Comparative genomic analysis of clinical Acinetobacter nosocomialis isolates from Terengganu, Malaysia led to the discovery of a novel tetracycline-resistant plasmid.

OBJECTIVES: To analyse the genome sequences of four archival Acinetobacter nosocomialis clinical isolates (designated AC13, AC15, AC21 and AC25) obtained from Terengganu, Malaysia in 2011 to determine their genetic relatedness and basis of antimicrobial resistance. METHODS: Antimicrobial susceptibility profiles of the A. nosocomialis isolates were determined by disk diffusion. Genome sequencing was performed using the Illumina NextSeq platform. RESULTS: The four A. nosocomialis isolates were cefotaxime resistant whereas three isolates (namely, AC13, AC15 and AC25) were tetracycline resistant. The carriage of the blaADC-255-encoded cephalosporinase gene is likely responsible for cefotaxime resistance in all four isolates. Phylogenetic analysis indicated that the three tetracycline-resistant isolates were closely related, with an average nucleotide identity of 99.9%, suggestive of nosocomial spread, whereas AC21 had an average nucleotide identity of 97.9% when compared to these three isolates. The tetracycline-resistant isolates harboured two plasmids: a 13476 bp Rep3-family plasmid of the GR17 group designated pAC13-1, which encodes the tetA(39) tetracycline-resistance gene, and pAC13-2, a 4872 bp cryptic PriCT-1-family plasmid of a new Acinetobacter plasmid group, GR60. The tetA(39) gene was in a 2 001 bp fragment flanked by XerC/XerD recombination sites characteristic of a mobile pdif module. Both plasmids also harboured mobilisation/transfer-related genes. CONCLUSIONS: Genome sequencing of A. nosocomialis isolates led to the discovery of two novel plasmids, one of which encodes the tetA(39) tetracycline-resistant gene in a mobile pdif module. The high degree of genetic relatedness among the three tetracycline-resistant A. nosocomialis isolates is indicative of nosocomial transmission.

Draft Genome Sequences of Two Acinetobacter soli Clinical Isolates from a Tertiary Hospital in Terengganu, Malaysia.

We report the draft genome sequences of Acinetobacter soli AC1511 and AC15148, which were isolated from a tertiary hospital in Terengganu, Malaysia, in 2015. AC1511 was assembled into 43 contigs with a total genome size of 3,320,693 bp, whereas AC15148 was 3,260,687 bp over 47 contigs.

Antimicrobial susceptibility, serotype distribution, virulence profile and molecular typing of piliated clinical isolates of pneumococci from east coast, Peninsular Malaysia.

Pilus has been recently associated with pneumococcal pathogenesis in humans. The information regarding piliated isolates in Malaysia is scarce, especially in the less developed states on the east coast of Peninsular Malaysia. Therefore, we studied the characteristics of pneumococci, including the piliated isolates, in relation to antimicrobial susceptibility, serotypes, and genotypes at a major tertiary hospital on the east coast of Peninsular Malaysia. A total of 100 clinical isolates collected between September 2017 and December 2019 were subjected to serotyping, antimicrobial susceptibility test, and detection of pneumococcal virulence and pilus genes. Multilocus sequence typing (MLST) and phylogenetic analysis were performed only for piliated strains. The most frequent serotypes were 14 (17%), 6A/B (16%), 23F (12%), 19A (11%), and 19F (11%). The majority of isolates were resistant to erythromycin (42%), tetracycline (37%), and trimethoprim-sulfamethoxazole (24%). Piliated isolates occurred in a proportion of 19%; 47.3% of them were multidrug-resistant (MDR) and a majority had serotype 19F. This study showed ST236 was the most predominant sequence type (ST) among piliated isolates, which was related to PMEN clone Taiwan19F-14 (CC271). In the phylogenetic analysis, the piliated isolates were grouped into three major clades supported with 100% bootstrap values. Most piliated isolates belonged to internationally disseminated clones of S. pneumoniae, but pneumococcal conjugate vaccines (PCVs) have the potential to control them.

Complete Genome Sequencing of Acinetobacter baumannii AC1633 and Acinetobacter nosocomialis AC1530 Unveils a Large Multidrug-Resistant Plasmid Encoding the NDM-1 and OXA-58 Carbapenemases.

Carbapenem-resistant Acinetobacter spp. are considered priority drug-resistant human-pathogenic bacteria. The genomes of two carbapenem-resistant Acinetobacter spp. clinical isolates obtained from the same tertiary hospital in Terengganu, Malaysia, namely, A. baumannii AC1633 and A. nosocomialis AC1530, were sequenced. Both isolates were found to harbor the carbapenemase genes blaNDM-1 and blaOXA-58 in a large (ca. 170 kb) plasmid designated pAC1633-1 and pAC1530, respectively, that also encodes genes that confer resistance to aminoglycosides, sulfonamides, and macrolides. The two plasmids were almost identical except for the insertion of ISAba11 and an IS4 family element in pAC1633-1, and ISAba11 along with relBE toxin-antitoxin genes flanked by inversely orientated pdif (XerC/XerD) recombination sites in pAC1530. The blaNDM-1 gene was encoded in a Tn125 composite transposon structure flanked by ISAba125, whereas blaOXA-58 was flanked by ISAba11 and ISAba3 downstream and a partial ISAba3 element upstream within a pdif module. The presence of conjugative genes in plasmids pAC1633-1/pAC1530 and their discovery in two distinct species of Acinetobacter from the same hospital are suggestive of conjugative transfer, but mating experiments failed to demonstrate transmissibility under standard laboratory conditions. Comparative sequence analysis strongly inferred that pAC1633-1/pAC1530 was derived from two separate plasmids in an IS1006-mediated recombination or transposition event. A. baumannii AC1633 also harbored three other plasmids designated pAC1633-2, pAC1633-3, and pAC1633-4. Both pAC1633-3 and pAC1633-4 are cryptic plasmids, whereas pAC1633-2 is a 12,651-bp plasmid of the GR8/GR23 Rep3-superfamily group that encodes the tetA(39) tetracycline resistance determinant in a pdif module.IMPORTANCE Bacteria of the genus Acinetobacter are important hospital-acquired pathogens, with carbapenem-resistant A. baumannii listed by the World Health Organization as the one of the top priority pathogens. Whole-genome sequencing of carbapenem-resistant A. baumannii AC1633 and A. nosocomialis AC1530, which were isolated from the main tertiary hospital in Terengganu, Malaysia, led to the discovery of a large, ca. 170-kb plasmid that harbored genes encoding the New Delhi metallo-ß-lactamase-1 (NDM-1) and OXA-58 carbapenemases alongside genes that conferred resistance to aminoglycosides, macrolides, and sulfonamides. The plasmid was a patchwork of multiple mobile genetic elements and comparative sequence analysis indicated that it may have been derived from two separate plasmids through an IS1006-mediated recombination or transposition event. The presence of such a potentially transmissible plasmid encoding resistance to multiple antimicrobials warrants vigilance, as its spread to susceptible strains would lead to increasing incidences of antimicrobial resistance.

Biocide susceptibilities and biofilm-forming capacities of Acinetobacter baumannii clinical isolates from Malaysia.

INTRODUCTION: Acinetobacter baumannii is a Gram-negative nosocomial pathogen that has the capacity to develop resistance to all classes of antimicrobial compounds. However, very little is known regarding its susceptibility to biocides (antiseptics and disinfectants) and capacity to form biofilms, particularly for Malaysian isolates. AIM: To determine the susceptibility of A. baumannii isolates to commonly-used biocides, investigate their biofilm-forming capacities and the prevalence of biocide resistance and biofilm-associated genes. METHODOLOGY: . The minimum inhibitory concentration (MIC) values of 100 A. baumannii hospital isolates from Terengganu, Malaysia, towards the biocides benzalkonium chloride (BZK), benzethonium chloride (BZT) and chlorhexidine digluconate (CLX), were determined by broth microdilution. The isolates were also examined for their ability to form biofilms in 96-well microplates. The prevalence of biocide resistance genes qacA, qacE and qacDE1 and the biofilm-associated genes bap and abaI were determined by polymerase chain reaction (PCR). RESULTS: Majority of the A. baumannii isolates (43%) showed higher MIC values (> 50 µg/mL) for CLX than for BZK (5% for MIC > 50 µg/mL) and BZT (9% for MIC > 50 µg/mL). The qacDE1 gene was predominant (63%) followed by qacE (28%) whereas no isolate was found harbouring qacA. All isolates were positive for the bap and abaI genes although the biofilm-forming capacity varied among the isolates. CONCLUSION: The Terengganu A. baumannii isolates showed higher prevalence of qacDE1 compared to qacE although no correlation was found with the biocides' MIC values. No correlation was also observed between the isolates' biofilm-forming capacity and the MIC values for the biocides.

Prevalence and antimicrobial susceptibilities of Acinetobacter baumannii and non-baumannii Acinetobacters from Terengganu, Malaysia and their carriage of carbapenemase genes.

A total of 153 non-repeat Acinetobacter spp. clinical isolates obtained in 2015 from Hospital Sultanah Nur Zahirah (HSNZ) in Terengganu, Malaysia, were characterized. Identification of the isolates at species level was performed by ribosomal DNA restriction analysis (ARDRA) followed by sequencing of the rpoB gene. The majority of the isolates (n=128; 83.7 %) were A. baumannii while the rest were identified as A. nosocomialis (n=16), A. calcoaceticus (n=5), A. soli (n=2), A. berezeniae (n=1) and A. variabilis (n=1). Multidrug resistance (MDR) was most prevalent in A. baumannnii (66.4 %) whereas only one non-baumannii isolate (A. nosocomialis) was MDR. The blaOXA-23 gene was the predominant acquired carbapenemase gene (56.2 %) and was significantly associated (P<0.001) with carbapenem resistance. However, no significant association was found for carbapenem resistance and isolates that contained the ISAba1-blaOXA-51 configuration.

Acinetobacter spp. Infections in Malaysia: A Review of Antimicrobial Resistance Trends, Mechanisms and Epidemiology.

Acinetobacter spp. are important nosocomial pathogens, in particular the Acinetobacter baumannii-calcoaceticus complex, which have become a global public health threat due to increasing resistance to carbapenems and almost all other antimicrobial compounds. High rates of resistance have been reported among countries in Southeast Asia, including Malaysia. In this review, we examine the antimicrobial resistance profiles of Acinetobacter spp. hospital isolates from Malaysia over a period of nearly three decades (1987-2016) with data obtained from various peer-reviewed publications as well as the Malaysian National Surveillance on Antibiotic Resistance (NSAR). NSAR data indicated that for most antimicrobial compounds, including carbapenems, the peak resistance rates were reached around 2008-2009 and thereafter, rates have remained fairly constant (e.g., 50-60% for carbapenems). Individual reports from various hospitals in Peninsular Malaysia do not always reflect the nationwide resistance rates and often showed higher rates of resistance. We also reviewed the epidemiology and mechanisms of resistance that have been investigated in Malaysian Acinetobacter spp. isolates, particularly carbapenem resistance and found that blaOXA-23 is the most prevalent acquired carbapenemase-encoding gene. From the very few published reports and whole genome sequences that are available, most of the Acinetobacter spp. isolates from Malaysia belonged to the Global Clone 2 (GC2) CC92 group with ST195 being the predominant sequence type. The quality of data and analysis in the national surveillance reports could be improved and more molecular epidemiology and genomics studies need to be carried out for further in-depth understanding of Malaysian Acinetobacter spp. isolates.