Nursing Home as a Reservoir of Carbapenem-Resistant Acinetobacter baumannii

Abstract

Acinetobacter baumannii is an increasingly common pathogen in healthcare settings globally. It is frequently resistant to multiple antimicrobial agents and there are recent reports on strains that are pandrug resistant. The aim of the study was to characterize the mechanisms of carbapenem resistance of A. baumannii strains from a nursing home in Zagreb and to genotype the strains. Antimicrobial susceptibility testing was performed by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI). PCR was used to detect genes encoding carbapenemases of groups A, B, and D and extended-spectrum β-lactamases. Genotyping of the strains was performed by rep-PCR. All strains were found to be resistant to ceftazidime, cefotaxime, ceftriaxone, cefepime, piperacillin/tazobactam, imipenem, meropenem, and ciprofloxacin. All, but one strain, were resistant to gentamicin. PCR revealed bla_OXA-23 genes in 14, bla_OXA-24 in 5, and bla_VIM in 11 strains. All strains positive for bla_V_IM genes coharbored bla_OXA-23 genes. The 14 strains with OXA-23 belonged to ICL II, whereas the 5 strains positive for bla_OXA-24 belonged to ICL I. In contrast to hospitals where OXA-24/40-like β-lactamases and OXA-58 were the most prevalent, OXA-23-like β-lactamases are the dominant group in the nursing home. OXA-58-like β-lactamase, which is the most widespread group, was not found. Acquisition of bla_MBL genes in A. baumannii strains was observed. Rep-PCR identified two clones. Two strains A10 and A13 were alocated to a novel sequence type ST 637. Nursing homes can act as a source of dissemination of bla_OXA and bla_MBL genes in the environment and the possible influx to the hospital environment.

Introduction

Acinetobacter baumannii is an increasingly common pathogen in healthcare settings globally.⁵⁸ It can infect the respiratory tract, blood, soft tissues, urinary tract, and central nervous system.⁴⁰ These organisms are frequently resistant to multiple antimicrobial agents, and there are recent reports on strains that are pandrug resistant.⁴⁵ Genotypic characterization showed that clusters of highly similar strains occurred, which are designated as International clonal lineages (ICL) I, II, and III.⁷³ Recently, more lineages have been reported. Carbapenem-resistant strains have been reported worldwide.⁷³ Resistance to carbapenems can be due to impaired permeability or alternation in penicillin-binding proteins.^35,51 However, β-lactamase-mediated resistance is the most common mechanism of carbapenem resistance in this species. Carbapenemases found in Acinetobacter belong to the molecular Ambler class A (KPC),⁵⁵ class B (metallo-β-lactamases [MBLs] of IMP, VIM, SIM, or NDM family),^12,28,33,50 or class D (OXA enzymes) recently known as CHDL (carbapenem-hydrolyzing class D oxacillinases).^9,24,26 OXA enzymes of Acinetobacter are divided into five phylogenetic groups: acquired enzymes OXA-23-like, OXA-24/40-like, OXA-58-like, OXA-143-like, and OXA-51-like enzymes, which are intrinsic in A. baumannii.^9,24,26 Enzymes belonging to the OXA-51 group are naturally occurring β-lactamases of A. baumannii and are normally expressed at low levels, but can be overexpressed as a consequence of the ISAba1 location upstream of the genes.^24,57 ISAba1 can also be located upstream of the other bla_OXA genes and promotes their expression.⁵⁷ Recently, extended-spectrum β-lactamases (ESBLs), such as PER, VEB, IBC, SHV, or CTX-M enzymes, which confer resistance to expanded-spectrum cephalosporins, have been reported in A. baumannii.^6,7,44,48,52 The first outbreak of carbapenem-resistant isolates in Croatia has been documented in the Split University Hospital.²⁰ The analysis of carbapenem resistance mechanisms found that carbapenem resistance was mediated by hyperproduction of OXA-51 due to the ISAba1 location upstream of the genes.²¹ Later, the studies of carbapenem resistance in Croatia have reported the occurrence of OXA-72-producing isolates in the Split and Zagreb University Hospitals.^18,22 In 2008 and 2009, carbapenem-resistant isolates of A. baumannii emerged with increasing frequency in most hospital centers in Croatia and became an important public health problem. These observations gave rise to a multicenter study conducted in northern Croatia and Istria in 2009–2010. The study found OXA-24-like and OXA-58-like enzymes to be the dominant mechanism of carbapenem resistance in Croatia.⁶⁶ In 2013, the emergence of carbapenem-resistant A. baumannii was observed in a nursing home in Zagreb (Godan). Nursing homes are known to be an important reservoir of multiresistant bacteria, including methicillin-resistant Staphylococcus aureus, ESBL, and AmpC-producing organisms.^40,58 The aim of the study was to characterize the mechanisms of carbapenem resistance of A. baumannii strains from a nursing home and to genotype the isolates.

Materials and Methods

Bacterial isolates

Nineteen isolates resistant to carbapenems were collected in the Godan nursing home in Zagreb from various clinical specimens from March 1, 2013, till April 24, 2014. The isolates were identified by conventional biochemical testing and the Vitek2 (bioMerieux) automated system.

Antimicrobial susceptibility testing

The antimicrobial susceptibility to a wide range of antibiotics (piperacillin alone and combined with tazobactam, sulbactam/ampicillin, ceftazidime, cefotaxime, ceftriaxone, cefepime, imipenem, meropenem, gentamicin, amikacin, ciprofloxacin, and colistin) was determined by the broth microdilution method in Mueller-Hinton broth in 96-well microtiter plates according to the Clinical and Laboratory Standards Institute (CLSI) guidelines.¹⁰ Pseudomonas aeruginosa ATCC 27853 was used as the quality control strain. Minimum inhibitory concentrations (MICs) of imipenem and meropenem were determined also by agar dilution with addition of cloxacillin (200 mg/L) and sodium chloride to determine the effect of chromosomal AmpC β-lactamase on the susceptibility to carbapenems and to detect the OXA-58 group, which is susceptible to inhibition with sodium chloride, respectively.⁵³ The susceptibility to tigecycline was determined by Vitek and by the E-test. Isolates were defined as multidrug resistant (MDR) according to Magiorakos et al.³⁹

E-test MBL strips (AB Biodisk) and the combined disk test with EDTA were used for detection of MBL.^2,67 ESBLs were detected by the combined disk test with cephalosporins and clavulanic acid according to CLSI with addition of cloxacillin in the medium (200 mg/L)¹⁰ to inhibit the chromosomal AmpC β-lactamase, which can antagonize the synergistic effect with clavulanate.

CarbAcineto NP test

CarbAcineto NP test was performed to determine hydrolysis of imipenem as described previously.¹⁶

Molecular characterization of β-lactamases

The presence of the genes encoding KPC,⁵⁶ MBLs of IMP, VIM, SIM, and NDM series,^12,32,33,72 OXA β-lactamase (bla_OXA-51-like, bla_OXA-23-like, bla_{OXA-24/40-like}, bla_OXA-58-like, and bla_OXA-143-like) genes,⁷⁰ and ESBLs of TEM, SHV, CTX-M, PER, and GES family^{3,6,7,44,46,47,48,52} was determined by PCR as previously described. The strain positive for CTX-M β-lactamases was subjected to PCR with primers specific for groups 1, 2, 8, 9, and 25.⁷¹ The amplicons of the selected representative strains were column purified (QIAquick PCR purification kit; Inel Medicinska Tehnika) and subjected to sequencing in the Macrogen sequencing service (South Korea) with the same primers used for PCR to determine the identity of the enzyme. Sequence alignment analysis was done online by utilizing the BLAST Program (www.ncbi.nlm.nih.gov). Reference strains producing OXA-23-like, OXA-24/40-like, OXA-58-like, and OXA-143-like β-lactamases were used as positive control strains for PCR. The strains were kindly provided by Dr. Paul Higgins, University of Cologne, Germany. The reference strains positive for CTX-M-15, CTX-M-2, and CTX-M-9 were provided by Dr. Neil Woodford (Health Protection Agency, London, UK), while reference strains positive for SHV-1 and TEM-1 were obtained by Prof. Adolf Bauernfeind (Max von Pettenkofer Institute, Munich, Germany). The genetic context of bla_OXA-51, bla_OXA-23, and bla_OXA-24 genes was determined by PCR mapping with primers for ISAba1 combined with forward and reverse primers for bla_OXA-51, bla_OXA-23, and bla_OXA-24 as described previously.⁶⁴ Primers for conserved sequences 5′-CS and 3′-CS were used to amplify class 1 integron.

Characterization of plasmids

To determine if acquired oxacillinase genes were plasmid encoded, plasmid DNA of the strains with acquired oxacillinases (Table 1) was extracted with the Macherey-Nagel extraction minikit (Macherey Nagel) according to the manufacturer's instructions. The incompatibility group was determined by PCR-based replicon typing (PBRT) according to Bertini et al.⁵

Table 1.

Antibiotic Susceptibility, Phenotypic Tests for Detection of ESBLs and MBLs, β-Lactamase Content, and Sequence Groups of Acinetobacter Baumannii Strains from the Nursing Home

				Phenotypic tests					MIC (mg/L)
Strain no.	Protocol no.	Specimen	Date of isolation	ESBL test	MBL test	β-lactamase content	SG/ICL	ISAba/bla_OXA-51	CAZ	CTX	CRO	FEP	SAM	TZP	IMI	MEM	GM	AMI	CIP	COL	TIG
2	148205	Urine	3.9.2013	−	+	OXA-23, OXA-51	1 (II)	+	>128	>128	>128	>128	4	>128	32	32	16	4	32	1	0.5
3	157652	Urine	9.9.2013	−	+	OXA-23, OXA-51	1 (II)	+	>128	>128	>128	>128	4	>128	32	32	16	1	64	0.5	0.5
4	152622	Wound swab	10.09.2013	−	+	OXA-24, OXA-51,	2 (I)	+	>128	>128	>128	>128	2	>128	>128	>128	2	4	32	0.12	1
5	157195	Wound swab	18.9.2013	−	+	OXA-23, OXA-51, VIM	1 (II)	+	>128	>128	>128	>128	32	>128	64	>128	64	16	>128	1	4
						TEM-1
6	172547	Urine	14.10.2013	−	+	OXA-23, OXA-51, VIM	1 (II)	−	>128	>128	>128	>128	64	>128	64	>128	>128	32	64	2	4
						TEM-1
7	196698	Urine (catheter)	4.07.2013	−	+	OXA-51, OXA-24	2 (I)	+	>128	>128	>128	>128	>128	>128	>128	>128	32	32	64	1	1
8	114270	Wound swab	01.07.2013	−	+	OXA-51, OXA-24	2 (I)	+	>128	>128	>128	>128	8	>128	>128	>128	32	64	>128	0.12	1
10	39061	Urine (catheter)	1.3.2013	−	+	OXA-24, OXA-51	2 (I)	+	>128	>128	>128	>128	4	>128	>128	>128	32	8	>128	1	2
11	077158	Urine	20.4.2013	−	+	OXA-23, OXA-51, VIM	1 (II)	+	>128	>128	>128	>128	64	>128	64	>128	64	16	>128	0.5	4
						TEM-1
12	143984	Urine (catheter)	26.08.2013	−	+	OXA-23, OXA-51, VIM	1 (II)	+	>128	>128	>128	>128	16	>128	64	>128	64	16	64	1	4
						TEM-1, CTX-M-15
13	042639	Tracheal aspirate	06.03.2013	−	+	OXA-51, OXA-24	2 (I)	+	>128	>128	>128	>128	4	>128	>128	>128	>128	16	>128	0.5	1
14	198050	Wound swab	25.11.2013	−	+	OXA-23, OXA-51, VIM	1 (II)	+	>128	>128	>128	>128	64	>128	>128	>128	64	64	32	0.5	4
						TEM-1
15	206518	Wound swab	12.12.2013	−	−	OXA-23, OXA-51	1 (II)	+	>128	>128	>128	>128	>128	>128	64	64	>128	64	16	0.12	4
						TEM-1
17	213516	Urine	12.12.2013	−	+	OXA-23, OXA-51, VIM, TEM-1	1 (II)	+	>128	>128	>128	>128	32	>128	64	64	>128	4	>128	0.25	2
18	212264	Nasal swab	18.12.2013	−	+	OXA-23, OXA-51, VIM, TEM-1	1 (II)	+	>128	>128	>128	>128	32	>128	64	64	>128	8	>128	0.25	2
19	2902	Urine	08.01.2014	−	+	OXA-23, OXA-51, VIM	1 (II)	+	>128	>128	>128	>128	32	>128	>128	>128	>128	8	>128	0.25	4
20	9434	Urine	24.01.2014	−	+	OXA-23, OXA-51, VIM, TEM-1	1 (II)	+	>128	>128	>128	>128	64	>128	>128	>128	>128	32	>128	0.12	4
21	55026	Urine	4.4.2014	−	+	OXA-23, OXA-51, VIM, TEM-1	1 (II)	+	>128	>128	>128	>128	64	>128	>128	>128	>128	32	64	0.12	2
22	65503	Urine	24.4.2014	−	+	OXA-23, OXA-51, VIM, TEM-1	1 (II)	+	>128	>128	>128	>128	32	>128	>128	>128	>128	16	32	0.12	4

AMI, amikacin; CAZ, ceftazidime; CIP, ciprofloxacin; COL, colistin; CRO, ceftriaxone; CTX, cefotaxime; ESBL, extended-spectrum β-lactamases; FEP, cefepime; GM, gentamicin; ICL, international clonal lineage; IMI, imipenem; MBL, metallo-β-lactamases; MEM, meropenem; MIC, minimum inhibitory concentration; SAM, sulbactam/ampicillin; SG, sequence group; TG, tigecycline; TZP, piperacillin/tazobactam.

Molecular typing of isolates

Sequence groups (SGs 1–3) corresponding to ICL I–III determination were performed according to the procedure described by Turton et al.⁶³

Isolates were further investigated by rep-PCR (DiversiLab System bioMerieux) following the manufacturer's instructions. Amplified products were loaded into microfluidic chips and electrophoresis performed in the Agilent 2100 Bioanalyzer (Agilent Technologies). The resulting virtual gels were analyzed with the DiversiLab Software using the modified Kullback–Leibler statistical method to determine distance matrices and the unweighted pair-group method with arithmetic averages (UPGMA) to create dendrograms. Isolates that clustered above 95% were considered related.²³

Pulsed-field genotyping of ApaI-digested genomic DNA was performed on 18 strains with a CHEF-DRIII system (Bio-Rad)³⁰; the images were processed using the Gel-Compar software, and a dendrogram was computed after band intensity correlation using global alignment with 1.5% optimization and tolerance and UPGMA clustering. The strains were considered to be clonally related if they showed more than 80% similarity of their pulsed-field gel electrophoresis (PFGE) patterns.⁶⁰ Selected strains (A2, A5, A10 and A13) were subjected to multilocus sequence typing (MLST) according to Diancourt.¹⁴

Results

Bacterial isolates

Twelve isolates originated from urine samples, including catheters, five from wound swabs, and one from a tracheal aspirate and nasal swab, respectively. All patients were previously hospitalized in one of the hospital centers in Zagreb.

Susceptibility testing

All isolates were found to be resistant to ceftazidime, cefotaxime, ceftriaxone, cefepime, piperacillin/tazobactam, imipenem, meropenem, and ciprofloxacin as shown in Table 1. Only one strain was susceptible to gentamicin. A high resistance rate was observed for sulbactam/ampicillin (68%, 13/19). Amikacin preserved good activity with 73% of the susceptible strain (12/19). No resistance to colistin was observed. Addition of either cloxacillin or sodium chloride in the medium did not lower the MICs of carbapenems, indicating that chromosomal AmpC β-lactamases were not responsible for elevated MICs of carbapenems and pointing to the absence of the OXA-58 group. Colistin was the most potent antibiotic with MIC₉₀ of 1 mg/L. MICs of tigecycline ranged from 0.5 to 4 mg/L with Vitek testing, indicating susceptibility according to CLSI (resistance breakpoint 16 mg/L). EUCAST does not have breakpoint values for tigecycline for A. baumannii. The E-test yielded slightly lower MIC values confirming susceptibility. All A. baumannii isolates showed the MDR phenotype since they were resistant to at least one agent in more than three antimicrobial classes (Table 1).

Detection and characterization of β-lactamases

The combined disk test with addition of clavulanic acid revealed no production of ESBL as shown in Table 1. The E-test and combined disk test with EDTA were positive in 18 strains, indicating production of MBL. PCR identified the bla_OXA-23 genes in 14, bla_OXA-24 in 5, and bla_VIM in 11 strains as shown in Table 1. Seven strains positive in the combined disk test with EDTA, but negative for bla_MBL genes (strains 2, 3, 4, 7, 8, 10, 13), were subjected to the CarbAcineto NP test to determine the hydrolysis of imipenem. The test was positive in five of the seven strains (4, 7, 8, 10, 13). Strains positive for bla_V_IM genes coharbored bla_OXA-23 genes. Sequencing of bla_OXA-23 and bla_OXA-24 amplicons from representative strains revealed the presence of bla_OXA-23 (strain 2) and bla_OXA-72 genes (strain 4). ISAba1 was found upstream of bla_OXA-51 genes. It was also associated with bla_OXA-23, but not with bla_OXA-24 genes. Bla_CTX-M-15 and bla_TEM-1 genes were found in 1 and 11 strains, respectively, as shown in Table 1. Attempts to amplify class 1 integron were unsuccessful.

Characterization of plasmids

Electrophoresis of plasmid extractions revealed no visible before and after digestion with EcoRI endunuclease. PBRT was positive for the group 2 plasmid (aci2-replicase gene), which was previously found on pACICU1 in all strains, and additionally OXA-24/40-positive strains harbored plasmids belonging to group 6 (aci6-replicase gene) originally found on pACICU2.

Genotyping

Fourteen strains with OXA-23 belonged to ICL II (SG 1), whereas five strains positive for bla_OXA-24 belonged to ICL I (SG 2).

Rep-PCR identified two clusters: one large cluster comprising 13 strains positive for OXA-23 with additional VIM in the majority of strains (strains 2, 3, 5, 6, 12, 14, 15, 17, 18, 19, 20, 21, 22) belonging to ICL II and one small cluster containing strains positive for OXA-24 (strains 4, 7, 8, 10) belonging to ICL I. Two strains were singletons (strain 11 and strain 13) as shown in Fig. 1. PFGE confirmed the existence of two clones and two singleton isolates identified by rep-PCR as shown in Fig. 2. The strains A2 and A5 were found to belong to ST487 while strains A10 and A13 were alocated to a novel sequence type ST637.

FIG. 1.

Rep-PCR dendrogram of Acinetobacter baumannii strains. Date of isolation and specimen type are shown. Similarity threshold of 97% was applied to define a clone (no difference in banding pattern).

FIG. 2.

Pulsed-field gel electrophoresis dendrogram of A. baumannii strains. Date of isolation and specimen type are shown. Similarity threshold of 80% was applied to define a clone (difference in maximum three bands).

Discussion

The study found the nursing home to be an important reservoir of colistin-only susceptible isolates of A. baumannii. Nineteen isolates from a nursing home in Zagreb showed a high level of resistance to carbapenems associated with production of either OXA-23-like or OXA-24-like β-lactamases combined with MBLs in some strains. In contrast to hospitals where OXA-24/40-like and OXA-58-like β-lactamases were the most prevalent, OXA-23 like β-lactamases are the dominant group of CHDL in the nursing homes. OXA-58-like β-lactamases, which are the most widespread of CHDL in hospitals, were not found. Aquistion of bla_MBL genes in A. baumannii strains was observed. Nursing homes can act as a source of dissemination of bla_OXA genes in the environment and the possible influx to the hospital environment. The majority of strains were resistant to all other antibiotics, including sulbactam/ampicillin. The CarbAcineto NP test was positive in five of the seven strains phenotypically positive for MBLs, but yielding no PCR products with primers specific for MBLs, indicating hydroysis of imipenem. The possible explanation is production of a novel type of MBL. Two strains with a negative CarbAcineto NP test probably gave a false-positive result in the inhibitor-based test. Similar observation of the false-positive MBL test in strains positive for CHDL was previously reported by Vilalon et al.⁶⁵ The possible explanation is that, in the presence of EDTA, oxacillinase changes to a less active state, leading to a drastic reduction in MIC or augmentation of the inhibition zone.⁶

OXA-23 β-lactamase was found in a majority of isolates combined with VIM MBL. OXA-23 was the first carbapenem-hydrolyzing oxacillinase reported in 1986, in the United Kingdom, as ARI-1.⁴⁹ Later, it was renamed to OXA-23. It spread all over the world, but most reports originate from Europe and Far East.^41,62 It is the dominant type of CHDL in Bulgaria,⁵⁹ United Kingdom,^11,62 Germany,⁵⁶ Italy,^42,43 Poland,²⁹ China,⁶⁸ and many other countries⁶² and is often associated with nosocomial outbreaks.⁷⁴ However, there are no reports on outbreaks associated with OXA-23 from nursing homes. Since all patients were previously hospitalized in one of the hospital centers in Zagreb, it was hypothesized that the strain was imported from the hospital. However, OXA-23 was reported from only two hospital centers in Zagreb in the multicenter study conducted in 2009–2010.⁶⁶

The OXA-24/40 group is the second group of CHDL among the strains tested, and this group originated from the Iberian Penninsula,^8,13,37,54 but was later identified in the Czech Republic,⁴⁵ United States,³⁶ and recently in Bulgaria.⁶¹ In contrast to our isolates, the isolates from Spain and the Czech Republic belonged to ICL II. Interestingly, Croatian hospital isolates producing OXA-24/40 β-lactamase were also uniformly assigned to ICL II^18,22,66 and thus it could be concluded that a new clone emerged in the nursing home that was not imported from the hospital environment. Sequencing of the representative bla_OXA-24 amplicon revealed the presence of OXA-72 β-lactamase. OXA-72 was previously reported in the University Hospital Zagreb and Split in Croatia.^18,22,66 OXA-72 β-lactamase with similar properties was previously reported in France,⁴ South Korea,³⁴ Taiwan,³⁸ China,⁶⁸ and Brazil.⁶⁹ In contrast to our results, they proved the plasmid location of the bla_OXA-72 gene. MBLs of the VIM family were recently reported in Italy and India.^1,17,50 The aquisition of MBL genes can further complicate the therapy. The production of class A β-lactamases as a cause of carbapenem resistance was ruled out.

The strains possessing OXA-23 and VIM uniformly belonged to ICL II. Other European studies have already demonstrated the spread of EU clone II isolates and the association of carbapenem resistance with these isolates^19,45,72 The presence of ISAba1 upstream of bla_OXA-51 and bla_OXA-23 probably increased the expression of the genes and the level of carbapenem resistance as reported previously.⁵⁷

This situation in long-term care facilities is reminiscent to that in hospitals where ICL II clones have overtaken the previous ICL I clones because of broader antibiotic resistance. Previous data on A. baumannii genotypes in nursing homes are not available in bibliography to make comparisons. The switch from OXA-58 to OXA-23 clones was also previously reported elsewhere. The possible explanation is selective advantage associated with higher carbapenemase activity of the OXA-23 group and consequent selection of such clones in the environment where carbapenems are often prescribed.⁷³

There were two clusters found among isolates from the nursing home in Zagreb: one large with strains possessing OXA-23 and VIM-1 and the other smaller with OXA-24/48. It is likely to assume that these first two strains were actually introduced to a nursing home from a hospital and were later propagated among patients with chronic diseases due to cross-infection. Within the cluster, a certain degree of diversification was observed. The fact that genotypically related strains persisted during a longer period raises the possibility that these strains, as representatives of a possible outbreak, may have persisted, unnoticed in different facilities in the nursing home, and served as a source of patient contamination and infection. Good correlation between three typing methods, rep-PCR, PFGE, and SG, was observed. However, rep-PCR and PFGE are more suitable methods for outbreak analysis, while MLST allows comparison between isolates from a different center and is the most portable method, but is expensive and time-consuming because it requires sequencing of seven housekeeping genes.^14,25,27

Contaminated urinary catheters were the most important source of carbapenem-resistant A. baumannii. Plasmid characterization found multiple plasmids belonging to different incompatibility groups. Previous studies found OXA-58-like and OXA-24/40-like positive isolates to belong to groups 2 and 6 as reported by Bertini et al.⁵

Interestingly, 11 of the 14 sulbactam/ampicillin-resistant isolates carried the bla_TEM-1 gene, suggesting that in A. baumannii resistance to sulbactam my rely on additional mechanisms besides TEM-1 overexpression, although this was the most important mechanism as reported previously.³¹

The E-test and combined disk test for detection of MBLs were positive in 18 strains, but PCR revealed VIM-1 MBL in only 11 strains. It is possible that seven strains harbor some rare type of MBL, which was not sought in this study. The combined disk test for detection of ESBLs was negative, but PCR identified CTX-M β-lactamases in one isolate. It is well known that phenotypic tests are not reliable in detection of ESBLs in A. baumannii because chromosomal AmpC β-lactamase can antagonize the synergistic effect with clavulanic acid and lead to false-negative results. There are few reports in bibliography on CTX-M β-lactamases in A.baumannii.⁵²

Based on susceptibility testing, colistin and tigecycline could be recommended as the antibiotic of choice for the treatment of infections caused by MDR A. baumannii in our long-term care facilities. However, tigecyline is not recommended for urinary tract infections due to its low concentrations in urine.

In conclusion, hygiene measures, such as general cleaning of the nursing home, wearing gloves and gowns by the staff, adequate number of staff, screening of patients who were previously hospitalized upon admission, and isolation of colonized patients, should be employed^15,73 to reduce the spread of carbapenemase-positive A. baumannii among nursing home residents.

Footnotes

Acknowledgment

The authors thank Stjepan Katić and Dubravko Šijak for technical assisance with PFGE. The results were, in part, presented at 24th ECCMID, 2014, Barcelona, Spain. We thank Pasteur Institute for assigning a novel sequence type.

Disclosure Statement

There are no competing financial interests.

References

Amudham

M.S.

, Sekar

, Kamalanathan

, and Balaraman

2012. Bla_I_MP and bla_VIM mediated carbapenem-resistance in Pseudomonas and Acinetobacter species in India. J. Infect Ctries., 6:757–762.

Arakawa

, Shibata

, and Shibayama

2000. Convenient test for screening metallo-β-lactamase-producing Gram-negative bacteria by using thiol compounds. J. Clin. Microbiol., 38:40–43.

Arlet

, Brami

, Decre

, Flippo

, Gaillot

, and Lagrange

P.H.

1995. Molecular characterization by PCR restriction fragment polymorphism of TEM β-lactamases. FEMS Microbiol. Lett., 134:203–208.

Barnaud

, Zihoune

, Ricard

J.D.

, Hippeaux

M.C.

, Eveillard

, Dreyfuss

, and Branger

2010. Two sequential outbreaks caused by multidrug-resistant Acinetobacter baumannii isolates producing OXA-58 and OXA-72 oxacillinase in an intensive care unit in France. J. Hosp. Infect., 76:358–360.

Bertini

, Poirel

, Mugnier

, Villa

, Nordmann

, and Caratoli

2010. Characterization and PCR-based replicon typing of resistance plasmids in Acinetobacter baumannii. Antimicrobial. Agents Chemother., 54:4168–4177.

Bonnin

R.A.

, Potron

, Poirel

, Lecuyer

, Neri

, and Nordmann

2011. PER-7, an extended-spectrum β-lactamase with increased activity toward broad-spectrum cephalosporins in Acinetobacter baumannii. Antimicrob. Agents Chemother., 55:2424–2427.

Bonnin

R.A.

, Rotimi

V.O.

, Al Hubail

, Gasiorovski

, Al Sweih

, Nordmann

, and Poirel

2013. Wide dissemination of GES-type carbapenemases in Acinetobacter baumanii isolates in Kuwait. Antimicrob. Agents Chemother., 57:183–185.

Bou

, Oliver

, and Martinez-Beltran

2000. OXA-24, a novel class D β-lactamase with carbapenemase activity in an Acinetobacter baumannii clinical strain. Antimicrob. Agents Chemother., 44:1556–1561.

Brown

, and Amyes

2006. OXA β-lactamase in Acinetobacter: the story so far. J. Antimicrob. Chemother., 57:1–3.

10.

Clinical and Laboratory Standards Institute. 2013. Performance Standards for Antimicrobial Susceptibility Testing; 23rd Informational Supplement. Wayne, PA: CLSI, M100-S23.

11.

Coelho

J.M.

, Turton

J.F.

, Kaufmann

M.E.

, Glover

, Woodford

, Warner

, Palepou

M.F.

, Pike

, and Pitt

T.L.

2006. Ocurrence of carbapenem-resistant Acinetobacter baumannii clones at multiple hospitals in London and Southeast England. J. Clin. Microbiol., 44:3623–3627.

12.

Cornaglia

, Riccio

M.L.

, Mazzariol

, Lauretti

, Fontana

, and Rossolini

, G.M. 1999. Appearance of IMP-1 metallo-β-lactamase in Europe. Lancet, 353:899–900.

13.

Da Silva

G.J.

, Quinteria

, Bertolo

, Sousa

J.C.

, Galego

, Duarte

, and Peixe Portugese Resistance Study Group L.. 2004. Long-term dissemination of an OXA-40 carbapenemase producing Acinetobacter baumannii in the Iberian Peninsula. J. Antimicrob. Chemother., 54:255–258.

14.

Diancourt

, Passet

, Nemec

, Dijkshoorn

, and Brisse

2010. The population structure of Acinetobacter baumannii: expanding multiresistant clones from an ancestral susceptible genetic pool. PLoS One., 54:e10034.

15.

Dijkshoorn

, Nemec

, and Seifert

2007. An increasing threat in hospitals: multidrug-resistant Acinetobacter baumannii. Nat. Rev. Microbiol., 5:939–951.

16.

Dortet

, Poirel

, Errera

, and Nordman

2014. CarbAcineto NP test for rapid detection of carbapenemase-producers in Acinetobacter spp. J. Clin. Microbiol., 52:2359–2364.

17.

El-Ageery

, and Al-Hazmi

S.S.

2014. Microbiology and molecular detection of VIM-1 metallo-β-lactamase-producing Acinetobacter baumannii. Eur. Rev. Med. Pharmacol. Sci., 18:965–970.

18.

Franolić-Kukina

, Bedenić

, Budimir

, Herljević

, Vraneš

, and Higgins

2011. Clonal spread of carbapenem-resistant OXA-72 positive Acinetobacter baumannii in a Croatian university hospital. Int. J. Infect. Dis., 15:e706–e709.

19.

Gogou

, Pournaras

, Giannouli

, Voulgari

, Piperaki

E.T.

, Zarilli

, and Tsakris

2011. Evolution of multidrug-resistant Acinetobacter baumannii clonal lineages: a 10 year study in Greece (2000–2009). J. Antimicrob. Chemother., 66:2767–2772.

20.

Goić-Barišić

, Bedenić

, Tonkić

, Katić

, Kalenić

, and Punda-Polić

2007. Molecular characterisation of carbapenem resistant Acinetobacter baumannii in different Intensive Care Units in University Hospital Split, Croatia J. Chemother., 19:462–464.

21.

Goić-Barišić

, Bedenić

, Tonkić

, Novak

, Katić

, Kalenić

, Punda-Polić

, and Towner

K.J.

2009. Occurrence of OXA-107 and ISAba 1 in carbapenem-resistant isolates of Acinetobacter baumannii from Croatia. J. Clin. Microbiol., 47:3348–3349.

22.

Goić-Barišić

, Towner

K.J.

, Kovačić

, Šiško-Kraljević

, Tonkić

, Novak

, and Punda-Polić

2011. Outbreak in Croatia caused by a new carbapenem-resistant clone of Acinetobacter baumannii producing OXA-72 carbapenemase. J. Hospit. Infect., 77:368–369.

23.

Healy

M.J.

, Huong

, Bittner

, Lising

, Frye

, Raza

, Schrock

, Manry

, Renwick

, Nieto

, Wood

, Versalovic

, and Lupski

J.R.

2005. Microbial DNA typing by automated repetitive-sequence-based PCR. J. Clin. Microbiol., 43:199–207.

24.

Heritier

, Poirel

, Fournier

P.E.

, Claverie

J.M.

, Raoult

, and Nordmann

2005. Characterization of the naturally occurring oxacillinase of Acinetobacter baumannii. Antimicrob. Agents Chemother., 49:4174–4179.

25.

Higgins

, Hujer

A.M.

, Hujer

K.M.

, Bonomo

R.A.

, and Seifert

2012. Interlaboratory reproducibility of Diversilab rep-PCR typing and clustering of Acinetobacter baumannii isolates. J. Med. Microbiol., 61:137–141.

26.

Higgins

, Poirel

, Lehmann

, Nordmann

, and Seifert

2009. OXA-143, a novel carbapenem-hydrolyzing class D β-lactamase in Acinetobacter baumannii. Antimicrob. Agents Chemother., 53:5035–5038.

27.

Higgins

P.G.

, Janssen

, Fresen

, Wisplinghof

, and Seifert

2012. Molecular epidemiology of Acinetobacter baumannnii bloodstream isolates obtained in United States from 1995 to 2004 using rep-PCR and multilocus sequence typing. J. Clin. Microbiol., 50:3493–3500.

28.

Hrabak

, Stolobova

, Studentova

, Fridrichova

, Chudackova

, and Zemlickova

2012. NDM-1 producing Acinetobacter baumannii isolated from a patient repatriated to the Czech Republic from Egypt, July 2011. Euro. Surveill., 17:pii 20085.

29.

Izedbski

, Fiett

, Hryniewitz

, and Gniadkowski

2012. Molecular analysis of Acinetobacter baumannii isolates from invasive infections in 2009 in Poland. J. Clin. Microbiol., 50:3813–3815.

30.

Kaufman

M.E.

1998. Pulsed-field gel electrophoresis. In Woodfor

and Johnsons

(eds.), Molecular Bacteriology. Protocols and Clinical Applications, 1st edition. Humana Press, Inc., Totowa, NJ, pp. 33–51.

31.

Krizova

, Poirel

, Nordmann

, and Nemec

2013. TEM-1 β-lactamase as a source of resistance to sulbactam in clinical strains of Acinetobacter baumannii. J. Antimicrob. Chemother., 68:2786–2791.

32.

Lauretti

, Riccio

M.L.

, Mazzariol

, Cornaglia

, Amicosante

, Fontana

, and Rossolini

G.M.

1999. Cloning and characterization of bla_VIM, a new integron-borne metallo-β-lactamase gene from Pseudomonas aeruginosa clinical isolate. Antimicrob. Agents Chemother., 43:1584–1590.

33.

Lee

, Jum

J.H.

, Yong

, Lee

H.M.

, Kim

H.D.

, Docquier

J.D.

, Rossolini

G.M.

, and Chong

2005. Novel acquired metallo-β-lactamase gene, bla(_SIM-1) in a class 1 integron from Acinetobacter baumannii clinical isolates from Korea. Antimicrob. Agents Chemother., 49:4485–4491.

34.

Lee

, Kim

M.N.

, Choi

T.Y.

, Lee

, Whang

D.H.

, Yong

, Chong

, Woodford

, Livermore

D.M.

, and Group

Konsar

. 2009. Wide dissemination of OXA-type carbapenemases in clinical Acinetobacter spp. Isolates from South Korea. Int. J. Antimicrob. Agents, 33:520–524.

35.

Livermore

D.M.

, and Woodford

2006. The β-lactamase threat in Enterobacteriaceae, Pseudomonas and Acinetobacter. Trends Microbiol., 14:413–420.

36.

Lolans

, Rice

T.W.

, Munoz-Price

, and Quinn

J.P.

2006. Multiple outbreaks of carbapenem-resistant Acinetobacter baumannii isolates producing the carbapenamase OXA-40. Antimicrob. Agents Chemother., 50:2941–2945.

37.

Lopez-Otsoa

, Gallego

, Towner

K.J.

, Tysall

, Woodford

, and Livermore

D.M.

2002. Endemic carbapenem resistance associated with OXA-40 carbapenemase among Acinetobacter baumannii isolates from a Hospital in Northern Spain. J. Clin. Microbiol., 40:4741–4743.

38.

P.L.

, Doumith

, Livermore

D.M.

, Chen

T.P.

, and Woodford

2009. Diversity of carbapenem resistance mechanisms in Acinetobacter baumannii from a Taiwan hospital: spread of plasmid-borne OXA-72 carbapenemase. J. Antimicrob. Chemother., 63:641–647.

39.

Magiorakos

A.P.

, Srinivasan

, Carey

R.B.

, Carmeli

, Alagas

M.E.

, Giske

C.G.

, Harbarth

, Hindler

J.F.

, Kahlmeter

, Olsson-Liljequist

, Paterson

D.L.

, Rice

L.B.

, Stelling

, Struelens

M.J.

, Vatopoulos

, Weber

J.T.

, and Monnet

D.L.

2012. Multidrug-resistant, extensively drug-resistant and pandrug-resista.nt bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin. Microbiol. Infect., 18:268–281.

40.

Medina

, and Carmeli

2010. The pivotal role of long-term care facilities in the epidemiology of Acinetobacter baumannii: another brick in the wall. Clin. Infect. Dis., 50:1617–1618.

41.

Mendes

, Bell

J.M.

, Turnidge

J.D.

, Castahneira

, and Jones

R.N.

2009. Emergence and widespread dissemination of OXA-23, OXA-24/40 and OXA-58 carbapenemases among Acinetobacter spp. in Asia-pacific nations: report from the SENTRY Surveillance Program. J. Antimicrob. Chemother., 63:55–59.

42.

Mendes Rem Spabu

, Desphande

, Castanheira

, Jones

R.N.

, and Fadda

2009. Clonal dissemination of two clusters of Acinetobacter baumannii producing OXA-23 and OXA-58 in Rome, Italy. Clin. Microbiol. Infect., 15:588–592.

43.

Minadri

, D'Arezzo

, Antuneus

L.C.

, Pourcel

, Principe

, Petrosillo

, and Visca

2012. Evidence of diversity among epidemiologically related carbapenemase producing Acinetobacter baumannii strains belonging to International Clonal Lineage II. J. Clin. Microbiol., 50:590–597.

44.

Naas

, Namdri

, Reglier-Poupet

, Poyart

, and Nordmann

2007. Panresistant extended-spectrum β-lactamase SHV-5-producing Acinetobacter baumannii from New York City. J. Antimicrob. Chemother., 60:1174–1176.

45.

Nemec

, Križova

, Maixnerova

, Diancourt

, van der Rejden

T.L.

, Brisse

, van den Broek

, and Dijkshorn

2008. Emergence of carbapenem resistance in Acinetobacter baumannii in the Czech Republic is associated with the spread of multidrug-resistant strains of European clone II. J. Antimicrob. Chemother., 62:484–489.

46.

Nüesch-Inderbinen

M.T.

, Hächler

, and Kayser

F.H.

1996. Detection of genes coding for extended-spectrum SHV β-lactamases in clinical isolates by a molecular genetic method, and comparison with the E test. Eur. J. Clin. Microbiol. Infect. Dis., 15:398–402.

47.

Pagani

, Mantengoli

, Migliavacca

, Nucleo

, Pollini

, Spalla

, Daturi

, Romero

, and Rossolini

G.M.

2004. Multifocal detection of multidrug-resistant Pseudomonas aeruginosa producing PER-1 extended-spectrum β-lactamase in Northern Italy. J. Clin. Microbiol., 42:2523–2529.

48.

Pasteran

2006. Emergence of PER-2 and VEB-1 in Acinetobacter baumannii in the America. Antimicrob. Agents Chemother., 50:3222–3224.

49.

Paton

R.H.

, Miles

R.S.

, Hood

, and S. Amyes

G.B.

1993. ARI-1: β-lactamase mediated imipenem resistance in Acinetobacter baumannii. Int. J. Antimicrob. Agents, 2:81–88.

50.

Perilli

, Pelegrini

, Calenza

, Segatore

, and Amicosante

2011. First report from Italy of bla_VIM-1 and bla_TEM-1 genes in Pseudomonas putida and Acinetobacter baumannii isolated from wastewater. J. Chemother., 23:181–182.

51.

Poirel

, and Nordmann

2006. Carbapenem resistance in Acinetobacter baumannii: mechanisms and epidemiology. Clin. Microb. Infect., 12:826–836.

52.

Potron

, Munoz-Price

L.S.

, Nordamnn

, Cleary

, and Poirel

2011. Genetic features of CTX-M-15-producing Acinetobacter baumanii from Haiti. Antimicrob. Agents Chemother., 55:54396–54398.

53.

Pournaras

, Markogiannakis

, Ikonomidis

, Kondyli

, Bethimouti

, Maniatis

A.N.

, Legakis

N.J.

, and Tsakris

2006. Outbreak of multiple clones of imipenem-resistant Acinetobacter baumannii isolates expressing OXA-58 carbapenemase in an intensive care unit. J. Antimicrob. Chemother., 57:557–561.

54.

Quinteira

, Grosso

, Ramus

, and Peixe

2007. Molecular epidemiology of imipenem-resistant Acinetobacter haemolyticus and Acinetobacter baumannii isolates carrying plasmid-mediated OXA-40 from a Portuguese Hospital. Antimicrob. Agents Chemother., 51:3465–3466.

55.

Robledo

I.E.

, Aquino

E.E.

, Sante

M.I.

, Santana

J.L.

, Oterko

D.M.

, Leon

C.F.

, and Vazqueiz

G.J.

2010. Detection of KPC in Acinetobacter spp in Puerto Rico. Antimicrob. Agents Chemother., 43:1354–1357.

56.

Schleicher

, Higgins

P.G.

, Wisplinghoff

, Korber-Irrgang

, Kresken

, and Seifert

2012. Molecular epidemiology of Acinetobacter baumannii and Acinetobacter nosomialis in Germany over a 5-year period (2005–2009). Clin. Microbiol. Infect., 19:737–742.

57.

Segal

, Garny

, and Elisha

B.G.

2005. Is ISAbaI customized for Acinetobacter?. FEMS Microbiol. Lett., 243:425–429.

58.

Sengstock

D.M.

, Thyagrajan

, Apalara

, Mira

, Chopra

, and Kaye

K.S.

2010. Multidrug-resistant Acinetobacter baumannii: an emerging pathogen among older adults in community hospitals and nursing homes. Clin. Infect. Dis., 50:1611–1616.

59.

Stoeva

, Higgins

, Bojkova

, and Seifert

2008. Clonal spread of carbapenem-resistant OXA-23-positive Acinetobacter baumannii in a Bulgarian university hospital. Clin. Microbiol. Infect., 14:723–726.

60.

Tenover

, Arbeit

, Goering

R.V.

, Mickelsen

P.A.

, Murray

B.E.

, Persing

D.H.

, and Swaminthan

1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol., 33:2233–2239.

61.

Todorova

, Velinov

, Ivanov

, Dobreva

, and Kantardijev

2014. First detection of OXA-24 carbapenemase-producing Acinetobacter baumannii in Bulgaria. World J. Microbiol. Biotechnol., 30:1427–1430.

62.

Towner

K.J.

, Levi

, and Vlassiadi

on behalf of the ARPAC Steering Group. 2007. Genetic diversity of carbapenem-resistant isolates of Acinetobacter baumannii in Europe. Clin. Microbiol. Infect., 14:161–167.

63.

Turton

J.F.

, Gabriel

S.N.

, Valderrey

, Kaufmann

M.E.

, and Pitt

T.L.

2007. Use of sequence based typing and multiplex PCR to identify clonal lineages of outbreak strains of Acinetobacter baumannii. Clin. Microbiol. Infect., 13:807–815.

64.

Turton

J.F.

, Ward

M.E.

, Woodford

, Kaufmann

M.E.

, Pike

, Livermore

D.M.

and Pitt

T.L.

2006. The role of ISAba1 in expression of OXA carbapenemase genes in Acinetobacter baumannii. FEMS Microbiol. Lett., 258:72–77.

65.

Vilalon

, Valdezete

, Medina-Pascual

M.J.

, Curasco

, Vindel

, and Saez-Nieto

J.A.

2011. Epidemiology of the Acinetobacter-derived cephalosporinase, carbapenem-hydrolyzing oxacillinase and metallo-beta-lactamase genes, and of common insertion sequences, in epidemic clones of Acinetobacter baumannii. J. Antimicrob. Chemother., 68:550–553.

66.

Vranić-Ladavac

, Bedenić

, Minandri

, Ištok

, Frančula-Zaninović

, Ladavac

, and Visca

2014. Carbapenem-resistance and acquired class D carbapenemases in Acinetobacter baumannii from Croatia 2009–2010. Eur. J. Clin. Microbiol. Infect. Dis., 33:471–478.

67.

Walsh

T.R.

, Bolmstrom

, and Gales

2002. Evaluation of new E test for detecting metallo-β-lactamases in routine clinical testing. J. Clin. Microbiol., 40:2755–2759.

68.

Wang

, Guo

, Sun

, Wang

, Yang

, Chen

, Xu

, and Zhu

2007. Molecular epidemiology of clinical isolates of carbapenem-resistant Acinetobacter spp. from Chinese Hospitals. Antimicrob. Agents Chemother., 51:4022–4208.

69.

Werneck

J.S.

, Picao

R.C.

, Carvalhaes

C.G.

, Cardoso

J.P.

, and Gales

2010. OXA-72-producing Acinetobacter baumannii in Brazil: a case report. J. Antimicrob. Chemother., 66:452–454.

70.

Woodford

, Ellington

M.J.

, Coelho

J.M.

, Turton

J.F.

, Ward

M.E.

, Brown

, Amyes

S.G.

, and Livermore

D.M.

2006. Multiplex PCR for genes encoding prevalent OXA carbapenemases. Int. J. Antimicrob. Agents, 27:351–353.

71.

Woodford

, Fagan

E.J.

, and Ellington

M.J.

2006. Multiplex PCR for rapid detection of CTX-M β-lactamases. J. Antimicrob. Chemother., 57:154–155.

72.

Yong

, Toleman

M.A.

, Giske

C.G.

, Cho

H.S.

, Sundman

, Lee

, and Walsh

T.R.

2009. Characterization of a new metallo-β-lactamase gene, bla_NDM-1, and a nove erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob. Agents Chemother., 53:5046–5054.

73.

Zarilli

, Pournaras

, Giannouli

, and Tsakris

2013. Global evolution of multidrug-resistant Acinetobacter baumannii clonal lineages. Int. J. Antimicrob. Agents, 41:11–19.

74.

Zong

, Lu

, Valenzuela

J.K.

, Partridge

S.R.

, and Iredell

2008. An outbreak of carbapenem-resistant Acinetobacter baumannii producing OXA-23 β-lactamase. Int. J. Antimicrob. Agents, 31:50–54.