Multidrug-resistant and epidemic clones of Escherichia coli from natural beds of Venus clam.

Epidemic Escherichia coli clones have been recovered in marine sediment along the coast of Marche, an Adriatic region in central Italy. In the present study, E. coli strains from the clam Chamelea gallina, sampled from seven natural beds in the same area, were detected. Selected E. coli isolates from all sampling sites were screened for antimicrobial susceptibility, genetic diversity and correlation. The majority (60%) belonged to phylogroups A or B1, 31% to the other groups (B2, C, D, E, F), 8% to cryptic clades, and 1% were untypable. Moreover, 33.3% of isolates were resistant to at least one drug and 11% were multidrug resistant (MDR). The most common resistance was to tetracycline, ampicillin, and streptomycin. No clonality was detected, but the strains' high genetic heterogeneity pointed at multiple sources of microbiological contamination. MLST analysis found potentially pathogenic and even epidemic MDR strains in clams collected in class A (ST746 and ST46) and class B (ST393, ST58 and ST131) areas, indicating that strains of clinical origin are detectable in clams. These data highlight that eating raw or lightly cooked clams may pose a health risk if purification is not performed or is ineffective.

Antibiotic pressure can induce the viable but non-culturable state in Staphylococcus aureus growing in biofilms.

OBJECTIVES: Staphylococcal biofilms are among the main causes of chronic implant-associated infections. We have recently suggested that their transformation into viable but non-culturable (VBNC) forms (i.e. forms capable of resuscitation) could be responsible for the recurrent symptoms. This work aims to establish whether Staphylococcus aureus biofilms can give rise to VBNC forms capable of being resuscitated in suitable environmental conditions, the role of different stressors in inducing the VBNC state and the conditions favouring resuscitation. METHODS: S. aureus 10850 biofilms were exposed to different concentrations of antibiotic (vancomycin or quinupristin/dalfopristin) and/or to nutrient depletion until loss of culturability. The presence of viable cells and their number were examined by epifluorescence microscopy and flow cytometry. Gene expression was measured by real-time PCR. Resuscitation ability was tested by growth in rich medium containing antioxidant factors. RESULTS: Viable subpopulations were detected in all non-culturable biofilms. However, viable cell numbers and gene expression remained constant for 150 days from loss of culturability in cells from antibiotic-exposed biofilms, but not in those that had only been starved. Resuscitation was obtained in rich medium supplemented with 0.3% sodium pyruvate or with 50% filtrate of a late-log culture. CONCLUSIONS: Our findings demonstrate that S. aureus can enter the VBNC state in infectious biofilms. The presence of vancomycin or quinupristin/dalfopristin can inadvertently induce a true VBNC state or its persistence in S. aureus cells embedded in biofilms, supporting previous findings on the role of staphylococcal biofilms in recurrent infections.

Epidemic Escherichia coli ST131 and Enterococcus faecium ST17 in coastal marine sediments from an Italian beach.

Fecal indicator bacteria (FIB) are used worldwide to assess water quality in coastal environments, but little is known about their genetic diversity and pathogenicity. This study examines the prevalence, antimicrobial resistance, virulence, and genetic diversity of FIB isolated from marine sediments from a central Adriatic seaside resort. FIB, recovered from 6 out of 7 sites, were significantly more abundant at sampling stations 300 m offshore than close to the shore. Escherichia coli accounted for 34.5% of fecal coliforms, and Enterococcus faecalis accounted for 32% of enterococci. Most isolates (27% of E. coli and 22% of enterococci) were recovered from the sediments that had the highest organic content. Multidrug-resistant E. coli (31%) and enterococci (22%) were found at nearly all sites, whereas 34.5% of E. coli and 28% of enterococci harboring multiple virulence factors were recovered from just two sites. Pulsed-field gel electrophoresis typing showed wide genetic diversity among isolates. Human epidemic clones ( E. coli ST131 and Enterococcus faecium ST17) were identified for the first time by multilocus sequence typing in an area where bathing had not been prohibited. These clones were from sites far removed from riverine inputs, suggesting a wide diffusion of pathogenic FIB in the coastal environment and a high public health risk.

New sequence types and multidrug resistance among pathogenic Escherichia coli isolates from coastal marine sediments.

The spread of antibiotic-resistant microorganisms is widely recognized, but data about their sources, presence, and significance in marine environments are still limited. We examined 109 Escherichia coli strains from coastal marine sediments carrying virulence genes for antibiotic susceptibility, specific resistance genes, prevalence of class 1 and 2 integrons, and sequence type. Antibiotic resistance was found in 35% of strains, and multiple resistances were found in 14%; the resistances detected most frequently were against tetracycline (28%), ampicillin (16.5%), trimethoprim-sulfamethoxazole (13%), and streptomycin (7%). The highest prevalence of resistant strains was in phylogenetic group A, whereas phylogroup B2 exhibited a significantly lower frequency than all the other groups. Sixty percent of multiresistant strains harbored class 1 or 2 integrase genes, and about 50% carried resistance genes (particularly dfrA and aadA) linked to a class 1 integron. Multilocus sequence typing of 14 selected strains identified eight different types characteristic of extraintestinal pathogens and three new allelic combinations. Our data suggest that coastal marine sediment may be a suitable environment for the survival of pathogenic and antimicrobial-resistant E. coli strains capable of contributing to resistance spread via integrons among benthic bacteria, and they highlight a role for these strains in the emergence of new virulent genotypes.

Extraintestinal Escherichia coli carrying virulence genes in coastal marine sediments.

Despite the recognized potential of long-term survival or even growth of fecal indicators bacteria (FIB) in marine sediments, this compartment is largely ignored by health protection authorities. We conducted a large-scale study over approximately 50 km of the Marche coasts (Adriatic Sea) at depths ranging from 2 to 5 m. Total and fecal coliforms (FC) were counted by culture-based methods. Escherichia coli was also quantified using fluorescence in situ hybridization targeting specific 16S rRNA sequences, which yielded significantly higher abundances than culture-based methods, suggesting the potential importance of viable but nonculturable E. coli cells. Fecal coliforms displayed high abundances at most sites and showed a prevalence of E. coli. FC isolates (n = 113) were identified by API 20E, additional biochemical tests, and internal transcribed spacer-PCR. E. coli strains, representing 96% of isolates, were then characterized for genomic relatedness and phylogenetic group (A, B1, B2, and D) of origin by randomly amplified polymorphic DNA and multiplex-PCR. The results indicated that E. coli displayed a wide genotypic diversity, also among isolates from the same station, and that 44 of the 109 E. coli isolates belonged to groups B2 and D. Further characterization of B2 and D isolates for the presence of 11 virulence factor genes (pap, sfa/foc, afa, eaeA, ibeA, traT, hlyA, stx(1), stx(2), aer, and fyuA) showed that 90% of B2 and 65% of D isolates were positive for at least one of these. Most of the variance of both E. coli abundance and assemblage composition (>62%) was explained by a combination of physical-chemical and trophic variables. These findings indicate that coastal sediments could represent a potential reservoir for commensal and pathogenic E. coli and that E. coli distribution in marine coastal sediments largely depends upon the physical and trophic status of the sediment. We conclude that future sampling designs aimed at monitoring the microbiological quality of marine coastal areas should not further neglect the analysis of the sediment and that monitoring of these environments can be improved by including molecular methods as a complement of culture-based techniques.

Nosocomial outbreak of systemic candidosis associated with parenteral nutrition.

Eight patients in two surgical units developed systemic candidosis during a 40-day period from June 5 to July 13, 1987 (in five cases Candida albicans was identified). Three of them died. All cases belonged to a group of 27 patients receiving parenteral nutrition (PN), while among the 108 patients who did not receive PN, no cases were observed (p = .000001). Candida was cultured from two PN bags administered to the cases. A specialized nutrition nurse was responsible for the PN compounding and for maintaining administration sets in the two wards involved. An epidemiological investigation, in which 19 uninfected patients who had had PN were used as controls, showed no significant difference between cases and controls except that lipids were more frequently added to bags administered to cases (p = .0005). Furthermore, the bags administered to cases contained a higher average number of multidose constituents (p = .0008) when the comparison was focused on the two days before the onset of symptoms. Given the favorable medium provided by lipids, even a low level contamination of PN solutions during compounding and/or administration could have been responsible for the exposure of cases to multidose vials suggests, although not conclusively, that an extrinsic contamination occurred during compounding. Six isolates of C albicans were available from four cases. C albicans was cultured from the pharyngeal swabs of two physicians and three nurses, including the specialized nutrition nurse.

Clinical distribution and antibiotic sensitivities of staphylococcal strains isolated over an eight-month period.

A total of 842 staphylococci isolated from clinical material over an eight-month period and regarded as probable pathogens were identified according to lyogroup. Almost half the isolates belonged to lyogroups other than lyogroup I (Staphylococcus aureus), suggesting that coagulase-negative staphylococci are increasingly involved in human infections. All isolates were tested for sensitivity to 12 antibiotics. A greater resistance was observed in non-lyogroup I isolates, which again suggests a pathogenic significance of coagulase-negative staphylococci. Only lyogroup I strains, however, were obtained more frequently from clinical isolates than from healthy human skin. The distribution of the isolates in each lyogroup according to their clinical source is reported.

Genotypic characterization of a nosocomial outbreak of VanA Enterococcus faecalis.

Despite growing concern about vancomycin-resistant enterococci (VRE) as nosocomial pathogens, especially in the United States, in Italy VRE still represent an uncommon and occasional experience for most diagnostic laboratories. We report a genotypic characterization of the first reported nosocomial outbreak of VRE in Italy. Some experiments, including plasmid analysis and pulsed-field gel electrophoresis (PFGE) assays, aimed at investigating the genetic relatedness of the VRE isolates. Other experiments, based on hybridization and polymerase chain reaction (PCR) assays, aimed at characterizing the vancomycin resistance determinants. Over a 6-month period, 21 VRE, all identified as Enterococcus faecalis, were isolated from eight patients (all treated earlier with glycopeptide antibiotics) in a neurosurgical intensive care unit. All isolates had the same biochemical profile and antibiotic susceptibility pattern, including high-level resistance to aminoglycosides and vancomycin and teicoplanin MICs of 256 and 128 micrograms/ml, respectively. Three plasmids, one strongly hybridizing with a vanA probe, were detected in all but the last of the 21 VRE isolates. The last isolate of the cluster lacked the smallest of the three plasmids. Similar restriction profiles were obtained after plasmid DNA digestion with several endonucleases, with minor differences appreciated only in the first and last isolates. Analysis of genomic DNA restriction fragment patterns by PFGE confirmed that the reported cluster of VRE isolations was due to a single nosocomial strain of E. faecalis, despite some modifications in plasmid DNA at the beginning and at the end of the outbreak. Completely different PFGE patterns were yielded by vancomycin-susceptible E. faecalis strains isolated during the same period from inpatients in the same intensive care unit. Hybridization experiments with vanA and vanS-vanH probes and DNA amplification assays using 14 PCR primer pairs specific for vanA cluster genes (vanR, vanS, vanH, vanA, and vanY), orf1, orf2, vanB, and vanC showed identical organization of resistance determinants in all epidemic VRE isolates. This organization appeared to be the same as that described for Tn1546 in VanA prototype strain E. faecium BM4147.

An electron microscopic study of clinical and laboratory-derived strains of teicoplanin-resistant Staphylococcus haemolyticus.

Staphylococcal resistance to glycopeptides (which involves more teicoplanin than vancomycin) is uncommon and largely confined to Staphylococcus haemolyticus, an emerging nosocomial pathogen with a tendency to develop antibiotic resistance. In this study, six S. haemolyticus strains, including two isogenic pairs of teicoplanin-susceptible/-resistant strains and two resistant clinical isolates, were used in a morphologic and morphometric electron microscope investigation. Cells from both clinical and laboratory-derived teicoplanin-resistant strains exhibited abnormally roughened, irregular outlines when observed by transmission electron microscopy. However, no significant differences in cell wall thickness resulted from morphometric analysis when the susceptible/resistant cells of the two isogenic pairs were compared. By scanning electron microscopy, an abnormally roughened, blistered surface was associated with teicoplanin-resistant cocci. A certain variability was noted between strains, not clearly related to the resistance level. In freeze-fracture investigations, a higher number per square micrometer of intramembrane particles, more significant in the E than in the P membrane fracture face, was observed in the laboratory-derived resistant clones as compared to susceptible parent strains. Further studies are needed to understand the cause-effect relation between these ultrastructural alterations and staphylococcal resistance to teicoplanin (but not to vancomycin).

Export of Bordetella pertussis serotype 2 and 3 fimbrial subunits by Escherichia coli.

Bordetella pertussis serotype 2 and 3 fimbrial subunits were expressed and exported in Escherichia coli using the recently described expression/secretion vector pCGV1. Two protease deficient E. coli strains (CAG629 and EC538) and two periplasmic-leaky mutants (AE84064 and A593) were transformed with the different constructs and, after thermal induction, proteins present in the various cellular compartments were analyzed by Western blot. The results obtained with the two types of fimbrial subunits were generally the same: a recombinant protein of the expected molecular mass (19.2 kDa) was present in the periplasm of the leaky mutants and of CAG629 strain (Ion protease- and heat shock protease-deficient). Only the expression of the recombinant fimbrial subunits by the tolB A593 mutant resulted in protein release into the extracellular medium. These results indicate that the use of hybrid plasmids based on pCGV1 in combination with the tolB mutant constitute an efficient system for the export of recombinant proteins.

Selection of resistance to glycopeptide antibiotics in staphylococci.

Clinical strains belonging to ten Staphylococcus species were investigated for their abilities to develop single-step resistance in vitro to vancomycin and teicoplanin. Surviving clones were only recovered from strains of three species, namely S. aureus, S. epidermidis, and S. haemolyticus. A similar ratio of grown to plated cells (approximately 1 x 10(-8)) was mostly obtained from strains of S. aureus and S. epidermidis. Higher ratios (1 x 10(-6) to 1 x 10(-7)) were obtained from strains of S. haemolyticus, especially when exposed to teicoplanin. When tested for susceptibility, many survivors exhibited vancomycin and teicoplanin minimum inhibitory concentrations (MICs) below the drug concentration used for in vitro selection, probably due to an inoculum effect in the plating procedure. However, MICs were particularly high in many clones of S. haemolyticus (up to 12.8 microg/ml for vancomycin, and > or = 102.4 microg/ml for teicoplanin).

Detection of viable but non-culturable staphylococci in biofilms from central venous catheters negative on standard microbiological assays.

Viable bacteria were sought in 44 Maki-negative biofilms from central venous catheters (CVCs) using epifluorescence microscopy after live/dead staining. Thirty (77%) samples contained viable but non-culturable (VBNC) cells; the majority were positive on real-time PCR specific for Staphylococcus epidermidis (one also for Staphylococcus aureus). Viable cells were significantly (p<0.01) associated with CVCs from febrile patients, three of whom showed S. epidermidis-positive blood cultures, suggesting that CVC-associated biofilms can be reservoirs for staphylococci in the VBNC state. The possible role of VBNC staphylococci in persistent infections related to medical devices requires further investigation.

Bacteria associated with the rapid tissue necrosis of stony corals.

The rapid tissue necrosis (RTN) is a common disease of both wild and captive stony corals, which causes a fast tissue degradation (peeling) and death of the colony. Here we report the results of an investigation carried out on the stony coral Pocillopora damicornis, affected by an RTN-like disease. Total abundance of prokaryotes in tissue samples, determined by epifluorescence microscopy, was significantly higher in diseased than in healthy corals, as well as bacterial counts on MB2216 agar plates. Further experiments performed by fluorescent in situ hybridization using a 16S rDNA Vibrio-specific probe showed that vibrios were significantly more abundant in diseased than in healthy corals. Accordingly, bacterial counts on TCBS agar plates were higher in diseased than in healthy tissues. 16S rDNA sequencing identified as Vibrio colonies from diseased tissues only. Cultivated vibrios were dominated by a single ribotype, which displayed 99% of similarity with Vibrio harveyi strain LB4. Bacterial ribotype richness, assessed by terminal-restriction fragment length polymorphism analysis of the 16S rDNA, was significantly higher in diseased than in healthy corals. Using an in silico software, we estimated that a single terminal restriction fragment, putatively assigned to a Vibrio sp., accounted for > 15% and < 5% of the total bacterial assemblage, in diseased and healthy corals respectively. These results let us hypothesize that the RTN in stony corals can be an infectious disease associated to the presence of Vibrio harveyi. However, further studies are needed to validate the microbial origin of this pathology.

VanA-type enterococci from humans, animals, and food: species distribution, population structure, Tn1546 typing and location, and virulence determinants.

VanA-type human (n=69), animal (n=49), and food (n=36) glycopeptide-resistant enterococci (GRE) from different geographic areas were investigated to study their possible reservoirs and transmission routes. Pulsed-field gel electrophoresis (PFGE) revealed two small genetically related clusters, M39 (n=4) and M49 (n=13), representing Enterococcus faecium isolates from animal and human feces and from clinical and fecal human samples. Multilocus sequence typing showed that both belonged to the epidemic lineage of CC17. purK allele analysis of 28 selected isolates revealed that type 1 was prevalent in human strains (8/11) and types 6 and 3 (14/15) were prevalent in poultry (animals and meat). One hundred and five of the 154 VanA GRE isolates, encompassing different species, origins, and PFGE types, were examined for Tn1546 type and location (plasmid or chromosome) and the incidence of virulence determinants. Hybridization of S1- and I-CeuI-digested total DNA revealed a plasmid location in 98% of the isolates. Human intestinal and animal E. faecium isolates bore large (>150 kb) vanA plasmids. Results of PCR-restriction fragment length polymorphism and sequencing showed the presence of prototype Tn1546 in 80% of strains and the G-to-T mutation at position 8234 in three human intestinal and two pork E. faecium isolates. There were no significant associations (P>0.5) between Tn1546 type and GRE source or enterococcal species. Virulence determinants were detected in all reservoirs but were significantly more frequent (P<0.02) among clinical strains. Multiple determinants were found in clinical and meat Enterococcus faecalis isolates. The presence of indistinguishable vanA elements (mostly plasmid borne) and virulence determinants in different species and PFGE-diverse populations in the presence of host-specific purK housekeeping genes suggested that all GRE might be potential reservoirs of resistance determinants and virulence traits transferable to human-adapted clusters.

Glycopeptide resistance in coagulase-negative staphylococci.

Coagulase-negative staphylococci (CNS) were the first organisms in which acquired glycopeptide resistance was recognized. Ever since the early reports, it has been apparent that resistance to teicoplanin is more common than that to vancomycin and that resistance occurs mostly in species such as Staphylococcus haemolyticus and Staphylococcus epidermidis. The minimum inhibitory concentrations (MICs) of teicoplanin for CNS usually fall over a wide range, and, especially in some methicillin-resistant isolates of the two above-mentioned species, they can reach and even exceed the resistance breakpoint, whereas vancomycin MICs tend to remain more stable over a narrower range within the limits of susceptibility. CNS strains intermediately susceptible and even resistant not only to teicoplanin but also to vancomycin have, however, been isolated, most frequently from patients subjected to prolonged glycopeptide treatment. Laboratory detection of glycopeptide-resistant CNS may be problematic, mainly because susceptibility tests, particularly those for teicoplanin, are influenced by various technical factors, and agar diffusion tests may yield false susceptibility data. In studies with experimental glycopeptides, some molecules have exhibited improved in vitro activity compared with teicoplanin and vancomycin, but these encouraging microbiological findings have not usually been followed by in vivo trials. Stepwise and single-step exposure to teicoplanin and vancomycin has allowed stable clones for which glycopeptide MICs are increased to be obtained from susceptible CNS strains, particularly strains of Staphylococcus haemolyticus and Staphylococcus epidermidis. In these studies, resistance to teicoplanin was generally easier to obtain than resistance to vancomycin, and the levels of teicoplanin resistance were higher. Population studies have demonstrated the usually heterogeneous nature of glycopeptide resistance in CNS. Although glycopeptide-resistant CNS have been shown to differ in several features from their glycopeptide-susceptible counterparts, the exact mechanism of staphylococcal glycopeptide resistance remains unknown.

In vitro activities of three semisynthetic amide derivatives of teicoplanin, MDL 62208, MDL 62211, and MDL 62873.

MDL 62208, MDL 62211, and MDL 62873 are three semisynthetic amide derivatives of teicoplanin (MDL 62208 is an amide of teicoplanin aglycone, MDL 62211 is an amide of the teicoplanin A2 complex, and MDL 62873 is the corresponding derivative of peak A2-2 of the complex). The three semisynthetic glycopeptides were evaluated for in vitro antibacterial activity in comparison with the parent drug (teicoplanin) and vancomycin. A variety of gram-positive bacteria of clinical origin, whose species were carefully determined and that included 428 staphylococci (207 methicillin susceptible and 221 methicillin resistant), 41 streptococci, 82 enterococci, 43 strains of Listeria monocytogenes, 10 JK coryneform bacteria, and 67 anaerobes belonging to the genera Clostridium, Propionibacterium, Peptostreptococcus, and Eubacterium, were tested. The only resistances to MDL 62208, MDL 62211, and MDL 62873 were encountered with vancomycin- and teicoplanin-resistant enterococci. All of the other test strains, including some teicoplanin-resistant coagulase-negative staphylococci of the species Staphylococcus haemolyticus and Staphylococcus epidermidis, were highly susceptible to the three teicoplanin amides. Only minor differences in activity were observed among MDL 62208, MDL 62211, and MDL 62873, whereas the three experimental compounds were usually found to be more potent than teicoplanin or vancomycin (especially against staphylococci, with differences mostly ranging from 2- to 16-fold). The MBC-to-MIC ratios varied depending on the organisms, with the highest ratios usually observed for enterococci and listeriae. Overall, the MBC-to-MIC ratios yielded by the teicoplanin analogs were slightly greater than those yielded by teicoplanin or vancomycin.

News & notes: adhesiveness of Bacteroides fragilis strains isolated from feces of healthy donors, abscesses, and blood.

Bacteroides fragilis strains attached to oral epithelial cells (ECs) and the cell line Intestine 407 and associated with human phagocytes with different efficiencies depending on their source. The 58%, 75%, and 40% of strains isolated from feces, abscesses, and blood respectively adhered to ECs with good efficiency (11-40 bacteria/cell). Of the strains from feces and abscesses, 17% and 20% exhibited a high adherence (>40 bacteria/cell); however, none of the blood isolates presented this property. Similar results were obtained with the cell line Intestine 407 and human phagocytes. Of the isolates from feces, abscesses, and blood, 20%, 56%, and 71% respectively also exhibited hemagglutination ability, indicating that this property is a virulence trait more frequently present among pathogenic isolates than in commensal strains.