Emergence of carbapenem-resistant ST131 Escherichia coli carrying bla OXA-244 in Germany, 2019 to 2020

The dissemination of carbapenem-producing Gram-negative bacteria is a major public health concern. We report the first detection of OXA-244-producing ST131 O16:H5 Escherichia coli in three patients from two tertiary hospitals in the south-west of Germany. OXA-244 is emerging in Europe. Because of detection challenges, OXA-244-producing E. coli may be under-reported. The emergence of carbapenem resistance in a globally circulating high-risk clone, such as ST131 E. coli is of clinical relevance and should be monitored closely.

The dissemination of carbapenem-producing Gramnegative bacteria is a major public health concern. We report the first detection of OXA-244-producing ST131 O16:H5 Escherichia coli in three patients from two tertiary hospitals in the south-west of Germany. OXA-244 is emerging in Europe. Because of detection challenges, OXA-244-producing E. coli may be underreported. The emergence of carbapenem resistance in a globally circulating high-risk clone, such as ST131 E. coli is of clinical relevance and should be monitored closely.
Escherichia coli of the ST131 lineage is considered as a successful and emerging high-risk pandemic multidrug-resistant E. coli strain [1,2]. Typically, most ST131 E. coli are resistant to third-generation cephalosporins but remain susceptible to carbapenems [1]. We detected three OXA-244-producing ST131 E. coli from patient samples in two tertiary hospitals in the southwest of Germany between January 2019 and June 2020.
The aim of our study was to investigate the genetic diversity of the emerging OXA-244-producing E. coli in the Rhine-Neckar region using whole-genome sequencing.
Black squares: presence, grey squares: absence of antimicrobial resistance genes; red font and red squares: carbapenemase genes.  C. Minimum-spanning tree based on the core genome of all sequenced E. coli in this study. Potential transmission clusters are indicated by the grey circles with SNP differences over the core genome in blue. There was no indication of patient-to-patient transmission (3,413 genes, 108,017 polymorphic sites). Numbers in square brackets indicate the number of isolates belonging to the MLST.

Molecular and microbiological characteristics of OXA-244producing Escherichia coli
Between January 2019 and June 2020, we identified 50 E. coli with phenotypic carbapenem resistance, of which 41 carried a carbapenemase. Nine of the 41 carried bla  , which belonged to three clonal lineages ST38 (n = 5), ST131 (n = 3) and ST167 (n = 1). The isolate belonging to ST167 haboured two carbapenemase genes, bla NDM-5 and bla OXA-244 . Relevant clinical and microbiological characteristics of the nine patients are summarised in Table 1.
The presence of genotypic antibiotic resistance determinants is summarised in Figure 1. Antibiotic susceptibility of all bla OXA-244 is displayed in Table 2 Consistent with published data, the bla OXA-244 genes are most likely to have been integrated into the chromosome because sequencing coverage of the blaOXA-244containing contigs was lower than the overall average sequencing coverage (Figure 2A and 2B) [5,15].
Seven of nine isolates were susceptible to meropenem as indicated by the low MIC in two different AST methods (Tables 1 and 2). One isolate (ST167, P9) carried both bla OXA-244 and bla NDM-5 so that high MIC values for carbapenem were expected. However, the isolate from P3 exhibited an unusually high MIC for meropenem for an OXA-244 producer in both AST methods (≥ 16 mg/L in VITEK and 6 mg/L in E-test) (Tables 1 and 2), for reasons we could not explain. Nevertheless, all nine isolates exhibited positive results in the phenotypic carbapenem inactivation assay (CIM) using meropenem disk (10 µg) with a 2 h inactivation step [16]. Our findings suggest that CIM may be a reliable method to detect OXA-244 producers and should be validated in further studies.

Potential origin and nosocomial transmission of OXA-244-producing ST131 Escherichia coli
SNP analysis to evaluate the clonal relationship of the isolates suggested two potential transmission clusters of patients P1-P2 with five SNP and P4-P5-P8 with 15-24 SNP ( Figure 2C). Patient P1 was colonised with bla OXA-244 E. coli on admission. There was no recent travel exposure so that community acquisition in Germany was possible. P2 stayed in the same ward as P1 with some temporal overlap. P2 was born in the hospital and acquired the colonisation with ST131 OXA-244-producing E. coli during the hospital stay. Nosocomial transmission is a very likely source of acquisition as suggested by the identical genotypic and phenotypic resistance of both isolates of P1 and P2 ( Figure 1 and Table 2). P3 was in a different hospital than P1 and P2. The lack of epidemiological link is consistent with the genomic analysis, which did not indicate transmission. P3 had had contact with the healthcare system in Libya and was initially screened negative on admission in Germany. The bla OXA-244 E. coli was detected in subsequent screenings. However, we cannot fully rule out importation because the sensitivity of the detection method is limited [15].
In the ST38 cluster, there was no epidemiological overlap so that a nosocomial patient-to-patient transmission event is unlikely. Nevertheless, community transmissions caused by clonal dissemination of bla OXA-244 -positive ST38 E. coli in Germany cannot be entirely ruled out [17].

Discussion
The increased incidence in Europe of communityacquired infections with E. coli carrying OXA-244 is of public health relevance as reflected by the rapid risk assessment by the European Centre for Disease Prevention and Control (ECDC) at the beginning of 2020 [18]. Recently, several federal states in Germany reported a rise in detection of community-acquired infections with ST38 OXA-244-producing E. coli [17]. Similar observations have been reported in other European countries [4,5,[19][20][21].
In Germany and other neighbouring countries in Europe, bla OXA-244 is predominantly found in ST38 E. coli [4,17,19,21,22]. Surveillance data from Denmark and France reported the presence of bla OXA-244 in other clonal groups (ST10, ST38, ST69, ST167, ST10, ST361 and ST 3268) [21,23], but to the best of our knowledge the presence of bla OXA-244 in ST131 E. coli in Europe has not been reported before. Besides being responsible for serious extra-intestinal infections, the development of resistance to carbapenems in the ST131 E. coli clonal lineage, is particularly worrisome as carbapenems are often the last line of therapy for life-threatening infections [2,24]. There are no systematic data on the prevalence of carbapenemase-producing Gram-negative bacteria in the Rhine-Neckar region. However, our data suggest a low prevalence of 0.5% (131/27,387 screened patients in the Heidelberg University Hospital in 2019), which is consistent with published data [25].
Peirano et al. reported that the global incidence of carbapenemase-producing E. coli ST131 O25b:H4 of the fimH30/virotype C lineage is increasing, with bla KPC as the most common carbapenem-resistance determinant [2]. In contrast, our E. coli ST131 has the serotype O16:H5 with bla OXA-244 that belongs to the fimH41/virotype C lineage [26]. Although the major lineage of the highly virulent ST131 belongs to the serotype O25b:H4 and fimH30, a murine infection model suggested that ST131 O16:H5 fimH41 is comparable to the H30 lineage in virulence and lethality [27], which implies that the emergence of carbapenems resistance in the H41 ST131 lineage is equally relevant.
Our study has limitations, the detection of OXA-244 producing E. coli is a major diagnostic challenge owing to its low level of phenotypic resistance to carbapenems; therefore OXA-244 producers may be underreported. Nevertheless, our finding suggests that a simple phenotypic assay for carbapenem inactivation combined with routine WGS may be useful to detect low carbapenemase producers, such as OXA-244. In addition, the epidemiological data of our patients were limited so that the exact origin of the OXA-244-producing ST131 E. coli in this study cannot be fully elucidated.

Conclusion
The emergence and dissemination of virulent and dominant E. coli clones with resistance to last-line antibiotics is a public health concern. Our findings emphasise the necessity of adequate surveillance measures and warrant further studies on the epidemiology and transmission dynamics of carbapenem-resistant E. coli both in the hospital and community setting.

Ethical statement
Data and isolates were collected and characterised in accordance to the German Infection Protection Act. The local ethical committee was consulted for the usage of clinical data for scientific purposes and granted waiver of informed consent (S-474/2018).