Respiratory carriage of hypervirulent Klebsiella pneumoniae by indigenous populations of Malaysia.

Klebsiella pneumoniae is a Gram-negative Enterobacteriaceae that is classified by the World Health Organisation (WHO) as a Priority One ESKAPE pathogen. South and Southeast Asian countries are regions where both healthcare associated infections (HAI) and community acquired infections (CAI) due to extended-spectrum ß-lactamase (ESBL)-producing and carbapenem-resistant K. pneumoniae (CRKp) are of concern. As K. pneumoniae can also exist as a harmless commensal, the spread of resistance genotypes requires epidemiological vigilance. However there has been no significant study of carriage isolates from healthy individuals, particularly in Southeast Asia, and specially Malaysia. Here we describe the genomic analysis of respiratory isolates of K. pneumoniae obtained from Orang Ulu and Orang Asli communities in Malaysian Borneo and Peninsular Malaysia respectively. The majority of isolates were K. pneumoniae species complex (KpSC) 1 K. pneumoniae (n = 53, 89.8%). Four Klebsiella variicola subsp. variicola (KpSC3) and two Klebsiella quasipneumoniae subsp. similipneumoniae (KpSC4) were also found. It was discovered that 30.2% (n = 16) of the KpSC1 isolates were ST23, 11.3% (n = 6) were of ST65, 7.5% (n = 4) were ST13, and 13.2% (n = 7) were ST86. Only eight of the KpSC1 isolates encoded ESBL, but importantly not carbapenemase. Thirteen of the KpSC1 isolates carried yersiniabactin, colibactin and aerobactin, all of which harboured the rmpADC locus and are therefore characterised as hypervirulent. Co-carriage of multiple strains was minimal. In conclusion, most isolates were KpSC1, ST23, one of the most common sequence types and previously found in cases of K. pneumoniae infection. A proportion were hypervirulent (hvKp) however antibiotic resistance was low.

The phenotypes and genotypes associated with biofilm formation among methicillin-susceptible Staphylococcus aureus (MSSA) isolates collected from a tertiary hospital in Terengganu, Malaysia.

Methicillin-susceptible Staphylococcus aureus (MSSA) is an important nosocomial pathogen worldwide. This study aims to investigate the in vitro biofilm-forming ability of clinical MSSA isolated from various sources in the main public tertiary referral hospital in Terengganu, Malaysia and to detect the presence of biofilm-associated and regulatory genes among these isolates. A total of 104 MSSA isolates [pus (n = 75), blood (n = 24), respiratory secretions (n = 2), eye (n = 2), and urine (n = 1)] were investigated for slime production and biofilm formation using Congo red agar and crystal violet microtitre plate, respectively. Fifteen MSSA isolates with varying degrees of biofilm formation were selected for validation via a real-time cell analyser. All isolates were screened for microbial surface components recognising adhesive matrix molecules (MSCRAMM) and accessory gene regulator (agr) using polymerase chain reaction assay. A total of 76.0% (79/104) isolates produced slime layer, while all isolates developed biofilm as follows: 52.8% (55/104) strong biofilm producers, 40.4% (42/104) intermediate biofilm producers, and 6.7% (7/104) weak biofilm producers. A total of 98.1% (102/104) isolates carried at least one of the screened MSCRAMM gene(s) with the eno gene detected at the highest rate (87.5%, 91/104), while the sasG gene was significantly associated with strong biofilm production (p = 0.015). Three agr groups, 1, 2, and 3, were detected among the MSSA isolates with a predominance of agr-3 (32.7%, 34/104). In conclusion, biofilm formation varied greatly among clinical MSSA isolates, and the presence of sasG gene and agr-1 may play important role in initiating MSSA infections via biofilm formation.

relBE toxin-antitoxin system as a reliable anti-biofilm target in Pseudomonas aeruginosa.

AIMS: The ability of the pathogenic bacterium Pseudomonas aeruginosa to produce biofilms has made it more difficult to treat its infections with current antibiotics. Several genes are involved in biofilm production, and toxin-antitoxin (TA) loci have been reported to be responsible for the regulation of biofilm-associated genes. This study was aimed at evaluating various TA loci in P. aeruginosa to find a reliable target in order to disrupt biofilm formation. METHODS AND RESULTS: Thirty clinical isolates of P. aeruginosa were assessed for biofilm production as well as the presence of various TA loci in their genomes. The relBETA locus was present in all 30 P. aeruginosa isolates but its expression was not detectable in isolates that did not show biofilm production. Quantitative real-time -PCR (q-PCR) also demonstrated that the expression of relBE was higher in isolates with stronger biofilm-producing capability. Knocking out the relBE locus in one biofilm-producing P. aeruginosa isolate led to the cessation of biofilm-producing capacity in that isolate and eliminated the expression of ndvB, which is among the genes involved in biofilm production. CONCLUSIONS: These results inferred the involvement of relBE TA locus in the regulation of biofilm production in P. aeruginosa and indicated the possibility of relBE as an anti-biofilm target for this pathogen.

GNAT toxins of bacterial toxin-antitoxin systems: acetylation of charged tRNAs to inhibit translation.

GCN5-related N-acetyltransferase (GNAT) is a huge superfamily of proteins spanning the prokaryotic and eukaryotic domains of life. GNAT proteins usually transfer an acetyl group from acetyl-CoA to a wide variety of substrates ranging from aminoglycoside antibiotics to large macromolecules. Type II toxin-antitoxin (TA) modules are typically bicistronic and widespread in bacterial and archael genomes with diverse cellular functions. Recently, a novel family of type II TA toxins was described, which presents a GNAT-fold and functions by acetylating charged tRNA thereby precluding translation. These GNAT toxins are usually associated with a corresponding ribbon-helix-helix-fold (RHH) antitoxin. In this issue, Qian et al. describes a unique GNAT-RHH TA system, designated KacAT, from a multidrug resistant strain of the pathogen, Klebsiella pneumoniae. As most type II TA loci, kacAT is transcriptionally autoregulated with the KacAT complex binding to the operator site via the N-terminus region of KacA to repress kacAT transcription. The crystal structure of the KacT toxin is also presented giving a structural basis for KacT toxicity. These findings expand our knowledge on this newly discovered family of TA toxins and the potential role that they may play in antibiotic tolerance and persistence of bacterial pathogens.

Nasal colonisation, antimicrobial susceptibility and genotypic pattern of Staphylococcus aureus among agricultural biotechnology students in Besut, Terengganu, east coast of Malaysia.

BACKGROUND: This study aimed to profile the antimicrobial susceptibility and presence of resistance and virulence genes of methicillin-susceptible Staphylococcus aureus (MSSA) and MRSA nasal carriage, by means of genotypic analyses, in students of a tertiary institution in the state of Terengganu, east coast of Malaysia. METHODS: A total of 370 agricultural biotechnology students from Universiti Sultan Zainal Abidin in Besut, Terengganu, were enrolled in this study. Antimicrobial susceptibility profiles were evaluated by standard methods. PCR detection of resistance and virulence genes was performed on S. aureus that were methicillin-resistant, macrolide-lincosamide-streptogramin B (MLSB )-positive phenotype and/or positive for the leukocidin (pvl) gene followed by staphylococcal cassette chromosome mec (SCCmec), staphylococcal protein A (spa) and accessory gene regulator (agr) typing. RESULTS: One hundred and nineteen of 370 students carried S. aureus (32%); 18 of the isolates were MRSA (15%). Erythromycin resistance was detected in 20% (24/119) of which 15% (18/119) were MRSA and 5% (6/119) MSSA. Among the 24 erythromycin-resistant isolates, D-test was positive in 29% (7/24) displaying inducible MLSB , whereas the remaining 71% (17/24) showed constitutive MLSB phenotypes. Nine (7.6%) of 119 isolates were pvl positive: 44% MRSA (4/9) and 56% MSSA (5/9). Staphylococcal surface protein sasX gene was present in 92% of MRSA and 8% of MSSA isolates. The majority of MRSA isolates were agr type I (15/18; 83%). Five spa types identified with spa t037 were predominant, followed by spa types (t304 and t8696) as newly reported Malaysian MRSA in a community setting. CONCLUSION: The presence of MRSA with SCCmec of hospital-associated features and globally recognised spa types in community setting is worrisome. Furthermore, the presence of MLSB strains among multidrug-resistant (MDR) S. aureus with sasX as well as pvl-positive isolates highlights the potential risk of a community setting in facilitating the dissemination of both virulence and resistance determinants.

Neutralization of Bacterial YoeBSpn Toxicity and Enhanced Plant Growth in Arabidopsis thaliana via Co-Expression of the Toxin-Antitoxin Genes.

Bacterial toxin-antitoxin (TA) systems have various cellular functions, including as part of the general stress response. The genome of the Gram-positive human pathogen Streptococcus pneumoniae harbors several putative TA systems, including yefM-yoeBSpn, which is one of four systems that had been demonstrated to be biologically functional. Overexpression of the yoeBSpn toxin gene resulted in cell stasis and eventually cell death in its native host, as well as in Escherichia coli. Our previous work showed that induced expression of a yoeBSpn toxin-Green Fluorescent Protein (GFP) fusion gene apparently triggered apoptosis and was lethal in the model plant, Arabidopsis thaliana. In this study, we investigated the effects of co-expression of the yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic A. thaliana. When co-expressed in Arabidopsis, the YefMSpn antitoxin was found to neutralize the toxicity of YoeBSpn-GFP. Interestingly, the inducible expression of both yefMSpn antitoxin and yoeBSpn toxin-GFP fusion in transgenic hybrid Arabidopsis resulted in larger rosette leaves and taller plants with a higher number of inflorescence stems and increased silique production. To our knowledge, this is the first demonstration of a prokaryotic antitoxin neutralizing its cognate toxin in plant cells.

The dnd operon for DNA phosphorothioation modification system in Escherichia coli is located in diverse genomic islands.

BACKGROUND: Strains of Escherichia coli that are non-typeable by pulsed-field gel electrophoresis (PFGE) due to in-gel degradation can influence their molecular epidemiological data. The DNA degradation phenotype (Dnd(+)) is mediated by the dnd operon that encode enzymes catalyzing the phosphorothioation of DNA, rendering the modified DNA susceptible to oxidative cleavage during a PFGE run. In this study, a PCR assay was developed to detect the presence of the dnd operon in Dnd(+) E. coli strains and to improve their typeability. Investigations into the genetic environments of the dnd operon in various E. coli strains led to the discovery that the dnd operon is harboured in various diverse genomic islands. RESULTS: The dndBCDE genes (dnd operon) were detected in all Dnd(+) E. coli strains by PCR. The addition of thiourea improved the typeability of Dnd(+) E. coli strains to 100% using PFGE and the Dnd(+) phenotype can be observed in both clonal and genetically diverse E. coli strains. Genomic analysis of 101 dnd operons from genome sequences of Enterobacteriaceae revealed that the dnd operons of the same bacterial species were generally clustered together in the phylogenetic tree. Further analysis of dnd operons of 52 E. coli genomes together with their respective immediate genetic environments revealed a total of 7 types of genetic organizations, all of which were found to be associated with genomic islands designated dnd-encoding GIs. The dnd-encoding GIs displayed mosaic structure and the genomic context of the 7 islands (with 1 representative genome from each type of genetic organization) were also highly variable, suggesting multiple recombination events. This is also the first report where two dnd operons were found within a strain although the biological implication is unknown. Surprisingly, dnd operons were frequently found in pathogenic E. coli although their link with virulence has not been explored. CONCLUSION: Genomic islands likely play an important role in facilitating the horizontal gene transfer of the dnd operons in E. coli with 7 different types of islands discovered so far.

Expression of the Streptococcus pneumoniae yoeB chromosomal toxin gene causes cell death in the model plant Arabidopsis thaliana.

BACKGROUND: Bacterial toxin-antitoxin systems usually comprise of a pair of genes encoding a stable toxin and its cognate labile antitoxin and are located in the chromosome or in plasmids of several bacterial species. Chromosomally-encoded toxin-antitoxin systems are involved in bacterial stress responses and activation of the toxins usually leads to cell death or dormancy. Overexpression of the chromosomally-encoded YoeB toxin from the yefM-yoeB toxin-antitoxin locus of the Gram-positive bacterium Streptococcus pneumoniae has been shown to cause cell death in S. pneumoniae as well as E. coli. RESULTS: Induction of a YoeB-GFP fusion protein using a 17-ß-estradiol-inducible plant expression system in Arabidopsis thaliana Col 0, was lethal in all T2 progeny. Examination of plants by fluorescent confocal microscopy showed GFP fluorescence in all parts of the leaves at 24 hours after 17-ß-estradiol induction, continuing up to plant death. Quantitative RT-PCR analysis revealed that the expression of the yoeB toxin gene peaked at 3 days after induction with 17-ß-estradiol, coinciding with the onset of visible effects on the plants. Moreover, we detected DNA laddering in the transgenic plants at 24 hours after yoeB induction, indicative of apoptosis. CONCLUSIONS: Expression of the YoeB toxin from Streptococcus pneumoniae is lethal in Arabidopsis. We believe this is the first report of a toxin from a bacterial toxin-antitoxin system functioning in plants. The results presented here mark an important milestone towards the development of a cell ablation based bio-containment strategy, which may be useful for functional studies and for the control of spread of transgenic plants.

Whole genome sequencing of methicillin-resistant Staphylococcus aureus clinical isolates from Terengganu, Malaysia, indicates the predominance of the EMRSA-15 (ST22-SCCmec IV) clone.

Despite the importance of methicillin-resistant Staphylococcus aureus (MRSA) as a priority nosocomial pathogen, the genome sequences of Malaysian MRSA isolates are currently limited to a small pool of samples. Here, we present the genome sequence analyses of 88 clinical MRSA isolates obtained from the main tertiary hospital in Terengganu, Malaysia in 2016-2020, to obtain in-depth insights into their characteristics. The EMRSA-15 (ST22-SCCmec IV) clone of the clonal complex 22 (CC22) lineage was predominant with a total of 61 (69.3%) isolates. Earlier reports from other Malaysian hospitals indicated the predominance of the ST239 clone, but only two (2.3%) isolates were identified in this study. Two Indian-origin clones, the Bengal Bay clone ST772-SCCmec V (n = 2) and ST672 (n = 10) were also detected, with most of the ST672 isolates obtained in 2020 (n = 7). Two new STs were found, with one isolate each, and were designated ST7879 and ST7883. From the core genome phylogenetic tree, the HSNZ MRSA isolates could be grouped into seven clades. Antimicrobial phenotype-genotype concordance was high (> 95%), indicating the accuracy of WGS in predicting most resistances. Majority of the MRSA isolates were found to harbor more than 10 virulence genes, demonstrating their pathogenic nature.

Prevalence and characterization of verotoxigenic-Escherichia coli isolates from pigs in Malaysia.

BACKGROUND: Postweaning diarrhea caused by pathogenic Escherichia coli, in particular verotoxigenic E. coli (VTEC), has caused significant economic losses in the pig farming industry worldwide. However, there is limited information on VTEC in Malaysia. The objective of this study was to characterize pathogenic E. coli isolated from post-weaning piglets and growers with respect to their antibiograms, carriage of extended-spectrum beta-lactamases, pathotypes, production of hemolysins and fimbrial adhesins, serotypes, and genotypes. RESULTS: PCR detection of virulence factors associated with different E. coli pathotypes (ETEC, EPEC, EHEC, and VTEC) revealed that VTEC was the only pathotype identified from six swine farms located at north-western Peninsular Malaysia. A low prevalence rate of VTEC was found among the swine samples (n = 7/345) and all 7 VTEC isolates were multidrug resistant. Five of these isolates from different hosts raised in the same pen were likely to be of the same clone as they shared identical sero-pathotypes (O139:H1, VT2e/α-hly/F18), resistance profiles and DNA fingerprinting profiles. Two other serotypes, O130: H26 (n = 1) and O168: H21 (n = 1) carrying virulence factors were also identified. O168: H21 is possibly a new serotype as this has not been previously reported. CONCLUSIONS: The occurrence of VTEC with infrequently encountered serotypes that are multidrug resistant and harbouring virulence factors may be of public health concern. The detection of possible clones in this study also showed that the combination of different typing tools including phenotyping and genotyping methods is useful for molecular epidemiologic surveillance and studies.

Type II bacterial toxin-antitoxins: hypotheses, facts, and the newfound plethora of the PezAT system.

Toxin-antitoxin (TA) systems are entities found in the prokaryotic genomes, with eight reported types. Type II, the best characterized, is comprised of two genes organized as an operon. Whereas toxins impair growth, the cognate antitoxin neutralizes its activity. TAs appeared to be involved in plasmid maintenance, persistence, virulence, and defence against bacteriophages. Most Type II toxins target the bacterial translational machinery. They seem to be antecessors of Higher Eukaryotes and Prokaryotes Nucleotide-binding (HEPN) RNases, minimal nucleotidyltransferase domains, or CRISPR-Cas systems. A total of four TAs encoded by Streptococcus pneumoniae, RelBE, YefMYoeB, Phd-Doc, and HicAB, belong to HEPN-RNases. The fifth is represented by PezAT/Epsilon-Zeta. PezT/Zeta toxins phosphorylate the peptidoglycan precursors, thereby blocking cell wall synthesis. We explore the body of knowledge (facts) and hypotheses procured for Type II TAs and analyse the data accumulated on the PezAT family. Bioinformatics analyses showed that homologues of PezT/Zeta toxin are abundantly distributed among 14 bacterial phyla mostly in Proteobacteria (48%), Firmicutes (27%), and Actinobacteria (18%), showing the widespread distribution of this TA. The pezAT locus was found to be mainly chromosomally encoded whereas its homologue, the tripartite omega-epsilon-zeta locus, was found mostly on plasmids. We found several orphan pezT/zeta toxins, unaccompanied by a cognate antitoxin.

Multidrug-Resistant Methicillin-Resistant Staphylococcus aureus Associated with Hospitalized Newborn Infants.

Multidrug resistance (MDR) is a significant challenge in healthcare management, and addressing it requires a comprehensive approach. In this study, we employed a combination of phenotypic and genotypic approaches, along with whole genome sequencing (WGS) to investigate five hospital-associated MDR methicillin-resistant Staphylococcus aureus (MRSA) strains that were isolated from newborn infants. Our analysis revealed the following for the MDR-MRSA strains: SauR31 was resistant to three antimicrobial classes; SauR12, SauR91 and SauR110 were resistant to four antimicrobial classes; and SauR23 exhibited resistance to seven classes. All the MDR-MRSA strains were capable of producing slime and biofilms, harbored SCCmec type IV, and belonged to different spa types (t022, t032, and t548), with varying profiles for microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) and virulence genes. The WGS data for the MDR SauR23 and SauR91 strains revealed that most of the antimicrobial resistance genes were present in the chromosomes, including blaZ, mecA, norA, lmrS, and sdrM, with only the ermC gene found in a small (<3 kb) plasmid. The presence of MDR-MRSA strains among neonates raises public concern, hence implementation of multifaceted interventions is recommended to address this issue. In addition, metadata is needed to improve the investigation of antimicrobial resistance genes in MDR isolates.

Complete Genome Sequence and Analysis of a ST573 Multidrug-Resistant Methicillin-Resistant Staphylococcus aureus SauR3 Clinical Isolate from Terengganu, Malaysia.

Methicillin-resistant Staphylococcus aureus (MRSA) is a World Health Organization-listed priority pathogen. Scarce genomic data are available for MRSA isolates from Malaysia. Here, we present the complete genome sequence of a multidrug-resistant MRSA strain SauR3, isolated from the blood of a 6-year-old patient hospitalized in Terengganu, Malaysia, in 2016. S. aureus SauR3 was resistant to five antimicrobial classes comprising nine antibiotics. The genome was sequenced on the Illumina and Oxford Nanopore platforms and hybrid assembly was performed to obtain its complete genome sequence. The SauR3 genome consists of a circular chromosome of 2,800,017 bp and three plasmids designated pSauR3-1 (42,928 bp), pSauR3-2 (3011 bp), and pSauR3-3 (2473 bp). SauR3 belongs to sequence type 573 (ST573), a rarely reported sequence type of the staphylococcal clonal complex 1 (CC1) lineage, and harbors a variant of the staphylococcal cassette chromosome mec (SCCmec) type V (5C2&5) element which also contains the aac(6')-aph(2″) aminoglycoside-resistance genes. pSauR3-1 harbors several antibiotic resistance genes in a 14,095 bp genomic island (GI), previously reported in the chromosome of other staphylococci. pSauR3-2 is cryptic, whereas pSauR3-3 encodes the ermC gene that mediates inducible resistance to macrolide-lincosamide-streptogramin B (iMLSB). The SauR3 genome can potentially be used as a reference genome for other ST573 isolates.

The Plasmidomic Landscape of Clinical Methicillin-Resistant Staphylococcus aureus Isolates from Malaysia.

Methicillin-resistant Staphylococcus aureus (MRSA) is a priority nosocomial pathogen with plasmids playing a crucial role in its genetic adaptability, particularly in the acquisition and spread of antimicrobial resistance. In this study, the genome sequences of 79 MSRA clinical isolates from Terengganu, Malaysia, (obtained between 2016 and 2020) along with an additional 15 Malaysian MRSA genomes from GenBank were analyzed for their plasmid content. The majority (90%, 85/94) of the Malaysian MRSA isolates harbored 1-4 plasmids each. In total, 189 plasmid sequences were identified ranging in size from 2.3 kb to ca. 58 kb, spanning all seven distinctive plasmid replication initiator (replicase) types. Resistance genes (either to antimicrobials, heavy metals, and/or biocides) were found in 74% (140/189) of these plasmids. Small plasmids (<5 kb) were predominant (63.5%, 120/189) with a RepL replicase plasmid harboring the ermC gene that confers resistance to macrolides, lincosamides, and streptogramin B (MLSB) identified in 63 MRSA isolates. A low carriage of conjugative plasmids was observed (n = 2), but the majority (64.5%, 122/189) of the non-conjugative plasmids have mobilizable potential. The results obtained enabled us to gain a rare view of the plasmidomic landscape of Malaysian MRSA isolates and reinforces their importance in the evolution of this pathogen.

Genome sequence of Acinetobacter baumannii AC12, a polymyxin-resistant strain isolated from Terengganu, Malaysia.

Acinetobacter baumannii is a major cause of nosocomial infection worldwide. We report the draft genome sequence of A. baumannii AC12, a multidrug-resistant nosocomial strain with additional resistance to carbapenems and polymyxin. The genome data will provide insights into the genetic basis of antimicrobial resistance and its adaptive mechanism.

Genome sequence of multidrug-resistant Escherichia coli EC302/04, isolated from a human tracheal aspirate.

Escherichia coli is an important etiologic agent of lower respiratory tract infections (LRTI). Multidrug-resistant E. coli EC302/04 was isolated from a tracheal aspirate, and its genome sequence is expected to provide insights into antimicrobial resistance as well as adaptive and virulence mechanisms of E. coli involved in LRTI.

Outbreak-associated Vibrio cholerae genotypes with identical pulsotypes, Malaysia, 2009.

A cholera outbreak in Terengganu, Malaysia, in November 2009 was caused by 2 El Tor Vibrio cholerae variants resistant to typical antimicrobial drugs. Evidence of replacement of treatable V. cholerae infection in the region with antimicrobial-resistant strains calls for increased surveillance and prevention measures.

Epidemiology and Characteristics of Elizabethkingia spp. Infections in Southeast Asia.

Elizabethkingia spp. is a ubiquitous pathogenic bacterium that has been identified as the causal agent for a variety of conditions such as meningitis, pneumonia, necrotizing fasciitis, endophthalmitis, and sepsis and is emerging as a global threat including in Southeast Asia. Elizabethkingia infections tend to be associated with high mortality rates (18.2-41%) and are mostly observed in neonates and immunocompromised patients. Difficulties in precisely identifying Elizabethkingia at the species level by traditional methods have hampered our understanding of this genus in human infections. In Southeast Asian countries, hospital outbreaks have usually been ascribed to E. meningoseptica, whereas in Singapore, E. anophelis was reported as the main Elizabethkingia spp. associated with hospital settings. Misidentification of Elizabethkingia spp. could, however, underestimate the number of cases attributed to the bacterium, as precise identification requires tools such as MALDI-TOF MS, and particularly whole-genome sequencing, which are not available in most hospital laboratories. Elizabethkingia spp. has an unusual antibiotic resistance pattern for a Gram-negative bacterium with a limited number of horizontal gene transfers, which suggests an intrinsic origin for its multidrug resistance. Efforts to prevent and further understand Elizabethkingia spp. infections and limit its spread must rise to this new challenge.

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.

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.