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.

A rapid, antibiotic susceptibility test for multidrug-resistant, Gram-negative bacterial uropathogens using the biochemical assay, DETECT.

The increasing prevalence of extended spectrum ß-lactamases (ESBLs) and plasmid-mediated AmpC (pAmpC) ß-lactamases among Enterobacterales threatens our ability to treat urinary tract infections (UTIs). These organisms are resistant to most ß-lactam antibiotics and are frequently multidrug-resistant (MDR). Consequently, they are often resistant to antibiotics used to empirically treat UTIs. The lack of rapid diagnostic and antibiotic susceptibility tests (AST) makes clinical management of UTIs caused by such organisms difficult, as standard culture and susceptibility assays require several days. We have adapted a biochemical detection assay, termed dual-enzyme trigger-enabled cascade technology (DETECT) for rapid detection of resistance (time-to-result of 3 h) to other antibiotics commonly used in treatment of UTIs. DETECT is activated by the presence of CTX-M and pAmpC ß-lactamases. In this proof-of-concept study, the adapted DETECT assay (AST-DETECT) has been performed on pure-cultures of Klebsiella pneumoniae and Escherichia coli (48 isolates) expressing ESBL or pAmpC ß-lactamases to perform AST for ciprofloxacin (sensitivity 96.9%, specificity 100%, accuracy 97.9%) nitrofurantoin (sensitivity 95.7%, specificity 91.7%, accuracy 94%) and trimethoprim/sulfamethoxazole (sensitivity 83.3%, specificity 100%, accuracy 89.4%). These results suggest that AST-DETECT may be adapted as a potential diagnostic platform to rapidly detect multidrug-resistant E. coli and K. pneumoniae that cause UTI.

A Dual Enzyme-Based Biochemical Test Rapidly Detects Third-Generation Cephalosporin-Resistant CTX-M-Producing Uropathogens in Clinical Urine Samples.

Extended-spectrum ß-lactamase (ESBL)-producing Gram-negative bacteria (GNB) are increasingly identified as the cause of both community and healthcare-associated urinary tract infections (UTIs), with CTX-Ms being the most common ESBLs identified. CTX-M-producing GNB are resistant to most ß-lactam antibiotics and are frequently multidrug-resistant, which limits treatment options. Rapid diagnostic tests that can detect ESBL-producing GNB, particularly CTX-M producers, in the urine of patients with UTIs are needed. Results from such a test could direct the selection of appropriate antimicrobial therapy at the point-of-care (POC). In this study, we show that a chromogenic, dual enzyme-mediated amplification system (termed DETECT [dual-enzyme trigger-enabled cascade technology]) can identify CTX-M-producing GNB from unprocessed urine samples in 30 minutes. We first tested DETECT against a diverse set of recombinant ß-lactamases and ß-lactamase-producing clinical isolates to elucidate its selectivity. We then tested DETECT with 472 prospectively collected clinical urine samples submitted for urine culture to a hospital clinical microbiology laboratory. Of these, 118 (25%) were consistent with UTI, 13 (11%) of which contained ESBL-producing GNB. We compared DETECT results in urine against a standard phenotypic method to detect ESBLs, and polymerase chain reaction and sequencing for CTX-M genes. DETECT demonstrated 90.9% sensitivity and 97.6% specificity (AUC, 0.937; 95% confidence interval, 0.822-1.000), correctly identifying 10 of 11 urine samples containing a clinically significant concentration of CTX-M-producing GNB (including Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis). Our results demonstrate the clinical potential of DETECT to deliver diagnostic information at the POC, which could improve initial antibiotic selection.

Prevalence of ß-Lactam Drug-Resistance Genes in Escherichia coli Contaminating Ready-to-Eat Lettuce.

Thirty-four Escherichia coli isolates from 91 ready-to-eat lettuce packages, obtained from local supermarkets in Northern California, were genotyped by multilocus sequence typing, tested for susceptibility to antimicrobial agents, and screened for ß-lactamase genes. We found 15 distinct sequence types (STs). Six of these genotypes (ST1198, ST2625, ST2432, ST2819, ST4600, and ST5143) have been reported as pathogens found in human samples. Twenty-six (76%) E. coli isolates were resistant to ampicillin, 17 (50%) to ampicillin/sulbactam, 8 (23%) to cefoxitin, and 7 (20%) to cefuroxime. blaCTX-M was the most prevalent ß-lactamase gene, identified in eight (23%) isolates. We identified a class A broad-spectrum ß-lactamase SED-1 gene, blaSED, reported by others in Citrobacter sedlakii isolated from bile of a patient. This study found that fresh lettuce carries ß-lactam drug-resistant E. coli, which might serve as a reservoir for drug-resistance genes that could potentially be transmitted to pathogens that cause human infections.

Escherichia coli from Commercial Broiler and Backyard Chickens Share Sequence Types, Antimicrobial Resistance Profiles, and Resistance Genes with Human Extraintestinal Pathogenic Escherichia coli.

Escherichia coli recovered from poultry, and extraintestinal pathogenic E. coli (ExPEC), responsible for most cases of urinary tract infection (UTI) and bloodstream infection (BSI) in humans, may share genetic characteristics, suggesting that poultry are a potential source of ExPEC. Here, we compared E. coli isolated from commercial broiler and backyard chickens (n = 111) with ExPEC isolated from patients with community- or hospital-acquired UTI or BSI (n = 149) from Southeast Brazil. Isolates were genotyped by multilocus sequence typing, tested for susceptibility to antimicrobial agents, and screened for ß-lactamase genes. We found that 10 genotypes were shared among poultry and human isolates: sequence type (ST) 10, ST48, ST58, ST88, ST90, ST93, ST131, ST602, ST617, and ST1018. Thirty-five (23%) ExPEC and 35 (31%) poultry E. coli isolates belonged to the shared STs. ST58 and ST88 isolates from human and poultry sources shared identical antimicrobial resistance profiles. blaTEM-1 was the most prevalent ß-lactamase gene, identified in 65 (92%) of 71 ExPEC and 29 (67%) of 43 poultry E. coli that tested positive for ß-lactamase genes. Commercial broiler chicken isolates shared the extended-spectrum ß-lactamase (ESBL) genes blaCTX-M-2,blaCTX-M-8, and blaSHV-2 with human isolates; backyard chicken isolates lacked ESBL genes. In conclusion, several genotypic and phenotypic characteristics were shared between human and poultry E. coli; this suggests that there is potential for transmission of E. coli and antimicrobial resistance genes from poultry to humans, perhaps through environmental contamination, direct contact, or consumption. Additional research is needed to understand the potential direction and pathways of transmission.

A Trimethoprim Conjugate of Thiomaltose Has Enhanced Antibacterial Efficacy In Vivo.

Trimethoprim is one of the most widely used antibiotics in the world. However, its efficacy is frequently limited by its poor water solubility and dose limiting toxicity. Prodrug strategies based on conjugation of oligosaccharides to trimethoprim have great potential for increasing the solubility of trimethoprim and lowering its toxicity, but they have been challenging to develop due to the sensitivity of trimethoprim to chemical modifications, and the rapid degradation of oligosaccharides in serum. In this report, we present a trimethoprim conjugate of maltodextrin termed TM-TMP, which increased the water solubility of trimethoprim by over 100 times, was stable to serum enzymes, and was active against urinary tract infections in mice. TM-TMP is composed of thiomaltose conjugated to trimethoprim, via a self-immolative disulfide linkage, and releases 4'-OH-trimethoprim (TMP-OH) after disulfide cleavage, which is a known metabolic product of trimethoprim and is as potent as trimethoprim. TM-TMP also contains a new maltodextrin targeting ligand composed of thiomaltose, which is stable to hydrolysis by serum amylases and therefore has the metabolic stability needed for in vivo use. TM-TMP has the potential to significantly improve the treatment of a wide number of infections given its high water solubility and the widespread use of trimethoprim.

Monoclonal antibody-mediated detection of CTX-M ß-lactamases in Gram-negative bacteria.

Gram-negative bacteria (GNB) that express CTX-M ß-lactamases have become a serious threat to the clinical management of GNB infections. While antibody-based platforms have been successfully used in research settings to study and detect other ß-lactamases-including SHV, CMY, and TEM enzymes-there is currently a lack of antibody-based tools to detect the CTX-M enzymes. Here we describe the development of an anti-CTX-M sandwich ELISA based on a pair of monoclonal antibodies (mAbs)-mAb 6101-33 and mAb 6101-19-used as the capture and detection antibody, respectively. This antibody pair detected CTX-M variants from group 1 (CTX-M-15), group 2 (CTX-M-2), group 8 (CTX-M-8), and group 9 (CTX-M-14) that were expressed by a training set of clinical GNB isolates. The limit of detection for this sandwich ELISA was 30ng of recombinant CTX-M-15, and CTX-Ms expressed by 106 lysed CFU of GNB. When tested against a blinded panel of 78 clinical isolates, the sandwich ELISA demonstrated a sensitivity of 96% and a specificity of 100%. The mAb pair did not cross-react with bacteria that contained other ß-lactamases, including TEM, SHV, OXA, KPC, NDM, CMY, and DHA. In conclusion, we developed a highly sensitive and specific sandwich ELISA, capable of detecting CTX-M enzyme production in GNB pathogens.

Wild birds and urban pigeons as reservoirs for diarrheagenic Escherichia coli with zoonotic potential.

In order to describe the role of wild birds and pigeons in the transmission of shiga toxigenic Escherichia coli (STEC) and enteropathogenic Escherichia coli (EPEC) to humans and other animals, samples were collected from cloacae and oropharynx of free-living wild birds and free-living pigeons. Two STEC (0.8%) and five EPEC strains (2.0%) were isolated from wild birds and four EPEC strains (2.0%) were recovered from pigeons. Serogroups, sequence types (STs) and virulence genes, such as saa, iha, lpfA O113, ehxA, espA, nleB and nleE, detected in this study had already been implicated in human and animal diseases. Multidrug resistance (MDR) was found in 25.0% of the pigeon strains and in 57.0% of the wild bird strains; the wild birds also yielded one isolate carrying extended-spectrum ß-lactamases (ESBLs) gene bla CTX-M-8. The high variability shown by PFGE demonstrates that there are no prevalent E. coli clones from these avian hosts. Wild birds and pigeons could act as carriers of multidrug-resistant STEC and EPEC and therefore may constitute a considerable hazard to human and animal health by transmission of these strains to the environment.

Captive and free-living urban pigeons (Columba livia) from Brazil as carriers of multidrug-resistant pathogenic Escherichia coli.

Thirty Escherichia coli isolates from captive and free-living pigeons in Brazil were characterised. Virulence-associated genes identified in pigeons included those which occur relatively frequently in avian pathogenic E. coli (APEC) from commercial poultry worldwide. Eleven of 30 E. coli isolates from pigeons, belonging mainly to B1 and B2 phylogenetic groups, had high or intermediate pathogenicity for 1-day-old chicks. The frequency of multi-drug resistant (MDR) E. coli in captive pigeons was relatively high and included one isolate positive for the extended-spectrum ß-lactamase (ESBL) gene blaCTX-M-8. Pulsed field gel electrophoresis (PFGE) showed high heterogeneity among isolates. There is potential for pigeons to transmit antibiotic resistant pathogenic E. coli to other species through environmental contamination or direct contact.

Frequencies of virulence genes and pulse field gel electrophoresis fingerprints in Escherichia coli isolates from canine pyometra.

Escherichia coli is the most common bacterial agent isolated from canine pyometra. The frequencies of 24 virulence genes and pulsed field gel electrophoresis (PFGE) profiles were determined for 23 E. coli isolates from cases of canine pyometra in Brazil. The frequencies of virulence genes were 91.3% fimH, 91.3% irp-2, 82.6% fyuA, 56.5% iroN, 47.8% traT, 39.1% usp, 34.8% sfaD/E, 34.8% tsh, 30.4% papC, 30.4% hlyA, 26.1% papGIII, 26.1% cnf-1, 21.7% papE/F, 21.7% iss, 17.4% iutA, 17.4% ompT, 17.4% cvaC, 17.4% hlyF, 17.4% iucD, 13.0% iucC, 13.0% astA, 4.3% papGII, 0% afaB/C and 0% papGI. The high frequency of yersiniabactin (fyuA and irp2) and salmochelin (iroN) genes suggests that iron uptake systems might be important in the pathogenesis of canine pyometra. PFGE profiles of 19 isolates were heterogeneous, confirming that E. coli isolates from canine pyometra are unlikely to be epidemic clones.

Escherichia coli Strains Isolated from the Uteri Horn, Mouth, and Rectum of Bitches Suffering from Pyometra: Virulence Factors, Antimicrobial Susceptibilities, and Clonal Relationships among Strains.

Pyometra is recognized as one of the main causes of disease and death in the bitch, and Escherichia coli is the major pathogen associated with this disease. In this study, 70 E. coli isolates from the uteri horn, mouth, and rectum of bitches suffering from the disease and 43 E. coli isolates from the rectum of clinically healthy bitches were examined for the presence of uropathogenic virulence genes and susceptibility to antimicrobial drugs. DNA profiles of isolates from uteri horn and mouth in bitches with pyometra were compared by REP, ERIC, and BOX-PCR. Virulence gene frequencies detected in isolates from canine pyometra were as follows: 95.7% fim, 27.1% iss, 25.7% hly, 18.5% iuc, and 17.1% usp. Predominant resistance was determined for cephalothin, ampicillin, and nalidixic acid among the isolates from all sites examined. Multidrug resistance was found on ∼ 50% pyometra isolates. Using the genotypic methods some isolates from uteri, pus, and saliva of the same bitch proved to have identical DNA profiles which is a reason for concern due to the close relationship between household pets and humans.

Intestinal parasites inside public restrooms and buses from the city of Uberlândia, Minas Gerais, Brazil.

The objective of this study was to describe the occurrence of intestinal parasites inside public restrooms and buses from a Brazilian city. Sample material was obtained using a transparent adhesive tape. Thirty two public restrooms were investigated and two (6.25%) were contaminated with helminth eggs (Ascaris lumbricoides and Enterobius vermicularis). Of the sixteen different bus lines, three (18.7%) were found to harbor eggs of E. vermicularis. Public restrooms and buses can be an important source of parasite transmission and sanitary education could be improved by using these points.

Intestinal parasites inside public restrooms and buses from the city of Uberlândia, Minas Gerais, Brazil / Parasitas intestinais no interior de sanitários públicos e ônibus na cidade de Uberlândia, Minas Gerais, Brasil

The objective of this study was to describe the occurrence of intestinal parasites inside public restrooms and buses from a Brazilian city. Sample material was obtained using a transparent adhesive tape. Thirty two public restrooms were investigated and two (6.25%) were contaminated with helminth eggs (Ascaris lumbricoides and Enterobius vermicularis). Of the sixteen different bus lines, three (18.7%) were found to harbor eggs of E. vermicularis. Public restrooms and buses can be an important source of parasite transmission and sanitary education could be improved by using these points.

O objetivo do presente estudo foi descrever a ocorrência de parasitas intestinais em sanitários públicos e ônibus de uma cidade do Brasil. As amostras foram obtidas utilizando-se fita adesiva transparente. Trinta e dois sanitários públicos foram investigados e dois (6,25%) estavam contaminados com ovos de helmintos (Ascaris lumbricoides e Enterobius vermicularis). Das 16 diferentes linhas de ônibus, três (18,7%) foram positivas para ovos de E. vermicularis. Sanitários públicos e ônibus podem ser uma importante via de transmissão de parasitas e a educação sanitária pode ser aperfeiçoada por meio do uso destes pontos.