Microbiology of Hafnia alvei. / Microbiología de Hafnia alvei.

Hafnia alvei is a Gram-negative facultatively anaerobic bacillus that constitutes part of the human gut flora. Until recently, H. alvei strains could be mistakenly identified by conventional methods, miniaturisation or automatic systems as members of the Serratia, Escherichia, Citrobacter, Yokenella, Obesumbacterium or Salmonella genera. Consequently, molecular techniques were required for their definitive identification in the clinical laboratory. In addition, a new Hafnia species, H. paralvei, has recently appeared, which undoubtedly includes many of the strains reported in the literature as H. alvei. Alrhough H. alvei isolation from human clinical specimens remains uncommon, the development of drug resistance due to this species is emerging and it is likely that this organism will gain increasing importance in the future. Moreover, although H. alvei shares some virulence mechanisms with other Gram-negative enteropathogens, little is known about the factors that contribute to its pathogenesis in humans. The present article reviews the current identification methods, antimicrobial resistance and virulence factors of this bacterium.

Microbiología de Hafnia alvei / Microbiology of Hafnia alvei

Hafnia alvei es un bacilo gramnegativo facultativamente anaeróbico que constituye parte de la flora intestinal humana. Hasta hace poco, las cepas de H. alvei, que se identificaban por métodos convencionales, por miniaturización o por sistemas automáticos, podían confundirse fácilmente con miembros de los géneros Serratia, Escherichia, Citrobacter, Yokenella, Obesumbacterium o Salmonella, por lo que era necesario realizar técnicas moleculares para su identificación definitiva en la práctica clínica. Además, recientemente se ha introducido una nueva especie de Hafnia, H. paralvei, que sin duda incluye a muchas de las cepas previamente identificadas en la literatura como H. alvei. Aunque el aislamiento de H. alvei de muestras clínicas humanas sigue siendo poco frecuente, han aumentado los artículos científicos que evidencian un aumento de la resistencia a los antibióticos en esta especie y es probable que este organismo gane cada vez más importancia en el futuro. Además, aunque H. alvei comparte algunos mecanismos de virulencia con otros enteropatógenos gramnegativos, se sabe poco sobre los factores que contribuyen a su patogénesis en humanos. El presente artículo revisa los métodos de identificación actuales, la resistencia a los antimicrobianos y los factores de virulencia de esta bacteria

Hafnia alvei is a Gram-negative facultatively anaerobic bacillus that constitutes part of the human gut flora. Until recently, H. alvei strains could be mistakenly identified by conventional methods, miniaturisation or automatic systems as members of the Serratia, Escherichia, Citrobacter, Yokenella, Obesumbacterium or Salmonella genera. Consequently, molecular techniques were required for their definitive identification in the clinical laboratory. In addition, a new Hafnia species, H. paralvei, has recently appeared, which undoubtedly includes many of the strains reported in the literature as H. alvei. Alrhough H. alvei isolation from human clinical specimens remains uncommon, the development of drug resistance due to this species is emerging and it is likely that this organism will gain increasing importance in the future. Moreover, although H. alvei shares some virulence mechanisms with other Gram-negative enteropathogens, little is known about the factors that contribute to its pathogenesis in humans. The present article reviews the current identification methods, antimicrobial resistance and virulence factors of this bacterium

Profiling of VOCs released from different salivary bacteria treated with non-lethal concentrations of silver nitrate.

Considering the shortcomings related to antibiotics usage, the introduction of other bacteriostatic and bactericidal agents that present synergetic effects or standalone properties is urgently needed. AgNO3 is an important bactericidal agent, which imparts various functions on bacteria dependent on its concentration. Therefore, an understanding of its mechanisms of action in infinitesimal concentrations plays an important role which can ultimately lead to AgNO3 involvement in the pharmaceutical industry. The monitoring of VOC (volatile organic compound) profiles emitted by bacteria is a simple method to assess changes occurring in bacterial metabolism. In this study, VOCs of Hafnia alvei, Pseudomonas luteola and Staphylococcus warneri cultures were analyzed both in the absence and in the presence of three concentrations of AgNO3. Headspace solid-phase microextraction gas chromatography/mass spectrometry (HS-SPME-GC/MS) was employed for extraction and analysis. After supplementation with AgNO3, changes in the emitted fingerprints were investigated. Odorants associated with mouth-related and systemic diseases, like dimethyl trisulfide, indole (halitosis) and 2-hexanone (celiac disease), were also affected by addition of AgNO3. Statistical tests proved discrimination between obtained profiles with more that 90% variability. Moreover, physiological states of bacteria after dosage with various concentration of stressing agent were investigated and explained by the mechanisms of action.

Effects of Sulfide Flavors on AHL-Mediated Quorum Sensing and Biofilm Formation of Hafnia alvei.

In this study, 10 different sulfide flavor compounds commonly used as food additives were screened for antiquorum-sensing activity. Among these, diallyl disulfide (DADS) and methyl 2-methyl-3-furyl disulfide (MMFDS) were found to exert the strongest inhibition against violacein production in Chromobacterium violaceum 026, the tested biosensor strain. DADS and MMFDS also inhibited the growth of Hafnia alvei H4, yielding MIC values of 48 and 41.6 mM, respectively. In addition, DADS and MMFDS also inhibited the ability of H. alvei H4 to produce acyl-homoserine lactone as demonstrated by the reduced level of C6-HSL in the supernatant of DADS-treated culture. At concentrations corresponding to 1/4 MIC, DADS, and MMFDS inhibited the swarming ability of H. alvei H4 by 73.50% and 76.43%, respectively, while having virtually no effect on cell growth. The same concentrations of DADS and MMFDS also completely inhibited the formation of biofilm. These antiquorum sensing effects of DADS and MMFDS involved changes in the expression of the quorum-sensing genes luxI and luxR. Quantitative RT-PCR analysis showed that the mRNA levels of both genes were significantly reduced by DADS and MMDFS at concentrations below their MICs. However, further test using a mutant strain of H. alvei lacking luxR (ΔluxR) revealed significant reduction in luxI mRNA level upon treatment of the strain with DADS or MMDFS, but no change in luxR mRNA level occurred when a luxI-lacking mutant (ΔluxI) was treated with these compounds. The result therefore suggested that the antiquorum-sensing effect of DADS and MMFDS against H. alvei H4 might operate mainly through the inhibition of luxI expression in the cells. PRACTICAL APPLICATION: The sulfide flavors compounds used in this paper are commonly used in food processing in China and are listed in the national standard of Chinese food additives GB2760-2014. The application of sulfide flavors in food processing can enhance aroma and prevent food spoilage.

[A descriptive study of Hafnia alvei isolated from stool samples: an approach to its clinical assessment]. / Descriptivo de Hafnia alvei aisladas en coprocultivo: aproximación a su valoración en clínica.

OBJECTIVE: The importance in human diarrhoeal disease of Hafnia alvei is unclear. The objective of the study was to describe the population which was isolated H. alvei in stool cultures and the therapeutic management of these cases in our Health Area. METHODS: A descriptive retrospective study was carried out in 2014 and 2015. Epidemiological, clinical, treatment and evolution variables were collected in the computerized clinical history. RESULTS: A collection of 7,290 stool specimens were processed, 3,321 in 2014 and 3,969 in 2015, of which 58 (1.7%) and 53 (1.3%) were positive for H. alvei, respectively. A 60.4% of samples were isolated in women. The mean age was 38.68 years. A 68.5% of samples were from primary care. In 71.2% there was related clinic, diarrhoea in 57.7%. In 75.7% of the cases there was not associated underlying disease. A 43.2% of the cases received treatment. A 66.7% of treated patients came from Primary Care. The mean duration of treatment was 8 days. The evolution was favourable in 85.4% of the cases treated. All strains were susceptible to ciprofloxacin and trimethoprim/sulfamethoxazole. CONCLUSIONS: More evidence is needed to support H. alvei as a cause of gastroenteritis.

Inhibition of Low-Grade Inflammation by Anthocyanins after Microbial Fermentation in Vitro.

The anti-inflammatory effects of anthocyanins (ACNs) on vascular functions are discussed controversially because of their low bioavailability. This study was performed to determine whether microorganism (MO)-fermented ACNs influence vascular inflammation in vitro. Therefore, MO growth media were supplemented with an ACN-rich grape/berry extract and growth responses of Escherichia coli, E. faecalis and H. alvei, as well as ACN fermentation were observed. MO supernatants were used for measuring the anti-inflammatory effect of MO-fermented ACNs in an epithelial-endothelial co-culture transwell system. After basolateral enrichment (240 min), endothelial cells were stimulated immediately or after 20 h with TNF-α. Afterwards, leukocyte adhesion, expression of adhesion molecules and cytokine release were measured. Results indicate that E. coli, E. faecalis and H. alvei utilized ACNs differentially concomitant with different anti-inflammatory effects. Whereas E. coli utilized ACNs completely, no anti-inflammatory effects of fermented ACNs were observed on activated endothelial cells. In contrast, ACN metabolites generated by E. faecalis and H. alvei significantly attenuated low-grade stimulated leukocyte adhesion, the expression of adhesion molecules E-selectin, VCAM-1 and ICAM-1 and cytokine secretion (IL-8 and IL-6), as well as NF-κB mRNA expression with a more pronounced effect of E. faecalis than H. alvei. Thus, MO-fermented ACNs have the potential to reduce inflammation.

Occurrence of urinary tract infection in adolescent and adult women of shanty town in Dhaka City, Bangladesh.

BACKGROUND: Urinary tract infection (UTI) is commonly experienced by women of various age groups especially elderly ones. We planned to find out the prevalent microbial strains causing UTI in slum inhabitant adolescent and adult women in Dhaka City, Bangladesh. METHODS AND MATERIALS: Urine sample was collected from 462 UTI suspected female subjects. Pathogenic bacteria were identified using standard microbiological tests, and antimicrobial sensitivity profiles of the pathogens were determined. RESULTS: Bacteriuria was present in 9% of the subjects. A higher incidence (16.8%) of UTI was noted among adult women aged above 19 years. Escherichia coli (69%), Streptococcus spp. (15%) and Pseudomonas aeruginosa (7%) were more frequently isolated from the urine samples compared to Enterococcus faecalis (3%), Staphylococcus aureus (2%), Klebsiella pneumoniae (2%) and Hafnia alvei (2%). The E. coli isolates showed complete resistance to commonly used drugs, and 58% of these isolates were multidrug resistant (MDR). Minimum Inhibitory Concentration (MIC) values for ciprofloxacin ranged between 64µg/ml and 512µg/ml, and the Minimum Bactericidal Concentration (MBC) values against the isolates were 128µg/ml or above. Isolated strains of E. coli exhibited equal extent of ciprofloxacin resistance irrespective of the presence or absence of plasmid in them. CONCLUSION: The extent of drug resistance among the uropathogens if ignored may render them uncontrollable. This study suggests regular monitoring of drug resistance phenotype of the UTI pathogens to reduce the morbidity of female UTI patients and offer better treatment strategy in the healthcare sectors of Bangladesh.

Elaboration of highly hydrophobic polymeric surface--a potential strategy to reduce the adhesion of pathogenic bacteria?

Different polymeric surfaces have been modified in order to reach a high hydrophobic character, indeed the superhydrophobicity property. For this purpose, polypropylene and polystyrene have been treated by RF or µwaves CF4 plasma with different volumes, the results were compared according to the density of injected power. The effect of pretreatment such as mechanical abrasion or plasma activation was also studied. The modified surfaces were shown as hydrophobic, or even superhydrophobic depending of defects density. They were characterized by measurement of wettability and roughness at different scales, i.e. macroscopic, mesoscopic and atomic. It has been shown that a homogeneous surface at the macroscopic scale could be heterogeneous at lower mesoscopic scale. This was associated with the crystallinity of the material. The bioadhesion tests were performed with Gram positive and negative pathogenic strains: Listeria monocytogenes, Pseudomonas aeruginosa and Hafnia alvei. They have demonstrated an antibacterial efficiency of very hydrophobic and amorphous PS treated for all strains tested and a strain-dependent efficiency with modified PP surface being very heterogeneous at the mesoscopic scale. Thus, these biological results pointed out not only the respective role of the surface chemistry and topography in bacterial adhesion, but also the dependence on the peaks and valley distribution at bacteria dimension scale.

Impact on disinfection efficiency of cell load and of planktonic/adherent/detached state: case of Hafnia alvei inactivation by plasma activated water.

This paper describes the effects of initial microbial concentration and planktonic/adherent/detached states on the efficiency of plasma-activated water. This disinfecting solution was obtained by treating distilled water with an atmospheric pressure plasma produced by gliding electric discharges in humid air. The inactivation kinetics of planktonic cells of Hafnia alvei (selected as a bacterial model) were found to be of the first order. They were influenced by the initial microbial concentration. Efficiency decreased when the initial viable population N(0) increased, and the inactivation rate k(max) was linearly modified as a function of Log(10) (N(0)). This relation was used to compare planktonic, adherent, and detached cells independently from the level of population. Bacteria adhering to stainless steel and high-density polyethylene were also sensitive to treatment, but at a lower rate than their free-living counterparts. Moreover, cells detached from these solid substrates exhibited an inactivation rate lower than that of planktonic cells but similar to adherent bacteria. This strongly suggests the induction of a physiological modification to bacteria during the adhesion step, rendering adherent--and further detached--bacteria less susceptible to the treatment, when compared to planktonic bacteria.

Invasion and intracellular survival of Hafnia alvei strains in human epithelial cells.

AIMS: The aim of this study was to investigate the invasion and intracellular survival of different Hafnia alvei strains in HeLa cells. METHODS AND RESULTS: We performed different experiments on the bacterial invasion of different strains of H. alvei into the HeLa cell line using gentamicin protection assays and immunofluorescence. We also report the time course of cell internalization and the effects of inhibitors on the invasion of H. alvei. Levels of invasion varied depending on the conditions (strain, time and inoculum size) used. CONCLUSIONS: This study revealed that H. alvei strains were able to enter and persist in a human epithelial cell line. SIGNIFICANCE AND IMPACT OF THE STUDY: Our in vitro findings highlight the possibility that some H. alvei strains may exploit nonprofessional phagocytes or nonphagocytic cells to spread in vivo, which may be important for the persistence and establishment of an asymptomatic carrier state.

Graft versus host disease-related Hafnia alvei colonization and probable infection.

We describe the case of a graft versus host disease (GvHD) patient, in whom Hafnia alvei was cultured as a single organism, and at high bacterial counts from stool samples, from the onset of the disease until its resolution. This case is a further example of the contentious role of this species in causing human intestinal disease. Furthermore, it focuses on enteric damage by GvHD as a risk factor for acquiring H. alvei colonization, and probably infection.

Antibiotic resistance and adherence properties of Hafnia alvei clinical isolates: a 19-month study in the hospital of Orléans, France.

We studied the epidemiology of antibiotic resistance and adherence properties among all Hafnia alvei clinical isolates collected from August 2003 to February 2005 from patients hospitalized in the hospital of Orléans, France. The isolates were typed by random amplified polymorphic DNA (RAPD), screened for antibiotic resistance and bacterial adherence to A549 respiratory and T24 bladder cells. Six intestinal, 3 respiratory, and 8 isolates from different body sites were collected. A total of 12 RAPD profiles were found, demonstrating a high genetic diversity. All the isolates were resistant to amoxicillin + clavulanate and cephalothin and sensitive to aminoglycosides, fluoroquinolones, cefepime and imipenem. Six isolates had a high-level and constitutive cephalosporinase production phenotype. Three independent isolates were resistant or had intermediate sensitivity to nalidixic acid, sulfonamide and trimethoprim or chloramphenicol. All the isolates adhered efficiently to the two cell lines with a higher effectiveness of adherence to bladder cells. The respiratory isolates adhered more efficiently to epithelial cells than intestinal isolates. No relationship was found between antibiotic resistance phenotypes, adherence properties, and RAPD types. In conclusion, H. alvei is an unusual nosocomial pathogen with little acquired antibiotic resistance able to adhere to human epithelial cells from different human body compartments.

Natural antimicrobial susceptibility patterns and biochemical identification of Escherichia albertii and Hafnia alvei strains.

Bangladeshi diarrheagenic Hafnia alvei-like strains have been described recently as the new species Escherichia albertii (Int J Syst Evolut Microbiol. 2003;53:807-810). The natural susceptibility of 21 E. albertii and 76 H. alvei strains to 69 antimicrobial agents was examined, applying a microdilution procedure in IsoSensitest broth (for all the strains) and cation-adjusted Mueller-Hinton broth (for some strains). Examining the phenotypic features of both taxa with commercial identification systems and conventional tests, a database for an accurate biochemical separation of E. albertii from H. alvei was also established. Both taxa were naturally sensitive or sensitive and of intermediate susceptibility to aminoglycosides, acylureidopenicillins, ticarcillin, several cephalosporins, carbapenems, aztreonam, quinolones, folate pathway inhibitors, and nitrofurantoin. They were naturally resistant to tetracycline, penicillin G, oxacillin, all macrolides except for azithromycin, lincosamides, streptogramins, glycopeptides, rifampicin, and fusidic acid. Taxon-related differences in natural susceptibility affecting clinical assessment criteria were seen with doxycycline, minocycline, aminopenicillins, some cephalosporins, azithromycin, and fosfomycin. E. albertii was more susceptible than H. alvei to these agents and was naturally sensitive to all beta-lactams (except for penicillin G and oxacillin), azithromycin, and fosfomycin. H. alvei was naturally resistant or of intermediate susceptibility to all tetracyclines, amoxicillin, amoxicillin-clavulanate, ampicillin-sulbactam, narrow-spectrum cephalosporins, azithromycin, and fosfomycin. Motile malonate-negative Hafnia strains (indicating genospecies 2 of the H. alvei complex) were less susceptible to some cephalosporins than nonmotile, malonate-positive hafniae (indicating genospecies 1). Proline deaminase, hydroxyproline amidase, tripeptidase, chitinase, Voges-Proskauer reaction, and assimilation of histidine as well as acid production from glycerol, rhamnose, and xylose were suitable tests to separate strains of E. albertii from those of the H. alvei complex. Although out of the scope of this study, it should be noted that several strains of E. albertii showed acquired resistances to some penicillins and antifolates.

Conjugative plasmid mediated inducible nickel resistance in Hafnia alvei 5-5.

Hafnia alvei 5-5, isolated from a soil-litter mixture underneath the canopy of the nickel-hyperaccumulating tree Sebertia acuminata (Sapotaceae) in New Caledonia, was found to be resistant to 30 mM Ni(2+) or 2 mM Co(2+). The 70-kb plasmid, pEJH 501, was transferred by conjugation to Escherichia coli, Serratia marcescens, and Klebsiella oxytoca. Transconjugant strains expressed inducible nickel resistance to between 5 and 17 mM Ni(2+), and cobalt resistance to 2 mM Co(2+). A 4.8-kb Sal- EcoRI fragment containing the nickel resistance determinant was subcloned, and the hybrid plasmid was found to confer a moderate level of resistance to nickel (7 mM Ni(2+)) even to E. coli. The expression of nickel resistance was inducible by exposure to nickel chloride at a concentration as low as 0.5 mM Ni(2+). By random Tn phoA'-1 insertion mutagenesis, the fragment was shown to have structural genes as well as regulatory regions for nickel resistance. Southern hybridization studies showed that the nickel-resistance determinant from pEJH501 of H. alvei 5-5 was homologous to that of pTOM9 from Alcaligenes xylosoxydans 31A.

Extraintestinal infection due to Hafnia alvei.

The aim of this study was to establish the clinical features of extraintestinal infections caused by Hafnia alvei. Over a 5-year period (1994-1998), data were collected regarding inpatients (n = 8) with nosocomial (n = 5) or community-acquired (n = 3) infections caused by Hafnia alvei. The mean age of the patients was 47 +/- 21 years. Three patients had hospital-acquired urinary tract infections. Hafnia alvei also caused community-acquired cholangitis, cholecystitis, appendicitis, psoas abscess and prosthetic endocarditis. Hafnia alvei was susceptible to amoxicillin/clavulanic acid and to first-generation cephalosporins in two cases. Susceptibility to aminoglycosides, imipenem, cotrimoxazole, ciprofloxacin, piperacillin and cefotaxime was very good (8/8). Four patients required invasive treatment.