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We constructed a phylogenetic analysis of class A beta-lactamases and found that the blaCTX-Ms have been mobilized to plasmids approximately 10 times more frequently than other class A beta-lactamases. We also found that the blaCTX-Ms are descended from a common ancestor that was incorporated in ancient times into the chromosome of the ancestor of Kluyvera species through horizontal transfer. Considerable sequence divergence has occurred among the descendents of that ancestral gene sequence since that gene was inserted. That divergence has mainly occurred in the presence of purifying selection, which indicates a slow rate of evolution for blaCTX-Ms in the pre-antimicrobial drug era. [ABSTRACT FROM AUTHOR]

Factors that affect the resistance rates for an organism-drug combination in a given hospital also might influence resistance rates for other organism-drug combinations. We examined correlations between resistance prevalence in non-intensive care inpatient areas of 41 hospitals participating in phase 3 (1998-1999) of Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology). We focused on statistically significant (P<.05) Pearson correlation coefficients for methicillin-resistant Staphylococcus aureus, coagulase-negative staphylococci, vancomycin-resistant enterococci, and resistance to third-generation cephalosporins, imipenem, and fluoroquinolones in Escherichia coli, Klebsiella pneumoniae, Enterobacter species, and Pseudomonas aeruginosa. Resistance prevalence rates in individual hospitals were not strongly correlated among gram-positive organisms, and few correlations were seen between rates in gram-positive and gram-negative organisms. More frequent significant associations were found among resistance rates for gram-negative organisms. Resistance to third-generation cephalosporins in K. pneumoniae was significantly correlated with the majority of other sentinel antimicrobial-resistant organisms. High prevalence of this organism may serve as a marker for more generalized resistance problems in hospital inpatient areas. Copyright © 2002 Infectious Diseases Society of America [ABSTRACT FROM AUTHOR]

Background: Patient-specific risk factors for acquisition of vancomycin-resistant enterococci (VRE) among hospitalized patients are becoming well defined. However, few studies have reported data on the institutional risk factors, including rates of antimicrobial use, that predict rates of VRE. Identifying modifiable institutional factors can advance quality-improvement efforts to minimize hospital-acquired infections with VRE.Objective: To determine the independent importance of any association between antimicrobial use and risk factors for nosocomial infection on rates of VRE in intensive care units (ICUs).Design: Prospective ecologic study.Setting: 126 adult ICUs from 60 U.S. hospitals from January 1996 through July 1999.Patients: All patients admitted to participating ICUs.Measurements: Monthly use of antimicrobial agents (defined daily doses per 1000 patient-days), nosocomial infection rates, and susceptibilities of all tested enterococci isolated from clinical cultures.Results: Prevalence of VRE (median, 10%; range, 0% to 59%) varied by type of ICU and by teaching status and size of the hospital. Prevalence of VRE was strongly associated with VRE prevalence among inpatient non-ICU areas and outpatient areas in the hospital, ventilator-days per 1000 patient-days, and rate of parenteral vancomycin use. In a weighted linear regression model controlling for type of ICU and rates of VRE among non-ICU inpatient areas, rates of vancomycin use (P < 0.001) and third-generation cephalosporin use (P = 0.02) were independently associated with VRE prevalence.Conclusions: Higher rates of vancomycin or third-generation cephalosporin use were associated with increased prevalence of VRE, independent of other ICU characteristics and the endemic VRE prevalence elsewhere in the hospital. Decreasing the use rates of these antimicrobial agents could reduce rates of VRE in ICUs. [ABSTRACT FROM AUTHOR]

Context: Ceftriaxone, an expanded-spectrum cephalosporin, is an antimicrobial agent commonly used to treat severe Salmonella infections, especially in children. Ceftriaxone-resistant Salmonella infections have recently been reported in the United States, but the extent of the problem is unknown.Objectives: To summarize national surveillance data for ceftriaxone-resistant Salmonella infections in the United States and to describe mechanisms of resistance.Design and Setting: Case series and laboratory evaluation of human isolates submitted to the Centers for Disease Control and Prevention from 17 state and community health departments participating in the National Antimicrobial Resistance Monitoring System (NARMS) for enteric bacteria between 1996 and 1998.Patients: Patients with ceftriaxone-resistant Salmonella infections between 1996 and 1998 were interviewed and isolates with decreased ceftriaxone susceptibility were further characterized.Main Outcome Measures: Exposures and illness outcomes, mechanisms of resistance.Results: The prevalence of ceftriaxone-resistant Salmonella was 0.1% (1 of 1326) in 1996, 0.4% (5 of 1301) in 1997, and 0.5% (7 of 1466) in 1998. Ten (77%) of the 13 patients with ceftriaxone-resistant infections were aged 18 years or younger. The patients lived in 8 states (California, Colorado, Kansas, Massachusetts, Maryland, Minnesota, New York, and Oregon). Nine (82%) of 11 patients interviewed did not take antimicrobial agents and 10 (91%) did not travel outside the United States before illness onset. Twelve of the 15 Salmonella isolates with ceftriaxone minimum inhibitory concentrations of 16 microg/mL or higher were serotype Typhimurium but these isolates had different pulsed-field gel electrophoresis patterns. Thirteen of these 15 isolates collected between 1996 and 1998 were positive for a 631-base pair polymerase chain reaction product obtained by using primers specific for the ampC gene of Citrobacter freundii.Conclusions: Domestically acquired ceftriaxone-resistant Salmonella has emerged in the United States. Most ceftriaxone-resistant Salmonella isolates had similar AmpC plasmid-mediated resistance. [ABSTRACT FROM AUTHOR]

Resistance is an emerging problem in human medicine and the effects of resistance are being noted on an ever-increasing scale. Whether it is treatment of nosocomial bacteremia in New York City or community-acquired dysentery in Central Africa, multiresistant organisms are diminishing our ability to control the spread of infectious diseases. Clearly, the rate at which resistant organisms develop is not solely a function of the use of antimicrobials in humans, but is also highly influenced by the use of these agents in veterinary medicine, animal husbandry, agriculture, and aquaculture, as has been emphasized at recent meetings sponsored by organizations such as Rockefeller University and the American Society for Microbiology, and in the report on bacterial resistance recently issued by the US Office of Technology Assessment. We have entered an era where both physicians and patients must take on the responsibility to use antimicrobials wisely and judiciously. Just as in the days at the turn of the century when the public was an integral part of establishing quarantines for infectious diseases, now again the public's cooperation must be sought for this latest threat to public health. The multiresistant organisms of the 1990s are a grim warning of the possibility of the postantibiotic era. [ABSTRACT FROM AUTHOR]

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.)

Infections caused by multidrug-resistant Gram-negative organisms have become a global threat. Such infections can be very difficult to treat, especially when they are caused by carbapenemase-producing organisms (CPO). Since infections caused by CPO tend to have worse outcomes than non-CPO infections, it is important to identify the type of carbapenemase present in the isolate or at least the Ambler Class (i.e., A, B, or D), to optimize therapy. Many of the newer beta-lactam/beta-lactamase inhibitor combinations are not active against organisms carrying Class B metallo-enzymes, so differentiating organisms with Class A or D carbapenemases from those with Class B enzymes rapidly is critical. Using molecular tests to detect and differentiate carbapenem-resistance genes (CRG) in bacterial isolates provides fast and actionable results, but utilization of these tests globally appears to be low. Detecting CRG directly in positive blood culture bottles or in syndromic panels coupled with bacterial identification are helpful when results are positive, however, even negative results can provide guidance for anti-infective therapy for key organism-drug combinations when linked to local epidemiology. This perspective will focus on the reluctance of laboratories to use molecular tests as aids to developing therapeutic strategies for infections caused by carbapenem-resistant organisms and how to overcome that reluctance., Competing Interests: Author FT was employed by Cepheid., (Copyright © 2021 Tenover.)

In vitro MICs and in vivo pharmacodynamics of ceftazidime and cefepime human-simulated regimens (HSR) against modified carbapenem inactivation method (mCIM)-positive Pseudomonas aeruginosa isolates harboring different OXA-10-like subtypes were described. The murine thigh model assessed ceftazidime (2 g every 8 h [q8h] HSR) and cefepime (2 g and 1 g q8h HSR). Phenotypes were similar despite possessing OXA-10-like subtypes with differing spectra. Ceftazidime produced ≥1-log 10 killing in all isolates. Cefepime activity was dose dependent and MIC driven. This approach may be useful in assessing the implications of β-lactamase variants.

This study characterized the mechanisms of carbapenem resistance in gram-negative bacteria isolated from patients in Yola, Nigeria. Whole genome sequencing (WGS) was performed on 66 isolates previously identified phenotypically as carbapenem-non-susceptible. The patterns of beta-lactamase resistance genes identified were primarily species-specific. However, bla NDM-7 and bla CMY-4 were detected in all Escherichia coli and most Providencia rettgeri isolates; bla NDM-7 was also detected in 1 Enterobacter cloacae. The E. coli and E. cloacae isolates also shared bla OXA-1, while bla OXA-10 was found in all P. rettgeri, one Pseudomonas aeruginosa and 1 E. coli. Except for Stenotrophomonas maltophilia isolates, which only contained bla L1 , most species carried multiple beta-lactamase genes, including those encoding extended-spectrum beta-lactamases, AmpC and OXA in addition to a carbapenemase gene. Carbapenemase genes were either class B or class D beta-lactamases. No carbapenemase gene was detected by WGS in 13.6% of isolates., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The rapid detection and characterization of carbapenemases in isolates of Enterobacterales are crucial for precise antibiotic administration and infection control. This article reports the findings from a parallel evaluation of the NG-Test Carba 5 (NG Biotech, Guipry, France) and Xpert Carba-R (Cepheid, Sunnyvale, CA) assays in the detection and differentiation of five carbapenemases [imipenem-resistant phenotype (IMP), Klebsiella pneumoniae carbapenemase, New Delhi metallo-β-lactamase (NDM), oxacillin-hydrolyzing β-lactamase (OXA)-48-like, and Verona integron-encoded metallo-β-lactamase] or the genes that encode them. A total of 122 isolates recovered from blood cultures and 106 positive blood culture broth (BCB) specimens, including 134 Klebsiella pneumoniae, 54 Escherichia coli, 27 Enterobacter cloacae, 8 Klebsiella oxytoca, 2 Klebsiella aerogenes, and 3 Citrobacter freundii, were collected from two tertiary hospitals (Xi'an, China). Using PCR sequencing techniques, 89 isolates and 29 BCB specimens were determined to be Enterobacterales harboring carbapenem-resistance genes. In comparison to the PCR sequencing results, the specificities with both the NG-Test Carba 5 and Xpert Carba-R assays were 100%; the sensitivities were 92.1% and 100%, respectively, for recovered isolates and 79.3% and 100% for BCB specimens. The NG-Test Carba 5 missed eight NDM, four OXA-48-like, and one IMP β-lactamases in specimens containing two or three carbapenemase types. In summary, the NG-Test Carba 5 assay may yield false-negative results if isolates or BCB specimens contain two or three carbapenemases., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Background: Gram-negative organisms harbouring carbapenem resistance genes (CRGs) are spreading globally, including in Gulf Cooperation Council (GCC) countries. However, relatively few data are available about carriage of CRGs in hospitalized patients in this region., Aim: To determine prevalence of CRG carriage and risk factors for colonization among patients in GCC hospitals., Methods: Rectal swabs were obtained from ∼50 intensive care unit (ICU) patients from each of 11 hospitals in five GCC countries between March and November 2019. The swabs were tested for the presence of bla KPC , bla NDM , bla VIM , bla IMP , and bla OXA-48 CRG using a commercial polymerase chain reaction test. Data on risk factors for colonization were collected and analysed., Findings: Of 529 specimens screened, 138 (26.1%) were positive for one or more CRGs. The positivity rates among the hospitals ranged from 8.0% to 67.3%; ∼20% of the positive specimens harboured ≥2 CRGs. The most common CRG detected was bla OXA-48 , which was present in 82 specimens (15.5%). Additional CRGs included bla NDM , bla VIM , bla KPC , and bla IMP either alone or in combination. Overall, 31.1% of patients on antibiotics on admission to the ICU were positive for CRGs compared to 16.5% not on antibiotic therapy (P < 0.001). CRG detection was also more common among patients aged >65 years (P = 0.027) and increased with hospital length of stay (P = 0.025)., Conclusion: The rate of CRGs detected in hospitalized patients in GCC countries varied considerably. Prior antibiotic exposure, increasing age, and prolonged length of stay were associated with CRG detection., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Rapid detection of carbapenemase-producing organisms is clinically desirable for hospital infection control and antibiotic stewardship. In this multicenter study, the Xpert Carba-R assay was evaluated for detection of the five carbapenemase genes (bla KPC , bla NDM , bla IMP , bla OXA-48 , and bla VIM ) in 2404 nonduplicate rectal swabs of admitted inpatients and 521 Gram-negative isolates from four tertiary hospitals in China, compared with the reference growth-based method with DNA sequence analysis of colonies. All suspected false-positive results in rectal swabs were resolved by supplementary sequencing from broth cultures. A total of 197 bla KPC , 171 bla NDM , 142 bla IMP , 6 bla VIM , and 5 bla OXA-48 genes were detected by Xpert Carba-R in 417 rectal swabs, with overall positive and negative percentage agreements ranging from 94.5% to 100% and from 94.8% to 99.9%, respectively. Notably, 17.5% (263/1500) of inpatients had rectal colonization with carbapenem-nonsusceptible organisms detected in intensive care units, and 63.1% (166/263) were Xpert Carba-R positive. Among the 469 carbapenem-nonsusceptible and 52 carbapenem-susceptible isolates examined, 373 were Enterobacteriaceae, 55 were Pseudomonas aeruginosa, and 93 were Acinetobacter baumannii. Compared with the reference isolate sequencing, overall positive and negative percentage agreements were 99.7% and 98.0%, respectively. The intra-assay and interassay coefficient of variability values were both <2%. Thus, we show that Xpert Carba-R assay provides good reproducibility and reliable results for detection and differentiation of five carbapenemase genes in both rectal swabs and clinical isolates., (Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Objectives: Many multidrug-resistant Gram-negative bacilli (MDR-GNB) harbour multiple β-lactamases. The aim of this study was to assess the impact of multiple β-lactamase carriage on the accuracy of susceptibility tests and extended-spectrum β-lactamase (ESBL) and carbapenemase confirmation methods., Methods: A total of 50 MDR-GNB, of which 29 carried multiple β-lactamases, underwent broth microdilution (BMD) and disk diffusion (DD) testing as well as confirmation tests for ESBLs and carbapenemases. Whole-genome sequencing (WGS) was used for β-lactamase gene identification., Results: Categorical agreement of BMD and DD testing results ranged from 86.5 to 97.7% for 10 β-lactam agents. BMD and DD algorithms for ESBL detection were highly variable; 6 of 8 positive strains carried an ESBL plus a carbapenemase or an AmpC enzyme, which may confound antimicrobial selection. The sensitivity and specificity of the modified carbapenem inactivation method (mCIM) were both 100%, whilst mCIM and EDTA-modified carbapenem inactivation method (eCIM) when used together to differentiate serine from metallo-β-lactamase carriage were both 96%. Xpert® Carba-R results (in vitro diagnostic test) were consistent with WGS results. Predicting phenotypic carbapenem resistance from WGS data overall showed 100% specificity but only 66.7% sensitivity for Enterobacterales isolates that were non-susceptible to imipenem and meropenem., Conclusions: Multiple β-lactamases in MDR-GNB does not impact DD results, the utility of mCIM/eCIM tests, or Xpert Carba-R results. However, ESBL algorithms produced inconsistent results and predicting carbapenem resistance from WGS data was problematic in such strains., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Approximately 15-20% of the S. aureus genome contains mobile genetic elements that can cause discrepancies between phenotypic and genotypic identification methods. Three blood culture bottles (each from a different patient) that showed discordant results, were shown to contain 2 S. aureus isolates after additional subcultures. One bottle had MRSA and MSSA that by DNA sequence analysis differed only by 31 kb; however, the deletions encompassed parts of SCCmec including mecA and SCC M1 . The second bottle contained MRSA and MSSA that differed by 124 kb; the MSSA was missing the entire SCCmec and spa regions. The last bottle contained 2 MRSA, one with ACME II disrupting SCCmec and a 24 bp spa deletion. The deletions in SCCmec and the other elements gave rise to the discrepancies between molecular and the original culture results. Such discrepancies should prompt a search for additional strains in the blood culture bottle., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The growing prevalence and diversity of carbapenemase producers among carbapenem-resistant Pseudomonas aeruginosa (CRPA) isolates warrants an expansion of detection capabilities. The purpose of this study was to evaluate the performance of the commercially available Xpert Carba-R (Carba-R) and the research-use-only Xpert Carba-R NxG (Carba-R NxG) in a global collection of P. aeruginosa The challenge set included 123 P. aeruginosa clinical isolates from 12 countries. Isolates were previously categorized via PCR or whole-genome sequencing. Carbapenemase classes tested include VIM, IMP, NDM, SPM, KPC, and GES. Non-carbapenemase (non-CP)-harboring isolates were also tested (negative control). Isolates were tested using the Carba-R NxG and the Carba-R tests per the manufacturer's instructions. Carba-R NxG testing was completed by Cepheid (Sunnyvale, CA), blinded to genotype. Both assays gave negative results for all non-CP isolates and positive results for all VIM, NDM, and KPC isolates. An improvement in IMP detection among isolates was observed (100% detection by Carba-R NxG versus 58% by Carba-R). All SPM and GES isolates, targets not present in commercially available Carba-R, were positive by Carba-R NxG. Two isolates harbored both VIM and GES, while a third isolate contained VIM and NDM. The Carba-R NxG identified both targets in all 3 isolates, while the Carba-R was negative for both GES-containing isolates. Overall, the Carba-R NxG successfully categorized 100% of isolates tested compared with 68% for its predecessor. The Carba-R NxG will expand the detection spectrum of the current Carba-R assay to include SPM, GES, and expanded IMP variants, increasing the global utility of the test., (Copyright © 2020 American Society for Microbiology.)

Background: Healthcare-associated infections (HCAIs) continue to be a major cause of morbidity and mortality. Many HCAI pathogens, including multidrug-resistant organisms (MDROs), colonize the gastrointestinal tract., Aim: To determine the frequency of MDRO carriage in patients who do and do not harbour toxigenic Clostridioides difficile in their stools., Methods: Stool specimens received from nine US laboratories were cultured using media selective for C. difficile, Staphylococcus aureus, vancomycin-resistant enterococci (VRE), and carbapenem-resistant Gram-negative organisms (CROs). Specimens and isolates were also tested by polymerase chain reaction (PCR). Bacterial isolates underwent susceptibility testing and genotyping., Findings: Among 363 specimens, 175 yielded toxigenic C. difficile isolates spanning 27 PCR ribotypes. C. difficile (TCD + ) stools harboured an additional 28 organisms, including six CROs (3.4%), of which two (1.1%) were carbapenemase-producing organisms (CPOs), 19 VRE (10.9%), and three meticillin-resistant S. aureus isolates (MRSA, 1.7 %). Stools that were culture negative for toxigenic C. difficile (TCD - ) yielded 26 organisms, including four CROs (2.1%), 20 VRE (10.6), and two MRSA (1.1%). Excluding C. difficile, no significant differences were seen in the rates of the MDROs between TCD + and TCD - specimens., Conclusion: Overall, 15.4% of the TCD + stools and 11.2% of the TCD - stools carried at least one non-C. difficile MDRO pathogen, indicating that multiple MDROs may be present in the gastrointestinal tracts of patients, including those that harbour C. difficile., (Copyright © 2020 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.)

Objectives: This study aimed to identify carbapenem-resistant Gram-negative bacteria from clinical specimens of patients in Yola, Nigeria., Methods: Routine clinical specimens were screened for the presence of carbapenem-resistant Gram-negative bacteria using chromogenic agar plates. Susceptibility of all presumptive isolates to carbapenems was tested by MIC and disk diffusion methods. Real-time PCR was used to test for the presence of carbapenemase genes., Results: Screening of 1741 clinical specimens yielded 119 (6.8%) presumptive carbapenem-resistant Gram-negative bacteria. Antimicrobial susceptibility testing confirmed carbapenem resistance in 105 of these isolates. New Delhi metallo-β-lactamase (bla NDM ) gene was detected in 26 isolates and Verona integron-encoded metallo-β-lactamase (bla VIM ) gene was detected in four. The mechanism of resistance could not be identified in approximately two thirds of the carbapenem-resistant isolates., Conclusion: While bla NDM and bla VIM accounted for 28.6% of the resistance seen, further molecular-based studies are needed to characterise the other mechanisms of carbapenem resistance in these isolates., (Copyright © 2019 International Society for Antimicrobial Chemotherapy. Published by Elsevier Ltd. All rights reserved.)

PCR ribotyping and antimicrobial susceptibility testing were used to characterize 940 Clostridioides (Clostridium) difficile isolates collected from 26 U S. hospitals over three time periods from 2011 to 2017. The proportion of ribotype (RT) 027 isolated during the three surveys decreased significantly over time from 31% in 2011-2012, to 22% in 2013-2014, and to 14% in 2015-2017 (p < 0.001 and p = 0.010, respectively), while we observed an increase in prevalence of RT106, that rose from 7% in our first survey to 19% of isolates in our last survey (p < 0.001). In addition, both RT056 and RT002 rose from 3% to 10% (p < 0.001). The proportions of all other ribotypes remained steady over time, and RT014/020 was the third most common strain type in our convenience sample in the final survey. Overall, resistance to moxifloxacin, rifampin, and vancomycin decreased during our studies, mainly due to the decline in RT027 isolates. A decrease in moxifloxacin resistance and an increase in tetracycline resistance were found among RT027 strains isolated in the last survey. Although the proportion of RT027 isolates declined, multidrug resistance among this ribotype continues to be common., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Molecular diagnostic tests can be used to provide rapid identification of staphylococcal species in blood culture bottles to help improve antimicrobial stewardship. However, alterations in the target nucleic acid sequences of the microorganisms or their antimicrobial resistance genes can lead to false-negative results. We determined the whole-genome sequences of 4 blood culture isolates of Staphylococcus aureus and 2 control organisms to understand the genetic basis of genotype-phenotype discrepancies when using the Xpert MRSA/SA BC test ( in vitro diagnostic medical device [IVD]). Three methicillin-resistant S. aureus (MRSA) isolates each had a different insertion of a genetic element in the staphylococcal cassette chromosome (SCC mec )- orfX junction region that led to a misclassification as methicillin-susceptible S. aureus (MSSA). One strain contained a deletion in spa , which produced a false S. aureus -negative result. A control strain of S. aureus that harbored an SCC mec element but no mecA (an empty cassette) was correctly called MSSA by the Xpert test. The second control contained an SCC M1 insertion. The updated Xpert MRSA/SA BC test successfully detected both spa and SCC mec variants of MRSA and correctly identified empty-cassette strains of S. aureus as MSSA. Among a sample of 252 MSSA isolates from the United States and Europe, 3.9% contained empty SCC mec cassettes, 1.6% carried SCC M1 , <1% had spa deletions, and <1% contained SCC mec variants other than those with SCC M1 These data suggest that genetic variations that may interfere with Xpert MRSA/SA BC test results remain rare. Results for all the isolates were correct when tested with the updated assay., (Copyright © 2019 Tenover et al.)

Staphylococcus aureus strains that possess a mecA gene but are phenotypically susceptible to oxacillin and cefoxitin (OS-MRSA) have been recognized for over a decade and are a challenge for diagnostic laboratories. The mechanisms underlying the discrepancy vary from isolate to isolate. We characterized seven OS-MRSA clinical isolates of six different spa types from six different states by whole-genome sequencing to identify the nucleotide sequence changes leading to the OS-MRSA phenotype. The results demonstrated that oxacillin susceptibility was associated with mutations in regions of nucleotide repeats within mecA Subinhibitory antibiotic exposure selected for secondary mecA mutations that restored oxacillin resistance. Thus, strains of S. aureus that contain mecA but are phenotypically susceptible can become resistant after antibiotic exposure, which may result in treatment failure. OS-MRSA warrant follow-up susceptibility testing to ensure detection of resistant revertants., (Copyright © 2019 Goering et al.)

Surveillance of circulating microbial populations is critical for monitoring the performance of a molecular diagnostic test. In this study, we characterized 31 isolates of Streptococcus agalactiae (group B Streptococcus [GBS]) from several geographic locations in the United States and Ireland that contain deletions in or adjacent to the region of the chromosome that encodes the hemolysin gene cfb , the region targeted by the Xpert GBS and GBS LB assays. PCR-negative, culture-positive isolates were recognized during verification studies of the Xpert GBS assay in 12 laboratories between 2012 and 2018. Whole-genome sequencing of 15 GBS isolates from 11 laboratories revealed four unique deletions of chromosomal DNA ranging from 181 bp to 49 kb. Prospective surveillance studies demonstrated that the prevalence of GBS isolates containing deletions in the convenience sample was <1% in three geographic locations but 7% in a fourth location. Among the 15 isolates with chromosomal deletions, multiple pulsed-field gel electrophoresis types were identified, one of which appears to be broadly dispersed across the United States., (Copyright © 2019 American Society for Microbiology.)