Unusual Diversity of Acquired β-lactamases in Multidrug-Resistant Pseudomonas aeruginosa Isolates in a Mexican Hospital

Abstract

Aims: To investigate the presence of extended spectrum and metallo β-lactamases (MBLs) in Pseudomonas aeruginosa isolates which are resistant to imipenem and ceftazidime that were isolated in a hospital in Mexico. Results: Pulsed-field gel electrophoresis (PFGE) revealed the presence of four clonal types among the 14 isolates. All these genes were found either alone or simultaneously in the P. aeruginosa strains in the following five different arrangements: <bla_GES-5>; <bla_GES-5, bla_VIM-11>; <bla_GES-5, bla_VIM-2, bla_VIM-11>; <bla_GES-5, bla_OXA-2>; and <bla_GES-5, bla_VIM-2, bla_VIM-11, and bla_OXA-2>. Class 1 integrons were detected and contained the cassettes bla_GES-5 and bla_OXA-2, but not that of bla_VIM. bla_VIM genes occurred only in the chromosome, while bla_GES-5 was located in the chromosome and in the plasmids. Conclusions: To our knowledge, this is the first description of P. aeruginosa strains simultaneously producing the VIM-2 and VIM-11 variants, and the combination of GES-5 and MBL carbapenemases, which determines a major challenge for the clinical microbiology laboratory and a remarkable epidemiological risk for the nosocomial spread of multidrug-resistant determinants.

Introduction

Ceftazidime and imipenem are useful antibiotics in the treatment of infections caused by Pseudomonas aeruginosa. However, clinical isolates often show resistance to these due to the acquisition of genes encoding for extended-spectrum β-lactamases (ESBLs) and/or metallo-β-lactamases (MBLs). Of particular interest are the GES β-lactamases, an ESBL family with some variants, such as GES-5, showing carbapenem hydrolysis.¹ Genes encoding for β-lactamases may be located in integrons, transposons, prophages, and genomic islands, and can be mobilized from the chromosome to a plasmid and vice versa.²⁵

Clinical isolates of P. aeruginosa producing acquired β-lactamases such as GES-5 have caused outbreaks in several hospitals in different regions of the world, such as China,³³ South Africa,¹⁵ Brazil,²⁰ and Spain.³² VIM-family MBLs were first described in Europe in 1999 and since that time, specific reports on VIM-11¹⁹ and VIM-13,¹⁴ as well as on other VIM variants, have been made worldwide, at present reaching a family with 32 subtypes¹⁸ (nomenclature is in accordance with that provided by G. Jacoby on the Lahey website available at www.lahey.org/studies/other.asp#table1]). The simultaneous presence of genes encoding MBLs and/or ESBLs has been described in P. aeruginosa.³² In Mexico, bla_VIM-2 was found in Escherichia coli isolates in 2009¹⁷ and simultaneously with bla_IMP-15 in P. aeruginosa clinical isolates in 2010.²⁴ In addition, a bla_VIM-23 variant was identified in an Enterobacter cloacae isolate (GenBank accession number GQ242167).

The aim of this study was to determine the diversity of β-lactamases in a group of multidrug-resistant (MDR) isolates of P. aeruginosa involved in nosocomial infections in Mexico in 2004, demonstrating the novel combination bla_GES-5 and bla_VIM-11, as well as the previously reported bla_VIM-2 and bla_OXA-2.

Materials and Methods

Biological material

Fourteen imipenem- and ceftazidime-resistant P. aeruginosa isolates were studied.⁵ These isolates were obtained from cases of nosocomial infections at the Intensive Care Service, Hospital of Infectious Diseases, La Raza National Medical Center, Mexican Institute of Social Security (IMSS) in Mexico City, from August to November 2004. Bacteriological identification was performed with the BBL Crystal^® system (Becton, Dickinson and Company), and three isolates were selected at random to confirm identification at the molecular level, analyzing their 16S rDNA gene partial sequence (results not shown).

Molecular typing and susceptibility to antibiotics

The clonal relationship of isolates was studied by pulsed-field gel electrophoresis (PFGE), as described by Spencker et al.²⁹ Genomic DNA was subjected to digestion with SpeI. Macrorestriction patterns of DNA were interpreted under the criteria established by Tenover et al.³⁰ and the dendrogram-derived degree of genetic relatedness. The dendrogram was derived from the PFGE results from the matrix of correlation distances by using the Jaccard similarity coefficient with the Dendro-UPGMA program (http://genomes.urv.cat/UPGMA).¹⁰

Susceptibility to 21 antibiotics (amikacin, azlocillin, aztreonam, carbenicillin, cefepime, cefoperazone, ceftazidime, ceftizoxime, ceftriaxone, ciprofloxacin, imipenem, levofloxacin, meropenem, mezlocillin, netilmicin, norfloxacin, ofloxacin, piperacillin, ticarcillin, ticarcillin/clavulanic acid, and tobramycin) was assessed by the disk diffusion method utilizing filter paper discs and following the recommendations of the Clinical and Laboratory Standard Institute (CLSI).⁶

The MBL phenotype was detected with E-test metallo-β-lactamase strips (E-test MBLs; AB Biodisk) employed according to the manufacturer's instructions. ESBL detection was performed with the double-disk synergy test (DDST) with ceftazidime and amoxicillin/clavulanate with a 10–20 mm separation.¹³

Detection, characterization, and cloning of acquired β-lactamases

To have a biochemical approximation to the potentially acquired β-lactamases, crude protein extracts were prepared by sonication for determination of isoelectric focusing (IEF) using Phast gels (pH gradient, 3–9) in a Phast System apparatus (Pharmacia AB). The extracts from four control strains of the collection of the Son Dureta Hospital were subjected in parallel to electrophoresis CTX-M-15 producing Klebsiella pneumoniae (isoelectric point [pI], 8.6),⁹ GES-1, GES-5, and OXA-2 producing P. aeruginosa (pI, 5.9, 5.8, and 7.7, respectively),³² TEM-1 and CTX-M-14 producing E. coli (pI, 5.4 and 8.0, respectively),⁹ and SHV-12 producing E. coli (pI, 8.2).³² For genetic characterization, polymerase chain reactions (PCRs) were performed using as a template the genomic DNA obtained with the DNeasy Tissue kit (Qiagen), as well as specific primer sequences previously reported to identify β-lactamase genes, including GIM, IMP, SIM, SPM, and VIM,^12,23 GES,³² and OXA²⁷ (Table 1). Amplicons obtained by PCR and cloned DNA fragments were sequenced in ABI PRISM 3100 automated equipment^® (PE Applied Biosystems). The resulting sequences were then compared with those available at GenBank (www.ncbi.nih.gov/BLAST).

Table 1.

Details of Primers: Genes Targeted, Primer Denomination (Forward-Followed by Reverse Primer), Sequence, and Expected Amplicon Length

Target-name	Primer	Sequence	Product size (bp)	Reference
bla_GIM-1	GIM-F	AGAACCTTGACCGAACGCAG	753	4
	GIM-R	ACTCATGACTCCTCACGAGG
bla_IMP	IMP-F	CTACGGCAGCAGAGTCTTTG	587	27
	IMP-R	AACCAGTTTTGCCTTACCAT
bla_SIM-1	SIM1-F	TACAAGGGATTCGGCATCG	551	16
	SIM1-R	TAATGGCCTGTTCCCATGTG
bla_SPM	SPM (5′)	CCTACAATCTAACGGCGACC	629	4
	SPM (3′)	TCCGCCGTGTCCAGGTATAAC
bla_VIM	VIM-F	AGTGGTGAGTATCCGACAG	500	9
	VIM-R	ATGAAAGTGCGTGGAGAC
bla_VIM-2	VIM-2-A	ATGTTCAAACTTTTGAGTAAG	801	20
	VIM-2-B	CTACTCAACGACTGAGCG
bla_GES-5	GES5inic-F (5′)	CACTCTGCATATGCCTCGGA	Variable	31
	GESint-R (3’)	GCCGTTGTATACACCGCTAC
bla_GES-5-aadA1	HIaadA1-R^a	CAAGAATGTCATTGCGCTGCC	Variable	This study
IntI-qacΔE1	INT-R-I	CGCAGTGGCGGTTTTCAT	Variable	9
	qacE-R	ATTATGACGACGCCGAGTC
aacA4-bla_OXA-2	IntF-F (5′)^a	GTACTTGCCAAGCGTTTTA	1,500	This study
	IntR-F (3′)^a	CCTTTGCAAATAGCTCATAC
intI	INT-F	CTCTCACTAGTGAGGGGC	1,010	9
	INT-R	ATGAAAACCGCCACTGCG
intI2	IntI2-L (5′)	GTACTTGCCAAGCGTTTTA	789	18
	IntI2-R (3′)	CACGGATATGCGACAAAAAGGT
intI3	HS207 (5′)	GGAATTCCTGCATGAACAGGTATAACGG	1,044	6
	HS208 (3′)	CGGGACCCGCAGCCGGGCGACAAGGCA

Used only for sequencing.

When the IEF assay showed bands with a pI corresponding to β-lactamases reported in the literature but whose presence was not detected by PCR, cloning of the involved gene was attempted. Cloning was performed in E. coli XL1-Blue or E. coli DH5-α utilizing the CaCl₂ method with ligation reactions of pUCP24 and total DNA digested with EcoRI and BamHI.²⁶ The transformants were selected on Luria Bertani (LB) agar plates containing 5 mg/L gentamicin and 30 mg/L ampicillin. Since the potential presence of multiple integrons simultaneously in the same isolate may interfere with PCR and sequencing experiments, cloning was carried out to detect integrons and/or genetic regions containing resistance genes. ESBL and MBL activity was analyzed in the obtained transformants as previously described (DDST and E-test MBLs). Minimal inhibitory concentration (MIC) to the antibiotics piperacillin, piperacillin/tazobactam, ceftazidime, cefepime, imipenem, meropenem, gentamicin, tobramycin, amikacin, ciprofloxacin, and colistin was determined with E-test (AB Biodisk).

Characterization of the involved integrons

The search for integrons of classes 1, 2, and 3 that potentially contain β-lactamase genes and/or other resistance determinants was carried out by PCR, employing as a target the conserved regions of genes encoding for different integrases.^7,12,21 Detection of class 1 integrons was achieved after obtaining an amplicon of 1,010 bp that was confirmed by sequencing. Subsequently, to identify the cassettes present in the integrons, PCR amplification was performed for the integron-variable region located between genes intI1 and qacEΔ.^{9
,
21} The amplicons obtained in this case had a size ranging from about 800 and 1,400 bp. Finally, the integrons amplified directly from the genomic DNA of P. aeruginosa isolates and those identified from recombinant pUC24 clones were sequenced.

Characterization of genetic elements carrying the transferable β-lactamases

B203(B), 204(B1), and 208(D) were the only clinical isolates that were shown to have plasmids. Electroporation of the plasmid DNA in P. aeruginosa PAO1 was carried out.²⁸ The transformants were selected on LB agar plates and added together with ceftazidime at a concentration of 16 mg/L. The analysis of resistance transfer in the transformed clones was tested by E-test (AB Biodisk) for piperacillin, piperacillin/tazobactam, ceftazidime, cefepime, imipenem, meropenem, gentamicin, tobramycin, amikacin, and ciprofloxacin, interpreting the results according to CLSI. β-lactamase phenotypic tests were conducted as previously described.

A series of hybridization analyses was performed to determine the localization of genes encoding β-lactamases in the bacterial genome. Southern hybridization analyses were carried out according to the manufacturer's instructions (GE Healthcare), utilizing as hybridization probes internal fragments of bla_GES, or bla_VIM and genomic or plasmid DNA obtained through Genopure Plasmid Maxi kit (Roche Diagnostics) from clinical isolates and recombinant clones.

Results

Molecular typing and susceptibility to antibiotics

PFGE analysis allowed grouping the 14 MDR clinical isolates of P. aeruginosa into four types designated A to D (containing from one to six isolates each); and four subtypes: A1, A2, B1, and C1 (Fig. 1). Eleven isolates showed resistance to nearly all (≥20/21) of the antibiotics tested and were distributed among the four PFGE types. Two isolates (from clusters B and D) demonstrated the ESBL phenotype by DDST, whereas the MBL E-test yielded a positive result in seven isolates (Table 2).

FIG. 1.

Analysis of Pseudomonas aeruginosa clinical isolates by pulsed-field gel electrophoresis (PFGE). (A) Clonal diversity was observed among 14 strains under study (clue strain [type of pattern]). PAO1, P. aeruginosa PAO1. (B) Dendrogram of the relationships between PFGE results constructed from the matrix of correlation distances with a cophenetic correlation coefficient of 0.96 (Jaccard coefficient) with the Dendro-UPGMA program.

Table 2.

Phenotypic and Genotypic Characterization of Multiresistant Pseudomonas aeruginosa Clinical Isolates

Strain (PFGE pattern)	Clinical isolates antibiotic resistance	Antibiotics resistant/total	ESBL phenotype	ESBL genotype	MBL phenotype	MBL genotype
201(A)	AMK, AZL, AZT, CAR, CAZ, CEF, CIP, CTX, CTZ, FEP, IMP, LEV, MER, MEZ, NET, NOR, OFX, PIP, TIC, TIC/CLAV and TOB		−	GES-5	−	N.D.
201a(A)			−	GES-5	+	VIM-11
211(A)			−	GES-5	+	VIM-2 VIM-11
207(A2)			−	GES-5 OXA-2	−	N.D.
212(A2)		21/21	−	GES-5 OXA-2	+	VIM-2
203(B)			−	GES-5	+	VIM-11
B203(B)			+	GES-5	+	VIM-11
215(B)			−	GES-5	+	VIM-11
210(C)			−	GES-5	−	N.D.
204(B1)	AMK, AZL, CAR, CAZ, CEF, CIP, CTX, CTZ, FEP, IMP, LEV, MER, MEZ, NET, NOR, OFX, PIP, TIC, TIC/CLAV and TOB		+	GES-5 OXA-2	−	N.D.
		20/21
208(D)			+	GES-5 OXA-2	+	VIM-2 VIM-11
214(C1)	AMK, CTX, CTZ, IMP, NET, OFX	6/21	−	N.D.	−	N.D.
173(A1)	CEF, CTX, IMP, NET, TOB	5/21	−	N.D.	−	N.D.
206(C)	CEF, CTX, IMP, NET, TOB	5/21	−	N.D.	−	N.D.

The highlighted text indicates strains representing each of the four clonal groups.

N.D., not detected; PFGE, pulsed-field gel electrophoresis; ESBL, extended-spectrum β-lactamases; MBL, metallo-β-lactamase; AMK, amikacin; AZL, azlocillin; AZT, aztreonam; CAR, carbenicillin; CAZ, ceftazidime; CEF, cefoperazone; CIP, ciprofloxacin; CTX, ceftizoxime; CTZ, ceftriaxone; FEP, cefepime; IMP, imipenem; LEV, levofloxacin; MER, meropenem; MEZ, mezlocillin; NET, netilmicin; NOR, norfloxacin; OFX, ofloxacin; PIP, piperacillin; TIC, ticarcillin; TIC/CLAV, ticarcillin-clavulanate; TOB, tobramycin.

Characterization of acquired β-lactamases

Strains 212(A2), 204(B1), 206(C), and 208(D) were selected as representative of each PFGE clonal type to detect the production of β-lactamases by the IEF technique. Two bands with a pI of 5.8 and 7.7 suggested the presence of GES-5 and OXA-2 β-lactamases, respectively. Since no pI corresponding to MBLs could be observed, we decided to do specific MBL gene amplification on all the strains. The search was positive for the MBL gene bla_VIM for seven isolates, and each bla_VIM fragment was sequenced either directly or from recombinant E. coli clones. An analysis of the corresponding amino-acid sequences revealed the presence of VIM-2 and VIM-11 variants (Fig. 2). Interestingly, two strains showed both variants of VIM. Moreover, bla_GES-5 was detected by PCR and sequencing in 11 strains, as well as bla_OXA-2, which was present in four strains (Table 2).

FIG. 2.

Comparison of a 168 amino-acid region of the lactamase B superfamily domain of VIM from seven clinical isolates of P. aeruginosa. The reference sequences to compare with were VIM-2 (ACV91880) and VIM-11 (ABL61511). The amino-acid conservation identities are plotted to a VIM-2 as a dot, as well as by numbering. Reference and clinical isolates sequence titles are shown on the left. P. aeruginosa isolates present VIM-2 and VIM-11.

Characterization of the integrons involved

Only six strains of P. aeruginosa showed class 1 integrons, carrying mainly the gene cassettes encoding the resistance to aminoglycosides. However, bla_VIM was not detected. The bla_GES-5 and bla_OXA-2 genes detected in diverse integrons presented a different arrangement. The organization of integrons in 173(A1), 212(A2), 206(C), and 210(C) showed an identity with intI1 [integrase], aacA7 (GenBank accession number: AM988778) [resistant to aminoglycosides], and qacΔE1 [codifying resistance to quaternary ammonium salts]; the integron 204(B1) contained genes intI1, aadA6 (GenBank accession number: FJ752628), and qacΔE1; organization of the integron in 208(D) presented gene sequences corresponding to intI1, aacA4 (GenBank accession number: GQ202693), aadA7 (GenBank accession number: DQ899757), bla_OXA-2 [resistant to oxacillin], and qacΔE1 (Fig. 3).

FIG. 3.

Schematic representation of the class 1 integrons identified in strains of P. aeruginosa. (a) Organization of integrons in 173(A1), 212(A2), 206(C), and 210(C) showed an identity to intI1 (integrase), aacA7 (GenBank accession number: AM988778) (resistant to aminoglycosides), and qacΔE1 (codifying resistance to quaternary ammonium salts); (b) array of one integron in 204(B1) presented an identity to the following genes: intI1, aadA6 (GenBank accession number: FJ752628), and qacΔE1; (c) organization of the integron in 208(D) presented gene sequences corresponding to the following: intI1, aacA4 (GenBank accession number: GQ202693), aadA7 (GenBank accession number: DQ899757), bla_OXA-2 (resistant to oxacillin), and qacΔE1 (d) second integron in 204(B1) located in the recombinant plasmid pUC204B presented gene sequences corresponding to intI1, bla_GES-5, aadA (Gen Bank accession number: JN253504), and qacΔE1.

To study the genetic regions associated with the resistance, total DNA obtained from the isolates 173(A1), 212(A2), B203(B), 204(B1), 206(C), 210(C), and 208(D) was digested with BamHI and EcoRI and ligated to pUCP24 for cloning in E. coli; however, we only obtained the recombinants pUC203B and pUC204B, with both a DNA insert of ∼3.4 kb and an ESBL-positive MBL-negative phenotype (Table 3). The recombinant plasmids were used as templates for the search for class 1 integrons. After amplification and sequencing of pUC204B, a class 1 integron was identified as carrying genes intI1, bla_GES-5, aadA1, and qacΔE1.

Table 3.

Results of Resistance to Antibiotics and β-Lactamase Phenotype in the Recombinant Plasmids pUC204B and pUCB203B; and Strains with Native Plasmids 204PA, 208PA, and B203PA

		MIC (μg/ml) ^a
Receiver	Plasmid	CAZ	FEP	PIP	PIP-TZ	IMP	MER	CIP	COL	GEN	TOB	AMK	MBL	ESBL
Escherichia coli XL1-blue	No	0.5	0.25	1	1	0.125	0.016	0.064	0.064	0.25	0.38	0.75	−	−
	pUC204B	12	0.5	4	1	0.125	0.012	0.047	0.064	12	0.5	1	−	+
	pUCB203B	>256	2	8	2	0.25	0.094	0.032	0.064	8	3	4	−	+
Pseudomonas aeruginosa	Native plasmid
PAO1	No	2	1.5	3	2	1	0.25	0.094	1.5	2	0.75	3	−	−
	204PA	>256	48	48	24	1.5	0.38	0.125	2	4	32	64	−	+
	208PA	96	12	>256	128	1	0.5	0.094	2	1.5	1	3	−	+
	B203PA	>256	24	256	256	1.5	0.38	0.125	2	2	1	4	−	+

Cutoff values (μg/ml) for antibiotics: CAZ^R≥32; FEP^R≥32; PIP^R≥128; PIP-TZ^R≥128; IMP^R≥16; MEM^R≥16; CIP^R≥4; COL^R≥8; TOB^R≥8; AMK^R≥64. GEN was the selection marker.

MIC of CAZ, FEP, PIP, PIP-TZ, IMP, MER, CIP, COL, GEN, TOB, AMK, were determined by E-test.

MIC, minimal inhibitory concentration; PIP-TZ, piperacillin-tazobactam; CIP, ciprofloxacin; COL, colicistine; GEN, gentamicin.

bla_GES and bla_VIM distribution in MDR strains of P. aeruginosa

P. aeruginosa PAO1 was electroporated with native plasmids to obtain three recombinants B203PA, 204PA, and 208PA, which showed a positive phenotype for ESBL, and a negative phenotype for MBL, in addition to presenting resistance to ceftazidime and the transfer of other resistance markers, and susceptibility to imipenem (Table 3).

A Southern hybridization assay was performed to localize bla_GES in the chromosome and/or plasmid transferred to P. aeruginosa PAO1. Eleven strains previously positive by PCR to bla_GES-5 demonstrated a chromosomal location; in addition, the study on native plasmids obtained from B203(B), 204(B1), and 208(D) confirmed the plasmidic localization of the integrons. On the other hand, bla_VIM was found to have chromosomal localization in all the strains (Fig. 4).

FIG. 4.

Location of the genes of the lactamases VIM and GES in total and plasmid DNA in strains of P. aeruginosa. (a) Hybridization of bla_VIM in undigested plasmid DNA of P. aeruginosa, strains B203, 204, 208, and 211 (lanes 1–4) and total DNA undigested (lanes 6–9). Lane 5, 1 Kb plus DNA ladder (Invitrogen). The arrows indicate the positive signal for bla_VIM in the total DNA of the four strains. (b) Hybridization of bla_GES in the isolate 204 from total DNA (lane 1) and plasmid DNA (lane 2). The arrows indicate a positive signal for bla_GES.

Discussion

Typing of P. aeruginosa by PFGE led to the identification of four different types circulating in the Intensive Care Unit for a period of 3 months, showing the coexistence of different strains within a short period, which is similar to reports of outbreaks by MDR P. aeruginosa in other countries.⁸

A plasmid containing a class 1 integron with a cassette containing the bla_GES-5 sequence was isolated from the 204PA transformant clone type. In addition, in the same clinical strain-type source, 204(B1), a copy of this gene, was found in the chromosome, suggesting that bla_GES-5 could be found at first to be associated with a plasmid and subsequently integrated into the bacterial chromosome. The bla_GES-5 gene has been reported in South America²⁰ and Canada,²² thus the results presented here indicate an expansion of the presence of this gene to another country of the Americas.

The VIM-2 MBL was first described in P. aeruginosa in Europe by Poirel et al. in 2000²³ and since then, there have been reports in different regions of the world.³¹ In Mexico, the detection of the bla_VIM-2 gene in isolates of P. aeruginosa was documented in 2009¹¹ and 2010.²⁴ On the other hand, the VIM-11 MBL has undergone a very different distribution, because it was first described in Argentina by Pasteran et al. in 2005¹⁹ and to date, there is solely one additional report of this type of MBL in strains of P. aeruginosa collected in India.³ Interestingly, in two of the seven strains, more than one bla_VIM variant was observed, which included a bla_VIM-2 and a bla_VIM-11. To date, P. aeruginosa strains coproducing two variants of VIM β-lactamase have only been reported by Bogaerts et al. in 2008.²

The β-lactamase genes are mainly associated with class 1 integrons localized in plasmids or chromosomes. This study did not detect bla_VIM genes linked with integrons and plasmids; however, to rule out the presence of bla_VIM genes in these elements, it would be necessary to determine the regions flanking these genes in the P. aeruginosa strains studied. Nevertheless, and exceptionally, resistant cassettes could be found without a transmissible element, as has been shown by Toleman et al.,³¹ who demonstrated the absence of a transmissible element in upstream or downstream regions of the bla_SPM-1 gene.

The presence of bacteria carrying ß-lactamases that can cause outbreaks in hospitals in Mexico appears to be an emerging problem. In a previous study, Garza-Ramos et al.¹¹ reported the isolation of VIM-2 and OXA-2 β-lactamases in P. aeruginosa from the hospital environment. However, in this study, the simultaneous presence of the genes bla_OXA-2, bla_GES-5, bla_VIM-2, and bla_VIM-11 is shown. The isolates of this study were recovered in 2004, which is close to 2005 when Pasteran et al.¹⁹ recovered strains with the gene bla_VIM-11 in Argentina. The results indicate that β-lactamase-carrying bacteria have been circulating in Mexico since several years.

To our knowledge, this is the first study that reports the presence of bla_GES-5 in P. aeruginosa strains from Mexico and Central America, in addition to the particular finding that several P. aeruginosa clinical strains simultaneously produced bla_VIM-2 and/or bla_VIM-11.The coexistence of several and variable β-lactamases in a single strain is, to our knowledge, reported here for the first time, adding further complexity to the epidemiology of transferable resistance in P. aeruginosa. In addition, the detection of two VIM variants through the approach followed suggests the possibility that this circumstance might be more common than thought, because the detection of a VIM variant may mask the presence of other, additional β-lactamases. It is important to highlight the epidemiological risk posed by these strains, due to the potential spread of resistance genes, especially when these strains possess β-lactamases of a different family that can be transferred horizontally.

Footnotes

Acknowledgments

J.C.-V. received a graduate studies fellowship and support to carry out a research stay at Hospital Universitario Son Dureta, Palma de Mallorca, Spain, from CONACyT, México and I.P.N., México. R.M.R.-A. and G.A.-O. are recipients of COFAA-IPN and EDD-IPN fellowships. This work was supported by SIP-IPN-México grants 20091358 and 20101151. C.J. and A.O. are supported by the Ministerio de Ciencia e Innovación of Spain and Instituto de Salud Carlos III, through the Spanish Network for the Research in Infectious Diseases (REIPI C03/14 and RD06/0008).

Disclosure Statement

No competing financial interests exist.

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Unusual Diversity of Acquired β-lactamases in Multidrug-Resistant Pseudomonas aeruginosa Isolates in a Mexican Hospital

Abstract

Introduction

Materials and Methods

Biological material

Molecular typing and susceptibility to antibiotics

Detection, characterization, and cloning of acquired β-lactamases

Characterization of the involved integrons

Characterization of genetic elements carrying the transferable β-lactamases

Results

Molecular typing and susceptibility to antibiotics

Characterization of acquired β-lactamases

Characterization of the integrons involved

blaGES and blaVIM distribution in MDR strains of P. aeruginosa

Discussion

Footnotes

Acknowledgments

Disclosure Statement

References

bla_GES and bla_VIM distribution in MDR strains of P. aeruginosa