Rapid identification and mapping of insertion sequences in Escherichia coli genomes using vectorette PCR

BACKGROUND: Insertion sequences (IS) are small DNA segments capable of transposing within and between prokaryotic genomes, often causing insertional mutations and chromosomal rearrangements. Although several methods are available for locating ISs in microbial genomes, they are either labor-intensive or inefficient. Here, we use vectorette PCR to identify and map the genomic positions of the eight insertion sequences (IS1, 2, 3, 4, 5, 30, 150, and 186) found in E. coli strain CGSC6300, a close relative of MG1655 whose genome has been sequenced. RESULTS: Genomic DNA from strain CGSC6300 was digested with a four-base cutter Rsa I and the resulting restriction fragments ligated onto vectorette units. Using IS-specific primers directed outward from the extreme ends of each IS and a vectorette primer, flanking DNA fragments were amplified from all but one of the 37 IS elements identified in the genomic sequence of MG1655. Purification and sequencing of the PCR products confirmed that they are IS-associated flanking DNA fragments corresponding to the known IS locations in the MG1655 genome. Seven additional insertions were found in strain CGSC6300 indicating that very closely related isolates of the same laboratory strain (the K12 isolate) may differ in their IS complement. Two other E. coli K12 derivatives, TD2 and TD10, were also analyzed by vectorette PCR. They share 36 of the MG1655 IS sites as well as having 16 and 18 additional insertions, respectively. CONCLUSION: This study shows that vectorette PCR is a swift, efficient, reliable method for typing microbial strains and identifying and mapping IS insertion sites present in microbial genomes. Unlike Southern hybridization and inverse PCR, our approach involves only one genomic digest and one ligation step. Vectorette PCR is then used to simultaneously amplify all IS elements of a given type, making it a rapid and sensitive means to survey IS elements in genomes. The ability to rapidly identify the IS complements of microbial genomes should facilitate subtyping closely related pathogens during disease outbreaks

Electron Transfer Precedes ATP Hydrolysis during Nitrogenase Catalysis

The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s−1, 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s−1, 25 °C), (ii) ATP hydrolysis (kATP = 70 s−1, 25 °C), (iii) Phosphate release (kPi = 16 s−1, 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s−1, 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein–protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Feox(ADP)2 protein and the reduced MoFe protein

Negative Cooperativity in the Nitrogenase Fe Protein Electron Delivery Cycle

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen (N2) to two ammonia (NH3) molecules through the participation of its two protein components, the MoFe and Fe proteins. Electron transfer (ET) from the Fe protein to the catalytic MoFe protein involves a series of synchronized events requiring the transient association of one Fe protein with each αβ half of the α2β2 MoFe protein. This process is referred to as the Fe protein cycle and includes binding of two ATP to an Fe protein, association of an Fe protein with the MoFe protein, ET from the Fe protein to the MoFe protein, hydrolysis of the two ATP to two ADP and two Pi for each ET, Pi release, and dissociation of oxidized Fe protein-(ADP)2 from the MoFe protein. Because the MoFe protein tetramer has two separate αβ active units, it participates in two distinct Fe protein cycles. Quantitative kinetic measurements of ET, ATP hydrolysis, and Pi release during the presteady-state phase of electron delivery demonstrate that the two halves of the ternary complex between the MoFe protein and two reduced Fe protein-(ATP)2 do not undergo the Fe protein cycle independently. Instead, the data are globally fit with a two-branch negative-cooperativity kinetic model in which ET in one-half of the complex partially suppresses this process in the other. A possible mechanism for communication between the two halves of the nitrogenase complex is suggested by normal-mode calculations showing correlated and anticorrelated motions between the two halves

Antimicrobial resistance in Streptococcus pneumoniae: a retrospective analysis of emerging trends in the United Arab Emirates from 2010 to 2021

IntroductionAlthough pneumococcal conjugate vaccines (PCV) have been effective in reducing the burden of Streptococcus pneumoniae infections, there is a paucity of data on the relationship with antimicrobial resistance (AMR) trends in the Arabian Gulf region. This study was carried out to assess S. pneumoniae resistance trends in the United Arab Emirates (UAE) where PCV-13 vaccination was introduced in 2011.MethodsRetrospective analysis of S. pneumoniae demographic and microbiological data collected as part of the national AMR surveillance program from 2010 to 2021 was carried out. A survey of reporting sites and hand searching of annual reports of local health authorities was carried out to identify data on S. pneumoniae serotypes as this is not included in the AMR surveillance database.ResultsFrom 2010 to 2021, 11,242 non-duplicate S. pneumoniae isolates were reported, increasing from 324 in 2010 to 1,115 in 2021. Factoring in annual increment in the number of surveillance sites, the number of isolates per site showed an upward trajectory from 2015 to 2018 and declined in 2020 with the onset of the pandemic. The majority of isolates (n/N = 5,751/11,242; 51.2%) were from respiratory tract specimens with 44.5% (n/N = 2,557/5,751) being nasal colonizers. Up to 11.9% (n/N = 1,337/11,242) were invasive pneumococcal disease (IPD) isolates obtained from sterile site specimens including blood (n = 1,262), cerebrospinal (n = 52), pleural (n = 19) and joint (n = 4) fluid; and were predominantly from pediatric patients. The downward trend for amoxicillin and for penicillin G at the non-meningitis and meningitis as well as oral penicillin breakpoints was statistically significant. In contrast, increasing trends of resistance were seen for levofloxacin, moxifloxacin, trimethoprim/sulfamethoxazole and erythromycin. IPD and non-IPD isolates showed similar demographic and AMR trends. None of the surveillance sites carried out S. pneumoniae serotyping and handsearching of annual reports did not yield this information.ConclusionThe increasing trend of pneumococcal disease and AMR with emergence of isolates with MDR phenotype despite is of concern. In the absence of S. pneumoniae serotyping the role of non-vaccine serotypes in driving this pattern remains unknown. There is an urgent need for serotype, genomic and AMR surveillance of S. pneumoniae isolates in the UAE

Carbapenem resistant Enterobacterales in the United Arab Emirates: a retrospective analysis from 2010 to 2021

BackgroundCarbapenem-resistant Enterobacterales (CRE) are spreading in the United Arab Emirates (UAE) where their dissemination is facilitated by international travel, trade, and tourism. The objective of this study is to describe the longitudinal changes of CRE as reported by the national AMR surveillance system of the UAE.MethodsIn this study, we retrospectively describe CRE isolated from 317 surveillance sites, including 87 hospitals and 230 centers/clinics from 2010 to 2021. The associated clinical, demographic, and microbiological characteristics are presented by relying on the UAE national AMR surveillance program. Data was analyzed using WHONET microbiology laboratory database software (http://www.whonet.org).ResultsA total of 14,593 carbapenem resistant Enterobacterales were analyzed, of which 48.1% were carbapenem resistant Klebsiella pneumoniae (CRKp), 25.1% carbapenem resistant Escherichia coli (CREc), and 26.8% represented 72 other carbapenem resistant species. Carbapenem resistant strains were mostly associated with adults and isolated from urine samples (36.9% of CRKp and 66.6% of CREc) followed by respiratory samples (26.95% for CRKp) and soft tissue samples (19.5% for CRKp). Over the studied period carbapenem resistance rates remained high, especially in K. pneumoniae, and in 2021 were equivalent to 67.6% for imipenem, 76.2% for meropenem, and 91.6% for ertapenem. Nevertheless, there was a statistically significant decreasing trend for imipenem and meropenem resistance in Klebsiella species (p < 0.01) while the decrease in ertapenem resistance was non-significant. Concerning E. coli, there was a statistically significant decreasing trend for meropenem and imipenem resistance over the 12 years, while ertapenem resistance increased significantly with 83.8% of E. coli exhibiting ertapenem resistance in 2021. Resistance rates to ceftazidime and cefotaxime remained higher than 90% (in 2021) for CRKp and cefotaxime rates increased to 90.5% in 2021 for CREc. Starting 2014, resistance to colistin and tigecycline was observed in carbapenem resistant Enterobacterales. CRE were associated with a higher mortality (RR: 6.3), admission to ICU (RR 3.9), and increased length of stay (LOS; 10 excess inpatient days per CRE case).ConclusionThis study supports the need to monitor CRE in the UAE and draws attention to the significant increase of ertapenem resistance in E. coli. Future surveillance analysis should include a genetic description of carbapenem resistance to provide new strategies

Methicillin resistant Staphylococcus aureus in the United Arab Emirates: a 12-year retrospective analysis of evolving trends

Introduction: Methicillin resistant Staphylococcus aureus (MRSA) is a major contributor to the global burden of antimicrobial resistance (AMR). As MRSA continues to evolve, the need for continued surveillance to evaluate trends remains crucial. This study was carried out to assess MRSA trends in the United Arab Emirates (UAE) based on analysis of data from the national AMR surveillance program. Methods: We carried out a 12-year (2010–2021) retrospective analysis of MRSA demographic and microbiological data collected as part of the UAE national AMR surveillance program. Participating centers from across the country routinely submit AMR surveillance data collected by trained personnel to the National AMR Surveillance Committee, where data is analyzed using a unified WHONET platform. Data on non-duplicate isolates associated with clinical infections were obtained and included in the analysis. Results: A total of 29,414 non-duplicate MRSA isolates associated with clinical infections were reported between 2010 and 2021 (2010: n = 259; 2021: n = 4,996). MRSA represented 26.4% of all S. aureus (n = 111,623) isolates identified during the study period. In 2010, among the S. aureus isolates with reported oxacillin testing, 21.9% (n/N = 259/1,181) were identified as MRSA and this showed an increase to 33.5% (n/N = 4,996/14,925) in 2021. Although there was variation in the distribution of MRSA across the seven emirates of the country, most had an upward trend. Patient demographics reflected a male preponderance, with most being adults and from the outpatient setting. Isolates were mostly from skin and soft tissue infection specimens (72.5%; n/N = 21,335/29,414). Among the inpatients (N = 8,282), a total of 3,313 MRSA isolates were from specimens obtained ≤ 48 h after admission indicative of community acquired infection. Increasing resistance trends were observed for most antibiotics including ciprofloxacin, levofloxacin, moxifloxacin, erythromycin, gentamicin, trimethoprim-sulfamethoxazole, and quinupristin/dalfopristin. Low levels of resistance (0.0–0.8%) were sustained for linezolid except for 2015, 2016, and 2017 with 2.5, 2.6, and 2.9%, respectively. No confirmed vancomycin resistance was reported. Conclusion: The increasing trend of MRSA isolates associated with clinical infections in the hospital and community settings is a concern. Continued monitoring including incorporation of genomic surveillance and infection control measures are recommended to stem the dissemination

Carbapenem resistant Enterobacterales in the United Arab Emirates: a retrospective analysis from 2010 to 2021

Background: Carbapenem-resistant Enterobacterales (CRE) are spreading in the United Arab Emirates (UAE) where their dissemination is facilitated by international travel, trade, and tourism. The objective of this study is to describe the longitudinal changes of CRE as reported by the national AMR surveillance system of the UAE. Methods: In this study, we retrospectively describe CRE isolated from 317 surveillance sites, including 87 hospitals and 230 centers/clinics from 2010 to 2021. The associated clinical, demographic, and microbiological characteristics are presented by relying on the UAE national AMR surveillance program. Data was analyzed using WHONET microbiology laboratory database software (http://www.whonet.org). Results: A total of 14,593 carbapenem resistant Enterobacterales were analyzed, of which 48.1% were carbapenem resistant Klebsiella pneumoniae (CRKp), 25.1% carbapenem resistant Escherichia coli (CREc), and 26.8% represented 72 other carbapenem resistant species. Carbapenem resistant strains were mostly associated with adults and isolated from urine samples (36.9% of CRKp and 66.6% of CREc) followed by respiratory samples (26.95% for CRKp) and soft tissue samples (19.5% for CRKp). Over the studied period carbapenem resistance rates remained high, especially in K. pneumoniae, and in 2021 were equivalent to 67.6% for imipenem, 76.2% for meropenem, and 91.6% for ertapenem. Nevertheless, there was a statistically significant decreasing trend for imipenem and meropenem resistance in Klebsiella species (p < 0.01) while the decrease in ertapenem resistance was non-significant. Concerning E. coli, there was a statistically significant decreasing trend for meropenem and imipenem resistance over the 12 years, while ertapenem resistance increased significantly with 83.8% of E. coli exhibiting ertapenem resistance in 2021. Resistance rates to ceftazidime and cefotaxime remained higher than 90% (in 2021) for CRKp and cefotaxime rates increased to 90.5% in 2021 for CREc. Starting 2014, resistance to colistin and tigecycline was observed in carbapenem resistant Enterobacterales. CRE were associated with a higher mortality (RR: 6.3), admission to ICU (RR 3.9), and increased length of stay (LOS; 10 excess inpatient days per CRE case). Conclusion: This study supports the need to monitor CRE in the UAE and draws attention to the significant increase of ertapenem resistance in E. coli. Future surveillance analysis should include a genetic description of carbapenem resistance to provide new strategies

Epidemiology of vancomycin-resistant enterococci in the United Arab Emirates: a retrospective analysis of 12 years of national AMR surveillance data

Introduction: Enterococci are usually low pathogenic, but can cause invasive disease under certain circumstances, including urinary tract infections, bacteremia, endocarditis, and meningitis, and are associated with peritonitis and intra-abdominal abscesses. Increasing resistance of enterococci to glycopeptides and fluoroquinolones, and high-level resistance to aminoglycosides is a concern. National antimicrobial resistance (AMR) surveillance data for enterococci from the Middle East and North Africa (MENA) and the Gulf region is scarce. Methods: A retrospective 12-year analysis of N = 37,909 non-duplicate diagnostic Enterococcus spp. isolates from the United Arab Emirates (UAE) was conducted. Data was generated by routine patient care during 2010–2021, collected by trained personnel and reported by participating surveillance sites to the UAE National AMR Surveillance program. Data analysis was conducted with WHONET. Results: Enterococcus faecalis was the most commonly reported species (81.5%), followed by Enterococcus faecium (8.5%), and other enterococci species (4.8%). Phenotypically vancomycin-resistant enterococci (VRE) were found in 1.8% of Enterococcus spp. isolates. Prevalence of VRE (%VRE) was highest for E. faecium (8.1%), followed by E. faecalis (0.9%). A significant level of resistance to glycopeptides (%VRE) for these two species has been observed in the majority of observed years [E. faecalis (0–2.2%), 2010: 0%, 2021: 0.6%] and E. faecium (0–14.2%, 2010: 0%, 2021: 5.8%). Resistance to fluoroquinolones was between 17 and 29% (E. faecalis) and was higher for E. faecium (between 42 and 83%). VRE were associated with higher patient mortality (RR: 2.97), admission to intensive care units (RR: 2.25), and increased length of stay (six excess inpatient days per VRE case), as compared to vancomycin-susceptible Enterococcus spp. Discussion: Published data on Enterococcus infections, in particular VRE-infections, in the UAE and MENA region is scarce. Our data demonstrates that VRE-enterococci are relatively rare in the UAE, however showing an increasing resistance trend for several clinically important antibiotic classes, causing a concern for the treatment of serious infections caused by enterococci. This study also demonstrates that VRE were associated with higher mortality, increased intensive care unit admission rates, and longer hospitalization, thus poorer clinical outcome and higher associated costs in the UAE. We recommend the expansion of current surveillance techniques (e.g., local VRE screening), stricter infection prevention and control strategies, and better stewardship interventions. Further studies on the molecular epidemiology of enterococci are needed

Emergence of highly resistant Candida auris in the United Arab Emirates: a retrospective analysis of evolving national trends

The Centers for Disease Prevention and Control lists Candida auris, given its global emergence, multidrug resistance, high mortality, and persistent transmissions in health care settings as one of five urgent threats. As a new threat, the need for surveillance of C. auris is critical. This is particularly important for a cosmopolitan setting and global hub such as the United Arab Emirates (UAE) where continued introduction and emergence of resistant variant strains is a major concern. The United Arab Emirates has carried out a 12 years of antimicrobial resistance surveillance (2010–2021) across the country, spanning all seven Emirates. A retrospective analysis of C. auris emergence from 2018–2021 was undertaken, utilising the demographic and microbiological data collected via a unified WHONET platform for AMR surveillance. Nine hundred eight non-duplicate C. auris isolates were reported from 2018–2021. An exponential upward trend of cases was found. Most isolates were isolated from urine, blood, skin and soft tissue, and the respiratory tract. UAE nationals nationals comprised 29% (n = 186 of 632) of all patients; the remainder were from 34 other nations. Almost all isolates were from inpatient settings (89.0%, n = 809). The cases show widespread distribution across all reporting sites in the country. C. auris resistance levels remained consistently high across all classes of antifungals used. C. auris in this population remains highly resistant to azoles (fluconazole, 72.6% in 2021) and amphotericin. Echinocandin resistance has now emerged and is increasing annually. There was no statistically significant difference in mortality between Candida auris and Candida spp. (non-auris) patients (p-value: 0.8179), however Candida auris patients had a higher intensive care unit (ICU) admission rate (p-value \u3c0.0001) and longer hospital stay (p \u3c 0.0001) compared to Candida spp. (non-auris) patients. The increasing trend of C. auris detection and associated multidrug resistant phenotypes in the UAE is alarming. Continued C. auris circulation in hospitals requires enhanced infection control measures to prevent continued dissemination