Screening for β-lactamases.

All 743 isolates collected in U.S. hospitals during 2013 displaying the CLSI ESBL phenotypic criteria as described above were tested for β-lactamase-encoding genes using the microarray-based assay Check-MDR CT101 kit (Check-points, Wageningen, Netherlands). The assay was performed according to the manufacturer's instructions. This kit has the capabilities to detect CTX-M groups 1, 2, 8 + 25, and 9, TEM wild-type (WT) and ESBL, SHV WT and ESBL, ACC, ACT/MIR, CMY II, DHA, FOX, KPC, and NDM-1. The most common mutations that expand the spectrum of TEM and SHV enzymes are detected, and these mutations include 104K, 164S/C/H, or 123S for TEM and 138S, 238A, and 240K for SHV.

All isolates displaying a ceftazidime-avibactam MIC of >4 μg/ml were screened for the presence of metallo-β-lactamase and serine-carbapenemase-encoding gene families bla_IMP, bla_VIM, bla_NDM, bla_KPC, bla_OXA-48, bla_GES, bla_IMI, bla_NMC-A, and bla_SME by PCR as previously described (12). Amplicons were sequenced on both strands, and results were analyzed using the Lasergene software package (DNASTAR, Madison, WI). Amino acid sequences were compared with those available through the internet using NCBI/BLAST. These isolates were also submitted to a simplified protein extraction, and the hydrolysis of the extracts was measured against ceftazidime, meropenem, and imipenem as previously described (13).

Occurrence of β-lactamase-producing isolates in 2013 and activity of ceftazidime-avibactam and comparators.

Among Enterobacteriaceae isolates from 2013 (n = 8,836), the CLSI epidemiological screening criteria for the ESBL phenotype (9) were observed among 743 isolates (12.5% of 5,943 isolates belonging to targeted species), including 368 E. coli isolates (12.3% of the overall samples for this species), 298 K. pneumoniae isolates (17.0%), 38 K. oxytoca isolates (8.0%), and 39 P. mirabilis isolates (5.7%). These isolates were screened against several common β-lactamase-encoding genes.

A total of 307 isolates were positive for CTX-M group 1 (here called “CTX-M-15-like” and including CTX-M-1, CTX-M-15, and CTX-M-3 among others), and this was the most prevalent β-lactamase detected. The CTX-M-15-like enzyme was detected among all four organisms. This β-lactamase was observed alone in 130 strains or in 14 combinations with 1 to 4 other β-lactamase-encoding genes/families, including KPC (10 isolates) (Table 3). Isolates harboring CTX-M-15-like β-lactamase-encoding genes without carbapenemases or other CTX-M groups (n = 284) displayed elevated MIC values for cephalosporins, but ceftazidime-avibactam was very active against these isolates (MIC_50/90, 0.06/0.25 and 0.12/0.5 μg/ml, respectively). Meropenem (MIC_50/90, ≤0.06/≤0.06 μg/ml), tigecycline (MIC_50/90, 0.12/0.5 μg/ml), colistin (MIC_50/90, 0.5/0.5 μg/ml), and ceftazidime-avibactam were the most active agents tested against these strains (Table 3).

TABLE 3

Enzymes alone and in combinations detected among isolates collected during 2013 in U.S. hospitals^a

β-Lactamase(s) and enzyme(s)	No. of isolates producing enzyme(s) shown
	Total	E. coli	K. pneumoniae	K. oxytoca	P. mirabilis
Carbapenemases
KPC	2	2
KPC, CMY II, SHV WT, TEM WT	2		2
KPC, CTX-M group 1, CMY II, SHV WT, TEM WT	1		1
KPC, CTX-M group 1, SHV ESBL, SHV WT, TEM WT	1		1
KPC, CTX-M group 1, SHV WT	5		5
KPC, CTX-M group 1, SHV WT, TEM WT	3		3
KPC, FOX, TEM WT	1			1
KPC, SHV ESBL	1		1
KPC, SHV ESBL, SHV WT	2		2
KPC, SHV ESBL, SHV WT, TEM WT	42		42
KPC, SHV WT	10		10
KPC, SHV WT, TEM WT	44		44
KPC, TEM WT	6	3	2	1
ESBLs
CTX-M group 1	130	127	1	1	1
CTX-M group 1, CMY II	1	1
CTX-M group 1, CTX-M group 9	1	1
CTX-M group 1, CTX-M group 9, TEM WT	2	2
CTX-M group 1, SHV ESBL	3		3
CTX-M group 1, SHV ESBL, SHV WT	1		1
CTX-M group 1, SHV ESBL, SHV WT, TEM WT	3		3
CTX-M group 1, SHV ESBL, TEM WT	1				1
CTX-M group 1, SHV WT	22		22
CTX-M group 1, SHV WT, TEM WT	55		55
CTX-M group 1, TEM WT	78	67		3	8
CTX-M group 9	57	55			2
CTX-M group 9, SHV ESBL, SHV WT, TEM WT	1		1
CTX-M group 9, SHV WT	4		4
CTX-M group 9, SHV WT, TEM WT	5		5
CTX-M group 9, TEM ESBL	1	1
CTX-M group 9, TEM WT	39	35			4
SHV ESBL	8		5	3
SHV ESBL, SHV WT	38		38
SHV ESBL, SHV WT, TEM WT	11		11
SHV ESBL, TEM WT	4	2		2
TEM ESBL	11	7		1	3
Transferable AmpC
ACT/MIR, TEM WT	1		1
CMY II, SHV ESBL, SHV WT, TEM WT	1		1
CMY II, SHV ESBL, TEM WT	1	1
CMY II, SHV WT	3		3
CMY II, TEM WT	25	20			5
CMY II	20	17			3
DHA, SHV WT	1		1
FOX	1				1
FOX, SHV WT	3		3
FOX, TEM WT	1				1
Narrow-spectrum enzymes
SHV WT	24		24
SHV WT, TEM WT	2		2
TEM WT	18	12			6
Negative results	46	15	1	26	4

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^aCombinations were grouped according to the most relevant enzyme present.

KPC serine-carbapenemases were very prevalent, being observed among 120 isolates, most of them K. pneumoniae (113 isolates) (Table 3), but also including E. coli (5 isolates) and K. oxytoca (2 isolates). KPC-encoding genes were detected alone or in the presence of narrow-spectrum enzymes (SHV and/or TEM) in 62 isolates, and combinations with transferable cephalosporinases, CTX-M and SHV ESBL were also noted (Table 3). KPC producers were very resistant to all agents tested (Table 2), and ceftazidime-avibactam (MIC_50/90, 0.5/2 μg/ml) and tigecycline (MIC_50/90, 0.5/1 μg/ml) were the most active antimicrobial agents against these isolates. Colistin (MIC_50/90, 0.5/8 μg/ml) displayed activity against 83.1% of the KPC-producing isolates according to EUCAST breakpoints. One KPC-producing isolate had ceftazidime-avibactam MIC result of >32 μg/ml, and further investigations demonstrated that this K. pneumoniae isolate produced KPC-2 and VIM-4. This isolate, belonging to sequence type 258 (ST258), also carried genes encoding CMY-2 and narrow-spectrum TEM and SHV and had overexpression of the efflux pump AcrAB-TolC and reduced expression of porin OmpK35- and OmpK37-encoding genes (M. Castanheira, L. M. Deshpande, J. C. Mills, R. N. Jones, S. G. Jenkins, and A. N. Schuetz, submitted for publication).

CTX-M group 9 enzymes and the CTX-M-14-like β-lactamase, the most common variant detected in U.S. hospitals within this group (6), were detected among 110 isolates: 94 E. coli, 10 K. pneumoniae, and 6 P. mirabilis isolates (Table 3). The CTX-M-14-like enzyme was the only β-lactamase detected among 57 isolates, and in 39 isolates, a combination of this enzyme and the TEM WT was observed (Table 3). Ceftazidime, piperacillin-tazobactam, meropenem, and tigecycline inhibited >80% (83.0 to 100.0%) of the CTX-M-14-like enzyme-producing isolates according to the CLSI breakpoint criteria, and colistin inhibited 94.3% of the isolates when the EUCAST breakpoint was applied. Ceftazidime-avibactam (MIC_50/90, 0.12/0.25 μg/ml; highest MIC, 0.5 μg/ml) displayed good activity against these strains (Table 2).

SHV enzymes with an extended spectrum of activity (SHV ESBLs) were detected among 118 isolates, and the vast majority of isolates (109/118) were K. pneumoniae (Table 3). SHV ESBL enzymes were found in all bacterial species, and SHV-12, SHV-5, SHV-7, SHV-2, and SHV-30 (in this order), are the most common SHV ESBL types reported in these U.S. hospitals (6). SHV ESBLs were detected in various combinations with other β-lactamases (Table 3). For the 61 isolates in which an SHV ESBL was the only extended-spectrum enzyme detected, meropenem (MIC_50/90, ≤0.06/≤0.06 μg/ml), ceftazidime-avibactam (MIC_50/90, 0.25/0.5 μg/ml) (Table 1), and tigecycline (MIC_50/90, 0.25/1 μg/ml) were the most active agents (data not shown).

Transferable cephalosporinases (plasmidic AmpCs) were detected among 62 isolates, and 54 strains displayed a positive result for the CMY II probe (here, “CMY-2-like”; 39 E. coli, 7 K. pneumoniae and 8 P. mirabilis isolates) (Table 3). All CMY-2-like enzyme-positive isolates that did not carry carbapenemases or CTX-M enzymes (n = 48) were susceptible to meropenem and tigecycline (100.0% susceptible according to CLSI breakpoint criteria). Ceftazidime-avibactam (MIC_50/90, 0.12/0.5 μg/ml) (Table 1), meropenem (MIC_50/90, ≤0.06/0.12 μg/ml) (data not shown), and tigecycline (MIC_50/90, 0.12/1 μg/ml) were the most active compounds against these isolates.

Other β-lactamases were also detected in smaller numbers, as follows: FOX (6 isolates), TEM ESBL (12 isolates), and one K. pneumoniae isolate each producing DHA and ACT/MIR. Additionally, narrow-spectrum enzymes of the SHV WT (all K. pneumoniae isolates [a ubiquitous enzyme in this species]) and TEM WT were detected among 284 and 348 isolates, respectively (Table 3). Forty-six strains had negative results for all β-lactamases tested. The majority of these β-lactamase-negative isolates were K. oxytoca isolates that might hyperproduce OXY (K1) and were not evaluated in this study, but E. coli, K. pneumoniae, and P. mirabilis isolates displaying borderline MIC values (≤2 μg/ml) for the β-lactams used for the ESBL screening criteria were also noted. Among these isolates displaying negative β-lactamase screening results, one P. mirabilis isolate had a ceftazidime-avibactam MIC of >32 μg/ml; this strain was isolated from an intraabdominal infection in a medical center located in West Roxbury, MA. Screening for all carbapenemases, including metallo-β-lactamases followed up by ceftazidime hydrolysis displayed negative results for the presence of β-lactamases (data not shown).

Enterobacteriaceae isolates are an important cause of infections, and production of β-lactamases, including ESBLs, transferable cephalosporinases, and carbapenemases among these organisms has become a matter of great concern (1, 2, 14) since in most instances these resistance determinants are disseminated by plasmids and other mobile genetic elements carrying resistance genes for other antimicrobial classes.

A high prevalence of CTX-M- and KPC-producing isolates has been noticed in certain areas of the United States, but ESBL phenotype rates and the occurrence of isolates producing these enzymes are very heterogeneous (6, 7). However, due to the constant dissemination of these isolates and transfer of patients among hospitals, treatment options for severe infections caused by these MDR organisms need to be available.

According to recent publications, ceftazidime-avibactam is expected to play a role in the empirical monotherapy of invasive infections suspected to be caused by resistant Enterobacteriaceae pathogens and also potentially as therapy of KPC-producing Enterobacteriaceae infection (14, 15). The in vitro results from this study support this prediction, since ceftazidime-avibactam demonstrated very good activity against most isolates in this large collection of Enterobacteriaceae isolates recovered over 3 years in over 70 hospitals throughout the country. Additionally, as demonstrated for isolates collected in 2012 (7), ceftazidime-avibactam was very active against recent isolates producing the most common β-lactamases detected in U.S. hospitals, including CTX-M and KPC variants. The potent Gram-negative spectrum of activity of ceftazidime-avibactam, including activity against resistant organisms, demonstrates that it warrants further study in difficult-to-treat serious infections where resistant Gram-negative bacteria may occur.