Unravelling AmpC Beta-Lactamases in Escherichia coli: Mechanisms, Resistance Patterns, and Implications for Diagnostic Strategies Evert Pieter Marie den Drijver
Unravelling AmpC Beta-Lactamases in Escherichia coli: Mechanisms, Resistance Patterns, and Implications for Diagnostic Strategies PhD thesis, Utrecht University, the Netherlands ISBN: 978-94-6483-486-4 Cover design: Annika van Rooij based on Achnatherum (Lasiagrostis) splendens. Drawing, analysis and description were done by Univ. Prof. Dr. Erwin Lichtenegger (1928-2004) and Univ. Prof. Dr. Lore Kutschera (1917-2008) *, leader of Pflanzensoziologisches Institut, Klagenfurt, (now in Bad Goisern, Austria). Use with permission of the copyright holder. Provided by thesis specialist Ridderprint, ridderprint.nl Printing: Ridderprint Layout and design: Tara Schollema, persoonlijkproefschrift.nl © Evert Pieter Marie den Drijver, 2023
Unravelling AmpC Beta-Lactamases in Escherichia coli: Mechanisms, Resistance Patterns, and Implications for Diagnostic Strategies Analyseren van AmpC Beta-Lactamasen in Escherichia coli: Mechanismen, Resistentiepatronen en Implicaties voor Diagnostische Strategieën (met een samenvatting in het Nederlands) Proefschrift ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector magnificus, prof. dr. H.R.B.M. Kummeling, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op dinsdag 5 december 2023 des middags te 12.15 uur door Evert Pieter Marie den Drijver geboren op 17 januari 1987 te Gorinchem
Promotor: Prof. dr. J.A.J.W. Kluijtmans Copromotor: Dr. J.J. Verweij Beoordelingscommissie: Prof. dr. P.H.M. Savelkoul Prof. dr. J.A. Stegeman Prof. dr. J.A.G. van Strijp Prof. dr. C.M. Vandenbroucke-Grauls Prof. dr. R.J.L. Willems (voorzitter)
5 Paranimfen Pepijn Huizinga Jordy Coolen
“Gras groeit niet sneller door aan de sprietjes te trekken.” Marcel van Roosmalen, 2012
Table of contents Chapter 1 General introduction 11 Chapter 2 AmpC beta-lactamases: epidemiology, infection control and treatment 19 Chapter 3 Decline in AmpC beta-lactamase-producing Escherichia coli in a Dutch teaching hospital (2013-2016) 33 Chapter 4 Development of an algorithm to discriminate between plasmid- and chromosomal-mediated AmpC beta-lactamase production in Escherichia coli by elaborate phenotypic and genotypic characterization 49 Chapter 5 Detection of AmpC beta-lactamases in Escherichia coli using different screening agars 79 Chapter 6 Limited genetic diversity of blaCMY-2-containing IncI1-pST12 plasmids from Enterobacteriaceae of human and broiler chicken origin in the Netherlands 95 Chapter 7 Genome-wide analysis in Escherichia coli unravels a high level of genetic homoplasy associated with cefotaxime resistance 117 Chapter 8 In silico estimates of plasmid copy number are associated with increased resistance to cefotaxime, ceftazidime, and piperacillin/ tazobactam in CMY-2-producing Escherichia coli 145 Chapter 9 Summary and general discussion 161 Closing pages 169 References 189
Chapter 1 General introduction
12 Chapter 1 Antibiotic resistance, what is it and why is it a global problem? Infectious diseases have plagued humanity since ancient times, presenting a significant challenge. For centuries, little was understood about their causes, let alone finding cures. Among the microorganisms responsible for these diseases, bacteria have played a crucial role in mortality and morbidity. From the devastating plagues of Yersinia pestis in the Middle Ages to the widespread cholera outbreaks in the 19th century, bacterial infections have been a leading cause of death and despair throughout history (Glatter and Finkelman 2021; Waldman, Ronald and Claeson, Mariam 2023). A pivotal moment in the fight against infectious diseases occurred in the 19th century with the discovery that bacteria were the root cause of many common infections. This breakthrough was made possible by the pioneering work of Robert Hermann Koch, one of the foremost scientists in the field of microbiology (Münch 2003). Using Koch’s postulates, which provided a framework for establishing the pathogenicity of bacteria, scientists were able to confirm their role as disease-causing agents. This understanding laid the foundation for the development of antimicrobial therapeutics to combat infectious diseases. The first breakthrough in this regard came in 1910 with the discovery of arsphenamine, also known as salvarsan, as a cure for syphilis (Hutchings, Truman, and Wilkinson 2019; Christensen 2021). Shortly thereafter, the antibiotic effect of sulphonamides was uncovered, leading to their widespread use as antibiotics. One of the most well-known discoveries in the field was made by Alexander Fleming in 1928 when he observed the inhibitory effect of penicillin, produced by the fungus Penicillium chrysogenum, on the growth of Staphylococcus aureus bacteria he was studying (Lobanovska and Pilla 2017). Although the antimicrobial properties of many soil microorganisms, such as actinomycetales, were known before Fleming’s discovery, the identification, purification, and stabilization of penicillin marked a monumental scientific breakthrough in the medical and pharmaceutical realms (Clardy, Fischbach, and Currie 2009). Many widely used antibiotics, including cephalosporins and carbapenems, belong to the same class as penicillin—beta-lactams. These beta-lactam antibiotics play a crucial role in the treatment of bacterial infections. For instance, in the Netherlands, cephalosporins and carbapenems are the preferred antimicrobial agents for septicaemia (Sieswerda et al. 2020). The discovery of penicillin, a significant antibiotic, has been accompanied by a drawback—the emergence of antibiotic resistance (Lobanovska and Pilla 2017; Zaman
13 General introduction et al. 2017). Even before its clinical usage began in the 1940s, resistance to penicillin was observed. This phenomenon was initially detected in bacteria that had been exposed to sublethal doses of penicillin over time. In 1940, Abraham et al. discovered that certain bacteria, like Escherichia coli, produce an enzyme called penicillinase that can destroy penicillin (Abraham and Chain 1940). The clinical impact of resistance became evident as the rate of penicillin resistance in S. aureus rose rapidly, reaching over 80% by the late 1960s (Lowy 2003). This meant that nearly 4 out of 5 patients with S. aureus infections were no longer responsive to the first-choice antibiotic (Lobanovska and Pilla 2017; Lowy 2003). Fortunately, new broad-spectrum antibiotics and resistance inhibitors (e.g., clavulanic acid) were subsequently discovered (Docquier and Mangani 2018). Currently, we have a range of antimicrobial agents from various groups, such as quinolones and lipopeptides, although the development of new antibiotic groups has slowed down (Garcia-Bustos, Cabañero-Navalón, and Salavert Lletí 2022; Ventola 2015). The slow pace of antibiotic development raises concerns, particularly in light of the rapid increase in antibiotic resistance rates over the past two decades, particularly among gram-negative bacteria (Ventola 2015; Plackett 2020; Paterson 2006). Among these, the Enterobacterales order holds significant clinical importance (Paterson and Bonomo 2005). Comprising a large group of gram-negative rod-shaped bacteria, Enterobacterales are commonly found in the human gut and are associated with prevalent bacterial infections, such as biliary and urinary tract infections (Janda and Abbott 2021). Septicaemia caused by Enterobacterales is a frequently encountered and potentially fatal complication if not effectively treated (Bone 1993). While many beta-lactam antibiotics, such as cephalosporins and carbapenems, traditionally exhibit susceptibility against most Enterobacterales, the rising rates of beta-lactam resistance within this bacterial order pose a challenge to treatment and have led to decreasing cure rates (Kang et al. 2005; Pop-Vicas and Opal 2014). A major cause of beta-lactam resistance is the presence of enzymes called betalactamases (D M Livermore 1995). These enzymes diminish the effectiveness of betalactam antibiotics by hydrolysing their molecular structure. Specifically, they break open the beta-lactam ring of the antibiotic, rendering its antimicrobial activity inactive. One example of a beta-lactamase is penicillinase. There exists a wide array of different beta-lactamases, some exerting a greater impact on antibiotic resistance than others. Certain beta-lactamases are capable of hydrolysing only narrow-spectrum antibiotics like penicillin, while others can destroy broad-spectrum antibiotics like cephalosporins and carbapenems. 1
14 Chapter 1 Among the frequently encountered beta-lactamases are the extended spectrum betalactamases (ESBLs). This group comprises numerous variants (e.g., TEM, SHV, CTX-M, etc.), all of which possess the ability to hydrolyse the broader-spectrum cephalosporin antibiotics (David M. Livermore 2008). The escalating prevalence of ESBLs in the past two decades has significantly influenced the clinical use of cephalosporins (David M. Livermore et al. 2007). In certain countries, resistance due to ESBLs has reached alarming levels, necessitating the restriction of cephalosporins as the first-line treatment for common infections. Consequently, more broad-spectrum antibiotics like carbapenems and quinolones are employed. However, the efficacy of these broad-spectrum antibiotics may be limited as resistance to these agents rapidly emerges due to their widespread use. What is an “AmpC beta-lactamase”? The AmpC beta-lactamase is a bacterial enzyme that primarily targets specific betalactam antibiotics, especially cephalosporins (Jacoby 2009). It is prevalent among gramnegative bacteria, particularly in the Enterobacterales order. AmpC beta-lactamase production leads to resistance against commonly used beta-lactam antibiotics, and it is often unaffected by beta-lactamase inhibitors, distinguishing it from other ESBLs. The AmpC beta-lactamase belongs to molecular class C and is regulated by pathways involved in cell-wall degradation. Mutations in the regulatory gene ampR result in overexpression of ampC, causing broader resistance to cephalosporins, including thirdgeneration cephalosporins like ceftriaxone, cefotaxime, and ceftazidime. Additionally, mutations primarily in the ampD gene can lead to constitutive overexpression of ampC, resulting in long-lasting antibiotic resistance, particularly in species like Enterobacter cloacae complex and Citrobacter freundii (Kohlmann, Bähr, and Gatermann 2018). In most Enterobacterales, AmpC enzymes are inducible, but certain species like E. coli and Shigella spp. express ampC at low levels due to the absence of the ampR gene. However, mutations in the promoter/attenuator region of the ampC gene can lead to hyperproduction of the AmpC beta-lactamase (Caroff N, Espaze E, Gautreau D, Richet H 2000; Tracz et al. 2007). Figure 1 shows the sequence of the E. coli ATCC 25922 ampC promoter/attenuator region. Although most AmpC hyperproducers show relatively low resistance to third-generation cephalosporins, alterations in the AmpC beta-lactamase or changes in membrane permeability can contribute to increased resistance (Nordmann and Mammeri 2007; Martínez-Martínez 2008).
15 General introduction Various types of plasmid-encoded ampC genes have been identified, with blaCMY-2 (beta-lactamase type CMY-2) being the most common in the Netherlands (E. Ascelijn Reuland et al. 2015; E. Den Drijver et al. 2018). These genes vary in their hydrolysing capability and are associated with specific bacterial species, such as blaCMY-2 in E. coli and Salmonella spp. and blaDHA in Klebsiella spp (Philippon, Arlet, and Jacoby 2002). Multiple studies have investigated the epidemiology of plasmid-encoded ampC genes in the Netherlands, but information on trends remains limited. Figure 1. Sequence of the E. coli ATCC 25922 ampC promoter/attenuator region. Sequence regions based on Tracz et al. with numbering according to Jaurin et al. and (Jaurin et al. 1981; Tracz et al. 2007). How to detect AmpC beta-lactamases Detection and differentiation of plasmid-encoded AmpC and chromosomal-encoded AmpC genes pose challenges due to their coexistence. This difficulty is amplified in the presence of ESBL. The Dutch guideline for detection of highly-resistant microorganisms recommends initial screening for plasmid AmpC by assessing resistance to cephamycins, using a cefoxitin minimal inhibitory concentrations (MICs) of 8 mg/L or higher and elevated MICs for cefotaxime, ceftriaxone, or ceftazidime (MIC >1 mg/L), as indicators of AmpC production (J.A.J.W Kluytmans et al. 2021). Confirmation tests involve inhibitory tests using cloxacillin or boric acid and various disc diffusion or gradient strip methods that compare zone differences between third-generation cephalosporins with or without an inhibitor. 1
16 Chapter 1 Molecular confirmation tests, such as multiplex PCRs, microarrays, and whole genome sequencing (WGS), are often required to specifically identify plasmid-encoded ampC genes and differentiate them from chromosomal ampC. WGS allows for the detection of promoter/attenuator mutations in E. coli and the examination of different plasmid families. WGS facilitates cluster analysis and confirmation of outbreaks by integrating cluster analysis results with epidemiological data (Quainoo et al. 2017). In settings where molecular diagnostics may not be accessible, the implementation of a practical algorithm for distinguishing ampC genotypes in E. coli through phenotypic susceptibility testing can be valuable. Notably, utilizing cefotaxime minimal inhibitory concentrations obtained from gradient test results demonstrated a high level of accuracy in predicting the ampC genotype. The utilization of diverse screening media in prevalence studies introduces variability in the interpretation of prevalence data. While specific media for screening ESBL are available, the options for screening AmpC-producing Enterobacterales are limited. Although there are media with increased cephamycin concentrations, the effectiveness of these media has been insufficiently studied. The effectiveness of media with increased cephamycin concentrations requires further study, and standardization of screening strategies using antibiotic enrichment broth is yet to be established. What are the sources of AmpC-producing Enterobacterales? Plasmids are extrachromosomal DNA elements that can be transferred between bacteria, often carrying antibiotic resistance genes. Plasmid-based resistance poses a significant challenge to infection control, with a particular focus on Enterobacterales carrying plasmid-mediated AmpC beta-lactamases (pAmpC-E). Certain plasmid families, such as IncA/C, IncB/O/K, and IncI, are commonly associated with blaCMY-2, the prevalent AmpC resistance gene (Accogli et al. 2013; Alessandra Carattoli et al. 2018). Notably, IncI plasmids, including the prevalent IncI-ST12 sequence type, have been detected in diverse sources, including human clinical samples, traveller rectal carriage samples, livestock samples, and dog samples (Lorme et al. 2018; Hansen et al. 2016). The transmission mechanisms of these blaCMY-2-carrying IncI plasmids remain to be fully understood. The origin of the promoter/attenuator mutations that lead to ampC hyperexpression in E. coli is difficult to confirm due to the phenomenon of convergent evolution, where mutations can independently reoccur in multiple isolates and separate lineages. This process, known as homoplasy, is potentially influenced by selective pressure from the
17 General introduction use of antibiotics or antiseptics. Homoplasy is a biological phenomenon characterized by the independent occurrence or recurrence of similar traits or genetic changes in different organisms or lineages, irrespective of their genetic relatedness (Wake, Wake, and Specht 2011). It refers to the parallel evolution of similar features, mutations, or genetic variations across diverse populations. This phenomenon poses challenges in determining the true origin or evolutionary history of a particular trait or mutation, as it can arise through convergent evolution rather than through shared ancestry. Is AmpC-mediated resistance a significant concern? Antibiotic resistance resulting from the production of broad-spectrum beta-lactamases in Gram-negative bacteria presents a formidable challenge in both clinical and community settings (Murray et al. 2022). The global dissemination of ESBL-producing variants of E. coli has severely restricted treatment options, and the emergence of AmpC betalactamases is also causing a notable impact, albeit to a lesser extent, by compromising the effectiveness of broad-spectrum penicillins and third-generation cephalosporins. The resistance exhibited by AmpC beta-lactamases to beta-lactamase inhibitor combinations, such as clavulanic acid and tazobactam is of particular concern. Various hypotheses have been proposed to explain this phenomenon, one of which suggests that an elevation in the plasmid copy number of blaCMY-2 containing plasmids could be a contributing factor (Kurpiel and Hanson 2012). Aims of this thesis The main objectives of this thesis are to delve into various aspects of AmpC-related antimicrobial resistance in Enterobacterales. Firstly, the thesis aims to assess the prevalence of rectal colonization by AmpC-producing Enterobacterales in the Netherlands and identify potential trends in colonization rates over time. Secondly, the thesis seeks to optimize the detection of AmpC genes in E. coli through the use of selective media and phenotypic characterization techniques, such as determining minimal inhibitory concentrations of specific antibiotics. The obtained results contribute to the development of a screening strategy that utilizes the phenotype of E. coli to predict the underlying genotype. 1
18 Chapter 1 The third objective focuses on investigating the relatedness between plasmids containing the blaCMY-2 gene in epidemiologically linked and unrelated Enterobacterales isolates from humans and livestock. The aim is to explore the feasibility of accurately distinguishing related samples from unrelated ones based solely on plasmid sequencing data. The fourth aim of this thesis is to examine whether mutations occurring in the ampC promoter/attenuator region of E. coli are homoplastic and whether these homoplastic mutations are associated with cefotaxime resistance. This investigation sheds light on the relationship between specific mutations and antibiotic resistance. The fifth aim of this thesis was to compare the sequencing depth between chromosomal household genes and plasmid-encoded scaffolds containing the blaCMY-2 gene, as well as to utilize the ratio as an estimated plasmid copy number. This analysis aimed to provide insights into the abundance of plasmids carrying the blaCMY-2 gene within the studied isolates. Furthermore, the relationship between the estimated plasmid copy number and the minimal inhibitory concentrations of cefotaxime, ceftazidime, and piperacillin-tazobactam was investigated. This examination allowed for a deeper understanding of the association between plasmid presence and the level of resistance to these specific antibiotics.
Chapter 2 AmpC beta-lactamases: epidemiology, infection control and treatment Evert den Drijver, Jaco J. Verweij, Jan A.J.W. Kluytmans Adapted from: Tijdschrift voor Infectieziekten, 18 (1), March 2023
20 Chapter 2 Summary Antibiotic resistance is an increasing problem. Particularly in gram-negative bacteria, there is a wide variety of resistance mechanisms affecting different antibiotics, e.g. the beta-lactam group. Besides the already common extended spectrum beta-lactamases (ESBL), the AmpC beta-lactamases make a significant contribution to resistance in gram-negative bacteria. Some of these resistance mechanisms are already intrinsically present on the bacterium’s chromosome, but can also be transferred from bacteria to bacteria on plasmids. The latter may be a significant factor in the spread and increase of antibiotic resistance in healthcare. This article has been written to provide more insight into the background of AmpC beta-lactamases, as well as epidemiology and diagnostics. Hopefully, it can provide tools for microbiologists or clinical infectious disease specialists for the diagnosis, treatment and prevention of further transmission of this resistance mechanism.
21 AmpC beta-lactamases: epidemiology, infection control and treatment Introduction A well-known healthcare-related problem among patients in hospitals and nursing homes is the increasing antibiotic resistance in gram-negative bacteria (David M. Livermore et al. 2007). Extended spectrum beta-lactamase (ESBL)-producing gram-negative bacteria within the order Enterobacterales, such as Escherichia coli and Klebsiella pneumoniae, are increasingly found worldwide. Due to the resistance caused by the beta-lactamases, frequently used beta-lactam antibiotics are no longer effective. This limits the options for adequate treatment for the patient, because empirical therapy for bloodstream infections in most Dutch hospitals is based on the use of beta-lactam antibiotics. For example, second- and third-generation cephalosporins such as cefuroxime, ceftriaxone or ceftazidime occupy an important place in the Netherlands as the first-choice treatment for sepsis, as described in the SWAB guideline Sepsis 2020 (Sieswerda et al. 2020). Although to a lesser extent than ESBL, acquired AmpC beta-lactamases have emerged as a potential threat to the utility of broad-spectrum penicillins and thirdgeneration cephalosporins (Jacoby 2009). Acquired AmpC beta-lactamases are encoded on plasmids and are transferable between different bacteria species. Detecting AmpC production in Enterobacterales can be challenging and guidelines and protocols on the detection and infection prevention are still scarce. In this review, the background of the resistance mechanism and possible detection methods of plasmid-encoded AmpC are described. A summarized overview of the epidemiology of plasmid-encoded AmpC in the Netherlands is provided as well. Furthermore, some descriptions of outbreaks with plasmid AmpC are given with possible guidance on infection prevention regarding the spread of AmpC producing Enterobacterales. Finally, the treatment options for infections with AmpC producing Enterobacterales are summarized. Background of AmpC The existence of the AmpC beta-lactamase in E. coli has been known since the 1940s of the twentieth century (Jacoby 2009). In the 1960s, the name AmpC was first used in scientific literature for a specific mutant of a penicillinase regulated by the ampA (Jacoby 2009). This type of penicillinase was later found to be different from other known penicillinases such as blaTEM-1. In the Ambler classification, the AmpC betalactamases are classified in a separate class C (Bush and Jacoby 2010). The most notable feature of the AmpC beta-lactamases is that they can hydrolyse cephalosporins and cephamycins, such as cefoxitin and cefotetan. This last type of beta-lactam antibiotic cannot be hydrolysed by ESBLs, such as the SHV or CTX-M beta-lactamases (Jacoby 2
22 Chapter 2 2009). Fourth generation cephalosporins such as cefepime are an exception, as they are not hydrolysed by AmpC beta-lactamases. A second difference to most ESBLs is that commonly used beta-lactamase inhibitors such as clavulanic acid and tazobactam have very little or no effect on the AmpC activity. Cloxacillin and avibactam do have a good inhibitory effect on AmpC beta-lactamases. This characteristic of AmpC can be used in diagnostics (see paragraph Detection of plasmid AmpC ) (Jacoby 2009; J.A.J.W Kluytmans et al. 2021). Ever since the first studies on AmpC beta-lactamase in E. coli, this class of beta-lactamases has been found in a multitude of Enterobacterales and other Gramnegative bacteria over the years. For example, AmpC enzymes have been detected in Pseudomonas aeruginosa and Acinetobacter baumannii complex (Jacoby 2009). Although different AmpC beta-lactamases are very similar in enzyme structure, the genetic sequence differs per variant and species. The phenotype can also be very diverse. This is partly related to the phenomenon of induction and derepression of the AmpC beta-lactamase. AmpC production is related to cell wall degradation in gram-negative bacteria. As soon as more degradation products, such as 1,6-anhydromuropeptides, are released during the degradation of the cell wall, the expression of the ampC gene increases via a transcription regulator (AmpR). A second enzyme, called AmpD amidase, can initiate an alternative pathway in the cell wall degradation cycle that can inhibit ampC expression. Normally this phenomenon is balanced, but the administration of beta-lactam antibiotics increases the cell wall degradation and therefore causes an elevated ampC expression and subsequently the AmpC beta-lactamase production. This phenomenon is called induction, resulting in the typical resistant phenotype. Once the supply of cell wall degradation products decreases again, the expression of the ampC gene and the beta-lactamase production will return to the original level (Jacoby 2009). However, mutations can occur in the ampR or ampD gene, resulting in an adaptation in the enzyme structure. The cycle remains in a continuously increased status and in such cases, the beta-lactam resistance will be maintained without an increased supply of cell wall degradation products. This phenomenon is known as derepression and is mainly seen in Enterobacter spp, Klebsiella aerogenes and Citrobacter freundii (Jacoby 2009; Kohlmann, Bähr, and Gatermann 2018). This group of Enterobacterales is often referred to as “group II Enterobacterales”. When these mutants are selected out, for example under antibiotic pressure, the beta-lactam resistance remains constant and the microorganism can no longer be adequately treated with third-generation cephalosporins. In recent decades it has been discovered that ampC genes can also be encoded on plasmids (Jacoby 2009; Philippon, Arlet, and Jacoby 2002). Plasmids are circular
23 AmpC beta-lactamases: epidemiology, infection control and treatment strands of DNA apart from the chromosomal DNA of the bacterium. They can be transferred from bacterium to bacterium by horizontal transfer. Several types of plasmidencoded ampC (pampC) genes have been detected in Enterobacterales species often referred to as “group I Enterobacterales” (including E. coli, K. pneumonia, P. mirabilis, Salmonella enteritidis), with blaCMY-2 being the most common pampC resistance gene in the Netherlands (E. Ascelijn Reuland et al. 2015). Other less commonly isolated pampC genes are other variants of blaCMY, as well as variants that differ more from blaCMY, e.g. blaDHA, blaACC, blaACT, blaMIR, blaMOX and blaFOX (Philippon, Arlet, and Jacoby 2002) (see Table 1.). All of these different pampC genes are originally derived from chromosomal ampC genes. For example, the blaCMY originates from the C. freundii chromosome, but several mutations have resulted in a great diversity of variants of the blaCMY gene (Jacoby 2009). Depending on the variant of AmpC beta-lactamase the hydrolysing capacity varies (Philippon, Arlet, and Jacoby 2002). This leads to different phenotypes per variant of ampC gene (see Table 1). The blaCMY-2 gene most common in the Netherlands generally leads to increased minimum inhibitory concentrations (MICs) for ceftriaxone, ceftazidime and cefoxitin (Philippon, Arlet, and Jacoby 2002; Coolen et al. 2019). However, in isolates with blaDHA this hydrolysing activity is less prominent, so that the effect on ceftriaxone or ceftazidime MICs may be less pronounced (Coolen et al. 2019). Moreover, the blaDHA gene is inducible when exposed to different antibiotics, e.g., imipenem. The hydrolysing effect can increase under the influence of these antibiotics (Jacoby 2009). Another example is the blaACC gene, which has the specific effect that it cannot hydrolyse cephamycins (e.g., cefoxitin) (Jacoby 2009; Philippon, Arlet, and Jacoby 2002). This is unique, as the hydrolysis of this antibiotic group is considered typical for AmpC beta-lactamases and the increased cephamycin MICs are used in many diagnostic algorithms (J.A.J.W Kluytmans et al. 2021; Martinez and Simonsen 2017). It seems that certain pampC genes are more common in certain species of the group 1 Enterobacterales. For example, blaCMY-2 is more commonly detected in E. coli and Salmonella spp and blaDHA in Klebsiella spp (Rodríguez-Guerrero et al. 2022). Furthermore, which variant is most prevalent can differ regionally. For example, in North-western Europe, the blaCMY-2 gene is most commonly detected in human and veterinary samples, while the blaDHA gene is more often found in equivalent samples in East Asia (e.g., South Korea and Japan) (Rodríguez-Guerrero et al. 2022). 2
24 Chapter 2 Table 1. Different beta- lactamase families with possible original species, number of variants and expected phenotype ( ↑↑ = strongly increased MIC, ↑ = moderately increased MIC, ↓ = low MIC) 3,4 AmpC betalactamase family Probably from chromosome of species Genetic similarity chromosomal gene Number of variants Phenotype 1 blaCMY -2 family Citrobacter freundii 96% n =171 Third-generation cephalosporins ↑↑, cephamycins (blaCMY -13 inducible) 2 blaDHA family Morganella morgagnii 99% n =29 Third-generation cephalosporins ↑, cephamycins (inducible) 3 blaACC family Hafnia alvei 99% n = 7 Third-generation cephalosporins ↑, cephamycins 4 blaACT /MIR family Enterobacter cloacal complex 98-99% n = 83 (blaACT ), n = 22 (blaMIR ) Third-generation cephalosporins ↑, cephamycins (blaACT inducible) 5 blaFOX family Aeromonas caviae 99% n=16 Third-generation cephalosporins ↓, cephamycins ↑↑ 6 blaMOX/CMY-1 family Aeromonas hydrophila 80-82% n=14 (blaMOX), n=6 (blaCMY-1) Third-generation cephalosporins ↑, cephamycins In certain species within the group 1 Enterobacterales, e.g., E. coli and Shigella spp, the production of AmpC beta-lactamase is not only encoded on plasmids, but can also be mediated by hyperexpression of a chromosome-encoded ampC gene (campC) (Jacoby 2009; Tracz et al. 2007). Normally, campC is only expressed at a low level in E. coli, but mutations in the promoter/attenuator region of the campC gene lead to hyperproduction of the chromosome-encoded AmpC beta-lactamase. The presence of these “chromosomal AmpC hyperproducers” complicates the detection of pampC genes in E. coli when
25 AmpC beta-lactamases: epidemiology, infection control and treatment using only phenotypic assays (Coolen et al. 2019). Although most chromosomal AmpC hyperproducing E. coli appear to have lower MICs for third-generation cephalosporins, there is evidence that changes in the AmpC beta-lactamase or changes in membrane permeability can lead to an increased cephalosporin resistance (Coolen et al. 2019; Nordmann, Poirel, and Nordmann 2007). Co-expression of ESBL and pampC genes in the same isolate can be even more challenging to detect using phenotypic assays because this detection is dependent on genotypic confirmation (J.A.J.W Kluytmans et al. 2021; Martinez and Simonsen 2017). Plasmid-based resistance is considered a greater threat regarding infection control than clonal transmission of chromosome-encoded resistance genes. Therefore, the focus within infection control is mainly on pAmpC producing Enterobacterales and less on the AmpC hyperproducing Enterobacterales. Certain types of plasmids are associated with specific pampC genes. Common plasmid families related to blaCMY-2 are IncA /C-, IncB /O/K and IncI (Alessandra Carattoli et al. 2018; Pietsch et al. 2018). In particular, IncI plasmid families are increasingly found in combination with blaCMY-2. IncI-ST12 is one of the most common plasmid sequence types (Pietsch et al. 2018; Roer et al. 2019). Strikingly, the IncI plasmids harbouring blaCMY-2 are found in several domains, such as human clinical samples, rectal carrier samples from travellers, veterinary samples and pet samples (Lorme et al. 2018; Hansen et al. 2016; Roer et al. 2019; E. P. M. Den Drijver et al. 2020). It is not yet known whether the elevated prevalence of these IncI plasmids is due to the presence of a common clone or more efficient transfer mechanisms among bacteria. Detection of Plasmid AmpC Detection of plasmid encoded AmpC is based on phenotypic and genotypic testing. Due to the coexistence of both chromosome and plasmid encoded ampC genes, it can be difficult to distinguish them. The presence of ESBL can make detection even more difficult. The recently updated NVMM guideline for BRMO detection recommends screening and confirmation of plasmid AmpC at Enterobacterales (J.A.J.W Kluytmans et al. 2021). The high pAmpC resistance phenotype to cephamycins is often used as a first screening criterion. A cefoxitin MIC >8 mg/L in combination with elevated MICs for cefotaxime, ceftriaxone or ceftazidime (MIC >1 mg/L) may indicate the presence of AmpC production (J.A.J.W Kluytmans et al. 2021). The confirmation tests are based on the aforementioned inhibitory capacity of cloxacillin or boric acid. Various disc diffusion tests and gradient strips are available, which confirm the presence of AmpC production based on zone difference between e.g., a third-generation cephalosporin with or without an inhibitor (J.A.J.W Kluytmans 2
26 Chapter 2 et al. 2021). Figure 1. shows an example of an AmpC and ESBL confirmation test based on disk diffusion. Figure 1. Example of a disk confirmation test for AmpC and ESBL (Mast® D68C, Mast Group Ltd., Bootle, United Kingdom). Disk A: Cefpodoxime 10µg, Disk B Cefpodoxime 10µg + ESBL Inhibitor, Disk C Cefpodoxime 10µg + AmpC Inhibitor, Disk D Cefpodoxime 10µg + ESBL Inhibitor + AmpC Inhibitor. Figure 1a. no AmpC if no ESBL, Figure 1b. AmpC positive, Figure 1c. ESBL positive, 1d AmpC and ESBL positive. (photo C. Verhulst, Microvida, Breda, Netherlands) The confirmation of AmpC production in various species within the Enterobacterales cannot differentiate the expression of a plasmid and a chromosome encoded ampC gene. In many cases, it will be necessary to perform a molecular confirmation test, which specifically determines the presence of plasmid encoded ampC genes. Various multiplex PCRs and microarrays have been developed (J.A.J.W Kluytmans et al. 2021). In recent years, whole genome sequencing has taken flight, making it possible to analyse the entire chromosome and plasmids of the bacterium. This has the additional advantage
27 AmpC beta-lactamases: epidemiology, infection control and treatment that promoter/attenuator mutations in E. coli can be confirmed and the presence of the different plasmid families can be examined. Moreover, whole genome sequencing allows is that cluster analysis can be performed and outbreaks can be confirmed based on the combination of this cluster analysis and epidemiological data (Quainoo et al. 2017). However, the reconstruction of full plasmid sequences may necessitate the utilization of multiple sequencing methods, resulting in a time-consuming and labour-intensive analysis. Screening with specific media for AmpC producing Enterobacterales, a common method for other resistance presences such as ESBL, is currently still limited. Although there are producers of media that contain elevated cephamycin concentrations, the number of comparative studies on the effectiveness is limited. The use of a specific antibiotic enrichment broth as a screening strategy has not yet been standardized. The lack of guidelines on screening strategies makes the diversity of screening methods in prevalence studies wide, which influences their heterogeneity. When interpreting prevalence data, it is therefore important to take into account the possible differences in screening methods. Epidemiology of plasmid encoded AmpC in the Netherlands Within the Netherlands, various studies have been conducted into the prevalence of plasmid encoded AmpC producing Enterobacterales (see Table 2). Most studies have focused on rectal or perineal carriage in humans. In general, the prevalence of AmpC producing Enterobacterales rectal carriage in the Netherlands is considered low. In the general population, the prevalence of plasmid AmpC producing E. coli varies between 0.2% and 1.3% (E. Ascelijn Reuland et al. 2015; Van Hoek et al. 2015; van den Bunt et al. 2017). In the two studies of carrier status in hospital patients within the Netherlands, the prevalence was not significantly higher (0.7% to 0.9%) (E. Den Drijver et al. 2018; X. Zhou et al. 2017). Few studies have looked at trends over time. A 2013-2016 study did not find a significant increase in plasmid levels AmpC producing E. coli (E. Den Drijver et al. 2018). Prevalence studies of plasmid AmpC-producing Enterobacterales in clinical isolates, for example from blood cultures, are scarcely performed in the Netherlands. Voets et al. stated that in a collection of isolates from urine cultures and blood cultures with 3rd generation cephalosporin resistance, plasmid AmpC was the cause of the resistance in 5% of E. coli and 4% of K. pneumoniae (Voets et al. 2013). More recent prevalence data of plasmidal AmpC-producing Enterobacterales in clinical isolates are absent in the Netherlands. Outside of the Netherlands, the prevalence of plasmid encoded AmpC producing Enterobacterales is significantly higher (Rodríguez-Guerrero et al. 2022). 2
28 Chapter 2 Table 2. Prevalence studies of AmpC producing Enterobacterales based on rectal carriage Author Year Study population Source Screened species Prevalence ( pAmpC / cAmpC ) Prevalence per pAmpC gene 1 Hoek et al. 2015 (21) 2011 General population (n=1033) Straighten out the rectum Enterobacterales E. coli pAmpC 0.39% P. agglomerans 0.10% blaCMY -2 0.29% blaDHA -1 0.10% blaCMY -48 0.10% 2 Reuland et al. 2015 (22) 2011 General population (n=550) Faeces Enterobacterales E. coli pAmpC 1.27% blaCMY -2 1.09% blaDHA -1 0.18% 3 Zhou et al., 2017 (24) 2012-2013 Hospital (n=445) rectal smear E. coli E. coli pAmpC 0.67% P. mirabilis pAmpC 0.22% blaCMY 0.67% blaDHA 0.22% 2012-2013 General population (n=400) rectal smear E. coli E. coli pAmpC 0.25% blaCMY 0.25% 4 Van den Bunt et al. 2016 (23) 2013-2015 General population (n=1004 children) faeces Enterobacterales E. coli pAmpC 0.40% K. pneumonia pAmpC 0.10% blaCMY -2 0.40% blaDHA -1 0.10%
29 AmpC beta-lactamases: epidemiology, infection control and treatment Table 2. Continued. Author Year Study population Source Screened species Prevalence ( pAmpC / cAmpC ) Prevalence per pAmpC gene General population (n=995 adults) faeces Enterobacterales E. coli pAmpC 0.20% K. pneumonia pAmpC 0.10% blaCMY -2 0.20% blaDHA -1 0.10% 5 Den Drijver et al. 2018 (25) 2013-2016 Hospital (n=2126) rectal smear E. coli, K. pneumoniae E. coli pAmpC 0.90% cAmpC 1.46% blaCMY 0.85% blaDHA 0.05 % cAmpC 1.46% 2
30 Chapter 2 Outbreaks with Plasmid AmpC Within the Netherlands outbreaks with plasmid encoded AmpC producing Enterobacterales have not yet been described. However, transmission can occur within hospitals which has been described in other countries as summarized below. One of the first reports of an outbreak was made by Papanicolaou et al. in 1990 of a blaMIR producing K. pneumoniae in Rhode Island, USA (n=11 patients) (Papanicolaou, Medeiros, and Jacoby 1990). A larger outbreak with a blaACC producing K. pneumoniae was later described in France, where a cluster of 57 patients was detected between 1999 and 2003 (Ohana et al. 2005). Clusters of both blaDHA and blaCMY-2 producing K. pneumoniae were identified at a liver transplant facility in Japan (Matsumura et al. 2015). Interestingly, eight of the blaDHA positive and one of the blaCMY-2 positive isolates showed carbapenem resistance, probably due to a combination of AmpC production and changes in membrane permeability. In 2013, Wendorf et al. reported a small outbreak (n=7) of E. coli carrying a blaCMY-2 variant gene in the United States. It concerned patients who had undergone endoscopic retrograde cholangiopancreatography, which suggested an association with contaminated endoscopes (Wendorf et al. 2015). AmpC and Infection Prevention Guidelines on infection prevention and plasmid AmpC-producing Enterobacterales are scarce. Currently, plasmid encoded AmpC is not yet specified in the WIP guidelines ‘High resistant micro-organisms in hospitals” and “‘High resistant micro-organisms in nursing homes, residential care centres and facilities for small-scale living for the elderly’’ (Werkgroep Infectiepreventie et al. 2018; 2014). However, given the similarity to ESBL-producing Enterobacterales, it is expected that comparable measures will be effective against the spread of AmpC producing Enterobacterales. A revision of this guideline is currently under review by the Partnership for Infection Prevention Guidelines. In the revised version, plasmid AmpC-producing Enterobacterales are not classified as highly resistant microorganisms in routine care settings. However, it is recommended to monitor nosocomial transmission, and in the event of an outbreak of plasmid AmpC-producing Enterobacterales, additional interventions are advised to prevent further spread (Severin, J.A. et al. 2023). Treatment recommendations for AmpC- producing Enterobacterales Treating infections of AmpC producing Enterobacterales can be complicated due to their resistance to many of the first-choice beta-lactam antibiotics. Both penicillins
31 AmpC beta-lactamases: epidemiology, infection control and treatment with beta-lactamase inhibitor combinations (such as amoxicillin-clavulanic acid) and third-generation cephalosporins are in most cases ineffective. Often it will be necessary to switch to a carbapenem or another group of antibiotics, such as quinolones. In some cases, the MIC values of third-generation cephalosporins are still below the resistance breakpoint in Enterobacterales with campC genes. Nonetheless, resistance to these agents can still occur due to derepression during treatment. Based on the study by Kohlmann et al, the risk of derepression appears to be different per species. Enterobacter cloacae complex isolates have a greater chance of developing resistance than Morganella morgagnii (Kohlmann, Bähr, and Gatermann 2018). That is why a distinction can be made per campC expressing species as to whether or not third-generation cephalosporins can be safely used when treating infections with AmpC producing Enterobacterales with low cephalosporin MIC values. A fourth-generation cephalosporin such as cefepime could be an alternative, but is currently used only to a limited extent in the Netherlands (Tamma et al. 2019). Beta-lactamase inhibitors such as tazobactam and avibactam may also be an alternative therapy. In the Merino-II study, no significant difference was seen in clinical outcomes between the treatment with carbapenems and piperacillintazobactam, although microbiological failure occurred significantly more often in the piperacillin-tazobactam group (Stewart et al. 2021). Clinical comparative studies have mainly been performed with therapeutics that need parenteral administration. Data on oral treatment with 3rd generation cephalosporins (e.g., ceftibuten) of infections with chromosomal AmpC-producing Enterobacterales are scarce, although a recent study of a new oral combination preparation (e.g. ceftibuten/VNRX-7145) shows effectiveness in urinary tract infections (Karlowsky, Hackel, and Sahm 2022). As this new combination drug is not yet available in the Netherlands, oral treatment with, for example, quinolones or trimethoprim-sulfamethoxazole will depend on the resistance pattern and (local) antibiotic guidelines. It is unknown if Enterobacterales containing pampC genes can be treated similarly, when measured MICs are below breakpoint level. Comparative clinical studies such as with chromosomal AmpC are lacking. The NVMM guideline “Laboratory detection of high resistant microorganisms (BRMO)” recommends blocking the result for the antibiotic in question, reporting it as resistant, warning of unclear therapeutic effect, or prescribing only in consultation with a clinical microbiologist or infectious disease specialist (J.A.J.W Kluytmans et al. 2021). Future studies on the optimal treatment of Enterobacterales containing different plasmids AmpC variants are needed. 2
32 Chapter 2 Conclusion The presence of plasmid AmpC in Enterobacterales may affect the patient’s treatment options. Although the prevalence of plasmid AmpC producing Enterobacterales in the Netherlands is lower than the prevalence of ESBL-producing Enterobacterales, it is important to be aware of this resistance mechanism. Since empirical treatment of infections in the Netherlands is currently based on the 2nd and 3rd generation cephalosporins, detection of AmpC- beta-lactamase producing Enterobacterales is critical, so that any increase in this resistance mechanism can be halted at an early stage. Attention to the detection of AmpC producing Enterobacterales and limiting the spread of this resistance mechanism can ensure that antibiotic resistance within the Netherlands remains limited in the future. Directions for practice • The presence of plasmid AmpC- producing Enterobacterales may affect a patient’s treatment due to related resistance to commonly used beta-lactam antibiotics. • Diagnostics for the presence of plasmid AmpC deviates from the diagnosis for the presence of ESBL in Enterobacterales, but can be confirmed with specific phenotypic and genotypic tests in most laboratories • Although plasmid AmpC- producing Enterobacterales are currently still limited in the Netherlands, attention to prevalence and transmission is desirable to prevent further spread
Chapter 3 Decline in AmpC beta-lactamase-producing Escherichia coli in a Dutch teaching hospital (2013-2016) Evert den Drijver, Jaco J. Verweij, Carlo Verhulst, Stijn Oome, Joke Soer, Ina Willemsen, Eefje J. A. Schrauwen, Marjolein F. Q. Kluytmans—van den Bergh, Jan A. J. W. Kluytmans Adapted from: PLoS ONE. 2018;13 (10): https://doi.org/10.1371/journal.pone.0204864
34 Chapter 3 Abstract Objective The objective of this study is to determine the prevalence of rectal carriage of plasmid- and chromosome-encoded AmpC beta-lactamase-producing Escherichia coli and Klebsiella spp. in patients in a Dutch teaching hospital between 2013 and 2016. Methods Between 2013 and 2016, hospital-wide yearly prevalence surveys were performed to determine the prevalence of AmpC beta-lactamase-producing E. coli and Klebsiella spp. rectal carriage. Rectal swabs were taken and cultured using an enrichment broth and selective agar plates. All E. coli and Klebsiella spp. isolates were screened for production of AmpC beta-lactamase using phenotypic confirmation tests and for the presence of plasmid-encoded AmpC (pAmpC) genes. E. coli isolates were screened for chromosome-encoded AmpC (cAmpC) promoter/attenuator alterations. Results Fifty (2.4%) of 2,126 evaluable patients were identified as rectal carrier of AmpC betalactamase-producing E. coli. No carriage of AmpC beta-lactamase producing Klebsiella spp. was found. Nineteen (0.9%) patients harboured isolates with pAmpC genes and 30 (1,4%) patients harboured isolates with cAmpC promoter/attenuator alterations associated with AmpC beta-lactamase overproduction. For one isolate, no pAmpC genes or cAmpC promotor/attenuator alterations could be identified. During the study period, a statistically significant decline in the prevalence of rectal carriage with E. coli with cAmpC promotor/attenuator alterations was found (p = 0.012). The prevalence of pAmpC remained stable over the years. Conclusions The prevalence of rectal carriage of AmpC-producing E. coli and Klebsiella spp. in patients in Dutch hospitals is low and a declining trend was observed for E. coli with cAmpC promotor/attenuator alterations.
35 Decline in AmpC beta-lactamase-producing E. coli in a Dutch teaching hospital Introduction Antibiotic resistance caused by broad-spectrum beta-lactamase production in Gramnegative bacteria is a well-known problem in clinical settings and in the community. Extended-spectrum beta-lactamases (ESBL) in Enterobacteriaceae are generally accepted as a major cause of beta-lactam resistance (David M. Livermore et al. 2007; Jan A. J. W. Kluytmans et al. 2013; Willemsen et al. 2015). Willemsen et al studied the epidemiology of ESBL rectal carriage between 2010 and 2014 in the same teaching hospital. Although the annual prevalence of ESBL was stable, a decline was seen in the proportion of certain ESBL groups, mainly CTX-M-1-1. In addition to ESBL, AmpC beta-lactamases are increasingly recognized as a growing and clinically relevant problem (Jacoby 2009; Rodríguez-Baño et al. 2012; Park et al. 2013; Vanesa Pascual et al. 2015; V Pascual et al. 2016). Most studies have focused on the dissemination of mobile genetic elements encoding these beta-lactamases (Rodríguez-Baño et al. 2012; Park et al. 2013; V Pascual et al. 2016). However, in certain species (e.g. Escherichia coli and Shigella spp.), AmpC beta-lactamase production is not only plasmid-encoded, but can also be caused by chromosomal hyperproduction due to mutations within the promoter/attenuator region (Jacoby 2009; Jørgensen et al. 2010; Alonso et al. 2016). However, little is known on the carriage of either plasmid-encoded (pAmpC) or chromosome-encoded AmpC (cAmpC) Enterobacteriales in hospitalised patients in the North-Western European region and no studies have been performed over a multiple year period. Moreover, screening methods for ESBL, such as ESBL selective media, may not always be optimal to screen for AmpC-producing Enterobacteriales. The present study describes the prevalence of rectal carriage with AmpC beta-lactamase-producing E. coli and Klebsiella spp. in patients in Dutch hospitals during a four-year period. Materials and methods Sample collection and phenotypical AmpC testing Four yearly point prevalence surveys (PPS) were performed in the Amphia Hospital from 2013 to 2016 in the months October or November. All hospitalised patients, including patients on dialysis and day-care, were screened for AmpC carriage using rectal swabs (Eswab, Copan, Italy). After vortexing, the swab was plated on Blood Agar plate (growth control, performed since 2011) and the liquid Amies eluent was inoculated in selective tryptic soy broth, containing cefotaxime (0.25 mg/L) and vancomycin (8 mg/L) (TSB3
36 Chapter 3 VC) and incubated for 18–24 hours (35–37°C). In 2013, broths were subcultured on a MacConkey agar containing cefotaxime 1 mg/L (Mediaproducts, Groningen, The Netherlands). In 2014, a switch to a more selective double MacConkey agar plate (containing on one side cefotaxime 1 mg/L, cefoxitin 8 mg/L and on the other side ceftazidime 1 mg/L, cefoxitin 8 mg/L, Mediaproducts, Groningen, the Netherlands) was made to improve sensitivity and specificity of the screening. Broths were simultaneously subcultured on both sides of an EbSA agar plate (AlphaOmega, ‘s-Gravenhage, Netherlands). The Extended Beta-Lactamase Screening Agar (EbSA) plate consists of a split MacConkey agar plate containing ceftazidime (1.0 mg/L) on one side and cefotaxime (1.0 mg/L) on the other side. Both sides contain cloxacillin (400 mg/L) and vancomycin (64 mg/L) for inhibition of AmpC beta-lactamase-producing bacteria and Gram-positive bacteria, respectively. Subsequently, the plates were incubated for 18–24 hours (35–37°C). AmpC producing isolates found in 2013, were retrospectively cultured on the new selective AmpC agar to confirm if they would have been detected using the new agar plate. For all oxidase-negative isolates that grew on either side of the selective agar plates, species identification was performed by MALDI-TOF (bioMérieux, Marcy l’Etoile, France). The presence of AmpC in all E. coli and Klebsiella spp. isolates was phenotypically confirmed using the D68C AmpC & ESBL Detection Set (Mastdiscs, Mastgroup Ltd, Bootle, United Kingdom) and interpreted according to manufacturer’s instructions (Ingram et al. 2011; Nourrisson et al. 2015). The presence of ESBL in isolates with a MIC of > 1 mg/L for ceftazidime and/or cefotaxime was phenotypically confirmed with the combination disk diffusion method for cefotaxime, ceftazidime, and cefepime with and without clavulanic acid (Rosco, Taastrup, Denmark)) and interpreted according to manufacturer’s instructions. Minimal inhibitory concentration (MIC) values for cefotaxime (CTX), ceftazidime (CAZ) and cefoxitin (FOX) were measured using the gradient on a strip method (E-test, bioMérieux, Marcy l’Etoile, France). Genetic confirmation of phenotypically confirmed isolates All phenotypically confirmed E. coli and Klebsiella spp. isolates were screened for the presence of pampC genes using the microarray check MDR CT103 according to the manufacturer’s instructions (Check-Points, Wageningen, the Netherlands) (Cuzon et al. 2012). In addition, all phenotypically confirmed E. coli isolates were subjected to Sanger sequencing of the promoter/attenuator region of the cAmpC gene using M-13 tailed primers as described by Corvec et al. (Stephane Corvec et al. 2002). The obtained sequences of each isolate were assembled and aligned against the promoter/attenuator
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