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MOLECULAR DIAGNOSTICS IN MICROBIOLOGY
Resistance mechanisms in Enterobacteriaceae SALLY R. PARTRIDGE Centre for Infectious Diseases and Microbiology, The University of Sydney, Westmead Hospital and Westmead Millennium Institute, Westmead, NSW, Australia
Summary Enterobacteriaceae are responsible for a large proportion of serious, life-threatening infections and resistance to multiple antibiotics in these organisms is an increasing global public health problem. Mutations in chromosomal genes contribute to antibiotic resistance, but Enterobacteriaceae are adapted to sharing genetic material and much important resistance is due to ‘mobile’ resistance genes. Different mobile genetic elements, which have different characteristics, are responsible for capturing these genes from the chromosomes of a variety of bacterial species and moving them between DNA molecules. If transferred to plasmids, these resistance genes are then able to be transferred ‘horizontally’ between different bacterial cells, including different species, and well as being transferred ‘vertically’ during cell division. Carriage of several resistance genes on the same plasmid enables a bacterial cell to acquire multi-resistance in a single step and means that spread of one resistance gene may be co-selected for by use of antibiotics other than those to which it confers resistance. Many different mobile genes conferring resistance to each class of antibiotic have been identified, complicating detection of the factors responsible for a particular resistance phenotype, especially when changes in chromosomal genes may also confer or contribute to resistance. Understanding the mechanisms of antibiotic resistance, and the means by which these mechanisms can evolve and disseminate, is important for developing ways to efficiently track the spread of resistance and to optimise treatment. Key words: Aminoglycosides, antibiotic resistance, b-lactams, fluoroquinolones, insertion sequence, integron, plasmid, transposon. Received 11 November 2014, revised 28 January, accepted 28 January 2015
INTRODUCTION The use of antibiotics to treat bacterial infections is a key component of modern medicine but antibiotic resistance, particularly multi-resistance, in the Enterobacteriaceae is an increasing global problem, with strains resistant to most (or even all) available antibiotics emerging. Much of the resistance in Enterobacteriaceae is due to ‘mobile’ genes captured from various source species by different mobile genetic elements and transferred to plasmids, which can then move between cells, including of different species and indeed even different genera. However, mutations in chromosomal genes are also important in conferring or enhancing resistance to certain classes of antibiotics, including some newly introduced antibiotics and older antibiotics that are being revisited for use against multiresistant organisms. Print ISSN 0031-3025/Online ISSN 1465-3931 DOI: 10.1097/PAT.0000000000000237
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The variety of different genes that can contribute to resistance to each class of antibiotic in the Enterobacteriaceae can make identifying the gene(s) responsible for a particular phenotype challenging. This review aims to provide a summary of the characteristics of resistance genes in Enterobacteriaceae, with an emphasis on mobile resistance to the most clinically important antibiotics.
MOBILE ELEMENTS INVOLVED IN ANTIBIOTIC RESISTANCE Acquired ‘resistance’ genes found in pathogenic bacteria appear to be derived from the chromosomes of a number of other bacterial species, where they may originally have had other functions.1 Various types of mobile genetic elements have captured these genes and the capturing element may provide a more powerful promoter and/or capture may separate the gene from a regulatory system on the chromosome, resulting in increased or constitutive expression. Each particular resistance gene generally appears to have been captured by one particular mobile element (Tables 1–3), presumably because capture events are rare and if selected for, the successful partnership then becomes dominant.2 Antibiotic pressure may result in selection of mutations that result in increased resistance or extension of the spectrum of antibiotics covered. Three main types of mobile genetic element (insertion sequences, transposons and the gene cassette/integron system; Fig. 1) are known to be involved in capturing resistance genes and moving them between DNA molecules in the same cell, e.g., from the chromosome to a plasmid or between plasmids, while plasmids can transfer resistance genes between bacterial cells, including different species. Insertion sequences (IS) Most IS3 are delineated by inverted repeats (IR), short identical or almost identical sequences in opposite orientation, which flank little more than a transposase (tnpA) gene. These IR are designated left (IRL) and right (IRR) relative to the direction of transcription of tnpA (Fig. 1). The encoded transposase protein recognises the IR and moves the IS to a new location by a ‘cut and paste’ or ‘copy and paste’ mechanism, the details of which vary for different IS families.3 Many IS create staggered cuts in the two DNA strands during the transposition process, which when repaired result in short direct repeats (DR; 2–14 bp, characteristic for each IS family) flanking the new insertion. DR are also referred to as target site duplications (TSD), although most IS do not appear to ‘target’ particular sequences. A pair of closely-related IS, in either relative orientation, can capture and move themselves and the region between them, which may include a resistance gene, as a composite
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RESISTANCE MECHANISMS IN ENTEROBACTERIACEAE
Table 1
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Main types and variants of b-lactamases in Enterobacteriaceae Associated with resistance to
Class A blaTEM blaSHV§ blaCTX-M-1 group blaCTX-M-9 group blaCTX-M-2 group blaCTX-M-8 group blaCTX-M-25 group blaKLUC blaKPCjj blaGES Class B blaIMP-1-like blaIMP-2-like blaIMP-4-like blaVIM-1-like blaNDM Class C blaCMY-2-like blaDHA blaCMY-1-like/MOX blaACC blaACTô blaMIRô blaFOX Class D blaOXA-10-like blaOXA-30-like blaOXA-48-like blaOXA-181-like
APP*
3GC*
CPM{
Minor variants
Some Some No No No No No No No No
Some Some Yes Yes Yes Yes Yes Yes Yes Yes
No No No No No No No No (Yes) Some
Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
z
Mobile element
Source
>200 >150 53 47 23 3 10 4 21 26
Tn3-like IS26 ISEcp1 ISEcp1, ISCR1 ISCR1 ISEcp1, IS10 ISEcp1 ISEcp1 Tn4401 Gene cassette
Unknown Klebsiella pneumoniae Kluyvera ascorbata Kluyvera georgiana Kluyvera ascorbata Kluyvera georgiana Kluyvera georgiana Kluyvera cryocresens Unknown Unknown
(Yes) (Yes) (Yes) (Yes) Yes
7 5 3 14 15
Gene cassette Gene cassette Gene cassette Gene cassette ISAba125
Unknown Unknown Unknown Unknown Unknown
Yes Yes Yes Yes Yes Yes Yes
No No No No No No No
101 23 17 5 36 18 12
ISEcp1 ISCR1 ISCR1 ISEcp1 ? ISCR ISApu2
Citrobacter freundii Morganella morganii Aeromonas hydrophila Hafnia alvei Enterobacter asburiae Enterobacter cloacae Aeromonas caviae
Some No No No
No No (Yes) (Yes)
11 7 9 2
Gene cassette Gene cassette Tn1999 ISEcp1
Unknown Unknown Shewanella Shewanella xiamenensis
*
APP, anti-pseudomonal penicillins (b-lactam þ b-lactamase inhibitor); 3GC, third-generation cephalosporin; some, inhibitor resistant or extended-spectrum variants. { CPM, carbapenems; (yes), an enzyme that is considered a carbapenemase but only gives noticeable carbapenem resistance if e.g. combined with porin defects. GES variants with 170Asp (2, 13) or 170Ser (4-6, 14-16, 18, 20-21, 24) have been shown or would be expected to confer CPM resistance, GES-1, 3, 7–12, 17, 19, 22–23 (170Gly) would not. Some AmpC and ESBL enzymes have been found to confer CPM resistance if porin defects are present. z Variant numbers are from http://www.lahey.org/Studies/, January 2015. § Klebsiella pneumoniae may carry two blaSHV variants, one chromosomal and one plasmid-borne. jj KPC are numbered 2–22. KPC-1 is no longer used, as an error was found in the original sequence. ô blaACT and blaMIR genes are related enough to be picked up by the same set of primers, but seem to be derived from different Enterobacter species.
transposon. For some types of IS, a single copy is able to capture and mobilise an adjacent resistance gene. ISEcp1 appears to miss its IRR end during transposition and use an alternative sequence situated further downstream, creating 5 bp DR flanking the entire ISEcp1 ‘transposition unit’.4 ISCR
elements5 are related to IS1294-like elements that have been demonstrated to transpose by a rolling circle replication mechanism.6 Replication starts at oriIS (downstream of the tnpA gene, also known as rcr, for rolling circle replicase) and should end at terIS, upstream of rcr. If replication continues past terIS
Table 2 Main mobile genes conferring resistance to clinically-important aminoglycosides in Enterobacteriacaeae
Table 3
Acetyltransferases aacA4/aac(60 )-Ib aacA4/aac(60 )-IIa aacC1/aac(3)-Ia aac(3)-II Adenylyltranseferases aadB/ant(200 )-1a Phosphorylases aphA6 aphA15 16S rRNA methylases armA rmtB rmtC rmtF
Resistance to
Mobile element
AMK TOB GEN TOB GEN GEN TOB
Gene cassette Gene cassette Gene cassette IS26?
GEN TOB
Gene cassette
AMK (AMK)
ISAba14 Gene cassette
AMK AMK AMK AMK
GEN GEN GEN GEN
TOB TOB TOB TOB
ISCR1 ISCR3? ISEcp1 ISCR3-like
Main types of PMQR determinants in Enterobacteriaceae Variants
Mobile element
Source
qnrA qnrB2 qnrB1 qnrB4 qnrB6 qnrB10 qnrB19
7
Shewanella Citrobacter Citrobacter Citrobacter Citrobacter Citrobacter Citrobacter
qnrD qnrS1-like qnrS2-like qnrS5-like qnrVC aac(60 )-Ib-cr qepA oqxAB
2 6 2 1 6 2 2 1
ISCR1 ISCR1 IS3000, IS26 ISCR1 ISCR1 ISCR1 ISEcp1 or small plasmid Small plasmid IS26? ? ? Gene cassette Gene cassette ISCR3?, IS26? IS26
algae spp. spp. spp. spp. spp. spp.
Unknown Vibrio splendidus Unknown Unknown Vibrio? Unknown Unknown Klebsiella pneumoniae
AMK, amikacin; GEN, gentamicin; TOB, tobramycin.
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A B
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C. freundii chromosome
ISEcp1 transposition unit
to antibiotic resistance and have 25 bp IR, four tni transposition genes and a res site and create 5 bp DR. They are known to target the res regions of Tn21 and relatives.10 The distinction between IS and Tn can become blurry, as ‘passenger’ genes have now been identified in relatives of elements traditionally thought of as IS, while some elements closely related to Tn carry no such genes.11
ampR blaCMY-2 ATTTC
ATTTC ISEcp1 blaCMY-2
C
CGTTCAGCA Composite transposon
CGTTCAGCA
IS1999
IS1999 blaOXA-48
TATAC
D
TATAC
Tn3 subfamily transposon tnpA
tnpR blaTEM-52
E ISCR1 insertion
ISCR1 qnrB2
F
Class 1 integron IRi 5'-CS | Cassette array |3'-CS
IRt
DR
DR |blaIMP-4|qacG|aacA4|catB3| sul1
G
tniB∆
tniA
IRmer
IRtnp mer (mercury resistance) tnpA
tnpR
Tn21 subfamily transposon
Resistance gene
res site
Other gene Inverted repeat (IR) Promoter
attI site attC site
Fig. 1 The main types of mobile elements associated with resistance genes in Enterobacteriaceae. (A) The blaCMY-2 region of the Citrobacter freundii chromosome and (B) part of the same region found in an ISEcp1 transposition unit on a plasmid, illustrating gene capture. (C) The composite transposon Tn1999 carrying blaOXA-48. (D) A Tn2-like transposon carrying the blaTEM-52 ESBL gene, an example of a Tn3 subfamily transposon. (E) A gene captured by ISCR1 and inserted in a class 1 integron (F) with four cassettes and a partial tni region inserted in the res site of a Tn21-subfamily transposon (G). Sequences of DR are shown. All examples have been detected in bacterial DNA sequences, except for the exact combination of gene cassette array and Tn21-like transposon shown in F and G.
the adjacent region can be captured, apparently as a circular molecule proposed to insert by homologous recombination.5 Mobilisation of resistance genes by a single copy of the very common IS26 has also recently been demonstrated.7 Transposons (Tn) A Tn is a larger element that is essentially an IS that carries captured resistance or other genes internally. Tn implicated in movement of resistance genes generally belong to the Tn3 or Tn21 subfamilies.8,9 In addition to a large (3 kb) tnpA gene these Tn include a resolvase gene (tnpR) and its cognate resolution (res) site, usually have 38 bp IR and create 5 bp DR. The two subfamilies differ in the arrangement of tnpA, tnpR and res (Fig. 1). During transposition a cointegrate is formed from the original DNA molecule carrying the Tn and a recipient molecule, separated by two copies of the Tn. This cointegrate is resolved into two molecules, each with a copy of the Tn, by TnpR-mediated recombination in the res site. Tn5053/Tn402-like transposons are also important in relation
Integrons and gene cassettes A gene cassette is a small mobile element consisting of little more than a (usually promoterless) gene and a recombination site (attC) and can exist in a circular form.12 Gene cassettes are captured by larger elements called integrons by recombination between attC and the integron recombination site (attI), catalysed by the integrase encoded by the intI gene of the integron.13 The integron also provides a promoter (Pc) for expression of the captured cassette genes, which are all inserted in the same orientation, and integration of multiple cassettes can create cassette arrays conferring multi-resistance. Integrons have been divided into classes, defined on the basis of the relatedness of their IntI protein sequences, and the most common type in clinical isolates are class 1 integrons. The first examples to be identified appear to have been created by capture of the intI1/attI1/Pc components by a Tn5053/Tn402 family transposon and replacement of part of the tni transposition region by a sequence defined as the 30 -conserved segment (30 -CS),14 preventing self-transposition. More rarely, examples with intI1/attI1/Pc and a complete tni region are found (mostly in Pseudomonas aeruginosa) and these are able to move themselves. The IR of both types of In/Tn unit are designated IRi (integrase end) and IRt (tni end) and the region between IRi and the recombination point in the attI1 site is defined as the 50 -CS. PCR with different primers in the 50 -CS and 30 -CS is frequently used to amplify cassette arrays and identify resistance genes. In so-called ‘complex’ class 1 integrons an ISCR1 element and associated resistance gene(s) are found between partially duplicated copies of the 30 -CS. The known preference for insertion of Tn5053/Tn402-like transposons in Tn21-like res sites also means that transposition-defective class 1 integrons can ‘piggyback’ on larger Tn.
RESISTANCE TO b-LACTAM ANTIBIOTICS The b-lactam group of antibiotics encompasses penicillins, cephalosporins, carbapenems, penems and monobactams. They irreversibly bind to the pencillin binding proteins (PBP) required for the final crosslinking (transpeptidation) step in the synthesis of peptidoglycan for cell wall construction. The most clinically important are the third-generation cephalosporins, ‘anti-pseudomonal’ b-lactam/b-lactamase inhibitor combinations, and carbapenems. The most common reason for resistance to b-lactam antibiotics in the Enterobacteriaceae is the expression of b-lactamase enzymes (encoded by bla genes) that hydrolise the b-lactam ring, resulting in inactivation. Many different types of b-lactamase can confer resistance to each of the most clinically-important b-lactam types and a single amino acid difference may affect the phenotype conferred (Table 1). b-lactamases have been grouped in different ways, the simplest being division into classes A–D.15 Class A b-lactamases Class A includes ‘narrow spectrum’ enzymes, most notably the blaTEM and blaSHV groups, that confer resistance to penicillins
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RESISTANCE MECHANISMS IN ENTEROBACTERIACEAE
and early cephalosporins. ‘Extended-spectrum’ (ESBL) and inhibitor-resistant variants, with 1–3 amino acid changes at a few key positions, began to appear after introduction of third-generation cephalosporins and b-lactamase inhibitors.16 Other Class A b-lactamases intrinsically have this ‘extendedspectrum’ phenotype, e.g., the CTX-M family. CTX-M enzymes can be divided into six groups (-1, -2, -8, -9, -25, -KLUC) with 96% identity within each group. The -cr (ciprofloxacin resistance) variant of the aac(60 )-Ib (aacA4) gene encodes an enzyme that can N-acetylate the piperazinyl substituent of ciprofloxacin and is associated with low-level fluoroquinolone resistance but reduced resistance to aminoglycosides.69 All currently known gene cassettes encoding this variant have T329/Leu102, a mutation at position 514 (GAT to TAT; Asp164Tyr) and one of two mutations at position 283 (TGG to AGG or CGG, giving Trp87Arg). Two plasmid-encoded efflux pumps are also relevant to fluoroquinolone resistance. The qepA gene encodes a proton antiporter efflux pump of the major facilitator superfamily (MFS) and can remove quinolones from the cell.70 The OqxAB efflux pump contributes to resistance to olaquindox (used as a growth promoter in animals) as well as fluoroquinolones and chloramphenicol.71 The oqxAB genes were initially identified on a plasmid,72 having apparently been captured from the K. pneumoniae chromosome.
RESISTANCE TO TRIMETHOPRIM AND SULPHONAMIDES Trimethoprim and sulphonamides are used in combination (cotrimoxazole) to target two successive steps in the folate synthesis pathway. Sulphonamide resistance in Enterobacteriaceae is associated with three different genes (sul1, sul2, sul3) encoding drug-resistant dihydropteroate synthases. sul1 is part
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of the 30 -CS found in the most common type of class 1 integrons in clinical isolates. At least 19 different dfrA and >8 different dfrB gene cassettes, encoding two different types of trimethoprim-resistant dihydrofolate reductases, have been identified although some, e.g., dfrA12 and dfrA17, appear much more common than others.12 A few other dfrA genes are mainly associated with ISCR1 or ISCR2 elements. The presence of a dfr cassette, or a dfrA gene associated with ISCR1, in a class 1 integron that has sul1 in the 30 -CS can confer resistance to cotrimoxazole. sul3 is also associated with a type of class 1 integron that lacks the 30 -CS and that to date is only known to be associated with a limited selection of gene cassettes, but these can include dfrA12.12 sul2 is associated with ISCR2 or located close to fragments of Tn5393 that include the strAB genes conferring streptomycin resistance, with no obvious link to dfr genes, but these may be found on the same plasmid.
RESISTANCE TO TIGECYCLINE Tigecycline, a glycylcycline, inhibits bacterial protein synthesis by blocking entry of amino-acyl tRNA molecules into the ribosomal A-site, preventing incorporation of amino acids into elongating peptide chains. It is related to tetracyclines, but appears not to be a substrate for tetracycline-specific efflux pumps, encoded by plasmid-borne tetA genes in Enterobacteriaceae, apparently due to its larger side chain. Although tigecycline was introduced relatively recently Enterobacteriaceae isolates resistant to this antibiotic have already been reported, due to overexpression of other efflux pumps including AcrAB-TolC and OqxAB. The AcrAB-TolC efflux pump, present in most Enterobacteriaceae, belongs to the resistance-nodulation-division (RND) family of transporters. It consists of the AcrA periplasmic adaptor, the AcrB cytoplasmic membrane transporter protein and the TolC outer membrane channel. AcrAB expression is regulated by a specific transcriptional repressor, AcrR, and by global regulatory systems. Mutations leading to tigecycline resistance appear to occur in different regulatory genes. For example, in E. coli AcrAB expression may be upregulated by mutations affecting MarR, part of the mar (multiple antibiotic resistance) regulon, that activate MarA, lifting the repression caused by AcrR.73 In K. pneumoniae mutations in ramR, encoding a specific transcriptional repressor expression of the RamA transcription factor, can cause increased AcrAB expression.74 An IS5 insertion in the promoter region of the kpgABC operon, proposed to encode an efflux pump, has also been reported in relation to tigecycline resistance in K. pneumoniae,75 while in other isolates this resistance remains unexplained,76 suggesting that further tigecycline resistance mechanisms may be identified.
RESISTANCE TO COLISTIN/POLYMYXIN Polymyxins, cyclic cationic decapeptides bound to a fatty acid chain, were originally discovered in the 1940s. They target the negatively-charged lipid moiety of lipopolysaccharide (LPS) of the outer membrane, displacing divalent cations (Ca2þ, Mg2þ) that stabilise the LPS. This disrupts the integrity of the outer membrane, causing leakage of intracellular contents and cell death. Routine use of polymyxins was discontinued in the 1980s due to common and serious side effects (nephrotoxicity, neurotoxicity), but the need to find ways of treating multidrugresistant Gram-negative infections has led to a recent revival in
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their use. Polymyxins B and E (colistin), which differ by a single amino acid, are used clinically, with colistin being the most commonly used in recent years. In Enterobacteriaceae, polymyxin resistance can arise by a variety of LPS modifications,77 e.g., addition of 4-amino4-deoxy-l-arabinose (L-Ara4N) to the phosphate group of Lipid A, which reduces the negative charges available for interactions. L-Ara4N is synthesised and added to LPS by proteins encoded by the arn (for AraN synthesis) operon, previously called the pmrHFIJKLM (for polymyxin resistance) or pbg operon.78 Expression of this operon is under the control of the PmrA/PmrB and PhoP/PhoQ two-component regulatory systems that allow bacteria to alter gene expression in response to environmental changes, such as ion concentrations and pH. In each case a sensor histidine kinase (PmrB or PhoQ) located in the cytoplasmic membrane can phosphorylate and activate the regulator (PmrA or PhoP). Activated PmrA increases expression of the arn operon, while PhoP enhances expression of another protein PmrD, which maintains activated PmrA by inhibiting dephosphorylation.78 Mutations in pmrA and pmrB have been detected in E. coli.79 In K. pneumoniae carrying a blaKPC gene colistin resistance has arisen during treatment due to mutations in pmrB80 or insertional inactivation of the mgrB gene, which encodes a negative-feedback regulator of PhoQ/ PhoQ.81 Other LPS modifications, efflux pump alterations, overexpression of porins and increased production of capsule polysaccharide can also contribute to polymyxin resistance.77
RESISTANCE TO FOSFOMYCIN Fosfomycin, another older antibiotic being re-examined in the era of multi-resistance, inhibits bacterial cell wall biosynthesis by alkylating an active site cysteine residue of the MurA enzyme, preventing formation of the peptidoglycan precursor N-acetylmuramic acid. Several fosfomycin resistance mechanisms are already known. FosA-, FosB-, and FosX-type enzymes add glutathione, L-cysteine, or a hydroxyl group, respectively, to the oxirane ring of fosfomycin, inactivating this antibiotic. Several gene cassette-borne fos genes have been identified but most have been found only in P. aeruginosa. The exception is the single known example of fosC2 from E. coli,82 which encodes glutathione S-transferase activity. The fosA3 gene, associated with IS26 and first reported in Japan82 has also been found in Korea83 and is increasingly reported in China.84
RESISTANCE PLASMIDS AND MULTI-RESISTANCE Plasmids that carry resistance genes may either be large conjugative plasmids, which carry the genes encoding all of the machinery required to transfer themselves between bacterial cells, or smaller mobilisable plasmids that may be co-transferred by conjugative plasmids. These plasmids generally consist of a ‘backbone’ that encodes plasmid functions such as replication, stability and conjugation, into which variable ‘accessory’ regions are inserted. Plasmid backbones were originally classified into ‘Inc’ groups based on experimentally-defined mutual incompatibility, i.e., plasmids in the same Inc group cannot stably co-exist in the same cell and one will be lost. PCR-based typing of replicons (PBRT)85 or genes encoding the relaxase protein required for mobilisation/conjugation (DPMT)86 can be used to classify plasmids, but each only samples part of the backbone.
Accessory regions, including those encoding antibiotic resistance, may occupy a large proportion of the plasmid. The adverse effects of disrupting essential plasmid functions means that a mobile element that has become inserted without incapacitating a plasmid may then become a target of insertion of other mobile elements and associated resistance genes, so that these elements are often found clustered together in complex multi-resistance regions (MRR).2 Subsequent movement of resistance genes between DNA molecules may then be facilitated by other nearby mobile elements or by homologous recombination between repeated elements, which may also lead to rearrangements in MRR. Clustering of resistance genes in this way means that certain combinations of genes frequently travel together and that use of one antibiotic may select for maintenance of genes giving resistance to unrelated antibiotic classes. For example, E. coli ST131 isolates often carry blaCTX-M-15 in large, complex MRR on IncF-type plasmids. These related MRR can carry different combinations of the blaTEM-1, aac(3)-II, tetA(A) (tetracycline resistance) and either both aac(60 )-Ib-cr and blaOXA-30 or neither of these genes. Differences can be explained by the actions of the various mobile elements associated with these genes and/or homologous recombination. Identifying whether aac(3)-II (gentamicin resistance), in particular, is present may be clinically relevant. Knowledge of gene associations may help in tracking important genes that do not always express a strong resistance phenotype. For example, in Sydney, an aac(3)-II gene (gentamicin and tobramycin resistance) was consistently identified on the same IncL/M plasmid as blaIMP-4,87 which only gives a truly carbapenem-resistant phenotype in the presence of OmpK defects.88 This allowed more efficient tracking of the silent spread of blaIMP-4, by pre-screening isolates on gentamicin and testing only those that were resistant for blaIMP-4 by PCR. Identification of blaIMP-4 by screening also indicated when neither carbapenems, which select for isolates that have become fully resistant, nor gentamicin should be prescribed.89 Klebsiella pneumoniae ST258 isolates that are commonly associated with carriage of blaKPC can be divided into two subtypes that appear to carry different sets of plasmids and resistance genes and apparently have different levels of resistance to carbapenems,90 possibly due to porin defects.29 The common association of blaKPC with the amikacin resistance variant of aacA4 may be clinically important, but in this case the blaKPC and aacA4 genes are found on different plasmids in both ST258 types, so do not necessarily always travel together. Tracking of sets of resistance genes, rather than individual genes, therefore may be important for epidemiological, infection control and/or treatment purposes, but identifying the best sets of targets to use can be challenging and may require initial detailed characterisation of a small number of isolates. Combinations of PCR tests for individual genes can be employed, but multiplexed methods such as multiplex PCR/reverse line blot33,91 can simultaneously cover more targets in multiple isolates economically and efficiently. Microarray-type and highly multiplexed PCR methods are also available, but may be unnecessary if known gene associations can be taken into account.
KEY MESSAGES Resistance mechanisms include hydrolysis or modification of antibiotics, protection or replacement of antibiotic
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RESISTANCE MECHANISMS IN ENTEROBACTERIACEAE
targets, decreased antibiotic entry and/or increased efflux or modification of the outer membrane. Much important resistance in Enterobacteriaceae is due to mobile resistance genes that can transfer between different cells and to different species on plasmids. Each mobile resistance gene is generally closely associated with one particular mobile element (IS, Tn or the gene cassette/integron system) which may also enhance expression. Resistance genes and mobile elements tend to cluster in large, complex multi-resistance regions on plasmids, allowing simultaneous transfer of multi-resistance and co-selection Mutations in chromosomal genes are important for resistance to some antibiotic classes, including some now being employed as last-line antibiotics, such as polymyxins and tigecycline. A large number of different genes may confer resistance to each antibiotic class or subclass and distinguishing them for epidemiological or treatment purposes is likely to require different levels of gene typing.
Conflicts of interest and sources of funding: The author states that there are no conflicts of interest to disclose. Address for correspondence: Dr Sally Partridge, Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, 176 Hawkesbury Road, Westmead, NSW 2145, Australia. E-mail: [email protected]. gov.au
References 1. Martinez JL, Fajardo A, Garmendia L, et al. A global view of antibiotic resistance. FEMS Microbiol Rev 2009; 33: 44–65. 2. Partridge SR. Analysis of antibiotic resistance regions in Gram-negative bacteria. FEMS Microbiol Rev 2011; 35: 820–55. 3. Chandler M, Mahillon J. Insertion sequences revisited. In: Craig NL, Craigie R, Gellert M, Lambowitz AM, editors. Mobile DNA II. Washington DC: ASM Press, 2002: 305–366. 4. Poirel L, Lartigue MF, Decousser JW, Nordmann P. ISEcp1B-mediated transposition of blaCTX-M in Escherichia coli. Antimicrob Agents Chemother 2005; 49: 447–50. 5. Toleman MA, Bennett PM, Walsh TR. ISCR elements: novel gene-capturing systems of the 21st century? Microbiol Mol Biol Rev 2006; 70: 296– 316. 6. Tavakoli N, Comanducci A, Dodd HM, Lett MC, Albiger B, Bennett P. IS1294, a DNA element that transposes by RC transposition. Plasmid 2000; 44: 66–84. 7. Harmer CJ, Moran RA, Hall RM. Movement of IS26-associated antibiotic resistance genes occurs via a translocatable unit that includes a single IS26 and preferentially inserts adjacent to another IS26. MBio 2014; 5: e01801– 1814. 8. Grindley NDF. The movement of Tn3-like elements: transposition and cointegrate resolution. In: Craig NL, Craigie R, Gellert M, Lambowitz AM, editors. Mobile DNA II. Washington DC: ASM Press, 2002: 272–302. 9. Grinsted J, de la Cruz F, Schmitt R. The Tn21 subgroup of bacterial transposable elements. Plasmid 1990; 24: 163–89. 10. Kamali-Moghaddam M, Sundstro¨m L. Transposon targeting determined by resolvase. FEMS Microbiol Lett 2000; 186: 55–9. 11. Siguier P, Gagnevin L, Chandler M. The new IS1595 family, its relation to IS1 and the frontier between insertion sequences and transposons. Res Microbiol 2009; 160: 232–41. 12. Partridge SR, Tsafnat G, Coiera E, Iredell JR. Gene cassettes and cassette arrays in mobile resistance integrons. FEMS Microbiol Rev 2009; 33: 757– 84. 13. Mazel D. Integrons: agents of bacterial evolution. Nat Rev Microbiol 2006; 4: 608–20. 14. Gillings M, Boucher Y, Labbate M, et al. The evolution of class 1 integrons and the rise of antibiotic resistance. J Bacteriol 2008; 190: 5095–100. 15. Bush K, Jacoby GA. Updated functional classification of b-lactamases. Antimicrob Agents Chemother 2010; 54: 969–76. 16. Bradford PA. Extended-spectrum b-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001; 14: 933–51.
283
17. D’Andrea MM, Arena F, Pallecchi L, Rossolini GM. CTX-M-type b-lactamases: A successful story of antibiotic resistance. Int J Med Microbiol 2013; 303: 305–17. 18. Woodford N, Turton JF, Livermore DM. Multiresistant Gram-negative bacteria: the role of high-risk clones in the dissemination of antibiotic resistance. FEMS Microbiol Rev 2011; 35: 736–55. 19. Nordmann P, Cuzon G, Naas T. The real threat of Klebsiella pneumoniae carbapenemase producing bacteria. Lancet Infect Dis 2009; 9: 228–33. 20. Ambler RP, Coulson AF, Frere JM, et al. A standard numbering scheme for the Class A b-lactamases. Biochem J 1991; 276: 269–70. 21. Frase H, Shi Q, Testero SA, Mobashery S, Vakulenko SB. Mechanistic basis for the emergence of catalytic competence against carbapenem antibiotics by the GES family of b-lactamases. J Biol Chem 2009; 284: 29509–13. 22. Cornaglia G, Giamarellou H, Rossolini GM. Metallo-b-lactamases: a last frontier for b-lactams? Lancet Infect Dis 2011; 11: 381–93. 23. Johnson AP, Woodford N. Global spread of antibiotic resistance: the example of New Delhi metallo-b-lactamase (NDM)-mediated carbapenem resistance. J Med Microbiol 2013; 62: 499–513. 24. Jacoby GA. AmpC b-lactamases. Clin Microbiol Rev 2009; 22: 161–82. 25. Nelson EC, Elisha BG. Molecular basis of AmpC hyperproduction in clinical isolates of Escherichia coli. Antimicrob Agents Chemother 1999; 43: 957–9. 26. Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: the phantom menace. J Antimicrob Chemother 2012; 67: 1597–606. 27. Wang XD, Cai JC, Zhou HW, Zhang R, Chen GX. Reduced susceptibility to carbapenems in Klebsiella pneumoniae clinical isolates associated with plasmid-mediated b-lactamase production and OmpK36 porin deficiency. J Med Microbiol 2009; 58: 1196–202. 28. Martı´nez-Martı´nez L. Extended-spectrum b-lactamases and the permeability barrier. Clin Microbiol Infect 2008; 14 (Suppl 1): 82–9. 29. Partridge SR, Ginn AN, Wiklendt AM, et al. Emergence of blaKPC carbapenemase genes in Australia. Int J Antimicrob Agents 2013; 45: 130–6. 30. Clancy CJ, Chen L, Hong JH, et al. Mutations of the ompK36 porin gene and promoter impact responses of ST258, KPC-2-producing Klebsiella pneumoniae strains to doripenem and doripenem-colistin. Antimicrob Agents Chemother 2013; 57: 5258–65. 31. Turnidge JD, Gottlieb T, Mitchell DH, Coombs GW, Pearson JC, Bell JM. Hospital-onset Gram-negative Surveillance Program annual report, 2011. Commun Dis Intell Q Rep 2014; 38: E49–53. 32. Turnidge JD, Gottlieb T, Mitchell DH, Coombs GW, Daly DA, Bell JM. Community-onset Gram-negative Surveillance Program annual report, 2012. Commun Dis Intell Q Rep 2014; 38: E54–8. 33. Ginn AN, Zong Z, Wiklendt AM, et al. Limited diversity in the gene pool allows prediction of third-generation cephalosporin and aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae. Int J Antimicrob Agents 2013; 42: 19–26. 34. Ellem J, Partridge SR, Iredell JR. Efficient direct ESBL detection by multiplex real-time PCR: accurate assignment of phenotype using a limited set of genetic markers. J Clin Microbiol 2011; 49: 3074–7. 35. Peleg AY, Franklin C, Bell J, Spelman DW. Emergence of IMP-4 metallob-lactamase in a clinical isolate from Australia. J Antimicrob Chemother 2004; 54: 699–700. 36. Thomas L, Espedido B, Watson S, Iredell J. Forewarned is forearmed: antibiotic resistance gene surveillance in critical care. J Hosp Infect 2005; 60: 291–3. 37. Leung GH, Gray TJ, Cheong EY, Haertsch P, Gottlieb T. Persistence of related bla-IMP-4 metallo-beta-lactamase producing Enterobacteriaceae from clinical and environmental specimens within a burns unit in Australia - a six-year retrospective study. Antimicrob Resist Infect Control 2013; 2: 35. 38. Peirano G, Lascols C, Hackel M, Hoban DJ, Pitout JD. Molecular epidemiology of Enterobacteriaceae that produce VIMs and IMPs from the SMART surveillance program. Diagn Microbiol Infect Dis 2014; 78: 277– 81. 39. Poirel L, Lagrutta E, Taylor P, Pham J, Nordmann P. Emergence of metallob-lactamase NDM-1-producing multidrug-resistant Escherichia coli in Australia. Antimicrob Agents Chemother 2010; 54: 4914–6. 40. Fernando GA, Collignon PJ, Bell JM. A risk for returned travellers: the ‘‘post-antibiotic era’’. Med J Aust 2010; 193: 59. 41. Sidjabat H, Nimmo GR, Walsh TR, et al. Carbapenem resistance in Klebsiella pneumoniae due to the New Delhi metallo-b-lactamase. Clin Infect Dis 2011; 52: 481–4. 42. Blyth CC, Pereira L, Goire N. New Delhi metallo-beta-lactamase-producing Enterobacteriaceae in an Australian child who had not travelled overseas. Med J Aust 2014; 200: 386. 43. Shoma S, Kamruzzaman M, Ginn AN, Iredell JR, Partridge SR. Characterization of multidrug-resistant Klebsiella pneumoniae from Australia carrying blaNDM-1. Diagn Microbiol Infect Dis 2014; 78: 93–7. 44. Espedido BA, Steen JA, Ziochos H, et al. Whole genome sequence analysis of the first OXA-48-producing outbreak-associated Klebsiella pneumoniae isolates: the resistome and in vivo evolution. PLoS One 2013; 8: e59920.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
284
PARTRIDGE
45. Sidjabat HE, Kennedy K, Silvey A, Collignon P, Paterson DL. Emergence of blaOXA-181-carrying ColE plasmid in Klebsiella pneumoniae in Australia. Int J Antimicrob Agents 2013; 41: 294–6. 46. Coatsworth NR, Huntington PG, Hardiman RP, Hudson BJ, Fernandes CJ. A case of carbapenemase-producing Klebsiella pneumoniae in Australia. Pathology 2012; 44: 42–4. 47. Chang L. Investigation of a nosocomial outbreak of KPC-producing Klebsiella pneumoniae. Pathology 2014; 46 (Suppl 1): S46. 48. Williamson DA, Heffernan H. The changing landscape of antimicrobial resistance in New Zealand. NZ Med J 2014; 127: 41–54. 49. Heffernan HM, Woodhouse RE, Pope CE, Blackmore TK. Prevalence and types of extended-spectrum b-lactamases among urinary Escherichia coli and Klebsiella spp. in New Zealand. Int J Antimicrob Agents 2009; 34: 544–9. 50. Freeman JT, Williamson DA, Heffernan H, Smith M, Bower JE, Roberts SA. Comparative epidemiology of CTX-M-14 and CTX-M-15 producing Escherichia coli: association with distinct demographic groups in the community in New Zealand. Eur J Clin Microbiol Infect Dis 2012; 31: 2057–60. 51. Williamson DA, Sidjabat HE, Freeman JT, et al. Identification and molecular characterisation of New Delhi metallo-b-lactamase-1 (NDM-1)- and NDM-6-producing Enterobacteriaceae from New Zealand hospitals. Int J Antimicrob Agents 2012; 39: 529–33. 52. Williamson DA, Heffernan H, Sidjabat H, et al. Intercontinental transfer of OXA-181-producing Klebsiella pneumoniae into New Zealand. J Antimicrob Chemother 2011; 66: 2888–90. 53. Shaw KJ, Rather PN, Hare RS, Miller GH. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol Rev 1993; 57: 138–63. 54. Ramirez MS, Tolmasky ME. Aminoglycoside modifying enzymes. Drug Resist Updat 2010; 13: 151–71. 55. Novick RP, Clowes RC, Cohen SN, Curtiss R 3rd, Datta N, Falkow S. Uniform nomenclature for bacterial plasmids: a proposal. Bacteriol Rev 1976; 40: 168–89. 56. Hanau-Berc¸ot B, Podglajen I, Casin I, Collatz E. An intrinsic control element for translational initiation in class 1 integrons. Mol Microbiol 2002; 44: 119–30. 57. Rather PN, Munayyer H, Mann PA, Hare RS, Miller GH, Shaw KJ. Genetic analysis of bacterial acetyltransferases: identification of amino acids determining the specificities of the aminoglycoside 60 -N-acetyltransferase Ib and IIa proteins. J Bacteriol 1992; 174: 3196–203. 58. Lambert T, Ploy MC, Courvalin P. A spontaneous point mutation in the aac(60 )-Ib0 gene results in altered substrate specificity of aminoglycoside 60 -N-acetyltransferase of a Pseudomonas fluorescens strain. FEMS Microbiol Lett 1994; 115: 297–304. 59. Casin I, Hanau-Bercot B, Podglajen I, Vahaboglu H, Collatz E. Salmonella enterica serovar Typhimurium blaPER-1-carrying plasmid pSTI1 encodes an extended-spectrum aminoglycoside 60 -N-acetyltransferase of type Ib. Antimicrob Agents Chemother 2003; 47: 697–703. 60. Wachino J, Arakawa Y. Exogenously acquired 16S rRNA methyltransferases found in aminoglycoside-resistant pathogenic Gram-negative bacteria: an update. Drug Resist Updat 2012; 15: 133–48. 61. Aldred KJ, Kerns RJ, Osheroff N. Mechanism of quinolone action and resistance. Biochemistry 2014; 53: 1565–74. 62. Hopkins KL, Davies RH, Threlfall EJ. Mechanisms of quinolone resistance in Escherichia coli and Salmonella: recent developments. Int J Antimicrob Agents 2005; 25: 358–73. 63. Strahilevitz J, Jacoby GA, Hooper DC, Robicsek A. Plasmid-mediated quinolone resistance: a multifaceted threat. Clin Microbiol Rev 2009; 22: 664–89. 64. Jacoby GA, Griffin CM, Hooper DC. Citrobacter spp. as a source of qnrB alleles. Antimicrob Agents Chemother 2011; 55: 4979–84. 65. Jacoby G, Cattoir V, Hooper D, et al. qnr gene nomenclature. Antimicrob Agents Chemother 2008; 52: 2297–9. 66. Poirel L, Rodriguez-Martinez JM, Mammeri H, Liard A, Nordmann P. Origin of plasmid-mediated quinolone resistance determinant QnrA. Antimicrob Agents Chemother 2005; 49: 3523–5. 67. Wang M, Guo Q, Xu X, et al. New plasmid-mediated quinolone resistance gene, qnrC, found in a clinical isolate of Proteus mirabilis. Antimicrob Agents Chemother 2009; 53: 1892–7. 68. Cattoir V, Poirel L, Mazel D, Soussy CJ, Nordmann P. Vibrio splendidus as the source of plasmid-mediated QnrS-like quinolone resistance determinants. Antimicrob Agents Chemother 2007; 51: 2650–1. 69. Robicsek A, Strahilevitz J, Jacoby GA, et al. Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med 2006; 12: 83–8.
Pathology (2015), 47(3), April
70. Yamane K, Wachino J, Suzuki S, et al. New plasmid-mediated fluoroquinolone efflux pump, QepA, found in an Escherichia coli clinical isolate. Antimicrob Agents Chemother 2007; 51: 3354–60. 71. Hansen LH, Jensen LB, Sorensen HI, Sorensen SJ. Substrate specificity of the OqxAB multidrug resistance pump in Escherichia coli and selected enteric bacteria. J Antimicrob Chemother 2007; 60: 145–7. 72. Hansen LH, Johannesen E, Burmolle M, Sorensen AH, Sorensen SJ. Plasmid-encoded multidrug efflux pump conferring resistance to olaquindox in Escherichia coli. Antimicrob Agents Chemother 2004; 48: 3332–7. 73. Keeney D, Ruzin A, McAleese F, Murphy E, Bradford PA. MarA-mediated overexpression of the AcrAB efflux pump results in decreased susceptibility to tigecycline in Escherichia coli. J Antimicrob Chemother 2008; 61: 46–53. 74. Hentschke M, Wolters M, Sobottka I, Rohde H, Aepfelbacher M. ramR mutations in clinical isolates of Klebsiella pneumoniae with reduced susceptibility to tigecycline. Antimicrob Agents Chemother 2010; 54: 2720–3. 75. Nielsen LE, Snesrud EC, Onmus-Leone F, et al. IS5 element integration, a novel mechanism for rapid in vivo emergence of tigecycline nonsusceptibility in Klebsiella pneumoniae. Antimicrob Agents Chemother 2014; 58: 6151–6. 76. Sheng ZK, Hu F, Wang W, et al. Mechanisms of tigecycline resistance among Klebsiella pneumoniae clinical isolates. Antimicrob Agents Chemother 2014; 58: 6982–5. 77. Olaitan AO, Morand S, Rolain JM. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria. Front Microbiol 2014; 5: 643. 78. Falagas ME, Rafailidis PI, Matthaiou DK. Resistance to polymyxins: Mechanisms, frequency and treatment options. Drug Resist Updat 2010; 13: 132–8. 79. Quesada A, Porrero MC, Tellez S, Palomo G, Garcia M, Dominguez L. Polymorphism of genes encoding PmrAB in colistin-resistant strains of Escherichia coli and Salmonella enterica isolated from poultry and swine. J Antimicrob Chemother 2015; 70: 71–4. 80. Cannatelli A, Di Pilato V, Giani T, et al. In vivo evolution to colistin resistance by PmrB sensor kinase mutation in KPC-producing Klebsiella pneumoniae is associated with low-dosage colistin treatment. Antimicrob Agents Chemother 2014; 58: 4399–403. 81. Cannatelli A, D’Andrea MM, Giani T, et al. In vivo emergence of colistin resistance in Klebsiella pneumoniae producing KPC-type carbapenemases mediated by insertional inactivation of the PhoQ/PhoP mgrB regulator. Antimicrob Agents Chemother 2013; 57: 5521–6. 82. Wachino J, Yamane K, Suzuki S, Kimura K, Arakawa Y. Prevalence of fosfomycin resistance among CTX-M-producing Escherichia coli clinical isolates in Japan and identification of novel plasmid-mediated fosfomycinmodifying enzymes. Antimicrob Agents Chemother 2010; 54: 3061–4. 83. Lee SY, Park YJ, Yu JK, et al. Prevalence of acquired fosfomycin resistance among extended-spectrum b-lactamase-producing Escherichia coli and Klebsiella pneumoniae clinical isolates in Korea and IS26-composite transposon surrounding fosA3. J Antimicrob Chemother 2012; 67: 2843–7. 84. Hou J, Yang X, Zeng Z, et al. Detection of the plasmid-encoded fosfomycin resistance gene fosA3 in Escherichia coli of food-animal origin. J Antimicrob Chemother 2013; 68: 766–70. 85. Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 2005; 63: 219–28. 86. Alvarado A, Garcillan-Barcia MP, de la Cruz F. A degenerate primer MOB typing (DPMT) method to classify gamma-proteobacterial plasmids in clinical and environmental settings. PLoS One 2012; 7: e40438. 87. Espedido BA, Partridge SR, Iredell JR. blaIMP-4 in different genetic contexts in Enterobacteriaceae isolates from Australia. Antimicrob Agents Chemother 2008; 52: 2984–7. 88. Espedido BA, Thomas LC, Iredell JR. Metallo-b-lactamase or extendedspectrum b-lactamase: a wolf in sheep’s clothing. J Clin Microbiol 2007; 45: 2034–6. 89. van Hal SJ, Wiklendt A, Espedido B, Ginn A, Iredell JR. Immediate appearance of plasmid-mediated resistance to multiple antibiotics upon antibiotic selection: an argument for systematic resistance epidemiology. J Clin Microbiol 2009; 47: 2325–7. 90. Wright MS, Perez F, Brinkac L, et al. Population structure of KPCproducing Klebsiella pneumoniae isolates from midwestern U. S. hospitals. Antimicrob Agents Chemother 2014; 58: 4961–5. 91. Kong F, Gilbert GL. Multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB)–a practical epidemiological and diagnostic tool. Nat Protoc 2006; 1: 2668–80.
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MOLECULAR DIAGNOSTICS IN MICROBIOLOGY
Resistance mechanisms in Enterobacteriaceae SALLY R. PARTRIDGE Centre for Infectious Diseases and Microbiology, The University of Sydney, Westmead Hospital and Westmead Millennium Institute, Westmead, NSW, Australia
Summary Enterobacteriaceae are responsible for a large proportion of serious, life-threatening infections and resistance to multiple antibiotics in these organisms is an increasing global public health problem. Mutations in chromosomal genes contribute to antibiotic resistance, but Enterobacteriaceae are adapted to sharing genetic material and much important resistance is due to ‘mobile’ resistance genes. Different mobile genetic elements, which have different characteristics, are responsible for capturing these genes from the chromosomes of a variety of bacterial species and moving them between DNA molecules. If transferred to plasmids, these resistance genes are then able to be transferred ‘horizontally’ between different bacterial cells, including different species, and well as being transferred ‘vertically’ during cell division. Carriage of several resistance genes on the same plasmid enables a bacterial cell to acquire multi-resistance in a single step and means that spread of one resistance gene may be co-selected for by use of antibiotics other than those to which it confers resistance. Many different mobile genes conferring resistance to each class of antibiotic have been identified, complicating detection of the factors responsible for a particular resistance phenotype, especially when changes in chromosomal genes may also confer or contribute to resistance. Understanding the mechanisms of antibiotic resistance, and the means by which these mechanisms can evolve and disseminate, is important for developing ways to efficiently track the spread of resistance and to optimise treatment. Key words: Aminoglycosides, antibiotic resistance, b-lactams, fluoroquinolones, insertion sequence, integron, plasmid, transposon. Received 11 November 2014, revised 28 January, accepted 28 January 2015
INTRODUCTION The use of antibiotics to treat bacterial infections is a key component of modern medicine but antibiotic resistance, particularly multi-resistance, in the Enterobacteriaceae is an increasing global problem, with strains resistant to most (or even all) available antibiotics emerging. Much of the resistance in Enterobacteriaceae is due to ‘mobile’ genes captured from various source species by different mobile genetic elements and transferred to plasmids, which can then move between cells, including of different species and indeed even different genera. However, mutations in chromosomal genes are also important in conferring or enhancing resistance to certain classes of antibiotics, including some newly introduced antibiotics and older antibiotics that are being revisited for use against multiresistant organisms. Print ISSN 0031-3025/Online ISSN 1465-3931 DOI: 10.1097/PAT.0000000000000237
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The variety of different genes that can contribute to resistance to each class of antibiotic in the Enterobacteriaceae can make identifying the gene(s) responsible for a particular phenotype challenging. This review aims to provide a summary of the characteristics of resistance genes in Enterobacteriaceae, with an emphasis on mobile resistance to the most clinically important antibiotics.
MOBILE ELEMENTS INVOLVED IN ANTIBIOTIC RESISTANCE Acquired ‘resistance’ genes found in pathogenic bacteria appear to be derived from the chromosomes of a number of other bacterial species, where they may originally have had other functions.1 Various types of mobile genetic elements have captured these genes and the capturing element may provide a more powerful promoter and/or capture may separate the gene from a regulatory system on the chromosome, resulting in increased or constitutive expression. Each particular resistance gene generally appears to have been captured by one particular mobile element (Tables 1–3), presumably because capture events are rare and if selected for, the successful partnership then becomes dominant.2 Antibiotic pressure may result in selection of mutations that result in increased resistance or extension of the spectrum of antibiotics covered. Three main types of mobile genetic element (insertion sequences, transposons and the gene cassette/integron system; Fig. 1) are known to be involved in capturing resistance genes and moving them between DNA molecules in the same cell, e.g., from the chromosome to a plasmid or between plasmids, while plasmids can transfer resistance genes between bacterial cells, including different species. Insertion sequences (IS) Most IS3 are delineated by inverted repeats (IR), short identical or almost identical sequences in opposite orientation, which flank little more than a transposase (tnpA) gene. These IR are designated left (IRL) and right (IRR) relative to the direction of transcription of tnpA (Fig. 1). The encoded transposase protein recognises the IR and moves the IS to a new location by a ‘cut and paste’ or ‘copy and paste’ mechanism, the details of which vary for different IS families.3 Many IS create staggered cuts in the two DNA strands during the transposition process, which when repaired result in short direct repeats (DR; 2–14 bp, characteristic for each IS family) flanking the new insertion. DR are also referred to as target site duplications (TSD), although most IS do not appear to ‘target’ particular sequences. A pair of closely-related IS, in either relative orientation, can capture and move themselves and the region between them, which may include a resistance gene, as a composite
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RESISTANCE MECHANISMS IN ENTEROBACTERIACEAE
Table 1
277
Main types and variants of b-lactamases in Enterobacteriaceae Associated with resistance to
Class A blaTEM blaSHV§ blaCTX-M-1 group blaCTX-M-9 group blaCTX-M-2 group blaCTX-M-8 group blaCTX-M-25 group blaKLUC blaKPCjj blaGES Class B blaIMP-1-like blaIMP-2-like blaIMP-4-like blaVIM-1-like blaNDM Class C blaCMY-2-like blaDHA blaCMY-1-like/MOX blaACC blaACTô blaMIRô blaFOX Class D blaOXA-10-like blaOXA-30-like blaOXA-48-like blaOXA-181-like
APP*
3GC*
CPM{
Minor variants
Some Some No No No No No No No No
Some Some Yes Yes Yes Yes Yes Yes Yes Yes
No No No No No No No No (Yes) Some
Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes
Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes
z
Mobile element
Source
>200 >150 53 47 23 3 10 4 21 26
Tn3-like IS26 ISEcp1 ISEcp1, ISCR1 ISCR1 ISEcp1, IS10 ISEcp1 ISEcp1 Tn4401 Gene cassette
Unknown Klebsiella pneumoniae Kluyvera ascorbata Kluyvera georgiana Kluyvera ascorbata Kluyvera georgiana Kluyvera georgiana Kluyvera cryocresens Unknown Unknown
(Yes) (Yes) (Yes) (Yes) Yes
7 5 3 14 15
Gene cassette Gene cassette Gene cassette Gene cassette ISAba125
Unknown Unknown Unknown Unknown Unknown
Yes Yes Yes Yes Yes Yes Yes
No No No No No No No
101 23 17 5 36 18 12
ISEcp1 ISCR1 ISCR1 ISEcp1 ? ISCR ISApu2
Citrobacter freundii Morganella morganii Aeromonas hydrophila Hafnia alvei Enterobacter asburiae Enterobacter cloacae Aeromonas caviae
Some No No No
No No (Yes) (Yes)
11 7 9 2
Gene cassette Gene cassette Tn1999 ISEcp1
Unknown Unknown Shewanella Shewanella xiamenensis
*
APP, anti-pseudomonal penicillins (b-lactam þ b-lactamase inhibitor); 3GC, third-generation cephalosporin; some, inhibitor resistant or extended-spectrum variants. { CPM, carbapenems; (yes), an enzyme that is considered a carbapenemase but only gives noticeable carbapenem resistance if e.g. combined with porin defects. GES variants with 170Asp (2, 13) or 170Ser (4-6, 14-16, 18, 20-21, 24) have been shown or would be expected to confer CPM resistance, GES-1, 3, 7–12, 17, 19, 22–23 (170Gly) would not. Some AmpC and ESBL enzymes have been found to confer CPM resistance if porin defects are present. z Variant numbers are from http://www.lahey.org/Studies/, January 2015. § Klebsiella pneumoniae may carry two blaSHV variants, one chromosomal and one plasmid-borne. jj KPC are numbered 2–22. KPC-1 is no longer used, as an error was found in the original sequence. ô blaACT and blaMIR genes are related enough to be picked up by the same set of primers, but seem to be derived from different Enterobacter species.
transposon. For some types of IS, a single copy is able to capture and mobilise an adjacent resistance gene. ISEcp1 appears to miss its IRR end during transposition and use an alternative sequence situated further downstream, creating 5 bp DR flanking the entire ISEcp1 ‘transposition unit’.4 ISCR
elements5 are related to IS1294-like elements that have been demonstrated to transpose by a rolling circle replication mechanism.6 Replication starts at oriIS (downstream of the tnpA gene, also known as rcr, for rolling circle replicase) and should end at terIS, upstream of rcr. If replication continues past terIS
Table 2 Main mobile genes conferring resistance to clinically-important aminoglycosides in Enterobacteriacaeae
Table 3
Acetyltransferases aacA4/aac(60 )-Ib aacA4/aac(60 )-IIa aacC1/aac(3)-Ia aac(3)-II Adenylyltranseferases aadB/ant(200 )-1a Phosphorylases aphA6 aphA15 16S rRNA methylases armA rmtB rmtC rmtF
Resistance to
Mobile element
AMK TOB GEN TOB GEN GEN TOB
Gene cassette Gene cassette Gene cassette IS26?
GEN TOB
Gene cassette
AMK (AMK)
ISAba14 Gene cassette
AMK AMK AMK AMK
GEN GEN GEN GEN
TOB TOB TOB TOB
ISCR1 ISCR3? ISEcp1 ISCR3-like
Main types of PMQR determinants in Enterobacteriaceae Variants
Mobile element
Source
qnrA qnrB2 qnrB1 qnrB4 qnrB6 qnrB10 qnrB19
7
Shewanella Citrobacter Citrobacter Citrobacter Citrobacter Citrobacter Citrobacter
qnrD qnrS1-like qnrS2-like qnrS5-like qnrVC aac(60 )-Ib-cr qepA oqxAB
2 6 2 1 6 2 2 1
ISCR1 ISCR1 IS3000, IS26 ISCR1 ISCR1 ISCR1 ISEcp1 or small plasmid Small plasmid IS26? ? ? Gene cassette Gene cassette ISCR3?, IS26? IS26
algae spp. spp. spp. spp. spp. spp.
Unknown Vibrio splendidus Unknown Unknown Vibrio? Unknown Unknown Klebsiella pneumoniae
AMK, amikacin; GEN, gentamicin; TOB, tobramycin.
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278
A B
Pathology (2015), 47(3), April
PARTRIDGE
C. freundii chromosome
ISEcp1 transposition unit
to antibiotic resistance and have 25 bp IR, four tni transposition genes and a res site and create 5 bp DR. They are known to target the res regions of Tn21 and relatives.10 The distinction between IS and Tn can become blurry, as ‘passenger’ genes have now been identified in relatives of elements traditionally thought of as IS, while some elements closely related to Tn carry no such genes.11
ampR blaCMY-2 ATTTC
ATTTC ISEcp1 blaCMY-2
C
CGTTCAGCA Composite transposon
CGTTCAGCA
IS1999
IS1999 blaOXA-48
TATAC
D
TATAC
Tn3 subfamily transposon tnpA
tnpR blaTEM-52
E ISCR1 insertion
ISCR1 qnrB2
F
Class 1 integron IRi 5'-CS | Cassette array |3'-CS
IRt
DR
DR |blaIMP-4|qacG|aacA4|catB3| sul1
G
tniB∆
tniA
IRmer
IRtnp mer (mercury resistance) tnpA
tnpR
Tn21 subfamily transposon
Resistance gene
res site
Other gene Inverted repeat (IR) Promoter
attI site attC site
Fig. 1 The main types of mobile elements associated with resistance genes in Enterobacteriaceae. (A) The blaCMY-2 region of the Citrobacter freundii chromosome and (B) part of the same region found in an ISEcp1 transposition unit on a plasmid, illustrating gene capture. (C) The composite transposon Tn1999 carrying blaOXA-48. (D) A Tn2-like transposon carrying the blaTEM-52 ESBL gene, an example of a Tn3 subfamily transposon. (E) A gene captured by ISCR1 and inserted in a class 1 integron (F) with four cassettes and a partial tni region inserted in the res site of a Tn21-subfamily transposon (G). Sequences of DR are shown. All examples have been detected in bacterial DNA sequences, except for the exact combination of gene cassette array and Tn21-like transposon shown in F and G.
the adjacent region can be captured, apparently as a circular molecule proposed to insert by homologous recombination.5 Mobilisation of resistance genes by a single copy of the very common IS26 has also recently been demonstrated.7 Transposons (Tn) A Tn is a larger element that is essentially an IS that carries captured resistance or other genes internally. Tn implicated in movement of resistance genes generally belong to the Tn3 or Tn21 subfamilies.8,9 In addition to a large (3 kb) tnpA gene these Tn include a resolvase gene (tnpR) and its cognate resolution (res) site, usually have 38 bp IR and create 5 bp DR. The two subfamilies differ in the arrangement of tnpA, tnpR and res (Fig. 1). During transposition a cointegrate is formed from the original DNA molecule carrying the Tn and a recipient molecule, separated by two copies of the Tn. This cointegrate is resolved into two molecules, each with a copy of the Tn, by TnpR-mediated recombination in the res site. Tn5053/Tn402-like transposons are also important in relation
Integrons and gene cassettes A gene cassette is a small mobile element consisting of little more than a (usually promoterless) gene and a recombination site (attC) and can exist in a circular form.12 Gene cassettes are captured by larger elements called integrons by recombination between attC and the integron recombination site (attI), catalysed by the integrase encoded by the intI gene of the integron.13 The integron also provides a promoter (Pc) for expression of the captured cassette genes, which are all inserted in the same orientation, and integration of multiple cassettes can create cassette arrays conferring multi-resistance. Integrons have been divided into classes, defined on the basis of the relatedness of their IntI protein sequences, and the most common type in clinical isolates are class 1 integrons. The first examples to be identified appear to have been created by capture of the intI1/attI1/Pc components by a Tn5053/Tn402 family transposon and replacement of part of the tni transposition region by a sequence defined as the 30 -conserved segment (30 -CS),14 preventing self-transposition. More rarely, examples with intI1/attI1/Pc and a complete tni region are found (mostly in Pseudomonas aeruginosa) and these are able to move themselves. The IR of both types of In/Tn unit are designated IRi (integrase end) and IRt (tni end) and the region between IRi and the recombination point in the attI1 site is defined as the 50 -CS. PCR with different primers in the 50 -CS and 30 -CS is frequently used to amplify cassette arrays and identify resistance genes. In so-called ‘complex’ class 1 integrons an ISCR1 element and associated resistance gene(s) are found between partially duplicated copies of the 30 -CS. The known preference for insertion of Tn5053/Tn402-like transposons in Tn21-like res sites also means that transposition-defective class 1 integrons can ‘piggyback’ on larger Tn.
RESISTANCE TO b-LACTAM ANTIBIOTICS The b-lactam group of antibiotics encompasses penicillins, cephalosporins, carbapenems, penems and monobactams. They irreversibly bind to the pencillin binding proteins (PBP) required for the final crosslinking (transpeptidation) step in the synthesis of peptidoglycan for cell wall construction. The most clinically important are the third-generation cephalosporins, ‘anti-pseudomonal’ b-lactam/b-lactamase inhibitor combinations, and carbapenems. The most common reason for resistance to b-lactam antibiotics in the Enterobacteriaceae is the expression of b-lactamase enzymes (encoded by bla genes) that hydrolise the b-lactam ring, resulting in inactivation. Many different types of b-lactamase can confer resistance to each of the most clinically-important b-lactam types and a single amino acid difference may affect the phenotype conferred (Table 1). b-lactamases have been grouped in different ways, the simplest being division into classes A–D.15 Class A b-lactamases Class A includes ‘narrow spectrum’ enzymes, most notably the blaTEM and blaSHV groups, that confer resistance to penicillins
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RESISTANCE MECHANISMS IN ENTEROBACTERIACEAE
and early cephalosporins. ‘Extended-spectrum’ (ESBL) and inhibitor-resistant variants, with 1–3 amino acid changes at a few key positions, began to appear after introduction of third-generation cephalosporins and b-lactamase inhibitors.16 Other Class A b-lactamases intrinsically have this ‘extendedspectrum’ phenotype, e.g., the CTX-M family. CTX-M enzymes can be divided into six groups (-1, -2, -8, -9, -25, -KLUC) with 96% identity within each group. The -cr (ciprofloxacin resistance) variant of the aac(60 )-Ib (aacA4) gene encodes an enzyme that can N-acetylate the piperazinyl substituent of ciprofloxacin and is associated with low-level fluoroquinolone resistance but reduced resistance to aminoglycosides.69 All currently known gene cassettes encoding this variant have T329/Leu102, a mutation at position 514 (GAT to TAT; Asp164Tyr) and one of two mutations at position 283 (TGG to AGG or CGG, giving Trp87Arg). Two plasmid-encoded efflux pumps are also relevant to fluoroquinolone resistance. The qepA gene encodes a proton antiporter efflux pump of the major facilitator superfamily (MFS) and can remove quinolones from the cell.70 The OqxAB efflux pump contributes to resistance to olaquindox (used as a growth promoter in animals) as well as fluoroquinolones and chloramphenicol.71 The oqxAB genes were initially identified on a plasmid,72 having apparently been captured from the K. pneumoniae chromosome.
RESISTANCE TO TRIMETHOPRIM AND SULPHONAMIDES Trimethoprim and sulphonamides are used in combination (cotrimoxazole) to target two successive steps in the folate synthesis pathway. Sulphonamide resistance in Enterobacteriaceae is associated with three different genes (sul1, sul2, sul3) encoding drug-resistant dihydropteroate synthases. sul1 is part
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of the 30 -CS found in the most common type of class 1 integrons in clinical isolates. At least 19 different dfrA and >8 different dfrB gene cassettes, encoding two different types of trimethoprim-resistant dihydrofolate reductases, have been identified although some, e.g., dfrA12 and dfrA17, appear much more common than others.12 A few other dfrA genes are mainly associated with ISCR1 or ISCR2 elements. The presence of a dfr cassette, or a dfrA gene associated with ISCR1, in a class 1 integron that has sul1 in the 30 -CS can confer resistance to cotrimoxazole. sul3 is also associated with a type of class 1 integron that lacks the 30 -CS and that to date is only known to be associated with a limited selection of gene cassettes, but these can include dfrA12.12 sul2 is associated with ISCR2 or located close to fragments of Tn5393 that include the strAB genes conferring streptomycin resistance, with no obvious link to dfr genes, but these may be found on the same plasmid.
RESISTANCE TO TIGECYCLINE Tigecycline, a glycylcycline, inhibits bacterial protein synthesis by blocking entry of amino-acyl tRNA molecules into the ribosomal A-site, preventing incorporation of amino acids into elongating peptide chains. It is related to tetracyclines, but appears not to be a substrate for tetracycline-specific efflux pumps, encoded by plasmid-borne tetA genes in Enterobacteriaceae, apparently due to its larger side chain. Although tigecycline was introduced relatively recently Enterobacteriaceae isolates resistant to this antibiotic have already been reported, due to overexpression of other efflux pumps including AcrAB-TolC and OqxAB. The AcrAB-TolC efflux pump, present in most Enterobacteriaceae, belongs to the resistance-nodulation-division (RND) family of transporters. It consists of the AcrA periplasmic adaptor, the AcrB cytoplasmic membrane transporter protein and the TolC outer membrane channel. AcrAB expression is regulated by a specific transcriptional repressor, AcrR, and by global regulatory systems. Mutations leading to tigecycline resistance appear to occur in different regulatory genes. For example, in E. coli AcrAB expression may be upregulated by mutations affecting MarR, part of the mar (multiple antibiotic resistance) regulon, that activate MarA, lifting the repression caused by AcrR.73 In K. pneumoniae mutations in ramR, encoding a specific transcriptional repressor expression of the RamA transcription factor, can cause increased AcrAB expression.74 An IS5 insertion in the promoter region of the kpgABC operon, proposed to encode an efflux pump, has also been reported in relation to tigecycline resistance in K. pneumoniae,75 while in other isolates this resistance remains unexplained,76 suggesting that further tigecycline resistance mechanisms may be identified.
RESISTANCE TO COLISTIN/POLYMYXIN Polymyxins, cyclic cationic decapeptides bound to a fatty acid chain, were originally discovered in the 1940s. They target the negatively-charged lipid moiety of lipopolysaccharide (LPS) of the outer membrane, displacing divalent cations (Ca2þ, Mg2þ) that stabilise the LPS. This disrupts the integrity of the outer membrane, causing leakage of intracellular contents and cell death. Routine use of polymyxins was discontinued in the 1980s due to common and serious side effects (nephrotoxicity, neurotoxicity), but the need to find ways of treating multidrugresistant Gram-negative infections has led to a recent revival in
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their use. Polymyxins B and E (colistin), which differ by a single amino acid, are used clinically, with colistin being the most commonly used in recent years. In Enterobacteriaceae, polymyxin resistance can arise by a variety of LPS modifications,77 e.g., addition of 4-amino4-deoxy-l-arabinose (L-Ara4N) to the phosphate group of Lipid A, which reduces the negative charges available for interactions. L-Ara4N is synthesised and added to LPS by proteins encoded by the arn (for AraN synthesis) operon, previously called the pmrHFIJKLM (for polymyxin resistance) or pbg operon.78 Expression of this operon is under the control of the PmrA/PmrB and PhoP/PhoQ two-component regulatory systems that allow bacteria to alter gene expression in response to environmental changes, such as ion concentrations and pH. In each case a sensor histidine kinase (PmrB or PhoQ) located in the cytoplasmic membrane can phosphorylate and activate the regulator (PmrA or PhoP). Activated PmrA increases expression of the arn operon, while PhoP enhances expression of another protein PmrD, which maintains activated PmrA by inhibiting dephosphorylation.78 Mutations in pmrA and pmrB have been detected in E. coli.79 In K. pneumoniae carrying a blaKPC gene colistin resistance has arisen during treatment due to mutations in pmrB80 or insertional inactivation of the mgrB gene, which encodes a negative-feedback regulator of PhoQ/ PhoQ.81 Other LPS modifications, efflux pump alterations, overexpression of porins and increased production of capsule polysaccharide can also contribute to polymyxin resistance.77
RESISTANCE TO FOSFOMYCIN Fosfomycin, another older antibiotic being re-examined in the era of multi-resistance, inhibits bacterial cell wall biosynthesis by alkylating an active site cysteine residue of the MurA enzyme, preventing formation of the peptidoglycan precursor N-acetylmuramic acid. Several fosfomycin resistance mechanisms are already known. FosA-, FosB-, and FosX-type enzymes add glutathione, L-cysteine, or a hydroxyl group, respectively, to the oxirane ring of fosfomycin, inactivating this antibiotic. Several gene cassette-borne fos genes have been identified but most have been found only in P. aeruginosa. The exception is the single known example of fosC2 from E. coli,82 which encodes glutathione S-transferase activity. The fosA3 gene, associated with IS26 and first reported in Japan82 has also been found in Korea83 and is increasingly reported in China.84
RESISTANCE PLASMIDS AND MULTI-RESISTANCE Plasmids that carry resistance genes may either be large conjugative plasmids, which carry the genes encoding all of the machinery required to transfer themselves between bacterial cells, or smaller mobilisable plasmids that may be co-transferred by conjugative plasmids. These plasmids generally consist of a ‘backbone’ that encodes plasmid functions such as replication, stability and conjugation, into which variable ‘accessory’ regions are inserted. Plasmid backbones were originally classified into ‘Inc’ groups based on experimentally-defined mutual incompatibility, i.e., plasmids in the same Inc group cannot stably co-exist in the same cell and one will be lost. PCR-based typing of replicons (PBRT)85 or genes encoding the relaxase protein required for mobilisation/conjugation (DPMT)86 can be used to classify plasmids, but each only samples part of the backbone.
Accessory regions, including those encoding antibiotic resistance, may occupy a large proportion of the plasmid. The adverse effects of disrupting essential plasmid functions means that a mobile element that has become inserted without incapacitating a plasmid may then become a target of insertion of other mobile elements and associated resistance genes, so that these elements are often found clustered together in complex multi-resistance regions (MRR).2 Subsequent movement of resistance genes between DNA molecules may then be facilitated by other nearby mobile elements or by homologous recombination between repeated elements, which may also lead to rearrangements in MRR. Clustering of resistance genes in this way means that certain combinations of genes frequently travel together and that use of one antibiotic may select for maintenance of genes giving resistance to unrelated antibiotic classes. For example, E. coli ST131 isolates often carry blaCTX-M-15 in large, complex MRR on IncF-type plasmids. These related MRR can carry different combinations of the blaTEM-1, aac(3)-II, tetA(A) (tetracycline resistance) and either both aac(60 )-Ib-cr and blaOXA-30 or neither of these genes. Differences can be explained by the actions of the various mobile elements associated with these genes and/or homologous recombination. Identifying whether aac(3)-II (gentamicin resistance), in particular, is present may be clinically relevant. Knowledge of gene associations may help in tracking important genes that do not always express a strong resistance phenotype. For example, in Sydney, an aac(3)-II gene (gentamicin and tobramycin resistance) was consistently identified on the same IncL/M plasmid as blaIMP-4,87 which only gives a truly carbapenem-resistant phenotype in the presence of OmpK defects.88 This allowed more efficient tracking of the silent spread of blaIMP-4, by pre-screening isolates on gentamicin and testing only those that were resistant for blaIMP-4 by PCR. Identification of blaIMP-4 by screening also indicated when neither carbapenems, which select for isolates that have become fully resistant, nor gentamicin should be prescribed.89 Klebsiella pneumoniae ST258 isolates that are commonly associated with carriage of blaKPC can be divided into two subtypes that appear to carry different sets of plasmids and resistance genes and apparently have different levels of resistance to carbapenems,90 possibly due to porin defects.29 The common association of blaKPC with the amikacin resistance variant of aacA4 may be clinically important, but in this case the blaKPC and aacA4 genes are found on different plasmids in both ST258 types, so do not necessarily always travel together. Tracking of sets of resistance genes, rather than individual genes, therefore may be important for epidemiological, infection control and/or treatment purposes, but identifying the best sets of targets to use can be challenging and may require initial detailed characterisation of a small number of isolates. Combinations of PCR tests for individual genes can be employed, but multiplexed methods such as multiplex PCR/reverse line blot33,91 can simultaneously cover more targets in multiple isolates economically and efficiently. Microarray-type and highly multiplexed PCR methods are also available, but may be unnecessary if known gene associations can be taken into account.
KEY MESSAGES Resistance mechanisms include hydrolysis or modification of antibiotics, protection or replacement of antibiotic
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targets, decreased antibiotic entry and/or increased efflux or modification of the outer membrane. Much important resistance in Enterobacteriaceae is due to mobile resistance genes that can transfer between different cells and to different species on plasmids. Each mobile resistance gene is generally closely associated with one particular mobile element (IS, Tn or the gene cassette/integron system) which may also enhance expression. Resistance genes and mobile elements tend to cluster in large, complex multi-resistance regions on plasmids, allowing simultaneous transfer of multi-resistance and co-selection Mutations in chromosomal genes are important for resistance to some antibiotic classes, including some now being employed as last-line antibiotics, such as polymyxins and tigecycline. A large number of different genes may confer resistance to each antibiotic class or subclass and distinguishing them for epidemiological or treatment purposes is likely to require different levels of gene typing.
Conflicts of interest and sources of funding: The author states that there are no conflicts of interest to disclose. Address for correspondence: Dr Sally Partridge, Centre for Infectious Diseases and Microbiology, Westmead Millennium Institute, 176 Hawkesbury Road, Westmead, NSW 2145, Australia. E-mail: [email protected]. gov.au
References 1. Martinez JL, Fajardo A, Garmendia L, et al. A global view of antibiotic resistance. FEMS Microbiol Rev 2009; 33: 44–65. 2. Partridge SR. Analysis of antibiotic resistance regions in Gram-negative bacteria. FEMS Microbiol Rev 2011; 35: 820–55. 3. Chandler M, Mahillon J. Insertion sequences revisited. In: Craig NL, Craigie R, Gellert M, Lambowitz AM, editors. Mobile DNA II. Washington DC: ASM Press, 2002: 305–366. 4. Poirel L, Lartigue MF, Decousser JW, Nordmann P. ISEcp1B-mediated transposition of blaCTX-M in Escherichia coli. Antimicrob Agents Chemother 2005; 49: 447–50. 5. Toleman MA, Bennett PM, Walsh TR. ISCR elements: novel gene-capturing systems of the 21st century? Microbiol Mol Biol Rev 2006; 70: 296– 316. 6. Tavakoli N, Comanducci A, Dodd HM, Lett MC, Albiger B, Bennett P. IS1294, a DNA element that transposes by RC transposition. Plasmid 2000; 44: 66–84. 7. Harmer CJ, Moran RA, Hall RM. Movement of IS26-associated antibiotic resistance genes occurs via a translocatable unit that includes a single IS26 and preferentially inserts adjacent to another IS26. MBio 2014; 5: e01801– 1814. 8. Grindley NDF. The movement of Tn3-like elements: transposition and cointegrate resolution. In: Craig NL, Craigie R, Gellert M, Lambowitz AM, editors. Mobile DNA II. Washington DC: ASM Press, 2002: 272–302. 9. Grinsted J, de la Cruz F, Schmitt R. The Tn21 subgroup of bacterial transposable elements. Plasmid 1990; 24: 163–89. 10. Kamali-Moghaddam M, Sundstro¨m L. Transposon targeting determined by resolvase. FEMS Microbiol Lett 2000; 186: 55–9. 11. Siguier P, Gagnevin L, Chandler M. The new IS1595 family, its relation to IS1 and the frontier between insertion sequences and transposons. Res Microbiol 2009; 160: 232–41. 12. Partridge SR, Tsafnat G, Coiera E, Iredell JR. Gene cassettes and cassette arrays in mobile resistance integrons. FEMS Microbiol Rev 2009; 33: 757– 84. 13. Mazel D. Integrons: agents of bacterial evolution. Nat Rev Microbiol 2006; 4: 608–20. 14. Gillings M, Boucher Y, Labbate M, et al. The evolution of class 1 integrons and the rise of antibiotic resistance. J Bacteriol 2008; 190: 5095–100. 15. Bush K, Jacoby GA. Updated functional classification of b-lactamases. Antimicrob Agents Chemother 2010; 54: 969–76. 16. Bradford PA. Extended-spectrum b-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001; 14: 933–51.
283
17. D’Andrea MM, Arena F, Pallecchi L, Rossolini GM. CTX-M-type b-lactamases: A successful story of antibiotic resistance. Int J Med Microbiol 2013; 303: 305–17. 18. Woodford N, Turton JF, Livermore DM. Multiresistant Gram-negative bacteria: the role of high-risk clones in the dissemination of antibiotic resistance. FEMS Microbiol Rev 2011; 35: 736–55. 19. Nordmann P, Cuzon G, Naas T. The real threat of Klebsiella pneumoniae carbapenemase producing bacteria. Lancet Infect Dis 2009; 9: 228–33. 20. Ambler RP, Coulson AF, Frere JM, et al. A standard numbering scheme for the Class A b-lactamases. Biochem J 1991; 276: 269–70. 21. Frase H, Shi Q, Testero SA, Mobashery S, Vakulenko SB. Mechanistic basis for the emergence of catalytic competence against carbapenem antibiotics by the GES family of b-lactamases. J Biol Chem 2009; 284: 29509–13. 22. Cornaglia G, Giamarellou H, Rossolini GM. Metallo-b-lactamases: a last frontier for b-lactams? Lancet Infect Dis 2011; 11: 381–93. 23. Johnson AP, Woodford N. Global spread of antibiotic resistance: the example of New Delhi metallo-b-lactamase (NDM)-mediated carbapenem resistance. J Med Microbiol 2013; 62: 499–513. 24. Jacoby GA. AmpC b-lactamases. Clin Microbiol Rev 2009; 22: 161–82. 25. Nelson EC, Elisha BG. Molecular basis of AmpC hyperproduction in clinical isolates of Escherichia coli. Antimicrob Agents Chemother 1999; 43: 957–9. 26. Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: the phantom menace. J Antimicrob Chemother 2012; 67: 1597–606. 27. Wang XD, Cai JC, Zhou HW, Zhang R, Chen GX. Reduced susceptibility to carbapenems in Klebsiella pneumoniae clinical isolates associated with plasmid-mediated b-lactamase production and OmpK36 porin deficiency. J Med Microbiol 2009; 58: 1196–202. 28. Martı´nez-Martı´nez L. Extended-spectrum b-lactamases and the permeability barrier. Clin Microbiol Infect 2008; 14 (Suppl 1): 82–9. 29. Partridge SR, Ginn AN, Wiklendt AM, et al. Emergence of blaKPC carbapenemase genes in Australia. Int J Antimicrob Agents 2013; 45: 130–6. 30. Clancy CJ, Chen L, Hong JH, et al. Mutations of the ompK36 porin gene and promoter impact responses of ST258, KPC-2-producing Klebsiella pneumoniae strains to doripenem and doripenem-colistin. Antimicrob Agents Chemother 2013; 57: 5258–65. 31. Turnidge JD, Gottlieb T, Mitchell DH, Coombs GW, Pearson JC, Bell JM. Hospital-onset Gram-negative Surveillance Program annual report, 2011. Commun Dis Intell Q Rep 2014; 38: E49–53. 32. Turnidge JD, Gottlieb T, Mitchell DH, Coombs GW, Daly DA, Bell JM. Community-onset Gram-negative Surveillance Program annual report, 2012. Commun Dis Intell Q Rep 2014; 38: E54–8. 33. Ginn AN, Zong Z, Wiklendt AM, et al. Limited diversity in the gene pool allows prediction of third-generation cephalosporin and aminoglycoside resistance in Escherichia coli and Klebsiella pneumoniae. Int J Antimicrob Agents 2013; 42: 19–26. 34. Ellem J, Partridge SR, Iredell JR. Efficient direct ESBL detection by multiplex real-time PCR: accurate assignment of phenotype using a limited set of genetic markers. J Clin Microbiol 2011; 49: 3074–7. 35. Peleg AY, Franklin C, Bell J, Spelman DW. Emergence of IMP-4 metallob-lactamase in a clinical isolate from Australia. J Antimicrob Chemother 2004; 54: 699–700. 36. Thomas L, Espedido B, Watson S, Iredell J. Forewarned is forearmed: antibiotic resistance gene surveillance in critical care. J Hosp Infect 2005; 60: 291–3. 37. Leung GH, Gray TJ, Cheong EY, Haertsch P, Gottlieb T. Persistence of related bla-IMP-4 metallo-beta-lactamase producing Enterobacteriaceae from clinical and environmental specimens within a burns unit in Australia - a six-year retrospective study. Antimicrob Resist Infect Control 2013; 2: 35. 38. Peirano G, Lascols C, Hackel M, Hoban DJ, Pitout JD. Molecular epidemiology of Enterobacteriaceae that produce VIMs and IMPs from the SMART surveillance program. Diagn Microbiol Infect Dis 2014; 78: 277– 81. 39. Poirel L, Lagrutta E, Taylor P, Pham J, Nordmann P. Emergence of metallob-lactamase NDM-1-producing multidrug-resistant Escherichia coli in Australia. Antimicrob Agents Chemother 2010; 54: 4914–6. 40. Fernando GA, Collignon PJ, Bell JM. A risk for returned travellers: the ‘‘post-antibiotic era’’. Med J Aust 2010; 193: 59. 41. Sidjabat H, Nimmo GR, Walsh TR, et al. Carbapenem resistance in Klebsiella pneumoniae due to the New Delhi metallo-b-lactamase. Clin Infect Dis 2011; 52: 481–4. 42. Blyth CC, Pereira L, Goire N. New Delhi metallo-beta-lactamase-producing Enterobacteriaceae in an Australian child who had not travelled overseas. Med J Aust 2014; 200: 386. 43. Shoma S, Kamruzzaman M, Ginn AN, Iredell JR, Partridge SR. Characterization of multidrug-resistant Klebsiella pneumoniae from Australia carrying blaNDM-1. Diagn Microbiol Infect Dis 2014; 78: 93–7. 44. Espedido BA, Steen JA, Ziochos H, et al. Whole genome sequence analysis of the first OXA-48-producing outbreak-associated Klebsiella pneumoniae isolates: the resistome and in vivo evolution. PLoS One 2013; 8: e59920.
Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited.
284
PARTRIDGE
45. Sidjabat HE, Kennedy K, Silvey A, Collignon P, Paterson DL. Emergence of blaOXA-181-carrying ColE plasmid in Klebsiella pneumoniae in Australia. Int J Antimicrob Agents 2013; 41: 294–6. 46. Coatsworth NR, Huntington PG, Hardiman RP, Hudson BJ, Fernandes CJ. A case of carbapenemase-producing Klebsiella pneumoniae in Australia. Pathology 2012; 44: 42–4. 47. Chang L. Investigation of a nosocomial outbreak of KPC-producing Klebsiella pneumoniae. Pathology 2014; 46 (Suppl 1): S46. 48. Williamson DA, Heffernan H. The changing landscape of antimicrobial resistance in New Zealand. NZ Med J 2014; 127: 41–54. 49. Heffernan HM, Woodhouse RE, Pope CE, Blackmore TK. Prevalence and types of extended-spectrum b-lactamases among urinary Escherichia coli and Klebsiella spp. in New Zealand. Int J Antimicrob Agents 2009; 34: 544–9. 50. Freeman JT, Williamson DA, Heffernan H, Smith M, Bower JE, Roberts SA. Comparative epidemiology of CTX-M-14 and CTX-M-15 producing Escherichia coli: association with distinct demographic groups in the community in New Zealand. Eur J Clin Microbiol Infect Dis 2012; 31: 2057–60. 51. Williamson DA, Sidjabat HE, Freeman JT, et al. Identification and molecular characterisation of New Delhi metallo-b-lactamase-1 (NDM-1)- and NDM-6-producing Enterobacteriaceae from New Zealand hospitals. Int J Antimicrob Agents 2012; 39: 529–33. 52. Williamson DA, Heffernan H, Sidjabat H, et al. Intercontinental transfer of OXA-181-producing Klebsiella pneumoniae into New Zealand. J Antimicrob Chemother 2011; 66: 2888–90. 53. Shaw KJ, Rather PN, Hare RS, Miller GH. Molecular genetics of aminoglycoside resistance genes and familial relationships of the aminoglycoside-modifying enzymes. Microbiol Rev 1993; 57: 138–63. 54. Ramirez MS, Tolmasky ME. Aminoglycoside modifying enzymes. Drug Resist Updat 2010; 13: 151–71. 55. Novick RP, Clowes RC, Cohen SN, Curtiss R 3rd, Datta N, Falkow S. Uniform nomenclature for bacterial plasmids: a proposal. Bacteriol Rev 1976; 40: 168–89. 56. Hanau-Berc¸ot B, Podglajen I, Casin I, Collatz E. An intrinsic control element for translational initiation in class 1 integrons. Mol Microbiol 2002; 44: 119–30. 57. Rather PN, Munayyer H, Mann PA, Hare RS, Miller GH, Shaw KJ. Genetic analysis of bacterial acetyltransferases: identification of amino acids determining the specificities of the aminoglycoside 60 -N-acetyltransferase Ib and IIa proteins. J Bacteriol 1992; 174: 3196–203. 58. Lambert T, Ploy MC, Courvalin P. A spontaneous point mutation in the aac(60 )-Ib0 gene results in altered substrate specificity of aminoglycoside 60 -N-acetyltransferase of a Pseudomonas fluorescens strain. FEMS Microbiol Lett 1994; 115: 297–304. 59. Casin I, Hanau-Bercot B, Podglajen I, Vahaboglu H, Collatz E. Salmonella enterica serovar Typhimurium blaPER-1-carrying plasmid pSTI1 encodes an extended-spectrum aminoglycoside 60 -N-acetyltransferase of type Ib. Antimicrob Agents Chemother 2003; 47: 697–703. 60. Wachino J, Arakawa Y. Exogenously acquired 16S rRNA methyltransferases found in aminoglycoside-resistant pathogenic Gram-negative bacteria: an update. Drug Resist Updat 2012; 15: 133–48. 61. Aldred KJ, Kerns RJ, Osheroff N. Mechanism of quinolone action and resistance. Biochemistry 2014; 53: 1565–74. 62. Hopkins KL, Davies RH, Threlfall EJ. Mechanisms of quinolone resistance in Escherichia coli and Salmonella: recent developments. Int J Antimicrob Agents 2005; 25: 358–73. 63. Strahilevitz J, Jacoby GA, Hooper DC, Robicsek A. Plasmid-mediated quinolone resistance: a multifaceted threat. Clin Microbiol Rev 2009; 22: 664–89. 64. Jacoby GA, Griffin CM, Hooper DC. Citrobacter spp. as a source of qnrB alleles. Antimicrob Agents Chemother 2011; 55: 4979–84. 65. Jacoby G, Cattoir V, Hooper D, et al. qnr gene nomenclature. Antimicrob Agents Chemother 2008; 52: 2297–9. 66. Poirel L, Rodriguez-Martinez JM, Mammeri H, Liard A, Nordmann P. Origin of plasmid-mediated quinolone resistance determinant QnrA. Antimicrob Agents Chemother 2005; 49: 3523–5. 67. Wang M, Guo Q, Xu X, et al. New plasmid-mediated quinolone resistance gene, qnrC, found in a clinical isolate of Proteus mirabilis. Antimicrob Agents Chemother 2009; 53: 1892–7. 68. Cattoir V, Poirel L, Mazel D, Soussy CJ, Nordmann P. Vibrio splendidus as the source of plasmid-mediated QnrS-like quinolone resistance determinants. Antimicrob Agents Chemother 2007; 51: 2650–1. 69. Robicsek A, Strahilevitz J, Jacoby GA, et al. Fluoroquinolone-modifying enzyme: a new adaptation of a common aminoglycoside acetyltransferase. Nat Med 2006; 12: 83–8.
Pathology (2015), 47(3), April
70. Yamane K, Wachino J, Suzuki S, et al. New plasmid-mediated fluoroquinolone efflux pump, QepA, found in an Escherichia coli clinical isolate. Antimicrob Agents Chemother 2007; 51: 3354–60. 71. Hansen LH, Jensen LB, Sorensen HI, Sorensen SJ. Substrate specificity of the OqxAB multidrug resistance pump in Escherichia coli and selected enteric bacteria. J Antimicrob Chemother 2007; 60: 145–7. 72. Hansen LH, Johannesen E, Burmolle M, Sorensen AH, Sorensen SJ. Plasmid-encoded multidrug efflux pump conferring resistance to olaquindox in Escherichia coli. Antimicrob Agents Chemother 2004; 48: 3332–7. 73. Keeney D, Ruzin A, McAleese F, Murphy E, Bradford PA. MarA-mediated overexpression of the AcrAB efflux pump results in decreased susceptibility to tigecycline in Escherichia coli. J Antimicrob Chemother 2008; 61: 46–53. 74. Hentschke M, Wolters M, Sobottka I, Rohde H, Aepfelbacher M. ramR mutations in clinical isolates of Klebsiella pneumoniae with reduced susceptibility to tigecycline. Antimicrob Agents Chemother 2010; 54: 2720–3. 75. Nielsen LE, Snesrud EC, Onmus-Leone F, et al. IS5 element integration, a novel mechanism for rapid in vivo emergence of tigecycline nonsusceptibility in Klebsiella pneumoniae. Antimicrob Agents Chemother 2014; 58: 6151–6. 76. Sheng ZK, Hu F, Wang W, et al. Mechanisms of tigecycline resistance among Klebsiella pneumoniae clinical isolates. Antimicrob Agents Chemother 2014; 58: 6982–5. 77. Olaitan AO, Morand S, Rolain JM. Mechanisms of polymyxin resistance: acquired and intrinsic resistance in bacteria. Front Microbiol 2014; 5: 643. 78. Falagas ME, Rafailidis PI, Matthaiou DK. Resistance to polymyxins: Mechanisms, frequency and treatment options. Drug Resist Updat 2010; 13: 132–8. 79. Quesada A, Porrero MC, Tellez S, Palomo G, Garcia M, Dominguez L. Polymorphism of genes encoding PmrAB in colistin-resistant strains of Escherichia coli and Salmonella enterica isolated from poultry and swine. J Antimicrob Chemother 2015; 70: 71–4. 80. Cannatelli A, Di Pilato V, Giani T, et al. In vivo evolution to colistin resistance by PmrB sensor kinase mutation in KPC-producing Klebsiella pneumoniae is associated with low-dosage colistin treatment. Antimicrob Agents Chemother 2014; 58: 4399–403. 81. Cannatelli A, D’Andrea MM, Giani T, et al. In vivo emergence of colistin resistance in Klebsiella pneumoniae producing KPC-type carbapenemases mediated by insertional inactivation of the PhoQ/PhoP mgrB regulator. Antimicrob Agents Chemother 2013; 57: 5521–6. 82. Wachino J, Yamane K, Suzuki S, Kimura K, Arakawa Y. Prevalence of fosfomycin resistance among CTX-M-producing Escherichia coli clinical isolates in Japan and identification of novel plasmid-mediated fosfomycinmodifying enzymes. Antimicrob Agents Chemother 2010; 54: 3061–4. 83. Lee SY, Park YJ, Yu JK, et al. Prevalence of acquired fosfomycin resistance among extended-spectrum b-lactamase-producing Escherichia coli and Klebsiella pneumoniae clinical isolates in Korea and IS26-composite transposon surrounding fosA3. J Antimicrob Chemother 2012; 67: 2843–7. 84. Hou J, Yang X, Zeng Z, et al. Detection of the plasmid-encoded fosfomycin resistance gene fosA3 in Escherichia coli of food-animal origin. J Antimicrob Chemother 2013; 68: 766–70. 85. Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 2005; 63: 219–28. 86. Alvarado A, Garcillan-Barcia MP, de la Cruz F. A degenerate primer MOB typing (DPMT) method to classify gamma-proteobacterial plasmids in clinical and environmental settings. PLoS One 2012; 7: e40438. 87. Espedido BA, Partridge SR, Iredell JR. blaIMP-4 in different genetic contexts in Enterobacteriaceae isolates from Australia. Antimicrob Agents Chemother 2008; 52: 2984–7. 88. Espedido BA, Thomas LC, Iredell JR. Metallo-b-lactamase or extendedspectrum b-lactamase: a wolf in sheep’s clothing. J Clin Microbiol 2007; 45: 2034–6. 89. van Hal SJ, Wiklendt A, Espedido B, Ginn A, Iredell JR. Immediate appearance of plasmid-mediated resistance to multiple antibiotics upon antibiotic selection: an argument for systematic resistance epidemiology. J Clin Microbiol 2009; 47: 2325–7. 90. Wright MS, Perez F, Brinkac L, et al. Population structure of KPCproducing Klebsiella pneumoniae isolates from midwestern U. S. hospitals. Antimicrob Agents Chemother 2014; 58: 4961–5. 91. Kong F, Gilbert GL. Multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB)–a practical epidemiological and diagnostic tool. Nat Protoc 2006; 1: 2668–80.
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