Sabrina Montaña

Genetic Variability of AdeRS Two-Component System Associated with Tigecycline Resistance in XDR-Acinetobacter baumannii Isolates

AbstractThe emergence of tigecycline resistance has increased in the last years. Although tigecycline-resistant Acinetobacter baumannii isolates were described all over the world, few reports regarding the molecular basis of this resistance are available. It has been recognized that the overexpression of AdeABC efflux pump is related to the tigecycline-resistant phenotype. In 37 clinical A. baumannii isolates we first determined the tigecycline-resistant phenotype and then, within a selected group, we analyzed the sequence of the adeRS operon, which is involved in the expression of the AdeABC efflux pump. Nucleotide sequence analysis of adeR and adeS showed the presence of 5 and 16 alleles, respectively. nThese results expose a high genetic variability in both genes, the adeS gene being more susceptible to genetic variation. The presence of 2 AdeR and 2 AdeS new variants were reported. Two of the new AdeRS variants were present in the intermediate and the resistant tigecycline A. baumannii isolates, suggesting a putative role in the development of the observed phenotype. More studies need to be addressed to determine the role of the genetic variability observed in the adeRS operon.n

Presence of New Delhi metallo- β-lactamase gene (NDM-1) in a clinical isolate of Acinetobacter junii in Argentina

In the last few years, Acinetobacter infections caused by members of this genus other than baumannii have been recognized as a result of the implementation of new technologies in diagnostic laboratories. Acinetobacter junii is an atypical human pathogen that has been mainly associated with bacteraemia in neonates and paediatric oncology patients. Some cases of meningitis, peritonitis, ocular infection and septicaemia caused by A.xa0junii have been reported [1]. Moreover, many recently published reports of Acinetobacter spp. harbouring blaNDM suggested Acinetobacter as the source and cause of spread for this threatening carbapenemase [2], [3], [4], [5], [6]. The identification of blaNDM-1 was recently described in A.xa0junii clinical isolates from China [6], [7]. n nHere we report the presence of a clinically significant A.xa0junii blaNDM-1 positive in a 38-year-old woman who was admitted to the emergency department with a fever and leg ulcers with signs of infection. She presented a history of bipolar disorder, hypothyroidism, obesity and chronic necrotizing vasculitis. She had received treatment with corticosteroids and rituximab several months before. Fine-needle puncture aspiration of the ulcers was performed, and empiric treatment with piperacillin/tazobactam at 4.5xa0g/6 hours was administered intravenously plus vancomycin at 1xa0g/12 hours administered intravenously. n nAspiration samples from the infected ulcers were cultured and grew Enterobacter cloacae after 24 hours of incubation. The isolate was carbapenem susceptible, and the presence of extended β-lactamase activity was detected by Clinical and Laboratory Standards Institute (CLSI) guidelines. Considering the antibiotic susceptibility report, the antimicrobial therapy was changed to ertapenem at 1xa0g/24 hours, which resulted in the patient recovering well. After 8 days, she became febrile; therefore, the central venous catheter was removed, one blood culture was obtained and vancomycin was added to the antimicrobial therapy. n nAfter 18 hours of incubation, the growth of a Gram-negative, nonfermenting, rod-shaped bacterium, originally identified as Acinetobacter spp., was identified from both catheter tip and blood culture samples via conventional biochemical tests. The Acinetobacter spp. isolate 23910 was further identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) MS (Bruker Daltonics), rpoB amplification and sequencing to arrive to the species level. MALDI-TOF identified the strain as A.xa0junii, with a score of 2.34. This result was confirmed with rpoB sequence analysis, which showed 99% identity with A.xa0junii strain CIP 107470 (accession no. {type:entrez-nucleotide,attrs:{text:DQ207483,term_id:78099445,term_text:DQ207483}}DQ207483, previously named as A.xa0grimontii). The antibiotic susceptibility test was performed using the Phoenix Automated Microbiology System (Becton Dickinson, Franklin Lakes, NJ, USA) using panel NMIC/ID 92 (Gram-negative susceptibility card). The minimum inhibitory concentration (MIC) results were interpreted using the CLSI categories. The MIC results for the tested antibiotics were as followed (μg/mL): ampicillin >16; ampicillin/sulbactam 8/4; piperacillin/tazobactam 16/4; cefazolin >8, cephalotin >16; cefoxitin >16; ceftriaxone >4; ceftazidime >16; cefepime >16; ertapenem >1; imipenem >8; meropenem >8; amikacin ≤8; gentamicin ≤2; colistin ≤1; trimethoprim/sulfamethoxazole ≤0.5/9.5; ciprofloxacin 1; levofloxacin ≤1; fosfomycin >64. These results revealed that Aj23910 was susceptible to the following: ampicillin/sulbactam, piperacillin/tazobactam, amikacin, gentamicin, colistin, trimethoprim/sulfamethoxazole, ciprofloxacin and levofloxacin. It was resistant to ampicillin, cefazolin, ceftriaxone, cefoxitin, ceftazidime, cefepime, ertapenem, meropenem, imipenem and fosfomycin-G6P. n nAfter the antimicrobial susceptibility report for this strain, the antimicrobial therapy was changed again to ampicillin/sulbactam at 1.5xa0g/6 hours administered intravenously. The clinical finding—the same as A.xa0junii isolate recovered from the blood culture and from the catheter tip culture—was interpreted as a bacteraemia associated with venous central catheter. n nIn order to test for the presence of metallo-β-lactamase (MBL), we performed disk diffusion assays and a double-disk assay using an EDTA/SMA disk (1900/750xa0μg per disk, respectively) (Laboratorios Britania, Buenos Aires, Argentina) and an imipenem disk (placed 15xa0mm from each other). This assay showed synergism between carbapenem and EDTA/SMA disks, which suggests the presence of a putative MBL present in Aj23910. Considering the results, we decided to search for the most widespread MBL genes by PCR amplification. Total DNA extraction was performed according to the manufacturers instructions (Promega, Madison, WI, USA). We carried out PCR reactions using previously described primers to determine the presence of blaVIM, blaIMP, blaNDM and blaSPM genes [4]. The reactions were performed using GoTaq enzyme according to the manufacturers instructions (Promega). We obtained positive results for the amplification of blaNDM-1 in the Aj23910 strain. Nucleotide sequencing and sequence analysis of the positive amplification showed 100% identity with blaNDM1. We also obtained positive results for ISAba125 and aphA6 genes, which were previously reported in the same genetic context as NDM [3], [4], [8]. PCR reactions revealed the link and proximity of these genes to blaNDM-1. Positive PCR products (blaNDM-1 F- ISAba125F, blaNDM-1 R-ISAba125F, blaNDM-1 R-aphA6F, aphA6F- ISAba125F and aphA6F-ISAba125R) were sequenced. The sequence analyses confirmed the presence of aphA6-ISAba125–blaNDM-1 association. n nIn addition, conjugation assays were performed to see if blaNDM-1 was plasmid located. Briefly, Aj23910 and Escherichia coli J53-2xa0cells grown with agitation in Luria Bertani (LB) broth were mixed (1:10 and 5:10 donor:recipient) and incubated for 18 hours at 30°C. Transconjugant cells were selected on LB agar supplemented with sodium azide (150xa0μg/mL) and ampicillin (100xa0μg/mL) and were incubated overnight at 37°C. The negative results from the conjugation assay suggests that blaNDM-1 is codified in a nonconjugative element. Further studies are required to determine whether the gene possessed a chromosome location or a nonconjugative element location. n nThe NDM-1 carbapenemase has been dramatically spread among Gram-negative bacilli, thus imposing a new challenge on the health system to fight bacterial infections. n nThese data expand the number of Acinetobacter species harbouring blaNDM-1. The wide existence of Acinetobacter harbouring and dispersing this carbapenemase emphasizes the importance of non–previously recognized pathogens as reservoirs of dangerous resistance determinants. These resistance determinants can be later easily transferred to other menacing pathogens.

Genetic analysis of a PER-2-producing Shewanella sp. strain harbouring a variety of mobile genetic elements and antibiotic resistance determinants

The objective of this study was to investigate the molecular mechanisms explaining the multidrug-resistant (MDR) phenotype found in a novel clinical Shewanella sp. strain (Shew256) recovered from a diabetic patient. Whole-genome shotgun sequencing was performed using Illumina MiSeq-I and Nextera XT DNA library. De novo assembly was performed with SPAdes. RAST Server was used to predict the open-reading frames and the predictions were confirmed using BLAST. Further genomic analysis was carried out using average nucleotide identity (ANI), ACT (Artemis), OrthoMCL, ARG-ANNOT, ISfinder, PHAST, tRNAscan-SE, plasmidSPAdes, PlasmidFinder and MAUVE. PCR and plasmid extraction were also performed. Genomic analysis revealed a total of 456 predicted genes unique to Shew256 compared with other Shewanella genomes. Moreover, the presence of different resistance genes, including blaPER-2, was found. A complex class 1 integron containing the ISCR1 gene, disrupted by two putative transposase genes, was identified. Furthermore, other resistance genes, a transposon containing aph(3), insertion sequences, phages and non-coding RNAs were also found. In conclusion, evidence of acquisition of resistance genes and mobile elements that could explain the MDR phenotype were observed. This Shewanella sp. represents a prime example of how antibiotic resistance determinants can be acquired by uncommon pathogens.

Widespread dispersion of the resistance element tet(B)::ISCR2 in XDR Acinetobacter baumannii isolates.

Acinetobacter baumannii is a significant nosocomial pathogen often associated with extreme drug resistance (XDR). In Argentina, isolates of A. baumannii resistant to tetracyclines have accounted for more than 40% of drug-resistant isolates in some hospitals. We have previously reported the dispersion of the tet(B) resistance element associated with the ISCR2 transposase in epidemiologically unrelated A. baumannii isolates recovered from 1983 to 2011. This study extends this surveillance to 77 recent (2009-2013) XDR A. baumannii isolates with different levels of minocycline susceptibility. Isolates were examined by a pan-PCR assay, which showed six different amplification patterns, and specific PCRs were used for the confirmation of the the ΔISCR2-tet(B)-tet(R)-ISCR2 element. The tet(B) gene was present in 66 isolates and the ISCR2 element in 68 isolates; the tet(B) gene was associated with ISCR2 in all tet(B)-positive isolates. We conclude that this element is widespread in XDR A. baumannii isolates from Argentina and could be responsible for the emergence of tetracycline resistance in recent years.

Draft Genome Sequence of Empedobacter (Formerly Wautersiella) falsenii comb. nov. Wf282, a Strain Isolated from a Cervical Neck Abscess

ABSTRACT Empedobacter (formerly Wautersiella) falsenii comb. nov. strain Wf282 was isolated from a cervical neck abscess sample from an 18-year-old female patient. The isolate was resistant to many antibiotics, including meropenem and colistin. The total DNA from the multidrug-resistant E. falsenii comb. nov. Wf282 clinical isolate was sequenced.

The Genetic Analysis of an Acinetobacter johnsonii Clinical Strain Evidenced the Presence of Horizontal Genetic Transfer

Acinetobacter johnsonii rarely causes human infections. While most A. johnsonii isolates are susceptible to virtually all antibiotics, strains harboring a variety of β-lactamases have recently been described. An A. johnsonii Aj2199 clinical strain recovered from a hospital in Buenos Aires produces PER-2 and OXA-58. We decided to delve into its genome by obtaining the whole genome sequence of the Aj2199 strain. Genome comparison studies on Aj2199 revealed 240 unique genes and a close relation to strain WJ10621, isolated from the urine of a patient in China. Genomic analysis showed evidence of horizontal genetic transfer (HGT) events. Forty-five insertion sequences and two intact prophages were found in addition to several resistance determinants such as blaPER-2, blaOXA-58, blaTEM-1, strA, strB, ereA, sul1, aacC2 and a new variant of blaOXA-211, called blaOXA-498. In particular, blaPER-2 and blaTEM-1 are present within the typical contexts previously described in the Enterobacteriaceae family. These results suggest that A. johnsonii actively acquires exogenous DNA from other bacterial species and concomitantly becomes a reservoir of resistance genes.

A taxonomically unique Acinetobacter strain with proteolytic and hemolytic activities recovered from a patient with a soft tissue injury.

ABSTRACT A taxonomically unique bacterial strain, Acinetobacter sp. A47, has been recovered from several soft tissue samples from a patient undergoing reconstructive surgery owing to a traumatic amputation. The results of 16S rRNA, rpoB, and gyrB gene comparative sequence analyses showed that A47 does not belong to any of the hitherto-known taxa and may represent an as-yet-unknown Acinetobacter species. The recognition of this novel organism contributes to our knowledge of the taxonomic complexity underlying infections caused by Acinetobacter.

Preferential carriage of class 2 integrons in Acinetobacter baumannii CC113 and novel singletons.

Our understanding of the distribution of integrons associated with multidrug resistance in Acinetobacter baumannii isolates around the world remains incomplete. The association between the class 1 and 2 integron A. baumannii-positive isolates (n = 60), recovered since 1982 from 11 Argentinean hospitals, and the circulating lineages, was investigated. While class 2 integrons were highly significantly associated with clonal lineage CC113B/CC79P (P = 0·009) and novel singletons (P = 0·001), class 1 integrons were found not to be associated with CC109B/CC1P or other lineages. The study reveals a differential distribution of class 2 integrons in lineages, and suggests that the prevalence of intI2 in Argentina is related to the emergence of novel singletons in recent years and to the abundance of CC113B/CC79P, which has been the local dominant lineage for several decades.

Genomics helps to decipher the resistance mechanisms present in a Pseudomonas chlororaphis strain recovered in an HIV patient.

S. Montaña, T. Lazzaro, S. Uong, K. Place, A. Iriarte, C. V. Ocampo, C. Vay and M. S. Ramírez 1) Instituto de Microbiología y Parasitología Médica (IMPaM, UBACONICET), Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina, 2) Centre for Applied Biotechnology Studies, Department of Biological Science, California State University Fullerton, Fullerton, CA, USA, 3) Dpto de Desarrollo Biotecnológico, Instituto de Higiene, Facultad de Medicina, UdelaR, Montevideo, Uruguay, 4) Sanatorio Mater Dei, Ciudad Autónoma de Buenos Aires, Buenos Aires and 5 Laboratorio de Bacteriología, Departamento de Bioquímica Clínica, Hospital de Clínicas José de San Martín, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Ciudad Autónoma de Buenos Aires, Argentina

Whole-genome analysis and description of an outbreak due to carbapenem-resistant Ochrobactrum anthropi causing pseudo-bacteraemias

Ochrobactrum anthropi, a rare human pathogen, has been isolated predominantly from patients with catheter-related bacteraemia and rarely from other infections. In 2016, six cases of pseudo-bacteraemia caused by carbapenem-resistant O. anthropi isolates were recovered from an Argentinian hospital. The resistant phenotype exposed by the isolates caught our attention and led to an extensive epidemiologic investigation. Here we describe the characterization of a carbapenem-resistant O. anthropi outbreak whose probable cause was by contaminated collection tubes. The genome analysis of one strain revealed the presence of various resistant determinants. Among them, a metal-dependent hydrolase of the β-lactamase superfamily I, phnP, was found. Lately the recovery of unusual multidrug-resistant pathogens in the clinical setting has increased, thus emphasizing the need to implement standardized infection control practice and epidemiologic investigation to identify the real cause of hospital outbreaks.