Dissemination of High-Risk Clones Enterobacterales among Bulgarian Fecal Carriage Isolates

The gastrointestinal tract is an important reservoir of high-risk Enterobacteria clones and a driver of antimicrobial resistance in hospitals. In this study, patients from six hospitals in four major Bulgarian towns were included in this study. Overall, 205 cefotaxime-resistant isolates (35.3%) of Enterobacterales order were detected in fecal samples among 580 patients during the period of 2017–2019. ESBL/carbapenemase/plasmidic AmpC producer rates were 28.8%, 2.4%, and 1.2%, respectively. A wide variety of ESBLs: CTX-M-15 (41%), CTX-M-3 (24%), CTX-M-27 (11%), and CTX-M-14 (4%) was found. The carbapenemases identified in this study were New Delhi metalo-β-lactamase (NDM)-1 (5.4%) and Klebsiella carbapenemase (KPC)-2 (1.5%). Most NDM-1 isolates also produced CTX-M-15/-3 and CMY-4 β-lactamases. They belonged to ST11 Klebsiella pneumoniae clone. The epidemiology typing revealed three main high-risk K. pneumoniae clones (26%)—ST11, ST258, and ST15 and five main Escherichia coli clones—ST131 (41.7%), ST38, ST95, ST405, and ST69. Sixty-one percent of ST131 isolates were from the highly virulent epidemic clone O25b:H4-ST131. Phylotyping revealed that 69% of E. coli isolates belonged to the virulent B2 and D groups. Almost all (15/16) Enterobacter isolates were identified as E. hormaechei and the most common ST type was ST90. Among all of the isolates, a high ESBL/carbapenemases/plasmid AmpC (32.4%) prevalence was observed. A significant proportion of the isolates (37%) were members of high-risk clones including two pan-drug-resistant K. pneumoniae ST11 NDM-1 producing isolates. Due to extensive antibiotic usage during COVID-19, the situation may worsen, so routine screenings and strict infection control measures should be widely implemented.


Bacterial Isolates-Collection and Isolation
The study was conducted in six hospitals-University Multiprofile Hospital for active treatment (UMHAT), Varna; UMHAT, Plovdiv; UMHAT, Pleven, and three hospitals in Sofia during the period of December 2017-June 2019. The fecal samples were obtained from patients (>48 h hospitalization) during the routine diagnostic process and were additionally inoculated on selective MacConkey agar with 1 mg/L cefotaxime and on Chromagar TM KPC (Becton Dickinson, Springfield, IL, USA). Bacterial isolates were identified using routine biochemical identification and were confirmed by VITEK (bioMérieux, Salt Lake City, UT, USA) or Phoenix (Becton Dickinson, Springfield, IL, USA). Hsp60 sequencing [12] was used for species identification of Enterobacter spp. and Klebsiella oxytoca isolates.

Phenotypic ESBL and Carbapenemases Detection
Presumptive ESBL production was detected with the double-disk synergy method [14]. Potential inducible AmpC producers were detected on the basis of antagonism between disks ceftazidime or cefotaxime and amoxicillin/clavulanic acid. In case of nonsusceptibility to carbapenems or/and growth on selective Chromagar TM KPC media (CHRO-Magar, Paris, France), a phenotypic confirmation of carbapenemase production was performed by the KPC, MBL(Metallo-β-lactamase), and OXA-48 Kit (Liofilchem, Roseto degli Abruzzi, Italy).

Isoelectric Focusing and Bioassay
Production and number of β-lactamases were detected and analyzed by analytical isoelectric focusing (IEF) according to the method of Mathew [15] with modifications [16] with Multifor II aparatus (Amersham Biosciences, Freiburg, Germany). The immobilized pH gradient polyacrylamid gel was made with ampholyte Pharmalyte 3-10 (Amersham Biosciences, Freiburg, Germany). β-lactamases were separated on the basis of their pH in the specific isoelectric points (pI). Nitrocefin (500 mg/L) was used to stain the β-lactamase's bands. The hydrolytic activity of individual β-lactamase bands after the isoelectric focusing was assessed by a bioassay as previously described [16]. Two consecutive agar overlays were loaded onto the gel: the first overlay containing the respective β-lactam (cefotaxime 2 mg/L or imipenem 0.5 mg/L) and following 2 h incubation at 35-37 • C, by a second agar overlay containing the susceptible indicator strain E. coli K12:W3110 RifR lac/-/(1.2 × 10 7 CFU/mL). After overnight incubation at 37 • C, growth of the indicator strain on the gel localized the β-lactamase band by which the β-lactam had been inactivated.

ERIC, MLST Typing, and Phylotyping
Clonal relatedness was investigated by ERIC PCR and Multilocus Sequence Typing (MLST). For Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR, ERIC-1 and -2a primers were used as described previously [21]. Genetic similarity was determined using Dice coefficient as similarity measure and the unweighted pair group method with arithmetic mean (UPGMA) (http://genomes.urv.cat/UPGMA/), last accessed on 30 July 2022. A clone was defined as isolates showing 80% similarity.
Pasteur scheme was used for K. pneumoniae MLST typing and Achtman scheme was applied for E. coli. For E. coli isolates, the assignment to allelic numbers and sequence types (STs) was performed according to the MLST database (https://bigsdb.web.pasteur. fr/ecoli/ecoli.html), last accessed on 30 July 2022. Detection of specific O25b-ST131 clone was performed with allele-specific PCR for pabB gene as previously described [22].
For K. pneumoniae isolates, protocols and assignment to allelic numbers and sequencetypes (STs) were carried out as described in the MLST database (Pasteur Institute, Paris, France; http://bigsdb.web.pasteur.fr/klebsiella/klebsiella.html), last accessed on 30 July 2022. A clonal complex was defined as a group of two or more independent isolates that shared six identical alleles.
For E. cloacae complex isolates, primers, protocols, and assignment to allelic numbers and sequence-types (STs) were carried out as described in the MLST database (https: //pubmlst.org/organisms/enterobacter-cloacae), last accessed on 30 July 2022.
Phylotyping was applied for E. coli isolates as previously described [23].
The phenotypic test with meropenem and meropenem/EDTA disks was positive in 11 carbapenem-resistant K. pneumoniae isolates, suggesting class B carbapenemase production (zone around the disk with EDTA is ≥5 mm). Three isolates (two K. pneumoniae and one E. coli) demonstrated increased zones of inhibition by the disk containing meropenem and phenylboronic acid, suggesting class A (KPC) carbapenemase activity.

Molecular-Genetic Identification of β-lactamase
PCR and sequencing revealed the presence of genes, encoding ESBL in 167 (28.8%) isolates, obtained from 580 patients. In one isolate, only bla SHV-1 gene was detected (which suggests possible SHV-1 hyperproduction). Three cefotaxime-resistant isolates, susceptible to carbapenems and with positive DDST did not produce a positive PCR reaction with any of the used ESBL group-specific primers.
The prevalence of ESBL producers in Sofia hospitals was significantly lower (19.7%, 49 ESBL producers from 249 patients) than in the other locations, 35.6% (118 ESBL producers/331 patients) (p < 0.0001). The ESBL producers among the isolates of E. coli and K. pneumoniae  (Table 1).
bla CTX-M-15 was more often detected among E. coli, E. hormachei, C. freundii complex, and M. morganii isolates. Almost all members of CTX-M-9 family (bla CTX-M-9,-14,-27 ), except one isolate that showed bla CTX-M-14 , were produced by E. coli strains. In contrast, bla CTX-M-3 was the prevailing ESBL among K. pneumoniae and K. michiganensis isolates. One K. pneumoniae isolate showed mixed sequences (GA/GT, both A and G in a codon at position 238, GAT is a codon for aspartic acid (specific for bla CTX-M-3 ), GGT is a codon for glycine (specific for bla CTX-M-15 )). All K. pneumoniae isolates were bla SHV positive. Of them, 29 representative isolates were sequenced, and bla SHV-1 (n = 21) and bla SHV-11 (n = 8) were identified. In 71 isolates, bla TEM was found and, later, 10 of them were confirmed by sequencing as bla TEM-1 .
All tested isolates showed only one type of ESBL or/and carbapenemases (have only one CTX or IMP hydrolyzing band) and did not show cefotaxime hydrolyzing bands corresponding to TEM and SHV ESBL β-lactamases. Three isolates with unidentified enzyme type gave bands with pI8.0 (two isolates) and pI9.2 (one isolate). They were without CTX hydrolytic activity.
Among 16 ESBL positive E. hormaechi, C. freundii complex and M. morganii isolates, 5 isolates were tested, and a single cefotaxime hydrolyzing band was detected, pIs corresponded with sequenced enzymes (Table S2).
E. cloacae isolates (n = 16) demonstrated 12 ERIC clusters and 6 ST types. The most common ST was ST90, associated with CTX-M-15 production (Table 4). Table 2. Distribution of MLST types in 65 cefotaxime-resistant K. pneumoniae isolates according to the center and the β-lactamase production.

Discussion
This study reveals a moderate rate of fecal colonization with ESBL producers (28.8%) among Bulgarian patients during the period 2017-2019. The rate of third-generation cephalosporinresistant isolates (mostly due to production of ESBL/carbapenemase/plasmid AmpC) was high (35.3%), but lower than the rate observed in a pilot study on hospital fecal carriage in Varna city, Bulgaria in 2015 (42.5%) [25]. A possible reason could be the multicenter design of the present study. It represents the β-lactamase fecal carriage among Bulgarian patients, hospitalized in three centers in the capital city of Sofia and three other hospitals in main Bulgarian cities (Varna, Plovdiv, and Pleven). In addition, in this study, the rate of ESBLproducing isolates among hospitalized patients in Sofia was significantly lower compared with that in patients from the other centers. The ESBL fecal prevalence in the whole patient group (n = 580) was similar to that in Portuguese hospitals (24%) [26] and higher than that in French hospitals (17.7%) [27]. The observed frequency in our study was much lower than the rate reported from Africa and Asia (>50% in China, Chad, and Egypt) [28]. Our findings are in concordance with the high incidence of third-generation cephalosporin-resistant invasive E. coli and K. pneumoniae isolates (38.6% and 75.7%, respectively) in Bulgaria in 2019, which was the highest in Europe and showed a stable trend during the last 5 years (https://www.ecdc.europa.eu/sites/default/files/documents/Additional-tables-EUEEApopulation-weighted-mean-2019.pdf), last accessed on 10 August 2022. We also found increased nonsusceptibility rates in the tested ESBL fecal isolates to aminoglycosides (50-68%) and quinolones (63-68%) in comparison to the rates found in 2015 (21-57% for aminoglycosides and 15-27% for quinolones) [25]. A similar trend was detected for carbapenem resistance-7% during the period of 2017-2019 and 3% in 2015 [25].
An important finding in the current study is the detection of carbapenemase-producing isolates. Although the detection rate was low (2.4%), it is an indicator that the enteric tract may act as a reservoir for these problematic bacteria. The carbapenem resistance in the present collection of fecal isolates was associated with NDM-1 and KPC-2, detected in K. pneumoniae isolates and in a single E. coli isolate. All carbapenem-producing isolates were obtained from hospitalized patients in Sofia. Interestingly, the frequency of ESBL producers (19.7%) was not high among these patients. The coproduction of NDM-1, CTX-M-15/-3, and CMY-4 enzymes in nine K. pneumoniae isolates is a possible explanation for the high resistance rates, identified in the NDM-1 producing isolates, two of them being pandrug-resistant.
The rates of carbapenemase producers in Europe have steadily increased during the last years in both clinical and intestinal isolates [2,[30][31][32][33][34][35]. The fecal colonization with carbapenemase producers is of high clinical importance because of its possible prolonged persistence over time (387 days) [36].
ST15 is another high-risk K. pneumoniae clone which has commonly been associated with the production of ESBLs, mainly CTX-M-15 [3,7,44], but also is involved in KPC dissemination [30]. In the present study, only four carbapenem-susceptible isolates were identified as ST15 producing CTX-M-15 ESBL, confirming the results in Bulgaria in the last years [25].
Other intestinal multidrug-resistant K. pneumoniae clones found in this study were ST37 and ST17. These clones showed a sustained persistence in Bulgaria. These ST have been already identified in clinical and fecal isolates, associated with CTX-M-15 and KPC-2 production [7,26,45]. ST37 and ST17 have been reported as important worldwide distributed MDR clones. Moreover, they can easily acquire mobile elements that carry carbapenemase genes, providing them an additional advantage [3,7,30].
In the present study, ST353 was presented by a high number of isolates (19%), mainly obtained from patients hospitalized in Plovdiv University Hospital(A). ST353 isolates were associated mainly with CTX-M-3 production. This ST type has been rarely reported. In studies from China and Colombia, authors reported ST353 isolates, producing KPC and OXA-48 [46,47]. ST1198 is also a rare ST. Interestingly, both ST types showed relatedness to ST37, (two-allele difference). A further investigation of ST353 is needed, because of its possible potential to be a high-risk clone.
Five high-risk clones were observed among E. coli isolates with ST131 being the predominant, found in 41.7%. The second most common ST was ST38 (10%). ST405, ST95, and ST69 were presented by single isolates only. The isolates from these high-risk clones represented 57% (n = 59) of all the studied E. coli isolates. All of them belonged to the B2 or D phylogroups. This is an important finding as these phylogroups are associated with a prolonged fecal carriage [48]. This will increase the possibility for members of the high-risk clones to cause both community-and healthcare-associated infections such as urinary tract and bloodstream infections.
Our results showed that the gut could be an important reservoir of highly resistant and virulent bacteria in hospitals. This could increase the incidence of infection, as some reports showed high similarity between fecal isolates and clinical isolates: the report was for transplant patients [55]. In the time of increased antimicrobial usage during the COVID-19 pandemic, an increased frequency of such bacteria could be expected, as the previous antibiotic treatment is one of the most important factors for ST131 dissemination [4,49]. Regular follow-up studies are of great importance.
In our study, E. coli ST38, belonging to D phylogroup, was the second most commonly isolated type, associated with production of a wide range of ESBLs (CTX-M-15, CTX-M-3, CTX-M-14, and CTX-M-27) and the plasmid AmpC enzyme DHA-1. This type has also been reported as a high-risk clone, causing both nosocomial and community-acquired infections, mainly urinary tract infections [4,7]. Another high-risk clone identified in this study is ST95, presented as a single CTX-M-14 producing isolate, also belonging to the virulent B2 phylogroup. The representatives of ST95 have demonstrated an increased virulence and have often caused urinary and bloodstream infection [51]. The detection of ST95 is an indicator for its stable persistence in Bulgarian patients as it was identified in fecal isolates in 2015 [25].
ST69 is an interesting lineage from the D phylogroup, identified in two isolates of E. coli. Recently, it has been found that representatives of ST69 clone carry an intact locus of enterocytes effacement (LEE), coding second bacterial type III secretion systems involved in the pathogenesis of Gram-negative infections [56]. In our study, three CTX-M-15 producing isolates of E. coli were identified as ST405 (D phylogroup). This clone was previously detected in Bulgaria as a carrier of NDM-1 carbapenemases [57]. E. coli ST405 is an emerging urosepsis pathogen, reported to carry bla CTX-M , bla NDM , and a number of virulent genes comparable with O25b:H4-ST131 [58].
In addition to the isolates that represent highly virulent clones, we also detected E. coli isolates belonging to B1 and A phylogroups, which have been reported as commensal gut bacteria [10]; the observed isolates belonged mostly to CC10 and CC155 (11 isolates).
In the group of Enterobacter isolates, ST90 producing CTX-M-15 was detected as the dominant clone. Although no high-risk clones have been defined in Enterobacter spp. so far, ST90 isolates, producing different carbapenemases, were detected in many countries: VIM-1 in Greece, IMP-4 in Canada and UK, and NDM-1 in Romania [59]. Polish authors reported a wide ST90 hospital dissemination [60]. So, we can assume that ST90 E. cloacae complex can be a candidate for an international high-risk clone.
The fecal colonization with ESBL and/or carbapenemase producers from high-risk international clones, associated with significant virulence and invasive potential and multidrug or pandrug resistance, can be an important reservoir not only for difficult to treat nosocomial infections, but also for wide dissemination in the community. Given that this study was performed before the COVID-19 pandemic, we can assume that the increased antibiotic usage during the last three years has further worsened the situation.

Conclusions
In conclusion, a high rate of fecal colonization (32.3%) with ESBL/carbapenemase/plasmid AmpC producers among patients in Bulgarian hospitals was found. A high rate of ESBL producers (28.8%) was detected with a relatively low rate of carbapenemase producers (2.4%). Seventy six isolates (59 E. coli and 17 K. pneumoniae) were members of high risk clones (37%).Twenty six percent of the K. pneumoniae isolates were representatives of high-risk clones such as ST11, ST258 and ST15 K. pneumoniae isolates. A very worrying finding was the detection of two ST11 pandrug resistant isolates, coproducing NDM-1, CMY-4, and CTX-M-15. Among E. coli, five high risk clones (57% of E. coli isolates) were found (ST131, ST38, ST405, ST69, and ST95). The investigated isolates were producers of a wide range of β-lactamases-CTX-M-15, CTX-M-3, CTX-M-27 (reported for the first time in Bulgaria), CTX-M-14, NDM-1, KPC-2, and plasmid AmpC DHA-1 and CMY-2 enzymes. The detected high frequency of ESBL/carbapenemases-producing enteric bacteria before COVID-19 and the dramatically increased selective pressure during the pandemic period will negatively impact the antimicrobial resistance in clinically significant bacterial species. Further studies should closely monitor the future trends. The routine screening for colonization with MDR bacteria at hospital admission and during the hospital stay, especially in high-risk departments, as well as strict infection control measures should be widely implemented in Bulgarian hospitals to limit the further dissemination of problematic multidrug-resistant bacteria.