Conjugation-Mediated Transfer of Antibiotic-Resistance Plasmids Between Enterobacteriaceae in the Digestive Tract of Blaberus craniifer (Blattodea: Blaberidae).

Cockroaches, insects of the order Blattodea, seem to play a crucial role in the possible conjugation-mediated genetic exchanges that occur among bacteria that harbor in the cockroach intestinal tract. The gut of these insects can be thought of as an effective in vivo model for the natural transfer of antimicrobial resistance plasmids among bacteria. In our study, we evaluated the conjugation-mediated horizontal transfer of resistance genes between Escherichia coli and other microorganisms of the same Enterobacteriaceae family within the intestinal tract of Blaberus craniifer Burmeister, 1838 (Blattodea: Blaberidae). Different in vivo mating experiments were performed using E. coli RP4 harboring the RP4 plasmid carrying ampicillin, kanamycin, and tetracycline resistance genes as the donor and E. coli K12 resistant to nalidixic acid or Salmonella enterica serovar Enteritidis IMM39 resistant to streptomycin as the recipients. The RP4 plasmid was successfully transferred to both recipients, producing E. coli K12-RP4 and S. Enteritidis IMM39-RP4 transconjugants. Conjugation frequencies in vivo were similar to those previously observed in vitro. The transfer of the RP4 plasmid in all transconjugants was confirmed by small-scale plasmid isolation and agar gel electrophoresis, suggesting that the intestinal tract of cockroaches is an effective in vivo model for natural gene transfer. Our results confirm that cockroaches allow for the exchange of antimicrobial resistance plasmids among bacteria and may represent a potential reservoir for the dissemination of antibiotic-resistant bacteria in different environments. These findings are particularly significant to human health in the context of health care settings such as hospitals.

Ecological behaviour of three serogroups of Legionella pneumophila within a model plumbing system.

Three Legionella pneumophila strains isolated from water samples and belonging to serogroups (sgs) 1, 6 and 9 were analysed for their capacity to colonise an experimental model simulating a domestic hot water distribution system. Ecological factors that could influence the persistence of the sgs such as intracellular life within protozoan hosts and bacterial interference by the production of antagonistic compounds were also studied. Viable counts of L. pneumophila increased both in the planktonic and in the sessile phases. Sg 6 showed a marked prevalence during the whole experiment and exhibited the highest host infection efficiency. Sg 1 was significantly less represented, but showed the highest capacity to reproduce in the protozoan hosts. Sg 9 was poorly represented and less adapted to intracellular life. Among the 14 bacteria constantly isolated in the system, five (35.7%) produced antagonistic substances against Legionella, with differences according to the bacterial strain and L. pneumophila sgs.

Influence of Legionella pneumophila and other water bacteria on the survival and growth of Acanthamoeba polyphaga.

We investigated in solid medium, in water microcosm co-cultures and by light and transmission electron microscopy the influence of Legionella pneumophila Lp-1, Pseudomonas aeruginosa ATCC 27853, Burkholderia cepacia ATCC 25416 and Pseudomonas fluorescens SSD35 on the growth and survival of Acanthamoeba polyphaga. The infection with L. pneumophila was microscopically characterized by the presence of few bacteria inside protozoa at 4th h, and by the beginning of disruptive effects in late phase of trial. In water microcosm studies, performed at different temperature, the more significant interactions were observed at 30°C. In these conditions, L. pneumophila caused a marked reduction in trophozoite and cyst counts from the 4th day until the end of incubation (11 days). B. cepacia showed, by microscopic observation, few and generally single rods within protozoan phagosomes and caused a light reduction of trophozoite viability and cyst formation in co-cultures. A more invasive type of endocytosis, characterized by an early invasion with the presence of a high bacteria number inside amoebae, was observed for Pseudomonas strains. P. fluorescens produced a violent lysis of the host, whereas P. aeruginosa did not cause lysis or suffering. These results underline that water bacteria other than legionella are capable of intracellular survival in Acanthamoeba, influencing the protozoa viable cycle.

Detection of bacteriocin production and virulence traits in vancomycin-resistant enterococci of different sources.

AIM: Three hundred and two enterococci were isolated from food, animal and clinical samples in order to evaluate the incidence of vancomycin-resistant enterococci (VRE) and bacteriocin, cytolysin, haemolysin, gelatinase production. METHODS AND RESULTS: Among the isolates, 27 (8.9%) were VRE, and 17 (63%) of these showed, by the deferred antagonism method, bacteriocin production against gram-positive and some gram-negative indicators. Eight bacteriocin producers displayed by polymerase chain reaction an enterocin structural gene: six Enterococcus faecium the Enterocin A, two Enterococcus faecalis the Enterocin P genes. The enterocins AS-48, 31, L50 and 1071A/B genes were not found. Regarding the virulence factors, two VRE produced gelatinase and seven were haemolytic. Gelatinase gelE gene was found in 19 strains and cytolysin cylL(L) gene in eight. Among the strains showing the cylL(L) gene, only two E. faecalis expressed a beta-haemolysis. CONCLUSIONS: Our results showed the persistence of VRE in food, animal and clinical samples. Many of these strains displayed antibacterial activity and sometimes different components of virulence, which could emphasize their pathogenicity. SIGNIFICANCE AND IMPACT OF THE STUDY: This work indicates the need of a constant monitoring of enterococci in order to assess their possible pathogenic properties. The strains of interest in the food industry or used as probiotics should be tested for antibiotic resistance and virulence traits.

Glycopeptide-resistance transferability from vancomycin-resistant enterococci of human and animal source to Listeria spp.

AIMS: The glycopeptide-resistance transferability from vancomycin-resistant enterococci (VRE) of clinical and animal origin to different species of Listeria was investigated. METHODS AND RESULTS: Of 36 matings, performed on membrane filter, the glycopeptide resistance was successfully transferred in six attempts, five with donors of animal origin and only one with donors from clinical source. The acquired glycopeptide resistance in Listeria transconjugants was confirmed by the presence of the conjugative plasmid band and by the amplification of the 732-bp fragment of vanA gene in transferred plasmids. CONCLUSIONS: Despite the lower number of bacteria used in this study, the source of enterococci influenced the outcome of mating. Moreover transferred VanA plasmid induced a different expression in Listeria transconjugants, suggesting that gene expression might be influenced by species affiliation of recipients. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data strengthen the opinion that enterococci are an important source of resistance genes for Listeria via the transfer of movable genetic elements. As these strains are commonly found in the same habitats, a horizontal spread of glycopeptide resistance in Listeria spp. could be possible.

Study of two bacteriocins produced by Enterococcus casseliflavus and Ent. faecalis.

AIMS: The antimicrobial activity of two plasmid-borne bacteriocins produced by Enterococcus casseliflavus IM 416K1 and Ent. faecalis IM 388C and their mating transferability were studied. METHODS AND RESULTS: Both bacteriocins showed antibacterial activity against taxonomically related micro-organisms and Listeria monocytogenes but differ for heat sensitivity, antimicrobial titre, molecular size and class of affiliation. The transferability by mating of the antibacterial properties from producers to Enterococcus faecalis JH2-2 revealed that the bacteriocin-phenotype was linked in both strains to genes located on a 34 MDa plasmid. This result was confirmed by loss of antibacterial activity and immunity after curing treatment. CONCLUSIONS: Restriction analysis has shown a different profile of the two conjugative plasmids. Enterocin 416K1 and Enterocin 388C could represent natural antilisterial agents to use in food technology. SIGNIFICANCE AND IMPACT OF THE STUDY: The transferability of the 34 MDa conjugative plasmids might be considered a possibility for the study of bacteriocins expression in bacterial hosts different from the native strains.

Bacteriocin-producing Enterococcus casseliflavus IM 416K1, a natural antagonist for control of Listeria monocytogenes in Italian sausages ("cacciatore").

The bacteriocinogenic Enterococcus casseliflavus IM 416K1 (Bac+) isolated from Italian sausages or its bacteriocin Enterocin 416K1, with strong anti-listerial activity, were used in trials to evaluate the effect on Listeria monocytogenes NCTC 10888 in artificially inoculated Italian sausages ("cacciatore"). In trials with Enterocin 416K1 added, L. monocytogenes showed a significant reduction as compared to the control inoculated with L. monocytogenes alone. The elimination of L. monocytogenes was only obtained in sausages added with E. casseliflavus IM 416K1 Bac+.

Survival of an Aeromonas hydrophila in an artificial mineral water microcosm.

The survival capacity of an Aeromonas hydrophila strain (named SB14) isolated from mineral water was investigated in an artificial mineral water microcosm. The bacterial count of this microorganism was compared with two strains of other species from aquatic environments (Pseudomonas fluorescens SSD and Pseudomonas putida SSC) and a bacterium indicative of faecal pollution (Escherichia coli ATCC 25922). Among the strains, all added to sterile Pyrex glass flasks (1 l) to yield a final bacterial count of about 5 x 10(6) CFU/ml, A. hydrophila SB14 showed a quite strong survival capacity (150 days), even though the Pseudomonas strains were better adapted to this habitat (more than 240 days). E. coli ATCC 25922 was the least well fitted to survive and was no longer detected after 70 days. When A. hydrophila SB14 was inoculated together with one or two of the above strains, its survival appeared to be dependent on interaction with other organisms. A marked decrease in survival by 30 days, possibly due to antagonistic interaction, was observed when this microorganism was associated with E. coli ATCC 25922, and an increase by 30 and 60 days, possibly due to commensalic interaction, was obtained when A. hydrophila SB14 was inoculated with P. fluorescens SSD or P. putida SSC, respectively.

Enterocin 416K1, an antilisterial bacteriocin produced by Enterococcus casseliflavus IM 416K1 isolated from Italian sausages.

Enterococci (118) from Italian sausages were tested for the production of antimicrobial substances. Of these, 7.6% showed antibacterial activity against one or several closely related microorganisms used as indicators. Enterococcus casseliflavus IM 416K1 in particular produced a bacteriocin (Enterocin 416K1) with strong anti-listerial antagonistic activity. The bacteriocin withstood heating at 90 degrees C for 120 min and storage at 4 degrees C for 6 months. The mode of action was identified as bactericidal. The crude activity of Enterocin 416K1 was linked to a molecule with an apparent molecular weight smaller than 5 kDa. Plasmid analysis of E. casseliflavus IM 416K1 revealed the presence of four plasmids with different molecular weights (34, 11, 7 and 3.3 MDa). All the Bac- variants produced by curing experiments showed loss of the single plasmid of 34 MDa. Bacteriocin activity and immunity production may be linked to genes located on that same plasmid.

Detection and preliminary characterization of a bacteriocin (plantaricin 35d) produced by a Lactobacillus plantarum strain.

Lactic acid bacteria (134) from Italian sausages were tested for the production of antimicrobial substances (bacteriocins). Six percent of these showed antibacterial activity against one or several closely related microorganisms used as indicators. Lactobacillus plantarum 35d in particular produced a bacteriocin of high activity (320 AU ml(-1)) and a wide range of antimicrobial activity including S. aureus, L. monocytogenes, and A. hydrophila. The bacteriocin withstood heating at 80 degrees C for 120 min and storage at 4 degrees C for 6 months. The mode of action was identified as bactericidal. The apparent molecular weight of the bacteriocin extracted with n-butanol was estimated to be 4.5 kDa.

Virulence profiles and other biological characters in water isolated Aeromonas hydrophila.

Thirty water isolates of A. hydrophila were tested for potential virulence profiles, antibiotic resistance and Bacteriocin-Like Substances (BLS) production. Cytotoxic activity was present in all strains tested, 87% were hemolytic and 70% adhesive. Lysine decarboxylase reactions (LDC) positivity was correlated with virulence factors: 100% versus cytotoxicity, 84% versus adherence, 76% versus hemolytic activity. The correlation was also present in the LDC-negative strains. Hemolytic and cytotoxic activities were frequently associated: high cytotoxicity, corresponding to high hemolytic activity and vice versa. The in vitro susceptibility of A. hydrophila to 28 antibacterial agents showed that cefotaxime was the most active beta-lactam antibiotic, and Cefuroxime inhibited 90% of the strains. Isolates were resistant to Penicillin G, Ampicillin, Carbenicillin, Amoxicillin, Cephalotin and Cefaclor. Tetracycline, Chloramphenicol, Nitrofurantoine, the quinolones and the aminoglycosides (except Streptomycin) were consistently active. BLS production never emerged against closely-related microorganisms. On the contrary A. hydrophila presented a heteroinhibitory activity against non-taxonomically related genera such as Listeria spp. (L. seeligeri NCTC 11856, L. welshimeri NCTC 11857, L. ivanovii NCTC 11846) and S. aureus ATCC 25923. Although a large number of strains showed virulence determinants together with other biological characters such as antibiotic resistance and BLS production, it was not possible to relate these factors to the observed plasmids.

Antimicrobial properties and morphological characteristics of two Photorhabdus luminescens strains.

The biological properties of two Photorhabdus luminescens isolates (MU1 and MU2) of environmental source and the activity of antimicrobial agar diffusible agents (AADA) produced by the same are reported. With regard to cultural features, two variant forms for P. luminescens MU1 and three for P. luminescens MU2 (including an intermediate phase I-like form) have been found. These three forms differ in biological and biochemical properties: beta-lactamase, urease, bioluminescence and antimicrobial agar diffusible substance production associated with the phase I form, were less evident in the intermediate phase I-like MU2 and were absent in phase II form. Antimicrobial activity was present in both strains, with the production of a large amount of a diffusible compound with a wide spectrum of action against bacteria of other genera; a reduced activity against correlated species was also observed. Examination by electron microscopy of MU1 and MU2 purified broth cultures revealed the presence of particles belonging to the class of the phage tail-like bacteriocins, described in recent studies as responsible for antibacterial activity against correlated bacteria, a result never confirmed "in vitro". A plasmid of 21 Mdal was observed in all the form variants of P. luminescens MU2, suggesting that plasmids are not involved in the transition from primary to secondary phase; no plasmid was detected in P. luminescens MU1.

Streptococcus mutans: classification in bacteriocin-types.

A sample of S. mutans bacteriocins was studied to obtain a useful outline of strain typing since their synthesis has proved stable and not under plasmidial control. The inhibiting effectiveness against 9 oral streptococci and the sensitivity of mutacins produced by 49 S. mutans strains to heat, chloroform and proteasic activity were evaluated. On the basis of our results the producing strains are classified into five different types. We examine the possibility of obtaining a useful typing with bacteriocins and we discuss the choice of the most suitable number of indicators to arrange the strains in a limited cluster number for epidemiological purpose, or to classify freshly isolated S. mutans strains into bacteriocin-types.

Chlamydia trachomatis infections in asymptomatic women. Results of a study employing different staining techniques.

A total of 300 cervical smears randomly collected from asymptomatic women in a mass-screening program for the detection of cervical carcinoma was investigated for Chlamydia trachomatis infection by the use of Papanicolaou and immunofluorescence staining. Features of chlamydial infection detected in 18 cases by Papanicolaou-stained smears were confirmed in 11 cases with immunofluorescence; not a single case that was negative in the Papanicolaou-stained smears was positive by immunofluorescence. The presence of Chlamydia in the Papanicolaou-stained smears in ten cases, including two cases that were negative by immunofluorescence, was also proven by either immunoperoxidase staining or in situ hybridization. On the other hand, either immunoperoxidase or in situ hybridization gave false-negative results in two of the ten cases. Therefore, the combined use of different techniques demonstrated that false-negative results occurred with all techniques, except with Papanicolaou-stained smears, whose sensitivity is apparently the highest.

[Presence of bacteria belonging to the genus Yersinia in surface and ground water]. / Presenza di batteri appartenenti al genere Yersinia in acque superficiali e di falda.

Yersinia strains in surface waters and ground waters. After reviewing national and international writing regarding the prevalence of yersiniae in surface waters and ground waters, the Authors report data about a research carried out in the province of Modena (Italy). Over a period of 16 months (february 1986-may 1987) 89 water samples were examined (55 samples collected from rivers and streams and 34 from wells). During every sampling session two samples were collected; one was taken in a sterile bottle (1000 cm3), one by means of the Moore tampon, allowed to float in the stream for 48 h. Yersinia strains were isolated from 37% of the water samples; 33 samples gave a positive result. Forty one strains were isolated on the whole; filtration method by Millipore membrane appeared the most suitable technique in order to obtain a good recovery rate. Y. enterocolitica appeared the most represented species (43.8%), followed by Y. intermedia (21.9%) and Y. fredericksenii (17.1%). Five atypical strains were isolated, while no strain belonging to the species Y. kristensenii has been evidenced. No relevant difference in Yersinia presence appeared between surface waters and ground waters. No human pathogenic strain has been evidenced and all the isolates appeared belonging to environmental biogroups, serogroups and phage-types.

Production of bacteriocin-like substances by human oral streptococci.

A sample of human streptococci (mainly Streptococcus mutans species) from dental plaques was examined in order to evaluate the production frequency and activity spectrum of bacteriocin-like substances (mutacins). 89% of the 55 Streptococcus mutans strains produced substances with a wide spectrum of antibacterial activity. The bacteriocins produced showed a marked inhibitory activity against Gram-positive bacteria. Among the Gram-negative species tested, only Neisseria sicca was inhibited by 25% of Streptococcus mutans strains. Also, 16 strains belonging to oral streptococci other than Streptococcus mutans, were examined for their inhibitory capacity against the same indicator. The authors stress the importance of mutacins production in oral ecology and Streptococcus mutans pathogenicity.

In vitro antibacterial activity of enoxacin, a new pyridonecarboxylic acid.

The antibacterial activity of enoxacin was determined against 1015 strains of Gram-positive and Gram-negative bacteria, mainly freshly isolated from clinical specimens. The minimum inhibitory concentrations (MIC 50-75-90) were determined in comparison to three commonly used antibiotics: ampicillin, cefotaxime and gentamicin. Enoxacin has shown a broad spectrum of action and antibacterial activity in general higher that than of three currently available antibiotics. The antibacterial activity seems similar to that of other quinolones of second generation.

Conjugal plasmids in group D streptococci.

159 strains of group D streptococci isolated from clinical specimens were examinated for plasmids content. Our objective was to study some characters carried by plasmids: drug-resistance, hemolysins and bacteriocin activity. 73,6% of the strains were antibiotic resistant and in 69% of these, the drug-resistance was transferable by conjugation. In mating of S. faecalis subsp. zymogenes strains we could also isolate three different types of transconjugants in hemolytic activity. The interpretation of this observation was facilitated by the research of bacteriocin activity. We also classified the bacteriocins found in our strains into different types.

Plasmids in Streptococcus faecalis subsp. zymogenes: transferability and molecular properties.

Seven strains of Streptococcus faecalis subsp. zymogenes were studied. Plasmids originally harboured by these bacteria, codifing for drug resistance, hemolysin and gelatinase activity, were transferred to a recipient strain ( JH2 -2) by conjugation. Studies on bacteria in mating showed the formation of protoplasmatic bridges between donors and recipients; these bridges should provide for the passage of genetic material. Kinetics of acquisition of the transferable resistance were carried out in liquid medium. Plasmids harboured in donor and transconjugant strains, responsable for antibiotic-resistance, for hemolysin and gelatinase activity, were analyzed later by agarose gel electrophoresis. These analysis permitted us to demonstrate the presence of many plasmids, some conjugative with a high molecular weight and others small and non-conjugative.