Emergence of VIM-1 Metallo-β-Lactamase-Producing Escherichia coli in a Neonatal Intensive Care Unit

Abstract

A carbapenem-resistant Escherichia coli isolate was recovered from rectal swab of a 12-day-old female neonate, which was admitted to a Greek neonatal intensive care unit (NICU). Phenotypic testing, polymerase chain reaction assays with sequencing, and plasmid analysis revealed that the isolate harbored a plasmid-mediated bla_VIM-1 metallo-β-lactamase gene. The appearance of a metallo-β-lactamase-producing E. coli in NICU is worrisome. Further surveys are needed to determine whether such Enterobacteriaceae may also be spreading in other NICUs.

Introduction

Carbapenems constitute the most potent antibiotics of the β-lactam group and are commonly used as a last resort treatment against multidrug-resistant gram-negative bacteria. Carbapenem-hydrolyzing enzymes have over the past decade spread not only among the nonfermenting gram-negative bacteria^13,18,23 but also among different genera of the Enterobacteriacae family,^7,14,15 thus posing a serious public health issue. Carbapenemase production has been mainly attributed to the production of molecular class B metallo-β-lactamases (MBLs), usually of the IMP and VIM categories, which confer various levels of resistance to all β-lactams, including carbapenems with the exception of aztreronam; are not inhibited by class A β-lactamase inhibitors; and are predominately encoded by genes carried in class 1 integrons.^13,21 The MBLs that have disseminated within the Enterobacteriaceae family are usually hospital-acquired^6,7,10,14,15 and rarely encountered in the community setting.^16,20 Interestingly enough, it would appear that children and particularly neonates are rarely colonized or infected by such gram-negative bacteria.^2,4 In this article, we aim to report the emergence of VIM-1 MBL-producing Escherichia coli in a neonate admitted to our neonatal intensive care unit (NICU).

Case report

A 12-day-old female neonate was admitted from home to the level III–IV, 30-bed, university-affiliated NICU of Aglaia Kyriakou Pediatric Hospital, Athens, due to fever (38.5°C) of a few hours duration and failure to feed properly. The neonate was born with a cesarean section after a 38-week gestation and had normal weight (3,220 g) and Apgar score. She was breastfeeding normally. Two members of the neonate's family also presented with fever and nasal discharge. Neither the neonate nor the mother had been previously exposed to antibiotics. With the exception of fever, nasal discharge, and a pre-existing heart murmur due to a patent foramen ovale, there were no other abnormal findings on clinical examination. Blood work was indicative of viral infection, whereas blood cultures obtained at this time did not produce a pathogen. Rectal and nasopharyngeal surveillance swabs were taken upon admission. The rectal specimen produced an E. coli isolate that was resistant to carbapenems. Administration of antibiotic treatment was not deemed necessary; the neonate became afebrile within 24 hours and was discharged 2 days after her admission.

Methods

Bacterial identification and susceptibility assays

The E. coli isolate that was recovered from the rectal swab was evaluated. Species identification was preformed with the Vitek 2 automated identification system (bioMérieux, Marcy l'Étoile, France) and confirmed with the API 20E system (bioMérieux). Susceptibility to antibiotics was further assessed using the disk diffusion method on Mueller-Hinton agar plates (Oxoid, Ogdensburg, New York), and results were interpreted according to Clinical and Laboratory Standards Institute (CLSI) criteria.³ MICs for imipenem and meropenem were also evaluated by agar dilution³ and by using the Etest method (AB Biodisk, Solna, Sweden).

Phenotypic testing

Preliminary screening for the presence of a carbapenemase was performed with the modified Hodge test.¹ The MBL Etest (AB Biodisk) and the combined-disk test with imipenem and ethylenediaminetetraacetic acid were used to screen for the production of a class B MBL carbapenemase.⁵ The phenotypic detection of a class A MBL carbapenemase was also evaluated with boronic acid potentiation disk tests using different antibiotic substrates.¹⁷ ESBL production was tested using the CLSI confirmatory test.³

Polymerase chain reaction assays and sequencing

The E. coli isolate was screened for β-lactamase genes by polymerase chain reaction (PCR) amplification using a panel of primers for the detection of MBLs (VIM and IMP types), KPCs, and ESBLs, including SHV, TEM, and CTX-M enzymes. The consensus primers and conditions used for amplification were as previously reported (Table 1). Plasmid-mediated AmpCs were screened in single PCR reactions for each gene using primers and conditions described previously.¹² The PCR products thar were subjected to direct sequencing were purified using ExoSAP-IT reagent (USB Corporation, Cleveland, OH) and used as templates for sequencing on both strands with an ABI Prism 377 DNA sequencer (Applied Biosystems, Foster City, CA).

Table 1.

Sequences of the Primers Used for amplification and/or Sequencing

Gene type	Primer	Sequences (5′ to 3′)	Size of the amplicon (bp)	Use	Reference
VIM	VIM-F	5′-AGTGGTGAGTATCCGACAG-3′	261	Amplification	18
	VIM-R	5′-ATGAAAGTGCGTGGAGAC-3′
	VIM-1FVIM-1R	5′-TTATGGAGCAGCAACGATGT-3′5′-CAAAAGTCCCGCTCCAACGA-3′	920	Amplification and sequencing	23
KPC	KPC-F1KPC-R1	5′-TCGCTAAACTCGAACAGG-3′5′-TTACTGCCCGTTGACGCCCAATCC-3′	780	Amplification and sequencing	9
SHV	SHV-A (forward)	5′-ACTGAATGAGGCGCTTCC-3′	220	Amplification	19
	SHV-B (reverse)	5′-CGCACCCCGCTTGCT-3′
TEM	OT-1 (forward)	5′-TTGGGTGCACGAGTGGGTTA-3′	465	Amplification	19
	OT-2 (reverse)	5′-TAATTGTTGCCGGGAAGCTA-3′
CTX-M	CT-F (forward)CT-R (reverse)	5′-GGTTAAAAAATCACTGCG-3′5′-TTACAAACCGTCGGTGA-3′	873	Amplification and sequencing	19
IMP-1	IMP-1FIMP-1R	5′-TGAGCAAGTTATCTGTATTC-3′5′-TTAGTTGCTTGGTTTTGATG-3′	740	Amplification and sequencing	23
IMP-2	IMP-2F	5′-GGCAGTCGCCCTAAAACAAA-3′	737	Amplification	23
	IMP-2R	5′-TAGTTACTTGGCTGTGATGG-3′

Conjugation experiments and plasmid analysis

The potential for conjugational transfer of carbapenem resistance was examined in biparental matings using E. coli strain 26R793 (lac⁻, Rif^R) as the recipient strain. Conjugation assays were preformed using Luria broth cultures. Donor and recipient cells were mixed in a ratio of 1:5 and transconjugant clones were screened on Mac Conkey agar plates containing rifampicin (150 μg/ml) and amoxicillin (40 μg/ml) or ertapenem (0.5 μg/ml). The colonies that grew on the selecting medium where picked up. MICs were determined by agar dilution and β-lactamase genes were sought by PCR amplification. Plasmid extraction on the clinical isolates and the transconjugants was preformed by using an alkaline lysis protocol and E. coli 39R861 as a control strain.

Results

The E. coli isolate that was retrieved from the neonate was highly resistant to imipenem and meropenem with MICs >32 μg/ml for both carbapenems. It was also resistant to various penicillins, β-lactam/inhibitor combinations, expanded-spectrum cephalosporins, amikacin, netilmicin, tobramycin, and trimethoprim-sulfamethoxazole, while remained susceptible to gentamicin, ciprofloxacin, and aztreonam. The modified Hodge test showed the production of a carbapenemase. Further, the MBL E-test and the combined-disk test were positive for the production of class B carbapenemase, whereas the boronic acid potentiation disk tests yielded negative results for the production of class A carbapenemase. The CLSI confirmatory test for ESBL production was also negative. In accordance with these results, amplification of the β-lactamase genes confirmed the presence solely of the bla_VIM gene, which was subsequently identified by sequencing analysis as bla_VIM-1.

Plasmid analysis of VIM-1-producing E. coli isolate revealed the presence of only one plasmid of ∼120 kb, which was self-transferable in conjugation experiments and conferred resistance to β-lactams, including carbapenems in the recipient strain. Resistance to aminoglycosides (amikacin, netilmicin, and tobramycin) was also cotransferred. DNA extracted from plasmid bands of E. coli isolate and its transconjugant yielded positive results for bla_VIM gene, confirming that the bla_VIM gene was located on the 120 kb plasmid.

Discussion

MBL-producing pathogens present a healthcare problem with serious consequences in antimicrobial treatment and patients' outcome.^13,21 However, these pathogens are almost exclusively recovered from the adult population, and there are limited data showing their possible occurrence among children as well as neonates. Characteristically, a large population-based surveillance study of infections caused by carbapenem-resistant Pseudomonas aeruginosa isolates revealed the absence of MBL-producing pathogens among neonates, children, and young adults in difference with the situation among the elderly hospitalized population.⁸

The literature is limited of MBL-positive isolations in the pediatric population. In that respect, VIM-producing Pseudomonas spp. isolates have described among hospitalized children in a single institution in Poland and characteristically the MBL-harboring strains have persisted for extended periods due to MBL gene fixation in the chromosome.¹¹ Also, in a recent study, VIM-1-producing Enterobacteriaceae isolates that exhibited low-level resistance to carbapenems were recovered in a tertiary care hospital in Spain from a patient population, which was mostly pediatric.² This population was characterized by severe underlying diseases that involved long-term hospitalization, major surgery, and immunosuppressive therapy.² In the neonatal population, a VIM-2-producing P. aeruginosa has been recovered from a female neonate institutionalized at the local children's hospital in Warsaw, Poland.²² Four VIM-2-producing Klebsiella oxytoca isolates were also detected in neonatal patients hospitalized in a pediatric hospital in Portugal.⁴

Recently, a number of studies from Southern European countries have reported the sporadic detection of MBL-producing E. coli isolates from adults, with hospital-acquired infections or colonization.^6,10,14,15 Most cases were attributed to severe underlying diseases, a prolonged hospital stay, or antimicrobial therapy. The present study for the first time describes the recovery of an MBL-producing E. coli isolate in the neonatal population. Our report deals with an otherwise healthy neonate, which had not been exposed to carbapenems or to other antibiotics and had not been hospitalized postbirth. It is most likely that the neonate has been colonized during her delivery at the maternity hospital. Further, in contrast to previous reports describing MBL-possessing E. coli isolates,^{6,10,14,15,20} in this particular VIM-1-positive isolate, high-level resistance to imipenem and meropenem with MICs >32 μg/ml was detected. This finding is indicative of the serious therapeutic problems that are likely to arise in the future. The need for the implementation of surveillance screening, of measures for controlling the spread of such multidrug-resistant microorganisms, and of a more rational antibiotic use cannot be overemphasized.

Footnotes

Disclosure Statement

No competing financial interests exist.

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