Summary
Introduction: Interest in fosfomycin, an old antibiotic, has been reignited because of the use of its intravenous formulation (fosfomycin sodium) in resistant infections. The aim of this study was to screen for carbapenem resistance in Enterobacterales isolates from the bloodstream, and determine the frequency of carbapenamases types and fosfomycin resistance genes in carbapenem-resistant Enterobacterales (CRE) strains.
Materials and Methods: Enterobacterales isolates from the bloodstream of liver transplant patients aged 18 years and older were screened for carbapenem resistance between 2017 and 2019. In isolates that were resistant to at least one carbapenem antibiotic were further screened for fosfomycin susceptibility. Carbapenem susceptibility was tested for by the E-test, fosfomycin susceptibility was tested for by the agar dilution methods, and evaluated in accordance with European Committee on Antimicrobial Susceptibility Testing criteria. The frequency of OXA-48, NDM, KPC, VIM, and IMP type carbapenemases and fosA, fosA3, and fosC2 fosfomycin resistance genes were screened for using the polymerase chain reaction method.
Results: A total of 115 Enterobacterales isolates from bloodstream infections were obtained. Carbapenem resistance was detected in 34 (29.3%) isolates, 41.2% of them were Escherichia coli and 58.8% of them were Klebsiella pneumoniae. Out of the 34 isolates, 61.8% produced carbapenemases, and OXA-48 was the most common type of carbapenemase. The fosfomycin resistance rate was 73.5%. Among the 34 carbapenem-resistant isolates, the frequency of the fosA gene was 5.9%. The genes fosA3 and fosC2 were not detected in any isolates. FosA + OXA-48 and fosA + NDM genes were detected in CRE isolates.
Conclusion: This is the first study reporting on the screening for fosfomycin resistance genes in blood isolates of liver transplant patients in Turkey. Despite high fosfomycin resistance, detection of only two resistance genes reveals that there may be fosfomycin resistance due to other resistance mechanisms.
Introduction
Sepsis and bacteremia, in particular, are complications causing high mortality in patients that have undergone liver transplantation and immunosuppressive treatments. In liver transplant patients, the most common microorganisms causing infections are enterococci, Gram-negative bacilli, and, rarely, other bacteria and candida strains[1]. Antibacterial resistance occurs in Enterobacterales by mutation, selection, or when sensitive bacteria gain resistance genes from other bacteria through conjugation or transduction[2]. The increase in multidrug-resistant (MDR) Gram-negative bacteria is causing difficulties in managing these patients.
Fosfomycin phosphoenolpyruvate stops the synthesis of peptidoglycan, one of the basic components of the cell wall, causing cell lysis and death of bacteria. Fosfomycin has been well-known as an oral antibiotic for the treatment of uncomplicated urinary tract infections. However, thanks to the intravenous formulation (fosfomycin sodium), it is now widely used for the treatment of vancomycin-resistant enterococcus, methicillin-resistant Staphylococcus aureus, and MDR Gram-negative bacterial infections[3]. Becuase of its antimicrobial, antioxidant, and anti-inflammatory effects, fosfomycin is a candidate for reducing sepsis-induced lung damage[4]. In Enterobacterales strains, genes that are mainly responsible for fosfomycin resistance are fosA (1–10), fosL1-2, and fosC2, which codes for glutathione transferase[5]. These fos genes are currently of low to medium effectiveness in spreading fosfomycin resistance. Nevertheless, their occurrence in portable plasmids might render them one of the most effective agents in spreading fosfomycin resistance. Moreover, these genes co-occur with other genes that cause resistance to other groups of antibiotics, thereby enhancing the outbreak of MDR strains[6].
This study aimed to screen for fosfomycin susceptibility and fos resistance genes in carbapenem-resistant Enterobacterales (CRE) strains from positive blood cultures of liver transplant patients at the Liver Transplantation Institute, İnönü University. Currently, there are only a few studies on fosfomycin susceptibility and resistance genes in Turkey. To address the aim of this study, we used a gold standard method, the agar dilution method, to analyze for fosfomycin susceptibility and polymerase chain reaction (PCR) to detect fosA, fosA3, fosC2 resistance genes. Finally, sequence analyses were performed to validate resulting isolates with resistant genes.
Methods
Patient Population and Bacteria
Between January 2017 and December 2019, we examined blood cultures of patients over the age of 18 who were hospitalized at the liver transplant institute and who had liver transplant. A total of 106 patients and 115 isolates with Enterobacterales blood infection were analyzed. Isolates that were resistant to at least one of the carbapenem antibiotics were included in the study. The Centers for Disease Control and Prevention defines CRE as Enterobacterales that are resistant to at least one of the carbapenem antibiotics or produce a carbapenemase. According to this definition, these isolates were considered CRE[7]. The strains were detected at different stages of bacteremia and only one of the strains with similar types and phenotypic resistance in the same attack were included in the study. Fosfomycin resistance genes and carbapenamase genes were screened for in 33 patients and 34 CRE isolates.
The blood culture analyses were performed in Render BC128 fully automated blood culture systems in compliance with the manufacturer’s instructions (Shandong Huifa Electronics Technology Co., Jinan, Shandong, China). The isolates were typed according to “Matrix-Assisted Laser Desorption/Ionization Time of flight, Mass Spectrometry” (VITEK® MS, bioMérieux, Marcy l’Etoile, France)[8].
The European Committee on Antimicrobial Susceptibility Testing (EUCAST) v12.0 guideline breakpoints were used to screen the isolates for antimicrobial susceptibility[9]. The gradient strip test (E-test®, bioMérieux) was conducted to screen for carbapenem susceptibility[10]. Colistin susceptibility of isolates was screened for by broth microdilution method. Susceptibility to other antibiotics was investigated by the Kirby Bauer disk diffusion method[11].
The Preparation of Isolates
Bacterial isolates were stored at -80 °C until further analyzes. Before the study, subculturing was performed using blood agar with incubation of 18 to 24 h at 35 °C.
The Determination of Fosfomycin Susceptibility
The agar dilution method, which is the gold standard method according to EUCAST, was used to examine the susceptibility of the isolates to fosfomycin. The threshold minimum inhibitory concentration (MIC) for fosfomycin in EUCAST is 32 mg/l; thus, the strains with MIC ≤32 values were regarded as susceptible, and those with MIC of >32 values as resistant. The fosfomycin agar dilution test was carried out in Mueller-Hinton agar with a series of dilutions achieved by adding fosfomycin antibiotic powder (Sigma-Aldrich, St. Louis, MO, USA) and 25 mg/l glucose-6-phosphate (Sigma-Aldrich, St. Louis, MO, USA). The control strain, E. coli ATCC25922, was used in each study.
The DNA Extraction of Isolates and Screening for fosA, fosA3, fosC2, and Carbapenemase Genes by PCR
DNA isolation of bacteria isolates was performed using a QIAamp DNA midi kit (Qiagen, Hilden, Germany) on a QIAsymphony automated DNA extraction machine (Qiagen, Hilden, Germany). The DNA extracts were kept at -20 °C until they were used PCR.The fosA3, fosA, and fosC2 resistance genes were examined by the in-house PCR method. To do so, the following primers were utilized: F-5’-ATC TGT GGG TCT GCC TGT CGT-3’, R-5’-ATG CCC GCA TAG GGC TTCT-3’ for fosA, F-5’-GGC ATT TTA TCA GCA GT-3’, R-5’-AGA CCA TCC CCT TGT AG-3’ for fosA3 and F-5’-CGA GCC AAG ATT ACT GT-3’, R-5’-AAC GAT TCC AAA CGA CT-3’ for fosC[12]. The presence of blaIMP, blaVIM, blaOXA-48, blaKPC, and blaNDM genes of the isolates was determined by the in-house multiplex PCR method. For the carbapenemase genes, the primers used were those described by Poirel et al.[13].
DNA amplification was carried out by a thermocycler, GeneAmp PCR System 9700 (Applied Biosystems, Waltham, MA, USA). The amplification process for fosA was as follows: the first denaturation for 5 min at 94 °C, 30 cycles and denaturation for 30 s at 94 °C, annealing for 30 s at 59.5 °C, 1-min primer extension at 72 °C, and final extension for 10 min at 72 °C. Primer unification was set to 57.5 °C for fosA3 and 50.5 °C for fosC2[12, 14]. The amplification cycles for carbapenemase genes were as follows: 10 min at 94 °C and 36 cycles of amplification consisting of 30 s at 94 °C, 40 s at 52 °C, and 50 s at 72 °C, with 5 min at 72 °C for the final extension.
The amplicons were electrophoresed for 1 h at 100V in 1.5% agarose gel and stained by ethidium bromide before their UV images were taken using a Kodak Gel Logic 200 (1708x1280, Kodak Company, USA).
Molecular Typing of the CRE Strains
The clonal relationships between the isolates of the patients (n=34) were investigated by performing an arbitrary primer sequence-based PCR (AP-PCR). AP-PCR analysis was performed according to the protocol described by Grundmann et al.[15] with a minor modification. Briefly, AP-PCR reaction mixture (50 μl) contained 100 ng of genomic DNA, 100 pmol of M13 primer (5’-GAG GGT GGC GGT TCT-3’), 0.5 unit of Taq DNA polymerase (Vivantis Technologies, Malaysia), 200 μmol deoxynucleoside triphosphate mix, and 10X amplification buffer. Band profiles were analyzed using the GelCompar II software system (version 6.5; Applied Maths, Sint-Martens-Latem, Belgium). The dice correlation coefficient was used to calculate similarity for band analysis, and the UPGMA (“Unweighted Pairwise Grouping Mathematical Avenging” mathematical mean grouping of unweighted pairs) method was used for cluster analysis.
The Validation of Amplified Items by Sequence Analyses
The sequence analyzes for amplicons were made with Bigdye Terminator V3.1 cycle sequencing by ABI Prism 310 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA). The obtained fosA gene sequences were aligned by the Ugene software (http://ugene.unipro.ru). In order to screen for fosA subtypes, GenBank and Basic Local Alignment Search Tool (http://blast.ncbi.nlm.nih.gov/Blast) included in National Center for Biotechnology Information were consulted.
Statistical Analysis
All data obtained in the study were analyzed using the Statistical Packages for the Social Sciences (SPSS) version 26.0 (IBM SPSS®, Armonk, New York, USA). Mean values (±standard deviation) and median values for quantitative variables and numbers (percentages/rates) were used for qualitative variables. The Fisher’s Exact test was used to compare categorical variables, and a p value of <0.05 (two-sided) was considered statistically significant.
Results
Table 1 shows the demographic and clinical characteristics of the 33 patients included in this study.
The median number of days (minimum-maximum) from transplantation to diagnosis of CRE bacteremia in the 33 patients was 64.5 (1-3469) days. The overall case fatality rate in the first month after liver transplantation was 15.2% (5/33) and the one year case fatality rate was 30.3% (10/33). The case fatality rates in the first 28 days, one year, and two years after the diagnosis of bacteremia were 24.2% (8/33), 45.5% (15/33), and 48.5% (16/33), respectively.
The most common cause for bacteremia was an intraabdominal infection, which accounted for 85.3% (29/34) of the cases. Intraabdominal infections included biliary system infections, surgical infections, and peritonitis. Three cases were diagnosed with urinary tract infection, and two were diagnosed with pneumonia.
Results for Antibiotic Susceptibility of Isolates
The 34 isolates comprised E. coli (41.2%) and K. pneumoniae (58.8%), and all strains were ertapenem resistant. The prevalence of meropenem resistance was 14.3% in E. coli and 75% in K. pneumoniae. The prevalance of imipenem resistance in E. coli and K. pneumoniae was 14.3% and 45%, respectively. Table 2 shows in vitro susceptibility to antibiotics studied in here.
Of the 34 isolates, 21 (61.8%) produced carbapenemases. OXA-48 was the most common carbapenemase produced by E. coli (8/8, 100%) and K. pneumoniae (11/13, 84.6%). The distribution of carbapenemases among E. coli and K. pneumoniae isolates is presented in Table 3. The prevalence of carbapenemase-producing gene was 55.6% (5/9) in fosfomycin-susceptible isolates and 64% (16/25) in fosfomycin-resistant isolates. There was no significant difference between fosfomycin susceptibility and carbapenemase gene positivity (p=0.704). The AP-PCR results detected no dominant epidemic strain in the 34 CRE isolates.
Of all the isolates included in our study, 73.5% were resistant to fosfomycin. In the E. coli isolates, the rate of fosfomycin susceptibility was 64.3%, whereas no fosfomycin-susceptible isolate was found in the K. pneumoniae isolates (p<0.001). Figure 1 shows the resulting MIC values.
MIC: 32 mg/L (cut-off fosfomycine in European Committee on Antimicrobial Susceptibility Testing).
MIC: Minimum inhibitory concentration
All isolates were screened for the fosA, fosA3, and fosC2 resistance genes by PCR, and isolates that were positive for any of the resistant genes were validated by sequence analysis. The fosA gene was was detected in two (5.8%) isolates but fosA3 and fosC2 were not present in any isolate. The two fosA gene positive isolates were K. pneumoniae isolates, and the MIC values of fosfomycin for these isolates were 512 and >512. In addition, one of the isolates with the fosA gene was OXA-48 positive and the other was NDM positive. Figure 2 shows gel electrophoresis images of strains that were positive for fosA. The presence of the fosA resistance gene in these two isolates was confirmed by sequence analyses.
Discussion
Bacteremia is one of the critical complications that causes morbidity or mortality in solid organ transplanted patients. During the first year after liver transplantation, the rate of bacteremia is about 10-23%[16]. Carbapenem resistance in Enterobacterales isolates has recently increased, and most of Enterobacterales cause nosocomial infections. This kind of resistance raises challenges in treating the bacterial infection using antimicrobials and causes a high level of mortality. In a multi-centered cohort study focusing on CRE infections, 13% of the cases had bacteremia, K. pneumoniae was observed in 57%, and E. coli occurred in 12% of the isolates[17]. In our study, the distribution of the isolates was 58.8% K. pneumoniae and 41.2% E. coli in the liver transplant patients with bacteremia.
The rate of mortality is higher among patients with MDR bacterial infections than in those without[18]. Between 2015 and 2021, a total of 1206 patients over 18 years of age underwent liver transplantation in our hospital. The survival rate one month after the transplantation was 90% and it dropped to 79.4% after one year. One month and one year survival rates were lower in the group of patients with CRE bacteremia. Our findings suggest that circulatory infections caused by resistant bacteria like CRE bacteremia increase the rate of mortality. However, other causes such as unsolvable surgical complications, long-term stays in care units/hospitals, and long-term use of antibiotics can not be ruled out. Therefore, further studies are required to to investigate the causes of high mortality.
Jafarpour et al.[19] reported the occurrence of urinary infections (22%), intraabdominal and surgical infections (20.6%), pneumonia (20.6%), sepsis (19.1%), primary circulatory infection (2.2%) and other infections (8%) in liver transplant patients with bacterial infections. Another study reported aprevalence of 10.2% for pneumonia, 69.4% for intraabdominal and biliary system infections, and 20.4% for urinary infections in liver transplant patience with Entrobacterales infections[20]. Our findings indicate that the most common cause of infections was intraabdominal infections accounting for 85.4% of the cases.
A previous study reported antibiotic resistance rates of imipenem (97.2%), ertapenem (95.8%), meropenem (90.8%), and amikacin (24.1%) in E. coli isolates; and ertapenem (100%), imipenem (99.4%), meropenem (95.3%), and amikacin (41.8%) in K. pneumoniae isolates[21]. In a study on KPC positive K. pneumoniae isolates, meropenem, fosfomycin, amikacin, and colistin resistance rates were 100%, 41%, 76% and 65% respectively[22]. In our study, the rates of resistance in K. pneumoniae isolates were: fosfomycin (100%), ertapenem (100%), meropenem (75%), imipenem (45%), amikacin (41.2%), and colistin (5.6%). In E. coli isolates, the rates for resistance were: ertapenem (100%), fosfomycin (35.7%), meropenem (14.3%), imipenem (14.3%), amikacin (7.7%), and colistin (0%). Antibiotic resistance rates obtained in this study were similar to those of other studies, and resistance rates are higher in K. pneumoniae than in E. coli.
Antibiotic options to treat resistant bacterial infections are gradually becoming limited for critical patients, including solid organ transplant recipients. Intravenous fosfomycin (fosfomycin sodium) has a broad antibacterial effect on Gram-negative and positive bacteria, and when it is used with other antibiotics, such as beta-lactam, aminoglycoside, and glycopeptide, a synergistic effect might arise[23]. Clinical and microbiological responses have been reported when fosfomycin was used in combination treatment regimes[24, 25]. It is now widely accepted that fosfomycin is most effective when used as a part of antibitoc combination regimes to treat extensive-drug resistance (XDR) Enterobacterales infections in critical patients[26].
In KPC-producing K. pneumoniae isolates, fosfomycin resistance rates of 36.4% and 60.8% have been reported[27, 28]. A recent study by Zarakolu et al.[29] reported that from CRE from the blood cultures, fosfomycin susceptibility was 95.1% in E. coli and 69.4% in K. pneumoniae. Carbapenemase genes were detected in 88.1% of CRE isolates, and OXA-48 was the most common carbapenemase type. In another multi-centered study conducted in Turkey, in KPC-producing Enterobacterales isolates, the prevalence of fosfomycin resistance was 43.7%, and the rates for Klebsiella spp. and E. coli were 46.8% and 21.1%, respectively. OXA-48 is the most frequently detected type of carbapenemases[30]. In a study of E. coli isolates from Iran, OXA-48 and VIM were the most reported types of carbapenamases[31]. In another study, the most common VIM carbapenemase genes were detected in CRE isolates[32]. From the findings of these studies, we might conclude that fosfomycin resistance rates are higher in Klebsiella strains in CRE. Emerging fosfomycin resistance of carbapenem-resistant K. pneumoniae isolates is of concern. When all isolates were separately evaluated by agar dilution for fosfomycin resistance, fosfomycin resistance had a prevalence of 35.7% in E. coli, 100% in K. pneumoniae, and 73.5% in all the isolates combined. In our study, 61.8% of the isolates produced carbapenemases, and OXA-48 was the most common type. OXA-48 carbapenemase is endemc in Turkey. The higher prevalence of isolates with fosfomycin resistance than in previous studies might be due to our special study population, which included liver transplant patients with many risk factors like long-term hospital stays and intensive antimicrobial treatment exposure.
The resistance against fosfomycin, which is an important alternative treatment, has increased dramatically. The effect of fos genes synthesizing fosfomycin-inactivating enzymes on the resistance and spread of fosfomycin seems to be at a low to medium level. However, the occurrence of fos genes in mobile plasmids might be one of the effective means by which fosfomycin resistance spreads. Moreover, the co-occurrence of fos genes with other antibiotic resistance genes might increase XDR strains[6]. In Egypt, Elshimy et al.[33] examined E. coli isolates and detected the fosA resistance gene. In another study on the fosA gene in fosfomycin-resistant K. pneumoniae isolates, the rate of fosA was 40%[34]. Among the fosfomycin-resistant isolates in the carbapenem-resistant K. pneumoniae, no fosA and fosC2 genes were detected apart from 15% fosA3[27]. In a similar study, it was reported that the fosA3 gene had a prevalence of 77.2% and the fosA gene had a prevalence of 1.8%, but the fosC2 gene was absent[35]. A previous study in Iran reported that the fosA, fosA3, and fosC2 resistance genes were absent in all the isolates[36]. In other studies on fosfomycin-resistant E. coli isolates, Wachino et al.[37] reported that fosA3 was present in 20% and fosC2 in 10% of the isolates, Mueller et al.[38] reported that fosA3 occurred in 23.5%, Loras et al.[39] reported that fosA3 occurred in 5.1% of the isolates. In East Asian countries, the fosA3 gene had a higher prevalence. It was concluded that this result was caused by the bacteria in the animals in that region carrying and spreading this resistance gene[40].
In Turkey, there are only a few studies so far on fosfomycin resistance genes. In a study by Nigiz et al., fosA3 was detected in only one of 16 fosfomycin-resistant isolates, and fosA and fosC2 genes were not detected[41]. Another study reported absence of fosA, fosA3, and fosC2 resistance genes in bacterial isolates[42]. A recent study on E. coli and K. pneumoniae proliferating in blood cultures, reported that the fosA3 gene was not detected in the fosfomycin-resistant strains[29]. In this study, fosA was detected in 10% of all K. pneumoniae isolates and in 5.8% of total strains. No fosA3 and fosC2 genes were detected in any isolates. The isolates with the fosA gene were phenotypically fosfomycin-resistant, and had a high MIC value. In addition, one of the isolates with the fosA gene was OXA-48 positive and the other one was NDM positive. The recent detection of fos resistance genes in in Turkey suggests that plasmid-mediated fosfomycin resistance may increase in the near future in our country as well as in the whole world.
Study Limitations
The generalizability of the results is limited due to the fact that the study was conducted from a single center and to a specific patient group. In addition, the study was also limited by the small number of cases used.
Conclusion
Only two fosA resistance genes were detected in the carbapenem-resistant isolates studied but the prevalence of fosfomycin resistance was higher. These findings suggest that antibiotic resistance can be spread through other resistance mechanisms besides fos genes. This is the first study in Turkey of fosfomycin susceptibility and resistance genes on blood culture isolates of liver transplanted patients. Further studies are recommended to investigate treatment response and mortality rates after fosfomycin-based combined treatments for CRE-infected patients.
Ethics
Ethics Committee Approval: This study was retrospectively approved by Health Sciences, İnönü University, as a non-invasive clinical study and numbered 2020/1103, date: 13.10.2020.
Informed Consent: Informed consent was not obtained as only study isolates were tested, and electronic data of patients were used without identity information.
Peer-review: Externally peer-reviewed.
Authorship Contributions
Surgical and Medical Practices: Y.G., E.S.T., Concept: Y.G., E.S.T., Design: Y.G., E.S.T., B.O., Y.B., Data Collection or Processing: Y.G., E.S.T., Analysis or Interpretation: Y.G., E.S.T., Y.B., Literature Search: Y.G., Y.B., S.Y., Writing: Y.G.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that this study received no financial support.
