Concurrent Occurrence of bla_ampC Families and bla_CTX-M Genogroups and Association with Mobile Genetic Elements ISEcp1, IS26, ISCR1, and sul1-Type Class 1 Integrons in Escherichia coli and Klebsiella pneumoniae Isolates Originating from India

Abstract

Cefoxitin-resistant Escherichia coli (n = 109) and Klebsiella pneumoniae (n = 16) isolates collected from patients in India in 2009 to 2010 were screened for bla_ampC families and mobilizing elements (ISEcp1, IS26, ISCR1, and sul-1-type class 1 integrons) and their association with bla_ampC and for the occurrence of class A beta-lactamases (BLs) (CTX-M, TEM, and SHV). The concurrent occurrences of two distinct AmpC families (bla_CIT and bla_EBC) and of class A with class C beta-lactamase were observed. All but one of the isolates harboring CTX-M extended-spectrum BLs (ESBLs) were carrying bla_CTX-M genogroup 1; the remaining isolate carried bla_CTX-M genogroup 9. The mobilizing elements occurred in different combinations in the study isolates.

TEXT

AmpC beta-lactamases (BLs) were originally described as chromosomal, inducible enzymes in members of Enterobacteriaceae; however, the plasmid-mediated AmpC beta-lactamases (pMAmpCs) were first discovered in the late 1980s (6). Since then, they have been detected in different regions around the globe (11, 13). pMAmpCs are assumed to be less common than extended-spectrum BLs (ESBLs) in Escherichia coli and Klebsiella isolates, but their importance should be noted, since they impart a broader spectrum of resistance, especially when present along with other classes of ESBLs. The data regarding the worldwide distribution and prevalence of AmpC-mediated resistance are fragmentary compared to those regarding ESBLs. This may be due to the limited number of surveillance studies performed and the fact that laboratories often face difficulties in accurately detecting these resistance mechanisms (5). Reports from molecular studies in India analyzing such a resistance mechanism are fragmentary (2, 4, 11). Notably, none of the researchers from around the globe have reported the simultaneous occurrences of these two bla_ampC families on the plasmid isolated from a single bacterial strain. Similarly, reports describing the simultaneous occurrences of two bla_CTX-M genogroups are also fragmentary (9).

Clinical isolates of E. coli (n = 109) and K. pneumoniae (n = 16) demonstrating resistance to cefoxitin from clinical samples of in-patients undergoing routine microbiological examination were studied during 2009 to 2010 in the Department of Microbiology at the Jawaharlal Nehru Medical College and Hospital, Aligarh Muslim University, Aligarh, India. Further determinations of antibiotic susceptibility to the antibiotics (as shown in Fig. 1) were performed. To detect AmpC enzymes phenotypically, all cefoxitin-resistant isolates were subjected to the modified three-dimensional extract test (MTDET) as described previously (12).

Fig 1.

Antibiotic resistance profile of the cefoxitin-resistant isolates obtained from various hospital wards. Antibiotic key: Cs, cefoperazone; Cfx, cefixime; Ce, cefotaxime; Ca, ceftazidime; Ci, ceftriaxone; Cfp, cefpirome; Cpm, cefepime; Ao, aztreonam; Gf, gatifloxacin; Of, ofloxacin; G, gentamicin; Ak, amikacin; I, imipenem.

bla_ampC genes were detected in all isolates on monoplex PCR (the primers span the universal region of the ampC gene) as described by Féria et al. (3). Further, all 125 isolates were tested by multiplex PCR to detect pMAmpCs by the method described by Pérez-Pérez and Hanson (7). Isolates were screened for mobile genetic elements (ISEcp1, IS26, ISCR1, and sul-1-type class 1 integrons) as previously described (10). The isolates characterized as carrying pMAmpCs (n = 44) were also screened for the presence of class A ESBLs, bla_CTXM, bla_TEM, and bla_SHV by the use of monoplex PCR. Further, the isolates were characterized for the specific CTX-M genogroup by multiplex PCR as described previously (10). PCR amplicons were sequenced, and the results were compared to sequences in the GenBank database at the National Centre for Biotechnology Information BLAST network (1) using the BLAST search tool in Chromous 2.33 software. Percent identity to the best hits was noted, and the respective sequences were further aligned, using Clustal W software (http://www.genome.jp/tools/clustalw/).

The detailed results for the antibiotic resistance rates and patterns observed in the various wards of the tertiary care hospital where these isolates were obtained are shown in Fig. 1. When phenotypic detection to determine the presence of AmpC beta-lactamase was performed, 76 (60.8%) isolates were noted as AmpC producers whereas 16 (12.8%) and 33 (26.4%) were categorized as AmpC nonproducers and as intermediate with respect to AmpC production, respectively.

In monoplex PCR, 79.2% (n = 99) of the isolates were found to harbor bla_ampC genes (Table 1). When comparing phenotypic and genotypic results, it was observed that, of 65 E. coli isolates characterized as AmpC producers by MTDET, only 57 showed amplification for bla_ampC. Among 29 AmpC-intermediate isolates, bla_ampC was found in 23; however, 11 of 15 E. coli isolates categorized as AmpC nonproducers showed the presence of the bla_ampC gene. Somewhat similar results were observed for Klebsiella isolates. bla_ampC was found in only 7 of 11 AmpC producers, and none of the AmpC-intermediate Klebsiella isolates showed the presence of bla_ampC, whereas 1 Klebsiella isolate that was phenotypically categorized as an AmpC nonproducer showed the presence of bla_ampC. In multiplex PCR, 44 (35.2%) of 125 cefoxitin-resistant isolates were found to be positive for pMAmpCs. Thirty-eight isolates were E. coli, and six were K. pneumoniae. Two distinct bands (PCR amplicons) were obtained for 31 isolates (27 E. coli and 4 K. pneumoniae).

Table 1.

Percentages and numbers of bla_ampC genes (detected by monoplex PCR) and plasmid-mediated ampC genes (pMAmpCs; detected by multiplex PCR) in E. coli and K. pneumoniae isolates studied

Bacterial isolate	Total no. of isolates studied	% (n) of isolates blaampC positive by monoplex PCR	% (n) of pMAmpC isolates with indicated gene(s) (as detected by multiplex PCR)
			Total	CIT only	EBC only	CIT and EBC
E. coli	109	83.49 (91)	34.86 (38)	1.83 (2)	8.25 (9)	24.77 (27)
K. pneumoniae	16	50.00 (8)	37.50 (6)	6.25 (1)	6.25 (1)	25.00 (4)
Total	125	79.20 (99)	35.2 (44)	2.4 (3)	8 (10)	24.80 (31)

In the isolates showing two bands in multiplex PCR, the upper band corresponded to the CIT family. Upon performing monoplex PCR, an amplified product of 450 bp corresponding to the CIT family was obtained, whereas the lower band obtained at 200 bp corresponded to the expected position of the FOX family. On conducting monoplex PCR for the FOX family, no amplification product was obtained. To determine exactly what the lower fragment represented, monoplex PCR was conducted for all families of AmpC β-lactamases that produced an amplified product of 302 bp that corresponded to the EBC family. Alternatively, in three isolates (2 E. coli and 1 K. pneumoniae), amplification was observed for the CIT family only. Ten (9 E. coli and 1 K. pneumoniae) of 44 isolates displayed a single-band correspondence to EBC in multiplex PCR. When monoplex PCR was performed using EBC primers, the amplicon of 302 bp was observed, proving it to be representative of a member of the EBC family. Detailed results are shown in Table 1.

In order to analyze the presence of these genes on a plasmid, PCR was performed using purified plasmid as a template DNA (according to a method reported earlier) (10). Isolates showing two bands (of CIT and EBC) when a lysate was used as a template for the PCR cycle also displayed a similar amplification pattern in experiments where purified plasmid was used as a template. However, when monoplex PCR was performed for CIT and EBC, amplification was observed for the CIT family only. This indicates the presence of bla_EBC on the chromosome but not on the plasmids; however, for two isolates, bands were observed that corresponded to both the CIT and EBC families, denoting the presence in these isolates of both bla_CIT and bla_EBC on the plasmids. Isolates showing a single band for EBC in multiplex PCR when a lysate was used as a template did not show any amplification in multiplex PCR when a plasmid was used as a template; however, the same isolates displayed EBC bands in monoplex PCRs performed using lysate or purified plasmids.

Mobile elements ISEcp1, IS26, and ORF513 and sul-1 integrons were detected in 32/44 (72.72%), 17/44 (38.63%), 32/44 (72.72%), and 35/44 (79.54%) isolates, respectively (shown in Table 2). Highly similar frequencies of occurrence of these genetic elements were observed in the three bla groups (CIT plus EBC, CIT alone, and EBC alone). The isolates were also analyzed in reference to various combinations of observed genetic elements, and ISEcp1 plus ORF513 plus sul-1 integrons was found to be the most common combination, followed by ISEcp1 plus IS26 plus ORF513 plus sul-1 integrons. The frequencies of occurrence of mobile element combinations were similar in the three bla groups (Table 2).

Table 2.

Frequencies of single mobile genetic elements and combinations of mobile genetic elements in the study isolates

Occurrence of blaampC(s) (no. of isolates)	% (n) of total isolates with indicated mobile genetic element(s)				% (n) of isolates with indicated combinations of mobile genetic elements
	ISEcp1	IS26	ORF513	sul-1	ISEcp1 + IS26	ISEcp1 + ORF513	ISEcp1 + sul-1	IS26 + ORF513	IS26 + sul- 1	ORF513 + sul-1	ISEcp1 + IS26 + ORF513	ISEcp1 + ORF513 + sul-1	IS26 + ORF513 + sul-1	ISEcp1 + sul-1 + IS26	ISEcp1 + IS26 + ORF513 + sul-1	ISEcp1 only	IS26 only	ORF513 only	sul-1 only	No genetic element
blaCIT (3)	66.7 (2)	33.33 (1)	33.33 (1)	66.7 (2)						33.33 (1)				33.33 (1)		33.33 (1)
blaEBC (10)	60 (6)	50 (5)	80 (8)	80 (8)			10 (1)	10 (1)		10 (1)	10 (1)	20 (2)	10 (1)		20 (2)				10 (1)
blaCIT and blaEBC (31)	77.42 (24)	35.48 (11)	74.19 (23)	80.65 (25)	3.23 (1)		3.23 (1)			9.68 (3)		32.26 (10)	3.23 (1)	12.9 (4)	19.35 (6)	6.45 (2)		6.45 (2)		3.23 (1)

Detailed results with respect to the frequencies of occurrence of class A ESBLs (bla_CTX-M, bla_TEM, and bla_SHV) in the pMAmpC-harboring isolates are illustrated in Fig. 2. When analyzed for co-occurrence, bla_CTX-M was found to be associated with all isolates showing the presence of bla_CIT or bla_EBC alone; however, co-occurrence was noticed in 90.32% isolates harboring both bla_CIT and bla_EBC. bla_TEM and bla_SHV together were almost equally distributed in isolates harboring only bla_EBC and in isolates harboring bla_CIT plus bla_EBC (60% and 61.29%, respectively). bla_TEM and bla_SHV were noticed in 100% of isolates showing the presence of bla_CIT alone (Fig. 2).

Fig 2.

Co-occurrence of various bla_ampC families with class A ESBLs.

The CTX-M-harboring isolates were further characterized for CTX-M genogroups, and all isolates but one were observed to carry CTX-M genogroup 1. Sequencing of representative isolates revealed the presence of bla_CTX-M-15. The remaining single isolate was found to carry CTX-M of genogroup 9; further sequencing of the isolate revealed the presence of bla_CTX-M-9-like alleles. The nucleotide sequences determined in this work were submitted to GenBank (see below).

During the present study, the CIT family and the EBC family were observed at frequencies of 27.20% and 32.8%, respectively, while simultaneous occurrences of CIT and EBC were observed in 24.80% of the isolates. Since two or more plasmid-mediated AmpC families were observed simultaneously in the respective isolates, this suggests an alarming state of affairs, because the present situation indicates that antibiotic resistance genes are now accumulating in individual organisms. To date, various bla genes of different molecular classes (CTX-M, SHV, TEM, CMY, and metallo-β-lactamase) have been reported (8, 10); however, to the best of our knowledge, this is the first report describing the occurrence of two AmpC types in a single strain. Notably, the isolates harboring the bla_ampC also carried the genes for class A ESBLs (bla_CTX-M, bla_TEM, and bla_SHV) in different combinations. This is troublesome due to the fact that simultaneous occurrences of class A and class C beta-lactamases cause broad-spectrum antibiotic resistance, including resistance to extended-spectrum cephalosporins as well as aztreonam. It should also be kept in mind that the presence of bla_ampC together with porin loss could also lead to carbapenem resistance.

The present report is also unique in that it analyzes the battery of associated mobile elements and the frequencies of their occurrence, specifically, the occurrence of various combinations. sul-1-type class 1 integrons were observed to be the most common genetic elements associated with Enterobacteriaceae, followed by ORF513. IS26 was observed to be the least frequent; however, variations in the insertion positions of IS26 elements (evident from the variable sizes of the IS26 amplicons) are noteworthy and suggest frequent rearrangements within the genetic environment of Enterobacteriaceae. ISEcp1 plus ORF513 plus sul-1 integrons was the most common combination, followed by ISEcp1 plus IS26 plus ORF513 plus sul-1 integrons, in bla_ampC-harboring Indian isolates. Since the first report of the detection of bla_CTX-M-15 in India, it is now considered the most common CTX-M type worldwide. Moreover, CTX-M types from genogroup 1 (specifically, CTX-M-15) were reported as the only types existing in India. In the present study, CTX-M genogroup 1 was observed to be the most prevalent group and sequencing of its representative isolates indicated the presence of the CTX-M-15 type. However, it is also reported here that the occurrence of the CTX-M-9-like (genogroup 9) beta-lactamase was observed for the first time in Northern India, in a town called Aligarh, located only 132 km from New Delhi, where the occurrence of CTX-M-15 was reported, also for the first time. Of even more interest, the isolate bearing bla_CTX-M-9 was also carrying bla_CTX-M-15, bla_SHV, bla_TEM, and bla_ampC (bla_CIT group only). Upon analyzing the genetic environment of the CTX-M genogroup 1-harboring isolates, it was once again determined that ISEcp1 plus ORF513 plus sul-1 integrons was the most common combination, followed by ISEcp1 plus IS26 plus ORF513 plus sul-1 integrons. Upon analysis of the genetic environment of the isolate carrying bla_CTX-M-9, the presence of a combination of ISEcp1 plus IS26 plus sul-1 integrons was discovered.

In a nutshell, this is the first report describing the occurrence of two AmpC families in a single strain, especially on plasmids. Detection of CMY-2 and of CTX-M-9 (observed in northern India) is reported for the first time. The occurrence of newer resistance mechanisms, the concurrent occurrences of multiple bla genes, and the frequent association of a large number of genetic elements, termed “cocktail bugs” here, clearly indicates the emergence of a complex resistance mechanism in Enterobacteriaceae. This is troublesome and warrants serious public health concern.

Nucleotide sequence accession numbers.

Nucleotide sequences were submitted to GenBank under the following accession numbers: for the sul 1-type class 1 integron, accession no. JF918432; for the bla_CTX-15-like allele, accession no. JF918433; for the bla_CMY-2-like allele, accession no. JF918434; and for the bla_MIR-1-like allele, accession no. JF918435.