Genetic traits and transmission of antimicrobial resistance characteristics of cephalosporin resistant Escherichia coli in tropical aquatic environments.

This study investigates the genetic traits and transmission mechanisms of cephalosporin-resistant Escherichia coli in tropical aquatic environments in Singapore. From 2016 to 2020, monthly samples were collected from wastewater treatment plants, marine niches, community sewage, beaches, reservoirs, aquaculture farms, and hospitals, yielding 557 isolates that were analyzed for antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) using genomic methods. Findings reveal significant genotypic similarities between environmental and hospital-derived strains, particularly the pandemic E. coli ST131. Environmental strains exhibited high levels of intrinsic resistance mechanisms, including mutations in porins and efflux pumps, with key ARGs such as CMY-2 and NDM-9 predominantly carried by MGEs, which facilitate horizontal gene transfer. Notably, pathogenic EPEC and EHEC strains were detected in community sewage and aquaculture farms, posing substantial public health risks. This underscores the critical role of these environments as reservoirs for multidrug-resistant pathogens and emphasizes the interconnectedness of human activities and environmental health.

Bacterial Epidemiology and Antimicrobial Resistance Profiles of Bloodstream Infections Caused by Negative Bacteria in Children's: A Multicenter Study in China (2016-2022).

Objective: Aim to investigate the pathogens distribution and drug resistance of gram-negative bacteria causing bloodstream infection (BSIs) in Infectious Disease Surveillance of Pediatric from 2016 to 2022. The prevalence of four important drug resistance phenotypes was studied: difficult-to-treat resistance, fluoroquinolone resistance, carbapenem resistance, and extended-spectrum cephalosporin resistance, and to provide reference basis for preventing and treating BSIs diseases in children. Methods: Strain identification and antimicrobial susceptibility tests were independently performed at each hospital. Data were analyzed using Whonet 5.6 and GraphPad Prism 8 software. The Mann-Whitney U-test was used to examine and compare temporal changes. Results: A total of 39977 BSIs strains were isolated, with 27.1% of the negative bacteria causing BSIs (10824 strains). The highest bacteria detected were E. coli and S. maltophilia in the neonatal and pediatric groups. The detection rate of carbapenem-resistant-K. pneumoniae (CRKPN) in neonate group was 31.4%, significantly increased compared with pediatric group, whose detection rate was 24.7%. The rates of resistance to levofloxacin and trimethoprim/sulfamethoxazole were significantly lower in neonatal groups than pediatric groups in BSIs caused by K. pneumoniae. To imipenem and meropenem were 3.6% and 3.9% among neonatal isolates, which was lower than 4.7% and 5.8 among pediatric BSIs caused by E. coli. Isolated from neonatal BSIs caused by A. baumannii showed lower resistance ratios to all the agents tested than those from pediatric. However, only the prevalence of piperacillin/tazobactam resistance was statistically lower than that in pediatric BSIs caused by P. aeruginosa. The average detection rates of carbapenem resistance, extended-spectrum cephalosporin resistance, and fluoroquinolone resistance for K. pneumoniae and E. coli were 28.1%,41.4%,11.6% and 4.0%,24.3%,31.1%, respectively. Conclusion: The detection rate of gram-negative pathogens showed an increasing trend among the bloodstream infection. The detection rate of CRKPN assumed a downward trend in 2018. There are differences types of pathogens between the neonatal group and the pediatric group, The detection rate of CRKPN in the neonate group was significantly higher than pediatric group. The first average detection rates for carbapenem resistance, extended-spectrum cephalosporin resistance, and fluoroquinolone resistance were obtained for A. baumannii, K. pneumoniae, and Escherichia coli, respectively. Those data showed a high level of antimicrobial resistance, which has posed an urgent threat to Children's health, suggested that effective monitoring of antimicrobial resistance and antimicrobial stewardship among children in China are required.

Genetic landscape of ESBL producing international clone ST410 of Escherichia coli from pediatric infections in Shenzhen, China.

Background: The emergence of ESBLs producing cephalosporin-resistant Escherichia coli isolates poses a threat to public health. This study aims to decipher the genetic landscape and gain insights into ESBL-producing E. coli strains belonging to the high-risk clone ST410 from pediatric patients. Methods: 29 E. coli ST410 isolates were collected from young children and subjected to antimicrobial susceptibility testing, Whole-genome sequencing (WGS), serotype analysis, MLST, ESBL genes, virulence genes, and plasmid profiling. Results: Antimicrobial susceptibility testing demonstrated a high level of resistance to cephalosporins followed by aminoglycoside, sulfonamide, carbapenem and penicillin group of antibiotics. However, n=20/29 shows MDR phenotype. Phylogenetic group B2 (n=15) dominated, followed by group D (n=7), group A (n=4), and group B1 (n=3). Serotyping analysis identified O1:H7 (n=8), O2:H1 (n=6), O8:H4 (n=5), O16:H5 (n=4), and O25:H4 (n=3). Other serotypes identified included O6:H1, O15:H5, and O18:H7 (n=1 each). The most commonly detected ESBL genes were bla CTX-M, (n=26), followed by bla TEM (n=23), and bla SHV (n=18). Additionally, bla OXA-1 (n=10), bla OXA-48 (n=5), bla KPC-2 (n=3), bla KPC-3 (n=2), bla NDM-1 (n=4), bla NDM-5 (n=1), bla GES-1 (n=2), bla GES-5 (n=1), and bla CYM-1 (n=3). Notable virulence genes identified within the ST410 isolates included fimH (n=29), papC (n=24), hlyA (n=22), and cnf1 (n=18), among others. Diverse plasmids were observed including IncFIS, IncX4, IncFIA, IncCol, IncI2 and IncFIC with transmission frequency ranges from 1.3X10-2 to 2.7X10-3. Conclusion: The ST410 clone exhibited a complex resistance profile, diverse serotypes, the presence of specific resistance genes (ESBL genes), virulence gene repertoire, and diverse plasmids. The bla CTX-M was the most prevalent ESBL gene detected.

Phylogenetics and Mobilization of Genomic Traits of Cephalosporin-Resistant Escherichia coli Originated from Retail Meat.

Contaminations with cephalosporin-resistant Escherichia coli across the food chain may pose a significant threat to public health because those antimicrobials are critically important in human medicine. The impact of the presented data is especially significant concerning Poland's role as one of the leading food producers in the EU. This work aimed to characterize the genomic contents of cephalosporin-resistant Escherichia coli (n = 36) isolated from retail meat to expand the official AMR monitoring reported by EFSA. The ESBL mechanism was predominant (via blaCTX-M-1 and blaSHV-12), with the AmpC-type represented by the blaCMY-2 variant. The strains harbored multiple resistance genes, mainly conferring resistance to aminoglycosides, sulfonamides, trimethoprim, tetracyclines. In some isolates, virulence factors-including intimin (eae) and its receptor (tir) were detected, indicating significant pathogenic potential. Resistance genes showed a link with IncI1 and IncB/O/K/Z plasmids. Cephalosporinases were particularly linked to ISEc9/ISEc1 (blaCTX-M-1 and blaCMY-2). The association of virulence with mobile elements was less common-mostly with IncF plasmids. The analysis of E. coli isolated from retail meat indicates accumulation of ARGs and their association with various mobile genetic elements, thus increasing the potential for the transmission of resistance across the food chain.

Evolution of the Antibiotic Resistance Levels, Multi-Resistance Patterns, and Presence of Antibiotic Resistance Genes in E. coli Isolates from the Feces of Breeding Hens during the Rearing Period.

The food chain acts as an entry point for antibiotic resistance to reach humans and environment. Because of the importance of the poultry sector, we investigated the prevalence and evolution of antibiotic resistance in Escherichia coli isolates from a series of 14,500 breeding hens and their farm environment during the rearing period. Samples included meconium from one-day-old breeders and fecal samples and boot swabs from the breeding sheds of pullets and adult hens. All E. coli isolates from one-day-old chicks, 77% from feces and 61% from boot swabs, were resistant to at least one antibiotic. Cefotaxime and multi-drug resistance in fecal isolates decreased during the rearing period from 41.2% and 80.8% in one-day-old chicks to 3.8% and 33.8% in adults. All genes studied were detected in E. coli from feces and boot swabs, the most common being blaTEM (75%), blaSHV (72%), and qnrB (67%). blaCMY-2 was detected in 100% of one-day-old breeders. The combination of at least one cephalosporin and one quinolone resistance gene was detected in 68.7% of fecal and boot swab isolates. Our results highlight the need to monitor the prevalence of antibiotic resistance on farms and to take appropriate measures to reduce the risk to public and environmental health.

Changes in the Phenotypes of Salmonella spp. in Japanese Broiler Flocks.

Salmonella infections represent a leading cause of foodborne illnesses; resistance to third-generation cephalosporins (TGCs), which are a first-choice antimicrobial for treating human Salmonella enteritis, has become a serious public health concern worldwide. Because the consumption of undercooked chicken meat products is a major cause of foodborne salmonellosis in Japan, we conducted three surveys at different periods between 2017 and 2022, with the cooperation of four abattoirs (two in Eastern and two in Western Japan). The first survey was conducted at abattoir A, which is located in Eastern Japan. Salmonella was detected in 84.4% of broiler flocks tested (27/32); among them, all the TGC-resistant isolates obtained from one farm (farm FA) were identified as S. Infantis. Salmonella was recovered from 62.5% of breast meat samples (20/32), with one case suggesting cross-contamination. The second survey was conducted at three other abattoirs to examine the prevalence of TGC-resistant Salmonella, in both Western (abattoirs B and C) and Eastern (abattoir D) Japan. Salmonella was detected in 90.6% of broiler flocks examined (29/32). TGC-resistant S. Infantis was isolated from 2 flocks until 2018 and not thereafter. Subsequently, isolates were identified as TGC-susceptible S. Schwarzengrund in both regions. The third survey was performed at abattoir A to elucidate whether there were changes in the phenotypes. Of the 11 broiler flocks introduced from farm FA, 10 were positive for Salmonella (90.9%); all the isolates were S. Schwarzengrund susceptible to TGC. This study shows that TGC-susceptible S. Schwarzengrund has replaced the resistant phenotypes among broiler flocks in both Eastern and Western Japan. Although chicken meat products could be cross-contaminated with Salmonella during the slaughtering process, reducing the prevalence of Salmonella in broiler flocks remains important to decrease Salmonella enteritis in humans.

Pbp4 provides transpeptidase activity to the FtsW-PbpB peptidoglycan synthase to drive cephalosporin resistance in Enterococcus faecalis.

Enterococci exhibit intrinsic resistance to cephalosporins, mediated in part by the class B penicillin-binding protein (bPBP) Pbp4 that exhibits low reactivity toward cephalosporins and thus can continue crosslinking peptidoglycan despite exposure to cephalosporins. bPBPs partner with cognate SEDS (shape, elongation, division, and sporulation) glycosyltransferases to form the core catalytic complex of peptidoglycan synthases that synthesize peptidoglycan at discrete cellular locations, although the SEDS partner for Pbp4 is unknown. SEDS-bPBP peptidoglycan synthases of enterococci have not been studied, but some SEDS-bPBP pairs can be predicted based on sequence similarity. For example, FtsW (SEDS)-PbpB (bPBP) is predicted to form the catalytic core of the peptidoglycan synthase that functions at the division septum (the divisome). However, PbpB is readily inactivated by cephalosporins, raising the question-how could the FtsW-PbpB synthase continue functioning to enable growth in the presence of cephalosporins? In this work, we report that the FtsW-PbpB peptidoglycan synthase is required for cephalosporin resistance of Enterococcus faecalis, despite the fact that PbpB is inactivated by cephalosporins. Moreover, Pbp4 associates with the FtsW-PbpB synthase and the TPase activity of Pbp4 is required to enable growth in the presence of cephalosporins in an FtsW-PbpB-synthase-dependent manner. Overall, our results implicate a model in which Pbp4 directly interacts with the FtsW-PbpB peptidoglycan synthase to provide TPase activity during cephalosporin treatment, thereby maintaining the divisome SEDS-bPBP peptidoglycan synthase in a functional state competent to synthesize crosslinked peptidoglycan. These results suggest that two bPBPs coordinate within the FtsW-PbpB peptidoglycan synthase to drive cephalosporin resistance in E. faecalis.

Epidemiology of bloodstream infections caused by extended-spectrum cephalosporin-resistant Escherichia coli and Klebsiella pneumoniae in Switzerland, 2015-2022: secular trends and association with the COVID-19 pandemic.

BACKGROUND: The association between the COVID-19 pandemic and the incidence of invasive infections caused by multidrug-resistant organisms remains a topic of debate. AIM: To analyse the national incidence rates of bloodstream infections (BSI) caused by Escherichia coli (EC) and Klebsiella pneumoniae (KP) with extended-spectrum cephalosporin resistance (ESCR) in two distinct regions in Switzerland, each exhibiting varying antimicrobial resistance patterns and that were impacted differently by the pandemic. METHODS: Data was analysed from positive blood cultures prospectively collected by the nationwide surveillance system (ANRESIS) from January 1st, 2015, to August 31st, 2022. To explore the potential relationship between COVID-19 patient occupancy and ESCR incidence rates, an in-depth analysis was conducted over the two-year pandemic period from April 1st, 2020, to March 30th, 2022, using Quasi-Poisson and logistic regression analyses. FINDINGS: During the study period, 40,997 EC-BSI and 8537 KP-BSI episodes were collected and reported to ANRESIS by the participating hospitals. ESCR was observed in 11% (N = 4313) of E. coli and 8% (N = 664) of K. pneumoniae, respectively. A significant reduction in ESCR-EC BSI incidence occurred during the pandemic in the region with the highest COVID-19 incidence. Conversely, ESCR-KP BSI incidence initially fell considerably and then increased during the pandemic in both regions, though this effect was not statistically significant. No association between hospital occupancy from COVID-19 patients and these trends was observed. CONCLUSION: In the early phase of the COVID-19 pandemic, a decrease in ESCR rates was observed, particularly in ESCR-EC BSI within the most heavily impacted region.

Changing epidemiology and antimicrobial resistance of bacteria causing bacteremia in Taiwan: 2002-2020.

Bacteremia is associated with significant morbidity and mortality. The emergence of bacteria with antimicrobial resistance (AMR) has further exacerbated the poor outcomes associated with bacteremia. The Taiwan Surveillance of Antimicrobial Resistance (TSAR) program was established in 1998 to monitor bacterial epidemiology and antimicrobial resistance trends across all patient types and age groups. Between 2002 and 2020, a total of 14,539 non-duplicate bacteremia isolates were collected biennially from 29 hospitals during the months of July-September as part of the TSAR program. The three most common bacteremia agents were Escherichia coli (31%), Staphylococcus aureus (13.6%), and Klebsiella pneumoniae (12.7%) overall. However, there was a steady increase in the proportions of E. coli and Enterococcus faecium isolated from bacteremia cases (both P < 0.001), while the proportions of Acinetobacter spp. decreased. Regarding antimicrobial resistance, there was a notable increase in rates of third-generation cephalosporin and fluoroquinolone non-susceptibility among E. coli and K. pneumoniae, while the rates of carbapenem non-susceptibility were elevated but remained milder in these two species, especially in E. coli. Of concern is the alarming increase in vancomycin resistance among E. faecium, rising from 10.0% in 2004 to 47.7% in 2020. In contrast, the prevalence of methicillin-resistant S. aureus has remained stable at 51.2% overall. In conclusion, E. coli, with increasing third-generation cephalosporin and fluoroquinolone resistance, is the predominant cause of bacteremia in Taiwan during the 18-year surveillance. The escalating proportion of E. faecium in bacteremia, coupled with a concurrent upsurge in vancomycin resistance, presents a therapeutic challenge in the recent decade. IMPORTANCE: AMR surveillance not only enables the identification of regional variations but also supports the development of coordinated efforts to combat AMR on a global scale. The TSAR has been a biennial, government-endorsed, multicenter study focusing on pathogens isolated from inpatients and outpatients in Taiwan hospitals since 1998. Our report presents an 18-year comprehensive analysis on blood isolates in the 2002-2020 TSAR program. The study highlights an alarming increase in the proportion of E. faecium causing bacteremia accompanied by elevated vancomycin resistance. It is worth noting that this trend differs from the observations in the United States and China. Understanding the composition of bacteria causing bacteremia, along with their prevalence of antimicrobial resistance, holds significant importance in establishing healthcare and research priorities. Additionally, this knowledge serves as a critical factor in evaluating the effectiveness of preventive interventions.

Addressing Sexually Transmitted Infections Due to Neisseria gonorrhoeae in the Present and Future.

It was in the 1800s when the first public publications about the infection and treatment of gonorrhoea were released. However, the first prevention programmes were only published a hundred years later. In the 1940s, the concept of vaccination was introduced into clinical prevention programmes to address early sulphonamide resistance. Since then, tons of publications on Neisseria gonorrhoeae are undisputed, around 30,000 publications today. Currently, the situation seems to be just as it was in the last century, nothing has changed or improved. So, what are we doing wrong? And more importantly, what might we do? The review presented here aims to review the current situation regarding the resistance mechanisms, prevention programmes, treatments, and vaccines, with the challenge of better understanding this special pathogen. The authors have reviewed the last five years of advancements, knowledge, and perspectives for addressing the Neisseria gonorrhoeae issue, focusing on new therapeutic alternatives.

ESBL-Type and AmpC-Type Beta-Lactamases in Third Generation Cephalosporin-Resistant Enterobacterales Isolated from Animal Feces in Madagascar.

Third generation cephalosporin-resistant (3GCR) Enterobacterales are known to be prevalent in Madagascar, with high colonization or infection rates in particular in Madagascan patients. Extended spectrum beta-lactamases (ESBLs) have been reported to be the predominant underlying resistance mechanism in human isolates. So far, little is known on antimicrobial resistance and its molecular determinants in Enterobacterales and other bacteria causing enteric colonization of Madagascan wild animals. To address this topic, swabs from 49 animal stool droppings were collected in the Madagascan Tsimanapesotsa National Park and assessed by cultural growth of bacterial microorganisms on elective media. In addition to 7 Acinetobacter spp., a total of 31 Enterobacterales growing on elective agar for Enterobacterales could be isolated and subjected to whole genome sequencing. Enterobacter spp. was the most frequently isolated genus, and AmpC-type beta-lactamases were the quantitatively dominating molecular resistance mechanism. In contrast, the blaCTX-M-15 gene, which has repeatedly been associated with 3GC-resistance in Madagascan Enterobacterales from humans, was detected in a single Escherichia coli isolate only. The identification of the fosfomycin-resistance gene fosA in a high proportion of isolates is concerning, as fosfomycin is increasingly used to treat infections caused by multidrug-resistant bacteria. In conclusion, the proof-of-principle assessment indicated a high colonization rate of resistant bacteria in stool droppings of Madagascan wild animals with a particular focus on 3GCR Enterobacterales. Future studies should confirm these preliminary results in a more systematic way and assess the molecular relationship of animal and human isolates to identify potential routes of transmission.

Clinical characteristics and drug resistance of Klebsiella pneumoniae infections at different systems / 中国基层医药

Objective:To investigate the clinical characteristics and drug resistance of Klebsiella pneumoniae infections at different systems, providing laboratory reference for the rational use of antibiotics in clinical practice.Methods:The clinical characteristics and drug resistance of patients with Klebsiella pneumoniae infections in respiratory, urinary, hematologic, and other systems who received treatment in Wuzhou Red Cross Hospital from January 2015 to December 2021 were analyzed.Results:From 2015 to 2021, there were 3 496 cases of Klebsiella pneumoniae infections in the hospital, among which the respiratory system was most affected with 2 250 strains (64.34%). The sex ratio of patients with Klebsiella pneumoniae infections at different systems was statistically significant ( χ2 = 266.77, P < 0.001). The respiratory system and hematological system were more commonly infected by Klebsiella pneumoniae in men, while the urinary system was more commonly infected in women. The sex ratio of patients with Klebsiella pneumoniae infections at other systems were similar. The age distribution of patients with Klebsiella pneumoniae infections at different systems was significantly different ( χ2 = 176.54, P < 0.001). Klebsiella pneumoniae infections of the respiratory, urinary, and hematological systems were the most common in people aged > 60-80 years, while Klebsiella pneumoniae infections in other systems were mainly found in people aged > 18-60 years. There were significant differences in the distribution of departments among different Klebsiella pneumoniae infection systems ( χ2 = 1 415.30, P < 0.001). The ICU had the highest incidence of Klebsiella pneumoniae infections in the respiratory system, while the department of internal medicine had the highest incidence of Klebsiella pneumoniae infections in the urinary and hematological systems, and the department of surgery had the highest incidence of Klebsiella pneumoniae infections in other parts of the body. The resistance rate of Klebsiella pneumoniae to common antibiotics for the hematologic system was lower than that of the other three infection systems. For infections in the respiratory system, urinary system, and other body parts, the resistance rates of Klebsiella pneumoniae to amikacin, meropenem, imipenem, and piperacillin/tazobactam were all below 10%, while the resistance rate to tobramycin was below 20%, and the resistance rate to cephalosporins was around 30%. Conclusion:The proportion of Klebsiella pneumoniae infections in different body parts varies by gender, age, and department. For Klebsiella pneumoniae infections in the hematological system, the resistance rate to commonly used antibiotics is lower than that for infections in the other three systems. In contrast, Klebsiella pneumoniae infections in the respiratory system, urinary system, and other systems have a particularly high resistance rate to third- and fourth-generation cephalosporins but are still sensitive to piperacillin/tazobactam and carbapenem antibiotics.

Risk Factors for Recurrence of Community-Onset Urinary Tract Infections Caused by Extended-Spectrum Cephalosporin-Resistant Enterobacterales.

Background: Extended-spectrum cephalosporin-resistant Enterobacterales (ESCrE) are an increasingly important cause of community-onset urinary tract infections (UTIs), including recurrent infections. We evaluated risk factors for recurrence among patients with community-onset ESCrE UTI. Methods: This retrospective cohort study included adults with community-onset ESCrE UTI in the Duke University Health System from April 2018 through December 2021. ESCrE UTI recurrence by the same species was assessed 14-180 days (ie, 6 months) after completion of antibiotic treatment. We evaluated the relationships between candidate risk factors and time to recurrence using Cox proportional hazards regression models. Results: Among 1347 patients with community-onset ESCrE UTI, 202 (15.0%) experienced recurrent infection during the 6-month follow-up period. Independent risk factors for recurrence included neurogenic bladder (adjusted hazard ratio [aHR], 1.8 [95% confidence interval {CI}, 1.2-2.6]; P = .005), prior history of UTI (aHR, 2.4 [95% CI, 1.7-3.3]; P < .001), and fluoroquinolone nonsusceptibility of the index UTI (aHR, 1.5 [95% CI, 1.1-2.1]; P = .02). Klebsiella pneumoniae infection was associated with recurrence in univariate analysis (HR, 1.6 [95% CI, 1.1-2.1]; P = .007) but not multivariate analysis (aHR, 1.4 [95% CI, 1.0-1.9]; P = .06). Inappropriate initial or definitive antibiotic therapy was not predictive of ESCrE UTI recurrence. Conclusions: Recurrence of community-onset ESCrE UTI was common and associated with several patient and pathogen-level risk factors. Future studies should evaluate microbial risk factors for recurrence and improve the management of ESCrE UTI.

Distribution of blaCTX-M-gene variants in E. coli from different origins in Ecuador.

The increasing abundance of extended spectrum (ß-lactamase (ESBL) genes in E. coli, and other commensal and pathogenic bacteria, endangers the utility of third or more recent generation cephalosporins, which are major tools for fighting deadly infections. The role of domestic animals in the transmission of ESBL carrying bacteria has been recognized, especially in low- and middle-income countries, however the horizontal gene transfer of these genes is difficult to assess. Here we investigate blaCTX-M gene diversity (and flanking nucleotide sequences) in E. coli from chicken and humans, in an Ecuadorian rural community and from chickens in another location in Ecuador. The blaCTX-M associated sequences in isolates from humans and chickens in the same remote community showed greater similarity than those found in E. coli in a chicken industrial operation 200 km away. Our study may provide evidence of blaCTX-M transfer between chickens and humans in the community.

Secrets of getting started: Regulation of the first committed step of peptidoglycan synthesis by protein phosphorylation in Enterococcus and other Gram-positive bacteria.

Regulation of the first committed step of peptidoglycan precursor synthesis by MurA-enzyme homologs has recently taken center stage in many different bacteria. In different low-GC Gram-positive bacteria, regulation of this step has been shown to be regulated by phosphorylation of homologs of the IreB/ReoM regulatory protein by PASTA-domain Ser/Thr-protein kinases. In this issue, Mascari, Little, and Kristich determine this regulatory pathway and its links to resistance to cephalosporin ß-lactam antibiotics in the major human pathogen, Enterococcus faecalis (Efa). Unbiased genetic selections identified MurAA (MurA-family homolog) as the downstream target of IreB regulation in the absence of the IreK Ser/Thr-protein kinase. Physiological and biochemical approaches, including determination of MICs to ceftriaxone, Western blotting of MurAA cellular amounts, isotope incorporation into peptidoglycan sacculi, and thermal-shift binding assays of purified proteins, demonstrated that unphosphorylated IreB, together with proteins MurAB (MurZ-family homolog), and ReoY(Efa) negatively regulate MurAA stability and cellular amount by the ClpCP protease. Importantly, this paper supports the idea that ceftriaxone stimulates phosphorylation of IreB, which leads to increased cellular MurAA amount and precursor pathway flux required for E. faecalis cephalosporin resistance. Overall, findings in this paper significantly contribute to understanding variations of this central regulatory pathway in other low-GC Gram-positive bacteria.

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.

Dynamics of Extended-spectrum Beta-lactamase-producing, Third-generation Cephalosporin-resistant and Tetracycline-resistant Escherichia coli in Feedlot Cattle With or Without Tylosin Administration.

The impact of in-feed use of tylosin in feedlot cattle on Gram-negative foodborne bacteria is unknown. We evaluated the effect of continuous in-feed tylosin use on the concentration and prevalence of tetracycline-resistant (TETr)-, third-generation cephalosporin-resistant (3GCr)-, and extended-spectrum ß-lactamase-producing (ESBLs) E. coli in feedlot cattle. A cohort of weaned calves (10 animals/group) were randomized to receive a feed ration with or without tylosin. Fecal samples, regularly collected over the entire feeding period, and pen surface and feed samples, collected at the end of the feeding period, were cultured on selective media. Enumeration and binary outcomes were analyzed by mixed effects linear regression or logistic regression, respectively, using treatment and days on feed as fixed factors, and animal ID as a random variable. Tylosin supplementation did not affect the fecal concentrations of TETrE. coli or fecal prevalence of 3GCrE. coli. However, cattle in the tylosin group were 1.5 times more likely (Odds ratio = 1.5: 95% confidence interval: 1.1-2.0) to harbor ESBLs E. coli than the control cattle. Regardless of tylosin treatment, fecal concentrations of TETrE. coli and the prevalence of 3GCr- and ESBLs-E. coli increased over time. Tylosin-supplemented feed did not affect the prevalence of TETrE. coli; 3GCr and ESBLs-E. coli were not detected from the feed samples. Most of the 3GCr- and ESBLs-E. coli isolates carried the blaCTX-M-15 gene, widely detected among ESBLs-E. coli human isolates. In summary, although in-feed tylosin use in feedlot cattle did not select for TETr- and 3GCr-E. coli, it increased the likelihood of detecting ESBL-producing E. coli. Furthermore, the study indicated that the feedlot production setting gradually increases the levels of E. coli resistant to the critically and/or important antibiotics for public health, indicating an increased risk of their dissemination beyond the feedlot environment.

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.

The CREPE Score: A Predictive Tool for Third-Generation Cephalosporin-Resistant Enterobacterales Pneumonia in Community Settings.

Purpose: To evaluate risk factors and develop a prediction score for community-acquired pneumonia caused by third-generation cephalosporin-resistant Enterobacterales (3GCR EB-CAP). Patients and Methods: A retrospective study was conducted by reviewing the medical records of patients hospitalized with community-acquired pneumonia caused by Enterobacterales (EB-CAP) between January 2015 and August 2021 at Srinagarind Hospital, Khon Kaen University, Thailand. Logistic regression was used to analyze clinical parameters associated with 3GCR EB-CAP. The coefficients of significant parameters were simplified to the nearest whole number for a prediction score, called the CREPE (third-generation Cephalosporin Resistant Enterobacterales community-acquired Pneumonia Evaluation). Results: A total of 245 patients with microbiologically confirmed EB-CAP (100 in the 3GCR EB group) were analyzed. Independent risk factors for 3GCR EB-CAP included in the CREPE score were (1) recent hospitalization within the past month (1 point), (2) multidrug-resistant EB colonization (1 point), and (3) recent intravenous antibiotic use (2 points for within the past month or 1.5 points for between one and twelve months). The CREPE score had an area under the receiver operating characteristic curve (ROC) of 0.88 (95% CI 0.84-0.93). Using a cut-off point of 1.75, the score had a sensitivity and specificity of 73.5% and 84.6%, respectively. Conclusion: In areas with high prevalence of EB-CAP, the CREPE score can assist clinicians in selecting appropriate empirical therapy and reducing overuse of broad-spectrum antibiotics.