Epidemiological Profile and Detection of Resistance Genes in Bloodstream Infection in Cancer Patients: High Occurrence of Metallo-β-lactamases in Enterobacteriales


 Bloodstream infections remain one of the most common major complications in cancer patients. The aim of the study was to describe the etiology, phenotypic and molecular epidemiology of ICS isolates from cancer patients. Method: identification and the resistance profile were carried out using the automated biochemical method Vitek 2®. The presence and genes resistant to carbapenemases blaIMP, blaVIM, blaGIM, blaSIM, blaSPM, blaKPC, blaNDM, genes oxacilinase blaOXA-48, blaOXA-58, and the presence of ESBL genes blaSHV, blaCTX, blaTEM for Gram-negatives, as well as, mecA, vanA and vanB for Gram-positives were investigated in blood culture isolates. Result: Escherichia coli and Klebsiella pneumoniae were the most frequent pathogens. The serine-β-lactamase gene blaOXA-48 was the most frequent, followed by MβL blaSIM. blaTEM and blaCTX were the most common among ESBL. The mecA and vanA genes were found in Staphylococcus spp. and Enterococcus faecium, respectively. Candida spp showed high resistance to voriconazole and fluconazole. Conclusion: measures to prevent and control the spread of resistance genes are essential to reduce the risks of morbidity and mortality.

The DNA was extracted according to the protocol of Inácio et al., (2016) 22 , using the extraction buffer (CTAB 2%, NaCl 1.5 M, 100 mM Tris-HCl, 20 mM EDTA, polyvinylpyrrolidone 1%) previously heated to 65 ° C . The cell wall was broken through mechanical agitation (speed 5.5 m / s for 30 seconds) in FastPrep® (BIO 101, Farmingdale, New York, USA) and incubated for 10 minutes at 65°C. The DNA was recovered from a sequential treatment based on alcohol-isoamyl chloroform (24: 1), isopropanole ethanol (70%). Finally, 50µl of MiliQ autoclaved water was added, and the sample was incubated at 37°C for 20min for DNA elution. The extraction products were assembled in para lm (5µL of the extraction product, 2µL GLB and 2 µL GelRed), using the Lambda DNA marker as standard. The DNA was subjected to electrophoresis on 1.5% agarose gel, in a horizontal electrophoresis tank, containing the Tris-Acetate-EDTA (TAE) buffer, with a voltage of 100v for 20min. Subsequently, the DNA was analyzed for quality and quantity in a transilluminator. The genomic material was kept at 4°C until the time of analysis.

Identi cation of fungal isolates
The species were identi ed from the partial sequencing of the D1/D2 domain of the LSU (28S) region of the rDNA using primers NL1 (5'-GCATATCAATAAGCGGAGGAAAAG -3') and NL4 (5'-GGTCCGTGTTTCAAGACGG -3') 23 . The sequencing samples had the PCR products puri ed (GenJET PCR Puri cation -Fermentas, UK), and the sequencing was performed on the Sequencing Platform-LABCEN / CCB (UFPE), according to internal protocols of the partner laboratory.
Identi cation and Antimicrobial Susceptibility Test (TSA) The identi cation and antimicrobial susceptibility test (TSA) were carried out by phenotypic methods by automation. The automated identi cation tests (Vitek ® ) based on the fermentation of carbohydrates, followed the interpretation of the Clinical and Laboratory Standards Institute (CLSI) manual, and the TSA interpretation followed the Brazilian Committee on Antimicrobial Susceptibility Testing (BrCAST) protocol for interpretation of the results.
Extraction of plasmid and chromosomal DNA from bacterial isolates All bacterial isolates were collected from positive blood cultures and subjected to DNA extraction and detection of resistance genes. From the cultures of the isolates, on BHI agar, the DNA was extracted using the PROMEGA ReliaPrep ™ kit (Promega®, São Paulo, Brazil) according to the manufacturer's protocol. The A conventional PCR assay was performed for 55 isolates recovered from 45 patients to detect three families of ESBL genes. The detection of bla SHV , bla TEM , bla CTX-M resistance genes was performed in all Gram-negative bacterial isolates. And the detection of the mcr-1 gene was carried out in everyone who was resistant to multiple drugs. PCR for detection of ESBL was performed using the following protocol for ampli cation; initial denaturation 95ºC to 5 minutes, followed by 30 cycles of denaturation 95ºC to 1 minute, annealing 60ºC to 1 minute and extension 72ºC to 1 minute. A nal extension step was performed at 72ºC at 10 minutes, for each of the studied genes.
And eight families of the metallo-β-lactamase (MβL) genes, (bla KPC , bla GES , bla NDM, bla IMP , bla VIM , bla SPM , bla GIM and bla SIM ) were investigated for the detection of carbapenemases, performed in all Gram-negative bacterial isolates. Using the following protocol for ampli cation; initial denaturation 95ºC at 5 minutes, followed by 25 cycles of denaturation 95ºC at 1 minute, ringing at 1 minute (according to the ringing temperature for each gene) and extension 72ºC at 1 min.
A nal extension step was performed at 72ºC at 10 minutes, table 1.
The strains were also subjected to PCR to detect class D carbapenemases, such as oxacillinase (bla OXA-48 , bla OXA-58 ), using the following protocol for ampli cation; initial denaturation 94ºC at 5 minutes, followed by 30 cycles of denaturation 94ºC at 45 seconds, annealing 52ºC at 45 seconds and extension 72ºC at 1 minutes. A nal extension step was performed at 72ºC at 6 minutes, as shown in table 1.

PCR conditions for identi cation of the mcr-1 gene
The investigation of the mcr-1 gene was performed using the primers described by Lima et al., (2017) 31 . The ampli cation reactions were prepared in a volume of 25 µL per tube, comprising: 1µL of genomic DNA, 1.0U of the Taq DNA polymerase enzyme (Promega), 2 mM of each dNTP, 2.5 mM of MgCl2 and 3.5 pmol of the primers. The ampli cation was performed under the following thermal cycling conditions: 94ºC for 10 minutes followed by 30 cycles of 94ºC for 30 seconds, 58ºC for 30 seconds and 72ºC for 2 minutes, with a nal extension of 72ºC for 10 minutes. The PCR products were visualized on a 1% agarose gel stained with ethidium bromide.
The ampli ed PCR products were stained with ethidium bromide and visualized by electrophoresis on 1% agarose gels, using the System L-Pix EX photographic documentation system (LoccusBiotechnology, Brazil).
The consensus strings were edited using the Sequencher 4.7 program and then submitted to the BLAST tool from GenBank (National Center of Biotechnology Information, http://www.ncbi.nlm.nih.gov) to search for similar strings. The obtained sequences were aligned with con dence sequences deposited in the database and analyzed phylogenetically with the aid of the MEGA-x program.

Detection of pathogens in the bloodstream
Were recovered 55 clinical isolates from blood culture samples from 45 cancer patients, 38 Gram-negative, 13 Gram-positive and four yeast. Of the BGN, the most frequent were Escherichia coli (36,8%) and Klebsiella pneumoniae (28,9%). Among the Gram-positive, the most frequent were Staphylococcus aureus (38,5%) and Staphylococcus epidermidis (23%). Four patients had infections due to Candida species (graphic 1). Polymicrobial bloodstream infection was identi ed in three cases. As Escherichia coli, Klebsiella pneumonia, Candida parapslosis. Coagulase negative Staphylococcus (SCN) and Candida tropicalis. And nally, Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae.
In general, when analyzing the percentage of sensitivity of BGN, we observed that 78,9% were sensitive to amikacin and meropenem, and 76,3% to gentamicin and 68,4% to ertapenem.
Resistance to clindamycin, sulfamethoxazole / trimethoprim and levo oxacin were observed in S. aureus and S.epidermidis. Resistance to amikacin and vancomycin was observed only in Enterococcus faecium (table 3).
Of the 55 isolates, 28 were MDR (50,9%), while six (10,9%) bacterial isolates were sensitive to all antibiotics tested and three (5,45%) of the isolated pathogens were resistant to all classes of antibiotics tested.
Of the Gram-positive bacteria, ve were S. aureus, three were coagulase-negative Staphylococcus (three were S. epidermidis, one S warneri and one Streptococcus salivarus, an unidenti ed SCN), and two Enterococcus spp. The presence of the mecA gene was detected in 6/13 (46,15%) isolates, Staphylococcus aureus and coagulase negative Staphylococcus (S. epidermidis) and Enterococcus faecalis. Only one Enterococcus faecium isolate had the vanA gene (1/13) (7,7%). The vanB gene was not detected in any of the studied isolates (table 4).

Detection of fungal ICS
Four species of Candida spp. were found in this population. Resistance to uconazole was observed in 3/4 isolates. One Candida tropicalis isolate presented caspofungin as a sensitive dose dependent. All species showed resistance to voriconazole (table 5).

Discussion
The phenotypic and molecular characteristics of 55 clinical isolates rescued from blood cultures of patients with malignant neoplasms, allowed to observe a higher frequency of BGN (69%) greater than CGP (23,63%), in addition to 7,2% of yeasts. BGNs have been implicated as the main cause of ICS in cancer patients 32,33 .
The most commonly found BGN were Escherichia coli (25,4%), Klebsiella pneumoniae (20%) and Pseudomonas aeruginosa (12,7%), as well as reported in recent studies 34,35,36,37 . Evidence has suggested the phenomenon of bacterial translocation, where enterobacteria cross the intestine wall, migrating into the bloodstream, causing bacteremia in patients receiving cancer chemotherapy 38,39  The occurrence of polymicrobial infections in three cases was associated with neutropenia and the presence of a catheter. In two cases, they were associated with MDR and in one case, an operative procedure was performed. Presence of neutropenia, recent invasive procedures and devices have been identi ed as risk factors for polymicrobial infection of the bloodstream, highlighting that MDR isolates have been more commonly found in polymicrobial infection 44,45 .
Polymicrobial infections were responsible for 5,45% (3/55) of these cases, corroborating the study of ICS in cancer patients in India 46 .
The occurrence of β-lactamase resistance genes has been reported in isolates of polymicrobial bloodstream infection from a patient with acute myeloid leukemia in Brazil, including bla TEM e bla SIM in P. aeruginosa, bla TEM , bla CTX and bla OXA-48 in E coli, bla CTX , bla KPC , bla NDM , bla SIM and bla OXA-48 in K.
BGNs showed greater resistance to aminopenicillins, fourth generation cephalosporins, uoroquinolones, second and rst generation cephalosporins and aminoglycosides, in agreement with the results of a study in India carried out with 66 cases of bloodstream infection 40 . Increased resistance to carbapenems, combinations of beta-lactamase inhibitors, beta-lactamase, aminoglycosides, uoroquinolones and cephalosporins (including high resistance to cefepime) has been observed 41 .
Our study showed (46,15%) of MRSA, including Staphylococcus aureus and Staphylococcus coagulase negative (MRCoNS) species, respectively. Recent studies have observed resistance to methicillin in Staphylococcus spp ranging from 38,4% to 93% 48,7 . Resistance to methicillin has been more frequent in SCN compared to S. aureus 49,50 .
And the occurrence of vancomycin-resistant Enterococcus was 7,7%, similar to that found by Bhat et al., (2021) 40 . The prevalence of vancomycin-resistant enterococci (VRE) has become stable or decreasing after 2012 42 .
MDR infections are worrisome phenomena, particularly in cancer patients 17 , due to low therapeutic options and, consequently, a higher risk of mortality 51 . Among the isolated pathogens, the general frequency of MDR was 50,9% (28/55). The main isolated MDR bacteria were E. coli and K. pneumoniae, which present results in agreement with other studies 49,41 .
A high prevalence of MβL (94,7%) was detected, which seems to express a much higher frequency than those presented by other studies 51,54 . Carbapenemresistant enterobacteriaceae (CRE) has been increasingly prevalent in cancer patients and associated with ineffective empirical therapy 55 . The mechanism of resistance to carbapenems is hydrolysis by carbapenemases that are encoded in plasmids and are highly transmissible. Resistance to carbapenem can also be attributed to mutations or other modi cations that alter the level of production or binding a nity of penicillin-binding proteins 56 . This problem re ects the use of different carbapenems in hospitals, in addition to varying according to geographic location 56 .
The most frequently detected carbapenemase was bla OXA-48 (81,5%). A study from Egypt with cancer patients also found a high prevalence of bla  (68,88%) 54 . Another study involving several European countries showed that E. coli and Klebsiella pneumoniae were the main producers of carbapenemases (KPC, NDM, OXA-48-like or VIM) 19 .
The frequency of the blaoxa-48 gene was detected in 70,83% and 92% in isolates of P. aeruginosa and A. baumannii, respectively in Iran 57 .
Other studies have reported the occurrence of genes that encode subgroups of OXA carbapenemases, including the bla OXA-58 gene 58,59 . The OXA-48 enzyme leads to resistance to carbapenems, limiting therapeutic options, organisms that produce OXA-48 and are intrinsically resistant to colistin when infecting patients can be fatal, cause high mortality 60 .
We detected a high frequency of bla SIM 27/38 (71%) in enterobacteciales. The frequency of bla SIM-1 in Egypt was 48% 61 . And in Brazil, 66.6% were reported in human and animal samples 62 .
We highlight the presence of the bla SPM gene in Pseudomonas spp isolates, as has already been found 28 and in A. baumanni 63 , who described the rst report of the bla SPM-1 gene in Acinetobacter baumannii in Brazil.
Although MRSA is frequent in ICS in the hospital population 64 , few studies mention the occurrence of MRSA in ICS of cancer patients 65 . The frequency of the mecA gene, in which all showed phenotypic resistance to methicillin, was 46,1% (6/13). However, the prevalence of MRSA in cancer patients in hospitals in Turkey was 50%, harboring the mecA genotype 66 . The vanA gene was detected in an isolate of Enterococcus faecium rescued from the bloodstream of a patient with colon cancer, whose phenomenon of bacterial translocation through the permeability of the injured colon mucosa may have acted as a risk factor for the event 67 .
Candida species were found in four samples (7,2%). Candida spp. is the most commonly isolated yeast in ICS 68 . Candida tropicalis was the most frequent species in our study, being commonly found in cancer patients 69  Our result showed resistance to voriconazole in all Candidas spp. The rate of azole resistance among C. tropicalis has been signi cant compared to rates of resistance to micafungin 71 . The antifungal caspofugin showed dose-dependent susceptibility in Candida tropicalis (1/4). Caspofungin has been reported to have potent activity against Candida spp. resistant to uconazole 72 .
The growing scenario related to the emergence of Candida species resistant to uconazole and voriconazole is a worrying scenario. A study in Australia warns of the emergence of azole resistance among C. tropicalis 73 . Voriconazole has been reported as an important agent in the treatment of hyaline fungi and effective for infections caused by Candida species, including those that are resistant to uconazole 74 .
Azole resistance can arise through several mechanisms, including overexpression or alteration of the drug target, positive regulation of drug transporters or cellular alterations that reduce drug toxicity or allow tolerance to drug-induced stress. The activation of membrane-associated e ux pumps that modulate resistance through ABC transporters and main facilitator (MFS), allowing resistance to multiple drugs 75 .
Thus, as the emergence of drug-resistant fungi, the combination of antibiotics with antifungals has been explored to enhance the treatment, as colistin could increase the antifungal activity of uconazole 76 .
The worldwide dissemination of resistant isolates, combined with the few available therapeutic options, has negatively in uenced the treatment and prognosis of patients 77 . Species of Candida albicans, especially emerging ones, have signi cantly in uenced the clinical course of patients around the world. Currently, the most recent guidelines for the treatment of fungal infections have recommended uconazole as a primary therapeutic option to combat these infections. However, there have been increasing reports of increased resistance inherent in this drug that can pose a global health threat 78 .
Antimicrobial resistance is of great concern mainly in the population group vulnerable to infections due to chemotherapy itself or due to factors attributed to the history of cancer. Measures to prevent and control the spread of resistance genes are essential to reduce the risks of morbidity and mortality.

Interest con icts
All authors declare no con ict of interest.

Availability of data and materials
All data generated or analyzed during this study is included in this published article.