Molecular epidemiology and insights into the genomes of Acinetobacter calcoaceticus - Acinetobacter baumannii complex

The genus Acinetobacter is amongst the most common causes of nosocomial bacterial infections. Even though Acinetobacter baumannii is the most frequent identified species causing a wide range of infections in humans, other species such as Acinetobacter pittii, Acinetobacter nosocomialis, Acinetobacter haemolyticus, Acinetobacter johnsonii, Acinetobacter lwoffii and Acinetobacter ursingii are sporadically observed as nosocomial pathogens. The four species that show similar phenotypes were grouped together as “Acinetobacter calcoaceticus – A. baumannii (ACB) complex”, i. e. A. baumannii, A. nosocomialis, A. pittii and less clinically important A. calcoaceticus. Owing to the high complexity within Acinetobacter genus, it is difficult to differentiate Acinetobacter into species level and these organisms are frequently misidentified. Acinetobacter spp. exhibit a great propensity to acquire antimicrobial resistance determinants and rapidly develop multidrug-resistant (MDR) phenotypes especially resistance to carbapenems, the last resort of antimicrobials to treat Acinetobacter infections. A high prevalence and endemic situations of carbapenem-resistant A. baumannii (CRAB) have been observed in multiple geographical areas such as Asia-Pacific and South America where predominant A. baumannii clonal lineages were noted. The international clone (IC) 2 is recognised as the most successful clone of A. baumannii causing outbreaks and persisting in hospital environments worldwide, particularly in Asia. Despite the increased amount of research on Acinetobacter epidemiology, potential virulence and evolution, little is known about the factors that may have contributed to the success of this well-known clone, IC2. The broad objectives of my PhD are to 1) develop a rapid method to assist in species identification of Acinetobacter non-baumannii, 2) determine the molecular epidemiology of A. baumannii, primarily from Thailand and 3) describe the genome of A. baumannii IC2 isolates from Thailand and compare these genome data with A. baumannii IC2 isolates from Japan, Malaysia and Singapore. Firstly, a multiplex PCR was developed to detect intrinsic oxacillinases encoding genes (blaOXAs), which assisted in rapid identification of multiple Acinetobacter species including A. lwoffii/Acinetobacter schindleri, A. johnsonii, A. calcoaceticus, A. haemolyticus and Acinetobacter bereziniae. Additionally, 30 novel blaOXAs variants were identified in this study. The investigation of Acinetobacter spp. collected worldwide revealed that acquired-type blaOXAs disseminated globally in Acinetobacter spp. as opposed to A. baumannii causing carbapenem resistance particularly in A. pittii. Carbapenem resistance was also observed in A. pittii from Australia and Thailand. Through the genomes of the two A. pittii strains ST119 and a novel ST655, several classes of antimicrobial resistance genes including a novel blaOXA-421, blaOXA-23, blaOXA-96, blaOXA-10, blaIMP-4, blaVEB-7, blaCRAB-2, floR, cmlA1, aar-2 and dfrA10 were identified. This emphasises the importance of A. pittii as an impending multidrug resistance pathogen in this region. Secondly, to explore an endemic situation of multidrug-resistant Acinetobacter spp., 300 non-repetitive ACB complex isolates, mainly A. baumannii from the largest tertiary hospital in Thailand were characterised for their molecular epidemiology and antimicrobial resistance mechanisms. Of these, 270 isolates were carbapenem-resistant and 92.2% resistant to amikacin. A. baumannii IC2 was the dominant clone of A. baumannii (80%) and blaOXA-23-like was detected in most of CRAB isolates. The genomes of 13 representative isolates of A. baumannii (n=11), A. nosocomialis (n=1) and A. pittii (n=1) were analysed via whole genome sequencing. Antimicrobial resistance island, AbaR4-type containing Tn2006, was found in all CRAB isolates. armA was the only 16S rRNA methylase gene found that caused resistance to amikacin (located within Tn1548) and in close proximity to macrolide resistance genes (mphE and msrE). csu locus, bap, bfmRS and pga locus associated with biofilm formation were found in all IC2 isolates in addition to the typical set of antimicrobial resistance genes; blaOXA-23, blaOXA-66, blaADC, strA, strB and tetB in this clone. Therefore, the presence of arrays of antimicrobial resistance genes and biofilm-related loci may contribute to the spread and persistence of A. baumannii IC2 in this hospital. Lastly, comparative genome analysis was performed on 21 representative Acinetobacter spp. isolates from Thailand and other countries, i.e. Japan, Malaysia and Singapore. The range of the CRAB genome size was 3.9 - 4.1 Mb with GC content approximately 39%. Interestingly, the susceptible A. baumannii genome size was 3.7-3.8 Mb. The size difference was due to the absence of resistance genes and regions for bacterial competition and biofilm formation in susceptible isolates. The genetic contexts of key antimicrobial resistance genes and resistance islands were investigated. Our study confirmed that the antimicrobial resistance genes and other genomic features of IC2 isolates were homogenous. The diversity was mainly found in the composition of the antimicrobial resistance genes of IC2 isolates and locus for capsule synthesis (K locus). The genome and composition of antimicrobial resistance genes between IC2 isolates from Thailand and all IC2 from other countries were closely related. Two IC2 carbapenem-susceptible isolates from Japan harboured less antimicrobial resistance genes (blaOXA-66, strA, strB, sul2 and tetB). The locus for the outer core of lipid A-core moiety (OC locus) was generally conserved - OCL1 was a common type within IC2. Such variations observed of the K locus may impact in the difficulties to generate human immune response to A. baumannii. Lastly, one key feature of IC2 was the integrity of all chromosomal regions for type VI secretion system (T6SS) and biofilm formation (csu). In contrast, these regions in non-IC2 isolates were disrupted. This may indicate the incapability of non-IC2 isolates to persist in the hospital environment. The study of various aspects of carbapenem-resistant Acinetobacter spp. in our region has revealed specific insights into this pathogen locally and globally. These include the species identification, molecular epidemiology and the genome of Acinetobacter spp. Genomic analysis has described all necessary attributes of the superiority of IC2 to spread further under antimicrobial pressure and harsh environments. Further research is required to understand greater detail of these many unique findings.