Search

We analyzed the whole genome sequences (WGS) and antibiograms of 35 Enterobacter isolates, including E. hormaechei and E. asburiae , and the recently described E. bugandensis , E. kobei , E. ludwigii , and E. roggenkampii species. Isolates were obtained from human blood and urinary tract infections in patients in the United States. Our goal was to understand the genetic diversity of antimicrobial resistance genes and virulence factors among the various species. Thirty-four of 35 isolates contained an AmpC class bla ACT allele; however, the E. roggenkampii isolate contained bla MIR-5 . Of the six Enterobacter isolates resistant to ertapenem, imipenem, and meropenem, four harbored a carbapenemase gene, including bla KPC or bla NDM . All four isolates were mCIM-positive. The remaining two isolates had alterations in ompC genes that may have contributed to the resistance phenotype. Interpretations of cefepime test results were variable when disk diffusion and automated broth microdilution results were compared due to the Clinical Laboratory and Standards Institute use of the "susceptible dose-dependent" classification. The diversity of the bla ACT alleles paralleled species identifications, as did the presence of various virulence genes. The classification of recently described Enterobacter species is consistent with their resistance gene and virulence gene profiles.

Purpose: Donor-derived infection (DDI) has become an important factor affecting the prognosis of lung transplantation patients. The risks versus benefits of using donor organs infected with multidrug-resistant organisms (MDRO), especially carbapenem-resistant organisms (CRO), are frequently debated. Traditional microbial culture and antimicrobial susceptibility testing at present fail to meet the needs of quick CRO determination for donor lungs before acquisition. In this study, we explored a novel screening method by using Xpert ® Carba-R assay for CRO in donor lungs in a real-time manner to reduce CRO-associated DDI mortality., Methods: This study was registered on chictr.org.cn (ChiCTR2100053687) on November 2021. In the Xpert Carba-R screening group, donor lungs were screened for CRO infection by the Xpert Carba-R test on bronchoalveolar fluid (BALF) before acquisition. If the result was negative, donor lung acquisition and subsequent lung transplantation were performed. In the thirty-five potential donors, nine (25.71%) with positive Xpert Carba-R results in BALF were declined for lung transplantation. Twenty-six recipients and the matching CRO-negative donor lungs (74.29%) were included in the Xpert Carba-R screening group. In the control group, nineteen recipients underwent lung transplants without Xpert Carba-R screening. The incidence and mortality of CRO-associated DDI were collected and contrasted between the two groups., Results: Multivariate analysis showed that CRO-related death due to DDI within 60 days was significantly lower in the Xpert Carba-R screening group than that in the control group (OR = 0.05, 95% CI 0.003-0.74, p = 0.03)., Conclusion: Real-time CRO screening of donor lungs before transplantation at the point of care by the Xpert Carba-R helps clinicians formulate lung transplantation strategies quickly and reduces the risk of subsequent CRO infection improving the prognosis of lung transplantation., (© 2023. The Author(s).)

Objectives: We investigated the amino acid substitutions in the GES family of ESBLs that were most likely to be involved in the evolution of carbapenemase activity., Methods: To identify the substitutions that are functionally important, we analysed the evolutionary history of the GES β-lactamases using an alignment and phylogeny to identify sites in GES that show evidence of positive selection and the selected phenotypes., Results and Conclusions: Data indicate that the substitutions G170S and G243A are associated with carbapenemase activity. The substitutions Q43E, E104K and T237A are most likely associated with ESBL activity., (© The Author(s) 2024. Published by Oxford University Press on behalf of British Society for Antimicrobial Chemotherapy.)

Importance: Early identification of complicated urinary tract infections caused by ESBL-producing Enterobacterales has the potential to limit the use of carbapenems to those patients without alternative antibiotic options and avoid the empirical use of carbapenems in patients without ESBL-producing bacteria. The purpose for such a test will differ by setting and ESBL prevalence rates. Countries with low ESBL rates and cephalosporins as empiric treatment (e.g., The Netherlands) will need a rule-in test to decide to use carbapenems, while countries with high ESBL rates and empiric carbapenem treatment will need a rule-out test for ESBLs to de-escalate therapy early. Anyway, such as a test would-at least theoretically-improve patient care and reduce selective pressure for the emergence of carbapenem resistance., Competing Interests: This Radboudumc researcher-initiated study was supported in kind with free testing cartridges by Cepheid. I.A.T. is an employee of Cepheid, and F.C.T. is a former Cepheid employee.

Molecular carbapenem-resistance testing, such as for the presence of carbapenemases genes, is commonly implemented for the detection of carbapenemase-producing Enterobacterales. Carbapenemase-producing P. aeruginosa is also associated with significant morbidity and mortality, although; prevalence may be underappreciated in the United States due to a lack of carbapenemase testing. The present study sought to compare hands-on time, cost and workflow implementation of carbapenemase gene testing in Enterobacterales and P. aeruginosa isolates versus sending out isolates to a public health laboratory (PHL) for testing to assess if in-house can provide actionable results. The time to carbapenemase gene results were compared. Differences in cost for infection prevention measures were extrapolated from the time of positive carbapenemase gene detection in-house versus PHL. The median time to perform carbapenemase gene testing was 7.5 min (range 5-14) versus 10 min (range 8-22) for preparation to send isolates to the PHL. In-house testing produced same day results compared with a median of 6 days (range 3-14) to receive results from PHL. Cost of in-house testing and send outs were similar ($46.92 versus $40.53, respectively). If contact precautions for patients are implemented until carbapenemase genes are ruled out, in-house testing can save an estimated $76,836.60 annually. Extension of in-house carbapenemase testing to include P. aeruginosa provides actionable results 3-14 days earlier than PHL Standard Pathway testing, facilitating guided therapeutic decisions and infection prevention measures. Supplemental phenotypic algorithms can be implemented to curb the cost of P. aeruginosa carbapenemases testing by identifying isolates most likely to harbour carbapenemases.

Infections due to antimicrobial resistant gram-negative bacteria cause significant morbidity and mortality in sub-Saharan Africa. To elucidate the molecular epidemiology of antimicrobial resistance in gram-negative bacteria, we characterized beta-lactam and fluoroquinolone resistance determinants in Escherichia coli , Klebsiella pneumoniae, and Pseudomonas aeruginosa isolates collected from November 2017 to February 2018 (Period 1) and October 2021 to January 2022 (Period 2) in a tertiary medical center in north-eastern Nigeria. Whole genome sequencing (WGS) was used to identify sequence types and resistance determinants in 52 non-duplicate, phenotypically resistant isolates. Antimicrobial susceptibility was determined using broth microdilution and modified Kirby-Bauer disk diffusion methods. Twenty sequence types (STs) were identified among isolates from both periods using WGS, with increased strain diversity observed in Period 2. Common ESBL genes identified included bla CTX-M , bla SHV, and bla TEM in both E. coli and K. pneumoniae . Notably, 50% of the E. coli in Period 2 harbored either bla CTX-M-15 or bla CTX-M-1 4 and phenotypically produced ESBLs. The bla NDM-7 and bla VIM-5 metallo-beta-lactamase genes were dominant in E. coli and P. aeruginosa in Period 1, but in Period 2, only K. pneumoniae contained bla NDM-7 , while bla NDM-1 was predominant in P. aeruginosa . The overall rate of fluoroquinolone resistance was 77% in Period 1 but decreased to 47.8% in Period 2. Various plasmid-mediated quinolone resistance (PMQR) genes were identified in both periods, including aac(6 ' )-Ib-cr , oqxA/oqxB , qnrA1 , qnrB1 , qnrB6 , qnrB18, qnrVC1 , as well as mutations in the chromosomal gyrA , parC and parE genes. One E. coli isolate in Period 2, which was phenotypically multidrug resistant, had ESBL bla CTX-M-15, the serine carbapenemase, bla OXA-181 and mutations in the gyrA gene. The co-existence of beta-lactam and fluoroquinolone resistance markers observed in this study is consistent with widespread use of these antimicrobial agents in Nigeria. The presence of multidrug resistant isolates is concerning and highlights the importance of continued surveillance to support antimicrobial stewardship programs and curb the spread of antimicrobial resistance.

A total of 199 Gram-negative bacterial isolates from urinary tract infections and 162 from bloodstream infections were collected from 12 healthcare systems throughout the United States between May 2021 and August 2022. The isolates, phenotypically non-susceptible to 2nd or 3rd generation cephalosporins or carbapenems, were characterized through antimicrobial susceptibility testing and whole genome sequence analysis to obtain a broad snapshot of beta-lactamase-mediated resistance among these two sample types. Overall, 23 different carbapenemase genes were detected among 13 species (20.5% of isolates). The bla KPC-3 and bla KPC-2 subtypes were the most common carbapenemase genes identified, followed by bla NDM and the co-carriage of two different bla OXA carbapenemases by Acinetobacter baumannii isolates. All carbapenemase-producing A. baumannii isolates were mCIM negative. Extended-spectrum beta-lactamase genes were identified in 66.2% of isolates; bla CTX-M-15 was the most common. AmpC genes, both plasmid and chromosomal, were detected in 33.2% of isolates. Importantly, 2.8%, 8.3%, and 22.2% of bla KPC -positive organisms were susceptible to ertapenem, imipenem, and meropenem, respectively. The correlation between broth microdilution and disk diffusion results was high for most drugs except cefepime, where the detection of resistance was statistically lower by disk diffusion. Thus, there were gaps in the accuracy of susceptibility testing for some mechanisms of resistance.

Infections caused by multi-drug resistant Enterobacterales (MDR-E) are difficult to treat and cause significant mortality, especially in developing countries. This study characterized the phenotypic and genotypic profiles of 49 randomly selected beta-lactam resistant MDR-E previously isolated from patients being managed in hospitals in Nigeria using whole genome sequencing. The study isolates exhibited 85.5% resistance to 3rd generation cephalosporins and 65.3% resistance to carbapenems. The bla TEM-1B (29, 59.2%) , bla CTX-M-15 (38, 77.6%) , and bla NDM-1 (17, 51.5%) were the most common penicillinase, ESBL, and carbapenem resistant genes across isolates, respectively. Seventeen (45%) of bla CTX-M-15 was carried on the insertion sequence ISEc9 while bla NDM-1 (11, 64.7%) were associated with ISEc33. None of the 21 plasmids detected were associated with β-lactamase genes. Higher resistance rates were found in E. coli ST-88 (n = 2) and the high-risk ST-692 (n = 2). For Klebsiella species, the high-risk clones ST-476 (n = 8) and ST-147 (n = 3) predominated and had higher phenotypic resistance rates and higher number of AMR genes. The mechanisms and pattern of antibiotic resistance differ from patterns previously described with isolates harbouring a wide range of AMRGs. The detection of several chromosomally mediated carbapenemases in our study also represents a significant finding that warrants further investigation to better understand its' implications for clinical practice and public health. The selected MDR-Es were found to be pan-susceptible to tigecycline and had very low resistance to fosfomycin, suggesting a potential for these as empiric treatments. A surveillance approach incorporating both conventional laboratory techniques and modern molecular techniques is essential for the comprehensive characterization of the emergence and dissemination of antimicrobial resistance in Enterobacterales infections within Nigeria., (© 2023. The Author(s).)

Background: Carbapenemase-producing gram-negative organisms continue to be a significant healthcare concern and a therapeutic challenge. Members of the genus Citrobacter have emerged as increasingly multidrug resistant and versatile healthcare-associated pathogens. In this study we investigated five KPC-producing Citrobacter freundii isolates, from the same patient, that presented unusual phenotypic characteristics including false susceptibility to carbapenems detection by culture-based methods., Methods: The isolates were tested for antimicrobial susceptibility using broth microdilution and disk diffusion. Production of serine carbapenemase was confirmed with the mCIM (modified carbapenem inactivation method) test. Genotypes were determined by PCR and whole genome sequencing analysis., Results: The five isolates were susceptible to meropenem by broth microdilution and presented varying colonial morphologies and levels of susceptibility to carbapenems by multiple phenotypic methods, despite being positive for carbapenemase production by mCIM and positive for bla KPC by PCR. Whole genome sequence analysis showed that three of the five highly related isolates harbor an additional gene cassette, including bla CARB-2 , ant(2''), aadA2, dfrA19, catB3, cmlA1, mph(E), msr(E), and qnrA1. The presence of these genes explains the difference in phenotypes observed., Conclusion: Failure to detect and completely eradicate the carbapenemase-producing C. freundii in the urine with ertapenem therapy, likely due to the presence of a heterogeneous population, resulted in the phenotypic and genotypic adaptations of the organism as it disseminated to the bloodstream and kidneys. The fact that carbapenemase-producing C. freundii can elude detection by phenotypic methods and can so easily acquire and transfer resistance gene cassettes is of concern., (© 2023. The Author(s).)

Colonization with multidrug-resistant organisms (MDROs) is a risk factor for subsequent infection. Surveillance for MDROs, including methicillin-resistant Staphylococcus aureus , vancomycin-resistant enterococci, extended-spectrum beta-lactamase-producing Enterobacterales, and carbapenemase-producing organisms, is commonly conducted in hospitals to prevent spread of MDROs, in part to reduce the potential for additional infections. Although colonization is a risk factor for infection, data on colonization with various MDROs are often not considered when selecting anti-infective therapy. There are conflicting data on the strength of the positive and negative predictive values of the colonization test results to guide therapeutic strategies. Defining therapeutic strategies for patients with complicated or drug-resistant infections or to select antimicrobial prophylaxis before performing prostate biopsies often falls under the purview of the antimicrobial stewardship team. Should colonization data, which are often present in the patient's medical record from routine infection prevention measures, be reviewed before selecting therapy for infections or for prophylaxis? In this perspective, we will explore the intersection of infection control and antimicrobial stewardship activities., (© The Author(s) 2023. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Rapid and accurate detection of carriers of carbapenemase-producing organisms (CPO) in hospitalized patients is critical for infection control and prevention. This study aimed to evaluate a pooling strategy for the detection of carbapenem resistance genes (CRG) in multiple specimens using the Xpert Carba-R test. Two rectal swabs each were collected from 415 unique patients. One swab was tested by Carba-R on the five specimen-pooled strategy. The other swab was tested individually by culture followed by DNA sequence analysis for CRG as the reference. At the first 5:1 pooling testing, 22 of 83 pools were positive, which yielded 34 positives from individual specimens when positive pools were subsequently retested. All individual specimens in the 61 negative pools were retested as negative by Carba-R. Among the 34 Carba-R-positive samples, 30 and four were positive and negative, respectively, by culture and sequencing. The remaining 381 Carba-R-negative specimens were also negative by culture and sequencing. Overall sensitivity, specificity, positive predictive value, and negative predictive value of the 5:1 pooled screening were 100.0% (95% confidence interval [CI] = 85.9% to 100%), 99.0% (95% CI = 97.2% to 99.7%), 88.2% (95% CI = 71.6% to 96.2%), and 100.0% (95% CI = 98.8% to 100%), respectively. Using the 5:1 pooling strategy, our study completed CRG screening in 414 patients with 193 reagents with significant cost savings. The 5:1 pooling strategy using the Carba-R test showed a potential method for screening CRG from rectal swabs with good sensitivity and decreased cost.

Objectives: Pseudomonas aeruginosa is one of the leading causes of healthcare-associated infections globally. High-risk carbapenemase-encoding P. aeruginosa clones are disseminating in many regions. The aim of this study was to learn more about the lineages and mechanisms of resistance of P. aeruginosa circulating in Peru., Methods: A total of 141 carbapenemase-producing isolates recovered from hospitalized and ambulatory patients in Lima were sequenced and analyzed to infer their lineages through whole-genome sequence typing (wgST) and to identify their antimicrobial resistance genes., Results: wgST identified nine sequence types (STs); ST111 and ST357 were the most frequently encountered (44.0% and 38.3%, respectively), followed by ST179 (8.5%), with the remaining six detected only sporadically. Among ST357 isolates, 96.3% carried the novel bla IMP-93 allele, whereas the remainder harbored bla IMP-74 . 74.2% of ST111 isolates co-harbored bla IMP-18 and bla VIM-2 , while the rest carried either of these genes individually . All other ST lineages carried a single carbapenemase, which was either bla IMP-16 , bla IMP-74 , or bla VIM-2 ., Conclusion: Our study shows that the high-risk P. aeruginosa clones ST357, which harbors the novel bla IMP-93 , and ST111, which carries bla IMP-18 and bla VIM-2 , have apparently become endemic in the region., Competing Interests: Competing interests Isabella A. Tickler and Fred C. Tenover are employees of Cepheid. Anne E. Obradovich has received research funding from Cepheid. The other authors report no conflicts of interest., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Carbapenem-resistant Pseudomonas aeruginosa (CR-PA) is a major healthcare-associated pathogen worldwide. In the United States, 10-30% of P. aeruginosa isolates are carbapenem-resistant, while globally the percentage varies considerably. A subset of carbapenem-resistant P. aeruginosa isolates harbour carbapenemases, although due in part to limited screening for these enzymes in clinical laboratories, the actual percentage is unknown. Carbapenemase-mediated carbapenem resistance in P. aeruginosa is a significant concern as it greatly limits the choice of anti-infective strategies, although detecting carbapenemase-producing P. aeruginosa in the clinical laboratory can be challenging. Such organisms also have been associated with nosocomial spread requiring infection prevention interventions. The carbapenemases present in P. aeruginosa vary widely by region but include the Class A beta-lactamases, KPC and GES; metallo-beta-lactamases IMP, NDM, SPM, and VIM; and the Class D, OXA-48 enzymes. Rapid confirmation and differentiation among the various classes of carbapenemases is key to the initiation of early effective therapy. This may be accomplished using either molecular genotypic methods or phenotypic methods, although both have their limitations. Prompt evidence that rules out carbapenemases guides clinicians to more optimal therapeutic selections based on local phenotypic profiling of non-carbapenemase-producing, carbapenem-resistant P. aeruginosa . This article will review the testing strategies available for optimizing therapy of P. aeruginosa infections.

Staphylococcal cassette chromosome mec (SCC mec ) represents a sequence of clear clinical and diagnostic importance in staphylococci. At a minimum the chromosomal cassette contains the mecA gene encoding PBP2a but frequently also includes additional antibiotic resistance genes (e.g., ermA and aadC ; macrolide and aminoglycoside resistance, respectively). Certain regions within SCC mec elements are hot spots for sequence instability due to cassette-specific recombinases and a variety of internal mobile elements. SCC mec changes may affect not only cassette stability but the integrity of adjacent chromosomal sequences (e.g., the staphylococcal protein A gene; spa ). We investigated SCC mec stability in methicillin-resistant Staphylococcus aureus (MRSA) strains carrying one of four SCC mec types cultured in the absence of antimicrobial selection over a 3-month period. SCC mec rearrangements were first detected in cefoxitin-susceptible variants after 2 months of passage, and most commonly showed precise excision of the SCC mec element. Sequence analysis after 3 months revealed both precise SCC mec excision and a variety of SCC mec internal deletions, some including extensive adjacent chromosomal loss, including spa . No empty cassettes (i.e., loss of just mecA from SCC mec ) were observed among the variants. SCC mec stability was influenced both by internal mobile elements (IS 431 ) as well as the host cell environment. Genotypically similar clinical isolates with deletions in the spa gene were also included for purposes of comparison. The results indicate a role for host-cell influence and the IS 431 element on SCC mec stability.

The application of molecular detection methods for bacterial pathogens has dramatically improved the outcomes of septic patients, including those with methicillin-resistant Staphylococcus aureus (MRSA) infections. Molecular methods can be applied to a variety of clinical specimens including nasal swabs, growth in blood culture bottles, and wounds. While data show that the overall accuracy of molecular tests for MRSA is high, results can be confounded by the presence of multiple staphylococcal species in a specimen, insertions and deletions of DNA in and around the Staphylococcal Cassette Chromosome mec (SCC mec ) element, and point mutations in mecA . Herein, we explore the complexities of molecular approaches to MRSA detection and the instances where phenotypic methods should be pursued to resolve discrepancies between genotypic and phenotypic results.

Background: We aimed to evaluate the clinical performance of the GeneXpert ® (Xpert) CT/NG assay for the detection of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) using urine and cervical swabs collected from patients in China., Methods: This study was conducted from September 2016 to September 2018 in three Chinese urban hospitals. The results from the Xpert CT/NG test were compared to those from the Roche cobas ® 4800 CT/NG test. Discordant results were confirmed by DNA sequence analysis., Results: In this study, 619 first void urine (FVU) specimens and 1,042 cervical swab specimens were included in the final dataset. There were no statistical differences between the results of the two tests for the detection of CT/NG in urine samples ( p  > 0.05), while a statistical difference was found in cervical swabs ( p  < 0.05). For CT detection, the sensitivity and specificity of the Xpert test were 100.0% (95%CI = 96.8-99.9) and 98.3% (95%CI = 96.6-99.2) for urine samples and 99.4% (95%CI = 96.5-100.0) and 98.6% (95%CI 97.5-99.2) for cervical swabs, respectively. For NG detection, the sensitivity and specificity of the Xpert test were 99.2% (95%CI = 94.9-100.0) and 100.0% (95%CI = 99.0-100.0) for urine and 100% (95%CI = 92.8-100.0) and 99.7% (95%CI = 99.0-99.9) for cervical swabs, respectively., Conclusion: The Xpert CT/NG test exhibited high sensitivity and specificity in the detection of CT and NG in both urine and cervical samples when compared to the reference results. The 90-min turnaround time for CT and NG detection at the point of care using Xpert may enable patients to receive treatment promptly., Competing Interests: FT and YT are employees of Cepheid, the commercial manufacturer of the Xpert® CT/NG test. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Han, Shi, Jiang, Le, Qin, Xiong, Zheng, Tenover, Tang and Yin.)