Spread of multidrug-resistant Acinetobacter baumannii isolates belonging to IC1 and IC5 major clones in Rondônia state

Abstract

In Brazil, carbapenem-resistant A. baumannii (CRAB) is a critical pathogen showing high carbapenem resistance rates. Currently, there is little epidemiological data on A. baumannii isolated in the Northern Brazilian region. Herein, this study aimed to characterize the resistance mechanisms of CRAB isolates recovered from hospitalized patients in the state of Rondônia in 2019. Most of CRAB were considered as extensively drug-resistant, and some of them showed high MICs for minocycline. Only polymyxins showed a satisfactory activity. All isolates carried bla_OXA-23 and were included in 14 distinct clusters, with the predominance of clonal group A (29%). The IC1 was the most frequent clonal group, followed by IC5 and IC4. Here, we firstly reported the epidemiological scenario of CRAB in the state of Rondônia, located in the Brazilian Amazon region. The high frequency of CRAB presenting XDR phenotype is of great concern, due to limited therapeutical options, especially in the actual pandemic scenario, in which we observed an overcrowding of ICU beds. Such results are essential to better characterize the epidemiology of CRAB in the entire Brazilian territory.

Introduction

Currently, carbapenem-resistant Acinetobacter baumannii (CRAB) is considered one of the main nosocomial pathogens causing serious infections worldwide; it has been responsible for outbreaks of difficult control associated with high mortality rates [1, 2]. For these reasons, the World Health Organization (WHO) pointed CRAB as a critical priority pathogen for research and development of new antimicrobials [3]. Additionally, data released by the Brazilian National Health Surveillance Agency (ANVISA) showed that Acinetobacter spp. was the fourth most frequent pathogen causing primary central line catheter-associated bloodstream infections (ICS) in adult intensive care units (ICU) in 2019, among which carbapenems and polymyxins resistance reached 79.5% and 4.7%, respectively [4]. The high carbapenem resistance rates observed in this pathogen occur mainly due to the production of acquired carbapenem-hydrolyzing class D β-lactamases (CHDL) [1, 5]. The widespread of extensively drug-resistant (XDR) A. baumannii occurs mainly through pandemic strains belonging to international clones (IC), which have several characteristics that facilitate their survival and dissemination into nosocomial environment [6]. In Brazil, the high carbapenem resistance rates are mainly associated to the spread of epidemic clones belonging to IC7 followed by IC1 and IC4 carrying bla_OXA-23 and/or bla_OXA-72 [7–10]. In addition, studies conducted in the cities of São Luis and Cuiabá located in the Northern and Midwestern regions, respectively, reported the occurrence of CRAB carrying bla_KPC-2,-3 [11, 12] and bla_NDM-1 [13]_.

To date, there is a scarce data reporting the epidemiology of CRAB in the Brazilian Northern region, especially in Rondônia state. Herein, this study aimed to characterize the resistance mechanisms and genetic relatedness of CRAB isolates recovered from a tertiary hospital located in Porto Velho, Rondônia state.

Methods

Bacterial strains and species identification

Twenty-seven CRAB isolates recovered in 2019 from a tertiary hospital located in the city of Porto Velho, Rondônia state, Brazil, were evaluated. The identification at species level were confirmed in triplicate by Matrix-Assisted Laser Desorption Ionization - Time of Flight - Mass Spectrometry (MALDI-TOF MS) technique using Microflex LT spectrometer and Biotyper^TM 3.3 software package (Bruker Daltonics^TM, MA, USA), according to manufacturer’s recommendations.

Antimicrobial susceptibility testing

Minimal inhibitory concentrations (MICs) for ceftriaxone, ceftazidime, cefepime, aztreonam, imipenem, meropenem, ciprofloxacin, levofloxacin, amikacin, gentamicin, and minocycline (Sigma-Aldrich, St. Louis, USA) were determined by agar dilution, except for polymyxin B, colistin, and tigecycline (Sigma-Aldrich, St. Louis, USA). The susceptibility to these compounds was evaluated using the Mueller-Hinton cation-adjusted broth microdilution method according to the ISO 20776-1. Pseudomonas aeruginosa ATCC^® 27853^TM and Escherichia coli ATCC^® 25922^TM were tested as quality control strains. The MICs for imipenem, meropenem, ciprofloxacin, levofloxacin, amikacin, gentamicin, polymyxin B, and colistin were interpreted according to the Brazilian Committee on Antimicrobial Susceptibility Testing (BrCAST/EUCAST) (http://brcast.org.br /). For tigecycline, the available Pk/Pd data breakpoints of BrCAST/EUCAST (S ≤ 0.5 and > 0.5 μg/mL) were used (http://brcast.org.br/).

Detection of carbapenemase encoding genes

All CRAB isolates were screened for genes encoding for serine β-lactamases of classes A (bla_KPC-like) and D (bla_OXA-23-like, bla_OXA-58-like, bla_OXA-24/40-like, bla_OXA-143-like, bla_OXA-235-like, and bla_OXA-48-like), and for metallo-β-lactamases (bla_IMP-like, bla_VIM-like, bla_SPM-like, bla_GIM-like, bla_SIM-like, and bla_NDM-like) by PCR followed by DNA sequencing using specific primers, as previously published [14–16].

Molecular typing

To establish the clonal relatedness of CRAB isolates, rep-PCR was performed using the primer pairs REP-1 (5′-IIIGCGCCGICATCAGGC-3′) and REP-2 (5′-ACGTCTTATCAGGCCTAC-3′), as previously described [17]. Fingerprint patterns were normalized and analyzed using the BioNumerics software v.6.0 (Applied Maths, Sint-Martens-Latem, Belgium), and a dendrogram was generated by unweighted pair group with a mathematical average (UPGMA) cluster analyses algorithm using 1.5% Dice correlation coefficient and 1.5% tolerance. The identification of ICs was determined by sequencing of bla_OXA-51-like genes, as previously proposed [18].

Results and discussion

Most CRAB isolates (n = 11; 40.7%) were recovered from blood (Figure 1) and tracheal aspirate (n = 7; 25.9%). All CRAB isolates were resistant to aminoglycosides (MIC₉₀, > 256 μg/mL; Table 1). High MICs are observed for extended-spectrum cephalosporins (MIC₉₀, > 256 μg/mL), as shown in Table 1. All isolates were also resistant to fluoroquinolones (MIC₉₀, > 64 and 64 μg/mL ciprofloxacin and levofloxacin, respectively), except for one isolate (26PVH) that showed MIC of 0.125 μg/mL for ciprofloxacin (Figure 1). We observed that despite levofloxacin (MIC_50, 16 μg/mL) being at least four-fold more potent than ciprofloxacin (MIC_50, 64 μg/mL), 100% of the isolates were also resistant to this agent. Only polymyxins showed in vitro activity against all CRAB isolates (MIC₉₀ ≤ 0.25 μg/mL for both), corroborating with previous Brazilian reports [7–10, 19]. Nonetheless, it is worth mentioning that current studies demonstrating a change in the Latin American scenario, with the increase in the polymyxin-resistance rates, have been recently published [20]. Besides, it has also been reported that most of polymyxin-resistant A. baumannii isolates recovered in distinct Brazilian hospitals presented the XDR phenotype and belonged to the major clonal groups, mainly ST79/IC5 [21]. Fourteen CRAB isolates (51.9%) were classified as resistant to tigecycline (MIC₉₀, 1 μg/mL), while four CRAB isolates belonging to IC1, IC4, and IC5 presented a decreased susceptibility to minocycline (MICs 4–8 μg/mL) (Figure 1), contrasting with results of previous studies that reported a superior activity of minocycline compared to that of tigecycline against MDR or XDR A. baumannii isolates recovered in the Southeastern region of Brazil [8, 10, 21], as well as worldwide [22, 23]. Corroborating with our results, decreased susceptibility to minocycline (100 to 83.9%) against 3367 A. calcoacetius-A. baumannii complex isolates observed in Latin America was reported during a 15-year period (2001 to 2016, respectively) [20].

Fig. 1.

Genetic relatedness, IC typing, and minimal inhibitory concentration to 15 antimicrobial agents of 27 carbapenem-resistant A. baumannii isolates recovered in a tertiary hospital located in the city of Porto Velho, Rondônia state, Brazil. The underlined MIC values indicated those categorized as resistant according to BrCAST/EUCAST (http://brcast.org.br/). All CRAB isolates carried the acquired CHDL encoding gene bla_OXA-23

Table 1.

Antimicrobial susceptibility profile of 27 carbapenem-resistant A. baumannii isolates recovered from hospitals located in Rondônia state, Brazil^a

Antimicrobials		MIC (μg/mL)			Category (%)b
		MIC50	MIC90	Range	S	R
β-lactams
	Ceftazidime	> 256	> 256	32–> 256	-	-
	Ceftriaxone	> 256	> 256	> 256–> 256	-	-
	Cefepime	256	> 256	64–> 256	-	-
	Imipenem	64	64	16–64	0.0	100.0
	Meropenem	64	64	32–64	0.0	100.0
Fluoroquinolones
	Ciprofloxacin	> 64	> 64	8–> 64	3.7	96.3
	Levofloxacin	16	64	8–64	0.0	100.0
Aminoglycosides
	Amikacin	256	> 256	64–> 256	0.0	100.0
	Gentamicin	256	> 256	32–> 256	0.0	100.0
Tetracyclines
	Minocycline	0.5	8	≤ 0.25–8	-	-
	Tigecycline	0.25	1	0.25–1	48.1	51.9
Polymyxins
	Colistin	≤ 0.25	≤ 0.25	≤ 0.25–2	100	0.0
	Polymyxin B	≤ 0.25	≤ 025	≤ 0.25–1	100	0.0

^aMIC, minimal inhibitory concentration; S, susceptible; R, resistant

^bAntimicrobial susceptibility results interpreted according to BrCAST/EUCAST breakpoints (http://brcast.org.br/). For tigecycline, the available Pk/Pd data breakpoints (S ≤0.5 and >0.5 μg/mL). (-) Breakpoints not available for BrCAST

All CRAB isolates carried the acquired CHDL encoding gene bla_OXA-23. None of the other carbapenemases encoding genes searched was found. Such result is expected, since bla_OXA-23 is widely distributed in CRAB isolates recovered in distinct Brazilian geographic regions [8, 9, 19]. Although OXA-23 is the most frequent carbapenemase found among Brazilian A. baumannii isolates, it has been observed an increased in the frequency of OXA-72-producing A. baumannii in Brazil [7, 21]. In fact, OXA-72-producing CRAB has been reported in Roraima and Pará, Northern Brazil, states [24, 25].

Genotyping using the rep-PCR technique revealed the occurrence of 14 clusters (Figure 1), being 29% of the CRAB isolates grouped cluster A (n = 8/29.6%). Regarding the IC typing (Figure 1), 44% of the CRAB isolates belonged to IC1 (n = 12; 44.5%), followed by IC5 (n = 11; 40.7%) and IC4 (n = 4; 14.8%). Our results corroborated with previous studies conducted in Brazil, which showed that IC1 and IC5 were also predominant in the Northern region of Brazil [24, 26, 27]. These major clones are routinely associated with the MDR and XDR phenotype [8, 21, 28], and, particularly, the IC5 has been linked to high mortality rates [19].

In conclusion, we report for the first time the epidemiological scenario of CRAB in the Brazilian state of Rondônia, which proved to be similar to the other states of the country, with a predominance of IC1 and IC5 among OXA-23-producing CRAB isolates. The high frequency of XDR A. baumannii in such state is worrisome, especially in the actual pandemic scenario, in which we observed an overcrowding of ICU beds, where CRAB has been commonly found as the etiological agent of serious infections, as demonstrated by Shinohara et al. [29], which reports the occurrence of an outbreak by CRAB in an intensive care unit of a hospital in southern Brazil.

Funding

We are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for providing grants to T.B.V., C.S.N., and F.F.S. (PNPD), and to the National Council for Science and Technological Development (CNPq) for providing grant to A.C.G. (Process number: 312066/2019).

Declarations

Conflict of interest

A.C.G. has recently received research funding and/or consultation fees from bioMérieux Eurofarma, MSD, Pfizer, Sandoz, United Medical and Zambon. Other authors have nothing to declare. This study was not financially supported by any Diagnostic/Pharmaceutical company.