Systematic Review of Plasmid AmpC Type Resistances in Escherichia coli and Klebsiella pneumoniae and Preliminary Proposal of a Simplified Screening Method for ampC
Abstract
Beta-lactamase (BL) production is a major public health problem. Although not the most frequent AmpC type, AmpC-BL is increasingly isolated, especially plasmid AmpC-BL (pAmpCBL). The objective of this study was to review information published to date on pAmpC-BL in Escherichia coli and Klebsiella pneumoniae, and on the epidemiology and detection methods used by clinical microbiology laboratories, by performing a systematic review using the MEDLINE PubMed database. The predictive capacity of a screening method to detect AmpC-BL using disks with cloxacillin (CLX) was also evaluated by studying 102 Enterobacteriaceae clinical isolates grown in CHROMID ESBL medium with the addition of cefepime (FEP), cefoxitin (FOX), ertapenem (ETP), CLX, and oxacillin with CLX. The review, which included 149 publications, suggests that certain risk factors (prolonged hospitalization and previous use of cephalosporins) are associated with infections by pAmpC-BL-producing microorganisms. The worldwide prevalence has increased over the past 10 years, with a positivity rate ranging between 0.1 and 40%, although AmpC was only detected when sought in a targeted manner. CMY-2 type has been the most prevalent pAmpC-BLproducing microorganism. The most frequently used phenotypic method has been the double-disk synergy test (using CLX disks or phenyl-boronic acid and cefotaxime [CTX] and ceftazidime) and the disk method combined with these inhibitors. In regard to screening methods, a 1- g oxacillin disk with CLX showed 88.9% sensitivity, 100% specificity, 100% positive predictive value (PPV), 98.9% negative predictive value (NPV), and 98.9% validity index (VI). This predictive capacity is reduced with the addition of extended-spectrum beta-lactamases, showing 62.5% sensitivity, 100% specificity, 100% PPV, 93.5% NPV, and 94.1% VI. In conclusion, there has been a worldwide increase in the number of isolates with pAmpC-BL, especially in Asia, with CMY-2 being the most frequently detected pAmpC-BL-producing type of microorganism. Reduction in its spread requires routine screening with a combination of phenotypic methods (with AmpC inhibitors) and genotypic methods (multiplex PCR). In conclusion, the proposed screening technique is an easy-to-apply and inexpensive test for the detection of AmpC-producing isolates in the routine screening of multidrugresistant microorganisms.
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Citation: Rodríguez-Guerrero, E.; Callejas-Rodelas, J.C.; Navarro-Marí, J.M.; Gutiérrez-Fernández, J. Systematic Review of Plasmid AmpC Type Resistances in Escherichia coli and Klebsiella pneumoniae and Preliminary Proposal of a Simplified Screening Method for ampC. Microorganisms 2022,10, 611. https://doi.org/10.3390/ microorganisms10030611 Academic Editor: Mariagrazia Perilli Received: 14 February 2022 Accepted: 8 March 2022 Published: 14 March 2022 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). microorganisms Article Systematic Review of Plasmid AmpC Type Resistances in Escherichia coli and Klebsiella pneumoniae and Preliminary Proposal of a Simplified Screening Method for ampC Enrique Rodríguez-Guerrero 1, Juan Carlos Callejas-Rodelas 2, JoséMaría Navarro-Marí1 and JoséGutiérrez-Fernández 1,2,* 1Laboratory of Microbiology, Virgen de las Nieves University Hospital & ibs.Granada—Instituto de Investigación Biosanitaria de Granada, Avda. de las Fuerzas Armadas 2, 18014 Granada, Spain; [email protected] (E.R.-G.); josem.navarr[email protected] (J.M.N.-M.) 2Department of Microbiology, School of Medicine, University of Granada & ibs.Granada—Instituto de Investigación Biosanitaria de Granada, Avenida de la Investigación 11, 18016 Granada, Spain; [email protected] *Correspondence: [email protected] Abstract: Beta-lactamase (BL) production is a major public health problem. Although not the most frequent AmpC type, AmpC-BL is increasingly isolated, especially plasmid AmpC-BL (pAmpCBL). The objective of this study was to review information published to date on pAmpC-BL in Escherichia coli and Klebsiella pneumoniae, and on the epidemiology and detection methods used by clinical microbiology laboratories, by performing a systematic review using the MEDLINE PubMed database. The predictive capacity of a screening method to detect AmpC-BL using disks with cloxacillin (CLX) was also evaluated by studying 102 Enterobacteriaceae clinical isolates grown in CHROMID ESBL medium with the addition of cefepime (FEP), cefoxitin (FOX), ertapenem (ETP), CLX, and oxacillin with CLX. The review, which included 149 publications, suggests that certain risk factors (prolonged hospitalization and previous use of cephalosporins) are associated with infections by pAmpC-BL-producing microorganisms. The worldwide prevalence has increased over the past 10 years, with a positivity rate ranging between 0.1 and 40%, although AmpC was only detected when sought in a targeted manner. CMY-2 type has been the most prevalent pAmpC-BLproducing microorganism. The most frequently used phenotypic method has been the double-disk synergy test (using CLX disks or phenyl-boronic acid and cefotaxime [CTX] and ceftazidime) and the disk method combined with these inhibitors. In regard to screening methods, a 1µ g oxacillin disk with CLX showed 88.9% sensitivity, 100% specificity, 100% positive predictive value (PPV), 98.9% negative predictive value (NPV), and 98.9% validity index (VI). This predictive capacity is reduced with the addition of extended-spectrum beta-lactamases, showing 62.5% sensitivity, 100% specificity, 100% PPV, 93.5% NPV, and 94.1% VI. In conclusion, there has been a worldwide increase in the number of isolates with pAmpC-BL, especially in Asia, with CMY-2 being the most frequently detected pAmpC-BL-producing type of microorganism. Reduction in its spread requires routine screening with a combination of phenotypic methods (with AmpC inhibitors) and genotypic methods (multiplex PCR). In conclusion, the proposed screening technique is an easy-to-apply and inexpensive test for the detection of AmpC-producing isolates in the routine screening of multidrugresistant microorganisms. Keywords: plasmidic AmpC betalactamase; Enterobacteriaceae; multi-resistant bacteria diagnosis; screening 1. Introduction The loss of susceptibility to beta-lactam antibiotics in Gram-negative bacteria is an emerging problem worldwide and is mainly attributable to the production of beta-lactamases, especially extended-spectrum (ESBL) and AmpC type (AmpC-BL) beta-lactamases and carbapenMicroorganisms 2022,10, 611. https://doi.org/10.3390/microorganisms10030611 https://www.mdpi.com/journal/microorganisms
Microorganisms 2022,10, 611 2 of 24 emases [ 1 ]. The CESPM group (Citrobacter freundii, Klebsiella aerogenes,Enterobacter cloacae , Serratia marcescens ,Providencia stuartii, and Morganella morganii) comprises common Enterobacteriaceae species responsible for nosocomial and community infections produced by inducible chromosomal AmpC beta-lactamases. However, other more prevalent species in patients, such as Escherichia coli and Klebsiella pneumoniae, can present these enzymes with plasmid gene encoding (pAmpC-BL). There has been limited research on pAmpC-BL in comparison to the more frequently detected ESBLand carbapenemase-producing microorganisms [ 1 – 3 ]. There has been no estimate of the true worldwide prevalence of pAmpC-BLproducing microorganisms [ 4 ], and no consensus on the most effective laboratory technique for their detection [ 5 ]. AmpC-BL-producing microorganisms can also be associated with other types of resistance, highlighting the coexistence of AmpC-BL and ESBL [3]. Colonization of the intestine and larynx may serve as an important reservoir for resistance genes [ 6 , 7 ] of the microorganisms that inhabit them, and can be a risk factor for infection. There is a need for simple, effective, easy-to-apply, and inexpensive techniques to screen for these pathogens in the digestive tract of infected or colonized patients. ESBLproducing Enterobacteriaceae colonies can be detected by various techniques, including the use of CHROMID ESBL (bioMérieux, France). This transparent medium contains cefpodoxime and other substances that inhibit Gram-positive bacteria growth, and chromogenic substrates that presumptively identify genera and species according to their color (pink/burgundy for E. coli; blue/green for Klebsiella,Enterobacter,Serratia, or Citrobacter; and light to dark brown for Proteae) [ 8 ]. The inclusion of cefoxitin (FOX), cefepime (FEP), and ertapenem (ETP) disks on CHROMID ESBL medium has been proposed for the presumptive identification of ESBLand/or carbapenemase-producing microorganisms through their resistance to these antibiotics, and a halo diameter breakpoint of 16 mm has proven diagnostically useful [ 9 ]. However, the diagnostic performance can be further improved by the addition of other antibiotic disks to reveal the possible presence of AmpC-BL. The production of carbapenemase, ESBL, and AmpC-BL is frequently studied in episodes of colonization by multi-resistant Gram-negative bacteria, and the addition of ETP, FOX, FEP, and cloxacillin (CLX) disks to this medium may offer a simple and effective method for this purpose. CLX shows greater activity against AmpC-BL-producing Enterobacterales but lesser activity against carbapenemaseor ESBL-producing Enterobacterales or carbapenemase-producing Pseudomonas spp. and A. baumannii; hence, CLX disks may be useful to detect the presence of microorganisms with AmpC [10]. Tests using cultures for the detection of colonies of resistant microorganisms offer an advantage over PCR tests because they detect viable microorganisms and facilitate their recovery, avoiding their loss. Unfortunately, no commercial culture tests are available for the simultaneous detection of Gram-negative microorganisms with different mechanisms of resistance to β -lactam antibiotics. The objectives of the present study were: carry out a systematic review of epidemiological information on pAmpC-BL in K. pneumoniae and E. coli species; and evaluate the behavior of microorganisms with AmpC-BL in the ChromID ® ESBL medium by using the disk diffusion test with FOX, FEP, ETP, and CLX disks. 2. Material and Methods 2.1. Systematic Review The PubMed ® database was searched using the search term “(AmpC [Title/Abstract]) AND (Plasmid [Title/Abstract])”. Review inclusion criteria were: (i) study on the conceptualization of AmpC type resistances; (ii) study on the epidemiology and clinical relevance of these resistances from 2010 onwards; and (iii) study on their detection. Exclusion criteria were: (i) study of isolation of bacterial species other than K. pneumoniae or E. coli; (ii) study of their isolation in food production chains, animals, farms, aqueous media, and other environmental settings; (iii) language other than English or Spanish; and iv) inability to access the text. The search yielded 1001 publications published up to 7 January 2021; 395 of these met the eligibility criteria, and 149 were finally included in the review.
Microorganisms 2022,10, 611 3 of 24 2.2. Behavior of Enterobacteriaceae with AmpC-BL in ChromID®ESBL Medium, Using the Disk Diffusion Test with CLX Disks A retrospective study was conducted in the Microbiology Department of our hospital in Granada (Spain), which covers a population of around 440,000 inhabitants. It included all Enterobacterales strains (67 from rectal swabs and 35 from urine cultures) detected during February 2021 with suspicion of colonization or urinary tract infection by ESBL or carbapenemase microorganisms in individuals aged >14 years). Species were grouped according to their resistance mechanisms, as defined by EUCAST 2021 criteria [ 11 ], finding: 47 with ESBL (25 E. coli, 19 K. pneumoniae, 1 C. freundii, 1 Proteus mirabilis , and 1 Klebsiella oxytoca); 39 with the carbapenemases oxacillinase (OXA) (16 K. pneumoniae, 11 E. cloacae , and 1 E. coli), Klebsiella pneumoniae carbapenemase (KPC) (6 K. pneumoniae), and Verona integron-encoded metallo-beta-lactamase (VIM) (5 K. pneumoniae); 9 with AmpC (5 M. morganii, 2Kluyvera intermedia , 1 E. cloacae, and 1 K. aerogenes); and 7 with ESBL and AmpC (6 E. cloacae and 1 K. pneumoniae). Isolates were identified using the MicroScan system (Beckman Coulter, Brea, CA, USA) and mass spectrometry (Maldi-Tof ® , Bruker Daltonik GmbH, Bremen, Germany). Resistances were characterized with the MicroScan microdilution system, followed, when appropriate, by carbapenemase determination using the Rapidec ® Carba NP colorimetric test (BioMerieux, Marcy l’Etoile, France) and immunochromatography (NG5-Test Carba, NG Biotech, Guipry, France). The carbapenemase-producing type was confirmed by the Andalusian Molecular Typing Laboratory of the Spanish PIRASOA Program using mass sequencing (Illumina Inc, San Diego, CA, USA), CLC Genomics Workbench v10 software (Qiagen), ResFinder (Lyngby, Denmark) (https://cge.cbs.dtu.dk/services/ResFinder, (accessed on 30 November 2021)), and CARD (Hamilton, ON, Canada) (https://card.mcmaster.ca/, (accessed on 30 November 2021)) databases. ESBL production was defined by resistance to cefotaxime (CTX) and/or ceftazidime (CAZ) and synergy with clavulanic acid (CLAV) and FEP, and by susceptibility to amoxicillin/CLAV, piperacillin/tazobactam, FOX, and carbapenems. AmpC production was defined by synergy with cloxacillin [ 3 ] (with gradient test, cefotetan/cefotetan-CLX E-Test (CTT/CXT), Liofilchem ® —MIC Test Strip Technical Sheet AmpC), resistance to FOX, amoxicillin/clavulanic acid/CLAV, piperacillin/tazobactam, and CTX and/or CAZ, with an increased minimal inhibitory concentration (MIC) in the presence of CLAV and susceptibility to FEP and carbapenems. A 0.5 McFarland suspension of each isolate was prepared from colonies grown on lamb blood agar (Becton Dickinson, Franklin Lakes, NJ, USA). Next, a sterilized swab was soaked with the homogenized suspension, excess liquid was removed, and it was uniformly seeded on one half of the plate on CHROMID ESBL medium, streaking the bacterial load on the other half with a calibrated inoculation loop. FEP (30 µ g, Becton Dickinson), FOX (30 µ g, Becton Dickinson), ETP (10 µ g, Becton Dickinson), and CLX (in two variants [test 1 and test 2] to detect AmpC) disks were then placed equidistantly on the seeded area for growth/inhibition measurement with a separation of 1.5 cm between each. The medium was then incubated at 37 ◦ C, with readings at 24 h. Test 1 used a sterile paper disk (BBL TM TAXO TM Blank Paper Disks, Becton Dickinson) with a diameter of 13 mm located at the center of the plate with the addition of 20 µ L CLX (50 mg/mL) (Sigma-Aldrich, Madrid, Spain); and Test 2 used a disk with 1 µ g oxacillin (Becton-Dickinson) with addition of 10 µ L CLX (50 mg/mL). IBM SPSS Statistics 19 and Microsoft Excel 2019 were used for statistical analyses. Calculations were made of the diagnostic value of the presence of synergy when applying Tests 1 and 2. 3. Results 3.1. Systematic Review 3.1.1. Worldwide AmpC-BL Epidemiology Three AmpC-BL categories have been described: chromosomal type AmpC-BL with inducible expression; chromosomal type AmpC-BL with stable derepression and noninducible expression (enzyme hyperproduction by mutations in AmpC regulating genes);
Microorganisms 2022,10, 611 4 of 24 and plasmid type AmpC-BL (encoded by genes in transfer plasmids) (12). The former enzymes are expressed constitutively and at low concentrations in Citrobacter spp., Enterobacter spp. ,Serratia spp., Morganella spp., and Providencia spp. (CESPM group), and in Pseudomonas aeruginosa [ 5 , 12 , 13 ]. However, exposure of these bacterial species to certain beta-lactams can lead to hyperexpression of the encoding gene and elevated production of cAmpC-BL, with the expression of AmpC-BL being inducible. This is attributed to mutations that affect the enzyme responsible for regulating the AmpC-BL gene [ 3 ]. Its constitutive expression at low concentrations has also been documented in other bacteria, such as E. coli, and in Shigella spp., but it is not inducible in these cases because the chromosomal genes of the enzyme lack the natural promoter (ampR). Nonetheless, Pfeifer et al. (2010) described cases of resistance to cephalosporins in E. coli mediated by the inducible expression of cAmpC-BL, caused by mutations that increased expression of the enzyme [ 14 ]. The rise in plasmid type AmpC-BL over the past few years has been described as an epidemic by some authors [ 12 ]. In 1989, it was discovered that ampC genes may be transmittable by plasmids [ 4 , 15 ], after the finding in South Korea of an isolate highly resistant to FOX, designated plasmid CMY-1 [ 15 ]. pAmpC-BLs have traditionally been described in Enterobacteriaceae and other Gram-negative bacilli [ 3 , 5 ] (Table 1). The various plasmid AmpC families have been grouped as follows: CIT group of C. freundii (including LATand some CMY-, such as CMY-2 and BIL); EBC group of Enterobacter spp. (MIR-1, ACT-1); DHA group of M. morganii (DHA-1, DHA-2); ACC group of H. alvei (highlighting ACC-1); MOX group of Aeromonas spp. (MOXand the rest of CMY); and FOX group, observing a very close genetic relationship between these plasmids and their chromosomal origins [4,16]. Table 1. Isolation of the first pAmpC-BLs (modified by Jacoby, 2009) [3]. pAMPC-BL Enzyme Country of Discovery Year of Isolation First Species in Which It Was Isolated Chromosomal Origin Species % Similarity (with Respect to the Chromosomal Gene) CMY-1 South Korea 1989 K. pneumoniae A. hydrophila 82 CMY-2 Greece 1996 K. pneumoniae C. freundii 96 MIR-1 USA 1990 K. pneumoniae E. cloacae 99 MOX-1 Japan 1993 K. pneumoniae A. hydrophila 80 LAT-1 Greece 1993 K. pneumoniae C. freundii 95 FOX-1 Argentina 1994 K. pneumoniae A. caviae 99 DHA-1 Saudi Arabia 1997 S. enteriditis M. morganii 99 ACT-1 USA 1997 K. pneumoniae E. asburiae 98 ACC-1 Germany 1999 K. pneumoniae H. alvei 99 CFE-1 Japan 2004 E. coli C. freundii 99 pAmpC-BL: plasmid AmpC beta-lactamases. Species that can express pAmpC-BL include K. pneumoniae,Salmonella,E. coli, P. mirabilis , and C. freundii, in which these genes are constitutively expressed at high concentrations [ 15 ]. All pAmpC-BLs are expressed constitutively, except for DHA-1, ACT-1, DHA-2, and CMY-13 enzymes. These have been described as inducible because the plasmids that contain them include not only the encoding ampC genes for the enzyme but also ligated ampR genes. These genes are transcription factors responsible for decreasing or increasing the expression of inducible ampC genes depending on the cofactor that interacts with AmpR [ 3 , 5 , 13 , 15 , 17 , 18 ]. Since the discovery of this type of plasmid, it has been repeatedly observed that the most frequently recorded ampC gene worldwide is CMY-2 [7,14,17–20], followed by DHA-1 [18]. Enzymes in the CIT group (CMY-like) are predominant in E. coli, while enzymes of the DHA family predominate in Klebsiella spp. [19–23].
Microorganisms 2022,10, 611 5 of 24 As in the case of Gram-negative bacteria families, AmpC-BL-producing microorganisms produce various types of infection, both nosocomial and community-acquired: urinary tract infections (UTIs, E. coli being the most frequently isolated pathogen in this type of infection), intra-abdominal infections, pneumonias, and soft tissue infections, among others [ 23 ]. The literature describes various risk factors associated with infections by pAmpC-BL-carrier microorganisms, observing that they do not differ from those described for ESBLs [ 24 ], associating AmpC-BL acquisition with previous hospitalization even more than ESBL acquisition [25]. Independent risk factors have been reported for infection by pAmpC-BL-producing microorganisms, including the previous receipt of fluoroquinolones [ 2 , 26 , 27 ] and cephalosporins such as cephamycin and FOX [ 2 , 3 , 26 – 28 ], demonstrating the possible therapeutic failure of using cephalosporins as empirical treatment (30) even being considered as an independent risk factor [ 2 , 25 ]. In addition, not all AmpC plasmid families are associated with the same mortality rate; thus, according to the report of Pai et al. (2004), infections produced by AmpC-BL DHA-1 have a higher mortality rate than those produced by CMY-2 [ 29 ]. Other risk factors are prolonged hospitalization [ 2 – 4 , 28 , 30 ] or patient institutionalization (in the study by Rodríguez-Baño et al. (2015)) [ 25 ], more than 50% of infections were associated with community outbreaks, especially in patients with associated health care), hospitalization in intensive care units (ICUs), and use of central and urinary catheters [ 3 , 25 , 28 ], mainly in the case of nosocomial infections. Age, presence of diabetes mellitus, hospital admission, institutionalization in care homes, and the use of urinary catheters were associated with community-acquired infections [2]. The worldwide epidemiology of pAmpC-BLs was evaluated by classifying studies according to their target population in the following three groups: hospital (isolates of patients admitted to hospital centers, including ICUs and health institutions), community (isolates from community sampling, studies performed in primary care, infections acquired in the community, and non-hospitalized patients), and hospital and community. The prevalence of AmpC in the reviewed literature was evaluated by considering the total number of isolates in each study. Tables 2–5display data were obtained from Europe, America, Africa, and countries in Asia, Oceania, and the Middle East, respectively. PRESENCE IN EUROPE (Table 2). Major regional differences can be observed in the percentage of pAmpC-BL positivity. It has remained relatively low in Europe over the past 10 years, ranging from 0.06% Denmark (2010) [ 31 ] to 2.6% in Holland (2014) [30,32] . However, higher percentages of positivity have occasionally been observed, such as Holland in 2012 with 5% [ 33 ], Spain in 2018 with 14.2% [ 34 ], and Germany in 2020 with 11.9% positivity. These higher positivity rates may result from differences in screening method or study population (36). For instance, resistance of clinical isolates to carbapenems was used in the 2018 Spanish study [ 34 ] while the 2012 study in Holland evaluated isolates with reduced susceptibility to FOX [ 33 ]. pAmpC-BL positivity in E. coli is generally less frequent in Europe than that observed in other parts of the world, with percentages of 0.06% reported in Denmark (2010) [ 31 ], 0.46% in France (2010) [ 35 ], 0.73% in Holland and Germany (2017) [ 36 ], 1.28% in Portugal (2019) [ 37 ], and 2.4% in Holland (2018) [ 38 ]. The prevalence was slightly higher (7.55%) in the study by Findlay et al. (2020) in England, because all of the clinical isolates evaluated showed CTX resistance, used as a screening method [ 39 ]. An elevated prevalence of pAmpC-BL was also obtained in Ireland (19%) because the isolates had a AmpC phenotype (positivity in the phenotypic detection procedure) [ 40 ]. In the same way, the elevated percentage (12.5%) described in Switzerland (2013) [ 41 ] was obtained in isolates with resistance to third-generation cephalosporins. Reports on the positivity of K. pneumoniae isolates have varied among European countries, with findings of 0.5% in France (2012) [42], 0.47% in Holland (2012) [33], and 1.04% in Portugal (2019) [37]. PRESENCE IN AMERICA (Table 3). Our search of the literature retrieved few publications on the prevalence of pAmpC-BL in America. In general, the global prevalence of pAmpC-BL has been relatively low in the USA, ranging between 1.3% in 2016 [ 32 ] and 3.42% in 2019 [ 52 ]. Reports on the prevalence of pAmpC-BL in E. coli have ranged
Microorganisms 2022,10, 611 6 of 24 widely between 2.23%, as described by Tamma et al. (2019) [ 52 ], and 16.33%, reported by Park et al. (2012), who studied the presence of pAmpC-BL in FOX-resistant isolates [ 53 ]. In Mexico, Paniagua-Contreras et al. (2018) found a higher prevalence (23.7%) of AmpC-BL among E. coli isolates [54]. PRESENCE IN AFRICA (Table 4). Reports on the prevalence of pAmpC-BL have varied widely among African countries. E. coli percentages have ranged from 0.50 in Tanzania (2016) [ 56 ] and 0.59% in Morocco (2013) [ 57 ] to 14.68% in Egypt (2014) [ 58 ], with reports of 4.23% in Nigeria (2014) [ 59 ] and 10.86% in Mozambique (2021) [ 60 ]. Likewise, the percentages of K. pneumoniae isolates have ranged between 0.88 in Morocco (2013) [ 57 ] and 3.97% in Libya (2017) [ 61 ]. Overall, the highest prevalence rates of pAmpC-BL have been described in Uganda (2014) [ 60 ], with a rate of 39.6% among FOX-resistant isolates; in Egypt (2014) with 18.8% [58]; and in Nigeria (2014) with 11.23% [59]. PRESENCE IN ASIA, OCEANIA, AND THE MIDDLE EAST (Table 5). These regions have reported the highest prevalence rates of pAmpC-BL isolation. The rate of K. pneumoniae ranges from 0.01% in Japan (2010) [ 21 ] to a very high rate of 44.95% in a Hong Kong study (2016) [ 71 ]. Elevated rates have also been described in India (2010 and 2012) [ 72 , 73 ] with 13.14% and 13.27% positivity, respectively; Pakistan (2013) [ 74 ], with 12.37% positivity among isolates with ESBL phenotype; and China (2015) [ 75 ], with 31.50% positivity among multi-drug resistant K. pneumoniae isolates. Positivity rates in E. coli have ranged from 0.07% in Japan (2010) [ 21 ] to India (2010 and 2012) [ 72 , 73 ] with 24.57% and 24.89% positivity, respectively. 3.1.2. Phenotypic Detection Methods Since the discovery of AmpC type resistances several decades ago, the approach to their detection has been controversial, attributable to the lack of clear guidelines from CLSI or EUCAST. This has led to an underdiagnosis of AmpC-BLs, contributing to underestimation of the prevalence and global spread of this type of resistance. In 2018, Conejo et al. (2018) called for an improvement in the phenotypic detection of AmpC-BLs in Spanish clinical laboratories [ 97 ]. The ability of a laboratory to detect both types of AmpC resistance is essential, and the detection of pAmpC-BLs is of vital epidemiological importance, given their transmission and dissemination capacity and their association with outbreaks of community and nosocomial infections [ 17 , 98 ]. AmpC-BL detection is especially difficult in microorganisms that can produce chromosomal and plasmid AmpC-BL (e.g., E. coli). In these cases, the presence of a plasmid needs to be investigated to monitor their spread more closely [ 99 ] and address the clinical, therapeutic, epidemiological, and organizational repercussions, including the isolation of infected patients. For the laboratory detection of these resistances, the presence of pAmpC-BLs should be surveilled in species without chromosomal AmpCs that have proven able to disseminate these enzymes, mainly K. pneumonia [ 100 ]. The behavior of antibiotics against pAmpCBL-producing microorganisms is characterized by a decreased susceptibility to oxyiminocephalosporins (e.g., CTX or CAZ) and methoxy-cephalosporins (e.g., FOX), and a susceptibility to fourth-generation cephalosporins (e.g., FEP) [ 17 , 101 ]. Therefore, isolates showing some of these characteristics in the antibiogram should be suspected of pAmpC-BL production when there is no other apparent cause. However, there have been multiple reports of AmpCproducing bacteria that appeared susceptible to both oxyimino-cephalosporins and FOX in the antibiogram [18,19,24,97,99,102–106]. This may be attributable to a so-called “inoculum effect” (susceptibility in vitro at low microorganism concentrations but inefficacy in vivo) [24,106] or to the conventional consideration of these isolates as susceptible to cephalosporins in vitro according to now-outdated CLSI or EUCAST cutoff points [ 103 , 106 ]. It should be borne in mind that pAmpC-BL-producing isolates occasionally present with more than one beta-lactamase and are multidrug resistant [ 4 , 75 , 94 , 101 ]. The presence of AmpC can mask the coexistence of ESBL, hampering differentiation of the two resistances [ 22 , 79 , 99 , 105 , 107 , 108 ], because the two enzyme groups are hydrolytically very similar, except that AmpC-BL is not inhibited by CLAV, with reports of an increased cephalosporin MIC in its presence [107].
Microorganisms 2022,10, 611 7 of 24 Table 2. Epidemiology of pAmpC-BLs in Europe (2010–2020). Author (Reference) Year of Population Study Year of Publication Country of Target Population Population (H/C) aSpecific Conditions nAmpC (%) b Global cGenetic Identification Most Frequent AmpC Enzymes E. coli K.pneumoniae Jørgensen et al. [31] 2006 2010 Denmark H ECI 74 0.06 - - PCR/WGS CMY-2 Courpon-Claudinon et al. [35]2005 2010 France H 3GCR 1051 0.46 - - PCR/WGS CMY-2 Illiaquer et al. [42] 2007–2009 2012 France H KPI 1505 - 0.50 - PCR/WGS DHA-1 Voets et al. [33] 2009 2012 Holland C ESBL 636 3.93 0.47 5.03 PCR/WGS CMY-2 Miróet al. [43] 2009 2013 Spain H EI 100,132 0.69 1.02 0.64 PCR/WGS CMY-2 Seiffert et al. [41] 2011 2013 Switzerland H/C ECI 611 12.50 - - PCR/WGS CMY-2 Gude et al. [44] 2008–2010 2013 Spain H EI - - - 0.56 PCR/WGS CMY-2 Galán-Sánchez et al. [45]2011–2012 2014 Spain H/C ECI - 0.78 - - PCR/WGS CMY-2 Reuland et al. [30] 2007 2014 Holland H 3GCR 503 - - 2.60 PCR CMY-2 Jones-Dias et al. [46] 2004–2008 2014 Portugal H 3GCR 124 - - 0.80 PCR/WGS CMY-2 Reuland et al. [47] 2011 2015 Holland C EI 550 1.30 - - PCR CMY-2 Ibrahimagi´c et al. [48]2009–2010 2015 Bosnia and Herzegovina H/C ESBL 85 - - 8.23 PCR CMY-2 Alonso et al. [49] 2010–2011 2016 Spain H/C ECI 21,563 1.10 - - PCR/WGS CMY-2 Li et al. [40] 2011–2012 2015 Ireland H 3GCR 95 19 - - PCR/WGS CIT group Pascual et al. [50] 2010–2011 2016 Spain H/C 3GCR 841 2.02 - - PCR/WGS CMY-2 Zhou et al. [36] 2012–2013 2017 Holland/Germany H/C EI 1087 0.73 - - PCR/WGS CMY-2 Gómara et al. [34] 2013–2014 2018 Spain H CR 63 - - 14.2 PCR CIT group Den Drijver et al. [38] 2013–2016 2018 Holland H EI 2126 2.40 - - PCR CMY-2 Ribeiro et al. [37] 2010–2016 2019 Portugal H 3GCR 1246 1.28 1.04 2.60 PCR/WGS DHA-1 Findlay et al. [39] 2017–2018 2020 England C 3GCR 225 7.55 - - PCR/WGS DHA-1 Rohde et al. [51] 2014–2015 2020 Germany C 3GCR 828 - - 11.90 PCR/WGS CMY-2 a Type of population studied: Hospital (H)/Community (C). b Percentage positivity for AmpC among all isolates evaluated in the study. c Percentage global positivity that includes species other than E. coli and K. pneumoniae and/or does not differentiate between cAmpC-BL and pAmpC-BL. PCR: polymerase chain reaction; WGS: whole genome sequencing; ECI: E. coli isolates; 3GCR: third-generation cephalosporin-resistant; EI: Enterobacteriaceae isolates; KPI: K. pneumoniae isolates; ESBL: extended spectrum beta-lactamase; CR: carbape nemase res istant.
Microorganisms 2022,10, 611 8 of 24 Table 3. Epidemiology of pAmpC-BLs in America (2010–2020). Author (Reference) Year of Population Study Year of Publication Country of Target Population Population (H/C) aSpecific Conditions nAmpC (%) b Global cGenetic Identification Most Frequent AmpC Enzymes E. coli K.pneumoniae Park et al. [53] 2008–2012 2012 USA H 3GCR 300 16.33 - - PCR/WGS CMY-2 Suwantarat et al. [32] 2014–2015 2016 USA H EI 854 - - 1.30 PCR/WGS CMY-2 Logan et al. [55] 2011–2015 2016 USA H MDR 225 14.22 - - PCR/WGS CMY-2 Paniagua-Contreras et al. [54]Data not available 2018 Mexico C ECI 194 23.70 - - PCR CIT group Tamma et al. [52] 2014–2015 2019 USA H EI 1,929 2.23 0.88 3.42 PCR CMY-2 a Type of population studied: Hospital (H)/Community (C). b Percentage of positivity for AmpC among all isolates evaluated in the study. c Percentage global positivity that includes species other than E. coli and K. pneumoniae and/or does not differentiate between cAmpC-BL and pAmpC-BL. PCR: polymerase chain reaction; WGS: whole genome sequencing; ECI: E. coli isolates; 3GCR: third-generation cephalosporin-resistant; EI: Enterobacteriaceae isolates; MDR: multidrug resistant. Table 4. Epidemiology of pAmpC-BLs in Africa (2010–2020). Author (Reference) Year of Population Study Year of Publication Country of Target Population Population (H/C) aSpecific Conditions nAmpC (%) b Global cGenetic Identification Most Frequent AmpC Enzymes E. coli K. pneumoniae Ogbolu et al. [62] 2005–2007 2011 Nigeria H EI 134 - - 4.50 PCR/WGS DHA-1 Barguigua et al. [63] 2010 2013 Morocco C ECI 1,174 0.59 - - PCR/WGS CIT group Barguigua et al. [57] 2010–2011 2013 Morocco C KPI 453 - 0.88 - PCR/WGS EBC group Yusuf et al. [59]Data not available 2014 Nigeria H/C EI 543 4.23 3.50 11.23 - - Helmy et al. [58] 2011–2012 2014 Egypt H EI 143 14.68 2.09 18.18 CIT group Nakaye et al. [64] 2013 2014 Uganda H 3GCR 293 - - 39.60 PCR EBC group Gharout-Said et al. [65]2005–2010 2015 Algeria H EI 922 - - 1.60 PCR/WGS CMY-4 Chérif et al. [66] 2006–2009 2015 Tunisia H EI 11,393 - - 0.59 PCR/WGS CMY-2 Tellevik et al. [56] 2010–2011 2016 Tanzania H/C EI 603 0.50 - - PCR/WGS CMY-2 Zorgani et al. [61] 2013–2014 2017 Libya H EI 151 1.98 3.97 5.96 PCR CIT group Tanfous et al. [67] 2002–2011 2018 Tunisia H KPI 128 - 2.30 - PCR/WGS CMY-4 Tanfous et al. [68] 2002–2013 2018 Tunisia H ESBL 128 - 2.34 - PCR/WGS CMY-4
Microorganisms 2022,10, 611 9 of 24 Table 4. Cont. Author (Reference) Year of Population Study Year of Publication Country of Target Population Population (H/C) aSpecific Conditions nAmpC (%) b Global cGenetic Identification Most Frequent AmpC Enzymes E. coli K. pneumoniae Rensing et al. [69] 2013 2019 Egypt H/C EI 225 1.45 0.97 2.91 PCR CIT group Mohamed et al. [70] 2018 2020 Egypt C EI 440 2.04 2.04 4.09 PCR/WGS DHA-1 Estaleva et al. [60] 2015 2021 Mozambique H/C ECI 230 10.86 - - PCR/WGS FOX/MOX a Type of population studied: Hospital (H)/Community (C). b Percentage positivity for AmpC among all isolates evaluated in the study. c Percentage global positivity that includes species other than E. coli and K. pneumoniae and/or does not differentiate between cAmpC-BL and pAmpC-BL. PCR: polymerase chain reaction; WGS: whole genome sequencing; ECI: E. coli isolates; 3GCR: third-generation cephalosporin-resistant; EI: Enterobacteriaceae isolates; KPI: K. pneumoniae isolates; ESBL: extended spectrum beta-lactamase. Table 5. Epidemiology of pAmpC-BLs in Asia, Oceania, and the Middle East (2010–2020). Author (Reference) Year of Population Study Year of Publication Country of Target Population Population (H/C) aSpecific Conditions nAmpC (%) b Global cGenetic Identification Most Frequent AmpC Enzymes E. coli K. pneumoniae Yoo et al. [76] 2008–2009 2010 South Korea H EI 276 1,81 16.66 - PCR DHA-1 Yamasaki et al. [21] 2002–2008 2010 Japan H/C EI 22,869 0.07 0.01 0.13 PCR/WGS CMY-2 Singtohin et al. [77] 2005–2006 2010 Thailand H EI 2,712 1.62 0.29 1.91 PCR CMY-2 Mohamudha et al. [72]2008 2010 India H EI 175 24.57 13.14 44.57 - - Mohamudha et al. [73]2009–2010 2012 India H EI 241 24.89 13.27 38.17 PCR DHA-1 Manoharan et al. [78] 2007–2008 2012 India H 3GCR 312 - - 15.38 PCR CIT group Matsumura et al. [79] 2010 2012 Japan H ECI 1,327 1.73 - - PCR/WGS CMY-2 Gupta et al. [80] 2008–2009 2012 India H KPI 100 - 32 - PCR CMY-2 Sasirekha et al. [81] 2008 2012 India H EI 90 4.44 3.33 7.77 - -
Microorganisms 2022,10, 611 16 of 24 with CTX MIC > 1 mg/L as non-susceptible. Another shortcoming of phenotypic methods is that they are not all equally effective at detecting all AmpC families [ 20 , 137 ], and Ingram et al. (2011) found that inhibitors such as CLX or BA are more sensitive to DHA than CMY [114]. Proposed phenotypic methods that have demonstrated greatest diagnostic usefulness are those based on inhibitors. Thus, acceptable sensitivity and specificity values (>90%) are obtained using the double-disk synergy method with CLX + FOX [ 20 , 30 , 114 ] and the AmpC disk method [ 114 , 122 ]. The AmpC E-test has obtained the worst results, possibly because it contains CTT, showing lower sensitivity but higher specificity to detect AmpC [114]. The rapid fluorogenic method (1.5 h) recently developed by Park et al. (2020) has demonstrated high sensitivity and specificity to detect pAmpC-BL. It utilizes an antimicrobial bound to a fluorogenic substance that emits fluorescence in the presence of a beta-lactamase capable of its hydrolyzation. This method can also be combined with direct diagnostic techniques (Vitek2 ® or MALDI-TOF ® ) for the rapid detection of the bacterial species [ 138 ]. This represents an advancement in the development of novel methods for pAmpC-BL detection. The coexistence of ESBL and AmpC poses a diagnostic challenge, given that ESBL resistance may go unnoticed in AmpC-producing organisms by presenting this resistance to CLAV. It is also possible that FOX resistance may be produced by a combination of ESBL production and reduced external membrane permeability [ 120 ]. A possible solution to the diagnostic challenge posed by the coexistence of ESBL and AmpC is to include FEP, which is not affected by the presence of AmpC, in ESBL screening along with CLAV [ 17 , 99 ]. In cases of ESBL and AmpC coexistence, BA is a more diagnostically valuable inhibitor [ 128 ]. Song et al. (2007) [ 139 ] modified the CLSI ESBL detection technique, which uses CLAV, by adding BA to CTX or CAZ disks and to CTX/CAZ disks with CLAV, producing CTX/CAZ+BA disks and CTX/CAZ+CLAV+BA disks. An increase of ≥ 3 mm in the halo of the CTX/CAZ+CLAV+BA versus CTX/CAZ+BA disk is considered positive for ESBL [113,135,140]. Genotypic methods are considered the gold standard techniques for the detection of AmpC resistances [ 3 , 30 , 114 ] and are able to differentiate between chromosomal and plasmid AmpC-BLs [ 98 ]. However, most of them are expensive, technically complex, and time-consuming methods that are mainly used in research and are reserved for doubtful cases in a clinical setting (e.g., E. coli isolates) [ 140 ]. Another major drawback is that they can detect ampC genes that are already known but not new mutations or AmpC families [16,98,112,115,132]. Based on the data gathered in this review, we developed an algorithm similar to that depicted in Figure 3. Accordingly, the presence of AmpC-BL should be suspected when there is resistance to cephamycins (MIC > 8 mg/L for FOX) and oxyimino-cephalosporins (pattern of resistance to CTX or CAZ). This helps avoid a search for AmpC-BL in ESBLproducing bacteria, which would meet the second but not the first criterion because they are susceptible to FOX. A phenotypic method should then be applied to confirm the presence of AmpC, with the double-disk synergy test being the most highly recommended approach. Finally, a PCR or a genotypic analysis should be carried out in doubtful cases to verify the presence/absence of AmpC-BL encoding genes, bearing in mind that some isolates present positivity in the double-disk method that even PCR cannot detect [102,135].
Microorganisms 2022,10, 611 17 of 24 Microorganisms 2022, 10, x FOR PEER REVIEW 16 of 23 Figure 3. Diagnostic algorithm for the phenotypic detection of AmpC-BL (based on data from the review). MIC: minimum inhibitory concentration; FOX: cefoxitin; CAZ: ceftazidime; BA: boronic acid; CLX: cloxacillin; PCR: polymerase chain reaction. Regarding our screening proposal (see Section 2.2) for the phenotypic detection of AmpC-producing Enterobacteriaceae, test 2 obtained the best results. In comparison, Reuland et al. [30] obtained 91% sensitivity and 96% specificity using the double-disk synergy method with CLX in 66 isolates with reduced susceptibility to FOX and third-generation cephalosporins, and 85% sensitivity and 95% specificity when they applied the same method but with BA [30]. The use of CTT disks alone, and with the addition of BA as phenotypic confirmation, was recommended in a study with 635 isolates of Enterobacteriaceae not susceptible to FOX (MIC ≥ 32 mg/dL) [141]. In another study using the doubledisk synergy method to test 255 isolates, the addition of CLX to a FOX disk, obtaining the best predictive values when the halo increase ≥4 mm, was considered positive, achieving 95% sensitivity and 95% specificity [20]. Finally, Polsfuss et al. used the double-disk synergy method in 305 isolates and described 97.2% sensitivity and 100% specificity [114]. With regard to other techniques, Black et al. used the AmpC disk to screen 140 isolates not susceptible to FOX and obtained 100% sensitivity and 98% specificity [122]. Ingram et al. compared different screening and confirmatory methods in a study of 246 isolates, concluding that the screening method with the AmpC disk obtained the best result, offering 95% sensitivity and 98% specificity [109]. In a sample of 125 pAmpC-BL-positive isolates, the CTT/CXT E-Test showed 98.6% sensitivity and 35.4% specificity, while the AmpC disk method obtained slightly lower sensitivity (96%) but higher specificity (58%) values [44]. Hence, the results achieved with test 2 are comparable to the best results described for screening phenotypic tests, with the added advantage of integrating this fourth disk in the screening test with FEP, FOX, and ETP disks, previously proposed by our group for the detection of microorganisms with ESBL and/or carbapenemase [9]. Test 2 yields lower values in the presence of ESBL. As noted above, the coexistence of AmpC and ESBL production hampers the phenotypic detection of both resistance mechanisms, and phenotypic methods are recommended with the addition of CLAV (AmpC inducer and ESBL inhibitor) and even genotypic methods for a definitive diagnosis [17]. Song et al. used BA as AmpC inhibitor in their study of 182 isolates, comparing CTX/CA/BA disks with CTX and/or CAZ/CA/BA with CAZ disks as well as CTX/CA/BA disks with CTX/BA disks and/or CAZ/CA/BA disks with CAZ/BA disks, reporting that both approaches markedly improved sensitivity and specificity values in comparison to the utilization of CA alone [139]. However, a review proposed the double-disk synergy Figure 3. Diagnostic algorithm for the phenotypic detection of AmpC-BL (based on data from the review). MIC: minimum inhibitory concentration; FOX: cefoxitin; CAZ: ceftazidime; BA: boronic acid; CLX: cloxacillin; PCR: polymerase chain reaction. Regarding our screening proposal (see Section 2.2) for the phenotypic detection of AmpC-producing Enterobacteriaceae, test 2 obtained the best results. In comparison, Reuland et al. [ 30 ] obtained 91% sensitivity and 96% specificity using the double-disk synergy method with CLX in 66 isolates with reduced susceptibility to FOX and third-generation cephalosporins, and 85% sensitivity and 95% specificity when they applied the same method but with BA [ 30 ]. The use of CTT disks alone, and with the addition of BA as phenotypic confirmation, was recommended in a study with 635 isolates of Enterobacteriaceae not susceptible to FOX (MIC ≥ 32 mg/dL) [ 141 ]. In another study using the double-disk synergy method to test 255 isolates, the addition of CLX to a FOX disk, obtaining the best predictive values when the halo increase ≥4 mm, was considered positive, achieving 95% sensitivity and 95% specificity [ 20 ]. Finally, Polsfuss et al. used the double-disk synergy method in 305 isolates and described 97.2% sensitivity and 100% specificity [ 114 ]. With regard to other techniques, Black et al. used the AmpC disk to screen 140 isolates not susceptible to FOX and obtained 100% sensitivity and 98% specificity [ 122 ]. Ingram et al. compared different screening and confirmatory methods in a study of 246 isolates, concluding that the screening method with the AmpC disk obtained the best result, offering 95% sensitivity and 98% specificity [ 109 ]. In a sample of 125 pAmpC-BL-positive isolates, the CTT/CXT E-Test showed 98.6% sensitivity and 35.4% specificity, while the AmpC disk method obtained slightly lower sensitivity (96%) but higher specificity (58%) values [ 44 ]. Hence, the results achieved with test 2 are comparable to the best results described for screening phenotypic tests, with the added advantage of integrating this fourth disk in the screening test with FEP, FOX, and ETP disks, previously proposed by our group for the detection of microorganisms with ESBL and/or carbapenemase [9]. Test 2 yields lower values in the presence of ESBL. As noted above, the coexistence of AmpC and ESBL production hampers the phenotypic detection of both resistance mechanisms, and phenotypic methods are recommended with the addition of CLAV (AmpC inducer and ESBL inhibitor) and even genotypic methods for a definitive diagnosis [ 17 ]. Song et al. used BA as AmpC inhibitor in their study of 182 isolates, comparing CTX/CA/BA disks with CTX and/or CAZ/CA/BA with CAZ disks as well as CTX/CA/BA disks with CTX/BA disks and/or CAZ/CA/BA disks with CAZ/BA disks, reporting that both approaches markedly improved sensitivity and specificity values in comparison to the utilization of CA alone [ 139 ]. However, a review proposed the double-disk synergy method using CTX and CAZ with and without the addition of CLX as the optimal phenotypic
Microorganisms 2022,10, 611 18 of 24 confirmation test for AmpC in the presence of ESBL [ 119 ]. The diagnostic usefulness of test 2 in these isolates is enhanced by increasing the amount of CLX on the oxacillin disk. Limitations Studies with larger samples of AmpC-producing isolates are required to obtain more accurate predictive values. In common with other phenotypic techniques, our method cannot differentiate between the presence of plasmid or chromosomal AmpC except when the isolate is known to produce plasmid AmpC producer alone, as in the case of K. pneumoniae . In addition, the detection of pAmpC-BL is hampered by confluence with other resistance mechanisms such as ESBL production, porin loss, or E. coli producing ESAC beta-lactamases. Reference methods used for the detection of AmpC producers were the cefotetan/cefotetanCLX E-Test and an increased CTX and CAZ MIC in the presence of CLAV, because the means required for genotypic identification of the AmpC resistance mechanism were not available. 5. Conclusions There has been an increase in pAmpC-BL-producing isolates over the past 10 years, especially in the Asian continent, and CMY-2 producers are the most frequently responsible. Prevention of their spread requires the implementation of routine surveillance procedures that combine phenotypic and genotypic approaches (multiplex PCR). Among phenotypic screening methods, double-disk synergy and AmpC disk methods can be especially recommended for their predictive capacity. Our proposed screening method, which involves the addition of CLX on an oxacillin disk, is an easy-to-use and inexpensive test for the detection of AmpC-producing isolates, especially when there is no other resistance mechanism. Moreover, it can be combined on a single plate with a screening method for the detection of Enterobacteriaceae with ESBL and/or carbapenemases through the addition of FOX, FEP, and ETP disks. Author Contributions: Conceptualization, J.G.-F.; Investigation, E.R.-G., J.C.C.-R. and J.G.-F.; Methodology, E.R.-G.; Supervision, J.G.-F.; Writing–original draft, E.R.-G. and J.C.C.-R.; Writing–review & editing, J.M.N.-M.and J.G.-F. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in the main text. Conflicts of Interest: The authors declare no conflict of interest. References 1. Livermore, D.M. Antibiotic resistance during and beyond COVID-19. JAC-Antimicrob Resist 2021 ,3(Suppl. 1), i5–i16. [CrossRef] [PubMed] 2. Delgado-Valverde, M.; Sojo-Dorado, J.; Pascual, A.; Rodríguez-Baño, J. Clinical management of infections caused by multidrugresistant Enterobacteriaceae.Ther. Adv. Infect. Dis. 2013,1, 49–69. [CrossRef] [PubMed] 3. Jacoby, G.A. AmpC beta-lactamases. Clin. Microbiol. Rev. 2009,22, 161–182. [CrossRef] [PubMed] 4. Philippon, A.; Arlet, G.; Jacoby, G.A. Plasmid-determined AmpC-type beta-lactamases. Antimicrob Agents Chemother. 2002 ,46, 1–11. [CrossRef] [PubMed] 5. Walther-Rasmussen, J.; Høiby, N. Plasmid-borne AmpC beta-lactamases. Can. J. Microbiol. 2002 ,48, 79–93. [CrossRef] [PubMed] 6. Soria-Segarra, C.; Delgado-Valverde, M.; Serrano-García, M.L.; López-Hernández, I.; Navarro-Marí, J.M.; Gutiérrez-Fernández, J. Infections in patients colonized with carbapenem-resistant Gram-negative bacteria in a medium Spanish city. Rev. Esp. Quimioter. Publ. Of. Soc. Esp. Quimioter. 2021,34, 50–58. [CrossRef] [PubMed] 7. Cano-Martín, E.; Portillo-Calderón, I.; Pérez-Palacios, P.; Navarro-Marí, J.M.; Fernández-Sierra, M.A.; Gutiérrez-Fernández, J. A Study in a Regional Hospital of a Mid-Sized Spanish City Indicates a Major Increase in Infection/Colonization by CarbapenemResistant Bacteria, Coinciding with the COVID-19 Pandemic. Antibiotics 2021,10, 1127. [CrossRef] [PubMed]
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