A small Acinetobacter plasmid carrying the tet39 tetracycline resistance determinant

Sir, 
 
Tetracycline can still be used for treatment of infections caused by Gram-negative organisms that lack resistance genes. In Acinetobacter baumannii tetracycline resistance is often caused by tetA(A) and tetA(B), which encode efflux pumps,1,2 and occasionally by tet(M), which encodes a ribosomal protection protein.1 In addition, a novel determinant, tet39 (tetA39-tetR39), was found in tetracycline-resistant Acinetobacter strains recovered from freshwater fish farms in Denmark3 and Thailand4 and from a clinical Acinetobacter calcoaceticus/baumannii complex isolate recovered from human urine in 1986.3 The tetA39 gene encodes an efflux pump that was reported to confer resistance to tetracycline but not minocycline.3 
 
In strains belonging to global clone 1 (GC1), the tetA(A) gene is part of the AbaR resistance island5 while in GC2 isolates the tetA(B) gene is found in an AbGRI1 resistance island.6 However, there is little information about the tetracycline resistance determinants and their context in strains that do not belong to the two major global clones. Here, we have examined the cause of tetracycline resistance in RCH52, a clinical multiply antibiotic-resistant A. baumannii strain recovered prior to 2010 in a Queensland hospital. 
 
RCH52 was found to be resistant to ampicillin, ceftazidime, ticarcillin/clavulanic acid, imipenem, meropenem, streptomycin, spectinomycin, sulphonamides, trimethoprim, kanamycin, gentamicin and tetracycline. The genome of RCH52 was sequenced using the Illumina Hiseq platform and assembled as described previously,7 generating 60 contigs. RCH52 was ST729 (Institut Pasteur scheme), a novel single locus variant of ST3 (rpoB4 in ST729 differs from rpoB3 in ST3 by a single base pair) and RCH52 therefore belongs to the European clone III. To the best of our knowledge this is the first report of the European clone III in Australia. ResFinder 2.1 (https://cge.cbs.dtu.dk//services/ResFinder/) was used to identify resistance genes, and RCH52 contains aphA1b, aacC2, aadA1, floR, cmlA1, arr-2, sul1, sul2, dfrA14, oxa10, blaTEM, oxa23 (encoding a variant of OXA-23 differing by four amino acids) and the tet39 determinant, accounting for the resistances observed. The comM gene is uninterrupted and ISAba1 was not found upstream of the ampC gene. 
 
The tet39 determinant was found on an 11 kb contig that was shown to be circular using PCR followed by sequencing. The plasmid carrying tet39, named pRCH52-1 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"KT346360","term_id":"924867386","term_text":"KT346360"}}KT346360; Figure ​Figure1),1), is 11146 bp. Plasmid DNA was isolated and electroporated into A. baumannii ATCC 17978, which is tetracycline susceptible. Transformants were selected on L-agar supplemented with 20 mg/L tetracycline (transformation frequency = 4.5 × 107 transformants/μg of DNA). None of the transformants grew on L-agar containing other antibiotics that RCH52 was resistant to, indicating that only the tetracycline resistance had transferred into the recipient cells. Primers tet39-F (5′-GCAGCTAATGCCCATACCAT-3′) and tet39-R (5′-GCCTTTTGCGTTGTTACCAT-3′) were designed to amplify a 219 bp internal fragment of the tetA39 gene. This PCR generated the expected product for all of the transformants tested. Although it was reported that tet39 does not confer resistance to minocycline,3 transformants tested here exhibited reduced susceptibility to minocycline, with inhibition zone diameters of 18 mm compared with 26 mm for ATCC 17978. 
 
 
 
Figure 1. 
 
Linearized map of pRCH52-1 compared with other plasmids. (a) Comparison of the original tet39 region found in pLUH5605 with the corresponding region found in pRCH52-1 and pAB1H8. (b) pRCH52-1 compared with the cryptic plasmids pAb-G7-1 and pD72-1 seen ... 
 
 
 
The original study showed that tet39 was located on plasmids but only a 3727 bp fragment of one plasmid (named here pLUH5605) was sequenced (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"AY743590","term_id":"58200477","term_text":"AY743590"}}AY743590). This sequence, the only complete tet39 sequence found in the GenBank non-redundant database, includes tetA39, tetR39 and two ORFs (Figure ​(Figure1a),1a), one of which encodes a replication initiation protein.3 Only 2360 bp of the pLUH5605 sequence was present in pRCH52-1 (Figure ​(Figure1a).1a). To explore the distribution of the tet39 determinant in Acinetobacter strains, the whole genome sequence database of NCBI was explored using the sequences of pRCH52-1 and pLUH5605. Twenty-one Acinetobacter strains belonging to different species were found to contain tet39 (Table S1, available as Supplementary data at JAC Online). However, the tet39 region appears to be in different contexts in all but one A. baumannii strain, AB1H8 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"ANNC01000048","term_id":"452972063","term_text":"ANNC01000048"}}ANNC01000048). AB1H8 appears to include a plasmid that contains a Rep that is 97% identical in terms of amino acids to the pLUH5605 RepA (Figure ​(Figure1a).1a). Hence, discrete boundaries surrounding tet39 were not found. 
 
pRCH52-1 encodes a replication initiation protein Rep that belongs to the Rep_3 superfamily (pfam01051) and differs from the Rep found in pLUH5605 by 43%. The pRCH52-1 Rep is identical to the Rep protein found in a strain belonging to the A. calcoaceticus/baumannii complex (NCBI Reference Sequence number {"type":"entrez-protein","attrs":{"text":"WP_000845850","term_id":"446768594","term_text":"WP_000845850"}}WP_000845850). Thereafter, the closest Rep is RepAci7 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"GU978996","term_id":"306476522","term_text":"GU978996"}}GU978996), with 96% amino acid identity. 
 
pRCH52-1 also encodes a TonB-dependent transporter homologue. These outer membrane proteins bind and transport siderophores, vitamin B12, nickel complexes and carbohydrates.8 This gene is also present in several cryptic plasmids of A. baumannii,6,7,9,10 including pAb-G7-1 and pD72-1 from GC1 and GC2 strains, respectively. However, the amino acid sequence of Rep encoded by pRCH52-1 differs from those in pAb-G7-1 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"KJ586856","term_id":"596334985","term_text":"KJ586856"}}KJ586856) and pD72-1 (GenBank accession number {"type":"entrez-nucleotide","attrs":{"text":"KM051986","term_id":"668729060","term_text":"KM051986"}}KM051986) by 24% and 21%, indicating that the tonB gene, which is a potential virulence determinant, is widely distributed. 
 
The tet39 determinant is widespread in Acinetobacter species. It has also been found in other species of Gram-negative and Gram-positive bacteria recovered from a polluted Nigerian river.11 Its location on plasmids in clinical isolates would facilitate the spread of tetracycline resistance amongst Acinetobacter strains, leading to further restriction of treatment options.

substitutions previously reported to mediate ceftaroline resistance (Table 1). Two isolates (ASARM167 and A38) belonging to ST22 (epidemic MRSA-15) had an E239K substitution in PBP2a. ASARM167 was isolated from a patient with bacteraemia at Cambridge University Hospitals NHS Foundation Trust (CUH) in 2008 and A38 was isolated from a canine wound infection in 2006 treated in Wiltshire, south-west England. 8 Phylogenetic analysis of these two isolates based on core genome SNPs placed them in different clades separated by .120 SNPs (data not shown), indicating that the E239K mutation arose independently in these two isolates. The third isolate (ASARM130) had the N146K substitution in PBP2a, belonged to ST241 (CC8) and was isolated from a patient with bacteraemia at CUH in 2007. This isolate was also noted to have an N204K substitution, which has not been reported previously in isolates with the N146K substitution. 2,3 The effect of these PBP2a substitutions on the ceftaroline resistance phenotype was evaluated for these three isolates using the disc diffusion assay based on EUCAST guidelines 10 and the Etest (bioMérieux, Lyon, France) according to the manufacturer's instructions. Two isolates were susceptible and one was resistant to ceftaroline by disc diffusion, but all three isolates were susceptible by Etest (Table 1). Although isolates with the N146K substitution have been reported previously to have an MIC of 0.5 mg/L (susceptible), all previously reported isolates with E239K had an MIC of ≥2 mg/L (resistant). 2,4 The lack of association between a resistant phenotype and the N146K substitution indicates that secondary chromosomal mutations are likely to be involved, as reported previously. 4,7 The three study isolates were cultured before the clinical introduction of ceftaroline into clinical practice in the USA in 2010 and Europe in 2012, demonstrating that these are natural variants of PBP2a that occur (albeit at low prevalence) even without pressure from ceftaroline use. All previously reported isolates with PBP2a substitutions mediating ceftaroline resistance belonged to CC5 and CC8, which has led to the suggestion that these two lineages might be more prone to such mutations. Our findings suggest that they probably occur in multiple MRSA lineages including the pandemic CC22 lineage, which is important in many parts of the world including Australia and the Middle East and is the dominant MRSA lineage in the UK and much of Europe.

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None to declare.  Sir, Tetracycline can still be used for treatment of infections caused by Gram-negative organisms that lack resistance genes. In Acinetobacter baumannii tetracycline resistance is often caused by tetA(A) and tetA(B), which encode efflux pumps, 1,2 and occasionally by tet(M), which encodes a ribosomal protection protein. 1 In addition, a novel determinant, tet39 (tetA39-tetR39), was found in tetracycline-resistant Acinetobacter strains recovered from freshwater fish farms in Denmark 3 and Thailand 4 and from a clinical Acinetobacter calcoaceticus/baumannii complex isolate recovered from human urine in 1986. 3 The tetA39 gene encodes an efflux pump that was reported to confer resistance to tetracycline but not minocycline. 3 In strains belonging to global clone 1 (GC1), the tetA(A) gene is part of the AbaR resistance island 5 while in GC2 isolates the tetA(B) gene is found in an AbGRI1 resistance island. 6 However, there is little information about the tetracycline resistance determinants and their context in strains that do not belong to the two major global clones. Here, we have examined the cause of tetracycline resistance in RCH52, a clinical multiply antibiotic-resistant A. baumannii strain recovered prior to 2010 in a Queensland hospital.
RCH52 was found to be resistant to ampicillin, ceftazidime, ticarcillin/clavulanic acid, imipenem, meropenem, streptomycin, spectinomycin, sulphonamides, trimethoprim, kanamycin, gentamicin and tetracycline. The genome of RCH52 was sequenced using the Illumina Hiseq platform and assembled as described previously, 7 generating 60 contigs. RCH52 was ST729 (Institut Pasteur scheme), a novel single locus variant of ST3 (rpoB4 in ST729 differs from rpoB3 in ST3 by a single base pair) and RCH52 therefore belongs to the European clone III. To the best of our knowledge this is the first report of the European clone III in Australia. ResFinder 2.1 (https://cge.cbs.dtu.dk//services/ ResFinder/) was used to identify resistance genes, and RCH52 contains aphA1b, aacC2, aadA1, floR, cmlA1, arr-2, sul1, sul2, dfrA14, oxa10, bla TEM , oxa23 (encoding a variant of OXA-23 differing by four amino acids) and the tet39 determinant, accounting for the resistances observed. The comM gene is uninterrupted and ISAba1 was not found upstream of the ampC gene.
The tet39 determinant was found on an 11 kb contig that was shown to be circular using PCR followed by sequencing. The plasmid carrying tet39, named pRCH52-1 (GenBank accession number KT346360; Figure 1), is 11146 bp. Plasmid DNA was  isolated and electroporated into A. baumannii ATCC 17978, which is tetracycline susceptible. Transformants were selected on L-agar supplemented with 20 mg/L tetracycline (transformation frequency ¼ 4.5×10 7 transformants/mg of DNA). None of the transformants grew on L-agar containing other antibiotics that RCH52 was resistant to, indicating that only the tetracycline resistance had transferred into the recipient cells. Primers tet39-F (5 ′ -GCAGCTAATGCCCATACCAT-3 ′ ) and tet39-R (5 ′ -GCCTTTTGC GTTGTTACCAT-3 ′ ) were designed to amplify a 219 bp internal fragment of the tetA39 gene. This PCR generated the expected product for all of the transformants tested. Although it was reported that tet39 does not confer resistance to minocycline, 3 transformants tested here exhibited reduced susceptibility to minocycline, with inhibition zone diameters of 18 mm compared with 26 mm for ATCC 17978.
The original study showed that tet39 was located on plasmids but only a 3727 bp fragment of one plasmid (named here pLUH5605) was sequenced (GenBank accession number AY743590). This sequence, the only complete tet39 sequence found in the GenBank non-redundant database, includes tetA39, tetR39 and two ORFs (Figure 1a), one of which encodes a replication initiation protein. 3 Only 2360 bp of the pLUH5605 sequence was present in pRCH52-1 (Figure 1a). To explore the distribution of the tet39 determinant in Acinetobacter strains, the whole genome sequence database of NCBI was explored using the sequences of pRCH52-1 and pLUH5605. Twenty-one Acinetobacter strains belonging to different species were found to contain tet39 (Table S1, available as Supplementary data at JAC Online). However, the tet39 region appears to be in different contexts in all but one A. baumannii strain, AB1H8 (GenBank accession number ANNC01000048). AB1H8 appears to include a plasmid that contains a Rep that is 97% identical in terms of amino acids to the pLUH5605 RepA (Figure 1a). Hence, discrete boundaries surrounding tet39 were not found.
pRCH52-1 encodes a replication initiation protein Rep that belongs to the Rep_3 superfamily ( pfam01051) and differs from the Rep found in pLUH5605 by 43%. The pRCH52-1 Rep is identical to the Rep protein found in a strain belonging to the A. calcoaceticus/baumannii complex (NCBI Reference Sequence number WP_000845850). Thereafter, the closest Rep is RepAci7 (GenBank accession number GU978996), with 96% amino acid identity.
The tet39 determinant is widespread in Acinetobacter species. It has also been found in other species of Gram-negative and Gram-positive bacteria recovered from a polluted Nigerian river. 11 Its location on plasmids in clinical isolates would facilitate the spread of tetracycline resistance amongst Acinetobacter strains, leading to further restriction of treatment options.