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HOSPITAL EFFLUENTS AS A RESERVOIR OF BETA-LACTAMASE- AND CARBAPENEMASE- PRODUCING ENTEROBACTERIACEAE

The aim of this study was to determine the presence of beta-lactamase(bla) producing Enterobacteriaceae in hospital effluent samples from two level II and III hospitals in Lima, Peru. The resistance profile of the isolated bacteria was identified and characterized using the MicroScan system for 18 antimicrobials, and the presence of extended spectrum beta-lactamases (ESBL) (blaCTX-M, blaSHV blaTEM, blaPER) and carbapenemases (blaKPC, blaNDM, blaVIM, blaIMP) resistance genes was determined by conventional PCR. Thirty-two isolates were identified (20 Enterobacteriaceae and 12 gram-negative bacteria). All the isolated bacteria showed multidrug resistance. ESBL (blaTEM) and carbapenemase (blaKPC, blaIMP) genes were found in samples from the hospitals that we evaluated. The release of these microorganisms to public areas and the lack of treatment of the hospital effluents could be an important public health problem.


INTRODUCTION
Multi-drug resistance in bacteria is considered a health problem, due to the high mortality and the lack of therapeutic options (1) . Previous studies have shown that hospital effluents contain antibiotics and multi-drug resistant bacteria (2,3) , especially enterobacteria that produce betalactamases with the capacity to hydrolyze penicillins, cephalosporins, monobactams, carbapenems and beta-lactamase inhibitors (4) . In this sense, hospital effluents are a major reservoir of bacteria with antimicrobial resistance.
Unlike other countries, where hospital effluents are treated in a specialized process (5) , only five hospitals in Peru treat their solid waste, but not their liquid waste (6) . In most cases, hospital effluents flow directly into nearby rivers where people are contaminated by using the water to wash their clothes or for personal hygiene (7) . Therefore, the aim of this research was to determine the presence of extended-spectrum betalactamase (ESBL)-and carbapenemase-producing bacteria in the hospital effluents of two hospitals in Lima and to characterize their resistance profile, to provide evidence that can be used to promote control and biosafety measures.
Motivation for the study: Hospital effluents containing antibiotics and multi-drug resistant bacteria are not treated, and it could result into a public health problem.

Main findings:
The isolated enterobacteria and gram-negative bacteria showed multidrug resistance. ESBL genes (bla TEM ) and carbapenemases (bla KPC and bla IMP ) were found in effluents from level II and III hospitals in Lima.

Implications:
The high presence of enterobacteria and multidrug-resistant gram-negative bacteria that produce bla TEM , bla KPC and bla IMP genes in hospital effluents is alarming and should lead to the implementation of wastewater treatment systems before discharge into the sewer system.

THE STUDY
This was a descriptive cross-sectional study conducted at the Laboratory for Research in Molecular Biology of the Universidad Peruana Unión (LIBM-UPeU). Hospital effluent samples were obtained from two hospitals in Lima.
The level II Huaycán Hospital, located in the district of Ate has emergency and hospitalization services and receives about 647,000 people (8) . The level III Hipólito Unanue Hospital located in the district of El Agustino has outpatient, emergency, hospitalization, and surgery services; and during 2018, about 200,000 medical attentions were carried out in this hospital (9) .
During June and July 2019, Wastewater effluent samples were collected prior to disposal into the sewerage system. Two samples were obtained in 100 mL sterile bottles for each hospital with a difference of 30 minutes between both collections at the same location. Subsequently, they were transported in cold chain (2-8 °C) to the laboratory. The collected samples were diluted with distilled water (1:50 dilution) and 1 mL was inoculated in triplicate in selective media for bacteria with phenotype resistant to penicillins, third-generation broad-spectrum cephalosporins and monobactams (medium, CHROMagar ESBL) and in selective media for bacteria with phenotype resistant to carbapenemases (medium, CHROMagar mSupercarba), according to the manufacturer's instructions (10) . Then, the colonies were reseeded on MacConkey agar, four strains per plate, and incubated at 37 °C for 24 hours. This was done to isolate the colonies and identify them.
Identification and susceptibility profiling was carried out using the MicroScan® automated system (AutoScan-4) and the use of panels for gram-negative bacteria (Dade MicroScan®), following the manufacturer's instructions (10) . Eighteen antimicrobials were used for each strain. The minimum inhibitory concentration (MIC) values were used to interpret the antibiotic resistance profile according to the cut-off points recommended by the Clinical and Laboratory Standards Institute 2020 (11) . Multidrug resistance was defined as the detection of a phenotype resistant to at least one antimicrobial from three or more families (12) . DNA extraction was performed by the bacteria-specific silica gel column-based method with the innuPREP Bacteria DNA kit (Analytikjena, Germany), following the manufacturer's protocol (13) . The isolated DNA was stored at -20 °C until amplification of each gene by conventional PCR.
For the detection of ESBL genes (bla CTX-M , bla SHV , bla TEM , bla PER ) and carbapenemases (bla KPC , bla NDM , bla VIM , bla IMP ), we used conventional PCR in an endpoint thermal cycler (Bio-Rad, USA) (14,15) . The primers used for this procedure are detailed in Table 1. The amplification products were visualized by electrophoresis in 2% agarose gels at 120v for 55 minutes, according to the protocol standardized at LIBM-UPeU.
The results were processed with Labpro software and exported to Microsoft Excel where they were subsequently analyzed. We obtained the number of isolates identified by hospital. A heat map was prepared with the antibiotic resistance profile of the isolated bacteria for each hospital and for the bla gene profile (11) .
The study was classified as risk-free research and did not require informed consent. Data management was carried out under strict confidentiality parameters. This research

DISCUSSION
In this study, all the isolated enterobacteria and gram-negative bacteria were multidrug resistant. Amikacin and tigecycline were the most sensitive antibiotics, whereas 70%100% of the isolates were resistant to ampicillins, cephalosporins, monobactams and carbapenemics. ESBL (bla TEM ) and

III (n = 9) Carbapenemases
Extended-spectrum beta-lactamases II (n = 7) III (n = 9) carbapenemase genes (bla KPC and bla IMP ) were found in level II and level III hospitals, with a higher frequency in the latter level.
Regarding the resistance profile, all enterobacteria and gram-negative bacteria showed resistance to beta-lactam antibiotics, with multidrug resistance in 100% of the isolates.
This differs from the description in a hospital in Brazil, where multidrug resistance was found to be 33.3%, with 38.0% resistance to cefoxitin; 27.0% to ceftazidime; 22.0% to cefepime, 13.0% to imipenem, 11.0% to meropenem and 44.0% to aztreonam (16) . It also differs from another study in to ciprofloxacin (17) .   trimethoprim/sulfamethoxazole, and ciprofloxacin, which occurs mainly because of the plasmid transmission mechanism in these bacteria (16) . Carbapenemics-associated genes were detected in almost 28.6% and 44.4% of the isolates from level II and III hospitals, respectively, with the bla KPC gene predominating in both.
In the study from Brazil, the presence of the bla KPC gene associated with ESBL was also detected (16) . On the other hand, in a university hospital in Switzerland, the researchers found the presence of the OXA-48 gene associated with Escherichia coli and Citrobacter freundii (18) . However, our study highlights the presence of the bla IMP gene associated with Pseudomonas spp.
A higher number of multidrug resistant enterobacteria was found in level III hospital, which could be due to the high demand of a population of more than two million inhabitants in the central and eastern districts of Lima (19) .
In contrast, level II hospital, which cares for a population of approximately 200,000 inhabitants (20) and has fewer specialties, had a lower incidence of enterobacteria.
The limitation found in this study was only one sampling point of hospital effluents was accessible; two more sampling points should have been considered, which correspond to points where urban wastewater is mixed with hospital effluent and urban wastewater already mixed with hospital effluents from the public sewer system (17) . Likewise, the results could not be extrapolated to hospitals of other levels of care and in other provinces of Peru. However, according to the review carried out, this is the first study that identifies the phenotype and resistance genes of enterobacteria from effluents from two Peruvian hospitals.
In conclusion, phenotypic multidrug resistance was found in all isolates. Likewise, we found genotypic resistance to ESBL genes (bla TEM ) and carbapenemases (bla KPC and bla IMP ), with a greater presence in level III hospital. We recommend to carry on further studies on multidrug resistance of bacteria present in hospital effluent wastewater from hospitals of different levels (I, II, III and IV) in Peru to determine the microbiological quality of hospital effluents, and to implement hospital wastewater treatment systems.