Molecular Genetic Epidemiology of an Emerging Antimicrobial-Resistant Klebsiella pneumoniae Clone (ST307) Obtained from Clinical Isolates in Central Panama

Klebsiella pneumoniae has been among the main pathogens contributing to the burden of antimicrobial resistance (AMR) in the last decade, and K. pneumoniae AMR strains predominantly cluster in the ST258 clonal complex. However, ST307 is emerging as an important high-risk clone. In Central America, there have been few studies on the molecular epidemiology of the K. pneumoniae strains involved in infections. Materials and Methods: We conducted an epidemiological study in three reference hospitals in the central region of Panama, using isolates of K. pneumoniae involved in infections, and identifying their AMR profile, associated clinical risk factors, and molecular typing using a multilocus sequence typing (ST) scheme. Results: Six STs were detected: 307 (55%), 152, 18, 29, 405, and 207. CTX-M-15- and TEM-type beta-lactamases were identified in 100% of ESBL-producing strains; substitutions in gyrA Ser83Ile and parC Ser80Ile were identified in all ST307s; and in ST152 gyrA Ser83Phe, Asp87Ala, and parC Ser80Ile, the qnrB gene was detected in all strains resistant to ciprofloxacin. Conclusions: We present the first report on ST307 in three reference hospitals in the central region of Panama, which is a high-risk emerging clone and represents a public health alert for potential difficulties in managing K. pneumoniae infections in Panama, and which may extend to other Central American countries.


Introduction
Klebsiella pneumoniae is an opportunistic enterobacterium that is responsible for infections in susceptible populations, such as the elderly, neonates, and patients with diabetes or immunosuppressed states, as well as healthcare-associated infections [1]. K. pneumoniae represents one of the main pathogens contributing to the burden of antimicrobial resistance (AMR) and associated deaths [2], which is why the World Health Organization (WHO) includes K. pneumoniae with AMR in the list of Priority 1 (critical group) pathogens resistant to antibiotics [3].
K. pneumoniae strains with AMR mainly belong to certain sequence types (STs) that represent high-risk international clones. In the last decade, hospital outbreaks have been predominantly attributed to isolates belonging to the clonal complex 258 (i.e., ST258, ST11, and ST512) [10]. However, ST307 has emerged in different parts of the world, with involvement in hospital outbreaks in Africa, Asia, Europe, and the Americas [11]. In Latin America, ST307 has been described in the last decade in countries such as Colombia [12], Brazil [13,14], Mexico [15], and Ecuador [16].
K. pneumoniae ST307 is frequently associated with AMR, especially because it tends to carry plasmid-mediated ESBL CTX-M-15 and carbapenemases, which hydrolyze carbapenems. These plasmids also carry genes conferring resistance to aminoglycosides and quinolones, emerging globally as an important AMR organism [4]. The most commonly identified carbapenemases in ST307 are KPC-2, KPC-3, OXA-48, and NDM-1. In addition, resistance to combined beta-lactam inhibitors, such as ceftazidime/avibactam and colistin, has been reported [17]. Previous studies have identified chromosome-and plasmid-encoded mechanisms of colistin resistance [18,19]. Currently, in Central America, knowledge of the epidemiological and molecular characteristics of the circulating K. pneumoniae strains remains limited. A study conducted on K. pneumoniae isolates carrying blaKPC carbapenemase collected between 2006 and 2015 in various Central American countries, including Panama, identified the high-risk clone ST258 [20].
It is known that a better understanding of the epidemiology of circulating K. pneumoniae strains is crucial to identify the factors contributing to the spread of resistance genes and is essential for the development of specific strategies for infection prevention, control, and new therapeutic strategies [21]. The purpose of this study was to characterize, through molecular epidemiology, the strains of K. pneumoniae isolated in the clinical laboratories of hospitals in the central region of Panama during 2018-2019 and involved in infections in patients prior to the COVID-19 pandemic.

Study Design
We conducted a prospective epidemiological study between October 2018 and November 2019 in three reference hospitals in central Panama: Hospitals A and B located in the Azuero region, and Hospital C located in the province of Veraguas. The three hospitals represent the main centers providing medical and laboratory care in central Panama.

K. pneumoniae Isolates
During the study period, we included K. pneumoniae samples that (a) were isolated from various outpatient and inpatient samples within the first 48 h of admission, (b) were collected as part of routine patient care procedures, and (c) showed resistance to at least one of the antibiotics routinely tested in hospital clinical microbiology laboratories. In vitro antimicrobial activity was determined using the Vitek2 system (BioMérieux; Marcy l'Etoile, France). The test results were interpreted according to the breakpoints defined by the Clinical Laboratory Standards Institute (CLSI) [22].
A technical sheet was completed anonymously for each sample collected, recording the following risk factors: age, sex, hospitalization for 2 or more days in the previous 90 days, antibiotic treatment in the previous 90 days, personal history of immunosuppressive therapy, home wound care, hemodialysis within the previous 90 days, and outpatient chemotherapy.

Statistical Analyses
Data was recorded in MS Excel (The Microsoft Corporation; Redmond, WA, USA). Data analyses were conducted in Stata v. 11.0 (StataCorp, LLC; College Station, TX, USA). We calculated descriptive statistics and estimates with their respective 95% confidence intervals (CIs). We used Fisher's exact test to compare proportions, and Mann-Whitney's U test to compare medians, setting alpha to 0.05 for statistical significance.

Discussion
K. pneumoniae strains with AMR in the last decade were mainly grouped into certain types of high-risk international clones, predominantly belonging to the clonal complex 258 (i.e., ST258, ST11, and ST512) [11]. However, ST307 is emerging as an important AMR clone. In this study, through molecular analysis with MLST, we identified the presence of the emerging clone ST307 in 55% of K. pneumoniae strains isolated between 2018 and 2019 in three hospitals in the central region of Panama.
ESBL-producing strains of K. pneumoniae have increased in frequency and severity worldwide, causing an impact on the prolongation of hospital stays and delays in appropriate antibiotic therapy, which have led to an increase in healthcare costs [2,30]. We observed that ESBL-producing K. pneumoniae strains mostly belonged to ST307 (67%), identifying the blaCTX-M-15 gene in all ESBL-carrying strains. It has been reported that the movement of plasmids between different species and lineages of enterobacteria represents an important source of AMR; an example of this is the pandemic clone of Escherichia coli ST131, which contributes to the dissemination of ESBL genes (blaCTX-M-15) among Enterobacteriaceae [6,31]. This pandemic clone E. coli ST131, which is a carrier of CTX-M-15, was identified in a recent study in clinical isolates responsible for infections in outpatients and hospitalized patients in Panama, which suggests a high prevalence among Enterobacteriaceae [32].
Most of the global isolates of ST307 described in the literature carry the blaCTX-M-15 gene on FIB-like plasmids and contain several additional AMR determinants responsible for resistance to aminoglycosides, quinolones (qnrB1), and other antimicrobial agents [11,33,34]. These data coincide with our findings, where all ST307 strains were resistant to ciprofloxacin and 83% to gentamicin. Our genetic analyses showed that the qnrB gene was detected in 100% of the ST307 strains, and the QRDR substitutions were observed in gyrA Ser83Ile and parC Ser80Ile in all strains with ST307. Sequencing studies of K. pneumoniae ST307 [11] described that the clone ST307 emerged around 1994 and consists of two deep branching lineages. One lineage containing the gyrA Ser83Ile and parC Ser80Ile substitutions has shown a global distribution and the other lineage, also containing an additional gyrA D87N substitution, has only been present in Texas, United States. One report [11] also proposed there was genomic evidence of between-country movement of patients infected or colonized with isolates of ST307 that belonged to the global lineage. Our data showed that the substitutions observed in the QRDR of the ST307 identified in this study corresponded to the global lineage. This being the first report of clone ST307 in Panama, its origin is not clear. It is plausible that a sensitive strain has acquired a plasmid-carrying blaCTXM-15 from other Enterobacteriaceae (e.g., E. coli ST131), as has previously been identified [32,35], or that a strain of ST307 was introduced before 2018 from another source, which could partly explain the fact that it was distributed in the three participating hospitals, and in different hospitalization wards, such as surgery, internal medicine, the intensive care unit, and outpatient wards.
In a recent study conducted in Colombia with K. pneumoniae isolates, the SHV enzyme (SHV-11 or SHV-1) was identified in all strains studied, as well as other TEM enzymes such as TEM-11 and TEM-1. These data are consistent with the present study, where SHV and TEM were identified in all strains with the ESBL phenotype [12].
In Latin America, the percentage of ESBL-producing K. pneumoniae strains is estimated at 24.7%, a percentage that has increased in the last decade, surpassed only by the Asia-Pacific region [5]. This scenario represents a notable problem, as it demands the use of broader-spectrum antimicrobials, such as carbapenems, resistance to which has already spread worldwide. Correlations between the use of carbapenems in hospitalized patients and the development of resistance have been demonstrated, even after up to 3 months of treatment [36]. The Latin American Antimicrobial Resistance Surveillance Network (ReLAVRA) published a growing trend in K. pneumoniae resistant to carbapenems, with resistance rates reaching an average of 21% [37]. ST307 associates with blaCTX-M-15; however, sufficient indexed literature supports that this lineage could acquire and spread carbapenemases (blaKPC, blaNDM, blaOXA-48, blaOXA-48, blaOXA-181, and blaGES-5) [34]. In Latin America, the first ST307 described was in Colombia in 2015 in a strain of K. pneumoniae carrying blaKPC-2 [12], but it has also been described in Brazil [13,14], Mexico [15], and Ecuador [16]. ST307 behaves as an emerging high-risk clone, whose genetic characteristics contribute to its adaptation to the hospital environment, as well as its potential to acquire carbapenemase-carrying plasmids. The rational use of antibiotics and surveillance are essential to counteract the high potential for plasmid acquisition.
The small number of samples is a noteworthy limitation of this study. The sample size was due, first, to the infrequent request for cultures by the attending physicians and, second, to the limited human and infrastructure resources for processing cultures in clinical laboratories in central Panama.
Limitations aside, this study makes unprecedented contributions to the knowledge of the microbiology and molecular genetic epidemiology of K. pneumoniae strains in Panama and Central America by identifying several STs, including the emerging clone ST307. This draws our attention to the potential difficulties in the treatment of infections originating in hospitals and the community, as well as to the importance of knowing the composition and distribution of antibiotic resistance genotypes, as an important step to establish public policies aimed at limiting the impact of AMR K. pneumoniae infections.