Molecular Epidemiology of Global Carbapenemase-Producing Citrobacter spp. (2015–2017)

Diego Nobrega; Gisele Peirano; Yasufumi Matsumura; Johann D D Pitout

doi:10.1128/spectrum.04144-22

. 2023 Feb 27;11(2):e04144-22. doi: 10.1128/spectrum.04144-22

Molecular Epidemiology of Global Carbapenemase-Producing Citrobacter spp. (2015–2017)

Diego Nobrega ^a,^✉, Gisele Peirano ^b,^c, Yasufumi Matsumura ^d, Johann D D Pitout ^b,^c,^e

Editor: Ahmed Babiker^f

PMCID: PMC10101073 PMID: 36847542

ABSTRACT

The emergence of carbapenem resistance is a significant public health concern. The rate of infections caused by carbapenemase-producing Citrobacter spp., particularly C. freundii, is increasing. Concomitantly, comprehensive global genomic data on carbapenemase-producing Citrobacter spp. are scarce. We used short read whole-genome sequencing to describe the molecular epidemiology and international distribution of eighty-six carbapenemase-producing Citrobacter spp. obtained from two surveillance programs (2015 to 17). The common carbapenemases were KPC-2 (26%), VIM-1 (17%), IMP-4 (14%) and NDM-1 (10%). C. freundii and C. portucalensis were the principal species. C. freundii consisted of multiple clones obtained mainly from Colombia (with KPC-2), the United States (with KPC-2, -3), and Italy (with VIM-1). Two dominant C. freundii clones were identified: ST98 was linked with bla_IMP-8 from Taiwan and bla_KPC-2 from the United States, and ST22 was linked with bla_KPC-2 from Colombia and bla_VIM-1 from Italy. C. portucalensis consisted mainly of two clones: ST493 with bla_IMP-4 which was limited to Australia, and ST545 with bla_VIM-31 which was limited to Turkey. Class I integron (In916) with bla_VIM-1 was circulating between multiple sequence types (STs) in Italy, Poland, and Portugal. In73 with bla_IMP-8 was circulating between various STs in Taiwan, while In809 with bla_IMP-4 was circulating between different STs in Australia. The global carbapenemase-producing Citrobacter spp. population is dominated by diverse STs with different characteristics and varied geographical distribution and thus requires continued monitoring. Ongoing genomic surveillance should use methodologies able to distinguish between C. freundii and C. portucalensis.

IMPORTANCE Citrobacter spp. are gaining recognition as important causes of hospital-acquired infections in humans. Among Citrobacter spp., carbapenemase-producing strains are cause of utmost concern to health care services globally due to their ability to resist therapy with virtually any beta-lactam antibiotic. Here, we described the molecular characteristics of a global collection of carbapenemase-producing Citrobacter spp. C. freundii and C. portucalensis were the most common species among Citrobacter spp. with carbapenemases from this survey. Importantly, C. portucalensis was misidentified as C. freundii when using Vitek 2.0/MALDI-TOF MS (matrix-assisted laser desorption/ionization–time of flight mass spectrometry) phenotypic identification, which has important implications for future surveys. Among C. freundii, we identified two dominant clones: ST98 with bla_IMP-8 from Taiwan and bla_KPC-2 from the United States, and ST22 with bla_KPC-2 from Colombia and bla_VIM-1 from Italy. As for C. portucalensis, the dominant clones consisted of ST493 with bla_IMP-4 from Australia and ST545 with bla_VIM-31 from Turkey.

KEYWORDS: carbapenemase-producing Enterobacterales, Citrobacter spp., molecular epidemiology, population-based surveillance, carbapenemase

INTRODUCTION

The continuous rise in infections caused by carbapenemase-producing Enterobacterales (CPE) is a cause of utmost concern to health care services globally (1). Infections caused by CPE are linked to high mortality rates in humans because these isolates are resistant to virtually all beta-lactam antibiotics and often harbor genetic determinants that confer resistance against other drug classes such as aminoglycosides and fluoroquinolones (2). CPEs often contain various plasmid-borne resistance genes, including KPCs, NDMs, and OXA-48-like carbapenemases (3).

Among the CPE, Klebsiella pneumoniae and Escherichia coli represent the main clinical load of infections (3). Nevertheless, Citrobacter spp. are gaining recognition as important causes of nosocomial infections (4). Recent evidence has suggested that the rate of infections caused by carbapenemase-producing Citrobacter spp., particularly C. freundii, is increasing (5, 6). In general, carbapenemase-producing Citrobacter spp. populations are genetically diverse (6), but a few sequence types (STs) such as ST22 and ST19 have recently emerged and demonstrated the potential to become dominant clones in health care settings (5).

To date, information regarding the molecular epidemiology of carbapenemase-producing Citrobacter spp., including its genetic diversity and genetic mechanisms of carbapenem resistance, is limited to regional or countrywide studies. From an epidemiological standpoint, such assessments are valuable to specific geographical areas, but fall short in providing a broader overview from a global perspective, which includes a description of global dominant STs and genetic mechanisms of carbapenem resistance. Here, we described the molecular characteristics of a global collection of carbapenemase-producing Citrobacter species isolates obtained systematically from two global surveillance programs. We report the geographical distribution of STs and provide an in-depth assessment of molecular mechanisms associated with carbapenem resistance in carbapenemase-producing Citrobacter spp.

RESULTS

Global distribution of carbapenemases.

Overall, high (≥20%), intermediate, or resistant (i.e., not susceptible [NS]) rates were found for ertapenem (100%), piperacillin-tazobactam (97%), meropenem (96%), ceftriaxone (93%), ceftazidime (92%), cefepime (92%), and trimethoprim-sulfamethoxazole (59%), followed by tetracycline (33%). Low NS rates (<10%) were found for ciprofloxacin (6%), gentamicin (8%), tobramycin (8%), and amikacin (2%). Isolates were susceptible to tigecycline and colistin. Gentamicin and tobramycin NS rates were associated with aac(3′)-IIa and amikacin rates with armA, rmtC, and rmtD2.

Overall, range of carbapenems MICs were similar for all STs. However, MICs were lower for OXA-48 isolates compared to other carbapenemases (OXA-48 [n = 4]: ertapenem = 2 to 32 μg/mL, imipenem = 2 to 16 μg/mL, and meropenem = 1 to 8 μg/mL; other carbapenemases [n = 82]: ertapenem = 16 to >64 μg/mL, imipenem = 8 to 32 μg/mL, and meropenem = 2 to 32 μg/mL). We detected 81 Citrobacter spp. which were positive for a single carbapenemase and 5 isolates harboring 2 different carbapenemases (Table S2). These isolates were obtained from the following countries: Argentina, Australia, Austria, Belgium, Brazil, Colombia, Egypt, Hungary, Italy, Jordan, Philippines, Poland, Portugal, Serbia, South Africa, Spain, Taiwan, Thailand, Turkey, and the United States. The most common carbapenemase groups included KPCs (n = 29), VIMs (n = 21), and IMPs (n = 20), followed by NDMs (n = 13) and OXA-48-like (n = 8). The OXA-48-like carbapenemases were identified as OXA-48. The most frequent individual carbapenemases consisted of KPC-2 (n = 22), VIM-1 (n = 15), IMP-4 (n = 13), and NDM-1 (n = 9) (Table S2). There was evidence of clustering at the continent level: KPCs were the most frequently detected carbapenemases in the Americas, whereas NDMs and IMPs were prevalent in Southeast Asia and Oceania, respectively (Fig. 1). VIMs were more frequent in Europe and OXA-48 in Turkey (Fig. 1).

FIG 1 — Global distribution of carbapenemase-positive *Citrobacter* spp., with an enlarged Mediterranean insert (map created with MapChart [https://www.mapchart.net/index.html]).

Species and sequence types.

The following 6 species were identified (Table S2, Fig. 2): Citrobacter freundii (n = 51), Citrobacter portucalensis (n = 20), Citrobacter koseri (n = 10), Citrobacter farmeri (n = 3), Citrobacter amalonaticus, and Citrobacter braakii (n = 1 each). The C. portucalensis isolates were reported as C. freundii using MALDI-TOF MS (matrix-assisted laser desorption/ionization–time of flight mass spectrometry).

The most common carbapenemase among C. freundii (n = 51) was KPC-2 (27%), followed by VIM-1 (25%), IMP-8 (14%), KPC-3 (12%), NDM-1 (10%), IMP-4 (8%), NDM-7 (4%), and OXA-48 (2%). C. freundii consisted of 20 different STs which included two dominant clones, namely, ST22 (20%) and ST98 (22%) (Table 1, Fig. 2). The ST22 isolates (n = 10) showed a global distribution but clustered in Italy (n = 3 with VIM-1) and Colombia (n = 4 with KPC-2/3) (Fig. 1). The ST98 isolates (n = 11) also showed a global distribution but clustered in Taiwan (n = 5 with IMP-8) and the United States (n = 3 with KPC-2/3) (Fig. 1).

TABLE 1.

Characteristics of dominant sequence types among carbapenemase-positive Citrobacter spp.

Characteristic	ST98 (n = 11)	ST22 (n = 10)	ST493 (n = 9)	ST545 (n = 5)	Other STs^b (n = 51)	All STs (n = 86)
Geographic location	Global^c	Global^d	Australia	Turkey	Global^e	Global
Citrobacter species	freundii	freundii	portucalensis	portucalensis	Multiple^f	Multiple
Carbapenemases, n (%)
KPC-2	2 (18.2%)	4 (40%)	0 (0%)	0 (0%)	16 (31.4%)	22 (25.6%)
KPC-3	1 (9.1%)	2 (20%)	0 (0%)	0 (0%)	3 (5.9%)	6 (7%)
NDM-1	0 (0%)	1 (10%)	0 (0%)	0 (0%)	8 (15.7%)	9 (10.5%)
NDM-5	0 (0%)	0 (0%)	0 (0%)	0 (0%)	1 (2%)	1 (1.2%)
NDM-7	1 (9.1%)	0 (0%)	0 (0%)	0 (0%)	2 (3.9%)	3 (3.5%)
VIM-1	2 (18.2%)	3 (30%)	0 (0%)	0 (0%)	9 (17.6%)	14 (16.3%)
VIM-4	0 (0%)	0 (0%)	0 (0%)	0 (0%)	1 (2%)	1 (1.2%)
VIM-31	0 (0%)	0 (0%)	0 (0%)	1 (20%)	0 (0%)	1 (1.2%)
IMP-4	0 (0%)^a	0 (0%)^a	9 (100%)	0 (0%)^a	4 (7.8%)	13 (15.1%)
IMP-8	5 (45.5%)	0 (0%)^a	0 (0%)^a	0 (0%)	2 (3.9%)	7 (8.1%)
OXA-48	0 (0%)	0 (0%)	0 (0%)	0 (0%)	4 (7.8%)	4 (4.7%)
KPC-3 + VIM-1	0 (0%)	0 (0%)	0 (0%)	0 (0%)	1 (2%)	1 (1.2%)
VIM-31 + OXA-48	0 (0%)^a	0 (0%)^a	0 (0%)^a	4 (80%)	0 (0%)	4 (4.7%)
Other β-lactamases, n (%)
CTX-M^g	1 (9.1%)	2 (20%)	0 (0%)	0 (0%)	9 (17.6%)	12 (14%)
OXA^h	4 (36.4%)^a	2 (20%)^a	8 (88.9%)	0 (0%)^a	16 (31.4%)	30 (34.9%)
TEM-1	8 (72.7%)	2 (20%)^a	9 (100%)	0 (0%)^a	18 (35.3%)	37 (43%)
SHV-12	2 (18.2%)	3 (30%)	0 (0%)	0 (0%)	7 (13.7%)	12 (14%)

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Significantly different from the dominant sequence type(s) at the 5% level.

Other STs (n) include the following: ST112 (4), ST396 (4), ST11 (3), ST63 (3), ST686 (3), ST900 (3), ST116 (2), ST415 (2), ST535 (2), ST544 (2), ST8 (1), ST18 (1), ST21 (1), ST85 (1), ST91 (1), ST95 (1), ST111 (1), ST214 (1), ST523 (1), ST527 (1), ST528 (1), ST539 (1), ST548 (1), ST549 (1), ST550 (1), ST690 (1), ST710 (1), ST854 (1), ST899 (1), ST901 (1), ST902 (1), ST903 (1), and ST904 (1).

Taiwan (5), United States (3), Philippines (1), Poland (1), and Spain (1).

Colombia (4), Italy (3), Argentina (1), Austria (1), and Thailand (1).

Italy (11), Colombia (8), United States (7), Australia (5), Philippines (5), Argentina (2), Taiwan (2), Thailand (2), Belgium (1), Brazil (1), Egypt (1), Hungary (1), Jordan (1), Portugal (1), Serbia (1), South Africa (1), and Spain (1).

C. freundii (30), C. koseri (10), C. portucalensis (6), C. farmeri (3), C. amalonaticus (1), and C. braakii (1).

CTX-M-15 (6), CTX-M-12 (4), CTX-M-9 (2).

OXA-1 (16), OXA-9 (8), OXA-10 (3), OXA-2 (2), OXA-1 + OXA-10 (1).

The most common carbapenemase among C. portucalensis (n = 20) was IMP-9 (45%), followed by VIM-31 (25%), OXA-48 (20%), NDM-1 (10%), and 5% for VIM-4 and NDM-7, respectively. C. portucalensis consisted of 6 different STs which included two dominant clones, namely, ST493 (45%) and ST545 (25%) (Table 1, Fig. 2). The ST493 isolates (n = 9) with IMP-4 were obtained from Australia and the ST545 isolates (n = 5) with VIM-31 were obtained from Turkey.

The most common carbapenemase among C. koseri (n = 10) was KPC-2 (n = 6) followed by KPC-3, NDM-1, NDM-5, and OXA-48 (1 isolate each). C. koseri consisted of 8 different STs with a global distribution (Table 1, Fig. 2). C. farmeri (n = 3) belonged to ST686 and was obtained from Italy (n = 2 with VIM-1) and the Philippines (n = 1 with NDM-1). C. braakii (n = 1) was obtained from Spain, belonged to ST548, and contained OXA-48. C. amalonaticus was obtained from Italy, belonged to ST710, and contained OXA-48.

Dominant sequence types: antimicrobial resistance determinants and plasmid replicon types.

C. freundii ST98 were positive for IMP-8 (46%, 5 isolates), KPC-2 (18%, 2 isolates), VIM-1 (18%, 2 isolates), KPC-3 (9%, 1 isolate), and NDM-7 (9%, 1 isolate). The frequency of IMP-8 was higher in ST98 compared to other dominant STs (Table 1). IMP-8 positive ST98 were obtained from Taiwan and contained IncFII replicon types, which were absent among IMP-8-negative ST98. ST98 were positive for other antimicrobial resistance (AMR) determinants, including aac(6′)-Ib-cr, aadA1, dfrA14, sul1, and others (Table S3).

C. freundii ST22 were positive for KPC-2 (40%, 4 isolates), VIM-1 (30%, 3 isolates), KPC-3 (20%, 2 isolates), and NDM-1 (10%, 1 isolate) (Table 1). The three VIM-1 positive ST22 isolates were obtained from Italy and were positive for aacA4, aphA15, aadA1b, catB2, dfrA1, sat2, and aadA1. Two of the three VIM-1-positive isolates contained IncC replicons that were absent in VIM-1 negative ST22. No replicons were detected in the remaining isolate. ST22 with KPCs were obtained from Colombia (n = 4), Argentina, and Austria (n = 1 each) (Table 1). Different replicons were detected in KPC-positive ST22, including IncFII, IncX3, IncX5, and IncN (2 isolates each). Most of the KPC-positive ST22 also contained other AMR determinants, including mphA (5 out of 6 isolates), tetD, arr3, and sul2 (4 isolates each). In addition, most ST22 (80%) were positive for aac(6′)-Ib-cr. The NDM-1-positive ST22 was obtained from Thailand and contained qnrB4, tetA, sul1, sul2, aac(6′)-Ib-cr, and aac(3′)-IIa, catB3, mphA, and arr-3 resistance genes (Table S3). The same isolate contained IncN2 and IncR replicons.

C. portucalensis ST493 isolates were positive for IMP-4, obtained from Australia, and contained qacG2, aacA4, and catB3. ST493 isolates were positive for IncA/C, IncFII, IncL, and IncHI2 replicons (Table 1). ST493 also contained tetD, mphE, msrE, sul1, dfrA19, dfrA12, aph(6′)-1d, aadA2, bla_TEM-1, bla_OXA-1, and catB3. The frequency of these resistance determinants was higher in ST493 than in other dominant STs (Table S3).

C. portucalensis ST545 isolates were positive for VIM-31 and obtained from Turkey. Four of 5 isolates also harbored bla_OXA-48. All ST545 isolates were positive for IncFIB, IncFII, IncHI1A, IncHI1B, IncL/M and IncN replicons. In comparison to other dominant STs, ST545 had increased frequencies of AMR determinants, including aadA2, dfrA12, sul1, sul2, mphA, qnrB17, and tetD (Table S3).

The frequencies of other β-lactamases among Citrobacter spp. were as follows: CTX-Ms, 14%; OXAs (non-OXA-48), 35%; TEM-1, 43%; and SHV-12, 14% (Table 1). OXAs were more frequent among ST493 isolates and TEM-1 was more frequent in ST98 and ST493 (Table 1).

Carbapenemase gene flanking regions.

Due to the limitations of short-read sequencing (7), analyses of the immediate carbapenemase gene flanking regions and plasmids harboring carbapenemase genes were insufficient for some of the isolates. We were able to characterize the immediate carbapenemase gene flanking regions for 16/29 isolates with bla_KPC, 13/13 isolates with bla_NDM, 8/8 isolates with bla_OXA-48, and all the class I integrons containing bla_IMP (n = 20) and bla_VIM (n = 21).

The bla_KPC-2 (n = 9) was situated within Tn4401a in a single C. koseri isolate and within Tn4401b in 8 Citrobacter species isolates which belonged to different species and STs. The bla_KPC-3 (n = 7) was situated within Tn4401a (n = 3) and Tn4401b (n = 4) that belonged to different Citrobacter species and STs. The bla_NDM were located on truncated Tn125 elements and situated downstream of ISAba125 among all the isolates. The bla_NDM upstream regions showed significant diversities with various IS family insertions (e.g., IS3, IS5, IS6, IS30 and IS91). All the bla_OXA-48 were situated within Tn1999.2.

The IMP-4-positive isolates (n = 13) were obtained from Australia and consisted of C. freundii ST396 (n = 4) and C. portucalensis ST493 (n = 9). The bla_IMP-4 (12/13) was situated in the class 1 integron In809 that also contained qacG2, aacA4, catB3 (Fig. 3). The remaining bla_IMP-4 was situated in a novel class 1 integron that also contained qacG2, aacA4, bla_OXA-1, catB3, and arr-2 (Fig. 3). Unfortunately, due to the absence of sequence data on the integron 5′ and 3′ ends, a novel integron number was not assigned for this structure.

FIG 3 — Diagram of integrons detected in carbapenemase-positive *Citrobacter* spp. The array of promoterless gene cassettes is indicated by arrows.

The IMP-8-positive isolates (n = 7) were obtained from Taiwan and consisted of C. freundii STs, namely, ST98 (n = 5), ST8 (n = 1), and ST528 (n = 1). The bla_IMP-8 was situated in the class 1 integron In73 among the different STs. In73 also contained aacA4 and catB3 (Fig. 3).

The VIM-1-positive isolates (n = 15) consisted of multiple STs (n = 10) and were obtained from Italy (n = 11), and 1 each from Poland, Portugal, Spain, and the United States. The majority of bla_VIM-1 (n = 9) were situated in a class 1 integron In916 and were obtained from Italy (i.e., ST22, ST112, ST523, ST686), Poland (i.e., ST98), and Portugal (i.e., ST95). In916 also contained aacA4, aphA15, aadA1b, and catB2. The remaining VIM-1 genes (n = 6) were situated in class integrons In179 and In1209 (Fig. 3).

The VIM-31-positive isolates (n = 5) were obtained from Turkey and consisted of C. portucalensis ST545. The bla_VIM-31 gene was situated in the class 1 integron In669 that also contained aacA4. The VIM-4 (n = 1) was present in C. portucalensis ST63 from Hungary and was situated in In1433 which also contained aacA7 and smr2 (Fig. 3).

DISCUSSION

The KPC-producing Citrobacter spp. from this survey were mainly from Colombia (41%) and the United States (31%); VIMs were mainly from Italy (52%) and Turkey (24%), IMPs were mainly from Australia (65%) and Taiwan (35%), NDMs were mainly from the Philippines (46%), and the OXA-48-like carbapenemases were mainly obtained from Turkey (50%). These results are similar to those of previously reported global CPE surveys (8).

VIM and IMP metallo-β-lactamases are rare among CPEs, especially within Klebsiella spp. and E. coli (9). CPE isolates with bla_IMP are endemic in Japan, Taiwan, and Australia (3, 10) while CPE with bla_VIM are mainly found in Italy and Greece (4, 11). Among the Citrobacter spp. from this study, VIMs and IMPs were the 2nd and 3rd most common carbapenemases. VIMs were found in Citrobacter spp. from Turkey and Italy while IMPs were limited to Taiwan and Australia. The Turkish isolates (n = 5) were positive for bla_VIM-31 and belonged to a single clone (C. portucalensis ST545) that contained the class I integron In669. Italian isolates (n = 11) were positive for bla_VIM-1, belonged to multiple C. freundii STs, and contained In916. In916 was also found in Citrobacter spp. from Poland (ST98) and Portugal (ST95). This indicated that identical class I integrons with bla_VIM-1 are circulating between different STs in Italy, Poland, and Portugal. A similar scenario was previously reported with Enterobacter spp. harboring bla_VIM-1 within In916 from Spain, Greece, and Italy (12). The IMP-8 isolates from Taiwan belonged to ST8, ST98, and ST528 that harbored the identical class I integron In73. The Australian IMP-4-positive Citrobacter species isolates belonged to 2 clones, namely, ST396 and ST493, which contained the identical class I integron In809. This in-depth characterization of AMR isolates showed how genomic surveillance using whole-genome sequencing offered an unprecedented level of detail that clarified the underlying differences among CPEs from various countries.

The geographical distribution and carbapenemase types of the Citrobacter spp. from this study were different from those of carbapenemase-producing E. coli obtained from the same surveillance programs over identical periods (2). The most common carbapenemases from the E. coli survey were OXA-181 and NDM-5, while VIMs and IMPs were rare. The Citrobacter spp. results from this survey showed some similarities with carbapenemase-producing K. pneumoniae (13) and Enterobacter cloacae complex (12) obtained from the same surveillance programs. The K. pneumoniae with KPC-2 was obtained mainly from Colombia and the isolate with KPC-3 was mainly from the United States. The E. cloacae complex isolates consisted mainly of VIM-1 isolates obtained from Italy and Greece. Citrobacter spp., K. pneumoniae (1), and Enterobacter spp. (14) are typically hospital pathogens, while E. coli is typically a community pathogen (15). This fact could be partly responsible for the different carbapenemase types and geographical distributions among these species.

C. freundii (60%) and C. portucalensis (23%) were the principal species among Citrobacter spp. with carbapenemases from this survey. C. portucalensis was first reported in 2017 from Portuguese aquatic samples (16) and later from Nigerian leafy vegetables (17). This bacterium is closely related to and often misidentified as C. freundii, especially when using phenotypic identification systems such as Vitek 2.0 and MALDI-TOF MS (18). C. portucalensis with bla_CTX-M-15 (19) and with bla_NDM-1 (18) were previously reported from Brazil (two environmental isolates) and China (one clinical isolate), respectively. In our study, MALDI-TOF MS identified C. portucalensis as C. freundii. C. portucalensis was mainly found in Australia and Turkey, but also in Egypt, Hungary, Italy, Jordan, and the Philippines. The C. freundii population was linked with various STs obtained from Colombia, the United States, and Italy. The population structure of C. portucalensis consisted mainly of two clones, namely, ST493 with IMP-4 (Australia) and ST545 with VIM-31 (Turkey). Our results indicate that the geographical and underlying molecular epidemiology is different between carbapenemase-producing C. freundii and C. portucalensis isolates. Therefore, future surveys should use methodologies that can distinguish between C. freundii and C. portucalensis isolates. This will emphasize the role of C. portucalensis in the global dissemination of carbapenemases among Citrobacter spp. We recommend that identification systems in clinical laboratories be updated to include the routine identification of C. portucalensis. This will enable the clinical microbiology community to determine the overall prevalence, clinical significance, and geographical distribution of this newly described Citrobacter species.

High-risk AMR clones such as K. pneumoniae ST258 (3) and E. coli ST410 (20) contribute significantly to the global spread of CPE. We observed a high diversity of carbapenemase-producing Citrobacter spp. clones that was represented by 37 STs, including 4 dominant clones representing >40% of the total Citrobacter spp. population. C. freundii ST98 and ST22 were global, whereas C. portucalensis ST493 and ST545 were limited to Australia and Turkey, respectively. The dominant STs were linked to different carbapenemase genes. The global carbapenemase Citrobacter spp. population is dominated by diverse STs with different characteristics and varied geographical distributions. ST22 and ST98 isolates with different carbapenemases (VIMs, KPC-2, NDM-1, OXA-48) were previously reported from Spain (21, 22), Tunisia (23) and Germany (24).

We provide pertinent information about the global distribution of carbapenemases and population structure among a large collection of carbapenemase-producing Citrobacter spp. This study has some limitations. We used short-read sequencing to characterize our collection, which limited our ability to fully reconstruct flanking regions and plasmids harboring carbapenemases. Additionally, many countries contributed few isolates, and statistical approaches to deal with clustered data failed to estimate country-adjusted rates of carbapenemases among Citrobacter spp. Countries contributing few isolates may not be fully representative of the population of carbapenemases in Citrobacter spp. from that region. The global distribution and prevalence of carbapenemases will be influenced by the clonal dissemination of isolates during nosocomial outbreaks that might have occurred during the surveillance period.

In summary, carbapenemase-producing Citrobacter spp. are a highly diverse group of bacteria that contain 4 dominant global clones. Carbapenemase-producing Citrobacter spp. are disseminated either clonally and polyclonally depending on the species, ST, and geographical location. We also demonstrated that the newly described C. portucalensis has a global distribution and is likely involved in some of the clonal outbreaks of carbapenemase-producing Citrobacter spp. Our findings greatly contribute to global and local surveillance activities, particularly those in lower- and middle-income countries. Our findings underline the increasing importance of Citrobacter spp. in the global dissemination of carbapenemases.

MATERIALS AND METHODS

Bacterial isolates.

We obtained isolates from two global surveillance programs (2015 to 2017), namely, the Merck Study for Monitoring Antimicrobial Resistance Trends (SMART) and the International Network for Optimal Resistance Monitoring (INFORM) programs. The SMART program includes Gram-negative isolates from intra-abdominal, lower respiratory tract, and urinary tract infections obtained from 55 countries, whereas the INFORM program collects isolates from blood, intra-abdominal, lower respiratory tract, skin, soft tissue, and urinary tract infections obtained from 42 countries. Participating countries are listed in the Appendix (Table S1). Medical centers contributed isolates regardless of their antimicrobial resistance profiles.

The two programs collect around 100 consecutive, nonrepetitive, and clinically relevant (based on local definitions) Gram-negative bacteria per year at each participating center. The isolates underwent phenotypic identification using MALDI-TOF MS (Vitek AMS; bioMérieux Vitek Systems Inc., Hazelwood, MO) and custom microdilution panel susceptibility testing using Clinical Laboratory and Standards Institute guidelines, as described previously (9, 25, 26). Overall, 87,182 Enterobacterales isolates were obtained from 2015 to 2017, of which 2,256 (2.6%) were identified as Citrobacter spp., and 112 (5%) tested nonsusceptible to meropenem (MIC ≥ 2 μg/mL). Meropenem-nonsusceptible Citrobacter spp. were screened for presence of carbapenemase genes (i.e., KPCs, NDMs, OXA-48-like, IMPs, VIMs, and GES), as described previously (9, 26). Citrobacter species isolates that were positive for carbapenemases (n = 86) were included in this study.

“Dominant” sequence types were defined as those representing >5% of the total population of Citrobacter spp. with carbapenemases (27).

Genomic analysis.

Carbapenemase-producing Citrobacter species isolates were sequenced on an Illumina NovaSeq SP with a 300-cycle run, 2 × 150 paired-end reads, and a minimum of 100× coverage. SPAdes 3.15 (28) de novo assembly generated draft genomes. TYGS (the Type [Strain] Genome Server) (29) and an average nucleotide identity (ANI) calculator (30) were used for species identification. The ANI cutoff score for genomes belonging to the same species was ≥95%. ResFinder 4.1 (31), PlasmidFinder 2.1 (32), and INTEGRALL (33), were accessed to identify antimicrobial resistance genes, plasmid replicons, and integron types in draft genomes, respectively. Multilocus sequence typing was done in silico at PubMLST (34). A whole-genome SNP-based phylogeny was built using kSNP 3.0 (35). The following reference genomes were included in our analysis: C. freundii ATCC 8090, C. portucalensis NCTC11104, C. braakii FDAARGOS_1421, C. koseri ATCC BAA-895, C. farmeri FDAARGOS_1423, and C. amalonaticus FDAARGOS_122. C. werkmanii UMH18 was selected as the out-species for this analysis.

Statistical analysis.

We used frequency tables to summarize presence of antimicrobial resistance genes, integron types, and plasmid replicon types for each dominant ST. Initially, we attempted to fit exact logistic regression models for clustered data considering isolates clustered within countries, but the models failed to converge. We then used Fisher’s exact tests to perform pairwise comparisons of each outcome between dominant STs. We adjusted P values for multiple comparisons for each individual outcome using the false discovery rate (36). We considered statistical significance at the 5% level. We used R version 4.1.2 for all analyses.

Ethics statement.

Ethics approval for this study was obtained through the University of Calgary Conjoint Health Research Ethics Board (REB17-1010).

Data availability.

The sequencing data were deposited in the NCBI database (BioProject ID PRJNA882828).

ACKNOWLEDGMENTS

This work was supported by a research grant from the JPIAMR/Canadian Institute Health Research program (no. 10016015) and the National Institutes of Health (no. 10028552).

We thank Merck and AstraZeneca for providing the SMART and INFORM isolates.

The authors have no conflicts of interest pertaining to this study or the writing of the manuscript.

Footnotes

Supplemental material is available online only.

Supplemental file 1

Tables S1 to S3. Download spectrum.04144-22-s0001.pdf, PDF file, 0.1 MB^{(129.4KB, pdf)}

Contributor Information

Diego Nobrega, Email: diego.nobrega@ucalgary.ca.

Ahmed Babiker, Emory University School of Medicine.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplemental file 1

Tables S1 to S3. Download spectrum.04144-22-s0001.pdf, PDF file, 0.1 MB^{(129.4KB, pdf)}

Data Availability Statement

The sequencing data were deposited in the NCBI database (BioProject ID PRJNA882828).

[B1] 1.Pitout JD, Nordmann P, Poirel L. 2015. Carbapenemase-producing Klebsiella pneumoniae, a key pathogen set for global nosocomial dominance. Antimicrob Agents Chemother 59:5873–5884. doi: 10.1128/AAC.01019-15. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2.Peirano G, Chen L, Nobrega D, Finn TJ, Kreiswirth BN, DeVinney R, Pitout JDD. 2022. Genomic epidemiology of global carbapenemase-producing Escherichia coli, 2015–2017. Emerg Infect Dis 28:924–931. doi: 10.3201/eid2805.212535. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3.Logan LK, Weinstein RA. 2017. The epidemiology of carbapenem-resistant Enterobacteriaceae: the impact and evolution of a global menace. J Infect Dis 215:S28–S36. doi: 10.1093/infdis/jiw282. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4.Arana DM, Ortega A, Gonzalez-Barbera E, Lara N, Bautista V, Gomez-Ruiz D, Saez D, Fernandez-Romero S, Aracil B, Perez-Vazquez M, Campos J, Oteo J, Spanish Antibiotic Resistance Surveillance Programme Collaborating Group . 2017. Carbapenem-resistant Citrobacter spp. isolated in Spain from 2013 to 2015 produced a variety of carbapenemases including VIM-1, OXA-48, KPC-2, NDM-1 and VIM-2. J Antimicrob Chemother 72:3283–3287. doi: 10.1093/jac/dkx325. [DOI] [PubMed] [Google Scholar]

[B5] 5.Yao Y, Falgenhauer L, Falgenhauer J, Hauri AM, Heinmuller P, Domann E, Chakraborty T, Imirzalioglu C. 2021. Carbapenem-resistant Citrobacter spp. as an emerging concern in the hospital-setting: results from a genome-based regional surveillance study. Front Cell Infect Microbiol 11:744431. doi: 10.3389/fcimb.2021.744431. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6.Babiker A, Evans DR, Griffith MP, McElheny CL, Hassan M, Clarke LG, Mettus RT, Harrison LH, Doi Y, Shields RK, Van Tyne D. 2020. Clinical and genomic epidemiology of carbapenem-nonsusceptible Citrobacter spp. at a tertiary health care center over 2 decades. J Clin Microbiol 58:e00275-20. doi: 10.1128/JCM.00275-20. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7.Arredondo-Alonso S, Willems RJ, van Schaik W, Schurch AC. 2017. On the (im)possibility of reconstructing plasmids from whole-genome short-read sequencing data. Microb Genom 3:e000128. doi: 10.1099/mgen.0.000128. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8.van Duin D, Doi Y. 2017. The global epidemiology of carbapenemase-producing Enterobacteriaceae. Virulence 8:460–469. doi: 10.1080/21505594.2016.1222343. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9.Kazmierczak KM, Karlowsky JA, de Jonge BLM, Stone GG, Sahm DF. 2021. Epidemiology of carbapenem resistance determinants identified in meropenem-nonsusceptible Enterobacterales collected as part of a global surveillance program, 2012 to 2017. Antimicrob Agents Chemother 65:e0200020. doi: 10.1128/AAC.02000-20. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10.Sidjabat HE, Townell N, Nimmo GR, George NM, Robson J, Vohra R, Davis L, Heney C, Paterson DL. 2015. Dominance of IMP-4-producing enterobacter cloacae among carbapenemase-producing Enterobacteriaceae in Australia. Antimicrob Agents Chemother 59:4059–4066. doi: 10.1128/AAC.04378-14. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11.Lascols C, Peirano G, Hackel M, Laupland KB, Pitout JD. 2013. Surveillance and molecular epidemiology of Klebsiella pneumoniae isolates that produce carbapenemases: first report of OXA-48-like enzymes in North America. Antimicrob Agents Chemother 57:130–136. doi: 10.1128/AAC.01686-12. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12.Peirano G, Matsumura Y, Adams MD, Bradford P, Motyl M, Chen L, Kreiswirth BN, Pitout JDD. 2018. Genomic epidemiology of global carbapenemase-producing Enterobacter spp., 2008–2014. Emerg Infect Dis 24:1010–1019. doi: 10.3201/eid2406.171648. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13.Peirano G, Bradford PA, Kazmierczak KM, Chen L, Kreiswirth BN, Pitout JD. 2017. Importance of clonal complex 258 and IncFK2-like plasmids among a global collection of Klebsiella pneumoniae with bla_KPC. Antimicrob Agents Chemother 61:e02610-16. doi: 10.1128/AAC.02610-16. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14.Stokes W, Peirano G, Matsumara Y, Nobrega D, Pitout JDD. 2022. Population-based surveillance of Enterobacter cloacae complex causing blood stream infections in a centralized Canadian region. Eur J Clin Microbiol Infect Dis 41:119–125. doi: 10.1007/s10096-021-04309-z. [DOI] [PubMed] [Google Scholar]

[B15] 15.Pitout JDD. 2021. Population dynamics of Escherichia coli causing bloodstream infections over extended time periods. mSphere 6:e0095621. doi: 10.1128/msphere.00956-21. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16.Ribeiro TG, Goncalves BR, da Silva MS, Novais A, Machado E, Carrico JA, Peixe L. 2017. Citrobacter portucalensis sp. nov., isolated from an aquatic sample. Int J Syst Evol Microbiol 67:3513–3517. doi: 10.1099/ijsem.0.002154. [DOI] [PubMed] [Google Scholar]

[B17] 17.Igbinosa EO, Rathje J, Habermann D, Brinks E, Cho GS, Franz C. 2018. Draft genome sequence of multidrug-resistant strain Citrobacter portucalensis MBTC-1222, isolated from uziza (Piper guineense) leaves in Nigeria. Genome Announc 6:e00123-18. doi: 10.1128/genomeA.00123-18. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B18] 18.Cao X, Xie H, Huang D, Zhou W, Liu Y, Shen H, Zhou K. 2021. Detection of a clinical carbapenem-resistant Citrobacter portucalensis strain and the dissemination of C. portucalensis in clinical settings. J Glob Antimicrob Resist 27:79–81. doi: 10.1016/j.jgar.2021.04.027. [DOI] [PubMed] [Google Scholar]

[B19] 19.Sellera FP, Fernandes MR, Fuga B, Fontana H, Vasquez-Ponce F, Goldberg DW, Monte DF, Rodrigues L, Cardenas-Arias AR, Lopes R, Cardoso B, Costa DGC, Esposito F, Lincopan N. 2022. Phylogeographical landscape of Citrobacter portucalensis carrying clinically relevant resistomes. Microbiol Spectr 10:e0150621. doi: 10.1128/spectrum.01506-21. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B20] 20.Chen L, Peirano G, Kreiswirth B, DeVinney R, Pitout J. 2022. Acquisition of genomic elements were pivotal for the success of Escherichia coli ST410. J Antimicrob Chemother 77:3399–3407. doi: 10.1093/jac/dkac329. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B21] 21.Villa J, Arana DM, Viedma E, Perez-Montarelo D, Chaves F. 2017. Characterization of mobile genetic elements carrying VIM-1 and KPC-2 carbapenemases in Citrobacter freundii isolates in Madrid. Int J Med Microbiol 307:340–345. doi: 10.1016/j.ijmm.2017.07.001. [DOI] [PubMed] [Google Scholar]

[B22] 22.Pulido MR, Garcia-Montaner A, Lopez-Cerero L, Fernandez-Cuenca F, Gutierrez-Fernandez J, Pascual A. 2022. Identification of a stable chromosomal tandem multicopy of bla_VIM-63, a new bla_VIM-2 carbapenemase. J Bacteriol 204:e0008822. doi: 10.1128/jb.00088-22. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B23] 23.Dziri R, Kuşkucu MA, Arfaoui A, Fethi M, Ifaoui S, Bellaaj R, Ouzari I, Saltoğlu N, Klibi N. 2022. Whole genome sequencing of a Citrobacter freundii strain isolated from the hospital environment: an extremely multiresistant NDM-1 and VIM-48 coproducing isolate. Microb Drug Resist 28:18–22. doi: 10.1089/mdr.2020.0417. [DOI] [PubMed] [Google Scholar]

[B24] 24.Schweizer C, Bischoff P, Bender J, Kola A, Gastmeier P, Hummel M, Klefisch FR, Schoenrath F, Fruhauf A, Pfeifer Y. 2019. Plasmid-mediated transmission of KPC-2 carbapenemase in Enterobacteriaceae in critically ill patients. Front Microbiol 10:276. doi: 10.3389/fmicb.2019.00276. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B25] 25.CLSI. 2022. Performance standards for antimicrobial susceptibility testing, p 362. Clinical and Laboratory Standards Institute, Wayne, PA. [Google Scholar]

[B26] 26.Karlowsky JA, Lob SH, Kazmierczak KM, Young K, Motyl MR, Sahm DF. 2020. In vitro activity of imipenem/relebactam against Enterobacteriaceae and Pseudomonas aeruginosa isolated from intraabdominal and urinary tract infection samples: SMART Surveillance United States 2015–2017. J Glob Antimicrob Resist 21:223–228. doi: 10.1016/j.jgar.2019.10.028. [DOI] [PubMed] [Google Scholar]

[B27] 27.Holland MS, Nobrega D, Peirano G, Naugler C, Church DL, Pitout JDD. 2020. Molecular epidemiology of Escherichia coli causing bloodstream infections in a centralized Canadian region: a population-based surveillance study. Clin Microbiol Infect 26:1554.e1–1554.e8. doi: 10.1016/j.cmi.2020.02.019. [DOI] [PubMed] [Google Scholar]

[B28] 28.Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. doi: 10.1089/cmb.2012.0021. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29.Meier-Kolthoff JP, Goker M. 2019. TYGS is an automated high-throughput platform for state-of-the-art genome-based taxonomy. Nat Commun 10:2182. doi: 10.1038/s41467-019-10210-3. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B30] 30.Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P, Tiedje JM. 2007. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 57:81–91. doi: 10.1099/ijs.0.64483-0. [DOI] [PubMed] [Google Scholar]

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PERMALINK

Molecular Epidemiology of Global Carbapenemase-Producing Citrobacter spp. (2015–2017)

Diego Nobrega

Gisele Peirano

Yasufumi Matsumura

Johann D D Pitout

Roles

ABSTRACT

INTRODUCTION