Genetic features of antimicrobial drug-susceptible extraintestinal pathogenic Escherichia coli pandemic sequence type 95.

IMPORTANCE: Despite the increasing prevalence of antibiotic-resistant Escherichia coli strains that cause urinary tract and bloodstream infections, a major pandemic lineage of extraintestinal pathogenic E. coli (ExPEC) ST95 has a comparatively low frequency of drug resistance. We compared the genomes of 1,749 ST95 isolates to identify genetic features that may explain why most strains of ST95 resist becoming drug-resistant. Identification of such genomic features could contribute to the development of novel strategies to prevent the spread of antibiotic-resistant genes and devise new measures to control antibiotic-resistant infections.

Genetic Predictive Factors for Nonsusceptible Phenotypes and Multidrug Resistance in Expanded-Spectrum Cephalosporin-Resistant Uropathogenic Escherichia coli from a Multicenter Cohort: Insights into the Phenotypic and Genetic Basis of Coresistance.

Antimicrobial resistance in urinary tract infections (UTIs) is a major public health concern. This study aims to characterize the phenotypic and genetic basis of multidrug resistance (MDR) among expanded-spectrum cephalosporin-resistant (ESCR) uropathogenic Escherichia coli (UPEC) causing UTIs in California patient populations. Between February and October 2019, 577 ESCR UPEC isolates were collected from patients at 6 clinical laboratory sites across California. Lineage and antibiotic resistance genes were determined by analysis of whole-genome sequence data. The lineages ST131, ST1193, ST648, and ST69 were predominant, representing 46%, 5.5%, 4.5%, and 4.5% of the collection, respectively. Overall, 527 (91%) isolates had an expanded-spectrum ß-lactamase (ESBL) phenotype, with blaCTX-M-15, blaCTX-M-27, blaCTX-M-55, and blaCTX-M-14 being the most prevalent ESBL genes. In the 50 non-ESBL phenotype isolates, 40 (62%) contained blaCMY-2, which was the predominant plasmid-mediated AmpC (pAmpC) gene. Narrow-spectrum ß-lactamases, blaTEM-1B and blaOXA-1, were also found in 44.9% and 32.1% of isolates, respectively. Among ESCR UPEC isolates, isolates with an ESBL phenotype had a 1.7-times-greater likelihood of being MDR than non-ESBL phenotype isolates (P < 0.001). The cooccurrence of blaCTX-M-15, blaOXA-1, and aac(6')-Ib-cr within ESCR UPEC isolates was strongly correlated. Cooccurrence of blaCTX-M-15, blaOXA-1, and aac(6')-Ib-cr was associated with an increased risk of nonsusceptibility to piperacillin-tazobactam, cefepime, fluoroquinolones, and amikacin as well as MDR. Multivariate regression revealed the presence of blaCTX-M-55, blaTEM-1B, and the ST131 genotype as predictors of MDR. IMPORTANCE The rising incidence of resistance to expanded-spectrum cephalosporins among Escherichia coli strains, the most common cause of UTIs, is threatening our ability to successfully empirically treat these infections. ESCR E. coli strains are often MDR; therefore, UTI caused by these organisms often leads to treatment failure, increased length of hospital stay, and severe complications (D. G. Mark, Y.-Y. Hung, Z. Salim, N. J. Tarlton, et al., Ann Emerg Med 78:357-369, 2021, https://doi.org/10.1016/j.annemergmed.2021.01.003). Here, we performed an in-depth analysis of genetic factors of ESCR E. coli associated with coresistance and MDR. Such knowledge is critical to advance UTI diagnosis, treatment, and antibiotic stewardship.

Genotypic analysis of uropathogenic Escherichia coli to understand factors that impact the prevalence of ß-lactam-resistant urinary tract infections in a community.

OBJECTIVES: To determine the contribution of specific uropathogenic Escherichia coli (UPEC) lineages, drug resistance genes, and plasmid incompatibility/replicon (Inc) groups to the prevalence of ß-lactam-resistant urinary tract infections (UTIs) in a university community. METHODS: Urine samples were consecutively collected and cultured over a 2-year period from patients presenting to a university health centre with symptoms of UTI. Isolated UPEC were subtyped by multilocus sequence typing and fimH typing, and tested by PCR and sequencing for ß-lactamase genes and plasmid Inc groups. RESULTS: Among 273 UPEC isolates, 85 (31%) were ampicillin-resistant (AMP-R) and 188 (69%) were susceptible to all ß-lactam drugs (AMP-S). Six lineages accounted for two-thirds of the isolates: ST95 (21%), ST69 (11%), ST420 (11%), ST73 (10%), ST127 (8%), and ST404 (3%). ST69 and ST404 were associated with AMP-R (P=0.003, P=0.0005), while ST420 and ST127 were associated with AMP-S (P<0.0001, P=0.027). ST95 contained four fimH types; the ST95/f-6 sublineage was more frequently identified among the AMP-R population (P = 0.009), while the ST95/f-47 sublineage was more frequently identified among the AMP-S population (P=0.007). The most common ß-lactamase gene was blaTEM, which was identified in 81 (95%) AMP-R isolates. IncFIB, IncFIA, and IncB/O type plasmids were the most commonly identified types, and were associated with ß-lactam resistance (P<0.001 for all). CONCLUSIONS: These observations indicate that the prevalence of ß-lactam-resistant UTIs in this community was largely determined by a limited set of circulating UPEC STs and sublineages, carrying TEM ß-lactamase genes that were likely encoded on one of three Inc type plasmids.

An Enzyme-Mediated Amplification Strategy Enables Detection of ß-Lactamase Activity Directly in Unprocessed Clinical Samples for Phenotypic Detection of ß-Lactam Resistance.

Biochemical assays that can identify ß-lactamase activity directly from patient samples have the potential to significantly improve the treatment of bacterial infections. However, current ß-lactamase probes do not have the sensitivity needed to measure ß-lactam resistance directly from patient samples. Here, we report the development of an instrument-free signal amplification technology, DETECT, that connects the activity of two enzymes in series to effectively amplify the activity of ß-lactamase 40 000-fold, compared to the standard ß-lactamase probe nitrocefin.

Stress-Adaptive Responses Associated with High-Level Carbapenem Resistance in KPC-Producing Klebsiella pneumoniae.

Carbapenem-resistant Enterobacteriaceae (CRE) organisms have emerged to become a major global public health threat among antimicrobial resistant bacterial human pathogens. Little is known about how CREs emerge. One characteristic phenotype of CREs is heteroresistance, which is clinically associated with treatment failure in patients given a carbapenem. Through in vitro whole-transcriptome analysis we tracked gene expression over time in two different strains (BR7, BR21) of heteroresistant KPC-producing Klebsiella pneumoniae, first exposed to a bactericidal concentration of imipenem followed by growth in drug-free medium. In both strains, the immediate response was dominated by a shift in expression of genes involved in glycolysis toward those involved in catabolic pathways. This response was followed by global dampening of transcriptional changes involving protein translation, folding and transport, and decreased expression of genes encoding critical junctures of lipopolysaccharide biosynthesis. The emerged high-level carbapenem-resistant BR21 subpopulation had a prophage (IS1) disrupting ompK36 associated with irreversible OmpK36 porin loss. On the other hand, OmpK36 loss in BR7 was reversible. The acquisition of high-level carbapenem resistance by the two heteroresistant strains was associated with distinct and shared stepwise transcriptional programs. Carbapenem heteroresistance may emerge from the most adaptive subpopulation among a population of cells undergoing a complex set of stress-adaptive responses.

Genomic Analysis of Factors Associated with Low Prevalence of Antibiotic Resistance in Extraintestinal Pathogenic Escherichia coli Sequence Type 95 Strains.

Extraintestinal pathogenic Escherichia coli (ExPEC) strains belonging to multilocus sequence type 95 (ST95) are globally distributed and a common cause of infections in humans and domestic fowl. ST95 isolates generally show a lower prevalence of acquired antimicrobial resistance than other pandemic ExPEC lineages. We took a genomic approach to identify factors that may underlie reduced resistance. We fully assembled genomes for four ST95 isolates representing the four major fimH-based lineages within ST95 and also analyzed draft-level genomes from another 82 ST95 isolates, largely from the western United States. The fully assembled genomes of antibiotic-resistant isolates carried resistance genes exclusively on large (>90-kb) IncFIB/IncFII plasmids. These replicons were common in the draft genomes as well, particularly in antibiotic-resistant isolates, but we also observed multiple instances of a smaller (8.3-kb) ampicillin resistance plasmid that had been previously identified in Salmonella enterica. Among ST95 isolates, pansusceptibility to antibiotics was significantly associated with the fimH6 lineage and the presence of homologs of the previously identified 114-kb IncFIB/IncFII plasmid pUTI89, both of which were also associated with reduced carriage of other plasmids. Potential mechanistic explanations for lineage- and plasmid-specific effects on the prevalence of antibiotic resistance within the ST95 group are discussed. IMPORTANCE Antibiotic resistance in bacterial pathogens is a major public health concern. This work was motivated by the observation that only a small proportion of ST95 isolates, a major pandemic lineage of extraintestinal pathogenic E. coli, have acquired antibiotic resistance, in contrast to many other pandemic lineages. Understanding bacterial genetic factors that may prevent acquisition of resistance could contribute to the development of new biological, medical, or public health strategies to reduce antibiotic-resistant infections.

Rapid induction of high-level carbapenem resistance in heteroresistant KPC-producing Klebsiella pneumoniae.

Enterobacteriaceae strains producing the Klebsiella pneumoniae carbapenemase (KPC) have disseminated worldwide, causing an urgent threat to public health. KPC-producing strains often exhibit low-level carbapenem resistance, which may be missed by automated clinical detection systems. In this study, eight Klebsiella pneumoniae strains with heterogeneous resistance to imipenem were used to elucidate the factors leading from imipenem susceptibility to high-level resistance as defined by clinical laboratory testing standards. Time-kill analysis with an inoculum as low as 3 × 10(6) CFU/ml and concentrations of imipenem 8- and 16-fold higher than the MIC resulted in the initial killing of 99.9% of the population. However, full recovery of the population occurred by 20 h of incubation in the same drug concentrations. Population profiles showed that recovery was mediated by a heteroresistant subpopulation at a frequency of 2 × 10(-7) to 3 × 10(-6). Samples selected 2 h after exposure to imipenem were as susceptible as the unexposed parental strain and produced the major outer membrane porin OmpK36. However, between 4 to 8 h after exposure, OmpK36 became absent, and the imipenem MIC increased at least 32-fold. Individual colonies isolated from cultures after 20 h of exposure revealed both susceptible and resistant subpopulations. Once induced, however, the high-level imipenem resistance was maintained, and OmpK36 remained unexpressed even without continued carbapenem exposure. This study demonstrates the essential coordination between blaKPC and ompK36 expression mediating high-level imipenem resistance from a population of bacteria that initially exhibits a carbapenem-susceptibility phenotype.

Association of class 1 and 2 integrons with multidrug-resistant Acinetobacter baumannii international clones and Acinetobacter nosocomialis isolates.

The Acinetobacter baumannii clonal complex 113/79 (CC113/79) and class 2 integrons predominate in Latin America; a relationship between these characteristics was explored. The presence of integrases was determined in successive hospital Acinetobacter isolates (163 A. baumannii isolates and 72 Acinetobacter nosocomialis isolates). Most isolates had integrons, but class 1 and 2 integrons were present significantly more often in CC109/1 and CC113/79, respectively. The high prevalence of CC113/79 in Latin America may account for the predominance of class 2 integrons.

Characterization of ESBL-producing Escherichia coli and Klebsiella pneumoniae from bloodstream infections in three hospitals in Bucharest, Romania: a preliminary study.

BACKGROUND: Our objective was to describe the distribution of extended-spectrum beta-lactamase (ESBL) genes found in Escherichia coli and Klebsiella pneumoniae isolates from hospital-acquired or community-onset bloodstream infections (BSIs) in Bucharest, Romania. METHODS: E. coli and K. pneumoniae isolates, resistant to at least one antimicrobial drug family, were analyzed for genes encoding ESBL by PCR and sequencing. The E. coli isolates were screened by an ST131 clone allele-specific PCR for the pabB gene. RESULTS: ESBL genes were found in 30 (35%) of 85 drug-resistant E. coli and 19 (70%) of 27 drug-resistant K. pneumoniae. Twenty-nine (97%) E. coli carried blaCTX-M-15. Nineteen (63%) E. coli carrying an ESBL gene belonged to the ST131 clonal group. The blaCTX-M-15 gene was found in 15 (79%) and blaSHV-12 in 12 (63%) K. pneumoniae isolates. CONCLUSION: In Bucharest CTX-M15 was the most frequently encountered ESBL in both E. coli and K. pneumoniae BSI isolates regardless of the setting of infections.

Clonal composition and community clustering of drug-susceptible and -resistant Escherichia coli isolates from bloodstream infections.

Multidrug-resistant Escherichia coli strains belonging to a single lineage frequently account for a large proportion of extraintestinal E. coli infections in many parts of the world. However, limited information exists on the community prevalence and clonal composition of drug-susceptible E. coli strains. Between July 2007 and September 2010, we analyzed all consecutively collected Gram-negative bacterial isolates from patients with bloodstream infection (BSI) admitted to a public hospital in San Francisco for drug susceptibility and associated drug resistance genes. The E. coli isolates were genotyped for fimH single nucleotide polymorphisms (SNPs) and multilocus sequence types (MLSTs). Among 539 isolates, E. coli accounted for 249 (46%); 74 (30%) of them were susceptible to all tested drugs, and 129 (52%) were multidrug resistant (MDR). Only five MLST genotypes accounted for two-thirds of the E. coli isolates; the most common were ST131 (23%) and ST95 (18%). Forty-seven (92%) of 51 ST131 isolates, as opposed to only 8 (20%) of 40 ST95 isolates, were MDR (P < 0.0001). The Simpson's diversity index for drug-susceptible ST genotypes was 87%, while the index for MDR ST genotypes was 81%. ST95 strains were comprised of four fimH types, and one of these (f-6) accounted for 67% of the 21 susceptible isolates (P < 0.003). A large proportion (>70%) of both MDR and susceptible E. coli BSI isolates represented community-onset infections. These observations show that factors other than the selective pressures of antimicrobial agents used in hospitals contribute to community-onset extraintestinal infections caused by clonal groups of E. coli regardless of their drug resistance.