Nosocomial Pluralibacter gergoviae Isolates Expressing NDM and KPC Carbapenemases Characterized Using Whole-Genome Sequencing

LETTER

Pluralibacter (formerly Enterobacter) gergoviae is recovered from environmental, industrial, and less frequently, clinical settings. It has been identified in dental plaque colonization (1) but is rarely associated with clinical disease. It is a known cause of cosmetic contamination (2) due to its resistance to parabens and other preservatives (2–4). Recalls have been reported from numerous hygiene products (5, 6). There are scattered reports of nosocomial outbreaks (7, 8) and opportunistic infections in immunocompromised hosts (9–11). Though significant antibiotic resistance is rare (12), there are published reports of highly resistant isolates, including bla_NDM in Iran (9), bla_IMP-positive isolates from an outbreak in Brazil (n = 26) (8), and three cases of bla_KPC-carrying isolates from the United States and Brazil (11, 13, 14). Carbapenem-resistant isolates have been identified in hospital wastewater studies from Saudi Arabia (15) and England (16).

The emergence of carbapenemases in environmental opportunistic pathogens is highly concerning. We describe the clinical, genetic, and infection control aspects of multidrug-resistant (MDR) P. gergoviae isolates recovered within the same week from two hospitalized patients.

Isolates were from two patients admitted to Brigham and Women’s Hospital (Boston, MA) in June of 2021. WGS was performed using a DNA prep kit (Illumina, Inc., California) and a 600-cycle v3 kit on a MiSeq instrument (Illumina). Multiplexed microbial SMRTbell libraries were prepared using the SMRTbell template prep kit 2.0 and sequenced on a PacBio Sequel IIe sequencer using a Sequel II sequencing kit 2.0 (PacBio, California). Raw data were demultiplexed with the Demultiplex Barcodes application, and de novo assembly was done using the microbial assembly in SMRTLink 10 (PacBio). SNPs were assessed using the FDA CFSAN pipeline (17). Antibiotic resistance genes (ARGs) and plasmids were identified using ResFinder (18) and PlasmidFinder (19, 20). Sequences were annotated using RAST (21). Sequence data were deposited at NCBI under BioProject numbers SAMN28861032 and SAMN28861031. Individual isolate SRA numbers are SRR19543005 (EXT00430872), SRR19543008 (EXT00430870).

Patient 1 was a 55-year-old woman with pulmonary fibrosis and no known history of MDR organisms (MDROs) or recent hospitalizations who underwent lung transplantation, later requiring tracheostomy. Then, 5 weeks postoperatively, P. gergoviae carrying bla_NDM grew from respiratory and urine cultures. Though asymptomatic, she received 2 weeks of cefiderocol. She remained colonized with P. gergoviae (BWH_1_P_GER).

Patient 2 was a 70-year-old man with no history of MDROs and minimal health care exposure who presented with an intraparenchymal hemorrhage requiring craniotomy, extraventricular drain placement, and tracheostomy. He developed central fevers and received vancomycin and cefepime, after which he developed increased secretions but no change in ventilator settings. Respiratory cultures grew P. gergoviae carrying bla_KPC (BWH_6_P_GER). He transitioned to comfort measures and expired. The contribution of P. gergoviae to his clinical symptoms was unclear.

An epidemiological assessment revealed no common location, providers, roommates, or ventilation equipment between these patients. There were no known FDA alerts/recalls for P. gergoviae-contaminated supplies within a year of these events.

Whole-genome sequencing (WGS) demonstrated that the genomes were ~5.4 Mb and >80% similar to the closest reference strain (NZ_CP020388). The two were broadly homologous across their lengths and were approximately 30,000 single nucleotide polymorphisms (SNPs) apart. Each isolate harbored 3 plasmids (Table 1): both had IncHl1B and IncA types, while BWH_6_P_GER and BWH_1_P_GER also had, respectively, an IncX3 and IncM plasmid. In addition to the carbapenemases, both isolates harbored several other β-lactamases, including a bla_CTX-M-15 in BWH_6_P_GER.

TABLE 1.

Antibiotic susceptibilities and associated genetic mutations from two clinical isolates of P. gergoviae^a

Class and drug	AST method	AST result (MIC, μg/mL) for:		Genotype ford:
		Patient 1 (strain BWH_P_GER_6)	Patient 2 (strain BWH_P_GER_1)	Patient 1	Patient 2
β-lactam
Amoxicillin-clavulanate	Vitek 2	≥32 (R)	≥32 (R)
Cefoxitin	Vitek 2	≥64 (R)	≥64 (R)
Ceftriaxone	Vitek 2	≥64 (R)	≥64 (R)
Ceftazidime	Vitek 2	≥64 (R)	≥64 (R)
Cefepime	Vitek 2	≥64 (R)	16 (R)
Ceftazidime-avibactam	E test	≥264 (R)	4 (S)
Piperacillin-tazobactam	Vitek 2	≥128 (R)	≥128 (R)
Ertapenem	Vitek 2	≥8 (R)	≥8 (R)
Meropenem	Vitek 2	≥16 (R)	≥16 (R)
Meropenem-vaborbactam	E test	32 (R)	32 (R)
Cefiderocol	KB (mm)	18 (S)	NA
				bla OXA-1 c	bla OXA-1
				bla NDM-1	bla KPC-4
				bla TEM-1A	bla TEM-1A
				bla CTX-M-15	bla SHV b
				blaSHV-12
Tetracycline
Tetracycline	Vitek 2	≥16 (R)	≥16 (R)	tet(D)
Amino-glycosides
Amikacin	Vitek 2	≥64 (R)	≤2 (S)
Tobramycin	Vitek 2	≥16 (R)	8 (I)
				aac(6′)-lb-crc	aac(6′)-lb-cr
				ant(3′')-Ia	ant(2′)-la
				armA	aph(3′)-la
				aph(3′)-VI
				aac(6′)-lb
Folate antagonists
Trimethoprim-sulfamethoxazole	Vitek 2	≥320 (R)	≤20 (S)
				sul1c	sul1
				dfrA14
Fluoroquinolones
Ciprofloxacin	Vitek 2	≥4 (R)	≥4 (R)
Levofloxacin	Vitek 2	4 (R)	1 (I)
				qnrS1	qnrS1
				qnrB1	aac(6′)-lb-cr6
				aac(6′)-lb-crc
Phenicol
Chloramphenicol	NA	NA	NA
				catB3c	catB3
				qacEdelta1
				msrE, cmlA1
Macrolides				ere(A), mph(E)
				mph(A)	mph(A)
Associated plasmids				IncHI1B/FIB (pBWH_6_304719)	IncHI1B/FIB (pBWH_1_257132)
				IncA (pBWH_6_195607)	IncA pBWH_1_231840
				IncX3 (pBWH_6_43728)	IncM1 pBWH_1_80199

^aSusceptibilities were performed using the Vitek-2 (GN59, bioMérieux, NC), Etest (bioMérieux), and disk diffusion, according to CLSI guidelines. Carbapenemase PCR was performed using Xpert Carba-R (Cepheid, California). Mutations are shading-coded based according to their associated plasmid. Plasmids names reflect the isolate number followed by the length in kilobases. R, resistant; S, susceptible; I, intermediate; NA, not applicable.

^bMost closely related to bla_SHV-105/bla_SHV-30/bla_SHV-189 but unable to differentiate.

^cGenes found on both InHI1B and IncA.

^dGenes that are bolded correspond with IncHI1B, gray shading corresponds with IncA, italics corresponds to IncX3, and underline corresponds to IncM1.

In both isolates, antibiotic resistance genes (ARGs) were exclusively on plasmids (Table 1). The IncA plasmids were closely related to p34998-239.973kb (CP012168.1), identified from Enterobacter hormaechei (22). pBWH_1_231840 contained an ~30-kb insertion which encoded bla_KPC4 as well as several other transposases and ARGs. The ~300-kb IncHl1b plasmids were homologous to pKP3301 (AP018748) from Klebsiella pneumoniae. While similar across large stretches of their backbones, pBWH_6_304719 did not harbor any ARGs, while pBWH_1_257132 harbored bla_NDM and bla_CTXM15 among others.

The IncM plasmid in BWH_1 carried genes conferring resistance to β-lactams, aminoglycosides, and fluoroquinolones and was 98% identical to one isolated in our institution from a bla_NDM-carrying Enterobacter cloacae in 2015 (CP020530) (23). The IncX plasmid in BWH_6_P_GER was nearly identical to one from Cronobacter sakazakii (MT759838.1).

We identified two contemporaneously admitted patients with no history of MDROs or carbapenem exposure from whom MDR P. gergoviae isolates were identified as likely colonizers. This is the second documented case of an NDM-producing Pluralibacter (9).

Epidemiological investigation did not yield a clear common exposure. Given the 30,000-SNP difference between these isolates, derivation from a single hospital-adapted lineage is unlikely.

To our knowledge, this is the first description of plasmids carried by P. gergoviae, all of which had close homologues identified from related enteric organisms (Enterobacter and Klebsiella). The IncA and IncH plasmids which carried the carbapenemase and other ARGs were conserved across much of their backbones.

Though P. gergoviae is rarely virulent, these cases demonstrate its potential to act as a vector for transmission of MDR plasmids to cocolonizing clinical strains. The host ranges of IncA (broad) and IncH1b (medium) plasmids described in this study raise concerns that P. gergoviae, as a member of wastewater, hygiene products, and other environmental flora, may facilitate horizontal exchange of high-risk ARGs.