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Genomic Analysis of two NDM-1 Providencia stuartii Strains Recovered from a Single Patient

In the last years, an increasing number of untreatable infections caused by drug-resistant microbes have impacted the health care system. Worldwide, infections caused by carbapenem-resistant (CR) Gram-negative bacilli have dramatically increased. Among the CR-Gram-negative bacilli, those producing carbapenemases, such as NDM-1, are the main concern. Different Enterobacterales harboring NDM-1 have been reported lately. Providencia stuartii, a member of the Morganellaceae family, is ubiquitous in the environment, but is also known to cause nosocomial infections. Here we describe the genomic analysis of two NDM-1- producing P. stuartii strains recovered from the same patient as well as other carbapenem resistant strains recovered from the same hospital. As a result of the genomic analysis thirteen resistance genes, including three to β-lactams (blaOXA-1, blaTEM-1, blaNDM-1), four to aminoglycosides (aphA6, aac(3)-IId, aac(2′)-Ia, aac(6′)-Ib-cr5), one to sulfonamides (sul1), two to chloramphenicol (catB3, catA3), one to rifampicin, one to bleomycin (ble), and one to tetracycline (tet(B)) were found. Moreover, a variety of mobile genetic elements, such as insertion sequences, plasmids and phage- related sequences, were found within P. stuartii genomes. The spread of carbapenem-resistant isolates remains a significant clinical and public health concern. Therefore, we considered that the detection of CR isolates is an essential step in addressing this problem.


Introduction
Carbapenem antibiotics are generally considered to be the last option in the treatment of patients with severe bacterial infections. The recent increase in the rates of carbapenemresistant Enterobacterales (CRE) among healthcare-associated Enterobacterales is a major concern [1][2][3]. Under those circumstances, CRE infections are associated with high mortality due to limited treatment options [1,3]. In addition, according to the CDC report Antibiotic Resistance Threats in the United States 2019, CRE was also found in the urgent threat category. Three groups of carbapenemases (KPC, NDM, and OXA-48) are currently considered to be the major β-lactamases with epidemiological and clinical significance [4,5]. Providencia species are members of the Morganellaceae family, among them, P. stuartii and P. rettgeri are the most common causes of infections in hospitalized patients, mainly urinary tract infections [6,7]. Moreover, cases of peritonitis, endocarditis, meningitis, bacteremia due to P. stuartii has been also reported [6,[8][9][10][11].
P. stuartii is intrinsically resistant to colistin and tigecycline, but carbapenem-resistant P. stuartii (CRPS) are Amparo Hoard, Sabrina Montaña have contributed equally to this work.
In the present study, we described the clinical data and the whole genome sequence (WGS) analysis of two NDM-1 CRPS strains from a 55-year-old patient with a history of chronic obstructive pulmonary disease, hypertension and diabetes. Moreover, we described the molecular characterization of two more CR isolates (Klebsiella pneumoniae and Pseudomonas aeruginosa) from the same patient together with others CR isolates (P. stuartii, P. rettgeri, Proteus mirabilis and K. pneumoniae) recovered from the same hospital.

Clinical Case
In April of 2018, the patient was admitted to the intensive care unit (ICU) due to cranial brain trauma requiring craniectomy with evacuation of the intracerebral hematoma plus surgical clipping of the sylvian aneurysm and placement of an external ventricular shunt. In June, the patient presented febrile registers associated with hypoventilation and fatigue, therefore blood cultures, catheterdrawn blood cultures and urine cultures were performed. K. pneumoniae (Kb846) grew in the two blood culture bottles and in the blood-culture drawn from the catheter. In addition, the growth of Providencia stuartii (Ps848) was obtained in one of two bottles of blood culture. In the urine culture, Pseudomonas aeruginosa (PaC850) and P. stuartii (Ps850) were isolated ( Table 1). The established treatment was with fosfomycin, meropenem and amikacin for 14 days. During treatment the patient evolved with increased blood sodium attributable to fosfomycin, so it was changed to tigecycline. With the exception of an oxygen requirement following a tracheostomy, the patient´s condition remained stable, steadily improving favorably without febrile records (Table 2).

Other Clinical CR Strains
At the same time, four other CR isolates were recovered. Two isolates were P. stuartii (Ps722) and P. rettgeri (Pr783), which were obtained from the surgery service. The other two isolates were a Proteus mirabilis isolate (Pm856) obtained from a urine culture of a dialyzed patient, and a K. pneumoniae isolate (Kb443) from a tracheal aspirate of a pediatric patient, were recovered (Table 1).

Antimicrobial Susceptibility Assay
The recovered isolates were characterized at species level by conventional biochemical tests and by the VITEK 2 System (bioMérieux). The susceptibility assay was performed using the VITEK 2 (AST-279 panel). The minimum inhibitory concentration (MIC) results were interpreted using the Clinical and Laboratory Standards Institute [20]. For the rapid detection of carbapenemases, a colorimetric method (Rapid Blue-Carba Test) was also performed [21]. A color change obtained after the reading limit time (120 min) was recorded as an indeterminate result. To study the presence of metallo-β-lactamases (MBL), a double-disc diffusion test using meropenem and imipenem (10 μg, OXOID, UK) was performed on Mueller Hinton agar using an 1900/750 μg EDTA/SMA disc (Laboratorios Britania, Argentina). Additionally, to detect the presence of serine or oxacillinase carbapenemases, the screening method using the KPC-MBL Kit Confirm ID Pack (Rosco Taastrup, Denmark) containing meropenem (10 μg), meropenem 10 μg + dipicolinic acid (Metalloβ-Lactamase inhibitor); meropenem 10 μg + cloxacillin (AmpC inhibitor); meropenem 10 μg + phenylboronic acid (KPC and AmpC inhibitor) was carried out [22].

General Molecular Techniques
To perform the molecular characterization of the isolates recovered, total DNA extraction was carried out using Wiz-ard® Genomic DNA Purification Kit according to manufacturer instructions (Promega, Madison, WI). PCR reactions using GoTaq® enzyme (Promega, Madison, WI) were carried out for the most common carbapenemase genes (bla IMP , bla VIM , bla KPC and bla NDM ), as well as, the presence of mobile elements related with them. Plasmid extractions were carried using two different techniques, the QIAprep Spin Miniprep Kit following manufacturer instructions (Qiagen Germantown, MD, USA) and a manual plasmid extraction design for megaplasmids [23,24].

Whole-genome Sequence and Sequences Analysis of Ps848 and Ps850 Clinical Isolates
Genomic DNA was extracted using a DNeasy Blood and Tissue kit from Qiagen (Qiagen Germantown, MD, USA). Whole-genome shotgun sequencing was performed using Illumina NextSeq550. De novo assembly was performed with SPAdes Assembler (version.3.1.0) [25] using a preassembly approach with Velvet [26]. The RAST (Rapid Annotation using Subsystem Technology) server was used to predict and annotate open reading frames [27], and BLAST (v.2.0) software was utilized to confirm the predictions. The Whole Genome Shotgun project has been deposited at GenBank under the accession JABLTB000000000 and JABLTC000000000 for Ps848 and Ps850, respectively. ARG-ANNOT [28] and The Resistance Gene Identifier (CARD-RGI) [29] were used to identify antibiotic resistance genes. The ISfinder and PHASTER 's softwares were used to identify insertion sequences and phages and prophages, respectively [30,31]. The RaxML software was used to predict the phylogenetic relation among the strains [32].

Antibiotic Susceptibility and Carbapenemase Identification in Carbapenem-Resistant Gram-Negative Strains
Antimicrobial susceptibility testing revealed that Ps848, Ps850 and Ps783 exhibited an extreme-drug resistant phenotype, where Ps850 and Ps783 were susceptible to amikacin and trimethoprim/sulfamethoxazole, and Ps848 was only susceptible to amikacin. Among Kb isolates, Kb443 was only susceptible to colistin and nitrofurantoin, while  13 13 Kb846 was resistant to all of the antibiotic tested. PaC850 was multidrug resistant with susceptibility to amikacin, gentamicin and colistin. Moreover, Pm856 and Pr783 were both susceptible to amikacin and Pr783 was also susceptible to ciprofloxacin ( Table 1). The four isolates obtained from the same patient showed a positive result for the Rapid Blue-Carba test: a weak positive result (light green color) for Ps848, Ps850 and PaC850 isolates between 60 and 90 min of incubation and a strong positive result for isolate Kb846 at 30 min of incubation were obtained. Moreover, a positive result was also obtained for the isolates Pm856 and Kb443. Also, an indeterminate result was obtained for the isolates Ps722 y Pr783 In addition, synergy between carbapenems and EDTA was observed for Ps848, Ps850, PaC850, Pr783, Ps722, Pm856 and Kb443 isolates. Additionally, the KPC-MBL Kit Confirm ID Pack showed the presence of MBL-type carbapenemase in Ps848, Ps850, PaC850, Pr783, Ps722, Pm856, Kb846 and Kb443 isolates. A halo difference of ≥ 5 mm in the case of meropenem + dipicolinic acid with respect to the meropenem 10 μg disc was observed. Instead, for isolate Kb846, a halo difference of ≥ 5 mm in the case of meropenem + phenylboronic acid with respect to the meropenem 10 μg disc was observed ( Table 1).

WGS of Ps848 and Ps850: Genome Features, Antibiotic Resistance Determinant, and Mobile Genetic Elements
The draft genome of Ps848 consists of 4,404,416 bp. The RAST server predicted 4379 protein-coding genes with a corresponding G + C content of 41.2%, whereas the draft genome of Ps850 consists of 4403,514 bp and the RAST server predicted 4382 protein-coding genes with a corresponding G + C content of 41.2% (Table 1).
Phylogenetic analysis was performed using RaxML and showed that both strains belong to the same clone (Fig. 1).
Analysis of the mobilome and horizontal gene transfer related features showed a remarkable similarity between the content of both genomes. Both strains carried genetic traits that evidenced the presence of several mobile genetic elements (MGEs): a CR-bearing plasmid, 8 different ISs (ISSf1, ISKpn13, IS1326, ISAba14, IS15, ISCfr1 and two copies of ISAba125) and several phage-related regions (two intact prophages, eight incomplete prophages and one questionable prophage). The sequences corresponding to the plasmid found in both Ps strains were highly similar to the Proteus cibarius plasmid p152 (AN CP047288) and the P. mirabilis pPm50 (AN MG516911). In-depth backbone analysis revealed that plasmid shares several traits with P. rettgeri plasmids p16-01,619-1 (AN KX832929) and pPrY2001 (AN NC_022589), which are pNDM-BJ01-like elements and thus far they have not been classified in an incompatibility group. P. stuartii Ps848 and Ps850 strains also encoded the same type I-F CRISPR-Cas system, which consisted of a cas operon surrounded by 2 CRISPR arrays of 7 and 11 identical repeat and spacer sequences Four additional CRISPR arrays with high confidence were found at different loci (Supplementary Table S1).

Analysis of bla NDM-1 genetic context and integrons found in Ps848 and Ps850.
A depth analysis was carried out to characterize the genetic context of bla NDM-1 gene, which was found to be plasmid located. The genetic context flanking the upstream and downstream (5′ and 3′ end) region of the bla NDM-1 gene was the same in both stains. The 5′ region was composed of the insertion sequence ISAba125 and aphA6 gene (Fig. 2a). Whereas the downstream (3′ end) region was composed of a bleomycin resistance gene (ble) and a phosphoribosylanthranilate isomerase (trpF). Moreover, another ISAba125 was 7,019 bp away (from the 3′ end of the bla NDM-1 until the 5′ end of ISAba125) (Fig. 2a).
Regarding to the others the others CR isolates found in the same hospital, Pm856, Kb443, Ps722, Pr783 exposed the presence of bla NDM-1 . bla TEM-1 gene was only found in Pm856 and Kb443. Moreover, a class 1 integron harboring the bla VIM-2 gene was found in Ps722, Pm856 and Kb443.

Discussion
In this work, we present the molecular characterization of two carbapenemase-producing P. stuartii and two more CR isolates (P. aeruginosa and K. pneumoniae) recovered from Fig. 1 Core-genome phylogeny of P. stuartii and P. rettgerii. Twenty P. stuartii genomes and eighteen P. rettgeri genomes was represented in the phylogenetic tree. P. stuartii and P. rettgeri available in GenBank database is indicate in red and blue, respectively. The isolates sequenced are indicate in black Fig. 2 a Genetic context of bla NDM-1 gene found in Ps848 and Ps850. b Schematic representation of the class 1 integron found in Ps848 and Ps850 a single patient. We also performed the molecular characterization of other CR isolates, recovered from the same period of time in the hospital (P. stuartii, P. rettgeri, P. mirabilis and K. pneumoniae). The presence of more than one CR Enterobacterales harboring plasmid-mediated NDM-1 in a single patient has been described in the literature [33,34]. Recently, Abdallah et al. also described a case of hospitalacquired pneumonia due to CR P. stuartii and K. pneumoniae in a single patient [35]. Among Providencia 's members, P. stuartii and P. rettgeri are the most common causes of infections in hospitalized patients, and are mainly associated with urinary tract infections. It has been also described in nosocomial outbreaks caused by multidrug resistant strains of P. stuartii [36][37][38][39]. In addition, P. stuartii clinical isolates harboring plasmids encoding different beta-lactamases have also been studied [12,13,40]. The WGS analysis of Ps850 and Ps848 exposed the presence of 13 resistance genes (bla OXA-1 , bla TEM-1 , bla NDM-1 , aphA6, aac(3)-IId, aac(2′)-Ia and aac(6′)-Ib-cr5, sul1, catB3, catA3, arr3, ble and tet(B)). These results are in accordance with previous reports that showed the presence of MDR P. stuartii clinical isolates [41][42][43]. Moreover, the presence of P. stuartii clinical isolates harboring bla NDM-1 gene was also described in the literature [12,13,35]. Among the other CR isolates analyzed Pm856, Kb443, Ps722 and Pr783 were also NDM-1 positive. Furthermore, in the literature the presence of P. mirabilis, K. pneumoniae and P. rettgeri harboring bla NDM-1 gene was also reported [44][45][46]. Similarly, the bla NDM-1 gene with the same 3′ end region found in Ps850 and Ps848 was previously reported present in plasmids in E. coli 3426 (AN LS992188) and C. freundii CRE3 (AN MK101346). While the same context (upstream and downstream) was previously reported in A. nosocomialis IEC38057 (AN MK053934) and A. baumannii (AN LC032101).
In addition, a variety of MGEs were also identified in Ps850 and Ps848 genomes, such as a resistance plasmid, ISs, several phage-related regions and a CRISPR-Cas type I-F. The analysis of the plasmids identified in Ps850 and Ps848, showed that they share several traits with P. rettgeri plasmids p16-01,619-1 and pPrY2001. As a common feature, they all encode the beta-lactamase NDM-1. Martinez et al., proposed that all P. rettgeri NDM positive strains harbor a putative conjugative transfer mechanism named Tra-Pre, which is not present in pNDM-BJ01-like plasmids from Acinetobacter spp [47]. We hypothesized that this novel plasmid backbone could have emerged as the consequence of different recombination events (homologous and nonhomologous) between coexisting plasmids in the clinical setting. Furthermore, a class 1 integron was also identified in both genomes. The same class 1 array was reported in K. pneumoniae WCHKP020030 (AN CP028791) and integrons with regions similar to VR-1 were also described in C. freundii AA535 and AA593 (AN MH594477 and MH594478, respectively). Only a few cases of a P. stuartii harboring class 1 integrons have been reported [16,48]. In addition, the presence of the class 2 integron and the Tn7 transposon associated genes were also found in the analyzed genomes. Barlow et al., previously described the presence of the intI2 gene in P. stuartii isolates not associated with the typical class 2 integron array. They also suggested that the intI2 gene present in their isolates could be part of a novel superintegron structure; however, further analysis needs to be done to confirm [49]. The presence of P. stuartii harboring a class 2 integron was scarcely described in the literature [49,50]. Regarding the other CR isolates PaC850, Kp846, Ps722, Pm856 and Kb443 harbored also class 1 integron. The spread of carbapenemase-producing isolates remains a significant clinical and public health concern. Therefore, we considered that the detection of carbapenemase production is an essential first step in combating this problem.