Emergence and Clonal Spread of KPC-2 Producing Clinical Klebsiella Aerogenes Isolates From a Teaching Hospital in Tucuman, Argentina.

Klebsiella aerogenes is a nosocomial pathogen associated with drug resistance and healthcare-associated infections. We pursued this study to investigate an outbreak of clinical carbapenem-resistant K. aerogenes(CRKA) in an argentinian tertiary hospital which persisted for 4 months despite aggressive infection control measures. The primary goals aimed to evaluate the molecular characteristics and the clonal relationships among the CRKA isolates. Methods We characterized CRKA isolates by multiplex PCR and PFGE. The information was integrated with clinical and epidemiologic data. Results The 14 CRKA strains were disseminated in an adult intensive care unit (50%) and ve different wards. In patients who received antimicrobial treatment, 8 staggered to directed treatment, mainly with amikacin(6/8) and/or carbapenemes(5/8). The overall mortality was 42.8%, and the attributed mortality to CRKA infection was 21.4%, strains showed high rates of resistance to most of the antimicrobials without resistance to Amikacin and Tigecycline, and carried the bla KPC-2 , bla SHV-2 and bla CTXM-15 genes. The PFGE indicated 2 distinct groups; 12/14 CRKA isolates associated with the dominant subgroup A and likely to be primarily responsible for the ﬁ rst isolation and subsequent dissemination in the hospital.


Introduction
Klebsiella aerogenes (formerly described as Enterobacter aerogenes) is a ubiquitous member of the Enterobacteriaceae family and a signi cant nosocomial pathogen associated with drug resistance and a wide variety of infections including pneumonia, bacteremia, urinary tract and surgical site infections 1,2 . K. aerogenes infections can arise endogenously (gastrointestinal ora) or be acquired from surroundings in the facility where the patient is admitted (horizontal transmission through colonized healthcare workers, contaminated devices/shared equipment, other patients etc.), with the most critical risk factor for acquiring infection being prolonged broad-spectrum antibiotic administration 3 . Risk factors for K. aerogenes infections include prolonged stay at healthcare facilities especially for patients who are immunosuppressed, on mechanical ventilation or harbor foreign devices 3 . Numerous hospital ward outbreaks in both pediatric and adult populations due to K. aerogenes have been described associated to a common source 4 or spread via patient-to-patient transmission [5][6][7] . A particularly high frequency of hospital ICU outbreaks was continually reported from Western Europe in a period between the 1990s and early 2000s, that were largely attributed to the spread and endemic establishment of a clonal K.
aerogenes strain harboring the extended-spectrum β-lactamase TEM-24 (bla TEM-24 ) 1,8 . Within the US and other regions across the globe, Klebsiella aerogenes has also been reported along with Klebsiella pneumoniae, Enterobacter cloacae and Escherichia coli to be among the frequently isolated carbapenem resistant Enterobacteriaceae 9,10 . K. aerogenes strains harboring plasmid-borne serine carbapenemases have been described in the US and worldwide, while metallo-β-lactamases and OXA-48 have been reported in Europe, Asia and Brazil 1,11 . However, the primary mechanisms underlying resistance to carbapenems in K. aerogenes are considered carbapenemase independent and attributed to chromosomal AmpC β-lactamase over-expression and mutations affecting membrane permeability 1 .
Considering the importance of K. aerogenes in causing healthcare-associated infections we pursued this study to investigate an outbreak of clinical carbapenem-resistant Klebsiella aerogenes (CRKA) in an argentinian tertiary hospital which persisted for 4 months despite aggressive infection control measures.
The primary goals aimed to evaluate the molecular characteristics and the clonal relationships among the CRKA isolates.

Materials And Methods
Setting, study design and K. aerogenes strains This study was conducted in a teaching hospital in Tucuman, Argentina (500 beds) with approximately 3000 admissions/day, with three 18-bed intensive care unit/s, serving the greater north area of Argentina.
A total of 14 K. aerogenes strains collected during June-September 2018 were selected for the study and corresponded to a single sequential and non-duplicated CRKA strain isolated during the course of hospitalization. The study was part of Hospital Infection Control Committee activities; as such, no informed consent was needed from the patients. Pertinent clinical and epidemiological information were obtained through review of patient medical records and the laboratory information system and are shown on Table 1. Clonal relationships among the CRKA isolates Molecular typing was performed by pulsed-eld electrophoresis (PFGE). Isolates were typed by PFGE of SpeI-digested total genomic DNA (TaKaRa, Tokyo, Japan), and the DNA fragments were separated by electrophoresis on 1% SeaKeam®Gold agarose (Lonza, Rockland, ME, United States) in 0.5X TBE (45 mM Tris, 45 mM boric acid, 1.0 mM EDTA; ph 8.0) buffer using the CHEF Mapper XA PFGE system (Bio-Rad, United States) at 6 V/cm2 and 14ºC, with alternating pulses at a 120º angle in a 5-20 s pulse time gradient for 19 h. DNA patterns were interpreted according to Tenover et al 14 . Strains were considered to be the same clone (type) if they showed ≥75% genetic identity, or fewer than three fragment differences on the PFGE pro les.

Results
In a 4-month period in 2018, there was an increased incidence of cultures for carbapenem-resistant K. aerogenes (CRKA) disseminated in an adult intensive care unit (50%) and ve different wards at the hospital.
A total of 14 patients colonized/infected with CRKA were studied, and their clinical/epidemiological characteristics as demographics and mortality data, unit location, carbapenem exposure, procedures, and surgical histories are shown on Table 1. Patients were admitted in a range of 0-44 days previous to the CRKA detection, in the majority were male patients (93.8%) with an average age of 50 years (23-69 years).
The rst identi ed case (Patient 1/ward 10) had a past medical history signi cant for intraabdominal infection ( Figure 1, Table 1). The temporal association of subsequent positive cultures from patients in the ICU 1 and other different wards at the hospital, prompted an outbreak investigation ( Figure 1). In response to the event, infection prevention implemented universal contact precautions, distribution of educational materials pertaining to hand hygiene and equipment disinfection were performed. Infected patients were cohorted to one side of the intensive care unit (ICU1) with dedicated nursing staff and antibiotic stewardship efforts were also reinforced during the study period. Despite interventions, 6 additional ICU1 patients had positive CRKA cultures between June and September 2018 ( Figure 1, Table   1). Of the total 14 positive cultures from unique patients, 10 were clinical specimens (one respiratory, three urinary, four soft tissue, one bone and one blood sample) and four were rectal surveillance cultures.
Epidemiological investigation of possible risk factors among the patients for developing CRKA infection/colonization was performed. K. aerogenes isolates from colonized or infected patients were also included in the study for context and comparison. Cases were not found to be associated with a speci c ward. After October 2018, no CRKA were isolated in either clinical or surveillance specimens. Active surveillance in the UCI was discontinued in the end of December 2018 and the outbreak was deemed to have subsided.
The scores of Mc Cabe and Charlson (excluding 2 patients whose information was not available) averaged 2.25 and 3.25, respectively, and indicate a population at medium/high risk of mortality associated with nosocomial complications.
According to CLSI (2014) and EUCAST breakpoints, there were high rates of resistance to most of the antimicrobials tested without resistance to Amikacin and Tigecycline. (Table 2).
To confirm the colistin resistance, broth microdilution (the gold standard method) was used to test the isolates, 4 (28,6%) showed resistance (MIC50= 2 µg/ml and MIC range 0,25-16 µg/ml) ( Table 2). All 14 isolates carried the bla KPC-2 as well as the bla SHV-2 and bla CTXM-15 genes. The bla TEM-24 gene was detected in 45% of the isolates and no PCR product was obtained for the other bla genes investigated. The mcr-1 gene was not detected in the colistin resistant isolates.
The PFGE results indicated that the CRKA isolates belonged to 2 distinct PFGE groups (types A and B), with type A being the most dominant including 12 of the K. aerogenes isolates (Figure 2). The four K. aerogenes isolates resistant to colistin belonged to clonal group A. In our study, the association of KPC-2 with other β-lactamases was evident in all the isolates.

Discussion
The rapid spread of KPC-producing enterobacteria is a major clinical and public health concern and continue epidemiological surveillance is necessary. These broad-spectrum β-lactamases are increasing in new locations worldwide, indicating an ongoing process 15 . The Pan American Health Organization (PAHO) reports that Argentina is one of the countries with the most "pandrug resistant" nosocomial isolations of Latin America 16 . Besides the numerous efforts made at local or national level to control the spread of these bacteria, the rapid dissemination of carbapenem-resistant enterobacteria constituted a clinically relevant problem of our region. Tucuman is situated, in the north of Argentina (NOA), within a multi border area limiting with Bolivia, Chile and Paraguay. Since 2006 an active monitoring for carbapenem-resistant Gram-negative bacteria detection is carried out in our Department.
This study was initiated in order to establish the molecular epidemiology of CRKA strains isolated from patients in our hospital following an outbreak event. During the period between June and September 2018 we informed the Infection prevention team regarding the extent of the transmission event and effectiveness of infection control interventions.
Focusing on the patients a high percentage had comorbidities and risk factors such as prolonged hospitalization and previous use of broad-spectrum antibiotics as potential risk factors for the CRKA acquisition. The average time of hospitalization was 16 days, patients were admitted in a range of 0-40 days previous to CRKA detection denoting the high hospital stay. The Intensive Care Unit was the most common site of acquisition, in line with previous reports [17][18][19] .
According to the revision of clinical histories empirical antimicrobial treatments applied were poor due to multiple reasons: multi-resistance of the strains studied, medical personnel lack of knowledge of the magnitude of the outbreak and limited availability of other therapeutic options (tigecycline and fosfomycin) in the nosocomial pharmacy. These data are in line with Hao et al., who also identi ed the presence of severe disease and prior use of antimicrobials, primarily cephalosporins and quinolones, as risk factors associated to KPC-2 K. aerogenes infection 17 .
The soft tissue and the urinary tract (30%) were the most common sources of clinical samples, followed by abdominal liquid (20%), while other authors reported respiratory samples and blood as the prevalent clinical sources 17,20 .
Antimicrobial susceptibility testing confirmed resistance to piperacillin/tazobactam, ciprofloxacin and carbapenemes in all isolates, results in line with Hao et al., 19 who reported similar resistance rates against piperacillin/tazobactam, ciprofloxacin and carbapenemes and higher than 75% to amikacin and gentamicin. None of the isolates presented resistance to tigecycline, results not in line with Tavares et al., 21 who reported approximately 60% of the K. aerogenes isolates as non-susceptible.
In this study, four isolates (28.6%) were found to be resistant to colistin, in coincidence with Tavares et al., 21 who reported 26,3% of colistin resistance between the K. aerogenes isolates also included in the predominant pulse type. Hao et al., 19 found only one isolate resistant to colistin; there were, indeed, some reports of colistin resistance in K. aerogenes during this time. Since colistin is generally considered a "last-line" antibiotic, this emerging resistance is highly concerning and worth highlighting. To our knowledge, this is the first report of K. aerogenes strains resistant to colistin in our region. All polymyxinresistant K. aerogenes isolates belonged to clone A. This is a worrisome fact (it may indicate the adaptation of this clone and possible spread) and mainly warrants a more judicious application of this antimicrobial agent.
In clinical CRKA strains, carbapenem resistance has been associated with either carbapenemase production or coupling of adaptive mutations affecting membrane permeability and AmpC hyperproduction, Szabó et al., in the study of the resistance mechanism in CRKA isolates, indicated that efflux pumps are not involved despite a previous report of their important role in carbapenem resistance and moreover, porin dysfunction is probably not the main mechanism of resistance in these isolates 22 . In Brazil the KPC enzymes have been reported in E. cloacae complex and K. aerogenes isolates 21,23 the same as in the present study, highlighting the prevalence of bla KPC in K. aerogenes isolates. Other authors included bla KPC-2 , bla KPC-3 , bla OXA-48 , bla NDM-6 and bla NMC-A genes in these isolates 17 .
The ESBL rate in the K. aerogenes detected was 100% and corresponds to CTX-M-15 and SHV-2. CTX-M-2is the most common subtype in our country 24 and is found in several species of Enterobacteriaceae 25,26 . However, recent studies in coincidence with us, have reported an increase in the number of Enterobacter species producing CTX-M-15 24,27 .
In the present study, isolates of K. aerogenes showed monoclonal spread in line with other authors 1,18,28 . The 12 CRKA strains in the dominant subgroup A were likely to be primarily responsible for the first isolation and subsequent dissemination in the hospital. The outbreak characteristic data showed prolonged hospitalization and previous use of broad-spectrum antibiotics as potential risk factors for the acquisition of CRKA across the hospitalized patients. In conclusion it is extremely important to perform phenotypic and genotypic identi cation of early genetic resistance mechanisms in these isolates, not only from infections sites but also from colonization, in order to prevent the spread of these MDR isolates, which may present different resistance genes.

Declarations Funding
There was no in uence of the funding organisation on analysis or interpretation of the described data.
There review was undertaken as an additional activity parallel to a PhD project.

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. Table12.docx