Predictability of Phenotype in Relation to Common β-Lactam Resistance Mechanisms in Escherichia coli and Klebsiella pneumoniae

Alex Agyekum; Alicia Fajardo-Lubián; Xiaoman Ai; Andrew N Ginn; Zhiyong Zong; Xuejun Guo; John Turnidge; Sally R Partridge; Jonathan R Iredell

doi:10.1128/JCM.02153-15

. 2016 Apr 25;54(5):1243–1250. doi: 10.1128/JCM.02153-15

Predictability of Phenotype in Relation to Common β-Lactam Resistance Mechanisms in Escherichia coli and Klebsiella pneumoniae

Alex Agyekum ^a, Alicia Fajardo-Lubián ^a,^✉, Xiaoman Ai ^a,^*, Andrew N Ginn ^a, Zhiyong Zong ^a,^*, Xuejun Guo ^a,^*, John Turnidge ^b,^c, Sally R Partridge ^a, Jonathan R Iredell ^a,^✉

Editor: N A Ledeboer

PMCID: PMC4844708 PMID: 26912748

Abstract

The minimal concentration of antibiotic required to inhibit the growth of different isolates of a given species with no acquired resistance mechanisms has a normal distribution. We have previously shown that the presence or absence of transmissible antibiotic resistance genes has excellent predictive power for phenotype. In this study, we analyzed the distribution of six β-lactam antibiotic susceptibility phenotypes associated with commonly acquired resistance genes in Enterobacteriaceae in Sydney, Australia. Escherichia coli (n = 200) and Klebsiella pneumoniae (n = 178) clinical isolates, with relevant transmissible resistance genes (bla_TEM, n = 33; plasmid AmpC, n = 69; extended-spectrum β-lactamase [ESBL], n = 116; and carbapenemase, n = 100), were characterized. A group of 60 isolates with no phenotypic resistance to any antibiotics tested and carrying none of the important β-lactamase genes served as comparators. The MICs for all drug-bacterium combinations had a normal distribution, varying only in the presence of additional genes relevant to the phenotype or, for ertapenem resistance in K. pneumoniae, with a loss or change in the outer membrane porin protein OmpK36. We demonstrated mutations in ompK36 or absence of OmpK36 in all isolates in which reduced susceptibility to ertapenem (MIC, >1 mg/liter) was evident. Ertapenem nonsusceptibility in K. pneumoniae was most common in the context of an OmpK36 variant with an ESBL or AmpC gene. Surveillance strategies to define appropriate antimicrobial therapies should include genotype-phenotype relationships for all major transmissible resistance genes and the characterization of mutations in relevant porins in organisms, like K. pneumoniae.

INTRODUCTION

Escherichia coli and Klebsiella pneumoniae are among the most important pathogens in community and hospital settings, and their increasing resistance to extended-spectrum (third- and fourth-generation) cephalosporin and carbapenem antibiotics is a major health threat. In Gram-negative bacteria, such as these, a variety of mechanisms may confer β-lactam resistance (1), but the spread of transmissible genes encoding extended-spectrum β-lactamases (ESBLs) (2), plasmid-mediated AmpC β-lactamases (pAmpCs) (3), and carbapenemases (4) is particularly significant. Carbapenems have been the antibiotics of choice for treating ESBLs and other multidrug-resistant strains, but carbapenem resistance is increasingly common (5). This is mostly attributed to the production of specific carbapenemases (6), but the expression of AmpC or ESBL enzymes in isolates with altered outer membrane porins is also associated with decreased susceptibility to carbapenems (7 –9). Mutations in major outer membrane porins may be required for clinically relevant carbapenem resistance in K. pneumoniae isolates expressing carbapenemases, such as KPC and OXA-48-like enzymes, which rarely elicit clinically important antibiotic resistance in E. coli (6).

The clinical definitions of antibiotic susceptibility are developed by the collection and analysis of MIC data and with consideration for the pharmacokinetics and pharmacodynamics of antimicrobials and the outcomes of treatment (10). One of the first steps is to establish the MIC distribution of naturally occurring strains that do not have acquired resistance mechanisms. The epidemiological cutoff (ECOFF), defining the upper end of the wild-type MIC distribution and serving as a reference value for acquired resistance, is selected from data collated from various sources and defined by various methods but does not specifically require the exclusion of all potentially relevant resistance traits (11).

The aim of this study was to define the usual MIC distributions of selected β-lactam antibiotics against E. coli and K. pneumoniae isolates carrying important examples of transmissible β-lactam resistance mechanisms. We chose the most common TEM gene, bla_TEM-1, the two most common plasmid-borne AmpC genes, the two most common bla_CTX-M-type ESBL genes, and bla_IMP metallo-β-lactamase genes for study.

MATERIALS AND METHODS

Bacterial isolates and identification of β-lactamase genes.

We selected unique clinical isolates of E. coli and K. pneumoniae collected from microbiology laboratories in Sydney between 2005 and 2013, with almost all coming from Westmead Hospital (see Tables S1 to S8 in the supplemental material). These were chosen on the basis of antimicrobial susceptibility (Phoenix automated susceptibility test NMIC-101; BD Diagnostic Systems, Sparks, MD, USA) and the presence of relevant antibiotic resistance genes, as determined by PCR for bla_TEM (12), plasmid AmpC genes (13), common ESBL genes (14), and for bla_IMP (15). Isolates were grouped according to the bla genes identified by multiplex PCR/reverse line blot (mPCR/RLB) (16, 17): bla_TEM (n = 33), bla_CMY-2-like genes (n = 23), bla_CMY-2-like plus bla_TEM (n = 18), bla_DHA (n = 28), bla_CTX-M (n = 116), or bla_IMP (n = 100). A control group (n = 60) was selected on the basis of (i) the absence of phenotypic resistance to β-lactam antibiotics and (ii) negative results for relevant gene targets in mPCR/RLB (16 –18). bla_CTX-M genes were further identified as group 1 or group 9 by specific PCR amplification (19) and as specific genes by sequencing. bla_TEM was amplified (12) and sequenced in all E. coli isolates categorized in the bla_TEM and bla_CMY-2-like plus bla_TEM groups. Additional PCR was performed to detect bla_OXA-48-like (20) and bla_NDM genes (21).

The quality control strains used for the antimicrobial susceptibility tests are listed in Table S9 in the supplemental material. K. pneumoniae ATCC 13883 (22) and E. coli ATCC 25922 (23) were used as controls for porin expression.

Antimicrobial susceptibility tests.

Susceptibilities to ticarcillin (TIC) (Sigma-Aldrich, St. Louis, MO, USA), cefotaxime (CTX) (A.G. Scientific, Inc., San Diego, CA, USA), ceftazidime (CAZ) (Sigma-Aldrich), ertapenem (ETP) (Sigma-Aldrich), imipenem (IPM) (Sigma-Aldrich), and meropenem (MEM) (A.G. Scientific, Inc.) were determined by microdilution in cation-adjusted Mueller-Hinton (MH) broth (Becton Dickinson) with inocula of 5 × 10⁵ CFU/ml, according to CLSI M07-A9 guidelines (24). All MICs were determined in triplicate on three separate occasions and the mode (with at least 6 of 9 data points in agreement) given as the final value. MIC distributions were compared with the EUCAST epidemiological cutoff (ECOFF) and clinical breakpoints for all antibiotics (25). Ticarcillin plus clavulanate (TIM) was measured by Etest (AB Biodisk, Solna, Sweden). Susceptibility to all antibiotics was measured in control isolates (see Table S1A and B in the supplemental material). CTX, ETP, TIC, and TIM were tested against isolates with bla_CMY-2-like and/or bla_TEM genes only (see Tables S2 to S4 in the supplemental material). CTX and ETP susceptibilities were measured in isolates with a bla_DHA gene (see Table S5 in the supplemental material). CTX, CAZ, and ETP were tested against isolates carrying bla_CTX-M (see Tables S6 and S7 in the supplemental material). Susceptibility to carbapenems (MEM, IPM, and ETP) was measured in isolates with a bla_IMP gene (see Table S8A and B in the supplemental material).

Plasmid analysis.

PCR-based replicon typing (PBRT) was performed, as previously described (26), with additional primers to detect FII, FII_K (27), and IncX subtypes (28) and the IncL/M plasmid variant known to carry bla_OXA-48 (29) (now designated IncL [30]).

Investigation of outer membrane porins.

Isolates were grown in high-osmolarity MH or low-osmolarity nutrient broth (NB) (Oxoid, Basingstoke, England). Bacteria were disrupted by sonication and outer membrane porins (OMPs) isolated with sarcosyl, as previously described (31). Samples were boiled, analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12% separating gels), and stained with Imperial protein stain (Thermo Scientific, Rockford, IL, USA). Porin genes and their promoter regions were amplified and sequenced using the primers listed in Table S10 in the supplemental material.

RESULTS

Detection of genes encoding β-lactamases and plasmid typing.

The mPCR/RLB results are given in Tables S1 to S8 in the supplemental material. No bla_NDM, bla_VIM, or bla_KPC carbapenemase genes were detected in any isolates. The control group comprised isolates that were susceptible to β-lactam antibiotics and negative for all β-lactamase genes tested (see Tables S1A and B in the supplemental material). The majority of resistant isolates harbored more than one resistance gene (see Tables S2 to S8 in the supplemental material). PCR and sequencing (12) revealed that all E. coli isolates containing bla_TEM encode a TEM-1 enzyme (see Tables S2 and S4 in the supplemental material; all were bla_TEM-1b, except JIE1805, with bla_TEM-1c). Sequencing of bla_CTX-M genes revealed bla_CTX-M-3 (E. coli, n = 3), bla_CTX-M-15 (E. coli, n = 27; K. pneumoniae, n = 43), bla_CTX-M-14 (E. coli, n = 12; K. pneumoniae, n = 24), bla_CTX-M-9 (E. coli, n = 2; K. pneumoniae, n = 3), and bla_CTX-M-24 (E. coli, n = 2).

All E. coli (50/50) and K. pneumoniae (50/50) isolates with bla_IMP also had bla_TEM, aac(3)-II, and aac(6′) genes; most (76/100) isolates also had a qnrB gene, and 96/100 isolates had an IncL/M replicon (see Table S8A and B in the supplemental material), consistent with the expected associations of bla_IMP-4 in local isolates (15). Although direct relationships between plasmid type and resistance genes were not experimentally determined, associations between other resistance genes and replicons identified in each strain by PBRT (26, 27, 32) were also as expected. In E. coli, IncI1 was common in isolates with a bla_CMY-2-like gene (33) (see Tables S3 and S4 in the supplemental material), and the IncFII replicon was commonly found in association with bla_CTX-M-15 (19) (see Table S6A in the supplemental material). In K. pneumoniae, IncFII_K-type plasmids were predominant and broadly distributed among resistant and susceptible isolates (see Tables S1B and S5 to S8 in the supplemental material), and an IncL/M variant, now designated IncL (30), was found in association with bla_OXA-48-like genes (see Table S7B in the supplemental material) (29).

Distribution of antibiotic resistance phenotypes associated with transmissible genes.

For all control isolates, the distribution of different carbapenem (IPM, ETP, and MEM) and β-lactam (TIC, CTX, and CAZ) antibiotic MICs were below the resistance cutoff value and comparable to wild-type EUCAST data (25) (see Table S1 in the supplemental material).

As expected, pAmpC genes (bla_CMY-2-like genes in E. coli and bla_DHA in K. pneumoniae) were associated with CTX resistance (Fig. 1A). The bla_CMY-2-like gene was less active against TIC, and the presence of a pAmpC gene did not increase resistance to TIC in the presence of the penicillinase gene bla_TEM-1. This is important, because more than half of the E. coli isolates carrying a bla_CMY-2-like gene also had bla_TEM-1 (see Tables S2 to S4 in the supplemental material). TIC MICs were reduced by inhibiting TEM (with clavulanate [CLA]) in isolates carrying bla_TEM-1, but bla_CMY-2-like genes are associated with clinically important TIC/TIM resistance.

The CTX and CAZ MIC distributions in isolates carrying a bla_CTX-M group 1 or 9 gene (Fig. 2A and B) illustrate the expected differences between the activities of the encoded enzymes in both bacterial species. The wild-type ECOFF for CTX (0.25 mg/liter) was exceeded in all isolates carrying bla_CTX-M (Fig. 2A), but the group 9 CTX-M enzymes did not always result in MICs above the CAZ ECOFF (0.5 mg/liter; Fig. 2B). Elevated CAZ MICs (≥32 mg/liter) were associated with bla_CTX-M-15 (Fig. 2B) but not bla_{CTX-M-9/14/24}, as expected (34, 35): a D240G substitution in the glycine-rich β3 loop of CTX-M enzymes present in CTX-M-15 (but not CTX-M-3) is thought to enhance the accessibility of CAZ as a substrate (35, 36).

With the exception of one E. coli isolate (JIE808; IPM MIC, 0.5 mg/liter) (Fig. 3B; see also Table S8A in the supplemental material), carbapenem MICs against isolates with bla_IMP were distributed above the EUCAST ECOFF value in E. coli and K. pneumoniae (Fig. 3; see also Table S8A and B in the supplemental material).

Twenty-five isolates (E. coli, n = 2; K. pneumoniae, n = 23) had an unexpectedly high ertapenem MIC (Table 1 and Fig. 1B, 2C, and 3C). Only one of these (K. pneumoniae JIE533; ETP MIC, 32 mg/liter) had the carbapenemase gene bla_IMP. Of the remaining 24 isolates, one E. coli isolate carrying a bla_CMY-2-like gene, two K. pneumoniae isolates carrying bla_DHA, and 11 K. pneumoniae isolates carrying bla_CTX-M-15 were resistant to ETP (MIC, 2 to 128 mg/liter) (Table 1; see also Tables S3, S5, and S6B in the supplemental material). Ten isolates (9 K. pneumoniae and 1 E. coli) carrying bla_CTX-M-9/14 were not susceptible to ETP and CAZ (Table 1; see also Table S7A and B in the supplemental material), which is noteworthy given the relatively poor activity of bla_CTX-M-9/14 genes against CAZ.

TABLE 1.

Description of mutations in ompK36 in K. pneumoniae isolates

Isolate by bla gene carried	MIC (mg/liter)		ompC/K36 mutation^b	OmpK36^c	Other bla gene(s)^d
Isolate by bla gene carried	ETP	CAZ^a	ompC/K36 mutation^b	OmpK36^c	Other bla gene(s)^d
bla_IMP
JIE533	32	NA	Premature stop codon. 181delG. Val61fsTer11	No	bla_TEM
bla_DHA
JIE2597	64	NA	IS903B, after nucleotide 137	No	bla_SHV-5/12
JIE3813	64	NA	Premature stop codon. 91_92insA. Leu31fsTer7	No	bla_CTX-M-15, bla_OXA-30
bla_CTX-M-15
JIE1309	8	NA	ISKpn26, 48 bp upstream ATG (46)	No
JIE1398^e	8	NA	Premature stop codon. 374G→A. Trp125Ter	No	bla_TEM, bla_OXA-30
JIE1462	8	NA	Gly134_Asp135dup	Yes	bla_TEM, bla_OXA-30
JIE1474	8	NA	Gly134_Asp135dup	Yes	bla_TEM, bla_OXA-30
JIE1505^e	16	NA	IS903, after nucleotide 126	No	bla_TEM
JIE1652	8	NA	Premature stop codon. 262delA. Arg88fsTer7	No	bla_TEM
JIE1703	8	NA	Ser332Pro	No	bla_OXA-30
JIE1983	4	NA	Premature stop codon. 371_383del. Ser124fsTer38	No	bla_OXA-30
JIE2038	8	NA	Gly134_Asp135dup	Yes	bla_TEM, bla_OXA-30
JIE2055	8	NA	Gly134_Asp135dup	Yes	bla_TEM, bla_OXA-30
JIE2157	64	NA	1009_1016del. Ile337fs	No	bla_OXA-30
bla_CTX-M-14
JIE1333	64	16	Gly134_Asp135dup	Yes	bla_TEM, bla_OXA-48
JIE1334	64	16	Gly134_Asp135dup	Yes	bla_TEM, bla_OXA-48
JIE1335	64	16	Gly134_Asp135dup	Yes	bla_TEM, bla_OXA-48
JIE1348	64	32	Gly134_Asp135dup	Yes	bla_TEM, bla_OXA-48
JIE1383	64	8	Gly134_Asp135dup	Yes	bla_TEM, bla_OXA-48
JIE1482	64	32	Gly134_Asp135dup	Yes	bla_TEM, bla_OXA-48
JIE2218	128	16	Gly134_Asp135dup	Yes	bla_TEM, bla_OXA-48
JIE2999^e	64	256	Premature stop codon. 508C→T. Gln170Ter	No
JIE2753	1	8	Val10Glu	No	bla_TEM

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CAZ MIC is only valid to predict ompK36 defects in isolates carrying bla_CTX-M-14. NA, not applicable.

ompK36 mutations. For the premature stop codon, mutations are described at the DNA and protein levels, respectively. ins, insertion; del, deletion; dup, duplication; →, substitution; fs, frameshift; Ter, stop codon. For example, for JIE533, 181delG. Val61fsTer11 indicates that there is a deletion of glutamine at position 181, causing a frameshift where valine 61 is the first amino acid affected, creating a new reading frame ending with a stop codon at position 11 (counting starts with the valine as amino acid 1). Val10Glu denotes that amino acid Val10 is changed to Glu.

SDS-PAGE analysis: yes, OmpK36 detected by SDS-PAGE; no, OmpK36 not detected by SDS-PAGE.

Genes detected by mPCR/RLB (16, 17) but not sequenced. bla_OXA-30 indicates a bla_OXA-30/1-like gene. bla_OXA-48 indicates a bla_OXA-48-like gene.

ompK35 was sequenced in these isolates. JIE1398 and JIE2999, intact OmpK35 sequence identical to accession no. WP_004141771.1. JIE1505, premature stop codon. 706insA. Tyr236fsTer2.

OmpK36 defects are associated with resistance to ETP and CAZ in K. pneumoniae.

The major nonspecific porins expressed in E. coli are the OmpF matrix porin and the OmpC osmoporin, with their equivalents being OmpK35 and OmpK36 in K. pneumoniae. Nutrients and other hydrophilic molecules, such as β-lactam antibiotics, diffuse through these channels, and changes in these pores are linked to decreases in β-lactam susceptibility (7 –9, 37 –39). Previously described changes include disruption of the porin gene or promoter by insertion sequences (IS), frameshifts, nonsense mutations, and insertions or duplications that increase electronegativity of the inner channel-exposed loop 3 region of OmpK porins (40 –45).

Of the 23 ETP-nonsusceptible K. pneumoniae isolates (Table 1 and Fig. 1B, 2C, and 3C), 10 isolates had an ompK36 defect (three isolates had IS interrupting the coding or promoter sequences, one isolate had a 24-bp deletion, and six isolates had point mutations resulting in a frameshift and early termination). Two different point mutations, Ser332Pro and Val10Glu, were present in JIE1703 and JIE2753, respectively (Table 1; see also Fig. S1 in the supplemental material). A Gly134Asp135 (GD) duplication in OmpK36 loop 3, commonly seen in sequence type 258 (ST258) isolates carrying bla_KPC (43, 46) and predicted to functionally constrict the inner channel of the porin, was present in the other 11 isolates (Table 1; see also Fig. S1 in the supplemental material). The ompC gene was intact in the two ETP-resistant E. coli isolates that we analyzed (JIE121 and JIE2558; the protein sequence was identical to a common OmpC, GenBank accession no. WP_000865538.1).

All 23 ETP-nonsusceptible K. pneumoniae isolates expressed an ∼32-kDa protein consistent with an OmpA-like protein (47) when cultured in MH (see Fig. S2A in the supplemental material). A protein band consistent with OmpK36 was also evident in the 11 K. pneumoniae isolates with the OmpK36 loop 3 GD duplication but was absent from the 10 isolates with gene disruptions (see Fig. S2A in the supplemental material) and from JIE1703 (Ser332Pro) and JIE2753 (Val10Glu). Like wild-type E. coli ATCC 25922, both ETP-resistant clinical E. coli isolates JIE121 and JIE2558 appeared to express OmpC (see Fig. S2A in the supplemental material).

OmpK35 was not evident on SDS-PAGE of membrane extracts from 23 ETP-nonsusceptible K pneumoniae isolates, including many with intact ompK35 (e.g., JIE1398 and JIE2999, in which the protein sequence was the same as that of a common OmpK35, GenBank accession no. WP_004141771.1). SDS-PAGE showed no correlation between the presence or absence of OmpK35 and ETP MICs (see Fig. S2B in the supplemental material). The OmpF sequences of E. coli JIE2558 (bla_CMY-2-like; ETP MIC, 8 mg/liter; see Table S3 in the supplemental material) and JIE121 (bla_CTX-M-14; ETP MIC, 2 mg/liter; see Table S7A in the supplemental material) are both indistinguishable from an intact and frequent OmpF protein, GenBank accession no. WP_000977905.1.

DISCUSSION

Local antibiotic-susceptible clinical isolates of E. coli and K. pneumoniae in which relevant antibiotic resistance genes have been excluded display a phenotype consistent with published EUCAST MIC data (25). Here, we show that acquired resistance genes are associated with similarly distributed MICs that are shifted along a drug concentration gradient as a completely predictable function of the relevant genotype, and that additive effects from other genes are also predictable and consistent.

Bacteria carrying acquired β-lactamases usually have a multiresistance phenotype (48, 49), and the distribution of associated plasmid types (see Tables S1 to S8 in the supplemental material) in our geographically localized study is consistent with those reported elsewhere, which is important for the generalization of these findings. The IncL/M replicon is strongly associated with bla_IMP-4 in this region (15), and IncL (30) was found in association with bla_OXA-48-like genes (see Table S7B in the supplemental material) (29). IncF plasmids, frequently found in clinical enterobacterial isolates associated with relevant antimicrobial resistance genes (48), were common in our study (IncFII in combination with IncFIA and/or FIB replicons in E. coli and FII_K in K. pneumoniae; see Tables S1 to S8 in the supplemental material).

The principal question is whether the usually recommended phenotypic screening methods are adequate for antibiotic resistance surveillance. In the case of common and important acquired AmpC (bla_CMY-2-like or bla_DHA) and ESBL (bla_CTX-M-15) genes in Enterobacteriaceae, a CTX MIC cutoff of >1 mg/liter (50, 51) is a good fit to our data (Fig. 1A and 2A). However, phenotypic screening for carbapenemase genes (e.g., bla_KPC, bla_NDM, and bla_IMP) in Enterobacteriaceae is more problematic, since MICs are often low, and automated systems are unreliable (52). The CLSI recommends ETP (MIC, >1 mg/liter) (50), while EUCAST suggests MEM (MIC, >0.12 mg/liter) to achieve an optimal balance of sensitivity and specificity (51). Our study confirms that MEM at a concentration of >0.12 mg/liter was more reliable than ETP, as some isolates carrying bla_IMP did not have an ETP MIC in excess of 1 mg/liter (Fig. 3). Susceptibility to MEM was tested down to a concentration of 0.004 mg/liter, with the lowest MIC in isolates carrying a bla_IMP gene being 0.5 mg/liter (Fig. 3; see also Table S1 in the supplemental material). As expected, sequencing revealed an intact ompK36 coding region and promoter sequence in all K. pneumoniae isolates carrying bla_IMP, with the single exception of JIE533 (ETP MIC, 32 mg/liter).

We therefore reviewed data from previously published local and international clinical isolates of E. coli (16, 17). A breakpoint for CTX of <2 mg/liter applied to these populations has a negative predictive value of >99.5% for acquired resistance mechanisms (including bla_CMY-2-like, bla_CTX-M, and bla_IMP) (16). Clinical isolates carrying a bla_OXA-48-like gene are typically highly CTX resistant due to cocarriage of an ESBL gene (often bla_CTX-M), but even E. coli with no other major resistance genes present can still exhibit MICs in excess of 0.25 mg/liter (53, 54). Taken together, our data suggest that a CTX breakpoint of 0.5 mg/liter, followed by an accurate PCR for key resistance genes (16), might also be considered a simple effective screen for transmissible ESBL, AmpC, or carbapenemase resistance mechanisms in Enterobacteriaceae without the need to add other agents, such as ETP or MEM. Lowering the CTX breakpoint to 0.5 mg/liter for screening would efficiently rule out almost all important transmissible β-lactam resistance with few or no false positives, although the sensitivity for detecting OXA-48/-181 and related enzymes should be further examined. In this study, the CTX MIC of 0.5 mg/liter in combination with our genetic testing identified all β-lactam resistance mechanisms studied (carbapenemases, CTX-M, and AmpC), with the exception of TEM, most examples of which (e.g., TEM-1) produce no CTX-R phenotype.

ETP MICs appear to be bimodally distributed in K. pneumoniae, and this seems to correlate with defects and variations in OmpK36, as previously reported in association with KPC-producing isolates, and with ESBL and AmpC-type β-lactamases (8, 40, 55, 56). We found a large diversity of OmpK36 defects in our set of ETP-resistant K. pneumoniae isolates, all of which were reported previously: frameshift mutations (43), IS (57), a premature stop codon (58, 59), and a GD duplication in loop 3 (44, 45, 60). OmpK36 was detected by SDS-PAGE only in those isolates with the GD duplication. In two isolates, JIE1703 and JIE2753, OmpK36 was not detected despite an intact promoter region and apparently normal coding sequence (Table 1; see also Fig. S1 in the supplemental material). JIE1703 had a point mutation in OmpK36 (Ser332Pro) that seems unlikely to be relevant. JIE2753 had a substitution in the hydrophobic core of the signal peptide (Val10Glu) that might affect the correct secretion of the protein (61, 62), but this was not explored further. Likewise, changes in regulatory factors that might affect porin expression (63) were not analyzed. The E. coli equivalent osmoporin gene ompC and promoter sequences in JIE121 (ETP MIC, 2 mg/liter) and JIE2558 (ETP MIC, 8 mg/liter) were intact, but the outer membrane protein profile of these isolates showed a pattern different from that of the wild-type strain ATCC 25922 (23) (see Fig. S2A in the supplemental material). Although detailed transcriptional and proteomic analyses are beyond the scope of this work, simple analysis of the association between MICs and the nucleotide sequences and apparent expression of the matrix porins OmpK35 (in K. pneumoniae) and OmpF (in E. coli) in SDS-PAGE revealed no apparent correlation of these proteins with MICs.

Except for a low-level endemicity of bla_IMP-4, classic carbapenemase genes, such as bla_NDM and bla_KPC, have been seen as sporadic imports to Australia (16, 19, 46), although a recent KPC outbreak (64) may now result in KPC becoming endemic in some Victorian hospitals. A surprising finding is that a high percentage of ETP nonsusceptibility in K. pneumoniae in our region is due to the combination of common bla_CTX-M (ESBL) genes with an OmpK36 variation (21 out of 70 K. pneumoniae isolates carrying a bla_CTX-M gene had a mutation in OmpK36). More than half of these isolates (11 out of 21) had the GD duplication in loop 3 (L3) of OmpK36 (Table 1; see also Fig. S1 in the supplemental material). The additional aspartate residue increases electronegativity in the eyelet of the porin channel, altering permeability to different molecules (45, 65), and this is expected to be especially true for large charged molecules, such as ETP or CAZ. This might be due to the fact that ETP reaches low concentrations in the periplasmic space, which further facilitates the activity of enzymes with weak carbapenemase activity (42, 66). Our most resistant isolates (ETP MIC, ≥64 mg/liter) have this mutation in combination with bla_OXA-48, encoding a true carbapenemase. Therefore, almost all (n = 22/23 isolates; Table 1) K. pneumoniae isolates with porin defects and an ESBL-type gene would be identified using the CDC definition of ETP resistance (MIC, ≥2 mg/liter) as carbapenem-resistant Enterobacteriaceae (CRE). We found that MEM at >0.12 mg/liter is more reliable for detecting a relevant transmissible gene; many isolates with the classical (IMP-type) carbapenemase do not have an ETP MIC in excess of 1 mg/liter (Fig. 3) (55).

The predominance of the GD duplication, also seen in association with bla_KPC (43, 46), and presumably excluding some large molecules, like ETP, without restricting smaller essential nutrients, is noteworthy. The complete loss of OmpK36 in K. pneumoniae also decreases β-lactam susceptibility but might have a greater impact on bacterial fitness, especially in those strains with defects in the other major porin, OmpK35 (38), which are commonly reported in clinical isolates (67).

In conclusion, we find that β-lactam MICs associated with specific transmissible resistance genes are normally distributed in these important bacterial species. In K. pneumoniae, any variation in this normal distribution is predictably linked to an OmpK36 defect or alteration. We suggest that OmpK36 alterations are highly likely to be present in any K. pneumoniae strain with an ETP MIC of >1 mg/liter and no recognized carbapenemase gene, and in any strain with a CAZ MIC of ≥8 mg/liter carrying a bla_CTX-M group 9 gene.

The increased deployment of genetic detection in screening and diagnosis makes it essential for us to efficiently assign isolates for further investigation and confidently exclude those that do not need it. Given that ompK36 genetic diversity in K. pneumoniae may affect the in vitro and in vivo responses to combinations that include carbapenems (44, 68, 69), these data also suggest that it would be useful to systematically define the phenotype-genotype relationship for all major transmissible resistance factors and characterize specific porin defects in organisms, like K. pneumoniae. It seems likely that appropriately targeted genetic methods can be effectively combined with key antibiotic screens to efficiently detect all important transmissible β-lactam resistance with a high degree of confidence. Quality control programs need to include key gene targets and be coordinated with systematic regular (e.g., yearly) surveillance of a sufficient number (e.g., 100 isolates of E. coli and K. pneumoniae of each key phenotype, e.g., by screening at 0.5 mg/liter CTX, 1 mg/liter ETP, or 0.25 mg/liter MEM) in order to minimize labor-intensive analysis of unexplained phenotypes. It is important to note that these breakpoint MICs are discussed in the context of the detection of resistance traits of concern and are quite different from clinical breakpoints, which are designed to help predict the risk of therapeutic failure. Systematic high-throughput sensitive screening will facilitate early recognition of newly arrived resistance genes and changes in resistance gene epidemiology, and it can be efficiently managed in a way that has been routine in other surveillance programs (e.g., of influenza viruses) for decades.

Supplementary Material

Supplemental material

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ACKNOWLEDGMENTS

This work was supported by NHMRC grant G1001021. A.F-.L. was supported by grant G1001021, A.N.G. by grant G1046886, and J.R.I. by grant G1002076. A.A. was supported by a scholarship from the Ghana Education Trust Fund (GetFund).

We declare no conflicts of interest.

Footnotes

Supplemental material for this article may be found at http://dx.doi.org/10.1128/JCM.02153-15.

REFERENCES

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[B1] 1.Pfeifer Y, Cullik A, Witte W. 2010. Resistance to cephalosporins and carbapenems in Gram-negative bacterial pathogens. Int J Med Microbiol 300:371–379. doi: 10.1016/j.ijmm.2010.04.005. [DOI] [PubMed] [Google Scholar]

[B2] 2.Cantón R, Coque TM. 2006. The CTX-M β-lactamase pandemic. Curr Opin Microbiol 9:466–475. doi: 10.1016/j.mib.2006.08.011. [DOI] [PubMed] [Google Scholar]

[B3] 3.Jacoby GA. 2009. AmpC β-lactamases. Clin Microbiol Rev 22:161–182. doi: 10.1128/CMR.00036-08. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4.Nordmann P, Naas T, Poirel L. 2011. Global spread of carbapenemase-producing Enterobacteriaceae. Emerg Infect Dis 17:1791–1798. doi: 10.3201/eid1710.110655. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5.Nordmann P, Dortet L, Poirel L. 2012. Carbapenem resistance in Enterobacteriaceae: here is the storm! Trends Mol Med 18:263–272. [DOI] [PubMed] [Google Scholar]

[B6] 6.Queenan AM, Bush K. 2007. Carbapenemases: the versatile β-lactamases. Clin Microbiol Rev 20:440–458. doi: 10.1128/CMR.00001-07. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B7] 7.Bradford PA, Urban C, Mariano N, Projan SJ, Rahal JJ, Bush K. 1997. Imipenem resistance in Klebsiella pneumoniae is associated with the combination of ACT-1, a plasmid-mediated AmpC β-lactamase, and the loss of an outer membrane protein. Antimicrob Agents Chemother 41:563–569. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8.Crowley B, Benedi VJ, Doménech-Sánchez A. 2002. Expression of SHV-2 β-lactamase and of reduced amounts of OmpK36 porin in Klebsiella pneumoniae results in increased resistance to cephalosporins and carbapenems. Antimicrob Agents Chemother 46:3679–3682. doi: 10.1128/AAC.46.11.3679-3682.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9.Padilla E, Alonso D, Doménech-Sánchez A, Gomez C, Pérez JL, Albertí S, Borrell N. 2006. Effect of porins and plasmid-mediated AmpC β-lactamases on the efficacy of β-lactams in rat pneumonia caused by Klebsiella pneumoniae. Antimicrob Agents Chemother 50:2258–2260. doi: 10.1128/AAC.01513-05. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10.Turnidge J, Paterson DL. 2007. Setting and revising antibacterial susceptibility breakpoints. Clin Microbiol Rev 20:391–408. doi: 10.1128/CMR.00047-06. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11.Turnidge J, Kahlmeter G, Kronvall G. 2006. Statistical characterisation of bacterial wild-type MIC value distributions and the determination of epidemiological cut-off values. Clin Microbiol Infect 12:418–425. doi: 10.1111/j.1469-0691.2006.01377.x. [DOI] [PubMed] [Google Scholar]

[B12] 12.Canica MM, Lu CY, Krishnamoorthy R, Paul GC. 1997. Molecular diversity and evolution of bla_TEM genes encoding β-lactamases resistant to clavulanic acid in clinical Escherichia coli. J Mol Evol 44:57–65. doi: 10.1007/PL00006121. [DOI] [PubMed] [Google Scholar]

[B13] 13.Pérez-Pérez FJ, Hanson ND. 2002. Detection of plasmid-mediated AmpC β-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 40:2153–2162. doi: 10.1128/JCM.40.6.2153-2162.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]

[B14] 14.Ellem J, Partridge SR, Iredell JR. 2011. Efficient direct extended-spectrum β-lactamase detection by multiplex real-time PCR: accurate assignment of phenotype by use of a limited set of genetic markers. J Clin Microbiol 49:3074–3077. doi: 10.1128/JCM.02647-10. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Predictability of Phenotype in Relation to Common β-Lactam Resistance Mechanisms in Escherichia coli and Klebsiella pneumoniae

Alex Agyekum

Alicia Fajardo-Lubián

Xiaoman Ai

Andrew N Ginn

Zhiyong Zong

Xuejun Guo

John Turnidge

Sally R Partridge

Jonathan R Iredell

Roles

Abstract

INTRODUCTION

MATERIALS AND METHODS