Resistance phenotype and virulence potential of Leclercia adecarboxylata strains isolated from different sources.

Introduction. Leclercia adecarboxylata is a member of Enterobacterales, often considered an opportunistic pathogen. Recent reports have highlighted L. adecarboxylata as an emerging pathogen harbouring virulence and resistance determinants.Gap statement. Little information exists on virulence and resistance determinants in L. adecarboxylata strains isolated from environmental, food, and clinical samples.Aim. To determine the presence of resistance and virulence determinants and plasmid features in L. adecarboxylata strains isolated from environmental, food, and clinical samples, as well as their phylogenetic relationship.Results. All strains tested showed resistance to ß-lactams and quinolones but were sensitive to aminoglycosides and nitrofurans. However, even though fosfomycin resistance is considered a characteristic trait of L. adecarboxylata, the resistance phenotype was only observed in 50 % of the strains; bla TEM was the most prevalent BLEE gene (70 %), while the quinolone qnrB gene was observed in 60 % of the strains. Virulence genes were differentially observed in the strains, with adhesion-related genes being the most abundant, followed by toxin genes. Finally, all strains carried one to seven plasmid bands ranging from 7 to 125 kbps and harboured several plasmid addiction systems, such as ParDE, VagCD, and CcdAB in 80 % of the strains.Conclusions. L. adecarboxylata is an important emerging pathogen that may harbour resistance and virulence genes. Additionally, it has mobilizable genetic elements that may contribute to the dissemination of genetic determinants to other bacterial genera.

The Enolase of the Haemophilus influenzae Mediates Binding to Collagens: An Extracellular Matrix Component.

Enolase proteins play a significant role as moonlighting proteins. In their role as surface-associated enolase, they have multiple functions as they interact with extracellular matrix proteins. Type I and III collagens are the major constituents of this extracellular matrix, and collagen is one of the targets of interaction with the enolase of many pathogens, thereby helping the colonization process and promoting the subsequent invasion of the host. This work aimed to determine the participation of non-typeable H. influenzae enolase as a collagen-binding protein. In this study, through the use of in vitro tests it was demonstrated that recombinant enolase of non-typeable H. influenzae (rNTHiENO) strongly binds to type I collagen. Using molecular docking, the residues that could take part in the interaction of non-typeable H. influenzae enolase-type I collagen (NTHiENO-Cln I) and non-typeable H. influenzae enolase-type III collagen (NTHiENO-Cln III) were identified. However, in vitro assays show that NTHiENO has a better affinity to interact with Cln I, concerning type Cln III. The interaction of NTHiENO with collagen could play a significant role in the colonization process; this would allow H. influenzae to increase its virulence factors and strengthen its pathogenesis.

Strain belonging to an emerging, virulent sublineage of ST131 Escherichia coli isolated in fresh spinach, suggesting that ST131 may be transmissible through agricultural products.

Food contamination with pathogenic Escherichia coli can cause severe disease. Here, we report the isolation of a multidrug resistant strain (A23EC) from fresh spinach. A23EC belongs to subclade C2 of ST131, a virulent clone of Extraintestinal Pathogenic E. coli (ExPEC). Most A23EC virulence factors are concentrated in three pathogenicity islands. These include PapGII, a fimbrial tip adhesin linked to increased virulence, and CsgA and CsgB, two adhesins known to facilitate spinach leaf colonization. A23EC also bears TnMB1860, a chromosomally-integrated transposon with the demonstrated potential to facilitate the evolution of carbapenem resistance among non-carbapenemase-producing enterobacterales. This transposon consists of two IS26-bound modular translocatable units (TUs). The first TU carries aac(6')-lb-cr, bla OXA-1, ΔcatB3, aac(3)-lle, and tmrB, and the second one harbors bla CXT-M-15. A23EC also bears a self-transmissible plasmid that can mediate conjugation at 20°C and that has a mosaic IncF [F(31,36):A(4,20):B1] and Col156 origin of replication. Comparing A23EC to 86 additional complete ST131 sequences, A23EC forms a monophyletic cluster with 17 other strains that share the following four genomic traits: (1) virotype E (papGII+); (2) presence of a PAI II536-like pathogenicity island with an additional cnf1 gene; (3) presence of chromosomal TnMB1860; and (4) frequent presence of an F(31,36):A(4,20):B1 plasmid. Sequences belonging to this cluster (which we named "C2b sublineage") are highly enriched in septicemia samples and their associated genetic markers align with recent reports of an emerging, virulent sublineage of the C2 subclade, suggesting significant pathogenic potential. This is the first report of a ST131 strain belonging to subclade C2 contaminating green leafy vegetables. The detection of this uropathogenic clone in fresh food is alarming. This work suggests that ST131 continues to evolve, gaining selective advantages and new routes of transmission. This highlights the pressing need for rigorous epidemiological surveillance of ExPEC in vegetables with One Health perspective.

Comparative Genomics of Pseudomonas aeruginosa Strains Isolated from Different Ecological Niches.

The Pseudomonas aeruginosa genome can change to adapt to different ecological niches. We compared four genomes from a Mexican hospital and 59 genomes from GenBank from different niches, such as urine, sputum, and environmental. The ST analysis showed that high-risk STs (ST235, ST773, and ST27) were present in the genomes of the three niches from GenBank, and the STs of Mexican genomes (ST167, ST2731, and ST549) differed from the GenBank genomes. Phylogenetic analysis showed that the genomes were clustering according to their ST and not their niche. When analyzing the genomic content, we observed that environmental genomes had genes involved in adapting to the environment not found in the clinics and that their mechanisms of resistance were mutations in antibiotic resistance-related genes. In contrast, clinical genomes from GenBank had resistance genes, in mobile/mobilizable genetic elements in the chromosome, except for the Mexican genomes that carried them mostly in plasmids. This was related to the presence of CRISPR-Cas and anti-CRISPR; however, Mexican strains only had plasmids and CRISPR-Cas. blaOXA-488 (a variant of blaOXA50) with higher activity against carbapenems was more prevalent in sputum genomes. The virulome analysis showed that exoS was most prevalent in the genomes of urinary samples and exoU and pldA in sputum samples. This study provides evidence regarding the genetic variability among P. aeruginosa isolated from different niches.

A Plasmid Carrying blaIMP-56 in Pseudomonas aeruginosa Belonging to a Novel Resistance Plasmid Family.

blaIMP and blaVIM are the most detected plasmid-encoded carbapenemase genes in Pseudomonas aeruginosa. Previous studies have reported plasmid sequences carrying blaIMP variants, except blaIMP-56. In this study, we aimed to characterize a plasmid carrying blaIMP-56 in a P. aeruginosa strain isolated from a Mexican hospital. The whole genome of P. aeruginosa strain PE52 was sequenced using Illumina Miseq 2 × 150 bp, with 5 million paired-end reads. We characterized a 27 kb plasmid (pPE52IMP) that carried blaIMP-56. The phylogenetic analysis of RepA in pPE52IMP and 33 P. aeruginosa plasmids carrying resistance genes reported in the GenBank revealed that pPE52IMP and four plasmids (pMATVIM-7, unnamed (FDAARGOS_570), pD5170990, and pMRVIM0713) were in the same clade. These closely related plasmids belonged to the MOBP11 subfamily and had similar backbones. Another plasmid (p4130-KPC) had a similar backbone to pPE52IMP; however, its RepA was truncated. In these plasmids, the resistance genes blaKPC-2, blaVIM variants, aac(6')-Ib4, blaOXA variants, and blaIMP-56 were inserted between phd and resolvase genes. This study describes a new family of plasmids carrying resistance genes, with a similar backbone, the same RepA, and belonging to the MOBP11 subfamily in P. aeruginosa. In addition, our characterized plasmid harboring blaIMP-56 (pPE52IMP) belongs to this family.

IncFIB plasmids carrying the resistance gene blaCTX-M-15 in ESBL-producing Escherichia coli clones from pediatric patients.

INTRODUCTION: The emergence of extended-spectrum ß-lactamases (ESBLs)-producing Escherichia coli clones are a public health concern worldwide. Scarce information does exist about the spread of ESBLs-producing E. coli in pediatric patients from developing countries. METHODOLOGY: E. coli strains were analyzed by multilocus-sequence-typing, pulsed-field-gel-electrophoresis and phylogenetic group. The antimicrobial-resistance genes were detected by PCR, and plasmid content by the PCR-based replicon-typing. Horizontal transfer was tested by conjugation and the location of the blaCTX-M-15 gene by Southern blot hybridization. RESULTS: Thirty-two cefotaxime-resistant E. coli were recovered. Eleven of them were ESBL-producing isolates, which were well characterized and ascribed to seven sequence types and five phylogroups. The ESBL CTX-M-15 was the most prevalent enzyme (9 of 11). Plasmids of variable sizes (40-220 kb) were visualized, and the incompatibility (Inc) group FIB plasmid-replicon was detected in the ESBL strains and transferred by conjugation in 45.45% of them. Plasmid-borne toxin-antitoxin systems were the most frequently detected systems, strongly associated to IncF plasmids. The CTX-M-15-encoding gene was located on IncFIB plasmids. CONCLUSIONS: Even though a small number of ESBL-producing strains was recovered, we evidenced that IncFIB plasmids carry the blaCTX-M-15 gene, highlighting the role of IncF-type plasmids in facilitating the spread and maintenance of ESBL-encoding genes, which further favors the rapid increase of the antimicrobial resistance dissemination in disease-causing E. coli strains in pediatric patients.

Structural Characterization of Haemophilus influenzae Enolase and Its Interaction with Human Plasminogen by In Silico and In Vitro Assays.

Haemophilus influenzae is the causal agent of invasive pediatric diseases, such as meningitis, epiglottitis, pneumonia, septic arthritis, pericarditis, cellulitis, and bacteremia (serotype b). Non-typeable H. influenzae (NTHi) strains are associated with localized infections, such as otitis media, conjunctivitis, sinusitis, bronchitis, and pneumonia, and can cause invasive diseases, such as as meningitis and sepsis in immunocompromised hosts. Enolase is a multifunctional protein and can act as a receptor for plasminogen, promoting its activation to plasmin, which leads to the degradation of components of the extracellular matrix, favoring host tissue invasion. In this study, using molecular docking, three important residues involved in plasminogen interaction through the plasminogen-binding motif (251EFYNKENGMYE262) were identified in non-typeable H. influenzae enolase (NTHiENO). Interaction with the human plasminogen kringle domains is conformationally stable due to the formation of four hydrogen bonds corresponding to enoTYR253-plgGLU1 (K2), enoTYR253-plgGLY310 (K3), and enoLYS255-plgARG471/enoGLU251-plgLYS468 (K5). On the other hand, in vitro assays, such as ELISA and far-western blot, showed that NTHiENO is a plasminogen-binding protein. The inhibition of this interaction using polyclonal anti-NTHiENO antibodies was significant. With these results, we can propose that NTHiENO-plasminogen interaction could be one of the mechanisms used by H. influenzae to adhere to and invade host cells.

Location of OprD porin in Pseudomonas aeruginosa clinical isolates.

Multidrug-resistant Pseudomonas aeruginosa is one of the main opportunistic pathogens causing severe infection. One of the mechanisms involved in the resistance to imipenem in clinical isolates is the loss of the OprD porin. Changes like substitutions, deletions, insertions, or mutations in the oprD gene can modify the conformation of OprD porin or inhibit its presence and generate resistance to carbapenems. The aim of this work was to obtain anti-OprD polyclonal antibodies and to determine by both immunofluorescence microscopy (IFI) and Western blot assays, the presence of the OprD porin in resistant-carbapenem P. aeruginosa strains with different changes in the oprD gene. Changes in the gene oprD were identified in clinical isolates of P. aeruginosa. When proteins were translated, several polymorphisms were found; however, these did not affect the presence of OprD porin (PCM25, PCM36, and PCM78). Also it was detected an insertion sequence ISPa1328 (PCM52) and a premature stop codon (PCM91), which inhibited the presence of the OprD porin. This study shows how changes in the oprD gene of P. aeruginosa clinical isolates affect the presence of the OprD porin detected by Western blot and indirect immunofluorescence assays using specific polyclonal anti-OprD antibodies generated in this work.

Antibiotic resistance mechanisms in Acinetobacter spp. strains isolated from patients in a paediatric hospital in Mexico.

OBJECTIVES: The aim of this study was to identify Acinetobacter spp. strains from paediatric patients, to determine their genetic relationship, to detect antibiotic resistance genes and to evaluate the role of efflux pumps in antibiotic resistance. METHODS: A total of 54 non-duplicate, non-consecutive Acinetobacter spp. isolates were collected from paediatric patients. Their genetic relationship, antibiotic resistance profile, efflux pump activity, antibiotic resistance genes and plasmid profile were determined. RESULTS: The isolates were identified as 24 Acinetobacter haemolyticus, 24 Acinetobacter calcoaceticus-baumannii (Acb) complex and 1 strain each of Acinetobacter junii, Acinetobacter radioresistens, Acinetobacter indicus, Acinetobacter lwoffii, Acinetobacter ursingii and Acinetobacter venetianus. The 24 A. haemolyticus were considered genetically unrelated. One strain was resistant to carbapenems, two to cephalosporins, two to ciprofloxacin and sixteen to aminoglycosides. The antibiotic resistance genes blaOXA-214 (29%), blaOXA-215 (4%), blaOXA-264 (8%), blaOXA-265 (29%), blaNDM-1 (4%), aac(6')-Ig (38%) and the novel variants blaOXA-575 (13%), blaTEM-229 (75%), aac(6')-Iga (4%), aac(6')-Igb (13%) and aac(6')-Igc (42%) were detected. Among 24 Acb complex, 5 were multidrug-resistant, carbapenem-resistant strains carrying blaOXA-51 and blaOXA-23; they were genetically related and had the same plasmid profile. Other species were susceptible. In some strains of A. haemolyticus and Acb complex, the role of RND efflux pumps was evidenced by a decrease in the MICs for cefotaxime, amikacin and ciprofloxacin in the presence of an efflux pump inhibitor. CONCLUSIONS: This study identified isolates of A. haemolyticus carrying new ß-lactamase variants and shows for the first time the contribution of efflux pumps to antibiotic resistance in this species.

Comparative genomics of a subset of Adherent/Invasive Escherichia coli strains isolated from individuals without inflammatory bowel disease.

There is increased evidence demonstrating the association between Crohn's Disease (CD), a type of Inflammatory Bowel Disease (IBD), and non-diarrheagenic Adherent/Invasive Escherichia coli (AIEC) isolates. AIEC strains are phenotypically characterized by their adhesion, invasion and intra-macrophage survival capabilities. In the present study, the genomes of five AIEC strains isolated from individuals without IBD (four from healthy donors and one from peritoneal liquid) were sequenced and compared with AIEC prototype strains (LF82 and NRG857c), and with extra-intestinal uropathogenic strain (UPEC CFT073). Non-IBD-AIEC strains showed an Average Nucleotide Identity up to 98% compared with control strains. Blast identities of the five non-IBD-AIEC strains were higher when compared to AIEC and UPEC reference strains than with another E. coli pathotypes, suggesting a relationship between them. The SNPs phylogeny grouped the five non-IBD-AIEC strains in one separated cluster, which indicates the emergence of these strains apart from the AIEC group. Additionally, four genomic islands not previously reported in AIEC strains were identified. An incomplete Type VI secretion system was found in non-IBD-AIEC strains; however, the Type II secretion system was complete. Several groups of genes reported in AIEC strains were searched in the five non-IBD-AIEC strains, and the presence of fimA, fliC, fuhD, chuA, irp2 and cvaC were confirmed. Other virulence factors were detected in non-IBD-AIEC strains, which were absent in AIEC reference strains, including EhaG, non-fimbrial adhesin 1, PapG, F17D-G, YehA/D, FeuC, IucD, CbtA, VgrG-1, Cnf1 and HlyE. Based on the differences in virulence determinants and SNPs, it is plausible to suggest that non-IBD AIEC strains belong to a different pathotype.

Resistome and a Novel blaNDM-1-Harboring Plasmid of an Acinetobacter haemolyticus Strain from a Children's Hospital in Puebla, Mexico.

Acinetobacter calcoaceticus-baumannii complex isolates have been frequently associated with hospital and community infections, with A. baumannii being the most common. Other Acinetobacter spp. not belonging to this complex also cause infections in hospital settings, and the incidence has increased over the past few years. Some species of the Acinetobacter genus possess a great diversity of antibiotic resistance mechanisms, such as efflux pumps, porins, and resistance genes that can be acquired and disseminated by mobilizable genetic elements. By means of whole-genome sequencing, we describe in the clinical Acinetobacter haemolyticus strain AN54 different mechanisms of resistance that involve blaOXA-265, blaNDM-1, aphA6, aac(6')-Ig, and a resistance-nodulation-cell division-type efflux pump. This strain carries six plasmids, of which the plasmid pAhaeAN54e contains blaNDM-1 in a Tn125-like transposon that is truncated at the 3' end. This strain also has an insertion sequence IS91 and seven genes encoding hypothetical proteins. The pAhaeAN54e plasmid is nontypable and different from other plasmids carrying blaNDM-1 that have been reported in Mexico and other countries. The presence of these kinds of plasmids in an opportunistic pathogen such as A. haemolyticus highlights the role that these plasmids play in the dissemination of antibiotic resistance genes, especially against carbapenems, in Mexican hospitals.

Adherent/invasive Escherichia coli (AIEC) isolates from asymptomatic people: new E. coli ST131 O25:H4/H30-Rx virotypes.

BACKGROUND: The widespread Escherichia coli clone ST131 implicated in multidrug-resistant infections has been recently reported, the majority belonging to O25:H4 serotype and classified into five main virotypes in accordance with the virulence genes carried. METHODS: Pathogenicity Islands I and II (PAI-I and PAI-II) were determined using conventional PCR protocols from a set of four E. coli CTXR ST131 O25:H4/H30-Rx strains collected from healthy donors' stool. The virulence genes patterns were also analyzed and compared them with the virotypes reported previously; then adherence, invasion, macrophage survival and biofilm formation assays were evaluated and AIEC pathotype genetic determinants were investigated. FINDINGS: Non-reported virulence patterns were found in our isolates, two of them carried satA, papA, papGII genes and the two-remaining isolates carried cnfI, iroN, satA, papA, papGII genes, and none of them belonged to classical ST131 virotypes, suggesting an endemic distribution of virulence genes and two new virotypes. The presence of PAI-I and PAI-II of Uropathogenic E. coli was determined in three of the four strains, furthermore adherence and invasion assays demonstrated higher degrees of attachment/invasion compared with the control strains. We also amplified intI1, insA and insB genes in all four samples. INTERPRETATION: The results indicate that these strains own non-reported virotypes suggesting endemic distribution of virulence genes, our four strains also belong to an AIEC pathotype, being this the first report of AIEC in México and the association of AIEC with healthy donors.

Characterization of antimicrobial resistance mechanisms in carbapenem-resistant Pseudomonas aeruginosa carrying IMP variants recovered from a Mexican Hospital.

PURPOSE: Pseudomonas aeruginosa infections in hospitals constitute an important problem due to the increasing multidrug resistance (MDR) and carbapenems resistance. The knowledge of resistance mechanisms in Pseudomonas strains is an important issue for an adequate antimicrobial treatment. Therefore, the objective was to investigate other antimicrobial resistance mechanisms in MDR P. aeruginosa strains carrying bla IMP, make a partial plasmids characterization, and determine if modifications in oprD gene affect the expression of the OprD protein. METHODOLOGY: Susceptibility testing was performed by Kirby Baüer and by Minimum Inhibitory Concentration (presence/absence of efflux pump inhibitor); molecular typing by Pulsed-field gel electrophoresis (PFGE), resistance genotyping and integrons by PCR and sequencing; OprD expression by Western blot; plasmid characterization by MOB Typing Technique, molecular size by PFGE-S1; and bla IMP location by Southern blot. RESULTS: Among the 59 studied P. aeruginosa isolates, 41 multidrug resistance and carbapenems resistance isolates were detected and classified in 38 different PFGE patterns. Thirteen strains carried bla IMP; 16 bla GES and four carried both genes. This study centered on the 17 strains har-boring bla IMP. New variants of ß-lactamases were identified (bla GES-32, bla IMP-56, bla IMP-62) inside of new arrangements of class 1 integrons. The presence of bla IMP gene was detected in two plasmids in the same strain. The participation of the OprD protein and efflux pumps in the resistance to carbapenems and quinolones is shown. No expression of the porin OprD due to stop codon or IS in the gene was found. CONCLUSIONS: This study shows the participation of different resistance mechanisms, which are reflected in the levels of MIC to carbapenems. This is the first report of the presence of three new variants of ß-lactamases inside of new arrangements of class 1 integrons, as well as the presence of two plasmids carrying bla IMP in the same P. aeruginosa strain isolated in a Mexican hospital.

Molecular diversity and conjugal transferability of class 2 integrons among Escherichia coli isolates from food, animal and human sources.

Integrons are genetic platforms able to excise, integrate and express antibiotic resistance gene cassettes (GCs). Here we investigated the complete genetic organisation, genetic environment, location and conjugative transferability of a collection of class 2 integrons carried by Escherichia coli strains from different sources (poultry/pork meat, animals and humans). PCR cartography was conducted to determine the genetic arrangement of the integrons, their physical linkage to Tn7 and chromosomal insertion at the attTn7 site. Clonal relatedness of specific isolates was determined by MLST and DO-PCR. Transferability of class 2 integrons was tested by conjugation. The resulting transconjugants were characterised by antimicrobial resistance genotyping, S1-PFGE and replicon typing. Although a limited diversity of GCs was shown, a high percentage of novel structures was identified owing to the integration of insertion sequence (IS) elements at different sites (IS3/IS4/IS5/IS21 families). Insertion of IS10 in the attI2 site of a class 2 integron, between Pc2B and Pc2C promoters, was likely mediated by a site-specific transposition event. Chromosomal insertion of integrons at attTn7 was confirmed in 80% of the isolates. Conjugation experiments demonstrated that 29% of class 2 integrons could be mobilised to E. coli CHS26, demonstrating that they can be located in conjugative/mobilisable elements at a low frequency. Reported structures evidence how class 2 integrons have evolved by the activity of integron integrases and the invasion of ISs. Since most of them are chromosomally located, dispersion is predominantly vertical, although conjugation events also contribute to the spread of class 2 integrons among bacterial communities.

Flagellar expression in clinical isolates of non-typeable Haemophilus influenzae.

PURPOSE: Haemophilus influenzae is a commensal organism found in the upper respiratory tract of humans. When H. influenzae becomes a pathogen, these bacteria can move out of their commensal niche and cause multiple respiratory tract diseases such as otitis media, sinusitis, conjunctivitis and bronchitis in children, and chronic obstructive pulmonary disease in adults. However, H. influenzae is currently considered a non-flagellate bacterium. METHODOLOGY AND RESULTS: In this study, 90 clinical isolates of H. influenzae strains (typeable and non-typeable) showed different degrees of the swarm-motility phenotype in vitro.Keys findings. One of these strains, NTHi BUAP96, showed the highest motility rate and its flagella were revealed using transmission electron microscopy and Ryu staining. Moreover, the flagellar genes fliC and flgH exhibited high homology with those of Actinobacillus pleuropneumoniae, Escherichia coli and Shigella flexneri. Furthermore, Western blot analysis, using anti-flagellin heterologous antibodies from E. coli, demonstrated cross-reaction with a protein present in NTHi BUAP96. CONCLUSION: This study provides, for the first time, information on flagellar expression in H. influenzae, representing an important finding related to its evolution and pathogenic potential.

Detection and Molecular Characterization of Escherichia coli Strains Producers of Extended-Spectrum and CMY-2 Type Beta-Lactamases, Isolated from Turtles in Mexico.

Multidrug-resistant bacteria are a growing problem in different environments and hosts, but scarce information exists about their prevalence in reptiles. The aim of this study was to analyze the resistance mechanisms, molecular typing, and plasmid content of cefotaxime-resistant (CTX(R)) Escherichia coli isolates recovered from cloacal samples of 71 turtles sheltered in a herpetarium in Mexico. CTX(R)-E. coli were recovered in 11 of 71 samples (15.5%), and one isolate/sample was characterized. Extended-spectrum ß-lactamase (ESBL)-producing E. coli isolates were detected in four samples (5.6%): two strains carried the blaCTX-M-2 gene (phylogroup D and ST2732) and two contained the blaCTX-M-15 gene (phylogroup B1 and lineages ST58 and ST156). The blaCMY-2 gene was detected by PCR in E. coli isolates of eight samples (9.8%) (one of them also carried blaCTX-M-2); these isolates were distributed into phylogroups A (n = 1), B1 (n = 6), and D (n = 1) and typed as ST155, ST156, ST2329, and ST2732. Plasmid-mediated quinolone resistance (PMQR) genes were detected in five isolates [aac(6')Ib-cr, qnrA, qnrB19, and oqxB]. From three to five replicon plasmids were detected among the strains, being IncFIB, IncI1, IncFrep, and IncK the most prevalent. ESBL or pAmpC genes were transferred by conjugation in four strains, and the blaCTX-M-15 and blaCMY-2 genes were localized in IncFIB or IncI1 plasmids by Southern blot hybridization assays. Class 1 and/or class 2 integrons were detected in eight strains with six different structures of gene cassette arrays. Nine pulsed-field gel electrophoresis patterns were found among the 11 studied strains. To our knowledge, this is the first detection of ESBL, CMY-2, PMQR, and mobile determinants of antimicrobial resistance in E. coli of turtle origin, highlighting the potential dissemination of multidrug-resistant bacteria from these animals to other environments and hosts, including humans.

First evidence of polar flagella in Klebsiella pneumoniae isolated from a patient with neonatal sepsis.

The genus Klebsiella belongs to the family Enterobacteriaceae, and is currently considered to be non-motile and non-flagellated. In the present work, 25 Klebsiella strains isolated from nosocomial infections were assessed for motility under different growth conditions. One Klebsiella isolate, KpBUAP021, demonstrated a swim-like motility phenotype. The K. pneumoniae genotype was confirmed by 16S rRNA and rpoB gene sequence analysis. Multilocus sequence typing analysis also revealed that the KpBUAP021 strain places it in the ST345 sequence type, and belongs to the phylogenetic Kpl group. Transmission electron microscopy and the Ryu staining technique revealed that KpBUAP021 expresses polar flagella. Finally, the presence of fliC, fliA and flgH genes in this K. pneumoniae strain was confirmed. This report presents the first evidence for flagella-mediated motility in a K. pneumoniae clinical isolate, and represents an important finding related to its evolution and pathogenic potential.

Identificación y tipificación de Haemophilus influenzae mediante PCR múltiple / Use of multiplex PCR for detection and identification of Haemophilus influenzae

Se diseñó una estrategia metodológica para la detección simultánea entre Haemophilus influenzae serotipo b (Hib) y no tipificable (HiNT), así como para discriminar entre H. influenzae, Moraxella catarrhalis y Streptococcus pneumoniae. El estudio se realizó en una recolección de 64 cepas, las cuales se sometieron a amplificación por PCR utilizando ADN genómico o directamente el cultivo bacteriano como plantilla. La PCR se realizó en dos fases: 1) PCR triple para discriminar entre H. influenzae y M. catarrhalis empleando oligonucleótidos específicos para los genes del ARNr 16S y de la neumolisina (Ply) para identificar S. pneumoniae; 2) PCR doble para la identificación simultánea de Hib y HiNT, utilizando oligonucleótidos específicos de los genes cap y hmw. Se logró la amplificación del gen ARNr 16S en las 64 cepas de H. influenzae, sin obtenerse amplificación en las cepas control. La secuencia Bex A se amplificó en 29 de 32 cepas de Hib, y la secuencia HMWA se amplificó en 29 de 30 cepas de HiNT. La amplificación con los oligonucleótidos específicos para M. catarrhalis y S. pneumoniae no mostró productos con ninguna cepa de H. influenzae. Estos resultados permiten proponer la aplicación de esta herramienta metodológica para discriminar entre M. catarrhalis y S. pneumoniae, e identificar H. influenzae directamente a partir de muestras clínicas.