Outbreak of CTX-M-15 Extended-Spectrum β-Lactamase-Producing Klebsiella pneumoniae ST394 in a French Intensive Care Unit Dedicated to COVID-19

Infections caused by extended-spectrum β-lactamase-producing Klebsiella pneumoniae (ESBL-KP) are constantly rising worldwide and are often reported as causative agent of outbreaks in intensive care units (ICUs). During the first wave of the COVID-19 pandemic, bacterial cross-transmission was thought unlikely to occur due to the reinforcement of hygiene measures and prevention control. However, we report here an ESBL-producing K. pneumoniae (ST394) isolate responsible for a nosocomial outbreak in an ICU dedicated to COVID-19 patients.


Introduction
Klebsiella pneumoniae is an important bacterial pathogen responsible for various infections, including nosocomial infections in intensive care units (ICUs). In addition, this bacterial species is a major cause of multidrug-resistant infections worldwide through the production of extended-spectrum β-lactamases (ESBLs) and carbapenemases [1,2].
In March 2020, a huge increase in the number of patients hospitalized in ICUs was observed in many European countries due to the spread of SARS-CoV-2. In France, specific wards dedicated to the management of COVID patients have been implemented in many hospitals. In these wards, infection control and hygiene measures were drastically upgraded (i.e., contact and respiratory precautions) to protect healthcare workers. In this context, the risk of emergence of a nosocomial outbreak was thought to be at its lowest level. However, we report here a large outbreak of CTX-M-15-producing Klebsiella pneumoniae in an ICU dedicated to COVID-19 infected patients between March and June 2020.

Description of the CTX-M-Producing K. pneumoniae Related Outbreak
Between 30 March and 20 June 2020, 101 patients have been admitted to the surgical ICU of Bicêtre Hospital (37 for severe COVID-19 and 64 for other reasons) and 34 patients were colonized and/or infected with a CTX-M-producing K. pneumoniae isolate ( Figure 1A). Temporal and geographic links were established between all patients ( Figure 1B). Among the 34 patients (including 24 patients COVID-19 positive), 18 patients (52.2%) were colonized only, but 16 (47.1%) were also infected (9 patients with sepsis, 4 with pneumonia, one with urinary tract infection, one with meningitidis, and one with surgical site infection). The origin of the nine sepsis was a pneumonia, a urinary tract infection, and a catheterrelated infection for three, two, and one patients, respectively, and was unknown for three patients. Eight of the 34 patients died during their ICU stay.

Bacterial Characterization and Clonal Relationship
Between 30 March and 20 June 2020, 83/515 rectal swab screenings were positive for ESBL-producing K. pneumoniae (from 34 patients). For all these strains, the NG-test CTX-M MULTI tests was positive, revealing the production of a CTX-M-type ESBL. All these strains possessed an identical resistance profile, with a resistance to third-and fourth-generation cephalosporins, aztreonam, piperacillin/tazobactam, amoxicillin/clavulanate, gentamicin, tobramycin, all fluoroquinolones, and co-trimoxazole according to CASFM ( Figure S1). These strains remained susceptible to carbapenems, cefoxitin, temocillin, and amikacin.
MLST analysis identified 29 strains belonging to the ST-394, and one to each ST-39, -45, -48, -147, and -429. On all 29 genomes of CTX-M-producing K. pneumoniae belonging to the ST-394, we used a core genome SNP-based (single-nucleotide polymorphism) approach to create a phylogenetic tree using the K. pneumoniae of the patient 1 as reference. Two strains were considered to be clonally related if they were separated by fewer than 50 SNPs along their common genome. The maximum number of SNPs observed between two strains was 43, confirming the cross-transmission of as a single strain ( Figure 2).

Bacterial Characterization and Clonal Relationship
Between 30 March and 20 June 2020, 83/515 rectal swab screenings were positive for ESBL-producing K. pneumoniae (from 34 patients). For all these strains, the NG-test CTX-M MULTI tests was positive, revealing the production of a CTX-M-type ESBL. All these strains possessed an identical resistance profile, with a resistance to third-and fourthgeneration cephalosporins, aztreonam, piperacillin/tazobactam, amoxicillin/clavulanate, gentamicin, tobramycin, all fluoroquinolones, and co-trimoxazole according to CASFM ( Figure S1). These strains remained susceptible to carbapenems, cefoxitin, temocillin, and amikacin.
MLST analysis identified 29 strains belonging to the ST-394, and one to each ST-39, -45, -48, -147, and -429. On all 29 genomes of CTX-M-producing K. pneumoniae belonging to the ST-394, we used a core genome SNP-based (single-nucleotide polymorphism) approach to create a phylogenetic tree using the K. pneumoniae of the patient 1 as reference. Two strains were considered to be clonally related if they were separated by fewer than

Virulence
Despite the ST-394 CTX-M-15 producing K. pneumoniae isolate resulted in mucoid colonies, the string test was negative for all 34 strains. In addition, none of the seven vir-

Virulence
Despite the ST-394 CTX-M-15 producing K. pneumoniae isolate resulted in mucoid colonies, the string test was negative for all 34 strains. In addition, none of the seven virulence factor genes associated with hypermucous Klebsiella (ybtS, mrkD, entB, rmpA, kfu, allS, and iutA) were found in the genome. Within the ST-394 CTX-M-15 producing K. pneumoniae, two operons involved in iron uptake were present: the ent, fep, and mrk operons. However, these operons are found in other ST of Klebsiella that were not particularly reported as hypervirulent.

Discussion
ESBL-producing K. pneumoniae is mainly transmitted from patient to patient directly by the medical staff's hands, or indirectly via the environment [22]. To prevent transmission, infection control measures, such as additional contact precautions and patient screening, must be implemented. In our study, we have highlighted the dissemination of a ST-394 CTX-M-15-producing K. pneumoniae in the ICU during the COVID-19 pandemic in France. Between 30 March and 20 June 2020, 29 patients were colonized and/or infected with this strain in the ICU. However, strains belonging to other STs (n = 5) did not disseminate in the ICU. Among the 29 ST-394 CTX-M-15 producing K. pneumoniae isolates, 16 (55.2%) were infected (including nine patients with sepsis) and no strain belonging to other STs caused infection. Geographic and temporal links were established between these patients. In addition, whole-genome sequencing analysis confirmed the dissemination of a unique strain of K. pneumoniae belonging to the ST-394. This particular ST was not described in any medical publication as being associated with ESBLs or being particularly virulent. In Europe, K. pneumoniae clinical isolates that produce CTX-M-15 are particularly found in the well-known "high-risk clones" of ST-307, ST-147, or ST-101 [23,24]. Several observations made in the ICU highlighted contributing factors linked to the pandemic context that can explain this massive dissemination of CTX-M-producing K. pneumoniae: surge of critically ill patients with a huge subsequent increase in workload, tight physical spaces and close proximity of patients in the common PAECU, increased pressure exerted on the healthcare system: recruitment of healthcare workers from other hospital services (less familiar with infection control measures), multiple medical and non-medical staff in contact with each patient, adoption of new safety measures such as the use of PPE (designed for self-protection, but also allowing the transmission of micro-organisms between patients as it could create a false sense of security among some users), and concomitant shortage of long-sleeved water-resistant gowns and hydroalcoholic solutions. Various measures were taken to stop this epidemic situation: (i) the reinforcement of gowns and hydroalcoholic solutions stock, (ii) the bleaching of siphons and sinks, and (iii) the education of medical and non-medical staff. The dissemination of this strain stopped quickly after the implementation of these measures. As previously reported in most of bla CTX-M-15 -carrying plasmids, bla OXA-1 , bla TEM-1 , and aac6'-Ib-cr genes were associated with bla CTX-M-15 in this ST-394 K. pneumoniae isolate [18,19,21,25]. Usually, only 6% to 7% of the patients colonized with ESBL-producing K. pneumoniae further develop an infection with the same strain [26]. Here, 16/34 (47.1%) patients were further infected with this carriage ST-394 CTX-M-15-producing K. pneumoniae isolate. This particularly high prevalence (7-fold more than expected) of infection among colonized patients led us to look for specific virulence factors. The most studied hypervirulent Klebsiella pneumoniae are known to possess at least few genes among ybtS, mrkD, entB, rmpA, kfu, allS, and iutA [27]. In addition, these hypervirulent clones are reported to form hypermucoid colonies that lead to a positive string test. Here, the string test remained negative, despite hypermucoid colonies. In addition, the genome analysis did not identify any particular virulence factor encoding gene among ybtS, mrkD, entB, rmpA, kfu, allS, and iutA. Of note, the operons ent and fep were found. The ent and fep operons encode enterobactin and ferrienterobactin, respectively. These two siderophores were reported to be strongly implicated in bacterial virulence [28]. Moreover, the mrk operon was also identified in this strain. This operon, and more precisely mrkCDF encoding anchoring for type II fimbriae, was previously reported to be involved in transmigration of the bacteria through the intestinal epithelium [29]. However, these virulence factors are present in most of the K. pneumoniae isolates including ST that are not particularly hypervirulent. Accordingly, further experiments should be performed to link the potential implication of these operons with the high incidence of bacteremia that occurred in patient firstly colonized with this ST-394 K. pneumoniae

Context
The ICU is a 35-bed service located in a large teaching hospital in a suburb of Paris in France, with a total of 953 beds. This surgical ICU is usually divided into six different parts located on the same floor: four critical care units (CCU) with five single rooms in each one, one intermediate-care unit (IMCU) with eight single rooms and one common sevenbed post anesthetic and emergency care unit (PAECU; admission and intensive care for in-hospital and out-of-hospital vital emergencies and high-risk surgical patients). All these units share the same hospital staff. All patients traditionally undergo rectal swab screening for ESBL-producing Enterobacterales at the admission into the ICU and then once a week. During the first wave of COVID-19 pandemic in France (March-June 2020), the entire ICU (except the eight ACU beds) has been dedicated to patients with severe COVID-19 and the capacity of the common PAECU was increased from seven to eighteen beds (by transiently transforming 11 non-ICU PAECU beds into ICU beds). Institutional protocols on personal protective equipment (PPE) donning and doffing were distributed to all healthcare providers, leading to the upgrade of infection control and hygiene measures including contact and respiratory precautions. From 30 March to 20 June 2020, a rise in the number of patients colonized or infected with ESBL-producing K. pneumoniae was observed.

Screening, Bacterial Isolates, and Susceptibility Testing
The screening for ESBL-producing Enterobacterales in fecal samples was performed by plating rectal swabs on a chromogenic and selective agar: ChromID ® ESBL (bioMérieux, Marcy-l'Etoile, France). For all Enterobacterales cultured on this screening medium, the bacterial identification was performed via MALDI-TOF mass spectrometry (Microflex, Bruker Daltonics, Bremen, Germany) and antimicrobial susceptibility testing was performed by disc diffusion according to EUCAST guidelines. An immunochromatographic assay NG-test CTX-M MULTI (NG-Biotech, Guipry, France) [30] was performed on all ESBL-producing K. pneumoniae isolates.
Single nucleotide polymorphism (SNP) analysis was performed on 34 whole genomes using the CSIphylogeny V1.4 server (www.cge.cbs.dtu.dk/services/CSIPhylogeny/, accessed on 3 November 2021) with parameters as follows: select min depth at SNP position at 10X, minimum distance between SNPs at 10 bp, and minimum SNP quality at 30. Phylogeny was performed using the CSIphylogeny v1.4 server and visualised using FigTree software v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/, accessed on 3 November 2021). A SNP matrix was built from data obtained on the CSIphylogeny, in order to compare the CTX-M-producing K. pneumoniae isolates.

Virulence
The high incidence of bacteremia in patients carrying this particular CTX-M-producing K. pneumoniae isolate (ST-394) led us to look for virulence factors. First, a string test was performed to identify a hypermucous phenotype. Then, whole genomes of the 34 strains were analyzed to identify the seven genes of virulence factors known to be expressed by known hypervirulent Klebsiella pneumoniae (ybtS, mrkD, entB, rmpA, kfu, allS, and iutA) [27].