Extended spectrum beta lactamase--mediated resistance among isolates of family Enterobacteriaceae

The emergence and spread of extended spectrum beta lactamase (ESBL) producing strains have led to questions regarding optimal therapy for infections especially empirical treatment of suspected gram negative infection. A total of400 isolates belonging to family enterobacteriaceae were isolated from various clinical samples and were studied for ESBL production by NCCLS (National Committee for Clinical Laboratory Standards) confirmatory test (using disc diffusion technique) and MIC brott dilution test. ESBL production was found in 61.72% of hospital isolates and 30.90% of outdoor isolates. The susceptibility of ESBL producers to imipenem and amikacin was found to be 100% and 63.38% respectively. A high degree of associated resistance to other antimicrobial was found in ESBL producers. Tests for detection of ESBLs and continuous monitoring of antimicrobial sensitivity pattern in individual setting is emphasized to minimise the emergence of drug resistant bacteria.     

Zinc-dependent carbapenemases in clinical isolates of family Enterobacteriaceae

Purpose: The emergence and spread of zinc-dependent carbapenem resistance has become a diagnostic challenge for clinical microbiologists. The objective of the present study was to screen zinc-dependent carbapenemase activity in clinical isolates of family Enterobacteriaceae. Materials and Methods: A total of 102 multidrug-resistant organisms (MDROs), non-repeat clinical isolates of family Enterobacteriaceae from two tertiary care centres in Delhi, were screened for carbapenemase production by a modified Hodge test (MHT) and additionally by a re-modified Hodge test, EDTA double disc synergy test, and combined disc test (or disc enhancement test) to determine zinc dependence of carbapenemases harbouring bacteria. Results: Of the total 102 clinical isolates (June through November 2010), 91 were from urine and 11 were from blood specimens. The isolates were obtained from patients visiting the outpatient department (18 isolates), admitted in non-ICU inpatient care units (74 isolates) and patients admitted in ICUs (4 isolates). MHT identified 92 (90.2%) isolates as carbapenemases producers. Among those found negative for MHT (n=10), metallo-beta-lactamases (MBLs) activity was demonstrated through the EDTA disc diffusion synergy test and the combined disc test in 8 and 9 isolates respectively. A total of 63 (61.7%) isolates demonstrated MBL activity despite in vitro sensitivity to Imipenem. Conclusions: The study demonstrated that supplementing the MHT with at least one of the screening methods increases the likelihood of picking up such isolates that may be missed by the MHT. The study also demonstrates the wide-spread presence of MBLs in Enterobacteriaceae members from patients visiting hospitals in east Delhi.     

Evaluation of the DMS Rapid E system for identification of clinical isolates of the family Enterobacteriaceae.

A total of 387 unique clinical isolates of the family Enterobacteriaceae were examined with the new DMS Rapid E gram-negative identification system (DMS Laboratories, Inc., Flemington, N.J.) and the API 20E procedure (Analytab Products, Plainview, N.Y.). Altogether, 376 strains (97.2%) were correctly identified to species level within 4 h with the DMS Rapid E system; 366 strains (94.6%) were correctly identified with the API 20E after overnight incubation.     

Phosphatase activity is a constant feature of all isolates of all major species of the family Enterobacteriaceae.

In this study we evaluated phosphatase activity in members of the family Enterobacteriaceae by conventional methods and by a novel method. The novel method is based on the formation of bright-green-strained colonies by phosphatase-positive, but not phosphatase-negative, strains in the presence of a phosphate substrate, such as phenolphthalein monophosphate or 6-benzoylnaphthyl phosphate (6-BNP), and methyl green. A total of 1,055 strains belonging to 65 different species of Enterobacteriaceae were tested for green staining of the colonies in the presence of methyl green and either phenolphthalein monophosphate or 6-BNP and for phosphatase activity by three different conventional methods. With the sole exception of one Leminorella richardii type strain, all isolates of all of the species formed green-stained colonies in the presence of the substrate 6-BNP. All strains were phosphatase positive by all of the conventional methods.     

Antigenic variation among recent Japanese isolates of bovine coronaviruses belonging to phylogenetically distinct genetic groups

Bovine coronaviruses (BCoVs) isolated in Japan consist of four genetic groups, as determined by phylogenetic analysis using the polymorphic region (aa 456–592) of the S glycoprotein gene. Japanese field isolates of BCoV, reference Kakegawa strain, and vaccine strain 66/H were analyzed for their antigenic properties by indirect immunofluorescence and neutralization testing. There were no significant differences observed among these BCoVs in direct immunofluorescence tests. However, antigenic differences were observed between BCoVs in the neutralization tests, although there was no clear indication of a distinct serotype. A monoclonal antibody, 4H4, against the Kakegawa strain belonging to group 1 lacked significant neutralizing activity for viruses of groups 2, 3, and 4. Therefore, we speculate that the genetic differences between these groups may have altered their antigenicity. Analysis of mutant viruses resistant to neutralization by 4H4 revealed that the antigenic site of the Kakegawa strain maps to amino acid position 284 of the S glycoprotein. This site is not homologous to a known antigenic site (aa 528) of the Quebec strain belonging to group 1, and it is not located in the conformational domain comprising domain I (aa 351–403) and domain II (aa 517–621). This amino acid constitutes a neutralization epitope of BCoV, which is distinct from aa 528 of the Quebec strain. These results indicate antigenic evolution of BCoV between the genetic groups circulating in Japan.     

Glycosidase profiles of members of the family Enterobacteriaceae.

A total of 712 strains representing 47 taxa of the family Enterobacteriaceae were tested for the ability to hydrolyze 14 4-methylumbelliferyl (4-MU)-linked substrates within 3 h of incubation. In addition to the well-known differentiation potential of the hydrolysis of 4-MU-beta-D-galactopyranoside, 4-MU-beta-D-glucuronide, and 4-MU-beta-D-xylopyranoside, the hydrolysis of some other fluorogenic substrates (e.g., 4-MU-beta-D-fucopyranoside, 4-MU-N-acetyl-beta-D-galactosaminide, and 4-MU-alpha-D-galactopyranoside) can also be used for species differentiation within the family Enterobacteriaceae.     

Distribution of the serine protease autotransporters of the Enterobacteriaceae among extraintestinal clinical isolates of Escherichia coli

Urinary tract infections continue to be among the most common extraintestinal diseases. Cystitis in women is by far the most common urinary tract infection; pyelonephritis in both sexes and prostatitis in men are more severe but less frequent complaints. Escherichia coli is by far the most common cause of urinary tract infection. It is believed that uropathogenic E. coli is adept at colonizing the urinary tract via the production of specific virulence factors. Recently, a novel virulence determinant, Vat, was described for the prototypical uropathogenic E. coli strain CFT073. Vat is a member of the SPATE (serine protease autotransporters of the Enterobacteriaceae) subfamily of the autotransporters. Previously, SPATEs have been described for all pathovars of E. coli, but until recently their presence had been noticeably absent in nonpathogenic E. coli. In this report we describe the prevalence and phylogenetic distribution of the SPATEs among uropathogenic E. coli and the ECOR collection, demonstrating an association between the presence of the SPATEs, including Vat, and uropathogenic E. coli phylogroups. In addition, we describe the distribution of SPATEs among nonpathogenic E. coli.     

Genotyping of outbreak-related and sporadic isolates of Clostridium difficile belonging to serogroup C.

Serogroup C of Clostridium difficile is the serogroup most frequently related to outbreaks. Fifty-six toxigenic serogroup C isolates of C. difficile were genotyped by ribotyping PCR (ribo-PCR), random amplified polymorphic DNA (RAPD) assay, and pulsed-field gel electrophoresis (PFGE). Thirty-five of the 56 isolates were recovered from four unrelated outbreaks (Belgium, 1987, 1992, and 1995; France, 1992 to 1993) 7 derived from a spatiotemporal cluster in Cotonou, Benin (1992), and 14 were sporadic isolates. The serogroup C reference strain, also isolated during an outbreak (Belgium, 1983), was genotyped too. Ribo-PCR, the RAPD assay, and PFGE generated 2, 5, and 11 major genotypes, respectively. Combination of the three methods finally yielded 13 general types, although ribo-PCR did not play any role in enhancing resolution. Three general types were recovered from all the isolates from the five outbreaks and the cluster, with two types being predominant. The 14 sporadic serogroup C isolates were divided into 11 overall genotypes. These results indicate that genotyping methods, and more particularly the combination of the RAPD assay and PFGE, can resolve genetic diversity within toxigenic, serogroup C C. difficile strains. Also, this study suggests that outbreak-related serogroup C strains are limited to a few genetically stable and apparently very widely (internationally and intercontinentally) distributed genotypes.     

Iron and virulence in the family Enterobacteriaceae

The ability of bacterial pathogens to acquire iron in the host is an essential component of the disease process. Pathogenic Enterobacteriaceae spp. may either scavenge host iron sources such as heme or induce high-affinity iron-transport systems to remove iron from host proteins. The ease with which iron is acquired from the host will be at least partially determined by the iron status of the host at the time of infection. In response to infection, mammalian hosts reduce serum iron levels and withhold iron from the invading microorganisms. Thus the competition for iron is an active process which influences the outcome of a host-bacterial interaction.     

High-level fluoroquinolone resistance in ophthalmic clinical isolates belonging to the species Corynebacterium macginleyi

The clinical importance of nondiphtherial Corynebacterium, a ubiquitous member of the normal human microflora of the skin and mucous membrane, for ocular surface infections has been recognized recently. We performed an antimicrobial susceptibility test with Etest strips for three fluoroquinolones (ciprofloxacin, norfloxacin, and levofloxacin) and a taxonomic analysis on 21 isolates of Corynebacterium from ophthalmic samples. Of these, 16 isolates were identified as C. macginleyi at the species level on the basis of 16S rRNA gene sequence comparisons. The remaining five isolates were determined to be C. mastitidis (four) or C. accolens (one). Eleven of the C. macginleyi isolates showed high levels of resistance to all of the fluoroquinolones tested, and one isolate was resistant to norfloxacin alone. An analysis of the amplified quinolone-resistance-determining regions of the gyrA genes revealed that a single amino acid substitution in position 83 of the gyrA product was sufficient to generate the norfloxacin resistance phenotype, and double mutations leading to amino acid changes in positions 83 and 87 were necessary for high-level resistance against the other fluoroquinolones. We conducted the first example of multilocus sequence typing (MLST) analysis on C. macginleyi. The MLST analysis grouped the majority of C. macginleyi isolates into a single lineage, and another molecular strain typing by random amplified polymorphic DNA fragment patterns supported the finding, indicating that a particular lineage of C. macginleyi is dominant on the human ocular surface. This type of population might be particularly adaptable to the milieu on the human ocular surface.     

First description of hemagglutination by a virus belonging to the family Dicistroviridae

Triatoma virus is the only virus whose genome has been sequenced and studied in triatomines. It belongs to the family Dicistroviridae. In order to detect whether TrV has the ability to agglutinate erythrocytes of domestic and laboratory animals, we performed a hemagglutination assay. Positive hemagglutination was found for red blood cells of guinea pigs. The HA assay could be used as a titration method, at least for purified viral particles obtained from triatomine stool. This is the first record of hemagglutinating properties for Dicistroviridae.     

Distribution of emm types among group A streptococcal isolates from Serbia

This is the first study concerning the molecular epidemiology of group A streptococcus in Serbia and includes 145 isolates from patients with various infections during the period 2001–2007. The emm types, superantigen profile and susceptibility pattern were determined. Among 31 emm types identified, the most prevalent were emm6, emm12, emm1, and emm58. All isolates showed uniform antimicrobial susceptibility to all tested antibiotics, with the exception of tetracycline and erythromycin (41% and 0.7% resistant strains, respectively). Significant heterogeneity of emm types was found, with a high frequency of emm6 and emm58, as well as a considerable prevalence of tetracycline resistance, and a low level of macrolide resistance.     

Comparison of Candida albicans strain types among isolates from three countries

Multi-locus sequence typing data for 217 Candida albicans isolates cultured since 1990 from blood and vaginal samples in Japan, England/Wales and the USA were analysed for geographically related variations. While no significant differences were found between distributions of diploid sequence types (DSTs) in blood vs. vaginal isolates, there were highly significant differences in the clade distributions of isolates from the three geographical sources. Clade 2 strains were predominantly isolates from England/Wales, while clade 3 strains came mainly from the USA. The isolates from Japan were highly prevalent among strains in clades 5-17, and provided the first example seen so far in C. albicans of an amino acid encoded by three separate codons. Within clade 1, the most commonly encountered clade for isolates from all three regions, 15 Japanese isolates and 1 English isolate formed a separate clonal cluster in eBURST analysis. A similarly well demarcated clonal cluster rich in isolates from Japan was also found among the clade 4 strains. The data suggest C. albicans undergoes localized evolution, but human movements and person-to-person spread considerably blur the boundaries of such evolution.     

Insights into new bacteriophages of Lactococcus garvieae belonging to the family Podoviridae

Lactococcus garvieae is an emerging pathogen responsible for lactococcosis, a serious disease in trout aquaculture. The identification of new bacteriophages against L. garvieae strains may be an effective way to fight this disease and to study the pathogen’s biology. Three L. garvieae phages, termed WP-1, WWP-2 and SP-2, were isolated from different environments, and their morphological features, genome restriction profiles and structural protein patterns were studied. Random cloning of HindIII-cut fragments was performed, and the fragments were partially sequenced for each phage. Although slight differences were observed by transmission electron microscopy, all of the phages had hexagonal heads and short non-contractile tails and were classified as members of the family Podoviridae. Restriction digestion analysis of the nucleic acids of the different phages revealed that the HindIII and AseI digests produced similar DNA fragment patterns. Additionally, SDS-PAGE analysis indicated that the isolated phages have similar structural proteins. The sequence BLAST results did not show any significant similarity with other previously identified phages. To the best of our knowledge, this study provides the first molecular characterization of L. garvieae phages.     

Identification of family Enterobacteriaceae using a three tube method.

Four hundred isolates of Gram Negative bacilli from different samples were tested by three tube method and conventional tests. An overall agreement between the two methods was 89.5%. Non-agreement was in respect of less stable tests like gas production and motility. This does not reflect adversely on the efficacy and suitability of the method. The three tube method is simple, rapid, economical and accurate, saving both time and material.     

Immunomodulatory properties of porins of some members of the family Enterobacteriaceae.

The outer membrane protein (OMP) preparation of Salmonella typhi was observed to have several immunomodulatory properties. Treatment of mice with an intraperitoneal injection of the OMP preparation enhanced both cellular and humoral responses of the mice to an unrelated antigen, a killed vaccine of Mycobacterium vaccae; both the delayed-type hypersensitivity (DTH) response and the antibody titers were enhanced. The predominant isotype of the antibody shifted from immunoglobulin G1 (IgG1) to IgG2a upon treatment with OMP. Treatment of mice with the OMP preparation improved the efficiency of in vitro antigen presentation by the peritoneal cells and also induced the cells to secrete interleukin-1. Treatment with the lipopolysaccharide (LPS) preparation of S. typhi had the opposite effect; i.e., the DTH response to M. vaccae was suppressed. Treatment with OMP neutralized the suppressive effects of LPS. The OMP preparation also had an enhancing effect on the innate immune mechanisms of the mice. Intraperitoneal injection of the OMP preparation enhanced the microbicidal activity of the peritoneal cells, and production of nitric oxide intermediates was stimulated. Injection of the OMP preparation into footpads of naive nonimmune mice induced a sustained hypersensitivity response that peaked at 24 h. Purified porins of the OMP preparation could induce both immunomodulation and hypersensitivity. Porins prepared from five different Salmonella strains and a strain of normal fecal Escherichia coli also exhibited immunomodulatory and hypersensitivity-inducing activities.     

Quality of powdered substitutes for breast milk with regard to members of the family Enterobacteriaceae.

Members of the family Enterobacteriaceae were cultured from 52.5% of 141 milk substitute infant formulas which were obtained in 35 countries. The concentration did not exceed a level of 1 CFU/g in any product. The species which were isolated most frequently were Enterobacter agglomerans, cloacae, Enterobacter sakazakii, and Klebsiella pneumoniae. If infections due to these organisms occur, it can be useful to include a check of the hygienic precautions which are taken during the preparation and storage of the formula. Milk powders without members of the Enterobacteriaceae might offer extra protection to the newborn if some multiplication does occur in the formula.     

Formation of crystalline deposits by several genera of the family Enterobacteriaceae.

Several species of bacteria from the family Enterobacteriaceae formed crystalline materials containing calcium when grown in a defined culture medium. Enterobacter aerogenes, Proteus vulgaris, Citrobacter freundii, and C. intermedius produced calcium pyrophosphate crystals. Edwardsiella tarda and Escherichia coli formed calcite III crystals, whereas Proteus mirabilis, Klebsiella pneumoniae, Providencia stuartii, and Serratia marcescens produced hydroxyapatite crystals. Several of these bacteria have been isolated from the kidneys of patients with kidney stones, indicating that microorganisms may be involved in the enucleation process of kidney stone formation.