Pasteur Multilocus Sequence Typing Research Articles

The context of blaOXA-23 gene in Iraqi carbapenem-resistant Acinetobacter baumannii isolates belonging to global clone 1 and global clone 2.

Of the genes conferring resistance to carbapenems in Acinetobacter baumannii, the blaOXA-23 gene is the most widely found across the world. The gene carrying blaOXA-23 transposons in A. baumannii isolates of global clones GC1 and GC2 is found worldwide. Here, we examined whether transposons play a role in the dissemination of the blaOXA-23 in globally distributed clones, GC1 and GC2 A. baumannii isolates from Iraq. The 119 non-repetitive A. baumannii isolates including 94 recovered from clinical specimens and 25 isolates from hospital environment between September 2021 and April 2022 from different medical centers located at various regions in Baghdad, Iraq. The global clones (GC) and the genes encoding carbapenem resistance, including blaOXA-23, blaOXA-24, and blaOXA-58 were identified using multiplex PCR assays. Antibiotic susceptibility testing was performed by the Kirby-Bauer disk diffusion susceptibility method. The transposons carrying blaOXA-23 were examined using PCR mapping. In cases when carbapenem susceptible A. baumannii isolates were found, they were subjected to E test, full length sequencing of blaOXA-Ab (blaOXA-51-like) and Institut Pasteur multi-locus sequence typing scheme. All but two isolates (92 clinical and 25 environmental) were identified carbapenem-resistant A. baumannii (CRAB). Of 117 CRAB isolates, 20 belong to GC1, 19 contained blaOXA-23; of them, 17 isolates harbored the blaOXA-23 located on Tn2006. Among the 46 CRAB belonging to GC2, 39 contained blaOXA-23; of them, 34 carried the blaOXA-23 located on Tn2006. The remaining GC1 and GC2 isolates, one GC1 as well as one GC2 isolate, were susceptible to imipenem, doripenem, and meropenem and considered carbapenem-susceptible A. baumannii (CSAB). Full-length sequencing of the blaOXA-Ab and MLST for the two CSAB isolates belonging to GC1 and GC2 confirmed that the GC1 isolate belongs to ST 623 and contained an allele that encodes an blaOXA-69 variant of the blaOXA-Ab while the GC2 belong to ST2 and carried an blaOXA-66 variant. This study provides evidence for the dissemination of blaOXA-23 on the Tn2006 in CRAB isolates in Baghdad, Iraq. It appears that this transposon is widespread in GC1 and 2 isolates as in the other parts of the world. Interestingly, one GC1 and one GC2 isolate from Iraq were found to be susceptible to carbapenem while the isolates belonging to GC1 and GC2 have so far rarely been found to be susceptible to carbapenem globally.

Phylogenomics of Acinetobacter species and analysis of antimicrobial resistance genes.

Non-baumannii Acinetobacter species are increasingly isolated in the clinical setting and the environment. The aim of the present study was to analyze a genome database of 837 Acinetobacter spp. isolates, which included 798 non-baumannii Acinetobacter genomes, in order to define the concordance of classification and discriminatory power of 7-gene MLST, 53-gene MLST, and single-nucleotide polymorphism (SNPs) phylogenies. Phylogenies were performed on Pasteur Multilocus Sequence Typing (MLST) or ribosomal Multilocus Sequence Typing (rMLST) concatenated alleles, or SNPs extracted from core genome alignment. The Pasteur MLST scheme was able to identify and genotype 72 species in the Acinetobacter genus, with classification results concordant with the ribosomal MLST scheme. The discriminatory power and genotyping reliability of the Pasteur MLST scheme were assessed in comparison to genome-wide SNP phylogeny on 535 non-baumannii Acinetobacter genomes assigned to Acinetobacter pittii, Acinetobacter nosocomialis, Acinetobacter seifertii, and Acinetobacter lactucae (heterotypic synonym of Acinetobacter dijkshoorniae), which were the most clinically relevant non-baumannii species of the A. baumannii group. The Pasteur MLST and SNP phylogenies were congruent at Robinson-Fould and Matching cluster tests and grouped genomes into four and three clusters in A. pittii, respectively, and one each in A. seifertii. Furthermore, A. lactucae genomes were grouped into one cluster within A. pittii genomes. The SNP phylogeny of A. nosocomialis genomes showed a heterogeneous population and did not correspond to the Pasteur MLST phylogeny, which identified two recombinant clusters. The antimicrobial resistance genes belonging to at least three different antimicrobial classes were identified in 91 isolates assigned to 17 distinct species in the Acinetobacter genus. Moreover, the presence of a class D oxacillinase, which is a naturally occurring enzyme in several Acinetobacter species, was found in 503 isolates assigned to 35 Acinetobacter species. In conclusion, Pasteur MLST phylogeny of non-baumannii Acinetobacter isolates coupled with in silico detection of antimicrobial resistance makes it important to study the population structure and epidemiology of Acinetobacter spp. isolates.

Genetic Diversity and New Sequence Types of Escherichia coli Coharboring β-Lactamases and PMQR Genes Isolated from Domestic Dogs in Central Panama.

β-lactamase-producing Escherichia coli are a widely distributed source of antimicrobial resistance for animals and humans. Little is known about the susceptibility profile and genetic characteristics of E. coli strains isolated from domestic dogs in Latin America. We report on a cross-sectional study that evaluated E. coli strains isolated from fecal samples of domestic dogs in central Panama. The extended-spectrum β-lactamase (ESBL), AmpC genes, and plasmid-mediated quinolone resistance were investigated. Molecular typing using Pasteur's multilocus sequence typing (MLST) was conducted. A total of 40 E. coli isolates were obtained, of which 80% (32/40) were resistant to at least one of the antibiotics tested, while 20% (8/40) were sensitive to all antibiotics analyzed in this study (p < 0.001). Forty percent of the strains were resistant to three or more antibiotics. The most common resistance was to tetracycline (45%) and ampicillin (30%) while 2.5% showed an ESBL phenotype. Antibiotic resistance genes were detected for one β-lactamase (blaTEM-1) and two plasmid-mediated quinolone resistance (PMQR) enzymes (qnrS and qnrB). In addition, mutations in the chromosomal AmpC gene were observed at positions -35, -28, -18, -1, and +58. Fourteen different sequence types (STs) were identified; the most frequent were ST399 and ST425 (12% each). ST3 and ST88, which have been previously identified in human clinical isolates, were also evidenced. Three new STs were found for the first time: ST1015, ST1016 (carrier of the blaTEM-1 gene), and ST1017 (carrier of the blaTEM-1, qnrS, and qnrB genes). In the intestinal strains of E. coli isolated from domestic dogs, there was a high frequency of resistance to antibiotics. The presence of genes from plasmids and chromosomal mutations that conferred antibiotic resistance, the identification of isolates previously reported in humans, and the genetic diversity of STs (including three that were newly identified) confirmed the determinants of resistance to antibiotics in the domestic dogs from central Panama.

Genomic and phylogenetic analysis of a multidrug-resistant Klebsiella pneumoniae ST15 strain co-carrying blaOXA-232 and blaCTX-M-15 recovered from a gallbladder infection in China

ObjectivesThe occurrence of OXA-232-producing carbapenem-resistant Klebsiella pneumoniae (CRKP) has been increasing in China during the last five years. The blaOXA-232-carrying CRKP strain's clonal propagation can readily lead to nosocomial epidemics. Here, we report the genome sequence of an OXA-232 and CTX-M-15 co-producing K. pneumoniae strain isolated from a gallbladder infection in China. MethodsThe genome sequence of K. pneumoniae S105 was determined using the Illumina NovaSeq 6000 platform. Antimicrobial resistance genes (ARGs), multilocus sequence typing (MLST) and plasmid replicons were identified using the BacWGSTdb server. The phylogenetic relationship between S105 and other K. pneumoniae strains was analysed using the core genome multilocus sequence typing (cgMLST) strategy. ResultsThe genomic sequence of K. pneumoniae S105 is made up of 111 contigs with a total length of 5 748 752 bp. According to the Pasteur MLST scheme, S105 belongs to sequence type (ST) 15. Fifteen ARGs were discovered in the genome, including the beta-lactam resistance genes blaOXA-232 and blaCTX-M-15. The blaOXA-232 gene was located in a ColKP3 plasmid. KL112 was anticipated to be the capsule and lipopolysaccharide serotype. A total of 73 phylogenetically related strains were found from 19 nations across four continents; 22 of them were from China, with 21 strains harbouring the blaOXA-232 gene, and the majority of them diverged by just 6–37 cgMLST alleles. ConclusionIn summary, we reported the genomic sequencing of a K. pneumoniae ST15 clinical strain co-carrying the blaOXA-232 and blaCTX-M-15 genes. The clonal dissemination of OXA-232-producing K. pneumoniae ST15 strains in China needs our attention.

Diversity of carbapenem-resistant Acinetobacter baumannii and bacteriophage-mediated spread of the Oxa23 carbapenemase.

Carbapenem-resistant Acinetobacter baumannii are prevalent in low- and middle-income countries such as Egypt, but little is known about the molecular epidemiology and mechanisms of resistance in these settings. Here, we characterize carbapenem-resistant A. baumannii from Alexandria, Egypt, and place it in a regional context. Fifty-four carbapenem-resistant isolates from Alexandria Main University Hospital (AMUH), Alexandria, Egypt, collected between 2010 and 2015 were genome sequenced using Illumina technology. Genomes were de novo assembled and annotated. Genomes for 36 isolates from the Middle East region were downloaded from GenBank. The core-gene compliment was determined using Roary, and analyses of recombination were performed in Gubbins. Multilocus sequence typing (MLST) sequence type (ST) and antibiotic-resistance genes were identified. The majority of Egyptian isolates belonged to one of three major clades, corresponding to Pasteur MLST clonal complex (CCPAS) 1, CCPAS2 and STPAS158. Strains belonging to STPAS158 have been reported almost exclusively from North Africa, the Middle East and Pakistan, and may represent a region-specific lineage. All isolates carried an oxa23 gene, six carried bla NDM-1 and one carried bla NDM-2. The oxa23 gene was located on a variety of different mobile elements, with Tn2006 predominant in CCPAS2 strains, and Tn2008 predominant in other lineages. Of particular concern, in 8 of the 13 CCPAS1 strains, the oxa23 gene was located in a temperate bacteriophage phiOXA, previously identified only once before in a CCPAS1 clone from the USA military. The carbapenem-resistant A. baumannii population in AMUH is very diverse, and indicates an endemic circulating population, including a region-specific lineage. A major mechanism for oxa23 dissemination in CCPAS1 isolates appears to be a bacteriophage, presenting new concerns about the ability of these carbapenemases to spread throughout the bacterial population.

Emergence and multi-lineages of carbapenemase-producing Acinetobacter baumannii-calcoaceticus complex from canine and feline origins.

The carbapenemase-producing Acinetobacter baumannii is an important opportunistic bacterium and frequently causes hospital-acquired infections in humans. It also has increasingly been reported in veterinary medicine. This study illustrates multiple clones of carbapenemase-producing A. baumannii disseminating and causing diseases in dogs and cats in Thailand. Between 2016 and 2020, 44 A. baumannii and two A. pittii isolates exhibiting imipenem resistance (MIC≥16 μg/mL) from diagnostic samples were characterized by Pasteur multilocus sequence typing (MLST), sequence grouping (SG), repetitive extragenic palindromic element (rep)-PCR fingerprint analysis and antimicrobial resistance (AMR) profiling. All isolates contained blaOXA-23 in the Tn2006 family, and A. baumannii showed the sequence type (ST) 16 (14/44), ST149 (12/44), ST25 (6/44), ST2 (4/44), ST1581 (3/44), ST23 (2/44), ST1575 (1/44) and ST1576 (1/44). DNA fingerprint analysis and SG illustrated clonal relationships in the STs and its single locus variants, and AMR gene profiles, including tetracycline and aminoglycoside resistance genes, showed minor variations in the clones. The findings suggest that blaOXA-23 has been spread in multiple clones of A. baumannii and A. pittii from canine and feline hosts. With the collection of multiple AMR genes and intrinsic resistance, antimicrobial options are limited for treatment, and pets can be a potential reservoir of extensively drug-resistant, carbapenemase-producing A. baumannii in the community. Epidemiological tracking by passive and active surveillance in animals, veterinary personnel and hospital environment and preventive measurements should be promoted to decrease the risk of infection and transmission to humans.

Analysis of blaCHDL Genes and Insertion Sequences Related to Carbapenem Resistance in Acinetobacter baumannii Clinical Strains Isolated in Warsaw, Poland

Acinetobacter baumannii is an important cause of nosocomial infections worldwide. The elucidation of the carbapenem resistance mechanisms of hospital strains is necessary for the effective treatment and prevention of resistance gene transmission. The main mechanism of carbapenem resistance in A. baumannii is carbapenemases, whose expressions are affected by the presence of insertion sequences (ISs) upstream of blaCHDL genes. In this study, 61 imipenem-nonsusceptible A. baumannii isolates were characterized using phenotypic (drug-susceptibility profile using CarbaAcineto NP) and molecular methods. Pulsed field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) methods were utilized for the genotyping. The majority of isolates (59/61) carried one of the following acquired blaCHDL genes: blaOXA-24-like (39/59), ISAba1-blaOXA-23-like (14/59) or ISAba3-blaOXA-58-like (6/59). Whole genome sequence analysis of 15 selected isolates identified the following intrinsic blaOXA-66 (OXA-51-like; n = 15) and acquired class D β-lactamases (CHDLs): ISAba1-blaOXA-23 (OXA-23-like; n = 7), ISAba3-blaOXA-58-ISAba3 (OXA-58-like; n = 2) and blaOXA-72 (OXA-24-like; n = 6). The isolates were classified into 21 pulsotypes using PFGE, and the representative 15 isolates were found to belong to sequence type ST2 of the Pasteur MLST scheme from the global IC2 clone. The Oxford MLST scheme revealed the diversity among these studied isolates, and identified five sequence types (ST195, ST208, ST208/ST1806, ST348 and ST425). CHDL-type carbapenemases and insertion elements upstream of the blaCHDL genes were found to be widespread among Polish A. baumannii clinical isolates, and this contributed to their carbapenem resistance.

Antibiotic induced biofilm formation of novel multidrug resistant Acinetobacter baumannii ST2121 clone.

The aim of this study was to identify antimicrobial resistance and virulence factor genes exhibited by multidrug resistant (MDR) Acinetobacter baumannii, to analyze biofilm formation and to investigate clonal subtypes of isolate. Whole genome sequencing was done by Illumina NovaSeq 6,000 platform and multilocus sequence typing (MLST) was performed by Oxford and Pasteur typing schemes. Influence of imipenem and levofloxacin on biofilm formation was investigated in 96-well plates at 3 replicates. The strain was found to carry OXA-23, OXA-51-like, AmpC and TEM-1 beta-lactamases. The sequence of the blaOXA-51-like gene has been identified as a blaOXA-66. According to Pasteur MLST scheme the strain displayed ST2 allelic profile. However, based on Oxford MLST scheme this strain represents the new ST2121, as the gdhB gene has a single allelic mutation namely, the gdhB-227. It was determined that MDR isolate carried bap, basABCDFGHIJ, csuA/BABCDE, bauABCDEF, plcD, pgaABCD, entE, barAB, ompA, abaIR, piT2EAFTE/AUBl, fimADT, cvaC, bfmR, bfmS virulence genes. In our study imipenem induced the highest biofilm formation at a concentration of 32 µg/ml and levofloxacin at a concentration of 16 µg/ml. In conclusion, we detected a new MDR A. baumannii ST2121 clone harboring blaOXA-66 gene that has been reported for the first time in Turkey.

Molecular Epidemiology of Acinetobacter calcoaceticus-Acinetobacter baumannii Complex Isolated From Children at the Hospital Infantil de México Federico Gómez.

The Acinetobacter calcoaceticus-baumannii (Acb) complex is regarded as a group of phenotypically indistinguishable opportunistic pathogens responsible for mainly causing hospital-acquired pneumonia and bacteremia. The aim of this study was to determine the frequency of isolation of the species that constitute the Acb complex, as well as their susceptibility to antibiotics, and their distribution at the Hospital Infantil de Mexico Federico Gomez (HIMFG). A total of 88 strains previously identified by Vitek 2®, 40 as Acinetobacter baumannii and 48 as Acb complex were isolated from 52 children from 07, January 2015 to 28, September 2017. A. baumannii accounted for 89.77% (79/88) of the strains; Acinetobacter pittii, 6.82% (6/88); and Acinetobacter nosocomialis, 3.40% (3/88). Most strains were recovered mainly from patients in the intensive care unit (ICU) and emergency wards. Blood cultures (BC) provided 44.32% (39/88) of strains. The 13.63% (12/88) of strains were associated with primary bacteremia, 3.4% (3/88) with secondary bacteremia, and 2.3% (2/88) with pneumonia. In addition, 44.32% (39/88) were multidrug-resistant (MDR) strains and, 11.36% (10/88) were extensively drug-resistant (XDR). All strains amplified the blaOXA-51 gene; 51.13% (45/88), the blaOXA-23 gene; 4.54% (4/88), the blaOXA-24 gene; and 2.27% (2/88), the blaOXA-58 gene. Plasmid profiles showed that the strains had 1–6 plasmids. The strains were distributed in 52 pulsotypes, and 24 showed identical restriction patterns, with a correlation coefficient of 1.0. Notably, some strains with the same pulsotype were isolated from different patients, wards, or years, suggesting the persistence of more than one clone. Twenty-seven sequence types (STs) were determined for the strains based on a Pasteur multilocus sequence typing (MLST) scheme using massive sequencing; the most prevalent was ST 156 (27.27%, 24/88). The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas I-Fb system provided amplification in A. baumannii and A. pittii strains (22.73%, 20/88). This study identified an increased number of MDR strains and the relationship among strains through molecular typing. The data suggest that more than one strain could be causing an infection in some patient. The implementation of molecular epidemiology allowed the characterization of a set of strains and identification of different attributes associated with its distribution in a specific environment.

Unravelling the genome sequence of NDM-1 and KPC-2 co-producing Klebsiella pneumoniae ST11 isolated from a bloodstream infection.

The resistance rate of Klebsiella pneumoniae to commonly used antibiotics has been increasing rapidly, causing serious concern among clinicians and microbiologists. Resistance to carbapenems in K. pneumoniae is increasing dramatically in Chinese hospitals. Here we report the genome sequence of an NDM-1 and KPC-2 co-producing K. pneumoniae (CRKP380) isolated from a bloodstream infection in Hangzhou, China. The whole genome sequence of strain CRKP380 was determined using an Illumina NovaSeq 6000 platform. Antimicrobial resistance genes (ARGs) were identified using the BacWGSTdb server. The phylogenetic relationship between strain CRKP380 and other K. pneumoniae strains isolated from Hangzhou currently deposited in the NCBI GenBank database was analysed using a core genome-based single nucleotide polymorphism strategy. Klebsiella pneumoniae CRKP380 was resistant to all antibiotics tested except tigecycline and colistin. The genome sequence of K. pneumoniae CRKP380 consisted of 75 contigs comprising 5 590 460 bp. According to the Pasteur multilocus sequence typing (MLST) scheme, CRKP380 belongs to ST11. Eight ARGs were identified in CRKP380, including two carbapenemase genes (blaKPC-2 and blaNDM-1). Ten phylogenetically-related K. pneumoniae strains from Hangzhou were identified with identical ARGs and the same capsular serotype KL105, but none of these strains carried the blaNDM gene. Here we report the genome sequence of a K. pneumoniae ST11 clinical strain co-carrying blaNDM-1 and blaKPC-2 from Hangzhou, China. The genome sequence of CRKP380 can be used as a reference sequence for future comparative genomic analysis, including the acquisition and mobilisation of carbapenem resistance genes.

Database for the ampC alleles in Acinetobacter baumannii.

Acinetobacter baumannii is a troublesome opportunistic pathogen with a high capacity for clonal dissemination. We announce the establishment of a database for the ampC locus in A. baumannii, in which novel ampC alleles are differentiated based on the occurrence of ≥ 1 nucleotide change, regardless of whether it is silent or missense. The database is openly accessible at the pubmlst platform for A. baumannii (http://pubmlst.org/abaumannii/). Forty-eight distinctive alleles of the ampC locus have so far been identified and deposited in the database. Isolates from clonal complex 1 (CC1), according to the Pasteur multilocus sequence typing scheme, had a variety of the ampC locus alleles, including alleles 1, 3, 4, 5, 6, 7, 8, 13, 14, 17, and 18. On the other hand, isolates from CC2 had the ampC alleles 2, 3, 19, 20, 21, 22, 23, 24, 26, 27, 28, and 46. Allele 3 was characteristic for sequence types ST3 or ST32. The ampC alleles 10, 16, and 25 were characteristic for CC10, ST16, and CC25, respectively. Our study points out that novel gene databases, in which alleles are numbered based on differences in their nucleotide identities, should replace traditional records that use amino acid substitutions to define new alleles.

Predominance of international clone 2 OXA-23-producing-Acinetobacter baumannii clinical isolates in Greece, 2015: results of a nationwide study.

In a previous nationwide study in Greece, OXA-58 was the sole carbapenemase present among carbapenem-resistant Acinetobacter baumannii (CRAB) isolated between 2000 and 2009. In this study, the antibiotic resistances, carbapenemase gene content and clonal relatedness of 194 single-patient CRAB clinical isolates collected randomly during 2015 from 11 tertiary hospitals located throughout Greece were investigated. Antimicrobial susceptibility was determined using commercial and dilution methods. PCR assays for carbapenemase genes were performed. Clonality was tested by a scheme based on two multiplex PCRs and single-locus blaOXA-51-like sequence-based typing. Furthermore, Pasteur's multilocus sequence typing (MLST) scheme and pulsed-field gel electrophoresis (PFGE) were applied to 31 selected representative isolates. The most active antibiotics were trimethoprim/sulfamethoxazole (SXT) (34.6% of isolates susceptible), minocycline (71.6%), colistin (72.7%) and tigecycline (MIC50/90 values, 1/2 mg/L). The blaOXA-23-like gene was identified in 188 isolates (96.9%), blaOXA-23-like together with blaOXA-58-like in 3 isolates (1.5%), blaOXA-58-like in 2 isolates (1.0%) and blaOXA-40-like in 1 isolate (0.5%). ISAba1 was found upstream of the blaOXA-23-like gene in all isolates. International clone (IC) 2 comprised 157 isolates (80.9%), IC1 comprised 36 isolates (18.6%) and ST78 comprised 1 isolate (0.5%). All IC2 and IC1 isolates tested by MLST were ST2 and ST1, respectively. Seven PFGE types were detected. IC2 isolates were resistant to more antibiotics than IC1, except for SXT. This nationwide study showed that CRAB isolates in Greek hospitals currently produce almost uniformly the OXA-23 carbapenemase and belong mainly to IC2 and, to a lesser extent, IC1. Of particular concern, colistin susceptibility is recently severely reduced.

OXA-23 and ISAba1–OXA-66 class D β-lactamases in Acinetobacter baumannii isolates from companion animals

Acinetobacter baumannii is recognised as a major pathogen of nosocomial infections that frequently show resistance to last-resort antimicrobials. To investigate whether A. baumannii from companion animals harbour carbapenem resistance mechanisms, 223 clinical isolates obtained from veterinary clinics between 2000 and 2013 in Germany were screened for carbapenem-non-susceptibility employing meropenem-containing Mueller–Hinton agar plates. Minimum inhibitory concentration (MIC) data were obtained using the VITEK®2 system. Assignment to international clones (ICs) was done by multiplex PCR or repetitive sequence-based PCR employing the DiversiLab system. Clonality was studied using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Genes encoding carbapenemases and aminoglycoside-modifying enzymes were detected by PCR. In three samples from dogs, carbapenem-resistant A. baumannii carrying the blaOXA-23 gene on plasmids and located on transposon Tn2008 were identified. The isolates belonged to sequence type ST1P (clonal complex CC1/IC1/pulsotype II) and ST10P (CC10/IC8/pulsotype IV) according to the Pasteur MLST scheme, and to ST231Ox (CC109) and ST585Ox (CC447) following the Oxford scheme. Insertion sequence ISAba1 was identified upstream of blaOXA-66 in 58 A. baumannii isolates. MLST referred them to ST2P (CC2/IC2/pulsotypes I and III), ST208Ox, ST350Ox and ST556Ox (all CC118), respectively. PFGE suggested nosocomial spread of these highly related strains, which frequently demonstrated a multidrug-resistant phenotype, in one veterinary clinic. These data show that A. baumannii from companion animals reveal resistance determinants and clonal lineages of strains globally emerging in humans. This suggests an interspecies transmission and warrants molecular surveillance of A. baumannii in veterinary clinics to mitigate its further spread.

A new trilocus sequence-based multiplex-PCR to detect major Acinetobacter baumannii clones

A collection of 163 Acinetobacter baumannii isolates detected in a large Brazilian hospital, was potentially related with the dissemination of four clonal complexes (CC): 113/79, 103/15, 109/1 and 110/25, defined by University of Oxford/Institut Pasteur multilocus sequence typing (MLST) schemes. The urge of a simple multiplex-PCR scheme to specify these clones has motivated the present study. The established trilocus sequence-based typing (3LST, for ompA, csuE and blaOXA-51-like genes) multiplex-PCR rapidly identifies international clones I (CC109/1), II (CC118/2) and III (CC187/3). Thus, the system detects only one (CC109/1) out of four main CC in Brazil. We aimed to develop an alternative multiplex-PCR scheme to detect these clones, known to be present additionally in Africa, Asia, Europe, USA and South America. MLST, performed in the present study to complement typing our whole collection of isolates, confirmed that all isolates belonged to the same four CC detected previously. When typed by 3LST-based multiplex-PCR, only 12% of the 163 isolates were classified into groups. By comparative sequence analysis of ompA, csuE and blaOXA-51-like genes, a set of eight primers was designed for an alternative multiplex-PCR to distinguish the five CC 113/79, 103/15, 109/1, 110/25 and 118/2. Study isolates and one CC118/2 isolate were blind-tested with the new alternative PCR scheme; all were correctly clustered in groups of the corresponding CC. The new multiplex-PCR, with the advantage of fitting in a single reaction, detects five leading A. baumannii clones and could help preventing the spread in healthcare settings.

Emergence in Taiwan of novel imipenem-resistant Acinetobacter baumannii ST455 causing bloodstream infection in critical patients.

Acinetobacter baumannii is one of the most important noscomial pathogens worldwide. The study aimed to use multilocus sequence typing (MLST) for epidemiological surveillance of A. baumannii isolates in Taiwan and analyze the clinical presentations and patients' outcomes. MLST according to both Bartual's PubMLST and Pasteur's MLST schemes was applied to characterize bloodstream imipenem-resistant A. baumannii (IRAB) infection in intensive care units in a medical center. A total of 39 clinical IRAB bloodstream isolates in 2010 were enrolled. We also collected 13 imipenem-susceptible A. baumannii bloodstream isolates and 30 clinical sputum isolates (24 IRAB and 6 imipenem-susceptible A. baumannii) for comparison. Clinical presentations and outcome of the patients were analyzed. We found that infection by ST455B/ST2P and inappropriate initial therapy were statistically significant risk factors for mortality. More than one third of the IRAB isolates belonged to ST455B/ST2P. Most ST455B/ST2P (80%) carried ISAba1-blaOXA-23, including 10 (66.7%) with Tn2006 (ISAba1-blaOXA-23-ISAba1) in an AbaR4-type resistance island. ST455B/ST2P appears to evolve from ST208B/ST2P of clonal complex (CC) 92B/CC2P. In this hospital-based study, A. baumannii ST455 accounted for 38.5% of IRAB bacteremia, with a high mortality of 86.7%. Approximately 85% of ST455B/ST2P bacteremia had a primary source of ventilation-associated pneumonia. We report the emergence in Taiwan of IRAB ST455B/ST2P, which is the current predominant clone of IRAB in our hospital and has been causing bacteremia with high mortality in critical patients.

Emergence in Taiwan of novel imipenem-resistant Acinetobacter baumannii ST455 causing bloodstream infection in critical patients.

Acinetobacter baumannii is one of the most important nosocomial pathogens worldwide. This study aimed to use multilocus sequence typing (MLST) for the epidemiological surveillance of A. baumannii isolates in Taiwan and analyze the clinical presentations and patients' outcome. MLST according to both Bartual's PubMLST and Pasteur's MLST schemes was applied to characterize bloodstream imipenem-resistant A. baumannii (IRAB) infection in intensive care units in a medical center. A total of 39 clinical IRAB bloodstream isolates in 2010 were enrolled. We also collected 13 imipenem-susceptible A. baumannii (ISAB) bloodstream isolates and 30 clinical sputum isolates (24 IRAB and 6 ISAB) for comparison. Clinical presentations and outcome of the patients were analyzed. We found that infection by ST455(B)/ST2(P) and inappropriate initial therapy were statistically significant risk factors for mortality. More than one-third of the IRAB isolates belonged to ST455(B)/ST2(P). Most ST455(B)/ST2(P) (80%) carried ISAba1-blaOXA-23, including 10 (66.7%) with Tn2006 (ISAba1-blaOXA-23-ISAba1) in an AbaR4-type resistance island. ST455(B)/ST2(P) appears to evolve from ST208(B)/ST2(P) of clonal complex (CC) 92(B)/CC2(P). In this hospital-based study, A. baumannii ST455 accounted for 38.5% of IRAB bacteremia, with a high mortality of 86.7%. Approximately 85% of ST455(B)/ST2(P)bacteremia had a primary source of ventilation-associated pneumonia. We report the emergence in Taiwan of IRAB ST455(B)/ST2(P), which is the current predominant clone of IRAB in our hospital and has been causing bacteremia with high mortality in critical patients.

Single-Locus-Sequence-Based Typing of bla OXA-51-like Genes for Rapid Assignment of Acinetobacter baumannii Clinical Isolates to International Clonal Lineages

ABSTRACT Single-locus bla OXA-51-like sequence-based typing (SBT) was evaluated for its ability to determine correctly sequence types (STs) in Acinetobacter baumannii clinical isolates, in comparison with the Pasteur's multilocus sequence typing (MLST) reference method and 3-locus sequence typing (3-LST). The comparative study was performed in 585 multidrug-resistant (MDR) A. baumannii clinical isolates recovered from 21 hospitals located throughout Greece, Italy, Lebanon, and Turkey. The isolates belonged to nine clonal complexes (CCs) that correspond to 12 distinct sequence types (STs) and to one singleton ST. These clonal lineages predominate worldwide among nosocomial MDR A. baumannii strains. The most common clone was CC2 (ST2 and ST45; n = 278 isolates) followed by CC1 (ST1 and ST20; n = 155), CC25 ( n = 65), ST78 ( n = 62), CC15 (ST15 and ST84; n = 9), CC10 ( n = 4), CC3 ( n = 4), CC6 ( n = 3), CC54 ( n = 3), and CC83 ( n = 2). Using the bla OXA-51-like SBT method, all 585 isolates of the study were typed and assigned correctly to the nine CCs and the singleton ST78. The 3-LST method was not able to classify isolates belonging to CC6, CC10, CC54, and CC83, which are not yet characterized in its database. The low-cost and convenient bla OXA-51-like SBT method, compared with 3-LST and MLST, discriminated all epidemic and sporadic lineages of our collection and could be effectively applied to type rapidly A. baumannii strains.