Genomic analysis reveals high virulence and antibiotic resistance amongst phage susceptible Acinetobacter baumannii

In this study, we examined the association between antimicrobial resistance, CRISPR/Cas systems and virulence with phage susceptibility in Acinetobacter baumannii and investigated draft genomes of phage susceptible multidrug resistant A. baumannii strains from Thailand. We investigated 230 A. baumannii strains using 17 lytic A. baumannii phages and the phage susceptibility was 46.5% (107/230). Phage susceptibility was also associated with resistance to numerous antibiotics (p-value < 0.05). We also found association between biofilm formation and the presence of ompA gene among phage susceptible A. baumannii strains (p-value < 0.05). A. baumannii isolates carrying cas5 or combinations of two or three other cas genes, showed a significant increase in phage resistance. Whole-genome sequences of seven phage susceptible A. baumannii isolates revealed that six groups of antibiotic resistance genes were carried by all seven phage susceptible A. baumannii. All strains carried biofilm associated genes and two strains harbored complete prophages, acquired copper tolerance genes, and CRISPR-associated (cas) genes. In conclusion, our data exhibits an association between virulence determinants and biofilm formation among phage susceptible A. baumannii strains. These data help to understand the bacterial co-evolution with phages.

Acinetobacter baumannii is a major cause of opportunistic infection, especially among immunocompromised patients. The emergence of multidrug-resistant A. baumannii (MDR-AB) and even the extensively drug-resistant A. baumannii (XDR-AB) has been increasing worldwide, and especially in Thailand and Nepal 1-3 . Thus, alternative treatments against A. baumannii infection is urgently needed. Bacteriophages (phages) are good candidates, specifically killing host bacteria resulting in minimal impact on bacterial normal flora and with no known critical side effects 4 . To use phages for therapy, it is important to identify broad host range phages that kill the highest possible number of strains of bacterial species. In addition, it is crucial to understand the host-phage susceptibility mechanism. One of the important mechanisms impacting host range specificity is phage adsorption 5 . This is a crucial step in the infection process, which represents the initial contact between virus and its host and requires phage receptors; outer membrane proteins (OMPs), lipopolysaccharides and teichoic acids 5 . Several phages use the outer membrane protein OmpA as a receptor to infect Gram negative bacteria 6,7 . Alterations to this molecule result in a decrease of bacterial virulence in phage resistance strains 8 . Previous studies reported a positive correlation of antibiotic resistance in A. baumannii with phage susceptibility 9,10 . Phage susceptibility represents an evolutionary trade-off in A. baumannii strains that were selected for antibiotic resistance, particularly in hospital environments with high antibiotic use 10 . However, the mechanism and genetic basis of phage susceptibility in A. baumannii is not completely understood. In this study, we aimed to determine the association between antimicrobial resistance and virulence with phage susceptibility in a large collection of A.
phages (Table 1) into four clusters (I, II, III, and IV), depending on their ability to infect 230 A. baumannii isolates (Fig. 1). The vPhT19, vPhT29 and vPhT44 phages belonged to cluster I and infected between 13.91 and 20.43% of bacterial hosts. The phages vPhT01, vPhT25, vPhT35 and vPhT55 were grouped into cluster II, showing between 3.91 and 7.83% infectivity. Phages vPhT02, vPhT04, and vPhT39 showed infectivity against 14.78-18.26% of A. baumannii isolates and belonged to cluster III. Cluster IV contained five phages (vPhT05, vPhT09, vPhT48, vPhT49 and vPhT52), infecting approximately 1.30-3.48% of A. baumannii. Overall phage susceptibility of the 230 A. baumannii strains was 46.5% (107/230). The bacteria were divided into three groups. Group 1 ( Fig. 1; green) were the bacteria susceptible to a variety of phages and were mostly infected by phages in cluster II, III and IV. In Group 2 ( Fig. 1; red) were the bacteria mostly susceptible to phages in cluster I. In Group 3 ( Fig. 1; blue) were the phage resistant bacteria. Among the 199 MDR-AB isolates, 105 (52.76%) were infected specifically by at least one phage. Most of the CR-AB (53.64%) and XDR-AB (71.43%) were infected specifically by at least one phage. Only two of twenty-eight of non MDR-AB (7.14%) were phage susceptible strains (Fig. 1). Of the 230 A. baumannii strains, 205 were isolated from Thailand and 25 were from Nepal. We found 52.2% (107/205) of A. baumannii isolates from Thailand were phage susceptible (Supplementary Table S1). Interestingly, we observed that all 17 phages isolated from Thailand did not infect the 25 Nepalese A. baumannii clinical isolates.

Discussion
In Thailand, the incidence of MDR-AB, CR-AB and XDR-AB infection has increased in the past decade 3,11 . Tigecycline and colistin are still last resort drugs of choice for treatment of multidrug-resistant A. baumannii. However, cases of colistin resistance in A. baumannii was reported in Thailand and can cause serious problems in treatment outcome 12 . Phage therapy is one potential candidate for the treatment of multidrug resistant bacteria. We found that 53.5% of A. baumannii strains isolated from six hospitals in Thailand and Nepal were resistant to all 17 phages tested. This is close to what was found previously by Thawal et al. in 2012, where approximately 51% of A. baumannii strains tested in this study were found to be phage resistant 13 . We found that A. baumannii strains were more sensitive to phages from the same geographic area, since the Thai phages could not lyse A. baumannii strains isolated from Nepal. This is also consistent with previously published investigation, where one study has reported that phage-host specificity was limited to specific geographic areas 14 R16 and R24). The phage resistant strains were derived from various sources and did not have a common stressor or environmental selector meaning they presented a diversity of REP-type patterns. In contrast, the phage susceptible strains that can infect by at least one phage may be caused by the selective pressure of adopting resistance to multiple antibiotics and limitation of phage immunity. Bacterial biofilm formation is one strategy mediating protection against phage lysis. However, our study showed positive association between biofilm formation and phage susceptibility. Interestingly, some studies . These data imply that OmpA could also be a receptor for A. baumannii phage adsorption. Bacteria have numerous strategies to prevent bacteriophage infection and cell lysis. The CRISPR/Cas systems are one of the phage immunity systems that are present in bacteria and enabling the organisms to respond to and eliminate invading genetic material 18   www.nature.com/scientificreports/ compared to strains isolated from outside the hospital environment 19 . PCR with cas-specific primers showed that 19% of the A. baumannii isolates had a CRISPR-Cas system. The majority (70.5%) of the cas positive strains were classified as phage resistant strains. The positive correlation between the presence of cas5 or combinations of two or three different cas genes and phage resistant strains was statistically determined (p-value < 0.05). This data supports a hypothesis that CRISPR/Cas systems are one of the important phage resistance mechanisms in A. baumannii. We analyzed whole genome sequence of Acinetobacter spp. deposited on National Center for Biotechnology Information (NCBI) database showed that these contain six cas genes including cas1, cas2, cas3, cas5, cas6 and cas9 20,21 . Cas1 and Cas2 proteins recognize invading phage nucleic acids and insert them into the CRISPR/Cas array as a spacer in order to transcribe into pre-CRISPR-RNA (crRNA) 22 , whereas, the Cas5 protein has RNase activity, resulting in inhibition of the transcription machinery in phages 18 . The cas6 gene encodes a type I-F CRISPR-associated endoribonuclease Cas6/Csy4 protein, which has an important role in the cleavage

Macrolide resistance ND mph(E), msr(E) mph(E), msr(E) mph(E), msr(E) mph(E), msr(E) mph(E), msr(E) mph(E), msr(E)
Phenicol resistance ND catB8 ND ND ND ND ND www.nature.com/scientificreports/ of the repeat sequences to yield mature CRISPR-RNAs (crRNAs) that complementary pair with invading phage nucleic acid bases, causing destruction of the target phage DNA 23 . Phage resistance may be related to lower virulence, making the resistant bacteria less virulent than nonphage-resistant strains 24 . We detected antibiotic resistance, CRISPR-associated (cas) genes and virulence genes among A. baumannii isolates using PCR and found association between phage susceptibility with antibiotic resistance, CRISPR-associated (cas) genes and virulence genes. Whole genome sequencing of virulence genes involving biofilm formation revealed that the seven phage susceptible A. baumannii in this study harbored various virulence genes linked to the ability to form biofilms, iron acquisition and bacterial secretion systems. cas genes (cas1, cas3-cas2, and cas6) were found in two of seven the phage susceptible A. baumannii genomes (AB003 and AB329). Antibiotic resistance genes involved sulphonamide, tetracycline, β-lactam, aminoglycoside, macrolide and phenicol resistance were found in seven phage susceptible A. baumannii genomes. In a previous study, we identified the acquired copper tolerance genes, copRS that respond to copper toxicity in the genome of A. baumannii 25 . These genes were identified in two of the seven phage susceptible strains and both strains also showed the copper tolerance phenotype. The correlation between phage susceptibility and regulating heavy metal toxicity is in agreement with the findings of Zhang et al., where plasmid-borne cadmium resistant determinants were associated with the susceptibility of Listeria monocytogenes to phages 26 . Antibiotic resistance genes and heavy metal tolerance genes can be disseminated within the microbial population by horizontal gene transfer mechanisms using plasmids and phages. Among 19 groups of plasmids identified in A. baumannii, in this study, we found plasmids GR1, GR2, GR6, and pRAY in phage susceptible A. baumannii. Plasmid GR6, linked to the dissemination via horizontal gene transfer by conjugation was detected in four strains of phage susceptible A. baumannii 27 . In contrast, phages are important genetic vehicles for transferring genetic information between bacteria via transduction 28 . All seven phage susceptible A. baumannii strains in our analysis carried prophage associated sequences on their chromosomes. However, only two strains harbored complete prophages. Prophages are directly related to genome diversity, evolution and strains variation as well as an association with the presence of antibiotic resistance genes and virulence genes 29,30 . In addition, prophages are responsible for gene disruption or translocation to phenotypic changes in their host and can introduction of pathogenicity determinants that contribute positively to bacterial fitness 29,31 .

Virulence genes
An analysis of the clonal relationship of the seven phage susceptible A. baumannii genomes from this study with 149 previously published A. baumannii genomes (Fig. 2.) revealed that strain AB003, isolated from hospital HE, was closely related to strains AB0057 (CP001182), NCTC13421 (LS483472), A85 (CP021782) and USA15 (CP020595). The three strains isolated from hospital HB (AB140, AB229 and AB135) all belonged to the same ST type and are very closely related with Malaysian and Chinese isolates AC30 (CP007577), AC29 (CP007535), XH731 (CP021321) and Aba (CP030083). The presence of closely related bacterial strains collected from Thailand, Malaysia and China might be descriptive that these organisms shared ancestry and emerged at the same time, caused by rapid dissemination of genetic material.
In conclusion, we found that phage susceptibility was associated with antibiotic resistance and virulence among A. baumannii strains. Moreover, in silico analysis showed that seven strains of phage susceptible A. baumannii carried at least six antibiotic resistance genes which included sulphonamide, tetracycline, β-lactam, aminoglycoside, macrolide, phenicol and several virulence genes involved biofilm formation. Thus, the data from this study can be used as essential information about phage therapy in the future.

Materials and methods
Bacteria and phages used in this study. We used 230 A. baumannii isolates collected from inpatient units of five hospitals in Thailand (HA-HE) and one hospital in Nepal (HF) as described by Niumsup et al.,Joshi et al.,and Leungtongkam et al. 2,3,32 (Supplementary Table S1). Seventeen A. baumannii phages used in this study were isolated from wastewater treatment plants of two hospitals (HE and HG) in Phitsanulok province, Thailand 9,33 (Table 1). A. baumannii strains used as the host for phage propagation are shown in Table 1 Antimicrobial susceptibility testing. Antimicrobial susceptibility testing was performed using disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (2017) with fifteen antibiotics; amikacin, cefepime, cefotaxime, cefoperazone/sulbactam, ceftazidime, ceftriaxone, ciprofloxacin, colistin, gentamicin, imipenem, meropenem, piperacillin/tazobactam, tetracycline, tigecycline and trimethoprim/sulfamethoxazole. Escherichia coli ATCC 25922 was used as a quality control strain. All isolates were defined as being MDR-AB, when there was resistance to more than three antibiotic classes, as carbapenemresistant A. baumannii (CR-AB), when there was resistance to carbapenems and as XDR-AB, when there was resistance to all antimicrobial agents tested except the polymyxin colistin, and tigecycline. Non MDR-AB classified as bacteria that non-resistant or less resistant than the three antibiotic group 34 . phages susceptibility of A. baumannii. Assessment of phage susceptibility was determined by spot test on all A. baumannii isolates. A colony of bacteria was suspended in 0.85% (w/v) NaCl to the equivalent of a 0.5 McFarland standard (1 × 10 8 CFU/ml). The suspensions were swabbed on to Trypticase Soy Agar (TSA). Phage suspensions (2 µl at 1 × 10 8 PFU/ml) were dropped into the bacterial lawn. Then, plates were incubated at 37 °C for 8 h. The result of spot test being clearance at the location of phage inoculation when the host was sensitive to phage ( Supplementary Fig. S1). All experiments were performed in duplicate.
Detection of CRISPR-associated (cas) genes. The bacterial protein sequences and GenBank nucleotide sequences of well-identified cas genes, detected in six example Acinetobacter spp., were used as templates to design the specific primers using Primer-BLAST program (https ://www.ncbi.nlm.nih.gov/tools /prime r-blast /) (Supplementary Table S3). The genomic DNA of all isolates in this study was extracted by a previously described boiling method 36  CRISPR-associated (cas) genes within bacterial genomes were detected by using CRISPRCasFinder programs and the prediction of prophage regions genomes were performed by using PHASTER 42,43 . Prophage hits with status "questionable" were scored as incomplete prophages. The virulence genes were examined by BlastN algorithm using collection of virulence genes as queries.
Statistical analysis. The Fisher's exact test was used to analyze phage susceptibility associated with the drug resistance patterns, biofilm formation, ompA gene, copper tolerance and CRISPR-associated (cas) genes. Data with a p-value < 0.05 were classed as statistically significant.