1

Detection of New Delhi Metallo-β-Lactamase (blaNDM-1) in Acinetobacter schindleri during 1

routine surveillance 2

Patrick McGann1*, Michael Milillo1, Robert J. Clifford1, Erik Snesrud1, Lindsay Stevenson2, 3

Michael G. Backlund2, Helen B. Viscount2, Reyes Quintero1, Yoon I Kwak1, Michael J. Zapor3, 4

Paige E Waterman1, Emil P Lesho1 5

1 Multidrug-resistant Organism Repository and Surveillance Network, Walter Reed Army 6

Institute of Research, Silver Spring, Maryland 7

2 Department of Pathology, Walter Reed National Military Medical Center, Bethesda 8

Maryland 9

3 Infectious Diseases Service, Walter Reed National Military Medical Center, Bethesda, 10

Maryland 11

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Running Title: blaNDM1 in Acinetobacter schindleri 13

Keywords: Acinetobacter schindleri, blaNDM-1, surveillance network 14

15

* Corresponding author 16

Patrick McGann, PhD 17

Multidrug-resistant Organism Repository and Surveillance Network 18

Walter Reed Army Institute of Research, Building 503, 2S35 19

Silver Spring, Maryland 20910, USA 20

Ph: (301) 319 9912 21

Fax (301) 319 9548 22

Email: patrick.mcgann@amedd.army.mil 23

Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00281-13 JCM Accepts, published online ahead of print on 3 April 2013

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Abstract. A carbapenem-resistant Alcaligenes faecalis was isolated from a surveillance swab of 24

a service member injured in Afghanistan. The isolate was positive for blaNDM by real-time PCR. 25

Species identification was re-evaluated on three identification systems, but was inconclusive. 26

Genome sequencing indicated the closest relative was Acinetobacter schindleri and blaNDM-1 was 27

carried on a plasmid that shared >99% identity with one identified in an Acinetobacter lwoffi. 28

The isolate also carried a novel chromosomally-encoded class D oxacillinase. 29

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In response to global concerns over the spread of blaNDM (1-3), the Multidrug-resistant Organism 30

Repository and Surveillance Network (MRSN) implemented routine monthly screening for this 31

gene in all carbapenem-resistant Gram-negative organisms in 2010 (4). From this surveillance 32

initiative, we previously described the first incidence of blaNDM-1 in the US military healthcare 33

system from a strain of Providencia stuartii, isolated from a local Afghan national treated at a 34

military facility in Afghanistan (5). Timely feedback was provided to the submitting facility, 35

which resulted in increased surveillance and enhanced infection control policies. 36

In June 2012, following a blast injury involving shrapnel in Afghanistan, a 22 year-old male 37

received cefazolin for routine prophylaxis and was evacuated to the U.S. via Germany. During 38

that escalation of care, a routine groin surveillance swab revealed mixed microbial flora. An 39

isolate identified as Alcaligenes faecalis using the BD Phoenix Automated Microbiology System 40

(BD Diagnostics Systems, Sparks, MD) displayed resistance to all tested B-lactam antibiotics 41

(Intermediate to Ceftriaxone), including the carbapenems and the monobactam aztreonam (Table 42

1). A Modified Hodge Test (MHT) was negative for meropenem. For ertapenem, the MHT was 43

positive but the clover leaf-like growth indentation of E.coli ATCC 25922 was significantly 44

reduced when grown with the test isolate, designated MRSN 10319, compared to growth 45

alongside the blaKPC-positive control strain Klebsiella pneumoniae ATCC BAA-1705. The 46

isolate was forwarded to the MRSN, a College of American Pathologists (CAP) certified 47

laboratory, for further evaluation. Identification was re-evaluated on three automated 48

identification systems: the VITEK 2 (bioMerieux, Durham, NC), the BD Phoenix, and the 49

Microscan Walkway (Siemens Healthcare Diagnostics Inc, Deerfield, IL). The MRSN employs 50

the three most common automated instruments as these instruments are used throughout the 51

Military Healthcare System (MHS) and discrepancies between their results have been noted (for 52

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a comprehensive review see (6)). Both VITEK 2 and Microscan identified the organism as 53

Acinetobacter lwoffi, whereas the Phoenix was in agreement with the initial identification of 54

Alcaligenes faecalis. 16S rRNA sequencing was performed (7) and indicated the isolate shared 55

99% identity with Acinetobacter schindleri/Acinetobacter johnsonii. Whole genome sequencing 56

(WGS) using an Ion Torrent PGM (Ion Torrent Systems, Inc., Guilford, CT), which provided 57

87X coverage of the 16S rRNA gene, demonstrated 99.5% identity to Acinetobacter schindleri 58

16S rRNA sequences deposited at GenBank (Figure 1A) (8). Phylogenetic analysis using the 59

rpoB gene sequence confirmed this identification (Figure 1B). 60

MRSN 10319 was tested by real-time PCR for carbapenemase genes (9), and was positive for 61

blaNDM. The gene was found to contain a single nucleotide polymorphism (guanine to adenine) at 62

position 468 compared to blaNDM-1, resulting in a synonymous mutation. The gene was located 63

on a 47.3 kB plasmid that shared >99% identity with pNDM-BJ02, a plasmid identified in an 64

isolate of Acinetobacter lwoffi cultured from the urine of a 62-year-old female patient in Beijing 65

in November 2010 (10). Hu and colleagues identified A. lwoffi by VITEK 2, but make no 66

mention of the 16S rRNA sequence to verify this result. In accordance with the discrepancies 67

noted in this report, Dortet and colleagues have noted that the VITEK 2 identifies rare species of 68

Acinetobacter, including A. schindleri, as A. lwoffi (11). 69

Plasmid pNDM-BJ02 has 46 open reading frames and there is no plasmid sequence in 70

Genbank that shares more than 15% homology with it. Furthermore, the plasmid cannot be 71

assigned to any of the described incompatibility groups using the PCR replicon typing method 72

developed by Carattolli and colleagues (12). The plasmid harbors a type IV secretion system 73

(T4SS) gene cluster and a single copy of aphA6, which encodes resistance to some 74

aminoglycosides. However, MRSN 10319 was susceptible to all aminoglycosides tested (Table 75

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1). Sequence comparison to pNDM-BJ02 suggests that the original promoter sequence for this 76

gene has been disrupted by an upstream transposition event as previously noted (10). Hu and 77

colleagues demonstrated that the plasmid had a relatively high transfer frequency (9.1 x 10-3 to 78

1.3 x 10-2 per donor cell) to E. coli J53 AziR, suggesting that this plasmid has a high propensity 79

for horizontal transmission (10). 80

Analysis of the chromosomal sequence revealed just a single locus with homology to 81

known antibiotic resistance genes. This locus encoded a class D oxacillinase that shares closest 82

homology to the recently described blaOXA-237 gene (13), but has 18 amino acid differences and 83

represents a novel blaOXA allele. The final nomenclature for this gene is currently being assigned 84

by the Lahey group (www.lahey.org; last accessed March 2013) and the complete gene sequence 85

has been deposited at GenBak (Accession number KC771279). There is no evidence (i.e. no 86

transposons or Insertion Sequences) in the surrounding genetic environment to suggest 87

horizontal acquisition of this gene. Based on the antibiotic profile of blaOXA-237 (13) and the lack 88

of any other antibiotic resistance genes, including other β-lactams, aztreonam resistance in this 89

strain is most likely due to this class D oxacillinase,. A complete analysis of the MRSN 10319 90

genome is ongoing and will provide further information. 91

This report highlights the limitations of automated identification systems when working 92

with unusual species. Commonly used clinical laboratory identification systems do not include A. 93

schindleri or A. johnsonii on identification panels (manufacturer literature), which can lead to 94

erroneous identification. Due to the high correlation between blaNDM carriage and the 95

Enterobacteriaceae, surveillance strategies for this gene have primarily focused on this group of 96

bacteria. However, given the association of this gene with highly promiscuous plasmids, as well 97

as documented horizontal dissemination of this gene, it is critical that surveillance efforts 98

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continue to test all carbapenem-resistant Gram-negative organisms for blaNDM. A number of good 99

techniques exist for detecting NDM-producing Enterobacteriaceae (14), but false-negative and 100

weakly-positive results have been observed in this Family with the popular MHT (15, 16). 101

Detection in Acinetobacter species remains a challenge due to the potential failure of many 102

techniques, including the MHT and Etest MBL strip (17). Bonnin and colleagues have suggested 103

that for carbapenem-resistant A. baumannii, isolates should first be screened using EDTA 104

inhibition-based techniques, followed by further PCR-based techniques in a reference laboratory 105

(17). We suggest that the same method be applied to all other carbapenem-resistant 106

Acinetobacter species isolated from clinical specimens. 107

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Acknowledgements 108

Major funding for this project was provided by the Global Emerging Infections Surveillance and 109

Response System (GEIS; Grant #C070912WR) and Defense Medical Research and Development 110

Program (DMRDP; Grant #D61I10J258). 111

Material has been reviewed by the Walter Reed Army Institute of Research. There is no 112

objection to its presentation. The opinions or assertions contained herein are the private views of 113

the authors and are not to be construed as official, or reflecting the views of the Department of 114

Defense. 115

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Table 1. Antibiotic susceptibility profile of A. schindleri MRSN 10319 177

Antibiotic MIC (µg/ml)1 Interpretation (µg/ml)2

Arbekacin ≤0.253 NA

Amikacin ≤8, S ≤16, 32, ≥64

Ampicillin-sulbactam >16/8, R ≤8/4, 16/8, ≥32/16

Aztreonam >164 NA

Cefepime >16, R ≤8, 16, ≥32

Ceftazidime >16, R ≤8, 16, ≥32

Ceftriaxone 32, I ≤8, 16-32, ≥64

Ciprofloxacin ≤0.5, S ≤1, 2, ≥4

Colistin 0.25, S5 ≤2, -, ≥4

Gentamicin ≤1, S ≤4, 8, ≥16

Imipenem >8, R ≤4, 8, ≥16

Levofloxacin ≤1, S ≤2, 4, ≥8

Meropenem ≥32, R6 ≤4, 8, ≥16

Piperacillin-tazobactam >64/4, R ≤16/4, 32/4-64/4, ≥128/4

Tetracycline ≤2, S ≤4, 8, ≥16

Tobramycin ≤1, S ≤4, 8, ≥16

Trimethoprim-sulfamethoxazole ≤0.5/9.5, S ≤2/38, -, ≥4/76

1 Determined using three automated systems (see text) except for arbekacin, colistin, ertapenem, 178

and meropenem. All results were consistent across the three instruments. The MICS and 179

resulting interpretations are presented using the Phoenix output for clarity. 180

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2 As recommended by the Clinical and Laboratory Standards Institute (CLSI) (18). No CLSI 181

interpretative guidelines are indicated by “NA”. 182

3 MICs for arbekacin represent the average of three independent microbroth dilution assays as 183

described (19). 184

4 No CLSI interpretive guidelines of aztreonam are available for Acinetobacter species. 185

5 Average of three independent Etest assay performed as described by the manufacturer 186

(bioMérieux). Etest results were consistent across replicates. 187

6 Ertapenem is not usually reported for Acinetobacter species due to intrinsic resistance but is 188

included due to the rarity of susceptibility data from A. schindleri. As meropenem is not reported 189

by the BD Phoenix Automated Microbiology System, MICs were performed in triplicate by Etest 190

as described by the manufacturer (bioMérieux). Meropenem MICs represents the average of 191

three independent Etests performed as described by the manufacturer (bioMérieux). No 192

variation in Etest results was evidenced. 193 on January 14, 2019 by guesthttp://jcm

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Figure 1. Dendrogram showing the relationship between A. schindleri MRSN 10319 and other 194

Acinetobacter species based on (A) 16S rRNA and (B) rpoB gene sequences. Acinetobacter 195

species 16S rRNA and rpoB gene sequences were retrieved from GenBank 196

(http://www.ncbi.nlm.nih.gov/pubmed: last accessed August 23rd 2012) and aligned using 197

MegAlign (DNASTAR, Madison, MI). Dendrograms, based on the number of nucleotide 198

changes were generated using the MegAlign program (DNASTAR). 199

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