Reciprocal regulation of enterococcal cephalosporin resistance by products of the autoregulated yvcJ-glmR-yvcL operon enhances fitness during cephalosporin exposure.

Enterococci are commensal members of the gastrointestinal tract and also major nosocomial pathogens. They possess both intrinsic and acquired resistance to many antibiotics, including intrinsic resistance to cephalosporins that target bacterial cell wall synthesis. These antimicrobial resistance traits make enterococcal infections challenging to treat. Moreover, prior therapy with antibiotics, including broad-spectrum cephalosporins, promotes enterococcal proliferation in the gut, resulting in dissemination to other sites of the body and subsequent infection. As a result, a better understanding of mechanisms of cephalosporin resistance is needed to enable development of new therapies to treat or prevent enterococcal infections. We previously reported that flow of metabolites through the peptidoglycan biosynthesis pathway is one determinant of enterococcal cephalosporin resistance. One factor that has been implicated in regulating flow of metabolites into cell wall biosynthesis pathways of other Gram-positive bacteria is GlmR. In enterococci, GlmR is encoded as the middle gene of a predicted 3-gene operon along with YvcJ and YvcL, whose functions are poorly understood. Here we use genetics and biochemistry to investigate the function of the enterococcal yvcJ-glmR-yvcL gene cluster. Our results reveal that YvcL is a DNA-binding protein that regulates expression of the yvcJ-glmR-yvcL operon in response to cell wall stress. YvcJ and GlmR bind UDP-GlcNAc and reciprocally regulate cephalosporin resistance in E. faecalis, and binding of UDP-GlcNAc by YvcJ appears essential for its activity. Reciprocal regulation by YvcJ/GlmR is essential for fitness during exposure to cephalosporin stress. Additionally, our results indicate that enterococcal GlmR likely acts by a different mechanism than the previously studied GlmR of Bacillus subtilis, suggesting that the YvcJ/GlmR regulatory module has evolved unique targets in different species of bacteria.

The spread of pESI-mediated extended-spectrum cephalosporin resistance in Salmonella serovars-Infantis, Senftenberg, and Alachua isolated from food animal sources in the United States.

The goal of this study is to investigate the origin, prevalence, and evolution of the pESI megaplasmid in Salmonella isolated from animals, foods, and humans. We queried 510,097 Salmonella genomes under the National Center for Biotechnology Information (NCBI) Pathogen Detection (PD) database for the presence of potential sequences containing the pESI plasmid in animal, food, and environmental sources. The presence of the pESI megaplasmid was confirmed by using seven plasmid-specific markers (rdA, pilL, SogS, TrbA, ipf, ipr2 and IncFIB(pN55391)). The plasmid and chromosome phylogeny of these isolates was inferred from single nucleotide polymorphisms (SNPs). Our search resolved six Salmonella clusters carrying the pESI plasmid. Four were emergent Salmonella Infantis clusters, and one each belonged to serovar Senftenberg and Alachua. The Infantis cluster with a pESI plasmid carrying blaCTX-M-65 gene was the biggest of the four emergent Infantis clusters, with over 10,000 isolates. This cluster was first detected in South America and has since spread widely in United States. Over time the composition of pESI in United States has changed with the average number of resistance genes showing a decrease from 9 in 2014 to 5 in 2022, resulting from changes in gene content in two integrons present in the plasmid. A recent and emerging cluster of Senftenberg, which carries the blaCTX-M-65 gene and is primarily associated with turkey sources, was the second largest in the United States. SNP analysis showed that this cluster likely originated in North Carolina with the recent acquisition of the pESI plasmid. A single Alachua isolate from turkey was also found to carry the pESI plasmid containing blaCTX-M-65 gene. The study of the pESI plasmid, its evolution and mechanism of spread can help us in developing appropriate strategies for the prevention and further spread of this multi-drug resistant plasmid in Salmonella in poultry and humans.

Association of cephalosporin resistance in intraoperative biliary cultures with surgical site infections in patients undergoing pancreaticoduodenectomy. A retrospective cohort study.

BACKGROUND: The aim of this study was to assess the incidence of bacterobilia at the time of a pancreaticoduodenectomy (PD) and the association of resistant bacteria in bile to surgical site infections (SSI). METHODS: This was a retrospective cohort study including patients undergoing PD in a single center between May 2016 and October 2020. Data of preoperative biliary drainage (PBD), intraoperative biliary cultures (IBC) and postoperative complications were analysed to assess the risk factors for resistant bacteria in IBC and SSIs. RESULTS: Of 361 patients included, 254 (70%) had undergone PBD. Second-generation cephalosporin resistant bacteria were found in IBC of 183 (64%) of all the patients. PBD was the only risk factor for second-generation cephalosporin resistance. The risk for second-generation cephalosporin resistance was more than 20-fold in patients with PBD [n = 170/254 (67%) (OR 22.58 (95% CI, 9.61-53.01), p < 0.001)] compared to patients who did not have PBD (n = 13/107 (12%)). Also, if the time between PBD and surgery was 2 months or more the second-generation cephalosporin resistance in IBC increased the risk for SSIs (OR 4.14 (95% CI, 1.18-14.51), p = 0.027). CONCLUSION: The second-generation cephalosporin resistance in IBC is common in patients who have undergone PBD. Broad-spectrum antibiotics in prophylaxis may be beneficial for these patients.

Cephalosporin resistance, tolerance, and approaches to improve their activities.

Cephalosporins comprise a ß-lactam antibiotic class whose first members were discovered in 1945 from the fungus Cephalosporium acremonium. Their clinical use for Gram-negative bacterial infections is widespread due to their ability to traverse outer membranes through porins to gain access to the periplasm and disrupt peptidoglycan synthesis. More recent members of the cephalosporin class are administered as last resort treatments for complicated urinary tract infections, MRSA, and other multi-drug resistant pathogens, such as Neisseria gonorrhoeae. Unfortunately, there has been a global increase in cephalosporin-resistant strains, heteroresistance to this drug class has been a topic of increasing concern, and tolerance and persistence are recognized as potential causes of cephalosporin treatment failure. In this review, we summarize the cephalosporin antibiotic class from discovery to their mechanisms of action, and discuss the causes of cephalosporin treatment failure, which include resistance, tolerance, and phenomena when those qualities are exhibited by only small subpopulations of bacterial cultures (heteroresistance and persistence). Further, we discuss how recent efforts with cephalosporin conjugates and combination treatments aim to reinvigorate this antibiotic class.

PASTA-kinase-mediated signaling drives accumulation of the peptidoglycan synthesis protein MurAA to promote cephalosporin resistance in Enterococcus faecalis.

The bacterial PASTA kinase, IreK, is required for intrinsic cephalosporin resistance in the Gram-positive opportunistic pathogen, Enterococcus faecalis. IreK activity is enhanced in response to cell wall stress, such as cephalosporin exposure. The downstream consequences of IreK activation are not well understood in E. faecalis, but recent work in other low-GC Gram-positive bacteria demonstrated PASTA kinase-dependent regulation of MurAA, an enzyme that performs the first committed step in the peptidoglycan synthesis pathway. Here, we used genetic suppressor selections to identify MurAA as a downstream target of IreK signaling in E. faecalis. Using complementary genetic and biochemical approaches, we demonstrated that MurAA abundance is regulated by IreK signaling in response to physiologically relevant cell wall stress to modulate substrate flux through the peptidoglycan synthesis pathway. Specifically, the IreK substrate, IreB, promotes proteolysis of MurAA through a direct physical interaction in a manner responsive to phosphorylation by IreK. MurAB, a homolog of MurAA, also promotes MurAA proteolysis and interacts directly with IreB. Our results therefore establish a connection between the cell wall stress sensor IreK and one critical physiological output to modulate peptidoglycan synthesis and drive cephalosporin resistance.

Implications of different waterfowl farming on cephalosporin resistance: Investigating the role of blaCTX-M-55.

We investigated the cephalosporin resistance of Escherichia coli from waterfowl among different breeding mode farms. In 2021, we isolated 200 strains of E. coli from waterfowl feces samples collected from Sichuan, Heilongjiang, and Anhui provinces. The key findings are: Out of the 200 strains, 80, 80, and 40 strains were isolated from waterfowl feces samples in intensive, courtyard, and outdoor breeding mode farms, respectively. The overall positive rate of the ESBL phenotype, detecting by the double disk diffusion method, was 68.00% (136/200). In particular, the rates for intensive, courtyard, and outdoor breeding modes were 98.75%, 36.25%, and 70.00%, respectively. Results of MIC test showed drug resistance rates in the intensive breeding mode: 100.00% for cephalothin, 38.75% for cefoxitin, 100.00% for cefotaxime, and 100.00% for cefepime. In courtyard breeding mode, the corresponding rates were 100.00%, 40.00%, 63.75%, and 45.00%, respectively. In outdoor breeding mode, the corresponding rates were 100.00%, 52.50%, 82.50%, and 77.50%, respectively. The PCR results for blaCTX-M, blaTEM, blaOXA, and blaSHV showed the detection rate of blaCTX-M was highest at 75.50%, with blaCTX-M-55 is the main subtype gene, followed by blaTEM at 73.50%. We screened 58 donor strains carrying blaCTX-M-55, including 52 strains from the intensive breeding mode. These donor bacteria can transfer different plasmids to recipient E. coli J53, resulting in recipient bacteria acquiring cephalosporin resistance, and the conjugational transfer frequency ranged from 1.01 × 10-5 to 6.56 × 10-2. The transferred plasmids remained stable in recipient bacteria for up to several days without significant adaptation costs observed. During molecular typing of E. coli with conjugational transfer ability, the blaCTX-M-55 was found to be widely present in different ST strains with several phylogenetic groups. In summary, cephalosporin resistance of E. coli carried by waterfowl birds in intensive breeding mode farm was significantly higher than in courtyard and outdoor mode farms. The blaCTX-M-55 subtype gene was the prevalent ARGs and can be horizontally transferred through plasmids, which plays a key role in the spread of cephalosporin drug resistance.

Mortality associated with third-generation cephalosporin resistance in Enterobacterales bloodstream infections at eight sub-Saharan African hospitals (MBIRA): a prospective cohort study.

BACKGROUND: Bacteria of the order Enterobacterales are common pathogens causing bloodstream infections in sub-Saharan Africa and are frequently resistant to third-generation cephalosporin antibiotics. Although third-generation cephalosporin resistance is believed to lead to adverse outcomes, this relationship is difficult to quantify and has rarely been studied in this region. We aimed to measure the effects associated with resistance to third-generation cephalosporins in hospitalised patients with Enterobacterales bloodstream infection in Africa. METHODS: We conducted a prospective, matched, parallel cohort study at eight hospitals across sub-Saharan Africa. We recruited consecutive patients of all age groups with laboratory-confirmed Enterobacterales bloodstream infection and matched them to at least one patient without bloodstream infection on the basis of age group, hospital ward, and admission date. Date of infection onset (and enrolment) was defined as the day of blood sample collection for culturing. Patients infected with bacteria with a cefotaxime minimum inhibitory concentration of 1 mg/L or lower were included in the third-generation cephalosporin-susceptible (3GC-S) cohort, and the remainder were included in the third-generation cephalosporin-resistant (3GC-R) cohort. The primary outcomes were in-hospital death and death within 30 days of enrolment. We used adjusted multivariable regression models to first compare patients with bloodstream infection against matched patients within the 3GC-S and 3GC-R cohorts, then compared estimates between cohorts. FINDINGS: Between Nov 1, 2020, and Jan 31, 2022, we recruited 878 patients with Enterobacterales bloodstream infection (221 [25·2%] to the 3GC-S cohort and 657 [74·8%] to the 3GC-R cohort) and 1634 matched patients (420 [25·7%] and 1214 [74·3%], respectively). 502 (57·2%) bloodstream infections occurred in neonates and infants (age 0-364 days). Klebsiella pneumoniae (393 [44·8%] infections) and Escherichia coli (224 [25·5%] infections) were the most common Enterobacterales species identified. The proportion of patients who died in hospital was higher in patients with bloodstream infection than in matched controls in the 3GC-S cohort (62 [28·1%] of 221 vs 22 [5·2%] of 420; cause-specific hazard ratio 6·79 [95% CI 4·06-11·37] from Cox model) and the 3GC-R cohort (244 [37·1%] of 657 vs 115 [9·5%] of 1214; 5·01 [3·96-6·32]). The ratio of these cause-specific hazard ratios showed no significant difference in risk of in-hospital death in the 3GC-R cohort versus the 3GC-S cohort (0·74 [0·42-1·30]). The ratio of relative risk of death within 30 days (0·82 [95% CI 0·53-1·27]) also indicated no difference between the cohorts. INTERPRETATION: Patients with bloodstream infections with Enterobacterales bacteria either resistant or susceptible to third-generation cephalosporins had increased mortality compared with uninfected matched patients, with no differential effect related to third-generation cephalosporin-resistance status. However, this finding does not account for time to appropriate antibiotic treatment, which remains clinically important to optimise. Measures to prevent transmission of Enterobacterales could reduce bloodstream infection-associated mortality from both drug-resistant and drug-susceptible bacterial strains in Africa. FUNDING: Bill & Melinda Gates Foundation.

Multisite Phosphorylation Regulates GpsB Function in Cephalosporin Resistance of Enterococcus faecalis.

Enterococci are normal human commensals and major causes of hospital-acquired infections. Enterococcal infections can be difficult to treat because enterococci harbor intrinsic and acquired antibiotic resistance, such as resistance to cephalosporins. In Enterococcus faecalis, the transmembrane kinase IreK, a member of the bacterial PASTA kinase family, is essential for cephalosporin resistance. The activity of IreK is boosted by the cytoplasmic protein GpsB, which promotes IreK autophosphorylation and signaling to drive cephalosporin resistance. A previous phosphoproteomics study identified eight putative IreK-dependent phosphorylation sites on GpsB, but the functional importance of GpsB phosphorylation was unknown. Here we used genetic and biochemical approaches to define three sites of phosphorylation on GpsB that functionally impact IreK activity and cephalosporin resistance. Phosphorylation at two sites (S80 and T84) serves to impair the ability of GpsB to activate IreK in vivo, suggesting phosphorylation of these sites acts as a means of negative feedback for IreK. The third site of phosphorylation (T133) occurs in a segment of GpsB termed the C-terminal extension that is unique to enterococcal GpsB homologs. The C-terminal extension is highly mobile in solution, suggesting it is largely unstructured, and phosphorylation of T133 appears to enable efficient phosphorylation at S80 / T84. Overall our results are consistent with a model in which multisite phosphorylation of GpsB impairs its ability to activate IreK, thereby diminishing signal transduction through the IreK-dependent pathway and modulating phenotypic cephalosporin resistance.

PhoPQ Regulates Quinolone and Cephalosporin Resistance Formation in Salmonella Enteritidis at the Transcriptional Level.

The two-component system (TCS) PhoPQ has been demonstrated to be crucial for the formation of resistance to quinolones and cephalosporins in Salmonella Enteritidis (S. Enteritidis). However, the mechanism underlying PhoPQ-mediated antibiotic resistance formation remains poorly understood. Here, it was shown that PhoP transcriptionally regulated an assortment of genes associated with envelope homeostasis, the osmotic stress response, and the redox balance to confer resistance to quinolones and cephalosporins in S. Enteritidis. Specifically, cells lacking the PhoP regulator, under nalidixic acid and ceftazidime stress, bore a severely compromised membrane on the aspects of integrity, fluidity, and permeability, with deficiency to withstand osmolarity stress, an increased accumulation of intracellular reactive oxygen species, and dysregulated redox homeostasis, which are unfavorable for bacterial survival. The phosphorylated PhoP elicited transcriptional alterations of resistance-associated genes, including the outer membrane porin ompF and the aconitate hydratase acnA, by directly binding to their promoters, leading to a limited influx of antibiotics and a well-maintained intracellular metabolism. Importantly, it was demonstrated that the cavity of the PhoQ sensor domain bound to and sensed quinolones/cephalosporins via the crucial surrounding residues, as their mutations abrogated the binding and PhoQ autophosphorylation. This recognition mode promoted signal transduction that activated PhoP, thereby modulating the transcription of downstream genes to accommodate cells to antibiotic stress. These findings have revealed how bacteria employ a specific TCS to sense antibiotics and combat them, suggesting PhoPQ as a potential drug target with which to surmount S. Enteritidis. IMPORTANCE The prevalence of quinolone and cephalosporin-resistant S. Enteritidis is of increasing clinical concern. Thus, it is imperative to identify novel therapeutic targets with which to treat S. Enteritidis-associated infections. The PhoPQ two-component system is conserved across a variety of Gram-negative pathogens, by which bacteria adapt to a range of environmental stimuli. Our earlier work has demonstrated the importance of PhoPQ in the resistance formation in S. Enteritidis to quinolones and cephalosporins. In the current work, we identified a global profile of genes that are regulated by PhoP under antibiotic stresses, with a focus on how PhoP regulated downstream genes, either positively or negatively. Additionally, we established that PhoQ sensed quinolones and cephalosporins in a manner of directly binding to them. These identified genes and pathways that are mediated by PhoPQ represent promising targets for the development of a drug potentiator with which to neutralize antibiotic resistance in S. Enteritidis.

Collateral sensitivity profiling in drug-resistant Escherichia coli identifies natural products suppressing cephalosporin resistance.

The rapid emergence of antimicrobial resistance presents serious health challenges to the management of infectious diseases, a problem that is further exacerbated by slowing rates of antimicrobial drug discovery in recent years. The phenomenon of collateral sensitivity (CS), whereby resistance to one drug is accompanied by increased sensitivity to another, provides new opportunities to address both these challenges. Here, we present a high-throughput screening platform termed Collateral Sensitivity Profiling (CSP) to map the difference in bioactivity of large chemical libraries across 29 drug-resistant strains of E. coli. CSP screening of 80 commercial antimicrobials demonstrated multiple CS interactions. Further screening of a 6195-member natural product library revealed extensive CS relationships in nature. In particular, we report the isolation of known and new analogues of borrelidin A with potent CS activities against cephalosporin-resistant strains. Co-dosing ceftazidime with borrelidin A slows broader cephalosporin resistance with no recognizable resistance to borrelidin A itself.

Dynamics of extended-spectrum cephalosporin resistance genes in Escherichia coli from Europe and North America.

Extended-spectrum cephalosporins (ESCs) are critically important antimicrobial agents for human and veterinary medicine. ESC resistance (ESC-R) genes have spread worldwide through plasmids and clonal expansion, yet the distribution and dynamics of ESC-R genes in different ecological compartments are poorly understood. Here we use whole genome sequence data of Enterobacterales isolates of human and animal origin from Europe and North America and identify contrasting temporal dynamics. AmpC ß-lactamases were initially more dominant in North America in humans and farm animals, only later emerging in Europe. In contrast, specific extended-spectrum ß-lactamases (ESBLs) were initially common in animals from Europe and later emerged in North America. This study identifies differences in the relative importance of plasmids and clonal expansion across different compartments for the spread of different ESC-R genes. Understanding the mechanisms of transmission will be critical in the design of interventions to reduce the spread of antimicrobial resistance.

Interspecies differences in clinical characteristics and risk factors for third-generation cephalosporin resistance between Escherichia coli and Klebsiella pneumoniae bloodstream infection in patients with liver cirrhosis.

This retrospective study aimed to clarify the interspecies differences in the clinical characteristics and risk factors of bloodstream infection (BSI) due to third-generation cephalosporin-resistant (3GC-R) Escherichia coli (EC) and Klebsiella pneumoniae (KP) in patients with liver cirrhosis (LC). KP BSI had more comorbidities and higher treatment failure rate than EC BSI. Non-alcoholic LC was a risk factor for treatment failure in EC, whereas it was not associated with KP. Risk factors for BSI due to 3GC-R strain were nosocomial infection in EC, and ß-lactam/fluoroquinolone treatment ≤ 30 days in KP. These results could help predict outcomes of BSI and improve clinical practice.

GpsB Promotes PASTA Kinase Signaling and Cephalosporin Resistance in Enterococcus faecalis.

Enterococci are opportunistic pathogens that can cause severe bacterial infections. Treatment of these infections is challenging because enterococci possess intrinsic and acquired mechanisms of resistance to commonly used antibiotics, including cephalosporins. The transmembrane serine/threonine PASTA kinase, IreK, is an important determinant of enterococcal cephalosporin resistance. Upon exposure to cephalosporins, IreK becomes autophosphorylated, which stimulates its kinase activity to phosphorylate downstream substrates and drive cephalosporin resistance. However, the molecular mechanisms that modulate IreK autophosphorylation in response to cell wall stress, such as that induced by cephalosporins, remain unknown. A cytoplasmic protein, GpsB, promotes signaling by PASTA kinase homologs in other bacterial species, but the function of enterococcal GpsB has not been previously investigated. We used in vitro and in vivo approaches to test the hypothesis that enterococcal GpsB promotes IreK signaling in response to cephalosporins to drive cephalosporin resistance. We found that GpsB promotes IreK activity both in vivo and in vitro. This effect is required for cephalosporins to trigger IreK autophosphorylation and activation of an IreK-dependent signaling pathway, and thereby is also required for enterococcal intrinsic cephalosporin resistance. Moreover, analyses of GpsB mutants and a ΔireK gpsB double mutant suggest that GpsB has an additional function, beyond regulation of IreK activity, which is required for optimal growth and full cephalosporin resistance. Collectively, our data provide new insights into the mechanism of signal transduction by the PASTA kinase IreK and the mechanism of enterococcal intrinsic cephalosporin resistance. IMPORTANCE Enterococci are opportunistic pathogens that can cause severe bacterial infections. Treatment of these infections is challenging because enterococci possess intrinsic and acquired resistance to commonly used antibiotics. In particular, enterococci are intrinsically resistant to cephalosporin antibiotics, a trait that requires the activity of a transmembrane serine/threonine kinase, IreK, which belongs to the bacterial PASTA kinase family. The mechanisms by which PASTA kinases are regulated in cells are poorly understood. Here, we report that the cytoplasmic protein GpsB directly promotes IreK signaling in enterococci to drive cephalosporin resistance. Thus, we provide new insights into PASTA kinase regulation and control of enterococcal cephalosporin resistance, and suggest that GpsB could be a promising target for new therapeutics to disable cephalosporin resistance.

CroR Regulates Expression of pbp4(5) to Promote Cephalosporin Resistance in Enterococcus faecalis.

Enterococcus faecalis is an opportunistic pathogen and a major cause of severe nosocomial infections. Treatment options against enterococcal infections are declining due to the resistance of enterococci to numerous antibiotics. A key risk factor for developing enterococcal infections is treatment with cephalosporin antibiotics, to which enterococci are intrinsically resistant. For susceptible organisms, cephalosporins inhibit bacterial growth by acylating the active site of penicillin-binding proteins (PBPs), key enzymes that catalyze peptidoglycan cross-linking. Two specific PBPs of enterococci, Pbp4(5) and PbpA(2b), exhibit low reactivity toward cephalosporins, allowing these PBPs to cross-link peptidoglycan in the presence of cephalosporins to drive resistance in enterococci, but the mechanisms by which these PBPs are regulated are poorly understood. The CroS/R two-component signal transduction system (TCS) is also required for cephalosporin resistance. Activation of CroS/R by cephalosporins leads to CroR-dependent changes in gene expression. However, the specific genes regulated by CroS/R that are responsible for cephalosporin resistance remain largely unknown. In this study, we characterized CroR-dependent transcriptome remodeling by RNA-seq, identifying pbp4(5) as a CroR regulon member in multiple, diverse lineages of E. faecalis. Through genetic analysis of the pbp4(5) and croR promoters, we uncovered a CroR-dependent regulatory motif. Mutations in this motif to disrupt CroR-dependent upregulation of pbp4(5) in the presence of cell wall stress resulted in a reduction of resistance to cephalosporins in E. faecalis, demonstrating that enhanced production of Pbp4(5) and likely other proteins involved in peptidoglycan biogenesis by the CroS/R system drives enterococcal cephalosporin resistance. IMPORTANCE Investigation into molecular mechanisms used by enterococci to subvert cephalosporin antibiotics is imperative for preventing and treating life-threatening infections. In this study, we used genetic means to investigate the functional output of the CroS/R TCS required for enterococcal resistance to cephalosporins. We found that enhanced production of the penicillin-binding protein Pbp4(5) upon exposure to cell wall stress was mediated by CroS/R and was critical for intrinsic cephalosporin resistance of E. faecalis.

In vitro selection of Neisseria gonorrhoeae unveils novel mutations associated with extended-spectrum cephalosporin resistance.

The emergence of Neisseria gonorrhoeae strains resistant to extended-spectrum cephalosporins (ESCs) is a worldwide concern because this class of antibiotics represents the last empirical treatment option for gonorrhea. The abusive use of antimicrobials may be an essential factor for the emergence of ESC resistance in N. gonorrhoeae. Cephalosporin resistance mechanisms have not been fully clarified. In this study, we mapped mutations in the genome of N. gonorrhoeae isolates after resistance induction with cefixime and explored related metabolic pathways. Six clinical isolates with different antimicrobial susceptibility profiles and genotypes and two gonococcal reference strains (WHO F and WHO Y) were induced with increasing concentrations of cefixime. Antimicrobial susceptibility testing was performed against six antimicrobial agents before and after induction. Clinical isolates were whole-genome sequenced before and after induction, whereas reference strains were sequenced after induction only. Cefixime resistance induction was completed after 138 subcultures. Several metabolic pathways were affected by resistance induction. Five isolates showed SNPs in PBP2. The isolates M111 and M128 (ST1407 with mosaic penA-34.001) acquired one and four novel missense mutations in PBP2, respectively. These isolates exhibited the highest minimum inhibitory concentration (MIC) for cefixime among all clinical isolates. Mutations in genes contributing to ESC resistance and in other genes were also observed. Interestingly, M107 and M110 (ST338) showed no mutations in key determinants of ESC resistance despite having a 127-fold increase in the MIC of cefixime. These findings point to the existence of different mechanisms of acquisition of ESC resistance induced by cefixime exposure. Furthermore, the results reinforce the importance of the gonococcal antimicrobial resistance surveillance program in Brazil, given the changes in treatment protocols made in 2017 and the nationwide prevalence of sequence types that can develop resistance to ESC.

Excreted Antibiotics May Be Key to Emergence of Increasingly Efficient Antibiotic Resistance in Food Animal Production.

At a time when antibiotic resistance is seemingly ubiquitous worldwide, understanding the mechanisms responsible for successful emergence of new resistance genes may provide insights into the persistence and pathways of dissemination for antibiotic-resistant organisms in general. For example, Escherichia coli strains harboring a class A ß-lactamase-encoding gene (blaCTX-M-15) appear to be displacing strains that harbor a class C ß-lactamase gene (blaCMY-2) in Washington State dairy cattle. We cloned these genes with native promoters into low-copy-number plasmids that were then transformed into isogenic strains of E. coli, and growth curves were generated for two commonly administered antibiotics (ampicillin and ceftiofur). Both strains met the definition of resistance for ampicillin (≥32 µg/mL) and ceftiofur (≥16 µg/mL). Growth of the CMY-2-producing strain was compromised at 1,000 µg/mL ampicillin, whereas the CTX-M-15-producing strain was not inhibited in the presence of 3,000 µg/mL ampicillin or with most concentrations of ceftiofur, although there were mixed outcomes with ceftiofur metabolites. Consequently, in the absence of competing genes, E. coli harboring either gene would experience a selective advantage if exposed to these antibiotics. Successful emergence of CTX-M-15-producing strains where CMY-2-producing strains are already established, however, requires high concentrations of antibiotics that can only be found in the urine of treated animals (e.g., >2,000 µg/mL for ampicillin, based on literature). This ex vivo selection pressure may be important for the emergence of new and more efficient antibiotic resistance genes and likely for persistence of antibiotic-resistant bacteria in food animal populations. IMPORTANCE We studied the relative fitness benefits of a cephalosporin resistance enzyme (CTX-M-15) that is displacing a similar enzyme (CMY-2), which is extant in E. coli from dairy cattle in Washington State. In vitro experiments demonstrated that CTX-M-15 provides a significant fitness advantage, but only in the presence of very high concentrations of antibiotic that are only found when the antibiotic ampicillin, and to a lesser extent ceftiofur, is excreted in urine from treated animals. As such, the increasing prevalence of bacteria with blaCTX-M-15 is likely occurring ex vivo. Interventions should focus on controlling waste from treated animals and, when possible, selecting antibiotics that are less likely to impact the proximal environment of treated animals.

Extended Spectrum ß-Lactamase Activity and Cephalosporin Resistance in Escherichia coli from U.S. Mid-Atlantic Surface and Reclaimed Water.

Phylogenetic distribution and extended spectrum ß-lactamase (ESBL) activity of Escherichia coli recovered from surface and reclaimed water in the mid-Atlantic U.S. were evaluated. Among 488 isolates, phylogroups B1 and A were the most and least prevalent, respectively. Water type, but not season, affected phylogroup distribution. The likelihood of detecting group A isolates was higher in reclaimed than pond (P < 0.01), freshwater river (P < 0.01) or brackish river (P < 0.05) water. Homogeneity in group distribution was lowest in pond water, where group B1 comprised 50% of isolates. Only 16 (3.3%) isolates exhibited phenotypic resistance to one or more cephalosporins tested and only four had ESBL activity, representing groups B1, B2 isolates, and D. Phylogroup was a factor in antimicrobial resistance (P < 0.05), with group A (8.7%) and D (1.6%) exhibiting the highest and lowest rates. Resistance to cefoxitin was the most prevalent. Multi- versus single drug resistance was affected by phylogroup (P < 0.05) and more likely in groups D and B1 than A which carried resistance to cefoxitin only. The most detected ß-lactam resistance genes were blaCMY-2 and blaTEM. Water type was a factor for blaCTX-M gene detection (P < 0.05). Phenotypic resistance to cefotaxime, ceftriaxone, cefuroxime and ceftazidime, and genetic determinants for ESBL-mediated resistance were found predominantly in B2 and D isolates from rivers and reclaimed water. Overall, ESBL activity and cephalosporin resistance in reclaimed and surface water isolates were low. Integrating data on ESBL activity and ß-lactam resistance among E. coli populations can inform decisions on safety of irrigation water sources and One Health. IMPORTANCE Extended spectrum ß-lactamase (ESBL) producing bacteria, that are resistant to a broad range of antimicrobial agents, are spreading in the environment but data remain scarce. ESBL-producing Escherichia coli infections in the community are on the rise. This work was conducted to assess presence of ESBL-producing E. coli in water that could be used for irrigation of fresh produce. The study provides the most extensive evaluation of ESBL-producing E. coli in surface and reclaimed water in the mid-Atlantic United States. The prevalence of ESBL producers was low and phenotypic resistance to cephalosporins (types of ß-lactam antibiotics) was affected by season but not water type. Data on antimicrobial resistance among E. coli populations in water can inform decisions on safety of irrigation water sources and One Health.

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

Treatment with ß-lactam antibiotics, particularly cephalosporins, is a major risk factor for Clostridioides difficile infection. These broad-spectrum antibiotics irreversibly inhibit penicillin-binding proteins (PBPs), which are serine-based enzymes that assemble the bacterial cell wall. However, C. difficile has four different PBPs (PBP1-3 and SpoVD) with various roles in growth and spore formation, and their specific links to ß-lactam resistance in this pathogen are underexplored. Here, we show that PBP2 (known to be essential for vegetative growth) is the primary bactericidal target for ß-lactams in C. difficile. PBP2 is insensitive to cephalosporin inhibition, and this appears to be the main basis for cephalosporin resistance in this organism. We determine crystal structures of C. difficile PBP2, alone and in complex with ß-lactams, revealing unique features including ligand-induced conformational changes and an active site Zn2+-binding motif that influences ß-lactam binding and protein stability. The Zn2+-binding motif is also present in C. difficile PBP3 and SpoVD (which are known to be essential for sporulation), as well as in other bacterial taxa including species living in extreme environments and the human gut. We speculate that this thiol-containing motif and its cognate Zn2+ might function as a redox sensor to regulate cell wall synthesis for survival in adverse or anaerobic environments.