A unique class of Zn2+-binding PBPs underlies cephalosporin resistance and sporogenesis in Clostridioides difficile

β-Lactam antibiotics, particularly cephalosporins, are major risk factors for C. difficile infection (CDI), the most common hospital acquired infection. These broad-spectrum antibiotics irreversibly inhibit penicillin-binding proteins (PBPs), essential enzymes that assemble the bacterial cell wall. Little is known about the C. difficile PBPs, yet they play central roles in the growth, infection, and transmission of this pathogen. In this study we discover that PBP2, essential for vegetative growth, is the primary bactericidal target for β-lactams in C. difficile. We further demonstrate PBP2 is insensitive to cephalosporin inhibition, revealing a key cause of the well-documented, but poorly understood, cephalosporin resistance in C. difficile. For the first time, we determine the crystal structures of C. difficile PBP2, which bears several highly unique features, including significant ligand-induced conformational changes and an active site Zn2+-binding motif that influences β-lactam binding and protein stability. Remarkably, this motif is shared in two other C. difficile PBPs essential for sporulation, PBP3 and SpoVD. While these PBPs are present in a wide range of bacterial taxa, including species in extreme environments and the human gut, they are mostly found in anaerobes, typically Firmicutes. The widespread presence of this convergently evolved thiol-containing motif and its cognate Zn2+ suggests it may function as a redox-sensor to regulate cell wall synthesis for survival in adverse environments. Collectively, our findings address important etiological questions surrounding C. difficile, characterize new elements of PBP structure and function, and lay the groundwork for antibiotic development targeting both C. difficile growth and sporulation.

Selection of AmpC β-lactamase variants and metallo-β-lactamases leading to ceftolozane/tazobactam and ceftazidime/avibactam-resistance during treatment of MDR/XDR Pseudomonas aeruginosa infections

Infections caused by ceftolozane/tazobactam and ceftazidime/avibactam-resistant P. aeruginosa infections are an emerging concern. We aimed to analyze the underlying ceftolozane/tazobactam and ceftazidime/avibactam resistance mechanisms in all MDR/XDR P. aeruginosa isolates recovered during one year (2020) from patients with a documented P. aeruginosa infection. Fifteen isolates showing ceftolozane/tazobactam and ceftazidime/avibactam resistance were evaluated. Clinical conditions, previous positive cultures and β-lactams received in the previous month were reviewed for each patient. MICs were determined by broth microdilution. MLSTs and resistance mechanisms were determined using short- and long-read WGS. The impact of PDCs on β-lactam resistance was demonstrated by cloning into an ampC -deficient PAO1 derivative (PAOΔC) and construction of 3D models. Genetic support of acquired β-lactamases was determined in silico from high-quality hybrid assemblies. In most cases, the isolates were recovered after treatment with ceftolozane/tazobactam or ceftazidime/avibactam. Seven isolates from different STs owed their β-lactam resistance to chromosomal mutations and all displayed specific substitutions in PDC: Phe121Leu and Gly222Ser, Pro154Leu, Ala201Thr, Gly214Arg, ΔGly203-Glu219 and Glu219Lys. In the other eight isolates, the ST175 clone was overrepresented (6 isolates) and associated with IMP-28 and IMP-13, whereas two ST1284 isolates produced VIM-2. The cloned PDCs conferred enhanced cephalosporin resistance. 3D PDC models revealed rearrangements affecting residues involved in cephalosporin hydrolysis. Carbapenemases were chromosomal (VIM-2) or plasmid-borne (IMP-28, IMP-13), and associated with class-1 integrons located in Tn402-like transposition modules. Our findings highlight that cephalosporin/ß-lactamase inhibitors are potential selectors of MDR/XDR P. aeruginosa strains producing PDC variants or metallo-ß-lactamases. Judicious use of these agents is encouraged.

Third-Generation Cephalosporin Resistance in Intrinsic Colistin-Resistant Enterobacterales Isolated from Retail Meat

Consumption of retail meat contaminated with antimicrobial-resistant (AMR) bacteria is a common route for transmitting clinically relevant resistant bacteria to humans. Here, we investigated the genotypic and phenotypic resistance profiles of intrinsic colistin-resistant (ICR) Enterobacterales isolated from retail meats. ICR Enterobacterales were isolated from 103 samples of chicken, 103 samples of pork, and 104 samples of beef purchased from retail shops in Japan, using colistin-containing media, and their antimicrobial susceptibility was examined. Serratia spp. (440 isolates) showed resistance to cefotaxime (19 isolates, 4.3%), tetracycline (15 isolates, 3.4%), and other antimicrobials (<1%). Hafnia spp. (136) showed resistance to cefotaxime (12 isolates, 8.6%), ceftazidime (four isolates, 2.9%), and tetracycline (two isolates, 1.4%). Proteus spp. (39) showed resistance to chloramphenicol (four isolates, 10.3%), sulfamethoxazole-trimethoprim (four isolates, 10.3%), cefotaxime (two isolates, 5.1%), kanamycin (two isolates, 5.1%), and gentamicin (one isolate, 2.6%). Cedecea spp. (22) were resistant to tetracycline (two isolates, 9.1%) whereas Morganella spp. (11) were resistant to tetracycline (four isolates, 36.4%) and chloramphenicol (one isolate, 9.2%). The resistance genes blafonA, blaACC, and blaDHA were detected in cefotaxime-resistant Serratia spp., Hafnia spp., and Morganella spp. isolates, respectively. This emergence of antimicrobial resistance in ICR Enterobacterales may pose a public health risk.

International High-Risk Clones Among Extended-Spectrum β-Lactamase–Producing Escherichia coli in Dhaka, Bangladesh

Background:Escherichia coli is a major extended-spectrum β-lactamase (ESBL)–producing organism responsible for the rapid spread of antimicrobial resistance (AMR) that has compromised our ability to treat infections. Baseline data on population structure, virulence, and resistance mechanisms in E. coli lineages from developing countries such as Bangladesh are lacking.Methods: Whole-genome sequencing was performed for 46 ESBL–E. coli isolates cultured from patient samples at the International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b)-Dhaka. Sequence data were analyzed to glean details of AMR, virulence, and phylogenetic and molecular markers of E. coli lineages.Results: Genome comparison revealed presence of all major high-risk clones including sequence type 131 (ST131) (46%), ST405 (13%), ST648 (7%), ST410 (4.3%), ST38 (2%), ST73 (2%), and ST1193 (2%). The predominant ESBL gene and plasmid combination were blaCTX–M–15 and FII-FIA-FIB detected in diverse E. coli phylogroups and STs. The blaNDM–5 (9%) gene was present in prominent E. coli STs. One (2%) mcr-1–positive ST1011 E. coli, coharboring blaCTXM–55 gene, was detected. The extraintestinal pathogenic E. coli genotype was associated with specific E. coli lineages. The single nucleotide polymorphism (SNP)-based genome phylogeny largely showed correlation with phylogroups, serogroups, and fimH types. Majority of these isolates were susceptible to amikacin (93%), imipenem (93%), and nitrofurantoin (83%).Conclusion: Our study reveals a high diversity of E. coli lineages among ESBL-producing E. coli from Dhaka. This study suggests ongoing circulation of ST131 and all major non-ST131 high-risk clones that are strongly associated with cephalosporin resistance and virulence genes. These findings warrant prospective monitoring of high-risk clones, which would otherwise worsen the AMR crises.

Increasing incidence and antimicrobial resistance in Escherichia coli bloodstream infections: a multinational population-based cohort study

Background Escherichia coli is an important pathogen in humans and is the most common cause of bacterial bloodstream infections (BSIs). The objectives of our study were to determine factors associated with E. coli BSI incidence rate and third-generation cephalosporin resistance in a multinational population-based cohort. Methods We included all incident E. coli BSIs (2014–2018) from national (Finland) and regional (Australia [Canberra], Sweden [Skaraborg], and Canada [Calgary, Sherbrooke, and western interior]) surveillance. Incidence rates were directly age and sex standardized to the European Union 28-country 2018 population. Multivariable negative binomial and logistic regression models estimated factors significantly associated with E. coli BSI incidence rate and third-generation cephalosporin resistance, respectively. The explanatory variables considered for inclusion in both models were year (2014–2018), region (six areas), age (< 70-years-old and ≥ 70-years-old), and sex (female and male). Results We identified 31,889 E. coli BSIs from 40.7 million person-years of surveillance. Overall and third-generation cephalosporin-resistant standardized rates were 87.1 and 6.6 cases/100,000 person-years, respectively, and increased 14.0% and 40.1% over the five-year study. Overall, 7.8% (2483/31889) of E. coli BSIs were third-generation cephalosporin-resistant. Calgary, Canberra, Sherbrooke, and western interior had significantly lower E. coli BSI rates compared to Finland. The significant association between age and E. coli BSI rate varied with sex. Calgary, Canberra, and western interior had significantly greater odds of third-generation cephalosporin-resistant E. coli BSIs compared to Finland. Compared to 2014, the odds of third-generation cephalosporin-resistant E. coli BSIs were significantly increased in 2016, 2017, and 2018. The significant association between age and the odds of having a third-generation cephalosporin-resistant E. coli BSI varied with sex. Conclusions Increases in overall and third-generation cephalosporin-resistant standardized E. coli BSI rates were clinically important. Overall, E. coli BSI incidence rates were 40–104% greater than previous investigations from the same study areas. Region, sex, and age are important variables when analyzing E. coli BSI rates and third-generation cephalosporin resistance in E. coli BSIs. Considering E. coli is the most common cause of BSIs, this increasing burden and evolving third-generation cephalosporin resistance will have an important impact on human health, especially in aging populations.

OmpF Downregulation Mediated by Sigma E or OmpR Activation Confers Cefalexin Resistance in Escherichia coli in the Absence of Acquired β-Lactamases.

Cefalexin is a widely used 1 st generation cephalosporin, and resistance in Escherichia coli is caused by Extended-Spectrum (e.g., CTX-M) and AmpC β-lactamase production and therefore frequently coincides with 3 rd generation cephalosporin resistance. However, we have recently identified large numbers of E. coli isolates from human infections, and from cattle, where cefalexin resistance is not β-lactamase mediated. Here we show, by studying laboratory selected mutants, clinical isolates, and isolates from cattle, that OmpF porin disruption or downregulation is a major cause of cefalexin resistance in E. coli . Importantly, we identify multiple regulatory mutations that cause OmpF downregulation. In addition to mutation of ompR , already known to downregulate OmpF and OmpC porin production, we find that rseA mutation, which strongly activates the Sigma E regulon, greatly increasing DegP production, which degrades OmpF, OmpC and OmpA. Furthermore, we reveal that mutations affecting lipopolysaccharide structure, exemplified by the loss of GmhB, essential for lipopolysaccharide heptosylation, also modestly activate DegP production, resulting in OmpF degradation. Remarkably, given the critical importance attached to such systems for normal E. coli physiology, we find evidence for DegP-mediated OmpF downregulation, gmhB and rseA loss of function mutation in E. coli isolates derived from human infections. Finally, we show that these regulatory mutations enhance the ability of group 1 CTX-M β-lactamase to confer reduced carbapenem susceptibility, particularly those mutations that cause OmpC in addition to OmpF downregulation.

Conjugative Plasmid-Mediated Extended Spectrum Cephalosporin Resistance in Genetically Diverse Escherichia coli from a Chicken Slaughterhouse

ESC-resistant E. coli isolates were collected from broiler chickens, a slaughterhouse, and retail meat to assess their dispersion and their involvement in cross-contamination. ESBL-/AmpC-producing E. coli were isolated during the slaughter process of all six investigated chicken flocks from scalding, feather removal, first conveyor, evisceration, second washing, third conveyor, and third washing areas, and from handling workers in the slaughterhouse. ESC-resistant E. coli isolates with the same pulsed-field gel electrophoresis type were found in the same site (scalding) on different sampling days. ESBL/AmpC-producing E. coli isolates were absent in the lairage area in the slaughterhouse, but present in the retail markets in 36.8% (7/19) of the chicken flocks. The blaCTX-M genes and blaCMY-2 were conjugated to recipient E. coli J53 in 67.5% (27/40) and 56.1% (23/41) of ESBL-producing and AmpC-producing E. coli isolates, respectively. The presence of the same conjugative plasmids was found in genetic diversity ESC-resistant E. coli colonies collected on different sampling days. Our study emphasizes that cross-contamination of ESBL/AmpC-producing E. coli in slaughterhouse has a crucial impact on the occurrence of ESC resistance in retail chicken meat.

Assessment of Phenotype Relevant Amino Acid Residues in TEM-β-Lactamases by Mathematical Modelling and Experimental Approval

Single substitutions or combinations of them alter the hydrolytic activity towards specific β-lactam-antibiotics and β-lactamase inhibitors of TEM-β-lactamases. The sequences and phenotypic classification of allelic TEM variants, as provided by the NCBI National Database of Antibiotic Resistant Organisms, does not attribute phenotypes to all variants. Some entries are doubtful as the data assessment differs strongly between the studies or no data on the methodology are provided at all. This complicates mathematical and bioinformatic predictions of phenotypes that rely on the database. The present work aimed to prove the role of specific substitutions on the resistance phenotype of TEM variants in, to our knowledge, the most extensive mutagenesis study. In parallel, the predictive power of extrapolation algorithms was assessed. Most well-known substitutions with direct impact on the phenotype could be reproduced, both mathematically and experimentally. Most discrepancies were found for supportive substitutions, where some resulted in antagonistic effects in contrast to previously described synergism. The mathematical modelling proved to predict the strongest phenotype-relevant substitutions accurately but showed difficulties in identifying less prevalent but still phenotype transforming ones. In general, mutations increasing cephalosporin resistance resulted in increased sensitivity to β-lactamase inhibitors and vice versa. Combining substitutions related to cephalosporin and β-lactamase inhibitor resistance in almost all cases increased BLI susceptibility, indicating the rarity of the combined phenotype.

Detection of Extended Spectrum Beta-lactamase Gene (CTX-M) among Representative Multidrug-Resistant Gram-negative Bacterial Isolates from Patients with Urinary Tract Infections

Urinary tract infection (UTI) is a huge public health problem and the emergence of extended spectrum-beta-lactamase producing bacterial pathogens increases the burden of infectious diseases in Nigeria. This study determined the current prevalence of cephalosporin resistance among Gram-negative bacteria isolated from patients with urinary tract infections between February 2018 and June 2018. This study was aimed to determine cephalosporin resistance prevalence among Gram-negative bacteria isolated from patients with urinary tract infections between February 2018 and June 2018. A total number of forty representative Gram-negative bacterial isolates namely Escherichia coli (n=14), Klebsiella pneumonia (n=9), Proteus mirabilis (n=12), and Klebsiella oxytoca (n=5) were subjected to polymerase chain reaction (PCR) to detect extended spectrum beta-lactamase (ESBL) genes using primers specific for blaTEM, blaSHV and blaCTX-M. The molecular evaluation indicated the presence of blaCTX-M gene in 20.0% of the tested organisms, while other ESBL genes variants were not detected. The organisms carrying the blaCTX-M gene included E. coli (n=3, 37.5%), K. pneumoniae (n=1, 12.5%), P. mirabilis (n=1, 12.5%),) and K. oxytoca (n=3, 37.5%). The presence of cephalosporin resistant Gram-negative bacteria among patients with UTI may constitute a serious threat to public health and efforts must be intensified to regulate the clinical use of the cephalosporins.