Susceptibility profiles of Enterobacteriaceae to temocillin, piperacillin/tazobactam and faropenem and characterisation of carbapenemase resistance mechanisms.

Vaughan E.S.1, Hobson J.A1,2 School of Pharmacy and Biomolecular Sciences, Liverpool John Moores University (1), Mast Group Ltd (2)

IntroductionThe prevalence of antibiotic resistance in Gram-negative pathogens has rapidly become an increasing concern in healthcare environments worldwide. In particular the effectiveness of carbapenems, often the last line of defence against ESBL and AmpC-producing isolates, is compromised by the development and rise of carbapenemase-producing-Enterobacteriaceae (CPE)1. Although they are considered rare, CPE continue to disseminate worldwide and remain extremely difficult to treat due to the often diverse range of resistance mechanisms they employ. In order for medical professionals to make a specific diagnosis of CPE, quick, simple and effective detection methods are a necessity. Specific detection methods allow tailored treatment to be provided to patients, helping to conserve and reserve valuable carbapenems, by ultimately reducing the risk of the dissemination of carbapenemases. Phenotypic assays are currently the most popular method of choice for detecting antibiotic resistance as they are well established, cost-effective and require only basic microbiological skills to carry out. Although they are not as rapid as molecular methods, requiring a minimum of 24 hours incubation period, they are the most standardized method available1. There is significant difficulty associated with the detection of CPE when performing first line panel testing in hospital laboratories, due to the diverse susceptibility patterns that vary between different types of carbapenemase enzymes (MBL, OXA, and KPC). The breakpoints that are advised by varying committees are not always reliable for the detection of CPE, and it has often been found that even modified carbapenem disc cut-offs recently proposed by CLSI and EUCAST fail to detect a substantial proportion of CPE isolates, predominantly OXA-482. Previous studies have established a range of surrogate markers, specifically temocillin, faropenem and piperacillin/tazobactam, which have proven useful in providing presumptive identification of CPE. High-level resistance was previously observed when performing susceptibility testing between CPE and temocillin, faropenem and piperacillin/tazobactam, either individually or in combination2, 3, 4. In this study, we aimed to verify the correlation between minimum inhibitory concentration (MIC) testing and susceptibility disc testing using temocillin against CPE, as previous studies have tended to focus on MIC methods. We also wanted to confirm that these surrogate markers remain consistent using BSAC methodology, and can be utilised effectively in routine laboratory testing.

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1) EUCAST . EUCAST guidelines for detection of resistance mechanisms and specific resistances of clinical and/or epidemiological importance. http://www. eucast.org/fileadmin/src/media/PDFs/EUCAST_files/Resistance_mechanisms/ EUCAST_detection_of_resistance_mechanisms_v1.0_20131211.pdf (Accessed 06th June 2014).

2) Huang T, Poirel L, Bogaerts P, Berhin C, Nordmann P, Glupczynsk Y. Temocillin and piperacillin/tazobactam resistance by disc diffusion as antimicrobial surrogate markers for the detection of carbapenemase-producing Enterobacteriaceae in geographical areas with a high prevalence of OXA-48 producers. J Antimicrob Chemother 2014; 69(2): 445-50.

3) Day KM, Pike R, Winstanley TG, Lanyon C, Cummings SP, Raza MW, Woodford N, Perry JD. Use of faropenem as an indicator of Carbapenemase activity in the Enterobacteriaceae. J Clin Microbiol 2013; 51:1881-1886.

4) van Dijk K, Scharringa J, Voets G, Voskuil WS, Fluit A.C., Rottier WC, Leverstein- van Hall MA, Cohen Stuart JWT. A novel phenotypic detection strategy for class A, B and OXA-48 carbapenemases in Enterobacteriaceae using temocillin 2013; ECCMID, Berlin, Germany.

5) Andrews, JM. Determination of minimum inhibitory concentrations. J Antimicrob Chemother 2001; 48: 5-16.

6) BSAC. BSAC Methods for Antimicrobial Susceptibility Testing, Version 12 May 2013. http://bsac.org.uk/wp-content/uploads/2012/02/Version-12-Apr-2013_ final1.pdf (Accessed 10th June 2014).

7) Andrews J, Jevons G, Walker R, Ashby J, Fraise AP. Temocillin susceptibility by BSAC methodology. J Antimicrob Chemother 2007; 60: 185–7.

MethodsIsolates

A total of 208 isolates were used, comprising 90 well-characterised isolates of known carbapenemase, ESBL or AmpC mediated resistance mechanism (Klebsiella pneumoniae n=55, Escherichia coli n=24, Enterobacter cloacae n=8, Citrobacter freundii n=2, Morganella morganii n=1) and 118 isolates with no such resistance mechanism (Escherichia coli n=77, Klebsiella pneumoniae n=21, Proteus mirabilis n=10, Citrobacter freundii n=6 Serratia spp. n=3, Morganella morganii n=1). The distribution of isolates and resistance mechanisms can be seen in figure 1. Prior to testing, all isolate identities were confirmed using the Mast Uri®System (Mast Group Ltd), as per the manufacturer’s instructions. Culture and maintenance of isolates

The isolates used in this study were obtained fresh from -70°C storage (Cryobank™ - Mast Group Ltd) and cultured onto Columbia agar (E&O Laboratories Ltd). All cultures were incubated for 18-20 hours at 35-37°C and were sub-cultured out to first generation (G1) before testing commenced. Isolates were sub-cultured to a maximum generation of G2 from the primary sub-culture during the two week period. Susceptibility testing

Minimum inhibitory concentration (MIC) for temocillin (EUMEDICA Pharmaceuticals) was determined for each isolate via agar dilution method5 in accordance with BSAC methodology6. The MIC range tested was from 0.25 to 128 mg/L in a serial two fold dilution series and prepared in Iso-Sensitest (IST) agar (Thermo Scientific). Temocillin MIC agar plates were produced using 100 ml of IST agar with 1 ml of desired temocillin dilution added. To ensure plate uniformity a measured 60 g of each agar dilution was poured into 120 mm square petri dishes. All organism suspensions were

produced to a density equivalent of a McFarland 0.5 standard and pipetted in triplicate into a 96-well microtiter plate. A sterile, electronic, 96-pin Mast Uri® Dot (Mast Group Ltd) inoculator was used to inoculate each MIC plate, with each pin transferring 0.3 µl of organism suspension. All plates were incubated for 18-20 hours at 35-37°C. Inoculation took place within 15 minutes after organism suspensions being made. Susceptibility disc testing (SDT) for each isolate was carried out in triplicate according to BSAC methodology6 using pre-poured ISA sensitivity test agar plates (E&O Laboratories Ltd) and susceptibility test discs (Mast Group Ltd) containing temocillin 30 µg (TEM30C), faropenem 10 µg (FARID)*, meropenem 10 µg (MEM10C), ertapenem 10 µg (ETP10C) and piperacillin/tazobactam combination containing piperacillin 75 µg and tazobactam 10 µg (PTZ85C). All organism suspensions were produced to a density equivalent to McFarland 0.5 standard and diluted and used within 15 minutes of preparation. Antibiotic discs were transferred to inoculated agar plates using a 6 place disc dispenser - Mast DiscMaster™ 3 (Mast Group Ltd). With each batch of testing performed (MIC and SDT), control strains Escherichia coli NCTC 10418 and Escherichia coli NCTC 12241 were used and performed as expected. All results were interpreted using breakpoints established by BSAC6. There are currently no breakpoints established for faropenem by BSAC. Zone diameters generated by all isolates against faropenem were recorded for interpretation as described previously3. *FARID discs are not yet commercially available.

ResultsTable 1. Distribution of zone diameters (ZD), according to inhibition diameter cut-offs and their performance, for the detection of CPE among isolates used in this study (n=205).

Table 2. Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) calculated according to the proposed combination cut-offs for the detection of CPE among isolates used in this study (n = 205).

Isolates

Fig. 1. Distribution of the differing resistance enzymes that are produced by isolates used in this study.

Key:R = ResistantS = SusceptibleI = IntermediateUTI = Urinary tract infection breakpointSYS = Systemic infection breakpoint

Zone diameters

Fig. 2. Distribution of zone diameters (mm) generated from ESBL, AmpC, ESBL and AmpC co-producing and CPE, and isolates derived from UTIs with no relevant resistance mechanism (wild type), for temocillin (30 µg) discs. UTI (R = ≤ 11 mm, S = ≥ 12 mm) and Systemic (R = ≤ 19 mm, S = ≥20 mm) breakpoints, according to BSAC6, are indicated on the graph.

Fig. 3. Distribution of zone diameters (mm) generated from ESBL, AmpC, ESBL and AmpC co-producing and CPE and isolates derived from UTIs with no relevant resistance mechanism (wild type), for faropenem (10 µg) discs. As there is no current breakpoint established by BSAC6 for faropenem, a suggested breakpoint of ≤ 16 mm is indicated on the graph, for identification of carbapenemase producers.

Fig. 4. Distribution of zone diameters (mm) generated from ESBL, AmpC, ESBL and AmpC co-producing and CPE, and isolates derived from UTIs with no relevant resistance mechanism (wild type), for piperacillin/tazobactam (85 µg) discs. Breakpoints, according to BSAC6, are indicated on the graph (R = ≤ 20 mm, I = 21-22 mm, S = ≥23 mm).

MIC vs. Zone diameter

Fig. 5. Distribution of inhibition zone diameter (mm) with corresponding minimum inhibitory concentration (MIC) value (mg/L) generated from ESBL, AmpC, ESBL and AmpC co-producing and CPE, and isolates derived from UTIs with no relevant resistance mechanism (wild type), for temocillin. UTI (R = ≤ 11 mm, S = ≥ 12 mm) zone diameter (ZD) breakpoints, according to BSAC6, are indicated on the graph, and correspond to 32 and 8 mg/L (MIC breakpoints) respectively.

DiscussionAs expected, all CPE tested other than those producing OXA-48 (n=39), formed no zone of inhibition around the FARID disc. When using faropenem in a BSAC based methodology it was apparent that the distinctive double zone associated with OXA-48 producing organisms, and reported by Day et al3, was absent. Additional testing confirmed that the organisms affected did revert to their expected appearance when the EUCAST test method was employed. As only a small sub-population of OXA-48 producers express resistance to faropenem it has been suggested (J D Perry, Newcastle Hospitals NHS Foundation Trust, personal communication) that the inoculum used in the BSAC method (100-fold lower than the EUCAST recommendation) renders this subpopulation undetectable. Although 97% of isolates of CPE gave zones of ≤15mm against FARID this nevertheless overlapped the zones produced by a small number of ESBL (n=4), AmpC (n=1) and ESBL/AmpC (n=2) co-producing organisms (Fig.3). Without the presence of the distinctive ingrowth, the utility of the faropenem disc in detection of CPE including OXA-48 producers is therefore reduced if the disc is to be employed by a laboratory using BSAC methodology. Parallel consideration of the zones produced by a PTZ85C combination disc (Fig. 4) considerably reduced the potential for false negative interpretation and using a combined algorithm of PTZ85C breakpoint of ≤20mm (current BSAC resistance breakpoint)6 and FARID breakpoint of ≤15mm, a sensitivity and specificity of 96 and 99%, respectively, is achieved for CPE detection (Table 2). Temocillin resistance has been proposed as a sensitive surrogate marker in CPE screening particularly for its value in discrimination of OXA-48 producers2. Although a previous study7 demonstrated a high degree of agreement between ZD and MIC for temocillin susceptibility by BSAC methodology, the organisms selected at that time did not include examples of CPE isolates. Extending this original work to include a selection of CPE (Fig. 2 & Fig. 5) confirmed that all OXA-48-CPE tested had MICs of >128mg/L and showed no zone of inhibition. High level temocillin resistance, defined as MIC to temocillin of >32mg/L and corresponding ZD against TEM30C of <11mm, has been advocated previously as a phenotypic indicator of OXA-48 production1. Since the MBL producers tested were also shown to demonstrate high level resistance within this definition and as temocillin retains useful activity against KPC producing isolates (94% of the isolates tested proved susceptible at the BSAC UTI breakpoint of S≥12mm), when using TEM30C in a screen for CPE it is also useful to separately identify KPC and MBL producers. Synergy between meropenem and phenyl boronic acid (PBA) and dipicolinic acid is commonly used for this confirmatory testing4. Interestingly, a number of characteristic resistance profiles can be seen when data from all three discs tested are analysed (Table 2). TEM30C used in conjunction with FARID and PTZ85C could additionally provide a useful level of discrimination of individual carbapenemase mechanisms prior to, or as an adjunct to, inhibitor based phenotypic confirmation. Producers of OXA-48 generally demonstrated complete resistance to TEM30C and a zone against FARID of ≥7mm and against PTZ85C of ≤20mm. Using these criteria, a sensitivity and specificity for identification of OXA-48 producers of 82 and 100%, respectively, is achieved. MBL producing organisms tended to show total resistance or a very small zone of ≤10mm against TEM30C, complete resistance to FARID and a zone of ≤20mm against PTZ85C - sensitivity and specificity for identification of MBL producers of 100 and 98% respectively. Finally it was possible to discriminate KPC producers as producing a zone against TEM30C of ≥11mm, and against PTZ85C of ≤20mm, with total resistance to FARID. This provided a sensitivity of 94% and a specificity of 100%.

ConclusionThese results support previous data and confirm that high-level resistance to faropenem and temocillin provide very sensitive and specific phenotypic surrogate markers of carbapenemase production in strains of Enterobacteriaceae. When used in a BSAC method, the combination of a proposed FARID cut off of ≤15mm should be coupled with observation of resistance to PTZ85C to remain predictive of carbapenemase production. Addition of a TEM30C disc then provides simple and useful three disc algorithms for phenotypic recognition and discrimination of class A, B and D carbapenemase production.

References

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

5

0

10

15

20

30

Num

ber o

f iso

late

s

Zone diameter (mm)

Temocillin (30 μg)

ESBL/AmpC

AmpC

ESBL

Wild type

KPC/ESBL

KPC

MBL

OXA-48

25

UTI SYS

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

5

10

15

20

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35

40

45

50

0

Num

ber o

f iso

late

s

Zone diameter (mm)

Faropenem (10 μg)

ESBL/AmpC

AmpC

ESBL

Wild type

KPC/ESBL

KPC

MBL

OXA-48

10

6

6

2

4

12

14

16

18

20

8

0

Num

ber o

f iso

late

s

Zone diameter (mm)

Piperacillin/tazobactam (85 μg)

ESBL/AmpC

AmpC

ESBL

Wild type

KPC/ESBL

KPC

MBL

OXA-48

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

R I S

6 10 11

5

10

15

20

25

30

0

Num

ber o

f iso

late

s

Disc zone diameter (mm)

2

4

8

16

32

64

>128

UTI SYS

Temocillin (30 µg) vs. MIC

12 14 15 16 17 18 19 20 21 22 23 24 25 26 27 2813 29 30 31 32 33 34 35 37 38 42

MIC (mg/L)

Distribution of resistance mechanisms producedby isolates used in this study

Isolates derivedfrom UTIs with

no relevantresistance

mechanism(Wild type)57%

ESBL & AmpCCo-producer

3%

AmpC7%

ESBL9%

KPC16%

MBL3%

OXA-485%

Combination cut-offs Sensitivity % Specificity % NPV % PPV %

FAR = ≤15 and PTZ ≤20 (Carbapenemase profile)

96 99 99 96

TEM = 6, FAR = ≥7 and PTZ = ≤20 (OXA profile)

82 100 99 100

TEM = ≤10, FAR = 6 and PTZ = ≤20 (MBL profile)

100 98 100 67

TEM = ≥11, FAR = 6 and PTZ = ≤20 (KPC profile)

94 100 99 100

Zone diameter cut-offs (mm) OXA-48 KPC MBL ESBL AmpC ESBL/AmpC

Co-producerWild type

(Total = 11)

(Total = 33)

(Total = 6)

(Total = 18)

(Total = 15)

(Total = 5)(Total

= 117)

Susceptibility breakpoints

TEM = ≥12 0 31 0 17 15 5 117

PTZ = ≤20 11 32 6 4 1 1 14

Modified cut-offs

FAR = 6 2 33 6 1 0 0 0

FAR = >6 but ≤16 8 0 0 4 3 3 2

TEM = >6 but ≤11 0 1 1 1 0 0 0

TEM = 6 11 1 5 0 0 0 0

Combination cut-offs

FAR = ≤15 and PTZ ≤20 (Carbapenemase profile) 10 32 6 2 0 0 0

TEM = 6, FAR = ≥7 and PTZ = ≤20 (OXA profile) 9 0 0 0 0 0 0

TEM = ≤10, FAR = 6 and PTZ = ≤20 (MBL profile) 2 1 6 0 0 0 0

TEM = ≥11, FAR = 6 and PTZ = ≤20 (KPC profile) 0 31 0 0 0 0 0

Did not meet any of the combination cut-off criteria 0 1 0 16 15 5 117