Whole-Genome Sequencing of ST2 A. baumannii Causing Bloodstream Infections in COVID-19 Patients.

A total of 43 A. baumannii strains, isolated from 43 patients affected by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and by bacterial sepsis, were analyzed by antimicrobial susceptibility testing. All strains were resistant to almost three different classes of antibiotics, including carbapenems and colistin. The whole-genome sequencing (WGS) of eight selected A. baumannii isolates showed the presence of different insertion sequences (ISs), such as ISAba13, ISAba26, IS26, ISVsa3, ISEc29, IS6100 and IS17, giving to A. baumannii a high ability to capture and mobilize antibiotic resistance genes. Resistance to carbapenems is mainly mediated by the presence of OXA-23, OXA-66 and OXA-82 oxacillinases belonging to OXA-51-like enzymes. The presence of AmpC cephalosporinase, ADC-25, was identified in all A. baumannii. The pathogenicity of A. baumannii was exacerbated by the presence of several virulence factors. The multi-locus sequence typing (MLST) analysis showed that all strains belong to sequence type 2 (ST) international clone.

1. Introduction

Acinetobacter baumannii belongs to a group of nosocomial pathogens, designated by the acronym “ESKAPE” (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.). These bacteria are the predominant cause of life-threatening nosocomial infections in critically ill and immunocompromised patients worldwide. ESKAPE pathogens have developed resistance mechanisms against almost all antimicrobial agents, including carbapenems, the last line of defense in clinical armamentarium [1,2,3,4]. A. baumannii is commonly associated with nosocomial infections including bacteremia, endocarditis, urinary tract infections (UTIs), meningitis, gastrointestinal and skin/wound infections [5,6,7,8]. However, community-acquired A. baumannii infections have been described, in particular, in people with comorbidities [9,10,11]. Several studies have reported that respiratory viral infections, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), predispose patients to bacterial co-infections and secondary infections [12]. Prolonged hospitalization in the intensive care unit (ICU) may be linked with increased possibility of developing bacterial co-infection, especially with multi-drug resistant (MDR) A. baumannii in critically ill coronavirus disease 2019 (COVID-19) patients. Generally, antibiotic resistance in A. baumannii is due to different mechanisms: (a) inactivation of antibiotics by enzymatic hydrolysis (i.e., β-lactamases), (b) reducing membrane permeability, (c) increasing efflux of antibiotics (i.e., overexpression of drug efflux pumps) and (d) mutations in antibiotic binding targets by genetic insertion sequences. Since 1990, carbapenems have been the mainstay antibiotics in the treatment of A. baumannii infections. Currently, carbapenem-resistant A. baumannii (CRAb) poses a global threat to human health. CRAb is emerging worldwide, and the majority of these isolates often show MDR or extensive drug resistant (XDR) patterns [13,14]. In 2018, the World Health Organization (WHO) considered A. baumannii a priority for the research and development of new antibiotics [3]. The presence of β-lactamases, in particular oxacillinases (OXAs), constitutes the main mechanism of resistance to carbapenems in A. baumannii. The main bla genes found in A. baumannii are represented by the presence of intrinsic chromosomal blaOXA-51-like genes and other groups of OXA acquired carbapenemases, including OXA-23-like, OXA-24/40-like, OXA-58-like, and OXA-143 enzymes [15]. In a large number of studies, ISAba1 has been identified in combination with different OXA-β-lactamases, including the blaOXA−51-like genes. This insertion sequence is located upstream in the opposite direction of blaOXA−51-like, acting as a promoter [16]. Among other β-lactamases, class A β-lactamases (e.g., PER-1, VEB-1, CTX-M-1, TEM-1), metallo β-lactamases (e.g., IMP, VIM, NDM) and AmpC cephalosporinases are prevalent in A. baumannii [17,18]. A. baumannii is also able to promote antibiotic resistance by virulence factors such as outer membrane protein modification, cell envelope factors, enzymes, quorum sensing, biofilm formation, motility and micronutrient acquisition systems [19]. Currently, very few therapeutic agents exist or are in development to treat MDR A. baumannii infections. Indeed, the ability of A. baumannii to persist and easily survive on biotic and abiotic surfaces and under dry conditions makes its treatment and eradication really complicated [20]. Because of the decrease in susceptibility to a wide range of antibiotics, the treatment options are frequently limited to synergistic use of these agents. Despite its nephrotoxicity, colistin, as monotherapy or in combination with other active compounds, has been used in critically ill patients [21]. Unfortunately, colistin-resistant Acinetobacter strains have rapidly emerged [22,23,24]. In recent times, cefiderocol, a novel siderophore cephalosporin, and durlobactam were approved for the treatment of MDR Gram-negative infections, including A. baumannii [25,26,27]. The aim of the present study was to explore, by whole-genome sequencing (WGS), antibiotic resistance genes (ARGs) and virulence factors of A. baumannii strains isolated from COVID-19 patients affected by sepsis and admitted to the ICU of Spirito Santo Hospital, Pescara, Central Italy.

2. Results

2.1. Minimal Inhibitory Concentrations (MICs)

A total of 43 A. baumannnii strains, isolated from positive blood cultures, were analyzed against a panel of antibiotics. All the strains showed the same antimicrobial susceptibility profile with a remarkable resistance to amikacin (MIC ≥ 64 mg/L), gentamicin (MIC ≥ 16 mg/L), ciprofloxacin (MIC ≥ 4 mg/L), meropenem (MIC > 8 mg/L), colistin (MIC > 8 mg/L) and trimethoprim–sulfamethoxazole association (MIC > 160 mg/L).

2.2. WGS and Multi-Locus Sequence Typing (MLST)

The WGS was performed on eight non-replicative A. baumannii strains isolated from patients admitted to ICU in different time periods. By molecular analysis we identified the sequence type (MLST), ARGs, virulence factors and mobile genetic elements (MGEs) harbored in the analyzed genome of A. baumannii. The genome size of the eight A. baumannii strains ranged from 3.8 to 4.0 Mb, and no plasmids were found. The MLST analysis using the Pasteur scheme revealed that all isolates belonged to the ST2 sequence type (Table 1).

Table 1

Antimicrobial resistance genes (ARGs) and mobile genetic elements (MGEs) in bloodstream (BSI) A. baumannii isolates.

Strain	Genome Size (bp)	MGEs	Β-Lactam Resistance	Aminoglycoside Resistance	Macrolide Resistance	Fluoroquinolone Resistance	Others
A. baumannii PE1 (ST2)	4,023,584	Tn6207ISEc29ISAba26IS26ISAba125	bla ADC-25 bla OXA-23 bla OXA-66	aadA1 aacA4 aph(3′)-VIa aac(6′)Ib-cr strB strA armA	mph(E) msr(E)	aac(6′)Ib-cr	sul1 tet(B) catB8
A. baumannii PE2 (ST2)	4,023,635	Tn6207ISAba125ISAba26ISVsa3	bla ADC-25 bla OXA-23 bla OXA-66	aadA1 aacA4 aph(3′)-VIa aac(6′)Ib-cr strB strA armA	mph(E) msr(E)	aac(6′)Ib-cr	sul1 tet(B) catB8
A. baumannii PE3 (ST2)	3,850,309	ISEc29ISAba125ISAba2ISAba13IS17IS6100	bla ADC-25 bla OXA-23 bla OXA-82	aadA1 aacA2 aacA4 aadB aph(3′)-Ic aac(3)-Ia armA	mph(E) msr(E)	aac(6′)Ib-cr	sul1 catB8
A. baumannii PE4 (ST2)	3,965,839	Tn6207ISVsa3ISEc29ISAba125ISAba26	bla ADC-25 bla OXA-23 bla OXA-66	aadA1 aacA4 strB strA armA	mph(E) msr(E)	aac(6′)Ib-cr	sul1 tet(B) catB8
A. baumannii PE5 (ST2)	3,829,825	ISEc29ISAba125ISAba2ISAba13IS17IS6100	bla ADC-25 bla OXA-23 bla OXA-82	aadA1 aacA2 aacA4 aadB aph(3′)-VIa aph(3′)-Ic aac(3)-Ia armA	mph(E) msr(E)	aac(6′)Ib-cr	sul1 catB8
A. baumannii PE6 (ST2)	4,041,529	Tn6207ISVsa3ISEc29ISAba125ISAba26	bla ADC-25 bla OXA-23 bla OXA-66	aadA1 aacA4 strB strA armA	mph(E) msr(E)	aac(6′)Ib-cr	sul1 tet(B) catB8
A. baumannii PE7 (ST2)	3,841,128	ISEc29ISAba125ISAba2ISAba13IS17IS6100	bla ADC-25 bla OXA-23 bla OXA-82	aadA1 aacA2 aacA4 aadB aph(3′)-VIa aph(3′)-Ic aac(3)-Ia armA	mph(E) msr(E)	aac(6′)Ib-cr	sul1 catB8
A. baumannii PE8 (ST2)	3,852,243	ISEc29ISAba125ISAba2ISAba13IS17IS6100	bla ADC-25 bla OXA-23 bla OXA-82	aadA1 aacA2 aacA4 aadB aph(3′)-Ic aac(3)-Ia armA	mph(E) msr(E)	aac(6′)Ib-cr	sul1 catB8

2.3. Antimicrobial Resistance Genes (ARGs)

The A. baumannii isolates showed a wide variety of antibiotic resistance genes, mobile genetic elements (e.g., IS, sequence insertion) and several virulence factors (Table 1).

2.3.1. β-Lactam Resistance Genes

The A. baumannii strains analyzed in this study harbored molecular class C and class D β-lactamases. Overall, blaADC-25 and blaOXA-23 genes were found in all isolates with a co-presence of blaOXA-66 (four out of eight isolates) or blaOXA-82 (four out of eight isolates) (Table 1). Both blaOXA-66 and blaOXA-82 belong to the constitutive blaOXA-51-type genes. The OXA-82 enzyme differs from OXA-66 only by one amino acid residue. The leucine 157 (L157) in OXA-66 is replaced by valine (V157) in OXA-82.

2.3.2. Aminoglycoside Resistance Genes

In 100% of A. baumannii, resistance to aminoglycosides was mediated by aadA1, aacA4 and armA genes with the presence of the bi-functional gene aac(6′)Ib-cr. The armA gene confers resistance to gentamicin and amikacin, and it is generally plasmid-mediated. In four isolates we found the presence of other genes conferring resistance to aminoglycosides, such as aph(3′)-IVa, aacA2, aadB, aac(3′)-Ic and aac(3)-Ia (Table 1).

2.3.3. Fluoroquinolone Resistance Genes

Resistance to fluoroquinolones is mediated by the aac(6′)Ib-cr gene, a bi-functional gene which confers resistance to both fluoroquinolones and aminoglycosides (Table 1).

2.3.4. Other Antimicrobial Resistance Genes

The mph(E) and msr(E) (macrolide resistance), sul1 (sulphonamide resistance), tet(B) (tetracycline resistance) and catB8 (chloramphenicol resistance) genes were also detected in all A. baumannii isolates. The strA/strB genes, which confer resistance to streptomycin, were identified in 50% of the analyzed strains (Table 1).

2.4. Mobile Genetic Elements (MGEs)

A wide variety of sequence insertions (ISs) and transposon elements were identified in all analyzed strains (Table 1). In detail, transposon Tn6207 was found in 3 A. baumannii isolates. Sequence insertion elements such as ISAba2 of 1308bp length (3 isolates), ISAba13 of 1039 bp length (4 isolates), ISAba26 of 1318 bp length (4 isolates), IS26 of 820 bp length (1 isolate), ISVsa3 of 977 bp (3 isolates), ISEc29 of 1325 bp (7 isolates), IS6100 of 880 bp (4 isolates) and IS17 of 1040 bp (4 isolates) were identified in the genome of A. baumannii.

2.5. Virulence Factors and Other Mechanisms of Resistance

Virulome analysis allowed to identify genes involved in biofilm (bap and pgaABCD locus) and pili (csu) formation. The ompA porin, lipid A synthesis (lpxABCDLM locus), biosynthesis of the LPS core (lpsB), serum resistance (pbpG), phospholipase C and D (plc and plcD), LysR-type transcriptional regulator as well as acinetobactin genes cluster (bau, bas, bar, ent) were found in all ST2 A. baumannii isolates (Table 2). The resistance-nodulation-cell division efflux pump genes (RND: adeFGH) were also found in the totality of the analyzed strains. The gene hemO, related to iron uptake, and the catalase gene (katA), which protects bacteria from superoxidants produced by leukocytes as a host defense mechanism, were also present in all sequenced strains.

Table 2

Virulence factors of the eight A. baumannii analyzed in this study.

Category	Virulence Factors	Related Genes
Adherence	Outer membrane protein	ompA
Biofilm formation	AdeFGH efflux pump	adeF; adeG; adeH
	Biofilm-associated protein	bap
	Csu fimbriae	csuA; csuB; csuC; csuD; csuE
	Polysaccharide poly-N-acetylglucosamine	pgaA; pgaB; pgaC; pgaD
Enzyme	Phospholipase C	plc
	Phospholipase D	plcD
Immune evasion	LPS	lpsB; lpxA; lpxB; lpxD; lpxL; lpxM
Iron uptake	Acinetobactin	barA; barB; basA; basB; basC; basD; basF; basG; basH; basI; basJ; bauA; bauB; bauC; bauD; bauE; bauF; entE;
	Heme utilization	hemO
Regulation	Quorum sensing	abaI; abaR
	Two-component system (BfmRS)	bfmR; bfmS
Serum resistance	PbpG (Penicillin-binding protein)	pbpG
Stress adaption	Catalase	katA

3. Discussion

In this study, we used WGS to retrospectively investigate the resistance mechanisms in carbapenem- and colistin-resistant A. baumannii isolates in COVID-19 patients admitted to the ICU of Spirito Santo Hospital, Pescara (Central Italy). Analysis of the sequenced genomes revealed that all isolates shared the same MLST sequence type (ST2), which is the representative ST type of global clone II [28]. To date, the international clone ST2 is the most dominant type globally detected in all A. baumannii genomes sequenced [29]. Several studies showed the dissemination of CRAb isolates harboring the blaOXA-23-like and belonging to ST2 from different countries [30]. As reported in previous studies, blaOXA-23 was the most widely reported gene, and blaOXA-66 was the most common variant of OXA enzymes [31]. In our strains, the OXA-23 class D carbapenemase was identified in association with OXA-66 or OXA-82. The OXA-66 and OXA-82, which differ each other by L167V substitution, belong to the OXA-51-like enzymes. The OXA-51 is intrinsically over-expressed and it is able to confer high resistance to carbapenems in A. baumannii isolates [32]. The increased antibiotic resistance in A. baumannii is largely due to the actions of mobile genetic elements, to the activation of intrinsic resistance mechanisms, such as the chromosomal β-lactamase blaADC, and to the presence of efflux pumps [33]. In our study we found the presence of an AmpC enzyme, the ADC-25 cephalosporinase, and the AdeFGH RND efflux pumps in all A. baumannii strains. To our knowledge, the AdeFGH RND efflux pumps seem to play a major role in acquired resistance and may also be associated with carbapenem non-susceptibility in A. baumannii [34,35].

The pathogenicity of A. baumannii isolates analyzed in the present study is exacerbated by the presence of the abaI/abaR quorum sensing system which is involved in morphology, growth characteristics, biofilm formation, motility, resistance and virulence of these microorganisms [36]. In addition, in our A. baumannii we found two-component signal transduction system BfmRS, which seem to be implicated in the control of various virulence-related traits and acting as a global modulator of A. baumannii physiology [37,38]. It is well known that, A. baumannii showing a loss of BfmS results in a significant reduction of motility [39]. A. baumannii represents a serious problem for public health because of its intrinsic and acquired antimicrobial resistance determinants and pathogenicity factors. The main types of mobile elements involved in the capture and mobilization of ARGs in Gram-negative pathogens, including A. baumannii, is represented by gene cassettes, transposons and sequence insertions (ISs) [40]. These mobile structures could disseminate antibiotic resistance determinants between pathogens compromising antibiotic treatment [41,42]. In the present study, we found a wide variety of ISs belonging to different IS families such as IS3 (ISAba2), IS4 (ISEc29), IS5 (IS17 and ISAba13), IS6 (IS26 and IS6100), IS30 (ISAba125), IS91 (ISVsa3) and IS256 (ISAba26). Generally, two copies of the same or related ISs can move resistance genes as part of a composite transposon, and some of the ISs include a strong promoter that drives expression of the captured gene [41]. The ISAba1 was first identified in A. baumannii upstream of blaOXA-23, blaOXA-51, blaOXA-58 and blaADC-genes, and it seems to be an important factor in the genetic plasticity of this microorganism [43,44]. The presence of ISAba1 upstream of blaOXA-51-like genes might represent a real mechanism of carbapenem resistance [45]. Insertion of ISAba125 in the blaOXA-23 promoter sequence has been reported to be associated with overexpression of blaOXA-23, blaOXA-51 and carbapenem resistance phenotypes in A. baumannii [46]. In all analyzed strains we also found gene structures indicating the presence of the Tn6207 transposon. Tn6207 is a composite transposon containing a tetracycline efflux pump and its regulator genes tetB and aminoglycoside resistance genes strB and strA.

Respiratory viral infections, such as SARS-CoV-2, predispose patients to bacterial co-infections and secondary infections [47,48,49,50]. This represents a high risk for hospitalized patients. In some patients we have ascertained the co-presence of other microorganisms such as K. pneumoniae. Bloodstream CRAb and K. pneumoniae remain one of the most common ICU-acquired infections [50,51,52].

4. Materials and Methods

4.1. Strains Selection and Identification

A total of 43 A. baumannii strains were isolated from bloodstream infection of 43 COVID-19 patients with evident clinical signs of bacterial sepsis. The patients were admitted to the ICU ward of Spirito Santo Hospital (Pescara, Central Italy). Overall, 32 (74%) patients were male and 11 (26%) were female, with an age ranging from 31 to 82 years old. These patients had comorbidities such as kidney disease, diabetes, hypertension, or heart disease, and in some of them a co-presence of E. faecalis, S. marcescens and K. pneumoniae was also ascertained. The blood sample collection was repeated with an interval of 2 days for each patient. All samples were transferred to clinical molecular laboratory and analyzed following methods according to Clinical and Laboratory Standards Institute (CLSI) guidelines [53]. One A. baumannii strain per patient was included in the study. The strains, selected from positive cultures, were grown in Cled agar plates (Oxoid, UK) at 37 °C, overnight, and identified by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry (Bruker Daltonics, Billerica, MA, USA).

4.2. Antimicrobial Susceptibility

Antimicrobial susceptibility was determined by the Phoenix system (Becton, Dickinson and Company, Sparks, MD, USA) at Clinical Microbiology and Virology Unit, Spirito Santo Hospital, Pescara, Italy. The MICs for amikacin, gentamicin, ciprofloxacin, meropenem, colistin and trimethoprim–sulfamethoxazole association were also performed by conventional broth microdilution procedures in Mueller–Hinton broth (Biolife Italiana, Milan, Italy) supplemented with calcium and magnesium (CAMHB), using an inoculum of 5 × 105 CFU/mL according to CLSI [54].

4.3. WGS

A total of eight representative A. baumannii clinical strains, named PE/1-PE/8, were included in the genome sequencing. Total DNA was extracted using MagMax Microbiome Ultra Nucleic Acid Isolation kit (Applied Biosystems and ThermoFisher Scientific, Monza, Italy), according to the manufacturer’s instructions. Library preparation was performed as previously reported [55,56]. Sequencing of the library was carried out on an Illumina MiSeq using a paired-end 600-cycle protocol with a read length of 300 bp. DRAGEN FastQC + MultiQC v3.6.3 (https://basespace.illumina.com/apps/10562553/DRAGEN-FastQC-MultiQC, access date: 31 January 2022) were used for quality control and sequences filtering. Paired-end reads were assembled with Velvet v1.2.10 (https://basespace.illumina.com/apps/8556549/Velvet-de-novo-Assembly, access date: 21 February 2022). Multi-locus sequence typing (MLST) on assembled A. baumannii genomes was performed utilizing the Pasteur scheme. This scheme includes the identification of seven internal housekeeping genes: citrate synthase (gltA), homologous recombination factor (recA), 60-kDa chaperonin (cpn60), elongation factor EF-G (fusA), CTP synthase (pyrG), 50S ribosomal protein L2 (rplB) and RNA polymerase subunit B (rpoB) [57]. ResFinder4.1 and MobileElementFinder 1.0.3 were used to detect acquired antimicrobial resistance genes and mobile genetic elements, respectively (http://www.genomicepidemiology.org, accessed on 28 February 2022). Virulence Factor Database (VFDB) was used for the detection of virulence genes (http://www.mgc.ac.cn/VFs/, accessed on 4 March 2022).

5. Conclusions

In the present study all A. baumannii were isolated from patients which developed a bloodstream infection. A detailed molecular characterization of resistomes, including ARGs and MGEs, and virulence factor analysis were performed on the eight ST2 A. baumannii clinical isolates. This WGS analysis remarks the importance to know the genetic background of clinical pathogens. The use of a rapid method, such as next-generation sequencing, to identify resistomes and virulomes in clinical settings could be useful for an effective antimicrobial stewardship program.