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.
Human serum albumin alters specific genes that can play a role in survival and persistence in Acinetobacter baumannii
In the past few decades Acinetobacter baumannii has emerged as a notorious nosocomial pathogen because of its ability to acquire genetic material and persist in extreme environments. Recently, human serum albumin (HSA) was shown to significantly increase natural transformation frequency in A. baumannii. This observation led us to perform transcriptomic analysis of strain A118 under HSA induction to identify genes that are altered by HSA. Our results revealed the statistically significant differential expression of 296 protein-coding genes, including those associated with motility, biofilm formation, metabolism, efflux pumps, capsule synthesis, and transcriptional regulation. Phenotypic analysis of these traits showed an increase in surface-associated motility, a decrease in biofilm formation, reduced activity of a citric acid cycle associated enzyme, and increased survival associated with zinc availability. Furthermore, the expression of genes known to play a role in pathogenicity and antibiotic resistance were altered. These genes included those associated with RND-type efflux pumps, the type VI secretion system, iron acquisition/metabolism, and ß-lactam resistance. Together, these results illustrate how human products, in particular HSA, may play a significant role in both survival and persistence of A. baumannii.
Clinical cases of VIM-producing Pseudomonas mendocina from two burned patients
Genomics helps to decipher the resistance mechanisms present in a Pseudomonas chlororaphis strain recovered in an HIV patient
Molecular characterization of KPC-2-positive Klebsiella pneumoniae isolates from a neurosurgical centre in Argentina.
Carbapenem-resistant Enterobacteriaceae is a growing concern worldwide. Klebsiella pneumoniae is an important nosocomial pathogen with a high capacity for nosocomial spread. We described the occurrence of plasmid-encoded KPC-2-harbouring K. pneumoniae isolates recovered from a neurosurgical centre in Argentina. The blaKPC-2 gene was surrounded by ISkpn6 and ISkpn7.
Comparison between disk diffusion and agar dilution methods to determine in vitro susceptibility of Corynebacterium spp. clinical isolates and update of their susceptibility.
Although Corynebacterium spp. are part of the microbiota of the skin and mucous membranes, human infections caused by Corynebacterium spp. have been reported and the multidrug resistance pattern of the recovered isolates was emphasised. Due to the usefulness of disk diffusion in daily practice, the purpose of this study was to compare disk diffusion with agar dilution to determine disk diffusion breakpoints and to review the antimicrobial susceptibility of the most frequent Corynebacterium spp. isolated in clinical samples.
Whole-Genome Analysis of an Extensively Drug-Resistance Empedobacter falsenii Strain Reveals Distinct Features and the Presence of a Novel Metallo-ß-Lactamase (EBR-2).
The spread of antibiotic resistance is rapidly threatening the effectiveness of antibiotics in the clinical setting. Many infections are being caused by known and unknown pathogenic bacteria that are resistant to many or all antibiotics currently available. Empedobacter falsenii is a nosocomial pathogen that can cause human infections. E. falsenii Wf282 strain was found to be resistant to many antibiotics, including carbapenems and colistin. Whole-genome shotgun sequencing of the strain was performed, and distinct features were identified. A novel metallo-β-lactamase, named EBR-2, was found, suggesting a potential role of E. falsenii as a reservoir of β-lactamases and other resistance determinants also found in its genome. The EBR-2 protein showed the highest catalytic efficiency for penicillin G as compared to meropenem and ampicillin and was unable to hydrolyze cefepime. The results described in this work broaden the current understanding of the role of β-lactamases in the Flavobacteriaceae family and suggest that E. falsenii Wf282 may be a reservoir of these novel resistance determinants.
Characterisation of OXA-258 enzymes and AxyABM efflux pump in Achromobacter ruhlandii
The aim of this study was to characterise OXA-258 variants and other features that may contribute to carbapenem resistance in Achromobacter ruhlandii
Ignavigranum ruoffiae, a rare pathogen that caused a skin abscess.
An 83-year-old male with a painless, ten-day-old, erythematous skin abscess on his left flank, which had showed a purulent discharge for 48 h, was admitted to the Emergency service. He was treated with cephalexin, disinfection with Codex water and spray of rifampicin. Five days later, surgical drainage of the abscess was proposed due to the torpid evolution of the patient. Samples were taken for culture, and antibiotic treatment with trimethoprim-sulfamethoxazole was established. The patient returned after 10 days showing healing of the abscess. Microbiological studies showed a few Gram-positive cocci present as single cells and short chains that grew after 72 h of incubation at 35 °C with CO2 on 5 % sheep blood agar. Colonies presented a strong sauerkraut odour. Initial biochemical test results were negative for catalase, aesculin and bile-aesculin, and positive for pyrrolidonyl arylamidase, leucine aminopeptidase and growth in 6.5 % NaCl broth, which prompted the preliminary identification of Facklamia species or I. ruoffiae. The positive result for arginine deamination and negative result for hippurate hydrolysis, failure to produce acid from mannitol, sucrose, sorbitol or trehalose, plus the distinctive sauerkraut odour identified the organism as I. ruoffiae. The phenotypic identification was confirmed by 16S rRNA gene sequence analysis. The strain seemed to be susceptible to the antimicrobials tested but had decreased susceptibility to carbapenems
The effect of sub-inhibitory concentrations of antibiotics on natural transformation in Acinetobacter baumannii
Effect of Host Human Products on Natural Transformation in Acinetobacter baumannii
Our previous data show that serum albumin can trigger natural transformation in Acinetobacter baumannii. However, extracellular matrix/basal membrane components, norepinephrine, and mucin did not have a significant effect on this process. Therefore, the effect of human products appears to be albumin specific, as both BSA and HSA have been shown to increase of natural transformation.
Amikacin: Uses, Resistance, and Prospects for Inhibition.
Aminoglycosides are a group of antibiotics used since the 1940s to primarily treat a broad spectrum of bacterial infections. The primary resistance mechanism against these antibiotics is enzymatic modification by aminoglycoside-modifying enzymes that are divided into acetyl-transferases, phosphotransferases, and nucleotidyltransferases. To overcome this problem, new semisynthetic aminoglycosides were developed in the 70s. The most widely used semisynthetic aminoglycoside is amikacin, which is refractory to most aminoglycoside modifying enzymes. Amikacin was synthesized by acylation with the l-(-)-γ-amino-α-hydroxybutyryl side chain at the C-1 amino group of the deoxystreptamine moiety of kanamycin A. The main amikacin resistance mechanism found in the clinics is acetylation by the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib], an enzyme coded for by a gene found in integrons, transposons, plasmids, and chromosomes of Gram-negative bacteria. Numerous efforts are focused on finding strategies to neutralize the action of AAC(6')-Ib and extend the useful life of amikacin. Small molecules as well as complexes ionophore-Zn+2 or Cu+2 were found to inhibit the acetylation reaction and induced phenotypic conversion to susceptibility in bacteria harboring the aac(6')-Ib gene. A new semisynthetic aminoglycoside, plazomicin, is in advance stage of development and will contribute to renewed interest in this kind of antibiotics.
First case of bacteraemia due to Acinetobacter schindleri harbouring blaNDM-1 in an immunocompromised patient
Clinically significant NDM-1-producing Acinetobacter schindleri has not yet been described in the literature. We report the first case of bacteraemia due to an A. schindleri strain harbouring blaNDM-1 recovered from an immunocompromised patient. Our report reinforces the fact that NDM-1 can easily be acquired by Acinetobacter species.
Unusual presentations of Comamonas kerstersii infection
The association of Comamonas kerstersii with peritonitis resulting from the presence of perforated appendix has previously been described by our research team. In the present study, we describe the isolation of this microorganism from two forms of unusual presentations of C. kerstersii infection not previously described in the literature: localized intra-abdominal infection (psoas abscess) and pelvic peritonitis.
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.
Light Modulates Metabolic Pathways and Other Novel Physiological Traits in the Human Pathogen Acinetobacter baumannii
Light sensing in chemotrophic bacteria has been relatively recently ascertained. In the human pathogen Acinetobacter baumannii, light modulates motility, biofilm formation, and virulence through the blue-light-sensing-using flavin (BLUF) photoreceptor BlsA. In addition, light can induce a reduction in susceptibility to certain antibiotics, such as minocycline and tigecycline, in a photoreceptor-independent manner. In this work, we identified new traits whose expression levels are modulated by light in this pathogen, which comprise not only important determinants related to pathogenicity and antibiotic resistance but also metabolic pathways, which represents a novel concept for chemotrophic bacteria. Indeed, the phenylacetic acid catabolic pathway and trehalose biosynthesis were modulated by light, responses that completely depend on BlsA. We further show that tolerance to some antibiotics and modulation of antioxidant enzyme levels are also influenced by light, likely contributing to bacterial persistence in adverse environments. Also, we present evidence indicating that surfactant production is modulated by light. Finally, the expression of whole pathways and gene clusters, such as genes involved in lipid metabolism and genes encoding components of the type VI secretion system, as well as efflux pumps related to antibiotic resistance, was differentially induced by light. Overall, our results indicate that light modulates global features of the A. baumannii lifestyle. IMPORTANCE The discovery that nonphototrophic bacteria respond to light constituted a novel concept in microbiology. In this context, we demonstrated that light could modulate aspects related to bacterial virulence, persistence, and resistance to antibiotics in the human pathogen Acinetobacter baumannii In this work, we present the novel finding that light directly regulates metabolism in this chemotrophic bacterium. Insights into the mechanism show the involvement of the photoreceptor BlsA. In addition, tolerance to antibiotics and catalase levels are also influenced by light, likely contributing to bacterial persistence in adverse environments, as is the expression of the type VI secretion system and efflux pumps. Overall, a profound influence of light on the lifestyle of A. baumannii is suggested to occur.
Matrix-assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a Reliable Tool to Identify Species of Catalase-negative Gram-positive Cocci not Belonging to the Streptococcus Genus
The results obtained suggest that MALDI-TOF MS has the potential of being an accurate tool for Catalase-negative GPC identification even for those species with difficult diagnosis as Helcococcus, Abiotrophia, Granulicatella, among others. Nevertheless, expansion of the library, especially including more strains with different spectra on the same species might overcome potential "intraspecies" variability problems. Moreover, a decrease of the identification scores for species and genus-level identification must be considered since it may improve the MALDI-TOF MS accuracy.
Antimicrobial susceptibility of clinical isolates of Actinomyces and related genera reveals an unusual clindamycin resistance among Actinomyces urogenitalis strains
Patterns of antimicrobial susceptibility in Actinomyces and related genera are very limited in the literature. Data of predominant susceptibility profiles could contribute to the establishment of an accurate empirical treatment.
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.
Whole-Genome Comparative Analysis of Two Carbapenem-Resistant ST-258 Klebsiella pneumoniae Strains Isolated during a North-Eastern Ohio Outbreak: Differences within the High Heterogeneity Zones.
Klebsiella pneumoniae has become one of the most dangerous causative agents of hospital infections due to the acquisition of resistance to carbapenems, one of the last resort families of antibiotics. Resistance is usually mediated by carbapenemases coded for by different classes of genes. A prolonged outbreak of carbapenem-resistant K. pneumoniae infections has been recently described in northeastern Ohio. Most strains isolated from patients during this outbreak belong to MLST sequence type 258 (ST258). To understand more about this outbreak two isolates (strains 140 and 677), one of them responsible for a fatal infection, were selected for genome comparison analyses. Whole genome map and sequence comparisons demonstrated that both strains are highly related showing 99% average nucleotide identity. However, the genomes differ at the so-called high heterogeneity zone (HHZ) and other minor regions. This study identifies the potential value of the HHZ as a potential marker for K. pneumoniae clinical and epidemiological studies.
Serum Albumin and Ca2+ Are Natural Competence Inducers in the Human Pathogen Acinetobacter baumannii
The increasing frequency of bacteria showing antimicrobial resistance (AMR) raises the menace of entering into a postantibiotic era. Horizontal gene transfer (HGT) is one of the prime reasons for AMR acquisition. Acinetobacter baumannii is a nosocomial pathogen with outstanding abilities to survive in the hospital environment and to acquire resistance determinants. Its capacity to incorporate exogenous DNA is a major source of AMR genes; however, few studies have addressed this subject. The transformation machinery as well as the factors that induce natural competence in A. baumannii are unknown. In this study, we demonstrate that naturally competent strain A118 increases its natural transformation frequency upon the addition of Ca(2+)or albumin. We show that comEA and pilQ are involved in this process since their expression levels are increased upon the addition of these compounds. An unspecific protein, like casein, does not reproduce this effect, showing that albumin's effect is specific. Our work describes the first specific inducers of natural competence in A. baumannii Overall, our results suggest that the main protein in blood enhances HGT in A. baumannii, contributing to the increase of AMR in this threatening human pathogen.
Reservoir of antimicrobial resistance determinants associated with horizontal gene transfer in clinical isolates of the genus Shewanella
Although Shewanella is usually considered an environmental genus, different clinical infections have appeared in recent years (4, 7). Treatment of such infections is difficult due to the lack of knowledge concerning the natural antimicrobial resistance as well as the recommended antibiotic treatment of their infections (11). The aim of our study was to investigate the antimicrobial resistance mechanisms acquired by this genus in the nosocomial environment.
All isolates identified as Shewanella spp. (n = 10) by the use of standard biochemical tests were collected in a public hospital of Argentina during 2005 and 2006. In Argentina, the relative frequency of isolates positively identified as harboring Shewanella spp. among infections by nonfermentative Gram-negative bacilli (NFGNB), excluding the important pathogens Acinetobacter baumannii and Pseudomonas aeruginosa, is only 2.5% of the total number of isolates. Amplification and sequencing of the 16S rRNA gene was used to identify the isolates (4, 12). Three isolates were identified as Shewanella putrefaciens and seven as Shewanella algae (Table 1).
All isolates identified as Shewanella spp. (n = 10) by the use of standard biochemical tests were collected in a public hospital of Argentina during 2005 and 2006. In Argentina, the relative frequency of isolates positively identified as harboring Shewanella spp. among infections by nonfermentative Gram-negative bacilli (NFGNB), excluding the important pathogens Acinetobacter baumannii and Pseudomonas aeruginosa, is only 2.5% of the total number of isolates. Amplification and sequencing of the 16S rRNA gene was used to identify the isolates (4, 12). Three isolates were identified as Shewanella putrefaciens and seven as Shewanella algae (Table 1).
Tn7::In2-8 dispersion in multidrug resistant isolates of Acinetobacter baumannii from Chile
Acinetobacter baumannii is considered an important pathogen in our hospital environment having a well-known capacity to acquire different mechanisms of antibiotic resistance. Previous studies in our laboratory had exposed the high dispersion of class 2 integrons in this species. In the present study, we analyzed 7 multiresistant intI2 positive A. baumannii isolates, 6 of which were found to harbour the Tn7::In2-8 element. Our results demonstrate the unusually high distribution of Tn7::In2-8 among different A. baumannii clones from Chile, suggesting a particular behavior of these elements at geographical level.
A naturally competent Acinetobacter baumannii clinical isolate as a convenient model for genetic studies
Acinetobacter baumannii A118 was isolated from a patient's blood culture. It is susceptible to several antibiotics, is naturally competent, and supports replication and stable maintenance of four plasmid replicons. A. baumannii A118 took up a fluorophore-labeled oligonucleotide analog. These characteristics make this isolate a convenient model for genetic studies.
Novel insights about class 2 integrons from experimental and genomic epidemiology
In order to contribute to the knowledge of the architecture and epidemiology of class 2 integrons, we performed a class 2 integron molecular survey in which we analyzed 726 isolates in two bacterial populations from environmental and nonepidemiologically related clinical samples, respectively, collected from 1982 to 2007. We recovered the intI2 gene from 130 of 726 isolates, most of which were clinical isolates, and only 1 (a psychrophilic Pseudomonas sp.) was from a water sample. Unlike the widespread distribution of class 1 integrons within Gram-negative bacilli, only Acinetobacter baumannii and Enterobacter cloacae harbored class 2 integrons at a high frequency in our collection. Class 2 integrons with six novel cassette arrays were documented. Characterization of the transposition module of Tn7, the genetic platform in which class 2 integrons have always been reported, showed tns modules with a mosaic genetic structure. A bioinformatic analysis performed with the tns genes present in sequence databases, the finding of intI2 not associated with tns genes, and the genetic examination of novel tns-like genes found in three isolates indicated the possibility of the independent evolution of the two components related to horizontal gene transfer, the class 2 integrons and the Tn7 transposons.
First case of human infection due to Pseudomonas fulva, an environmental bacterium isolated from cerebrospinal fluid
We report the first case of human infection due to Pseudomonas fulva. P. fulva caused acute meningitis following the placement of a drainage system in a 2-year-old female. Additionally, the isolate displayed a VIM-2 carbapenemase in a class 1 integron context.