein genes and the multidrug efflux systems genes were restored in Δpk1-C. In conclusion,the lost of ppk1 gene can affect the expression of the outer membrane protein and multidrug efflux systems encoding genes of ESBLs-UPEC, which increase the sensitivity of ESBLs-UPEC to various drugs.
Humans ; beta-Lactamases/metabolism* ; Uropathogenic Escherichia coli/metabolism* ; Urinary Tract Infections ; Plasmids ; Membrane Proteins/genetics* ; Escherichia coli Infections ; Microbial Sensitivity Tests ; Anti-Bacterial Agents/pharmacology*

To investigate the effect and the mechanism of ppk1 gene deletion on the drug susceptibility of uropathogenic Escherichia coli producing extended-spectrum beta-lactamases (ESBLs-UPEC). The study was an experimental study. From March to April 2021, a strain of ESBLs-UPEC (genotype was TEM combined with CTX-M-14) named as UE210113, was isolated from urine sample of the patient with urinary tract infection in the Laboratory Department of Guangzhou Eighth People's Hospital, meanwhile its ppk1 gene knock-out strain Δpk1 and complemented strain Δpk1-C were constructed by suicide plasmid homologous recombination technique, which was used to study the effect of ppk1 gene on ESBLs-UPEC drug sensitivity and its mechanism. The drug susceptibility of UE210113, Δpk1, and Δpk1-C were measured by Vitek2 Compact System and broth microdilution method. The quantitative expression of ESBLs, outer membrane protein and multidrug efflux systems encoding genes of UE210113, Δpk1 and Δpk1-C were performed by using qRT-PCR analysis. By using two independent sample Mann-Whitney U test, the drug susceptibility results showed that, compared with UE210113 strain, the sensitivities of Δpk1 to ceftazidime, cefepime, tobramycin, minocycline and cotrimoxazole were enhanced (Z=-2.121,P<0.05;Z=-2.236,P<0.05;Z=-2.236,P<0.05;Z=-2.121,P<0.05), and the drug susceptibility of Δpk1-C restored to the same as which of UE210113 (Z=0,P>0.05). The expression levels of ESBLs-enconding genes bla_TEM and bla_CTX-M-14 in Δpk1 were significantly down-regulated compared with UE210113, but the expression was not restored in Δpk1-C. The expression of outer membrane protein gene omp F in Δpk1 was significantly up-regulated, while the expression of omp A and omp C were down-regulated. The results showed that the expression of multidrug efflux systems encoding genes tol C, mdt A and mdtG were down-regulated in Δpk1 compared with UE210113. The expression of all of the outer membrane protein genes and the multidrug efflux systems genes were restored in Δpk1-C. In conclusion,the lost of ppk1 gene can affect the expression of the outer membrane protein and multidrug efflux systems encoding genes of ESBLs-UPEC, which increase the sensitivity of ESBLs-UPEC to various drugs.
Humans ; beta-Lactamases/metabolism* ; Uropathogenic Escherichia coli/metabolism* ; Urinary Tract Infections ; Plasmids ; Membrane Proteins/genetics* ; Escherichia coli Infections ; Microbial Sensitivity Tests ; Anti-Bacterial Agents/pharmacology*

The widespread of tigecycline resistance gene tet(X4) has a serious impact on the clinical efficacy of tigecycline. The development of effective antibiotic adjuvants to combat the looming tigecycline resistance is needed. The synergistic activity between the natural compound β-thujaplicin and tigecycline in vitro was determined by the checkerboard broth microdilution assay and time-dependent killing curve. The mechanism underlining the synergistic effect between β-thujaplicin and tigecycline against tet(X4)-positive Escherichia coli was investigated by determining cell membrane permeability, bacterial intracellular reactive oxygen species (ROS) content, iron content, and tigecycline content. β-thujaplicin exhibited potentiation effect on tigecycline against tet(X4)-positive E. coli in vitro, and presented no significant hemolysis and cytotoxicity within the range of antibacterial concentrations. Mechanistic studies demonstrated that β-thujaplicin significantly increased the permeability of bacterial cell membranes, chelated bacterial intracellular iron, disrupted the iron homeostasis and significantly increased intracellular ROS level. The synergistic effect of β-thujaplicin and tigecycline was identified to be related to interfere with bacterial iron metabolism and facilitate bacterial cell membrane permeability. Our studies provided theoretical and practical data for the application of combined β-thujaplicin with tigecycline in the treatment of tet(X4)-positive E. coli infection.
Humans ; Tigecycline/pharmacology* ; Escherichia coli/metabolism* ; Reactive Oxygen Species/therapeutic use* ; Plasmids ; Anti-Bacterial Agents/metabolism* ; Escherichia coli Infections/microbiology* ; Bacteria/genetics* ; Microbial Sensitivity Tests

China ; epidemiology ; Escherichia coli Infections ; epidemiology ; microbiology ; Escherichia coli O157 ; classification ; isolation & purification ; metabolism ; Humans ; Shiga Toxin 1 ; biosynthesis ; isolation & purification ; Shiga Toxin 2 ; biosynthesis ; isolation & purification ; Sorbitol

OBJECTIVETo study the role of outer membrane protein T (OmpT) in the pathogenesis of uropathogenic Escherichia.coli.

METHODSIn cultured human bladder epithelial cell line 5637, we examined the adhesion ability of wild-type (CFT073), ompT gene knockout (COTD), and revertant (pST) strains of E.coli to the cells and the extracellular matrix (ECM). The expressions of the adhesion gene iha and virulence gene iroN were detected by real-time PCR. Murine models of urinary tract infection with the 3 strains were established to evaluate the bacterial burden of the bladder and kidney tissue and bacterial counts in blood. We also detected the expressions of interleukin-6 (IL-6) and IL-8 in the bladder and kidney tissues of the mice.

RESULTThe COTD strain showed a significantly lower cell adhesion rate than CFT073 strain [(4.62∓0.39)% vs (8.81∓1.13)%, P<0.05] with also a lower ECM-adhesion rate [(4.95∓0.59)% vs (8.85∓0.79)%, P<0.05]. The mRNA expressions of iha and iroN in CFT073 strain were 2.1 and 3.8 times that of COTD strain. In the mouse model, the mean bacterial load of CFT073 strain in the bladder tissue was 6.36∓0.06, significantly greater than that of COTD (6.01∓0.07) and revertant (6.29∓0.06) strains (P<0.05); the bacterial load of CFT073 strain in the kidney tissue was also significantly higher than that of COTD strain (6.25∓0.05 vs 5.87∓0.06, P<0.05). In mice infected with the wild-type, knockout, and revertant strains, the detection rates of IL-6, which were identical to those of IL-8, in the inflammatory bladder and kidney tissues were 60%, 12.5%, and 50%, respectively.

CONCLUSIONSOmpT may regulate the expression of the adhesion gene iha and the transferrin gene iroN to affect the adhesion of uropathogenic E.coli to host cells.

Animals ; Bacterial Adhesion ; Bacterial Load ; Bacterial Outer Membrane Proteins ; metabolism ; Cell Line, Tumor ; Escherichia coli Infections ; pathology ; Escherichia coli Proteins ; metabolism ; Gene Knockout Techniques ; Humans ; Inflammation ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Kidney ; microbiology ; Mice ; Peptide Hydrolases ; metabolism ; Receptors, Cell Surface ; metabolism ; Urinary Bladder ; microbiology ; Urinary Tract Infections ; microbiology ; pathology ; Uropathogenic Escherichia coli ; pathogenicity

The use of intrauterine devices (IUDs) have been widespread since the 1960s. In 2002, the World Health Organization estimated that approximately 160 million women worldwide use IUDs. However, IUDs are associated with short-term complications such as vaginal bleeding, pelvic discomfort, dyspareunia and pelvic infection. Herein, we report the case of a woman who had recurrent urinary tract infection (UTI) due to the use of an IUD, even after treatment. The patient developed four episodes of UTI within a seven-month period after IUD insertion. During each episode of UTI, extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli (E. coli) was cultured from the patient’s midstream urine. The IUD was finally removed, and culture of the removed IUD was positive for ESBL-producing E. coli. An infected IUD as a source of recurrent UTI should be considered in women with IUD in situ who develop recurrent UTI even after treatment.
Adult ; Anti-Bacterial Agents ; therapeutic use ; Escherichia coli Infections ; drug therapy ; etiology ; Female ; Humans ; Intrauterine Devices ; adverse effects ; Microbial Sensitivity Tests ; Recurrence ; Treatment Outcome ; Urinary Tract Infections ; drug therapy ; etiology ; microbiology ; Uropathogenic Escherichia coli ; enzymology ; beta-Lactamases ; metabolism

PURPOSE: Extended spectrum beta-lactamases (ESBLs) are cephalosporinases that confer resistance to a wide variety of oxyimino cephalosporins and create serious therapeutic problems. In addition, the quinolone resistance qnr genes are becoming increasingly prevalent in clinical isolates, some of which also produce ESBL. This study was designed to evaluate the occurrence and genotypic distribution of ESBL producing Escherichia coli (E. coli) and Klebsiella pneumoniae (K. pneumoniae) as well as the prevalence and distribution of qnr genes in ESBL-producing isolates in a tertiary care hospital in Korea. MATERIALS AND METHODS: We tested a total of 111 ESBL-producing isolates of E. coli and K. pneumoniae, which were collected at Kyung Hee Medical Center from November 2006 to June 2008. ESBL production was determined by the Clinical and Laboratory Standards Institute (CLSI) ESBL confirmatory test. The cefotaxime and ceftazidime resistance of the ESBL-producers were transferred to azide-resistant E. coli J53 by conjugation. The presence and identity of ESBL and qnr genes were determined by polymerase chain reaction (PCR) and nucleotide sequencing. RESULTS: The prevalence of ESBLs was 17.7% (297/1,680) of E. coli and 26.5% (240/904) of K. pneumoniae in our hospital during the study periods. Of the 111 collected isolates, 69 isolates were E. coli and 42 isolates were K. pneumoniae. The most prevalent ESBL genotype was CTX-M15. Among the ESBL-producing isolates, 4 E. coli (5.8%) and 17 K. pneumoniae (40.5%) contained qnr genes. qnrB4 was the most frequent type in both E. coli and K. pneumoniae. CONCLUSION: CTX-M15 was the most frequently encountered ESBL. In addition, a high prevalence of qnr genes among ESBL-producing K. pneumoniae was identified in this study.
Azides/pharmacology ; Bacterial Proteins/*metabolism ; Cefotaxime/pharmacology ; Ceftazidime/pharmacology ; Drug Resistance, Multiple, Bacterial/genetics ; Escherichia coli/drug effects/*enzymology ; Escherichia coli Infections/*microbiology ; Escherichia coli Proteins/*metabolism ; Humans ; Klebsiella Infections/*microbiology ; Klebsiella pneumoniae/drug effects/*enzymology ; Korea ; Microbial Sensitivity Tests ; Polymerase Chain Reaction ; beta-Lactamases/*metabolism

The 987P fimbriae of enterotoxigenic Escherichia coli (ETEC) mediates adhesive interactions with brush border vesicle (BBV) of the intestinal epithelial cells from the neonatal piglets. By adhering to intestinal epithelial cells, producing localized multiplication, the 987P ETEC can progress to mucosal surface colonization and concomitant effective enterotoxin delivery. To identify the receptors for the 987P, BBV proteins from piglet intestinal villous epithelial cells were separated by SDS-PAGE and analyzed by Ligand blot, protein bands with a set of 32-35 kD recognized by the 987P fimbriae were subjected to in gel proteolysis with trypsin. The tryptic fragments were separated by microbore reversed phase HPLC(RP-HPLC), samples shown to contain one major peak by MALDI-MS were submitted to Edman sequencing, three peptides were sequenced successfully and the all of three peptides matched the sequences of human or porcine histone H1 proteins. Porcine histone H1 proteins isolated from both piglet intestinal epithelial cells and BBV demonstrated the same SDS-PAGE migration pattern and 987P-binding properties as the 987P-specific protein receptors from piglet intestinal brush border did. The above results indicated that the 987P protein receptors are piglet BBV-derived Histone H1 proteins.
Adhesins, Escherichia coli ; metabolism ; Amino Acid Sequence ; Animals ; Enterotoxigenic Escherichia coli ; metabolism ; pathogenicity ; Escherichia coli Infections ; microbiology ; veterinary ; Fimbriae Proteins ; metabolism ; Fimbriae, Bacterial ; chemistry ; Histones ; genetics ; metabolism ; Host-Pathogen Interactions ; Intestinal Mucosa ; metabolism ; Molecular Sequence Data ; Receptors, Cell Surface ; genetics ; metabolism ; Swine

To examine the genetic background of avian pathogenic Escherichia coli (APEC) that affects virulence of this microorganism, we characterized the virulence genes of 101 APEC strains isolated from infected chickens between 1985~2005. Serotypes were determined with available anti-sera and median lethal doses were determined in subcutaneously inoculated chicks. The virulence genes we tested included ones encoding type 1 fimbriae (fimC), iron uptake-related (iroN, irp2, iucD, and fyuA), toxins (lt, st, stx1, stx2, and vat), and other factors (tsh, hlyF, ompT, and iss). Twenty-eight strains were found to be O1 (2.0%), O18 (3.0%), O20 (1.0%), O78 (19.8%), and O115 (2.0%) serotypes. The iroN (100%) gene was observed most frequently followed by ompT (94.1%), fimC (90.1%), hlyF (87.1%), iss (78.2%), iucD (73.3%), tsh (61.4%), fyuA (44.6%), and irp2 (43.6%). The strains were negative for all toxin genes except for vat (10.9%). All the strains were classified into 27 molecular pathotypes (MPs). The MP25, MP19, and MP10 pathotypes possessing iroN-fimC-ompT-hlyF-iucD-tsh-iss-irp2-fyuA (22.8%), iroN-fimC-ompT-hlyF-iucD-tsh-iss (21.8%), and iroN-fimC-ompT-hlyF-iss (11.9%) genotypes, respectively, were predominant. Redundancy of iron uptake-related genes was clearly observed and some strains were associated with higher mortality than others. Therefore, strains with the predominant genotypes can be used for diagnosis and vaccine.
Animals ; Bacterial Proteins/genetics/metabolism ; Chickens ; Escherichia coli/*classification/genetics/*pathogenicity ; Escherichia coli Infections/epidemiology/microbiology/*veterinary ; Gene Expression Regulation, Bacterial/physiology ; Phylogeny ; Poultry Diseases/epidemiology/*microbiology ; Republic of Korea/epidemiology ; Virulence

Cows infected with Escherichia (E.) coli usually experience severe clinical symptoms, including damage to mammary tissues, reduced milk yield, and altered milk composition. In order to investigate the host response to E. coli infection and discover novel markers for mastitis treatment, mammary tissue samples were collected from healthy cows and bovines with naturally occurring severe E. coli mastitis. Changes of mammary tissue proteins were examined using two-dimensional gel electrophoresis and label-free proteomic approaches. A total of 95 differentially expressed proteins were identified. Of these, 56 proteins were categorized according to molecular function, cellular component, and biological processes. The most frequent biological processes influenced by the proteins were response to stress, transport, and establishment of localization. Furthermore, a network analysis of the proteins with altered expression in mammary tissues demonstrated that these factors are predominantly involved with binding and structural molecule activities. Vimentin and alpha-enolase were central "functional hubs" in the network. Based on results from the present study, disease-induced alterations of protein expression in mammary glands and potential markers for the effective treatment of E. coli mastitis were identified. These data have also helped elucidate defense mechanisms that protect the mammary glands and promote the pathogenesis of E. coli mastitis.
Animals ; Cattle ; Electrophoresis, Gel, Two-Dimensional/veterinary ; Escherichia coli/*physiology ; Escherichia coli Infections/genetics/immunology/microbiology/*veterinary ; Female ; Mammary Glands, Animal/*immunology/pathology ; Mastitis, Bovine/*genetics/immunology/microbiology ; Proteome/*genetics/metabolism ; *Proteomics