Dissemination of a Multidrug-Resistant VIM-1- and CMY-99-Producing Proteus mirabilis Clone in Bulgaria

Abstract

The aim of this study was to analyze the beta-lactamases and the molecular epidemiology of 19 clinically significant isolates of Proteus mirabilis with decreased susceptibility to imipenem, which have been collected from seven hospitals, located in different Bulgarian towns (Sofia, Varna, and Pleven). The isolates were obtained from blood, urine, tracheal and wound specimens. One additional isolate from hospital environment was included. Susceptibility testing, conjugation experiments, and plasmid replicon typing were carried out. Beta-lactamases were characterized by isoelectric focusing, PCR, and sequencing. Clonal relatedness was investigated by RAPD and PFGE. Integron mapping was performed by PCR and sequencing. All isolates showed a multidrug-resistance profile, but remained susceptible to piperacillin/tazobactam, cefepime, meropenem, and fosfomycin. They produced identical beta-lactamases, namely: TEM-1, VIM-1, and CMY-99. PCR mapping revealed that the bla_VIM-1 gene was part of a class 1 integron that additionally included the aac(6′)-I, dhfrA1, and ant(3″)-Ia genes. In addition, 17 of the isolates carried the armA gene. Conjugation experiments and plasmid replicon typing were unsuccessful. The isolates were clonally related according to RAPD and PFGE typing. This study reveals the nationwide distribution of a multidrug-resistant P. mirabilis clone producing VIM-1 and CMY-99 along with the presence of different aminoglycoside resistance mechanisms.

Introduction

Proteus mirabilis isolates are responsible for a wide range of community- and hospital-acquired infections of the urinary tract, wounds, burns, abdomen, and the blood stream. Wild-type isolates lack intrinsic chromosomal beta-lactamase genes and are susceptible to many antibiotics except to tetracyclines, colistin, and nitrofurantoin.¹ However, antibiotic resistance due to the production of different acquired beta-lactamases including SHV-, TEM-, CTX-M-type ESBLs, and AmpC type enzymes have been increasingly reported in this species.^2–4

Acquired cephalosporinases of the AmpC type found in P. mirabilis isolates from different parts of the world (France, Italy, Poland, and Korea) include variants of the ACC-, CMY-, and DHA-lineages.^5–8 Representatives of the CMY-2/LAT-lineage (derived from the chromosomal AmpC enzyme of Citrobacter freundii) are the most common AmpC variants.^3,9 Production of AmpC-type enzymes is associated with resistance to penicillins, beta-lactam/beta–lactamase inhibitor combinations, and cephalosporins (except to cefepime and cefpirome).⁶

Furthermore, reports on P. mirabilis producing carbapenemases of various functional groups appeared within the last years. A VIM-type metallo-beta-lactamase in P. mirabilis was first described in Greece.¹⁰ Later, outbreaks with VIM-producing isolates were reported in Italy, Poland, and Spain.^3,9,11

Recently, P. mirabilis simultaneously producing VIM- and CMY-type enzymes were identified in Greece³ and in Bulgaria.¹² In Bulgaria, isolates of P. mirabilis with reduced susceptibility to carbapenems were isolated in different parts of the country within the last years.

The aim of this study was to investigate the clonal relatedness and the mechanisms of beta-lactam resistance in a collection of imipenem-nonsusceptible P. mirabilis isolates from seven hospitals in three Bulgarian towns.

Materials and Methods

Bacterial isolates

Between 2012 and 2015, seven hospitals in three Bulgarian towns were asked to collect nonduplicate clinical P. mirabilis isolates that are resistant or intermediate resistant to imipenem and/or meropenem. The number of P. mirabilis meeting those criteria respectively the number of all P. mirabilis were 4/60 for center 1 (Second Multiprofile Active Treatment Hospital, Sofia); 55/613 for center 2 (2013 only, Emergency Medical Institute “Pirogov,” Sofia); 2/45 for center 3 (Hospital “Torax,” Sofia); 5/717 for center 4 (Medical Institute - Ministry of the Interior, Sofia); 5/125 for center 5 (University Hospital “Ivan Rilski,” Sofia); 2/455 for center 6 (University Hospital; Varna); 1/437 for center 7 (University Hospital; Pleven).

The prevalence of imipenem-nonsusceptible P. mirabilis varied between 0.2% and 9% by center. Nineteen of those 74 isolates were sent for further investigation (Table 1). They had been isolated between February 2012 and July 2015 from blood, urine, tracheal and wound specimens of nonduplicate patients. An additional isolate (1053) recovered during infection control screening procedures of the hospital environment from the respiratory equipment, used for the patient from whom isolate 1051 was obtained, was included (Table 1). Two of the patients had been treated at the ambulatory units of the respective hospitals (center 3 and 4) (Table 1). Species identification was performed by routine biochemical identification procedures or API 20 E (bioMérieux, Marcy l′Etoile, France) or Crystal Enteric/NF identification system (Becton Dickinson, Sparks, MD).

Table 1.

Characteristics of Proteus mirabilis Isolates

Isolate	Hospital/ward	Date of isolation	Specimen	RAPD-type	PFGE	armA-PCR	MIC [mg/L] ^a
							AUG	TZP^b	CAZ	CTX	FEP	FOX	IMP	MER	TOB	GEN	AMK	CIP	LEV	SXT	CHL
567	1/ICU	02.2012	Tracheal lavage	A		Pos	>32	2	32	128	4	32	4	1	>128	>128	>64	>64	>8	>4/76	>32
568	1/INT	04.2012	Urine	A		Pos	>32	4	32	128	4	32	4	1	>128	>128	>64	>64	>8	>4/76	>32
569	1/INT	05.2013	Urine	A	a1	Pos	>32	4	16	64	2	32	4	1	>128	>128	>64	>64	>8	>4/76	>32
570	2/ICU	04.2013	Tracheal lavage	A		Pos	>32	4	32	128	4	32	4	1	>128	>128	>64	>64	>8	>4/76	>32
571	2/ICU	05.2013	Tracheal lavage	A	a1	Pos	>32	8	32	128	4	16	8	0.5	>128	>128	>64	>64	>8	>4/76	>32
572	2/Burn	05.2013	Wound	A		Pos	>32	4	32	128	4	16	4	1	>128	>128	>64	>64	>8	>4/76	>32
573	2/Burn	06.2013	Wound	A	a1	Pos	>32	2	32	128	2	32	2	1	>128	>128	>64	>64	>8	>4/76	>32
574	2/SG	12.2013	Bile	A		Pos	>32	4	32	128	2	32	4	1	>128	>128	>64	>64	>8	>4/76	>32
576	3/ICU	06.2013	Urine	A	a1	Pos	>32	2	32	128	8	16	4	1	>128	>128	>64	>64	>8	>4/76	>32
577	3/AMB	08.2013	Urine	A		Pos	>32	4	16	64	2	16	2	1	>128	>128	>64	>64	>8	>4/76	>32
679	6/PS	03.2014	Urine	A	a1	Neg	>32	4	16	64	1	16	2	1	16	1	4	>64	>8	>4/76	>32
795	7/U	04.2014	Urine	A		Pos	>32	8	32	128	4	16	4	1	>128	>128	>64	>64	>8	>4/76	>32
890	1/ICU	05.2014	Urine	A	a1	Neg	>32	8	32	128	4	32	2	0.5	16	2	4	>64	>8	>4/76	>32
1034	5/ICU	02.2015	Urine	A	a1	Neg	>32	2	16	64	4	16	2	1	8	1	2	>64	>8	>4/76	>32
1037	4/AMB	01.2015	Tracheal lavage	A	a2	Pos	>32	4	16	64	8	16	4	1	>128	>128	>64	>64	>8	>4/76	>32
1050	5/ICU	07.2015	Tracheal lavage	A	a2	Pos	>32	4	32	128	4	32	4	0.5	>128	>128	>64	>64	>8	>4/76	>32
1051	5/ICU	07.2015	Tracheal lavage	A		Pos	>32	4	32	128	4	32	4	1	>128	>128	>64	>64	>8	>4/76	>32
1052	5/ICU	07.2015	Tracheal lavage	A		Pos	>32	4	32	128	4	32	4	1	>128	>128	>64	>64	>8	>4/76	>32
1053	5/ICU	07.2015	Respiratory equipment	A		Pos	>32	4	32	128	4	32	4	1	>128	>128	>64	>64	>8	>4/76	>32
1054	5/ICU	07.2015	Blood	A		Pos	>32	4	32	128	4	32	4	1	>128	>128	>64	>64	>8	>4/76	>32

MIC values in bold are in the intermediate or resistant range.

Tazobactam was used at a fixed concentration of 4 mg/L.

ICU, intensive care unit; Burn, burn unit; INT, internal ward; SG, surgery; AMB, ambulatory care unit; PS, psychiatry; U, urology; AUG, amoxicillin-clavulanate; TZP, piperacillin-tazobactam; CAZ, ceftazidime; CTX, cefotaxime; FEP, cefepime; FOX, cefoxitin; IMP, imipenem; MER, meropenem; TOB, tobramycin; GEN, gentamicin; AMK, amikacin; CIP, ciprofloxacin; LEV, levofloxacin; SXT, sulfamethoxazole/trimethoprim; CHL, chloramphenicol.

Antimicrobial susceptibility testing and phenotypic carbapenemase screening

Minimal inhibitory concentrations (MICs) were determined by broth microdilution method and interpreted according to the CLSI guidelines.¹³ Susceptibility to fosfomycin was determined by disc diffusion method and the results were analyzed according to Pasteran et al.¹⁴

The phenotypic detection of carbapenemase production was performed using the modified Hodge test according to the CLSI guidelines and disk synergy tests with boronic acid, dipicolinic acid, EDTA, or cloxacillin in combination with imipenem and meropenem according to the EUCAST guidelines (www.eucast.org/resistance_mechanisms/) using the KPC/Metallo-beta-lactamase and OXA-48 Confirm Kit (ROSCO Diagnostica A/S, Taastrup, Denmark).

Isoelectric focusing and bioassay

Beta-lactamase production was analyzed by isoelectric focusing (IEF) as described previously.¹⁵ The hydrolytic activity of individual beta-lactamase bands was assessed by bioassay.¹⁶

Conjugation experiments and plasmid replicon typing

Conjugative plasmid transfer was performed on Mueller–Hinton agar using Escherichia coli K12:W₃₁₁₀ resistant to rifampicin as recipient strain. For the selection of transconjugants MacConkey agar containing 2 mg/L ceftazidime or cefotaxime or 0.5 mg/L meropenem and 50 mg/L rifampicin was prepared.

The isolates were analyzed for the presence of the following plasmid replicon types according to the scheme of Carattoli et al: FIA, FIB, FIC, HI1, HI2, I1-Iγ, L/M, N, P, W, T, A/C, K, B/O, X, Y, F, FIIA, U, and R.^17,18

Molecular analyses

PCR was used to screen for bla_TEM, bla_SHV, bla_CTX-M,¹⁹ bla_CMY-2-like,¹² bla_VIM, bla_IMP, bla_KPC, bla_NDM, and bla_OXA-48 genes.²⁰ Sequencing of PCR-products was performed with primers binding outside the coding regions^12,19 at Eurofins MWG operon (Ebersberg, Germany). The nucleotide and deduced amino acid sequences were analyzed and multiple alignments were performed using Chromas Lite 2.01 (Technelysium Pty Ltd., Brisbane, Australia) and DNAMAN version 8.0 Software (Lynnon BioSoft, Vaudreuil-Dorion, Canada).

Whole-cell DNA was prepared using the DNA Swab Isolation Spin Kit (AppliChem GmbH, Darmstadt, Germany) and used in random amplified polymorphic DNA analysis (RAPD) with RAPD primer 5 and Ready-To-Go-RAPD Analysis Kit (GE Healthcare, Little Chalfont, United Kingdom). PFGE typing was performed for nine isolates representing different centers and resisto-types with the Chef Genomic DNA Plug Kit (BioRad Laboratories, Hercules, CA), and macrorestriction with NotI (New England BioLabs, Inc., Ipswich, MA). PFGE was carried out using the CHEF Gene Navigator PFGE apparatus (Pharmacia-LKB, Uppsala, Sweden) with the following running conditions: 170V, pulse with switch time from 2.2 to 54.2 seconds for 22 hours, 14°C. The clonal relatedness was evaluated according to Tenover et al.²¹

The armA, rmtB, and rmtC genes were detected by multiplex PCR as described previously by Doi and Arakawa.²²

Detection and mapping of class 1 integron were carried out by PCR combining primers binding to conserved 5′-CS and 3′-CS sequences and primers specific for bla_VIM, dhfrI, and aadA genes as described previously.^23,24 After amplification, the segments obtained from five isolates were sequenced and analyzed with DNAMAN version 8.0 Software (Lynnon BioSoft, Vaudreuil-Dorion, Canada) and the Basic Local Alignment Search Tool of the National Center for Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

Results

Antimicrobial susceptibility

Results of antimicrobial susceptibility testing are shown in Table 1. All isolates were resistant or intermediate resistant to amoxicillin/clavulanic acid, ceftazidime, cefotaxime, cefoxitin, imipenem, tobramycin, ciprofloxacin, levofloxacin, sulfamethoxazole/thrimethoprim, and chloramphenicol. All isolates were susceptible to piperacillin/tazobactam, cefepime, meropenem, and fosfomycin and only three of them to gentamicin and amikacin.

Phenotypic carbapenemase screening

The modified Hodge test, the imipenem-EDTA synergy test, and the meropenem-dipicolinic acid synergy test were positive, suggesting the production of a metallo-beta-lactamase. The cloxacillin combination disk had inhibitory activity in all the isolates suggesting the presence of class C enzymes.

IEF and bioassay

IEF showed the presence of three bands at isoelectric points (pIs) of 5.1, 5.4, and 9.2 (data not shown). The beta-lactamase with the pI of 5.4 showed no cefotaxime or imipenem hydrolyzing activity. For the beta-lactamase focusing at a pI of 5.1 weak hydrolysis of imipenem could be observed. The enzyme with pI of 9.2 displayed cefotaxime hydrolysis.

Conjugation experiments and replicon typing

Attempts to transfer the beta-lactam resistance determinants by conjugation and to identify plasmid replicons by PCR-based replicon typing were unsuccessful.

Molecular analyses

All isolates demonstrated positive PCR-results with CMY-2-group-, VIM-, TEM-, and SHV-primers. Sequencing of PCR-products revealed the presence of bla_TEM-1, bla_VIM-1, and bla_CMY-99 corresponding to the enzymes with the pIs of 5.4, 5.1, and 9.2, respectively. The SHV-gene found was identified as bla_SHV-12, however, no corresponding beta-lactamase could be detected by IEF.

For the 17 isolates resistant to gentamicin and amikacin, the presence of the 16S rRNA methylase (armA) gene was shown, in contrast to the three isolates that were susceptible to gentamicin and amikacin. Genes encoding the methylases RmtB and RmtC were not detected.

For all isolates RAPD typing revealed identical RAPD patterns. The clonality was confirmed by PFGE, showing highly similar DNA-fingerprints with one band difference forming two subclones (Table 1). Furthermore, it was shown that one of the previously reported VIM-1- and CMY-99-producing P. mirabilis isolates (915) recovered in 2011 in the Alexandrovska University hospital in Sofia¹² showed a PFGE pattern identical to the “a1” pattern of this study. The integron mapping revealed a common class 1 integron with the bla_VIM-1 gene inserted as the first cassette at the 5′-CS site after the aatI1 recombination site. The gene cassette order downstream of bla_VIM-1 gene was as follows: cassettes encoding for aminoglycoside acetyltransferase of the AAC (6′)-I group, dihydrofolate reductase DHFRA1, and aminoglycoside nucleotidyltransferase ANT (3″)-Ia. Comparison with database entries revealed identical integron structures (JQ690541, GU724870)^25,26 in Greek P. mirabilis isolates and high similarity with an integron named In-e541 found on a plasmid of a Greek Klebsiella pneumoniae isolate (GU585907) by Miriagou et al.²⁷

Discussion

P. mirabilis is one of the commonly isolated species from the family Enterobacteriaceae and an etiological agent of a wide range of community acquired and nosocomial infections, for example, urinary tract, skin and soft tissue, abdominal, and bloodstream infections. This study reveals the prolonged persistence of multidrug-resistant, clonal P. mirabilis isolates in Bulgaria. All the isolates produced TEM-1, VIM-1, and CMY-99. The bla_VIM-1 gene was part of a class 1 integron containing further antibiotic resistance genes.

The first report on a CMY-enzyme produced by P. mirabilis dates back to 1998.²⁸ Since then CMY-producing P. mirabilis have been predominantly reported from Europe; particularly with CMY-2 in Spain,⁹ CMY-16 in Greece and Italy, and various variants (CMY-4, -12, -14, -15, -38, -45) in Poland.³

VIM-producing P. mirabilis have been detected for the first time in Greece, where they soon were distributed endemically.^10,11 Later on they also appeared in Italy and Spain.^3,9,29 Up to now there are three reports on the coproduction of CMY- and VIM-enzymes in P. mirabilis. Two of them describe isolates from Greece^3,30 producing the beta-lactamases TEM-1, VIM-1, and CMY-16. This is a combination very similar to that found in this study, namely TEM-1, VIM-1, and CMY-99, which has already been described for three Bulgarian isolates recovered in 2011 and 2012.¹² CMY-99 is closely related to CMY-16 differing by only one amino acid exchange.

In this study the bla_VIM-1 gene was the first gene cassette located in a class 1 integron followed by cassettes encoding for AAC (6′)-I, DHFRA1, and ANT (3″)-Ia. Comparison of our sequences with database entries revealed identical integron structures with that described by Papagiannitsis et al.²⁵ (JQ690541) and Drieux et al.²⁶ (GU724870) in P. mirabilis isolates from Greece respectively from a patient, which had been transferred from a Greek to a French hospital. Both authors found the VIM-1-harboring integron structure located on self-transmissible plasmids. In contrast, Vourli et al.¹⁰ and Tsakris et al.¹¹ found identical structures (no sequence database entries available) to be chromosomally located. We may suppose that the Bulgarian P. mirabilis clone could be a result of further diversification of the Greek isolates with identical VIM-1-encoding class I integron structure and closely related CMY-variants. However, this remains to be elucidated.

This study documents the capability of the detected P. mirabilis clone to persist for years within the healthcare settings of the country. The first appearance of representatives of this clone has been previously reported¹² among patients hospitalized in the Alexandrovska University hospital in Sofia. The first patient of this study (isolate 567) was transferred from the Alexandrovska University hospital to the center 1. Later identical isolates appeared sporadically in five centers in Sofia and two centers in Pleven and Varna. At the end of the investigation period isolates belonging to this clone were detected in the neurosurgery intensive care unit in center 5. There, identical P. mirabilis strains were isolated from the tracheal aspirates of three patients. Microbiological investigation of the hospital environment recovered one P. mirabilis isolate from a respiratory equipment. We have no epidemiological data explaining the route of transmission of the strains between the hospitals. According to Tsakris et al., the most sufficient route for dissemination of the VIM-producing P. mirabilis in Greece could be prolonged carriage after hospitalization.¹¹ These authors reported sporadic isolation of clonal VIM-1-producing P. mirabilis in the community. Persisting reservoirs in hospitals or the community may play a further role in ongoing occurrence of the VIM-1-producing isolates in Bulgaria. Our results suggest a great potential for dissemination and persistence of this clone over time. During the preparation of the article, four additional P. mirabilis isolates with the same resisto-type and positive PCR results for CMY- and VIM-group enzymes, two in center 4, one in center 5, and one in center 6, have been detected, showing their continuing presence.

Treatment of infections caused by isolates of this clone is very problematic due to their multidrug resistance profile. Only the MICs for piperacillin/tazobactam, cefepime, meropenem, and fosfomycin were in the “susceptible” range for all isolates. Some authors suggest a carbapenem (preferentially meropenem) combined with a suitable aminoglycoside as the first choice for primary and secondary blood stream infections caused by carbapenemase-producing isolates, in case the meropenem MIC of the infecting isolate is ≤4 mg/L.³¹ This rule is widely accepted, but in case of P. mirabilis blood stream infection some authors reported treatment failure using meropenem even if the MICs indicated susceptibility.³² They report a successful usage of amikacin, but in combination with cefepime in a case of P. mirabilis bacteremia. In this study the patient with positive blood culture was treated successfully with piperacillin/tazobactam as verified by negative blood cultures 1 month later. Furthermore, most of the investigated isolates additionally produced the ArmA 16S rRNA methylase conferring high level resistance to all aminoglycosides, which excludes amikacin as a treatment option. Three of our isolates lacking the ArmA 16S rRNA methylase remained susceptible to amikacin and gentamicin. However, taking into account the presence of a gene encoding the aminoglycoside acetyltransferase AAC (6′)-I, which affects tobramycin and amikacin and the EUCAST expert rules³³ it should be recommended to report the isolate as intermediate to amikacin.

Similar to several reports,^3,10,29,30 the Bulgarian P. mirabilis isolates showed, despite the production of VIM-1, relatively low carbapenem MIC values, with meropenem MICs of 0.5 to 1 mg/L in the susceptible range and imipenem MICs classified as intermediately resistant remaining near the cutoff. This underlines the possible difficulties in detection of such isolates.

Our results demonstrate the persistence of clonally related, multidrug-resistant P. mirabilis isolates over a period of 4 years in Bulgarian healthcare settings. The multidrug-resistant phenotype of the isolates and the location of some of the identified resistance markers on genetic elements, which may be inserted both in the chromosome and in plasmids, which, in turn, are transferrable to other enterobacterial species, indicate the high risk of further spread of those resistance determinants and the need of implementing intervention strategies.

Dedication

The work is dedicated to the memory of Adolf Bauernfeind, who passed away recently.

Footnotes

Disclosure Statement

The authors declare no conflicts of interest.

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