Carbapenem-Resistant Pseudomonas aeruginosa Infection and Mixed Infections Are Risk Factors for Poor Outcome After Lung Transplant.

OBJECTIVES: In this study, we analyzed the effects of carbapenem-resistant Pseudomonas aeruginosa infection and mixed infection on the perioperative prognosis of lung transplant recipients and studied statistics on antibiotic resistance in P aeruginosa. MATERIALS AND METHODS: This was a retrospective casecontrol study. We collected data on lung transplant recipients with combined lower respiratory tract P aeruginosa infection within 48 hours after lung transplant at the China-Japan Friendship Hospital from August 2018 to April 2022. We grouped recipients according to P aeruginosa resistance to carbapenem antibiotics and summarized the clinical characteristics of carbapenem-resistant P aeruginosa infection. We analyzed the effects of carbapenemresistant P aeruginosa infection and mixed infections on all-cause mortality 30 days after lung transplant by Cox regression. We used the Kaplan-Meier method to plot survival curves. RESULTS: Patients in the carbapenem-resistant P aeruginosa group had a higher all-cause mortality rate than those in the carbapenem-sensitive P aeruginosa group at both 7 days (6 patients [22.3%] vs 2 patients [4.5%]; P = .022) and 30 days (12 patients [44.4%] vs 7 patients [15.9%]; P = .003) after lung transplant. In multivariate analysis, both carbapenemresistant P aeruginosa infection and P aeruginosa combined with bacterial infection were independent risk factors for death 30 days after transplant in lung transplant recipients (P < .05). In subgroup analysis, carbapenem-resistant P aeruginosa combined with bacterial infection increased the risk of death 30 days after transplant in lung transplant recipients compared with carbapenem-sensitive P aeruginosa combined with bacterial infection (12 patients [60%] vs 6 patients [19.4%]; P < .001). CONCLUSIONS: Combined lower respiratory tract carbapenem-resistant P aeruginosa infection and P aeruginosa combined with bacterial infection early after lung transplant increased the risk of 30-day mortality after lung transplant.

Genomic context as well as sequence of both psr and penicillin-binding protein 5 contributes to ß-lactam resistance in Enterococcus faecium.

Penicillin-binding protein 5 (PBP5) of Enterococcus faecium (Efm) is vital for ampicillin resistance (AMP-R). We previously designated three forms of PBP5, namely, PBP5-S in Efm clade B strains [ampicillin susceptible (AMP-S)], PBP5-S/R (AMP-S or R), and PBP5-R (AMP-R) in clade A strains. Here, pbp5 deletion resulted in a marked reduction in AMP minimum inhibitory concentrations (MICs) to 0.01-0.09 µg/mL for clade B and 0.12-0.19 µg/mL for clade A strains; in situ complementation restored parental AMP MICs. Using D344SRF (lacking ftsW/psr/pbp5), constructs with ftsWA/psrA (from a clade A1 strain) cloned upstream of pbp5-S and pbp5-S/R alleles resulted in modest increases in MICs to 3-8 µg/mL, while high MICs (>64 µg/mL) were seen using pbp5 from A1 strains. Next, using ftsW ± psr from clade B and clade A/B and B/A hybrid constructs, the presence of psrB, even alone or in trans, resulted in much lower AMP MICs (3-8 µg/mL) than when psrA was present (MICs >64 µg/mL). qRT PCR showed relatively greater pbp5 expression (P = 0.007) with pbp5 cloned downstream of clade A1 ftsW/psr (MIC >128 µg/mL) vs when cloned downstream of clade B ftsW/psr (MIC 4-16 µg/mL), consistent with results in western blots. In conclusion, we report the effect of clade A vs B psr on AMP MICs as well as the impact of pbp5 alleles from different clades. While previously, Psr was not thought to contribute to AMP MICs in Efm, our results showed that the presence of psrB resulted in a major decrease in Efm AMP MICs. IMPORTANCE: The findings of this study shed light on ampicillin resistance in Enterococcus faecium clade A strains. They underscore the significance of alterations in the amino acid sequence of penicillin-binding protein 5 (PBP5) and the pivotal role of the psr region in PBP5 expression and ampicillin resistance. Notably, the presence of a full-length psrB leads to reduced PBP5 expression and lower minimum inhibitory concentrations (MICs) of ampicillin compared to the presence of a shorter psrA, regardless of the pbp5 allele involved. Additionally, clade B E. faecium strains exhibit lower AMP MICs when both psr alleles from clades A and B are present, although it is important to consider other distinctions between clade A and B strains that may contribute to this effect. It is intriguing to note that the divergence between clade A and clade B E. faecium and the subsequent evolution of heightened AMP MICs in hospital-associated strains appear to coincide with changes in Pbp5 and psr. These changes in psr may have resulted in an inactive Psr, facilitating increased PBP5 expression and greater ampicillin resistance. These results raise the possibility that a mimicker of PsrB, if one could be designed, might be able to lower MICs of ampicillin-resistant E. faecium, thus potentially resorting ampicillin to our therapeutic armamentarium for this species.

Altered PBP4 and GdpP functions synergistically mediate MRSA-like high-level, broad-spectrum ß-lactam resistance in Staphylococcus aureus.

Infections caused by Staphylococcus aureus are a leading cause of mortality worldwide. S. aureus infections caused by methicillin-resistant Staphylococcus aureus (MRSA) are particularly difficult to treat due to their resistance to next-generation ß-lactams (NGBs) such as methicillin, nafcillin, and oxacillin. Resistance to NGBs, which is alternatively known as broad-spectrum ß-lactam resistance, is classically mediated by PBP2a, a penicillin-binding protein encoded by mecA (or mecC) in MRSA. Thus, presence of mec genes among S. aureus spp. serves as the predictor of resistance to NGBs and facilitates determination of the proper therapeutic strategy for a staphylococcal infection. Although far less appreciated, mecA-deficient S. aureus strains can also exhibit NGB resistance. These strains, which are collectively termed as methicillin-resistant lacking mec (MRLM), are currently being identified in increasing numbers among natural resistant isolates of S. aureus. The mechanism/s through which MRLMs produce resistance to NGBs remains unknown. In this study, we demonstrate that mutations that alter PBP4 and GdpP functions, which are often present among MRLMs, can synergistically mediate resistance to NGBs. Furthermore, our results unravel that this novel mechanism potentially enables MRLMs to produce resistance toward NGBs at levels comparable to those of MRSAs. Our study provides a fresh new perspective about alternative mechanisms of NGB resistance, challenging our current overall understanding of high-level, broad-spectrum ß-lactam resistance in S. aureus. It thus suggests reconsideration of the current approach toward diagnosis and treatment of ß-lactam-resistant S. aureus infections. IMPORTANCE: In Staphylococcus aureus, high-level, broad-spectrum resistance to ß-lactams such as methicillin, also referred to as methicillin resistance, is largely attributed to mecA. This study demonstrates that S. aureus strains that lack mecA but contain mutations that functionally alter PBP4 and GdpP can also mediate high-level, broad-spectrum resistance to ß-lactams. Resistance brought about by the synergistic action of functionally altered PBP4 and GdpP was phenotypically comparable to that displayed by mecA, as seen by increased bacterial survival in the presence of ß-lactams. An analysis of mutations detected in naturally isolated strains of S. aureus revealed that a significant proportion of them had similar pbp4 and GGDEF domain protein containing phosphodiesterase (gdpP) mutations, making this study clinically significant. This study not only identifies important players of non-classical mechanisms of ß-lactam resistance but also indicates reconsideration of current clinical diagnosis and treatment protocols of S. aureus infections.

Understanding the effects of sub-inhibitory antibiotic concentrations on the development of ß-lactamase resistance based on quantile regression analysis.

AIMS: Quantile regression is an alternate type of regression analysis that has been shown to have numerous advantages over standard linear regression. Unlike linear regression, which uses the mean to fit a linear model, quantile regression uses a data set's quantiles (or percentiles), which leads to a more comprehensive analysis of the data. However, while relatively common in other scientific fields such as economic and environmental modeling, it is infrequently used to understand biological and microbiological systems. METHODS AND RESULTS: We analyzed a set of bacterial growth rates using quantile regression analysis to better understand the effects of antibiotics on bacterial fitness. Using a bacterial model system containing 16 variant genotypes of the TEM ß-lactamase enzyme, we compared our quantile regression analysis to a previously published study that uses the Tukey's range test, or Tukey honestly significantly difference (HSD) test. We find that trends in the distribution of bacterial growth rate data, as viewed through the lens of quantile regression, can distinguish between novel genotypes and ones that have been clinically isolated from patients. Quantile regression also identified certain combinations of genotypes and antibiotics that resulted in bacterial populations growing faster as the antibiotic concentration increased-the opposite of what was expected. These analyses can provide new insights into the relationships between enzymatic efficacy and antibiotic concentration. CONCLUSIONS: Quantile regression analysis enhances our understanding of the impacts of sublethal antibiotic concentrations on enzymatic (TEM ß-lactamase) efficacy and bacterial fitness. We illustrate that quantile regression analysis can link patterns in growth rates with clinically relevant mutations and provides an understanding of how increasing sub-lethal antibiotic concentrations, like those found in our modern environment, can affect bacterial growth rates, and provide insight into the genetic basis for varied resistance.

ESBL-Escherichia coli extracellular vesicles mediate bacterial resistance to ß-lactam and mediate horizontal transfer of blaCTX-M-55.

OBJECTIVES: Extracellular vesicles (EVs) have become the focus of research as an emerging method of horizontal gene transfer. In recent years, studies on the association between EVs and the spread of bacterial resistance have emerged, but there is a lack of research on the role of EVs secreted by extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli in the spread of ß-lactam resistance. Therefore, the aim of this study was to investigate the role of EVs in the transmission of ß-lactam resistance. METHODS: In this study, the role of EVs in the transmission of ß-lactam resistance in E. coli was evaluated by the EVs-mediated bacterial resistance to ß-lactam antibiotics test and the EVs-mediated blaCTX-M-55 transfer experiments using EVs secreted by ESBL-E. coli. RESULTS: The results showed that ESBL-EVs were protective against ß-lactam antibiotic-susceptible bacteria, and this protective effect was dependent on the integrity of the EVs and showed dose- and time-dependent effects. At the same time, ESBL-EVs can also mediate the horizontal transmission of blaCTX-M-55, and EVs-mediated gene transfer is selective, preferring to transfer in more closely related species. CONCLUSIONS: In this study, we demonstrated the important role of EVs in the transmission of ß-lactam resistance in chicken ESBL-E. coli, and evaluated the risk of EVs-mediated horizontal gene transfer, which provided a theoretical basis for elucidating the mechanism of EVs-mediated resistance transmission.

Antimicrobial resistance, ß-lactamase genotypes, and plasmid replicon types of Shiga toxin-producing Escherichia coli isolated from different animal hosts.

AIMS: The primary objective of this study was to analyze antimicrobial resistance (AMR), with a particular focus on ß-lactamase genotypes and plasmid replicon types of Shiga toxin-producing Escherichia coli (STEC) strains originating from various animal hosts. METHODS AND RESULTS: A total of 84 STEC strains were isolated from cattle (n = 32), sheep/goats (n = 26), pigeons (n = 20), and wild animals (n = 6) between 2010 and 2018 in various regions of Iran. The Kirby-Bauer susceptibility test and multiple polymerase chain reaction (PCR) panels were employed to elucidate the correlation between AMR and plasmid replicon types in STEC isolates. The predominant replicon types were IncFIC and IncFIB in cattle (46.8%), IncFIC in sheep/goats (46.1%), IncA/C in pigeons (90%), and IncP in wild animals (50%). STEC of serogroups O113, O26, and O111 harbored the IncFIB (100%), IncI1 (80%), and IncFIC + IncA/C (100%) plasmids, respectively. A remarkable AMR association was found between ciprofloxacin (100%), neomycin (68.7%), and tetracycline (61.7%) resistance with IncFIC; amoxicillin + clavulanic acid (88.8%) and tetracycline (61.7%) with IncA/C; ciprofloxacin (100%) with IncFIB; fosfomycin (85.7%) and sulfamethoxazole + trimethoprim (80%) with IncI1. IncI1 appeared in 83.3%, 50%, and 100% of the isolates harboring blaCTX-M, blaTEM, and blaOXA ß-lactamase genes, respectively. CONCLUSIONS: The emergence of O26/IncI1/blaCTX-M STEC in cattle farms poses a potential risk to public health.

Monitoring viruses and beta-lactam resistance genes through wastewater surveillance during a COVID-19 surge in Suwon, South Korea.

The present study reports data on a long-term campaign for monitoring SARS-CoV-2, norovirus, hepatitis A virus, and beta-lactam resistance genes in wastewater samples from a wastewater treatment plant during COVID-19 surge in Suwon, South Korea. Real-time digital PCR (RT-dPCR) assays indicated 100 % occurrence of all but hepatitis A virus and blaNDM gene in influent wastewater samples. CDC-N1 assay detected SARS-CoV-2 in all influent samples with an average log-transformed concentration of 5.1 ± 0.39 and the highest level at 6.02 gene copies/L. All samples were also positive for norovirus throughout the study with a mean concentration 5.67 ± 0.65 log10 gene copies/L. On the contrary, all treated wastewater (effluent) tested negative for both viruses' genetic materials. Furthermore, plasmid-mediated AmpC ß-lactamases (PABLs) genes blaDHA, blaACC, and blaFOX, extended-spectrum ß-lactamases (ESBLs) genes blaTEM and blaCTX, and Klebsiella pneumoniae carbapenemase (blaKPC) gene were measured at average concentrations of 7.05 ± 0.26, 5.60 ± 0.35, 7.82 ± 0.43, 8.38 ± 0.20, 7.64 ± 0.29, and 7.62 ± 0.41 log10 gene copies/L wastewater, respectively. Beta-lactam resistance genes showed strong correlations (r), the highest being 0.86 for blaKPC - blaFOX, followed by 0.82 for blaTEM - blaCTX and 0.79 for blaTEM - blaDHA. SARS-CoV-2 RNA occurrence in the wastewater was strongly associated (r = 0.796) with COVID-19 cases in the catchment during the initial study period of six months. A positive association of the SARS-CoV-2 RNA with the prevalence of COVID-19 cases showed a promising role of community-scale monitoring of pathogens to provide considerable early signals of infection dynamics. High concentrations of beta-lactam resistance genes in wastewater indicated a high concern for one of the biggest global health threats in South Korea and the need to find control measures. Moreover, antibiotic-resistance genes in treated wastewater flowing through water bodies and agricultural environments indicate further dissemination of antibiotic resistance traits and increasing microbial antibiotic resistance.

Epidemiological investigation and ß-lactam antibiotic resistance of Riemerella anatipestifer isolates with waterfowl origination in Anhui Province, China.

Riemerella anatipestifer (R. anatipestifer) is a highly pathogenic and complex serotypes waterfowl pathogen with inherent resistance to multiple antibiotics. This study was aimed to investigate the antibiotic resistance characteristics and genomic features of R. anatipestifer isolates in Anhui Province, China in 2023. A total of 287 cases were analysed from duck farms and goose farms, and the R. anatipestifer isolates were subjected to drug resistance tests for 30 antimicrobials. Whole genome sequencing (WGS) and bioinformatics analysis were performed on the bacterial genomes, targeting the ß-lactam resistance genes. The results showed that a total of 74 isolates of R. anatipestifer were isolated from 287 cases, with a prevalence of 25.8%. The antimicrobial susceptibility testing (AST) revealed that all the 74 isolates were resistant to multiple drugs, ranging from 13 to 26 kinds of drugs. Notably, these isolates showed significant resistance to aminoglycosides and macrolides, which are also commonly used in clinical practices. Data revealed the presence of several ß-lactamase-related genes among the isolates, including a novel blaRASA-1 variant (16.2%), the class A extended-spectrum ß-lactamase blaRAA-1 (12.2%), and a blaOXA-209 variant (98.6%). Functional analysis of the variants blaRASA-1 and blaOXA-209 showed that the blaRASA-1 variant exhibited activity against various ß-lactam antibiotics while their occurrence in R. anatipestifer were not common. The blaOXA-209 variant, on the other hand, did not perform any ß-lactam antibiotic resistance. Furthermore, we observed that blaRAA-1 could undergo horizontal transmission among different bacteria via the insertion sequence IS982. In conclusion, this study delves into the high prevalence of R. anatipestifer infection in waterfowl in Anhui, China. The isolated strains exhibit severe drug resistance issues, closely associated with the prevalence of antibiotic resistance genes (ARG). Additionally, our research investigates the ß-lactam antibiotic resistance mechanism in R. anatipestifer.

Exposure to trace levels of live seaweed-derived antibacterial 2,4,6-tribromophenol modulates ß-lactam antibiotics resistance in Vibrio.

Non-antibiotic substances have been found to contribute to the spread of antibiotic resistance. Bromophenols (BPs) are special anti-bacterial substances obtained from seaweed. This study explored the modulatory effect of trace BPs from a live seaweed on the antibiotic resistance of pathogenic Vibrio (V.) strains. A hydroponic solution of Ulva fasciata was found to contain trace levels (9-333 µg L-1) of 2,4,6-tribromophenol (TBP), a typical BP. TBP at a concentration of 165 µg L-1 significantly increased the inhibition zone diameter of widely used ß-lactam antibiotics (amoxicillin and ampicillin) against V. alginolyticus M7 (Va. M7) and V. parahaemolyticus M3 (Vp. M3) as well as reduced the minimum inhibitory concentration by 2-4 fold against Va. M7. Whole genome re-sequencing analysis demonstrated that Va. M3 (53-60) had more mutant genes than Vp. M7 (44) in ß-lactam resistance pathway. Transcriptome sequencing analysis, along with verification through RT-qPCR, further showed that oligopeptide permease (opp) was the only differentially expressed gene (DEG) among the mutated genes in the ß-lactam resistance pathway. The opp transport activity and membrane permeability of Vibrio were both enhanced at 165 µg L-1 of TBP, and the ability of biofilm formation was also decreased. Thus, antibiotics resistance improvement of Vibrio by TBP was potentially related with the promoted opp transport activity, membrane permeability and inhibited biofilm formation.

Molecular characterization of antimicrobial resistance in Brachyspira species isolated from UK chickens: Identification of novel variants of pleuromutilin and beta-lactam resistance genes.

Brachyspira species are Gram negative, anaerobic bacteria that colonise the gut of many animals, including poultry. In poultry, Brachyspira species can be commensal (B. innocens, B. murdochii, 'B. pulli') or pathogenic (B. pilosicoli, B. intermedia, B. alvinipulli or rarely B. hyodysenteriae), the latter causing avian intestinal spirochaetosis (AIS). Antimicrobial therapy options for treatment is limited, frequently involving administration of the pleuromutilin, tiamulin, in water. In this study 38 Brachyspira isolates from chickens in the UK, representing both commensal and pathogenic species, were whole genome sequenced to identify antimicrobial resistance (AMR) mechanisms and the minimum inhibitory concentration (MIC) to a number of antimicrobials was also determined. We identified several new variants of blaOXA in B. pilosicoli and B. pulli isolates, and variations in tva which led to two new tva variants in B.murdochii and B.pulli. A number of isolates also harboured mutations known to encode AMR in the 16S and 23S rRNA genes. The percentage of isolates that were genotypically multi-drug resistance (MDR) was 16%, with the most common resistance profile being: tetracycline, pleuromutilin and beta-lactam, which were found in three 'B. pulli' and one B. pilosicoli. There was good correlation with the genotype and the corresponding antibiotic MIC phenotypes: pleuromutilins (tiamulin and valnemulin), macrolides (tylosin and tylvalosin), lincomycin and doxycycline. The occurrence of resistance determinants identified in this study in pathogenic Brachyspira, especially those which were MDR, is likely to impact treatment of AIS and clearance of infections on farm.

Hospital outbreak of Carbapenem-resistant acinetobacter baumannii in the context of local facility transmission.

Carbapenem-resistant Acinetobacter baumannii are of increasing concern in the health care setting. We describe a cluster of 9 cases in hospitalized patients over a 3-month period that reflected ongoing community transmission from high-risk facilities. Robust surveillance and knowledge of local epidemiology are critical to mitigating onward transmission in the health care setting.

The integrative and conjugative element ICECiPOL15 mediates horizontal transfer of ß-lactam resistance gene in Chryseobacterium indoltheticum POL15.

OBJECTIVES: The dissemination of antibiotic resistance genes (ARGs) from the environment, including agricultural sources, is of increasing concern. In this study, we examined the antibiotic resistance profile and genomic sequence of a strain of Chryseobacterium indoltheticum obtained from an agricultural location. METHODS: The multidrug-resistant bacterial strain POL15 was isolated from the wastewater of a livestock farm in China. Whole-genome sequencing was performed followed by bioinformatics analyses to identify integrative and conjugative elements (ICEs) and ARGs. Mating assays were performed to analyse ICE transferability. RESULTS: Whole-genome sequencing and annotation showed that the genome of POL15 encodes ARGs. Additionally, an ICE named ICECiPOL15, which carries a class C ß-lactamase-encoding gene blaAQU, was identified in the POL15 genome. Genes encoding an integrase, an excisionase, a relaxase, a type IV coupling protein and conjugative transposon proteins involved in a type IV secretion system were also identified in ICECiPOL15. Sequence alignment revealed that ICECiPOL15 might have evolved from other Chryseobacterium species. The horizontal transferability of ICECiPOL15 was demonstrated by mating experiments between C. indoltheticum POL15 and Escherichia coli DL21. CONCLUSIONS: This study represents the first characterization of a mobilizable antibiotic resistance ICE in a species of C. indoltheticum and provides evidence that C. indoltheticum strains could be important reservoirs and vehicles for ARGs on livestock farms.

Filling knowledge gaps related to AmpC-dependent ß-lactam resistance in Enterobacter cloacae.

Enterobacter cloacae starred different pioneer studies that enabled the development of a widely accepted model for the peptidoglycan metabolism-linked regulation of intrinsic class C cephalosporinases, highly conserved in different Gram-negatives. However, some mechanistic and fitness/virulence-related aspects of E. cloacae choromosomal AmpC-dependent resistance are not completely understood. The present study including knockout mutants, ß-lactamase cloning, gene expression analysis, characterization of resistance phenotypes, and the Galleria mellonella infection model fills these gaps demonstrating that: (i) AmpC enzyme does not show any collateral activity impacting fitness/virulence; (ii) AmpC hyperproduction mediated by ampD inactivation does not entail any biological cost; (iii) alteration of peptidoglycan recycling alone or combined with AmpC hyperproduction causes no attenuation of E. cloacae virulence in contrast to other species; (iv) derepression of E. cloacae AmpC does not follow a stepwise dynamics linked to the sequential inactivation of AmpD amidase homologues as happens in Pseudomonas aeruginosa; (v) the enigmatic additional putative AmpC-type ß-lactamase generally present in E. cloacae does not contribute to the classical cephalosporinase hyperproduction-based resistance, having a negligible impact on phenotypes even when hyperproduced from multicopy vector. This study reveals interesting particularities in the chromosomal AmpC-related behavior of E. cloacae that complete the knowledge on this top resistance mechanism.

Structural basis of metallo-ß-lactamase resistance to taniborbactam.

The design of inhibitors against metallo-ß-lactamases (MBLs), the largest family of carbapenemases, has been a strategic goal in designing novel antimicrobial therapies. In this regard, the development of bicyclic boronates, such as taniborbactam (TAN) and xeruborbactam, is a major achievement that may help in overcoming the threat of MBL-producing and carbapenem-resistant Gram-negative pathogens. Of concern, a recent report has shown that New Delhi MBL-9 (NDM-9) escapes the inhibitory action of TAN by a single amino acid substitution with respect to New Delhi MBL-1 (NDM-1), the most widely disseminated MBL. Here, we report a docking and computational analysis that identifies that "escape variants" against TAN can arise by disruption of the electrostatic interaction of negative charges in the active site loops of MBLs with the N-(2-aminoethyl)cyclohexylamine side chain of TAN. These changes result in non-productive binding modes of TAN that preclude reaction with the MBLs, a phenomenon that is not restricted to NDM-9. This analysis demonstrates that single amino acid substitutions in non-essential residues in MBL loops can unexpectedly elicit resistance to TAN.

Synergistic effect of secondary metabolites isolated from Pestalotiopsis sp. FKR-0115 in overcoming ß-lactam resistance in MRSA.

Six aromatic secondary metabolites, pestalone (1), emodin (2), phomopsilactone (3), pestalachlorides B (4), C (5), and D (6), were isolated from Pestalotiopsis sp. FKR-0115, a filamentous fungus collected from white moulds growing on dead branches in Minami Daito Island. The efficacy of these secondary metabolites against methicillin-resistant Staphylococcus aureus (MRSA) with and without meropenem (ß-lactam antibiotic) was evaluated using the paper disc method and broth microdilution method. The chemical structures of the isolated compounds (1-6) were characterised using spectroscopic methods, including nuclear magnetic resonance and mass spectrometry. All six isolated compounds exhibited synergistic activity with meropenem against MRSA. Among the six secondary metabolites, pestalone (1) overcame bacterial resistance in MRSA to the greatest extent.

Clostridioides difficile canonical L,D-transpeptidases catalyze a novel type of peptidoglycan cross-links and are not required for beta-lactam resistance.

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea worldwide with significant morbidity and mortality. This organism is naturally resistant to several beta-lactam antibiotics that inhibit the polymerization of peptidoglycan, an essential component of the bacteria cell envelope. Previous work has revealed that C. difficile peptidoglycan has an unusual composition. It mostly contains 3-3 cross-links, catalyzed by enzymes called L,D-transpeptidases (Ldts) that are poorly inhibited by beta-lactams. It was therefore hypothesized that peptidoglycan polymerization by these enzymes could underpin antibiotic resistance. Here, we investigated the catalytic activity of the three canonical Ldts encoded by C. difficile (LdtCd1, LdtCd2, and LdtCd3) in vitro and explored their contribution to growth and antibiotic resistance. We show that two of these enzymes catalyze the formation of novel types of peptidoglycan cross-links using meso-diaminopimelic acid both as a donor and an acceptor, also observed in peptidoglycan sacculi. We demonstrate that the simultaneous deletion of these three genes only has a minor impact on both peptidoglycan structure and resistance to beta-lactams. This unexpected result therefore implies that the formation of 3-3 peptidoglycan cross-links in C. difficile is catalyzed by as yet unidentified noncanonical Ldt enzymes.

The LysR-type transcription factor LsrB regulates beta-lactam resistance in Agrobacterium tumefaciens.

Agrobacterium tumefaciens is a plant pathogen, broadly known as the causal agent of the crown gall disease. The soil bacterium is naturally resistant to beta-lactam antibiotics by utilizing the inducible beta-lactamase AmpC. Our picture on the condition-dependent regulation of ampC expression is incomplete. A known regulator is AmpR controlling the transcription of ampC in response to unrecycled muropeptides as a signal for cell wall stress. In our study, we uncovered the global transcriptional regulator LsrB as a critical player acting upstream of AmpR. Deletion of lsrB led to severe ampicillin and penicillin sensitivity, which could be restored to wild-type levels by lsrB complementation. By transcriptome profiling via RNA-Seq and qRT-PCR and by electrophoretic mobility shift assays, we show that ampD coding for an anhydroamidase involved in peptidoglycan recycling is under direct negative control by LsrB. Controlling AmpD levels by the LysR-type regulator in turn impacts the cytoplasmic concentration of cell wall degradation products and thereby the AmpR-mediated regulation of ampC. Our results substantially expand the existing model of inducible beta-lactam resistance in A. tumefaciens by establishing LsrB as higher-level transcriptional regulator.

Adaptive ß-lactam resistance from an inducible efflux pump that is post-translationally regulated by the DjlA co-chaperone.

The acquisition of multidrug resistance (MDR) determinants jeopardizes treatment of bacterial infections with antibiotics. The tripartite efflux pump AcrAB-NodT confers adaptive MDR in the polarized α-proteobacterium Caulobacter crescentus via transcriptional induction by first-generation quinolone antibiotics. We discovered that overexpression of AcrAB-NodT by mutation or exogenous inducers confers resistance to cephalosporin and penicillin (ß-lactam) antibiotics. Combining 2-step mutagenesis-sequencing (Mut-Seq) and cephalosporin-resistant point mutants, we dissected how TipR uses a common operator of the divergent tipR and acrAB-nodT promoter for adaptive and/or potentiated AcrAB-NodT-directed efflux. Chemical screening identified diverse compounds that interfere with DNA binding by TipR or induce its dependent proteolytic turnover. We found that long-term induction of AcrAB-NodT deforms the envelope and that homeostatic control by TipR includes co-induction of the DnaJ-like co-chaperone DjlA, boosting pump assembly and/or capacity in anticipation of envelope stress. Thus, the adaptive MDR regulatory circuitry reconciles drug efflux with co-chaperone function for trans-envelope assemblies and maintenance.

Ultrafast detection of ß-lactamase resistance in Klebsiella pneumoniae from blood culture by nanopore sequencing.

Aim: This study aimed to assess the ultra-fast method using MinION™ sequencing for rapid identification of ß-lactamase-producing Klebsiella pneumoniae clinical isolates from positive blood cultures. Methods: Spiked-blood positive blood cultures were extracted using the ultra-fast method and automated DNA extraction for MinION sequencing. Raw reads were analyzed for ß-lactamase resistance genes. Multilocus sequence typing and ß-lactamase variant characterization were performed after assembly. Results: The ultra-fast method identified clinically relevant ß-lactamase resistance genes in less than 1 h. Multilocus sequence typing and ß-lactamase variant characterization required 3-6 h. Sequencing quality showed no direct correlation with pore number or DNA concentration. Conclusion: Nanopore sequencing, specifically the ultra-fast method, is promising for the rapid diagnosis of bloodstream infections, facilitating timely identification of multidrug-resistant bacteria in clinical samples.

Klebsiella pneumoniae is a bacterium that can cause infections in the blood. These infections can be severe, especially if K. pneumoniae is not susceptible to antibiotics ('antibiotic resistant'). Tools that can detect this resistance are important. In this study, we tested one such tool called MinION™ with blood samples. In 1 h, we were able to identify the bacteria within the sample and their resistance. This type of testing would help clinicians to give the best treatment to patients. More studies are needed to prove the usefulness of MinION for processing samples from real patients.