Genetic Characterization of ST195 and ST365 Carbapenem-Resistant Acinetobacter baumannii Harboring bla OXA-23 in Guangzhou,China

Abstract

We investigated the distribution of resistance genes and the clonal relationships among carbapenem-resistant Acinetobacter baumannii isolates from the intensive care unit wards of two hospitals in Guangzhou, China. From 2012 to 2013, 57 A. baumannii isolates were obtained from blood cultures from two hospitals in Guangzhou. The antibiotic resistance profiles were determined by using the Vitek2 system and Etest strips. PCR was used to detect the genes encoding OXA-type carbapenemases and metallo-β-lactamases and the presence of ISAba1 upstream of the bla_OXA-51-like gene and the bla_OXA-23-like gene. Multilocus sequence typing (MLST) and sequence-based typing of bla_OXA-51-like genes (SBT-bla_OXA-51-like genes) were performed to analyze the genetic relationship of the isolates. Among the 57 isolates, 46 were carbapenem-resistant A. baumannii (CRAB) isolates. The bla_OXA-51-like gene was identified in all 57 isolates, while the bla_OXA-23-like gene was present in all 46 CRAB isolates. The MLST analysis grouped the A. baumannii isolates into five existing sequence types (STs) and five new STs. Fifty-two isolates belonged to the worldwide spread of clonal complex 92 (CC92), among which ST195 and ST365 were the most common STs. The MLST data and SBT-bla_OXA-51-like genes showed that all isolates harboring the major bla_OXA-51-like alleles, such as bla_OXA-66, belonged to CC92.

Introduction

Acinetobacter baumannii is considered to be an important opportunistic pathogen that primarily causes nosocomial outbreaks worldwide, especially in intensive care units (ICUs).^6,22 Such infections are often attributed to the resistance of the isolates to antimicrobial agents, including carbapenems. Carbapenem resistance has become far more common among A. baumannii isolates in Chinese hospitals in recent years. According to data from the CHINET (antimicrobial resistance surveillance networks in China), the resistance of A. baumannii isolates to imipenem and meropenem increased from 31.5% and 40.7% in 2004–2005 to 65.2% and 66.2% in 2011, respectively.^27,30

Resistance to carbapenems in A. baumannii is primarily mediated by the production of carbapenem-hydrolyzing class D oxacillinase (CHDL). There are four groups of CHDLs identified in A. baumannii; one is the intrinsic chromosomal OXA-51-like β-lactamase and three are acquired CHDLs, represented by the OXA-23-like, OXA-24/OXA-40-like, and OXA-58-like β-lactamases.²⁴

The increased detection of carbapenem-resistant A. baumannii (CRAB) strains in hospital settings is mainly linked to the global dissemination of a number of highly successful clones, such as international clonal lineages I, II, and III (ICL-I, ICL-II, and ICL-III).^6,22 Many studies reported that A. baumannii isolates producing OXA-23 and belonging to ICL-II, in particular those that are defined as ST92 by multilocus sequence typing (MLST), are widespread throughout China.^10,12,13,18

Currently, two large national investigations have shown that ST92,^10,18 the founder of CC92, was the predominant ST in China; however, isolates corresponding to ST75 and ST208, single locus variants (SLVs) of ST92, have increasingly emerged and spread nationwide recently.^26,31 Other SLVs of ST92, ST191, and ST195 have been identified primarily in East China and South China, respectively.^5,15

Evans et al. have performed the sequence-based typing of bla_OXA-51-like genes (SBT-bla_OXA-51-like genes) and showed that bla_OXA-66, bla_OXA-69, and bla_OXA-71 are the predominant members of closely related bla_OXA-51-like subgroups, which are associated with sequence groups (SG) I, II, and III, respectively.⁸ Other studies also indicated that the SBT-bla_OXA-51-like genes could rapidly type the A. baumannii strains into three ICLs when compared with repetitive-sequence-based PCR (rep-PCR) and MLST.^11,29 One study has revealed that bla_OXA-66 was the most prevalent bla_OXA-51-like allele in China, but the correlation between bla_OXA-51-like alleles with some common molecular typing methods has not been investigated.²⁵

This study investigated 57 A. baumannii isolates from blood cultures collected from two teaching hospitals in Guangzhou to define their epidemiological traits and the genetic basis of antibiotic resistance.

Materials and Methods

Bacterial isolates and susceptibility testing

In this study, 57 A. baumannii isolates were obtained from blood cultures, between May 2012 and November 2013, in the ICUs of two university hospitals (designated A and B) in the Guangzhou urban area. Hospitals A and B are tertiary care academic medical centers with 1,600 and 1,000 beds, respectively, and the distance between the two hospitals is ∼9 km. Only one isolate from each patient was assessed. The clinical samples were collected as part of the standard patient care regimen, and informed consent for the use of clinical data was obtained from the patients. Initial identification was performed with the Vitek2 system (bioMérieux, Marcy L'Etoile, France), and final identification was performed using the sequence analysis of the 16S-23S rRNA intergenic spacer region.³

The susceptibilities of the isolates to 10 antimicrobial agents, including ampicillin, ampicillin/sulbactam, piperacillin, piperacillin/tazobactam, ceftriaxone, ceftazidime, cefepime, tobramycin, gentamicin, and ciprofloxacin, were tested by using the Vitek2 system with an AST-GN04 card and interpreted according to the Clinical and Laboratory Standards Institute guidelines (M100-S23, 2013).⁴ Minimum inhibitory concentrations (MICs) of imipenem, meropenem, and polymicin were determined by the use of Etest strips, which are in accordance with the 2013 CLSI guidelines. Escherichia coli ATCC 25922 and Klebsiella pneumoniae ATCC 700603 were used as control strains.

Screening for resistance genes

PCR analyses were performed for the detection of OXA carbapenemase genes (bla_OXA-23-like, bla_OXA-24-like, bla_OXA-51-like, and bla_OXA-58-like), ISAba1/bla_OXA-51-like complex, ISAba/bla_OXA-23-like complex, and metallo-β-lactamase genes (bla_IMP, bla_VIM, bla_GIM, bla_SPM, bla_SIM, and bla_NDM) as previously described.^7,21,28

Genotyping by MLST

MLST was performed according to the protocols described on the A. baumannii MLST database website (http://pubmlst.org/abaumannii/) using primers that were modified as previously described.^2,10 The resulting sequences were assigned to sequence types (STs) using the tools on the A. baumannii MLST webpage. eBURST (version 3, http://eburst.mlst.net/) was used to assign STs to CCs and to assess the genetic relationship with the definition of the groups sharing alleles at ≥6 of 7 loci.⁹

Sequence-based typing of bla_OXA-51-like genes

A single-locus bla_OXA-51-like sequence-based typing (SBT) was performed to analyze the genetic diversity of the OXA-51-like gene of the study isolates as described by Pournaras et al.¹⁷

Results

Antibiotic resistance profiles

A total of 57 A. baumannii isolates were recovered from the ICU wards of two hospitals, hospital A (n=44) and hospital B (n=13), during this study period. Among those isolates, 46 isolates were CRAB isolates, while 11 isolates were susceptible to both imipenem and meropenem (MIC ≤4 mg/L).

All 46 CRAB isolates were resistant to both imipenem and meropenem. However, all CRAB isolates were susceptible to polymicin. Six of the 11 carbapenem-susceptible A. baumannii (CSAB) isolates showed a common antibiotic susceptibility profile characterized by resistance to ampicillin, ampicillin-sulbactam, piperacillin, ceftriaxone, ceftazidime, tobramycin, gentamicin, aztreonam, and ciprofloxacin, as well as intermediates of piperacillin/tazobactam and cefepime. The remaining five CSAB isolates were resistant to ampicillin only, an intermediate of ceftriaxone, and susceptible to 11 antimicrobial compounds (Table 1).

Table 1.

Susceptibility of the 57 Acinetobacter baumannii Isolates

	CSAB isolates (n=11)		CRAB isolates (n=46)
Antimicrobial	S n (%)	R n (%)	S n (%)	R n (%)
Ampicillin	0 (0)	11 (100)	0 (0)	46 (100)
Ampicillin/sulbactam	5 (45.5)	6 (54.5)	0 (0)	46 (100)
Piperacillin	5 (45.5)	6 (54.5)	0 (0)	46 (100)
Piperacillin/tazobactam	5 (45.5)	1 (9.1)	0 (0)	46 (100)
Ceftriaxone	0 (0)	6 (54.5)	0 (0)	46 (100)
Ceftazidime	5 (45.5)	6 (54.5)	0 (0)	46 (100)
Cefepime	5 (45.5)	1 (9.1)	0 (0)	46 (100)
Imipenem	11 (100)	0 (0)	0 (0)	46 (100)
Meropenem	11 (100)	0 (0)	0 (0)	46 (100)
Gentamicin	4 (36.4)	7 (63.6)	0 (0)	46 (100)
Tobramycin	4 (36.4)	7 (63.6)	0 (0)	46 (100)
Ciprofloxacin	5 (45.5)	6 (54.5)	0 (0)	46 (100)
Polymyxin	46 (100)	0 (0)	46 (100)	0 (0)

CRAB, carbapenem-resistant A. baumannii; CSAB: carbapenem-susceptible A. baumannii; R, resistant; S, susceptible.

Genetic and functional basis of carbapenem resistance

PCR detection of the metallo- and carbapenem-hydrolyzing OXA-type β-lactamase genes was positive only for bla_OXA-51-like and bla_OXA-23-like. The bla_OXA-51-like gene was identified in all 57 A. baumannii isolates, while the bla_OXA-23-like gene was present in all 46 CRAB isolates.

The insertion sequence, ISAba1, was found upstream of the bla_OXA-23-like gene in 45 CRAB isolates harboring this gene, with imipenem MICs from 16 to ≥32 mg/L. In contrast, eight isolates contained the ISAba1-activated bla_OXA-51-like gene. Among the six isolates also carrying ISAba-bla_OXA-23-like, one isolate was positive for ISAba1-bla_OXA-51-like only and one isolate had bla_OXA-23-like without the ISAba1 element (Table 2). In accordance with the previously published study, there was a clear correlation between the presence of ISAba1 upstream of the bla_OXA-23-like and the high level of resistance to carbapenem.²¹

Table 2.

Correlation Between the Genetic Basis of Carbapenem Resistance

		MIC₅₀	MIC₉₀
OXA-type determinant and genetic arrangement	No. of isolates (resistant to IMP/MEM)	IMP/MEM	IMP/MEM
bla _OXA-51-like	10 (0/0)	0.5/0.5	1/0.5
bla_OXA-51-like+ISAba1-bla_OXA-23-like	39 (39/39)	32/32	64/64
ISAba1-bla_OXA-51-like	1 (0/0)	4/4	4/4
ISAba1-bla_OXA-51-like+bla_OXA-23-like	1 (1/1)	64/32	64/32
ISAba1-bla_OXA-51-like+ISAba1-bla_OXA-23-like	6 (6/6)	32/32	64/64

IMP, imipenem; MEM, meropenem; MIC, minimum inhibitory concentration.

Multilocus sequence typing

The molecular characterization of 57 A. baumannii isolates by MLST identified 10 distinct STs, including 5 novel STs (designated STn1 to STn5). ST195 and its double-locus variant (DLV), ST365, were the predominant STs comprising 54.4% and 19.3%, respectively. Interestingly, the five novel ST isolates belonged to CSAB isolates. Notably, the worldwide dissemination of ST92, especially in China, was previously reported, which showed low prevalence in the present study (five isolates) and susceptibility to carbapenem. In contrast, both the predominant ST195 and ST365 correspond to CRAB.

In the present study, eBURST analysis indicated a total of 52 isolates belonging to ST195, ST365, ST92, ST381, ST75, and STn1, which were clustered into the globally disseminated CC92. The remaining five isolates belonged to four singletons (Table 3).

Table 3.

Characteristic of the 57 Acinetobacter baumannii Isolates

	ST profile
ST	gltA	gyrB	gdhB	recA	cpn60	gpi	rpoD	CC	No. (%) of isolates	Hospitals (No. of isolates)	bla_OXA-51-like allele (No. of isolates)	Imipenem MIC (mg/L)
195	1	3	3	2	2	96	3	92	31(54.4)	A(21),B(10)	bla_OXA-66(31)	≥16
365	1	15	3	2	2	59	3	92	11(19.3)	A(9),B(2)	bla_OXA-66(11)	≥16
92	1	3	3	2	2	7	3	92	5(8.8)	A(5)	bla_OXA-66(5)	0.25–4
381	1	81	3	2	2	16	3	92	3(5.3)	A(3)	bla_OXA-66(3)	≥16
75	1	3	3	2	2	11	3	92	1(1.8)	A(1)	bla_OXA-66(1)	16
STn1	1	3	3	2	2	154	3	92	1(1.8)	A(1)	bla_OXA-66(1)	0.25
STn2	1	34	80	28	35	117	45	Singleton	2(3.5)	A(2)	bla_OXA-217(2)	0.5
STn3	33	12	59	11	32	55	5	Singleton	1(1.8)	A(1)	bla_OXA-380(1)	1
STn4	36	57	62	31	22	10	4	Singleton	1(1.8)	A(1)	bla_OXA-64(1)	0.5
STn5	33	12	40	26	32	11	5	Singleton	1(1.8)	B(1)	bla_OXA-65(1)	1

ST, sequence type.

SBT-bla_OXA-51-like genes

According to SBT, five bla_OXA-51-like alleles were identified in our study; among these, bla_OXA-66 was the most dominant bla_OXA-51-like allele, which accounted for 52/57 (91.2%), followed by bla_OXA-217 (2/57, 3.5%), bla_OXA-64 (1/57, 1.8%), bla_OXA-65 (1/57, 1.8%), and bla_OXA-380 (1/57, 1.8%). All isolates carrying bla_OXA-66 belonged to CC92, whereas the isolates belonging to the four singletons each carried a unique bla_OXA-51-like variant (Table 3).

Discussion

Our data showed that except for OXA carbapenemase genes, no metallo-β-lactamase genes, including genes for VIM, IMP, NDM, GIM, SPM, and SIM, were detected. The most common carbapenem resistance mechanism found in this study was either ISAba1-bla_OXA-23-like or the coexistence of the ISAba1-bla_OXA-51-like+ISAba1-bla_OXA-23-like genetic platforms. One isolate carrying ISAba1-bla_OXA-51-like only showed reduced susceptibility to carbapenems (MIC=4 mg/L), suggesting ISAba1-driven overexpression of the intrinsic bla_OXA-51-like gene as previously described.²¹

Our report indicated that CC92 was the most prevalent clonal complex identified in 52 isolates from the two hospitals. However, ST92, the predicted founder of CC92 that was reported to be one of the most epidemic STs in multiple provinces of China,¹⁸ was only detected in five isolates in this study. Recently, several studies have revealed that ST75, ST208, ST191, and ST195 (but not ST92) were the most common STs discovered in different hospitals across China.^{5,15,26,31,32} In this study, ST195 was the most commonly observed ST, accounting for 31/57 (54.4%) isolates, followed by ST365, a DLV of ST92, which was only identified from 1 isolate in China previously,¹⁸ and identified in 11/57 (19.3%) isolates. ST195 and ST365 have frequently been identified in Southeast Asian countries, including Malaysia, Thailand, and Vietnam.^14,19 Li et al. first reported that ST195 was predominantly identified in a teaching hospital in Guangzhou.¹⁵ In the present study, ST195 has been detected in two hospitals in Guangzhou, suggesting that ST195 may be successfully disseminated in this city. These findings may suggest that the current epidemic of CC92 strains in China represents the clonal expansion of progenitor strains (ST92 possessed bla_OXA-23), which might be due to different antibiotic usage habits in different hospitals in China.

bla_OXA-66 has been reported as the most prevalent bla_OXA-51-like variant in China,²⁵ Japan,²⁰ Croatia,²³ Saudi Arabia,¹ and Portugal¹⁶; however, the correlation between the bla_OXA-51-like variants with MLST typing has not been investigated, although they observed that CC92 was the most common clonal complex in these studies. Recently, several studies have investigated the single-locus bla_OXA-51-like sequence-based typing (SBT) in comparison with other typing methods, where it was shown that SBT corresponded to MLST, DiversiLab rep-PCR typing, and sequence group typing.^8,11,17,29 Zander et al. have investigated the association between bla_OXA-51-like variants and DiversiLab clonal lineages and they confirmed that the isolates harboring bla_OXA-66 corresponded to international clone 2 (IC2).²⁸ Pournaras et al.¹⁷ and Hamouda et al.¹¹ have compared SBT with the Pasteur Institute MLST scheme and Bartual MLST scheme, respectively. The SBT analysis data correlated well with the data from both MLST schemes with respect to the identification of the International lineages I, II, and III. According to the MLST analysis, the isolates harboring the major bla_OXA-51-like alleles, bla_OXA-66, bla_OXA-69, and bla_OXA-71, fell within the three major European clonal lineages (currently named the International clonal lineage). Furthermore, it was shown that the same bla_OXA-51-like allele was detected in different STs (bla_OXA-66 to ST2 and ST45; bla_OXA-69 to ST1 and ST20), which were included in the same CC, and no case was observed where different bla_OXA-51-like alleles were detected in the same ST. In our study, 52 isolates carrying bla_OXA-66 were assigned to six distinct STs, which clustered into CC92, while the remaining isolates belonged to four singletons where each carried a single bla_OXA-51-like allele. Our findings indicate that the isolates harboring bla_OXA-23 and bla_OXA-66 belong to CC92, which may be the most common clone in Guangzhou, China.

Footnotes

Acknowledgments

This research was funded by the Project for Key Medicine Discipline Construction of Guangzhou Municipality (grant No. 2013-2015-07), the National Science and Technology Major Projects of China (No. 2012ZX10004213-005), the Sci-tech Research Project of Guangzhou Municipality, China (grant Nos. 2011J4300061), and the Medical Sci-tech Research Projects of Guangzhou Municipality, China (grant Nos. 20121A021019 and 20131A011107).

Disclosure Statement

No competing financial interests exist.

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2012. Clonal spread of carbapenem non-susceptible Acinetobacter baumannii in an intensive care unit in a teaching hospital in China. Ann. Lab. Med., 32:413–419.

Genetic Characterization of ST195 and ST365 Carbapenem-Resistant Acinetobacter baumannii Harboring bla OXA-23 in Guangzhou,China

Abstract

Introduction

Materials and Methods

Bacterial isolates and susceptibility testing

Screening for resistance genes

Genotyping by MLST

Sequence-based typing of blaOXA-51-like genes

Results

Antibiotic resistance profiles

Genetic and functional basis of carbapenem resistance

Multilocus sequence typing

SBT-blaOXA-51-like genes

Discussion

Footnotes

Acknowledgments

Disclosure Statement

References

Sequence-based typing of bla_OXA-51-like genes

SBT-bla_OXA-51-like genes