Outbreak of KPC-2 producing Serratia marcescens MDR in a tertiary hospital in the Midwest region, Brazil

About 700,000 deaths per year worldwide are caused of Healthcare-related infections (HAI). Result in significant patient morbidity and mortality, being is ranked the fourth leading cause of death in Brazil. Usually, HAI are caused by multidrug-resistance microorganisms (MDR), as well as Serratia marcescens, that is associated with healthcarerelated infections causing high patient mortality and morbidity. This paper describes an outbreak of S. marcescens MDR carrier gene blaKPC-2 in a university hospital, with the need to broadly combat horizontal transmission of this bacterium between patients, as well as the need to optimize treatment with other antimicrobial classes due to resistance of this microorganism to polymyxins. Ten isolates were analyzed for the presence of carbapenemic resistance gene blaKPC-2. This gene was detected in all isolates, with a corresponding high patient mortality rate, highlighting the importance of its molecular detection, which corroborates the resistance of S. marcescens to carbapenem antibiotics. The detection of this gene is necessary due to the importance to broadly combat horizontal transmission of this bacterium between patients, as well as the need to optimize treatment with other antimicrobial agents due to its resistance to polymyxins.


Introduction
Healthcare-related infections (HAI) are considered a serious medical and social problem, resulting in significant patient morbidity and mortality. They are caused by multidrug-resistance microorganisms (MDR), causing about 700,000 deaths per year worldwide, and may reach ten million by the year 2050 (IACG, 2019). In developing countries, HAI may be up to 20 times higher than in developed countries (Allegranzi et al., 2011). In Brazil, HAI occupy the fourth leading cause of death (Silva & Lincopan, 2012).
Serratia marcescens (S. marcescens) can be isolated in the skin, tracheal secretion, urine and feces of critically ill patients, also found in contaminated invasive medical devices, intravenous and topical solutions, liquid soaps and intensive care unit (ICU) air conditioning, favoring the emergence of colonization of patients and hospital outbreaks (Polilli, et al., 2011). According to Quezada-Aguiluz et al. (2020), this opportunistic pathogen of interest to public health has been neglected, even with high rates of antimicrobial resistance.
S. marcescens stands out for its intrinsic and acquired resistance to a variety of antibiotic classes, including β-lactams, aminoglycosides, quinolones, macrolides and polypeptides (Sandner-Miranda et al., 2016). Thus, the drugs that present greater efficacy in the treatment of infections caused by multiresistant Gram-negative bacteria are the carbapenems however, there has been an increase in resistance rates to these antibiotics in recent years in this group (Cristina et al., 2019), which can be explained by the fact that carbapenemase (KPC) genes are often inserted through conjugative plasmids, which makes S. marcescens resistant to carbapenems, causing great concern for the reduction of therapeutic options in AIH (Silva et al., 2015).
This study describes the outbreak of S. marcescens MDR carrier gene blaKPC-2 in a university hospital, with the need to broadly combat horizontal transmission of this bacterium between patients, as well as the need to optimize treatment with other antimicrobial classes due to resistance of this microorganism to polymyxins.

Methodology
A total of ten isolates were obtained from ten ICU patients, medical and surgical wards of a tertiary public university hospital in Cuiabá, Mato Grosso, Brazil's Midwestern Region, which has 118 beds. Samples were collected from patients as hospital routine between September and December 2018 ( Table 1)  Genomic DNA extraction from isolates was performed by the phenol-chloroform method according to Sambrook and Russel (2004). The extracted DNAs were submitted to PCR of the KPC-2 carbapenemase resistance gene. The oligonucleotide sequence used was that described by Yigiti et al. (2001). Subsequently, the PCR product was purified with the GE Healthcare Life Sciences illustra ExoProStar Kit and used in the sequencing reaction along with the BigDye Terminator Ready Reaction Cycle Sequencing (Applied Biosystems) in auto sequencer 3500 Genetic Analyzer (Applied Biosystems).

Results and Discussion
Of the ten patients, six were male (60%) and four female (40%). The average age of the patients was 58.8 (± 18.56) years. The clinical data of the patients are described in Table 1, and the patients presented severe intercurrent diseases demonstrating the opportunistic infection character of S. marcescens (Polilli et al., 2011).
In this study, the mortality rate was 40% (4/10), similar to other reports that varied from 14 to 60% (Arslan et al., 2010;Milisavljevic et al,. 2004), requiring a larger study relating the search for resistance genes of isolates and mortality, as HAI along with antimicrobial resistance, especially carbapenem resistance, increase patient mortality and morbidity rates, as well as their costs and length of stay (Kaleem et al., 2010).
The best result was glycylcyclines (tigecycline) with only 20% resistance, however the occurrence of SdeXY-HasF efflux pumps in S. marcescens decreases their activity as well as gallbladder excretion that may make treatment difficult. of urinary infections (Hornsey, 2010;Peleg & Hooper, 2010).
Only one patient was resistant to all classes of antimicrobials. In addition to this, two patients had intermediate sensitivity to tigecycline and one patient with intermediate sensitivity to gentamicin, gentamicin and tigecycline being the only antimicrobials to which isolates were susceptible. All patients who died had susceptibility to gentamicin and tigecycline, except one patient who had intermediate sensitivity to gentamicin (Table 2).  (Silva et al. 2015).
The bacteria of the Enterobacteriaceae family that extended-spectrum β-lactamase producing and resistant to carbapenem antibiotics have been classified by the World Health Organization (WHO) as critical pathogens for which further research is urgently needed (Tacconelli et al., 2018). In Brazil, other genes such as blaIMP-10, blaSME-4 and blaTEM1 have also been described, emphasizing the need for investigation of carbapenemic resistance genes due to the great importance of this antimicrobial class in the treatment of, especially in a healthcare setting where carbapenems are primarily used as a last-line treatment for gram-negative MDR bacterial infections HAI (Cayô et al. 2017;Margate et al. 2015;Silva et al. 2015). Source: Personal archive.
Due to the intrinsic resistance of S. marcescens to polymyxins (Silva et al. 2015;Denervaud-Tendon et al., 2017), finding the presence of carbapenemases becomes essential for the choice of the most appropriate antibiotics in order to achieve therapeutic success.

Conclusion
We observed that S. marcescens isolates from this study caused high mortality with high resistance profile. Thus, genomic studies should be performed in order to clarify whether such microorganisms are clonal in Cuiabá, alerting to the importance of S. marcescens in the Midwest region and possibly in other regions of Brazil. It is concluded that rapid detection methods should be performed in reference health units to verify early detection of resistance genes and to improve antimicrobial treatment, reducing the horizontal transfer of antimicrobial resistance genes.

Approvals and ethical statements
The research project was approved by the Research Ethics Committee of the Júlio Muller University Hospital, and was registered with the National System of Ethical Evaluation of Human Research Projects (CAAE 17331119.2.0000.5541).