Providencia stuartii with VIM-1 metallo-β-lactamase

P. stuartii, carbapenems, resistance, MBLs

Sir,

Providencia stuartii is a frequent cause of urinary tract infections in hospitalized and nursing home patients with long-term indwelling urinary catheters. Also, this opportunistic pathogen is occasionally implicated in other types of hospital-acquired infections. Clinical strains of P. stuartii are commonly resistant to aminopenicillins and early generation cephalosporins due to production of an inherent cephalosporinase. Furthermore, they can acquire plasmids encoding class A extended-spectrum β-lactamases (ESBLs) such as TEM, CTX-M and VEB conferring resistance to newer cephalosporins.^1–3 In this study, a ceftazidime-resistant strain of P. stuartii producing VIM-1 metallo-β-lactamase (MBL) is described. bla_VIM-1 was carried by a self-transferable plasmid.

P. stuartii 323/07 was derived from a patient in the ICU of ‘Eugenidion’ General Hospital in Athens. The patient (male, 72 years old) was intubated due to severe ischaemic stroke of the left brain hemisphere and was requiring mechanical ventilation since 10 October 2006. He was transferred to our ICU on 19 October with a lung infection due to aspiration pneumonia. After being stabilized, a new septic event with fever, purulent secretions and haemodynamic instability emerged on 19 January 2007, while on treatment with meropenem and linezolid. Cultures of blood and bronchial secretions yielded both Pseudomonas aeruginosa and Acinetobacter baumannii, and antimicrobial treatment was switched to ceftazidime and colistin with no clinical improvement. A multiresistant but ciprofloxacin-susceptible strain of P. stuartii was isolated from bronchial secretions on 29 January and colistin was replaced by ciprofloxacin. The latter regimen was apparently effective, resulting in a significant clinical improvement within 1 week.

MICs of β-lactams were determined by an agar dilution technique. Susceptibility status to non-β-lactams drugs was assessed by a disc diffusion method. P. stuartii 323/07 exhibited resistance or decreased susceptibility to penicillins, penicillin/inhibitor combinations, ceftazidime and cefotaxime. MICs of carbapenems, aztreonam and cefepime were within the susceptibility range (Table 1). The strain was also resistant to gentamicin, tobramycin, netilmicin, co-trimoxazole, tetracycline and chloramphenicol. Amikacin and fluorinated quinolones were active against P. stuartii 323/07.

Table 1.

Antibiotic resistance phenotypes of P. stuartii 323/07 and an E. coli transconjugant clone

Antibiotic	MICs (mg/L) for
	P. stuartii 323/07	E. coli (pPS-v1)	E. coli K12
Ampicillin	>128	>128	2
Amoxicillin/CLAa	>128	>128	1
Piperacillin	64	64	0.5
Piperacillin/TAZb	32	32	0.5
Ceftazidime	64	>128	0.25
Ceftazidime/CLAa	64	—	—
Ceftazidime/Ro 48-1220c	64	—	—
Cefotaxime	32	64	<0.12
Cefepime	1	2	<0.12
Aztreonam	0.25	<0.12	<0.12
Imipenem	1	2	<0.12
Meropenem	0.5	0.5	<0.12

^aCLA, clavulanic acid (2 mg/L).

^bTAZ, tazobactam (4 mg/L).

^cInhibitor at a concentration of 4 mg/L.

The strain was negative for ESBL production in the double disc synergy test. Also, addition of either Ro 48-1220 or clavulanic acid did not reduce the MIC of ceftazidime (Table 1), indicating that the observed phenotype was not due to production of an ESBL or a cephalosporinase. However, P. stuartii 323/07 was positive in the double disc test based on synergy between imipenem and EDTA. PCR assays using total bacterial DNA as template and primers specific for bla_VIM-1 were positive. Isoelectric focusing of β-lactamases showed that P. stuartii 323/07 produced an enzyme with a pI of 5.1, similar to that of VIM-1. A β-lactamase with an apparent pI of 8.5 was also detected. This was probably the chromosomal AmpC of P. stuartii. Conjugal transfer of resistance to β-lactam antibiotics to an Escherichia coli K12 recipient strain highly resistant to rifampicin was carried out in mixed broth cultures and selection on media containing rifampicin and ampicillin. Resistance to β-lactams as well as aminoglycosides and co-trimoxazole was readily transferred at a frequency of 10⁻⁴ transconjugants per donor cell. E. coli transconjugants were positive for bla_VIM-1 by PCR. Agarose gel electrophoresis of plasmid DNA preparations from P. stuartii 323/07 and an E. coli transconjugant indicated transfer of a single plasmid (pPS-v1) of ∼60 kb that was positive for bla_VIM-1 in hybridization experiments performed as described previously.⁴ PCR mapping was carried out using combinations of oligonucleotide primers specific for the 5′ and 3′ conserved sequences of class 1 integrons as well as bla_VIM-1 and various resistance genes. These experiments showed that bla_VIM-1 was the first gene cassette of an integron that also contained—from 5′ to 3′—aacA4, dhfrI, aadA and sulI, similar to In-e541 described previously in Klebsiella pneumoniae and E. coli (GenBank accession no. AY339625).⁵ pPS-v1 belonged to the incompatibility (Inc) group N as determined by a PCR-based Inc-typing method. It also exhibited a PstI-generated restriction profile similar to that of a group of IncN bla_VIM-1-carrying plasmids that have been spread mainly among K. pneumoniae (data not shown).⁶

The higher resistance levels to various β-lactams, including imipenem, of the E. coli transconjugant clones when compared with P. stuartii 323/07 (Table 1) suggested differences in the production of VIM-1. To validate this hypothesis, hydrolytic activities of bacterial cell extracts against imipenem were determined by spectrophotometry. Extracts from E. coli (pPS-v1) expressed approximately 5-fold higher activity than P. stuartii 323/07 that was consistent with the β-lactam MIC differences. Additionally, transconjugants were resistant to amikacin, whereas P. stuartii 323/07 appeared susceptible to this drug, probably indicating differences also in the expression of the aacA4 gene cassette.

To the best of our knowledge, this is the first description of a P. stuartii strain producing a VIM-type MBL. This finding underscores the ongoing spread of VIM-1 in the flora of Greek hospitals as well as the important role of IncN, bla_VIM-1-carrying plasmids in this process.

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