Antibiotic Resistances of Enterobacteriaceae with Chromosomal Ampc in Urine Cultures: Review and Experience of a Spanish Hospital

Citation: Rodríguez-Guerrero, E.; Cabello, H.R.; Expósito-Ruiz, M.; Navarro-Marí, J.M.; Gutiérrez-Fernández, J. Antibiotic Resistances of Enterobacteriaceae with Chromosomal Ampc in Urine Cultures: Review and Experience of a Spanish Hospital. Antibiotics 2023,12, 730. https://doi.org/10.3390/ antibiotics12040730 Academic Editor: Mario Varcamonti Received: 8 March 2023 Revised: 30 March 2023 Accepted: 6 April 2023 Published: 8 April 2023 Copyright: © 2023 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/). antibiotics Article Antibiotic Resistances of Enterobacteriaceae with Chromosomal Ampc in Urine Cultures: Review and Experience of a Spanish Hospital Enrique Rodríguez-Guerrero 1, Horacio Requena Cabello 2, Manuela Expósito-Ruiz 3, JoséMaría Navarro-Marí1and JoséGutiérrez-Fernández 1,2,* 1Laboratory of Microbiology, Virgen de las Nieves University Hospital & Biosanitary Research Institute of Granada (ibs.GRANADA), 18016 Granada, Spain; [email protected] (E.R.-G.); [email protected] (J.M.N.-M.) 2Department of Microbiology, School of Medicine, University of Granada & Biosanitary Research Institute of Granada (ibs.GRANADA), 18016 Granada, Spain; [email protected].es 3Unit of Biostatistics, Department of Statistics, School of Medicine, University of Granada & Biosanitary Research Institute of Granada (ibs.GRANADA), 18016 Granada, Spain; [email protected] *Correspondence: [email protected] Abstract: The Enterobacteriaceae Citrobacter freundii,Enterobacter cloacae,Klebsiella aerogenes,Morganella morganii,Providencia stuartii, and Serratia marcescens (CESPM group) produce numerous urinary tract infections (UTIs) which are difficult to treat due to their high multiresistance rate. The objectives of this study were to carry out a systematic review of antibiotic resistances by UTIs and to determine changes over time in urine cultures from a reference hospital in southern Spain. The literature was searched for European data on the resistance rates of each microorganism, and a retrospective cross-sectional descriptive study was performed in samples with suspicion of UTI from patients in Virgen de las Nieves University Hospital (Granada, Spain) between 2016 and the first half of 2021. Among 21,838 positive urine cultures, 1.85% were caused by E. cloacae, 0.77% by M. Morganii, 0.65% by K. aerogenes, 0.46% by C. freundii, 0.29% by P stuartii, and 0.25% by S. marcescens. The lowest resistance rates by microorganism were: E. cloacae to amikacin (3.47%) and imipenem (5.28%); M. morganii to piperacillin–tazobactam (1.79%), cefepime (4.76%), and tobramycin (7.74%); K. aerogenes to tobramycin (3.55%), gentamicin (4.25%), trimethoprim–sulfamethoxazole (4.96%), imipenem (5.75%), and cefepime (6.43%); C. freundii to imipenem (no resistance), nitrofurantoin (1.96%), fosfomycin (2.80%), and ertapenem (6.12%); P. stuartii to cefepime (3.28%) and ceftazidime (3.28%); and S. marcescens to gentamicin (1.8%), ciprofloxacin (3.64%), cefepime (3.70%), piperacillin–tazobactam (3.70%), and trimethoprim–sulfamethoxazole (5.45%). In our setting, CESMP Enterobacteriaceae showed the lowest resistance to piperacillin–tazobactam, cefepime, imipenem, gentamicin, and colistin, which can therefore be recommended for the empirical treatment of UTIs. The COVID-19 pandemic may have had a clinical impact in relation to the increased resistance of E. cloacae and M. morgani to some antibiotics. Keywords: antibiotic resistances; urinary tract infections; chromosomal AmpC beta-lactamases; Enterobacteriaceae 1. Introduction Urinary tract infections (UTIs) are highly frequent [ 1 ], particularly among sexually active women of childbearing age [ 1 , 2 ]. It has been estimated that one-third of Primary Care visits are for infections, 10% of which are UTIs [ 3 ], and this does not take into account the numerous UTIs treated by self-medication or in hospital emergency departments, where 22% of treated infections are UTIs, the second most frequent after respiratory infections [ 1 ]. UTIs are the most common infection in hospitals, in which 80% are related to vesical catheters or other devices [4]. Antibiotics 2023,12, 730. https://doi.org/10.3390/antibiotics12040730 https://www.mdpi.com/journal/antibiotics Antibiotics 2023,12, 730 2 of 14 UTIs are generally of low severity, but they are highly prevalent. In Spain, over four million women aged between 20 and 40 years report acute cystitis every year, and onequarter of them have relapses [ 1 ]. Severe complications can be caused by these infections, including sepsis or pyelonephritis, especially in vulnerable patients [ 5 ]. This is of special relevance, given changes in the profile of patients with UTIs towards older age groups with more comorbidities and risk factors for multiresistant microorganisms [1]. There has been a global increase in antibiotic resistance rates over the past few years, mainly in Gram-negative bacilli. Among these, the main UTI producer is Escherichia coli [ 4 ]. However, there is increasing interest in a group of Enterobacteriaceae responsible for 10% of nosocomial and community UTIs, known as the CESPM group (Citrobacter freundii,Klebsiella aerogenes,Enterobacter cloacae,Serratia marcescens,Providencia stuartii, and Morganella morganii). They are characterized by naturally producing chromosomal AmpCinducible beta-lactamases [ 6 – 8 ], unlike Enterobacteriaceae which produce plasmid-origin AmpC beta-lactamases, for which various diagnostic methods are available [9]. According to the review by Jacoby et al. [ 8 ], AmpC beta-lactamases confer resistance to penicillin and cephalosporins (including cefotaxime, ceftazidime, or ceftriaxone). Although these proteins produce the weak hydrolysis of cefepime and carbapenems, their effectiveness is impaired by the presence of efflux pumps or pore loss on the external membrane. The production of these beta-lactamases can be induced by exposure to different beta-lactams, including penicillin, ampicillin, amoxicillin, cefazolin, cefoxitin, and imipenem. Beta-lactamase inhibitors such as clavulanic acid have been found to produce a paradoxical increase in AmpC. UTIs are generally treated empirically, and active surveillance studies by microbiology laboratories are required to select the appropriate empirical approach. Resistance rates vary widely over time and among geographic regions, and it is essential to possess information on the resistance patterns of microorganisms in each setting. Clinicians need to know current resistance rates and recent changes to support their decision making. The prescription of antibiotics increased during the COVID-19 pandemic, undermining the struggle against multiresistant microorganisms [ 10 , 11 ]. A large proportion of the mortality from this virus has been attributed to bacterial overinfections [ 12 ] that favor the overutilization of antibiotics [ 12 – 14 ], especially of beta-lactams which may induce AmpC overexpression. The objectives of this study were to determine changes in the antibiotic resistance of UTIs produced by microorganisms of the CESPM group in a systematic review of the literature and to compare findings with data on clinical isolates obtained from urine cultures in our hospital between 2016 and the first half of 2021, assessing the possible impact of the COVID-19 pandemic on resistance rates. 2. Material and Methods 2.1. Systematic Review The MEDLINE (PubMed) database was searched using the search terms “urinary tract infection” and “antibiotic resistance”, along with the full scientific names of the different species. Inclusion criteria were a publication date between 1 January 2010 and 30 June 2021; publication in Spanish, Portuguese, Italian, English, or French; and the provision of data on the antibiotic resistance rates of microorganisms. Exclusion criteria were the analysis of non-UTI samples; no separation of data between UTI-causing isolates and those responsible for other types of infections; and information from outside Europe (including Russia and Turkey). After the application of the eligibility criteria, four articles were retrieved on E. cloacae, six on M. morganii, three on K. aerogenes, three on C. freundii, and two on S. marcescens. No articles on P. stuartii met the eligibility criteria. 2.2. Analysis of data from the Virgen de las Nieves Hospital (HUVN) of Granada (Southern Spain) A retrospective cross-sectional descriptive study was performed in consecutive urine samples with a suspected diagnosis of UTI processed by the hospital microbiology lab- Antibiotics 2023,12, 730 3 of 14 oratory between 1 January 2016 and 30 June 2021. No exclusion criteria were applied. Sample processing always followed the standard laboratory protocol of our hospital [ 15 ]. Matrix-assisted laser desorption ionization (MALDI) Biotyper (Bruker Daltonics, Billerica, MA, USA) or MicroScan (Beckman Coulter, Barcelona, Spain) systems were employed to identify microorganisms grown in culture, and MicroScan was used to evaluate their antibiotic susceptibility. The minimum inhibitory concentration (MIC) was recorded for each antibiotic. Isolates were categorized as susceptible, intermediate, or resistant to antibiotics in accordance with Clinical and Laboratory Standards Institute (CLSI) recommendations for each year until 2019, and thereafter, in accordance with recommendations of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for each year. Information on urine sample origin, microorganism, and patient age was collected from the MODULAB ® system used by the Public Health System of Andalusia to support electronic clinical records. Data on UTI episodes were stratified by sex, age ( ≤ 14 years, 15–64 years, and ≥ 65 years), and origin (hospitalized vs. community). No clinical information was gathered to analyze clinical factors related to the presence of microorganisms. The percentage resistance to the different antibiotics was compared by sex, age, and origin using Pearson’s chi-square test, applying Fisher’s exact test when chi-square test conditions were not met ( ≤ 20% of expected frequencies <5). R 4.4.1 software was used for data analyses, and p< 0.05 was considered significant. 2.3. Ethical Considerations Ethical Approval The study protocol was carried out in accordance with the Declaration of Helsinki [ 16 ]. This was a non-interventional study with no additional investigation to routine procedures. Biological material was only used for standard infection diagnostics ordered by the attending physician. There was no additional sampling or modification of the routine sampling protocol of the laboratory. Data analyses were based on an anonymous database. For these reasons, ethics committee approval was considered unnecessary according to national guidelines. The Clinical Microbiology Clinical Management Unit of the University Hospital Virgen de las Nieves of Granada (Spain) granted permission to access and use the data. 2.4. Informed Consent The study protocol was carried out in accordance with the Helsinki Declaration [ 16 ]. Data analyses were performed using an anonymous database. Therefore, approval was considered unnecessary according to the guidelines of our country (Law on Data Protection -Organic Law 15/1999 of 13 December on the protection of data of a personal nature, available online: https://www.boe.es/buscar/doc.php?id=BOE-A-1999-23750 (accessed on 30 June 2021)). 3. Results 3.1. Global Prevalence The HUVN microbiology laboratory processed 74,106 urine samples for suspicion of UTI between 1 January 2016 and 30 June 2021, with 21,838 (29.47%) testing positive. Table 1displays the number of clinical isolates and the percentage of positive urine cultures per microorganism. E. cloacae was isolated in 405 patients, representing 1.85% of positive urine cultures, while S. marcescens was isolated in 55 patients, representing 0.25% of positive urine cultures. Antibiotics 2023,12, 730 4 of 14 Table 1. Number of clinical isolates and percentage of those positive for tested microorganisms each year. Year (N◦Positive Cultures) Microorganisms 2016 (n= 3811) 2017 (n= 4581) 2018 (n= 3851) 2019 (n= 4201) 2020 (n= 3654) 2021 (n= 1740) Total (n= 21,838) E. cloacae 50 (1.31) 74 (1.62) 68 (1.77) 88 (2.09) 88 (2.41) 37 (2.13) 405 (1.85) M. morganii 34 (0.89) 37 (0.81) 29 (0.75) 31 (0.74) 26 (0.71) 11 (0.63) 168 (0.77) K. aerogenes 23 (0.6) 24 (0.52) 21 (0.55) 37 (0.88) 23 (0.63) 13 (0.75) 141 (0.65) C. freundii 12 (0.31) 18 (0.39) 25 (0.65) 26 (0.62) 14 (38) 5 (0.29) 100 (0.46) P. stuartii 12 (0.31) 19 (0.41) 13 (0.34) 8 (0.19) 7 (0.19) 5 (0.29) 64 (0.29) S. marcescens 12 (0.31) 8 (0.17) 6 (0.16) 19 (0.45) 8 (22) 2 (0.11) 55 (0.25) Table 2lists the number and percentage of clinical isolates per microorganism according to patient sex and age and sample origin and type. E. cloacae (p= 0.001) and S. marcescens (p= 0.019) were more frequently detected in males, while C. freundii (p= 0.006) was more frequently isolated in females. All microorganisms were more frequent in the hospital setting, except for P. stuartii, which was more prevalent in community samples. Table 2. Number and percentage of clinical isolates for each category. Variables E. cloacae K. aerogenes C. freundii P. stuartii M. morganii S. marcescens Gender Man 252 (62.22) 70 (49.65) 41 (41) 29 (45.31) 94 (55.95) 39 (70.91) Woman 153 (37.78) 71 (50.35) 59 (59) 35 (54.69) 74 (44.05) 16 (29.09) Age Children 37 (9.14) 11 (7.8) 4 (4) - 15 (8.93) 6 (10.91) Adults 153 (37.78) 57 (40.43) 25 (25) 25 (25) 45 (26.79) 20 (36.36) Elderly 215 (53.09) 73 (51.77) 71 (71) 71 (71) 108 (64.29) 29 (52.73) Healthcare Community 172 (42.47) 69 (48.94) 41 (41) 38 (59.38) 77 (45.83) 25 (45.45) Hospitable 233 (57.53) 72 (51.06) 59 (59) 26 (40.63) 91 (54.17) 30 (54.55) Type of urine sample Cleancatch midstream technique 193 (47.65) 86 (60.99) 63 (63) 22 (34.38) 96 (57.14) 33 (60) Permanent catheterization 85 (20.99) 21 (14.89) 17 (17) 23 (35.94) 25 (14.88) 9 (16.36) Urinary catheter 104 (25.68) 32 (22.69) 18 (18) 18 (28.13) 45 (26.79) 11 (20) Nephrostomy catheter 11 (2.71) 1 (0.71) 1 (1) 1 (1.56) - 2 (3.64) Pediatric urine collection bag 12 (2.96) 1 (0.71) 1 (1) - 2 (1.19) - 3.2. Enterobacter cloacae 3.2.1. Systematic Review Tables S1 and S2 (Supplementary Material) list the four studies selected for review by year of publication. They report on a total of 948 clinical isolates of E. cloacae with antibiogram. Although some antibiotics yielded resistance rates <10% (imipenem–relebactam, meropenem, doripenem, levofloxacin, and colistin), many were not effective in vitro against >50% of clinical isolates. 3.2.2. HUVN Laboratory Study This study gathered 405 clinical isolates of Enterobacter cloacae with antibiogram. Table S3 exhibits the annualized general resistance rates, and Tables S4–S15 (Supplementary Material) show the results by category. Antibiotics 2023,12, 730 5 of 14 Statistical analysis by year revealed significantly increased resistances to cefuroxime (p< 0.001), ceftazidime (p< 0.001), cefepime (p< 0.001), piperacillin–tazobactam (p= 0.004), tobramycin (p< 0.001), gentamicin (p< 0.001), ciprofloxacin (p< 0.001), levofloxacin (p< 0.001), nitrofurantoin (p< 0.001), and trimethoprim–sulfamethoxazole (p< 0.001). Resistance to nalidixic acid was also increased (p= 0.003), reversing a previous downward trend. Lower resistance rates (<10%) were observed to amikacin (3.47%) and imipenem (5.28%). Resistance rates were higher in the hospital versus community setting against ticarcillin (p= 0.043), cefuroxime (p= 0.017), cefotaxime (p= 0.006), ceftazidime (p= 0.017), cefepime (p= 0.001), piperacillin–tazobactam (p= 0.003), tobramycin (p= 0.001), gentamicin (p< 0.001), nalidixic acid (p= 0.009), levofloxacin (p= 0.006), and trimethoprim–sulfamethoxazole (p= 0.003). In addition, resistance to ticarcillin (p= 0.015) and gentamicin (p= 0.034) was higher in females than in males. Higher resistance rates were observed in adults than in the elderly or children against ceftazidime (p< 0.001), cefepime (p= 0.027), piperacillin–tazobactam (p= 0.039), tobramycin (p= 0.016), gentamicin (p= 0.001), nalidixic acid (p= 0.032), ciprofloxacin (p= 0.009), levofloxacin (p< 0.001), and trimethoprim–sulfamethoxazole (p= 0.001). Higher resistance rates were recorded in adults and the elderly than in children against fosfomycin (p= 0.049), while isolates with intermediate susceptibility to nitrofurantoin were more frequent (p= 0.049) in children than in adults or the elderly. For piperacillin–tazobactam and ciprofloxacin, the respective cutoff points of MIC 16 and 0.5 mg/dL correspond to areas of technical uncertainty (ATUs) according to EUCAST 2022, and these were observed for piperacillin–tazobactam (9.45%) and ciprofloxacin (4.45%) in the present sample. Figure 1depicts the upward trend over the years, especially between 2019 and 2021, in the percentage resistance of the antibiotics most frequently prescribed to treat UTIs caused by E. cloacae. Antibiotics 2023, 12, x FOR PEER REVIEW 6 of 15 Figure 1. General annualized resistances (%) of Enterobacter cloacae during 2016–2021 against the antibiotics most frequently used to treat UTIs. FEP = cefepime; TZP = piperacillin–tazobactam; CIP = ciprofloxacin; FOF = fosfomycin; NIT = nitrofurantoin; SXT = trimethoprim–sulfamethoxazole. 3.3. Morganella morganii 3.3.1. Systematic Review Table S16 (Supplementary Material) lists the six studies selected for review, reporting on a total of 431 clinical isolates of M. morganii in Europe with antibiogram. Only two antibiotics had resistance rates >30%: amoxicillin–clavulanic acid (92.23%) and ampicillin (95.39%). 3.3.2. HUVN Laboratory Study The laboratory identified 168 clinical isolates of M. morganii during the study period. Table S17 displays the annualized general resistance rates, and Tables S18–S29 (Supplementary Material) show the results by category. No statistically significant differences were found by sex or by sample type or origin. The only statistically significant between-year difference was a major increase in resistance (p = 0.007) to cefuroxime during 2019 (96.67%), 2020 (96.15%), and 2021 (90.91%). The lowest resistance rates were against piperacillin–tazobactam (1.79%), cefepime (4.76%), and tobramycin (7.74%). Resistance rates to imipenem were higher in adults than in the elderly or children (p = 0.016), higher in the elderly than in adults, and higher in adults than in children against nalidixic acid (p < 0.001), ciprofloxacin (p = 0.016), and trimethoprim–sulfamethoxazole (p = 0.040), respectively. ATUs were observed for ciprofloxacin (7.23%) but not for piperacillin–tazobactam. Figure 2 depicts the upward trend over the years, especially between 2020 and 2021, in the percentage resistance of the antibiotics most frequently prescribed to treat UTIs caused by M. morganii. We highlight the higher annualized general resistance of fosfomycin in comparison to the other antibiotics shown. 0 10 20 30 40 50 60 70 80 90 100 2016 2017 2018 2019 2020 2021 Percentage Year FEP TZP CIP FOF NIT SXT Figure 1. General annualized resistances (%) of Enterobacter cloacae during 2016–2021 against the antibiotics most frequently used to treat UTIs. FEP = cefepime; TZP = piperacillin–tazobactam; CIP = ciprofloxacin; FOF = fosfomycin; NIT = nitrofurantoin; SXT = trimethoprim–sulfamethoxazole. 3.3. Morganella morganii 3.3.1. Systematic Review Table S16 (Supplementary Material) lists the six studies selected for review, reporting on a total of 431 clinical isolates of M. morganii in Europe with antibiogram. Only two Antibiotics 2023,12, 730 6 of 14 antibiotics had resistance rates >30%: amoxicillin–clavulanic acid (92.23%) and ampicillin (95.39%). 3.3.2. HUVN Laboratory Study The laboratory identified 168 clinical isolates of M. morganii during the study period. Table S17 displays the annualized general resistance rates, and Tables S18–S29 (Supplementary Material) show the results by category. No statistically significant differences were found by sex or by sample type or origin. The only statistically significant between-year difference was a major increase in resistance (p= 0.007) to cefuroxime during 2019 (96.67%), 2020 (96.15%), and 2021 (90.91%). The lowest resistance rates were against piperacillin–tazobactam (1.79%), cefepime (4.76%), and tobramycin (7.74%). Resistance rates to imipenem were higher in adults than in the elderly or children (p= 0.016), higher in the elderly than in adults, and higher in adults than in children against nalidixic acid (p< 0.001), ciprofloxacin (p= 0.016), and trimethoprim–sulfamethoxazole (p= 0.040), respectively. ATUs were observed for ciprofloxacin (7.23%) but not for piperacillin–tazobactam. Figure 2depicts the upward trend over the years, especially between 2020 and 2021, in the percentage resistance of the antibiotics most frequently prescribed to treat UTIs caused by M. morganii. We highlight the higher annualized general resistance of fosfomycin in comparison to the other antibiotics shown. Antibiotics 2023, 12, x FOR PEER REVIEW 7 of 15 Figure 2. General annualized resistances (%) of Morganella morganii between 2016 and 2021 against the antibiotics most frequently used to treat UTIs. FEP = cefepime; TZP = piperacillin–tazobactam; FOF = fosfomycin; SXT = trimethoprim–sulfamethoxazole. 3.4. Klebsiella aerogenes 3.4.1. Systematic Review Table S30 (Supplementary Material) lists the three studies selected for review, reporting on 270 clinical isolates of Klebsiella aerogenes in Europe. The resistance rates were not high, only being >30% against ceftazidime (32.51%), ceftriaxone (50%), and ceftolozane–tazobactam (42.9%). The resistance rates to cefepime (4.63%) and carbapenems (4.7%) were very low. 3.4.2. HUVN Laboratory Study The laboratory identified 141 isolates of Klebsiella aerogenes in samples with suspicion of UTI received during the study period. Table S31 lists the annualized general resistance rates, and Tables S32–S43 (Supplementary Material) show the results by category. A significantly decreased annualized resistance rate was only observed for imipenem (p = 0.019). The lowest resistance rates were against tobramycin (3.55%), gentamicin (4.25%), trimethoprim–sulfamethoxazole (4.96%), imipenem (5.75%), and cefepime (6.43%). The resistance rates were higher for males versus females against cefuroxime (p < 0.0001), cefixime (p = 0.038), cefotaxime (p = 0.003), ceftazidime (p = 0.003), cefepime (p = 0.033), piperacillin–tazobactam (p = 0.004), and fosfomycin (p = 0.031). The resistance rates were higher in hospital versus community samples against ticarcillin (p = 0.009), cefuroxime (p = 0.017), cefixime (p = 0.035), cefotaxime (p < 0.001), ceftazidime (p < 0.001), and piperacillin–tazobactam (p = 0.003). No differences were detected among age groups. ATUs were observed for piperacillin–tazobactam (13.47%) and ciprofloxacin (2.84%). Figure 3 depicts the downward trend over the years, especially between 2020 and 2021, in the percentage resistance of the antibiotics most frequently prescribed to treat UTI caused by K. aerogenes. 0 10 20 30 40 50 60 70 80 90 100 2016 2017 2018 2019 2020 2021 Percentage Year FEP TZP GEN FOF CIP SXT Figure 2. General annualized resistances (%) of Morganella morganii between 2016 and 2021 against the antibiotics most frequently used to treat UTIs. FEP = cefepime; TZP = piperacillin–tazobactam; FOF = fosfomycin; SXT = trimethoprim–sulfamethoxazole. 3.4. Klebsiella aerogenes 3.4.1. Systematic Review Table S30 (Supplementary Material) lists the three studies selected for review, reporting on 270 clinical isolates of Klebsiella aerogenes in Europe. Antibiotics 2023,12, 730 7 of 14 The resistance rates were not high, only being >30% against ceftazidime (32.51%), ceftriaxone (50%), and ceftolozane–tazobactam (42.9%). The resistance rates to cefepime (4.63%) and carbapenems (4.7%) were very low. 3.4.2. HUVN Laboratory Study The laboratory identified 141 isolates of Klebsiella aerogenes in samples with suspicion of UTI received during the study period. Table S31 lists the annualized general resistance rates, and Tables S32–S43 (Supplementary Material) show the results by category. A significantly decreased annualized resistance rate was only observed for imipenem (p= 0.019). The lowest resistance rates were against tobramycin (3.55%), gentamicin (4.25%), trimethoprim–sulfamethoxazole (4.96%), imipenem (5.75%), and cefepime (6.43%). The resistance rates were higher for males versus females against cefuroxime (p< 0.0001), cefixime (p= 0.038), cefotaxime (p= 0.003), ceftazidime (p= 0.003), cefepime (p= 0.033), piperacillin–tazobactam (p= 0.004), and fosfomycin (p= 0.031). The resistance rates were higher in hospital versus community samples against ticarcillin (p= 0.009), cefuroxime (p= 0.017), cefixime (p= 0.035), cefotaxime (p< 0.001), ceftazidime (p< 0.001), and piperacillin–tazobactam (p= 0.003). No differences were detected among age groups. ATUs were observed for piperacillin–tazobactam (13.47%) and ciprofloxacin (2.84%). Figure 3depicts the downward trend over the years, especially between 2020 and 2021, in the percentage resistance of the antibiotics most frequently prescribed to treat UTI caused by K. aerogenes. Antibiotics 2023, 12, x FOR PEER REVIEW 8 of 15 Figure 3. General annualized resistances (%) of Klebsiella aerogenes between 2016 and 2021 against the antibiotics most frequently used to treat UTIs. FEP = cefepime; TZP = piperacillin–tazobactam; GEN: gentamicin CIP = ciprofloxacin; FOF = fosfomycin; NIT = nitrofurantoin; SXT = trimethoprim– sulfamethoxazole; CST = colistin. 3.5. Citrobacter freundii 3.5.1. Systematic Review Table S44 (Supplementary Material) lists the three studies selected for review, reporting on the resistance of C. freundii in urine cultures. The resistance rates were only >30% against ceftazidime (33.13%), ceftriaxone (38.5%), and ceftolozane–tazobactam (30.8%). No tested isolate was resistant to carbapenems or colistin. 3.5.2. HUVN Laboratory Study The laboratory identified 107 clinical isolates of C. freundii during the study period. Table S45 exhibits the annualized general resistance rates, and Tables S46–S57 (Supplementary Material) show the results by category. All isolates were susceptible to imipenem, and low resistance rates were observed against nitrofurantoin (1.96%), fosfomycin (2.80%), ertapenem (6.12%), colistin (8%), gentamicin (8.41%), piperacillin–tazobactam (9.35%), and tobramycin (9.35%). No significant differences in resistance rates were found among years or age groups. The resistance rates were higher in females versus males against cefixime (p = 0.013) and in hospital versus community samples against cefuroxime (p = 0.014), cefotaxime (p=0.016), and ceftazidime (p = 0.005). ATUs were observed for piperacillin–tazobactam (8.05%) and ciprofloxacin (3.74%). Figure 4 depicts the upward trend in percentage resistance to ciprofloxacin, nitrofurantoin, and fosfomycin and the downward trend in resistance to trimethoprim–sulfamethoxazole, gentamicin, and cefepime between 2020 and 2021. 0 10 20 30 40 50 60 70 2016 2017 2018 2019 2020 2021 Percentage Year FEP TZP GEN CIP FOF NIT SXT CST Figure 3. General annualized resistances (%) of Klebsiella aerogenes between 2016 and 2021 against the antibiotics most frequently used to treat UTIs. FEP = cefepime; TZP = piperacillin–tazobactam; GEN: gentamicin CIP = ciprofloxacin; FOF = fosfomycin; NIT = nitrofurantoin; SXT = trimethoprim– sulfamethoxazole; CST = colistin. 3.5. Citrobacter freundii 3.5.1. Systematic Review Table S44 (Supplementary Material) lists the three studies selected for review, reporting on the resistance of C. freundii in urine cultures. The resistance rates were only >30% against ceftazidime (33.13%), ceftriaxone (38.5%), and ceftolozane–tazobactam (30.8%). No tested isolate was resistant to carbapenems or colistin. Antibiotics 2023,12, 730 8 of 14 3.5.2. HUVN Laboratory Study The laboratory identified 107 clinical isolates of C. freundii during the study period. Table S45 exhibits the annualized general resistance rates, and Tables S46–S57 (Supplementary Material) show the results by category. All isolates were susceptible to imipenem, and low resistance rates were observed against nitrofurantoin (1.96%), fosfomycin (2.80%), ertapenem (6.12%), colistin (8%), gentamicin (8.41%), piperacillin–tazobactam (9.35%), and tobramycin (9.35%). No significant differences in resistance rates were found among years or age groups. The resistance rates were higher in females versus males against cefixime (p= 0.013) and in hospital versus community samples against cefuroxime (p= 0.014), cefotaxime (p=0.016), and ceftazidime (p= 0.005). ATUs were observed for piperacillin–tazobactam (8.05%) and ciprofloxacin (3.74%). Figure 4depicts the upward trend in percentage resistance to ciprofloxacin, nitrofurantoin, and fosfomycin and the downward trend in resistance to trimethoprim–sulfamethoxazole, gentamicin, and cefepime between 2020 and 2021. Antibiotics 2023, 12, x FOR PEER REVIEW 9 of 15 Figure 4. General annualized resistances (%) of Citrobacter freundii between 2016 and 2021 against the antibiotics most frequently used to treat UTIs. FEP = cefepime; TZP = piperacillin–tazobactam; GEN: gentamicin CIP = ciprofloxacin; FOF = fosfomycin; NIT = nitrofurantoin; SXT = trimethoprim– sulfamethoxazole. 3.6. Providencia stuartii 3.6.1. Systematic Review No study was traced on the resistance of P. stuartii in urine cultures in Europe. 3.6.2. HUVN Laboratory Study The laboratory identified 64 isolates of P. stuartii during the study period. Table S58 displays the annualized general resistance rates, and Tables S59–S70 (Supplementary Material) show the results by category. No significant differences were found by age, sex, sample, or year. No resistance was observed to ertapenem or piperacillin–tazobactam, while the resistance rates were low against cefepime (3.28%) and ceftazidime (3.28%) but much higher against imipenem (19.64%). ATUs were observed for ciprofloxacin (17.46%) but not for piperacillin–tazobactam. 3.7. Serratia marcescens 3.7.1. Systematic Review Table S71 (Supplementary Material) exhibits the two studies selected for review, which reported resistance rates that were relatively low, observing a rate of >30% against colistin alone (98.03%). 3.7.2. HUVN Laboratory Study The laboratory identified 55 clinical isolates of Serratia marcescens during the study period. Table S72 lists the annualized general resistance rates, and Tables S73–S84 (Supplementary Material) show the results by category. The resistance against cefoxitin only increased (p < 0.001) over the years, reaching 100% in 2020 and 2021. No statistically significant changes were observed against the other antibiotics studied. All isolates were susceptible to imipenem, and the resistance rates were very low against gentamicin (1.8%), ciprofloxacin (3.64%), cefepime (3.70%), piperacillin–tazobactam (3.70%), and trimethoprim–sulfamethoxazole (5.45%). ATUs were observed for piperacillin–tazobactam (3.70%) but not for ciprofloxacin. 0 10 20 30 40 50 60 2016 2017 2018 2019 2020 2021 Percentage Year FEP TZP GEN CIP FOF NIT SXT Figure 4. General annualized resistances (%) of Citrobacter freundii between 2016 and 2021 against the antibiotics most frequently used to treat UTIs. FEP = cefepime; TZP = piperacillin–tazobactam; GEN: gentamicin CIP = ciprofloxacin; FOF = fosfomycin; NIT = nitrofurantoin; SXT = trimethoprim– sulfamethoxazole. 3.6. Providencia stuartii 3.6.1. Systematic Review No study was traced on the resistance of P. stuartii in urine cultures in Europe. 3.6.2. HUVN Laboratory Study The laboratory identified 64 isolates of P. stuartii during the study period. Table S58 displays the annualized general resistance rates, and Tables S59–S70 (Supplementary Material) show the results by category. No significant differences were found by age, sex, sample, or year. No resistance was observed to ertapenem or piperacillin–tazobactam, while the resistance rates were low against cefepime (3.28%) and ceftazidime (3.28%) but much higher against imipenem (19.64%). ATUs were observed for ciprofloxacin (17.46%) but not for piperacillin–tazobactam. Antibiotics 2023,12, 730 9 of 14 3.7. Serratia marcescens 3.7.1. Systematic Review Table S71 (Supplementary Material) exhibits the two studies selected for review, which reported resistance rates that were relatively low, observing a rate of >30% against colistin alone (98.03%). 3.7.2. HUVN Laboratory Study The laboratory identified 55 clinical isolates of Serratia marcescens during the study period. Table S72 lists the annualized general resistance rates, and Tables S73–S84 (Supplementary Material) show the results by category. The resistance against cefoxitin only increased (p< 0.001) over the years, reaching 100% in 2020 and 2021. No statistically significant changes were observed against the other antibiotics studied. All isolates were susceptible to imipenem, and the resistance rates were very low against gentamicin (1.8%), ciprofloxacin (3.64%), cefepime (3.70%), piperacillin–tazobactam (3.70%), and trimethoprim–sulfamethoxazole (5.45%). ATUs were observed for piperacillin–tazobactam (3.70%) but not for ciprofloxacin. 4. Discussion 4.1. Antibiotic Resistances of Chromosomal AmpC-Producing Enterobacteriaceae E. cloacae Cutoff points for E. cloacae followed EUCAST [ 17 , 18 ] guidelines in two studies and recommendations of the CLSI [ 19 ] and the Comitéde l’antibiogramme de la SociétéFrançaise de Microbiologie (CA-SFM) [ 20 ] in one study each. Resistance rates for E. cloacae were lower in our hospital than in the systematic review against all antibiotics except for cefepime, tobramycin, and colistin, which showed slightly higher resistance rates in our setting. Resistance rates were higher in one study than in the others, possibly because it only included the elderly [ 20 ]. An increase in resistance rates against numerous antibiotics has been detected over the past few years, which may be related to a wider prescription of antibiotics during the COVID-19 pandemic. Comparisons with data from the same laboratory in previous years [ 19 ] reveal an increase in resistance to fosfomycin (from 28 to 32.51%) and cefepime (20 to 26.48%). One of the largest reductions in the resistance rate was against gentamicin (18 to 10.62%) and imipenem (8 to 5.28%), while no major differences were observed for the other antibiotics. According to these findings, the lowest resistance rates (<10%) were against amikacin (3.47%) and imipenem (5.28%), which may therefore be the best choice for the empirical treatment of E. cloacae, with colistin (12.67%) being another possible option. 4.2. M. morganii Cutoff points for M. morganii followed CLSI guidelines in all studies [ 19 , 21 – 23 ] except for two that followed EUCAST recommendations [ 17 , 24 ]. In comparison to the studies in the review, isolates detected in the HUVN laboratory had higher resistance rates against cefotaxime, imipenem, gentamicin, fosfomycin, ciprofloxacin, and trimethoprim– sulfamethoxazole but lower rates against ceftazidime, cefepime, piperacillin–tazobactam, and tobramycin. The resistance rate against imipenem was higher in adults than in the elderly or children but showed a general trend towards a reduction (29.41 to 9.09%) over the past few years. There was an increase in the resistance to cefuroxime, cefotaxime, ceftazidime, cefepime, tobramycin, and nitrofurantoin in 2020 and 2021, possibly attributable to a greater exposure to at-home and oral versus hospital and intravenous treatments. Resistance rates to fluroquinolones and trimethoprim–sulfamethoxazole were higher in the elderly than in adults or children. A comparison with data from the same laboratory in previous years [19] revealed a marked increase in the resistance of M. morganii to gentamicin, fosfomycin, nalidixic acid, ciprofloxacin, imipenem, and trimethoprim–sulfamethoxazole, especially to fosfomycin (from 4 to 80.61%), nalidixic acid (18 to 46.95%), and trimethoprim– sulfamethoxazole (14 to 33.33%). In contrast, resistance rates decreased against tobramycin (22 to 7.74%), piperacillin–tazobactam (19 to 1.79%), and cefepime (10 to 4.76%).