Emergence of ST63 Pandrug-Resistant Acinetobacter pittii Isolated From an AECOPD Patient in China

Acinetobacter sp. is among the ESKAPE organisms which represent the major nosocomial pathogens that exhibited a high resistance rate. A. pittii, frequently associated with antimicrobial resistance particularly to carbapenems, is one of the most common Acinetobacter species causing invasive infection. Pandrug resistant A. pittii has rarely been reported. Here, we report the case of a patient with acute exacerbations of chronic obstructive pulmonary disease three years after double lung transplantation and developed severe pneumonia associated with pandrug resistant A. pittii infection. Phenotypic and genomic characteristics of this pandrug resistant isolate (17-84) was identified, and the mechanisms underlying its resistance phenotypes were analyzed. Isolate 17-84 belonged to ST63, carried a non-typable and non-transferable plasmid encoding multiple acquired resistance genes including carbapenemase gene bla OXA-58. Point mutations and acquired resistance genes were identified which were associated with different drug resistance phenotypes. To our knowledge, this is the first detailed phenotypic and genomic characterization of PDR A. pittii causing severe infections in clinical settings. Findings from us and others indicate that A. pittii could serve as a reservoir for carbapenem determinants. The emergence of such a superbug could pose a serious threat to public health. Further surveillance of PDR A. pittii strains and implementation of stricter control measures are needed to prevent this emerging pathogen from further disseminating in hospital settings and the community.


INTRODUCTION
Acinetobacter species are among the high-priority nosocomial pathogens which could cause severe infections among immunocompromised patients (Howard et al., 2012). Infections caused by Acinetobacter spp. could be untreatable as they exhibit the potential to develop resistance to a wide range of antibiotics, particularly carbapenems (Wong et al., 2019). Carbapenem resistance in Acinetobacter spp. is mainly caused by the production of carbapenem-hydrolyzing class D blactamases (CHDLs) including OXA-23-like, OXA-58-like, and OXA-24-like enzymes (Ayibieke et al., 2020). Acinetobacter pittii, formerly termed genomospecies 3 of the A. calcoaceticus-A. baumannii complex, is a close relative of A. baumanii which is increasingly recognized as a significant cause of hospitalacquired infections (Chusri et al., 2014). A recent multicenter investigation in Japan suggested A. pittii was the most common species causing invasive Acinetobacter infection (Kiyasu et al., 2020). The terms multidrug resistance (MDR), extensive drug resistance (XDR) and pandrug resistance (PDR) were used worldwide to define the non-susceptibility level of isolates, which are resistant to at least one agent in three or more antimicrobial categories, to at least one agent in all but two or fewer categories and to all agents in all categories, respectively (Magiorakos et al., 2012). MDR A. pittii including those resistant to antibiotics quinolones, carbapenems, etc., was reported across continents, yet PDR A. pittii was rarely reported (Gu et al., 2015;Brasiliense et al., 2019;Chen et al., 2019a;Cosgaya et al., 2019;Chopjitt et al., 2021). Here, we report the case of a patient with acute exacerbations of chronic obstructive pulmonary disease (AECOPD) three years after double lung transplantation (DLT) and developed severe pneumonia associated with PDR A. pittii infection. Currently available therapeutic options for such isolates remained limited and relied largely on antibiotics recently approved by the Food and Drug Administration (FDA) and European Medicines Agency (EMA) such as eravacycline, rifabutin and cefiderocol (Livermore et al., 2016;Trecarichi et al., 2019). This severe situation called for urgent action to prevent the healthcare-associated transmission of such PDR A. pittii.

Patient Information and Strain Isolation
In 2017, a 59-year-old male patient was admitted to hospital (Day 0) with a diagnosis of AECOPD. The patient underwent DLT three years before hospitalization and regular medical examinations revealed no abnormalities after DLT. On Day 2, the patient was referred to an intensive care unit (ICU) due to the development of severe symptoms complicated with type 2 respiratory failure. A CT scan showed inflammations and fibrosis, reduced transparency and patchy shadows in both lungs, inferring occurrence of bilaterally pleural effusion. He received mechanical ventilation and other supporting treatments. On Day 20, Stenotrophomonas maltophilia, Acinetobacter sp. and Aspergillus sp. strains were recoverable from the sputum sample. On Day 25, a sputum culture became positive for carbapenem-resistant Acinetobacter sp. Amphotericin B, imipenem, meropenem and polymyxin B were introduced. A tracheal aspiration culture on Day 34 was positive for carbapenem-and colistin-resistant Acinetobacter sp. From Day 35, lung infection worsened with giant pulmonary bulla and gradual decline in leukocytes is continued. The patient discharged following critical illness and was transferred to another medical center on Day 41. A sputum culture performed on Day 35 flagged positive for carbapenem-and colistin-resistant Acinetobacter sp. (isolate 17-84).

Species Identification, Antimicrobial Susceptibility Testing, and Conjugation Assays
The species of the isolate was identified using the matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF MS) (Bruker Daltonik GmbH, Bremen, Germany) and verified with the whole genome sequencing results. Antimicrobial susceptibility of 15 commonly used antibiotics (amikacin, gentamicin, kanamycin, meropenem, imipenem, ertapenem, cefotaxime, ampicillin, aztreonam, ciprofloxacin, colistin, tetracycline, tigecycline, trimethoprimsulfamethoxazole, azithromycin) was tested using broth dilution method and interpreted according to the Clinical Laboratory Standards Institute (CLSI) guidelines, with the exception of tigecycline and colistin (CLSI 2020). The breakpoint of colistin was interpreted according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) recommendations (EUCAST, 2020). No interpretive criteria are available for tigecycline from CLSI, EUCAST or FDA. Plasmid transferability was tested by conjugation using a rifampicin-resistant mutant of A. baumannii ATCC 17978 as the recipient strain. Presumptive transconjugants were selected using Mueller-Hinton II agar plates supplemented with meropenem (0.5 mg/L) and rifampicin (600 mg/L).

Whole Genome Sequencing and Bioinformatics Analysis
Genomic DNA was extracted from overnight cultures by using the PureLink Genomic DNA Mini Kit (Invitrogen, Carlsbad, CA, USA). Whole genome sequencing was performed using both the Illumina HiSeq (Illumina, San Diego, CA) and the Oxford nanopore MinION (Oxford, UK) platforms. Hybrid assembly of both sequencing reads was conducted using Unicycler (v 0.4.4) (Wick et al., 2017). Complete genome sequence was annotated by the RAST tool and edited manually (Overbeek et al., 2013). Multilocus sequence typing (MLST) was conducted using MLST v2.1 (Seemann, 2021). Acquired antibiotic resistance genes were identified by ResFinder 4.1 (Bortolaia et al., 2020). Plasmid replicons were analyzed using the A. baumannii PCR-based replicon typing (AB-PBRT) scheme (Bertini et al., 2010). Insertion sequences (ISs) are identified using ISfinder (Siguier et al., 2006). Plasmid map was plotted using DNAPlotter v1.11 (Carver et al., 2009). Mutations in the pmrCAB and lpxACD operons which were associated with polymyxin resistance were determined by aligning their amino acid sequences of strain 17-84 with that of a polymyxin-susceptible strain A. pittii ST220 (NZ_CP029610) (Zhang et al., 2018). Point mutations conferring resistance to quinolones (gyrA/B and parC/E) were detected by comparing the coding sequences in the 17-84 genome with the corresponding sequences in the genome of an ST63 quinolonesusceptible A. pittii strain in our collection (data not shown).
Isolate 17-84 contained a 3,914,011 bp chromosome and a 108,715 bp plasmid designated as p17-84_OXA. It belonged to ST63 A. pittii (Pasteur Scheme). Resistance genes including bla ADC-25 and bla OXA-51 both not associated with insertion sequences were identified on the chromosome of isolate 17-84. bla ADC-25 and bla OXA-51 -like genes were both naturally occurring b-lactamase-encoding genes in Acinetobacter spp. which does not contribute to intrinsic b-lactam resistance unless an insertion sequence such as ISAba1 is located upstream (Zhou et al., 2020).
Previous studies have reported the mutations on different chromosome-encoded genes associated with quinolone, polymyxin and tetracycline resistance among Acinetobacter spp. In isolate 17-84, quinolone resistance-associated mutations in topoisomerase parC (S84L), DNA gyrase gyrA (S81L) and gyrB (A414T) genes were detected. Among these, gyrA (S81L) and parC (S84L) target site mutations have been demonstrated to lower the affinity for quinolone, while the function of A414T mutation in gyrB remained to be investigated (Hujer et al., 2009). The major mechanisms of resistance to colistin in Acinetobacter sp. included complete loss of lipopolysaccharide (LPS) resulting from mutations in lpxACD genes, and phosphoethanolamine addition to LPS mediated through mutations in the pmrCAB operon (Beceiro et al., 2014). Mutations in the pmrC gene including Q143L, L146F, Q148K, I171V, and L258S were detected in strain 17-84 and whether these mutations contributed to the colistin resistance phenotype remined to be studied. No acquired tetracycline resistance determinant was observed in strain 17-84, and its resistance to tetracycline could be associated with the presence of diverse efflux pump genes (adeDE, adeN-adeIJK, adeL-adeFGH, adeRS-adeAB) belonging to the resistancenodulation-division (RND) family that were reported to confer multidrug resistance (Pagdepanichkit et al., 2016). The function of these efflux pump genes warrant further verification.
Plasmid p17-84_OXA contained 132 ORFs with a GC content of 39.6%. It was 100% identical to the 105,591 bp plasmid pOXA58_100004 (CP027249) from an A. pittii strain isolated from Sichuan province in China at 90% coverage. Compared with bla ADC-25 and bla OXA-51 genes were also detected on the chromosome of the genome, but not in association with an IS.
pOXA58_100004, plasmid p17-84_OXA has lost the floR resistance gene and gained a~9 Kb fragment encoding hypothetical proteins. p17-84_OXA and pOXA58_100004 both carried multiple antimicrobial resistance genes, including aph(3')-VIb, aac(3')-IId, bla OXA-58 , bla PER-1 , sul2, msr(E), mph(E) and strA (2 copies) and strB genes ( Figure 1). aph(3')-VIb and aac(3)-IId confer aminoglycoside resistance by encoding O-phosphotransferase-type and Nacetyltransferase-type aminoglycoside-modifying enzymes, respectively. bla OXA-58 and bla PER-1 confer b-lactam resistance, with the former encoding carbapenem-hydrolysing class D blactamase oxacillinase OXA-58. bla OXA-58 was bracketed by insertion sequences ISAba3 with the genetic structure of ISAba3bla OXA-58 -ISAba3. ISAba3 drove the overexpression of bla OXA-58 , which is associated with imipenem resistance (Nguyen et al., 2020). sul2 confers resistance to sulfonamide. msr(E) and mph(E) are associated with macrolide resistance. strA and strB genes confer resistance to streptomycin. p17-84_OXA was non-typeable by AB-PBRT and non-transferable by conjugation under the experimental conditions. Infections caused by MDR or PDR A. baumannii have been widely acknowledged, whereas non-baumannii Acinetobacter spp. infections are rarely reported in China (Yang et al., 2012;Zarrilli et al., 2013;Al Atrouni et al., 2016). The phenotypical species identification methods available to most diagnostic laboratories were not able to accurately differentiate the species belonging to the A. baumannii complex (Vijayakumar et al., 2019). The technological limitations in species discrimination have led to underestimation of non-baumannii Acinetobacter species in human infections (Pailhories et al., 2018). This could have led to the rare report of infections caused by PDR A. pitti previously, even though the pandrug-resistance phenotype in A. baumannii complex is common in some clinical settings (Karakonstantis et al., 2020a;Karakonstantis et al., 2020b). According to a recent study from a French hospital, A. pittii was isolated more frequently associated FIGURE 1 | Circular plasmid map of p17-84_OXA. Pink, blue, and green arrows indicated the antimicrobial resistance genes, mobile elements and other ORFs, respectively. Antimicrobial resistance genes carried by the plasmid were labelled. with bloodstream infections than A. baumannii, highlighting the importance of A. pittii in clinical settings (Pailhories et al., 2018). Chen et al. reported the co-occurrence of two carbapenemase genes, bla OXA-58 and bla NDM-1 , on a single plasmid in A. pittii (Chen et al., 2019b). Yang et al. (Yang et al., 2012) reported an outbreak of ST63 carbapenem-resistant A. pittii which carried a 45-kb novel bla NDM-1 -bearing plasmid in a Chinese ICU. This evidence suggested ST63 A. pittii was readily transmissible and has the capacity to acquire antimicrobial determinants and become competent, which could pose serious threat on public health. In vivo emergence of resistance to last resort antimicrobials including tigecycline and colistin was reported to be quite common in Acinetobacter spp (Karakonstantis, 2020). The fitness cost associated with acquired resistance and the virulence of PDR Acinetobacter sp. remained a major issue for medical treatment (Karakonstantis, 2020).Besides, differentiating A. baumannii complex infection from colonization remains difficult and further complicated particularly in polymicrobial infections such as the case in the current study (Karakonstantis and Kritsotakis, 2021). We acknowledge the limitation that the antimicrobial resistance profiles of strain 17-84 to recently approved antibiotics including eravacycline, cefiderocol and rifabutin, which are potential last resort treatment options, were not tested (Karakonstantis et al., 2020c;Luna et al., 2020). A further study on the treatment of infections caused by such PDR Acinetobacter sp. is underway to provide more insights into the therapeutic scheme.
In conclusion, we reported a case of severe pneumonia due to A. pittii infection in a patient with AECOPD three years after DLT. Phenotypic and genomic characterization indicated the A. pittii strain belonged to ST63, harbored a bla OXA-58 -bearing MDR plasmid, carried resistance-associated point mutations and efflux pump genes, and exhibited a PDR phenotype. To our knowledge, this is the first detailed characterization of PDR A. pittii causing severe infections in clinical settings. Attentions should be paid to this emerging pathogen to prevent it from further disseminating in hospital settings and the community.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/supplementary material. The complete genome sequence of Acinetobacter pittii strain 17-84 was deposited in GenBank with accession numbers CP059479 (chromosome) and CP059478 (plasmid p17-84_OXA) under BioProject accession PRJNA648287.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
LYang and ND collected and characterized the strain and participated in manuscript writing. ND, CX, and LYe performed the whole genome sequencing, bioinformatics analysis and the conjugation assay. SC designed and supervised the study, interpreted the data, and wrote the manuscript. All authors contributed to the article and approved the submitted version.