Clonal Transmission of Gram-Negative Bacteria with Carbapenemases NDM-1,VIM-1,and OXA-23/72 in a Bulgarian Hospital

Abstract

We characterized 72 isolates with reduced susceptibility to carbapenems (50 Acinetobacter spp., 13 Proteus mirabilis, five Escherichia coli, one Morganella morganii, one Enterobacter cloacae, one Providencia rettgeri, and one Pseudomonas aeruginosa) from a hospital in Sofia, Bulgaria. Different β-lactamase genes were identified by polymerase chain reaction and sequencing. Bacterial strain typing was performed by enzymatic macrorestriction and pulsed-field gel electrophoresis (PFGE) typing as well as multilocus sequence typing for selected isolates. The majority of Acinetobacter baumannii (46/50) and one Acinetobacter pittii isolate harbored carbapenemase genes bla_OXA-23 or bla_OXA-72; two A. baumannii contained both genes. PFGE typing of all A. baumannii showed the presence of nine different clones belonging to eight sequence types ST350, ST208, ST436, ST437, ST449, ST231, ST502, and ST579. Molecular characterization of the remaining isolates confirmed the presence of one NDM-1-producing E. coli-ST101 clone (five isolates) and one P. mirabilis clone (13 isolates) with VIM-1 and CMY-99. Furthermore, NDM-1 was identified in P. rettgeri and M. morganii and VIM-2 in the P. aeruginosa isolate. The permanent introduction of OXA-23/72 carbapenemase-producing A. baumannii clones into the hospital and the repeated occurrence of one VIM-1-producing P. mirabilis and one NDM-1-producing E. coli-ST101 clone over a period of more than 1 year is of concern and requires intensified investigations.

Introduction

Within the genus Acinetobacter, the species Acinetobacter baumannii is a worldwide emerging nosocomial pathogen. In the past 10 years, the increasing resistance of this species to various antibiotics is a serious threat of public health.¹ Of special concern are A. baumannii strains with resistance to all clinically relevant β-lactams, including carbapenems and antibiotics of other classes; only colistin is one of the few treatment options in case of an infection.² Carbapenem resistance in A. baumannii is mainly due to production of different OXA-type carbapenemases, for example, OXA-23, OXA-58, and OXA-72. Furthermore, the overexpression of the chromosomal bla_OXA-51-like gene in A. baumannii mediated by insertion sequence ISAba1 results in reduced susceptibility to imipenem and meropenem.³ Typing studies revealed the presence of several international clones (ICs, formerly named worldwide clones or European clones) that caused outbreaks and spread successfully in hospitals worldwide.^4,5

Apart from multidrug-resistant Acinetobacter, carbapenemase-producing Enterobacteriaceae are an increasing problem and undetected fecal carriage enables the cross transmission in healthcare settings.⁶ The most prevalent carbapenemases are NDM, VIM, KPC, and OXA-48 and their success is based on horizontal transfer of the carbapenemase genes within the different enterobacterial species or clonal transfer of carbapenemase-producing strains facilitated by deficiencies in hygiene management and insufficient screening efficacy in healthcare settings.^3,6 Due to the great variety of carbapenemase-producing strains of different species, molecular typing is often essential for outbreak identification and evaluation of the ways of transmission of resistant bacteria or their resistance genes. The aim of the present study was the molecular characterization of 72 Gram-negative isolates with reduced susceptibility to carbapenems that were collected in a period of 15 months in a Bulgarian hospital.

Materials and Methods

Study setting and isolate collection

The military medical academy (MMA) is a multiprofile hospital with 800 beds and it is one of the national centers for treatment of patients with trauma, respiratory diseases, and liver transplantations. The MMA consists of eight surgery units, two intensive care units (ICUs), and two cabinets for outpatients (no pediatrics). Military and civilians were admitted in the ratio of 1:3. The patients are usually managed in open wards with four patients in a room. In ICUs the patients are treated also in open wards, except the patients after the liver transplantation, who are served in separate boxes.

From October 2013 to November 2014 Enterobacteriaceae isolates (n = 21) with reduced susceptibility to carbapenems (13 Proteus mirabilis, five E. coli, one Morganella morganii, one Enterobacter cloacae, and one Providencia rettgeri) were identified in seven wards of the MMA. Furthermore, 50 Acinetobacter spp. isolates with reduced susceptibility to carbapenems were collected from September 2014 to January 2015 in the same hospital. The Acinetobacter spp. were isolated mainly from respiratory specimen; the Enterobacteriaceae were mainly from urine samples. In the urine sample of one patient a carbapenem-resistant Pseudomonas aeruginosa strain was detected additionally to P. mirabilis. Furthermore, two P. mirabilis isolates were found in urine samples of another patient taken at a distance of 16 days. Species identification and a first antibiotic susceptibility testing were performed using an automated system (VITEK 2; bioMérieux, Marcy-l'Étoile, France). In the end of January 2015, all isolates were sent to the Robert Koch Institute (RKI; Germany) for further strain characterization, including resistance gene identification and evaluation of their genetic relationship.

Phenotypic and genotypic characterization of bacterial isolates

In the RKI laboratory, antimicrobial susceptibilities of the isolates were confirmed again by Etest (ertapenem, meropenem, imipenem) and VITEK 2 system (card AST-N248) using EUCAST breakpoints (version v 5.0). Acinetobacter species were determined by polymerase chain reaction (PCR) and sequencing of the rpoB gene and 16S rRNA sequencing, respectively.^7,8 The presence of various β-lactamase genes (bla_OXA-23-like, bla_OXA-40-like, bla_OXA-58-like, ISAba1+bla_OXA-51-like, bla_VIM-like, bla_IMP-like, bla_NDM-like, bla_OXA-48-like, bla_NDM-like, bla_{CTX-M-1-2-9group}, bla_TEM-like, bla_SHV-like, bla_CMY-like, bla_DHA-like, bla_{OXA-1-2-9-10group}, bla_PER-like, bla_GES-like, and bla_VEB-like) was tested by PCR and sequencing using previously described primers.^9–11 Additionally, a PCR screening for genes contributing to resistance to fluoroquinolones and aminoglycosides (aac(6′)Ib-like, qnrA/B/S-like, armA, rmtB,) was performed as described in different studies.^12–14

Bacterial strain typing of A. baumannii isolates was performed by ApaI-macrorestriction followed by pulsed-field gel electrophoresis (PFGE). Further typing included assignment to the important ICs 1–3 (formerly named European clones I-III) by multiplex-PCR and bla_OXA-51-like gene sequencing.^5,15 Finally, multilocus sequence typing (MLST) was performed according to the Oxford scheme (http://pubmlst.org abaumannii/). Bacterial strain typing of E. coli isolates was performed by XbaI-macrorestriction followed by PFGE, PCR-based identification of phylogenetic groups was done as previously described,¹⁶ and MLST analyses of E. coli were performed using the database http://mlst.warwick.ac.uk/mlst/dbs/Ecoli. PFGE typing of P. mirabilis was done after SmaI-macrorestriction.¹⁷ All PFGE results were interpreted according to the criteria of Tenover et al. 2000.¹⁸

Results

Analysis of the Acinetobacter spp. isolates

The majority of Acinetobacter spp. isolates was resistant to piperacillin/tazobactam (50/50), ciprofloxacin (48/50), sulfamethoxazole/trimethoprim (46/50), amikacin (35/50), and gentamicin (29/50). Resistance to imipenem and meropenem was confirmed for 45 of the 50 isolates. All isolates remained susceptible to colistin (50/50) and 86% to minocycline (43/50). Species identification of Acinetobacter spp. isolates revealed the presence of 47 A. baumannii, one Acinetobacter seifertii, one Acinetobacter radioresistens, and one Acinetobacter pittii. The A. pittii isolate and nearly all A. baumannii isolates (44/47) were resistant to imipenem and meropenem (>8 mg/L) and harbored carbapenemase genes bla_OXA-23 (38 A. baumannii and the A. pittii), bla_OXA-72 (n = 4), or both genes (n = 2). Additionally to these carbapenemases, three isolates harbored PER-1 extended-spectrum β-lactamase (ESBL) and six isolates were positive for β-lactamase TEM-1 (Table 1). Three A. baumannii and the A. seifertii isolate showed slightly increased MIC for imipenem and meropenem (1–2 mg/L) and harbored no carbapenemase gene. The A. radioresistens strain harbored carbapenemase gene bla_OXA-23, but without insertion sequences ISAba1 or ISAba4 upstream of this gene, and it was susceptible to imipenem and meropenem as described in a previous study.¹⁹

Table 1.

Detection of Acinetobacter baumannii Clones from September 2014 to January 2015 in a Bulgarian Hospital

PFGE-type (n)	ST^a (n)	IC^b (n)	Ward (n)	Material (n)	Beta-lactamases (n)	September 14	October 14	November 14	December 14	January 15
A1 (19)	ST350/ST2 (19)	IC 2 (19)	ICU (3); ARC (16)	Rs (16); urine (2); blood (1)	OXA-23 (16); OXA-23 + OXA-72 (2); -^c (1)	+(4)	+(7)	+(2)	+(4)	+(2)
A2 (4)	ST436/ST2 (4)	IC 2 (4)	ICU (2); ARC (1); HPS (1)	Rs (4)	OXA-23 (4)	+(1)	+(3)	−	−	−
A3 (8)	ST208/ST2 (8)	IC 2 (8)	ICU (5); ARC (3)	Rs (5); urine (1); nose (1); abd. ex. (1)	OXA-23 (5); OXA72 (3)	−	+(7)	+(1)	−	−
A4 (2)	ST208/ST2 (2)	IC 2 (2)	ICU (1); ARC (1)	Rs (2)	OXA-23 + TEM-1 (2)	−	+(1)	+(1)	−	−
A5 (4)	ST437/ST2 (4)	IC 2 (4)	ICU (3); ARC (1)	Rs (4)	OXA-23 + PER-1 (3); OXA-23 (1)	−	−	−	+(3)	+(1)
A6 (4)	ST449/ST20 (4)	IC 1 (4)	ICU (2); ARC (2)	Wound (3); rs (1)	OXA-23, TEM-1 (4)	−	+(3)	+(1)	−	−
A7 (4)	ST231/ST1 (4)	IC 1 (4)	ICU (1); ARC (3)	Rs (4)	OXA-23 (3); -^c (1)	+(2)	−	+(1)	+(1)	−
A8 (1)	ST502/ST2 (1)	IC 3 (1)	ARC (1)	Rs (1)	OXA-72	−	−	−	+(1)	−
A9 (1)	ST579/ST2 (1)	IC 2 (1)	URO (1)	Urine	-^c (1)	−	+(1)	−	−	−

sequence type Oxford scheme/Pasteur scheme.

International clone (formerly named European clone).

resistant only to ertapenem, for imipenem and meropenem MIC = 1–2 mg/L; rs, respiratory material.

(n), number of isolates; abd. ex., abdominal exudate; ICU, intensive care unit; ARC, anesthesiology and resuscitation clinic respiratory diseases; HPS, hepato-pancreatic surgery.

IC, international clones; PFGE, pulsed-field gel electrophoresis.

PFGE typing and PCR-based typing of all 50 A. baumannii indicated the presence of nine different clones belonging mainly to the IC 2 (36/45; 80%). Using MLST, we differentiated eight STs (ST350 n = 19, ST208 n = 10, ST231 n = 4, ST449 n = 4, ST436 n = 4, ST437 n = 4, ST502 n = 1, and ST579 n = 1). Especially, the ST350 isolates (PFGE-type A1) were detected over the complete collection period of five months, including two isolates with carbapenemase gene combination bla_OXA-23 ₊ ₇₂ (Table 1).

Analysis of Enterobacteriaceae and P. aeruginosa isolates

The 22 non-Acinetobacter isolates were resistant to cefotaxime, ceftazidime, cefepime, aztreonam, ciprofloxacin, levofloxacin, amikacin, tobramycin, nitrofurantoin, tigecycline, and sulfamethoxazole/trimethoprim. With exception of the E. cloacae isolate all these isolates were also resistant to imipenem, meropenem, and gentamicin. The 13 P. mirabilis isolates, the M. morganii and the P. rettgeri isolates were naturally resistant to colistin, but remained susceptible to piperacillin/tazobactam and fosfomycin.

Molecular screening of the 22 non-Acinetobacter isolates revealed the presence of carbapenemase NDM-1 in one P. rettgeri, one M. morganii, and in the five E. coli isolates. Carbapenemase gene bla_VIM-2 was found in one P. aeruginosa and bla_VIM-1 in 13 P. mirabilis isolates. The single E. cloacae isolate did not produce any carbapenemase and was resistant only to ertapenem, with slightly elevated MIC values for imipenem and meropenem (1 mg/L). The 22 isolates co-harbored various resistance genes, for example, encoding plasmid-mediated AmpC β-lactamases (CMY-99, CMY-4, CMY-6, DHA-1), ESBL (CTX-M-15, VEB-1-like, SHV-12), and further β-lactamases (TEM-1, OXA-1, OXA-2), as well as genes contributing to resistance to fluoroquinolones and aminoglycosides, for example, aac(6′)Ib-cr, qnrA1, qnrB1, aacA4, armA, and rmtB (Table 2).

Table 2.

Carbapenem-Resistant non-Acinetobacter Isolates Collected from November 2013 to 2014 in One Bulgarian Hospital

Isolate No.	Ward	Species	Material	Isolation date	Beta-lactamases	Other resistance genes	Pg, ST Escherichia coli	PFGE-type
52/15	ICU	Proteus mirabilis	Wound	Nov 2013	VIM-1; CMY-99, SHV-12, TEM-1, OXA-9nf	aacA4, armA	nt	Pm-1
53/15	NSG	P. mirabilis	Urine	Dec 2013	VIM-1; CMY-99, SHV-12, TEM-1, OXA-9nf	aacA4, armA	nt	Pm-1
54/15	TOR	P. mirabilis	Urine	Jan 2014	VIM-1; CMY-99, SHV-12, TEM-1, OXA-9nf	aacA4, armA	nt	Pm-1
55/15	TOR	P. mirabilis	Urine	Feb 2014	VIM-1; CMY-99, SHV-12, TEM-1, OXA-9nf	aacA4, armA	nt	Pm-1
56/15	URO	P. mirabilis	Urine	Feb 2014	VIM-1; CMY-99, SHV-12, TEM-1, OXA-9nf	aacA4, armA	nt	Pm-1a
57/15	ICU	M. morganii	Ur. cat.	Mar 2014	NDM-1, DHA-1, CTX-M-15, OXA-1	aacA4; aac(6′)Ib-cr	nt	Pm-2
58/15	ABS	P. mirabilis	Wound	Apr 2014	VIM-1; CMY-99, SHV-12, TEM-1, OXA-9nf	aacA4, armA	nt	Pm-1
59/15	ICU	P. mirabilis	Wound	Apr 2014	VIM-1; CMY-99, TEM-1, OXA-9nf	aacA4, armA	nt	Pm-1b
60/15	ICU	P. mirabilis	Urine	May 2014	VIM-1; CMY-99, SHV-12, TEM-1, OXA-9nf	aacA4, armA	nt	Pm-1c
61/15	ICU	P. mirabilis	Rs	May 2014	VIM-1; CMY-99, SHV-12, TEM-1, OXA-9nf	aacA4, armA	nt	Pm-1
62/15	ICU	P. mirabilis	Rs	Jun 2014	VIM-1; CMY-99, SHV-12, TEM-1, OXA-9nf	aacA4, armA	nt	Pm-1
63/15	TRA	P. mirabilis	Urine	Jun 2014	VIM-1; CMY-99, SHV-12, TEM-1, OXA-9nf	aacA4, armA	nt	Pm-1
64/15-1^a	ICU	P. mirabilis	Urine	Jun 2014	VIM-1; CMY-99, SHV-12, TEM-1, OXA-9nf	aacA4, armA	nt	Pm-1
64/15-2^a	ICU	Pseudomonas aeruginosa	Urine	Jun 2014	VIM-2	—	nt	Pa-1
65/15	ABS	P. mirabilis	No data	Nov 2014	VIM-1, TEM-1, SHV-12, CMY-99, OXA-9nf	aacA4, armA	nt	Pm-1
69/15	ICU	E. coli	Urine	Oct 2013	NDM-1, CMY-4,CTX-M-15, TEM-1, OXA-2	rmtB	B1, ST101	Ec-1a
70/15	ICU	E. coli	Rs	Dec 2013	NDM-1, CMY-4,CTX-M-15, TEM-1, OXA-2	rmtB	B1, ST101	Ec-1
71/15	ICU	P. rettgeri	Urine	Mar 2014	NDM-1, VEB-1like^b, CMY-6, OXA-1	aacA4; qnrB1; qnrA1, armA	nt	nt
73/15	ICU	E. coli	Urine	Nov 2014	NDM-1, CMY-4,CTX-M-15, TEM-1, OXA-2	rmtB	B1, ST101	Ec-1
74/15	ICU	E. coli	Urine	Nov 2014	NDM-1, CMY-4,CTX-M-15, TEM-1, OXA-2	rmtB	B1, ST101	Ec-1
75/15	ICU	E. coli	Urine	Nov 2014	NDM-1, CMY-4,CTX-M-15, TEM-1, OXA-2	rmtB	B1, ST101	Ec-1
76/15	RES	E. cloacae	Rs	Feb 2014	CTX-M-15, TEM-1, OXA-1	aac(6′)Ib-cr; qnrB1	nt	nt

isolates from the same patient–the urine sample contained one enterobacterial species (P. mirabilis) and P. aeruginosa

S131T substitution in comparison with VEB-1; nf, nonfunctional due to one point mutation in bla_OXA-9 resulting in a premature STOP-codon; nt, not tested; rs, respiratory material; ur. cat., urethral catheter; ICU, Intensive Care Unit; NSG, neurosurgery; TOR, thoracic surgery; URO, urology; ABS, abdominal surgery; TRA, traumatology; RES, respiratory diseases; Pg, phylogenetic group E. coli; ST, sequence type E. coli (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli/).

PFGE analyses confirmed repeated occurrence of a VIM-1-producing P. mirabilis clone in 12 patients (13 isolates) from November 2013 to 2014. In the same period of time a multidrug-resistant NDM-1-producing E. coli clone of sequence type ST101 (phylogenetic group B1) was detected in five ICU patients. Both clones were mainly the cause of nosocomial urinary tract infections (11/17 patients) or were involved in wound infections (n = 3) and respiratory infections (n = 3). Several of these patients died due to severe underlying diseases or multimorbidity; the age of the patients ranged from 21 to 91 years (median age 61.6 years; supporting information Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/mdr).

Discussion

Resistance to carbapenems in A. baumannii is a serious problem worldwide with resistance rates >50% in many countries, for example, Greece and Mexico.^1,20 Ten years ago, Bulgarian local surveillance systems detected A. baumannii carbapenem resistance rates of 58%/74% in 2005/2006 and a few studies reported OXA-23 or OXA-24-like carbapenemase-producing A. baumannii clones in Bulgarian hospitals.^21–23 In 2006 a 3-month study in the MMA showed the permanent presence of one clone with OXA-23 or OXA-58 carbapenemase.²¹ In the present study, we identified eight different clones with OXA-23 and/or OXA-72 carbapenemase in a period of 5 months (September 2014–January 2015). Two isolates of clones 1 and 7 and another isolate (clone 9) were carbapenemase negative and showed slightly elevated MIC for imipenem and meropenem (1–2 mg/L) probably due to intrinsic mechanisms like porin loss.^15,24 OXA-58 carbapenemase was not found in our isolates confirming data on replacement of OXA-58 producers in favor of OXA-23 from other countries.²⁴ The additionally identified PER-1 ESBL in three isolates of our study collection has been described as uncommon, but internationally widespread in A. baumannii and Enterobacteriaceae, and was previously reported in a clinical isolate from Bulgaria.^25–27

One A. baumannii-ST350 clone was found in two wards (19 patients) over the whole study period, whereas several isolates of the other clones occurred 2–3 months in different wards (Table 1). ST350, like the majority (38/47) of our A. baumannii isolates, belonged to the IC 2 and was detected before only in a few isolates in China, the Netherlands, and the USA (http://pubmlst.org abaumannii/). In contrast, sequence types ST436, ST437, and ST208 are common and widespread and have been reported from many countries worldwide, for example, Romania, Japan, and Arab countries.^28–31 Widespread, but less common are the IC 1 isolates, including the related sequence types ST231 and ST449 (one allele difference) that were described from Russia, Brazil, and the USA (http://pubmlst.org/ abaumannii/).^32,33 The occurrence of all these different A. baumannii clones in two or more wards in the hospital over a period of 2–5 months indicates their continuous introduction from outside followed by further clonal spread within the hospital. There are neither data on colonization with carbapenem-resistant A. baumannii in the healthy population in Southeast Europe, nor data on the epidemiological situation in other Bulgarian hospitals, but the long-time presence of that A. baumannii-ST350 clone in the study hospital indicated that the transfer of colonized patients between hospitals might be a source for the spread of resistant clones.

Apart from carbapenem-resistant A. baumannii, we identified three other species (M. morganii, E. coli, and P. rettgeri) that were positive for various β-lactamases, including carbapenemases, AmpC enzymes, and ESBLs. The ESBL-positive (CTX-M-15) E. cloacae strain was ertapenem-resistant, but carbapenemase-negative possibly due to modification or loss of outer membrane proteins in combination with β-lactamase production.³ The NDM-1-producing P. rettgeri strain is a rare finding, but single isolates have been described from several countries, for example, Nepal, Israel, or Brazil.³⁴ The combination of additional resistance genes (bla_CMY-6, bla_VEB-1like, bla_OXA-1, aacA4, qnrB1, qnrA1, armA) is unique in our isolate, although a related combination (bla_CMY-4, bla_VEB-1, bla_OXA-10, aacA4, qnrA1) was described in a previous study in a NDM-1-negative P. rettgeri.³⁵ Further resistance gene combinations were detected in the present study in one NDM-1-producing M. morganii strain (bla_DHA-1, bla_CTX-M-15, bla_OXA-1, aacA4, aac(6′)Ib-cr) and five NDM-1-producing E. coli isolates (bla_CMY-4, bla_CTX-M-15, bla_OXA-2, rmtB). In many studies, the bla_NDM-1 gene was found to be located on various plasmids alone or with other resistance genes, and the independent acquisition of those plasmids results in a great variety of resistance gene combinations.³⁶

Remarkably, the NDM-1-producing E. coli-ST101 clone that caused nosocomial urinary tract infections in five ICU patients from October 2013 to November 2014 was described 1 year before (March–September 2012, 12 patients) in the MMA.³⁷ NDM-1-producing E. coli-ST101 isolates have been described from different countries worldwide (e.g., UK, Pakistan, Australia, Canada, and Germany) indicating a possible association that has to be further studied in future.³⁸ Furthermore, the P. mirabilis clone with VIM-1 and CMY-99 was found earlier (November 2011–January 2012) in three patients of another hospital in Sofia.³⁹ Therefore, both clones—the NDM-1-producing E. coli-ST101 and the VIM-1-producing P. mirabilis—seem to be present in hospitals in Sofia over several years and their spread by transfer of patients between the different hospitals is highly probable. Several patients were previously hospitalized in other Bulgarian hospitals (Table S1), but unfortunately, information of travel history or ethnic background was not available enabling an assessment of a possible introduction of resistant clones from abroad. The open wards and possible lack in continuous hygiene training due to missing infection control personnel could facilitate the spread of these clones within the hospital; this is under discussion in the MMA. However, it remains to be elucidated if colonized patients in the hospital are the only reservoir of these strains or if there are other reservoirs outside the hospitals (patients in rehabilitation centers, nursing home residents, or healthy persons of the community).

In conclusion, this study confirmed the repeated occurrence of several OXA carbapenemase-producing clones of A. baumannii within 5 months in a Bulgarian hospital and further confirmed the presence of an NDM-1-producing E. coli-ST101 clone and one VIM-1/CMY-99-producing P. mirabilis clone in the same hospital over a period of 1 year. For the most A. baumannii clones, a successive import from outside and subsequent clonal transfer within the hospital is probable. Of major concern is the long-time detection of the two carbapenemase-producing E. coli and P. mirabilis clones indicating either their permanent presence in the hospital environment or a reservoir outside the hospitals. The multidrug resistance of both clones complicates the therapy in case of infections. Intensified hygiene measurements and continuous molecular surveillance in and outside the clinical settings are essential to control the further spread of these bacteria.

Footnotes

Acknowledgments

The authors extend special thanks to Sibylle Müller-Bertling, Christine Günther, and Evelyn Skiebe for excellent technical assistance.

Disclosure Statement

No competing financial interests exist.

References

Llaca-Diaz

J.M.

, Mendoza-Olazaran

, Camacho-Ortiz

, Flores

, and Garza-Gonzalez

. 2012. One-year surveillance of ESKAPE pathogens in an intensive care unit of Monterrey, Mexico. Chemotherapy., 58:475–481.

Garnacho-Montero

, Amaya-Villar

, Ferrándiz-Millón

, Díaz-Martín

, López-Sánchez

J.M.

, and Gutiérrez-Pizarraya

. 2015. Optimum treatment strategies for carbapenem-resistant Acinetobacter baumannii bacteremia. Expert Rev. Anti. Infect. Ther., 13:769–777.

Livermore

D.M.

, and Woodford

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

Higgins

P.G.

, Dammhayn

, Hackel

, and Seifert

. 2010. Global spread of carbapenem-resistant Acinetobacter baumannii. J. Antimicrob. Chemother., 65:233–238.

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.

Viau

, Frank

K.M.

, Jacobs

M.R.

, Wilson

, Kaye

, Donskey

C.J.

, Perez

, Endimiani

, and Bonomo

R.A.

. 2016. Intestinal carriage of carbapenemase-producing organisms: current status of surveillance methods. Clin. Microbiol. Rev., 29:1–27.

Nemec

, Musílek

, Maixnerová

, De Baere

, van der Reijden

T.J.

, Vaneechoutte

, and Dijkshoorn

. 2009. Acinetobacter beijerinckii sp. nov. and Acinetobacter gyllenbergii sp. nov., haemolytic organisms isolated from humans. Int. J. Syst. Evol. Microbiol., 59:118–124.

Weisburg

W.G.

, Barns

S.M.

, Pelletier

D.A.

, and Lane

D.J.

. 1991. 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol., 173:697–703.

Gröbner

, Linke

, Schütz

, Fladerer

, Madlung

, Autenrieth

I.B.

, Witte

, and Pfeifer

. 2009. Emergence of carbapenem-non-susceptible extended-spectrum-beta-lactamase (ESBL)-producing Klebsiella pneumoniae isolates at the university hospital of Tübingen, Germany. J. Med. Microbiol., 58:912–922.

10.

Pfeifer

, Wilharm

, Zander

, Wichelhaus

T.A.

, Göttig

, Hunfeld

K.P.

, Seifert

, Witte

, and Higgins

P.G.

. 2011. Molecular characterization of bla_NDM-1 in an Acinetobacter baumannii strain isolated in Germany in 2007. J. Antimicrob. Chemother., 66:1998–2001.

11.

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.

12.

Pfeifer

, Matten

, and Rabsch

. 2009. Salmonella enterica serovar Typhi with CTX-M beta-lactamase, Germany. Emerg. Infect. Dis., 15:1533–1535.

13.

Park

C.H.

, Robicsek

, Jacoby

G.A.

, Sahm

, and Hooper

D.C.

. 2006. Prevalence in the United States of aac(6′)-Ib-cr Encoding a Ciprofloxacin-Modifying Enzyme. Antimicrob. Agents Chemother., 50:3953–3955.

14.

Doi

, and Arakawa

. 2007. 16S ribosomal RNA methylation: emerging resistance mechanism against aminoglycosides. Clin. Infect. Dis., 45:88–94.

15.

Zander

, Nemec

, Seifert

, and Higgins

P.G.

. 2012. Association between β-lactamase-encoding bla(OXA-51) variants and DiversiLab rep-PCR-based typing of Acinetobacter baumannii isolates. J. Clin. Microbiol., 50:1900–1904.

16.

Clermont

, Bonacorsi

, and Bingen

. 2000. Rapid and simple determination of the Escherichia coli Phylogenetic Group. Appl. Environ. Microbiol., 66:4555–4558.

17.

Schultz

, Haenni

, Mereghetti

, Siebor

, Neuwirth

, Madec

J.Y.

, Cloeckaert

, and Doublet

. 2015. Survey of multidrug resistance integrative mobilizable elements SGI1 and PGI1 in Proteus mirabilis in humans and dogs in France, 2010–13. J. Antimicrob. Chemother., 70:2543–2546.

18.

Tenover

F.C.

, Arbeit

R.D.

, Goering

R.V.

, Mickelsen

P.A.

, Murray

B.E.

, Persing

D.H.

, and Swaminathan

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

19.

Savov

, Pfeifer

, Wilharm

, Trifonova

, Todorova

, Gergova

, Borisova

, and Kjoseva

. 2015. Isolation of Acinetobacter radioresistens from a clinical sample in Bulgaria. J. Global Antimicrob. Res. DOI: 10.1016/j.jgar.2015.10.008.

20.

Gogou

, Pournaras

, Giannouli

, Voulgari

, Piperaki

E.T.

, Zarrilli

, 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.

21.

Stoeva

, Higgins

P.G.

, Savov

, Markovska

, Mitov

, and Seifert

. 2009. Nosocomial spread of OXA-23 and OXA-58 beta-lactamase-producing Acinetobacter baumannii in a Bulgarian hospital. J. Antimicrob. Chemother., 63:618–620.

22.

Stoeva

, Higgins

P.G.

, Bojkova

, and Seifert

. 2008. Clonal spread of carbapenem-resistant OXA-23-positive Acinetobacter baumannii in a Bulgarian university hospital. Clin. Microbiol. Infect., 14:723–727.

23.

Todorova

, Velinov

, Ivanov

, Dobreva

, and Kantardjiev T.

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

24.

Zarrilli

, Pournaras

, Giannouli

, and Tsakris

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

25.

Strateva

, Todorova

, Ouzounova-Raykova

, and Mitov

. 2008. Emergence of a PER-1 extended-spectrum beta-lactamase-producing Acinetobacter baumannii clinical isolate in Bulgaria. J. Chemother., 20:391–392.

26.

Bonnin

R.A.

, Poirel

, Licker

, and Nordmann

. 2011. Genetic diversity of carbapenem-hydrolysing ß-lactamases in Acinetobacter baumannii from Romanian hospitals. Clin. Microbiol. Infect., 17:1524–1528.

27.

Bae

I.K.

, Jang

S.J.

, Kim

, Jeong

S.H.

, Cho

, and Lee

. 2011. Interspecies dissemination of the bla gene encoding PER-1 extended-spectrum β-lactamase. Antimicrob. Agents Chemother., 55:1305–1307.

28.

Gheorghe

, Novais

, Grosso

, Rodrigues

, Chifiriuc

M.C.

, Lazar

, and Peixe

. 2015. Snapshot on carbapenemase-producing Pseudomonas aeruginosa and Acinetobacter baumannii in Bucharest hospitals reveals unusual clones and novel genetic surroundings for bla_OXA-23. J. Antimicrob. Chemother., 70:1016–1020.

29.

Asai

, Umezawa

, Iwashita

, Ohshima

, Ohashi

, Sasaki

, Hayashi

, Matsui

, Shibayama

, Inokuchi

, and Miyachi

. 2014. An outbreak of bla_OXA-51-like- and bla_OXA-66-positive Acinetobacter baumannii ST208 in the emergency intensive care unit. J. Med. Microbiol., 63:1517–1523.

30.

Zowawi

H.M.

, Sartor

A.L.

, Sidjabat

H.E.

, Balkhy

H.H.

, Walsh

T.R.

, Al Johani

S.M.

, AlJindan

R.Y.

, Alfaresi

, Ibrahim

, Al-Jardani

, Al Salman

, Dashti

A.A.

, Johani

, and Paterson

D.L.

. 2015. Molecular epidemiology of carbapenem-resistant Acinetobacter baumannii isolates in the Gulf Cooperation Council States: dominance of OXA-23-type producers. J. Clin. Microbiol., 53:896–903.

31.

Lopes

B.S.

, Al-Agamy

M.H.

, Ismail

M.A.

, Shibl

A.M.

, Al-Qahtani

A.A.

, Al-Ahdal

M.N.

, and Forbes

K.J.

. 2015. The transferability of blaOXA-23 gene in multidrug-resistant Acinetobacter baumannii isolates from Saudi Arabia and Egypt. Int. J. Med. Microbiol., 305:581–588.

32.

Solomennyi

, Goncharov

, and Zueva

. 2015. Extensively drug-resistant Acinetobacter baumannii belonging to the international clonal lineage I in a Russian burn intensive care unit. Int. J. Antimicrob. Agents., 45:525–528.

33.

Martins

, Martins

I.S.

, de Freitas

W.V.

, de Matos

J.A.

, Magalhães

A.C.

, Girão

V.b.

, Dias

R.C.

, de Souza

T.C.

, Pellegrino

F.L.

, Costa

L.D.

, Boasquevisque

C.H.

, Nouér

S.A.

, Riley

L.W.

, Santoro-Lopes

, and Moreira

B.M.

. 2012. Severe infection in a lung transplant recipient caused by donor-transmitted carbapenem-resistant Acinetobacter baumannii. Transpl. Infect. Dis., 14:316–320.

34.

Tada

, Miyoshi-Akiyama

, Dahal

R.K.

, Sah

M.K.

, Ohara

, Shimada

, Kirikae

, and Pokhrel

B.M.

. 2014. NDM-1 Metallo-β-Lactamase and ArmA 16S rRNA methylase producing Providencia rettgeri clinical isolates in Nepal. BMC Infect. Dis., 14:56. Doi: 10.1186/1471-2334-14-56.

35.

Aibinu

I.E.

, Pfeifer

, Ogunsola

, Odugbemi

, Koenig

, and Ghebremedhin

. 2011. Emergence of β-lactamases OXA-10, VEB-1 and CMY in Providencia spp. from Nigeria. J. Antimicrob. Chemother., 66:1931–1932.

36.

Tijet

, Richardson

, MacMullin

, Patel

S.N.

, and Melano

R.G.

. 2015. Characterization of multiple NDM-1-producing Enterobacteriaceae isolates from the same patient. Antimicrob. Agents Chemother., 59:3648–3651.

37.

Poirel

, Savov

, Nazli

E, A.

, Trifonova

, Todorova

, Gergova

, and Nordmann

. 2014. Outbreak caused by NDM-1- and RmtB-producing Escherichia coli in Bulgaria. Antimicrob. Agents Chemother., 58:2472–2474.

38.

Mushtaq

, Irfan

, Sarma

J.B.

, Doumith

, Pike

, Pitout

, Livermore

D.M.

, and Woodford

. 2011. Phylogenetic diversity of Escherichia coli strains producing NDM-type carbapenemases. J. Antimicrob. Chemother., 66:2002–2005.

39.

Schneider

, Markovska

, Marteva-Proevska

, Mitov

, Markova

, and Bauernfeind

. 2014. Detection of CMY-99, a novel acquired AmpC-Type β-lactamase, and VIM-1 in Proteus mirabilis isolates in Bulgaria. Antimicrob. Agents Chemother., 58:620–621.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.04 MB