Characteristics of Escherichia coli Urine Isolates and Risk Factors for Secondary Bloodstream Infections in Patients with Urinary Tract Infections.

Escherichia coli is responsible for more than 80% of all incidences of urinary tract infections (UTIs). We assessed a total of 636 cases of patients with E. coli UTIs occurring in June 2019 in eight tertiary hospitals in South Korea for the traits of patients with E. coli UTIs, UTI-causative E. coli isolates, and risk factors associated with bloodstream infections (BSIs) secondary to UTIs. Antimicrobial susceptibility testing was conducted using the disc diffusion method, and the genes for extended-spectrum beta-lactamases (ESBLs) and plasmid-mediated ampC genes were screened by using PCR and sequencing. Multilocus sequence typing and virulence pheno-/genotyping were carried out. A total of 49 cases developed BSIs. The E. coli urine isolates primarily comprised sequence type 131 (ST131) (30.0%), followed by ST1193, ST95, ST73, and ST69. Three-quarters of the ST131 H30Rx isolates possessed the blaCTX-M-15-like gene, whereas 66% of H30R and 50% of H41 isolates possessed the blaCTX-M-14-like gene. All the ST1193 isolates showed biofilm formation ability, and three-quarters of the ST73 isolates exhibited hemolytic activity with high proportions of papC, focG, and cnf1 positivity. The prevalence of the ST131 H41 sublineage and its abundant CTX-M possession among the E. coli urine isolates were noteworthy; however, no specific STs were associated with bloodstream invasion. For BSIs secondary to UTIs, the papC gene was likely identified as a UTI-causative E. coli-related risk factor and urogenital cancer (odds ratio [OR], 12.328), indwelling catheter (OR, 3.218), and costovertebral angle tenderness (OR, 2.779) were patient-related risk factors. IMPORTANCE Approximately half of the BSIs caused by E. coli are secondary to E. coli UTIs. Since the uropathogenic E. coli causing most of the UTIs is genetically diverse, understanding the risk factors in the E. coli urine isolates causing the BSI is important for pathophysiology. Although the UTIs are some of the most common bacterial infectious diseases, and the BSIs secondary to the UTIs are commonly caused by E. coli, the assessments to find the risk factors are mostly focused on the condition of patients, not on the bacterial pathogens. Molecular epidemiology of the UTI-causative E. coli pathogens, together with the characterization of the E. coli urine isolates associated with the BSI secondary to UTI, was carried out, suggesting treatment options for the prevalent antimicrobial-resistant organisms.

Characterisation of successive Acinetobacter baumannii isolates from a deceased haemophagocytic lymphohistiocytosis patient.

In this study, 38 Acinetobacter baumannii isolates successively isolated from blood, skin swabs and tracheal aspirates from a single patient who died from haemophagocytic lymphohistiocytosis were investigated. The isolates were collected between March 2012 and August 2012. A. baumannii genotypes were determined by multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE). In vitro antimicrobial susceptibility testing was performed and colistin heteroresistance and persistence were evaluated. The structure of AbaR resistance islands was explored, and serum sensitivity was determined. Based on MLST analysis, all 38 A. baumannii isolates showed the same sequence type (ST138). However, PFGE analysis showed that isolates from blood samples belonged to different genotypes depending on the isolation time: whilst blood isolates obtained at the early stages showed restriction patterns similar to those of isolates from other sources, isolates obtained at later stages exhibited a distinct pattern. All isolates were resistant to imipenem, cefepime, ciprofloxacin and piperacillin/tazobactam. Five isolates from tracheal aspirates and one from a skin swab were resistant to polymyxins, and two isolates from skin swabs and one from another source were non-susceptible to tigecycline. All colistin-susceptible isolates showed heteroresistance to colistin, and four were persisters. Isolates from blood showed higher survival rates against human serum than those from other sources. This study shows that the patient was infected with more than one A. baumannii strain. Heteroresistance, persistence or evasion of the innate immune response may explain the failure of antimicrobial treatments in this patient.

Preservation of Acquired Colistin Resistance in Gram-Negative Bacteria.

Colistin-resistant mutants were obtained from 17 colistin-susceptible strains of Acinetobacter baumannii, Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli. The stability of colistin resistance in these mutants was investigated. Three of four colistin-resistant P. aeruginosa mutants recovered colistin susceptibility in colistin-free medium; however, colistin-susceptible revertants were obtained from only one strain each of A. baumannii and E. coli. No susceptible revertants were obtained from K. pneumoniae mutants.

Differential expression of two-component systems, pmrAB and phoPQ, with different growth phases of Klebsiella pneumoniae in the presence or absence of colistin.

We explored whether expression of pmrAB, pmrD, and phoPQ is dependent on growth phase with or without colistin exposure in colistin-resistant Klebsiella pneumoniae strains. In four colistin-resistant K. pneumoniae strains, the expression of pmrAB, pmrD, and phoPQ was evaluated at mid-log, late-log, and stationary phases in the absence or presence of colistin, by qRT-PCR. The expression pattern in the presence of colistin was different from that in the absence of colistin: overall, pmrAB, pmrD, and phoPQ expressed the highest at the stationary phase in the absence of colistin, but the expression of pmrD and phoPQ decreased with the growth in the presence of colistin. Exposure to colistin might change the expression patterns of two-component regulatory systems in colistin-resistant K. pneumoniae strains.

Improved PCR for identification of Pseudomonas aeruginosa.

The aim of the present study was to develop a noble and specific marker for a quantitative polymerase chain reaction (PCR) assay for the species-specific detection of Pseudomonas aeruginosa based on the O-antigen acetylase gene. It is an important challenge to characterize populations of the bacterium P. aeruginosa, an opportunist by virtue of its physiological and genetic adaptability. However, molecular and serological methods currently available for sensitive and specific detection of P. aeruginosa are by no means satisfactory because there have been critical defects in the diagnosis and identification of P. aeruginosa strains in that these assays also detect other Pseudomonas species, or do not obtain amplified products from P. aeruginosa strains. Therefore, a primer set was designed based on the O-antigen acetylase gene of P. aeruginosa PA01 because it has been known that this gene is structurally diverse among species. The specificity of the primer set was evaluated using genomic DNA from six isolates of P. aeruginosa, 18 different species of Pseudomonas, and 23 other reference pathogenic bacteria. The primer set used in the PCR assay amplified a 232-bp amplicon for only six P. aeruginosa strains. The assay was also able to detect at least 1.41 × 10(3) copies/µl of cloned amplified target DNA using purified DNA, or 2.7 × 10(2) colony-forming unit per reaction when using calibrated cell suspension. In conclusion, this assay can be applied as a practical diagnostic method for epidemiological research and the sanitary management of water with a low level or latent infection of P. aeruginosa.

Rapid and Specific Detection of Burkholderia glumae in Rice Seed by Real-Time Bio-PCR Using Species-Specific Primers Based on an rhs Family Gene.

In this study, we developed a reliable, quick, and accurate quantitative polymerase chain reaction (qPCR) assay to detect grain rot caused by Burkholderia glumae in rice seed. The control of bacterial grain rot is difficult, and the only practical methods for disease management rely on the use of pathogen-free seed, appropriate culture practices, and resistant cultivars. Therefore, the specific detection of this pathogen in seed is essential for effective control of the disease. However, other Burkholderia spp. are also detected by currently available molecular and serological methods. In this study, we exploited the available genome sequence information in public databases to develop specific PCR primers for accurate diagnosis of B. glumae. An SYBR Green real-time PCR primer set was designed based on the rhs family gene (YD repeat protein) of B. glumae BGR1 because these genes are structurally diverse. The specificity of the primers was evaluated using purified DNA from 5 isolates of B. glumae, 6 different species of Burkholderia, and 18 other reference pathogenic bacteria. The assay was able to detect at least one genome equivalent of cloned amplified target DNA using purified DNA or 1 CFU per reaction when using calibrated cell suspension. This method is rapid and reliable and has great potential for analyzing large numbers of samples.

Quantitative Real-Time Polymerase Chain Reaction Assay for Detection of Pectobacterium wasabiae Using YD Repeat Protein Gene-Based Primers.

The aim of this study was to develop a quantitative polymerase chain reaction (qPCR) assay for specific detection of Pectobacterium wasabiae using a primer pair based on the YD repeat protein gene for amplification of a 140-bp DNA fragment from infected wasabi (Wasabia japonica), a member of the crucifer family. The soft rot caused by P. wasabiae is an emerging disease that is present in many wasabi-producing areas. However, specific and reliable methods for identifying the pathogen are not available. Therefore, a qPCR primer set for accurate diagnosis of P. wasabiae was developed from publically available genome sequences. A SYBR Green qPCR primer set was designed based on a YD repeat protein gene of P. wasabiae WPP163 because it is known that this gene is structurally diverse among species, pathovars, or subspecies. The specificity of the primer set was evaluated using genomic DNA from 5 isolates of P. wasabiae, 5 different species of Pectobacterium, and 16 other pathogenic reference bacteria. The primer set used in the PCR assay successfully amplified a 140-bp amplicon for all five P. wasabiae strains. No amplification was obtained from 29 other pathogenic bacteria. The assay was also able to detect at least two genomic DNA, or 3 CFU per reaction, when using calibrated cell suspension.

Dissemination of ST131 and ST393 community-onset, ciprofloxacin-resistant Escherichia coli clones causing urinary tract infections in Korea.

OBJECTIVE: Ciprofloxacin-resistant Escherichia coli is growing concern in clinical settings. In this study, we investigated the distribution of virulence determinants and phylogenetic groups among community-onset, ciprofloxacin-resistant E. coli isolates causing urinary tract infections (UTIs) in Korea. In addition, the evidence of clonal spread in the community was also examined. METHODS: From November 2006 to August 2007, 543 community-onset E. coli isolates causing UTIs were collected as part of a multicenter surveillance study. In vitro susceptibility testing was performed using broth microdilution method. Distribution of virulence determinants and phylogenetic groupings were examined. In addition, multilocus sequence typing (MLST) analysis was performed. RESULTS: In vitro antimicrobial susceptibility testing revealed that 154 isolates (28.4%) were ciprofloxacin-resistant. Of these, 129 ciprofloxacin-resistant E. coli isolates were further characterized. As a result of phylogenetic subgrouping, we found that phylogenetic subgroup D was the most predominant (46 isolates, 35.7%), followed by B2 (44 isolates, 34.1%), A (21 isolates, 16.3%), and B1 (18 isolates, 14.0%). MLST analysis showed 48 sequence types (STs). The most prevalent ST was ST131 (32 isolates, 24.8%), followed by ST393 (23 isolates, 17.8%). While all ST131 isolates belonged to phylogenetic subgroup B2, which is known to be a highly virulent, all ST393 isolates belonged to subgroup D. ST131 and ST393 showed different profiles of virulence factors; papA, papG allele III, and traT genes were significantly more prevalent in ST131 than in ST393 (p values, <0.001). CONCLUSIONS: Based on genotyping, it is suggested that epidemic and virulent ciprofloxacin-resistant E. coli clones such as ST131 and ST393 have disseminated in Korea. However, the diversity of CTX-M genes in ST131 isolates may indicate that ESBL genes have been acquired independently or several ESBL-producing, ciprofloxacin-resistant E. coli clones may have disseminated in the Korean community.