Genomic study of Acinetobacter baumannii strains co-harboring bla OXA-58 and bla NDM-1 reveals a large multidrug-resistant plasmid encoding these carbapenemases in Brazil.

Introduction: Acinetobacter baumannii contributes significantly to the global issue of multidrug-resistant (MDR) nosocomial infections. Often, these strains demonstrate resistance to carbapenems (MDR-CRAB), the first-line treatment for infections instigated by MDR A. baumannii. Our study focused on the antimicrobial susceptibility and genomic sequences related to plasmids from 12 clinical isolates of A. baumannii that carry both the blaOXA-58 and bla NDM-1 carbapenemase genes. Methods: Whole-genome sequencing with long-read technology was employed for the characterization of an A. baumannii plasmid that harbors the bla OXA-58 and blaNDM-1 genes. The location of the bla OXA-58 and bla NDM-1 genes was confirmed through Southern blot hybridization assays. Antimicrobial susceptibility tests were conducted, and molecular characterization was performed using PCR and PFGE. Results: Multilocus Sequence Typing analysis revealed considerable genetic diversity among bla OXA-58 and bla NDM-1 positive strains in Brazil. It was confirmed that these genes were located on a plasmid larger than 300 kb in isolates from the same hospital, which also carry other antimicrobial resistance genes. Different genetic contexts were observed for the co-occurrence of these carbapenemase-encoding genes in Brazilian strains. Discussion: The propagation of bla OXA-58 and bla NDM-1 genes on the same plasmid, which also carries other resistance determinants, could potentially lead to the emergence of bacterial strains resistant to multiple classes of antimicrobials. Therefore, the characterization of these strains is of paramount importance for monitoring resistance evolution, curbing their rapid global dissemination, averting outbreaks, and optimizing therapy.

Comparative analysis of the antimicrobial resistance and virulence traits in ESBL-producing-Klebsiella pneumoniae ST307 strains colonizing the gastrointestinal tract and causing a fatal bloodstream infection in a leukemia patient.

Klebsiella pneumoniae is an opportunistic pathogen that can colonize the gastrointestinal tract (GIT) of humans. The mechanisms underlying the successful translocation of this pathogen to cause extra-intestinal infections remain unknown, although virulence and antimicrobial resistance traits likely play significant roles in the establishment of infections. We investigated K. pneumoniae strains isolated from GIT colonization (strains Kp_FZcol-1, Kp_FZcol-2 and Kp_FZcro-1) and from a fatal bloodstream infection (strain Kp_HM-1) in a leukemia patient. All strains belonged to ST307, carried a transferable IncF plasmid containing the blaCTX-M-15 gene (pKPN3-307 TypeA-like plasmid) and showed a multidrug-resistance phenotype. Phylogenetic analysis demonstrated that Kp_HM-1 was more closely related to Kp_FZcro-1 than to the other colonizing strains. The Kp_FZcol-2 genome showed 81 % coverage with the Kp_HM-1 246,730 bp plasmid (pKp_HM-1), lacking most of its putative virulence genes. Searching public genomes with similar coverage, we observed the occurrence of this deletion in K. pneumoniae ST307 strains recovered from human colonization and infection in different countries. Our findings suggest that strains lacking the putative virulence genes found in the pKPN3-307 TypeA plasmid are still able to colonize and infect humans, highlighting the need to further investigate the role of these genes for the adaptation of K. pneumoniae ST307 in distinct human body sites.

Occurrence of extended-spectrum ß-lactamases-producing Escherichia coli isolates over gradient pollution in an urban tropical estuary.

Bacterial resistance to antimicrobials is a global public health problem that surpasses the human context and can be increased by pollution. However, the lack of systematic monitoring of resistance in some aquatic matrices, such as tropical estuaries, makes it unknown whether its occurrence is associated with anthropogenic pollution in these environments. Therefore, we investigated the occurrence of extended-spectrum beta-lactamases (ESBLs) producing Escherichia coli as a resistance indicator for 12 consecutive months at three representative points of a pollution gradient in Guanabara Bay (GB), Brazil. Sixty-six E. coli strains were selected from 72 samples of GB waters in the presence of ceftriaxone (8 µg mL-1 ) and identified by MALDI-TOF MS. Of the 66, 55 (83.3%) strains were ESBL producers. They carried beta-lactamase/ESBL genes, with the predominance of blaCTX-M (54, 98.2%), especially the blaCTX-M-1,2 allele (49.1%). These strains were detected frequently (81.8%) from the point with the highest pollution levels. Furthermore, the marker for Class 1 integron, intI1 gene, was detected in 54.5% of ESBL producers. These data suggest an association between antimicrobial-resistant E. coli and sewage pollution in aquatic environments raising concerns about the possible risks of human exposure to these waters and fish consumption.

Description of a novel IncP plasmid harboring blaKPC-2 recovered from a SPM-1-producing Pseudomonas aeruginosa from ST277.

The high rates of carbapenem resistance among Brazilian Pseudomonas aeruginosa isolates are mainly associated with the clone ST277 producing the carbapenemase SPM-1. Here, the complete genetic composition of a IncP plasmid harboring blaKPC-2 in isolates of this endemic clone carrying chromosomal blaSPM-1 was described using whole genome sequencing. These results confirm the association of these two carbapenemases in ST277 and also describe the genetic composition of a novel blaKPC-2-plasmid. Considering the fact that this association occurs in a high-risk clone, monitoring the dissemination of this plasmid should be a public health concern.

Description and comparative genomic analysis of a mcr-1-carrying Escherichia coli ST683/CC155 recovered from touristic coastal water in Northeastern Brazil.

Polymyxin resistance is an emerging health issue aggravated by mcr dissemination among Enterobacterales recovered from various sources. Commensal Escherichia coli plays a key role in the spread of antimicrobial resistance in community settings and is likely to spread silently. It may transfer resistance genes to pathogenic bacteria in the gastrointestinal tract and the environment, and may cause difficult-to-treat infections, especially in immunocompromised patients. Unraveling actors disseminating resistance to last-resort antimicrobials might support the future development of control measures. Here we report the occurrence of a commensal ST683/CC155 colistin-resistant mcr-1.1-harboring E. coli (JP24) obtained from touristic coastal water. JP24's genome was sequenced and comparatively analyzed with other genomes from ST683/CC155 isolated worldwide and with mcr-carrying isolates recovered from various sources in Brazil. Besides mcr-1, JP24 carried blaCTX-M-8, tet(A), tet(34), dfrA12, sul2, sul3, aph(3')-Ia, aph(3')-IIa, aadA1, aadA2, cmlA1, Inu(G), mef(B) and mdf(a). mcr-1 and blaCTX-M-8 were transferable by IncX4 and IncI1/Iγ plasmids, respectively. Tree-based phylogeny of the ST683/CC155 isolates core genome revealed two larger clades. E. coli JP24 was grouped into a subclade together with an isolate from Thailand (ERR4221036), both carrying mcr-1. The core genome-based tree of the isolates carrying mcr-1 from Brazil revealed proximity with E. coli ECEST9 recovered from a mangrove also located in Northeastern Brazil. Accessory genome-based tree clustered most environmental isolates apart from the clinical ones and remained JP24 closer to ECEST9. High sequence conservation was observed between mcr-1-harboring plasmids detected in different species and reservoirs in Brazil and other countries. In addition to recreational coastal waters being potential sources for community exposure to antimicrobial-resistant bacteria, our findings reinforce a more prominent role of horizontal gene transfer, other than clonal expansion, in mcr dissemination in the community.

Prevalence and antimicrobial susceptibility of Gram-negative bacilli in subgingival biofilm associated with periodontal diseases.

BACKGROUND: This cross-sectional study aimed to determine the prevalence and antimicrobial susceptibility of Gram-negative bacilli (GNB) isolated from subgingival biofilm of individuals with different periodontal conditions. METHODS: Subgingival biofilm was obtained from 362 individuals with periodontal health (PH) (n = 83), gingivitis (n = 74), and periodontitis (n = 205), cultivated in broth and selective media. Isolated strains were identified by mass spectrometry. Antimicrobial susceptibility was determined by the Clinical and Laboratory Standards Institute disk diffusion guidelines. Production of extended-spectrum beta-lactamase (ESBL) and carbapenemases were evaluated by double disk synergy test and spectrophotometric detection of imipenem hydrolysis, respectively. ESBL and carbapenemase encoding genes were surveyed by Polymerase chain reaction (PCR). Differences among groups were examined by Chi-square, Kruskal-Wallis or Mann-Whitney tests. RESULTS: GNB were isolated from 36.2% of all subgingival biofilm samples, with a significantly greater prevalence and species diversity (P < 0.001) in patients with periodontitis (45.9%) compared with individuals with PH (24.1%) and gingivitis (22.9%). Pseudomonas aeruginosa (27.5%), Enterobacter cloacae (16.8%), and Enterobacter asburiae (10.7%) were the most predominant species. Resistance/reduced sensitivity to at least 1 antimicrobial was detected in 60% of the strains, but only 4.6% were multidrug resistant. Serratia marcescens, E. cloacae, and Enterobacter kobei presented high rates of intrinsic resistance (>40%) to amoxicillin-clavulanate and first/second-generations of cephalosporins. One strain of Klebsiella pneumoniae isolated from periodontitis was resistant to imipenem, but no ESBL encoding genes or ESBL phenotype was detected. CONCLUSION: High prevalence and diversity of GNB, with low susceptibility to ß-lactams are observed in the subgingival microbiota associated with periodontitis.

Characterization of an emergent high-risk KPC-producing Klebsiella pneumoniae lineage causing a fatal wound infection after spine surgery.

Surgical site infections in instrumented posterior lumbar interbody fusion surgery are normally due to gram-positive bacteria, but gram-negative bacteria can cause infections in cases involving lower lumbar interventions as its closer to the perianal area. Here we report an uncommon fatal wound infection caused by a multidrug-resistant Klebsiella pneumoniae after an elective spine surgery. In silico analysis revealed that LWI_ST16 belonged to ST16, an emergent international clone notable for its increased virulence potential. We also observed that this strain carried a conjugative IncF plasmid encoding resistance genes to beta-lactams (blaKPC-2 and blaOXA-1), tetracycline (tetA), aminoglycosides and fluoroquinolones (aac(6')-Ib-cr). The carbapenemase encoding gene blaKPC-2 was located on a Tn4401e transposon previously characterized to increase blaKPC expression. LWI_ST16 is a strong biofilm producer on polystyrene and capable of forming tower-like structures on a titanium device like the one inserted in the patient's spine. Our findings strengthen the valuable contribution of continuous surveillance of multidrug-resistant and high-risk K. pneumoniae clones to avoid unfavourable clinical outcomes.

NDM-1-encoding plasmid in Acinetobacter chengduensis isolated from coastal water.

BACKGROUND: Acinetobacter spp. may cause difficult-to-treat nosocomial infections due to acquisition of carbapenemases, including New Delhi metallo-ß-lactamase (NDM). This genus has been pointed out as a possible actor in the early dissemination of blaNDM, and this gene has been documented in a variety of species. OBJECTIVE: Here we describe an Acinetobacter chengduensis (isolate FL51) carrying blaNDM recovered from coastal water in Brazil. METHODS: In vitro techniques included antimicrobial susceptibility and minimum inhibitory concentration tests, PCR, plasmid profile and matting-out/transformation assays. In silico approaches comprised comparative genomic analyses using appropriate databases. RESULTS: FL51 grew at room temperature in a variety of culture media, excluding MacConkey. It showed resistance to all beta-lactams tested and to ciprofloxacin. blaNDM-1 was identified, and a single replicon was observed in plasmid profile. In silico DNA hybridization revealed Acinetobacter FL51 as being Acinetobacter chengduensis. blaNDM-1 was flanked upstream by ISAba14-aphA6-ISAba125 and downstream by bleMBL-trpF-Δtat, inserted in a 41,068 bp non typeable plasmid named pNDM-FL51. This replicon showed high coverage and identity with other sequences present in plasmids deposited on the GenBank database, recovered almost exclusively from Acinetobacter spp., associated with hospital settings and animal sources. CONCLUSION: We described a recently described environmental Acinetobacter species carrying a plasmid-borne blaNDM associated with a Tn125-like structure. Our findings suggest that replicon may play an important role in blaNDM dissemination among distinct settings within this genus and may support the theory of blaNDM emergence from an environmental Acinetobacter.

Acquisition of antimicrobial resistance determinants in Enterobacterales by international travelers from a large urban setting in Brazil.

BACKGROUND: Antimicrobial resistance is increased by international mobility. We present data about intestinal colonization of travelers departing from a middle-income country. METHODS: Travelers were recruited from 2015 to 2019, collected an anal stool specimen and answered a questionnaire before and after travel. Enterobacterales isolates were investigated for antimicrobial resistance; extended-spectrum beta-lactamase (ESBL) and carbapenemase production; plasmid-encoded cephalosporinases (pAmpC), plasmid-mediated quinolone resistance (PMQR) and mcr genes by PCR and sequencing; and association with travel related variables. RESULTS: Among 210 travelers, 26 (12%) carried multidrug-resistant Enterobacterales (MDR-E) and 18 (9%) ESBL-producing Enterobacterales (ESBL-E) before travel, with an increased prevalence from 1% to 11% over the study years. Acquisition of MDR-E and ESBL-E occurred in 59 (32%) and 43 (22%) travelers, respectively, mostly blaCTX-M-15 carrying Escherichia coli. One traveler acquired one isolate carrying blaOXA-181 gene, and two others, isolates carrying mcr-1. PMQR were detected in 14 isolates of returning travelers. The risk of MDR-E acquisition was higher in Southeast Asia and the Indian subcontinent, and after using antimicrobial agents. CONCLUSION: We describe an increasing pre-travel prevalence of ESBL-E colonization in subjects departing from this middle-income country over time. Travel to known risk areas and use of antimicrobial agents during travel were associated with acquisition of MDR-E. Travel advice is critical to mitigating this risk, as colonization by MDR-E may raise the chances of antimicrobial-resistant infections.

Frequency and diversity of Stenotrophomonas spp. carrying blaKPC in recreational coastal waters.

Stenotrophomonas can survive in a wide range of environments and is considered an opportunistic pathogen. Because of its intrinsic resistance to beta-lactams, this genus is considered irrelevant in studies addressing the environmental spread of antimicrobial resistance genes of medical importance. Consequently, studies on environmental Stenotrophomonas carrying acquired carbapenemase-encoding genes are scarce, though not inexistent. Here, we investigated the frequency and diversity of Stenotrophomonas spp. carrying genes encoding carbapenemases of medical relevance in coastal waters with distinct pollution degrees over one year. Among 319 isolates recovered, 220 (68.9%) showed blaKPC. The frequency of blaKPC-positive Stenotrophomonas spp. was not correlated with thermotolerant counts in coastal waters evaluated. All isolates were susceptible to minocycline, levofloxacin, and trimethoprim-sulfamethoxazole. PFGE typing of 101 blaKPC-positive isolates revealed 55 pulsotypes with 5 subtypes, all of which carried the blaKPC-2 variant. Interspecies differentiation of pulsotypes' representatives revealed 55 isolates belonging to the S. maltophilia complex (91.7%) and 5 S. acidaminiphila (8.3%). The blaKPC-2 gene was more frequently harbored on transposable elements found in enterobacteria of clinical origin, especially Tn4401b. Even though beta-lactams are no therapeutic options to treat Stenotrophomonas infections, the occurrence of a highly relevant antimicrobial resistance determinant harbored on mobile genetic elements in a diverse collection of these ubiquitous microorganisms is noteworthy. Therefore, Stenotrophomonas may act as acceptor, stable reservoirs, and potential vectors of antimicrobial resistance in environmental settings, especially aquatic matrices, and should not be neglected.

CTX-M- and pAmpC-Encoding Genes Are Associated with Similar Mobile Genetic Elements in Escherichia coli Isolated from Different Brands of Brazilian Chicken Meat.

In this study we characterized the genetic environment of blaCTX-M and blaCMY-2 genes carried by 46 Escherichia coli isolates obtained from 20 chicken carcasses produced by five different brands in Brazil, including exporters and antibiotic-free-certified producers, purchased between 2010 and 2014. Similar plasmids characterized according to size and incompatibility group (Inc) were identified in E. coli belonging to different MLST-ST collected, regardless of carcass brand or production system. Hybridization assays with transconjugant strains revealed that blaCMY-2 gene (n = 19) was located on 85 kb plasmids of IncB/O, IncI1, IncFIB, or nontypeable groups. blaCTX-M-8 (n = 9) was located on 90 kb IncI1 plasmids. blaCTX-M-2 (n = 14) was inserted in class 1 integrons and conjugated only by one isolate in a 125 kb IncP plasmid. blaCTX-M-15 (n = 1), rarely described in isolates from food-producing animals in South America, was characterized by whole genome sequencing of transconjugant; the gene was carried in a 49.3 kb IncX1 plasmid. Sequencing of bla gene-flanking regions indicated the association of these genes with previously described insertion sequences. These results suggest that conserved genetic environments are related to ESBL and pAmpC genes in the Brazilian chicken production chain.

Diversity of clonal types of Klebsiella pneumoniae causing infections in intensive care neonatal patients in a large urban setting.

BACKGROUND: Klebsiella infections are reported from neonatal intensive care units (NICUs) worldwide, but data on their incidence and genetic diversity remain scarce. OBJECTIVE: We determined the incidence and genetic diversity of Klebsiella infections in NICU patients in Rio de Janeiro. METHODS: This was a prospective study including newborns admitted to NICU in three hospitals during April 2005-November 2006 and March 2008-February 2009. Klebsiella pneumoniae isolates were genotyped by multilocus sequence typing (MLST) and extended spectrum ß-lactamases (ESBL) were characterized. RESULTS: Klebsiella infections occurred in 38 of 3984 patients (incidence rate, 9.5/1000 admissions); 14 (37%) of these 38 newborns died. Two clonal groups, CC45 and CC1041, caused 11 cases (42% of K. pneumoniae infection). Ten (32%) of the isolates causing infection produced ESBL, 9 of which (83%) carried blaCTX-M-15, all belonging to clonal complex (CC) 45 and CC1041. Nine of these ESBL-producing isolates were confined to only one of the NICUs. MAJOR CONCLUSIONS: The high incidence of Klebsiella infections in NICU in Rio de Janeiro appeared to be due to a combination of frequent sporadic infections caused by multiple K. pneumoniae genotypes and small outbreaks caused by dominant multidrug-resistant clones.

qnrD-harboring plasmids in Providencia spp. recovered from food and environmental Brazilian sources.

QnrD is a plasmid-mediated quinolone resistance (PMQR) determinant first reported in clinical Salmonella enterica isolates from China, located on nonconjugative plasmids of 4270 bp. Since then, the qnrD gene has been mostly found on plasmids around 2683 bp in Proteus and Morganella genera. However, Providencia spp. strains carrying qnrD-harboring plasmids have only been reported among clinical samples, in France and China. In this paper we describe two plasmids carrying qnrD in Providencia spp. isolated from Brazilian food and coastal waters. These plasmids present high coverage and identity with those recovered in France. Our results emphasize the relevance of the Proteeae tribe as reservoirs of qnrD and include P. rettgeri as a possible environmental carrier of this gene.

Staphylococcus saprophyticus Recovered from Humans, Food, and Recreational Waters in Rio de Janeiro, Brazil.

Staphylococcus saprophyticus is an important agent of urinary tract infection (UTI) in young women, but information about this pathogen in human microbiota and in common environment is lacking. The aim of this study was to characterize S. saprophyticus isolates from genitoanal microbiota of 621 pregnant women, 10 minas cheese packs, and five beaches in Rio de Janeiro city and compare PFGE profiles of these isolates with five UTI PFGE clusters described in this city. We investigated 65 S. saprophyticus isolates from microbiota, 13 from minas cheese, and 30 from beaches and 32 UTI isolates. Antimicrobial resistance was determined by disk diffusion, MIC by agar dilution, and PCR. Erythromycin-resistance genes erm(C), msr(A), msr(B), mph(C), and lin(A) were found in 93% of isolates. Trimethoprim-sulfamethoxazole resistance correlated with dfrG or dfrA genes. Three cefoxitin-resistant isolates carried the mecA gene. All isolates obtained from cheese were susceptible to all antimicrobial agents. Six of 10 pregnant women with >1 isolate had monoclonal colonization. Isolates from pregnant women shared 100% similarity with UTI PFGE cluster types A and E obtained almost 10 years previously, suggesting temporal persistence of S. saprophyticus. Antimicrobial resistance of beach isolates reflected the profiles of human isolates. Taken together, results indicate a shared source for human and environmental isolates.

Updated Multiplex PCR for Detection of All Six Plasmid-Mediated qnr Gene Families.

Plasmid-mediated qnr genes have been reported in bacteria worldwide and are widely associated with other relevant determinants of resistance in multiresistance plasmids. Here, we provide an update on a previously described multiplex PCR in order to detect all six qnr families (including qnrA, qnrS, qnrB, qnrC, qnrD, and qnrVC) described until now. The proposed method makes possible the screening of these genes, reducing cost and time, and it may demonstrate an underestimated prevalence of the latest variants described.

Mechanisms of carbapenem resistance in endemic Pseudomonas aeruginosa isolates after an SPM-1 metallo-ß-lactamase producing strain subsided in an intensive care unit of a teaching hospital in Brazil.

Carbapenem-resistance mechanisms are a challenge in the treatment of Pseudomonas aeruginosa infections. We investigated changes in P. aeruginosa carbapenem-resistance determinants over a time period of eight years after the emergence of São Paulo metallo-ß-lactamase in a university hospital in Rio de Janeiro, Brazil. Patients admitted to the intensive care unit (ICU) were screened for P. aeruginosa colonisation and followed for the occurrence of infections from April 2007 to April 2008. The ICU environment was also sampled. Isolates were typed using random amplified polymorphic DNA, pulsed-field gel electrophoresis and multilocus sequence typing. Antimicrobial susceptibility was determined by disk diffusion and E-test, production of carbapenemases by a modified-CarbaNP test and presence of carbapenemase-encoding genes by polymerase chain reaction. Non-carbapenemase resistance mechanisms studied included efflux and AmpC overexpression by PAßN and cloxacillin susceptibility enhancement, respectively, as well as oprD mutations. From 472 P. aeruginosa clinical isolates (93 patients) and 17 isolates from the ICU environment, high genotypic diversity and several international clones were observed; one environment isolate belonged to the blaSPM-1 P. aeruginosa epidemic genotype. Among isolates from infections, 10 (29%) were carbapenem resistant: none produced carbapenemases, three exhibited all non-carbapenemase mechanisms studied, six presented a combination of two mechanisms, and one exclusively displayed oprD mutations. Carbapenem-resistant P. aeruginosa displayed a polyclonal profile after the SPM-1 epidemic genotype declined. This phenomenon is connected with blaSPM-1 P. aeruginosa replaced by other carbapenem-resistant pathogens.

Mechanisms of carbapenem resistance in endemic Pseudomonas aeruginosa isolates after an SPM-1 metallo-β -lactamase producing strain subsided in an intensive care unit of a teaching hospital in Brazil

Carbapenem-resistance mechanisms are a challenge in the treatment of Pseudomonas aeruginosa infections. We investigated changes in P. aeruginosa carbapenem-resistance determinants over a time period of eight years after the emergence of São Paulo metallo-β-lactamase in a university hospital in Rio de Janeiro, Brazil. Patients admitted to the intensive care unit (ICU) were screened for P. aeruginosa colonisation and followed for the occurrence of infections from April 2007 to April 2008. The ICU environment was also sampled. Isolates were typed using random amplified polymorphic DNA, pulsed-field gel electrophoresis and multilocus sequence typing. Antimicrobial susceptibility was determined by disk diffusion and E-test, production of carbapenemases by a modified-CarbaNP test and presence of carbapenemase-encoding genes by polymerase chain reaction. Non-carbapenemase resistance mechanisms studied included efflux and AmpC overexpression by PAβN and cloxacillin susceptibility enhancement, respectively, as well as oprD mutations. From 472 P. aeruginosa clinical isolates (93 patients) and 17 isolates from the ICU environment, high genotypic diversity and several international clones were observed; one environment isolate belonged to the blaSPM-1 P. aeruginosa epidemic genotype. Among isolates from infections, 10 (29%) were carbapenem resistant: none produced carbapenemases, three exhibited all non-carbapenemase mechanisms studied, six presented a combination of two mechanisms, and one exclusively displayed oprD mutations. Carbapenem-resistant P. aeruginosa displayed a polyclonal profile after the SPM-1 epidemic genotype declined. This phenomenon is connected with blaSPM-1 P. aeruginosa replaced by other carbapenem-resistant pathogens.

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.